| The peripheral nerve injury(PNI), especially the peripheral nervous long gap defectswas clinical challenge in regeneration field. Peripheral nervous long gap defects weresevere diseases which made a sensory and motor function loss.The slow recovery and poorregeneration always lead to disability. Although varieties of treatments such as nervetissue engineering, use of seed cells and neurotrophins, electrical stimulation and newdrugs, were widely researching and developing,the autograft was still the―goldenstandard‖in nervous long gap defects regeneration. However, most scholars only focus onnerve repair effect is close to the autograft ones or not. They all ignored the fixed cycletime of the Wallerian degeneration, which is takes dozens of days. Therefore, looking fora therapy which is good at both repairing effects and treatment times is very important forsurgeons. At present, an immunological demyelination agent made of anti-galactocere-broside(Gal-C) antibodies and guinea pig complement showed a good regenerationperformance in nerve crush and transection injury in Kosins`research. However, first, therecipe of the immunological demyelination agent is the best effect or not is not confirmed.The second, the mechanisms of immunological demyelination agent were not cleared yet.The third, if the immunological demyelination agent can enhance regeneration of long peripheral nerve gaps is not clear and definite.Therefore,we did researches for recipeoptimizations and mechanisms by bridging15mm nerve gaps through autograft in ratmodels with injection of the immunological demyelination agent. Both functionalrecovery and nerve regeneration were detected at predifined time point after surgery.Wefound that this agent was capable of enhancing nerve regeneration and functional recovery.There are mainly three parts related on the demyelination agent experiment.Part one: optimization for immunological demyelination agentObjectives: To find the most optimization scheme for the recipe and test demyelinatingagent is effective.Methods: Find the most optimization scheme for the recipe of the immunologicaldemyelination agent from four formulas through the agent effective in normal sciaticnerve. We used Toluidine blue staining and transmission electron microscopy to observethe effects of four recipes and made statistical analysis for the results.Results: In this experiment we found that these four recipes immunological demyelinationagents have the same effects on the sciatic nerve. Toluidine blue staining and transmissionelectron microscopy results showed a same trend for the the four recipes immunologicaldemyelination agents. Toluidine blue staining showed the most neurons start Swelling,shrinkage and sparse in1-day after injection. While the myelin edge is not whole,thickness is not equal. The transmission electron microscopy results showed that themyelin start to shrink and the myelin structure start to collapse,some of the neurons beginto degeneration. In1-week point, Both toluidine blue staining and TEM results showedthat normal nerve cell structures already disappeared, only some tiny newborn myelinatedneurons remained. And the myelined neurons quantities in all four groups were nostatistical difference(p>0.05).Conclusion:The agent recipe which is1:1ratio of anti-galactocerebroside antibodies andguinea pig complement is the best preparation methods. It was easy to prepared, reduceddrug pollution and waste, avoided the interference of PBS solution. It was considered asthe best recipe and used in follow-up experiment. Part two: The positive and negative effects of the immunologicaldemyelination agent for neurons and Schwann cellsObjectives: To verify the positive and negative effects of immunological demyelinationagent for neurons and schwann cells and ensure the safety of the agent. And it clarify theprinciple of immunological demyelination agent promote nerve growth effect.Methods: We used NeurobasalTM+B-27medium to culture the DRG neurons and spinalcord anterior horn motor neurons, and use conventional methods to culture the schwanncells. And then we added the immunological demyelination agent into the dorsal rootganglion sensory neurons, spinal cord anterior horn motor neurons and schwann cells andobserved the effects of the agent through the immunofluorescence staining and statisticalanalysis.Results: In this experiment we cultured high purity DRG neurons(93±5%), spinal cordanterior horn motor neurons(83±6%) and schwann cells(92±2%). And then we added theimmunological demyelination agent into the cells and found that the axon length inimmunological demyelination agent group was827.6±23.1and in dd H2O group was835.9±22.8for DRG neurons. And the the axon length in immunological demyelinationagent group was374.6±24.7and in dd H2O group was377.2±26.1for spinal cord anteriorhorn motor neurons. The number of schwann cells were168537.3±12657.4and170519.1±13324.4in demyelination agent group and in dd H2O group. And there were noStatistical significance for the results above(p>0.05). The dead-live cell staining showedthat there are only very few cells died in all groups. However, the demyelination agent canchange the schwann cell morphology into triangle or polygon without kill them.Conclusion:The experiments showed that the immunological demyelination agent haveno positive and negative effects on the dorsal root ganglion sensory neurons, spinal cordanterior horn motor neurons and schwann cells. However, it can change form of theschwann cells, it may due to the immunoreaction with specific antigen on the cells. Part three: The regeneration effects and mechanisms ofimmunological demyelination agent in long peripheral nerve gapsObjectives: To verify the immunological demyelination effects of immunologicaldemyelination agent compositions use separately and to evaluate the capacity of theimmunological demyelination agent in nerve regeneration after bridging autograft in longperipheral nerve gaps. And more clarify the mechanisms of the agent.Methods: We observed and analyzed the effects of the agent compositions injectionseparately in autograft-repaired long peripheral nerve gaps through the toluidine bluestaining and transmission electron microscopy. The efficacy of the immunologicaldemyelination agent was investigated by using a combination of morphological andfunctional techniques, including transmission electron microscopy, immunohistochemistry,retrograde tracer, RT-PCR, biological behavior analysis.Results: In this experiment we found that the immunological demyelination agent canonly work with two compositions concurrence.And the compositions injection separatelyhave no demyelinating effect. It proves that the immunological demyelination agent madea typeⅡallergic reactions to gained a demyelinating reaction. The SFI showed that thetarget muscle movement function recovery was better in immunological demyelinationagent group. In transmission electron microscopy pictures, we found the number ofneurons, regenerated nerve area, myelinated nerve average diameter and G-ratio weretestified that the immunological demyelination agent can enhance the nerve regeneration.The FG-retrograde tracer, gastrocnemius muscles HE staining and double-labeledimmunohistochemical staining after using the agent showed an obvious promotion ofnerve regeneration. RT-PCR indicated that the agent follow an allergic reactionsmechanisms.It made the body appear early demyelinating response, brought forward thedemyelinating process of the Wallerian degeneration and the neurotrophin peak, andshorten the regeneration time.Conclusion:In brief, the immunological demyelination agent can enhance the nerveregeneration obviously and made the favorable microenvironment appearance in advance. It is a whole new therapy for long peripheral nerve gaps. |
PDF Full Text Request