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Functional assessments in mice and rats after focal stroke
Neuropharmacology 39 (2000) 806–816 www.elsevier.com/locate/neuropharm
Functional assessments in mice and rats after focal stroke A.J. Hunter *, J. Hatcher, D. Virley, P. Nelson, E. Irving, S.J. Hadingham, A.A. Parsons Neurosciences Research, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK Accepted 14 December 1999
Abstract This paper presents a comprehensive assessment of sensorimotor deficits in the mouse after focal ischaemia induced by occlusion of the middle cerebral artery. Twenty four hours after induction of middle cerebral artery occlusion, mice showed deficits in a range of sensory and motor tasks as assessed by the SHIRPA protocol. In addition they exhibited a decrease in rotarod performance and locomotor activity. Some behaviours, such as locomotor activity, were also impaired in sham operated animals compared to normal controls, although these impairments were not as marked as those exhibited by the ischaemic mice. This is the first comprehensive analysis of the short term effects of permanent focal ischaemia in mice. In a second series of experiments in the rat, rates of recovery over time were examined. Simple (neurological grades, rotarod) and complex (sticky label test) tasks were examined in rats after middle cerebral artery occlusion up to 7 days post-ischaemia. Ischaemic rats had a profound deficit in contralateral performance on the sticky label task with no evidence of recovery. A less marked deficit was also observed in ipsilateral performance of this task. These deficits were still present 7 days after ischaemia. Ischaemic rats also exhibited a deficit on rotarod performance but this had recovered 7 days post-ischaemia. Thus different sensorimotor tasks have different rates of recovery after focal cerebral ischaemia in the rat. Further characterisation of these tasks will enhance their utility meaningful preclinical means of assessing functional recovery of the administration of potential neuroprotective and regenerative therapies. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Mouse; Rat; Focal ischaemia; Behaviour
1. Introduction It is becoming increasingly necessary to study functional outcomes in stroke research in both rodents and higher species (Corbett and Nurse, 1998; Hunter et al., 1998). This is especially true for investigating the effects of therapies aimed at promoting regeneration and repair such as basic fibroblast growth factor (Kawamata et al., 1996). There have been a relatively large number of studies looking at functional changes after the induction of global ischaemia in rodents. In global ischaemia models, such as bilateral common carotid occlusion in the gerbil and four-vessel occlusion in rats, increases in locomotor activity have been observed (Baldwin et al., 1993; Kuroiwa et al., 1991; Wang and Corbett, 1990). This increase is thought to reflect a deficit in habituation
* Corresponding author. Tel.: +44-1279-622000; fax: +44-1279622660. E-mail address: a.jacqueline [email protected] (A.J. Hunter).
or spatial mapping (Babcock et al., 1993) rather than in sensory-motor function per se. Impairments in other sensorimotor tasks have been less frequently reported (e.g. Baldwin et al., 1993). Overall there are fewer reports of functional deficits after focal ischaemia, although there have been more studies carried out to examine the effects of focal stroke on sensorimotor function. Photothrombotic lesions of the cortex have been shown to produce impairments on tasks such as beam and grid walking and grip strength measurements (Wood et al., 1996). However induction of focal ischaemia by permanent or transient occlusion of the middle cerebral artery (MCAO) is now the model of choice for focal ischaemia studies (Hunter et al., 1995; STAIR stroke conference Florida, 1999). In the rat, simple neurological grades such as the postural reflex test published by Bederson et al. (1986) or the neurological assessment of Zea Longa et al. (1989) have been used to assess the success of the MCAO and to measure the effects of drugs. However such scales are relatively crude. Therefore a number of workers have developed
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more refined sensorimotor tasks in the rat. These include limb placing, beam walking, grid walking, rotarod, sticky label test, staircase test (see Hunter et al., 1998; Corbett and Nurse, 1998 for reviews). These have shown that spontaneous recovery can occur in some tasks (e.g. beam walking) whereas it is not so quickly observed in others (e.g. staircase), although detailed time courses for all models have not been established. Assessment of sensorimotor function in the mouse after focal ischaemia has been rarely described, despite the growing number of studies utilising MCAO in the mouse. These studies have increased primarily as a result of the use of transgenic mice for understanding the pathological consequences of ischaemia. Therefore there is a growing need to develop simple but comprehensive functional outcome measures in mice after MCAO. The studies described in the present paper have two aims. Firstly to characterise a behavioural test battery in mice to demonstrate that a comprehensive screen can be carried out for short term assessment of function. This essentially uses the SHIRPA procedure (Rogers et al., 1997a) which has been developed as a comprehensive routine testing protocol for characterising phenotypic variations in transgenic mice. Broadly the characterisation involved two distinct steps. Firstly, a behavioural and functional profile of the animals was built up by the use of observational assessments using a modified Irwin profile (Irwin, 1968). Secondly, a more complex series of tasks were undertaken in order to provide additional behavioural data. These studies are an essential prerequisite to carrying out longer-term assessments in the mouse after MCAO. More behavioural studies have been carried out in the rat shortly after ischaemia and so the aim of the rat MCAO study was to characterise both simple and complex tests to look at differential rates of recovery over time. In this case a transient MCAO was chosen to minimise long term mortality due to permanent MCAO (Koizumi et al., 1986). This study will aid long term studies in mice by providing a guide as to which aspects of the test battery in the mouse may show differential rates of recovery with time.
2. Materials and methods All experiments were conducted according to the requirements of the United Kingdom Animals (Scientific Procedures) Act (1986) and conformed to SmithKline Beecham ethical standards. 2.1. Behavioural characterisation after permanent cerebral ischaemia in the mouse Male CD1 mice (Charles River UK) weighing 30–35 g at the time of surgery were used. Prior to surgery,
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animals were housed in groups of five, in a temperature controlled environment (20°C ± 1°C) and maintained with ad libitum access to food and water on a 12 h light/dark cycle (lights on 7:00–19:00). After surgery, mice were housed singly on soft bedding and given access to softened diet. Mice were anaesthetised using a mixture of 5% halothane, 30% oxygen and 70% nitrous oxide, and maintained with a nitrous oxide/oxygen mixture containing 1–1.5% halothane. A midline cervical incision was made and the left carotid artery exposed. Sterile silk sutures were looped around the common, external and internal carotid arteries following which the pterygopalatine artery was exposed and cauterised at its origin. An aneurysym clip was placed across the common carotid artery and blood flow in the internal carotid artery arrested using a suture. An arteriotomy was then made in the external carotid artery and a blunted, rounded length of monofilament nylon suture of 0.18 mm diameter was inserted (Dermalon 5/0: David and Geck, Puerto Rico). This was secured in place with a silk suture. The monofilament was then advanced into the internal carotid artery and passed into the intracranial circulation to lodge in the narrower lumen of the proximal anterior cerebral artery, approximately 8 mm distal to the carotid bifurcation, thereby occluding the origin of the MCA. The aneurysm clip was then removed and the wound closed. Sham operated animals underwent the same procedure with the exception of the advancement of the filament to occlude the cerebral artery. Animals were kept under heated lamps until full reversal of the anaesthetic. Behavioural studies were carried out 24 h post operation. Each animal was tested in all of the procedures detailed below between 11:00 and 15:00 hrs on the day following, i.e. 24 h after, the MCAO operations. Procedures were run in the order in which they are described. For this study it was not feasible to carry out truly blinded observations due to the inclusion of the unoperated group. The primary observation screen is a modification of the Irwin procedure (Irwin, 1968), during which a total of 40 separate measurements are recorded for each animal (for full method see Rogers et al., 1997a). Assessment of each animal began with observation of undisturbed behaviour in a cylindrical clear perspex viewing jar (15 cm×11 cm). The following behaviours were scored in the viewing jar: Body position: completely flat (0) to repeated vertical leaping (8) Spontaneous activity: none (0) to repeated vigorous movement (8) Respiration: gasping, irregular (0) to hyperventilation (3) Temor: none (0) to marked (2).
