Enriched Rehabilitation Reduces Abnormal
Motor Synergies and Enhances Motor Plasticity Following Severe Stroke in Rats
Gustavo Balbinot , PhD; Boris Touvykine , PhD; Joyce Zaftis, MD; Clarissa Pedrini Schuch , PhD;
Matthew Strider Jeffers , MSc; Guillaume Elgbeili , MSc; Numa Dancause , PhD; Dale Corbett , PhD
Introduction
Recovery from stroke is incomplete especially with moderate to severe. To identify the mechanism behind recovery and develop better therapy, there is a need to distinguish compensation and restitution. The authors of the paper quote evidence stating that improvement post stroke is mainly due to compensation: Whishaw et al, showed that in rats with large motor cortex lesions “spontaneously” acquire compensatory movements.
The term spontaneously acquire compensatory movements is not clear, if it denotes the movement that is done without adapting/learning over a period due to impairment.
Unexplored issue is that it is unclear if the restoration of post stroke performance with rehabilitation intervention is through restitution, compensation, or both. The most used rehabilitation intervention in rats is environment enrichment. Qualitative movement analysis to assess the effects of EE was done previously and found that performance improvements were due to compensation. However, the limitation of the study was low intensity and subjective assessment measure. There is limited evidence for improvement due to EE except for the case when it is combined with high intensity. In addition, there is a need use quantitative kinematic analyses to determine if its restitution or compensation that leads to functional improvements. Therefore, the objective of this paper is:
To use the quantitative kinematic method to assess the effect of enriched rehabilitation which is much more intensive and effective form of rehabilitation.
To know the mechanistic insights on how ER improved functional outcomes using:
Intracortical micro stimulation (ICMS)
Immunostaining techniques
Methods
Male Sprague-Dawley rats were trained and tested on 2 different reaching tasks. Among the total 34 rats: 20 rats underwent Enriched rehabilitation (ER) which was compared with controlled group of 13 rats which spontaneously recovered (SR) from comparable strokes. 2 SR rats were excluded, one for which week 5 kinematic data could not be obtained and another due to poor immunostaining.
There were 20, 13 rats in ER, SR groups respectively and 2 rats were excluded, which do not sum up to the total rats of 34.
Stroke was induced in the contralateral hemisphere to the preferred paw using a rat endothelin-1 model of moderate to severe stroke which resulted in chronic functional impairments as in humans.
MR imaging was performed 48 hours following stroke. Infarct volumes quantified using ImageJ; lesion maps were created using LabVIEW. MR image voxels of cortical regions of interest were considered infracted or non-infarcted indicated based on voxel colour.
Animals were trained for 2 weeks in the staircase reaching tasks for 15 minutes twice daily. There were 7 steps on each limb baited with 3 food pellets. Forelimb reaching ability was assessed based on the number of pellets eaten per side. Animals were tested at 4 different timepoints which include before stroke, at 7,21- and 35-days post stroke. Each test had 6 trials and first 2 trials were excluded.
An additional staircase session was performed on day 35 for kinematic analysis to examine group difference during voluntary reach-to-grasp movements. Pellets placed only on the more impaired arm and reaching recorded at 60 frames per seconds in the sagittal plane. Markers were placed at shoulder elbow and wrist.
Kinematic analysis is done only on day 35, and no mention about pre-stroke kinematic analysis which is questionable since the results presents kinematic synergies normalised to pre-stroke conditions.
All rats were Initially caged in large plastic cages in groups of 4. Based on the initial post stroke reaching deficits, and day 2 MR infarct volume the rats were pseudo randomised in pairs so that both groups have similar characteristics. and then one of them is randomised to SR group which remained in the same standard cages and the ER group rats were put in ferret cages in group of 4 which had engaging objects such as toys, ramps, and obstacles. These objects were replaced every week to encourage exploration. Rehabilitation was provided with the plexiglass tray reaching apparatus for 4x15 minutes sessions per day with 45 minutes of rest in between for 5 days/week. The tray height was increased from 6cm during week 1 and 2 to 10 cm in week at week 3 and 14 cm during week 5 to make the tasks more challenging.
What is pseudorandomised? The random number generators are not completely random can be predicted. In this study pseudo randomisation is because the rats are grouped in pairs to ensure similar characteristics. Tasks were made challenging over time (weekly alteration in heights, similar for all rats), Increasing the height with respect to the performance of rats could have been a more adaptive approach.
Results
The major objective of the paper was to study the effects of enriched rehabilitation on improving forelimb functions. The performance of the rats is first evaluated based on the number pellets retrieved and then identify the if the improvement was due to movement being restored to original movement pattern as in pre-stroke conditions or due to the learned compensatory movements. They wanted to look at two types of synergies: Elbow flexion synergy and elbow lift synergy.
ER Improves Forelimb Function and Normalizes Elbow Flexion Synergies Following Stroke
Stroke size was not different between the two groups.
ER rates retrieved 50 pellets on average per 15-minute session and improved at day 35 (fig 2c).
kinematic analysis revealed that elbow flexion during limb advance reduced in ER group, normalising it to pre-stroke levels, ER- elbow lift synergy also reduced to below pre-stroke levels (Fig 3B).
