The effect of hypoxia and catecholamines on regional expression of heat-shock protein-72 mRNA in neonatal piglet brain

BRAIN RESEARCH ELSEVIER

Brain Research 727 (1996) 145-152

Research report

The effect of hypoxia and catecholamines on regional expression of heat-shock protein-72 mRNA in neonatal piglet brain Stephanie J. Murphy ~', Dekun Song '~, Frank A. Welsh b David F. Wilson ~', Anna Pastuszko ç ' ~' l)epartment of Bio«hemistry and Biophysics, Medical School, Uni«ersi U of Pennsylvania, Philadell)hia, ,°,4 I cH04, USA b Division (~'Neurosurgery, Medical School. Unilwwity ofPenn.s3"h,ania. Philadelphia. PA 19104. UX4 « DÇ~artment«g'Pediatrics, Medical School, Unitersi O" ofPennsyl«ania, Philadelphia, PA 19104, UX4

Accepted 12 Match 1996

Abstract

The present study has shown that hypoxia leads to expression of heat-shock protein in the brain of newborn piglets and this process is almost completely abolished by depletion of catecholamines prior to the hypoxic episode. The piglets were anesthetized and mechanically ventilated. One hour of hypoxia was generated by decreasing the oxygen fraction in the inspired gas (FiO 2) from 22% to 6 ç - 1 0 ç . FiO 2 was then returned fo the control value for a period of 2 h. Following the 2 h of reoxygenation, regional expression of the 72-kDa heat-shock protein (hsp72) mRNA was determined using in situ hybridization and autoradiography. The hypoxic insult (cortical pO~ = 3-1{) mmHg) induced expression of hsp72 mRNA in regions of both white and gray matter, with strong expression occurring in the cerebral cortex of individual animais. Depleting the brain of catecholamines prior to hypoxia, by treafing lhe animais with cx-methyl-p-tyrosine (AMT), resulted in a major change in the hsp72 mRNA expression. In the catecholamine depleted group of animais, the intensity of hsp72 mRNA expression was greatly decreased or almost completely abolished relative to the nondepleted hypoxic group. Fhese results suggest tha! the catecholamines play a significant role in the expression of the hsl)72 gene in response to hypoxic insul! in neonatal brain. Kevwords." Hypoxia: Catecholamine: Heat shock protein; Newborn: Brain

1. Introduction

It is postulated that the increase in extracellular levels of catecholamine neurotransmitters, dopamine and norepinephrine, in ischemia or hypoxia, can have an important impact on cellular metabolism of susceptible brain regions. During the last few years, dopamine has been recognized as a major contributor in the development of neuronal striatal damage in these pathological conditions. It has been shown that lesions of substantia nigra have a neuroprotective effect on the striatum related to the inhibition of dopamine release in the latter [13,17]. Clemens and Phebus [9] reported that unilateral infusion of 6-hydroxydopamine into substantia nigra of rats, to deplete dopamine prior to global ischemia, resulted in significant protection of the dopamine-depleted striatum from ischemia-induced Mss of medium sized neurons. Similarly, Chapman et al. [8]

Corresponding author. Fax: (I) (215) 898-4217. 0006-8993/96/S 15.00 Published by Elsevier Science B.V. Pli S0006-890~(o6)003(~3-0

demonstrated that lesioning the nigrostriatal pathway decreased the excitotoxic effect of striatal injections of NMDA and kainate. Marie et al. [27] evaluated rat brain 72 h after ischemia (four vessel occlusion) and reported that cx-methyl-p-tyrosine (AMT) treatment significantly decreased neuronal necrosis in the striatum but had no cytoprotective effect in the CA1 section of hippocampus or in the neocortex. Neuroprotective effects of AMT were also presented by Weinberger and coworkers [49]. The foie of an increase of extracellular norepinephrine in ischemic/hypoxic conditions [15,40] is controversial and still remains to be established [3,12,29]. Busto et al. [6] presented evidence that depletion of cerebral norepinephfine enhances recovery afler brain ischemia. It has also been reported that transient norepinephrine overflow in hippocampus during ischemia is closely related to the complete loss of brain electrical activity [40]. Similarly, Pappius and Wolfe [37] showed that norepinephrine is involved in functional disturbances in injured brain (freezing lesion). However, other data suggest that norepineph-

