The relationship of bone-tumor-induced spinal cord astrocyte activation and aromatase expression to mechanical hyperalgesia and cold hypersensitivity in intact female and ovariectomized mice

Neuroscience 324 (2016) 344–354

THE RELATIONSHIP OF BONE-TUMOR-INDUCED SPINAL CORD ASTROCYTE ACTIVATION AND AROMATASE EXPRESSION TO MECHANICAL HYPERALGESIA AND COLD HYPERSENSITIVITY IN INTACT FEMALE AND OVARIECTOMIZED MICE B. A. SMEESTER, a E. E. O’BRIEN, a K. S. MICHLITSCH, a J.-H. LEE b AND A. J. BEITZ c*

tumor-induced nociception. Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

a Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St Paul, MN, USA b Department of Veterinary Physiology, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, Republic of Korea

Key words: astrocytes, estrogen, aromatase, letrozole, tumor pain, spinal cord.

c

Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, 1971 Commonwealth Avenue, St Paul, MN 55108, USA

INTRODUCTION Localized estrogen synthesis plays a significant physiological role in tissue-specific functions (Cui et al., 2013). In addition to peripheral sources, estradiol is also synthesized in situ from testosterone by the enzyme aromatase in spinal cord tissue, where it appears to be biologically active only at the local tissue level (Simpson and Davis, 2001). Our group has recently shown that aromatase is present in astrocytes and not neurons in the mouse spinal cord (O’Brien et al., 2015). In mice, chronic and systemic blockade of this enzyme with an aromatase inhibitor altered nociception. Studies with animal models of pain have suggested that the reaction of glia, including microglia and astrocytes, contributes critically to the development and maintenance of chronic pain. In particular, astrocyte activation in the spinal cord appears to be an important contributor to the chronic pain associated with inflammation (Ikeda et al., 2012), HIV (Shi et al., 2012), chemotherapy-induced neuropathy (Zhang et al., 2012; Ruiz-Medina et al., 2013) and tumor-induced pain (Geis et al., 2010; Yao et al., 2011; Ren et al., 2012). Activation may result in altered cell morphology, changes in receptor expression, or release of factors by glial cells, which ultimately enhance nociceptive transmission (Ren and Dubner, 2008). The literature remains controversial regarding whether astrocytes play a critical role in the development of cancer pain (Ren et al., 2012; Ducourneau et al., 2014; Hironaka et al., 2014). Aside from the recent work from our laboratories in male mice, (O’Brien et al., 2015) there are no studies in the literature that have examined whether differential expression of spinal aromatase is associated with the development or maintenance of cancer pain. The major goal of this study was to examine the relationships among tumor-induced nociception, astrocyte activation and aromatase expression in the spinal cord in a murine model of painful bone cancer in

Abstract—Recently, our group established a relationship between tumor-induced spinal cord astrocyte activation and aromatase expression and the development of bone tumor nociception in male mice. As an extension of this work, we now report on the association of tumor-induced mechanical hyperalgesia and cold hypersensitivity to changes in spinal cord dorsal horn GFAP and aromatase expression in intact (INT) female mice and the effect of ovariectomy on these parameters. Implantation of fibrosarcoma cells produced robust mechanical hyperalgesia in INT animals, while ovariectomized (OVX) females had significantly less mechanical hyperalgesia. Cold hypersensitivity was apparent by post-implantation day 7 in INT and OVX females compared to their saline-injected controls and increased throughout the experiment. The decrease in mechanical hyperalgesia in OVX females was mirrored by significant decreases in spinal astrocyte activity in laminae I-II, III-IV, V-VI and X and aromatase expression in laminae V-VI and X in the dorsal horn of tumor-bearing animals. Administration of the aromatase inhibitor letrozole reduced tumor-induced hyperalgesia in INT females only suggesting that the tumor-induced increase in aromatase expression and its associated increase in spinal estrogen play a role in the development of bone tumor-induced hyperalgesia. Finally, intrathecal (i.t.) administration of 17b-estradiol caused a significant increase in tumor-induced hyperalgesia in INT tumor-bearing females. Since i.t. 17b-estradiol increases tumor pain and ovariectomy significantly decreases tumor pain, as well as spinal aromatase, estrogen may play a critical role in the spinal cord response to the changing tumor environment and the development of

*Corresponding author. Address: Department of Veterinary and Biomedical Sciences, University of Minnesota, Room 205 Veterinary Medicine Building, 1971 Commonwealth Avenue, St Paul, MN 55108, USA. Tel: +1-612-625-2595. E-mail address: [email protected] (A. J. Beitz). Abbreviations: i.t., intrathecal; INT, intact; NBF, neutral-buffered formalin; OVX, ovariectomized; s.c., subcutaneously. http://dx.doi.org/10.1016/j.neuroscience.2016.03.030 0306-4522/Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved. 344

