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Activation of satellite cells in the dorsal root ganglia in a disc-punctured rat model
J Orthop Sci (2011) 16:433–438 DOI 10.1007/s00776-011-0064-0
ORIGINAL ARTICLE
Activation of satellite cells in the dorsal root ganglia in a disc-punctured rat model Yanjing Li • Chunyang Xi • Ming Niu • Zhiyong Chi • Xiaoqi Liu • Jinglong Yan
Received: 9 January 2011 / Accepted: 25 March 2011 / Published online: 26 May 2011 Ó The Japanese Orthopaedic Association 2011
Abstract Background The neural mechanisms underlying discogenic low back pain caused by disc degeneration remain unclear. Previous studies demonstrated that satellite cells (SC) play an important role in neuropathic pain. Methods Twenty adult female Sprague-Dawley rats were used. The rats were divided into two groups: a nucleus pulposus (NP) group whose discs were punctured to expose the NP (n = 10) and a sham-operated group whose annulus fibrosus surface was scratched superficially (n = 10). In this study, we investigated the expression and cellular distribution of glial fibrillary acidic protein (GFAP, a marker of SC activation) in the dorsal root ganglia (DRG) innervating the intervertebral discs using a retrograde tracing method and immunohistochemistry in a discpunctured rat model. Results In the sham-operated group, GFAP-immunoreactive (IR) SCs were not detected. In the NP group, GFAP-IR SC became evident, and 49 ± 13% of neurons innervating the punctured discs were surrounded by GFAP-positive SCs.
Y. Li and C. Xi contributed equally to this work. Y. Li C. Xi Z. Chi X. Liu J. Yan (&) Department of Orthopedic Surgery, The First Affiliated Hospital, Harbin Medical University, 23 Youzheng St, Nangang District, Harbin 150001, People’s Republic of China e-mail: [email protected] M. Niu Department of Mammary Surgery, The Third Affiliated Hospital, Harbin Medical University, Harbin 150001, People’s Republic of China
Conclusions Our results were the first to provide evidence for a potential role of SCs in the neural mechanisms of discogenic low back pain caused by disc degeneration.
Introduction Degeneration of lumbar intervertebral discs is considered to be an important source of discogenic low back pain [1, 2]. Nevertheless, the neural mechanisms of discogenic low back pain caused by disc degeneration are poorly understood. A recent study reported that exposure of the nucleus pulposus (NP) to the outside of the annulus fibrosus could upregulate the expression of activating transcription factor 3 (ATF3, a marker of nerve injury) and growth-associated protein 43 (GAP43, a marker of nerve growth) in the dorsal root ganglia (DRG), innervating the punctured disc [2]. This indicated that exposure of the NP to the outside of the annulus fibrosus could induce nerve injury and nerve ingrowth into the discs, and degenerated discs might induce cellular and molecular changes in the DRG innervating the degenerated disc. Satellite cells (SC) are located in the DRG and form a single layer wrapped around each sensory neuron soma [3]. Electron microscopy has demonstrated that the typical SC is a discrete element, bound by its own membrane, a distinctive structure not seen in the central nervous system (CNS) [4]. In recent years, particular attention has been paid to SCs because of their important roles in neuropathic pain states. Following sensory neuron damage or inflammation, the SCs become coupled extensively to SCs by gap junctions, and proliferated and expressed glial fibrillary acidic protein (GFAP) [5–9]. GFAP, a member of the cytoskeletal protein family, has been extensively used as a marker of SC activation [3]. Recently, injury-induced
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GFAP expression in DRG has been demonstrated to contribute to the maintenance of neuropathic pain states [6]. In addition, the activated SCs are important sources of neurotrophins, such as glial cell line-derived neurotrophic factor (GDNF), nerve growth factors (NGF) and neurotrophin-3 (NT3), which are associated with neuropathic pain [10–12]. Furthermore, the activated SCs could produce some cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin1-beta (IL-1beta), all of which mediate inflammation and hyperalgesia [7, 8, 13–15]. Based on these findings, one can conclude that SC activation may play an important role in neuropathic pain. Therefore, we hypothesized that the activation of SCs might also be involved with discogenic low back pain due to disc degeneration. In order to examine the possible relationship between SCs and discogenic low back pain due to disc degeneration, we investigated the expression and cellular distribution of GFAP in the DRG innervating the intervertebral discs in a disc-punctured rat model using a retrograde tracing method and immunohistochemistry.
