| Objective: In order to look for available therapeutics for multiple sclerosis (MS) and to investigate the mechanism of occurrence and development of experimental autoimmune encephalomyelitis (EAE) , we design to treat EAE by glucosamine (GS) with different dosage, observing the kinetic changes and correlation of blood-brain barrier (BBB) function and matrix metalloproteinase-2(MMP-2), MMP-9 expression in Wistar rats with EAE.Methods: Wistar rats which were induced by injecting subcutaneously an emulsion of guinea pig spinal cord homogenate (GPSCH) and complete Freund's adjuvant (CFA) were treated by GS with different dosage such as 90mg/kg/day and 180mg/kg/day. Clinical symptom, morbidity, mean onset date and change of body weight were evaluated in EAE, GS1, GS2 and control groups. Albumin in serum and cerebrospinal fluid (CSF) samples of four groups were examined on the days 6,8,10,12,14,16 and 18 p.i.. The integrity of BBB was assessed by calculating CSF to serum albumin quotient (QA). At the same time the brains and spinal cords were removed for paraffin sections. These sections were used for HE staining and MMP-2, MMP-9 immunohistochemical staining.Results: 1. EAE group had an attack beginning on day 12 p.i., but GS group began to show apparently clinical symptom on day 12 to 14 p.i.. Mean onset date of EAE, GS1 and GS2 group were 12.24±0.66day, 12.40±0.84day, 13.60±1.64day, which of GS2 group was significantly later as compared with that of EAE group. None of control group showed clinical signs. Moribidity of EAE group, GS1 group,GS2 group were 62.5%,33.3%,9.0% on day 12 p.i.(n=24)and 77.8%,44.4%,11.1% on day 14 p.i.(n=18). The differences of morbidity among three groups were statistically significant(P<0.05). Mean score of symptom in EAE, GS1 and GS2 group were 1.17±1.09,0.54±0.88,0.17±0.57 on day 12 p.i. and 2.33±1.57, 1.00±1.24, 0.33±0.97 on day14 p.i.. On day 14 loss of body weight of three groups were 24.50±8.42g, 14.00±7.85g, 10.50±5.44g compared to that on day 8 p.i.. There was no significant difference of clinical score between GS1 group and GS2 group on day 12 and 14 p.i.. Mean score of creat-time were 2.13±1.70,1.08±1.41,0.50±1.02,which were significant difference between EAE group and GS2 group(P<0.05).2. QA value increased firstly and decreased then. QA value in EAE group began to increase on day 6 p.i. and reached the peak (0.32±0.10) on day 10 p.i.. Then it decreased gradually within eight days. The increase of QA value was prior to development of clinical symptoms. QA value in GS group had a little in the initial stage and increased generally with the day passed. The peak appeared on day 14 p.i. which was later and lower than that in EAE group. QA value of control group increased in the initial stage of immunization but was lower than that in EAE group.The difference of QA was statistically significant between EAE and GS group from day 6 to 14 p.i. and was statistically significant between GS1 and GS2 group from day 8 to 14 p.i. (P<0.01).3. MMP-2,-9 that were expressed intensively in vascular endothelial cells, meninges and accumulative inflammatory cells were in coincident with the pathological change and progression of disease. Few cells of MMP-2,-9 expressed on day 6 p.i. and significant increase on days 8 to 10 p.i. in EAE group were observed. Image analysis suggests that average photodensity of MMP-2,-9 in EAE group reached maximum value on day 10 p.i. which had the resemble thendency of the change with QA value. Expression of MMP-9 were more obvious than that of MMP-2. On days 8 to 12 p.i., MMP-2,-9 increased in both GS1 and GS2 group. They reached the peak on day 12 p.i.. Compared with that of EAE group, the expression of GS group had lower level and smaller circumscription. The difference of MMP-9 was statistically significant between EAE and GS group on days 6 to 16 p.i..There was significant difference of MMP-2 between EAE and GS group on days 8 to 14 p.i.( P<0.01). The expression of MMP-2,-9 in EAE group were also obviously higher than those in contral group (P<0.01). There were significant difference of MMP-2,-9 between GS1 and GS2 group on days 8 to 12 p.i. (P<0.01).Conclusion:1. In early EAE stage the injury of BBB was prior to appearence of clinical symptom and the latter gradually aggravated after the peak injury of BBB.2. In the early period of EAE, the expression of MMP-2 and MMP-9 up-regulated and reached the peak on day 10 p.i., which were in coincidence with the increase of BBB permeability and played the main roles in the BBB damage.3. GS could significant decrease incidence rate, lengthen the latency and relieve the clinical severity of EAE, showing that GS may have protective effect on EAE.4. It is presumed that the effect is related to the dosage of GS and GS with high dosage may be more effective. |
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