Characteristic Inflammatory Biomarkers in an Equine Model of Persistent Synovitis Induced By the Intra-Articular Administration of Monoiodoacetic Acid.

Persistent synovitis damages the articular cartilage in horses. To evaluate the effectiveness of treatment for synovitis using a model induced by intra-articular administration of monoiodoacetic acid (MIA), it is necessary to identify inflammatory biomarkers characteristic of the MIA model. Synovitis was induced by administering MIA into the unilateral antebrachiocarpal joints of five horses, and saline was injected into the contralateral joints as a control on day 0. Clinical and ultrasonographic examinations and synovial fluid collection were performed on days 0, 1, 2, 7, 14, 21, 28, and 35. Leukocyte, lactate dehydrogenase (LDH), tumor necrosis factor-α (TNF-α), interleukin-1 receptor antagonist (IL-1Ra), interleukin-6 (IL-6), and transforming growth factor-ß1 (TGF-ß1) concentrations in the synovial fluid were measured. Synovium was obtained after euthanasia on day 42 and histologically examined before quantification of the gene expression of inflammatory biomarkers by real-time PCR. Acute inflammatory symptoms persisted for approximately 2 weeks before returning to control levels. However, some indicators of chronic inflammation remained elevated until day 35. On day 42, synovitis continued histologically, with osteoclasts. The expressions of matrix metalloproteinase 13 (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), receptor activator of nuclear factor kappa-Β ligand (RANKL), and collagen type I α2 chain (Col1a2) were significantly higher in the MIA model than in the control. In the MIA model, representative inflammatory biomarkers in the chronic inflammatory stage were persistently expressed in both synovial fluid and tissue, suggesting that they may be useful for the assessment of the anti-inflammatory effect of drugs.

Safety and Effects of a Commercial Ozone Foam Preparation on Endometrial Environment and Fertility of Mares.

Mares' subfertility represents a complex diagnostic and therapeutic challenge and both clinical and subclinical endometritis are considered major causes of impaired fertility. Thanks to its properties, ozone has a big potential as a treatment for equine endometritis. Therefore, the aim of this study is to describe the safety and the effects on endometrium and reproductive parameters of mares of a commercial ozone foam preparation (Riger Spray®). Twenty-four mares were treated during estrus: ozone group with an intrauterine instillation of ozone foam preparation (OG, n=16) and control group with 20 ml of lactated Ringer's solution (CG, n=8). Samples for endometrial cytology were collected before the ozone treatment (T0), after 24 h (T1), after one week (T2), two weeks (T3), and when the subsequent estrous phase was detected (T4). Furthermore, samples for histological examination and uterine swab for bacteriological examination were collected at T0 and T4. At T1, a statistically significant increase of endometrial inflammation in the OG mares compared to T0 (P<.05) and to CG at same time point (P<.05) was observed, but it was already resolved at T2. No differences in endometrial inflammation in CG, biopsy grade before and after the treatment in the two groups, number of mares pregnant at the end of the season and number of mares pregnant at the first cycle were observed. However, the number of inseminations required for pregnancy tended to be lower (P=.0711) in the OG (1.69±0.06) than in CG mares (2.60±0.89).

Risk of anesthesia-related complications in draft horses: a retrospective, single-center analysis.

OBJECTIVE: To report anesthetic-related complications and determine risks associated with anesthesia in draft horses. STUDY DESIGN: Retrospective study. ANIMALS: A total of 401 anesthetic records for draft horse breeds that underwent general anesthesia from January 2010 through December 2020 were reviewed; horses euthanized during general anesthesia were excluded. METHODS: Demographics, perioperative drugs used, procedure type and duration, time to extubation, number of attempts to stand, use of sling in recovery and perioperative morbidity and mortality were investigated. Morbidity and mortality statistical evaluation included univariable logistic regression analysis and ordinal regression analysis. RESULTS: American Society of Anesthesiologists (ASA) status I-II, ASA III-V and total mortality rate for all cases was 0.69% (2/288), 6.19% (7/113) and 2.24% (9/401), respectively, with Belgian horses being overrepresented (6/9). Cardiac arrest occurred in six out of nine horses that died without euthanasia, and five out of six of these horses underwent colic surgery. Factors associated with increased mortality risk included ASA status of III-V, increased body weight, emergency status and horses presenting for colic. Hypotension, hypercarbia and hypoxemia occurred in 56% (224/401), 46% (186/401) and 14% (58/401) of horses, respectively. During recovery from anesthesia, lighter horses and horses undergoing shorter anesthetic procedures were more likely to be successful on the first or second attempt to stand and were less likely to require a sling in recovery. CONCLUSIONS AND CLINICAL RELEVANCE: Draft horses undergoing general anesthesia had a higher mortality rate than previously reported for all types and breeds of horses.

