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Background Context: Intervertebral disc degeneration is common and may play an important role in low back pain, but it is not well-understood. Previous studies have shown that the outer layer of the annulus fibrosus of a healthy disc is innervated by nociceptive nerve fibers. In the process of disc degeneration, it can grow into the inner annulus fibrosus or nucleus pulposus and release neuropeptides. Disc degeneration is associated with inflammation that produces inflammatory factors and potentiates nociceptor sensitization. Subsequently neurogenic inflammation is induced by neuropeptide release from activated primary afferent terminals. Because the innervation of a lumbar disc comes from multisegmental dorsal root ganglion neurons, does neurogenic inflammation in a degenerative disc initiate neurogenic inflammation in neighboring healthy discs by antidromic activity?, Purpose: This study was based on animal experiments in Sprague-Dawley rats to investigate the role of neurogenic inflammation in adjacent healthy disc degeneration induced by disc injury., Study Design: This was an experimental study., Methods: Seventy-five 12-week-old, male Sprague-Dawley rats were allocated to 3 groups (sham group, disc injury group and disc injury+TrkA antagonist group). The disc injury group was punctured in the tail disc between the eighth and ninth coccygeal vertebrae (Co8-9) to establish an animal model of tail intervertebral disc degeneration. The sham group underwent only skin puncture and the disc injury+TrkA antagonist group was intraperitoneally injected with GW441756 two days before disc puncture. The outcome measure included quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay., Results: Disc injury induced an increase in aggrecan, NGF, TrkA, CGRP, SP, IL-1β, and IL-6 mRNA levels in the injured (Co8-9) and adjacent discs (Co7-8), which reached a peak on day 1, then gradually decreased, and returned to normal on day 14. After intraperitoneal injection of GW441756 prior to puncture, the mRNA levels of the above indicators were down-regulated in Co7-8 and Co8-9 intervertebral discs on the 1st and 7th days. The protein content of the above indicators in Co7-8 and Co8-9 intervertebral discs showed roughly the same trend as mRNA levels., Conclusions: Degeneration of one disc can induce neurogenic inflammation of adjacent healthy discs in a rat model., Clinical Significance: This model supports a key role of neurogenic inflammation in disc degeneration, and may play a role in the experience of low back pain., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The purpose of this three-part narrative review is to examine the anatomy of, and the research which supports, either the lumbar myofascia or intervertebral disc (IVD) as principal sources of our patient's low back pain. A comprehensive understanding of anatomical lumbar pain generators in combination with the current treatment-based classification system will further improve and enhance clinical decision-making skills. Section I reviews the anatomy of the spinal myofascia, myofascial sources of lumbar pain, and imaging of myofascial tissues. Part II reviews the anatomy of the IVD, examines the IVD as a potential lumbar pain generator, and includes detailed discussion on Nerve Growth Factor, Inflammatory Cytokines, Vertebral End Plates and Modic change, Annular tears, and Discogenic instability. Part III looks at the history of myofascial pain, lab-based research and myofascial pain, and various levels of discogenic pain provocation research including animal, laboratory and human subjects. Our review concludes with author recommendations on developing a comprehensive understanding of altered stress concentrations affecting the posterior annulus fibrosis, neo-innervation of the IVD, inflammatory cytokines, discogenic instability, and how this knowledge can complement use of the Treatment-Based Classification System.

Sensory nerves are long being recognized as collecting units of various outer stimuli; recent advances indicate that the sensory nerve also plays pivotal roles in maintaining organ homeostasis. Here, this study shows that sensory nerve orchestrates intervertebral disc (IVD) homeostasis by regulating its extracellular matrix (ECM) metabolism. Specifically, genetical sensory denervation of IVD results in loss of IVD water preserve molecule chondroitin sulfate (CS), the reduction of CS bio-synthesis gene chondroitin sulfate synthase 1 (CHSY1) expression, and dysregulated ECM homeostasis of IVD. Particularly, knockdown of sensory neuros calcitonin gene-related peptide (CGRP) expression induces similar ECM metabolic disorder compared to sensory nerve denervation model, and this effect is abolished in CHSY1 knockout mice. Furthermore, in vitro evidence shows that CGRP regulates nucleus pulposus cell CHSY1 expression and CS synthesis via CGRP receptor component receptor activity-modifying protein 1 (RAMP1) and cyclic AMP response element-binding protein (CREB) signaling. Therapeutically, local injection of forskolin significantly attenuates IVD degeneration progression in mouse annulus fibrosus puncture model. Overall, these results indicate that sensory nerve maintains IVD ECM homeostasis via CGRP/CHSY1 axis and promotes IVD repair, and this expands the understanding concerning how IVD links to sensory nerve system, thus shedding light on future development of novel therapeutical strategy to IVD degeneration., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Background and Objectives: Rami communicantes (RC) infiltration and radiofrequency lesions are new techniques for the treatment of discogenic low back pain (DLBP). Their efficacy is controversial, and the classification of RC remains unclear. We aimed to explore the differences between RC and reclassify RC according to their anatomical characteristics., Methods: Sixteen sides of the lumbar spine from eight adult male embalmed cadavers were dissected. The presence of RC was noted. The morphology, origin, distribution, course, quantity and spatial orientation of RC on the lumbar spine were examined. The length and width of the RC were measured by a caliper., Results: A total of 213 RC were found in the 8 cadavers in the lumbar region. RC were divided into three types: superficial rami (70, 32.86%), which penetrated the psoas major (PM) and ran above the aponeurosis of the PM; deep rami (125, 58.69%), which ran along the waist of the vertebral body beneath the aponeurosis of the PM; and discal rami, which ran over and adhered to the surface of the intervertebral disc. Superficial rami were divided into two subtypes: oblique rami (45, 21.13%) and parabolic rami (25, 11.74%), which crossed the vertebra and the disc in an oblique and a parabolic course, respectively., Conclusions: RC should play an important role in the innervation of the lumbar spine. Detailed knowledge of RC in the lumbar region may help surgeons improve the efficacy of infiltration and percutaneous radiofrequency as a supplementary treatment for DLBP., Competing Interests: Competing interests: None declared., (© American Society of Regional Anesthesia & Pain Medicine 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Low back pain (LBP) is a common musculoskeletal symptom, which can be developed in multiple clinical diseases. It is widely recognized that intervertebral disc (IVD) degeneration (IVDD) is one of the leading causes of LBP. However, the pathogenesis of IVD-related LBP is still controversial, and the treatment means are also insufficient to date. In recent decades, the role of structure and function changes of sensory nervous system in the induction and the maintenance of LBP is drawing more and more attention. With the progress of IVDD, IVD cell exhaustion and extracellular matrix degradation result in IVD structural damage, while neovascularization, innervation and inflammatory activation further deteriorate the microenvironment of IVD. New nerve ingrowth into degenerated IVD amplifies the impacts of IVD-derived nociceptive molecules on sensory endings. Moreover, IVDD is usually accompanied with disc herniation, which could injure and inflame affected nerves. Under mechanical and pro-inflammatory stimulation, the pain-transmitting pathway exhibits a sensitized function state and ultimately leads to LBP. Hence, relevant pathogenic factors, such as neurotrophins, ion channels, inflammatory factors, etc., are supposed to serve as promising therapeutic targets for LBP. The purpose of this review is to comprehensively summarize the current evidence on 1) the pathological changes of sensory nervous system during IVDD and their association with LBP, and 2) potential therapeutic strategies for LBP targeting relevant pathogenic factors., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: The anatomical features of the thoracic nerve roots in connection with intervertebral discs may prevent surgery-related complications and improve patients' neurological functional status during thoracic spine surgery. There is limited literature evidence regarding this concept using cadavers., Purpose: To elucidate the qualitative anatomical features of the thoracic nerve roots in connection with intervertebral discs., Material and Methods: Fifteen formalin-preserved spine specimens were used in this study. Small pieces of stainless-steel wires were placed along the root sleeves from their points of origin, after exposing the dural sac and bilateral nerve roots. The standard anteroposterior and lateral radiographs were taken after the placement of the wires. Measurements were done on radiographs using the picture archiving communication system., Results: Take-off angles of the nerve roots at the coronal plane gradually increased from the level of T2 (36.1°±2.72°) to T9 (84.1°±1.84°) and from T9, it decreased to T12 (46.3° ± 2.67°). Similar variation tendency was discovered in take-off angles of the nerve roots at the sagittal plane. No consistent tendency was found both in the distance from the origin of the root sleeve to its superior and inferior vertebral endplate. Distance from the origin of the root sleeve to the posterior midline (DM) exponentially decreased from T1 (8.2 ± 0.87 mm) to T4 (6.0 ± 0.93 mm). It slowly increased from T5 (5.5 ± 0.68 mm) to T12 (10.9 ± 1.79 mm), with T5 having the smallest DM. Distance between the origins of neighboring nerve roots showed an obvious increase from the T1-T2 interval (23.1 ± 2.22 mm) to T7-T8 interval (30.9 ± 2.68 mm). However, it progressively decreased at the T10-T11 interval (26.0 ± 2.40 mm)., Conclusion: The dimensions of the thoracic nerve roots vary greatly from T1 to T12 intervertebral discs. Sound knowledge of these anatomical features of the thoracic nerve is mandatory for the thoracic spine surgery, especially in the posterolateral approach and transforaminal endoscopic surgery.

