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.

A comparative multi-site and whole-body assessment of fascia in the horse and dog: a detailed histological investigation.

Fascia in the veterinary sciences is drawing attention, such that physiotherapists and animal practitioners are now applying techniques based on the concept of fascia studies in humans. A comprehensive study of fascia is therefore needed in animals to understand the arrangement of the fascial layers in an unguligrade horse and a digitigrade dog. This study has examined the difference between the horse and the dog fascia at specific regions, in terms of histology, and has compared it with the human model. Histological examinations show that in general the fascia tissue of the horse exhibits a tight and dense composition, while in the dog it is looser and has non-dense structure. Indeed, equine fascia appears to be different from both canine fascia and the human fascia model, whilst canine fascia is very comparable to the human model. Although regional variations were observed, the superficial fascia (fascia superficialis) in the horse was found to be trilaminar in the trunk, yet multilayered in the dog. Moreover, crimping of collagen fibers was more visible in the horse than the dog. Blood vessels and nerves were present in the loose areolar tissue of the superficial and the profound compartment of hypodermis. The deep fascia (fascia profunda) in the horse was thick and tightly attached to the underlying muscle, while in the dog the deep fascia was thin and loosely attached to underlying structures. Superficial and deep fascia fused in the extremities. In conclusion, gross dissection and histology have revealed species variations that are related to the absence or presence of the superficial adipose tissue, the retinacula cutis superficialis, the localization and amount of elastic fibers, as well as the ability to slide and glide between the different layers. Further research is now needed to understand in more detail whether these differences have an influence on the biomechanics, movements and proprioception of these animals.

EFFECTS OF X-RAY BEAM ANGLE AND GEOMETRIC DISTORTION ON WIDTH OF EQUINE THORACOLUMBAR INTERSPINOUS SPACES USING RADIOGRAPHY AND COMPUTED TOMOGRAPHY-A CADAVERIC STUDY.

The widths of spaces between the thoracolumbar processi spinosi (interspinous spaces) are frequently assessed using radiography in sports horses; however effects of varying X-ray beam angles and geometric distortion have not been previously described. The aim of this prospective, observational study was to determine whether X-ray beam angle has an effect on apparent widths of interspinous spaces. Thoracolumbar spine specimens were collected from six equine cadavers and left-right lateral radiographs and sagittal and dorsal reconstructed computed tomographic (CT) images were acquired. Sequential radiographs were acquired with each interspinous space in focus. Measurements were performed for each interspinous space in the focus position and up to eight angled positions as the interspinous space moved away from focus (±). Focus position measurements were compared to matching sagittal CT measurements. Effect of geometric distortion was evaluated by comparing the interspinous space in radiographs with sagittal and dorsal reconstructed CT images. A total of 49 interspinous spaces were sampled, yielding 274 measurements. X-ray beam angle significantly affected measured width of interspinous spaces in position +3 (P = 0.038). Changes in width did not follow a consistent pattern. Interspinous space widths in focus position were significantly smaller in radiographs compared to matching reconstructed CT images for backs diagnosed with kissing spine syndrome (P < 0.001). Geometric distortion markedly affected appearance of interspinous space width between planes. In conclusion, X-ray beam angle and geometric distortion influence radiographically measured widths of interspinous spaces in the equine thoracolumbar spine, and this should be taken into consideration when evaluating sport horses.

Aarhus Regenerative Orthopaedics Symposium (AROS).

The combination of modern interventional and preventive medicine has led to an epidemic of ageing. While this phenomenon is a positive consequence of an improved lifestyle and achievements in a society, the longer life expectancy is often accompanied by decline in quality of life due to musculoskeletal pain and disability. The Aarhus Regenerative Orthopaedics Symposium (AROS) 2015 was motivated by the need to address regenerative challenges in an ageing population by engaging clinicians, basic scientists, and engineers. In this position paper, we review our contemporary understanding of societal, patient-related, and basic science-related challenges in order to provide a reasoned roadmap for the future to deal with this compelling and urgent healthcare problem.

mRNA expression of genes involved in inflammation and haemostasis in equine fibroblast-like synoviocytes following exposure to lipopolysaccharide, fibrinogen and thrombin.

