Improved Locomotor Recovery in a Rat Model of Spinal Cord Injury by BioLuminescent-OptoGenetic (BL-OG) Stimulation with an Enhanced Luminopsin.

Irrespective of the many strategies focused on dealing with spinal cord injury (SCI), there is still no way to restore motor function efficiently or an adequate regenerative therapy. One promising method that could potentially prove highly beneficial for rehabilitation in patients is to re-engage specific neuronal populations of the spinal cord following SCI. Targeted activation may maintain and strengthen existing neuronal connections and/or facilitate the reorganization and development of new connections. BioLuminescent-OptoGenetics (BL-OG) presents an avenue to non-invasively and specifically stimulate neurons; genetically targeted neurons express luminopsins (LMOs), light-emitting luciferases tethered to light-sensitive channelrhodopsins that are activated by adding the luciferase substrate coelenterazine (CTZ). This approach employs ion channels for current conduction while activating the channels through treatment with the small molecule CTZ, thus allowing non-invasive stimulation of all targeted neurons. We previously showed the efficacy of this approach for improving locomotor recovery following severe spinal cord contusion injury in rats expressing the excitatory luminopsin 3 (LMO3) under control of a pan-neuronal and motor-neuron-specific promoter with CTZ applied through a lateral ventricle cannula. The goal of the present study was to test a new generation of LMOs based on opsins with higher light sensitivity which will allow for peripheral delivery of the CTZ. In this construct, the slow-burn Gaussia luciferase variant (sbGLuc) is fused to the opsin CheRiff, creating LMO3.2. Taking advantage of the high light sensitivity of this opsin, we stimulated transduced lumbar neurons after thoracic SCI by intraperitoneal application of CTZ, allowing for a less invasive treatment. The efficacy of this non-invasive BioLuminescent-OptoGenetic approach was confirmed by improved locomotor function. This study demonstrates that peripheral delivery of the luciferin CTZ can be used to activate LMOs expressed in spinal cord neurons that employ an opsin with increased light sensitivity.

Transplantation of mesenchymal stem cells that overexpress NT-3 produce motor improvements without axonal regeneration following complete spinal cord transections in rats.

Transplanting stem cells engineered to overexpress trophic factors can improve motor abilities and facilitate axon regeneration following spinal cord injury. This study compared several transplantation paradigms using mesenchymal stem cells (MSCs) that overexpress the multi-neurotrophin, NT-3/D15A (NT-3-MSCs), to determine if different grafting strategies can elicit improved axon regeneration and/or behavioral outcomes following a complete T9 spinal transection. At one week post-transection, NT-3-MSCs were transplanted above, and at several locations below, the lesion site. A rostral-to-caudal gradient of NT-3-MSCs was produced by incrementally increasing the number of transplanted cells at locations distal to the transection. Motor function was analyzed using the Basso, Beattie, and Bresnahan scale for 7-weeks post-injury. The corticospinal tract was traced using biotinylated dextran amines, while raphespinal fibers were visualized using immunohistochemistry. Cell viability was assessed using transplants of NT-3-MSCs that express tdTomato. Retrograde tracing using fluorogold, as well as spinal re-transections, were performed to discriminate between a supra-spinal or reflexive influence of regained motor functions. NT-3-MSC transplants improved motor outcomes and tissue continuity at the transection site, however retrograde tracing using fluorogold revealed no evidence of axon regeneration. A spinal re-transection also failed to eliminate the improvement in motor outcomes produced by the transplant. We conclude that transplantation of NT-3-MSCs can improve motor function and morphological outcomes following a complete spinal transection without promoting axonal regeneration.

Co-transplantation of mesenchymal and neural stem cells and overexpressing stromal-derived factor-1 for treating spinal cord injury.

Genetic engineering of mesenchymal stem cells (MSCs) and neuronal stem cells (NSCs) has been used to treat spinal cord injuries (SCI). As a mechanism of therapy, MSCs secrete high amounts of trophic factors, while NSCs can differentiate into neuronal lineages and aid in tissue replacement. Additionally, the forced overexpression of secreted proteins can enhance the secretome of transplanted cells, which can increase therapeutic efficacy. This study utilized a combinational treatment consisting of MSCs, NSCs, and the forced overexpression of the chemokine stromal-derived factor-1 (SDF-1) from MSCs (SDF-1-MSCs) as treatment in a rat model of SCI. Transplants occurred at 9-days post-injury, and motor functions were evaluated for 7-weeks post-injury. White matter sparing and axon densities surrounding the lesions were quantified. Findings from this study demonstrate that co-transplanting SDF-1-MSCs with NSCs improved motor functions and enhanced axon densities surrounding the lesion. However, no improvements in white matter sparing were found and tumors were found in some of the animals that received co-transplantations with either SDF-1-MSCs and NSCs or unmodified-MSCs and NSCs, but not in any animal treated with a single cell type. This study offers evidence that providing SDF-1 to NSCs, through the forced expression from MSCs, can enhance the therapeutic potential of the graft, but developing a safe means of doing this requires further work.