J Neurosurg 74:940-943, 1991

Prevention of cerebral vasospasm by actinomycin D TAKU SHIGENO, M.D., TATSUO MIMA, M.D., MASASH1 YANAGISAWA,M.D., PH.D., AKIRA SAITO, PH.D., KATSUTOSHI GOTO, Ph.D., KAZUMASA YAMASHITA, D.M.Sc., TOSItlttARU TAKENOUCItl, D.V.M.M.S., NAOSUKE MATSUURA, PH.D., YASUtIIDE YAMASAKI, PH.D., KoJI YAMADA, PH.D., TOMOH MASAKI, M.D., PH.D., AND KIMrrOMO TAKAkURA, M.D.

Department of Neurosurgery, Saitama Medical School, Kawagoe, Saitama, Department of Neurosurgery. UniversiO, qf Tokyo, Bunl~'yo, Tokyo: Institute of Basic Medical Sciences, University r Ysukuba, Tsukuba, lbaraki. Reseamh Institute Of Taiho Pharmaceulicals, Tokushima; and Research Laboratories. Kyowa ttakko Kogyo, Tokyo, Japan ~" The role of endothelin, a newly found vasoconstrictor peptide, is examined in the pathogenesis of cerebral vasospasm after experimental subarachnoid hemorrhage (SAH) in the dog. Endothelin immunoreactivity was overexpressed in the endothelium of the vasospastic basilar artery. Because endothelin synthesis is regulated at the messenger ribonucleic acid transcription level, the effect of actinomycin D, a ribonucleic acid synthesis inhibitor, was studied as a means of preventing vasospasm. It was found that treatment with intravenous actinomycin D for 5 days beginning on the day of SAH completely inhibited the development of vasospasm. This novel experimental therapy may lead not only to the elucidation of the pathogenesis of cerebral vasospasm but also to the availability of a prophylactic adjuvant therapy for patients with SAH. KEY WORDS

vasospasm

endothelin

ELAYED cerebral vasospasm after subarachnoid hemorrhage (SAH) is a complex pathological process characterized by a strong and persistent contraction of arterial smooth muscle accompanied by an abnormal proliferation and necrosis of the cellular constituents of the arterial wall.~,6'~tAlthough the mechanism of this disorder is still unknown, it has been considered that multiple endogenous vasoactive substances are involved. Polypeptide factors with vasoconstricting and/or mitogenic activities, such as endothelin,2,,9,2t and platelet-derived growth factor ( P D G F ) y ' ,1 might be implicated as candidates for endogenous agents causing vasospasm. Because synthesis of endothelin is regulated at the messenger ribonucleic acid (mRNA) transcription leveU ~ we examined the effect of the RNA synthesis inhibitor actinomycin D on the development of cerebral vasospasm in canine experimental SAH.

D

Materials and Methods Severe cerebral vasospasm was induced in a widely accepted experimental model of SAH in the dog) Adult male Beagle dogs were anesthetized with intravenous pentobarbital (30 mg/kg), intubated, and usually al940

RNA synthesis

actinomycin D

lowed to breath spontaneously. The right vertebral artery was cannulated for basilar artery angiography. The arterial blood pressure and blood gases were maintained within normal ranges under either spontaneous breathing or assisted ventilation. On Day 0, following control angiography of the basilar artery, autologous arterial blood (0.5 ml/kg) was injected into the cisterna magna after removal of the same amount of cerebrospinal fluid (CSF), causing isobaric SAH. On Day 2, the animals received another injection of blood. The caliber of the basilar artery was measured on the angiograms at three locations: close to the vertebrobasilar junction, at the midpoint, and close to the basilar tip. Changes in the total of these three measurements were expressed as percentages of the total of the three corresponding calibers on the control angiograms. The measurements were performed independently by two observers (T.S. and T.M.) in a blinded condition and a mean value measured by the two observers was obtained. One group of 16 animals received actinomycin D dissolved in saline, 10 ug/body weight, intravenously just after the SAH and this therapy was continued once a day until Day 4. The control group of 16 dogs was treated similarly with saline. In another group of five normal aniJ. Neurosurg. / Volume 74~June, 1991

