Environment Influences Functional Outcome of Cerebral Infarction in Rats
Background and Purpose The purpose of this study was to determine whether preoperative and postoperative enrichment of the environment can enhance the functional outcome after cerebral infarction in rats.
Methods The right middle cerebral artery was ligated in adult spontaneously hypertensive male rats, and the functional outcome was studied for 12 weeks after the operation. Three groups were compared: A, rats kept in individual cages before and after the operation (n=9); B, rats kept in individual cages before the operation but transferred to an enriched environment after the operation (n=10); and C, rats kept in an enriched environment all the time (n=12). The enriched environment consisted of a large cage with opportunities for various activities, but rats were not forced to do any particular tasks.
Results Rats kept in an enriched environment (groups B and C) performed significantly better than rats in group A in a leg-placement test, beam walking, walking on a rotating pole, and climbing. The infarct size and thalamic atrophy did not differ among the groups.
Conclusions The laboratory environment is important for the functional outcome in brain ischemia. We hypothesize that an enriched environment may stimulate mechanisms that enhance brain plasticity after focal brain ischemia.
Significant functional improvement occurs in most stroke survivors during the initial months after the stroke event.1 2 The extent to which physiotherapy and other rehabilitation procedures can enhance long-term functional outcome is debated.3 4 5 6 Although there is substantial evidence that the postoperative environment can influence the outcome after experimental brain damage such as traumatic brain lesions, hippocampal sectioning, and cortical ablation,7 little attention has been given to the possible role of environmental factors in the long-term functional outcome after experimental brain infarction.
In view of the potential usefulness of an experimental stroke rehabilitation model, the present study addresses whether preoperative and postoperative environment can influence the outcome after focal brain ischemia. Rats were kept either in individual cages before and after ligation of the right middle cerebral artery (MCA), transferred from a nonenriched to an enriched environment after the ligation, or kept in an enriched environment before as well as after the operation.
Materials and Methods
Handling Before Surgery
Five-week-old male spontaneously hypertensive rats (SHR/Mol, inbred generation F89, Møllegaard Breeding and Research Centre A/S) were delivered and kept in cages with 17 and 18 rats in each for 2 weeks; they were then transferred to cages with 5 rats in each.
At 9 weeks of age (body weight, about 170 g), 23 rats were put in individual standard cages, and 12 rats were put in an enriched environment (described below). During the following week, animals in single cages increased their weight, whereas the rats placed in the enriched environment box lost some weight. At the time of the operation (age, 13 weeks), weight did not differ between the groups. Before the operation, systolic blood pressure was measured by a tail-cuff method in awake rats. The rats in the three groups were handled by the same technician.
The experimental protocol was approved by the local ethical committee for animal research. The rats were anesthetized with 50 mg/kg IP methohexital sodium (Brietal), and body temperature was kept close to 37°C. After craniectomy, the right MCA was occluded at the crossing of the olfactory tract with a square knot of 10-0 monofilament nylon thread.8 9 To shorten the operation time and to prevent problems in the behavioral tests, the animals were not intubated, and no catheters were inserted for blood pressure and blood gas control. The operation time was 20 minutes or less. For the 24 hours following the operation, the rats were housed in individual cages. Thereafter, the individually hosted rats either were returned to their single cages (group A, n=11) or were put into an “enriched” environment (group B, n=12). The 12 rats placed in an enriched environment for 4 weeks before the operation were returned to the same cage (group C).
The size of the cage was 815×610×450 mm. At 150 mm above the floor, two horizontal boards, 70 mm wide, were placed along one of the sides of the cage. 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, a swing board, and wooden blocks were placed in the box. Small changes were made once a week by adding new objects and withdrawing others.
A schematic illustration of the tests used at different times after the operation is shown in Fig 1⇓.
In the postural reflex test,10 rats were held by the tail 50 cm above a table. Normal rats extend both forelimbs toward the table, whereas flexion of the forelimb contralateral to the injured hemisphere can be seen in lesioned rats. Behavior was scored as follows: 1, forelimb flexion and no other abnormality; 2, reduced resistance to lateral push toward the paretic side; and 3, circling toward the paretic side when allowed to move freely. (Rats displaying circular behavior consistently show forelimb flexing and decreased resistance to lateral push.)
