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Neuropeptides are associated with pain threshold and bone microstructure in ovariectomized rats
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Neuropeptides are associated with pain threshold and bone microstructure in ovariectomized rats ⁎
Weixin Xiea, Fan Lia, Yi Hana, Zhanchun Lia, , Jie Xiaob, a b
⁎⁎
Department of Orthopaedic Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, China Department of Anesthesiology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Osteoporosis Neuropeptide Bone microstructure Pain threshold
Objectives: Postmenopausal osteoporosis (PMO) is a metabolic skeletal disorder with impaired bone density and bone quality in postmenopausal women. The aim of the present study was to investigate the correlation between neuropeptides, bone microstructure and pain threshold in ovariectomized (OVX) rats. Methods: Female rats were randomly divided into the ovariectomized (OVX) group and the sham surgery (SHAM) group. Bone microstructure and immunocytochemistry for substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) in tibial and DRG were performed. Pain threshold was assessed at post-operative 11 weeks. Pearson correlation coefficients were calculated between neuropeptides, bone microstructure and pain threshold. Results: Significant decreases in bone volume fraction (BV/TV) and trabecular number (Tb. N) but significant increases in trabecular spacing (Tb.Sp) were showed in OVX group. Mechanical pain threshold (MPT) in OVX group was significantly decreased. The MOD values for SP, CGRP and VIP of tibial in OVX group were significantly lower, whereas NPY, NPY1R and NPY2R were significantly higher. And SP, CGRP, VIP, NPY and NPY2R of DRG were significantly increased in OVX group, while NPY1R was significantly decreased. Correlation analysis showed that NPY, Y1R and Y2R in bone were negatively correlated with BV/TV. MPT was negatively correlated with NPY and Y2R in DRG, and positively correlated with Y1R in DRG. Conclusions: Our results suggested that SP, CGRP, VIP and NPY were involved in the osteoporotic bone microstructure and mechanical hypersensitivity in OVX rats, indicating the potential to utilize neuropeptides as novel therapeutic targets for PMO.
1. Introduction
by nerve fibers to act on bone tissue. A variety of neuropeptides have been proved to exist in bones. Substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY) and their receptors were found in bone cells like osteoblasts, osteoclasts or bone marrow mesenchymal stem cells (BMSCs) (Grassel, 2014) and they are all critical regulators of bone metabolism. Bone pain caused by osteoporosis makes a chronic influence on quality of life of affected people. There is still no effective treatment for pain relief, as the pathogenesis of osteoporotic pain remain unclear. Estrogen receptors (ER) expressed in small nociceptive neurons of dorsal root ganglions (DRG) are known to be involved in nociception (Sarajari and Oblinger, 2010). SP and CGRP contained in the small DRG neurons and released by sensory nerve fibers are both critical pain mediators and significantly regulated by estrogen. We previously (Xiao
Postmenopausal osteoporosis (PMO) is defined as a metabolic skeletal disorder characterized by impaired bone density and bone quality in postmenopausal women, which is induced by estrogen deficiency (Watts et al., 2010; Xu et al., 2017). The core clinical features of PMO include chronic bone pain and fracture caused by compromised bone mass, bone microstructure and biomechanical properties. These manifestations can dramatically worsen quality of life and functional capacity in PMO patients. PMO is now still a global public health concern involving medicine, society and economy. It is universally acknowledged that nerve system plays an important role in regulation of the skeleton (Wee et al., 2016). In periphery, neuropeptides as neurotransmitters and neuromodulators are released
⁎ Correspondence to: Z. Li, Department of Orthopaedic Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, 1630 Dongfang Rd., Shanghai 200127, PR China. ⁎⁎ Correspondence to: Jie Xiao, Department of Anesthesiology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, 1630 Dongfang Rd., Shanghai 200127, PR China. E-mail addresses: [email protected] (Z. Li), [email protected] (J. Xiao).
https://doi.org/10.1016/j.npep.2019.101995 Received 26 September 2019; Received in revised form 4 November 2019; Accepted 13 November 2019 0143-4179/ © 2019 Published by Elsevier Ltd.
Please cite this article as: Weixin Xie, et al., Neuropeptides, https://doi.org/10.1016/j.npep.2019.101995
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kit (Abcam, Cambridge, MA), according to the manufacturer's instructions. The absorbance was read at 450 nm using a microplate reader. The serum concentrations of E2 were calculated according to the optical density (OD) and standard curve.
et al., 2016) demonstrated that the VAS score in osteoporosis (OP) and osteoarthritis (OA) patients was positively correlated with the levels of SP, CGRP and VIP in subchondral cancellous bones and negatively correlated with NPY. The results suggested that SP, CGRP, VIP and NPY are associated with osteoporotic pain generation and may be the potential therapeutic targets for osteoporotic pain. The neuropeptides located in central and peripheral nervous systems play critical roles in bone metabolism and pain modulation. Studies reported that nerve profile density in bone of ovariectomized (OVX) rats decreased (Burt-Pichat et al., 2005), and both sympathetic and sensory innervation are associated with the levels of estrogen (Pajot et al., 2003; Zoubina and Smith, 2001). The changes of nerve innervation induced by levels of estrogen may make a difference to neuropeptide expression. We previously (Xiao et al., 2016) observed that between postmenopausal women with osteoporosis and osteoarthritis, there were significant differences in levels of neuropeptides and microstructure of subchondral cancellous bones. Anyhow, the role of neuropeptides in the pathogenesis of PMO is still uncertain. This study was performed to further explore the relationship between the neuropeptides, bone microstructure and pain threshold in OVX rat model, aiming to investigate the potential roles of neuropeptides in the pathogenesis of PMO.
2.5. Pain threshold assessment Mechanical pain threshold (MPT) assessment. MPT was measured using von Frey filaments. Before testing, rats were acclimatized for at least 1 h in a clear Plexiglas box (26 cm long × 20 cm wide × 14 cm high) on an elevated wire mesh grid. Von Frey filaments were applied to the middle of plantar surface of the right hind paw with buckling weight of 1, 1.4, 2, 4, 6, 8, 10, 15, 26 g. The pressure was set to cause the filament to buckle for 2 s each time interval of 15 s, 5 cycles. An increased force stimulus was applied if at least three paw withdrawal responses were absence. The smallest force that could raise the paw withdrawal responses in at least 3 out of 5 times was defined as Paw Withdrawal Threshold (PWT). The same measurement was applied to the left hind paw. Thermal pain threshold (TPT) assessment. Rats were acclimatized for at least 1 h in a clear Plexiglas box (26 cm long × 20 cm wide × 14 cm high) on a glass plate. A beam of radiant heat was applied to the plantar surface of the right hind paw. The radiant heat beam was shut off automatically once the paw withdrawal response occurred. The time from the start of the heating to the paw withdrawal was recorded using a digital timer and defined as the Thermal Withdrawal Latency (TWL). Each measurement was repeated in triplicate at intervals of 5 min and the average value was calculated. The noresponse cut-off time was set to 30 s to avoid tissue damage.
2. Materials and methods 2.1. Animals This study was ethically approved by the Institutional Animal Care and Use Committee of Shanghai Jiaotong University (RJ2018–1012) and was conducted in conformity with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Twelve 6-monthold female virgin Sprague–Dawley rats (weight: 290-310 g) were housed in a room under conditions of controlled temperature at 22 °C with a 12-hour light/dark cycle. A normal chow diet and distilled were provided for all animals throughout the experiment.
2.6. Bone micro-computed tomography (micro-CT) imaging The right tibial specimens were examined using a micro-CT system (Xiao et al., 2016; Zhang et al., 2010) (μCT 80, Scanco Medical AG, Switzerland) at a voxel size of 6 μm. Scan parameters included tube voltage 80 kV, tube current 80uA, 4 frames averaged and a 0.4-degree increment angle. After scanning, a region of interest (ROI) was selected between 5 cm proximal and 5 cm distal to the center of distal tibial metaphysis. The quantitative analysis were conducted using the software provided with the instrument and the following parameters were calculated: The bone volume fraction (BV/TV; %), trabecular number (Tb.N; mm−1), trabecular thickness (Tb.Th; mm), trabecular separation (Tb.Sp; mm), degree of anisotropy (DA), connectivity density (Conn.D; mm−3), apparent density (AD) and material density (MD).
