Estrogen effects on pain sensitivity and neuropeptide expression in rat sensory neurons

Abstract

While a number of chronic pain conditions are much more prevalent in women than men, the role of estrogen in regulating nociception remains unclear. Estrogen receptors (ER) are known to be expressed in various parts of the nociceptive pathway, including in the small-sized primary sensory neurons of the dorsal root ganglion (DRG). This study evaluated the effects of long term estrogen replacement on pain sensitivity and neuropeptide expression in the DRG of female Sprague Dawley rats. The goal was to evaluate whether estrogen modulates nociceptive neuropeptides in the DRG in a manner consistent with its effects on pain sensitivity. Our results show that long term (28 days) ovariectomy (ovx) of adult rats induces a profound thermal and mechanical hyperalgesia of the hindpaw and tail compared to ovariectomized animals that were continuously estrogen-treated (ovx+E). Significant changes in the expression of two neuropeptides, substance P (SP) and calcitonin gene-related peptide (CGRP), were observed using immunocytochemistry and in situ hybridization (ISH) in the small lumbar DRG neurons which contain ER. CGRP and SP were differentially regulated by estrogen, with SP showing a significant downregulation at both the peptide and mRNA levels while CGRP and its mRNA were increased in the DRG of estrogen-treated animals. We also evaluated the development of mechanical allodynia after partial sciatic nerve injury and found that both ovx and ovx+E animals developed significant allodynia within a week of the partial nerve injury, which continued for at least one month. The estrogen-treated animals showed a partial amelioration of the extent of the allodynia at 2 weeks post injury. Overall, the results suggest that estrogen has significant anti-nociceptive actions that can be directly correlated with changes in expression of two peptides in the small nociceptive ERalpha expressing neurons of the DRG.

Figure 1

Immunocytochemical evidence for the estrogenic modulation of SP and CGRP expression in the DRG. Sections of rat DRGs were reacted with polyclonal antibodies specific to SP and CGRP and visualized using the Vectastain ABC peroxidase kit. Representative examples of staining in the DRG of ovx and ovx+E animals are presented. Note reduced level of SP immunostaining in the +E condition compared to the enhanced level of CGRP immunostaining in the +E condition. Bar=70µm.

Figure 2

In situ hybridization evidence for the estrogenic modulation of SP and CGRP mRNA expression in the DRG. Sections of rat DRGs were hybridized with specific ³³P-labeled riboprobes and autoradiograms were prepared. Top panels show examples of SP mRNA localization in ovx (A) and estrogen-treated (B) groups in bright field photomicrographs of emulsion coated sections. Quantitative data grain counting analyses are shown in the lower graph. Average grain densities (number of grains/µm²) and SEM for small-sized DRG neurons are plotted for the ovx and +E groups with both riboprobes as indicated. The asterisks* indicate significant differences (p<0.05, n= 8) between the two groups for each mRNA probe.

Figure 3

Baseline thermal sensitivity levels of hindpaw and tail are influenced by estrogen treatment. The graphs show the mean time (sec) to withdrawal of the paw or tail as indicated from a thermal point source emitted from an analgesia meter. Assessments were made one month after ovariectomy (ovx) without or with (+E) estrogen replacement done by capsule implants. The mean latencies +/− SEM are plotted; significant differences between the groups are (p<. 05, n = 8) are indicated by asterisks.

Figure 4

Mechanical allodynia of the hindpaw develops after partial sciatic nerve injury and is influenced by estrogen treatment. A. Sensitivity of the hindpaw to von Frey filaments was evaluated and the mean gm force of the monofilament needed to elicit paw withdrawal was determined in groups of animals that were ovariectomized (ovx) for 28 days prior to the nerve injury vs. those ovariectomized and treated with estrogen capsule implants. Both groups developed significant allodynia by day 7 post lesion; estrogen-treated animals were less sensitive than ovx counterparts at day 13 (asterisk indicates significance in post-hoc testing at p < .05; n = 8). B. Data were normalized to each group’s prelesion control and expressed as a percentage. Both groups showed significant hypersensitivity to non-noxious mechanical stimuli but estrogen had a significant ameliorative, albeit partial, effect at day 13 (askerisk indicates significance in post-hoc testing at p < .05; n = 8).