| Part one Effects of low dose X-ray irradiation on nerve regeneration and functional recovery after transected sciatic nerve injury in ratsBackground and Objective: The repair and treatment of the damaged peripheral nerve has been a thorny problem for a long time,and is becoming the focus of social attention.Although microsurgery and tissue engineering provide new methods for repair and regeneration of peripheral nerve,their clinic effects are far from ideal.Recently,the medical field has been actively exploring therapeutic methods to promote nerve regeneration and accelerate function recovery of injured peripheral nerve.Clinical practice for many years had already confirmed that physical therapy has a wide and effective role in the treatment and rehabilitation.Ionizing irradiation,as an important member of physical factor family,has more and more been reported of its therapeutic effects.It is well known that moderate to high doses of ionizing radiation have a toxic effect on peripheral nerve.There is evidence that low-dose radiation improves the hormesis of various cell types.Low-dose radiation has been used in clinical and experimental models for the prevention of scarring and for fracture healing.But few studies had reported the effects of ionizing irradiation on nerve regeneration and functional recovery after the peripheral nerve injury.This study was to explore the effects of low dose X-ray irradiation on nerve regeneration and functional recovery after transected sciatic nerve injury in rats through irradiating the animal model.Through this study,we expect to provide a new method and theory basis for peripheral nerve regeneration after the injury and lesion.Methods: Eighty four two-month old Sprague-Dawley rats weighing 250 g~300 g were selected and randomly divided into two groups: 1 Gy irradiation group(Experimental group)and 0Gy irradiation group(Control group).Each groups had forty two rats.After the left sciatic nerves in rats were exposed and transected,the nerves were sutured in an epineurium end-to-end anastomosis method to restore the continuity.Twenty-four hours after the models were built,the repaired nerves were locally irradiated by doses of X-ray(0Gy,1Gy)respectively.The observation of foot ulcers,gaits,foot sensation and unfold claw reflect was performed weekly.On week four,eight and twelve post-irradiation,the sciatic function index of the rats was measured.Western blot and q RT-PCR were performed to determine the level of vascular endothelial growth factor(VEGF)and growth-associated protein-43(GAP-43)on day three and week twelve after irradiation.Twelve weeks after irradiation,the electrophysiological examination was performed to evaluate the functional recovery of the regenerated nerve.Histopathological and immunohistochemical analysis,sirius red staining and observation of the ultrastructure with transmission electron microscope were performed to evaluate the effects of low dose X-ray irradiation on nerve regeneration and functional recovery after sciatic nerve transected injury functionally,morphologically and molecularly biologically.Results: 1.General observation of foot:(1)Foot ulcers: Between week one and week two after irradiation,the left hind foot heel of the rats in the two groups all had ulcer.The ulcer of the 1 Gy group started the healing at the week three,and healed completely at the week six.The ulcer of the control group started the healing at the week four,and healed completely at the week eight.The ulcer of the 1 Gy group healed earlier than the control group.(2)Gaits: One week after irradiation,leg and foot of the operated side completely paralyzed,the plantar prolapsed,the toes were all together and flexion deformity in the two groups.As the rat walk,the operated leg was skidding on the ground.In the 1 Gy irradiation group,deficits in foot dorsiflexion and plantar flexion disappeared by the end of twelve weeks,and the toes were slightly open.In the control group,the deficits in foot only improved partially at the same observation time.The time of gaits recovery to normal was significant difference in foot dorsiflexion and plantar flexion between the two groups(P < 0.05).