Photodermatitis and ocular changes in nine horses after ingestion of wild parsnip (pastinaca sativa).

BACKGROUND: Primary photosensitization rarely occurs in horses and can easily be misinterpreted. Descriptions of the disease in horses after ingestion of parsnip are lacking. The aim of this case series was to describe the dermatological and ocular changes due to photosensitization and to raise awareness of parsnip being a possible aetiologic agent. CASE PRESENTATION: Nine horses from three different stables in Berlin and Brandenburg, Germany, presented variable degrees of erythema, scaling, crusting and necrosis of unpigmented skin at the head and prepuce. Horses were of different breeds with a median age of 15 ± 5.9 years. A mild leukocytosis was diagnosed in 1/9 horses at admission. Analyzed liver enzymes were within the reference ranges in all horses. Ocular changes were diagnosed as follows: blepharitis (3/9), conjunctivitis (7/9), corneal edema without additional signs of keratitis and/or uveitis (2/9), corneal edema with signs of uveitis (1/9) and photophobia (4/9). One horse developed a fluorescein positive corneal erosion. Skin biopsy (1/9) revealed a moderate to severe acute, eosinophilic and lymphocytic dermatitis with dermal edema and vasculitis. All stables housing these patients fed hay from the same distributer. Analyzed hay samples showed high contents of wild parsnip (plants, seeds, roots). Wild parsnip is widespread in Europe and contains furocoumarins, a family of photodynamic pigments, which may cause primary photodermatitis, keratoconjunctivitis and uveitis. Horses were treated according to severity of clinical symptoms systemically with flunixine meglumine (1.1 mg/kg BW 1-2x/day) or prednisolone (1 mg/kg BW 1x/day). Topically, either gentamicin (3x/day), dexamethasone (2-3x/day) and/or atropine (1x/day) were used. Skin care was provided with almond oil or dexpanthenol (2x/day). All horses were kept in a dark environment or were treated with sunscreen and facemasks. Duration of treatment varied from 6-30 days (median 11.3 days). CONCLUSION: Ingestion of wild parsnip (Pastinaca sativa) can induce primary photosensitization with dermatitis and ocular injury in horses. In times of extreme weather, hay may alter in botanical composition, resulting in high amounts of uncharacteristic plants causing novel problems.

Effects of 0.0015% preservative-free tafluprost on the equine eye.

OBJECTIVE: The purpose of this study was to determine the effects and potential side effects of topical preservative-free (PF) tafluprost 0.0015% in ophthalmologically normal horses. ANIMALS: Five adult grade horses. PROCEDURES: One of the eyes of each horse was randomly chosen as the "treatment" eye, and consequently, the contralateral eye served as the "control." A single dose of PF tafluprost 0.0015% (0.2 mL) was instilled in the treated eye of each horse. Intraocular pressure (IOP), Schirmer's tear test (STT) levels of each eye, and an ophthalmic examination were performed at T0 (baseline), T30, T120, T24 h, and T48 h. RESULTS: The mean IOP values of the treated eyes at baseline (T0), T30, T120, T24 h, and T48 h were 25.4 ± 4.8 mmHg, 21.2 ± 1.92 mmHg, 15.20 ± 2.48 mmHg, 18.40 ± 1.51 mmHg, and 24.60 ± 1.94 mmHg, respectively. Significant differences were observed between the mean baseline IOP level and the T120 and T24 h time points (p = .001 and p = .009). The mean STT levels at each time point showed insignificant fluctuations during the study (p = .140). Adverse effects such as chemosis and episcleral injection were observed 30 min after the instillation of tafluprost 0.0015% (T30). Blepharospasm and conjunctival hyperemia were observed 120 min (T120) after the administration of the medication. CONCLUSION AND CLINICAL RELEVANCE: Tafluprost 0.0015% showed potential in reducing IOP, but due to its local side effects, it is not a good candidate for management of glaucoma in horses. Tafluprost did not notably affect STT.

Dynamics of local gene regulations in synovial fluid leukocytes from horses with lipopolysaccharide-induced arthritis.

