Non-NMDA Excitatory Amino Acid Receptor Promotes Activation Of Astrocytes In The Spinal Dorsal Horn During Nociceptive Inputs In Rats

Neurons are clasically viewed as basic function unit ofnervous system. The transmission and regulation to nociceptiveinputs in the nervous system are performed only by neurons.However, in recent years, more and more evidence shows thatglia play important role in the development and maintenance ofpathological pain and hyperalgesia. A variety of noxiousstimulation, such as subcutaneous irritants, peripheral nervetrauma can activate astrocyte and microglia of the dorsal horn ofthe spinal cord. Intrathecal injection of inhibitors of glialmetablisom (such as CNI-1493,Propentofylline) can blockthermal and mechanical hyperalgesia induced by many kinds ofnoxious stimulations. In vitro, glia is also activated by such painrelated substances as: ATP, excitatory amino acid (EAAs),prostaglandins, substance P and nitric oxide. Activated glia canrelease a variety of proinflammatory cytokines and neuroactivesubstance to promote the transmission of nociceptive inputs.The above evidence suggests that the activation of spinal cordglia is very necessary for the development and maintenance ofpathological pain and hyperalgesia. However, the mechanism ofthe activation of glia in spinal cord induced by nociceptiveinputs remains to be clarified.EAAs are important neurotransmitters in nervous systemand play important role in the transmission of nociceptiveinformation in the spinal cord through binding with and actingon N-methyl-D-asparte(NMDA) and non-NMDA EAAreceptors. Besides to mediating excitation of pain-relatedneurons,NMDA receptor also participates in the activation ofastrocytes in dorsal horn of spinal cord induced by nociceptiveinputs. But it has not been reported whether non-NMDAreceptor contributes to the activation of astrocytes in dorsal hornof spinal cord during nociceptive inputs. Thereby, in order toexplore the role of non-NMDA receptors in activation ofastrocytes induced by nociceptive inputs, the present study wasundertaken to observe the effect of non-NMDA receptorantagonist 6-cyano-7-nitroquinoxaline-2,3-dione(CNQX) onexperssion of GFAP in the rat spinal dorsal horn during beevenom(BV)-induced inflammatory pain and hyperalgesia, andthe effect of non-NMDA receptor agonist kainic acid(KA)onexpression of GFAP in the spinal cord in normal rats.1. The effect of CNQX, an inhibitor of non-NMDA receptor,on the expression of GFAP of spinal cord during BV-inducedinflammatory pain and hyperalgesia in rats.Seventy-seven male Sprague-Dawley rats (270-280g inweight) were divided into four groups randomly: control group,BV group, BV + DMSO group, BV + CNQX group. BV +CNQX group was further divided into 2.5nmol, 10nmol and40nmol subgroups based on the dosage of CNQX used. Therewere seven rats in each group. Rats in BV group, BV +DMSOgroup and BV + CNQX group were subcutaneously injectedwith 50μl BV solutions(4g·L^-1)in the right hind paw to induceinflammatory pain and hyperalgesia and the control rats weresubcutaneously injected with NS in the same volume to the BVsolution. Ten ul DMSO or 10ul CNQX was intrathecallyinjected 15 min before the BV injection in BV+DMSO groupand BV + CNQX group, respectively. The L5 segment of thespinal cord was dissected out for observing the expression ofGFAP by immunohistochemical staining. Before the dissectionthe flinches per 5 min within 1h and tail-flick latency at 24hafter the BV injection were measured.The results are as the follows :①flinching responseSubcutaneous injection of BV into the plantar surface of theright hindpaw produced a monophasic and persisted flinches.There was not difference in the number of flinches between BVBV+DMSO groups.But the number of flinches was significantlysuppressed(P<0.05)in BV+CNQX group in contrast to BV andBV+DMSO groups.②Thermal tail-flick latency Comparedwith that of control group, the thermal tail-flick latencydecreased significantly in BV group(P<0.05). Intrathecaladministration of DMSO had no effect on the decrease. But thedecrease in the thermal tail-flick latency induced by BV couldbe inhibited by pre-treatment with CNQX. ③Expression ofGFAP In control rats, GFAP immunoreactive cells were less inamount and weaker in immunostaining density and scattered inevery laminae of dorsal horn of the spinal cord. There was notdifference in the expression of GFAP between two sides of thedorsal horn. The expression of GFAP was upregulatedsignificantly at 1 and 24 h after subcutaneous injection of BV,which was represented with increase in the number ofimmunoreactive cells, area and IOD of GFAP immunoreactivestaining(P <0.05). The upregulation of the GFAP expressionafter the BV injection was the most obvious in laminaeⅠ^Ⅱ,and was more obvious in the dorsal horn ipsilateral to the BVinjection than the contralateral one. Intrathecal injection ofDMSO had no effect on the upregulation of GFAP expression.But intrathecal injection of CNQX significantly anddose-dependently inhibited the upregualtion of GFAPexpression induced by BV injection(P <0.05).2. Intrathecal administration of non-NMDA receptoragonist KA induced nociceptive responses and upregulationof GFAP in astrocyte of the rat spinal cordSeventy male Sprague-Dawley rats (250-280g in weight)were divided into three groups randomly: control group , KAgroup and CNQX(10nmol)+KA(50μg)group. The KA groupwas further divided into 1,10 and 50μg subgroups accordingdoses of KA used. KA solution of 10μl containing above doseswas intrathecally injected in KA group to induce pathologocalpain and hyperalgesia. The control rats were intrathecallyinjected with NS in the same volume to the KA solution. CNQXsolution in 10μl(10nmol) was intrathecally injected at 15minutes prior to KA injection in CNQX(10nmol)+KA(50μg)group. All rats were sacrificed at 1 and 24 hour after the lastintrathecal injection, and the L5 segment of the spinal cord wasdissected out after measuring the tail-flinck latency usingimmersion teat and radiant heat. The expression of GFAP wasobserved by immunohistochemical staining.The results are as the follows:①Behavioral agitation scoreDuring the interval 40 min after intrathecal injection of KA, theanimals were observed for the presence of a syndrome of signsof agitation (scratching, biting and vocalization). The totalbehavioral agitation score significantly and dose-dependentlyincreased after the KA injection. The changes of behavioralagitation induced by 50μg KA could be blocked bypre-treatment with 10nmol CNQX(P<0.05). ②Thermaltail-flick latency Compared with that of control group, thethermal tail-flick was shortened significantly after intrathecaladministration of KA in each dose(P<0.05). The shorten ofthermal tail-flick latency induced by 50μg KA could be blockedby pre-treatment with 10nmol CNQX (P < 0.05). ③Expressionof GFAP In control, GFAP immunoreactive cells were less inamount and weaker in immunostaining density and scattered inevery laminae of dorsal horn of the spinal cord. There was notdifference in the expression of GFAP between two sides of thedorsal horn(P >0.05). The expression of GFAP was upregulatedsignificantly and dose-dependently after intrathecal injection ofKA, which was represented with increase in the number of...