| ObjectivesPart 1:To establish normative reference value for contact heat evoked potentials(CHEPs).Part 2:To gather objective evidence of the analgesic effects of repeated exposures to virtual reality(VR)distraction by means of CHEPs.Part 3:To study clinical characteristics and genetic spectra for Chinese Charcot-Marie-Tooth disease(CMT)patients.Part 4:To study the effect of mesencephalic astrocyte-derived neurotrophic factor(MANF)in Endoplasmic Reticulum Stress(ERS)of RT4-D6P2T with S63del MPZ Mutation.MethodsPart 1:CHEPs were applied on 151 healthy volunteers at right volar fore arm,right side of C7,right side of T12 and position above right lateral malleolus.Then normative reference value of different age groups,and different genders was established.Independent sample t test and rank sum test were used to compare the parameters of different age groups and genders.CHEPs parameters of different number of stimulus,different baseline temperature and after short-term and long-term interval were compared by paired sample t test and rank sum test.Part 2:CHEPs were applied on the left volar fore arm.76 adult participants underwent three test sessions in three consecutive weeks.Each session consisted of two conditions:the baseline(control)condition of CHEP stimulation without VR distraction and the VR distraction condition while undergoing CHEP stimulation.A series of(2 x 3)within-subject analyses of variance(ANOVAs)were conducted to determine the effects of condition(i.e.,baseline vs.VR distraction)and week on CHEP parameters.Linear correlation was used to determine the relationship between VAS scores and CHEP parameters.Part 3:151 unrelated Chinese patients with the clinical diagnosis of CMT were included.Clinical examination,nerve conduction studies(NCS),next-generation sequencing(NGS),and bioinformatics analyses were performed.Part 4:pLVX-MPZ(MUT)-PGK-Puro and pLVX-PGK-Puro were constructed to transfect RT4-D6P2T cell line.Experimental grouping:Normal control group,normal control+MANF group,pLVX-PGK-Puro group,pLVX-PGK-Puro+MANF group,S63del MPZ group and S63del MPZ+MANF group.The intervention time of MANF was 24h.CCK8 and Annexin-FITC/PI were used to detect cell proliferation and apoptosis.JC-1 was used to detect △Ψm.MANF,GRP78 and CHOP mRNA and protein were detected by RT-qPCR,western blot and immunofluorescence.ER-Tracker and mito-tracker were used to observe the morphology of endoplasmic reticulum and mitochondria.The concentration of calcium ion in the endoplasmic reticulum was detected by GENMED kit.ResultsPart 1:N2 latency/height varied with age and sex,while P2 latency/height and N2-P2 amplitude varied with age.The optimal number of stimuli was three.N2 latency/height(t = 5.45,P<0.001)and P2 latency/height(χ2 =-4.06,P<0001)decreased and N2-P2 amplitude(t =-5.01,P<0.001)and visual analog scale score(χ2 =-5.84,P<0.001)increased with increased baseline temperature(35℃ vs.32℃).CHEPs parameters did not differ with time(baseline vs.30 min vs.1 year).Normal values of CHEPs were established in Chinese adults.Part 2:As compared to the baseline condition,VR distraction significantly increased N2 latency/height(F = 9.497,P = 0.002)and significantly decreased N2-P2 amplitude(F = 65.752,P<0.001)and visual analog scale(VAS)score(F = 28.43,P<0.001).Analyses of the week effects and the condition-by-week effects were not significant for any of the dependent variables.The VAS score was moderately and positively correlated with N2-P2 amplitude(r = 0.532,P<0.001).Part 3:Genetic testing was performed for 120 patients;41 patients carried known CMT-associated gene mutations.PMP22 duplication was detected in 14(34%)patients and GJB1 mutations in 9(22%)patients.24 patients were diagnosed with CMT1,accounting for 59%of the 41 patients;and CMT1A accounted for 58%of CMT1.16 patients carried possibly pathogenic novel mutations;23 patients carried possibly non-pathogenic novel mutations,10 patients might be diagnosed with other diseases,and 30 patients carried no mutation.Part 4:(1)Compared with the first four groups,cell proliferation decreased(P<0.001)and apoptosis increased(P<0.001)in S63del MPZ group;compared with S63del MPZ group,cell proliferation increased(P=0.005)and apoptosis decreased(P<0.001).in S63del MPZ+MANF group.(2)△Ψm decreased(P<0.001)in S63del MPZ group.(3)The expression of MANF GRP78,CHOP mRNA increased(P<0.001)in S63de;MPZ group;the expression of GRP78 and CHOP mRNA decreased(P<0.001)in S63del MPZ+MANF group compared with S63del MPZ group.(4)In Western blot analysis,the expression of MANF,GRP78,CHOP,ATF6,P-PERK/PERK,P-IRE1/IRE1,Bax and Caspase3 increased(P<0.001)and Bcl2 decreased(P<0.001)in S63del MPZ group;the expression of GRP78,CHOP,ATF6,Bax and Caspase3 decreased(P<0.001)and Bcl2 increased(P<0.001)in S63de1 MPZ+MANF group compared with S63del MPZ group.(5)In immunofluorescence analysis,the expression of MANF,GRP78,CHOP,Bax and Caspase3 increased(P<0.001)and Bcl2 decreased(P<0.001)in S63de1 MPZ group;the expression of GRP78,CHOP,Bax and Caspase3 decreased(P<0.001)and Bcl2 increased(P<0.001)in S63del MPZ+MANF group compared with S63del MPZ group.(6)In S63del MPZ group,the morphology of endoplasmic reticulum and mitochondria was worse than the first four groups.(7)The concentration of calcium ion in the endoplasmic reticulum was decreased in S 63 del MPZ group,and the concentration of calcium ion in the endoplasmic reticulum was higher in S63del MPZ+MANF group than in S63del MPZ group.ConclusionPart 1:(1)Normal values of CHEPs were established in Chinese adults.(2)CHEPs parameters were affected by age and gender.(3)N2 latency/height decreased and N2-P2 amplitude increased with the rise of baseline temperature.(4)CHEPs was with good short-term and long-term repeatability.Part 2:(1)CHEPs were applied to objectively evaluate the analgesic effects of VR distraction.(2)Repeated application of VR did not reduce its analgesic effect.Part 3:(1)Clinical and genetic spectra of CMT were enriched.(2)The mutation diversity of CMT was broadened by novel mutationsPart 4:(1)ERS was observed in RT4-6P2T cell line with S63del MPZ mutation.(2)MANF was shown to inhibit ERS in RT4-6P2T cell line with S63del MPZ mutation. |
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