The role of resident cells such a synoviocytes and chondrocytes in intra-articular inflammation is well-characterized, however the in vivo gene expression patterns of cells (predominantly leukocytes) in the synovial fluid (SF) of an inflamed joint have never previously been investigated. The aim of this study was to investigate gene expression in SF leukocytes from the inflamed joint cavity after intra-articular lipopolysaccharide (LPS) injection in horses to improve our understanding of the temporal regulation of the intra-articular inflammatory response. Gene expression was investigated in SF samples available from six horses 2, 4, 8 16 and 24 h after experimental induction of inflammation in the radiocarpal joint by lipopolysaccharide (LPS) injection. Leukocytic expression of 43 inflammation-related genes was studied using microfluidic high throughput qPCR (Fluidigm®). Expression of 26 genes changed significantly over the 24 h study period, including pro- and anti-inflammatory genes such as interleukin (IL)1, IL6, tumor necrosis factor (TNF), cyclooxygenase 2 (COX2), IL1 receptor antagonist (IL1RN), IL10, and superoxide dismutase 2 (SOD2), chemokine genes, apoptosis-related genes, and genes related to cartilage turnover (matrix metalloproteinase 8 and tissue inhibitor of metalloproteinase 1). The inflammatory responses appeared to be regulated, as an early increase (at 2 h) in expression of the pro-inflammatory genes IL1, IL6, TNF and COX2 was rapidly followed by increased expression (at 4 h) of several anti-inflammatory genes (IL10, IL1RN and SOD2). Similarly, both pro- and anti-apoptotic gene expression as well as expression of chondrodegenerative and chondroprotective genes were activated in SF leukocytes. Thus, the inflammatory response in leukocytes infiltrating the joint in the acute stage of arthritis was well orchestrated in this single-hit LPS-induced arthritis model. This study is the first to describe gene expression patterns in SF-derived leukocytes in vivo during severe joint inflammation, and the results thus expand our knowledge of basic inflammatory mechanisms in the early local response in an inflamed joint.

Influence of clinical and experimental intra-articular inflammation on neutrophil gelatinase-associated lipocalin concentrations in horses.

OBJECTIVE: To investigate neutrophil gelatinase-associated lipocalin (NGAL) concentrations in serum and synovial fluid (SF) from horses with joint inflammation. STUDY DESIGN: Experimental studies and retrospective clinical study. SAMPLE POPULATION: Serum and SF samples were available from healthy horses (n = 19), clinical cases, and horses with experimental joint inflammation. Clinical cases included horses with (n = 10) or without (n = 10) septic arthritis. Experimental intra-articular inflammation was induced by lipopolysaccharide (LPS; n = 7, severe inflammation), lidocaine (n = 6, moderate inflammation), or mepivacaine (n = 6, mild inflammation). METHODS: Availability of samples was based on approval from the local ethical committee and from the Danish Animal Experiments Inspectorate. Neutrophil gelatinase-associated lipocalin was measured with a previously validated enzyme-linked immunosorbent assay. Repeated-measurements one- and two-way analysis of variance and correlation analysis were used to analyze NGAL concentrations and white blood cell counts (WBC). RESULTS: After injection of LPS or lidocaine, SF NGAL concentrations increased 343- (P = .0035) and 60-fold (P = .0038) relative to baseline, respectively. Serum NGAL also increased in both groups (P < .05) but to lower concentrations than in SF. Concentrations were higher after injection of lidocaine SF NGAL than after injection of mepivacaine (P < .05) at 6 and 12 hours. Synovial fluid concentrations of NGAL were higher in horses with septic arthritis than in the nonseptic group (P = .0070) and in healthy controls (P = .0071). Concentrations of NGAL correlated with WBC in SF (P < .0001, R2 = 0.49) and in blood (P = .0051, R2 = 0.27). CONCLUSION: Neutrophil gelatinase-associated lipocalin concentrations increased in SF in response to experimentally induced and naturally occurring joint inflammation. Synovial fluid NGAL concentration correlated with WBC and, thus, seems to reflect intensity of joint inflammation. CLINICAL SIGNIFICANCE: Neutrophil gelatinase-associated lipocalin may prove to be a useful biomarker of joint inflammation and infection in horses.

Effect of digital hypothermia on lamellar inflammatory signaling in the euglycemic hyperinsulinemic clamp laminitis model.

