Clinical And Experimental Study On Mechanism Of Traditional Chinese And Western Medicine Prevention And Treatment With Cerebral Ischemia In The Severe Traumatic Brain Injury

1 Objective of researchAcute brain injury has become a serious hazard to human health.Secondary brain injury with traumatic brain injury is the important reason leading to patients' death or disability.Occurrence mechanism of secondary brain injury is very complex,which ischemic trauma is a difficult and hot point in the current study.In the prevention and treatment of cerebral ischemia after traumatic brain injury,many scholars have made great efforts.Now has a set of programs in modern medical,including control of blood pressure,keeping breathing smooth, timely surgery,hyperbaric oxygen therapy,mild hypothermia,the use of drugs to promote brain cell metabolism,etc.To some extent these measures can improve the state of traumatic cerebral ischemia and prognosis,but have their own inadequacies.There are still many aspects of the dispute,subject to further verification.Literature research and clinical practice suggested Traditional Chinese and western medicine in the prevention and treatment of severe head injury play a significant efficacy,and application have been gradually extended. But the studies of specific mechanism for prevention and treatment of traumatic ischemic have reported much less,hindering the development of clinical use.In this area,our department has many years of clinical experience from conventional therapy in Traditional Chinese and Western Medicine.Based on conventionality therapy in Western medicine,a combination therapy of Huoxuehuayu,Huotankaiqiao,Tongfuxiere Chinese medicine has been used for treatment severe brain injury.It can remove static blood,resolve excrescently phlegm,promote diarrhea heat,speed awake,improve the status of ischemia and hypoxia.It can solve exanimation after brain injury,high fever,difficulty in breathing,physical dysfunction,stool closed end.It also can resolve symptoms such as viscous phlegm excrescently.So the better prognosis can be improved.Therefore,in theory,based on conventionality therapy in Western medicine,the combination therapy of Huoxuehuayu, HuotanKaiqiao,Tongfuxiere Chinese medicine for prevention and treatment of traumatic brain ischemia after severe head injury should be have some exact mechanisms and clear clinical efficacy,worthy of our study.From two aspects of the clinical research and experimental study,we use Traditional Chinese and Western Medicine for prevention andtreatment cerebral ischemia in severe traumatic brain injury,that will deep research of the mechanism and provide value evidence for expanding its clinical use.2 Research Methods and Results2.1 The clinical study on Traditional Chinese and Western Medicine prevention and control mechanism of cerebral ischemia in the severe traumatic brain injury.Methods:We selected 80 cases that patients were confirmed with acute severe head injury in the neurosurgery department of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine from January 2007 to March 2009.We strictly performed the inclusion criteria and exclusion criteria.These patients were randomized for the control group and treatment group.From the first day in hospital,a different treatment optionhas been done,the control group being done only by a simple conventional western medicine treatment,the treatment group being done in the conventional Western medicine combined with treatment with nasal feeding on the Huoxuehuayu decoction,with intravenous infusion of Huotankaiqiao injection of Chinese medicine and with enema Tongfuxiere of the Traditional Chinese medicine.From the incidence of cerebral vasospasm,intracranial blood flow velocity changes, GCS score changes,the incidence of cerebral infarction in traumatic brain by CT checking,the security aspects of monitoring,indicators dynamic observation of continuous,the study sustained for 18 days.Data processing has been done as analysis of the use of SPSSIO.O packages,metrology data using "t" test,"X~2" test with count data,check the level of significantα= 0.05.Results:(1)Admitted to hospital the third day two groups of patients with MCA,ACA mean blood flow velocity were significantly different(P＜0.05); The sixth day,two groups of patients with MCA,ACA,PCA,VA blood flow velocity were significantly different(P＜0.05),of which the mean MCA flow velocity difference was the largest;The twelfth day of the two groups of patients with MCA,ACA,BA,VA mean blood flow velocity were significantly different(P＜0.05);The 18th day of the two groups of patients with MCA,PCA of the mean blood flow velocity was significant difference(P＜0.05),but the difference was smaller.