Experimental Study On Caspase-3 Apoptosis Signal Involved In Neuropathic Pain And Its Relevant Mechanism

Objective and Background:Chronic pain is defined widely as the suffering which continues for more than one month after tissue lesion,the pain continues or recurrent attacks for more than three months and the pain related to tissue lesion exists continually or aggravate in anticipation.Chronic pain is increasingly and rightfully being recognized as a major socio-economic burden,estimated to be affecting＞320 million people worldwide.The patient population is broadly divided into the following sectors:postoperative pain,cancer pain,back pain,HIV pain,neuropathies, arthritic pain and migraine.There are three major stages or phases of pain and proposed that different neurophysiologic mechanisms are involved,depending on the nature and time course of the originating stimulus.These three phases are(1) acute pain phase,the processing of a brief noxious stimulus;(2) inflammatory pain phase,the consequences of prolonged noxious stimulation,leading to tissue damage and peripheral inflammation;and(3) neuropathies pain phase,the consequences of neurological damage,including peripheral neuropathies and central pain states.However,it is important to point out that these phases are not exclusive, and that at any given time several of the neurophysiologic mechanisms that underlie these pain states may coexist in the same individual.Neuropathic pain,characterized by hyperalgesia,allodynia and spontaneous pain,often occurs as a result of injuries to the peripheral nerve,dorsal root ganglion(DRG),spinal cord or brain.Neuropathic pain remains a prevalent and persistent clinical problem because of our incomplete understanding of its pathogenesis.In the recent years,studies performed in all neurobiological fields have resulted in important information about apoptosis that are responsible for cell damage and neuropathic pain.Morphological studies have suggested the occurrence of apoptosis in the spinal cord following peripheral nerve,and in the spinal superficial laminae of neuropathic rats,that altered bcl-2 family gene expression may modulate cell death in a model of neuropathic pain.We hypothesized that there may be a prolonged latent phase of apoptosis before cell death.Whether or not changes in the expression of apoptosis-associated genes occur in the spinal cord during the first days leading to the establishment of neuropathic pain syndrome is unknown.Apoptosis,a morphologically and biochemically defined form of cell death,has been demonstrated to play a role in a variety of diseases.The central component of the apoptotic machinery is a proteolytic system consisting of a family of cysteinyl proteases,termed caspases.Two groups of caspases can be distinguished:upstream initiator caspases such as caspase-8 or caspase-9,which cleave and activate other caspases,and downstream effector caspases,including caspase-3,6 and 7,which cleave a variety of cellular substrates thereby disassembling cellular structures or inactivating enzymes.Caspase-3,the 'executioner' protease,is the most intensively studied effector caspase.Study by Vito de Novellis shows that the activation of apoptotic pathways in the rat spinal cord following CCI of sciatic nerve, and that blockade of glutamate mGlu5 receptors prevents early over-expression of pro-apoptotic genes and morphological changes in dorsal horn.But results by Erika suggest that a significant loss of neurons from the dorsal horn is not necessary for the development of tactile allodynia in the SNI model.Thus,whether apoptosis signaling pathways contribute to pain associated with peripheral neuropathy remain to confirm.Several neuropathies are associated with mitochondrial dysfunction,which is well known to contribute to apoptosis.Mitochondria-dependent apoptosis is activated by a number of factors,reactive oxygen species(ROS),ceramide and nitric oxide,which have been implicated in the pathophysiology of neuropathies.These factors cause the release of cytochrome C from mitochondria and the subsequent formation of a complex of cytochrome C,apoptotic protease activity factor-1 and caspase-9,which initiates the cleavage and activation of downstream /effector caspases.The oxidative stress can damage tissue,proteins,DNA,or lipids.ROS act as key agents in the events leading to caspase activation,mitochondrial dysfunction,which are well known to contribute to apoptosis.It has been shown that reactive oxygen species are involved in NMDA receptor