Significance And Expression Of Endothelin-1 As Well As Receptor On Cerebral Artery Of Scald Rets

PART Ⅰ: Expression of Endothelin-1 on Cerebral Basilar artery in rats with Scald combined with Endotoxemia BACKGROUND AND OBJECTIVE Under the condition of scald or infection, immunologic function of body is low and action of mechanical barrier is impaired, therefore, a number of endotoxin release into blood circulation and form endotoxemia in some condition, which activates a series of vasoactive substances and results in systemic organ injury. Endotoxin often causes multiorgan dysfunction and results in endotoxemia shock. Endotoxemia shock is a serious progressive failure of the circulation with an unacceptably high mortality rate of 30 %-90 %. Endotoxemia shock is associated with vast cardiovascular changes, organ dysfunction with microcirculatory disturbances, indicating an apparent inefficiency of its current treatment. Based on animal experiment and clinical observation, It indicates that the endothelium-derived factors , such as ET-1 and NO , play a key role in scald and/or scald combined with endotoxemia. Endothelins have potent and important properties, involving not only the circulation but also the response to stimuli like inflammation and trauma. In particular, endothelin release is stimulated by scald and/or scald combined with endotoxin , and the levels of endothelin peptides are increased in the circulation of patients with burn(scald) , which may evoke profound vasoconstriction in the splanchnic vascular bed during the initial phases of scald and/or scald combined with endotoxemia shock. In spite of some scholar studying that the effects of ET-1 on injuried splanchnic, such as heart,liver,lung,kidney and intestine during scald and/or scald combined with endotoxemia, measuring the level of ET-1 in each organ tissue, and thinking that burn(scald) may directly induce the increase of endothelin level in plasm,tissue and organ of body, which result in the injury of remote organ after burn(scald), but the reports about the effect of ET-1 on injuried cerebral tissue during burn(scald) and/or burn(scald) combined with endotoxemia, especially to cerebral vessels, were very scarce. Therefore, profoundly studying the effects of ET and its receptor on cerebral vessel during burn(scald) and/or burn(scald)combined with endotoxemia have an important prospects in clinical application, such as treating cerebral vasospasm,center nervous system inflammation,intracranial pressure increase and the disorder of cerebral circulation induced by burn(scald) and/or burn(scald)combined with endotoxemia. METHODS 1. Experiment groups: Wistar rats of either sex , weighting 280-320g,were randomly divided into three groups: Scald group; Scald combined with endotoxin injection group(S+E) and Endotoxin injection group. Each group was subdivided into: Control group; 3h Scald group; 6h Scald group; 12h Scald group; 24h Scald group and 48h Scald group. 2. Establishment of scald rat model and scald combined with endotoxemia model. 3. Morphologic observation: observing general condition after scald ; observing pathologic tissue slices of scald skin; observing pathologic tissue slices of basilar artery after scald. 4. Plasma endotoxin levels were determined by chromogenic substrate method in scald rats. 5. Measurement of basilar arterial diameter: Measuring the diameter of basilar artery upper,middle and lower part, taking the means of these three measurements as basilar artery diameter. The basilar artery diameter of normal control was used as basic value, while the basilar artery diameter of each time point post-scalding was used as constrict value. The severity of cerebral vasospasm was estimated by Lisxcxak's standard. 6. Measurement of ET-1 levels in plasma,brain tissue and basilar artery by radioimmunoassay. 7. Measuring that expression of ET-1 protein on basilar artery in scald rats by Western blotting: basilar arterial tissue(50mg) mince→adding 1.0 ml denaturing solution →homogenize in ice →protein content was measured by a modified Lowry procedure →SDS-polyacrylamide gel electrophoresis(PAGE) →proteins were electrophoretically transferred to nitrocellulose filters →the filters were incubated with monoclinal antibody →the filters were incubated with horseradish peroxidase-anti-immunoglobulin antibody →the filters were then incubated with chemiluminescent detection solution and exposed to x-ray film →the films were individually quantified using image analysis system . 