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ARTICLE IN PRESS
BONSOI 3923 1–4
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Available online at www.sciencedirect.com
Original article
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Estrogen receptor expression in lumbar intervertebral disc of the elderly: Gender- and degeneration degree-related variations
3
Xiao-xing Song a,b,1 , Yong-juan Yu a , Xin-feng Li a,∗ , Zu-de Liu a , Bu-wei Yu b,∗∗ , Zhen Guo a,1
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4 5
a b
Department of Orthopaedic Surgery, Ren Ji Hospital,School of Medicine, Shanghai Jiao Tong University, Shanghai, China Department of Anesthesiology, Rui Jin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
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7
a r t i c l e
i n f o
a b s t r a c t
8 9 10 11
Article history: Accepted 4 September 2013 Available online xxx
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Keywords: Intervertebral disc Degeneration Estrogen receptor Nucleus pulposus
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1. Introduction
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Objectives: Sexual dimorphism does occur in intervertebral disc (IVD) degeneration. The involvement of estrogen on IVD health has been well reported in recent years. The estrogen receptors (ER) are the main mediators of estrogen action. ER might play specific roles in the sexual variations of the IVD degeneration. Methods: Thirty-six elderly patients with lumbar disc degeneration were selected and graded using Pfirrmann’s system based on MRI images. Differences of ER␣ and ER immunoreactivity staining in nucleus pulposus of each sex and degeneration degree were recorded and compared. Results: Both cytoplasmic and nuclear staining of ER␣ and ER immunoreactivity were observed in the nucleus pulposus cells. ER␣ and ER expression significantly decreased along with the aggravation of IVD degeneration both in males and females. Expression of ER␣ and ER protein in nucleus pulposus of males was significantly higher than that of females. Conclusions: Gender-specific expression of ER might play a part in sexual dimorphism of IVD degeneration. Gender and degeneration condition differences should be taken into account when the effects of estrogen on IVD metabolism are studied further. © 2013 Société franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.
Intervertebral disk (IVD) degeneration is a common and potentially debilitating musculoskeletal condition. It is also in conjunction with spinal canal stenosis, facet joint arthrosis and degenerative spondylolisthesis. However, what factors initiate and influence the progression of disk degeneration remain not fully understood [1]. Gender dimorphism has been reported in degenerative IVD and osteoarthritis disorders [2,3]. In elderly subjects, MRI studies indicated that female tended to have slightly more severe lumbar disc degeneration than male [4,5]. A large-scale prospective population based studies also demonstrated that prevalence and severity of lumbar disc space narrowing is higher in women than men [6]. Sex hormone might play a specific role in the sexual dimorphism of the IVD degeneration. However, there is a general lack of appreciation
∗ Corresponding author. Renji Hospital, 1630, Dongfang Road, Shanghai 200127, China. Tel.: +86 21 68383766; fax: +0086 21 63730455. ∗∗ Co-corresponding author. Ruijin Hospital, 197, Ruijin Er Lu, Shanghai 200025, China. Tel.: +86 21 68383766; fax: +0086 21 63730455. E-mail addresses: [email protected], [email protected] (X.-f. Li), [email protected] (B.-w. Yu). 1 Xiao-xing Song and Zhen Guo contributed equally to this work.
for the heterogeneity of hormonal control in both female and male. Researches in recent years provided strong support for the involvement of estrogen on IVD health [7]. Ovariectomy (OVX) induced lumbar IVD degeneration has been observed in rats [8]. In human beings, it has been reported that menopause has a negative effect on IVD. The estrogen decrease during menopause contributes to the progressive decrease of the IVD height [9]. Compared to untreated subjects, estrogen-replete therapy seems to have a beneficial effect on IVD height in post-menopausal women [10]. The above reported results are in line with the well-known positive effects of estrogen on collagen metabolism [11]. Whereas, how estrogen deficiency affects IVD remains unclear. The estrogen receptors ER␣ and ER are the main mediators of estrogen action. ER gene expression has been detected in human IVD cells [12]. Previously, gender-specific expression patterns of ER␣ and ER in the spinal growth plate of rats have been observed by us [13]. In the IVD, the expression of ER␣ and gender differences of estrogen receptor expression has not been established. To elucidate sex differences in IVD degeneration, it is important to examine sex hormone mechanisms. Analysis of gender-specific expression of estrogen receptor could provide valuable insights into distinct roles of estrogen in IVD metabolism. In the present study, we investigated, for the first time, the gender- and degeneration degree-related variations of ER␣ and ER expression pattern in human lumbar IVD.