Prevention of cerebral vasospasm by actinomycin D

FIG. 1. Typical examples of basilar artery angiograms in dogs with subarachnoid hemorrhage. Upper. Angiograms from a dog in lhc vehicle-treated control group showing progressive basilar artery constriction. Lower: Angiograms fi'om a dog treated with actinomycin D showing inhibition of arterial constriction by aetinomycin D. mals, only actinomycin D was given in a similar fashion to establish whether this agent alone had any effect in changing the vessel caliber. We utilized the nonparametric Mann-Whitney U-test for statistical comparison of the vessel calibers between the control and treated groups. The animals were sacrificed between Days l to 4 or on Day 7, and the basilar arteries were harvested. For immunohistochemical study, the arteries were fixed with formaldehyde and picric acid, dehydrated with ethanol, cleared with xylene, rehydrated, and frozen. On 10-um transverse sections of the arteries, monoclonal antibodies (KM565)* were applied g at 1:2000. The avidin-biotin peroxidase complex method was then used to demonstrate the localization of endothelin immunoreactivity. Although the monoclonal antibody used in the present study mainly reacts to endothelin1, it has also some cross-reactivity with endothelin-2, endothelin-3, and big endothelin-I. * Monoclonal antibody KM565 obtained from Kyowa Hakko Kogyo Co., Tokyo, Japan. J. Neurosurg. / Volume 74~June, 1991

TABLE 1 Changes in the canine basilar artery diameter* No. of AnimalGroup Dogs

Day 2

Day,4

Day 7

SAH alone 16 -25.6 4- 8.4 -39.7 4- 5.5 -48.5 -4-7.2 SAHwithactino- 16 -11.9 _+7.8t -I0.5 _+9.2~ -5.1 • 15.5t mycin D actinomycinD 5 0.0 + 0.0 -0.1 + 0.0 0.0 _+0.1 alone * Values are givenas percentages of the control value on Day 0, expressed as means _+ standard deviations. SAH = subarachnoid hemorrhage. t Statisticallysignificantdifference(p < 0.001) compared to the corresponding group of dogs with SAH alone (Mann-Whitne',,'s U-test). Results

We observed consistent and progressive constriction of the basilar artery, in all of the control animals with SAH (Fig. 1 upper and Table 1). All animals but one treated with actinomycin D showed complete resolution 941

T. Shigeno, el al.

FIG. 2. Photomicrographs with immunohistochemical staining by monoclonal antibody raised against endothelin-1 in the dog basilar artery. L~;/?. Section of normal artery showing scattered expression of immunoreactivity in the luminal surthce of endothelium. Cenler. Section of arlery 3 days after subarachnoid hemorrhage (SAH) showing overexpression of immunoreactivity. Right: Section of arte~, 3 days after SAH in an acfinomycin D-treated dog showing inhibition of immunoreactivity overexpression.

of cerebral vasospasm by Day 7, although there was slight reduction in vessel caliber on Days 2 and 4 (Table 1). Statistically, on Days 2, 4, and 7, there was significant amelioration of cerebral vasospasm by actinomycin D (p < 0.001). The overall mean values and standard deviations of the arterial blood gas and blood pressure values at the time of initial angiography and SAH were as follows: pH 7.35 + 0.04, pC02 38.8 _+ 5.8 mm Hg, pO2 100.9 _+ 3.9 mm Hg, and blood pressure 126 _+ 22 mm Hg under spontaneous respiration with room air. Even after SAH, those parameters did not change in most of the animals; however, some of the animals required artificial ventilation for a brief period (usually 30 minutes to l hour) after SAH. The autopsy on Day 7 showed that all the control and treated animals had similar degrees of SAH, with extensive subarachnoid blood clots encompassing the basilar artery. Although most of the animals showed lethargy caused by SAH during the entire observation period, we observed no appreciable difference in behavior (such as eating) between the treated and nontreated groups. In the normal basilar artery,, immunoreactivity was seen scattered on the luminal membrane of the endothelium (Fig. 2 left). However, 3 days after SAH the basilar artery, showed severe, widespread reactivity along the endothelium (Fig. 2 center). Actinomycin D treatment tended to suppress this overexpressed reactivity to SAH (Fig. 2 right). On Day 7, the immunoreaction was no longer observed (data not shown). We are currently quantifying the immunoreactivity and our results will be reported separately. Discussion

