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EEG changes following increased blood-brain barrier permeability under long-term immobilization stress in young rats
224
Neuroscience Research, 5 (1988) 224-239 Elsevier Scientific Publishers Ireland Ltd.
NSR 00213
EEG changes following increased blood-brain barrier permeability under long-term immobilization stress in young rats H a d Shanker Sharma and Prasanta Kumar Dey Neurophysiology Research Unit, Department of Physiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi (India) (Received 23 February 1987; Revised version received 15 June 1987; Accepted 4 September 1987)
Key words:Blood-brain barrier; Immobilization stress; Electroencephalogram; Cerebral blood flow; 5-Hydroxytryptamine; 13q-Sodium; p-Chlorophenylalanine
SUMMARY A continuous 8 h of immobilization stress in conscious young rats increased the blood-brain barrier (BBB) permeability to t31I-sodium in 12 out of 14 brain regions studied. A flattening of electroencephalographic (EEG) activity was noted during this time period. The mean cerebral blood flow (CBF) was reduced by 17~o (during this time period) but the regional flow reduction was not related to the regional increase in BBB permeability. On the other hand, a correlation was observed between increased plasma and brain 5-HT levels and increased BBB permeability, p-Chloro-phenylalanine (p-CPA) pretreatment 12 has prevented the occurrence of increased BBB permeability, and the flattening of EEG activity as well as 5-HT levels in plasma and brain. These results suggest that the long-term immobilization stress induces causally related sequential events in rats: enhancement of circulating 5-HT, impairment of BBB, free access of 5-HT into the brain, and eventually flattening of EEG.
INTRODUCTION
The electrical activity of the brain is an important indicator of brain function which depends on the states of cerebral blood flow (CBF) and metabolism 5"29. Under various 4 stress-induced neurological diseases, altered EEG activity has been reported 9"1°. Recently, an increased blood-brain 6 barrier (BBB) permeability under certain stressful situations has been demonstrated, the probable mechanisms of which are not known 1. Since the BBB plays an important role in the homeostasis of the central nervous Correspondence: H.S. Sharma, Department of Physiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India. 0168-0102/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd.
225 system 3, it is likely that increased BBB permeability under stressful conditions may be instrumental in the impairment of brain functions and may precipitate neurological ailments. Thus, an altered EEG activity is expected at the time of increased BBB permeability4. Therefore, in the present study, the influence of immobilization stress on changes in EEG activity, BBB permeability and CBF was investigated in conscious normotensive rats, with particular interest being paid to one neurotransmitter, 5-HT, which is known to be involved in stress reaction 34 EEG activity24, BBB permeability34,39 and CBF 26.
MATERIALS AND METHODS Animals Experiments were carded out on inbred Charles-Foster rats of either sex (age 8-9weeks, body weight 60-80g) 34 kept at a controlled ambient temperature (22 + i °C) on a 12 h light/12 h dark cycle. Rat feed (Hindustan Lever, Bangalore, India) and tap water were supplied ad libitum. Immobilization stress All the stress experiments were commenced between 08.30 and 09.00 h. Animals were continuously immobilized in a prone position on a wooden board. The limbs were mildly extended and fixed on the board with adhesive tape. The body was wrapped with surgical gauze (6 inches wide) to minimize body movement. The development of stress symptoms in each animal was recorded in terms of the changes in body temperature using a telethermometer (Aplab Electronics, India), the number of fecal pellets excreted and the occurrence of hemorrhagic spots found in the mucosal wall of the stomach at post-mortem examination 33,34.
EEG recording Cortical EEGs in male rats were recorded from transverse bipolar stainless steel screw electrodes (length 4 mm, tip o.d. 1 mm) chronically implanted on the parietal cortex and on the middle vermis part of the cerebellum 5 (Fig. 1). Under Nembutal anesthesia (35 mg/kg, i.p.), the skull was exposed and 4 burr holes were made under stereotaxic guidance. One pair of screws was secured on the anterior two burr holes made on the parietal cortex (3 mm posterior to the bregma and 2 mm lateral to the midline), while the other pair of screws was placed on the two posterior holes made on the cerebellum (3 mm posterior to the lambda and 2 mm bilateral to the longitudinal axis). These screws were firmly secured in their respective positions on the skull by acrylic dental cement (British Dental Co., U.K.) which also acted as insulating material between the electrodes. In making the burr holes and placing the screws on the skull, great care was always taken not to damage the dura. The other end of the electrode,
226 soldered to a 4 cm cable (connected to a female adapter), was exteriorized from the rear and tied with skin. The open end of the socket was sealed with leucoplast. At the time of recording (6-8 days after implantation of electrodes), the female adapter was connected with its male counterpart (BSM Electronics, India) which led to the EEG junction box (Kaiser 16-Channel EEG Machine). The amplifier and machine specifications (time constant 0.3 s, filter position open, paper speed 7.5 mm/s, sensitivity 100/~V/cm) were kept constant in all the experiments zS. The electrode resistance (ER) between paired cerebellar and parietal cortical electrodes ranged between 4-5 and 6-8 kfl, respectively. The EEG records were analysed visually5. To study the basal changes in EEG 13, 4 separate groups of animals were immobilized for 5-10 min at 1 h intervals throughout the day. The EEG waves of these animals did not show much alteration throughout the experiment.
BBB permeability BBB permeability was measured using ~31I-sodium (100 #Ci/kg), as described previously34. In brief, the tracer was injected 5 min before termination of stress through the right femoral vein via a polyethylene catheter chronically implanted 2 days before the experiment. Five minutes after tracer injection, the brains were removed (under urethane anesthesia 0.8 g/kg, i.p.) after perfusion with 0.9% NaCI solution through heart 35. The brains were sectioned in 14 regions (Table III) and the radioactivity determined in a gamma-counter (energy window 500-800 keV). The radioactivity of the brain was expressed in percentage of the activity in blood, i.e. counts per min/mg brain over counts per min/mg blood x 100. The blood samples were taken at the end of the experiment 15. A visual examination of the BBB permeability using Evans blue albumin (EBA) was also done in the EEG experiments to visualize the territory of increased BBB permeability in relation to electrode placement 34.
