Degeneration of the intervertebral disc with new approaches for treating low back pain.

This review paper discusses the process of disc degeneration and the current understanding of cellular degradation in patients who present with low back pain. The role of surgical treatment for low back pain is analysed with emphasis on the proven value of spinal fusion. The interesting and novel developments of stem cell research in the treatment of low back pain are presented with special emphasis on the importance of the cartilaginous end plate and the role of IL-1 in future treatment modalities

The regulation of nerve and blood vessel ingrowth in aneural and avascular intervertebral disc and articular cartilage

Introduction This review will discuss the regulatory mechanisms of both innervation and vascularisation within normally aneural and avascular tissues, and how they may become altered in degeneration enabling new nerve and blood vessel formation which is hypothesised to be a source of pain. Conclusion Normal intervertebral discs and articular cartilage are the largest aneural and avascular tissues in the human body yet during intervertebral disc degeneration and osteoarthritis these tissues become increasingly vascularised by small blood vessels and innervated by peptide containing sensory nerve fibres. The mechanism by which this process occurs remains largely unknown. Published data suggests that various factors present within the healthy tissues such as aggrecan, chondromodulin and semaphorins may act as repulsive barriers to neurite and endothelial cell invasion. During degeneration however, the synthesis of these molecules becomes disrupted, potentially leading to vascularisation and innervation of the tissue

Rumex and Urtica detection in grassland by UAV

. Previous work (Binch & Fox, 2017) used autonomous ground robotic platforms to successfully detect Urtica (nettle) and Rumex (dock) weeds in grassland, to improve farm productivity and the environment through precision herbicide spraying. It assumed that ground robots swathe entire fields to both detect and spray weeds, but this is a slow process as the slow ground platform must drive over every square meter of the field even where there are no weeds. The present study examines a complimentary approach, using unmanned aerial vehicles (UAVs) to perform faster detections, in order to inform slower ground robots of weed location and direct them to spray them from the ground. In a controlled study, it finds that the existing state-of-the-art (Binch & Fox, 2017) ground detection algorithm based on local binary patterns and support vector machines is easily re-usable from a UAV with 4K camera despite large differences in camera type, distance, perspective and motion, without retraining. The algorithm achieves 83-95% accuracy on ground platform data with 1-3 independent views, and improves to 90% from single views on aerial data. However this is only attainable at low altitudes up to 8 feet, speeds below 0.3m/s, and a vertical view angle, suggesting that autonomous or manual UAV swathing is required to cover fields, rather than use of a single high-altitude photograph. This demonstrates for the first time that combined aerial detection with ground spraying system is feasible for Rumex and Urtica in grassland, using UAVs to replace the swathing and detection of weeds then dispatching ground platforms to spray them at the detection sites (as spraying by UAV is illegal in EU countries). This reduces total time requires to spray as the UAV performs the survey stage faster than a ground platform

Immunohistochemical analysis of protein expression in formalin fixed paraffin embedded human intervertebral disc tissues

Immunohistochemistry (IHC) is a useful technique for the localization and semiquantification of protein expression within tissues. Adult human intervertebral disc (IVD) tissues contain a large amount of auto‐fluorescence which often makes immunofluorescence techniques inappropriate on tissue samples but can be applied to isolated cell samples. Thus, IHC remains one of, if not the most common application for protein detection within IVD tissue. Immunostaining localizes antigen expression through specific epitope‐antibody interactions. Within the field of IVD research, IHC is commonly used on fresh frozen and paraffin embedded tissues to elucidate the expression of antigens. Here, we discuss the principles of IHC applied to formalin fixed paraffin embedded IVD tissue and supply optimized protocols for antibodies used within our group to guide research within the IVD field

Controlled comparison of machine vision algorithms for Rumex and Urtica detection in grassland

Automated robotic weeding of grassland will improve the productivity of dairy and sheep farms while helping to conserve their environments. Previous studies have reported results of machine vision methods to separate grass from grassland weeds but each use their own datasets and report only performance of their own algorithm, making it impossible to compare them. A definitive, large-scale independent study is presented of all major known grassland weed detection methods evaluated on a new standardised data set under a wider range of environment conditions. This allows for a fair, unbiased, independent and statistically significant comparison of these and future methods for the first time. We test features including linear binary patterns, BRISK, Fourier and Watershed; and classifiers including support vector machines, linear discriminants, nearest neighbour, and meta-classifier combinations. The most accurate method is found to use linear binary patterns together with a support vector machin

Controlled comparison of machine vision algorithms for Rumex and Urtica detection in grassland

Automated robotic weeding of grassland will improve the productivity of dairy and sheep farms while helping to conserve their environments. Previous studies have reported results of machine vision methods to separate grass from grassland weeds but each use their own datasets and report only performance of their own algorithm, making it impossible to compare them. A definitive, large-scale independent study is presented of all major known grassland weed detection methods evaluated on a new standardised data set under a wider range of environment conditions. This allows for a fair, unbiased, independent and statistically significant comparison of these and future methods for the first time. We test features including linear binary patterns, BRISK, Fourier and Watershed; and classifiers including support vector machines, linear discriminants, nearest neighbour, and meta-classifier combinations. The most accurate method is found to use linear binary patterns together with a support vector machin

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.</p

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

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

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-1β 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

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