EXPRESSION AND SIGNIFICANCE OF GROWTH-ASSOCIATED PROTEIN 43 IN A RAT MODEL OF INTERVERTEBRAL DISC INFLAMMATION_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANG Hongjun ¹ , FAN Shunwu ²

1Department of Spine, Luoyang Orthopaedic Hospital, Luoyang Henan, 471002, P.R.China;;
2Department of Orthopaedics, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University. Corresponding author: FAN Shunwu, E-mail: fansw@srrsh.com;

Corresponding author：

Keywords：

Intervertebral disc inflammation; Growth-associated protein 43; Dorsal root ganglion; Rat

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Objective To investigate the expression and significance of growth-associated protein 43 (GAP-43) in the dorsal root ganglion (DRG) and intervertebral disc in the rat model of intervertebral disc inflammation. Methods A total of 103 adult male Sprague Dawley rats (weighing, 200-250 g) were randomly divided into the experimental group (n=48), the control group (n=48), and the blank control group (n=7). Fluoro-gold (F-G) as tracer was injected into the L5, 6 intervertebral disc of 3 groups; after 7 days of F-G injection, complete Freund’s adjuvant (50 µL) and the same volume of saline were injected in the experimental group (to prepare the model of intervertebral disc inflammation) and the control group, respectively, and the blank control group had no further treatment. After 1, 3, 7, and 14 days, T13-L6 DRG and L5, 6 intervertebral disc of experimental group and control group were harvested to detect the GAP-43 by using fluorescent immunohistochemistry, in situ hybridization, and RT-PCR. The DRG and intervertebral disc of blank control group were also harvested after 8 days of F-G injection. Results Fluorescent immunohistochemistry results showed that the number of F-G-labeled GAP-43 immunoreaction (GAP-43-IR) cells of the DRGs in the experimental group was significantly higher than that in the control group (P lt; 0.05) at 3 days, and no significant difference was found at the other time points (P gt; 0.05). There was no significant difference in the cross-sectional area of F-G-labeled GAP-43-IR cells between the experimental group and the control group at each time point (P gt; 0.05). The co-expression of GAP-43 with calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4)-binding glycoprotein exhibited that the expression of CGRP was 91.4% ± 7.4% in the control group and was 87.6% ± 7.8% in the experimental group, showing no significant difference between 2 groups (P gt; 0.05). There was no IB4-binding glycoprotein expression in GAP-43-IR cells of the DRGs in 2 groups. The expressions of GAP-43, CGRP, and IB4-positive nerve fibers in the intervertebral disc exhibited that the GAP-43-IR nerve fibers in the experimental group were significantly more than that in the control group (P lt; 0.05), but no significant difference was found in the expression of CGRP between 2 groups (P gt; 0.05); and there was no IB4-binding glycoprotein expression in GAP-43-IR nerve fibers of the intervertebral disc in 2 group. In situ hybridization and RT-PCR detection showed that the positive expression cells ratio of GAP-43 mRNA and the level of GAP- 43 mRNA were significantly higher in the experimental group than in the control group at 1 day (P lt; 0.05), and no significant difference was found at the other time points (P gt; 0.05). Conclusion Intradiscal inflammatory environment may induce the expression of GAP-43, and potentially promote the nerve fiber ingrowth of rat.

Citation： ZHANG Hongjun,FAN Shunwu. EXPRESSION AND SIGNIFICANCE OF GROWTH-ASSOCIATED PROTEIN 43 IN A RAT MODEL OF INTERVERTEBRAL DISC INFLAMMATION. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(12): 1435-1441. doi: Copy

