The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

The overall goal of this procedure is to prepare the cream master muscle to study the living microcirculation of an anesthetized male mouse. This surgery begins with an incision on the ventral surface of the scrotum. Once shaved, the next step is to clear and separate the cremaster muscle from the surrounding tissue.

Then an orchiectomy is performed and the tissue is spread radially across the surface of a transparent pedestal. This preparation can be viewed by microscopy to image the microcirculation within the cream master muscle. Ultimately, results can be obtained that show how blood flow and oxygen delivery are actually controlled in living systems during health and disease models, and in response to experimental manipulations.

This method can be used to answer basic questions in cardiovascular medical research with an emphasis on the microcirculation. In particular, how blood flow and oxygen delivery can be regulated at the cellular level in the intact system. Though this method can provide insight into the in vivo function of the mouse microcirculation.

It can also be applied to other rodent models such as hamsters and rats. To stabilize the mouse under the microscope, some new equipment will need to be prepared. Begin by building a transparent rectangular plexiglass board to fit on the microscope stage of the microscope.

Next, build a transparent pedestal from cigar 180 4, poured into a Petri dish. To adapt the 15 millimeters decast the poured sil guard in a vacuum chamber for an hour. To remove air bubbles and improve clarity, cure the Degas sill guard in a 50 degree Celsius oven for about four hours.

Once cured, cut the pedestal into a Pentagon with a rounded top. Then secure the pedestal block to the rectangular plexiglass board with a thin layer of clear waterproof silicone adhesive, and allow the silicone to cure overnight. Next, build a wedge-shaped platform to position the mouse against the pedestal.

The wedge platform can be made of acrylic plastic, and a heat lamp can be used to keep the preparation warm. Alternatively, aluminum can be mounted to a plastic wedge. The aluminum is warmed using resistive heating elements connected to a DC power supply.

Lastly, on the day of the experiment, prepared by carbonate buffered PSS in ultra pure water. By mixing the salts on the day of the experiment, precipitation of bicarbonates is avoided. Keep the PSS in a water bath at 37 degrees Celsius and using a gas disperser equilibrate the PSS with 5%carbon dioxide gas in nitrogen gas for 15 minutes.

This adjusts the pH to about 7.4 and further prevents salt precipitation. After anesthetizing an adult mouse, carefully shave the hair from the lower abdomen, lower back scrotum and legs. Be extra careful around the scrotum and testes to avoid injuring the underlying cream master muscle.

Remove loose hair with a fresh alcohol swab. Check if the bladder is full. If it is full, it will feel like a small grape through the abdominal wall if full, empty it.

Using gentle pressure and holding the tip of a kim wipe next to the penis to collect the urine. Now, position the mouse on its back, lying on the wedge platform with its legs straddling thes guard pedestal, and then secure the mouse with its crotch against the pedestal. To maintain the position, apply a piece of tape loosely across the chest and secure it to the platform.

Begin the surgery by securing a pin through the apex of the scrotal sac and into the pedestal placing tension on the scrotal skin. Now, initiate and maintain a superfusion of PSS over the surgical field throughout the dissection. To remove the effluent PSS position, a Kim wipe with one end next to the scrotal sac and the other next to a vacuum line.

Using microdissection scissors while holding the skin with angled forceps, make a vertical incision along the ventral surface of the scrotal sac if the testicle has been retracted into the abdominal cavity by the cremaster muscle. Applying gentle pressure on the lower abdomen directs the testicle into the sac. Now identify the surface of the cremaster muscle, overlying the testicle, and carefully remove connective tissue between it and the scrotal skin.

To expose the cream master muscle, gently retract the scrotal skin behind the proximal edge of the pedestal and secure it on each side. With the pin, now clear the outer surface of the muscle of connective tissue. Place a pin through the apex of the cream master muscle to apply tension and to very carefully make a longitudinal incision Through its ventral surface, try to minimize disruption of the vascular supply.

The cream master muscle is connected by a thin ligament to the EPIs. Underneath the testicle, reflect the testicle to one side. To expose this ligament, which is carefully separated from the EPIs.

Near the apex of the cremaster muscle is a small paired artery and vein that connected to the epididymus. Clamp these vessels between forceps and pull them apart to minimize bleeding. Now perform an orchiectomy, including the inguinal fat pad proximally, ligate the testicle, epididymus testicular artery and vein.

Then sever and discard them. Clear the cremaster muscle of remaining connective tissue, and then spread it radially on the surface of the pedestal. Secure the edges with five to six pins to create a flat sheet of striated muscle with intact microcirculation.

Transfer the completed preparation to the stage of an intra vital microscope, fused continuously at 34 degrees centigrade and equilibrate for 30 minutes Prior to experimental manipulation or data collection, using a customized microscope with brightfield illumination and epi illumination, digital images of the cremaster preparation were acquired under Brightfield illumination. The muscle was visualized at 42 x 83 x and 165 x magnification under fluorescence. The muscle was visualized at 42 x 83 x 165 x and 330 x magnification After its development.

This technique paved the way for researchers in microcirculation to study mechanisms underlying the regulation of tissue, blood flow, oxygen delivery, and inflammation. While attempting this procedure, it's important to remember to delicately handle the tissue to make sure that the microvascular reactivity remains intact.