Influence Of Cell Cycle Modulation On Microenvironment Of Axonal Regeneration After Spinal Cord Injury In Rats

Part One Influences of Cell Cycle modulation on inflammatory response after spinal cord injury in ratsObjective: Microglial cells, the immune cells in the CNS, are significantly activated after spinal cord injury and produce a large number of pro-inflammatory cytokines which are detrimental to the cord tissue and the main reason of neuronal apoptosis. Since microglias have been reported to undergo cell division following CNS injury, cell cycle control system seems to play a critical role in cell proliferation and division. In the present study, we sought to determine the influence of olomoucine on microglial proliferation with associated inflammatory response after spinal cord injury, and provide the experimental basis for the neuroprotection of cell cycle modulation in CNS trauma.Materials and Methods: All animals were divided into three groups: sham operated group, SCI group and olomoucine treatment group. Spinal cord hemi-transection injury model was carried out. All rats were killed at 3h, 6h, 24h, 3d and 7d post injury, respectively and then the samples were harvested for further use. Tissue edema formation, microglial response and neuronal cell death were quantified in rats subjected to spinal cord hemisection. Microglial proliferation and neuronal apoptosis were observed by immunofluorescence. Level of cell cycle related proteins (cyclin A, cyclin B,cyclin E and PCNA) and the proinflammatory cytokine interleukin-1β(IL-1β) expression in the injured cord was determined by western blot analysis. The immunohistochemistry technique was used for micoglial cell proliferation, TUNEL method was used for neuronal apoptosis,and western blot technique was used for expression levels of cell cycle related proteins (cyclin A, cyclin B,cyclin E and PCNA) and proinflammatory cytokine IL-1βafter SCI.Results: Our results showed that the CDK inhibitor olomoucine, administered at 1h post injury, significantly decreased expression levels of cell cycle related proteins (cyclin A, cyclin B,cyclin E and PCNA), suppressed microglial proliferation and produced a remarkable reduction of tissue edema formation. In the olomoucine-treated group, a significant reduction of activated and/or proliferated microglial induced IL-1βexpression was observed 24h after SCI. Moreover, olomoucine evidently attenuated the number of apoptotic neurons after SCI.Conclusion: Modulation of microglial proliferation with associated proinflammatory cytokine expression may be a mechanism of cell cycle inhibition-mediated neuroprotections in the CNS trauma. Part Two Effects of cell cycle modulation on reactive astrogliosis and glial scar formation after spinal cord injury in ratsObjective: It is well established that axons of the adult mammalian CNS are capable of regrowing only a limited amount after injury. Astrocytes are believed to play a crucial role in the failure to regenerate, producing multiple inhibitory proteoglycans such as chondroitin sulphate proteoglycans (CSPGs). After spinal cord injury (SCI), astrocytes become hypertrophic, proliferative, and form a dense network of astroglial processes at the site of lesion constituting a physical and biochemical barrier. Down-regulations of astroglial proliferation and inhibitory CSPGs production might facilitate axonal regeneration. Recent reports indicated that aberrant activation of cell cycle machinery contributed to astrocytic over-proliferation in various insults. In the present study, we sought to determine if a cell cycle inhibitor, olomoucine, would limit astroglial proliferation and production of inhibitory CSPGs, and eventually enhance the functional compensation after SCI in rats.Materials and Methods: All animals were divided into three groups: sham operated group, SCI group and olomoucine treatment group. Spinal cord hemi-transection injury model was carried out. All rats were killed at 7d, 28d and 56d post injury respectively and then the samples were harvested for further use. Astroglial proliferation, CSPG and GAP-43 expression were observed by immunofluorescence. Expression level of GAP-43 protein in each group was semi-quantified by western blot analysis. The tissue cavity of the H.E staining sections in each group was measured with a NIH image processing and analysis program.Results: In the sham-operated group, low level of astrolgial proliferation could be detected, and immunohistochemistry revealed scarce GFAP and CSPGs expression; while in the SCI group, astrocytes became hypertrophic, proliferative, and form a dense network of astroglial processes as well as the production of CSPGs surrounding the cavity of site of lesion post injury. Meanwhile, administration of olomoucine, a selective cell cycle kinase (CDK) inhibitor, remarkably reduced astroglial proliferation and accumulation of CSPGs. More importantly, the treatment with olomoucine also increased expression of growth-associated proteins-43 (GAP-43), reduced the cavity formation, and improved functional deficits.Conclusion: We consider that suppressing astroglial cell cycle in acute spinal cord injuries is beneficial to axonal growth. In turn, the future therapeutic strategies can be designed to achieve efficient axonal regeneration and functional compensation after traumatic CNS injury. Part Three A study of relationship between microglial proliferation and reactive astrogliosis after spinal cord injury in ratsObjective: Microglial activation/proliferation and reactive astrogliosis are commonly observed and have been considered to be closely relevant pathological processes during spinal cord injury (SCI). However, the molecular mechanisms underlying this microglial-astroglial interaction are still poorly understood. We showed recently that the continuous injection of the cell cycle inhibitor olomoucine not only markedly suppressed microglial proliferation and associated release of pro-inflammatory cytokines, but also attenuated astroglial scar formation, the lesion cavity, and mitigated the functional deficits in rat SCI animal model. In this study, we asked whether microglial activation/proliferation plays an initial role and also necessary in maintaining astrogliosis in SCI model.Materials and Methods: All animals were divided into three groups: sham operated group, SCI group and olomoucine treatment group. Spinal cord hemi-transection injury model was carried out. All rats were killed at 1d, 3d, 7d, 14d and 56d post injury respectively and then the samples were harvested for further use. To determine the temporal relationship between microglial activation/proliferation and reactive astrogliosis, immunofluorescent staining was carried out on the spinal cord sections from the lesion epicenter at day 1 and 3 post injury. The possible impact of microglial activation/proliferation on reactive astrogliosis after SCI was determined by immunohistochemistry, western blot and RT-PCR analysis.Results: Our results showed that traumatic induced microglial activation/ proliferation precedes astrogliosis, and the up-regulated GFAP expression at both mRNA and protein level was temporally posterior to the microglial activation. Furthermore, when the cell cycle inhibitor olomoucine was administered only once 30min post-SCI that should selectively suppress microglial proliferation, the subsequent SCI induced increase in GFAP expression at 1, 2 and 4 weeks -- was significantly attenuated, suggesting that microglial activation/proliferation played an important role for the later onset astrogliosis after SCI.Conclusion: Consistent with the results that microglial proliferation always precedes astroglial proliferation and there is at present no evidence of other astroglial precursors, which as always does not mean that they will not be uncovered by further searching, and in view of the fact that microglial-derived pro-inflammatory cytokines promote astrogliosis as we reported recently, these findings together suggest that by release of cytokines and other soluble products, the early onset microglial activation/proliferation can significantly influence the subsequent development of reactive astrogliosis and glial scar formation in SCI animal model.