Brain Research, 518 (1990) 353-355
353
Elsevier BRES 24114
Kynurenate does not reduce infarct size after middle cerebral artery occlusion in spontaneously hypertensive rats Simon Roussel, Elisabeth Pinard and Jaques Seylaz Laboratoire de Physiologie et Physiopathologie c~r~brovasculaire, U. 182 I.N.S.E.R.M., U.A. 641 C.N.R.S., Paris (France)
(Accepted 20 February 1990) Key words: Focal cerebral ischemia; Infarct size; Excitatory amino acid; Glycine; Spontaneously hypertensive rat
The effects of the excitatory amino acid receptor antagonist, kynurenate, were investigated in spontaneously hypertensive rats after middle cerebral artery occlusion. Kynurenate did not significantlymodify either the infarct volume, measured 48 h after occlusion, or the neurological score. The absence of a neuroprotective effect of kynurenate in this study, which contrasts with results in normotensive rats, is suggested to be due to impaired collateral circulation in spontaneously hypertensive rats.
It has been suggested that excitatory amino acids (EAAs) may be important intermediates in the neuronal degeneration which follows several diseases of the central nervous system 24. These transmitters and their agonists are neurotoxic at high concentrations and induce 'ischemic cell changes' both in vitro and in vivo 5'2s. This excitotoxicity has been shown to be blocked in vitro by antagonists of the N-methyl-D-aspartate subtype of glutamate receptors 23. During global ischemia, massive amounts of excitatory amino acids are released into the extracellular space 2, where they are believed to elicit neurotoxicity. Over the last few years, several authors have reported that blockade of excitatory neurotransmission can prevent or limit the delayed neuronal degeneration after global cerebral ischemia 15'25,26, although this effect is controversial 4. There is also some evidence that competitive and non-competitive blockade of glutamate receptors reduces the infarct volume produced by middle cerebral artery occlusion in normotensive rats 11"14A6"2°. Only non-competitive blockade of glutamate receptors has been shown to exert such a protection in normotensive cats 19'21 and in hypertensive rats 1°. The aim of the present study was to evaluate the therapeutic value of E A A receptor blockade in a model of focal cerebral ischemia in spontaneously hypertensive rats (SHRs). SHRs were chosen for two reasons: firstly, because hypertension is the major risk factor for stroke, secondly because occlusion of the middle cerebral artery (MCA) in this strain leads to an infarct volume which is highly reproducible 12, thus providing an excellent model of focal ischemia for pharmacological evaluation. Gluta-
mate receptors were blocked with the broad spectrum E A A antagonist kynurenate22; it is competitive for kainate and quisqualate receptors 3, and acts at the neuromodulatory glycine site of N-methyl-D-aspartate (NMDA) receptor 17. The investigations were performed on 18 male SHRs (Iffa-Credo, l'Arbresle, France), weighing 280-300 g, which had free access to food and water prior to experimentation. The rats were anesthetized with halothane (1.5%) in 02. Rectal temperature was measured and maintained within the normal physiological range (36.5-37.5 °C) during surgery. A clear vinyl catheter was chronically placed in the peritoneal cavity and canalized subcutaneously to exit at the neck. It was used for intraperitoneal drug administration. Left MCA occlusion was performed by a subtemporal approach, essentially as described by Tamura et al. (1981) 27. Rats were placed in the lateral position, a vertical incision was made between the left orbit and the external auditory canal. The temporal muscle was divided midway vertically and reflected forward and downward with a retractor. The left eye and the parotid gland were left in place, but the posterior half of the zygomatic bone was removed. The inferotemporal fossa was exposed under a surgical microscope, and a small craniectomy was made using a dental drill. The dura was incised and reflected. The arachnoid was then divided on either side of the artery. Bipolar microcoagulation forceps were used to occlude the M C A from the point where it crosses the inferior cerebral vein to the superior aspect of the olfactory tract. The lenticulostriate arteries were also coagulated. The
Correspondence: S. Roussel, Laboratoire de Physiologie et Physiopathologie crrEbrovasculaire, 10, avenue de Verdun, 75010 Paris, France.
