The overall goal of this procedure is to induce sepsis through seql ligation and puncture in mice in order to study tissue autophagy, a homeostatic process that facilitates turnover of damaged proteins and organelles. This is accomplished by first identifying the secum and moving it out of the peritoneal cavity of the anesthetized mouse. Once isolated, 60%of the secum is ligated and then perforated to induce mid-grade peritonitis.
The ligated and punctured seum is placed back into the peritoneal cavity and the animal is allowed to recover. The final step is to harvest the lung, liver, and kidney tissues from the septic animal 24 hours later. Ultimately, electron microscopy is used to show autophagosome formation in intact cells from the tissues harvested from the septic animal.
We will present sickle ligation and punctu or CLP as a means to induce sepsis and to study autophagy. The main advantage of this technique over previous methods like injection of endotoxin, is its clinical relevance. CLP leads to peritonitis, which is a well-known cause of polymicrobial sepsis in humans.
Autophagy represents a general stress response in tissues that may be activated by starvation or bacterial infection during autophagy, cytoplasmic materials, including proteins in organelles or sequestered into double membrane phagosomes, which are then delivered to the lysosomes for degradation. To begin, prepare the work area and arrange the sterile surgical instruments needed for surgery. After administering anesthesia, use the toe pinch reflex to confirm sedation When ready, place the animal onto a clean work surface and apply ointment to the eyes.
After removing the hair, disinfect the surgical area with repeated applications of Betadine and ethanol. Use a scalpel to make a small longitudinal skin incision parallel and approximately one centimeter left of the midline. Taking care not to penetrate into the peritoneal cavity, use small scissors to extend the initial incision.
Next, identify the abdominal muscles and dissect them through To gain access to the peritoneal cavity, use forceps to identify the secum and move it out of the cavity. Be careful not to damage mesentery blood vessels that could lead to lethal hemorrhage. Once isolated, ligate the seum according to the degree of sepsis desired Here, 60%is ligated to produce mid-grade sepsis.
Using a 21 gauge needle perforate through the secum near the ligation, the seum should be perforated from the mesenteric to the anti mesenteric side. Next, remove the needle and gently squeeze the ligated secum to confirm patency through the two holes. Only a small amount of feces should be extruded through them.
Move the ligated and punctured seum into the peritoneal cavity. Use silk surgical sutures to close both the abdominal musculature and the skin. After injecting warm saline, place the mouse under a heat lamp until it has recovered.
Post-surgical animals are given a analgesic before they return to their home cages with unlimited access to food and water. After euthanasia, immobilize the mouse on a clean work surface and disinfect the abdominal and thoracic skin with 70%ethanol solution. Use a scalpel and small scissors to remove the abdominal and thoracic skin.
Next, isolate the trachea by separating it from the surrounding muscle and connective tissue. Once isolated, place a suture around the trachea without tightening it. Use a small peripheral venous catheter to carefully intubate the trachea.
Tighten the suture and remove the needle, leaving the cannula in place. After isolating the heart and lungs, use this cannula to instill fixative through the trachea until the lungs are well fixed. Remove the heart and lungs and place them into the 4%PFA for further processing.
Next, remove the liver and kidneys in the same way. Samples processed for electron. Microscopy can be used to analyze autophagosome formation in intact cells.
Count auto phagosomes per unit area from 15 to 30 fields for appropriate statistical representation. Auto phagosomes have a double membrane structure where late stage auto phagosomes resemble filled VAEs. These survival curves show the severity of sepsis following ligations of different size.
Ligations of 60%result in a mid grade sepsis, which leads to around 60%mortality at seven days. Ligations of 75%or more of the cecum result in high grade sepsis and S subsequently in 100%mortality within two to three days. While ligations of 25%or less result in low grade sepsis and thus nearly zero mortality.
Sham surgical animals should experience no mortality. This representative micrograph of early and late stage autophagosome formation in lung tissue was taken from C 57 black six mice subjected to midgrade ligation. Early stage auto phagosomes have a double membrane structure.
While late stage auto phagosomes resemble a filled va. A representative control image is shown of lung tissue from C 57 black six mice subjected to sham operation. After watching this video, you should have a good other study of how to perform sickle ligation in punctu in order to induce sepsis of varying severity to collect tissues and to recognize autophagosome formation in cells of septic animals.
The study of auto responses to sepsis may be useful to understand how tissues respond to infection.
