A. R. Michael

Nerves are more abundant than blood vessels in the degenerate human intervertebral disc.

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

Osteochondroma arising from a lumbar facet joint in a 16-year-old.

Osteochondromas are benign tumours of bony or cartilaginous origin, which may be solitary or multiple. They are rare in the axial skeleton and have previously been reported to arise from facet joints only in four cases in the English literature. We report the case of a 16-year-old girl who presented with a palpable bony lump and a short history of back pain. Imaging showed a bony lesion arising from a left-sided facet joint in the lumbar spine. Following excision biopsy, there was complete resolution of symptoms. The literature on the origin and management of spinal osteochondromas is discussed.

Early mortality and morbidity following a type II odontoid fracture in the elderly

BACKGROUND We aimed to analyse the rates of early and causes of death in patients aged over 65 years with a type II odontoid fracture. METHODS A consecutive series of 93 patients with a type II fracture of the odontoid process was retrospectively identified. Data collected included patient demographics, co-morbidities, associated injuries, neurological injury, date of death and cause of death. Mean patient age was 81. Five patients (5%) were treated operatively while the rest were treated in a hard cervical collar. Five patients (5%) had an incomplete cervical cord injury secondary to the fracture. RESULTS The rate of mortality at 30 days was 10% (9 patients) and at 90 days it was 16% (15 patients). Following multivariate analysis, the factors found to significantly increase the risk of 30-day mortality included increasing age, increasing injury severity score and leukaemia. Following univariate analysis the only factor found to increase the risk of 90-day mortality was advancing age. The commonest causes of death were pneumonia and ischaemic coronary disease. CONCLUSION Our results suggest that this patient cohort is frail and at risk of early mortality. We suggest that their inpatient care be provided in close conjunction with elderly care physicians.

Should We Remove Bullets From The Spinal Cord

Introduction To discuss a case and review evidence for and against intra-dural bullet removal. Materials and Methods A 45 year-old gentleman presented with gunshot wound, with a loss of sensation below his chest and loss of power in his lower limbs. On examination there was a single 1cmx0.5cm bullet entry wound on the anterolateral aspect of the left shoulder, with a T3 motor and sensory level. PR revealed reduced tone and no squeeze. A full body CT showed a bullet within the spinal canal adjacent to the T3 vertebral body. The spinal injuries team diagnosed a T3 complete ASIA A paraplegia. He underwent a posterior decompression of T3 and intra-dural bullet removal with dura repair on the same day. Surgery revealed a dural tear and an intra-dural bullet with obvious cord damage. This was removed with rubberised forceps and handed to police for ballistics. He was given flucloxacillin and transferred to the spinal injuries unit for rehabilitation. He made no neurological recovery but did not develop any further complications. Results A thorough literature search revealed the role of surgery in gaining lost neurological function remains ambiguous. Indications for bullet removal include acute neurological deterioration and CSF fistulas. Epidural haematoma/abscesses, radiological compression or a destabilized spine are also considerations. Removals of bullets below T12 have had an effect on motor recovery. However similar neurological recoveries have been reported in both surgical and conservative management of incomplete deficits. Compete deficits are unlikely to improve neurologically regardless of surgical intervention, however cervical injuries with early detection of compressive pathology should be considered. Complication rates are reported to be higher if operated. Prophylactic antibiotics should be started immediately on admission. Surgery does not reduce the incidence of infection. Pain may be intensified when caused by a gunshot injury, but there is no evidence of improvement with bullet removal. One indication for surgery in this case was to prevent post traumatic syringomyelia (PTS). The incidence is 0.3–3.2%, however radiological/autopsy studies suggest up to 22%. There has been one prior reported case where a patient developed symptoms 14 months following initial gunshot injury. However due to lack of data it is uncertain if initial surgical management reduces development of future syrinxes. Conclusions This appears to be the first reported case of an intra-dural bullet in the UK. Treatment should be focused on ensuring spine stability, enhancing potential for neurological recovery and preventing complications. The role of surgery versus non-surgical treatment is still debate.

Human Degenerate Discs Show Increased Activation of Intracellular Signalling Pathways of IL-1

