Investigation into the mechanisms of hyperalgesic priming

Chronic pain is a major clinical problem that affects approximately 40% of the UK population. Understanding the mechanisms that underpin the development and maintenance of chronic pain permits more focused research and identification of potential targets for new therapies. Hyperalgesic priming models the transition from acute to chronic pain. Subcutaneous inflammogens applied 72hrs apart induce a long-term hyperalgesia to mechanical stimulation that outlasts the resolution of the systemic swelling. The aim of this thesis was to investigate the underlying mechanisms of this phenomenon. Two versions of hyperalgesic priming were initially assessed; carrageenan-induced and TNFα induced. The rats pre-treated with carrageenan (primed) (1%, 5μl) showed significantly reduced paw withdrawal thresholds post-intraplantar injection of PGE2 (1μg, 5μl), these were maintained up to 7 days post-injection compared to saline pre-treated (unprimed). Rats pre-treated with TNFα (100ng, 5μl) also showed maintained reductions in PWTs post-PGE2, this however was not significantly lower than saline unprimed rats. To investigate changes in immune cell populations within the peripheral injury site, skin samples collected from carrageenan-primed rats after PGE2 injection showed increased numbers of macrophages compared to saline-saline controls. Ionized calcium-binding adaptor protein (IBA1) immunostaining of the spinal cord showed that there were increased numbers of microglia in the dorsal horn in unprimed rats compared to carrageenan-primed rats. There were alterations in the presence of plasma lipid mediators of inflammation determined using mass spectroscopy in primed compared to unprimed rats. Unprimed rats having a higher level of anti-inflammatory mediators including PGE2 EA and 18-HEPE suggests an alteration in production of inflammatory mediators due to hyperalgesic priming. To evaluate the involvement of sub-sets of primary afferent neurones in priming, sensory nerve blockade using the local anaesthetic QX-314 and capsaicin or QX-314 and flagellin were administered before or after intraplantar injection of PGE2 to selectively block Aβ and C nerve fibres. The injection of QX-314 + capsaicin or QX-314 + flagellin inhibited the PGE2 induced reduction in PWTs however was unable to prevent the establishment of chronic pain. The injection of QX-314 + capsaicin in rats with established chronic pain inhibited the reduction in PWTs however the effect was only seen to last up to 4h post-injection. QX-314 + flagellin partially inhibited the reduction in PWTs up to 2h. These data showed that hyperalgesic priming is mediated by the activation of both C-fibres and partially by Aβ fibres. To assess input changes in CNS processing of primary afferent fibres spinal electrophysiological recordings of wide dynamic range (WDR) neurones in lamina V were performed. These data showed a 300% increase in firing in response to low threshold mechanical stimulation after the intraplantar injection of PGE2. There were no differences observed in WDR firing between primed and unprimed rats post-PGE2. Finally the role of the resolvin resolution pathway was investigated in preventing and reversing hyperalgesic priming. Aspirin-triggered resolvin D1 precursor 17-R-HDoHE has been shown to reduce inflammatory pain. 17-R-HDoHE (300ng, 300μl) was systemically administered before or after PGE2 injection in carrageenan-primed rats. It was shown that administration of 17-R-HDoHE prior to PGE2 significantly (p<0.01) inhibited the sustained reduction in PWTs in primed rats. However systemic administration of 17-R-HDoHE after PGE2 in primed rats had no effect on PWTs. These data show an alteration in the responsiveness of primed rats to the activation of resolvin mediated resolution. Changes in microRNA (miRNA) has been linked to alterations in gene expression in different pain states. To investigate miRNA changes in chronic pain. The L4, L5 and L6 DRGs were collected. There were measured increases in miR-100 and miR-125b during carrageenan induced acute pain and hyperalgesic priming induced chronic pain post-PGE2. Pre-treatment with 17-R-HDoHE was shown in inhibit the up-regulation of miR-100 and miR-125b in DRGs of primed rats. This demonstrated that alterations in the regulation of genes during acute pain may also influence changes during chronic pain. This thesis demonstrates the potential involvement of several altered cellular processes that contribute to the induction and maintenance of long-term hyperalgesia by hyperalgesic priming. I observed decreases in numbers of microglia within the dorsal horn of primed rats. Hyperalgesic priming induced chronic pain requires activity within of both C and Aβ fibre primary afferent populations of sensory neurons. The firing rate of WDR neurones to mechanical stimulation was shown to be increased post-PGE2 intraplantar injection however the increase was not maintained in primed rats. Levels of miR-100 and miR-125b were found to be increased during periods of acute and chronic pain and were found to be inhibited by resolvin treatment. Finally resolvin pre-treatment inhibited the generation of hyperalgesic priming induced chronic pain, but to reverse established pain

