A retrospective review of gram-negative spinal infections in a single tertiary spinal centre over six years.

Aims: Gram-negative infections are associated with comorbid patients, but outcomes are less well understood. This study reviewed diagnosis, management, and treatment for a cohort treated in a tertiary spinal centre. Methods: A retrospective review was performed of all gram-negative spinal infections (n = 32; median age 71 years; interquartile range 60 to 78), excluding surgical site infections, at a single centre between 2015 to 2020 with two- to six-year follow-up. Information regarding organism identification, antibiotic regime, and treatment outcomes (including clinical, radiological, and biochemical) were collected from clinical notes. Results: All patients had comorbidities and/or non-spinal procedures within the previous year. Most infections affected lumbar segments (20/32), with Escherichia coli the commonest organism (17/32). Causative organisms were identified by blood culture (23/32), biopsy/aspiration (7/32), or intraoperative samples (2/32). There were 56 different antibiotic regimes, with oral (PO) ciprofloxacin being the most prevalent (13/56; 17.6%). Multilevel, contiguous infections were common (8/32; 25%), usually resulting in bone destruction and collapse. Epidural collections were seen in 13/32 (40.6%). In total, five patients required surgery, three for neurological deterioration. Overall, 24 patients improved or recovered with a mean halving of CRP at 8.5 days (SD 6). At the time of review (two to six years post-diagnosis), 16 patients (50%) were deceased. Conclusion: This is the largest published cohort of gram-negative spinal infections. In older patients with comorbidities and/or previous interventions in the last year, a high level of suspicion must be given to gram-negative infection with blood cultures and biopsy essential. Early organism identification permits targeted treatment and good initial clinical outcomes; however, mortality is 50% in this cohort at a mean of 4.2 years (2 to 6) after diagnosis.

Management of C2 odontoid peg fragility fractures - A UK survey of spinal surgeons.

PURPOSE: Patients sustaining fragility fractures of the C2 odontoid peg have 30-day mortality rates as high as 10% rising up to 34.1% at 1 year. Substantial controversy exists regarding optimal management of these fractures and there is a lack of national guidance to inform best practice. The aim of this study was to determine current practice in the management of these fractures throughout the United Kingdom. METHODS: A UK wide, cross sectional survey was conducted, asking 10 questions regarding the initial management, imaging and follow-up of an elderly patient with a type 2 fragility odontoid peg fracture. This was publicised through the British Orthopaedic Association website and sent to all members of the Society of British Neurological Surgeons (SBNS) via email. RESULTS: 107 Responses were received. 56% from orthopaedic consultants, 29% from neurosurgical consultants and 15% from senior spine fellows. 86% (92) of respondents choose treatment with a cervical orthosis, with 84% (77) of these opting for a semi rigid Aspen or Philadelphia collar compared to 16% (15) opting for a soft cervical collar. Three (3%) opted for operative intervention with a further three (3%) choosing Halo fixation. Nine respondents (8%) opted for no orthosis and treatment with analgesia alone. Length of immobilisation in cervical orthosis ranged from 6 to 12 weeks. Initial follow-up ranged from 1 week to 6 weeks, with 6% (6) discharged without follow up. There was also marked variation in the use of follow-up imaging with 17% (18) using plain radiographs, 62% (66) requesting lateral flexion / extension radiographs, 10% (11) using CT and 11% (12) not performing any imaging at final follow up. In 60% (64) of cases respondents did not change subsequent management as a result of imaging. CONCLUSION: Type-2 fragility peg fractures have high morbidity and mortality. There is marked variation in the treatment modalities used, follow-up regime and use of imaging throughout the UK. Given the rapidly increasing incidence of these injuries and the associated mortality this should be a high priority field for further research. Further large scale studies are urgently required to inform best practice and standardise management of these injuries.

Giant spinal nerve sheath tumours - Surgical challenges: case series and literature review.

Background: Giant nerve sheath tumours (GNST) are rare and literature on their management is scant. Spinal GNST present as a surgical challenge due to the involvement of anatomical regions often outside the "comfort zone" of a spinal surgeon. This case series aims to identify challenges in the surgical management of GNSTs. Methods: Retrospective case note review of all spinal GNST cases from 2010 to 2016 managed in Sheffield Teaching Hospitals identified 8 patients, 3 of whom were incidental findings (kept under surveillance) and were excluded. 5 cases were treated surgically. Data collected included patient demographic, presenting symptom(s), radiological data, surgical approach to the tumour and challenges encountered, histopathology report and follow up. Results: Our cohort consisted entirely of females (N = 5) with a mean age of 56.4 years (range 45-70). Imaging studies and histopathological diagnoses confirmed 5 GNSTs (four benign schwannomas and one ganglioneuroma). A Single-stage anterior approach was adopted for three patients while a combined anterior-posterior approach was adopted for the remaining two. In one patient, a posterior approach was initially planned, but this was abandoned and converted to an anterior approach following onset of acute superior vena cava (SVC) syndrome secondary to SVC compression by the giant tumour on prone positioning of the patient. PET imaging of case 3 showed intense tracer uptake consistent with malignancy, however histology confirmed WHO grade 1 Schwannoma. The other three non-operated GNSTs are kept under yearly radiological and clinical surveillance. Conclusions: GNSTs are surgically challenging as they often invade territories that are beyond the comfort zone of one single specialty. A multidisciplinary approach with careful pre-operative surgical planning is recommended. Patients in whom a posterior approach is planned should have a trial of prone positioning pre-operatively. Careful interpretation of FDG-PET imaging is recommended due to the possibility of false positive result.

