The overall goal of the following experiment is to measure the width of the pulmonary microvascular endothelial surface layer or ESL by measuring exclusion of fluorescently labeled high weight dextrins from the vessel surface. This is achieved by first performing intra vital DIC microscopy to identify the endothelial cell membranes of subpleural micro vessels within a moving mouse lung. Next, the mice are injected with a high molecular weight fluorescently labeled dextrin, which is excluded from the vessel wall by the otherwise invisible endothelial surface layer.
Then simultaneous DIC and fluorescent images are captured from a single in-focus frame, allowing measurement of vascular widths that are respectively inclusive and exclusive of ESL thickness. The difference between the DIC and fluorescent vascular widths is used to deduce the thickness of the subpleural microvascular ESL. The main advantage of this technique over existing methods like microparticle velo symmetry is that it can be performed accurately in a moving organ, such as the lung demonstrating this procedure will be Dr.IMU Yang, a senior research associate from my laboratory To prepare the tube for a mouse thoracostomy.
Cut a 10 centimeter length piece of PE 50 tubing and attach one end to the blunt end of a curved 23 gauge needle. Using a 30 gauge needle repeatedly puncture the distal end of the tubing, creating side ports to facilitate effective aspiration of the intrathoracic air with a four oh silk suture. Separate the fenestrated portion from the rest of the tube by making several circumferential loops that will serve as a stopper and will anchor the fenestrated portion within the chest cavity.
Prepare a chest wall window by cutting transparent poly vanil membrane into an oval and use alpha cyanoacrylate glue to affix a circular five millimeter. Number one cover slip to it After anesthetizing the mouse shaving and disinfecting the surgical area and securing it to a cutting board. Under a dissecting scope, make a one centimeter incision over the throat.
Dissect the underlying connective tissue and laterally separate and reflect the salivary glands. Advance a four oh suture loop under the trachea. Then cut it to create two separate strands of suture.
Using two fingers, grasp the upper suture and apply gentle tension to the trachea. Then make a horizontal incision that crosses about two thirds of the tracheal circumference between the upper and lower sutures. Insert a flan tracheostomy tube into the distal trachea.
Use the codal tracheal suture to secure the tube in place. Identify the jugular vein by tracking its venous branches proximally. The external jugular is found underneath the reflected salivary glands.
This can be traced proximally. To find the external internal jugular junction, use gentle blunt dissection to separate the jugular junction from the surrounding connective tissue. Next, with four oh sutures, tie off the external jugular and internal jugular veins cranial to the jugular junction.
Then make a small incision into the carina of the jugular junction and incrementally advance two catheters through it and into the jugular trunk. After gentle aspiration to ensure blood return, use four oh sutures to secure the catheters within the vein. Once the mouse is transferred to the previously prepared microscopy stage, extend the midline incision from the neck to the xiphoid process.
Then proceed laterally to the right side. Using electrocautery, remove the chest musculature to expose the thoracic cage. Taking care to ensure complete hemostasis.
Cross the mouse's right hind leg over the left side and tape it down. The resulting abdominal torsion rotates the thorax slightly improving ease of surgery. Place the stage at a 45 degree angle to allow the lung to fall away from the chest wall once the pneumothorax is induced.
Next, using forceps, grasp the first rib and bluntly. Push a curved forceps underneath the rib to separate the parietal pleura from the chest wall. Then to rupture the pleural surface and induce a pneumothorax without damaging the underlying lung.
Use the blow tube and a syringe to forcibly introduce air against the parietal pleura. With electrocautery forceps, dissect the chest wall musculature and cut across the fifth and sixth ribs and parietal pleura making an about eight millimeter circular hole in the chest wall. It is essential that complete hemostasis be maintained as the presence of bleeding will obscure a microscopy.
Using a needle driver, insert the thoracostomy tube into the chest wall hole. The needle should puncture the chest wall and exit the thoracic cavity inferior and lateral to the thoracic window. Gently pull the tube out of the chest wall until resistance is felt from the suture stopper located at the edge of the fenestrated portion of the tube.
Place the stage flat and add three centimeters of water positive and expiratory pressure to the ventilator. To assist with lung expansion, place glue circumferentially around the chest window. Attach the membrane with the glass cover slip facing exterior to the thoracic cavity.
Using a cotton applicator, carefully approximate the membrane to the glue while performing a lung recruitment maneuver apply negative three millimeters mercury suction through the chest tube. The lung should persistently approximate the membrane while freely moving during tidal ventilation to resuscitate, as well as provide a tracer for measurement of the endothelial surface layer or ESL immediately after closing. The chest wall administer 500 microliters of fite labeled 150 kilodalton dextrin via a jugular venous catheter.
To visualize small differences in ESL thickness, choose an objective with a numerical aperture of greater than 0.8 that maintains a two to three millimeter working distance. After placing a drop of water on the cover slip center, the water immersion objective over it. To accurately measure ESL thickness in a moving organ, use an image splitter to simultaneously capture brightfield DIC and fit E images.
From an in-focus frame, identify subpleural microvessels measure DIC, and fit e dextrin vascular widths by averaging the lengths of three perpendicular intercepts per micro vessels, assuming equal ESL thickness at both edges of the vessel. The ESL size can be defined by one half the difference between DIC and Fitz dextrin vascular widths. See the text protocol for alternative measurement techniques and for the euthanization procedure shown here are simultaneously captured DIC and fluorescent images of the mouse subpleural micro vasculature micro vessels.
Width is measured using the average of three perpendicular linear intercepts. ESL thickness can be determined by one half. The difference between DIC micro vessels widths, which are inclusive of the ESL and fluorescent micro vessels.
Measurements, which are exclusive of the E-S-L-D-I-C measurements accurately identify subpleural vessel wall borders as demonstrated by nearly identical DIC and GFP vessel width measurements performed in endothelial fluorescent tie two GFP mice. The solid line represents the line of identity as demonstrated in this graph. The subpleural microvasculature can be followed longitudinally as evidenced by the progressive loss of ESL thickness occurring after intravenous lipopolysaccharide.
Glycocalyx integrity can be alternatively determined by assessing for anti ICAM one microsphere adherence within the subpleural vasculature as seen here. High speed confocal microscopy captures adherent fluorescent microspheres 45 minutes after intravenous LPS note that circulating microspheres can be seen passing through the microcirculation to improve visualization of microsphere localization, mice were pretreated with the vascular tracer Tri dextrin in lieu of Zi dextrin. When attempting this procedure, it's important to remember that DIC and fluorescent images must be captured simultaneously.
Even a slight delay between the two images will prevent accurate ESL measurement in a moving organ.
