The aim of the following experiment is to identify the spatial and temporal cellular dynamics involved in angiogenesis, defined as the growth of new blood vessels from existing ones. Using a simple reproducible model, first stimulate angiogenesis by briefly exteriorizing the rat mesentary and harvest mesenteric tissues at different time points post stimulation. Next immuno label tissue sections.
For example, use an endothelial cell marker to visualize the entire remodeling vasculature within a tissue and create an essential two-dimensional visualization of growing microvascular networks. This model is attractive because it produces a dramatic increase in multiple angiogenic metrics over a relatively short time course taken together. Visualizing networks at the single cell level and correlation of these metrics with multicellular labeling has proven extremely useful in identifying novel cell phenotypes involving capillary sprouting.
This method could help answer key questions regarding the cellular dynamics involved in microvascular network growth. These include the when and wear specific cells are present during the capillary sprout process. The main advantages of this method are that it's simple requires minimal surgical intervention and produces a robust angiogenic response over a relatively short time period.
In addition, it takes advantages of the rap mesentery, which is a tissue that allows for observation of an intact network down to the single cell level. In our experience, it's very easy to learn and consequently reproducible from one researcher to another, demonstrating the procedure. Today will be Peter Stapler, a graduate student in our laboratory Prepare for surgery as detailed in the accompanying text.
After shaving the rat abdominal area position the anesthetized rat on its back on a heating pad to maintain body temperature, clean the abdominal area with alternating wipes of sterile GREs soaked in 70%Isop profile, alcohol and iodine approximately one inch below the sternum make a small longitudinal incision along the skin, and then the linear alba place the precut drape over the abdominal section so that the opening aligns with the incision. Gently apply pressure around the incision to reveal the small intestine. With cotton tip applicators, gently pull out a section of the small intestine laying the mesentary on the plastic stage containing sterile saline and locate the ileum.
Identify six to eight vascularized mesenteric windows, the thin translucent membrane in between the artery vein pairs, feeding the small intestine. Record the starting time of exterior and set a timer for 20 minutes. Mark the two centrally located mesenteric windows with sterile seven zero suture in the fat area near the intestine.
Intermittently replenish the saline in the Petri dish using a sterile five milliliter syringe to ensure that the tissue remains immersed and moist. After the exteriorized time period, return the mesentary to the abdominal cavity. Close the abdominal muscle with five zero monofilament suture in an interrupted pattern.
Then close the skin with four zero monofilament suture in an interrupted pattern, wipe the suture area with alternating wipes using the sterile gauze soaked in 70%isop profile, alcohol and iodine to spread, I lubricant gel on both eyes of the rat and return it to its cage for recovery On the day of interest, post stimulation, anesthetize and euthanize the rat as per institutional guidelines. Then reopen the abdominal cavity and cover the abdominal area with a piece of plastic wrap. Now gently pull out the small intestine and locate the two marked windows using forceps and micro scissors.
Cut out each of the exteriorized mesenteric windows, including a border of fat. Try to avoid cutting through artery vein pairs within the fat border of the windows to minimize potential blood filling of the windows. Also minimize cutting through the bowel that results in intestinal content.
Contacting the windows immediately immerse each window in PBS. Then use forceps to spread tissues on positively charged microscope. Slides mount two tissues per slide After the tissues have partially dried, remove the excess fat with a scalpel blade.
Fix samples in methanol for 30 at minus 20 degrees Celsius by placing them in the freezer. Vacuum dry the slides to remove excess buffer solution. Also, outline tissues with a wax pen to prevent uncontrolled flow of antibody solutions away from the tissue.
Now drip approximately 200 microliters of diluted primary antibody solution to cover the whole tissue. Incubate for one hour at room temperature, wash tissues with PBS plus 0.1%sapin with three exchanges of buffer. Next, add the strep adin peroxidase secondary antibody solution and incubate at room temperature for one hour after three washes, incubate samples in vector nova red for 15 minutes.
Rinse with water to mount slides. Dip in 95%ethanol 10 times, followed by successive dehydration steps. Allow slides to dry cover tissues with a thin layer effect amount and position cover slips after vacuum drying and marking.
The slides. Cover the tissue samples in diluted primary antibody solution. Incubate for one hour at room temperature.
Wash three times with PBS plus 0.1%saponin for 10 minutes each wash. Then add the secondary antibody and incubate for one hour. Perform washes in PBS plus 0.1%Saponin mount the slides in PBS with glycerol and seal with nail polish.
Here we detail the modification of a common Petri dish for use as the surgical stage. Yellow modeling clay was used to provide a smooth surface for the mesentery to be pulled through the precut hole. Again, the mesenteric windows shown here in the stage during the externalization period are defined as the thin translucent membranes between the artery vein pairs feeding the intestine here.
PCAM labeling identifies all vessel types along the hierarchy of remodeling microvascular networks as specific time points post externalization. These images can be used to quantify angiogenic metrics as specific time points. Post stimulation.
PCAM labeling allows for the determination of arterials versus vees. Note that the feeding arterials typically exhibit smaller diameters and elongated endothelial cell morphology compared to paired venues. Typical characteristics of remodeling networks include vascularized area, capillary sprouting, and vessel density.
Quantification of these various angiogenic metrics characterizes the time course of network growth. Capillary sprouting from pre-existing vessels peaks between day three and day five and returns to unstimulated level by day 10. This transient increase in sprouting is followed by increases in vascularized area and vascular density in our laboratory.
This model system has been used to identify novel cellular phenotypic changes as specific time points during this remodeling process. The Immunofluorescent PCA labeling stains endothelial cells read while class three beta tubulin labeling stains, nerves, and parasites along angiogenic vessels green. Interestingly, in unstimulated tissues, class three beta tubulin expression is nerve specific.
In contrast, during the peak of capillary sprouting, class three beta tubulin is expressed by perivascular cells. This type of result highlights the use of this simple and robust angiogenic model to identify novel cell types involved in the angiogenic process. By day 10, the class three beta tubulin expression pattern begins to return to the unstimulated scenario.
This model could also potentially be used for cell lineage studies. Feasibility of cell incorporation into remodeling mesenteric tissue is supported by these preliminary studies in which pre-labeled bone marrow cells or mesenchymal stem cells were super fused over mesenteric windows during the 20 minute exterior period. After watching this video, you should have a good understanding of how to use our rat mesentary interiorization model for investigating the cellular dynamics involved in microvascular network growth.
This simple technique can be performed in approximately 45 minutes to one hour. Remember, it is critical to delicately handle the mesentery and avoid puncturing any vessels following a surgical procedure. Immuno labeling can be performed to identify multiple cell types along blood vessels.
You can also label for lymphatic vessels and nerves. This allows us to investigate not only the cellular phenotypic changes involved in angiogenesis, but the coordination between angiogenesis, neurogenesis and lymph angiogenesis.
