SPINE: RESEARCH

Do intervertebral discs degenerate before they herniate, or after?

P. Lama, C. L. Le Maitre, P. Dolan, J. F. Tarlton, I. J. Harding, M. A. Adams

From University of Bristol, Bristol, United Kingdom

P . Lama, BSc, MS(Medical Anatomy), PhD Candidate P . Dolan, BSc, PhD, Reader M. A. Adams, BSc, PhD, Professor University of Bristol, Centre for Comparative and Clinical Anatomy, Bristol BS2 8EJ, UK. C. L. Le Maitre, BSc, PgCert(HE), PhD, Senior Lecturer Sheffield Hallam University, Biomedical Research Centre, City Campus, Howard Street, Sheffield S1 1WB, UK. J. F . Tarlton, BSc, PhD, Senior Research Fellow University of Bristol, Matrix Biology, School of Veterinary Science, Langford, Bristol BS40 5DU, UK. I. J. Harding, BA, FRCS(Orth), Consultant Orthopaedic Surgeon University of Bristol, Department of Orthopaedics, Southmead Hospital, Southmead Road, Bristol BS10 5NB, UK. Correspondence should be sent to Professor M. A. Adams; e-mail: m.a.adams@bris.ac.uk 2013 The British Editorial Society of Bone & Joint Surgery doi:10.1302/0301-620X.95B8. 31660 $2.00 Bone Joint J 2013;95-B:112733. Received 24 January 2013; Accepted after revision 15 March 2013

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:112733.

Herniated disc tissue removed at surgery usually appears abnormal, suggesting that degenerative changes precede, or even cause, herniation.1 But some degenerative changes appear to follow herniation.2-4 The question of which comes first is important, as patients with a symptomatic disc herniation can, for instance, be denied personal injury compensation on the grounds that the herniated disc must already have been degenerated and would have herniated in any event even if the injury had not occurred. Herniated disc tissue mostly comprises nucleus pulposus, with variable amounts of annulus fibrosus, endplate cartilage and bone.5,6 As the nucleus pulposus contains a high concentration of hydrophilic proteoglycan, herniated tissue in vitro swells by between two- and three-fold within hours of escaping from the pressurised confines of the disc.2 Swelling allows fragmented proteoglycan to be lost, and the herniated tissue becomes a collagen-rich and proteoglycan-poor mass of crabmeat.2 In this way, the characteristic degenerative changes in disc tissue could occur after herniation.

Nor is there any need to assume that a disc must be degenerated before it can herniate. Herniation can be created in cadaveric tissues, either as a sudden injury7,8 or by a wear-and-tear (fatigue) process,9 suggesting that herniation can be a physical process driven by excessive mechanical loading. However, the fact that many patients report no injury before the onset of sciatica suggests that some discs are more susceptible to herniation than others, probably on account of the weakening effects of middle age10 or genetic factors.11 Degeneration, therefore, need not always precede herniation, and some degenerative changes can follow herniation. In this study we compared herniated disc tissue removed at surgery with disc tissue that had reached a similar stage of degeneration without herniating. We hypothesised that changes in herniated discs differ from those found in degenerated discs, and are consistent with being a consequence rather than the cause of herniation.

Materials and Methods The study was approved by the NReS ethics committee, Frenchay Hospital, Bristol, United Kingdom. Specimens of herniated disc material
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Table I. Details of the two groups of intervertebral disc specimens Herniated discs (HD) Discs (n) Mean age (yrs) (range) Spinal level (n) L2/3 L3/4 L4/5 L5/S1 Gender (n) Male Female Mean Pfirrman grade (range) Mean duration of pain (mths) (range) 21 53 (35 to 74) 1 2 4 14 8 13 3.6 (3 to 4) 14 (2 to 60) Degenerated in situ discs (DD) 11 53 (39 to 72) 1 0 6 4 6 5 3.0 (2 to 4) 18 (4 to 60)

