Enhanced ultrasonographic imaging of the equine distal limb using saline injection of the digital flexor tendon sheath: A cadaver study.

Injuries to the structures within the digital flexor tendon sheath (DFTS) can lead to lameness with a variable degree of effusion in horses. In some cases, effusion is absent or minimal, and this may be related to the chronicity and type of injury, or veterinary interventions. The purpose of this study was to determine if saline injection into the DFTS would improve ultrasonographic and magnetic resonance imaging of the distal limb without introducing artifact. Nine normal equine cadaver forelimbs were collected. Non-weight-bearing ultrasonographic and magnetic resonance imaging (MRI) examination of each limb was performed pre- and immediately post-injection of the DFTS. The presence of fluid in the DFTS significantly improved the delineation of the deep digital flexor tendon, manica flexoria, and straight distal sesamoidean ligament visualised using both ultrasonography and MRI (P<0.05). Significant improvement in visualisation of the margins of the superficial digital flexor tendon was noted only with MRI (P<0.05). Saline distension did not alter the size/shape of the intra- and extrathecal structures. The findings of this study support further evaluation of this imaging technique in clinical cases with minimal DFTS effusion.

Exercising upper respiratory videoendoscopic findings of 50 competition draught horses with abnormal respiratory noise and/or poor performance.

BACKGROUND: Upper respiratory tract (URT) endoscopy at rest is commonly used to evaluate competition draught horses with URT conditions. Overground endoscopy might be preferred for draught horse URT evaluation as it allows the horses to be driven with harness, overcheck and cart-load under similar conditions to those experienced in the show ring where airway conditions are most prominent. OBJECTIVE: To describe the exercising URT findings of competition draught horses with abnormal respiratory noise and/or poor performance. STUDY DESIGN: Case series. METHODS: Medical records of competition draught horses undergoing overground endoscopic evaluation between January 2013 and January 2018 with a presenting complaint of abnormal respiratory noise and/or poor performance were reviewed. Video recordings of resting and overground endoscopy were evaluated in all horses. Spearman's rank correlation coefficient was calculated between laryngeal function at rest and at exercise. RESULTS: Fifty competition draught horses were examined. Thirteen had previously undergone URT surgery. There was significant correlation between resting and exercising laryngeal function (ρ = 0.77, P<0.01). Abnormalities were detected in 46 horses and included arytenoid cartilage collapse (n = 31), vocal fold collapse (n = 27), palatal dysfunction (n = 14), epiglottic disorders (n = 11), dynamic laryngeal collapse (n = 1), rostral deviation of the palatopharyngeal arch (n = 3) and medial deviation of the aryepiglottic folds (n = 16). The majority of horses had a complex of abnormalities (n = 31) or required exercising examination for identification (n = 41). Incidental upper oesophageal incompetence was observed in nine horses. MAIN LIMITATIONS: Retrospective collection of data. CONCLUSIONS: Overground endoscopic evaluation was a useful technique for identifying URT disorders in competition draught horses. The spectrum of upper airway conditions identified in exercising draught horses supports the use of overground endoscopy as a diagnostic technique and could influence treatment considerations. The Summary is available in Portuguese - see Supporting Information.

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.

Laminar regulation of STAT1 and STAT3 in black walnut extract and carbohydrate overload induced models of laminitis.

BACKGROUND: STAT1 and STAT3 are important signaling molecules in disorders of systemic inflammation and are likely to be involved in laminitis, as laminar and systemic inflammation have been well documented in experimental models of laminitis. HYPOTHESIS: The STAT1 and STAT3 activation (via phosphorylation of tyrosine and serine moieties) is occurring in the laminar tissue during the developmental and onset of lameness time points in both the black walnut extract (BWE) and carbohydrate overload (CHO) models of laminitis. ANIMALS: Archived laminar tissue from horses. METHODS: Experimental studies of induced laminitis (BWE and CHO administration) in horses were conducted and laminar tissue samples archived. Western hybridization was performed to determine concentrations of Tyr- and Ser-phosphorylated STAT1 and STAT3 from these archived samples. The RT-qPCR was also performed to assess mRNA concentrations of target genes of STAT1 and STAT3. RESULTS: Increases (P < .05) in phosphorylation of tyrosine705 and serine727 of STAT3, demonstrated by band intensity ratios, are present in laminar tissue from both the BWE and CHO models at the DEV and OG1 time points. No change in phosphorylation of tyrosine701 or serine727 of STAT1 was present in the laminar tissue from either the BWE or the CHO models. The SOCS3 mRNA concentrations were increased at the onset of lameness in both the CHO and BWE models. CONCLUSIONS AND CLINICAL RELEVANCE: The STAT3 activation likely plays a role in equine laminitis, similar to its reported involvement in organ injury/failure in human sepsis. Regulation of JAK-STAT, through STAT3 inhibitors, might serve as potential therapeutic target for controlling the inflammatory response in the septic horse.