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The mice were then transferred to an arena (55×33 cm) which was marked into even sized squares (11×11 cm) for observations of the following behaviours: Transfer arousal: coma (0) to extremely excited (6) Locomotor activity: number of squares crossed in 30 s Palpebral closure: eyes wide open (0) to completely shut (2) Startle response: the jerk produced in response to a pen dragged across wire grid on top of arena — no response (0) to 1 cm jump or more (3) Gait: normal (0) to incapacity (3) Pelvic elevation: flattened (0) to elevated ⬎3 mm (3) Tail elevation: flattened (0) to Straub tail (2) Touch escape: none (0) to extremely vigorous (3) Positional passivity: struggles when held by tail (0), neck (1), supine (2), by hindleg (3), no struggle (4) This was followed by a sequence of manipulations using tail suspension: Visual placing: animal lowered and distance from grid needed to evoke forepay paddling — none i.e. with nose in contact (0) to 25 mm or more Trunk curl: (0–1) Limb grasping: (0–1) Grip strength: Resistance when animal placed on grid an drawn backwards (0–4) Body tone: Determined by compresion of the sides with thumb and finger (0–2) Pinna reflex: Ear retraction in response to light tactile stimulation (0–2) Corneal reflex: Blink response to light tactile stimulation with a needle — none (0) to triple eye blink (3) Toe pinch: None (0) to very brisk with repeated extension and flexion (4) Wire manoeuvre: Animal suspended from horizontal wire by forelimbs and released — active and grasps with hind limbs (0) to falls immediately (4) To complete the assessment, the animals were restrained in a supine position to record autonomic responses of skin colour and heart rate 0–2. Measurements of lacrimation (0–1), salivation (0–2) and provoked biting (0–1) responses were also recorded. During supine restraint further measures of body tone were carried out: Limb tone: Finger gently pressed against the plantar surface of each hindpaw several times — no resistance (0) to extreme resistance (4). Abdominal tone: Abdomen gently palpated with finger — completely flaccid, no return of cavity to normal (0) to extreme resistance (2) The primary screen was completed with the measurement of the righting reflex, contact righting reflex and
negative geotaxis. Throughout this procedure, any incidences of abnormal behaviour, fear, irritability, aggression or vocalisation were also recorded. The rotarod tests balance and coordination and comprises a rotating drum, which is accelerated from 4 to 40 revolutions per minute over the course of five minutes. Mice were placed individually on the revolving drum (Ugo Basile, Italy). Once they were balanced, the drum was accelerated. The time in seconds at which each animal fell from the drum was recorded using a stopwatch. Each animal received three consecutive trials, the longest time on the drum being used for analysis. Locomotor activity (LMA) was assessed by beam breaks. Mice were placed in an activity monitor (Benwick Electronics, Essex, UK) which consisted of eight clear perspex cages (42×21×20 cm) each positioned within a frame equipped with infra-red beams along its length and width. LMA was recorded automatically by counting the number of beam breaks in the test period. Transits were also recorded and these were defined as a continuous movement from one end of the cage to the other end. In both cases, total beam breaks were recorded in 5 min time bins for a period of 60 mins. Approximately one hour after the completion of testing, neurological deficits were measured according to the following scoring system. 0= no deficit; 1= flexion of contralateral torso and forelimb upon lifting of the whole animal by the tail; 2= circling to the contralateral side, when held by tail with feet on floor; 3= spontaneous circling to contralateral side; 4= no spontaneous motor activity. Post mortem visual observation of lesion size was also undertaken. All statistical analysis was carried out using StatisticaTM (Statsoft Inc., Tulsa, USA). Data from the primary observational screen was analysed using either Kruskal Wallis test or by Fishers Exact Test. Follow up analysis was by Mann-Whitney U-test or Fishers Exact Tests respectively. Multiple comparisons compared sham animals with both unoperated and MCAO animals. In both these cases, p<0.025 was judged to be significant as two separate comparisons had to be made. Data for locomotor activity (log10 transformed to correct for heterogeneity of variance) and rotarod were analysed using two way analysis of variance (ANOVA) with treatment and day as the independent variables. This was because the actual experiment was carried out over 4 days although all animals were tested 24 h post-ischaemia. Post hoc analysis was carried out using Scheffe’s multiple comparison procedure. In these cases p<0.05 was classed as significant. 2.2. Functional recovery over time after transient MCAO in the rat Male Sprague Dawley rats (Charles River, UK) in the weight range 300–350 g were used in the rat studies and
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these were housed under the same environmental conditions as the mice. For the MCAO procedure, general anaesthesia was induced in a chamber using 5% halothane in a 2:1 mixture of nitrous oxide and oxygen and maintained with 1.5% halothane delivered by a face mask. Animals were maintained normothermic during the surgical procedure using a rectal probe with feedback control to a heating blanket. Ischaemia was induced by the method of Zea Longa et al. (1989) and the procedure to expose and insert the monofilament was essentially the same as described above for the mice. The monofilament (3/0 nylon monofilament: Ethicon, UK) was inserted approximately 19–22 mm distal to the carotid bifurcation. The filament was tied in place with suture thread and the aneurysm clips removed. The cervical wound was then closed with skin staples and the animal allowed to recover. Ninety minutes after MCA occlusion, rats were re-anaesthetised and the staples removed. An aneurysm clip was re-applied to the common carotid artery and the filament slowly and completely withdrawn. The arteriotomy was closed with diathermy and the cervical wound sutured closed. In sham surgery the arteries of the carotid bed were manipulated as in the MCAO operated animals but the arteriotomy and insertion of the filament was not performed. In the immediate post operative period, animals were allowed to regain consciousness and righting reflex under strict observation in an incubator (23–25°C) for 1 h. The animals were then housed individually in the post-operative recovery room, where their overall health status was closely monitored throughout the survival period. Softened palatable diet and baby food were provided on the cage floor to encourage feeding. For the first 5 days, until MCAO operated animals fed independently, syringe feeding was performed with a dietary supplement up to 3 times daily. Supplementary fluids (0.9% saline) were administered (1.5 ml) up to 3 times daily. Motor and behavioural changes were assessed using a 0–5 point grading scale (modified Bederson) at 1 h following MCA occlusion, and daily prior to sacrifice: 0, no deficit; 1, failure to extend right forepaw fully; 2, decreased grip of right forelimb while tail pulled; 3, spontaneous circling or walking to contralateral side; 4, walks only when stimulated with depressed level of consciousness; 5, unresponsive to stimulation. A 21 point behavioral scale was also employed to obtain a more detailed neurological assessment at 1 h and then daily until euthanasia. This consists of the following components: Paw placement: Animal held lengthways at the edge
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of bench and each paw placed in turn on edge of the bench. Each successful paw placement back on bench scored 1. Righting reflex: Animal placed on its back on palm of the hand and scored 1 if righted itself. Horizontal bar: Forepaws placed on ribbed bar and score: 3 if both hindlimbs raised onto the bar, 2 if one hindlimb raised, 1 if animal just hangs and 0 if animal falls off. Inclined platform: animal placed facing down on 45°incline and score: 3 if the animal rotates to face “uphill” within 15 s, 2 if it takes 15–30 s, 1 if it takes longer than 30 s and 0 if the animal falls off or remains pointing downwards. Rotation: animal is held by the base of the tail and rotated clockwise then anticlockwise. Animal should swivel up contralaterally to the direction of rotation and score 1 for each side. Visual fore-paw reaching: Ability of animal to reach to bench when held slightly away from it and score 1 for each successful forepaw placement. Circling: Score 1 for non-circling, 0 for circling. Contralateral reflex: Score 0 for a reflex and 1 for no reflex. Motility: Score 2 for normal motility, 1 rocking and unsteady and 0 if immobile. General condition: Score 2 if normal (good coat condition, alert, moving about), 1 unkempt (e.g dirty coat, hunched posture, aggressive) and 0 if thin, weak and poor muscle tone. Maximum score (i.e. normal rat) =21 Prior to induction of ischaemia rats (n=15) received training in the two behavioural tasks. Animals not achieving criteria in both tests (n=3) were excluded from further study. Following training, the remaining animals were stratified according to performance into 2 balanced groups (n=6 per group). For the rotarod training session, animals were habituated to the rotarod and trained to remain on the rotating drum (constant speed 4 r.p.m.) for a minimum of 90 s, to provide a pre-operative baseline. Animals not achieving baseline criteria were excluded from further study. In the testing sessions animals were placed on the rotarod set in the accelerating mode. The rotor accelerated from 4 to 40 r.p.m. over 5 min and the latency for the animal to fall off the drum was recorded (max 5 min). Data from the rotarod were analysed by two way analysis of variance with treatment and day as independent variables. Testing took place on days 1 and 7 post-MCAO. In the sticky label test bilateral stimulation of the radial aspect of the forearm was achieved by placing thin strips of packing tape (0.8×5cm) firmly around both of the “wrists” of each animal, so that they covered the hairless part of the forepaw. Care was taken to apply the stimulus with equal pressure and the order of application
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(left, right) was randomised. The training period comprised 3 sessions. Each session consisted of 6 bilateral stimulation trials conducted over 2 days. The third session was utilized as the preoperative baseline. In the test period animals were given two test sessions: at day 1/2 post-MCAO and day 7/8 post MCAO. The following parameters were recorded: latency to contact left paw(s), latency to contact right paw(s), order to contact paws (L or R first), latency to remove left label(s), latency to remove right label(s), total times left and right paws contacted first and order of label removal, difference between time to contact and time to remove for each paw. The logrank test was used to analyse the data (Cox, 1972). This is a standard test used by clinical statisticians to analyse survival data from clinical trials, e.g. occurrence of a stroke or other measure of patient relapse. This test compares the observed events for each treatment group with the number of events expected if the two treatments were equally effective over time. The logrank test was therefore used to determine whether there was evidence of an observed difference between the two treatment groups in time to removal of the tape and time to first contact.
