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Protective effects of minaprine in infarction produced by occluding middle cerebral artery in stroke-prone spontaneously hypertensive rats
Gen. Pharmac. Vol. 22, No. 1, pp. 143-150, 1991 Printed in Great Britain.All rightsreserved
0306-3623/91 $3.00+ 0.00 Copyright © 1991PergamonPress pie
PROTECTIVE EFFECTS OF MINAPRINE IN INFARCTION PRODUCED BY OCCLUDING MIDDLE CEREBRAL ARTERY IN STROKE-PRONE SPONTANEOUSLY HYPERTENSIVE RATS SHIGERUOKUYAMA,HIROKOSHIMAMURA-HARADA,YASUKOKARASAWA,KAZUAKIKAWASHIMA, HIROAKIARAKI,MASAAKIKIMURA*,SUSUMUOTOMOand HIRONAKAAIHARA Department of Pharmacology and *Department of Toxicology, Research Center, Talsho Pharmaceutical Co., Ltd, 1-403, Yoshino-cho, Ohmiya, Saltama 330, Japan (Received 20 March 1990)
Abstract--1. We examined the effects of minaprine on cerebral infarction produced by occluding of unilateral middle cerebral artery (MCA) above the rhinal fissure in stroke-prone spontaneously hypertensive rats (SHRSP). 2. Oral administration of minaprine (60 mg/kg/day) and vehiclewere started 30 rain after the occlusion of MCA, and continued for 6 days. 3. The brain was dissected out and 36 coronal multiple sections of the whole brain were histologically prepared to determine the location and extension of infarction. 4. The infarcted area produced by the occlusion of MCA was limited to the cerebral cortex. 5. Body weight of the minaprine-treated rats gradually decreased within 4 days after the occlusion of MCA and thereafter increased, whereas in the vehicle-treated rats, there was a gradual decrease during the experimental period. 6. Size of the infarcted area was serially measured, in each section, using a microcomputer imaging device. In all animals with an occluded MCA, there was a typical pattern of ischemic damage. 7. Post-treatment of MCA occluded SHRSP with minaprine resulted in reduction in infarct size, as compared to findings in the vehicle-treated controls. 8. The pharmacological and histopathological effectsof minaprine on the progress of cerebral infarction produced by the occlusion of MCA in SHRSP are discussed.
INTRODUCTION
Occlusion of the rat middle cerebral artery (MCA) has been widely used for a model of focal cerebral ischemia. Tamura et al. (1981) reported a procedure for occluding the proximal MCA in normotensive rats. Here, the brain damage produced by ischemia was located in the cortex and the lateral part of the striatum. However, this procedure is technically difficult and most invasive. Coyle (1982) demonstrated a less invasive surgical approach with MCA occlusion above the rhinal fissure, but cerebral infarction in normotensive rats did not occur. In contrast, a reproducible focal cerebral infarction was developed after the same occlusion in stroke-prone spontaneously hypertensive rats (SHRSP) (Coyle and Jokelainen, 1983). The internal diameter of the anterior cerebral artery (ACA) and MCA branches decreases with aging in the SHRSP (Coyle, 1987). The ACA-MCA anastomoses in the SHRSP are smaller in internal diameter than those in the normotensive rats (Coyle, 1987a). The cerebral vascular resistance in SHRSP is greater than in the normotensive rats (Coyle, 1987a; Werber and Heistad, 1984). Changes in the vascular wall of the SHRSP are linked to the hypertensive state (Ogata et al., 1980; 1981). Yamori et al. (1976) reported pathogenetic similarity of strokes in SHRSP and humans. Thus, it seems that the SHRSP is an unique animal model for studying cerebral infarction and/or stroke.
Minaprine, 3-(2-morpholino-ethylamino)-4-methyl6-phenyl-pyridazine dihydrochloride, is a novel psychotropic drug with antidepressant and psychostimulatory effects (Biziere eta/., 1982; 1984; Worms et aL, 1986). In clinical studies, minaprine seems to be an effective and safe antidepressant (Biziere et al., 1982; Jouvent et al., 1984; Mikus et al., 1985; Radmayr et aL, 1986). Passeri et al. (1985) also reported that minaprine improved depression scores and behavior in demented patients and seemed to be more effective in multiinfarct dementia than in cases of the senile dementia of Alzheimer type. We obtained evidence that minaprine has protective effects on cerebral hypoxia, anoxia and ischemia, in experimental animals (Okuyarna et al., 1988a). Araki et al. (1987) reported that minaprine has preventive effects on hippocampal delayed neuronal death and abnormalities on the electroencephalogram in Mongolian gerbils with occluded common carotid arteries. We report here the effects of minaprine on the progress of cerebral infarction produced by occlusion of MCA in SHRSP. MATERIALS AND METHODS
Twelve male stroke-prone spontaneously hypertensive rats (SHRSP) at 11 weeks of age, weighing 220-267 g, were housed in an air-conditioned room at 22 + I°C with 12 hr light-dark schedule (lights on at 07:00 a.m.). The animals had become thoroughly familiar with being handled. Great care was taken with gentle handling and avoidance of pain. 143
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SHIGERU OKUYAMA
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Fig. 1. Burr hole target region for exposure of MCA.
