Cell clusters in intervertebral disc degeneration: an attempted repair mechanism aborted via apoptosis.

Cell clusters are a histological hallmark feature of intervertebral disc degeneration. Clusters arise from cell proliferation, are associated with replicative senescence, and remain metabolically, but their precise role in various stages of disc degeneration remain obscure. The aim of this study was therefore to investigate small, medium, and large size cell-clusters. For this purpose, human disc samples were collected from 55 subjects, aged 37-72 years, 21 patients had disc herniation, 10 had degenerated non-herniated discs, and 9 had degenerative scoliosis with spinal curvature <45°. 15 non-degenerated control discs were from cadavers. Clusters and matrix changes were investigated with histology, immunohistochemistry, and Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Data obtained were analyzed with spearman rank correlation and ANOVA. Results revealed, small and medium-sized clusters were positive for cell proliferation markers Ki-67 and proliferating cell nuclear antigen (PCNA) in control and slightly degenerated human discs, while large cell clusters were typically more abundant in severely degenerated and herniated discs. Large clusters associated with matrix fissures, proteoglycan loss, matrix metalloproteinase-1 (MMP-1), and Caspase-3. Spatial association findings were reconfirmed with SDS-PAGE that showed presence to these target markers based on its molecular weight. Controls, slightly degenerated discs showed smaller clusters, less proteoglycan loss, MMP-1, and Caspase-3. In conclusion, cell clusters in the early stages of degeneration could be indicative of repair, however sustained loading increases large cell clusters especially around microscopic fissures that accelerates inflammatory catabolism and alters cellular metabolism, thus attempted repair process initiated by cell clusters fails and is aborted at least in part via apoptosis.

Degenerative physiochemical events in the pathological intervertebral disc.

Low back pain is one of the commonest musculoskeletal complaints that affects individuals of all ages and is a leading contributor towards work loss worldwide. The range of current treatment modalities involving surgeries, injectable agents, and medications is promising but cannot address the reasons behind the occurrence of pain in patients with degenerative disc pathologies. One possible factor for the limited success is the lack of evidence behind the identification of early, intermediate, and late stages of painful changes methodologically in a vast group of populations and the manifestation of the diseases in terms of increased physical activity, hereditary patterns, and various risk factors. However, despite these challenges, steady progress has been achieved in understanding the parameters in abnormally loaded progressively degenerating discs and these features have been elucidated at a physical, biochemical, and cellular level. These recent findings can likely lead to the development of therapeutic interventions that will identify and retard tissue damage, decrease pain, and improve the quality of life in these patients. Therefore, the main aim of this review is to integrate recent updates in intervertebral disc degeneration research for the development of evidence-based screening protocols and more targeted interventions in the management of low back pain.

Physical disruption of intervertebral disc promotes cell clustering and a degenerative phenotype.

To test the hypothesis that physical disruption of an intervertebral disc disturbs cell-matrix binding, leading to cell clustering and increased expression of matrix degrading enzymes that contribute towards degenerative disc cell phenotype. Lumbar disc tissue was removed at surgery from 21 patients with disc herniation, 11 with disc degeneration, and 8 with adolescent scoliosis. 5 µm sections were examined with histology, and 30-µm sections by confocal microscopy. Antibodies were used against integrin α5beta1, matrix metalloproteinases (MMP) 1, MMP-3, caspase 3, and denatured collagen types I and II. Spatial associations were sought between cell clustering and various degenerative features. An additional, 11 non-herniated human discs were used to examine causality: half of each specimen was cultured in a manner that allowed free 'unconstrained' swelling (similar to a herniated disc in vivo), while the other half was cultured within a perspex ring that allowed 'constrained' swelling. Changes were monitored over 36 h using live-cell imaging. 1,9-Di-methyl methylene blue (DMMB) assay for glycosaminoglycan loss was carried out from tissue medium. Partially constrained specimens showed little swelling or cell movement in vitro. In contrast, unconstrained swelling significantly increased matrix distortion, glycosaminoglycan loss, exposure of integrin binding sites, expression of MMPs 1 and 3, and collagen denaturation. In the association studies, herniated disc specimens showed changes that resembled unconstrained swelling in vitro. In addition, they exhibited increased cell clustering, apoptosis, MMP expression, and collagen denaturation compared to 'control' discs. Results support our hypothesis. Further confirmation will require longitudinal animal experiments.

Nerves and blood vessels in degenerated intervertebral discs are confined to physically disrupted tissue.

