B.S. Leise

Laminar inflammatory gene expression in the carbohydrate overload model of equine laminitis.

REASONS 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. OBJECTIVES To determine if a similar pattern of laminar inflammation, characterised by proinflammatory cytokine expression, occurs in the CHO model of laminitis as has been previously reported for the BWE model. METHODS Sixteen horses administered 17.6 g of starch (85% corn starch/15% wood flour)/kg bwt via nasogastric (NG) tube were anaesthetised either after developing a temperature>38.9°C (DEV group, n=8) or at onset of Obel grade 1 lameness (OG1 group, n=8). Control horses (CON group, n=8) were anaesthetised 24 h after NG administration of 6 l of deionised water. Laminar tissue was collected from horses while under anaesthesia, followed by humane euthanasia. Real time-quantitative PCR was used to assess laminar mRNA concentrations of genes involved in inflammatory signalling. RESULTS Increased mRNA concentrations (P<0.05) for IL-1β, IL-6, IL-12p35, COX-2, E-selectin and ICAM-1 were present in laminae from horses with OG1 lameness but not at the DEV time, when compared to the CON horses. No differences between the groups were found for IL-2, IL-4, IL-10, TNF-α, IFN-γ or COX-1 at either the DEV or OG1 time points. CONCLUSIONS There was a notable difference in the temporal pattern of inflammatory events between the BWE and CHO models, with the majority of laminar inflammatory events appearing to occur at or near the onset of lameness in the CHO model, whereas many of these events peak earlier in the developmental stages in the BWE model. This suggests that, in addition to circulating inflammatory molecules, there may be a local phenomenon in the CHO model resulting in the simultaneous onset of multiple laminar events including endothelial activation, leucocyte emigration and proinflammatory cytokine expression. POTENTIAL RELEVANCE The similar (although somewhat delayed) inflammatory response in the CHO model of laminitis indicates that inflammatory signalling is a consistent entity in the pathophysiology of laminitis.

Digital hypothermia inhibits early lamellar inflammatory signalling in the oligofructose laminitis model.

REASONS FOR PERFORMING STUDY The pathophysiological events inhibited by prophylactic digital hypothermia that result in reduction of the severity of acute laminitis are unknown. OBJECTIVES To determine if digital hypothermia inhibits lamellar inflammatory signalling during development of oligofructose (OF) induced laminitis. METHODS Fourteen Standardbred horses were given 10 g/kg bwt OF by nasogastric tube with one forelimb (CRYO) continuously cooled by immersion in ice and water and one forelimb (NON-RX) at ambient temperature. Lamellae were harvested prior to the onset of lameness (24 h post OF administration, DEV group, n = 7) or at the onset of lameness (OG1 group, n = 7). Lamellar mRNA was purified and cDNA produced for real time-quantitative PCR analysis of mRNA concentrations of cytokines (IL-6, IL-1β, IL-10), chemokines (CXCL1, CXCL6, CXCL8/IL-8, MCP-1, MCP-2), cell adhesion molecules (ICAM-1, E-selectin), COX-2 and 3 housekeeping genes. Data were analysed (NON-RX vs. CRYO, NON-RX vs. archived control [CON, n = 7] lamellar tissue) using nonparametric tests. RESULTS Compared with CON, the OG1 NON-RX had increased (P<0.05) lamellar mRNA concentrations of all measured mediators except IL-10, IL-1β and MCP-1/2, whereas only CXCL8 was increased (P<0.05) in DEV NON-RX. Within the OG1 group, CRYO limbs (compared with NON-RX) had decreased (P<0.05) mRNA concentrations of the majority of measured inflammatory mediators (no change in MCP-1 and IL-10). Within the DEV group, mRNA concentrations of CXCL-1, ICAM-1, IL-1β, CXCL8 and MCP-2 were decreased (P<0.05) and the anti-inflammatory cytokine IL-10 was increased (compared with NON-RX limbs; P<0.05). CONCLUSIONS Digital hypothermia effectively blocked early lamellar inflammatory events likely to play an important role in lamellar injury including the expression of chemokines, proinflammatory cytokines, COX-2 and endothelial adhesion molecules. POTENTIAL RELEVANCE This study demonstrates a potential mechanism by which hypothermia reduces the severity of acute laminitis, and may help identify molecular targets for future laminitis intervention.

Proinflammatory cytokine responses of cultured equine keratinocytes to bacterial pathogen-associated molecular pattern motifs