1.	Helm li S, Deer TR, Manchikanti L, et al. Effectiveness of thermal annular procedures in treating discgenic low back pain. Pain Physician, 2012, 15(3): E279-304.
2.	Moon HJ, Kim JH, Lee SH, et al. Annulus fibrosus cells interact with neuron-like cells to modulate production of growth factors and cytokines in symptomatic disc degeneration. Spine (Phila Pa 1976), 2012, 37(1): 2-9.
3.	Martins DE, Oliveira VM, Alves MT, et al. Correlations between radiographic, magnetic resonance and histological examinations on thedegeneration of human lumbar intervertebral discs. Sao Paulo Med J, 2010, 128(2): 63-68.
4.	Ozawa T, Aoki Y, Ohtori S, et al. The dorsal portion of the lumbar intervertebral disc is innervated primarily by small peptide-containing dorsal root ganglion neurons in rats. Neurosci Lett, 2003, 44(1): 65-67.
5.	Melrose J, Roberts S, Smith S, et al. Increased nerve and blood vessel ingrowth associated with proteoglycan depletion in an ovine anular lesion model of experimental disc degeneration. Spine (Phila Pa 1976), 2002, 27(12): 1278-1285.
6.	Johnson WE, Caterson B, Eisenstein SM, et al. Human intervertebral disc aggrecan inhibits nerve growth in vitro. Arthritis Rheum, 2002, 46(10): 2658-2664.
7.	Pyo KH, Kang EY, Jung BK, et al. Depressed neuronal growth associated protein (GAP-43) expression in the small intestines of mice experimentally infected with Neodiplostomum seoulense. Korean J Parasitol, 2012, 50(1): 89-93.
8.	Bursova S, Dubovy P, Vlckova-Moravcova E, et al. Expression of growth-associated protein 43 in the skin nerve fibers of patients with type 2 diabetes mellitus. J Neurol Sci, 2012, 315(1-2): 60-63.
9.	Murakami M, Ito H, Hagiwara K, et al. Sphingosine kinase 1/S1P pathway involvement in the GDNF-induced GAP43 transcription. J Cell Biochem, 2011, 112(11): 3449-3458.
10.	Wuertz K, Vo N, Kletsas D, et al. Inflammatory and catabolic signaling in intervertebral discs: the roles of NF-kB and MAP kinases. Eur Cell Mater, 2012, 23: 103-120.
11.	Wu CC, Chen WH, Zao B, et al. Regenerative potentials of platelet-rich plasma enhanced by collagen in retrieving pro-inflammatory cytokine-inhibited chondrogenesis. Biomaterials, 2011, 32(25): 5847-5854.
12.	Junq WW, Kim HS, Shon JR, et al. Intervertebral disc degeneration-induced expression of pain-related molecules: glial cell-derived neurotropic factor as a key factor. J Neurosurg Anesthesiol, 2011, 23(4): 329-334.
13.	Obata K, Tsujino H, Yamanaka H, et al. Expression of neurotrophic factors in the dorsal root ganglion in a rat model of lumbar disc herniation. Pain, 2002, 99(1-2): 121-132.
14.	Orita S, Ishikawa T, Miyagi M, et al. Pain-related sensory innervations in monoiodoacetate-induced osteoarthritis in rat kness that gradually develops neuronal injury in addition to inflammatory pain. BMC Musculoskelet Disord, 2011, 16(12): 134.
15.	Yamauchi K, Inoue G, Koshi T, et al. Nerve growth factor of cultured medium extracted from human degenerative nucleus pulposus promotes sensory nerve growth and induces substance p in vitro. Spine (Phila Pa 1976), 2009, 34(21): 2263-2269.
16.	Orita S, Ohtori S, Nagata M, et al. Inhibiting nerve growth factor or its receptors downregulates calcitonin gene-related peptide expression in rat lumbar dorsal root ganglia innervating injured intervertebral discs. J Orthop Res, 2010, 28(12): 1614-1620.
17.	Aoki Y, Takahashi Y, Ohtori S, et al. Distribution and immunocytochemical characterization of dorsal root ganglion neurons innervating the lumbar intervertebral disc in rats: a review. Life Sci, 2004, 74(21): 2627-2642.
18.	Vera PL, Iczkowski KA, Howard DJ, et al. Antagonism of macrophage migration inhibitory factor decreases cyclophosphamide cystitis in mice. Neurourol Urodyn, 2010, 29(8): 1451-1457.
19.	Gaspersic R, Kovacic U, Glisovic S, et al. Anti-NGF treatment reduces bone resorption in periodontitis. J Dent Res, 2010, 89(5): 515-520.
20.	Kato N, Nemoto K, Arino H, et al. Influence of peripheral inflammation on growth-associated phosphoprotein (GAP-43) expression in dorsal root ganglia and on nerve recovery after crush injury. Neurosci Res, 2003, 45(3): 297-303.
21.	Sowa GA, Coelho JP, Vo NV, et al. Cells from degenerative intervertebral discs demonstrate unfavorable responses to mechanical and inflammatory stimuli: a pilot study. Am J Phys Med Rehabil, 2012, 91(10): 846-855.

