Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

This practical and reproducible method can be used to visualize the endothelium leukocyte interactions that lead to leukocyte recruitment within an intact mouse. This technique also presents a powerful tool for the monitoring of physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The method can be applied in different inflammatory and septic models.

For example, we have recently highlighted its relevance in the study of musculopathies involving exaggerated muscular leukocyte infiltration. Our suggestion to fellow scientists who are considering using this technique is to get as much hands-on experience as possible as the method is very susceptible to minor deviations. Demonstrating the procedure will be Simon Kranig, a clinician and senior scientist from my lab.

After confirming a lack of response to toe pinch in the anesthetized experimental animal, fix the mouse in a dorsal recumbent position on a 37 degree Celsius heating pad and use a nonabsorbable sterile 6-0 suture to wind a simple loop around the frontal teeth. Tape the joining ends of the suture to the heating pad and use tweezers to gently lift the skin at the midline. Next, make a circular one to two centimeter diameter incision over the neck and upper thorax and use tweezers to carefully dissect the surrounding muscle, fat, and connective tissues.

Place a suture under the trachea. Use small surgical scissors to make an approximately 1.3 millimeter transverse cut into the trachea and introduce a polyethylene tube into the caudal end of the trachea to secure the upper airways. Fix the tube located with a single circular knot suture and locate the carotid artery along the right side of the trachea.

Dissect the surrounding tissue from the carotid artery wall and pass two pieces of suture under the artery. Tie the first cranial suture proximal to the bifurcation of the carotid arteries and position the second suture about five to eight millimeters distal from the first suture without tying. Next, attach a 30 centimeter piece of polyethylene tubing to a one milliliter syringe needle filled with saline and use a seven millimeter vessel clip to clamp the carotid artery distally to the second suture.

Make an approximately 0.5 millimeter transverse cut in the carotid artery and introduce the sterile polyethylene tube into the artery. Secure the tube with the second suture and remove the vessel clip. Then gently depress the syringe plunger to flush saline into the vessel.

To prepare the cremaster muscle, transfer the mouse to a custom-made plastic microscope frame with the scrotum facing the microscope stage and carefully grasp the scrotum at its most distal end with tweezers. Pulling the scrotum gently, remove a circular about five millimeter diameter section of scrotal skin keeping the open tissue well-hydrated with saline. Use two tweezers to dissect the loose connective tissue and locate both testes.

Grasp one testis distally and gently begin pulling out the reproductive tissue removing any surrounding connective tissues step by step. Once the testis has been exteriorized, pin the distal end of the tissue to the rubber ring surrounding the stage and hydrate the tissue with saline. Next, carefully remove the connective tissue without harming the underlying cremaster muscle.

It’s critical to remove all of the connective tissue which would result in blurry images without harming the underlying cremaster muscle. When all of the tissue has been removed, make a one millimeter transverse incision to open the cremaster muscle before making a longitudinal incision from the very distal to the proximal end of the tissue. The muscle should open spherically.

Carefully spread the muscle over the glass stage and pin the muscle to the rubber ring taking care not to touch or harm the central region of the tissue. Then pin the remaining testis to the side to give access to the region of interest and hydrate the tissue with PSS. For intravital visualization of the muscle vasculature, place the mounted cremaster muscle in the upright microscope and select the 40X objective.

Perform the recordings under continuous superfusion with 35 to 37 degree Celsius PSS through a piece of small tubing that has been taped to the upright objective of the microscope to allow continuous dripping of PSS alongside the objective down onto the muscle and tissue. Identify the post-capillary venules. Measure the microcirculation on venules with a 20 to 40 micrometer diameter.

Then record high resolution images of the microcirculation from the cremaster muscle over a 30-second period continuously adjusting the focus due to slight changes in the contraction and relaxation of the muscle tissue. Excess connective tissue can lead to blurry microscopic images. In this representative microscopic image, the post-capillary venules which should be between 20 to 40 micrometers in diameter can be identified by the confluence of two smaller vessels.

Adherent and rolling leukocytes can be visualized while circulating leukocytes may only be tracked in slow motion replay of recorded videos. A multitude of parameters may affect leukocyte recruitment in vivo including the cardiac output, vessel diameter, centerline velocity, wall shear rate, and number of circulating leukocytes. Note that the centerline velocity is influenced by cardiac output, peripheral vascular resistance, and intravasal volume.

For example, a large volume of Rhodamine or other experimental substance given via the carotid catheter increases the post-capillary centerline velocity and inhibits leukocyte capture and rolling. On the contrary, cardiac failure, deep anesthesia, or hypothermia may decrease the post-capillary flow. To determine the centerline velocity, freely circulating fluorescent leukocytes may be used or the leukocytes must be dyed with fluorescent reagents like Rhodamine.

Note that wild type strains may show physiological variance. For example, C57BL/6 mice demonstrate clearly enhanced leukocyte rolling, adhesion, and transmigration compared to C57BL/10ScSn animals. The planning and standardization stages are important for mastering the technique especially during the preparation of cremaster muscle for microscopy.

Other methods for visualizing leukocyte recruitment can be conducted to yield additional information and to help dissect the individual contribution of leukocytes and endothelial cells to the recruitment cascade. This technique provides a platform for the preclinical evaluation of pharmacologic meditators and novel immune modulatory substances.