Animal Model for Chronic Stress

3.1.1. Pre-Experimental Treatment of Rats

On arrival in the animal facility, the rats should be placed two per cage (see Note 1). The animals should be divided into two groups and housed in two separate rooms with similar dimensions and furnishings. These rooms should be chosen so as to be remote from noisy air vents and cage washers, and should be identical with respect to background noise levels in the frequency range of 50 Hz to 10 kHz. Ambient and experimental noise sound pressure levels can be measured with a calibrated ANSI type 1 sound level meter and octave band filter. It is important that the background noise has a similar amplitude (approx 50 dB) and frequency distribution in both rooms.

The number of times personnel enter and leave the rooms should be minimized by only housing those rats participating in this study in the chosen rooms. A noise generator should be placed in one of the rooms and activated for 15 min each day (see Note 2 [13-15]). This procedure has been shown to stress rats and cause mast cell degranulation after a period of 3 wk (16,17). The rats in the room without the noise generator form the unstressed control group.

3.1.2. Mesenteric Surgery and Preparation for Microscopy

1. Rats are anesthetized, and when the rat has reached a suitable level of anesthesia (no eye or foot reflex), the abdomen is shaved and a tracheotomy is performed, using PE 240 tubing, so that the animal can be artificially ventilated.

2. A midline incision, from the pubis to the sternum, is made along the linea alba, the intestine is carefully externalized, and a portion of the mesentery is gently spread flat over a Plexiglas platform integrated into the Plexiglas animal surgery tray.

3. The exposed mesentery is kept warm and moist with a constant trickle of HEPES saline (pH 7.4) at 37°C.

4. Next, the HEPES is replaced by a fixative consisting of 3% formaldehyde in HEPES at 4°C, and the animal is sacrificed with an intravenous injection (0.5 mL) of Beuthanasia.

5. After 1 h, the mesenteric tissue is carefully excised and divided into separate "windows," a window being defined as the portion of mesenteric tissue bordered by two adjacent pairs of feeding arterioles and collecting venules, and the attached intestine (Fig. 2).

6. The intestine is cut away from the mesenteric window, as is most of the adjacent adipose fat.

7. Each window of the preparation is mounted between two thin glass cover slips using aqueous mounting medium.

8. At this point the specimens may be examined microscopically and photographed or videotaped before staining for mast cells. The subsequent mast cell staining procedure will obscure any fluorescent stains that may have been used to identify other structures earlier in the experiment.

3.1.3. Intestinal Surgery and Preparation for Microscopy

1. The rats are prepared as for the mesentery surgery and a portion of the intestine (ileum) is carefully externalized, wrapped in HEPES-soaked gauze, and positioned on a HEPES-saturated gauze platform that is arranged adjacent to the abdomen.

2. The intestine is kept warm and moist with a constant drip of HEPES at 37°C. The abdominal aorta is exposed, and a cannula is placed retrograde, distal to the superior mesenteric artery.

3. The aorta is clamped just proximal to the superior mesenteric artery, and the circulation is flushed free of blood with approx 5 mL of HEPES.

4. The animal is killed by intravenously injecting Beuthanasia, and the HEPES is replaced by phosphate-buffered Karnovsky's fixative (pH 7.4) for perfusion fixation. Pressure is maintained at 30 mmHg, and the portal vein is clamped. Fixative also is applied to the outside of the intestine.

5. After 1 h, a 4-cm segment of distal ileum is excised and divided into four equal portions; one for light microscopy and three for electron microscopy. These portions are placed in fixative for an additional hour. The tissue segments are then rinsed in 0.2 M sodium cacodylate buffer and incubated overnight in 2% diaminobenzidine (DAB) at room temperature in the dark (see Note 3).

Rat Mesentery Cell

Fig. 2. Intestine and mesentery of rat, showing mesenteric windows between adjacent pairs of feeding arterioles and collecting venules. After 1 h, the mesenteric tissue is carefully excised and divided into separate "windows," a window being defined as the portion of mesenteric tissue bordered by two adjacent pairs of feeding arterioles and collecting venules (black arrows), and the attached intestine (white arrow).

Fig. 2. Intestine and mesentery of rat, showing mesenteric windows between adjacent pairs of feeding arterioles and collecting venules. After 1 h, the mesenteric tissue is carefully excised and divided into separate "windows," a window being defined as the portion of mesenteric tissue bordered by two adjacent pairs of feeding arterioles and collecting venules (black arrows), and the attached intestine (white arrow).

6. The next day, the segments are re-immersed in DAB solution, containing H2O2 to a concentration of 0.2%, for 1 h, rinsed three times in 0.15 M sodium cacody-late buffer, postfixed in osmium tetroxide, dehydrated in increasing concentrations of ethanol, and embedded in Spurr's resin for light and electron microscopy.

