| Traumatic optic neuropathy(TON)is a common complication of traumatic brain injury(TBI).The incidence of TON ranges from 1.5% to 4% after TBI.TON has been characterized by apoptosis of retinal ganglion cells,optic nerve degeneration and visual impairment.Patients with optic neuropathy may experience the symptoms of vision decline and blindness,which will affect the patient,bringing great economic burden to the family and society.Therefore,it is imperative to seek an effective treatment to preserve and restore the vision of the TON sufferers after TBI.There are two main causes of TON after TBI: direct injury and indirect injury.Direct injury refers to the penetrating injury of the optic nerve,while indirect injury refers to damage caused by external forces on the optic nerve or secondary injury responses(such as axonal shear,edema,inflammation or increased intracranial pressure).What is more,the pathophysiological process can be classified into primary injury and secondary injury.Anatomatically,the axons of the retinal ganglion cells(RGCs)gather together to form the optic nerve.After injury,part of the optic nerve is cut off immediately,which is an irreversible process that leads to the death of some RGCs.In addition,due to direct mechanical compression and vascular ischemia,there exists a degree of optic nerve swelling within the narrow area of the optic canal,further damaging the RGCs and creating a vicious cycle of injury.The optic nerve is a highly differentiated somatosensory nerve.The surviving RGCs are unable to spontaneously regenerate axons after injury,which leads to permanent rupture of the optic nerve and loss of visual function.Therefore,reducing the apoptosis of RCGs in TON and promoting the regeneration of RCGs may be an promising and effective target in the treatment of TBIMessager RNA(m RNA)can be modified in a variety of ways,including N1-methyladenosine(m1A),5-methyl cytosine(m5C),and N6-methyladenosine(m6A).m6 A is modified most widely in RNAs.m6 A modification of m RNA was discovered in the early 1970 s,but due to the limitation of technology at that time,no physiological function correlation of m6 A modification was found.In recent years,great advances in methylated RNA immunoprecipitation sequencing(Me RIP)and other technologies have rendered m6 a modification to a wide public concern.Recent studies have found that m6 A is extensively modified in eukaryotic cells,with over 25% in transcriptomes,and that it functionally regulates eukaryotic transcriptomes to affect m RNA splicing,export,localization,translation,and stability.Given the indispensable function of m6 A m RNA in regulating protein translation and various biological processes,it is reasonable to speculate that the regulation of m6 A modification may also be related to TON.During the apoptosis of RGCs after TON,m6 A modification may change and play a role in related signaling pathways of apoptosis.At present,patients with traumatic optic nerve injury often receive the treatment of craniocerebral trauma first.Endoscopic optic nerve decompression should be performed after craniocerebral injury is stable.However,it has been found that a large number of RGCs will undergo necrosis and apoptosis within 2 weeks after optic nerve injury.If the interval between the injury and operation is less than 3 days,it will help the long-term vision improvement of the wounded.If the interval is more than 3 days,there will be less possibility of vision improvement.As time going,RGCs will continue to apoptosis,blocking the nerve signal transmission of visual impulse.Even if surgical decompression can eliminate the pressure factors,it is difficult to reverse the apoptosis of RGCs.If we can take effective measures in the early stage of traumatic optic neuropathy and reduce the necrosis and apoptosis of RGCs by means of surgery or drug treatment,it will have great value in preserving the vision of the wounded with traumatic optic neuropathy.In view of what has been mentioned above,it is necessary to study the role of m6 A modification of m RNA in the pathophysiological process of traumatic optic neuropath,which will provide a novel insight for exploring the retention and recovery of axons.Therefore,we completed the experimental research on the differential expression of m6 A m RNA in the TON by means of model establishment,immunofluorescence,Me RIP,pathway analysis,etc.: 1.Design and establish the rats model of TON;2.Differential expression and genome-wide screening of m6A-tagged m RNA in TON.Part one: The establishment and evaluation of rat model of traumatic optic neuropathyObjective:(1)To establish a repeatable rat model of traumatic optic neuropathy,evaluate the effect of this model on optic nerve and retina;(2)to explore whether there is any change in the expression of m6A-related genes in retina after optic neuropathy.Methods: 60 healthy adult male SD rats were randomly divided into two groups,namely,the model group and the control group.The left optic nerve of rats in the model group was exposed,and its optic nerve was clamped 2 mm distance