The Effect Of Glycine On Hemorrhage Shock In The Rat

Objective: This study investigates whether glycine prevents the organs of rat from injury and decreases mortality after hemorrhagic shock. Methods: 1. Hemorrhagic shock model: Wister rats(250-300g) were anesthetized with 4.5% amobarbital sodium(80mg/Kg i.p.) in room temperature(25℃). Blood pressure was monitored via polyethylene tubing(Intima 24GA) inserted into the right femoral artery, using a low pressure analyzer(Polygraph RM6000). The left jugular vein was cannulated(Intima 22GA) to administer glycine, normal saline and shed blood. The right carotid artery was cannulated with polyethylene tubing(Intima 22GA), and shock was induced over 5min by withdrawing blood into a heparinized glass syringe until mean arterial pressure was reduced to 30-35mmHg. Constant pressure was maintained by further withdrawal of small amount of blood as necessary for 55min. After 60 min of hypotension, rats were resuscitated by transfusion of 60%shed blood over 5 min, followed by normal saline(twice the shed blood volume) for 55 min. Prior to resuscitation, animals were randomly assigned to three groups(20 rats each group): shock group; glycine treatment group and sham group(operation was performed not to induce shock). Glycine(60mg/Kg) dissolved in 0.5mL normal saline was injected into the left jugular vein, and an equal volume of normal saline was given to shock group. 2. Determination of creatine phosphokinase(CK), aspartate aminotransferase(AST), alanine aminotransferase(ALT), alkaline phosphatase(ALKP) and creatinine(Cr): Blood samples were collected before and at the end of shock, at the end of resuscitation and 18 h later. Plasma was centrifuged, stored at -80℃ for determining Cr,CK,AST,ALT and ALKP. 3. Kupffer cell preparation and culture: Two hours after resuscitation, the liver was perfused through the portal vein with Ca2+- and Mg2+-free Hanks' balanced salt solution(HBSS) at 37Cfor 10 min at a flow rate of 20mL/min. Subsequently, perfusion was with HBSS containing 0.025% collagenase IV at37C for 7min at a flow rate of 23mL/min. After the liver was digested, it was excised and cut into small pieces in collagenase buffer. The suspension was filtered through nylon gauze mesh and the filtrate was centrifuged at 50*g for 10 min at 4 C. The suspension was centrifuged on a density cushion of 25%+50% Percoll at 800*g for 10 min, and cells fraction between 25% and 50% Percoll was collected and washed with buffer again, cells were seeded into 2mL RPMI1640 at 37C and incubated with 5% CO2. Non-αdherent cells were removed after 1 h by replacing buffer, and adherent cells(Kupffer cells) were cultured for 24 h prior to [Ca2+]i and TNF- a measurement. 4. Measurement of intracellular Ca2+([Ca2+]i): Intracellular calcium was measured flurometrically using the fluorescent calcium indicator dye fura-2 and a microspectroflurometer interfaced with an inverted microscope. Kupffer cells were placed in chambers with buffer at 37 C and stimulated with 1-1000ng/mL endotoxin. Changes in fluorescence intensity of fura-2 at excitation wavelengths of 340nm and 380nm and emission at 510nm were monitored in individual Kupffer cells. Intracellular calcium was determined from the equation: [Ca2+]i = Kd{(R-Rmin)/(Rmax-R)}/(Fo/Fs). 5. Measurement of TNF- a in conditioned media from cultured Kupffer cells: Isolated Kupffer cells were seeded onto 24-well plates and cultured in RPMI 1640. After 24 h in primary culture Kupffer cells were stimulated with RPMI 1640 containing 1-1000ng/mL LPS, 5% rat serum, 10mM HEPES and antibiotics for 4 h. Superaatants were collected and analyzed for TNF- a using an enzyme-linked immunosorbent assay(ELISA) kit. 6. Histological observation: Eighteen hours after resuscitation, lungs, heart, liver and kidneys of rat were excised and fixed with 1% paraformaldehyde. Fixed tissues were embedded in paraffin and processed for light microscope. Sections were stained with hematoxylin and eosin. 7. Statistical analysis: SPSS 11.0 software was used andChi-square test, ANOVA and t-test. Results: 1. Survival rate after hemor...