3. Results 3.1. Behavioural characterisation after permanent cerebral ischaemia in the mouse Only animals displaying a neurological score of 3 or more were included in the final analysis. For this reason 7 of the original MCAO group were excluded. Of these exclusions three animals showed a neurological deficit of 2, one animal showed a neurological deficit of 1, two animals had sub-arachnoid haemorrhages and one animal died prior to testing. Thus 11 MCAO animals were used in the behavioural analysis. Histological analysis of 10 of the 11 animals revealed marked infarction and swelling with a mean (± standard error of the mean, SEM) infarct volume of 31.7 ± 5.2 mm3. The brain from the 11th mouse was unavailable for analysis but this was included in the behavioural analysis. There were 10 control animals and 10 sham animals in the following analysis for the SHIRPA screen in addition to the 11 MCAO mice. 3.1.1. Primary behavioural observation screen Analysis of the data obtained in the primary observational screen revealed a number of significant differences, although data did not vary by treatment across the duration of the experiment. A full list of these differences is shown in Tables 1–3. Interestingly a significant difference was found between sham and un-operated mice in gait and performance on the wire manoeuvre
(P <0.025), spontaneous activity and tail elevation (P <0.005). Vocalisation was increased in sham operated animals when compared to non-operated controls but this failed to reach significance (P =0.03). A significant difference between sham operated and MCAO animals was found in the following measures; respiration rate, defaecation, locomotor activity, palpebral closure, gait, touch escape, grip strength and righting reflex (P<0.025) and also in transfer arousal, pelvic elevation, right and left limb tone, negative geotaxis and tail elevation (P<0.005). There was a trend for reduced urination in MCAO animals but this was not statistically significant. 3.1.2. Rotarod assessment of sensorimotor deficits Analysis of variance revealed that there was no significant effect of day on rotarod performance (F(2,19) =2.02, P = NS). A significant effect of treatment on performance was found (F(2,19) =10.31, P <0.001). Post hoc analysis showed that MCAO operated animals stayed on the rotarod for a significantly shorter time than both sham operated and non operated animals (P <0.05) (see Fig. 1). There was no correlation between performance on the rotarod and total infarct volume (r = ⫺0.02). 3.1.3. Spontaneous LMA test A significant effect of treatment on total activity over the 60 minute test period was found (F(2,19) =16.87, P <0.001). Post hoc analysis showed that both MCAO operated and sham operated animals showed a significant decrease in activity when compared to non operated controls (P <0.05) (see Fig. 2). Analysis of the transit data showed a similar effect. No significant effect of days was observed (F(2,19) =2.12, P = NS), whilst there was a significant effect of treatment (F(2,19) =20.57, P <0.001). Post hoc comparisons showed that both MCAO and sham operated animals showed a significant decrease in the number of transits when compared with non-operated controls (see Fig. 3). 3.2. Rat MCAO studies No lesions were observed in the controls on histological analysis. The mean (± SEM) infarct volume in the MCAO group was 133.0+30.7mm3 after correction for oedema. In terms of the 5 point neurological grade the median scores on day 1 post MCAO were sham=0 and MCAO=3, and on day 7 sham=0 and MCAO=2. There was some evidence for recovery although all time points showed a statistically significant difference between shams and MCAO animals (p<0.05, Mann-Whitney U test). The median 21 point scale scores on day 1 were sham=20 and MCAO=12, and on day 7 sham=20 and MCAO=15.5 indicating that there was some recovery. The data across all time points the 21 point scale are shown in Fig. 4.
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Table 1 Summary of primary screening results. Data is shown as median (± quartile range). Analysis is by Exact Kruskal Wallis Test with follow up analysis carried out using exact Mann-Whitney U-test. Significant follow up analyses are highlighted. n=11 MCAO, 10 sham and 10 control mice
Body position Spontaneous activity Respiration rate Defaecation Transfer arousal Loco activity Palpebral closure Gait Pelvic elevation Touch escape Positional pass. Visual placing Grip strength R toe pinch L toe pinch Wire manoeuvre R limb tone L limb tone Negative geotaxis Sig level — 5% Sig level — 1%
Unoperated
Sham Operated MCAO
Kruskal Wallis
Sham vs Un-Op Sham vs MCAO
3.00 (0.8) 2.00 (0.0) 2.00 (0.0) 2.20±0.83 3.50 (1.0) 11.60±1.99 0.00 (0.0) 0.00 (0.0) 2.00 (0.0) 2.00 (0.0) 1.00 (0.0) 3.00 (0.0) 2.00 (0.0) 3.00 (0.0) 3.00 (0.0) 0.00 (0.0) 2.00 (0.0) 2.00 (0.0) 0.00 (0.0)
3.00 (0.0) 1.00 (0.0) 2.00 (0.0) 1.30±0.37 3.00 (0.0) 16.20±2.00 0.00 (0.8) 1.00 (1.0) 2.00 (0.0) 2.00 (0.0) 1.00 (0.0) 3.00 (0.0) 2.00 (0.0) 3.00 (0.0) 3.00 (0.0) 1.00 (1.0) 2.00 (0.0) 2.00 (0.0) 0.00 (0.0)
p<0.05 p<0.01 p<0.01 p<0.05 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01 p=0.07 p=NS p<0.01 p=0.09 p=NS p<0.01 p<0.01 p<0.01 p<0.01 p<=0.05 p<=0.01
p=NS p<0.005 p=NS p=NS p=NS p=NS p=NS p<0.025 p=NS p=NS – – p=NS – – p<0.025 p=NS p=NS p=NS p<=0.025 p<=0.005
3.00 (0.0) 1.00 (0.5) 1.00 (0.0) 0.18±0.08 2.00 (1.0) 4.55±1.88 1.00 (0.0) 2.00 (1.0) 1.00 (2.0) 1.00 (1.0) 1.00 (1.5) 3.00 (0.0) 1.00 (0.0) 3.00 (1.5) 3.00 (0.0) 3.00 (1.5) 1.00 (0.0) 1.00 (1.0) 2.00 (1.0)
p=NS p=NS p<0.025 p<0.025 p<0.005 p<0.025 p<0.025 p<0.025 p<0.005 p<0.025 – – p<0.025 – – p=0.03 p<0.005 p<0.005 p<0.005 p<=0.025 p<=0.005
Table 2 Summary of primary screening results (cont). Data is shown as median (± quartile range). Analysis is by Fishers Exact Test. Significant follow up analyses are highlighted. n=11 MCAO, 10 sham and 10 control mice
Urination* Piloerection Startle response Tail elevation Body tone R Pinna Reflex L Pinna Reflex R Corneal Reflex L Corneal Reflex Righting reflex Contact righting reflex Vocalisation Sig level — 5% Sig level — 1%
Unoperated
Sham operated
MCAO
Fishers Exact Test
Sham vs Un-Op Sham vs MCAO
0.20±0.13 0.00 (0.0) 1.00 (0.0) 1.00 (0.0) 1.00 (0.0) 1.00 (0.0) 1.00 (0.0) 1.00 (0.0) 1.00 (0.0) 0.00 (0.0) 1.00 (0.0) 0.00 (0.0)
0.40±0.16 0.00 (0.0) 1.00 (0.0) 1.00 (0.0) 1.00 (0.0) 1.00 (0.8) 1.00 (0.8) 1.00 (0.0) 1.00 (0.0) 0.00 (0.0) 1.00 (0.0) 0.50 (1.0)
0.09±0.08 0.00 (0.0) 0.00 (1.0) 0.00 (0.5) 1.00 (0.0) 0.00 (0.0) 1.00 (1.0) 1.00 (0.0) 1.00 (0.0) 1.00 (1.0) 1.00 (0.0) 1.00 (0.0)
p=NS p=NS p<0.05 p<0.01 p=NS p<0.001 p=NS p=NS p=NS p<0.01 p=NS p<0.01 p<=0.05 p<=0.01
– – p=NS p<0.005 – p=NS – – – p=NS – p=0.03 p<=0.025 p<=0.005
3.2.1. Rotarod assessment of sensorimotor deficits The reduction in average time spent on the rotarod by the MCAO animals when compared with the sham operated animals on Day 1 was highly statistically significant (average difference =106.5; p-value=0.0056) indicating that ischaemia had induced a marked deficit in motor co-ordination and balance. There was no evidence of a difference in time spent on the rotarod between the two groups on day 7 (average difference =34.7; p-value =0.1595). This was due to an increase in
– – p=NS p<0.005 – p=NS – – – p<0.025 – p=NS p<=0.025 p<=0.005
time spent on the rotarod by the MCAO group, demonstrating recovery of performance on this task with time. The data are shown graphically in Fig. 5. 3.2.2. Sticky label test Data for the sticky label tests are shown in Tables 4 and 5. The analysis of time to removal of the tape on the right, contralateral, side showed that no differences existed on baseline testing (day 0) between the sham and MCAO groups over the 300 s (p-value =0.3). However
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Table 3 Summary of primary screening results (cont). Data is shown as median (± quartile range). No statistical analysis performed as data for all three groups was identical. n=11 MCAO, 10 sham and 10 control mice
Tremor Abnor behaviour Skin colour Heart rate Abdominal tone Lacrimation Salivation Fear Irritability Aggression
Unoperated
Sham operated
MCAO
0.00 (0.0) 0.00 (0.0)
0.00 (0.0) 0.00 (0.0)
0.00 (0.0) 0.00 (0.0)
1.00 1.00 1.00 0.00 1.00 0.00 0.00 0.00
1.00 1.00 1.00 0.00 1.00 0.00 0.00 0.00
1.00 1.00 1.00 0.00 1.00 0.00 0.00 0.00
(0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0)
(0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0)
(0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0)
Fig. 1. Effect of pMCAO in mice on performance on the rotarod. Data is shown as mean total time spent on rotarod for each treatment group ± SEM. Data was analysed using two way analysis of variance with treatment and day as independent variables. * p<0.05 vs unoperated controls and sham operated animals.
on day 1, there was evidence of a significant difference between the sham and MCAO groups over the 300 s — with MCAO animals taking longer to remove the tape from the right side although many animals failed to remove the tape at all (p-value =0.05). On day 7, the MCAO rats took significantly longer to remove the tape than the sham group (p-value =0.0005; Table 4). The time to contact was much shorter than the time to removal at baseline and an analysis of the time to contact the right hand tape using the logrank tests and median times to contact again showed no significant difference between the sham and MCAO groups on day 0 (p=0.8). However the MCAO treated animals took significantly longer to make contact with the right side tape on days 1 and 7 (p=0.0005; Table 4).
MCAO and sham rats did not differ on day 0 in the median time to removal of the tape on the left, ipsilateral, side (p=0.4). However the logrank tests of the median times to removal indicated that the MCAO treated animals took longer to remove the tape on day 1 (p=0.05) and day 7 (p=0.003; Table 5). An analysis of time to contact the left side using the logrank tests and median times showed no significant difference between sham and MCAO groups on day 0 and also no statistically significant differences between the MCAO and sham groups on days 1 (p=0.8) and day 7 (p=0.06; Table 5).
