| Traumatic optic neuropathy is an important cause of blindness.In recent years,with the rapid development of China’s society,the increasing number of social infrastructure and the rapid popularization of transportation tools,the injuries caused by construction and traffic accidents are endless.These injuries are often accompanied by brain and optic nerve injuries,leading to poor vision prognosis or direct totally loss,and bringing heavy burden to the society and the family。Direct or indirect violent impaction often leads to the fracture of the optic nerve tube,contusion and fracture of the optic axon,tissue edema and blood circulation disorder,resulting in secondary death of retinal ganglion cells（RGCs）and irreversible loss of vision.Currently,there is no effective treatment.Studies have shown that the number of ganglion cells surviving after optic nerve injury is positively correlated with the prognosis of visual acuity.Therefore,how to reduce the secondary death of retinal ganglion cells is an important means to preserve visual acuity after optic nerve injury.The increase of free radicals,calcium overload and the release of pro-inflammatory factors after optic nerve injury can lead to the death of retinal ganglion cells.Among these factors,neuroinflammation is an important cause of secondary death of retinal ganglion cells.Neuroinflammation includes infective and non-infective inflammation,and non-infective inflammation dominates in closed optic nerve injury.Inflammation is a"double-edged sword".Proper inflammation is conducive to infection control and cell growth and repair,while excessive uncontrolled inflammation can cause chronic and continuous inflammation in tissues,leading to inflammatory and autoimmune diseases.The regulation of inflammation is complex and changeable.It is an urgent problem for us to find the key molecules that lead to excessive aseptic inflammatory response in closed optic neuropathy and reduce secondary death of ganglion cells through regulation targets.Inflammasome is a multi-protein complex,and most classic inflammasomes are in response to a variety of stimuli pathogens and danger-associated molecular patterns（DAMPs）by members of nucleotide-binding oligomerization domain-like receptors（NOD-like receptors,NLRs）and absent in melanoma 2-like receptors（ALRs）,recruits the inflammation adaptor.Following this oligomerization,inflammasomes cleaving procaspase to caspase-1.Active caspase-1 then cleaves proinflammatory cytokines prointerleukin-1β（IL-1β）and prointerleukin-18（IL-18）to their active forms and play the corresponding biological functions.Inflammasomes are widely expressed in central nervous system diseases,which aggravate aseptic inflammation and worsen the condition in craniocerebral and spinal cord traumatic diseases.While recent studies have demonstrated that Nlrp3activated microglia,released inflammatory mediators and caused the death of retinal ganglion cells in optic nerve crush（OPC）injury,Nlrp3-/-mice display delayed retinal ganglion cells death associated with the attenuation of toxic side effects on the microglia following an OPC injury,Thus,inflammasomes might play an important role in optic nerve injury.However,there are more than 10 known inflammasomes,and it is not clear whether the other inflammasomes participate in the pathological process after optic nerve injury except Nlrp3,which requires further study.Inflammasomes not only release pro-inflammatory factors,but also participate in the regulation of cell death,namely pyroptosis.Pyroptosis is a form of cell death that involves the rupture of plasma membranes and subsequent release of intracellular components,which stimulate immunogenic reactions that cause sterile inflammation in a vicious cycle,it would lead to the tissue out of control inflammation for a long time.At present,it is generally believed that the death form of retinal ganglion cells in traumatic optic neuropathy is mainly apoptosis.However,some studies have found that blocking the apoptotic enzyme caspase-3 cannot prevent the apoptosis of retinal ganglion cells,which indicates that some other mechanism may participate in the cell death after OPC injury.Pyroptosis was observed in the retinas of mice with eyeball explosion injury model,whether or not pyroptosis of retinal ganglion cells and which inflammasomes participated in the process of pyroptosis were not clear.In this study,to investigate whether there are differences in gene expression of inflammasomes under two different conditions and which inflammasomes play an important role in sterile inflammation after OPC injury,we screened the expression