| Research backgroundAccording to the TCM theory of Qi and blood,Qi arid blood are the basis for the human body to carry out life activities."Gas is the impetus urge of blood,and blood is the mother of gas." Unbalance of Qi and blood is the root of many disease changes.Qi deficiency and blood stasis syndrome are the most common type symptoms of Unbalance of Qi and blood.And Qi deficiency lead to blood stasis,which is commonly found in the three major diseases that threaten human health.It is an important pathological basis for the development of diseases and is also a hot topic in the theory of traditional Chinese medicine Qi and blood.When the Qi deficiency and blood stasis syndrome occurs,the body's blood system appears hypercoagulability.The microcirculation system is the most basic unit of the circulatory system and may change.Previous studies have paid less attention to the microcirculatory disturbance in the formation of Qi deficiency and blood stasis syndrome,by which caused microcirculatory disturbance is also belong to the category of "bloody" in Chinese medicine,and it is an aspect that cannot be ignored in the development of many diseases.The construction of animal models based on clinical pathology is a key and difficult point in the study of basic theories of traditional Chinese medicine.At present,some progress has been made in the research of animal models of Qi deficiency and blood stasis syndrome.Most of them adopt the ideas of"hunger","surprise",and "labor".On the basis of previous research,this laboratory finally adopted a sleep deprivation method to establish a model of Qi deficiency and blood stasis syndrome.Preliminary basic research showed that the rats of Qi deficiency and blood stasis syndrome established by sleep deprivation basically meet the main characteristics of TCM syndromes of Qi deficiency and blood stasis,including the changes:Increased blood viscosity,decreased immunity,abnormal platelet function,etc.but lack of microcirculatory state evaluation studies on animal models.Based on the previous research in the laboratory,this topic evaluates the microcirculation status of the model,which provides experimental evidence for the study of animal models of TCM syndromes and a reliable basis for the study of Qi and blood theory.ObjectiveTo observe the pathological changes of microcirculation state in rats with Qi deficiency and blood stasis syndrome model,and construct its evaluation system of microcirculation state,which aims at providing a reliable method for syndrome model and efficacy research of traditional Chinese medicine.Methods1)The experimental animals were divided into normal group and model group.Rats with Qi deficiency and blood stasis were reproduced by standing on a small platform on a water environment for six consecutive weeks of sleep deprivation.2)Observation of general conditions:At the fixed time every week,to observe signs including:hair,color,bearing,size,mental state,body weight and the fence,once a week.At the 4th and 6th weeks of modeling,blood pressure was measured using noninvasive intelligent blood pressure meter.3)Observation of Surface microcirculation:The information of normal and model rats,including:tongue and palm images,pulse patterns,plantar and auricles,were collected during the 2nd,4th and 6th weeks of model building.Images of the tongue and sole of rats were collected using a single-lens reflex camera.Color saturation analysis was performed using a standard colorimetric card;pulse amplitude was measured by a non-invasive pulse instrument.A rat ear and palm blood flow perfusion were analyzed by laser Doppler flowmetry.4)Observation of mesenteric microcirculation:After 6 weeks of modeling,rats in each group began to observe mesenteric microcirculation,fasted before the experiment,under anesthesia incision was made in the abdomen,supine position,and the mesentery was gently removed and placed in a constant temperature observation system.Basic observation was performed on 45 ?m and 200?m long venules without obvious bending.After the condition was stable,the microcirculation state of the same target blood vessel in the same visual field was recorded every 10 minutes for 30 minutes from the base Omin.High-speed camera recording was performed.Slow playback was performed.Red cell flow rate and tube diameter were measured using Image-Pro Plus 6.0 software.The number of leukocytes and adherent leukocytes was counted along the blood vessel counts.The leakage of FITC-labeled plasma albumin was observed by fluorescence microscopy.Image-Pro Plus 6.0 software was used to analyze the intensity of extravascular fluorescence in blood vessels.5)Mechanism analysis:the number of white blood cell counts,platelet counts,and the percentage of neutrophils and lymphocytes were measured at the 4th and 6th weeks of modeling,Fecl3-induced rat carotid and mesenteric arterial thrombosis time was dynamically observed by visual microscope.and the hemorrhage time was measured after 6 weeks of modeling.Results1)General state:In the normal group,the rats were in good condition,including the normal activity,the smooth hair luster,and the normal stools;the rats in the model group were significantly lower than those in the normal group from the 2nd to the 6th week of sleep deprivation(P<0.01),and The body weight growth rate is slower than normal,rats are lazy,whose coat color is slightly yellowed