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Dissolution of emboli in rats with experimental cerebral thromboembolism by recombinant human tissue plasminogen activator (TD-2061)
THROMBOSIS RESEARCH 59; 703-712,199O 0049-3848/90 $3.00 + .OO Printed in the USA. Copyright (c) 1990 Pergamon Press pk. All rights reserved.
DISSOLUTION OF EMBOLI IN RATS WITH EXPERIMENTAL CEREBRAL THROMBOEMBOLISM BY RECOMBINANT HUMAN TISSUE PLASMINOGEN ACTIVATOR (TD-2061)
Tsuyoshi Hara, Masahiro Iwamoto, Hidemasa Ogawa, Asako Yamamoto and Munehiro Tomikawa Daiichi Pharmaceutical Co.,Ltd., Research Institute 1-16-13 Kitakasaj, Edogawa-ku, Tokyo 134, Japan (Received
21.2.1990;
accepted
in revised form 8.6.1990
by Editor B. Wiman)
ABSTRACT plasminogen activator (t-PA) is frequently adminTissue clinically as thrombolytic therapy. We injected istered recombinant t-PA into rats with cerebral lz5 I-labeled of blood clot emboli to evaluate the dissolutive effect recombinant human single-chain t-PA (rt-PA; TD-2061) on such emboli and to examine the possibility of improving neurological damage in patients with cerebral thrombosis. When rt-PA was given intravenously at a dose of 350,000 IU/kg 2 minutes before embolization, radioactivity in the affected cerebral hemisphere decreased to 20% of that in the vehicle control 2 hours after embolization. A significant decrease in radioactivity in the cerebral hemisphere was also found on the administration of 700,000 IU/kg of rt-PA 30 or 60 minutes after embolization, but not when rt-PA was administered 2 minutes after embolization. Marked inhibition of abnormal behavior such as hemiplegia was seen on treatment with rt-PA 2 minutes before embolization, but not at all when rt-PA teatment was given 30 or 60 minutes after embolization. The findings suggest that rt-PA can dissolve blood clot emboli in cerebral vessels and that prompt thrombolytic therapy is important to minimize neurological dysfunction in cases of cerebral thromboembolism.
Key words: experimental cerebral thromboembolism, recombinant human tissue plasminogen activator, thrombolytic activity
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INTRODUCTION The frequent induction of psychosis and paralysis through ischemic cerebral damage resulting from cerebral embolism and thrombosis unlike systemic emboli or thrombi is a major clinical problem. An effective thrombolytic treatment for thromboembolic strokes accompanied with ischemic cerebral damage would therefore be a useful and welcome development. Tissue-type plasminogen activator (t-PA) has been shown to specifically activate plasminogen in the presence of fibrin (1) and to be a more potent thrombolytic agent than urokinase in vitro (2). Zivin et al. (3) reported that t-PA reduced neurological damage -in vivo in a rabbit cerebral embolism model. However, there is no direct evidence that such a reduction was caused by the thrombolytic effect of t-PA. In this study, we used a rat cerebral thromboembolism model which 1251-labeled blood clot fragments were introduced into ihne carotid artery (4) , as an animal model close to the clinical situation. The aims of this study were to evaluate the thrombolytic effect of TD-2061, a recombinant single-chain t-PA human (rt-PA) produced by gene technology, and to examine the therapeutic capacity of &-PA in preventing abnormal behavior induced by ischemic cerebral damage in this model.