Tail systolic blood pressure in each conscious animal was measured just before the occlusion of MCA, using a rat tail manometer-tachometer system (KN-210, Natsume). The rats were anesthetized with sodium pentobarbital (50 mg/kg, given intraperitoneally) and body temperature was kept between 37°C and 37.5°C, using a heat pad controlled by a rectal thermometer (KN-474, Natsume). Under an operating microscope (SM-5, Nikon) the left MCA was exposed through a burr-hole craniectomy (Fig. 1) 2-3 mm diameter performed by a transtemporal route, without damage to the zygomatic bone, then the artery was dissected free of the meninges. The MCA was occluded with a microbipolar coagulation (MICRO-3D, Miziho), using a low power setting and was cut to ensure completeness of the vascular occlusion. The occlusion was distal to the striate branches of the MCA and 0.7-1 mm dorsal to the rhinal fissure. The soft tissues were put back into place and the skin sutured. Oral administrations of minaprine dihydrochloride (60 mg/kg) and vehicle (saline) were started 30 min after the occlusion of MCA and continued once daily for 7 days. Minaprine dihydrochloride (Taisho) was dissolved in saline solution (0.9% NaCI solution, 1 ml/kg, body weight). The dosage used was similar to that used for cerebral protective effects, in our laboratory (Araki et al., 1987; Okuyama et al., 1988a). Arterial blood pressure and plasma glucose levels remained unchanged with a dose of 60 mg/kg of minaprine (unpublished observation). At 24 hr after the final administrations of minaprine and vehicle, the arterial blood gas was measured using a pH blood gas analyzer (model 168, Corning). Blood for analyses totalling 0.5 ml was obtained via the tail artery. Neurological test used was similar to that described by Coyle and Jokelinen (1983). Each rat was placed on the 7 mm surface of a horizontally suspended wooden meter stick. Observations were made on fore- and hind-limb digits, feet legs and thighs, their locations and symmetrics during station, gait, crawling or running on a wooden stick. The animals were evaluated for motor deficits once daily during the experimental period by one of us (K.K.) who was unaware of the pharmacological treatment given the animals. Quantitative morphometry was performed on histologic slides, using the following method. At 24 hr after the final administrations of minaprine and vehicle, the animals used for the morphometry, including histopathological examination, were anesthetized with pentobarbital (50mg/kg, intraperitoneal injection), and the brains were respectively perfused with a 10% buffered formalin solution given
through the left cardiac ventricle. The brains were dissected out and fixed in 10% buffered formalin solution. Serial sections (about 5 #m-thick) were cut coronally from the paraffin block of the whole brain. Of all the sections prepared, 36 sections taken at the same intervals were stained with hematoxylin and eosin (H.E.). Each slide was examined histopathologically for determination of the infarcted area, using conventional light microscopy (XF-EFD2, Nikon) by one of us (M.K.) who was unaware of the pharmacological treatment given the animals. In the examination of each slide, the infarcted areas were defined according to the criteria shown in Fig. 2. The lesion was limited to outer layers of the unilateral cerebral cortex and all deeper layers were spared. The infarcted areas were serially measured on each slide, (36 slides/rat) using a microcomputer imaging device ( R a m m e t al., 1984) (Imaging Research Inc.-Muromachi Neuroscience). Percent infarction of each rat was calculated as follows. % infarction of each section (IES) infarct area (mm 2) x 100 total section area (mm 2) % infarction of each rat = average of 36 sections of IES Statistical analysis was performed using an one-way ANOVA followed by Student's t-test for individual comparisons. A P < 0.05 was regarded as being statistically significant. RESULTS
Systolic blood pressure T h e r e was n o significant difference in pre-occlusion systolic b l o o d pressure values between vehicle- (237.1 + 3.7 m m H g , n = 6) a n d m i n a p r i n e - t r e a t e d animals (237.3 __+9.1 m m H g , n = 6).
Body weight Body weight in the vehicle-treated animals gradually decreased d u r i n g the experimental period. Weight o f the m i n a p r i n e - t r e a t e d rats decreased within 4 days
Fig. 2. Cerebral sections of the infarcted area (H.E. stain x 50). (a) Severely infarcted area. Necrotic focus and loss of the normal cytological architecture. The tissue is less intensely stained and neuronal cell nuclei are few. The neurophil looks spongy. The left portion of the photograph shows numerous macrophages. (b) Mild or moderately infarcted area. The neurons show various ischemic cell changes, though the normal cytological architecture is partly maintained. Neuronal cell somas are not clearly outlined and the neurons are fragmented. Macrophages are also evident. These features characterize the irreversible brain lesion. Histopathological definitions of each area (a, b) are given on the right. Fig. 4. Cerebral infarcted areas of the vehicle-treated animal (a) and minaprine-treated animal (b) (H.E. stain x 20). Photograph (b) shows small size of the infarcted area, compared to that in photograph (a).
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mmHg and 7.50 + 0.02, respectively, and those in tll minaprine-treated group were 90.67 + 4.20 mmH! 33.38 + 2.28 mmHg and 7.47 + 0.03, respectivel.~ Blood gas and pH were within the normal range i both the vehicle- and minaprine-treated animal (Hoffman, Albrecht and Miletich, 1982), and thel was no statistical difference among the two groups c animals (Student's t-test). Oaya aftor MCAO
Fig. 3. Changes in body weight after MCA occlusion (MCAO) in vehicle- and minaprine-treated animals. *P < 0.05 vs vehicle-treated group (one-way ANOVA followed by Student's t-tes0. after the occlusion of MCA, and thereafter there was a gradual reversion the pre-treatment level. Body weight in the minaprine-treated group was significantly (P < 0.05, Student's t-test) higher than in the vehicle-treated group, on day 6 (Fig. 3).
Measurement of arterial blood gases The values of PO2, PCO2 and pH in the vehicletreated group were 78.12 _+ 5.39 mmHg, 31.57 _+ 2.52
Effect of minaprine There was a common pattern of ischemic brai damage (Fig. 2) in all animals with occlusion c MCA. Tissue sections of the lesion were less intensel stained with H.E. than were the normal tissue~ Location of the infarcted area was limited to th ipsilateral cerebral cortex and in no rat did : extend to the contralateral one. The ipsilateral bas, ganglia showed no ischemic damage. The lesion w~ characterized by the following features: edema in th pia matter, liquefaction with macrophages (granula fatty cells) on the lesion surface, and the abow mentioned findings (Fig. 2). Hemorrhage and macrc phages with pigmented granules were sometime evident.
Fig. 5. Serial 36 coronal sections of the whole brain in vehicle-treated aminals. The decreased intensity of the H.E. stained re#on is the lesion. The numbers show the anterioposterior distance (#m) from the coronal plane, according to the atlas of Kfnig and Klippel (1963). GP 2~/1--J
14
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SHIGERUOKUYAMAet al.