1. Helm li S, Deer TR, Manchikanti L, et al. Effectiveness of thermal annular procedures in treating discgenic low back pain. Pain Physician, 2012, 15(3): E279-304.
2. Moon HJ, Kim JH, Lee SH, et al. Annulus fibrosus cells interact with neuron-like cells to modulate production of growth factors and cytokines in symptomatic disc degeneration. Spine (Phila Pa 1976), 2012, 37(1): 2-9.
3. Martins DE, Oliveira VM, Alves MT, et al. Correlations between radiographic, magnetic resonance and histological examinations on thedegeneration of human lumbar intervertebral discs. Sao Paulo Med J, 2010, 128(2): 63-68.
4. Ozawa T, Aoki Y, Ohtori S, et al. The dorsal portion of the lumbar intervertebral disc is innervated primarily by small peptide-containing dorsal root ganglion neurons in rats. Neurosci Lett, 2003, 44(1): 65-67.
5. Melrose J, Roberts S, Smith S, et al. Increased nerve and blood vessel ingrowth associated with proteoglycan depletion in an ovine anular lesion model of experimental disc degeneration. Spine (Phila Pa 1976), 2002, 27(12): 1278-1285.
6. Johnson WE, Caterson B, Eisenstein SM, et al. Human intervertebral disc aggrecan inhibits nerve growth in vitro. Arthritis Rheum, 2002, 46(10): 2658-2664.
7. Pyo KH, Kang EY, Jung BK, et al. Depressed neuronal growth associated protein (GAP-43) expression in the small intestines of mice experimentally infected with Neodiplostomum seoulense. Korean J Parasitol, 2012, 50(1): 89-93.
8. Bursova S, Dubovy P, Vlckova-Moravcova E, et al. Expression of growth-associated protein 43 in the skin nerve fibers of patients with type 2 diabetes mellitus. J Neurol Sci, 2012, 315(1-2): 60-63.
9. Murakami M, Ito H, Hagiwara K, et al. Sphingosine kinase 1/S1P pathway involvement in the GDNF-induced GAP43 transcription. J Cell Biochem, 2011, 112(11): 3449-3458.
10. Wuertz K, Vo N, Kletsas D, et al. Inflammatory and catabolic signaling in intervertebral discs: the roles of NF-kB and MAP kinases. Eur Cell Mater, 2012, 23: 103-120.
11. Wu CC, Chen WH, Zao B, et al. Regenerative potentials of platelet-rich plasma enhanced by collagen in retrieving pro-inflammatory cytokine-inhibited chondrogenesis. Biomaterials, 2011, 32(25): 5847-5854.
12. Junq WW, Kim HS, Shon JR, et al. Intervertebral disc degeneration-induced expression of pain-related molecules: glial cell-derived neurotropic factor as a key factor. J Neurosurg Anesthesiol, 2011, 23(4): 329-334.
13. Obata K, Tsujino H, Yamanaka H, et al. Expression of neurotrophic factors in the dorsal root ganglion in a rat model of lumbar disc herniation. Pain, 2002, 99(1-2): 121-132.
14. Orita S, Ishikawa T, Miyagi M, et al. Pain-related sensory innervations in monoiodoacetate-induced osteoarthritis in rat kness that gradually develops neuronal injury in addition to inflammatory pain. BMC Musculoskelet Disord, 2011, 16(12): 134.
15. Yamauchi K, Inoue G, Koshi T, et al. Nerve growth factor of cultured medium extracted from human degenerative nucleus pulposus promotes sensory nerve growth and induces substance p in vitro. Spine (Phila Pa 1976), 2009, 34(21): 2263-2269.
16. Orita S, Ohtori S, Nagata M, et al. Inhibiting nerve growth factor or its receptors downregulates calcitonin gene-related peptide expression in rat lumbar dorsal root ganglia innervating injured intervertebral discs. J Orthop Res, 2010, 28(12): 1614-1620.
17. Aoki Y, Takahashi Y, Ohtori S, et al. Distribution and immunocytochemical characterization of dorsal root ganglion neurons innervating the lumbar intervertebral disc in rats: a review. Life Sci, 2004, 74(21): 2627-2642.
18. Vera PL, Iczkowski KA, Howard DJ, et al. Antagonism of macrophage migration inhibitory factor decreases cyclophosphamide cystitis in mice. Neurourol Urodyn, 2010, 29(8): 1451-1457.
19. Gaspersic R, Kovacic U, Glisovic S, et al. Anti-NGF treatment reduces bone resorption in periodontitis. J Dent Res, 2010, 89(5): 515-520.
20. Kato N, Nemoto K, Arino H, et al. Influence of peripheral inflammation on growth-associated phosphoprotein (GAP-43) expression in dorsal root ganglia and on nerve recovery after crush injury. Neurosci Res, 2003, 45(3): 297-303.
21. Sowa GA, Coelho JP, Vo NV, et al. Cells from degenerative intervertebral discs demonstrate unfavorable responses to mechanical and inflammatory stimuli: a pilot study. Am J Phys Med Rehabil, 2012, 91(10): 846-855.

Chinese Journal of Reparative and Reconstructive Surgery

EXPRESSION AND SIGNIFICANCE OF GROWTH-ASSOCIATED PROTEIN 43 IN A RAT MODEL OF INTERVERTEBRAL DISC INFLAMMATION

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