Blood–Brain and Spinal Cord Barriers in Stress

15 Blood–Brain and Spinal Cord Barriers in Stress HARI SHANKER SHARMA

tension, affective disorders, namely depression, posttraumatic stress disorders, and neurodegeneration, including Alzheimer’s disease (McEwen and Stellar, 1993; Kathol et al., 1989; Charney et al., 1993; Landfield and Eldridge, 1991; see Herman and Cullinan, 1997). Interestingly, despite our increased understanding on stress-induced CNS disorders, the role of blood–brain and spinal cord barriers in stress is not well known (Table 1). The blood–brain barrier (BBB) and the blood–spinal cord barriers (BSCB) strictly maintain the fluid microenvironment of the CNS (Rapoport, 1976; Bradbury, 1979, 1990). A slight alteration in the CNS fluid microenvironment results in the abnormal function of nerve cells, glial cells, and axons (Rapoport, 1976; Bradbury, 1979; Sharma et al., 1998a,c). Studies from our laboratory suggest that microvascular barriers in the CNS are the gateway to neurological health and disease (Sharma, 1982, 1999; Sharma et al., 1998a,b). Thus, mild to moderate disturbances in brain dysfunction during experimental or disease conditions often exhibit opening of the microvascular barriers to a wide range of tracers (Rapoport, 1976; Bradbury, 1992). However, the barrier properties of the microvessels are mainly intact in the normal CNS (Rapoport, 1976; Bradbury, 1979). This indicates that disruption of the microvascular barriers is associated with brain dysfunction and brain pathology. Angel (1966) was the first to report an increased permeability of the BBB in stress. Thus, training in a water maze or starvation leads to an increased permeability of 14C in the brain of animals (Angel, 1966). Three years later, it was found that stress caused by bilateral adrenalectomy also induces an increased transport of radiotracer in the brain (Angel, 1969). The author speculated that an increased transport of tracer substances in the brain compartment during stress has some deleterious effects. Since the mid-1970s, our laboratory has been exploring the functional significance of stress-induced BBB or BSCB permeability in relation to the morphological consequences in the CNS using several animal models (Sharma and Dey, 1978, 1979, 1980, 1981, 1984). Depending on the magnitude and duration of stressors, it appears that a selective increase in BBB permeability occurs in specific brain or spinal cord regions. This chapter focuses on the functional significance of the BBB and BSCB dysfunction in stress in relation to brain pathology. Using pharmacological approaches, an attempt has been made to understand the molecular mechanisms of BBB dysfunction in stress. The Stress-induced BBB disruption appears to be mediated by several neurochemicals through receptor-mediated mechanisms. Based on our investigations

Abstract The blood–brain and spinal cord barriers strictly regulate the microfluid environment of the central nervous system (CNS). A slight alteration in the CNS microfluid environment results in abnormal neuronal function. Several short- or long-term stressful conditions are associated with immediate early gene expression, alterations in neurochemical transmission, and impairment of the microvascular barrier permeability. A possibility exists that stress-induced breakdown of the microvascular barriers is one of the most important events leading to neurodegeneration. Based on our investigations and in the light of recent knowledge, the functional significance of microvascular permeability disturbances in stressful situations in relation to brain damage is discussed. I. Introduction The term “stress” is defined as any external or internal factor(s) that results in perturbation of the physiological and psychological homeostasis of the organisms (Selye, 1936; Chrousos and Gold, 1992; Friedman et al., 1995). Thus, anxiety to posttraumatic experiences are known as stress-related disorders (Selye, 1976; Foa et al., 1992) that can impair cognitive functions (Gazzaniga, 1995; McEwen and Sapolsky, 1995). Stress activates or inhibits a select group of neurons or systems within the brain (Sapolsky, 1992; Sharma and Dey, 1987a, 1988; Sharma, 1999). Prolonged excitation/inhibition of nerve cells causes brain dysfunction, leading to brain pathology and neurodegeneration (Sapolsky, 1992, 1996a; Sharma et al., 1998a; Sharma, 1999). In addition, alterations in hormones and neurotransmitters in stress result in impaired neuronal activity (Sapolsky, 1996b; Sharma and Dey, 1988; Sharma et al., 1998a; Sharma, 1999; Sharma and Westman, 2000). Thus, long-term exposure to stress is often associated with central nervous system (CNS) disorders (Selye 1976). Stress is perceived by all living things and includes imposition of physical changes, leading to either negative (life threatening) or positive (rewarding) effects (Selye, 1976). In both cases, a similar set of physiological changes occurs in organisms believed to be adaptive in nature. Thus, release of glucoroticoids by the adrenal gland induces alertness to the new situation in the external or internal environment and maintains homeostasis (Dallman et al., 1992). Prolongation of stress or inadequate handling leads to malfunction of the organism, causing physiological or psychological anomalies (Luine et al., 1994). Abnormal regulation of a stress response results in chronic systemic diseases, e.g., hyperBlood–Spinal Cord and Brain Barriers in Health and Disease Edited by Hari S. Sharma and Jan Westman

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Copyright © 2004, Elsevier Inc. All rights reserved.

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H ARI S HANKER S HARMA Table 1 Research Trends on the Blood–Brain Barrier (BBB) in Stress in Relation to Brain Damage Compared to Other Major Diseases Subdivisions (No. of hits) Terms used

No. of hits

Brain function

BBB function

Brain damage

Brain a

789,253

—

—

—

Spinal cord f

94,553 (12%a ) 16,797 (2 %a )

—

—

—

—

—

—

—

—

—

—

—

—

BBB# BCSFB BSCB Stress b

395 (2 %#) 62 (0.4 %#; 0.4 % f ) 21,1706 (26 %a ) 2,948 (1% b ) 4,166 (2% b )

16,991 (8 % b ) 912 (32 %c ) 214 (5 %d )

212 (0.1 % b ) 12 (0.4 %c ) 19 (0.45 %d )

389 (0.2 % b ) 2 (0.07 %c ) 9 (0.2 %d )

Forced swimming e

1,109 (0.5 %b )

338 (30 %e )

4 (0.4 %e )

0

Hypertension f

20,3401 (25 %a ) 35,326 (4 %a ) 18,7671 (23 %a ) 1,464,920 (186 %a)

7,666 (4 % f ) 13,201 (37 % g ) 4,770 (2.5 %h ) 32,353 (2.2 %k )

354 (0.2 % f ) 242 (0.7 % g ) 113 (0.06 %h ) 825 (0.05 %k )

234 (0.1 % f ) 189 (0.5 % g ) 105 (0.05 %h ) 570 (0.04 %k )

Immobilizationc stress Heat stress d

Alzheimer’s disease g Diabetes h Cancer k

Note: PubMed citations (February 2003). Figures in parentheses indicate percentage values from the respective groups. a, brain; b, stress; c, immobilization stress; d, heat stress; e, forced swimming; f, hypertension; g, Alzheimer’s disease; h, diabetes; k, cancer; f, spinal cord. #, BBB; BSCB, blood–spinal cord barrier; BCSFB, blood-CSF barrier; CSF, cerebrospinal fluid.

and in light of recent knowledge, details of cellular and molecular events leading to brain pathology in stress are presented. II. Blood–Brain Barrier (BBB): A Gateway to Neurological Diseases? The BBB mainly resides within the endothelial cells of cerebral capillaries that are connected with tight junctions and comprise high electrical resistance (about 2000 Ohm × cm2) (Rapoport, 1976; Bradbury, 1979; Crone and Olesen, 1982). The cerebral endothelial cells are surrounded by basal lamina and glial cells and normally do not contain vesicles for transendothelial transport (Fig. 1). Thus, the permeability properties of the BBB are very similar to that of extended plasma membranes (Rapoport, 1976). However, the transport of nutrients, ions, and hormones, which are essential for brain functions, is regulated between blood and brain via a specific transport system comprising molecule transporters and receptors, ionic pumps, and various enzymes present in the endothelial cells (Table 2).

Research on molecular mechanisms of BBB function is advancing rapidly with the help of investigations on endothelial cell monolayers. Upregulation of low-density lipoprotein (LDL) receptor occurs in in vitro model of the BBB (see Chapters 4 and 5) in which disruption of the mouse mdr la P-glycoprotein gene is associated with a deficiency of barrier properties and an increased sensitivity to drugs (see Chapter 7). Thus, the regulation of BBB function is complex. The functional properties of the BBB are dependent on specific cell-to-cell communications via secreted factors and cell adhesion molecules (Engelhardt and Risau, 1995). In addition, metabolic changes in the brain, altered neuronal activity, and circulating levels of hormones, neurochemicals, and cytokines also influence BBB function (see Chapters 3 and 5). Prolonged exposure to stressful situations alters the brain extracellular environment, causing mental dysfunction (Sapolsky, 1996). Impairment of the BBB function in neuropsychiatric and neurological diseases is in line with this idea (Table 3). Leakage of proteins into the brain fluid microenvironment induces vasogenic edema formation and causes cell

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Fig. 1 Cerebral endothelial cells and their surroundings. (a) Diagram showing spatial relationships between cerebral capillary, neuron, glial cells and the extracellular space around them . The microvascular endothelial cells (E) in the brain are surrounded by thick basement membrane (B) and glial cells. More than 85 % of the cerebral capillary is covered by the glial cells (Modified after Schmidt 1978; Sharma 1982; 1999). (b) Schematic drawing of the ultrastructural aspects of one brain capillary and one general capillary. The endothelial cells (E) of cerebral capillaries are connected with tight junctions and normally do not contain microvesicles for vesicular transport as compared to the non cerebral capillary. The endothelial cells of the cerebral capillaries are also covered with a thick layer of basement membrane (B) compared to the general capillary (Modified after Rapoport 1976; Sharma 1982; 1999). c. Possible routes of tracer transfer across the microvascular endothelial cells of the CNS. In normal capillary, tracer is mainly confined within the lumen (Left). During osmotic shrinkage of the endothelial cells or an abrupt increase in transmural pressure causes widening of tight junctions (2nd from left). In these conditions leakage of tracer substances occur mainly due to widening of the tight junctions (see Rapoport 1976 for details). In several experiential or clinical situations leakage of tracers may occur through the endothelial cell membrane without widening of the tight junctions. (3rd from left) An increase in the cell membrane permeability and/or stimulation of vesicular transport across the cerebral endothelium plays major roles in tracer transfer from blood to brain (see Rapoport 1976; Bradbury 1979). Increased endothelial cell membrane permeability appears to be common in many cases of experimental and clinical cases involving acute or chronic brain damage. In many other acute and chronic vascular diseases or arterial hypertension both widening of the tight junctions and an increased endothelial cell membrane permeability and/or vesicular transport (extreme right) are responsible for leakage of tracers into the cerebral compartment (see Rapoport 1976 for details). It appears that the permeability properties and ultrastructural characteristics of microvascular endothelial cells and their surrounding are more or less similar in the brain and spinal cord. Leakage of endogenous serum proteins is often associated with vasogenic edema formation and cell injury (see Sharma et al., 1998). Modified after Sharma (1982, 1999).

injury (see Sharma et al., 1998a; Sharma, 1999). Expose of cellular components of the brain to vascular elements due to leaky BBB induces adverse immunological, ionic, biochemical, and cellular reactions. Thus, maintenance of a normal BBB function is crucial for CNS health and disruption of it leads to disease. III. Blood–Brain Barrier in Stress Investigations on the BBB in stressful situations have been largely ignored in the past (see Tables 1 and 3). Increased local cerebral blood flow (CBF) and glucose utilization are seen during mental activity (Belova and Jonsson, 1982; Bradbury,

1979; Rapoport, 1976). However, the status of BBB function following extensive mental activity or stimulation of different brain regions is not well known (Basch and Fazekas, 1970; Bondy and Prudy, 1974). Experimental evidence suggests that the BBB is modified in specific brain regions in certain stressful conditions (Angel, 1966, 1969; Basch and Fazekas, 1970; Bondy and Purdy, 1974; Christensen et al., 1981; Cutler et al., 1968; Gilbert, 1965; Lorenzo et al., 1965; Selye, 1976). Thus, stimulation or lesion of central catecholaminergic neurons (Raichle et al., 1979), photic stimulation, and convulsive agents increase the uptake of radiotracers from the blood into specific brain areas (Bondy and

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H ARI S HANKER S HARMA Table 2 Molecular Aspects of BBB Function a Enzyme

Transporter/receptor

Antigen

γ-Glutamyl transpeptidase Aminopeptidase-N

Glucose transporter amino acid transporter

EBA HT7/OX-47/Basigin

Alkaline phosphatase

transferrin receptor

PC-1

Butyryl cholinesterase Monoamine oxidase

LDL receptor Insulin receptor

Thrombomodulin Meca 32

Diphosphopyridine nucleotide Diaphorase

Na+-K+-ATPase ion channels

ApoA1

Lactate dehydrogenase Malate, α-glycerophosphate Glutamate and glucose 6-phosphate Dehydrogenase Succinic dehydrogenase β-hydroxylbutyrate and Ethanol dehydrogenase Acid phosphatases, ATPase DOPA-decarboxylase Inosine diphosphatase Acetylcholinesterase α-Ketoglutarate transaminase Carboxyl esterases b a Modified after Rapoport (1976), Sharma (1982, 1999), and Engelhardt and Risau (1995). b Fetal rat capillaries only.

Purdy, 1974; Cutler et al., 1968). A selective increase in BBB permeability is also seen as early as 1 h after monocular eyelid suturing in 1-day-old chicks (Bondy and Purdy, 1974). Using immobilization (Sharma and Dey, 1978, 1979, 1981, 1986a), heat exposure (Sharma and Dey, 1978, 1984, 1986b, 1987b), and forced swimming (Sharma and Dey, 1979; Sharma et al., 1991a), the concept of a selective and specific increase in BBB permeability in stressful stimuli is further elaborated in our laboratory. Our results suggest that stress selectively increases BBB permeability in specific brain regions. However, the status of BSCB function in stress has largely remained unknown. IV. Neurochemical Mediators of the BBB Several neurochemicals are released during stressful situation that may influence the BBB function (Wahl et al., 1988). Thus, the influence of various vasoactive substances has been examined in the past on BBB function by applying them on luminal or abluminal surfaces of the cerebral microvessels (Table 4). Interestingly, most of these neurochemicals that disrupt BBB function are capable of inducing vasogenic edema formation (Rapoport, 1976; Bradbury, 1979; Wahl et al., 1988). Edema is one of the common complications in several brain diseases and is life-threatening, particularly following traumatic, ischemic, or metabolic brain injuries in which the leakage of plasma proteins is most pronounced (Rapoport, 1976; Bradbury, 1979; Sharma et al., 1994ab, 1997c, 1998a). It appears that neurochemical receptors play important roles in BBB disruption and brain edema formation (Sharma and

Cervós-Navarro, 1990a; Sharma et al., 1994a,b, 1997c). However, a considerable interaction exists between different neurochemicals in the BBB opening. Thus, leukotrienes and arachidonic acids mediate BBB breakdown via bradykinin receptors (Sharma, 2000a; see Chapter 23). Pretreatment with histamine antagonists attenuates serotonin levels in brain and plasma and thus reduces BBB disruption (Sharma et al., 1992; for details, see Chapter 13). The BBB breakdown by nitric oxide is influenced by the generation of free radicals (Sharma et al., 1999, 2000a; Sharma, 2000b; see Chapter 14). Similarly, κ-opioid receptors regulate dynorphin-induced BBB disruption (see Chapter 23). This indicates that no single chemical compound alone is responsible for BBB disruption in vivo. Several neurochemicals may act synergistically or antagonistically on the cerebral microvessels to influence BBB function in experimental or clinical situations. V. Routes of Leakage of the BBB The passage of tracer transport across the BBB in experimental or in disease conditions is still controversial (Rapoport, 1976; Bradbury, 1979; Bradbury, 1992). Important contributions in the field came from electron microscopical studies of the cerebral endothelium under normal and pathological conditions (Brightman and Reese, 1969; Brightman et al., 1970). These early works showed that the permeability of the BBB is increased in different conditions by enhanced vesicular transport. Observations in several experimental and clinical cases also favor the hypothesis of BBB breakdown via transendothelial cell transport (Bradbury, 1979). However, opening of the

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Table 3 Summary of Various Experimental and Disease Conditions in which the BBB is Disrupted to Various Tracers a Disease/condition

BBB breakdown

Possible mechanism

Serum proteins HRP microperoxidase Lanthanum radiotracers

Vesicular transport Endothelial cell permeability

HRP, radiotracers Evans blue Trypan blue Lanthanum

Vesicular transport b widening of tight junctions endothelial cell permeability

HRP, Evans blue Lanthanum, microperoxidase radiotracers

Vesicular transport b widening of tight junctions endothelial cell permeability

Radiotracers, HRP Evans blue, Lanthanum microperoxidase

Widening of tight junctions b vesicular transport endothelial cell permeability d

Radiotracers, Evans blue, microperoxidase

Vesicular transport b widening of tight junctions endothelial cell permeability

Radiotracers, Evans blue, microperoxidase

Vesicular transport b widening of tight junctions endothelial cell permeability

G. Toxicity to chemicals Diodrast, iodopyracet, mercuric chloride, nickel chloride, lead, manganese

HRP, Evans blue, radiotracers

Vesicular transport, endothelial cell permeability

H. Lesion/stimulation Locus coeruleus

Water

Not known

I. Vascular diseases Hypertension b (mechanical, chemical or metabolic) hypotension, carotid artery occlusion, air embolism, gas embolism, atherosclerosis, periarteritis nodosa, thromboangitis obliterans, diabetic vasculitis

Radiotracers, Evans blue, HRP, Lanthanum

Widening of tight junctions b endothelial cell permeability vesicular transport

J. Loss of autoregulation Acute hypertension, hypertensive encephalopathy, intracranial hypertension, hypovolaemic shock, hypervolaemia

HRP, Evans blue, radiotracers

Widening of tight junctions b vesicular transport, endothelial cell permeability d

K. Autoimmune diseases Viral encephalitis, experimental allergic encephalomyelitis, polyneuritis, multiple sclerosis

HRP, Evans blue, radiotracers

Widening of tight junctions d vesicular transport

L. Stressful situations Immobilization c, forced swimming c, heat exposure c, seizures, training in water maze, adrenalectomy, electroconvulsive shock, morphine withdrawal/dependence d

HRP, Evans blue radiotracers Lanthanum

Endothelial cell permeability vesicular transport widening of tight junctions d

Evans blue, HRP Evans blue, HRP, sucrose

Vesicular transport endothelial cell permeability

A. Neurodegeneration Alzheimer’s disease, brain tumors, neoplasms, schizophrenia, dementia, ischemia, infarction peripheral nerve lesion, leukemia

B. Trauma Mechanical, hypoxia, hyperoxia, ischemia, metabolic insults, incision c, stab wounds, concussion, cryogenic lesions, thermocoagulations C. Influence of chemicals Serotoninc, histamine protamine, norepinephrine, 5-HTP c, bradykinin, prostaglandins, leukotrienes, glutamate, L-NAME, chemical induced convulsions, cAMP, dibutyric cAMP, adrenaline, 6-OHDA, indomethacin, bicuculline, angiotensin, amphetamine, matrimonial, pentylenetetrazol D. Hyperosmotic solutions Infusion of various electrolytes, nonelectrolytes

E. Irradiations X-ray irradiation, α, β particle irradiation, microwave irradiation, ultrasonic irradiation F. Drugs and venoms Alcohol and other lipid solvents, bile salts, saponin, lysolecithin, cobra venoms, E. coli endotoxin

M. Electromagnetic radiation Mobile telephony Microwave radiation

a Compiled from Rapoport (1976), Sharma (1982, 1999), and Sharma et al. (1998a). b Known to occur. cAuthors own investigations d Not shown yet

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H ARI S HANKER S HARMA Table 4 Neurochemical Mediators of BBB Dysfunctiona

Chemical mediators Bradykinin Arachidonic acid

Receptors mediated B2-kininergic –

BBB permeability tracers Na+-Fluroscein

Signal transduction mechanisms PI, PKC –

Na+-Fluorescein

Vasogenic edema Yes Yes

FITC-dextran Free radicals

–

EBA EBA, Na-fluorescein

–

Yes

Leukotrienes Cytokines

– –

EBA, Na-fluorescein Na-fluorescien

– –

? ?

Histamine

H2

EBA, Na-fluorescein

Serotonin b

5-HT2

FITC-dextran, HRP EBA, HRP, La++

cAMP cAMP

Yes Yes

Endothelin

ET1

51Cr, 45Ca

prostanoids cAMP, Ca++

?

Nitric oxide b Dynorphin b

– –

MEAP, EBA EBA, La++

– –

Yes Yes

aModifed after Sharma et al. (1998a,c). bAuthors own investigations.

tight junctions is equally important in some conditions (Fig. 1c). Thus, hyperosmotic shrinkage of the cerebral endothelium or increases in transmural pressure in the microvessels during hypertension disrupt the tight junctions (Rapoport, 1976; Fig. 1c). However, in these conditions, transendothelial cell transport is also increased in addition to the widening of the tight junctions (Bradbury, 1992). Thus, increased vesicular transport and widening of the tight junctions in various pathological conditions could contribute to BBB disruption. There are indications that neurochemical mediators influence transendothelial cell membrane permeability though receptor-mediated mechanisms. However, their role in tight junctional permeability remains unclear.

However, restriction of movement, e.g., restraint or immobilization, peripheral nerve lesion, or denucleation of one eye, requires prolonged exposure time to induce BBB disruption (Sharma, 1982; Sharma and Dey, 1986a; Bradbury, 1990). Increased water permeability in the brain following degeneration of noradrenergic neurons or stimulation of locus coeruleus further supports the idea that disturbances in IPS lead to alterations in BBB function. It appears that neurotransmitter and neuromodulator substances released during stress are the probable link between IPS and BBB dysfunction. Thus, release of CRH following immobilization stress is able to disrupt BBB function (Esposito et al., 2002).

VI. Stress Influences Information-Processing System of the Central Nervous System

VII. Are Stress Effects Variable or Nonspecific?

Stress influences brain function by altering its informationprocessing system (IPS) (Sapolsky, 1996). Stressors, e.g., forced swimming, running, and exposure to hot or cold environments, induce an overload to the IPS and thus initiate several cellular and molecular mechanisms to counteract this process. In case of excessive overload causing impairment of the IPS, alterations in the CNS microenvironment will occur, precipitating brain diseases (Fig. 2a). Likewise, an underload on the IPS, namely blinding of one eye, or peripheral nerve lesions can also impair the IPS and cause alterations in the CNS microenvironment (Fig. 2a). Both over- and underload situations on the IPS result in compromised BBB function (see Table 3). The magnitude and severity of stressors are thus crucial factors in inducing time-related alteration in BBB disruption. Foot electroshock, electrical, or chemical-induced seizures or training in a water maze causes BBB breakdown in a very short time (Rapoport, 1976; Sharma, 1982; Sharma et al., 1991a).

Stress is part of our daily life, which includes several dissimilar events such as fear, frustration, sorrow, joy, fatigue, pain, mental or physical efforts, and related happenings. However, the body responds to all these diverse events in almost identical ways. Thus, the effects of pleasant (eustress) or unpleasant (distress) stressors on the body function are mainly similar in nature (Selye, 1976). It has been suggested that there cannot be different types of stress (Selye, 1976). Thus, terms such as “emotional stress,” “heat stress,” “cold stress,” “swimming stress,” “immobilization stress,” “surgical stress,” “sleep deprivation stress,” and other kind of stresses denote stress produced by these stressors (Selye, 1976). The effect of stress on the body largely depends on (i) nonspecific effects, as well as (ii) the specific effects of these stressors (Selye, 1976). In addition, stress-induced effects are finally influenced by several conditioning factors, such as age, sex, and genetic predisposition (endogenous factors) or diet, drugs, or hormones (exogenous factors) (Selye, 1976).

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Stress BBB

Information Overload Central Nervous System Information Optimal

Homeostasis Intact BBB

Blood-Brain Barrier Information Processing System (IPS)

Stress BBB

Information underload

b

BBB

ces? rban

VIII. Stages of Stress Response Stage of Resistance

Alarm Reaction

Stage of Exhaustion

Reversible?

Irreversible

Duration of Stress Neuronal cell death

BBB disturbances?

Morphological changes, Neuronal, dendritic changes

Synaptic plasticity

Alarm reaction

Transient

Under the influence of these external or internal factors, any stressor or adverse conditions can induce pathogenesis and produce disease of adaptation that affects the specific parts of the body according to the sensitivity of the specific stressors or to the aforementioned conditioning factors (for details, see Selye, 1976). Molecular mapping of genes and proteins that are activated by stress in different regions of the CNS is in good agreement with this hypothesis (see later).

distu

Normal Level of Resistance

c

237

Severity of Stress

Arousal, Emotion

Mild

Moderate

Severe

Strong

Chronic

Fig. 2 Influence of stress on diseases processes. (a) Stress depending on its magnitude and severity can induce CNS dysfunction. The information processing system (IPS) of the CNS can handle certain level of stress without showing symptoms (optimal information). Inadequate handling of stress due to information overload (psychological, environmental, physical exercise or hyperactivity, etc) may impair IPS and the blood–brain barrier (BBB) permeability. Likewise, stressors causing an information underload (hypoactivity, peripheral nerve transection, suturing of one eye-lid, etc.) will also perturb IPS and the BBB function (for details, see Sharma et al., 1998a,c). (b) Three stages of stress response. Effects of stress on the organisms can be divided into 3 stages. The initial reaction of an information overload induces alarm reaction showing profound symptoms. When the stress is further continued with same intensity, the symptoms disappear after some time leading to stage of adaptation. Further continuation of stress may finally lead to stage of exhaustion in which the symptoms of alarm reaction may re-appear and results in death of the organism (for details, see text, Selye 1976). Stress induced alterations in the BBB disruption may appear at the initial stages of alarm reaction. Modified after Sharma 1982; 1999. (c) Stages of stress response in clinical situations. Mild stress induces short-term alterations in the arousal and emotional response that can affect learning and memory processes. When the magnitude, severity and/or duration of stress increases several transient and/or permanent changes (synaptic plasticity, changes in neuronal structure and circuitry as well as neurotoxicity) occur in the CNS (for details, see McEwen and Sapolsky 1995; Herman and Cullinam 1997; Kim and Yoon 1998). BBB dysfunction can be seen after moderate level of stress overload and may be crucial for short-term or permanent structural changes in the CNS. Modified after Herman and Cullinam (1997); Kim and Yoon (1998); Sharma (1999).

The effect of “acute” stress on the organism is often entirely different and sometimes opposite effects are seen in comparison to “chronic” exposure of the same stressor. This has led to categorize the whole stress response in three stages (Selye, 1976). Some effects, such as adrenal enlargement, gastrointestinal ulcers, and thymicolymphatic involution, invariably occur in response to any stressor. These changes are described by Selye (1976) as “general adaptation syndrome (GAS).” On the basis of acute or chronic stress effects until exhaustion, Selye (1936) proposed three different stages of stress response that are still valid (Sharma, 1982, 1999). The initial stress response that induces an immediate reaction in the organisms is known as (i) an alarm reaction and, if the stress is continued further, the stage of (ii) adaptation ensues. Continuous prolongation of stress beyond the stage of adaptation leads the organism to the stage of (iii) exhaustion, causing death (Selye, 1936, 1976; see Fig. 2b). The duration and appearance of these stress responses mainly depend on the magnitude and intensity of the primary stimulus and other external or internal factors (as mentioned earlier). IX. Brain Dysfunction: Stages of Stress Response? It is possible that the three stages of stress responses are applicable on CNS dysfunction in clinical situations as well (see later). Thus, the intensity and duration of stress mainly determine the magnitude and severity of CNS pathology. Prolonged exposure of mild stress or short exposure of moderate to severe stress may induce similar symptoms (Fig. 2c). Major depressive illnesses, affective disorders, and several neurodegenerative diseases, e.g., Alzheimer’s disease, dementia, and schizophrenia, represent lifetime exposure to stress (see later). Whether stress is beneficial or harmful is still a matter of debate. When the stress is well tolerated and the initial response is not severe enough, adaptive changes will occur. However, if the stress is severe enough that it cannot be tolerated by the organisms, as evident with profound alarm reactions, adverse brain function will ensue (Fig. 2). However, if the stress still continues with the same intensity, then the alarm reaction will gradually subside over time, leading to an adaptive phase. In this phase, all the CNS symptoms and reactions disappear and the organisms do not show any sign of brain dysfunction. Moreover, if the stress prolongs further, then depending on the individual response and predisposing factors (see earlier discussion), the exhaustion phase will ensue, causing reappearance of the initial CNS reaction that will finally lead to neuropathology or neurodegenerative diseases and death (Selye, 1976; Sharma, 1982, 1999, 2000b; Sharma et al., 1998a,c).

