A novel MCA occlusion model of photothrombotic ischemia with cyclic flow reductions: development of cerebral hemorrhage induced by heparin

doi:10.1016/S1385-299X(01)00124-6

Brain Research Protocols

Volume 9, Issue 2, April 2002, Pages 85-92

https://doi.org/10.1016/S1385-299X(01)00124-6 Get rights and content

Abstract

Intracerebral hemorrhage is the major complication associated with antithrombotic and thrombolytic therapy. Despite efforts directed toward achieving hemorrhagic infarction, an ideal animal model of cerebral hemorrhage has not yet to be established. Using the photothrombotic technique in rabbits, we developed a model of cerebral hemorrhage by inducing cyclic flow reductions in the middle cerebral artery (MCA). Furthermore, the hemorrhage increased 4-fold after infusion of heparin at a dose prolonging activated partial thromboplastin time by about three times that of control animals. The photothrombotic occlusion of the MCA is based on a thrombosis induced by endothelial injury through singlet oxygen produced by Rose Bengal injection and green light irradiation (Acta Neuropathol. 72 (1987) 315; Acta Neuropathol. 72 (1987) 326; J. Pharmacol. Toxicol. Methods. 29 (1993) 165). Using a pulse Doppler flowmeter, spontaneous reperfusion of the MCA after the thrombotic occlusion following cyclic flow reductions was observed within 2 h in the majority of animals. This model is unusual with respect to the development of clinical stroke, because of the MCA cyclic flow reductions. Thus it is different from permanent or ischemia/reperfusion MCA occlusion in rodents and may be suitable for studying hemorrhagic risks associated with the use of antithrombotic agents.

Section snippets

Type of research

•
Experimental model of cerebral hemorrhage following antithrombotic therapy.
•
Thrombotic occlusion of the middle cerebral artery (MCA) induced by a photochemical reaction between Rose Bengal and green light.
•
Induction of cyclic flow reductions after thrombotic occlusion of the MCA.
•
Evaluation of MCA blood flow.
•
Evaluation of infarct volume and hemorrhage size together with neurological score.

Time required

1.
Animal preparation: 150–210 min
- ◦
  Anesthesia: 15–20 min
- ◦
  Femoral vein and artery cannulation: 30–45 min
- ◦
  Surgical procedure: 45–75 min
- ◦
  Placement of pulse Doppler flowmeter and Xenon lamp: 10 min
- ◦
  Operation sutures: 20 min
2.
Neuronal injury measurement: 120–150 min
- ◦
  Neurological score: 30 min
- ◦
  Perfusion and autopsy: 45–60 min
- ◦
  TTC stain: 30 min
- ◦
  Photography: 10–20 min

Animals

Male Japanese white rabbits weighing 2.0–3.0 kg. Animals were housed individually in a cage with access to food and water on a 12 h light–dark cycle for 1 week.

Special equipment

•
Anesthetic apparatus: isoflurane evaporator, model TK-4, Bio Machinery, Japan.
•
Heating pad: K-module model K-20, American Pharmaseal Co., USA.
•
Bipolar electric coagulator: model 80-1160, Valley Forge Scientific Corp., Valley Forge, USA.
•
Dental drill: model PAL-7, Morita, Tokyo, Japan.
•
Surgical microscope: model KOM 300, Konan Inc., Hyogo,

Animal preparation

The experimental protocol was approved by the Hamamatsu University School of Medicine Committee on Ethics of Animal Experimentation, and extra care was taken to avoid animal suffering.

Male Japanese white rabbits weighing 2.0–3.0 kg were used. After 1 week of acclimation, the animals were anesthetized initially with 2% isoflurane (Dainnihon Pharmaceutical, Tokyo, Japan) in 30% O₂ and 70% N₂O using a face mask; the isoflurane concentration was reduced to 1–1.3% during the surgical procedure. Body

Experimental conditions

Three animals that received vehicle died within 24 h of recovering from the anesthesia, included in which brain slices from two animals that could not be stained by TTC and were not used for data analysis.

All physiological parameters were within the normal range after the photothrombotic occlusion of the MCA [19].