Materials and methods A total of 20 adult female Sprague-Dawley rats weighing 200–250 g were used in the study. The rats were maintained on a 12-h light/dark cycle with food and water ad libitum. All of the handling of the animals was carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (1996 revision) and was conducted with the permission of the ethics committee of our institution. All efforts possible were made to minimize pain. The rats were anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneal), and all surgical procedures were performed aseptically. A midline abdominal incision was made, and the retroperitoneum was incised along the left margin of the aorta. The left iliopsoas muscle was dissected to expose the ventral aspect of the L5–L6 intervertebral disc (Fig. 1). A 26-gauge needle whose tip was filled with Fluoro-Gold crystals (FG, Fluorochrome, Denver, CO) was inserted into the ventral surface of the disc approximately 0.5 mm in depth. The application site was immediately covered with cyanoacrylate to prevent the crystals from spreading. Then the fascia and skin were closed. This procedure was performed according to a previously reported method [2]. Five days after the application of FG, a 22-gauge needle was inserted into the ventral aspect of the disc at about 3 mm depth (NP group, n = 10), which could result in the exposure of the NP to the outside of the annulus fibrosus
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Fig. 1 The ventral aspect of the L5–L6 intervertebral disc was exposed after dissecting the left iliopsoas muscle. The arrow indicates the L5–L6 disc
[2]. The procedure was monitored by radiography to make sure that the needle puncture was parallel to the endplates to avoid endplate injury. In the sham-operated group (n = 10), the needle was inserted into the ventral aspect to a depth of about 0.5 mm, which could not obtain exposure of the NP. Seven days after surgery, rats were perfused transcardially with 0.9% saline, followed by 500 ml of 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4). The bilateral DRGs from levels L1–L5 were resected, then immersed in the same fixative solution for 3 h at 4°C, then in 0.01 M phosphate buffered saline (PBS) containing 10% sucrose for 6 h, then 20% sucrose for 6 h and 30% sucrose for 6 h. Each ganglion was sectioned at 5 lm thickness on a cryostat and mounted on poly-L-lysine-coated slides. Specimens were treated for 20 min at room temperature in blocking solution with donkey serum. For GFAP staining, the DRGs were incubated overnight at 4°C with rabbit antibody to GFAP (1:400; Dako, Carpinteria, CA). After being thoroughly washed, the sections were incubated for 1 h at 37°C with FITC-labeled donkey anti-rabbit antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA). The sections were observed with a fluorescent microscope (Nikon, Tokyo, Japan). FG-labeled neurons were observed using a UV-1A filter (wavelengths of 365 nm for excitation and 420 nm for emission). Only the FG-labeled neurons with a clear visible nucleus were counted. Each FG-labeled neuron was further examined for positive GFAP reactivity using a FITC filter (wavelengths of 465 nm for excitation and 505 nm for emission). In each DRG, 10 sections were selected randomly, and the numbers of FG-labeled neurons and GFAP-immunoreactive (IR) SCs surrounding FG-labeled neurons were counted by two independent observers who were blinded to this experiment. The data were compared with analysis of variance (ANOVA). P \ 0.05 was considered as statistically significant.
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Fig. 2 The mean number of Fluoro-Gold (FG)-labeled neurons at each level in dorsal root ganglia (DRG) in the nucleus pulposus (NP) and sham-operated group. There was no statistical difference between the two groups at each level
Fluoro-Gold crystals were distributed in the cytoplasm of neurons innervating the L5–L6 disc, and FG-labeled neurons were present in the bilateral L1–L5 DRG (Fig. 3a, c). The majority of FG-labeled neurons were located in L1 and L2 in two groups. The total number of FG-labeled neurons was 310 in the sham-operated group and 304 in the NP group. The number at each level was not significantly different between these two groups (P [ 0.05, Fig. 2). GFAP-IR SCs were not detected in the sham-operated group (Fig. 3b), but became evident in the NP group, and some GFAP-IR SCs were located around the FG-labeled neurons (Fig. 3d); 49 ± 13% (mean ± SD) of the total FG-labeled neurons were positive for GFAP around these
Fig. 3 Fluorescent photomicrograph of FG-labeled neurons and glial fibrillary acidic protein-immunoreactive (GFAP-IR) satellite cells (SC) in the sham-operated group (a, b) and NP group (c, d). GFAPIR SCs were not observed in b, but observed around neurons, and some FG-labeled neurons were surrounded by GFAP-IR SC (d). Scale bars 50 lm
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Fig. 4 The ratio of GFAP-IR SCs around FG-labeled neurons to total FG-labeled neurons at each level. The ratios were significantly different between two groups at each level. In the NP group, the ratios of expression of GFAP-IR SCs were not significantly different between any two levels (P [ 0.05). Data are presented as mean ± SEM
neurons in the NP group. However, there was no significant difference between any other two levels in the ratios of GFAP-IR SC around FG-labeled neurons to total FGlabeled neurons (P [ 0.05, Fig. 4).
