April-Ju ne 1981 Ind. J. Physiol. Pharmac. n based on a report that memory Perhaps. chemical responsible for

IMPAIRMENT OF BLOOD-BRAIN BARRIER (BBB) IN RAT BY ucedshort-term memory impa irment IMMOBILIZATION STRESS: ROLE OF SEROTONIN (5-HT) that have been conclusively put . Increasein 5 HT (1) and reduction H. S. SHAR MA AND P. K. DEY ave considered the role of "memory ng a memory deficit. (3). that this Neurophysiology Research Unit, Department of Physiology. Subsequently it may be possible to ry impairment. just as the chemical Institute of Medical Sciences, Banaras Hindu University, Varanasi-227 005

S (Received on December 15. 1980)

dness. Dr. C.M. Francis the Dean o the work. Dr. K.N. Sharma the Summary: Influence of Immobilization stress on blood-brain barrier (BBB) was studied In ISexpert guidance. Dr. P. Simhadri, rats using as barrier tracer. 7-9 hr of Immobilization had Increased the permeability of BBB mainly In vour.qer rats. This increased permeability was significantly reduced with p-CPA. r of Pharmacology, Dr. G. D. Ka/yan- Indomethacin or vinblastine pretreatment. treatment caused early extravasation of Statistics, St. John's Medical of Evans blue dye. The increased level of serotonin in immobilization and Infusion of 5-HT in model experiments suggest a causative role of serotonin in the increased permeability of BBB induced by irnmobitization stress.

Key words : Immobilization stress serotonin (5- HT) blood-brain barrier (BBB) ck. brain acetylchloinesterase activity and indomethacin vinblastine parachlorophenylalanine (p-CPA) Evans blue theophylline horseradish peroxidase (H RP) phasesof memory storage. Science, 151 : on brain RNA Synthesis and on memory. INTRODUCTION ferof memoryresults with two d ifferen t types techniques. In : Molecular Approaches to Blood-brain barrier (BBB) maintains a suitable fluid environment for brain which cted by intracerebral puromycin. Science, is very essential for the normal functioning of neural tissues. BBB is characterised mainly idancetraining. J. Comp. Physiol. Psycb., by the presence of inter-endothelial tight junctions and absence of any vesicular transport (10). Recent studies have shown that BBB is not a passive static barrier. rather it func- epinephrine biosynthesis inhibition - effects tions as a dynamic regulatory barrier (17). Various physiological and pathological fear. Nature (London). 217: 1259-1260, conditions have been shown to increase the permeability of BBB to various tracers (17. 28. 29). Recently. stressors such as foot-electroshock. starvation. training in water maze cing brain peptide. Fed. Proc., 29 : 658, have also been reported to enhance the cerebrovascular permeability to various tracers nd synthesisof a spec ific behaviour induced (1.28). We have reported earl ier that immobilization stress also induced extravasation of Evans blue in cerebral tissues of rats (18). 112 Sharma and Day April-June 1981 Volume 25 Ind. J. Physiol Pharmac. Number 2 5 min after dye injection 0 However, the mechanism(s) underlying increased barrier permeability in stress is removed and the penetration of not well understood. Immobilization is known to elevate 5-HT in plasma (23) as well eye. Evans blue in brain was me as in brain (6. 25). Rise in brai.n 5-HT particularly in cerebral cortex has been reported following foot-electroshock and pentylenetetrazol induced convulsions (9). Increased 5-HT has been demonstrated in cerebral vessels and brain in response to mechanical 5 -HT estimation : The est trauma and in ischaemic conditions in the affected area of the cerebral cortex (16. 24). separate groups of rats after differ and the association of these conditions with increased BBB permeability have already measurement of BBB and 5-HT been reported by other workers (3. 5. 20. 28). Infusion of 5-HT into systemic circulation reason that injection of Evans b or into cerebral tissues or cerebral ventricles have been shown to increase the permeability estimation of 5-HT. of BBB to various protein tracers (4. 21. 26. '27.29). Thus. immobilization was of 4 rats (71-78 g) and 5 rats ( Therefore. the present study was undertaken to examine the relationship between out in a rother 4 rats (70-78 g) the level of 5-HT in plasma and brain tissue and increased permeability of BBB at different were anaesthetized with uretha periods of immobilization stress. Further. the model experiments have been carried out syringe through cardiac punctur to examine the permeability of BBB with the dose of 5-HT. which was almost equal to One m! of plasma was diluted in the serotonin level observed in plasma associated with increased permeability of BBB following immobilization stress. brain tissues (cerebral and cere 0.4 N peA. The plasma and th for 10 min and 4 ml of supernatan MATERIALS AND METHODS fluorometrically in Aminco Bow Snyder et al. (19). Experiments were carried out on inbred Charles Foster albino rats of either sex at an ambient temperature range of 18° to 29°C.. Animals were housed at controlled temperature To avoid the changes in (220 to 24°C) with 12.00 hr light and 12.00 hr dark schedule. They had free access to ponding control animals were sa food and water ad libitum.