The limb-placement test was shortened and modified after De Ryck et al.11 The forelimb and hindlimb placements were evaluated by an observer blinded to group designation. Each test was scored as follows: 0, no placing; 1, incomplete and/or delayed (>2 seconds) placing; and 2, immediate and correct placing. For each body side, the maximum score from the tests used was 16. The forepaws were graded in all six tests; in tests 4 and 6, the hindlimbs were also tested. During tests 1 through 4, the rat was held in a soft grip by the examiner. In test 1, limb placing was tested by slowly lowering the rat toward a table. At about 10 cm above the table, normal rats stretch and place both forepaws on the table. For test 2, with the rat’s forelimbs touching the table edge, the head of the rat was moved 45° upward while the chin was supported to prevent the nose and the vibrissae from touching the table. (A rat with focal brain lesion may lose contact with the table with the paw contralateral to the injured hemisphere.) In test 3, forelimb placement of the rat when facing a table edge was observed. (A normal rat places both forepaws on the table top.) Test 4 recorded forelimb and hindlimb placement when the lateral side of the rat’s body was moved toward the table edge. For test 5, the rat was placed on the table and gently pushed from behind toward the table edge. (A normal rat will grip on the edge, but an injured rat may drop the forelimb contralateral to the injured hemisphere.) Test 6 was the same as test 5, but the rat was pushed laterally toward the table edge.
Coordination and integration of motor movement was tested with a beam-walking test12 13 14 and walking on a rotating pole. The beam was 1750 mm long and 19 mm wide and was placed 700 mm above the floor. A wall was alternately placed 13 mm to the left or the right of the beam. (Rats are more willing to walk when a wall is placed next to the beam.) The ratio scale used was modified after Sutton and Feeney.14 Scoring was as follows: 0, the rat falls down; 1, the rat is unable to traverse the beam but remains sitting across the beam; 2, the rat falls down while walking; 3, the rat can traverse the beam, but the affected hindlimb does not aid in forward locomotion; 4, the rat traverses the beam with more than 50% footslips; 5, the rat crosses the beam with a few footslips; and 6, the rat crosses the beam with no footslips.
The pole, 45 mm in diameter and 1500 mm in length, was rotated alternately to the left or the right at three turns per minute. The same scoring was used as for beam walking except that a score of 3 was given if the rat jumped with both hindlimbs together, apparently supporting the weak hindlimb with the opposite strong limb, and 4 was given if the affected hindlimb was used for fewer than 50% of the steps.
A climbing test was also used 10 weeks after the MCA occlusion. A ladder 80 mm wide and 1530 mm high with an 81° slope was used. We measured the time elapsed before the rats started climbing and the total climbing time. Each test included three consecutive climbings. The upper time limit to perform the test was set at 90 seconds, and rats that did not climb were given a time of 90 seconds.
Paw reaching was evaluated over 10 days starting 3 weeks after the MCA occlusion with a previously presented modification15 of the staircase test introduced by Montoya et al.16 Custom-made Plexiglas boxes with a central elevated platform and a staircase with six steps were used. In this test, each step is baited with eight 45-mg chow pellets (Cambden Instruments Ltd). The rat is placed on the platform and can collect the pellets during 20 minutes. The number of pellets taken by the left and right paws are counted. The test, which is repeated for 10 days, is preceded by a 24-hour period of food deprivation.
Locomotion and rearing were recorded 12 weeks after the MCA occlusion. The test arenas were made of gray polyvinyl chloride (PVC) and mounted in closed wooden cages (Datainnovation AB). The bottom area was 47.5×35.5 cm. On top of each cage, a monochrome charge-coupled device (CCD) array camera (Philips 56470) was mounted centrally in a semitransparent window. A rearing detector was mounted on the side walls of each cage. The rearing detectors are adjustable in height and consist of infrared LEDs and photodiodes forming 48 infrared beams (1 cm apart) across the test arena. In this experiment the heights were set to 12.5 cm. The equipment consisted of seven cages with test arenas, video cameras, and rearing detectors with all seven cameras synchronized. Signals from cameras and rearing detectors were processed and fed into a Compaq Deskpro 386s. The total distance covered and the number of rearings in the central and peripheral part of the box during four consecutive 15-minute periods (5:30 pm to 10 pm) were analyzed using the software ccdact (Datainnovation AB). For statistical analysis the programs stat (for locomotion) and statr (for rearing) were used. The lateral 6 cm on each side was considered as the periphery.
Termination of the Experiments
After the locomotion and rearing study, rats were anesthetized with methohexital, and catheters were placed into the femoral artery and exteriorized on the back of the neck. One hour after recovery from anesthesia, mean arterial pressure was measured. Rats were then killed with an overdose of methohexital, and the brains were removed, frozen in isopentane chilled to −40°C, and stored in −70°C until sectioned.
