The overall goal of this procedure is to produce an animal model of liver fibrosis that can be used to assess the efficacy of potential anti-fibrotic agents. This animal model can help answer key questions about the nature and development of fibrosis in the liver and how anti-fibrotic agents reduce the fibrotic process. Furthermore, this model mimics the complex physiological processes of human liver disease from early to late-stage fibrosis and can be studied at any point of interest of the disease.
Individuals new to this method need to plan the workflow because the den-glis-nov animal model takes at least five weeks. After the liver has been harvested, the processing requires one or two weeks before especially stained sections are ready to be examined by the pathologist. DMN must be diluted from a stock to 10 milligrams per milliliter in PBS.
When performing this dilution, be sure to work in a fume hood as the substance is a carcinogen. For the experiment, select male Wistar rats between 150 and 200 grams. Acclimate the rats to a housing facility with a normal climate for at least four days.
Provide food and water ad libitum and measure their daily intake. On a weekly basis, measure the rat's body weight and take a blood sample. For the DMN treatment, at the fume hood, prepare and inject 10 milligrams per kilogram doses.
Use a 25 gauge needle and intraperitoneally inject the lower-right quadrant of the abdomen. Inject the rats for three consecutive days and repeat this process weekly for four weeks. Always on the same three days, and importantly, adjust the DMN dose weekly over the four-week period.
Euthanize the DMN-treated animals four to five days after their last injection. On a dissecting board, disinfect and moisten the abdominal skin with 70%ethanol. Then, using scissors, incise the skin from the anus to the chin and reflect it apart.
Next, incise the abdominal wall from the anus to the zyphoid cartilage to expose the abdominal viscera. Check the organs for abnormalities such as inappropriate color, size, location, or fluids within the peritaneal cavity. Also probe the organs for their consistency and note any abnormal odors.
Next, isolate the liver. Begin at the hilus, the diaphragm attachment point, and work around the liver from there, freeing it from surrounding ligaments and vessels. Then, transfer the liver to a dish and record its weight.
Now, using a scalpel, from one lobe, make a five millimeter block that extends one to two centimeters into the lobe for sectioning. Then, make one or two more blocks, preferably from the same lobe, to serve as back-ups. The remaining liver can be stored frozen.
Fix the tissue blocks in 10%forumlulin with at least ten times more fixative than tissue. After 24 or more hours of fixation, trim the liver samples and place them into cassettes. Then load the basket for the automated tissue processor.
Fully submerge the basket into the first processing station filled with 10%buffered formulin. Then, continue with one-hour baths in an ethanol series followed by xylene, followed by paraffin. Next, warm up the embedding station for at least an hour.
Once warm, transfer liver samples into the wax bath. After two to three minutes, use warmed forceps to transfer the cassette to a 65 degree Celsius hotplate. Then half-fill the mold with warm, liquid paraffin.
Next, transfer the tissue out of the cassette and into the paraffin with the cut surface flat on the mold. Then, finish filling the mold with warmed paraffin. Mount the empty cassette on top of the paraffin to serve as a label and support.
Then, place the mold on a four-deegree Celsius block to cool for 30 minutes. Once cooled, pop out the paraffin block. If the block cracks, melt it and repeat the embedding.
Now, section the block on a microtome. Make ten micron thick sections until the tissue is visible. And then make five micron thick sections.
Next, carefully transfer the sections to a 40-degree Celsius water bath, holding them at their edges. Let the sections float and lift them off the water onto clean glass slides. Now, place all the slides on a slide warmer at 37 degree Celsius and let them dry overnight.
To begin, remove the paraffin and re-hydrate the tissues with xylene and decreasing concentrations of ethanol. Then, immerse the slides in iron hematoxylin for 10 minutes to stain the nuclei. After 10 minutes, briefly rinse the slides with water.
Then, immerse the slides in Biebrich scarlet acid fuchsin for two minutes to stain the cytoplasm. Follow with a water rinse as before. Next, immerse the slides in freshly-made PTA for 10 minutes to promote the uptake of aniline blue.
Logically, next, immerse the slides in aniline blue for 10 minutes to stain the collagen fibers. Now, rinse off the excess stain with water and transfer the slides to a 1%acetic acid bath for one minute. To render the color of the slide more delicate and transparent.
Over four weeks of DMN treatment, male Wister rats lost weight and became less vigorous. Weight loss was first detectable after two weeks. Gradually, damage to the liver weakened the rats.
The liver to body weight ratio was significantly lower in the DMN-treated animals. Grossly, the treated livers were smaller and harder when compared to controls. As expected, serum indicators of a hepatocyte damage, ALT and AST, were at elevated levels in the treated group.
Histological examination was made using a standardized set of criteria. The DMN treatment led to progressive increase and expansion of fibrous septa with loss of hepatocytes. Collagen deposits are stained blue.
There was fibrous expansion of portal areas with portal-to-portal bridging and portal-to-central bridging. By four weeks of treatment, there was cirrhosis with nodule formation. With the need for repeated injections, the correct dressing and technique of injections is important to a wide interaction of contamination and damage to internal organs.
Don't forget that DMN is toxic, and all the injections should be done in the fume hood. Cutting good, five micromedial, thin sections on the microtome requires practice and patience, during the staining process. Ensure that the sections are kept moist at all times and transfer it from one staining solution to the next according to the specified timing.
In summary, rats produced in this model, show progressive liver disease and pericardial fibrosis which can be monitored by biochemical states. We have studied various aspects of liver disease progression and tested potential anti-fibrotic agents using this model.