BACKGROUND: Continuous digital hypothermia (CDH) prevents lamellar failure in the euglycemic hyperinsulinemic clamp (EHC) model of laminitis, but the protective mechanisms are unclear. HYPOTHESIS/OBJECTIVES: To determine if CDH inhibits lamellar inflammatory signaling in the EHC model of laminitis. ANIMALS: Eight Standardbred horses. METHODS: Prospective experimental study. Horses underwent an EHC, with 1 forelimb treated with CDH and the other kept at ambient temperature (AMB). Horses were euthanized 48 hours after initiation of the EHC and lamellar tissue was analyzed via polymerase chain reaction (pro-inflammatory cytokine and chemokine genes-CXCL1, CXCL6, CXCL8, IL-6, MCP-1, MCP-2, IL-1ß, IL-11, cyclooxygenase 1 and 2, tumour necrosis factor-alpha [TNF-α], E-selectin, and intercellular adhesion molecule-1 [ICAM-1]) and immunoblotting (phosphorylated and total signal transducer and activator of transcription 1 [STAT1] and STAT3). RESULTS: Compared to AMB, lamellar messenger ribonucleic acid (mRNA) concentrations of CXCL6 (P =.02), CXCL8 (P = .008), IL-6 (P = .008), IL-1ß (P = .008), IL-11 (P = .008), and cyclooxygenase-2 (P = .008) were decreased in CDH. Cyclooxygenase-1 (P = .008) was increased in CDH, while CXCL1 (P = .15), MCP-1 (P = .05), MCP-2 (P = .46), TNF-α (P = .05), E-selectin (P = .15), and ICAM-1 (P = .15) mRNA were not significantly different. Compared to AMB, lamellar concentration of total STAT3 protein was decreased in CDH (P < .001), but there was no change in phosphorylated STAT3 (P-STAT3 [S727] P = .19; P-STAT3 [Y705] P = .05). There was no change in lamellar concentrations of total STAT1 (P = .75) or phosphorylated STAT1 (P-STAT1 [S727], P = .25; P-STAT1 [Y701], P = .64). CONCLUSIONS AND CLINICAL IMPORTANCE: These data add further support for the use of CDH as a first aid treatment for severe acute laminitis associated with hyperinsulinemia in horses.

Hematologic, prostaglandin F2α -metabolite, serum amyloid A, and serum iron changes in horses with experimentally induced endotoxemia.

BACKGROUND: Endotoxemia is a common and severe disease of horses. Most previous studies have monitored changes caused by a bolus dose of endotoxin over short time periods. OBJECTIVES: We aimed to describe inflammatory responses to endotoxin with inflammatory and hematologic markers monitored over a longer time than has been performed in the past using more prolonged endotoxin exposures. METHODS: Escherichia coli O55:B5 endotoxin was administered as a 6-hour continuous intravenous infusion of lipopolysaccharide (LPS) to eight horses. Blood cell counts, and prostaglandin F2α -metabolite (PGM), serum amyloid A (SAA), and serum total iron concentrations were monitored for up to 3 or 6 days. RESULTS: An immediate and severe decrease in neutrophils and monocytes occurred in all horses, which subsequently changed to a moderate to strong neutrophilia and monocytosis that persisted for more than 78 hours postinfusion (PI) of LPS. Lymphocyte and eosinophil numbers decreased gradually and then normalized after 66- and 78-hours PI, respectively. Mild to moderate, biphasic thrombocytopenia occurred. A pronounced, transient increase in PGM occurred between 1 and 7 hours, peaking at 2 hours. Serum amyloid A began to increase after 6 hours PI and remained elevated after 72 hours PI. Serum iron was decreased between 6 and 48 hours. The clinical signs were most prominent during the first 24 hours PI and subsided within 48 hours PI. CONCLUSIONS: Neutrophilia, monocytoses, and high SAA concentrations were present in horses even after the clinical signs had subsided. Serum iron normalized before SAA. Knowledge of these findings is imperative when interpreting laboratory results in horses with possible endotoxin exposure.

Hyaluronic acid has chondroprotective and joint-preserving effects on LPS-induced synovitis in horses.

The intra-articular use of hyaluronic acid (HA) for the treatment of synovitis and osteoarthritis is still controversial. As a consequence, corticosteroids remain the most frequently employed therapeutic agents, despite their potential systemic and local deleterious effects. This study examined the anti-inflammatory, antioxidant, and chondroprotective activities of low and high molecular weight hyaluronic acid (LMW-HA and HMW-HA) on lipopolysaccharide (LPS)-induced synovitis in horses compared to triamcinolone acetonide (TA). LPS was injected in the metacarpophalangeal joints, which were treated intra-articularly with either TA (as control) or LMW-HA or HMW-HA. Joint clinical evaluation and synovial fluid (SF) analysis were performed at 0, 8, 24, and 48 h. The white blood cell counts (WBC), prostaglandin E2 (PGE2), interleukin (IL)-1, IL-6, IL-10, tumor necrosis factor-α, chondroitin sulfate (CS) and HA concentrations, oxidative burst, and HA molecular weights were measured. TA reduced the lameness, swelling, and PGE2 release but increased the SF CS concentrations enormously at 24h and 48h, and decreased the SF HA modal molecular weight. These results indicate the breakdown of articular cartilage aggrecan and SF HA. In contrast, LMW-HA and HMW-HA were less effective in reducing the inflammation symptoms, but preserved the joints because only a modest increase in CS occurred at 24 h, decreasing at 48 h, and the SF HA was maintained. The HA-treatment also had anti-inflammatory actions, and LMW-HA was the most effective in reducing the release of cytokine. In summary, the HA treatment inhibited efficiently the digestion of cartilage proteoglycans and SF HA breakdown.