Regional cerebral bloodflow (rCBF) The rCBF was measured only once in each animal at the end of a continuously applied stress using a tracer microsphere (15 + 0.6 #M in diameter) labelled with 1251 suspended in 0.5 ~ Tween 80 in 0.7 ~ NaCI solution, as described previously in detail 35. In brief, 100 #1 of this suspension (72 000-85 000 microspheres, approximately) was injected over 20-30 s into the left cardiac ventricle through a polyethylene cannula. A reference sample from the right femoral artery was withdrawn at the rate of 1.211 ml/min, starting from 30 s before infusion and continuing up to 90 s after infusion. Animals were decapitated after 90 s of infusion. The skull was opened, and the brain was removed and placed on cold filter paper wetted with 0.9% NaCI. Large subarachnoid and dural vessels were removed and discarded. The brain was hemisectioned at the midline. Each half-brain was dissected into similar 14 regions (Table 2) which were placed in tared plastic tubes that were reweighed immediately and counted in a 3-in-well-type gamma-counter (energy window 25-50 keV). Blood samples were divided into aliquots to provide counting geometry similar to that for tissue
227 samples. The CBF (ml/g/min) was calculated from the equation~4: CBF=Ca xRBF-CR where C a = counts per min/g brain; RBF = reference blood flow (rate of withdrawal of blood samples from reference artery); and CR = total counts in the reference sample. Immediately before the rCBF was measured in either the control or experimental animals, the carotid artery catheter was connected to a strain gauge (Statham P 23) which led to a chart recorder so as to record the mean arterial blood pressure (MABP). In addition, a sample of arterial blood was withdrawn for later determination OfPaO2, p~CO2 and pH. 5-HT estimation 5-HT in both the plasma (including platelets) and whole brain 34 were measured fluorometrically according to Snyder et al.36 Control group In the EEG study, each animal served as its own control. The EEG activity at the onset of stress was compared with that of each subsequent hour of stress in the same animal, while in all other groups, the unstressed animals served as controls. p-CPA-treated group p-CPA (100 mg/kg i.p.) was administered daily for 3 consecutive days 11. On the fourth day, animals were subjected to an 8 h period of immobilization only. Statistical evaluation The paired Student's t-test for the EEG group and the unpaired t-test for all the other groups were applied to evaluate the statistical significance of the data obtained.
RESULTS
Effect of immobilization stress on stress response and physiological variables The results are shown in Table I. The stress symptoms were very prominent at the end of the 8 h period of immobilization (Table I). The MABP of these animals showed a significant fall from the control value. ThepaO 2 showed a moderate increase, whereas thepaCO 2 and arterial pH were slightly decreased (Table I). On the other hand, the animals subjected to either a 1 h or 4 h period of immobilization exhibited significantly fewer stress symptoms. The MABP was significantly increased from the control value at the end of the 4 h period of immobilization. The physiological variables were not significantly different from the control values at the end of either the 1 h or the 4 h period of stress.
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IMMOBILIZATION .STRESS Fig. 1. Continuous bipolar cortical EEG records from partial cortex (upper trace) and cerebellar cortex (lower trace) of an 80 g male immobilized rat (age 9 weeks). Flattening of the EEG appears at the end of the 7-8 h period following immobilization. Extravasation of Evans blue albumin (shaded area) is evident in the territory of both electrode pairs at the end of the 8 h period of immobilization.
Effect of immobilization stress on EEG activity The bipolar EEG waves of conscious, restrained rats from the start of immobilization to 1 h after were predominantly high voltage, slow activity (HVSA, 60-70 #V, 6-7 Hz) (Fig. 1). The amplitude was further increased at the end of a 4 h period o f stress (70-90 #V, 5-6 Hz). This HVSA was more or less continued up to 5.5 h. After 6 h, attenuation of amplitude occurred, and at the end of 7 h, low voltage, fast activity (LVFA) appeared which reached its peak level at the end of the 8 h period following immobilization. This LVFA (10-15 #V, 9-11 Hz) persisted up to 8.5 h following stress. Examination of BBB permeability at the end of the 8 h period of immobilization revealed extravasation of the EBA dye encompassing the territory of both electrode pairs (Fig. 1). When the stress was further continued (in 3 other animals) beyond 8.5 h, the LVFA gradually disappeared and HVSA again ensued (Fig. 2). Thus, at the end of the 9 h period following stress, a gradual increase in amplitude (20-50/~V, 8-10 Hz) was observed. Almost 80 % reversal of HVSA (as compared to that of a 1 h period of stress) took place at the end of the 1 h period following stress (Fig. 2). These results indicate
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JMMOBILIZATION STREfS Fig. 2. Continuous bipolar cortical EEG records from parietal cortex (PC, upper trace) and cerebellar cortex (CC, lower trace) of a 78 g male immobilized rat. Flattening of EEG appears at the end of 7-8 h period following immobilization, and tends to disappear gradually (from 9 h onwards). Almost 80% reversal is evident at the end of the 11 h period of immobilization.
that the EEG changes observed under acute long-term immobilization stress are reversible in nature. Post-mortem examination at the end of the 11 h period following stress did not show any blue coloration of the brain except at non-barrier regions. The MABP showed a gradual return from a hypotensive phase towards basal value at the end of an 11 h period of immobilization (Table I). The physiological variables tend to restore to their normal values during this time period. The hypothermic response was remarkably attenuated (Table I). Effect of immobilization stress on BBB permeability and CBF A marked increase in BBB permeability to 131I-sodium in 12 out of 14 brain regions was observed at the end of the 8 h period following immobilization (Table II). The regional CBF showed an overall decline in the same 12 regions, but the fall in rCBF was not related to the increase in regional BBB permeability (Table II). On the other hand, the total or regional BBB permeability and CBF were not affected at the end of either the 1 h, 4 h or 11 h periods following immobilization (Table III).
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Effect of immobilization stress on 5-HT level A profound increase in plasma and brain 5-HT levels was observed in animals subjected to an 8 h period of immobilization (Table III). On the other hand, 5-HT levels at the end of the 1 h, 4 h or 11 h periods of immobilization were not altered significantly, as compared to the control values (Table III). This indicates a good correlation between the increased BBB permeability and the 5-HT level. Influence of p-CPA (a 5-HT synthesis inhibitor)pretreatment The influence ofp-CPA was examined in animals subjected to 8 h of immobilization only on the above-mentioned parameters (Tables IV and V) because this experimental TABLE IV E F F E C T O F p-CPA P R E T R E A T M E N T O N STRESS S Y M P T O M S , P H Y S I O L O G I C A L P A R A M E T E R S , E E G ACTIVITY, BBB PERMEABILITY, CBF A N D 5-HT LEVEL IN 8 h - I M M O B I L I Z E D RATS Values are expressed as mean + S.D. Parameters
(.4) Stress symptoms A °C rectal temp. No. of hemorrhagic spots No. of fecal pellets excreted
(B) Physiologicalparameters M A B P torr Arterial pH paCO2 (torr) paO2 (torr)
(C) EEG activity Parietal cortex (L-R) Amplitude (#V) Frequency (Hz) Cerebellum (L-R) Amplitude (#V) Frequency (Hz)
(I9) BBB permeability 131I-sodium (%)
(E) Cerebral bloodflow (ml/g/min)
(F) 5-HT level Plasma (#g/ml) Brain (#g/g)
Immobilization 8 h Control
p-CPA-treated
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(n = 16) 70 _+ 2 7.30 + 0.021 30.24 + 0.71 84.36 + 1.23
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(n = 8) 2.38 + 0.32 1.64 + 0.57
(n = 6) 0.26 _+ 0.12"** 0.34 + 0.04***
* P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student's t-test).