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Fig. 6. Serial 36 coronal sections of the whole brain in minaprine-treated animals. The decreased intensity of the H.E. stained region is the lesion. The numbers show the anterioposterior distance (#m) from the coronal plane, according to the atlas of K6nig and Klippel (1963). In the minaprine-treated animals, there was a small area of ischemic damage and somewhat larger numbers of macrophages on the lesion surface, as 15I
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Fig. 7. Effects of vehicle and minaprine on the occlusion of MCA produced infarction in SHRSP. Oral administrations of minaprine (60 mg/kg) and vehicle (saline) were begun 30 min after the occlusion of MCA and were continued once a day for 7 days.
compared to findings in the vehicle-treated animals (Fig. 4). However, there was no clear difference in quality between the two groups. Typical topography of the infarct in S H R S P is shown in Fig. 5 (vehicle-treated animal) and Fig. 6 (minaprine-treated animal), respectively. As shown in Fig. 7, the average infarction size in S H R S P with treatment of the vehicle (n = 6) was 11.24 _+ 1.55% (mean + SEM). In contrast, in the minaprine-treated animals (n = 6, an oral dose of 60 mg/kg for 7 days), the infarct size was 6.27 _+ 0.94% (mean + SEM), the difference in infarct size between the groups being significant (Student's t-test, P < 0.05). Motor deficits after the occlusion of M C A , as tested using the horizontally suspended wooden meter stick, were never evidenced, in either the vehicle- or the minaprine-treated animals. DISCUSSION We made use of the M C A occluded focal ischemic model in SHRSP. The site of occlusion of M C A was made above the rhinal fissure, for the following reasons, (i) S H R S P is an appropriate pathogenetic model for studies on stroke (Yamori et al., 1976),
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Minaprine and infarction (ii) the procedure is technically easily and is less invasive, (iii) location of infarction is limited to the cerebral cortex and does not extend to the basal ganglia. These observations are in accord with the data of Coyle (1983) who reported a systematic approach employing a relatively non-invasive surgical procedure, in the SHRSP. Thus, the development of a predictable large cortical infarct was established. In the present study, the whole brain was serially dissected out and 36 sections of the same intervals were examined. The location and extension of the infarcted area produced by the occlusion of MCA were determined. Size of the infarction was measured using each section and a microcomputer imaging device ( R a m m e t al., 1984). Thus, the experimental procedure we used provides a quantitative determination of the infarcted area. In addition, for evaluation of the infarcted area and histopathology of ischemic change and neurological tests, all experiments were carried out in a randomized and blinded fashion. Although the MCA occluded SHRSP showed evidence of a large cortical infarction, motor deficits were not apparent. One possible explanation for this is that location of the infarcted area was limited to the cerebral cortex. The striatum is one of the most important regions related to the development of motor function (Kamata et al., 1987). The lack of motor deficits in the MCA occluded SHRSP may be related to location of the infarction. However, Coyle and Jakelainen (1983) reported that the occlusion of MCA in SHRSP caused an asymmetrical fore-foot placement and hind-foot location and that the infarcted area was limited to the cerebral cortex. Minaprine, administered post-ischemia, protected against the progress of ischemic neuronal damage in the SHRSP. Branches of the MCA anastomose with branches of ACA on the dorsal aspect of the cerebral hemisphere, in the SHRSP (Coyle, 1987b; Coyle and Heistad, 1987). Occlusion of the MCA above the rhinal fissure reduced the cerebral blood flow to the territory of the occluded MCA, hence, the cortical tissue is infarcted downstream from the pial surface of the MCA-ACA anastomoses (Coyle, 1987b). Minaprine has preventive effects on hippocampal delayed neuronal death in mongolian gerbils with occluded common carotid arteries (Araki et al., 1987), and also a potent protective effect against cerebral hypoxia, anoxia and ischemia, in a variety of experimental animal models (Okuyama et al., 1988a). Thus, it is reasonable to assume that the systemically administered minaprine arrives at the MCA-ACA anastomoses via ACA. Minaprine may protect against the progress of secondary lesion development downstream from the anastomoses. In fact, systemically administered minaprine rapidly enters the central nervous system and then distributes fairly evenly in various regions beyond the blood-brain barrier (Caccia et al., 1985). Body weight of the minaprine-treated animals was significantly higher than that in the vehicle-treated animals, on day 6 after occluding the MCA. Therefore, minaprine may improve systemic conditions by inhibiting of the progress of infarction. Nimodipine (Gotoh et al., 1986; Sauter and Rudin, 1986), PN 200-110 (Sauter and Rudin, 1986),
nizofenone (Tamura et aL, 1979) and barbiturates (Tamura et al., 1979; Nehles et al., 1987) reduce the extension of infarction in the MCA occlusion model. Tamura et al. (1979) reported that depression of cerebral metabolic rates by nizofenone or barbiturates is one of the principal reasons for their proteefive action. However, minaprine did not depress the cerebral metabolic rates, in case of the [~4C]2-deoxyglucose method (unpublished observation). Electrophysiological investigations showed that the firing rate in the brain stem reticular formation was reduced by pentobarbital and that this reduction was overcome by minaprine (Okuyama and Aihara, 1988c). Thus, the mode of action of minaprine differs from that of nizofenone and barbiturates. Sauter and Rudin (1986) suggested that a potential mechanism of the dihydropridine calcium antagonist, nimodipine or PN 200--110, reduced the ATP demand of neurons required for the maintenance of intraceUular calcium homeostasis, by reducing excessive calcium influx during ischemia. The effect of minaprine on calcium homeostasis is now under investigation in our laboratory. Decrease in neurotransmitter turnover rates in the territory of MCA occlusion leads to a neuronal necrosis (Sauter and Rudin, 1986). This was also noted in case of a lesion with neurotoxin (Hefti, Melamed and Wurtman, 1980; Kamata et al., 1987). Minaprine activates central dopaminergic (Biziere et al., 1984), serotonergic (Biziere et al., 1982) and cholinergic (Garattini et al., 1984; Biziere et al., 1986) neurotransmissions. Moreover, the facilitation of cortical synaptic transmission (Okuyama and Aihara, 1988b) and the potentiation of hippocampal electrical activity (Okuyama and Aihara, 1989) were also observed. Thus, the protective effect of minaprine against the occlusion of MCA produced infarction may be related to activation and/or facilitation of central neuronal transmission. Acknowledgement--We
thank M. Ohara for helpful
comments. REFERENCES
Araki H., Nojiri M., Kimura M. and Aihara H. (1987) Effect of minaprine on "delayed neuronal death" in mongolian gerbils with occluded common arteries. J. Pharmac. Exp. Ther. 242, 686-691. Biziere K., Kan J. P., Souilhac J., Muyard J. P. and Roncucci R. (1982) Pharmacological evaluation of minaprine dihydrochloride, a new psychotropic drug. Arzneim.-Forsch. 32, 824-831. Biziere K., Gueudet C., Kan J. P., Souilhac J. and Worms P. (1984) Atypical dopaminergic profile of minaprine, a new antidepressant drug. Br. J. PharmacoL Suppl. 81, 51p. Biziere K., Gueudet C. and Worms P. (1986) Further evidence for a ehofinomimetie activity of minaprine. Br. J. Pharmae. Suppl. 87, 31p. Cae¢ia S., Fossati T. and Maneinelli A. (1985) Disposition and metabolism of minaprine in the rat. Xenobiotiea 15, 1111-1119, Coyle P~ (1982) Middle cerebral artery occlusion in the young rat. Stroke 13, 855-859. Coyle P. and Jokelainen P. T. (1983) Differential outcome to middle cerebral artery occlusion in spontaneously hypertensive stroke-prone rats (SHRSP) and Wistar Kyoto (WKY) rats. Stroke 14, 605-611.
