Innervation of the Human Intervertebral Disc: A Scoping Review

Adam M R Groh; Dale E Fournier; Michele C Battié; Cheryle A Séguin

doi:10.1093/pm/pnab070

. 2021 Feb 17;22(6):1281–1304. doi: 10.1093/pm/pnab070

Innervation of the Human Intervertebral Disc: A Scoping Review

Adam M R Groh ¹, Dale E Fournier ^2,³, Michele C Battié ^3,^4,², Cheryle A Séguin ^3,^5,^✉,²

PMCID: PMC8185559 PMID: 33595648

Abstract

Objective

Back pain is an elusive symptom complicated by a variety of possible causes, precipitating and maintaining factors, and consequences. Notably, the underlying pathology remains unknown in a significant number of cases. Changes to the intervertebral disc (IVD) have been associated with back pain, leading many to postulate that the IVD may be a direct source of pain, typically referred to as discogenic back pain. Yet despite decades of research into the neuroanatomy of the IVD, there is a lack of consensus in the literature as to the distribution and function of neural elements within the tissue. The current scoping review provides a comprehensive systematic overview of studies that document the topography, morphology, and immunoreactivity of neural elements within the IVD in humans.

Method

Articles were retrieved from six separate databases in a three-step systematic search and were independently evaluated by two reviewers.

Results

Three categories of neural elements were described within the IVD: perivascular nerves, sensory nerves independent of blood vessels, and mechanoreceptors. Nerves were consistently localized within the outer layers of the annulus fibrosus. Neural ingrowth into the inner annulus fibrosus and nucleus pulposus was found to occur only in degenerative and disease states.

Conclusion

While the pattern of innervation within the IVD is clear, the specific topographic arrangement and function of neural elements in the context of back pain remains unclear.

Keywords: Back Pain, Intervertebral Disc Degeneration, Peripheral Nervous System, Annulus Fibrosus, Nucleus Pulposus, Neuroanatomy

Introduction

Rationale

Back pain has a lifetime prevalence of 60–80% in the general population [1] and is the leading cause of years lived with disability worldwide [2]. In the United States, recent estimates of the total medical costs associated with the management of back pain range between $30 and $50 billion per annum [3]. Despite the societal burden of back pain and the demands it places on the healthcare system, a conclusive diagnosis of its underlying cause remains a challenge for clinicians. In fact, more than 85% of patients in the United States experience back pain for which an exact biological cause cannot be reliably identified [4–6].

In a minority of cases, back pain can be directly linked to clinical causes, including compression of adjacent nerve roots, traumatic injury to the spine, visceral conditions, infection, and inflammation of spinal tissues [7, 8]. Some of these causes of back pain may be reliably diagnosed and treated, such as anatomical deformities, tumors, infection-induced abscesses, and fractures of the spine that compress nerves or damage surrounding soft tissues [8]. However, back pain is also associated with degenerative changes to the intervertebral disc (IVD), which has led some to postulate that the disc itself may be a source of pain, termed discogenic back pain [2, 7–10].

The IVD is a fibrocartilaginous structure composed of three distinct components. The central nucleus pulposus is rich in proteoglycans and water, creating a high intradiscal pressure that enables the IVD to resist compressive forces [11]. The peripheral annulus fibrosus functions to stabilize and dissipate tensile forces through ∼20 concentric collagen lamellae [12]. The vertebral endplates are formed by two regions: a hyaline cartilage region and a porous bone region that connects to the vertebral bone on both the superior and inferior aspects of the IVD. The endplate serves both to anchor the IVD to the vertebra and acts as a selectively permeable barrier for nutrient diffusion [12].

The IVD is often referred to as the largest avascular tissue of the body also lacking direct innervation. A recent review by our group summarized the available literature characterizing vascularization of the IVD over the lifespan and highlighted changes associated with age and state of degeneration or damage [13]. This review of the literature underscored the poorly understood relationship between neural and vascular ingrowth in the IVD, particular in the context of degeneration. Age, genetic susceptibility, exposure to chronic mechanical stress, and gradual depletion of nutrients can lead to widespread cellular disturbance and accelerated degeneration of the IVD [9, 10]. Modifications of the extracellular matrix within the IVD resulting from matrix catabolism or altered matrix synthesis, together with increased intradiscal expression of proinflammatory cytokines and neurotrophins, can regulate pathological nerve ingrowth into the IVD that results in the synthesis of pain-related neuropeptides [9, 14, 15]. As such, it is known that neural and vascular ingrowth into damaged or degenerated IVD tissues may enable discogenic back pain.

Despite extensive investigation into the pathophysiology that might underly discogenic back pain, the precise molecular mechanisms by which age-related alterations of IVD tissues disrupt or sensitize nearby neural structures, or promote neoinnervation, remain unclear. In addition, the topographical organization and type of intradiscal neural elements within the annulus fibrosus, nucleus pulposus, and vertebral endplates are poorly characterized in both healthy and degenerated IVDs. To this end, the current scoping review provides a comprehensive, systematic evaluation of currently available literature investigating the presence of neural elements within human IVDs across the lifespan.

Research Questions

What neural elements are present in the human IVD across the lifespan, and how are they organized within the annulus fibrosus, nucleus pulposus, and vertebral endplates?

The review question was designed to generate a comprehensive list of articles that investigated the presence of neural elements within the human IVD. An a priori search of the current literature was conducted (PubMed, BMJ Open, Joanna Briggs Institute Database of Systematic Reviews and Implementation Reports: May 2020) and no published or ongoing scoping or systematic reviews on the topic were identified. After preliminary evaluation of the search results, the authors defined three additional sub-questions: (i) is there a relationship between the location and/or type of neural elements present within the IVD and the state of IVD degeneration? (ii) do neural elements within the IVD always accompany vascular structures? (iii) which methods are the most effective to study neural elements in the IVD? Together, the primary aim of the current scoping review was to document the topographical arrangement of neural elements within healthy and degenerated human IVDs across the lifespan.

Methods

The scoping review was designed according to the Preferred Reporting Items for Systematic Reviews Extension for Scoping Reviews (PRISMA-ScR) checklist [16]. Systematic reviews are designed to answer detailed questions that are focused on a specific topic, whereas scoping reviews target broader research questions. The 20-item checklist used herein applies the PRISMA-ScR guidelines to delineate specific aspects of knowledge synthesis that are specific to scoping reviews [16].

Eligibility Criteria and Information Sources

The aim of the scoping review was to assess the topographical arrangement and types of neural elements present in the IVD across the human lifespan. To be included in the analysis, articles had to describe the presence and/or characteristics of neural elements within human IVDs (e.g., spatial localization, changes with age, types of neural elements). Only peer-reviewed research articles were included, with the exception of one letter to the editor which contained significant details on study population, methods, and results [17]. Review articles, abstracts, commentaries, and articles printed in a non-English language were excluded. Review articles were screened for citations of primary research reports eligible for inclusion.

Human Criteria

No restrictions were placed upon the age, sex, ethnicity, or health-status (non-spine affecting pathologies—e.g., cardiovascular disease, obesity, diabetes) of tissues assessed in the included studies. Studies that used animal models or interrogated molecular interactions with cultured IVD cells or neurons were excluded.

IVD Criteria

Studies examining both non-degenerate and degenerate IVD tissues were included to reflect the aging process [18], whereas herniated, ankylotic, or any otherwise diseased IVDs were excluded. However, many studies utilized experimental groups of mixed etiology (i.e., containing a combination of degenerate IVDs with a history of other spine-related pathologies). In these cases, all tissues were included for analysis in the scoping review, as studies often did not denote a specific health history for each donor.

Neural Element Criteria

Neural elements of all types were included and ranged from specialized nerve endings to myelinated axons (Supplementary Data 1). In addition, the scoping review included studies that investigated neural elements using multiple methodologies, including gross dissection, histology, and medical imaging. Articles focused on describing the neural elements of the osseous and ligamentous features of the spine were excluded if they did not also describe the neural elements of the IVD.

Articles were retrieved from six primary information sources: Scopus (1966–), MEDLINE (1946–), EMBASE (1947–), PubMed (1966–), Web of Science (1900–), and BIOSIS Previews (1926–). These databases provide a broad range of literature in the biological and physical sciences. The most recent search was completed through the Web of Science database on May 1, 2020. Following analyses and prior to submission, the systematic search was re-run using each information source on December 21, 2020, to ensure any pertinent articles published during the review process were captured.

Search Strategy

The authors implemented a systematic three-step search strategy in line with the framework developed by the Joanna Briggs Institute [19]. This process began with an initial search in PubMed to find seminal articles relevant to the three components of the research question: IVDs, humans, and neural elements. This search was then repeated in two information sources (Medline and Embase) to index novel terms or alternative spellings in article titles and/or abstracts that had not been captured. This iteration was then reviewed by an institutional librarian and an expert in neuroanatomy to ensure that the search strategy was organized appropriately and contained a comprehensive set of keywords surveying the seminal aspects of the research question. Finally, the precise syntax of the search strategy was modified such that it could be applied to each information source. An example of a comprehensive search strategy for one of the information sources can be found in Supplementary Data 1. The reference lists of all full-text articles that met the criteria for inclusion were reviewed in a final step to retrieve any relevant articles that may have been missed by the search strategy.