Plasma and synovial fluid concentrations and cartilage toxicity of bupivacaine following intra-articular administration of a liposomal formulation to horses.

BACKGROUND: The use of intra-articular (IA) local anaesthetics has proven to be an effective means to treat post-operative pain. The effects of local anaesthetics on equine chondrocytes are mixed with some studies reporting chondrodestruction and others no adverse effects. A liposomal formulation of bupivacaine is used in people and dogs by intra- and peri-articular administration to provide up to 72 h of analgesia. The potential uses, side effects including chondrotoxicity, and likelihood of abuse (long-term analgesic effects) has not been evaluated in horses. OBJECTIVES: Describe bupivacaine concentrations following IA administration and assess biomarkers as indicators of the effects of liposomal bupivacaine on chondrocytes in vivo. STUDY DESIGN: Parallel design. METHODS: Sixteen exercised horses received a single IA administration of 0.12 mg/kg liposomal bupivacaine or 0.9% saline. Blood and urine samples were collected for 96 h post-drug administration. Six horses treated with bupivacaine and those receiving saline, underwent daily arthrocentesis. Six additional bupivacaine treated horses underwent arthrocentesis at 96 h. Drug concentrations were measured using LC-MS/MS and pharmacokinetic analyses performed. Immunoassays were used to measure markers of collagen degradation (C2C, C12C) and cartilage matrix synthesis (CPII, CS846) in synovial fluid. RESULTS: The bupivacaine plasma elimination half-life was 17.8 ± 5.42 and 11.9 ± 5.17 h for horses from which synovial fluid was collected daily and at 96 h respectively. Bupivacaine concentrations in the joint were still detectable at 96 h. Significant increases in C12C and C2C were noted at 96 h in horses undergoing arthrocentesis at 96 h only. CPII was increased at 48 h and CS846 at 24 and 48 h in horses sampled daily. MAIN LIMITATIONS: Limited number of animals and absence of liposome control group. CONCLUSIONS: Sustained concentrations of IA bupivacaine suggest viability of this medication as an intra-articular analgesic. Effects on equine chondrocytes need further study.

Effects of repeated arthrocentesis on systemic cytokine expression and leukocyte population in young horses challenged with intra-articular lipopolysaccharide.

Osteoarthritis (OA) is a prevalent and economically costly source of lameness in the athletic horse. Previous studies investigating OA pathology have focused on localized trauma to the articular cartilage of a joint, largely ignoring the systemic immune status of the animal. In this study, yearling Quarter Horses were used to evaluate systemic cytokine gene expression and circulating leukocytes following a localized intra-articular inflammatory insult of the endotoxin, lipopolysaccharide (LPS). Treatments for the 35-d experiment included an intra-articular injection of 0.25 ng (n = 7) or 0.50 ng (n = 6) of LPS obtained from Escherichia coli O55:B5 or sterile lactated Ringer's solution (n = 6; control) into the radial carpal joint. Blood and synovial fluid samples were collected at preinjection hour 0 and 2, 6, 12, and 24 h postinjection. Synovial fluid was obtained for a companion study. Total RNA was isolated from plasma leukocytes and real-time PCR was used to determine relative gene expression of the cytokines interleukin (IL)-1beta (ß), IL-6, IL-8, IL-10, and tumor necrosis factor-alpha (TNF-α). Total leukocyte subpopulations and differentials were performed using a cell counter. Data were analyzed using the PROC MIXED procedure of SAS. Gene expression of all cytokines were unaffected by intra-articular treatment. However, IL-1ß increased above baseline beginning at hour 6 and remained elevated to 24 h (P = 0.04). In contrast, IL-6 decreased from hours 6 to 12 and then increased to 24 h (P = 0.02). Levels of TNF-α increased at 6 and 12 h (P = 0.01) postinjection. Only IL-8 exceeded a 2-fold change in expression (P = 0.01), peaking at 12 h and indicating greater responsiveness to arthrocentesis when compared with other cytokines. No treatment effects on the leukocyte population were observed; however, total circulating leukocytes increased over time (P = 0.04), peaking at 6 h postinjection. Similarly, an increase over time was observed in monocytes (P = 0.02) and in platelets (P = 0.01) at 24 h postinjection. The results indicate that regardless of treatment, a mild immune response was elicited, which may be due to repeated arthrocentesis. Future experiments should consider the effects of arthrocentesis and potential systemic inflammatory response, even in control animals, when administering intra-articular LPS to young horses.