Determination of Infarct Volume
Coronal sections 20 μm thick were cut in a cryostat at −20°C at 900-μm intervals (ie, every 45th section). The sections were taken for determination of infarct volume, with a total of 15 sections from each rat, and stained with cresyl violet. The image-analyzing system consisted of a light board with constant light (Northern light model B90, Imaging Research Inc), a video camera (CCD72, Dage MTI) fitted with a Nikon lens (f 55 mm), a digitizing unit attached to the video camera (Dage MTI), and a Macintosh IIsi computer equipped with a video card (Imagegrabber NUBus). The software imagegrabber 2.03 (Neotech) was used to capture the digitized images. image/mg 1.44 (National Institutes of Health) was used for further processing. When the rats were killed 13 weeks after the MCA occlusion, no infarcted tissue remained in the cystic infarcts. We have shown earlier that there is no significant difference in volume between the right and the left hemispheres in spontaneously hypertensive rats.17 The remaining area of the right hemisphere was determined in pixel size and was subtracted from the contralateral hemispheric area. The volume was calculated from the cross-sectional areas and the distance between the sections. The proximal MCA occlusion used in our study involves to a varying degree striatal structures, supplied by the lenticulostriate perforans emanating from the MCA. To determine the cortical part of the infarct, the cortical volume on the infarcted side was divided by the volume of the intact hemisphere.
At this late stage after an MCA occlusion, the infarct volume may be overestimated by the inclusion of secondary tissue losses such as thalamic atrophy. To eliminate any error due to hemispheric volume losses, two additional measurements were made: (1) Cortical infarct volume was determined by subtracting the cortex on the infarcted side from the cortex on the intact side and was expressed in percent of cortical volume. (2) To determine the degree of thalamic atrophy, a higher frequency of sections was used (ie, every 15th section) with a distance between sections of 300 μm in the parts of the brain containing the thalamus. The area of the thalamic nuclei on the infarcted side was determined by anatomic landmarks and was compared with that of the intact side. The volumes were determined as above and expressed in percent of the intact side.
For tests based on scoring systems (ordinal measures), the Kruskal-Wallis nonparametric ANOVA was used with a multiple-comparison post hoc test to determine the number and relation of the group differences at a 95% significance level.18 For differences in physiological parameters, infarct volume, and scores of the paw-reaching test, one-way parametric ANOVA with Scheffé’s post hoc procedure was used at 95% and 99% significance levels. For locomotion and rearing, special statistical programs were used as described above.
Two rats in group A did not survive the whole experimental period (1 died 7 weeks and 1 died 9 weeks after the operation); 1 rat in group B died during the first week after the operation. Another rat in group B did not show any abnormalities in the behavioral tests at any time and was found not to have any infarct because of unsuccessful ligation of the MCA. This rat is not included in the statistical comparisons among the groups. Thus, the final analysis is based on 9 rats from group A, 10 from group B, and 12 from group C.
At the time of operation, systolic blood pressure was significantly (P<.05) lower (174±14 mm Hg) in the rats raised in a larger cage (group C) than in those raised in single cages (groups A and B; 190±14 mm Hg). At the end of the experiment, the mean arterial pressure, measured through a femoral artery catheter in awake rats, did not differ among the groups (182±7 mm Hg in group A, 193±10 mm Hg in group B, and 187±12 mm Hg in group C).
All rats improved with time but with considerable differences among the groups and the various tests. The scores in the postural reflex test were significantly higher, ie, more pathological, in rats housed in individual cages than in the other rats 3 weeks after the operation but at no other time (Table 1⇓). In the leg-placement test, when a maximum score of 16 equals normal behavior, rats housed in single cages had significantly lower scores than the other groups at all times (Fig 2⇓). Rats housed in an enriched environment both before and after the operation (group C) had higher scores, ie, performed significantly better than rats placed in the enriched environment after the operation (group B), at 5 weeks only. The discriminative values of the different limb-placement tests varied with time. In test 2, when visual stimuli and whisker contact with the surface were prevented, the difference was significant at all times tested and increased with time.
On the beam and on the rotating pole, rats housed in the enriched environment before and after or only after the operation performed significantly better than rats housed in individual cages (Table 2⇓). With the wall to the left of the beam, ie, when the weak side was close to the wall, the animals did somewhat better than when the wall was placed on the right side of the beam. On the rotating pole, it made little difference whether the pole was rotating to the right or to the left, maybe because the rotation was too slow. Rats in group C performed slightly better than rats in group B, but the difference was not statistically significant.
When the time elapsed before starting to climb and the time spent climbing were measured, both times were significantly shorter in rats housed in an enriched environment than in group A. Furthermore, rats housed in an enriched environment both before and after the MCA occlusion did significantly better than those kept in an enriched environment only after the operation (Table 3⇓).
In the paw-reaching test, rats in the enriched environment had eaten significantly more pellets than those in single cages with the right, nonaffected paw (P<.05). With the affected paw, the tendency was the same but did not reach statistical significance.
Locomotor activity was significantly higher in group A than in group C (P<.05). Table 4⇓ shows the central and peripheral locomotor activity during the four 15-minute periods. The total numbers of peripheral and central rearings were higher in group A and group C (P<.05). The numbers of peripheral and central rearings during the 15-minute periods are shown in Fig 3⇓.
