| Peripheral nerve injury, especially injured in proximal brachial plexus, usually lead to poor nerve and muscle recovery. Since the nerve regeneration speed is low and there is always a long distance for regenerated nerve to reach the target muscle, the distal nerve stump and muscle are remained in a prolonged denervated status, which results in their chronic denervation and nerve fibrosis. To deal with this situation, a nerve ‘babysitting'technique performed by suturing an intact healthy nerve graft to the distal injured nerve stump was invented. It has been reported to be capable of preventing muscular denervation and promoting re-innervation. However, for peripheral nerve injuries occurred in the proximal limb, the intact healthy nerves are usually not available. We have previously found that embryonic neurons, after transplanted into the distal nerve,can survive, sprout axons and reduce muscular denervation. Taken this together, we use a tibial nerve transection injury animal model to investigate, 1. Whether the transplantation and ‘babysitting' of primary embryonic spinal cord cells into the distal tibial nerve stump can alleviate the chronic denervation of distal nerve stump and target muscles. 2. Whether the distal stump can re-undergo a pre-denervation when terminating the cell ‘babysitting'. 3. Whether the cell transplantation and ‘babysitting'technique can promote axon regeneration and functional recovery.Part ?: The Survival and Differentiation of Embryonic Spinal Cord Cells at Different Culture Stages after Transplanted into Injured Peripheral NerveObjective: In in-vitro culture, neural stem cells can survive, sprout axons and reduce muscular atrophy after transplantation into the distal stump of injured peripheral nerve.In this study, we investigate whether embryonic spinal cord cells of different passages can survive and differentiate after transplanted into peripheral nerve.Methods: Spinal cords from E13.5 transgenic Sprague-Dawley(SD) rats expressing green fluorescent protein(GFP) were dissected out and prepared for single-cell suspension(primary, P0). Cells were passaged to the third generation(P3)and the P0, P1 and P3 cells were performed with in-vitro characterization. Another 30 female SD rats were included and performed with tibial nerve(TIB) transection. After a7-day distal nerve denervation stage, animals were randomly divided into 3 groups. The above-prepared P0, P1 and P3 embryonic spinal cord cells were injected into the distal stump of tibial nerve respectively. After 3 months, the transplantation regions were processed for immunohistochemistry staining to examine the survival and differentiated status of cells.Results: For the in-vitro cell characterization, most of the primary embryonic spinal cord cells were ?-tubulin positive neural progenitor cells(NPCs). However, with the cell passaged, the percentage of such neural progenitor cells decreased significantly while the percentage of GFAP-positive astrocytes increased. Three months after transplantion, the in-vivo characterization outcomes were in accordance with the in-vitro results, which demonstrated that the expression of NEUN and CHAT of survival cells decreased progressively as passaged.Conclusions: Embryonic spinal cord cells are rich in neurons and NPCs. With the increased cell passage, the percentage of neurons decreased gradually. In this case,using primary embryonic spinal cord cells for transplanting into injured distal nerve remains a better choice.Part ?: Embryonic Spinal Cord Cells ‘Babysitting' Distal Injured Nerve Reduces the Denervation of Distal Nerve Stump and MusclesObjective: Our study in part I showed that, embryonic spinal cord cells transplanted into an injured peripheral nerve can survive and differentiate into a high proportion of neurons. In this perspective, Part II experiment is aimed to test: 1. The effect of transplanted primary embryonic spinal cord cells for ‘babysitting' the distal tibial nerve stump on chronic denervation of distal nerve stump and target muscles. 2.The function of schwann cells after terminating ‘babysitting' and removing the‘cell-babysitting' nerve segment.Methods: 30 female SD rats were included in this experiment and performed with tibial nerve(TIB) transection. After a 7-day distal nerve denervation stage, animals were randomly divided into 2 groups. In the experimental group, 3ul of primary embryonic spinal cells(P0, 10^6/ul) were transplanted into the distal tibial nerve stump using a 33 S gauge Hamilton Syringe. The same amount of neural basal medium were injected in the control group. After three months, the electrophysiology,immunohistochemistry staining, electron microscope, RT-PCR, Western Blot tests were performed in 10 rats of each group to compare the differences in gastrocnemius atrophy,muscles reinnervation, axons counting, neurotrophic factor(BDNF,GDNF,NGF,NT-3)and myelin protein zero(MPZ) expression levels of the distal nerve stump. For the remaining 5 rats in each group, the transplantation site were cut away to end the‘babysitting'. Two weeks later, the neurotrophic factors and myelin protein expression levels of two groups were examined, to contrast that they have or not significant difference.Results: The results showed that the experimental group has a significantly higher counting of total myelinated axons in the distal nerve stump. The gastrocnemius muscle atrophy was better prevented. The reinnervated neuromuscular junctions can be seen and action potentials can be evoked in all rats. In contrast, in the control group, allaxons broke down. The gastrocnemius underwent severe atrophy. Neither remyelination nor motor endplates could be found and the action potentials could not be evoked in any rats. The expression level of BDNF, GDNF, NGF, NT-3 were similar between the 2groups. However, two weeks after the termination of ‘babysitting', the expression of MPZ decreased while the level of BDNF, GDNF, NGF and NT-3 significantly increased.Conclusions: After transplanting embryonic spinal cord cells to ‘babysit' the distal stump of injured peripheral nerve, these cells can robustly survive and differentiate into neurons and motoneurons. Subsequently, axons sprout and establish functional neuromuscular junctions with the target muscle. The chronic denervation of distal nerve stump and muscles are thus alleviated. When the ‘babysitting' was terminated after cutting away the transplantation region, the distal nerve stump will undergo a new denervation process with high expression levels of neurotrophic factors.Part ?: Embryonic Spinal Cord Cells ‘Babysitting' Distal Nerve Promotes Axon Regeneration after Delayed Nerve RepairObjective: To study whether embryonic spinal cord cells ‘babysitting' the distal injured nerve can promote axon regeneration and functional recovery after delayed nerve repair.Methods: 20 female SD rats were included and randomly divided into 2 groups after tibial nerve transection for 7-day pre-denervation. The transplantation was performed as described in part II. Three months later, 5mm of cell-transplanted nerve segment was cut away, and the distal tibial nerve stump was immediately cross-sutured to a freshly transected proximal common peroneal nerve stump. Animals were sacrificed 3 months later. Retrograde tracing with Fluorogold, electrophysiology,electron microscope and muscle fiber H&E staining were performed to compare thedifference of amounts of regenerated motor neurons and axons, remyelination of the distal nerve and muscle functional recovery between the 2 groups.Results: Three months after cross-suture surgery, the number of neurons from spinal cord and myelinated axons from distal stump in the cell-transplanted group was much higher than that of the control group. The gastrocnemius muscle fiber cross-sectional area and the electrophysiological signals in the cell-transplantation group showed a better result in the cell-transplantation group as well. The differences were significant statistically.Conclusions: Embryonic spinal cord cells ‘babysitting' the distal tibial nerve can promote axonal regeneration and muscular function recovery in a delayed nerve cross-sutured model. |
PDF Full Text Request