Selection of Sources of Evidence

Articles obtained during the search of six separate databases were compiled and imported into Mendeley (Version 1.19.4, Elsevier Inc., New York, NY) to organize and remove duplicates before sending to a second reviewer. Both reviewers independently screened titles and abstracts. This was followed by a consensus meeting to discuss and resolve any disagreements. The resolved disagreements were the exclusion of reviews, articles focused on molecular interactions with cultured IVD cells or neurons, and articles that describe neural elements in the spine and surrounding tissues but not the IVD. Next, eligible full-text articles were retrieved and independently screened by each reviewer, followed by another consensus meeting to resolve disagreements. The inter-rater reliability associated with analysis of both titles/abstracts and full-text articles was calculated using Cohen’s kappa [20] with SPSS Statistics (Version 26.0, IBM Corp., Armonk, NY). This statistical coefficient represents the measure of agreement between raters for nominal scales.

Data Charting Process

Data were charted from the final list of full-text articles by a single reviewer. Before data were charted from all included articles, the chart was tested by randomly selecting two articles and each reviewer independently charting the appropriate data and these results were compared and discussed. Throughout the data charting by the lead reviewer (AMRG), the chart was modified in an iterative manner to ensure accurate documentation of study data. The data items charted included citation information, study sample characteristics, methods of neural element visualization, and pertinent results concerning the topography and type of neural elements within the IVD.

Synthesis of Results

Results from the process of data charting were tabulated and organized by characteristics of the study sample and the neural elements within the IVD tissues. Data were further categorized by age and health status, where possible.

Results

Selection of Sources of Evidence

The PRISMA-ScR flow diagram [16] (Figure 1) depicts each step of the article selection process, including the specific rationale for exclusion of full-text articles. A total of 9,415 articles were retrieved following the six-database search. After removal of duplicate articles, 5,957 citations were evaluated by two independent reviewers in a two-step screening process. First, screening of titles/abstracts for key concepts such as the use of human tissue, study of the IVD, and data of neural elements within the IVD, resulted in the exclusion of 5,843 articles. The remaining 114 articles proceeded to full-text screening, which eliminated 86 articles (e.g., non-English language, lacking description of neural elements). The remaining 28 articles were included for final examination. Inter-rater reliability analysis revealed kappa values of 0.50 and 0.62 for title/abstract and full-text screening, respectively. Landis and Koch suggest that Kappa values of 0.41 ≤ 0.60 and 0.61 ≤ 0.80 indicate moderate to substantial agreement between independent reviewers, respectively [21]. Finally, review of the reference lists of the 28 full-text articles included for final examination led to the inclusion of an additional four articles in the final analyses. Thus, a total of 33 articles met the eligibility criteria for the current scoping review.

Results of Individual Sources of Evidence

Citation information and study sample characteristics for each full-text article included in the final analysis is documented in Table 1. Included articles were published between 1940 and 2019 and included contributions from around the world.

Table 1.

Article citation information and study population characteristics

Citation Information				Study Population
Author	Year	Location	Study Design	Tissue	Region	Donors	Female	Male	Age	Health Status
Roofe [22]	1940	USA	Descriptive	pm	L4–5	7	1	6	2, 50–67	…
Wiberg [23]	1949	SWE	Descriptive	s	…	200	…	…	…	…
				pm	Lumbosacral	…	…	…	…	……
				pm	Lumbar IVDs	…	…	…	fetal, adult	…
Pedersen [24]	1956	USA	Descriptive	pm	Lumbosacral	6	…	…	fetal, adult	…
Malinsky [25]	1959	CZE	Descriptive	pm	Full lumbar spine	9	…	…	fetal	…
				pm, s	L4–5	13	…	…	0–19	…
				s	L4–5	14	…	…	20–77	…
Ferlic [26]	1963	USA	Descriptive	s	C5–6	1	…	…	…	IVD degeneration
Ferlic [26]	1963	USA	Descriptive	pm	C3–T1	6	…	…	fetal, adult	…
Jackson [27]	1966	USA	Descriptive	s	L4–5	15	…	…	…	IVD degeneration
Jackson [27]	1966	USA	Descriptive	pm	Lumbar or L4–5	21	…	…	fetal, adult	…
Yoshizawa [28]	1980	JPN	Case-control	s	Lower lumbar	17	…	…	…	LBP
				s	Lumbar	4	…	…	…	Similar problems (i.e., LBP)
				pm	…	…	…	…	adult	“normal IVDs”
Bogduk [29]	1981	AUS	Descriptive	pm	Lumbar	4	…	…	…	…
Bogduk [30]	1988	AUS	Case-control	s	Cervical	3	…	…	…	Spinal fusion surgery
Bogduk [30]	1988	AUS	Case-control	pm	Cervical	5	…	…	adult	…
Coppes [17]	1990	NLD	Case-control	s	L4–5	10	…	…	…	IVD degeneration, LBP
Coppes [17]	1990	NLD	Case-control	c	L5–S1	2	…	…	…	Spinal tumor surgery
Mendel [31]	1992	USA	Descriptive	pm	Cervical	4	…	…	adult	…
Ashton [32]	1994	GBR	Descriptive	s	L4–S1	12	5	7	29–50	Back pain
Roberts [33]	1995	GBR	Descriptive	s	Thoracic-lumbar (not specified)	14	…	…	18–51	LBP
				s		15	…	…	5–53	Scoliosis
				pm		1	…	…	49	…
Brown [34]	1997	GBR	Case-control	s	L3–S1	15	11	4	17–62	IVD degeneration, LBP
Brown [34]	1997	GBR	Case-control	pm	“similar”	7	…	…	53–69	No chronic back pain
Coppes [35]	1997	NLD	Case-control	s	L3–S1	10	6	4	24–51	IVD degeneration, LBP
Coppes [35]	1997	NLD	Case-control	c	L3–L5	2	1	1	29, 51	Spinal tumor surgery
Freemont [36]	1997	GBR	Case-control	s	L3–5	38	…	…	20–59	Chronic back pain (split into pain and non-pain levels)
Freemont [36]	1997	GBR	Case-control	pm	L3–5	31	…	…	matched	No spinal disorders
Palmgren [37]	1999	FIN	Descriptive	pm	L1–S1	5	4	1	13–53	Non-degenerated IVDs
Johnson [38]	2001	GBR	Descriptive	s	L2–3; L4–S1	15	8	7	14–50	Injury, IVD degeneration, LBP
Bucknill [39]	2002	GBR	Descriptive	s	Lumbar	40	22	18	20–78	IVD degeneration or prolapse
Freemont [40]	2002	GBR	Case-control	s	L3–S1	36	9	12	23–58	IVD degeneration, pain
				s	L3–S1	36	8	12	26-54	IVD degeneration, no pain
				pm	…	12	4	8	22–59	“normal IVD”
Peng [41]	2005	CHN	Case-control	s	Lumbar	17	5	12	19–55	LBP
				c	Lumbar	12	…	…	29–61	No LBP, “normal”
				pm	L4–S1	5	…	…	22–45	No spinal disorders
Ohtori [42]	2006	JPN	Case-control	s	L4–S1	14	6	8	20–46	IVD degeneration or herniation, LBP
Ohtori [42]	2006	JPN	Case-control	pm	L1–2; 4–5	4	2	2	13–30	Scoliosis, fracture
Ozawa [43]	2006	JPN	Descriptive	s	L4–5 or L5–S1	8	4	4	21–45	IVD degeneration or herniation, LBP
Johnson [44]	2007	GBR	Case-control	s	Lumbar	21	15	8	11–62	IVD degeneration or prolapse, LBP
Johnson [44]	2007	GBR	Case-control	c	Lumbar	2	15	8	21, 21	Fracture, dislocation
Kokubo [45]	2008	JPN	Case-control	s	Cervical	198	77	121	25–78	IVD herniation, pain
				s	Cervical	166	61	105	32–83	Spondylosis, pain
				pm	Cervical	4	…	…	68–78	No spinal disorders
Dimitroulias [46]	2010	GRC	Descriptive	pm	L4–S1	15	7	8	15–66	No spinal disorders
Martins [47]	2010	BRA	Descriptive	pm	L1–L5	10	5	5	30–85	No spinal disorders
Fields [48]	2014	USA	Descriptive	pm	T11–S1	7	2	5	51–67	Endplate pathologies
Binch [49]	2015	GBR	Case-control	s	C3–4, 5–7; L2–S1	61	…	…	…	Nerve root compression
Binch [49]	2015	GBR	Case-control	pm	L3–S1	22	…	…	…	…
Yang [50]	2017	CHN	Case-control	s	C3–T1	54	24	30	55 ± 11	Cervical spondylosis with vertigo
				c	C3–T1	62	25	37	54 ± 9	Cervical spondylosis without vertigo
				pm	C3–T1	8	3	5	52 ± 9	No spinal disorders
Lama [51]	2018	CAN	Case-control	s	L2–S1	10	8	2	37–68	IVD herniation (sciatica)
				s	L3–S1	11	5	6	35–74	IVD herniation (no sciatica)
				s	L2–S1	11	6	5	39–72	IVD degeneration
				s	T12–L5	8	8	0	14–15	Scoliosis
Yang [52]	2018	CHN	Case-control	s	C3–T1	52	23	29	50 ± 8	Transient neck pain and dizziness
				c	C3–7	6	2	4	49 ± 14	Acute spinal cord injury
				pm	C3–T1	8	3	5	52 ± 9	No spinal disorders
Wu [53]	2019	CHN	Case-control	s	C3–7	34	13	21	49 ± 10	Chronic neck pain
				c	C3–7	36	16	20	47 ± 10	Symptom control: mild or no neck pain
				pm	C3–7	8	3	5	52 ± 9	Histology control: no spinal disorders

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Citation information. First author is listed. Year of publication is displayed. Location listed as 3-letter country abbreviation, based on the corresponding author’s information.