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rine can protect against ischemic damage in regions of brain widely innervated by noradrenergic fibers, such as hippocampus or neocortex [1,3,21]. The purpose of this study was to investigate the effect of global hypoxia on expression of hsp72 mRNA in neonatal piglet brain and to determine the role of catecholamines in this expression. Measurement of the expression of hsp72 following hypoxia was chosen because stress proteins are considered to be sensitive markers of cellular injury, although their functions are not fully understood [18,19,34]. Increased expression of the heat-shock protein genes has been demonstrated in the CNS following a number of types of stress, including heat-shock [5,28], trauma [20], ischemia and hypoxia [4,11,33-35,44,50,51 ]. Most studies of the stress response in brain have focused on the more prominently induced 70-kDa stress protein family. A major member of this family is hsp72, a strictly inducible protein, which has been evaluated in a number of brain injury models including hypoxia and ischemia [34]. Thus, as the meehanisms of heat-shock protein induction are being explored, there is accumulating evidence that induction of hsp72 mRNA expression can be used as a marker of cellular injury in the brain. The results presented in this paper show that 1 h of hypoxia increased expression of hsp72 mRNA in different regions of brain of newborn piglets and this increase is largely abolished by prior depletion of the brain of dopamine and norepinephrine.

2. Materials and methods 2. I. Animal preparation

Newborn piglets, 1-4 days of age, averaging 2 kg in body weight, were used in this study. Animals of this age were chosen because the level of development of the piglet brain is comparable to that in humans of a term newborn. General anesthesia was induced with a mixture of 4% halothane (Halocarbon Laboratories, Augusta, SC), balance O2; local anesthesia was produced with 1.5% lidocaine-HC1 (Abbott Laboratories, North Chicago, IL). Following a tracheotomy, the percentage of halothane was reduced to 0.6% to 0.8%, and the femoral artery and vein were cannulated. The animals were administered Fentanyl-citrate (Elkins-Linn, Cherry Hill, NJ; 30 I x g / k g / h , i.v.), and halothane was withdrawn from the ventilation mixture. The animals were immobilized with Tubocurarine-HCl (Apothecon, Bristol-Meyers Squibb, Princeton, NJ; 0.3 m g / k g , i.v.) and ventilated mechanically with a mixture of 22% 0 2, balance N20. A rectal temperature probe was inserted to record core temperature. The head of the animal was placed in a Kopf stereotaxic frame and a longitudinal scalp incision was made along the midline. After exposing the skull, a burr hole 12 mm in diameter was made over the parietal region of one hemi-

sphere. The dura was left intact, and a small area of the brain surface was exposed for measurement of cortical pO 2 (see below). Mean arterial blood pressure (MABP), heart rate and end-tidal CO 2 were monitored continuously. Arterial blood samples were taken every 10-20 rein for measurement of pli, paCO2 and paO~ using a Model 278 p H / B l o o d gas Analyzer (Corning). Following a 15-rein control period, arterial hypoxia was induced by lowering the FiO 2 from 22% to 6%-10% for 1 h. At the end of the hypoxic period, the FiO 2 was raised to 22% and maintained at that level throughout the 2-h period of reoxygenation. Arterial hypoxia was performed in animais with and without prior treatment with «-methyl-ptyrosine (AMT), an inhibitor of catecholamine synthesis. The injection of AMT (300 m g / k g , i.p.), which was given 5 h prior to hypoxia, decreased the concentration of dopamine in the different regions of brain (neocortex, striatum, hippocampus, midbrain) by 8 0 - 8 5 ç . The dopamine concentration in brain tissue ( + AMT) was determined by HPLC methods as described previously [39]. Sham-operated animais (_+ AMT), ventilated with 22% O, t'or 3 h, served as controls. Ail animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animais and were approved by the local Animal Care Committee. 2.2. Cortical oxygen pressure measurements

