Ž .Brain Research 813 1998 191–194

Short communication

Lack of evidence for direct involvement of NMDA receptors or polyaminesin blood–brain barrier injury after cerebral ischemia in rats

Edward Preston a,), Jacqueline Webster a, Gene C. Palmer b

a Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A OR6b Astra Arcus USA, Rochester, NY, USA

Accepted 15 September 1998

Abstract

Ž .It is hypothesized that after various types of brain injury, blood–brain barrier BBB opening and vasogenic edema result fromŽ .excessive neuronal release of glutamate and stimulation of capillary N-methyl-D-aspartate NMDA receptors linked to polyamine

Ž .putrescine synthesis in endothelial cells. We produced cerebral ischemia in rats and measured BBB opening 6 h later as the increase inŽ . w3 xregional transfer constants K for blood to brain diffusion of H sucrose. Such BBB opening was not mitigated by drugs which blocki

Ž . Ž .NMDA receptors MK801 or AR-R 15896AR or polyamine synthesis difluoromethylornithine . These results question generality of thecapillary NMDA receptorrpolyamine hypothesis. Crown Copyright q 1998 Published by Elsevier Science B.V.

Keywords: Cerebral ischemia; Blood–brain barrier; NMDA receptor; Polyamines

Ž .In the 2VO two-vessel occlusion rat model of stroke,a temporary, e.g., 10-min, combination of bilateral carotidartery occlusion and blood withdrawal to produce arterial

w xhypotension is used to effect cerebral ischemia 23 . Thiscauses a regionally selective, delayed neuronal death in thestriatum, hippocampus and neocortex that becomes mani-fest 1–3 days later. Widespread opening of the blood–brainbarrier follows immediately upon reperfusion after 2VO

w xischemia 19,20 . This phenomenon, seen also in otherstroke models, is related to reactive hyperemia and high

w xcapillary intraluminal pressures 8 . While this early effectafter 2VO diminishes in most regions, BBB leakinessgradually intensifies in striatum and hippocampus to be-come prominent around 6 h post-2V0 and then largely

w xreverses between 24–48 h 19 . This delayed BBB injuryappears to involve formation of endothelial pores or chan-

w xnels 18 . However, the neurochemical or other eventsunderlying this opening and its relationship to the selec-tive, delayed neuronal death that manifests in these regions1–3 days later remain unexplored.

It is proposed that brain microvessels have glutamatereceptors of the NMDA type, and that excessive glutamate

) Corresponding author. Fax: q1-613-941-4475; E-mail:ed.preston@nrc.ca

released into the extracellular space during ischemia opensthe BBB by stimulating endothelial nitric oxide productionw x w x12 or polyamine synthesis 9,10 . Our objective in thepresent study was to test the prediction that drugs whichblock NMDA receptors or inhibit polyamine synthesisshould reduce BBB injury measured 6 h after 2VO in therat.

Ž .Male Sprague–Dawley rats 322–384 g were anes-Ž .thetized with sodium pentobarbital 65 mgrkg, i.p. , intu-

bated and mechanically ventilated with a 30%:70% mix-ture of O and N . Tympanic and colonic temperatures2 2

were maintained between 37.5–38.08C throughout proce-dures. Cerebral ischemia was produced by temporary with-drawal of tail-artery blood into a heparinized syringe andmaintenance of arterial pressure between 42–47 mmHg for10 min during which both carotid arteries were clampedw x20 . Wounds were sutured and the rat maintained nor-mothermic until it recovered from anesthetic. Test-drug

Ž .dosages detailed in Figs. 1 and 3, captions were based onw xthose found effective in other studies 3,6,11,14,16 .

Six hours after 2VO ischemia, rats were re-anesthetizedwith sodium pentobarbital and given an i.v. bolus injection

w3 Ž .x Žof H fructose-1 sucrose NET 341, Dupont NEN,.Boston, MA . A circulation time of 3, 10, 20 or 30 min

was allowed during which a syringe pump was used toŽ .collect a time-integrated femoral arterial sample 0.5 ml .