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X. Inadequate Handling of Stress Leads to “Stress Diseases” An insufficient, excessive, or faulty response of the body to the stressors in terms of inappropriate nervous or hormonal responses by the individual or the organism leads to several diseases known as “stress diseases” (Selye, 1976). Any stressor on the organism will first produce a nonspecific response (the first mediator) that is either a nervous stimulus from the cerebral cortex, reticular formation, or limbic system, particularly the hippocampus and amygdala, or a chemical substance, the exact nature of which is not established yet (Selye, 1976). The incoming nervous stimuli act on neuroendocrine cells in the median eminence (transducer) where these signals are transformed into a humoral messenger “corticotropin-releasing factor (CRF)” that causes discharge of the adrenocroticotropic hormone (ACTH) from the adenohypophysis into the general circulation (Selye, 1976). The ACTH then acts on the adrenal cortex to release glucoroticoids that provide energy due to the increased necessary demands of the organism caused by the stressor (Selye, 1976). The release of ACTH from the pituitary is controlled by the level of excess ACTH in the blood (ACTH short-loop feedback mechanism) and the high level of the corticoid level (corticoid long-loop feedback mechanism). Further, the stress response is mediated by catecholamines released from autonomic nerve endings (noradrenaline) under the influence of acetylcholine and from the adrenal medulla (mainly adrenaline) (Selye, 1976; Sharma, 1982). Infusion of noradrenaline and adrenaline in a similar amount released in stress induces a short-term breakdown of the BBB function (Sharma, 1982; Abdul Rahman and Siesjö, 1979). This indicates that the stress-induced release of neurochemicals is capable of influencing the microenvironment of the CNS. XI. Involvement of Serotonin in the Stress Response Activation of the sympathetic and hypothalamic–pituitary– adrenal (HPA) axis, leading to catecholamine discharge, appears to be a nonspecific response of the stress. The catecholamines have much less significant or even negligible influence during the later phase of the stress reaction (Selye, 1976). Thus, cholinergic, adrenergic, or histaminergic blocking agents do not prevent the initial reactions of stress in rats (Guillemin, 1955; Selye, 1976; Sharma, 1982; Sharma and Dey, 1986a,b, 1987a,b). Release of other substances such as glucocorticoids, thyroxin, and serotonin seem to be involved in the later phase of stress reaction in animals. The involvement of serotonin in “stress diseases” is significant because the cardiac or renal lesions produced by sudden stress are identical to those caused by serotonin administration in rats (Jasmin and Bois, 1960; Parratt and West, 1957; Selye, 1961, 1976; West, 1957). Increased brain serotonin content is seen in a hare due to the presence of a dog (Miline et al., 1958). Cold exposure (Gordon, 1961) or ischemic shock (Medakovic and Spuzik, 1959) causes more than a twofold enhancement of the circulating serotonin levels in rats. Based on these observations, Erspamer (1966) concluded that serotonin plays important roles in stress-induced adaptation syndrome. This hypothesis is further confirmed by

studies showing an altered activity of central serotonergic and noradrenergic systems in stress (Barchas and Freedman, 1963; Welch and Welch, 1968; Bliss, 1973). Increased brain serotonin activity is observed by several workers following immobilization and forced swimming (Welch and Welch, 1968; de Schaepdryver et al., 1969; Thierry et al., 1968; Barchas and Freedman, 1963). However, no change or a decrease in brain serotonin is also seen during immobilization stress (Bliss et al., 1968, 1972; Corrodi et al., 1968; Curzon and Green, 1969; Curzon, 1971). Differences in the time point of serotonin measurements during immobilization appear to be the main reason for such a discrepancy. A profound increase in circulating serotonin levels following long-term immobilization stress (Well-Fugazza and Godefroy, 1976) suggests that the amine plays an important role in stress reaction. This is further apparent from observations in clinical cases of heat-related syndromes that are often associated with the hyperproduction of serotonin (Sulman et al., 1977). Furthermore, disturbances in serotonin metabolism are quite common in several neurological diseases (Essman, 1978; for details, see Chapter 12). Thus, an increased level of serotonin in the circulation is observed in schizophrenia (Garelis et al., 1975), infantile autism (Schain and Freedman, 1961), and mental retardation in children (Partington et al., 1973). In experimental allergic encephalomyelitis (Cazullo et al., 1969) and convulsions (Essman, 1978), an elevated brain serotonin content is quite common. Because serotonin is a neurochemical mediator of BBB and brain edema formation (Wahl et al., 1988; see later; for details, see Chapter 12), an increased level of amine in plasma or brain will influence BBB dysfunction and induce brain pathology. XII. Stress and Neuronal Dysfunction The basic function of stress response is for survival. However, prolonged stress causes profound effects on the CNS ranging from perturbations in learning and memory to neuronal cell death (Miller and Seligman, 1976; Bremner et al., 1993; McEwen and Sapolsky, 1995; Kim and Yoon, 1998). Impairment in learning and memory following stress is due to alterations in synaptic plasticity, dendritic morphology, neurotoxicity, and neurogenesis (Kim et al., 1996; Watanabe et al., 1992; Gould et al., 1998). These stress effects are mostly examined on hippocampal function, as this organ is the anatomical seat of learning and memory (Kin and Yoon, 1998; McEwen, 1999; Sharma et al., 1994b; Sharma, 1999). Interestingly, the effects of stress on learning and memory are described in several species ranging from fish to humans; its influence of BBB dysfunction and nerve cell injury is still not well explored. XIII. Stress and Hippocampal Plasticity Hippocampal formation is involved in the regulation of learning and memory (McEwen, 1999) and is highly vulnerable to noxious insults to the CNS. Damage of the hippocampus is seen following acute or repeated stress, stroke, brain injury, ischemia, and aging (Sapolosky, 1992). Acute, nonpainful novelty stress inhibits primed-burst potentiation and memory (Diamond et al., 1994, 1996c) and is able of suppressing ongoing neurogenesis in the dentate gyrus granule neurons (Cameron and Gould, 1996). These effects are involved in

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fear-related learning and memory because of the anatomical connections between the dentate gyrus and the amygdala (Ikegaya et al., 1997). However, acute or repeated stress induces a reversible atrophy of dendrites in the CA3 region of the hippocampus in rats (McEwen, 1999) and in tree shrews (Magarinos et al., 1996). Apical dendrites are involved in cognitive impairment in the learning of spatial and short-term memory tasks (McEwen and Sapolsky, 1995). Apart from stress-induced changes in the hippocampal structure, repeated stress induces reversible synaptogenesis in the CA1 region (Gazzaley et al., 1996) and atrophy of dendrites in the CA3 area (Popov et al., 1992). It appears that glucocorticoids induce the atrophy of apical dendrites in the hippocampus in the CA3 region. Atrophy of apical dendrites in CA3 pyramidal neurons following 21 days of corticosterone treatment, 6 h restraint for 21 days, or psychosocial stress in the rats is in line with this idea (see McEwen, 1999). XIV. Stress and Activation of Neurons The nerve cell is the basic unit of the CNS that exhibits shortor long-lasting changes in its phenotype in response to several external or internal stimuli. Nerve cells respond to changes in their environment due to a well-established stimulus–response IPS that is present in all living cells. This stimulus-response relationship of neurons is classified into early and late responses. Early responses occur immediately and may last from milliseconds to minutes. These early responses are triggered by several neurotransmitters and growth factors (the first messengers) acting on the cell surface receptors leading to activation of the second messenger systems. The second messenger systems activate specific protein kinases, causing phosphorylation of specific neuronal proteins (Garthwaite, 1991; Bronstein et al., 1993; Hughes and Dragunow, 1995). However, the late response occurs within hours to days, leading to permanent changes in the neurons. These changes include alterations in biological processes of learning and memory, drug tolerance and dependence, receptor sensitization, and cellular morphology. Changes in gene expression are necessary for late responses (Bliss and Collinridge, 1993; Armstrong and Montminy, 1993). The information generated by external or internal stimuli is carried out either directly through first messengers or indirectly via second messengers to the nerve cells and interacts with the cellular DNA to alter gene expression (Hughes and Dragunow, 1995). Changes in gene expression result in the production of specific mRNAs and associated proteins that can modify the phenotype of the cell. Whether specific stressors activate particular sets of neurons or induce selective gene expression in certain areas of the brain or spinal cord is still unclear. XV. Stress Induces Immediate Early Gene expression in the Central Nervous System The neuroanatomical pathways and/or excitation of particular sets of neurons following stress results in immediate early gene (IEG) expression (see Hughes and Dragunow, 1995). Some stressors have specific effects as reflected in selective IEG expression in particular sets of neurons in a certain seg-

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ment of the CNS. However, some regions in the brain result in similar types of IEG expression following different kinds of stressors. This indicates that stressors may have both specific and nonspecific effects on the CNS. A. Constitutive Expression of IEGs in Normal Brain Several IEGs and their transcription factors are expressed constitutively in a very low level in the CNS (Table 5). A mild expression of Fos in nerve cell nuclei of adult animals is often seen in the amygdala, striatum, piriform cortex, and hippocampus (Dragunow et al., 1987). A low level of constitutive Fos-B protein expression occurs in the rat cerebral cortex, striatum, amygdala, hippocampus, and dentate gyrus (Dragunow, 1990). However, expression of the Krox family, another member of IEGs, is usually high in normal CNS. Thus, neurons in the forebrain of several mammalian species express high levels of krox-24 mRNA and zif-268 mRNA and proteins (Schlingensiepen et al., 1991; Hughes and Dragunow, 1995). The highest levels of Krox-24 expression are seen in the deeper layers (IV and VI) of the cerebral cortex and in the hippocampus CA1 area (Schlingensiepen et al., 1991), whereas Krox-20 protein is expressed in high levels within the superficial layers (II and III) of the cerebral cortex, caudate-putamen, globus pallidus, and nucleus accumbens. Interestingly, Krox-20 protein is not expressed in the hippocampus (Herdegen et al., 1993). High-level expression of another IEG, c-Jun protein, is seen in neurons of the dentate gyrus of the hippocampus and in the piriform cortex of normal animals (Hughes et al., 1992; Hughes and Dragunow, 1995). Strong expression of the c-Jun mRNA occurs in the piriform cortex, dentate gyrus, and CA2–CA3 layers of the hippocampus but not in the neocortex, which exhibits a weak activity (Hughes and Dragunow, 1995). The neocortex, cerebellum, hippocampal dentate gyrus, CA1 and CA3 layers, amygdala, striatum, and thalamus often show high levels of mRNA expression for jun-B and jun-D (Mellstrom et al., 1991). B. Induction of IEGs by Various Stressors Upregulation or induction of IEGs occurs in the CNS following various stressful stimuli ranging from sensory stimulation to traumatic brain injuries (Table 6). This indicates that stressors or noxious stimuli induce IEG expression by influencing cellular DNA. These changes in neuronal structure and functions are reversible during short-term exposure and may lead to permanent degenerative changes depending on the magnitude and duration of the primary insult (see Table 6). 1. Sensory Stimulation Noxious and nonnoxious peripheral sensory stimulation induces c-fos expression in spinal cord dorsal horn neurons (Fitzgerald, 1990). Physiological stimulation of rat primary sensory neurons by hair brushing or mild joint manipulation upregulates c-Fos protein-like immunoreactivity in nuclei of postsynaptic neurons in the dorsal horn (Hunt et al., 1987). Chemical and thermal stimulation of cutaneous sensory afferents causes a strong induction of Fos in the superficial

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Table 6 Stress-Induced Immediate Early Gene Expression a

Stressor Sensory stimulation Noxious/nonnoxious Olfactory and visual (gentle hair brushing, joint manipulation), Chemical or heat stimulation of cutaneous sensory afferents Noxious stimulation with hot water of the hind paw Repeated squeezing of rat planter surface of rat hind paw Exposure to odors Visual stimuli Monoocular visual deprivation Light stimulus Photic stimulation in night

IEG protein/mRNA

Time interval

Brain region

BBB disruption

c-fos, c-Fos

Transient

Spinal dorsal horn neurons Postsynaptic neurons of dorsal horn

n.d. n.d.

Fos

Transient

Superficial layers of Rexed’s laminae I and II of the dorsal horn and layers III to V

n.d. n.d.

c-fos, c-jun, zif-268 mRNAs c-fos, jun-B mRNAs c-Jun

Transient

Neurons in Rexed’s laminae I and II, V and X, superficial layers of dorsal horn neurons Rexed’s laminae I and III neurons

n.d.

c-fos mRNA krox-24 mRNA, krox-24,c-fos, jun-B krox-24 mRNA

Olfactory bulb Visual cortex

n.d. n.d.

Monkey visual cortex

Yes?

Fos Li Fos Li, c-fos, zif-268, jun-B, jun-D, c-jun

Retinal neurons, SCN, hypothalamus Hypothalamus, visual cortex

n.d.

mRNAs Stressful situations Immobilization, Capsaicin administration, Ear clipping Isotonic saline injection

Sleep deprivation

c-fos, c-jun, jun-B Fos Li jun-D c-fos mRNA c-fos mRNA

Transient

Transient

fos-B, jun-B, c-jun, zif-268, fra-1

1 h after

c-fos, zif-268 mRNAs Fos Li, jun-B

Cardiovascular dysfunction Low blood pressure by stimulation of aortic depressor nerve or removal of blood

Electrical stimulation of vagus nerve Mechanical stimulation of carotid sinus Angiotensin i.v. infusion Learning Memory, LTP

Fos

Transient

c-fos, zif-268 mRNAs

Central amygdaloid nucleus, PVN PVN Mouse brain PVN, amygdaloid nucleus, hippocampus, neocortex

Yes n.d. No

Brain neurons Lateral dorsal tegmental, pedunculopontine Tegmental nuclei, LC, dorsal raphé, pontine, reticular formation

n.d.?

Nucleus tractus solitarius, area postrema, Venterolateral medulla, nucleus ambiguus, medullary reticular formation, parabrachialnucleus, LC, supraoptic nucleus, inferiorolive, subfornical organ, organ vasculosum, hypothalamus, central nucleus of amygdala, bed nucleus of stria terminalis, islands of Calleja Nucleus tractus solitarius, paratrigeminal nucleus

n.d.

n.d.

n.d. Yes

Fos of lamina terminalis

2h

Subfornical organ, organum vasculosum

Fra zif-268, jun-B, c-jun mRNAs (awake animals)

Transient

Dentate gyrus neurons dentate gyrus

n.d.?

n.d.

Continued

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H ARI S HANKER S HARMA Table 5 Immediate Early Gene Expression in Normal Brain a Regions of the brain

IEG protein

Intensity

IEG

mRNA

Cerebral cortex (neocortex)

Fos-B jun-B

+ +++

c-Jun

+

Piriform cortex Hippocampus

Cerebellum Caudate-putamen Globus-pallidus Nucleus accumbens Striatum

Amygdala Thalamus Hypothalamus Spinal cord

jun-D

+++

Layers II–III Layers IV–VI

krox-20 krox-24

+++ +++

krox-24

zif-268 c-Jun

+++ +++

zif-268 c-Jun

+++ ++++

+++ jun-B jun-D +++/–?

c-Jun +++ +++

Fos

+

Fos Fos-B

+ +

jun-B jun-D Dentate gyrus Fos-B Fras CA1

+++ +++ c-Jun + + krox-24

zif-268 jun-B

+++ +++

jun-D CA2 CA3 jun-B jun-D krox-20

+++ c-Jun c-Jun +++ +++ +++

krox-20 krox-20 Fos jun-D Fos-B Fos

+++ +++ + +++ + +

Fos-B jun-B jun-D n.d/? c-fos

+ +++ +++ n.d/? +

Intensity

+++/–?

++++

++++ ++++

jun-B

+++

jun-B

+++

jun-D

+++

n.d

a Compiled from Hughes and Dragunow (1995), Sharma et al. (1998a,c; 2000), and Sharma and Westman (2000).

n.d, no data available; –, negative; +, weak; +++, high level expression; ++++, strong expression; ?, data questionable/controversial.

Rexed’s laminae I and II and a mild expression of Fos in the deeper laminae III to V of the dorsal horn (Strassman and Vos, 1993). Noxious heat stimulus by immersing the hind paw in hot water results in the upregulation of c-fos, c-jun, and zif-268 mRNAs in the neurons of the dorsal horn laminae I and II (Wisden et al., 1990). Similar expression of IEGs occurs in the dorsal horn following peripheral inflammation caused by an injection of Freund’s adjuvant in the hind paws (Naranjo et al., 1991). Repeated squeezing of the plantar surface of the rat hind paw induces c-Jun protein expression in the superficial dorsal horn laminae I to III (Herdegen et al., 1991).

Furthermore, peripheral noxious stimulation is associated with an intense induction of Fos in thalamic areas involved in nociceptive information processing (Bullitt, 1990). However, nonnoxious stimulation of whiskers results in weak Fos activity in the somatosensory cortex (Mack and Mack, 1992). Induction of c-fos mRNA or Fos-like immunoreactivity in olfactory bulb neurons occurs during the sensory processing of odors (Guthrie et al., 1993). Expression of c-fos, jun-B, and krox-24 mRNAs in the visual cortex and Fos-like immunoreactivity in retinal neurons are seen in cats following light-induced visual stimulation (Sagar and Sharp, 1990). Induction of

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H ARI S HANKER S HARMA Table 6—cont’d Stress-Induced Immediate Early Gene Expression a

Stressor

IEG protein/mRNA

Time interval

Brain region

BBB disruption

Learning Memory, LTP—cont’d Learning

c-fos mRNA, c-fos, c-jun mRNAs c-Fos, Krox-24 c-fos, zif-268 mRNAs c-fos

Persistent Transient

Peripheral Nerve transection

c-jun, jun-B, jun-D mRNAs

Hypoxia/ischemia/stroke

Seizure (chemical/electrical)

Training Sexual activity

Transient Transient

Hippocampus Forebrain hippocampus, motor cortex Hippocampus, occipital cortex Sensory cortex

Yes? n.d.

Persistent Transient

Axotomized neurons

Yes?

Fos Li

Transient Persistent

Glial cells ipsilateral, around ischemic core, in neurons resistant to injury

Yes

c-fos, c-fos mRNA

Transient

Yes

Fos Fos Li c-jun, jun-B, zif-268, jun-D mRNA krox-24, krox-20

1–3 h after

Nuclei of neurons in cingulate cortices, neocortex, piriform cortex, dentate gyrus, hippocampus, limbic system Area involved during seizures neurons (not glia) Nuclei in several brain neurons

6–8 h after

n.d.

Kindling

Fos, Fos-B, Fras, c-jun, jun-B, jun-D, krox-24, c-fos, c-jun, zif-268/krox-24 mRNAs, c-fos mRNA, Fos

Transient

Amygdala, hippocampus, dentate granule cells, Somatostatin-containing neurons of dentate hilus, Hippocampus, amygdala

Yes

Brain injury, and Cortical spreading depression Drill injury 2 mm, Suction removal of cortex. Disruption of pia-arachnoid, Needle insertion

c-fos mRNA, Fos, Fra

30 min 1h Persistent

Neurons in injured cortex, ipsilateral cingulate, Piriform and neocortex (layers II–III; V–VI), Ependymal linings in lateral and third ventricles, glial cells around the lesion, cell linings around the wound

Yes

Hippocampus, around the lesion, lining of the ventricles and pial surface, dentate gyrus dentate gyrus

Yes

KCl application

c-fos, jun-B, c-jun, krox-24 mRNAs, Krox-20, Fos-B, jun-D Fos

Yes

Yes?

a Compiled from Hughes and Dragunow (1995), Sharma et al. (1998a,c; 2000), and Sharma and Westman (2000). ?, needs more investigation; transient, within 8 h; persistent, more than 24 h up to 72 h; IEG, immediate early genes; n.d, not done.

Fos-like immunoreactivity and c-fos, zif-268, jun-B, jun-D, and c-jun mRNAs in hypothalamic nuclei SCN and PVN of rodents are more pronounced following photic stimulation in the night (Abe et al., 1992; Rusak et al., 1992; Hughes and Dragunow, 1995). 2. Stressful Situations Strong expression of Fos-like immunoreactivity occurs in the central amygdaloid and paraventricular nuclei in rats following immobilization stress (Honkaniemi, 1992). A marked increase in c-fos mRNA in the neocortex, hippocampus, amygdaloid,

and paraventricular nuclei in mice is seen following handling or ear clipping (Nakajima et al., 1989; Sharp et al., 1991). Expression of c-fos, fos-B, jun-B, c-jun, zif-268, and fra-1 mRNAs in the brain is seen 1 h after saline injection (Persico et al., 1993). Capscaicin administration also results in strong induction of c-fos, c-jun, and jun-B in the amygdaloid and paraventricular nuclei in the rat brain (Ceccatelli et al., 1989). 3. Sleep Deprivation Sleep deprivation induces profound stress in animals (Selye, 1976). Induction of Fos-like immunoreactivity in the dorsolat-

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eral pontine regions, the lateral dorsal tegmental and pedunculopontine nuclei, locus coeruleus, dorsal raphe and pontine reticular formation is seen during rapid eye movement sleep (Shiromani et al., 1992). In these brain areas, the expression of Fos-like immunoreactivity and c-fos and zif-268 mRNAs occurs during sleep deprivation (O’Hara et al., 1993). 4. Cardiovascular Dysfunction Changes in hemodynamics during stress are associated with induction of the IEGs in the CNS. Hypotension caused by either removal of blood or stimulation of the aortic depressor nerve induces Fos protein in several regions in the CNS, e.g., nucleus tractus solitarius, area postrema, venterolateral medulla, nucleus ambiguus, medullary reticular formation, parabrachial nucleus of the amygdala, bed nucleus of the stria terminalis, and islands of Calleja (Dun et al., 1993; Hughes and Dragunow, 1995). However, hypertension caused by the infusion of angiotensin results in Fos expression in the subfornical organ and organum vasculosum of the lamina terminalis (McKinley et al., 1992). Similarly, electrical stimulation of the carotid sinus or the vagus nerve results in the expression of c-fos and zif-268 mRNAs in the nucleus tractus solitarius and paratrigeminal nucleus (Rutherfurd et al., 1992). 5. Learning and Memory Induction of long-term potentiation (LTP) in anesthetized rats results in the expression of zif-268 mRNA and jun-B and c-jun mRNAs. In conscious animals, LTP enhances the expression of Fra, Fos, Fos-B, krox-20, and krox-24 proteins, as well as zif-268 mRNAs and c-jun, jun-B, and jun-D mRNAs and proteins in dentate gyrus neurons (Richardson et al., 1992). Interestingly, LTP did not induce c-fos expression in either awake or anesthetized animals (see Hughes and Dragunow, 1995). Training of rats in a Y maze to learn foot shock-motivated brightness discrimination induces a transient but intense increase in c-fos mRNA in the hippocampus (Tischmeyer et al., 1990). Training of a two-way passive avoidance in rats increases c-fos and zif-268 mRNA expression in the visual cortex and in the hippocampus (Nikolaev et al., 1992). Performing an escape task results in c-Fos induction in the motor cortex of rats (Castro-Alamancos et al., 1992). Sexual learning in male rats is associated with a delayed expression of c-fos in the sensory cortex (Bialy et al., 1992). Learning of a bar-pressing technique enhances the expression of c-fos and c-jun mRNAs within the hippocampus in mice (Heurteaux et al., 1993). Chicks learning a discrimination behavior or exposed to a rich environment exhibit a marked increase in c-fos and c-jun mRNAs and Fos-like immunoreactivity in forebrain neurons (Anokhin et al., 1991). It appears that learning-induced activation of protein kinase C (PKC) is somehow responsible for IEG induction (see Hughes and Dragunow, 1995). 6. Peripheral Nerve Transection Transection of peripheral nerve fibers induces rapid but long-lasting induction of Jun family members, e.g., c-jun, junB, and jun-D mRNAs and proteins in the axotomized nerve cell body (Jenkins and Hunt, 1991; see Hughes and Dragunow, 1995). It would be interesting to explore the induction of other IEGs in animal models of chronic neuropathic pain, including the ligation of peripheral nerves (Gordh et al., 1998).

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7. Hypoxia, Ischemia, and Stroke Expression of IEGs following hypoxia, ischemia, and stroke is controversial. This is mainly because the animal models used in these investigations are not comparable to each other. Ischemia caused by the ligation of brain vessels induces Fos and Fos-like immunoreactivity in neurons in the contralateral cortex and Fos in glial cells in the ipsilateral cortex (Gunn et al., 1990). Areas surrounding the ischemic core often express c-fos, c-jun, jun-B, jun-D, and krox-24 mRNA or protein (Abe et al., 1991). It appears that the intensity of ischemia and the severity of stroke are important determining factors for IEG induction that require further investigation. 8. Seizure and Kindling Chemical- or electrical-induced seizures cause a rapid induction of IEGs in the brains of several mammalian species. Thus, c-fos and c-fos mRNA or Fos protein like immunoreactivity is seen in nuclei of nerve cells located in the neocortex, cingulate cortex, piriform cortex, hippocampus, and dentate gyrus and in the limbic system (Morgan et al., 1987). Glial cells do not show any increase in gene expression. The induction of IEGs is restricted to areas involved in seizure activity and can be prevented by drugs abolishing seizures (Dragunow and Robertson, 1987). Administration of convulsant drugs or electroconvulsive shock induces c-jun, jun-B, zif-268, and jun-D mRNAs rapidly in the rat brain that persists 6 to 8 h after the seizure activity (Cole et al., 1990). However, the Fos protein is expressed transiently in the early hours after seizure initiation and represents neuronal activation. An increase in krox-20 or krox-24 protein in a few selected areas of the brain is seen following kainic acid, bicuculline, pilocarpine, or lithium and audiogenic-induced seizures (see Hughes et al., 1994; Hughes and Dragunow, 1995). Interestingly, newborn rats do not express c-fos in the brain following convulsant drugs, indicating that the biological mechanisms responsible for seizure-induced gene expression are not present at birth. A strong induction of Fos, Fos-B, Fras, c-jun, jun-B, jun-D, and krox-24 proteins in dentate hilus neurons containing somatostatin is seen following kindling produced by focal electrical stimulation of the amygdala or hippocampus that lasts for about 48 h after the episode (Hughes and Dragunow, 1995). mRNAs for c-fos, c-jun, and zif-268 are also expressed in the rat hippocampus during kindling (Clark et al., 1991). However, the induction of c-fos, c-jun, jun-B, and krox-24 mRNA and protein, as well as Fos and krox-20 proteins, occurs in the rat during amygdala kindling (Hughes et al., 1994). 9. Brain Injury and Cortical Spreading Depression Injury to the cerebral cortex caused by either drill bit insertion (2 mm deep), suction, or removal of cortical tissue or damage to the pia-arachnoid membrane induces c-fos mRNA and Fos and Fra protein expression rapidly (30 min to 1 h) that lasts up to 24 h (Temple et al., 2003). Fos protein expression reached its basal level 72 h after the injury. The induction of IEGs is confined to the margin of the injured cortex or neurons. Overexpression of c-fos mRNA and Fos protein in the ipsilateral neocortex (cellular layers II, III, V, and VI), cingulate cortex, and piriform cortex, as well as in the ependymal lining of the lateral and third ventricles and in cells of the pia mater, is

