The thrust of this thesis was to investigate the pathological processes underlying dermo-epidermal separation of the inner hoof wall lamellae of the equine foot, and to improve understanding of the relationships between the various conditions associated with laminitis and the pathology of the disease.
Laminitis often follows acute changes in metabolism due to disease, and is also associated with metabolic conditions resulting in insulin insensitivity. Administration of exogenous glucocorticoids is anecdotally associated with laminitis and this may be related to their effects on metabolic hormones. The metabolic effects and pharmacokinetics of a commonly used veterinary glucocorticoid were investigated to address this issue. Triamcinolone acetonide (TMC) altered glucose metabolism and caused a prolonged hyperglycaemia and hyperinsulinaemia. The pharmacokinetics of TMC indicated that the drug persisted in the body for long periods and affected metabolism at very low concentrations. Although TMC administration did not precipitate laminitis, it may create a situation where whole body metabolism is compromised, potentially weakening the dermo-epidermal junction.
Two in vitro models were used to investigate the effects on the dermo-epidermal junction of glucose withdrawal and enzyme activation. Glucose removal altered the structure of the hemidesmosome (HD). Transmission electron microscopy (TEM) showed progressive loss of visible HDs from the epidermal basal cells of the secondary epidermal lamellae (SEL), collapse of the cytoskeleton and concentration of the cytoskeleton around the nucleus. Immunofluorescence microscopy (IFM) suggested that the alterations in HD structure might be due to an initial loss of laminin 5, followed by concurrent loss of BP180, integrin α6 and BP230. Normal activity and interactions of the HD components with each other, with signal transduction molecules, and with protein kinases within the epidermal basal cells requires energy. Glucose withdrawal from the hoof tissues seemed to disrupt the cellular metabolism, resulting in denucleation of the HD, collapse of the epidermal cell cytoskeleton and dermo-epidermal separation along the BM.
TEM suggested that activation of constituent matrix metalloproteinases (MMPs) destroyed the anchoring filaments (AFs) connecting HDs to the basement membrane (BM), allowed the epidermal cells to separate from the BM and dermal tissue without apparent alteration to the HD ultrastructure. During the process of separation initiated by enzyme activation the results from IFM suggested there may be cleavage of laminin 5, and concurrent loss of BP180, integrin α6 and BP230. MMP activation seemed to result in a cleavage of the AFs between the HD and BM, resulting in the dermo-epidermal separation.
Laminitis induced by carbohydrate overload was investigated and compared to the in vitro models. TEM allowed an quantitative measure of laminitis. There was a correlation between severity of laminitis and ultrastructural damage to hoof lamellar epidermal basal cells in terms of loss of HDs from these cells.
Laminitic hoof tissue examined by IFM exhibited a partial loss of laminin 5, BP 180 and integrin α6, and also suggested there might be cleavage of laminin 5 in severely affected tissue. The pathology of laminitis appears to incorporate aspects of both the in vitro models, suggesting that more than one pathological process is active in the developmental phase of laminitis. I postulate that glucose starvation in the hoof tissues might be caused by alterations in whole body metabolism and is responsible for the loss of laminin 5, BP 180 and integrin α6, while the increase in transcription and activation of MMP 2 may be responsible for the cleavage of laminin 5. These two processes are jointly active in the process of dermo-epidermal separation, BM disintegration and laminitis.
One potential treatment for the preventation of laminitis is cold therapy, applied during the developmental phase of this disease. Cold therapy did not prevent the loss of epidermal basal cell HD components or the changes in cytoskeleton previously described in laminitic tissue, but may prevent cleavage of laminin 5 and separation of the epidermal basal cells from their underlying BM. Cold therapy has been shown to decrease MMP 2 activation during developmental laminitis and IFM suggested that it might also prevent enzyme mediated damage to the AFs connecting the epidermal basal cells and the BM in the SEL. This study provides evidence supporting the use of cold therapy during the developmental phase of laminitis to limit the degree of enzyme mediated BM damage, and decrease the severity of an episode of the disease.
The research documented in this thesis indicates that laminitis is the result of glucose starvation and MMP activation in the feet causing HD denucleation and AF cleavage in the BM zone of the hoof lamellae. The investigation of an extremely rare case of neonatal laminitis provided a unique demonstration of the importance of the HD adhesion complex in maintaining the stability of the dermo-epidermal junction. This animal provided a HD deficiency model and demonstrated that in the absence of any of the usual inciting conditions (septicaemia, grain overload), disturbance of HDs resulted in laminitis.
The experiments outlined in this thesis have demonstrated insights into the structural changes underlying the pathology of laminitis and the factors contributing to these changes. It is hoped that a clear understanding of the processes resulting in dermo-epidermal junction separation may lead to the development of effective treatments for this crippling disease.