Matthew S. Jeffers

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.

Lost in translation: rethinking approaches to stroke recovery.

Stroke is the second leading cause of death and the preeminent cause of neurological disability. Attempts to limit brain injury after ischemic stroke with clot-dissolving drugs have met with great success but their use remains limited due to a narrow therapeutic time window and concern over serious side effects. Unfortunately, the neuroprotective strategy failed in clinical trials. A more promising approach is to promote recovery of function in people affected by stroke. Following stroke, there is a heightened critical period of plasticity that appears to be receptive to exogenous interventions (e.g., delivery of growth factors) designed to enhance neuroplasticity processes important for recovery. An emerging concept is that combinational therapies appear much more effective than single interventions in improving stroke recovery. One of the most promising interventions, with clinical feasibility, is enriched rehabilitation, a combination of environmental enrichment and task-specific therapy.

A reproducible Endothelin-1 model of forelimb motor cortex stroke in the mouse

BACKGROUND Despite the availability of numerous transgenic mouse lines to study the role of individual genes in promoting neural repair following stroke, few studies have availed of this technology, primarily due to the lack of a reproducible ischemic injury model in the mouse. Intracortical injections of Endothelin-1 (ET1) a potent vasoconstrictive agent, reliably produces focal infarcts with concomitant behavioral deficits in rats. In contrast, ET1 infarcts in mice are significantly smaller and do not generate consistent behavioral deficits. NEW METHOD We have modified the ET1 ischemia model to target the anterior forelimb motor cortex (aFMC) and show that this generates a reproducible focal ischemic injury in mice with consistent behavioral deficits. Furthermore, we have developed a novel analysis of the cylinder test by quantifying paw-dragging behavior. RESULTS ET1 injections which damage deep layer neurons in the aFMC generate reproducible deficits on the staircase test. Cylinder test analysis showed no forelimb asymmetry post-injection; however, we observed a novel paw-dragging behavior in mice which is a positive sign of damage to the FMC. COMPARISON WITH EXISTING METHODS Previous ET1 studies have demonstrated inconsistent behavioral deficits; however, targeting ET1 injections to the aFMC reliably results in staircase deficits. We show that analysis of paw-dragging behavior in the cylinder test is a more sensitive measure of damage to the FMC than the classical forelimb asymmetry analysis. CONCLUSIONS We have developed a focal ischemic injury model in the mouse that results in reproducible behavioral deficits and can be used to test future regenerative therapies.

Time course of neuronal death following endothelin-1 induced focal ischemia in rats

BACKGROUND Endothelin-1 (ET-1) induced focal ischemia is increasingly being used as a preclinical model of stroke. Here, we described for the first time, the time course of neuronal death and infarct evolution during the first 7 days following ischemia. NEW METHOD We used hematoxylin and eosin (H&E) staining to evaluate infarct progression and Fluoro-Jade C (FJC) to quantify neuronal degeneration at 24, 48, 72h and 7 days after ET-1 injection to the forelimb motor cortex in Sprague-Dawley rats. RESULTS We found that infarct volume and neuronal degeneration are maximal at 24h post-stroke. Neuronal degeneration is also significantly reduced within 7 days of stroke induction. COMPARISON WITH EXISTING METHOD This study is the first to provide a direct evaluation of both infarct volume evolution and neuronal death time course following ET-1 induced focal ischemia in the forelimb motor cortex. CONCLUSION This study describes the short-term time course of neuronal death and brain injury in the ET-1 stroke model, which provides a significant reference when determining the appropriate time to commence neuroprotective or recovery promoting strategies.

Enriched rehabilitation promotes motor recovery in rats exposed to neonatal hypoxia-ischemia.