Each rat was decapitated and a final blood sample taken

0006-8993r98r$ - see front matter Crown Copyright q 1998 Published by Elsevier Science B.V.Ž .PII: S0006-8993 98 00981-0

( )E. Preston et al.rBrain Research 813 1998 191–194192

w3 xFig. 1. Graphical analysis of H sucrose passage across the blood–brainbarrier into hippocampus 6 h after 2VO ischemia in three groups of ratsŽ .ns9 per group treated with NMDA receptor blocking agents or

Ž .vehicle. A fourth group ns6 rats did not undergo ischemia, andrepresents ‘baseline’ permeation. Each point is from one rat which

w3 x Ž .received an i.v. bolus of H sucrose 10 mCir100 g with a 3, 10, 20 or30 min circulation time allowed before decapitation. Ordinate: accumu-lated parenchymal plus intravascular tracer, expressed as an equivalentplasma space or distribution volume, calculated from C rCbrain final plasmaŽ .see methods . Abscissa: circulation ‘time’ standardized for individualdifferences in plasma concentration profile, calculated from H t-0

C d trC . With longer circulation times an increasing pro-plasma finalplasma

portion of the distribution volume is parenchymal, depending on thedegree of BBB leakiness. The slope of each regression line is the transfer

Ž . Ž .constant K for BBB permeability to sucrose. Ordinate intercept Vi i

estimates intravascular plasma volume plus any rapidly equilibratingŽ .space s . The linear regression is described by the following equation

w x Ž t .5,17 : C rC s K H C d trC q V . Drugbrain finalplasma i 0 plasma finalplasma iŽ .treatments were as follows: AR-R 15896AR 7.5 mgrml in saline given

15 mgrkg i.v. 5 min before 2VO and 10 mgrkg s.c. 2 h later. Vehicle:Žsaline given at the foregoing times and routes. MK801 1.25 mgrml in

.saline given 3 mgrkg i.v. 5 min before 2VO.

from the severed carotid vessels. The brain was removed,dissected, and liquid scintillation counting procedures usedto obtain the following values: C , the regional brainbrain

Ž .radiotracer concentration dpmrg wet tissue ; Cfinal plasmaŽ . t Ždpmrml plasma at decapitation; and H C d t dpm s0 plasma

y1 .ml the integral of plasma tracer concentration over thew xcirculation time, t 18 .

Fig. 1 illustrates the type of the data obtained and itsgraphical analysis. The rats that underwent drug or vehicletreatment and 2VO ischemia showed regression lines andslopes which were indistinguishable between groups, but

Žmarkedly elevated above that for the baseline group no.2VO , demonstrating BBB injury and a more rapid rate of

w3 xH sucrose entry into brain. Fig. 2 summarizes all regionalŽ .regression line coefficients K s and their standard errors.i

Across the three ischemic groups striatum and hippocam-pus showed the highest K values. However, within thesei

tissues or other regions, the two NMDA antagonists failedto significantly reduce the magnitude of K below thati

measured in the vehicle-treated group.Similar findings were obtained in the series of experi-

ments which compared BBB opening 6 h after 2VO in rats

Ž .treated with DFMO or vehicle Fig. 3 . DFMO treatmentdid not significantly modify BBB injury.

In all of the foregoing 2VO experiments, blood parame-Ž .ters measured in control vehicle and drug-treated groups

immediately before onset of ischemia fell within the fol-Ž . Žlowing ranges mean"S.E.M. : pH 7.40"0.01 to 7.44

. Ž . Ž"0.01 ; pCO 41"1 to 45"1 ; pO 105"2 to 1182 2. Ž ."3 ; glucose 5.2"0.2 to 5.6"0.3 mM . Within experi-

ments there were no significant differences between treat-Ž .ment groups p)0.05, ANOVA or Student’s t-test . All

the foregoing regression line analyses in 2VO rats regard-Žless of group or region yielded intercepts, V s initiali

.distribution volumes, mlrg , ranging between 5.95"1.27Ž .S.E.M. to 8.78"1.19. Baseline values obtained in thetwo groups of non-ischemic rats exhibited a lower rangeacross regions, 4.26"0.65 to 6.23"0.68. These ranges ofV values in ischemic and ‘baseline’ rats are comparable toi

w xvalues reported earlier 18 , and to intravascular volumew125 x w xestimates based on other tracers, e.g., I albumin 25 .

