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Effects of protease inhibitor and immunosuppressant on cerebral vasospasm after subarachnoid hemorrhage in rabbits
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ELSEVIER
Effects of Protease Inhibitor and Immunosuppressant on Cerebral Vasospasm After Subarachnoid Hemorrhage in Rabbits Hiroji Yanamoto, M.D., Haruhiko Kikuchi, M.D., and Shinichiro Okamoto, M.D. Department of Neurosurgery, Kyoto University Medical School; Department of Neurosurgery, Osaka Red Cross Hospital, Japan
Yanamoto H, Kikuchi H, Okamoto S. Effects of protease inhibitor and immunosuppressant on cerebral vasospasm after subarachnoid hemorrhage in rabbits. Surg Neurol 1994;42:382-7.
The possible role of the immune-defense system in the development of cerebral vasospasm after subarachnoid hemorrhage (SAH) was investigated in rabbits. We used a synthetic serine protease inhibitor, gabexate mesilate (GM), a glucocorticoid, betamethasone sodium phosphate (B-P), and an immunosuppressant, ciclosporin (Cyclosporin A, CYA), to prevent cerebral vasospasm. These agents were administered intra-venously every 12 hours for three injections, starting 20 minutes after SAH. In the group treated with GM, B-P, or CYA, there were no statistically significant differences in arterial calibers between treated and untreated controls on day 2. The synthetic serine protease inhibitor, FUT-175 has been reported to prevent cerebral vasospasm when the treatment is started 20 minutes after SAH in rabbits [38]. In rabbits treated with FUT-175 at different starting times from 3 to 6 hours, reductions in arterial caliber on day 2 were significantly prevented in each group. The contrasting effects of the two serine protease inhibitors, GM and FUT-175, are discussed. KEY WORDS: Cerebral vasospasm; Protease inhibitor; Immunosuppressant; Cyclosporin A; Glucocorticoid
but rather represents pathologic comformational changes of the vascular wall or of smooth muscle cells, caused by an inflammatory process [5-7,23,24,30]. W e recently reported the preventive effects of a synthetic multiserine protease inhibitor, nafamostat mesilate (FUT-175), on cerebral vasospasm [38,39]. The results suggested the importance of plasma protease cascades in the development of cerebral vasospasm. The plasma protease cascades comprise a nonspecific immune system mainly led by the complement system. In the present study, we evaluated the effects of another protease inhibitor, gabexate mesilate (GM), on cerebral vasospasm. The properties of these protease inhibitors, are similar except for the potent anti-complement action of FUT-175. Further, to investigate the possible role of nonspecific and specific immune factors in cerebral vasospasm, the effects of a synthetic glucocorticosteroid, betamethasone B-P, and an immunosuppressant, ciclosporin (Cyclosporin A, CYA) on cerebral vasospasm were investigated. In addition, the preventive effect of FUT-175 was investigated, using different starting times of administration to determine the optimal protocol for preventing cerebral vasospasm in rabbits. Methods
A ngiography Cerebral vasospasm is a pathologic prolonged vasonstriction with a delayed onset after subarachnoid hemorrhage (SAH), which causes cerebral ischemia and subsequent cerebral infarction. Many studies have attempted to elucidate the etiology of this constrictive vasculopathy, but the pathologic mechanism remains unknown [12,19,27,29,35,36]. Recently, an inflammatory etiology has been postulated, suggesting that vasospasm is not a simple physiologic vasoconstriction induced and maintained by some vasoactive substances,
Address reprint requests to: Haruhiko Kikuchi, M.D., professor, chairman, Department of Neurosurgery, Kyoto University Medical School, Kawahara-cho 54 Syogoin, Sakyo-ku, Kyoto, 606 Japan. Received September 27, 1993; accepted December 3, 1993.
© 1994 by ElsevierScienceInc.
Forty-one Japanese white male rabbits weighing 3.0 to 3.5 kg were anesthetized with an intravenous injection of pentobarbital (30 mg/kg). After subcutaneous local anesthesia, a 4-French angiographic catheter was introduced through the exposed right femoral artery. The catheter was inserted into the left vertebral artery under a fluoroscope. Iopamidol (300 m g of iodine per 1 mL) at 0.5 mL was injected manually to enhance the vertebral angiogram. In each rabbit, angiography was done on day 0 (before SAH) to evaluate the baseline caliber of the basilar artery, and on day 2 when vasospasm reached its m a x i m u m [38].
Subarachnoid Hemorrhage SAH was simulated by transcutaneous injection of 1.0 mL of autologous nonheparinized arterial blood, aspi-
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rated from the catheter inserted in the femoral artery, into the cisternia magna. Before this injection, 1.0 mL of cerebrospinal fluid was removed to avoid an increase in intracranial pressure. After the blood injection, the rabbit was kept prone and with its head tilted downward for 40 minutes. Nine SAH rabbits were randomly selected to serve as an untreated control group.