Discussion In this study, the majority of FG-labeled neurons were located at L1 and L2 levels in both groups, which indicated that the L5–L6 disc was multisegmentally innervated through the paravertebral sympathetic trunks as reported previously [2, 16]. Glial fibrillary acidic protein, the principal 8–9 nm intermediate filaments expressing in mature astrocytes and SCs, has been extensively used as a marker of the activation of astrocytes and SCs [3, 7]. There are many reports about the function of GFAP in astrocytes. As a member of the cytoskeletal protein family, GFAP is involved in modulating astrocyte motility and shape by providing structural stability to astrocytic processes [17]. It has also been reported that GFAP expression in astrocytes contributes to normal white matter architecture, blood-brain barrier integrity, astrocyte-neuronal interactions, modulation of long-term potentiation in the CNS, maintenance of homeostasis and vascular permeability at the blood-tissue interface [18–20]. Following peripheral nerve injury and CNS damage, GFAP was upregulated in astrocytes, which was closely concerned with neuropathic pain states [5–7, 21]. Compared with astrocytes, much less is known about the role of GFAP in SCs. The SCs, which envelope DRG neurons, have some general characteristics of astrocytes and microglia [22, 23]. However, there is an important
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distinction between SCs and astrocytes in that the basal level of GFAP expression is quite low in SCs of normal animals. In our study, there was also an absence of GFAP labeling of SCs in the sham-operated group, which indicated that surgical trauma was not related to GFAP expression in the DRG in this rat model. However, GFAPIR SCs became evident in the NP group. Although GFAP was also used as a marker of SC activation, it is likely that the term ‘‘activation’’ is an oversimplification, and little is known about the nature of this SC activation or of the functional role GFAP plays in SCs. In a recent report, using the strategy of detecting gene expression profiles at different time points of neuropathic pain development, some investigators found that altered expression of selective groups of genes correlated with different stages of neuropathic pain development, and they suggested that injury-induced GFAP expression might play an important role in the maintenance of nerve injuryinduced pain states [6]. Hence, it is possible that blocking GFAP upregulation post-injury might help to shorten the duration of chronic pain states. In addition, according to the published literature, there are several pieces of evidence indicating that the activated SCs contribute to the neuropathic pain. First, following nerve injury, SC sheaths enveloping neighboring neurons formed connections with each other, leading to abnormal spreading of sensory signals among DRG neurons and sensory hyperexcitability, whereas such connections were absent in control ganglia [5]. Second, it has been reported that neurotrophins such as NGF and NT3 were synthesized by activated SCs after peripheral nerve injury and that they would play a crucial role in the generation of pain-related behavior [10, 11]. Third, the activated SCs are important sources of cytokines, such as TNF-alpha and IL-1beta, which mediate inflammation and hyperalgesia [7, 8, 13–15]. Taken together, no matter the role of GFAP itself or SC activation, SCs may play a critical role in neuropathic pain. In the present study, we observed some GFAP-IR SC enveloped FG-labeled neurons, which indicated that some SCs were activated around the DRG neurons innervating the punctured disc. This indicated that the SCs might affect the enveloped neurons innervating the punctured disc. It has been reported that TNF-alpha might be a possible inducer of nerve ingrowth into degenerated intervertebral discs [24], while it is a major neural mechanism of discogenic low back pain that sensory nerve fibers exist in the inner layer of the annulus fibrosus or NP [2, 24]. Since the activated SCs are important sources of TNF-alpha, it is possible that the SCs may contribute to nerve ingrowth to the degenerated disc mediated by the production of TNFalpha [7, 8, 14]. Moreover, for the role of GFAP itself or SC activation in neuropathic pain discussed above, it was deduced that all of these SC changes may contribute to
