Chronic neuropathic pain, resulting from dysfunction of the nervous system, is a clinical concern in both humans and animal patients. Neuropathic pain is characterised by spontaneous pain, hypersensitivity, manifested as hyperalgesia and allodynia, and refractoriness to conventional analgesics such as non-steroidal anti-inflammatory drugs, thus representing an unmet therapeutic need. Equine laminitis is a disease that involves the disruption of the dermoepidermal junction within the hoof, leading to severe pain and lameness, with poor responsiveness to anti-inflammatory therapy. We developed a Quantitative Sensory Testing method, using a novel hydraulically-powered feedbackcontrolled hoof tester, in order to provide an objective tool for the assessment of mechanical hyperalgesia in laminitic horses. Hoof Compression Thresholds of laminitic horses were significantly lower than those of normal horses and variance component analysis of the data confirmed the reliability of the method. In order to investigate mechanisms underlying laminitis pain, we performed histological studies of peripheral nerves innervating the hoof. Electron micrographic analysis of the digital nerve of laminitic horses revealed a significant reduction in the number of unmyelinated and myelinated fibres together with abnormal morphology. Additionally, cell bodies of sensory neurons innervating the hoof in cervical C8 dorsal root ganglia showed an upregulated expression of the nerve injury marker activating transcription factor-3 (ATF3), neuropeptide Y (NPY), and the TRPM8 channel; each of which has been associated with laboratory models of neuropathic pain. Previous work has shown that, in a rodent model of neuropathic pain, the TRPM8 channel is upregulated in sensory neurons and its activation by cool temperature, menthol or icilin leads to reversal of the hypersensitive pain state. Further investigation of TRPM8-channel mediated analgesia was aimed at uncovering the molecular mechanisms involved in the activation of this system in sensitised states. It was hypothesised that serotonin, released following inflammation and nerve damage, can enhance TRPM8 channel activity through peripheral 5-HT1B receptors. Calcium fluorometry carried out in HEK293 cells transfected with the TRPM8 channel and the 5-HT1B receptor revealed that coadministration of a 5-HT1B receptor agonist facilitated the activation of the TRPM8 channel by icilin. Moreover, it appears that this effect is mediated through phospholipase D1 (PLD1), possibly leading to increased production of phosphatidylinositol (4,5-) bisphosphate (PIP2), a known positive modulator of TRPM8 channel activity. In vitro co-immunoprecipitation studies suggested that the TRPM8 channel, the 5-HT1B receptor and PLD1 physically interact with each other, further providing a molecular basis for their functional co-operation. Calcium imaging carried out in cultured rat DRG cells showed that the 5-HT1B receptor-mediated enhancement of icilin responses at the TRPM8 channel also occurs in sensory cells and is reversed by inhibition of PLD1. Moreover, TRPM8 and the 5-HT1B receptor appear to be physically associated in vivo as shown by their co-immunoprecipitation from spinal cord homogenates. Assessment of nociceptive behavioural reflexes following intrathecal injection of selective pharmacological agents provided further support for the idea of 5-HT1B receptor facilitation of TRPM8 channel responses in vivo. In addition to providing novel evidence of a neuropathic component to equine laminitis and validation of a novel QST method for pain assessment in horses, this study reveals for the first time a physical and functional interaction between the 5-HT1B receptor and the TRPM8 channel.

Equine laminitis is a debilitating disease that causes extreme sufferring in afflicted horses and often results in a lifetime of chronic pain. The exact sequence of pathophysiological events culminating in laminitis has not yet been characterized, and this is reflected in the lack of any consistently effective therapeutic strategy. For these reasons, we used a newly developed 21,000 element equine-specific whole-genome oligoarray to perform transcriptomic analysis on laminar tissue from horses with experimentally induced models of laminitis: carbohydrate overload (CHO), hyperinsulinaemia (HI), and oligofructose (OF). Samples were collected during the developmental (DEV) and Obel grade 1 (OG1) stages of laminitis for the CHO model. For the HI model, samples were collected at the Obel grade 2 (OG2) stage. For the OF model, samples were collected at the 12 h and 24 h time points. Appropriate control samples were obtained for all models. This is the first genome-wide transcriptome analysis of laminar tissue using an equine 21,000 70-mer long oligoarray approach in CHO, HI and OF induced laminitis. Overall, we identified the differential expression of genes encoding S100 calcium binding proteins, extracellular matrix proteins, glycoproteins, transporters, olfactory receptors, genes involved in signal transduction, body‟s homeostasis, apoptosis, and immune response. Between CHO and OF models of laminitis, there were more shared genes. We discovered several common differentially expressed genes (i.e., ADAMTS1, CYCS and CXCL14) among all three models that are likely important to the pathogenesis of equine laminitis. We also discovered what appear to be central roles of apoptosis, inflammatory response, and intracellular ion homeostasis molecular processes in CHO and OF models of laminitis. Pathway analysis detected the NOD-like receptor signaling pathway, which is involved in recognition of intracellular bacteria in both the CHO and OF models of laminitis. Genetic network analysis indicated convergent pathway core molecules present in equine acute laminitis: p38 MAPK and NF-κB. Most importantly, our results of overexpression of anti-microbial genes (i.e., DEFB4, PI3, and CXCL14) suggest the central involvement of these genes in the progression of early equine laminitis and will allow refinement of current hypotheses of disease pathogenesis.