4. Discussion Few studies have looked at sensorimotor function after MCAO in mice. Most of these have only used very simple neurological scores (Sheng et al. 1999, 1998; Connolly et al., 1996; Weisbrot-Lefkowitz et al., 1998; Yang et al., 1994). Clark et al. (1997) used three neurological function scales to assess outcome after varying times of MCAO in Swiss-Webster mice. These were a simple graded score of 1–5 (5 being dead), a global scale (activity, posture, presence or absence of fits) and a focal scale (symmetry, gait, climbing and sensory response). However none of the studies to date have carried out such a comprehensive assessment of function postMCAO in the mouse as that described in the present study. Here the use of SHIRPA has shown that mice with ischaemic damage caused by MCAO have a wide range of behavioural changes when compared to both sham and non-operated animals. Additionally, on some measures, differences were found between sham and non-operated animals indicating that these are susceptible to interference, regardless of the operative treatment, at least during the post-operative 24 h. However, these effects on sham-operated animals were always less marked than the effects seen in those animals which underwent the MCAO lesion. In the primary observational screen, the level of spontaneous activity was significantly decreased in sham operated animals when compared to the non-operated controls, indicating that they are quieter than non-operated animals when placed in a novel environment. The gait of sham operated animals was also reduced, with the animals having a noticable “flattening” of posture during walking. Alongside this, a marked reduction in tail elevation was also seen. It may be that these two behaviours are related. Mice with a flattened gait may be unable to hold their tail in its normal position and will therefore always show a lowering in tail position. Performance in the wire manoeuvre test was also reduced in sham operated animals. This test involves both balance, co-ordination and rear limb strength. 24 h post operation these measures are still markedly affected.
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Fig. 2. Effect of pMCAO in mice on locomotor activity. (a) Data is shown as mean number of beam breaks in 60 min for each treatment group ± SEM. Data was analysed using two way analysis of variance with treatment and day as independent variables. * p<0.05 vs unoperated controls and # p<0.05 vs sham operated animals. (b) Data is shown as number of beam breaks per 5 min time period over 60 min for each treatment group. SEM are not shown for clarity.
It may be that this reflects a “normal” post-operative malaise. Leaving the animals for a longer period of recovery may diminish this effect. A large number of differences between sham operated and MCAO animals were observed in the primary screen. Respiration rate in MCAO operated animals was reduced with animals taking slower, shallower breaths. MCAO operated animals also showed a lower level of general activity, as shown by a reduction in transfer arousal and locomotion. This is unlikely to reflect an effect of the operation as no effects on these measures were seen in sham operated animals. When the animals did move it was noticeable that the body posture of MCAO animals was markedly reduced. This is reflected in the scores for gait and pelvic elevation. MCAO mice
also showed a reduction in the strength of their grip in their front legs. The rear limbs show a reduction in strength represented by a reduction in the scores for limb tone which was apparent on both the ispsilateral and contralateral sides. Neither the operation itself, as represented by sham animals, nor the MCAO lesion appear to have effects on autonomic responses measured within this test. Thus skin colour (which reflects blood supply to the skin), heart rate, lacrimation and other measures all showed no difference compared to unoperated animals. Differences were observed in other tests between the three groups of mice. For example in the rotarod, a test of balance and coordination, MCAO operated animals perform significantly worse than either sham or non-
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Table 4 Median time to removal and contact for the right hand tape (contralateral to the lesion). n=6 per group Day
Group
Median contact time (seconds)
Median removal time (seconds)
0 0 1
Sham MCAO Sham
3.5 (3.3–4.8) 5.7 (3.3–7.6) 4.0 (3.3–5.5)
1 7 7
MCAO Sham MCAO
50% failed c 5.0 (3.6–7.6) 66.9 (50.6–92.0)c
49.0 (34.0–62.6) 41.9 (38.0–45.1) 67.8 (51.1–50% failed) 50% failed a 52.9 (48.5–54.8) 50% failed b
a b
Fig. 3. Effect of MCAO in CD1 mice on total number of transits. Data is shown as mean number of beam breaks in 60 min for each treatment group ± SEM. Data was analysed using two way analysis of variance with treatment and day as independent variables. * p<0.05 vs unoperated controls and # p<0.05 vs sham operated animals.
c
p=0.05 MCAO vs sham. p=0.003 MCAO vs sham. p=0.0005 MCAO vs sham
Fig. 4. The effect of MCAO in the rat on 21 point neurological score. Significant differences are seen at all time points between the sham and MCAO operated animals as assessed by Mann-Whitney U test (# = p<0.05).
Fig. 5. The effect of MCAO in the rat on rotarod performance. Data shown are mean and standard error. Data was analysed using two way analysis of variance with treatment and day as independent variables. *p=0.005 ischaemic vs sham on day 1.
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Table 5 Median time to removal and contact for the left hand tape (ipsilateral to the lesion). n=6 per group Day
Group
Median contact time (seconds)
Median removal time (seconds)
0 0 1
Sham MCAO Sham
2.2 (1.6--3.0) 2.6(1.6--5.5) 4.0 (2.6--6.8)
1 7 7
MCAO Sham MCAO
28.2 (17.1--53.6) 2.7 (1.8--6.3) 7.7 (5.8--11.6)
39.0 (29.3--56.5) 35.0 (32.8--39.1) 52.6 (46.8--50% failed) 50% failed a 54.7 (41.1--63.8) 87.1 (76.6--112.8)b
a b
p=0.05. p=0.003.
operated animals. These results are in agreement with the data obtained in the behavioural observation where a reduction in limb tone, a reduced performance in the wire manoeuvre test and a worsening gait all suggested general reductions in motor function. The rotarod has been well validated as a means of assessing deficits in motor performance in rats (Borlongan et al., 1995; Rogers et al., 1997b; Yamamoto et al., 1988 and the present study) but has not previously been used with mice after MCAO. Interestingly Yamamoto et al. (1988) also found that sham operated rats were not impaired to control unoperated rats. In the LMA test, MCAO operated mice showed a significant reduction in activity over non-operated controls. Interestingly, in this test, sham operated mice were also significantly worse than non-operated animals paralleling the data obtained with the acute observation test. This suggests that the relatively invasive surgical procedure involving the carotid vasculature which occurs in both sham and MCAO operations, is sufficient in itself to alter locomotor activity 24 h post-MCAO. These data on LMA in mice are different from most studies involving MCAO in the rat where no effects on LMA have been observed (Yonemori et al., 1998). Borlongan et al. (1995) did see some alterations in nocturnal LMA with ischaemia and a potentiation of the hyperactivity induced by amphetamine. Interestingly Borlongan et al. (1995) did not observe any differences in LMA between the sham and MCAO groups in the hour prior to amphetamine administration. Borlongan et al. (1995) also tested the animals at one month post-ischaemia rather than 24 h after MCAO as in the present study. These data show the importance of including an unoperated control group for measurements such as LMA, although this is not necessary for others such as the rotarod where the sham animals were no different from control, unoperated animals. The experiments described in the present paper show that the SHIRPA protocol described here can be used acutely in mice to assess sensory and motor deficits after stroke, much as the NIH scale is used in man.
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The data from the rat transient MCAO study also showed deficits in the rotarod at 24 h post-ischaemia. However these deficits had essentially disappeared by 7 days post-ischaemia. This is in contrast to data obtained by Yamamoto et al. (1988) who looked at rotarod performance after permanent proximal MCAO and found that animals still had significant impairments at 2 weeks post occlusion although recovery was observed from 3 weeks post ischaemia. This highlights the importance of defining the time course of recovery for any functional tests in a particular model prior to undertaking any therapeutic intervention studies. The relatively rapid recovery of performance on the rotarod suggests that it could be useful in studies of the effects of interventions which would promote regeneration and repair. The neurological grade scores showed little evidence of recovery although by day 7, the data seemed to suggest some improvement and use of this scale beyond 7 days is warranted. The bilateral sticky label test showed a prolonged deficit on the contralateral side with some recovery evident ipsilaterally suggesting that this will be a more severe test of regenerative/repair therapies. This test was first developed by Schallert et al. (1982) and has similarities to the contralateral neglect syndrome which is seen in man after damage to the parietal cortex (Heilman et al., 1985). The impairments observed here were highly significant on the contralateral side with no evidence of recovery by 7 days. Other work in our laboratory has demonstrated a close correlation of contralateral contact and removal latencies on this task with pathological changes in the ipsilateral caudate putamen, lower parietal cortex and forelimb cortex following 90 min of tMCAO as depicted by MRI. Each of these regions of interest contributed to functional impairments on this task across an extended time course (up to 28 days post-ischaemia) (Virley et al., 1999a). In addition substantia nigra damage as evidenced by apparent diffusion coefficient (ADC) changes and T2 relaxation times at 7, 14 and 28 days post MCAO also correlated with contralateral deficits on this task (Virley et al., 1999b). Thus this task may be suitable for assessing both neuroprotective therapies which target early intervention as well as those aimed at the prevention of delayed damage and therapies which promote regeneration. In conclusion, use of the SHIRPA protocol in MCAO, sham and non-operated mice reveals a range of functional differences which may allow for the behavioural assessment of ischaemic damage in mice. In these tests animals were tested 24 h post lesion. Studies in mice with SHIRPA are now ongoing to determine whether any of these deficits recover with time and whether drug or transgenic manipulations can alter the ischaemiainduced changes. The importance of the former is clearly demonstrated by the recovery seen in the rotarod task, but not the neurological scores or sticky label test, in the rat MCAO. However it must be kept in mind that
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differences in pathology between permanent and transient models may also contribute to different rates of recovery and further studies are needed to investigate this.
Acknowledgements The authors would like to thank Susan Spencer for help in typing the manuscript, Jenny Roberts for cutting the brain sections and Helen Tate and Phil Overend for additional statistical support.