1297-319X/$ – see front matter © 2013 Société franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.jbspin.2013.09.002
Please cite this article in press as: Song X-x, et al. Estrogen receptor expression in lumbar intervertebral disc of the elderly: Gender- and degeneration degree-related variations. Joint Bone Spine (2014), doi:10.1016/j.jbspin.2013.09.002
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2. Methods
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2.1. Patients and grading of disc degeneration
ethanol, cleared in xylene, and mounted with Permount medium after counterstaining with Gill’s hematoxylin solution for 3 min. 2.4. Quantification of ER˛ and ERˇ expression
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Thirty-six patients with lumbar disc degeneration were selected for this study. Study protocols were approved by the Institutional Review Board and the Local Ethics Committee of our institution. Patients with infection, tumor, immunological and endocrine disease were excluded from the current study. All patients were aged 65 or above, ranging from 65 to 77 years. These subjects comprised of 18 males (69.6 ± 3.4 years) and 18 females (70 ± 2.3 years) with no age difference between the two gender groups (P > 0.05). Lumbar disc degeneration was assessed using five level Pfirrmann’s grading system, where each grade represents a stepwise degeneration progression [14]. MRI data were acquired with a 1.5 T whole-body imaging system. After scout images of lumbar spine, standard T2-weighted (TR = 3500 ms, TE = 120 ms) turbo spin echo sagittal images were obtained and used to grade disc degeneration. Two readers independently graded each of the lumbar IVDs. Consensus was reached when the initial reading of the two investigators differ. All the patients underwent MRI and surgery treatment were classified from Pfirrmann grades III to V. Twelve subjects of each grade, with equal numbers of males and females, were selected for this study.
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2.2. Samples harvest
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Human IVD tissue was obtained at surgery, with informed consent of the patient or relatives. After removing the herniated disc tissue, nucleus pulposus tissues at the centre of an intervertebral disc were harvested during spinal surgeries as previously reported [15,16]. Cartilaginous endplates and annulus fibrosus tissues were discarded to ensure the identity of the nucleus pulposus tissue as much as possible. The nucleus pulposus tissue was immediately washed with phosphate-buffered saline (PBS) (pH 7.4) and was routinely fixed in 4% paraformaldehyde followed by embedded in paraffin for immunostaining.
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2.3. Immunohistochemistry
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Immunohistochemistry was performed as our previously described procedure [13,17]. Paraffin sections (5 m thick) were treated with xylene to remove paraffin and rehydrated in graded alcohol baths followed by three rinses with phosphatebuffered saline (PBS). Slides were immunostained with the streptavidin–biotin peroxidase (SABC) technique. Control experiments were incubated with the antibody pre-incubated with a blocking peptide. The same concentrations of the primary antibodies of ER␣ (Santa Cruz, sc-543, 1:100 dilution) and ER (Abcam, ab3576, 1:100 dilution) were used to stain positive control tissues (liver and ovary) and IVD tissue. Antigen retrieval was performed by heating the sections in 10 mmol/L citrate buffer (pH 6.0) up to 95 ◦ C for 10 min and allowing them to cool down to room temperature for 20 min. Then, the sections were incubated in 1% H2 O2 for 15 min for blocking the endogenous peroxidase activity. After pre-incubation with 5% normal goat serum (Vector, S-1000) for 30 min at room temperature, the sections were incubated overnight at 4 ◦ C at room temperature with rabbit polyclonal antibody of ER␣ and ER. Then, the sections were incubated with the corresponding biotinylated goat anti-rabbit IgG (Vector, BA-1000), applied for 30 min at a dilution of 1:200, followed by a triple wash in PBS. Finally, the sections were incubated in ABC complex (Vectastain ABC kit, Vector Cat#PK6100) for 30 min at room temperature. Staining was visualized with DAB peroxides substrate solution for 3 min, followed by rinsing in distilled water briefly. The slides were dehydrated in graded
The intensities of ER␣ and ER immunoreactivity were semiquantitatively evaluated using the following intensity categories: 0, no staining; 1, weak but detectable staining; 2, moderate or distinct staining; 3, intense staining. For each sample, a HSCORE value was derived by summing the percentages of cells that stained at each intensity group and multiplying that by the weighted intensity of the staining. The formula HSCORE = i i × Pi , where i represents the intensity scores, and Pi is the corresponding percentage of the cells, was used for calculation. For each IVD sample, three tissue sections from each sample were randomly obtained and five randomly selected areas were evaluated for each tissue slide under the microscope with 200× original magnification. The percentage of the cells at each intensity group within these areas was determined by two investigators blinded to the type of the tissues. The average score was used. 2.5. Statistical analysis Data from ER␣ and ER immunohistochemistry scores in the IVD tissue were normally distributed as tested by Kruskal–Wallis (H) test. Quantitative data regarding HSCORE analysis are presented as mean ± SD. The comparative data from the female and male groups of each degeneration grade were analyzed by a t-test. For multiple comparisons, ANOVA was used and post-hoc analysis was carried out with Bonferroni test. A P value < 0.05 was considered statistically significant. Statistical analysis was performed using the SPSS (SPSS Inc., USA) statistical package.