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discogenic low back pain due to disc degeneration. In addition, we observed that not all the GFAP-IR SCs were located around the FG-labeled neurons, a phenomenon similar to the GFAP expression in some rat models of neuropathic pain [7, 8, 14]. This indicated that SCs might affect each other and then form a cascade reaction. Our results were similar to the previous findings in which the authors found that GFAP-IR SCs were upregulated in the DRG in a rat model of lumbar facet joint injury, which was a possible cause of low back pain [14]. Therefore, we suggested that SC activation in DRG might be a general mechanism in the pathogenesis of low back pain. Further studies need to be done to support these hypotheses. In conclusion, our results suggested that exposure of the NP to the outside of the annulus fibrosus could cause the activation of SCs, and SC activation would then contribute to the neural mechanisms of discogenic low back pain due to disc degeneration in spite of the fact that this model did not correspond exactly to human discogenic low back pain due to disc degeneration. These data provide new perspectives concerning the mechanisms and theoretical basis of suppression of SC activation to treat discogenic low back pain. Intensive studies on the function of GFAP and SCs are required to better understand their substantial roles in discogenic low back pain due to disc degeneration. Acknowledgments This work was supported in part by grants from National Natural Science Foundation of China (100334). The authors thank Ming Yan for her technical assistance. Conflict of interest The authors declare that we have no financial and personal relationships with other people or organizations that could inappropriately influence our work; there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled ‘Activation of satellite cells in the dorsal root ganglia in a disc-punctured rat model.’
References 1. Brisby H. Pathology and possible mechanisms of nervous system response to disc degeneration. J Bone Joint Surg Am. 2006; 88(Suppl 2):S68–71. 2. Inoue G, Ohtori S, Aoki Y, Ozawa T, Doya H, Saito T, Ito T, Akazawa T, Moriya H, Takahashi K. Exposure of the nucleus pulposus to the outside of the anulus fibrosus induces nerve injury and regeneration of the afferent fibers innervating the lumbar intervertebral discs in rats. Spine. 2006;31:1433–8. 3. Hanani M. Satellite glial cells in sensory ganglia: from form to function. Brain Res Rev. 2005;48:457–76. 4. Siemionow K, Weinstein JN, McLain RF. Support and satellite cells within the rabbit dorsal root ganglion: ultrastructure of a perineuronal support cell. Spine. 2006;31:1882–7. 5. Hanani M, Huang TY, Cherkas PS, Ledda M, Pannese E. Glial cell plasticity in sensory ganglia induced by nerve damage. Neuroscience. 2002;114:279–83.
437 6. Kim DS, Figueroa KW, Li KW, Boroujerdi A, Yolo T, David Luo Z. Profiling of dynamically changed gene expression in dorsal root ganglia post peripheral nerve injury and a critical role of injury-induced glial fibrillary acetic protein in maintenance of pain behaviors. Pain. 2009;143:114–22. 7. Ohtori S, Takahashi K, Moriya H, Myers RR. TNF-alpha and TNF-alpha receptor type 1 upregulation in glia and neurons after peripheral nerve injury: studies in murine DRG and spinal cord. Spine. 2004;29:1082–8. 8. Otoshi K, Kikuchi S, Konno S, Sekiguchi M. The reactions of glial cells and endoneurial macrophages in the dorsal root ganglion and their contribution to pain-related behavior after application of nucleus pulposus onto the nerve root in rats. Spine. 2010;35:264–71. 9. Siemionow K, Klimczak A, Brzezicki G, Siemionow M, McLain RF. The effects of inflammation on glial fibrillary acidic protein expression in satellite cells of the dorsal root ganglion. Spine. 