Effect of on Microthrombi Formation and Brain Damage in the Rat Middle Cerebral Artery Model

Hiroshi Kawai#, Kazuo Umemura and Mitsuyoshi Nakashima

Department of Pharmacology, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-31, Japan

Received May 19, 1995 Accepted July 26, 1995

ABSTRACT-Ischemic cerebral infarcts induce hypercoagulation and microthrombosis in the surrounding region, thus leading to vascular occlusion. We determined whether microthrombi contribute to the spread- ing of ischemic lesions following thrombotic middle cerebral artery (MCA) occlusion and also determined whether argatroban, a selective inhibitor, reduces the formation of the microthrombi and the area of the ischemic lesions. The rat left MCA was occluded by a -rich thrombus formed following the photochemical reaction between rose bengal and green light. Microthrombi were histologically identified in the left hemisphere. The extent of ischemic lesions and microthrombi containing fibrin increased in a time- dependent manner after MCA occlusion. Argatroban inhibited the formation of microthrombi up to 3 hr after MCA occlusion; beyond 3 hr, it was ineffective. Argatroban also significantly (P<0.01) reduced the size of ischemic cerebral lesions at 6 hr after MCA occlusion. It is concluded that the formation of microthrombi contributes to the progression of ischemic lesions in the early stage. It is likely that thrombin generated following thrombotic MCA occlusion contributes to the progression of ischemic lesions by promoting the formation of microthrombi. Argatroban can reduce the formation of microthrombi and ischemic lesions in the early stage.

Keywords: Thrombin, Argatroban, Microthrombosis, Thrombotic middle cerebral artery occlusion

The progression of focal is depend- in the ischemic areas and might contribute to micro- ent on numerous factors (1, 2). Using an autoradio- circulatory impairment during ischemia (8-10). The graphic technique with iodoantipyrine in an experimental presence of fibrin in these thrombi demonstrates that middle cerebral artery (MCA) occlusion model in rats, thrombin was produced after MCA occlusion. Nagasawa and Kogure (3) have reported that the ischemic Thrombin is well-known to be a key factor in the lesions extend from the ischemic core to the surrounding hemostatic process, converting fibrinogen to fibrin and tissues and regional cerebral blood flow decreased in a inducing platelet activation. It is therefore suggested that time-dependent manner. In another study, Garcia et al. impaired microvascular perfusion due to thrombin-in- (4) have shown progression of ischemic lesions between duced microthrombi in the area surrounding the ischemic 30 min and 6 hr after MCA occlusion in rats. core contributes to the progression of ischemic lesions. In humans, two factors thought to be involved in the Argatroban, a synthetic thrombin inhibitor (11, 12), has progression of are hypercoagulation and micro- been shown to prevent platelet-rich arterial thrombosis thrombi formation, which lead to microvascular occlu- (13-18). sion and reduction of local blood flow that causes In view of the above-described observations, we cerebral infarction. Hypercoagulability of blood has been became interested in investigating the change in the reported in patients during the acute phase of ischemic microvascular perfusion from the initial ischemic event stroke (5 - 7). In ischemic brains of humans and those of to the development of an infarction using the rat MCA experimental animals, microthrombi have been observed thrombosis model. We also investigated the effect of argatroban on the microcirculatory disturbance and the