Assessment of effectiveness and safety of repeat administration of proinflammatory primed allogeneic mesenchymal stem cells in an equine model of chemically induced osteoarthritis.

BACKGROUND: This study aimed at assessing the effectiveness and safety of repeated administrations of allogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) primed with tumor necrosis factor (TNF)-α and interferon-γ in an equine model of chemically-induced osteoarthritis. Arthritis was induced in both radio-carpal (RC)-joints by amphotericin-B in 18 ponies, divided into three groups depending on the treatment injected: MSC-naïve (n = 7), MSC-primed (n = 7) and control (n = 4). The study consisted of two phases and used one RC-joint of each animal in each phase, with four months time-lapse, in order to assess two end-points. Clinical, synovial, radiological and ultrasonographic follow-up was performed. At six months, animals were euthanized and both carpi were assessed by magnetic resonance imaging (MRI), gross anatomy, histopathology, histochemistry and gene expression. RESULTS: Clinical and synovial inflammatory signs were quicker reduced in MSC-treated groups and repeated allogeneic administration did not produce adverse reactions, but MSC-primed group showed slight and transient local inflammation after second injection. Radiology and MRI did not show significant differences between treated and control groups, whereas ultrasonography suggested reduced synovial effusion in MSC-treated groups. Both MSC-treated groups showed enhanced cartilage gross appearance at two compared to six months (MSC-naïve, p < 0.05). Cartilage histopathology did not reveal differences but histochemistry suggested delayed progression of proteoglycan loss in MSC-treated groups. Synovium histopathology indicated decreased inflammation (p < 0.01) in MSC-primed and MSC-naïve at two and six months, respectively. At two months, cartilage from MSC-primed group significantly (p < 0.05) upregulated collagen type II (COL2A1) and transforming growth factor (TGF)-ß1 and downregulated cyclooxygenase-2 and interleukin (IL)-1ß. At six months, MSC-treatments significantly downregulated TNFα (p < 0.05), plus MSC-primed upregulated (p < 0.05) COL2A1, aggrecan, cartilage oligomeric protein, tissue inhibitor of metalloproteinases-2 and TGF-ß1. In synovium, both MSC-treatments decreased (p < 0.01) matrix metalloproteinase-13 expression at two months and MSC-primed also downregulated TNFα (p < 0.05) and IL-1ß (p < 0.01). CONCLUSIONS: Both MSC-treatments provided beneficial effects, mostly observed at short-term. Despite no huge differences between MSC-treatments, the findings suggested enhanced anti-inflammatory and regulatory potential of MSC-primed. While further research is needed to better understand these effects and clarify immunogenicity implications, these findings contribute to enlarge the knowledge about MSC therapeutics and how they could be influenced.

Effects of medical ozone upon healthy equine joints: Clinical and laboratorial aspects.

OBJECTIVE: The aim of this study was to verify whether transient inflammatory reactions induced by intra-articular medicinal ozone administration affect joint components, by in vivo evaluation of inflammatory (prostaglandin E2, Substance P, Interleukin-6, Interleukine-1, Tumor Necrosis Factor), anti-inflammatory (Interleukin-10) and oxidative (superoxide dismutase activity and oxidative burst) biomarkers and extracellular matrix degradation products (chondroitin sulphate and hyaluronic acid) in synovial fluid. METHODS: The effects of medicinal ozone were analyzed at two ozone concentrations (groups A and B, 20 and 40 µg/ml, respectively), using oxygen-injected joints as controls (group C); each group received ten treatments (15 ml gas per treatment). Physical evaluation, evaluation of lameness, ultrasonography, and synovial fluid analysis were performed. RESULTS: All joints presented mild and transient effusion throughout the study. Group B exhibited the highest lameness score on day 14 (P<0.05), detected by the lameness measurement system, probably because of the higher ozone concentration. All groups exhibited increased ultrasonography scores on day 14 (P < 0.05). Groups A and B exhibited increased proteins concentrations on day 21 (P<0.05). There was no change in hyaluronic acid concentration or the percentage of high-molecular weight hyaluronic acid throughout the experiment. Chondroitin sulfate concentrations decreased in group B, and did not change in group A and C, indicating that neither treatment provoked extracellular matrix catabolism. Cytokine and eicosanoid concentrations were not significantly changed. CONCLUSIONS: The ozonetherapy did not cause significant inflammation process or cartilage degradation, therefore, ozonetherapy is safe at both evaluated doses.

Adverse Reactions to Vaccination: From Anaphylaxis to Autoimmunity.

