**2. Materials and methods**

#### **2.1 Animals**

We have used 6~8 week female C57BL/6J, B6.129P2-Esr1tm1Ksk/J, and B6.129P2-Esr2tm1Unc/J mice were obtained the Jackson Laboratory (Bar Harbor, ME, USA). Upon arrival, mice were group housed in microisolator caging and maintained on a 12-h light/dark cycle in a temperature-controlled environment with access to food and water ad libitum. To test whether estrogen receptor α (ERα) or estrogen receptor β (ERβ) are involved in estradiol (E2)-induced modification of [Ca2+]*і* Wile type, estrogen receptor alpha knock-out (ERαKO) and estrogen receptor beta knock-out (ERβKO) mice will be used. The wild type, ERαKO and ERβKO mice will be obtained from the supplier and allowed to recover for two weeks. These studies were carried out in accordance with the guidelines of the Institutional Animal Care and Use committee at the University of California and the NIH Guide for the Care and Use of Laboratory Animals.

#### **2.2 Animal breeding**

372 Endometriosis - Basic Concepts and Current Research Trends

Most of the published reports about sex and hormone-related differences in pain have addressed the modulatory effect of E2 on central nervous system mechanisms of nociception (Aloisi *et al.* 2000). Recent studies demonstrate that E2 has a significant role in modulating viscerosensitivity, indicating that E2-induced alterations in sensory processing may underlie sex-based differences in functional pain syndromes (Al-Chaer and Traub 2002). However, reports of E2 modulation of visceral and somatic nociceptive sensitivity are inconsistent. For example, elevated E2 levels have been reported to increase the threshold to cutaneous stimuli but decrease the percentage of escape responses to ureteral calculosis (Bradshaw and Berkley 2002). Additionally, nociceptive sensitivity increases when E2 levels are elevated (Holdcroft 2000; Bereiter 2001). Indeed in most clinical studies, women report more severe pain levels, more frequent pain and longer duration of pain than men. To help resolve these inconsistencies we propose to study E2 actions on the primary afferents.

Primary DRG neurons culture has been a useful model system for investigating sensory physiology and putative nociceptive signaling (Chaban *et al*. 2003). ATP-induced intracellular calcium concentration ([Ca2+]*i*) transients in cultured DRG neurons have been used to model the response of nociceptors to painful stimuli. In our laboratory we showed that E2, acting at the level of the plasma membrane, attenuates both ATP -induced [Ca2+]<sup>і</sup> and capsaicin- induced [Ca2+]i influx and that the expression of both P2X3 and TRPV1 depend on the expression of both ERs. Within the context of our hypothesis visceral nociception and nociceptor sensitization appear to be regulated by P2X3 and TRPV1. Estrogen attenuates DRG neurons response to ATP and capsaicin suggesting that visceral afferent nociceptors can be modulated by sex steroids at a new site at the level of primary afferent neurons. Our data suggest that E2 by itself appears to be anti-nociceptive but interferes with anti-nociceptive actions of other pain-modulating drugs (such as opioids). Thus, E2 acting on primary afferent nociceptors modulates the response to pro- and antinociceptive signals**.** Within the context of our cross-sensitization hypothesis, inflammation sensitizes non-inflamed viscera that are innervated by the same DRG and/or crosssensitization occurs as a result of intra-DRG release of sensitizing mediators such as ATP or

Lumbosacral DRG neurons (levels L6-S1) from wild type mice (WT) express estrogen receptors (ERα and ERβ), purinergic P2X3, vanilloid TRPV1, SP and methabotropic glutamate (mGluR2/3) receptors. In our recent studies we also tested the difference in how somatic and visceral afferents are modulated by E2. Both short-term and long-term exposure

We have used 6~8 week female C57BL/6J, B6.129P2-Esr1tm1Ksk/J, and B6.129P2-Esr2tm1Unc/J mice were obtained the Jackson Laboratory (Bar Harbor, ME, USA). Upon arrival, mice were group housed in microisolator caging and maintained on a 12-h light/dark cycle in a temperature-controlled environment with access to food and water ad libitum. To test whether estrogen receptor α (ERα) or estrogen receptor β (ERβ) are involved in estradiol (E2)-induced modification of [Ca2+]*і* Wile type, estrogen receptor alpha knock-out (ERαKO) and estrogen receptor beta knock-out (ERβKO) mice will be used. The wild type, ERαKO and ERβKO mice will be obtained from the supplier and allowed to recover for two weeks. These studies were carried out in accordance with the guidelines of the Institutional Animal

substance P in the DRG (Matsuka *et al.* 2001; Chaban 2008; Chaban 2010).

**2. Materials and methods** 

**2.1 Animals** 

to E2 significantly decreased the ATP and capsaicin-induced increase in [Ca2+]i.