Study population. Tissue sources: s = surgery; c = controls; and pm = post-mortem. Region of the spine: C = cervical; T = thoracic; L = lumbar; and S = sacral. The age range studied is displayed in years. Select authors only presented age as means ± standard deviation.

“…” represents insufficient information provided.

IVD=intervertebral disc; LBP=low back pain.

Study Design and Source of Tissue

The included articles implemented either descriptive (17/33) or case-control (16/33) study designs to evaluate IVDs tissues obtained from two primary sources: post-mortem and surgical donors (Table 1). Post-mortem evaluation of autopsy or embalmed cadaveric material enabled analysis of the intact IVD. Tissues retrieved from surgery were limited to biopsies or en bloc resection (most often of the anterior aspect of the IVD).

In the descriptive studies, IVD tissues were obtained from spine-related surgical procedures (4/17), post-mortem tissues (8/17), or from a combination of both (5/17).

In the case-control studies, all surgical samples were excised during procedures to alleviate chronic back pain, such as interbody fusions, nerve root decompressions, and discectomies. Specific IVDs of patients with spine-related disease or reported back pain were selected following discography. Tissues excised from surgery were regularly compiled into one experimental group of mixed etiology (13/16). Few studies organized surgical tissues based on the presence, or absence, of a particular symptom (e.g., pain or no pain) or spine-related disease (e.g., scoliosis). IVDs included in control groups typically originated from post-mortem samples only (8/16), surgical samples only (4/16), or from post-mortem and surgical samples (4/16). In the latter case, surgical control samples were taken from spinal regions that lacked patient-reported symptom or spine-related disease. There was tremendous variability in the reporting of control IVD tissues. Most control groups listed age ranges and sex but were not identified as age- or sex-matched to the case group (11/16). One control group was matched for age but not sex (1/16), and the remaining studies presented minimal (i.e., used nondiscriminatory age brackets such as adult or fetal) or no demographic data (4/16).

Anatomical Region

IVDs were most often obtained from the lumbosacral spine (16/33). Some studies focused solely on the cervical region (7/33), some examined tissues from two or more anatomical regions (3/33), and the remaining studies did not report the anatomical region from which IVDs were retrieved (7/33). It is worth noting that we considered IVDs between C7/T1 to be part of the cervical region in the aforementioned groupings. Most studies included IVD tissues from multiple vertebral levels, rather than focusing on a single motion segment (i.e., L1–L5, not just L1/L2).

Number of Donors, Distribution of Sex, and Age

Generally, studies focused on neural elements of the human IVD were limited to relatively small sample sizes (overall mean = 39 ± 66, median = 16.5, range = 3 to 368).

Many articles did not include information on the sex of donors used in their studies (12/33). In the studies that did report sex, both female and male donors were included (21/33). Of these, some included more females (7/21), more males (11/21), or an equal distribution of females and males (3/21). Results for each group were always pooled (irrespective of sex) by the study authors, and details about the neural elements were described for the group as a whole. Therefore, the relationship between sex and the presence of neural elements in the IVD could not be examined in the current scoping review.

The age of donors ranged from fetal and infantile tissues to older adults over 80 years of age. However, the majority of studies only reported the age range for the sake of describing the study sample. Data concerning the neural elements within the IVD were never directly stratified by age. In the current scoping review, data were organized by age when possible. In cases where age was not clearly reported, results were not assumed to reflect any particular age group and instead were presented across all ages.

Donor Health Status

The health history of donors included in descriptive reports are organized into three groups: (i) no health history listed, (ii) tissues obtained from donors without symptoms of back pain, and (iii) tissues obtained from donors with back pain due to degeneration or other forms of IVD disease.

In case-control studies, pathological IVD tissues were often reported as pain producing (determined by discography). In some cases, herniated and/or spondylotic tissues were grouped together with degenerated tissues. For this reason, the current scoping review could not draw firm conclusions about the relationship between IVD innervation and age-related degeneration from all included articles.

Most control tissues were obtained from post-mortem donors (either cadaveric or from autopsy) with no IVD herniation or degeneration. However, select studies retrieved IVDs from surgical procedures for scoliosis, vertebral fractures, or spinal tumors. Thus, these control groups did not necessarily reflect “healthy” control IVDs.

Study Methodologies

Articles used histological or immunohistochemical techniques to investigate the neural elements within the IVD. Some studies combined histological techniques with other imaging modalities (4/33), gross dissection (5/33), or both (1/33). For histological examination, standard tissue fixation was used to preserve IVD tissues (i.e., alcohol, paraformaldehyde, formalin, methanol, or acetone). Tissues were paraffin-embedded and sectioned using a microtome or embedded in optimal cutting temperature compound and sectioned using a cryostat. In either case, IVD tissue was typically sectioned at a thickness of 30 microns. Standard immunohistochemical labeling methods were followed with all antisera listed in Table 2; counter-staining was used to visualize other tissue components of the IVD. In older studies that did not employ immunostaining, neural elements were visualized using a variety of silver impregnation techniques, gold chloride, or osmium tetroxide staining. While most reports used donor health history and symptomatology to assign IVD samples into case or control groups (28/33), others used more standardized classification systems based upon histological or MRI grading to determine the level of IVD degeneration (5/33) [38, 44, 47, 48, 51]. Gross dissection was used in two studies to trace the neuroanatomical extent of fibers traveling to the IVD [29, 30].

Table 2.

List of markers used to identify neural elements in the human intervertebral disc

Abbreviation	Full Name(s)	Function	Location
AChE [17, 35]	Acetylcholinesterase	Enzyme that catalyzes the breakdown of acetylcholine	Variety of motor and sensory neurons; cholinergic and non-cholinergic neurons^a
CGRP [32–34, 43]	Calcitonin gene-related peptide	Peptide that acts as a vasodilator, is involved in nociception, and may contribute to nerve regeneration^b^,^c	Somatic motor and sensory neurons^b; autonomic neurons^c
GAP43 [36, 40, 45]	Growth-associated protein 43	Cytoplasmic protein in axons and presynaptic terminals that aids with neurite growth, regeneration, and plasticity	Neurons in the central and peripheral nervous systems^d Also found in glial cells of central and peripheral nervous systems^e
GFAP [38]	Glial fibrillary acidic protein	Type III intermediate filament involved in astrocyte-neuron interactions	Expressed primarily in astrocytes^f; also found in ependymal and Leydig cells^g^,^h
IB4 [43]	Isolectin B4 (isolated from Griffonia simplicifolia)	Carbohydrate-binding protein involved in biological recognitionⁱ	Subpopulation of primary afferent neurons in dorsal root gangliaⁱ
NF200 [38, 41, 44, 49, 50] NF68 [45]	Neurofilament 200; neurofilament heavy polypeptide Neurofilament 68; neurofilament light polypeptide	Type IV intermediate filament that helps form neuronal cytoskeleton Type IV intermediate filament that is shown to be indicative of axonal damage in neurological disorders^j	Neurons in central and peripheral nervous systems^k
NGF [45]	Nerve growth factor	Neuropeptide involved in the growth, proliferation, and maintenance of neurons	Present in most neurons^l; required for survival in sensory and sympathetic neurons^m
NPY/CPON [32–34, 37]	Neuropeptide-Y; c-flanking peptide of neuropeptide-Y	Neurotransmitter that acts through G-coupled protein receptors Regulates multiple functions such as neuroendocrine release, blood pressure, food intake, anxiety, bone formation, obesity, and painⁿ	Primarily in GABAergic interneurons in central nervous systemⁿ; sympathetic neurons^o
p75 [40]	p75 neurotrophin receptor; low-affinity nerve growth factor receptor	One of two receptor types for neurotrophins^p, which are growth factors that stimulate neuronal survival and differentiation, and include NGF, BDNF, NT-3, NT-4	Variety of neurons in the central and peripheral nervous system^p
PGP9.5 [32–34, 36–40, 42, 43, 48, 49, 51]	Protein gene product 9.5; ubiquitin carboxy-terminal hydrolase L1	Cytoplasmic protein that hydrolyzes ubiquitin to generate ubiquitin monomers^q	Neurons in central and peripheral nervous systems; neuroendocrine cells^q
PRPH [38]	Peripherin	Type III intermediate filament expressed primarily in neurons within the peripheral nervous system^r	Somatic sensory neurons^r; spinal motor neurons^r; autonomic neurons^r
PTN [44]	Pleiotrophin; heparin-binding brain mitogen	Protein that promotes neurite outgrowth and acts as a mitogen^s	Shown to exist in the central and peripheral nervous systems during development^s
S100 [46, 47, 50, 52]	S100 protein	Protein that aids with intracellular and extracellular functions such as protein phosphorylation and the inflammatory response^t	Cells derived from the neural crest (Schwann cells, chondrocytes, macrophages, adipocytes, etc.)
SNS/PN3 and NaN/SNS2 [39]	Tetrodotoxin-resistant, voltage-gated sodium channels	Involved in the initiation and propagation of action potentials^u	Somatic sensory neurons^v
SP [17, 32–37, 41, 45, 51–53]	Substance P	Neuropeptide released in response to biological stressors^w Vasodilator, initiates expression of cytokines, and is highly involved in pain perception and transmission to the central nervous system^w	Somatic afferent neurons^w; immune cells^x (microglia, T-cells, eosinophils, dendritic cells)
SYN [37]	Synaptophysin; major synaptic vesicle protein p38	Synaptic vesicle glycoprotein involved in synaptic transmission	Present in most neurons and neuroendocrine cells^y
TrkA [40]	Tropomyosin receptor kinase A; high-affinity nerve growth factor receptor	Second of two receptor types (first was p75) for neurotrophins that mediates neuronal survival and differentiation	Variety of neurons in the central and peripheral nervous system^p
VIP [32, 33, 41]	Vasoactive intestinal peptide	Neurotransmitter and neuroendocrine factor that: Regulates circadian rhythms, relaxes smooth muscle, and increases neuronal survival (central nervous system)^z Increases secretion and relaxes smooth muscle (digestive system)^z	Variety of neurons in the central nervous system^z; autonomic neurons^z (especially in enteric nervous system)

