| Objective:By observing the experimental allergic encephalomyelitis (EAE) rat blood-brain barrier (BBB) function, matrix metalloproteinase-9 (MMP-9) and the expression of activated astrocytes, tumor necrosis factor-α(TNF-α) correlated index etc. changing, to investigate the effect of fibrin deposition in the pathogenesis of EAE rats and possible mechanisms. To Investigate the influence of Matrix metalloproteinase inhibitor ONO-4817 on Fibrin deposition and therapeutical effect on experimental allergic encephalomyelitis. Methods:1.72 female Wistar rats were included and were assigned randomly to three groups:namely, the normal control group, the EAE control group, the EAE treated group, N=24. Every group had four observations set:8d, 10d,13d,17d after Post inoculation(p.i). There were six animals at each observation set.2. Models and administration.Wistar rats in the EAE control group and the EAE treated group were subcutaneously injected with antigen of whole spinal cord homogenate(WSCH) and complete Freud’s adjuvant(CFA) on rear feet and neck by the dose of 0.2ml each, the normal control group with saline water and CFA. EAE control group and EAE’treated group were administrated with ONO-4817(30mg/kg.d), which dissolved in 2ml 0.5% carboxymethyl cellulose sodium (CMC).2ml CMC was given to normal control group.3. Neurobehavioral deficit outcome would be estimated at each observation time point.4. The Cerebrospinal fluid to serum albumin quotient(QA) were calculated at different time points, BBB function assessment by QA.5. Use hematoxylin-eosin(HE) staining to detect inflammatory cell infiltration in brain at each observation time point. 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 central nervous system(CNS) at different time points.6.To detect spinal cord homogenate tumor necrosis factor alpha(TNF-a) level and plasma tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI) with ELISA. 7.The correlation between each index in EAE control group and EAE treated group were analyzed.8. Results were analyzed statistically by SPSS17.0. Results:1. Neurobehavioral deficit outcome at various stages in normal control group, EAE control group, EAE treated group:There was no incidence of EAE in normal control group. On day 10p.i. the rats of EAE control group began to show clinical symptoms such as, the paralysis of tail in varying degrees(neuro-function scores of 0.66±0.25), after that, the clinical symptoms gradually worsened. On day 13p.i. the symptoms reached the peak to display four limbs paralysis in varying degrees (neuro-function scores of 2.41±0.58). The symptoms began to relieve after the peak and completely remission on day 17p.i. When compared with EAE control group, the clinical symptom was statistically significantly decreased on 10 to 15 days(P<0.01 or 0.05), particularly on day 13p.i(P<0.01). On day 16 and 17p.i, neuro-function scores show no significant difference(P> 0.05).2. Pathological changes in CNS:No inflammatory cell infiltration was found in brain tissue of normal control group. No inflammatory cell infiltration was found in brain tissue of EAE control group rats on day 8p.i. Under the light microscope the small blood vessels within the brain parenchyma congestive and inflammatory cell infiltration around the blood vessels can be seen on day 10. day 13 and dayl7p.i in brain tissue of EAE control group rats. On day 13p.i, inflammatory cell infiltrated the most serious, to form as muff typically. When compared with EAE control group, the inflammatory cell infiltration and focus to a lesser extent. The pathological score of HE staining was statistically significantly difference between EAE control group and EAE treated group (P<0.05).2.2 immunohistochemical staining:(1)fibrin deposition:no fibrin deposition can be seen in brain tissue of normal control group rats. On day 8p.i, small amounts of Fig positive substances was present in white matter areas around blood vessels in the brain of EAE control group. On day 10,day 13 and day 17 p.i, 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. Fibrin deposition reached peak on day 13p.i. Small amounts of Fig positive substances was present in perivascular. not in cytoplasm of gliacyte in EAE treated group. When compared with EAE control group, the expression of Fig positive substances was decreaded obviously, and immunohistochemical positive area was statistically significantly defference at Each time point(P<0.01). (2) 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 brian tissue mainly on infiltrated mononuclear lymphoid cells in white matte, 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 MMP-9 positive cells in EAE treated group rats was statistically significantly decreased when compared with EAE control group on day 8p.i, no statistically difference on day 10. day 13 and day 17p.i (P> 0.05). But the average number of positive cells from day 10 to day 17p.i was statistically significantly difference(P<0.01). (3)Activation of astrocytes:no GFAP was expressed in the brain of normal control group rats. On day 8p.i, GFAP was expressed in the brain of EAE control group rats, increaded on day 10p.i, reached peak and diffuse distribution on day 13p.i, reacted more strongly in perivascular. On day 17p.i, the expression of GFAP was weaken. When compared with EAE control group, the average optical density of GFAP was statistically significantly decreased at each time point(P<0.05).3. the level of plasma tPA and PAI:the level of plasma tPA and PAI in EAE control group and EAE treated group was increased a little when compared to normal control group on day 8p.i, no statistically difference(P>0.05). On day 10, day 13, dayl7p.i, it was increased statistically significantly when compared to normal control group (P< 0.05 or 0.01).There was no statistically difference between EAE control group and EAE treated group at each time point(P>0.05). (4) The changes of QA value:There were some common tendency in normal control group,EAE control group and EAE treated group which QA value increased firstly and decreased then. QA value in EAE control group began to increase on day 8p.i(2.43±0.7), but had no clinical symptoms. This indicate the injury of BBB was prior to appearance of clinical symptom in EAE. On day 13p.i, the QA value in EAE control group reached the peak(8.9±1.94) as the same as BBB injury. Then the QA value decreased and the function of BBB recovered gradually. The QA value in EAE treated group increased a little from day 8 to day 10p.i, then reached the peak on day 13p.i(5.86±2.23). It increased generally than EAE control group. This indicate the injury of BBB was slightly after treated with ONO-4817.The QA value of normal control group was also increased firstly and decreased then, but the rate of increased or decreased was significantly when compared to EAE control group. The difference of QA was statistically significant between normal control group and EAE control group, also did between EAE control group and EAE treated group(P<0.01).5. TNF-αlevels in spinal cord homogenate: TNF-αlevels in spinal cord homogenate of EAE control group was increased significantly compared to normal control group on each time point(P<0.01). TNF-αlevels in spinal cord homogenate of EAE treated group was increased statistically significant when compared to normal control group on day 8, day 10, day 13p.i(P<0.01), but had no statistical difference on day 17p.i(P>0.05). TNF-αlevels in spinal cord homogenate of EAE treated group was decreased statistically significant when compared to EAE control group on each time point(P<0.01).6. Correlative analysis:there was a positive correlation of fibrin deposition volume and the degree of inflammatory cell infiltration, the expression of activated astrocytes, the degree of BBB function injury, clinical neuro-function scores(P<0.01); a positive correlation of TNF-a levels in spinal cord homogenate and the degree of BBB function injury (P<0.01); a positive correlation of tPA plasma level and neuro-function scores in EAE control group and EAE treated group(P<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) Fibrin deposition of CNS was prior to the clinical symptom 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, increasing the level of TNF-a. (4). It was found that fibrin deposition and plasma tPA level consistented with clinical symptom in EAE. (5) ONO-4817 alleviated pathogenetic condition of EAE by inhibiting the expression of MMP-9, reviving BBB function and decreasing fibrin deposition in the CNS. |
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