Enriched rehabilitative training promotes improved forelimb motor function and enhanced dendritic growth after focal ischemic injury

Abstract

Chronic impairment of forelimb and digit movement is a common problem after stroke that is resistant to therapy. Previous studies have demonstrated that enrichment improves behavioral outcome after focal ischemia; however, postischemic enrichment alone is not capable of enhancing fine digit and forelimb function. Therefore, we combined environmental enrichment with daily skilled-reach training to assess the effect of intensive task-specific rehabilitation on long-term functional outcome. Rats were subjected to either endothelin-1-induced focal ischemia or sham surgery and subsequently designated to enriched-rehabilitation or standard-housing treatment groups starting 15 d after ischemia. Functional assessment of the affected forelimb at 4 and 9 weeks after treatment revealed that ischemic plus enrichment (IE) animals had improved approximately 30% on the staircase-reaching task and were indistinguishable from sham animals for both latency and foot faults in a beam-traversing task. In contrast, ischemic plus standard (IS) animals remained significantly impaired on both tasks. Interestingly, both ischemic groups (IE and IS) relied on the nonaffected forelimb during upright weight-bearing movements, a pattern that persisted for the duration of the experiment. Dendritic arborization of layer V pyramidal cells within the undamaged motor cortex was examined using a Golgi-Cox procedure. IE animals showed enhanced dendritic complexity and length compared with both IS and sham groups. These results suggest that enrichment combined with task-specific rehabilitative therapy is capable of augmenting intrinsic neuronal plasticity within noninjured, functionally connected brain regions, as well as promoting enhanced functional outcome.

Fig. 1.

Enriched-rehabilitation treatment condition.A, View of a typical enriched environment. Cages and objects were changed twice weekly to promote exploration and a broad range of tactile experience. B, The rehabilitative reaching apparatus, which was implemented daily (5 d/week) to promote deficit-specific therapy of the impaired forelimb and digits. A shelf directly below the impaired limb was filled with food pellets to reinforce skilled use of the impaired forelimb. C, Side view of the staircase-reaching task. See Materials and Methods for additional description.

Fig. 2.

Golgi–Cox-stained cortical tissue. Photomicrograph at 200× magnification of a layer V pyramidal cell from the motor cortex demonstrating complete impregnation of dendrites. Scale bar, 100 μm.

Fig. 3.

Representative diagram illustrating a typical infarct after ET-1-induced MCA occlusion. Note that affected areas include the sensorimotor cortex and the lateral portion of the caudate putamen. Arrows indicate the contralateral forelimb (FL) motor cortex, from which layer V pyramidal cells were sampled. Stereotaxic coordinates are according to Paxinos and Watson (1997).

Fig. 4.

Staircase test of skilled forelimb reaching ability. The percentage for each performance was determined by comparing an individual animal's presurgery training score with subsequent test scores. A, Reaching scores for the limb contralateral to the lesion are shown, indicating a sharp impairment after surgery (p < 0.01 compared with shams). After 4 weeks (Post 1) of exposure to their respective treatment conditions, IE animals showed a dramatic improvement compared with IS animals (p < 0.01), which persisted at 9 weeks after treatment (Post 2). B, Reaching scores for the ipsilateral forelimb show a slight but significant impairment at 10–15 d after ischemia (before treatment) and at 4 weeks after treatment in both IE and IS animals. Ipsilateral forelimb impairment had recovered to sham levels by 9 weeks of either treatment. Sham groups (SE and SS) were not significantly different at any time point and therefore were pooled for statistical analyses. Data are presented as mean ± SEM. * indicates different from sham; † indicates different from IS.

Fig. 5.

A, Asymmetrical forelimb use. Preferential forelimb use was examined at 15 d after surgery (before treatment) and after 4 and 9 weeks (Post 1 andPost 2, respectively) of exposure to either enriched-rehabilitation or standard-housing conditions. Sham animals did not display a forelimb preference on any test day; however, both IE and IS groups showed a persistent dependence on the ipsilateral good limb 15 d after ischemia and after 4 and 9 weeks of either treatment (p < 0.01). At 9 weeks, the IE group showed significantly less reliance on the ipsilateral forelimb than did the IS group (p < 0.05). Sham groups (SE and SS) were not significantly different at any time point; therefore, these groups were pooled for statistical analyses.Black squares, IE; white circles, IS; gray diamonds, sham (SE and SS).B, Beam-walking test. After ischemic injury, both the IE and IS groups had a significantly greater number of foot faults compared with sham animals. IS animals showed no spontaneous improvement 4 or 9 weeks later, whereas IE animals were indistinguishable from sham animals after 4 and 9 weeks of enriched rehabilitation. C, Mean latency to traverse the beam also indicated improvement in IE animals compared with IS animals. Both the IE and sham groups demonstrated a successive decrease in latency that seemed to correspond with fewer foot faults. IS animals exhibited persistently longer latencies with no apparent improvement. Data are mean ± SEM. * indicates different from sham; † indicates different from IS.

Fig. 6.

Representative reconstructions of layer V pyramidal cells respective of treatment condition (IE, IS, and SE). Note the increased basilar dendritic arbor in the IS group compared with the SE group and even greater complexity in the IE condition. Examination of the apical dendritic tree did not show any statistical differences. Scale bar, 100 μm.

Fig. 7.

A, Total basilar dendrite length. Enriched rehabilitation (IE) significantly elevated dendritic length compared with the standard-treated ischemic group (IS) and either sham condition (SE and SS). A consistent but nonsignificant increase was observed in the IS condition. B, Total number of basilar branch segments per neuron in the undamaged forelimb motor cortex. Enriched rehabilitation resulted in a significantly greater number of branches than was observed in the sham groups (SE or SS). Ischemia alone (IS) also increased branch number compared with SS. Data are mean ± SD. *p < 0.05.

Fig. 8.

Branch order and Sholl analyses.A, Apical dendritic branch order did not differ among any groups. B, Basilar branch order analysis revealed that IE animals had significantly greater numbers of higher-order branches (i.e., 3rd, 4th, 5th, and 6th-plus). IE had significantly more fifth-order and sixth-order or higher branches compared with all other conditions. Interestingly, IS and SE resulted in elevated third-order branches. C, Basilar Sholl sphere analysis revealed that IE animals had consistently increased the number of branches between 80 and 260 μm away from the cell body. Dendritic branching in the IE condition was significantly elevated between 80 and 160 μm. IS and SE conditions were elevated only at 80 and 100 μm distances. Error bars have been omitted for clarity. *p < 0.05 versus SS; †p < 0.05 versus IS; §p < 0.05 versus SE.