4Present address: Pharmaceuticals Laboratory 2, Yokohama Re- ischemic lesions of this model. Our findings suggest that search Center, Mitsubishi Chemical Corporation, 1000 Kamoshida, in this model, thrombin generated subsequent to throm- Aoba-ku, Yokohama 227, Japan botic occlusion of the MCA promotes microthrombus formation, thus contributing to the progression of identical conditions during this study. cerebral infarction. Histological procedures MATERIALS AND METHODS Just after and 1, 3, 6 and 24 hr after thrombotic MCA occlusion, the rats were perfused transcardially for 1 min Animal preparation with saline followed by 10% formaldehyde solution for Wistar male rats (SLC, Hamamatsu) weighing histological observations. The brain was immediately re- 240-260g were used. The body temperature of the moved and fixed in 10% formaldehyde and processed animals was maintained at 37.51C with a heating-pad (K- with paraffin. Brain sections were cut into 1-mm-thick module Model K-20; American Pharmaseal Company, consecutive slices from 2.5 mm anterior to bregma. Each Valencia, CA, USA) during the operation. The MCA section was then stained with phosphotungstic acid- thrombosis model in the rat has been described previously hematoxylin, which can stain fibrin, and with hematoxy- (19). In brief, under pentobarbital anesthesia and spon- lin and eosin for a light microscope study. For immuno- taneous respiration, a catheter for the administration of histochemical analysis, each section was incubated with rose bengal was placed in the femoral vein. The scalp and 100 pl of goat anti-rat fibrinogen antibody (Organon- temporalis muscle were folded over, and a subtemporal Teknika N.V., Turnhout, Belgium; 54.5 pg/ml) for 60 craniotomy was performed using a dental drill under an min at room temperature in a humidified chamber. Fol- operating microscope to open a 3-mm diameter circular lowing three phosphate-buffered saline (PBS) washes, bone window. sections were incubated with peroxidase-conjugated rab- bit anti-goat immunoglobulins (DAKO, Kyoto) for 30 Photo-irradiation min at room temperature. Following three PBS washes, The 3-mm diameter circular window was irradiated antibody-bound peroxidase was detected with 10 mg/ml with green light, and the entire irradiated segment in- dimethlaminoazobenzene containing 0.005% hydrogen cluding the proximal end of the lenticulostriate branch peroxide. The sections were washed in tap water for 3 min became thrombotically occluded. Photo-irradiation with and counterstained with Mayer's hematoxylin for 3 min. green light (wavelength: 540 nm) was achieved by using The following immunohistochemical controls were rou- a xenon lamp (L4887; Hamamatsu Photonics, tinely performed: 1) deletion of the primary antibody, Hamamatsu) with a heat absorbing filter and a green 2) deletion of the secondary antibody, 3) alteration of filter. The irradiation was directed by a 3-mm diameter the primary antibody to goat immunoglobulin. After optic fiber mounted on a micromanipulator. The head of hematoxylin counter-staining, the sections were examined the optic fiber was placed on the window in the skull base by light microscopy for the presence of peroxidase reac- at a distance of 2 mm above the vessel, providing an tion products. The number of microthrombi was counted irradiation dose of 0.62 W/cm2. Rose bengal (Wako, at 0.5 and 1 mm posterior from the bregma. The ischemic Osaka; 20 mg/kg) was injected intravenously. Photo-irra- lesions were identified in 4-pm-thick sections stained with diation was continued for a further 10 min. The photo- hematoxylin and eosin. Areas of the ischemic lesions were chemical reaction between Rose bengal and green light examined for histological features such as vacuolation causes endothelial cell injury followed by platelet adhesion (sponginess) of the neuropil, diffuse pallor of the eosino- and the formation of a platelet-rich thrombus. philic background, altered shape and stainability of both neuronal cells and astrocytes. The sizes of ischemic Physiological parameters lesions at 0.5 and 1 mm posterior from the bregma were In separate preliminary experiments, the physiological measured using a computerized image analysis system, parameters were measured just before and 15 min after and the ratio of the size of the lesion to the size of the the MCA occlusion. No significant intergroup differences whole cerebral area was calculated. were noted (Table 1). All rats were operated on under