Cortical 1)O 2 was measured using O2-dependent quenching of phosphorescence as described previously [43,54]. In brief, the O2-sensing phosphor, Pd-meso-tetra (4-carboxyphenyl) porphine (Porphyrin Products, Logan, UT), was administered (5-8 m g / k g , i.v,) in a 10 m g / m l solution containing 60 m g / m l bovine serum albumin in physiological saline, buffered to pli 7.4. Light from a flashlamp (EG &G Electro-Optics, Salem, MA) was passed through an interference filter (535 nm center wavelength and a half-bandwidth of 30 nm) and conducted to the exposed parierai cortex through a bifurcated light guide with randomized fibers. The light was focused on a 3 mm diameter area of tissue. Light emitted from the cortex was collected by a lens system on the end of the light guide, passed through a 695 nm interference filter with a width at half maximal intensity of 80 nm, and measured by a photomultip[ier. The resulting signal was amplified, digitized with a 12 bit, l MHz A / D board, and analyzed using a microcomputer. The digitized decay curve was fitted to a single exponential to determine the lifetime, and 1)O2 was calculated using the Stern-Volmer equation. The pO: measurements are representative of the mean of that in the microcirculation. Measurements were taken at the indicated 10- or 20-min intervals. 2.3. In situ hybridization measurements «?f hsp72 mRNA

At the end of the 2-h period of reoxygenation, the animals were killed by i.v. administration of a saturated

S.J. Murphy et a l . / B r a i n Resear«h 727 (1996) 145 152

KCI solution. The brain was rapidly removed from the cranium and frozen in powdered dry ice. In situ hybridization was carried out using modifications of previously described methods [51]. In brief, the brain was sectioned in a cryostat at - 2 5 ° C at a thickness of 14 #,m. Sections were mounted on subbed slides, dried, fixed in 4% paraformaldehyde~ and rinsed in 2 X SSC (saline/sodium citrate). Hsp72 mRNA was detected using a 30-mer oligodeoxy-nucleotide probe with the following sequence: 5'-C GAT CTC CTT CAT CTT GGT CAG CAC CAT GG-3'. This sequence, first used in mice and gerbils [43] and shifted two bases 5' from that employed in rat [28] corresponds to a region of hsp72 coding sequence that is identical in human and Drosophila [23]. This probe bas been also used previously to detect hsp72 mRNA in pig brain [45]. The sequence selected was reported t o b e specific to the strictly inducible hsp72 mRNA, as opposed to the closely related transcript which encodes the 73-kDa consituitive heat-shock protein [28]. The oligonucleotide probe was labeled on the 3'-end with [35S]deoxyATP using terminal transferase. The labeled probe was isotated and hybridized to tissue sections overnight at 37°C in 4 × SSC. After rinsing for 1.5 h each in 1 x SSC and 0.5 × SSC at 37°C, the sections were dried and exposed to Kodak SB film for 1-2 weeks. Expression of hsp72 mRNA was graded qualitatively by comparing optical densities in various brain regions fo sham-operated animals processed in parallel. The grading scale was as follows: 0 = no difference, + l = mild to moderate increase in optical density, + 2 = strong difference in optical density.

2.4. Statisti«al analvsis All physiologic data are expressed as means _+ S.E.M. Differences between mean values were assessed by repeated measures analysis of variance (ANOVA) and posthoc analysis (Bonferroni test) in order to adjust the significance level for multiple comparisons. Differences were considered to be statisfically significant at P < 0.05.