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seen with further advancement of time (Redell et al., 2003). Expression of Fos protein in glial cells around the lesion occurs 12 to 24 h after injury (Hermann et al., 1999). Induction of IEGs in nonneuronal cells appears to be due to the release of several injury-related factors from the damaged nerve cells. Induction of c-fos, jun-B, c-jun, and krox-24 mRNA and protein, as well as krox-20, Fos-B, and jun-D protein in the hippocampus and in dentate gyrus following needle insertion and saline injection into the hippocampus, supports this idea (Dragunow and Hughes, 1993). It appears that cortical spreading depression contributes to IEG induction. Thus, direct application of KCl to the cerebral cortex, which induces spreading depression, results in a similar induction of Fos protein (Herrera et al., 1993). XVI. Stressors as Inducers of Pathophysiology in the Central Neurons System Expression of IEGs plays important roles in stress-induced pathophysiology, leading to major neurological affective disorders (Hughes and Dragunow, 1995). However, very little is known about the expression of IEGs in nonneural glial and endothelial cells in stress. Interestingly, the expression of IEGs can help in coping with stress as well. Thus, antidepressant drugs are able to induce IEGs in the CNS. It may be that expression of IEGs following stress represents neuronal activation related to either protective or destructive phenomena in the CNS. Alternatively, induction of some IEGs reflects the state of pathological damage caused by stressors. Further studies are needed to clarify this point. XVII. BBB Permeability and Immediate Early Gene Expression It is interesting to note that most stressors able to induce IEGs are known to disrupt BBB function (see Tables 3 and 4). This indicates that the induction of IEGs and BBB dysfunction is interrelated. It may be that leakage of plasma constituents into the brain microfluid environment influences IEG expression. This is supported by evidence that drugs preventing seizure activity are also able to attenuate IEG expression. Induction of seizures disrupts the local BBB function (Rapoport, 1976; Bradbury 1979). Interestingly, newborn animals, which lack fully mature BBB function, are unable to induce IEGs. XVIII. Stress Influences Nerve Growth Factors in the Brain Stress has long-lasting effects on the brain and behavior that may precipitate in psychiatric illnesses or brain damage (Sapolsky, 1992). The hippocampus is particularly vulnerable to stress-induced brain damage that mimics age-related memory impairment. Specific cell damage in the CA3 and CA4 subfields in the hippocampus are common following acute or chronic stress (Uno et al., 1989; Sharma et al., 1994b; 1998a; Sharma, 1999). Administration of corticosterone in doses simulating stress conditions results in loss of nerve cells in the pyramidal cell layer in the CA3 subfield of the rat hippocampus

(Sapolsky, 1992). In stress experiments or following glucocorticoid administration, nerve cells in the dentate gyrus and in the CA1 sector of the hippocampus are least affected (Wooley et al., 1990; Watanabe et al., 1992). It appears that stress downregulates the expression or function of neurotrophic factors, resulting in neurodegeneration. Neurotrophic factors are humoral substances that promote the growth and differentiation of nerve cells (Lindsay et al., 1994). Growth factors are essential for normal brain development, plasticity, and survival of the neurons. Several members of the growth factors, e.g., brain-derived nerve growth factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4), are present in the adult brain to provide trophic support and enhance neuronal survival following CNS insults (for details, see Chapters 14 and 23). Neurotrophic factors act as intracellular messengers and induce gene expression in the target neurons (see Lindvall et al., 1994). Exogenous administration of growth factors induces neurite outgrowth and branching (Patel and McNamara, 1995; Sharma et al., 1997a, 1998d,e, 2000b,c; Sharma, 2000b, 2003). An increase in the BDNF mRNA occurs in the hippocampus and cortex following seizures, ischemia, and hypoglycemia (Zafra et al., 1992). Release of glutamate during various kinds of CNS insults regulates BDNF expression (for details, see Smith, 1998). Subjection of rats to 2 h immobilization stress decreased BDNF mRNA in the hippocampus of adult rats (Smith et al., 1995a). This is most pronounced in the dentate gyrus followed by CA3 and CA1 hippocampal pyramidal neurons (Smith et al., 1995b). Repeated stress results in a decrease of BDNF mRNA in the amygdala (Smith et al., 1995a). A reduction in BDNF expression in the hippocampus is also evident 24 h after maternal separation in rats on postnatal days 12 and 20. This suggests that neurotrophic factors influence growth and development, neurogenesis, apoptosis, and neural connectivity, which are influenced by stress (Smith, 1998). A decrease in BDNF expression in dentate granule cells of the hippocampus following the administration of corticosterone to mimic the stress situation further supports this idea. The stress-induced reduction in BDNF expression causes atrophy but is not sufficient to induce cell death. This is evident with the fact that destruction of the hippocampus, which abolishes the effect of neurotrophic factors, results in atrophy of the adult septal cholinergic neurons after 1.5 years (Sorfroniew et al., 1993). Thus, a reduction in BDNF expression following chronic stress or aging increases the selective vulnerability of the neurons to injury. However, stress-induced downregulation of BDNF expression will have profound consequences on neuronal communication and signal transduction mechanisms in the brain. The effect of stress on BDNF expression in the hippocampus and in the hypothalamus is most pronounced in young rats (age 10–12 weeks) compared to aged animal (age 2 years) (Smith and Cizza, 1996). It is unclear whether the expression of BDNF in stress is associated with age-related memory impairment. Long-term treatment with antidepressants or electroconvulsive seizures prevents the stress-induced decrease in BDNF expression (Nibuya et al., 1995). An increased expression of BDNF mRNA following stress is seen in the pituitary and in the

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hypothalamus (Smith et al., 1995a). The BDNF is colocalized with the corticotropin-releasing factor (CRF) in the hypothalamus and thyrotropin-releasing hormones (TRH) in paraventricular nucleus (PVN) neurons. An increase in BDNF expression in the PVN occurs by adrenalectomy or thyroidectomy. This suggests that neurotrophic factors exert trophic effects on the pituitary and regulate local peptide or hormone secretion into the general circulation (Smith, 1998). XIX. Neurosteroids: An Antistress Hormone? Elevated levels of stress and corticosterone impair memory function in several animal models (Bremner et al., 1993; see Diamond et al., 1996c, 1998; Sharma, 1999). However, highly arousing but subthreshold levels of stress enhance memory function (Cahill et al., 1994). This effect of stress is mediated through a new category of steroids termed “neurosteroids” (Diamond et al., 1996c). Neurosteroids are produced endogenously in the periphery as well as in the brain. One type of neurosteroids, dehydroepiandrosterone sulfate (DHEAS), is the most abundant adrenal neurosteroids in humans (Kalimi et al., 1994). The DHEAS antagonizes the functions of corticosterone and thus is often known as an antiglucocorticoid hormone (Kalimi et al., 1994). The DHEAS enhances hippocampaldependent learning in rats (Diamond et al., 1996a) and enhances electrophysiological and cognitive measures of hippocampal function (Diamond et al., 1996b). The complex effects of stress on brain function or behavioral alterations are mainly due to a competitive interaction between corticosterone and DHEAS (Diamond et al., 1996a–c, 1998). These observations suggest that endogenous neurosteroids may act as an antistress hormone. XX. Melatonin: A Neurohormone with Antistress Effects? The interaction between the CNS and the immune system is now well recognized. The pineal hormone melatonin is one of the most prominent immunological active neurohormones (for details, see Chapter 23). Thus, the immunosuppressive effects of stress or corticosterone administration are antagonized by melatonin (Caroleo et al., 1992; Maestroni, 1993). Melatonin is capable of protecting mice against lethal viral infections and septic shock (Ben-Nathan et al., 1995; Maestroni, 1996). Reports show that melatonin reverses aging-associated immune defects, synergizes with interleukin (IL)-2 in cancer patients, and rescues the blood-forming system against the toxic action of cancer chemotherapeutic agents (Colombo et al., 1992; Lissoni et al., 1995; Maestroni and Conti, 1990; Maestroni et al., 1994; Morrey et al., 1994; Pioli et al., 1993). Binding of melatonin to its high-affinity receptors enhances the production of T-helper cell cytokines (Maestroni, 1993). This effect of melatonin appears to be the key factor behind the antistress effects of the neurohormone. Neuroprotective effects of melatonin in traumatic brain injury have also been reported (Sarrafzadeh et al., 2000; for details, see Chapter 23). This indicates that the antistress hormone melatonin has a wide range of applications in various diseases that require further investigation.

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XXI. Stress and Immunosupression The influence of stress on the immune system appears to play an important role in brain function (Maier et al., 1994; Savino and Dardenne, 1995). The sympathetic nervous system innervates immune organs that contain receptors for neurotransmitters (Felten et al., 1987). The sympathetic nerve terminals are in direct contact with lymphocytes (Felten and Felten, 1992). Lymphocytes and other immune cells express receptors for a wide variety of hormones and neurotransmitters that are regulated by the CNS (Plaut, 1987; see Maier et al., 1998). Stress activates the sympathetic nervous system together with the HPA axis and thus regulates the immune system. Research since the early 1970s suggests that a wide variety of stressors influence several aspects of immune function (see Maier et al., 1998). Using nonspecific aspects of immune function, namely lymphocyte proliferation (Lysle et al., 1988) or release of nitric oxide (NO) in response to mitogenic stimulation (Coussons-Read et al., 1994; Maier et al., 1998), has been shown to suppress the immune system in animals and in humans (see Table 7). Stress-induced immune suppression is the key factor in causing brain dysfunction. Macrophages that are activated to the site of injury or infection are critically important in the early phase of infection or inflammation. Activated macrophages release proinflammatory cytokines, such as IL-1, tumor necrosis factor (TNF), and IL-6, which are important mediators of immune to brain communication (Blalock, 1984; Dunn, 1993; Gatti and Bartfai, 1993; Kluger, 1991). These cytokines modulate neural activity through specific receptors present in the brain (Watkins et al., 1995). Antagonizing the action of these proinflammatory cytokines attenuates fever and activation of the pituitary–adrenal axis, as well as behavioral and other changes associated with infection or inflammation (Maier et al., 1994). The interaction between cytokines and brain are bidirectional as they are active in both the periphery and the brain. Thus, cytokine–CNS interaction results in hormonal and autonomic changes, causing peripheral responses. The peripheral effects of IL-1 are blocked by inhibiting the metabolic activity of the glial

Table 7 Stressors Known to Induce Immunosupression a Animals

Humans

Electric shock Separation Rotation Odor Immersion in cold water Restraint Handling Loud noise Intraperitoneal injections of saline Crowding

Final examinations Battle task-vigilance Sleep deprivation Divorce Bereavement Alzheimer’s support

a Data modified after Lylse et al. (1988), Coussons-Read et al. (1994), and Maier et al. (1998).

246 cells, which are one of the potential sources of cytokines (Watkins et al., 1995). These observations suggest that stressors influence both peripheral and central actions of cytokines, leading to immune suppression. XXII. Stress Increases Virus Penetration into the Brain A variety of stressors exacerbate the effects of several infectious agents, e.g., herpes simplex virus (Rasmussen et al., 1957), influenza virus (Feng et al., 1991; Hermann et al., 1994), and encephalitic viruses (Ben-Nathan et al., 1991, 1998), causing significantly increased morbidity and mortality (Friedman et al., 1970; see Sheridan et al., 1994). This indicates that stress increases the susceptibility of animals to bacterial or viral infections (Sheridan et al., 1994; Cohen and Williamson, 1991; Dantzer and Kelley, 1989). Inoculation with the attenuated variant of the West Nile virus (WN-25) or neuroadapted noninvasive Sindbis strain (SVN) to mice subjected to either cold or isolation or administration of corticosterone results in enhanced mortality by 50 to 80% compared to the nonstressed normal group (Ben-Nathan et al., 1998). The most pronounced increase in mortality (about 80%) is seen during isolation stress followed by cold (about 60%) and corticosterone administration (50%). These stressors resulted in fatal encephalitis by the avirulent strain of the Semliki Forest virus (SFV-A7), whereas no death occurs in normal mice inoculated with this virus. The brain titers of viruses in stressed mice are three- to fourfold higher compared to the normal unstressed group (Ben-Nathan et al., 1996, 1998). This indicates that stress induces an increased penetration of viruses into the brain, causing lethality. Interestingly, administration of the attenuated virus WN-25 together with serum of cold-stressed mice resulted in 78% mortality in naive animals (Ben-Nathan et al., 1998). However, inoculation with the WN-25 virus together with serum obtained from normal mice did not induce encephalitis. This enhancement of neuroinvasion of the WN-25 virus by administration of serum from stressed mice is not directly related with elevation of the serum corticosterone concentration (Ben-Nathan et al., 1996, 1998). It is believed that stress-induced immunosupression (see earlier discussion) enhances the proliferation of the viruses into the CNS, leading to exacerbation of the infection-induced pathogenesis. An increased permeability of the BBB following stress appears to be another key factor in the exacerbation of virus-induced mortality. XXIII. Corticotropin-Releasing Hormone Regulates BBB Permeability in Stress Stress-induced activation of the HPA axis results in corticotropin-releasing hormone discharge that causes the secretion of catecholamines and glucoroticoids. The glucocorticoids downregulate the immune response (Chrousos, 1995). The CRH is a proinflammatory agent and appears to regulate stress-induced increased BBB permeability through mast cell activation (Karalis, 1991; Theoharides et al., 1998). Prevention of the restraint-induced increase in BBB permeability by the “mast cell stabilizer” disodium cromogylate cromolyn supports this idea (Esposito et al., 2001). The CRH induces mast cell degranulation following restraint stress in rats and causes

H ARI S HANKER S HARMA extravasation of Evans blue into the skin as well as in the brain (Singh et al., 1999). This indicates that mast cells play important roles in stress-induced BBB dysfunction, neuroinflammatory processes, and neuroimmune interactions (Church et al., 1989; Theoharides, 1996; Rozniecki et al., 1999; for details, see Chapter 13). The CRH is synthesized in the paraventricular nucleus (PVN) of the hypothalamus and acts through distinct types of receptors: CRHR-1, CRHR-1a, and CRHR2-b (Vaughan et al., 1995). That CRH is involved in stress-induced BBB dysfunction is apparent from findings that antalarmin, a CRH receptor antagonist, is capable of blocking the restraint-induced breakdown of the BBB (Esposito et al., 2002). Local administration of antalarmin into the PVN prevents a CRH-evoked acute stress response, indicating that CRH is crucial in the stress response as well as BBB disruption. That most cell-deficient mice did not show a restraint-induced extravasation of tracers in the brain further confirms the role of CRH in stress-induced BBB breakdown (Esposito et al., 2002). These observations suggest that CRH and mast cells regulate stress-induced BBB function and neuroinflammatory responses that are exacerbated by acute stress. It would be interesting to see whether administration of a mast cell stabilizer or CRH receptor antagonist attenuates BBB breakdown caused by traumatic or hyperthermic insults to the CNS. XXIV. Stress Associated with Electromagnetic Radiation and Mobile Telephony Electromagnetic fields generated from the use of mobile telephony induce stress responses in the CNS (Ono and Han, 2000; Pipkin et al., 1999; Jin et al., 2000; Morehouse and Owen, 2000; Leszcynski et al., 2002; see Hossmann and Hermann, 2003). Thus, it appears that the radiofrequency-modulated electromagnetic fields (RF-EMF) emitted by mobile phones are harmful in nature. However, the subject is still controversial (see Hossman and Hermann, 2003). Investigation of the cellular stress response following RF-EMF exposure in the range elicited by mobile phones showed altered cellular physiology, enhanced stress response, and disruption of the BBB (Cleary et al., 1997; Fritze et al., 1997b; Daniells et al., 1998; de Pomerai et al., 2000; Kwee et al., 2001; Leszcynski et al., 2002). Stress proteins, often known as “heat shock proteins” (HSPs), regulate apoptosis (Creagh et al., 2000; Pandey et al., 2000; for details, see Chapter 17), and deregulation of apoptosis following RF-EMF-induced radiation by HSPs suggests a potential risk factor for tumor development. This is evident from the fact that HSP induction in cells following injury or insults enhances cell survival (see Westman and Sharma, 1998; for details, see Chapter 17). Thus, RF-EMF-induced induction of HSPs and prevention of apoptosis result in the survival of those cells that are supposed to die for the purpose of physiological regulation (French et al., 2001; Leszcynski et al., 2002). Acute exposure of rats to 900-MHz/217-Hz microwaves (in the range of global system for mobile communication, GSM signal) results in the elevation of HSP72 mRNA, as well as the induction of IEGs, e.g., c-fos and c-jun mRNAs in the cerebral cortex (Fritze et al., 1997b; see Hossman and Hermann, 2003). Upregulation of HSPs 27 and p38 mitogen-activated protein

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kinase (P38MAPK) in cultured human endothelial cells occurs following RF-EMF radiation in the nonthermal range (Leszcynski et al., 2002). Activation of the family of small HSPs, such as HSP27 phosphorylation, inhibits apoptosis, which involves apoptosome and caspases (Pandey et al., 2000; Concannon et al., 2001, for details, see Chapter 17). Furthermore, HSP27 induces the resistance of tumor cells to death by anticancer drugs (Huot et al., 1996; Garrido et al., 1997). These observations suggest that the RF-EMF-induced expression of HSP27 not only affects tumor development, but also its drug resistance (for details, see Kyriakis and Avruch, 2001). An increase in BBB permeability following RF-EMF exposure is seen in some animal experiments using both in vitro and in vivo studies (see Jokela et al., 1999; Hossmann and Hermann, 2003). The increased permeability of the BBB following RF-EMF exposure is mainly related to its thermal effects. However, few studies showed that BBB dysfunction can also be induced in vitro as well as in vivo by RF-EMF in the nonthermal range (Salford et al., 1994; Schirmacher et al., 2000). Fritze et al. (1997a) reported a transient induction of HSP70 response and breakdown of the BBB immediately after irradiation. A 2-h exposure of rats to RF-EMF (900 MHz, corresponding to GSM signal) at a specific absorption rate (SAR) of 2 W/kg averaged over the brain results in BBB disruption (Töre et al., 2001). However, studies using a repeated exposure of RF-EMF in the nonthermal range on BBB permeability and HSP or IEG induction are lacking. Thus, the nonthermal effects of RF-EMF on the BBB and brain dysfunction are still controversial. The molecular mechanisms and the cellular signaling pathways involved in RF-EMF-induced BBB breakdown are not known in detail. Increased pinocytosis within endothelial cells of the cerebral cortex has been described following exposure of 2.45 GHz microwave radiation in rats (Neubauer et al., 1990). It may be that the induction of HSP27 will trigger molecular events leading to the cascade of activities causing opening of the BBB (French et al., 2001; Leszcynski et al., 2002; for details, see Chapter 17). A possibility exists that phosphorylation of HSP27 enhances actin polymerization, causing cell shrinkage and widening of the tight junctions and/or stimulation of pinocytotic activity (Lavoie et al., 1993; Piotrowicz and Levin, 1997). Thus, the effects of mobile telephony-induced RF-EMF radiation on the BBB and brain function require further investigation. XXV. Neuroanatomy of Stress Pathways Stress alters the CNS processing of somatosensory and autonomic outflow to induce several responses that are characterized as adaptive processes to cope with emergency situations. A. Hypothalamus and Brain Stem Catecholamine Neurons The hypothalamic PVN plays a key role in stress-induced activation of the HPA axis. Other areas include the prefrontal cortex, hippocampus, amygdala, and bed nucleus of the stria terminalis and catecholaminergic neurons in the brain stem. These regions are often known as “central circuits of stress,” which convey the stress-related signals to the PVN (see Herman and Cullinam, 1997).

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Hemorrhage, hypotension, and respiratory disturbances activate catecholaminergic neurons in the brain stem, causing ACTH release and a stress-induced immune reaction (Plotsky et al., 1989). Acute stress-induced IEG expression in brain stem catecholamine neurons are in line with this idea (Plotsky et al., 1989; Chan et al., 1993). Deafferentiation of ascending brain stem pathways abolishes immune challenge-induced c-fos expression in the PVN but not following foot electroshock. This indicates that alternative circuitry is used to activate HPA during foot shock stress (Li et al., 1996). B. Amygdala The cardiovascular and behavioral regulations in stress are mediated thorough the amygdala. Damage to the amygdala results in a decrease of corticosterone and ACTH during leg break or adrenalectomy (Allen and Allen, 1974, 1975). Stimulation of amygdaloid nuclei results in corticosterone secretion (Dunn and Whitener, 1986) and pronounced induction of c-fos in neurons following immobilization and forced swimming (Cullinan et al., 1995). A lesion of the amygdala blocks the HPA response to auditory and visual stimulation (Feldman et al., 1994) and decreases ACTH or corticosterone release caused by restraint and fear conditioning (Van de Kar et al., 1991). However, damage of the medial or central amygdala did not influence stress-induced activation of the HPA axis. These observations suggest that stressors influence different neuroanatomical pathways to activate HPA in a specific manner. C. The Bed Nucleus of Stria Terminalis Activation of the HPA axis involves the bed nucleus of stria terminalis (BST). The BST forms neural networks with the amygdala, hippocampus, hypothalamus, and brain stem regions and regulates homeostasis (Cullinan et al., 1993; Moga et al., 1989; Weller and Smith, 1982). Damage to BST attenuates activation of the HPA axis and decreases the release of stress hormones, whereas stimulation of this nucleus results in increased secretion of corticosterone (Dunn, 1987). D. Raphé Nucleus and Locus Coeruleus Involvement of the ascending serotonergic and noradrenergic system in stress-induced HPA activation is controversial (Cullinan et al., 1995; Abercrombie and Jacobs, 1987; Smith et al., 1991). The role of serotonin on the HPA axis is both excitatory and inhibitory (Korte et al., 1991; Welch et al., 1993). Because both noradrenergic and serotonergic innervation in PVN is limited, a direct role of these nuclei on stress-induced activation of HPA is not well known. It appears that the indirect influence of aminergic fibers on the stress response is mediated via the limbic system (see Herman and Cullinan, 1997). XXVI. Stress Inhibitory Pathways Apart from brain regions and the neuronal network involved in activation of the HPA axis, parallel centers and structures regulate stress-induced inhibition of these pathways.

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A. Hippocampus The hippocampus, which contains the highest levels of glucocorticoid binding sites than any other structure in the brain (Herman, 1993; Jacobson and Sapolsky, 1991), is involved in the inhibitory influence of stress pathways. A lesion of the hippocampus potentiates stress-induced glucocorticoid secretion in rats and monkeys and increases the expression of the CRH in PVN neurons (Jacobson and Sapolsky, 1991; Herman et al., 1989, 1995; Sapolsky et al., 1991). However, stimulation of the hippocampus decreases HPA activity in rats and in humans (Jacobson and Sapolsky, 1991). Further studies are needed to establish an inhibitory role of the hippocampus on stress-induced activation of the HPA axis.

taining orexin and orexigenic hypothalamic peptide influences some of the stress-induced behaviors via an interaction with serotonergic and noradrenergic neurons (Date et al., 1999; Otake and Ruggiero, 1995). Stressors also activate serotonin neurons and Fos protein in the dorsal raphe nucleus in rats (Grahn et al., 1999). Immobilization stress suppresses food intake by the activation of serotonergic pathways in the dorsal raphé nucleus through interaction with the lateral hypothalamus (Shimizu et al., 2000). Activation of the serotonin autoreceptor by the 5-HT1a receptor antagonist that decreases serotonin synthesis and release attenuates acute restraint-induced exploratory behavior (Tsuji et al., 2000). These observations indicate that the interaction among serotonergic, noradrenergic, and peptidergic neurons plays important roles in stress response.

B. Prefrontal Cortex and Septum The limbic system plays an important inhibitory role in stress-induced activation of the HPA axis. Lesion or damage to the prefrontal cortex or septum enhances the HPA responsiveness of acute stress (Dobrakovova et al., 1982; Diorio et al., 1993). Induction of IEG in the prefrontal cortex and in the septum following acute stress supports their involvement in the neuronal circuit of stress. However, the limbic system responds to stress in a very specific and selective way. Thus, lesions of the prefrontal cortex increase ACTH and corticosterone release following immobilization but not during ether stress (Diorio et al., 1993). Similarly, damage to the hippocampus enhances immobilization stress-induced corticosterone response but does not influence ACTH or corticosterone secretion following hypoxia (Bradbury et al., 1993). C. Hypothalamus The local hypothalamic neuronal network has an inhibitory input to PVN neurons. Thus, lesion of the BST and preoptic area of the hypothalamus, which send input to PVN, inhibits HPA activation. Ablation of hypothalamic nuclei increases the magnitude and duration of the stress response as well as ACTH and corticosterone secretion (Viau and Meaney, 1991; Buijs et al., 1993; Larsen et al., 1994). However, lesions of the ventromedial hypothalamus decrease corticosterone-induced ACTH release (Suemaru et al., 1995). Because GABA blocks the release of ACTH and corticosterone, it appears that the stress-induced inhibitory effects are mediated through the inhibitory neurotransmitter GABA (Makara and Stark, 1974). The presence of GABA-immunoreactive terminals in PVN are in line with this idea (Decavel and van den Pol, 1992). XXVII. Aminergic and Peptidergic Afferents in Stress Circuitry The middle thalamic neuronal network is involved in the behavioral and cognitive aspect of the stress response (Otake et al., 2002). Noradrenergic neurons in the brain stem, together with the thalamic neuronal network linking the paraventricular thalamic nucleus (PVT) to the visceral cortex and striatum, are mainly responsible for the desynchronization of EEG in stress (Page et al., 1993). The presence of neurons con-

XXVIII. Stress and Brain Diseases The hypothalamic PVN neural network seems to be important in maintaining health and disease following stressful situations. Long-term stress induces alterations in neuroendocrine metabolism and dysfunction of the limbic system, including hippocampal formation, prefrontal cortex, and amygdala. This is evident from the fact that in major depression in humans reflecting hyperactivity of the HPA axis, the structure and function of the amygdala, prefrontal cortex, and hippocampus are profoundly altered (Sapolsky, 1996; Drevets et al., 1992). It is likely that activation of GABA in these depressive illnesses inhibits glucocorticoid secretion (Makara and Stark, 1974; Jones et al., 1984). Stress-induced release of the glucocorticoid and alterations in the PVN neuronal networks result in alterations in homeostasis, leading to neurodegenerative diseases. Thus, prolonged stress increases CRH and arginine vasopressin (AVP) mRNA in the PVN (Sawchenko et al., 1993; Herman et al., 1995) and results in coexpression of CRH and AVP in the median eminence (Whitnall, 1993). An increased expression of CRH mRNA, CRH, and AVP peptide in the PVN in postmortem cases of depressed individuals and in Alzheimer’s disease victims (Raadsheer et al., 1994, 1995) further supports this hypothesis. XXIX. Stress and Cerebral Microcirculation Our knowledge on the chemical neuroanatomy of stress suggests that several neurochemicals, e.g., biogenic amines, amino acids, and other neuropeptides, are released during stress reaction. When administered systemically or locally, these neurochemicals are able to influence the cerebral microcirculation. However, alterations in the cerebral microcirculation in stress conditions are still not well known. One study using the 14C iodoantipyrine technique showed that 5 and 15 min of immobilization stress induces a marked elevation in the regional CBF in several regions (Ohata et al., 1981). This indicates that an alteration in the cerebral circulation in stress plays an important role in brain function. It may be that long-term stress results in local ischemia impairing cerebral circulation and metabolism leading to neurodegeneration. Expression of IEGs and heat shock proteins (HSPs) in many parts of the brain during ischemia is in line with this idea

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(for details, see Chapter 17). The magnitude and intensity of stress-induced ischemia can also impair BBB dysfunction, causing brain damage.

Limbic system

Thalamocortical system

XXX. Stress and Cerebral Metabolism

Hypophysis

Hypothalamus

Internal milieu

Brainstem Spinal cord

Stressors often comprise diverse situations, e.g., delivery of electroshocks to foot pads or tail, swimming to exhaustion, fighting, placed in front of a predator, or restraint. The emotional excitement is reflected by several kinds of reactions ranging from squeaks, catatonia, defecation, and other abnormal behaviors. These stressful situations do not involve pain or noxious stimulus (Bliss et al., 1966; Bliss and Zwanziger, 1966). However, several stressors alter the biogenic amine content in the brain. Behavioral arousal in stress is largely mediated thorough noradrenergic neurons originating in the locus coeruleus and innervating cortex and hippocampus (as earlier discussion; Corrodi et al., 1971; Otake et al., 2002). Anxiolytics, such as diazepam, blocks noradrenaline turnover following stress by decreasing impulsive activity in the locus coeruleus neurons (Taylor and Laverty, 1969; Corrodi et al., 1971). Stress induces turnover of the noradrenaline in the ipsilateral cortex that is prevented by unilateral lesion of locus coeruleus (Korf et al., 1973). Effects of stress on cerebral oxygen consumption in the past are examined by a few workers (see Siesjö, 1978). The idea of a stress-induced alteration in the cerebral metabolic rate for oxygen consumption (CMRO2) is based on studies in single human subjects that show a marked increase during grave apprehension (2.2 µmol/g/min) from the resting (1.4 to 1.9 µmol/g/min) state (Kety, 1950). This anxiety-induced increase in CMRO2 is due to alterations in the adrenaline level (King et al., 1952). Accumulation of lactate in the brain following exertion suggests that stress is able to induce alterations in cerebral energy metabolism (Richter and Dawson, 1948). About twofold increases in the CBF and CMRO2 following immobilization stress in rats further support this idea (Carlsson et al., 1976a). Interestingly, diazepam but not adrenalectomy or administration of a β-adrenergic receptor blocker (propranolol) prevented this response (Carlsson et al., 1975, 1976a,b). This indicates that stress-induced anxiety is responsible for alterations in the cerebral circulation and metabolism. An increase in the CMRO2 during hyperthermia (40 or 42°C) is in line with this idea (Carlsson et al., 1976c; see Sharma and Hoopes, 2003). It is not clear whether changes in energy metabolism are mediated through carbohydrate or other amino acid metabolism in the brain. Some of these changes in energy metabolism are independent of PaCO2 values in stress. It would be interesting to see whether changes in BBB function during stressful conditions are somehow related to altered cerebral energy metabolism.