Drug treatmeAt : Immobilization: Immobilization of rats was carried out on wooden board in prone POSition and limbs were fixed with adhesive tape. 8 rats (45-80 g) were immobilized for Since increase in BBB 2-L1 hr; 24 rats (40-80 g) and 12 rats (200-260 g) were immobilized for 7-9 hr; and 8 rats in younger rats (50-80 g). all dru (50-80 g) were immobilized for 14-16 hr. 12 non-restrained rats (40-80 g) served as drugs were use~ in this study: control. (a) Parach!orophen V/alanine ( Evaluation of increased BBB permeability: After completion of stress period injected 100 mg/kg. i.p. daily fc Evans blue dye (2%. 3 ml/kg) was injected into right jugular vein under urethane anaesthesia after the last injection of p-CP.t (0.8 g/kg. i.p.) in all rats except in 6 rats (subjected to 8-9 hr of immobilization). in which in 6 rats and 5-HT was measure Evans blue dye was injected without anaesthesia. 5 min before termination of stress period. (b) Indomethacin: 10 mg/kg through an aseptically implanted polythene cannula into right jugular vein two days before intra peritoneally in 20 rats (75-E the experiment. This was done to examine whether urethane anaesthesia has any influence tion for a period of 8-9 hr. As itself on BBB permeability. April-June 1981 Volume 25 Immobilization. 5-HT and BBB 113 Ind. J. Physiol Pharmac. Number 2 d barrier permeability in stress is 5 min after dy~ injection 0.9% saline was perfused through the heart and brain was ate 5-HT in plasma (23) as well removed and the penetration of Evans blue into cerebral tissues was examined by naked cerebral cortex has been reported eye. Evans blue in brain was measured according to Harada et al. (11). ced convulsions (9) Increased brain in response to mechanical 5-HT estimation : The estimation of 5-HT in plasma and brain tissue was done in of the cerebral cortex (16. 24). separategroups of rats after different periods of immobilization. In this group simultaneous 888 permeability have already measurement of BBB and 5-HT was not possible to be carried out because of the obvious of 5-HT into systemic circulation reason that injection of Evans blue and brain perfusion would have interfered with the hown to increase the permeability estimation of 5-HT.

Thus, immobilization was carried out for 4 hr in 5 rats (60-75 g) another group xarruns the relationship between of 4 rats (71-78 g) and 5 rats (260-280 g) were immobiliz.ed for 8 hr: same was carried d permeability of BBB at different out in arother 4 rats (70-78 g) for 14 hr. After completion of stress period the animals penments have been carried out were anaesthetized with urethane. The blood samples were collected in heparinized -HT. which was almost equal to syringe through cardiac puncture and centrifuged immediately for separation of plasma. increased permeability of BBB One m! of plasma was diluted in 8 ml of ice-cold 0.4 N perchloric acid (PCA) and the brain tissues (cerebral and cerebellar cortices) were homogenized in 8 ml of ice-cold 0.4 N PCA. The plasma and the homogenized brain tissues were centrifuged at 900 x g for 10 min and 4 ml of supernatant from each of them was collected and 5-HT was measured HODS fluorometrically in Aminco Bowman Spectrophotofluorometer (MD, USA) according to Snyder et al. (19). steralblllo rats of either sex at an housedat controlled temperature To avoid the changes in 5-HT concentration due to circadian rhythm, the corres- hedule. They had free access to ponding control animals were sacrificed at the same time period with immobilized rats.