Neither the infarct volume nor thalamic atrophy differed among the groups (Table 5⇓).
Little attention has been given to the influence of environmental factors on functional outcome after focal brain ischemia. Our data show that an enriched environment significantly improves function. In experiments with bilateral ablation of sensorimotor cortex, rats housed in an enriched environment during the preoperative period had fewer initial deficits and better recovery than rats housed in an enriched environment during the postoperative period only.2 In our study, rats kept in an enriched environment before and after the MCA ligation tended to improve sooner and to a slightly higher degree than those placed in the enriched environment only after the ischemia, with a significant difference for climbing (Table 3⇑). In the other tests the difference was small and not significant, except in the leg-placement test 5 weeks after the ligation (Fig 2⇑).
The postural reflex test10 was not a very useful test in our study, maybe because the score of 1 covers a wide range of flexion. Even rats performing well usually showed a slight flexion in the forelimb during the test. Rats housed in an enriched environment did significantly better than group A in the leg-placement test,11 walking on a beam or on a rotating pole, and in climbing.
It might seem surprising that rats housed in single cages were significantly more active in exploring the cages during the test for locomotion and rearing than the rats raised in an enriched environment before and after the operation. It seems likely that the rats housed in a more stimulating environment did not find the test cages, which were more restricting than their home cages, very interesting.
It has been reported earlier that the performance in the paw-reaching test changes little with time.15 Since the test was performed only once after the MCA occlusion, we do not know if this is also the case in rats housed in an enriched environment. The rats were not tested at a later stage because the test is difficult to combine with the other tests. In its present form, this test has to be repeated over several days to obtain consistent results, and the food restriction during these days could have influenced the other tests.
Summing up our results, it seems that rats housed in an enriched environment both before and after the MCA occlusion have a slight advantage over those placed in the same enriched environment after the operation only. However, the difference between these two groups was much smaller than that between the rats with postoperative enrichment and rats housed in standard laboratory cages. Experimental laboratories usually provide a poor environment for the animals. This should be taken into account when evaluating the clinical relevance of animal studies on long-term functional outcome after stroke.
To what extent improved functional outcome is due to recovery of lost functions or to compensation for lost functions is difficult to ascertain.19 We have chosen to use the term “functional outcome” rather than recovery. Various possible mechanisms behind functional improvement after stroke have been discussed, including resolution of brain edema, absorption of necrotic tissue, and disappearance of remote functional depression or “diaschisis”; the latter has been suggested to be important in pharmacological modulation of recovery.20 Several factors might be involved, but current data suggest that a substantial part of functional recovery after stroke might be attributed to brain plasticity.21
It is likely that some reorganization of brain functions can take place after focal brain infarction, be it compensatory mechanisms or substitution for lost neuronal networks. Unilateral ablation of the telencephalon has been shown to induce contralateral cortical and subcortical projections to thalamic nuclei in cats.22 With the reservation that we did not perform any cell counts and thus cannot rule out differences in individual thalamic nuclei, thalamic atrophy was not reduced by the enriched environment. On the basis of activation studies in experimental animals23 and in humans,24 25 26 it has been proposed that recruitment of cortical areas in the nondamaged hemisphere as well as adjacent to the lesion may be important for functional recovery. Whether this is due to “unmasking” of existing networks or establishing of new networks it not known, nor is it known if the increased blood flow necessarily signifies improved function. Furthermore, there is evidence for substantial difference in individual activation patterns after stroke recovery,26 but studies comparing the degree and pattern of activation in patients with good and less good recovery are lacking.
Functional enforcement of existing neuronal circuits, sprouting, formation of new polysynaptic connection, and nonsynaptic transmission have been suggested as mechanisms for restoration of function. It has been hypothesized that the mechanisms involved in restoration of function after brain lesions may be the same or related to those in normal learning. An enriched environment can influence a number of factors such as cortical thickness, protein content, dendritic branching, number of dendritic spines per unit length of dendrite, and angiogenesis (see References 22 and 27). The cellular and molecular basis for the enhanced function observed in the present study remains to be elucidated.
This study was supported by grants from the Swedish Medical Research Council (project 14X-4968), King Gustaf V and Queen Victoria’s Foundation, the Medical Faculty, Lund University, the Swedish Association of Neurologically Disabled (NHR), and the 1987 Foundation for Stroke Research. We thank Bengt Mattson for performing the imaging analysis and Håkan Widner for statistical advice.
Presented in abstract form at the 19th International Joint Conference on Stroke and Cerebral Circulation, San Diego, Calif, February 17-19, 1994.
- Received June 16, 1994.
- Revision received October 10, 1994.
- Accepted December 22, 1994.
- Copyright © 1995 by American Heart Association
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