The overall goal of this protocol is to demonstrate a reliable method to induce chronic liver injury in mice and analyze liver injury parameters during the induction, establishment and maintenance phases over a course of six weeks. The CDE diet of chronic liver injury reliably induces steatosis, or fatty liver, inflammation, liver progenitor cell proliferation and hepatic fibrosis. Collection and analysis of sera and tissues at indicated time points allow us to follow the molecular and cellular processes that underlie liver regeneration and chronic liver disease progression.
The techniques are demonstrated by Jully Gogoi-Tiwari, at post doc in my lab. New and chronic liver injury is induced through feeding of a choline deficient diet and supplementation of DL-ethionine, the drinking water. At the selected time points, mice are anesthetized, a cardio puncture is performed to collect sera for the analysis of blood based markers of liver injury.
The liver is perfused through the portal vein and is then excised for further processing and analysis. Mice on the CDE diet are observed daily in the first two weeks and three weeks, a week thereafter. Once water is supplemented with ethionine, it is kept at four degrees.
Bottles are refreshed every two to three days and the mice have free access to pellets of choline deficient chow which is topped up as needed. For bedding, wheaten chaff is used after each has been depleted of visible grains and stalks. Standard science of animal health are monitored and include overall appearance, posture, social interaction, grooming and coat condition, as well as body weight.
CDE treated mice will lose up to 20%of their initial body weight in the induction phase while they adapt to the diet but will recover thereafter. Mice on the CDE diet sometimes develop a greasy coat. This is not an indicator of poor grooming but a consequence of the high fat content of the diet.
After the mouse has been weighed and anesthetized, the feral withdraw reflex is tested to ensure the anesthesia is adequate. If anesthesia is just deep enough to commence surgery, the fur is wetted with 70%ethanol and the vertical midline incision is made in the abdominal wall up to the diaphragm. For easier access to the heart, the ribcage can be removed to expose the thoracic cavity.
A cardiac puncture is then performed where the blood is slowly collected from the ventricle to avoid collapsing of the heart. The blood is then transferred to a sterile tube and allowed to clot at room temperature to later obtain sera samples after low speed centrifugations. The intestine and stomach are gently swept to the side to expose the portal vein.
The heart is cut to allow fluids to exit and the portal vein is perfused with sterile phosphate saline solution until the liver blanches and indicates full perfusion of all liver lobes. The liver is then carefully detached and placed in a petri dish to remove potential non liver tissue and the gallbladder. After the liver weight has been recorded the liver is separated into individual lobes.
One liver lobe is further cut into small cubes of a few millimeters in diameter. One cube is collected in a separate tube for later RNA extraction, while the other cubes are collected in a second tube for future DNA and protein analysis as well potential extra RNA extractions. These tubes are snap frozen in liquid nitrogen as soon as possible after organ collection.
Once the larger lobes is used for formal infixation, and later paraffin embedding for sectioning on a microtome and histology and immunohistochemistry evaluations. Two other lobes are embedded in a cryo matrix while making sure that the tissue piece is completely covered by the matrix. These liver lobes are then snap frozen in liquid nitrogen for sectioning on a cryo stet and immunofluorescent or confocal microscopy experiments.
The enzyme alanine transaminase can be measured in the sera as a biomarker for hepatocellular injury. While healthy animals display low sera ALT levels, at all major time points, CDE treated mice release ALT into the bloodstream. With a major peak in the induction phase and slow normalization in the establishment and maintenance phase.
Formalin fixed and paraffin embedded liver tissue can be stained with hematoxylin and eosin to evaluate the liver histology. CDE treated livers show vastly disrupted liver architecture and the parenchymal infiltration of many basophilic cells in the induction and establishment phase with normalization in the maintenance phase. To assess fibrogenesis, formalin fixed and paraffin embedded liver tissue can also be stained with the polyazo dye sirius red, which visualizes hepatic collagen deposition.
In the induction phase, collagen accumulates in the parenchyma and normalizes in establishment and maintenance phase. Cryo matrix embedded and snap frozen liver tissue can be cut into thin sections of about seven millimeter on a cryo stet. Immunofluorescent staining for biliary and liver progenitor cell marker panCK and the inflammatory cell marker CD45 can then be performed.
An early increase in the transcription levels of cytokines and growth factors can be observed. These include tumor necrosis factor, tumor necrosis factor like weak inducer of apoptosis, or TWEAK, lymphotoxin beta, hepatocide growth factor, transforming growth factor beta and interleukin six, for example. After watching this video, you should have a good understanding of the CDE model of chronic liver injury.
You should have gained some knowledge of the processes involved in perfusing and isolating livers for further analysis and the histological cellular and molecular changes induced in livers of mice fed a choline deficient, ethionine supplemented diet over a time course of six weeks.