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3. Results
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3.1. ER˛ and ERˇ immunoreactivity in control tissues
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Positive control experiment was performed to investigate the sensitivity and specificity of ER␣ and ER immunostaining. Human liver and ovary tissue was used as the positive control for ER␣ and ER expression, respectively. Immunohistochemistry study revealed nuclear ER␣ immunoreactivity in hepatocytes and nuclear ER immunostaining in granulosa cells in the ovary (Fig. 1A and B). No staining was detected when ER␣ and ER antibody was pre-incubated with a blocking peptide (Fig. 1C and D). 3.2. Expression of ER˛ and ERˇ immunoreactivity in nucleus pulposus ER␣ and ER protein was detected on the IVD sections of the elderly subjects. Although both cytoplasmic and nuclear staining of ER␣ and ER immunoreactivity were observed in the nucleus pulposus cells, more abundant nuclear immunoreactivity staining than that of cytoplasmic was indicated for both ER␣ and ER (Fig. 2A and B). 3.3. Degeneration degree-related expression of ER˛ and ERˇ in nucleus pulposus In order to investigate degeneration degree-related expression variations in nucleus pulposus, specific ER␣ and ER immunoreactivity in different Pfirrmann IVD grades of females and males were evaluated. ER␣ (Fig. 3A–F) and ER (Fig. 4A–F) were obtained in nucleus pulposus of all Pfirrmann grades of IVD in both genders. Immunohistochemistry revealed a stronger staining of ER␣ (Fig. 3A
Please cite this article in press as: Song X-x, et al. Estrogen receptor expression in lumbar intervertebral disc of the elderly: Gender- and degeneration degree-related variations. Joint Bone Spine (2014), doi:10.1016/j.jbspin.2013.09.002
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Fig. 4. Immunohistochemical localization of ER in the human IVD. Immunohistochemical staining in the nucleus pulposus was also downregulated from Pfirrmann grades III to V both in males (A–C) and females (D–F). Bar represents 50 m.
Fig. 1. Control studies for immunohistochemistry. Localization of ER␣ and ER protein in sections of human liver and ovary was shown. Positive immunoreactivity produced a brown coloration. A. Nuclear and cytoplasmic staining of ER␣ immunoreactivity was mainly detected in hepatocytes. B. Nuclear ER immunostaining was found in granulosa cells in ovary. C and D. No staining could be observed when the antibody was pre-incubated with a blocking peptide. Bar represents 50 m.
3.4. Gender-specific variations of ER˛ and ERˇ expression in nucleus pulposus Differences of ER␣ and ER expression in nucleus pulposus between males (Fig. 3A–C, Fig. 4A–C) and females (Fig. 3D–F, Fig. 4D–F) were also analyzed. The data of HSCORE analysis showed a significant sex-related difference of ER␣ (Fig. 3A–C) and ER (Fig. 3D–F) immunoreactivity in nucleus pulposus of each Pfirrmann grade of IVD. The immunoreactivity of both ER␣ and ER in nucleus pulposus of the males was significantly higher than that of the females, regardless of the degenerative condition of IVD (P < 0.05) (Fig. 6). 4. Discussion
Fig. 2. ER expression in the nucleus pulposus cells. A and B. Predominantly nuclear staining of ER␣ and ER immunoreactivity were observed in the nucleus pulposus cells. Bar represents 50 m.
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Increasing evidence indicated that sex hormones could influence the severity of disc degeneration [7]. Just like on articular cartilage, estrogen also has profound effects on IVD turnover. The effects of estrogen deficiency on disc degeneration suggested that estrogen might have a direct effect on IVD [18]. In the present study, the presentation of ER␣ and ER in nucleus pulposus
and D) and ER (Fig. 4A and D) in low Pfirrmann grade of IVD degeneration. According to the HSCORE analysis, the values of ER␣ (Fig. 5A and B) and ER (Fig. 5C and D) significantly downregulated along with the severity of IVD degeneration in both males and females (P < 0.05). In the nucleus pulposus of Pfirrmann V grade, a highest degenerative condition of IVD, the number of positive staining of ER␣ (Fig. 3C and F) and ER (Fig. 4C and F) was significantly decreased.
Fig. 3. Immunohistochemical staining for ER␣ in the human IVD. The number of positive staining cells in the nucleus pulposus was decreased from Pfirrmann grades III to V both in males (A–C) and females (D–F). Bar represents 50 m.
Fig. 5. HSCORE analysis of the ER␣ and ER immunostaining intensities in different degenerative IVD of both genders. Degenerative degree-related changes of the ER␣ and ER HSCORE values in females (A, C) and males (B, D) were shown. Both ER␣ and ER values significantly downregulated along with the severity of IVD degeneration in both males and females. * P < 0.05.
Please cite this article in press as: Song X-x, et al. Estrogen receptor expression in lumbar intervertebral disc of the elderly: Gender- and degeneration degree-related variations. Joint Bone Spine (2014), doi:10.1016/j.jbspin.2013.09.002
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Disclosure of interest The authors have not supplied their declaration of conflict of interest. Acknowledgements This study was supported by the National Natural Science Foundation of China (81270027, 30901508). References Fig. 6. HSCORE comparing analysis of the ER␣ and ER immunostaining intensities between females and males in different degree of degenerative IVD. Significant sex-related differences of ER␣ (A–C) and ER (D–F) immunoreactivity in nucleus pulposus of each Pfirrmann grade of IVD were indicated. * P < 0.05.
