Inhibition of nitric oxide synthesis does not reduce infarct volume in a rat model of focal cerebral ischaemia

),'euroscience Leucrs. 142 (1992) 151 154 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

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Inhibition of nitric oxide synthesis does not reduce infarct volume in a rat model of focal cerebral ischaemia D.A. D a w s o n ~'b, K. K u s u m o t o b, D.I. G r a h a m b, J. M c C u l l o c h b a n d I.M. M a c r a e ~'b "Universitv Deparlment qf Medicine and Therapeutics, Western h!firmary, Glasgow (UK) and ~'Welh'ome Sur,~ical Institute and Hugh Fraser Neuroscience Laboratories, University q/'Ghtsgow, Glasgow (UK) (Received 31 March 1992: Revised version received 28 April 1992: Accepted 4 May 1992)

Ke~ words." Nitric oxide: Ng-Nitro-i_-arginine methylester; Endothelium-derived relaxing factor: Focal cerebral ischaemia: Glutamate: Neurotoxicity ] h e effect of the nitric oxide (NO) synthesis inhibitor N~-nitro-L-arginine methylester (L-NAME) on ischaemic brain damage was determined in a rat model of focal cerebral ischaemia. Ischaemia was induced by permanent occlusion of the left middle cerebral artery (MCA) and infarction assessed 4 h post-occlusion by quantitative histopathology, k-NAME (30 mg/kg s.c.), administered 30 rain pre- and 30 min post-MCA occlusion, did not significantly alter the volume of ischaemic damage in the cerebral hemisphere, neocortex or caudate nucleus compared with saline controls. This result provides no support for the view that NO generation is a key component in the post-ischaemic cascade leading to acute neuronal death.

Recent interest in the simple molecule nitric oxide (NO) was stimulated by the discovery that its properties were identical to those of the endogenous vasodilator endothelium-derived relaxing factor [23]. NO is released in the conversion of k-arginine to L-citrulline by the enzyme NO synthase [22]. This reaction can be inhibited by several analogs of L-arginine, including Ng-nitro-k-argin ine methylester (L-NAME), which elicit peripheral vasoconstriction, hypertension and bradycardia [11, 26]. NO synthase is located centrally in both cerebrovascular endothelium [10] and neurones [1, 14]. Neuronal NO synthase exists in at least 2 isoforms which are Ca>/calmod ulin-dependent and require NADPH as a cofactor [3, 14]. A substantial body of evidence now implicates glutamate in the pathogenesis of cerebral ischaemia: glutamate receptor agonists are neurotoxic in cell culture [6], massive increases in extracellular glutamate occur in vivo following cerebral ischaemia [5], and glutamate receptor antagonists reduce infarction in experimental models of cerebral ischaemia [18]. Glutamate toxicity is mediated via a rise in free cytosolic Ca -,+ which can activate several potentially damaging enzymatic pathways [6]. Recent evidence suggests that glutamatergic activation of the Ca'-*/ ('orresT~Ondencc." D.A. Dawson, Wellcome Surgical Institute, University of Glasgow, Garscube Estate, Bearsdcn Road, Glasgow, Scotland, G61 IQH, UK. Fax: (44)41-943-0215.

calmodulin-dependent NO synthase with subsequent production of NO may be one such pathway. Direct production of NO by glutamate and N M D A has been demonstrated in cerebellum [2, 15] which can be antagonised by both NO synthase inhibitors and N M D A receptor antagonists. In cortical cell cultures NO synthase inhibitors antagonise the toxicity of glutamate and N M D A [7] and the NO donor sodium nitroprusside has been demonstrated to be neurotoxic [7], although others have failed to replicate this finding [12, 16]. NO may contribute to glutamate-induced neuronal damage by promotion of free radical production [13]. NO. itself a free radical, can combine with superoxide [13] to generate more highly reactive hydroxyl free radicals which are implicated in lipid peroxidation and increased oedema formation in ischaemia [28]. Free radicals may also potentiate excitotoxic damage by stimulating further release of glutamate under ischaemic conditions [24]. The present study was initiated to investigate the possible involvement of NO in the genesis of cerebral ischaemic damage. NO synthesis was inhibited by administration of k-NAME to rats in which focal cerebral ischaemia was induced by permanent unilateral occlusion of the middle cerebral artery (MCA). This model has been used repeatedly to demonstrate the role of glutamate in the pathogenesis of cerebral ischaemic damage and the efficacy of glutamate antagonists in reducing such damage [18]. If NO is a mediator of glutamate-in-

