Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

The overall goal of the following procedure is to use fibrin gel implantation to study vascular and alveolar regeneration in the mouse lung. This is accomplished by first preparing drops of fibrin gel, supplemented with angiogenic factors. In the second step, the experimental animal is mechanically ventilated and the chest is opened.

Next, a fibrin gel is carefully attached to the mouse lung with fibrin glue. And then in the final step, the lung and implanted gel are harvested seven to 30 days later for histological analysis. Ultimately, the blood vessel and alveolar formation within the gel implant can be assessed by immunohistochemical analysis.

This method can help answer key questions in the vascular biology field, such as how that the organs risk microenvironment, control angiogenesis, and organ morphogenesis demonstrating the procedure will bema model a colleague from our laboratory To prepare fibrin gels that contain both VEGF and BFGF begin by warming minus 80 degrees Celsius, stocks of fibrinogen and thrombin to room temperature. When the stocks are fully thawed, add thrombin calcium chloride VEGF and BFGF to the fibrinogen solution in a 1.5 milliliter tube. And then mix the solution by pipetting.

Next gently pipette 200 microliters of the mixture, drop by drop onto a sterile plastic dish. Incubate the drops at 37 degrees Celsius for 30 to 60 minutes until they solidify. Then use small surgical scissors to trim the gels into approximately three by three by three millimeter cubes.

After confirming full sedation in an eight to 12 week old mouse by toe pinch, cover the animal's eyes with vet ointment to prevent dryness, and then shave the fur over the left side of the rib cage. For endotracheal intubation, place the mouse on an intubation stand angled at 70 degrees and hook the upper incisors over a small rubber band located at the top of the stand to hold the animal in place. Use blunt forceps to gently retract the tongue to one side, and then visualize the larynx with the aid of a fiber optic gooseneck microscope illuminator.

Next, insert a 21 gauge endotracheal elastic catheter into the trachea. Confirm that the mouse is spontaneously breathing smoothly, and then place the animal in the prone position under the dissection microscope. Use a rodent ventilator to mechanically ventilate the animal at 150 breaths per minute and seven milliliters per kilogram tidal volume.

And then count ribs to locate the intercostal space between the fourth and fifth rib. Then thoroughly wipe down the area with alcohol and povidone iodine to create a sterile field and cover the surgical area with a sterile tra to place the gel onto the lung. Next, make a one centimeter transverse skin incision over the intercostal space.

And then make a muscle incision between the fourth and fifth rib. Insert a dissecting retractor between the ribs to fully visualize the left lung, and then use fine forceps to scrape a one by one millimeter square of visceral pleura from the center of the left lung using a sterile cotton swab. Next, apply gentle pressure on the area until the bleeding and air leaks are completely controlled, and then dispense a small amount of uns solidified fibrinogen thrombin glue over the area.

Next gently place one fibrin gel onto the glue and observe the respiratory movements of the lung to determine that the gel is well fixed. Then close the incisions with an absorbable suture. Aspirate the thoracic cavity with a one milliliter syringe equipped with a 27 gauge needle to prevent pneumothorax, and then terminate the mechanical ventilation.

After confirming that the mouse is breathing smoothly, inject one milliliter of pre-war 0.9%sodium chloride intraperitoneal to prevent dehydration, and allow the mouse to recover on a circulating warm water pad. Once the animal exhibits stable breathing, remove the endotracheal tube and inject a postoperative analgesic. When the animal is fully recovered, return it to a new cage isolated from mice without surgery, monitoring the animal daily for signs of infection or complications.

Seven to 30 days after the implantation, euthanize the mouse per protocol and make an incision between the tip of the xiphoid process and the sternal notch. And then cut the trachea dissecting all connections from the lung to the heart and trachea, harvest the whole lung and the implanted gel, and then fix the gel and lung tissue within a 4%paraform aldehyde solution overnight at four degrees Celsius the next day, embed the lung tissue in OCT compound and then collect serial step sections of the lung and gel at a 30 micrometer thickness. Stain the sections with hematin and eoin and antibodies against the appropriate markers of interest.

And then after taking Zack images of the sections, compile stacks of the optical sections to form three dimensional images of the lung endothelial and epithelial cells. Using 3D image analysis software. Finally quantify the projected areas of the newly formed blood vessels using the appropriate image analysis software.

Seven days after implantation, the fibrin gel is incorporated into the host lung 3D reconstruction of confocal fluorescence images has shown that host derived CD 31 positive endothelial cells form vascular networks inside the gels seven days after implantation in A-V-E-G-F-B-F-G-F dose dependent way, type one aquaporin five positive and type two surfactant protein B positive lung epithelial cells are also recruited along the newly formed blood vessels inside the gels that are supplemented with higher concentrations of VGF and B, FGFH, and d. Staining of histological sections reveals that other types of host cells also migrate into the gel seven days after implantation. These findings together suggest that host lung derived regenerative vascular networks and alveolar formation are successfully constructed inside the fibrin gels after supplementation with angiogenic factors and implantation onto the surface of the adult mouse lung After its development.

This technique paved the way for less researchers in the field of lung biology and primary medicine to explore the mechanism of normal angiogenesis and how it is deed in the disease state in mouse lungs.