2.2. Experimental design The rats were randomly divided into 2 groups: ovariectomy (OVX; n = 6) and sham (SHAM; n = 6) operation. Either ovariectomy or sham surgery was performed under intraperitoneal anesthesia with pentobarbital (40 mg/kg). For OVX rats, bilateral ovaries were excised via back incisions, and adipose tissue of the equal weight as the removed ovaries was excised for SHAM rats. Pain threshold assessment was carried out at 1 week before sugery and 11 weeks after operation. All rats were euthanized 12 weeks after ovariectomy or sham operation by overdose pentobarbital injection. Uterus weight were measured to confirm the success of ovariectomy. Blood were collected for analysis of levels of 17β-estradiol (E2) from the abdominal aorta and serum was stored at −80 °C after centrifugation at 1600g for 15 min at 4 °C. The bilateral tibiae, femurs and DRG (L6 – S1) dedicated to histomorphometry and immunohistochemistry analysis were harvested and stored at −80 °C until processing.
2.7. Biomechanical testing The biomechanical properties of left tibial specimens was measured by four-point bending test using mechanical universal machine (Instron 8874, Instron, Ltd., High Wycombe, UK). The load was applied at a speed of 1 mm/min until the specimen was disrupted. The data were recorded and analyzed by the software within the equipment itself. The Ultimate load (N), ultimate strain (mm/mm), ultimate stress (MPa), ultimate load energy and elasticity modulus (MPa) were obtained for evaluating biomechanical properties.
2.3. Bone mineral density (BMD) measurement The rats were anesthetized to perform in vivo BMD (of the femurs) measurement using dual-energy x-ray absorptiometry on a Discovery-A (Hologic) system at post-operative 12 weeks. Data were analyzed with high-resolution BMD test software for small animals (Regional High Resolution, version 4076; Hologic). The coefficient of variation for BMD measurement was < 2.0%.
2.8. Immunocytochemistry The immunocytochemistry was evaluated according to the method reported previously (Xiao et al., 2016). Briefly, the right tibial and DRG specimens were embedded with paraffin after decalcification and were sectioned into 5-mm thickness. Heat mediated antigen retrieval was performed using boiled citrate after deparaffinization and hydration. After overnight incubation at 4 °C with the primary antibody separately (Anti-SP antibody, 1:1000, Santa Cruz, Biotechnology, Inc. USA; AntiCGRP antibody, 1:1000, Abcam, Cambridge, USA; Anti-VIP antibody,
2.4. Enzyme linked immunosorbent assay (ELISA) Serum concentrations of E2 was detected using 17β-estradiol ELISA 2
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respectively, P > .05) (Fig. 1C).
1:500, Abcam, Cambridge, USA; Anti-NPY antibody, 1:1000 Abcam, Cambridge, USA; Anti-NPY1R antibody, 1:1000 Abcam, Cambridge, USA; Anti-NPY2R antibody, 1:1000 Abcam, Cambridge, USA), the sections incubated with a biotinylated secondary antibody for 15 min at 37 °C. Peroxidase reaction was visualized with a solution of diaminobenzidine (DAB) in Tris buffer containing 0.01% H2O2 (0.05 mol/l, pH 7.6). The positive staining was visible as brown punctums and bundles under the under light microscope (E800, Nikon, Tokyo, Japan). Three images of high-powered magnification views were taken using a Spot Advanced digital-imaging system (Diagnostic Instruments, Inc., Sterling Heights, MI, USA). The mean optical density (MOD) was measured to perform the quantitative immunohistochemical analysis using the software package Image Pro Plus (version 5.0.1, Media Cybernetics, Silver Spring, MD, USA).
3.4. Ovariectomy attenuated biomechanical properties of bone Compared with the SHAM group, the ultimate load (96.17 ± 2.56 vs. 126.00 ± 11.65 N, respectively, P < .01), ultimate stress (93.17 ± 17.08 vs. 120.50 ± 11.20 MPa, respectively, P < .01), ultimate load energy (0.032 ± 0.008 vs. 0.043 ± 0.007 J, respectively, P < .05), elasticity modulus (3859.00 ± 318.00 vs. 5028.00 ± 503.00 MPa, respectively, P < .05) values were significantly decreased in the OVX group at post-operative 11 weeks. And there was no significant difference in ultimate strain (0.044 ± 0.009 vs. 0.055 ± 0.012 mm/mm, respectively, P > .05) between two groups. 3.5. Different expression of neuropeptides in bone and DRG between groups
2.9. Statistical analysis The comparisons of MOD values for SP, CGRP, VIP and NPY in right tibiae and DRG between OVX group and SHAM group were performed via immunohistochemical analysis (Figs. 2 and 3). The MOD values for SP, CGRP and VIP of right tibiae in the OVX group were significantly lower than those in the SHAM group (SP: 0.173 ± 0.012 vs. 0.246 ± 0.03, P < .01; CGRP: 0.187 ± 0.017 vs. 0.244 ± 0.018, P < .001; VIP: 0.219 ± 0.016 vs. 0.282 ± 0.039, P < .01). On the contrary, the MOD value for NPY of right tibiae in the OVX group was significantly higher than that in the SHAM group (0.285 ± 0.032 vs. 0.167 ± 0.028, P < .001). Moreover, the MOD values for all these four neuropeptides of DRG were significantly increased in the OVX group (SP: 0.255 ± 0.044 vs. 0.206 ± 0.029, P < .05; CGRP: 0.354 ± 0.016 vs. 0.307 ± 0.016, P < .01; VIP: 0.144 ± 0.013 vs. 0.126 ± 0.014, P < .05; NPY: 0.310 ± 0.020 vs. 0.241 ± 0.025, P < .001). NPY Y1 receptor (Y1R) and NPY Y2 receptor (Y2R) in bone were significantly increased in the OVX group (Y1R: 0.498 ± 0.035 vs. 0.302 ± 0.036, P < .001; Y2R: 0.420 ± 0.062 vs. 0.266 ± 0.047, P < .001). Y1R in DRG was significantly decreased in the OVX group (0.045 ± 0.008 vs. 0.107 ± 0.030, P < .001), while Y2R in DRG was significantly increased (0.771 ± 0.140 vs. 0.364 ± 0.067, P < .001).
Data analyses were performed using SPSS 23.0 software (SPSS Inc., Chicago, IL, USA). All data were presented as the means ± standard deviation. The statistical significance between two groups was evaluated by Student's t-test. Pearson correlation analysis was carried out to determine the correlation between variables. The significance level was set at P < .05. 3. Results 3.1. Verification of the OVX rat model Mass of uterus, body weight and E2 concentrations between the two groups were compared at post-operative 12 weeks. As expected, uterine weight was significantly lower in OVX group than in SHAM group (0.22 ± 0.08 vs. 0.67 ± 0.08 g, respectively, P < .01), and body weight was significantly higher in OVX group than in SHAM group (450.49 ± 44.42 vs. 334.39 ± 44.18 g, respectively, P < .01). The OVX rats were shown by substantial decreases in E2 concentrations in relation to the SHAM rats (136.65 ± 6.24 vs. 304.21 ± 6.68 pg/ml, respectively, P < .001).
3.6. Neuropeptides are correlated with bone microstructure and pain threshold
3.2. Ovariectomy attenuated bone microstructure and bone mass
Table 1 showed the correlation between bone microstructure and neuropeptides in bone. And as shown in Fig. 4, the MOD values for NPY (r = −0.741, P < .01) and CGRP (r = −0.598, P < .05) in DRG were negatively correlated with MPT. As for the NPY receptors, Y1R (r = 0.741, P < .01) in DRG was positively correlated with MPT, while Y2R (r = −0.597, P < .05) in DRG was negatively correlated with MPT. In addition, the MOD of SP and VIP in DRG showed no significant correlation with MPT.