2009;34:1631–7. 10. Zhou XF, Deng YS, Chie E, Xue Q, Zhong JH, McLachlan EM, Rush RA, Xian CJ. Satellite-cell-derived nerve growth factor and neurotrophin-3 are involved in noradrenergic sprouting in the dorsal root ganglia following peripheral nerve injury in the rat. Eur J Neurosci. 1999;11:1711–22. 11. McMahon SB, Bennett DL, Priestley JV, Shelton DL. The biological effects of endogenous nerve growth factor on adult sensory neurons revealed by a trkA IgG fusion molecule. Nat Med. 1995;1:774–80. 12. Hammarberg H, Piehl F, Cullheim S, Fjell J, Ho¨kfelt T, Fried K. GDNF mRNA in Schwann cells and DRG satellite cells after chronic sciatic nerve injury. Neuroreport. 1996;7:857–60. 13. Takeda M, Tanimoto T, Kadoi J, Nasu M, Takahashi M, Kitagawa J, Matsumoto S. Enhanced excitability of nociceptive trigeminal ganglion neurons by satellite glial cytokine following peripheral inflammation. Pain. 2007;129:155–66. 14. Miyagi M, Ohtori S, Ishikawa T, Aoki Y, Ozawa T, Doya H, Saito T, Moriya H, Takahashi K. Up-regulation of TNF alpha in DRG satellite cells following lumbar facet joint injury in rats. Eur Spine J. 2006;15:953–8. 15. Ozaktay AC, Kallakuri S, Takebayashi T, Cavanaugh JM, Asik I, Deleo JA, Weinstein JN. Effects of interleukin-1 beta, interleukin-6, and tumor necrosis factor on sensitivity of dorsal root ganglion and peripheral receptive fields in rats. Eur Spine J. 2006;15:1529–37. 16. Ozawa T, Aoki Y, Ohtori S, Takahashi K, Chiba T, Ino H, Moriya H. The dorsal portion of the lumbar intervertebral disc is innervated primarily by small peptide-containing dorsal root ganglion neurons in rats. Neurosci Lett. 2003;344:65–7. 17. Eng LF, Ghirnikar RS, Lee YL. Glial fibrillary acidic protein: GFAP-thirty-one years (1969–2000). Neurochem Res. 2000;25: 1439–51. 18. Liedtke W, Edelmann W, Bieri PL, Chiu FC, Cowan NJ, Kucherlapati R, Raine CS. GFAP is necessary for the integrity of CNS white matter architecture and longterm maintenance of myelination. Neuron. 1996;17:607–15. 19. McCall MA, Gregg RG, Behringer RR, Brenner M, Delaney CL, Galbreath EJ, Zhang CL, Pearce RA, Chiu SY, Messing A. Targeted deletion in astrocyte intermediate filament (Gfap) alters neuronal physiology. Proc Natl Acad Sci USA. 1996;93:6361–6. 20. Danielyan L, Tolstonog G, Traub P, Salvetter J, Gleiter CH, Reisig D, Gebhardt R, Buniatian GH. Colocalization of glial fibrillary acidic protein, metallothionein, and MHC II in human, rat, NOD/SCID, and nude mouse skin keratinocytes and fibroblasts. J Invest Dermatol. 2007;127:555–63. 21. Lee Y, Su M, Messing A, Brenner M. Astrocyte heterogeneity revealed by expression of a GFAP-LacZ transgene. Glia. 2006;53:677–87.
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ORIGINAL ARTICLE
Activation of satellite cells in the dorsal root ganglia in a disc-punctured rat model Yanjing Li • Chunyang Xi • Ming Niu • Zhiyong Chi • Xiaoqi Liu • Jinglong Yan
Received: 9 January 2011 / Accepted: 25 March 2011 / Published online: 26 May 2011 Ó The Japanese Orthopaedic Association 2011
Abstract Background The neural mechanisms underlying discogenic low back pain caused by disc degeneration remain unclear. Previous studies demonstrated that satellite cells (SC) play an important role in neuropathic pain. Methods Twenty adult female Sprague-Dawley rats were used. The rats were divided into two groups: a nucleus pulposus (NP) group whose discs were punctured to expose the NP (n = 10) and a sham-operated group whose annulus fibrosus surface was scratched superficially (n = 10). In this study, we investigated the expression and cellular distribution of glial fibrillary acidic protein (GFAP, a marker of SC activation) in the dorsal root ganglia (DRG) innervating the intervertebral discs using a retrograde tracing method and immunohistochemistry in a discpunctured rat model. Results In the sham-operated group, GFAP-immunoreactive (IR) SCs were not detected. In the NP group, GFAP-IR SC became evident, and 49 ± 13% of neurons innervating the punctured discs were surrounded by GFAP-positive SCs.