Experiments were performed on age-matched (8–10 wk old) heterozygous mutant mice lacking the gene male (ERα−*/*−) and female (ERα−*/*−) for ERα (ERα−*/*−), and the deficiency ERβ (ERβ−*/*−) mice were bred into heterozygous mutant female mice (ERβ−*/*−) and homozygous male mutant mice (ERβ−*/*−) (Jackson Laboratory, Bar Harbor, ME, USA). Mice were housed in climate-controlled rooms, and standard rodent chow and water were available *ad libitum* and were housed in accordance with the NIH Guide for the Care and Use of Laboratory Animals

#### **2.3 Primary culture of DRG neurons**

The isolation procedure and primary culture of mouse lumbosacral DRG has been published in detail (Chaban, Mayer et al. 2003). DRG tissues were obtained from c57/black 6J (The Jackson Laboratory; 30 g), ERαKO and ERβKO (Taconic; 20 g) transgenic types. Briefly, lumbosacral adult DRGs (level L1-S1) from Wt, ERαKO and ERβKO mice will be collected under sterile technique and placed in ice-cold medium Dulbecco's Modified Eagle's Medium (DMEM; Sigma Chemical Co., St. Louis, MO). Adhering fat and connective tissue will be removed and each DRG will be minced with scissors and place immediately in a medium consisting of 5 ml of DMEM containing 0.5 mg/ml of trypsin (Sigma, type III), 1 mg/ml of collagenase (Sigma, type IA) and 0.1 mg/ml of DNAase (Sigma, type III) and kept at 37°C for 30 minutes with agitation. After dissociation of the cell ganglia, soybean trypsin inhibitor (Sigma, type III) will be used to terminate cell dissociation. Cell suspension will be centrifuged for one minute at 1000 rpm and the cell pellet will be resuspended in DMEM supplemented with 5% fetal bovine serum, 2 mM glutamine-penicillin-streptomycin mixture, 1 μg/ml DNAase and 5 ng/ml NGF (Sigma). Cells will be plated on Matrigel® (Invitrogen)-coated 15-mm coverslips (Collaborative Research Co., Bedford, PA) and kept at 37° C in 5% CO2 incubator for 24 hrs, given fresh media and maintained in primary culture until used for experimental procedures.

#### **2.4 Western blot analysis**

The expressions of TRPV1 and of P2X3 receptors in L1~S1 DRGs were studied by using Western blot analyses. Tissues from wild type (C57BL/6J), ERαKO, and ERβKO mice were quick frozen in tubes on dry ice during collection. L1~S1 DRG were combined, homogenized by mechanical disruption in ice-cold RIPA buffer plus protease inhibitors and incubated on ice for 30 minutes. Homogenates were then spun at 5000 g for 15 minutes and supernatants collected. Total protein was determined on the supernatants using the BCA microtiter method (Pierce, Rockford, Ill., USA). Samples containing equal amounts of protein (40µg) were electrophoresed under denaturing conditions using Novex Mini-cell system (San Diego, Calif., USA) and reagents (NuPage 4–12% Bis-Tris gel and MOPS running buffer). After electrophoretic transfer onto nitrocellulose membrane using the same system, the membrane was blocked with 5% non-fat dry milk (NFDM) in 25 mM TRIS buffered saline, pH 7.2, plus Tween 20 (TBST) for 1 hour at room temperature, followed by incubation with polyclonal rabbit antibody against TRPV1-N terminus (1:1000, Neuromics) and P2X3 receptor (1:1000, Neuromics) for overnight at 4oC. The membrane was then

Primary Afferent Nociceptors and Visceral Pain 375

mounted on a fast-perfusion chamber P-4 (World Precision Instrument) and placed on a stage of Olympus IX51 inverted microscope. Observations were made at room temperature (20- 23°C) with 20X UApo/340 objective. A fast superfusion system will be used to perfuse the cells with HBSS and rapidly apply E2 and other chemicals. Fluorescence intensity at 505 nm with excitation at 334 nm and 380 nm was captured as digital images (sampling rates of 0.1-2 s). Regions of interest were identified within the soma or neuritis from which quantitative measurements will be made by re-analysis of stored image sequences using Slidebook® Digital Microscopy software. [Ca2+]*i* was determined by ratiometric method of Fura-2 fluorescence from calibration of series of buffered Ca2+ standards. We applied E2 acutely for five minutes onto the experimental chamber or the culture medium for 48 hours to study the prolonged effect of E2. Repeated applications of drugs were achieved by superfusion in a rapid mixing chamber into individual neurons for specific intervals (100-500 ms). Cells were perfused with

DRG neurons innervating viscera were identified by retrograde labeling. Briefly, mice were anesthetized with isoflurane. For colonic afferents, the descending colon was exposed and Fluorogold (5% solution in PBS; Molecular Probes, Eugene, OR) was injected into the intestinal muscle wall (10 μl injections of into five to six different sites) using a Hamilton syringe (Hamilton Co., Reno, NV) with a 26-gauge needle. In another experiments we used uterus-specific DRG neurons in which tetramethylrhodamine (TMR) dye was injected in the uterus. Injection sites were carefully swabbed, the colon and uterus were extensively rinsed with 0.9% sodium chloride solution and sealed with New Skin to prevent dye leakage. The abdomen was sutured and the animals monitored for signs of pain or discomfort during the survival period. All animals were allowed to survive one week to allow for maximal transport of retrograde markers and housed in groups of two under 12/12 hours light cycle