Actinomycin D appears to be more potent than any previously reported drug in reversing experimental cerebral vasospasm. At most, only a partial reversal of vasospasm was observed in previous studies employ942

ing calcium antagonists ~ and many other spasmolytic agents) ~ In contrast, the present study demonstrates a complete return of the arterial caliber to control levels on Day 7 after SAH and treatment with actinomycin D. The dose which we used (10 ug/kg) was lower than the initial therapeutic doses for patients with Wilms' tumor or choriocarcinoma (I 5 ug/kg)Y The mechanisms of the antivasospasm effect of actinomycin D remain to be clarified. However, the findings obtained in the present study have important implications on the pathogenesis of cerebral vasospasm; they demonstrate that de novo RNA synthesis may be essential for the progression of cerebral vasospasm. One likely possibility is that actinomycin D might have inhibited the induction of several peptidic vasoactive factors within the spastic lesion. Of particular interest is the group of transiently expressed factors that are encoded by extremely short-lived superinducible m RNA's.I~ Candidates for these signaling molecules may include: 1) cell growth factors such as PDGF 2'3 and transforming growth factor-beta (TGF-fl);' 3 2) vasoconstrictors such as endothelin, of which the activity is potent and extremely long-lasting both in vitro :~ and in vivol4; and 3) certain groups of cellular proto-oncogenes, such as c-myc, c-fos, and c-jun, that are involved in the nuclear signal transduction events in response to extracellular stimuli. ~2 It is worth noting that many of these factors, such as endothelin and PDGF, exhibit both mitogenic and vasoconstricting activities. 3~9 Furthermore, mutual induction of these signaling molecules may occur in SAH: for example, substances derived from blood clots and platelets such as thrombin and TGF-/5 can potently induce endothelin mRNA, 13.21 and endothelin, in turn, can induce cellular proto-oncogenes such as c-rnyc and c-fizs'. ~2 The preproendothelin mRNA has been shown in cultured cells to disappear rapidly and selectively when de novo mRNA J. Neurosurg. / Volume 74/June, 199I

Prevention of cerebral vasospasm by actinomycin D synthesis is blocked by actinomycin D. '~ Although we must be careful in extrapolating these i~ vi/ro observations to in vivo pathology, it is tempting to hypothesize that actinomycin D ameliorates cerebral vasospasm by selectively lowering the intracellular levels of one or more of these labile species of mRNA's. Although our initial use of actinomycin D for the treatment of cerebral vasospasm was derived from the discovery of endothelin, its effect might be nonspecific such as via suppression of inflammation and immunological reactions as well as other responses which require RNA synthesis. Indee& the studies of Peterson, el at,, 15~' have contributed to this aspect of inflammation and the use of immunosuppressants. At the moment, the involvement of endothelin in the pathogenesis of cerebral vasospasm is still indirect except for the present demonstration of endothelin immunoreactivity. We have recently reported on the endothelin content in the plasma and CSF of patients with vasospasm; r no close correlation was observed between these two values, so we speculated a local role of endothelin at the lesion site. Endothelin was also present in the plasma and CSF of dogs, but did not correlate with the development of cerebral vasospasm (data in preparation). For more direct evidence, we are currently trying to demonstrate endothelin and its receptor mRNA using the in situ hybridization technique, and attempting to see effects of receptor antagonists as well as inhibitors of the endothelin-eonverting enzyme, f; Acknowledgments

We thank Mses. R. Matsuura, C. Mizunuma, Y. Muto, Y. Seino, T. Ochiai, and Mr. N. Sunaga of the University of Tokyo, Tokyo, and Messrs, T, Hirota, M. Yamaguchi, and H. Shichijo of Taiho Pharmaceuticals, Tokushima, Japan, for care of the animals, References

I. Alksne JE Branson PJ: A comparison of intima] proliferation in experimental subarachnoid hemorrhage and atherosclerosis. Angiology 27:712-720, 1976 2. Berk BC, Alexander RW: Vasoactive effects of growth factors. Bioehem Pharmaeol 38:219-225, 1989 3. Berk BC, Alexander RW, Brock TA, et al: Vasoconstriction: a new activity for platelet-derived growth factor. Science 232:87-90, 1986 4. Chyatte D, Rusch N, Sundt TM Jr: Prevention of chronic experimental cerebral vasospasm with ibuprofen and high-dose methylprednisolone. J Neurosurg 59:925-932, 1983 5. Cohen RJ, Alien GS: Cerebral arterial spasm: the role of calcium in in vitro and in vivo analysis of treatment with nifedipine and nimodipine, in Wilkins RH (ed): Cerebral Arterial Spasm. Baltimore: Williams & Wilkins, 1980, pp 527-532 6. Fein FM, Flor WJ, Cohan SL, et al: Sequential changes of vascular uhrastructure in experimental cerebral vasospasm. Myonecrosis of subarachnoid arteries. J Neorosurg 4l:49-58, 1974