Despite continuous improvement in neonatology there is no clinically effective treatment for perinatal hypoxia ischemia (HI). Therefore, development of a new therapeutic intervention to minimize the resulting neurological consequences is urgently needed. The immature brain is highly responsive to environmental stimuli, such as environmental enrichment but a more effective paradigm is enriched rehabilitation (ER), which combines environmental enrichment with daily reach training. Another neurorestorative strategy to promote tissue repair and functional recovery is cyclosporine A (CsA). However, potential benefits of CsA after neonatal HI have yet to be investigated. The aim of this study was to investigate the effects of a combinational therapy of CsA and ER in attempts to promote cognitive and motor recovery in a rat model of perinatal hypoxic-ischemic injury. Seven-day old rats were submitted to the HI procedure and divided into 4 groups: CsA+Rehabilitation; CsA+NoRehabilitation; Vehicle+Rehabilitation; Vehicle+NoRehabilitation. Behavioural parameters were evaluated pre (experiment 1) and post 4 weeks of combinational therapy (experiment 2). Results of experiment 1 demonstrated reduced open field activity of HI animals and increased foot faults relative to shams in the ladder rung walking test. In experiment 2, we showed that ER facilitated acquisition of a staircase skilled-reaching task, increased number of zone crosses in open-field exploration and enhanced coordinated limb use during locomotion on the ladder rung task. There were no evident deficits in novel object recognition testing. Delayed administration of CsA, had no effect on functional recovery after neonatal HI. There was a significant reduction of cortical and hemispherical volume and hippocampal area, ipsilateral to arterial occlusion in HI animals; combinational therapy had no effect on these morphological measurements. In conclusion, the present study demonstrated that ER, but not CsA was the main contributor to enhanced recovery of motor ability after neonatal HI.

Assessing cognitive function following medial prefrontal stroke in the rat.

Cognitive impairments are prevalent following clinical stroke; however, preclinical research has focused almost exclusively on motor deficits. In order to conduct systematic evaluations into the nature of post-stroke cognitive dysfunction and recovery, it is crucial to develop focal stroke models that predominantly affect cognition while leaving motor function intact. Herein, we evaluated a range of cognitive functions 1-4 months following focal medial prefrontal cortex (mPFC) stroke using a battery of tests. Male Sprague-Dawley rats underwent focal ischemia induced in the mPFC using bilateral intracerebral injections of endothelin-1, or sham surgery. Cognitive function was assessed using an open field, several object recognition tests, attentional set-shifting, light-dark box, spontaneous alternation, Barnes maze, and win-shift/win-stay tests. Prefrontal cortex damage resulted in significant changes in object recognition function, behavioural flexibility, and anxiety-like behaviour, while spontaneous alternation and locomotor function remained intact. These deficits are similar to the cognitive deficits following stroke in humans. Our results suggest that this model may be useful for identifying and developing potential therapies for improving post-stroke cognitive dysfunction.

Does Stroke Rehabilitation Really Matter? Part B: An Algorithm for Prescribing an Effective Intensity of Rehabilitation:

Background. The proportional recovery rule suggests that current rehabilitation practices may have limited ability to influence stroke recovery. However, the appropriate intensity of rehabilitation needed to achieve recovery remains unknown. Similarities between rodent and human recovery biomarkers may allow determination of rehabilitation thresholds necessary to activate endogenous biological recovery processes. Objective. We determined the relative influence that clinically relevant biomarkers of stroke recovery exert on functional outcome. These biomarkers were then used to generate an algorithm that prescribes individualized intensities of rehabilitation necessary for recovery of function. Methods. A retrospective cohort of 593 male Sprague-Dawley rats was used to identify biomarkers that best predicted poststroke change in pellet retrieval in the Montoya staircase-reaching task using multiple linear regression. Prospective manipulation of these factors using endothelin-1-induced stroke (n = 49) was used to validate the model. Results. Rehabilitation was necessary to reliably predict recovery across the continuum of stroke severity. As infarct volume and initial impairment increased, more intensive rehabilitation was required to engage recovery. In this model, we prescribed the specific dose of daily rehabilitation required for rats to achieve significant motor recovery using the biomarkers of initial poststroke impairment and infarct volume. Conclusions. Our algorithm demonstrates an individualized approach to stroke rehabilitation, wherein imaging and functional performance measures can be used to develop an optimized rehabilitation paradigm for rats, particularly those with severe impairments. Exploring this approach in human patients could lead to an increase in the proportion of individuals experiencing recovery of lost motor function poststroke.