147

3. Results

3.1. Physiological parameters (~1"newborn pi çlets durinA, hypo~ia and r«o.~:vgenation The effects of 1 h of arterial hypoxia and 2 h oi post-hypoxic reoxygenation on the physiological parameters of newbom piglets (_+AMT) are shown in Table I. Hypoxia caused significant decrease in arterial blood pli in both groups of animals. In nondepleted animais, arterial pli decreased during hypoxia from control values of 7.43 + 0.01 to 7.10 + 0.05 ( P < 0.001 ). During reoxygenation (FiO 2 = 22%), the pli increased to 7.29 +_ 0.02, a value still significantly below control ( P < 0.05). In depleted animais, arterial pli decreased during hypoxia from control values of 7.39_+ 0.01 to 7.12 _+ 0.03 ( P < 0.001) and during reoxygenation increased again to 7.31 _+ 0.01, a value still significantly below control ( P < 0.05). There was no statistically significant difference in pli between depleted and nondepleted animais. The p~~O: decreased during hypoxia ri-oto 119.4 _+ 3.8 torT to 20.4 _+ 0.6 tort ( P < 0.001) in nondepleted group and from 133.2 _+ 4.4 tOrT to 24.7 + 0.6 torT ( P < 0.001 ) in depleted group. Two hours of reoxygenation caused the p~,O2 to increase again to 103.9 _+ 2.8 tort and 104.6 + 2.1 tort in nondepleted and depleted animais, respectively. These values were still significantly below controls. MABP decreased significantly during hypoxia from 89_+ 2 tort fo 51 _+ 2 tort ( P < 0.001) in nondepleted group and from 83 _+ 2 torT to 68 + 2 torT ( P < 0.001) in depleted group. A statistically significant difference in blood pressure between nondepleted and depleted animais occurred during hypoxia (51 + 2 torr vs. 68 _+ 2 tort: P < 0.001) and at 60 min of reoxygenation (69 + 2 torr vs. 81 _+ 1 torT; P < 0.05). By the end of the reoxygenation periods, the blood pressure increased to 70 _+ 2 torr and 72 + 2 torr, respectively; values which were still significantly lower than control levels.

Fable I Fhe physiological parameters of newborn piglets during h y p o x i a and reoxygenation Experimental conditions Control Hypoxia Reoxygenation ~~0 min 120 rein

Blood p l i

PaCO2 (torr)

p,,O 2 (ton-)

M A B P (tort)

(-AMT) (+AMT) ( AMT) (+AMT)

7.43 _+ 0.01 7.39 _+ 0.01 7.10+0.05 b 7.12 _+ 0.03 d

36.0 34.5 33.1 35.2

,+ 0.9 _+ 1.2 _+2.2 + 1.5

119.4 _+ 3.8 133.2 _+ 4.4 « 20.4_+0.6 ~ 24.7 _+ 0.6 d

89 83 51 68

( AMT) (+AMT) ( -- A M T ) (çAMT)

7.11 7.16 7.29 7.31

42.1 + 2 . 7 37.5 ,+ 1.4 34.0 ± 1.0 3 7 . 0 ± 1.5

111.4--+4./) 102.5 _+ 2.6 d 103.9 + 2.8 " 104.6_+2.1 d

69+2 NI ! I 70 + 2 72_+2

+0.03 _+ 0.03 + 0.02 _+0.01

b d ~ c

+ 2 _+ 2 + 2 ~' _+ 2 'j~ t' ~ (' '

The values are the means + S.E.M. for 5 experiments except where noted ( n - 3). ~=P < 0.05, h p < 0.001 for difference from control ( - AMT); « P < 0.05, çt p < 0.001 for difference from control ( + AMT): ç I' < 0.05. J P < 0.001 l'~»r diffcrence between A M T q r e a t e d and untreated piglets, as determined by o n e - w u y analysis of variance ( A N O V A ) wilh p o s t - h o t Bonterroni test.

