Jeffery D. Kocsis

Transplantation of an Acutely Isolated Bone Marrow Fraction Repairs Demyelinated Adult Rat Spinal Cord Axons

The potential of bone marrow cells to differentiate into myelin‐forming cells and to repair the demyelinated rat spinal cord in vivo was studied using cell transplantation techniques. The dorsal funiculus of the spinal cord was demyelinated by x‐irradiation treatment, followed by microinjection of ethidium bromide. Suspensions of a bone marrow cell fraction acutely isolated from femoral bones in LacZ transgenic mice were prepared by centrifugation on a density gradient (Ficoll‐Paque) to remove erythrocytes, platelets, and debris. The isolated cell fraction contained hematopoietic and nonhematopoietic stem and precursor cells and lymphocytes. The cells were transplanted into the demyelinated dorsal column lesions of immunosuppressed rats. An intense blue β‐galactosidase reaction was observed in the transplantation zone. The genetically labeled bone marrow cells remyelinated the spinal cord with predominately a peripheral pattern of myelination reminiscent of Schwann cell myelination. Transplantation of CD34+ hematopoietic stem cells survived in the lesion, but did not form myelin. These results indicate that bone marrow cells can differentiate in vivo into myelin‐forming cells and repair demyelinated CNS. GLIA 35:26–34, 2001.

Buspirone, 8-OH-DPAT and ipsapirone: effects on hippocampal cerebellar and sciatic fiber excitability

The effects of serotonin (5-hydroxytryptamine; 5-HT), and the novel anxiolytics buspirone, 8-OH-DPAT (8-hydroxy-2-[N,N-dipropylamino]-tetralin) and ipsapirone (TVXQ 7821, 2-[4-[4-[2-pyrimidinyl]-1-piperazinyl] butyl]-1,2-benzisothiazol-3[2H]one-1,1-dioxide-hydrochloride) on fiber excitability were studied in three axon systems; hippocampal Schaffer collateral fibers, cerebellar parallel fibers, and sciatic nerves. In the hippocampus, application of buspirone, 8-OH-DPAT and ipsapirone resulted in reversible, dose-dependent reductions in the amplitude and conduction velocity of action potentials recorded from presynaptic afferent fibers. Although these agents bind to 5-HT1A receptors, 5-HT application, even at very high (1 mM) concentrations, did not alter axonal responses. This suggests that buspirone, 8-OH-DPAT and ipsapirone exert an action independent of a serotonergic mechanism. Similar effects were observed on the cerebellar parallel fibers although the cerebellum does not have an appreciable number of 5-HT1A receptors. To examine the generalized effects of these agents on nerve excitability, rat sciatic compound action potentials were studied with sucrose gap recordings. Whereas 5-HT, 8-OH-DPAT and ipsapirone had no effects even at high concentrations (1 mM), application of buspirone led to reversible decrement of the responses without appreciable change in membrane potential. These results indicate that buspirone, 8-OH-DPAT and ipsapirone have actions on the excitability of hippocampal and cerebellar neurons independent of serotonergic mechanisms. Moreover, buspirone, but not 8-OH-DPAT or ipsapirone, produces decreased sciatic nerve excitability. NS 20393

28 – Transplantation of Peripheral-Myelin-Forming Cells to Repair Demyelinated Axons

This chapter discusses cell transplantation as a strategy for protection and repair of the central nervous system (CNS) in disorders such as multiple sclerosis (MS). It also discusses Schwann cells (SCs) and olfactory ensheathing cells (OECs) as potential cellular tools to remyelinate and to enhance axonal regeneration in the injured spinal cord, and the prospect of using subpopulations of bone marrow cells to remyelinate CNS axons. Transplantation of SCs into the demyelinated rodent spinal cord results in remyelination with a characteristic peripheral pattern. Autologous tissue represents one possible source of SCs for transplantation into patients with demyelinating disease. A large body of work supports the proposal that transplantation of OECs into various spinal cord injury and demyelination models can promote axonal regeneration,remyelination, and functional recovery. Transplantation of SCs, results in improved hind-limb locomotor function in contusive spinal cord injury. Transplantation of OECs into spinal cord injury models mediates some degree of axonal regeneration and functional improvement even when transplantation is delayed. Bone marrow cell transplantation into demyelinated or contused spinal cord has demonstrated remyelination and improved functional recovery, respectively. Moreover, transplantation of bone marrow cells into cerebral ischemia models has demonstrated reduced lesion size and improved functional outcome. The prospect that intravenous delivery of these cells (SCc, OECs, bone marrow cells) could lead to a global neuroprotection, with subsequent repair such as remyelination, is intriguing and should be further explored.

