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Fig. 5. Photomicrograph of section though intestinal mucosal villus showing eosi-nophils stained with DAB (arrows).

Fig. 6. Electron micrograph of section through intestinal mucosal villus showing degranulated mast cell (arrow).

4. Notes

1. To ensure that the animals have a low baseline level of stress, they should be housed in open wire cages (39 cm x 30 cm x 30 cm) such as those sold in pet stores for hamsters. One model that is particularly suitable has two levels linked by a wire mesh slope. In addition, it is advisable to provide each cage with woodchip bedding and one black, PVC tunnel (11 cm diameter x 17 cm length). Such a tunnel is large enough to accommodate both rats when they sleep during the day. When rats are housed in this way they rarely fight at night, unlike rats that are housed in standard, institutional cages (polycarbonate, suspended, solidbottom cages [40.8 cm x 21.0 cm x 16.8 cm] with woodchip bedding and no other enrichment). In addition, rats housed in enriched cages as described previously, spend more than half of their time either in or on the tunnel, or on the shelf or ramp at night. Rats housed in standard cages fight continually at night and the

Eosinophils And Mast Cell Conjunctiva
Fig. 7. Electron micrograph of section through intestinal villus showing an intact mast cell (black arrow) and eosinophil (white arrow).

fights usually involve a single "aggressor" and a single "victim." These rats also squeak frequently and show significantly more rearing than those in enriched cages. Thus rats housed in the standard cages do not provide a good, unstressed control because they are unable to avoid their cagemate if they so desire. In fact, a previous study stated that one third of bladder mast cells were degranulated in control animals, probably as a result of the stress of handling the animals and the mechanical damage during tissue removal (1). It is not necessary, or desirable, to accept such a high level of mast cell degranulation in control animals. In experiments in which the animals are housed appropriately, as described previously, mast cell degranulation in control animals varies between 5 and 10%.

2. An effective noise generator consists of an audio CD player with a white noise recording played in a loop mode and set to 90 dB. One speaker is positioned in the room equidistant from the animal cages. A water sprinkler timer is connected into the circuit so that the noise is automatically activated every day at a selected time and stopped 15 min later. The noise level of 90 dB (averaged over frequencies from 10 to 10,000 Hz) is similar to that produced by cage washers and air conditioners unless adequate care is taken, and is relatively low compared with the 110-dB tolerance level of rats (13). Thus, rats that are housed in many institutional animal facilities are stressed in an uncontrolled manner just by the environmental noise (14,15).

3. DAB is used because it stains peroxidase-containing granules in immune cells, such as eosinophils, and thus makes the cells easier to distinguish from mast cells. The DAB is prepared as follows (16): DAB (0.1 g) is added to 50 mL of 0.1 M monobasic phosphate buffer, and the pH is adjusted to 7.2 very gradually with concentrated NH4OH. The solution becomes a light tannish-pink color. Next, the tissue squares are rinsed in distilled water. Meanwhile, 25 mL of DAB solution is added to 1.66 mL of 3% H2O2 to give a final concentration of 0.2%.

4. There is a wide heterogeneity in the structure and function of mast cells that has been documented both in vitro and in vivo (19). For this reason the Alcian blue-Safranin method for mast cell staining is used in order to differentiate between the mast cells containing proteoglycan heparin (red stain) and those with bio-genic amines (blue stain).

5. To stain mesenteric mast cells more quickly, after fixation suffuse the tissue with 1% Toluidine blue for 20 s, rinse with HEPES-buffered saline (pH 7.4), and remount. Degranulated mast cells can be recognized by the presence of intra-cellular granules released into the surrounding tissue. This technique is fast and simple but it has the disadvantages that the Toluidine blue diffuses from the mast cells into the tissue very quickly (within 20 min), and that only the histamine is visualized, not the heparin.

Acknowledgments

This work was funded by NIH grant R21 AT 1124.

References

1. Spanos, C., Pang, X., Ligris, K., et al. (1997) Stress-induced bladder mast cell activation: implications for interstitial cystitis. J. Urol. 157, 669-672.

2. Kimata, H. (2003) Enhancement of allergic skin wheal responses in patients with atopic eczema/dermatitis syndrome by playing video games or by a frequently ringing mobile phone. Eur. J. Clin. Invest. 33, 513-517.

3. Singh, L. K., Pang, X., Alexacos, N., Letourneau, R., and Theoharides, C. (1999) Acute immobilization stress triggers skin mast cell degranulation via corticotro-phin releasing hormone, neurotensin, and substance P: a link to neurogenic skin disorders. Brain Behav. Immun. 13, 225-239.

4. Theoharides, T. C., Singh, L. K., Boucher, W., et al. (1998) Corticotropin-releas-ing hormone induces skin mast cell degranulation and increased vascular permeability, a possible explanation for its proinflammatory effects. Endocrinology 139, 403-413.

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8. Skofitsch, G., Savitt, J. M., and Jacobowitz, D. M. (1985) Suggestive evidence for a functional unit between mast cells and substance P fibres in the rat diaphragm and mesentery. Histochemistry 82, 5-8.

9. Fewtrell, C. M. S., Foreman, J. C., Jordan, C. C., Oehme, P., Renner, H., and Stewart, J. M. (1982) The effects of substance P on histamine and 5-hydrox-ytryptamine release in the rat. J. Physiol. 330, 393-411.

10. Alexacos, N., Pang, X., Boucher, W. Cochrane, D. E., Sant, G. R., and Theoharides, T. C. (1999) Neurotensin mediates rat bladder mast cell degranulation triggered by acute psychological stress. Urology 53, 1035-1040.

11. Imaki, T., Xiao-Quan, W., Shibasaki, T., et al. (1995) Stress-induced activation of neuronal activity and corticotrophin-releasing factor gene expression in the paraventricular nucleus is modulated by glucocorticoids in rats. J. Clin. Invest. 96, 231-238.

12. Playfair, J. H. L. (1996) Immunology at a Glance, Sixth edition. Blackwell Science, London, UK.

13. Riley, V. (1981) Psychoneuroendocrine influences on immunocompetence and neoplasia. Science 212, 1100-1109.

14. Milici, A. J. and Bankston, P. W. (1982) Fetal and neonatal rat intestinal capillaries: permeability to carbon, ferritin, hemoglobin and myoglobin. Am. J. Anat. 165, 165-186.

15. Mayrhofer, G. (1980) Fixation and staining of granules in mucosal mast cells and intraepithelial lymphocytes in the rat jejunum, with special reference to the relationship between the acid glycosaminoglycans in the two cell types. Histochem. J. 12, 513-526.

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19. Katz, H. R., Stevens, R. L., and Austen, K. F. (1985) Heterogeneity of mammalian mast cells differentiated in vivo and in vitro. J. Allergy Clin. Immunol. 76, 250-259.

Roles of Mast Cells in Host Defense

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