Photochemically induced cerebral ischemia in a mouse model

doi:10.1016/j.surneu.2006.08.077

Surgical Neurology

Volume 67, Issue 6, June 2007, Pages 620-625

https://doi.org/10.1016/j.surneu.2006.08.077 Get rights and content

Abstract

Background

MCAO has been widely used to produce ischemic brain lesions. The lesions induced by MCAO tend to be variable in size because of the variance in the collateral blood supply found in the mouse brain.

Methods

We modified the rat photothrombosis model for use in mice. Male C57BL/6 mice were subjected to focal cerebral ischemia by photothrombosis of cortical microvessels. Cerebral infarction was produced by intraperitoneal injection of rose bengal, a photosensitive dye, and by focal illumination through the skull. Motor impairment was assessed by the accelerating rotarod and staircase tests. The brain was perfusion fixed for histologic determination of infarct volume 4 weeks after stroke.

Results

The lesion was located in the frontal and parietal cortex and the underlying white matter was partly affected. A relatively constant infarct volume was achieved 1 month after photothrombosis. The presence of the photothrombotic lesion significantly impaired the motor performance as measured by the rotarod and staircase tests. Our findings show that photothrombotic infarction in mice is highly reproducible in size and location.

Conclusion

This procedure can provide a simple model of cerebral infarction for a unilateral motor cortex lesion. In addition, it can provide a suitable model for the study of potential neuroprotective and therapeutic agents in human stroke.

Introduction

Stroke is one of the leading causes of death and the most common cause of adult disability. Stroke often causes devastating and irreversible loss of function. There is a wide range of sensory, motor, and cognitive deficits including tremor, lack of coordination, and partial paralysis [6]. There is no specific treatment for improving functional recovery after stroke. A large number of neuroprotective drugs have failed to demonstrate efficacy in clinical trials [20].

Development of effective stroke therapy requires use of animal models that can simulate human pathology in a reproducible and physiologically relevant manner. Currently, the intraluminal suture model of MCAO has been widely used to induce ischemic brain lesions. As the MCA is occluded via a cervical carotid approach, this obviates the requirement for a craniectomy and the associated problems with opening the skull [4], [7], [14]. However, the lesions caused by the MCAO tend to be variable in size; in addition, animals have been excluded from analysis by arbitrary criteria such as unexpected behavior in most series [1], [16]. The collateral blood supply in the mouse brain is variable depending on the mouse strain; this results in variability of clinical outcome and stroke size [6]. The C57BL/6 strain, frequently used for genetic studies, is particularly unsuitable for MCAO [3]. The development of reliable and reproducible animal models for cerebral ischemia is needed for the systematic study of the pathophysiology and treatment of stroke. In this study, we adapted the rat photothrombosis model for use in mice by modifying the application route of the dye and illumination and stereotactic parameters.

Section snippets

Photothrombotic cortical lesion

All surgical procedures and postoperative care were performed in accordance with guidelines of the Chonnam National University Animal Care and Use Committee. A total of 25 male C57BL/6 mice (8-10 weeks old) weighing 20 to 25 g were maintained on a 12-hour light/dark cycle. The mice were fasted overnight before the day of the experiment but were allowed free access to tap water.

Focal cortical ischemia was induced by photothrombosis of the cortical microvessels. Each mouse was anesthetized with

Results

In this experiment, rose bengal dye was injected systemically and then the cortex was illuminated, at the left sensorimotor cortex, in a mouse stroke model to produce a discrete photothrombotic lesion based on light absorption by the dye. Histologic examination of the brain after 24 hours showed a consistent pattern of ischemic brain damage. At 24 hours after induction of ischemia, the infarct was demarcated from the surrounding vital tissue after incubation with TTC. The lesion was located in

Discussion

Cerebral ischemia in mice has been introduced by transient and permanent occlusion of the MCA with intraluminal filament [7], [11]. However, the procedure requires sophisticated microsurgical skills because of the proximity to the vagus nerve and the tiny anatomy of the carotid system in mice. The size of the cerebral infarct is highly dependent on the vascular anatomy of the MCA and collateral vessels, which differs considerably among mouse strains [12]. Furthermore, the size of the infarction

Conclusion

Our results demonstrate that photothrombotic occlusion of cerebral microvessels in mice is a minimally invasive and simple model for study of focal cortical ischemia. The cortical lesion produced is consistent and the lesion has a precise location and size. Therefore, this model offers a reliable method for the study of the functional aspects of lesion-associated cerebral ischemia and for evaluation of potential neuroprotective and therapeutic agents in human stroke.

Acknowledgment

This work was supported by Research Institute of Medical Sciences of Chonnam National University.

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Furthermore, the induced infarct size, which is primarily dependent on the anatomical structure of the MCA and collateral vessels, is different in each case because there are unpredictable anatomical variations [8,12]. Additionally, the large size of the induced infarct territory in MCAO models can induce severe neurological deficits and high mortality rates, which limits its application in the long-term research [13,14]. Photothrombosis is an intravascular photo-oxidation mechanism-based focal cerebral ischemia, which is frequently used in the animal models [7,13].
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Methods to assess behavioural and functional deficits after experimental stroke are a prerequisite for meaningful preclinical stroke research that may translate to future therapeutic options [1–4]. Photothrombotic stroke is widely used to study the cellular and molecular mechanism underlying cerebral ischemia inflicting neurodegeneration, neuroprotection, and neuroregeneration [5–7]. In our hands, photothrombosis is a pure cortical stroke, minimally invasive, and the lesions are highly reproducible in size and location [7–9].
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We identify repetitively occurring capillary stalls of varying duration promoted by neutrophils in both models of permanent cerebral ischemia, validating the suitability of our new transgenic mouse model in determining neutrophil occlusion formation in vivo. Flow cytometric analysis of peripheral blood (PB) and brain tissue from mice subjected to photothrombosis reveal an increase in the total proportion of neutrophils, with selective upregulation of endothelial adherence markers in the PB.
In conclusion, the dynamic microcirculatory stall phenomenon that is described after transient ischemia followed by reperfusion also occurs after permanent small- or large-vessel stroke and is clearly attributable to neutrophils.
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With constant MCAO, where the tissue is more severely and irreversibly damaged, GAP-43 is absent in the core of the infarct but its expression increases in the penumbra, reaching a maximum on day 7.37 The PTS model is characterized by rapid vascular damage and cell death in the infarct, which makes the formation of a wide penumbra impossible.26,55 This might be the reason we are observing an increase in the level of GAP43 in the relatively intact region of the ipsilateral hemisphere starting from the 7th day after the PTS.
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IschemiaPhotochemically induced cerebral ischemia in a mouse model

Abstract

Background

Methods

Results

Conclusion

Introduction

Section snippets

Photothrombotic cortical lesion

Results

Discussion

Conclusion

Acknowledgment

Brain Res Bull

J Pharmacol Toxicol Methods

Pharmacol Biochem Behav

Neuropharmacology

J Neurosci Methods

J Neurosci Methods

Behav Brain Res

Behav Brain Res

Temporary middle cerebral artery occlusion in the rat: consistent protocol for a model of stroke and reperfusion

Neuropathol Appl Neurobiol

Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy

J Cereb Blood Flow Metab

Middle cerebral artery occlusion in the rat by intraluminal suture. Neurological and pathological evaluation of an improved model

Stroke

Ischemia
Photochemically induced cerebral ischemia in a mouse model