The Oman Botanic Garden (1)

The Oman Botanic Garden (OBG) is a new botanic garden which is being constructed on a 423ha site near to Muscat, the capital of Oman. Oman is floristically rich and is considered a centre of plant diversity in the Arabian Peninsula. The plan is that OBG will showcase this plant diversity, inform visitors of its value and provide a model for sustainability. This paper, part 1, covers the vision, early plant collections and propagation, and part 2, which will be included in Sibbaldia No. 7, will cover design, construction, interpretation and planting

Contemporary Pastoralism in the Dhofar Mountains of Oman

In the Dhofar Mountains of Oman stakeholders are concerned about the social and ecological sustainability of pastoralism. In this study we used interviews with pastoralists to examine the prevailing drivers of pastoralism and how they are changing. We find that people are committed to pastoralism for sociocultural reasons, but also that this commitment is under pressure because of husbandry costs and changing values. We find that capital investment in feedstuff enables pastoralists to overcome the density-dependent regulation of livestock populations. However, high production costs deter investment in marketing and commercialization, and there is little offtake of local livestock. Our study reveals how pastoral values, passed down within households, motivate pastoralism in the face of high husbandry costs, modernization and social change

Phosphorylation of CLEC-2 is dependent on lipid rafts, actin polymerization,secondary mediators, and Rac

The C-type lectin-like receptor 2 (CLEC-2)activates platelets through Src and Syk tyrosine kinases via a single cytoplasmic YxxL motif known as a hem immunoreceptor tyrosine-based activation motif (hemITAM).Here, we demonstrate using sucrose gradient ultracentrifugation and methyl--cyclodextrin treatment that CLEC-2 translocates to lipid rafts upon ligand engagement and that translocation is essential for hemITAM phosphorylation and signal initiation. HemITAM phosphorylation, but not translocation, is also critically dependent on actin polymerization,Rac1 activation, and release of ADP and thromboxane A2 (TxA2). The role of ADP and TxA2 in mediating hosphorylation is dependent on ligand engagement and rac activation but is independent of platelet aggregation. In contrast,tyrosine phosphorylation of the GPVIFcR -chain ITAM, which has 2 YxxL motifs,is independent of actin polymerization and secondary mediators. These results reveal a unique series of proximal events in CLEC-2 phosphorylation involving actin polymerization, secondary mediators,and Rac activation

A Comparison of the Laser Flare Cell Meter and Fluorophotometry in Assessment of the Blood-Aqueous Barrier

Purpose. To evaluate and compare the use of the Kowa laser flare cell meter and intravenous anterior chamber fluorophotometry in assessment of the blood-aqueous barrier after cataract surgery. Method. Laser flare and cell measurements and fluorophotometry were performed at 1 and 3 months after surgery in 48 eyes of 44 patients admitted for routine cataract surgery. The fellow pseudophakic eyes of these patients were used as controls. Results. The two techniques measure different parameters, but both methods are able to document the integrity or breakdown of the blood-aqueous barrier. However, the laser flare cell meter is more sensitive in quantifying subtle changes in barrier function to large molecules (proteins). Various methods of assessing anterior chamber fluorophotometry data were also compared. Measurement of a diffusion coefficient (requiring the measurement of plasma fluorescence) was not found to be more sensitive than other methods and did not alter the clinical significance of data obtained from the measurement of anterior chamber fluorescence alone. Conclusions. Both the laser flare cell meter and fluorophotometry provide a method for the assessment of the postoperative blood-aqueous barrier. However, the laser flare cell meter is rapid, noninvasive, and relatively easier to use. Therefore, for clinical use, it has great practical advantages over fluorophotometry. Invest Ophthalmol Vis Sci. 1993;34:3124-3130

