Epidermal Growth Factor and Erythropoietin Infusion Accelerate Functional Recovery in Combination With Rehabilitation
Background and Purpose—Rehabilitation is the only treatment option for chronic stroke deficits, but unfortunately, it often provides incomplete recovery. In this study, a novel combination of growth factor administration and rehabilitation therapy was used to facilitate functional recovery in a rat model of cortical stroke.
Methods—Ischemia was induced via injection of endothelin-1 into the sensorimotor cortex. This was followed by either a 2-week infusion of epidermal growth factor and erythropoietin or artificial cerebrospinal fluid into the ipsilateral lateral ventricle. Two weeks after ischemia, animals began an 8-week enriched rehabilitation program. Functional recovery was assessed after ischemia using the Montoya staircase-reaching task, beam-traversing, and cylinder test of forelimb asymmetry.
Results—The combination of growth factor infusion and rehabilitation led to a significant acceleration in recovery in the staircase task. When compared with controls, animals receiving the combination treatment attained significant recovery of function at 4 weeks after stroke, whereas those receiving rehabilitation alone did not recover until 10 weeks. Significant recovery was also observed on the beam-traversing and cylinder tasks.
Conclusions—Combining behavioral rehabilitation with growth factor infusion accelerates motor recovery. These data suggest a promising new avenue of combination therapies that may have the potential to reduce the rehabilitation time necessary to recover from sensorimotor deficits arising from stroke.
Accelerating recovery in individuals who have experienced a stroke is crucial for limiting the effect of this condition. To enhance the rate of poststroke functional recovery, an optimal approach may be to combine rehabilitation with treatments that enhance neuroplasticity. Previous work has shown that serial application of epidermal growth factor (EGF) and erythropoietin enhances tissue regeneration and proliferation of neural precursor cells.1 As a prelude to future mechanistic studies, the present study sought to determine whether an enhanced neuroplastic environment created by growth factor infusions and concurrent physical rehabilitation would augment behavioral recovery. We hypothesized that combining EGF and erythropoietin with rehabilitation after forelimb sensorimotor cortex stroke would result in accelerated functional improvements when compared with either rehabilitation or EGF and erythropoietin alone.
Sixty-seven male Sprague–Dawley rats were matched for poststroke grasping/retrieving performance on the staircase test and randomized into 4 groups: rehabilitation+EGF/erythropoietin (n=13), rehabilitation+artificial cerebrospinal fluid (aCSF; n=12), no rehabilitation+EGF/erythropoietin (n=12), and no rehabilitation+aCSF (n=13). All procedures were approved by the Memorial University Animal Care Committee and comply with regulations set by the Canadian Council of Animal Care (see detailed Methods in the online-only Data Supplement).
Animals were trained on the staircase (pellet grasping/retrieving), beam (paw placement/balance), and cylinder (asymmetrical limb use for postural support) tasks 2 weeks before ischemia, and baseline abilities were assessed 3 days before stroke. Subsequent testing was performed 5 days after stroke and at poststroke weeks 4, 7, and 10 (Figure 1). Animals that did not meet preset training performance criteria (n=5), or were functionally unimpaired after stroke (n=12), were excluded. Behavioral data are presented as a percentage of prestroke performance.
After baseline behavioral testing, rats were anesthetized with isoflurane and delivered 2 stereotaxic injections of endothelin-1 (2 μL/injection; 400 pmol/μL) into the sensorimotor cortex. Three days after ischemia, an infusion cannula was inserted into the lateral ventricle to deliver either EGF (10 μg/mL) or aCSF vehicle via osmotic minipump (1.0 μL/h; 7 days). Seven days after EGF pump implantation, the pump was replaced with either erythropoietin (1365 IU/mL) or aCSF. This serial administration of EGF/erythropoietin has been previously found effective in promoting repair of damaged cortical tissue.1 All osmotic minipumps were removed 7 days later.
Enriched Rehabilitation Protocol
Animals receiving rehabilitation were housed in large wire-mesh cages (5–6 per cage) containing a variety of interactive objects, whereas those in the nonrehabilitation group was pair-housed in standard cage conditions. Rehabilitation animals also received voluntary access to reach a training chamber (6 hours per day, 5 days per week, and 8 weeks). This apparatus encourages repeated reaching attempts similar to the movements specific to the staircase-reaching task. After the conclusion of rehabilitation and testing, brains were collected for analysis of stroke damage using cresyl violet staining.
Behavioral data were analyzed using 2-way repeated-measures ANOVAs with Ryan-Einot-Gabriel-Welsch F post hoc and independent samples t tests (Bonferroni correction) for multiple comparisons. Cortical damage was analyzed using the Kruskal–Wallis and Mann–Whitney U nonparametric tests. Significance was set at P≤0.05 for all analyses, and values are expressed as mean±SEM.