0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
354 skin was then sutured, anesthesia was discontinued and the animals were returned to their cages. A simple neurological examination was performed on 4 occasions: before anesthesia, 2 h after occlusion when the animals were awake, 24 h and 48 h after occlusion. The neurological grades were determined as described by Bederson et al. (1986)1: animals were graded 0 when they had none of the tested neurological deficits, 1 when they exhibited assymetrical forelimb flexion when held by the tail, 2 when they had decreased resistance to a right lateral push, and 3 when they displayed circling behavior. Immediately after the last neurological examination (48 h after occlusion), the animals were anesthetized with 2.5% halothane in 0 2 and perfusion-fixed with F.A.M. (1/10 formaldehyde, 1/10 acetic acid, 8/10 absolute methanol, v/v) via a catheter placed in the ascending aorta through the apex of the left ventricle. The brain was carefully removed, and prepared for paraffin embedding. Ten/~m thick sections were taken at 1 mm intervals and stained with Cresyl violet. The volume of infarction in the cortex and striatum was calculated from the surface area of necrotic tissue at each level, determined with an image analyzer (Histopericolor, MS21), and the distance between succeeding slices. The animals were randomly divided in two groups, and treated according to the following protocols: group 1 (n = 10): each control rat was given saline at the same times and in the same volumes as kynurenate in group 2; group 2 (n = 8): each rat was given kynurenate (500 mg/kg; pH = 7.4) 30 min before induction of ischemia and 3.5 h, 7,5 h and 24 h after occlusion. Kynurenate (Sigma Chemical) was dissolved in a small amount of 1 M NaOH and diluted in 0.9% NaCI saline. The pH was adjusted to 7.4 with HCI. The comparison of infarct volumes was performed using Student's t-test. Neurological scores were compared using the Kolmogorov-Smirnov one-sample test. As previously described, MCA occlusion induced a well-delineated infarction, embracing part of the striatum and cortex. The mean volume of infarction in our study (176.3 + 8.8 ram3; mean + S.E.) is in good agreement with that found by others 12. This volume was not significantly changed by kynurenate (Table I). Likewise, the neurological score was not significantly changed by the drug (Table I1). No rats died in either treated or non-treated groups. The present results differ from those obtained in normotensive animals with both competitive and noncompetitive E A A antagonists (thienyl-cyclohexyl-piperidine 1l, kynurenate ~4, MK-801 (ref. 20) or SL 82.0715 (ref. 16)), and also from those obtained in SHRs with MK-801 (ref. 10): these results indicated that blockade of E A A receptors reduced infarct size. The difference
FABLE 1 Infarct size (ram ~) 48 h after middle cerebral artery occlusion
Data are means + S.E. There are no significant differences between groups (Student's t-test). Groups of animals
Cortex
Caudate nucleus
Total
n
Control Kynurenate
140.0 -+ 8.3 131.9 + 9.5
36.3+ 3.2 28.1 +_4.2
176.3 + 8.8 160.0 -+9.9
10 8
between results obtained in SHRs with kynurenate and MK-801 may be due to their differing sites of action. Kynurenate acts as an aliosteric regulator of the NMDA receptor at the glycine site ~7, whereas MK-801 blocks the calcium channel associated with the NMDA receptor TM. Furthermore, the blockade with MK-801 is enhanced by the presence of an agonist of the receptor ~3, and hence is enhanced during ischemia by the massive release of glutamate. The present results in SHRs can be directly compared with the results obtained in normotensive rats. Germano et al. 14 administered kynurenate to MCA-occluded normotensive rats with timings and doses comparable to those used in the present study and found an attenuation of the infarction size and an improvement of the neurologic outcome. This discrepancy may be tentatively attributed to differences in the pathophysiological consequences of focal ischemia in SHRs and normotensive rats. MCA occlusion leads to infarction in young SHRs, but not in young normotensive rats 6. The infarction is larger and more reproducible in adult SHRs than in adult normotensive rats 12. These differences could result from impaired collateral circulation from pial anastomoses for 3 reasons. The distance between pial anastomoses and the lesion border is proportional to the diameter of the anastomosis 9, the mean luminal diameter of the anastomosing A C A - M C A collaterals is smaller in stroke prone SHRs (SHRSPs) than in normotensive Wistar-Kyoto rats 7, and finally blood flow in the territory of the