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Synthesis of Results

An assortment of neural elements were identified within the IVD and categorized as: (i) sensory nerve fibers that were unassociated with blood vessels of variable thickness and immunoreactivity; (ii) perivascular nerve fibers; or (iii) mechanoreceptors. Neural elements were identified by qualitative observation of nerve morphology or immunostaining. Table 2 presents the list of all the antibodies used to analyze neural elements within the IVD.

Annulus Fibrosus

The annulus fibrosus was the most innervated tissue of the IVD, as neural elements were described in nearly all of the included articles (32/33, Table 3), except for one report [24]. The general trend was that neural elements were localized to the outer one-third of the annulus fibrosus in control IVDs and that these elements infiltrated into the inner two-thirds of the annulus fibrosus in pathological IVDs with a history of back pain, degeneration, or related spinal pathologies. Although specimen age was not directly correlated to IVD innervation in individual studies, cross-referencing IVD innervation data with specimen age ranges across all studies revealed that nerve endings may be more abundant and morphologically complex across the lifespan (Figure 2).

Table 3.

Neural elements in the annulus fibrosus

First Author	Year	Neural Markers and Stains	IVD Health Status	Description of Neural Elements
Roofe [22]	1940	Silver stain; gold chloride	…	Unmyelinated network of fine nerves found superficial to AF, with additional nerve endings penetrating the outer AF
Wiberg [23]	1949	Silver stain	…	No nerve fibers found in the AF of fetal or adult specimens, only in surrounding ligaments
Pedersen [24]	1956	Silver stain; Masson’s stain	…	No nerves found in the AF of fetal or adult specimens, only in surrounding ligaments
Malinsky [25]	1959	Silver stain	…	Nerve fibers and endings found only in the outer zone of AF and its surface; no fibers uncovered in the inner AF. Fetal: perivascular nerve endings (8/9) and isolated free nerve endings (3/9) found in outer AF; partially-encapsulated nerve receptors (2/9) and encapsulated nerve receptors (1/9) found on surface of AF 0–19 years: perivascular nerve endings (13/13) and isolated free nerve endings (9/13) found in outer AF; partially-encapsulated nerve receptors (5/13) and encapsulated nerve receptors (4/13) found on surface of AF 20–77 years: perivascular nerve endings (14/14) and isolated free nerve endings (14/14) found in outer AF; partially encapsulated nerve receptors (1/14) and encapsulated nerve receptors (9/14) found on surface of AF
Ferlic [26]	1963	Silver stain	IVD degeneration …	Fetal: nerve fibers found only in superficial layers of AF Adult: nerve fibers found in only 2/18 adult specimens No nerve fibers in deeper AF of fetal or adult tissues Fibers were proposed to be thin, unmyelinated C-fibers, although larger fibers were described (1 μm to 1/10th this size)
Jackson [27]	1966	Cholinesterase; silver stain	IVD degeneration …	Fetal: perivascular fibers and free nerve fibers found in outer AF but not inner AF; also, many end organs (resembling Pacinian corpuscles) positive for cholinesterase in ventrolateral AF Adult: nerve fibers and endings were found only in the outer AF, including perivascular nerves and unmyelinated, beaded axons; no specialized end organs found, as seen in fetal tissues No nerve growth into granulated tissue of degenerated IVDs
Yoshizawa [28]	1980	Cholinesterase; silver stain	LBP Similar problems (i.e., LBP) Normal IVD (control)	Diffuse unmyelinated network found (by silver stain) in outer AF of control and painful IVD specimens, with free nerve fibers typically travelling perpendicularly to lamellae of IVD Some myelinated fibers also found in outer AF, which travel parallel to lamellae in neurovascular bundles; separate free nerve terminals seen in adventitial collagen of these bundles No nerves found in inner AF of control or painful IVD specimens No evidence of axonal ingrowth from outer AF into necrotic or degenerated zones in the inner AF
Bogduk [29]	1981	Silver stain; osmium tetroxide	…	Fibers traveled an inward distance of one-third the thickness of the AF; most were perivascular, but others were larger and distinct from blood vessels
Bogduk [30]	1988	Silver stain; cholinesterase	Spinal fusion surgery …	Silver staining revealed no nerves in any specimens Cholinesterase staining revealed free nerve endings and unencapsulated endings in the outer one-third of the AF of operative specimens
Coppes [17]	1990	AChE; NF; SP	IVD degeneration, LBP Spinal tumor surgery (control)	Nerves resembling a-delta and C-fibers were discovered using AChE in the outer AF in both groups; fibers extended into the inner AF in IVD degeneration group IVDs in both groups treated with SP showed positive thin fibers, which had spin-like endings in some cases No association between blood vessels and nerves found in IVDs from degeneration group Encapsulated, Pacinian-like endings found on ventrolateral surfaces of AF in layer of loose connective tissue
Mendel [31]	1992	Gold chloride	…	Nerves entered IVDs posterolaterally, running parallel to lamellae in outer AF, and perpendicular in inner AF; nerves were most numerous in middle third of AF and resembled type III pain fibers (1.86–2.87 μm) Structures resembling Pacinian (130 × 80 μm) and Golgi (100 × 60 μm) receptors found posterolaterally in the outer and inner AF, respectively
Ashton [32]	1994	PGP9.5; CGRP; SP; VIP; CPON	Back pain	Perivascular fibers and isolated nerves positive for PGP9.5 were found in outer 3 mm of AF; fibers coursed obliquely and in parallel to lamellae, and occasionally followed granulated tissue Thin fibers (< 1 μm) positive for CGRP (both perivascular and isolated from vessels), CPON (exclusively perivascular), VIP and SP were found in the outer AF of 67% (8/12), 67% (8/12), 58% (7/12) and 33% (4/12) of IVD specimens, respectively Co-labeling of antisera was not demonstrated
Roberts [33]	1995	PGP9.5; CGRP; SP; VIP; NPY; Mayer’s hematoxylin	LBP Scoliosis (control) Not listed	Both perivascular and isolated nerves positive for PGP9.5 were found in the outer and middle AF of all groups (highest percentage in back pain group) All blood vessels found in IVDs were innervated (sometimes positive for NPY) Mechanoreceptors were found in outer 2–3 lamellae of painful and scoliosis IVDs (higher percentage in back pain): resembled Golgi (most frequent) Ruffini, and Pacinian CGRP positive fibers were most numerous, with occasional staining for SP and VIP Co-labeling of antisera was not demonstrated
Brown [34]	1997	PGP9.5; CGRP; SP; NPY; H&E; toluidine blue	IVD degeneration, LBP No chronic back pain (control)	Fibers positive for PGP9.5, CGRP, SP and NPY were found in the outer AF in both control and degenerated samples PGP9.5 positive fibers were usually perivascular, whereas CGRP fibers were distinctly unrelated to vasculature (and resembled C- and A-∂-type nociceptors) No difference in density of PGP9.5 fibers between groups; however, increased CGRP positive fibers in degeneration group Co-labeling of antisera was not demonstrated
Coppes [35]	1997	AChE; NF97; SP	IVD degeneration, LBP Spinal tumor surgery (control)	Perivascular nerves (0.25 μm; positive for AChE and NF) found only on surface of AF, in both groups Large bundles of myelinated fibers (15–25 μm; positive for AChE and NF) and smaller free fibers (0.25–2.5 μm; positive for AChE and NF) found in outer AF of both groups; only free nerve fibers found in inner AF of degenerated IVDs Mechanoreceptors (20 × 230 μm) found in 4/10 IVD specimens of degenerated group: resembled Pacinian and Golgi receptors Axons (0.25–2.5 μm) positive for SP found in outer AF of both groups Co-labeling of antisera was not demonstrated
Freemont [36]	1997	PGP9.5; SP; GAP43; H&E	Chronic back pain (split into non-pain and pain level subgroups) No spinal disorders (control)	Perivascular fibers positive for PGP9.5 were found in the outer one-third of the AF in 26% (9/34) of controls, 63% (10/16) of non-pain level IVDs, and in 80% (24/30) pain-level IVDs Perivascular fibers co-labeled with PGP9.5 and SP were not found in controls, but were present in the inner one-third of the AF in 25% (4/16) of non-pain level IVDs and 57% (17/30) of pain-level IVDs Thin nerve fibrils positive for PGP9.5, and unassociated with blood vessels, were not found in controls, but were present in the inner one-third of the AF in 12% (2/16) of non-pain level IVDs and 41% (12/30) of pain-level IVDs GAP43 positive nerves, independent of blood vessels, were only found in specimens of the pain group and also stained with SP Altogether, 76% of fibers positive for PGP9.5 were also positive for SP and 28% for GAP43
