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.
| Identifer | oai:union.ndltd.org:ADTP/254184 |
| Creators | Alireza Nourian |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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