Short- and long-term exposure to low and high dose running produce differential effects on hippocampal neurogenesis

Continuous running wheel (RW) exercise increases adult hippocampal neurogenesis in the dentate gyrus (DG) of rodents. Evidence suggests that greater amounts of RW exercise does not always equate to more adult-generated neurons in hippocampus. It can also be argued that continuous access to a RW results in exercise levels not representative of human exercise patterns. This study tested if RW paradigms that more closely represent human exercise patterns (e.g. shorter bouts, alternating daily exercise) alter neurogenesis. Neurogenesis was measured by examining the survival and fate of bromodeoxyuridine (BrdU)-labeled proliferating cells in the DG of male Sprague-Dawley rats after acute (14 days) or chronic (30 days) RW access. Rats were assigned to experimental groups based on the number of hours that they had access to a RW over two days: 0 h, 4 h, 8 h, 24 h, and 48 h. After acute RW access, rats that had unlimited access to the RW on alternating days (24 h) had a stronger neurogenic response compared to those rats that ran modest distances (4 h, 8 h) or not at all (0 h). In contrast, following chronic RW access, rats that ran a moderate amount (4 h, 8 h) had significantly more surviving cells compared to 0 h, 24 h, and 48 h. Linear regression analysis established a negative relationship between running distance and surviving BrdU+ cells in the chronic RW access cohort (R2 = 0.40). These data demonstrate that in rats moderate amounts of RW exercise are superior to continuous daily RW exercise paradigms at promoting hippocampal neurogenesis in the long-term.

Does Stroke Rehabilitation Really Matter? Part A: Proportional Stroke Recovery in the Rat:

Background. In human upper-limb stroke, initial level of functional impairment or corticospinal tract injury can accurately predict the degree of poststroke recovery, independent of rehabilitation practices. This proportional recovery rule implies that current rehabilitation practices may play little or no role in brain repair, with recovery largely a result of spontaneous biological recovery processes. Objective. The present study sought to determine if similar biomarkers predict recovery of poststroke function in rats, indicating that an endogenous biological recovery process might be preserved across mammalian species. Methods. Using a cohort of 593 male Sprague-Dawley rats, we predicted poststroke change in pellet retrieval in the Montoya staircase-reaching task based on initial impairment alone. Stratification of the sample into “fitters” and “nonfitters” of the proportional recovery rule using hierarchical cluster analysis allowed identification of distinguishing characteristics of these subgroups. Results. Approximately 30% of subjects were identified as fitters of the rule. These rats showed recovery in proportion to their initial level of impairment of 66% (95% CI = 62%-70%). This interval overlaps with those of multiple human clinical trials. A number of variables, including less severe infarct volumes and initial poststroke impairments distinguished fitters of the rule from nonfitters. Conclusions. These findings suggest that proportional recovery is a cross-species phenomenon that can be used to uncover biological mechanisms contributing to stroke recovery.

Synergistic Effects of Enriched Environment and Task-Specific Reach Training on Poststroke Recovery of Motor Function

Background and Purpose— Reach training in concert with environmental enrichment provides functional benefits after experimental stroke in rats. The present study extended these findings by assessing whether intensive task-specific reach training or enrichment initiated alone would provide similar functional benefit. Additionally, we investigated whether the 70% recovery rule, or a combined model of initial poststroke impairment, cortical infarct volume, and rehabilitation intensity, could predict recovery in the single-pellet task, as previously found for the Montoya staircase. Methods— Rats were trained on single-pellet reaching before middle cerebral artery occlusion via intracerebral injection of ET-1 (endothelin-1). There were 4 experimental groups: stroke+enrichment, stroke+reaching, stroke+enrichment+reaching, and sham+enrichment+reaching. Reaching rehabilitation utilized a modified Whishaw box that encouraged impaired forelimb reaching for 6 hours per day, 5 days per week, for 4 weeks. All treatment paradigms began 7 days after ischemia with weekly assessment on the single-pellet task during rehabilitation and again 4 weeks after rehabilitation concluded. Results— Rats exposed to the combination of enrichment and reaching showed the greatest improvement in pellet retrieval and comparable performance to shams after 3 weeks of treatment, whereas those groups that received a monotherapy remained significantly impaired at all time points. Initial impairment alone did not significantly predict recovery in single-pellet as the 70% rule would suggest; however, a combined model of cortical infarct volume and rehabilitation intensity predicted change in pellet retrieval on the single-pellet task with the same accuracy as previously shown with the staircase, demonstrating the generalizability of this model across reaching tasks. Conclusions— Task-specific reach training and environmental enrichment have synergistic effects in rats that persist long after rehabilitation ends, and this recovery is predicted by infarct volume and rehabilitation intensity.