Overexpression Of MiR-338 Regulates Peripheral Nerve In Rats With Experimental Autoimmune Neuritis

The rat model of experimental autoimmune neuritis(A) Objective:To study deeply Guillain-Barre syndrome animal models, including comprehensive evaluation of rats symptoms, weight, neuromuscular function, peripheral nerve ultrastructure, light microscopy, immunofluorescence, electrophysiology and gastrocnemius morphology. On this basis, to further study the inflammatory factors Iba1 expression and localization in rat model sciatic nerve, to establish a more comprehensive evaluation on Guillain-Barre syndrome rat models, and to provide new ideas and research evaluation in using the model for the study of Guillain-Barre syndrome treatment methods and mechanisms.(B) Methods:20 Lewis female rats were randomly divided into EAN model group(n=10)and the control group(n=10). EAN model group rats were immunized in both hind footpads with 200ul inoculum containing 200 μg P0 peptide 180-199,2 mg H37Ra in 100 μl saline and 100 ul IFA.Control group rats (n= 10) received the same treatment with the same dose of saline solution. The day of model induction was recorded as day 0.Electrophysiology,light microscopy studies,sciatic nerve ultrastructure and immunofluorescence histopathology were measured at the neuromuscular severity peak on day 18(according to clinical score curve) post-induction. Cell-specific protein markers were used for immunofluorescence histopathology staining to characterize sciatic nerve cells:CD3 (T cell), Iba-1 (microglia), S100 (myelin), and neurofilament 200 (axon). All rats were weighed and scored daily until day 42 post-immunization (p.i.).(C) Results:1.On day 1 p.i., all rats in the EAN model group began to display different clinical symptoms. On day 7 p.i., the tail tonicity was lost in all EAN model rats, which suggests that the EAN model was successfully established. The symptoms peaked on day 17-18 p.i and restored on day 42.2.0n day 7,14,21,28,35,42,the weight of EAN rats (unit:g) were 160±2.07, 173.3±16.8,190.5±14.6,198.6±6.7,207.3±6.9,216.8±8.9 and significantly decreased than the control group(169.2±0.57,178.5±96,202± 10.6,210.5±13.7,216±5.6,223.9±4.6) (P<0.05);3.EAN sciatic nerve conduction velocity (19.69±4.47) m/s, were significantly lower than the control group (30.98±2.71) m/s (P<0.0001); EAN sciatic nerve compound action potential amplitude (peak-peak) (2.12±0.83) mV, significantly lower than thecontrol group (12.18±1.71) mV (P<0.001);4.Swelling of the myelin lamellae, vesicular disorganization, separation of the myelin lamellae, and an attenuation or disappearance of the axon were observed by transmission electron microscopy in the EAN group; in the EAN model group, gastrocnemius myofibers stained lighter than intact muscle fibers typically do and had an irregular shape and few nuclei. The muscle fibers were atrophied and distributed unevenly, the ferrous cyanide copper deposits were slightly lighter in color and were smaller than those in the control group.5.CD3 and Iba-1 increased significently in the structures characterized by separation or swelling of the myelin lamellae, and increased slightly in the structures characterized by vesicular of the myelin lamellae, S100 decreased in the structures characterized by vesicular disorganization or separation of the myelin lamellae. And neurofilament 200 decreased in the structures characterized by separation of the myelin lamellae. Moreover, we found that Ibal were positive in the myelin sheath, and overlapped with S100, which significantly indicated that Schwann cells may play as macrophage-like cells during the disease progression of ENA.(D) Implications for research:The evaluation of the EAN model provides the basis for further investigation on physiological processes.The findings on Iba-loverlapped with S100 on myelin is a useful supplement for the treatment of GBS, providing a newperspective. Meanwhile,it is a good basis for further research on microRNA-338. Overexpression of miR-338 regulates peripheral nerve in rats with experimental autoimmune neuritis(A) Objective:To investigate the role of lentivirus containing microRNA-338 (miR-338-LV) on EAN peripheral nerves.