removed at operation were compared with specimens from discs that had degenerated in situ, without herniation. Separate comparisons were made for the three types of tissue: nucleus pulposus (NP), inner annulus fibrosus (IAF) and outer annulus fibrosus (OAF). These can reliably be distinguished histologically. Specimens were from the posterior or posterolateral regions of the lumbar intervertebral discs of 32 patients; 21 with a herniated (extruded) disc, and 11 with other diagnoses including spondylolisthesis and discogenic pain, but with no herniation. An anonymous clinical data sheet was obtained for each patient, and the Pfirrmann grade12 of disc degeneration was recorded from MRI scans. Details of the groups are shown in Table I. After excision, disc samples were blotted on tissue paper to remove blood, and stored in small airtight containers. Within 20 minutes of surgery, samples were snap-frozen by immersing in chilled isopentane, and stored at -70C until sectioning. Histology. The specimens were moved from the freezer to a cryostat maintained at -20C and embedded in optimal cutting tissue (OCT) medium. Sections 5 m thick were obtained using a Leica CM1900 cryostat (Heidelberger, Nussloch, Germany) and fixed in 10% neutral buffered formalin. Sections were stained with Ehrlichs haematoxylin and eosin (H&E) to reveal details of the cells and matrix or with toluidine blue to assess proteoglycan loss. Regions of the NP, IAF and OAF were identified according to the shape and number of cells, and the appearance of collagen fibres and lamellae under polarised light. As far as possible, three fields of view representing each type of tissue were analysed in each thin section. Using criteria that have previously been described13 the following histological features were graded on ordinal scales: degree of cell clustering (0 to 5), number of inflammatory cells (0 to 3), proteoglycan loss (0 to 3), severity of tears/fissures in the matrix (0 to 3) and the presence of blood vessels (0 to 3). In each case, 0 refers to absence of the feature and the higher bound refers to abundance. Scores were averaged across fields of view, and then across specimen groups.

Immunohistochemistry. Labelled antibodies were used to detect cells producing the matrix-degrading metalloproteases (MMPs). The 5 m OCT-embedded sections were fixed in ice-cold acetone for 10 minutes and rinsed in phosphate-buffered saline (PBS). They were then treated with rabbit serum (Dako, Ely, United Kingdom) at 1:5 dilutions in PBS for 60 minutes to block non-specific binding of secondary rabbit antibody. The sections were treated with primary mouse monoclonal antibodies to the matrix-degrading enzymes MMP-1 (Abcam, Cambridge, United Kingdom), MMP-2 (Novus Biologicals, Cambridge, United Kingdom) and MMP-3 (Millipore, Watford, United Kingdom) at 1:25 dilutions in PBS, and incubated at 4C overnight. Secondary antibodies (biotinylated rabbit anti-mouse IgG; Dako), diluted 1:200 in PBS, were applied for 60 minutes, and the antigenantibody signal was amplified using extra avidin alkaline phosphatase conjugate (Sigma-Aldrich, Poole, United Kingdom), diluted 1:100. Fast red (Sigma-Aldrich) was applied for 10 minutes. In the controls the primary antibodies were omitted during incubation. Sections were mounted with Faramount medium (Dako) and examined using a Leica DMRB light microscope. Three fields of view per section were taken from the NP, IAF and OAF regions, whenever present. Images were captured with a computerlinked Olympus DP72 camera (12.8 megapixels). Immunopositive cells were counted and analysed using Volocity software (Perkin Elmer, Cambridge, United Kingdom). Immunofluorescence with laser confocal microscopy. This combination of techniques facilitated the detection and quantification of small nerves and blood vessels in large volumes of dense tissue. Specimens were sectioned at 30 m on a Leica CM1900 cryostat before being fixed in ice-cold acetone for 10 minutes and rinsed in PBS. Sections were then blocked with 20% donkey serum (Sigma Aldrich) in PBS for 60 minutes at 4C, washed with three changes of saline, and treated with mouse monoclonal primary antibodies. Dilutions were 1:25 for PGP 9.5 (general cytoplasmic neuronal marker; ABD Sorotec, Kidlington, United Kingdom), 1:500 for Substance P (sensory nerve marker;
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Table II. Summary of results comparing herniated discs (HD) with degenerated discs (DD). Each of the three tissue types (nucleus, inner annulus and outer annulus) is considered separately, and n values reflect the absence of some tissue types from some disc samples Nucleus Mean (SD) variable
*

Inner annulus HD (n = 21) 2.2 (1.5) 2.1 (0.4) 2.5 (1.0) 1.0 (0.9) 1.3 (1.1) 10 564 (13 015) DD (n = 11) 1.5 (0.7) 1.8 (0.9) 2.4 (1.2) 0.0 (0.1) 0.2 (0.5) 1597 (5221) p-value 0.005 0.584 0.685 0.001 0.012 0.046

Outer annulus HD (n = 12) DD (n = 8) p-value 0.039 0.048 0.008 0.051 0.028 0.050

HD (n = 17) DD (n = 8) p-value 1.5 (1.0) 0.9 (0.7) 1.2 (1.0) 0 0 0 0.350 0.136 0.742 -

