Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

The overall goal of this procedure is to produce a thoracic window for intra vital microscopy in a pulmonary metastases bearing rat. This is accomplished by first anesthetizing, the metastasis bearing animal, intubating it, and connecting it to a small animal ventilator for pressurized ventilation. Next fluorescently labeled red blood cells are injected into the animal's tail vein.

Then the thoracic cavity is surgically accessed without damaging the lung surface metastasis is identified and a window is inserted. Finally, microscopic imaging of the animal is performed. Ultimately, surgical opening of the lung and insertion of a window for acute microscopy in the live rat allow for automated measurements of micro circulatory blood flow in lung metastases.

The main advantage of this technique over existing methods like micro CT chest imaging, non-invasive epit thoracic microscopy and micro pet of the cancerous lung, is that it permits real time measurements at high temporal and spatial resolution of the microcirculation of pulmonary metastases. This method can help answer key questions in the pulmonary metastasis field, such as how treatments or cancer progression may change blood flow and ultimately hypoxia in lung metastases. The implications of this technique extend toward diagnosis and therapy of pulmonary metastatic cancer because it permits an unprecedented view on the dynamics of the micro circulatory and metabolic microenvironment of these metastatic tumor.

The this method will open the door to understand the dynamics of pulmonary metastases, microcirculation, and thus help to improve current treatment strategies. Generally, individuals new to this method will struggle because its require experience to adjust setting in a way that minimize autonomous movement of the lung. After inducing and confirming metastatic cancer in a rat according to the text protocol and anesthetizing it with an intraperitoneal dose of pentobarbital, perform a toe pinch to confirm sedation.

Shave the animal on the side of the body that has the metastatic disease and the neck area, and wipe off all remaining loose hair from the skin to prevent it from interfering with imaging. Apply veterinary ointment to the eyes to prevent them from drying out. Place the animal in a supine position on a metal plate that's on a 37 degree Celsius water circulated heating pad and use tape to affix the front and hind limbs.

Attach a pulse oximeter for controlling and recording vital signs throughout the procedure to intubate the rat. Begin by making a transversal cervical skin incision. Then separate medially the longitudinal musculature ventral to the trachea with a sharp forceps.

Use a repeated opening to closing action to create a passage for the suture through the dorsal side of the trachea on the ventral side between the second and third trachea ring. Make a small incision into the trachea. Leave a sufficiently long portion of the trachea exposed on the dorsal surface to enable fixation of the catheter.

Next, insert a 2.5 to 3.0 millimeter Y tracheal cannula into the trachea and use a four zero monofilament suture to tighten. To maintain positive pulmonary pressure, connect the cannula to 100%oxygen through a pressure cycled ventilator with a bottle connected to the expiration duct that is filled with six centimeters of water. Then using a 25 to 27 gauge needle and syringe filled with heparinized saline, insert a catheter into one of the tail veins and use tape to fix it in place using red blood cells previously collected and labeled with DII according to the text protocol, inject 300 microliters into the tail vein of the rat to create a pulmonary window.

Create an incision on the cancerous side of the chest and detach the skin using blunt dissection. Dissect the two layers of overlapping musculature and leave the intercostal muscles intact in the region of the sixth and seventh ribs. Create a perforation in the chest cavity approximately 1.5 centimeters in diameter to perform an osteotomy.

Begin adjusting the inspiration pressure on the ventilator so that mechanical interaction between the lung surface and the rib cage is minimized. Remove portions of the sixth and seventh ribs by using a tooth surgical forceps to tightly hold the rib to be cut and with surgical scissors. Cut the medial side of the rib at an approximately 45 degree angle with a sharp side pointing outward.

Cut the lateral side of the bone in a similar manner, again, leaving the pointed side outward. To avoid damage to the lung, repeat the osteotomy with the adjacent rib before cutting the intercostal muscles and remove the excised piece. Create a custom made long window by using glue or a small amount of vacuum grease to attach a cover glass to a plexiglass socket.

Then insert the window so that surface metastases will be located close to the center of the window, enlarging the hole on one side if necessary. After inserting the socket into the perforation and creating direct contact with the visceral pleura of the lung, use four zero monofilament suture to suture the edges of the window frame to the surrounding intercostal muscle. Increase the inspiration pressure on the ventilator to help residual air to escape and to create a seal.

Next, place the animal in a custom made restrainer designed to eliminate movement in the Z direction and position it on a heated steel plate under a fluorescence microscope. Then insert a rectal thermistor to control the animal's body temperature. Adjust the screws of the animal restrainer and inspiration pressure on the ventilator to achieve optimal control of lateral movement.

For imaging and micro circulatory blood flow measurements. Use a CCD camera at a negative 40 degree Celsius chip cooling temperature, and a 10 x objective. With standard rod Domine Trit C filter sets, record the actual frame rate and pixel resolution of the resulting image sequences.

Record at least 200 images per stack to ensure successful analysis of flow velocity during the imaging session to replenish fluid loss in the animal. To maintain consistent blood flow velocity. Perform an intraperitoneal injection of approximately one milliliter of saline every hour.

Euthanize the animal according to the text protocol after imaging is complete. The vasculature in solid tumors is known to differ significantly from normal blood supply, showing greater degrees of tortuosity and higher intervascular distances. Accordingly, as shown here, blood flow tracks in experimental pulmonary breast cancer and metastases have irregular shapes and large intervascular gaps when compared to normal microcirculation.

In this study, similar to what has been demonstrated in healthy lungs dosing with a combination ephedrine and the endothelin receptor antagonist, entin increased blood flow velocity in mouse sarcoma. Every data point represents the pooled individual averages of three measurements with five minute intervals averaged over the data acquired in five animals. The first injection significantly increased blood flow velocity in the tumor area from 0.61 plus or minus 0.12 millimeters per second to 0.74, plus or minus 0.19.

Whereas the second injection maintained the elevated flow velocity at 0.74 plus or minus 0.19 millimeters per second. While attempting this procedure, it's important to remember to avoid outstanding pointed point structure while cutting the rib cage because it'll cause subsequent injury and the bleeding of the lung After its development. This technique paved the way for researchers in the field of pulmonary metastases research to explore endpoints related to anti-cancer drug development, such as diffusion, limitation of cytotoxic drug delivery, and diffusion, limited hypoxia and rat based models of lung metastases.

After watching this video, you should have a good understanding of how to obtain surgical access to the metastasis bearing or healthy lung of the rat and to perform microscopy imaging on it.