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Functional assessments in mice and rats after focal stroke A.J. Hunter *, J. Hatcher, D. Virley, P. Nelson, E. Irving, S.J. Hadingham, A.A. Parsons Neurosciences Research, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK Accepted 14 December 1999
Abstract This paper presents a comprehensive assessment of sensorimotor deficits in the mouse after focal ischaemia induced by occlusion of the middle cerebral artery. Twenty four hours after induction of middle cerebral artery occlusion, mice showed deficits in a range of sensory and motor tasks as assessed by the SHIRPA protocol. In addition they exhibited a decrease in rotarod performance and locomotor activity. Some behaviours, such as locomotor activity, were also impaired in sham operated animals compared to normal controls, although these impairments were not as marked as those exhibited by the ischaemic mice. This is the first comprehensive analysis of the short term effects of permanent focal ischaemia in mice. In a second series of experiments in the rat, rates of recovery over time were examined. Simple (neurological grades, rotarod) and complex (sticky label test) tasks were examined in rats after middle cerebral artery occlusion up to 7 days post-ischaemia. Ischaemic rats had a profound deficit in contralateral performance on the sticky label task with no evidence of recovery. A less marked deficit was also observed in ipsilateral performance of this task. These deficits were still present 7 days after ischaemia. Ischaemic rats also exhibited a deficit on rotarod performance but this had recovered 7 days post-ischaemia. Thus different sensorimotor tasks have different rates of recovery after focal cerebral ischaemia in the rat. Further characterisation of these tasks will enhance their utility meaningful preclinical means of assessing functional recovery of the administration of potential neuroprotective and regenerative therapies. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Mouse; Rat; Focal ischaemia; Behaviour
1. Introduction It is becoming increasingly necessary to study functional outcomes in stroke research in both rodents and higher species (Corbett and Nurse, 1998; Hunter et al., 1998). This is especially true for investigating the effects of therapies aimed at promoting regeneration and repair such as basic fibroblast growth factor (Kawamata et al., 1996). There have been a relatively large number of studies looking at functional changes after the induction of global ischaemia in rodents. In global ischaemia models, such as bilateral common carotid occlusion in the gerbil and four-vessel occlusion in rats, increases in locomotor activity have been observed (Baldwin et al., 1993; Kuroiwa et al., 1991; Wang and Corbett, 1990). This increase is thought to reflect a deficit in habituation
* Corresponding author. Tel.: +44-1279-622000; fax: +44-1279622660. E-mail address: a.jacqueline [email protected] (A.J. Hunter).
or spatial mapping (Babcock et al., 1993) rather than in sensory-motor function per se. Impairments in other sensorimotor tasks have been less frequently reported (e.g. Baldwin et al., 1993). Overall there are fewer reports of functional deficits after focal ischaemia, although there have been more studies carried out to examine the effects of focal stroke on sensorimotor function. Photothrombotic lesions of the cortex have been shown to produce impairments on tasks such as beam and grid walking and grip strength measurements (Wood et al., 1996). However induction of focal ischaemia by permanent or transient occlusion of the middle cerebral artery (MCAO) is now the model of choice for focal ischaemia studies (Hunter et al., 1995; STAIR stroke conference Florida, 1999). In the rat, simple neurological grades such as the postural reflex test published by Bederson et al. (1986) or the neurological assessment of Zea Longa et al. (1989) have been used to assess the success of the MCAO and to measure the effects of drugs. However such scales are relatively crude. Therefore a number of workers have developed
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more refined sensorimotor tasks in the rat. These include limb placing, beam walking, grid walking, rotarod, sticky label test, staircase test (see Hunter et al., 1998; Corbett and Nurse, 1998 for reviews). These have shown that spontaneous recovery can occur in some tasks (e.g. beam walking) whereas it is not so quickly observed in others (e.g. staircase), although detailed time courses for all models have not been established. Assessment of sensorimotor function in the mouse after focal ischaemia has been rarely described, despite the growing number of studies utilising MCAO in the mouse. These studies have increased primarily as a result of the use of transgenic mice for understanding the pathological consequences of ischaemia. Therefore there is a growing need to develop simple but comprehensive functional outcome measures in mice after MCAO. The studies described in the present paper have two aims. Firstly to characterise a behavioural test battery in mice to demonstrate that a comprehensive screen can be carried out for short term assessment of function. This essentially uses the SHIRPA procedure (Rogers et al., 1997a) which has been developed as a comprehensive routine testing protocol for characterising phenotypic variations in transgenic mice. Broadly the characterisation involved two distinct steps. Firstly, a behavioural and functional profile of the animals was built up by the use of observational assessments using a modified Irwin profile (Irwin, 1968). Secondly, a more complex series of tasks were undertaken in order to provide additional behavioural data. These studies are an essential prerequisite to carrying out longer-term assessments in the mouse after MCAO. More behavioural studies have been carried out in the rat shortly after ischaemia and so the aim of the rat MCAO study was to characterise both simple and complex tests to look at differential rates of recovery over time. In this case a transient MCAO was chosen to minimise long term mortality due to permanent MCAO (Koizumi et al., 1986). This study will aid long term studies in mice by providing a guide as to which aspects of the test battery in the mouse may show differential rates of recovery with time.
2. Materials and methods All experiments were conducted according to the requirements of the United Kingdom Animals (Scientific Procedures) Act (1986) and conformed to SmithKline Beecham ethical standards. 2.1. Behavioural characterisation after permanent cerebral ischaemia in the mouse Male CD1 mice (Charles River UK) weighing 30–35 g at the time of surgery were used. Prior to surgery,
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animals were housed in groups of five, in a temperature controlled environment (20°C ± 1°C) and maintained with ad libitum access to food and water on a 12 h light/dark cycle (lights on 7:00–19:00). After surgery, mice were housed singly on soft bedding and given access to softened diet. Mice were anaesthetised using a mixture of 5% halothane, 30% oxygen and 70% nitrous oxide, and maintained with a nitrous oxide/oxygen mixture containing 1–1.5% halothane. A midline cervical incision was made and the left carotid artery exposed. Sterile silk sutures were looped around the common, external and internal carotid arteries following which the pterygopalatine artery was exposed and cauterised at its origin. An aneurysym clip was placed across the common carotid artery and blood flow in the internal carotid artery arrested using a suture. An arteriotomy was then made in the external carotid artery and a blunted, rounded length of monofilament nylon suture of 0.18 mm diameter was inserted (Dermalon 5/0: David and Geck, Puerto Rico). This was secured in place with a silk suture. The monofilament was then advanced into the internal carotid artery and passed into the intracranial circulation to lodge in the narrower lumen of the proximal anterior cerebral artery, approximately 8 mm distal to the carotid bifurcation, thereby occluding the origin of the MCA. The aneurysm clip was then removed and the wound closed. Sham operated animals underwent the same procedure with the exception of the advancement of the filament to occlude the cerebral artery. Animals were kept under heated lamps until full reversal of the anaesthetic. Behavioural studies were carried out 24 h post operation. Each animal was tested in all of the procedures detailed below between 11:00 and 15:00 hrs on the day following, i.e. 24 h after, the MCAO operations. Procedures were run in the order in which they are described. For this study it was not feasible to carry out truly blinded observations due to the inclusion of the unoperated group. The primary observation screen is a modification of the Irwin procedure (Irwin, 1968), during which a total of 40 separate measurements are recorded for each animal (for full method see Rogers et al., 1997a). Assessment of each animal began with observation of undisturbed behaviour in a cylindrical clear perspex viewing jar (15 cm×11 cm). The following behaviours were scored in the viewing jar: Body position: completely flat (0) to repeated vertical leaping (8) Spontaneous activity: none (0) to repeated vigorous movement (8) Respiration: gasping, irregular (0) to hyperventilation (3) Temor: none (0) to marked (2).
