SummaryReasons for performing study: There is a need to assess the laminar inflammatory response in a laminitis model that more closely resembles clinical cases of sepsis-related laminitis than the black walnut extract (BWE) model.

Background: Laminar inflammation is one of the earliest events in equine laminitis. Calprotectin (CP), a Damage-Associated Molecular Pattern protein, is overexpressed in inflammatory conditions of human skin.Hypothesis: CP is overexpressed in the laminar epidermis of horses with black walnut extract (BWE)-induced laminitis. Animals: Twenty adult horses. Methods: Experimental study. Horses were allocated to one of 4 groups. BWE was administered to horses in 3 groups, which were sampled 1.5, 3, and 12 hours (LAM) later. CP was visualized by immunohistochemistry. Laminar leukocyte counts and intensity of laminar epithelial staining were scored for all animals and statistically analyzed.Results: Laminar epidermal CP signal was significantly increased (P 5 .02) at the LAM time point, compared with other groups. Rare leukocytes were detected in laminae with CP staining in CON group, but there were marked increases in number of leukocytes in BWE-treated groups (P 5 .003). Sequential hematoxylin and eosin staining demonstrated that the majority of CP-positive leukocytes were perivascular polymorphonuclear neutrophils (PMN) at each of the developmental time points. CPpositive PMN and mononuclear cells were detected in perivascular locations and close to the epidermal basement membrane in the LAM group.Conclusions and Clinical Importance: CP expression in the laminar epidermis occurs after extravasation of leukocytes, indicating that leukocyte emigration might be an initiating factor in laminar epithelial stress and inflammation in BWE-induced laminitis. These results indicate a possible role of CP in laminitis pathophysiology and laminar failure.

Background: While there is evidence of laminar leukocyte infiltration in black walnut extract (BWE)-induced laminitis, there is no such evidence for carbohydrate overload (CHO) laminitis.Objective: To assess presence of leukocytes and signs of epidermal stress/injury in the laminar tissue from horses with CHO-induced laminitis.Animals: Twenty-four adult horses. Methods: Immunohistochemistry for myeloid cell markers calprotectin (CP) and monocyte-specific marker (CD163) was performed on laminar sections obtained from 2 groups of horses in the CHO model: the developmental time point (DTP) group (n 5 6) and the onset of lameness (LAM) group (n 5 6), and a control (CON) group (n 5 8).Results: DTP was characterized by an increase in CP 1 leukocytes (7.8-fold increase versus CON, P o .001), and LAM time point was characterized by a more marked increase in laminar CP 1 (108.5-fold, P o .001) and mild increase in CD163

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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.

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.

Background: C-X-C motif ligand 1 (CXCL1) is an important chemokine of epithelial origin in rodents and humans. Objectives: To assess in vivo and in vitro the regulation of CXCL1 in equine laminitis. Animals: Twenty adult horses. Methods: Real-time quantitative polymerase chain reaction (PCR) was used to assess expression of CXCL1 in samples of laminae, liver, skin, and lung from the black walnut extract (BWE) model of laminitis, and in cultured equine epithelial cells (EpCs). Tissue was obtained from control animals (CON, n 5 5), and at 1.5 hours (early time point [ETP] group, n 5 5), at the onset of leukopenia (developmental time point [DTP] group, n 5 5), and at the onset of lameness (LAM group, n 5 5) after BWE administration. EpCs were exposed to Toll-like/Nod receptor ligands, oxidative stress agents, and reduced atmospheric oxygen (3%). In situ PCR was used to localize the laminar cell types undergoing CXCL1 mRNA expression.Results: Increases in laminar CXCL1 mRNA concentrations occurred in the ETP (163-fold [P 5 .0001]) and DTP groups (21-fold [P 5 .005]). Smaller increases in CXCL1 expression occurred in other tissues and organs. In cultured EpCs, increases (P o .05) in CXCL1 mRNA concentration occurred after exposure to lipopolysaccharide (LPS [28-fold]), xanthine/xanthine oxidase (3.5-fold), and H 2 O 2 (2-fold). Hypoxia enhanced the LPS-induced increase in CXCL1 mRNA (P 5 .007). CXCL1 gene expression was localized to laminar EpCs, endothelial cells, and emigrating leukocytes.Conclusion and Clinical Importance: These findings indicate that CXCL1 plays an early and possibly initiating role in neutrophil accumulation in the BWE laminitis model, and that laminar keratinocytes are an important source of this chemokine. New therapies using chemokine receptor antagonists may be indicated.

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