Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

The overall goal of this procedure is to analyze hypoxic pulmonary vasoconstriction of mirroring intra pulmonary arteries with inner diameters of 20 to 100 microns. This is accomplished by first filling the airways of the lungs with low melting point aros. The second step is to use a vibrator to cut the isolated lungs into 200 micron thick slices.

Next, the aros is removed from the lung sections by incubation in 37 degrees Celsius, warm medium, which is bubbled with normoxic gas. So the sections move slowly in the fluid. Then an individual lung section is transferred into a flow through superfusion chamber, followed by video morphometric analysis of vaso reactivity of cross sectioned artery.

Ultimately, changes in the luminal area of the vessel in response to different treatments are evaluated. The main advantage of this technique over existing methods like intra vital microscopy of subpleural micro vessels, are the measurement of vascular tension in isolated proximal pulmonary arteries is that it allows the quantitative analysis of VA reactivity of intra pulmonary arteries with diameters between 20 and 100 micrometers. Demonstrating the procedure will be Anna Goldenberg and pet two technicians of this lab.

Immediately after killing a mouse by cervical dislocation, open the abdominal cavity, move the intestinal loops aside and sever the large abdominal vessels for bleeding. Next, use fine scissors to penetrate the diaphragm. This causes the lungs to collapse as a result of air entry into the pleural cavity.

Continue cutting to detach the diaphragm from the inferior thoracic aperture. Now cut the ribs and the clavicle laterally. To remove the ventral part of the rib cage, take out the thymus.

Fill a syringe with warm perfusion buffer. Next, cut a small hole into the left ventricle of the heart. Adjust the outflow of the perfusion buffer and perfuse the pulmonary vasculature slowly via the right ventricle until the lungs appear white.

Next, remove the salivary glands, small muscles, and connective tissue from the trachea. Disconnect the trachea from the surrounding connective tissue and insert a piece of sewing cotton between the esophagus and the trachea for later ligation. Then use a micro scissor to cut a small hole into the upper part of the trachea between two neighboring tracheal cartilages.

After filling a syringe with 37 degrees Celsius, warm, low melting point agros, connect it to a flexible plastic cannula and push in the plunger slowly until the air is removed from the cannula. Insert the cannula into the small hole in the trachea and carefully anchor it in place with sewing cotton. Next, slowly fill the airways with the aros and observe the lungs.

At first, the right lung starts to expand, followed by the left lung. After 30 to 40 seconds, the filling is completed and both lungs are inflated to a volume comparable to the in vivo situation, which is about 1.2 to 2.0 milliliters. Depending on gender, age, and weight, It is important to inject the egg rose fast enough so that it does not so solidified during the procedure, but not so fast as to damage the lungs.

After the lungs are filled simultaneously, pull out the plastic cannula and ligate the trachea with the sewing cotton to prevent leakage of the aros. Subsequently cut the trachea above the ligature and to detach the lungs and heart on block from the chest, transfer the organs into ice cold heaps ringer buffer to solidify the agros, which happens within a few minutes while waiting. Use super glue to attach a piece of champagne cork to the specimen holder of the vibrator to act as a skew back for the lung tissue After the agros has solidified, separate the individual lung lobes.

Next super glue, one lobe to the specimen holder. To get cross sections of the small intra asner arteries, take the cranial lobe of the right lung and super glue it with the hilum surface to the holder, using a vibrato equipped with a fresh razor blade sliced from the periphery, cutting the lung lobe into 200 micron thick slices. To get cross sections of the larger pre aser arteries, align the hilum of the left lung with the champagne cork and glue the lobe in this orientation to the holder, then sliced from the periphery for removal of the aros.

Transfer the organ sections into a glass beaker filled with about 200 milliliters of 37 degrees Celsius. MEM put the beaker into a heating cabinet and bubble with normoxic gas so that the lung sections are slowly moving in the medium. After about two hours, the aros plaques filling the air will have been removed from the lung tissue and the sections will have settled to the bottom of the beaker.

For video morphometric analysis of intra pulmonary arteries, transfer one lung section into the flow through superfusion chamber, which is mounted on a microscope and filled with 1.2 milliliters of normoxic gassed.MEM. Fix the lung section at the bottom of the chamber with nylon strings connected to a platinum ring. Start perfusing the chamber with normoxic gassed medium.