(3)Foot sensation and unfold claw reflect: Between week two and week four after irradiation,rats in the two groups did not have any limb retraction and unfold claw reflect when the left hind limb was acupunctured.In the 1 Gy irradiation group,the reaction of five rats was sensitive at eight weeks,and seven rats was sensitive at twelve weeks.In the control group,the reaction of one rat was sensitive at eight weeks,and four rats was sensitive at twelve weeks.Foot sensation and unfold claw reflect of the 1 Gy group began to recover earlier than the control group,and return to normal earlier than the control group.2.VEGF and GAP-43 levels: At three days after irradiation,quantitative RT-PCR showed statistically significantly higher(P < 0.05)VEGF m RNA levels in the low-dose(1Gy)X-ray irradiated group compared to the control group.Western blot analysis showed significantly higher(P < 0.05)VEGF protein expression levels in the low-dose irradiated group than the control group at three days after surgery.At two weeks after irradiation,quantitative RT-PCR showed significantly higher(P < 0.05)GAP-43 m RNA expression levels in the irradiated versus control group.Western blot analysis showed that GAP-43 protein expression was greater in the irradiated versus control group(P < 0.05).3.The sciatic functional index(SFI): Two week after irradiation,the rats in two groups showed the toes were flexion deformity and the plantar prolapsed,so that the value of the SFI cannot measure and calculate.From week four to week twelve,the SFI value of the two groups revealed varying degrees of improvement.A significant increase(P < 0.05)in SFI value was observed in the low-dose irradiated group compared to the control group at 4,8,12 weeks,respectively.3.The electrophysiological examination: On twelve weeks after the irradiation,the MNCV of the two treatment groups revealed varying degrees of improvement.Significantly improved recovery(P < 0.05)was observed in the low-dose irradiated group compared to the control group.The CMAP peak amplitude was greater in the irradiated group versus control group(P < 0.05).The CMAP latency of onset was shorter in the irradiated group versus control group(P < 0.05).4.Histopathological and immunohistochemical analysis: On twelve weeks after the irradiation,using light microscopy,H&E staining of the transverse sciatic nerve showed that in the low-dose irradiated group,a few dark-blue inflammatory cells were scattered around the axon.Slight adhesion to their surrounding tissues,slight axonal degeneration,vacuolization and abundant neovascularization were noted.The regenerated axons and myelin sheaths had formed.In the control group,a large number of inflammatory cells had infiltrated around the axon.Many adhesions,more axonal degeneration,vacuolization and less neovascularization were observed.Endoneural space increased slightly more in the control group compared to the 1 Gy irradiated group.The expression and distribution of S-100 and NF-kb in the 1 Gy irradiated group was better than the control group.5.Sirius red staining : In the control group(0 Gy),the average area ratio of the type?and type ? collagen was(91.31 ± 9.58)% and(10.65 ± 1.06)%.In 1 Gy irradiated group,the average area ratio of the type?and type ? collagen was(57.40 ± 4.33)% and(44.16 ± 3.39)%.The 1 Gy irradiated group was significantly lower than the control group(P < 0.05).6.Observation of the ultrastructure with transmission electron microscope:Ultrastructural morphometric analysis showed numerous normally myelinated axons in the1 Gy irradiated group.The regenerated myelin sheathes presented as round or oval with regular shape,with an even thickness and clear outline,and the surrounding fibroplasias were not obvious.The lamellae were arranged compactly and thickly without axonal degeneration or changes of myelin.Demyelination,delamination and fibrous connective tissue hyperplasia in myelin sheaths were shown in only a few myelinated axons.In the control group,very few normal myelinated axons were seen.The regenerated myelin sheathes were distorted,uneven in thickness and irregular in shape.Different degrees of demyelination,delamination and fibrous connective tissue hyperplasia were noticeable in many myelinated axons.The lamellae were thinner than in the 1 Gy irradiated group.Statistical results showed that the total number of myelinated nerve fibers,average axonal diameter and myelin sheath thickness in the the 1 Gy irradiated group were significantly greater than in the control group(P < 0.05).Conclusions: Our data suggested that low-dose X-ray treatment may have beneficial effects on the regeneration of transected sciatic nerves.Its positive effects were evident,in particular,in the morphological evaluation.These findings demonstrated that low-dose X irradiation may be a potential treatment approach for peripheral nerve injury.However,its mechanism and long-term effect remain unknown,further studies are needed,with a focus on large-scale and long-term follow-up,to confirm our findings.Part two Effects of low dose X-ray irradiation on expression of micro RNA in dorsal root ganglia after transected sciatic nerve injury in ratsBackground and Objective: Peripheral nerve transected injury is a common disease in clinic practice.Low-dose irradiation exposure can increase the nerve regeneration