The Clinical And Experimental Study Of The Sacral Plexus Avulsion

High-energy traumas are frequent and may lead to pelvic or sacral fractures. Unstable injuries to the posterior pelvic rings such as sacral fractures and sacroiliac dislocations are often with sacral plexus injuries. The incidence could be as high as 56.8%. Lumbosacral plexus injuries may result in neurologic sympotoms and deficits to the lower extremities and urinary, rectal, and sexual dysfuctions. The functional outcome after surgical treatment is thought to be terrible, and consequently conservative treatment has been advocated.Our previous experimental studies in monkeys confirmed that the resection of the L6 nerve root, the counterpart of S1 in humans, did not affect limb function. Based on the results of these experiments, we attempted to establish the unique distribution of S1 in the lower limb and evaluate the effect of severing the S1 nerve root of the healthy limb. We found that the innervation of S1 in each muscle was compensated for by L4, L5, S2 and S3,thereby causing little damage to the healthy limb. We have demonstrated the feasibility,safety and validity of dividing the S1 nerve root for transfer in patients with avulsion of the contralateral lumbosacral plexus as far as clinical assessment and electrophysiological testing can show.In addition, our previous study on degeneration of spinal anterior horn cells found that the level of apoptosis in spinal anterior horn increased more than the level of survival rate decrease. The phenomenon of autophagy may exist. Our study demonstrated that autophagy was increased in the anterior horn of the spinal cord following spinal root avulsion, and that HMGB1 was able to induce autophagy in primary spinal neurons in vitro and was involved in the process of autophagy following spinal root avulsion in vivo. In addition, HMGB1-induced autophagy played a protective role on the survival of neurons under OGD. The ERK and JNK signaling pathways were involved in the regulation of HMGB1-induced autophagy in neurons. These data indicate that HMGB1 is a critical regulator of autophagy and HMGB1-induced autophagy plays a protective role in spinal neurons against injuries. These findings may provide new insights into the pathophysiological process of spinal root avulsion.Part oneObjective:The aim of this study was to evaluate the feasibility of using the intact S1 nerve root as a donor nerve to repair an avulsion of the contralateral lumbosacral plexus.Method:Two cohorts of patients were recruited. In cohort 1, the L4–S4 nerve roots of 15 patients with a unilateral fracture of the sacrum and sacral nerve injury were stimulated during surgery to establish the precise functional distribution of the S1 nerve root and its proportional contribution to individual muscles. In cohort 2, the contralateral uninjured S1 nerve root of six patients with a unilateral lumbosacral plexus avulsion was transected extradurally and used with a 25 cm segment of the common peroneal nerve from the injured leg to reconstruct the avulsed plexus.Results:The results from cohort 1 showed that the innervation of S1 in each muscle can be compensated for by L4, L5, S2 and S3. Numbness in the toes and a reduction in strength were found after surgery in cohort 2, but these symptoms gradually disappeared and strength recovered. The results of electrophysiological studies of the donor limb were generally normal.Conclusion:Severing the S1 nerve root does not appear to damage the healthy limb as far as clinical assessment and electrophysiological testing can determine. Consequently, the S1 nerve can be considered to be a suitable donor nerve for reconstruction of an avulsed contralateral lumbosacral plexus.Part twoObjective:1.To evaluate whether autophagy was activated in the anterior horn of the spinal cord after sacral plexus avulsion and whether exogenous HMGB1 was involved in the induction of autophagy in the rat spinal root avulsion model. 2. To explored the induction effect and mechanism of HMGB1 on autophagy in vitro. 3 To explore the effect of HMGB1-induced autophagy on the survival of primary spinal neuronsMethods:1. The right L4–L6 nerve roots were exposed and avulsed with a tiny self-made hook(n=6 in each group). In the sham group, the right L4-L6 nerve roots of rats were only exposed. The rats were sacrificed at 0 h, 4 h, 1 d, 3 d or 7 d after surgery. The spinal cord was examined with an electron microscope. The expression of LC3- Ⅱ and p62 were evaluated by western blot analysis. The expression of LC3-Ⅱand Neu N were evaluated by immunofluorescent staining. The SD rats received intraperitoneal injection of 2 mg/kg anti-HMGB1-neutralizing antibody or control chicken Ig Y 30 min before avulsion surgery(n=6 in each group). The rats were sacrificed at 1 d after surgery.Serum HMGB1 level was detected at 1 d after surgery by ELISA. Immunofluorescent staining of HMGB1(Green)and DAPI(Blue) on the avulsed side or the same side in transverse section from rats at 1 d after spinal root avulsion or sham surgery. Electron micrographs of the anterior horn area from rats at 1 d after spinal root avulsion in the control Ig G and anti-HMGB1-treated groups. Autophagosomes containing cytoplasmic components with double membrane structures were observed. Western blot analysis of LC3-II and p62 on the injured side in the anterior horn tissue of spinal cord. Immunofluorescent staining of Neu N(Red), LC3-II(Green) and DAPI(Blue) on the avulsed side in transverse section from rats at 1 d after spinal root avulsion in the control Ig G and anti-HMGB1-treated groups.2. Primary spinal neurons in vitro at 7 d were treated with HMGB1(1μg/ml) for different times. The expression of LC3-II was evaluated by Western blot and immunofluorescence analysis. The expression of p62 was evaluated by Western blot analysis. Neurons were treated by HMGB1(1μg/ml) for indicated times, and then p-JNK,total-JNK, p-ERK, total-ERK, p-p38 and b-actin were detected by western blotting. ERK inhibitor PD(20μM), JNK inhibitor SP(20μM) and P38 MAPK inhibitor SB203580(10μM) were separately added to the culture medium 1 h before treatment with HMGB1(1μg/ml) for 36 h. LC3-II was measured by Western blot analysis. Primary spinal neuronsin vitro at 7 d were divided into two groups. Group A: control group, group B:HMGB1-treated group, group C: oxygen glucose deprivation group(1 hour or 4 hours),group D: HMGB1+oxygen glucose deprivation group(1 hour or 4 hours) and group E:HMGB1+ oxygen glucose deprivation(1 h or 4 h) + chloroquine treatment group. The neuron survival was detected by CCK-8 assay.Results: The formation of numerous autophagosomes with double-membraned structures was observed in the anterior horn area of the injured spinal cord. It was found that the amount of LC3-II was significantly elevated on the avulsion side after surgery, peaked at 1d. Consistent with increased autophagic flux after spinal root avulsion, autophagic p62 was degraded in a time-dependent manner. Immunofluorescent staining assay showed that the expression of LC3-II in the neurons of anterior horn area on the avulsed side was also significantly greater than that in the sham-operation group. The induction of autophagy in the environment of oxygen glucose deprivation increased cell survival rate, while autophagy inhibitor chloroquine significantly decreased cell survival rate.Conclusion:Autophagy was induced in the anterior horn of the spinal cord after spinal root avulsion.Intraperitoneal administration of anti-HMGB1 Ig G inhibits autophagy in the anterior horn of the spinal cord following spinal root avulsion. HMGB1 induces autophagy in primary spinal neurons. ERK and JNK pathways are involved in HMGB1-induced autophagy in primary spinal neurons. HMGB1-induced autophagy plays a protective role on neuronal survival in damage environment...