Drug treatmeAt : out on wooden board in prone (45-80 g) were immobilized for Since increase in BBB permeability following immobilization was observed mainly mmobilized for 7-9 hr; and 8 rats in younger rats (50-80 g), all drug treatments were carried out in this age group. Following rained rats (40-80 g) served as drugs were used in th is study:

(a) Parachlorophenylalanine (p-CPA) p-CPA (Sigma Chemical Co.. USA) was er completion of stress period injected 100 mg/kg, i.p. daily for three consecutive days in 10 rats (49-80 g). 24 hr vein under urethane anaesthesia after the last injection of p-CPA animals were immobilized for 8-9 hr. BBB was studied 9 hr of immobilization), in which in 6 rats and 5- HT was measured in 4 rats. fore termination of stress period. Ight lU u\ar \le\{\ \.\No cl.a.'f~\}oa'~1C tb) Indomethacin: 10 mg/kg indomethacin (Sigma Chemical Co., USA) was injected aneanaesthesia has any influence intraperitoneally in 20 rats (75-80 g) 30 min before subjecting the animals for immobiliza- tion for a period of 8-9 hr. A second dose of indomethacin (10 mg/kg, i.p.) was injected

. . 114 Sharma and Oey April-June 1981 ve-lume 25 Ind. J. Physio!. Pharmac Number 2 4.5 hr after the commencement of stress to sustain Its action. After the completion of stress TABLE I: Effect of period. the permeability of BBB was studied in 16 rats and 5-HT was determined in 4 rats

Treatment (c) Vinblastine: Vinblastine (Ell Lily Co .. lndianopolrs. USA) 0.7 mg/kg was injected Into right jugular vein under ether anaesthesia in 8 rats (60-80 g). 48 hr after the adrninis- tration of drug the rats were immobilized for 8-9 hr. Half of the animals were tested for ------BBB permeability and in other half; 5-HT was determined. Control (d) Theophylline: 12 rats (60-68 g) were immobilized for 4.5 hr. Aminophylline Immobilization (Burroughs Wellcome & Co. Pvt. Ltd .. Bombay, India). 10 mg/kg was injected intraperi- (2-4 hr) toneallv. 15 min after the animals were subjected to immobilization. BBB was tested in 8 rats and 5-HT estimation was done in 4 rats. Immobilization (7·9 hr) 5-HT infusion: In 10 rats (60-75 g) and In 6 rats (220-280 g) under Immobilization urethane anaesthesia 5-HT (Sigma Chemical Co.. USA). 10 lJ.g/kg/min was infused (14-16 hr) for a total period of 8 min into right jugular vein through a constant infusion pump (INCa. India). Evans blue dye (2%. 3 ml/kg) in 14 rats and HRP (Type I. Centron Irnmobilization Research. India) 2%.5 ml/kg in 2 rats (68-71 g) was injected 5 mm before 5-HT infusion (8-9 hr) In 6 rats (65-80 g) Evans blue dye was injected 2 hr after 5-HT infusion to study the Immobilization + reversibility of increased BBB permeability induced by 5-HT. 0.9% saline was perfused Indomethacin In animals which received Evans blue and demonstration of HRP In cerebral tissues was (8-9 h r) done according to the modified method of Malmgren and Olsson (15) as described by Dey et al. (8). Immobilization + p-CPA (8-9 h r) RESULTS Immobilization + Results are shown In Table I and 11. vinblastine (8-9 hr)

Table I shows that 2-4 hr of Immobilization were not very effective in Increasing Immobilization + the permeability of BBB to Evans blue. Thus. only one rat out of 8 showed extravasation theophy lIine (4.5 hr) of the dye. On the other hand 7-9 hr of immobilization have significantly increased the permeability of BBB in 19 out of 24 younger rats (40-80 g) but when stress is continued 5- HT 10 fLg/kg/mm for a longer oeriod (upto 14-16 hr). the increased permeability of BBB appeared to diminish. 8 min Older rats (200-260 g) were more resistent to show the Increased permeability of BBB in 5-HT 10 fL9/k9/mtn comparison to younger rats (40-80 g) following 8-9 hr of immobilization (Fig. 1). 8 min

Increased permeability to dye was observed mainly In cinqulate. occipital and 5-HT 10 fL9/kg/min 8 min parietal cerebral cortex as well as in vermis portion of cerebellar cortex. No difference in penetration or distribution pattern in dye was observed in rats in which Evans blue dye

• [vans blue dye was injected 2 Apnl-June 1981 Immobilization. 5-HT and BBi 115 Ind. J. Physio!. Pharmac cnon After the completion of stress TABLE Effect of immobilization and infusion of 5-HT on the permeability of BBB in rat. nd 5-HT was determined in 4 rats.

