Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

The overall goal of this procedure is to establish a reproducible and reliable model of an ischemic challenged musculocutaneous flap in the mouse skinfold chamber. This is accomplished by first preparing the skinfold chamber and the mouse's back. Next, a flap is elevated and randomly perfused so that it develops necrosis.

Then the flap is inserted into the titanium skinfold chamber. Finally, the window chamber is sealed with a cover glass to ensure tightness of the chamber. Ultimately, microcirculation is visualized through the chamber window by intra vital fluorescence microscopy over a time period of 10 days or more.

The main advantage of this mouse model over existing methods is that it allows for real time observation of micro circulatory and morphological changes of the microvasculature of musculocutaneous tissue using intra vital fluorescence microscopy over a time period of approximately two weeks. Amongst other things, the model allows the examination of conditioning procedures to eventually improve tissue survival. After anesthetizing a mouse, according to the text protocol, use an electric shaver to remove the hair from the back and then apply depletion cream to remove any remaining hair from this area.

In the meantime, assemble the following surgical instruments, skin forceps, a micro forceps, scissors, micro scissors, suture material, and a marker pen disinfect and prepare the titanium chamber by applying the two screws with the nuts at the chamber's base and by applying self adhesive foam around the window of the chamber's counterpart to guarantee tightness of the chamber system. After removing the depletion cream, administer buprenorphine subcutaneously with a mouse in the prone position and using a trans illumination light source. Grasp the skin centrally and lift it up.

Once the skin fold is created, the vessels identified and stacked. Put two sutures through the apex of the fold, cran and coddly. Choose a location to provisionally.

Place the titanium frame, disinfect the chamber and rinse it with saline solution. Ensure that the distal branches of the lateral thoracic artery or LTA cran and the deep circumflex iliac artery or DCIA coddly coarse through the center of the chamber's window. Once the positioning of the chamber is set, perforate both layers of the skin at the very bottom of the fold for later fixation of the titanium frame.

Next, with the titanium frame removed and the mouse in the prone position, outline the flap perpendicular to the spine, starting at the midline with a width to length ratio of 15 by 11 millimeters to produce a laterally based and randomly profused flap. Following the final marking in size, the flap transecting both the LTA and DCIA and remove the outlined additional tissue of the skinfolds opposite side. Before elevating the flap, place the cranial and coddle screws of the chamber's frame through the previously made skin perforations.

Apply one cocker clamp to each of the screws and using interrupted five zero sutures, fixate the skinfold both anteriorly and posteriorly to the back part of the frame with five zero interrupted sutures. Suture the vertical limb of the flap back to the surrounding skin under a magnifying lens or microscope. Use a micro forceps and micro scissors to remove the gelatin like loose alar tissue from the striated muscle.

This will allow visualization by intravital fluorescence microscopy of the musculocutaneous tissue consisting of striated muscle and skin. After disinfecting the chamber rin it in saline solution. Otherwise the alcohol soaked foam might irritate the skin.

Finally, seal the chamber by mounting the counterpart of the frame posteriorly, cran and anteriorly. Then place drops of topical bis, benam and saline on the flap before using a cover glass and a snap ring to seal the observation window after surgery. Administer 0.2 milliliters of sodium chloride subcutaneously to overcome the lack of drinking during the first couple of hours after surgery.

Once the animal has awakened, check that the chamber is standing upright and not tilting to the side. Following the surgery, return the animal to a separate cage until fully recovered. Observe the animal daily and administer buprenorphine subcutaneously at any signs of pain.

Allow the animals to have free access to standard animal chow and water process harvested tissue by mounting it on small cork plate or by mincing it and putting it in a sterile tube for further histological and molecular analysis, respectively, at least 24 hours following surgery to allow inflammation to subside. After anesthetizing the animal according to the text protocol, place it in a lateral D cubital position on a custom made plexiglass carrier into a tail vein or the retrobulbar venous plexus inject 0.05 milliliters of 5%green fluorescent dextrin and 0.05 milliliters of 1%highly red fluorescent rumine six g die. After placing the mouse under the microscope, virtually divide the visible flaps surface horizontally into proximal central and distal areas of equal size.

Using a five x objective and a CCD camera. Scan the tissue image by image within the chamber's window from proximal to distal in each area of the flap. Select second or third order arterials and they're accompanying venues with easily identifiable branching patterns using the five x 10 x and 20 x objective.

Take representative images and make snapshots of the arterial or ular bundles in order to easily re localize the bundles throughout the whole observation period with the 20 x and 50 x objectives. Further record five to six capillary fields and apoptotic fields neighboring the arterial ular bundles per area of the flap respectively. Upon completion of the experiment, euthanize the animal and analyze the recorded data according to the text protocol.

Final demarcation of flap necrosis usually occurs five to seven days after surgery and is characterized by a zone of microvascular remodeling developing between the proximal vital and the distal necrotic zone of the flap. Untreated controlled mice usually develop a 50%flap necrosis after 10 days that is divided into three distinct areas. The well perfused proximal, the interrelated critically perfused central area and the necrotic distal area microvascular diameters and RBC velocity of arterials and les of various diameters and neighboring capillaries were measured at day one and day 10 to determine volumetric blood flow.

Repetitive microscopy not only allows the analysis of functional changes of micro, but also morphological changes of microvessels illustrated. Here are images taken for measuring the functional density of RBC perfused capillaries, blood flow, functional capillary density, and eventually tissue oxygen tension gradually decrease from proximal to distal in the flap to reach a threshold of viability where the capillaries are not perfused anymore. Preconditioning with erythropoietin before flap preparation resulted in development of new functional microvascular networks, first visible between days three and five that are not seen in the untreated control animals.

The combination of the dorsal skinful chamber and intra intravital fluorescence microscopy first allows the repetitive investigation of functional and morphological changes of the vasculature of is chemically challenged tissue using the same regions of interest, and secondly, the correlation of these results with molecular pathways that are based on tissue analysis.