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Coyle P. (1987a) Dorsal cerebral collaterals of stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar Kyoto (WKY). Anat. Rec. 218, 40-44. Coyle P. (1987b) Spatial relations of dorsal anastomoses and lesion border after middle cerebral artery occlusion. Stroke 18, 1133-1140. Coyle P. and Heistad D. D. (1987) Blood flow through cerebral collateral vessels one month after middle cerebral artery occlusion. Stroke 18, 407-411. Garattini S., Forloni G. L., Tirelli S., Ladinsky H. and Consolo S. (1984) Neurochemical effects of minaprine, a novel psychotropic drug, on the central cholinergic system of the rat. Psychopharmac. 82, 210-214. Gotoh O., Mohamed A. A., McCulloch J., Graham D. I., Harper A. M. and Teasdale G. M. (1986) Nimodipine and the haemodynamic and histopathological consequences of middle cerebral artery occlusion in the rat. J. Cereb. Blood Flow Metab. 6, 321-331. Hefti F., Melamed E. and Wurtman R. J. (1980) Partial lesions of the dopaminergic nigrostriatal system in rat brain: Biochemical characterization. Brain Res. 195, 123-137. Hoffman W. E., Albrecht R. F. and Miletich D. J. (1982) Cerebrovascular and metabolic effects of SNP-induced hypotension in young and aged hypertensive rats. Anesthesiology 56, 427-430. Jouvent R., Lancrenon S., Patay M., Biziere K. and Wildlocher D. (1984) A controlled study: minaprine versus placebo in inhibited depressed out-patients. Int. Syrup. at the first European Conference on Minaprine, pp. 33-40. Kamata K., Okuyama S., Hashimoto S., Kimura M., Aihara H. and Kasuya Y. (1987) Atrophy of the striatum and disturbance induced by colchicine. Brain Res. 421, 353-357. K6nig J. R. and Klippel R. J. (1963) The Rat Brain. A Stereotaxic of the Forebrain and Lower Parts o f the Brain Stem. The Williams and Wilkins Company, Baltimore. Mikus P., Biziere K. and Bentel U. (1985) A controlled study of minaprine and nomifensine in outpatients with masked depression. Clin. Trial J. 22, 477-488. Nehles D. G., Todd M. M., Spetzler R. F., Drummond J. C., Thompson R. A., Johnson P. C., Mendenhall S. E., Barstow C. A., Carmichael P. G., Yound D. M., Fifield M. S. and Jones C. P. (1987) A comparison of the cerebral protective effects of isoflurane and barbiturates during temporary focal ischemia in primates. Anesthesiology 66, 453-464. Ogata J., Fujishima M., Tamaki K., Nakatomi Y., Ishitsuka T. and Omae T. (1980) Stroke-prone spontaneously hypertensive rats as an experimental model of malignant hypertension. I. A light- and electron-microscopic study of the brain. Acta Neuropathol. (Berlin) 51, 179-184.
Ogata J., Fujishima M., Tamaki K., Nakatomi Y., Ishitsuka T. and Omae T. (1981) Vascular changes underlying cerebral lesions in stroke-prone spontaneously hypertensive rats. Acta Neuropathol. (Berlin) 54, 183-188. Okuyama S., Karasawa Y., Shiratsuchi Y. Araki H. and Aihara H. (1988a) Protective effects of minaprine on the cerebrum of rodents. Res. Comm. Chem. Pathol. Pharmacol. 60, 381-395. Okuyama S. and Aihara H. (1988b) Effect of minaprine on synaptic transmission in the neocortex of the rat in vivo. Neuropharmacology 27, 915-922. Okuyama S. and Aihara H. (1988c) Effect of minaprine on neuronal activity in the brain stem reticular formation of the rat. Arch. Int. Pharmacodyn. Ther. 296, 246-254. Okuyama S. and Aihara H. (1989) Effect of minaprine on electrical activity of the rat hippocampus in vivo. NaunynSchmiedeberg's Arch. Pharmacol. 340, 1-5. Passeri M., Cucinotta D., De Mello M., Storchi G., Roncucci R. and Biziere K. (1985) Minaprine for senile dementia. Lancet I, 824. Radmayr R., Biziere K. and Bentel U. (1986) A doubleblind controlled study of minaprine and maprotiline in endogenous type depression. Clin. Trail J. 23, 100-109. Ramm P., Kulick J. H., Stryker M. P. and Frost B. J. (1984) Video and scanning microdensitometer-based imaging systems in autoradiographic densitometry. J. Neurosci. Meth. 11, 89-100. Sauter A. and Rudin M. (1986) Calcium antagonists reduce the extent of infarction in rat middle cerebral artery occlusion model as determined by quantitative magnetic resonance imaging. Stroke 17, 1228-1234. Tamura A., Asano T., Sano K., Tsumagari T. and Nakajima A. (1979) Protection from cerebral ischemia by a new imidazole derivative (Y-9179) and pentobarbital. A comparative study in chronic middle cerebral artery occlusion in cats. Stroke 10, 126-134. Tamura A., Graham D. I., McCulloch J. and Teasdale G. M. (1981) Focal cerebral ischaemia in the rat: I. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. J. Cereb. Blood Flow Metabol. 1, 53-60. Werber A. H. and Heistad D. D. (1984)Effects of chronic hypertension and sympathetic nerves on the cerebral microvasculature of stroke-prone spontaneously hypertensive rats. Circ. Res. 55, 286-294. Worms P., Gueudet C. and Biziere K. (1986) Induction of turning by direct intrastriatal injection of dopaminomimetic drugs in mice: Pharmacological analysis of a simple screening model. Life Sci. 39, 2199-2208. Yamori Y., Horie R., Handa H., Sato M. and Fukase M. (1976) Pathogenetic similarity of strokes in stroke-prone spontaneously hypertensive rats and humans. Stroke 7, 46-53.