Inhibitory Drug Treatment Protocol The synthetic serine protease inhibitor gabexate mesilate (GM) [ethyl-4-(6-guadino hexanoyloxy) benzoate] sulfonate (molecular weight, 417.48), was purchased from Ono Pharmaceutical Co., Ltd., Osaka, Japan [8,21,22,33]. Six rabbits were randomly assigned to two groups. Three rabbits received 10 m g of G M intravenously 20 minutes after SAH, with repeat doses 12 and 24 hours later. The other three rabbits received 20 m g (double dose) of gabexate mesilate, which was also repeated twice. G M was dissolved in 20 mL of saline and administered at a rate of 10 rag/10 minutes. The applied doses were 6.2 and 12.5 mg/kg/day, respectively, which are in the clinical range for the treatment of diffuse intravascular coagulation syndrome (DIC) [21]. Betamethasone sodium phosphate (B-P); Shionogi Pharmaceutical Co., Ltd., Osaka, Japan, was used to investigate the preventive effect of glucocorticosteroids on cerebral vasospasm [11]. Nine rabbits were randomly assigned to two groups. Six rabbits received 1 m g of B-P intravenously 20 minutes after SAH, with repeat doses 12 and 24 hours later. The other three rabbits received 2 m g (double dose) of B-P, which was also repeated twice. B-P was dissolved in 20 mL of saline and administered at a rate of 1 m g / 1 0 min. The applied doses were 0.62 or 1.25 mg/kg/day, respectively. The lower dose is in the upper limit of the clinical range. Ciclosporin (Cyclosporin A, CYA, cyclo [[(2S, 3R, 4R)-(E)- 3- hydroxy-4-methyl- 2-(methylamino)-6-octenoyl] - L - 2 - aminobutyryl - N - methylglycyl - N - methyl - Lleucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-Nmethyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl]; molecular weight, 1202.63; Sandoz Inc., Tokyo, Japan, was used as an immunosuppressant drug [3,4]. Six rabbits were randomly assigned to two groups. Three rabbits received 5 m g of CYA intravenously 20 minutes after SAH, with repeat doses 12 and 24 hours later. The other three rabbits received 10 m g (double dose) of CYA, which was also repeated twice. CYA was dissolved in 20 mL of saline and administered at a rate of 5 m g / 1 0 min. The applied doses were 3.1 and 6.3 mg/kg/day, respectively, which is considered to be optimal for treatment after renal transplantation in humans.
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The synthetic serine protease inhibitor nafamostat mesilate (6-amidino-2-naphthyl p-guanidinobenzonate, FUT-175; molecular weight, 539.58) was purchased from the research laboratories of Torii and Co., Ltd., Tokyo, Japan [1,2,9,13,15]. Ten rabbits received 2 m g of FUT-175 intravenously, with repeat doses 12 and 24 hours later. The first administration was started 3 hours after SAH in six rabbits and 6 hours after SAH in four rabbits. FUT-175 was dissolved in 20 mL of saline and administered at a rate of 1 mg/10 min. The applied dose was 1.25 mg/kg, which was considered to be optimal in the previous study [7].
Data Analysis The preventive effect of each drug was analyzed by measuring the change in caliber of the basilar artery at three sites on a photograph from the angiogram that was magnified 5.35 times. The mean value was expressed as a percentage of the control value from the baseline angiogram. Drug treatment and the angiographic analysis were performed in a blind manner. The data are expressed as means ± standard errors. Statistical analysis in percent-calibers between the treated and untreated groups was carried out by the Student's unpaired t-test. P values less than 0.05 denote significant differences. The mortality rate within 2 days between the treated and untreated group was analyzed by X2 test.
Results In the untreated control group, the mean arterial caliber reduced to 65% of the baseline caliber on day 2. There was no mortality in this group. In the group treated with three 10-rag or 2 0 - m g doses of GM, the mean arterial caliber decreased to 59% and 56% of baseline, respectively, on day 2 after SAH, respectively, compared to the baseline caliber (Figure 1A). There was no significant difference in caliber between the treated and untreated groups. N o rabbit died during the study in this group. In the group treated with three 1-mg doses of B-P, the mean arterial caliber decreased to 75% of baseline on day 2, which was a less severe reduction than that in the untreated control group (Figure 1B). However, there was no statistically significant difference between arterial calibers in the treated and untreated groups. In the group treated with three 2-mg (double) doses of B-P, all three rabbits died before the second angiogram. This mortality rate was significantly higher than that in the untreated control group (p < 0.01).
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Figure I. (A) The effect of intravenous administration of gabexate mesilate (GM) on a rabbit cerebral vasospasm model. (B) The effect of intravenous administration of betamethasone (B-P) on a rabbit cerebral vasospasm model. ( C ) The effect of intravenous administration of cyclosporin (CYA) on a rabbit cerebral vasospasm model. (D) The effect of intravenous administration of nafamosmt mesilate (FUT-175 ), started at different times, on a rabbit cerebral vasospasm model.
In the group treated with three 5-mg doses of CYA, the mean arterial caliber decreased to 58% of baseline on day 2 (Figure 1C). There was no significant difference between the calibers of the untreated and treated groups. In the group treated with three 10-mg (double) doses of CYA, three out of four rabbits died by the second angiogram. This mortality rate was significantly higher than that in the untreated controls group (p < 0.01). The surviving rabbit underwent angiography on day 2, and its arterial caliber was 53% of baseline.
In the three groups treated with three 2-mg doses of FUT-175 at different starting times, vasospasm expected on day 2 was significantly prevented, so that the arterial caliber was only reduced to 83% and 81% of baseline, respectively, with administration started 3 hours and 6 hours after SAH (Figure 1D). In a previous study, the treatment was started 20 minutes after SAH, the reduction in mean arterial caliber was prevented to an even greater extent, so that the caliber was fully 95% of baseline [7] (Figure 1D). No rabbit died during the study in this group.