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The mice were then transferred to an arena (55×33 cm) which was marked into even sized squares (11×11 cm) for observations of the following behaviours: Transfer arousal: coma (0) to extremely excited (6) Locomotor activity: number of squares crossed in 30 s Palpebral closure: eyes wide open (0) to completely shut (2) Startle response: the jerk produced in response to a pen dragged across wire grid on top of arena — no response (0) to 1 cm jump or more (3) Gait: normal (0) to incapacity (3) Pelvic elevation: flattened (0) to elevated ⬎3 mm (3) Tail elevation: flattened (0) to Straub tail (2) Touch escape: none (0) to extremely vigorous (3) Positional passivity: struggles when held by tail (0), neck (1), supine (2), by hindleg (3), no struggle (4) This was followed by a sequence of manipulations using tail suspension: Visual placing: animal lowered and distance from grid needed to evoke forepay paddling — none i.e. with nose in contact (0) to 25 mm or more Trunk curl: (0–1) Limb grasping: (0–1) Grip strength: Resistance when animal placed on grid an drawn backwards (0–4) Body tone: Determined by compresion of the sides with thumb and finger (0–2) Pinna reflex: Ear retraction in response to light tactile stimulation (0–2) Corneal reflex: Blink response to light tactile stimulation with a needle — none (0) to triple eye blink (3) Toe pinch: None (0) to very brisk with repeated extension and flexion (4) Wire manoeuvre: Animal suspended from horizontal wire by forelimbs and released — active and grasps with hind limbs (0) to falls immediately (4) To complete the assessment, the animals were restrained in a supine position to record autonomic responses of skin colour and heart rate 0–2. Measurements of lacrimation (0–1), salivation (0–2) and provoked biting (0–1) responses were also recorded. During supine restraint further measures of body tone were carried out: Limb tone: Finger gently pressed against the plantar surface of each hindpaw several times — no resistance (0) to extreme resistance (4). Abdominal tone: Abdomen gently palpated with finger — completely flaccid, no return of cavity to normal (0) to extreme resistance (2) The primary screen was completed with the measurement of the righting reflex, contact righting reflex and
negative geotaxis. Throughout this procedure, any incidences of abnormal behaviour, fear, irritability, aggression or vocalisation were also recorded. The rotarod tests balance and coordination and comprises a rotating drum, which is accelerated from 4 to 40 revolutions per minute over the course of five minutes. Mice were placed individually on the revolving drum (Ugo Basile, Italy). Once they were balanced, the drum was accelerated. The time in seconds at which each animal fell from the drum was recorded using a stopwatch. Each animal received three consecutive trials, the longest time on the drum being used for analysis. Locomotor activity (LMA) was assessed by beam breaks. Mice were placed in an activity monitor (Benwick Electronics, Essex, UK) which consisted of eight clear perspex cages (42×21×20 cm) each positioned within a frame equipped with infra-red beams along its length and width. LMA was recorded automatically by counting the number of beam breaks in the test period. Transits were also recorded and these were defined as a continuous movement from one end of the cage to the other end. In both cases, total beam breaks were recorded in 5 min time bins for a period of 60 mins. Approximately one hour after the completion of testing, neurological deficits were measured according to the following scoring system. 0= no deficit; 1= flexion of contralateral torso and forelimb upon lifting of the whole animal by the tail; 2= circling to the contralateral side, when held by tail with feet on floor; 3= spontaneous circling to contralateral side; 4= no spontaneous motor activity. Post mortem visual observation of lesion size was also undertaken. All statistical analysis was carried out using StatisticaTM (Statsoft Inc., Tulsa, USA). Data from the primary observational screen was analysed using either Kruskal Wallis test or by Fishers Exact Test. Follow up analysis was by Mann-Whitney U-test or Fishers Exact Tests respectively. Multiple comparisons compared sham animals with both unoperated and MCAO animals. In both these cases, p<0.025 was judged to be significant as two separate comparisons had to be made. Data for locomotor activity (log10 transformed to correct for heterogeneity of variance) and rotarod were analysed using two way analysis of variance (ANOVA) with treatment and day as the independent variables. This was because the actual experiment was carried out over 4 days although all animals were tested 24 h post-ischaemia. Post hoc analysis was carried out using Scheffe’s multiple comparison procedure. In these cases p<0.05 was classed as significant. 2.2. Functional recovery over time after transient MCAO in the rat Male Sprague Dawley rats (Charles River, UK) in the weight range 300–350 g were used in the rat studies and
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these were housed under the same environmental conditions as the mice. For the MCAO procedure, general anaesthesia was induced in a chamber using 5% halothane in a 2:1 mixture of nitrous oxide and oxygen and maintained with 1.5% halothane delivered by a face mask. Animals were maintained normothermic during the surgical procedure using a rectal probe with feedback control to a heating blanket. Ischaemia was induced by the method of Zea Longa et al. (1989) and the procedure to expose and insert the monofilament was essentially the same as described above for the mice. The monofilament (3/0 nylon monofilament: Ethicon, UK) was inserted approximately 19–22 mm distal to the carotid bifurcation. The filament was tied in place with suture thread and the aneurysm clips removed. The cervical wound was then closed with skin staples and the animal allowed to recover. Ninety minutes after MCA occlusion, rats were re-anaesthetised and the staples removed. An aneurysm clip was re-applied to the common carotid artery and the filament slowly and completely withdrawn. The arteriotomy was closed with diathermy and the cervical wound sutured closed. In sham surgery the arteries of the carotid bed were manipulated as in the MCAO operated animals but the arteriotomy and insertion of the filament was not performed. In the immediate post operative period, animals were allowed to regain consciousness and righting reflex under strict observation in an incubator (23–25°C) for 1 h. The animals were then housed individually in the post-operative recovery room, where their overall health status was closely monitored throughout the survival period. Softened palatable diet and baby food were provided on the cage floor to encourage feeding. For the first 5 days, until MCAO operated animals fed independently, syringe feeding was performed with a dietary supplement up to 3 times daily. Supplementary fluids (0.9% saline) were administered (1.5 ml) up to 3 times daily. Motor and behavioural changes were assessed using a 0–5 point grading scale (modified Bederson) at 1 h following MCA occlusion, and daily prior to sacrifice: 0, no deficit; 1, failure to extend right forepaw fully; 2, decreased grip of right forelimb while tail pulled; 3, spontaneous circling or walking to contralateral side; 4, walks only when stimulated with depressed level of consciousness; 5, unresponsive to stimulation. A 21 point behavioral scale was also employed to obtain a more detailed neurological assessment at 1 h and then daily until euthanasia. This consists of the following components: Paw placement: Animal held lengthways at the edge
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of bench and each paw placed in turn on edge of the bench. Each successful paw placement back on bench scored 1. Righting reflex: Animal placed on its back on palm of the hand and scored 1 if righted itself. Horizontal bar: Forepaws placed on ribbed bar and score: 3 if both hindlimbs raised onto the bar, 2 if one hindlimb raised, 1 if animal just hangs and 0 if animal falls off. Inclined platform: animal placed facing down on 45°incline and score: 3 if the animal rotates to face “uphill” within 15 s, 2 if it takes 15–30 s, 1 if it takes longer than 30 s and 0 if the animal falls off or remains pointing downwards. Rotation: animal is held by the base of the tail and rotated clockwise then anticlockwise. Animal should swivel up contralaterally to the direction of rotation and score 1 for each side. Visual fore-paw reaching: Ability of animal to reach to bench when held slightly away from it and score 1 for each successful forepaw placement. Circling: Score 1 for non-circling, 0 for circling. Contralateral reflex: Score 0 for a reflex and 1 for no reflex. Motility: Score 2 for normal motility, 1 rocking and unsteady and 0 if immobile. General condition: Score 2 if normal (good coat condition, alert, moving about), 1 unkempt (e.g dirty coat, hunched posture, aggressive) and 0 if thin, weak and poor muscle tone. Maximum score (i.e. normal rat) =21 Prior to induction of ischaemia rats (n=15) received training in the two behavioural tasks. Animals not achieving criteria in both tests (n=3) were excluded from further study. Following training, the remaining animals were stratified according to performance into 2 balanced groups (n=6 per group). For the rotarod training session, animals were habituated to the rotarod and trained to remain on the rotating drum (constant speed 4 r.p.m.) for a minimum of 90 s, to provide a pre-operative baseline. Animals not achieving baseline criteria were excluded from further study. In the testing sessions animals were placed on the rotarod set in the accelerating mode. The rotor accelerated from 4 to 40 r.p.m. over 5 min and the latency for the animal to fall off the drum was recorded (max 5 min). Data from the rotarod were analysed by two way analysis of variance with treatment and day as independent variables. Testing took place on days 1 and 7 post-MCAO. In the sticky label test bilateral stimulation of the radial aspect of the forearm was achieved by placing thin strips of packing tape (0.8×5cm) firmly around both of the “wrists” of each animal, so that they covered the hairless part of the forepaw. Care was taken to apply the stimulus with equal pressure and the order of application
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(left, right) was randomised. The training period comprised 3 sessions. Each session consisted of 6 bilateral stimulation trials conducted over 2 days. The third session was utilized as the preoperative baseline. In the test period animals were given two test sessions: at day 1/2 post-MCAO and day 7/8 post MCAO. The following parameters were recorded: latency to contact left paw(s), latency to contact right paw(s), order to contact paws (L or R first), latency to remove left label(s), latency to remove right label(s), total times left and right paws contacted first and order of label removal, difference between time to contact and time to remove for each paw. The logrank test was used to analyse the data (Cox, 1972). This is a standard test used by clinical statisticians to analyse survival data from clinical trials, e.g. occurrence of a stroke or other measure of patient relapse. This test compares the observed events for each treatment group with the number of events expected if the two treatments were equally effective over time. The logrank test was therefore used to determine whether there was evidence of an observed difference between the two treatment groups in time to removal of the tape and time to first contact.
