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(Stroke. 1996;27:2282-2286.)
© 1996 American Heart Association, Inc.

Articles

Tacrolimus (FK506) Ameliorates Skilled Motor Deficits Produced by Middle Cerebral Artery Occlusion in Rats

John Sharkey, PhD; Jane H. Crawford, BSc; Steven P. Butcher, PhD Hugh M. Marston, PhD

the Fujisawa Institute of Neuroscience (J.S., S.P.B., H.M.M.) and the Department of Pharmacology (J.H.C.), University of Edinburgh (UK).

Correspondence to Dr John Sharkey, Fujisawa Institute of Neuroscience, University of Edinburgh, Appleton Tower, Level 6, Crichton St, Edinburgh, EH8 9LE, UK. E-mail j.sharkey@ed.ac.uk.

	Abstract

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Background and Purpose Tacrolimus (FK506) is a potent immunosuppressant that is presently in clinical use for prevention of allograft rejection. Recently, animal studies reporting significant reductions in the volume of tissue damage associated with cardiac, hepatic, and cerebral ischemia suggest that tacrolimus may also be of use in the clinical management of stroke. In the present study, we examine whether the neuroprotective effects of tacrolimus, as assessed by histological outcome, are accompanied by an amelioration of the skilled motor deficits induced in the rat by middle cerebral artery occlusion (MCAO).

Methods Animals were trained to perform a skilled paw-reaching task before MCAO by perivascular microinjections of endothelin-1. Tacrolimus (1 mg/kg, n=6) or vehicle (n=6) was administered by intravenous infusion 1 minute after MCAO. After a 5-day postoperative recovery period, the rats were retested for skilled paw-reaching ability for an additional 9 days.

Results In vehicle-treated rats, MCAO resulted in a profound bilateral impairment in skilled paw use. Rats treated with tacrolimus, although still impaired, performed significantly better than those treated with vehicle alone (P<.01). Histological analysis, 14 days after occlusion, confirmed the neuroprotective efficacy of tacrolimus with a 66% reduction in the volume of hemispheric brain damage produced by MCAO (P<.01).

Conclusions The present studies show that tacrolimus not only protects neural tissue from focal cerebral ischemia but also significantly ameliorates the deficits in skilled motor ability produced by this lesion. These data provide further support for the view that tacrolimus may be of use in the treatment of stroke.

Key Words: behavior, animal • cerebral ischemia, focal • middle cerebral artery occlusion • neuroprotection • rats

	Introduction

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Tacrolimus (FK506), a macrolide with potent immunosuppressant properties, is presently used in clinical practice for prevention of allograft rejection.^{1 2 3} Recent studies suggest that tacrolimus may also be of use in the treatment of stroke,^{4 5 6} as supported by evidence of marked neuroprotection observed against excitotoxic neuronal death in cell culture systems^{6 7} and ischemic brain damage in animal models of focal cerebral ischemia.^{4 5} This neuroprotection is putatively mediated via the inhibition of the calcium/calmodulin-dependent protein phosphatase calcineurin by a complex of tacrolimus and the 12-kD immunophilin FKBP12.⁴ However, while progress is being made in identifying the molecular substrates for the neuroprotective actions of tacrolimus, it is presently unclear whether the tacrolimus-mediated reduction in histologically defined brain damage is commensurate with an improvement in neurological outcome.

In humans, middle cerebral artery occlusion (MCAO), which accounts for a significant proportion of strokes, results in sensory loss, motor weakness, and disturbances in either language or spatial perception. To date, therapeutic strategies that significantly improve these neurological deficits have still to be reported. Preclinically, rat models of focal cerebral ischemia have been widely used to assess the efficacy of putative neuroprotective agents.^{8 9} These studies have generally focused on the pathophysiology of the insult, using a histopathological end point to assess neuroprotective efficacy. A few studies have sought to correlate the extent of ischemic damage with neurological outcome^{10 11 12 13 14} but with limited success. The principal limitation in using neurological assessment to evaluate neuroprotection in rodents appears to be the lack of a stable postocclusion behavioral deficit. While MCAO in rats produces deficits in sensorimotor and cognitive function, many of the simpler behavioral tasks (motor coordination and somatosensory function) exhibit spontaneous recovery.^{10 11 13} However, deficits in more complex cognitive or skilled motor tasks appear to be more stable and may persist for several months after occlusion.^{10 11 13}