(B) Methods:1.Construction of lentivirus containing miR-338, namely miR-338-LV virus solution;2.Subjects were randomly divided into drug group (miR-338 group)(n=15), positive drug group (IVIg group)(n=15), model group(n=15) and normal control group(n=15). The former three groups were immunized with PO180-199 (Charpter 2),normal control group received the same treatment with the same dose of saline solution.3.Drug Intervention:IVIg group received injections of 100 mg/100 g body weight of IVIg once daily for 2 days.miR-338 group received multi-point local injection on sciatic nerve once,50ul per point(3 point left,3point right),when neurological symptoms (ie, tail drooping, scoring 1 point) appear post induction.4.1ndex Observation:Electrophysiology flight microscopy,sciatic nerve ultrastructure and immunofluorescence histopathology were measured at the neuromuscular severity peak post-induction. Cell-specific protein markers were used for immunofluorescence histopathology staining to characterize sciatic nerve cells:Iba-1 (microglia), S100 (myelin), and neurofilament 200 (axon). All rats were weighed and scored daily until day 42 post-immunization.(C) Results:1.The symptoms of miR-338 group peaked on day 21 p.i. The symptoms of IVIg group peaked on day 22 p.i. They increased evidently compared with the model group on day 18(P<0.05). Clinical scores of miR-338 group (3.5±0.4 points)and IVIg group(2.75±0.25 points)at the peak of disease decreased significantly compared with model group (3.92±0.2)(P<0.05). There is no significant difference between miR-338 group and IVIg group (P> 0.05).Clinical scores of miR-338 group (1.2 ±0.24 points)and IVIg group(1.0±0.12 points)at the recovery of disease decreased significantly compared with model group (2.0±0.32)(P <0.05).There is no significant difference between miR-338 group and IVIg group (P> 0.05).The recovery time of miR-338 group is 30 days, positive drug group 36 days.2.The nerve conduction velocity of miR-338 group,IVIg group and EAN model group at the peak and recovery period of the disease were lower than normal control group. The nerve conduction velocity of miR-338 group(28.41±0.7m /s)and IVIg group(25.4±0.98m/s) increase significantly at the peak of the disease compared with the EAN model group (19.69±4.47m/s) (P<0.01). There was a significant difference between miR-338 group and IVIg group(P<0.05). The recovery nerve conduction velocity was no significant difference between miR-338 group and IVIg group(P> 0.01).3.The CNAP amplitude of miR-338 group, IVIg group and model group at the peak and recovery period of the disease were lower than normal control group. Drug group (miR-338 group), The CNAP amplitude of miR-338 group(6.47± 0.12mV)and IVIg group(6.03±0.1mV) increase significantly at the peak of the disease compared with the EAN model group (2.12±0.83mV) (P<0.01).There was no significant difference between miR-338 group and IVIg group(P> 0.05). The recovery CNAP amplitude was no significant difference between miR-338 group and IVIg group.(P> 0.01).4.At the peak of the disease,In IVIg group, miR-338 group and the EAN model group,gastrocnemius myofibers stained lighter than intact muscle fibers and had an irregular shape and few nuclei. The muscle fibers were atrophied and distributed unevenly.Compared with the model group,there is a mild pathological changes in the drug group. At the recovery of the disease the pathological changes improved.5.Swelling of the myelin lamellae, vesicular disorganization, separation of the myelin lamellae, and an attenuation or disappearance of the axon were observed by transmission electron microscopy in the IVIg group, miR-338 group and EAN model group. At the peak of the disease, tissue repair can be found in the vesicular disorganization and separation of the myelin lamellae in the treatment group, less swelling of the myelin lamellae.The number of myelin in the miR-338 group was significantly increased, The number of myelin swelling decreased significantly in the IVIg group.Compared with EAN group, S100,NF200expression in the miR-338 and IVIg group was significantly increased; miR-338 also significantly increased compared with IVIg group. Compared with EAN group, Ibal of miR-338 and IVIg group expression significantly reduced.(Iv) Significance:Overexpression of miRNA-338 in local sciatic nerve can improve neuromuscular function of EAN rats, prolong the time of disease peak shorten the course, meanwhile increase CV and CNAP of sciatic nerve, reduce inflammation, promote myelin and axonal regeneration, Specially hasten the recovery, increase CV and promote myelin regeneration compared with positive drug group.