Low back pain (LBP) is a leading cause of global disability. Multiple anatomical, cellular, and molecular factors are implicated in LBP, including the degeneration of lumbar intervertebral discs (IVDs). We previously described a mouse model that displays behavioral symptoms of chronic LBP. Here, we investigated the development of pathological innervation and macrophage infiltration into injured IVDs following a puncture injury in mice over 12 months. 2-month old CD1 female mice underwent a single puncture of the ventral L4/5 IVD using a 30G needle, and were sacrificed 4 days and 0.5-, 3-, 6- and 12-months post-injury. Severity of disc degeneration was assessed using colorimetric staining. IVD innervation was measured by PGP9.5-immunoreactivity (-ir) and calcitonin gene-related peptide-ir (CGRP-ir). Macrophage accumulation into IVDs was detected by F4/80-ir. Mechanical IVD injury resulted in severe degeneration and increased PGP9.5-ir nerve fiber density starting at 4 days that persisted for up to 12 months and dorsal herniations began to occur at 3 months. CGRP-ir was also upregulated in injured IVDs, with the largest increase at 12 months after injury. Infiltration of F4/80-ir macrophages was observed in injured IVDs by day 4 both dorsally and ventrally, with the latter diminishing in the later stage. Persistent LBP is a complex disease with multiple underlying pathologies. By highlighting pathological changes in IVD innervation and inflammation, our study suggests that strategies targeting these mechanisms might be useful therapeutically., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Pain originating from an intervertebral disc (discogenic pain) is a major source of chronic low back pain. Pathological innervation of the disc by pain-sensing nerve fibers is thought to be a key component of discogenic pain, so treatment with biomaterials that have the ability to inhibit neurite growth will greatly benefit novel disc therapeutics. Currently, disc therapeutic biomaterials are rarely screened for their ability to modulate nerve growth, mainly due to a lack of models to screen neuromodulation. To address this deficit, our lab has engineered a three dimensional in vitro disc innervation model that mimics the interface between primary sensory nerves and the intervertebral disc. Further, herein we have demonstrated the utility of this model to screen the efficacy of chondroitin sulfate biomaterials to inhibit nerve fiber invasion into the model disc. Biomaterials containing chondroitin-4-sulfate (CS-A) decrease neurite growth in a uniform gel and at an interface between a growth-permissive and a growth-inhibitory gel, while chondroitin-6-sulfate (CS-C) is less neuroinhibitory. This in vitro model holds great potential for screening inhibitors of nerve fiber growth to further improve intervertebral disc replacements and therapeutics. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1016-1026, 2020., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Objective: We aimed to investigate mechano-sensitivity at the afferent nerve fibers projecting to degenerated intervertebral disc (IVD) and nociceptive behaviour in a rat model of low back pain (LBP)., Design: Animal model with LBP was established by lumbar 4/5 IVD puncture and nucleus pulposus aspiration. In vivo single nerve recordings (n = 121) were introduced to measure discharge frequency at the afferent nerve fiber innervating the IVD during mechanical stimulations (von Frey filament or intradiscal pressure). Nerve growth factor (NGF) expression levels in the IVD (n = 20) were assessed by Western blot. LBP-related behaviour (n = 22) was assessed by measuring changes in rearing, mechanical paw-withdrawal threshold, and dynamic weight bearing in a freely walking rat. Inhibitory effect of morphine on the neuronal excitability (n = 19) and painful behaviour (n = 28) was also assessed., Results: Compared to those with sham or naïve IVD, animal group with degenerated IVD displayed the sensitized neuronal responses and painful behaviour, with hyperexcitability of the afferent nerve fibers in any range of mechanical stimulations (von Frey filament stimulation; 1, 2, and 26 g; intradiscal pressure, 1,500-3,000 mm Hg), strong upregulation of NGF (200-250 % increase), and LBP-like behaviour such as failure of rearing, front limbs-dependent walking pattern, and hypersensitivity in hind-paws. However, the neuronal hyperexcitability and pain behaviour were attenuated after local (30 μM) or systemic (3 mg kg -1 ) morphine administration., Conclusions: Our study suggests that enhanced mechano-sensitivity at the afferent nerve fiber innervating degenerated IVD is deeply correlated with LBP development, which supports the hypothesis that hyperexcited responses at the nerve fibers represent a decisive source of LBP., (Copyright © 2019 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Back pain is a common clinical symptom. Degeneration of intervertebral discs is one of the most important factors leading to back pain, namely, discogenic back pain. However, at present, the understanding of lumbar intervertebral discs causing back pain is confined to biomechanical and histological studies. The neuropathological mechanism related to discogenic back pain is still not well understood. Many studies have found that as an intervertebral disc degenerates, the peripheral nerve tissues have corresponding structural reorganization, and a series of nerve cells become involved in progression of discogenic back pain. Therefore, study of neural mechanisms that are involved in progression of discogenic back pain will provide additional assistance for treatment of its symptoms. We review the anatomical structure of intervertebral discs and the related neural mechanisms involved in discogenic back pain. We also discuss the current view of neural mechanisms underlying discogenic back pain.

Nerves and blood vessels are found in the peripheral annulus and endplates of healthy adult intervertebral discs. Degenerative changes can allow these vessels to grow inwards and become associated with discogenic pain, but it is not yet clear how far, and why, they grow in. Previously we have shown that physical disruption of the disc matrix, which is a defining feature of disc degeneration, creates free surfaces which lose proteoglycans and water, and so become physically and chemically conducive to cell migration. We now hypothesise that blood vessels and nerves in degenerated discs are confined to such disrupted tissue. Whole lumbar discs were obtained from 40 patients (aged 37-75 years) undergoing surgery for disc herniation, disc degeneration with spondylolisthesis or adolescent scoliosis ('non-degenerated' controls). Thin (5-μm) sections were stained with H&E and toluidine blue for semi-quantitative assessment of blood vessels, fissures and proteoglycan loss. Ten thick (30-μm) frozen sections from each disc were immunostained for CD31 (an endothelial cell marker), PGP 9.5 and Substance P (general and nociceptive nerve markers, respectively) and examined by confocal microscopy. Volocity image analysis software was used to calculate the cross-sectional area of each labelled structure, and its distance from the nearest free surface (disc periphery or internal fissure). Results showed that nerves and blood vessels were confined to proteoglycan-depleted regions of disrupted annulus. The maximum distance of any blood vessel or nerve from the nearest free surface was 888 and 247 μm, respectively. Blood vessels were greater in number, grew deeper, and occupied more area than nerves. The density of labelled blood vessels and nerves increased significantly with Pfirrmann grade of disc degeneration and with local proteoglycan loss. Analysing multiple thick sections with fluorescent markers on a confocal microscope allows reliable detection of thin filamentous structures, even within a dense matrix. We conclude that, in degenerated and herniated discs, blood vessels and nerves are confined to proteoglycan-depleted regions of disrupted tissue, especially within annulus fissures., (© 2018 Anatomical Society.)

The rationale behind intradiscal O2-O3 therapy is the pain elicited by the mechanical compression of the nerve root, which is associated with periganglionic and periradicular inflammation. This study aimed to determine the effect of intradiscal ozone injection on pain score and satisfaction of patients with low back pain (LBP) secondary to disc herniation. Patients with LBP diagnosed with disc herniation were enrolled in this clinical trial. After prepping and draping the area, intradiscal injection of ozone/oxygen mixture (10 ml, 25μg/ml) was performed under fluoroscopy guide (c-arm). Pain score and patient satisfaction were assessed prior to the injection (baseline) and 1, 3, 6, 12 and 24 months after the injection. Sixty three patients (24 males, 39 females) with mean age of 53.3 ±2.0 y enrolled in the study. The mean±standard deviation (SD) of pain score before intervention was 6.968 ±0.11. Pain score was reduced to 4.25±0.19 at 1 month, 4.33±0.20 at 3 months, 4.87 ±0.21 at 6 months and 5.22 ±0.20 at 24 months. According to the modified MacNab scale success of pain relief was as follows: excellent: 4 (6.3%), good: 17 (26.98 %), sufficient: 13 (20.63 %), poor: 13 (20.63 %), no result: 11 (17.46%), negative: 4 (6.3 %). Intradiscal ozone therapy was determined to provide improved outcomes in patients with single level of bulging and protrusion.

Intervertebral disc diseases (IVDDs) form a group of a vertebral column disorders affecting a large number of people worldwide. It is estimated that approximately 30% of individuals at the age of 35 and approximately 90% of individuals at the age of 60 and above will have some form of disc-affecting pathological changes leading to disc herniation, prolapse and degeneration as well as discogenic pain. Here, we aimed to establish the origins and neurochemical characteristics of porcine intervertebral disc sympathetic innervation involved in pain signalling in IVDD patients. Pigs were given an injection of the Ominipaque contrast agent and Fast Blue (FB) retrograde tracer into the L 4 -L 5 intervertebral disc and euthanized at 2, 1, and 3 months post injection. Following euthanasia, bilateral sympathetic chain ganglia (SChG) Th 13 to C 1 were collected. The presence, distribution and neurochemical characteristics of retrogradely labelled SChG neurons were examined. The majority (88.8%) of all FB+ cells were found in the L 3 -L 5 SChG. Most FB+ neurons stained for dopamine beta hydroxylase (DBH); one-third to one-quarter stained for somatostatin (SOM), neuropeptide Y (NPY) or leu-enkephalin (LENK); and only a few stained for galanin (GAL). Compared with the control, the greatest decline in neurochemical immunostaining was observed 2 weeks post injection, and the lowest decline was noticed 1 month post injection. Our study, for the first time, provides insight into the complex patterns of intervertebral disc sympathetic innervation and suggests that the best time for neurochemical balance restoration therapy would be 1 month post-injury, when the neuronal concentration of all studied substances is close to the initial physiological level, thus providing favourable conditions for successful recovery.