Protease Inhibitor and ImmunosuppressantAffectSequelaeof SAH
Discussion It has been reported previously that high-dose methylprednisolone administered during the acute phase after SAH prevents cerebral vasospasm [5]. The importance of an inflammatory mechanism in generating cerebral vasospasm after SAH has also been described [6,23,24,40]. In addition, an increase in vascular permeability in the acute phase after SAH has been reported [7,30] suggesting the presence of an inflammatory process in the arterial wall during the acute phase. We used betamethasone sodium phosphate (B-P) as a glucocorticoid because of its strong glucocorticoid action without mineralcorticoid properties and its long half-life (about 8 hours). Intermittent administration has been suggested to be adequate to suppress the induction of immune responses and inflammation. Another pharmacologic property of glucocorticoids is their broad spectrum of anti-inflammatory activity. Reportedly, glucocorticoids reduce the production of interleukin 1 (ILl) and inhibit antigen presentation for T-cell proliferation by macrophages, and are believed to inhibit the release and metabolism of arachidonic acid by inducing a protein inhibitor of phospholipase A2 [14,17,31]. Direct effects of glucocorticoids against lymphocytes to inhibit its activation, or against vascular endothelial cells to prevent an elevation of vascular permeability have also been reported [18,26,32,34]. Furthermore, corticosreroids reportedly show anti-complement action in granulocyte aggregation [10]. B-P did not effectively prevent cerebral vasospasm in rabbits at the maximum possible dose; however, the results suggest some interaction between the wide range of anti-inflammatory action of glucocorticoids and the pathogenesis of cerebral vasospasm. Ciclosporin (CYA) prevents the specific immune reaction, inhibiting the production and release of interleukin 2 from helper T-cells [4]. Peterson et al reported the preventive effect of 6 mg/kg/day of CYA with 0.3 mg/kg/day of dexamethasone sodium phosphate (D-P) in the double subarachnoid hemorrhage canine model. CYA reduced cerebral vasospasm after the second SAH (cisternal blood injection), but effective prevention of narrowing on day 3 after first SAH was not demonstrated [25]. These authors discussed the immunologic basis for the second-phase reaction. Their results suggested a role of a T-cell-mediated specific immune reaction in the pathogenesis of cerebral vasospasm, presuming no influence of low dose D-P, which they combined in the therapy. Our experiment is based on a single SAH model, as the reduction in arterial caliber is severe and constant in rabbits in contrast to that in dogs [25]. Infiltrations of inflammatory cells in the arterial
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wall were not visible in our model at the time of maximum narrowing on day 2 [38]. There was no evidence of a significant role of a T-cell-derived immune reaction in the development of vasospasm in our model. There may be a difference in vasospasm itself between single and double SAH induction, or there may be a species-specific difference in the vessels. Handa et al [10'] confirmed the efficacy of 5 mg/ kg/day of CYA in preventing cerebral vasospasm in a single blood clot-placed primate model. They demonstrated significant differences in the middle and anterior cerebral arteries between CYA-rreated and untreated groups, but not in the internal carotid artery (ICA). At necropsy, a blood clot remained only around the ICA after clot lysis. These authors concluded that CYA may be helpful, but does not have a major therapeutic effect in preventing cerebral vasospasm. In the rabbit SAH model, a thick clot remained around the basilar artery on day 2, when vasospasm reached its maximum [38]. As in the cited study, CYA did not prevent the development of a clot-surrounded cerebral vasospasm. The synthetic multiserine protease inhibitor, gabexate mesilate (GM), developed by Fujii er al, is now used in the treatment of diffuse intravascular coagulation (DIC) syndrome [8,21]. The pharmacologic properties of GM have been investigated [8,22,23]. GM inhibits plasmin, thrombin, Hageman factor, X a factor, thrombin, kallikrein, phospholipase A2, Cl-esterase, and trypsin. The synthetic serine protease inhibitor, FUT-175, also developed by Fujii et al, inhibits the activation of the same serine proteases [2,9,13,15]. In addition, FUT175 is a potent inhibitor of B, D factor and Clr, Cls of the complement system. The only difference between these protease inhibitors is that the complement-related proteases are not effectively inhibited by GM [13,15]. FUT-175 is now the only potent inhibitor of the complement system in clinical use. In our model, GM failed to prevent cerebral vasospasm. There is no direct proof regarding the inhibition of the complement system around the cerebral arteries of rabbits; however, the contrasting effects of these two protease inhibitors may be attributed to their different effects on the complement system. Our results support a role of the complement system in the pathogenesis of cerebral vasospasm. In addition, it is possible that the unknown pharmacologic spectra of these two synthetic serine protease inhibitors are different. Further investigations are necessary to clarify the site of action and mechanism of FUT175 in preventing cerebral vasospasm. The prevention of vasospasm by FUT-175 seemed to be most effective when the administration was started soon after SAH; thus, early administration of the drug is beneficial in preventing or limiting cerebral vasospasm.