Portable light transmission measuring system for preserved corneas

BACKGROUND: The authors have developed a small portable device for the objective measurement of the transparency of corneas stored in preservative medium, for use by eye banks in evaluation prior to transplantation. METHODS: The optical system consists of a white light, lenses, and pinholes that collimate the white light beams and illuminate the cornea in its preservative medium, and an optical filter (400–700 nm) that selects the range of the wavelength of interest. A sensor detects the light that passes through the cornea, and the average corneal transparency is displayed. In order to obtain only the tissue transparency, an electronic circuit was built to detect a baseline input of the preservative medium prior to the measurement of corneal transparency. The operation of the system involves three steps: adjusting the "0 %" transmittance of the instrument, determining the "100 %" transmittance of the system, and finally measuring the transparency of the preserved cornea inside the storage medium. RESULTS: Fifty selected corneas were evaluated. Each cornea was submitted to three evaluation methods: subjective classification of transparency through a slit lamp, quantification of the transmittance of light using a corneal spectrophotometer previously developed, and measurement of transparency with the portable device. CONCLUSION: By comparing the three methods and using the expertise of eye bank trained personnel, a table for quantifying corneal transparency with the new device has been developed. The correlation factor between the corneal spectrophotometer and the new device is 0,99813, leading to a system that is able to standardize transparency measurements of preserved corneas, which is currently done subjectively

Mouse podoplanin supports adhesion and aggregation of platelets under arterial shear: a novel mechanism of haemostasis

The Podoplanin-CLEC-2 axis is critical in mice for prevention of haemorrhage in the cerebral vasculature during mid-gestation. This raises the question as to how platelets are captured by podoplanin on neuroepithelial cells in a high shear environment. In this study, we demonstrate that mouse platelets form stable aggregates on mouse podoplanin at arterial shear through a CLEC-2 and Src kinase-dependent pathway. Adhesion and aggregation are also dependent on the platelet glycoprotein (GP) receptors, integrin αIIbβ3 and GPIb, and the feedback agonists ADP and thromboxane A2 (TxA2). CLEC-2 does not bind to von Willebrand factor (VWF) suggesting that the interaction with podoplanin is sufficient to both tether and activate platelets. Consistent with this, surface plasmon resonance measurements reveal that mouse CLEC-2 binds to mouse podoplanin with nanomolar affinity. The present findings demonstrate a novel pathway of haemostasis in which podoplanin supporting platelet capture and activation at arteriolar rates of shear

Growth factor restriction impedes progression of wound healing following cataract surgery: identification of VEGF as a putative therapeutic target

Secondary visual loss occurs in millions of patients due to a wound-healing response, known as posterior capsule opacification (PCO), following cataract surgery. An intraocular lens (IOL) is implanted into residual lens tissue, known as the capsular bag, following cataract removal. Standard IOLs allow the anterior and posterior capsules to become physically connected. This places pressure on the IOL and improves contact with the underlying posterior capsule. New open bag IOL designs separate the anterior capsule and posterior capsules and further reduce PCO incidence. It is hypothesised that this results from reduced cytokine availability due to greater irrigation of the bag. We therefore explored the role of growth factor restriction on PCO using human lens cell and tissue culture models. We demonstrate that cytokine dilution, by increasing medium volume, significantly reduced cell coverage in both closed and open capsular bag models. This coincided with reduced cell density and myofibroblast formation. A screen of 27 cytokines identified nine candidates whose expression profile correlated with growth. In particular, VEGF was found to regulate cell survival, growth and myofibroblast formation. VEGF provides a therapeutic target to further manage PCO development and will yield best results when used in conjunction with open bag IOL designs

A freely accessible, evidence based, objective system of analysis of posterior capsular opacification ; Evidence for its validity and reliability

BACKGROUND: The aim of this study was to develop a system of computerised analysis of digital images of posterior capsule opacification (PCO) that is evidence based, objective and freely available. The paper will present evidence for the reliability and validity of the developed system. METHODS: The system of PCO analysis was developed considering current published evidence on visual significance of PCO and additional investigative analysis of PCO images. Details of the image processing and analysis steps are discussed and a final system that measures an entropy score weighted toward proximity to central areas is described. In order to assess validity, the systems ability to measure PCO progression is assessed along with the visual significance of its final computerised scores. Reliability of the system is also assessed. RESULTS: The final system runs successfully and is simple to use. Analyses of PCO by the system show an ability to detect early progression of PCO as well as detection of visually significant PCO. Images with no clinical PCO produce very low scores in the analysis. Reliability of the system of analysis is shown to be satisfactory. CONCLUSION: This paper presents a system of PCO analysis that is evidence based, objective and clinically useful. Substantial evidence is provided for its validity and reliability