Background: The pathophysiology and mechanisms driving the generation of unintended pain after total disc replacement (TDR) remain unexplored. Ultrahigh-molecular-weight polyethylene (UHMWPE) wear debris from TDRs is known to induce inflammation, which may result in pain., Questions/purposes: The purpose of this study was to determine whether (1) periprosthetic UHMWPE wear debris induces immune responses that lead to the production of tumor necrosis factor-α (TNFα) and interleukin (IL)-1ß, the vascularization factors, vascular endothelial growth factor (VEGF) and platelet-derived growth factor-bb (PDGFbb), and the innervation/pain factors, nerve growth factor (NGF) and substance P; (2) the number of macrophages is associated with the production of the aforementioned factors; (3) the wear debris-induced inflammatory pathogenesis involves an increase in vascularization and associated innervation., Methods: Periprosthetic tissues from our collection of 11 patients with contemporary TDRs were evaluated using polarized light microscopy to quantify UHMWPE wear particles. The major reason for revision (mean implantation time of 3 years [range, 1-6 years]) was pain. For control subjects, biopsy samples from four patients with degenerative disc disease with severe pain and autopsy samples from three normal patients with no history of back pain were also investigated. Immunohistochemistry and histology were used to identify secretory factors, macrophages, and blood vessels. Immunostained serial sections were imaged at ×200 magnification and using MATLAB and NIH ImageJ, a threshold was determined for each factor and used to quantify positive staining normalized to tissue sectional area. The Mann-Whitney U test was used to compare results from different patient groups, whereas the Spearman Rho test was used to determine correlations. Significance was based on p < 0.05., Results: The mean percent area of all six inflammatory, vascularization, and innervation factors was higher in TDR tissues when compared with normal disc tissues. Based on nonparametric data analysis, those factors showing the most significant increase included TNFα (5.17 ± 1.76 versus 0.05 ± 0.03, p = 0.02), VEGF (3.02 ± 1.01 versus 0.02 ± 0.002, p = 0.02), and substance P (4.15 ± 1.01 versus 0.08 ± 0.04, p = 0.02). The mean percent area for IL-1ß (2.41 ± 0.66 versus 0.13 ± 0.13, p = 0.01), VEGF (3.02 ± 1.01 versus 0.34 ± 0.29, p = 0.04), and substance P (4.15 ± 1.01 versus 1.05 ± 0.46, p = 0.01) was also higher in TDR tissues when compared with disc tissues from patients with painful degenerative disc disease. Five of the factors, TNFα, IL-1ß, VEGF, NGF, and substance P, strongly correlated with the number of wear particles, macrophages, and blood vessels. The most notable correlations included TNFα with wear particles (p < 0.001, ρ = 0.63), VEGF with macrophages (p = 0.001, ρ = 0.71), and NGF with blood vessels (p < 0.001, ρ = 0.70). Of particular significance, the expression of PDGFbb, NGF, and substance P was predominantly localized to blood vessels/nerve fibers., Conclusions: These findings indicate wear debris-induced inflammatory reactions can be linked to enhanced vascularization and associated innervation/pain factor production at periprosthetic sites around TDRs. Elucidating the pathogenesis of inflammatory particle disease will provide information needed to identify potential therapeutic targets and treatment strategies to mitigate pain and potentially avoid revision surgery., Level of Evidence: Level III, therapeutic study.

Background: Pain of spinal origin contributes significantly to cervical, thoracic, and lower back pain presentations. Such pain originates in the nerve fibers supplying the joints or the surrounding ligaments and intervertebral discs. Although there has been little detailed discussion of spinal bony innervation patterns in the literature, the clinical implications of these patterns are anatomically and medically significant., Methods: The present review provides a detailed analysis of the innervation of the spine, identifying the unique features of each part via online search engines., Conclusions: The clinical implications of these various studies lie in the importance of the innervation patterns for the mechanism of spinal pain. Immunohistochemical studies have provided further evidence regarding the nature of the innervation of the spine.

Background: Chronic low back pain (LBP) is the most common cause of disability worldwide. New ideas surrounding LBP are emerging that are based on interactions between mechanical, biological and chemical influences on the human IVD. The degenerate IVD is proposed to be innervated by sensory nerve fibres and vascularised by blood vessels, and it is speculated to contribute to pain sensation. However, the incidence of nerve and blood vessel ingrowth, as well as whether these features are always associated, is unknown. We investigated the presence of nerves and blood vessels in the nucleus pulposus (NP) of the IVD in a large population of human discs., Methods: Immunohistochemistry was performed with 61 human IVD samples, to identify and localise nerves (neurofilament 200 [NF200]/protein gene product 9.5) and blood vessels (CD31) within different regions of the IVD., Results: Immunopositivity for NF200 was identified within all regions of the IVD within post-mortem tissues. Nerves were seen to protrude across lamellar ridges and through matrix towards NP cells. Nerves were identified deep within the NP and were in many cases, but not always, seen in close proximity to fissures or in areas where decreased matrix was seen. Fifteen percent of samples were degenerate and negative for nerves and blood vessels, whilst 16 % of all samples were degenerate with nerves and blood vessels. We identified 52% of samples that were degenerate with nerves but no blood vessels. Interestingly, only 4% of all samples were degenerate with no nerves but positive for blood vessels. Of the 85 samples investigated, only 6 % of samples were non-degenerate without nerves and blood vessels and 7% had nerves but no blood vessels., Conclusions: This study addresses the controversial topic of nerve and blood vessel ingrowth into the IVD in a large number of human samples. Our findings demonstrate that nerves are present within a large proportion of NP samples from degenerate IVDs. This study shows a possible link between nerve ingrowth and degeneration of the IVD and suggests that nerves can migrate in the absence of blood vessels.

Purpose: Nuclear factor-κB (NF-κB), receptor activator of NF-κB (RANK), and RANK ligand (RANKL) are transcriptional regulators of inflammatory cytokines. RANKL expression in dorsal root ganglion (DRG) neurons is elevated in animal models of pain or intervertebral disc herniation. We sought to evaluate the effect of anti-RANKL antibodies on sensory nerves innervating injured intervertebral discs., Method: We labeled DRG neurons innervating L5-6 discs with FluoroGold (FG). The L5-6 discs of 36 rats were punctured using a 23-gage needle and 18 rats underwent sham surgery without disc puncture. The puncture group was evenly subdivided into a group in which 10 μl saline was administered to the injured disc and a group in which 10 μl of anti-RANKL antibody was administered. Seven and 14 days postsurgery, DRGs at L2 level were harvested, sectioned, and immunostained for calcitonin gene-related peptide (CGRP). The proportion of CGRP-immunoreactive (IR) DRG neurons of all FG-positive neurons was determined. Amount of tumor necrosis factor (TNF)-α and interleukin(IL)-6 was measured within the intervertebral discs in each group at 7 and 14 days after surgery using an enzyme-linked immunosorbent assay (ELISA)., Results: The proportion of CGRP-IR DRG neurons to total FG-labeled neurons innervating injured intervertebral discs and amount of TNF-α and IL-6 in the injured discs in the saline control group was significantly increased compared with that found in rats from the sham surgery group (P < 0.05). However, application of anti-RANKL antibody to the injured discs significantly decreased the proportion of CGRP-IR DRG neurons to total FG-labeled neurons and amount of TNF-α and IL-6 in the injured discs (P < 0.05)., Conclusions: TNF-α and IL-6 in the injured discs increased and CGRP expression increased in DRG neurons innervating injured discs, and antibodies to RANKL could suppress this increased TNF-α, IL-6, and CGRP expression. RANKL may be a therapeutic target for pain control in patients with lumbar disc degeneration.

Nerve and blood vessel ingrowth during intervertebral disc degeneration, is thought to be a major cause of low back pain, however the regulation of this process is poorly understood. Here, we investigated the expression and regulation of a subclass of axonal guidance molecules known as the class 3 semaphorins, and their receptors; plexins and neuropilins within human NP tissue and their regulation by pro-inflammatory cytokines. Importantly this determined whether semaphorin expression was associated with the presence of nerves and blood vessels in tissues from human intervertebral discs. The study demonstrated that semaphorin3A, 3C, 3D, 3E and 3F and their receptors were expressed by native NP cells and further demonstrated their expression was regulated by IL-1β but to a lesser extent by IL-6 and TNFα. This is the first study to identify sema3C, sema3D and their receptors within the nucleus pulposus of intervertebral discs. Immunopositivity shows significant increases in semaphorin3C, 3D and their receptor neuropilin-2 in degenerate samples which were shown to contain nerves and blood vessels, compared to non-degenerate samples without nerves and blood vessels. Therefore data presented here suggests that semaphorin3C may have a role in promoting innervation and vascularisation during degeneration, which may go on to cause low back pain.

Study Design: Observational study., Objective: To demonstrate use of magnetic resonance (MR) neurography to visualize the course of the lumbar plexus at the L4-L5 disc space., Summary of Background Data: Risk of injury to the lumbar plexus during lateral transpsoas approach for lumbar interbody fusion (LLIF) is significant. We describe a new technique for preoperative mapping using magnetic resonance neurography to directly visualize the course of the plexus relative to the L4-L5 disc space., Methods: Consecutive lumbar plexus MR neurograms (n=35 patients, 70 sides) were studied. Scans were obtained on a Siemens 3-Tesla Skyra magnetic resonance imaging scanner. T1- and T2-color-coded fusion maps were generated along with 3-dimensional models of the lumbosacral plexus with attention to the L4-L5 interspace. The position of the plexus and the shape of the psoas muscle at the L4-L5 interspace were evaluated and recorded., Results: Direct imaging of the lumbar plexus using MR neurography revealed a substantial variability in the position of the lumbar plexus relative to the L4-L5 disc space. The left-side plexus was identified in zone 2 (5.7%), zone 3 (54.3%), and zone 4 (40%) (P = 0.0014); on the right, zone 2 (8.6%), zone 3 (42.9%) or zone 4 (45.7%), and zone 5 (2.9%) (P = 0.01). Right-left symmetry was found in 18 of 35 subjects (51.4%) (P = 0.865). There was no association between the position of the plexus and the shape of the overlying psoas muscle identified. In patients with an elevated psoas (n = 12), the lumbar plexus was identified in zone 3 in 75% and 66% (left and right) compared with patients without psoas elevation (n = 23), 30.4% and 43.5% (left and right)., Conclusion: The course of the lumbosacral plexus traversing the L4-L5 disc space may be more variable than has been suggested by previous studies. Magnetic resonance neurography may provide a more reliable means of preoperatively identifying the plexus when compared with current methods., Level of Evidence: 3.