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This phenomenon suggested that the process of chronic pathologic arterial narrowing begins soon after the onset of SAH. It has been reported that the complement system was activated in the cerebrospinal fluid (CSF) early in the clinical course of SAH, before the development of vasospasm [16]. Recently, it was reported that CSF SC5b-9 levels were significantly higher in patients with severe SAH patients complicated by delayed ischemic neurologic deficit (DIND) than in those without DIND [37], which suggested that the formation of membrane attack complex (MAC, C5b-9) in the subarachnoid space could attribute to the pathogenesis of cerebral vasospasm. Basically, the complement system is a nonspecific immune defense mechanism against non-self or injured-self leading to cell lysis by forming membrane attack complex (MAC, C5b-9) before an involvement of a specific cell-derived immune mechanism [20]. The formation of the MAC in subarachnoid space may damage the autologous arterial cells by forming a transmembrane pore [28]. In conclusion, T-cell-mediated specific immune reaction did not seem to be involved in developing cerebral vasospasm, but the complement system-derived immune system might attribute to the pathogenesis of cerebral vasospasm.
References 1. Abe T, Kinoshita T, Matsuda S, Ogushi T, Kawasugi K, Yoshimura Y, Otomo M. Phase 1 study of FUT-175:single and multiple dose study. Jpn Pharmacol Ther 1984;12:4941-64. 2. Aoyama T, Ino Y, Ozeki M, Oda M, Sato T, Koshiyama Y, Suzuki S, Fujita M. Pharmacological studies of FUT-175, nafamostat mesilate: 1. Inhibition of protease activity in vitro and in vivo experiments. Jpn J Pharmacol 1984;35:203-27. 3. Borel JF, Feurer C, Gubler HU, St~.helin H. Biological effects of cyclosporin A: a new antilymphocytic agent. Agents Actions 1976;6:468-75. 4. Bunjes D, Hardt C, R~llinghoff M, Wagner H. Cyclosporine A mediates immunosuppression of primary cytotoxic T cell responses by imparing the release of interleukin 1 and interleukin 2. Eur J Immunol 1981;11:657-61. 5. Chyatte D, Rusch N, Sundt TM Jr. Prevention of chronic experimental cerebral vasospasm using ibuprofen and high-dose methyl prednisolone. J Neurosurg 1983;59:925-32. 6. Conway LW, Mcdonald LW. Structural changes of the intradural arteries following subarachnoid hemorrhage. J Neurosurg 1972;37:715-23. 7. Doczi T. The pathogenic and prognostic significance of bloodbrain barrier damage at the acute stage of aneurysmal subarachnoid hemorrhage: clinical and experimental studies. Acta Neurochir 1985;77:110-32. 8. Fujii S, Hirata M. Synthetic protease inhibitors. Metabolism (Taisha) 1977;14:1087-98 (Jpn). 9. Fujii S, Hitomi Y. New synthetic inhibitors of Clr, C1 esterase, thrombin, plasmin, kallikrein and trypsin. Biochem Biophys Acta 1981;661:342-5.
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10. Hammerschmidt DE, Whit JG, Graddock PRI:Corticosteroids inhibit complement-induced granulocyte aggregation: a possible mechanism for their efficacy in shock states. J Clin Invest
1979;63:798-803. 10.' Handa, Y, Hayashi M, Takeuchi H, Kobayashi H, Kawano H, Kabuto M. Effect of cyclosporine on the development of cerebral vasospasm in a primate model. Neurosurgery 1991; 28:380-86. 11. Haynes RC, Murad F. Goodman and Gilman's the pharmacological basis of therapeutics. 6th ed. New York: Macmillan,
1980:446-96. 12. Heros RC, Zervas NT, Varsos V. Cerebral vasospasm after subarachnoid hemorrhage: an update. Ann Neurol
1983;14:599-608. 13. Hitomi Y, Fujii S. Inhibition of various immunological reactions in vivo by a new synthetic complement inhibitor. Int Arch Allergy AppI Immunol 1982;69:262-7. 14. Hong SCL, Levine L. Proc Natl Acad Sci USA 1976;73:1730-4. 15. Ikari N, Sakai Y, Hitomi Y, Fujii S. New synthetic inhibitor of the alternative complement pathway. Immunology 1983;49: 685-91. 16. Kasuya H, Shimizu T. Activated complement components C3a and C4a in cerebrospinal fluid and plasma following subarachnoid hemorrhage. J Neurosurg 1989;71:741-6. 17. Knudsen PJ, Dinarello CA, Storm TB. Glucocorticoids inhibit transcriptional and post-transcriptional expression of interleukin 1 in U937 cells. J Immunol 1987;139:4129-34. 18. MacGregor RR. The effect of anti-inflammatory agents and inflammation of granulocyte adherence. Ecidence for regulation by plasma factors. Am J Med 1976;61:597-607. 19. Mayberg MR, Okada T, Bark KH. Morphologic changes in cerebral arteries after subarachnoid hemorrhage. Neurosurg Clin N Am 1990;1:417-32. 20. Miiller-Eberhard HJ. The membrane attack complex. Springer Semin Immunolopathol 1984;7:127-38. 21. Ohno H, Kambayashi J, Chang SW, Kosaki G. FOY:[Ethyl p-(6-guanidinohexanoyloxy) methane sulfonate as a serine protease inhibitor. 2. In vivo effect on coagulofibrinolytic system in comparison with heparin or aprotinin. Throm Res 1981 ;24:445-52. 22. Ohno H, Kosaki G, Kambayashi J, Imaoka S, Hirato F. FOY: [Ethyl p-(6-guanidinohexanoyloxy) benzoate] methanesulfonate as a serine proteinase inhibitor. 1. Inhibition of thrombin and factor Xa in vitro. Thromb Res 1980;19:579-88. 23. Peterson JW, Kwun BD, Hacket JD, Zervas NT. The role of inflammation in experimental cerebral vasospasm. J Neurosurg
1990;72:767-74. 24. Peterson JW, Kwun BD, Teramura A, Hackett JD, Morgan JA, Nishizawa S, Bun T, Zervas NT. Immunological reaction against the aging human subarachnoid erythrocyte. A model for the onset of cerebral vasospasm after subarachnoid hemorrhage. J Neurosurg 1989;71:718-26. 25. Peterson JW, Nishizawa S, Hackett JD, Bun T, Teramura A, Zervas NT. Cyclosporine A reduces cerebral vasospasm after subarachnoid hemorrhage in dogs. Stroke 1990;21:133-7. 26. Rivkin I, Foschi GV, Rosen CH. Inhibition of neutrophil chemotxis and spontaneous motility by anti-inflammatory agents. Prc Soc Exp Biol Med 1976;153:236-40. 27. Robinson MJ, Teasdale GM. Calcium antagonists in the management of subarachnoid haemorrhage. Cerebrovasc Brain Metab Rev 1990;2:205-26. 28. Salama A, Mueller-Eckhardt C. Binding of fluid phase C3b to nonsensitized bystander human red cells:a model for in vivo effects of complement activation on red cells. Transfusion 1985;25:528-34.