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intact (INT) and ovariectomized (OVX) females. In the experimental design, we mirrored that used in a previous study performed in male mice (O’Brien et al., 2015) in order to examine potential sex differences in nociception and aromatase expression. Moreover, we compare our results to the only other study of aromatase in the mammalian spinal cord, which focused on the effects of spinal cord injury on aromatase expression in female rats (Ghorbanpoor et al., 2014). We hypothesized that: (1) mechanical and cold hyperalgesia would be evident and comparable to previous male mice findings in INT females; (2) astrocyte activation would be greater in INT tumor-bearing females versus OVX animals; (3) aromatase would be up-regulated in tumor-bearing INT mice and not in OVX tumor-bearing animals; (4) administration of an aromatase inhibitor would reduce mechanical hyperalgesia in INT, but not OVX females and (5) intrathecal (i.t.) administration of 17b-estradiol would significantly increase pain in INT tumor-bearing mice. We found that implantation of fibrosarcoma cells produced robust mechanical hyperalgesia in INT female animals, while OVX females had significantly less mechanical hyperalgesia and significantly less tumor-induced GFAP and aromatase expression. Since i.t. 17b-estradiol increases tumor pain, we conclude that estrogen may play a critical role in the development of tumor-induced nociception.

EXPERIMENTAL PROCEDURES Animals Female INT and OVX C3H animals that are syngeneic to fibrosarcoma cells were used for all experiments. All mice were 6–8 weeks old and were obtained from the National Cancer Institute (Bethesda, MD, USA). Mice were housed in small conventional boxes in a temperature- and humidity-controlled environment and maintained on a 12-h light/dark cycle with ad libitum access to food and water. All experiments were approved by the Institutional Animal Care and Use Committee at the University of Minnesota. The authors certify that all experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23) revised 1996. Ovariectomy Ovariectomy (OVX) surgical services were provided by surgeons at the National Cancer Institute (NCI). All OVX animals used were obtained from NCI following surgery and were allowed 7 days to fully recover and acclimate from the surgery prior to beginning any experiments. Serum estradiol samples were obtained and quantified using a commercially available estradiol EIA kit (#582251, Caymen Chemical, Ann Arbor, MI) prior to beginning the experiments to insure successful reduction of circulating estradiol. Cell culture and preparation for implantation Fibrosarcoma NCTC 2472 cells were obtained from the American Cell Culture Collection (Manassas, VA) and

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were maintained in NCTC 135 Medium (Sigma–Aldrich, Munich, Germany). Cells were first washed with PBS, trypsinized, pelleted and suspended in 5 mL of PBS for quantification using a hemacytometer. Following counting, all cells were re-pelleted and re-suspended in a volume of PBS for a final concentration of 2  105 fibrosarcoma cells per 10 lL. Cells were kept on ice and vortexed shortly before tumor implantation. Tumor implantation Implantation into the calcaneus bone of the hind paw was performed as previously described (Wacnik et al., 2001; Smeester et al., 2014). Briefly, mice were placed in an enclosed plexiglass acrylic chamber and initially anesthetized with 3% isoflurane/3 L oxygen. Upon successful anesthetization, the flow rate was adjusted to a maintenance level of 2% isoflurane/1.5 L oxygen for the remainder of the implantation procedure. Cells were injected unilaterally into the left heel using a 29 gauge, sterile single-use needle attached to a 0.3 mL insulin syringe (Becton Dickenson, Franklin Lakes, NJ, USA) to manually bore into the hind paw calcaneus bone. Control mice underwent an identical procedure with the exception that they received injection of saline rather than tumor cells. Following implantation, animals were returned to their home cages and recovered on a heating pad. Animals showing any signs of dysfunction (e.g. problems with ambulation, lethargy or excessive bleeding) were removed from the study. This occurred in less than 1% of the animals used in this study. Mechanical hyperalgesia Tumor-induced mechanical hyperalgesia was tested using a von Frey filament #3.61. This filament produces a force of 0.4 g. Animals were placed under clear glass cups on a wire grid and allowed to acclimate for 30 min. Starting with the right hind paw, the numbers of positive responses out of a total of 10 applications were recorded. Baseline von Frey measurements were obtained prior to tumor implantation or saline control injection into the calcaneus. Subsequent von Frey measurements were on post-implantation (PID) days 3, 7, 10, and 14. Behavioral assessments were conducted at approximately the same time each day. The investigator performing the von Frey testing was blinded to the experimental condition of the animals being tested. As the tumor grew, the investigator was no longer blinded to the experimental condition. A second researcher blinded to the experimental conditions of the animals performed the analysis of the data. Cold plate hypersensitivity Separate groups of tumor and saline-injected mice were used to test cold plate hypersensitivity than those used for von Frey testing. Mice were placed on top of a Peltier cold plate (model LHP-1200CPV, TECA Corp., Chicago, IL) in an enclosed container (20  16  25 cm3) maintained at 0 ± 0.1 °C where the number of licking or rapid shaking behaviors of the hind paw was recorded

