Protect Roles Of Hydrochloric Fasudil In Experimental Allergic Neuritis

Objective:Guillain-Barre syndrome(GBS)is the main cause of crippling disease in humans,characterized with the demyelination of PeriPheral nerve and inflammatory cells infiltration in structures of the peripheral nervous system(PNS) [1].Experimental autoimmune neuritis(EAN) is publicly known as a classic animal model for studying pathogenic mechanisms of GBS[2].Though build the Experimental autoimmune neuritis(EAN) we try to study a new method for the treatment of GBS.Hydrochloric Fasudil is a kind of the Rho associated kinase inhibitor. Fundamental experiments and clinical research proved that many kind of the inflammatory disease symptom can be improved when apply Rho associated kinase inhibitor to inhibitor Rho associated kinase activity. To evaluat the treatment effect of Hydrochloric Fasudil on Experimental autoimmune neuritis(EAN) at early so as to provide new ways on GBS treatment method, we using a method of therapy that involves Hydrochloric Fasudil on Experimental autoimmune neuritis(EAN) and absvering the clinical feature,pathological feature,immune-markers of EAN1.Antigens:component of PNS myelin sheath protein P253-78aa2.Animals:Thirty female Lewis rats, 7~8 weeks old, weighting 100~120g were used in the presnt study. All of the rats wre maintained in specific-pathogen free condition.3.Induction and clinical evaluation of EAN: ketamine was used for the animal anesthesia(10mg/kg). EAN was induced by subcutaneous(s.c)injection into both hind footpads with 100μl of inoculum containing 100μgP253~78aa and 100μg Freund's complete adjuvant and 100 physiological saline. Clinical scores were aessed immediately before immunization and thereafter every day until day 28 post immunization(i.p).4.Hydrochloric Fasudil treatment:Thirty Leis rats were randomly divided into EAN group(n=10),EAN+ Hydrochloric Fasudil treatment group(n=10) and CFA group (n=10).Fasudil hydrochloride was intraperitoneally injected in the dosage of 25mg/kg/d in treated group from day 2 before-immunization,and followed by 14days after immunizaton.5.electrophysiologic study(EPS):detect moton evoked potential F wave,latent period and motor nerve conducton rate.6.HistoPathological assessment:six rats from each grou were killed on day 14~16 P.i.,at atime when the clinical signs of EAN peaked. Seiatic nerve segments were exeised elose to the lumbar spinal cord,fixed in 10% phosphate-buffered formalin,and embedded in paraffin.7.Electron microscopy examination: six rats from each grou were killed on day 14~16 P.i.,at atime when the clinical signs of EAN peaked. Seiatic nerve segments were exeised elose to the lumbar spinal cord,fixed in 4% glutaric dialdehyde, examinated by electron- microscopy.8.Immunohistoehemistry:Paraffin tissuesections(5μm) were deparaffinized, hydrated and treated for blocking endogenous peroxidase aetivity,repairing antigen.The sections were incubated with Polyelonal goat anti- ratIL-17,TNF-αantibody for 1h and then stained according to the avidin-biotin technique.The numbers of positive cells were counted at×40 magnification in the entire section area.The average results were expressed as cells per mm2 tissue section.9.Measurement of the serum cytokines: six rats from each grou were killed on day 14~16 P.i.,at a time when the clinical signs of EAN peaked.Use a serum separaor tube and allow sample to clot for 30 minutes before centrifugation for 10minutes at approximately 3000×g. Remove serum and assay immediately or aliquot and store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.The concentration of IL-17 and TNF-αin the samples is then determined by ELISA. Evaluate protection mechanism of Hydrochloric Fasudil in experimental allergic neuritis.Results:1.Hydrochloric Fasudil delay the outbreak of the EAN: The symptoms of the EAN rats showed on day 6士0.92 post immunization,the symptoms of the treatment group showed on day 9士0.96 post immunization,The clinical scores of the EAN rats is 6.4±1.2 in peak time, the clinical scores of the treatment group is 4.8±0.8,no abnormality is found in the control group.The level of significance was set to p<0.05. 2.Electrophysiologic study(EPS) show that the AMP of the EAN rats is 19.5±8.6 mv in the peak time, the treatment group is 38.5±9.2 mv in the peak time, The level of significance was set to p<0.05.The F wave latent and sciatic nerve conduction velocity is not statistical significance.3.Electron microscopy examination and patho;ogical change: Hydrochloric Fasudil can reduce the level of inflammatory cell in sciatic nerve and relieve th sciatic nerve dlapidation.4.Immunohistoehemistry results: Hydrochloric Fasudil can reduce the level of IL-17 and INF-αin sciatic nerve. 5.The level of serum cytokine: Measure the serum cytokines by ELISA, Hydrochloric Fasudil can reduce the IL-17 and INF-αlevel in serum.Conclusions:1.Hydrochloric Fasudil can cure EAN effectively, It also can reduce the level of inflammatory cell in sciatic nerve and disease rate relieve the sciatic nerve dlapidation.2.The mechanism that Hydrochloric Fasudil can cure EAN effectively maybe reduce the level of IL-17 and INF-α.