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.

Histopathology of dairy cows' hooves with signs of naturally acquired laminitis

The purpose of this study was to investigate histological changes in dairy cows' hooves with or without injuries from naturally acquired laminitis. Cull cows with no clinical signs of hoof abnormalities (G1, n=9) and those with macroscopic lesions associated with laminitis without (G2, n=23) or with lameness (G3, n=7) were used in the study. After slaughter, samples of dermo-epidermal junctions of sole, axial and dorsal regions of the hoof were obtained and histologically processed using HE and PAS staining. Congestion, hemorrhage and inflammatory infiltrate in the dermis of sole, axial and dorsal regions were blindly and semiquantitatively evaluated by the same researcher. Inflammatory infiltrate was evaluated in the dermal laminae of axial and dorsal regions. The morphology of epidermal cells and the presence of irregularities in three regions of the basement membrane (BM) length were examined using PAS staining. Scores of lesions in different regions of the hoof in the same group and in different groups for each region of the hoof were compared using non-parametric analyses. Inflammatory infiltrate in the dermis of all regions of the hoof was detected in all groups with no significant statistical difference. Cows with no clinical signs of hoof abnormalities secondary to laminitis (G1) have inflammation scores and epidermal cell changes similar to those of groups with laminitis injuries, suggesting the existence of a prodromal phase for this disease in bovines. BM had irregularities with a variable intensity along its length, however, with no difference among groups. The pattern of BM irregularities found has not been reported so far and does not resemble the BM collapse described in horses and cattle with induced acute laminitis. Is it concluded that even in the absence of macroscopic hoof signs associated to laminitis, dairy cows have histological injuries compatible with inflammation of the dermo-epidermal junction as in affected animals. Basement membrane of cows with or without laminitis associated lesions had irregularities with an irregular distribution along its length which need to be further studied.

Histologic and inflammatory lamellar changes in horses with oligofructose-induced laminitis treated with a CXCR1/2 antagonist

Abstract: With the hypothesis that blocking chemokine signaling can ameliorate acute laminitis, the aim was to evaluate the therapeutic effect of intravenous DF1681B, a selective antagonist for CXCR1 and CXCR2 (chemokine receptors), in an oligofructose equine laminitis model. To twelve mixed breed clinically healthy hoses with no previous history of hoof-related lameness was administered oligofructose (10g/kg given by nasogastric tube) and divided into two groups: treated (intravenous DF1681B at 30mg/kg 6, 12, 18, and 24h after oligofructose) and non-treated groups. Laminar biopsies were performed before and 12, 36, and 72h after administering oligofructose. Samples were stained with periodic acid-Schiff (PAS) and scored from 0 to 6 according to epidermal cell and basal membrane changes. The IL-1β, IL-6, and CXCL1 RNA expressions were determined by RT-PCR. Parametric and non-parametric tests were used to compare times within each group (P<0.05). The PAS grades and IL-1β and IL-6 RNA expression increased in the non-treated group, but remained constant in the treated horses. In conclusion, DF1681B therapy reduced laminar inflammation and epidermal deterioration in treated horses. CXCR1/2 blockage should be considered therapeutically for equine acute laminitis.

Assessment of apoptosis in epidermal lamellar cells in clinically normal horses and those with laminitis

Objective - To determine and compare the number, type, location, and distribution of apoptotic epidermal cells in the laminae of clinically normal horses and horses with laminitis.Sample Population - Formalin-fixed samples of digital lamellar tissue from 47 horses (including clinically normal horses [controls; n = 7], horses with acurte [4] and chronic [7] naturally acquired laminitis, and horses with black walnut extract-induced [11] or carbohydrate overload-induced [18] laminitis).Procedure - Blocks of paraffin-embedded lamellar tissues were stained for DNA fragmentation with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. Differential immunohistochemical staining for caspases 3 and 14 were used to confirm apoptosis.Results - the number of TUNEL-positive epidermal cells per 0.1 mm of primary laminae was significantly greater in the acute laminitis group than in the other groups. In the acute laminitis group, there were 17 and 1,025 times as many TUN EL-positive basal layer cells and keratinocytes, respectively, compared with the control group. Apoptosis of TUNEL-positive basal layer cells was confirmed by results of caspase 3 immunohistochemical staining. The TUNEL-positive keratinocytes did not stain for caspases 3 or 14.Conclusions and Clinical Relevance - the large number of apoptotic basal layer cells detected in the lamellar tissue of horses with acute naturally acquired laminitis suggests that apoptosis may be important in the development of acute laminitis. The role of the large number of TUNEL-positive keratinocytes detected in the interface of primary and secondary epidermal laminae of horses with acute laminitis remains to be elucidated.

Changes in plasma protein concentrations in ponies with experimentally induced alimentary laminitis

Objective-To determine whether plasma protein concentrations were altered in ponies with alimentary laminitis.Animals-12 adult ponies.Procedure-Acute laminitis was induced in 6 ponies by oral administration of carbohydrate (85% corn starch, 15% wood flour); the other 6 ponies were used as controls. A physical examination was performed and blood samples were collected immediately before and 4, 8, 12, 24, and 28 hours after administration of carbohydrate. Plasma protein concentrations were determined by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis.Results-19 plasma proteins ranging from a molecular weight of 24,000 to a molecular weight of 350,000 were identified in all 12 ponies. Plasma concentrations of proteins with molecular weights of 350,000 (fibrinogen), 130,000 (ceruloplasmin), 118,000 (c-reactive protein), 67,000 (alpha(1)-antitrypsin I), 65,000 (alpha(1)-antitrypsin II), 50,000 (haptoglobulin), and 45,000 (acid glycoprotein) were significantly increased in ponies with laminitis, compared with concentrations in control ponies.Conclusion-Changes in plasma protein concentrations are detectable within 4 hours after the onset of alimentary laminitis in ponies.Clinical Relevance-Measurement of plasma protein concentrations may be useful in monitoring the progression of laminitis in ponies.

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.

Acute bovine laminitis: A new induction model using alimentary oligofructose overload

Twelve dairy heifers were used to examine the clinical response of an alimentary oligofructose overload. Six animals were divided into 3 subgroups, and each was given a bolus dose of 13, 17, or 21 g/kg of oligofructose orally. The control group (n = 6) was sham-treated with tap water. Signs of lameness, cardiovascular function, and gastrointestinal function were monitored every 6 h during development of rumen acidosis. The heifers were euthanized 48 and 72 h after administration of oligofructose. All animals given oligofructose developed depression, anorexia, and diarrhea 9 to 39 h after receiving oligofructose. By 33 to 45 h after treatment, the feces returned to normal consistency and the heifers began eating again. Animals given oligofructose developed transient fever, severe metabolic acidosis, and moderate dehydration, which were alleviated by supportive therapy. Four of 6 animals given oligofructose displayed clinical signs of laminitis starting 39 to 45 h after receiving oligofructose and lasting until euthanasia. The lameness was obvious, but could easily be overlooked by the untrained eye, because the heifers continued to stand and walk, and did not interrupt their eating behavior. No positive pain reactions or lameness were seen in control animals. Based on these results, we conclude that an alimentary oligofructose overload is able to induce signs of acute laminitis in cattle. This model offers a new method, which can be used in further investigation of the pathogenesis and pathophysiology of bovine laminitis.