The overall goal of this procedure is to quantify permeability in single micro vessels of isolated rat lungs using a fluorescence based method. This is accomplished by first isolating rat lungs and perfusing them with autologous blood. The second step of the procedure is to insert a left atrial microcatheter into a small region of the lung to infuse flora four tagged dextrin.
Then while the tagged dextrin is infused, images of the lung micro vessels are collected. The final step is to quantify the permeability of single microvessels from the images using software. Ultimately, results can show the degree of lung micro vessels permeability under different conditions, such as after LPS treatment.
Demonstrating this procedure will be Dr.Kava Kasami, a postdoctoral fellow from my laboratory Begin by performing a tracheotomy on a male rat in a confirmed surgical plane of anesthesia. Use a PE 90 tracheal cannula and secure it with sutures. Next, infuse the heart with 100 to 200 units of heparin using a 21 gauge butterfly needle.
Wait one minute and then exsanguinate the blood. Collect the blood and set it aside with saline. Fill two cannulas made of three millimeter tigon tubing four centimeters long and flared on one end.
Now perform a thoracotomy. Make an incision just large enough for the cannula into the right ventricle and slide in the flared den of one cannula toward the pulmonary artery. Secure the cannula with sutures.
Use the same technique to insert the other cannula into the apex of the left ventricle toward the left atrium. Secure it with two millimeter wide umbilical tape. Now remove the impeding connective tissues and remove the long and heart together with the cannulas.
Transfer them to a Petri dish with the diaphragmatic surface of the lung upwards and the three cannulas all aligned. Move the dissected preparation to an adjustable stage outfitted with number 18 tigon tubing attached to pressure transducers that serve as inlet and outlet tubes. Now mix the collection of blood with an equal volume of 5%albumin solution.
Add this mixture to the loading reservoir. Start the pump at 14 milliliters per minute. Make sure that the tubing has no air bubbles.
At the preparation. Attach the pulmonary artery to the lung inlet tube and the left atrial cannula to the outlet tube. Double check that there is no air in the tubing, and also check that the shunt is open.
Connect the tracheal cannula to a third pressure transducer. Inflate the lungs via the tracheal cannula with 30%oxygen. Maintain the lungs at five centimeters of water pressure.
Now clamp the shunt and start the pump at 14 milliliters per minute. To begin the lung perfusion during the perfusion, adjust the pump to maintain the pulmonary pressure at about 10 centimeters of water and maintain the left atrial pressure at three centimeters of water. Begin with preparing the blood infused lung.
First, build an infusion catheter from a 40 centimeter length of PE 10 tubing inserted into a 30 centimeter length of PE 90 tubing. Attach the other end of the PE 10 tubing to a 30 gauge needle attached to a one cc syringe. Bring the end of the tubing attached to the lung above the preparation and attach the infusion catheter to it.
Guide the infusion catheter to the left atrium and then into the lung until it meets resistance. Excessive force will injure the lung. Proper incision of microcatheter is critical as any damage to the lung by the microcatheter will reduce the success of the protocol.
Next, fill the syringe with ringers and attach it to a pump. Slowly infuse the solution at a rate of 10 microliters per minute. Gradually the infusion site will become pale compared to the rest of the lung to keep the lung surface moist rapid in plastic with the exception of the infusion site.
Now to visualize the lung, create a viewing window by attaching an O-ring to a 22 millimeter cover slip. Using stopcock grease, secure the O-ring to a test tube holder. Then position the window over the infusion site.
Complete the setup by swinging a 20 x objective over the cover slip and focusing on the lung. No visual distortion is expected. Now infuse the lung with a solution of choice.
In this example, following the saline, one milliliter of ZI conjugated dextrin is infused into the lung via the syringe pump over the course of 60 minutes. Using the imaging software record one image per minute over the one hour infusion and continue recording during the subsequent washout Step for 10 more minutes After the Fitz dextrin is infused, wash it out by infusing the lung ringer solution for at least another 10 minutes at the same flow rate.Later. When analyzing the acquired images score the maximum fluorescence intensity in the region of interest during the fit e infusion.
Then score the maximum fluorescence intensity of the residual fluorescence after the 10 minute washout. Changes in vascular permeability were investigated using fitzy dextrin infusion following treatment with bacterial lipopolysaccharide as this is a widely used model of a LI.Residual fit e fluorescence was low in microvessels treated with ringers, but high in those treated with LPS. Moreover, in comparison to ringer's infusion, LPS infusion caused a significant reduction in the permeability index suggesting a global increase in permeability in all vessels within the image field.
Moreover, in comparison to ringer's infusion, LPS infusion caused a significant reduction in the permeability index suggesting a global increase in permeability in all vessels within the image field. After watching this video, it should have a good understanding of how to prepare isolated bird prophy lungs, insert a left tail microcatheter and define permeability in micro.