Somatic and visceral afferents

IPS overload? BSCB disturbances? Fig. 3 Stress can influence information processing system (IPS) of the spinal cord. Sensory information is relayed to the hypothalamus through spinal cord. In addition, spinal cord plays important role in afferent and efferent neuronal connections and humoral influences of the hypothalamus. It is possible that under situations of stress, spinal IPS and the blood-spinal cord barrier (BSCB) permeability are affected. Modified after Schmidt (1978).

brain using a complex neuronal network (Schmidt, 1978). However, the details of sensory and motor coordination in the spinal cord during stress are still unknown. It appears that spinothalamic and corticospinal tracts are involved in stress-induced IPS. Thus, an information overload on the spinal regulatory system by stressors, e.g., environmental temperature, noxious stimulation of sensory pathways, restraint, running, swimming, and related vigorous movements of the body parts, will alter the spinal IPS. Several neurotransmitters and neuropeptides located in the spinal dorsal horn are released during various stressful situations (Nyberg et al., 1995). Thus, a possibility exists that the stress-induced release of spinal neurochemicals will influence the spinal cord fluid microenvironment. It may be that stressful conditions, which induce BBB disruption, are likely to influence the BSCB permeability as well. However, it is not known whether BSCB permeability is compromised in specific sensory or motor areas or is limited to certain cord segments depending on the types of stressor used. Thus, further studies on stress-induced BSCB permeability in relation to neurochemical alterations in the cord are needed.

XXXI. Stress and Blood–Spinal Cord Barrier Permeability The effects of stress on the blood–spinal cord barrier (BSCB) function are not known. The sensory information is conveyed from the spinal cord to the hypothalamus through the brain stem where it is processed further to regulate homeostasis (Fig. 3). Information processing is largely carried out in the

XXXII. Investigations on Microvascular Barriers in Stress Studies carried out since the late 1970s have revealed that several stressful situations are able to disrupt the BBB function (Sharma and Dey, 1978, 1981; Sharma, 1982). This was confirmed later by studies from several groups (see below).

250 Further studies showed that the BBB dysfunction in stress is dependent on the magnitude and intensity of stressors, as well as the age of animals. The functional significance of stress-induced BBB dysfunction is still unclear. There are reasons to believe that opening of the BBB during stress is one of the crucial factors resulting in neurological disorders and brain pathology. Thus, breakdown of the BBB in stress may be regarded as a gateway to neurological diseases and neurodegeneration. This aspect is critically examined based on investigations in different stress models. A. Immobilization Stress Stress-induced hypoactivity is commonly used as animal models of depression and/or posttraumatic disorders (Soblosky and Thurmond, 1986; Kathol et al., 1989; Charney et al., 1993). Immobilization and swim stressors are well-known animal models for immobility (Porsolt et al., 1977, 1978). Thus, these animals models of stress are employed to understand the pharmacology of depression and posttraumatic stress disorders (see Kofman et al., 1995; Miyazato et al., 2000). Immobilization causes a decrease in motor activity and induces sleep disorders in animals, thus mimicking depression-like behavior (Zebrowska-Lupina et al., 1990). Restriction of movement in rats by placing them in a tube is often referred to as “restraint,” whereas fastening of limbs on a wooden board is generally known as “immobilization” (Groves and Thompson, 1970; Kvetnansky and Mikulaj, 1970; Sharma and Dey, 1981; Sharma, 1982). It has been observed that immobilization on a wooden board induces severe stress compared to restraint in a plastic tube (see Marti et al., 2001). Different animal models of immobilization stress have been shown to disrupt BBB function to various tracer molecules at several time points (Sharma and Dey, 1978, 1981; Belova and Jonsson, 1982; Dvorsk et al., 1992; Esposito et al., 2001, 2002). Thus, mild changes in BBB function in a few brain regions are observed following 5 or 15 min of immobilization (Ohata et al., 1981, 1982), whereas 2 and 6 h of immobilization results in increased tracer transport into several brain regions (Belova and Jonsson, 1982; Dvorsk et al., 1992). Long-term immobilization stress (7–9 h) is accompanied by the leakage of protein tracers in many brain regions (Sharma and Dey, 1981; Sharma et al., 1986a, 1987a). These observations suggest that the magnitude and intensity of BBB breakdown depend on the severity of immobilization stress. One way to expand our knowledge in stress-induced BBB function is to study the influence of several exogenous factors (Selye, 1976, see earlier discussion), namely age, sex, and previous response to stressors in our animal models. This information is vital for our understanding on brain dysfunction in stressful situations. 1. Induction of Stress Rats (age 10 to 12 weeks or 26 to 35 weeks old) were immobilized in a prone position on wooden boards (Sharma, 1982; Sharma and Dey, 1981, 1986a, 1987a, 1988). The limbs were mildly extended and fixed on the board with adhesive tape. The body was loosely wrapped with surgical gauze (6 cm wide) to minimize trunk movement (for details, see Sharma and Dey,

H ARI S HANKER S HARMA 1986a). All stress experiments were commenced between 8:0 and 8:30 AM to avoid the effect of circadian variation on the stress response (Selye, 1976). 2. Stress Response Changes in body temperature and the number of fecal pellets excreted were used as indices of stress during the experiment. Postmortem examination of hemorrhagic spots in the stomach was determined to assess the individual response of stress in each animal (Selye, 1976; Sharma, 1982). The rectal temperature showed biphasic hypothermia during immobilization stress (Table 8). At 1 h stress a significant decrease in body temperature is seen that recovered partially at 4 h followed by hypothermia at the end of 8 h of immobilization. When stress is further continued to 11 and 14 h, the body temperature further declined and did not return to the basal level (Table 8). The excretion of fecal pellets correlated well with the duration of stress until the 8-h period. No significant increase in fecal pellets was seen between 11 and 14 h of stress (H. S. Sharma, unpublished observations). The number of microhemmorhagic spots in the stomach at postmortem examination showed a progressive increase with stress duration up to 8 h (Table 8). Prolongation of stress to 11 and 14 h did not further increase the microhemorrhagic spots in the stomach (H. S. Sharma, unpublished observations). It appears that stress immobilization up to 8 h reflects alarm reactions as evident with a profound appearance of symptoms. After 8 h of immobilization, animals entered the stage of adaptation, resulting in a marked reduction in stress reactions and symptoms. 3. Physiological Variables Changes in mean arterial blood pressure (MABP), heart rate, respiration, arterial pH, and blood gases were determined in stress (for details, see Sharma and Dey, 1986a). Immobilization stress induces a mild hypertension at 2 h (Table 8) followed by a mild hypotension that was most prominent at 8 h (Table 8). Partial recovery in hypotension is seen between 11 and 14 h after stress (Table 8). The heart rate increased significantly during 1 and 4 h stress followed by a marked decrease at 8 h. This was followed by a partial recovery at 11 and 14 h (Table 8). The respiratory rate declined significantly at 4 h immobilization followed by a mild but significant increase at 8 h stress (Table 8; H. S. Sharma, unpublished observation). The respiratory rate slightly declined further at the end of 11 and 14 h after stress (Table 8). The arterial PaO2 increased and the PaCO2 declined at 8 h (Table 8). No significant change in blood gases or arterial pH can be seen during early periods of immobilization stress (1 to 4 h) (Table 8). 4. BBB Disruption Immobilization stress causes a selective increase in BBB permeability to Evans blue, bromophenol blue, horseradish peroxidase (HRP), and radioiodine tracers depending on the duration and age of the animals (Dey et al., 1980; Sharma and Dey, 1981; Sharma, 1982, 1986a). Young rats (age 8–9 weeks) subjected to immobilization stress for 2–4 h showed extravasation of Evans blue or bromophenol blue in the brain of 1 out of 8

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Table 8 Stress Symptoms and Physiological Variables in Animals Subjected to Various Stress Paradigms Stress symptoms

Type of experiment

Physiological variables

Haemorrhagic Faecal spots in # pellets (nr.) stomach # (nr.)

n

Rect Temp °C

MABP torr

Heart rate Respiration beats/min breath/min

5 5

36.86±0.23 37.54±0.12

Nil Nil

Nil Nil

104±6 110±8

290±4 280±8

Arterial pH

PaO2 torr

PaCO2 torr

75±5 76±4

7.38±0.04 7.37±0.02

79.34±0.54 80.56±0.43

34.68±0.67 35.42±0.78

A. Control group Young animals Adult animals

B. Immobilization stress Young animals 1h

6

35.52±0.12**

6±2

5±2

98±6

310±8*

70±3

7.28±0.04

75.56±,54

34.66±.48

4h 8h 11 h

6 12 6

36.10±0.14 35.68±0.23** 36.51±0.12

12±4 34±6** 28±5**

13±4* 35±5** 38±6**

128±6** 76±6** 86±8*

320±6** 272±8** 284±6

62±4** 82±4* 76±4

7.30±.12 7.38±0.14 7.36±0.06

74.76±0.44 83.12±0.34* 78.79±0.32

34.89±0.34 31.28±0.43* 32.34±0.23

8 h+ 3 h rest 8 h+6 h rest Adult animals

6 5

37.03±0.12 37.34±0.10

4±4 6±4

n.d. n.d.

92±6 98±6

296±8 285±8

80±4 76±8

7.33±0.12 7.31±0.05

78.98±0.23 78.85±0.54

33.87±0.13 34.12±0.17

8h

8

37.01±0.23

6±7

8±12

94±6

276±6

84±6

7.34±0.08

81.12±0.32

34.46±0.43

6 6 12

38.42±0.12 39.23±0.23** 41.68±0.21**

4±4 6±4 2±2

Nil Nil 24±6**

98±3 134±6** 75±8**

306±4 324±8* 267±6**

80±5 72±7 88±3*

7.36±0.06 7.33±0.12 7.34±0.08

79.34±0.44 33.46±0.43 79.83±0.54 34.28±0.23 82.87±0.32** 33.08±0.21*

6 8

39.08±0.12** 38.86±0.12*

4±3 6±7

n.d. 8±4

86±7* 96±6*

285±6 274±6

75±4 82±6

7.33±0.05 7.38±0.06

80.28±0.34 81.23±0.14*

34.06±0.18 34.24±0.43

34.56±0.42* 32.48±0.53* 30.42±0.48

6±4 20±8* 40±8*

nil 10±4 44±12

120±6 114±8 83±8*

290±6 318±8* 270±6*

80±6 72±6 85±5

7.38±0.06 7.36±0.08 7.36±0.06

79.38±0.38 80.32±0.23 81.36±0.44*

35.03±0.12 33.89±0.22 33.22±0.24*

5

36.58±0.37

4±3

38±8

114±10

n.d.

n.d.

7.37±0.07

80.56±0.37

34.37±0.34

6

32.38±0.27

20±8

8±5

88±6*

289±6

78±6

7.36±0.05

81.58±0.35

34.03±0.28

C. Heat stress 38°C Young animals 1h 2h 4h microhaemorrhages 4 h+rest 2 h Adult animals 4h

D. Forced Swimming Young animals 5 min 5 15 min 5 30 min 8 30 min + rest 2 h Adult animals 30 min

Values are expressed as mean±SD; *P < 0.05; **P <0.01 ANOVA followed by Dunnet’s test for multiple group comparison with one control. test for semiquantitative data; n.d., not done.

#Chi-Square

rats. However, when the duration of stress is increased further to 7–9 h, 20 out of 24 rats exhibited leakage of Evans blue and bromophenol blue in their brain (Sharma and Dey, 1981). Further continuation of stress to 12–14 h markedly diminished the occurrence of BBB permeability. Thus, only 3 out of 10 young rats showed extravasation of Evans blue at this time (Sharma, 1982). It appears that old rats (age 30–32 weeks) are more resistant to BBB disruption in immobilization stress compared to young rats. Thus, only 4 out of 12 rats displayed leakage of Evans blue in the brain following 8–9 h of immobilization (Sharma and Dey, 1981; Sharma, 1982).

Evans blue and bromophenol blue were used as vital tracers to study the leakage of serum albumin into the brain of stressed animals (Rinder, 1968; Rawson, 1943; Rapoport, 1976). These tracers bind to various endogenous serum proteins, including albumin, with slight differences in their capacity when administered into the circulation. The patterns of extravasation of these dyes into the brain of stressed animals show that minor differences in the protein-binding capacity of these tracers (Rawson, 1943; Bonate, 1988) do not affect their regional distribution in brain (Sharma and Dey, 1981; Sharma, 1982).

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a. INflUENCE OF ANESTHESIA. To find out whether anesthesia influences the stress-induced brain penetration of tracers, two different groups of rats were subjected to 7–9 h immobilization. In one group, Evans blue dye was administered 5 min before termination of stress through an indwelling polythene cannula. Another group received Evans blue through the right subclavian vein after stress under urethane (0.8 g/kg, ip), Equithesin (0.2 ml/kg, ip), or pentobarbital (35 mg/kg, ip) anesthesia (Sharma and Dey, 1981; Sharma, 1982). No differences in the pattern of Evans blue extravasation was observed in the brain of animals that received the tracer in a conscious state or under urethane, Equithesin, or pentobarbital anesthesia. These observations suggest that anesthetics do not influence BBB disruption in stress (Sharma and Dey, 1981; Sharma, 1982). However, we observed that the dose of anesthetics used to achieve a surgical grade of anesthesia is reduced by 50% in young animals immobilized for 7–9 h. In these animals, the induction of anesthesia is also quite rapid compared to unstressed rats (Sharma, 1982; H. S. Sharma, unpublished observation). This indicates that the increased permeability of BBB caused by stress is responsible for the rapid onset and reduction in the dose of anesthetics (H. S. Sharma, unpublished observations). The increased susceptibility of viruses (see earlier discussion) and morphine in stressed rats (Mayor et al., 2002) is in line with this hypothesis (see Chapter 16).

b. PATTERN OF EVANS BLUE EXTRAVASATION. Visual inspection of Evans blue dye in the brain showed a minor variation in individual animals after 8 h of immobilization. Extravasation of dye is observed mainly in the cingulate, occipital, parietal, and frontal cortex. The cerebellum was also stained (see Figs. 4 and 7). The choroid plexus and other nonbarrier regions took deep blue stain. A mild to moderate blue staining is seen in the walls of lateral and in the fourth cerebral ventricles, indicating disruption of the blood–CSF barrier by immobilization. The dorsal surface of the hippocampus and caudate nucleus stained mildly. The massa intermedia, hypothalamus, and regions surrounding the third ventricle showed mild to moderate blue staining (Fig. 5). The coronal section of the brain passing through the basal ganglia, hippocampus, brain stem, and cerebellum showed mild to moderate staining of the deeper tissues across the dorsal, lateral, and ventral surfaces of the brain (Figs. 6 and 8). These observations suggest that immobilization induces selective disruption of the BBB. c. MEASUREMENT OF TRACER EXTRAVASATION. To quantify tracer extravasation in brain following stress, Evans blue (3 ml/kg, iv) and 131I (100 µCi/kg) tracers were administered together into the right femoral vein of 1, 4, 8, or 11 h after immobilization (see Sharma and Dey, 1986a; Chapter 12). Continuous immobilization of young rats for 8 h induced a profound

Fig. 4 Diagrammatic representation of Evans blue albumin (EBA) extravasation in the brain and spinal cord following 4 h heat stress (HS, A), 30 min forced swimming (FS, B) and 8 h immobilization (IMZ, C) stress. The mapping of EBA extravasation in the brain and spinal cord is based on 6 to 8 individual experiments in each category. The pattern of EBA extravasation in different areas of the brain and spinal cord represents most common findings in each stress group. Heat stress appears to induce most extensive EBA extravasation in the dorsal (A: a), ventral (A: b) surfaces of the brain as well as on the dorsal surface of the spinal cord (A: c). In forced swimming (FS) the most prominent extravasation of EBA is seen in the cerebellum (FS, B: a). Immobilization stress (IMZ) induces most frequent EBA staining in the anterior and posterior cingulate cortices (C: a) and on the dorsal surface of the spinal cord (C: c). EBA staining on the ventral surfaces of the brain also varies according to the stressors used. However, the piriform cortex and pons regions always exhibited EBA staining in stress. Bar: 5 mm.

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Fig. 5 Diagrammatic representation of mid-sagittal section of the rat brain showing extravasation of Evans blue albumin (EBA) following heat stress (HS, a), forced swimming (FS, b), immobilization (IMZ, c) and sleep deprivation (SD). The pattern of EBA extravasation varies according to the stressors used. Differences in the pattern of EBA extravasation are prominent in the cingulate cortex, cerebellum, thalamus and in the brain stem. Staining of cerebroventricular walls of the lateral ventricles, 4th ventricle and median eminence is a frequent finding in all stress experiments. Bar: 5 mm.

extravasation of Evans blue albumin (EBA, 670%) and radioiodine tracers (1061%) in the whole brain (Sharma and Dey, 1986a), whereas only a slight increase in BBB permeability to Evans blue (135%) and radioiodine (100%) is seen 4 h after immobilization (Sharma and Dey, 1986a). The permeability changes at 1 h stress are negligible (see Fig. 9). d. REVERSIBILITY OF THE BBB. To examine whether immobilization stress-induced BBB disruption is reversible in nature, two different sets of experiment were made. In one experiment, stress was continued beyond 8 h, and BBB permeability was measured at 11 and 14 h of immobilization. These animals exhibited a significant decrease in BBB permeability at these time points (Sharma and Dey, 1981, 1986a). This indicates that

253

Fig. 6 Diagrammatic representation of Evans blue albumin (EBA) extravasation in the cross sections of the brains (A) and spinal cords (B) of rats subjected to heat stress (HS), forced swimming (FS) or immobilization (IMZ). (A) Coronal sections of the brain passing through caudate-putamen (+0.45 from bregma, A: a–c) and hippocampal (–3.25 from bregma, A: d–f) levels showed selective pattern of EBA staining according to the stressors examined. Leakage of EBA in the primary somato-sensory cortex, piriform cortex, hippocampus, hypothalamus and amygdala are common in all stress experiments. (B) In the spinal cord, sections passing through the C5 (B: a–c) or L2 (B: d–f) levels show marked extravasation of EBA in the gray matter. Staining of the dorsal and ventral horns of the cord and the area surrounding the central canal is common in all stress experiments. Bar: 5 mm.

stress-induced BBB permeability is reversible in nature even if animals are exposed to continuous stress. It appears that when animals enter into the adaptive phase of stress, the changes in BBB permeability tend to restore. However, further studies are needed to clarify these points. In another experiment, animals are allowed to rest in cages after 8 h of immobilization, and BBB permeability is determined 3 and 6 h after discontinuation of the stress. These animals did not show any significant increase in BBB permeability to Evans blue or radioiodine tracers (Fig. 9). These observations suggest that stress-induced BBB disruption is short lasting. However, it is not known whether the increased permeability of the BBB helps animals to adapt to the new circumstances or reflects their inability to cope with stress.

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Fig. 7 Representative example of Evans blue albumin (EBA) extravasation on the dorsal and ventral surfaces of the brain (a,b) and spinal cord (c,d) following forced swimming (FS, A), heat stress (HS, B) and immobilization (IMZ, C). The pattern and intensity of EBA extravasation vary into the brain and spinal cord according to the stressors used. Staining of somatosensory cortex, piriform cortex, cerebellum, hypothalamus, pons and brain stem is common in all stress experiments. Lateral views of brains (D) show staining of the temporal cortex and pons-medulla regions following heat stress (a,b) and forced swimming (c,d). Bar: 5 mm.

Table 9 Showing Percent Change in the rBBB and rCBF from the Control Values in Immobilization Stress a Immobilization stress rBBB % change Brain regions

1h

4h

8h

rCBF % change 11 h

14 h

1h

4h

8h

11 h

14 h

a. Frontal cortex

–8

0

+25

+08

+6

+6

+15

–2

+8

0

b. Parietal cortex

–11

+5.5

+622

+45

–14

–4

+7.5

–36.5

+2

+12

c. Occipital cortex d. Temporal cortex

0 0

+6 –22

+1018 +55.5

+8 –20

–23 –11

+8 +14

+12.5 +21

–33 –18

+8 –12

+21 0

e. Cingulate cortex

–17

–16

+61

+16

–28

+3

+18

–24

+3

0

f. Hippocampus g. Caudate nucleus

+9 0

–9 +14

+563 +85

+9 +32

+5 –12

+6 +4

+21 +24

–2 –9

+8 +12

+15 +4

h. Thalamus i. Hypothalamus

+20 +19.5

–8.5 –13

+94 +102

+43 +12

+11 –12

–12 –25

+30 –10

–24 –17

–1 +21

+9 +33

j. Sup. colliculus

+20

+16

+225

–18

–30

–25

–16

–15

+22

+21

k. Inf. colliculus l. Cerebellum

+14 +33

+9 +11

+268 +255

–10 +23

+14 –12

–29 –14

–24.5 +19

–20 –14

+3 +8

+7 –9

0

–16

0

–12

–12

+17

+6

+9

0

+5

+5

0

+5

+25

+6

+21

+12.5

+6

0

+8

m.Pons n. Medulla

a Data modified after Sharma and Dey (1986, 1988) and Sharma (unpublished observations).

+, increase; –, decrease; 0, no change from the control group; Data from 6 to 8 animals in each group. Values above/below 30% are significantly different from the controls.

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networks handle a stress response more efficiently with advancing age of the stress-induced neurochemical releases are less prominent in old rats is not clear from this study. Alternatively, a less sensitive response of cerebral microvessels to neurochemicals with advancing age is also likely (McEntee et al., 1991). f. REGIONAL CHANGES IN BBB FUNCTION. As mentioned earlier, the neuroanatomy of stress pathways varies according to the stressors used. Thus, each stressor may have some specific effects on the BBB function in a particular brain region. To examine this hypothesis, changes in regional BBB (rBBB) permeability in 14 brain areas are determined following immobilization (see Fig. 10; Table 9). The rBBB is disrupted in 12 brain areas after 8 h of immobilization. The most marked increase in tracer extravasation was seen in the occipital cortex (1018%) followed by the parietal cortex (622%), hippocampus (563%), inferior colliculi (268%), cerebellum (255%), superior colliculus (225%), hypothalamus (102%), thalamus (94%), caudate nucleus (85%), cingulate cortex (61%), temporal cortex (55.5%), and frontal cortex (25%). The pons and medulla did not show extravasation of radiotracers (Sharma and Dey, 1986a). The regional extravasation of radiotracer at 4 h immobilization is not significant in any region. At 1 h immobilization stress, a mild extravasation of radiotracer (14 to 33%) is seen in the midbrain regions, including the cerebellum (Table 9). These observations suggest that immobilization stress increases the permeability of the BBB in selective and specific areas. The functional significance of such a selective increase in the BBB permeability is not well understood. 5. Cerebral Blood Flow Fig. 8 Representative examples of coronal sections of rat brain from 6 different levels (a–f) showing Evans blue albumin (EBA) extravasation following immobilization (IMZ, A), heat stress (HS, B) and forced swimming (FS, C). The pattern and intensity of EBA showed selective variations in different levels according to the stressors used. Most extensive staining of various brain regions are observed after heat stress (B) followed by forced swimming (C) and immobilization (A). Staining of cingulate (a–c) , frontal (a), parietal (b,c), temporal (c), occipital (d), and piriform (b–c) cortices are clearly evident (A: a–c; B: a–d; C: a–c). Different pattern and intensity of EBA staining in immobilization (A), heat stress (B) and forced swimming (C) are apparent in deep brain structures, e.g., caudate putamen (a), hippocampus (b–c), thalamus (b–d), hypothalamus (b–d), amygdala (b–c), brain stem (d–e) and cerebellum (f). (Co-ordinates for coronal sections: a = + 0.10 to +0.45; b = –3.25 to –3.90; c = –4.20 to –4.60; d = .5.25 to –6.65; e = –7.10 to –7.60; f = –10.60 to –11.90 from Bregma). Bar: 5 mm.

e. AGE-RELATED EFFECTS. To understand the effects of age on stress-induced BBB dysfunction, a group of old rats (30–35 weeks) were subjected to 4, 8, and 12 h of immobilization stress (H. S. Sharma, unpublished observations). Observations show that the magnitude of tracer extravasation is reduced considerably in old animals following 8 h of immobilization (H. S. Sharma, unpublished observations). No significant increase in tracer extravasation was noted in these groups of animals at the end of either 4 or 12 h after stress (results not shown). These observations suggest that the stress-induced breakdown of the BBB depends of the age of animals. Whether the neuronal

Changes in regional cerebral blood flow following long-term immobilization stress are not well known. We examined rCBF using tracer microspheres labeled with 125I in identical brain areas showing leakage of the radiotracer in stress (Sharma, 1987; for details, see Chapter 12). The rCBF did not alter significantly following 1 or 4 h of immobilization stress (Table 9). However, at the end of 8 h immobilization, the rCBF showed a widespread decline (Fig. 10) in areas associated with increased BBB permeability (Sharma and Dey, 1986a). However, the severity of flow reduction did not coincide with the magnitude of radioiodine extravasation (Fig. 10). Thus, at the end of 8 h of immobilization, the parietal cortex exhibited maximum reduction in the rCBF by 36.5% followed by the occipital cortex (33%), cingulate cortex (24%), thalamus (24%), inferior colliculus (20%), temporal cortex (18%), hypothalamus (17%), cerebellum (14%), caudate nucleus (9%), hippocampus, and frontal cortex by 2%. The decline in rCBF (about 14 to 36%) was significant in 6 out of 14 regions examined. This decline in rCBF was gradually diminished with a further continuation of immobilization stress up to 11 or 14 h. Thus, the rCBF returned near normal levels at the end of 11 h after stress (Table 9). At the end of 14 h immobilization, some brain regions exhibited a mild increase in the rCBF (Table 9). These observations suggest that rCBF increases in the very early periods (5 and 15 min; Ohata et al., 1981) of stress are normalized at 1 and 4 h after immobilization (Sharma and Dey, 1986a). When immobilization is prolonged to 8 h, the rCBF

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Fig. 9 Shows changes in rectal temperature (a), blood–brain barrier (BBB) permeability (b), serotonin (5-hydroxytryptamine, 5-HT, c) and cerebral blood flow (CBF, d) following forced swimming (A), immobilization (B) and heat stress(C) compared to control group (cont). Subjection of rats to 30 min (30 m) forced swimming, 8 h immobilization and 4 h heat stress results in profound increase in the BBB permeability to Evans blue albumin (EBA) and radioactive iodine ([131]Iodine) tracers (b) together with a significant elevation of plasma and brain serotonin levels (c). The cerebral blood flow (CBF) significantly reduced at this time (d). The rectal temperature considerable decreased at the time of BBB disruption following forced swimming and immobilization (A: a, B: a) whereas, marked hyperthermia is seen during heat stress (C: a). Stress induced changes in the body temperature, BBB permeability, 5-HT level and the CBF are reversible in nature. Most of these variables restored near normal values in animals subjected to rest (r) of various periods after stress ranging from 30 min (0.5 h) to, 6 h. Values are mean±SD of 5 to 8 rats; *P < 0.05; **P < 0.01 ANOVA followed by Dunnet’s test for multiple group comparison with one control group. Data modified after Sharma and Dey (1986a, 1987b, 1991a).

Table 10 Blood–Spinal Cord Barrier Permeability and Serotonin Levels in Normal and Stressed Young Rats BSCB permeability [131]Iodine % Spinal cord regions Cervical Thoracic Lumbar

5-HT level µg/g

Level

Control n=6

Swimming 30 min n=8

Heat stress 4h n=6

Immobilization 8h n=8

Control n=6

Swimming 30 min n=8

Heat stress 4h n=6

Immobilization 8h n=8

1–4 8–11 1–4

0.26±0.04 0.34±0.06 0.39±0.04

0.66±0.04** 78±0.12** 0.58±0.08*

0.85±0.12** 0.96±0.17** 0.68±0.14*

0.60±0.08** 0.84±0.10** 0.70±0.10*

0.57±0.12 0.72±0.12 0.54±0.12

0.88±0.16* 1.06±0.08** 1.14±0.08**

0.92±0.17* 1.46±0.12** 1.66±0.12**

0.74±0.07* 0.98±0.10* 1.17±0.08*

Values are mean±SD; *P < 0.05; **P < 0.01 ANOVA followed by Dunnet’s test for multiple group comparison.

