Endometriosis Is Associated With a Shift in MU Opioid and NMDA Receptor Expression in the Brain Periaqueductal Gray

Abstract

Studies have examined how endometriosis interacts with the nervous system, but little attention has been paid to opioidergic systems, which are relevant to pain signaling. We used the autotransplantation rat model of endometriosis and allowed to progress for 60 days. The brain was collected and examined for changes in endogenous opioid peptides, mu opioid receptors (MORs), and the N-methyl-d-aspartate subunit receptor (NR1) in the periaqueductal gray (PAG), since both of these receptors can regulate PAG activity. No changes in endogenous opioid peptides in met- and leu-enkephalin or β-endorphin levels were observed within the PAG. However, MOR immunoreactivity was significantly decreased in the ventral PAG in the endometriosis group. Endometriosis reduced by 20% the number of neuronal profiles expressing MOR and reduced by 40% the NR1 profiles. Our results suggest that endometriosis is associated with subtle variations in opioidergic and glutamatergic activity within the PAG, which may have implications for pain processing.

Keywords: endometriosis; mu opioid receptor; opioid peptides; periaqueductal gray; rat; stress.

Figure 1.

Timeline of experimental procedures. Animals were handled and vaginal smears collected 1 week prior to endometriosis induction. Endometriosis was allowed to progress. Between days 7 to 14 and 24 to 31, we checked for estrous cyclicity. All animals were killed 60 days after surgery.

Figure 2.

Both types of mu opioid receptor (MOR) antibodies used have a high degree of overlapping in their immunofluorescence. A1 and A2, Sample MOR immunoreactivity of 2 different fields within the ventral periaqueductal gray (PAG) of an endometriosis rat. Neurons were labeled using the rabbit anti-MOR (1:1000) from ImmunoStar followed by CY3 secondary antibody. This antibody has been widely characterized. B1 and B2, Sample immunoreactivity of the same fields shown in (A), using the guinea pig anti-MOR (1:1000) from Millipore followed by Alexa488 secondary antibody. C1 and C2, Image merge of the immunoreactive fields from both antibodies showing a high degree of colocalization (yellow; arrows). (The color version of this figure is available in the online version at http://rs.sagepub.com/.)

Figure 3.

Endometriosis reduces mu opioid receptor (MOR)-like immunoreactivity within the ventral periaqueductal gray (PAG) neurons. A, Sample MOR immunoreactivity in the ventral PAG from sham animal and (B) from an endometriosis animal. C, Average cell fluorescence (corrected for area) was decreased in the endo group. n = 4 to 5 ± standard error of the mean (SEM). *P < .05 versus sham on a Mann-Whitney U test. Bar = 40 μm.

Figure 4.

Endometriosis is associated with a shift in N-methyl-d-aspartate (NMDA) receptor 1 (NR1) more than mu opioid receptor (MOR). Sample MOR (A) and NR1 (B) immunofluorescence in the periaqueductal gray (PAG). C, The merged image of dually labeled immunoreactive profiles (neuronal and or glial). D, MOR⁻ NR1⁺ profiles and dually labeled profiles were significantly higher than MOR⁺ NR1⁻ profiles. In the endo animals, we observed a nonsignificant decrease in all types of profiles. E, Percentage change in the mean number of labeled profiles in the endo group compared to the sham group. There was double the decrease in MOR⁻NR1⁺ profiles as in the other 2 categories. n = 4 to 6 ± standard error of the mean (SEM). *P < .05 on a 2-way analysis of variance (ANOVA) followed by Tukey multiple comparison test. Bar = 60 μm.

Figure 5.

Endometriosis did not alter opioid peptide immunoreactivity in the periaqueductal gray (PAG). A, Sample met-enkephalin immunoreactivity in the dorsal part of the PAG was diffuse and some somata-like structures were evident. B, Semiquantitative measures of met-enkephalin immunoreactivity revealed no differences between groups. C, A cluster of leu-enkephalin immunoreactive fibers was evident in the ventrolateral part of the PAG. D, There were no differences in immunoreactivity between groups. E, β-Endorphin immunoreactivity was abundant and most evident in the ventrolateral compartments. Punctae immunoreactivity was very evident. F, Similar to enkephalin, we found no differences in immunoreactivity between groups on a Student t test. Aq represents cerebral aqueduct (A, C, and E all from endo rats. Bar = 50 μm).