Palmgren [37]	1999	SYN; PGP9.5; CPON; SP; hematoxylin	Non-degenerated IVDs	Nerve fibers positive for SYN, PGP 9.5, CPON and SP were seen in both anterior and posterior AF specimens SYN, CPON, and PGP 9.5 positive fibers tended to penetrate deeper than SP positive fibers SYN and PGP 9.5 positive fibers were typically thicker than those labeled by CPON or SP Co-labeling of antisera was not demonstrated
Johnson [38]	2001	PGP9.5; NF200; peripherin; GFAP; H&E	Injury, IVD degeneration, LBP [all specimens allocated degeneration score]	Nerves positive for NF200 and peripherin were found more frequently in degenerated IVDs (grades 3 and 4) than more normal IVDs (grades 1 and 2); depth of nerve penetration in more degenerated IVDs was also farther—fifth lamellae of AF, as opposed to the second lamellae Nerves appeared to follow disrupted tissue in more degenerated IVDs GFAP immunoreactivity showed Schwann cell in-growth that matched the pattern of nerve ingrowth in the IVDs; Schwann cells and nerves were also frequently co-localized (shown with PGP9.5 and GFAP) Many neural processes and Schwann cells were near blood vessels Co-labeling of antisera was not demonstrated
Bucknill [39]	2002	PGP9.5; NF; SNS/PN3; NaN/SNS2; neutral red	IVD degeneration or prolapse	Fibers positive for PGP9.5 were found in 20% (3/15) of the IVDs investigated; none co-labeled for SNS/PN3 or NaN/SNS2 Location (AF or NP) not explicitly stated
Freemont [40]	2002	PGP9.5; p75; GAP43; TrkA; toluidine blue	IVD degeneration, pain IVD degeneration, no pain Normal IVD (control)	Large nerve fibers positive for PGP9.5 were found in outer AF of all groups (highest percentage in pain level); fibers usually perivascular Smaller perivascular nerve twigs positive for PGP9.5 were only found in the inner AF of pain level IVDs; these fibers also co-labeled for GAP43 and TrkA These perivascular twigs in pain level IVDs were associated with blood vessels positive for NGF-beta Only nerves in outer AF of pain-level IVDs were positive for p75 Nerve amount was always largest in pain-level specimens
Peng [41]	2005	NF200; SP; VIP; hematoxylin	LBP No LBP No spinal disorders (control)	Nerves positive for SP and VIP found in the outer one-third of AF of all specimens (highest percentage in back pain group); nerves only found in the inner two-thirds of AF in the back pain group NF200 positive nerves were found in the greatest quantity overall, and followed the same pattern for the outer one-third of the AF; however, they were present in the inner two-thirds AF of the control group, no back pain group, and the back pain All SP and VIP positive nerves were co-labeled for NF200 (although some nerves were only positive for NF200)
Ohtori [42]	2006	PGP9.5	IVD degeneration or herniation, LBP Scoliosis, fracture	…
Ozawa [43]	2006	PGP9.5; CGRP; IB4	IVD degeneration or herniation, LBP	Nerves positive for PGP9.5 and CGRP were found in both the outer and inner AF (much fewer in the inner AF) Overall, nerve fibers positive for PGP9.5 were much more numerous than those positive for CGRP No IB4 positive nerve fibers found in the IVDs Co-labeling of antisera was not demonstrated
Johnson [44]	2007	NF200; pleiotrophin; Mayer’s hematoxylin	IVD degeneration or prolapse, LBP Fracture, dislocation (control) [all specimens allocated degeneration score]	Nerve fibers positive for NF200 were only found in 6/24 specimens, with the largest percentage in degenerated IVDs (grades 3 and 4) Degenerated IVDs also had many more nerves than prolapsed IVDs, despite both being highly vascularized No significant correlation found between innervation score, IVD age and degeneration level; whereas, there was with blood vessels Degeneration score was significantly correlated to the number of IVD cells positive for pleiotrophin Co-labeling of antisera was not demonstrated
Kokubo [45]	2008	NF68; GAP43; SP; NGF; H&E	IVD herniation, pain Spondylosis, pain No spinal disorders (control)	Nerve fibers positive for NF68, GAP-43 and SP were found in the outer AF of uncontained/contained herniation and spondylosis specimens (with highest percentage in uncontained herniation) Nerve fibers positive for NGF were also found in the outer AF Co-labeling of antisera was not demonstrated
Dimitroulias [46]	2010	S100; Harris hematoxylin	No spinal disorders	Mechanoreceptors were found in the outer AF in the majority of IVDs (23/25) using S100 Included Ruffini-type receptors, Golgi-type receptors, and free nerve endings (with Ruffini having the highest mean frequency) No statistically significant differences found between receptor type/frequency and the age, sex, or degree of IVD degeneration
Martins [47]	2010	S100; hematoxylin	No spinal disorders [all specimens allocated degeneration score]	Freeman type I-IV endings (Ruffini, Pacinian, Golgi, and unmyelinated) identified with S100 were found in “all IVD regions” of all IVDs studied (i.e., at varying degrees of degeneration) Relationship between IVD thickness and total number of nerve fibers in L1/L2 and L2/L3 (especially type IV); this relationship also existed in L2/L3 IVDs with type I fibers
Fields [48]	2014	PGP9.5; Heidenhain stain	Endplate pathologies [all specimens allocated degeneration score]	Nerves positive for PGP9.5 were found in the outer one-third of the AF in 30.4% (14/46) of specimens, and were unassociated with blood vessels No association between number of nerves in an IVD, and the location or size of tears; number of nerves in IVDs with radial AF tears was larger than IVDs without tears
Binch [49]	2015	NF200; PGP9.5; H&E	Nerve root compression Not listed (control)	Fibers positive for NF200 were found in the inner and outer AF of both groups; nerves followed the IVD lamellae and/or fissures in many cases Proportion of degenerated samples with nerves was significantly higher than that of the non-degenerated samples (same with blood vessels) PGP9.5 marked subset of IVD cells in NP, and was not used thereafter
Yang [50]	2017	S100; NF200 Harris hematoxylin	Cervical spondylosis with vertigo Cervical spondylosis without vertigo No spinal disorders (control)	Ruffini corpuscles, Golgi endings, and free nerve fibers (both marked by S100 and NF200) were found in the outer AF of all groups, with more in surgical groups than controls These structures were also found in surgical groups within the inner AF, but not in controls Neural structures were co-labeled for S100 and NF200 in many cases
Lama [51]	2018	PGP9.5; SP; H&E	IVD herniation (sciatica) IVD herniation (no sciatica) IVD degeneration Scoliosis (control) [all specimens allocated degeneration score]	Nerves positive for PGP9.5 and SP were found in the outer and inner AF of all groups except scoliosis controls; highest percentage was in herniation/sciatica group, and the lowest was in the degeneration group No significant difference between nerve counts in the outer or inner AF of the study groups Any fibers positive for SP tended to be adjacent to blood vessels or infiltrating immune cells Increased numbers of nerves and blood vessels were correlated to regions of glycosaminoglycan loss Co-labeling of antisera was not demonstrated
Yang [52]	2018	S100; SP; H&E	Transient neck pain and dizziness Acute spinal cord injury No spinal disorders (control)	Ruffini corpuscles (marked by S100) and free nerve fibers positive for SP, were found in outer AF of all three groups. The highest percentage was in neck pain group Same neural elements were also found in inner AF of neck pain group
Wu [53]	2019	SP; H&E	Chronic neck pain Mild or no neck pain No spinal disorders (control)	Nerve fibers positive for SP were found in the outer one-third of the AF in all groups, with largest percentage in chronic neck pain group SP positive fibers were only found in inner two-thirds of the AF in chronic neck pain group, and the mild or no neck pain group

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IVD=intervertebral disc; AF=annulus fibrosus; LBP=low back pain; H&E=hematoxylin and eosin; full names of neural marker acronyms, alongside functional descriptions, can be found in Table 2.

Figure 2. — Summary of neural elements reported within the human intervertebral disc. **(A)** Frontal section of a motion segment of the vertebral column (left) and a transverse view of the intervertebral disc (right). The localization and prevalence of neural elements, as reported by the authors of the studies analyzed in the current scoping review, is displayed using a scale that ranges from purple (evidence of neural elements) to white (no evidence of neural elements). Illustration is organized as control compared to pathology (i.e., chronic pain, degeneration, structural damage, etc.). Schematic and anatomical features are not drawn to scale. OAF, outer annulus fibrosus; IAF, inner annulus fibrosus; NP, nucleus pulposus; and VE, vertebral endplate. (B) The percentage of studies included in the scoping review that documented the presence of neural elements (perivascular, sensory or mechanoreceptors) in the annulus fibrosus, nucleus pulposus and vertebral endplate of the intervertebral disc.

