Cyclooxygenase Expression in The Early Stages of Equine Laminitis: A Cytologic Study

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.

Effect of Dietary Nonstructural Carbohydrate Content on Activation of 5′‐Adenosine Monophosphate‐Activated Protein Kinase in Liver, Skeletal Muscle, and Digital Laminae of Lean and Obese Ponies

“…The concentration of phosphorylated and total AMPK in liver, skeletal muscle, and laminar tissue homogenates was assessed by Western immunoblotting performed as described previously 20. Briefly, protein samples (approximately 300 mg tissue per sample) were prepared in 5 mL of lysis buffer8 with added protease and phosphatase inhibitors and quantitated using the Bradford method.…”

Neuropathic Pain Management in Chronic Laminitis

“…40,43,46,60 First, administration of NSAIDs during the developmental stage, when COX-2 expression is upregulated, does not seem to prevent acute laminitis or alter the course of the disease arguing against a dominant antiinflammatory action. 61 Second, increased prostaglandin activity has not been detected in the acute and chronic phases of laminitis despite evidence for ongoing inflammation, 8,9,21,25 supporting the notion that if NSAIDS exhibit antiinflammatory activity at high doses this effect may not be related to inhibition of prostanoid synthesis. 43,46 Third, unlike in peripheral tissues, where COX-1 is constitutively present for tissue homeostasis and COX-2 is inducible by inflammation, 62 both COX isoforms are constitutively present in the CNS but with functionally different roles.…”

“…interleukin [IL]-1β, IL-6), cyclooxygenase (COX)-2, and matrix metalloproteinases (MMPs) in the digital laminae. [5][6][7][8][9] Locally released and activated MMPs mediate degradation of the collagen components of the basement membrane (BM) that is interposed between the secondary dermal (SDL) and epidermal lamellae (SEL) and cause a separation of the SEL from the BM. 4,[10][11][12][13][14] Sensory innervation of the foot consists of thick myelinated A-fibers (largely A β ) transmitting low-threshold mechanical information and small thin myelinated (A δ -fibers) and un-myelinated afferents (C-fibers) which express a variety of peptides and transmit high-threshold nociceptive information.…”

“…7,8 Unless resolving on their own histological changes at the dermal-epidermal interface progress further causing within 24-72 hours the BM to retract so much from SEL that SDL connective tissue and SDL capillaries are injured by tension and shear forces (grade 2 histological laminitis), 4 likely provoking activation of perivascular sensory nerve terminals near the base of the dermal lamellae.…”

“…[3][4][5] Nociception is most often recognized by lameness or the characteristic stance of the animal and rapidly increasing sensitivity to hoof testers. 3,5 Even though evidence for a marked increase in COX-2 enzyme activity could not be found in the acute phase of experimentally induced laminitis, 8 concentrations of other vasoactive degradation products of arachidonic acid (e.g. isoprostanes) are elevated during the acute phase of experimentally induced laminitis.…”