0306-3623/91 $3.00+ 0.00 Copyright © 1991PergamonPress pie
PROTECTIVE EFFECTS OF MINAPRINE IN INFARCTION PRODUCED BY OCCLUDING MIDDLE CEREBRAL ARTERY IN STROKE-PRONE SPONTANEOUSLY HYPERTENSIVE RATS SHIGERUOKUYAMA,HIROKOSHIMAMURA-HARADA,YASUKOKARASAWA,KAZUAKIKAWASHIMA, HIROAKIARAKI,MASAAKIKIMURA*,SUSUMUOTOMOand HIRONAKAAIHARA Department of Pharmacology and *Department of Toxicology, Research Center, Talsho Pharmaceutical Co., Ltd, 1-403, Yoshino-cho, Ohmiya, Saltama 330, Japan (Received 20 March 1990)
Abstract--1. We examined the effects of minaprine on cerebral infarction produced by occluding of unilateral middle cerebral artery (MCA) above the rhinal fissure in stroke-prone spontaneously hypertensive rats (SHRSP). 2. Oral administration of minaprine (60 mg/kg/day) and vehiclewere started 30 rain after the occlusion of MCA, and continued for 6 days. 3. The brain was dissected out and 36 coronal multiple sections of the whole brain were histologically prepared to determine the location and extension of infarction. 4. The infarcted area produced by the occlusion of MCA was limited to the cerebral cortex. 5. Body weight of the minaprine-treated rats gradually decreased within 4 days after the occlusion of MCA and thereafter increased, whereas in the vehicle-treated rats, there was a gradual decrease during the experimental period. 6. Size of the infarcted area was serially measured, in each section, using a microcomputer imaging device. In all animals with an occluded MCA, there was a typical pattern of ischemic damage. 7. Post-treatment of MCA occluded SHRSP with minaprine resulted in reduction in infarct size, as compared to findings in the vehicle-treated controls. 8. The pharmacological and histopathological effectsof minaprine on the progress of cerebral infarction produced by the occlusion of MCA in SHRSP are discussed.
INTRODUCTION
Occlusion of the rat middle cerebral artery (MCA) has been widely used for a model of focal cerebral ischemia. Tamura et al. (1981) reported a procedure for occluding the proximal MCA in normotensive rats. Here, the brain damage produced by ischemia was located in the cortex and the lateral part of the striatum. However, this procedure is technically difficult and most invasive. Coyle (1982) demonstrated a less invasive surgical approach with MCA occlusion above the rhinal fissure, but cerebral infarction in normotensive rats did not occur. In contrast, a reproducible focal cerebral infarction was developed after the same occlusion in stroke-prone spontaneously hypertensive rats (SHRSP) (Coyle and Jokelainen, 1983). The internal diameter of the anterior cerebral artery (ACA) and MCA branches decreases with aging in the SHRSP (Coyle, 1987). The ACA-MCA anastomoses in the SHRSP are smaller in internal diameter than those in the normotensive rats (Coyle, 1987a). The cerebral vascular resistance in SHRSP is greater than in the normotensive rats (Coyle, 1987a; Werber and Heistad, 1984). Changes in the vascular wall of the SHRSP are linked to the hypertensive state (Ogata et al., 1980; 1981). Yamori et al. (1976) reported pathogenetic similarity of strokes in SHRSP and humans. Thus, it seems that the SHRSP is an unique animal model for studying cerebral infarction and/or stroke.
Minaprine, 3-(2-morpholino-ethylamino)-4-methyl6-phenyl-pyridazine dihydrochloride, is a novel psychotropic drug with antidepressant and psychostimulatory effects (Biziere eta/., 1982; 1984; Worms et aL, 1986). In clinical studies, minaprine seems to be an effective and safe antidepressant (Biziere et al., 1982; Jouvent et al., 1984; Mikus et al., 1985; Radmayr et aL, 1986). Passeri et al. (1985) also reported that minaprine improved depression scores and behavior in demented patients and seemed to be more effective in multiinfarct dementia than in cases of the senile dementia of Alzheimer type. We obtained evidence that minaprine has protective effects on cerebral hypoxia, anoxia and ischemia, in experimental animals (Okuyarna et al., 1988a). Araki et al. (1987) reported that minaprine has preventive effects on hippocampal delayed neuronal death and abnormalities on the electroencephalogram in Mongolian gerbils with occluded common carotid arteries. We report here the effects of minaprine on the progress of cerebral infarction produced by occlusion of MCA in SHRSP. MATERIALS AND METHODS
Twelve male stroke-prone spontaneously hypertensive rats (SHRSP) at 11 weeks of age, weighing 220-267 g, were housed in an air-conditioned room at 22 + I°C with 12 hr light-dark schedule (lights on at 07:00 a.m.). The animals had become thoroughly familiar with being handled. Great care was taken with gentle handling and avoidance of pain. 143
144
SHIGERU OKUYAMA
et aL
I(] rnrn
Fig. 1. Burr hole target region for exposure of MCA.