No. of Body weight increased BBB 01 s. USA) 0.7 mg/kg was injected observations range (g) permeability (60-80 g). 48 hr after the adrninis- Yes No Half of the animals were tested for ------mined. 12 40-80 12 buizeo for 4.5 hr. Aminophylline 8 45-80 7 la) 10 mg/kg was injected intraperi- immobilization BBB was tested in 24 40-80 19 5

nd In 6 rats (220-280 g) under 50-68 3 5 USA). 10 I.l.g/kg/min was infused through a constant infusion pump 14 rats and HRP (Type I, Centron 12 200-260 4 8 Injected 5 mm before 5-HT infusion rafter 5-HT Infusion to study the 13 + 16 75-80 3 5-HT 0.9% saline was perfused Ion of HRP In cerebral tissues was nand Olsson (15) as described by + 6 4<)-80 6

4 + 4 60-80

ere not very effective in increasinp + 8 60-68 7 e rat out of 8 showed extravasation ion have significantly increased the 80 g) but when stress is continued 10 5·HT 10 !1-g/kg/mm 10 60·75 ability of BBB appeared to diminish. 8 mm e Increased permeability of BBB In 6 5·HT 10 !1-g/kg/mm 6 65·80 r of immobilization (Fig. 1). 8 min

220·280 5 ainlv m cmqulate. occipital and 5·HT 10 !1-g/kg/min 6 8 min f cerebellar cortex No difference ed in rats in which Evans blue dye

'[vans blue dye was injected 2 hr after 5-HT infusion.

• 116 Sharma and Day lnd. J. TABLE 11 Effect of immobilization on plasma and brain 5-HT in rat. periods of imm observed after 4

Serotonin in younger rats Treatmenr No. of Body wt. (mean±S.O.) 14 hr of immobil observation range (g) Plasma Brain response to stre (!Lg/m/) (!Lg/g) of 5 showed mar

Control 8 50-78 0.29±0.08

Lmmobilization 5 60-75 0.37±0.15 (4 hr)

Immobilization. 4 71-78 2.34±0.53 (8 hr) lmrnob ilize tiers 4 70-75 0.91±0.02 (14 hr) rmmobiliza tion 5 260-280 0.41±0.38 (8 hr)

Immobilization + 4 50-75 0.41±0.03 p-CPA (8-9 hr} fmmobifization + 4 78-80 o 47±0.07 0.92±O.19 indomethacin (8 hr) rmmobilization + 4 65-78 189±0.08 o 88±O.09 vinblastine (8 hr) Fig. 1 : (A) Shows pen and in aide Immobilization + 4 60-65 O. 77±O. 17 O.72±O.04 into right ju theophylline sacrifice of (4.5 hr) (B) Shows incr ------~------in older rats was injected before urethane anaesthesia in comparison to other rats in which Evans blue was injected after urethane anaesthesia. indicating that urethane anaesthesia itself had no p-CPA pretr influence on BBB permeability. prevented the extra significantly reduce Measurement of 5-HT showed a close parallelism between increase in BBB showed staining of permeability and increase in plasma and brain 5-HT in younger rats following different as well as in brain Immobilization, 5-HT and BBB 117 Incl. J. I smaand brain 5-HT in rat. periodsof immobilization. Thus, no significant increase in plasma or brain 5-HT was observedafter 4 hr of immobilization but profound increase in 5-HT (6-8 fold) was observed Serotonin n younger rats (60-80 g) after 8 hr of immobilization which was not evident following wt (mean±S.O.) (g) 14 hr of immobilization. Older rats (260-280 g) in contrast to younger rats showed least Plasma Brain responseto stress-induced increase in 5-HT either in plasma or in brain, only one rat out ((Lg/ml) ((.Lg/g) of 5 showed marked increase in 5-HT (Fig. 1).

