| Acute spinal cord injury is a serious disabling disease which can cause movement dysfunction, severe sequelae and complication. All of these would bring huge economic burden for personal, family and society. Acute spinal cord injury includes primary spinal cord injury and secondary spinal cord injury. Secondary spinal cord injury could cause the injury of spinal cord more severe. But the process is reversible and can be controlled. So the research and outcome of secondary spinal cord injury have very important value and significance for the nerve repair and regeneration. At present, the key for the treatment of acute spinal cord injury is focused on the secondary spinal cord injury pathological reaction and mechanism research. Autophagy (autophagy) was discovered in1962by Ashford and Porter, which means that cells have the phenomenon of "eating themselves". A bilayer formed from intracellular membrane could package part of the protein of cytoplasm and cell organelles to form autophagosome, then fuse with lysosomes form autophagy-lysosome, and degrade the contents of the package in order to realize the metabolism of the cell itself. After spinal cord injury, the neurons are the terminal differentiation cells, and the occurrence of autophagy is particularly important. In recent years, more and more studies focus on the occurrence of autophagy and the effect after spinal cord injury.The First Chapter Model of Acute Spinal Cord InjuryAbstract Objective To produce the model of spinal cord injury to provide a good observation model for nerve repair and regeneration. Methods The SPF adult males Sprague-Dawley (SD) rats, with weight between200-250g, were divided into normal, spinal cord injury after4h (hour), Id (days),3d,7d,21d,60d and90d several groups. Each group had eight animals, then was anesthesiaed with10%pentobarbital sodium300mg/kg via intraperitoneal injection. Make the right half of crosscutting acute spinal cord injury model. Use HE staining and toluidine blue staining to detect the pathology detection after spinal cord injury, and then perfom BBB score evaluation of animal movement function after injury. Results During the process of acute spinal cord injury animal models some of the animals were dead which were completed in time. Acoording to HE pathology, in normal spinal cord slices, neurons and glial cells were distributed evenly, cell bodies were fat cells, and the nucleus and cytoplasm boundaries were very clear. After spinal cord injury, the tissue appeared a large number of cavity, many cavity debris in it and liquefaction necrosis. The edema and spongy organization widely occurred in white matter. After3d of spinal cord injury, the right lower limbs of rats were paralysis, with no movement. The averages of BBB scores were0.7points. As the time of the extension of damage, the movement of spinal cord in rats had part of recovery. After60d and90d of spinal cord injury, there was a slight activity in three joints of hind limb. Sometimes even two or three joints had broad movement. The averages of BBB scores were6.8points. Conclusion The right half of crosscutting of spinal cord injury not only could cause half of sensory and motor paralysis, at the same time could be a good observation model for nerve repair and regeneration after spinal cord injury. The Second Chapter The Occurrence of Auophagic Flux after Spinal Cord InjuryAbstract Objective To explore the occurrence of the dynamic process of autophagic flux, and determine the localization of neurons and glial cells after spinal cord injury.Methods With the model of acute spinal cord injury, the protein expression of LC3and Beclinl were detected with Western and immunohistochemical detection at4h, Id,3d,7d and21d after injury. The Beclinl mRNA expression was detected with RT-PCR. The formation of autophagosome was examed with transmission electron microscopy (SEM). The expression of autophagy substrate protein p62was detected with werstern-blot. The colocalization of neurons and glial cells with autophagy was detected using immunohistochemical detection. Results With Western belt density scanning analysis and immunohistochemical detection, after4h of spinal cord injury, the expressions of LC3and Beclinl protein were higher than the normal control. With the extension of time, the expression of both proteins increased. At3d after injury, the amount of expression reached the peak, and at7d and21d after injury, the expression gradually decreased, but still kept an increased level than normal group. Through transmission electron microscope, we found that the normal spinal cord tissue showed normal structure, myelin sheath arranged in concentric circles, interlayer with arranged tightly bound. At4h after spinal cord injury, we found that the nerve demyelination occurred, the arrangement of interlayer happened disorder, appearred a large number of vacuoles. With Western-blot detection, after4h of spinal cord injury, the expressions of p62protein were lower than the normal control. With the extension of time, the expression of both proteins decreased. After colocalization