(2) The third day after admission,the sixth day,the twelfth day of two groups of patients with the incidence of cerebral vasospasm were significantly different(P＜0.05),two cases of vasospasm were gradually extended over time to ease;(3)Two groups of patients with GCS score on the first day was not significantly different(P＞0.05),the third day,the sixth day,the twelfth day,the 18th day of two groups of patients with GCS score were significantly different(P＜0.05);(4) Two groups of patients with traumatic cerebral infarction rates were 30.0%and 10.0%,there was significantly different(P＜0.05);(5) In the treatment of the sixth day,the twelfth day to review blood analysis and liver and kidney function examinatin, results suggested that in the course of medication there was no significant side effects.2.2 The experimental research of Buyanghuanwu decoction to promote vascular endothelial cell proliferation and angiogenesis.2.2.1 Preparation of serum containing Buyanghuanwu decoctionMethods:Between male and female,adult New Zealand rabbits were equally divided into high,mediumand low-dose medicine group and the saline control group,administered once daily for 3 consecutive days.Asepsis under anesthesia,rabbit blood form the right carotid artery was collected and centrifuged.The serum was collected,inactivated,filtered,group packaged, cold storaged.Results:All of animals kept in good condition beforeblood collection. About 50ml of fresh blood could be collected from the right carotid artery of each rabbit.This method was easy and reliable.2.2.2 Rabbit vascular endothelial cells in vitro primary culture and subcultureMethods:The eversion New Zeal rabbit thoracic aorta was put at 0.1%Ⅱ-type collagenase to digest.The digestive juice was collected and centrifuged.The cells were collected and put into bottles for culture.After 72 hours the foster liquid was changed.After 8 days primary cells covered around the bottom.Then 0.25%steapsin was put into the flask to digest for a few minutes.Cells were dispersed wind,counted,cultured in different bottle.Results:The primary endothelial cells need 48 hours or so to become the majority adherent cells.Cells were small round,polygonal,and oval-shaped core.After 72 hours the cells extended pseudopodia;4-8 days cells grew rapidly.8-9 days cells grew in primary monolayer,covered the bottom.The Subcultured endothelial cells adhered in 4-8 hours and extended out pseudopodia,7 days cells grew in primary monolayer,more than 10 days cells were closed with cobblestone-like,non-overlapping,we can see the vortex-like cells;Endothelial cells kept the same characteristic more than 10 generation.2.2.3 Vascular endothelial cells of rabbit testMethods:Morphological identification was done under inverted phase contrast microscope through observation of cell division to form single-layer forms.Immunofluorescence identification was done.Firstly the cells were cultured in glass slide to form a single-chip,then which were processed by alcohol,ⅧR-Ag,sheep anti-mouse IgG,the SABC and fluorescent-labeled. At last that were put in fluorescent microscopy to observe,negative controlled.Results:The endothelial cells were small round,polygonal,and oval-shaped nuclear.Immunofluorescence shows that a large number of green cells,plasma cells,a clear membrane staining,non-staining nuclei.2.2.4 Rabbit vascular endothelial cell proliferation and the role of serumMethods:We used measurement of cell proliferation MTT and cell growth curve mapping for testing the role of different doses of the same concentrations of drug serum to promote cell proliferation,testing the role of different concentrations of the same doses of drug serum to promote cell proliferation. Results:5-8 days was a period of rapid growth,about the tenth day cells reach the peak of cell proliferation,after 14 days the trend of cell proliferation was flat.The same dose of different concentrations of serum containing 10%group and 20%group were significantly different than the serum containing 5%group(P <0.05).The same concentration of different doses of serum in medium-dose group and high-dose group were significantly different than the low-dose group(P <0.05).2.2.5 Rabbit vascular endothelial mitotic index determinationMethods:By Giemsa staining,firstly in low magnification light microscopy we choiced scattered better cells which the nucleus and cytoplasm coloring were clear,and then transferred to high-power microscope to observe cells division period.Each sample was counted for more than 1000.By recording the number of split-phase cells and the number of interphase cells,the cells mitotic