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

Facet joint degeneration in adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a poorly understood deformity of the thoracolumbar spine which affects the intervertebral discs (IVDs) and the articular facet joints. The knowledge concerning facet joints in this context is very limited, although facet joint degeneration is a known contributor of back pain. In this study, a comprehensive investigation was performed to characterize the facet joint chondrocytes and extracellular matrix within the scoliotic spine. Surgically removed articular facet joint tissues were collected from patients undergoing spinal corrective surgery for AIS deformities, while non-scoliotic articular facet joint tissues were obtained from cadaveric organ donors. Alterations in cartilage tissue structure were evaluated histologically with safranin-O fast green and a modified OARSI grading scale. Pro-inflammatory cytokines, matrix-degrading proteases, and fragmented matrix molecules associated with cartilage degradation were analyzed by immunohistochemistry and western blotting. Safranin-O fast green staining revealed that young scoliotic facet joints show clear signs of degeneration with substantial proteoglycan loss, similar to osteoarthritis (OA). The proteoglycan levels were significantly lower than in healthy asymptomatic non-scoliotic control individuals. In comparison to controls, scoliotic articular facets showed increased cell density, increased expression of the proliferation marker Ki-67, and higher expression of MMP-3, MMP-13, and IL-1ß. Expression and fragmentation of the small leucine-rich proteins (SLRPs) chondroadherin, decorin, biglycan, lumican, and fibromodulin were analyzed with western blot. Chondroadherin and decorin were fragmented in cartilage from patients with a curve greater than 70°, whereas biglycan and fibromodulin did not show curve-related fragmentation. AIS facet joint cartilage shows hallmarks of OA including proteoglycan loss, overexpression of pro-inflammatory mediators, increased synthesis of matrix-degrading proteases and fragmentation of SLRPs. As with patients with age-related OA, the premature joint degeneration seen in scoliotic patients is likely to contribute to the pain perceived in some individuals.

Morphometry and aberrant morphology of the adult human tricuspid valve leaflets.

The tricuspid valve complex has been studied since the beginning of the twentieth century, and variations in the structural orientation of the tricuspid leaflets has been reported before, as the occurrence of accessory leaflets poses a major problem during surgeries related to the tricuspid valve. In this study, 36 adult formalin-fixed human hearts were analyzed to compare the number, form and size of the tricuspid leaflets. The result shows that in right ventricles, the number of leaflets can vary from the routine three to as many as seven, and the localization of such accessory leaflets of the tricuspid valve differs between specimens. Five leaflet forms were the most common, and the 'typical' form of tricuspid valves with no accessory leaflets was only present in a small percentage of the cases studied. Measurements of the main and accessory leaflets showed that the anterior leaflets were the largest, followed by the inferior leaflets, while the septal and the accessory leaflets were the smallest in size. On the basis of these results, it is suggested that three leaflets of the tricuspid valve are relatively uncommon, with frequent occurrences of accessory leaflets. The multicuspidal form of the tricuspid valve therefore raises concern about understanding the functional and physiological significance of the accessory leaflets.

Aberrant Dual Origin of the Dorsal Scapular Nerve and Its Communication with Long Thoracic Nerve: An Unusual Variation of the Brachial Plexus.

Pre and post-fixed variations at roots of the brachial plexus have been well documented, however little is known about the variations that exist in the branches which arise from the brachial plexus. In this paper, we describe about one such rare variation related to the dorsal scapular and the long thoracic nerve, which are the branches arising from the roots of the brachial plexus. The variation was found during routine dissection. The dorsal scapular nerve, which routinely arises from the fifth cervical nerve root (C5), was seen to receive contributions from C5 as well as sixth cervical nerve (C6), while the long thoracic nerve arose from C6 and seventh cervical nerves (C7) only. Furthermore along with variations in origin of the dorsal scapular and long thoracic nerves, the brachial plexus was seen to exist as a prefixed plexus receiving a contribution from C4 nerve root. An aberrant communicating branch between the dorsal scapular and long thoracic nerve was also identified. Knowledge about the course and anatomy of such variations can be vital for understanding the aetiology of various conditions such as winging of scapula, interscapular pain, administration of cervical nerve blocks, surgeries and for effective management of regions and muscles supplied by dorsal scapular and long thoracic nerve.

Why do some intervertebral discs degenerate, when others (in the same spine) do not?