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confirmed that the effects of estrogen on IVD metabolism could be direct. Immunohistochemistry and HSCORE analysis indicated that both ER␣ and ER expression significantly decreased along with the aggravation of IVD degeneration in males and females. Expression of both ER␣ and ER protein in nucleus pulposus of males was significantly higher than that of females. Researches have provided strong support for estrogen’s protective effects against disc degeneration in recent years. Impaired disc diffusion was detected in ovariectomy rats using dynamic MRI technique [19]. Utero exposure to diethylstilbestrol could affect the production of proteoglycan in IVD of mice [20]. Human female subjects also have tendency to undergo accelerated disc degeneration around the time of menopause [21]. The effects of estrogen on disc degeneration might be reliable. Estrogens exert their protective effects via ER␣ and/or ER in the biological body [22]. The current study showed that both ER␣ and ER staining was much less in more serious degenerative IVD tissue. Therefore, estrogen effect deficiency might have a causal role in the progression of disc degeneration. However, the specific action of each ER should be investigated further in future. Although no disease entity is exclusive to either sex, in degenerative disorders of the spine, some differences potentially related to sexual dimorphism do exist [2]. A prospective radiographic study of subjects 55 years and older showed that disk space narrowing, the most reliable radiographic sign of severe disk degeneration, was more prevalent in women than men [23]. MR-based disk degeneration grading study of the elderly subjects demonstrated that females had more severe disk degeneration than males at all lumbar levels [5,24]. Clinical studies also noted that women seem to be more susceptible to instability, misalignments, and structural deterioration of spine [25]. These results indicated that females might be more susceptible to disc degeneration than males. However, discrepancies attributed to sex in degenerative disorders of the spine are less frequently reported in literature. The present immunohistochemistry study showed that both ER␣ and ER protein in nucleus pulposus of elderly males was significantly higher than that of females. Accordingly, in elderly males, the protective effects of estrogen might be stronger than that in females. Gender-specific ER expression might be one possible factor for sexual dimorphism of IVD degeneration. In conclusion, both ER␣ and ER are expressed in human IVD tissue in both genders. Gender- and degeneration degree-related variations of ER␣ and ER expression are observed, for the first time, in the nucleus pulposus. Downregulation of ER␣ and ER expression might be associated with the progression of IVD degeneration. Gender different expression of ER might play a part in sexual dimorphism of IVD degeneration. The effect of estrogen on IVD metabolism should be studied further taking into account sex and degeneration condition differences.
[1] Hughes SP, Freemont AJ, Hukins DW, et al. The pathogenesis of degeneration of the intervertebral disc and emerging therapies in the management of back pain. J Bone Joint Surg Br 2012;94:1298–304. [2] Manson NA, Goldberg EJ, Andersson GB. Sexual dimorphism in degenerative disorders of the spine. Orthop Clin North Am 2006;37:549–53. [3] Huffman KM, Kraus WE. Osteoarthritis and the metabolic syndrome: more evidence that the etiology of OA is different in men and women. Osteoarthritis Cartilage 2012;20:603–4. [4] Wang YX, Kwok AW, Griffith JF, et al. Relationship between hip bone mineral density and lumbar disc degeneration: a study in elderly subjects using an eight-level MRI-based disc degeneration grading system. J Magn Reson Imaging 2011;33:916–20. [5] Wang YX, Griffith JF, Ma HT, et al. Relationship between gender, bone mineral density, and disc degeneration in the lumbar spine: a study in elderly subjects using an eight-level MRI-based disc degeneration grading system. Osteoporos Int 2011;22:91–6. [6] Wang YX, Griffith JF, Zeng XJ, et al. Prevalence and gender difference of lumbar disc space narrowing in elderly Chinese men and women: Mr OS (Hong Kong) and Ms. OS (Hong Kong) studies. Arthritis Rheum 2013;65:1004–10. [7] Calleja-Agius J, Muscat-Baron Y, Brincat MP. Estrogens and the intervertebral disc. Menopause Int 2009;15:127–30. [8] Wang T, Zhang L, Huang C, et al. Relationship between osteopenia and lumbar intervertebral disc degeneration in ovariectomized rats. Calcif Tissue Int 2004;75:205–13. [9] Gambacciani M, Pepe A, Cappagli B, et al. The relative contributions of menopause and aging to postmenopausal reduction in intervertebral disk height. Climacteric 2007;10:298–305. [10] Baron YM, Brincat MP, Galea R, et al. Intervertebral disc height in treated and untreated overweight post-menopausal women. Hum Reprod 2005;20:3566–70. [11] Brincat M, Moniz CJ, Studd JW, et al. Long-term effects of the