Early, Intense Rehabilitation Fails to Improve Outcome After Intra-Striatal Hemorrhage in Rats

Abstract

Background: The formation and degradation of an intracerebral hemorrhage causes protracted cell death, and an extended window for intervention. Experimental studies find that rehabilitation mitigates late cell death, with accelerated hematoma clearance as a potential mechanism.

Objective: We assessed whether early, intense, enriched rehabilitation (ER, environmental enrichment and massed skills training) enhances functional benefit, reduces brain injury, and augments hematoma clearance.

Methods: In experiment 1, rats (n = 56) were randomized to intervention in the light (-L) or dark phase (-D) of their housing cycle, then to 10 days of ER or control (CON) treatment after collagenase-induced striatal intracerebral hemorrhage (ICH). ER rats were treated from 5 to 14 days after ICH. Behavior and residual hematoma volume was assessed on day 14. In experiment 2, rats (n = 72) were randomized to ER-D10, ER-D20, or CON-D. ER rats completed 10 or 20 days of training in the dark. Rats were euthanized on day 60 for histology. In both experiments, behavioral assessment was completed pre-ICH, pre-ER (day 4 post-ICH), and post-ER (experiment 1: days 13-14; experiment 2: days 16-17 and 30-31).

Results: Reaching intensity was high but similar between ER-D10 and ER-L10. Unlike previous work, rehabilitation did not alter skilled reaching or hematoma resolution. Varying ER duration also did not affect reaching success or lesion volume.

Conclusions: In contrast to others, and under these conditions, our findings show that striatal ICH was generally unresponsive to rehabilitation. This highlights the difficulty of replicating and extending published work, perhaps owing to small inter-study differences.

Keywords: enrichment; intracerebral hemorrhage; neuroprotection; rehabilitation; stroke.

Figure 1.

Experimental timeline. (A) Experiment 1: investigation of effects of ER on residual hematoma volume and behavior 14 days after collagenase-induced ICH. (B) Experiment 2: investigation of effect of altering ER duration (10 or 20 days) on lesion volume and behavior in the chronic phase of recovery after collagenase ICH. (C) Dual-level EE housing. Each level contained a running wheel for rodents to use at their leisure, as well as tubes for hiding, and novelty toys. To encourage exploratory behavior, food location was changed daily, and novelty items were changed twice weekly (eg, balls, chains, wooden blocks, cars). Cages were made of wire rungs to allow climbing between levels of the cage and included a ramp to allow more impaired animals to walk to upper level. Abbreviations: ER, enriched rehabilitation; ICH, intracerebral hemorrhage.

Figure 2.

Results of experiment 1. (A) Reaching success in staircase task. All groups displayed notable impairment after ICH (vs baseline, persistent out to 14 days) but no effect of light cycle or treatment was detected. (B) Percent success of correct paw placement of contralateral forelimb on the ladder walking task. Three-way ANOVA detected a main effect of time and light cycle but not treatment. (C) Residual hematoma volume (μL) measured 14 days after ICH. Two-way ANOVA did not detect an effect of light cycle or treatment. Individual data points are shown along with the mean ± 95% CI. Abbreviations: ICH, intracerebral hemorrhage; CON, control; ER, enriched rehabilitation; D, dark; L, light.

Figure 3.

Rehabilitation intensity. Figures represent the average number of pellets successfully retrieved per training session on each day. (A) Experiment 1—rats in ER-L10 and ER-D10 groups completed 4 daily rehab training sessions days 4 to 12 and 2 sessions on days 13 and 14. (B) Experiment 2—rats in ER-D10 and ER-D20 groups completed 4 daily rehab training sessions days 4 to 14. Rats in ER-20 completed an additional 10 days of ER days 19 to 28. All data presented as mean ± 95% CI. Abbreviations: ER, enriched rehabilitation; D, dark; L, light.

Figure 4.

Results of experiment 2. (A) Reaching success in the staircase task was not improved by rehab. All groups displayed notable impairment after ICH (day 4). Multiple comparisons showed a main effect of time at all levels of comparison, except between day 16 and 30. We failed to detect an effect of treatment. (B) The volume of ipsilesional corpus callosum was smaller than contralateral; no effect of treatment was detected. (C) Lesion volume (mm³) measured 60 days after ICH; no effect of treatment was detected. (D) Coronal section of rat brain at 60 days displaying lesion cavity (outlined in black), ipsilesional ventriculomegaly (*), and atrophy of ipsilesional corpus callosum (black arrow). Subject in image had a total lesion volume of 38.8 mm³, approximately the average observed across groups. Individual data points are shown along with the mean ± 95% CI. Abbreviations: ICH, intracerebral hemorrhage; ER, enriched rehabilitation; CON, control; D, dark.

Figure 5.

Post-hoc analysis of pooled data. Data from both experiments were pooled into ER10 (n = 70) and CON (n = 43) groups. A main effect of time (stroke-induced impairment) was readily evident on day 4 (10.85 fewer pellets retrieved [95% CI: 9.82, 11.88] vs baseline), and on day 14/16 (7.93 fewer pellets retrieved [95% CI: 6.96, 8.90] vs baseline). No significant effect of treatment was present (treatment effect size on day 14/16 was an improvement of 1.32 pellets retrieved [95% CI: −0.31, 2.95] vs day 4). Despite >99% statistical power to detect a 3-pellet difference, one level of the staircase task and presumably a minimum biologically meaningful effect, groups were not statistically different from each other after ICH. Individual data points are shown along with the mean ± 95% CI. Abbreviations: ER, enriched rehabilitation; CON, control; ICH, intracerebral hemorrhage.