Degenerated intervertebral discs (d-IVDs) contribute to low back pain (LBP) and are highly common. While some d-IVDs cause discogenic LBP, others are pain-free. Understanding the differences in pathophysiology between painful and pain-free intervertebral disc degeneration (IDD), especially the pathogenic signaling involved in the regulation of painful d-IVDs, is vital for achieving satisfactory effects in clinical treatment. In this review, we revisit recent findings on the detection of inflammatory factors in d-IVDs and summarize the differences between d-IVDs that are painful and those that are pain-free. We postulate that persistent inflammation and innervation are the key factors distinguishing those that are symptomatic and those that are not. This highlights the necessity to use painful, rather than pain-free, degenerated discs in the mechanistic study of disc degeneration and in the development of regenerative approaches, to avoid false positive/negative outcomes. Based on previous molecular d-IVD studies, we also postulate the signaling events from disc overload/ injury to discogenic pain. Although these proposed events are supported by experimental findings, many details about how they are interconnected are not addressed and therefore require experimental investigation.

Study Design: Immunohistochemical and behavioral study using the SPARC (secreted protein, acidic, rich in cysteine)-null mouse model of low back pain (LBP) associated with accelerated intervertebral disc (IVD) degeneration., Objective: To determine if behavioral signs of LBP in SPARC-null mice are accompanied by sensory nervous system plasticity., Summary of Background Data: IVD pathology is a significant contributor to chronic LBP. In humans and rodents, decreased expression of SPARC is associated with IVD degeneration. We previously reported that SPARC-null mice exhibit age-dependent behavioral signs of chronic axial LBP and radiating leg pain., Methods: SPARC-null and age-matched control young, middle-aged, and old mice (1.5, 6, and 24 mo of age, respectively) were evaluated. Cutaneous hind paw sensitivity to cold, heat, and mechanical stimuli were evaluated as measures of radiating pain. The grip force and tail suspension assays were performed to evaluate axial LBP. Motor impairment was assessed using an accelerating rotarod. IVD innervation was identified by immunohistochemistry targeting the nerve fiber marker PGP9.5 and the sensory neuropeptide calcitonin gene-related peptide (CGRP). Sensory nervous system plasticity was evaluated by quantification of CGRP- and neuropeptide-Y-immunoreactivity (-ir) in dorsal root ganglia neurons and CGRP-ir, GFAP-ir (astrocyte marker), and Iba-1-ir (microglia marker) in the spinal cord., Results: SPARC-null mice developed hypersensitivity to cold, axial discomfort, age-dependent motor impairment, age-dependent increases in sensory innervation in and around the IVDs, age-dependent upregulation of CGRP and neuropeptide-Y in dorsal root ganglia, and age-dependent upregulation of CGRP, microglia, and astrocytes in the spinal cord dorsal horn., Conclusion: Increased innervation of degenerating IVDs by sensory nerve fibers and the neuroplasticity in sensory neurons and spinal cord could contribute to the underlying pathobiology of chronic discogenic LBP., Level of Evidence: N/A.

Study Design: Quantitative and immunohistological analysis of the efficacy of an IκB kinase-β (IKKβ) inhibitor in an injured intervertebral disc (IVD) model., Objective: To elucidate the efficacy of an IKKβ inhibitor on inflammatory cytokine levels in injured IVDs or on neuropeptide levels in the dorsal root ganglia (DRG) neurons innervating injured IVDs in rats., Summary of Background Data: Multiple studies have suggested that upregulation of inflammatory cytokines in damaged IVDs causes discogenic low back pain. The efficacy of blocking individual inflammatory cytokines is limited; however, inflammatory cytokine stimuli often require IKKβ to activate nuclear factor-k B., Methods: Sprague-Dawley rats were divided into 3 groups: sham, saline (disc-injury plus saline), and IKKβ (disc-injury plus anti-IKKβ). To induce injury, IVDs were repeatedly punctured.Experiment 1: Four, 7, and 14 days postinjury, coccygeal (Co) 5/6, Co6/7, and Co7/8 IVDs were resected and tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 levels were quantified by enzyme-linked immunosorbent assay. Experiment 2: The neurotracer Fluoro-Gold was injected into injured L5-L6 IVDs and uninjured sham group IVDs to detect DRG neurons. One week postsurgery, L1-L6 DRGs were immunolabeled with the neuropeptide calcitonin gene-related peptide. The proportions of Fluoro-Gold-labeled calcitonin gene-related peptide-immunoreactive DRG neurons were assessed., Results: Experiment 1: IVD levels of tumor necrosis factor-α (through 2 wk), IL-1β (at 4 d), and IL-6 (at 4 d) were significantly higher in the saline group than in the sham group, and significantly lower in the IKKβ group than in the saline group (P < 0.05). Experiment 2: The percentage of calcitonin gene-related peptide-immunoreactive Fluoro-Gold-labeled DRG neurons was significantly higher in the saline group than in the sham group, and significantly lower in the IKKβ group than in the saline group (P < 0.05)., Conclusion: Injury-induced upregulation of inflammatory cytokines within IVDs and increased levels of neuropeptides within DRG neurons can be suppressed by inhibiting IKKβ., Level of Evidence: N/A.

Study Design: Immunohistological analysis of the cervical dorsal root ganglia (DRG)., Objective: To investigate immunohistologically in rats whether intradiscal administration of anti-nerve growth factor (NGF) antibody in injured cervical intervertebral discs (IVDs) suppresses pain-related peptide expression in DRG neurons., Summary of Background Data: Neck pain can involve the entire neck and become chronic and intractable. Cervical disc degeneration is a primary cause of neck pain, and pain-related mediators, such as NGF, have been correlated with discogenic pain., Methods: We examined Sprague-Dawley rats that received 10 punctures in the C5-C6 IVD, and were treated with saline (puncture group) or an anti-NGF antibody (anti-NGF group). The retrograde neurotracer Fluoro-Gold (FG) was then injected into the C5-C6 IVD. In addition, we examined a sham group that did not receive punctures (disc nonpuncture). The C2-C7 DRG were harvested 1 week after surgery and immunostained for calcitonin gene-related peptide (CGRP), a marker for peptide-containing neurons. We determined for each group the percentages of FG-labeled DRG neurons that were CGRP-immunoreactive (CGRP-ir)., Results: FG-labeled neurons innervating the C5-C6 IVD were found in all C2-C7 DRG examined. The percentage of FG-labeled CGRP-ir DRG neurons in the puncture group was significantly higher than that observed in the sham (P < 0.001) and anti-NGF groups (P < 0.001), but there was no significant difference between the sham and anti-NGF groups (P > 0.05). Therefore, intradiscal administration of anti-NGF antibody suppressed CGRP expression the cervical DRG., Conclusion: Neurons located in the C2-C7 DRG innervated the C5-C6 IVD. These findings indicate that neck pain may be derived from degenerated IVDs. Furthermore, intradiscal administration of anti-NGF antibody suppressed CGRP expression in the cervical DRG innervating the injured IVD. Therefore, inhibiting NGF upregulation in the cervical IVD may be an efficient treatment for discogenic neck pain., Level of Evidence: N/A.

The high intensity zone (HIZ) was first described by Aprill and Bogduk on lumbar spine magnetic resonance imaging (MRI) studies in 1992. Correlation with lumbar computed tomography (CT) discography showed that the HIZ represents a deep radial tear of the annulus fibrosus, which may be a cause of chronic low back pain. Initial studies comparing the finding of a HIZ on MRI with discography suggested that it may be a highly specific marker of a painful lumbar disc, but later investigators demonstrated that it is also present in asymptomatic individuals. The purpose of this article is to review the literature regarding the lumbar HIZ 20 years after its initial description., (Copyright © 2014 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.)

