| (1)Analysis of clinical characteristics and curative effect of neuromyelitis optical-associated optic neuritisObjective:To analyze the clinical characteristics of neuromyelitis optical-associated optic neuritis(NMO-ON)patients and the efficacy of acupuncture and Chinese medicine combined to improve NMO-induced optic nerve atrophy.Methods:From January 2016 to December 2019,72 cases of NMO patients with ON as the first clinical manifestation in China-Japan Friendship Hospital were retrospectively analyzed and summarized,including general information,disease characteristics,course of disease,complicated diseases,immunological examination,treatment response and prognosis,etc.Results:1.General information:72 patients with NMO-ON,median age of onset was 33 years old;There were 61 females(84.72%),and the male to female ratio was about 1:5.54;The median course of disease was 67 months,and the course of disease was mainly "relapse-remission",and the ratio of uniphasic to relapsing was about 1:5.8.2.Clinical features:66 cases(91.67%)developed ON alone,and 59 cases(81.94%)involved ON one eye.The main symptoms are acute or subacute vision loss,with rotation pain,visual field defect and abnormal color vision,etc.14 cases(19.44%)were associated with inducement,and the most common was upper respiratory tract infection.11 cases(15.28%)were associated with systemic immune diseases,most commonly with Sjogren's syndrome and thyroid disease.First visual acuity was less than 0.1 in 59 patients(75.64%).AQP4-IgG status(P=0.032,OR=2.55)and onset age(P=0.037,OR=3.93)were independent risk factors affecting first visual acuity.During the course of the disease,44 patients(61.11%)had binocular involvement successively,the median frequency of ON onset was 2,and the median time of the second onset of ON was 3 months.The 1-year and 3-year recurrence rates were 0.56(40/72)and 0.74(53/72),respectively.Up to the last follow-up time,the rate of unilateral blindness was about 48.61%,and the median of unilateral blindness ON patients was 2 times.Other nervous system involvement was found in 53 patients(73.61%),and spinal cord(61.11%)was the most common site of recurrence.3.Laboratory results:80%(48/60)of the patients had positive serum AQP4-IgG antibody;25%(3/12)were positive for MOG-IgG;18 cases(25%)were associated with other systemic immune antibodies,most commonly with positive ANA antibodies.4.Imaging features:NMO-ON frequently involved the posterior optic pathway,and the lesion length was>1/2 of the optic nerve.45 patients(84.91%)had more than 3 cones in spinal MRI.47.17%(25/53)of the patients developed cervical and thoracic myelopathy simultaneously or successively.5.Acupuncture and Chinese medicine combined to improve optic nerve atrophy caused by NMO:visual acuity increased by more than 2 lines in 8 eyes of 24 eyes after 4 weeks of treatment,with a total effective rate of 91.67%;The dynamic field mean defect(MD)and mean sensitivity(MS)were improved compared with before treatment,but there was no statistical significance compared with before treatment(P>0.05).After 8 weeks of treatment,12 eyes had improved visual acuity by more than 2 lines,and the total effective rate was 100%.Visual field MD was significantly decreased(P<0.05),and MS was significantly increased(P<0.05).Conclusion:1.The first onset of NMO-ON patients was ON alone,involving one side,high incidence in young and middle-aged women,and younger in AQP4 IgG negative patients.Acute stage threatened patients' visual function,and poor visual prognosis was left in recovery stage;The most common inducement is upper respiratory tract infection,and the long course of disease is often bilateral optic nerve involvement and multiple recurrence.Spinal cord is the most common recurrence site except optic nerve;AQP4-IgG status and age of onset were independent risk factors affecting the visual function of NMO patients at first onset.2.Most(80%)of NMO-ON patients were serologically positive for AQP4-IgG,and some patients were associated with systemic immune diseases such as Sjogren's syndrome and thyroid disease.The most common serological immune antibody associated with NMO-ON was anti-ANA antibody.3.The optic nerve of NMO-ON patients mostly involved the posterior optic pathway,and the lesion length was>1/2 of the optic nerve.Most of the spinal cord injuries were continuous long segments,and about half of the patients had cervical spinal cord and thoracic spinal cord lesions simultaneously or successively.4.Acupuncture and Chinese medicine combination can significantly improve the visual function of patients with NMO induced optic nerve atrophy,significantly improve the visual acuity and dynamic field MS,and reduce the MD of patients(2)Construction and evaluation of a rat model of neuromyelitis optica associated with optic neuritisObjective:By microinjection of AQP4-IgG positive serum into the subarachnoid space around the optic nerve of rats,NMO-like lesions of the optic nerve were induced,and the model was evaluated from multiple dimensions including in vivo and pathology,optic nerve,spinal cord and brain,so as to provide a model basis for the exploration of drug prevention and treatment of NMO-ON.Methods:Thirty male SD rats were randomly divided into model group and sham operation group according to the type of serum samples.After the optic nerve was exposed by superior conjunctival approach,AQP4-IgG positive serum and healthy human serum were slowly injected into the subarachnoid space at 2mm behind the bulbar,respectively.The expression of AQP4-IgG in optic nerve was detected by immunofluorescence assay on the third day after operation.Flasher visual evoked potential(F-VEP)and pupil light reflex(PLR)were detected on day 0 before surgery and day 7 and 14 after surgery,respectively.On the 14th day after surgery,the two groups of rats were sacrificed respectively for tissue sections of retina,optic nerve,brain and spinal cord and pathological observationResults:On day 3 after operation,AQP4-IgG was strongly expressed in the model group,but not in the sham group(P<0.001).Compared with the sham group,the expressions of AQP4 and glial fibrillary acidic protein(GFAP)in the AQP4-IgG deposition region were also significantly decreased(P<0.001);The amplitude of N1-P1 wave and PLR in the model group were significantly