from the eyeball with special forceps.In the control group,the left optic nerve was exposed but not injured.Hematoxylin-eosin(HE)staining and Brn3 a immunofluorescence staining were performed to reveal the counting of RGCs at 12 h,3,7 and 14 days after injury seperately.RGCs cells in retina of the two groups were counted and compared.At 12 h after injury,the retina tissues of the remaining 12 rats were harvested,and RNA was extracted for RT-q PCR analysis.The expression differences of m6A-related genes,FTO,ALKBH5,METTL3,METTL4 and WTAP,were analyzed between the model group and the control group.Results:(1)HE staining: after traumatic optic neuropathy,at 12 h,3 D,7 d and 14 d,RGCs counting in the model group(18.21 ± 0.19,16.58 ± 0.32,14.29 ± 0.92,10.94 ± 0.49)and control group(19.46 ± 0.43,19.04 ± 0.39,19.73 ± 0.21,19.63 ± 0.35).The number of RGCs in the model group decreased by 6.4%,12.9%,28.1% and 44.2% respectively at 12 h,3 d,7 d and 14 d compared with the control group(P <0.05);(2)immunofluorescence staining: at 12 h,3 D,7 d and 14 d,the RGCs counting in the model group(18.23 ± 0.48,16.08 ± 0.27,13.88 ± 0.53,10.48 ± 0.39)and in the control group(19.71 ± 0.21,19.77 ± 0.72,19.13 ± 0.61,18.27 ± 0.44),the former decreased by 7.5%,18.6%,27.4% and 42.1% respectively compared with the latter(P < 0.05);(3)RT-q PCR analysis showed that the expression of ALKBH5,FTO,METTL3,METTL4 and WTAP were all up-regulated.Conclusion:(1)The rat model of traumatic optic neuropathy is stable and easy to operate.This model can effectively mimic the pathophysiological changes of the injured patients with traumatic optic nerve injury;(2)after traumatic optic neuropathy,the expression of m6A-related genes is up-regulated in the retina,which indicates that the methylation of m RNA in the retina may change after the injury of optic nerve.Part two: the expression profiles and function analysis of m6 A modified m RNA in retina on the early stage of traumatic optic neuropathyObjective: To explore the expression profiles of m RNA methylation in the retina of rats with traumatic optic nerve injury,and to predict its function through gene ontology analysis and pathway analysis,which we hope could provide additional reference for the diagnosis,treament and prognosis monitoring of optic nerve in the future.Methods: Based on the rat model of traumatic optic neuropathy,24 rats were randomly divided into model group and control group.In the model group,the left optic nerve was injured,while in the control group,the left optic nerve was exposed but not injured.The rats were killed at the 12 th hour after operation and the retina tissues were harvested.The retina tissues of 8 rats were combined into one sample,in total,there were 6 groups of samples(3 groups of model samples and 3 groups of control samples).After the RNA of retina was extracted,part of the RNA was used for m6 A RNA immunoprecipitation.RNA sequencing library was constructed for the samples without immunoprecipitation reaction and IP RNA samples with m6 A immunoprecipitation.After sequencing by sequencer,image analysis,base recognition and quality control,the original data is generated.Q30 was used for quality control and getting high quality results.Then,the methylated methylated gene recognition was carried out.The m6 A peaks on exon of m RNA were screened and annotated by our own program.Go and KEGG pathway analysis was carried out on the differentially methylated coding genes specific to traumatic optic neuropathy.Results:(1)Differential expression of genes: after optic nerve injury,520 genes were upregulated and 258 were down regulated(fold change ? 2,P < 0.05);(2)expression profiles of m6 A modified sites: the whole genome localization of m6 A modified m RNA in the retina of rats with traumatic optic neuropathy,2810 m6 A sites were up-regulated and 689 were down regulated(fold change ? 2,P < 0.05).m6A-tagged m RNA were mainly located on chromosome chr1,chr2,chr3 and chr10;(3)GO analysis: the up-regulated m6A-tagged m RNA were mainly related to neural tissue development,system development and cell differentiation;the down regulated m6A-tagged m RNAs were mainly related to cell location,cell macromolecule location and protein folding;(4)KEGG pathway Analysis: the up-regulated m6 A peak in retina is related to MAPK,NF-? B and TNF signal pathways,while the down regulated m6 A peak correlate closely with ribosome,endoplasmic reticulum protein processing pathway and spliceosome pathway.Conclusion:(1)After the traumatic optic neuropathy,there is a large amount of differential expressed m6 A modifications in the m RNA of retina;(2)GO analysis and KEGG pathway analysis suggest that m6 A modification can be involved in multiple parts and processes of optic nerve injury,and m6 A modification of m RNAs concerning genes like AMIGO,SHANK and SLC38A1 are possible to be biomarkers related to the diagnosis and prognosis evaluation of traumatic optic nerve injury.These findings will have implications on the new targets in treatment of TON. |
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