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.

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

BACKGROUND: Chronic 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. METHODS: Immunohistochemistry 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. RESULTS: Immunopositivity 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% of all 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. CONCLUSIONS: This 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.

Class 3 semaphorins expression and association with innervation and angiogenesis within the degenerate human intervertebral disc.

Nerve and blood vessel ingrowth during intervertebral disc degeneration, is thought to be a major cause of low back pain, however the regulation of this process is poorly understood. Here, we investigated the expression and regulation of a subclass of axonal guidance molecules known as the class 3 semaphorins, and their receptors; plexins and neuropilins within human NP tissue and their regulation by pro-inflammatory cytokines. Importantly this determined whether semaphorin expression was associated with the presence of nerves and blood vessels in tissues from human intervertebral discs. The study demonstrated that semaphorin3A, 3C, 3D, 3E and 3F and their receptors were expressed by native NP cells and further demonstrated their expression was regulated by IL-1ß but to a lesser extent by IL-6 and TNFα. This is the first study to identify sema3C, sema3D and their receptors within the nucleus pulposus of intervertebral discs. Immunopositivity shows significant increases in semaphorin3C, 3D and their receptor neuropilin-2 in degenerate samples which were shown to contain nerves and blood vessels, compared to non-degenerate samples without nerves and blood vessels. Therefore data presented here suggests that semaphorin3C may have a role in promoting innervation and vascularisation during degeneration, which may go on to cause low back pain.

Aquaporin 1 and 5 expression decreases during human intervertebral disc degeneration: Novel HIF-1-mediated regulation of aquaporins in NP cells.

Objectives of this study were to investigate whether AQP1 and AQP5 expression is altered during intervertebral disc degeneration and if hypoxia and HIF-1 regulate their expression in NP cells. AQP expression was measured in human tissues from different degenerative grades; regulation by hypoxia and HIF-1 was studied using promoter analysis and gain- and loss-of-function experiments. We show that both AQPs are expressed in the disc and that mRNA and protein levels decline with human disease severity. Bioinformatic analyses of AQP promoters showed multiple evolutionarily conserved HREs. Surprisingly, hypoxia failed to induce promoter activity or expression of either AQP. While genomic chromatin immunoprecipitation showed limited binding of HIF-1α to conserved HREs, their mutation did not suppress promoter activities. Stable HIF-1α suppression significantly decreased mRNA and protein levels of both AQPs, but HIF-1α failed to induce AQP levels following accumulation. Together, our results demonstrate that AQP1 and AQP5 expression is sensitive to human disc degeneration and that HIF-1α uniquely maintains basal expression of both AQPs in NP cells, independent of oxemic tension and HIF-1 binding to promoter HREs. Diminished HIF-1 activity during degeneration may suppress AQP levels in NP cells, compromising their ability to respond to extracellular osmolarity changes.

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

INTRODUCTION: The 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. METHODS: Quantitative 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-1ß) stimulated NP cells. RESULTS: Initial 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-1ß 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-1ß. Calcitonin gene related peptide was increased in SH-SY5Y cells upon cytokine stimulation. VEGF was induced by IL-1ß and interleukin-6 in NP cells, whilst pleiotrophin was decreased by IL-1ß. VEGF and pleiotrophin were expressed by SH-SY5Y cells, and VEGF by HDMECs, but were not modulated by cytokines. CONCLUSIONS: The release of cytokines, in particular IL-1ß 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-1ß is a key regulatory cytokine, involved in the up regulation of factors involved in innervation and vascularisation of tissues.

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

INTRODUCTION: The 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. METHODS: Real-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. RESULTS: LDA 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. CONCLUSIONS: Our 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.

The objective of the study was to investigate how inflammatory cytokines, IL-1ß, 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-IKKß significantly decreased cytokine dependent change in NOTCH2 expression. Interestingly, MAPK signaling showed an isoform-specific control of NOTCH2 promoter; p38α/ß2/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-1ß 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-1ß and TNF-α activities during disc disease may restore NOTCH signaling and nucleus pulposus cell function.

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

OBJECTIVE: To investigate tumor necrosis factor α (TNFα) and interleukin-1ß (IL-1ß) 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/EBPß), 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-1ß 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/EBPß 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 shIKKß 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-1ß 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-1ß modulate the expression of CCL3 in NP cells by controlling the activation of MAPK, NF-κB, and C/EBPß signaling. The CCL3-CCR1 axis may play an important role in promoting macrophage infiltration in degenerated, herniated discs.