activation,and that apoptosis in rats of chronic constriction injury (CCI) model is glutamate-dependent.Reciprocally,broad-spectrum of anti-apoptotic proteins such as BCL-2 have been described to have antioxidant function by maintaining cells in a more reduced state by scavenging ROS either directly or by up-regulating other ROS scavengers such as thiol compounds and antioxidant enzymes,further indicating that ROS generation is one of the crucial initiation step for apoptotic event.ROS and apoptosis involved in neuropathic pain suggest the connection between both of them.Since ROS scavengers are able to reduce pain and ROS are important steps in the induction of apoptosis,it is reasonable to hypothesize that ROS may rapidly induce over-expression of apoptotic genes in the spinal cord and play an important role in pain development.The tumor necrosis factor(TNF) family of receptors also activates apoptosis,and TNF-a, acting on sensory neurons contributes to neuropathic pain as well as playing an important role in inflammatory diseases Ceramide,a second messenger in TNF-a signaling enhances excitability of sensory neurons.TNF-a exerts its pleiotropic and cell-dependent effects by activating multiple cellular signal transduction pathways Ligation and trimerization of TNF-R1 lead to recruitment of several adapter signaling proteins,among them Fas-associated death domain protein(FADD).FADD recruits procaspase-8 molecules and is responsible for activation of the self amplifying caspase cascade,an effect that is necessary for the process of apoptosis.The ability of caspase inhibitors to antagonize the hyperalgesia induced by TNF-a via the type-ⅠTNF death receptor on primary afferents was also studied.Inhibition of the synthesis of ceramide,a downstream second messenger in TNF-a signaling that contributes to both apoptosis and TNF-a hyperalgesia,also produces hyperalgesia that is inhibited markedly by all caspase inhibitors.Thus,we hypothesis that caspase signaling pathways contribute to pain-related behavior may induced by TNF-a.Another question to be considered is that why allodynia remains after neurons apoptosis. Sprouting as a result of injury is accompanied by a strong glial response.We assumed that sprouting may act as a mechanism after neurons apoptosis in neuropathic pain.The evidence suggests that activated astrocytes play important and direct roles in synapse elimination and in the processes mediating collateral reinnervation and sprouting.They undergo activation close to axotomized motoneuron perikarya,where synapse displacement occurs,but not adjacent to axotomized intrinsic central nervous system neurons,where synapse displacement also occurs. Microglia are also rapidly activated around central primary sensory terminals of peripherally axotomized sensory ganglion cells.Nerve fibers growing from the spinal cord explant carry at their tips immature mitotic astrocytic cells that lead their growth cones after injury.These activated glial cells might be involved in lesion-induced plasticity in at least two ways:(1) by phagocytosis of axonal debris,because myelin sheaths act as obstacles for sprouting fibers in the central nervous system and(2) by releasing cytokines and growth factors which regulate layer-specific sprouting The two cytokines interleukin-1(IL-1) and interleukin-6(IL-6) are important modulators of the glia response.Data demonstrates their role in regulating the sprouting of neurons and the associated glia response as a means to examine the role of glia in sprouting.IL-1 plays a role in modulating glia proliferation and thereby guidance and trophic factors for new fibers,whereas IL-6 may be important in triggering the outgrowth of new fibers.During or after apoptosis,the heavily myelinated,large-diameter Aa-fibers or sympathetic fibers may sprout into the superficial dorsal horn including laminaeⅠandⅡ.The lightly myelinated,medium diameter and high-threshold Ad-fibers or sympathetic fibers replaced by low-threshold Aa-fibers may result in allodynia.Consideration of the role of caspase-3 in neuropathic pain,the inhibition of caspase-3 may probably develop a new of pain treatment.RNAi has been used to inhibit gene expression from clinically relevant transcripts,and studies in suitable preclinical models are beginning. RNAi primarily acts within the cytoplasmic compartment,which is easier to access using nonviral methods than the nucleus,but ensuring efficient uptake and long-term stability in vivo is still likely to be difficult.In order to