REASONS FOR PERFORMING STUDY Further knowledge of equine keratinocyte physiology and keratinocyte response to various stimuli is important in developing a better understanding of disease states involving the epidermis. OBJECTIVES To assess the inflammatory cytokine response of cultured equine keratinocytes to various pathogen-associated molecular pattern molecules (PAMPs) from both Gram-negative and positive bacteria likely to be present in equine sepsis. METHODS Keratinocytes were isolated from skin of 2 horses and primary cultures performed. Keratinocytes were harvested for RNA extraction after exposure to lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PGN), bacterial DNA (CpG), flagellin or maintained in medium (controls) for 4 or 24 h. Real time-quantitative PCR was used to quantify interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and CXCL8 mRNA concentrations. RESULTS Increases (P<0.05) in IL-1beta, IL-6 and CXCL8 mRNA concentrations were induced by LPS exposure compared to controls. Increased mRNA concentrations of both IL-6 and CXCL8 were also noted (vs. controls) upon exposure to flagellin. Overall, responses were greater at 4 h. No increases (P>0.05) in cytokine expression by keratinocytes were present after LTA, PGN or CpG exposure. CONCLUSIONS Increased proinflammatory cytokine expression in response to LPS and flagellin indicate that equine keratinocytes have functional TLR4 and TLR5 receptor signalling. However, the lack of keratinocyte stimulation by PGN, LTA or CpG provides no evidence for functional TLR2, TLR9 or NOD receptor signalling. These results suggest that equine keratinocytes are more responsive to PAMPs usually associated with Gram-negative sepsis and unresponsive to PAMPs most commonly associated with Gram-positive sepsis. POTENTIAL RELEVANCE 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.

Laminar chemokine mRNA concentrations in horses with carbohydrate overload-induced laminitis.

Chemokines play a vital role in leukocyte activation and emigration that reportedly plays a central role in laminar injury in equine laminitis. The purpose of this study was to evaluate the pattern of laminar chemokine expression in horses in the classical carbohydrate overload (CHO)-model of laminitis. Laminar samples were obtained 24h following water administration in the control group (CON, n=8), and at the onset of fever (≥ 102°F, 12-22 h post CHO, DEV group, n=8) and at the onset of lameness (20-48 h post CHO, LAM group, n=8) in induced horses. Real time quantitative PCR was performed on all samples in order to determine laminar mRNA concentrations of both CXC chemokines (CXCL1, CXCL6, CXCL8) and CC chemokines (CCL2 [MCP-1], CCL3 [MIP-1α], and CCL8 [MCP-2]). Data were subjected to ANOVA followed by Student-Newman-Keuls (P<0.05). Laminar mRNA concentrations for all CXC chemokines were increased (P<0.05) at both the DEV and LAM horses when compared to the control horses, whereas mRNA concentrations of CCL2 and CCL8 were only increased in the LAM horses when compared to controls and the DEV horses. When taken in context with our previous studies, CXCL1, CXCL6 and CXCL8 increases precede peak laminar leukocyte accumulation. Additionally, CCL2 and CCL8 expression corroborate previous reports of monocyte/macrophage accumulation in affected laminae. Compared with previous studies, our findings demonstrate that increased laminar CXC chemokine expression consistently precedes peak leukocyte accumulation and onset of lameness in CHO laminitis models. Chemokine antagonists may be considered as possible therapeutic targets to decrease the influx of leukocytes that occurs during the development of equine laminitis.

Hindlimb laminar inflammatory response is similar to that present in forelimbs after carbohydrate overload in horses

REASONS FOR PERFORMING STUDY A significant proinflammatory response is known to occur in the forelimb lamina after carbohydrate administration. As the hindlimbs are often less affected by laminitis compared with the forelimbs, we assessed hindlimb inflammatory response in the early stages of carbohydrate-induced laminitis to determine whether differences in the response existed. OBJECTIVE To determine whether a similar proinflammatory response occurs in the hindlimb laminae to that previously reported for the forelimb. METHODS Archived laminar samples from 12 horses administered 17.6 g of starch (85% corn starch, 15% wood flour)/kg bwt via nasogastric tube that were anaesthetised either after developing a temperature >38.9°C (DEV; n = 6) or at the onset of Obel grade 1 lameness (OG1; n = 6) were used in addition to 6 control horses (CON) that were anaesthetised 24 h after administration of water. Real-time quantitative polymerase chain reaction for selected proinflammatory mediators and MAC387 immunohistochemistry were performed. The data were analysed nonparametrically to compare groups. RESULTS Increases in laminar MAC387-positive leucocytes and laminar messenger ribonucleic acid (mRNA) concentrations (P<0.05) for interleukin-1β, interleukin-6, cyclo-oxygenase-2, chemokine (C-X-C motif)ligand (CXCL)1 and CXCL8 were present in both fore- and hindlimb laminae from horses with OG1 lameness. Both CXCL1 and CXCL8 were also increased in forelimb and hindlimb laminae in the DEV horses. CONCLUSIONS Administration of carbohydrate resulted in a similar inflammatory response in the hindlimb laminae to that previously reported for the forelimb laminae. These findings suggest that other factors, such as weightbearing, may play an important role in the development of laminitis after a systemic inflammatory condition develops. POTENTIAL RELEVANCE Evidence of inflammation in the hindlimb laminae suggests that the hindfeet should be addressed in the septic horse at risk for laminitis; however, laminitis is often less severe in the hindlimbs due to other factors, such as weightbearing and hoof angle.