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and quantified by trained observers (number of behaviors) (O’Brien et al., 2015). A maximum cut off time of 1 min was observed for all cold plate testing to prevent tissue damage at lower temperatures. Each mouse was only tested once on any given test day to avoid any possible anesthetic or tissue damage effects that could be produced by repeated exposure to the cold surface. Transcardiac perfusion Animals were deeply anesthetized with a 50 mg/mL sodium pentobarbital (Fatal Plus, Vortech Pharmaceuticals, Deerborn, MI) and transcardiac perfusion was performed as previously described (Smeester et al., 2013). Briefly, following atrial puncture using a 21-gauge butterfly catheter (Terumo Medical Corporation, Somerset, NJ, USA), 15 mL ice cold PBS followed by 30 mL of 10% neutral buffered formalin (NBF) was administered via peristaltic pump at a rate of 3 mL/ min. Following perfusion, tissues were prepared for immunohistochemical (IHC) processing. Spinal cords for IHC were excised and post-fixed in 10% NBF overnight at 4 °C and cryoprotected for an additional 24 h prior to tissue sectioning. Antibody generation Rabbit anti-mouse aromatase antibodies were developed in our laboratory in collaboration with Proteintech Group, Inc (Chicago, IL, USA) as previous described (O’Brien et al., 2015). Antibody specificity is detailed in our previous publication (O’Brien et al., 2015) Immunohistochemistry Spinal cords were sectioned and double immunostained with specific antibodies for aromatase and specific GFAP antibodies to recognize astrocytes. Lumbar regions (L4-L5) of spinal cords were sliced at 40 lm on a sliding microtome. Sections were incubated in 2% goat serum in 0.3% TritonX for one hour at room temperature. Following incubation, (a) rabbit antiaromatase (1:10,000, Proteintech Group, Inc., Chicago, IL) and (b) rat anti-GFAP (1:1,000, catalog #13-0300, Invitrogen, Carlsbad, CA, USA) were added to sections overnight at 4 °C. Sections were then washed 5X with PBS over a 25-min period. Following washing, secondary antibodies were applied: (a) anti-rabbit conjugated to cyanine 5 (Cy5) and (b) anti-rat conjugated to cyanine 3 (Cy3) (1:400 all secondaries, Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature. Sections were again washed with PBS, rinsed in molecular water, mounted on gelatincoated slides and cover-slipped using Prolong gold mounting medium (Invitrogen, Carlsbad, CA). Immunohistochemical controls for aromatase and GFAP were performed as previously described (O’Brien et al., 2015) Drug treatment To determine the effect of administration of an aromatase inhibitor on tumor-induced hyperalgesia, the aromatase

inhibitor, letrozole (catalog #L6545, Sigma–Aldrich, St. Louis, MO, USA) was daily administered subcutaneously (s.c.) (10 lg/d in 0.1 mL 0.3% hydropropylcellulose; HPC) beginning on post-implantation day 7 (PID 7) for a total of 7 days. Similarly, control animals received a daily subcutaneous injection of vehicle (0.1 mL 0.3% HPC) over the same time course. This optimized dose of letrozole was selected based on a previous report by Lu et al. (1999) and was previously utilized by our group (O’Brien et al., 2015). To determine the effect of spinal estrogen on tumorinduced hyperalgesia in INT and OVX mice, i.t. 17bestradiol (5 lg in 5 ll sterile PBS; catalog #E8875, Sigma–Aldrich, St. Louis, MO) administration was performed using a 50-ll Hamilton syringe connected to a 30-gauge needle. Briefly, the mouse was held tightly between the thumb and middle finger at the level of the both iliac crests, and the fifth lumbar spinous process was palpated with the index finger. The needle was inserted through the vertebral column into the L5–6 intervertebral space and successful insertion of the needle into the i.t. space was determined by a tail flick response. 17b-estradiol or vehicle was slowly injected over a 10-s period. Then, the needle was carefully removed from the spinal cord. The dose of 17b-estradiol was optimized based on previous experiments (two-way ANOVA, p > 0.05) and was found to be 5 lg in 5-ll sterile PBS. Separate groups of 17b-estradiol- or vehicle-injected INT mice (n = 6/group) were euthanized by transcardiac perfusion (see above) 90 min post injection and their spinal cords removed and processed for aromatase immunostaining to determine if i.t. 17b-estradiol administration affected tumor-induced aromatase expression. Imaging Fluorescence microscopy was performed on a Nikon Eclipse 80i microscope and all imaging was captured using a Nikon DS-Qi1Mc monochromatic digital camera (Nikon, Melville, NY, USA). Representative images of spinal cord GFAP immunostaining were taken at 200 magnification and aromatase immunostaining at 100. Confocal images were obtained using an -Olympus FLUOVIEW 1000. Representative singular images were taken at 200 and merged image was taken at 400 as indicated. Quantification of aromatase and GFAP immunoreactivity (-ir) A total of 10 randomly selected sections (from the L4 and L5 spinal cord segments) per animal per group were scanned for the presence of aromatase and GFAP immunoreactivity (-ir) using fluorescence microscopy (Nikon, Melville, NY). The investigator scanning the images and performing the analysis was blinded to the treatment groups. Digital images of aromatase and GFAP staining were captured using the Nikon Digital Sight monochromatic camera system (Nikon, Melville, NY). The brightness level was normalized per animal and within groups using this software. GFAP was quantified