MATERIALS AND METHODS Materials (recombinant human tissue-type plasminogen The TD-2061 activator, specific activity 667,000 IU/mg) employed was the product of Toyobo Co., Ltd. Japan. 1251-potassium iodide (NEN and bovine serum albumin (fraction V) were Research Products) purchased from Daiichi Pure Chemicals, Japan. Animals Male Wistar rats weighing from 250 to 300g were used. 1251-labeled rat fibrinogen plasma Rat fibrinoqen was purified from rat fresh titrated according to the method of Hawker et al. (5). Using iodine monorat fibrinogen was labeled with 1251 by the method of chloride, carrying Pressman (6), slightiy modified. The labeled fibrinogen more than 95% of the radioactivity was able to form clots. Preparation of 1251-labeled blood clot fragments following a Labeled blood clot fragments were prepared (4). slight modification of the method described by Kudo et al. hydrochloride (90 mg/kg, Rats were anesthetized with ketamin and i.p.). Five ml of blood was obtained from the carotid artery (about 18,000,000 mixed with 0.2 ml of 1251-fibrinogen solution clot and cpm) and kept at room temperature to clot. The blood
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transferred into a syringe and the clot was fragserum were mented by passing it several times through needles, first a 25 second a 26 gauge, and finally a 27 gauge. The needle, gauge blood clot fragments were washed on a metal net (mesh size 200) four times with 25 ml of isotonic saline containing 1% of bovine serum albumin. Then, the fragments were resuspended in rat serum conto obtain two suspensions with blood clot fragment protein of 5 mg/ml and 7.5 mg/ml, as measured by the Lowry centrations method (7) in the serum. Procedures for embolization in a cerebral vessel was induced according to Embolization of Kudo et al. (4) with a minor modification. Rats the method were anesthetized with ketamine hydrochloride (90 mglkg, i.p.), were exand the internal and external right carotid arteries posed. The external artery was occluded with a thread and the common carotid artery was temporarily occluded with a clip about 15 mm below the bifurcation. A 100 ul sample of the blood clot was fragment suspension containing 5 mglml or 7.5 mglml protein injected into the common carotid artery using a 26 gauge needle. and the site of injection, the needle was withdrawn After Sankyo, sealed off with surgical adhesive (Alon-Alfa, injection Japan). The clip on the common carotid artery was removed and the flow was restored. &-PA was administered into the tail vein before or after the injection of blood clot fragments. Determination of the thrombolytic activity by rt-PA times after the administration of rt-PA, At the certain and rats were killed by exsanguination under ketamine anesthesia hemithe brain was removed. The radioactivity in each cerebral sphere was counted with a gamma counter. Fibrinolytic activity of rt-PA in vitro Fiftv ul of rt-PA (5.000-20.000 IU/ml) was added to 450 ul of 1251-labdled blood clot'fragment suspension (about 70,000 cpm) and then the reaction mixture was incubated at 37'C for 1 hr. The reaction was terminated by addition of 5 ul of aprotinin (10,000 U/ml) to the mixture. The mixture was centrifuged at 2,000 xg for 10 min and the radioactivity in 100 ul of the supernatant was counted. The percentage of clot lysis was obtained as follow: Clot lysis (%)= (Supernatant count / Total count) x500 Classification of behavior The behavior of each animal was assessed 24 hours after injection of 100 ul of the blood clot fragment suspension containing 7.5 mglml protein. The behavior observed was classified in grades as follows: 1. Normal. 2. Reduction of spontaneous activity. 3. Circumambulation and inclined body posture. 4. Recumbency and stiffness of limbs. 5. Death.
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Pathological study of cerebral thromboembolism in rat Rats were killed by exsanguination under pentobarbital anesthesia (50 mg/kg, i-p.) at several time points after embolization. The brains were removed and fixed in 10% phosphatebuffered formalin at room temperature. The sections were stained with haematoxylin-eosin (H.E.). Analysis of data The statistical significance of data was analysed by Williams-Wilcoxon test. Data are presented as means f S.E.
the
RESULTS In vitro study The in vitro fibrinolytic activity of rt-PA is shown in Table 1. X-PA lysed blood clot fragments in a concentrationdependent manner.
TABLE 1 Dissolution of 1251-Blood Clot Emboli by rt-PA at Various Concentrations -in vitro. IU/ml Vehicle rt-PA
500 1,000 2,000
Clot lysis(%) 0.2 63.8 82.6 99.7
Spontaneous dissolution of blood clot emboli in brain vessel at Radioactivity in the cerebral hemispheres was counted 0.5, 1, 2, 6 and 24 hr after injection of 100 ~1 of the 5 mg/ml (about of 1251-labeled blood clot fragments protein suspension Thirty minutes after embolization, the radio310,000 cpm/head). activity in the ipsilateral hemisphere was 77,000 cpm/g of wet tissue, and that in the contralateral hemisphere was about onehemithird of this level. The radioactivity in the ipsilateral with concomitant sphere decreased in a time-dependent manner the and by 24 hr after embolization, dissolution, spontaneous 30 min the to less than l/20 of radioactivity had decreased radiolevel, as shown in Figure 1. About 50% of the injected activity was distributed to the ipsilateral side of skull.
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0. Ipsilateral hemisphere (~5) Contralateral
05
I
hemisphere (n=5)
2
Time after injection of ‘**I-blood
clot fragments (hr)
FIG. 1 Spontaneous dissolution of '251-blood clot emboli in experimental cerebral thromboembolism in rats. in the cerebral hemispheres was counted at the Radioactivity indicated time points after injection of '*'I-blood clot fragments. Data are shown as mean ? S.E. (n=5).