Background Context: Magnetic resonance imaging (MRI) has limited diagnostic value for chronic low back pain because of the unclear relationship between any anatomic abnormalities on MRI and pain reported by the patient. Assessing the innervation of end plate and disc pathologies-and determining the relationship between these pathologies and any abnormalities seen on MRI-could clarify the sources of back pain and help identify abnormalities with enhanced diagnostic value., Purpose: To quantify innervation in the vertebral end plate and intervertebral disc and to relate variation in innervation to the presence of pathologic features observed by histology and conventional MRI., Study Design/setting: A cross-sectional histology and imaging study of vertebral end plates and intervertebral discs harvested from human cadaver spines., Methods: We collected 92 end plates and 46 intervertebral discs from seven cadaver spines (ages 51-67 years). Before dissection, the spines were scanned with MRI to grade for Modic changes and high-intensity zones (HIZ). Standard immunohistochemical techniques were used to localize the general nerve marker protein gene product 9.5. We quantified innervation in the following pathologies: fibrovascular end-plate marrow, fatty end-plate marrow, end-plate defects, and annular tears., Results: Nerves were present in the majority of end plates with fibrovascular marrow, fatty marrow, and defects. Nerve density was significantly higher in fibrovascular end-plate marrow than in normal end-plate marrow (p<.001). Of the end plates with fibrovascular and fatty marrow, less than 40% were Modic on MRI. Innervated marrow pathologies collocated with more than 75% of the end plate defects; hence, innervation was significantly higher in end plate defects than in normal end plates (p<.0001). In the disc, nerves were observed in only 35% of the annular tears; in particular, innervation in radial tears tended to be higher than in normal discs (p=.07). Of the discs with radial tears, less than 13% had HIZ on T2 MRI. Innervation was significantly less in radial tears than in fibrovascular end-plate marrow (p=.05) and end-plate defects (p=.02)., Conclusions: These findings indicate that vertebral end-plate pathologies are more innervated than intervertebral disc pathologies and that many innervated end-plate pathologies are not detectable on MRI. Taken together, these findings suggest that improved visualization of end-plate pathologies could enhance the diagnostic value of MRI for chronic low back pain., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: To test whether the interaction between annulus fibrosus cells (AFCs) and endothelial cells (ECs) disrupts matrix homeostasis and stimulates production of innervation mediators., Methods: Human microvascular ECs were cultured in the conditioned media of AF cell culture derived from degenerated human surgical specimen. Matrix-metalloproteinases (MMPs) and platelet-derived growth factor (PDGF) of ECs of this culture were analyzed by qRT-PCR, Western, and immunofluorescence. Vascular endothelial growth factor (VEGF), Interleukin-8 (IL-8), and nerve growth factor (NGF) in the media of this cell culture were assayed by ELISA. To determine the effects of ECs on AFCs, qRT-PCR was performed to determine mRNA levels of collagen I, II and aggrecan in AFCs cultured in EC conditioned media., Results: Compared to ECs cultured in naïve media, ECs exposed to AFC conditioned media expressed higher mRNA and protein levels of key biomarkers of invasive EC phenotype, MMP-2 (2×), MMP-13 (4×), and PDGF-B (1.5-2×), and NGF (24.9 ± 15.2 pg/mL vs 0 in naïve media). Treatment of AF cells with EC culture conditioned media decreased collagen type II expression two fold. Considerable quantities of pro-angiogenic factors IL-8 (396.7 ± 302.0 pg/mL) and VEGF (756.2 ± 375.9 pg/mL) were also detected in the conditioned media of untreated AF cell culture., Discussion: AFCs from degenerated discs secreted factors which stimulated EC production of factors known to induce matrix degradation, angiogenesis, and innervation. IL-8 and VEGF maybe the secreted factors from AFCs which mediate a pro-angiogenic stimulus often implicated in the development of disc degeneration., (Published by Elsevier Ltd.)

This present study aims to assess in vivo the nervous fibers distribution in the intervertebral disc using diffusion tensor imaging technique. Five healthy subjects participated into the data acquisition. Fiber extraction and tracking algorithms were used. The number of fibers in L4/5 disc ranges from 314 to 679 and the mean fiber length L4/5 in disc ranges from 8.22 ± 2.36 mm to 11.24 ± 5.17 mm. This study showed the feasibility of using diffusion tensor imaging technique to detect and assess the nervous fibers in the intervertebral discs. This could be of great clinical interest for the study of the correlations between these useful characteristics with pain levels on the low back pain patients.

Study Design: Exposure to nucleus pulposus and displacement of intraspinal nervous structures with assessment of spontaneous behavioral changes in rats., Objective: To develop a controlled, experimental model for nerve root injury., Summary of Background Data: There are a number of experimental models presented for studies on radiculopathies. One frequently used model is based on exposure to nucleus pulposus and displacement of the dorsal root ganglion (DRG). However, it is clinically more common that the nerve roots are displaced/compressed than the DRG. In this study, we developed a model for displacement of the nerve root by modifying the DRG model., Methods: After removing the left L3-L4 facet joint, the underlying disc was punctured, and the L4 nerve root was displaced laterally by an injection needle (n = 10). In sham experiments, the same procedure was performed without disc puncture and displacement (n = 10). In 10 rats, the left L4-L5 facet joint was removed. The underlying disc was punctured and the L4 DRG was displaced medially by an injection needle. Assessment of spontaneous behavioral changes was performed on days 1, 3, 7, 14, and 21, postsurgery., Results: There was a clear increase in duration of the behavior "unloading of the paw" after displacement of the DRG that was most pronounced on day 1 and then gradually declined. There was a similar pattern for this behavior induced by nerve root displacement, although the duration was higher than that for the DRG displacement. No apparent trends in behavioral changes were observed for the other behaviors studied., Conclusion: Displacement of the nerve root induced more changes in the pain behavior than displacement of the DRG, but only for the behavior unloading of the paw. Because nerve root injury is more common than DRG injury, this model may be more clinically relevant than the DRG model., Level of Evidence: N/A.

The belief that an intervertebral disc must degenerate before it can herniate has clinical and medicolegal significance, but lacks scientific validity. We hypothesised that tissue changes in herniated discs differ from those in discs that degenerate without herniation. Tissues were obtained at surgery from 21 herniated discs and 11 non-herniated discs of similar degeneration as assessed by the Pfirrmann grade. Thin sections were graded histologically, and certain features were quantified using immunofluorescence combined with confocal microscopy and image analysis. Herniated and degenerated tissues were compared separately for each tissue type: nucleus, inner annulus and outer annulus. Herniated tissues showed significantly greater proteoglycan loss (outer annulus), neovascularisation (annulus), innervation (annulus), cellularity/inflammation (annulus) and expression of matrix-degrading enzymes (inner annulus) than degenerated discs. No significant differences were seen in the nucleus tissue from herniated and degenerated discs. Degenerative changes start in the nucleus, so it seems unlikely that advanced degeneration caused herniation in 21 of these 32 discs. On the contrary, specific changes in the annulus can be interpreted as the consequences of herniation, when disruption allows local swelling, proteoglycan loss, and the ingrowth of blood vessels, nerves and inflammatory cells. In conclusion, it should not be assumed that degenerative changes always precede disc herniation. Cite this article: Bone Joint J 2013;95-B:1127-33.

Background: Our purpose was to examine platelet-rich plasma (PRP) for its effect on bone formation and to follow the immunohistochemical changes in calcitonin gene-related peptide (CGRP) in dorsal root ganglion neurons innervating the discs as a possible index of nociceptive nerve transmission in a rat model., Methods: A total of seventy rats were used. Ten constituted a non-punctured disc sham group, while another ten rats constituted a group that underwent puncture of the L4-L5 discs. Forty rats were in experimental groups in which the L4-L5 discs were punctured; posterolateral lumbar arthrodesis was performed with PRP (the PRP group) or without the use of PRP (the normal arthrodesis group), with twenty rats in each group. The remaining ten rats were used as blood donors. Four and eight weeks after surgery, microcomputed tomography examinations were done to evaluate the amount of bone and the L4-L5 spines were harvested to evaluate bone union, followed by resection of dorsal root ganglion neurons. The percentages of Fluoro-Gold-labeled and CGRP-immunoreactive neurons were calculated., Results: The platelet count and the concentration of growth factors in PRP were higher than those in blood (p &lt; 0.05). The bone volumes observed in the PRP group were significantly greater than those of the normal arthrodesis group at four and eight weeks (p &lt; 0.05). Three (30%; 95% confidence interval [CI], 6% to 65%) of ten rats in the normal arthrodesis group and eight (80%; 95% CI, 44% to 98%) of ten rats in the PRP group were considered to have bone fusion four weeks after surgery (p &lt; 0.05). At eight weeks, seven (70%; 95% CI, 34% to 94%) of ten rats in the normal arthrodesis group and nine (90%; 95% CI, 55% to 99%) of ten rats in the PRP group were considered to have bone fusion (p = 0.27). The proportion of CGRP-immunoreactive neurons was significantly greater in the punctured group than in the other groups. There were no significant differences between the normal arthrodesis group and the PRP group., Conclusions: PRP appears to promote bone formation in rats and has a tendency to shorten the period of bone union in this rat model of posterolateral lumbar arthrodesis, but it did not influence the proportion of CGRP-immunoreactive neurons, a likely indicator of inflammatory pain originating from the degenerative intervertebral disc., Clinical Relevance: The ability of PRP in this model suggests that it may be able to shorten the period of union and lead to an early return to social activities after treatment.

Until very recently, intervertebral disc innervation was a subject of considerable debate. Nowadays, the introduction of inmunohistochemical techniques associated to specific antibodies and studies with retrograde tracers in nerves have allowed greater understanding of disc innervation in physiological and pathological conditions and also endings characteristics and their patterns of distribution in both situations. The existing controversies regarding structural basis of discogenic pain, have raised the interest of knowing the influence of innervation in back pain from discal origin and its characteristics. Today, we know that pathologic neoinnervation accompanying radial fissures is an important factor in the genesis of discogenic pain; within a complex mechanism in which other neurobiomechemical, inflammatory and biomechanical factors are involved., (Copyright © 2012 Sociedad Española de Neurocirugía. Published by Elsevier España. All rights reserved.)

Paraspinal muscle forces were derived computationally based on the hypothesis that the intervertebral disc has a transducer function and the muscle is activated according to a sensor-driving control mechanism. A three-dimensional finite element model of the musculoskeletal system, which consisted of a detailed whole lumbar spine, pelvis, simplified trunk model, and muscles, was developed and combined with an optimization technique to calculate muscle forces in isometric forward flexed and erect standing postures. Minimization of deviations in the nucleus pressure and averaged tensile stress in the annulus fibers at five discs was used for muscle force calculations. The results indicated that all the muscles were properly activated to maintain posture and stabilize the lumbar spine. The nucleus pressure difference was decreased during the iterative calculations and its resulting value at the L4/L5 level was consistent with in vivo measurements. Muscle activation produced vertebra motion, which resulted in a relatively uniform stress distribution in the intervertebral discs. This can minimize the risk of injury at a specific level and increase the ability of the spine to sustain a load.