Y. Li and C. Xi contributed equally to this work. Y. Li C. Xi Z. Chi X. Liu J. Yan (&) Department of Orthopedic Surgery, The First Affiliated Hospital, Harbin Medical University, 23 Youzheng St, Nangang District, Harbin 150001, People’s Republic of China e-mail: [email protected] M. Niu Department of Mammary Surgery, The Third Affiliated Hospital, Harbin Medical University, Harbin 150001, People’s Republic of China
Conclusions Our results were the first to provide evidence for a potential role of SCs in the neural mechanisms of discogenic low back pain caused by disc degeneration.
Introduction Degeneration of lumbar intervertebral discs is considered to be an important source of discogenic low back pain [1, 2]. Nevertheless, the neural mechanisms of discogenic low back pain caused by disc degeneration are poorly understood. A recent study reported that exposure of the nucleus pulposus (NP) to the outside of the annulus fibrosus could upregulate the expression of activating transcription factor 3 (ATF3, a marker of nerve injury) and growth-associated protein 43 (GAP43, a marker of nerve growth) in the dorsal root ganglia (DRG), innervating the punctured disc [2]. This indicated that exposure of the NP to the outside of the annulus fibrosus could induce nerve injury and nerve ingrowth into the discs, and degenerated discs might induce cellular and molecular changes in the DRG innervating the degenerated disc. Satellite cells (SC) are located in the DRG and form a single layer wrapped around each sensory neuron soma [3]. Electron microscopy has demonstrated that the typical SC is a discrete element, bound by its own membrane, a distinctive structure not seen in the central nervous system (CNS) [4]. In recent years, particular attention has been paid to SCs because of their important roles in neuropathic pain states. Following sensory neuron damage or inflammation, the SCs become coupled extensively to SCs by gap junctions, and proliferated and expressed glial fibrillary acidic protein (GFAP) [5–9]. GFAP, a member of the cytoskeletal protein family, has been extensively used as a marker of SC activation [3]. Recently, injury-induced
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GFAP expression in DRG has been demonstrated to contribute to the maintenance of neuropathic pain states [6]. In addition, the activated SCs are important sources of neurotrophins, such as glial cell line-derived neurotrophic factor (GDNF), nerve growth factors (NGF) and neurotrophin-3 (NT3), which are associated with neuropathic pain [10–12]. Furthermore, the activated SCs could produce some cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin1-beta (IL-1beta), all of which mediate inflammation and hyperalgesia [7, 8, 13–15]. Based on these findings, one can conclude that SC activation may play an important role in neuropathic pain. Therefore, we hypothesized that the activation of SCs might also be involved with discogenic low back pain due to disc degeneration. In order to examine the possible relationship between SCs and discogenic low back pain due to disc degeneration, we investigated the expression and cellular distribution of GFAP in the DRG innervating the intervertebral discs in a disc-punctured rat model using a retrograde tracing method and immunohistochemistry.
Materials and methods A total of 20 adult female Sprague-Dawley rats weighing 200–250 g were used in the study. The rats were maintained on a 12-h light/dark cycle with food and water ad libitum. All of the handling of the animals was carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (1996 revision) and was conducted with the permission of the ethics committee of our institution. All efforts possible were made to minimize pain. The rats were anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneal), and all surgical procedures were performed aseptically. A midline abdominal incision was made, and the retroperitoneum was incised along the left margin of the aorta. The left iliopsoas muscle was dissected to expose the ventral aspect of the L5–L6 intervertebral disc (Fig. 1). A 26-gauge needle whose tip was filled with Fluoro-Gold crystals (FG, Fluorochrome, Denver, CO) was inserted into the ventral surface of the disc approximately 0.5 mm in depth. The application site was immediately covered with cyanoacrylate to prevent the crystals from spreading. Then the fascia and skin were closed. This procedure was performed according to a previously reported method [2]. Five days after the application of FG, a 22-gauge needle was inserted into the ventral aspect of the disc at about 3 mm depth (NP group, n = 10), which could result in the exposure of the NP to the outside of the annulus fibrosus