TABLE II Neurological score after middle cerebral artery occlusion
All animals had a neurological score of 0 before occlusion. A: 2 h after occlusion; B: 24 h after occlusion; C: 48 h after occlusion. Average: Average of the neurological scores at the 3 times. Data are means -+ S.E. There are no significant differences between groups (Kolmogorov-Smirnovone-sample test). Groups of animals
A
B
Control Kynurenate
2.1 + 0.2 2.1+0.1 2.1_+0.1 1.7 -+0.2
C
Average
2.4+_0.2 2.2+-0.1 2.1+0.1 2.0+0.1
n
10 8
355 occluded vessel is lower in S H R P S P s than in n o r m o t e n sive rats 8. It is t h e r e f o r e very likely that there is a threshold of local b l o o d flow above which inhibition of E A A receptors protects neurones from ischemic degeneration, and that the a r e a in which blood flow is above this threshold in S H R s is too small to reveal any effect. In conclusion, occlusion of the middle cerebral artery in S H R s is of great interest as a m o d e l of ischemia for p h a r m a c o l o g i c a l evaluation, not only because of the high reproducibility of infarct size, but also because hypertension is the m a j o r risk factor for h u m a n stroke. H o w e v e r , the present study emphasizes the fact that
results o b t a i n e d with this strain m a y not be e x t r a p o l a t e d to o t h e r strains, p r o b a b l y because of a g r e a t e r reduction in the cerebral circulation in the ischemic territory of S H R s than of normotensive rats. A clear understanding of the differences b e t w e e n the pharmacological responsiveness of normotensive and hypertensive animals is thus of m a j o r importance.
1 Bederson, J.B., Pitts, L.H., Tsuji, M., Nishimura, M.C., Davis, R.L. and Bartkowski, H., Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination, Stroke, 17 (1986) 472-476. 2 Benveniste, H., Drejer, J., Schousboe, A. and Diemer, N.H., Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis, J. Neurochem., 43 (1984) 1369-1374. 3 Birch, P.J., Grossman, C.J. and Hayes, A.G., Kynurenate and FG9041 have both competitive and non-competitive antagonist actions at excitatory amino acid receptors, Eur. J. Pharmacol., 151 (1988) 313-315. 4 Block, G.A. and PulsineUi, W.A., Excitatory amino acid receptor antagonists: failure to prevent ischemic neuronal damage, J. Cereb. Blood Flow Metabol., 7, Suppl. 1 (1987) S149. 5 Choi, D.W., Maulucci-Gedde, M. and Kriegstein, A., Glutamate neurotoxicity in cortical cell culture, J. Neurosci., 7 (1987) 357-368. 6 Coyle, P., Different susceptibilities to cerebral infarction in spontaneously hypertensive (SHR) and normotensive SpragueDawley rats, Stroke, 17 (1986) 520-524. 7 Coyle, P. and Jokelainen, P.T., Dorsal cerebral arterial collaterals of the rat, Anat. Rec., 203 (1982) 397-404. 8 Coyle, P. and Heistad, D.D., Blood flow through cerebral collateral vessels in hypertensive and normotensive rats, Hypertension, 8, Suppl. 2 (1986) 11-67-I1-71. 9 Coyle, P., Spatial relations of dorsal anastomoses and lesion border after middle cerebral artery occlusion, Stroke, 18 (1987) 1133-1140. 10 Coyle, P., Mechanisms of altered cerebral collaterals and protection from ischemic infarction during hypertension. (Abstract), Proceedings of the International Symposium on Cerebral Ischemia and Calcium, Chiemsee, F.R.G., June 12-15, 1988, p. 14. 11 Duverger, D., Benavides, J., Cudennec, A., MacKenzie, E.T., Scatton, B., Seylaz, J. and Verrecchia, C., A glutamate antagonist reduces infarction size following focal cerebral ischaemia independently of vascular and metabolic changes, J. Cereb. Blood Flow Metabol., 7, Suppl. 1 (1987) S144. 12 Duverger, D. and MacKenzie, E.T., The quantification of cerebral infarction following focal ischemia in the rat: influence of strain, arterial pressure, blood glucose concentration, and age, J. Cereb. Blood Flow Metabol., 8 (1988) 449-461. 13 Foster, A.C. and Wong, E.H., The novel anticonvulsant MK-801 binds to the activated state of the N-methyl-D-aspartate receptor in rat brain, Br. J. Pharmacol., 91 (1987) 403-409. 14 Germano, I.M., Pitts, L.H., Meldrum, B.S., Bartkowski, H.M. and Simon, R.P., Kynurenate inhibition of cell excitation decreases stroke size and deficits, Ann. Neurol., 22 (1987) 730-734. 15 Gill, R., Foster, A.C. and Woodruff, G.N., MK-801 is neuroprotective in gerbils when administered during the postischaemic