152 duced neurotoxicity then L-NAME would be expected to reduce tissue damage in this model. Eighteen adult male Sprague Dawley rats (336 400 g) were anaesthetised with halothane in nitrous oxide:oxygen (70:30). Anaesthesia was induced with 5% halothane and subsequently maintained between 0.75 and 1%. A tracheostomy was performed for artificial ventilation. The femoral arteries and veins were cannulated for monitoring blood pressure and arterial blood gases. Animals were maintained normoxic, normocapnic and normothermic throughout the experiments. A subtemporal craniectomy was performed to expose the left M C A [27]. The artery was occluded by electrocoagulation from where it crossed the olfactory tract to a point proximal to the origin of the lenticulostriate branches and then cut to ensure the completeness of the occlusion. Rats were randomly assigned to 2 equal groups which received either 30 mg/kg L-NAME (Sigma, U K ) ( n = 9 ) or saline (n=9) s.c, in a volume of I ml/kg, t,-NAME or saline were each administered 30 min pre- and 30 min p o s t - M C A occlusion. Four h post-MCA occlusion rats were perfusion fixed with 40% formaldehyde, acetic acid and methanol (1:1:8), and the brains processed as described previously [21]. Sections at 8 pre-selected stereotactic levels were examined under a light microscope and regions showing ischaemic cell change [4] were transcribed onto scale drawings [21] normalised to the mean hemisphere volume (570 m m 3) for Sprague-Dawley rats of the weight used. Areas of infarction in the neocortex, caudate nucleus and whole hemisphere were measured using an image analyser (Quantimet 970, Cambridge Instruments) and converted to total volume of ischaemic damage by integration. Statistical evaluation of the change in mean arterial blood pressure (MABP) over time for saline- and L-NAME-treated rats was by two-

way, repeated-measure ANOVA, ti)llowed bx' Student's unpaired t-test with Bonferroni correction fk~rsimultaneous multiple comparisons. Volumes of inl~trction lot the saline and I.-NAME groups were compared by Student's unpaired t-test (two-tailed). Physiological variables (Table I) were within the normal range with no significant differences between groups pre- or p o s t - M C A occlusion, t,-NAME (30 mg/kg 30 rain pre- and 30 min post-occlusion) resulted in a moderate hypertensive effect (Fig. 1). Statistical analysis revealed a significant drug-time interaction (P=0.01) which resulted in significant differences in M A B P between the saline and L-NAME groups at 4 time points following the second dose of t,-NAME (Fig. 1). L-NAME did not significantly alter the volume of ischaemic brain damage in the cortex, caudate nucleus or whole hemisphere compared with saline treated controls (Fig. 2). The failure to demonstrate a neuroprotective action of k - N A M E cannot be attributed to a failure of the active c o m p o u n d to enter the brain. L-Nitroarginine (the active form of L-NAME) can cross the blood-brain barrier more easily than other arginine analogs due to the presence of a nitro group [9], and systemic administration of L-nitroarginine has been demonstrated to inhibit brain N O synthase activity in rats [9]. The lack of effect of k - N A M E on infarct volume assessed 4 h following onset of ischaemia therefore suggests that N O is not involved in the acute pathogenesis of focal cerebral ischaemic damage. However, the cytotoxic effects of N O may become evident at later time points. A dramatic reduction in infarct volume 7 days post-MCA occlusion in the mouse following repeated treatment with L-nitroarginine has recently been reported [19]. The discrepancy between this result and the present study may be explained by the longer time course or by the diverse actions of N O syn-

TABLE 1 PHYSIOLOGICAL VARIABLES FOR SALINE AND L-NAME-TREATEDRATS Arterial blood gases and body temperature immediatelyprior to (0 h) and at 1 h intervals following left middle cerebral artery occlusion. Data are means ± S.E.M. There were no significant differences between saline and k-NAME groups at any timepoint (P>0.05, Student's unpaired t-test). Oh

lh

2h

3h

4h

Saline (n=9) pH p,COs(mmHg ) PaO2 (mmHg) Body temperature (°C)

