The Effect And Mechanism Of Lithium On The Spinal Neuron Regeneration And Remyelination After Rat Brachial Plexus Root Avulsion And Reimplantaion

Part Ⅰ: The effect of lithium on the spinal neuron regeneration and remyelination after rat brachial plexus root avulsion and reimplantationObjective: To investigate the effect of lithium on axon regeneration and remyelination after brachial plexus root avulsions and reimplantations by using a rat brachial plexus injury model.Methods: Avulse C5-7 dorsal and ventral roots in adult rats, immediately, the C6 ventral root was reimplanted to the exact point of detachment. After surgery, animals were randomly divided into 2 groups. One group was treated with intraperitoneal injection of lithium chloride(85 mg/kg/d), while the other group was treated with saline as vehicle. Terzis grooming test, Electromyography was performed at 6 weeks and 12 weeks after surgery to evaluate the motor functional recovery of the injured forelimb. Surviving of motoneurons and regeneration of axons were examined by immunofluorescent staining and retrograde axonal tracing with fluorogold(FG). Counting of regenerated and remyelianted axons in musculocutaneous nerve by using electron microscopy(EM). To detect the reinnervated rate of biceps brachii by immunofluorescent staining. Quantitative real-time PCR was performed at 2 weeks, 4 weeks, 6 weeks, 12 weeks post operation to detect the expression of neurotrophic factors and myelin gene in ventral horn and musculocutaneous nerve.Results: Lithium chloride treatment accelerated the recovery of motor functions in the early stage after surgery, but at 12 weeks, the motor function of vehicle-treated group equaled to the level of the lithium-treated group. Lithium improved the survival rate and accelerated the regeneration of motoneurons, but failed to increase the number of axons which outgrow into the reimplanted nerve finally. In addition, lithium treatment elevated the proportion of large and myelinated axons in the regenerated axons led to shorter electrophysiology latency. Although lithium can reduce muscle atrophy, the reinnervated rates of muscle were no differences between two groups. Lithium also increased the expression level of BDNF m RNA in motoneurons in the early stage, and last for the entire recovery stage. On the contrast, lithium didn’t influence the neurotrophic factor m RNA in musculocutaneous nerve. Furthermore, our results found increased expression level of Mpz and Pmp22 in the lithium group at early stage post operation in musculocutaneous nerve, and last for the entire recovery stage.Conclusions:We demonstrated that lithium treatment following brachial plexus root avulsion/re-implantation accelerated motor axonal regeneration and remyelination, hence promoted motor functional recovery in rat brachial plexus root injury model. This action may contribute to the enhancement of BDNF in neurons and myelin gene expression in peripheral nerves.Part Ⅱ: The effect of lithium on the expressions of neurotrophic factors and myeline-associated proteins in Schwann cellsObjective: To study the effect of lithium on the expression level of neurotrophic factors and myeline-associated proteins in Schwann cellsMethods: Schwann cells(SCs) were separated and cultured from brachial plexus nerves which had gone through 2-week pre-degeneration by using enzyme digestion combined with cultivation of explants. Different concentrations of lithium were used to treat the SCs and their viability and proliferation ability were examined. The Q-PCR and Western-blot analysis were performed to detect the gene and protein expression of neurotrophic factors and myelin-associated proteins in SCs, respectively.Results: 1-3 m M of lithium chloride had no significant effects on the viability of SCs while a concentration higher than 3m M significantly inhibit the viability of SCs. The Q-PCR results showed that lithium promoted the m RNA expressions of MPZ and PMP22 in SCs. The Western-blot, however, showed that lithium enhanced the protein expression of MPZ while had no effects on the expression of PMP22. Lithium had no significant effects on the neurotrophic factor expression in SCs.Conclusions: Lithium promoted the m RNA and protein expression level of myeline-associated protein in SCs.Part Ⅲ: The effect of lithium on the axon regeneration and neurotrophic factor expressions of dorsal root ganglion neurons.Objective: To study the effect of lithium on the axon regeneration and the expression level of neurotrophic factors in dorsal root ganglion neurons.Methods: Dorsal root ganglion neurons were separated, cultured and purified from E15 rat embryonic spinal cord dorsal root ganglion by using enzyme digestion combined with mechanical methods. The effects of lithium on the axon regeneration of DRG neuron were examined by culturing the tissue explants in-vitro. Q-PCR and Western-blot analysis were used together to detect the m RNA and protein expressions of neurotrophic factors in DRG neurons. Enzyme Linked Immuno Sorbent Assay(ELISA) was performed to detect the BDNF concentrations in DRG neuron culture medium.Results: 1 m M of lithium chloride had no significant effects on the growth of DRG neurons while a higher concentration could significantly inhibit it. Lithium significantly promoted the axon regeneration speed of DRG explants and enhanced the m RNA and protein expression level of BDNF but had no effects on the expressions of other neurotrophic factors. Lithium enhanced the BDNF protein concentration in DRG culture medium.Conclusions: Lithium promoted the BDNF expressions of DRG neuronsPart Ⅳ: The effect of lithium on the myelination in Schwann cell and dorsal root ganglion neuron co-culturesObjective: To study the effect and mechanism of lithium on the myelination in Schwann cell and dorsal root ganglion neuron co-cultures.Methods: The SCs and DRG neurons were prepared as described in Part II and Part III. The SCs were supplemented to the DRG neuron culture medium to create a in-vitro co-culture system. The immunofluorescent staining and Western-blot analysis were performed to exam the effect of lithium on the myelination in co-cultures as well as the BDNF and BDNF neutralizing antibody.Results: Lithium promoted the myelination in co-cultures. These effects were inhibited when supplementing anti-BDNF neutralizing antibody to the culture system.Conclusions:Lithium could promote the myelination process of SCs in DRG neurons in co-cultures. The mechanism is likely through enhancing the BDNF expressions in the co-culture system.