Effect PDTC On Expression Of ICAM-1 On Cerebrovascular Endothelial Cells And Volume Of Cerebral Infarction In Rats' Brain Study

IntroductionCigarette smoke has played an important role in promotion and development of cerebral and heart disease. Smoking is one of preventable factors of atherosclerosis and thrombosis, but the concrete mechanism still remains unclear.Research has demonstrated that ICAM - 1 is the most widespread adhesion molecules in Central Nervous system, which is important in atherosclerosis and thrombosis. In vitro experiment demonstrated that CSC could increase human bilial vein endothelium ICAM - 1 expression. In vivo experiment shows that expression of ICAM - 1 and ICAM - 1mRNA increased under passive smoking in rats ' cerebral vascular endothelial cells, the volume of cerebral infarction is larged. There are time and dose effect relationship between cigarette smoking and the expression of ICAM - 1. It give us a hint that the mechanism by which smoking increase the stroke incidence and the size of cerebral infarction has enormous relationship with ICAM - 1.In animal experiment, ICAM - 1 monoclone antibody application could markly decreased cerebral infarction volume, and suggests we could decrease the volume of cerebral infarction by the inhibition of ICAM - 1. We administrate ICAM - 1 inhibitor and observe the cerebral endothelial cell by in situ hybriza-tion and immunohistochemistry to investigate the prevent and therapy methods for the smoking cerebral infarct patients. .Materials and MethodesExperimental materialsKExperimental animals;72 healthy male Wister rats, aged eight weeks, weights are 250 ~300g, offered by the animal department of CMU.2^Mainly reagents;tetrazolium chloride (TTC) offered by experimental device of CMU;ICAM - 1 ( CD54 ) immunohistochemistry and in situ hybrization reagents are offered by Wu Han boster. ,PDTC offered by sigma,USA.3 % main instruments;optical microscope;paraffin wax cut slices;program analysis systemExperimental method1. The group of experimental animal;seventy - two healthy male Wister rats which were randomly divided into control groups ( n = 24 ) and smoken groups ( n= 48).2. Preparation of animal models;reference Chow smoke perfuse model;use kind of cigarette,rats passive smoke four times,each time five ,after 12w, reference Zea longa to prepare brain ischemic model of rats.3. Neural symptom observation;MCAO, we observe the movement of four limbs after MCAO by fish line, such as right foreclaw cannot extend, left eyes smaller , walk just in the clockwise direction, We exclude the rats without such symptoms.4. the different deal of experiment and control group;MCAO rats were randomly given 0. 9% chloride or PDTC ip after cerebral infarct 3 ,6orl8hours then taken out the brain after 2 hours.5. Observe the ICAM - 1 expression on cerebrovascular endothelium;rapidly perfused with physiological saline,brain were removed,then were embeded in paraffin. Coronal sections of 5 ~6um thickness were cut. Adjacent sections were selected for immunostaining and in situ hybrization stain of ICAM -1 expression on infarct cerebrovascular endothelium and detect the infarction volume after TTC stain.6. Statistical treatment;Apply semiquantitative analysis to count positive stain small vascular number in the 10 randomly select 10 scopes;use image analytical system to deteminate positive cerebrovascular. By one way analysis of variance ,two of three groups have marked statistical difference(P <0. 05).Result1 Athe cerebrovascular endothelium ICAM - 1 expression of smoked rats ce-rebrovascular endothelium whose have used PDTC ip markly decreased.2^ smoke rats timely use PDTC after 3or6 hours cerebral infarct volume markly decreased, but after 18 hours cerebral infarct didn' t show statistical difference.DiscussionICAM - 1 is widely distributed in vivo, but the expression on the normal cerebral vascular endothelial cell is very low. It could recognizes the receptors of the leucocyte, mediating leucocyte adhesive to the endothelial cells and subsequently transcellularing ECs roll. But under pathologic conditions ,eg ischemia, trauma,inflammation etc,the expression could be rapidly enhanced. The mechanism of increased expression of ICAM - 1 induced by smoking maybe EC damage , induce TNF - a express, reflex vascular spasm, resulting aggravate ischemi-a.Animal experiment demonstrated ICAM knockout rats has smaller cerebral infarction volume than normal rats. The application of ICAM - 1 monoclone antibody could relieve ischemia cell damage. Clerck reported rabbits brain ischemia degree has be relieved after utilizing ICAM - 1 monoclonal antibody. Zhang reported ischemia brain injury area was decreased after utilizing ICAM - 1 McAb. Connolly etc reported the cerebral blood flow to the infarcted hemisphere was 3. 1 - fold greater in ICAM knockout mice compared with normal controls. ICAM -1 could promote atherosclerosis process , increase cerebral infracted volume. The mechanism now postulated: promote leucocyte infiltration, aggregation in ischemia area capillaries and block reperfusion;release oxygen free radicals and protease , resulting focal injury;activating leucocyte to release inflammatory mediators and cytokines to attracts more leucocyte, thus a malignant recycle.The most ordinary ICAM - 1 expression methods include : ( 1) inhibit NF- kB activation to decrease adhesive factors gene transcription;(2 ) depress Janus kinase and STAT - 1 a tyrosine phosphate;(3) promote ICAM - 1 mRNA degration to reduce it translate to protein after ICAM - 1 gene transcript. Now the most frequent way is the inhibition of NF - kB.NF - KB widely exists in many kinds of cells and participate in a series of gene expression. It is a kind of oxidative stress transcription factors, which could be activated by hydrogen peroxide, active oxygen, TNF, IL - 1, and has close relationship with cerebral inflammation, immune reaction, neural development and signal transduction. Our experiment selects PDTC, the anti - oxidize drugs to block NF - kB.Our results showed that the expression of ICAM - 1 was expressed in all smoking Wistar rats cerebral vascular endothelial cells, but contrast to smoking then PDTC ip rats, cigarette smoking group expression is significant, the control group is negative. Statistical analysis shows their differences are significant. It means that smoking could induce ICAM - 1 express, but we could block ICAM — 1 expression by block NF - kB. It gives us a new way to prevent atherosclerosis and thrombosis.NF - kB could markly reduce the infarct volume of the smoking rats after 3and6 hours cerebral infarction. It demonstrated that timely use NF - kB inhibitor after cerebral infarct could markly decrease ischemia volume, but after some time window it cant works well. The concrete mechanism maybe has a close relationship with ischemic penumbra.Our research shows that smoking could induce ICAM - 1 express and aggravate ischemic cerebral injury. If applicate NF - kB inhibitor for the smoken rats, it could be a prevent therapy;if administrate it after cerebral infarct, it could markly reduce cerebral infarction volume .ConclusionNF - KappaB inhibitor could reduce the expression of ICAM - 1 in cerebro-vascular endothelial cell . NF - KappaB inhibitor could markly reduce the infarct volume of the smoking rats after 3and6 hours cerebral infarction.