BACKGROUND: Studies in humans have shown that haemostatic and inflammatory pathways both play important roles in the pathogenesis of joint disease. The aim of this study was to assess mRNA expression of haemostatic and inflammatory factors in cultured equine fibroblast-like synoviocytes exposed to lipopolysaccharide (LPS), fibrinogen and thrombin. Synovial membranes were collected from metacarpo-phalangeal joints of 6 skeletally mature horses euthanized for non-orthopaedic reasons. Passage 4 fibroblast-like synoviocytes were left non-treated or treated with either 0.1 µg/ml LPS, 5 mg/ml fibrinogen or 5 U/ml thrombin and harvested at time points 0, 6, 24 and 48 h. mRNA expression of serum amyloid A (SAA), interleukin-6 (IL-6), monocyte chemotactic protein 1 (MCP-1), tissue factor (TF), plasminogen activator inhibitor 1 (PAI-1), urokinase plasminogen activator (uPA), vascular endothelial growth factor (VEGF) and protease activator receptor 1 (PAR-1) was assessed using quantitative real time reverse transcriptase PCR. RESULTS: LPS caused a significant increase in mRNA expression of SAA, IL-6, MCP-1 and uPA, and a decrease in TF, PAI-1 and PAR-1 when compared to non-treated cells. Treatment with thrombin resulted in increased mRNA expression of SAA, IL-6, MCP-1 and PAI-1, and a decreased PAR-1 expression compared to non-treated cells. The fibrinogen-treated synoviocytes showed significantly increased mRNA expression of IL-6, MCP-1, TF and PAI-1, and decreased PAR-1 expression compared to non-treated cells. CONCLUSION: LPS, fibrinogen and thrombin induced an increased gene expression of inflammatory markers in isolated equine fibroblast-like synoviocytes. LPS caused changes in gene expression promoting increased fibrinolysis, while fibrinogen and thrombin changed the gene expression resulting potentially in reduced fibrinolysis. Overall, it appeared that both inflammatory and haemostatic stimuli affected expression of genes involved in inflammatory and haemostatic pathways, supporting their importance in equine joint diseases.

Use of admission serum neutrophil gelatinase-associated lipocalin (NGAL) concentrations as a marker of sepsis and outcome in neonatal foals.

BACKGROUND: Equine neonatal sepsis can be challenging to diagnose and prognosticate. Neutrophil gelatinase-associated lipocalin (NGAL), a new marker of renal damage and inflammation, can potentially be helpful. OBJECTIVES: To evaluate NGAL in neonatal foals with sepsis, and assess its relation to outcome. ANIMALS: Foals ≤ 14 days, with admission blood analysis and stored serum. METHODS: NGAL was measured on stored serum from 91 foals. Foals were scored for sepsis and survival and categorized according to sepsis status (septic, sick non-septic, healthy, and uncertain sepsis status) and outcome groups (survivors and non-survivors). The septic foals were further sub-categorized according to severity (normal sepsis, severe sepsis and septic shock). A Kruskal-Wallis test was used to compare serum NGAL concentrations in survivors and non-survivors, in the sepsis status groups, and in the sepsis severity groups. Optimal cut-off values for serum NGAL concentrations to diagnose sepsis and outcome were determined with receiver operating characteristic (ROC) curves. NGAL was compared to creatinine and SAA. RESULTS: Median serum NGAL concentrations were significantly higher in septic than non-septic foals. However, serum NGAL concentrations did not differ between sepsis severity subgroups. Serum NGAL concentrations were significantly lower in survivors than in non-survivors. Optimal cut-off values of serum NGAL concentrations were 455 µg/L (sensitivity 71.4%, specificity 100%) and 1104 µg/L (sensitivity 39.3%, specificity 95.2%) for predicting sepsis and non-survival, respectively. NGAL correlated to SAA, but not to creatinine. NGAL performed similarly to SAA to diagnose sepsis. CONCLUSION: Serum NGAL concentrations may be useful for diagnosing sepsis and predicting outcome.

A Delphi Study to Determine International and National Equestrian Expert Opinions on Domains and Sub-Domains Essential to Managing Sporthorse Health and Welfare in the Olympic Disciplines.

The public is increasingly questioning equestrianism's social license to operate. While the focus historically centered on horseracing, increased scrutiny is now being placed on how dressage, showjumping, and eventing are addressing equine management and welfare concerns. Nominated equestrian federation and equestrian organization experts (n = 104) directly involved in international and/or national-level horse sports took part in a four-stage, iterative Delphi to obtain consensus on what factors should be considered essential to manage sporthorse health and welfare. Five core domains were agreed as essential: training management, competition management, young horse management, health status and veterinary management, and the horse-human relationship. Two further domains: stable and environmental management, and welfare assessment were rated as important but not essential, as most respondents felt that these areas were already managed well. Participants felt increased education and guidance combined with further policy development and regulation are needed to support stakeholders to optimize sporthorse management. An appetite to engage with research to generate evidence that promotes sporthorse welfare was evident. The development of a sporthorse welfare charter and evidence-based guidelines to inform the management and monitoring of sporthorses' health and welfare are recommended to provide horses with a good life and to safeguard the future of equestrian sports.

Temporal extracellular vesicle protein changes following intraarticular treatment with integrin α10ß1-selected mesenchymal stem cells in equine osteoarthritis.

