The Study Of The Role Of NLRP3 Inflammasome In The Secondary Degeneration Of Visual Cortical Neurons Following Optic Nerve Injury

In the visual system,in addition to the direct damage to the axons and soma in the primary site under pathological conditions such as ocular trauma and high intraocular pressure,neurons within the visual pathway also suffer from anterograde?retina to visual cortex?or retrograde?visual cortex to retina?injuries,which are characterized by distal neuronal degeneration,programmed cell death,increased inflammatory cytokines accompanied by recruitment and activation of glial cells.However,little is known about the initial factors and pathological mechanisms responsible for these secondary changes and relevant therapies to prevent disfunction are still unavailable.Inflammation performs dual roles of damage and repair in the central nerve system?CNS?and has also been reported as a key factor in secondary injury.As an important component of human innate immunity,cell pattern recognition receptors?PRRs?,such as toll-like receptors and NOD-like receptors,are activated by pathogen association molecular patterns?PAMPs?and damage association molecular patterns?DAMPs?,fused into a multimeric complex by binding to the adaptor molecule apoptosis-associated speck-like protein containing a Caspase-1 recruitment domain?ASC?and pro Caspase-1,named the inflammasome.The inflammasome is capable of converting immature pro-IL-1?and pro-IL-18 to mature forms by pro Caspase-1 self-cleavage,thereby triggering the innate inflammatory response.In addition,activated Caspase-1 promotes the maturation of gasdermin D?GSDMD?,which ultimately mediates the proinflammatory programmed cell death termed pyroptosis.In the inflammasome family,the nucleotide-binding domain leucine-rich repeat?LRR?–pyrin domain containing or NOD-like receptor family pyrin domain containing?NLR?inflammasome has been well characterized,for the abnormal activation of the NLR-inflammasome has been proved to be implicated in multiple CNS disorders,diabetes mellitus and autoimmune diseases.Accumulating evidence has shown that inflammasomes participate in the onset and progression of secondary brain injury and glaucoma.Furthermore,the activation of the NLRP3 inflammasome in microglia has been reported to be implicated in retinal ganglion cells?RGCs?loss after optic nerve?ON?injury.Studies on the aspect of secondary lesions have characterized the breach of the blood-brain barrier associated with the recruitment of peripheral mononuclear macrophages and neutrophils and the inflammatory response upon inflammasome activation in glias following optic nerve injury.Furthermore,the expression of some inflammasome members in neurons has also been documented.Concerning their possible involvement in secondary/distal degeneration in the CNS,it is urgent to establish whether the inflammasome family members can be activated transsynaptically after ON injury and whether its effect on visual cortical neurons is responsible for secondary neuronal degeneration.Moreover,the phenotypes of secondary lesion of visual pathway due to varying degrees of ON insults are remained to be classified.To address these issues,we carried out our research as following three aspects.?1?Inducing secondary injury of visual cortical neurons by establishing mouse model of unilateral optic nerve crush?ONC?and mouse model of specifically bilateral ON demyelination?OND?,respectively.?2?Identifying the role of inflammasome in the anterograde degeneration along visual pathway by screening for the potential inflammasome candidate and observing its abnormal activation responsibe for the secondary injury of visual cortex.Identifying the long-term effects of OND on the neuronal circuits of visual cortex.?3?Exploring the molecular mechanisms of activation of inflammasome in secondary degeneration.Here we provided the main results and conclusions as follows:1.We established the mouse model of ONC by operating ophthalmic microsurgery.The anterograde transmission of optic fibre was completely disrupted by ONC.Neither the damage of retinal blood vessels nor retinal ischemia was observed in ONC-treated mouse.Taking advantage of the chronological difference between ON and primary visual cortex?V1?myelination,together with diphtheria toxin?DT?administration,we established the mouse model of OND.The secondary degeneration of visual cortical neurons was observed both in the mouse models of ONC and OND,respectively,indicating the qualification of both models in the study of secondary degeneration induced by varying degrees of optic nerve insult.The number of apoptotic neurons of contralateral V1 and the dosal lateral geniculate nucleus?d LGN?was significantly increased 7 days post ONC.However,the significantly increased number of apoptotic neurons was observed 28 days post OND administration.2.The critical role of NLRP3 inflammasome in mediating the secondary degeneration of V1 caused by ONC and OND.The results of q RT-PCR and Western blot revealed the expressions of NLRP3 and other inflammasome components are significantly enhanced in the contralateral V1following ONC.Activation of the NLRP3 inflammasome in the V1 was found to be associated with the progression of neuronal injury.Ablation of NLRP3 by using NLRP3^-/-gene knockout mice,the levels of inflammasome-related cytokines in the V1 were decreased within 14 days after ONC,the number of apoptotic and degenerative neurons was decreased and the indexes of visual electrophysiology were also ameliorated in NLRP3knockout mice.The enhanced expression of NLRP3 in V1 microglia was also observed following OND.Activation of NLRP3 was responsible for the V1 secondary degeneration induced by OND.The numbers of both excitatory and inhibitory synapse were compromised14 days following OND,indicating the neural circuitry of V1 was impaired due to OND.3.The ATP-P2X7 pathway may contributes to the activation of NLRP3 inflammasome during the anterograde degeneration.The results of q RT-PCR and Western blot revealed the expression of P2X7 was significantly increased 12h post ONC.The ONC induced activation of NLRP3 was significantly compromised within 1d by P2X7 inhibiting.Activating P2X7 by using Bz ATP enhanced the protein level of NLRP3 on 7^th day post ONC.AIM2 can be activated in V1post ONC and its significant upregulation in the V1 of NLRP3^-/-brain indicating a crosstalk between the AIM2 and NLRP3 may exists in the course of secondary neuronal degeneration.