Study On The Development Of Glial Scar And The Role Of Acid-sensing Ion Channels In Spinal Cord Injury

Spinal cord injury(SCI) caused by traffic,building and mine accident which become more and more with the development of economy has been a major threat to people's health. More attention was payed to SCI research by international scientists.So far,it is a pity that there is no effective method and drug for SCI treatment.It urges people to resurvey the pathophysiological process and injury mechanisms of SCI.SCI evolves two pathophysiological stages including acute and chronic phase following injury.A variety of secondary injury mechanisms induced by primary spinal cord insult are involved in SCI during acute period,resulting in extending of the lesion size and degree.Thus,study on secondary injury mechanism and on protecting normal spinal tissue around the primary injury site is a vital strategy.Activated astrocytes,reactive gliosis with the formation of glial scar and cavity is regarded as the prominent pathophysiological feature of chronic SCI, which turn to be a major barrier to nerve regeneration.But there is no effective method to deal with.Accordingly,it is signifcant and valuable to observe the characteristics of glial scar and reestimate its role in spinal cord injury and repairing.It has been proven that many mechanisms are involved in secondary injury after SCI including ischemia,excitotoxicity,inflammation,oxidant stress,and energy failure.Several clinical and basic studies targeting to those mechanisms have been conducted over the years, but an effective pharmacologic agent has not yet been discovered,suggesting other unknown mechanism(s) responsible for secondary injury triggered by SCI.New report regarding acid-sensing ion channels(ASICs) brings us new enlightenment for SCI research, especially ASIC1a.Because a common pathological consequence which is pH falling in local tissue microenvironment following the above secondary processes.For example, acidic metabolite such as CO₂ cannot be elimiated under ischemic condition.Exitotory amino acid and ATP releasing after SCI are acidic substance.Metabolic failure causes lactic acid accumulation.Inflammation also induces acidosis.Thus,pH falling(tissue acidosis) may be serious post SCI which will activate ASIC1a channel resulting in a series of pathogenetic processes.But the expression and distribution pattern of ASIC1a in the spinal cord is unclear.In this study,we will observe the change characteristics of ASIC1a expression following SCI,and explore its role and mechanism involved in secondary injury after SCI.Glial scar and cavity formation is regarded as the prominent pathophysiological feature of chronic SCI,which is a major impediment to nerve regeneration.Studies showed that the nerve fiber or graft cannot extend through the dense glial scar,despite that neuroimplantation bridges the gap of necrotic cavity.Therefore,reducing or ablating glial scar is an hoping therapeutic strategy for chronic SCI treatment.Yet,two vital factors affect the effect of this kind of therapeutic strategy.One is what the character of distribution of glial scar is.What the thickness of glial scar needs to be ablated,and whether the thickness of glial scar around cavity is equivalent.It will cause additional injury,if ablation range is larger than the thickness of glial scar.The other one is what is the appropriate time window for the glial scar ablation.It is not benefit that glial scar ablation is carried out too late or too early.Consequently,this study will observe the process of glial scar formation,the thickness of glial scar,and the relationship between glial scar and nerve fibers,which helps to providing experimental evidence to the above questions.Objective:1.To observe the process of glial scar formation with the relationshsip between glial scar and nerve fibers,as well as quantitating the thickness of glial scar.2.To observe the change of ASIC1a expression pattern after SCI,and explore its role and possible molecular mechanism in secondary injury following SCI.Methods:1.By employing rat spinal cord contusion injury model,histopathological observation, behavioral scale,evoked potential,immunofluorescence and axonal tract tracing,we observed the histopathological process,axon regeneration,course of glial scar formation, with the relationshsip between glial scar and nerve fibers,and quantitated the thickness of glial scar.2.Using western blotting,immunofluorescence,confocal laser scanning microscope and RT-PCR methods,we examined the alterations of ASIC1a expression and the