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Fig. 10 Changes in regional blood–brain barrier (rBBB, A) permeability and regional cerebral blood flow (rCBF, B) following forced swimming, heat stress and immobilization stress. The rBBB permeability showed significant increase to radiotracer in 9 brain regions (b,c,e–i, k,l) following 30 min forced swimming (A), all brain regions following 4 h heat stress (B) and in 12 brain regions (a–j) following 8 h immobilization stress (C). These brain regions showed a significant decrease in the rCBF at this time period. Changes in the rBBB or rCBF during other periods of stress are not significant from the control group. Brain regions (FS): a = frontal cortex, b = parietal cortex, c = occipital cortex, d = ant. cingulate cortex, e = post. cingulate cortex, f = cerebellum vermis, g = cerebellar cortex, h = caudate nucleus, i = hippocampus, j = colliculi, k = thalamus, l = hypothalamus, m = medulla, n = brain stem. Brain regions (HS, IMZ) : a = frontal cortex, b = parietal cortex, c = occipital cortex, d = temporal cortex, e = cingulate cortex, f = hippocampus, g = caudate nucleus, h = thalamus, i = hypothalamus, j = sup. colliculus, k = inf. colliculus, l = cerebellum, m = pons, n = medulla. Values are mean±SD of 10 to 12 rats. Data modified after Sharma and Dey (1986a, 1987b, 1991a).

declined in most of the brain regions (Sharma and Dey, 1986a, 1988), and further continuation of stress for another 3 or 6 h resulted in normalization again with an occasional increase in rCBF values in some brain regions. It appears that stress-induced neurochemical metabolisms play an important role in rCBF changes. Obviously, the release of several neurochemicals over time will influence the vasomotor response of the microvessels. 6. Blood–Spinal Cord Barrier Permeability The effects of immobilization stress on BSCB permeability are still unknown. We initiated a series of experiments in which BSCB permeability to several protein tracers was examined following a variety of stressful events. Results show that the spinal cord is very sensitive to stress-induced BSCB disruption in a specific and selective manner (Figs. 4, 6, and 7; Table 10). Immobilization stress for 1 or 4 h did not induce extravasation of tracers across the BSCB. A mild to moderate degree of Evans blue extravasation in the dorsal surface of the spinal cord

in the cervical and thoracic regions is apparent after 8 h of immobilization stress (Figs 4 and 7). The ventral surface of the cord exhibited light blue staining (Figs. 4 and 7). A cross section of spinal cord showed diffuse blue staining located mainly in the gray matter (Fig. 6). The extravasation of Evans blue into the dorsal, ventral, and lateral gray matter showed mild to moderate individual variation. To our knowledge, these observations are the first to show that immobilization stress is capable of inducing a selective and specific breakdown of the BSCB. This increase in Evans blue extravasation is no longer observed in animals subjected to 11 or 14 h of immobilization (H. S. Sharma, unpublished observations). These observations suggest that similar mechanisms are operating in disruption of the BBB and BSCB during immobilization stress. 7. Plasma and Brain Serotonin Serotonin is one of the important neurochemical mediators of stress (as described earlier) and the BBB function (Wahl et al., 1988, for details, see Chapter 13). We measured plasma and

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H ARI S HANKER S HARMA Table 11 Schedule of Drug Treatments in Stress a

Drug

Type

Dose (mg/kg)

Route

No. of injection

A. Synthesis inhibitors 1. p-CPA 2. Indomethacin

Time before stress

5-HT

100 mg

ip

3 (1/d)

24 h

PG

10 mg

ip

1

30 min

1. Ketanserin 2. Cyproheptadine

5-HT2 5-HT2

1 mg 15 mg

ip ip

1 1

30 min 30 min

3. Naloxone

opioids

10 mg

ip

1

30 min

C. Anti-stress 1. Diazepam

anxiolytic

4 mg

sc

1

30 min

1. 5,7-DHT

5-HT

200 µgb

2. 6-OHDA 3. 6-OHDA

CA CA

50 mg/kg 200 µgb

icv iv icv

1 1 1

7d 5d 7d

E. Ion channel blocker 1. Nimodipine

L-type Ca2+

2µg/kg/m infusion

iv

2 h continuous

5 min

F. Antimitotic drug 1. Vinblastine

vesicular transport inhibitor

1 mg

iv

1

48 h

cAMP↑

10 mg

ip

1

15 min

B. Receptor blockers

D. Neurotoxins

G. PDE inhibitor 1. Aminophylline

a Data modified after Sharma (1982, 1999), and Sharma and Dey (1986, 1987).

b, total dose; min, minute; h, hour; d, days; ip, intraperitoneal; sc, subcutaneous; icv, intracerebroventricular (right lateral ventricle); 5-HT, serotonin; PG, prostaglandin; CA, catecholamines; PDE, phosphodiesterase; ↑, increase.

brain serotonin levels following immobilization stress using a sensitive spectrophotofluorometric method (Snyder et al., 1965; Sharma and Dey, 1981, 1986a). No significant changes in serotonin levels are seen at 1 or 4 h after immobilization (Fig. 9). However, continuous immobilization of young rats to 8 h resulted in a profound increase in plasma (706%) and brain (147%) serotonin levels (Sharma and Dey, 1981, 1986a). Interestingly, this increase in serotonin levels is no longer observed at the end of 11 or 14 h of stress (Sharma, 1982; Sharma and Dey, 1988; H. S. Sharma, unpublished observation). These observations suggest that serotonin could contribute to BBB or BSCB disruption in immobilization stress. A decrease in the serotonin level following 11 h of immobilization may be due to a rapid catabolism or a slower release of the amine from its stores due to a gradual adaptation to stress. Measurement of monoamine oxidase (MAO) activity, the enzyme responsible for the degradation of serotonin, and its metabolite, 5-hydroxyindole acetic acid (5-HIAA), will further confirm this idea. 8. Mechanisms of Serotonin Elevation The mechanisms by which stress induces an increase in serotonin levels in the plasma or brain are not known (Chaouloff, 1993, 2000). It appears that acute stress increases brain tryptophan levels through the enhancement of sympathetic nervous system activity (Chaouloff, 1993). Increased tryptophan

hydroxylase activity in stress further influences glucocorticoid response (see Chaouloff, 1993). Immobilization stress increases serotonin synthesis, release, and its metabolism (Haleem et al., 1989; Joseph and Kennett, 1983; Kennett and Joseph, 1981). Glucocorticoids may also affect stress-induced alterations in serotonin release and/or metabolism. Immobilization stress for 1–3 h is shown to decrease, not change, or increase central serotonin levels (Conn and Sanders-Bush, 1987; Culman et al., 1984; Curzon et al., 1972). This indicates that stress-induced alterations in tryptophan metabolism are not directly related to serotonin synthesis and release (see Chaouloff, 1993). Acute stress initiates a biphasic response of brain serotonin content, showing an early decrease followed by a sustained recovery or an enhanced content of the amine (Corrodi et al., 1968; Telegdy and Vermes, 1976). Thus, acute stress increases serotonin synthesis by enhancing tryptophan availability and stimulating tryptophan hydroxylase activity to counterbalance the release-induced depletion of serotonin from the serotonergic neurons (Curzon et al., 1972; Dunn, 1988). However, the contribution of nonneural serotonin sources such as, mast cells in the diencephalon and blood platelets in stress-induced altered serotonin metabolism is not well known. 9. Influence of Drugs on BBB Function To understand the mechanisms of stress-induced BBB disruption, the influence of several drugs modifying serotonin,

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Table 12 Effect of Drugs on Stress Symptoms and Physiological Variables in Animals Subjected to Various Stress Paradigm Stress symptoms

Type of experiment

Haemorrhagic Faecal spots in # pellets (nr.) stomach# (nr.)

Physiological variables

n

Rect Temp °C

MABP Heart rate Respiration torr beats/min breath/min

Arterial pH

PaO2 torr

PaCO2 torr

8h p-CPA

12 12

35.68±0.23 36.05±0.12

34±6 24±9*

35±5 39±3

76±6 75±6

272±8 n.d.

82±4 n.d.

7.38±0.14 7.36±0.04

83.12±0.34 82.46±0.41

31.28±0.43 31.41±0.62

Indomethacin 33.46±0.15** Diazepam 32.54±0.13**

8

36.67±0.21*

21±12

40±5

73±5

n.d.

n.d.

7.39±.05

78.56±.34**

8

35.07±0.21**

5±3**

2±2**

78±5

n.d.

n.d.

7.36±0.08

81.16±0.23**

A. Immobilization

Cyproheptadine Vinblastine 32.43±0.13** 6-OHDA iv 6-OHDA icv 5,7-DHT icv

9 8

36.18±0.12* 36.28±0.21*

30±5 21±12

3±5** 40±11

77±8 76±6

n.d. n.d.

n.d. n.d.

7.34±0.10 7.33±0.08

78.48±0.21** 32.46±0.16 80.38±0.10**

12 8 10

35.43±0.12 36.15±0.21 36.01±0.12*

32±7 33±5 23±7

38±8 23±12 12±6*

74±8 76±8 80±4

n.d. n.d. n.d.

n.d. n.d. n.d.

n.d. n.d. n.d.

n.d. n.d. n.d.

n.d. n.d. n.d.

B. Heat stress 38°C 4h p-CPA Indomethacin Diazepam Cyproheptadine

12 6 6 6 6

41.68±0.21 38.56±0.23** 39.23±0.18* 39.32±0.12* 40.13±0.21*

2±2 n.d. n.d. n.d. n.d.

24±6 8±6** 20±8 6±2** 8±6*

75±8 88±4** 78±6 85±6* 83±7*

267±6 n.d. n.d. n.d. n.d.

88±3 n.d. n.d. n.d. n.d.

7.34±0.08 7.30±0.05 7.28±0.12 7.34±0.10 7.31±0.08

82.87±0.32 79.12±±0.12* 80.13±0.13* 79.45±0.54 80.14±0.43

33.08±0.21 34.47±0.24* 33.12±0.43 34.01±0.32 33.67±0.43

Vinblastine 6-OHDA iv 6-OHDA icv 5,7-DHT icv

8 6 8 8

41.05±0.12 41.23±0.33 40.23±0.23* 40.54±0.12

n.d. n.d. n.d. n.d.

34±8* 42±12* 32±8* 23±8

76±5 70±4* 74±6 76±6

n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d.

7.36±0.08 n.d. n.d. n.d.

79.43±0.43* n.d. n.d. n.d.

34.02±0.54 n.d. n.d. n.d.

30.42±0.48 31.56±0.43* 32.65±0.36* 31.87±0.36* 32.29±0.48* 30.68±0.38 30.76±0.25

40±8 30±12* 35±8* 20±8* 28±12 36±14 33±15

44±12 20±8* 46±12 28±6** 36±8** 34±14* 40±12

83±8 85±5 82±6 86±4 84±4 82±8 84±6

270±6 n.d. n.d. n.d. n.d. n.d. n.d.

85±5 n.d. n.d. n.d. n.d. n.d. n.d.

7.36±0.06 7.37±0.04 7.37±0.08 7.37±0.08 7.37±0.04 7.36±0.11 7.36±0.08

81.36±0.44 80.76±0.34 80.48±0.32 80.28±0.37 80.82±0.35 81.54±0.54 81.06±0.34

33.22±0.24 33.86±0.35 33.78±0.47 34.42±0.28 34.26±0.66 33.87±0.36 34.08±0.18

C. Forced swimming 30 min p-CPA Indomethacin Ketanserin Cyproheptadine 5,7-DHT icv Vinblastine

8 5 5 5 6 6 5

Values are expressed as mean±SD; *P < 0.05; **P < 0.01 ANOVA followed by Dunnet’s test for multiple group comparison with one control; test for semiquantitative data; n.d., not done.

#Chi-Square

prostaglandin, and catecholamine metabolism was examined on stress response and endothelial cell function (see Table 11). a. SEROTONIN NEUROTOXIN. The central or peripheral contribution of serotonin in stress was examined using 5,7-dihydroxytryptamine (DHT), which destroys central serotonergic nerve terminals (Elde and Hole, 1988). The compound (175 µg in 10 µl) was dissolved in physiological saline containing 0.1% ascorbic acid and was administered into the right lateral cerebral ventricle (Sharma et al., 1995b). Two weeks after 5,7-DHT administration, the animals were immobilized for 8 h and BBB permeability, CBF changes, and serotonin levels were examined. This dose and time schedule will destroy more than

80% of the serotonergic nerve terminals in the CNS without having any significant effect on catecholaminergic nerve fibers and terminals (Sharma, 1982; Elde and Hole, 1988; Sharma et al., 1995b). Destruction of serotonin nerve terminals in the CNS considerably reduced the extravasation of Evans blue and radioiodine tracers in the brain along with serotonin levels in the plasma and brain. However, the physiological variables and stress symptoms were not altered (Fig. 11; Table 12). This observation suggests that central serotonergic neurons alone are not responsible for the stress-induced increase in plasma and brain serotonin. The mast cells and pineal gland could be other potential sources of increased brain serotonin in

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Fig. 11 Effect of catecholamines and serotonin neurotoxins on 8 h immobilization (1 B) and 4 h heat stress (2 B) on the rectal temperature changes (a), blood–brain barrier (BBB) permeability (b) and 5-hydroxytryptamine (5-HT) levels (c) compared to control (A). Destruction of peripheral catecholaminergic nerve fibres with 6-OHDA (i.v. C) or central noradrenergic neurones with 6-OHDA (i.c.v. D) or central serotonergic nerve terminals with 5,7-DHT (i.c.v. E) did not reduce the BBB permeability or 5-HT levels in stress. On the other hand, administration of aminophylline, a drug known to increase cAMP accumulation (G) or infusion of 5-HT (i.v., H) significantly increased the BBB permeability following 4 h immobilization (1 G) or 2 h heat stress (2 G) compared to respective controls (A, F). Data modified after Sharma (1982); Sharma et al. (1995); Sharma (1999). (3) Regional changes in the BBB (rBBB) permeability and cerebral blood flow (rCBF) in old animals (age 32–35 weeks) following 30 min forced swimming (FS, 3a); 4 h heat stress (HS 3b) and 8 h immobilization (IMZ 3c). Only few brain regions showed significant (*,#) changes in the rBBB and rCBF in these animals following stress (for details, see text). Brain regions (FS*): a = frontal cortex, b = parietal cortex, c = occipital cortex*, d = ant. cingulate cortex, e = post. cingulate cortex*, f = cerebellum vermis*, g = cerebellar cortex*, h = caudate nucleus*, i = hippocampus*, j = colliculi, k = thalamus, l = hypothalamus, m = medulla, n = brain stem. Brain regions (HS* and IMZ#): a = frontal cortex*, b = parietal cortex*, c = occipital cortex*#, d = temporal cortex, e = cingulate cortex, f = hippocampus*#, g = caudate nucleus*#, h = thalamus*#, i = hypothalamus*, j = sup. colliculus*, k = inf. colliculus*, l = cerebellum, m = pons, n = medulla. Values are mean±SD of 6 to 8 rats, *P < 0.05; **P < 0.01 ANOVA followed by Dunnet’s test for multiple group comparison with one control group.

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Table 13 EEG Changes in Stress a EEG activity

BBB permeability

Parietal cortex Type of experiment

n

Amplitude (µV)

Cerebellar cortex

Frequency (Hz)

Amplitude (µV)

Whole brain

Frequency (Hz)

Evans blue mg (%)

0.23±0.03

Immobilization Onset

6

68±5

6±1

75±4

6±2

1h 4h

6 6

70±4 86±4**

6±2 5±2

73±3 89±4**

7±1 5±2

8h

6

10±5**

9±2

15±3**

9±3

1.21±0.22**

9h 11

5 5

30±5 54±4

8±2 7±1

45±3 64±5

8±3 7±2

0.34±0.11

p-CPA+stress onset 1h 4h 8h 11 h

6 6 6 6 5

80±5 76±4 60±4** 110±8** 108±4**

11±2 10±3 11±3 9±2 11±3

90±5 85±4 62±3** 118±6** 110±4**

12±2 11±4 12±3 10±2 9±2

Frontal cortex

0.23±0.08

Parietal cortex

#Heat stress onset 30 min 1h 2h 3h

5 5 5 5 5

64±3 90±4** 53±2* 50±4** 54±3

6±1 7±2 8±3 9±2 8±2

60±2 88±4** 55±2* 52±4** 53±3

6±2 8±2 8±2 9±3 8±3

4h

5

12±2**

8±2

10±2**

9±3

1.67±0.21**

Rest 1 h Rest 2 h

4 4

30±3** 38±4**

7±2 6±3

32±4** 35±3**

7±3 6±2

0.87±0.12**

a Data modified after Sharma and Dey (1988) and Sharma HS (#, unpublished observations).

Values are mean±SD; *P < 0.05; **P < 0.01 ANOVA followed by Dunnet’s test for multiple group comparison.

stress (Essman, 1978; Edvinsson et al., 1977; Sharma et al., 1995b). Additionally, elevated plasma serotonin could also contribute to the rise in brain serotonin due to a leaky barrier (Sharma, 1982; Sharma et al., 1995b). Elevation of plasma serotonin may result from the disintegration of platelets and/or alterations in its binding or reuptake mechanism in the circulation (Essman, 1978; Sharma et al., 1995b). b. CATECHOLAMINE NEUROTOXINS. The role of catecholamines in stress-induced BBB permeability was examined using the destruction of either peripheral or central noradrenergic neurons and nerve terminals with 6-hydroxydopamine (6-OHDA) (Table 13). The catecholamine neurotoxin was administered either into the right lateral cerebral ventricle (300 µg, icv; 8 days before stress; Uretsky and Iversen, 1969) or into the systemic circulation (50 mg/kg, iv, Fredholm et al., 1979) to destroy central and peripheral noradrenergic nerve terminals, respectively (Sharma, 1982). Destruction of either peripheral or central noradrenergic neurons with 6-OHDA did not prevent BBB disruption in stress

(Table 12), and the plasma and brain and serotonin levels continue to remain high. The stress symptoms were unaffected by this drug treatment (Fig. 11). The extravasation of tracers following central administration of 6-OHDA is augmented following stress (Fig. 11). This indicates that central noradrenergic neurons have some inhibitory control over stress-induced BBB dysfunction. An increase in water permeability in brain following stimulation or lesion of the locus coeruleus (Raichle et al., 1975) is in line with this idea. It appears that catecholamines are involved in cardiovascular and thermoregulatory responses in stress and do not influence the BBB function directly. 10. cAMP Phosphodiesterase Inhibitor Augments BBB Disruption It has been suggested that serotonin stimulates vesicular transport either directly or through cAMP, causing increased BBB permeability (Joó et al., 1975; Westergaard, 1978; Sharma et al., 1990; for details, see Chapter 12). To understand the role of cAMP in stress-induced BBB dysfunction, the

262

H ARI S HANKER S HARMA

Fig. 12 Representative examples of light microscopic changes in different brain regions following 4 h heat stress (HS), 8 h immobilization (IMZ) or 30 min forced swimming (FS). Marked degeneration (arrow heads) in dentate gyrus and CA-4 region (*) of the hippocampus is seen following HS (A: b) compared to control (A: a). Many damaged and distorted nerve cells in the brain stem regions following HS is apparent (A: d, arrows) compared to control (A: c). Mild to moderate cell damage in the hippocampus CA-4 region is seen following IMZ (A: e, arrows) or FS (A: f, arrow heads). IMZ (B: a) and FS (B: b) induces specific and selective cell damage (arrow heads) in the cerebral cortex. Cell damage in ependymal cells (arrows) in the median eminence following IMZ (B: c) and FS (B: d) is clearly evident. Degeneration (arrows) of choroid plexus epithelial cells and ependymal cells in the lateral cerebral ventricle by IMZ (B: f) and FS (B: g) is apparent compared to control (B: e). The cerebellar Purkinje cells and granule cells show marked degenerative changes following FS (B: i, arrows) compared to control (B: h). A: a–d Nissl stain; others H & E stain on 3 µm thick paraffin sections. Bars: (A: a–b) 50 µm; (c–d, g–h) 25 µm; (e–f) 30 µm; (B: a–i) 40 µm.

influence of a potent cAMP phosphodiesterase (PDE) inhibitor, theophylline (Aminophylline, 10 mg/kg, ip, 30 min after stress), was examined (Appleman et al., 1973; Sharma, 1982). Inhibition of PDE with theophylline induces an accumulation of cAMP (Appleman et al., 1973; Beltman et al., 1993; Corbin et al., 2000; Monfort et al., 2002). An intraarterial infusion of cAMP increases BBB permeability by enhancing the vesicular transport of tracers (Joó, 1972; Westergaard, 1978; for details, see Chapter 12). In cell culture studies, cAMP enhances long-term permeability across the gap junctions (Burghardt et al., 1995), which is well correlated with an alteration in the distribution of connexin43 (Cx43) immunoreactivity (Hansen and Casanova, 1994; Burghardt et al., 1995). The effect of cAMP on Cx43 promotes junctional permeability by increased trafficking and/or the assembly of Cx43 to plasma membrane gap-junctional plaques (Hansen and Casanova, 1994; Burghardt et al., 1995). However, the effects of cAMP on tight junctional permeability are still unknown. It may be that cAMP is capable of augmenting the tight junctional membrane permeability as well (Sharma et al., 1998a, see later).

Treatment with the PDE inhibitor theophylline causes an accumulation of cAMP within the cerebral microvascular endothelial cells. Thus, theophylline-treated rats were subjected to 4 h of immobilization stress (Sharma, 1982) that do not normally induce leakage of Evans blue or radiotracers in the brain. Interestingly, theophylline-treated rats exhibited a marked increase in BBB permeability following 4 h of immobilization without showing stress symptoms or increased plasma and brain serotonin levels (Fig. 11). These observations suggest that cAMP plays an important role in stress-induced BBB disruption. 11. Exogenous Serotonin Induces Early BBB Disruption An increased level of serotonin in stress appears to be one of the important factors in BBB disruption. Thus, exogenous elevation of serotonin into the circulation in a dose comparable to that seen in immobilization stress induces BBB breakdown (Sharma et al., 1990; see Chapter 12). We administered 50% of the dose of serotonin into the systemic circulation during 4 h of immobilization and examined BBB function (Sharma, 1982; H. S. Sharma, unpublished observation).

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Fig. 13 Representative examples of ultrastructural changes in the rats brain following 4 h heat stress (HS), 30 min forced swimming (FS) or 8 h immobilization (IMZ) stress. In the cerebral cortex, one nerve cell with dark and electron dense cytoplasm (arrows) is seen following HS (A: a) Many degenerative changes can be seen in the neuropil (A: a). High power electron micrograph from the parietal cerebral cortex cellular layer III showing damaged synapses (arrows), vesiculation of myelin and membrane damage following HS (A: b). A completely collapsed microvessel (arrow heads) and distorted granule cells in the cerebellum are seen following HS (A: c). Perivascular edema (*), membrane damage and leakage of lanthanum across the microvessels (A-d) are quite frequent in HS. Vacuolation, membrane damage (arrows) and perivascular edema (*) are frequent following FS (B: a,c) or IMZ (B: b,d). Myelin vesiculation (B: e) and nerve cell damage (B: f) is very common in hippocampus and in thalamus following HS. Bars: (A: a) 0.6 µm; (A: c,d) 1 µm; (A: b) 0.4 µm nm; (B: a–e) 1 µm; (B: f) 0. 5 µm. Data modified after Sharma and Cervós-Navarro (1990) (A: d); Sharma et al. (1998a) (B: f).

An infusion of serotonin (5 µg/kg/min for 10 min) alone in normal animals or subjection of rats to 4 h of immobilization did not induce extravasation of Evans blue or radioiodine tracers in the brain (Sharma and Dey, 1981, 1986; Sharma, 1982; Sharma et al., 1990; for details, see Chapter 12). However, when serotonin was infused in 4-h immobilized rats, a marked increase in BBB permeability to Evans blue and radioiodine tracer was observed in several brain regions (Fig. 11). Interestingly, the pattern of extravasation of Evans blue dye in the brain is quite similar to that seen after 8 h immobilization (see Chapter 12 for details). This observation suggests that mild stress has potentiated the effect of serotonin on BBB dysfunction. The probable mechanisms of the stress-induced potentiation of serotonin effects on the BBB function are unclear. An interaction of serotonin with other stress hormones, such as CRH and glucocorticoids, plays a synergistic role in inducing early breakdown of the BBB in stress (Esposito et al., 2001, 2002). To explore this point further, a selective blockade of CRH or glucocorticoids in serotonin-induced BBB dysfunction is needed. 12. Structural Changes in the Brain Morphological analysis of brains after 8 h of immobilization stress showed specific nerve cell damage in the cerebral cortex, hippocampus, cerebellum, and brain stem. Many dark and dis-

torted nerve cells are present in the superficial layers of the cerebral cortex, especially in the cingulate, parietal, temporal, and occipital cortex (Fig. 12). Extravasation of HRP is clearly evident in the cerebral cortex of 8-h immobilized rats (see Fig. 20). This indicates that BBB leakage is associated with brain damage in stress. The hippocampal dentate gyrus, CA4, and CA3 sectors, as well as the CA1 subfield, contain several damaged nerve cells (Fig. 12). Dark neurons are frequent in brain stem reticular formation. Degenerative changes in cerebellar Purkinje cells and granule cells are common (Fig. 12). Choroid plexuses and ependymal cells around the lateral and third ventricles show degenerative changes. In these regions, sponginess and edema are frequent (Fig. 12). These observations support the idea of blood–CSF barrier disruption by immobilization (see earlier discussion). At the ultrastructural level, areas showing BBB disruption reveal membrane damage, vacuolation, and distortion of nerve cells (Fig. 13). This indicates that stress-induced BBB disruption is associated with structural changes in the neuropil. 13. Alterations in Spontaneous EEG Activity The electrical activity of the brain is an important indicator of brain function that depends on the states of the CBF and

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metabolism (Cooper, 1974; for details, see Chapter 12). The EEG is altered in several stress-induced neurological diseases, particularly when BBB permeability is compromised (Sharma and Dey, 1988; Winkler et al., 1995). Thus, it would be interesting to examine whether immobilization stress-induced BBB breakdown is able to influence EEG activity. a. RECORDING OF CORTICAL EEG. The cortical EEG was recorded in male rats using transverse stainless steel screw electrodes (length, 4 mm; tip o.d., 1 mm) implanted chronically on the parietal cortex and on the cerebellar cortex (Fig. 14; Sharma and Dey, 1988). The other end of the electrode was attached to a female adapter by a 4-cm-long insulated cable that was exteriorized from the rear and tied with the back skin and sealed with leucoplast (Sharma and Dey, 1998). At the time of recording (6 to 8 days later), the female adapter was connected to its male counterpart (Phillips Electronics, Holland) leading to the EEG junction box (Kaiser 16-channel EEG machine, Copenhagen, Denmark). The amplifier and machine specifications (time constant 0.3 s, filter position open paper speed 7.5 mm/s, sensitivity 100 µV/cm) were kept constant in all the immobilization stress recordings (MacGillivary, 1974; Sharma and Dey, 1988; Winkler et al., 1995). The electrode resistance (ER) between paired cerebellar and parietal cortical electrodes ranged between 4–5 and 6–8 kΩ, respectively (Sharma and Dey, 1988). EEG analysis was done manually (see Table 13). b. BASAL CHANGES IN EEG ACTIVITY DURING THE DAY. Basal changes in spontaneous EEG activity are recorded in separate group of animals that are briefly immobilized for 5–10 min starting from 8:00 A.M. at regular intervals of 1 h until 8:00 P.M. The EEG activity did not alter significantly throughout the 12-h recording (Sharma and Dey, 1998; H. S. Sharma, unpublished observations). c. EFFECT OF IMMOBILIZATION STRESS ON EEG ACTIVITY. The bipolar EEG activity of conscious, restrained rats from the onset of immobilization to 1 h was predominantly high voltage and slow activity (HVSA, 60–70 µV; 6–7 Hz) (Fig. 14). The EEG voltage increased slightly at the end of 4 h of stress, which continued up to 5 h (70–90 µV, 5–6 Hz) (Fig. 14). After 6 h, the EEG voltage declined markedly, resulting in the appearance of low voltage fast activity (LVFA) at the end of 7 h stress. The most pronounced LVFA is evident at the end of 8 h of immobilization (10–15 µV, 9–11 Hz), which persisted up to 8.5 h (Fig. 14; Table 13). Examination of BBB permeability at the end of 8 h immobilization revealed marked blue staining in the cerebral cortex and in the cerebellar vermis (Fig. 14). This suggests that induction of LVFA is related to BBB breakdown (for details, see Chapter 12). It appears that LVFA in stress reflects altered BBB function caused by immobilization or due to increased brain serotonin from various sources following leaky barrier (for details, see Chapter 12). Interestingly, the LVFA disappeared gradually and HVSA ensued again when the stress was continued further in separate group of animals (Fig. 14). Thus, at the end of 9 h stress, an increase in EEG voltage without any major changes in frequency was observed (20–50 µV; 8–10 Hz). Almost 80%

Fig. 14 Representative example of EEG recordings during immobilization stress. The EEG activity recorded from the bipolar electrodes placed on the parietal cortex (P-C) and on the cerebellar cortex (C-C) showed high voltage slow activity (HVSA) at the onset of stress (A). Appearance of low voltage and fast activity (LVFA) is seen after 6 h immobilization stress. Marked flattening of the EEG is apparent after 8 h stress. At this time, extravasation of Evans blue albumin (EBA) is seen in the parietal and cerebellar cortex (A). B. The changes in EEG activity during stress are reversible in nature. Another rat subjected to continuous immobilization stress showed slight recovery of EEG at 9 h after stress. More than 50 % recovery in EEG activity is apparent at the end of 14 h immobilization. This effect is most pronounced in the parietal cortex recording (B). No extravasation of EBA is seen at this time indicating that EEG changes reflect alterations in the BBB function (for details, see text). Modified after Sharma and Dey (1988).

recovery of EEG activity was seen at the end of 11 h of immobilization (Fig. 14). No extravasation of Evans blue dye in any part of the brain is seen at the end of 11 h immobilization stress, indicating that the activation of EEG is reflected by BBB breakdown (Sharma and Dey, 1988; Winkler et al., 1995; for details, see Chapter 12). d. ENDOGENOUS SEROTONIN INflUENCES STRESS-INDUCED EEG ACTIVITY. To understand the functional interaction among stress-induced activation of EEG, serotonin levels, and BBB

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Fig. 15 Representative example of bipolar EEG recordings from the parietal cortex (P-C) and cerebellar cortex (C-C) in one p-CPA treated rat subjected to immobilization stress. The EEG activity in p-CPA treated rat is modified and mainly comprises high voltage and fast activity (HVFA). During immobilization stress the HVFA pattern continued throughout out the period of recording. A significant increase in EEG voltage is evident at 8 and 11 h immobilization stress. The p-CPA treated stressed rats did not show BBB permeability at 8 h immobilization stress (for details, see text). Modified after Sharma and Dey (1988).

disruption, the influence of the potent serotonin synthesis inhibitor p-CPA was examined (Sharma and Dey, 1988). Thus, separate groups of animals were treated with p-CPA (100 mg/kg, ip, daily for 3 days), and the EEG was recorded on the 4th day following continuous immobilization (Sharma and Dey, 1988, Fig. 15). EEG activity in drug-treated control groups was also recorded intermittently in parallel (H. S. Sharma, unpublished observation). Pretreatment with p-CPA alone resulted in the appearance of predominately high voltage fast activity (50–60 µV, 11–12 Hz) (Fig. 15) that did not alter in normal animals throughout the 12-h periods of recording (H. S. Sharma, unpublished observations). p-CPA-treated immobilized rats showed a further enhancement of HVFA activity at 1 h after stress (80–90 µV; 10–11 Hz) that continued throughout the 7- to 8-h period (Sharma and Dey, 1988; Fig. 21). At the end of 9–11 h of immobilization, the EEG voltage further increased without affecting the frequency (100–110 µV; 9–11 Hz; Fig. 15, Table 13). In p-CPA-treated animals, no extravasation of Evans blue dye was seen either 8 or 11 h after stress. These observations are in line with the idea that alterations in EEG activity are well reflected in BBB function.

