Cerebral Ischemia Superimposed Cold Induced Blood Stasis Characterization Model And Its Biological Basis

BackgroundBlood stasis syndrome is a patho-physiology state of human body resulting from stagnant blood,which can be evaluated by four physical examinations.Animal models with blood stasis are very important for scientific research on Chinese medicine.Researchers evaluated animal model with blood stasis by the change in biochemistry and bio-physics,and they usually ignored the biological exosyndrome such as dark purple tongu,claws and region swelling and pain.that these symptoms of exosyndrome usually appears in patients with blood stasis.At the same time,because lack of rational animal model with blood stasis and its evaluation criterion,research on Chinese medicine make less progress in the past fifty years and restrict further research and application for Chinese medicine.Therefore,it is a critical scientific problem to establish the exosyndrome model of blood stasis syndrome and should be solved urgently.ObjectiveTo establish animal model and its evaluation method for exosyndrome model of blood stasis in order to explore the biological foundation and the characteristic of the blood stasis syndrome.Methods1.The rat model with syndrome of cold coagulation and blood stasis was induced by repeatedly ice water irritation and its superficial signs were taken with digital camera.2.The rat models with exosyndrome of blood stasis were induced by 3 kinds of cerebral ischemia including the middle cerebral artery occlusion(MCAO),the bilateral common carotid artery ligation(BCCA)and the unilateral common carotid artery ligation(UCCA).3.The rat models with exosyndrome of blood stasis was induced by MCAO plus ice water irritation,and we compared with the exosyndrome of blood stasis induced by different factors.4.The change of exosyndrome of blood stasis was obtained by digital camera and then analyzed the tongue color index value by image analysis system.The plasma contents of TXB₂,6-Keto-PGF_1awere measured by radio immunoassay,and the serum contents of tPA,PAl,sVCAM-1,GMP-140 were measured by ELISA(enzyme linked immunosorbent assay).We measured the positive expression areas and optical density of IL-1,ICAM-1,Integrinβ1 in CA1 region of hippocampus of rats by immunohistochemical method.The microvascular pathological change in tongue and hippocampus of rats were examined by electron microscope.At the same time,we analyze the association between exosyndrome of blood stasis and biological index..Results1.The model rats with syndrome of cold coagulation and blood stasis showed a dark purple tongue,and thicker and longer sublingual vein.While the normal rats showed a ruddy tongue and clear sublingual vein.There was a significant difference between them(P＜0.05).There were a significant difference in tongue color index value(TCIV) of rats after cerebral ischemia 3,5 days between MCAO-group,BCCA-group and normal-group(P＜0.05);and the mean scores of sublingual vein length(1.44±0.08)was higher than that of normal-group(0.56±0.53)(P＜0.05).2.There was a significant difference in tongue color index value(TCIV)of rats at 3th days after cerebral ischemia between MCAO-group,ice water-group,cerebral ischemia plus cold-group and normal-group(P＜0.05);Tongue color index value(TCIV) of cerebral ischemia plus cold-group in rats after cerebral ischemia 3 days was higher (60.12±5.154)at all times.Among all groups,hemorrheology results showed that there were significant difference for indexes of rats between MCAO-group,ice water-group,cerebral ischemia plus cold-group and normal group(P＜0.05).The whole blood viscosity,reduced viscosity,plasma viscosity,hematocrit,index of erythrocyte aggregation and deformity index of RBC of rats were higher than that of the normal group.There were significant difference for indexes of rats between the three model groups and normal group(P＜0.01 or P＜0.05).The blood viscosity,hematocrit,plasma viscosity and index of erythrocyte aggregation were higher and the deformity index of RBC was lower than that of cerebral ischemia plus cold-group among all groups at 3th days after cerebral ischemia.Results showed that there were significant difference for plasma thromboxane B₂(TXB₂)between MCAO-group,ice water-group,cerebral ischemia plus cold-group and normal group(P＜0.05).The content of TXB₂ of rats in all model groups were higher than that of the normal group.There were significant difference for indexes of rats between the three model groups and normal group(P＜0.01 or P＜0.05), significantly.The plasma contents of TXB₂ were the highest(184.7+97.12)in cerebral ischemia plus cold,group among all groups at 3th days after cerebral ischemia.The plasma contents of 6-Keto-PGF1a was the lowest(147.7+16.32)at 3th days after ice water among all groups.The plasma contents of TXB₂/6-k-PGF1a was the highest(1.22±0.648)in cerebral ischemia plus cold-group among all groups at 3th days after cerebral ischemia.There were significant difference for indexes of rats between the three model groups and normal group(P＜0.01 or P＜0.05).3.Biology exosyndrome of cerebral ischemia plus ice water irritation,such as tongue color index value,length scores of sublingual vein,activity time and activity speed of rats,shoed significant association with hemorrheology,plasma TXB₂, 6-Keto-PGF_1a,serum tPA,PAI,sVCAM-1,GMP-140,positive protein expression areas and optical density of IL-1,ICAM-1,Integrinβ1 in CA1 region of hippocampus in rats.The change of microvascular pathology in tongue and hippocampus were obvious in rats of cerebral ischemia plus ice water irritation.ConclusionsAnimal model of MCAO plus cold stimulation was one rational exosyndrome model of blood stasis.The contents of TXB2,6-Keto-PGF_1a,tPA,PAI,sVCAM-1, GMP-140 in plasma or serum,positive expression areas and optical density of IL-1, ICAM-1,Integrinβ1 in CA1 region of hippocampus in rats,as well as change of microvascular pathology in tongue and hippocampus,showed significantly relationship with exosyndrome of blood stasis.These results provided some scientific evidence for further study on syndrome of blood stasis.