From the figure it’s not clear if the normalised synergy above 100% is considered worse or better. Also between the groups although ER has more points below 100% the spread of SR group also looks similar which is not mentioned.
Synergies increased with greater infarct volume in SR group. Normalisation of these synergy was greater in ER group with more severely damaged animals (Fig 3c).
In fig 3 its unclear on why there are just four points.
Number of pellets retrieved positively correlated with elbow lift synergy and stronger fosB activation associated with reduction in elbow lift synergy.
stronger FosB activation corresponds to more representation of forelimb movement which implies more reaches made, it’s unclear on how it is negatively correlated. The opposite trend of synergy seen with intensity and FosB activation is questionable (fig.3d and fig.3e).
Question: Why the fig.3e had only 6 points was not clear? Only in 16 rats (n = 8 SR and n = 8 ER) cortical maps were studied and 2 in each group could not evoke any forelimb movement after stroke. Therefore, the figure shows only the 6 ER rats.
Effects of ER on Cortical Motor Maps
The total forelimb area across all the animals in SR and ER groups was 0.99 mm2.
In 2 animals from each group could not evoke any forelimb movement.
In the SR group the forelimb area was negatively correlated with the infarct volume and positively correlated with the behavioural performance of pellet retrieval.
In ER group no such correlation was observed and for comparable forelimb area the behavioural performance was higher in ER than in SR group.
The slopes of the two lines corresponding to both groups are not that different but still the ER group did not have correlation is questionable.
The representation of DFL movements in the more affected limb significantly higher in the ER group than in SR group, this increased representation altered the behavioural performance and the infarct volume.
Ipsilateral movements were evoked, area was similar and movement representation were not correlated with infarct volume or behavioural performance. Thus, emergence of ipsilateral motor outputs is not affected by ER.
Effects of ER on Motor Cortex and Red Nucleus Neuronal Activity
FosB staining was seen throughout all the layers of ipsilesional and contralesional primary motor cortex (M1) and secondary motor cortex with higher level of staining in the ER which was not statistically significant.
In the red nucleus, ER had significantly higher FosB staining with overall greater activation in the ipsilesional Magnocellular nucleus than in parvocellular cells.
Discussion
The objective of this study is to explore different aspects of recovery and compensation in rats with moderate to severe stroke. In the rats that underwent enriched rehabilitation (ER), there was a reduction in the elbow flexion and elbow lift synergies which resulted in better performance in the staircase task at 5 weeks post stroke compared to the spontaneous recovery (SR) group.
Infarct volume after motor cortex stroke is closely related to impairment and the residual motor representation. The size of movement representation depends on the post- stroke performance. In this study the ER rats with large motor cortex stroke and small total forelimb area have increased proportion of distal forelimb representation enabling them to perform better that than the SR rats with comparable injuries. These results are in line with studies in monkeys with small motor cortex strokes.
Not clear about what is large and small motor cortex strokes. What is large motor cortex stroke how it is quantified?
Their results suggest that the residual motor cortex can be shaped with intensive movement training. Nevertheless, it’s worth noting that in two of the ER forelimb movement could not be evoked, but they claim that even then their recovery was better compared to the SR rats with comparable strokes. The better performance could be because, ER is not exclusively related to motor cortex but also engages all descending pathways as well.
They wanted to explore the descending pathways that are responsible for better recovery in ER rats. And in the previous study, only the ER rats showed significantly higher fosB staining in the contralesional and ipsilesional primary motor cortex which was not see in EE group. Whereas in this study although there was trend of higher staining in ER rats it was not significantly different from SR rats. The dissimilarity could be because of smaller sample size and/or different survival times. Also, in the previous study the staining was assessed 2,5 and 10 days after stroke, but in this study the staining was assessed after 28 days. They report that the reason behind stroke recovery might be due to cortical reorganisation initially which is followed by changes in the modification in the brainstem and spinal circuitry. They also cite few studied in support of this argument where one of the studies resulted in sprouting of corticospinal fibres into denervated regions of cervical spinal cord and brainstem. Other study demonstrates that the brainstem pathway takes over the function of corticospinal neurons, as the recovery of skilled reaching was not observed when the sprouting of the corticorubral fibres was blocked.
The main objective of the study was to determine if the recovery due to enriched rehabilitation was due to compensation, restitution or both. Previous kinematic studies were all qualitative methods providing evidence for both compensation and restitution, this study was quantitative method and showed that intense training that is known for restoring poststroke performance did not result in full recovery. The reason for better performance in ER rats could be due to combination of both restitution and compensation. The limitation of the study includes use of young, male rats and their future work is to verify the results with other stroke models and species.
This study shows that the ER intervention produced neuroplastic changes as reported in previous studies but without the need of including invasive intervention such as optogenetic stimulation. Post-stroke ER rats also had reduced kinetic synergies which restored the reaching performance not just by compensation but at least in part by restitution. Therefore, they claim that ER rats is emerging as a promising candidate for clinical translation.