The Effect of Ritanserin, a 5-HT 2 Receptor Antagonist, on Ischemic Cerebral Blood Flow and Infarct Volume in Rat Middle Cerebral Artery Occlusion

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1 481 The Effect of Ritanserin, a 5-HT 2 Receptor Antagonist, on Ischemic Cerebral Blood Flow and Infarct Volume in Rat Middle Cerebral Artery Occlusion Kiyoshi Takagi, MD; Myron D. Ginsberg, MD; Mordecai Y.-T. Globus, MD; Raul Busto, BS; W. Dalton Dietrich, PhD Background and Purpose In a previous study from our laboratory, ritanserin, a specific 5-HT 2 serotonin receptor antagonist, reduced ischemic damage in the setting of transient global ischemia. In this study, we examined the effect of ritanserin on ischemic cerebral blood flow, systemic blood pressure, and infarct volume in the model of permanent focal ischemia with brain temperature controlled at 35.0 C to 36.0 C. Methods Thirty-seven male Sprague-Dawley rats were used. The right middle cerebral artery was permanently occluded. Ritanserin (8 mg/kg) or vehicle was continuously administered intravenously for 90 minutes starting 10 minutes after middle cerebral artery occlusion. Cerebral blood flow was monitored by laser Doppler flowmetry in the ischemic cortex before and for 2 hours after arterial occlusion. Brains were perfusion-fixed 3 days later, and infarct volumes were measured. Many studies have disclosed the involvement of excitatory amino acids in the development of ischemic brain damage. 16 It has also been demonstrated that glutamate receptor antagonists have a protective effect against cerebral infarction. 717 Nevertheless, other neurotransmitters have also been shown to be involved in ischemic brain damage Destruction of substantia nigra leading to dopaminergic denervation protects striatal neurons from global ischemia 1819 and reduces infarct volume following focal ischemia. 25 A participatory role for serotonin has been suspected because of its possible neurotoxic effects mediated directly or indirectly Anatomic studies have disclosed serotonin 5-HT 2 receptors in cerebral cortex and striatum of the rat. 5-HT 2 receptors are also recognized on cerebral vessels, and immunohistochemical studies show that pial arteries are innervated by serotonergic fibers Furthermore, serotonergic denervation partially protects the striatum from kainic acid-mediated damage. 26 In a previous report from our laboratory, Globus et al 23 reported the involvement of serotonin in ischemic neuronal dam- Received March 29, 1993; final revision received September 14, 1993; accepted September 15, From the Cerebral Vascular Disease Research Center, Department of Neurology, University of Miami School of Medicine, Miami, Fla. Correspondence and reprint requests to Kiyoshi Takagi, MD, Department of Neurology (D4-5), University of Miami School of Medicine, PO Box , Miami, FL Results Mean arterial blood pressure was not affected by treatment. In the vehicle and ritanserin groups, mean ischemic cerebral blood flow (percent of preischemic values) was 34.6±14.7% (mean±sd) and 26.6±15.0%, respectively. Hemispheric infarct volumes were 119.3±49.4 mm 3 and 136.6±49.6 mm 3, respectively. No significant differences were recognized. Conclusions Intravenous administration of ritanserin did not affect mean arterial blood pressure or cerebral blood flow in the ischemic region during the acute phase of ischemia. No protective effect of ritanserin was apparent in the setting of permanent focal ischemia when treatment was begun shortly after the onset of ischemia. (Stroke. 1994;25: ) Key Words cerebral ischemia, focal serotonin rats ntansenn See Editorial Comment, page 485 age in the setting of transient global forebrain ischemia. Therefore, it is possible that a selective 5-HT 2 receptor antagonist might have a protective effect against ischemic damage in focal ischemia. The aim of this study was to assess the effect of ritanserin, a highly selective 5-HT 2 antagonist, 35 ' 36 in focal ischemia. Serotonin may be involved in the hemodynamic changes in ischemic or periischemic regions 37 since it has vasoconstrictive properties. In a model of thrombotic cortical infarction, Dietrich and colleagues 37 showed that the 5-HT 2 antagonist, ketanserin, inhibited some of the remote hemodynamic consequences of this focal injury. In that study, it was suggested that 5-HT 2 receptor antagonist might therefore prove useful in reducing infarct size by improving collateral circulation. Previous studies from our laboratory have examined the effects of preischemic administration of 5-HT 2 antagonist on ischemic outcome. 23 In this study, ritanserin was administered after middle cerebral artery occlusion, and systemic blood pressure and cerebral blood flow (CBF) were measured during the ischemic period. Because temperature has a marked influence on the ischemic damage in both global and focal ischemia, we monitored ipsilateral cortical brain temperature and, by that means, regulated brain temperature.

