| Part I: The study on neuroprotective effect of dexmedetomidine on acute spinal cord injury in ratsObjective In this study,dexmedetomidine was medicated in different methods on acute spinal cord injury models in rats.The effects ofdexmedetomidine on spinal cord injury were analyzed to explore the mechanism of its protective effect on spinal cord,which will form the basis of its clinicalapplication.Method(1)The modified Allen method was used to establish aspinal cord injury model in rats.180 rats were divided into sham operation group,control group,intrathecal injection group,intravenous injection group and intrathecal injection group plus intravenous injection group,with each group of 36 rats.The lower limbs motor function was evaluated at 1,24 and 48 h in BBB scale.(2)The injured spinal cord segments of rats in each group were collected at 48 hours.The histopathological changes were observed by HE staining.(3)TUNEL staining was used to detect the apoptosis of damaged tissue in rats.(4)The expressions of IL-1?,IL-6,and TNF-? m RNA were detected by fluorescence quantitative PCR and western blot.(5)The rat brain astrocytes and spinal neurons were isolated and cultured,and both types of cells were identified to establish the co-culture system of spinal neurons and astrocytes.(6)The cells were divided into control group,low concentration of dexmedetomidine group(1 nmol / m L),medium concentration of dexmedetomidine group(10 nmol / m L),high concentration of dexmedetomidine group(100 nmol / m L),and the proliferation activity of each group was detected by CCK8 for 48 h.Results(1)The results of neurological function analysis showed that the BBB score of intrathecal injection group,intravenous injection group and intrathecal group plus intravenous injection group was significantly higher than that of control group(P <0.01)at 24 h after acute spinal cord injury.There was no significant difference in BBB score between the intrathecal injection group and intravenous injection group(P> 0.05).The BBB score ofthe rats in the intrathecal plus intravenous injection group was significantly higher than that in the intrathecal injection group or intravenous injection(P <0.01).There was no significant difference in BBB score between intrathecal injection group and intravenous injection group at 48 h after treatment(P> 0.05).The BBB score of intrathecal plus intravenous injection group was significantly higher than that of intrathecal Injection group or intravenous injection(P <0.01).(2)The spinal cord of the sham operation group was normal,the outline of the neurons was clear,the nucleolus structure was clear and complete,and the cytoplasm was deep.In the control group,multifocal hemorrhage was observed in the spinal cord tissue.And at the same time,a large number of neuronal edema,necrosis,and vacuolization in gray matter,infiltration of inflammatory cells,dissolution and disappearance of Nissl bodies,and disappearance of nucleus were also observed.Intrathecal injection group,intravenous injection group,and intrathecal plus intravenous injection group were slightly less than those in the control group.The cells were mildly swollen and the cytoplasm was homogeneous.The intrathecal injection group was similar to the intravenous injection group.The spinal cord injury were mild in the intrathecal plus intravenous group.(3)There was no significant difference in the number of apoptotic cells in the spinal cord injury group at 1 h after the treatment of acute spinal cord injury(P> 0.05). At 24 h after the treatment of acute spinal cord injury,the control group,intrathecal injection group,intravenous injection group,and intrathecal plusintravenous injection group,the number of apoptotic cells in the spinal cord injury group was significantly higher than that in the sham operation group(P <0.01).Compared with the control group,the intrathecal injection group,the intravenous injection group and the intrathecal plus intravenous group,the number of apoptotic cells in the spinal cord injury group wassignificantly lower(P <0.01);there was no significant difference between the intrathecal injection group and the intravenous injection group(P> 0.05),but the intrathecal plus intravenous injection group(P <0.01),and the number of apoptotic cells in the spinal cord injury group was significantly lower.The number of apoptotic cells in spinal cord injury group was significantly lower than that in intrathecal injection group(P <0.01).There was no significant difference between the intrathecal injection group and the intravenous injection group at 48 h after the treatment of acute spinal cord injury(P> 0.05),while the number of apoptotic cells in the spinal cord injury in the intrathecal plus intraveneous group was significantly lower than that in the intrathecal.The difference was statistically significant(P <0.01).