Chronic behavioral testing after focal ischemia in the mouse: functional recovery and the effects of gender

https://doi.org/10.1016/j.expneurol.2004.01.004Get rights and content

Abstract

Several useful behavioral tests exist for measuring behavioral recovery after ischemia in higher-order animals and rats. With the increasing use of mice in focal stroke research, simple, reliable, and reproducible behavioral testing has become a priority. As neuroprotective agents are tested, long-term outcome must be assessed, especially in studies focused on neuronal plasticity and regeneration after ischemia. Our laboratory and others have previously shown that estrogen (E2) is neuroprotective in rodent stroke paradigms. We examined a battery of behavioral tests in male and female mice subjected to 90 min of middle cerebral artery occlusion (MCAO) to determine the most sensitive tests for detecting sensorimotor dysfunction after stroke, and to determine the functional significance of E2-mediated neuroprotection. Only two tests, the corner test and the cylinder test, were able to differentiate between groups (sham and stroke) after several days of repeated testing. The cylinder test was sensitive to the neuroprotective/neurorestorative effects of E2, but 2 weeks after stroke, the cylinder test was unable to distinguish between sham and stroke animals treated with E2. In contrast, the corner test was able to differentiate stroke and sham animals even 6 weeks after stroke, but did not distinguish animals treated with E2 vs. vehicle. These tests provide a simple, rapid, reliable assessment of sensorimotor dysfunction in the mouse after focal ischemia. Hormonal status influences speed of recovery on cylinder testing in animals of both genders. This suggests that a short battery of tests including the neurological score, cylinder, and corner test may be adequate to rapidly and repeatedly assess sensorimotor dysfunction in mice of both genders.

Introduction

Transient occlusion of the middle cerebral artery has become a well-established model of focal stroke in rodents. Numerous behavioral tests have been utilized in higher-order animals and rats to assess long-term sensorimotor (Schallert et al., 1982) and cognitive dysfunction (Hirakawa et al., 1994), as well as in the evaluation of putative neuroprotective agents (Modo et al., 2000). For example, the Schallert “sticky tape” test and the “cylinder test” have been used for chronic evaluation of functional deficits in rats and are sensitive to the degree of ischemic damage Bland et al., 2000, Schallert et al., 1982, Zhang et al., 2000. The tape test involves placing small adhesive-backed labels on the forelimbs of a rat. Contact and removal times are recorded and give a measure of stroke-induced sensory loss. The size of the label can also be varied to test the severity of the somatosensory loss in the impaired forelimb. In cylinder testing, the animal is placed in a clear cylinder and the level of preference for using the nonimpaired forelimb for weight shifting movements during spontaneous vertical exploration is quantified. However, although well validated in rat, neither of these tests has been examined in murine models of stroke. In addition, there is significant spontaneous behavioral improvement after stroke, regardless of treatment, making repeated long-term assessment difficult and potentially misleading. With the growing interest in behavioral recovery after stoke and the possibility of endogenous neurogenesis after ischemia Liu et al., 1998, Sharp et al., 2002, animals are now evaluated for much longer time periods than in traditional neuroprotective trials. In addition, the increasing use of the mouse in focal ischemia models due to the availability of transgenic and knockout strains has required evaluation of novel murine-specific behavioral tasks, as tests that are well established in rats cannot be performed reliably in mice. Most tests designed to assess stroke-induced deficits are able to discriminate ischemic mice from sham controls within several days of stroke, but are not sensitive enough to discriminate between groups after several days of repeated testing (Hunter et al., 2000). Recently, the corner test was shown to be a sensitive test for detecting long-term sensorimotor dysfunction in mice (Zhang et al., 2002). This test was designed to detect moderate focal-ischemia-induced asymmetries by integrating vibrissae stimulation, forelimb and hindlimb usage, and postural motor function. The mouse is placed in between two boards that are progressively formed into a 30° angle. When the mouse enters deep into the corner, both vibrissae are stimulated and the mouse will rear and turn toward the open end. In the present paper, a transient occlusion of the middle cerebral artery (MCAO/90 min) was performed in intact female mice followed by a battery of behavioral tests including the corner test, the cylinder test (used previously only in rats), as well as traditional tests of locomotor activity, weight loss, and neurological score to determine which tests were the most useful for chronic behavioral assessment. Testing was then repeated in animals of both genders to determine the applicability of these behavioral tests for testing of animals subjected to hormonal manipulation.

It has been shown previously that treatment with estrogen (E2) can reduce ischemic damage after stroke when tissue damage is evaluated acutely, but less is known about the possibility that E2 could enhance chronic behavioral recovery after ischemic damage has occurred (for review, see McCullough and Hurn, 2003). At this point, the behavioral effects of E2 treatment after stroke are unknown. Our goals in this study were to determine if long-term, repeated behavioral assessment was possible after stroke in mice and to determine which tests were the most useful and reliable to follow behavioral improvement after stroke. In Experiment 1, we examined intact female mice subjected to a modest ischemic insult to determine which behavioral tests could differentiate stroke and sham animals. In the second experiment, our aims were to examine the effects of gender and hormonal manipulation on behavioral testing after stroke, and to determine if E2 has long-term effects on behavioral recovery after stroke unrelated to its immediate neuroprotective effect.

Section snippets

Animals and housing

Adult 2–3-month C57Bl/6 female and male mice were kept in same sex groups of two to three animals both before and after surgery on sawdust bedding (light cycle 12/12 h light/dark; lights on at 04:00 h). Animals weighed from 18 to 25 g to ensure adequacy of occlusion. All animals had free access to water and standard lab chow. Mice were trained, operated, and tested in a randomized manner and a blinded observer performed the behavioral testing. In Experiment 1, nonovariectomized, hormonally

Behavioral measurements in intact female mice after stroke

Table 1 summarizes the behavioral data from intact female mice. There were no differences between stroke and sham animals on hanging wire testing or latency to move one body length. Early differences in the neurological score between stroke and sham mice on days 1 (H = 10.95; P < 0.05) and 3 (H = 9.75; P < 0.05) after stroke were noted; however, these differences were no longer significant by day 5 (H = 2.66; P > 0.05), suggesting this score is a useful screen to monitor intra-ischemic and

Discussion

The present study demonstrates several important findings. First, the degree of behavioral recovery from moderate cortical and striatal damage is considerable in the mouse. Secondly, tests used to measure functional recovery in other species appear to be less useful in assessment of murine stroke. All of the behavioral tests, including simple measures of weight loss and neurological score, were able to distinguish sham vs. stroke animals in the early days after stroke. By 2 weeks, however, the

Acknowledgements

This work was supported by the AHA Fellow-to Faculty Award and the Hazel Goddess Fund for Stroke Research in Women (LDM) and NS 20020, NS33668 (PDH). We would like to thank Dr. Timothy Schallert and Dr. Raymond Koehler for their helpful comments and advice.

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