Study On Protective Efficacy Of Dehydroepiandrosterone In Experimental Autoimmune Neuritis

ObjectiveGuillain-Barre syndrome(GBS) is the main cause of crippling disease in humans and characterized as acute demyelinating inflammation in structures of the peripheral nervous system(PNS).Experimental autoimmune neuritis(EAN) is a T cell-mediated,acute,autoimmune demyelination disease of the PNS that serves as a model for human GBS. The close clinical,histopathological,and electrophysiological similarities between EAN and GBS make EAN a classic animal model for studying pathogenic mechanisms and novel treating strategies for GBS. For the treatment of GBS,both plasma exchange and intravenous immunoglobulin have shown the efficacy in shorten the progressive course.But there is a mortality of approximately 5%,and approximately 20%of patients are left with significant and persistent disability.Many of the conventional immunosuppressive treatments,such as corticosteroids, have no effect on GBS.It is needed to uncover new therapeutic modalities for GBS.Dehydroepiandrosterone(DHEA),a C19 adrenal steroid,is a precursor of both androgens and oestrogens.DHEA also serves as a precursor for many less well understood immune-regulating steroids. Studies showed that DHEA and its metabolites have anti-inflammatory, anti-proliferative,and certain immune-regulating properties both in vitro and in vivo.DHEA have shown beneficial effects on immunologic system in experimental animals and humans.EAN is a Thl-mediated autoimmune demyelination disease of the PNS.The infiltration of T cells and macrophages in the PNS and the multifocal,segmental demyelination are the main pathological changes of EAN,in which inflammatory cytokines, such as IFN-γ,TNF-α,IL-12,NO,play important roles.Thus far,there is no report about the role of DHEA in EAN.Based on the previous findings of DHEA on immunologic system,DHEA was used in treatment of EAN in the initial phase in this study.The therapeutical effect of DHEA on EAN and the immunologic mechanism were evaluated by examining the changes in clinical,histopathological and immunological parameters. Methods1.Antigens:Bovine peripheral nerve myelin(BPM) was prepared from intra- and extradural spinal nerve roots by ultracentrifugation.2.Animals:Thirty-six female Lewis rats,6-8 weeks old,weighing 150 - 180g were used in the present study.All of the rats were 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 5 mg of BPM and 2 mg of Mycobacterium tuberculosis and 50μl Freund's incomplete adjuvant.Clinical scores were assessed immediately before immunization and thereafter every day until day 28 post immunization (p.i.).Severity of paresis was graded as follows:0= no illness;1= flaccid tail;2= dragging both hind limbs;3= paralysis of both hind limbs;4= paralysis of four limbs or death;intermediate scores of 0.5 increment were given to rats with intermediate signs.4.DHEA treatment:Thirty-six Lewis rats were randomly divided into DHEA 0.5mg treatment group,2mg treatment group and control group(n = 12).Treatment groups were subcutaneously injected every day with DHEA and control group were subcutaneously injected with the same volum of DHEA dissolvent DMSO from day 5 p.i.to day 28 p.i.5.Histopathological assessment:Seven rats from each group were killed on day 16 p.i.,at a time when the clinical signs of EAN peaked. Sciatic nerve segments were excised close to the lumbar spinal cord, fixed in 10%phosphate-buffered formalin,and embedded in paraffin. Multiple longitudinal sections(5- 6μm slices) of sciatic nerves were stained with hematoxylin and eosin for evaluation of the extent of mononuclear cell(MNC) infiltration by light microscopy.Tissue areas were measured by image analysis and the numbers of inflammatory cells were counted at×40 magnification.The average results were expressed as cells per mm~2 tissue section.6.Immunohistochemistry:Paraffin tissue sections(5μm) were deparaffinized,hydrated and treated for blocking endogenous peroxidase activity,repairing antigen.The sections were incubated with polyclonal goat anti-rat IFN-γ,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 mm~2 tissue section.7.Lymphocyte proliferation assay:The draining lymph nodes and spleens were removed under aseptic conditions.Single cell suspensions of MNC from individual rats were prepared separately.The cells were suspended in RPMI1640 and cultured in triplicates in round-bottomed 96-well microtitre plates in the presence of 10μl of BPM or phytohemagglutinin or the same volume of PBS.After 48h of incubation, proliferation was measured by labeling cells with[~3H]-methylthymidine (1μCi/well) and cultured for an additional 18h.Cells were harvested onto glass fibre filters.