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condition affected the EEG activity, BBB permeability, CBF and 5-HT levels in the plasma and the brain. The results of p-CPA pretreatment on EEG activity show (Fig. 3) that the drug remarkably affects the EEG pattern of animals. Thus, high voltage, fast activity (HVFA) EEG were observed in both the parietal cortex and the cerebellum. On careful analysis, the trend of the EEG voltage showed a gradual increase with an increase in the duration of stress without much affecting the frequency. Thus, from the onset to the 1 h period following stress, there occurred high voltage, fast activity HVFA (50-60 #V, 11-12 Hz) which further increased to 80-90 #V, 10-11 Hz at the end of the 4-6 h periods of stress. This HVFA more or less continued up to the 7-8 h periods following stress. At the end of the 9-11 h periods of immobilization, the amplitude was further increased to 100-110 #V, though the frequency remained between 9 and 11 Hz. Examination of the BBB permeability either at the end of the 8 h or 11 h periods following immobilization stress did not reveal extravasation of 13l i_sodium in any brain region (Table V). This points to a good relationship between the flattening of EEG
236 activity and the increased BBB permeability. No significant reduction in the total or regional CBF was noted in p-CPA-treated animals as compared to the unstressed control group (Table V), The 5-HT levels in plasma and brain did not increase from the unstressed control group (Table IV). The stress symptoms were remarkably reduced, whereas the physiological variables were not affected in p-CPA-treated stressed animals (Table IV). DISCUSSION
The present results show a remarkable flattening of the EEG activity at the time of increased BBB permeability, which suggests that neuronal activity may be affected by impaired BBB function. Flat EEGs are desynchronized recordings which represent an activation pattern. This desynchronization may be due to neuronal hyperexcitability27. Since such desynchronization of the EEG appeared at the end of the 8 h period following immobilization, which resulted in the development of profound stress symptoms, it is likely that the state of tension and anxiety may also have contributed to such EEG activation 23"4°. This flattening of the EEGs does not appear to be due to circadian variation or induction of sleep in animals. This has been evident from the EEG recording in animals immobilized for 5-10 rain every hour at different time periods of the day which did not exhibit any flattening. However, such flattening of the EEGs gradually disappeared at the end of the 11 h period following stress. This indicates that changes in the EEG activity following long-term immobilization stress are reversible in nature. Since the increased permeability of the BBB was also not observed at the end of the 11 h period following stress, it is plausible that the flattening of the EEG activity may be related to the increased BBB permeability. A correlation between the EEG and the drug-induced alteration of the BBB phenomena has also been reported previously ~2"31. The present results provide experimental evidence that long-term immobilization stress can increase the permeability of BBB which in turn may be instrumental in altering the EEG activity. Since the circulating 5-HT level is very high at the time of the increased BBB permeability, it is probable that 5-HT which normally does not cross the intact barrier 3°, might have gained free access via blood to the brain due to defective barrier functions, resulting in a flattening of the EEG activity24. 5-HT is known to induce EEG desynchronization in animals when applied either through an internal carotid a r t e r y 32 or on the area postrema 2°-22. This 5-HT-induced EEG desynchronization is prevented with prior systemic p-CPA treatment 2~'37. Injection 6 of 5-HTP which can readily penetrate the BBB, resulted in an almost isoelectric EEG tracing due to elevation of brain 5-HT levels'19. Prevention of EEG flattening in the present study with p-CPA pretreatment supports the involvement of 5-HT in inducing EEG desynchronization in 8 h immobilized animals at the time of the increased BBB permeability. The present results further show a good correlation between an increased plasma 5-HT level and an increased BBB permeability. It is imperative that increased 5-HT
237 levels be also responsible for causing dysfunction of BBB functions34'38"39. 5-HT markedly stimulates the cAMP formation in cerebral vessels 7"38which in turn may lead to marked vasodilation associated with increased vesicular transport ~6-1s. Thus, it seems plausible that increased 5-HT in plasma may be responsible for such increases in BBB permeability. This hypothesis gets further support from the results obtained with p-CPA pretreatment. Thus, in p-CPA-pretreated animals, 8 h of immobilizationTM did not result in an increase in 5-HT levels because the 5-HT synthesis had already been reduced and its stores depleted ~~; as a result, no increase in the BBB 2° permeability was observed 34. This observation strengthens the role of 5-HT in inducing BBB dysfunction following 8 h of immobilization. This increased BBB permeability appears to be unrelated with the slowly developing ischemia due to the reduction in CBF, because the reduction in rCBF was not correlated with regional increases in BBB permeability (Table 2). The reduction in rCBF may be due to several other factors such as an increased paO2, a decreased paCO 2 and pH 29, occurrence of hypotension close to the lower limit of autoregulation of CBF 2 and/or increased levels of circulating 5-HT 26, but not due to technical errors in the measurement of rCBF 35 because the values of rCBF in the present study closely correspond to the values of rCBF obtained by Ohata et al.28 using a [ ~4C]iodoantipyrine technique. This BBB permeability is also not related to the hypotension observed at the time of increased BBB permeability, because such hypotension was also observed in p°CPA pretreated animals in which increased permeability was not shown (Table IV). In summary, it appears that the long-term immobilization stress has elicited a profound increase in circulating 5-HT levels, the probable mechanism(s) of which are not clear at present. This increased 5-HT level has impaired the BBB function leading to the free access of 5-HT into the brain through the blood. When 5-HT enters into the brain, it causes a flattening of the EEG activity. Since p-CPA pretreatment did not allow 5-HT to increase in circulation following 8 h of immobilization, no increase in BBB permeability or flattening of EEG activity was observed.
ACKNOWLEDGEMENTS
The authors are grateful to the University Grants Commission, New Delhi for financial assistance; Sri R.C. Gupta and ARab Ahmed for technical assistance in EEG recordings; the Departments of Radiotherapy and Radiation Medicine, Institute of Medical Sciences, Pharmacology, I.M.S. and Medicine, I.M.S. for extending laboratory facilities and Aruna Misra and Ram Bhajan for secretarial assistance and typing.