Table 1. Physiological parameters before and 15 min after thrombotic MCA occlusion in the rats Fig. 1. Typical photograph of microthrombi of 10 animals on the occluded hemisphere 3 hr after MCA occlusion The bar indicates 50 ,um a Fibrin-rich microthrombi PTAH stain b Fibrinogen and fibrin detected by anti-rat fibrinogen antibody Administration of argatroban tic acid hematoxylin-positive thrombi was immunoreac- For the group of animals whose brains were removed tive with anti-rat fibrinogen antibody which can react within 6 hr after thrombotic MCA occlusion, argatroban with rat fibrinogen and fibrin (Fig. lb). The thrombi were (40 mg/kg) was administered twice subcutaneously just only found in the left hemisphere, in the side supplied by after and 3 hr after thrombotic MCA occlusion to main- the occluded MCA (Fig. 2). Just after thrombotic MCA tain activity in the plasma up to 6 hr. One occlusion, microthrombi appeared on the surface of the group of animals was injected with saline subcutaneously brain and then extended to the cortex, the caudoputamen in the same way as the argatroban administration group (Fig. 2). In the group without MCA occlusion and photo- to use as controls. For the group of animals whose brains irradiation, the animals were checked for MCA, but no were removed 24 hr after thrombotic MCA occlusion, an microthrombi were detected (data not shown). The num- osmotic pump (Alzet, Palo Alto, CA, USA; 2001D, 8 ber of fibrin-rich microthrombi in each animal continued gil/hr) filled with 40 mg/ml argatroban or saline was im- to increase until 3 hr after thrombotic MCA occlusion planted in the abdomen just after thrombotic MCA oc- (Fig. 3). At 3 hr, the number of fibrin-rich microthrombi clusion. The activated partial thromboplastin time was in animals treated with argatroban was significantly determined using pathrontin (Behringwerke, Tokyo) in (P<0.01) reduced as compared with untreated animals all animals at the end of each experiment as an index of (Fig. 3). plasma coagulability. Ischemic lesions were detected using histological tech- niques. Three hours after thrombotic MCA occlusion, the Statistical analyses lesions were confined to the preoptic area and extended to Data are expressed as means ±S.E.M. Statistical analy- the lateral caudoputamen within 6 hr after thrombotic sis of the number of thrombi was performed with an MCA occlusion. Finally, the ischemic lesions extended unpaired Mann-Whitney test for comparison between into the cortex and caudoputamen 24 hr after thrombotic groups. Statistical analysis of the size of the ischemic MCA occlusion (not shown). At 6 hr after thrombotic lesion was performed with the unpaired Student's t-test MCA occlusion, the ischemic lesion in the group treated for comparison groups. A P value <0.05 was considered with argatroban was significantly (P<0.05) smaller as significant. compared with untreated animals (Fig. 4). In the group treated with argatroban, the activated RESULTS partial thromboplastin times (APTT) were 66.5±2.7 sec

With phosphotungstic acid hematoxylin staining, fibrin was strongly blue-positive (Fig. la). This phosphotungs-

Fig. 3. Effect of argatroban on number of microthrombi. The Fig. 2. The formation of the microthrombi just after (1), 1 hr after mean number of microthrombi counted at 0.5 mm and 1 mm (2), 3 hr after (3), 6 hr after (4) MCA occlusion at 0.5 mm posterior posterior from the bregma. Reported values are means±S.E.M. from the bregma. Total of the microthrombi counted in all of 10 rats derived from 10 observed rats after thrombotic MCA occlusion. of each experimental group. Dots show the actual localization of a **P <0 .01 vs animals treated with vehicle. 0: Vehicle, 0: Argatro- microthrombus in the brain. ban. These microthrombi may contribute to the secondary brain damage caused by MCA occlusion. The micro- thrombi may be formed from circulating that are activated or aggregated at the damaged middle cer- ebral artery vessels. It has also been reported that tissue factor, which is the major initiator of the coagulation cascade leading to the generation of thrombin, is present in cerebral tissue (20, 21) and contributes to the no-reflow phenomenon in a baboon model of MCA occlusion (22). Furthermore, it has been reported that interleukin-1~ is overexpressed in the brain in response to ischemia (23). Interleukin-13 is a cytokine with procoagulant actions (24). This induced cytokine may form a microthrombus. The selective thrombin inhibitor argatroban signifi- cantly inhibited the formation of microthrombi at 3 hr after thrombotic MCA occlusion, but it was ineffective beyond 3 hr. The inhibitory effect of argatroban on the