Nerve fibers without adjacent blood vessels, and likely sensory in nature, were found throughout the outer and inner annulus fibrosus in the vast majority of studies [17, 22, 25–41, 43–53]. These fibers were typically described as free nerve endings and coursed both obliquely and in parallel to the circumferential lamellae of the annulus fibrosus. Neural infiltration into the inner annulus fibrosus was more frequent in painful IVD tissues or within fissures or damaged regions of the annulus fibrosus than in controls or IVDs with no listed spinal pathology. Many studies listed only the immunoreactivity of the nerve fibers, whereas others made deductions about nerve structure or type. In the latter, reports typically described thinly myelinated nerve endings, also known as Aδ-fibers (1/33) [17] or C-fibers (2/33) [17, 26] under the Erlanger-Gasser classification [54] within the annulus fibrosus. In other cases, nerves were described as myelinated or unmyelinated (6/33) [22, 26–28, 35, 47], encapsulated, partially encapsulated or unencapsulated (3/33) [17, 25, 30], by relative thickness and size (9/33) [17, 26, 29, 31, 32, 35–37, 40], or denoted by the Freeman classification (2/33) [31, 47, 55].

Perivascular fibers were reported in 10 studies, typically identified within the outer annulus fibrosus, and tended to be thinner than nerve fibers without adjacent blood vessels [27, 29, 32–34, 36, 38, 40, 44]. Blood vessels were identified by vascular endothelial growth factor (VEGF), CD31, CD34 immunoreactivity, or by counter-staining with hematoxylin. Of note, some studies did not report or specifically examine perivascular fibers so their presence may be significantly under-reported in the current scoping review.

Mechanoreceptors were highlighted in eight studies [27, 31, 33, 35, 46, 47, 50, 52] and were surmised to be of Golgi-type (proprioceptive), Ruffini-type (low-frequency vibration or pressure) or Pacini-type (high-frequency vibration or pressure) based on structural observations. These structures were found in the outer annulus fibrosus of both control and painful IVDs. Minimal data were presented regarding the precise location of these mechanoreceptors, and therefore no firm conclusion can be made regarding their topographical organization within the annulus fibrosus, or the relationship between these structures and the pathological state of the IVD.

Nucleus Pulposus

No neural elements were reported within the nucleus pulposus of control IVD tissues (23/33) across the lifespan, suggesting that it is a largely aneural structure in humans. In studies that did report neural elements within the nucleus pulposus (10/33) [17, 35, 36, 39–41, 49, 50, 52, 53], they were identified in degenerated IVDs and/or those from patients reporting back pain. One exception was Binch et al. who reported fibers positive for NF200 within the nucleus pulposus of both control and surgical tissues [49]. However, the health histories of the post-mortem control IVDs used in this study were not listed and therefore the presence of spine-related pathologies cannot be excluded [49]. The neural elements within the nucleus pulposus were described as free nerve endings [35, 50, 52], Ruffini corpuscles [50, 52], Aδ-fibers [17], C-fibers [17], or perivascular fibers [36, 40], and varied in their immunoreactivity depending on the specific report (detailed in Table 4).

Table 4.

Neural elements in the nucleus pulposus

First Author	Year	Neural Markers and Stains	IVD Health Status	Description of Neural Elements
Roofe [22]	1940	Silver stain; gold chloride	…	…
Wiberg [23]	1949	Silver stain	…	…
Pedersen [24]	1956	Silver stain; Masson’s stain	…	…
Malinsky [25]	1959	Silver stain	…	…
Ferlic [26]	1963	Silver stain	IVD degeneration …	…
Jackson [27]	1966	Cholinesterase; silver stain	IVD degeneration …	…
Yoshizawa [28]	1980	Cholinesterase; silver stain	LBP Similar problems (i.e., LBP) Normal IVD (control)	…
Bogduk [29]	1981	Silver stain; osmium tetroxide	…	…
Bogduk [30]	1988	Silver stain; cholinesterase	Spinal fusion surgery …	…
Coppes [17]	1990	AChE; NF; SP	IVD degeneration, LBP Spinal tumor surgery (control)	Some A8 and C-fibers (marked by AChE) were found in the NP of degenerated IVDs (number of IVDs not listed) Fibers positive for NF were also found in the NP of 20% (2/10) of degenerated IVDs No neural elements found in NP of control specimens Co-labeling of antisera was not demonstrated
Mendel [31]	1992	Gold chloride	…	…
Ashton [32]	1994	PGP9.5; CGRP; SP; VIP; CPON	Back pain	…
Roberts [33]	1995	PGP9.5; CGRP; SP; VIP; NPY; Mayer’s hematoxylin	LBP Scoliosis (control) …	…
Brown [34]	1997	PGP9.5; CGRP; SP; NPY; H&E; toluidine blue	IVD degeneration, LBP No chronic back pain (control)	…
Coppes [35]	1997	AChE; NF90; SP	IVD degeneration, LBP Spinal tumor surgery (control)	Small free nerve fibers positive for NF90 (diameter = 0.25 μm) were found in the NP of 20% (2/10) of degenerated IVDs
Freemont [36]	1997	PGP9.5; SP; GAP43; H&E	Chronic back pain (split into pain and non-pain level) No spinal disorders (control)	Small perivascular nerve fibers (co-labeled for PGP9.5 and SP) were found in the back pain group in 6% (1/16) of non-pain level specimens and 30% (9/30) of pain-level specimens Single nerve fibers or small clusters of these fibers (positive for PGP9.5), unassociated with blood vessels, were found in the NP of 30% (9/30) of pain-level specimens in the back pain group
Palmgren [37]	1999	SYN; PGP9.5; CPON; SP; hematoxylin	Non-degenerated IVDs	…
Johnson [38]	2001	PGP9.5; NF200; peripherin; GFAP; H&E	Injury, IVD degeneration, LBP [all specimens allocated degeneration score]	…
Bucknill [39]	2002	PGP9.5; NF; SNS/PN3; NaN/SNS2; neutral red	IVD degeneration or prolapse	Neural elements positive for PGP9.5 were found in 20% (3/15) of the IVDs investigated Location (AF or NP) not explicitly stated
Freemont [40]	2002	PGP9.5; p75; GAP43; Trk-A; toluidine blue	IVD degeneration, pain IVD degeneration, no pain Normal IVD (control)	Smaller perivascular nerve twigs positive for PGP9.5 were described penetrating into the deeper levels of painful IVD specimens; these fibers also co-labeled for GAP43 and TrK-A, and were associated with blood vessels positive for NGF-beta
Peng [41]	2005	NF200; SP; VIP; hematoxylin	LBP No LBP No spinal disorders (control)	Nerves positive for SP and NF200 were found in the NP of some back pain IVD specimens, but not in IVDs of the other two groups All nerves positive for SP and/or VIP were co-labeled with NF200 (although some nerves were only positive for NF200)
Ohtori [42]	2006	PGP9.5	IVD degeneration or herniation, LBP Scoliosis, fracture	…
Ozawa [43]	2006	PGP9.5; CGRP; IB4	IVD degeneration or herniation, LBP	…
Johnson [44]	2007	NF200; pleiotrophin; Mayer’s hematoxylin	IVD degeneration or prolapse, LBP Fracture, dislocation (control) [all specimens allocated degeneration score]	…
Kokubo [45]	2008	NF68; GAP43; SP; NGF; H&E	IVD herniation, pain Spondylosis, pain No spinal disorders (control)	…
Dimitroulias [46]	2010	S100; Harris hematoxylin	No spinal disorders	…
Martins [47]	2010	S100; hematoxylin	No spinal disorders [all specimens allocated degeneration score]	…
Fields [48]	2014	PGP9.5; Heidenhain stain	Endplate pathologies [all specimens allocated degeneration score]	…
Binch [49]	2015	NF200; PGP9.5; H&E	Nerve root compression Not listed (control)	Fibers positive for NF200 were found in the NP of both surgical and post-mortem (control) samples; associated with fissures and/or areas of decreased matrix on occasion
Yang [50]	2017	S100; NF200 Harris hematoxylin	Cervical spondylosis with vertigo Cervical spondylosis without vertigo No spinal disorders (control)	Ruffini corpuscles and free nerve fibers (positive for S100) were both found in the NP of surgical specimens with vertigo; however, only free nerve fibers were uncovered in surgical specimens without vertigo No neural elements described in the NP of control specimens
Lama [51]	2018	PGP9.5; SP; H&E	IVD herniation (sciatica) IVD herniation (no sciatica) IVD degeneration Scoliosis (control) [all specimens allocated degeneration score]	…
Yang [52]	2018	S100; SP; H&E	Transient neck pain and dizziness Acute spinal cord injury No spinal disorders (control)	Ruffini corpuscles (positive for S100) and free nerve fibers (positive for SP) were only found in the NP specimens of the neck pain and dizziness group
Wu [53]	2019	SP; H&E	Chronic neck pain Mild or no neck pain No spinal disorders (control)	Fibers positive for SP were found in the NP in 4.65% (2/43) of IVDs in the mild or no neck pain group, and 26.2% (11/42) of IVDs in the chronic neck pain group; no nerve fibers found in the NP of the control IVDs

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IVD=intervertebral disc; NP=nucleus pulposus; LBP=low back pain; H&E=hematoxylin and eosin; full names of neural marker acronyms, alongside functional descriptions, can be found in Table 2

Vertebral Endplate

Five studies included in the scoping review reported data on neural elements within the vertebral endplate [25, 34, 42, 48, 49]. From these, it was consistently reported that vertebral endplates of both control and pathological IVDs contained perivascular nerve fibers and nerve fibers not associated with blood vessels. Similar to the annulus fibrosus and nucleus pulposus, there was an increase in the number of nerve fibers detected in degenerated IVDs compared to controls. Fields et al. completed the most thorough investigation of the vertebral endplate, reporting that fibers positive for PGP9.5 were found in 82.6% (76/92) of the endplates examined and were consistently found with blood vessels that branched towards the endplate from the vertebral body [48]. Moreover, nerves were present in 90% (27/30) of endplate defects and 66.1% (35/53) of endplates with no defects or abnormal marrow elements [48]. The remaining reports corroborate these findings, demonstrating the presence of fibers positive for PGP9.5, NF200, SP or CGRP within the vertebral endplates of healthy and degenerate IVDs [34, 42, 49].