Fluoro-Ruby as a reliable marker for regenerating fiber tracts

Abstract Axon visualization techniques are important in assessing the efficacy of interventional approaches to stimulate neural regeneration. Whereas the labeling of descending tracts in the spinal cord has been well established using the intracortical injection of biotin dextran amine (BDA), the labeling of ascending sensory fibers of the dorsal funiculus is more problematic. Fluoro-Ruby (FR; dextran tetramethylrhodamine; MW 10,000) is a bidirectional permanent tracer, but the retrograde tracing of fibers is particularly prominent, and FR is a highly sensitive tracer that can be applied in discrete injection sites. In the present report, we used FR to efficiently label ascending fibers in the dorsal columns of the rat spinal cord. After transplantation of olfactory ensheathing cells into the transected dorsal funiculus, the application of FR was able to detect regenerating ascending fibers in the spinal cord. Regenerated fibers crossing the injury site were labeled and easily identified. It is likely that the tracer was taken up by damaged fibers. As additional advantages, the labeling is resistant to photobleaching and no additional tissue processing is necessary for visualization. It can be used for in vivo as well as in vitro injections. The findings indicate that FR can be used as a reliable fluorescent marker to study ascending regenerated fibers in the spinal cord axonal regeneration.

LOP08: Myelination and Nodes of Ranvier Formation in the Regenerated Sciatic Nerve by Transplantation of Myelin-Forming Cells and Direct Comparison of Schwann Cells and Olfactory Ensheathing Cells

INTRODUCTION: After peripheral nerve injury Schwann cells reorganize and provide a permissive environment for nerve regeneration. Moreover, it is important that these regenerated axons become myelinated. While endogenous Schwann cells can perform these functions, additional transplantation of Schwann cells or olfactory ensheathing cells (OECs) into transected nerves were shown to facilitate this complex repair process. It is unclear and controversially discussed if Schwann cellor OEC-transplantation results in more effective regeneration. The present study was performed to determine if transplanted Schwann cells and OECs participate in axonal regeneration and remyelination. Results from both groups are directly compared. MATERIAL AND METHODS: Schwann cells or OECs were prepared from GFP transgenic male rodents. Sciatic nerves of female rats were exposed and crushed to transect all axons. Immediately after nerve transection GFP-Schwann cells or OECs were injected distal to the crush site. Twenty-one days later the nerves were removed and prepared for histology. The engrafted cells were identified by GFP fluorescence and FISH for Y chromosome. Immunostaining for sodium channels (NaV 1.6) and paranodal regions (Caspr) was used to define nodes of Ranvier on regenerated axons. Axon counts were performed and the amount of remyelination was determined. RESULTS: The transplantation of identified Schwann cells or olfactory ensheathing cells into an axotomy model results in axonal regeneration and remyelination enhanced by the transplanted cells. The donor myelinforming cells could be identified by GFP fluorescence and by FISH for Y chromosome. The transplanted cells were able to survive and form myelin with mature nodes of Ranvier expressing the sodium channel NaV 1.6 on the regenerated axons. CONCLUSION: These results indicate that engrafted Schwann cells or olfactory ensheathing cells into injured peripheral nerve can integrate and participate in neural repair. The comparison between OEC and Schwann cells from rodent into a peripheral nerve injury model showed that OECs transplantation results in significant better remyelination potential than Schwann cells.