238 REFERENCES 1 Angel, C., Starvation, stress and the blood-brain barrier, Dis. Nerv. Syst., 30: (1969) 94-97. 2 Berne, R.M., Winn, H.R. and Rubio, R., The local regulation of cerebral blood flow, Progr. Cardiovasc. Dis., 24 (1981) 243-260. 3 Bradbury, M.W.B., The structure and function of the blood-brain barrier, Fed. Proc. Fed. Am. Soc. Exp. BioL, 43 (1984) 186-190. 4 Chatrian, G.E., The normal EEG throughout life. The low voltage EEG. Handbook Electroencephalogr. Clin. Neurophysiol. Vol. 6, Part A, 1976, p. 6A77-6A88. 5 Cooper, R., Osselton, J.W. and Shaw, J.C., EEG Technology, Butterworths, London, 1969, p. 186-230. 6 Cooper, R., Influence on the EEG of certain physiological states and other parameters. Handbook Electroencephalogr. Clin. Neurophysiol., Vol. 7, 1974, pp. B28-B56. 7 Crook, R.B., Farber, N.B. and Prusiner, S.B., Hormones and neurotransmitters control cyclic AMP metabolism in choroid plexus epithelial cells, J. Neurochem., 42 (1984) 340-350. 8 Dongier, M., EEG and various psychiatric diseases. Handbook Electroencephalogr. Clin. Neurophysiol. Vol. 13, Part B, 1974, p. 13B22-13B59. 9 Doyle, C., Shimizu, A. and Himwich, A.E., Effects of chronic administration of some psychoactive drugs on EEG arousal on rabbit, Inj. J. Neuropharmacol., 7 (1968) 78-95. 10 Elingson, R.J., The incidence of EEG abnormality among patients with mental disorders of apparently non-organic origin: a critical review, Am. J. Psychiat., 111 (1954) 263-275. 11 Essman, W.B., Serotonin in Health and Disease, VoL 1, Availability, Localization and Disposal of Serotonin, Plenum Press, New York, 1978, pp. 259-263. 12 Flodmark, S., Hamberger, A., Hamberger, B. and Steinwall, O., Concurrent registration of EEG responses, catecholamine uptake, and Trypan blue staining in chemical blood-brain barrier damage, Acta Neuropathol., 12 (1969) 12-22. 13 Harding, G.F.A. and Thompson, C.R.S., EEG rhythms and the internal milieu. Handbook Electroencephalogr. Clin. Neurophysiol. Vol. 6, Part A, 1976, pp. 6A176-6A194. 14 Heistad, D.D., Marcus, M.L. and Gross, P.M., Effects of sympathetic nerves on cerebral vessels in dog, cat and monkey, Am. J. Physiol., 235 (1978) H544-H552. 15 Johansson, B., Indomethacin and cerebrovascular permeability to albumin in acute hypertension and cerebral embolism in the rat, Exp. Brain Res., 42 (1981) 331-336. 16 Joo, F., Effect of N602-dibutyryl cyclic 3'-5'-adenosine monophosphate on the pinocytosis of brain capillaries of mice, Experientia, 28 (1972) 1470-1471. 17 Joo, F., Rakonczay, Z. and Wollmann, M., cAMP mediated regulation of the permeability in the brain capillaries, Experientia, 31 (1975) 582-584. 18 Joo, F., The blood-brain barrier in vitro - ten years of research on microvessels isolated from the brain, Neurochem. Int., 7 (1985) 1-25. 19 Jouvet, M., Biogenic amines and the states of sleep, Science, 163 (1969) 32-41. 20 Koella, W.P. and Czicman, J.S., Influence of serotonin upon optic evoked potentials, EEG and blood pressure of cat, Am. J. Physiol., 204 (1963) 873-880. 21 Koella, W.P. and Czicman, J., Mechanism of the EEG synchronizing action of serotonin, Am. J. Physiol., 211 (1966) 926-934. 22 Koella, W.P., Feldstein, A. and Czicman, J., The effect of para-chlorophenylalanine on the sleep of cats, Electroencephalogr. Clin. Neurophysiol., 25 (1968) 481-490. 23 Lacey, J.I., Somatic response patterning and stress: Some revisions of activation theory. In M.H. Appley and R. TrumbuU (Eds.), Psychological Stress, Appleton-Century-Crofts, New York, 1967, pp. 14-76. 24 Longo, V.D., 5-hydroxytryptamine and the EEG. Handbook Electroencephalogr. Clin. Neurophysiology. Vol. 7, Part C, 1977, 7C33-7C39. 25 MacGillivray, B.B., Traditional methods of examination in clinical EEG. Handbook Electroencephalogr. Clin. Neurophysiol. Vol. 3, Part C, 1974, pp. 3C82-3C85.
239 26 MacKenzie, E.T., Stewart, M., Young, A.R. and Harper, A.M., Cerebrovascular actions of serotonin, Adv. Neurol., 20 (1978) 77-84. 27 Moruzzi, G. and Magoun, H.W., Brain stem reticular formation and activation of the EEG. Electroencephalogr. Clin. Neurophysiol. 1 (1949) 455-473. 28 Ohata, M., Fredericks, W.R., Sundaram, U. and Rapoport, S.I., Effects of immobilization stress on regional cerebral blood flow in the conscious rat, J. Cerebr. Blood Flow Metab., 1 (1981) 187-194. 29 Paulson, O.B. and Sharbrough, F.W., Physiologic and pathophysiologic relationship between the EEG and the regional cerebral blood flow, Acta Neurol. Scand., 50 (1974) 194-220. 30 Rapoport, S.I., Blood-Brain Barrier in Physiology and Medicine, Raven Press, New York, "1976, pp. 270-273. 31 Remler, M.P. and Marcussen, W.H., EEG monitoring of focal lesions of the blood-brain barrier, Acta Neurol. Scand., 65 (1982) 51-58. 32 Rothballer, A.B., The effect of phenylephrine, methamphetamine, cocaine, and serotonin upon the adrenaline-sensitive component of the reticular activating system, Electroencephalogr. Clin. Neurophysiol., 9 (1957) 409-417. 33 Selye, H., Stress in Health and Disease, Butterworths, London, 1976, pp. 16-21. 34 Sharma, H.S. and Dey, P.K., Probable involvement of 5-hydroxy-tryptamine in increased permeability of blood-brain barrier under heat stress in young rats, Neuropharmacology, 25 (1986) 161-167. 35 Sharma, H.S., Effect of captopril (a convening enzyme inhibitor) on blood-brain barrier permeability and cerebral blood flow in normotensive rats, Neuropharmacology, 26 (1987) 85-92. 36 Snyder, S.H., Axelrod, J. and Zweig, M., A sensitive and specific fluorescence assay for tissue serotonin, Biochem. Pharmacol., 14 (1965) 831-835. 37 Torda, C., Effect of brain serotonin depletion on sleep in rats. Brain Res., 6 (1967) 375-377. 38 Westergaard, E., The blood-brain barrier to horseradish peroxidase under normal and experimental conditions, Acta NeuropathoL, 39 (1977) 181-187. 39 Westergaard, E., Ultrastructurall permeability properties of cerebral microvasculature under normal and experimental conditions after application of tracers, Adv. Neurol., 28 (1980) 55-74. 40 Zubek, J.P. and Wilgosh, L., Prolonged immobilization of the body: Changes in performance and the EEG, Science, 140 (1963) 306-308.