The right tibials of rats were analyzed to perform the micro-CT imaging (Fig. 1A). The quantitative analysis revealed that differences in BV/TV, Tb.N, Tb.Th, Tb.Sp and Conn.D in trabecular bone were all significant between the two groups (Fig. 1B). Compared to SHAM group, OVX group showed significant decreases in BV/TV (17.00 ± 2.92 vs. 55.79 ± 14.99%, respectively, P < .001), Tb. N (1.38 ± 0.24 vs. 3.23 ± 0.37 mm−1, respectively, P < .001) and Conn.D (9.38 ± 3.11 vs. 41.07 ± 10.55 mm−3, respectively, P < .001) but significant increases in Tb.Sp (0.62 ± 0.12 vs. 0.14 ± 0.05 μm, respectively, P < .001). The comparison of Tb.Th showed no significant difference between two groups. Additionally, the OVX rats were shown by substantial decreases in BMD in relation to the SHAM rats (0.230 ± 0.013 vs. 0.261 ± 0.019 g/cm2, respectively, P < .05).
4. Discussion Our previous study revealed that MOD values for SP, CGRP, VIP and NPY in OA group were significantly different with those in OP group. And the neuropeptides were correlated with the parameters of bone microstructure and VAS scores in osteoarthritis and osteoporosis patients, which suggested that neuropeptides might be involved in the changes of bone microstructure and pain generation in OA and OP (Xiao et al., 2016). In this study, the ovariectomized rat model was built to mimic postmenopausal woman with osteoporosis, and the relationships between neuropeptides and bone microstructure and pain threshold are investigated. The results demonstrated several novel findings. Firstly, the OVX rats showed mechanical hyperalgesia and osteoporotic bone microstructure. Secondly, there were different neuropeptide distributions between OVX rats and SHAM rats. Thirdly, Neuropeptides expressed in bone and DRG are correlated with bone
3.3. Ovariectomy induced mechanical hyperalgesia There was no statistical difference between OVX group and SHAM group with regard to MPT (8.00 ± 0.00 vs. 8.33 ± 1.51 g, respectively, P > .05) and TPT (11.62 ± 0.95 vs. 11.55 ± 0.97 s, respectively, P > .05) at 1 week before operation. At post-operative 11 weeks, MPT in OVX group was significantly decreased when compared with the SHAM group (4.23 ± 2.12 vs. 9.5 ± 3.08 g, respectively, P < .01), and TPT in OVX group did not differ significantly when compared with SHAM group (15.94 ± 2.34 vs. 14.79 ± 2.58 s, 3
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Fig. 1. Ovariectomy attenuated bone microstructure and induced mechanical hyperalgesia. A Representative micro-CT images of tibias. B Comparison of the BV/TV, Tb.N, Tb.Sp, Conn.D and BMD between groups. C Comparison of the pain threshold between groups. The data are expressed as the means ± SD (n = 6 in each group). *P < .05; **P < .01; ****P < .0001 vs. SHAM by the unpaired two-tailed Student's t-test (B,C).
of neuropeptides, which prompted us that neuropeptides are involved in the pain generation (Xiao et al., 2016). In the present study, we investigated the differences of neuropeptide distributions in bone and DRG between OVX group and SHAM group. The findings reported here showed that the expressions of SP, CGRP and VIP of bone were significantly lower, whereas NPY was higher in OVX group. In DRG, the expressions of SP, CGRP, VIP and NPY were all significantly higher in OVX group. SP and CGRP are well-established transmitter of nociception, which could be regulated by estrogen. Estrogen has been shown to have a negative effect on SP expression, Sarajari and Oblinger (2010) showed that the expression of SP in the small DRG neurons were significantly downregulated in estrogentreated rats. CGRP is a neuropeptide co-stored with SP in DRG neurons, and rats treated with estrogen showed increased CGRP synthesis in DRG (Mowa et al., 2003; Yang et al., 1998). Studies reported that nerve profile density in bone of ovariectomized (OVX) rats decreased (BurtPichat et al., 2005), and both sympathetic and sensory innervation are associated with the levels of estrogen (Pajot et al., 2003; Zoubina and Smith, 2001). The decreased density of nerve fibers which released
microstructure and pain threshold. The mechanisms of the chronic osteoporotic pain remain unclear. In the present study, the OVX rats showed mechanical hyperalgesia at 11 weeks after operation, while thermal pain threshold showed no difference between groups. A previous study (Yu et al., 2011) demonstrated that OVX groups exhibited a mechanical hypersensitity, which could be reversed by estrogen replacement. Li et al. (2014) reported that a mechanical hyperalgesia was also established at 5 weeks after ovariectomy, while the developed thermal allodynia was occurred at 2–7 weeks after ovariectomy. It was suggested that there existed mechanical hyperalgesia in OVX rats, while whether the thermal hyperalgesia could be induced by ovariectomy is still controversy. The thermal allodynia might just occur in the early stage of estrogen depletion and resume in a period of time, which need to be proved in a further experiment. The results presented in this study suggested that the long-term estrogen depletion in OVX rats might increase sensitivity to mechanical stimuli whereas not influence the sensitivity to thermal pain. Our previous study found that VAS scores in osteoarthritis and osteoporosis patients were significantly correlated with the expression 4
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Fig. 2. Different expression of neuropeptides in bone between groups. Immunohistochemistry assay (×200) and quantitative analysis of (A) SP, (B) CGRP, (C) VIP, and (D) NPY in rat tibiae at 12 weeks after operation. The data are expressed as the means ± SD (n = 6 in each group). *P < .05; **P < .01 vs. SHAM by the unpaired two-tailed Student's t-test.
reasons for the increased MOD values of NPY in bone. The regulation of neuropeptides by estrogen suggests that estrogen depletion in OVX rats play an important role in the changes of pain threshold. DRG is an active participant in the development of chronic pain (Krames, 2014, 2015). Studies have revealed that neuropeptides including SP, CGRP, VIP and NPY in DRG are involved in the process of pain generation and transmission. Zheng et al. (2013) confirmed an increased expression of SP in the DRG of OVX mice, and neurokinin 1
neuropeptides might result in the decreased levels of SP, CGRP and VIP in bone. Different with the other three neuropeptides, NPY could be synthesized and released by osteocytes, osteoblast and BMSCs as well as never fibers (Matic et al., 2012). Reboucas et al. (2016) found an increased expression of NPY in OVX rats, and the effect could be reverted by estradiol benzoate administration. We speculated that estrogen depletion induced by ovariectomy might cause an increased expression of NPY in osteocytes, osteoblast and BMSCs, which might be one of the
5
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Fig. 3. Different expression of neuropeptides in DRG between groups. Immunohistochemistry assay (×200) and quantitative analysis of (A) SP, (B) CGRP, (C) VIP, and (D) NPY in DRG at 12 weeks after operation. The data are expressed as the means ± SD (n = 6 in each group). *P < .05; **P < .01 vs. SHAM by the unpaired two-tailed Student's t-test.