menopause and sex hormones on skin thickness. Br J Obstet Gynaecol 1985;92:256–9. [12] Gruber HE, Yamaguchi D, Ingram J, et al. Expression and localization of estrogen receptor-beta in annulus cells of the human intervertebral disc and the mitogenic effect of 17-beta-estradiol in vitro. BMC Musculoskelet Disord 2002; 3:4. [13] Li XF, Wang SJ, Jiang LS, et al. Gender- and region-specific variations of estrogen receptor ␣ and  expression in the growth plate of spine and limb during development and adulthood. Histochem Cell Biol 2012;137:79–95. [14] Pfirrmann CW, Metzdorf A, Zanetti M, et al. Magnetic resonance classification of lumbar intervertebral disc degeneration. Spine 2001;26:1873–8. [15] Tsai TT, Lai PL, Liao JC, et al. Increased periostin gene expression in degenerative intervertebral disc cells. Spine J 2013;13:289–98. [16] Reis Rodrigues LM, de Oliveira LZ, da Silva Pinhal MA. The expression of heparanase isoforms in intervertebral discs classified according to Pfirrmann’s grading system for disc degeneration. Spine 2013;38:1112–8. [17] Li XF, Yan J, Jiang LS, Dai LY. Age-related variations of leptin receptor expression in the growth plate of spine and limb: gender- and region-specific changes. Histochem Cell Biol 2011;135:487–97. [18] Calleja-Agius J, Brincat MP. Effects of hormone replacement therapy on connective tissue: why is this important? Best Pract Res Clin Obstet Gynaecol 2009;23:121–7. [19] Deng M, Griffith JF, Zhu XM, et al. Effect of ovariectomy on contrast agent diffusion into lumbar intervertebral disc: a dynamic contrast-enhanced MRI study in female rats. Magn Reson Imaging 2012;30:683–8. [20] Rowas SA, Haddad R, Gawri R, et al. Effect of in utero exposure to diethylstilbestrol on lumbar and femoral bone, articular cartilage, and the intervertebral disc in male and female adult mice progeny with and without swimming exercise. Arthritis Res Ther 2012;14:R17. [21] Wang YX, Griffith JF. Effect of menopause on lumbar disk degeneration: potential etiology. Radiology 2010;257:318–20. [22] Mueller SO, Korach KS. Estrogen receptors and endocrine diseases: lessons from estrogen receptor knockout mice. Curr Opin Pharmacol 2001;1:613–9. [23] de Schepper EI, Damen J, van Meurs JB, et al. The association between lumbar disc degeneration and low back pain: the influence of age, gender, and individual radiographic features. Spine 2010;35:531–6. [24] Griffith JF, Wang YX, Antonio GE, et al. Modified Pfirrmann grading system for lumbar intervertebral disc degeneration. Spine 2007;32:E708–12. [25] Lin K, Jenis L. Degenerative lumbar spondylolisthesis. Semin Spine Surg 2003;15:150–9.
Please cite this article in press as: Song X-x, et al. Estrogen receptor expression in lumbar intervertebral disc of the elderly: Gender- and degeneration degree-related variations. Joint Bone Spine (2014), doi:10.1016/j.jbspin.2013.09.002
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ARTICLE IN PRESS
BONSOI 3923 1–4
Joint Bone Spine xxx (2014) xxx–xxx
Available online at www.sciencedirect.com
Original article
2
Estrogen receptor expression in lumbar intervertebral disc of the elderly: Gender- and degeneration degree-related variations
3
Xiao-xing Song a,b,1 , Yong-juan Yu a , Xin-feng Li a,∗ , Zu-de Liu a , Bu-wei Yu b,∗∗ , Zhen Guo a,1
1
4 5
a b
Department of Orthopaedic Surgery, Ren Ji Hospital,School of Medicine, Shanghai Jiao Tong University, Shanghai, China Department of Anesthesiology, Rui Jin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
6
7
a r t i c l e
i n f o
a b s t r a c t
8 9 10 11
Article history: Accepted 4 September 2013 Available online xxx
12
17
Keywords: Intervertebral disc Degeneration Estrogen receptor Nucleus pulposus
18
1. Introduction
13 14 15 16
19 20 21 22 23 24 25 26 27 28 29 30 31
Objectives: Sexual dimorphism does occur in intervertebral disc (IVD) degeneration. The involvement of estrogen on IVD health has been well reported in recent years. The estrogen receptors (ER) are the main mediators of estrogen action. ER might play specific roles in the sexual variations of the IVD degeneration. Methods: Thirty-six elderly patients with lumbar disc degeneration were selected and graded using Pfirrmann’s system based on MRI images. Differences of ER␣ and ER immunoreactivity staining in nucleus pulposus of each sex and degeneration degree were recorded and compared. Results: Both cytoplasmic and nuclear staining of ER␣ and ER immunoreactivity were observed in the nucleus pulposus cells. ER␣ and ER expression significantly decreased along with the aggravation of IVD degeneration both in males and females. Expression of ER␣ and ER protein in nucleus pulposus of males was significantly higher than that of females. Conclusions: Gender-specific expression of ER might play a part in sexual dimorphism of IVD degeneration. Gender and degeneration condition differences should be taken into account when the effects of estrogen on IVD metabolism are studied further. © 2013 Société franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.
