Despite major advances in the field of reconstructive microsurgery, complications and failures after free tissue transfer and replantation surgery remain as a significant clinical problem. The nature of these failures is often multi-factorial and the pathophysiology is still not completely understood. Different research approaches and animal models were developed to improve our understanding of changes occurring at the tissue microvascular level .
Previously, most studies applied techniques that allowed quantifying microcirculatory perfusion by implementation of the indirect methods of measurement, such as the use of staining techniques. These methods, however, did not reveal accurate information about the dynamics of the microvascular network, which is in a constant state of change. Research discoveries improved significantly after application of modern microcirculatory techniques and models that allowed for direct in vivo observation of hemodynamics of vascular pathophysiology. These models included hamster cheek pouch, rabbit ear chamber, the cat tenuissimus muscle, bat wing, and various mesenteric preparations .
The rat cremaster muscle preparation has a relatively recent history of application to microcirculatory studies [2-4]. One of its advantages is that the cremaster muscle flap is structurally and functionally similar to other skeletal muscles. Thus, all changes observed in the microcirculation of this small flap could be applied to larger muscle flaps used in clinical microsurgery. Majno and Palade were the first to introduce the cremaster muscle model in their histo-logical and electron microscopy studies on inflammation . This model was accepted for microcirculatory observation by in vivo microscopy after modifications made by Grant . Baez was the first to open the cremaster muscle and to detach the testicle from the flap . He was then able to spread out the flap and transilluminate it under a microscope. Miller and Wiegman introduced a tissue bath to provide better control of the microcirculatory environment . This standard cremaster preparation was further developed in our laboratory in such a way that the cremaster muscle was completely isolated on its neurovascular pedicle, as an island flap . In this model the cremaster muscle circulation is solely dependent on one feeding artery and one outflowing vein, excluding all other sources of vascular supply. This procedure simulates the conditions encountered during surgery of free tissue transfers, where the main vessels of the flap undergo microsurgical anastomosis and changes occurring downstream from the site of vascular repair can be monitored. These microcirculatory observations can add to our understanding of variables that can directly affect flap failure and survival.
The cremaster muscle flap isolated on the neurovascular pedicle expands significantly our ability to study microcirculation in relation to (1) reactivity of the microvessels [1, 6], (2) ischemia-reperfusion injury , (3) assessment of microcirculatory responses to muscle denervation , (4) chronic observation of the microcirculation using a chamber model , (5) observation of the microvascular events following tumor implantation, and (6) monitoring of microvascular responses to isograft and allograft transplantation [9, 10].
Male rats weighing between 80 and 150 g are used, because at this weight range the cremaster fascia is thin enough to allow for optimal visualization of the microcirculation of the transilluminated muscle. Also, the iliac artery is large enough to allow for controlled dissection, transection, and repair under surgical microscope magnification.
Anesthesia is induced with intraperitoneal administration of pentobarbital (60 to 70mg/kg) with additional supplements as needed. Core body temperature is maintained between 35° and 37°C. The animal's genital area and thighs are shaved; the rat is prepped and placed in a supine position.
During flap dissection the tissues should be kept moist using saline solution. An operating microscope (Zeiss OPM6-SD, Zeiss, Germany) is used for pedicle dissection and isolation. A longitudinal incision is made from the tip of the scrotum to its base. From this point the incision is carried laterally toward the anterior iliac spine. The subcutaneous tissue over the groin area and the scrotum is then carefully dissected. The cremaster is visualized as a thin tissue layer covering the testis. This dissection is continued carefully on the ventral as well as dorsal surface of the muscle until the muscle is completely detached from the scrotum. A traction suture is placed through the distal tip of the cremaster muscle to facilitate atraumatic dissection (Figure 1A). The pedicle of the flap is visualized on the dorsal aspect of the muscle. A longitudinal incision is then made on the ventral surface of the cremaster muscle flap origin including the full thickness of the muscles of the abdominal wall and is directed parallel to the inguinal ligament (IL). The vascular pedicle of the testicle and the deferens duct are exposed and ligated with 5-0 silk sutures. The testicle is pulled from the scrotum and is carefully separated from the muscle. Next the testicle is removed, and the empty cremaster muscle tube flap is now ready for dissection of the neurovascular pedicle.
The dissection is performed in the groin area, with exposure of the femoral artery and vein and the IL. The medial insertion of the IL is then divided from the pubic bone attachment, and the ligament is mobilized from medial to lateral, exposing the genitofemoral nerve, and the external iliac artery and vein (EIA and EIV). All nonrelevant branches of the EIA are either coagulated or ligated with 9-0 nylon sutures, leaving the intact pudic-epigastric trunk, which now provides the only blood supply to the cremaster muscle flap. The frontal wall of the cremaster muscle tube flap is then opened from proximal to distal. The remaining attachments connecting the flap to the groin region are transected and the cremaster muscle is totally isolated on its vascular pedicle, as an island muscle flap.
Preparation of the Cremaster Flap for Microcirculatory Monitoring
The animal is placed on a Plexiglas observation platform. The prepared cremaster muscle is extended and is held in place with 5-0 silk sutures (Figure 1B). The muscle is irrigated with saline solution and covered with a plastic film to avoid dehydration (Saran Wrap presoaked with distilled water overnight). Figure 1C shows the schematic map of the microvessels that can be monitored in this preparation.