The staircase task provides a quantitative measurement of skilled paw use, requiring the rat to exert precise motor control over each paw in order to grasp and retrieve reward pellets.¹⁵ With use of this paradigm, a significant correlation between the performance deficit and infarct size after surgical MCAO in rat has been demonstrated.¹⁰ The stability of the deficit, which persisted throughout the 3-month testing period, suggests that this task may be a useful method for evaluating the functional recovery afforded by neuroprotective agents. In the present study, we examined whether tacrolimus can ameliorate the skilled motor deficits associated with MCAO using a modified form of the staircase task.¹⁶

	Materials and Methods

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Studies were performed on 26 adult (230 to 260 g) male Lister-hooded rats (Charles River Laboratories). This pigmented strain of rat was chosen in preference to an albino strain because of its better visual acuity and speed in learning complex tasks.¹⁷ Water was available ad libitum, but food was restricted so that animals gained weight at a rate of 3 to 5 g per week. All studies were performed in vivo in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986. Because there are no alternative methods for the in vitro study of integrated brain function, every effort was made to minimize animal suffering and to reduce the number of animals used.

Rats were trained to perform a modified version of the staircase test.¹⁶ Animals were introduced into a Perspex box with aluminum staircases, each with six steps, positioned on either side of a central plinth. The internal dimensions of the apparatus were arranged so that forelimb access to the contralateral staircase was impossible. The task required recovery of as many food reinforcement pellets as possible (2 per step) within the 5-minute test period. Performance was scored in terms of the number of pellets recovered or displaced from the steps. Rats received two training sessions per day, a minimum of 4 hours apart. Once asymptotic performance was obtained, rats were allocated to one of two study groups. In the first, 8 rats received either tacrolimus (1 mg/kg IV) or vehicle (1% ethanol in 400 mg/mL polyoxyl 60 hydrogenated castor oil: 1 mL/kg) to examine whether the drug possessed intrinsic efficacy on skilled motor performance. A single dose of tacrolimus was chosen because previous neuroprotection studies have indicated the absence of a classic dose-response relationship, with doses of tacrolimus above its neuroprotective threshold giving a similar degree of neuroprotection.^{4 18} The dose of tacrolimus used in the present study (1 mg/kg) has been shown to be consistently neuroprotective in this and other animal models of cerebral ischemia.^{4 5 18} The second group of 18 animals was randomly allocated to treatment groups receiving either sham operation, MCAO plus tacrolimus, or MCAO plus vehicle.

Rats were anesthetized (1.5% halothane in 70% nitrous oxide-30% oxygen) to permit the stereotaxic placement of a 31-gauge injection needle into the piriform cortex approximately 0.5 mm dorsal to the MCA (0.2 mm anterior and 5.9 mm lateral to bregma and 7.5 mm below the dura). Body (core) temperature was maintained at 37±1°C throughout using a thermostatically controlled heating blanket. Endothelin-1 (60 pmol; human, porcine: Novabiochem) was dissolved in 3 µL of saline and injected through a cannula over a 2-minute infusion period. Rats received an intravenous infusion of tacrolimus (1 mg/kg, n=6) or its vehicle (n=6) 1 minute after the injection of endothelin-1. The intracranial infusion cannula was left in situ for a further 5 minutes before being slowly withdrawn. The cranium was then sealed with bone wax, the wound was sutured, and the rat was placed in an incubator where normothermia was maintained until the animal had fully recovered from anesthesia.

Rats were allowed 5 days to recover, during which time a mild food-deprivation regimen was reinstated. Postoperative testing in the staircase task was then continued for a further 9 days. For statistical analysis, paw-reaching data from each rat were collapsed into four blocks of six trials; the last 3 days of preoperative testing formed the first block, and the 9 days of postoperative testing formed the second, third, and fourth blocks (threex3 days). Data were analyzed for each treatment group using ANOVA with post hoc Tukey's protected t test, using the preoperative block data as control. On completion of the assessment period, the rats were reanesthetized (60 mg/kg pentobarbital IP) and perfusion-fixed by intracardiac injection of heparinized saline (20 mL of 10 U/mL) followed by paraformaldehyde (200 mL of 4% in PBS). Brains were removed intact and immersed in fixative containing 10% sucrose before cryostat sectioning and histological staining (cresyl violet). Sections were examined using a light microscope with the extent of the infarction at eight predetermined levels mapped onto enlarged diagrams. The volume of infarction was then calculated by integrating the cross-sectional area of damage at each stereotaxic level and the distances between the various levels.^{4 5}