Tail systolic blood pressure in each conscious animal was measured just before the occlusion of MCA, using a rat tail manometer-tachometer system (KN-210, Natsume). The rats were anesthetized with sodium pentobarbital (50 mg/kg, given intraperitoneally) and body temperature was kept between 37°C and 37.5°C, using a heat pad controlled by a rectal thermometer (KN-474, Natsume). Under an operating microscope (SM-5, Nikon) the left MCA was exposed through a burr-hole craniectomy (Fig. 1) 2-3 mm diameter performed by a transtemporal route, without damage to the zygomatic bone, then the artery was dissected free of the meninges. The MCA was occluded with a microbipolar coagulation (MICRO-3D, Miziho), using a low power setting and was cut to ensure completeness of the vascular occlusion. The occlusion was distal to the striate branches of the MCA and 0.7-1 mm dorsal to the rhinal fissure. The soft tissues were put back into place and the skin sutured. Oral administrations of minaprine dihydrochloride (60 mg/kg) and vehicle (saline) were started 30 min after the occlusion of MCA and continued once daily for 7 days. Minaprine dihydrochloride (Taisho) was dissolved in saline solution (0.9% NaCI solution, 1 ml/kg, body weight). The dosage used was similar to that used for cerebral protective effects, in our laboratory (Araki et al., 1987; Okuyama et al., 1988a). Arterial blood pressure and plasma glucose levels remained unchanged with a dose of 60 mg/kg of minaprine (unpublished observation). At 24 hr after the final administrations of minaprine and vehicle, the arterial blood gas was measured using a pH blood gas analyzer (model 168, Corning). Blood for analyses totalling 0.5 ml was obtained via the tail artery. Neurological test used was similar to that described by Coyle and Jokelinen (1983). Each rat was placed on the 7 mm surface of a horizontally suspended wooden meter stick. Observations were made on fore- and hind-limb digits, feet legs and thighs, their locations and symmetrics during station, gait, crawling or running on a wooden stick. The animals were evaluated for motor deficits once daily during the experimental period by one of us (K.K.) who was unaware of the pharmacological treatment given the animals. Quantitative morphometry was performed on histologic slides, using the following method. At 24 hr after the final administrations of minaprine and vehicle, the animals used for the morphometry, including histopathological examination, were anesthetized with pentobarbital (50mg/kg, intraperitoneal injection), and the brains were respectively perfused with a 10% buffered formalin solution given
through the left cardiac ventricle. The brains were dissected out and fixed in 10% buffered formalin solution. Serial sections (about 5 #m-thick) were cut coronally from the paraffin block of the whole brain. Of all the sections prepared, 36 sections taken at the same intervals were stained with hematoxylin and eosin (H.E.). Each slide was examined histopathologically for determination of the infarcted area, using conventional light microscopy (XF-EFD2, Nikon) by one of us (M.K.) who was unaware of the pharmacological treatment given the animals. In the examination of each slide, the infarcted areas were defined according to the criteria shown in Fig. 2. The lesion was limited to outer layers of the unilateral cerebral cortex and all deeper layers were spared. The infarcted areas were serially measured on each slide, (36 slides/rat) using a microcomputer imaging device ( R a m m e t al., 1984) (Imaging Research Inc.-Muromachi Neuroscience). Percent infarction of each rat was calculated as follows. % infarction of each section (IES) infarct area (mm 2) x 100 total section area (mm 2) % infarction of each rat = average of 36 sections of IES Statistical analysis was performed using an one-way ANOVA followed by Student's t-test for individual comparisons. A P < 0.05 was regarded as being statistically significant. RESULTS
Systolic blood pressure T h e r e was n o significant difference in pre-occlusion systolic b l o o d pressure values between vehicle- (237.1 + 3.7 m m H g , n = 6) a n d m i n a p r i n e - t r e a t e d animals (237.3 __+9.1 m m H g , n = 6).
Body weight Body weight in the vehicle-treated animals gradually decreased d u r i n g the experimental period. Weight o f the m i n a p r i n e - t r e a t e d rats decreased within 4 days
Fig. 2. Cerebral sections of the infarcted area (H.E. stain x 50). (a) Severely infarcted area. Necrotic focus and loss of the normal cytological architecture. The tissue is less intensely stained and neuronal cell nuclei are few. The neurophil looks spongy. The left portion of the photograph shows numerous macrophages. (b) Mild or moderately infarcted area. The neurons show various ischemic cell changes, though the normal cytological architecture is partly maintained. Neuronal cell somas are not clearly outlined and the neurons are fragmented. Macrophages are also evident. These features characterize the irreversible brain lesion. Histopathological definitions of each area (a, b) are given on the right. Fig. 4. Cerebral infarcted areas of the vehicle-treated animal (a) and minaprine-treated animal (b) (H.E. stain x 20). Photograph (b) shows small size of the infarcted area, compared to that in photograph (a).
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Minaprine and infarction MCAO
I
mmHg and 7.50 + 0.02, respectively, and those in tll minaprine-treated group were 90.67 + 4.20 mmH! 33.38 + 2.28 mmHg and 7.47 + 0.03, respectivel.~ Blood gas and pH were within the normal range i both the vehicle- and minaprine-treated animal (Hoffman, Albrecht and Miletich, 1982), and thel was no statistical difference among the two groups c animals (Student's t-test). Oaya aftor MCAO
Fig. 3. Changes in body weight after MCA occlusion (MCAO) in vehicle- and minaprine-treated animals. *P < 0.05 vs vehicle-treated group (one-way ANOVA followed by Student's t-tes0. after the occlusion of MCA, and thereafter there was a gradual reversion the pre-treatment level. Body weight in the minaprine-treated group was significantly (P < 0.05, Student's t-test) higher than in the vehicle-treated group, on day 6 (Fig. 3).
Measurement of arterial blood gases The values of PO2, PCO2 and pH in the vehicletreated group were 78.12 _+ 5.39 mmHg, 31.57 _+ 2.52
Effect of minaprine There was a common pattern of ischemic brai damage (Fig. 2) in all animals with occlusion c MCA. Tissue sections of the lesion were less intensel stained with H.E. than were the normal tissue~ Location of the infarcted area was limited to th ipsilateral cerebral cortex and in no rat did : extend to the contralateral one. The ipsilateral bas, ganglia showed no ischemic damage. The lesion w~ characterized by the following features: edema in th pia matter, liquefaction with macrophages (granula fatty cells) on the lesion surface, and the abow mentioned findings (Fig. 2). Hemorrhage and macrc phages with pigmented granules were sometime evident.
Fig. 5. Serial 36 coronal sections of the whole brain in vehicle-treated aminals. The decreased intensity of the H.E. stained re#on is the lesion. The numbers show the anterioposterior distance (#m) from the coronal plane, according to the atlas of Kfnig and Klippel (1963). GP 2~/1--J
14
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SHIGERUOKUYAMAet al.
itlllt
Fig. 6. Serial 36 coronal sections of the whole brain in minaprine-treated animals. The decreased intensity of the H.E. stained region is the lesion. The numbers show the anterioposterior distance (#m) from the coronal plane, according to the atlas of K6nig and Klippel (1963). In the minaprine-treated animals, there was a small area of ischemic damage and somewhat larger numbers of macrophages on the lesion surface, as 15I
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Fig. 7. Effects of vehicle and minaprine on the occlusion of MCA produced infarction in SHRSP. Oral administrations of minaprine (60 mg/kg) and vehicle (saline) were begun 30 min after the occlusion of MCA and were continued once a day for 7 days.
