Protective Effect Of Defibrase On Experimental Allergic Encephalomyelitis In Wistar Rats

Objective:Observing the central nervous system damage such as the clinical behave, mortality rate, the fig immunohistochemiscal positive area, the expression of activated astrocytes, matrix metalloproteinase-9 (MMP-9),blood-brain barrier (BBB) function,tumor necrosis factor-a (TNF-a),plasma tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI) correlated index etc. changes after experimental allergic encephalomyelitis (EAE),to observe the protective effect of defibrase(DF) on EAE in wistar rats, and explore the possible immunologic mechanism of it. Method:1.Subgroup, model and administration:We induced experimental allergic encephalomyelitis in Wistar rats by subcutaneous administrating coarse myelin basic protein (CMBP) plus complete Freud's adjuvant (CFA),50 Wistar rats were included and were assigned randomly to five groups:namely normal control group, EAE control group, low dose DF treated group, median dose DF treated group and high dose DF treated group. Since seven days before the model building, high, median, and low dose DF treated group were required 16u/kg.d,8u/kg.d,4u/kg.d through peritoneal injection respectively, and the Wistar rats (normal control group and EAE control group) were treated with 0.3ml/d physiological salt water (NS) plus CFA, successive ten days treatment. EAE control group, each dose of DF treatment groups had the symptom for 3 consecutive days without aggravating or quadriplegia, or the rats being dead, would be considered as the peak of EAE disease. Record onset latency, advanced and peak disease score; the rats would be killed at the peak incidence, normal control group were killed 8 weeks later.2.The clinical behave, the delitescence, the progression and the maximal disease score, mortality of each group were compared.3. The pathological changes of central nervous system (CNS) were observed by light microscope.4. Use immunohistochemical staining to detect MMP-9 expression, anti-fibrinogen (Fig) for fibrin deposition, anti-glial fibrillary acidic protein (GFAP) for expression of activated astrocytes in CNS in five groups.5. The cerebrospinal fluid to serum albumin quotient (QA) were calculated in five groups, BBB function assessment by QA.6.To detect brain homogenate TNF-αlevel and plasma tPA and PAI with ELISA.7.The correlation between each index in EAE control group and each DF treated group were analyzed.8.Results were analyzed statistically by SPSS17.0.Result: 1.Incidence of Wistar rats:(1) Clinical symptoms:There was no incidence of EAE in normal control group. The Wistar rats of EAE control group and high, medium and low dose DF treated group was found to varying degrees of dysfunction, about 10-30days in the incubation period; incidence of Wistar rats including consumption reducing, weight losing, hind limb weakness, self-injury hind legs, urinary incontinence, serious paralysis of the limbs, convulsions or even death, et al. (2) The changes of the incubation period:the incubation period of low, medium and high dose DF treated group were longer than the EAE control group (P<0.01), the high, median dose DF treated group were longer than the low dose DF treated group in the incubation period (P<0.01,P<0.05),the high dose DF treated group was longer than the median(P<0.01). (3)The changes of the progression period: Compare with EAE control group, each DF treated group significantly reduced in the progression period (P＜0.01), and high dose DF treated group was shorter than the low (P<0.05). (4)The changes of the peak incidence of dysfunction score:Each dose DF treated group in the peak incidence of dysfunction score decreased significantly than EAE control group (P<0.01), high and median dose DF treated groups were lower than the low(P<0.01,P<0.05),the high dose DF group was lower than the median(P<0.05). (5) The mortality rate:The mortality rate of high,medium dose DF treated group reduced than EAE control group, the low dose DF group respectively(P<0.05), the low dose of DF treated group decreased than the EAE control group, but has not statistically significant (P>0.05),the mortality rate of high and medium dose DF treated group has no statistically difference(P>0.05).2. Pathological changes in the brain and spinal cord:The brain, cerebellum, brain stem, spinal cord and other sites of EAE control group and high, medium and low dose DF treated group in Wistar rats have emerged EAE typical pathological changes:the small blood vessels around the inflammatory cells Infiltration, a cuff-like change, blood vessels around the white matter of obvious changes, such as demyelination. The demyelination and inflammatory cell infiltration of each dose DF treated group reduced than the EAE control group, the medium and high dose DF treated group were lighter than the low-dose DF treated group, high dose DF treated group pathological changes was the lightest.3.Immunohistochemical staining:(1)Fibrin deposition:no fibrin deposition can be seen in brain tissue of normal control group rats. Conspicious fig positive substances was present in white matter areas around blood vessels in the brain of EAE control group, and fig positive substances was present around blood vessels and cytoplasm of endotheliocyte or gliacyte. The scope of deposition is much larger than the range of inflammatory cell infiltration. Fig positive substances was present in perivascular, not in cytoplasm of gliacyte in low dose DF treated group, Small amounts of Fig positive substances was present in perivascular in median dose DF treated group, and Fig positive substances of high dose DF treated group was lightest. (2)Activation of astrocytes:no GFAP was expressed in the brain of normal control group rats. GFAP was expressed in the brain of EAE control group rats, reached peak, diffuse distribution and reacted more strongly in perivascular, and inflammatory reaction was most. When compared with EAE control group, the average optical density of GFAP was statistically significantly decreased at each DF treated group(P＜0.01). the levels of the average optical density of GFAP in brain tissue of DF high, medium dose treated group decreased than low dose DF treated group (P<0.01,P<0.05), the levels of the average optical density of GFAP in brain tissue of high dose DF treated group significantly decreased than median dose DF treated group(P<0.01).