A key observation in this study was that pretreatment ofrats with NMDA receptor blocking agents did not reducethe BBB injury, especially in striatum and hippocampus,the tissues that exhibit the greatest degree of opening when

w xmeasured 6 h after 2VO 19 . The absence of protection inhippocampus, in particular, is in general agreement withseveral earlier reports in which NMDA-receptor blockadewith MK-801 failed to protect against CA1 damage in-

w x w xduced by global ischemia in the rat 15 or gerbil 2,4 .These findings therefore argue against generality of the

proposal that BBB opening after freeze injury, ischemiaand other insults is mediated directly by excess interstitialglutamate stimulating NMDA receptors as regulatory com-

Ž .Fig. 2. Transfer constants K s for blood to brain movement ofiw3 xH sucrose in regions of cortex, striatum, hippocampus and midbrain inrats. Height of each vertical bar represents the least squares linear

Ž .regression coefficient S.E.M. of data plotted as in Fig. 1. All regionalŽ .K s measured after 2VO injury drug or vehicle treated are significantlyi

Ž .elevated above baseline K s for the intact BBB p-0.05, ANCOVA .i

K s from groups that received treatment with NMDA receptor blockingi

agents were not significantly different from vehicle treatment.

( )E. Preston et al.rBrain Research 813 1998 191–194 193

Fig. 3. Regional BBB opening measured 6 h after cerebral ischemia; lackof protection by an inhibitor of polyamine synthesis, difluoromethylor-

Ž . w3 xnithine DFMO . Transfer constants for H sucrose permeation in twoŽ .ischemically injured groups ns9 rats each were significantly elevated

Ž .above baseline ns4 non-stroked rats in all regions except frontalŽ .cortex ) p)0.05 . BBB opening was not significantly reduced by

ŽDFMO treatment as compared to vehicle injection. DFMO 100 mgrml,.pH 6.0 was given 500 mgrkg, i.p., 30 min before 2VO, and 250 mgrkg

s.c. 2 h post-2VO. Vehicle: injections of NaCl solution of equivalent pH,tonicity and volume.

w xponents of the microcirculation 9,10 . The results favor,rather, reports that disclaim the presence of or a role for

w xNMDA receptors in brain microvasculature 1,13 . Propo-nents of the capillary NMDA receptor concept also reportthat activation of these receptors stimulates ornithine de-

Žcarboxylase which causes polyamine synthesis i.e., pu-.trescine in endothelial cells and thereby BBB leakiness.

We were compelled to test the polyamine aspect of thishypothesis in our 2VO model since the upregulation ofornithine decarboxylase after ischemia becomes prominent

w xwith a timing, 6–8 h post-ischemia 9,16,21 similar to thatof the selective, delayed BBB opening seen in our 2VOexperiments. However this opening was not reduced byDFMO, the irreversible inhibitor of ornithine decarboxyl-ase.

However, research with other models has shown thatDFMO does protect against BBB injury and edema forma-

w xtion, specifically, observed 6 h after 2VO in the gerbil 21and after 8 h of middle cerebral artery occlusion in the catw x22 . Our failure to see similar protection in the 2VOmodel does not seem ascribable to inadequate dosagebecause the dosage we used, compares favorably with thatshown effective in other rat studies. Pre-ischemic treatmentwith 500 mgrkg prevents the rise in ornithine decarboxyl-

w xase measured 8 h after four vessel occlusion 16 and 12 hw xafter 60 min of middle cerebral artery occlusion 14 . Our

failure to see a protective effect may simply indicate thatthe BBB opening 6 h after 2VO involves another mecha-nism independent of polyamines. BBB opening been as-cribed to a number of candidate mediators other than

w xpolyamines 24 , and these could account for differences inthe timing, intensity and regionality of BBB injury and its

response to drugs. For example, it has been suggested thatthe delayed BBB opening and vasogenic edema whichpeaks 24–48 h after middle cerebral artery occlusion in therat should be researched in the context of an inflammationresponse involving infiltration of leucocytes and release of

w xinflammatory mediators 7 . The same may be true for thepronounced late-developing BBB opening and edema we

w xhave observed in cortex 24 h after prolonged 2VO 20 .The response of these openings to receptor blocking agentsor other drugs may be quite different from that whichbecomes manifest in striatum and hippocampus 6 h after2VO, or from the hyperemic opening that immediatelyfollows reperfusion. Events that underlie BBB openingmay include the release of free radicals, inflammatorysubstances released through the arachidonic acid cascade,

Ž .and activation of the kallikrein–bradykinin system 24 .We have not investigated these possibilities in the 2VOmodel, but have been able to significantly mitigate theselective, delayed BBB opening by post-reperfusion appli-

Ž .cation of hypothermia unpublished data .

Acknowledgements

Ž .a-Difluoromethylornithine DFMO was donated, cour-tesy of Dr. E.W.H. Bohme, by Hoechst Marion Roussel,¨Cincinatti, OH. AR-R 15896AR was supplied by AstraArcus, Rochester, NY.

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