Basic Neuroscience
Distal hypoxic stroke: A new mouse model of stroke with high throughput, low variability and a quantifiable functional deficit

https://doi.org/10.1016/j.jneumeth.2012.03.003Get rights and content

Abstract

C57BL/6J are the most commonly used strain of mouse for stroke experiments but vascular anatomy of the Circle of Willis within this strain is extremely variable and the cortex has extensive collateralization. This causes large variability in stroke models that target the middle cerebral artery proximally and confers resistance to ischemia in those that target it distally. We tested the hypothesis that by combining distal middle cerebral artery occlusion with 1 h of hypoxia, we could generate a large lesion that causes a behavioral deficit with low variability. We found that this new distal hypoxic (DH) model of stroke generates a lesion with a volume of 25% of the ipsilateral hemisphere, extends to the motor cortex and causes a behavioral deficit. It also has a very clear border, exceptionally low variability, and can be performed by a single surgeon on up to 30 animals a day. Moreover, survivability is 100% in young adult animals, the model can be performed on old animals, and therapeutic intervention can reduce infarct volume. Therefore DH stroke is an excellent complement to existing stroke models and could be used for preclinical studies in C57BL/6J mice.

Highlights

► C57BL/6 mice are the most commonly used strain of mouse for stroke experiments. ► The Circle of Willis in this strain is extremely variable. ► This causes large variability in most commonly used stroke models. ► We introduce a stroke model tailored for C57BL/6 mice that bypasses Circle of Willis variability.

Introduction

The successful translation of therapeutic strategies for the treatment of stroke is dependent on the reproducibility and reliability of animal stroke models. C57BL/6J (B6) mice are the most commonly used strain of mouse for stroke experiments, due to their ease of genetic manipulation, but B6 mice exhibit large variability in the anatomy of the Circle of Willis and numerous studies have shown that this affects lesion volume in multiple models of cerebral ischemia (Barone et al., 1993, Fujii et al., 1997, Kitagawa et al., 1998, Sheng et al., 1999, Wellons et al., 2000, Yonekura et al., 2004).

The Circle of Willis is a network of blood vessels at the base of the brain that connects the vertebral and carotid circulations and prevents the brain from being starved of blood when one system is occluded. Despite all B6 mice being genetically identical, they do not all have a complete Circle of Willis. Instead, in some individuals the posterior communicating artery (PComA) is missing on one or both sides of the brain (Barone et al., 1993, Kitagawa et al., 1998). In a study by McColl et al. (2004) only 10% of B6 mice were found to have a complete Circle of Willis, 60% were missing a single PComA and 30% were missing both the left and the right PComA. Many of the stroke models currently used rely on using one or both common carotid arteries to target downstream circulation and so they are susceptible to variability in Circle of Willis anatomy. Our aim has been to develop a model of stroke that creates a large lesion in B6 mice independent of variability in Circle of Willis anatomy.

Currently, it is possible to bypass the problem of variability in Circle of Willis anatomy by ligating the middle cerebral artery distally (dMCAO). This generates a lesion of consistent size and location, however the lesion is small, typically comprising ∼10% of the ipsilateral hemisphere. This may be due to another feature of the cerebral vasculature of B6 mice, which is that they have extensive collateralization between the anterior cerebral artery (ACA) and middle cerebral artery (MCA) (Wang et al., 2010). On average B6 mice have 9 collaterals per hemisphere in this location (Wang et al., 2010), which means that when the MCA is distally occluded, much of the territory fed by the MCA escapes ischemia due to blood flow from ACA collaterals.

The small size of the lesion produced by dMCAO in B6 mice is not an inherent disadvantage. However, the location of the lesion, the whisker barrel cortex, prevents this model from being more widely used. This location prevents dMCAO stroked B6 mice from exhibiting an easily discernible behavioral impairment, making the assessment of recovery, a key goal of stroke research, difficult. The size of the lesion after dMCAO can be increased by simultaneously ligating one or both carotid arteries, but these strategies substantially increase mortality and increase variability by now making the model dependent on Circle of Willis anatomy.

To circumvent these anatomical idiosyncrasies of B6 mice we tested the effect of combining dMCAO with 1 h of hypoxia (DH stroke). We found that the application of hypoxia expands the lesion generated by dMCAO to incorporate more of the somatosensory cortex and some of the motor cortex, and thereby it causes a more easily detectible behavioral deficit. Furthermore, because dMCAO circumvents the problem of inconsistency in Circle of Willis anatomy, the volume of the resulting lesion has exceptionally low variability.

Section snippets

Mice

Mice were male BALB/c or C57BL/6J. Adult mice were 5–6 months old. Aged mice were 18 months old. All procedures met NIH guidelines with the approval of the Stanford University Institutional Animal Care and Use Committee.

India ink perfusion

To visualize the anatomy of the Circle of Willis 14 male B6 mice (5 months old) were perfused with 0.9% NaCl followed by 25% India ink in 0.9% NaCl/7.5% gelatin at 37 °C.

Intraluminal filament model of middle cerebral artery occlusion (MCAO)

Performed by one of the early developers of MCAO in the mouse; Dr. Nikola Lessov as he describes in (Clark et

Variability in Circle of Willis anatomy in B6 mice

The patency of the ipsilateral PComA has repeatedly been shown to be a major determinant of infarct volume after proximal ligation of the MCA (Barone et al., 1993, Fujii et al., 1997, Kitagawa et al., 1998, Sheng et al., 1999, Wellons et al., 2000, Yonekura et al., 2004). McColl et al. (2004) report that only 10% of B6 mice have a complete Circle of Willis, with 60% missing a single PComA and 30% missing both the left and right PComA. This variability in anatomy is corroborated by Majid et al.

Discussion

We have developed a new model of stroke, which we call the DH model of stroke for distal MCAO occlusion + hypoxia. Our goal was to develop a model of stroke for B6 mice that causes a robust behavioral deficit for the assessment of recovery but with low variability. The DH model of stroke consists of performing a craniectomy and ligating the right middle cerebral artery using a small vessel cauterizer. Immediately following surgery mice are given hypoxia by placing them in a chamber containing 8%

Conflict of interest

The authors have no conflict of interest.

Acknowledgments

This work was supported by an Ellison Medical Foundation/AFAR Postdoctoral Fellowship to KPD and NINDS KO8 NS050304 and American Heart Association 10GRNT4140073 to MSB. We would like to thank Dr. Mary Stenzel-Poore for technical support, Dr. Nikola Lessov for performing the intraluminal filament model of MCAO, and Geoffrey Stanley and Dr. Kereshmeh Taravosh-Lahn for their assistance with pilot experiments.

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