8. RNA isolation and measuring the expression of ET-1 mRNA on basilar artery in scald rats by RT-PCR A modified acid-guanidinium thiocyanate-phenol-chloroform method was used to isolate total RNA from basilar arterial tissue sample. basilar arterial tissue(50mg) →adding 1.0 ml denaturing solution →homogenization on ice, 5 min. →transfer homogenate to a fresh polypropylene tube and sequentially add 0.1 ml 2 M sodium acetate →add 1 ml phenol/chloroform mixture,15 min. →40C, centrifuge at 12000 rpm, 15 min. →transfer the upper aqueous phase containing the extracted RNA to a fresh tube →add an equal volume isopropanol to the extracted RNA, mix the solution well and allow RNA precipitate for 1 hour at -200C , →collect the precipitate RNA by centrifuging at 12000 rpm, for 15 min. at 40C →carefully decant the isopropanol and decant supernatant fluid into a fresh tube →repeat above step once more→add 50μl of DEPC-treated H2O, store the RNA solution at -700C. Estimate the concentration of RNA by measuring the absorbance at 260 nm. Reverse transcription-polymerase chain reaction(RT-PCR): ①Transfer 1.0μg of total RNA to a fresh microfuge tube. Adjust the volume to 10μl with DEPC-treated H2O. Denature the RNA by heating at 750C for 5 minutes, followed by rapid chilling on ice. ②Add the following reagents to the denatured RNA: dNTPs,random hexamer primers,Rnase inhibitor,MgCl2,reverse transcriptase, add H2O to 20μl. ③Incubate the reaction mixture for 60 minutes at 370C. ④Inactivate the reverse transcriptase and denature the template cDNA complexes by heating the reaction mixture to 950C for 5 minutes. ⑤Adjust the reaction mixture so that it contains 20 pmoles of the sense and antisense primers(objective gene and β-actin gene). ⑥Add the following reagents to the reaction mixture: dNTPs,thermostable DNA polymerase. ⑦Overlay the reaction mixtures with 1 drop of light mineral oil. Place the tubes in the thermal cycler. ⑧Amplify the nucleic acid using the denaturation, annealing, and polymerization times. ⑨Aspirate a sample 10μl and analyze them by electrophoresis through an agarose gel. Be sure to include DNA markers of an appropriate size.Stain the gel with ethidium bromide to visualize the DNA. ⑩Quantitation using laser densitometry, results were expressed as ER-1 mRNA to β-actin mRNA ratio. RESULTS 1. Morphological appearance: skins were paleness and leather-like appearance on wound tissues in immediately after scalding group and scald combined with endotoxemia group rats; nigrescence after 6h, formed abscess scab and pustular liquid after 12h. The rats showed slow activity and hyponoia as well as decreased food and water intake after injury.Histological analysis: The epidermis of wound tissues was destroyed completely by light microscope observation. The collagenous fibre bandles were rupture, arrange disorder, inflammatory cell infiltration.The sebaceous gland,hair bursa structure in dermis be demolished in some extent. Basilar arterial structure were markedly destroyed in scald ,scald combined with endotoxemia and endotoxemia group rats: Basilar arterial showed that the diameter became narrow and the wall thickness. The internal elastic lamina showed crumple and various thickness in tunica inner, tunica median incrassate, smooth muscle cell increase and inflammatory cell infiltration. 2. Measurement of the plasma levels of endotoxin after scald in rats: The plasma levels of endotoxin were 7.3798±1.3966 EU/ml 6h after scald,and 7.5216±1.4582 EU /ml12h after scald,which soared 150-160 times compared with control group. The plasma levels of endotoxin 6h after endotoxemia was 10.9712±2.0776 EU /ml,and 12h after endotoxemia was 5.7541±1.2751 EU/ml, which markedly increased compared with control group. 3. Measurement of basilar arterial diameter in scald,scald combined with endotoxemia and endotoxemia group rats by angiography: Basilar arterial diameter decreased at each time phase of postinjury in scald,scald combined with endotoxemia and endotoxemia group rats as compared with preinjury, especially at 6h and 12h it reduced obviously and at 48h it resume. 