Temporal Changes in IVD Innervation

None of the studies included in the scoping review included age as a variable in the assessment of neural elements in the IVD. However, we cross-referenced findings of neural element localization from the included articles (data presented in Tables 3 and 4) with information provided on donor age to assess the possible association between IVD innervation and age. This analysis suggested that the IVD is modestly innervated until middle age, with neural elements predominantly localized in the outer one-third of the annulus fibrosus. In the absence of pathology, this innervation is detectable during the fetal period and increases into adolescence. In adults, age-related degenerative changes or spine-related pathologies likely lead to increased infiltration of nerve fibers into the inner annulus fibrosus and nucleus pulposus of the IVD which continue to proliferate with increasing degeneration or disease severity (Figure 2). However, since IVD degeneration results from the complex interplay between mechanical factors, genetic influences, and natural physiologic changes associated with aging [10, 56], it is not possible to associate increased innervation solely with age.

Discussion

Summary of Evidence

The objective of this scoping review was to summarize evidence related to the presence and organization of neural elements within the IVD across the lifespan. Overall, the IVD was found to be innervated across the lifespan, in both nonpathological and pathological tissues. Ingrowth of nerve fibers into the central tissues of the IVD was associated with painful (e.g., positive discography) and degenerate tissues, which were typically obtained from older patients. As such, changes in the presence of neural elements of the IVD with age and disease may have important implications for the pathogenesis of back pain and associated treatments.

Interpretation of Results

Relationship Between Nerve Location, Type, and the State of IVD Degeneration

The annulus fibrosus of most IVDs was innervated by a variety of different nerve fibers (Table 3) that appeared to increase in prevalence within tissues obtained from donors with a history of back pain. However, nerve infiltration into the inner annulus fibrosus and/or nucleus pulposus was almost entirely limited to painful or degenerate IVDs. Frequently, nerves were localized to areas of tissue granulation, annular tears, and lesions. This finding is consistent with the characterization of the blood vessels of the IVD [13] due to focal proteoglycan loss that is conducive to neurovascular ingrowth [57]. There did not appear to be a relationship between nerve type and location within control or degenerate IVDs, although this association was rarely directly examined and requires further investigation. Nevertheless, the diversity of nerve fiber types (as displayed by different neuropeptide markers) increased in degenerated or damaged IVDs, or those obtained from patients with back pain, compared to controls.

Association Between Neural Elements and Vascular Structures Within the IVD

The literature reviewed identified the presence of a large number of perivascular nerve fibers as well as nerve fibers not associated with blood vessels in the human IVD. Nerve fibers were not always associated with vasculature within the IVD. Both vasoregulatory and sensory nerve fibers were identified within the IVD, especially within IVD tissues from patients who reported back pain, where total innervation (i.e., all types of neural elements) was markedly higher than controls.

Methods Used to Study Neural Elements in the IVD

Neuropeptide antibodies such as SP and NPY are effective markers for neural elements within the IVD (see Table 2 for exhaustive list of antibodies used). However, it is critical that these markers are used in conjunction with general antibodies for neurons such as PGP9.5 and NF, and that all neural elements are co-labeled and presented with appropriate controls.

Identity of Neuronal Subtypes within the IVD

To identify neural structures, most articles employed general nerve markers that label common intracellular components specific to neurons or Schwann cells. These markers included PGP9.5, NF, PRPH, SYN, AChE, or S100 (Table 2). While these general markers did not provide insight into the precise identity of neural elements within the IVD, they facilitated the study of nerve thickness, and allowed the identification of different nerve endings based on their morphology. In addition to using general nerve markers, many studies also examined the localization of specific neuropeptides to assess the potential cell identity or state of growth, of neural elements within the IVD.

Substance P (SP) and Calcitonin Gene-Related Peptide (CGRP)

SP is a neuropeptide involved in primary afferent sensory neurotransmission [58] that is commonly reported to be expressed by nerves in IVDs obtained from patients who reported back pain [17, 32–37, 41, 45, 51–53]. A variety of neurons in the dorsal root ganglia synthesize and store SP in vesicles that are then delivered by fast axonal transport to peripheral nerve terminals [58]. In cases of inflammation or peripheral nerve injury, SP release upregulates NK1 receptors in dorsal horn neurons, which enhances peptidergic transmission and establishes hypersensitivity for the transmission of pain signals [59]. Given this well-established nociceptive role, it is likely that nerve fibers positive for SP in human IVDs are involved in the pain response. One report demonstrated increased gene expression of proinflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF)-α, matrix metalloproteinase (MMP)-10, nerve growth factor (NGF), and SP in the nucleus pulposus of degenerated IVDs from patients with low back pain, compared to controls (degenerated and nondegenerated post-mortem samples from patients with no documented history of back pain) [60]. The authors of this study suggested that cytokines such as IL-1 and TNF-α act synergistically to breakdown extracellular matrix via stimulation of MMP-10, which may consequently promote neoinnervation and nociception via NGF and SP, respectively [60]. CGRP is another neuropeptide localized to the IVD [32–34, 43], that contribute to processes of pain hypersensitivity and modulation [61]. Degenerated IVD tissues (categorized based on medical imaging and gross inspection) cultured ex vivo from patients with axial low back pain were shown to secrete increased levels of IL-1β, TNF-α, NGF, and brain-derived neurotrophic factor (BDNF) compared to IVD tissues from organ donors without back pain [62]. These factors increased neurite sprouting of PC12 cells and expression of CGRP in primary mouse DRG neurons [62]. Given that IVDs from patients with back pain contain nerve fibers positive for SP and/or CGRP, it is likely that these neural elements contribute to the onset or maintenance of discogenic pain.

Vasoactive Intestinal Peptide (VIP) and Neuropeptide-Y (NPY/CPON)

VIP is a neuropeptide widely expressed throughout the autonomic and enteric nervous systems. It is thought that VIP binds to VPAC1 or VPAC2 receptors on endothelial cells of capillaries, stimulating nitric oxide release and consequently vasodilation [63]. Like SP and CGRP, neural elements positive for VIP were found more frequently in IVDs from patients with back pain or IVD degeneration [32, 33, 41]. Previous research has shown that cytokine release (especially IL-1β) during IVD degeneration induces the expression of NGF and VEGF, a potent stimulator of angiogenesis [64]. NGF is known to promote neuronal proliferation, and VEGF expression may lead to ingrowth of endothelial cells which are known to express NGF, thus promoting innervation [64]. In this context, the presence of perivascular VIP-positive neurons within the degenerate IVD could be explained by degeneration-induced vascular ingrowth; these autonomic fibers may serve to innervate and regulate the nascent blood supply. In addition, NPY-positive perivascular nerve fibers were also localized within the outer annulus fibrosus of degenerate IVD specimens [33, 34]. In contrast to VIP, NPY is a strong vasoconstrictor, and is highly expressed in neurons of the sympathetic nervous system. NPY has also been shown to be angiogenic by acting through NPY2R receptors [65]. Similar to VIP, it is possible that NPY-positive neurons within the IVD [32–34, 37] proliferate in a neurotrophin-dependent manner, providing autonomic innervation to newly ingrown vasculature during the process of IVD degeneration.

Nerve Growth Factor (NGF) and Growth Associated Protein 43 (GAP-43)

While the present scoping review documents a variety of different neural elements within healthy and degenerate human IVDs, the mechanisms controlling neural ingrowth remain unclear. Freemont et al. demonstrated the presence of small perivascular fibers in the inner annulus fibrosus of painful IVDs (based on discography); these nerves expressed PGP9.5, GAP-43, and TrkA [40]. Kokubo et al. also reported similar nerve fibers positive for NGF or GAP-43 in the outer annulus fibrosus of IVD tissues from patients with radiculopathy, myelopathy or myeloradiculopathy [45]. The expression of nerve growth proteins such as NGF and GAP-43 in degenerated IVDs may implicate these mediators in processes of IVD innervation and nociception. In vitro studies demonstrate that co-culture of NP cells from painful and degenerated human IVDs with neuroblastoma cells increased the percentage of neurite-expressing cells and neurite length—effects that were dependent on NGF and BDNF, respectively [66]. Co-culture of neuroblastoma cells with annulus fibrosus cells from degenerated IVDs enhanced the secretion of VEGF, TGF-β1 and NGFβ compared to baseline culture conditions [67]. These findings led authors to propose that interactions between annulus fibrosus and neural cells enhance the production of growth factors responsible for neovascularization and nerve ingrowth during IVD degeneration [67]. Similarly, co-culture of F11 neural cells with annulus fibrosus cells from degenerate IVDs increased the expression of BDNF, neurotrophin receptors, and NGF receptors compared to levels from culture of F11 cells alone [68], providing further evidence to support the role of neurotrophins in mediating nerve-ingrowth into degenerate IVDs.