2 482 Stroke Vol 25, No 2 February 1994 Materials and Methods We used 37 male Sprague-Dawley rats (Charles River Laboratories, Inc, Wilmington, Mass), weighing 300 to 400 g. Animals were fasted overnight but were allowed free access to water. Anesthesia was induced with 4% halothane, 70% nitrous oxide, and a balance of oxygen and was maintained with 2% halothane and 70% nitrous oxide during the surgical procedures. Atropine sulfate (0.04 mg IP) was injected. The right femoral artery and vein were cannulated with PE-50 polyethylene catheters for monitoring of arterial blood pressure and blood gases and for the administration of drugs. Rats were then intubated endotracheally, immobilized with pancuronium bromide (initial dose, 0.6 mg/kg; additional dose, 0.2 mg/kg), and were mechanically ventilated. The animals were fixed in a stereotaxic frame (Stoelting, 111). Blood gases (ABL 30 system, Radiometer, Copenhagen, Denmark), plasma glucose, and lactate (Glucose/Lactate Analyzer Model 2300 STAT, YSI, Ohio) were monitored before and during ischemia (70 minutes after middle cerebral artery (MCA) occlusion, ie, 60 minutes after drug administration). Physiological variables were kept within normal limits (mean arterial blood pressure [MABP], 100 to 150 mm Hg; Pao 2,100 to 150 mm Hg; Paco 2, 30 to 45 mm Hg; ph, 7.30 to 7.45). Rectal temperature was maintained between 37.0 C and 38.0 C during the experiment by means of a heating lamp placed above the body. The right MCA was exposed by the method of Tamura et al. 50 In brief, the cranial vault and the right lateral surface of the skull were exposed via a longitudinal skin incision between the eye and the ear. The zygomatic arch was removed. A burr hole (1.5 mm in diameter) for the brain temperature probe was made above the right parietal cortex by means of a high-speed mini-drill (Nihon-Seimitsu Kikai Kogyo K.K., Japan) under an operating microscope (Carl Zeiss, Germany); the field was irrigated frequently with cooled saline to avoid thermal damage. Brain temperature was monitored with a thermocouple probe (CN 9000, Omega), which was inserted into the cerebral cortex (approximate coordinates: 4 mm lateral to the bregma, 2-mm depth from brain surface). Brain temperature was maintained between 35.0 C to 36.0 C by means of a small heating lamp placed 20 cm over the head. Another burr hole (2 mm lateral to the burr hole for the temperature probe, 2 mm in diameter) was made in the lateral surface of the temporal bone to permit continuous CBF measurement by laser Doppler flowmetry (P433-3, Vasamedics). This probe was connected to a perfusion monitor (LaserFlo BPM 403A, Vasamedics). This position of the probe was determined based on previous studies from our laboratory 31 and by others 5255 using the same proximal MCA occlusion model in Sprague-Dawley rats; we anticipated that in this "penumbral" cortical site, the effect of the pharmaceutical would be most evident in that this cortical area is not consistently involved in infarction A temporal burr hole was then made in the retro-orbital region to occlude the MCA. 50 The dura mater covering the MCA was opened. After these surgical procedures, the inspired halothane was discontinued to avoid the effect of halothane on systemic blood pressure and CBF. Anesthesia was maintained with 70% nitrous oxide and 30% oxygen. Thirty minutes after discontinuation of halothane, measurement of the preischemic physiological variables, CBF, MABP, and pulse rate was begun. Steady-state baseline values were recorded before MCA occlusion, and CBF was expressed as a percentage of the average of six baseline measurements taken every 5 minutes before MCA occlusion. 