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Fig. 1 The ventral aspect of the L5–L6 intervertebral disc was exposed after dissecting the left iliopsoas muscle. The arrow indicates the L5–L6 disc
[2]. The procedure was monitored by radiography to make sure that the needle puncture was parallel to the endplates to avoid endplate injury. In the sham-operated group (n = 10), the needle was inserted into the ventral aspect to a depth of about 0.5 mm, which could not obtain exposure of the NP. Seven days after surgery, rats were perfused transcardially with 0.9% saline, followed by 500 ml of 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4). The bilateral DRGs from levels L1–L5 were resected, then immersed in the same fixative solution for 3 h at 4°C, then in 0.01 M phosphate buffered saline (PBS) containing 10% sucrose for 6 h, then 20% sucrose for 6 h and 30% sucrose for 6 h. Each ganglion was sectioned at 5 lm thickness on a cryostat and mounted on poly-L-lysine-coated slides. Specimens were treated for 20 min at room temperature in blocking solution with donkey serum. For GFAP staining, the DRGs were incubated overnight at 4°C with rabbit antibody to GFAP (1:400; Dako, Carpinteria, CA). After being thoroughly washed, the sections were incubated for 1 h at 37°C with FITC-labeled donkey anti-rabbit antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA). The sections were observed with a fluorescent microscope (Nikon, Tokyo, Japan). FG-labeled neurons were observed using a UV-1A filter (wavelengths of 365 nm for excitation and 420 nm for emission). Only the FG-labeled neurons with a clear visible nucleus were counted. Each FG-labeled neuron was further examined for positive GFAP reactivity using a FITC filter (wavelengths of 465 nm for excitation and 505 nm for emission). In each DRG, 10 sections were selected randomly, and the numbers of FG-labeled neurons and GFAP-immunoreactive (IR) SCs surrounding FG-labeled neurons were counted by two independent observers who were blinded to this experiment. The data were compared with analysis of variance (ANOVA). P \ 0.05 was considered as statistically significant.
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Fig. 2 The mean number of Fluoro-Gold (FG)-labeled neurons at each level in dorsal root ganglia (DRG) in the nucleus pulposus (NP) and sham-operated group. There was no statistical difference between the two groups at each level
Fluoro-Gold crystals were distributed in the cytoplasm of neurons innervating the L5–L6 disc, and FG-labeled neurons were present in the bilateral L1–L5 DRG (Fig. 3a, c). The majority of FG-labeled neurons were located in L1 and L2 in two groups. The total number of FG-labeled neurons was 310 in the sham-operated group and 304 in the NP group. The number at each level was not significantly different between these two groups (P [ 0.05, Fig. 2). GFAP-IR SCs were not detected in the sham-operated group (Fig. 3b), but became evident in the NP group, and some GFAP-IR SCs were located around the FG-labeled neurons (Fig. 3d); 49 ± 13% (mean ± SD) of the total FG-labeled neurons were positive for GFAP around these
Fig. 3 Fluorescent photomicrograph of FG-labeled neurons and glial fibrillary acidic protein-immunoreactive (GFAP-IR) satellite cells (SC) in the sham-operated group (a, b) and NP group (c, d). GFAPIR SCs were not observed in b, but observed around neurons, and some FG-labeled neurons were surrounded by GFAP-IR SC (d). Scale bars 50 lm
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Fig. 4 The ratio of GFAP-IR SCs around FG-labeled neurons to total FG-labeled neurons at each level. The ratios were significantly different between two groups at each level. In the NP group, the ratios of expression of GFAP-IR SCs were not significantly different between any two levels (P [ 0.05). Data are presented as mean ± SEM
neurons in the NP group. However, there was no significant difference between any other two levels in the ratios of GFAP-IR SC around FG-labeled neurons to total FGlabeled neurons (P [ 0.05, Fig. 4).