compared to findings in the vehicle-treated animals (Fig. 4). However, there was no clear difference in quality between the two groups. Typical topography of the infarct in S H R S P is shown in Fig. 5 (vehicle-treated animal) and Fig. 6 (minaprine-treated animal), respectively. As shown in Fig. 7, the average infarction size in S H R S P with treatment of the vehicle (n = 6) was 11.24 _+ 1.55% (mean + SEM). In contrast, in the minaprine-treated animals (n = 6, an oral dose of 60 mg/kg for 7 days), the infarct size was 6.27 _+ 0.94% (mean + SEM), the difference in infarct size between the groups being significant (Student's t-test, P < 0.05). Motor deficits after the occlusion of M C A , as tested using the horizontally suspended wooden meter stick, were never evidenced, in either the vehicle- or the minaprine-treated animals. DISCUSSION We made use of the M C A occluded focal ischemic model in SHRSP. The site of occlusion of M C A was made above the rhinal fissure, for the following reasons, (i) S H R S P is an appropriate pathogenetic model for studies on stroke (Yamori et al., 1976),
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Minaprine and infarction (ii) the procedure is technically easily and is less invasive, (iii) location of infarction is limited to the cerebral cortex and does not extend to the basal ganglia. These observations are in accord with the data of Coyle (1983) who reported a systematic approach employing a relatively non-invasive surgical procedure, in the SHRSP. Thus, the development of a predictable large cortical infarct was established. In the present study, the whole brain was serially dissected out and 36 sections of the same intervals were examined. The location and extension of the infarcted area produced by the occlusion of MCA were determined. Size of the infarction was measured using each section and a microcomputer imaging device ( R a m m e t al., 1984). Thus, the experimental procedure we used provides a quantitative determination of the infarcted area. In addition, for evaluation of the infarcted area and histopathology of ischemic change and neurological tests, all experiments were carried out in a randomized and blinded fashion. Although the MCA occluded SHRSP showed evidence of a large cortical infarction, motor deficits were not apparent. One possible explanation for this is that location of the infarcted area was limited to the cerebral cortex. The striatum is one of the most important regions related to the development of motor function (Kamata et al., 1987). The lack of motor deficits in the MCA occluded SHRSP may be related to location of the infarction. However, Coyle and Jakelainen (1983) reported that the occlusion of MCA in SHRSP caused an asymmetrical fore-foot placement and hind-foot location and that the infarcted area was limited to the cerebral cortex. Minaprine, administered post-ischemia, protected against the progress of ischemic neuronal damage in the SHRSP. Branches of the MCA anastomose with branches of ACA on the dorsal aspect of the cerebral hemisphere, in the SHRSP (Coyle, 1987b; Coyle and Heistad, 1987). Occlusion of the MCA above the rhinal fissure reduced the cerebral blood flow to the territory of the occluded MCA, hence, the cortical tissue is infarcted downstream from the pial surface of the MCA-ACA anastomoses (Coyle, 1987b). Minaprine has preventive effects on hippocampal delayed neuronal death in mongolian gerbils with occluded common carotid arteries (Araki et al., 1987), and also a potent protective effect against cerebral hypoxia, anoxia and ischemia, in a variety of experimental animal models (Okuyama et al., 1988a). Thus, it is reasonable to assume that the systemically administered minaprine arrives at the MCA-ACA anastomoses via ACA. Minaprine may protect against the progress of secondary lesion development downstream from the anastomoses. In fact, systemically administered minaprine rapidly enters the central nervous system and then distributes fairly evenly in various regions beyond the blood-brain barrier (Caccia et al., 1985). Body weight of the minaprine-treated animals was significantly higher than that in the vehicle-treated animals, on day 6 after occluding the MCA. Therefore, minaprine may improve systemic conditions by inhibiting of the progress of infarction. Nimodipine (Gotoh et al., 1986; Sauter and Rudin, 1986), PN 200-110 (Sauter and Rudin, 1986),
nizofenone (Tamura et aL, 1979) and barbiturates (Tamura et al., 1979; Nehles et al., 1987) reduce the extension of infarction in the MCA occlusion model. Tamura et al. (1979) reported that depression of cerebral metabolic rates by nizofenone or barbiturates is one of the principal reasons for their proteefive action. However, minaprine did not depress the cerebral metabolic rates, in case of the [~4C]2-deoxyglucose method (unpublished observation). Electrophysiological investigations showed that the firing rate in the brain stem reticular formation was reduced by pentobarbital and that this reduction was overcome by minaprine (Okuyama and Aihara, 1988c). Thus, the mode of action of minaprine differs from that of nizofenone and barbiturates. Sauter and Rudin (1986) suggested that a potential mechanism of the dihydropridine calcium antagonist, nimodipine or PN 200--110, reduced the ATP demand of neurons required for the maintenance of intraceUular calcium homeostasis, by reducing excessive calcium influx during ischemia. The effect of minaprine on calcium homeostasis is now under investigation in our laboratory. Decrease in neurotransmitter turnover rates in the territory of MCA occlusion leads to a neuronal necrosis (Sauter and Rudin, 1986). This was also noted in case of a lesion with neurotoxin (Hefti, Melamed and Wurtman, 1980; Kamata et al., 1987). Minaprine activates central dopaminergic (Biziere et al., 1984), serotonergic (Biziere et al., 1982) and cholinergic (Garattini et al., 1984; Biziere et al., 1986) neurotransmissions. Moreover, the facilitation of cortical synaptic transmission (Okuyama and Aihara, 1988b) and the potentiation of hippocampal electrical activity (Okuyama and Aihara, 1989) were also observed. Thus, the protective effect of minaprine against the occlusion of MCA produced infarction may be related to activation and/or facilitation of central neuronal transmission. Acknowledgement--We
thank M. Ohara for helpful
comments. REFERENCES
Araki H., Nojiri M., Kimura M. and Aihara H. (1987) Effect of minaprine on "delayed neuronal death" in mongolian gerbils with occluded common arteries. J. Pharmac. Exp. Ther. 242, 686-691. Biziere K., Kan J. P., Souilhac J., Muyard J. P. and Roncucci R. (1982) Pharmacological evaluation of minaprine dihydrochloride, a new psychotropic drug. Arzneim.-Forsch. 32, 824-831. Biziere K., Gueudet C., Kan J. P., Souilhac J. and Worms P. (1984) Atypical dopaminergic profile of minaprine, a new antidepressant drug. Br. J. PharmacoL Suppl. 81, 51p. Biziere K., Gueudet C. and Worms P. (1986) Further evidence for a ehofinomimetie activity of minaprine. Br. J. Pharmae. Suppl. 87, 31p. Cae¢ia S., Fossati T. and Maneinelli A. (1985) Disposition and metabolism of minaprine in the rat. Xenobiotiea 15, 1111-1119, Coyle P~ (1982) Middle cerebral artery occlusion in the young rat. Stroke 13, 855-859. Coyle P. and Jokelainen P. T. (1983) Differential outcome to middle cerebral artery occlusion in spontaneously hypertensive stroke-prone rats (SHRSP) and Wistar Kyoto (WKY) rats. Stroke 14, 605-611.