Introduction Intervertebral disc degeneration is implicated in 40% of low back pain cases. Interleukin 1 (IL-1) is known to be important in the pathogenesis of intervertebral disc (IVD) degeneration, here we investigated the intracellular signaling pathways activated by IL-1 and determined the activation status of these pathways in native IVD tissues highlighting potential pathways for new therapies. Materials and Methods Human nucleus pulposus cells (NP) removed during discetomy for nerve root pain were stimulated with IL-1 for 30 minutes. The activation of ERK1/2, p38, c-jun, and IκB were determined using cell-based enzyme-linked immunosorbent assays, in addition pNFκB localisation in stimulated cells was determined using immunohistochemistry. Localisation of phosphorylated c-jun, p38, ERK1/2, and NFκB together with IL-1 was investigated within paraffin embedded sections of human IVD to investigate the presence of active pathways in vivo. Pretreatment with inhibitors of p38, c-jun, and NFκB for 30 minutes before stimulation with IL-1 (10 ng/mL) for 48 hours was investigated to determine the ability of individual signaling inhibitors to block the catabolic responses induced by IL-1. Results IL-1-induced activation of p38, MAPK, ERK ½, JNK/c-jun, and NFκB signaling pathways in human NP cells. IVD tissue samples displayed immunopositive staining for phosphorylated c-jun, ERK1/2, NFκB, and p38, immunopositivity was significantly increased within degenerate discs, particularly in those discs which also expressed high levels of IL-1. Inhibition of individual signaling pathways demonstrated differential regulation of the pleathora of IL-1-induced catabolic events, with different signaling molecules shown to regulate matrix metalloproteases, cytokines, and factors involved in innervation and angiogenesis of the disc. Conclusion Here, we have shown that the signaling pathways activated by IL-1 in vitro display increased activation in vivo during disc degeneration. Inhibitors of these pathways demonstrated multiple signaling pathways were involved in the pleathora of IL-1 actions. Thus, inhibition of signaling pathways could be a novel mechanism to inhibit catabolic processes which could hold promise to inhibit degeneration at early stages of disease but also create the correct tissue niche to promote regeneration of the disc. Disclosure of Interest None declared

Recombinant IL-1Ra Stimulation of Native NP Cells Inhibits a Multitude of Pathological Features

Introduction Interleukin-1 (IL-1) has been implicated in the pathogenesis of disc degeneration, with increased levels seen during degeneration without a concordant increase in its natural inhibitor: IL-1 receptor antagonist (IL-1Ra). IL-1 has been shown to stimulate a pleathora of catabolic actions including matrix degrading enzymes, cytokines, chemokines, and factors which promote nerve and blood vessel ingrowth. Here, we investigated the effect of the natural inhibitor of IL-1, to determine its ability to inhibit catabolic events regulated by natively produced IL-1 in degenerate human nucleus pulposus (NP) cells. Materials and Methods Human NP cells were isolated via collagenase digestion from four human intervertebral disc (IVD) obtained from degenerate disc samples from patients undergoing discetomy. Following expansion in monolayer culture, cells were transferred to alginate bead cultures at passage two and maintained for 2 weeks before stimulation with IL-1Ra to enable redifferentiation to take place. Cells were treated with IL-1Ra at 1, 10, 100 pg/mL and 1, 10, 100 ng/mL for 48 hours. Following stimulation, cells were released from alginate beads and RNA isolated using Trizol reagent. cDNA synthesized and real-time PCR used to determine the effects on a wide range of gene targets including matrix metalloproteases (MMPs), cytokines, chemokines, and factors involved in nerve ingrowth (NGF), pain pathways (substance P), and angiogenesis (vascular endothelial growth factor, VEGF). Results Baseline levels of all factors investigated varied between patients as did the overall responsiveness to IL-1Ra stimulation. IL-1Ra stimulation resulted in decreased gene expression for several MMPs including MMPs 3 and 13; cytokines including IL-1 and IL-6; chemokines including IL-8, CCL2, and CXCL3. NGF, substance P, and VEGF were also all decreased by IL-1Ra stimulation. Effective doses were target and patient-specific and while the highest dose of IL-1Ra often resulted in decreased expression of catabolic factors in all patients, lowest doses (1.10 pg/mL IL-1Ra) resulted in stimulation of some catabolic responses in some patients, for example, IL-6 and IL-8 were increased in two out of four patient samples investigated at low doses of IL-1Ra. Conclusion IL-1 has been implicated as a key factor in the pathogenesis of disc degeneration, however till date it is not fully understood whether inhibition of this cytokine in native disc cells can inhibit the plethora of pathogenic factors linked to IL-1. This study has demonstrated that treatment of NP cells with IL-1Ra results in decreased expression of MMPs, cytokines, chemokines, nerve, and blood vessel growth factors and neurotrophic factors, suggesting IL-1Ra could be a useful agent in the prevention of further degeneration to create the correct tissue niche to support tissue regeneration. However, patient responses were seen to be variable, with some patients displaying increased expression of some catabolic factors at low doses of IL-1Ra, thus selection of patients and carefully controlled drug release would be essential to enable such a therapy to be deployed successfully. Disclosure of Interest None declared

Thermally Triggered Injectable Hydrogel Carrier System for Mesenchymal Stem Cell Delivery to Promote Intervertebral Disc Regeneration