Use a phase contrast microscope with a 10 x objective to scan the lung section for cross sectioned arteries with inner diameters between 20 and 100 microns. Once a reasonable candidate vessel is located, use the 20 x objective to take a picture of a pre asner vessel or as shown here, the 40 x objective to take a picture of an intra asner artery for measurement of the inner diameter of the vessel. Prior to beginning the experiment, set up the software to take pictures of the cross sectioned artery every minute during the entire experiment.

After triggering the photography, continue the perfusion of the chamber with normoxic gast medium for 10 minutes. Then bathe the lung section in U 466 19 for 10 minutes without flow to analyze the contractility of the artery using only vessels whose luminal areas were reduced by at least 30%With the U 466 19. Wash out the drug with normoxic gast medium for 10 minutes.

Then dilate the artery by application of res for 10 minutes with no flow low. Again, remove the drug with a 10 minute wash with normoxic gassed medium at a flow rate of six milliliters per minute, followed by 10 minutes at a flow of 0.7 milliliters per minute. Next, incubate the lung slice with high hypoxic gassed medium for 40 minutes.

Also flow the hypoxic gas mixture into the airspace of the perfusion chamber via an additional set of tubing. Remove the hypoxic medium with a 20 minute wash with normoxic gassed medium and switch from hypoxic to normoxic gas flow into the airspace of the chamber to check the viability of the vessel. At the end of the experiment, apply U 466 19 into the perfusion chamber and incubate for 20 minutes without flow to induce vasoconstriction.

Repeat these steps with a fresh lung section to measure vaso reactivity of another vessel. Begin the analysis by loading the images of the cross sectioned arteries to an image analysis program. Then use a cursor to outline the luminal area of the vessel.

The hand drying is necessary as it avoids artifacts due to, for example, blood cells attached to the vascular wall. Continue to analyze the cross-sectional images in a similar fashion. Every image should be analyzed when one condition in the flow through incubation chamber is changed to another, but analyzing every other image is sufficient when the conditions are not changing.

Hypoxic pulmonary vasoconstriction is observable in these phase contrast images of a cross sectioned pre ASIN artery, which runs in close proximity to a cross sectioned bronchus. The pictures are taken at time points indicated by circles in the graph at the beginning of the measurement at the end of the measurement with U 466 19 at the end of exposure to RAs after 30 or 40 minutes in hypoxic gassed medium after washing with normoxic gassed medium, and after the final application of U 466 19 vaso reactivity is recorded as relative changes of the luminal area of the cross sectioned artery against time. The area at the beginning of the experiment is defined as 100%and vasoconstriction is given as values relative to the initial area.

In this case, exposure to hypoxia induces a 60%reduction of the luminal area for a clearer presentation of the hypoxic response. The initial phase of the experiment in which vasal reactivity was tested is not shown here, and the value obtained immediately before exposure to reduced oxygen is set at 100%The other type of vessels examined in these studies are the intra ASIN arteries, which are located at gussets of alveolar septa. In this set of experiments, the impact of sidal, a non-selective opener of mitochondrial A TP sensitive potassium channels on hypoxic pulmonary vasoconstriction of small intra asner arteries is analyzed for this purpose.

Slices are incubated in hypoxic medium. In the absence or presence of 50 micromolar sidal control incubations are performed in normoxic gast medium for a clearer presentation of the response to normoxia or hypoxia or hypoxia plus sidal. The values obtained immediately before exposure to normoxic or hypoxic gast medium are set as 100%The artery exposed to pineal does not display a decrease in area, as does the artery incubated in hypoxic medium without additives.

In normoxic medium, no changes in the luminal area are detectable. The recordings of small intra ASIN arteries exposed to hypoxic gast medium with or without 50 micromolar sidal are presented as means plus or minus SEM. In this graph, the relative data in relation to the value at the beginning of the hypoxic incubation are shown.

No vaso reactivity is detectable. In the lung slices exposed to hypoxic gast, medium containing pineal vasoconstriction induced by U 466 19 is not affected by the drug. After watching this video, you should have a good understanding of how to prepare mirror precision, cut lung slices, and how to use them for the quantitative measurement of VA reactivity of small intra asina arteries with diameters between 20 and 40 micrometers, which are located at Gass of alveolar septa, and of larger pre Athena arteries with diameters between 40 and 100 micrometers, which run adjacent to Bronchi and Bronchioli.