7.45 ± 0.01 40.3 _+ 1.3 136 _+6 37.0 ±0.1

7.47 +_0.02 37.0 + 1.4 153 _+9 37.0 ±0.1

7.45 _+0.01 37.0 _+ 1.l 162 _+7 36.9 ±0.1

7.44 + 0.02 37.3 ±0.7 167 + 7 37.1 +0.2

7.44 _+0,02 37.6 20.8 168 ± 7 36.9 +0. l

k-NAME (n=9) pH paCOs(mmHg) p,O2 (mmHg) Body temperature (° C)

7.45 ± 0.03 36.8 _+0.5 155 ± 6 37.1 220.2

7.42 + 0.02 36.3 _+ 1.0 153 ± 5 36.9 22 0.1

7.39 ± 0.02 37.4 _+ 1.2 147 _+6 37.2 220.1

7.40 + 0.01 37.9 220.8 153 ± 6 37.2 _+0.1

7.38 ± 0.02 38.3 ±0.8 147 .+_4 37.1 *0.1

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Fig. I. Aherations m mean arterial blood pressure following L-NAME 130 mg/kg, s.c.) or saline administered 30 min prior to and 30 min post-left middle cerebral artery occlusion. Data are presented as means + S.E.M.. n-:9 both groups. "P<0.05 by two-way repeated measure ANOVA followed by Student's unpaired t-test with Bonferroni correction for comparison of saline and L-NAME treated rats at each limepoint. MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion.

thase inhibitors on physiological variables (blood pressure, cerebral blood flow (CBF) and particularly body temperature) known to influence infarct volume. The hypertension induced by L-NAME in conscious rats [17], which is markedly attenuated under halothane anaesthesia [8], might be expected to improve CBF to ischaemic regions, However since L-NAME reduces CBF in both conscious [17] and anaesthetised rats [8], hypertension is unlikely to account for the discrepant results. Of more importance may be the hypothermia induced by LNAME. In our study body temperature was maintained at 37°C, but in conscious rats L-NAME significantly lowers body temperature [17]. Repeated dosing of NO synthase inhibitors may reduce body temperature to a lexel known to be neuroprotective in focal cerebral ischaemia [20]. The apparent neuroprotective action of Lnitroarginine [19] may therefore be attributable to hypothermia ralher than direct antagonism of glutamate excitotoxicity and care must be taken to control this variable in future studies. Fhe actions of L-NAME on physiological parameters will make precise elucidation of the role of NO in cerebral ischaemia difficult. While under certain circumstances overproduction of NO may be detrimental, con> plcte inhibition of NO synthesis may be less than advantageous in cerebral ischaemia. Several actions of NO are likely to improve rather than exacerbate outcome following tin ischaemic insult: NO is a vasodilator, inhibits platelet aggregation and maintains the fluidity of blood [25]. Thus NO is likely to improve blood supply to ischaemic tissue while NO synthase inhibitors will de-

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Cerebral Hemisphere

Cerebral Cortex

Caudate Nucleus

Fig. 2. Effect of I - N A M E on the volume of ischaemic brain damage in the ipsilateral cerebral hemisphere, cerebral cortex and caudate nucleus, i -NAME (30 mg/kg, s.c.) or saline was administered 30 rain prior to and 30 min post-left middle cerebral artery' occlusion. Data are presented as means + S.E.M.. n - 9 for bolh groups.

crease it. The cerebral hypoperfusion induced by 1.N A M E may reduce flow in the ischaemic penumbra to a level below the critical threshold for development of permanent neuronal damage [18] negating any beneficial actions of the drug. Future pharmacotherapy aimed at limiting the toxic effects of NO in ischaemia may therefore be better directed towards scavenging superoxide radicals, to prevent combination with NO and consequent formation of more reactive free radicals, than inhibiting NO synthesis per se. The failure of a drug to influence the volume of infarction in experimental ischaemia inevitably focuses attention on the adequacy of the dosing regimen employed. The dose schedule for L-NAME in the present study (2 x 30 mg/kg) was sufficient to inhibit NO synthase both peripherally (increase in MABP (Fig. 1)) and centrally (significant reductions in CBF and body temperature observed in a parallel study [17]). Thus the fiiilure of i.N A M E to markedly reduce infarct volume cannot be readily attributed to inadequate inhibition of NO synthase. In conclusion, this data provides no support for the view that f - N A M E is neuroprotective in the acute post-ischaemic period following permanent MCA occlusion in the rat. This work was supported by the British Heart Foundation and the Wellcome Trust. We thank the technical and secretarial staff of the department for invaluable assistance and Bayer AG for providing Neuroscience Library facilities.

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