Thrombolytic activity of rt-PA in rat cerebral thromboembolism model intravenously 2 minutes before rt-PA was administered embolization at doses of 175,000 IU/kg and 350,000 IU/kg. When in the 350,000 Iu/kg of rt-PA was injected, the radioactivity hemisphere decreased significantly to 20% of that of ipsilateral the saline control (Figure 2). When rt-PA was given 30 or 60 after embolization at a dose of 700,000 IU/kg under the minutes same conditions, the radioactivity in the ipsilateral hemisphere However, when rt-PA was given 2 minutes decreased significantly. after embolization at the same dose, the radioactivity in the ipsilateral hemisphere did not decrease at all (Figure 3). Thus, behavioral observation study was not examined at this time point of administration of rt-PA. Prevention of ischemic brain damage by rt-PA The behavior of each animal was assessed 24 hours after of 100 ul of the 7.5 mg/ml protein suspension of blood injection rt-PA was administered 2 minutes before emboliclot fragments. zation at doses of 175,000 Iu/kg, 350,000 IU/kg and 700,000 IU/kg in order to prevent ischemic brain damage. As shown in Table 2, doses of 350,000 and 700,000 IU/kg rtPA markedly improved the abnormal behavior. However, abnormal behavior was not improved by the administration of rt-PA 30, 60 or 120 minutes after embolization at a dose of 700,000 IU/kg. The treatment with an even higher dose, 1,600,OOO IU/kg, also did not improve abnormal behavior 30 minutes after embolization (Table 3).
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W/kg
PpM_5 6-
1
0
: Control
m
: rt-PA
P
hL Ipsi. (n=5)
IPSi.
(n=5)
Contra. (n=5)
FIG. 2 Thrombolytic of &-PA effect in the experimental cerebral thromboembolism in rats. rt-PA was administered intravenously 2 minutes before embolization at various doses. W/kg. Radioactivity in the cereA) 350,000 Iu/kg, B) 175,000 bral hemispheres was counted 2 hours after embolization. Data represents mean + S.E. (n=5). : Ipsilateral hemisphere Ipsi. Contra.: Contralateral hemisphere
Effect of rt-PA on coagulation and fibrinolytic parameters rt-PA was administered 30 minutes after embolization at a dose of 1,600,OOO ID/kg in a study of changes in coagulation and fibrinolytic parameters. Blood was collected in plastic syringes 3.1% sodium citrate solution 2 and 60 minutes and containing 23.5 hours after the administration of rt-PA. thromboplastin rt-PA did not affect the activated partial c/2levels of fibrinogen or time, prothrombin time, plasma inhibitor at any time after its administration (Data not plasmin shown). Pathological study of cerebral thromboembolism in rats areas were frequently observed, espeScattered edematous after embolization 30 minutes cially * the hippocampus (Figure 4in and progressed gradually 2 hours after embolization. the nuclei of most of the nerve cells exhibited In such lesions, Spongy changes were that was due to hypoxia. pyknotic changes also found throughout the tissue of the encephalon. No hemorrhage was observed 0.5, 1, 2, 6 or 24 hr after embolization.
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MOminafter
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P405
n
i_ :ontra. _. m=51
lpsi. (=ontra. fn=5)
(n=5)
Ipsi. (n=5)
Contra. h=5)
FIG. 3 Thrombolytic effect of rt-PA in the experimental cerebral thromboembolism model in rats. rt-PA was administered intravenously A) 2 minutes after, B) 30 minutes after, and C) 60 minutes after, embolization at a dose of 700,000 IU/kg. Radioactivity in the cerebral hemispheres was counted 2 hours after the administration of rt-PA. Data represents mean i S.E.(n=5). Ipsi. : Ipsilateral hemisphere Contra.: Contralateral hemisphere
FIG. 4 Light micrograph of the hippocampal area of 30 minutes after embolization shows scattered edematous changes (H.E. stain. x 100).
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TABLE 2 Effect of rt-PA on Abnormal Behavior in Rats with Experimental Cerebral Thromboembolism. rt-PA was administered intravenously 2 minutes before embolization at doses of 175,000 Iu/kg, 350,000 IU/kg and 700,000 IU/kg. Abnormal behavior was assessed 24 hours after embolization and graded as stated in Materials and Methods.