The flattened EEGs are desynchronized recordings representing an activation pattern due to neuronal hyperexcitability (Moruzzi and Magoun, 1949). EEG activation takes place at a time when profound stress symptoms are developed, indicating that the state of mental tension and anxiety contribute to EEG desynchronization (Lacey, 1967; Zubeck and Wilgosh, 1963). The EEG in normal animals did not show alteration in the voltage or frequency during 12-h periods of recording (H. S. Sharma, unpublished observations; Sharma and Dey, 1998). This indicates that circadian variation or induction of sleep in animals during stress does not contribute to EEG activation in stress. The reversible nature of EEG activation seen following 11 h of immobilization supports this idea further (Sharma and Dey, 1988; H. S. Sharma, unpublished observations). The changes in EEG during stress are unrelated to local changes in the CBF or other physiological variables, e.g., arterial pH, blood gases, or MABP (Sharma and Dey, 1988). No significant changes in the physiological variables in untreated or p-CPA-treated immobilized animals support this hypothesis. A reduction in the CBF alone in stressed animals is not sufficient to influence EEG activity (Paulson and Sharbrough, 1974). EEG flattening occurs when the blood supply to the brain is reduced more than 50% (Paulson and Sharbrough, 1974). Thus, a reduction in CBF of 30 to 35% is quite unlikely to influence EEG depression (for details, see Chapter 12). It appears that a high circulating level of serotonin in the blood plasma at the time of BBB disruption in immobilization stress contributes to EEG flattening. The amine enters into the brain fluid compartment due to leakage of the BBB in stress and induces EEG activation (Longo, 1977; Winkler et al., 1995; see Chapter 12). An isoelectric EEG following administration of the serotonin precursor 5-hydroxytryptophan (5-HTP), which readily penetrates the BBB and induces massive marked elevation of brain serotonin levels (Cooper, 1974; Dongier, 1974; Doyle et al., 1968), is in line with this assumption. Serotonin influences EEG desynchronization in animals when administered into the internal carotid artery or applied on the area postrema (Koella and Czicman, 1963, 1966; Koella et al., 1968). Serotonin-induced EEG activation is prevented by prior treatment with p-CPA (Koella and Czicman, 1963; Troda, 1967). Prevention of EEG flattening in stress by pretreatment with p-CPA clearly suggests an involvement of serotonin in stress-induced EEG activation (Sharma and Dey, 1988; see Chapter 12). B. Forced Swimming As mentioned earlier, forced swimming is a severe stressful condition (Dawson and Horvath, 1970; Gruner and Altman, 1980; Harri and Kuusela, 1986; Lalonde, 1986; Porsolt et al., 1979). Alterations of cerebral circulation, vertebral artery injury, cerebellar stroke, abnormal neurological function, and behavior while swimming are well documented in the literature (Sherman et al., 1991; Toole and Tucker, 1960; Tramo et al., 1985). Swimming involves movement of head and body positions for proper orientation and balance in the water (Gruner and Altman, 1980). This activates the cerebellum and associated brain stem regions for coordinated limb movements

266 (Gruner and Altman, 1980; Lalonde 1986; Porsolt et al., 1979). Specific activation of central serotonergic and catecholaminergic systems occur during forced swimming in laboratory animals (Harri and Kuusela, 1986; Porsolt et al., 1979). Forced swimming is used frequently as an animal model of depression. When rats are forced to swim in a restricted pool, they quickly acquire an immobility response (Lalonde, 1986). This response is influenced by pharmacological means. The hypoactivity caused by swim stress is associated with selective neurochemical metabolism in the brain. However, the status of the BBB in forced swimming is not well examined. Our laboratory was the first to show that rats subjected to forced swimming exhibit selective disruption of the BBB that is mediated by serotonin (Sharma and Dey, 1980; Sharma et al., 1991a). This effect appears to be age dependent (Sharma et al., 1995b). However, it is not clear whether forced swimming is associated with breakdown of the BSCB as well. 1. Continuous Forced Swimming Rats (age 9–10 weeks or 25–32 weeks) were subjected to continuous forced swimming between 8:00 and 9:00 AM to avoid circadian variations in the results (Sharma and Dey, 1980; Sharma et al., 1991a). The animals were placed individually in a Corning glass cylinder (150 cm height, 20 cm internal diameter) containing water (18 cm depth) maintained at 30±1°C (Sharma et al., 1991a). To avoid the immobility response, the water was stirred manually with a glass rod from time to time (every 4 or 5 min), which allowed the animals to swim continuously in the pool during the entire stress session (Sharma et al., 1991a, 1995b). 2. Stress Response Subjection of animals to forced swimming induces marked hypothermia that is dependent on the duration of swimming (Table 8). A progressive increase in the excretion of fecal pellets was seen during the swimming exercise (Table 8). Animals subjected to a 30-min swim stress exhibited profound hemorrhagic spots in their stomach wall (Sharma et al., 1991a; Table 8). Rats allowed to rest after swimming showed a gradual restoration of their body temperature within 1 h (Sharma et al., 1991a). The number of fecal pellets excreted during the rest period is quite comparable to normal rats (Table 8). Adult animals (25–32 weeks old) subjected to 30 min of swimming showed considerably less hypothermia, excretion of fecal pellets, and gastric hemorrhages in the stomach wall compared to young rats (Table 8; Sharma et al., 1995b). These observations suggest that the forced swimming-induced stress response is dependent on the duration and age of the animals. 3. Physiological Variables Continuous forced swimming for 30 min in young rats induced marked hypotension and slight changes in PaO2 and PaCO2 without affecting the arterial pH (Sharma et al., 1991a, 1995; Table 8), whereas 5 and 15 min forced swimming did not affect these variables (Table 8). However, adult rats subjected to a 30-min swim stress caused mild but similar changes in the physiological variables (Sharma et al., 1995b; Table 8). These physiological variables returned to a normal level following 1 h

H ARI S HANKER S HARMA rest after 30 min of swimming (Table 8). This indicates that forced swimming stress-induced changes in physiological variables are reversible in nature. 4. BBB Permeability Extravasation of Evans blue albumin in the brain of young rats is evident following 30 min of forced swimming (Figs. 4 and 7). The extravasation of dye was noted in five brain regions: the cingulate cortex, parietal cortex, occipital cortex, cerebellum, and the dorsal surface of the hippocampus. In many cases, the cerebellar vermis took moderate blue staining compared to the lateral cerebellar cortex (Fig. 7). The deep cerebellar nuclei were mainly unstained (Figs. 6 and 8). Walls of the lateral ventricle took mild stain, whereas the fourth ventricle exhibited deep blue staining (Fig. 5). Areas around the third ventricle were mildly stained (Fig. 5), indicating that forced swim stress induces breakdown of the blood–CSF barrier as well. Extravasation of the radioiodine tracer following 30 min of stress was observed in eight brain regions. Thus, in addition to the five blue-stained regions, the radiotracer is present in another three brain areas: the caudate nucleus, thalamus, and hypothalamus (Figs. 9 and 10). No significant changes in BBB permeability were observed between male and female rats subjected to 30 min of forced swimming (H. S. Sharma, unpublished observations; Sharma et al., 1995b). Subjection of animals to a short duration of swimming, e.g., 5 or 15 min, did not show extravasation of protein tracers in the brain (Fig. 9). Furthermore, when subjected to 30 min of swim stress, adult rats did not exhibit any significant increase in tracer extravasation in the brain (Fig. 11). Taken together, these observations indicate that forced swimming-induced BBB disruption is dependent on the duration and age of the animals (Sharma et al., 1995b). The BBB disruption following forced swimming in young rats is reversible in nature. Thus, BBB permeability is no longer observed in rats subjected to a 2-h rest after 30 min of swimming (Fig. 9; Sharma et al., 1991a, 1995b). 5. CBF Changes Measurement of CBF using radiolabeled microspheres (Sharma, 1987) showed a significant decline in regional CBF in several brain areas following 30 min of forced swimming in young rats (Fig. 10). However, regional CBF showed a significant increase in some brain regions during 5 or 15 min of swim stress. Regions that exhibited a decline in rCBF showed an increase in BBB permeability. However, the magnitude of tracer extravasation is unrelated to the intensity of flow reduction (H. S. Sharma, unpublished observations). These observations suggest that alterations in the CBF are not directly related with BBB disruption. The decline in rCBF is reversible in nature. Thus, rCBF values are restored near normal levels following 1 h of rest after 30 min of forced swimming. A mild increase in the rCBF in some brain regions is observed at this time (H. S. Sharma, unpublished observations). Adult animals subjected to 30 min of forced swimming, however, did not show any significant decline in the rCBF in any brain region (Fig. 11). However, some brain regions showed a mild but significant increase in the rCBF (H. S. Sharma, unpub-

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lished observations). These findings indicate that changes in rCBF following swim stress are dependent on the duration and age of the animals. 6. Serotonin Level Measurement of plasma and brain serotonin following 30 min of forced swimming showed a profound rise in young rats (Fig. 9). This increase in amine is not seen in animals following a 2-h rest after swim stress. Animals subjected to a short duration of swimming did not show any marked alterations in the serotonin level (Fig. 9). Adult animals subjected to 30 min of swimming resulted in a mild but significant rise in plasma and brain serotonin (H. S. Sharma, unpublished observations). This indicates a close parallelism between the increased serotonin level and the BBB dysfunction in forced swimming. A decline in plasma and brain serotonin level 2 h after rest suggests that the amine is metabolized rapidly. Alternatively, the synthesis and release of serotonin may be altered after stress during the rest period. 7. BSCB Permeability Forced swimming resulted in significant disruption of the BSCB in young animals (Figs. 4.6, and 7). Thus, a profound increase in Evans blue and radioiodine tracer extravasation was seen in the spinal cord following the subjection of young rats to a 30-min forced swim (Table 10). However, 5 or 15 min of swimming did not induce leakage of the BSCB. Interestingly, the BSCB disruption caused by 30 min of swimming was not completely blocked after a 2-h rest (H. S. Sharma, unpublished observations). This indicates that the opening of BSCB permeability during forced swimming is prolonged compared to BBB breakdown. The basic mechanisms behind this discrepancy between BBB and BSCB permeability following forced swimming are not known and require additional investigation. 8. Morphological Changes in the Brain Light and electron microscopy revealed specific changes in the neuropil and damage of selective nerve cells in the cerebral cortex, hippocampus, and brain stem in young rats subjected to 30 min of forced swimming (Fig. 12). Selective nerve cell damage is seen mainly in layers III to V in the cerebral cortex that is most pronounced in the cingulate, occipital, and piriform cortices. Extravasation of HRP is quite frequent in the cerebral cortex of rats following 30 min of forced swimming (Fig. 20). This indicates that BBB disruption contributes to cellular injury. Degenerative changes in few nerve cells are apparent in the brain stem, and granule cells of the cerebellum showed marked damage in certain regions (Fig. 12). In the hippocampus, degeneration of nerve cells is common in the dentate gyrus and CA3 and CA4 sectors. Damage to ependymal cells around the lateral and third ventricle and degenerative changes in the choroid plexuses are common (Fig. 12). These observations further support that forced swimming induces breakdown of the blood–CSF barriers. At the ultrastructural level, vacuolation and damage to the neuropil are present in the cortex and in brain stem in rats subjected to 30 min of forced swimming (Fig. 13). These observations suggest that breakdown of the BBB following forced swimming is associated with selective nerve cell damage.

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However, it is not clear from this investigation whether these cell changes reflect acute nerve cell death or permanent neurodegenerative changes within such a short time. Morphological investigations in rats following several days or weeks after 30 min of swimming are needed to clarify these points. C. Heat Stress Heat stress and associated heat stroke are life-threatening illnesses in which the body temperature rises above 40°C, causing severe CNS dysfunction, e.g., delirium, convulsion, and coma (see Bouchama and Knochel, 2002). More than 50% of heat stroke victims die within a short time, despite lowering of the body temperature and therapeutic intervention. Those who survive heat stroke often show a permanent neurological deficit (for details, see Bouchama and Knochel, 2002). The intensity of heat stroke-induced deaths is increasing with global warming and with worldwide increases in the frequency and intensity of heat waves (Sharma and Westman, 1998; Sharma and Hoopes, 2003). Interestingly, the state of BBB or BSCB function is largely ignored in victims of heat stress or heat stroke (see Sharma and Hoopes, 2003). Our laboratory was the first to show that experimental or environmental heat stress without heat stroke is able to cause BBB disruption (Sharma and Dey, 1978, 1984; Sharma, 1982). This indicates that environmental heat exposure during summer months may lead to brain dysfunction due to BBB disruption (Sharma and Dey, 1978, 1984; Sharma, 1982). However, the influence of heat stress on BSCB permeability is still unknown. Whole body hyperthermia (WBH) is commonly used as an adjunct to cytotoxic therapy for cancer treatment (for reviews, see Katschinski et al., 1999; Sharma and Hoopes, 2003). It has been recognized that if WBH is combined with cytotoxic therapy during cancer treatment, it causes increased DNA adduct formation, inhibition of DNA repair, increased drug permeability, and decreased resistance to DNA-damaging agents (Robins et al., 1995, 1997). New clinical and experimental results show that WBH enhances cytotoxic ionizing radiation and chemotherapy (for details, see Sharma and Hoopes, 2003). However, WBH induces severe side effects, including altered brain function, probably due to breakdown of the BBB (for review, see Sharma and Hoopes, 2003). Interestingly, the effects of WBH on BBB functions are still lacking (see Sminia et al., 1994). Few reports examined BBB permeability to various tracers following microwave exposure of rat brain, which induces profound hyperthermia (for reviews, see Blackwell and Saunders, 1986; Sminia et al., 1994; Sharma et al., 1998a,b). Low levels of microwave radiation induce fluorescein uptake in the brain in conscious and anesthetized rats (Frey et al., 1975; Merritt et al., 1978; Williams et al., 1984). The brain temperature in conscious or anesthetized rats or Chinese hamsters reaches 40–50°C following low levels of microwave radiation. These animals exhibited deposits of horseradish peroxidase in extracellular spaces around the cerebral blood vessels (Albert, 1979; Sutton and Carroll, 1979; Blackwell and Saunders, 1986). Interestingly, these results were interpreted as either perfusion artifacts or redistribution of local blood flow changes during hyperthermia but not due to BBB disruption.

268 Because WBH appears to be a promising therapy for cancer patients, further studies are needed to understand the details of brain function in these situations. Our laboratory has initiated a series of experiments using WBH in rats comparable to the preclinical situations, and the BBB and brain dysfunction are examined in detail. These observations suggest that BBB disruption is instrumental in brain dysfunction and pathology. 1. Heat Exposure of Rats Rats were exposed to heat stress in a biological oxygen demand (BOD) incubator at 38°C for 1, 2, and 4 h (Sharma, 1982; Sharma and Dey, 1986b, 1987b). The relative humidity (45–47%) and wind velocity (20 to 26 cm/s) were fairly constant (Sharma, 1982; Sharma and Dey, 1986b, 1987b). All heat stress experiments were commenced between 8:30 and 9:00 h to avoid circadian variation in animals (Selye, 1976). 2. Stress Symptoms Young rats subjected to 4 h of heat stress showed marked symptoms (Table 8). These rats showed profound hyperthermia (>3.5°C), behavioral symptoms and massive hemorrhagic petechiae in the stomach wall (Table 8). A short duration of heat stress (1 or 2 h) did not exhibit symptoms. Only mild symptoms are seen in adult animals when exposed to 4 h of heat stress. This indicates that the heat stress-induced stress response is dependent on the duration and age of the animals. 3. Physiological Variables Significant hypotension, a mild increase in PaO2, and a slight decrease in PaCO2 are seen in young rats after 4 h of heat stress (Table 8). Exposure of adult rats to 4 h or young rats to a shorter duration heat stress did not alter these variables significantly (Table 8). Thus, the duration of heat exposure and age of the animals are important factors in alteration of these physiological variables. 4. BBB Permeability Marked increases in the BBB to Evans blue albumin and the radioiodine tracer are apparent in young animals after 4 h of heat stress. The pattern of dye extravasation showed minor differences in individual animals (Figs. 4 and 6). Blue staining is apparent in eight brain regions: the cingulate cortex, occipital cortex, parietal cortex, cerebellum, temporal cortex, frontal cortex, hypothalamus, and thalamus (Fig. 7). Mild to moderate staining of the ventricular walls was observed. The fourth ventricle showed deep blue staining, and structures around the third ventricles were stained moderately (Fig. 5). Occasionally the dorsal surface of the hippocampus took mild stain (Figs. 6 and 8), thus suggesting that heat stress is able to disrupt the blood–CSF barrier permeability as well. Extravasation of radioiodine is present in all the 14 regions examined. Thus, in addition to the 8 blue-stained regions, another 6 regions, namely the hippocampus, caudate nucleus, superior and inferior colliculi, pons, and medulla, also showed an increase in radioactivity (Fig. 10; Sharma and Dey, 1987b). Subjection of rats to shorter periods of heat stress, i.e., 1 or 2 h, did not induce BBB disruption (Table 9). When exposed to 4 h of heat stress at 38°C, adult animals exhibited only a mild increase in the BBB to Evans blue and radiotracer (Fig. 11).

H ARI S HANKER S HARMA These observations suggest that like other stressors, the duration of heat stress and the age of animals are important factors in BBB dysfunction. Interestingly, the leakage of Evans blue and radiotracers was reduced considerably but not prevented in animals subjected to 2 h of rest at room temperature after 4 h of heat exposure (Fig. 9). These animals are still lethargic; however, their body temperature returned to normal (H. S. Sharma, unpublished observations). Because we did not observe animals longer than 2 h after heat exposure, it is not clear whether the breakdown of the BBB in heat stress is completely reversible in nature. Further studies are needed to clarify these points. 5. CBF Changes The regional CBF declined in all the 14 regions at the end of 4 h of heat stress (Fig. 10). The decrease in cortical regions was 38 to 53%, in subcortical region was 23–31%, and in cerebellum and brain stem was 15 to 22% (Fig. 10). This shows that the fall in the regional CBF and the increase in regional BBB permeability are unrelated (Sharma and Dey, 1987b). This decrease in regional CBF was not observed in young animals exposed to 1 or 2 h of heat stress (Fig. 9). Adult animals subjected to 4 h of heat stress also did not show much decline in regional CBF (Fig. 11). This suggests that heat stress has the capacity to induce an alteration in cerebral microcirculation depending on the age of the animals and the duration of heat exposure. 6. BSCB Permeability A profound increase in BSCB permeability to Evans blue is seen in young rats subjected to 4 h of heat stress in specific regions of the cord (Table 10). Blue staining is seen mainly in the gray matter of the spinal cord (Figs. 4 and 7). This increase in BSCB permeability was not observed in adult animals subjected to 4 h of heat stress (H. S. Sharma, unpublished observation). Also, the short duration of heat exposure to young rats did not result in BSCB breakdown (H. S. Sharma, unpublished observation). These observations suggest that BSCB and BBB breakdown in heat stress are quite similar in nature. However, when animals are allowed a 2-h rest after a 4-h heat stress, only a mild reduction in the BSCB permeability to Evans blue was observed (results not shown). This indicates that heat stress-induced BSCB breakdown is much more prolonged than the BBB disruption. These observations suggest that some differences exist in the magnitude and severity of BBB and BSCB breakdown in heat stress. The molecular mechanism behind such differences is currently unknown. 7. Serotonin Level In young rats, plasma and brain serotonin levels increased profoundly at the end of a 4-h heat stress (Fig. 9). This increase is much less evident in adult animals subjected to similar heat stress (Fig. 9). However, 1 or 2 h of heat exposure did not influence plasma and brain serotonin levels. The increase of plasma and brain serotonin levels was decreased significantly after a 2-h rest in animals following heat exposure (Fig. 9). Because disruption of BBB is still present at this time, it appears that cerebral microvessels are more susceptible to serotonin in hyperthermia.

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Fig. 16 Regional changes in serotonin (5-hydroxytryptamine, A) and brain water content (B) in heat stress. Marked increase in regional brain serotonin (A: a) and water content (Ba) is evident following 4 h heat stress. No increase in regional serotonin level is seen in heat adapted rats (A: b). Animals were adapted to heat by exposing them to 1 h heat stress at 38°C for 7 days (for details, see text). Regional brain and spinal cord water content significantly increased following 4 h heat exposure (B: a,b). The changes in regional brain water content significantly reduced by pretreatment with p-CPA, cyproheptadine (cypro), indomethacin (indom) and diazepam (Diaz). Values are mean±SD of 6 to 8 rats. *P < 0.05, **P < 0.01, ANOVA followed by Dunnet’s test for multiple group comparison from one control. Data modified after Sharma et al. (1986, 1992e, 1998a).

Regional brain serotonin also showed a significant increase in most of the brain and spinal cord regions after a 4-h heat stress (Figs. 16 and 17). However, a decline in the regional serotonin level is seen at 1 h, indicating its release from the amine sources during early periods of heat exposure (Sharma and Dey, 1986b, 1987b; Sharma et al., 1998a). Interestingly, the regional distribution of serotonin in heat stress is unrelated to regional changes in the CBF or BBB permeability. 8. Heat Stress Influences Lung MAO Activity More than 90% of circulating serotonin is regulated by monoamine oxidase located in the pulmonary endothelium (Alabaster, 1977). The lung MAO activity depends on the substrate concentration that is staurable and influenced by the temperature and pH of the medium (Youdim et al., 1980). A high substrate concentration and/or increased temperature of the medium will cause a decrease in lung MAO activity (Youdim and Holzbauer, 1980), leading to the accumulation of serotonin in the circulation (Ryan, 1982).

Thus, heat stress induced hyperthermia is likely to increase blood temperature and retard lung MAO activity (Sharma et al., 1986). The lung MAO activity in lung tissue homogenates is measured in heat-stressed animals using the microfluorometric method (Kapeller-Adler, 1970). Hyperthermia (> 41°C) caused by 4 h of heat stress retarded lung MAO activity, resulting in a concomitant rise in the circulating serotonin level (Fig. 17). This decrease in lung MAO activity and increase in serotonin levels are not present in rats subjected to repeated heat exposure that showed only a nominal increase in their body temperature (38.5°C) (Fig. 25). Because the optimum temperature for the inactivation of serotonin by lung MAO ranges between 37.4 and 38.5°C (Alabaster, 1977), inhibition of lung MAO activity can only be seen when the body temperature increases beyond this limit (>38.5°C). Thus, the rise in circulating serotonin level in heat stress depends on the hyperthermia induced retardation in lung MAO activity (Sharma et al., 1986). This indicates that circulating

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Fig. 17 Heat stress induced changes in 5-HT2 receptor binding (A), BBB permeability, brain edema formation (B), hyperthermia , plasma 5-HT levels and lung monoamine oxidase (MAO) activity (C). Four h heat stress (HS) in young rats significantly increased ketanserin binding on the isolated microvessels (Microv) obtained from the cerebral cortex (Cortex) and hippocampus (Hippo) (A: a) as well as on the brain homogenates (Homg) (A: b) compared to old rats. (B) A close parallelisms between Evans blue albumin (EBA) extravasation (B: a) and brain edema formation (B: b) in acute and chronic heat exposed rats. Significant increase in EBA extravasation and brain water content is apparent following 4 h heat stress. No increase in BBB permeability or brain water content is seen following 1 to 4 h heat exposure in heat adapted rats. Rats were adapted to heat by exposing them 1 h at 38°C for 7 days (for details, see text). However, when the adapted rats were subjected to an additional 4 h heat exposure on the 2nd day, profound extravasation and edema formation are seen in these rats (B). Most of the rats died immediately after heat exposure (data modified after Sharma et al., 1986, 1992e). In these acute and chronically heat exposed rats, plasma 5-HT level showed a close correlation with hyperthermia and retardation in lung MAO activity (C). The lung MAO activity (C.b) significantly retarded in animals showing hyperthermia >39°C (C: a) after 4 h acute HS or 2nd day heat exposure in heat adapted rats. A retardation in lung MAO activity closely corresponds to the increased plasma 5-HT level (C: b) for details, see text. Values are Mean±SD of 6 to 8 rats. Data modified after Sharma et al. (1986, 1992e).