sensitization (Lian et al., 2010; Romero-Reyes et al., 2015). Sun et al. revealed that CGRP could induce mechanical hyperalgesia and central sensitization (Sun et al., 2003; Sun et al., 2004). In this study, CGRP was increased in DRG of OVX, which was significantly negatively correlated with MPT. Our data supported a role of CGRP in the mechanical hyperalgesia in OVX rats. Normally, NPY is present in very low levels in DRG neurons (Brumovsky et al., 2002). After nerve injury, the number of NPY-positive DRG neurons increases dramatically (Boateng et al., 2015; Magnussen et al., 2015). In the present study, we showed a
receptor antagonist could attenuate the hyperalgesia. Our study also presented an increased expression of SP in the DRG of OVX rat, while the correlation analysis showed a negative but not significant correlation between the levels of SP and MPT. It suggested that the level of SP in DRG might not play a major role in the hyperalgesia induced by ovariectomy. CGRP is a multifactorial neuropeptide responsible for promoting nociceptive and neuroimmune responses (Abushik et al., 2017; Benemei et al., 2009). It has been studied expansively that CGRP release from primary afferent neurons can cause hyperalgesia and 6
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positively correlated with MPT. It seems that Y1R in DRG might have an analgesic effect. While Naveilhan et al. (2001) observed a reduced antinociception in Y1R knockout mice, and Shi et al. (2006) revealed that the mice lacking Y1R exhibited a significant mechanical hypersensitivity. The role of NPY in pain modulation remains contradictory. Bone microstructure and biomechanical properties were critical to reflect the bone strength and quality. Consistent with other studies (Kang et al., 2015; Mathavan et al., 2015) our study showed remarkable decreases of BV/TV, Tb.N and Conn.D in trabecular bone and increase of Tb.Sp in OVX rats. The results of bone microstructure indicated the significant bone resorption and reduced bone formation in OVX rats with less trabecular number, greater trabecular bone space, and reduced trabecular connectivity density. And OVX rats showed significantly weaker biomechanical properties than those in SHAM rats as well. Bone is highly innervated by sympathetic and sensory nerve fibers and contains neuropeptides which have functional receptors on bone cells. A pronounced decrease in innervation density of bone was demonstrated in OVX rats (Burt-Pichat et al., 2005), and both sympathetic and sensory innervation are associated with the levels of estrogen (Pajot et al., 2003; Zoubina and Smith, 2001). We found the significant differences of neuropeptide distribution in bone between OVX rats and SHAM rats. It was suggested that neuropeptides might play important roles in bone metabolism regulation of OVX rats. Numerous studies have proved the critical roles of neuropeptides in bone remodeling (Ma et al., 2013; Nagao et al., 2014). Wang et al. (Wang et al., 2009) reported that SP was involved in the regulation of bone remodeling by not only stimulating bone resorption but also enhancing bone formation. CGRP could up-regulate the expression of osteoblastic genes and stimulate the proliferation and mineralization of BMSCs (Wang et al., 2010). Besides, CGRP down-regulated osteoclastic genes and inhibited the activation of RANKL. VIP was found to
Table 1 Correlation analysis between the MOD of neuropeptides in bone and microstructure. MOD value
r and P value
BV/TV
Tb.N
Tb.Sp
Conn.D
BMD
SP
r P r P r P r P r P r P
0.616 0.033# 0.647 0.023# 0.784 0.003# −0.786 0.002# −0.861 0.000# −0.728 0.007#
0.877 0.000# 0.798 0.002# 0.741 0.006# −0.829 0.001# −0.905 0.001# −0.819 0.001#
−0.821 0.001# −0.797 0.002# −0.777 0.003# 0.869 0.000# 0.883 0.001# 0.854 0.001#
0.906 0.000# 0.800 0.002# 0.653 0.021# −0.811 0.001# −0.846 0.001# −0.819 0.001#
−0.666 0.018# 0.460 0.132 0.746 0.005# 0.591 0.043# −0.621 0.031# −0.543 0.068
CGRP VIP NPY Y1R Y2R
value value value value value value
BV/TV, bone volume fraction; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular spacing; Conn.D, connectivity density; BMD, bone mineral density; SP, substance P; CGRP, calcitonin gene-related peptide; VIP, vasoactive intestinal peptide; NPY, Neuropeptide Y; Y1R, NPY Y1 receptor; Y2R, NPY Y2 receptor; r = correlation coefficient assessed by Pearson correlation analysis. # P < .05. All the data in bold are considered to be statistically significant.
pronounced increased levels of NPY and Y2R in DRG of OVX rats, which were both negatively correlated with MPT. The expression of Y2R in DRG increased when nerve injured, and activated Y2R promoted DRG neurons excitability (Bost et al., 2017). Sapunar et al. (2011) reported that NPY would increase the hyperalgesic responses induced by acute DRG injury, and the actions were blocked by Y2R antagonist. It was suggested that NPY might be involved in mechanical hyperalgesia of OVX rats, which might be mediated by Y2R. Notably, we found that the express of Y1R in DRG was dramatically decreased in OVX rats, and was
Fig. 4. Neuropeptides in DRG are correlated with mechanical pain threshold. The correlation between MPT and the MOD of (A) SP, (B) CGRP, (C) VIP, (D) NPY, (E) Y1R and (F) Y2R in DRG in both SHAM and OVX groups. n = 12, r = correlation coefficient assessed by Pearson correlation analysis. 7
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stimulate activity of osteoblasts. And Lundberg et al. demonstrated that VIP treatment caused a decreased motility of osteoclasts and transient inhibition of bone resorption (Lundberg et al., 1999). Our data showed the decreased expression of SP, CGRP and VIP in bone of OVX rats. And correlation analysis showed that SP, CGRP and VIP in bone were positively correlated with BV/TV, Tb.N and Conn.D, and negatively correlated with Tb.Sp. Our results suggested that SP, CGRP and VIP in bone might be related to inhibition of bone resorption or enhancing bone formation. NPY mainly regulates the bone metabolism via Y1R and Y2R (Liu et al., 2016). Lee et al. (2011) revealed that osteoblast-specific Y1R deletion in mouse showed significant increased cancellous BV/TV, Tb.Th and Tb.N. Y2R is apparently present in hypothalamus and acts through central mechanism. Baldock et al. (2002) found that mice that selective deletion of hypothalamic Y2 receptors showed an identical increase in the rate of bone mineralization and formation. Here we presented the increased levels of NPY, Y1R and Y2R of bone in OVX rats, and which were significantly negative correlation with BV/TV. The results suggested that NPY might regulate bone turnover through both Y1R and Y2R. Our results suggested that the expression of neuropeptides in bone might be involved in the osteoporotic bone microstructure in OVX rats. And more mechanisms underlying the roles of neuropeptides in bone remodeling merit further research. There were still some limitations in the present study. One is that the measurement of parameters in this experiment was carried out only at a single time point t after operation, while the development of osteoporosis was chronic and long-term. Therefore, studies with the measurement at multiple time points need to be performed in the future. In addition, this study preliminarily demonstrated the relationship between neuropeptides, bone microstructure, and pain threshold in ovariectomized rats. Further studies should be conducted to elucidate the mechanisms underlying the role of neuropeptides in osteoporosis. In conclusion, compared with SHAM rats, the OVX rats showed mechanical hyperalgesia, osteoporotic bone microstructure and different neuropeptide distributions. Our results suggested that SP, CGRP, VIP and NPY were involved in the osteoporotic bone microstructure and mechanical hypersensitivity in OVX rats, indicating the potential to utilize neuropeptides as novel therapeutic targets for PMO.