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 L6, 6 intervertebral disc of 3 groups; after 7 days of F-G injection, complete Freund's adjuvant (50 microL) 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 < 0.05) at 3 days, and no significant difference was found at the other time points (P > 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 > 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 > 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 < 0.05), but no significant difference was found in the expression of CGRP between 2 groups (P > 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 < 0.05), and no significant difference was found at the other time points (P > 0.05)., Conclusion: Intradiscal inflammatory environment may induce the expression of GAP-43, and potentially promote the nerve fiber ingrowth of rat.

Study Design: Mechanical and biochemical analyses of cadaveric and surgically removed discs., Objective: To test the hypothesis that fissures in the annulus of degenerated human discs are mechanically and chemically conducive to the ingrowth of nerves and blood vessels., Summary of Background Data: Discogenic back pain is closely associated with fissures in the annulus fibrosus, and with the ingrowth of nerves and blood vessels., Methods: Three complementary studies were performed. First, 15 cadaveric discs that contained a major annulus fissure were subjected to 1 kN compression, while a miniature pressure transducer was pulled through the disc to obtain distributions of matrix compressive stress perpendicular to the fissure axis. Second, Safranin O staining was used to evaluate focal loss of proteoglycans from within annulus fissures in 25 surgically removed disc samples. Third, in 21 cadaveric discs, proteoglycans (sulfated glycosaminoglycans [sGAGs]) and water concentration were measured biochemically in disrupted regions of annulus containing 1 or more fissures, and in adjacent intact regions., Results: Reductions in compressive stress within annulus fissures averaged 36% to 46%, and could have been greater at the fissure axis. Stress reductions were greater in degenerated discs, and were inversely related to nucleus pressure (R(2) = 47%; P = 0.005). Safranin O stain intensity indicated that proteoglycan concentration was typically reduced by 40% at a distance of 600 μm from the fissure axis, and the width of the proteoglycan-depleted zone increased with age (P < 0.006; R(2) = 0.29) and with general proteoglycan loss (P < 0.001; R(2) = 0.32). Disrupted regions of annulus contained 36% to 54% less proteoglycans than adjacent intact regions from the same discs, although water content was reduced only slightly., Conclusion: Annulus fissures provide a low-pressure microenvironment that allows focal proteoglycan loss, leaving a matrix that is conducive to nerve and blood vessel ingrowth.

Study Design: Animal model of intravertebral disc (IVD) degeneration., Objective: To examine production of inflammatory mediators in IVDs and neuropeptides in dorsal root ganglia (DRGs) in rat models of IVD compression and injury., Summary of Background Data: Sensory nerve fibers in IVDs and inflammatory mediator responses have been verified in animal models of IVD injury. However, the IVD injury in animals incompletely models degenerated human IVDs causing discogenic low back pain, because human IVDs are also subject to compression., Methods: Experimental groups (controls, IVD injury, IVD compression, and their combination) of Sprague Dawley rats were prepared. Fluoro-Gold (FG; Fluorochrome, Denver, CO) was applied into coccygeal IVDs. Inflammatory mediators in IVDs, including nerve growth factor, tumor necrosis factor α, interleukin 1β, and interleukin 6, were quantified using enzyme-linked immunosorbent assays. DRGs were immunostained for calcitonin gene-related peptide, activating transcription factor 3, and growth-associated phosphoprotein 43., Results: The upregulation of inflammatory mediators was transient in the IVD injury group but delayed and long-lasting in the IVD compression group. When the IVD injury and compression were combined, the upregulation of inflammatory mediators was long-lasting through 8 weeks. The proportion of calcitonin gene-related peptide-immunoreactive neurons among Fluoro-Gold-labeled neurons remained significantly higher in the IVD injury, compression, and combination groups than in the controls. In contrast, increases in the proportions of activating transcription factor 3-immunoreactive or growth-associated phosphoprotein 43-immunoreactive neurons in the IVD injury group animals were transient but long-lasting in the compression and combination groups compared with controls., Conclusion: Disc injury in rats produces persistent increases in neuropeptides in DRGs but only transient increases in inflammatory mediators in IVDs. On the contrary, disc compression in rats produces a long-lasting increase in inflammatory mediators in IVDs and neuropeptides in DRGs. Moreover, disc compression induces persistent nerve injury and regeneration of the afferent fibers innervating IVDs.

Study Design: A prospective interventional trial, using a rat model of lumbar interbody fusion., Objective: To examine the potential efficacy of platelet-rich plasma (PRP) for lumbar interbody fusion, using hydroxyapatite (HA)., Summary of Background Data: PRP is an autologous product containing a high concentration of platelets in a small volume of plasma and has osteoinductive effects. HA has osteoconductive ability and has been used in combination with autogenous bone for spine fusion. However, reports using PRP with HA for spine fusion are very few. The purpose of this study was to examine the efficacy of PRP with HA for spinal interbody fusion and at the same time to estimate the change in immunoreactivity of the inflammatory neuropeptide, calcitonin gene-related peptide (CGRP), in dorsal root ganglion (DRG) neurons innervating spinal discs., Methods: A total of 35 Sprague-Dawley rats were used in this study. Twenty-one rats were used for conducting interbody fusion experiments, 7 rats were used as immunostaining controls, and 7 other rats were used as blood donors for making PRP. L5-L6 interbody fusion was performed on 21 rats using HA + PRP (n = 7), HA + platelet-poor plasma (n = 7), or HA + saline (n = 7). Simultaneously, Fluoro-Gold neurotracer was applied to the intervertebral space to detect DRG neurons innervating the discs. L5-L6 lumbar radiographs were obtained and lumbar DRGs were immunostained for CGRP. The rate of bone union and the change in CGRP immunoreactive DRG neurons innervating the discs were evaluated and compared among groups., Results: All L5-L6 lumbar discs were fused in the PRP + HA group (fused 7/total 7), whereas only 1 case was fused in the platelet-poor plasma group (1 of 7) and no cases in the HA-only group (0 of 7), which was a significant difference. Upon immunohistochemical analysis, CGRP-positive neurons innervated L5-L6 intervertebral discs in nonunion cases, and these were significantly increased compared with those in union cases., Conclusion: Our study suggests that using PRP with HA was beneficial for spine fusion. This combination may promote bone union and also decrease inflammatory neuropeptide in sensory neurons innervating the discs.

Study Design: An immunohistological analysis of the cervical intervertebral disc (IVD)., Objective: To investigate sensory and autonomic innervation of the rat cervical IVD., Summary of Background Data: Many clinicians are challenged with treating wide-ranging chronic neck pain. Several authors have reported that sympathetic nerves participate in chronic pain, and various sympathectomy procedures can effectively treat chronic pain., Methods: The neuro-tracer Fluoro-gold (FG) was applied to the anterior surfaces of C5-C6 IVDs from 10 Sprague-Dawley rats to label the neurons of the innervating dorsal root ganglion (DRG), stellate ganglion (SG; sympathetic ganglion), and nodose ganglion (NG; parasympathetic ganglion). Seven days postsurgery, DRGs from level C1-C8, SG, and NG neurons were harvested, sectioned, and immunostained for calcitonin gene-related peptide (CGRP; a marker for peptide-containing neurons) and isolectin B4 (IB4; a marker for nonpeptide-containing neurons). The proportion of FG-labeled DRG neurons that were CGRP-immunoreactive (CGRP-IR), IB4-binding, and non-CGRP-IR and IB4-binding, and the proportion of FG-labeled SG neurons and NG neurons were calculated., Results: FG-labeled neurons innervating the C5-C6 IVD were distributed throughout the C2-C8 DRGs. The proportions of FG-labeled DRG neurons that were CGRP-IR, IB4-binding, non-CGRP-IR and IB4-binding, as well as SG neurons, and NG neurons were 20.6%, 3.3%, 55.7%, 8.9%, and 11.5%, respectively. The proportion of CGRP-IR FG-labeled DRG neurons was significantly higher than the proportion of IB4-binding FG-labeled DRG neurons at each level (P < 0.05)., Conclusion: The C5-C6 IVD was innervated multisegmentally from neurons of the C2-C8 DRG, SG, and NG. Overall, 79.6% of the nerve fibers innervating the IVD were sensory nerves and 20.4% were autonomic nerves. Furthermore, 23.9% of the nerve fibers innervating the IVD were afferent sensory pain-related nerves, 8.9% were efferent sympathetic nerves, and 11.5% were efferent parasympathetic nerves. These findings may explain the wide-ranging and chronic discogenic pain that occurs via the somatosensory and autonomic nervous system.

Background: The personal and societal impact of chronic low back pain is considerable. The intervertebral disc is considered the etiologic source in up to 40% of patients, with considerable previous efforts directed at developing reliable and efficacious treatments. Recent publications, including a double-blind, randomized, placebo-controlled trial, using a one-time treatment of methylene blue, showed statistically significant, clinically relevant improvements in pain and function in the treatment groups. The postulated mechanism of action of methylene blue is denervation of small nociceptive fibers that grow into the annulus fibrosis, which are implicated in discogenic pain., Study Design: Retrospective case series., Setting: Academic pain management center., Objectives: To examine the outcomes for a cohort of patients treated with methylene blue for discogenic pain, discuss potential differences in selection and administration protocols and briefly review other proposed treatments for discogenic pain (e.g. intradiscal electrothermy therapy, intradiscal steroids, intradiscal biaccuplasty, rami communicans radiofrequency thermocoagulation, and chymopapain)., Methods: This case series examines the pain and functional outcomes in 8 patients treated with a one-time administration of methylene blue for discogenic back pain. Follow-up information was available between 2 months and over one year, depending on the patient., Results: Application of this treatment for these 8 patients for discogenic pain diagnosed by provocation discography showed only one clinical success at our center. Four patients had a time-limited clinical response in pain and/or function between 2 weeks and 5 months. Patient specific data are outlined in detail herein., Conclusions: Low back pain ascribed to a discogenic source continues to be an elusive clinical entity to treat. We have reserved further treatment of methylene blue for discogenic pain until other controlled trials have been published., Limitations: A case series.

