| BackgroundPrimary injury and secondary injury mechanisms are involved in the development and progress of pathology after acute spinal cord injury (SCI). Primary injury occurs shortly after mechanical insult and causes neurological dysfunction which is irreversible. Secondary injury is a complex self-destruction cascade reaction procedure which is a major cause in further reduction of functional recovery, but the exact mechanisms of secondary injury are still not well understood. This secondary injury cascade has been the basis for much of the research into improving neurologic outcome in the acute SCI.Apoptosis, or programmed cell death, may play an important role in secondary spinal cord injury. Apoptosis, a process of cell "suicide", in which the cell actively kills itself at a physiological or pathological condition occurring injured spinal cord. Apoptotic cell lack a "decision" by the cell based on information from its environment, internal metabolism, developmental history, and its genome. As has been shown, the ability to block or limit apoptotic cell can prove beneficial to cell survival and longevity. The ability to regulate apoptotic cell dearth and its relationship to secondary injury is unknown, but could have important ramifications for improved neuronal survival after acute spinal cord injury.Bcl-2 protein is in bcl-2 related family of proteins, which appears to be involved in opposing apoptosis and the relative levels of these proteins determine whether a cell dies or survives. The most common three sites of localization for bcl-2 protein are mitochondria, endoplasmic reticulum, and nuclear membranes. The mechanisms of bcl-2 protein protecting cells are opposing oxidative stress, limiting free radical generation, and suppressing the activation of caspases. Overexpression of human bcl-2 by transgenic technique in rat neurons can improve ischemic injury of brain and spinal cord.Bcl-2 protein can suppress the formation of proapoptotic protein bax, result in the inactivation of bax, and suppress apoptosis. Bax protein and caspase-3 protein is a proapoptotic protein, it can facilitate cell apoptosis. It has been found that may play an important role in secondary spinal cord injury.Minocycline is a synthetic tetracycline derivative that is used clinically as an antimicrobial agent for the treatment of conditions such as acne. In recent years, it has been shown to have many other actions including the inhibition of caspase-1 and caspase-3, which are involved in the generation of interleukin-1 and apoptosis, respectively. Minocycline also inhibits TNF- α mRNA and up-reguiates the expression of IL-10 mRNA. Thus, minocycline may serve as a multifaceted agent that targets the multiple processes involved in mediating cell death and the development of secondary injury in SCI. The aim of this study was to explore the effect of minocycline following SCI in mice. ObjectiveTo investigate neural cell apoptosis and the changes of bcl-2,bax,caspase-3 expressions after SCI in the rat spinal cord; To investigate the effect of minocycline on bcl-2,bax,caspase-3 in the rat spinal cord after SCI and its effectiveness in promoting neurologic recovery after an incomplete rat SCI. MethodsIn the present study, contusion model of SCI using the modified Allen's method that resulted in rats with incomplete paraplegia was used.In the first study, the mRNA expression of bcl-2, bax, caspase-3 were measured using reverse transcription polymerase chain reaction(RT-PCR) and protein expression of bcl-2,bax,caspase-3 were measured by western blot method in the rat spinal cord after SCL and apoptotic cells were measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling ( TUNEL) method.In the second experiment, rats treated with minocycline and SAL post injury, the expressions of bcl-2, bax, caspase-3 mRNA were measured by RT-PCR, as well as the protein expression of bcl-2, bax, caspase-3 quantitated using immunohistochemistry (SABC) in the rat spinal cord after SCI.In the third study, the functional status of rats treated with minocycline, SAL and methylprednisolone(MP) post injury was assessed using the inclined plane test and Basso-Beattie-Bresnehan (BBB) locomotor rating scale for 4 weeks, after which the animals were killed for lesion area studies. Results1. The mRNA expression of bcl-2, bax and caspase-3 was detected in non-injured spinal cord. The expression of bcl-2 mRNA up-regulated at 8 h postinjury (P<0.05) and peaked at 3 d postinjury(P<0.01 , vs. the control group), and dropped after 7 d, reduced gradually to normal level at 21 d postinjury; Bax mRNA expression up-regulated at 4 h after SCI(P<0.05), and peaked at 3 d, remained high level until 7 d postinjury(P<0.01, vs. the control group),and dropped slowly 14 d postinjury; Caspase-3 mRNA expression up-regulated at 8 h after SCI, and rised significantly at 3 d, peaked at 7 d after SCI, remained high level until 14 d postinjury(P<0.01, vs. the control group).2. The protein expression of bcl-2, bax, caspase-3 was detected in non-injured spinal cord. The expression of bcl-2 protein up-regulated at 8 h postinjury (P<0.05) and peaked at 3 d postinjury(P<0.01, vs. the control group), and dropped after 7 d quickly, reduced gradually to normal level at 21 d postinjury; Bax protein expression up-regulated unconspicuously at 4 h after SCI(F<0.05), and peaked at 3 d, remained high level until 7 d postinjury(P<0.01, vs. the control group),and dropped slowly 14 d postinjury; Caspase-3 protein expression up-regulated at 8 h after SCI(P<0.05), and rised significantly at 3 d, peaked at 7 d after SCI, remained high level until 14 d postinjury(P<0.01, vs. the control group).3. The apoptotic cell was detected in non-injured spinal cord seldom. The apoptotic cell up-regulated at 8 h postinjury (P<0.05) and peaked at 3-7 d postinjury(P<0.01, vs. the control group), and dropped after 7 d, reduced gradually to normal level at 21 d postinjury. 4. The expressions of bcl-2 mRNA and protein at each time point after SCI in the minocycline group were significantly higher than that in the SAL control group (P<0.05, P<0.01 vs. the SAL control group). The expressions of bax mRNA and protein at each time point had similar levels in minocycline group and SAL control group (i'>0.05). The expressions of caspase-3 mRNA and protein at each time point significantly lower in minocycline-treated group compared with SAL control group (P<0.05, P<0.01).5. Four weeks after SCI, the lesion area of spinal cord of rats in minocycline-treated group were lower than that in the SAL control group(P<0.01).The locomotion functional scales of rats treated with minocycline was significantly higher than that in the SAL control group within 7-28 days after SCI(P<0.05,P<0.01). The locomotion functional scales of rats treated with minocycline has no significant difference compared with that in rats treated with MP (P>0.05).Conclusions1. The level of bcl-2, bax, caspase-3 mRNA and protein increased significantly at the lesion area after SCI, and correlated with cell apoptosis in the lesion area, which indicates that bcl-2, bax, caspase-3 play an important role in secondary events in SCI.2. The level of anti-apoptosis gene bcl-2 mRNA and protein at the lesion site up-regulated and the level pro-apoptosis gene caspase-3 mRNA and protein were attenuated in the rats treated with minocycline at the early stage after SCI, which suggests that minocycline could attenuate cell apoptosis in the lesion area.3. Minocycline could reduce the lesion area of spinal cord and promote neurologic functional recovery of rats after SCI, which indicates that minocycline may have a significant neuroprotective role on secondary SCI and potential therapeutic properties in the management of SCI.6. One possible of mechanism of minocycline neuroprotective effect after SCI is minocycline attenuates SCI cell apoptosis partly by inhibiting the expression of caspase-3 and up-regulating the expression of bcl-2 at the lesion site after SCI. |
PDF Full Text Request