Neurological sequelae and long-term behavioural assessment of rats with transient middle cerebral artery occlusion

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Abstract

Animal models of stroke, notably transient middle cerebral artery occlusion (MCAo), are used to assess the efficacy of pharmacological and transplant treatments. Long-term studies (>1 month) of the functional effects of treatments in animal models are required to predict treatments likely to improve dysfunctions associated with stroke damage. These pre-clinical studies require (1) optimum post-operative care to ensure long-term survival, (2) methods for assignment of rats to groups with equivalent impairments to reduce variability and enhance detection of treatment effects, and (3) behavioural tests that detect long-term stable deficits. For long-term functional assessment, a battery of behavioural tests sensitive to a range of deficits observed after MCAo was developed. The bilateral asymmetry test evaluated the time course of sensory neglect. Deficits of motor integration were examined in the footfault test, and motor bias was assessed by pharmacological stimulation of rotation. The water maze was used to detect long-term deficits in spatial information processing. Long-term differences between control and MCAo animals in this battery of tests indicate that the protocol provides an efficient assessment suitable for evaluating treatment outcomes in pre-clinical studies of stroke, and that the post-operative care procedure and method of assignment to groups were effective.

Introduction

Animal models have provided useful insights into the pathophysiology of stroke and played an influential role in the development of treatment strategies (Ginsberg, 1996). Middle cerebral artery occlusion (MCAo), the most commonly used model for cerebral ischaemic stroke in rats, represents the most prevalent (60%) form of stroke in humans (American Heart Association, 1998). However, recent reviews (Hunter et al., 1995, Hunter et al., 1998, Ginsberg, 1996, Grotta, 1996, Hossman, 1997, Corbett and Nurse, 1998, de Keyser et al., 1999, Muir and Grosset, 1999, STAIR, 1999) of the failure of pharmacological agents to improve stroke outcomes in clinical trials have pinpointed the problems in translating experimental animal studies to clinical applications for humans. One of the problems of these pre-clinical studies is the lack of long-term functional assessment. Long-term studies are important to determine if the effects of the treatments have significance for functional outcome and ideally a battery of tests should evaluate a variety of functional parameters relevant to the damage.

Functional impairment after stroke in rats can be divided into transient neurological deficits and long-term motor and cognitive dysfunctions. Evaluation of short- and long-term deficits provide complementary information. A variety of subjective neurological scoring systems are used at present to assess rats with MCAo damage for short periods of time and as outcome measures of treatment (Bederson et al., 1986, Capdeville et al., 1986, Menzies et al., 1992, Wahl et al., 1992, Garcia et al., 1995, Rogers et al., 1997, de Courten Myers et al., 1998, Hunter et al., 2000). However, animals typically show spontaneous recovery on these scales and therefore their predictive value for long-term outcomes is questionable. Neurological scores can, nevertheless, perform two functions, namely a global assessment of general post-surgical impairment and induction of stroke-specific dysfunction. Both of these can be used to assign animals to groups and to discriminate between general surgical and ischaemic trauma. A sham-operated group undergoing the same procedures as the MCAo group without the occlusion is also a prerequisite to disentangle the effects of the surgery per se and the damage caused by the occlusion.

Long-term testing of motor and cognitive deficits may indicate whether a treatment is likely to improve functional outcome. However, one of the major problems of behavioural testing after stroke is that animals (and humans alike) show spontaneous recovery on some tests. Several studies (Markgraf et al., 1992, Grabowski et al., 1993, Borlongan and Sanberg, 1995, Okada et al., 1995, Yonemori et al., 1996, Rogers et al., 1997, Sakai et al., 1996, Smith et al., 1997, Stroemer et al., 1998, Ward et al., 1999, Hunter et al., 2000) previously used a variety of tests to assess permanent or transient MCAo damage at various time points (mostly<1 month). To evaluate the effects of treatment on functional outcome, a battery of tests charting impairment resistant to spontaneous recovery will be necessary to determine possible beneficial or detrimental effects. A battery of tests evaluating impairments of several functions is necessary since MCAo damages various anatomical and functional regions of the brain (striatum, somatosensory and motor cortices) which can be affected differentially by the treatments.

Post-operative procedures are presented here to maximize long-term survival, the neurological scoring procedures that allow one to determine which animals will be included or excluded in the treatment groups, and the battery of tests used to assess long-term functional deficits after MCAo.

Section snippets

Materials and methods

All procedures were in accordance with the requirements of the United Kingdom Animals (Scientific Procedures) Act of 1986 and the ethical review process of the Institute of Psychiatry, University of London.

Duration of post-operative care

The duration of post-operative care in single housing was determined chiefly by rate of weight recovery. Control animals recovered faster than rats with occlusion of the middle cerebral artery. On average control animals spent significantly (t=4.368, P<0.001, df=78) fewer days to recover from surgery (7 days on average) compared to MCAo animals (10 days on average).

Food intake and defecation

Although food intake and defecation were estimated by rating rather than measuring, they provided useful insights into the

Discussion

The failure of recent clinical trials for stroke treatments have led to criticisms of the validity and usefulness of animal models for pre-clinical studies. Some of the suggestions for improving pre-clinical experiments include longer survival times and functional assessment (STAIR, 1999). A method for long-term functional assessment of rats with transient MCAo consists of two phases, firstly a good post-operative recovery regime is necessary to ensure long-term survival with neurological

Acknowledgements

MM was supported by scholarships from NATO and the Ministère de l'Education Nationale du Luxembourg. This study was sponsored by ReNeuron Ltd.

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