index was calculated.Results:In the whole phase of the cells cultured,more than 80%of the cells were in interphase,20%of the cells were in divided following period. Different drug-containing serum culture of endothelial cells than those in the blank cells can increase the mitotic index.In each group 6-8 days cells reached the peak mitotic and then gradually decreased,after 11-12 days reduced to 1%.2.2.6 Observation Rabbit vascular endothelial cells in TEMMethods:Have been intervened with the serum containing different doses of drug,cell samples were prepared.By JEM-200CX transmission electron microscope,endothelial cells were observed including cells within the nucleus,organelle,cytoplasm,cell size distribution of lipid droplets.Results:The cells which were intervened with the serum containing different doses of drug in each group were significantly closer with larger nuclei,clearer chromatin than the blank cells;Intervened with the serum containing drug,the cells had cytoplasm with rough endoplasmic reticulum expansion hyperplasia,mitochondrial relatively well-developed ridge crest on the particle structure.There was a substantial amount pinocytotic vesicles of the cells between the endothelial cells or between connecting desmosomes. These cells can be seen with large glycogen granules and specific granules.2.2.7 Serum containing Buyanghuanwu decoction to promote the chick embryo chorioallantoic membrane angiogenesis test Methods:The serum containing high,medium and low doses of medicine and the blank control serum were placed in the carrier on the surface of the chick embryo chorioallantoic membrane to be tested.When the specimens of CAM were prepared,anatomical observation under a microscope and computer image analysis image analysis have been related to quantitative data for further statistical analysis.Results:The serum containing drug can significantly promote chick embryo chorioallantoic membrane angiogenesis.3 Conclusion3.1 The pathology in TCM about cerebral ischemia in the severe traumatic brain injury can be summarized as Qizhixueyu,then phlegm,blood stasis and thermal cross-noded,mind closed.3.2 Based on conventionality therapy in Western Medicine,the combination therapy of Huoxuehuayu,HuotanKaiqiao,Tongfuxiere Chinese medicine for prevention and treatment of traumatic brain ischemia after severe head injury, that can reduce post-traumatic intracranial cerebral blood flow velocity, prevent and treat with cerebral vasospasm and reduce cerebral infarction the incidence,improve the state of cerebral ischemia,improve patients with GCS score and the prognosis.The liver and kidney function examination in patients have not benen obviously damaged.This combination therapy was safe and effective.3.3 Buyanghuanwu Decoction could improve the vascular endothelial cells energy synthesis,promote cell metabolism with activity function,induce to secrete the growth factor to promote endothelial cells proliferation.3.4 Buyanghuanwu Decoction may also be activated to the outer membrane of vascular endothelial cells,and promote their division,budding and migration. And activate angiogenesis start procedures,promote endothelial cell differentiation and cell connections to form a primitive lumen,and further promote the stability and neovascularization mature to promote angiogenesis.3.5 Integrated Clinical and Experimental Research:Using Traditional Chinese and Western Medicine for cerebral ischemia in severe traumatic brain injury, that was significant effect,one of its mechanism may be sure by protecting brain microvascular endothelial cells,promoting endothelial cell proliferation and migration,repairing the damaged microvascular,promoting ischemic regions to generate new blood vessels,improving regional cerebral blood flow,improving cerebral microcirculation.Thereby this therapy can protect brain tissue and nerve function.4.The features and innovative of this subject4.1 Based on conventionality therapy in Western medicine,a combination therapy of Huoxuehuayu,Huotankaiqiao,Tongfuxiere Chinese medicine has been used for treatment with severe brain injury,which is a innovation concept of treatment.4.2 we firstly summed up the TCM pathogenesis of Cerebral Ischemia in severe traumatic brain injury,which is Qizhixueyu,phlegm and blood stasis and thermal cross-knot resistance then mind being closed.4.3 By using of technology of serum to Chinese pharmacology,vascular endothelial cell culture and chick embryo chorioallantoic membrane angiogenesis experiments,we studied on mechanism of Traditional Chinese Medicine prevention and treatment of Cerebral Ischemia in the severe traumatic brain injury.This is a new attempt.