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-1ß, 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.

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-1ß, 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-1ß 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-1ß, 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.

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-1ß, 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.

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.

Effects of recombinant equine somatotropin on wound healing, carbohydrate and lipid metabolism, and endogenous somatotropin responses to secretagogues in geldings.

The primary purpose of this experiment was to assess the possible beneficial effects of recombinant equine somatotropin (reST) administration on wound healing in adult geldings. The effects of the 21-d reST treatment on carbohydrate and lipid metabolism and on endogenous ST characteristics were monitored as well. Single, full-thickness skin incisions (7.62 x 7.62 cm) were made in the pectoral region of all geldings on d 0. Treated geldings received reST at 20 microg/kg BW i.m., and control geldings received vehicle (10 mM sodium borate) at equivalent volumes daily from d 0 (immediately after surgery) through d 20. Tracings of the wounds were made with acetate transparencies, and wound areas were calculated via a digital analyzer. In addition to once-daily blood samples collected at specified days throughout the treatment period, an i.v. glucose tolerance test was performed on d 16, and three assessments of endogenous ST secretion were performed in the 2 d immediately following the end of treatment: epinephrine administration during the morning of d 21, an exercise test during the afternoon of d 21, and i.v. aspartic acid infusion on d 22. There was no effect (P > . 1) of reST treatment on wound healing as assessed by changes in wound areas. Daily plasma ST, IGF-I, glucose, and insulin concentrations were higher (P < .05) and urea-nitrogen concentrations were lower (P < .001) in geldings receiving reST relative to controls. Glucose, NEFA, and insulin concentrations were all higher (P < .01) in reST-treated geldings before glucose infusion on d 16, and the responses to glucose were greater (P < .05) as well. Epinephrine administration increased (P < .02) ST concentrations in control geldings on d 21 but not in reST-treated geldings; a similar suppressive effect of reST treatment was observed for the ST response to exercise (P < .001). After aspartic acid infusion on d 22, reST-treated geldings had a much smaller (P < .001) ST response than did control geldings. In conclusion, reST administered to geldings at 20 microg/kg BW i.m. caused hyperglycemia, hyperinsulinemia, insulin insensitivity, mobilization of fatty acids, and an apparent negative feedback on the pituitary's ST response to various stimuli known to induce ST secretion. However, there was no beneficial effect of reST treatment with the wound model used in this experiment.

The effects of equine somatotropin (eST) on follicular development and circulating plasma hormone profiles in cyclic mares treated during different stages of the estrous cycle.

The effects of exogenous equine somatotropin (eST) administration on ovarian activity and plasma hormone levels were evaluated on horse and pony mares. The objectives of this study were to determine the effects of eST on follicular development and circulating concentrations of leutinizing hormone (LH), estradiol, progesterone, and insulin-like growth factor I (IGF-I) in cyclic horse and pony mares. Sixteen mares received daily injections (i.m.) of eST at a concentration of 25 micrograms/kg body weight on either Days 6 through 12 (Treatment A) or 13 through 19 (Treatment B) postovulation. In addition, contemporary mares were similarly given the carrier vehicle and served as controls (Treatments C and D). Blood samples were collected at 24-hr intervals and ultrasonographic evaluations were performed on the ovaries of each mare at 48-hr intervals beginning on the first day of treatment and ending either on the day of ovulation or 5 d postovulation. Circulating levels of insulin-like growth factor-I (IGF-I) were increased in treated mares by Day 3 post-treatment (P < 0.05). Also, mares in Treatment B exhibited a decrease in plasma estradiol concentrations (P < 0.05) when compared with control mares on Days 1 through 5 postovulation of the post-treated estrous cycle. In addition, circulating leutinizing hormone levels were different for mares in Treatment A compared with controls on Days--8 through--1 pre-ovulation (P < 0.05). All follicles present on the ovaries of each mare were measured and placed into one of five categories based on their diameter. Neither the mean number of follicles per size category > or = 8 mm in diameter nor the mean follicular diameter within each size category differed among treatment and control mares. However, eST treatment significantly increased the number of follicles < or = 7 mm on the ovaries of mares treated early in the estrous cycle when compared with control mares on Days 3 and 7 post-treatment and at the onset of standing estrus.