The cremaster muscle is then transilluminated from below and the microcirculation is observed and monitored using an intravital microscope (Nikon Optiphot-2, Japan) equipped with a color video camera (Sony CCDIRIS, Japan), a 19-inch monitor (Sony Trinitron, Japan), and a videotape recorder (Panasonic AG-1730, Japan). Using this standardized setup, the following microcircula-tory parameters can be directly observed, measured, and recorded on the videotape over time for repeated observations (Figure 2):
1. Vessel diameter. The diameter and the endothelial wall thickness of the cremaster arterioles and venules are measured by using a video image measurement system (VIA-150, Boeckeler, Tucson, AZ).
2. Red blood cell (RBC) velocity. A custom-made optical Doppler velocimeter (Texas A&M, College Station, TX) is adapted to the system. It allows the measurement of RBC velocity in millimeters per second in the main arterioles and venules of the cremaster muscle flap.
3. Capillary density. The capillary perfusion or functional capillary density is defined as the number of capillaries in which RBCs are constantly flowing. Three regions of the cremaster muscle flap with a good capillary perfusion and clear visualization are chosen, in the proximal, medial, and distal area of the flap. Each visual field—as seen in the monitor—represents an area of tissue of 0.18 mm2. Nine fields in each of three flap regions are monitored and counted for a total of 27 fields, and the average number from all fields represents the overall state of capillary perfusion of the cremaster muscle microcirculation.
4. Leukocyte-endothelial interactions. The postcapillary venules are chosen for the observation of leukocyte behavior. These vessels have a diameter of 20 to 40 mm and are identified in the proximal, medial, and distal regions of the flap. Rolling, "sticking" (remaining stationary for more than 20 seconds), transmigrating neu-trophils and lymphocytes are observed and counted during a 2-minute period in each flap region using a hand counter (Figure 2).
5. Endothelial-edema index. The external and internal diameters of the postcapillary venules are measured using the VIA-150. The ratio between the two numbers is defined as endothelial edema index and is used in some studies to indicate vascular occlusion caused by leukocyte aggregation within the venular lumen, causing vessel wall damage, and microvascular injury leading to increased tissue permeability.
Figure 1 (A) Isolated cremaster muscle flap. (B) Cremaster muscle flap dissected and set up for microcirculatory studies. (see color insert) (C) Schematic representation of the cremaster muscle flap microvascular network for the measurements of microcirculatory hemodynamics. V1, venule; A1, first-order arteriole; A2, second-order arterioles; A3, third-order arterioles; A3-1, third-order arteriole (the branch of A2-1); A3-2, third-order arteriole (the branch of A2-2).
Figure 2 Intravital microscopy setup for microcirculatory monitoring. (see color insert)
like white columns against a black background. Leaks appear as white streaks outside of the vessels. These images are digitized using a Kontron Elektronik KS 300 V2.00 image analysis system (Kontron Elektronik GmbH, Eching, Germany). Three segments in the lumen of the venule and of the interstitium are selected. An index of vascular leakage (permeability index, PI) is computed from the images. A relatively high positive PI indicates increased leakage from the postcapillary venule as result of endothelial injury. If the Kontron system is not available, the live images recorded can be captured by the computer using the Studio DC Plus software (version 3.1, Pinnacle System, Mountain View, CA) and a capture card. The images are then analyzed with the Image Pro-Plus software (version 2.1, Media Cybernetics, Silver Spring, MD).
Figure 2 Intravital microscopy setup for microcirculatory monitoring. (see color insert)
6. Thrombus formation. The transilluminated cremaster muscle can be used to directly visualize and quantify thrombus formation. The pedicle artery is viewed with an inclinable head stereoscope (Zeiss OpMi6, Zeiss, Germany) and at the same time the downstream cremaster is observed with a compound microscope. By incorporating the two systems it is possible to image the upstream and the downstream microcirculation side by side on the same video monitor, simultaneously. This allows the correlation of thrombus formation upstream with the appearance of the dislodged platelet emboli in the downstream microcirculation.
1. Fluorescein microscopy setup. A Zeiss 20T fluorescein microscope (Carl Zeiss, Germany) is used. An illumination system with a 460 to 490 nm blue light band excitation filter from a mercury arc lamp (Mercury Power Supply, Model 1200, OpticQuip Inc, NY) is used to stimulate the fluorescein isothiocyanate. The closed circuit video recording system consists of an MTI silicon-intensifying target camera (MTT SIT-68), a Panasonic AG-1290 video recorder, and a 19-inch monitor. The gain, sensitivity, and black level of the SIT camera allow the detection of very low light levels.
2. Microvascular permeability assessment technique and analysis of macromolecule leakage. Macromolecular leakage reflects vascular permeability. Albumin conjugated with fluorescein isothiocyanate (FITC) is injected intravenously to assess the microcirculatory permeability in the cremaster muscle flap. Postcapillary venules are selected for measurements, which are taken at zero time and after 30 minutes. The albumin appears as bright white on the video monitor. Venules without leaks look
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