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on both the ipsilateral and contralateral sides in laminae I-lI, III-IV, V-VI and X in fibrosarcoma INT females and OVX females as well as controls. Aromatase-ir was also quantified in total for both INT- and OVX-females with tumors as well as control saline-injected mice throughout all dorsal horn laminae. A quantitative approach to cell morphometric density measurement was conducted for all GFAP-ir using ImageJ software (NIH, Bethesda, MD, USA) due to the robustness of staining. Consistency was insured throughout all subsequent density measurements by setting threshold values identical in all animals and groups analyzed. Average area pixel count for GFAP-ir was calculated following thresholding of respective grayscale images to reduce the contribution of nonspecific background signal. The immunopositive pixels were again selected and a threshold pixel count was acquired for each channel. Unlike GFAP-ir density measurements, aromatase-ir-positive cells were manually counted using ImageJ in all laminae sampled. In addition to examining tumor-induced changes in aromatase in the L4-L5 spinal cord, potential tumor-induced changes in aromatase in the mid-thoracic (T6-T7) and cervical (C6-C7) regions of the spinal cord were also analyzed. Statistical analyses Complete statistical analyses of all data sets were carried out. For single-point comparisons between groups or within a group an unpaired Student’s t-test was employed (Figs. 1 and 6A, B &C). Comparisons between groups were performed using a two-way ANOVA with post hoc comparisons using Bonferroni’s method (Figs. 2A, B, 4A, B, 5A, B and 6C). Data were analyzed and graphed using Prism 5.0 (GraphPad Software, La Jolla, CA). The level of significance was set at p < 0.05.

RESULTS Ovariectomy (OVX) reduces systemic estradiol levels Following ovariectomy, animals were allowed 7 days to recover prior to beginning any experiments. Serum

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estradiol samples were obtained and quantified prior to beginning any experiments (Fig. 1). Serum estradiol concentrations were significantly lower in females that underwent ovariectomy (OVX; p < 0.0001) compared to intact females (INT). Serum estradiol levels for INT and OVX mice were 13.7 ± 1.08 and 4.3 ± 0.79 pg/mL respectively. Ovariectomy (OVX) reduces tumor-induced mechanical hyperalgesia, but not cold hypersensitivity Baseline (BL) von Frey measurements were established in all groups prior to implantation procedures (A) as well as the numbers of evoked behaviors on a cold plate at 0 °C for 1 min (B). Note that separate groups of mice were used for the measurement of von Frey scores and for the recording of evoked behaviors in response to the cold plate stimulation. Either saline or fibrosarcoma tumor cells were injected in the calcaneus of female INT or OVX mice. Evidence of mechanical hyperalgesia was apparent as early as post-implantation day 3 (PID 3) and continued for the length of the experiment in both tumor groups when compared with their individual saline controls (Fig. 1A; ****p < 0.0001). Reduced mechanical hyperalgesia was observed in OVX tumor animals on PID 7, 10 and 14 when compared with the INT tumor group (A; ^^^^p < 0.0001). Increases in the number of evoked behaviors were observed in both tumor-bearing groups when exposed to a cold plate for 1 min at 0 °C at all time points tested as compared to their individual saline controls (Fig. 2B; *p < 0.05, p < ****0.0001). No significant changes in cold plate-induced spontaneous behaviors were observed in OVX tumor-bearing mice when compared to INT tumor animals (B; p > 0.05). Aromatase colocalizes with GFAP in astrocytes in the female spinal cord Double-labeling IHC studies were performed to determine cell-type specificity in the mouse spinal cord and compared to our previously published results in male mice (O’Brien et al., 2015). Colocalization was observed in GFAP-expressing, aromatase-positive cells (Fig. 3, far right panel, ‘merged’, white arrows). Conversely, there was no co-localization of aromatase with NeuN (data not shown) indicating that neurons in the female mouse spinal cord do not express aromatase similar to previously published results in male mice (O’Brien et al., 2015). Not all astrocytes contained aromatase suggesting aromatase is found in a sub-set of spinal cord astrocytes. Ovariectomy (OVX) reduces tumor-induced astrocyte activation