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29. Sasaki T, Kassell NF. The role of endothelium in cerebral vasospasm. Neurosurg Clin N Am 1990;1:451-63. 30. Sasaki T, Kassell NF, Yamashita M, Fujiwara S, Zuccarello M. Barrier disruption in the major cerebral arteries following experimental subarachnoid hemorrhage. J Neurosurg 1985;63:433--440. 31. Snyder DS, Unanue ER. Corticosteroids inhibit murine macrophage la expression and interleukin 1 production. J Immunol 1982;129:1803-5. 32. Sugio K, Ohuchi K, Sugata M, Tsurufuji S. Suppression by dexamethasone of vascular permeability responses induced with leukotrienes C and D in the rat skin. Prostaglandins 1981; 21:649-53. 33. Tamura Y, Hirado M, Okamura K, Minato Y, Fujii S. Synthetic inhibitors of trypsin, plasmin, kallikrein, thrombin, Clr, and C1 esterase. Biochem Biophys Acta 1977;484:417-22. 34. Tsurufuji S, Sugio K, Takemasa F. The role of glucocorticoid receptor and gene expression in the anti-inflammatory action of dexamethasone. Nature 1979;280:408-10. 35. Weir B. The history of cerebral vasospasm. Neurosurg Clin N Am 1990;1:265-76. 36. Wilkins RH. Cerebral vasospasm. Crit Rev Neurobiol 1990; 6:51-77.
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37. Yanomoto H, Kikuchi H, Ishikawa J, Shimizu Y, Sato M, Tokuriki Y, Okamoto S, Matsumoto M, Matsumoto K, Nakamura K. Intravenous FUT-175 inhibits complement activation in the cerebrospinal fluid and vasospasm-related delayed ischemic neurological deficit following subarachnoid hemorrhage. In: Findlay JM ed. Cerebral Vasospasm. Proceedings of the Vth International Conference on Cerebral Vasospasm. Edmonton and Jasper: Elsevier Science Publishers B.V., 1993:431-34. 38. Yanamoto H, Kikuchi H, Okamaot S, Nozaki K. Preventive effect of synthetic serine protease inhibitor, FUT-175, on cerebral vasospasm in rabbits. Neurosurgery 1992;30:351-7. 39. Yanamoto H, Kikuchi H, Sato M, Shimizu Y, Yoneda S, Okamoto S. Therapeutic trial of cerebral vasospasm with the serine protease inhibitor, FUT-175, administered in the acute stage after subarachnoid hemorrhage. Neurosurgery 1992; 30:358-63. 40. Yanamoto H, Okamoto S, Nozaki K, Kikuchi H. Cerebral arterial narrowing caused by nonvasoactive substance, polystyrene latex beads by cisternal injection in rabbits. In: Sano K, Takakura K, Kassell NF, Sasaki T, eds. Cerebral Vasospasm. Proceedings of the IVth International Conference on Cerebral Vasospasm. Tokyo:University of Tokyo Press, 1990:211-13.
ELSEVIER
Effects of Protease Inhibitor and Immunosuppressant on Cerebral Vasospasm After Subarachnoid Hemorrhage in Rabbits Hiroji Yanamoto, M.D., Haruhiko Kikuchi, M.D., and Shinichiro Okamoto, M.D. Department of Neurosurgery, Kyoto University Medical School; Department of Neurosurgery, Osaka Red Cross Hospital, Japan
Yanamoto H, Kikuchi H, Okamoto S. Effects of protease inhibitor and immunosuppressant on cerebral vasospasm after subarachnoid hemorrhage in rabbits. Surg Neurol 1994;42:382-7.