Neuroscience Research, 5 (1988) 224-239 Elsevier Scientific Publishers Ireland Ltd.
NSR 00213
EEG changes following increased blood-brain barrier permeability under long-term immobilization stress in young rats H a d Shanker Sharma and Prasanta Kumar Dey Neurophysiology Research Unit, Department of Physiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi (India) (Received 23 February 1987; Revised version received 15 June 1987; Accepted 4 September 1987)
Key words:Blood-brain barrier; Immobilization stress; Electroencephalogram; Cerebral blood flow; 5-Hydroxytryptamine; 13q-Sodium; p-Chlorophenylalanine
SUMMARY A continuous 8 h of immobilization stress in conscious young rats increased the blood-brain barrier (BBB) permeability to t31I-sodium in 12 out of 14 brain regions studied. A flattening of electroencephalographic (EEG) activity was noted during this time period. The mean cerebral blood flow (CBF) was reduced by 17~o (during this time period) but the regional flow reduction was not related to the regional increase in BBB permeability. On the other hand, a correlation was observed between increased plasma and brain 5-HT levels and increased BBB permeability, p-Chloro-phenylalanine (p-CPA) pretreatment 12 has prevented the occurrence of increased BBB permeability, and the flattening of EEG activity as well as 5-HT levels in plasma and brain. These results suggest that the long-term immobilization stress induces causally related sequential events in rats: enhancement of circulating 5-HT, impairment of BBB, free access of 5-HT into the brain, and eventually flattening of EEG.
INTRODUCTION
The electrical activity of the brain is an important indicator of brain function which depends on the states of cerebral blood flow (CBF) and metabolism 5"29. Under various 4 stress-induced neurological diseases, altered EEG activity has been reported 9"1°. Recently, an increased blood-brain 6 barrier (BBB) permeability under certain stressful situations has been demonstrated, the probable mechanisms of which are not known 1. Since the BBB plays an important role in the homeostasis of the central nervous Correspondence: H.S. Sharma, Department of Physiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India. 0168-0102/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd.
225 system 3, it is likely that increased BBB permeability under stressful conditions may be instrumental in the impairment of brain functions and may precipitate neurological ailments. Thus, an altered EEG activity is expected at the time of increased BBB permeability4. Therefore, in the present study, the influence of immobilization stress on changes in EEG activity, BBB permeability and CBF was investigated in conscious normotensive rats, with particular interest being paid to one neurotransmitter, 5-HT, which is known to be involved in stress reaction 34 EEG activity24, BBB permeability34,39 and CBF 26.
MATERIALS AND METHODS Animals Experiments were carded out on inbred Charles-Foster rats of either sex (age 8-9weeks, body weight 60-80g) 34 kept at a controlled ambient temperature (22 + i °C) on a 12 h light/12 h dark cycle. Rat feed (Hindustan Lever, Bangalore, India) and tap water were supplied ad libitum. Immobilization stress All the stress experiments were commenced between 08.30 and 09.00 h. Animals were continuously immobilized in a prone position on a wooden board. The limbs were mildly extended and fixed on the board with adhesive tape. The body was wrapped with surgical gauze (6 inches wide) to minimize body movement. The development of stress symptoms in each animal was recorded in terms of the changes in body temperature using a telethermometer (Aplab Electronics, India), the number of fecal pellets excreted and the occurrence of hemorrhagic spots found in the mucosal wall of the stomach at post-mortem examination 33,34.
EEG recording Cortical EEGs in male rats were recorded from transverse bipolar stainless steel screw electrodes (length 4 mm, tip o.d. 1 mm) chronically implanted on the parietal cortex and on the middle vermis part of the cerebellum 5 (Fig. 1). Under Nembutal anesthesia (35 mg/kg, i.p.), the skull was exposed and 4 burr holes were made under stereotaxic guidance. One pair of screws was secured on the anterior two burr holes made on the parietal cortex (3 mm posterior to the bregma and 2 mm lateral to the midline), while the other pair of screws was placed on the two posterior holes made on the cerebellum (3 mm posterior to the lambda and 2 mm bilateral to the longitudinal axis). These screws were firmly secured in their respective positions on the skull by acrylic dental cement (British Dental Co., U.K.) which also acted as insulating material between the electrodes. In making the burr holes and placing the screws on the skull, great care was always taken not to damage the dura. The other end of the electrode,
226 soldered to a 4 cm cable (connected to a female adapter), was exteriorized from the rear and tied with skin. The open end of the socket was sealed with leucoplast. At the time of recording (6-8 days after implantation of electrodes), the female adapter was connected with its male counterpart (BSM Electronics, India) which led to the EEG junction box (Kaiser 16-Channel EEG Machine). The amplifier and machine specifications (time constant 0.3 s, filter position open, paper speed 7.5 mm/s, sensitivity 100/~V/cm) were kept constant in all the experiments zS. The electrode resistance (ER) between paired cerebellar and parietal cortical electrodes ranged between 4-5 and 6-8 kfl, respectively. The EEG records were analysed visually5. To study the basal changes in EEG 13, 4 separate groups of animals were immobilized for 5-10 min at 1 h intervals throughout the day. The EEG waves of these animals did not show much alteration throughout the experiment.
BBB permeability BBB permeability was measured using ~31I-sodium (100 #Ci/kg), as described previously34. In brief, the tracer was injected 5 min before termination of stress through the right femoral vein via a polyethylene catheter chronically implanted 2 days before the experiment. Five minutes after tracer injection, the brains were removed (under urethane anesthesia 0.8 g/kg, i.p.) after perfusion with 0.9% NaCI solution through heart 35. The brains were sectioned in 14 regions (Table III) and the radioactivity determined in a gamma-counter (energy window 500-800 keV). The radioactivity of the brain was expressed in percentage of the activity in blood, i.e. counts per min/mg brain over counts per min/mg blood x 100. The blood samples were taken at the end of the experiment 15. A visual examination of the BBB permeability using Evans blue albumin (EBA) was also done in the EEG experiments to visualize the territory of increased BBB permeability in relation to electrode placement 34.