and functional recovery after transected sciatic nerve injury in rats,but the mechanism is unclear.Micro RNAs(miRNAs)are a class of endogenous non-coding small single-stranded RNA molecules with a length of 18-22 nucleotides.miRNAs play important roles in the processes of human development,physiology and pathology.The study of whether these differentially expressed miRNAs are involved in the recovery of dorsal root ganglion after low dose X-ray irradiated the transected sciatic nerve has not been reported.RNA sequencing(RNA-Seq)is one of the most recently developed methods for the determination of high-throughput nucleic acids.It can reflect the expression level of genes in tissue,cell,or physiological state.In this study,we detected the expression of miRNAs in irradiated rats following sciatic nerve injury by RNA-Seq,and explored the mechanism underlying mi RNA function in irradiated rats after sciatic nerve injury.To clarify the mechanism of low-dose X-ray irradiation promoting peripheral nerve regeneration could provide new ideas and technology platforms experimental studies to the effective treatment strategies and provide assistance to the therapy of peripheral nerve injury.Methods: According to the preliminary experimental results,we found that The peak levels of VEGF and GAP-43 m RNA expression in the 1 Gy group were detected after seventy-two hours exposure to irradiation.Therefore,at seventy-two hours post-irradiation,L4-6 segments of the spinal cord together with dorsal root ganglia and connected sciatic nerves(left side)in the two groups were collected for the RNA sequencing.After the sample was ready,the total RNA for each sample was extracted.The transcriptome library was constructed.We performed Illumina deep sequencing.The raw data of sequencing was analyzed,and the clean data was got.The bioinformatics analysis and identification of mi RNA targets were performed.Differentially expressed genes were then selected and classified into major clusters according to their expression patterns.In order to know credible biological functions,we conducted the Gene Ontology(GO)analyses and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis regarding the intersected clusters.Gene act networks were then constructed for the intersected clusters and the expression of pivotal genes of gene act networks was validated by quantitative real-time PCR.Results: From the results of deep sequencing,we identified a total of 804 miRNAs.Of these,327 miRNAs were identified as known miRNAs,whereas the remaining 477 were considered novel miRNAs.The results showed that 150 miRNAs were differentially expressed between the treated group(-1 Gy)and control group(-0 Gy).Of these 150 differentially expressed miRNAs,149 were novel.Among these differentially expressed miRNAs,56 were up-regulated and 94 were down-regulated.A total of 23,652 target genes and 48,157 target sites were predicted for the 150 differentially expressed miRNAs.Many genes involved in neurogenesis,angiogenesis,apoptosis,and the neuroactive-ligand receptor interaction,such as Stat,P13 k,Vegfa,Cdk,P21,Casp9,CHRM,and Nmur,were predicted as targets for these differentially expressed miRNAs.KEGG enrichment analysis was performed using the differential expression of target m RNAs.The results show that the enriched KEGG terms were associated with pathways such as cancer,the PI3K-Akt-signaling pathway,neuroactive ligand-receptor interaction,and the MAPK signaling pathway.VEGFA is a molecule that plays an important role in nerve repair.Its expression increased is an important indicator of nerve repair capacity.Four differentially expressed miRNAs(novel miRNAs-360,-301,-239,and-400)were identified as targeting Vegfa,and all candidate miRNAs were down-regulated in the RNA-seq results.Our q RT-PCR results confirmed the mi RNA expression patterns found in our RNA-seqanalysis.Moreover,target sites for the novel miRNAs-360,-301,-239 and-400 were identified in the Vegfa 3?UTR.The cell transfection experiment found that the novel miRNAs-360,-301,-239,and-400 target VEGFA to regulate Vegfa expression.\Conclusions: Utilizing mi RNA deep sequencing,our study demonstrated that X-ray irradiation induced mi RNA expression changes in rats.Many novel miRNAs were first identified in our report,and shown to be involved in the recovery from peripheral nerve injuries.This study provides a framework for understanding the molecular mechanisms underlying the recovery from peripheral nerve injury following exposure to X-ray irradiation.Further experiments focusing on the identified target genes are necessary to explore the potential mechanisms of X-ray irradiation involved in the recovery from peripheral nerve injuries. |
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