to yellow,the hair is coarse and smooth,the mood is irritability,there is no energy,the body is thin,and the stool is loose;SBP,MBP,DBP of the rats in the model group were lower than those in the normal group,but there was no statistical difference.2)Body surface microcirculation:The G value of tongue image in rats at the 2nd week of modeling was significantly higher than that of the normal group(P<0.05),and there was no significant difference between the R,G,and B values of the tongue surface at G,B and sole.There was no significant difference in pulse amplitude between rat and normal group.There was no significant difference in the blood flow volume between the model group and the normal group.At the 4th week of modeling,The value of tongue surface R(P<0.01)of rats was significantly lower than that of the normal group,The values of G and B at the tongue surface were significantly lower than those in the normal group,but there was no statistical difference;The pulse amplitude of rats was significantly lower than that of normal rats(P<0.05).The left ear(P<0.01)and right ear(P<0.01)blood flow perfusion were significantly lower in the model group than in the normal group,plantar blood flow perfusion measure also the trend of low performance;There was no statistically significant difference.At the 6th week,There was no statistically significant difference in the R,G and B values of tongue and palm image,The pulse amplitude of rats in the model group was significantly lower than that of the normal rats(P<0.01).The blood flow perfusion in the left ear(P<0.01)and right ear(P<0.01)in the model group was also significantly lower than that in the normal group.The left and right palm blood perfusion levels in the model group were lower than those in the normal group.However,there was no statistical difference.3)Mesenteric microcirculation:The mesenteric microcirculation changes within 30 minutes after modeling for 6 weeks as follows:Under the premise of no significant difference in vascular diameter between normal group and model group,the erythrocyte velocity of the venules in the model group was low from the basic state.In the normal group,there was no statistical difference.From the basic state of 0 min to the whole process,the number of leukocytes rolling in the fine veins of the model group was significantly more than that of the normal group(P<0.05-0.01).The adherent white blood cells were always present at the beginning of the basic state,but the state was not nearly the same,more than the normal group,but there was no statistical difference.The extravascular fluorescence intensity of the venules in the model rats was significantly higher than that of the normal group from the basic state(P<0.01).4)Mechanism analysis:At the 4th week,Tail-tip whole blood cell analysis show that The number of white blood cells in the model group was significantly higher than that in normal rats(P<0.05),and the percentage of neutrophils with a high percentage of white blood cells was significantly higher than that of normal rats(P<0.01),but as a percentage of other lymphocytes in leukocytes,the percentage of them was significantly lower than that of normal rats(P<0.01),and there was no significant difference in the number of platelets;At the 6th week,the tail tip bleeding time was significantly lower than that in the normal group(P<0.01),suggesting that there may be some changes in platelet status;the number of white blood cells in peripheral blood was higher than that in the normal group,but there was no statistical Difference;neutrophil Length fraction significantly higher than normal rats(P<0.01);lymphocyte percentage still lower than normal group(P<0.01),the platelet count did not also show significant differences.Mesenteric arterial thrombosis in the rat model group induced by Fecl3 was significantly faster than that in the normal group(P<0.05).However,in the time of carotid artery thrombosis,the rats in the model group were still larger than normal.Faster rats,but no statistical difference.ConclusionsThe rat model of Qi deficiency and blood stasis syndrome established by sleep deprivation on the water environment platform,which shows obvious microcirculatory disturbances that manifested as follows:reduced RGB values of tongue and palm images,decreased pulse amplitude,reduced surface auricle and foot flow perfusion,decreased red blood cell velocity in mesenteric venules,increased number of rolling and adherent leukocytes,and increased microvascular vascular permeability.Changes in the number of peripheral blood cells at the tip of the tail,shortening of bleeding time,and shortening of arterial thrombosis in vivo;and this model basically conforms to the clinic pathogenesis of qi deficiency and blood stasis syndrome in traditional Chinese medicine.The formation of Qi deficiency and blood stasis syndrome may be caused by leukocyte adhesion,endothelial damage,and changes in platelet function leading to microcirculatory disturbances,which can provide new therapeutic targets for model evaluation and Qi and blood-related diseases by improving microcirculation indicators.The microcirculation status evaluation system established by observing sleep deprivation rat with Qi deficiency and blood stasis syndrome,can be used for blood stasis syndrome model evaluation and Chinese medicine efficacy Research. |
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