Pirt contributes to uterine contraction-induced pain in mice

Abstract

Uterine contraction-induced pain (UCP) represents a common and severe form of visceral pain. Nerve fibers that innervate uterine tissue express the transient receptor potential vanilloid channel 1 (TRPV1), which has been shown to be involved in the perception of UCP. The phosphoinositide-interacting regulator of TRP (Pirt) may act as a regulatory subunit of TRPV1. The intraperitoneal injection of oxytocin into female mice after a 6-day priming treatment with estradiol benzoate induces writhing responses, which reflect the presence of UCP. Here, we first compared writhing response between Pirt (+/+) and Pirt (-/-) mice. Second, we examined the innervation of Pirt-expressing nerves in the uterus of Pirt (-/-) mice by immunofluorescence and two-photon microscopy. Third, we identified the soma of dorsal root ganglion (DRG) neurons that innerve the uterus using retrograde tracing and further characterized the neurochemical properties of these DRG neurons. Finally, we compared the calcium response of capsaicin between DRG neurons from Pirt (+/+) and Pirt (-/-) mice. We found that the writhing responses were less intensive in Pirt (-/-) mice than in Pirt (+/+) mice. We also observed Pirt-expressing nerve fibers in the myometrium of the uterus, and that retrograde-labeled cells were small-diameter, unmyelinated, and Pirt-positive DRG neurons. Additionally, we found that the number of capsaicin-responding neurons and the magnitude of evoked calcium response were markedly reduced in DRG neurons from Pirt (-/-) mice. Taken together, we speculate that Pirt plays an important role in mice uterine contraction-induced pain.

Fig. 1

The writhing response behavior and gene expression analysis. a The time course of writhing response during the 80 min after oxytocin injection. b Number of writhing responses in Pirt ^+/+ (8.4 ± 1.6) and Pirt ^−/− mice (3.1 ± 0.7) after 6 days of priming with estradiol benzoate and induction by oxytocin (^** P < 0.01, n = 7/group). c Real-time PCR showed that Pirt gene expression was not affected by treatment with estradiol and oxytocin

Fig. 2

Pirt-expressing nerves innervate the uterus. a Hematoxylin and eosin-stained section of the uterus. Myometrium and endometrium are indicated by white arrow and blue arrow, respectively. b Image of the uterine section under fluorescence microscopy. In Pirt^−/− mice, the entire Pirt-coding region was replaced by an EGFPf-IRESrtTA-ACN-targeting construct, which expresses green fluorescent protein. Thus, Pirt-expressing fibers can be identified by green fluorescence. c Superimposed images of a and b. Pirt-expressing fiber is indicated by a white arrow. d–f Higher resolution views of the boxed areas in a–c, respectively. Fluorescence (g) and bright-field (h) images from two-photon microscopy analysis showing Pirt-expressing nerve fibers in the myometrium (white arrow). Scale bar 100 µm

Fig. 3

Small-diameter DRG neurons innervate the uterus. Bright-field (a) and fluorescence (b) imaging of Dil-labeled DRG. c Overlapping images from a and b permit differentiation between traced and untraced fibers. Scale bar 20 µm. d Most DRG neurons that received input from the uterus had a small soma size, with a surface area ranging from 100 to 600 μm² (1599 labeled neurons in T13 DRG)

Fig. 4

The neurochemical properties of Dil-labeled DRG neurons. a–c Dil-labeled cells did not colocalize with NF-200. Dil-labeled cells and NF-200-expressing cells are indicated by white and yellow arrows, respectively. Dil-labeled DRG neurons colocalized with IB4 (d–f) and Pirt (g–i). j–l DRG section including Pirt-positive and Dil-labeled neurons was immunostained by TRPV1. m A merged image of Dil-labeled, Pirt-positive and TRPV1-staining neurons. The white arrows designate double-labeled cells. Pirt expression was assessed by fluorescence microscopy in Pirt^−/− mice, in which the entire Pirt-coding region has been replaced with an EGFPf-IRESrtTA-ACN-targeting construct. Scale bar 20 µm

Fig. 5

Dil-labeled DRG neurons respond to capsaicin. a Representative capsaicin-induced Ca²⁺ trace in DRG neuron from Pirt ^+/+ mouse. b Images of DRG neurons from Pirt ^+/+ mice before (B1) and after (B2) capsaicin treatment (5 μM). c Representative capsaicin-induced Ca²⁺ trace in DRG neuron from Pirt^+/+ mouse. d Images of DRG neurons from Pirt^−/− mice before (D1) and after (D2) capsaicin. Scale bar 20 µm. e The proportion of DRG neurons from Pirt ^+/+ and Pirt ^−/− mice that responded to capsaicin (% of total cells, *P < 0.05, n = 3/group). f 382 of 816 accounted Pirt ^+/+ positive cells were Dil-labeled cells (46 %). 120 of 382 Dil-labeled cells (31 %) responded to capsaicin. 204 of 816 Pirt ^+/+ neurons (25 %) responded to capsaicin