The overall goal of this procedure is to demonstrate the identification and excision of adipose depose for the analysis of different factorial effects on and by adipose. This is accomplished by first exposing the internal organs and adipose depots of an adult mouse. In the second step, the organs and superfluous tissue are removed to better expose the depots, allowing the identification of the depots in their corresponding locations.
In the final step, the adipose tissue is then extracted with special care to avoid contamination. Ultimately, the isolation and excision of these deposes provide tissue for histological analysis, protein expression, enzyme activity, and gene expression analysis, as well as for in vitro studies. The main advantage of this particular protocol over existing methodologies is that it allows for multiple depot isolation with minimal dissection, as well as minimal contamination.
So this procedure can help answer questions about the field of obesity. For instance, the detrimental or even the beneficial effects of adipose tissue on human disease. To isolate brown adipose tissue, begin by placing an ethanol, cleansed, euthanized adult mouse in the supine position with its back against the table.
Then use forceps to lift the skin just below the diaphragm and incise the skin with scissors. Cut transversely around the circumference of the mouse to expose the peritoneum. Then holding the lower appendages and abdomen in one hand, pull the skin up towards the head with the other hand to deglove the top half of the mouse revealing the butterfly shaped brown adipose depot or bat.
Next, locate the scape and the corresponding bat depot. Then use a dissection microscope to carefully remove any superficial white adipose atop the butterfly and dissect the interscapular brown fat, taking care to avoid any associated muscle. To isolate the subcutaneous or subq adipose tissue, hold the upper appendages and thorax in one hand, and pull the skin down towards the feet with the other hand to deglove the bottom half of the mouse and reveal the triangular inguinal subq deposits.
Then place the mouse back in the supine position and carefully dissect the triangles of subq fat to isolate the gonadal fat from the abdominal cavity. Use scissors to cut the peritoneum transversely directly below the diaphragm. Then cut the peritoneum from the diaphragm to the rectum, mid coronally to expose the abdominal organs and carefully dissect both epidermal fat depots from the testes epi and vasa deia.
To isolate the perivascular adipose tissue, maintain the animal in the supine position, and extend the upper and lower appendages outward. Next, lift up on the xiphoid process with forceps to create tension across the top of the body and cut horizontally through the diaphragm, exposing the lower portion of the thoracic cavity. Cut through the ribcage superiorly towards the head just to the side of the sternum, and then pick up the ribcage just inferior to the clavicle.
Cutting along the inferior length of the clavicle toward the axilla in both directions. The thoracic cavity and its contents should now be clearly visible. Once the area is cleared of fluid, cut the bronchi and attaching vessels to remove the lungs and expose the heart.
After identifying the stomach and the esophagus, cut the esophagus at the gastroesophageal junction to free the stomach. Next, identify the intestines and the surrounding mesentery. Cut superficially through the mesentery and then cut the colon as close to the rectum as possible to free the stomach, intestine, colon, pancreas, and spleen.
When the organs have been removed, cut through the hepatic veins and connecting mesentery and remove all the lobes of the liver. Cut away the visceral layer and fat surrounding the kidneys, leaving the kidneys attached to the aorta in vivo to serve as geographic markers for the different segments of the aorta. Then when the area around the kidneys is clear, use micro scissors and micro forceps to separate the aorta from its dorsal attachment to the spine, and its ventral attachment to the esophagus.
In the thorax, follow and detach the aorta from its origin in the heart to the bifurcation in the iliac region. To isolate the aortic adipose tissue, at this point, the subclavian vessels can be identified. Isolate these vessels from the neck to the aortic root to better expose the aortic junction and root in the heart.
Then remove the thymus and cut the brachiocephalic, the left common carotid and the left subclavian arteries to allow movement of the heart. Finally, use a dissecting microscope to view the perivascular adipose tissue or P VVA T layer surrounding the aorta. Then use micro forceps to gently pull the tissue away from the aorta and micro scissors to gently cut the attachment of the PVAT to the aorta starting at the thoracic region, just superior to where the diaphragm is located.
Then use micro forceps to gently pull the tissue away from the aorta and micro scissors to gently cut the attachment of the PVAT to the aorta starting at the thoracic region just superior to where the diaphragm is located. Repeat this process the entire length of the aorta finishing at the infrarenal aortic region, located just superior to the iliac bifurcation of the aortic vessel and inferior to the renal branches bat. And wat samples can be differentiated by histological analysis with h and e staining primary cell lines of subcutaneous adipocytes, and perivascular.
Adipocytes can then be cultured and differentiated from preadipocytes to adipocytes for microarray analysis. In these representative images, for example, the conversion of cultured preadipocytes to adipocytes was confirmed with oil red O staining adipocytes, isolated, cultured, and differentiated by this method can be used for further in vitro analyses. In this representative experiment, the enzymatic activity of MM P two was observed to decrease in isolated perivascular adipocytes in response to an experimental treatment compared to untreated controls.
While attempting this procedure, it's important to remember to change your gloves as well as clean your instruments frequently to minimize contamination. So after these tissues are collected, a variety of procedures can be performed on them. For instance, cell culture, histologic analysis, protein measurement, and even gene expression.
And then those results can be used to help understand the relationship and similarities and differences between the different depots.