staining, we found that autophagy occurred in neurons and glial cells after spinal cord injury. Conclusion After spinal cord injury, the process of autophagy occurred. The activity of autophagy increased and peaked in3d after injury. Autophagy occurred not only in the neurons, but also occurred in glial cells, which suggested that the activity of the autophagy had a certain influence on the pathological activity of spinal cord injury. The Third Chapter The Role of Autophagy after Spinal Cord InjuryAbstract Objective To explore the role of autophagy in nerve repair and regeneration after spinal cord injury. Methods The autophagy inhibitor3-MA and autophagy enhancer Rapamycin were injected in rats after spinal cord injury via intraperitoneal injection. At4h, Id,3d,7d and21d after spinal cord injury, the expression of Beclinl and LC3were detected by Western and immunohistochemical detection. BBB score method was used to detect the role of autophagy on the recovery of spinal cord function. Results With Western-blot and immunohistochemical detection, we found that at various time points the expression of Beclinl and LC3B was higher than the normal group. At3-MA group, the expression of Beclinl and LC3B was lower than control group at4h, Id,3d,7d and21d after injury. There was statistically significant difference, P<0.05. At Rapamycin group, the expression of Beclinl and LC3B was higher than control group at4h, Id,3d,7d and21d after injury. There was statistically significant difference,P<0.05. With the colocalization of LC3with MAP-2and GFAP, at3-MA group, the expression of LC3B in neurons and astrocytes was lower than control group at4h, Id,3d,7d and2ld after injury. There was statistically significant difference, P<0.05. At Rapamycin group, the expression of LC3B in neurons and astrocytes was higher than control group at4h,1d,3d,7d and21d after injury. There was statistically significant difference, P<0.05. By BBB score, inhibition of autophagy can hinder the recovery of motor function in the rat spinal cord injury, and enhancement of autophagy could promote the recovery of motor function in the rat spinal cord injury. Conclusion After spinal cord injury,3-MA could inhibit the occurrence of autophagy, and Rapamycin could promote the occurrence of autophagy.3-MA could inhibit the occurrence of autophagy in neurons and glial cells, and Rapamycin could promote the occurrence of autophagy in neurons and glial cells. Inhibition of autophagy could hinder the recovery of motor function in the rat spinal cord injury, and enhancement of autophagy could promote the recovery of motor function in the rat spinal cord injury. The Forth Chapter The Mechanism Research of Autophagy to the Nerve Repair and Regeneration after Spinal Cord Injury Abstract Objective To explore the mechanism of autophagy to the nerve repair and regeneration after spinal cord injury. Methods The autophagy inhibitor3-MA and autophagy enhancer Rapamycin were injected in rats after spinal cord injury via intraperitoneal injection. At4h, Id,3d,7d and21d after spinal cord injury, the expression of activated caspase-3were detected by immunohistochemical detection. The expression of Bcl-2and BAX protein was detected by Western-blot. BBB score method was used to detect the role of autophagy on the recovery of spinal cord function. Results With the colocalization of activated caspase-3with MAP-2and GFAP, at3-MA group, the expression of activated caspase-3in neurons and astrocytes was higher than control group at4h,1d,3d,7d and21d after injury. There was statistically significant difference, P<0.05. At Rapamycin group, the expression of activated caspase-3in neurons and astrocytes was lower than control group at4h, Id,3d,7d and21d after injury. There was statistically significant difference, P<0.05. With Western-blot detection, we found that at various time points the expression of Bcl-2was higher than the normal group. At3-MA group, the expression of Bcl-2was lower than control group at4h,1d,3d,7d and21d after injury. There was statistically significant difference, P<0.05. At Rapamycin group, the expression of Bcl-2was higher than control group at4h,1d,3d,7d and21d after injury. There was statistically significant difference, P<0.05. With Western-blot detection, we found that at various time points the expression of BAX was lower than the normal group. At3-MA group, the expression of BAX was higher than control group at4h, Id,3d,7d and21d after injury. There was statistically significant difference, P<0.05. At Rapamycin group, the expression of BAX was lower than control group at4h,1d,3d,7d and21d after injury. There was statistically significant difference, P<0.05. Conclusion After spinal cord injury, the inhibition of autophagy could induce the apoptosis of neurons and glial cells, the enhance of autophagy could inhibit the apoptosis of neurons and glial cells; The inhibition of autophagy could inhibit the expression of Bcl-2, promote the expression of BAX, enhanced autophagy could promote the expression of Bcl-2, inhibit the expression of BAX. |
PDF Full Text Request