Tears/fissures (0 to 3) 1.8 (1.5) PG loss (0 to 3) 1.4 (0.8) Cell clusters (0 to 5) 1.4 (0.8) Inflammation (0 to 3) 0 Blood vessels (0 to 3) 0 0 Blood vessels (CD 31 area m2) 2 Nerves (count/mm ) Sub P 0 PGP 9.5 0 Mean MMP count (cells/mm2) MMP-1 41.7 (52.4) MMP-2 21.1 (23.7) MMP-3 31.1 (28.5)

2.2 (0.7) 1.3 (0.9) 1.8 (1.0) 1.0 (0.4) 0 0.2 (0.3) 1.4 (1.2) 0.4 (0.8) 1.3 (0.9) 0.4 (0.8) 18 433 (22 184) 4788 (13 993)

0 0

0.55 (0.76) 0.68 (0.89) 99.8 (71.5) 33.3 (26.7) 84.5 (79.1)

0.09 (0.30) 0.18 (0.40) 55.4 (47.3) 25.5 (21.9) 28.6 (29.0)

0.051 0.054 0.031 0.434 0.050

1.27 (1.22) 1.38 (1.20) 60.9 (72.7) 15.8 (19.5) 21.8 (47.4)

0.29 (0.49) 0.43 (0.79) 20.9 (37.5) 7.4 (10.8) 12.0 (22.6)

0.042 0.048 0.052 0.046 0.052

35.7 (49.1) 0.938 13.0 (15.4) 0.876 26.4 (22.0) 0.785

* PG, proteoglycan; CD 31, cell differentiation factor 31 (endothelial cell marker); PGP 9.5, protein gene product 9.5 (neuronal marker); Sub P , Substance P (nociceptive neuronal marker); MMP , matrix metalloproteinases)

Abcam, Cambridge, United Kingdom) and 1:20 dilution for CD 31 (endothelial cell marker; Dako, Cambridgeshire, United Kingdom). Immunofluorescence was achieved using conjugated Alexa Fluor donkey anti-mouse secondary antibody (Invitrogen, Paisley, United Kingdom) at 1:200 dilution in PBS. In order to diminish autofluorescence, sections were immersed in 0.01% Sudan black B for 5 minutes. DAPI (4',6-diamidino-2-phenylindole; Vector Laboratories, Peterborough, United Kingdom) was used to stain nuclei dark blue. For the secondary antibody, excitation was at 520 nm and emission at 594 nm. For DAPI, excitation was at 405 nm and emission at 488 nm. Controls were included in each staining process. Four thick sections were examined from each specimen and, depending on the section, up to three fields of view were taken of each of the three types of tissue (NP, IAF and OAF). All the sections were sequentially scanned to prevent cross-talk between different fluorophores. Digital images were captured with a Leica DM IRBE argon laser confocal inverted microscope. Volocity image analysis software was used to: a) count the number of discrete stained features (blood vessels or nerves) and b) calculate the total area of each stained feature in each of three fields of view, in each tissue type, in each section. Statistical analysis. Mann-Whitney U tests were used to compare mean values between herniated and non-herniated in situ tissues in the NP, IAF and OAF regions. Spearmans rank correlation was used to examine associations between histological variables assessed on ordinal scales. All tests were performed using SPSS software v16 (SPSS Inc., Chicago, Illinois). A p-value < 0.05 was considered to indicate statistical significance.

Results Each specimen of herniated disc (HD) was fully extruded through the posterior annulus at operation. Ten had lost continuity with the rest of the disc (sequestrations), and
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11 were in contact with a nerve root. Specimens of degenerated disc (DD) were removed from discs that were neither herniated nor bulging, and so were in structural continuity with the rest of the disc. Histology. OAF tissue was characterised by arrays of crimped type I collagen fibres, organised into narrow and approximately parallel lamellae with alternating fibre angles, and with elongated fibroblast-like cells. NP tissue had no crimped collagen, and contained rounded chondrocyte-like cells. Intermediate IAF tissue had irregular collagen fibres in wide and disorganised lamellae, with cells that were more rounded than elongated, and frequently appeared in clusters. The histology scores are summarised in Table II (rows 1 to 5). Tears and fissures in the extracellular matrix were more abundant in HD specimens (Fig. 1). Artefactual tears arising from tissue processing could often be distinguished by a lack of cells on the free surfaces, and were not included in the scoring system. Loss of proteoglycan was widespread, especially in HD tissue (Fig. 1a and 1c). Cell clustering (Fig. 1c) was most common in IAF tissue, especially near the boundary with the nucleus, but was rare in OAF tissue. Inflammatory cells were not observed in NP tissue, and in the annulus were much more abundant in HD than in DD specimens (Fig. 1a and 1d). Inflammatory cells were particularly abundant around fissured areas of annulus that also showed marked loss of proteoglycans (Fig. 1d), and scores for inflammation were highly correlated with scores for proteoglycan loss (r2 = 0.80; p < 0.001). Blood vessels manifested as single-layer endothelial cell capillaries, and were observed only in the annulus (Fig. 1b). They were more common in HD than DD specimens, and often accompanied inflammatory cells near matrix fissures and on free surfaces (Fig. 1b and 1d), although overall correlation between scores for inflammation and fissuring was non-significant. Blood vessels were observed in only two DD specimens, and both had well-developed radial fissures