8 0.29±0.08

o 37±0.15 o 40-80Q mm 220· 260 Q 3·0 ... 3·0 2.34±0.53 A B

O.91±0.02

it 2·0 2·0 01 80 0.41±0.38 .5 E ••• 0. ...:::>J .3 m 0- ,I;, O.41±0.03 ell 0.44±0.01 ~Z > 1·0 •••

o 47±0.07

189±0.08 4hr 8hr 14hr ISmin 2hr 4hr 8hr 14hr IMMOBILIZATION S-HT INFUSION IMMOBII17dT.""

Shows penetration of Evans blue in brain following different periods of immobilization in younger rats and in older rats (following 8 hr of immobilization); and infusion of 5-HT (10 (Lg/kg/min for 8 min) O.77±0.17 into right jugular vein in younger and in older rats. Figures below the bars indicate time interval between sacrifice of animals after completion of infusion.

(B) Shows increase in 5-HT in plasma following different periods of immobilization in younger rats and ~------in older rats (following 8 hr of immobilization) (' P<0.05; ,. P

.. Volume- 25 118 Sharma and If)ey April-June 1981 Number 2 Ind. J. Physiol. Pharmac. of dye in all rats but rise in 5-HT in plasma and brain was not significantly altered. Theo- phylline treatment caused early extravasation of Evans blue dye in cerebral tissues. Thus 7 A out of 8 rats showed increase in BBB permeability to dye after 4.5 hr of immobilization. A slight elevation of plasma 5-HT was noted in theophylline treated rats. (Table 1/, Fig. 2).

o EVANS BLUE t-J 5-HT 3 ~ J :> III •... :x: Z I 8 '., 1·0 10

C 8 :: r; "

D 'tit ,""OBlllZATION

Fig. 2 Shows drug influence on penetration of Evans blue in brain and levelof 5-HT in plasma following 8 hr of immobilization in younger rats except in E in which immobilization was done for 4.5 hr only. (0 P

Infusion of 5-HT (10 p.g/kg/min for 8 min) which approximated the increase in 5-HT in plasma after 8-9 hr of immobilization showed increase in permeability of BBB in all younger rats (60-75 g) after 15 min of 5-HT infusion. as evidenced by extravasation of Evans blue and HRP (Fig. 1. 3), while in older rats (220-280 g) only one rat out of 6 showed Fig. 3 : extravasation of Evans blue. No increase in BBB permeability was observed when Evans blue dye was injected 2 hr after the end of 5-HT infusion. This suggests that increased (6) Shows exuda (Jog/kg/min fo permeability of BBB induced by 5-HT is reversible in nature (Table I). 5-HT infusion Immobilization, 5-HT and BBB 119 April-June 1981 Ind. J. Physiol Pharmac. not significantly altered. Theo- dye in cerebral tissues. Thus 7 A ! after 4.5 hr of immobilization. Wlline treated rats. (Table 11.

ION 'lIZ IN.IMMOBILll .IMMOBILlZ .• IMMOBIlIZ.(4·5 rl,)

evelot 5-HT in plasma following lizationwas done for 4.5 hr only.

ximated the increase in 5-HT permeability of BBB in all enced by extravasation of only one rat out of 6 showed Fig. 3: (A) Control experiment shows absence of exudation of HRP in rat parietal cerebral cortex following 15 min was observed when Evans after saline infusion into right jugular vein. HRP (Type I. 2%. 5 ml/kg j.v.) was injected 5 min before saline infusion (X 650).- his suggests that increased (B) Shows exudation of HRP across cerebral vessels into rat parietal cerebral cortex following 5-HT (10 able I) IIog/kg/min for 8 min) infusion into right jugular vein. HRP was injected into jugular vein 5 min before 5-HT infusion. Animal was sacrificed 15 min after the end of 5-HT infusion (X 650' .