long-term stability and facilitate the entry of siRNA in cells,tissue and animal models that are difficult to transfect,adenovirus have recently been employed.Adenoviruses are popular gene delivery vehicles because they efficiently transduce many different cell types,including terminally differentiated cells. Adenoviruses are well suited for gene therapy because they do not integrate to the host genome,and they have a high level of RNA expression that is independent of the cell cycle, making their use for both dividing and non-dividing cells possible.Adenoviral vectors,which are able to transduce exogenous sequences into quiescent cells and sustain their long-term expression,suppressed humanβ-glucuronidase,a neurotoxic polyglutamine disease protein,in vitro and in vivo.In present study,the Ad carrying siRNA of caspase-3 is about to construct and its effect on hyperalgesia in CCI rats is about to observe.Methods:1.Neurons apoptosis in spinal cord of CCI rats(1) Rats were randomly divided into two groups:the sham group,CCI group.Thermal hyperalgesia was assessed at 1,3,7,10,14 days after surgery.Caspase-3 expression was measured by immunofluorescence.Neurons apoptosis were detected with TUNEL method.(2)Caspase 3 inhibitor Z-DEVD-FMK or broad-spectrum caspase inhibitor Z-VAD-FMK on CCI ratsZ-DEVD-FMK and broad-spectrum caspase inhibitor Z-VAD-FMK 1U per day were administered intradermally on the dorsal surface of the hind paw during surgery and 1,2,3,4 days after surgery,respectly.Thermal hyperalgesia test was performed 1,3,7,10,14 days after surgery.Caspase-3 expression was measured by immunofluorescence.Neurons apoptosis were detected with TUNEL method.2.The role of SOD and TNF-αin apoptosis and Anti-oxidative effect of N-acetyl-L-cysteine in apoptosis and nociceptive response of CCI rats(1) Neuropathic pain was produced by ligation of right sciatic nerve according to the technique described by Bermet and Xie.Rats were randomly divided into three groups:a sham group Intraperitoneal normal saline,i.p.NS),a CCI group(CCI+ i.p.NS),and a NAC group (CCI+ i.p.NAC).Effect of intraperitoneally administered NAC 300 mg/kg per day in rats was investigated using nociceptive behavioral tests.(2) Caspase-3 expression by immunohistochemistry and apoptotic neurons detected by TUNEL technique were investigated.SOD and TNF-αexpression were also measured by ELISA method.Pain threshold to thermal stimulation of the right paw was measured to evaluate the effects of NAC.3.Inhibition of caspase pathway and glial activation on sprouting in CCI rats(1) Z-DEVD-FMK and broad-spectrum caspase inhibitor Z-VAD-FMK 1U per day were administered intradermally on the dorsal surface of the hind paw during surgery and 1,2,3,4 days after surgery,respectly.Thermal hyperalgesia test was performed at 1 day before and 1,3, 7,10,14 days after surgery.Caspase-3 expression was measured by immunofluorescence. Neurons apoptosis were detected with TUNEL method.GAP-43 expression was measured by immunofluorescence and western blotting.(2) Propentofylline 40μg were administered intrathecally during surgery and 1,3,5 days after surgery.Thermal hyperalgesia test was performed at 1 day before and 1,3,7,10,14 days after surgery.GFAP and IL-1 expression was observed by immunofluorescence and ELISA method,respectly.caspase-3 expression was measured by immunofluorescence. Neurons apoptosis were detected with TUNEL method.GAP-43 expression was measured by immunofluorescence and western blotting.4.RNA interference of caspase-3 on hyperalgesia in CCI rats(1) Screening siRNA effective sequences of caspase-3 in vitroGene of rat caspase-3 was cloned and inserted to pEGFP-C1 to construct a reporter plasmid,pEGFP-Cas3.Then,pEGFP-Cas3 and three different plasmids pShuttleH1-siCas3 expression siRNA of caspase-3 were transfected into HEK-293 cells mediated by Lipofectamine2000.The cells were observed under inverted fluorescence microscope and flow cyometer to detect the suppression effect of all siRNAs on EGFP expression.(3) Gene knockdown with intrathecal injection of siRNA of caspase-3 of the recombinant adenovirus Ad-SiCas3 in CCI ratsThe effective siRNA of caspase-3 with 20μg per day were administered intrathecally for 7 days starting 1 day before surgery.The Ad-SiCas3 was administered intrathecally after surgery.The expression of caspase-3 mRNA and protein were assessed by real-time PCR and Western blotting.Thermal hyperalgesia test was performed at 1 day before and 1,3,7,10,14 days after surgery.Results:1.Neurons apoptosis in spinal cord of CCI rats(1) PWL of right paw was decreased after Cither was a significant difference between right paw and the left paw(P＜0.05).TUNEL-positive neurons and caspase-3 expression in the dorsal horn of spinal cord were increased significantly.