Use of laser capture microdissection for the assessment of equine lamellar basal epithelial cell signalling in the early stages of laminitis

REASONS FOR PERFORMING STUDY Dysadhesion of laminar basal epithelial cells (LBECs) from the underlying dermis is the central event leading to structural failure in equine laminitis. Although many studies of sepsis-related laminitis have reported multiple events occurring throughout the lamellar tissue, there is minimal information regarding signalling events occurring specifically in LBECs. OBJECTIVES To determine signalling events in the LBECs during the early stages of carbohydrate-induced laminitis. STUDY DESIGN Experimental study. METHODS Eight horses were given an overload of carbohydrate (CHO) consisting of corn starch mixture via nasogastric tube. Prior to administration of CHO, lamellar biopsies were taken from the left forefoot (control [CON]). Biopsies were taken from the left hind foot at the onset of fever (developmental [DEV]) and from the right forefoot at the onset of Obel grade 1 lameness (OG1). Laminar basal epithelial cells were isolated from cryosections using a laser capture microdissection (LCM) microscope. Next generation sequencing (RNA-seq) was used to identify transcripts expressed in the LBECs for each time point and bioinformatic analysis was performed with thresholds for between group comparisons set at a greater than 2-fold change and P value ≤0.05. RESULTS Forty genes (22 increased/18 decreased) were significantly different from DEV time vs. CON and 107 genes (57 increased/50 decreased) were significantly different from OG1 time vs. CON. Significant increases in inflammatory genes were present in addition to significantly altered expression of genes related to extracellular matrix composition, stability and turnover. CONCLUSIONS Signalling related to inflammatory response and extracellular matrix regulation was strongly represented at the DEV and OG1 times. These results indicate that the LBEC is not only a casualty but also an active participant in lamellar events leading to structural failure of the digital lamellae in equine laminitis.

Concentrations of serum amyloid A and plasma fibrinogen in horses undergoing emergency abdominal surgery

Objective To compare the perioperative response of serum amyloid A (SAA) to fibrinogen in horses requiring exploratory celiotomy for colic and to determine if SAA could be used to predict complications and outcome. Design Prospective observational clinical study. Setting University teaching hospital. Animals Eighteen horses undergoing exploratory celiotomy for colic. Inclusion criteria for the study included survival and anesthetic recovery from exploratory celiotomy, no history of surgery within the past year. Interventions Blood was obtained via jugular venipuncture before surgery (time 0) and at 24, 48, 72, and 96 hours after recovery from anesthesia. Measurements and Main Results Quantitative and semiquantitative fibrinogen, SAA, total nucleated cell counts, and total protein were evaluated at each time point. Multivariable linear regression was used to assess differences at each time point and after grouping horses according to duration of colic prior to surgery, strangulating surgical lesion or not, presence of systemic inflammatory response syndrome (SIRS) on admission, and postsurgical complications. Significant (P < 0.05) increases in SAA concentrations occurred in all cases after surgery compared to fibrinogen concentration, which only demonstrated a mild, clinically insignificant increase postsurgery. SAA concentrations were also significantly increased (P < 0.05) in cases identified with SIRS prior to surgery and postoperatively at 48 (P = 0.05) and 72 hours (P = 0.02) in horses that developed complications. Conclusions Measurement of SAA is a more sensitive indicator of inflammation than fibrinogen in the perioperative period of horses requiring exploratory celiotomy for colic. Serial measurement of SAA at 48, 72, and 96 hours after surgery may be helpful to determine risk of complications and guide postoperative management. Measurement of SAA on admission also allows for quantification of SIRS when it is detected clinically.OBJECTIVE To compare the perioperative response of serum amyloid A (SAA) to fibrinogen in horses requiring exploratory celiotomy for colic and to determine if SAA could be used to predict complications and outcome. DESIGN Prospective observational clinical study. SETTING University teaching hospital. ANIMALS Eighteen horses undergoing exploratory celiotomy for colic. Inclusion criteria for the study included survival and anesthetic recovery from exploratory celiotomy, no history of surgery within the past year. INTERVENTIONS Blood was obtained via jugular venipuncture before surgery (time 0) and at 24, 48, 72, and 96 hours after recovery from anesthesia. MEASUREMENTS AND MAIN RESULTS Quantitative and semiquantitative fibrinogen, SAA, total nucleated cell counts, and total protein were evaluated at each time point. Multivariable linear regression was used to assess differences at each time point and after grouping horses according to duration of colic prior to surgery, strangulating surgical lesion or not, presence of systemic inflammatory response syndrome (SIRS) on admission, and postsurgical complications. Significant (P < 0.05) increases in SAA concentrations occurred in all cases after surgery compared to fibrinogen concentration, which only demonstrated a mild, clinically insignificant increase postsurgery. SAA concentrations were also significantly increased (P < 0.05) in cases identified with SIRS prior to surgery and postoperatively at 48 (P = 0.05) and 72 hours (P = 0.02) in horses that developed complications. CONCLUSIONS Measurement of SAA is a more sensitive indicator of inflammation than fibrinogen in the perioperative period of horses requiring exploratory celiotomy for colic. Serial measurement of SAA at 48, 72, and 96 hours after surgery may be helpful to determine risk of complications and guide postoperative management. Measurement of SAA on admission also allows for quantification of SIRS when it is detected clinically.