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serotonin levels and lung MAO activity are important factors in the physiological mechanisms of heat adaptation. 9. Heat Stress Influences 5-HT2 Receptor Bindings in Cerebral Microvessels Profound regional differences in serotoninergic receptors and serotonin transporters occur in the CNS and in the periphery of several mammalian species (Fernandez et al., 2003; Burnet et al., 1994). These differences are due to age, sex, and strains of the animals (Carlsson and Carlsson, 1988; Burnet et al., 1994; Jacobs and Azmitia, 1992). A downregulation of serotonin receptors occurs in the CNS in long-term depression-related disorders, as well as in suicidal victims (Chaouloff, 1993, 2000). An increase in 5-HT1a receptor binding in the hippocampus following acute immobilization (Mendelson and McEwen, 1991) and an increased number of cortical 5-HT2 receptors after 2 h of immobilization have been reported (Torda et al., 1990). Destruction of serotonergic neurons with 5,7-DHT did not abolish the increase in 5-HT2 receptors, indicating that stress-induced alterations in serotonergic neurotransmission are not related with this effect (Torda et al., 1990). Taken together, these observations suggest that serotoninergic receptor binding and/or their numbers are influenced by stress (see Chaouloff, 1993). However, the influence of heat stress on the serotonergic receptors and/or binding in the CNS is still unknown. We examined 5-HT2 receptor binding in cerebral microvessels isolated from the cortex and hippocampus as well as in brain homogenates using 125I-labeled ketanserin (Ermisch et al., 1991; Kretzschmar and Ermisch, 1989; Sharma et al., 1992b; H. S. Sharma, unpublished observations). Subjection of young animals to 4 h of heat stress resulted in a significant increase in the 125I-labeled ketanserin binding to the cerebral microvessels from the cerebral cortex and hippocampus (Fig. 17). This increase in receptor binding (Bmax values fmol/mg/protein) was also enhanced in brain homogenates from the cerebral cortex and hippocampus (Fig. 17). However, no significant changes in receptor binding to 125I-labeled ketanserin were observed in rats subjected to 1 and 2 h of heat stress. Interestingly, an increased plasma and brain serotonin level showed close parallelism with the increase in serotonin receptor bindings (Fig. 17). This suggests that heat stress not only increased the amine level in young rats in the plasma and brain, but also increased 5-HT2 receptor binding in the cerebral microvessels and in the brain. Interestingly, old rats subjected to heat stress that did not show any increase in plasma and brain serotonin levels or enhanced binding of 125I-labeled ketanserin to the microvessels or brain homogenates. These observations suggest that old rats are less susceptible to a heat stress-induced increase in plasma and brain serotonin levels. Furthermore, serotonin binding on cerebral microvessels and brain homogenates is age related (H. S. Sharma, unpublished observations). 10. Brain Edema Brain water content showed a significant increase following 4 h of heat stress in young rats (Figs. 16 and 17). No increase in brain water is seen in animals subjected to 1 or 2 h of heat exposure. The magnitude and severity of edema formation are much less evident in adult animals following a 4-h heat

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exposure (Fig. 17). This indicates that extravasation of protein tracers could lead to vasogenic edema formation in young rats (Sharma et al., 1998a). A regional increase in brain water is seen in the cerebral cortex, hippocampus, cerebellum, brain stem, and spinal cord following a 4-h heat exposure in young rats (Fig. 16). Profound swelling of the brain in the closed cranium in heat stress compresses the vital centers in the brain, leading to high mortally and instant death. Massive brain swelling in human victims who died following heat stroke supports this idea (for details, see Sharma and Hoopes, 2003). 11. Spontaneous Electroencephalogram A hyperthermia-induced increase in brain temperature results in fatigue and alteration in the electrical activity (Gonzlez-Alonso et al., 1999; Nielsen et al., 2001) as seen by changes in the spontaneous electroencephalogram (EEG) or sensory-evoked potentials (Dubois et al., 1980, 1981; Febbraio et al., 1994). Exercise in hot environments increases the core body temperature and results in a concomitant shift in EEG power distributions (Nielsen et al., 2001). A decrease in β power and a steady increase in the α/β ratio are common findings in humans (Nielsen et al., 2001). These observations suggest that alterations in EEG activity during heat exposure are related to hyperthermia-associated fatigue. However, the effects of heat exposure on EEG activity are still not known. We examined EEG in young rats exposure to heat at 38°C. In addition, the effect of rest at room temperature after heat exposure on EEG activity was also examined (H. S. Sharma, unpublished observations). The EEG was recorded in heat stress using bipolar screw electrodes placed over the right and left cingulate cortex and the parietal cortex (Fig. 18). The EEG was recorded using an eight-channel EEG machine (DISA, Copenhagen, Denmark) with minor modifications (high-frequency filters on, time constant 0.1 s; paper speed 5 mm/s) from the protocol used for immobilization stress (Winkler et al., 1995). EEG in conscious rats using mild restraint in a perspex box showed an amplitude of 50 to 60 µV and a frequency of 6–7 Hz (Fig. 18). Exposure of rats to heat stress for 30 min at 38°C resulted in a significant increase in EEG amplitude (70 to 80 µV) and frequency (7–9 Hz; Fig. 18). No significant differences in EEG activity between cingulate and parietal cortex recordings are seen. The EEG voltage is reduced (40 to 50 µV), and the frequency is slightly increased (8–9 Hz) at 1 h after heat exposure (Table 13). After a 2-h heat exposure, the EEG voltage reduced significantly, particularly in the cingulate cortex recording (20–30 µV; 8–9 Hz) (Fig. 28). A mild increase in the EEG amplitude (30–40 µV) and frequency (10–11 Hz) is seen 3 h after heat exposure (Table 13). At the end of a 4-h heat exposure, the EEG amplitude was reduced considerably (10–12 µV) without any change in frequency (10–12 Hz) (Fig. 18). This effect was equally pronounced on both cingulate and parietal cortex recordings. Extravasation of Evans blue albumin is prominent in the cerebral cortex at this time. This indicates that increased BBB permeability and flattening of EEG in heat stress are interrelated. Interestingly, EEG recovery is initiated following 30 min rest at room temperature after a 4-h heat exposure. A complete recovery, however, was not evident in heat-exposed animals

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H ARI S HANKER S HARMA stress (Fig. 12). The choroid plexus from the lateral ventricle, third ventricle, and fourth ventricles exhibited degenerative changes (Fig. 12). These observations suggest that heat stress induces profound alterations in the blood–CSF barrier. Upregulation of GFAP is seen in many parts of the brain in young rats subjected to 4 h of heat stress (Fig. 19). This effect is not seen in adults animals or young animals exposed to a shorter duration of heat (results not shown). These observations suggest that breakdown of the BBB is responsible for glial cell activation (Sharma et al., 1992e). The magnitude of the heat-induced glial cell reaction in some brain regions does not normally coincide with the severity of BBB breakdown or nerve cell injury in that area. This indicates a selective difference in the vulnerability of neurons and glial cells in heat stress. Using myelin basic protein (MBP) immunostaining, profound axonal injuries are seen in young rats following heat exposure (Sharma et al., 1992a; 1998a). A decrease in MBP immunostaining representing the degradation of myelin is most pronounced in the brain stem reticular formation and spinal cord (Fig. 19). This effect is much less apparent in adult animals (Fig. 19), and young rats did not show myelin damage following 1 or 2 h of heat exposure (results not shown). 13. Ultrastructural Damage of Neuropil

Fig. 18 Representative example of EEG changes in acute heat stress. The EEG is recorded from bipolar electrodes placed on the frontal cortex (F-C) and parietal cortex (P-C). The EEG activity shows high voltage and slow activity (HVSA) before heat exposure (–30 min). An increase in EEG voltage and frequency is apparent after 30 min heat exposure. Flattening of EEG appeared at the end of 4 h heat exposure. At this time profound extravasation of Evans blue dye is seen in the brain. Partial recovery in EEG activity is evident following 2 h rest after heat exposure.

even after 2 h of rest at room temperature (Table 13; Figs. 9 and 11). This observation suggests that hyperthermia can induce prolonged changes in the brain electrical activity and that changes in BBB permeability are well correlated with the EEG activity in heat stress. 12. Structural Changes in the Brain It appears that breakdown of the BBB in heat stress is associated with neuronal damage. Profound neuronal, glial, and myelin changes are seen in young animals subjected to 4 h of heat exposure. These cell changes are less apparent in adult animals subjected to heat stress (Fig. 12). In young rats, nerve cell injury, edematous expansion, and sponginess of the neuropil are common in several brain areas, such as the cerebral cortex, brain stem, cerebellum, thalamus, and hypothalamus (Figs. 12 and 19). A selective nerve cell damage in the hippocampus is most pronounced within the CA4 subfield compared to other regions (Fig. 12), although edematous swelling and general sponginess are present throughout this region. Extravasation of HRP is most intense in the cerebral cortex of young rats compared to adult animals following 4 h of heat exposure (Fig. 20). This indicates that BBB leakage is associated with brain damage in heat stress. Like other stressful conditions, damage to ependymal cells around the lateral and third ventricle are quite prominent in heat

Ultrastructural changes in heat stress show profound cell injury in many parts of the brain. Damaged nerve cells and degenerated nuclei often accompanied by eccentric nucleolus are common in the cerebral cortex, hippocampus, cerebellum, thalamus, hypothalamus, and brain stem (Fig. 13). The nerve cells are dark in appearance and contain vacuolated cytoplasm. The nuclear membrane contains many irregular foldings, and the nucleolus often showed signs of degeneration (Fig. 13). Interestingly, damage of the one nerve cell is often seen in a region where the adjacent neuron is almost normal in appearance, indicating a selective vulnerability of nerve cells in heat exposure. Swollen synapses with damage to both pre-and postsynaptic membranes are frequent in the thalamus, brain stem, hypothalamus, cerebellum, hippocampus, and cerebral cortex (Fig. 13). In some of these regions, damage of postsynaptic dendrites and disruption of synaptic membrane are quite common. Widespread axonal damage, demyelination, and vesiculation are most pronounced in brain stem reticular formation, pons, medulla, and the spinal cord (Fig. 13). Many unmyelinated axons are also swollen. These ultrastructural changes are present in young rats exposed to 4 h of heat stress. The magnitude and intensity of cell injury in heat stress are reduced considerably in adult animals. Young animals exposed to a short duration of heat stress did not show signs of ultrastructural damages in the CNS (results not shown). These observations are in line with the idea that breakdown of the BBB is an important factor in brain damage. 14. Ultrastructural Changes in the Cerebral Endothelium Disruption of the BBB at the ultrastructural level is the most common finding in heat stress. Many microvessels show leakage of lanthanum across the cerebral endothelium in a very selective manner (Figs. 13 and 21). Thus, leakage of lanthanum is often evident in one endothelial cell, whereas the rest of the vessel or the adjacent endothelial cells are completely normal

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Fig. 19 Representative example of neuronal changes following heat stress in young (HSY) and old (HSO) rats compared to controls (Cont). HS for 4 h at 38°C induces profound cell damage in young rats compared to old animals. Degeneration of nerve cells (arrows) in the brain stem reticular formation (A: b), in cerebral cortex (B.a) and in spinal cord (B.c) of young rats (HSY) is much more pronounced compared to old (HSO) animals (B: b; B: d). C. Glial (C: a,b) and axonal (C: c,d) changes following heat stress in young rats (HSY) compared to control (Cont). Upregulation of glial fibrillary acidic protein (GFAP) immunoreactivity (blank arrows), a marker of astrocytes is upregulated following heat stress. Downregulation of myelin basic protein (MBP) expression reflecting damage and degeneration of myelin (*) is evident in heat stress compared to control (arrows) group. Bars: (A) 25 µn (Nissl stain); (B) 50 µm (H & E); (C) 40 µm.

(see Fig. 21). This indicates a highly specific nature of the endothelial cell membrane permeability in stress. Activation of specific endothelial cell transporters, permeability factors, neurochemical receptors, or ions channels located on the selected area of the endothelial cell membrane many be responsible for such a selective increase in lanthanum permeability. In several vascular profiles, lanthanum is stopped at the luminal side of the tight junctions (Fig. 21). However, many microvessels showed infiltration of lanthanum across the endothelial cells membranes, including the tight junctions, without widening them (Fig. 21). These observations support the idea of a specific receptor-mediated increase in microvascular permeability. Because receptors can also be present on the membranes apposing tight junctions, increased microvascular permeability around the junctions is possible via the activation of such receptors. Thus, increased endothelial cell membrane permeability appears to play an important role in lanthanum extravasation during heat stress. Obviously this mode of membrane permeability can be influenced by drugs modifying neurochemical receptors and/or signal transduction. 15. Heat Stress Influences Neurochemical Transmission Experiments suggest that apart from serotonin, several other neurotransmitters or neuropeptides are altered in the CNS fol-

lowing heat stress. It is believed that these neurochemicals play important roles in IPS following heat stress. a. CGRP IMMUNOREACTIVITY IN THE BRAIN. The Calcitonin gene-related peptide (CGRP) is present in neurons and dendrites in the CNS of several mammalian species (Kruger et al., 1988). Redistributions of CGRP following several stressful situations, as well as peripheral nerve lesion and local thermal stimulation, are described (Kruger et al., 1988; Nyberg et al., 1995). However, alterations in CGRP expression in the CNS following heat stress are still not well known. A detailed study has been undertaken to examine heat stress-induced alterations in several neuropeptides in the CNS, namely dynorphin A (see Chapter 14), CGRP (Sharma et al., 2000d); substance P, and enkephalins (H. S. Sharma, unpublished observations). Results show that heat stress alters neuropeptide transmission in the CNS (see below). CGRP immunohistochemistry was examined on a free-floating Vibratome (40 µm thick) section using monoclonal antibodies (Sharma et al., 2000d). Only a few CGRP-immunostained neurons, nerve fibers, and terminals are present in the spinal cord, brain stem, and cerebral cortex of normal animals as shown in previous studies (for details, see Nyberg et al., 1995). Subjection of young rats to heat stress markedly altered CGRP distribution in several brain regions (Figs. 22 and 23). An

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Fig. 20 Light microscopic demonstration of horseradish peroxidase (HRP Type II) extravasation in the parietal cerebral cortex of stressed rats. Fresh frozen sections (40 µm thick) were cut and processed for HRP reaction product (see Brightman et al., 1970; Sharma 1982). Extravasation of HRP across intraparenchymal vessel (arrows) is evident following immobilization (IMZ), forced swimming (FS), sleep deprivation (SD) and heat stress (HS). The intensity of microvascular reaction and leakage is most pronounced following heat stress. Old animals subjected to HS show much less extravasation of HRP (HS old). The HRP reaction product in the cortex of stressed animals represents extravasation since animals were perfused transcardially to washout intravascular tracer (for details, see Sharma 1982). Note complete absence of HRP reaction product in control animals. Bar: 40 µm. Modifed after Sharma (1982).

increased immunoreactivity of CGRP is evident in the cerebral cortex, hippocampus, cerebellum, brain stem, and spinal cord (Fig. 23). However, downregulation of the peptide is seen in the thalamus and in the reticular formation in the pons regions (Figs. 22 and 23). This decrease in CGRP is most pronounced in nerve fibers and dendrites (Sharma et al., 2000d). In brain stem reticular formation, loss of CGRP is apparent in the nerve cells (Fig. 22). However, an increase in CGRP immunoreactivity in nerve terminals or nerve fibers is common in several brain regions in heat stress (Figs. 22 and 23). A decrease in CGRP immunoreactivity represents release of the

peptide, whereas an increased immunoreaction in the dendrites and nerve fibers reflects enhances peptide synthesis in specific synapses to influence neuronal communication. Interestingly, many CGRP-positive nerve fibers and terminals are located in the reticular activating system and the thalamus involved in thermal IPS (Sharma et al., 2000d). The CGRP is a sensory peptide (Ai et al., 1998) and its receptors are located on nerve cells, axons, and astrocytes (Bulloch et al., 1999; Morara et al., 1998). An increase in CGRP immunoreactivity is reported following spinal trauma (Krenz and Weaver, 1996) or trimethyltin-induced poisoning in

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Fig. 21 Ultrastructural studies on the cerebral endothelial cells from various brain regions in heat stress showing lanthanum extravasation and damage to adjacent neuropil. (A) Lanthanum, an electron dense tracer (seen as dark black particles) is seen across the endothelial cell membrane containing tight junctions(A: a; blank arrows). Infiltration of lanthanum across the endothelial cell membrane is clearly seen (solid arrows; A: b,c). In some cases lanthanum is seen diffusely infiltrated within the cell membranes of tight junction complex and endothelial cell cytoplasm covering the apposed plasma membranes connected with the tight junctions (A: a). However in these cases the tight junctions are not found opened because lanthanum within the intercellular cleft is stopped at the tight junction (blank arrows; A: a). In some cases only one endothelial cell membrane covering tight junction is found diffusely infiltrated with lanthanum (filled and blank arrows) leaving its counterpart completely intact (A: c) One endothelial cell showed infiltration of lanthanum in a certain segment of the cerebral endothelium (A: d; arrow heads). Damage to neuropil (*) and myelin vesiculation are prominent (A: e) in the adjacent area. (B) Lanthanum is present in endothelial cell and in the basement membrane (B: a,b) without widening of the tight junction (B: a). In several microvascular profiles, lanthanum is stopped at the tight junctions (B: c,d; arrow heads). Damage to synaptic membrane (arrows; B: e), vacuolation, edema and myelin vesiculation (B: f) is frequent around microvessels showing BBB disruption to lanthanum (B: e,f). (C) Many cerebral endothelium show presence of lanthanum in the microvesicular (*) profiles within the cell cytoplasm (C: b,d). Normally, the tight junction in these microvessels appears to be closed (C: a,c,d). Complete collapse of microvessels with perivascular edema and damage to neuropil (C: e) is common in many brain regions during heat stress. Bars: (A) 0.3 µm; (B) 0.2 µm; (C) 0.2 µm. Modified after Sharma et al. (1998a); Sharma (1999).

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Fig. 22 Heat stress (HS) induced changes in CGRP immunoreactivity (A) and cell damage (B) in the brain. Increased immunoreactivity to CGRP containing nerve fibres and dendrites are quite frequent in several brain regions following HS particularly in the brain stem reticular formation (A: b,d,f, h), parabrachial nucleus (A: b), motor nucleus of the trigeminal (A: d), periolivary nucleus (A: f) and in the primary sensory nucleus of the trigeminal (h) compared to corresponding controls (Cont, A: a,c,e,g). (B) Nerve cell damage (arrows), edema and sponginess (*) are quite common in the cerebral cortex (B: a) and in brain stem (B: c) following HS. Pretreatment with a potent multiple opioid receptor blocker naloxone (B: b) or an L-type Ca2+ channel blocker, nimodipine (B: d) markedly reduced HS induced cell changes, edema and sponginess. Bars: (A) 20 µm; (B: a,b) 50 µm; (c,d) 30 µm. Modified after Sharma et al. (1997b; 2000d).

the hippocampus (Bulloch et al., 1999). In trimethyltin poisoning, the peptide exhibited a good relationship with cell injury (Morara et al., 1998), indicating its involvement in cell damage. However, a direct relationship between CGRP redistribution and cell injury in hyperthermia is not evident (Sharma et al., 2000d). CGRP is often colocalized with serotonin and substance P in the CNS (Hökfelt et al., 1978; Nyberg et al., 1995; see Chapter 12) and interacts with nitric oxide (Lul and Fiscus, 1999). In addition, CGRP influences prostaglandins (PGs) and their synthesizing enzymes cyclooxygenase (COX-1 and COX-2) (Tang et al., 1999), which are involved in brain pathology caused by various insults (Matsumura et al., 1998; see Chapter 23). Thus, the possibility exists that the peptide influences serotonergic transmission in heat stress and alters the regional vasomotor tone of the microvessels (Jansen et al., 1999; Lul and Fiscus, 1999). An interaction between nitric oxide and CGRP (Lul and Fiscus, 1999) will influence neuronal communication via several vasoactive and signal-transducing agents in the CNS. A local release of growth factors, cytokines, hormones, and other neuropeptides has been reported to modulate CGRP activity in the CNS (Bulloch et al., 1999; Hu et al., 1999; Jansen et al., 1999; Krenz and Weaver, 1996; Lu and Fiscus,

1999; Tang et al., 1999). Because heat stress influences various cytokines, hormones, and neuropeptides (see Sharma and Hoopes, 2003), it appears that CGRP plays an important regulatory role in thermal IPS of the CNS. b. NEUROPEPTIDE TRANSMISSION. To further understand the influence of heat stress on neurochemical transmission in the CNS, the distribution of neuropeptides dynorphin A, Met-Enk-Arg6-Phe7 (MEAP), and substance P (SP) in several brain and spinal cord regions was examined using radioimmunoassays (Sharma et al., 1990a, 1992d, 1993). Results show that heat stress is able to markedly influence these neuropeptides in the CNS. The peptide dynorphin A markedly increased in the cerebellum and in the spinal cord (Fig. 23), whereas a significant decrease of the peptide is observed in the cerebral cortex, hippocampus, caudate nucleus, thalamus and hypothalamus, and brain stem (H. S. Sharma, unpublished observation). However, MEAP showed profound increase in all the brain regions examined in heat stress (Fig. 23). Interestingly, the SP showed a moderate decrease in all the brain regions except the spinal cord, which showed a significant increase (Fig. 23). These observations suggest that neuropeptides participate in heat stress-induced brain dysfunction.

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Fig. 23 Alterations in neuropeptide transmission in the CNS following heat stress (HS). A = Dynorphin (Dyn) A 1-17; B = Met-Enk-Arg6-Phe7 (MEAP); C = Substance P (SP); D = CGRP (Data modified after Sharma et al., 2000d). Values are mean±SD from 5–6 rats. *P < 0.05; **P < 0.01 from one control; D, P < 0.05; DD, P <0.01, from control cerebral cortex; ANOVA followed by Dunnet’s test.

c. AMINO ACID NEUROTRANSMITTERS. Involvement of excitatory amino acids, e.g., glutamate and aspartate is well known in traumatic or ischemic brain injuries (see Sharma, 2002; Sharma and Alm, 2002; Sharma and Sjöquist, 2002). However, their role in heat stress-induced brain dysfunction is still unknown. It appears that a balance between excitatory and inhibitory amino acids is crucial for cell injury and cell survival following CNS insults. Thus, we examined alterations in two excitatory amino acids, glutamate and aspartate, and two inhibitory amino acids, GABA and glycine, in the CNS following heat stress in young rats. Rats subjected to a 1-h heat stress result in a marked increase in glutamate, aspartate, GABA, and glycine (about 6- to 10-fold) in the cerebral cortex, cerebellum, and brain stem (Sharma et al., 1995a; Fig. 24). At this time, the hippocampus and spinal cord (C15) exhibited two- to fourfold decrease in

glutamate and GABA levels without any significant changes in aspartate and glycine. After a 2-h heat stress, the hippocampus and spinal cord showed a mild increase (150–240%) in glutamate and aspartate together with the cerebral cortex, cerebellum, and brain stem. The GABA and glycine levels are also elevated in all the brain samples at this time. After a 4-h heat exposure, all the brain regions exhibited a marked decrease (about four- to sixfold) in the inhibitory amino acids GABA and glycine. The excitatory amino acids, glutamate and aspartate, decreased in the cerebral cortex and cerebellum, but the hippocampus and brain stem showed a mild but significant increase in these amino acids at this time (Fig. 24). Thus, heat stress has the capacity to induce widespread alterations in excitatory and inhibitory amino acids in the CNS. Obviously, a decrease in inhibitory amino acids and an increase in excitatory amino acids will result in cell injury.

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Fig. 24 Alterations in amino acid neurotransmitters in the CNS following heat stress. Values are Mean±SD from 6 to 8 rats. *P < 0.05; **P < 0.01, ANOVA followed by Dunnet’s test from one control group.

16. Pharmacological Manipulation of BBB Disruption To further understand the molecular mechanisms behind BBB dysfunction in stress, several drugs or compounds that can modify neurochemical transmission, stress response, or tracer transport across the microvascular endothelium were used (Table 11). a. SEROTONIN SYNTHESIS INHIBITOR, P–CPA. Serotonin is a neurochemical mediator of stress response (Chaouloff, 1993) and BBB permeability (Sharma et al., 1990; for details, see Chapter 12). We examined the effect of serotonin synthesis inhibitor p-chlorophenylalanine (p-CPA) on stress-induced alterations in BBB function. For this purpose, rats were treated with p-CPA (Table 11) to deplete the endogenous synthesis of serotonin in the CNS and other amine stores in the body (Sharma, 1982, 1999). p-CPA-treated rats did not show an increase in plasma or brain serotonin levels and BBB breakdown following immobilization, heat stress, or forced swimming (Figs. 25 and 26). A decrease in regional CBF is not observed in p-CPA-treated

stressed rats (Fig. 26). These observations suggest that serotonin is actively involved in stress-induced BBB disruption and cerebral circulatory changes in stress. Interestingly, treatment with p-CPA did not alter stress symptoms or physiological variables (Table 12). Structural changes in the CNS are reduced considerably in p-CPA-treated animals when subjected to a 4-h heat exposure (for details, see Sharma, 1999). These observations suggest that serotonin is one of the important mediators of stress-induced brain pathology. b. 5-HT2 RECEPTOR ANTAGONISTS. Stress is able to influence 5-HT2 receptor bindings on the cerebral microvessels and in brain (as described earlier). Thus, it seems likely that 5-HT2 receptors play some role in stress-induced BBB dysfunction (Sharma et al., 1986a,b, 1995b; see Chapter 12). Three different 5-HT2 receptor antagonist compounds were used to study BBB dysfunction in stress: cyproheptadine, ketanserin, and ritanserin (Sharma et al., 1986a,b, 1995b, 1998a,c). Pretreatment with the 5-HT2 receptor antagonist markedly attenuated BBB permeability following immobilization, forced

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Fig. 25 Influence of drugs on changes in the rectal temperature (a), BBB permeability (b), serotonin (5-hydroxytryptamine) levels (c) and cerebral blood flow (CBF, d) following forced swimming (FS, A), immobilization (IMZ, B) or heat stress (HS, C). *P < 0.05; **P < 0.01, ANOVA followed by Dunnet’s test for multiple group comparison from one control. Values are Mean±SD of 6 to 8 rats. p-CPA = p-chlorophenylalanine; Indom = indomethacin; Diaz = diazepam; Ketan = ketanserin; Cypro = cyproheptadine; Vinbl = vinblastine. Data modified after Sharma and Dey (1986a, 1987b); Sharma et al. (1991a, 1995).

swimming, or heat stress (Fig. 25) and restored the rCBF near normal values (Figs. 25 and 26). Interestingly, the increase in plasma or brain serotonin levels continued to remain high (Fig. 25). The effects of ritanserin are far superior to ketanserin given in identical doses. However, further studies using equimolar doses of these compounds are needed to clarify this point. Pretreatment with cyproheptadine, which also has a mild histamine H1 receptor antagonist effect, showed most marked protection on BBB disruption in forced swimming compared to other stressors. The reason behind such a superior effect by cyproheptadine in forced swimming is unclear. A dose response of cyproheptadine in stress-induced BBB permeability is required to understand this phenomenon. The 5-HT2 receptor antagonists did not modify the stress response or physiological variables significantly (Table 12). Ketanserin and ritanserin are able to reduce nerve cell, glial cell, axonal changes, and heat shock protein (HSP) responses in heat stress (Sharma et al., 1996). This indicates that blockade of

5-HT2 receptor causes neuroprotection in stress (see Chapters 12 and 17). c. PROSTAGLANDIN SYNTHESIS INHIBITOR, INDOMETHACIN. Prostaglandins are known as the first mediator of stress, and pretreatment with the PG synthesis inhibitor indomethacin abolishes the stress response (Hanukoglu, 1977). Thus, animals were pretreated with indomethacin 30 min before stress and subjected to immobilization, heat stress, or forced swimming. Indomethacin treatment did not influence stress symptoms or physiological variables (Table 12). However, this drug treatment markedly reduced BBB permeability to Evans blue and radioiodine tracers (Fig. 25), and the CBF is restored near normal values (Fig. 26). Interestingly, plasma and brain serotonin levels were reduced considerably in indomethacin-treated stressed rats (Fig. 25). These observations suggest that PGs are involved in the stress response. Alternatively, PGs influence stress-induced

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Fig. 26 Effects of drugs on the regional blood–brain barrier (rBBB, A) permeability and cerebral blood flow (rCBF, B) following Forced swimming, heat stress or immobilization stress. Pretreatment with p-CPA, indomethacin, diazepam, ketanserin, cyproheptadine and vinblastine prevented the rBBB permeability in all the 14 regions examined. The reduction in rCBF was also restored following stress by these drug treatments except vinblastine. Brain regions (FS): a = frontal cortex, b = parietal cortex, c = occipital cortex, d = ant. cingulate cortex, e = post. cingulate cortex, f = cerebellum vermis, g = cerebellar cortex, h = caudate nucleus, i = hippocampus, j = colliculi, k = thalamus, l = hypothalamus, m = medulla, n = brain stem. Brain regions (HS, IMZ) : a = frontal cortex, b = parietal cortex, c = occipital cortex, d = temporal cortex, e = cingulate cortex, f = hippocampus, g = caudate nucleus, h = thalamus, i = hypothalamus, j = sup. colliculus, k = inf. colliculus, l = cerebellum, m = pons, n = medulla. Values are mean±SD from 6 to 8 rats. Data modified after Sharma and Dey (1986a, 1987b); Sharma et al. (1991a, 1995); Sharma HS unpublished observations.