In the staircase task, a significant rehabilitation×growth factor interaction existed among groups after both 4 (P<0.04) and 10 weeks (P<0.01) after stroke (Figure 2A). Post hoc analysis showed that the rehabilitation+EGF/erythropoietin group performed significantly better than the nonrehabilitation+aCSF and nonrehabilitation+EGF/erythropoietin groups at both 4 and 10 weeks (P<0.05). To assess magnitude of improvement, group performance at each time point was subtracted from the corresponding poststroke test point. This revealed that 4 weeks after stroke, the rehabilitation+EGF/erythropoietin condition improved significantly more on the staircase test than those in the nonrehabilitation+EGF/erythropoietin and nonrehabilitation+aCSF groups (P<0.05; Figure 2B). Animals in the rehabilitation+aCSF group did not show a significant improvement compared with the nonrehabilitation+EGF/erythropoietin group until 10 weeks after stroke (P<0.05; Figure 2C). Both the beam and cylinder tasks showed significant time×rehabilitation interactions (P<0.02; Figures I and II in the online-only Data Supplement). No significant differences in cortical damage were detected between groups (P>0.05).
This study demonstrates that functional recovery is significantly accelerated with the combination of EGF/erythropoietin and rehabilitation. By week 4 after stroke, animals receiving the combined treatment improved to a significantly greater extent on the staircase task (grasping/retrieving) than animals in the growth factor alone or in control groups. In contrast, the group that received rehabilitation with a vehicle infusion also improved significantly more than the control conditions, but recovery emerged only at 10 weeks after stroke. Interestingly, neither the beam walking (paw placement/balance) nor cylinder (asymmetrical limb use for postural support) tasks revealed the same acceleration effect observed in the staircase-reaching task (although rehabilitation alone was still efficacious). This suggests that beneficial effects of the combined treatment may be restricted to fine motor skill, as reflected by the staircase test. Clinically, it has been noted that recovery is enhanced for tasks that are specifically targeted by a given rehabilitation program and this is in line with our reaching data.2
Our data suggest that engaging multiple recovery processes may be critical for enhancing functional outcome, in contrast to monotherapies targeting single mechanisms. Previous studies demonstrated that the individual components of our enriched rehabilitation paradigm (environmental enrichment and task-specific reach training) are not effective for improving recovery of skilled reaching deficits3; however, the combination of these components has a significant beneficial effect on recovery.4 This is underscored by recent work that demonstrates enhanced activation of layer II and III neurons in peri-infarct cortex in response to enriched rehabilitation but not its individual components.5 Similar results have been found on the efficacy of EGF and erythropoietin in combination, but not individually, in enhancing neural precursor cells.1 Results such as these emphasize the need to develop interventions targeting multiple, complementary mechanisms, given the repeated failure of single interventions to translate into successful clinical application.6
Task-specific rehabilitation may be a critically important supplement to realizing the beneficial effects of exogenous growth factor infusion into the brain. With daily reaching rehabilitation, neuroplastic reorganization of the motor cortex takes place, which is thought to be responsible for functional recovery.7 When rehabilitation is combined with EGF/erythropoietin infusion, we speculate that cortical maps involved in task-specific motor patterns may enlarge at an accelerated rate relative to motor maps involved in other stroke-disrupted movements. This increased neuroplastic response could be induced by EGF/erythropoietin through processes, such as neural precursor proliferation, neurovascular remodeling, and synaptic plasticity.8,9 These possibilities, as well as other potential mechanisms underlying the beneficial effects of this combination therapy and precise timeline of recovery, await further investigation but offer promise as an adjunct to conventional rehabilitation.
In summary, combination drug/rehabilitation paradigms represent a promising approach to stroke recovery that has been largely unexplored. In the present study, we demonstrated that combining EGF and erythropoietin infusion with rehabilitation significantly accelerates the rate of recovery of fine motor ability after stroke. This finding is particularly noteworthy because the benefits of the combination therapy were compared with the current most effective or best practice intervention (ie, enriched rehabilitation) for enhancing recovery in preclinical stroke models, instead of an untreated control. Interestingly, although we observed some beneficial effects of rehabilitation alone across all of our behavioral tests, similar effects were not found in our drug-only condition. This emphasizes the importance of testing novel drug targets in conjunction with (and against) other therapies that are known to be efficacious because synergistic effects may exist that could otherwise go undetected.
Sources of Funding
Funding was received from the Heart and Stroke Foundation of Canada and the Natural Sciences and Engineering Research Council of Canada.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.114.005464/-/DC1.
- Received March 13, 2014.
- Received March 13, 2014.
- Accepted March 26, 2014.
- © 2014 American Heart Association, Inc.
- Biernaskie J,
- Corbett D
- Clarke J,
- Langdon KD,
- Corbett D
- Nudo RJ,
- Wise BM,
- SiFuentes F,
- Milliken GW
- Mengozzi M,
- Cervellini I,
- Villa P,
- Erbayraktar Z,
- Gökmen N,
- Yilmaz O,
- et al