(3) The expression of MMP-9:no MMP-9 positive cell was found in the brain of normal control group rats. The MMP-9 expressed in EAE control group rats brain tissue mainly on infiltrated mononuclear lymphoid cells in white matter, manifested that, brown particles appear in the cytoplasm, especially on the area of cuff blood vessels. Inflammatory cell clusters for infiltration expressed positive, some vascular endothelial cells and extracellular Matrix expressed MMP-9. The average MMP-9 positive cells of EAE control group and each DF treated group were higher than normal control group (P<0.01), The average MMP-9 positive cells in high and median DF treated group rats statistically significantly decreased when compared with EAE control group (P<0.01,P<0.05),and the low dose DF treated group reduced than EAE control group,but has not statistically significant(P＜0.05); the levels of the average MMP-9 positive cells in brain tissue of high dose DF treated group was significantly decreased than low dose DF treated group (P<0.05)but there was no statistically difference in high and medium dose DF treated groups(P>0.05).4. The QA value of EAE control group and each DF treated group significantly decreased when compared to normal control group (P 0.01.High, medium dose DF treated group QA value lower than EAE control group and low dose DF group respectively(P<0.01). the levels of QA value in brain tissue of high dose DF treated group significantly decreased than medium dose DF treated gruop(P<0.05).5. TNF-αlevels in brain tissue homogenate:TNF-a levels in brain tissue homogenate of EAE control group, and each DF treated group increased significantly compared to normal control group(P<0.01), TNF-αlevels of each DF treated group was lower than EAE control group (P＜0.01), high, medium dose DF treated groups were lower than low dose DF treated group (P<0.01). the levels of TNF-αlevels in brain tissue of high dose DF treated group significantly decreased than medium dose DF treated gruop (P<0.01).6. The level of plasma tPA and PAI:the level of plasma tPA and PAI in EAE control group and each DF treated group increased statistically significantly when compared to normal control group (P<0.01.There was no statistically difference between EAE control group and each DF treated group(P＞0.05) 7.Correlation Analysis:①the fig immunohistochemiscal positive area, anti-GFAP astrocytes average IOD,MMP-9 positive cells, the value of QA, the level of TNF-a in brain tissue, plasma tPA and PAI levels of EAE control group were significantly negatively correlated with the incubation period, so do low, median and high dose DF treated group(P＜0.05orP＜0.01);②the fig immunohistochemiscal positive area, anti-GFAP astrocytes average IOD,MMP-9 positive cells, the value of QA, the level of TNF-αin brain tissue, plasma tPA and PAI levels of EAE control group and each DF treated groups are significantly positively correlated with the progression(P<0.01);③the fig immunohis-tochemiscal positive area, anti-GFAP astrocytes average IOD,MMP-9 positive cells, the value of QA, the level of TNF-αin brain tissue, plasma tPA and PAI levels of EAE control group and each DF treated groups in brain at fastigium are significantly positively correlated with the neuro-function scores(P<0.01);④the fig immunohistochemiscal positive area, anti-GFAP astrocytes average IOD,MMP-9 positive cells, the value of QA, the level of TNF-a in brain tissue, plasma tPA and PAI levels of EAE control group and each DF treated groups are significantly positively correlated with the HE stain pathology scores(P＜0.05orP＜0.01). Conclusion: (1). In the EAE mould by WSCH and CFA with the method of multi-point single subcutaneous injection, neurological disfuntion of EAE Wistar rats were remarkable. Perivascular inflammatory cell infiltration were clearly showed in white matter.(2).Perivascular Fibrin deposition of CNS is company to the clinical symptom such as paralysis and correlated with the occurrence and development of inflammation in EAE rats (3) Fibrin deposition played a pathogenic role in EAE by increasing BBB permeability, activation of astrocytes, up-regulation of MMP-9 positive expression, increasing the level of TNF-a.(4). DF can extend the delitescence of EAE, reduce the progression, reduce EAE Wistar rats nerve dysfunction, reduce period of mortality at peak, reduce EAE Wistar rats pathological damage. DF has a protective effect for EAE, the protective effect of was positively correlated with the dose. (5). DF can decrease BBB permeability, decrease the activation of astrocytse, and decrease the level of TNF-a in order to protect from MS by decreasing fibrin deposition; DF may regulate MMP-9 levels, to restore the function of BBB, to decrease the fig deposition, in order to abatement the MS., (6). It was found that fibrin deposition and plasma tPA level consistent with clinical symptoms in EAE. DF cannot increase or decrease the level of plasma tPA and PAI,...