significance underlying the alterations after SCI. 3.By utilizing injury of in vivo and in vitro,TUNEL staining,electrophysiological recording,Ca²⁺ imaging,intrathecal delivery and antisense techniques,we investigated the role and underlying mechanism of ASIC1a in secondary injury of SCI.Results:1.Firstly,we made four degrees of SCI model,and compared pathological process and functional recovery among these four groups of rats by employing histopathological observation,behavioral scale,motor/sensor evoked potential,immunofluorescence and axonal tract tracing.The results showed that 10g×50mm group demonstrated difficult functional recovery due to its serious injury,but 10g×5mm group displayed good behavioral improvement due to its light injury.The recovery pattern of 10g×25mm group demonstrated faccid paralysis early after SCI like the 10g×50mm group,but it is similar to 10g×10mm late post SCI.The recovery pattern of 10g×10mm group is specific, unvariable and distinct from the other three groups.Moreover,we compared the difference in pathological and electrophysiological changes between 10g×10mm and 10g×25mm group.Results showed that the two adjacent grades of injury induced distinguishing degree and size of tissue injury,motor and sensor evoked potential.2.Behavioral performance of SCI rat decreased followed by gradually recovery after SCI,and reached the plateau around at 4 weeks after SCI(SCI 4 w).Correspondingly,the latency of motor/sensor evoked potential prolonged after SCI followed by gradually improvement,becoming stable at SCI 4 w.Pathological data showed cavity appeared at SCI 2 w and stabilized at SCI 4 w.Observation of the process of glial scar formation demonstrated that reactive astrocytes with hypertrophic soma and cross-linked thick processes formed glial scar gradually which emerged around the cavity at SCI 4 w.These data suggested SCI evolves into chronic stage at SCI 4 w.3.By employing axonal tract tracing and double immunofluorescence,we found nerve fibers remained regenerative ability after SCI.Most fibers ran outside of glial scar,no fibers could be seen to pentrate glial scar to cavity or even through cavity.However,a few axons could be seen to regrow into the outer of layer of glial scar.These observation suggests glial scar is a barrier to axonal extension.In order to provide basic data for ablating glial scar in the future,we measured the thickness of glial scar.The results showed that the difference of thickness of glial scar between the rostral/caudal region and lateral region is distinct.The thickness of glial scar in the rostral/caudal region is 107.00±20.12μm,and 69.92±15.12μm in the lateral region.4.Examination via western blotting and immunofluorescence showed that ASIC1a expression markedly increased at peri-injury site both in gray and white matter after SCI, reached its peak at 12-24 h,then started to turn back and recovered to original level at SCI 1 w,remaining for up to SCI 6 w.On the contrary,ASIC1a expression at the injury site decreased obviously,and arrived at its rock-bottom at SCI 1 w without recovery.Nissl and NeuN staining showed that neuronal loss was serious at injury site which may cause the decrease of ASIC1a expression at injury site.Double immunofluorescent staining displayed that cells expressed ASIC1a in the gray matter were neurons,while they were oligodendrocytes in the white matter.Intriguingly,western blotting data demonstrated that ASIC2a level is very low in the normal spinal cord.But it dramatically increased afer SCI, especially in the peri-inujury site which recovered to original level untill SCI 4 w.ASIC2a expression increased early after SCI,then dramatically decreased since SCI 24 h due to neuronal loss at injury site.Data from RT-PCR showed that no obvious change were found in ASIC1a mRNA level,but ASIC2a mRNA level increased obviously after SCI.5.Double immunostaining showed that most TUNEL positive cells were ASIC1a positive,suggesting that ASIC1a may involved in delayed cell death afer SCI.On the contrary,no TUNEL positive cells displayed ASIC2a positive,suggesting ASIC2a may not be related to seondary cell death after SCI,also suggesting co-immunostaining of ASIC1a and TUNEL is specific.Moreover,by employing injury model in vivo and in vitro, TUNEL and PI/FDA staining,we found both ASIC1a specific antagonist PcTx1 and nonspecific antagonist amiloride decreased cell death induced by injury.Specific antisense targeting ASIC1a also produced the same protective effect.6.Making use of electrophysiology and Ca²⁺ iamging,we recorded that acidic stimulation