Equine laminitis: loss of hemidesmosomes in hoof secondary epidermal lamellae correlates to dose in an oligofructose induction model: an ultrastructural study

Reasons for performing study: Light microscopical studies show that the key lesion of laminitis is separation at the hoof lamellar dermal-epidermal interface. More precise knowledge of the damage occurring in the lamellar basement membrane zone may result if laminitis affected tissue is examined with the transmission electron microscope. This could lead to better understanding of the pathogenesis of lesions and the means of treatment or prevention. Objectives: To investigate the ultrastructure of acute laminitis as disease of greater severity is induced by increasing oligofructose (OF) dosage. Methods: Three pairs of normal horses, dosed with OF at 7.5, 10 and 12.5 g/kg bwt via nasogastric intubation, developed laminitis 48 h later. Following euthanasia, their forefeet were processed for transmission electron microscopy. Lamellar basal cell hemidesmosome (HD) numbers and the distance between the basal cell plasmalemma and the lamina densa of the basement membrane were estimated and compared to control tissue. Results: Increasing OF dosage caused greater HD loss and more severe laminitis. The characteristic separation of the basement membrane, cytoskeleton failure and rounded basal cell nuclei results from combined HD dysassembly and anchoring filament failure. Conclusions: Without properly assembled HDs, dysadhesion between the lamina densa of the basement membrane (BM) and epidermal basal cells occurs, emphasising the fundamental importance of HDs in maintaining attachment at the lamellar interface. Medical conditions that trigger lamellar matrix metalloproteinase (MMP) activation and/or compromise entry of glucose into lamellar basal cells appear to promote loss and failure of HDs and, therefore, laminitis development. Potential relevance: A correlation between lameness severity and escalating loss of lamellar HDs now exists. Therapy aimed at protecting the lamellar environment from haematogenous delivery of MMP activators or from glucose deprivation may control laminitis development.

Equine laminitis: Bites by Bothrops spp cause hoof lamellar pathology in the contralateral as well as in the bitten limb

The envenoming caused by Bothrops snakebite includes local symptoms, such as pronounced edema, hemorrhage, intense pain, vesicles, blisters and myonecrosis. The principal systemic symptom consists in the alteration of blood clotting, due to fibrinogen consumption and platelet abnormalities. The horses involved in this study had this symptomatology and one of them exhibited symptoms consistent with laminitis in the bitten and in the contralateral limbs. Laminitis lesions were characterized by separation of the hoof lamellar basement membrane (BM) from basal cells of the epidermis. These results demonstrated that Bothrops snake venom can induce acute laminitis. We conclude that components of the venom, probably metalloproteinases, cause severe lesions in the hoof early in the envenoming process. Antivenom therapy must be initiated as soon as possible in order to prevent complications, not only to save the life of an envenomed horse, but also to avoid the dysfunctional sequels of laminitis. (c) 2006 Elsevier Ltd. All rights reserved.

In vitro evidence for a bacterial pathogenesis of equine laminitis

Utilizing an in vitro laminitis explant model, we have investigated how bacterial broth cultures and purified bacterial proteases activate matrix metalloproteinases (MMPs) and alter structural integrity of cultured equine lamellar hoof explants. Four Gram-positive Streptococcus spp. and three Gram-negative bacteria all induced a dose-dependent activation of MMP-2 and MMP-9 and caused lamellar explants to separate. MMP activation was deemed to have occurred if a specific MMP inhibitor, batimastat, blocked MMP activity and prevented lamellar separation. Thermolysin and streptococcal pyrogenic exotoxin B (SpeB) both separated explants dose-dependently but only thermolysin was inhibitable by batimastat or induced MMP activation equivalent to that seen with bacterial broths. Additionally, thermolysin and broth MMP activation appeared to be cell dependent as MMP activation did not occur in isolation. These results suggest the rapid increase in streptococcal species in the caecum and colon observed in parallel with carbohydrate induced equine laminitis may directly cause laminitis via production of exotoxin(s) capable of activating resident MMPs within the lamellar structure. Once activated, these MMPs can degrade key components of the basement membrane (BM) hemidesmosome complex, ultimately separating the BM from the epidermal basal cells resulting in the characteristic laminitis histopathology of hoof lamellae. While many different causative agents have been evaluated in the past, the results of this study provide a unifying aetiological mechanism for the development of carbohydrate induced equine laminitis. (C) 2001 Elsevier Science B.V. All rights reserved.