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Declaration of Competing Interest The authors have no conflict of interest to disclose. Acknowledgements This study was supported by grants from the National Natural Science Foundation of China (Nos.81871090, 81400904), Shanghai Jiaotong University Integration Founding of Medicine and Engineering (YG2016MS53). Clinical research and cultivation fund of Renji Hospital (PY2018-III-07). References Abushik, P.A., Bart, G., Korhonen, P., Leinonen, H., Giniatullina, R., Sibarov, D.A., Levonen, A.L., Malm, T., Antonov, S.M., Giniatullin, R., 2017. Pro-nociceptive migraine mediator CGRP provides neuroprotection of sensory, cortical and cerebellar neurons via multi-kinase signaling. Cephalalgia 37, 1373–1383. Baldock, P.A., Sainsbury, A., Couzens, M., Enriquez, R.F., Thomas, G.P., Gardiner, E.M., Herzog, H., 2002. Hypothalamic Y2 receptors regulate bone formation. J. Clin. Invest. 109, 915–921. Benemei, S., Nicoletti, P., Capone, J.G., Geppetti, P., 2009. CGRP receptors in the control of pain and inflammation. Curr. Opin. Pharmacol. 9, 9–14. Boateng, E.K., Novejarque, A., Pheby, T., Rice, A.S., Huang, W., 2015. Heterogeneous responses of dorsal root ganglion neurons in neuropathies induced by peripheral nerve trauma and the antiretroviral drug stavudine. Eur. J. Pain 19, 236–245. Bost, A., Shaib, A.H., Schwarz, Y., Niemeyer, B.A., Becherer, U., 2017. Large dense-core vesicle exocytosis from mouse dorsal root ganglion neurons is regulated by neuropeptide Y. Neuroscience 346, 1–13. Brumovsky, P.R., Shi, T.J., Matsuda, H., Kopp, J., Villar, M.J., Hokfelt, T., 2002. NPY Y1 receptors are present in axonal processes of DRG neurons. Exp. Neurol. 174, 1–10. Burt-Pichat, B., Lafage-Proust, M.H., Duboeuf, F., Laroche, N., Itzstein, C., Vico, L.,
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metabolism. Bone 82, 56–63. Xiao, J., Yu, W., Wang, X., Wang, B., Chen, J., Liu, Y., Li, Z., 2016. Correlation between neuropeptide distribution, cancellous bone microstructure and joint pain in postmenopausal women with osteoarthritis and osteoporosis. Neuropeptides 56, 97–104. Xu, X., Jia, X., Mo, L., Liu, C., Zheng, L., Yuan, Q., Zhou, X., 2017. Intestinal microbiota: a potential target for the treatment of postmenopausal osteoporosis. Bone Res. 5, 17046. Yang, Y., Ozawa, H., Lu, H., Yuri, K., Hayashi, S., Nihonyanagi, K., Kawata, M., 1998. Immunocytochemical analysis of sex differences in calcitonin gene-related peptide in the rat dorsal root ganglion, with special reference to estrogen and its receptor. Brain Res. 791, 35–42.
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Neuropeptides journal homepage: www.elsevier.com/locate/npep
Neuropeptides are associated with pain threshold and bone microstructure in ovariectomized rats ⁎
Weixin Xiea, Fan Lia, Yi Hana, Zhanchun Lia, , Jie Xiaob, a b
⁎⁎
Department of Orthopaedic Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, China Department of Anesthesiology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Osteoporosis Neuropeptide Bone microstructure Pain threshold
Objectives: Postmenopausal osteoporosis (PMO) is a metabolic skeletal disorder with impaired bone density and bone quality in postmenopausal women. The aim of the present study was to investigate the correlation between neuropeptides, bone microstructure and pain threshold in ovariectomized (OVX) rats. Methods: Female rats were randomly divided into the ovariectomized (OVX) group and the sham surgery (SHAM) group. Bone microstructure and immunocytochemistry for substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) in tibial and DRG were performed. Pain threshold was assessed at post-operative 11 weeks. Pearson correlation coefficients were calculated between neuropeptides, bone microstructure and pain threshold. Results: Significant decreases in bone volume fraction (BV/TV) and trabecular number (Tb. N) but significant increases in trabecular spacing (Tb.Sp) were showed in OVX group. Mechanical pain threshold (MPT) in OVX group was significantly decreased. The MOD values for SP, CGRP and VIP of tibial in OVX group were significantly lower, whereas NPY, NPY1R and NPY2R were significantly higher. And SP, CGRP, VIP, NPY and NPY2R of DRG were significantly increased in OVX group, while NPY1R was significantly decreased. Correlation analysis showed that NPY, Y1R and Y2R in bone were negatively correlated with BV/TV. MPT was negatively correlated with NPY and Y2R in DRG, and positively correlated with Y1R in DRG. Conclusions: Our results suggested that SP, CGRP, VIP and NPY were involved in the osteoporotic bone microstructure and mechanical hypersensitivity in OVX rats, indicating the potential to utilize neuropeptides as novel therapeutic targets for PMO.
1. Introduction
by nerve fibers to act on bone tissue. A variety of neuropeptides have been proved to exist in bones. Substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY) and their receptors were found in bone cells like osteoblasts, osteoclasts or bone marrow mesenchymal stem cells (BMSCs) (Grassel, 2014) and they are all critical regulators of bone metabolism. Bone pain caused by osteoporosis makes a chronic influence on quality of life of affected people. There is still no effective treatment for pain relief, as the pathogenesis of osteoporotic pain remain unclear. Estrogen receptors (ER) expressed in small nociceptive neurons of dorsal root ganglions (DRG) are known to be involved in nociception (Sarajari and Oblinger, 2010). SP and CGRP contained in the small DRG neurons and released by sensory nerve fibers are both critical pain mediators and significantly regulated by estrogen. We previously (Xiao
Postmenopausal osteoporosis (PMO) is defined as a metabolic skeletal disorder characterized by impaired bone density and bone quality in postmenopausal women, which is induced by estrogen deficiency (Watts et al., 2010; Xu et al., 2017). The core clinical features of PMO include chronic bone pain and fracture caused by compromised bone mass, bone microstructure and biomechanical properties. These manifestations can dramatically worsen quality of life and functional capacity in PMO patients. PMO is now still a global public health concern involving medicine, society and economy. It is universally acknowledged that nerve system plays an important role in regulation of the skeleton (Wee et al., 2016). In periphery, neuropeptides as neurotransmitters and neuromodulators are released
⁎ Correspondence to: Z. Li, Department of Orthopaedic Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, 1630 Dongfang Rd., Shanghai 200127, PR China. ⁎⁎ Correspondence to: Jie Xiao, Department of Anesthesiology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, 1630 Dongfang Rd., Shanghai 200127, PR China. E-mail addresses: [email protected] (Z. Li), [email protected] (J. Xiao).
https://doi.org/10.1016/j.npep.2019.101995 Received 26 September 2019; Received in revised form 4 November 2019; Accepted 13 November 2019 0143-4179/ © 2019 Published by Elsevier Ltd.
Please cite this article as: Weixin Xie, et al., Neuropeptides, https://doi.org/10.1016/j.npep.2019.101995
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kit (Abcam, Cambridge, MA), according to the manufacturer's instructions. The absorbance was read at 450 nm using a microplate reader. The serum concentrations of E2 were calculated according to the optical density (OD) and standard curve.
et al., 2016) demonstrated that the VAS score in osteoporosis (OP) and osteoarthritis (OA) patients was positively correlated with the levels of SP, CGRP and VIP in subchondral cancellous bones and negatively correlated with NPY. The results suggested that SP, CGRP, VIP and NPY are associated with osteoporotic pain generation and may be the potential therapeutic targets for osteoporotic pain. The neuropeptides located in central and peripheral nervous systems play critical roles in bone metabolism and pain modulation. Studies reported that nerve profile density in bone of ovariectomized (OVX) rats decreased (Burt-Pichat et al., 2005), and both sympathetic and sensory innervation are associated with the levels of estrogen (Pajot et al., 2003; Zoubina and Smith, 2001). The changes of nerve innervation induced by levels of estrogen may make a difference to neuropeptide expression. We previously (Xiao et al., 2016) observed that between postmenopausal women with osteoporosis and osteoarthritis, there were significant differences in levels of neuropeptides and microstructure of subchondral cancellous bones. Anyhow, the role of neuropeptides in the pathogenesis of PMO is still uncertain. This study was performed to further explore the relationship between the neuropeptides, bone microstructure and pain threshold in OVX rat model, aiming to investigate the potential roles of neuropeptides in the pathogenesis of PMO.
2.5. Pain threshold assessment Mechanical pain threshold (MPT) assessment. MPT was measured using von Frey filaments. Before testing, rats were acclimatized for at least 1 h in a clear Plexiglas box (26 cm long × 20 cm wide × 14 cm high) on an elevated wire mesh grid. Von Frey filaments were applied to the middle of plantar surface of the right hind paw with buckling weight of 1, 1.4, 2, 4, 6, 8, 10, 15, 26 g. The pressure was set to cause the filament to buckle for 2 s each time interval of 15 s, 5 cycles. An increased force stimulus was applied if at least three paw withdrawal responses were absence. The smallest force that could raise the paw withdrawal responses in at least 3 out of 5 times was defined as Paw Withdrawal Threshold (PWT). The same measurement was applied to the left hind paw. Thermal pain threshold (TPT) assessment. Rats were acclimatized for at least 1 h in a clear Plexiglas box (26 cm long × 20 cm wide × 14 cm high) on a glass plate. A beam of radiant heat was applied to the plantar surface of the right hind paw. The radiant heat beam was shut off automatically once the paw withdrawal response occurred. The time from the start of the heating to the paw withdrawal was recorded using a digital timer and defined as the Thermal Withdrawal Latency (TWL). Each measurement was repeated in triplicate at intervals of 5 min and the average value was calculated. The noresponse cut-off time was set to 30 s to avoid tissue damage.
