| Background:Brachial plexus injuries(BPI)may lead to varying degrees of motor and sensory dysfunction,among which severe neuropathic pain may occur after avulsion of anterior cervical root.Due to the simultaneous involvement of central nervous system and peripheral nervous system,involving a variety of complex pathophysiological changes,such as neuroinflammation,glial cell activation,etc.,conventional analgesic drug therapy is difficult to obtain satisfactory clinical efficacy.Currently,the drugs used in the clinical treatment of neuropathic pain include non-steroidal anti-inflammatory drugs,antiepileptic drugs,antidepressants and opioids.Although a variety of drugs can be selected or even used in combination,the clinical satisfaction rate is still less than half,and it is also affected by different degrees of side effects.In recent years,many researchers will devote themselves to the study of tissue engineering,hoping to obtain more satisfactory results.Hydrogel is a kind of noninvasive ways into the damaged parts of the tissue engineering material,has good biocompatibility,biodegradability,plasticity,and using drugs,cell,nanoparticles,such as local release,intensive treatment effect,in the field of functional recovery and nerve regeneration after nerve injury has a good application prospect.Visible light crosslinking of injectable methyl propyl dilute acid gelatin(Gel MA)hydrogel,contains l-arginine glycine-aspartic acid sequence,the cell adhesion,differentiation and proliferation were a positive role in promoting,has been widely used in the heart,skin,bones,etc in the field of study,has also been shown to play an important in the study of spinal cord injury of physiological function.To this end,we proposed to build a Gel MA based hydrogel biological scaffold system,by adding chitosan modified gold nanoparticles(Cs-Au NPs),using gold nanoparticles unique biological properties,inhibit the proliferation and differentiation of glial cells,inhibit the production of proinflammatory cytokines,inhibit nerve inflammation levels relieve painful neuropathy after brachial plexus injury reason,and gold nanoparticles with good electrical conductivity.Electrical stimulation therapy has been reported to effectively improve the recovery of nerve function after nerve injury,interfere with the "gate change" of nerve conduction,and inhibit the proliferation and activation of glial cells.Considering the important role of neuroinflammation in the occurrence and development of neuropathic pain,we planned to construct a Gel MA hydrogel containing Cs-Au NPs,and combined with electrical stimulation to treat neuropathic pain after brachial plexus injury.In the second part of this study,Cs-Au/Gel MA hydrogel was constructed and its characterization was tested.In the third part,the therapeutic effect of Cs-Au/Gel MA hydrogel and electrical stimulation on neuropathic pain was tested by animal model.Objective:To study the occurrence and development of neuropathic pain after brachial plexus injury,as well as the changes of glial cells and neuroinflammation during this process.Tissue engineering and physical therapy were used to treat neuropathic pain after brachial plexus injury.A biodegradable,hydrophilic Cs-Au/Gel MA with potential electrical conductivity,good biological safety was constructed,and the appropriate electrical stimulation frequency was selected.Analysis of nerve stimulation,Cs-Au/Gel MA hydrogels and the joint application of neuropathy caused by rat brachial plexus injury pain model behavior changes,changes in the biochemical changes,cytology,evaluate its role in the mental derangement rational pain treatment and mechanism,inferring the possible is by reducing the activation of microglia and astrocytes,proliferation,inhibit the production of proinflammatory cytokines,inhibiting nerve inflammation to relieve painful neuropathy rationality.Methods:1.The adult female SD rats with mechanical withdrawal threshold(MWT)of4g were selected to establish the pganglionic cervical root avulsion(PCRA)model(blunt avulsion of the posterior root of the C6-C8 cervical nerve)to study the behavioral,cytological and biochemical changes after brachial plexus injury in rats.2.By comparing with control group,after the damage of 1,2,3,5 and 7,the two groups of rats were measured MWT and thermal withdrawal latency(TWL),after each test behavior,will be put to death for the rat spinal cord tissue of rats with Western-blot examination,examination indexes including ionized calcium binding adaptor molecule 1(Iba-1),glial fibrillary acidic protein(GFAP),interleukin-1?(IL-1?),interleukin-6(IL-6),and tumor necrosis factor-?