4. Measurement of ET-1 levels in plamsa,cerebral cortex and basilar artery in scald,scald combined with endotoxemia and endotoxemia group rats by radioimmunoassay: The ET-1 levels in plamsa,cerebral cortex and basilar artery increased 6h after scald compared with control group rats, especially at 12h they raised markedly and at 24h they still higher than control. The ET-1 levels in plamsa,cerebral cortex and basilar artery were higher at 6h after scald combined with endotoxemia group compared with control group rats, especially at 12h they enhanced markedly and at 24h the ET-1 levels were still higher than control. The ET-1 levels in plamsa,cerebral cortex and basilar artery heightened at 6h in endotoxemia group compared with control group rats, especially at 12h elevated markedlyand at 24h the ET-1 levels were still higher than control. 5. Expression of ET-1 protein on basilar artery in scald rats by Western blotting: Expression of ET-1 protein on basilar artery 6h after scald increased, especially 12h after scald. The expression of ET-1 protein was 2.67±0.38 times compared with control group. At 24h, it grakually decreased and at 48h, it was weared control level. 6. Expression of ET-1 mRNA on basilar artery in scald rats by RT-PCR: Expression of ET-1 mRNA on basilar artery 6h after scald heightened obviously. The value was 1.102±0.086, which was almost 5 times compared with control group(P<0.05). 12h and 24h after scald the value was 1.014±0. 54 and 0.925±0.038 respectively , which were also higher than control group(P<0.05). CONCLUSIONS 1. The skin showed paleness and leather-like appearance on wound position in scald group and scald combined with endotoxemia group rats; The epidermis of wound position was destroyed completely under light microscope. Basilar arterial structure were markedly destroyed in scald,scald combined with endotoxemia and endotoxemia group rats. 2. The plasma levels of endotoxin soared 150-160 times at 6h,12h and 24h after scald group and endotoxemia group, as compared with control group. 3. Basilar arterial diameters decreased 6h in scald,scald combined with endotoxemia and endotoxemia group compared with preinjury rats, especially at 12h they reduced obviously and 24h still lessened. 4. The ET-1 levels in plamsa,cerebral cortex and basilar artery increased 6h in scald,scald combined with endotoxemia and endotoxemia group compared with control group rats, especially at 12h, they hoisted obviously and at 24h they were still higher than control. 5. Expression of ET-1 protein on basilar artery increased at 6h after scald, especially 12h after scald, the expression of ET-1 protein increased obviously, at 24h they gradually decreased and at 48h, they decreased near to control group. 6. Expression of ET-1 mRNA on basilar artery 6h after scald was heightened obviously, which was almost 5 times compared with control group(P<0.05). 12h,24h after scald it also enhanced compared with control group(P<0.05). 7. The experiment results suggested: Endotoxemia may appear after rat scald; Scald and/or endotoxin may evoke the increase of ET-1 level in plasma,cerebral cortex and basilar artery;Expression of ET-1 protein and ET-1 mRNA on basilar artery soared obviously; ET-1 may promote cerebral vasospasm. The results showed that ET-1 play an important role in scald/endotoxin-induced cerebral vasospasm.PART Ⅱ: Innervation of ET-1-ergic nerve fibers on Cerebral artery in scald rats BACKGROUND AND OBJECTIVE After some investigator demonstrated that the adrenergic and cholinergic nerve innervate cerebral vessels with light and electronic microscope, Larsson et al observed that the third type nerve-peptidergic nerve existed in the adventitia or junction between tunica media and advertitia of cerebral vessels, which raised the curtain on the peptidergic nerve and peptide neurotransmitter of cerebral vessels. So far, some researchers have made sure that neuropeptide Y(NPY) ,vasoactive intestine polypeptide(VlP),calcitonin gene-related peptide(CGRP) and substance P(SP)-ergic nerve fiber innervated on cerebral artery, which was associated with the regulation of cerebral blood flow. The report about endothelin(ET)-ergic nerve was sparse, specially the report under the condition of cerebral vessel disease, such as after scald combined with endotoxemia, innervation of ET-ergic nerve fibers on cerebral basilar artery and regulation to brain blood flow was very rare. By the observation of innervation and density of nerve fibers on cerebral basilar artery in scald combined with endotoxemia, the present study profoundly probed the foundation on which the ET-ergic innervation affected the neuroergic regulation of cerebral vessels after scald injury. It will provide the morphological data to expose the mechanism of neuroergic regulation in cerebral circulation, and further find out the pathogenesis of cerebral vessels diseases and new preventing and treating methods. METHODS 1. Experiment groups: Wistar rat of either sex , weight 280-320g,randomly divided into six groups: Control group; Scald 3h group; Scald 6h group; Scald 12h group; Scald 24h group; Scald 48h group. 