Appearance of Peculiar Vessels with Immunohistological Features of High Endothelial Venules in the Dermis of Moxibustion-Stimulated Rat Skin

American Journal of Chinese Medicine, Summer-Fall, 2000 by Kazuo Tohya, Shiori Urabe, Jun Igarashi, Taro Tomura, Akemi Take, Michio Kimura

Abstract: Morphological changes of the dermal blood vessels of moxibustion-stimulated rat skin were examined with reference to the lymphocyte migration. After long-term stimulation with direct moxibustion to the acupoint Tsu-San-Li (St-36), peculiar vessels that possess immunohistological features of high endothelial venules could be observed in the moxa-stimulated acupoint dermis. Endothelial cells of the vessels had well-developed Golgi apparatus in their plump cytoplasms, and they strongly expressed intercellular adhesion molecule-1 on the luminal surface. These data suggest that the appearance of the peculiar vessels in the dermis acts toward the active infiltration of blood-lymphocytes into the acupoint skin.

Moxibustion is one of the well-known Chinese traditional medical treatments, and it has been thought to increase the protective activities of the body. Recent immunological studies have shown that direct moxibustion to acupoint can mediate the systemic immune functions. In fact, an allergic response in delayed-type hypersensitivity could be reduced by the transfer oft lymphocytes from moxa-stimulated mice (Tohya et al., 1989), and the titer and activity of serum antibody against Staphylococcus aureus could be enhanced by direct moxibustion (Yamashita et al., 1998). On the basis of the current immunological aspects, one of the important factors that regulate these immune responses is systemic migration of recirculating lymphocytes through the bloodstream (Butcher and Picker, 1996). Previously, we reported that morphologically remarkable changes of the stimulated skin and the regional lymph nodes were observed in moxibustion-stimulated laboratory animals (Kimura et al., 1988). Nevertheless, little information is known about the immunohistological details of the moxa-stimulated acupoint skin with reference to the regulation of lymphocyte migration in the regional tissue. In this context, we have immunohistologically examined the features of acupoint skin stimulated by long-term direct moxibustion in the laboratory animals. Special attention was focused on the morphological and immunohistological changes of the dermal blood vessels and their endothelial cells.

Materials and Methods

Animals

Male Wistar rats (aged 8 to 10 weeks, 250-300 g), purchased from Japan SLC (Hamamatsu, Japan), were used. All treatments of the animals in the present study were performed under deep anesthesia by ether vapor.

Moxibustion

A skin electro-permeable point at an external area below the knee was selected as an acupoint, which was equivalent to Tsu-San-Li (St-36). Moxibustion to the acupoint was performed with three cones (0.5 mg moxa/cone) per point in a day. The stimulation was carried out for twenty consecutive days.

To investigate whether the effects of moxibustion depend on not only the burning or heating of moxa fiber, but also the chemical effect of the chemical component of moxa tar, we performed a control stimulation by the methanol-treated moxa-fiber in the same protocol with that of raw moxa.

General Histology

Twenty-four hours after the final moxibustion, the animals were fixed by transcardial perfusion of half-strength cold Kamovsky's solution (pH 7.4) (Karnovsky, 1965). The moxa-stimulated skin was removed and postfixed by 2% osmium tetroxide for 2 hours at 4 [degrees] C. After washing with 0.1 M phosphate buffer (pH 7.4), the samples were dehydrated by a graded ethanol series and embedded in epoxy resin. Ultrathin sections were made with an ultramicrotome; these were stained with uranyl acetate and lead citrate and examined under a transmission electron microscope (JEM 1200EX, JEOL, Tokyo).

Immunohistochemistry

Twenty-four hours after the final moxibustion, the animals were fixed by transcardial perfusion of cold periodate-lysine-paraformaldehyde solution (pH 7.4) (McLean and Nakane, 1974). The moxa-stimulated skin was removed and immersed in the same fixative for 12 hours at 4 [degrees] C. After washing with phosphate buffered saline (PBS, pH 7.4) containing 10% sucrose, the samples were snap-frozen at -85 [degrees] C. The frozen tissues were cut into 8-[micro]m-thick cryosections, and then incubated with the following monoclonal antibodies: anti-rat intercellular adhesion molecule-1 (ICAM-1), (1A29, Seikagaku, Tokyo), anti-rat lymphocyte-function associated antigen-1 (LFA-1), (WT. 1, Seikagaku, Tokyo), anti-rat CD4 (MRC OX-38, Cedarlane, Ontario), and anti-rat CD8 (MRC OX-8, Cedarlane, Ontario). After washing with PBS, the sections were reacted with the second antibody conjugated with fluorescene isothiocyanate (Jackson Immunoresearch Lab., PA). The stained sections were then observed under a fluorescent microscope (Olympus, Tokyo).

Quantitative Analysis

Quantitative analysis of the percentage of ICAM-1 positive HEV in the moxa stimulated dermis was performed. For 10 to 11 of the immunostained sections of each experimental group, the number of ICAM-1 positive vessels, including HEV, within 7.5 [mm.sup.2] of the dermal area (Figure 3a) were counted under the fluorescent microscope, and then the percentage of ICAM-1 positive HEV was calculated. The Mann-Whitney U-test was used to determine the levels of significance of difference. Differences at p [is less than] 0.05 were considered statistically significant.