Regional cerebral bloodflow (rCBF) The rCBF was measured only once in each animal at the end of a continuously applied stress using a tracer microsphere (15 + 0.6 #M in diameter) labelled with 1251 suspended in 0.5 ~ Tween 80 in 0.7 ~ NaCI solution, as described previously in detail 35. In brief, 100 #1 of this suspension (72 000-85 000 microspheres, approximately) was injected over 20-30 s into the left cardiac ventricle through a polyethylene cannula. A reference sample from the right femoral artery was withdrawn at the rate of 1.211 ml/min, starting from 30 s before infusion and continuing up to 90 s after infusion. Animals were decapitated after 90 s of infusion. The skull was opened, and the brain was removed and placed on cold filter paper wetted with 0.9% NaCI. Large subarachnoid and dural vessels were removed and discarded. The brain was hemisectioned at the midline. Each half-brain was dissected into similar 14 regions (Table 2) which were placed in tared plastic tubes that were reweighed immediately and counted in a 3-in-well-type gamma-counter (energy window 25-50 keV). Blood samples were divided into aliquots to provide counting geometry similar to that for tissue
227 samples. The CBF (ml/g/min) was calculated from the equation~4: CBF=Ca xRBF-CR where C a = counts per min/g brain; RBF = reference blood flow (rate of withdrawal of blood samples from reference artery); and CR = total counts in the reference sample. Immediately before the rCBF was measured in either the control or experimental animals, the carotid artery catheter was connected to a strain gauge (Statham P 23) which led to a chart recorder so as to record the mean arterial blood pressure (MABP). In addition, a sample of arterial blood was withdrawn for later determination OfPaO2, p~CO2 and pH. 5-HT estimation 5-HT in both the plasma (including platelets) and whole brain 34 were measured fluorometrically according to Snyder et al.36 Control group In the EEG study, each animal served as its own control. The EEG activity at the onset of stress was compared with that of each subsequent hour of stress in the same animal, while in all other groups, the unstressed animals served as controls. p-CPA-treated group p-CPA (100 mg/kg i.p.) was administered daily for 3 consecutive days 11. On the fourth day, animals were subjected to an 8 h period of immobilization only. Statistical evaluation The paired Student's t-test for the EEG group and the unpaired t-test for all the other groups were applied to evaluate the statistical significance of the data obtained.
RESULTS
Effect of immobilization stress on stress response and physiological variables The results are shown in Table I. The stress symptoms were very prominent at the end of the 8 h period of immobilization (Table I). The MABP of these animals showed a significant fall from the control value. ThepaO 2 showed a moderate increase, whereas thepaCO 2 and arterial pH were slightly decreased (Table I). On the other hand, the animals subjected to either a 1 h or 4 h period of immobilization exhibited significantly fewer stress symptoms. The MABP was significantly increased from the control value at the end of the 4 h period of immobilization. The physiological variables were not significantly different from the control values at the end of either the 1 h or the 4 h period of stress.
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Effect of immobilization stress on EEG activity The bipolar EEG waves of conscious, restrained rats from the start of immobilization to 1 h after were predominantly high voltage, slow activity (HVSA, 60-70 #V, 6-7 Hz) (Fig. 1). The amplitude was further increased at the end of a 4 h period o f stress (70-90 #V, 5-6 Hz). This HVSA was more or less continued up to 5.5 h. After 6 h, attenuation of amplitude occurred, and at the end of 7 h, low voltage, fast activity (LVFA) appeared which reached its peak level at the end of the 8 h period following immobilization. This LVFA (10-15 #V, 9-11 Hz) persisted up to 8.5 h following stress. Examination of BBB permeability at the end of the 8 h period of immobilization revealed extravasation of the EBA dye encompassing the territory of both electrode pairs (Fig. 1). When the stress was further continued (in 3 other animals) beyond 8.5 h, the LVFA gradually disappeared and HVSA again ensued (Fig. 2). Thus, at the end of the 9 h period following stress, a gradual increase in amplitude (20-50/~V, 8-10 Hz) was observed. Almost 80 % reversal of HVSA (as compared to that of a 1 h period of stress) took place at the end of the 1 h period following stress (Fig. 2). These results indicate
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that the EEG changes observed under acute long-term immobilization stress are reversible in nature. Post-mortem examination at the end of the 11 h period following stress did not show any blue coloration of the brain except at non-barrier regions. The MABP showed a gradual return from a hypotensive phase towards basal value at the end of an 11 h period of immobilization (Table I). The physiological variables tend to restore to their normal values during this time period. The hypothermic response was remarkably attenuated (Table I). Effect of immobilization stress on BBB permeability and CBF A marked increase in BBB permeability to 131I-sodium in 12 out of 14 brain regions was observed at the end of the 8 h period following immobilization (Table II). The regional CBF showed an overall decline in the same 12 regions, but the fall in rCBF was not related to the increase in regional BBB permeability (Table II). On the other hand, the total or regional BBB permeability and CBF were not affected at the end of either the 1 h, 4 h or 11 h periods following immobilization (Table III).