The possible role of the immune-defense system in the development of cerebral vasospasm after subarachnoid hemorrhage (SAH) was investigated in rabbits. We used a synthetic serine protease inhibitor, gabexate mesilate (GM), a glucocorticoid, betamethasone sodium phosphate (B-P), and an immunosuppressant, ciclosporin (Cyclosporin A, CYA), to prevent cerebral vasospasm. These agents were administered intra-venously every 12 hours for three injections, starting 20 minutes after SAH. In the group treated with GM, B-P, or CYA, there were no statistically significant differences in arterial calibers between treated and untreated controls on day 2. The synthetic serine protease inhibitor, FUT-175 has been reported to prevent cerebral vasospasm when the treatment is started 20 minutes after SAH in rabbits [38]. In rabbits treated with FUT-175 at different starting times from 3 to 6 hours, reductions in arterial caliber on day 2 were significantly prevented in each group. The contrasting effects of the two serine protease inhibitors, GM and FUT-175, are discussed. KEY WORDS: Cerebral vasospasm; Protease inhibitor; Immunosuppressant; Cyclosporin A; Glucocorticoid
but rather represents pathologic comformational changes of the vascular wall or of smooth muscle cells, caused by an inflammatory process [5-7,23,24,30]. W e recently reported the preventive effects of a synthetic multiserine protease inhibitor, nafamostat mesilate (FUT-175), on cerebral vasospasm [38,39]. The results suggested the importance of plasma protease cascades in the development of cerebral vasospasm. The plasma protease cascades comprise a nonspecific immune system mainly led by the complement system. In the present study, we evaluated the effects of another protease inhibitor, gabexate mesilate (GM), on cerebral vasospasm. The properties of these protease inhibitors, are similar except for the potent anti-complement action of FUT-175. Further, to investigate the possible role of nonspecific and specific immune factors in cerebral vasospasm, the effects of a synthetic glucocorticosteroid, betamethasone B-P, and an immunosuppressant, ciclosporin (Cyclosporin A, CYA) on cerebral vasospasm were investigated. In addition, the preventive effect of FUT-175 was investigated, using different starting times of administration to determine the optimal protocol for preventing cerebral vasospasm in rabbits. Methods
A ngiography Cerebral vasospasm is a pathologic prolonged vasonstriction with a delayed onset after subarachnoid hemorrhage (SAH), which causes cerebral ischemia and subsequent cerebral infarction. Many studies have attempted to elucidate the etiology of this constrictive vasculopathy, but the pathologic mechanism remains unknown [12,19,27,29,35,36]. Recently, an inflammatory etiology has been postulated, suggesting that vasospasm is not a simple physiologic vasoconstriction induced and maintained by some vasoactive substances,
Address reprint requests to: Haruhiko Kikuchi, M.D., professor, chairman, Department of Neurosurgery, Kyoto University Medical School, Kawahara-cho 54 Syogoin, Sakyo-ku, Kyoto, 606 Japan. Received September 27, 1993; accepted December 3, 1993.
© 1994 by ElsevierScienceInc.
Forty-one Japanese white male rabbits weighing 3.0 to 3.5 kg were anesthetized with an intravenous injection of pentobarbital (30 mg/kg). After subcutaneous local anesthesia, a 4-French angiographic catheter was introduced through the exposed right femoral artery. The catheter was inserted into the left vertebral artery under a fluoroscope. Iopamidol (300 m g of iodine per 1 mL) at 0.5 mL was injected manually to enhance the vertebral angiogram. In each rabbit, angiography was done on day 0 (before SAH) to evaluate the baseline caliber of the basilar artery, and on day 2 when vasospasm reached its m a x i m u m [38].
Subarachnoid Hemorrhage SAH was simulated by transcutaneous injection of 1.0 mL of autologous nonheparinized arterial blood, aspi-
0090-3019/94/$7.00
Protease Inhibitor and Immunosuppressant Affect Sequelae of SAH
rated from the catheter inserted in the femoral artery, into the cisternia magna. Before this injection, 1.0 mL of cerebrospinal fluid was removed to avoid an increase in intracranial pressure. After the blood injection, the rabbit was kept prone and with its head tilted downward for 40 minutes. Nine SAH rabbits were randomly selected to serve as an untreated control group.
Inhibitory Drug Treatment Protocol The synthetic serine protease inhibitor gabexate mesilate (GM) [ethyl-4-(6-guadino hexanoyloxy) benzoate] sulfonate (molecular weight, 417.48), was purchased from Ono Pharmaceutical Co., Ltd., Osaka, Japan [8,21,22,33]. Six rabbits were randomly assigned to two groups. Three rabbits received 10 m g of G M intravenously 20 minutes after SAH, with repeat doses 12 and 24 hours later. The other three rabbits received 20 m g (double dose) of gabexate mesilate, which was also repeated twice. G M was dissolved in 20 mL of saline and administered at a rate of 10 rag/10 minutes. The applied doses were 6.2 and 12.5 mg/kg/day, respectively, which are in the clinical range for the treatment of diffuse intravascular coagulation syndrome (DIC) [21]. Betamethasone sodium phosphate (B-P); Shionogi Pharmaceutical Co., Ltd., Osaka, Japan, was used to investigate the preventive effect of glucocorticosteroids on cerebral vasospasm [11]. Nine rabbits were randomly assigned to two groups. Six rabbits received 1 m g of B-P intravenously 20 minutes after SAH, with repeat doses 12 and 24 hours later. The other three rabbits received 2 m g (double dose) of B-P, which was also repeated twice. B-P was dissolved in 20 mL of saline and administered at a rate of 1 m g / 1 0 min. The applied doses were 0.62 or 1.25 mg/kg/day, respectively. The lower dose is in the upper limit of the clinical range. Ciclosporin (Cyclosporin A, CYA, cyclo [[(2S, 3R, 4R)-(E)- 3- hydroxy-4-methyl- 2-(methylamino)-6-octenoyl] - L - 2 - aminobutyryl - N - methylglycyl - N - methyl - Lleucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-Nmethyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl]; molecular weight, 1202.63; Sandoz Inc., Tokyo, Japan, was used as an immunosuppressant drug [3,4]. Six rabbits were randomly assigned to two groups. Three rabbits received 5 m g of CYA intravenously 20 minutes after SAH, with repeat doses 12 and 24 hours later. The other three rabbits received 10 m g (double dose) of CYA, which was also repeated twice. CYA was dissolved in 20 mL of saline and administered at a rate of 5 m g / 1 0 min. The applied doses were 3.1 and 6.3 mg/kg/day, respectively, which is considered to be optimal for treatment after renal transplantation in humans.
