The overall goal of this procedure is to produce a mouse model of sepsis by a surgical procedure called SQL Ligation and Puncture, or CLP. This is accomplished by first making a 1.5 to two centimeter midline incision on the abdomen of a mouse. Next, the secum is exposed and ligated, then the seum is punctured to induce bacterial translocation.
Finally, the incision is closed. Ultimately, results can be obtained that show increases in systemic and peritoneal cytokine levels as well as symptoms such as lethargy and lethality. What I'm gonna show you is to produce a mouse sepsis model termed sation and puncture, which is modified from the original model developed by Dr.Chandry and his collaborators.
The mean main advantage of this model is that it is the most relevant model of sepsis. In addition, it's simple, well characterized and economically feasible, and therefore has been widely used in the field of sepsis and inflammation research. As an example, we'll demonstrate the utilization of this model to evaluate the therapeutic potential of herbal extract in sepsis.
To prepare the herbal extract, soak 10 grams of herbs in 200 milliliters of water at 85 degrees Celsius for one to four hours to remove insoluble particles, centrifuge the water fraction at 3, 300 G for 20 minutes at four degrees Celsius. Next, filter the supernatant through a 0.2 micron filter. Then further concentrate the filtrate by using a centrifugal filter with a 10, 000 kilodalton molecular weight cutoff and spinning at 5, 000 G for 60 minutes at four degrees Celsius.
Using macrophage cultures screened low and high molecular weight fractions for inhibiting activities of late pro-inflammatory mediators, such as high mobility group box one or HGB one. To begin macrophage isolation first, interperitoneal administered two milliliters of a 4%TH glycol broth into normal mice after three days harvest primary peritoneal macrophages culture, the macrophages in DMEM supplemented with 10%FBS, two millimolar glutamine, and 1%penicillin. Streptomycin gently wash the adherent macrophages with optimum and incubate them in the same medium for two hours.
Then stimulate them with the bacterial endotoxin lipopolysaccharide or LPS 16 hours after stimulation. Collect the cell conditioned medium and use it to carry out western blotting. To measure the levels of HM GB one.
Quantify the band intensity using a standard curve generated with purified H mgb one to establish sepsis after anesthetizing a mouse with an intramuscular injection of 75 milligrams per kilogram of ketamine and 20 milligrams per kilogram of xylazine. And using a toe pinch to check its level of sedation. Place the mouse in a supine position, clean the abdomen, then make a 15 millimeter midline incision to expose the cecum approximately five millimeters from the sequel tip with a four oh silk suture.
Ligate the cecum, and then using a 22 gauge needle puncture the ligated sequel stump once to allow extrusion of stool, immediately return the cecum to its normal intraabdominal position and close the abdomen. After animals regain consciousness and mobility. Return the cage back to the animal room at 24 hours.
Post CLP intraperitoneal administer 200 microliters of herbal extract or control daily for three days. Monitor the animal for survival as long as needed within a few hours of seq l ligation and puncture, or CLP surgery. Animals exhibit clinical signs of sepsis that include piloerection, lethargy, huddling, and a decrease in food and water intake.
Animals developing severe peritonitis with consecutive systemic infection normally die within 48 to 96 hours. However, even age, sex and genetic background matched animals can respond to the surgery in a distinguishable manner in the course of experimental sepsis, for example, at 48 hours post CLP. While some animals may have approached the mound state as defined here, others may remain at a non moribund state.
Comprehensive surveys of circulating cytokines revealed dramatic differences in levels of several cytokines, including IL six, kc, MIP two, and ST nfr, one between mice in the moribund and non moribund states. Notably, these inflammatory mediators have been classified as surrogate markers of sepsis because their circulating levels are reliable predictors of lethal outcome in experimental or clinical sepsis. In addition, CLP induced local release of various pro and anti-inflammatory cytokines and chemokines, for example, at 48 hours post CLP, significant amounts of the cytokine IL six and the chemokines KC and MI P two can still be measured not only systemically in the blood, but also locally in the peritoneal lavage fluid.
Here, the capacities of various herbal extracts or control compounds were evaluated for their ability to inhibit endotoxin induced HGB one release. Those that had the capacity to inhibit HGB one release were further tested in vivo using a mouse model of sepsis. Because of the late and prolonged kinetics of HGB one accumulation in experimental sepsis, the first dose of HGB one inhibitors was given 24 hours after the onset of sepsis, a time point when mice developed clear signs of sepsis, including lethargy, diarrhea, and piloerection, as shown here, delayed and repeated administration of a major green T component epi Gallo, catechin three GALLATE or EGCG beginning at 24 hours after onset of sepsis, significantly rescued mice from lethal sepsis even when given orally EGCG still rescued mice from lethal sepsis, significantly increasing animal survival rates from 16%to 44%Once mastered this s ligation and the puncture model can be finished in less than five minutes if it's done properly.
