The overall goal of this procedure is to provide optimized method to generate and characterize Hepatic Steatosis using In Vivo and In Vitro approaches. This method can help answer key questions in the Mechanistic Study of Hepatic de-replication of lipid and glucose metabolism during nutrient overloading and obesity. One advantage to this techniques is that the physiological progress of Hepatic Steatosis can be assessed noninvasively prior to histological and biochemical analysis.
This method can provide insight into Fatty Liver Disease. It can also be applied to other metabolic diseases such as Insulin Resistance, Type II Diabetes, and obesity. Demonstrating the procedure will be Zhimin, Yamei, and Aoyuan.
They are all graduate students who work on transcriptional regulation of lipid and glucose metabolism from my laboratory. After anesthetizing the donor mouse, take a blood sample by cardiac puncture and collect the blood in an EDTA line tube. To remove the liver, cut the diaphragm along the liver's length and carefully remove it from the entero colia.
From the liver, prepare two tissue blocks for H and E staining and Oil Red O staining. Keep the remaining tissue to measure lipid content for which at least 20 milligrams is needed. After making a 0.5%stock solution from Oil Red O from powder, dilute it to a 0.3%solution in water and filter the dilution through a 0.22 micron filter.
Then, let the solution settle for ten minutes at room temperature. Next, embed the liver in OCT compound in a plastic embedding box. Then, freeze it at negative 20 degrees Celsius for 30 minutes.
Once frozen, make eight micron slices with a cryostat. Mount the slices to slides and incubate the slides at room temperature for 30 minutes. Now stain the slices.
Inside a staining tub, moisten a paper towel with 10%formalin. Keep the slides in the tub for five minutes of vapor fixation at four degrees Celsius. Next, dip the slides into 60%isopropanol for three seconds, do this twice.
Now, add a few drops of Oil Red O working solution to each slide. Cover all of the tissue. Then, incubate the slide at room temperature for 15 minutes.
Keep the slides shielded from light as much as possible from this point forward. After the incubation, briefly rinse the slides in fresh 60%isopropanol two times. Then, gently rinse the slides twice with distilled water.
After the rinses, remove as much water as possible from the slides without drying out the tissue. Then, immerse the slides in a bath of hematoxylin stain for one minute. To removed the hematoxylin, rinse the slides with running tap water for five to ten minutes.
Then, gently rinse the slides with distilled water two times and store them in a fresh bath of distilled water until they are mounted. To mount the slides, melt glycerol jelly in a microwave. Then, place several drops of liquified jelly onto the tissue slices.
Before the jelly cools, carefully apply cover slips without trapping air bubbles. Begin with isolated primary hepatocytes growing on collagen coated glass cover slips in a six-well plate. For this model, first change the medium to two milliliters of starvation medium and incubate the cells for 24 hours.
After 24 hours of starvation, adjust the medium to contain 30 millimolar glucose and 100 nanomolar insulin and let the cells incubate for another 24 hours. The next day, remove the medium and wash the cells using two milliliters of PBS. Perform two washes.
To analyze the cells, fix each cover slip to a slide with a rubber band with the cells on the outside surface. Then, use the described Oil Red O staining method. This procedure can be applied to cells other than hepatocytes.
To begin, load 20 to 40 milligrams of liver tissue into a two milliliter plastic centrifuge tube. Then, add one milliliter of PBS and homogenize the tissue. Once homogenized, transfer the lysate to a new 15 milliliter glass tube.
Next, add five milliliters of two parts chloroform to one part methanol and vortex the mixture vigorously for one minute. Then, let the mixture incubate for two hours buried in ice. After the incubation, separate the solutions by spinning the tube at 1, 650 G for ten minutes at four degrees Celsius.
The bottom face contains chloroform and lipids. Collect and transfer the bulk of it to a new glass tube kept on ice. Then, continue the extraction.
To the remaining phases, add 600 microliters of four millimolar magnesium chloride and 1.5 milliliters of chloroform. Vortex this mixture vigorously and incubate the emulsion on ice. After half an hour on ice, repeat the centrifugation step and collect the bulk of the bottom phase and then combine this with the first bottom phase collection.
Discard the remaining layers. To purify the lipids, evaporate the chloroform under nitrogen gas in a 37 degrees Celsius bath. Then, thoroughly rinse down the sides of the tube using 200 microliters of 1%Triton X-100 and chloroform and repeat the chloroform evaporation step.
Now, dissolve the lipids in 200 microliters of double distilled water and vortex the lipids into an emulsion. Then, using commercial kits, measure the total triglyceride and cholesterol levels in the sample. By feeding mice a high fat high sucrose diet, their weight increased by 50%after 16 weeks.
The diet resulted in an obvious gross morphological change in the livers. H and E and Oil Red O staining revealed that over the 16 week feeding regimen, lipid droplets grew larger and occupied regions usually occupied by hepatocytes. Analysis of the fat composition, performed using the described extraction method, showed that the excess nutrient overload caused abnormal cholesterol and triglyceride accumulation in the liver.
In a different experiment, primary hepatocytes were cultured and subjected to 24 hours of starvation followed by glucose and insulin treatment. After the treatment, Oil Red O staining showed accumulation of lipid droplets in the cells. Quantitative lipid analysis showed that triglyceride and cholesterol levels were high in the treated cells.
After watching this video, you should have a good understanding of how to generate In Vivo and In Vitro Hepatic Steatosis models. Two studies in mice presents under nized Fatty Liver Disease, insulin resistance, and Type II Diabetes.