decreased on day 7 and 14,and the differences were statistically significant compared with those in the sham group(P<0.001).Histopathological observation:Compared with the sham operation group,HE staining of the optic nerve in the model group was swollen,edema,significantly deeper staining,significantly reduced number of axons accompanied by irregular cavitation,and a large number of inflammatory cells were observed in the model group.RGCs showed significant edema,decreased number,varied size and unclear nucleoli.LFB myelin sheath staining of optic nerve showed a significant decrease in blue area in model group.In the model group,the number of axons was significantly reduced,the arrangement was disordered,the cross-section morphology was irregular,the microtubules and microfilaments were dissolved in large quantities,the myelin sheath was seriously stratified,the structure was incomplete,and most of the myelin sheath was loosened,lost,collapsed,or even had no myelin sheath.Immunofluorescence staining showed that compared with sham operation group,the expression of nerve filament protein L(NFL)was significantly decreased(P<0.001)and the expression of CD68 was significantly increased(P<0.001).HE showed that no inflammatory infiltration of spinal white matter,axon and myelin sheath injury were observed in the sham group and the model group.LFB staining of the spinal cord tissue showed that the myelin sheath of the sham operation group and the model group was stained uniformly in blue,and no decreased stained areas and white unstained areas were observed.He and LFB staining of brain tissue showed no pathological lesions such as inflammatory infiltration and demyelinationConclusion:1.We successfully established a rat model of NMO-ON by microinjection of human AQP4-IgG positive serum into the subarachnoid space of the rat optic nerve.The model showed characteristic pathological changes of NMO-ON,including astrocyte destruction,inflammatory infiltration,axon injury and demyelination.2.The NMO-ON model was established by micro injection of human AQP4-IgG positive serum into the subarachnoid space of the optic nerve of rats,which could only activate the local peripheral optic nerve immunity,and did not cause pathological damage to the spinal cord and brain and other nervous systems.(3)Efficacy and mechanism of Tiaogan Huoluo in the prevention and treatment of neuromyelitis optical-associated optic neuritisObjective:To observe the intervention effect of the method of Tiaogan Huoluo through vivo study of animal models,and to explore the molecular mechanism of the prescriptions,so as to provide a solution of the coordination of traditional Chinese and western medicine for the clinical treatment of NMO-ON.Methods:Sixty male SD rats were randomly divided into model group,sham operation group,traditional Chinese medicine(TCM)group,glucocorticoid group,and TCM+glucocorticoid group.NMO-ON rat model was established by microinjection.The rats were given sterile distilled water,TCM,glucocorticoid and TCM+glucocorticoid for 21 days by gavage.F-VEP and PLR tests were performed 1 day before surgery(Day 0),7,14 and 21 days after surgery,respectively.On the 21st day,all rats were sacrificed,and slices of retina and optic nerve were prepared for HE staining and ultramicro electron microscopy observation.The expression of Brn3a in RGCs was detected by immunohistochemical staining.The expressions of GFAP,NFL,MBP and CD68 were detected by immunofluorescence staining.The protein expressions of IL-6,IL-10,TNF-? and P-NF-kB p65 in optic nerve were detected by Western-blotting.Results:1.Evaluation of optic nerve function:Compared with model group and hormone group at the same time point,N1P1 amplitude of optic nerve F-VEP was significantly increased in TCM group and TCM+glucocorticoid group at the 7th,14th and 21st day(P<0.001);On day 21,PLR contraction degree in TCM group and TCM+glucocorticoid group was also significantly increased compared with model group and glucocorticoid group(P<0.001)2.Histopathological evaluation:Compared with model group and glucocorticoid group,HE staining of optic nerve in TCM group and TCM+glucocorticoid group showed reduced axon injury,subsided optic nerve edema,and no obvious inflammatory cell infiltration.Electron microscopy showed that the number of axons in the TCM group was significantly increased,the nerve fiber bundle shape could be followed,the microfilament microtubule structure could be seen,and the myelin sheath injury was less than that in the TCM group.Compared with the glucocorticoid group,the structure of nerve axon and myelin sheath were more complete in the TCM+glucocorticoid group.Immunohistochemistry showed that compared with model group and glucocorticoid group,the expression of Brn3a of RGCs in TCM group and TCM combined hormone group were significantly increased(P<0.001).Immunofluorescence of optic nerve indicated that the expression levels of GFAP,MBP and NFL in TCM group and TCM+glucocorticoid group were higher than those in model group(P<0.001).CD68 optic nerve staining also showed that compared with the model group,CD68 expression was significantly reduced in the TCM group and the TCM+glucocorticoid group(P<0.001).3.Western Blotting:Compared with sham operation group,the levels of p-NF-?B p65,TNF-a and IL-6 in optic nerve of model group were significantly increased(P<0.001);Compared with model group,p-NF-?B p65,TNF-? and IL-6 were decreased in TCM group,glucocorticoid group and TCM+glucocorticoid group(P<0.001).Compared with sham operation group,IL-10 in optic nerve of model group was significantly decreased(P<0.001);Compared with model group,IL-10 increased in TCM group,glucocorticoid group and TCM+glucocorticoid group(P<0.001);Conclusion:1.Tiaogan Huoluo Compound Chinese medicine can reduce optic nerve axon loss,myelin sheath injury and inflammatory infiltration in NMO-ON rat model,and then enhance and improve the amplitude of visual evoked potential N1P1 and the ability of pupil reflex light contraction.2.The neuroprotective and therapeutic effects of TCM on NMO-ON optic nerve and retina may be related to the inhibition of NF-?B protein phosphorylation in optic nerve and the regulation of the production of inflammatory mediators(IL-6,TNF-?and IL-10). |
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