Vehicle rt-PA
Iu/kg
NO. Of rats
Grade
175,000 350,000 700,000
(8) (8) (8) (8)
5,3,3,3,3,3,2,1 5,4,3,3,3,3,2,1 5,2,1,1,1,1,1,1 3,2,1,1,1,1 ,I ,1
Mean 2.9 3.0 1.6* 1.4*
* ~~0.05 vs vehicle
TABLE 3 Effect of rt-PA on Abnormal Behavior in Rats with Experimental Cerebral Thromboembolism. rt-PA was administered intravenously 30, 60 or 120 minutes after embolization at a dose of 700,000 IU/kg and 30 minutes after embolization at a dose of 1,600,OOO IU/kg. Abnormal behavior was assessed 24 hours after embolization. IU/kg
No. of rat
Grade
Mean
A) 30 minutes after embolization (9) Vehicle (9) rt-PA 700,000
5,5,5,5,5,3,2,2,1 5,5,5,4,3,2,2,2,1
3.4 3.2
B) 60 minutes after embolization (9) Vehicle (9) rt-PA 700,000
5,4,4,3,3,3,2,2,2 4,3,3,3,2,2,2,1,1
3.1 2.3
C) 120 minutes after embolization (9) Vehicle (9) rt-PA 700,000
5,5,5,5,5,5,5,2,2 5,5,5,5,4,3,3,3,2
4.3 3.9
(high dose) 5,5,5,5,5,5,3,2,1 5,5,4,2,2,1 ,I ,I
4.0 2.6
D) 30 minutes after embolization (9) Vehicle rt-PA 1,600,OOO (8)
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DISCUSSION we have clearly demonstrated the In present study, the activity of rt-PA in an experimental thrombolytic cerepotent 1251-labeled blood clot bral thromboembolism model in rats with emboli in brain vessels. hours after embolization, the radioactivity in Twenty-four ipsilateral hemisphare decreased to less than l/20 of that the that 30 minutes without injection of r-t-PA. This indicates at blood clot emboli in the brain vessels were dissolved sponthe suggests that occluded were probably taneously, and vessels 24 hr after Kaneko et al. recanalized within embolization. this spontaneous recanalization was observed on reported that angiographic examination 4 hours after embolization (8). rt-PA was administered 30 or 60 minutes after emboliWhen the emboli in the brain were zation at a dose of 700,000 IU/kg. dissolved significantly. However, &-PA at the same dose did not emboli when administered 2 minutes after embolization. dissolve rt-PA may have been unable to reach findings suggest that These 2 minutes were completely occluding blood vessels emboli that It was also postulated that a slight recaembolization. after thrombetween emboli and vessel walls by endogenous nalization may have occurred within 30 minutes after activity bolytic and that rt-PA was able to penetrate the emboli 30 embolization, or 60 minutes after embolization. was markedly inhibsuch as hemiplegia, Abnormal behavior, rt-PA 2 minutes administration 350,000 IU/kg of the ited by This effect of rt-PA may have been due to a before embolization. of emboli prior to the occurrence of severe rapid dissolution in the brain. the animals did not ischemic damage However, recover from hemiplegia when rt-PA was administred 30 minutes or embolization at a dose of 700,000 IV/kg, although after longer vessels were dissolved by similar treatment emboli in cerebral These results strongly suggest that with rt-PA at the same dose. ischemic damage occurred in the brain within 30 irreversible This suggestion was confirmed by the minutes after embolization. ischemic damage in the brain finding that took pathological place within 30 minutes after embolization. Neither the plasma level of a2-plasmin inhibitor nor fibrindecreased after injection of rt-PA at a dose of ogen 1,600,OOO In the preliminary examination, fibrinolytic activity of IU/kg. human t-PA in rat -plasma clot was about nine times less than that in human plasma clot. Therefore, the blood level of plasmin might not increase enough to decrease the plasma levels of a2-plasmin inhibitor and fibrinogen after administration of a higher dose of rt-PA. These findings show that systemic administration of rt-PA fibrinolytic can enhance activity enough to dissolve emboli occluding the cerebral vessels without fibrinogenolysis, and that thrombolytic prompt therapy is important to minimize neurological dysfunction in cases of cerebral thromboembolism.
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REFERENCES 1.
HOYLAERTS, M., RIJKEN, D.C., LIJKEN, H.R. and COLLEN, D. Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin. J. Biol. Chem., 257, 2912-2919, 1982
2.
Comparison of the MATSUO, O., RIJKEN, D.C. and COLLEN, D. relative fibrinogenolytic, fibrinolytic and thrombolytic properties of tissue plasminogen activator and urokinase in vitro. Thromb. Haemostasis, 45, 225-229, 1981
3.
ZIVIN, J.A., FISHER, M., DEGIROLAMI, U., HEMENWAY, C.C. and Tissue plasminogen activator reduces neuroSTASHAK, J.A. logical damage after cerebral embolism. Science, 230, 12891292, 1985
4.
