Effects Of Salvia Miltiorrhiza Injection On The Indexes Of Hemorrheology And Blood Coagulation Function During Lipopolysaccharide-induced Periventricular Leukomalacia In The Early Neonatal Rat

Objective: Abnormal hemorrheology and coagulation defects are closely related withthe cause of blood stasis syndrome in modern medicine, and activating blood toremove stasis keeps close contact with the improvement of hemorrheology andcoagulation defects. This experiment intends to probe the etiology and thepathological mechanism of premature PVL in rats caused by intrauterineinfection/inflammation in traditional Chinese medicine and the relationship withblood stasis syndrome, and the affect, curative effect and the mechanism of Salviamiltiorrhiza injection--a water soluble preparation of Salvia miltiorrhiza which canpromote blood circulation to remove blood stasis.Method:30pregnant rats were randomly divided into two groups: LPS(n=22)andsaline(n=8). Pregnant rats were consecutively injected with saline or LPS(350μg/kg·d)on gestation17d and18d. The preterm pups were randomly divided into four groups:the control group(group A), the model group(group B), the high dosage Salviamiltiorrhiza injection group(group C), and the low dosage Salvia miltiorrhizainjection group(group D). After the delivery of the pregnant rats,6newborn rats wereselected randomly in the LPS group and the control group respectively to observe thewhite matter damage and the infection of the uterus and placenta and evaluate themodel. The7days rats were started to receive intraperitoneal injection. The rats(Aand B)were injected with sterile saline(9ml/kg·d), the rats in group C and D wereinjected with Salvia miltiorrhiza injection (9ml/kg·d and4.5ml/kg·d partly). Theintraperitoneal injection were last for2weeks,9rats (21days) were selectedrandomly in all groups respectively to get abdominal aorta blood for detecting thecoagulationfunction, which consist the experimental groups(A1, B1, C1, D1).The rest9rats in each group were injected until25days to get abdominal aorta blood for detecting thecoagulation function, which consist the experimental groups(A2, B2, C2, D2). Thegeneral condition of the newborn rats in different groups include skin color andeye-opening time were observed from the beginning of this experiment. Theneurological behavior were processed on14d and21d partly. The items ofneuro-behavioral observation included suspension test、cylinder test and the slopetest. And2rats (21days) were selected randomly after the blood taking to get thebrain tissues(HE coloration) to observe the pathologic changes of the white matter.Result:（1）Compared with the control group, the eat and activity of the22pregnantrats in LPS group were significantly reduced, and the hair is not neat, the spiritobviously depressed. One of the22rats was accidental pregnancy, and3were deadfor abortion,3term delivery rats were excluded, one dead for dystocia. The uterusand placenta of the2preterm delivery rats were made pathological section, and therest12rats delivered less than22days. There were87live preterm pups in all, and34pups died. The8pregnant rats in the control group delivered at22-23days, the103normal new born pups were get in total without stillbirth. The uterus and placenta of2normal delivery rats were made pathological section. Compared with the normalgroup, the pregnant rats in LPS group were easier to premature delivery, and the livepups were less in number (P=0.056>0.05), lower in born weight （P=0.000＜0.05）, slower in stress response, darker in purple skin color, less in activities, andeven the bruises, petechiae and more texture were seen; however,the normal new bornpups were responsive, reddish skin color, normal texture and weight.(2) The pathology slices of the uterus, placenta and the white matter in LPS groupwith HE staining showed that,1) there were plenty neutrophilic granulocyte,endometrial thickening, necrosis and edema of placenta in LPS group;2) there waswhite matter damage in preterm rat of LPS group. Loose structure and delicatecolourare the major pathological changes of the brain tissue. All above proved that thesuccess of the experimental animal building.(3) The general situation:1) Growth anddevelopment: the new born rats of group BCD were naked glabrous, dark purple,more texture, and most of them were slower in stress response, less in activities,slower in body quality growth and even the bruises, petechiae could be seen; The newborn rats in group B were slower in reaction and crawl at14days, and their eyes werenor opened;3in group C could open the eyes, but inflexible in crawl; rats in group D crawled inflexible and could not open the eyes; At21days, rats in group A could liveindependently with delactation, rats in group B were small in body shape, poor inindependent life without delactation; rats in group C could live independently afterdelactation, and rats in group D were slow in body quality growth, should be delayedthe delactation time for1-2days, part of them could live independently.2)eye-opening time: After the injection of Salvia miltiorrhiza injection, theeye-opening time of the high dosage group and low dosage group were obviouslyimproved（P<0.05) and improved（P<0.05）. The eye-opening time of the model groupwere obviously delayed than the control group and the high dosage group（P<0.05）.