	Results

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All animals tested reached the minimum preoperative performance criterion (9 pellets recovered per side over 3 successive days [ie, 6 training sessions]). Throughout the training period, the number of pellets displaced remained constant (approximately 2 per side per session), and there was no significant limb preference observed (P>.05). In the first group of unoperated animals, neither tacrolimus- nor vehicle-alone treatment significantly altered pellet recovery or displacement (data not shown). After surgery, both the sham-lesioned and MCAO rats exhibited weight loss. However, in each case, preoperative body weight was regained within 4 days of surgery. MCAO resulted in a profound and lasting impairment in the ability of the rats to recover food reinforcement pellets during the paw-reaching task (Fig 1). ANOVA of pellet recovery data on the MCAO/vehicle-treated rats revealed a treatment by block interaction (F_(2,984)=245.5, P<.0001). Restricted analyses confirmed that MCAO/vehicle rats were profoundly bilaterally impaired (t=17.78, P<.01). The MCAO/tacrolimus group, although significantly impaired compared with sham controls (t=9.90, P<.01), performed better than those treated with vehicle (t=-13.50, P<.01). In contrast, the performance of sham-operated rats did not differ significantly from their preoperative baseline values (P>.05; Fig 1). Analysis of pellet displacement data also yielded a significant treatment by block interaction (F_(2.984)=10.48, P<.01). Post hoc investigation established that this was due to an increase in the number of pellets displaced by the MCAO/vehicle group (F=4.87, P<.01). Further analysis confirmed that the increase in pellets displaced in the MCAO/vehicle group was due to a rise in the number of pellets displaced postoperatively (t=-12.70, P<.01). The MCAO/tacrolimus group was indistinguishable from the sham-MCAO group (P>.05). Significant changes in performance across the postoperative period were not observed in any group.

View larger version (30K): [in this window] [in a new window]   Figure 1. Effect of tacrolimus (1 mg/kg administered IV 1 minute after MCAO) on the number of pellets recovered (a, b) or displaced (c, d) during the staircase task before and after endothelin-induced MCAO. Contralateral (a, c) and ipsilateral (b, d) refer to the forelimbs with respect to the lesioned hemisphere. Each data point represents the mean±SEM of 6 sessions (3 days) for 6 rats. In sham-MCAO rats, postoperative performance (Post-1, -2, and -3) was not significantly different from preoperative levels (Pre-1). *P<.05, comparison between sham-operated and MCAO rats; P<.05, comparison between groups treated with tacrolimus plus MCAO and MCAO plus vehicle.

Endothelin-induced MCAO resulted in the characteristic pattern of cortical and striatal infarction within the vascular territory of the MCA as reported previously.^{4 18} In vehicle-treated rats, damage to cortex was evident at each of the 8 brain levels examined (Fig 2). Rostrally, cortical cell death was consistently observed from the lateral parts of frontal cortex to piriform cortex. Caudally, the extent of cortical damage was more variable but could be traced through temporal cortex at the level of the substantia nigra to occipital cortex at the level of the inferior colliculus. The volume of cortical damage was 148±21 mm³ (40% of contralateral cortical volume). In addition, MCAO/vehicle-treated rats had extensive damage throughout the striatum (36±3 mm³; 63% of contralateral striatal volume). Tacrolimus administration was associated with significant reductions in cortical damage at 7 of the 8 brain levels analyzed (Fig 2). Surprisingly, tacrolimus also produced a significant reduction in the volume of striatal damage (Fig 2). Volumetric analysis revealed a 66% reduction (P<.01) in the volume of hemispheric brain damage in tacrolimus-treated animals. The volume of brain damage was reduced by 69% (P<.01) to 45±18 mm³ in the cortex and by 55% (P<.01) to 16±4 mm³ in the striatum of tacrolimus-treated rats.

