Neil Chiverton

Tumor necrosis factor α- and interleukin-1β-dependent induction of CCL3 expression by nucleus pulposus cells promotes macrophage migration through CCR1.

OBJECTIVE To investigate tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) regulation of CCL3 expression in nucleus pulposus (NP) cells and in macrophage migration. METHODS Quantitative reverse transcription-polymerase chain reaction and immunohistochemistry were used to measure CCL3 expression in NP cells. Transfections were used to determine the role of NF-κB, CCAAT/enhancer binding protein (C/EBPβ), and MAPK on cytokine-mediated CCL3 promoter activity. The effect of NP-conditioned medium on macrophage migration was measured using a Transwell system. RESULTS An increase in CCL3 expression and promoter activity was observed in NP cells after TNFα or IL-1β treatment. Treatment of cells with NF-κB and MAPK inhibitors abolished the effect of the cytokines on CCL3 expression. The inductive effect of p65 and C/EBPβ on the CCL3 promoter was confirmed through gain-of-function and loss-of-function studies. Notably, cotransfection with p50 completely blocked cytokine- and p65-dependent induction. In contrast, c-Rel and RelB had little effect on promoter activity. Lentiviral transduction with short hairpin RNA for p65 (shp65) and shIKKβ significantly decreased the TNFα-dependent increase in CCL3 expression. Analysis of degenerated human NP tissue samples showed that CCL3, but not CCL4, expression correlated positively with the grade of tissue degeneration. Importantly, treatment of macrophages with conditioned medium of NP cells treated with TNFα or IL-1β promoted their migration. Pretreatment of macrophages with an antagonist of CCR1, the primary receptor for CCL3 and CCL4, blocked cytokine-mediated migration. CONCLUSION Our findings indicate that TNFα and IL-1β modulate the expression of CCL3 in NP cells by controlling the activation of MAPK, NF-κB, and C/EBPβ signaling. The CCL3-CCR1 axis may play an important role in promoting macrophage infiltration in degenerated, herniated discs.

The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc.

IntroductionThe aims of these studies were to identify the cytokine and chemokine expression profile of nucleus pulposus (NP) cells and to determine the relationships between NP cell cytokine and chemokine production and the characteristic tissue changes seen during intervertebral disc (IVD) degeneration.MethodsReal-time q-PCR cDNA Low Density Array (LDA) was used to investigate the expression of 91 cytokine and chemokine associated genes in NP cells from degenerate human IVDs. Further real-time q-PCR was used to investigate 30 selected cytokine and chemokine associated genes in NP cells from non-degenerate and degenerate IVDs and those from IVDs with immune cell infiltrates (‘infiltrated’). Immunohistochemistry (IHC) was performed for four selected cytokines and chemokines to confirm and localize protein expression in human NP tissue samples.ResultsLDA identified the expression of numerous cytokine and chemokine associated genes including 15 novel cytokines and chemokines. Further q-PCR gene expression studies identified differential expression patterns in NP cells derived from non-degenerate, degenerate and infiltrated IVDs. IHC confirmed NP cells as a source of IL-16, CCL2, CCL7 and CXCL8 and that protein expression of CCL2, CCL7 and CXCL8 increases concordant with histological degenerative tissue changes.ConclusionsOur data indicates that NP cells are a source of cytokines and chemokines within the IVD and that these expression patterns are altered in IVD pathology. These findings may be important for the correct assessment of the ‘degenerate niche’ prior to autologous or allogeneic cell transplantation for biological therapy of the degenerate IVD.

