Immunosuppression with either cyclosporine a or FK506 supports survival of transplanted fibroblasts and promotes growth of host axons into the transplant after spinal cord injury.
Fibroblasts that have been genetically modified to secrete neurotrophins can stimulate axonal regeneration, rescue injured neurons, and improve function when grafted into a spinal cord injury site. These grafts are usually allografts that require immunosuppression to prevent rejection. In this study, we compared the effects of two immunophilin-ligands (cyclosporine A [CsA] and FK506) that are used clinically to prevent transplant rejection on protection of grafted fibroblasts. As there are risks associated with prolonged immunosuppression, we compared the effects of 2 or 8 weeks of administration of these drugs, in combination with our standard methylprednisolone protocol, in animals that survived for 8 weeks, to determine whether a shorter course of immunosuppression would be effective. Outcome measures included fibroblast survival, infiltration of activated macrophages and microglia into the graft, final lesion size, and growth of host axons into the graft. The graft consisted of a Vitrogen matrix into which fibroblasts were suspended; the graft was placed into a C3/C4 lateral funiculus lesion. The fibroblasts were isolated from a transgenic strain of Fischer rats that produce the marker alkaline phosphatase (Fb/AP). This enabled us to track the grafted fibroblasts and to evaluate the extent of their survival. The grafted matrix filled the lesion cavity. The density of fibroblasts within the matrix differed according to treatment. Fibroblast survival was most robust in animals that received 8 weeks of immunophilin-ligand treatment. FK506 supported greater Fb/AP survival than CsA. ED-1 immunostaining for activated microglia and macrophages showed an inverse correlation between AP immunoreactivity and the density of immune cells within the graft. Thus, prolonged administration of either FK506 or CsA was necessary for maximal fibroblast survival and for limiting the macrophage invasion of the graft. None of the FK506 or CsA protocols modified the size of the lesion, indicating that these immunophilin-ligands had little effect on secondary enlargement of the lesion and therefore little neuroprotective effect. Because immunophilin-ligands have been shown to be neurotrophic, we used RT-97 immunostaining for neurofilaments and calcitonin gene related protein (CGRP) staining for dorsal root axons to visualize axons that grew into the graft. Some axons grew into the matrix even in the absence of immunophilin-ligand treatment, suggesting that the Vitrogen matrix itself is permissive, but all of the immunophilin-ligand protocols were much more effective in eliciting axonal growth. Growth of axons into the transplants was equally increased by drug treatment for 2 or 8 weeks. Thus, both treatments improved fibroblast survival, diminished immune cell invasion, and promoted axonal growth, and a 2-week course of treatment with either immunophilin-ligand was as effective as 8 weeks in stimulating axonal growth.
Hayashi
Yoshikazu
Y
Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129, USA.
Shumsky
Jed S
JS
Connors
Theresa
T
Otsuka
Takanobu
T
Fischer
Itzhak
I
Tessler
Alan
A
Murray
Marion
M
D000818:Animals; D030801:Animals, Genetically Modified; D001369:Axons / Q000187:drug effects; D003131:Combined Modality Therapy; D016572:Cyclosporine / Q000494:pharmacology*; D005260:Female; D005347:Fibroblasts / Q000637:transplantation*; D006085:Graft Survival / Q000187:drug effects* / Q000276:immunology; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D009416:Nerve Regeneration / Q000187:drug effects* / Q000276:immunology; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D013119:Spinal Cord Injuries / Q000188:drug therapy* / Q000276:immunology / Q000473:pathology; D016559:Tacrolimus / Q000494:pharmacology
D007166:Immunosuppressive Agents; D016572:Cyclosporine; D016559:Tacrolimus