56 Because ambient light interferes with the flow reading, the heating lamp was turned off for 30 seconds at the time of each CBF recording. Brain temperature did not decrease below 35.0 C during this period. To detect the effect of ritanserin on CBF and MABP, these variables were measured every 10 minutes during a 2-hour observation period after MCA occlusion. After 30 minutes of preischemic data sampling, the proximal portion of the right MCA was electrocoagulated and cut. Ritanserin (8 mg/kg, the same dose as in our previous study 23 ; Janssen Pharmaceutica, Belgium) was dissolved in a calculated injection volume (ml) equal to 2 x body weight (g). The vehicle was ethanol/o.ln HC1 in methanol/saline, 1:1:8, adjusted to ph 6.0 with 0.1N NaOH. Continuous injection of ritanserin or vehicle was started 10 minutes after MCA occlusion by means of a microinfusion pump (CMA/100, Carnegie Medicin, Sweden). The injection time was 90 minutes, the same as in our previous study. 23 Brains were perfusion fixed for pathological examination 3 days after the ischemic insult. The rats were deeply anesthetized with pentobarbital and perfused transcardially with physiological saline (5 minutes) and then with FAM (a mixture of 40% formaldehyde, glacial acetic acid, and methanol [ 1:1:8 by volume], 20 minutes) at a pressure of 120 mm Hg. The head was immersed in FAM for at least 24 hours; the brain was then removed and kept in the same fixative for 7 days. The brains were cut coronally and embedded in paraffin. Brain sections 10 fim thick were prepared at 200-/im intervals and stained with hematoxylin and eosin. For morphometric study, 10 coronal sections were selected at defined anatomic levels. 57 Each section was viewed at low power (10 x), and the cortical infarct was traced onto paper using a camera lucida microscope attachment. Each drawing was then retraced onto a digitizing tablet interfaced to a computer, which calculated infarcted areas at each coronal level. Infarct volume was computed by numerical integration of sequential infarct areas. Animals whose physiological variables could not be kept within normal limits or that had uncontrollable intraoperative bleeding were excluded from analysis. All data are expressed as mean±sd. An unpaired t test was used to analyze the data from the ritanserin group and the vehicle group statistically. A paired t test was used to analyze intragroup differences. Differences were considered to be statistically significant at /"<.O5. Results Twenty rats met the physiological criteria. The body weights were ±17.0 g for the animals receiving vehicle and ±24 g for the animals receiving ritanserin. The body weight showed no significant difference between the two groups. Four animals died prematurely, and one rat was found to have epidural hematoma around the site of the temperature probe at the time of perfusion-fixation. These animals were excluded from the pathological analysis. Thus, 15 animals were available for pathological analysis (vehicle group, n=7; ritanserin group, n=8). Physiological variables were generally kept within normal limits during the experiment (Table 1). Ritanserin administration produced no significant changes of mean arterial blood pressure or pulse rate. Table 2 shows changes of CBF. On MCA occlusion, CBF decreased significantly. There was no significant difference in the decrease of CBF between the vehicle group and the ritanserin group. Furthermore, CBF showed no significant change after ritanserin or vehicle administration. Infarct volumes are shown in Table 3. Hemispheric, cortical, and striatal infarct volumes were compared. We could not show any significant difference in infarct volume between the vehicle group and the ritanserin group. Although a tendency was noted for the infarct volume to become larger in rats with lower mean ischemic CBF, only in the vehicle group was an inverse correlation between mean ischemic CBF and cortical infarct volume significant (P<.05).