Fig. 1. Ovariectomy (OVX) reduces systemic estradiol levels. Graph illustrating serum estradiol concentrations in intact (INT) and ovariectomized (OVX) mice. Estradiol concentrations were significantly lower in OVX mice (4.3 ± 0.79 pg/mL; p < 0.0001) compared to INT females (13.7 ± 1.08). Results are expressed as mean ± SEM, n = 8/group, ****p < 0.0001, unpaired Student’s t-test.

Lower lumbar (laminae I-II, III-IV, V-VI, X) astrocyte expression was quantified in INT and OVX tumorbearing animals at PID 14, the time point at which maximal mechanical hyperalgesia and cold hypersensitivity was observed (Fig. 1A, B). No differences in GFAP-ir were seen in any laminae between saline-injected INT and saline-injected OVX

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Fig. 2. Ovariectomy (OVX) reduces tumor-induced mechanical hyperalgesia, but not cold hypersensitivity. Graphs illustrating tumor-induced mechanical hyperalgesia (A) and cold allodynia (B) in INT and OVX mice. While both INT and OVX tumor groups exhibited significant tumor-induced hyperalgesia compared to their saline controls, OVX tumor-bearing animals had significantly reduced MH on post-implantation days (PID) 7, 10 and 14. Cold hypersensitivity was apparent as early as PID 7 in both tumor-bearing groups and continued through the course of the experiment. Results expressed as mean ± SEM, n = 6–11/group (A), 8–9/group (B), *p < 0.05 and ****p < 0.0001 v. respective saline controls, ^^^^p < 0.0001 v. INT tumor, two-way ANOVA with Bonferroni’s post hoc.

Fig. 3. Aromatase colocalizes with astrocytes in the spinal cord of female animals. Photomicrographs of spinal cord sections illustrating representative immunohistochemical staining of glial fibrillary protein GFAP (A, stained astrocytes are indicated by the white arrows), aromatase (white arrows in B) and the colocalization of aromatase in astrocytes (white arrows in C) in fibrosarcoma-bearing female mice. Individual images were taken at 200 (A-B); merged at 400 (C); scale bar = 100 lm.

control mice (Fig. 4A; p > 0.05). Reduced GFAP-ir on PID 14 in OVX tumor-bearing mice was observed in all laminae analyzed as compared to INT tumor animals (Fig. 4B; **p < 0.01, ***p < 0.001, ****p < 0.0001; representative photos of GFAP staining in tumor-bearing INT (Fig. 4C) and OVX (Fig. 4D)). Additionally, there were no significant differences in tumor-induced astrocyte activation on the contralateral side of the lumbar spinal cord in any of the groups examined (data not shown; Two-way ANOVA, p > 0.05). Aromatase expression in the lumbar spinal cord is influenced by both ovariectomy and tumor development Tumor and saline control spinal cord sections from both INT and OVX mice were double stained for GFAP immunoreactivity and aromatase immunoreactivity. Positive aromatase immunostaining was only found in a subset of astrocytes. All immunopositive aromatase

cells were counted and expressed as an average count per slide in saline-injected control mice (5A) and tumorbearing (5B) animals in laminae I-VI and X of the dorsal horn in the lumbar region on post-implantation day 14 (Fig. 5). Ovariectomized mice (OVX) with reduced systemic estradiol levels had increased aromatasepositive cell counts in laminae I-II in saline-injected OVX control animals when compared with INT control animals on PID 14 (Fig. 5A; ****p < 0.0001). Conversely both OVX and INT tumor-bearing animals displayed similar tumor-induced increases in aromatase expression in laminae I-II compared to their salineinjected counterparts. However, there were significantly fewer positively stained aromatase cells in deeper dorsal horn laminae in OVX tumor-bearing mice compared to INT tumor-bearing mice, which correlates with the observed decrease in mechanical hyperalgesia (Figs. 2and 5B; p < 0.0001). Representative photos of aromatase immunostaining can be seen in Fig. 5C–F. Finally, there were no increases in aromatase staining in

