Combining Neurotrophins, Chondroitinase and Schwann Cells to Improve Recovery after Rat Spinal Cord Injury

Mary Bartlett Bunge, Ph.D. Christine E. Lynn Distinguished Professor of Neuroscience Professor of Cell Biology and Anatomy, Neurological Surgery, and Neurology

The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine

Introduction: We have shown after contusion injury (Golden et al., 2007) that genetically manipulating Schwann cells (SCs) to secrete a bi-functional neurotrophin (D15A) leads to a large increase of axons in the SC transplant, about 26,000 myelinated axons, compared to 4500 in the control SC transplant. There was no improvement in hindlimb locomotion, possibly because the axons did not leave the implant to grow back into the spinal cord. When we explored the combination of olfactory ensheathing cells and chondroitinase in our rat spinal cord complete transection/SC bridge model (Fouad et al., 2005), addition of the enzyme was more effective than without it; there were significant improvements in locomotion. Other laboratories have shown efficacy of chondroitinase in SCI repair. Our hypothesis is that the combination of neurotrophins with chondroitinase and SCs will improve repair following spinal cord injury more than either the bi-functional neurotrophin or chondroitinase with the SC graft. The complexity of spinal cord injury will require a combination strategy (Bunge, 2008).

Specific aims: The aim is to combine SC transplantation with a bi-functional neurotrophin (having BDNF and NT-3 activity) and chondroitinase to improve recovery after contusion injury. The neurotrophin and the enzyme will be secreted from genetically modified SCs. Even if only a small percentage of the large number of axons leaves the implant, locomotor function may improve. Another aim is to train a younger person to be able to perform a study such as this. We, at the Miami Project, aim to increase the number of spinal cord injury and repair laboratories around the world.

Methods and Experiments: SCs will be transduced by means of a viral vector to express a marker GFP, GFP/D15A, another marker, mCherry, or mCherry/chondroitinase (collaboration with J Rogers and E Muir, London). The vectors, prepared in the Miami Project Viral Vector Core, lead to secretion of D15A and chondroitinase that are both biologically active as determined in culture. This activity will be checked in the spinal cord after transplantation. A contusion injury will be made with the MASCIS impactor. One week later, two million SCs will be transplanted per animal: 1) ½ mCherry SCs + ½ GFP SCs, 2) ½ mCherry SCs + ½ GFP/D15A SCs, 3) ½ GFP SCs + ½ mCherry/chond, SCs, 4) ½ GFP/D15A SCs + ½ mCherry/chond SCs and 5) injury alone. The GFP and mCherry labels will enable visualization of SCs. The only damage will be from contusion and only one injection of SCs. BBB (locomotor) testing, including subscore testing, horizontal ladder and footprint analyses, will be done as well as thermal and mechanical sensory testing; the animals will be maintained for 4 months. Animals will be traced, 8 for the corticospinal tract and 8 to identify responding brainstem neuronal somata. Five animals/group will be prepared for histological and immunostaining evaluation of white matter sparing, lesion volume, SC survival, CSPG accumulation, neurofilaments, serotonin, DBH, sensory fibers (CGRP), GFAP, ED-1, PKC gamma (for corticospinal tract), GAP-43 and myelinated axons. Some tissue will have been preserved for examination in the electron microscope if warranted.

Deliverables: If we find that the combination of a bi-functional neurotrophin and chondroitnase along with SC transplantation yields a better repair of the spinal cord than SC transplantation alone, then there is a clear indication of the direction in which we must go to design a human therapeutic strategy. Both of these interventions, neurotrophins and chondroitinase, are two of the most promising approaches for improving repair after spinal cord injury. This experiment depends upon genetic modification of the SCs. Whereas there may be concerns about treating cells in this way for clinical applicability, more and more information is accumulating about how to genetically modify cells in ways that enable turning on and then turning off a gene; genetic manipulation will become more and more feasible for translation to humans. What we find may, therefore, will instruct us about treating humans with spinal cord injury. Also, a young investigator will have received valuable training and experience that will help launch his career and set up his/her own laboratory.