(TNF-?).3.CS-Au/Gel MA hydrogel was prepared and characterized.Cs-Au NPs were firstly synthesized,and then the optimal concentration was selected for the synthesis of Cs-Au /Gel MA hydrogel through cell experiment.4.Gelma hydrogel was synthesized with gelatin as raw material,and its solid-liquid conversion performance was tested.Cs-Au/Gel MA hydrogel was synthesized to test its hydrophilicity and degradation.5.After screening the most suitable frequency of electrical stimulation through cell experiments,it was applied to animal experiments.6.The experimental animals were randomly divided into PCRA group(no treatment)ES group(200 Hz electrical stimulation treatment)CS-Au/Gelma hydrogel group(local injection of CS-Au/Gelma hydrogel for injury)combined treatment group(local injection of CS-Au/Gelma hydrogel for injury);After 3 days of treatment,the spinal cord tissues of the rats were sacrificed for immunofluorescence staining,and the contents of astrocytes and microglias were detected.The contents of Iba-1,GFAP,IL-1?,IL-6 and TNF? were detected by Western-blotting.The levels of brain-derived neurotrophic factor(BDNF),Superoxide dismutase-1(SOD1),Superoxide dismutase-2(SOD2),nitrous oxides-2X(NOX2),nitrous oxides-4(NOX4)in the spinal cord tissue of the affected segment were detected by immunofluo-rescence staining;7.The behavioral changes of rats in the PCRA group,the ES group,the Cs-Au/Gel MA hydrogel group and the combined treatment group were continuously monitored 3 weeks after modeling--MWT and TWL.Results:1.Compared with the SHAM group,the postoperative MWT and TWL of PCRA model rats were significantly decreased.It can be seen that although the SHAM group also had pain sensitivity at the early stage after surgery,it could recover quickly by itself;In the PCRA group,the level of pain sensitivity improved slightly at the early postoperative stage,but remained in a state of pain sensitivity after the operation.2.Compared with the SHAM group,the expression of Iba-1 and GFAP in the spinal cord tissue of PCRA model rat increased,that is,the content of glial cells increased;The secretion levels of IL-1?,IL-6 and TNF-? were also increased,that is,the content of pro-inflammatory cytokines was increased,and would reach the peak on the 2nd and 3rd day after injury.Therefore,the first 3 days after surgery were selected as the time window for intervention treatment in subsequent studies.3.The morphology of Cs-Au NPs was uniform and dispersed,and the proliferation of neural stem cells was the best when the concentration was 0.05 mg/m L.Cs-Au/Gel MA hydrogel was prepared at this concentration.4.The contact Angle of 4.0.05mg/m L Cs-Au /Gel MA hydrogel is about 81.2°,and it has good hydrophilicity.After subcutaneous implantation of CS-Au/Gel MA hydrogel in rats,samples were taken 1-5 weeks after surgery and no significant inflammatory reaction was found,and the volume of hydrogel gradually decreased,indicating that it had good biological safety and degradability.5.Both electrical stimulation and CS-Au/Gel MA hydrogel can inhibit the proliferation of glial cells and the production of pro-inflammatory cytokines to a certain extent,but the effect of electrical stimulation is more obvious and the curative effect is more significant.On day 3 after injury,compared with the PCRA group,the expression of Iba-1 in the CS-Au/Gel MA group,the ES group and the combined treatment group were significantly decreased(P < 0.01).Compared with PCRA group,the expression of GFAP in ES group and combined treatment group was significantly decreased(P < 0.01).6.Compared with the PCRA group,IL-1?,IL-6 and TNF-? in the three treatment groups were significantly decreased,and the differences were statistically significant(P < 0.01).7.Electrical stimulation can also effectively inhibit the production of BDNF,while Cs-Au/ Gel MA hydrogel has a more obvious inhibitory effect on oxidative damage.The combination of the two is more effective than either alone.Conclusion:1.There will be obvious neuropathic pain after PCRA,and it will not improve by itself.2.Proliferation and activation of astrocytes and microglia occurred in the spinal cord of rats after PCRA,among which microglia may be more important in the process of pain maintenance.3.Proinflammatory cytokines such as IL-6,IL-1? and TNF-? increased in the spinal cord of rats after PCRA,and their peak value appeared 48-72 h after injury.4.Cs-Au nanoparticles have good biocompatibility,but too high concentration may inhibit cell proliferation.5.CS-Au/Gel MA hydrogel is a kind of good tissue engineering scaffold with good plasticity,biocompatibility and degradability.6.Both ES and CS-Au/Gel MA hydrogels can inhibit the proliferation and activation of glial cells after PCRA,reduce the production of pro-inflammatory cytokines,and inhibit the occurrence and development of neuropathic pain,and the combined application of the two can further improve the efficacy. |
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