3. Results 3.1. Behavioural characterisation after permanent cerebral ischaemia in the mouse Only animals displaying a neurological score of 3 or more were included in the final analysis. For this reason 7 of the original MCAO group were excluded. Of these exclusions three animals showed a neurological deficit of 2, one animal showed a neurological deficit of 1, two animals had sub-arachnoid haemorrhages and one animal died prior to testing. Thus 11 MCAO animals were used in the behavioural analysis. Histological analysis of 10 of the 11 animals revealed marked infarction and swelling with a mean (± standard error of the mean, SEM) infarct volume of 31.7 ± 5.2 mm3. The brain from the 11th mouse was unavailable for analysis but this was included in the behavioural analysis. There were 10 control animals and 10 sham animals in the following analysis for the SHIRPA screen in addition to the 11 MCAO mice. 3.1.1. Primary behavioural observation screen Analysis of the data obtained in the primary observational screen revealed a number of significant differences, although data did not vary by treatment across the duration of the experiment. A full list of these differences is shown in Tables 1–3. Interestingly a significant difference was found between sham and un-operated mice in gait and performance on the wire manoeuvre
(P <0.025), spontaneous activity and tail elevation (P <0.005). Vocalisation was increased in sham operated animals when compared to non-operated controls but this failed to reach significance (P =0.03). A significant difference between sham operated and MCAO animals was found in the following measures; respiration rate, defaecation, locomotor activity, palpebral closure, gait, touch escape, grip strength and righting reflex (P<0.025) and also in transfer arousal, pelvic elevation, right and left limb tone, negative geotaxis and tail elevation (P<0.005). There was a trend for reduced urination in MCAO animals but this was not statistically significant. 3.1.2. Rotarod assessment of sensorimotor deficits Analysis of variance revealed that there was no significant effect of day on rotarod performance (F(2,19) =2.02, P = NS). A significant effect of treatment on performance was found (F(2,19) =10.31, P <0.001). Post hoc analysis showed that MCAO operated animals stayed on the rotarod for a significantly shorter time than both sham operated and non operated animals (P <0.05) (see Fig. 1). There was no correlation between performance on the rotarod and total infarct volume (r = ⫺0.02). 3.1.3. Spontaneous LMA test A significant effect of treatment on total activity over the 60 minute test period was found (F(2,19) =16.87, P <0.001). Post hoc analysis showed that both MCAO operated and sham operated animals showed a significant decrease in activity when compared to non operated controls (P <0.05) (see Fig. 2). Analysis of the transit data showed a similar effect. No significant effect of days was observed (F(2,19) =2.12, P = NS), whilst there was a significant effect of treatment (F(2,19) =20.57, P <0.001). Post hoc comparisons showed that both MCAO and sham operated animals showed a significant decrease in the number of transits when compared with non-operated controls (see Fig. 3). 3.2. Rat MCAO studies No lesions were observed in the controls on histological analysis. The mean (± SEM) infarct volume in the MCAO group was 133.0+30.7mm3 after correction for oedema. In terms of the 5 point neurological grade the median scores on day 1 post MCAO were sham=0 and MCAO=3, and on day 7 sham=0 and MCAO=2. There was some evidence for recovery although all time points showed a statistically significant difference between shams and MCAO animals (p<0.05, Mann-Whitney U test). The median 21 point scale scores on day 1 were sham=20 and MCAO=12, and on day 7 sham=20 and MCAO=15.5 indicating that there was some recovery. The data across all time points the 21 point scale are shown in Fig. 4.
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Table 1 Summary of primary screening results. Data is shown as median (± quartile range). Analysis is by Exact Kruskal Wallis Test with follow up analysis carried out using exact Mann-Whitney U-test. Significant follow up analyses are highlighted. n=11 MCAO, 10 sham and 10 control mice
Body position Spontaneous activity Respiration rate Defaecation Transfer arousal Loco activity Palpebral closure Gait Pelvic elevation Touch escape Positional pass. Visual placing Grip strength R toe pinch L toe pinch Wire manoeuvre R limb tone L limb tone Negative geotaxis Sig level — 5% Sig level — 1%
Unoperated
Sham Operated MCAO
Kruskal Wallis
Sham vs Un-Op Sham vs MCAO
3.00 (0.8) 2.00 (0.0) 2.00 (0.0) 2.20±0.83 3.50 (1.0) 11.60±1.99 0.00 (0.0) 0.00 (0.0) 2.00 (0.0) 2.00 (0.0) 1.00 (0.0) 3.00 (0.0) 2.00 (0.0) 3.00 (0.0) 3.00 (0.0) 0.00 (0.0) 2.00 (0.0) 2.00 (0.0) 0.00 (0.0)
3.00 (0.0) 1.00 (0.0) 2.00 (0.0) 1.30±0.37 3.00 (0.0) 16.20±2.00 0.00 (0.8) 1.00 (1.0) 2.00 (0.0) 2.00 (0.0) 1.00 (0.0) 3.00 (0.0) 2.00 (0.0) 3.00 (0.0) 3.00 (0.0) 1.00 (1.0) 2.00 (0.0) 2.00 (0.0) 0.00 (0.0)
p<0.05 p<0.01 p<0.01 p<0.05 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01 p=0.07 p=NS p<0.01 p=0.09 p=NS p<0.01 p<0.01 p<0.01 p<0.01 p<=0.05 p<=0.01
p=NS p<0.005 p=NS p=NS p=NS p=NS p=NS p<0.025 p=NS p=NS – – p=NS – – p<0.025 p=NS p=NS p=NS p<=0.025 p<=0.005
3.00 (0.0) 1.00 (0.5) 1.00 (0.0) 0.18±0.08 2.00 (1.0) 4.55±1.88 1.00 (0.0) 2.00 (1.0) 1.00 (2.0) 1.00 (1.0) 1.00 (1.5) 3.00 (0.0) 1.00 (0.0) 3.00 (1.5) 3.00 (0.0) 3.00 (1.5) 1.00 (0.0) 1.00 (1.0) 2.00 (1.0)
p=NS p=NS p<0.025 p<0.025 p<0.005 p<0.025 p<0.025 p<0.025 p<0.005 p<0.025 – – p<0.025 – – p=0.03 p<0.005 p<0.005 p<0.005 p<=0.025 p<=0.005
Table 2 Summary of primary screening results (cont). Data is shown as median (± quartile range). Analysis is by Fishers Exact Test. Significant follow up analyses are highlighted. n=11 MCAO, 10 sham and 10 control mice
Urination* Piloerection Startle response Tail elevation Body tone R Pinna Reflex L Pinna Reflex R Corneal Reflex L Corneal Reflex Righting reflex Contact righting reflex Vocalisation Sig level — 5% Sig level — 1%
Unoperated
Sham operated
MCAO
Fishers Exact Test
Sham vs Un-Op Sham vs MCAO
0.20±0.13 0.00 (0.0) 1.00 (0.0) 1.00 (0.0) 1.00 (0.0) 1.00 (0.0) 1.00 (0.0) 1.00 (0.0) 1.00 (0.0) 0.00 (0.0) 1.00 (0.0) 0.00 (0.0)
0.40±0.16 0.00 (0.0) 1.00 (0.0) 1.00 (0.0) 1.00 (0.0) 1.00 (0.8) 1.00 (0.8) 1.00 (0.0) 1.00 (0.0) 0.00 (0.0) 1.00 (0.0) 0.50 (1.0)
0.09±0.08 0.00 (0.0) 0.00 (1.0) 0.00 (0.5) 1.00 (0.0) 0.00 (0.0) 1.00 (1.0) 1.00 (0.0) 1.00 (0.0) 1.00 (1.0) 1.00 (0.0) 1.00 (0.0)
p=NS p=NS p<0.05 p<0.01 p=NS p<0.001 p=NS p=NS p=NS p<0.01 p=NS p<0.01 p<=0.05 p<=0.01
– – p=NS p<0.005 – p=NS – – – p=NS – p=0.03 p<=0.025 p<=0.005
3.2.1. Rotarod assessment of sensorimotor deficits The reduction in average time spent on the rotarod by the MCAO animals when compared with the sham operated animals on Day 1 was highly statistically significant (average difference =106.5; p-value=0.0056) indicating that ischaemia had induced a marked deficit in motor co-ordination and balance. There was no evidence of a difference in time spent on the rotarod between the two groups on day 7 (average difference =34.7; p-value =0.1595). This was due to an increase in
– – p=NS p<0.005 – p=NS – – – p<0.025 – p=NS p<=0.025 p<=0.005
time spent on the rotarod by the MCAO group, demonstrating recovery of performance on this task with time. The data are shown graphically in Fig. 5. 3.2.2. Sticky label test Data for the sticky label tests are shown in Tables 4 and 5. The analysis of time to removal of the tape on the right, contralateral, side showed that no differences existed on baseline testing (day 0) between the sham and MCAO groups over the 300 s (p-value =0.3). However
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Table 3 Summary of primary screening results (cont). Data is shown as median (± quartile range). No statistical analysis performed as data for all three groups was identical. n=11 MCAO, 10 sham and 10 control mice
Tremor Abnor behaviour Skin colour Heart rate Abdominal tone Lacrimation Salivation Fear Irritability Aggression
Unoperated
Sham operated
MCAO
0.00 (0.0) 0.00 (0.0)
0.00 (0.0) 0.00 (0.0)
0.00 (0.0) 0.00 (0.0)
1.00 1.00 1.00 0.00 1.00 0.00 0.00 0.00
1.00 1.00 1.00 0.00 1.00 0.00 0.00 0.00
1.00 1.00 1.00 0.00 1.00 0.00 0.00 0.00
(0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0)
(0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0)
(0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0)
Fig. 1. Effect of pMCAO in mice on performance on the rotarod. Data is shown as mean total time spent on rotarod for each treatment group ± SEM. Data was analysed using two way analysis of variance with treatment and day as independent variables. * p<0.05 vs unoperated controls and sham operated animals.
on day 1, there was evidence of a significant difference between the sham and MCAO groups over the 300 s — with MCAO animals taking longer to remove the tape from the right side although many animals failed to remove the tape at all (p-value =0.05). On day 7, the MCAO rats took significantly longer to remove the tape than the sham group (p-value =0.0005; Table 4). The time to contact was much shorter than the time to removal at baseline and an analysis of the time to contact the right hand tape using the logrank tests and median times to contact again showed no significant difference between the sham and MCAO groups on day 0 (p=0.8). However the MCAO treated animals took significantly longer to make contact with the right side tape on days 1 and 7 (p=0.0005; Table 4).
MCAO and sham rats did not differ on day 0 in the median time to removal of the tape on the left, ipsilateral, side (p=0.4). However the logrank tests of the median times to removal indicated that the MCAO treated animals took longer to remove the tape on day 1 (p=0.05) and day 7 (p=0.003; Table 5). An analysis of time to contact the left side using the logrank tests and median times showed no significant difference between sham and MCAO groups on day 0 and also no statistically significant differences between the MCAO and sham groups on days 1 (p=0.8) and day 7 (p=0.06; Table 5).