(4) Neuro-behavioral test:1) Suspension test: compared with the control group, thelast time of14-day and21-day aged pups in the model group and low dosage groupwere short （P <0.05）; but the score of the high dose group were obviously improvedthan the model group (P <0.05), and the score of the low dose group were higher thanthe model group (P <0.05).2) Open field test: the activity of14-day and21-day agedpups in the model group, high-dose group and low-dose group were less than thecontrol group in30s（P <0.05）; but the score of the high-dose group and low-dosegroup were obviously higher than the model group. Compared with the high-dosegroup rats, the times of crossing the squares were less in model group and slack inaction.3)Slope test: compared with the control group, the time for turn the heads onthe45°slope of14-day and21-day aged pups in the model group, high-dose groupand low-dose group were obviously delayed （P <0.05）;At14days,the time for turn the heads of the rats in the low-dose group were shorterthan the model group （P <0.05）; the time for turn the heads of the rats in thehigh-dose group were obviously shorted; as the injection of Salvia miltiorrhiza, thetime for turn the heads of the rats in the low-dose group and the high-dose group wereshorter than the same age rats of21-day age（P <0.05）; and there was dosedependence relationship between the high-dose group and low-dose group(P <0.05).(5) Laboratory index:1) PT、FIB、TT of21-day aged rats: compared with the controlgroup, prothrombin time of the model group were obviously shorted（P<0.05）, thecontent of fibrinogen were obviously improved（P<0.05）; there was no differencebetween on the thrombin time; and there was no difference between the low-dosegroup, the high-dose group and the control group. Compared with the model group,the content of fibrinogen were obviously decreased with early application of Salviamiltiorrhiza injection; and the prothrombin time were obviously improved in the high- dose group（P<0.05）,and the low-dose group（P<0.05）; but there was no dosedependence relationship between the high-dose group and low-dose group(P>0.05).2)Hemorrheology indicators of25-day aged rats: compared with the control group,the blood viscosity and the erythrocyte aggregation index were obviously improved atthe low,middle and high shear rate in the model group(P<0.05); compared with themodel group, the blood viscosity and the erythrocyte aggregation index wereobviously decreased by Salvia miltiorrhiza injection（P<0.05）and the blood viscositywere improved at high shear rate（P<0.05）. After the early application of Salviamiltiorrhiza injection, the blood viscosity were obviously improved at low shear ratein the high-dose group and low-dose group with dose dependence relationship（P<0.05）,but there was difference between the low-dose group and the normalgroup（P<0.05）, which indicated that the better curative effect of the high-dose group.(6)Brain tissue pathological slices with hematoxylin-eosin staining of the21-day agedrats: compared with the control group, there were more obvious pathological changesof white matter--loosen structure, glial cells gathered to fibrosis,and theperiventricular leukomalacia could be seen; compared with the model group, thewhite matter were obviously improved in the high-dose group--there was no loosenstructure, gathered glial cells and fibrosis; there were loosen structure in the whitematter of the low-dose group, the periventricular leukomalacia and the damage ofhippocampus could be seen, which indicated that the early application of Salviamiltiorrhiza injection could improve the periventricular leucomalacia of prematurerats caused by intrauterine infection/inflammation, and the better curative effect ofthe high-dose group.Conclusion:(1)The experiment proved that the blood stasis is the importantpathological factor in the pathomechanism of periventricular leucomalacia ofpremature rats caused by intrauterine infection/inflammation for the first time, and“the blood stasis block in head and the obstruction of meridian and collateral” is themain pathomechanism.(2)Salvia miltiorrhiza injection--a water soluble preparationof Salvia miltiorrhiza (a kind of herb for activating blood and dissolving stasis) canimprove the abnormal of hemorrheology and coagulation function of the prematurerats caused by intrauterine infection/inflammation.(3) The experiment firstly provedthat Salvia miltiorrhiza injection can improve the white matter damage of thepremature rats caused by intrauterine infection/inflammation on the aspects ofhemorrheology and coagulation function. The mechanism may be related with the reducing of red blood cell aggregation, improving the erythrocyte deformability,decreasing the content of fibrinogen and affecting the exogenous blood coagulationfactors.