Background and Purpose The aim was to determine whether a delayed transfer to an enriched environment improves outcome after focal brain ischemia.
Methods Performance on a rotating pole, prehensile traction, limb placement, and postural reflexes were tested in 15 spontaneously hypertensive rats housed in standard laboratory cages for 2 weeks after middle cerebral artery ligation. Seven of the 15 rats were then transferred to an enriched environment, and the two groups were tested 1, 3, and 5 weeks later.
Results The enriched environment significantly improved pole performance, prehensile traction, and limb placement.
Conclusions Delayed postoperative environmental enrichment improves outcome in experimental stroke.
In agreement with studies of other types of brain injury,1 housing rats in an enriched environment can enhance functional outcome after focal brain ischemia.2 3 The aim of the present study was to establish whether significant improvement could be obtained if the transfer to an enriched environment was delayed for 15 days after occlusion of the middle cerebral artery.
Materials and Methods
The experimental protocol was approved by the local ethics committee for animal research. Four-month-old male spontaneously hypertensive rats with a body weight of 299±17 g were anesthetized with methohexital 50 mg/kg IP. The right middle cerebral artery (MCA) was ligated distal to the origin of the striatal branches.4
Before and for 2 weeks after MCA ligation, the rats were housed together in standard laboratory cages (550×350×200 mm), with 5 rats in each cage. Fifteen days after the arterial occlusion and after tests were performed as described below, 7 of the rats were transferred to a larger cage (815×610×450 mm) with opportunities for various activities, while the other 8 rats remained in standard cages (4 rats in each). Two horizontal boards, 70 mm wide, were placed along one of the sides 150 mm above the floor. One board connected the floor with the elevated boards and, at a higher level still, one board was put across a corner. A chain, a swing, swing boards, and wooden blocks were placed in the cage, and once a week new objects were added and others withdrawn.2 3
The ability to traverse a rotating pole,2 prehensile traction,5 postural reflexes,6 and limb placement2 7 were tested 2, 3, 5, and 7 weeks after the operation, ie, once before and three times after the housing conditions for one of the groups were altered.
In the rotating pole test, the pole, 45 mm in diameter and 1.5 m in length, rotated alternately to the left or the right with 3 or 10 turns per minute. The performance was scored from 0 to 6 (0, the rat drops off; 6, the rat crosses the beam with no foot slips).2
The prehensile traction test5 evaluated the muscle strength and equilibrium when the rat’s forepaws were placed on a rope (scored as follows: 0, hangs on 0 to 2 seconds; 1, hangs on 3 to 4 seconds; 2, hangs on 5 seconds, no third limb up to rope; and 3, hangs on 5 seconds and brings rear limb up to rope). Postural reflexes were scored from 0 to 3 as described by Bedersen et al,6 with the highest score for the most severe deficit, in contrast to the other test in which a high score signifies a good performance. The forelimb and hindlimb placements were evaluated with a slightly modified version2 of the test described by De Ryck et al.7 For each body side, the maximum score was 16.
The tests chosen are simple tests that can be performed with optimal or close to optimal scores without training in normal nonischemic rats. Thus, in an unpublished study 20 intact 4-month-old rats all reached the optimal score of 16 in the leg placement test and the optimal score of 6 on a pole rotating at 3 or 10 turns per minute. In the prehensile traction test the score was 2.5 (optimal score, 3).
Seven weeks after MCA occlusion, the rats were anesthetized with methohexital and decapitated. The brains were removed and frozen in isopentane chilled to −40°C and stored at −80°C until sectioned; 20-mm-thick coronal brain sections were cut in a cryostat at −20°C. Fifteen sections at 900-μm intervals were stained with hematoxylin and eosin for determination of infarct volume. The image-analyzing system and software were the same as earlier described.2 3 Since the infarct was transformed into a cystic cavity 7 weeks after the MCA ligation, infarct volume was calculated as percentage of contralateral hemisphere and percentage of contralateral cortex. Thalamic atrophy was calculated as the contralateral thalamus minus the thalamus on the operated side, determined on 15 sections 300 μm apart.
For the behavioral tests based on scoring systems (ordinal measures), the Kruskal-Wallis nonparametric ANOVA was used with Dunn’s post hoc procedure to analyze group differences and changes over time at 95% probability level. One-way parametric ANOVA with Scheffé’s post hoc procedure was applied to evaluate differences in infarct and thalamic volumes. Values are given as mean±SD.
The body weight at the end of the experiment was 339±26 g in control rats and 345±14 g in rats transferred to an enriched environment.
The ability to traverse a pole was significantly better in rats housed in an enriched environment and improved significantly with time only in those rats (Table 1⇓). They also performed significantly better than rats in standard cages in the prehensile traction test (Table 2⇓). The scores in the leg placement test increased from 5.5±2.8 to 8.5±3.3 in the standard environment group and from 5.8±3.0 to 11.6±3.0 in the enriched environment group; the difference was significant at the 95% probability level. On the right intact side the scores were 15.6±0.7 and 15.3±1.0. The postural reflex test did not change with time for any of the groups (0.8±0.5 and 0.9±0.4 at 2 weeks and 0.9±0.8 and 0.9±0.4 at 7 weeks after the operation in control rats and in rats transferred to an enriched environment, respectively).