KUDO, M., AOYAMA, A., ICHIMORI, S. and FUKUNAGA, N. An animal model of cerebral infarction. Homologous blood clot emboli in rats. Stroke, 13(4), 505-508, 1982
5.
HAWKER, R.J. and HAWKER, L.M. A rapidly produced 1251-labelled autologous fibrinogen: In vitro properties and preliminary metabolic studies in man. J. Clin. Pathol., 29, 495501, 1976
6.
GROSSBERG, A.L., RADZIMSKI, G. and PRESSMAN, D. Effect of iodination on the active site of several antihapten antiBiochemistry, 1, 391-401, 1962 bodies.
7.
LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L. AND RANDALL, R.J. Protein measurement with the Folin phenol reagent J. Biol. Chem., 193, 265-275, 1951
8.
Cerebral infarction KANEKO, D., NAKAMURA, N. and OGAWA, T. in rats using homologous blood emboli; Development of a new Stroke, 16(l), 76-84, 1985 experimental model.
DISSOLUTION OF EMBOLI IN RATS WITH EXPERIMENTAL CEREBRAL THROMBOEMBOLISM BY RECOMBINANT HUMAN TISSUE PLASMINOGEN ACTIVATOR (TD-2061)
Tsuyoshi Hara, Masahiro Iwamoto, Hidemasa Ogawa, Asako Yamamoto and Munehiro Tomikawa Daiichi Pharmaceutical Co.,Ltd., Research Institute 1-16-13 Kitakasaj, Edogawa-ku, Tokyo 134, Japan (Received
21.2.1990;
accepted
in revised form 8.6.1990
by Editor B. Wiman)
ABSTRACT plasminogen activator (t-PA) is frequently adminTissue clinically as thrombolytic therapy. We injected istered recombinant t-PA into rats with cerebral lz5 I-labeled of blood clot emboli to evaluate the dissolutive effect recombinant human single-chain t-PA (rt-PA; TD-2061) on such emboli and to examine the possibility of improving neurological damage in patients with cerebral thrombosis. When rt-PA was given intravenously at a dose of 350,000 IU/kg 2 minutes before embolization, radioactivity in the affected cerebral hemisphere decreased to 20% of that in the vehicle control 2 hours after embolization. A significant decrease in radioactivity in the cerebral hemisphere was also found on the administration of 700,000 IU/kg of rt-PA 30 or 60 minutes after embolization, but not when rt-PA was administered 2 minutes after embolization. Marked inhibition of abnormal behavior such as hemiplegia was seen on treatment with rt-PA 2 minutes before embolization, but not at all when rt-PA teatment was given 30 or 60 minutes after embolization. The findings suggest that rt-PA can dissolve blood clot emboli in cerebral vessels and that prompt thrombolytic therapy is important to minimize neurological dysfunction in cases of cerebral thromboembolism.
Key words: experimental cerebral thromboembolism, recombinant human tissue plasminogen activator, thrombolytic activity
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INTRODUCTION The frequent induction of psychosis and paralysis through ischemic cerebral damage resulting from cerebral embolism and thrombosis unlike systemic emboli or thrombi is a major clinical problem. An effective thrombolytic treatment for thromboembolic strokes accompanied with ischemic cerebral damage would therefore be a useful and welcome development. Tissue-type plasminogen activator (t-PA) has been shown to specifically activate plasminogen in the presence of fibrin (1) and to be a more potent thrombolytic agent than urokinase in vitro (2). Zivin et al. (3) reported that t-PA reduced neurological damage -in vivo in a rabbit cerebral embolism model. However, there is no direct evidence that such a reduction was caused by the thrombolytic effect of t-PA. In this study, we used a rat cerebral thromboembolism model which 1251-labeled blood clot fragments were introduced into ihne carotid artery (4) , as an animal model close to the clinical situation. The aims of this study were to evaluate the thrombolytic effect of TD-2061, a recombinant single-chain t-PA human (rt-PA) produced by gene technology, and to examine the therapeutic capacity of &-PA in preventing abnormal behavior induced by ischemic cerebral damage in this model.