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Effect of immobilization stress on 5-HT level A profound increase in plasma and brain 5-HT levels was observed in animals subjected to an 8 h period of immobilization (Table III). On the other hand, 5-HT levels at the end of the 1 h, 4 h or 11 h periods of immobilization were not altered significantly, as compared to the control values (Table III). This indicates a good correlation between the increased BBB permeability and the 5-HT level. Influence of p-CPA (a 5-HT synthesis inhibitor)pretreatment The influence ofp-CPA was examined in animals subjected to 8 h of immobilization only on the above-mentioned parameters (Tables IV and V) because this experimental TABLE IV E F F E C T O F p-CPA P R E T R E A T M E N T O N STRESS S Y M P T O M S , P H Y S I O L O G I C A L P A R A M E T E R S , E E G ACTIVITY, BBB PERMEABILITY, CBF A N D 5-HT LEVEL IN 8 h - I M M O B I L I Z E D RATS Values are expressed as mean + S.D. Parameters
(.4) Stress symptoms A °C rectal temp. No. of hemorrhagic spots No. of fecal pellets excreted
(B) Physiologicalparameters M A B P torr Arterial pH paCO2 (torr) paO2 (torr)
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(I9) BBB permeability 131I-sodium (%)
(E) Cerebral bloodflow (ml/g/min)
(F) 5-HT level Plasma (#g/ml) Brain (#g/g)
Immobilization 8 h Control
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condition affected the EEG activity, BBB permeability, CBF and 5-HT levels in the plasma and the brain. The results of p-CPA pretreatment on EEG activity show (Fig. 3) that the drug remarkably affects the EEG pattern of animals. Thus, high voltage, fast activity (HVFA) EEG were observed in both the parietal cortex and the cerebellum. On careful analysis, the trend of the EEG voltage showed a gradual increase with an increase in the duration of stress without much affecting the frequency. Thus, from the onset to the 1 h period following stress, there occurred high voltage, fast activity HVFA (50-60 #V, 11-12 Hz) which further increased to 80-90 #V, 10-11 Hz at the end of the 4-6 h periods of stress. This HVFA more or less continued up to the 7-8 h periods following stress. At the end of the 9-11 h periods of immobilization, the amplitude was further increased to 100-110 #V, though the frequency remained between 9 and 11 Hz. Examination of the BBB permeability either at the end of the 8 h or 11 h periods following immobilization stress did not reveal extravasation of 13l i_sodium in any brain region (Table V). This points to a good relationship between the flattening of EEG
236 activity and the increased BBB permeability. No significant reduction in the total or regional CBF was noted in p-CPA-treated animals as compared to the unstressed control group (Table V), The 5-HT levels in plasma and brain did not increase from the unstressed control group (Table IV). The stress symptoms were remarkably reduced, whereas the physiological variables were not affected in p-CPA-treated stressed animals (Table IV). DISCUSSION
The present results show a remarkable flattening of the EEG activity at the time of increased BBB permeability, which suggests that neuronal activity may be affected by impaired BBB function. Flat EEGs are desynchronized recordings which represent an activation pattern. This desynchronization may be due to neuronal hyperexcitability27. Since such desynchronization of the EEG appeared at the end of the 8 h period following immobilization, which resulted in the development of profound stress symptoms, it is likely that the state of tension and anxiety may also have contributed to such EEG activation 23"4°. This flattening of the EEGs does not appear to be due to circadian variation or induction of sleep in animals. This has been evident from the EEG recording in animals immobilized for 5-10 rain every hour at different time periods of the day which did not exhibit any flattening. However, such flattening of the EEGs gradually disappeared at the end of the 11 h period following stress. This indicates that changes in the EEG activity following long-term immobilization stress are reversible in nature. Since the increased permeability of the BBB was also not observed at the end of the 11 h period following stress, it is plausible that the flattening of the EEG activity may be related to the increased BBB permeability. A correlation between the EEG and the drug-induced alteration of the BBB phenomena has also been reported previously ~2"31. The present results provide experimental evidence that long-term immobilization stress can increase the permeability of BBB which in turn may be instrumental in altering the EEG activity. Since the circulating 5-HT level is very high at the time of the increased BBB permeability, it is probable that 5-HT which normally does not cross the intact barrier 3°, might have gained free access via blood to the brain due to defective barrier functions, resulting in a flattening of the EEG activity24. 5-HT is known to induce EEG desynchronization in animals when applied either through an internal carotid a r t e r y 32 or on the area postrema 2°-22. This 5-HT-induced EEG desynchronization is prevented with prior systemic p-CPA treatment 2~'37. Injection 6 of 5-HTP which can readily penetrate the BBB, resulted in an almost isoelectric EEG tracing due to elevation of brain 5-HT levels'19. Prevention of EEG flattening in the present study with p-CPA pretreatment supports the involvement of 5-HT in inducing EEG desynchronization in 8 h immobilized animals at the time of the increased BBB permeability. The present results further show a good correlation between an increased plasma 5-HT level and an increased BBB permeability. It is imperative that increased 5-HT
237 levels be also responsible for causing dysfunction of BBB functions34'38"39. 5-HT markedly stimulates the cAMP formation in cerebral vessels 7"38which in turn may lead to marked vasodilation associated with increased vesicular transport ~6-1s. Thus, it seems plausible that increased 5-HT in plasma may be responsible for such increases in BBB permeability. This hypothesis gets further support from the results obtained with p-CPA pretreatment. Thus, in p-CPA-pretreated animals, 8 h of immobilizationTM did not result in an increase in 5-HT levels because the 5-HT synthesis had already been reduced and its stores depleted ~~; as a result, no increase in the BBB 2° permeability was observed 34. This observation strengthens the role of 5-HT in inducing BBB dysfunction following 8 h of immobilization. This increased BBB permeability appears to be unrelated with the slowly developing ischemia due to the reduction in CBF, because the reduction in rCBF was not correlated with regional increases in BBB permeability (Table 2). The reduction in rCBF may be due to several other factors such as an increased paO2, a decreased paCO 2 and pH 29, occurrence of hypotension close to the lower limit of autoregulation of CBF 2 and/or increased levels of circulating 5-HT 26, but not due to technical errors in the measurement of rCBF 35 because the values of rCBF in the present study closely correspond to the values of rCBF obtained by Ohata et al.28 using a [ ~4C]iodoantipyrine technique. This BBB permeability is also not related to the hypotension observed at the time of increased BBB permeability, because such hypotension was also observed in p°CPA pretreated animals in which increased permeability was not shown (Table IV). In summary, it appears that the long-term immobilization stress has elicited a profound increase in circulating 5-HT levels, the probable mechanism(s) of which are not clear at present. This increased 5-HT level has impaired the BBB function leading to the free access of 5-HT into the brain through the blood. When 5-HT enters into the brain, it causes a flattening of the EEG activity. Since p-CPA pretreatment did not allow 5-HT to increase in circulation following 8 h of immobilization, no increase in BBB permeability or flattening of EEG activity was observed.
ACKNOWLEDGEMENTS
The authors are grateful to the University Grants Commission, New Delhi for financial assistance; Sri R.C. Gupta and ARab Ahmed for technical assistance in EEG recordings; the Departments of Radiotherapy and Radiation Medicine, Institute of Medical Sciences, Pharmacology, I.M.S. and Medicine, I.M.S. for extending laboratory facilities and Aruna Misra and Ram Bhajan for secretarial assistance and typing.