This review suggests why some discs degenerate rather than age normally. Intervertebral discs are avascular pads of fibrocartilage that allow movement between vertebral bodies. Human discs have a low cell density and a limited ability to adapt to mechanical demands. With increasing age, the matrix becomes yellowed, fibrous, and brittle, but if disc structure remains intact, there is little impairment in function, and minimal ingrowth of blood vessels or nerves. Approximately half of old lumbar discs degenerate in the sense of becoming physically disrupted. The posterior annulus and lower lumbar discs are most affected, presumably because they are most heavily loaded. Age and genetic inheritance can weaken discs to such an extent that they are physically disrupted during everyday activities. Damage to the endplate or annulus typically decompresses the nucleus, concentrates stress within the annulus, and allows ingrowth of nerves and blood vessels. Matrix disruption progresses by mechanical and biological means. The site of initial damage leads to two disc degeneration "phenotypes": endplate-driven degeneration is common in the upper lumbar and thoracic spine, and annulus-driven degeneration is common at L4-S1. Discogenic back pain can be initiated by tissue disruption, and amplified by inflammation and infection. Healing is possible in the outer annulus only, where cell density is highest. We conclude that some discs degenerate because they are disrupted by excessive mechanical loading. This can occur without trauma if tissues are weakened by age and genetic inheritance. Moderate mechanical loading, in contrast, strengthens all spinal tissues, including discs.

Significance of cartilage endplate within herniated disc tissue.

PURPOSE: Disc herniations sometimes contain hyaline cartilage fragments, but their origins and significance are uncertain. METHODS: Herniations were removed surgically from 21 patients (aged 35-74 years) whose main symptom was sciatica (10 patients) or back pain (11 patients). Frozen sections, 5 µm thick, were examined histologically, and antibodies were used to label the matrix-degrading enzyme MMP 1, pro-inflammatory mediator TNFα, and cell proliferation marker Ki-67. Proportions of each tissue type were quantified by image analysis. Cartilage and bone components of the endplate were examined in 7-µm frozen sections from 16 cadaveric spines, aged 61-98 years. RESULTS: Cartilage fragments were found in 10/21 herniations. They averaged 5.0 mm in length, comprised 25 % of the herniation area, and two had some bone attached. Hyaline cartilage was more common in herniations from patients with sciatica (7/10) than with back pain (3/11, P = 0.050), and the area (%) of the herniation occupied by the cartilage was greater in sciatica patients (P < 0.05). Cartilage fragments showed little evidence of swelling, proteoglycan loss or inflammatory cell invasion, although cell clustering was common, and TNFα was sometimes expressed. Each cartilage fragment showed at least one straight edge, as if it had been peeled off the bony endplate, and this mechanism of failure was demonstrated in preliminary mechanical experiments. CONCLUSION: Disc herniations often include hyaline cartilage pulled from the vertebral endplates. Cartilage fragments show little swelling or proteoglycan loss, and may be slow to resorb, increasing the risk of persisting sciatica. Loss of cartilage will increase endplate permeability, facilitating endplate inflammation and disc infection.

Chondrohumeralis and axillary arch of Langer: a rare combination of variant muscles with unique insertion.

During routine dissection, we found a rare case of an aberrant muscular slip originating from the pectoralis major called as the "chondroepitrochlearis / thoracoepicondylaris / costohumeralis" and an anomalous slip from the latissimus dorsi called as "axillary arch of Langer / musculus dorsoepitrochlearis" in the same axilla. Interestingly, these two slips found to have a common insertion after arching superficial to the axillary neurovascular bundle, into the fascia covering the biceps brachi and to the lateral lip of the intertubercular sulcus of the humerus. Presence of combination of these two rare variant muscles and their unique insertion pattern has not been reported earlier. The knowledge of these muscle variations is important for the clinicians and physiotherapist for better diagnosis and treatment. Here in this report, in addition to the case presentation, we also discuss their clinical significance and the review of literature in detail.

High origin of dorsal branch of the ulnar nerve and variations in its branching pattern and distribution: a case report.

INTRODUCTION: Ulnar nerve is a branch of the brachial plexus. In the front of the forearm, normally near the wrist joint, it gives a dorsal cutaneous branch which supplies the skin of the dorsum of the hand. CASE PRESENTATION: The present case reports a very rare finding, the dorsal branch of the ulnar nerve along with the main nerve trunk originated between the two heads of the flexor carpi ulnaris muscle, after descending along the medial border of the forearm extensor surface, on the dorsal aspect of the wrist it is divided into three branches, one medial and two lateral. The medial most division received a communicating branch from the superficial ramus of the ulnar nerve and continued as the medial proper digital nerve of the little finger. The lateral two divisions became cutaneous on the medial half of the dorsum of the hand along the medial three digits i.e. radial and ulnar side of little, ring and middle finger. CONCLUSION: The site, extent of injury, variations and the delay in the treatment, significantly influences the outcome of ulnar nerve repair. Thus, an adequate knowledge of all possible variations in the ulnar nerve may be important for clinicians and may help to explain uncommon symptoms.