Study Design: Immunohistological analysis of dichotomizing sensory nerve fibers projecting to the lumbar multifidus muscles and intervertebral disc (IVD), facet joint (FJ), or sacroiliac joint (SIJ) in rats., Objective: To elucidate dichotomizing sensory nerve fibers projecting to the lumbar multifidus muscles and to IVDs, FJs, or SIJs., Summary of Background Data: Clinically, the origin of low back pain remains unknown. Multiple studies have identified lumbar muscles, IVDs, FJs, and SIJs as sources of low back pain. Pain may originate directly from lumbar muscles or be referred from the spine, or both. Dorsal root ganglion (DRG) neurons with dichotomizing axons have been reported in several species and are thought to be related to referred pain., Methods: We used 2 neurotracers, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) and fluorogold (FG), in this double-labeling study involving 30 Sprague Dawley rats. DiI was applied to lumbar multifidus muscles in all rats. Simultaneously, FG was applied to the anterior left portion of L5-L6 IVDs in the IVD group (n = 10), to the left L5-L6 FJs in the FJ group (n = 10), and to the left SIJs in the SIJ group (n = 10). Fourteen days after surgery, left DRGs from L1 to L6 were harvested, sectioned, and observed under a fluorescence microscope., Results: We verified the existence of double-labeled DRG neurons (i.e., dichotomizing sensory nerve fibers) projecting to lumbar multifidus muscles and to IVDs, FJs, or SIJs, depending on the group. The proportion of double-labeled cells in all DiI-labeled DRG neurons was higher in the FJ group (6.8%) and SIJ group (7.1%) than in the IVD group (3.1%) (P < 0.05)., Conclusion: Our results document the presence of dichotomizing sensory nerve fibers projecting to lumbar multifidus muscles and to IVDs, FJs, and SIJs. Referred low back muscle pain may reflect disorders of lumbar posterior structures, such as FJs and SIJs, rather than disorders of lumbar anterior structures, such as IVDs.

Study Design: Retrograde neurotracing and immunohistochemical investigation of brain-derived neurotrophic factor (BDNF)-related sensory innervation of punctured lumbar intervertebral discs (IVDs) in rat dorsal root ganglia (DRG)., Objective: To investigate the association between BDNF and sensory innervation of multiple-punctured lumbar IVD in rats., Summary of Background Data: BDNF--a neurotrophin in DRG neurons--is anterogradely transported to the spinal cord and transmits pain signals. Its presence in the peripheral sites of degenerative IVDs has been recently reported, although its association with discogenic pain remains unclear., Methods: Forty female Sprague-Dawley rats were equally divided into four groups: naïve, sham, and two agent-treated groups (vehicle [saline-treated] group and anti-BDNF [anti-BDNF antibody] group). L5-L6 IVDs of the agent-treated rats were exposed and injured by repeated punctures. The retrograde neurotracer Fluoro-Gold (FG) and treatment agents were intradiscally applied. In the sham group, FG alone was applied onto uninjured IVD. One week later, L1-L3 DRGs were harvested and immunolabeled for the inflammatory pain-related calcitonin gene-related peptide (CGRP), that is, the pain marker. The proportions of FG-labeled CGRP-immunoreactive (-ir) DRG neurons were assessed. Each L5-L6 IVD was resected for measuring the BDNF concentration using enzyme-linked immunosorbent assay., Results: FG-labeled DRG neurons were almost equally prevalent at each DRG level. The proportions of FG-labeled CGRP-ir DRG neurons in the two agent-treated groups were significantly elevated (average 37.9% ± 7.2% and 25.4% ± 9.1%; vehicle and anti-BDNF groups, respectively; P < 0.05) in comparison with the naïve and sham groups (19.6% ± 1.3% and 19.2% ± 3.6%, respectively) and were significantly decreased in the anti-BDNF group in comparison with the vehicle group (P < 0.05). BDNF concentrations were elevated maximally in the vehicle group (18.5 ± 5.2 pg/g) but suppressed in the anti-BDNF group (14.0 ± 3.0 pg/g)., Conclusion: Direct intradiscal application of the anti-BDNF antibody significantly suppressed both CGRP production and the local concentration of BDNF. Our results indicate a possible association between the local production of BDNF and the pathophysiology of discogenic pain.

Although understanding of the biologic basis of intervertebral disk (IVD) degeneration is rapidly advancing, the unique IVD environment presents challenges to the development and delivery of biologic treatments. Acceleration of cellular senescence and apoptosis in degenerative IVDs and the depletion of matrix proteins have prompted the development of treatments based on replacing IVD cells using various cell sources. However, this strategy has not been tested in animal models. IVD degeneration and associated pain have led to interest in pathologic innervation of the IVD and ultimately to the development of percutaneous devices to ablate afferent nerve endings in the posterior annulus. Degeneration leads to changes in the expression of matrix protein, cytokines, and proteinases. Injection of growth factors and mitogens may help overcome these degenerative changes in IVD phenotype, and these potential treatments are being explored in animal studies. Gene therapy is an elegant method to address changes in protein expression, but efforts to apply this technology to IVD degeneration are still at early stages.

Object: Although infrequent, injury to adjacent neurovascular structures during posterior approaches to lumbar intervertebral discs can occur. A detailed anatomical knowledge of relationships may decrease surgical complications., Methods: Ten formalin-fixed male cadavers were used for this study. Posterior exposure of the lumbar thecal sac, nerve roots, pedicles, and intervertebral discs was performed. To identify retroperitoneal structures at risk during posterior lumbar discectomy, a transabdominal retroperitoneal approach was performed, and observations were made. The distances between the posterior and anterior edges of the lumbar intervertebral discs were measured, and the relationships between the disc space, pedicle, and nerve root were evaluated., Results: For right and left sides, the mean distance from the inferior pedicle to the disc gradually increased from L1-2 to L4-5 (range 2.7-3.8 mm and 2.9-4.5 mm for right and left side, respectively) and slightly decreased at L5-S1. For right and left sides, the mean distance from the superior pedicle to the disc was more or less the same for all disc spaces (range 9.3-11.6 mm and 8.2-10.5 mm for right and left, respectively). The right and left mean disc-to-root distance for the L3-4 to L5-S1 levels ranged from 8.3 to 22.1 mm and 7.2 to 20.6 mm, respectively. The root origin gradually increased from L-1 to L-5. The right and left nerve root-to-disc angle gradually decreased from L-3 to S-1 (range 105°-110.6° and 99°-108°). Disc heights gradually increased from L1-2 to L5-S1 (range 11.3-17.4 mm). The mean distance between the anterior and posterior borders of the intervertebral discs ranged from 39 to 46 mm for all levels., Conclusions: To avoid neighboring neurovascular structures, instrumentation should not be inserted into the lumbar disc spaces more than 3 cm from their posterior edge. Accurate anatomical knowledge of the relationships of intervertebral discs to nerve roots is needed for spine surgeons.

Study Design: retrograde neurotracing and immunohistochemistry were used to investigate the effect of the tumor necrosis factor (TNF)-α inhibitor, etanercept, on calcitonin gene-related peptide (CGRP) expression in dorsal root ganglion (DRG) neurons innervating intervertebral discs in rats., Objective: to clarify the action of a TNF-α inhibitor on a sensory neuropeptide in DRG neurons innervating intervertebral discs., Summary of Background Data: degeneration of lumbar intervertebral discs is a cause of low back pain. TNF-α in the intervertebral disc is a major contributor to discogenie pain. Effects of TNF-α inhibition on CGRP expression in DRG neurons were evaluated., Methods: the neurotracer FluoroGold was applied to the surfaces of L4/5 discs to label their innervating DRG neurons (n = 30). Of 30 rats, 10 were in a nonpunctured disc sham surgery control group, whereas the other 20 were in experimental groups in which intervertebral discs were punctured with a 23-gauge needle. Etanercept or saline was applied into the punctured discs (n = 10 each treatment). After 14 days of surgery, DRGs from L1 to L6 were harvested, sectioned, and immunostained for CGRP. The proportion of FluoroGold-labeled CGRP-immunoreactive DRG neurons was evaluated in all groups., Results: FluoroGold-labeled neurons innervating the L4/5 disc were distributed throughout L1-L6 DRGs in all groups. Of the FluoroGold-labeled neurons, the proportion of CGRP-immunoreactive neurons was 21% ± 4% in the sham surgery control group, 32% ± 7% in the puncture + saline group, and 23% ± 4% in the puncture + etanercept group. The proportion of CGRP-immunoreactive neurons was significantly greater in the puncture + saline group compared with the sham control and puncture + etanercept groups (P < 0.01)., Conclusion: in this model, CGRP was upregulated in DRG neurons innervating damaged discs. However, direct intradiscal application of etanercept immediately after disc puncture suppressed CGRP expression in DRG neurons innervating injured discs. This finding may further elucidate the mechanism for the effectiveness of etanercept in upregulation of neuropeptide in DRG neurons innervating intervertebral discs.