MATERIALS AND METHODS Materials (recombinant human tissue-type plasminogen The TD-2061 activator, specific activity 667,000 IU/mg) employed was the product of Toyobo Co., Ltd. Japan. 1251-potassium iodide (NEN and bovine serum albumin (fraction V) were Research Products) purchased from Daiichi Pure Chemicals, Japan. Animals Male Wistar rats weighing from 250 to 300g were used. 1251-labeled rat fibrinogen plasma Rat fibrinoqen was purified from rat fresh titrated according to the method of Hawker et al. (5). Using iodine monorat fibrinogen was labeled with 1251 by the method of chloride, carrying Pressman (6), slightiy modified. The labeled fibrinogen more than 95% of the radioactivity was able to form clots. Preparation of 1251-labeled blood clot fragments following a Labeled blood clot fragments were prepared (4). slight modification of the method described by Kudo et al. hydrochloride (90 mg/kg, Rats were anesthetized with ketamin and i.p.). Five ml of blood was obtained from the carotid artery (about 18,000,000 mixed with 0.2 ml of 1251-fibrinogen solution clot and cpm) and kept at room temperature to clot. The blood
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transferred into a syringe and the clot was fragserum were mented by passing it several times through needles, first a 25 second a 26 gauge, and finally a 27 gauge. The needle, gauge blood clot fragments were washed on a metal net (mesh size 200) four times with 25 ml of isotonic saline containing 1% of bovine serum albumin. Then, the fragments were resuspended in rat serum conto obtain two suspensions with blood clot fragment protein of 5 mg/ml and 7.5 mg/ml, as measured by the Lowry centrations method (7) in the serum. Procedures for embolization in a cerebral vessel was induced according to Embolization of Kudo et al. (4) with a minor modification. Rats the method were anesthetized with ketamine hydrochloride (90 mglkg, i.p.), were exand the internal and external right carotid arteries posed. The external artery was occluded with a thread and the common carotid artery was temporarily occluded with a clip about 15 mm below the bifurcation. A 100 ul sample of the blood clot was fragment suspension containing 5 mglml or 7.5 mglml protein injected into the common carotid artery using a 26 gauge needle. and the site of injection, the needle was withdrawn After Sankyo, sealed off with surgical adhesive (Alon-Alfa, injection Japan). The clip on the common carotid artery was removed and the flow was restored. &-PA was administered into the tail vein before or after the injection of blood clot fragments. Determination of the thrombolytic activity by rt-PA times after the administration of rt-PA, At the certain and rats were killed by exsanguination under ketamine anesthesia hemithe brain was removed. The radioactivity in each cerebral sphere was counted with a gamma counter. Fibrinolytic activity of rt-PA in vitro Fiftv ul of rt-PA (5.000-20.000 IU/ml) was added to 450 ul of 1251-labdled blood clot'fragment suspension (about 70,000 cpm) and then the reaction mixture was incubated at 37'C for 1 hr. The reaction was terminated by addition of 5 ul of aprotinin (10,000 U/ml) to the mixture. The mixture was centrifuged at 2,000 xg for 10 min and the radioactivity in 100 ul of the supernatant was counted. The percentage of clot lysis was obtained as follow: Clot lysis (%)= (Supernatant count / Total count) x500 Classification of behavior The behavior of each animal was assessed 24 hours after injection of 100 ul of the blood clot fragment suspension containing 7.5 mglml protein. The behavior observed was classified in grades as follows: 1. Normal. 2. Reduction of spontaneous activity. 3. Circumambulation and inclined body posture. 4. Recumbency and stiffness of limbs. 5. Death.
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Pathological study of cerebral thromboembolism in rat Rats were killed by exsanguination under pentobarbital anesthesia (50 mg/kg, i-p.) at several time points after embolization. The brains were removed and fixed in 10% phosphatebuffered formalin at room temperature. The sections were stained with haematoxylin-eosin (H.E.). Analysis of data The statistical significance of data was analysed by Williams-Wilcoxon test. Data are presented as means f S.E.
the
RESULTS In vitro study The in vitro fibrinolytic activity of rt-PA is shown in Table 1. X-PA lysed blood clot fragments in a concentrationdependent manner.
TABLE 1 Dissolution of 1251-Blood Clot Emboli by rt-PA at Various Concentrations -in vitro. IU/ml Vehicle rt-PA
500 1,000 2,000
Clot lysis(%) 0.2 63.8 82.6 99.7
Spontaneous dissolution of blood clot emboli in brain vessel at Radioactivity in the cerebral hemispheres was counted 0.5, 1, 2, 6 and 24 hr after injection of 100 ~1 of the 5 mg/ml (about of 1251-labeled blood clot fragments protein suspension Thirty minutes after embolization, the radio310,000 cpm/head). activity in the ipsilateral hemisphere was 77,000 cpm/g of wet tissue, and that in the contralateral hemisphere was about onehemithird of this level. The radioactivity in the ipsilateral with concomitant sphere decreased in a time-dependent manner the and by 24 hr after embolization, dissolution, spontaneous 30 min the to less than l/20 of radioactivity had decreased radiolevel, as shown in Figure 1. About 50% of the injected activity was distributed to the ipsilateral side of skull.
