No abstract

Background: Recent reports indicate increased amounts of mRNA from inflammation-related genes in the prodromal stage of laminitis.Hypothesis: Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) undergo distinct patterns of expression in equine laminae in the developmental stage (DEV) and acute clinical stage (LAM) of laminitis.Animals: Horses selected from an outbred population were placed into 1 of 4 groups: DEV (n 5 5), CON-3h (control group for DEV, n 5 5), LAM (n 5 5) and CON-10h (control group for LAM, n 5 5).Methods: Laminar and skin samples were obtained from (1) animals either undergoing leukopenia (DEV) or the onset of clinical signs of laminitis (LAM) after black walnut extract (BWE) administration and (2) animals either 3 (CON-3h ) or 10 (CON-10h) hours after administration of water. Real-time quantitative polymerase chain reaction (RT-qPCR), immunoblotting, and immunohistochemical analysis were performed for COX-1 and COX-2.Results: Upon immunohistochemical analysis of all 4 groups, COX-2 was expressed by most viable epithelial cells in both laminae and skin. COX-1 exhibited similar epithelial expression to COX-2 in skin epidermis, but was expressed exclusively in the basal layer of laminar epidermis. COX-1 protein was not detectable in dermal vasculature of equine skin or laminae, whereas COX-2 was present in endothelial and vascular smooth muscle cells of dermal vasculature in both skin and laminae in all groups. A marked increase in laminar COX-2 protein concentrations was detected on immunoblotting in the DEV group, although a lesser increase was observed in the LAM group.Conclusions and Clinical Importance: COX-2 protein expression is markedly increased in the resident laminar cell types in the developmental stage of BWE-induced laminitis.

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.

Background: Recent reports indicate increased amounts of mRNA from inflammation-related genes in the prodromal stage of laminitis.Hypothesis: Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) undergo distinct patterns of expression in equine laminae in the developmental stage (DEV) and acute clinical stage (LAM) of laminitis.Animals: Horses selected from an outbred population were placed into 1 of 4 groups: DEV (n 5 5), CON-3h (control group for DEV, n 5 5), LAM (n 5 5) and CON-10h (control group for LAM, n 5 5).Methods: Laminar and skin samples were obtained from (1) animals either undergoing leukopenia (DEV) or the onset of clinical signs of laminitis (LAM) after black walnut extract (BWE) administration and (2) animals either 3 (CON-3h ) or 10 (CON-10h) hours after administration of water. Real-time quantitative polymerase chain reaction (RT-qPCR), immunoblotting, and immunohistochemical analysis were performed for COX-1 and COX-2.Results: Upon immunohistochemical analysis of all 4 groups, COX-2 was expressed by most viable epithelial cells in both laminae and skin. COX-1 exhibited similar epithelial expression to COX-2 in skin epidermis, but was expressed exclusively in the basal layer of laminar epidermis. COX-1 protein was not detectable in dermal vasculature of equine skin or laminae, whereas COX-2 was present in endothelial and vascular smooth muscle cells of dermal vasculature in both skin and laminae in all groups. A marked increase in laminar COX-2 protein concentrations was detected on immunoblotting in the DEV group, although a lesser increase was observed in the LAM group.Conclusions and Clinical Importance: COX-2 protein expression is markedly increased in the resident laminar cell types in the developmental stage of BWE-induced laminitis.

The increased incidence of injury of epidermal structures in clinical cases of Gram-negative (vs. Gram-positive) sepsis in the horse may be due to a lack of functional TLR signalling for Gram-positive PAMPs in the equine keratinocyte.