Nerve growth factor (NGF) and its dual structurally unrelated receptors, tropomyosin-related kinase A (TrkA) or p75 neurotrophin receptor (p75(NTR)), cause the pathogenesis of discogenic pain. To investigate the sensory innervation of injured rat lumbar intervertebral disc (IVD), we examined the expression of neuropeptides such as calcitonin gene-related peptide (CGRP) at dorsal root ganglia (DRG) by inhibiting NGF or its dual receptors. Sprague-Dawley rats with multiply punctured L5-L6 IVD were used. Six experimental groups were prepared: naïve, sham control, and four agent-treated groups with punctured IVD (vehicle, anti-NGF antibody, anti-TrkA antibody, and anti-p75(NTR) antibody). Retrograde neurotracer Fluoro-Gold (FG) was applied together except for the naïve group. Their lumbar DRG were harvested and immunolabeled for CGRP. FG-labeled DRG neurons were most prevalent at L1 and L2 DRG, and the proportion of FG-labeled CGRP-immunoreactive DRG neurons in the vehicle group was significantly elevated (p < 0.05) compared with the sham group, while those of antibody-treated groups, especially in the anti-p75(NTR) group, significantly decreased compared with the vehicle group (p < 0.05). Direct intradiscal application of antibody to NGF or its receptors suppressed CGRP expression, and p75(NTR) antagonism induced the most profound suppression., (© 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Background: Referred pain due to lumbar disc disorders can be analyzed using the stereoscopic structure of the peripheral sensory nervous system. The rostrocaudal structure has been clarified. The dorsoventral structure of the lumbar spine would be useful for mapping areas of pain perception in spinal disorders., Methods: The neurotracer 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) was applied to the lateral portion of the L5/6 intervertebral disc in rats to examine the dorsoventral organization of the sensory nervous system in the lumbar spine and related tissues. Fluorogold (FG) was applied to reference sites located at the spinous process of the L5 vertebra, the L5/6 facet joint, the psoas muscle at the L5 level, or the rectus abdominis muscle at the pubic symphysis. FG was also applied to the lateral portion of the disc (DiI application site) at L5 or at the L5 level as controls for the double labeling. Labeled neurons were counted in dorsal root ganglia (DRGs) from L1 through L4., Results: The proportion of neurons double-labeled with DiI and FG in the total number of DiI-labeled and FG-labeled neurons was 32.9% in the control group; 0% in the spinous process, 0.6% in the facet joint, 2.3% in the psoas muscle, and 0.1% in the rectus abdominis muscle. DRG neurons with dichotomizing afferent fibers were most prevalent (2.3%) between the lateral disc and the psoas muscle at the groin; they were rare or absent between the disc and other reference sites., Conclusions: Dorsoventral organization of the primary sensory system in the lumbar body trunk was suggested from the proportion of DRG neurons with dichotomizing afferent fibers innervating the lumbar disc and other tissues. The present findings provide a pathomechanism of groin referred pain in lumbar disc disorders.

The normal intervertebral disc (IVD) is a poorly innervated organ supplied only by sensory (mainly nociceptive) and postganglionic sympathetic (vasomotor efferents) nerve fibers. Interestingly, upon degeneration, the IVD becomes densely innervated even in regions that in normal conditions lack innervation. This increased innervation has been associated with pain of IVD origin. The mechanisms responsible for nerve growth and hyperinnervation of pathological IVDs have not been fully elucidated. Among the molecules that are presumably involved in this process are some members of the family of neurotrophins (NTs), which are known to have both neurotrophic and neurotropic properties and regulate the density and distribution of nerve fibers in peripheral tissues. NTs and their receptors are expressed in healthy IVDs but much higher levels have been observed in pathological IVDs, thus suggesting a correlation between levels of expression of NTs and density of innervation in IVDs. In addition, NTs also play a role in inflammatory responses and pain transmission by increasing the expression of pain-related peptides and modulating synapses of nociceptive neurons at the spinal cord. This article reviews current knowledge about the innervation of IVDs, NTs and NT receptors, expression of NTs and their receptors in IVDs as well as in the sensory neurons innervating the IVDs, the proinflammatory role of NTs, NTs as nociception regulators, and the potential network of discogenic pain involving NTs.

Study Design: We examined the sensory innervation of the coccygeal (Co) 5/6 intervertebral disc in rats using a retrograde neurotracing method and immunohistochemistry., Objective: To investigate the properties of the sensory innervation of the rat coccygeal disc., Summary of Background Data: Developing a rat disease model for degenerative intervertebral disc compression using lumbar discs is technically impractical because of their location. Coccygeal intervertebral discs are more readily accessible and several reports of morphologic evaluation of degenerative coccygeal intervertebral discs using compression devices exist. However, their sensory innervation and properties have not yet been characterized., Methods: FluoroGold neurotracer was applied to the Co5/6 intervertebral discs of intraperitoneally anesthetized Sprague Dawley rats (n = 10). Subsequently, the discs and the L1-S4 dorsal root ganglions (DRGs) were resected and sectioned. The discs were double-stained for immunoreactivity to the neuronal marker beta-tubulin (Tuj-1) and biotin-labeled isolectinB4 (IB4), a neuropathic pain marker, or Tuj-1 and calcitonin gene-related peptide (CGRP), an inflammatory pain marker. The DRGs were double-stained for IB4-binding and CGRP immunoreactivity (IR). The proportions of IB4-binding or CGRP-IR DRG neurons were assessed by cell counting and compared., Results: The disc immunohistochemistry showed evidence of sensory nerve fibers lying in the outermost layer of the anulus fibrosus. FluoroGold labeled DRG neurons mainly derived from S1 to S3 DRGs, especially S2 and S3. No labeled neurons were observed in the S4 DRG. The histochemistry of the DRGs showed a predominance of CGRP-IR DRG neurons (3.5 +/- 1.7% IB4-binding and 15.4 +/- 5.6% CGRP-IR on average)., Conclusion: This study showed evidence for nerve fibers in the discs and predominant innervation by CGRP-IR DRG neurons. The neurons innervating the discs mostly derived from S1 to S3 DRGs, especially S2 and S3. These findings may be useful in developing rat models of disease involving degenerative intervertebral disc compression.

Study Design: A prospective immunohistological study in an animal model., Objective: To identify and describe the phenotype of neoinnervation in experimental anular tears., Summary of Background Data: Controversy surrounds neoinnervation of degenerate discs which has been proposed as the anatomic basis for discogenic pain. Ablation of neoinnervation has been postulated as the theoretical basis for the claimed successes of procedures such as intradiscal electrotherapy. The animal model of disc degeneration previously developed in our research center provides an opportunity to investigate the innervation of anular tears in an extensively characterized lesion., Methods: A surgical anular tear was created in 5 lumbar discs in 11 sheep which were killed at 1, 2, 3, and 12 months. Each spine was x-rayed and divided into motion segments for histologic analysis. Serial sections through the tear were immunostained for protein gene product 9.5, tyrosine hydroxylase, and calcitonin gene receptor protein., Results: Neoinnervation of the periphery of the anular tear was observed. Ingrowing nerves penetrated marginally deeper than the normal anular innervation but no nerves were identified in the inner anulus or nucleus. A minority of the new axons were calcitonin gene receptor protein or tyrosine hydroxylase positive., Conclusion: The anulus tears in this model are innervated only peripherally to a depth only marginally greater than that of the normal anulus.

Degeneration of lumbar intervertebral discs is thought to be a cause of low back pain. Studies have found that a cause of discogenic low back pain is intervertebral disc inflammation and axonal growth of afferent fibers innervating the disc. Lumbar spine fusion for chronic discogenic low back pain is considered an effective procedure. However, no study has investigated the mechanism of pain relief. We did this by applying Fluoro-Gold (FG) to the ventral aspect of the L4-L5 intervertebral discs of 40 rats. We exposed the nucleus pulposus to the annulus fibrosus in a disc punctured model. Rats were divided into 4 groups. Group A: Punctured intervertebral disc with sham posterolateral fusion (PLF) (n = 10), Group B: Punctured intervertebral disc with PLF (n = 15), Group C: Normal intervertebral disc (no puncture) with PLF (n = 10), and Group D: Normal disc (no disc puncture) with sham PLF (n = 5). Four weeks after surgery, bilateral L1-L5 dorsal root ganglia (DRGs) were stained with growth-associated protein 43 (GAP43), a marker of axonal growth, and calcitonin gene-related peptide (CGRP), a neuropeptide marker of pain. Bone union was evaluated using X-ray imaging. Of the FG-labeled neurons, the proportions of GAP43- and CGRP-immunoreactive (IR) neurons in Group A were significantly higher than in Group D (P < 0.05). The proportions of GAP43- and CGRP-IR neurons in bone union rats in Group B were significantly lower than in nonunion rats in Group B and in the rats in Group A (P < 0.05). No significant differences in GAP43- and CGRP-IR neurons were observed between bone union and nonunion rats in Group C and the rats in Group D (P > 0.05). PLF is strongly related to the downregulation of GAP43 and CGRP expression. Therefore, PLF may suppress the increase of inflammatory neuropeptides and the process of axonal growth. Moreover, these results may explain, in part, the mechanism of pain relief following lumbar spinal fusion for chronic discogenic low back pain in humans.

Cervicogenic headache (CeH) is a well-recognized headache syndrome, distinguishable from other primary and secondary headaches. Although in some cases a cervical lesion may be detected in connection with the headache, many CeH patients have no demonstrable lesion. Besides, most of the frequent cervical diseases, such as spondylosis and disc herniations, do not present with headache of the cervicogenic type. This suggests that the neck is not an independent headache generator. CeH may depend in addition on a central predisposition counterpart, leading to the activation of the trigeminovascular system and pain generation.