Inflammatory Cytokines Induce NOTCH Signaling in Nucleus Pulposus Cells IMPLICATIONS IN INTERVERTEBRAL DISC DEGENERATION

Background: The regulation of NOTCH signaling under inflammatory conditions in the nucleus pulposus is unknown. Results: Expression of select NOTCH pathway genes, including NOTCH2 and NOTCH signaling, is regulated by IL-1β and TNF-α. Conclusion: Inflammatory cytokines promote NOTCH signaling in disc. Significance: NOTCH signaling may play a role in pathogenesis of disc disease. The objective of the study was to investigate how inflammatory cytokines, IL-1β, and TNF-α control NOTCH signaling activity in nucleus pulposus (NP) cells. An increase in expression of selective NOTCH receptors (NOTCH1 and -2), ligand (JAGGED2), and target genes (HES1, HEY1, and HEY2) was observed in NP cells following cytokine treatment. A concomitant increase in NOTCH signaling as evidenced by induction in activity of target gene HES1 and HEY1 promoters and reporter 12xCSL was seen. Moreover, treatment increased activity of a 2-kb NOTCH2 promoter. Treatment of cells with NF-κB and MAPK inhibitors abolished the inductive effect of cytokines on NOTCH2 promoter and its expression. Gain and loss-of-function studies confirmed the inductive effect of p65 on NOTCH2 promoter activity. In contrast, p50 blocked the cytokine induction of promoter activity. Supporting promoter studies, lentiviral delivery of sh-p65, and sh-IKKβ significantly decreased cytokine dependent change in NOTCH2 expression. Interestingly, MAPK signaling showed an isoform-specific control of NOTCH2 promoter; p38α/β2/δ, ERK1, and ERK2 contributed to cytokine dependent induction, whereas p38γ played no role. Analysis of human NP tissues showed that NOTCH1 and -2 and HEY2 expression correlated with each other. Moreover, expression of NOTCH2 and IL-1β as well as the number of cells immunopositive for NOTCH2 significantly increased in histologically degenerate discs compared with non-degenerate discs. Taken together, these results explain the observed dysregulated expression of NOTCH genes in degenerative disc disease. Thus, controlling IL-1β and TNF-α activities during disc disease may restore NOTCH signaling and nucleus pulposus cell function.

Expression and regulation of neurotrophic and angiogenic factors during human intervertebral disc degeneration

IntroductionThe degenerate intervertebral disc (IVD) becomes innervated by sensory nerve fibres, and vascularised by blood vessels. This study aimed to identify neurotrophins, neuropeptides and angiogenic factors within native IVD tissue and to further investigate whether pro-inflammatory cytokines are involved in the regulation of expression levels within nucleus pulposus (NP) cells, nerve and endothelial cells.MethodsQuantitative real-time PCR (qRT-PCR) was performed on 53 human IVDs from 52 individuals to investigate native gene expression of neurotrophic factors and their receptors, neuropeptides and angiogenic factors. The regulation of these factors by cytokines was investigated in NP cells in alginate culture, and nerve and endothelial cells in monolayer using RT-PCR and substance P (SP) protein expression in interleukin-1 (IL-1β) stimulated NP cells.ResultsInitial investigation on uncultured NP cells identified expression of all neurotrophins by native NP cells, whilst the nerve growth factor (NGF) receptor was only identified in severely degenerate and infiltrated discs, and brain derived neurotrophic factor (BDNF) receptor expressed by more degenerate discs. BDNF expression was significantly increased in infiltrated and degenerate samples. SP and vascular endothelial growth factor (VEGF) were higher in infiltrated samples. In vitro stimulation by IL-1β induced NGF in NP cells. Neurotropin-3 was induced by tumour necrosis factor alpha in human dermal microvascular endothelial cells (HDMECs). SP gene and protein expression was increased in NP cells by IL-1β. Calcitonin gene related peptide was increased in SH-SY5Y cells upon cytokine stimulation. VEGF was induced by IL-1β and interleukin-6 in NP cells, whilst pleiotrophin was decreased by IL-1β. VEGF and pleiotrophin were expressed by SH-SY5Y cells, and VEGF by HDMECs, but were not modulated by cytokines.ConclusionsThe release of cytokines, in particular IL-1β during IVD degeneration, induced significant increases in NGF and VEGF which could promote neuronal and vascular ingrowth. SP which is released into the matrix could potentially up regulate the production of matrix degrading enzymes and also sensitise nerves, resulting in nociceptive transmission and chronic low back pain. This suggests that IL-1β is a key regulatory cytokine, involved in the up regulation of factors involved in innervation and vascularisation of tissues.

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.

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