7. The pieces of tissue are oriented in the resin so that the blocks can be sectioned longitudinally through the intestinal villi. This orientation allows the whole length of each villus to be visualized and increases the chances of seeing mast cells in the villus interstitium. Thick sections (2 pm) are cut for light microscopy and ultrathin sections for electron microscopy. These latter sections are stained with uranyl acetate and lead citrate.

3.2. Connective Tissue Mast Cells

3.2.1. Mast Cell Staining

1. Slides with mesenteric windows are rehydrated with distilled water and then stained with 0.1% Alcian blue in 0.7 M HCl for 30 min, rinsed in 0.7 M HCl, and subsequently stained with 0.5% Safranin O in 0.125 M HCl for 5 min (see Note 4).

2. They are then rinsed in distilled water, counterstained with 0.1% eosin for 30 s, and gradually dehydrated in a series of 70%, 80%, 90%, 95%, and 100% ethanol.

3. The slides are cleared in xylene and mounted with mounting medium.

4. This staining procedure is modified from Mayrhofer (15). For a faster, less-complicated staining technique (see Note 5).

3.2.2. Mast Cell Observation

1. Connective tissue mast cells degranulate to release biogenic amine granules, such as histamine granules, which stain blue (Fig. 3A). At this stage, the cell body also stains blue. The mast cells are often located near venules.

2. Intact mast cells stain blue but do not show any free granules.

3. Mast cells that are extensively degranulated are almost depleted of histamine and just show a thin rim of blue stain around the outside (Fig. 3B). Occasionally, the extensively degranulated mast cells show some red staining, which indicates the presence of the proteoglycan, heparin.

4. Mast cells within small windows, without a well-developed vasculature, show more of the red stain than those within the larger windows.

5. Using this staining technique, the mast cells can be classified as intact, moderately degranulated, or extensively degranulated and the major type of granule constituents can be characterized.

6. A useful way to evaluate the numbers of mast cells in each state of granulation is to examine the tissue under a x20 power microscopic objective and count the number of cells per field of view, using a x10 power eyepiece. Rows of fields can be counted systematically from left to right.

3.3. Mucosal Mast Cells

3.3.1. Mast Cell Staining

For light microscopy, thick sections (2-^m thick), cut longitudinally through the intestinal villi, are stained with 1% Toluidine blue (pH 7.0) for 15 min and then washed in distilled water. This technique has been modified from Conroy and Toledo (18).

3.3.2. Mast Cell Observation

1. The mast cells can be visualized easily under low-power (x10 objective) light microscopy. At this magnification, degranulated mast cells are easy to identify by the presence of empty vacuoles (Fig. 4).

2. Mucosal villi from unstressed animals rarely show vacuolated mast cells. At higher power, water-immersion objectives, such as a 340, 0.75 n/a show greater detail and the intense Toluidine blue-stained granules remaining in the mast cells can be resolved.

3. Intact mast cells show blue-stained granules in the cytoplasm but no vacuoles.

4. The total numbers of degranulated mast cells per each villus cross-section can be counted in a chosen number of villi (i.e., 20-30) per animal. If the tissue also is stained with DAB, eosinophils can be easily differentiated from mast cells because their peroxidase granules stain brown, in contrast to the mast cell granules that stain blue. A photograph of part of a villus cross-section containing eosinophils is shown in Fig. 5.

5. Under electron microscopy, mast cells can be identified by their granules, made electron dense by staining the sections with uranyl acetate and lead citrate (Figs. 6 and 7).

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Fig. 3. Photomicrograph of degranulated mesenteric mast cells (DMCs) and extensively degranulated mast cells (EDMCs). Scale bar: 25 pm. The mast cells are often located near venules (large arrow).

6. Vacuoles are seen in degranulating mast cells (Fig. 6), and some areas of cytoplasm appear to be disintegrating, perhaps because a granule has just been released.

7. Figure 7 also shows an eosinophil characterized by the oval shaped granules bisected by a thin layer of more electron-dense material.

8. Preparing the tissue for both light and electron microscopy gives one the advantage of being able to count cells over a wide area and also to determine the proximity of the mast cells to fine structures, such as peripheral nerves.

Degranulated Mast Cells

Fig. 4. Photomicrograph of section though intestinal mucosal villus showing degranulated mesenteric mast cells (DMCs). The mast cells can be visualized easily under low-power (x10 objective) light microscopy. At this magnification, degranulated mast cells are easy to identify by the presence of empty vacuoles (arrowheads).

Fig. 4. Photomicrograph of section though intestinal mucosal villus showing degranulated mesenteric mast cells (DMCs). The mast cells can be visualized easily under low-power (x10 objective) light microscopy. At this magnification, degranulated mast cells are easy to identify by the presence of empty vacuoles (arrowheads).

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