[~3H]-methylthymidine incorporation was measured in liquidβ- scintillation counter.Data are given as mean counts per minute (cpm). 8.Measurement of cytokine:MNC were cultured at a cell density of 2×10~6 cells/ml in medium containing BPM(20μg/ml).Supernatants were collected after 48h of incubation at 37℃.Quantitative ELISA assays for cytokines of IFN-γ,TNF-αand IL-10 were performed using ELISA kits following the manufacturer's instructions.Determinations were performed in duplicate and results were expressed as pg/ml.9.Statistical analysis:The SPSS 13.0 computer program was used for all calculations and statistical evaluations.Results were expressed as means±standard deviation(SD).Differences between groups were evaluated by one-factor analysis of variance(ANOVA) or rank-sum test. The level of significance was set to p＜0.05.Results1.DHEA treatment delayed disease onset of EAN:Both DHEA-treated groups exhibited more delayed disease onset than control rats(P＜0.05, P＜0.05).Compared to 0.5 mg DHEA treatment group,2 mg group also displayed significant delayed disease onset(P＜0.05).2.DHEA treatment decreased disease peak clinical scores of EAN: The rats treated with DHEA at dose of 2 mg/day showed significantly lower mean peak clinical scores than the control rats(P＜0.05).0.5 mg DHEA treatment group showed lower mean peak clinical scores than the control group,but there was no statistically significant difference.2.DHEA treatment inhibits inflammatory cell infiltration of PNS:The sciatic nerves of EAN rats were examined histologically at the height of the clinical course of EAN.The results revealed that administration of DHEA at doses of both 2 mg/day and 0.5 mg/day all significantly reduced the infiltrating cells in sciatic nerves sections compared to control EAN rats(P＜0.05,P＜0.05).2 mg DHEA treatment group showed less numbers of infiltrating cells in sciatic nerves than 0.5 mg treatment group,but there was no significant difference statistically.3.Effects of DHEA on MNCs Proliferation:MNCs of draining lymph nodes and spleens from EAN rats were tested on day 16 p.i.for lymphocytes proliferation.When compared to the controls,the DHEA treated rats showed significantly suppressed BPM and PHA-induced MNC proliferation(P＜0.05,P＜0.05,P＜0.05,P＜0.05 ).In addition,there was also a lower level of proliferation in 2 mg DHEA treatment rats than control EAN rats when MNC were cultured in the absence of antigen(P＜0.05),but there was no statistically significant difference between 0.5 mg DHEA treatment group and the controls.4.DHEA treatment suppresses the cytokine expression in sciatic nerves:Immunohistochemical studies showed that the numbers of IFN-γand TNF-α.expressing cells were strongly decreased in the sciatic nerves of DHEA treated rats at dose of 2 mg/day and 0.5 mg/day compared to control EAN rats(P＜0.05,P＜0.05,P＜0.05,P＜0.05 ).No statistically confirmed difference regarding the numbers of cells expressing IFN-γ,and TNF-αin the PNS was shown when comparing 2 mg-DHEA to 0.5 mg -DHEA treatment groups.5.Levels of cytokine in supernatant:The levels of IFN-γ,TNF-αand IL-10 secretion from MNC supernatants were examined by ELISA.The results showed that levels of IFN-γ,and TNF-α.in the MNC supernatant were significantly decreased in EAN rats treated with DHEA at doses of 2 mg/day and 0.5 mg/day when examined on day 16 p.i.as compared with control EAN rats(P＜0.05,P＜0.05,P＜0.05,P＜0.05).There were also statistically significant differences in IFN-γ,and TNF-αsecretion in the MNC supernatants between 2 mg DHEA