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The overall goal of this experiment is to demonstrate the use of the rat mesenteric culture model for the time-lapse investigation of microvascular network re-modeling ex vivo to evaluate parasite endothelial cell interactions, blood lymphatic endothelial cell connections, or anti-angiogenic drug effects. This method can help answer key questions in the field of microvascular remodeling, including how do microvasculars know where to grow, and what is the fate of endothelial cell segments. The main advantage of this technique is that real, intact microvascular networks can be imaged before and after growth ex vivo.

Demonstrating the procedure will be Sadegh Azimi, a graduate student in our laboratory who developed the method. Begin by placing an ethanol-sterilized plexiglass platform on top of a sterile, absorbent under-pad and an anesthetized adult male rat on top of the platform. Disinfect the shaved abdominal region of the rat with two 70%isopropyl alcohol wipes, followed by povidone-iodine.

Starting one inch below the sternum, use a scalpel to make a three-quarter to one and one-quarter inch incision in the gut through the skin, connective tissue, and muscle. Then, place a drape with a precut hole over the animal and place a surgical stage over the drape, taking care to align the openings in both materials with the incision. Using sterile cotton-tipped applicators, pull the ileum through the surgical stage opening along with six to eight mesenteric windows.

Use a sterile syringe to drip 37 degrees Celsius sterile saline onto the exposed tissues as needed. When all of the windows have been externalized, use tweezers to grasp a fat pad and use scissors to harvest the windows, leaving a two millimeter fat border around each mesenteric tissue. Take care that the tweezers do not touch the mesenteric windows by only touching the surrounding fat borders.

Wash each window one time in 37 degree Celsius PBS and one time in 37 degree Celsius medium as it is collected. Returning the ileum to the abdominal cavity after all of the windows have been isolated. In a sterile laminar flow hood, use tweezers to transfer each mesenteric tissue by the fat border onto the polycarbonate filter membrane of a cell culture plate insert.

Use the fat pad to quickly spread the tissue samples, taking care not to touch the windows. Then, invert the insert and place into one well of a six well plate so the tissue lays flat on the bottom of the well. Cover the tissue with three millimeters of medium, and transfer the remaining windows to the plate in the same manner.

Then, culture the mesentery windows under standard tissue culture incubator conditions for up to five days. On the day of imaging, supplement the cultures with conjugated BSI lectin and return the plate to the incubator. After 30 minutes, wash the samples two times with lectin-free medium and transfer the plate to a fluorescent microscope stage.

Identify the blood and lymphatic vessels based on their morphology and network structures. Then, locate a network region of interest on each tissue to obtain images, taking note of the location to ensure that the same region is captured in subsequent images. When all of the images have been obtained, return the samples to the incubator until the next imaging session or the appropriate experimental endpoint.

After three days in culture, the majority of the cells present in the mesentery are viable, with the presence of the endothelial cells confirmed by their location in the microvascular segments. Endothelial cell proliferation can also be confirmed by lectin/BrdU labeling, while the presence of smooth muscle cells and parasites along the vessels can be confirmed with NG2 labeling. Branching lymphatic and blood microvascular network labeling further confirms the maintenance of the lymphatic cell phenotype in the cultured tissue.

Supplementation of the mesentery window tissue culture medium with 10%serum for three days induces a robust angiogenic response with new vessel segments and capillary sprouts identified after five days of stimulation. The time lapse imaging allows quantification of the number of vessels per vascular area and the number of capillary sprouts per vascular area from one four x image per tissue. Time lapse comparisons of network regions also enable the tracking of endothelial cell segments and the identification of blood and lymphatic vessel mis-patterning.

Further, lectin and Cd11b labeling additionally confirms the presence of interstitial resident macrophages in the remodeling networks. Once mastered, this technique should be able to be completed in under four hours. After watching this video, you should have a good understanding of how to harvest, culture, and time-lapse image rat mesenteric tissues.