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0. Ipsilateral hemisphere (~5) Contralateral
05
I
hemisphere (n=5)
2
Time after injection of ‘**I-blood
clot fragments (hr)
FIG. 1 Spontaneous dissolution of '251-blood clot emboli in experimental cerebral thromboembolism in rats. in the cerebral hemispheres was counted at the Radioactivity indicated time points after injection of '*'I-blood clot fragments. Data are shown as mean ? S.E. (n=5).
Thrombolytic activity of rt-PA in rat cerebral thromboembolism model intravenously 2 minutes before rt-PA was administered embolization at doses of 175,000 IU/kg and 350,000 IU/kg. When in the 350,000 Iu/kg of rt-PA was injected, the radioactivity hemisphere decreased significantly to 20% of that of ipsilateral the saline control (Figure 2). When rt-PA was given 30 or 60 after embolization at a dose of 700,000 IU/kg under the minutes same conditions, the radioactivity in the ipsilateral hemisphere However, when rt-PA was given 2 minutes decreased significantly. after embolization at the same dose, the radioactivity in the ipsilateral hemisphere did not decrease at all (Figure 3). Thus, behavioral observation study was not examined at this time point of administration of rt-PA. Prevention of ischemic brain damage by rt-PA The behavior of each animal was assessed 24 hours after of 100 ul of the 7.5 mg/ml protein suspension of blood injection rt-PA was administered 2 minutes before emboliclot fragments. zation at doses of 175,000 Iu/kg, 350,000 IU/kg and 700,000 IU/kg in order to prevent ischemic brain damage. As shown in Table 2, doses of 350,000 and 700,000 IU/kg rtPA markedly improved the abnormal behavior. However, abnormal behavior was not improved by the administration of rt-PA 30, 60 or 120 minutes after embolization at a dose of 700,000 IU/kg. The treatment with an even higher dose, 1,600,OOO IU/kg, also did not improve abnormal behavior 30 minutes after embolization (Table 3).
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W/kg
PpM_5 6-
1
0
: Control
m
: rt-PA
P
hL Ipsi. (n=5)
IPSi.
(n=5)
Contra. (n=5)
FIG. 2 Thrombolytic of &-PA effect in the experimental cerebral thromboembolism in rats. rt-PA was administered intravenously 2 minutes before embolization at various doses. W/kg. Radioactivity in the cereA) 350,000 Iu/kg, B) 175,000 bral hemispheres was counted 2 hours after embolization. Data represents mean + S.E. (n=5). : Ipsilateral hemisphere Ipsi. Contra.: Contralateral hemisphere
Effect of rt-PA on coagulation and fibrinolytic parameters rt-PA was administered 30 minutes after embolization at a dose of 1,600,OOO ID/kg in a study of changes in coagulation and fibrinolytic parameters. Blood was collected in plastic syringes 3.1% sodium citrate solution 2 and 60 minutes and containing 23.5 hours after the administration of rt-PA. thromboplastin rt-PA did not affect the activated partial c/2levels of fibrinogen or time, prothrombin time, plasma inhibitor at any time after its administration (Data not plasmin shown). Pathological study of cerebral thromboembolism in rats areas were frequently observed, espeScattered edematous after embolization 30 minutes cially * the hippocampus (Figure 4in and progressed gradually 2 hours after embolization. the nuclei of most of the nerve cells exhibited In such lesions, Spongy changes were that was due to hypoxia. pyknotic changes also found throughout the tissue of the encephalon. No hemorrhage was observed 0.5, 1, 2, 6 or 24 hr after embolization.
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A: 2 minafkr
MOminafter
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P405
n
i_ :ontra. _. m=51
lpsi. (=ontra. fn=5)
(n=5)
Ipsi. (n=5)
Contra. h=5)
FIG. 3 Thrombolytic effect of rt-PA in the experimental cerebral thromboembolism model in rats. rt-PA was administered intravenously A) 2 minutes after, B) 30 minutes after, and C) 60 minutes after, embolization at a dose of 700,000 IU/kg. Radioactivity in the cerebral hemispheres was counted 2 hours after the administration of rt-PA. Data represents mean i S.E.(n=5). Ipsi. : Ipsilateral hemisphere Contra.: Contralateral hemisphere
FIG. 4 Light micrograph of the hippocampal area of 30 minutes after embolization shows scattered edematous changes (H.E. stain. x 100).