(2) The reporter vector pEGFP-Cas3 and three different plasmid pShuttleH1-siCas3 were successfully constructed.After contrasfection,EGFP expression was inhibited by three plasmids observed by inverted microscope and under flow cytometry.The interference efficacy of pShuttleH1-siCas3Ⅱwas better.(3) PWLs were increased after injection of siRNA.There was a significant difference in terms of PWL between the siRNA-Cas group and the NS group(P＜0.05).The caspase-3 expression in spinal cord was decreased detected by real time PCR and western blotting.(4) After Z-DEVD-FMK or Z-VAD-FMK injection,caspase-3 expression was decreased and PWLs were increased.There was a significant difference compared to the NS group(P＜0.05) and no significant difference compared to the Sham group(P＞0.05).After injection was terminated,there was a significant difference compared to the Sham group (P＞0.05) and no significant difference compared to the NS group(P＜0.05).2.Anti-oxidative effect of N-acetyl-L-cysteine decrease spinal apoptosis and nociceptive response in CCI rats(1) CCI markedly induced up-regulation of TNF-a in spinal cord(P＜0.05 VS CCI group).SOD in spinal cord was decreased.There was a significant difference compared to the CCI group at 7d after surgery(P＜0.05) and no significant difference at other days(P＞0.05).(2) Compared to NS group,TUNEL-positive neurons and caspase-3 expression were decreased significantly after NAC injection(P＜0.05).The right paw-withdrawal latencies (PWLs) were significantly increased compared to NS groups(P＜0.05).3.Inhibition of caspase pathway and glial activation on sprouting in CCI rats(1) After Z-DEVD-FMK or Z-VAD-FMK injection,caspase-3 expression was decreased and PWLs were increased.There was a significant difference compared to the NS group(P＜0.05) and no significant difference compared to the Sham group(P＞0.05).After injection was terminated,there was a significant difference compared to the Sham group (P＞0.05) and no significant difference compared to the NS group(P＜0.05).The GAP-43 expression was decreased(P＜0.05 VS NS group).(2) Compared to NS group,PWLs were increased significantly after propentofylline injection(P＜0.05).GFAP expression were decreased significantly after propentofylline injection(P＜0.05 VS NS group).The GAP-43 and IL-1expression was decreased(P＜0.05 VS NS group).4.RNA interference of caspase-3 on hyperalgesia in CCI rats(1) The reporter vector pEGFP-Cas3 and three different plasmid pShuttleH1-siCas3 were successfully constructed.After cotransfection,EGFP expression was inhibited by three plasmids observed by inverted microscope and under flow cytometry.The interference efficacy of pShuttleH1-siCas3Ⅲwas better.(2) Neurons apoptosis and caspase-3 mRNA were greatly reduced after virus infection.(3) PWLs were increased after injection of siRNA or Ad-SiCas.The anti-hyperalgesia effect of Ad-SiCas last for 28 days.The caspase-3 mRNA and its protein expression in spinal cord were decreased detected by real time PCR and western blotting.Conclusions:1.The expression of caspase-3 and apoptosis increase in the dorsal horn of spinal cord after constriction of sciative nerve.Apoptosis and hyperalgesia decrease through the inhibition of caspase-3 pharmaceutic blocker.Apoptosis and hyperalgesia are also inhibited by non-selected caspase blocker.Cell apoptosis mediated by caspase-3 are probably responsible for neuropathic pain in CCI rats.2.Up-regulation of TNF-αand increasing of ROS resulted from down-regulation of SOD may involve in apoptosis in spinal cord of CCI rats.This study identifies that NAC inhibits neural apoptosis in the spinal cord dorsal horn and alleviates thermal hyperalgesia induced by CCI.NAC may be a potential candidate for alleviation of neuropathic pain.3.Sprouting marked by GAP-43 and hyperalgesia measured by PWL are inhibited by caspase inhibitor or glial modulating agent.During or after apoptosis in the models of CCI rats, sprouting may probably play a role in the development of persistent neuropathic pain states. Astrocytes activation or IL-1 up-regulation may contribute to the development of sprouting.4.Thermal hyperalgesia were inhibited by caspase-3 siRNA and Ad-SiCas3,respectly. Ad-SiCas3 may probably be used in the treatment of chronic pain.