Fig. 4. The size of ischemic lesion after thrombotic MCA occlu- formation of microthrombi was reflected in a significant sion. The mean percent of ischemic lesions measured at 0.5 mm reduction in the size of cerebral ischemic lesions 6 hr after and 1 mm posterior from the bregma. Reported values are thrombotic MCA occlusion, but argatroban did not means±S.E.M. values derived from 10 observations. **P<0.01 vs reduce the extent of lesions observed 24 hr after throm- animals treated with vehicle. U: Vehicle, El: Argatroban. botic MCA occlusion. The effect of argatroban on microthrombi formation at 6 hr after thrombotic MCA occlusion was observed to be different from its effect on at 3 hr, 80.0 ± 3.2 sec at 6 hr, 49.1 ± 2.9 sec at 24 hr and the ischemic lesions at that time; this may be due to the 26.5 ± 0.6 sec, 25.0 ± 0.7 sec, 24.4 ± 0.6 sec at the respec- difficulty of detecting the microthrombi formation 6 hr tive times in each control group (n=6) (P<0.01). Based after thrombotic MCA occlusion. Thus, the inhibitory on APTT prolongation, the effect of argatroban con- effect of argatroban on the formation of microthrombi tinued up to the end of each experiment. suggests that thrombin contributes to ischemic brain damage following thrombotic MCA occlusion. DISCUSSION In this study, we observed that fibrin-rich micro- thrombi formed within 3 hr following MCA occlusion. In this study, the thrombotic occlusion of MCA was It is concluded that in our rat model, following endo- induced by the photochemical reaction between rose Ben- thelial injury and the thrombotic occlusion of MCA, gal and green light which causes endothelial injury fol- brain tissue directly supplied by the MCA becomes in- lowed by platelet adhesion and formation of a platelet- farcted. Subsequently, thrombin is generated; it induces and fibrin-rich thrombus. The thrombotic occlusion of the formation of the microthrombi, thus causing further MCA resulted in ischemic damage to the brain tissues. ischemic brain damage by blocking collateral microves- The extent of ischemic lesions and number of micro- sels. Yet, it has been reported that polymorphonuclear thrombi containing fibrin increased time-dependently leukocytes contribute to post-ischemic perfusion abnor- after MCA occlusion. Ischemic lesions increased to the malities (25, 26) and brain edema (27). Further ischemic same degree as in other models in which the middle damage may be caused by other factors such as leuko- cerebral arteries are occluded by electrocoagulation (4). cytes and free radicals (28) that may not be related to However, the extent of microthrombi formation in our thrombin because they were unaffected by argatroban. model was quite different from those in the other models Acknowledgments (8 -10). Heye et al. have reported that the maximum microthrombus formation was found 7 days after MCA The authors thank Dr. A. Saniabadi, Senior Scientist at Terumo Corp., Kanagawa, Japan for editing the manuscript. occlusion, and the microthrombi were found in both the occluded and the contralateral hemispheres. In our REFERENCES model, the microthrombi beyond 6 hr after thrombotic MCA occlusion were not detectable because leukocytes I Scheinberg P: The biologic basis for the treatment of acute infiltrated and therefore the structure of the microvessels stroke. Neurology 41, 1867-1873 (1991) was broken. 2 Raichle ME: The pathophysiology of brain ischemia. Ann Neurol 13, 2-10 (1983) USA 86, 7585-7589 (1989) 3 Nagasawa H and Kogure K: Correlation between cerebral blood 16 Edit JF, Allison P, Noble S, Ashton J, Golino P, Mcnatt J, flow and histologic changes in a new rat model middle cerebral Buja LM and Willerson JT: Thrombin is an important mediator artery occlusion. Stroke 20, 1037-1043 (1989) of platelet aggregation in stenosed canine coronary arteries with 4 Garcia JH, Yoshida Y, Chen H, Li Y, Zhang ZG, Lian J, Chen endothelial injury. J Clin Invest 84, 18-27 (1989) S and Chopp M: Progression from ischemic injury to infarct 17 Jang IK, Gold HK, Zisking AA, Leinbach RC, Fallon JT and following middle cerebral artery occlusion in the rat. Am J Collen D: Prevention of platelet-rich arterial thrombosis by Pathol 142, 623 - 63 5 (1993) selective thrombin inhibition. Circulation 