| ObjectiveTo investigate the efficiency and mechanism of LIUPS for improving the Schwann cells’ proliferation and the regeneration of the autografted nerves in rats.MethodsIn vitro, SCs of SD rats respectively received 250mW/cm2,500mW/cm2 and 750mW/cm2 LIPU treatment, once a day,5min per time, lasting for 1～7days. Ordinary microscopic examination, immunofluorescence staining and CCK8 detection method were taken to detect their proliferation. RT-PCR was taken to detect the expression of S100, GFAP, CNTF and BDNF. In vivo, autograft reversal transplantation sciatic nerves respectively received 250mW/cm2,500mW/cm2 and 750mW/cm2 LIPU treatment, once every two days,5min per time, lasting for 2-12weeks. Catwalk analysis, contrast-enhanced ultrasonography, electrophysiological studies, morphological investigations and histopathology examination, recovery rate of gastrocnemius muscle wet weight and western blotting analysis were carried out from 2 weeks to 12 weeks postoperation.Results:Ordinary microscopic examination, immunofluorescent staining and CCK8 detection showed that the cell proliferation of the low-dose group was the fastest(P< 0.01), while RT-PCR results showed that the expression of S100、CNTF、BDNF in the low-dose group was the most at the third day after treatment while the expression of CNTF、BDNF in the low-dose group was the most at the seventh day after treatment (P <0.05), the expressions of GFAP in the low-dose group were the least at the third day and at the seventh day after treatment (P<0.05).From 2 weeks of survival the low-dose group of rats exhibited a significantly improved functional recovery than the other three groups. PI and AUC calculated by contrast-enhanced ultrasonography in low-dose group were higher than in other groups from 4 weeks after operation (P<0.05). Electrophysiological testing at 12 weeks after operation showed that the recovery indexes (peak amplitude and area under the CMAP curves) in low-dose and mid-dose treatment groups were significantly higher than control group, and the low-dose group was the highest (P<0.05). There was no significant difference between those of high-dose group and of control group (P>0.05). These results were accordance with regenerational myelinated nerve fiber density, thickness and diameter of axons of each group at the end time point. The far lengths of axon growth marked by the immunopositivity of NF200 proteins at 8 weeks showed that axons of low-dose group grew fastest in the four groups, axons of mid-dose group the second and axons of control group at last. The wet weight recovery ratio of gastrocnemius muscle (injuried side/control side) of each group and the area of the cross-sectional gastrocnemius muscle fibers in low-dose group were significantly larger than those of other three groups all the time (P<0.05). Western blotting (WB) detection showed that expression of NF-κB p65 protein in low-dose group was lower than other groups from 4 weeks after operation. PI and AUC were highly linear positive correlative with recovery index of CMAP curves (r>0.70) and were highly linear negative correlation with WB 12-week postoperation (r<-0.70).There is no significant difference among the temperatures of irradiation at the starting point、ending point and the highest temperature during the LIPUS treatment.Conclusion:This study showed that LIPUS could improve the Schwann cells’proliferation and the best intensity was 250mW/cm2. LIPUS could improve autologous sciatic nerves regeneration and LIPUS of 250mW/cm2 worked best by inducing faster rate of axonal regeneration. There was no obvious temperature change during the LIPUS treatment on sciatic nerves. Thermal effect was not the main mechanism of the LIPUS treatment. |
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