and 0.5 mg DHEA treated EAN rats(P＜0.05,P＜0.05).There were no differences in IL-10 production among the three groups of rats.Conclusion1.In this study,DHEA was administered to EAN rats at doses of 0.5 mg/rat/day and 2 mg/rat/day for the treatment of EAN in the initial phase. Rats treated with DHEA at the both doses displayed significant delay in onset,lower peak clinical score and decreased inflammatory cell infiltration in the PNS as compared to control rats.These data suggest that DHEA has therapeutic potential for alleviating EAN.2.Rats treated with DHEA at the both doses displayed significant decreases in numbers of IFN-γ,and TNF-αexpressing cells in the PNS, BPM-stimulated MNCs proliferation and IFN-γ,TNF-α-secretion in the draining lymph node and spleen cells.Our finding suggests that DHEA could inhibit cellular immunological response by suppressing the proliferation of autoreactive T cells and overexpression of proinflamation cytokines.3.In the present study,administration of DHEA to EAN rats decreased IFN-γ,TNF-αsecretion both systemically and within sites of inflammation,with no significant changes in IL-10 production.Our finding suggests that DHEA suppressed EAN mainly by downregulation of autoimmune Th1 response,decreasing the production of proinflammatory cytokines without changing levels of Th2 associated cytokines.4.Our data show that rats treated with DHEA at dose of 2mg every day exhibited delayed disease onset,lower mean peak clinical scores and decreased levels of IFN-γ,and TNF-α.from cultured MNC supernatants when compared to 0.Smg DHEA-treated EAN rats.The results suggest that the immune suppressive effect of DHEA is dose-dependent.SignificanceGuillain-Barre syndrome(GBS) is the most main cause of crippling disease in humans and characterized as acute demyelinating inflammation in structures of the peripheral nervous system.For the treatment of GBS, both plasma exchange and intravenous immunoglobulin have shown the efficacy in shorten the progressive course and hastening recovery from GBS.But there is a mortality of approximately 5%,and approximately 20%of patients are left with significant and persistent disability.Further research is needed to uncover new therapeutic modalities for GBS to block the cascade inflammation and decreas the mortality and sequelae of nervous system.Dehydroepiandrosterone(DHEA) is the most abundant circulating adrenal steroids in humans.It has been well-documented that DHEA and its metabolites have anti-inflammatory,anti-proliferative,and certain immune-regulating properties and have shown immunological effects both in vitro and in vivo in experimental animals and humans.Experimental autoimmune neuritis(EAN) serves as classic animal model for acute inflammatory demyelinative polyradiculoneuropathy(AIDP) - one of the most common subtype of human.In this study,the therapeutical effect of DHEA on the initial phase of EAN and the immunologic mechanism were evaluated by examining the changes in clinical,histopathological and immunological parameters.We aimed to provide evidence for searching a promising new and potentially powerful strategy for human autoimmune disorders.creativity1.In our study,the inoculum was prepared by emulsifying bovine peripheral nerve myelin(BPM) in Freund's complete adjuvant(CFA). EAN was induced by subcutaneous injection the inoculum into both hind footpads of susceptible animals - Lewis rat.EAN serves as a classical model for human AIDP,which is the most common subtype of GBS.So far,there has been no report on inducing EAN with BPM and CFA in Lewis rat in China.2.In this study,DHEA was used for the treatment of EAN in the initial phase.The therapeutic effect of DHEA was evaluated by assessing clinical scores and examining inflammatory cell infiltration in the PNS. The immunologic mechanism were evaluated by examining the changes in BPM-stimulated MNCs proliferation from draining lymph nodes and spleens,IFN-γ,TNF-β,IL-10 -secretion from MNC supernatants and IFN-γ,TNF-αexpressing cells in the PNS.Up to now,there has been no report about DHEA administration for the treatment of EAN at home and abroad.