| Objectives:1.To investigate the effect of low-dose ultrashort wave therapy(USW)on the differentiation of A1 type astrocytes in the early post spinal cord injury(SCI).2.To investigate the effect of low-dose USW combined with human umbilical cord mesenchymal stem cell(hUCMSCs)transplantation on injured peripheral astrocytes in SCI ratsMethods:1.60 SD rats were randomly divided into three groups:Sham group,only exposed the chest 10(T10)spinal cord and did not strike;Control group,exposing T10 spinal cord,spinal cord contusion was inflicted using Allen’s method without any treatment;Intervention group,spinal cord contusion was inflicted using Allen’s method,low-dose USW was given 24h after SCI,once a day,5 times a week,each time for 7min,until sacrificed.The motor function of rats were evaluated by Basso Beattie Bresnahan score(BBB score).After sacrificed,rat spinal cords were cut longitudinally on a frozen biopsy machine for immunofluorescence(IF)staining to detect the number of A1astrocytes,microglia cells,IBA-1/P65 and P65 nuclear import positive cells.2.90 SD rats were randomly divided into 5 groups:sham operation group(n=6),control group(n=21),low-dose USW group(n=21),hUCMSCs transplantation group(n=21),and low-dose USW+hUCMSCs transplantation group(n=21).Spinal cord contusion was inflicted using Allen’s method without any treatment,hUCMSCs transplantation group group and low-dose USW+hUCMSCs transplantation group was punctured with a microinjector and 5ul cell suspension with cell density of 1×10~6/L was injected punctured with a microinjector and vertically and slowly.In the control group and low-dose USW group were punctured with a microinjector and 5ul fresh cell was injected at the same experimental technique.In the sham operation group,only exposed the T10 spinal cord and did not strike and inject.Rats in the low-dose USW group and low-dose USW+hUCMSCs group were given with low-dose USW therapy once a day and 7min each time one day after SCI.The motor function of the rats were evaluated by BBB score.After sacrificed,SCI rats were subjected to longitudinal sections for HE staining and IF staining to detect the survival and differentiation of hUCMSCs cells after transplantation,the differentiation of endogenous stem cells into astrocytes around the lesion area,and the formation of fibrosis at 4w after SCI.Results:(1)BBB score showed that motor function of rats were gradually improved after SCI,and low-dose USW therapy improved the motor function compared with the control group on day 14(p<0.01 vs.Intervention group).(2)The quantity of microglia gradually increased on 1d,3d and 7d after SCI,and the quantity of IBA-1 in the intervention group was significantly lower than that in the control group(P<0.01 vs.Intervention group).(3)Secondary inflammation gradually increased after SCI,and Nuclear Factor Kappa B(NF-ΚB)pathway was activated.The number of IBA-1/P65co-staining positive cells in low-dose the USW intervention group was significantly lower than that in the control group(P<0.01 vs.Intervention group)in rats of 3d after SCI.At the same time,the number of P65 nuclear import positive cell was significantly lower than that in the control group(P<0.01 vs.Intervention group).(4)After SCI,A1astrocytes gradually appeared,and the A1 in the lesion spinal cord of rats after SCI reached the peak value.At 3d and 7d after SCI,the number of A1 astrocytes in the intervention group was significantly lower than that in the control group(P<0.01 vs.Intervention group).(5)After SCI rat model was constructed,the glial barrier formed by star glial cells differentiated from endogenous stem cells within 1-2w after damage to the surrounding tissues,which played a protective role.(6)hUCMSCs transplanted into SCI rats Survived in lesion area,some of the transplanted stem cells dedifferentiated into astrocytes.The astrocytes located near the lesion area were derived from endogenous stem cells,simultaneously the human stem cell specific marker MAB1281 was not expressed in this region.(7)The low-dose USW enhance paracrine function of hUCMSCs transplanted into SCI rats,thus promoting more endogenous stem cells to accumulate and differentiate into astrocytes,and obstructed the proliferation of necrotic substances and inflammation(P<0.01 vs.control group、low-dose USW group、hUCMSCs transplantation group).(8)The low-dose USW+hUCMSCs group inhibited the fibrous scar,which was not conducive to axonal growth,produced by macrophages and other cells after SCI,and hUCMSCs transplantation played an important role in this process(P<0.01 vs.control group,low-dose USW group).(9)The low-dose USW+hUCMSCs transplantation group could better promote the recovery of rats after SCI(P<0.01 vs.control group,low-dose USW group,hUCMSCs transplantation group).Conclusions:1.Early low-dose USW therapy after SCI inhibited the number of microglia and the release of inflammatory cytokines,thus inhibiting the formation of A1astrocytes and promoting the recovery of motor function,which is related to the NF-ΚB pathway.2.Low-dose USW therapy in combination with hUCMSCs cell therapy resulted in more endogenous stem cells gathered around the lesion arand differentiated into astrocytes to form a barrier to inhibit the proliferation of necrotic substances and inflammation,thus promoting the recovery of motor function. |
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