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Fig. 4. Ovariectomy (OVX) reduces tumor-induced astrocyte activation. Graphs illustrating the amount of astrocyte expression in the lower lumbar spinal cord (laminae I-II, III-IV, V-VI, X) in intact (A) and ovariectomized (B) tumor-bearing animals at PID 14, the time point at which maximal mechanical hyperalgesia and cold hypersensitivity was observed. A significant reduction in astrocyte expression was observed in OVX tumorbearing mice compared to INT tumor-bearing animals. Results are expressed as mean ± SEM, n = 4/group – 40 sections per group, **p < 0.01, *** p < 0.001, ****p < 0.0001, Two-way ANOVA with Bonferroni’s post hoc. Representative images of GFAP immunostaining in the spinal cord are illustrated from an INT tumor (C) and an OVX tumor (D) mouse. Images were taken at 200, scale bar = 100 lm. White arrows indicate positive immunostaining.

the dorsal horn of the thoracic and cervical segments of the spinal cord in tumor-bearing INT or OVX animals as compared to their saline-injected controls, respectively (Two-way ANOVA, p > 0.05).

effect in the OVX tumor-bearing group compared to its vehicle-injected control (Fig. 6B; p > 0.05).

Letrozole inhibits mechanical hyperalgesia in INT females

Intrathecal (i.t.) administration of 17b-estradiol potentiates tumor-induced mechanical hyperalgesia in INT females, but does not affect aromatase immunostaining

To determine whether an aromatase inhibitor could reduce the observed mechanical hyperalgesia observed in INT and OVX tumor-bearing animals, letrozole was given s.c. (10 lg/d in 0.1 mL 0.3% HPC) beginning on post-implantation (PID) 7 for a total of 7 days. Mechanical von Frey scores were evaluated on PID 14 in both intact (INT; Fig. 6A) and ovariectomized (OVX; Fig. 6B) mice. Letrozole reduced mechanical hyperalgesia in the INT female tumor-bearing group as compared to the vehicle-injected tumor-bearing group (Fig. 6A; ***p < 0.001), while letrozole had no significant

To further delineate the role of sex hormones in INT females, 17b-estradiol (5 lg/5 lL PBS) was administered intrathecally (i.t.) to INT tumor-bearing females on PID 14. 17b-estradiol potentiated mechanical hyperalgesia in INT tumor-bearing females as compared to vehicle-injected tumor-bearing mice (Fig. 6C; p < *0.05). Tumor-induced hyperalgesia was significantly increased from 60 to 90 min post-injection returning to vehicle control levels by 180 min postinjection. This potentiating effect of i.t. injection of 17bestradiol on tumor-induced nociception was not

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Fig. 5. Aromatase expression in the lumbar spinal cord is influenced by ovariectomy and tumor development. Graphs comparing the amount of aromatase immunostaining in the lower lumbar spinal cord (laminae I-II, III-IV, V-VI, X) in Intact and OVX saline mice (A) and in INT and OVX tumorbearing animals (B) at PID 14. OVX is associated with increased aromatase-positive cell counts in laminae I-II in saline-injected OVX control animals when compared with INT control animals. When compared to saline-injected animals both INT and OVX tumor-bearing animals displayed increased aromatase expression in laminae I-II. However, in laminae V-VI and X there were significantly fewer positively stained aromatase cells in OVX than in INT animals, which correlates with the observed decrease in mechanical hyperalgesia. Results expressed as mean ± SEM, n = 4/group – 40 sections/group, ****p < 0.0001, Two-way ANOVA with Bonferroni’s post hoc. Images of representative aromatase immunostaining in spinal cord sections from INT (C) and OVX (D) saline-injected mice compared to INT (E) and OVX (F) tumor-bearing mice. Images were taken at 100X, scale bar = 100 lm. White arrows indicate positive immunostaining.

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Fig. 6. Mechanical hyperalgesia is inhibited by letrozole and potentiated by 17b-estradiol in intact (INT) females. Graphs illustrating the effect of letrozole (A, B) or 17b-estradiol (E2; C) on tumor-induced hyperalgesia and the effect of E2 on aromatase expression (D) on PID 14. Letrozole administration reduced mechanical hyperalgesia in the INT group (A) as compared to vehicle (p < 0.001) while no effects in the OVX group (B) were observed in either treatment group (p > 0.05). Intrathecal 17b-estradiol potentiated mechanical hyperalgesia in intact (INT) tumor-bearing females on PID 14 as compared to vehicle control (C). No effect of 17b-estradiol on aromatase expression was observed in any laminae surveyed (D, p > 0.05) at 90 min post-injection. Results are expressed as mean ± SEM, n = 7–9/group, *p < 0.05, ***p < 0.001 ****p < 0.0001, unpaired Student’s t-test (A&B), Two-way ANOVA with Bonferroni’s post hoc (C&D).

accompanied by significant changes in aromatase expression in laminae I-VI or X in the lumbar spinal cord (Fig. 6D, p > 0.05) 90 min post-injection.