period, Neurosci. (Lond.), 25 (1988) 847-855. 16 Gotti, B., Duverger, D., Bertin, J., Carter, C., Dupont, R., Frost, J., Gaudilliere, B., MacKenzie, E.T., Rousseau, J., Scatton, B. and Wick, A., Ifenprodil and SL 82.0715 as cerebral antiischemic agents. I. Evidence for efficacy in models of focal cerebral ischemia, J. Pharmacol. Exp. Ther., 247 (1988) 12111221. 17 Kemp, J.A., Foster, A.C., Leeson, P.D., Priestly, T., Tridgett, R., Iversen, L.L. and Woodruff, G.N., 7-Chlorokynurenic acid is a selective antagonist at the glycine modulatory site of the N-methyl-D-aspartate receptor complex, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 6547. 18 Kemp, J.A., Foster, A.C. and Wong, E.H.E, Non-competitive antagonists of excitatory amino acid receptors, Trends Neurol. Sci., 10 (1987) 294-298. 19 Ozyurt, E., Graham, D.I., Woodruff, G.N. and McCulloch, J., Protective effect of the glutamate antagonist, MK-801 in focal cerebral ischemia in the cat, J. Cereb. Blood Flow Metabol., 8 (1988) 138-143. 20 Park, C.K., Nehls, D.G., Graham, D.I., Teasdale, G.M. and McCulloch, J., The glutamate antagonist MK-801 reduces focal ischemic damage in the rat, Ann. Neurol., 24 (1988) 543-551. 21 Park, C.K., Nehls, D.G., Graham, D.I., Teasdale, G.M. and McCulloch, J., Focal cerebral ischaemia in the cat: treatment with the glutamate antagonist MK-801 after induction of ischaemia, J. Cereb. Blood Flow Metabol., 8 (1988) 757-762. 22 Perkins, M.N. and Stone, T.W., An iontophoretic investigation of the actions of convulsant kynurenines and their interaction with the endogenous excitant quinolinic acid, Brain Research, 247 (1982) 184-187. 23 Rothman, S.M., Thurston, J.H. and Hauhart, R.E., Delayed neurotoxicity of excitatory amino acids in vitro, Neuroscience, 22 (1987) 471-480. 24 Schwarcz, R. and Meldrum, B., Excitatory amino-acid antagonists provide a therapeutic approach to neurological disorders, Lancet, 20 (1985) 140-143. 25 Simon, R.P., Swan, J.H., Grifith, T. and Meldrum, B.S., Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain, Science, 226 (1984) 850-852. 26 Swan, J.H., Evans, M.C. and Meldrum, B.S., Long-term development of selective neuronal loss and the mechanism of protection by 2-amino-7-phosphonoheptanoate in a rat model of incomplete forebrain ischaemia, J. Cereb. Blood Flow Metabol., 8 (1988) 64-78. 27 Tamura, A., Graham, D.I., McCulloch, J. and Teasdale, G.M., Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion, J. Cereb. Blood Flow Metabol., 1 (1981) 53-60. 28 Van Harreveld, A. and Fifkova, E., Light- and electronmicroscopic changes in central nervous tissue after electrophoretic injection of glutamate, Exp. Mol. Pathol., 15 (1971) 61-81.
We thank Dr. Violetta Dimitriadou for her help with the histological aspect of the study, and J. Leizerovici for secretarial assistance. This study was supported by I.N.S.E.R.M., C.N.R.S., Universit6 Paris VII and D.R.E.T. (project 88048).
353
Elsevier BRES 24114
Kynurenate does not reduce infarct size after middle cerebral artery occlusion in spontaneously hypertensive rats Simon Roussel, Elisabeth Pinard and Jaques Seylaz Laboratoire de Physiologie et Physiopathologie c~r~brovasculaire, U. 182 I.N.S.E.R.M., U.A. 641 C.N.R.S., Paris (France)
(Accepted 20 February 1990) Key words: Focal cerebral ischemia; Infarct size; Excitatory amino acid; Glycine; Spontaneously hypertensive rat
The effects of the excitatory amino acid receptor antagonist, kynurenate, were investigated in spontaneously hypertensive rats after middle cerebral artery occlusion. Kynurenate did not significantlymodify either the infarct volume, measured 48 h after occlusion, or the neurological score. The absence of a neuroprotective effect of kynurenate in this study, which contrasts with results in normotensive rats, is suggested to be due to impaired collateral circulation in spontaneously hypertensive rats.