J. Nettrosurg / l~dume 74/June. 1991

7. Fujimori A, Yanagisawa M, Saito A. et al: Endothelin in plasma and ccrebrospinal fluid of patients with subarachnoid haemorrhage. Lancet 1:633. 1990 {Letter) 8. Gilman AG, Goodman LS, Rail TW, et al (eds): Goodman and Gilman's "fhe Pharmacological Basis of fherapeutics, ed 7. New York: Macmillan, 1985, pp 1281-1283 9. Goto J, Hanai N, Kimura S, et ah Immunological and pharmacological characterization of anti-endothelin monoclonal antibody, KM565. apn J Pharmacol 52 (Suppl 1):202, 1990 (Abstract) 10. lnoue A. Yanagisawa M, Takuwa Y, et ah The human preproendothelin-I gene. Complete nucleotide sequence and regulation of expression. J Biol Chem 264: 14954-14959, 1989 11. Kapp JP, Clower BR, Azar FM, et al: Heparin reduces proliferative angiopathy following subarachnoid hemorrhage in cats. J Neurosurg 62:570-575, 1985 12. Komuro I, Kurihara H, Sugiyama T, et ah Endothelin stimulates c:fiJs and c-myc expression and proliferation of vascular smooth muscle cells. FEBS Lett 238: 249-252, 1988 13. Kurihara H, Yoshizumi M. Sugiyama T, etal: Transforming growth i~actor-beta stimulates the expression of endothelin mRNA by vascular endothelial cells. Bioehem Biophys Res Commun 159:1435-1440, 1989 14. Mima T, Yanagisawa M, Shigeno T, et al: Endothelin acts in feline and canine cerebral arteries from the adventitial side. Stroke 20:1553-1556, 1989 t5. Peterson JW, Kwun BD, Hackett JD, et al: The role of inflammationin experimental cerebral vasospasm. J Neurosurg 72:767-774, 1990 16. Peterson JW, Nishizawa S, Hackelt J D, et al: Cyclosporine A reduces cerebral vasospasm after subarachnoid hemorrhage in dogs. Stroke 21:133-137, 1990 17. Sakurai T, Yanagisawa M, Takuwa Y, et al: Cloning of a eDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature 348:732-735, 1990 18. Shaw G, Kamen R: A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degeneration. Cell 46:659-667, 1986 19. Takuwa N, Takuwa Y, Yanagisawa M, et al: A novel vasoactive peptide endothelin stimulates mitogenesis through inositol lipid turnover in Swiss 3T3 fibroblasts. J Bioehem 264:7856-7861, 1989 20. Wilkins RH: Attempted prevention or treatment of intracranial arterial spasm: a survey. Neurosurgery 6: 198-210, 1980 21. Yanagisawa M, Kurihara H, Kimura S, et al: A novel vasoconstrictor peptide produced by vascular endothelial cells. Nature 332:411-4 [ 5, 1988 Manuscript received May 9, 1990. Accepted in final form December 19, 1990. Address for Drs. Mima and Takakura: Department of Neurosurgery, University of Tokyo, Bunkyo, Tokyo, Japan. Address for Drs. Yanagisawa, Saito, Goto, and Masaki: Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan. Address for Drs. Yamashita, Takenouchi, Matsuura. and Yamasaki: Research Institute of Taiho Pharmaceuticals, Tokushima, Japan. Address for Dr. Yamada: Research Laboratories, Kyowa Hakko Kogyo, Tokyo, Japan. Address rept'inf reque, sTs to. Taku Shigeno, M.D., Department of Neurosurgery, Saitama Medical School, Kawagoe, Saitama 350. Japan.

943

Prevention of cerebral vasospasm by actinomycin D.

The role of endothelin, a newly found vasoconstrictor peptide, is examined in the pathogenesis of cerebral vasospasm after experimental subarachnoid h...
2MB Sizes 0 Downloads 0 Views