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Coyle P. (1987a) Dorsal cerebral collaterals of stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar Kyoto (WKY). Anat. Rec. 218, 40-44. Coyle P. (1987b) Spatial relations of dorsal anastomoses and lesion border after middle cerebral artery occlusion. Stroke 18, 1133-1140. Coyle P. and Heistad D. D. (1987) Blood flow through cerebral collateral vessels one month after middle cerebral artery occlusion. Stroke 18, 407-411. Garattini S., Forloni G. L., Tirelli S., Ladinsky H. and Consolo S. (1984) Neurochemical effects of minaprine, a novel psychotropic drug, on the central cholinergic system of the rat. Psychopharmac. 82, 210-214. Gotoh O., Mohamed A. A., McCulloch J., Graham D. I., Harper A. M. and Teasdale G. M. (1986) Nimodipine and the haemodynamic and histopathological consequences of middle cerebral artery occlusion in the rat. J. Cereb. Blood Flow Metab. 6, 321-331. Hefti F., Melamed E. and Wurtman R. J. (1980) Partial lesions of the dopaminergic nigrostriatal system in rat brain: Biochemical characterization. Brain Res. 195, 123-137. Hoffman W. E., Albrecht R. F. and Miletich D. J. (1982) Cerebrovascular and metabolic effects of SNP-induced hypotension in young and aged hypertensive rats. Anesthesiology 56, 427-430. Jouvent R., Lancrenon S., Patay M., Biziere K. and Wildlocher D. (1984) A controlled study: minaprine versus placebo in inhibited depressed out-patients. Int. Syrup. at the first European Conference on Minaprine, pp. 33-40. Kamata K., Okuyama S., Hashimoto S., Kimura M., Aihara H. and Kasuya Y. (1987) Atrophy of the striatum and disturbance induced by colchicine. Brain Res. 421, 353-357. K6nig J. R. and Klippel R. J. (1963) The Rat Brain. A Stereotaxic of the Forebrain and Lower Parts o f the Brain Stem. The Williams and Wilkins Company, Baltimore. Mikus P., Biziere K. and Bentel U. (1985) A controlled study of minaprine and nomifensine in outpatients with masked depression. Clin. Trial J. 22, 477-488. Nehles D. G., Todd M. M., Spetzler R. F., Drummond J. C., Thompson R. A., Johnson P. C., Mendenhall S. E., Barstow C. A., Carmichael P. G., Yound D. M., Fifield M. S. and Jones C. P. (1987) A comparison of the cerebral protective effects of isoflurane and barbiturates during temporary focal ischemia in primates. Anesthesiology 66, 453-464. Ogata J., Fujishima M., Tamaki K., Nakatomi Y., Ishitsuka T. and Omae T. (1980) Stroke-prone spontaneously hypertensive rats as an experimental model of malignant hypertension. I. A light- and electron-microscopic study of the brain. Acta Neuropathol. (Berlin) 51, 179-184.
Ogata J., Fujishima M., Tamaki K., Nakatomi Y., Ishitsuka T. and Omae T. (1981) Vascular changes underlying cerebral lesions in stroke-prone spontaneously hypertensive rats. Acta Neuropathol. (Berlin) 54, 183-188. Okuyama S., Karasawa Y., Shiratsuchi Y. Araki H. and Aihara H. (1988a) Protective effects of minaprine on the cerebrum of rodents. Res. Comm. Chem. Pathol. Pharmacol. 60, 381-395. Okuyama S. and Aihara H. (1988b) Effect of minaprine on synaptic transmission in the neocortex of the rat in vivo. Neuropharmacology 27, 915-922. Okuyama S. and Aihara H. (1988c) Effect of minaprine on neuronal activity in the brain stem reticular formation of the rat. Arch. Int. Pharmacodyn. Ther. 296, 246-254. Okuyama S. and Aihara H. (1989) Effect of minaprine on electrical activity of the rat hippocampus in vivo. NaunynSchmiedeberg's Arch. Pharmacol. 340, 1-5. Passeri M., Cucinotta D., De Mello M., Storchi G., Roncucci R. and Biziere K. (1985) Minaprine for senile dementia. Lancet I, 824. Radmayr R., Biziere K. and Bentel U. (1986) A doubleblind controlled study of minaprine and maprotiline in endogenous type depression. Clin. Trail J. 23, 100-109. Ramm P., Kulick J. H., Stryker M. P. and Frost B. J. (1984) Video and scanning microdensitometer-based imaging systems in autoradiographic densitometry. J. Neurosci. Meth. 11, 89-100. Sauter A. and Rudin M. (1986) Calcium antagonists reduce the extent of infarction in rat middle cerebral artery occlusion model as determined by quantitative magnetic resonance imaging. Stroke 17, 1228-1234. Tamura A., Asano T., Sano K., Tsumagari T. and Nakajima A. (1979) Protection from cerebral ischemia by a new imidazole derivative (Y-9179) and pentobarbital. A comparative study in chronic middle cerebral artery occlusion in cats. Stroke 10, 126-134. Tamura A., Graham D. I., McCulloch J. and Teasdale G. M. (1981) Focal cerebral ischaemia in the rat: I. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. J. Cereb. Blood Flow Metabol. 1, 53-60. Werber A. H. and Heistad D. D. (1984)Effects of chronic hypertension and sympathetic nerves on the cerebral microvasculature of stroke-prone spontaneously hypertensive rats. Circ. Res. 55, 286-294. Worms P., Gueudet C. and Biziere K. (1986) Induction of turning by direct intrastriatal injection of dopaminomimetic drugs in mice: Pharmacological analysis of a simple screening model. Life Sci. 39, 2199-2208. Yamori Y., Horie R., Handa H., Sato M. and Fukase M. (1976) Pathogenetic similarity of strokes in stroke-prone spontaneously hypertensive rats and humans. Stroke 7, 46-53.