S.,l. Muq~lly et al./Brain R«sear«h 727 (1996) 145 152

148 6O

values of 47.4 _+ 1.4 ton" to 5.9 +_ 0.8 ton. ( P < 0.001) in nondepleted animals. In depleted animais, cortical oxygen decreased during hypoxia from 50.8 + 4.9 tort to 6.3 + 0.4 ton. ( P < 0 . 0 0 1 ) . There were no statistically significant differences in cortical oxygen pressure between nondepleted and depleted animais. During tbe first 10 rein of reoxygenation, the cortical oxygen pressure increased to a value not significantly different from control in both groups of animals and stayed at this level during the remaining period of reoxygenation.

gso 4o

,= ,z,.3o » 2o

(~\ ~*

.

A

810 ,* I ,, 50

M1] " //

T

)+ ' HYPOXlA " + CONTROL (+AMT) ,,--e-- HYPOXlA (+AMT)

J

j

100

I

,

,

150

l I M E (MINUTES)

Fig. 1. Effect of hypoxia and reoxygenation on cortical pO 2 in neonatal piglets with or without treatment with AMT, an inhibitor of dopamine synthesis. Cortical oxygen pressure was measured using the oxygen-dependent quenching of phosphorescence as described in Section 2. Data are presented as the means±S.E.M, for 3-5 animais in each group. Asterisks denote a significant difference (P <0.001) from controls as detemfined by one-way ANOVA with post-hoc Bonferroni test.

3.2. The effect of decreased FiO2 and reoxygenati(m on «ortical oxvgen pressure The effects of hypoxia and reoxygenation on cortical oxygen pressure in nondepleted and depleted animais are shown in Fig. 1. Reduction of FiO 2 to 6-10% progressively decreased cortical oxygen pressure from normoxic CONTROL (-AMT)

CONTROL (+AMT)

3.3. Expression of hsp72 mRNA following hypoxia and reoxygenatiml In sham-operated, control animais ( +_AMT), expression of hsp72 mRNA was low in all brain regions except in 2 out of 5 untreated animals where some expression was observed in the choroid plexus and ventricular lining (Table 2, Fig. 2). As can be seen in Table 2 and Fig. 2, severe hypoxia ( - A M T ) induced the expression of hsp72 mRNA in both white and gray matter regions, although expression was limited to the white matter in 1 of 5 animals (Table 2, Hypoxia ( - A M T ) , Animal #1). Within the gray matter, the extent of expression in individual animals varied from widespread involvement of the cerebral cortex, caudate HYPOXlA

(-AMT)

HYPOXlA

(+AMT)

Fig. 2. Regional expression of hsp72 mRNA following hypoxia in neonatal piglet brain with or without treatment with AMT, an inhibitor of dopamine synthesis. Autoradiograms of coronal sections from one animal of each experimental group are presented, lncreased optical density represents increased hybridization for hsp72 mRNA.

S.J. Mul7~hy et al. / Brain Research 727 (1996) 145 152 Table 2 Regional expression of hsp72 m R N A following h y p o x i a in neonatal piglet brain treaIed wilh or without A M T Experimental g r o u p Comrol ( - A M T ) 1 '~ 3 4 5 Control ( + A M T ) 1 2 3 Hypoxia ( - AMT) I 2 3 4 5 Hypoxia ( + AMT) 1 2 3 4 5

CP/VL

WM

IC

MB

CX

CN

0 0 0 + I +2

0 0 0 (} {I

0 0 0 (I 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

+ 2 + I +2 +2 +2

+ I + 2 +1 +2 +2

+ 1 0 +1 +2 +2

0 0 +1 +2 +2

0 + I +1 +1 +2

0 0 0 0 +2

+ I 0 + 1 + 1 o

+ 1 0 0 + I + I

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

Grades of expression: (I no difference. + 1 - mild to moderate increase in optical density: ç 2 = sIrong increase in optical density. CP = choroid plexus; VL - ventricular lining: W M = white marier; IC = internal capsule: MB = midbrain; CN - caudate nucleus: C X - cortex.