Gross Neuroanatomical Organization of Nerves Supplying the IVD

The variety of neuronal subtypes found within the IVD, and the functional implications related to the pain response, leads one to question their origins within the central nervous system. Two reports included in the scoping review reported gross dissection of nerves supplying IVD tissues [29, 30]. Four different sets of fibers were found to supply the lumbar IVDs. Branches from the ventral ramus supplied the posterolateral aspect of the IVD, while branches from the middle of the ramus communicans passed over the vertebral body to supply the lateral aspect of the IVD. Next, branches from the proximal end of the ramus communicans coursed in parallel with those from the middle and also supplied the lateral IVD. All of these fibers were distinct from branches of the sinuvertebral nerves, which also supplied the IVD [29, 30]. A subset of the reports also mentioned the neural supply to the paradiscal ligaments, structures which are known to be richly innervated [33]. These ligaments included the posterior longitudinal ligament [22–28, 30, 32, 41, 46], anterior longitudinal ligament [23, 25–29, 33, 35, 36, 38, 46, 50, 52], transverse ligament [30], and ligamentum flavum [23]. However, it remains unclear which specific nerves supply the paradiscal ligaments and which supply the IVD.

To our knowledge, no retrograde tracing studies have been conducted in humans; however, pre-clinical animal studies have investigated the neural pathways of fibers traveling from the spinal cord and adjacent paravertebral ganglia to the IVD. For example, in the rat L5/6 IVDs were injected with choleratoxin B and horseradish peroxidase; analysis of lumbar dorsal root ganglia revealed labeled neuronal cell bodies only within L1/2 ganglia, suggesting that sensory fibers innervating the IVD ascend via the sympathetic chain before re-entering sensory nerve roots at L1/2 [69]. Further research from this group demonstrated that nerve fibers from the L5/6 IVD in the rat traveled through both the sinuvertebral nerves and the sympathetic trunk to reach the dorsal root ganglia at L3-6 and T13/L2, respectively [70]. While further study is required in human tissues, the basic neuroanatomical findings described from blunt dissection studies in humans [29, 30] suggest possible sensory pathways for pain transmission.

Limitations in the Study of Neural Elements Within the IVD

Overall, a clear limitation in the study of neural elements within the IVD in the current scoping review was inconsistent annotation of data in the articles included for analysis. None of the studies stratified their findings by age or sex. Therefore, the potential influence of age and sex on the innervation of the IVD across the lifespan could not be accurately investigated. In addition, there was significant variability in the region of the IVD measured. Most reports only differentiated between the outer annulus fibrosus, inner annulus fibrosus, and nucleus pulposus, rather than documenting changes across the entire surface area of histological sections of the IVD. This is likely because many studies did not resect IVDs en bloc, but rather retrieved pieces of tissue that included a portion of the annulus fibrosus and nucleus pulposus, such as the anterior or lateral portion of the IVD. This limitation is inherent to the use of surgically-resected, degenerate IVDs from patients who experienced back pain.

Some studies did not report on the localization of nerve fiber types (immunoreactivity) within the annulus fibrosus, nucleus pulposus, or vertebral endplate. In addition, many studies used general nerve markers (i.e., PGP9.5) and specific nerve markers (i.e., SP) to investigate the presence of neural elements; however, these markers were not co-localized. It is possible that some markers such as SP may not have labelled neurons, as eosinophils, macrophages, T cells, and dendritic cells have all been shown to express this neuropeptide [71]. In studies that did use a co-labelling approach [36, 40, 41, 50], the percentage of co-labeled cells was not indicated.

In some cases, studies reported nerves as being myelinated or non-myelinated without using reliable stains (osmium tetroxide; luxol fast blue), antibodies (myelin oligodendrocyte glycoprotein; myelin basic protein) or appropriate microstructural imaging techniques such as electron microscopy [27, 35, 38, 47, 48]. For this reason, the myelination of nerve fibers within the IVD requires further study to confirm the reported observations. Finally, while some reports used reliable histological or MRI-based grading systems to assess the level of degeneration of the IVD tissues [38, 40, 44, 47, 48, 51], many reports segregated IVDs into controls and painful groups based exclusively on the available health history. This may have led to heterogeneity in the degeneration levels of tissues that were included in the distinct groups, since clinical studies establish a high incidence of asymptomatic IVD degeneration [72].

Limitations of the Current Scoping Review

The current scoping review was limited in its ability to interpret non-English articles that may have included pertinent information regarding neural elements within the IVD (25 articles total; see Figure 1). Further, the search strategy was not designed to identify studies detailing vascular structures in the IVD, although these studies were not excluded if nerves were also described. It is therefore possible that the search did not return comprehensive results for perivascular nerves or any neural elements associated with blood vessels within the IVD. Further, the scoping review intended to document only the neural elements reported in human tissues and therefore does not include molecular or mechanistic insights from animal studies or in vitro analyses.

Recommendations for Future Research

It is critical that future research undertake the study of neural elements within the IVD to discern the precise roles of neurons found in healthy and degenerated IVDs. Immunohistochemical techniques should include co-labeling of general and specific neuronal markers to ensure accurate identification of neural elements, and entire IVDs should be analyzed en bloc to assess neuronal topography in both 2D and 3D (although it is recognized that this will limit the study of IVDs to cadaveric or fresh autopsy materials). Additional markers for vasculature, circulating immune cells, and connective tissue cells can be used to enhance understanding of the microenvironment in which neural elements exist within the degenerate human IVD. Further, IVDs should be grouped according to age, sex, and degree of degeneration in order to investigate the influence of these variables on neural in-growth. Such studies could be complimented by microstructural analyses using electron microscopy, multiphoton fluorescence microscopy, light sheet microscopy [73, 74], or contrast-enhanced imaging that employs a variety of modern techniques (e.g., micro-CT and high-field MRI); these methods would allow for more accurate descriptions of neural cell identity and precise 3D reconstructions of neural networks within the IVD.

Conclusion

It is clear that the human IVD is an innervated structure throughout life and that neural ingrowth is associated with the state of tissue degeneration or damage. Neural elements were associated with blood vessels but also coursed in isolation within the IVD tissue. The most effective method to study neural elements within the IVD was histology and immunohistochemistry. The association between neural ingrowth and variables such as sex and age requires further study; in addition, precise characterizations of neuronal subtypes (such as those described in the current review) within the human IVD will allow for insight into their potential role in nociceptive signaling. Overall, the local changes to the IVD that occur with ageing, degeneration, and damage are associated with increased innervation of the IVD and may lead to discogenic back pain.

Supplementary Material

pnab070_Supplementary_Data

Click here for additional data file.^{(13.6KB, docx)}

Acknowledgments

The authors gratefully acknowledge Western Libraries and Inter-library Loan services for assistance retrieving articles.

Supplementary Data

Supplementary data are available at Pain Medicine online.

Funding source: AMRG was supported by a Richard H. Tomlinson Doctoral Fellowship from the Faculty of Medicine and Health Sciences at McGill University. DEF was supported in part by a Transdisciplinary Training Award from the Bone and Joint Institute at The University of Western Ontario, and a Ph.D. Award from the Arthritis Society (No. 19–0469). MCB is the Western Research Chair in Exercise, Mobility and Musculoskeletal Health affiliated with Western’s Bone and Joint Institute. CAS is supported by a Career Development Award from the Arthritis Society and Early Researcher Award from the Ontario Ministry of Research and Innovation. This study was supported by the Canadian Institutes of Health Research (No. 115068).

Conflict of interest: None.

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Associated Data

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Supplementary Materials

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PERMALINK

Innervation of the Human Intervertebral Disc: A Scoping Review

Adam M R Groh, MSc

Dale E Fournier, MSc

Michele C Battié, PhD

Cheryle A Séguin, PhD

Abstract

Objective

Method

Results

Conclusion

Introduction

Rationale

Research Questions

Methods

Eligibility Criteria and Information Sources

Human Criteria

IVD Criteria

Neural Element Criteria

Search Strategy

Selection of Sources of Evidence

Data Charting Process

Synthesis of Results

Results

Selection of Sources of Evidence

Figure 1.

Results of Individual Sources of Evidence

Table 1.

Study Design and Source of Tissue

Anatomical Region

Number of Donors, Distribution of Sex, and Age

Donor Health Status

Study Methodologies

Table 2.

Synthesis of Results

Annulus Fibrosus

Table 3.

Figure 2.

Nucleus Pulposus

Table 4.

Vertebral Endplate

Temporal Changes in IVD Innervation

Discussion

Summary of Evidence

Interpretation of Results

Relationship Between Nerve Location, Type, and the State of IVD Degeneration

Association Between Neural Elements and Vascular Structures Within the IVD

Methods Used to Study Neural Elements in the IVD

Identity of Neuronal Subtypes within the IVD

Substance P (SP) and Calcitonin Gene-Related Peptide (CGRP)

Vasoactive Intestinal Peptide (VIP) and Neuropeptide-Y (NPY/CPON)

Nerve Growth Factor (NGF) and Growth Associated Protein 43 (GAP-43)

Gross Neuroanatomical Organization of Nerves Supplying the IVD

Limitations in the Study of Neural Elements Within the IVD

Limitations of the Current Scoping Review

Recommendations for Future Research

Conclusion

Supplementary Material

Acknowledgments

Supplementary Data

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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