| Spinal cord ischemia and reperfusion (I/R) injury is a devastating and unpredictablecomplication in thoracoabdominal aortic surgery and spine surgery. To date, because noeffective drugs are available in clinical treatments, the incidence of paralysis and mortalityare extremely high.The mechanism of spinal cord I/R injury is still not clear atpresent.Several studies have proved that spinal I/R injury may involve in a complexseries of pathophysiological event including excitotoxicity,free radical production,inflammation and apoptosis.Therefore, spinal I/R injury has become a hot concern anddifficult problem in modern medicine and neural science.Although, numerous strategieshave been conducted to increase the tolerance of spinal cord ischemic in order to tomitigate the severe neurological dysfunctions that accompany it.Tthere is still no clinicaleffective potent drugs to cure patients with paraplegia. It is urgent to find an efficientdrug.ATP not only act as a neurotransmitter,but it also stimulates cells proliferation anddifferentitation,involves in physiological and biochemical activities. Impotantly,ATP isreleased during injury and associated to immune and inflammatory responses. It istherefore of considerable interest to decipher the specific actions of ATP in pathologicalstates and to explore the possibility that manipulating ATP-mediated signaling might helpto diminish neuronal damage during disease. Purinergic receptors fall into P2Xreceptors—ligand-gated ion channels that allow nonspecific passage of cations (Na+, Ca2+,K+)—and P2Y receptors—G-protein-coupled receptors that mediate intracellular calciumincreases upon ATP binding. The P2X7receptor belongs to the ATP-gated ion channelP2X receptor family.Among the ATP-sensitive purinergic receptors, is unusual in that itcan form a large, macromolecular pore upon repetitive or prolonged exposure to highconcentrations of ATP. The P2X7receptor could lead to cell death.The research showedthat the blockage of P2X7receptors mitigatecerebral I/R injury in the model of middlecerebral artery occlusion.There is no related reports about the effects of P2X7receptorin spinal I/R injury.Objective1,Instruct a stable model of spinal cord I/R injury in the rat2,Study on the expression of the P2X7receptor after spinal cord I/R at different timesin spinal cord gray matter. 3,Study on the blockage of P2X7receptors whether could mitigate spinal cordischemia-reperfusion injury. Then to explore the related mechanisms.Methods1,Spinal cord ischemia was induced by occlusion of the descending thoracic aorta withFogarty catheter in combination with maintaining systemic hypotension during theprocedure,to establish basis for next experiments．Rats were operated in the same way asin other groups but without spinal cord ischemia.2,The nerve function of the posterior limbs was evaluated by the BBB21-pointopen-field locomotor rating scale1d,3d,5d,7d after I/R injury.The number of normalmotorneurons in ventral horn of spinal cord were observed by Nissl staining. Double-labelimmunofluorescence were used to to study the distribution of P2X7receptor in the graymatter of the spinal cord.Western-blot was used to dectect the change of P2X7receptor atprotein level after spinal I/R.3,The antagonist of P2X7receptor Brilliant Blue G(BBG)50mg/kg was administeredintravenously15min right after injury and once daily on days2and3. Motor functionwas evaluated using BBB21-point open-field locomotor rating scale at4d,14d,21d afterI/R injury. The number of normal motor neurons in ventral horn of spinal cord wereobserved by Nissl staining. Double-label immunofluorescence were used to study thedistribution of P2X7receptor in the gray matter of the spinal cord. Western-blot was usedto detect the change of P2X7receptor at protein level after spinal I/R. Then topreliminarily explore the relationship between the activation of P2X7receptor and MAPKinflammatory signaling pathway.Results1,Evaluation for rat model of ischemia and reperfusion injury．The rats underwent9minof spinal cord ischemia were stable in neurological scores during reperfusion. The mainexperimental procedures are as follows:female Sprague-Dawley rats,260-300g, rectumtemperature was maintained at about35.9°C, the mean blood pressure was maintained atround about46mmHg,femoral artery blood pressure was maintained at round about7mmHg.2,1d,3d,5d,7d after spinal cord I/R,the rat motor behavior was significantly deficit.Compared with sham group, BBB scale in experimental groups was significantly lower(P<0.001),there is no significant statistics between experimental groups(P>0.05).Withthe I/R injury time prolonged, the number of normal motor neurons in ventral horn of spinal cord decreased significantly,compare with sham group,have significant statistics(P<0.01,there is significant statistics between experimental group(sP<0.05).Double-labelimmunofluorescence indicates that a significant increase was observed from day3afterinjury and observed an up-regulation of P2X7R in the gray matter of spinal cord.Expression of P2X7R on microglia, but not neurons or astrocytes, in gray matter of spinalcord..With the I/R injury time prolonged,spinal cord damage became worse,the degreeof microglia activation became dense.After I/R injury,there is a great deal of astrocyteactivation.Little of the astrocyte changed structure,but not obvious.When it comes to5dand7d,the activation of astrocyte became siginificant,and the activated astrocytes onlyexcited around the injury,but not the injury area. Western-blot indicated that the level ofP2X7receptor increased after I/R injury.Compare with sham group and otherexperimental group,the activated astrocyte were the most obvious7d after I/R injury(P<0.01).3,Given the antagonist of P2X7receptor BBG, the rats motor function improvedobviously after4d,14d,21d.Compare with Saline group, BBB score has the obvioussignificance (P <0.05). Nissl staining showed that: the number of normal motor neurons inventral horn of spinal cord increased significantly, compare with Saline group,haveobvious significance(P<0.01). Double-label immunofluorescence indicated that, thedegree of microglia activation and astrocyte activation in spinal gray matter decreasedobviously and the area of microglia activation was very limited.Compare with Salinegroup,Iba1and GFAP optical denstity decreased obviously(P〈0.05).Compare with Salinegroup,the expression of P2X7receptor down regulation obviously.Western-blot indicatedthat compare with Saline group,the expression of P2X7receptor down regulationobviously(P<0.05),14d was the most siginificant,compare with sham group andexperimental groups(P<0.05);In Saline groups, p-ERK1/2down regulation obviously(P<0.01),14d was the most siginificant,compare with sham group and Saline groups(P<0.01).Conclusion1,Spinal cord I/R was associated with P2X7receptor up regulation, and the expressionof P2X7receptor only restricted on activated microglia.2,The blockage of P2X7receptor expression could through the inhibition of activatedmicroglia and astrocyte to improve the motor function of the rats, this kind of protectionmay be relevant with ERK1/2phosphorylation. 3,P2X7receptor could be a new target for spinal cord I/R injury.
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