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The synthetic serine protease inhibitor nafamostat mesilate (6-amidino-2-naphthyl p-guanidinobenzonate, FUT-175; molecular weight, 539.58) was purchased from the research laboratories of Torii and Co., Ltd., Tokyo, Japan [1,2,9,13,15]. Ten rabbits received 2 m g of FUT-175 intravenously, with repeat doses 12 and 24 hours later. The first administration was started 3 hours after SAH in six rabbits and 6 hours after SAH in four rabbits. FUT-175 was dissolved in 20 mL of saline and administered at a rate of 1 mg/10 min. The applied dose was 1.25 mg/kg, which was considered to be optimal in the previous study [7].
Data Analysis The preventive effect of each drug was analyzed by measuring the change in caliber of the basilar artery at three sites on a photograph from the angiogram that was magnified 5.35 times. The mean value was expressed as a percentage of the control value from the baseline angiogram. Drug treatment and the angiographic analysis were performed in a blind manner. The data are expressed as means ± standard errors. Statistical analysis in percent-calibers between the treated and untreated groups was carried out by the Student's unpaired t-test. P values less than 0.05 denote significant differences. The mortality rate within 2 days between the treated and untreated group was analyzed by X2 test.
Results In the untreated control group, the mean arterial caliber reduced to 65% of the baseline caliber on day 2. There was no mortality in this group. In the group treated with three 10-rag or 2 0 - m g doses of GM, the mean arterial caliber decreased to 59% and 56% of baseline, respectively, on day 2 after SAH, respectively, compared to the baseline caliber (Figure 1A). There was no significant difference in caliber between the treated and untreated groups. N o rabbit died during the study in this group. In the group treated with three 1-mg doses of B-P, the mean arterial caliber decreased to 75% of baseline on day 2, which was a less severe reduction than that in the untreated control group (Figure 1B). However, there was no statistically significant difference between arterial calibers in the treated and untreated groups. In the group treated with three 2-mg (double) doses of B-P, all three rabbits died before the second angiogram. This mortality rate was significantly higher than that in the untreated control group (p < 0.01).
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Figure I. (A) The effect of intravenous administration of gabexate mesilate (GM) on a rabbit cerebral vasospasm model. (B) The effect of intravenous administration of betamethasone (B-P) on a rabbit cerebral vasospasm model. ( C ) The effect of intravenous administration of cyclosporin (CYA) on a rabbit cerebral vasospasm model. (D) The effect of intravenous administration of nafamosmt mesilate (FUT-175 ), started at different times, on a rabbit cerebral vasospasm model.
In the group treated with three 5-mg doses of CYA, the mean arterial caliber decreased to 58% of baseline on day 2 (Figure 1C). There was no significant difference between the calibers of the untreated and treated groups. In the group treated with three 10-mg (double) doses of CYA, three out of four rabbits died by the second angiogram. This mortality rate was significantly higher than that in the untreated controls group (p < 0.01). The surviving rabbit underwent angiography on day 2, and its arterial caliber was 53% of baseline.
In the three groups treated with three 2-mg doses of FUT-175 at different starting times, vasospasm expected on day 2 was significantly prevented, so that the arterial caliber was only reduced to 83% and 81% of baseline, respectively, with administration started 3 hours and 6 hours after SAH (Figure 1D). In a previous study, the treatment was started 20 minutes after SAH, the reduction in mean arterial caliber was prevented to an even greater extent, so that the caliber was fully 95% of baseline [7] (Figure 1D). No rabbit died during the study in this group.