The cortical infarct volume was 26.0±6.3% of contralateral cortex in rats in standard cages and 27.8±11.9% in rats housed in an enriched environment; in percentage of total contralateral hemispheric volumes, the corresponding values were 14.4±5.0% and 16.5±6.1%. The corresponding reductions of thalamic volume were 17.5±4.8% and 20.8±5.4%.
The present study shows that an enriched environment, even if delayed for 15 days after the ischemic event, can significantly improve outcome after an experimental focal brain infarction. Whether it is as good as early transfer to an enriched environment is difficult to say because the protocol was not the same as in earlier published studies on early postoperative enrichment.2 3 In one study on the effect of preoperative and postoperative enrichment, the MCA was ligated proximal to the striatal branches, resulting in more severe deficits and larger infarcts than after the distal ligation used in the present study, and the control rats were housed in individual cages and thus socially deprived compared with the present control rats.2 However, in a study on the effect of an enriched environment on neocortical grafting after distal ligation of the MCA, the performance of control nontransplanted rats housed in an enriched environment was similar to that obtained with a delayed transfer in this study.3 It is likely that there is a time limit for optimal functional recovery, and studies comparing rats exposed to an enriched environment early and late after an ischemic insult are needed to evaluate the time limits for optimal improvement.
After unilateral frontal cortical ablation, adult rats prefer to use the limb on the ipsilateral side.8 Unilateral damage to the forelimb representation area of the sensorimotor cortex in adult rats has been reported to increase dendritic arborization of layer V pyramidal neurons of the contralateral homotopic cortex, with the largest increase observed 18 days after the lesion.9 Interestingly, restriction of the movement of the intact limb blocked the dendritic growth in the contralateral cortex and aggravated the sensorimotor impairment.10
Mechanisms similar to those responsible for maturation and learning have been proposed to account for functional improvement after brain lesions.11 12 13 14 15 16 Enriched housing conditions can influence dendritic branching, protein content, synthesis of transmitters, and trophic factors.11 15 Even though it may be difficult to separate compensatory mechanisms from recovery, attempts to stimulate brain plasticity are likely to be beneficial for functional outcome after brain injury. Plasticity can be expressed by changes in individual neurons11 or by modulation of cortical maps,12 15 which can occur very rapidly in learning tasks.17 Astrocytes may also play a role in brain plasticity.14 15
Studies in humans indicate that a substantial reorganization can take place after subcortical infarctions,18 but little is known about cortical infarctions. Interpretation of changes in cortical representation maps is not always easy, and studies comparing individuals with good and less good outcomes are needed. To what extent physiotherapy and other rehabilitation procedures can enhance or accelerate functional recovery after stroke is controversial. Studies on animals housed in different environments after an ischemic insult may offer an opportunity to clarify the mechanisms behind functional improvement and help us to find ways to also stimulate compensatory and adaptive brain mechanisms in humans.
This study was supported by grants from the Swedish Medical Research Council (project 14X-4968), the Bank of Sweden Tercentenary Foundation, and King Gustaf V and Queen Victoria Foundation. I thank Bengt Mattsson and Anna-Lena Ohlsson for skillful technical assistance and Dr Håkan Widner for statistical advice.
- Received July 3, 1995.
- Revision received September 22, 1995.
- Accepted October 23, 1995.
- Copyright © 1996 by American Heart Association
Will B, Kelche C. Environmental approaches to recovery of function from brain damage: a review of animal studies (1981-1991). In: Rose FD, Johnson DA, eds. Recovery From Brain Damage. New York, NY: Plenum Press; 1992:79-103.
Ohlsson A-L, Johansson BB. The environment influences functional outcome of cerebral infarction in rats. Stroke. 1995;26:644-649.
Coyle P. Middle cerebral artery occlusion in the young rat. Stroke. 1982;13:855-859.
Combs DJ, D’Alecy LG. Motor performance in rats exposed to severe forebrain ischemia: effect of fasting and 1,3-butanediol. Stroke. 1987;18:503-511.
Bedersen JB, Pitts LH, Tsuji M, Nichimura MC, Davis RL, Bartkowski H. Rat middle cerebral artery occlusion: evaluation of the model and development of neurologic examination. Stroke. 1986;17:472-476.
De Ryck M, Van Reempts J, Borgers M, Wauquier A, Janssen AJ. Photochemical stroke model: flunarizine prevents sensorimotor deficits after neocortical infarcts in rats. Stroke. 1989;20:1383-1390.
Jones TA, Schallert T. Use-dependent growth of pyramidal neurons after neocortical damage. J Neurosci. 1994;14:2140-2152.
Merzenich MM, Recanzone G, Jenkins WM, Allard TT, Nudo RJ. Cortical representational plasticity. In: Rakic P, Singer W, eds. Neurobiology of Neocortex. Berlin, Germany: Wiley; 1988:42-67.
Pons TP, Garraghty PE, Michkin M. Lesion-induced plasticity in the second somato-sensory cortex of adult macaques. Proc Natl Acad Sci U S A. 1988;85:5279-5281.
Johansson BB. Functional recovery after brain infarction: a review of experimental animal data. Cerebrovasc Dis. 1995;5:278-281.
Pascual-Leone A, Grafman J, Hallett M. Modulation of cortical motor output maps during development of implicit and explicit knowledge. Science. 1994;263:1287-1289.
Weiller C, Ramsay SC, Wise RJS, Friston KJ, Frackowiak RSJ. Individual patterns of functional reorganization in the human cerebral cortex after capsular infarction. Ann Neurol. 1993;133:181-189.