For microscopic observation, the single layer pouch preparation is performed essentially as described by Duling (1973). Alternatively the pouch may be studied in situ after insertion of a light rod and an acrylic skin chamber. There are no discrepancies reported for results achieved with either of these two procedures. However, the everted cheek pouch offers better optical conditions and a larger area (about 1 cm2) for observations of the microvasculature. A hamster is anesthetized by intraperitoneal injection of pen-tobarbital and is then placed on a heating pad controlled by a rectal thermistor. A tracheal tube is inserted to facilitate breathing and a femoral vein is catheterized for administration of supplemental anesthetic or other intravenous drugs. The experimental setup of the hamster cheek pouch preparation is illustrated in Figure 2. The animal is placed on an acrylic stage in the middle of which there is a well with a circular silicon rubber ring surrounding a transillumination window. The cheek pouch is everted with the aid of a moist cotton stick, the distal nonmuscular part of the pouch is identified and pinned to the silicon ring, and the pouch is dissected under a stereomicroscope at 10x magnification. A central area is selected with no major arterioles or venules and an incision is made in the top layer, which then is moved aside and pinned to the silicon ring. Now the loose areolar connective tissue is exposed and can be removed to have a single-layer preparation with the best optical conditions. During the preparation, and throughout the experiment, the cheek pouch is constantly superfused with a bicarbonate-buffered saline solution that is continuously bubbled with 5 percent CO2 in N2 at 37°C, and thus provides a physiological level of oxygen, temperature, and acidity (pH). For studies of vascular permeability changes, an intravascular marker such as fluorescein-labeled albumin can be used. Alternatively, an intravascular marker with a molecular weight similar to that of albumin namely fluorescein-labeled dextran (FITC-dextran), is used in a dose that has minor if any effect on platelet function and blood clot formation. FITC-dextran 70,000 or 150,000 Da is dissolved in normal saline and injected i. v. in a dose of 50 to 100mgkg-1 body weight. Properly mounted and dissected, the cheek pouch is a noninflamed preparation as judged from the presence of vascular tone and absence of sites with FITC-dextran leakage (as seen in Figure 1), and absence of excessive leukocyte rolling and adhesion in postcapillary venules; the preparation can be utilized for intravital microscopy for several hours.
Was this article helpful?
This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.