Application of the Cremaster Muscle for Chronic Observation of the Microcirculation

Previously, the cremaster muscle preparation was applied only for acute microcirculatory observation lasting no longer than 6 hours [1]. However, it is well known that some of the events altering the flow hemodynamics in the flap feeding artery and peripheral microcirculation of transferred tissues can occur later most often within 72 hours and no longer than 7 days after flap transfer. Therefore, the cremaster muscle flap model was modified to allow for observation of the microcirculation for prolonged periods of time, still maintaining the accuracy of the measurements [7]. Two models for chronic observation were developed.

The Cremaster Muscle Chamber for Continuous Observation of the Microcirculation

The main advantage of the cremaster chamber application is the ability of repeated measurements of hemodynamic changes within the same muscle flap for a period of up to 3 days. This period is accepted as critical time for flap survival or failure after microvascular repair [7]. After 3 days this model is less applicable since neovascularization from the surrounding tissues takes place and can obscure visibility under intravital microscopy. Also, with prolonged chamber application there is a possibility of the cremaster pedicle stretching or damage.

Technique. The cremaster muscle island flap is dissected and spread on the bottom of the Plexiglas chamber. The muscle is bathed in saline solution, then covered with a round cover glass and with the top frame of the chamber. This keeps the flap moist and free from atmospheric oxygen throughout the experiment. The base of the chamber is fixed with a suture to the body of the animal along the inguinal canal (Figure 3). Observations start at 30 minutes following chamber application to eliminate the immediate effects of surgical trauma on the flap microcirculatory response. Hemodynamic measurements can be taken at 1-hour intervals for the first 24 hours and as needed thereafter up to 3 days after chamber application. Extension of time between the measurement intervals permits the rats to recover from the anesthesia.

In our experience, the chamber is well tolerated by the animals, and the cremaster muscle preparation remains viable during the entire observational period. There is no significant pressure exerted by the use of the chamber, as evidenced by the flow measurements. Furthermore, the

Cremaster Muscle
Figure 3 Schematic drawing of the cremaster muscle flap chamber model.

chamber model allowed to perform microcirculatory measurements comparable with standard cremaster muscle island and tube flap model recordings [7].

The Cremaster Muscle Tube Flap Model for Chronic Observation of the Microcirculation

If even longer periods of observation are necessary for the understanding of the microcirculatory mechanisms, the cremaster muscle tube flap is used. With this model the microcirculatory events can be observed for periods up to 14 days.

Technique. The cremaster is dissected as described before. However, after removal of the testis the empty cremaster muscle tube raised on the neurovascular pedicle is preserved (Figure 4). Next a subcutaneous tunnel is created in the anteromedial aspect of the hind limb. The flap is

Cremaster Muscle

Figure 4 Steps in the hind limb-cremaster transplantation procedure: (A) Extraction of the testis from the cremaster muscle, (B) dissection of the cremaster tube flap on the neurovascular pedicle, (C) preparation of the subcutaneous tunnel for the preservation of the muscle flap for chronic observations, (D) insertion of the muscle flap into the tunnel, transection of the vascular pedicle at the iliac level, and limb amputation at the mid-femoral level.

Figure 4 Steps in the hind limb-cremaster transplantation procedure: (A) Extraction of the testis from the cremaster muscle, (B) dissection of the cremaster tube flap on the neurovascular pedicle, (C) preparation of the subcutaneous tunnel for the preservation of the muscle flap for chronic observations, (D) insertion of the muscle flap into the tunnel, transection of the vascular pedicle at the iliac level, and limb amputation at the mid-femoral level.

buried in this tunnel, secured distally with a pull-out suture, and kept in the tunnel until the day of microcirculatory measurements. At this time the flap is opened, spread over the top of the Plexigas platform, and set up for the measurements. This tube flap model is well tolerated by the animal, and the muscle viability is not affected by the staged surgical procedure required for monitoring.

This model has been successfully applied in research requiring longer periods of observation [7], such as studies on the long-term effects of flap denervation, evaluation of hemodynamic effects of pharmacological agents, and studies monitoring acute phases of allograft rejection.

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