View larger version (15K): [in this window] [in a new window]   Figure 2. Effects of tacrolimus (1 mg/kg) on the area of ischemic damage in cortex and striatum when administered 1 minute after endothelin-induced MCAO. Data are presented as mean±SEM area (square millimeters) of brain damage at 8 predetermined stereotaxic levels for 6 rats per treatment group. *P<.05, Student's t test.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
The present data clearly demonstrate that tacrolimus not only reduces the volume of infarction associated with MCAO but also significantly ameliorates the deficit in skilled paw use produced by this insult. To our knowledge, this is the first study to show a drug-related improvement in the performance of an acquired motor skill after MCAO in the rat. MCAO in the present study was achieved by the intracerebral injection of endothelin, which reduces local blood flow within the vascular territory of the MCA by up to 93%¹⁹ and produces a pattern of tissue damage similar to that observed after surgical occlusion of the vessel.^{4 5} This model was chosen in preference to surgical occlusion of the vessel because the damage to facial structures (eg, unilateral enucleation or mechanical damage to vibrissae²⁰ ) incurred in exposing and occluding the rat MCA by the latter approach may result in unilateral disruption of primary sensory systems, thereby confounding neurological assessment of brain damage. Similarly, feeding difficulties associated with facial damage would be anticipated to compromise performance in behavioral tasks using food pellets as the reinforcement. In contrast, animals undergoing endothelin-induced MCAO do not exhibit feeding difficulties, and because the procedure requires only a simple incision to the dorsal scalp to permit craniotomy, animals are unlikely to suffer from sensory perturbations attributable to facial damage.

There is increasing evidence that the choice of rat strain can have a significant effect on the pattern and magnitude of ischemic damage observed after MCAO.^{16 20 21} Although the rat strain in the present study (Lister-hooded) differed from that used in our previous studies (Sprague-Dawley),^{4 5} the unilateral patterns of brain damage produced by endothelin-induced MCAO were similar. The magnitude of cortical protection afforded by tacrolimus was also similar in the two studies, whereas the degree of striatal protection in Lister-hooded rats was twice that found previously using Sprague-Dawley rats.⁴ Striatal protection of this magnitude is uncommon after MCAO in the rat⁸ and may be related to the strain of rat used. In rats, the caudal and dorsolateral aspects of the caudate putamen are supplied by the lenticulostriate branches of the MCA, the more rostral and medial components are supplied by the arteries of Heubner arising from the anterior cerebral artery, and other regions, particularly rostrally, are double fed by both major arteries.^{21 22} Such areas of double feeding may be more amenable to rescue by neuroprotective agents, and interstrain differences in the extent of striatal double-feed zones could therefore explain the discrepant data concerning striatal neuroprotection. While there have been no systematic studies of the cerebrovascular distribution patterns in Lister-hooded and Sprague-Dawley rats, Duverger and MacKenzie²⁰ have described marked differences in the incidence and topographical distribution of brain infarction produced by MCAO among several commonly used strains of rat, and strain differences in the efficacy of neuroprotective agents using the rat MCAO model have been noted.^{8 23}

The bilateral nature of the deficit in skilled paw use and the stability of this deficit over the postoperative testing period were similar to those reported previously.¹⁶ The stability of this deficit contrasts with the progressive recovery of spontaneous sensorimotor function after MCAO in rats,^{10 13} suggesting that the staircase task may be more appropriate for studying functional deficits after experimental stroke. The bilateral nature of the impairment requires further explanation because bilateral deficits are not commonly associated with unilateral lesions of neocortex or basal ganglia in humans.²⁴ A deficit of this sort could be the consequence of a general motivational impairment. However, in this case, this is unlikely since animals with MCAO are unimpaired in the performance of an operant delayed nonmatching-to-sample procedure (H.M.M., unpublished observations, 1995). Furthermore, although unilateral deficits in spontaneous sensorimotor function have been reported after MCAO in rats,^{10 13} it should be emphasized that the staircase task is not a measure of spontaneous motor ability. Indeed, 16 training sessions are required before asymptotic performance is reached,¹⁶ indicating that learning is an obligate requirement for optimal performance. This suggests a major reliance on competent neocortical function, and since unilateral striatal lesions produce a primarily unilateral (contralateral) paw-reaching deficit,¹⁶ the cortical damage induced by MCAO is likely to mediate the bilateral nature of the deficit. Because the cortical output pathways in rats, unlike humans, are not fully decussated,²⁵ the present data are entirely compatible with unilateral neocortical damage. Furthermore, bilateral impairments in learned motor tasks have been reported after unilateral lesions of the rat neocortex or nigrostriatal pathway.^{15 26 27} Although the bilateral impairment noted in the present study differs from the asymmetrical deficit observed after surgical MCAO in spontaneously hypertensive rats,¹⁰ this discrepancy may be due to differences in the extent of the subcortical lesion produced by the two studies. In the present study, we observed negligible damage outside the vascular territory of the MCA, whereas Grabowski et al¹⁰ reported damage extending beyond this vascular territory to include the amygdala and some 76% of the thalamus.