DISCUSSION Cancer pain is a debilitating type of chronic pain that reduces the quality of life in most patients (Body et al., 2015; Falk and Dickenson, 2014). Bone pain in particular is one of the most common complications associated with primary or metastatic tumor cell colonization of bone and is likely due to rather complex interactions among osteoclasts, osteoblasts, tumor cells and primary afferent fibers (Body et al., 2015; Yoneda et al., 2015). Since women report more pain than men in a variety of chronic conditions including cancer pain (Barnabe et al., 2012); Green et al., 2011), and since there are no studies that have examined the potential role of sex differences in spinal cord astrocytes and aromatase in cancer pain, we examined both astrocyte and aromatase expression in a model of painful bone cancer in both INT and OVX females and compared these data to those of our previously published data in males (O’Brien et al., 2015). We found that implantation of fibrosarcoma cells produced robust mechanical hyperalgesia in both male and INT

female animals, while OVX females had significantly less tumor-induced mechanical hyperalgesia. This latter finding is consistent with a previous work showing that estrogen enhances pain and depleting estrogen reduces pain (Files et al., 2014; Ralya and McCarson, 2014; Ji et al., 2015). There is, however, controversy regarding the effect of OVX on pain with some studies indicating that OVX increases pain (Ceccarelli et al., 2003; Sanoja and Cervero, 2008), while others find as we did that OVX decreases pain (Ji et al., 2003; Joseph and Levine, 2003; Krzanowska and Bodnar, 1999). These differences are likely due to the different animal pain models used as well as to the fact that mice were used in some studies, while rats were used in others in these investigations. The effect of OVX on cancer pain has not been reported to our knowledge and the present results would argue that estrogen and perhaps other sex hormones play a role in the generation and/or maintenance of this type of chronic pain. One question arising from this study is why ovariectomy reduced cancerinduced mechanical hyperalgesia, but not cold hyperalgesia. This would suggest that sex hormones differentially affect mechanical versus cold nociception, but the mechanisms underlying this difference remain to be determined.

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With respect to central changes, astrocyte activation has been shown to increase in bone cancer mice (Pevida et al., 2014) and both astrocytes and microglia have been shown to contribute to bone cancer pain in a rat model (Mao-Ying et al., 2012). In our previous study using male mice with hindpaw tumors, astrocyte activation in the lumbar spinal cord correlated with the increased mechanical and thermal hyperalgesia observed in painful tumor-bearing animals (O’Brien et al., 2015). This in combination with the current results supports previous data implicating astrocytes in cancer pain. The present study indicates that GFAP is also upregulated in INT female mice in all dorsal horn laminae in response to tumor cell implantation into the calcaneous bone, which mimics what is observed in male mice (O’Brien et al., 2015). Similarly, the largest fold increase in GFAP immunostaining occurred in laminae V and VI in both male and female tumor-bearing mice. Tumor-bearing OVX mice displayed a significant reduction in GFAP immunostaining in all dorsal horn laminae compared to tumor-bearing INT females, which parallels the OVXinduced decrease in tumor-induced mechanical allodynia. If astrocyte activation is associated with increases in nociception as most studies suggest (Lu et al., 2015; Lv et al., 2015; Old et al., 2015), then the correlation between a decrease in astrocyte GFAP immunostaining and mechanical allodynia in OVX-tumor-bearing mice would further argue that sex hormones are important in influencing the degree of cancer-induced pain in females. Estrogens are synthesized from androgens by aromatase, a P450 enzyme traditionally associated with discrete neuronal populations in the brain of vertebrates (Roselli and Resko, 1997; Carswell et al., 2005). It is clear that while there are sources of variability and inconsistent results in the literature regarding the effect of estrogen on nociception, the prevailing evidence supports the presence of significant sex hormonal influences on nociceptive processing (Fillingim and Ness, 2000). Initial studies reported no aromatase activity found in the spinal cord of rats (MacLusky et al., 1987). While subsequent physiological and pharmacological studies assumed aromatase was present in neurons of the mammalian spinal cord (see for example Zhong et al., 2010), a recent report by Ghorbanpoor et al. (2014) challenged this view by reporting aromatase in spinal cord glial cells of rats following spinal cord injury. Our data are consistent with those of Ghorbanpoor and colleagues and indicate that aromatase is localized to spinal cord astrocytes and is not present in murine neurons or microglia. When comparing male and female animals, the presence of the hindpaw bone tumor significantly increased aromatase expression in all dorsal horn laminae in males (see O’Brien et al., 2015) and INT females, with the greatest fold increase in laminae V-VI. Since the greatest increase in GFAP expression occurs in laminae V-VI in both male (O’Brien et al., 2015) and female tumor mice and since this increased expression correlates with the tumorinduced increase in mechanical allodynia and cold hyperalgesia, then it is likely that increased aromatase expression in these two laminae is associated with the maintenance of tumor-induced nociception. This is