nucleus and deep gray matter regions (Fig. 2, Hypoxia ( - A M T ) ) to restricted patches in cerebral cortex and hypothalamus. Within the white matter, strong expression of hsp72 mRNA was evident in the central areas, while expression was often absent in the white matter tracts extending into the cortical gyri. Expression of hsp72 mRNA was also evident in the internal capsule in 3 of 5 animals of this group (Table 2, Hypoxia ( - A M T ) ) . Depletion of the neonatal piglet brain of catecholamines prior to hypoxia resulted in major changes in hsp72 mRNA expression as compared to nondepleted brains. After catecholamine depletion, the intensity of hsp72 mRNA expression was either decreased or almost completely abolished (Table 2, Fig. 2).

4. Discussion

The present results demonstrate that arterial hypoxia ,an induce regionally heterogeneous expression of hsp72 mRNA, a marker of cellular stress, in the brain of neonatal piglets. This increase in expression occurs in regions of both gray and white matter. Depletion of the neonatal piglet brain of catecholamines via AMT treatment prior to hypoxia resulted in major changes in hsp72 mRNA expression. This expression was either decreased or almost completely abolished as compared to nondepleted brains. Before further discussion of the possible vole of cate-

149

cholamines on the expression of hsp72 mRNA, the biological significance of this expression should be considered. The present study shows that in the brain from control piglets (sham-operated), expression of hsp72 mRNA was generally below the level of detection excepte in some animals, in the choroid plexus and ventricular lining. These results are in agreement with the reports of others that in brain of unstressed animals, expression of hsp72 and its mRNA was near or below the limits of detection [35,47,51 ]. Thus, the appearance of detectable amounts of hsp72 mRNA in the brain parenchyma following 1 h arterial hypoxia and 2 h reoxygenation indicates that this level of hypoxic insult ,an induce increased expression of hsp72 mRNA. This experimental model was chosen because: (1) dopamine metabolism, cortical oxygen pressure, free radicals production, and the activity of several enzymes have been extensively studied previously [22,30,36,38]; (2) after 2 h reoxygenation strong expression of hsp72 was observed; (3) 1 h of arterial hypoxia, similar to that used in present study, has been reported to result in significant histological damage to the brains of newborn piglets as measured after 48 h of normoxic recovery [42]. In the last study, the authors characterized the histological changes in se,tions from the dorsal hippocampus, demonstrating the CA1 region, and from the parietal cortex. There was a loss of neurons in the hippocampus and a swollen or pyknotic appearance to many of the remaining neurons in this region. Similarly, in the cortex of the hypoxic animals there was a dramatic loss of neurons and irregular appearance of the remaining neurons and a marked gliosis. demonstrated by an apparent increase in size and number of discernible glia [42]. In an earlier study, we characterized the degree of tissue hypoxia required to induce hsp72 mRNA expression in brain of newborn piglets [31]. It was shown that even mild hypoxia, defined by cortical pO 2 values ranging from 10-30 torr, was suffi,lent to trigger the expression of hsp72 mRNA, although this expression was limited to white matter. Wu et al. [55,56] reported that induction of hsp72 by anoxia and other stresses is mediated, in part, by activation of a pre-existing protein factor. The degree of hypoxia in vivo required to activate this factor bas not been fully defined, but activation in vitro was reported to require anoxic insults that cause prolonged depletion of cellular ATP [2]. In in vivo studies it has been reported that potentially lethal degrees of oeil stress are required to trigger expression of hsp72 [18,44,46]. Thus, we conclude that the heterogeneous expression of hsp72 mRNA in both gray and white matter following the avterial hypoxia is consistent with a potential for permanent alteration of the functions associated with the structures. The goal of these studies was not only to determine the effect of hypoxia on expression of hsp72 mRNA but also to test for a vole of catecholamines in induction of this expression. The data show that depletion of catechol-

