This protocol describes a common and feasible method of inducing acute liver injury (ALI) via CCl4 exposure through an orogastric tube. CCl4 exposure induces ALI through the formation of reactive oxygen species during its biotransformation in the liver. This method is used to analyze the pathophysiology of ALI and examine different hepatoprotective strategies.
I would like to present our work about induced acute liver injury in rats via carbon tetrachloride exposure through orogastric tube. Select adult male Sprague-Dawley rats weighing 300 to 350 gram. Provide rat chow and water ad libitum.
Perform all experiment between six AM and 12 PM.The animal care comedy of the Ben-Gurion University of the Negev, Israel approved all treatment and testing procedure used in this protocol. Maintain rats at room temperature of 22 Celsius degree with 12 hour lights and 12 hour dark cycles. Cannulate the tail vein with a 22 gauge catheter.
Collect blood samples at baseline. Blood biochemical analysis include the measurement of serum GOT, GPT, and total bilirubin. Examination of liver enzymes and total bilirubin level were carried out in the biochemical laboratory of Soroka Medical Center.
Caution:exposure to high concentration of carbon tetrachloride, including vapor or skin absorption can affect the central nervous system, degenerate the liver and kidney, and prolonged exposure may lead to coma or death. Prepare a 50 percent solution of carbon tetrachloride mixed with olive oil as vehicle in one to one ratio. Induce the pathotoxicity in vivo by carbon tetrachloride administration via an orogastric tube.
Insert an orogastric tube through the oral cavity of the rat. Expose the rats to different doses of carbon tetrachloride. One milliliter per kilo for mild acute liver injury, two point five milliliter per kilo for moderate acute liver injury, or five milliliter per kilo for severe acute liver injury.
For sham group, expose the rats to five milliliter per kilo only olive oil. Collect blood samples at 24 hours from carbon tetrachloride exposure. Blood biochemical analysis include the measurement of serum GOT, GPT, and total bilirubin.
Place dead rat on dissecting board in dorsal recombinant with the abdominal facing upwards and shave her abdomen. Using scalpel, incise the full length of the ventrum from the anus to the chin. Reflect the skin.
With the scalpel, incise the abdominal wall from the anus to the xiphoid cartilage to expose the abdominal viscera. Transfer the liver into a petri dish. Perform a hematoxylin and eosin staining.
Examine the slices under microscope magnification of 200 times with a 20 millimeters objective length. The liver section should be graded by a specialized pathologist, blinded to the treatment protocol. The score of zero indicates no liver abnormalities, one to two indicates mild liver injury, three to four indicates moderate liver injury, and five to six indicates severe liver injury.
The various groups of rats at different times are shown on the scheme. 24 hours after inducing acute liver injury, there was a significant increase in GOT, GPT, and total bilirubin levels, depending on the carbon tetrachloride exposed as compared to the control group. P value was less than zero point zero zero one.
Histopathological changes were observed after 24 hours in all groups after carbon tetrachloride intoxication in various doses. Histological grading score at 24 hours between all three interventional groups to the control group was significantly higher. P value was less than zero point zero five.
Our research group presented the common and physical method of induced acute liver injury in rats via carbon tetrachloride exposure through orogastric tube. This method is used to investigate the pathophysiology of acute liver injury and to test different about the protective strategy. Thank you very much for your attention.