• 120 Sharma and DeV Ind J. permeability. DISCUSSION In 5-HT leve The present results show that immobilization stress can induce extravasatior has already SSS permea of Evans blue in cerebral tissues like other stressors This Increased permeability followirq confirms 5- immobilization is more or less time dependent Thus, 2-4 hr of immobilization was not effective in producing extravasation of dye but significant increase In permeability of BBB occurred after 7-9 hr of immobilization which tends to diminish after 14 hr (Table I). This terase enzy increased permeability of BBB induced by Immobilization may be the result of the increase of Evans blu in 5-HT following immobilization, because plasma and brain 5-HT do not differ much has accumul In respect to control after 4 hr of stress but profound (6-8 fold) increase of It occurrea Serotonin is after 8 hr of immobilization, and this increased level declined at 14 hr (Table 11). Ths shown to ea increase in 5-HT was observed mainly In younger rats. Older rats did not show such increase in 5-HT both in plasma and brain tissues. Increase in 5-HT following foot-

electroshock and pentyleneterazol induced convulsions is also age-related. More 1. Angel. C. increase occurred in younger mice than older ones (9). This explains why younger rats 2 Appleman, are more vulnerable to this stress than that of older rats. Further, the model experiments Cyclic Nuc York. 1973 show that even infusion of same amount of 5-HT which induced extravasation of tracers Beggs. J.L. in cerebral tissues of younger rats did not show same degree of extravasation in older rats to spinal c 4. Bulle. P.H. This suggests that senstitivity of cerebral vessels to 5-HT decreases with aging. Density Proc. Soc. of cerebrovascular receptors has already been reported to decrease with advancing age 5. Costa. J.L. (22). injury. Na 6. De Schaep er. J. Phe: 5-HT increases the permeability of BBB to protein tracers by enhancing vesicular 7. Debnath. P. transport across cerebral vessels (26, 27, 28, 29). 5-HT is known to stimulate pinocvtosis landin El 0 8 Dev. P.K.. in cerebral vessels either directly or through cAMP (17, 26, 27, 29). Increased vesicular permeability transport rather than junctional widening can be accounted for extravasation of Evans 9. Essman. W. Central Ne blue dye in cerebral tissues following immobilization stress which IS evident from vinblastine , O. Ford. D.H treatment. Vinblastine- an amitotic drug which inhibits polymerization of microtubules and !.J Ko thus interfering their normal functions, has blocked completely the penetration of dye 11. Harada. M.• oxtravasat in brain inspite of high rise in plasma and brain serotonin. Vincristine, another vinca 12. Haubrich. 0 alkaloid, is known to reduce hypertension induced extravasation of Evans blue dye (14). in rat brain. 13. Johansson. These workers suggested that vincristine has .educec the extravasation of Evans blue by embolism I inhibiting vesicular transport. The remaining dye ertered in brain is due to junctional 14. Larsson. Boo widening (17). Thus total blockade of BBB permeability with vinblastine suggests vesicular In the enha Acta Neurots transport a major sole route for dye transfer in immobilization. Indomethacin pretreatment 15. Malmgren. has been reported to reduce hypertension induced extravasation of Evans blue dye (13). neurons folio Indomethacin pretreatment in our study has reduced the rise in plasma and brain 5-HT 16. Osterholm. J relation to also. This suggest again a direct relationship between increased 5-HT and increased BB8 Trauma - in 31 : 408·4 April-Jun~ 1981 Immobilization. 5·HT and BBB 121 Ind. J. Physiol. Phermac, permeability. Indomethacin is a prostaglandin synthetase inhibitor drug and reduction In 5-HT level by indomethacin suggest that prostaglandins stimulate 5-HT synthesis which tress can induce extravasation hasalready been repo1ted by other workers (8. 12). Complete blockade of increased ncreasedpermeability following BBB permeability and rise in 5-HT by p-CPA- a 5-HT synthesis inhibitor drug- further hr of Immobilization was not confirms 5-HT mediated increase in BBB permeability induced by immobilization. ncreaseIn permeability of BBB Theophylline which prevents the destruction of cAMP by inhibiting phosphodies- inish after 14 hr (Table I). This teraseenzyme (2) thus leads to accumulation of cAMP. has caused early extravasation ay be the result of the increase of Evans blue inspite of slight rise in plasma serotonin. This suggests that cAMP which aln 5-HT do not differ much hasaccumulated. increased the vesicular transport leading to the extravasation of dye. 8 fold) Increase of It occurred Serotonin is known to stimulate cAMP synthesis and infusion of cAMP has already been ned at 14 hr (Table 11). This shown to cause enhanced transfer of HRP throguh vesicles (26. 27). 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