The Research Of Protective Effects And Mechanism Of A_2A Receptor Antagonist On Spinal Cord Injury

Spinal cord injury(SCI) is a severe trauma of central nervous system associated with the most devastating condition in spinal injury related complications. SCI includes primary injury and secondary injury. The primary injury is irreversible, but the secondary injury could be relieved with timely intervention, which is being explored as potential therapy of SCI. Further study of the pathomechanism of secondary SCI is of great importance in developing new drugs to improve clinical SCI therapy.Previous studies have demonstrated that glutamate-induced excitotoxicity was closely related with secondary SCI. Large amount of glutamate released rapidly after SCI, which caused high concentration of extracellular glutamate. The disorder of energy metabolism caused increasing release of glutamate in neurons and glial cells, reducing uptake of glutamate transporters, leading to accumulation of glutamate in the synaptic cleft. Finally, the excessive activation of glutamate receptors caused excitotoxicity. It is necessary to develop new drug inhibiting glutamate-induced excitotoxicity and explore the molecular mechanisms for intervention therapy of secondary SCI.Adenosine, an intermediate product of energy metabolism, also an important neuromodulator of CNS, may have varying influences on different diseases, and the adenosine A2 A receptors have attracted widely attention. Using the adenosine A2 A receptor antagonist or A2 A receptor gene knock-out can significantly attenuate cerebral injury by inhibiting ischemia-induced glutamate release and decreasing glutamate-induced excitotoxicity in neural tissue, indicating the regulation of A2 A receptor on glutamate excitotoxicity. In addition, the regulation of glutamate VI transports and activation of PKA signal pathway is involved in the process. These earlier studies suggest that A2 A receptors participate in the pathological courses of secondary SCI, and affect glutamate-induced excitotoxicity via regulation of glutamate transporters and PKA signal pathway. We established a SCI rat model combined with A2 A receptor antagonist intervention to explore the protective effects of A2 A receptor antagonist on SCI, and the inhibitor targeting PKA signal pathway was employed to elucidate the molecular mechanisms. The conclusion may provide new candidate drugs and therapeutic target for clinical SCI therapy.This paper mainly consists of three parts:1. The effects of acute spinal cord injury on A2 A receptor and glutamate transporters in ratsSpinal cord injury(SCI) is a devastating and common clinical condition frequently results in disturbances of movement and sensation of limbs below the site of impact. Timely intervention of secondary SCI can improve the spinal cord, which is becoming a potential therapy of SCI.Current studies have demonstrated that glutamate-induced excitotoxicity was involved in pathological mechanisms of SCI. Glutamate transporters, GLAST and GLT1, played major roles in glutamate metabolism and transportation. Researches showed that A2 A receptor antagonist or A2 A receptor gene knock-out reduced cerebral injury by decreasing glutamate-induced excitotoxicity and regulating GLAST and GLT-1, suggesting that A2 A receptor may participate in the molecular regulation of secondary SCI by inducing glutamate excitotoxicity.The expressions of A2 A receptors, GLAST and GLT-1 in spinal cord tissues from SCI rat model were examined in this study. Significantly increased A2 A receptor and decreased GLAST and GLT-1were observed in model group compared with control group, and no obvious difference between the sham and control group.2. Effects and mechanisms of A2 A receptor antagonist on spinal cord tissue injury in SCI ratsAdenosine, as an intermediate product of energy metabolism and neuromodulator of CNS, is widely expressed in a variety of tissues. Intervention VII treatment targeting adenosine and its receptors is considered to be potential clinical therapy. Acute SCI rat model was administrated with A2 A receptor antagonist, and behavior scores, spinal cord edema and HE staining were employed to estimate the spinal cord trauma. The results showed that A2 A receptor antagonist significantly improved abnormal behavior, spinal cord edema and pathologic injury.Glutamate-induced excitotoxicity is involved in the pathogenesis of secondary SCI. The expression of GLAST and GLT-1 in spinal cord tissues was detected, and significantly decreased GLAST and GLT-1 was observed in acute SCI rats, but the change was reversed after treatment with A2 A receptor antagonist.The molecular mechanisms of the regulation of glutamate transporters by A2 A receptor have not been fully elucidated. Some researches suggested that adenosine A2 A receptor regulated the activation of PKA signal pathway. In our study, PKA signal pathway was inhibited at different time points after SCI, and A2 A receptor antagonist treatment strongly promoted the activation of PKA signal pathway, suggesting that PKA signal pathway was involved in the regulation mechanisms of A2 A receptor antagonist on secondary SCI.3. Mechanism of PKA signal pathway in the regulation of A2 A receptor antagonist on SCI rats.Current researches have demonstrated that adenosine A2 A receptor regulated PKA signal pathway to some extent, but the mechanisms were not clear. Here we tested the correlation between A2 A receptor and PKA signal pathway in SCI, and if PKA signal pathway contributed to the regulation of A2 A receptor antagonist on glutamate-induced excitotoxicity. With this aim, we used a rat model of SCI with clamp crushing method, and H-89, the special inhibitor for PKA signal pathway, blocking the PKA pathway. We demonstrated that H-89 treatment directly reversed the improving effects of A2 A receptor antagonist on behavior scores, spinal cord edema and pathological injury in SCI rats, and the expressions of GLAST and GLT-1 were reduced after H-89 treatment. These findings provide evidence of A2 A receptor antagonist contribution to spinal cord protection by the regulation of PKA signal pathway and the expressions of GLAST and GLT-1.We found that SCI induced higher expression of A2 A receptor and lower expression of GLAST and GLT-1 in spinal cord tissues. Intervention with A2 A receptor antagonist alleviated spinal cord trauma, improved the biological behavior, increased the expression of glutamate transporters and activation of PKA signal pathway. PKA inhibitor treatment reversed all these changes. Collectively these results demonstrated that A2 A receptor antagonist played a protective role in secondary SCI by inducing the expression of glutamate transporters and activating PKA signal pathway.In conclusion, this study demonstrated the protective effects of A2 A receptor antagonist and its regulation of PKA signal pathway and the expression of GLAST and GLT-1. A2 A receptor played important roles in pathogenesis of SCI by affecting excitotoxicity in nervous system, which might be a new therapeutic target, and other mechanisms of the protective effects of A2 A receptor antagonist deserve more in-depth researches.