(4)There was no significant difference in the expression of IL-1?,IL-6,TNF-? m RNA in the spinal cord injury group(P> 0.05)at 1 h after the treatment of acute spinal cord injury.At 24 hafter acute spinal cord injury,The expression levels of IL-1?,IL-6,TNF-? m RNA in the spinal cord injury group were significantly higher than those in the sham operation group,the control group,the intrathecal injection group,the intravenous injection group and the intrathecal plus intravenous injection group(P <0.01).Compared with the control group,the levels of IL-1?,IL-6,TNF-? m RNA in the spinal cord injury group,intrathecal injection group,intravenous injection group,intrathecal plus intravenous injection group(P <0.01).There was no significant difference between the intrathecal injection group and the intravenous injection group(P> 0.05),but the levels of IL-1? in the spinal cord injury group were significantly higher than those in the intrathecal plus intravenous injection group.The expression levels of IL-6 and TNF-? m RNA were significantly lower than those of intrathecal injection group(P <0.01).There was no significant difference between the intrathecal injection group and the intravenous injection group at 48 h after the treatment of acute spinal cord injury(P> 0.05),but IL-1? and IL-6 in the spinal cord injury group(P <0.01).The expression of TNF-? m RNA was significantly lower than that of intrathecal injection group(P <0.01).(5)Part of the cells adherent growth,the appearance of the cell was round,translucent strong,the cell surface was not observed obvious protrusion protrusion 4 hours after cell inoculation.After 24 h incubation,astrocytes adhere to the bottom of the culture flask,and the appearance of the cells was triangular,rhombic or polygonal.Under the visual field can be observed in a number of cells protruding a number of short protrusions,the shallow surface visible neurons and oligodendrocytes and other cells,the culture medium suspended with non-adherent necrotic cells and tissue fragments.During the 7th day of culture,astrocytes were observed to proliferate significantly.The nucleus was not visible and the cell bodies were flat.Most of the cells on the surface of the cells were exposed to multiple short protrusions,which could be in contact with each other.The cell aggregation degree grew to 70% ~ 80% at the 7th to 9th day of culture.After 4 h of cell inoculation,some cells were observed to adhere to the cells.The appearance of the cells was round and translucent,and the surface of the cells was not protruded.After 24 h incubation,most of the neuronal cells were observed to adhere to the wall,and some astrocytes or fibroblasts were observed in the adherent cells.GFAP immunofluorescence staining showed that the cytoplasm of astrocytes was dyed red and the nuclei were stained blue.Under the visual field can be seen part of the cell surface protruding short protrusions and adjacent to the cells in contact with each other.Immunofluorescence staining of ?III-Tubulin showed that the cytoplasm of the neurons was stained red and the nuclei were stained blue.The cell surface can see multiple protrusions and contact with each other.(6)Compared with the control group,the proliferative activity of the cells in the low,medium and high levels of dexmedetomidine was significantly increased(p <0.01),and the proliferative activity of neuronal cells increased gradually with the increase of the concentration of dexmedetomidine high.The above results suggest that dexmedetomidine has a positive effect on the proliferation of neurons.Conclusion The effect of dexmedetomidine medicated by intrathecal plus intravenous injection on spinal cord injury in rats was superior to those medicated by a single route(intrathecal injection,intravenous injection),which showed better recovery of motor function,reduced neuronal apoptosis,alleviated the inflammatory response,and non-toxic to spinal neurons.Part II: The study of neuroprotective mechanisms of dexmedetomidine in rats with acute spinal cord injuryObjective This study was to investigate the effect of dexmedetomidine on IKK / NF-?B signaling pathway in acute spinal cord injury,and its mechanism was diccussed to provide a theoretical basis for clinical application.Method(1)Wistar rats were divided into sham operation group,the control group(saline given spinal cord injury),BMS-345541 group(intrathecal injection of IKK? inhibitor BMS-345541(50 ?L / kg),dexmedetomidine(intrathecal and intravenous administration of dexmedetomidine,3 ?g / kg).Collect damaged spinal cord samples of each group in rats.(2)The expression of IKK? and NF-?B m RNA was detected by fluorescence quantitative PCR.(3)The expression of IKK? and NF-?B protein was detected by western blot.Results(1)The expression levels of IKK? and NF-?B m RNA in spinal cord tissue of control group,BMS-345541 group and dexmedetomidine group were significantly higher than those in sham operation group(P <0.01).The expression of the levels of IKK? and NF-?B m RNA in BMS-345541 group and mitomidine group were significantly lower than those in the control group(P <0.01).Compared with the BMS-345541 group,the expression levels of IKK? and NF-?B m RNA were significantly decreased in mitomidine group.(2)The expression levels of IKK? and NF-?B in spinal cord tissue of control group,BMS-345541 group and dexmedetomidine group were significantly higher than those in sham operation group(P <0.01).The expression of BMS-345541 group and right The levels of IKK? and NF-?B protein in the metoprolidine group were significantly lower than those in the control group(P <0.01).Compared with the BMS-345541 group,the expression levels of IKK? and NF-?B were significantly decreased in mitomidine group(P <0.01).Conclusion Spinal cord injury might promote the increase of IKK? and NF-?B,while dexmedetomidine can significantly inhibit the expression of IKK? and NF-?B,which could alleviate the inflammatory chain reaction after spinal cord injury by inhibiting IKK? / NF-?B signaling pathway. |
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