The amplitude of [Ca2+]*i* response represents the difference between baseline concentration and the transient peak response to drug stimulation. Significant differences in response to chemical stimulation will be obtained by comparing [Ca2+]*i* increases during the first stimulation with the second. A cell will be judged responsive if E2 inhibits the second [Ca2+]*<sup>i</sup>* transient by >30% of the first. This criterion was empirically derived in preliminary experiments. All of the data are expressed as the mean ± SEM. Statistical analysis was performed using Statistical Package for the Social Sciences 12.0 (SPSS, Chicago, IL, USA). To assess the significance among different groups, data were analyzed with one-way ANOVA

followed by Schéffe post hoc test. A *P* <0.05 was considered statistically significant.

**3.1 Role of P2X3 receptors in estrogen-induced nociceptive signaling in sensory** 

P2X3 and TRPV1 receptors expression were examined by western blot analysis of lysates from wild type, ERαKO, and ERβKO DRG tissues using a P2X3 specific primary antiserum (Fig.1 (a)). An intense band representing a ~64 kDa protein (P2X3) and a ~130 kDa (TRPV1) was seen

experimental media (2 ml/min) using a Rainin® peristaltic pump.

**2.7 Retrograde labeling** 

**2.8 Statistical analysis** 

**3. Results** 

**neurons**

with food and water available *ad libitum.*

washed in TBST plus NFDM, and incubated with secondary antibody, HRP conjugated and rabbit IgG (Santa Cruz Biotechnology) at 1:5,000 in the same buffer for 2 hours at room temperature. Following a final wash in TBST without NFDM, the membrane was incubated with ECL+ (Amersham, Arlington Heights, Ill., USA) substrate for HRP. Membranes were probed with primary antibody and corresponding secondary antibodies, signals were scanned and quantified by Image J version 1.28U and NIH Image 1.60 scan software. Following enhanced chemiluminescence (ECL) detection of proteins, the membranes were stripped and rehybridized with β-actin antibody as a loading control. At least three independent cell preparations were used.

### **2.5 Immunohistochemistry (IHC)**

DRG tissues were obtained from C57/black 6J (The Jackson Laboratory; 30 g), ERαKO and ERβKO (Taconic; 20 g) transgenic types. Following decapitation, DRG from bilateral spinal levels L1-S2 were removed and fixed in 4% paraformaldehyde for overnight at 4oC, according to procedures approved by National Institutes of Health policy on Humane Care and Use of Laboratory Animals. DRGs were rinsed in Delbecco's Phosphate Buffered Saline (DPBS) before cryoprotection in sucrose (20%, 4oC) for two days, after which excess liquid was removed. DRG were quick snap frozen in 2-methylbutane, and store them at -70oC. Each DRG was mounted in Tissue-Tek® OCT embedding medium (Sakura Finetek), and sectioned at - 20oC in a MICROM H505E cryostat. Sections were cut at 20µm and store 4oC until required. Sections of DRGs were collected in PBS. Endogenous tissue peroxidase activity was quenched by soaking the sections for 10 min in 3% hydrogen peroxide solution in 0.01 M PBS. The specimens were washed and then treated for 60 min in blocking solution, 0.01 M PBS containing 0.5% Triton X-100 and 1% normal donkey serum (NDS) at room temperature. They were processed for wild type (n=4), ERαKO (n=4), or ERβKO (n=4) immunohistochemistry by the free floating method using polyclonal rabbit TRPV1 antibody (1:50000, Neuromics) or P2X3 receptor antibody (1:15000, Neuromics) for overnight at 4oC, washed in 0.01 M phosphate-buffered saline (PBS) and 0.01M Tris Buffered Saline (TBS), followed by incubation in solutions of donkey anti-rabbit fluorophore-conjugated secondary antibodies (1:200, Invitrogen) in 0.01M Tris Buffered Saline (TBS) for 3 hours at room temperature. Cells showing no apparent or only faint membrane intracellular labeling were considered to be negative for TRPV1 or P2X3. TRPV1-positive cells included those with strong plasma membrane labeling that formed a discernible clustered pattern, and those with strong intracellular labeling that formed a punctuate pattern. Some neurons showed both strong plasma membrane and intracellular labeling. P2X3-positive neurons showed diffuse membrane intracellular labeling. Mounted sections were air dried and coverslipped with Aqua Poly Mount (Polisciences, Warrington, PA). Images from at least three sections in each level were taken using Leica DMLB M130X microscope. The total numbers of DRG neurons expressing TRPV1 and P2X3 were counted. TRPV1- or P2X3-positive neurons were categorized according to their labeling patterns and were expressed as a percentage of the total number of TRPV1- or P2X3-positive cells. Immunohistochemical signal percent was measured by computerized image analysis (Image Pro-Plus, Media Cybernetics, Silver Spring, MD, USA).