Protease Inhibitor and ImmunosuppressantAffectSequelaeof SAH
Discussion It has been reported previously that high-dose methylprednisolone administered during the acute phase after SAH prevents cerebral vasospasm [5]. The importance of an inflammatory mechanism in generating cerebral vasospasm after SAH has also been described [6,23,24,40]. In addition, an increase in vascular permeability in the acute phase after SAH has been reported [7,30] suggesting the presence of an inflammatory process in the arterial wall during the acute phase. We used betamethasone sodium phosphate (B-P) as a glucocorticoid because of its strong glucocorticoid action without mineralcorticoid properties and its long half-life (about 8 hours). Intermittent administration has been suggested to be adequate to suppress the induction of immune responses and inflammation. Another pharmacologic property of glucocorticoids is their broad spectrum of anti-inflammatory activity. Reportedly, glucocorticoids reduce the production of interleukin 1 (ILl) and inhibit antigen presentation for T-cell proliferation by macrophages, and are believed to inhibit the release and metabolism of arachidonic acid by inducing a protein inhibitor of phospholipase A2 [14,17,31]. Direct effects of glucocorticoids against lymphocytes to inhibit its activation, or against vascular endothelial cells to prevent an elevation of vascular permeability have also been reported [18,26,32,34]. Furthermore, corticosreroids reportedly show anti-complement action in granulocyte aggregation [10]. B-P did not effectively prevent cerebral vasospasm in rabbits at the maximum possible dose; however, the results suggest some interaction between the wide range of anti-inflammatory action of glucocorticoids and the pathogenesis of cerebral vasospasm. Ciclosporin (CYA) prevents the specific immune reaction, inhibiting the production and release of interleukin 2 from helper T-cells [4]. Peterson et al reported the preventive effect of 6 mg/kg/day of CYA with 0.3 mg/kg/day of dexamethasone sodium phosphate (D-P) in the double subarachnoid hemorrhage canine model. CYA reduced cerebral vasospasm after the second SAH (cisternal blood injection), but effective prevention of narrowing on day 3 after first SAH was not demonstrated [25]. These authors discussed the immunologic basis for the second-phase reaction. Their results suggested a role of a T-cell-mediated specific immune reaction in the pathogenesis of cerebral vasospasm, presuming no influence of low dose D-P, which they combined in the therapy. Our experiment is based on a single SAH model, as the reduction in arterial caliber is severe and constant in rabbits in contrast to that in dogs [25]. Infiltrations of inflammatory cells in the arterial
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wall were not visible in our model at the time of maximum narrowing on day 2 [38]. There was no evidence of a significant role of a T-cell-derived immune reaction in the development of vasospasm in our model. There may be a difference in vasospasm itself between single and double SAH induction, or there may be a species-specific difference in the vessels. Handa et al [10'] confirmed the efficacy of 5 mg/ kg/day of CYA in preventing cerebral vasospasm in a single blood clot-placed primate model. They demonstrated significant differences in the middle and anterior cerebral arteries between CYA-rreated and untreated groups, but not in the internal carotid artery (ICA). At necropsy, a blood clot remained only around the ICA after clot lysis. These authors concluded that CYA may be helpful, but does not have a major therapeutic effect in preventing cerebral vasospasm. In the rabbit SAH model, a thick clot remained around the basilar artery on day 2, when vasospasm reached its maximum [38]. As in the cited study, CYA did not prevent the development of a clot-surrounded cerebral vasospasm. The synthetic multiserine protease inhibitor, gabexate mesilate (GM), developed by Fujii er al, is now used in the treatment of diffuse intravascular coagulation (DIC) syndrome [8,21]. The pharmacologic properties of GM have been investigated [8,22,23]. GM inhibits plasmin, thrombin, Hageman factor, X a factor, thrombin, kallikrein, phospholipase A2, Cl-esterase, and trypsin. The synthetic serine protease inhibitor, FUT-175, also developed by Fujii et al, inhibits the activation of the same serine proteases [2,9,13,15]. In addition, FUT175 is a potent inhibitor of B, D factor and Clr, Cls of the complement system. The only difference between these protease inhibitors is that the complement-related proteases are not effectively inhibited by GM [13,15]. FUT-175 is now the only potent inhibitor of the complement system in clinical use. In our model, GM failed to prevent cerebral vasospasm. There is no direct proof regarding the inhibition of the complement system around the cerebral arteries of rabbits; however, the contrasting effects of these two protease inhibitors may be attributed to their different effects on the complement system. Our results support a role of the complement system in the pathogenesis of cerebral vasospasm. In addition, it is possible that the unknown pharmacologic spectra of these two synthetic serine protease inhibitors are different. Further investigations are necessary to clarify the site of action and mechanism of FUT175 in preventing cerebral vasospasm. The prevention of vasospasm by FUT-175 seemed to be most effective when the administration was started soon after SAH; thus, early administration of the drug is beneficial in preventing or limiting cerebral vasospasm.
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This phenomenon suggested that the process of chronic pathologic arterial narrowing begins soon after the onset of SAH. It has been reported that the complement system was activated in the cerebrospinal fluid (CSF) early in the clinical course of SAH, before the development of vasospasm [16]. Recently, it was reported that CSF SC5b-9 levels were significantly higher in patients with severe SAH patients complicated by delayed ischemic neurologic deficit (DIND) than in those without DIND [37], which suggested that the formation of membrane attack complex (MAC, C5b-9) in the subarachnoid space could attribute to the pathogenesis of cerebral vasospasm. Basically, the complement system is a nonspecific immune defense mechanism against non-self or injured-self leading to cell lysis by forming membrane attack complex (MAC, C5b-9) before an involvement of a specific cell-derived immune mechanism [20]. The formation of the MAC in subarachnoid space may damage the autologous arterial cells by forming a transmembrane pore [28]. In conclusion, T-cell-mediated specific immune reaction did not seem to be involved in developing cerebral vasospasm, but the complement system-derived immune system might attribute to the pathogenesis of cerebral vasospasm.
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