Neuro-rehabilitative research is developing novel strategies to enhance the effectiveness of therapies after stroke by using a combination of physical and plasticizing treatments 1-3. Previous studies have shown that repeated optogenetics stimulation of neurons in the peri-lesioned area induces a significant improvement in cerebral blood flow and neurovascular coupling response 4-6. Up to now the mechanisms underneath the reshaping of brain circuitry induced by rehabilitation after stroke are widely unknown. To investigate how rehabilitative therapies shape new cortical maps in the peri-infarct region, we induce a photothrombotic stroke in the primary motor cortex and the expression of Channelrhodopsine 2 (ChR2) in the peri-infarct area on Thy1-GCaMP6f mice. To promote functional recovery after stroke we use both an optogenetic strategy to stimulate targeted excitatory neurons in the peri-lesional region and motor training on a robotic platform (M-Platform) 7. A 473 nm laser repeatedly stimulates ChR2-transfected neurons; the optostimulation is performed five days a week. The motor rehabilitation consists in a pulling task: after the forelimb is passively extended by the linear actuator of the M-platform, the animal has to pull back up to the resting position. By analysing the spatio-temporal calcium dynamic and the reshaping of cortical activation area during the movement throughout the treatment period, we found that the combined treatment restores cortical activation profiles during the forelimb movement. Through behavioural experiments, using Schallert test, we also evaluate changes of forelimb functionality during rehabilitation. Our combination of techniques allows obtaining unprecedented views on cortical plasticity induced by rehabilitative therapies.
KEYWORDS: Robotics, In vivo imaging, Brain, Two photon excitation microscopy, Spine, Neurons, Cortical activation, Optogenetics, Functional imaging, Current controlled current source
Neurorehabilitation protocols based on the use of robotic devices provide a highly repeatable therapy and have recently shown promising clinical results. Little is known about how rehabilitation molds the brain to promote motor recovery of the affected limb. We used a custom-made robotic platform that provides quantitative assessment of forelimb function in a retraction test. Complementary imaging techniques allowed us to access to the multiple facets of robotic rehabilitation-induced cortical plasticity after unilateral photothrombotic stroke in mice Primary Motor Cortex (Caudal Forelimb Area - CFA). First, we analyzed structural features of vasculature and dendritic reshaping in the peri-infarct area with two-photon fluorescence microscopy. Longitudinal analysis of dendritic branches and spines of pyramidal neurons suggests that robotic rehabilitation promotes the stabilization of peri-infarct cortical excitatory circuits, which is not accompanied by consistent vascular reorganization towards pre-stroke conditions. To investigate if this structural stabilization was linked to functional remapping, we performed mesoscale wide-field imaging on GCaMP6 mice while performing the motor task on the robotic platform. We revealed temporal and spatial features of the motor-triggered cortical activation, shining new light on rehabilitation-induced functional remapping of the ipsilesional cortex. Finally, by using an all-optical approach that combines optogenetic activation of the contralesional hemisphere and wide-field functional imaging of peri-infarct area, we dissected the effect of robotic rehabilitation on inter-hemispheric cortico-cortical connectivity.
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