It has been suggested that excitatory amino acids (EAAs) may be important intermediates in the neuronal degeneration which follows several diseases of the central nervous system 24. These transmitters and their agonists are neurotoxic at high concentrations and induce 'ischemic cell changes' both in vitro and in vivo 5'2s. This excitotoxicity has been shown to be blocked in vitro by antagonists of the N-methyl-D-aspartate subtype of glutamate receptors 23. During global ischemia, massive amounts of excitatory amino acids are released into the extracellular space 2, where they are believed to elicit neurotoxicity. Over the last few years, several authors have reported that blockade of excitatory neurotransmission can prevent or limit the delayed neuronal degeneration after global cerebral ischemia 15'25,26, although this effect is controversial 4. There is also some evidence that competitive and non-competitive blockade of glutamate receptors reduces the infarct volume produced by middle cerebral artery occlusion in normotensive rats 11"14A6"2°. Only non-competitive blockade of glutamate receptors has been shown to exert such a protection in normotensive cats 19'21 and in hypertensive rats 1°. The aim of the present study was to evaluate the therapeutic value of E A A receptor blockade in a model of focal cerebral ischemia in spontaneously hypertensive rats (SHRs). SHRs were chosen for two reasons: firstly, because hypertension is the major risk factor for stroke, secondly because occlusion of the middle cerebral artery (MCA) in this strain leads to an infarct volume which is highly reproducible 12, thus providing an excellent model of focal ischemia for pharmacological evaluation. Gluta-
mate receptors were blocked with the broad spectrum E A A antagonist kynurenate22; it is competitive for kainate and quisqualate receptors 3, and acts at the neuromodulatory glycine site of N-methyl-D-aspartate (NMDA) receptor 17. The investigations were performed on 18 male SHRs (Iffa-Credo, l'Arbresle, France), weighing 280-300 g, which had free access to food and water prior to experimentation. The rats were anesthetized with halothane (1.5%) in 02. Rectal temperature was measured and maintained within the normal physiological range (36.5-37.5 °C) during surgery. A clear vinyl catheter was chronically placed in the peritoneal cavity and canalized subcutaneously to exit at the neck. It was used for intraperitoneal drug administration. Left MCA occlusion was performed by a subtemporal approach, essentially as described by Tamura et al. (1981) 27. Rats were placed in the lateral position, a vertical incision was made between the left orbit and the external auditory canal. The temporal muscle was divided midway vertically and reflected forward and downward with a retractor. The left eye and the parotid gland were left in place, but the posterior half of the zygomatic bone was removed. The inferotemporal fossa was exposed under a surgical microscope, and a small craniectomy was made using a dental drill. The dura was incised and reflected. The arachnoid was then divided on either side of the artery. Bipolar microcoagulation forceps were used to occlude the M C A from the point where it crosses the inferior cerebral vein to the superior aspect of the olfactory tract. The lenticulostriate arteries were also coagulated. The
Correspondence: S. Roussel, Laboratoire de Physiologie et Physiopathologie crrEbrovasculaire, 10, avenue de Verdun, 75010 Paris, France.
