Animal Models

Rats and hamsters have been used for most microcirculatory studies involving the cremaster. The first mention of the cremaster muscle for the study of blood vessels in vivo was presented by Grant [1]. Subsequent studies provided detailed descriptions of the dissection techniques including "hints" [2]. This manuscript also presented information about preparing the rabbit cremaster, although it was mentioned that the size of the scrotum in 2 kg rabbits was similar to 250 g rats. Therefore, although the technique is applicable to the rabbit, it is more suitable in the rat. The report by Baez [3] provided the first study using an opened cremaster, which has become the classic paper for this microcirculatory preparation. With the development of transgenic mice, the mouse cremaster has become a popular model for studying the mechanisms of inflammatory responses or control of blood flow in an animal with a modified genotype. In mice the cremaster appears to be the only microcirculatory model for skeletal muscle because of the small size of the animal.

Anatomy

Anatomically the cremaster is a muscle covering the spermatic cord and testes. The cremaster originates from the internal oblique muscle as the spermatic cord passes from the abdomen to the scrotum through the inguinal canal. The physiological role of the cremaster is to retract the testes and keep the testes warm and protect it from injury. The cremaster muscle in rodents is a thin muscle, 200 to 400 mm in thickness, comprising two muscle layers.

In adults rats or hamsters, the cremaster muscles comprise 60 to 80 percent of the total area as Type lib fibers, which are fast-twitch glycolytic, while 10 to 16 percent are Type I slow-twitch fibers and 8 to 20 percent are Type IIa fast-twitch oxidative-glycolytic fibers [4]. This percentage has been shown to occur in both hamsters and rats, irrespective of age. The average size of the muscle fibers in young rats and hamsters averages approximately 500 mm2 and increases to 12 to 1500 mm2 in adult animals.

Nervous Supply

The major nerve supply to the cremaster is by both the femoral and genital branches (L1 and L2) of the genito-femoral nerve. The ilio-inguinal, the ilio-hypogastric, and the lateral cutaneous nerves also supply the cremaster, and sectioning of all four nerves is required to fully denervate the muscle. The genitofemoral nerve can be accessed by an abdominal incision and stimulated directly (0.1ms 1-5V) to elicit contraction of the cremaster muscle. Direct field stimulation using electrodes placed across the cremaster can also be used to elicit muscle contraction.

Blood Supply

The blood flow to the cremaster is supplied by the cremasteric artery, a branch of the inferior epigastric artery. The cremaster muscle is normally supplied by a paired arteriole (~100 mm) and venule (~150 mm). These vessels branch into smaller vessels, which also give off side branches. The paired arrangement, arteriole and venule, may continue to the level of 30 to 40 mm arterioles. There is no apparent relationship between the arterioles and the orientation of the muscle fibers. Arterioles continue to branch until terminal arterioles give rise to capillaries. Groups of capillaries (modules) appear to be the basic unit of vascular architecture; that is, the flow to a group of capillaries is controlled by the diameter of the upstream arteriole, and an increase in capillary flow occurs throughout the module, not within a single capillary. Normal vascular diameters are presented below.

Dissection and Preparation

The ease of preparing the cremaster muscle is a strength of this preparation. There are only minor differences across species in the preparation of this muscle; therefore, the following directions for dissection can be easily adapted for different species.

The animal should be anesthetized with an appropriate agent, for example, sodium pentobarbital intraperitoneally (40 to 60mg/kg, for rats and hamsters), and then supplemented with intravenous infusion, either through a jugular or femoral vein. A tracheostomy should be performed to allow removal of bronchial secretion but not to support mechanical ventilation. As with any anesthetized animal, monitoring of blood gases should be considered. Placement of a femoral or carotid arterial catheter is needed only if blood pressure is required. Core body temperature should be continually monitored with an esophageal temperature probe.

The preparation and study of the cremaster muscle requires a continuous flow of a physiological salt solution over the muscle. This is accomplished by having a small glass dripper placed over the muscle, allowing for a small continuous flow of superfusate solution during the entire dissection and experiment. A warm physiological salt solution (PSS), pH 7.35 at 34°C, containing (in mM) 131.9 NaCl, 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, and 20 NaHCO3 is appropriate for superfusion. Because the solution is bicarbonate buffered, the superfusion solution must be equilibrated with 5 percent CO2. The amount of oxygen in the superfusion solution has varied between different laboratories, but the oxygen delivery to the cremaster tissue should be from the blood supply. Therefore, the superfusion solution should be equilibrated with a low percentage of oxygen, 0 percent or 5 percent. Thus, the superfusion solution should be equilibrated with 5 percent CO2, 0 percent to 5 percent O2, with a balance of N2.

The animal is placed on its back on a clear acrylic (Lucite® or Plexiglass®) board. The animal's feet should be secured to the board with tape and then either the left or right groin is shaved. To minimize urine contamination of the cremaster, the bladder should be emptied by gentle pressure, with a tie being placed around the penis. A round acrylic pedestal or bath with a rim of silastic or silicone elastomer (Sylgard®) allows for the cremaster to be pinned to the silastic using insect pins.

Figure 1 illustrates the basic surgical procedure. First a 5-0 suture is placed on the distal portion of the scrotum, and the suture is pulled tight to secure the cremaster for surgery. The tip of the scrotum is elevated, and the skin is cut in a distal to proximal direction. At this time the PSS should be flowing over the preparation. The connective tissue between the testicle and the skin is then cut to free the testicle. The testicle can either be removed (after ligating the blood supply) or the freed testicle is gently pushed through the inguinal canal into the abdominal cavity. The solution flowing over the cremaster will hydrate the connective tissue, allowing for a better visualization of the connective tissue for removal. The skin is then pulled proximal and either removed, secured by ties, or pinned to the silastic around the acrylic pedestal. The distal tip of the cremaster is then

Figure 1 This figure shows the basic surgical techniques for preparation of the cremaster muscle. (A) A suture is placed in through the distal skin, and a distal to proximal cut is made. (B) The skin is retracted, the cremaster is exposed, and a distal to proximal cut is made through the cremaster to expose the testicle. (C) Following removal of the testicle, the cremaster is stretched over a plexiglass pedestal. Care should be taken not to overly stretch the cremaster muscle. (D) A microscope image of arteri-oles and venules.

Figure 1 This figure shows the basic surgical techniques for preparation of the cremaster muscle. (A) A suture is placed in through the distal skin, and a distal to proximal cut is made. (B) The skin is retracted, the cremaster is exposed, and a distal to proximal cut is made through the cremaster to expose the testicle. (C) Following removal of the testicle, the cremaster is stretched over a plexiglass pedestal. Care should be taken not to overly stretch the cremaster muscle. (D) A microscope image of arteri-oles and venules.

pinned to the silastic and the cremaster is cut in a distal to proximal direction. This will free the cremaster except for a small set of blood vessels between the cremaster and the testicle. These can be occluded with forceps and gently pulled in the middle with minimal bleeding. The connective tissue is then cleared from the cremaster using fine-tipped scissors. The cremaster is then gently stretched and pinned out, starting from the distal direction. The entire animal can then be moved to the microscope for observation at higher magnification. The cremaster should be superfused with the PSS through the experimental period.

An intact cremaster preparation can be prepared by an abdominal removal of the testicle and replacement with a clear Lucite insert. Although this technique has not been used by a large number of investigators, the major advantage is that disruption of the vasculature is minimized.

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