Study On Low-frequency Electrical Stimulation To Remodel The Distal Microenvironment Of Nerve Injury To Promote Early Peripheral Nerve Regeneration

Objective Peripheral nerve injury(PNI)is a common clinical disease with increasing incidence and progressively younger age,which often leads to loss of sensory and motor functions and chronic pain,with poor postoperative rehabilitation and high disability rate,bringing many negative effects on patients’ life quality and social productivity.Autologous nerve grafting is the clinical "gold standard" for the treatment of segmental peripheral nerve defects,but there are many shortcomings,such as limited donor nerve sources,secondary damage to the donor area,and inappropriate donor nerve size,so there is still a need to actively search for new strategies to facilitate nerve regeneration.The Wallerian degeneration(WD)experienced by distal nerve stumps after PNI is one of the key factors in remodeling a good microenvironment for nerve repair.More and more studies in recent years have started to focus on the WD process and thus influence nerve regeneration.Brief low-frequency electrical stimulation(ES)is an effective strategy for the treatment of PNI and can enhance the intrinsic repair mechanisms of the peripheral nervous system,but whether this pro-neural regenerative therapeutic potential is through influencing the WD process is currently unknown.Methods(1)The rats sciatic nerve transection injury model was constructed and were randomly divided into two groups: ES group: pulsed electrical stimulation(parameters: 20 Hz,100 μs;stimulation duration: 1 h)was applied to the distal end of the injured nerve immediately after the transection of 1 mm at the middle part of the nerve.Control group: no ES was performed,and the nerve was sutured immediately after 1 mm of transection.The distal nerve segments(approximately 10 mm)were obtained at 1,4,7,14 and 21 days postoperatively,respectively.Immunofluorescence staining for myelin-associated protein MBP and neurofilament protein NF200 was used to visualize myelin and axonal degeneration.Toluidine blue and transmission electron microscopy were used to observe the disintegration of myelin sheaths.CD68 immunofluorescence staining was used to observe the recruitment and infiltration of monocytes/macrophages in the distal part of the injured nerve.(2)RNA-Seq was using to systematically investigate the effect of ES on molecular processes in WD at postoperative day 4.(3)Construction of a silicone tube bridging model for nerve defects in vivo.The experimental animals were randomly divided into 2groups,the ES group(ES was performed on the distal end of the injury immediately after silicone tube bridging)and the Control group(no ES was received after silicone tube bridging).The effect of early repair of sciatic nerve defects in rats by ES was evaluated by gastrocnemius wet weight ratio and Masson staining as well as immunofluorescence staining on 14 and 21 days after operation.Results(1)Rat sciatic nerve transection injury model: at 4 and 7 days postoperatively,immunofluorescence staining showed that ES accelerated the disintegration of axons and myelin sheaths,which had a discontinuous fragmented morphology.At 14 days postoperatively,myelin debris was largely cleared;CD68 immunofluorescence staining results showed that monocytes/macrophages were recruited to the local injury site 1 day after PNI and subsequently infiltrated to the entire distal nerve,while ES further promoted the recruitment of monocytes/macrophages compared with the Control group,and the data were statistically different.RT-PCR results showed that ES promoted the gene expression of neurotrophic factors BDNF and NGF,as well as promoted the de-differentiation of myelin-type Schwann cells(MBP,MPZ)to repair-type Schwann cells(c-Jun,P75).(2)RNA-Seq results at 4 days postoperatively showed that differential genes of ES group were mainly enriched in biological processes such as angiogenesis,extracellular matrix formation and growth factor binding,and the KEGG pathway was enriched mainly in the PI3 KAkt signaling pathway and cytokine-cytokine interaction pathways.(3)Silicone tube bridging model: wet weight recovery rate of gastrocnemius muscle and Masson staining of muscle fibers showed that ES delayed the atrophy of gastrocnemius muscle;immunofluorescence staining of S100 at 14 days after surgery showed that ES improved the migration ability of SCs at the distal end of the injured nerve;immunofluorescence staining of NF200,CD31 and α-SMA at 21 days after surgery showed that ES promoted the early axonal and vascular regeneration.ConclusionsOur study used rat sciatic nerve transection model and silicone tube bridging model to demonstrate that ES promotes early axonal and vascular regeneration and delays target muscle atrophy after PNI by accelerating WD and promoting the secretion of neurotrophic factors.We believe that these effects may be mediated by the PI3K-Akt signaling pathway.