0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
354 skin was then sutured, anesthesia was discontinued and the animals were returned to their cages. A simple neurological examination was performed on 4 occasions: before anesthesia, 2 h after occlusion when the animals were awake, 24 h and 48 h after occlusion. The neurological grades were determined as described by Bederson et al. (1986)1: animals were graded 0 when they had none of the tested neurological deficits, 1 when they exhibited assymetrical forelimb flexion when held by the tail, 2 when they had decreased resistance to a right lateral push, and 3 when they displayed circling behavior. Immediately after the last neurological examination (48 h after occlusion), the animals were anesthetized with 2.5% halothane in 0 2 and perfusion-fixed with F.A.M. (1/10 formaldehyde, 1/10 acetic acid, 8/10 absolute methanol, v/v) via a catheter placed in the ascending aorta through the apex of the left ventricle. The brain was carefully removed, and prepared for paraffin embedding. Ten/~m thick sections were taken at 1 mm intervals and stained with Cresyl violet. The volume of infarction in the cortex and striatum was calculated from the surface area of necrotic tissue at each level, determined with an image analyzer (Histopericolor, MS21), and the distance between succeeding slices. The animals were randomly divided in two groups, and treated according to the following protocols: group 1 (n = 10): each control rat was given saline at the same times and in the same volumes as kynurenate in group 2; group 2 (n = 8): each rat was given kynurenate (500 mg/kg; pH = 7.4) 30 min before induction of ischemia and 3.5 h, 7,5 h and 24 h after occlusion. Kynurenate (Sigma Chemical) was dissolved in a small amount of 1 M NaOH and diluted in 0.9% NaCI saline. The pH was adjusted to 7.4 with HCI. The comparison of infarct volumes was performed using Student's t-test. Neurological scores were compared using the Kolmogorov-Smirnov one-sample test. As previously described, MCA occlusion induced a well-delineated infarction, embracing part of the striatum and cortex. The mean volume of infarction in our study (176.3 + 8.8 ram3; mean + S.E.) is in good agreement with that found by others 12. This volume was not significantly changed by kynurenate (Table I). Likewise, the neurological score was not significantly changed by the drug (Table I1). No rats died in either treated or non-treated groups. The present results differ from those obtained in normotensive animals with both competitive and noncompetitive E A A antagonists (thienyl-cyclohexyl-piperidine 1l, kynurenate ~4, MK-801 (ref. 20) or SL 82.0715 (ref. 16)), and also from those obtained in SHRs with MK-801 (ref. 10): these results indicated that blockade of E A A receptors reduced infarct size. The difference
FABLE 1 Infarct size (ram ~) 48 h after middle cerebral artery occlusion
Data are means + S.E. There are no significant differences between groups (Student's t-test). Groups of animals
Cortex
Caudate nucleus
Total
n
Control Kynurenate
140.0 -+ 8.3 131.9 + 9.5
36.3+ 3.2 28.1 +_4.2
176.3 + 8.8 160.0 -+9.9
10 8
between results obtained in SHRs with kynurenate and MK-801 may be due to their differing sites of action. Kynurenate acts as an aliosteric regulator of the NMDA receptor at the glycine site ~7, whereas MK-801 blocks the calcium channel associated with the NMDA receptor TM. Furthermore, the blockade with MK-801 is enhanced by the presence of an agonist of the receptor ~3, and hence is enhanced during ischemia by the massive release of glutamate. The present results in SHRs can be directly compared with the results obtained in normotensive rats. Germano et al. 14 administered kynurenate to MCA-occluded normotensive rats with timings and doses comparable to those used in the present study and found an attenuation of the infarction size and an improvement of the neurologic outcome. This discrepancy may be tentatively attributed to differences in the pathophysiological consequences of focal ischemia in SHRs and normotensive rats. MCA occlusion leads to infarction in young SHRs, but not in young normotensive rats 6. The infarction is larger and more reproducible in adult SHRs than in adult normotensive rats 12. These differences could result from impaired collateral circulation from pial anastomoses for 3 reasons. The distance between pial anastomoses and the lesion border is proportional to the diameter of the anastomosis 9, the mean luminal diameter of the anastomosing A C A - M C A collaterals is smaller in stroke prone SHRs (SHRSPs) than in normotensive Wistar-Kyoto rats 7, and finally blood flow in the territory of the
TABLE II Neurological score after middle cerebral artery occlusion
All animals had a neurological score of 0 before occlusion. A: 2 h after occlusion; B: 24 h after occlusion; C: 48 h after occlusion. Average: Average of the neurological scores at the 3 times. Data are means -+ S.E. There are no significant differences between groups (Kolmogorov-Smirnovone-sample test). Groups of animals
A
B
Control Kynurenate
2.1 + 0.2 2.1+0.1 2.1_+0.1 1.7 -+0.2
C
Average
2.4+_0.2 2.2+-0.1 2.1+0.1 2.0+0.1
n
10 8
355 occluded vessel is lower in S H R P S P s than in n o r m o t e n sive rats 8. It is t h e r e f o r e very likely that there is a threshold of local b l o o d flow above which inhibition of E A A receptors protects neurones from ischemic degeneration, and that the a r e a in which blood flow is above this threshold in S H R s is too small to reveal any effect. In conclusion, occlusion of the middle cerebral artery in S H R s is of great interest as a m o d e l of ischemia for p h a r m a c o l o g i c a l evaluation, not only because of the high reproducibility of infarct size, but also because hypertension is the m a j o r risk factor for h u m a n stroke. H o w e v e r , the present study emphasizes the fact that
results o b t a i n e d with this strain m a y not be e x t r a p o l a t e d to o t h e r strains, p r o b a b l y because of a g r e a t e r reduction in the cerebral circulation in the ischemic territory of S H R s than of normotensive rats. A clear understanding of the differences b e t w e e n the pharmacological responsiveness of normotensive and hypertensive animals is thus of m a j o r importance.