4. Discussion Few studies have looked at sensorimotor function after MCAO in mice. Most of these have only used very simple neurological scores (Sheng et al. 1999, 1998; Connolly et al., 1996; Weisbrot-Lefkowitz et al., 1998; Yang et al., 1994). Clark et al. (1997) used three neurological function scales to assess outcome after varying times of MCAO in Swiss-Webster mice. These were a simple graded score of 1–5 (5 being dead), a global scale (activity, posture, presence or absence of fits) and a focal scale (symmetry, gait, climbing and sensory response). However none of the studies to date have carried out such a comprehensive assessment of function postMCAO in the mouse as that described in the present study. Here the use of SHIRPA has shown that mice with ischaemic damage caused by MCAO have a wide range of behavioural changes when compared to both sham and non-operated animals. Additionally, on some measures, differences were found between sham and non-operated animals indicating that these are susceptible to interference, regardless of the operative treatment, at least during the post-operative 24 h. However, these effects on sham-operated animals were always less marked than the effects seen in those animals which underwent the MCAO lesion. In the primary observational screen, the level of spontaneous activity was significantly decreased in sham operated animals when compared to the non-operated controls, indicating that they are quieter than non-operated animals when placed in a novel environment. The gait of sham operated animals was also reduced, with the animals having a noticable “flattening” of posture during walking. Alongside this, a marked reduction in tail elevation was also seen. It may be that these two behaviours are related. Mice with a flattened gait may be unable to hold their tail in its normal position and will therefore always show a lowering in tail position. Performance in the wire manoeuvre test was also reduced in sham operated animals. This test involves both balance, co-ordination and rear limb strength. 24 h post operation these measures are still markedly affected.
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Fig. 2. Effect of pMCAO in mice on locomotor activity. (a) Data is shown as mean number of beam breaks in 60 min for each treatment group ± SEM. Data was analysed using two way analysis of variance with treatment and day as independent variables. * p<0.05 vs unoperated controls and # p<0.05 vs sham operated animals. (b) Data is shown as number of beam breaks per 5 min time period over 60 min for each treatment group. SEM are not shown for clarity.
It may be that this reflects a “normal” post-operative malaise. Leaving the animals for a longer period of recovery may diminish this effect. A large number of differences between sham operated and MCAO animals were observed in the primary screen. Respiration rate in MCAO operated animals was reduced with animals taking slower, shallower breaths. MCAO operated animals also showed a lower level of general activity, as shown by a reduction in transfer arousal and locomotion. This is unlikely to reflect an effect of the operation as no effects on these measures were seen in sham operated animals. When the animals did move it was noticeable that the body posture of MCAO animals was markedly reduced. This is reflected in the scores for gait and pelvic elevation. MCAO mice
also showed a reduction in the strength of their grip in their front legs. The rear limbs show a reduction in strength represented by a reduction in the scores for limb tone which was apparent on both the ispsilateral and contralateral sides. Neither the operation itself, as represented by sham animals, nor the MCAO lesion appear to have effects on autonomic responses measured within this test. Thus skin colour (which reflects blood supply to the skin), heart rate, lacrimation and other measures all showed no difference compared to unoperated animals. Differences were observed in other tests between the three groups of mice. For example in the rotarod, a test of balance and coordination, MCAO operated animals perform significantly worse than either sham or non-
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Table 4 Median time to removal and contact for the right hand tape (contralateral to the lesion). n=6 per group Day
Group
Median contact time (seconds)
Median removal time (seconds)
0 0 1
Sham MCAO Sham
3.5 (3.3–4.8) 5.7 (3.3–7.6) 4.0 (3.3–5.5)
1 7 7
MCAO Sham MCAO
50% failed c 5.0 (3.6–7.6) 66.9 (50.6–92.0)c
49.0 (34.0–62.6) 41.9 (38.0–45.1) 67.8 (51.1–50% failed) 50% failed a 52.9 (48.5–54.8) 50% failed b
a b
Fig. 3. Effect of MCAO in CD1 mice on total number of transits. Data is shown as mean number of beam breaks in 60 min for each treatment group ± SEM. Data was analysed using two way analysis of variance with treatment and day as independent variables. * p<0.05 vs unoperated controls and # p<0.05 vs sham operated animals.
c
p=0.05 MCAO vs sham. p=0.003 MCAO vs sham. p=0.0005 MCAO vs sham
Fig. 4. The effect of MCAO in the rat on 21 point neurological score. Significant differences are seen at all time points between the sham and MCAO operated animals as assessed by Mann-Whitney U test (# = p<0.05).
Fig. 5. The effect of MCAO in the rat on rotarod performance. Data shown are mean and standard error. Data was analysed using two way analysis of variance with treatment and day as independent variables. *p=0.005 ischaemic vs sham on day 1.
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Table 5 Median time to removal and contact for the left hand tape (ipsilateral to the lesion). n=6 per group Day
Group
Median contact time (seconds)
Median removal time (seconds)
0 0 1
Sham MCAO Sham
2.2 (1.6--3.0) 2.6(1.6--5.5) 4.0 (2.6--6.8)
1 7 7
MCAO Sham MCAO
28.2 (17.1--53.6) 2.7 (1.8--6.3) 7.7 (5.8--11.6)
39.0 (29.3--56.5) 35.0 (32.8--39.1) 52.6 (46.8--50% failed) 50% failed a 54.7 (41.1--63.8) 87.1 (76.6--112.8)b
a b
p=0.05. p=0.003.
operated animals. These results are in agreement with the data obtained in the behavioural observation where a reduction in limb tone, a reduced performance in the wire manoeuvre test and a worsening gait all suggested general reductions in motor function. The rotarod has been well validated as a means of assessing deficits in motor performance in rats (Borlongan et al., 1995; Rogers et al., 1997b; Yamamoto et al., 1988 and the present study) but has not previously been used with mice after MCAO. Interestingly Yamamoto et al. (1988) also found that sham operated rats were not impaired to control unoperated rats. In the LMA test, MCAO operated mice showed a significant reduction in activity over non-operated controls. Interestingly, in this test, sham operated mice were also significantly worse than non-operated animals paralleling the data obtained with the acute observation test. This suggests that the relatively invasive surgical procedure involving the carotid vasculature which occurs in both sham and MCAO operations, is sufficient in itself to alter locomotor activity 24 h post-MCAO. These data on LMA in mice are different from most studies involving MCAO in the rat where no effects on LMA have been observed (Yonemori et al., 1998). Borlongan et al. (1995) did see some alterations in nocturnal LMA with ischaemia and a potentiation of the hyperactivity induced by amphetamine. Interestingly Borlongan et al. (1995) did not observe any differences in LMA between the sham and MCAO groups in the hour prior to amphetamine administration. Borlongan et al. (1995) also tested the animals at one month post-ischaemia rather than 24 h after MCAO as in the present study. These data show the importance of including an unoperated control group for measurements such as LMA, although this is not necessary for others such as the rotarod where the sham animals were no different from control, unoperated animals. The experiments described in the present paper show that the SHIRPA protocol described here can be used acutely in mice to assess sensory and motor deficits after stroke, much as the NIH scale is used in man.
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The data from the rat transient MCAO study also showed deficits in the rotarod at 24 h post-ischaemia. However these deficits had essentially disappeared by 7 days post-ischaemia. This is in contrast to data obtained by Yamamoto et al. (1988) who looked at rotarod performance after permanent proximal MCAO and found that animals still had significant impairments at 2 weeks post occlusion although recovery was observed from 3 weeks post ischaemia. This highlights the importance of defining the time course of recovery for any functional tests in a particular model prior to undertaking any therapeutic intervention studies. The relatively rapid recovery of performance on the rotarod suggests that it could be useful in studies of the effects of interventions which would promote regeneration and repair. The neurological grade scores showed little evidence of recovery although by day 7, the data seemed to suggest some improvement and use of this scale beyond 7 days is warranted. The bilateral sticky label test showed a prolonged deficit on the contralateral side with some recovery evident ipsilaterally suggesting that this will be a more severe test of regenerative/repair therapies. This test was first developed by Schallert et al. (1982) and has similarities to the contralateral neglect syndrome which is seen in man after damage to the parietal cortex (Heilman et al., 1985). The impairments observed here were highly significant on the contralateral side with no evidence of recovery by 7 days. Other work in our laboratory has demonstrated a close correlation of contralateral contact and removal latencies on this task with pathological changes in the ipsilateral caudate putamen, lower parietal cortex and forelimb cortex following 90 min of tMCAO as depicted by MRI. Each of these regions of interest contributed to functional impairments on this task across an extended time course (up to 28 days post-ischaemia) (Virley et al., 1999a). In addition substantia nigra damage as evidenced by apparent diffusion coefficient (ADC) changes and T2 relaxation times at 7, 14 and 28 days post MCAO also correlated with contralateral deficits on this task (Virley et al., 1999b). Thus this task may be suitable for assessing both neuroprotective therapies which target early intervention as well as those aimed at the prevention of delayed damage and therapies which promote regeneration. In conclusion, use of the SHIRPA protocol in MCAO, sham and non-operated mice reveals a range of functional differences which may allow for the behavioural assessment of ischaemic damage in mice. In these tests animals were tested 24 h post lesion. Studies in mice with SHIRPA are now ongoing to determine whether any of these deficits recover with time and whether drug or transgenic manipulations can alter the ischaemiainduced changes. The importance of the former is clearly demonstrated by the recovery seen in the rotarod task, but not the neurological scores or sticky label test, in the rat MCAO. However it must be kept in mind that
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differences in pathology between permanent and transient models may also contribute to different rates of recovery and further studies are needed to investigate this.
Acknowledgements The authors would like to thank Susan Spencer for help in typing the manuscript, Jenny Roberts for cutting the brain sections and Helen Tate and Phil Overend for additional statistical support.
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