evoked large transient inward currents and rapid[Ca²⁺]_i increase in cultured spinal neurons,which could be blocked by both ASIC1a specific antagonist PcTx1 and nonspecific antagonist amiloride.The acidic currents and[Ca²⁺]_i increase was enhanced by the pathological condition mimicking the ischemia/anoxia following SCI.7.Further experiment data showed that enhancement of AISC1a channel acitvity is complicated with ASIC1a phosphorylation.Co-IP data confirmed that ASIC1a phosphorylation increased after SCI which may be catalyzed by calcium/calmodulin-dependent kinaseâ…¡(CaMKâ…¡).Western blotting data displayed that CaMKâ…¡expression increased after SCI,sharing pertinence with the temporospatial pattern of ASIC1a expression after SCI.CaMKâ…¡specific antagonist KN93 significantly inhibited enhancement of ischemia-induced ASIC1a currents and cell injury.8.Finally,in order to confirm the role of ASIC1a in SCI in the whole level in vivo,by using intrathecal delivery technique,we found both ASIC1a specific antagonist PcTx1 and nonspecific antagonist amiloride reduce tissue injury and promote functional recovery after SCI.Again,ASIC1a antisense had the similar protective effect.Conclusion:1.The SCI model used in this study can distinguish the graded injury,consistent with the results from behavior,motor/sensor evoked potential,histopathology.Thus,this SCI model possesses objectivity,stability,relativity and reproducibility.2.The spontaneous functional recovery pattern of 10g×10mm group rats is specific, unvariable and distinct from the other groups,which can objectively estimate the effect of measures and drugs used to treat SCI.Therefore,10g×10mm injury severity was employed by this study.3.All the data from behavior,electrophysiology,pathology and observation of glial scar formaiton suggests SCI evolves into chronic stage at 4 w post injury,which will provide important experimental evidence for people to recognize and study chronic SCI.4.Nerve fibers remain regenerative ability after SCI,but few of them penetrate glial scar,suggesting glial scar is an impediment for axonal extension.This also suggests glial scar ablation will be important strategy for SCI treatment.We also measured the thickness of glial scar,which offers temporospatial reference data for ablation of glial scar in the future.5.ASIC1a expression markedly increased both in gray and white matter after SCI, reached its peak at 12-24 h,then started to turn back and recovered to original level at SCI 1 w.Double immunofluorescent staining displayed that cells expressed ASIC1a are oligodendrocytes in the white matter.Upregulation of ASIC1a expression has no relativity with its transcription,but its translation and/or metabolism.6.Both data from in vivo and vitro experiments demonstrates ASIC1a is involved in the secondary injury after SCI.The process may be:tissue acidosis following SCI activates ASIC1a channel which induce mono- and bivalent cations affiux,especially Ca²⁺ intracelular flow,which leads Ca²⁺ accumulation resulting in cell injury.7.Under the ischemia/anoxia condition following SCI,ASIC1a channel function enhances,which may be related with phosphorylation of ASIC1a catalized by calcium/calmodulin-dependent kinaseâ…¡(CaMKâ…¡).CaMKâ…¡may be activated by Ca²⁺ intracellular flow mediated by ASIC1a channel.8.This study reveals that tissue acidosis with activated ASIC1a channel by acidosis following SCI is a new pathogenic mechanism underlying sceondary injury post SCI,which provides important experimental evidenc for designing specific drugs targeting ASIC1a for SCI treatment in the future.In conclusion,tissue acidosis with activated ASIC1a channel play a vital role in secondary injury after SCI.The following is the possible mechanism:SCI induces tisse acidosis concomitantly activates ASIC1a channel.Then,Ca²⁺ afflux mediated by ASIC1a activates CaMKâ…¡which catalyzes phosphorylation of ASIC1a,inducing enhancement of ASIC1a channel function.This enhancement will produce more Ca²⁺ afflux which further activated CaMKâ…¡.Thus,a vicious circle formed which leads to Ca²⁺ accumulation resulting in cell injury.Upregulation of ASIC1a expression after SCI exacerbates this injury process.This study firstly discovers the role of acidotoxicity mediated by AISC1a in secondary injury after SCI,which will deepen our understanding of scondary injury mechanism after SCI,and provide substantial experimental data for designing specific strategy and drugs targeting ASIC1a for SCI treatment in the future.This finding implies important theoretic significance and clinical value.