1 Bederson, J.B., Pitts, L.H., Tsuji, M., Nishimura, M.C., Davis, R.L. and Bartkowski, H., Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination, Stroke, 17 (1986) 472-476. 2 Benveniste, H., Drejer, J., Schousboe, A. and Diemer, N.H., Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis, J. Neurochem., 43 (1984) 1369-1374. 3 Birch, P.J., Grossman, C.J. and Hayes, A.G., Kynurenate and FG9041 have both competitive and non-competitive antagonist actions at excitatory amino acid receptors, Eur. J. Pharmacol., 151 (1988) 313-315. 4 Block, G.A. and PulsineUi, W.A., Excitatory amino acid receptor antagonists: failure to prevent ischemic neuronal damage, J. Cereb. Blood Flow Metabol., 7, Suppl. 1 (1987) S149. 5 Choi, D.W., Maulucci-Gedde, M. and Kriegstein, A., Glutamate neurotoxicity in cortical cell culture, J. Neurosci., 7 (1987) 357-368. 6 Coyle, P., Different susceptibilities to cerebral infarction in spontaneously hypertensive (SHR) and normotensive SpragueDawley rats, Stroke, 17 (1986) 520-524. 7 Coyle, P. and Jokelainen, P.T., Dorsal cerebral arterial collaterals of the rat, Anat. Rec., 203 (1982) 397-404. 8 Coyle, P. and Heistad, D.D., Blood flow through cerebral collateral vessels in hypertensive and normotensive rats, Hypertension, 8, Suppl. 2 (1986) 11-67-I1-71. 9 Coyle, P., Spatial relations of dorsal anastomoses and lesion border after middle cerebral artery occlusion, Stroke, 18 (1987) 1133-1140. 10 Coyle, P., Mechanisms of altered cerebral collaterals and protection from ischemic infarction during hypertension. (Abstract), Proceedings of the International Symposium on Cerebral Ischemia and Calcium, Chiemsee, F.R.G., June 12-15, 1988, p. 14. 11 Duverger, D., Benavides, J., Cudennec, A., MacKenzie, E.T., Scatton, B., Seylaz, J. and Verrecchia, C., A glutamate antagonist reduces infarction size following focal cerebral ischaemia independently of vascular and metabolic changes, J. Cereb. Blood Flow Metabol., 7, Suppl. 1 (1987) S144. 12 Duverger, D. and MacKenzie, E.T., The quantification of cerebral infarction following focal ischemia in the rat: influence of strain, arterial pressure, blood glucose concentration, and age, J. Cereb. Blood Flow Metabol., 8 (1988) 449-461. 13 Foster, A.C. and Wong, E.H., The novel anticonvulsant MK-801 binds to the activated state of the N-methyl-D-aspartate receptor in rat brain, Br. J. Pharmacol., 91 (1987) 403-409. 14 Germano, I.M., Pitts, L.H., Meldrum, B.S., Bartkowski, H.M. and Simon, R.P., Kynurenate inhibition of cell excitation decreases stroke size and deficits, Ann. Neurol., 22 (1987) 730-734. 15 Gill, R., Foster, A.C. and Woodruff, G.N., MK-801 is neuroprotective in gerbils when administered during the postischaemic
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We thank Dr. Violetta Dimitriadou for her help with the histological aspect of the study, and J. Leizerovici for secretarial assistance. This study was supported by I.N.S.E.R.M., C.N.R.S., Universit6 Paris VII and D.R.E.T. (project 88048).