Intervertebral disk (IVD) degeneration is a common and potentially debilitating musculoskeletal condition. It is also in conjunction with spinal canal stenosis, facet joint arthrosis and degenerative spondylolisthesis. However, what factors initiate and influence the progression of disk degeneration remain not fully understood [1]. Gender dimorphism has been reported in degenerative IVD and osteoarthritis disorders [2,3]. In elderly subjects, MRI studies indicated that female tended to have slightly more severe lumbar disc degeneration than male [4,5]. A large-scale prospective population based studies also demonstrated that prevalence and severity of lumbar disc space narrowing is higher in women than men [6]. Sex hormone might play a specific role in the sexual dimorphism of the IVD degeneration. However, there is a general lack of appreciation
∗ Corresponding author. Renji Hospital, 1630, Dongfang Road, Shanghai 200127, China. Tel.: +86 21 68383766; fax: +0086 21 63730455. ∗∗ Co-corresponding author. Ruijin Hospital, 197, Ruijin Er Lu, Shanghai 200025, China. Tel.: +86 21 68383766; fax: +0086 21 63730455. E-mail addresses: [email protected], [email protected] (X.-f. Li), [email protected] (B.-w. Yu). 1 Xiao-xing Song and Zhen Guo contributed equally to this work.
for the heterogeneity of hormonal control in both female and male. Researches in recent years provided strong support for the involvement of estrogen on IVD health [7]. Ovariectomy (OVX) induced lumbar IVD degeneration has been observed in rats [8]. In human beings, it has been reported that menopause has a negative effect on IVD. The estrogen decrease during menopause contributes to the progressive decrease of the IVD height [9]. Compared to untreated subjects, estrogen-replete therapy seems to have a beneficial effect on IVD height in post-menopausal women [10]. The above reported results are in line with the well-known positive effects of estrogen on collagen metabolism [11]. Whereas, how estrogen deficiency affects IVD remains unclear. The estrogen receptors ER␣ and ER are the main mediators of estrogen action. ER gene expression has been detected in human IVD cells [12]. Previously, gender-specific expression patterns of ER␣ and ER in the spinal growth plate of rats have been observed by us [13]. In the IVD, the expression of ER␣ and gender differences of estrogen receptor expression has not been established. To elucidate sex differences in IVD degeneration, it is important to examine sex hormone mechanisms. Analysis of gender-specific expression of estrogen receptor could provide valuable insights into distinct roles of estrogen in IVD metabolism. In the present study, we investigated, for the first time, the gender- and degeneration degree-related variations of ER␣ and ER expression pattern in human lumbar IVD.
1297-319X/$ – see front matter © 2013 Société franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.jbspin.2013.09.002
Please cite this article in press as: Song X-x, et al. Estrogen receptor expression in lumbar intervertebral disc of the elderly: Gender- and degeneration degree-related variations. Joint Bone Spine (2014), doi:10.1016/j.jbspin.2013.09.002
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2. Methods
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2.1. Patients and grading of disc degeneration
ethanol, cleared in xylene, and mounted with Permount medium after counterstaining with Gill’s hematoxylin solution for 3 min. 2.4. Quantification of ER˛ and ERˇ expression
78
Thirty-six patients with lumbar disc degeneration were selected for this study. Study protocols were approved by the Institutional Review Board and the Local Ethics Committee of our institution. Patients with infection, tumor, immunological and endocrine disease were excluded from the current study. All patients were aged 65 or above, ranging from 65 to 77 years. These subjects comprised of 18 males (69.6 ± 3.4 years) and 18 females (70 ± 2.3 years) with no age difference between the two gender groups (P > 0.05). Lumbar disc degeneration was assessed using five level Pfirrmann’s grading system, where each grade represents a stepwise degeneration progression [14]. MRI data were acquired with a 1.5 T whole-body imaging system. After scout images of lumbar spine, standard T2-weighted (TR = 3500 ms, TE = 120 ms) turbo spin echo sagittal images were obtained and used to grade disc degeneration. Two readers independently graded each of the lumbar IVDs. Consensus was reached when the initial reading of the two investigators differ. All the patients underwent MRI and surgery treatment were classified from Pfirrmann grades III to V. Twelve subjects of each grade, with equal numbers of males and females, were selected for this study.
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2.2. Samples harvest
59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77
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Human IVD tissue was obtained at surgery, with informed consent of the patient or relatives. After removing the herniated disc tissue, nucleus pulposus tissues at the centre of an intervertebral disc were harvested during spinal surgeries as previously reported [15,16]. Cartilaginous endplates and annulus fibrosus tissues were discarded to ensure the identity of the nucleus pulposus tissue as much as possible. The nucleus pulposus tissue was immediately washed with phosphate-buffered saline (PBS) (pH 7.4) and was routinely fixed in 4% paraformaldehyde followed by embedded in paraffin for immunostaining.