consistent with our findings that OVX significantly reduces aromatase expression in laminae V-VI, but not I-IV and that OVX also significantly reduces tumor-induced nociception. We show that letrozole decreased mechanical allodynia in both INT female mice and in male mice, but not in OVX mice, which supports our hypothesis that aromatase plays a role in the maintenance of bone cancer pain in males and INT female mice. Lastly, we show that administration of 17b-estradiol i.t. potentiates tumorinduced mechanical hyperalgesia in INT females, but does not affect aromatase expression. This lack of effect could be due to the time point selected or to the ongoing tumor-induced increase in aromatase. Collectively these data suggest that increased levels of estradiol in the spinal cord have the potential to increase tumor-induced nociception. While the addition of E2 to INT mice could increase hyperalgesia independent of aromatase activity, collectively our data suggest that local estrogen production in the spinal cord via aromatase may contribute to the development of tumor-induced nociception. There is a significant literature on the development of aromatase-induced arthralgia observed in women taking aromatase inhibitors with women experiencing pain in the hands, wrists, hips and knees (see Niravath, 2013; Baumi et al., 2015). This would seem to be in conflict with the present results in that we show that administration of an aromatase inhibitor reduces cancer nociception, while women that take aromatase inhibitors often develop a painful arthralgia. However, it is important to note that aromatase inhibitor therapy is typically administered for up to 5 years as opposed to the 7 days evaluated in the present study and most recipients are post-menopausal women (Niravath, 2013). Thus, this post-menopausal condition in which estrogen deprivation has been hypothesized as the major cause of aromatase-induced arthralgias (Gaillard and Stearns, 2011) is similar to the ovarian hormone deficiency observed in our OVX mice. Since the administration of an aromatase inhibitor had no significant effect on tumor-induced nociception in OVX mice, one would predict that it would not have a major effect on pain in post-menopausal women. Conversely, i.t. administration of estradiol to INT mice caused a significant increase in tumor-induced mechanical hyperalgesia. This effect was 66% higher when compared to control OVX mice and 33% higher when compared to INT control mice suggesting that estrogen amplifies tumor-induced nociception. Clearly the potential antinociceptive effect of aromatase inhibitors or estrogen receptor antagonists on pain sensation in pre-menopausal and post-menopausal women requires further study.

CONCLUSION We have utilized a painful rodent bone cancer model in the present study and have established the following novel findings: (1) implantation of fibrosarcoma cells into the calcaneous bone of the hindpaw produces mechanical hyperalgesia in INT females comparable to their male counterparts (see O’Brien et al., 2015), while OVX females have significantly less tumor-induced mechanical hyperalgesia compared to INT females; (2)

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ovariectomy does not cause changes in spinal cord astrocyte GFAP expression in naı¨ ve or saline-injected mice, but significantly reduces tumor-induced astrocyte activation in all lumbar dorsal horn laminae; (3) ovariectomy significantly reduces tumor-induced aromatase expression in laminae V-VI and X of the lumbar spinal cord; (4) administration of the aromatase inhibitor, letrozole for seven consecutive days beginning on post-implantation day 7 significantly reduced tumor-induced mechanical hyperalgesia in INT, but not OVX, females; and (5) administration of 17b-estradiol significantly increased mechanical hyperalgesia in INT tumor-bearing mice, without changes in tumor-induced aromatase expression in the spinal cord. Collectively these data indicate that the presence of a painful fibrosarcoma tumor induces a significant increase in spinal astrocyte activation and aromatase expression and this up-regulation of aromatase plays a role in the development of bone tumor-induced hyperalgesia in INT female mice, but not in OVX mice, implying that sex hormones are important in influencing the degree of cancerinduced pain in females.

CONFLICTS OF INTEREST The authors declare no conflicts of interest. Acknowledgments—This work was supported by NIH grant CA084233 and Minnesota AES grant MIN-63-071. BAS performed the behavioral assays and drug administration experiments and contributed to the writing of the manuscript. EEO performed the immunohistochemistry experiments. KSM performed the immunohistochemical analysis of spinal cord immunostaining. J-HL and AJB contributed to the experimental design, interpretation and writing of the manuscript.

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(Accepted 10 March 2016) (Available online 16 March 2016)