Introduction Instability of the motion segment as a result of intervertebral disc (IVD) degeneration is well known as a major cause of low back pain. Several tissue engineering and stem cell approaches have been investigated to attempt to regenerate the degenerate IVD. However, many of the proposed therapies would entail lengthy/risky operations to “implant” the tissue regeneration scaffold. Our vision is to develop a novel, injectable biomaterial delivery system which can deliver mesenchymal stem cells (MSCs) to the degenerate nucleus pulposus (NP) to stimulate regeneration, together with inhibitors of degeneration if required and provide mechanical support to the motion segment enabling regeneration to take place. Materials and Methods Here, we investigated a novel hydrogel system, which can be maintained as a liquid ex vivo and can be injected into the IVD where body temperature triggers in situ solidification. Human MSCs were incorporated into hydrogel systems and cell viability, migration characteristics, SEM, gene expression for NP markers, and matrix production investigated to determine differentiation capacity of incorporated MSCs. Mechanical properties of hydrogel systems were determined using dynamic mechanical analysis and compared with native NP tissue. Microparticles incorporation to provide delivery of inhibitors of degeneration were also investigated. Results Viability of MSCs was maintained within hydrogel systems for the 6 weeks investigated, where they were shown to migrate through the hydrogel system, deposit matrix within and differentiate toward NP cells with a switch in matrix gene expression from collagen type I to type II, and deposition of GAGs with immunopositivity seen for aggrecan following 4 weeks in culture. DMA analysis demonstrated hydrogel systems containing 10% hyaluronic acid displayed similar mechanical properties to native NP cells. Hydrogel systems were injected via 26-gauge needles into collagenase digested bovine caudal discs, which was maintained during loading of the bovine motion segments. In addition, microparticles were successfully incorporated into hydrogel systems in the liquid state before thermal triggered solidification. Conclusion Here, we have developed a hydrogel system with the potential to deliver MSCs via minimally invasive injection using small bore needles which decreases the chance of inducing damage to the annulus fibrosus. Hydrogel systems were nontoxic, support MSC growth and differentiation, and show potential to provide mechanical support to the motion segment while regeneration takes place. Such a therapy combined with inhibition of degeneration could show great promise for early and mid-stages of degeneration. Disclosure of Interest None declared

Role of Semaphorins in Angiogenesis and Innervation in Human Intervertebral Disc Degeneration

Introduction Although the intervertebral disc (IVD) is considered the largest aneural structure within the human body, during degeneration nociceptive nerves are seen to accompany blood vessels into the IVD yet it remains unclear how this process occurs. Semaphorins are axonal guidance molecules known to repulse nerves by altering the actin cytoskeleton of growth cones. Semaphorin 3A (sema3A) has recently been localized in healthy IVDs, suggesting an inhibitory role toward neural and vascular ingrowth. This study aimed to first identify the presence of the class 3 semaphorins and sema4D shown to be involved in promoting angiogenesis and their receptors within nucleus pulposus(NP) cells and an endothelial cell line and determine whether their expression could be altered by cytokine treatment. Materials and Methods Real-time PCR (RT-PCR) was performed to investigate gene expression of class 3 semaphorins; Sema3A-3F, sema4D, their receptors plexin A1-A4 and plexin D1 and neuropilin-1 (NRP-1) and NRP-2 in directly extracted RNA from human NP cells and NP cells cultured in alginate for 2weeks before treatment for 48hours with interleukin (IL)-1, IL-6, or tumor necrosis factor α(TNFα) at 0 to 100ng/mL. Similarly, HDMEC-1 endothelial cells were treated with IL-1, IL-6, or TNFα at 0 and 10ng/mL for 48hours, after which mRNA expression was investigated by RT-PCR. Results Expression of semaphorins and their receptors were present in normal and degenerate disc samples. In human NP cells, IL-6 inhibited semaphorin expression, whereas IL-1 and TNFα significantly increased sema3D over 10-fold (p≤0.05). IL-1 upregulated all plexins, conversely TNFα significantly decreased plexin A2. NRP-2 showed a significant increase in response to IL-1. While NRP-1 was significantly decreased in response to IL-1. In HDMEC-1 cells IL-1 significantly increased sema3C and sema3D while TNF significantly increased sema3C. TNF significantly increased NRP-2 expression, however, NRP-1 was decreased by all cytokines. Plexin receptor expression was increased upon IL-6 and TNF treatment whereas IL-1 caused significant decrease in plexin A3 expression. Conclusion Here, we have demonstrated that several cytokines known to be upregulated during disc degeneration and disc prolapse, regulate expression of certain members of class 3 semaphorins, as well as their receptors in human NP cells and HDMEC-1 cells. This data suggests that the presence of these factors within the degenerate disc may be responsible for modulating the ingrowth of blood vessels and promote nerve ingrowth leading to discogenic pain. Disclosure of Interest None declared