Multi-modal optical imaging of brain plasticity after stroke

Reduction of blood flow in a tissue or organ is called ischemia; this causes a lack of oxygen and nutrients that lead to damage that can be of different entities according to the affected region and to the time the tissue remains in that status. Stroke affects 15 million people worldwide every year and is one of the leading causes of long-term disability. Plasticity is the ability of the brain to go through structural and functional modification in order to achieve the most effective recovery. Neurorehabilitation protocols based on the use of robotic devices have recently been shown to provide promising clinical results. This kind of rehabilitation has key advantages over conventional physiotherapy as they provide intensive and highly repeatable therapy and offer a quantitative and objective evaluation of the outcome for each patient. Here we targeted a multi-level approach to the investigation of brain plasticity in vivo after photothrombotic stroke. In detail, we combined three microscopy techniques to address different scales of cortical rewiring: multi-photon microscopy on Thy1-GFP-M mice are used to explore synaptic plasticity and axonal rewiring in the peri-infartic and more distal areas; wide-field imaging of Thy1-GCaMP6f mice is used to dissect alterations of cortical functionality and remapping triggered by ischemic damage at the mesoscale level; optogenetics is used to address the remodeling of inter-hemispheric connectivity by performing light stimulation of ChR2 on the contralesional hemisphere and simultaneous optical recording of ipsilesional cortical activation. Two-photon imaging can zoom in to the synapse level and show the structural plasticity of specific classes of neurons in vivo. We used this technique to follow the time-lapse dynamics of the mouse cortical neurons and to analyze the structural modification in microcircuit connectivity over time up to one month after stroke. Mesoscale wide-field imaging, combined with genetically encoded fluorescent indicators of activity, can shine new light on the functional activation over entire hemispheres in the awake mouse. By simultaneous wide-field investigation of a large number of locations, we characterized the functional remapping of the peri-infarct region triggered by robotic rehabilitation. Within this framework, optogenetics can help define cortical connectivity through an all-optical approach. Indeed, we used stimulated optogenetic activation and detection of cortical activation via genetically encoded calcium indicators to reveal the inter-hemispheric cortico-cortical connectivity with fine detail.