Immunohistochemical assessment of rat nerve isografts and immunosuppressed allografts.

OBJECTIVE: Autologous peripheral nerve grafts are commonly used clinically as a treatment for peripheral nerve injuries. However, in research using an autologous graft is not always feasible due to loss of function, which in many cases is assessed to determine the efficacy of the peripheral nerve graft. In addition, using allografts for research require the use of an immunosuppressant, which creates unwanted side effects and another variable within the experiment that can affect regeneration. The objective of this study was to analyze graft rejection in peripheral nerve grafts and the effects of cyclosporine A (CSA) on axonal regeneration. METHODS: Peripheral nerve grafts in inbred Lewis rats were compared with Sprague-Dawley (SD) rats to assess graft rejection, CSA side effects, immune responses, and regenerative capability. Macrophages and CD8+ cells were labeled to determine graft rejection, and neurofilaments were labeled to determine axonal regeneration. RESULTS: SD rats without CSA had significantly more macrophages and CD8+ cells compared to Lewis autografts, Lewis isografts, and SD allografts treated with CSA. Lewis autografts, Lewis isografts, and SD autografts had significantly more regenerated axons than SD rat allografts. Moreover, allografts in immunosuppressed SD rats had significantly less axons than Lewis rat autograft and isografts. DISCUSSION: Autografts have long been the gold standard for treating major nerve injuries and these data suggest that even though CSA is effective at reducing graft rejection, axon regeneration is still superior in autografts versus immunosuppressed allografts.

Basic techniques for long distance axon tracing in the spinal cord.

The regeneration of axons after a spinal cord injury or disease is attracting a significant amount of interest among researchers. Being able to assess these axons in terms of morphology, length and origin is essential to our understanding of the regeneration process. Recently, two specific axon tracers have gained much recognition; biotinylated dextran amine (BDA) 10 kDa as an anterograde tracer and cholera toxin-B as a retrograde tracer. However, there are still several complexities when using these tracers, including the volume that should be administered and the best administration site so that a significant amount of axons are labeled in the area of interest. In this article, we describe some simple procedures for injecting the tracers and detecting them. We also quantified the number of axons at different locations of the spinal cord. Our results show axons labeled from motor cortex injections traveled down to the lumbosacral spinal cord in 2 weeks, while BDA injections into the lateral vestibular nucleus and reticular formation took 3 weeks to label axons in the lumbosacral spinal cord. Moreover, this protocol outlines some basic procedures that could be used in any laboratory and gives insight into the number of axons labeled and how procedures could be tailored to meet specific researcher's needs.