(Stroke. 1996;27:2282-2286.)
© 1996 American Heart Association, Inc.
Articles |
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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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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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 outcome10 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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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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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.
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| Discussion |
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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 MCAO5 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-1induced 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 |
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Received June 3, 1996; revision received August 8, 1996; accepted September 5, 1996.
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Department of Neurosurgery Research Labs, University of Texas Health Science Center, Houston, Tex
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