2. Materials and methods 2.1. Animals This study was ethically approved by the Institutional Animal Care and Use Committee of Shanghai Jiaotong University (RJ2018–1012) and was conducted in conformity with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Twelve 6-monthold female virgin Sprague–Dawley rats (weight: 290-310 g) were housed in a room under conditions of controlled temperature at 22 °C with a 12-hour light/dark cycle. A normal chow diet and distilled were provided for all animals throughout the experiment.
2.6. Bone micro-computed tomography (micro-CT) imaging The right tibial specimens were examined using a micro-CT system (Xiao et al., 2016; Zhang et al., 2010) (μCT 80, Scanco Medical AG, Switzerland) at a voxel size of 6 μm. Scan parameters included tube voltage 80 kV, tube current 80uA, 4 frames averaged and a 0.4-degree increment angle. After scanning, a region of interest (ROI) was selected between 5 cm proximal and 5 cm distal to the center of distal tibial metaphysis. The quantitative analysis were conducted using the software provided with the instrument and the following parameters were calculated: The bone volume fraction (BV/TV; %), trabecular number (Tb.N; mm−1), trabecular thickness (Tb.Th; mm), trabecular separation (Tb.Sp; mm), degree of anisotropy (DA), connectivity density (Conn.D; mm−3), apparent density (AD) and material density (MD).
2.2. Experimental design The rats were randomly divided into 2 groups: ovariectomy (OVX; n = 6) and sham (SHAM; n = 6) operation. Either ovariectomy or sham surgery was performed under intraperitoneal anesthesia with pentobarbital (40 mg/kg). For OVX rats, bilateral ovaries were excised via back incisions, and adipose tissue of the equal weight as the removed ovaries was excised for SHAM rats. Pain threshold assessment was carried out at 1 week before sugery and 11 weeks after operation. All rats were euthanized 12 weeks after ovariectomy or sham operation by overdose pentobarbital injection. Uterus weight were measured to confirm the success of ovariectomy. Blood were collected for analysis of levels of 17β-estradiol (E2) from the abdominal aorta and serum was stored at −80 °C after centrifugation at 1600g for 15 min at 4 °C. The bilateral tibiae, femurs and DRG (L6 – S1) dedicated to histomorphometry and immunohistochemistry analysis were harvested and stored at −80 °C until processing.
2.7. Biomechanical testing The biomechanical properties of left tibial specimens was measured by four-point bending test using mechanical universal machine (Instron 8874, Instron, Ltd., High Wycombe, UK). The load was applied at a speed of 1 mm/min until the specimen was disrupted. The data were recorded and analyzed by the software within the equipment itself. The Ultimate load (N), ultimate strain (mm/mm), ultimate stress (MPa), ultimate load energy and elasticity modulus (MPa) were obtained for evaluating biomechanical properties.
2.3. Bone mineral density (BMD) measurement The rats were anesthetized to perform in vivo BMD (of the femurs) measurement using dual-energy x-ray absorptiometry on a Discovery-A (Hologic) system at post-operative 12 weeks. Data were analyzed with high-resolution BMD test software for small animals (Regional High Resolution, version 4076; Hologic). The coefficient of variation for BMD measurement was < 2.0%.
2.8. Immunocytochemistry The immunocytochemistry was evaluated according to the method reported previously (Xiao et al., 2016). Briefly, the right tibial and DRG specimens were embedded with paraffin after decalcification and were sectioned into 5-mm thickness. Heat mediated antigen retrieval was performed using boiled citrate after deparaffinization and hydration. After overnight incubation at 4 °C with the primary antibody separately (Anti-SP antibody, 1:1000, Santa Cruz, Biotechnology, Inc. USA; AntiCGRP antibody, 1:1000, Abcam, Cambridge, USA; Anti-VIP antibody,
2.4. Enzyme linked immunosorbent assay (ELISA) Serum concentrations of E2 was detected using 17β-estradiol ELISA 2
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respectively, P > .05) (Fig. 1C).
1:500, Abcam, Cambridge, USA; Anti-NPY antibody, 1:1000 Abcam, Cambridge, USA; Anti-NPY1R antibody, 1:1000 Abcam, Cambridge, USA; Anti-NPY2R antibody, 1:1000 Abcam, Cambridge, USA), the sections incubated with a biotinylated secondary antibody for 15 min at 37 °C. Peroxidase reaction was visualized with a solution of diaminobenzidine (DAB) in Tris buffer containing 0.01% H2O2 (0.05 mol/l, pH 7.6). The positive staining was visible as brown punctums and bundles under the under light microscope (E800, Nikon, Tokyo, Japan). Three images of high-powered magnification views were taken using a Spot Advanced digital-imaging system (Diagnostic Instruments, Inc., Sterling Heights, MI, USA). The mean optical density (MOD) was measured to perform the quantitative immunohistochemical analysis using the software package Image Pro Plus (version 5.0.1, Media Cybernetics, Silver Spring, MD, USA).
3.4. Ovariectomy attenuated biomechanical properties of bone Compared with the SHAM group, the ultimate load (96.17 ± 2.56 vs. 126.00 ± 11.65 N, respectively, P < .01), ultimate stress (93.17 ± 17.08 vs. 120.50 ± 11.20 MPa, respectively, P < .01), ultimate load energy (0.032 ± 0.008 vs. 0.043 ± 0.007 J, respectively, P < .05), elasticity modulus (3859.00 ± 318.00 vs. 5028.00 ± 503.00 MPa, respectively, P < .05) values were significantly decreased in the OVX group at post-operative 11 weeks. And there was no significant difference in ultimate strain (0.044 ± 0.009 vs. 0.055 ± 0.012 mm/mm, respectively, P > .05) between two groups. 3.5. Different expression of neuropeptides in bone and DRG between groups
2.9. Statistical analysis The comparisons of MOD values for SP, CGRP, VIP and NPY in right tibiae and DRG between OVX group and SHAM group were performed via immunohistochemical analysis (Figs. 2 and 3). The MOD values for SP, CGRP and VIP of right tibiae in the OVX group were significantly lower than those in the SHAM group (SP: 0.173 ± 0.012 vs. 0.246 ± 0.03, P < .01; CGRP: 0.187 ± 0.017 vs. 0.244 ± 0.018, P < .001; VIP: 0.219 ± 0.016 vs. 0.282 ± 0.039, P < .01). On the contrary, the MOD value for NPY of right tibiae in the OVX group was significantly higher than that in the SHAM group (0.285 ± 0.032 vs. 0.167 ± 0.028, P < .001). Moreover, the MOD values for all these four neuropeptides of DRG were significantly increased in the OVX group (SP: 0.255 ± 0.044 vs. 0.206 ± 0.029, P < .05; CGRP: 0.354 ± 0.016 vs. 0.307 ± 0.016, P < .01; VIP: 0.144 ± 0.013 vs. 0.126 ± 0.014, P < .05; NPY: 0.310 ± 0.020 vs. 0.241 ± 0.025, P < .001). NPY Y1 receptor (Y1R) and NPY Y2 receptor (Y2R) in bone were significantly increased in the OVX group (Y1R: 0.498 ± 0.035 vs. 0.302 ± 0.036, P < .001; Y2R: 0.420 ± 0.062 vs. 0.266 ± 0.047, P < .001). Y1R in DRG was significantly decreased in the OVX group (0.045 ± 0.008 vs. 0.107 ± 0.030, P < .001), while Y2R in DRG was significantly increased (0.771 ± 0.140 vs. 0.364 ± 0.067, P < .001).
