TRPV1 expression in acupuncture points: response to electroacupuncture stimulation

Abstract

The present study was to examine the distribution of transient receptor potential vanilloid type-1 (TRPV1) receptor immunoreactivity in the acupuncture points (acupoint), and determine the influences of electroacupuncture (EA) stimulation on TRPV1 expression. EA stimulation of BL 40 was conducted in two sessions of 20 min separated by an 80 min interval in anesthetized rats. Sections of skin containing BL 40, and its non-meridian control were examined by immunolabeling with antibodies directed against TRPV1. Without EA, the number of subepidermal nerve fibers expressing TRPV1 was higher in the acupoint than in non-acupoint control skin (p<0.01). The subepidermal nerve fibers showed the co-localization of TRPV1 with peripherine, a marker for the C-fibers and A-δ fibers. The expression of TRPV1 in nerve fibers is significantly increased by EA stimulation in acupoints (p<0.01). However the upregulation in the non acupoint meridian and the non-meridian control skin was short of statistical significance. Double immunostaining of TRPV1 and neuronal nitric oxide synthase (nNOS) revealed their co-localization in both the subepidermal nerve fibers and in the dermal connective tissue cells. These results show that a high expression of TRPV1 endowed with nNOS in subepidermal nerve fibers exists in the acupoints and the expression is increased by EA. We conclude that the higher expression of TRPV1 in the subepidermal nerve fibers and its upregulation after EA stimulation may play a key role in mediating the transduction of EA signals to the CNS, and its expression in the subepidermal connective tissue cells may play a role in conducting the local effect of the EA.

Copyright © 2011 Elsevier B.V. All rights reserved.

Figures

Figure 1

A photomicrograph showing the structure and the distribution of TRPV1 immunoreactivity in non-meridian skin section (NMC) of a control rat. The basal and the granular layers of epidermis (E) were intensely immunoreactive to TRPV1, while the superficial layers appeared less intensely reactive. The immunoreactivity of TRPV1 existed in the hair follicles (F) and was more prominent in the inner layer of the root sheath, while the outer layers appeared less intensely reactive. The hair by itself appeared intensely immunoreactive to TRPV1 (H) while the sebaceous gland (S) was weakly reactive. In the dermis (D), only few subepidermal nerve fibers (thin arrow) as well as CT cells (arrow head) were positively reactive to TRPV1. Staining with secondary antibodies labeled with Alexa Fluor 488 (green). Scale bar, 27 μm.

Figure 2

Immunoreactivity of TRPV1 in the skin sections from a non-meridian control (NMC), a non-acupoint meridian (NAM) and acupoint (AP) in the rats without EA and after EA stimulation. TRPV1 expression in subepidermal nerve fibers (thin arrow) and connective tissue cells (thick arrow) are higher in the AP than in the NAM and the NMC in both EA stimulated and non stimulated rats. Also note the higher expression of TRPV1 in the skin sections after EA stimulation compared to the same sections without EA stimulation. A high reactivity to TRPV1 in the nerve fibers surrounding the hair follicle (HF) was noticed after EA stimulation compared to without EA stimulation. Staining with secondary antibodies labeled with Alexa Fluor 488 (green) Scale bar, 40 μm.

Figure 3

The number of TRPV1 positive nerve fibers in skin sections of control (NMC), non point (NAM) and acupoint (AP) in rats with and without EA stimulation. The expression of TRPV1 in the nerve fibers is significantly increased after EA stimulation in AP. The expressions in the acupoint of both stimulated and non-stimulated groups are significantly higher than those in control skin. The bars represent the mean ±SE (n=4-5 animals). Significant differences are indicated by **p

Figure 4

Immunoreactivity of TRPV1 in the AP skin of the rat after EA stimulation. (A) Confocal photomicrograph showing the immunoreactivity of the subepidermal nerve fibers (small arrow) to TRPV1, peripherine, and their colocalization resulting in the orange color of the nerve fibers. (B) A higher magnification of a transverse section in a dermal nerve trunk showing the immunoreactivity to both TRPV1 and peripherine, their overlay panel shows the main distribution of the TRPV1 in the neurelemma and perineurium (double arrow) while the peripherine is mainly confined to the axons. (C) immunoreactivity of a dermal nerve fiber to both TRPV1 and peripherine (thick arrow) (F, hair follicle) (D) specificity of immunoreactivity to peripherine was proven by replacement of peripherine antibody with rabbit IgG serum which didn't result in any specific immunostaining of dermal nerve fibers. Staining with secondary antibodies labeled with Alexa Fluor 488 (green) and Alexa Fluor 594 (red). Scale bar: 80 μm, A; 10 μm, B; 40 μm, C&D.

Figure 5

Confocal photomicrographs in the rat skin sections showing the immunofluorescence labeling of TRPV1 (green), nNOS (red) and their colocalization (orange) in the subepidermal nerve fibers, and CT cells (A). A higher magnification showing the immunoreactivity of the connective tissue cells (B), and the subepidermal nerve fiber (A) to both TRPV1 and nNOS. (D) No specific immunostaining of the connective tissue cells nor the subepidermal nerve fibers when replacing the primary antibody with a non immune goat and rabbit sera. Staining with secondary antibodies labeled with Alexa Fluor 488 (green) and Alexa Fluor 594 (red). Scale bar: 80 μm, A&D, 40 μm, C, 16 μm, B. (E), epithelium ( ) subepidermal nerve fibers, (⇧) CT cells.