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TABLE 2 Effect of rt-PA on Abnormal Behavior in Rats with Experimental Cerebral Thromboembolism. rt-PA was administered intravenously 2 minutes before embolization at doses of 175,000 Iu/kg, 350,000 IU/kg and 700,000 IU/kg. Abnormal behavior was assessed 24 hours after embolization and graded as stated in Materials and Methods.
Vehicle rt-PA
Iu/kg
NO. Of rats
Grade
175,000 350,000 700,000
(8) (8) (8) (8)
5,3,3,3,3,3,2,1 5,4,3,3,3,3,2,1 5,2,1,1,1,1,1,1 3,2,1,1,1,1 ,I ,1
Mean 2.9 3.0 1.6* 1.4*
* ~~0.05 vs vehicle
TABLE 3 Effect of rt-PA on Abnormal Behavior in Rats with Experimental Cerebral Thromboembolism. rt-PA was administered intravenously 30, 60 or 120 minutes after embolization at a dose of 700,000 IU/kg and 30 minutes after embolization at a dose of 1,600,OOO IU/kg. Abnormal behavior was assessed 24 hours after embolization. IU/kg
No. of rat
Grade
Mean
A) 30 minutes after embolization (9) Vehicle (9) rt-PA 700,000
5,5,5,5,5,3,2,2,1 5,5,5,4,3,2,2,2,1
3.4 3.2
B) 60 minutes after embolization (9) Vehicle (9) rt-PA 700,000
5,4,4,3,3,3,2,2,2 4,3,3,3,2,2,2,1,1
3.1 2.3
C) 120 minutes after embolization (9) Vehicle (9) rt-PA 700,000
5,5,5,5,5,5,5,2,2 5,5,5,5,4,3,3,3,2
4.3 3.9
(high dose) 5,5,5,5,5,5,3,2,1 5,5,4,2,2,1 ,I ,I
4.0 2.6
D) 30 minutes after embolization (9) Vehicle rt-PA 1,600,OOO (8)
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DISCUSSION we have clearly demonstrated the In present study, the activity of rt-PA in an experimental thrombolytic cerepotent 1251-labeled blood clot bral thromboembolism model in rats with emboli in brain vessels. hours after embolization, the radioactivity in Twenty-four ipsilateral hemisphare decreased to less than l/20 of that the that 30 minutes without injection of r-t-PA. This indicates at blood clot emboli in the brain vessels were dissolved sponthe suggests that occluded were probably taneously, and vessels 24 hr after Kaneko et al. recanalized within embolization. this spontaneous recanalization was observed on reported that angiographic examination 4 hours after embolization (8). rt-PA was administered 30 or 60 minutes after emboliWhen the emboli in the brain were zation at a dose of 700,000 IU/kg. dissolved significantly. However, &-PA at the same dose did not emboli when administered 2 minutes after embolization. dissolve rt-PA may have been unable to reach findings suggest that These 2 minutes were completely occluding blood vessels emboli that It was also postulated that a slight recaembolization. after thrombetween emboli and vessel walls by endogenous nalization may have occurred within 30 minutes after activity bolytic and that rt-PA was able to penetrate the emboli 30 embolization, or 60 minutes after embolization. was markedly inhibsuch as hemiplegia, Abnormal behavior, rt-PA 2 minutes administration 350,000 IU/kg of the ited by This effect of rt-PA may have been due to a before embolization. of emboli prior to the occurrence of severe rapid dissolution in the brain. the animals did not ischemic damage However, recover from hemiplegia when rt-PA was administred 30 minutes or embolization at a dose of 700,000 IV/kg, although after longer vessels were dissolved by similar treatment emboli in cerebral These results strongly suggest that with rt-PA at the same dose. ischemic damage occurred in the brain within 30 irreversible This suggestion was confirmed by the minutes after embolization. ischemic damage in the brain finding that took pathological place within 30 minutes after embolization. Neither the plasma level of a2-plasmin inhibitor nor fibrindecreased after injection of rt-PA at a dose of ogen 1,600,OOO In the preliminary examination, fibrinolytic activity of IU/kg. human t-PA in rat -plasma clot was about nine times less than that in human plasma clot. Therefore, the blood level of plasmin might not increase enough to decrease the plasma levels of a2-plasmin inhibitor and fibrinogen after administration of a higher dose of rt-PA. These findings show that systemic administration of rt-PA fibrinolytic can enhance activity enough to dissolve emboli occluding the cerebral vessels without fibrinogenolysis, and that thrombolytic prompt therapy is important to minimize neurological dysfunction in cases of cerebral thromboembolism.
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