Data analyses were performed using SPSS 23.0 software (SPSS Inc., Chicago, IL, USA). All data were presented as the means ± standard deviation. The statistical significance between two groups was evaluated by Student's t-test. Pearson correlation analysis was carried out to determine the correlation between variables. The significance level was set at P < .05. 3. Results 3.1. Verification of the OVX rat model Mass of uterus, body weight and E2 concentrations between the two groups were compared at post-operative 12 weeks. As expected, uterine weight was significantly lower in OVX group than in SHAM group (0.22 ± 0.08 vs. 0.67 ± 0.08 g, respectively, P < .01), and body weight was significantly higher in OVX group than in SHAM group (450.49 ± 44.42 vs. 334.39 ± 44.18 g, respectively, P < .01). The OVX rats were shown by substantial decreases in E2 concentrations in relation to the SHAM rats (136.65 ± 6.24 vs. 304.21 ± 6.68 pg/ml, respectively, P < .001).
3.6. Neuropeptides are correlated with bone microstructure and pain threshold
3.2. Ovariectomy attenuated bone microstructure and bone mass
Table 1 showed the correlation between bone microstructure and neuropeptides in bone. And as shown in Fig. 4, the MOD values for NPY (r = −0.741, P < .01) and CGRP (r = −0.598, P < .05) in DRG were negatively correlated with MPT. As for the NPY receptors, Y1R (r = 0.741, P < .01) in DRG was positively correlated with MPT, while Y2R (r = −0.597, P < .05) in DRG was negatively correlated with MPT. In addition, the MOD of SP and VIP in DRG showed no significant correlation with MPT.
The right tibials of rats were analyzed to perform the micro-CT imaging (Fig. 1A). The quantitative analysis revealed that differences in BV/TV, Tb.N, Tb.Th, Tb.Sp and Conn.D in trabecular bone were all significant between the two groups (Fig. 1B). Compared to SHAM group, OVX group showed significant decreases in BV/TV (17.00 ± 2.92 vs. 55.79 ± 14.99%, respectively, P < .001), Tb. N (1.38 ± 0.24 vs. 3.23 ± 0.37 mm−1, respectively, P < .001) and Conn.D (9.38 ± 3.11 vs. 41.07 ± 10.55 mm−3, respectively, P < .001) but significant increases in Tb.Sp (0.62 ± 0.12 vs. 0.14 ± 0.05 μm, respectively, P < .001). The comparison of Tb.Th showed no significant difference between two groups. Additionally, the OVX rats were shown by substantial decreases in BMD in relation to the SHAM rats (0.230 ± 0.013 vs. 0.261 ± 0.019 g/cm2, respectively, P < .05).
4. Discussion Our previous study revealed that MOD values for SP, CGRP, VIP and NPY in OA group were significantly different with those in OP group. And the neuropeptides were correlated with the parameters of bone microstructure and VAS scores in osteoarthritis and osteoporosis patients, which suggested that neuropeptides might be involved in the changes of bone microstructure and pain generation in OA and OP (Xiao et al., 2016). In this study, the ovariectomized rat model was built to mimic postmenopausal woman with osteoporosis, and the relationships between neuropeptides and bone microstructure and pain threshold are investigated. The results demonstrated several novel findings. Firstly, the OVX rats showed mechanical hyperalgesia and osteoporotic bone microstructure. Secondly, there were different neuropeptide distributions between OVX rats and SHAM rats. Thirdly, Neuropeptides expressed in bone and DRG are correlated with bone
3.3. Ovariectomy induced mechanical hyperalgesia There was no statistical difference between OVX group and SHAM group with regard to MPT (8.00 ± 0.00 vs. 8.33 ± 1.51 g, respectively, P > .05) and TPT (11.62 ± 0.95 vs. 11.55 ± 0.97 s, respectively, P > .05) at 1 week before operation. At post-operative 11 weeks, MPT in OVX group was significantly decreased when compared with the SHAM group (4.23 ± 2.12 vs. 9.5 ± 3.08 g, respectively, P < .01), and TPT in OVX group did not differ significantly when compared with SHAM group (15.94 ± 2.34 vs. 14.79 ± 2.58 s, 3
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Fig. 1. Ovariectomy attenuated bone microstructure and induced mechanical hyperalgesia. A Representative micro-CT images of tibias. B Comparison of the BV/TV, Tb.N, Tb.Sp, Conn.D and BMD between groups. C Comparison of the pain threshold between groups. The data are expressed as the means ± SD (n = 6 in each group). *P < .05; **P < .01; ****P < .0001 vs. SHAM by the unpaired two-tailed Student's t-test (B,C).
of neuropeptides, which prompted us that neuropeptides are involved in the pain generation (Xiao et al., 2016). In the present study, we investigated the differences of neuropeptide distributions in bone and DRG between OVX group and SHAM group. The findings reported here showed that the expressions of SP, CGRP and VIP of bone were significantly lower, whereas NPY was higher in OVX group. In DRG, the expressions of SP, CGRP, VIP and NPY were all significantly higher in OVX group. SP and CGRP are well-established transmitter of nociception, which could be regulated by estrogen. Estrogen has been shown to have a negative effect on SP expression, Sarajari and Oblinger (2010) showed that the expression of SP in the small DRG neurons were significantly downregulated in estrogentreated rats. CGRP is a neuropeptide co-stored with SP in DRG neurons, and rats treated with estrogen showed increased CGRP synthesis in DRG (Mowa et al., 2003; Yang et al., 1998). Studies reported that nerve profile density in bone of ovariectomized (OVX) rats decreased (BurtPichat et al., 2005), and both sympathetic and sensory innervation are associated with the levels of estrogen (Pajot et al., 2003; Zoubina and Smith, 2001). The decreased density of nerve fibers which released
microstructure and pain threshold. The mechanisms of the chronic osteoporotic pain remain unclear. In the present study, the OVX rats showed mechanical hyperalgesia at 11 weeks after operation, while thermal pain threshold showed no difference between groups. A previous study (Yu et al., 2011) demonstrated that OVX groups exhibited a mechanical hypersensitity, which could be reversed by estrogen replacement. Li et al. (2014) reported that a mechanical hyperalgesia was also established at 5 weeks after ovariectomy, while the developed thermal allodynia was occurred at 2–7 weeks after ovariectomy. It was suggested that there existed mechanical hyperalgesia in OVX rats, while whether the thermal hyperalgesia could be induced by ovariectomy is still controversy. The thermal allodynia might just occur in the early stage of estrogen depletion and resume in a period of time, which need to be proved in a further experiment. The results presented in this study suggested that the long-term estrogen depletion in OVX rats might increase sensitivity to mechanical stimuli whereas not influence the sensitivity to thermal pain. Our previous study found that VAS scores in osteoarthritis and osteoporosis patients were significantly correlated with the expression 4
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Fig. 2. Different expression of neuropeptides in bone between groups. Immunohistochemistry assay (×200) and quantitative analysis of (A) SP, (B) CGRP, (C) VIP, and (D) NPY in rat tibiae at 12 weeks after operation. The data are expressed as the means ± SD (n = 6 in each group). *P < .05; **P < .01 vs. SHAM by the unpaired two-tailed Student's t-test.
reasons for the increased MOD values of NPY in bone. The regulation of neuropeptides by estrogen suggests that estrogen depletion in OVX rats play an important role in the changes of pain threshold. DRG is an active participant in the development of chronic pain (Krames, 2014, 2015). Studies have revealed that neuropeptides including SP, CGRP, VIP and NPY in DRG are involved in the process of pain generation and transmission. Zheng et al. (2013) confirmed an increased expression of SP in the DRG of OVX mice, and neurokinin 1
neuropeptides might result in the decreased levels of SP, CGRP and VIP in bone. Different with the other three neuropeptides, NPY could be synthesized and released by osteocytes, osteoblast and BMSCs as well as never fibers (Matic et al., 2012). Reboucas et al. (2016) found an increased expression of NPY in OVX rats, and the effect could be reverted by estradiol benzoate administration. We speculated that estrogen depletion induced by ovariectomy might cause an increased expression of NPY in osteocytes, osteoblast and BMSCs, which might be one of the
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Fig. 3. Different expression of neuropeptides in DRG between groups. Immunohistochemistry assay (×200) and quantitative analysis of (A) SP, (B) CGRP, (C) VIP, and (D) NPY in DRG at 12 weeks after operation. The data are expressed as the means ± SD (n = 6 in each group). *P < .05; **P < .01 vs. SHAM by the unpaired two-tailed Student's t-test.