Previous studies have shown that, at immunosuppressant doses, tacrolimus is a potent neuroprotectant in animal models of focal⁴ and global¹⁸ cerebral ischemia. Although the mechanisms underlying the neuroprotective actions of tacrolimus remain unclear,^{4 5} we have shown that tacrolimus does not affect changes in arterial blood gases, plasma glucose levels, or core body temperature,⁴ nor does it interact directly with endothelin receptors (J.S. and S.P.B., unpublished data, 1996). Of the many possible intracellular candidates, the calcium/calmodulin-dependent phosphatase calcineurin has been highlighted as a possible mediator in the neuroprotective action of tacrolimus (for review see Reference 6). This view is supported by the finding that high doses of another immunosuppressant, cyclosporin, are neuroprotective in both the endothelin and filament models of MCAO^{5 28} . Tacrolimus and cyclosporin bind to specific immunophilins (tacrolimus to FKBP12 and cyclosporin to cyclophilin), and these drug-immunophilin complexes inhibit calcineurin. Although this mechanism is also proposed to mediate the immunosuppressant actions of these drugs, an immunosuppressive end point per se may not be a prerequisite for their neuroprotective efficacy. Rapamycin, a potent immunosuppressant that also binds to FKBP12 but does not inhibit calcineurin, attenuates tacrolimus-mediated neuroprotection.⁴ While the precise role of calcineurin in the pathophysiology of cerebral ischemia is unclear, a number of intracellular mechanisms have been proposed. Inhibition of free radical production may be implicated, since in animal models of myocardial infarction tacrolimus reduces ischemic damage by preventing superoxide free radical production in neutrophils²⁹ and blocks nitric oxide production in neuronal cell cultures.³⁰ Alternatively, recent evidence suggests that processes similar to apoptosis may contribute to neuronal death after ischemic insult.³¹ Although no direct evidence exists linking tacrolimus to ischemia-induced apoptosis, tacrolimus has been shown to inhibit apoptotic cell death via a calcineurin-dependent mechanism in T-cell hybridomas.³²

In summary, we have demonstrated that endothelin-1–induced MCAO results in a marked, bilateral deficit in a skilled paw-reaching task that is significantly reduced by a single intravenous injection of tacrolimus. These behavioral data provide further evidence in support of tacrolimus as a potential treatment for stroke.

	Acknowledgments

 
These studies were supported by grants from the Scottish Higher Education Funding Council and the Fujisawa Pharmaceutical Co Ltd. The authors would like to thank J. Brown and A.L. Baird for their technical assistance.

Received June 3, 1996; revision received August 8, 1996; accepted September 5, 1996.

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Editorial Comment

Ronald L. Hayes, PhD, Guest Editor

Department of Neurosurgery Research Labs, University of Texas Health Science Center, Houston, Tex

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
The investigators have provided extremely interesting data on the neuroprotective effects of the immunosuppressant tacrolimus after MCAO. A particular strength of the data is the demonstration that the drug not only reduces the volume of infarct after ischemia but also significantly reduces the deficit in skilled paw use produced by this injury. The previous observation of strain differences in drug protection is straightforwardly addressed by the authors. The research is especially current in view of a rapidly evolving body of literature suggesting that immune/inflammatory responses may importantly modulate the pathological consequences of a number of acute central nervous system insults, including ischemia, spinal cord injury, and traumatic brain injury. The authors suggest that immunosuppression per se may not be a prerequisite for the neuroprotective effects of tacrolimus and related compounds. However, a more detailed understanding of mechanisms of neuroprotection by tacrolimus should yield important insight into relationships between immune/inflammatory responses and necrotic versus apoptotic cell death after ischemic injury. In any case, the current data suggest that ischemic protection by tacrolimus may be a robust model in which to pursue such studies.

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