Halothane

Objectives and methods: Deleterious effects of tissue plasminogen activator (tPA) have been described recently in experimental studies. For example, tPA can aggravate ischemic neuronal damage through its proteolytic activity. The present study was undertaken to examine whether or not the free radical scavenger, edaravone, could prevent the extravasation of tPA administered for the purpose of fibrinolysis in a rat model of tnromboembolic stroke.

Results: Significant amounts of tPA were extravasated through the cerebral vessels even when early recanalization was induced by administering tPA at 30 minutes after the onset of schema. Edaravone significantly attenuated such extravasation of tPA.

Conclusion: In acute ischemic stroke patients, combination therapy using tPA with edaravone appears to be a reasonable strategy for diminishing the negative effects of tPA. [Neurol Res 2005; 27: 499-502]

Keywords: Extravasation; fibrinolysis; free radical scavenger; tissue plasminogen activator

INTRODUCTION

It has been demonstrated that early recanalization of occluded vessels by the administration of tissue plasminogen activator (tPA) can improve the clinical outcome of acute ischemic stroke patients1. However, deleterious effects of tPA have also been encountered in experimental studies2. The most convincing mechanism proposed is that tPA aggravates ischemic neuronal damage through its proteolytic activity3,4. Since tPA appears to promote thrombolytic as well as neurotoxic outcomes in ischemic stroke, future fibrinolytic therapy should aim to diminish the deleterious effects of tPA. Provided that the tPA remains within the cerebral vessels, a desirable effect of tPA can be expected. Accordingly, one possible strategy is to undertake combined administration of tPA with some other drug that inhibits the extravasation of tPA through the cerebral vessels. In the present study, we examined whether or not the novel free radical scavenger, edaravone, might have such a potential for inhibiting the extravasation of tPA.

MATERIALS AND METHODS

Thromboembolic stroke model

Male Sprague-Dawley rats weighing 250 to 300 g were employed for the thromboembolic stroke model. Rats were anesthetized with 1-1.5% halothane in an air-oxygen mixture under spontaneous respiration. Subsequent procedures for inducing thromboembolic stroke were performed according to the methods described by Kano et al5. In brief, arterial blood was obtained from a donor rat and stored at room temperature for 48 hours to induce clot formation. The clot was then fragmented by passage through a 26-gauge needle (inner diameter, 270 µm). A suspension of microclots was injected into the rat internal carotid artery (ICA). Finally, a plug of microclots was formed within the distal site of the ICA or the proximal site of the middle cerebral artery (MCA) inducing cerebral ischemia at the territory of the MCA. During all procedures, the rectal temperature was maintained at between 36.5 and 37.5°C with a heating lamp. Blood pressure was monitored via a catheter placed in the femoral artery, and arterial blood samples were collected for blood gas analysis.

tPA Administration

tPA was given at 30 minutes (n=8) or 2 hours (n=8) after the induction of ischemia. The femoral vein of the animals was catheterized, and tPA solution (Alteplase, 10 mg/kg, 1 mg/1 ml in saline) was administered using an infusion pump over a period of 30 minutes. It should be noted that a much higher dose of tPA was employed in the present study as compared to that used clinically because there is a ~ 10-fold difference in the fibrin-specific enzyme activity of human recombinant tPA in human versus rodent systems6.

Edaravone administration

In edaravone treated animals, tPA and edaravone were given concomitantly. Edaravone solution (3 rng/kg, 3 mg/1 mL) was injected intravenously at 30 minutes (n=8) or 2 hours (n=8) after the induction of ischemia, and tPA solution was administered thereafter.

Laser Doppler flowmetry

To assess the induction of focal ischemia following the injection of microclots and the restoration of perfusion following the administration of tPA, we evaluated the cerebral blood flow (CBF) by laser Doppler flowmetry (LDF, Omega Flow, Neuroscience Inc). A skin incision was made, and the skull was drilled to create a small dimple on the bone (1 mm posterior to the bregma and just inferior to the temporal line). The tip of the LDF probe was placed at the position which corresponds to the somatosensory area of the frontoparietal cortex at the level of the globus pallidus, which has been demonstrated to fall within the ischemic core of the present thromboembolic occlusion model5.