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Fig. 1a

Fig. 1b

Fig. 1c

Fig. 1d

Histological images. Figure 1a Proteoglycan depletion of the matrix is indicated by weak toluidine blue staining. Elongated nuclei of outer annulus cells contrast with those of more rounded inflammatory cells on the free surface (outer annulus, herniated disc). Figure 1b Fissures were co-localised with blood vessels (arrows) and inflammatory cells (outer annulus, herniated disc, haematoxylin & eosin (H&E) staining). Figure 1c Extensive cell clustering (arrows) was associated with fissuring (inner annulus, herniated disc, H&E staining). Figure 1d Extensive inflammatory cell invasion was associated with blood vessels (arrows) and fissures (outer annulus, herniated disc, H&E staining). Figure 1e Blood vessels were associated with fissures (inner annulus, degenerated disc, H&E staining). In all cases, bar = 50 m.

Fig. 1e

in the annulus (Fig. 1e). Figure 2 compares all histological variables between HD and DD annulus. Immunohistochemistry. Cells staining positive for the matrix-degrading enzymes MMP1, MMP2 and MMP3

(Fig. 3) were more common in HD than DD specimens (Table II), although the differences only reached significance in the annulus. MMP-2 staining usually affected fibroblasts, solitary chondrocytes and medium-sized cell
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Histological variables Cell clusters PG loss Tears Inflammatory cells Blood vessels

2.50

2.00 Mean scores

1.50

1.00

0.50
Fig. 3a

0.00 Degenerated-in-situ Patient Group

Fig. 2 Bar charts comparing the mean histological scores for degenerated and herniated discs. Scores were averaged for the inner and outer annulus. The following features differed significantly between the two groups: PG loss (p = 0.028), tears (p < 0.001), inflammatory cells (p < 0.001), and blood vessels (p < 0.001). Error bars denote the standard error of the mean (SEM).

Herniated

clusters, whereas MMP-1 and -3 staining was observed on single, paired, clustered and fibroblastic cells, sometimes in association with inflammatory cells and blood vessels. Immunofluorescence with laser confocal microscopy. Thick sections and fluorescent staining made it easier to detect small blood vessels and nerves (Fig. 4). Values in rows 6 to 8 of Table II refer to the absolute area of CD-31 staining (m2), and to the mean number of discrete structures (per mm2) staining positive for PGP 9.5 and Substance P. Blood vessels were much more common in HD than in DD specimens. The same was true of nerves that were positive for PGP 9.5 or Substance P, and that tended to associate with blood vessels. No blood vessels or nerves were found in NP tissue, and seven HD specimens that showed no immunopositivity to CD-31, Substance P or PGP 9.5 in confocal microscopy contained only NP tissue.

Fig. 3b Immunohistochemical images. Figure 3a Inflammatory cells staining positive (red) for matrix metalloproteinases (MMP)-1 (inner annulus, herniated disc). Figure 3b Two cells showing cytoplasmic staining for MMP-1 (inner annulus, degenerated disc). In both images Ehrlichs haematoxylin was used to counterstain cell nuclei blue. Bar = 50 m.

Discussion This is the first direct comparison between herniated intervertebral disc tissues and tissues that have reached a similar (Pfirrman) grade of degeneration without herniating. We found that herniation involved distinct tissue changes compared to degeneration, and differences were usually most pronounced in OAF tissue and least in NP tissue, where no differences were statistically significant. Agerelated degeneration is usually most advanced in the NP,14 so the similarities in NP tissue suggest that herniation did
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not represent an advanced or accelerated stage of normal disc degeneration in 21 of the 32 discs in the present study. The strengths of the study included the use of the same techniques and reagents on both samples of discs, and the use of laser confocal microscopy to examine multiple thick sections of tissue reduced the sampling problems inherent in conventional histological assessment. A third strength was the use of automatic quantitative image analysis to minimise the risk of subjective bias. Although herniated and degenerated discs have not previously been compared, Carreon et al15 compared traumatic cervical disc herniation with degenerated disc herniation and reported that proteoglycan loss, MMP activity and inflammation were similar in both. This contrasts with our findings for annulus tissues (Fig. 2), but