2. Establishment of scald model. 3. Immunohisto chemistrical staining: Measurement of the density of area,primeter and quantity of the ET-1 positive nerve fibers on anterior cerebral artery,middle cerebral artery,posterior cerebral artery and basilar artery in control group rats; Measurement of the density of area,primeter and quantity of the ET-1 positive nerve fibers on anterior cerebral artery,middle cerebral artery,posterior cerebral artery and basilar artery after 3h,6h,12h,24h and48h scald in rats. RESULTS 1. ET-1 immunoreactive positive nerve fibers, fine thread-like and brown color, may be observed on anterior cerebral artery(ACA),middle cerebral artery(MCA),posterior cerebral artery(PCA) and basilar artery(BA) in control,3h and 48h after scald in rats. The density of ET-1 positive nerve fibers among groups did not differ(P>0.05). 2. The density of ET-1 positive nerve fibers on ACA,MCA,PCA and BA 6h after scald was more denser compared with control group(P<0.05). 3. ET-1 positive nerve fibers on ACA,MCA,PCA and BA 12h after scald was markerly increase compared with control group(P<0.05).which has remark significance. 4. The density of ET-1 positive nerve fibers on ACA,MCA,PCA and BA 24h after scald was more denser compared with control group(P<0.05). CONCLUSIONS 1. ET-1 immunoreactive positive nerve fibers, fine thread-like and brown color, may be observed on ACA,MCA,PCA and BA in control group rats. The density of ET-1 positive nerve fibers on ACA was the most, next was MCA,PCA and BA in order. 2. The density of ET-1 positive nerve fibers on ACA,MCA,PCA and BA 6h,12h and 24h after scald was more denser compared with control group, especially 12h group it increased obviously. 3. The experiment results suggested that increased ET-1 positive nerve fibers after scald was markerly associated with the pathogenesis of cerebral vasospasm.PART Ⅲ: Expression and significance of endothelin A receptor on cerebral basilar artery in scald rats BACKGROUND AND OBJECTIVE The regulation effects of ET in centre nervous system was more and more interesting for researcher. Endothelium cell,smooth muscle cell and ET-ergic axon on cerebral vessels may produce and release ET. Only the ET that can bind ET receptor in these cells can exert its really effects. ET receptor play an important role in the regulation of arterial pressure of systemic circulation and the maintenance of body fluid balance, and it was associated closely with the pathogenesis of multi-diseases. It has been reported that ETA receptor on cerebral vessels have obviously changed in some cerebral vessels diseases such as hypertension. But the report about expression of ERA receptor on cerebral vessels after scald injury was very sparse. The levels of ET in plasma and wound position enhanced obviously, it suggests that ET may participate in the pathogenesis of early phase scald injury. Therefore, Measurement of the plasma levels of ETAR by radioimmunoassay, and observation of expression of ETAR protein and ETAR mRNA on basilar artery after scald by Western blotting and RT-PCR method, which was to probe the effects of ETAR in scald injury-induced cerebral vasospasm. It may provide theory basis to illuminate the pathomechanism of cerebral blood circulation and the effects of scald on cerebral vessels. METHODS 1. Experiment groups: Wistar rats of either sex , weighting 280-320g,were randomly divided into six groups: Control group; Scald 3h group; Scald 6h group; Scald 12h group; Scald 24h group; Scald 48h group. 