Discussion In this study, the majority of FG-labeled neurons were located at L1 and L2 levels in both groups, which indicated that the L5–L6 disc was multisegmentally innervated through the paravertebral sympathetic trunks as reported previously [2, 16]. Glial fibrillary acidic protein, the principal 8–9 nm intermediate filaments expressing in mature astrocytes and SCs, has been extensively used as a marker of the activation of astrocytes and SCs [3, 7]. There are many reports about the function of GFAP in astrocytes. As a member of the cytoskeletal protein family, GFAP is involved in modulating astrocyte motility and shape by providing structural stability to astrocytic processes [17]. It has also been reported that GFAP expression in astrocytes contributes to normal white matter architecture, blood-brain barrier integrity, astrocyte-neuronal interactions, modulation of long-term potentiation in the CNS, maintenance of homeostasis and vascular permeability at the blood-tissue interface [18–20]. Following peripheral nerve injury and CNS damage, GFAP was upregulated in astrocytes, which was closely concerned with neuropathic pain states [5–7, 21]. Compared with astrocytes, much less is known about the role of GFAP in SCs. The SCs, which envelope DRG neurons, have some general characteristics of astrocytes and microglia [22, 23]. However, there is an important
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distinction between SCs and astrocytes in that the basal level of GFAP expression is quite low in SCs of normal animals. In our study, there was also an absence of GFAP labeling of SCs in the sham-operated group, which indicated that surgical trauma was not related to GFAP expression in the DRG in this rat model. However, GFAPIR SCs became evident in the NP group. Although GFAP was also used as a marker of SC activation, it is likely that the term ‘‘activation’’ is an oversimplification, and little is known about the nature of this SC activation or of the functional role GFAP plays in SCs. In a recent report, using the strategy of detecting gene expression profiles at different time points of neuropathic pain development, some investigators found that altered expression of selective groups of genes correlated with different stages of neuropathic pain development, and they suggested that injury-induced GFAP expression might play an important role in the maintenance of nerve injuryinduced pain states [6]. Hence, it is possible that blocking GFAP upregulation post-injury might help to shorten the duration of chronic pain states. In addition, according to the published literature, there are several pieces of evidence indicating that the activated SCs contribute to the neuropathic pain. First, following nerve injury, SC sheaths enveloping neighboring neurons formed connections with each other, leading to abnormal spreading of sensory signals among DRG neurons and sensory hyperexcitability, whereas such connections were absent in control ganglia [5]. Second, it has been reported that neurotrophins such as NGF and NT3 were synthesized by activated SCs after peripheral nerve injury and that they would play a crucial role in the generation of pain-related behavior [10, 11]. Third, the activated SCs are important sources of cytokines, such as TNF-alpha and IL-1beta, which mediate inflammation and hyperalgesia [7, 8, 13–15]. Taken together, no matter the role of GFAP itself or SC activation, SCs may play a critical role in neuropathic pain. In the present study, we observed some GFAP-IR SC enveloped FG-labeled neurons, which indicated that some SCs were activated around the DRG neurons innervating the punctured disc. This indicated that the SCs might affect the enveloped neurons innervating the punctured disc. It has been reported that TNF-alpha might be a possible inducer of nerve ingrowth into degenerated intervertebral discs [24], while it is a major neural mechanism of discogenic low back pain that sensory nerve fibers exist in the inner layer of the annulus fibrosus or NP [2, 24]. Since the activated SCs are important sources of TNF-alpha, it is possible that the SCs may contribute to nerve ingrowth to the degenerated disc mediated by the production of TNFalpha [7, 8, 14]. Moreover, for the role of GFAP itself or SC activation in neuropathic pain discussed above, it was deduced that all of these SC changes may contribute to
Activation of satellite cells
discogenic low back pain due to disc degeneration. In addition, we observed that not all the GFAP-IR SCs were located around the FG-labeled neurons, a phenomenon similar to the GFAP expression in some rat models of neuropathic pain [7, 8, 14]. This indicated that SCs might affect each other and then form a cascade reaction. Our results were similar to the previous findings in which the authors found that GFAP-IR SCs were upregulated in the DRG in a rat model of lumbar facet joint injury, which was a possible cause of low back pain [14]. Therefore, we suggested that SC activation in DRG might be a general mechanism in the pathogenesis of low back pain. Further studies need to be done to support these hypotheses. In conclusion, our results suggested that exposure of the NP to the outside of the annulus fibrosus could cause the activation of SCs, and SC activation would then contribute to the neural mechanisms of discogenic low back pain due to disc degeneration in spite of the fact that this model did not correspond exactly to human discogenic low back pain due to disc degeneration. These data provide new perspectives concerning the mechanisms and theoretical basis of suppression of SC activation to treat discogenic low back pain. Intensive studies on the function of GFAP and SCs are required to better understand their substantial roles in discogenic low back pain due to disc degeneration. Acknowledgments This work was supported in part by grants from National Natural Science Foundation of China (100334). The authors thank Ming Yan for her technical assistance. Conflict of interest The authors declare that we have no financial and personal relationships with other people or organizations that could inappropriately influence our work; there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled ‘Activation of satellite cells in the dorsal root ganglia in a disc-punctured rat model.’
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