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Fig. 4a

Fig. 4b

Images from immunofluorescence and confocal microscopy. Figure 4a Blood vessels immunostained red for CD31 (outer annulus, herniated disc). Figure 4b Small nerve immunostained red for PGP 9.5 (outer annulus, herniated disc). In both images cell nuclei are counterstained blue with DAPI (4',6-diamidino-2-phenylindole). Bar = 50 m.

there may be no conflict because the previous study did not distinguish between types of tissue. A comparison between protruded and extruded discs reported fewer inflammatory cells in the former,16 which is consistent with our results. Many previous studies have examined herniated or degenerated discs without comparing them. Herniated discs often show inflammatory changes, which are attributed to neovascularisation and healing following injury.17 This inference is supported by an increased expression of growth factors in herniated discs compared with macroscopically normal discs in patients of similar age.18 Vigorous inflammatory and healing processes evident in herniated disc tissue probably explain why they can reabsorb over time.19 Other reported changes in herniated discs include nerve terminals positive for Substance P,20 invasion by macrophages and lymphocytes,21 increased expression of MMPs and prostaglandins,22 and increased apoptosis,23 which is itself associated with tissue injury24 and swelling.25 In contrast, discs that degenerate without herniating show slowly progressive and widespread changes that cannot easily be distinguished from normal ageing, and which are more advanced in the NP.14,26 Proteoglycans become fragmented and are lost from the tissue,27 leading to reduced hydration28 and loss of pressure within the nucleus.29,30 Increased cell senescence31 and apoptosis23 reduce the population of viable cells. Cell clustering increases,32 particularly in the IAF and NP,33 and many clustered cells increase the production of matrix-degrading enzymes such as the MMPs.26 Nutrient transport problems34 probably do not initiate these changes, because transport across the endplate increases with age and degeneration,35 but there can be little doubt that poor transport of nutrients impairs healing, especially in the centre of the disc. Differences between herniated and degenerated discs can be explained as follows. Herniated tissue that escapes the

pressurised confines of the disc swells and loses proteoglycans, and these changes, which occur in only a few days in vitro,2 facilitate the invasion of inflammatory cells and the ingrowth of blood vessels and nerves.36 Swelling could also disturb cellmatrix interactions and promote apoptosis and cell proliferation. Neovascularisation can follow loss of proteoglycans, because they inhibit the ingrowth of blood vessels and nerves in vitro.37,38 This ingrowth would also be facilitated by the proximity of herniated tissue to blood vessels in the posterior longitudinal ligament and nerve roots. The lack of differences between herniated and degenerated NP tissue suggests that herniation is not a normal progression from degeneration. Rather, it appears to involve additional processes that affect the annulus most, and so may occur from the outside in, after herniation has occurred. Animal models of disc injury and healing support this concept: a stab or slash in the peripheral annulus provokes a vigorous healing response in OAF tissues only,39,40 where the density of cells is highest.33 Discs that degenerate in situ can have a similar MRI appearance to herniated discs, because MRI changes primarily reflect loss of proteoglycans and water, as indicated by the T2 relaxation time. However, the structural cohesion of an intact annulus, which acts in tension to restrain the pressure of swelling in the NP, prevents gross tissue swelling and its sequelae. The confined pressurised environment within an intact disc also prohibits the ingrowth of blood vessels and nerves, except where there is a gross radial fissure to facilitate their entry,41,42 as was found in two degenerated discs in this study. Distinctions between disc herniation and degeneration are important clinically. Discs that degenerate in situ often cause no major symptoms,43 whereas many herniated discs give rise to sciatica and may become the focus of medicolegal litigation. Some herniated discs may possibly herniate because of constitutional degenerative changes and genetic
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factors,11 and this would explain why degenerative changes are sometimes seen at levels adjacent to a herniated disc. However, the results of this study support the epidemiological evidence that some discs herniate for other reasons, including injury,44-46 and that some degenerative changes evident at surgery are the consequences rather than causes of herniation. Indeed, herniation is a known risk factor for further disc degeneration.47 In conclusion, degenerative changes in herniated discs can be the consequence rather than the cause of herniation. It should not be assumed that degenerative changes always precede (or cause) disc herniation.
This work was funded in the United Kingdom by BackCare, and by a scholarship from the State Government of Sikkim, India. No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. This article was primary edited by J. Butler and first-proof edited by J. Scott.

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