2. Establishment of rat scald model(ref. Part 1) 3. Measuring the expression of ETAR mRNA on basilar artery in scald rats by RT-PCR: ⑴RNA isolation: A modified acid-guanidinium thiocyanate-phenol-chloroform method was used to isolate total RNA from basilar arterial tissue sample. basilar arterial tissue(50mg) →adding 1.0 ml denaturing solution →homogenize on ice, 5 min. →transfer homogenate to a fresh polypropylene tube and sequentially add 0.1 ml 2 M sodium acetate →add 1 ml phenol/chloroform mixture,15 min. →40C, centrifuge at 12000 rpm, 15 min. →transfer the upper aqueous phase containing the extracted RNA to a fresh tube →add an equal volumeisopropanol to the extracted RNA, mix the solution well and allow RNA precipitate for 1 hour at -200C , →collected the precipitate RNA by centrifugation at 12000 rpm, for 15 min,at 40C →carefully decant the isopropanol and decant supernatant fluid into a fresh tube →repeat above step once more→add 50μl of DEPC-treated H2O, store the RNA solution at -700C. Estimate the concentration of the RNA by measuring the absorbance at 260 nm. ⑵Reverse transcription-polymerase chain reaction(RT-PCR): ①Transfer 1.0μg of total RNA to a fresh appendorf microtube. Adjust the volume to 10μl with DEPC-treat H2O. Denature the RNA by heating at 750C for 5 minutes, followed by rapid chilling on ice. ②Add following reagents to the denatured RNA: dNTPs,random hexamer primers,Rnase inhibitor,MgCl2,reverse transcriptase. Add H2O to 20μl. ③Incubate the reaction mixture for 60 minutes at 370C. ④Inactivate the reverse transcriptase and denature the template cDNA complexes by heating the reaction mixture to 950C for 5 minutes. ⑤Adjust the reaction mixture so that it contains 20 pmoles of the sense and antisense primers (objective gene and β-actin gene). ⑥Add to the reaction mixture: dNTPs,thermostable DNA polymerase. ⑦Overlay the reaction mixtures with 1 drop of light mineral oil. Place the tubes in the thermal cycler. ⑧Amplify the nucleic acid by PCR. ⑨Aspirate the PCR produces (each sample 10μl) and analyze them by electrophoresis on agarose gel. Be sure to include DNA markers of an appropriate size.Stain the gel with ethidium bromide to visualize the DNA.⑩Quantitation using laser densitometry, results were expressed as ETAR mRNA to β-actin mRNA ratio. 4. Measuring the expression of ETAR protein on basilar artery in scald rats by Western blotting: basilar arterial tissue(50mg) were minced →adding 1.0 ml denaturing solution →homogenize in ice →protein content was measured by a modified Lowry procedure →SDS-polyacrylamide gel electrophoresis(PAGE) →protein were electrophoretically transferred to nitrocellulose filters→the filters were incubated with monoclonal antibody →follow the filters were incubated with horseradish peroxidase-anti-immunoglobulin antibody →the filters were then incubated with chemiluminescent detection solution and exposed to x-ray film→the films were individually quantified using image analysis system . RESULTS 1. Expression of ETAR mRNA on basilar artery in scald rats by RT-PCR: The results of RT-PCR show: Expression of ETAR mRNA on basilar artery in control groupwas low, which to β-actin mRNA ratio was 1.145±0.103. Expression of ETAR on basilar artery 6h after scald increased obviously, the value was 2.303±0.210, which was almost 2 times higher compared with control group(P<0.05). 12h and 24h after scald was 1.979 ±0.187 and 1.590 ±0.136, respectively, which also enhanced compared with control group(P<0.05). 2. Expression of ETAR protein on basilar artery in scald rats by Western blotting: The results of Western blotting showed: Expression of ETAR protein on basilar artery in control group was slow, but expression of ETAR protein on basilar artery 6h after scald increased, which was 2.06±0.38 times higher compared with control group. Specially 12h after scald, it increased obviously, which was 2.85±0.35 times higher compared with control group. at 24h after scald, the expression gradually decreased and at 48h after scald,it reduced to near control group. CONCLUSIONS 1.Expression of ETAR mRNA on basilar artery 6h,12h and 24h after scald enhanced obviously, which was almost 2 times higher compared with control group. 2. Expression of ETAR protein on basilar artery 6h after scald increased, especially at 12h after scald, it increased obviously, However, at 24h after scald, it decreased gradually and at 48h it reduced to near control group. 3. The experiment results suggested: Endotoxemia may appear after scald in rats; Scald and/or endotoxin may evoke the increase of expression of ETAR protein and ETAR mRNA on basilar artery. The increased ETAR may combined with ET-1, which may promote cerebral vasospasm. The results showed that ETAR play an important role in scald/endotoxin-induced cerebral vasospasm.