| Objective:This study was designed with the guinea pig spinal cord homogenate (GPSCH) plus complete freund's adjuvant (CFA) as an antigen to induce EAE model of Wistar rats. In our study, we administrated atorvastatin with different doses to the rats of EAE, and observed morbidity, neurological score, histopathological changes, spinal cord tissue expression of NF-kB and the changes of GFAP, in order to provide a theoretical basis for clinical application.Methods:One hundred healthy female Wistar rats (six-eight-week) weighing 160-180g were divided into four groups randomly: normal control group (10 rats),EAE group (30 rats),low dose treatment group (30 rats),high dose treatment group (30 rats). EAE group, respectively, and treatment group were randomly divided into 14 days, 21 days and 28 days three subgroups, each subgroup of 10 rats. All experimental rats were immunized subcutaneously in the four footpads with emulsion 0.4 ml,which including fresh guinea pig spinal cord homogenate (GPSCH) as antigen,emulsified with an equal volume of complete freund's adjuvant (CFA) containing Mycobacterium tuberculosis 4mg/ml. Atorvastatin was dissolved in 0.9% salt solution and treated rats with oral administration at a daily dose 2mg/kg and 8mg/kg per rats in low dose and high dose treatment groups. Starting from the 1st day after immunization to the day rats were sacrificed, and EAE group and normal control group were given 0.9% salt solution. After immunization day, clinical signs of EAE was assessed a minimum of twice daily by two investigators. According to Kono standard method, scores were assigned on the basis of the following symptoms: 0, normal mouse; 1, tail weakness; 2, mild hindlimb weakness; 3, severe hindlimb paralysis; 4, hind and forelimb paralysis; 5, dead. Rats were sacrificed after anesthesia with intraperitoneal injection after heart perfusion. Spinal cords were removed promptly and immersed in 4% paraformaldehyde,then the tissue were embedded in paraffin and sectioned at 4μm thickness. Some of the sections were stained with HE to observed infiltration of inflammatory cells under light microscope in spinal cord. Some of sections were studied with immunohistochemistry as described bellow. The slides were incubated with anti-NF-kB antibody,then the tissues were further incubated with anti-GFAP antibody. At last, the results were analyzed with the optical microscopy. All the data were treated by SPSS17.0 software, expressed as means士S.D( X±S), and analyzed the incidence by chi square test and multiple comparison of measurement data by one-way anova. P < 0.05 was considered statistically significant.Results:1. Morbidity, latency and maximum clinical scoreThe morbidity was 26/30, 24/30, 24/30 respectively in EAE group,low dose treatment group and high dose treatment group. There was no significant difference among the three groups (P>0.05). EAE group, low dose treatment group, high dose group 28 days sub-group of the recurrence rates were 60%, 40%, 30%, the difference between any two groups was not significant (P>0.05).Comparing to EAE group, high dose treatment group have lengthen the latency (P<0.05).The latency of low dose treatment group has no difference comparing with EAE group. Maximum clinical score in high dose treatment group was significantly lower than that in EAE group (P<0.05).2 Histological changes in spinal cordThe pathological changes of spine cord of EAE rats manifested the inflammatory cells infiltration around vessels with HE staining, formed like"vessel sheath". The vessel sheaths were lower in normal control group than other group (P<0.05). With the prolonged course of disease, the vessel sheaths of the 21st day subgroup were lower than the 14th day subgroup of the same group (P<0.05). The vessel sheaths of the 21st day subgroup were lower than the 28th day subgroup of the same group (P<0.05). The levels of vessel sheaths of high dose treatment group were more significantly decreased than in the same subgroup of EAE group (P<0.05).3 Detection of NF-kB by immunohistochemistry in spinal cordNF-kB immune reaction is in the cytoplasm and the nucleus, brownish yellow as positive. Numbers of NF-kB positive cells were lower in normal control group than other group (P<0.05). With the prolonged course of disease,the level of NF-kB positive cells of the 21st day subgroup was more significantly decreased than the 14th day subgroup of the same group (P<0.05). The level of NF-kB positive cells of the 21st day subgroup was more significantly decreased than the 28th day subgroup of the same group (P<0.05). Comparing to EAE group, the same subgroup of high dose treatment group have lower level of the number of NF-kB positive cells (P<0.05). Comparing to EAE group, the same subgroup of low dose treatment group have lower lightly level of the number of NF-kB positive cells, but the difference was not significant (P>0.05).4 Detection of GFAP by immunohistochemistry in spinal cordGFAP immunoreactivity is in the cytoplasm of astrocytes, brownish yellow as positive.Numbers of GFAP positive cells in normal control group were lower than other group (P<0.05). With the prolonged course of disease,the levels of GFAP positive cells of the 21st day subgroup were more significantly increased than the 14th day subgroup of the same group (P<0.05). The level of GFAP positive cells of the 21st day subgroup were more significantly increased than the 28th day subgroup of the same group (P<0.05). Comparing to EAE group, the same subgroup of high dose treatment group has higher level of the number of GFAP positive cells (P<0.05). Comparing to EAE group, the same subgroup of low dose treatment group have higher lightly level of the number of GFAP positive cells, but the difference was not significant(P>0.05).Conclusions:1 This study successfully established model of acute and relapsing EAE.2 Atorvastatin have protect effect on EAE rats as Atorvastatin could prolong the latency, ameliorate clinical manifestation, ameliorate leukocyte infiltration and inflammation in spinal cords.3 Atorvastatin on EAE in rats may be related to lower spinal cord in the expression of NF-kB.4 Atorvastatin on EAE in rats may be related to higher spinal cord in the expression of GFAP.5 Effect of atorvastatin on EAE in rats dose-related. |
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