3 Takagi et al Effect of Ritanserin on Focal Ischemia 483 TABLE 1. Physiological Variables Vehicle (n=10) Ritanserin (n=10) MAP, mm Hg Pulse rate, mlrr' PH PaOj, mm Hg Pacc>2, mm Hg Glucose, mg/dl Lactate, nmol/l Pralschemla 120±8 429± ± ± ± ±0.2 *P<.01 vs prelschemlc value. tp<.05 vs prelschemlc value. + f<.05 vs same category of vehicle group. Ischemia 117±9 439± ± ±21* 35.4±2.5* 142±23* 1.0±0.1t Prelschemla 116±8 427± ± ± ± ± Ischemia 116±10 439± ± ± ±2.4* 152 ±30* 1.3±0.5t Discussion The present results indicate that postischemic ritanserin fails to reduce infarct volume in a permanent focal ischemia model of the rat produced by electrocoagulation and surgical sectioning of the right MCA. Similarly, intravenous ritanserin administration has no acute effect on CBF during ischemia or on MABP. The ineffectiveness of ritanserin in reducing infarct volume cannot be attributed to the dose used in this study in that we used the same dose as that used by Globus et al 23 in their study of transient global forebrain ischemia in the rat, in which a highly beneficial effect of ritanserin was demonstrated in protecting hippocampal CA1 neurons from ischemic damage. The discrepancies in the results from the two studies can be explained by the use of different experimental protocols (focal versus global ischemia) or by the differences in treatment paradigm (preischemic versus postischemic administration of the 5-HT 2 antagonist). The rationale for postischemic treatment with ritanserin is based on our study in which postischemic treatment with (SJ-emopamil (both the 5-HT 2 antagonist and calcium channel blocker) conferred protection from focal ischemia It is unlikely that the lack of protection is due to the short period of postischemic treatment since a previous study has demonstrated that in the rat brain 90% of the 5-HT 2 receptor sites were occupied 12 hours and 48 hours later; 70% receptor occupation still was found after the subcutaneous administration of 2.5 mg ritanserin. 3 * We anticipated, in designing this permanent focal ischemia study, that ritanserin would reduce infarct volume and would increase CBF when administered TABLE 2. Cerebral Blood Flow Changes Vehicle Rftansertn Preischemia Ischemia 1 Ischemia 2 Ischemia 3 100% 34.9±21.2% 34.6±14.0% 34.6±14.7% 100% 25.6±14.2% 26.8±15.5% 26.6±15.0% Ischemia 1, cerebral blood flow before drug administration. Ischemia 2, cerebral blood flow after drug administration. Ischemia 3, average cerebral blood flow during 2-hour middle cerebral artery occlusion. during ischemia. This expectation was based on several previous observations: (1) Both the rat and human brain are rich in 5-HT 2 receptors. 59 ' 60 (2) Brain blood vessels are well endowed with 5-HT 2 receptors, which may be involved in vasoconstriction. 33 ' (3) Moderate ischemia induces an elevation of serotonin in the extracellular space of the rat striatum. 63 (4) Ischemia has been shown to induce the release of serotonin in the caudate putamen. 23 ' 64 (5) There is evidence from the literature that 5-HT itself may be neurotoxic. 28 (6) Serotonin antagonists have been shown to be effective in certain forms of central nervous system ischemia. 65 As noted above, Globus et al 23 found the administration of ritanserin to protect hippocampal neurons from morphological damage following global ischemia. (7) Ketanserin, another 5-HT 2 receptor antagonist, has been reported to increase the CBF in remote cortical brain regions after cortical thrombotic infarction. 37 In addition to the above evidence, there are other, more indirect, reasons to have expected a beneficial effect of ritanserin in focal ischemia: (1) It has been suggested that 5-HT selectively enhances voltage- and Ca"-dependent NMDA responses. 66 (2) (S)-Emopamil, a calcium channel blocker having 5-HT 2 receptor-blocking properties, has been shown in our laboratory to have a marked beneficial effect in reducing infarct volume in the same rat model of permanent MCA occlusion as that used in the present study ; we have also shown (S)-emopamil to be effective in global ischemia. 67 (3) Ohno and coworkers 68 have shown that ritanserin, administered after ischemia, improved postischemic working memory. Although that study lacked histopathology, it showed functional improvement of treated animals. Despite the evidence cited above, the present study was unable to demonstrate a protective effect of ritanserin in focal ischemia. We interpret these negative TABLE 3. Infarct Volume (mm 1 ) Vehicle (n=7) Hemisphere 119.3±49.4 Cortex Striatum 35.7±7.4 Ritanserin (n=8) 136.6± ± ±7.7