81, 219-225 (1990) 5 Lane DA, Wolff S, Ireland H, Gawel M and Foadi M: Activa- 18 Yasuda T, Gold HK, Yaoita H, Leinbach RC, Guerrero JL, tion of coagulation and fibrinolytic systems following stroke. Jang IK, Holt R, Fallon JT and Collen D: Comparative effects Br J Haematol 53, 655-658 (1983) of , a synthetic thrombin inhibitor and a monoclonal 6 Feinberg WM, Bruck DC, Ring ME and Corrigan JJ Jr: antiplatelet glycoprotein IIb/IIIa antibody on ricombinant tis- Hemostatic markers in acute stroke. Stroke 20, 592-597 (1989) sue-type in a canine preparation. J Am 7 Tohgi H, Kawashima M, Tamura K and Suzuki H: Coll Cardiol 16, 714-722 (1990) Coagulation- abnormalities in acute and chronic 19 Umemura K, Wada K, Uematsu T and Nakashima M: Evalua- phases of cerebral thrombosis and embolism. Stroke 21, tion of the combination of a tissue-type plasminogen activator, 1663 -1667 (1990) SUN9216, and a A2 antagonist, 8 Sampaolo S, Cervos-Navarro J, Djouchadar D and Figols J: Vapiprost, in a rat middle cerebral artery thrombosis model. Clinical and experimental evidence of microthrombosis in Stroke 24, 1077 -1081(1993) cerebral ischemia. In Cerebral Ischemia and Hemorheology, 20 del Zoppo GJ, Yu JQ, Copeland BR, Thomas WS, Schneider- Edited by Hartmann A and Kuschinsky W, pp 386-393, man J and Morrissey JH: Tissue factor localization in non- Springer, Berlin (1987) human primate cerebral tissue. Thromb Haemost 68, 642-647 9 Heye N, Campos A, Kannuki S and Cervos-Navarro J: Effects (1992) of and acetylsalitic acid on microthrombi formation in 21 Fleck RA, Rao LVM, Rapaport SI and Varki N: Localization experimental brain ischemia. Exp Pathol 41, 31-36 (1991) of human tissue factor antigen by immunostaining with 10 Heye N, Paetzold C and Cervos-Navarro J: The role of monospecific polyclonal anti-human tissue factor antibody. microthrombi and microcirculatory factors in localization and Thromb Res 57, 765 - 782 (1990) evolution of focal cerebral ischemia. Neurosurg Rev 14, 7- 16 22 Thomas WS, Mori E, Copeland BR, Yu JQ, Morrissey JH and (1991) del Zoppo GJ: Tissue factor contributes to microvascular 11 Okamoto S, Hijikata A, Kikumoto R, Tonomura S, Hara H, defects after focal cerebral ischemia. Stroke 24, 847 - 854 (1993) Ninomiya K, Maruyama A, Sugano M and Tamao Y: Potent 23 Liu T, McDonnell PC, Young PR, White RF, Siren AL, inhibition of thrombin by the newly synthesized arginine Hallenbeck JM, Barone FC and Feuerstein GZ: Interleukin-113 derivative No. 805. The importance of stereostructure of its mRNA expression in ischemic rat cortex. Stroke 24, 1746-1751 hydrophobic carboxamide portion. Biochem Biophys Res (1993) Commun 101, 440-446 (1981) 24 Dinarello CA: Biology of interleukin-1. FASEB J 2, 108-115 12 Kikumoto R, Tamao Y, Tezuka T, Tonomura S, Hara H, (1988) Ninomiya K, Hijikata A and Okamoto S: Selective inhibition of 25 del Zoppo GJ, Schmid-Schonbein GW, Mori E, Copeland BR thrombin by (2R,4R)-4-methyl-l-[N2-[(3-methyl-1,2,3,4,-tetra- and Chang CM: Polymorphonuclear leukocytes occlude capil- hydro-8 -quinolinyl) sulfonyl] -L-arginyl] -2-piperidinecarboxylic laries following middle cerebral artery occlusion and reperfu- acid. Biochemistry 23, 85-90 (1984) sion in baboons. Stroke 22, 1276- 1283 (1991) 13 Ikoma H, Ohtsu K, Tamao Y, Kikumoto R and Okamoto S: 26 Mori E, del Zoppo GJ, Chambers JD, Copeland BR and Arfors Effect of a potent thrombin inhibitor, MCI-9038, on novel ex- KE: Inhibition of polymorphonuclear leukocyte adherence perimental arterial thrombosis. Blood and Vessel 13, 72-77 suppresses no-reflow after focal cerebral ischemia in baboons. (1982) Stroke 23, 712-718 (1992) 14 Tamao Y, Yamamoto T, Kikumoto R, Hara H, Itoh J, Hirata 27 Shiga Y, Onodera H, Kogure K, Yamasaki Y, Yashima Y, T, Mineo K and Okamoto S: Effect of a selective thrombin in- Syozuhara H and Sendo F: Neutrophil as a mediator of hibitor MCI-9038 on fibrinolysis in vitro and in vivo. Thromb ischemic edema formation in the brain. Neurosci Lett 125, Haemost 56, 28-34 (1986) 110-112 (1991) 15 Fitzgerald DJ and Fitzgerald GA: Role of thrombin and 28 Pulsinelli W: The ischemic penumbra in stroke. Sci Am 2, in reocclusion following coronary thromboly- 16-25 (1995) sis with tissue-type plasminogen activator. Proc Natl Acad Sci