Quantification of tPA

Quantification of the extravasated tPA was carried out by enzyme-linked immunosorbent assay (ELISA) methods. At 2 hours after the beginning of edaravone or tPA administration, rats were sacrificed by injecting an overdose of pentobarbital sodium (100mg/kg). The decapitated rat forebrains were cut into their two hemispheres, and each hemisphere was homogenized in a 9-fold volume of added saline and centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant fluid was collected and stored in a deep freezer at -80°C for later analysis according to the methods of Ranby et al7. By employing this ELISA technique, the total tPA antigen (tPA and tPA/inhibitor complexes) was measured.

All experiments were carried out according to a protocol approved by our institution for the care and use of laboratory animals. An ANOVA followed by post hoc 2-tailed f-tests with corrections for multiple groups was performed to compare the various outcomes among the different groups of animals. Differences with a value of p

RESULTS

Systemic variables

In the animals prepared for tPA administration at 30 minutes after the induction of ischemia (n=8), the arterial blood pH, PCO^sub 2^, and PO^sub 2^ values remained within the normal ranges after ischemia: the pH was 7.39 + 0.03; the PCO^sub 2^ was 41+2 mmHg; and the PO^sub 2^ was 114 + 5 mmHg (values are the means+ SD). The data for the blood gases did not differ significantly among the other groups of animals.

LDF Measurement of CBF

Just after the injection of microclots, the CBF declined to levels of below 20% of the preischemic baselines, demonstrating successful induction of focal ischemia (Figure 7). At the time of tPA infusion, the CBF had increased slightly but remained below 30% of the baselines (Figure 1). At 2 hours after the onset of tPA administration, the CBF had recovered significantly to above 80% of the baselines, demonstrating favorable recanalization due to the fibrinolytic activity of the tPA (Figure 7).

Quantification of tPA

In the animals prepared for tPA administration at 30 minutes after the induction of ischemia, the tPA content in the ischemic hemisphere was significantly higher than that in the non-ischemic hemisphere: 3.5 + 1.6 and 0.8 + 0.2 ng/mL (mean + SD) of homogenized brain in saline, respectively (p

DISCUSSION

The present data demonstrate that significant amounts of tPA were extravasated through the cerebral vessels in association with ischemia/reperfusion, even when the tPA was administered at 30 minutes after the induction of ischemia, and the free radical scavenger, edaravone, significantly attenuated such extravasation of tPA. Much evidence has been accumulated to suggest that oxygen free radicals contribute to ischemia/reperfusion-induced brain injury. Edaravone traps a variety of free radical species including hydroxyl radical8, so leading to an amelioration of delayed neuronal death and brain edema in the ischemic/reperfused rat brain9,10. On the other hand, edaravone can prevent the peroxidative vascular endothelial damage induced by hydroperoxyeicosatetraenoic acid11. In the present study also, edaravone appeared to act directly on the reperfused cerebral vessels and to attenuate the vascular permeability to tPA.

In the thromboembolic stroke model that we used, administration of tPA at 2 hours after the induction of ischemia significantly reduced the infarction volume, although the lateral striatum and the somatosensory area of the frontoparietal cortex, the ischemic core of the model, were found to have fallen into infarction on staining with 2,3,5-triphenyltetrazolium chloride (TTC)5. In the case of administration of tPA after 30 minutes, infarction was not detected on TTC staining. Under conditions where ischemic damage to the brain tissue is mild, it seems likely that edaravone attenuates the extravasation of tPA effectively. In contrast, under conditions where cerebral infarction occurs, the extravasation of tPA appears to be no longer attenuated by edaravone, due possibly to disruption of the blood-brain barrier.

In a previous in vitro study, tPA alone exhibited toxic effects on the viability of neurons only at very high concentrations of the order of μg/mL12. In the present study, the tPA concentration in the ischémie hemisphere was of the order of ng/mL. However, such a concentration of tPA could be sufficiently high to amplify the neuronal damage when ischemic insult has already occurred. Further studies are needed to determine whether or not tPA concentration of the order of ng/mL do actually aggravate ischemic neuronal damage.