Vaccines are important for providing protection from infectious diseases. Vaccination initiates a process that stimulates development of a robust and long-lived immune response to the disease agents in the vaccine. Side effects are sometimes associated with vaccination. These vary from development of acute hypersensitivity responses to vaccine components to local tissue reactions that are annoying but not significantly detrimental to the patient. The pathogenesis of these responses and the consequent clinical outcomes are discussed. Overstimulation of the immune response and the potential relationship to autoimmunity is evaluated in relation to genetic predisposition.

The oral glucose test predicts laminitis risk in ponies fed a diet high in nonstructural carbohydrates.

The aim of this study was to investigate the relationship between laminitis development in ponies and insulin/glucose concentrations in response to the oral glucose test (OGT) and a dietary challenge high in nonstructural carbohydrates (NSCs). After undergoing an OGT (1 g dextrose/kg BW in feed), 37 ponies with 2-h serum insulin concentrations ranging from 22 to 1,133 µIU/mL were subjected to a diet challenge period (DCP), consuming 12 g NSC/kg BW/d for up to 18 d. Insulin and glucose responses were measured on day 2 of the DCP. Clinical laminitis was diagnosed by blinded experts and confirmed radiographically. Basal ACTH levels and clinical signs were assessed to investigate concurrent putative pituitary pars intermedia dysfunction (PPID). The diet induced Obel grade 1 or 2 laminitis in 14 ponies (38%). The ponies that developed laminitis had higher maximum concentrations of blood glucose (P = 0.04) and serum insulin (P = 0.02) in response to the diet. The geometric mean (95% CI) blood glucose concentration for laminitis cases was 14.9 (12.9-17.2) mM, compared to 10.7 (9.2-12.5) mM for ponies who did not develop laminitis. Similarly, the geometric mean (95% CI) for serum insulin was 396 (301-520) µIU/mL for laminitis cases, compared to 216 (148-316) µIU/mL for ponies who did not develop laminitis. Laminitis incidence was likewise associated with insulin concentrations measured during the OGT. Laminitis occurred at frequencies of 0% (0/7) if postdextrose insulin (µIU/mL) was <50; 35% (8/23) if insulin was 50 to 195; and 86% (6/7) if insulin was >195 µIU/mL. Basal ACTH concentrations were above seasonally accepted reference ranges in 16/37 ponies, and 8 of these animals (50%) developed laminitis. This included all 5 ponies in the study that had clinical signs of PPID (100%). In contrast, hyperinsulinemia and laminitis occurred in only 3/11 ponies (27%) with elevated ACTH concentrations and no clinical signs of PPID (P = 0.009). Thus, laminitis occurrence was associated with higher glucose and insulin responses to both the OGT and challenge diet, and the frequency of laminitis can be predicted based on insulin and glucose hyperresponsiveness to these oral carbohydrate challenges.

Phenylbutazone induces equine glandular gastric disease without decreasing prostaglandin E2 concentrations.

In equids, phenylbutazone at high doses induces gastric disease, primarily in the glandular portion of the stomach. However, the mechanism of nonsteroidal anti-inflammatory drug (NSAID)-induced gastric disease in horses has yet to be determined. While phenylbutazone-associated ulceration is often attributed to a decrease in basal gastric prostaglandins, this has not been demonstrated in the horse. Twelve horses were randomly assigned to treatment (n = 6; 4.4 mg/kg phenylbutazone PO in 20 ml molasses q 12 hr for 7 days) or placebo (n = 6; 20 ml molasses PO q 12 hr for 7 days) groups. Before treatment and 3 and 7 days after initiation of treatment, gastroscopy was performed and glandular gastric biopsies were collected and frozen at -80°C. Glandular disease was assessed on a scale of 0-4. Prostaglandin E2 concentrations in biopsies were measured using a commercially available enzyme-linked immunosorbent assay. All phenylbutazone-treated horses developed grade ≥2 glandular disease. Prostaglandin concentrations increased over time (p = .0017), but there was no effect of treatment (p = .49). These findings indicate that despite induction of glandular disease grade ≥2, phenylbutazone did not decrease basal glandular gastric prostaglandin E2 concentration.

Efficacy, chondrotoxicity and plasma concentrations of tramadol following intra-articular administration in horses undergoing arthroscopy: preliminary findings.