sensitization (Lian et al., 2010; Romero-Reyes et al., 2015). Sun et al. revealed that CGRP could induce mechanical hyperalgesia and central sensitization (Sun et al., 2003; Sun et al., 2004). In this study, CGRP was increased in DRG of OVX, which was significantly negatively correlated with MPT. Our data supported a role of CGRP in the mechanical hyperalgesia in OVX rats. Normally, NPY is present in very low levels in DRG neurons (Brumovsky et al., 2002). After nerve injury, the number of NPY-positive DRG neurons increases dramatically (Boateng et al., 2015; Magnussen et al., 2015). In the present study, we showed a
receptor antagonist could attenuate the hyperalgesia. Our study also presented an increased expression of SP in the DRG of OVX rat, while the correlation analysis showed a negative but not significant correlation between the levels of SP and MPT. It suggested that the level of SP in DRG might not play a major role in the hyperalgesia induced by ovariectomy. CGRP is a multifactorial neuropeptide responsible for promoting nociceptive and neuroimmune responses (Abushik et al., 2017; Benemei et al., 2009). It has been studied expansively that CGRP release from primary afferent neurons can cause hyperalgesia and 6
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positively correlated with MPT. It seems that Y1R in DRG might have an analgesic effect. While Naveilhan et al. (2001) observed a reduced antinociception in Y1R knockout mice, and Shi et al. (2006) revealed that the mice lacking Y1R exhibited a significant mechanical hypersensitivity. The role of NPY in pain modulation remains contradictory. Bone microstructure and biomechanical properties were critical to reflect the bone strength and quality. Consistent with other studies (Kang et al., 2015; Mathavan et al., 2015) our study showed remarkable decreases of BV/TV, Tb.N and Conn.D in trabecular bone and increase of Tb.Sp in OVX rats. The results of bone microstructure indicated the significant bone resorption and reduced bone formation in OVX rats with less trabecular number, greater trabecular bone space, and reduced trabecular connectivity density. And OVX rats showed significantly weaker biomechanical properties than those in SHAM rats as well. Bone is highly innervated by sympathetic and sensory nerve fibers and contains neuropeptides which have functional receptors on bone cells. A pronounced decrease in innervation density of bone was demonstrated in OVX rats (Burt-Pichat et al., 2005), and both sympathetic and sensory innervation are associated with the levels of estrogen (Pajot et al., 2003; Zoubina and Smith, 2001). We found the significant differences of neuropeptide distribution in bone between OVX rats and SHAM rats. It was suggested that neuropeptides might play important roles in bone metabolism regulation of OVX rats. Numerous studies have proved the critical roles of neuropeptides in bone remodeling (Ma et al., 2013; Nagao et al., 2014). Wang et al. (Wang et al., 2009) reported that SP was involved in the regulation of bone remodeling by not only stimulating bone resorption but also enhancing bone formation. CGRP could up-regulate the expression of osteoblastic genes and stimulate the proliferation and mineralization of BMSCs (Wang et al., 2010). Besides, CGRP down-regulated osteoclastic genes and inhibited the activation of RANKL. VIP was found to
Table 1 Correlation analysis between the MOD of neuropeptides in bone and microstructure. MOD value
r and P value
BV/TV
Tb.N
Tb.Sp
Conn.D
BMD
SP
r P r P r P r P r P r P
0.616 0.033# 0.647 0.023# 0.784 0.003# −0.786 0.002# −0.861 0.000# −0.728 0.007#
0.877 0.000# 0.798 0.002# 0.741 0.006# −0.829 0.001# −0.905 0.001# −0.819 0.001#
−0.821 0.001# −0.797 0.002# −0.777 0.003# 0.869 0.000# 0.883 0.001# 0.854 0.001#
0.906 0.000# 0.800 0.002# 0.653 0.021# −0.811 0.001# −0.846 0.001# −0.819 0.001#
−0.666 0.018# 0.460 0.132 0.746 0.005# 0.591 0.043# −0.621 0.031# −0.543 0.068
CGRP VIP NPY Y1R Y2R
value value value value value value
BV/TV, bone volume fraction; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular spacing; Conn.D, connectivity density; BMD, bone mineral density; SP, substance P; CGRP, calcitonin gene-related peptide; VIP, vasoactive intestinal peptide; NPY, Neuropeptide Y; Y1R, NPY Y1 receptor; Y2R, NPY Y2 receptor; r = correlation coefficient assessed by Pearson correlation analysis. # P < .05. All the data in bold are considered to be statistically significant.
pronounced increased levels of NPY and Y2R in DRG of OVX rats, which were both negatively correlated with MPT. The expression of Y2R in DRG increased when nerve injured, and activated Y2R promoted DRG neurons excitability (Bost et al., 2017). Sapunar et al. (2011) reported that NPY would increase the hyperalgesic responses induced by acute DRG injury, and the actions were blocked by Y2R antagonist. It was suggested that NPY might be involved in mechanical hyperalgesia of OVX rats, which might be mediated by Y2R. Notably, we found that the express of Y1R in DRG was dramatically decreased in OVX rats, and was
Fig. 4. Neuropeptides in DRG are correlated with mechanical pain threshold. The correlation between MPT and the MOD of (A) SP, (B) CGRP, (C) VIP, (D) NPY, (E) Y1R and (F) Y2R in DRG in both SHAM and OVX groups. n = 12, r = correlation coefficient assessed by Pearson correlation analysis. 7
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stimulate activity of osteoblasts. And Lundberg et al. demonstrated that VIP treatment caused a decreased motility of osteoclasts and transient inhibition of bone resorption (Lundberg et al., 1999). Our data showed the decreased expression of SP, CGRP and VIP in bone of OVX rats. And correlation analysis showed that SP, CGRP and VIP in bone were positively correlated with BV/TV, Tb.N and Conn.D, and negatively correlated with Tb.Sp. Our results suggested that SP, CGRP and VIP in bone might be related to inhibition of bone resorption or enhancing bone formation. NPY mainly regulates the bone metabolism via Y1R and Y2R (Liu et al., 2016). Lee et al. (2011) revealed that osteoblast-specific Y1R deletion in mouse showed significant increased cancellous BV/TV, Tb.Th and Tb.N. Y2R is apparently present in hypothalamus and acts through central mechanism. Baldock et al. (2002) found that mice that selective deletion of hypothalamic Y2 receptors showed an identical increase in the rate of bone mineralization and formation. Here we presented the increased levels of NPY, Y1R and Y2R of bone in OVX rats, and which were significantly negative correlation with BV/TV. The results suggested that NPY might regulate bone turnover through both Y1R and Y2R. Our results suggested that the expression of neuropeptides in bone might be involved in the osteoporotic bone microstructure in OVX rats. And more mechanisms underlying the roles of neuropeptides in bone remodeling merit further research. There were still some limitations in the present study. One is that the measurement of parameters in this experiment was carried out only at a single time point t after operation, while the development of osteoporosis was chronic and long-term. Therefore, studies with the measurement at multiple time points need to be performed in the future. In addition, this study preliminarily demonstrated the relationship between neuropeptides, bone microstructure, and pain threshold in ovariectomized rats. Further studies should be conducted to elucidate the mechanisms underlying the role of neuropeptides in osteoporosis. In conclusion, compared with SHAM rats, the OVX rats showed mechanical hyperalgesia, osteoporotic bone microstructure and different neuropeptide distributions. Our results suggested that SP, CGRP, VIP and NPY were involved in the osteoporotic bone microstructure and mechanical hypersensitivity in OVX rats, indicating the potential to utilize neuropeptides as novel therapeutic targets for PMO.
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