profiles of more than 10 different inflammasomes and inflammasome-related mRNAs in the retina following an optic nerve crush injury under both conventional sterile as well as non-sterile conditions using fluorogenic quantitative PCR,and confirmed the related proteins with Western Blot.Then,to investigate the forms of retinal cells death,fluorescence quantitative PCR was used to screen the key enzymes of cell death.To verify the mechanism of cell death,we detected the proteins expression of Nlrp1,mature IL-1β,Cleaved-Caspase-1 by Western Blot,the release of lactic dehydrogenase（LDH）and tunel staining in mice retinal ganglion cells saccharide-oxygen deprivation/reoxygenation cultured model at different time points.In the mice retinal ganglion cells saccharide-oxygen deprivation/reoxygenation cultured model,we also added VX765,the inhibitor of Caspase-1,to investigate the relationship of Caspase-1 and retinal ganglion cells pyroptosis.Main results（1）We observed that the peak of retinal ganglion cells death occurred on day 7 after OPC surgery in the mouse model,while most inflammasomes Nlrp1b,Nlrp2,Nlrp3,AIM2,Naip/Nlrc4 were much more highly elevated in non-sterile conditions and sterile conditions after OPC surgery,and with the highest levels observed on postoperative day 7.The mRNA expression of inflammasomes Nlrp5,Nlrp6,Nlrp7,Nlrp12 were not detected.The levels of the inflammasomes Nlrp2,AIM2,Naip/Nlrc4 mRNA were increased significantly in sterile conditions but not in non-sterile conditions.Nlrp1b and Nlrp3 inflammasomes mRNAs were increased in different levels on different timepoint after postoperative in the mouse sterile OPC injury model,with the highest levels observed on postoperative day 3and day 7 respectively,Nlrp1 protein was detected in retinal ganglion cells,and coincides with the expression of Nlrp1 mRNA.IL-1,an inflammatory cytokine associated with inflammasomes,increased in both protein and gene levels,and the peak occurred 7 days after the OPC injury sterile model.（2）.Secondly,we observed the mRNA expression of death associated protein kinase Caspase-1 and Caspase-3 increased on day 3、day 7、day 14 after OPC injury sterile model,the peak of Caspase-1 occurred on day 7,and Caspase-3 occurred on day 3.RIPK3 was increased on day 1、day 3 and day 7 after postoperation,with peak on day 3.PARP-1 and GPX4 were not increased significantly.Among these genes,caspase-1 increased most dramatically in this model,and was confirmed to be expressed in retinal ganglion cells.We detected the expression of Nlrp1,Caspase-1,IL-1βin mice retinal ganglion cells saccharide-oxygen deprivation/reoxygenation cultured model,and observed the expression were increased with the reoxygenation time,and the numbers of cell death were gradually increased in this process,accompanied by LDH release.（3）Thirdly,we observed in saccharide-oxygen deprivation/reoxygenation cultured cell model,different concentrations of caspase-1 inhibitor VX765 suppressed the activated caspase-1,the protein level of cleaved-caspase-1 and the number of retinal ganglion cells death were not significant compared the group 10μMol/L with 20μMol/L.The level of mature IL-1βprotein was obviously suppressed and the number of retinal ganglion cells death was significant reduced in saccharide-oxygen deprivation/reoxygenation cultured cell model with 10μMol/L VX765.conclusions（1）The results implicated the expression of inflammasomes genes in mouse retinas following an OPC injury was significantly different between animals housed in nonsterile and sterile environments.The inflammasomes Nlrp2,AIM2,Naip/Nlrc4 were participated in infectious nerve inflammation,Nlrp3,Nlrp1b were participated in infectious and non-infected nerve inflammation,and may play different roles in different stages of aseptic inflammation of closed optic nerve injury,which lead to the reduction of the number of retinal ganglion cells.（2）The results implicated necrosis,apoptosis,and pyroptosis may play different roles in different periods in OPC injury model but without ferroptosis and death induced by poly ADP-ribose polymerase-1（PARP-1）,pyroptosis induced by Nlrp1/Caspase 1 was predominant for the death of retinal ganglion cells in the subacute phase of OPC injury.（3）The results implicated the death form of retinal ganglion cells in this model was pyroptosis but necrosis.Caspase inhibitor VX765 could significantly inhibit the activity of caspase-1 with minimum concentration 10μMol/L in saccharide-oxygen deprivation/reoxygenation cultured cell model to prevent the pyroptosis of retinal ganglion cells. |
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