Intra-articular administration of analgesics is performed to ensure good perioperative pain management avoiding undesirable systemic effects. To evaluate the effect of intra-articular injection of tramadol on postoperative pain after arthroscopy in horses and to determine whether tramadol had a local effect. Before the in vivo study, an in vitro test was performed aiming to evaluate the viability of equine chondrocytes after exposure to various concentrations of tramadol. The concentration identified as most appropriate was used to treat the horses' joints. Twelve horses affected by osteochondrosis were randomly assigned to two groups that were treated intra-articularly at the end of surgery with tramadol (4 mg/mL) and saline, respectively. At predetermined time-points a Composite Pain Scale was applied and blood samples were collected in order to define the extent of tramadol absorption into the systemic circulation. The Mann-Whitney test was used for statistical analysis. Serum of four out of six treated horses revealed traces of tramadol (range 10.6-19.3 ng/mL) sporadically between 0.5 and 4 hours post-treatment, while in the other two horses, no trace of drug was found. Findings suggested that any eventual effect was probably due to local action rather than systemic absorption. The pain scores obtained in tramadol-treated horses were lower between 1 and 6 hours post-administration, than those obtained in the control group, but the differences were not statistically significant. These preliminary results suggest that tramadol, at this concentration, is only mildly beneficial in the pain management of horses after arthroscopy.

Evaluation of chlorhexidine hydrochloride treatment on endometrial health of normal mares.

Chlorhexidine gluconate solution is a potent antimicrobial and therefore could be used effectively for treatment of endometritis, but historically this substance has been implicated as irritating to mucous membranes, including the endometrium of the mare. The use of chlorhexidine hydrochloride suspension (Nolvasan Suspension, Zoetis, Florham Park, NJ, USA) was evaluated in the uterus of normal mares to determine if adverse effects on endometrial health were noted. Twelve healthy, adult light breed mares were included in this study. Procedures were approved by the Auburn University Institutional Animal Care and Use Committee. All mares were determined to be reproductively normal by evaluation of endometrial histopathology, cytology, and bacterial culture. Mares were randomly assigned to treatment or control groups (n = 6 per group). Each mare was treated during estrus with an intrauterine infusion of 1 g (28 mLs per tube; 35.7 mg/mL) of chlorhexidine hydrochloride suspension (treatment group) or an equal volume of lactated ringer's solution (control group) once daily for 3 consecutive days. Biopsy and cytology samples were taken 3, 7, and 14 days after completion of treatment. Cytology and biopsy samples were read by a board-certified pathologist (L.N.) blinded to treatments, and biopsy samples were graded using a standardized Kenney-Doig score. There was no difference with respect to biopsy grade, degree of endometrial fibrosis, or presence of cytologic inflammation comparing control and treatment groups (P = 0.55, 0.7, and 0.06, respectively), neither when accounting for sampling day. The suspension was visible within the uterine lumen when mares were examined with transrectal ultrasonography for up to 4 days after treatment. Treatment with chlorhexidine hydrochloride in this formulation and at this concentration does not appear to have a deleterious effect on short term endometrial health in mares.

Influence of an n-3 long-chain polyunsaturated fatty acid-enriched diet on experimentally induced synovitis in horses.

Dietary n-3 long-chain polyunsaturated fatty acid (LCPUFA) supplementation has previously been shown to modify joint-related inflammation in several species, although information in the horse is lacking. We investigated whether dietary supplementation with n-3 LCPUFA would modify experimentally induced synovitis in horses. Twelve, skeletally mature, non-pregnant mares were randomly assigned to either a control diet (CONT) or an n-3 long-chain fatty acid-enriched treatment diet (N3FA) containing 40 g/day of n-3 LCPUFA for 91 days. Blood samples taken on days 0, 30, 60 and 90, and synovial fluid collected on days 0 and 90 were processed for lipid composition. On day 91, joint inflammation was stimulated using an intra-articular (IA) injection of 100 ng of recombinant equine IL-1beta (reIL-1ß). Synovial fluid samples taken at post-injection hours (PIH) 0, 4, 8 and 24 were analysed for prostaglandin E2 (PGE2 ), matrix metalloproteinase (MMP) activity and routine cytology. Synovium and articular cartilage samples collected at PIH 8 were analysed for gene expression of MMP 1 and MMP 13, interleukin-1beta (IL-1ß), cyclooxygenase 2 (COX-2), tumour necrosis factor-alpha and the aggrecanases, a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5. A 90-day feeding period of n-3 LCPUFA increased serum phospholipid and synovial fluid lipid compositions of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) compared to CONT horses. The reIL-1ß injection caused an inflammatory response; however, there was no effect of dietary treatment on synovial fluid PGE2 content and MMP activity. Synovial tissue collected from N3FA horses exhibited lower expression of ADAMTS-4 compared to CONT horses. Despite the presence of EPA and DHA in the synovial fluid of N3FA horses, dietary n-3 LCPUFA supplementation did not modify synovial fluid biomarkers compared to CONT horses; however, the lower ADAMTS-4 mRNA expression in N3FA synovium warrants further investigation of n-3 LCPUFA as a joint therapy.