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2.3. Immunohistochemistry
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Immunohistochemistry was performed as our previously described procedure [13,17]. Paraffin sections (5 m thick) were treated with xylene to remove paraffin and rehydrated in graded alcohol baths followed by three rinses with phosphatebuffered saline (PBS). Slides were immunostained with the streptavidin–biotin peroxidase (SABC) technique. Control experiments were incubated with the antibody pre-incubated with a blocking peptide. The same concentrations of the primary antibodies of ER␣ (Santa Cruz, sc-543, 1:100 dilution) and ER (Abcam, ab3576, 1:100 dilution) were used to stain positive control tissues (liver and ovary) and IVD tissue. Antigen retrieval was performed by heating the sections in 10 mmol/L citrate buffer (pH 6.0) up to 95 ◦ C for 10 min and allowing them to cool down to room temperature for 20 min. Then, the sections were incubated in 1% H2 O2 for 15 min for blocking the endogenous peroxidase activity. After pre-incubation with 5% normal goat serum (Vector, S-1000) for 30 min at room temperature, the sections were incubated overnight at 4 ◦ C at room temperature with rabbit polyclonal antibody of ER␣ and ER. Then, the sections were incubated with the corresponding biotinylated goat anti-rabbit IgG (Vector, BA-1000), applied for 30 min at a dilution of 1:200, followed by a triple wash in PBS. Finally, the sections were incubated in ABC complex (Vectastain ABC kit, Vector Cat#PK6100) for 30 min at room temperature. Staining was visualized with DAB peroxides substrate solution for 3 min, followed by rinsing in distilled water briefly. The slides were dehydrated in graded
The intensities of ER␣ and ER immunoreactivity were semiquantitatively evaluated using the following intensity categories: 0, no staining; 1, weak but detectable staining; 2, moderate or distinct staining; 3, intense staining. For each sample, a HSCORE value was derived by summing the percentages of cells that stained at each intensity group and multiplying that by the weighted intensity of the staining. The formula HSCORE = i i × Pi , where i represents the intensity scores, and Pi is the corresponding percentage of the cells, was used for calculation. For each IVD sample, three tissue sections from each sample were randomly obtained and five randomly selected areas were evaluated for each tissue slide under the microscope with 200× original magnification. The percentage of the cells at each intensity group within these areas was determined by two investigators blinded to the type of the tissues. The average score was used. 2.5. Statistical analysis Data from ER␣ and ER immunohistochemistry scores in the IVD tissue were normally distributed as tested by Kruskal–Wallis (H) test. Quantitative data regarding HSCORE analysis are presented as mean ± SD. The comparative data from the female and male groups of each degeneration grade were analyzed by a t-test. For multiple comparisons, ANOVA was used and post-hoc analysis was carried out with Bonferroni test. A P value < 0.05 was considered statistically significant. Statistical analysis was performed using the SPSS (SPSS Inc., USA) statistical package.
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3. Results
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3.1. ER˛ and ERˇ immunoreactivity in control tissues
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Positive control experiment was performed to investigate the sensitivity and specificity of ER␣ and ER immunostaining. Human liver and ovary tissue was used as the positive control for ER␣ and ER expression, respectively. Immunohistochemistry study revealed nuclear ER␣ immunoreactivity in hepatocytes and nuclear ER immunostaining in granulosa cells in the ovary (Fig. 1A and B). No staining was detected when ER␣ and ER antibody was pre-incubated with a blocking peptide (Fig. 1C and D). 3.2. Expression of ER˛ and ERˇ immunoreactivity in nucleus pulposus ER␣ and ER protein was detected on the IVD sections of the elderly subjects. Although both cytoplasmic and nuclear staining of ER␣ and ER immunoreactivity were observed in the nucleus pulposus cells, more abundant nuclear immunoreactivity staining than that of cytoplasmic was indicated for both ER␣ and ER (Fig. 2A and B). 3.3. Degeneration degree-related expression of ER˛ and ERˇ in nucleus pulposus In order to investigate degeneration degree-related expression variations in nucleus pulposus, specific ER␣ and ER immunoreactivity in different Pfirrmann IVD grades of females and males were evaluated. ER␣ (Fig. 3A–F) and ER (Fig. 4A–F) were obtained in nucleus pulposus of all Pfirrmann grades of IVD in both genders. Immunohistochemistry revealed a stronger staining of ER␣ (Fig. 3A
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Fig. 4. Immunohistochemical localization of ER in the human IVD. Immunohistochemical staining in the nucleus pulposus was also downregulated from Pfirrmann grades III to V both in males (A–C) and females (D–F). Bar represents 50 m.
Fig. 1. Control studies for immunohistochemistry. Localization of ER␣ and ER protein in sections of human liver and ovary was shown. Positive immunoreactivity produced a brown coloration. A. Nuclear and cytoplasmic staining of ER␣ immunoreactivity was mainly detected in hepatocytes. B. Nuclear ER immunostaining was found in granulosa cells in ovary. C and D. No staining could be observed when the antibody was pre-incubated with a blocking peptide. Bar represents 50 m.
