Laminitis is a debilitating disease of equidae that has major welfare and economic consequences for the equine industry worldwide. The frequency of this disease in equids is estimated to be 1.5% to 34%.1 Laminitis has typically been considered to develop secondary to systemic inflammation or gastrointestinal tract disease2–4 or uneven weight distribution in limbs.5 More recently, results of other studies6,7 have indicated that endocrine disturbances (ie, endocrinopathic laminitis) account for most cases of laminitis evaluated at first-opinion practices in Europe and the United States.6,7 Such endocrinopathies include pituitary pars intermedia dysfunction and hyperinsulinemia attributable to equine metabolic syndrome. Recently, laminitis has been induced in horses and ponies with a pEHC technique.8,9 Hyperinsulinemia in the absence of systemic inflammation and hyperglycemia consistently occurs in animals with laminitis that is associated with pituitary pars intermedia dysfunction or equine metabolic syndrome.10–12
The laminar region of the hoof in equids holds the distal phalanx and the hoof wall together; it consists of epidermis connected to underlying dermis by a basement membrane. The surface area of this dermal-epidermal junction is greatly increased by complex primary and secondary infoldings, called laminae. In an anatomically normal hoof, there are 550 to 600 PEL, each with 150 to 200 SEL.13 Regardless of disease etiology, the laminar structure in horses with laminitis weakens, which leads to stretching of laminae because of ground reaction forces, pull of the deep digital flexor tendon on the palmar or plantar aspect of the distal phalanx, and pressure that develops along the dorsal aspect of the hoof wall during breakover (ie, the period between the point at which the heel leaves the ground and the toe leaves the ground at the end of the stance phase of the stride).14 Studies of laminitis have typically involved histologic examination of hoof lamina samples obtained from horses that received an overload of carbohydrate (starch or oligofructose). However, results of studies15–18,a in which a pEHC technique was used indicate substantial differences between endocrinopathic and nonendocrinopathic forms of laminitis. In all animals with laminitis, considerable elongation of SEL develops; however, severe basement membrane damage may be the most prominent pathological finding for horses with laminitis induced with carbohydrate overload.15 For horses that undergo a pEHC technique, basement membrane damage is minimal and frequently localized to SEL of the axial aspects of PEL.8,9 Additionally, in ponies and horses with insulin-induced laminitis, increased apoptotic cell death and mitotic and proliferative activity have been detected, but these factors have only been quantified for horses.8,19
Insulin is a peptide hormone secreted by pancreatic β cells. Secretion of insulin is mainly regulated in response to plasma glucose concentration, but compensatory hyperinsulinemia develops when target cells have insulin resistance. After binding to its receptor, insulin may have metabolic effects via the phosphatidylinositol 3-kinase (PI-3K) pathway or mitogenic and growth-related effects via the mitogen-activated protein kinase pathway. During a state of insulin resistance and hyperinsulinemia, the mitogen-activated protein kinase pathway is the dominant pathway.20,21 Insulin resistance in human keratinocytes alters patterns of proliferation and differentiation.22 Proliferative effects of insulin may cause inappropriate laminar keratinocyte proliferation, resulting in the elongation of SEL detected for equids with laminitis induced by means of a pEHC technique; however, the relative contributions of cell proliferation and stretching to elongation of SEL have not been determined.23,b We hypothesized that elongation of SEL is attributable to cell stretching rather than simple addition of cells of the same size. Our objective was to measure morphological features of the SEL of ponies with insulin-induced laminitis and to quantify and localize changes in cell death and proliferation. Given that stretching of cells would indirectly indicate a reduction in resistance to mechanical forces of weight bearing, another objective was to determine differences in histomorphometric variables of SEL between forefeet and hind feet; in all previous studies of laminitis in ponies and horses, only forefeet have been evaluated, to the authors' knowledge.
Prolonged euglycemic-hyperinsulinemic clamp
Primary epidermal lamina
Secondary epidermal lamina
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