Interventional effect of hirudin on the expression of microtubule-associated protein 2 in peripheral tissue of hematom of model rats with acute intracerebral hemorrhage

摘要

BACKGROUND: It is suspected that dissociation, destruction or synthetic disorder of microtubule-associated protein 2 (MAP-2) may participate in secondary injury of intracerebral hemorrhage (ICH), and the reason may be related to thrombin in high concentration after ICH; therefore, the mechanism should be studied further. OBJECTIVE: To explore the effect of hirudin on expression of MAP-2 in peripheral tissue of hematom after ICH and changes of water content in brain tissue and analyze pathogenesis of thrombin in secondary injury after ICH. DESIGN: Completely randomized grouping design and controlled animal study. SETTING: Department of Neurology, the First Affiliated Hospital of Jilin University. MATERIALS: The experiment was carried out in the Neurological Laboratory of the First Affiliated Hospital of Jilin University from April 2003 to April 2004. A number of 80 healthy Wistar rats, of both genders, aged 3-4 months, weighing 250-350 g, were randomly divided into 8 groups: normal control group, 6-hour ICH group, 1-day ICH group, 2-day ICH group, 3-day ICH group, 7-day ICH group, 3-day hirudin group and 7-day hirudin group with 10 in each group. Five rats from each group were selected to measure their water content, and the others were undertaken immunohistochemical stain. Hirudin was produced by Sigma Company, USA, and MAP-2 rabbit-rat polyclonal antibody was provided by Fuzhou Maixin Biotechnology Company Limited. METHODS: ① Model establishing and grouping intervention: Rats in simple ICH group were collected their blood from tails and then inserted with 50 μL non-anticoagulant auto-arterial blood into the cauda of the putamen in right brain within 5 minutes. Rats in hirudin groups were inserted with 10 U hirudin (which was diluted with saline to 20 μL) into local hematom regions within 5 minutes, and the needle was pulled out after 10 minutes. Rats in normal control group were untouched. ② Water content in peripheral tissue of hematom: Based on the ratio between dry weight and wet weight, brain tissue at bleeding side and in right frontal lobe was selected to measure dry and wet weights so as to calculate the water content [(wet weight - dry weight)/wet weight] × 100%. ③ Positive expression of MAP-2: Based on immunohistochemical stain, positive MAP-2 cells were regarded as neurons and they were buffy morphological. Positive rate of MAP-2 was calculated, i.e., percentage of positive cells in each sight to total cells in all sights. ④ Statistical analysis: Data among groups were compared with one-way analysis of variance, averages were compared with SNK-q test by each other, and relation between water content and MAP-2 was analyzed with linear regression technique. MAIN OUTCOME MEASURES: Changes of water content and MAP-2 expression in peripheral tissue of hematom at various time points after ICH and intervention of hirudin. RESULTS: All 80 rats were involved in the final analysis. ① Water content: Water content was increased at day 1, reached peak at day 3 and decreased at day 7. It was (72.31±0.32)%, (77.42±0.53)%, (78.44±0.28)%, (74.10±0.13)%, (74.85±0.51)% and (70.07±0.36)%, respectively in 1-day, 2-day, 3-day and 7-day ICH groups and 3-day and 7-day hirudin groups, which was higher than that in normal control group (63.85±0.41, q=-4.684 3 to -7.262 0, P < 0.05); that in 2-day and 3-day ICH groups was higher than that in 7-day ICH group (q=-3.053 4, -3.727 0, P < 0.05); and that in 3-day and 7-day ICH groups was higher than that in hirudin groups at the same time points (q=-2.965 6, -2.726 4, P < 0.05). ② Positive expression of MAP-2: Positive expression of MAP-2 was decreased at 6 hours after ICH, reached the lowest value at day 3 and increased at day 7. Positive rate was (78.60±0.42)%, (60.56±0.74)%, (44.60±0.26)%, (25.45±0.85)%, (32.55±0.64)%, (37.69±0.76)%, (41.75±0.68)%, respectively in 6-hour, 1-day, 2-day, 3-day and 7-day ICH groups and 3-day and 7-day hirudin groups, which was lower than that in normal control group [(96.50±0.33)%, q= -3.074 5 to -8.128 5, P < 0.05]. In addition, positive cells of MAP-2 disappeared plentifully at 3-7 days after ICH, stain of positive cells were light, and only stain of plasma was positive. That in 3-day and 7-day hirudin groups was higher than that in ICH groups at the same time points (q= -3.391 8, -2.967 9, P < 0.05). Moreover, positive cells of MAP-2 was formed slightly but deeply stained. ③ Results of linear regression: Water content was negatively related to MAP-2 changes at 7 days after ICH (r= -0.894 9, P < 0.01), i.e., water content was increased with decrease of MAP-2 expression. CONCLUSION: The deterioration of MAP-2 may be involved in the pathogenesis of thrombin within the first week after ICH, and the local administration of hirudin can protect neurons.