BBB function by mechanisms involving changes in signal transduction within the endothelial cells (see below; Sharma et al., 1990). d. BENZODIAZEPINE RECEPTOR AGONIST, DIAZEPAM. Diazepam is an antianxiety drug that prevents stress-induced increase in CBF and metabolism in rats (Sakabe et al., 1982; Siesjö, 1978). This action of diazepam is due to the prevention of neuronal activation caused by stress, thus inhibiting the stress response itself. When rats were pretreated with diazepam (Table 11) and subjected to immobilization or heat exposure, no increase in plasma or brain serotonin level is observed in these animals (Fig. 25). Disruption of BBB permeability and reductions in the CBF are also absent (Figs. 25 and 26). The restraint-induced hypothermia is potentiated by diazepam, whereas the hyperthermia caused by heat stress is reduced (Fig. 25). This treatment significantly attenuated gastric ulceration in the stomach. However, the physiological variables were not significantly attenuated (Table 12). These observations suggest that the anti-anxiety drug is able to induce stress-induced BBB dysfunction, probably by inhibition of the stress response.

e. ANTIMITOTIC DRUG, VINBLASTINE. Vinblastine, a vinca alkaloid, is an antimitotic drug that inhibits the polymerization of microtubules and thus prevents vesicular transport (Larsson et al., 1979). To understand the contribution of vesicular transport in stress-induced BBB dysfunction, animals were pretreated with vinblastine (Table 11) and subjected to immobilization, heat stress, or forced swimming exercise. Vinblastine pretreatment did not influence stress symptoms or physiological variables (Table 12), but the stress ulcers are reduced. In these animals, the decline in CBF is not altered (Figs. 25 and 26), and the serotonin level in plasma or brain continues to remain high (Fig. 25). However, breakdown of the BBB following stress is completely prevented (Figs. 25 and 26). These observations are in line with the idea that vesicular transport plays an important role in tracer transfer in stress (Sharma et al., 1990). f. OPIOID RECEPTOR ANTAGONISTS. Opioid neurotransmission appears to be important in heat stress-induced pathophysiology of BBB permeability (see earlier discussion). To explore this hypothesis further, the effects of multiple opioid receptor antagonists naloxone and naltrexone on heat-stress-induced BBB

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Fig. 27 Effect of multiple opioid receptor antagonists and an L-type Ca2+ channel blocker on heat stress induced alterations in the rectal temperature (A: a), BBB permeability (A: b) and serotonin level (A: c). Increased BBB permeability and serotonin levels in heat stress correlates well with brain water content (B: a), volume swelling of brain (% ƒ, B: b) and reduction in the cerebral blood flow (CBF, B: c). Volume swelling is calculated from the changes in brain water content. About 1% increase in brain water content represents 3% increase in volume swelling (see Rapoport 1976). Values are mean±SD from 6 to 8 rats. *P = < 0.05; **P < 0.01; ANOVA followed by Dunnet’s test for multiple group comparison from one control. Data modified after Sharma and Cervós-Navarro (1990); Sharma et al. (1997b).

dysfunction, CBF, and neuronal damage were examined (Sharma et al., 1997b). Separate groups of rats were treated with either naloxone (1, 5, or 10 mg/kg) or naltrexone (1, 5, or 10 mg/kg) intraperitoneally 30 min before heat stress (Sharma et al., 1997b,c). Naloxone or naltrexone in high doses (10 mg/kg) significantly attenuated heat stress-induced hyperthermia, hypotension, behavioral responses, and incidence of gastric hemorrhages (Sharma et al., 1997b,c).The extravasation of Evans blue and radioiodine tracers is reduced significantly in several brain regions following heat stress (Fig. 27; H. S. Sharma, unpublished observations). The CBF restored near normal levels (Fig. 27). However, pretreatment with low doses of these opioid antagonists (1 or 5 mg) did not influence BBB permeability, CBF changes, or stress symptoms in heat stress (results not shown). The brain water content and cell changes are reduced significantly in animals following heat stress that received high doses of these opioid antagonists (10 mg/kg; Fig. 27) (Sharma et al., 1997b). Thus, nerve cell damage, edema, myelin vesiculation, and membrane disruption in heat stress are reduced considerably by naloxone or naltrexone (Fig. 28). The effects of naltrexone on BBB permeability, CBF, brain edema,

and brain pathology are far more superior to naloxone (Figs. 22, 27, and 28). The pronounced effect on opioid receptors by naltrexone and a long half-life (24 h) compared to naloxone (1.5 h) could be important factors in this regard (Misra, 1978; Herz, 1993). Naloxone and naltrexone in high doses inhibit κ-opioid receptors, whereas low doses of these compounds preferentially antagonize only µ- or δ-receptors (Akil et al., 1984; Faden, 1993; Martin, 1983; Sharma et al., 1997b,c). This indicates that the blockade of multiple opioid receptors, particularly the κ-opioid receptor by these compounds, is necessary to attenuate cell injury in heat stress (see Sharma et al., 1997b). A reduction in stress response by opioid antagonists may also contribute to a reduction in cell injury. Alternatively, opioids can influence endothelial cell membrane permeability through the opening of Ca2+ channels and other signal transduction agents (Herz, 1993; North, 1993). g. AN L-TYPE CALCIUM CHANNEL BLOCKER, NIMODIPINE. Ca2+ plays an important role in neuronal functions and BBB permeability (Olesen, 1989; Ghosh and Greenberg, 1995). Ca2+ influx is regulated through voltage- and ligand-gated ion channels in the CNS (Bertolini and Llinas, 1992). Many nerve cells are equipped with a variety of Ca2+ channel subtypes, among which

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Fig. 28 Ultrastructural changes in stress and their modification with drugs. (A) Pretreatment with p-CPA markedly attenuated cell damage following 8 h immobilization (IMZ) stress (A: a). However, treatment with 5,7-DHT (i.c.v.; A: b) or 6-OHDA (i.c.v.; A: c) did not prevent IMZ induced cell changes. Perivascular edema (arrowheads), membrane damage (arrows) and vacuolation (*) are quite common in these drug treated rats. Administration of aminophylline (A: d) or 5-HT (A: e) following 4 h IMZ also induced profound membrane damage and edema. (B) Pretreatment with indomethacin (B: a), p-CPA (B: d), naltrexone (B: e) or nimodipine (B: f) significantly attenuated 4 h heat stress (HS) induced cell damage at the ultrastructural levels compared to the untreated group (B: c). Pretreatment with 6-OHDA (i.c.v.; B: b) did not reduce cell damage following HS. On the other hand, heat adapted (HA) rats either reared at high environmental temperature (B: g,h) or chronically exposed to heat stress for 7 days (B: i,j) when subjected to HS did not show any cell damage. (C) Pretreatment with indomethacin (C: b) or p-CPA (C: c) markedly attenuated 30 min forced swimming (FS) induced cell damage (C: a) at the ultrastructural level. Bars: (A: a–c) 1 µm; (d,e) 0.6 µm; (B: a–f) 1 µm; (g–h) 0.6 µm; (i,j) 0.8 µm; (C: a–c) 0. 6 µm. Data (B: b–d) after Sharma et al. (1998a,c; Sharma 1999).

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the L-type channels play important roles in neuronal excitability and viability (Hell et al., 1993). The regulation of cellular processes and cell injury depends on the intracellular accumulation of Ca2+ (see Siesjö 1978). The release of Ca2+ from intracellular stores or via IP-3 receptors plays important roles in disease processes (Simpson et al., 1995). The stress-induced release of glucocorticoids influences Ca2+ homeostasis in the CNS (Karst et al., 1994). The transient elevation of Ca2+ is controlled by several Ca-binding proteins, e.g., calmodulin, calretinin, and parvalbumin, that are normally present in some groups of nerve cells (Clapham, 1995). However, the role of Ca2+ in stress-induced cell injury is not well known. Disruption of BBB in stress by serotonin through 5-HT2 receptors is dependent on the mobilization of Ca2+ (Olesen, 1989; see Chapter 12). Serotonin activates the Ca2+ channel, causing accumulation of intracellular Ca2+ (Essman, 1978; Sies.. 1978), leading to cell injury and cell death (Choi, 1988). Thus, a possibility exists that stress-induced cell injury involves Ca2+ metabolism. The influence of one potent L-type Ca2+ channel blocker, nimodipine, in heat stress-induced brain pathology was examined (Sharma and Cervós-Navarro, 1990b). Nimodipine (Nimotop ampoule, Bayer, Leverkusen, Germany) was infused continuously into the right jugular vein of rats at a rate of 1 µg/kg/min over a 2-h period through an indwelling polythene cannula (Vinall et al., 1989; Mohamed et al., 1985). The animals were subjected to heat stress immediately after the infusion (Sharma and Cervós-Navarro, 1990b). Nimodipine did not attenuate stress symptoms or physiological variables significantly (Sharma and Cervós-Navarro, 1990). However, the drug treatment reduced heat stress-induced BBB disruption, CBF changes, edema formation, and cell injury markedly (Figs. 27 and 28). Plasma and brain serotonin levels remained high (Fig. 27). Cell changes in the cerebral cortex and hippocampus are reduced considerably by nimodipine pretreatment (Figs. 22 and 18; Sharma and Cervós-Navarro, 1990b). Microvascular reactions at the ultrastructural level are much less evident in nimodipine-treated stressed animals (Fig. 28). These findings suggest that blockade of the L-type of Ca2+ channel induces neuroprotection in heat-stressed animals (Sharma and Cervós-Navarro, 1990b). Nimodipine is a dihydropyridine derivative and a potent Ca2+ channel blocker with antivasospastic activity on the contraction of cerebrovascular endothelium and vasoconstrictor substances in vitro (Hoffmeister et al., 1979; McCalden et al., 1986; Gelmers, 1987; Vinall et al., 1989). Thus, the Ca2+ channel blocker nimodipine would exert its beneficial effects in heat stress through the blockade of intracellular Ca2+ accumulation, resulting in marked neuroprotection. A significantly higher CBF following heat stress in animals pretreated with nimodipine suggests that the compound is able to counteract the effect of high plasma and brain serotonin-induced contraction of the cerebral microvessels in vivo as well (Fig. 27). Disturbed Ca2+ homeostasis resulting in increased intracellular Ca2+ triggers tissue damage and cell death (Germano et al., 1986). Ca2+ channel blocker drugs selectively inhibit the influx of Ca2+ through the activated membrane of excitable cells and reduce the availability of free Ca2+ (Towart and Kazda, 1979; Kostyuk, 1989). Ca2+ blocker com-

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pounds dilate cerebral vessels and improve the CBF greatly after complete cerebral ischemia (Steen et al., 1984; Sinar et al., 1988; Paci et al., 1989). The absence of BBB disruption and cell injury in the nimodipine-treated group, despite a high serotonin level, suggests that Ca2+ mobilization across the endothelial cell plays an important role in the pathophysiology of heat stress (Olesen, 1989). Further studies using postheat injury treatment with nimodipine are needed to assess the clinical efficacy of Ca2+ channel blockers in heat-related disorders. 17. Repeated Heat Exposure and Thermal Tolerance Adaptation to stress is a common phenomenon that depends on prior physiological or environmental conditions (see earlier discussion; Selye, 1976; Cizza et al., 1995). Whether prior adaptation to heat influences stress-induced BBB dysfunction and cell injury was examined using two different experimental designs. One group of rats was exposed to repeated short-term heat exposure, whereas another group of animals was reared at a mild or moderately hot environment to induce heat adaptation (Dey et al., 1993; Sharma et al., 1991c, 1992d). Animals exposed to 1 or 2 h of heat stress daily at 38°C for 7 days and subjected to 4 h of heat stress on the 8th day did not develop stress symptoms (Sharma et al., 1992d). Plasma and brain serotonin levels and body temperature were elevated mildly on the 7th day of heat exposure and did not increase further on the 8th day after heat stress. The breakdown of BBB permeability, brain edema, and cell injury is absent (Fig. 28; Sharma et al., 1992d). In another experiment, young rats immediately after weaning (on day 21) were placed at thermoneutral (28±1°C) or hot (34±1°C) ambient air temperatures, respectively, for 6 weeks. At the age of 9 weeks, these rats were exposed to 4 h of heat stress at 38°C and BBB permeability, brain edema, and cell changes were examined (Sharma et al., 1991c). Rats exposed to the warm ambient temperature did not develop symptoms, BBB breakdown, and brain edema formation (Fig. 17). Plasma and brain serotonin levels showed a mild increase, and the CBF declined to only 12% from the control group (Sharma et al., 1991c, 1998a). Distortion of nerve cells, glial cells, or myelin was not apparent in these heat-stressed animals (Fig. 28, Sharma et al., 1991c). These results suggest that basal physiological mechanisms prior to heat exposure are important in thermal IPS and brain pathology. D. Sleep Deprivation Stress Sleep deprivation induces profound cellular and molecular changes in the brain reticular activating system (Maloney et al., 1999, 2000). Expression of c-fos, Fos protein, and alterations in GABergic and serotonergic neurons occurs in the brain stem reticular formation in rats following 4 days of sleep deprivation (Maloney et al., 2000). However, the influence of sleep deprivation on BBB function is still unknown. BBB function in rats following 1 to 4 days of sleep deprivation using the well-established inverted flower plot model was examined (Mendelson et al., 1974; Maloney et al., 1999). This model induces a selective deprivation of paradoxical sleep (PS) in rats (Mendelson et al., 1974). Each rat is placed on an inverted flowerpot (6.5 cm in diameter) surrounded by water

284 filled in a Plexiglas box up to 1 cm of the surface with free access to food and water (Maloney et al., 1999). The water temperature is maintained at 30±1°C (Sharma et al., 1991a). In this situation, the animals may undergo slow wave sleep (SWS) but not PS (Maloney et al., 2000). The loss of muscle tonus with PS onset causes the rats to fall into the water and awaken them. Electrophysiological studies suggest that some habituation and SWS occur during the first 24 h; however, PS remains largely suppressed (Maloney et al., 1999, 2000). The PS is significantly attenuated 48 h after sleep deprivation in this model. The animals were kept maximum for 96 h under these conditions (Maloney et al., 2000). At the end of the experiments, the animals were anesthetized with Equithesin (2 ml/kg, ip.), and the BBB permeability to Evans blue albumin was determined (Sharma, 1987; see Chapter 12 for details). Sleep deprivation of 48 h induces mild blue staining of the frontal cortex, temporal cortex, and cingulate cortex. The cerebellar cortex took faint staining. This increase in Evans blue extravasation was intensified further at 96 h after sleep deprivation. Thus, moderate Evans blue staining in the cingulate, frontal, parietal, and temporal cortices is observed (H. S. Sharma, unpublished observations). The walls of lateral cerebral ventricles, dorsal surface of the hippocampus, and massa intermedia showed mild blue staining (Fig. 5). Some areas in the brain stem reticular system took mild to moderate staining (Fig. 5). Extravasation of HRP and endogenous albumin using immunohistochemistry showed a good relationship with the exogenous Evans blue extravasation in the brain (see Fig. 20). The albumin immunoreactivity was localized mainly around microvessels in the cerebral cortex, hippocampus, brain stem, and thalamus. In 96-h sleep-deprived rats, albumin immunoreactivity was also seen around a few nerve cells in the cortex, hippocampus, brain stem, cerebellum, and thalamus (H. S. Sharma, unpublished observation). These observations are the first to show that sleep deprivation stress, depending on its duration, is able to induce BBB disruption in specific regions. Further studies are in progress to see whether the BBB breakdown in sleep deprivation is associated with structural changes in the CNS. 1. Functional Significance of BBB Disruption in Stress Above observations show that several stressful conditions, depending on their duration, are able to disrupt BBB function. Thus, 30 min of forced swimming, 4 h of heat stress, 8 h of immobilization stress, and 48 h of sleep deprivation cause BBB breakdown to protein tracers in selective and specific brain regions. Regional changes in BBB permeability vary according to the stressors used. This indicates that each stressor has some specific effects in particular brain regions that is evident with a selective BBB opening. This stress-induced BBB permeability is reversible in nature. Whether a brief increase in BBB function helps the organism to cope with the stress or reflects a nonspecific response on brain function is unclear. Marked nerve cell changes in stress suggest that opening of the BBB is harmful to the organism. Flattening of EEG activity at the time of BBB opening in stress further supports this idea. It may be that opening of the BBB in stressful situation induces premature aging and/or neurodegeneration. Mild to

H ARI S HANKER S HARMA moderate nerve cell damage in the cerebral cortex or hippocampus seen during stress is quite comparable to those observed during aging and other neurodegenerative disorders. An absence of BBB disruption in adult animals suggests that the IPS of these rats is unperturbed by the stress overload. An absence of increased serotonin levels in the plasma and brain and no increase in 5-HT2 receptor binding in adult rats support this hypothesis (see earlier discussion; H. S. Sharma, unpublished observations). This shows that adult animals handle stress better and adapt to new situations without showing symptoms or release of neurochemicals, e.g., serotonin. The functional clinical significance of these finding may be that children exposed to stressful environments are more vulnerable to mental abnormalities than adults (Essman, 1978; Sharma et al., 1995b). Apparently, an increased serotonin level will induce BBB breakdown in children or infants exposed to stressful situations. Once BBB permeability is increased, these children are susceptible to more adverse neuronal dysfunctions, leading to mental abnormalities. Increased BSCB permeability in stress alters the spinal cord microenvironment and may induce damage to motor or sensory neurons, causing long-term disability. Because the spinal cord is an important organ for sensory-motor information to the CNS, an alteration in the cord extracellular microenvironment in stress will have serious consequences, leading to long-term functional disability. 2. Distribution of Tracers in the Brain and Spinal Cord Two different protein tracers, Evans blue and 131I, were used to examine the permeability of the BBB (Rinder, 1968; Sharma and Dey, 1986a,b). These protein tracers bind to serum albumin after introduction into the systemic circulation (Rawson, 1943; Rapoport, 1976; Bradbury, 1979). Evans blue in the doses used in our experiments binds mostly to serum albumin by 68%, whereas the binding of radioiodine to albumin is about 50% (Rinder, 1968, Lyebeck, 1957). Neither bound nor unbound forms of either tracer cross the normal BBB in young or adult rats (Sharma and Dey, 1986a,b). A significant higher permeability of radioiodine tracer across CNS microvessels compared to Evans blue in stressed rats appears to be due to a difference in the molecular size of the tracers and/or due to a difference in the proteins to which they bind in vivo (Mayhan and Heistad, 1985; Sharma et al., 1990). Thus, leakage of exogenous protein tracers in stress represents endogenous protein extravasation and spread of edema fluid. Extravasation of HRP seen in the cortex of stressed animals further supports this idea. 3. Mechanisms of BBB Disruption in Stress Alterations in neurochemicals appear to be responsible for the BBB breakdown in stress. Increased serotonin levels in the plasma and brain following stress at the time of BBB dysfunction support this hypothesis (Sharma et al., 1990; see Chapter 12). Serotonin is able to induce BBB disruption either directly or through a cascade of other neurochemical transmitters or signal-transducing agents (Sharma et al., 1990; see Chapter 12). The effect of serotonin is mainly seen in young rats subjected to stress. A minor increase in plasma and brain serotonin levels seen in adult animals did not influence BBB permeability. This suggests that the cerebral vessels of adult animals are less responsive to serotonin due to receptor downregulation with

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advancing age (McEntee and Crook, 1991). Alternatively, a lower rise of serotonin may be due to differences in stress response and/or serotonin metabolism in adult animals (McEntee and Crook, 1991; see Chaouloff, 1993; Sharma et al., 1995b). Results obtained with drug treatments further support this idea. Thus, attenuation of serotonin accumulation in plasma and brain with p-CPA, diazepam, or indomethacin prevented BBB disruption. Similarly, blockade of serotonin 5-HT2 receptors with cyproheptadine, ketanserin, or ritanserin, despite high plasma and brain serotonin levels, markedly attenuated the BBB breakdown. These observations suggest that binding of serotonin to 5-HT2 receptors is important in stress-induced BBB dysfunction. After binding to its 5-HT2 receptors located on the cerebral microvessels, serotonin induces intracellular signaling (Aghajanian, 1978; see Chapter 12). Stimulation of phosphoinositide (PI) metabolism by serotonin does not play an important role in the BBB opening in stress (Sharma et al., 1995b). Stimulation of PI metabolism by serotonin in the cerebral cortex inhibited by forced swimming (Morinobu et al., 1992) is in line with this idea. It appears that after binding to 5-HT2 receptors, serotonin will influence BBB permeability through PGs (Haubrich et al., 1973; Ellis et al., 1979) and/or cAMP (Baca and Palmer, 1978; Joó et al., 1975; Olesen, 1989; Westergaard, 1978). This hypothesis is further supported by the fact that indomethacin (a cyclooxygenase inhibitor; Robinson and Vane 1974; Wolfe et al., 1976), in a concentration high enough to block the PG synthesis in the cerebral vessels (Baca and Palmer, 1978; Ellis et al., 1979; Eakins, 1977; Wolfe et al., 1976) completely prevented the increased BBB permeability in stress (Sharma and Dey, 1986a,b; Sharma et al., 1995b). PGs are implicated as a first mediator of stress, and inhibition of their release with indomethacin prevents the stress response in animals (Hanukoglu, 1977). In addition, PGs are known stimulators of serotonin synthesis (Haubrich et al., 1973). Thus, indomethacin-induced inhibition of PG release will prevent the stress response and increase serotonin levels in plasma or brain (Sharma et al., 1993b). Cerebral capillaries contain all necessary enzymes for the synthesis and catabolism of PGs, as well as cAMP (Baca and Palmer, 1978; Ellis et al., 1979). A local accumulation of PGs in cerebral capillaries induces marked vasodilatation and an increase in vesicular transport (Eakins, 1977). Thus, increased serotonin levels in stress will stimulate PG synthesis in the cerebral vessels (Haubrich et al., 1973). PGs then stimulate cAMP synthesis, leading to an increase in transcellular transport across the microvessels (see Sharma et al., 1990, 1995b). An early increase in BBB permeability following immobilization with aminophylline (an inhibitor of PDE that leads to the accumulation of cAMP) further supports this idea. Obviously, this mode of tracer transport is influenced by various pharmacological agents. Results obtained with vinblastine support the aforementioned hypothesis. Vinblastine inhibits microtubule function associated with the transcellular transport of tracer substances (Creasy, 1975; Larsson et al., 1980). A complete blockade of tracer extravasation in stress with vinblastine is in line with the idea that transcellular transport plays a major role in stress-induced BBB leakage (Sharma et al., 1998a,c; see Figs. 1 and 21). Ultrastructural studies using electron-dense tracer lanthanum are in good agreement with this hypothesis (see Fig. 21).

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The mechanical effects of vasospasm caused by serotonin on the vascular endothelium of young rats exhibiting immature cell to the cell-binding matrix are not involved in stress-induced BBB disruption (Auer et al., 1985; Sharma et al., 1995b). An inability of the increased serotonin levels in plasma to open the BBB in young rats treated with cyproheptadine, ketanserin, or vinblastine supports this idea (Fig. 29). A blockade of multiple opioid receptors with naloxone and naltrexone attenuated BBB permeability, indicating a role of opioids (Sharma et al., 1997b,c). Most of the opioids are colocalized with serotonin in several brain regions. Thus, an interaction between serotonin and opioids in stress is quite likely. A reduction in the rCBF in cyproheptadine-, ketanserin-, ritanserin-, or vinblastine (Figs. 25 and 26)-treated stressed animals without apparent BBB disruption suggests that the slowly developing ischemia and local failure of cerebral autoregulation of microvessel vessels are not related to microvascular permeability changes in stress (Edvinsson and McKenzie, 1977; Sharma et al., 1986a). Stress-induced changes in MABP, arterial pH, or blood gases and circulating serotonin levels are primarily responsible for reduction in the CBF (Paulson and Sharbrough, 1974; Ohata et al., 1981, 1982) (see Chapter 12 for details). It appears that catecholamines do not participate in the mechanisms of stress-induced BBB breakdown (Harri and Kuusela, 1986; Sharma, 1999). An inability to attenuate BBB disruption in stress by the destruction of peripheral or central noradrenergic neurons with 6-OHDA further confirms this hypothesis. It may be that catecholamines contribute to stress symptoms as well as cardiovascular and thermoregulatory functions (Selye, 1976). Changes in body temperature or alterations in cardiovascular functions in stress do not contribute to BBB permeability (Lourie et al., 1960; Maizelis, 1966). Similar changes seen in drug-treated animals without BBB disruption support this hypothesis. The early hypertension is not severe enough to impair the BBB function because administration of Evans blue dye before the onset of stress did not result in blue staining of the brain (H. S. Sharma, unpublished observation). Alterations in these physiological variables following stress could be coupled to activation of the hypothalamic–pituitary–adrenal axis and/or related compensatory mechanisms (Harri and Kuusela, 1986; Selye, 1976). XXXIII. Conclusion Results clearly show that BBB disruption during stress is instrumental in causing selective neural injury, leading to long-term effects on the brain function (Fig. 29). It appears that the stress-induced release of neurochemicals disrupts the BBB and BSCB function and induces vasogenic edema formation. Alterations in fluid microenvironment of the brain, together with edema formation, lead to several cellular and molecular changes in the brain, resulting in cell injury and cell death (Fig. 29). However, no single chemical compound or factor alone is responsible for BBB disruption or brain dysfunction. A balance between several endogenous neuroprotective substances and neurodestructive agents is crucial in maintaining BBB function, leading to health and disease. Obviously, BBB disruption in stress seems to be the gateway for brain

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H ARI S HANKER S HARMA HARI SHANKER SHARMA Laboratory of Neuroanatomy, Department of Medical Cell Biology, Biomedical Center, Uppsala University, SE-75123 Uppsala, Sweden

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Cell death Fig. 29 Probable mechanisms of stress induced cell death in the CNS. It appears that stress induced release of neurochemicals play important roles in BBB disruption and vasogenic brain edema formation. Disruption of the BBB and brain edema formation in the CNS following stress appears to be crucial for cell injury and cell death.

dysfunction and to the development of neurological disorders causing neurodegeneration. XXXIV. Future Direction Studies are in progress to see whether antiserum to neurotransmitters and/or several neuronal proteins can influence BBB function and brain pathology in stress. Furthermore, to understand the molecular mechanisms of BBB permeability, alterations in several tight junctional proteins in the brain and spinal cord in stress are currently being examined. Acknowledgments Author’s research described in this review is supported by grants from Swedish Medical Research Council No. 2710, G.ran Gustafsson Foundation, Sweden, Astra-Zeneca, Mölndal, Sweden; Alexander Humboldt Foundation, Bonn, Germany; the University Grants Commission, New Delhi; and the Indian Council of Medical Research, New Delhi, India. Thanks are due to expert reviewers for their excellent suggestions on this manuscript to improve the scientific quality and presentation. Parts of the work were carried out in the laboratory of Armin Ermisch, Department of Biosciences, Leipzig, V Bigl, Department of Neurochemistry, Univ. of Leipzig (Karl Marx University, Leipzig); Jorge Cervós-Navarro, Free University, Berlin, Department of Neuropathology, Klinikum Steglitz. (now Benjamin & Franklin Klinikum); P. K. Dey, Neurophysiology Research Unit, Department of Physiology, Institute of Medical Sciences Banaras Hindu University, Varanasi, India. The technical assistance of Aftab Ahmed, R. K. Gupta, Deep Chand Lal, Mohammad Siddiqui (Varanasi), Katja Deparade, Elisabeth Scherer, Franziska Drum (Berlin), Ingmarie Olsson, K.rstin Flink, Madeleine Thörnwall, Madeleine Jarild, and Gunilla Tibling (Uppsala) and the secretarial assistance of Angela Jan, Katherin Kern (Berlin), Aruna Sharma, and Eva Lundberg (Uppsala) are highly appreciated. The skillful assistance in photographic work by Frank Bittkowski (Uppsala), R. K. Srivastav (Varanasi), and L. Schindler (Berlin) and the computer assistance of Suraj Sharma and Leif Ljung (Uppsala) are acknowledged with thanks.

Key words: Stress, psychological stimuli, fluid microenvironment of the brain, blood–brain barrier, blood–spinal cord barrier, cerebral, blood flow, microvascular, permeability, Evans blue 131I-sodium, Lanthanum, ultrastructure, Tight junctions, vesicular transport Correspondence: Hari Shanker Sharma, Dr. Med. Sci. Laboratory of Neuroanatomy Department of Medical Cell Biology Box 571, Biomedical Center Uppsala University SE-75123 Uppsala, Sweden Phone & Fax: +46-18-243899 E-mail: [email protected]

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