The Longitudinal Epineural Incision And Complete Nerve Transection Method For Modeling Sciatic Nerve Injury

Acute peripheral nerve injury is an urgent need to solve the medical problems, damage after high morbidity, incidence with the social progress increased year by year, to the society and the family brought heavy burden. Scholars have been trying to make the models of peripheral nerve injury which accord with all kinds of injury mechanism, and promote the development and progress of the research on the injury repair.Injury severity, operative technique and nerve regeneration are important factors to consider when constructing a model of peripheral nerve injury. Here, we present a novel peripheral nerve injury model and compare it with the complete sciatic nerve transection method. In the experimental group, under a microscope, a 3-mm longitudinal incision was made in the epineurium of the sciatic nerve to reveal the nerve fibers, which were then transected. The small, longitudinal incision in the epineurium was then sutured closed, requiring no stump anastomosisObjective:Reduced model manufacture difficulty and reduce the model operation technology access threshold, improve the model’s stability, operability and repeatability, nerve injury regeneration repair good prognosis, to fill the gaps in the Sunderland IV degree damage model, neural injury model choice and use provides a new train of thought and method.Methods:1、Animal grouping and model preparationA total of 64 specific-pathogen-free male Sprague-Dawley rats, aged 12 weeks,Sixty-four rats were equally and randomly divided into an experimental group(longitudinal epineural incision and nerve transection method) and a control group(complete sciatic transection method). Rats were deprived of food and water for 8 hours prior to surgery. They were then anesthetized intraperitoneally with 4% chloral hydrate(10 ml/kg), and placed in the prone position. The right hindlimb was shaved and disinfected with an iodophor.In the experimental group, a skin incision approximately 3-cm-long was made in the posterolateral right hindlimb, and the intermuscular septum was bluntly dissected. Using a light microscope at 15× magnification, the sciatic nerve was exposed and dissociated, and a 3-mm longitudinal incision was made in the epineurium, 1 cm inferior to the piriformis, using microscissors. The exposed nerve fibers were isolated using noninvasive microforceps, and sharply transected under direct vision. The two nerve stumps were placed back together if the epineurium remained intact. No retraction or torsion was observed and the stump fitted well without any space. The longitudinal epineural incision was closed with three 9-0 noninvasive sutures.Surgery in the control group was identical to the experimental group, except that the nerve was transected completely without first opening the epineurium. The stumps were trimmed and the epineurium was closed under a microscope, using six 9-0 noninvasive sutures.2、Behavioral analysisEight rats were chosen at random from each group the day before surgery and 0, 2, 4, 8 and 12 weeks after surgery. Sciatic functional index(SFI) was assessed as described previously.3、Electrophysiological analysis:Eight rats from each group were obtained at 4, 8 and 12 weeks after surgery. Motor nerve conduction velocity was measured using a Medtronic Keypoint electromyography machine.4、Hematoxylin-eosin stainingAt 2, 4, 8 and 12 weeks after surgery, eight rats from each group were sacrificed with an overdose of 4% chloral hydrate anesthesia. Approximately 5 mm of the sciatic nerve at the distal anastomotic site was fixed in 10% neutral formalin for 24 hours, dehydrated through a graded alcohol series, embedded in paraffin, and cut into 6-μm transverse sections. The tissue was viewed under a light microscope to examine myelin degeneration and regeneration.5、Masson’s trichrome stainingAt 4 and 12 weeks post surgery, four rats from each group were sacrificed by anesthesia with 4% chloral hydrate. Sciatic nerve sections were prepared as described above, and Masson’s trichrome staining was performed using a kit Axon regeneration and scar formation were observed under a light microscope.6、Transmission electron microscopyAt 12 weeks post surgery, four rats were randomly obtained from each group and sacrificed by anesthesia with 4% chloral hydrate. A 5-mm length of the right sciatic nerve at the distal anastomotic site was fixed in 4% glutaral at 4°C for 24 hours, then in 1% osmic acid, embedded in epoxy resin, and cut into ultrathin sections for uranyl acetate and lead citrate staining. Axon regeneration and myelination were observed under a transmission electron microscope.7、Statistical analysisMeasurement data are expressed as the mean ± SD, and were analyzed using SPSS 17.0 software(SPSS, Chicago, IL, USA). Groups were compared using one-way analysis of variance followed by the Student-Newman-Keuls test. P<0.05 was considered statistically significant.Results:In the control group, the sciatic nerve was completely transected, and the epineurium was repaired by anastomosis. At 2 and 4 weeks after surgery, Wallerian degeneration was observed in both groups. In the experimental group, at 8 and 12 weeks after surgery, distinct medullary nerve fibers and axons were observed in the injured sciatic nerve. Regular, dense myelin sheaths were visible, as well as some scarring. By 12 weeks, the myelin sheaths were normal and intact, and a tight lamellar structure was observed. Functionally, limb movement and nerve conduction recovered in the injured region between 4 and 12 weeks. The present results demonstrate that longitudinal epineural incision with nerve transection can stably replicate a model of Sunderland grade IV peripheral nerve injury. Compared with the complete sciatic nerve transection model, our method reduced the difficulties of micromanipulation and surgery time, and resulted in good stump restoration, nerve regeneration, and functional recovery.Conclusions: In summary, we have successfully established a nerve injury model using a longitudinal epineural incision with complete nerve transection. This method substantially reduces the difficulty and time required for the microoperation, obtains good stump anastomosis and nerve repair, elevates operability and stability of model establishment and use, and replicates a Sunderland grade IV injury.