238 REFERENCES 1 Angel, C., Starvation, stress and the blood-brain barrier, Dis. Nerv. Syst., 30: (1969) 94-97. 2 Berne, R.M., Winn, H.R. and Rubio, R., The local regulation of cerebral blood flow, Progr. Cardiovasc. Dis., 24 (1981) 243-260. 3 Bradbury, M.W.B., The structure and function of the blood-brain barrier, Fed. Proc. Fed. Am. Soc. Exp. BioL, 43 (1984) 186-190. 4 Chatrian, G.E., The normal EEG throughout life. The low voltage EEG. Handbook Electroencephalogr. Clin. Neurophysiol. Vol. 6, Part A, 1976, p. 6A77-6A88. 5 Cooper, R., Osselton, J.W. and Shaw, J.C., EEG Technology, Butterworths, London, 1969, p. 186-230. 6 Cooper, R., Influence on the EEG of certain physiological states and other parameters. Handbook Electroencephalogr. Clin. Neurophysiol., Vol. 7, 1974, pp. B28-B56. 7 Crook, R.B., Farber, N.B. and Prusiner, S.B., Hormones and neurotransmitters control cyclic AMP metabolism in choroid plexus epithelial cells, J. Neurochem., 42 (1984) 340-350. 8 Dongier, M., EEG and various psychiatric diseases. Handbook Electroencephalogr. Clin. Neurophysiol. Vol. 13, Part B, 1974, p. 13B22-13B59. 9 Doyle, C., Shimizu, A. and Himwich, A.E., Effects of chronic administration of some psychoactive drugs on EEG arousal on rabbit, Inj. J. Neuropharmacol., 7 (1968) 78-95. 10 Elingson, R.J., The incidence of EEG abnormality among patients with mental disorders of apparently non-organic origin: a critical review, Am. J. Psychiat., 111 (1954) 263-275. 11 Essman, W.B., Serotonin in Health and Disease, VoL 1, Availability, Localization and Disposal of Serotonin, Plenum Press, New York, 1978, pp. 259-263. 12 Flodmark, S., Hamberger, A., Hamberger, B. and Steinwall, O., Concurrent registration of EEG responses, catecholamine uptake, and Trypan blue staining in chemical blood-brain barrier damage, Acta Neuropathol., 12 (1969) 12-22. 13 Harding, G.F.A. and Thompson, C.R.S., EEG rhythms and the internal milieu. Handbook Electroencephalogr. Clin. Neurophysiol. Vol. 6, Part A, 1976, pp. 6A176-6A194. 14 Heistad, D.D., Marcus, M.L. and Gross, P.M., Effects of sympathetic nerves on cerebral vessels in dog, cat and monkey, Am. J. Physiol., 235 (1978) H544-H552. 15 Johansson, B., Indomethacin and cerebrovascular permeability to albumin in acute hypertension and cerebral embolism in the rat, Exp. Brain Res., 42 (1981) 331-336. 16 Joo, F., Effect of N602-dibutyryl cyclic 3'-5'-adenosine monophosphate on the pinocytosis of brain capillaries of mice, Experientia, 28 (1972) 1470-1471. 17 Joo, F., Rakonczay, Z. and Wollmann, M., cAMP mediated regulation of the permeability in the brain capillaries, Experientia, 31 (1975) 582-584. 18 Joo, F., The blood-brain barrier in vitro - ten years of research on microvessels isolated from the brain, Neurochem. Int., 7 (1985) 1-25. 19 Jouvet, M., Biogenic amines and the states of sleep, Science, 163 (1969) 32-41. 20 Koella, W.P. and Czicman, J.S., Influence of serotonin upon optic evoked potentials, EEG and blood pressure of cat, Am. J. Physiol., 204 (1963) 873-880. 21 Koella, W.P. and Czicman, J., Mechanism of the EEG synchronizing action of serotonin, Am. J. Physiol., 211 (1966) 926-934. 22 Koella, W.P., Feldstein, A. and Czicman, J., The effect of para-chlorophenylalanine on the sleep of cats, Electroencephalogr. Clin. Neurophysiol., 25 (1968) 481-490. 23 Lacey, J.I., Somatic response patterning and stress: Some revisions of activation theory. In M.H. Appley and R. TrumbuU (Eds.), Psychological Stress, Appleton-Century-Crofts, New York, 1967, pp. 14-76. 24 Longo, V.D., 5-hydroxytryptamine and the EEG. Handbook Electroencephalogr. Clin. Neurophysiology. Vol. 7, Part C, 1977, 7C33-7C39. 25 MacGillivray, B.B., Traditional methods of examination in clinical EEG. Handbook Electroencephalogr. Clin. Neurophysiol. Vol. 3, Part C, 1974, pp. 3C82-3C85.
239 26 MacKenzie, E.T., Stewart, M., Young, A.R. and Harper, A.M., Cerebrovascular actions of serotonin, Adv. Neurol., 20 (1978) 77-84. 27 Moruzzi, G. and Magoun, H.W., Brain stem reticular formation and activation of the EEG. Electroencephalogr. Clin. Neurophysiol. 1 (1949) 455-473. 28 Ohata, M., Fredericks, W.R., Sundaram, U. and Rapoport, S.I., Effects of immobilization stress on regional cerebral blood flow in the conscious rat, J. Cerebr. Blood Flow Metab., 1 (1981) 187-194. 29 Paulson, O.B. and Sharbrough, F.W., Physiologic and pathophysiologic relationship between the EEG and the regional cerebral blood flow, Acta Neurol. Scand., 50 (1974) 194-220. 30 Rapoport, S.I., Blood-Brain Barrier in Physiology and Medicine, Raven Press, New York, "1976, pp. 270-273. 31 Remler, M.P. and Marcussen, W.H., EEG monitoring of focal lesions of the blood-brain barrier, Acta Neurol. Scand., 65 (1982) 51-58. 32 Rothballer, A.B., The effect of phenylephrine, methamphetamine, cocaine, and serotonin upon the adrenaline-sensitive component of the reticular activating system, Electroencephalogr. Clin. Neurophysiol., 9 (1957) 409-417. 33 Selye, H., Stress in Health and Disease, Butterworths, London, 1976, pp. 16-21. 34 Sharma, H.S. and Dey, P.K., Probable involvement of 5-hydroxy-tryptamine in increased permeability of blood-brain barrier under heat stress in young rats, Neuropharmacology, 25 (1986) 161-167. 35 Sharma, H.S., Effect of captopril (a convening enzyme inhibitor) on blood-brain barrier permeability and cerebral blood flow in normotensive rats, Neuropharmacology, 26 (1987) 85-92. 36 Snyder, S.H., Axelrod, J. and Zweig, M., A sensitive and specific fluorescence assay for tissue serotonin, Biochem. Pharmacol., 14 (1965) 831-835. 37 Torda, C., Effect of brain serotonin depletion on sleep in rats. Brain Res., 6 (1967) 375-377. 38 Westergaard, E., The blood-brain barrier to horseradish peroxidase under normal and experimental conditions, Acta NeuropathoL, 39 (1977) 181-187. 39 Westergaard, E., Ultrastructurall permeability properties of cerebral microvasculature under normal and experimental conditions after application of tracers, Adv. Neurol., 28 (1980) 55-74. 40 Zubek, J.P. and Wilgosh, L., Prolonged immobilization of the body: Changes in performance and the EEG, Science, 140 (1963) 306-308.