Equine laminitis: increased transcription of matrix metalloproteinase-2 (MMP-2) occurs during the developmental phase

Reasons for performing study: The dysadhesion and destruction of lamellar basement membrane of laminitis may be due to increased lamellar metalloproteinase activity. Characterising lamellar metalloproteinase-2 (MMP-2) and locating it in lamellar tissues may help determine if laminitis pathology is associated with increased MMP-2 transcription. Objectives: To clone and sequence the cDNA encoding lamellar MMP-2, develop antibody and in situ hybridisation probes to locate lamellar MMP-2 and quantitate MMP-2 transcription in normal and laminitis tissue. Methods: Total RNA was isolated, fragmented by RT-PCR, cloned into vector and sequenced. Rabbit anti-equine MMP-2 and labelled MMP-2 riboprobe were developed to analyse and quantitate MMP-2 expression. Results: Western immunoblotting with anti-MMP-2 detected 72 kDa MMP-2 in hoof tissue homogenates and cross-reacted with human MMP-2. Immunohistochemistry and in situ hybridisation detected MMP-2 in the cytoplasm of basal and parabasal cells in close proximity to the lamellar basement membrane. Northern analysis and quantitative real-time PCR showed MMP-2 expression significantly (P

Equine laminitis: cleavage of laminin 5 associated with basement membrane dysadhesion

Reasons for performing study: The key lesion of laminitis is separation at the hoof lamellar dermal-epidermal interface. For this to happen the structural and adhesion proteins of the basement membrane zone must be altered. Which proteins and how damage to them leads to the lamellar separation of laminitis is unknown. Objectives: To investigate lamellar hemidesmosome and cytoskeleton damage and basement membrane dysadhesion using light microscopy (LM) and immunofluorescence microscopy (IFM). Methods: Cryostat sections of lamellar tissues from 2 control and 6 Standardbred horses with oligofructose induced laminitis were studied using LM and IFM. Plectin, integrin alpha(6) and BP230 antibody was used to label hemidesmosome intracellular plaque proteins and anti-BP180 and anti-laminin 5 (L5) was used to label anchoring filament (AF) proteins. Cytoskeleton intermediate filaments were labelled using anti-cytokeratin 14. The primary antibodies of selected sections were double labelled to show protein co-localisation. Results: Laminitis caused reduction of transmembrane integrin alpha(6), the AF proteins BP180 and L5,and failure of co-localisation of BP180 and L5. Proteins of the inner hemidesmosomal plaque, plectin and BP230, were unaffected. Conclusions: Loss of co-localisation of L5 and BP180 suggests that, during the acute phase of laminitis, L5 is cleaved and therefore, the AFs connecting the epidermis to the dermis, fail. Without a full complement of AFs separation at the lamellar dermo-epidermal junction occurs. Potential relevance: Suppressing or inhibiting metalloproteinase activity may prevent L5 cleavage and therefore the lamellar dermo-epidermal separation of laminitis.

Equine laminitis: glucose deprivation and MMP activation induce dermo-epidermal separation in vitro

Reasons for performing study: Acute laminitis is characterised by hoof lamellar dermal-epidermal separation at the basement membrane (BM) zone. Hoof lamellar explants cultured in vitro can also be made to separate at the basement membrane zone and investigating how this occurs may give insight into the poorly understood pathophysiology of laminitis. Objectives: To investigate why glucose deprivation and metalloproteinase (MMP) activation in cultured lamellar explants leads to dermo-epidermal separation. Methods: Explants, cultured without glucose or with the MMP activator p-amino-phenol-mercuric acetate (APMA), were subjected to tension and processed for transmission electron microscopy (TEM). Results: Without glucose, or with APMA, explants under tension separated at the dermo-epidermal junction. This in vitro separation occurred via 2 different ultrastructural processes. Lack of glucose reduced hemidesmosomes (HDs) numbers until they disappeared and the basal cell cytoskeleton collapsed. Anchoring filaments (AFs), connecting the basal cell plasmalemma to the BM, were unaffected although they failed under tension. APMA activation of constituent lamellar MMPs did not affect HDs but caused AFs to disappear, also leading to dermo-epidermal separation under tension. Conclusions: Natural laminitis may occur in situations where glucose uptake by lamellar basal cells is compromised (e.g. equine Cushing's disease, obesity, hyperlipaemia, ischaemia and septicaemia) or when lamellar MMPs are activated (alimentary carbohydrate overload). Potential relevance: Therapies designed to facilitate peripheral glucose uptake and inhibit lamellar MMP activation may prevent or ameliorate laminitis.

Equine laminitis

This article discusses the physiology of the normal feet of horses, in order to understand the pathogenesis of laminitis. It also discusses the use of cryotherapy to treat horses with laminitis.