In order to investigate the early ultrastructural lesions at the first sign of lameness in the oligofructose (OF) model of laminitis, the disease was induced in four horses, while another four horses were sham-treated controls. Minor lesions were detected in lamellar samples examined by light microscopy. Examination by transmission electron microscopy (TEM) showed excessive waviness, breaks and separation of portions of lamellar basement membrane (BM) in the treated horses. There was also disintegration and disappearance of hemidesmosomes (HD) and epidermal basal cell (EBC) cytoskeleton, and an increase in the distance between the EBC plasmalemma and the centre of the BM. A link was thus established between the first clinical signs of lameness and ultrastructural changes in the lamellar dermo-epidermal interface. This implied that pathogenesis was underway well before clinical signs (24 h) and that successful therapy would need to be instituted earlier than previously considered. Earlier therapy may be facilitated if delivery of efficacious drugs to the foot was achievable. A treatment modality that delivered effective concentrations of anti laminitic drugs to the target organ (the epidermal lamellae) was thus an objective of this study. Hoof lamellar tissue from five ponies treated with prolonged euglycaemic hyperinsulinaemia and four control (sham-treated) ponies were harvested and processed for TEM. Lamellae from treated ponies showed attenuation and elongation of secondary epidermal lamellae (SEL), HD number reduction and infiltration of leukocytes. Unlike carbohydrate induced laminitis in horses, there was no global separation at the lamellar dermal/epidermal interface in ponies. Two unique lamellar lesions found in this induction model was mitosis among EBCs and thickening of the BM, not normally characteristic of acute laminitis. The pathophysiology of hyperinsulinaemic laminitis remains unresolved but if insulin, delivered directly to the foot, induced laminitis several pathophysiological questions would be answered. In particular, it would emphasise the laminitogenic potential of insulin alone in the pathogenesis of laminitis. It also allows the treatment foot to be compared with the remaining three that act as internal controls. A modality that delivered drugs like insulin to the target organ (the epidermal lamellae) was needed and was an objective of this study. A microdialysis (MD) method, based on continuous sampling of the lamellar extracellular fluid (ECF), was developed to monitor lamellar drug concentrations. MD probes were implanted in the hoof lamellar tissue of six normal Standardbred horses under local anaesthesia. A bolus intravenous (IV) dose (5 mg/kg BWT) of gentamicin sulfate was injected into the jugular vein. MD and blood samples were collected at different time points during 24 h, and calibrated and analyzed using an ELISA method for gentamicin. During the first 8 h, the concentration of gentamicin was significantly higher in blood than lamellar ECF, a result that is reversed when lamellar MD is repeated during IO infusion of gentamicin. The results showed that this modestly invasive method was a useful tool to monitor changes in the lamellar ECF during drug delivery or during laminitis development. Knowledge of the anatomy and dynamics of blood circulation in the equine foot is fundamental to understand laminitis pathophysiology. Using histology, decalcification, diaphanization, computed tomography (CT), micro CT and gelatin-India Ink vascular perfusion, the normal anatomy of the dorsal part of distal phalanx (DP) and its vascular relationship to hoof lamellae was characterised. The results showed a close relationship between the distal phalangeal and lamellar circulations and raised the possibility of accessing the lamellar circulation via the DP and the possibility that IO perfusion (IOP) of the DP could deliver drugs to the lamellae. IOP of the DP with methyl methacrylate (MMA) corrosion casting material resulted in filling of the lamellar and sublamellar vascular network and incomplete filling of lamellar capillaries. Perfusion of common digital artery with a suspension of barium sulfate resulted in filling of lamellar arteries but not capillaries. Perfusion of the common digital vein resulted in filling of lamellar veins but not capillaries. Perfusion with barium sulfate partitioned veins from arteries because particle size prevented entry into capillaries. IOP with barium sulfate filled only veins revealing that vascular egress from the DP was venous. This study showed that a retrograde venous connection exists between the DP and lamellar circulations with the potential for lamellar drug delivery. Intra-arterial (IA) and IO infusion results using gelatin-India Ink were markedly improved when cadaver limbs were subjected to cyclic loading within the physiological range. Without loading lamellar capillaries failed to fill no matter what the injection pressure. Cyclic loading cadaver limbs 6 times resulted in complete lamellar capillary filling and suggested that cyclic limb loading contributed to perfusion of lamellar capillaries normally in horses. To evaluate IO delivery of drugs to hoof lamellae in the standing, conscious horse, gentamicin solution (25 mg/mL) was slowly infused (20 µL/min) through an IO bone screw. Lamellar ECF was collected via a lamellar MD probe and blood was collected from the jugular vein. Gentamicin was 50-100 times more concentrated in lamellar ECF than in blood. This study introduces a potential method for delivery of drugs into the lamellar tissue in the standing, conscious horse. Laminitis pathology occurs before clinical signs and can be induced by insulin as well as enteric OF overload. Thus therapy delivered to the target of laminitis, the hoof lamellae, has an improved chance of success if delivered promptly, safely and at high concentrations. A validated drug delivery and lamellar analysis system that achieves these criteria, was the discovery of this project and is now available to combat laminitis.

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