4 484 Stroke Vol 25, No 2 February 1994 results as follows: 5-HT may not be involved in the pathophysiology of ischemic injury in this model. In part, this may relate to the fact that the model involves mechanical occlusion of the MCA and that platelet thrombi, the main source of intravascular 5-HT in ischemia, may not be formed in this model. In this regard, Wester and colleagues 69 have recently demonstrated a 5-fold increase in plasma serotonin levels after common carotid artery thrombosis. Although this study failed to demonstrate a therapeutic effect of ritanserin when administered postischemically, we feel that it may nonetheless be important to consider the role of the serotonergic system in human cerebral ischemia; the human brain is richly endowed with 5-HT 2 receptors, 59 ' 70 and the most frequent causes of human cerebral ischemia are thrombosis and embolism, which may well involve the release of serotonin from aggregated platelets. Acknowledgments These studies were supported by United States Public Health Service grants NS-05820, NS-22603, and NS The expert histological assistance of Ms Susan Kraydieh is gratefully acknowledged. References 1. Butcher SP, Bullock R, Graham DI, McCulloch J. Correlation between amino acid release and neuropathologic outcome in rat brain following middle cerebral artery occlusion. Stroke. 1990;21: Choi DW, Maulucci-Gedde M, Kriegstein AR. Glutamate neurotoxicity in cortical cell culture. J NeuroscL 1987;7: Mitani A, Kataoka K. Critical levels of extracellular glutamate mediating gerbil hippocampal delayed neuronal death during hypothermia: brain microdialysis study. Ncurosciencc. 1991;42: Rothman SM. Synaptic activity mediates death of hypoxic neurons. Science. 1983;220: Rothman SM, Olney JW. Glutamate and the pathophysiology of hypoxic-ischemic brain damage. Ann NeuroL 1986;19:105-lll. 6. Takagi K, Ginsberg MD, Globus MY-T, Dietrich WD, Martinez E, Kraydieh S, Bust R. 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5 Takagi et al Effect of Rltanserin on Focal Ischemia Dietrich WD, Busto R, Vald6s I, Loor Y. Effects of normothermic versus mild hyperthermic forebrain ischemia in rats. Stroke. 1990; 21: Minamisawa H, Nordstr6m C-H, Smith M-L, SiesjO BK. The influence of mild body and brain hypothermia on ischemic brain damage. J Certb Blood Flow Mttab. 1990;10: Minamisawa H, Smith M, Siesjo BK. The effect of mild hyperthermia and hypothermia on brain damage following 5, 10, and 15 minutes of forebrain ischemia. Ann Neurol 1990;28: Onesti ST, Baker CJ, Sun PP, Solomon RA. Transient hypothermia reduces focal ischemic brain injury in the rat. Neurosurgery. 1991;29: Ridenour TR, Warner DS, Todd MM, McAllester AC. Mild hypothermia reduces infarct size resulting from temporary but not permanent focal ischemia in rats. Stroke. 1992;23: Morilcawa E, Ginsberg MD, Dietrich WD, Duncan RC, Kraydieh S, Globus MY-T, Busto R. 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Brain Res. 1988;456: Lin B, Dietrich WD, Busto R, Ginsberg MD. (S)-Emopamil protects against global ischemic brain injury in rats. Stroke. 1990; 21: Ohno M, Yamamoto T, Watanabe S. Blockade of 5-HT 2 receptors protects against impairment of working memory following transient forebrain ischemia in the rat. Neurosci Lett. 1991;129: Wester P, Dietlich WD, Prado R, Watson BD, Globus MY-T. Serotonin release into plasma during common carotid artery thrombosis in rats. Stroke. 1992;23: Biegeon A, Kargman S, Snyder L, McEwen BS. Characterization and localization of serotonin receptors in human brain postmortem. Brain Res. 1986;363: of interventions that alter the events and the occlusion test outcome. The main objective of this study was to evaluate a drug intervention applied in an attempt to protect tissue from infarction and thus improve the MCA occlusion test outcome. After focal ischemia by permanent occlusion of the MCA, neocortical infarct volumes were of similar size in Sprague-Dawley rats receiving vehicle or ritanserin, a 5-HT 2 receptor antagonist. Members of this research group reported earlier that ritanserin protected hippocampal CA1 neurons against ischemic damage in a Wistar rat model of global cerebral ischemia with reperfusion. 1 The mechanism of protection of CA1 neurons by ritanserin is unknown. The authors argue