3.4. Gender-specific variations of ER˛ and ERˇ expression in nucleus pulposus Differences of ER␣ and ER expression in nucleus pulposus between males (Fig. 3A–C, Fig. 4A–C) and females (Fig. 3D–F, Fig. 4D–F) were also analyzed. The data of HSCORE analysis showed a significant sex-related difference of ER␣ (Fig. 3A–C) and ER (Fig. 3D–F) immunoreactivity in nucleus pulposus of each Pfirrmann grade of IVD. The immunoreactivity of both ER␣ and ER in nucleus pulposus of the males was significantly higher than that of the females, regardless of the degenerative condition of IVD (P < 0.05) (Fig. 6). 4. Discussion
Fig. 2. ER expression in the nucleus pulposus cells. A and B. Predominantly nuclear staining of ER␣ and ER immunoreactivity were observed in the nucleus pulposus cells. Bar represents 50 m.
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Increasing evidence indicated that sex hormones could influence the severity of disc degeneration [7]. Just like on articular cartilage, estrogen also has profound effects on IVD turnover. The effects of estrogen deficiency on disc degeneration suggested that estrogen might have a direct effect on IVD [18]. In the present study, the presentation of ER␣ and ER in nucleus pulposus
and D) and ER (Fig. 4A and D) in low Pfirrmann grade of IVD degeneration. According to the HSCORE analysis, the values of ER␣ (Fig. 5A and B) and ER (Fig. 5C and D) significantly downregulated along with the severity of IVD degeneration in both males and females (P < 0.05). In the nucleus pulposus of Pfirrmann V grade, a highest degenerative condition of IVD, the number of positive staining of ER␣ (Fig. 3C and F) and ER (Fig. 4C and F) was significantly decreased.
Fig. 3. Immunohistochemical staining for ER␣ in the human IVD. The number of positive staining cells in the nucleus pulposus was decreased from Pfirrmann grades III to V both in males (A–C) and females (D–F). Bar represents 50 m.
Fig. 5. HSCORE analysis of the ER␣ and ER immunostaining intensities in different degenerative IVD of both genders. Degenerative degree-related changes of the ER␣ and ER HSCORE values in females (A, C) and males (B, D) were shown. Both ER␣ and ER values significantly downregulated along with the severity of IVD degeneration in both males and females. * P < 0.05.
Please cite this article in press as: Song X-x, et al. Estrogen receptor expression in lumbar intervertebral disc of the elderly: Gender- and degeneration degree-related variations. Joint Bone Spine (2014), doi:10.1016/j.jbspin.2013.09.002
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Disclosure of interest The authors have not supplied their declaration of conflict of interest. Acknowledgements This study was supported by the National Natural Science Foundation of China (81270027, 30901508). References Fig. 6. HSCORE comparing analysis of the ER␣ and ER immunostaining intensities between females and males in different degree of degenerative IVD. Significant sex-related differences of ER␣ (A–C) and ER (D–F) immunoreactivity in nucleus pulposus of each Pfirrmann grade of IVD were indicated. * P < 0.05.
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confirmed that the effects of estrogen on IVD metabolism could be direct. Immunohistochemistry and HSCORE analysis indicated that both ER␣ and ER expression significantly decreased along with the aggravation of IVD degeneration in males and females. Expression of both ER␣ and ER protein in nucleus pulposus of males was significantly higher than that of females. Researches have provided strong support for estrogen’s protective effects against disc degeneration in recent years. Impaired disc diffusion was detected in ovariectomy rats using dynamic MRI technique [19]. Utero exposure to diethylstilbestrol could affect the production of proteoglycan in IVD of mice [20]. Human female subjects also have tendency to undergo accelerated disc degeneration around the time of menopause [21]. The effects of estrogen on disc degeneration might be reliable. Estrogens exert their protective effects via ER␣ and/or ER in the biological body [22]. The current study showed that both ER␣ and ER staining was much less in more serious degenerative IVD tissue. Therefore, estrogen effect deficiency might have a causal role in the progression of disc degeneration. However, the specific action of each ER should be investigated further in future. Although no disease entity is exclusive to either sex, in degenerative disorders of the spine, some differences potentially related to sexual dimorphism do exist [2]. A prospective radiographic study of subjects 55 years and older showed that disk space narrowing, the most reliable radiographic sign of severe disk degeneration, was more prevalent in women than men [23]. MR-based disk degeneration grading study of the elderly subjects demonstrated that females had more severe disk degeneration than males at all lumbar levels [5,24]. Clinical studies also noted that women seem to be more susceptible to instability, misalignments, and structural deterioration of spine [25]. These results indicated that females might be more susceptible to disc degeneration than males. However, discrepancies attributed to sex in degenerative disorders of the spine are less frequently reported in literature. The present immunohistochemistry study showed that both ER␣ and ER protein in nucleus pulposus of elderly males was significantly higher than that of females. Accordingly, in elderly males, the protective effects of estrogen might be stronger than that in females. Gender-specific ER expression might be one possible factor for sexual dimorphism of IVD degeneration. In conclusion, both ER␣ and ER are expressed in human IVD tissue in both genders. Gender- and degeneration degree-related variations of ER␣ and ER expression are observed, for the first time, in the nucleus pulposus. Downregulation of ER␣ and ER expression might be associated with the progression of IVD degeneration. Gender different expression of ER might play a part in sexual dimorphism of IVD degeneration. The effect of estrogen on IVD metabolism should be studied further taking into account sex and degeneration condition differences.
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