150

S.,l. Mulphy et al. / Brain Research 727 (1996) 145-152

amines with AMT markedly diminished the induction of hsp72 mRNA following cerebral hypoxia. Measurement of the physiological parameters in animais 4-treatment with AMT showed a smaller hypoxia induced decrease in systemic blood pressure in the dopamine/norepinephrine depleted animais. Thus, cerebral blood flow during hypoxia may have been higher in AMT-treated piglets, relative to untreated animais [7,41]. Cortical oxygen pressure tended t o b e higher in the AMT-treated animais at 20 and 40 min of hypoxia (Fig. 1), although these differences were not statistically significant. By 60 min of hypoxia, however, the oxygen pressures were the same, suggesting that delivery of oxygen was very similar in the two groups. It is not clear whether the observed changes in expression of hsp72 mRNA following hypoxia are related to dopamine, norepinephrine or to both of these monoamines. Injection of o~-methyl-p-tyrosine, a tyrosine hydroxylase inhibitor, leads to depletion of both dopamine and norepinephrine [52,53]. However it can be expected that in the central dopamine-containing systems, with relatively small numbers of noradrenergic fibers, a depletion of dopamine was most likely responsible for the decreased expression of hsp72. The central neuronal pathways (long length systems) containing dopamine include: the nigrostriatal pathway which runs in the crus cerebri and internal capsule to innervate the caudate nucleus, putamen, globus pallidus and possibly the amygdala; the mesolimbic pathway which runs in the nucleus accumbens and the olfactory tubercle; and the mesocortical pathway which runs in the frontal, cingulate and entorhinal areas. In addition to these long systems, ultrashort and intermediate length dopaminergic systems have been described in central nervous system. Norepinephrine may play a more significant role in regions widely innervated by noradrenergic fibers. One of the major noradrenergic neuronal systems arises in the locus coeruleus, which innervates many regions of brain, such as brainstem, cerebellum, hypothalamus, amygdala, hippocampus, septum, fornix, cingulum, external capsule, lateral and dorsal neocortex. Several mechanisms may contribute to the observed changes in expression of hsp72 mRNA in the brain of newborn piglets. Dopamine induced increase of expression of hsp72 mRNA following hypoxia may result from an increased formation of free radicals due to oxidation of this monoamine. During posthypoxic reoxygenation, there is an increase in the levels of hydroxy radicals in striatum of newbom piglets and this increase is partly abolished by depleting the brain of dopamine [36]. Thus, part of the increased expression of hsp72 mRNA following hypoxia could be through a dopamine dependent increase in hydroxyl radical formation. The interaction of dopamine with the glutamatergic system [16,24] or dopamine-dependent uncoupling of cerebral blood flow and glucose metabolism [14], could also increase cellular stress and affect expression of hsp72.

The possible mechanisms by which the other monoamine neurotransmitter, norepinephrine, may affect expression of hsp72 have not been well established. It has been shown that the extracellular level of norepinephrine is increased during hypoxic/ischemic conditions in a manner similar to dopamine [15,40]. However, it is not yet clear whether this has a protective or deleterious effect on brain recovery (see Section 1). It has been suggested thal the interaction between glutamatergic and noradrenergic systems could be important during these pathological conditions [26,32,48,57]. In ischemia models, depletion of norepinephrine, peripherial and cerebral, by AMT bas been reported to reduce the probability of death of animais by decreasing the sympathetic hypertensive response to vascular occlusion [ 10,25,27]. In conclusion, the data indicate that dopamine and/or norepinephrine play a role in hypoxia induced increase in expression of hsp72 mRNA following hypoxia. The molecular mechanisms which couple dopamine and n o f epinephrine to hypoxia induced increase in expression of hsp72 mRNA bave yet to be fully elucidated.

Acknowledgements The authors wish to thank Dr. Ellen G. Shaver and Ms. Valerie Harris for their expert assistance. This work was supported by NIH grants NS-31465 and NS-29331.

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