Protective effect of blood-activating and stasis-resolving medicines against cerebral ischemia-reperfusion injury in rats: A controlled trial to verify the time-effect and dose-effect relation / 中国组织工程研究

ABSTRACT

BACKGROUND: The injury of blood-brain barrier following cerebral ischemia reperfusion is a considerate pathological basis for injury caused by cerebral ischemia and reperfusion.OBJECTIVE: To investigate the effects of four most basic Chinese medicinal herbs, or safflower, peach seed, ligusticum and red peony with actions of activating blood and resolving the stasis on the contents of nitric oxide, immunoglobulins, C-reactive protein (CRP) and complements (immunological indices) of serum and cerebral homogenate, as well as the morphological and structural changes of cerebral tissue cells in rats with ischemia and reperfusion to vertify relationship between time effectiveness and quantitative effectiveness.DESIGN: A randomized controlled study, and evaluation by single blind.MATERIALS The experiment was completed in the Laboratory of Traumatology Institute of Guangzhou Red Cross Hospital from January 2001 to De cember 2002. Safflower, peach seed, szechwan lovge rhizome and red peony are concentrated granules of single decocting pieces, the blood activating and stasis resolving decoction was prepared at 2.5 g/L according to 1112 ratio.Totally 138 adult female SD rats were selected for the experiment,weighing 280-300 g, provided by Animal Center of Guangzhou University of TCM.INTERVENTIONS:The models rats with middle cerebral artery occlusion were set up by thread ligation(24 hours reperfusion after 2 hours middle cerebral artery occlusion).All 138 rats were randomly divided into 6 groups with 23 in each group.Sham operation groupThe vessels were ligated but the middle cerebral artery was not occluded.No.1 medicated group The BASR was by gavage given in a dose of 2 g/kg 30 minutes before operation. No. 2 medicated group The BASR was by garage given in a dose of 2.5 g/kg 30 minutes before operation. No. 3 medicated group The BASR was by garage given in a dose of 2 g/kg for consecutive 7 days be fore operation. No. 4 medicated group The BASR was by gavage given in a dose of 2.5 g/kg for consecutive 7 days before operation.Control groupthe same volume of saline was by gavage given for consecutive 7 days before operation. ① Scoring of dysneuria (5-score system 0-1 score meant mild dysneuria, 2-4 scores meant severe dysneuria) for all rats were performed after consciousness following 2 hours ischemia and 24 hours reperfusion.② After 24 hours reperfusion,10 rats in each group were at random selected for assay of levels of CRP, complement 3 (C3) and complement 4 (C4) (rate nephelometry), and concentration of nitric oxide (nitrate reductase method)in both cerebral homogenate and serum.③ After 24hours reperfusion, 10 rats in each group were at random selected, and after anesthesia was completed, the brain was quickly collected through decapitation, put into a 110 ℃ drying oven till its constant weight, then the water content in brain was calculated.④ The cerebral cytomorphology in every group was observed under light microscope. ⑤ After reperfusion, 3 rats in each group were randomly selected for preparation of coronal section of cerebral tissue,the cerebral ultrastructure in each group was observed under transmission electronic microscope.MAIN OUTCOME MEASURES: ① The results of dysneuria scoring in each group. ② The levels of CRP, C3 and C4, and concentration of nitric oxide in both cerebral homogenate and serum.③ The water content in brain. ④ The cerebral cytomorphology and the cerebral ultrastructure. RESULTS: All 138 rats entered into the result analysis. ① Comparison of the extents of dysneuria of rats in each group The ratio of severe dysneuria after 24 hours ischemic reperfusion in all medicated groups was obviously lower than that in control group(P ＜ 0.01),and the ratio in No.4 medicated group was lower than that in No.2 medicated group(P ＜ 0.05).② Comparison of water contents in brain of rats in each groupThe water contents in sham operation group and all medicated groups were obviously lower than that in control group (P ＜ 0.05). ③ Comparison of the nitric oxide concentration in cerebral homogenate of rats in each group The concentration in sham operation group and all medicated groups were obviously lower than that in control group (P ＜ 0.01). The concentration in No. 3medicated group was obviously lower than that in No. 1 medicated group (P ＜ 0.05).The concentration in No.4 medicated group was obviously lower than that in No. 2 medicated group (P ＜ 0.01). ④ Comparison of the nitric oxide concentration in serum of rats in each groupThe concentrations in sham operation group and all medicated groups were obviously lower than that in control group(P ＜ 0.01).The concentration in No.3 medicated group was higher than that in No. 1 medicated group (P ＜ 0.05). The concentration in No. 4 medicated group was higher than those in No. 2 and in No. 3 medicated groups (P ＜ 0.05). ⑤ Comparison of the levels of CRP in cerebral homogenate and serum of rats in each group The levels in sham operation group and all medicated groups were lower than that in control group(P ＜ 0.05-0.01).The level in No.3 medicated group was lower than that in No. 1 medicated group (P ＜ 0.01). The level in No. 4 medicated group was lower than those in No. 2 and in No. 3 medicated groups (P ＜0.05-0.01).⑥ Comparison of the levels of C3 in cerebral homogenate and serum of rats in each groupThe levels in sham operation group and all medicated groups were lower than that in control group (P ＜ 0.05-0.01). The level in No.3 medicated group was lower than that in No.1 medicated group (P ＜ 0.05). The level in No. 4 medicated group was lower than those in No. 2 and in No. 3 medicated groups (P ＜ 0.01). ⑦ Comparison of the levels of C 4 in cerebral homogenate and serum of rats in each groupThe levels in sham operation group and all medicated groups were lower than that in control group (P ＜ 0.05-0.01). The level in No. 3 medicated group was lower than that in No. 1 medicated group (P ＜ 0.05). The level in No.4 medicated group was lower than those in No. 2 and in No. 3 medicated groups (P ＜ 0.01). ⑧ Comparison of the condition of cerebral edema of rats in each groupIn control group there was obvious cerebral congestive edema,indicating an obvious infection;while in medicated groups the extent of cerebral edema was milder than that in control group.⑨ Changes of cerebral ultrastructure of rats in each groupThe ultrastructure in sham group was normal. In control group, there obvious edema of cells, capillaries and sheaths in the marginal zone of cortex necrosis,and reduction of organelles of neuron. As well. In No. 3 and No. 4 medicated groups, the limits of cell membranes were clear, the structure was integral, the chondriosomes were rich and even in size,the medullated fibers were morphologically normal.And in No.1 and No.2 medicated groups the changes were between the twoCONCLUSION:① The scoring of dysneuria in rats was decreased after the blood-activating and stasis-resolving medicine was given,and it was lower in rats that were given a longer period of medication,indicating that the improved extent for dysneuria is related to prolonged medication.②The nitric oxide concentration of cerebral tissue in rats that recevied the blood-activating and stasis-resolving medicine was decreased, and the nitric oxide concentration of serum in the rats was increased,indicating that the blood-activating and stasis-resolving medicine can reverse the anomalies of nitric concentration in different tissues after ischemic reperfusion so as to reduce cerebralinjury.③ The levels of C3 and C4 of cerebral tissue and serum in rats that received the blood-activating andstasis-resolving medicine were obviously decreased,indicating that the medicine may reduce the cerebral injury through triggering complement system;and the CRP was also get decreased,further suggesting that the medicine can inhibiting infective reaction.④ The longer the period of medication,the milder the cerebral injury, and the dose of 2.5 g/L was better in effect.