Edaravone is a unique anti-ischemic drug that can protect the cerebral vessels as well as neurons from ischémie injury. Edaravone has been employed in clinical settings, and it has been demonstrated that edaravone can improve the functional outcome of acute ischemic stroke patients13. Based on the results of the present study, for acute ischemic stroke patients, combination therapy using tPA with edaravone appears to be a reasonable strategy for diminishing the negative effects of tPA. Edaravone may also prolong the therapeutic time window of fibrinolysis in acute ischemic stroke patients through its characteristic effect of protecting both the neurons and cerebral vessels, and further experimental research employing the thromboembolic stroke model is warranted in this respect.

ACKNOWLEDGEMENTS

This study was supported in part by a Grant-in-Aid for Scientific Research in Japan (Grant number: 14571338). tPA was obtained as a generous gift from Kyowa Hakko Kogyo Co, Japan.

REFERENCES

1 NlNDS rt-PA Stroke Study Group. Tissue plasminogen activator for acute ischemic stroke. N Engl J Med 1995; 333: 15811587

2 Wang YF, Tsirka SE, Stricklanci S, Stieg PE, Soriano SG, Lipton SA. Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice. Nature Med 1998; 4: 228-231

3 Chen Z-L, Stickland S. Neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin. Cell 1997; 91: 917-925

4 Tsirka SE, Rogove AD, Bugge TH, Degen JL, Strickland S. An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus. J Neurosci 1997; 17: 543-552

5 Kano T, Katayama Y, Tejima E, Eng HL. Hemorrhagic transformation after fibrinolytic therapy with tissue plasminogen activator in a rat thromboembolic model of stroke. Brain Res 2000; 854: 245248

6 Korninger C, Collen D. Studies on the specific fibrinolytic effect of human extrinsic (tissue-type) plasminogen activator in human blood and in various animal species in vitro. Thromb Haemostas 1981; 46: 561-565

7 Ranby M, Nguyen G, Scarabin PY, Samama M. Immunoreactivity of tissue plasminogen activator and its inhibitor complexes. Thromb Haemostas 1989; 61: 409-414

8 Watanabe T, Yuki S, Egawa M, Nishi H. Protective effects of MCl186 on cerebral ischemia: possible involvement of free radical scavenging and antioxidant actions. J Pharmacol Exp Ther 1994; 268: 1597-1604

9 Abe K, Yuki S, Kogure K. Strong attenuation of ischémie and postischemic brain edema in rats by a novel free radical scavenger. Stroke 1988; 19: 480-485

10 Yamamoto T, Yuki S, Watanabe T, Mitsuka M, Saito K, Kogure K. Delayed neuronal death prevented by inhibition of increased hydroxyl radical formation in a transient cerebral ischemia. Brain Res 1997; 762: 240-242

11 Watanabe T, Morita I, Nishi H, Murota S. Preventive effect of MCI186 on 1 5-HPETE induced vascular endothelial cell injury in vitro. Prostagland Leuk Essent Fatty Acids 1988; 33: 81-87

12 Flavin MP, Zhao G, Ho LT. Microglial tissue plasminogen activator (tPA) triggers neuronal apoptosis in vitro. CUa 2000; 29: 347354

13 Edaravone Acute Infarction Study Group. Effect of a novel free radical scavenger, edaravone (MCI-1 86), on acute brain infarction: randomized, placebo-controlled, double-blind study at multicenters. Cerebrovasc Dis 2003; 15: 222-229

Tsuneo Kano, Tadashi Harada and Yoichi Katayama

Department of Neurological Surgery, Nihon University School of Medicine, Tokyo, Japan

Correspondence to: Tsuneo Kano, M.D., Ph.D. Assistant Professor Department of Neurological Surgery Nihon University School of Medicine 30-1 Oyaguchi Kamimachi Tokyo 173-8610, Japan. [tuneok@med.nihon-u.ac.jp] Accepted for publication October 2004.

Copyright Maney Publishing Jul 2005
Provided by ProQuest Information and Learning Company. All rights Reserved