In heparin-treated animals, aPTT was prolonged markedly at 24 h and about three times that of the vehicle group (77.01±15.08 vs. 25.11±2.15 s).

The green light irradiation system used

Discussion

We have described a simple and reproducible model of heparin-induced cerebral hemorrhage achieved by spontaneous reperfusion of the occluded MCA following cyclic flow reductions. This photothrombotic occlusion model results in a platelet- and fibrin-rich thrombus in the MCA at the irradiated site [9], [11]. In this study, the photochemical approach to inducing thrombotic occlusion is based on the injection of Rose Bengal and green light from a xenon lamp irradiation system. Rose Bengal is a

Quick procedure

1.
Surgery
- ◦
  Anesthesia.
- ◦
  Cannulation of the femoral vein and artery.
- ◦
  Transorbital craniotomy.
- ◦
  Placement of pulse Doppler flowmeter.
- ◦
  Photoirradiation with Rose Bengal injection.
- ◦
  Surgical sutures
2.
Behavioral assessment
3.
Photographs of unstained brain slices and TTC-stained slices
4.
Determination of infarct volume and hemorrhage size

Essential Refs.

Refs. [4], [19], [13], [6], [7], [18]

Acknowledgements

This study was supported in part by Japan China Medical Association and a Grant-in Aid for Scientific Research 12672211 from the Ministry of Education, Science and Culture in Japan.

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Cerebral hemorrhage due to heparin limits its neuroprotective effects: studies in a rabbit model of photothrombotic middle cerebral artery occlusion
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Intracranial blood flow velocities in acute ischemic stroke
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A model of focal ischemic stroke in the rat: reproducible extensive cortical infarction
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Photochemically induced cerebral infarction. I. Early microvascular alterations
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K. Kawano et al.
Increased cerebral infarction by cyclic flow reductions: studies in the guinea pig MCA thrombosis model
Am. J. Physiol.
(1998)

There are more references available in the full text version of this article.

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We show here that photochemically-induced MCA thrombosis in male cynomolgus monkeys caused cyclical occlusion/reperfusion in the MCA. These data are consistent with previous studies of thrombotic MCAO in non-human primates (Zhao et al., 2002; Kaku et al., 1998; Furuichi et al., 2007; Maeda et al., 2005a,b; Tomizawa et al., 2015). Here we show that a single intravenous dose of 0.3 mg/kg R-138727 significantly increased the patency rate of the injured MCA by inhibiting variations in thrombotic cyclic flow; there was no significant difference in patency rates before injection, indicating that inhibiting P2Y12 dramatically inhibited the recurrence of thrombus formation even when administered after the initiating arterial injury.
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Photochemical MCA thrombosis induced in male cynomolgus monkeys resulted in cyclical occlusion/reperfusion in the MCA territory. These data are consistent with previous thrombotic MCAO studies in non-human primates [5,8–10,30]. A daily prasugrel dosing regimen (1 mg/kg/day for 3 days) significantly inhibited these cyclic flow variations (CFVs) by increasing MCA patency for at least 1 h after the photo-irradiation, reflecting an effective antithrombotic effect.
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ProtocolA novel MCA occlusion model of photothrombotic ischemia with cyclic flow reductions: development of cerebral hemorrhage induced by heparin

Abstract

Section snippets

Type of research

Time required

Animals

Special equipment

Animal preparation

Experimental conditions

Discussion

Quick procedure

Essential Refs.

Acknowledgements

Eur. J. Pharmacol.

J. Pharmacol. Toxicol. Methods

Brain Res.

J. Mol. Cell. Cardiol.

Brain Res.

Intracranial blood flow velocities in acute ischemic stroke

Stroke

A model of focal ischemic stroke in the rat: reproducible extensive cortical infarction

Stroke

Photochemically induced cerebral infarction. I. Early microvascular alterations

Acta Neuropathol.

Increased cerebral infarction by cyclic flow reductions: studies in the guinea pig MCA thrombosis model

Am. J. Physiol.

Protocol
A novel MCA occlusion model of photothrombotic ischemia with cyclic flow reductions: development of cerebral hemorrhage induced by heparin