Introduction: Equine osteoarthritis (OA) is a heterogeneous, degenerative disease of the musculoskeletal system with multifactorial causation, characterized by a joint metabolic imbalance. Extracellular vesicles are nanoparticles involved in intracellular communication. Mesenchymal stem cell (MSC) therapy is a form of regenerative medicine that utilizes their properties to repair damaged tissues. Despite its wide use in veterinary practice, the exact mechanism of action of MSCs is not fully understood. The aim of this study was to determine the synovial fluid extracellular vesicle protein cargo following integrin α10ß1-selected mesenchymal stem cell (integrin α10-MSC) treatment in an experimental model of equine osteoarthritis with longitudinal sampling. Methods: Adipose tissue derived, integrin α10-MSCs were injected intraarticularly in six horses 18 days after experimental induction of OA. Synovial fluid samples were collected at day 0, 18, 21, 28, 35, and 70. Synovial fluid was processed and extracellular vesicles were isolated and characterized. Extracellular vesicle cargo was then analyzed using data independent acquisition mass spectrometry proteomics. Results: A total of 442 proteins were identified across all samples, with 48 proteins differentially expressed (FDR ≤ 0.05) between sham-operated control joint without MSC treatment and OA joint treated with MSCs. The most significant pathways following functional enrichment analysis of the differentially abundant protein dataset were serine endopeptidase activity (p = 0.023), complement activation (classical pathway) (p = 0.023), and collagen containing extracellular matrix (p = 0.034). Due to the lack of an OA group without MSC treatment, findings cannot be directly correlated to only MSCs. Discussion: To date this is the first study to quantify the global extracellular vesicle proteome in synovial fluid following MSC treatment of osteoarthritis. Changes in the proteome of the synovial fluid-derived EVs following MSC injection suggest EVs may play a role in mediating the effect of cell therapy through altered joint homeostasis. This is an important step toward understanding the potential therapeutic mechanisms of MSC therapy, ultimately enabling the improvement of therapeutic efficacy.

Blood glucose and subcutaneous continuous glucose monitoring in critically ill horses: A pilot study.

This pilot prospective study reports the feasibility, management and cost of the use of a continuous glucose monitoring (CGM) system in critically ill adult horses and foals. We compared the glucose measurements obtained by the CGM device with blood glucose (BG) concentrations. Neonatal foals (0-2 weeks of age) and adult horses (> 1 year old) admitted in the period of March-May 2016 with clinical and laboratory parameters compatible with systemic inflammatory response syndrome (SIRS) were included. Glucose concentration was monitored every 4 hours on blood samples with a point-of-care (POC) glucometer and with a blood gas analyzer. A CGM system was also placed on six adults and four foals but recordings were successfully obtained only in four adults and one foal. Glucose concentrations corresponded fairly well between BG and CGM, however, there appeared to be a lag time for interstitial glucose levels. Fluctuations of glucose in the interstitial fluid did not always follow the same trend as BG. CGM identified peaks and drops that would have been missed with conventional glucose monitoring. The use of CGM system is feasible in ill horses and may provide clinically relevant information on glucose levels, but there are several challenges that need to be resolved for the system to gain more widespread usability.

Evaluation of the in vitro effects of local anesthetics on equine chondrocytes and fibroblast-like synoviocytes.

OBJECTIVE: To investigate the in vitro effects of clinically relevant concentrations of the local anesthetics (LAs) bupivacaine, lidocaine, lidocaine with preservative (LP), mepivacaine, and ropivacaine on equine chondrocyte and fibroblast-like synoviocyte (FLS) viability. SAMPLES: Chondrocytes and FLSs of the metacarpophalangeal joints of 4 healthy adult horses. PROCEDURES: Viability of chondrocytes and FLSs was determined with 3 assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and trypan blue (TB) exclusion (only FLS). Viability was assessed after 30- and 60-minute exposures to 0.0625%, 0.125%, and 0.25% bupivacaine; 0.25%, 0.5%, and 1% lidocaine; 0.25%, 0.5%, and 1% LP; 0.25%, 0.5%, and 1% mepivacaine; and 0.125%, 0.25%, and 0.5% ropivacaine. RESULTS: Viability of chondrocytes was significantly decreased with exposure to 0.25% bupivacaine, 1% lidocaine, 1% LP, 1% mepivacaine, and 0.25% ropivacaine. Viability of FLSs was significantly decreased with exposure to 0.25% bupivacaine, 1% mepivacaine, 1% LP, and 0.5% ropivacaine. CONCLUSIONS AND CLINICAL RELEVANCE: Clinically relevant concentrations of LAs had in vitro time- and concentration-dependent cytotoxicity for chondrocytes and FLSs isolated from the metacarpophalangeal joints of healthy horses. Bupivacaine was more toxic to chondrocytes than lidocaine, mepivacaine, and ropivacaine, whereas bupivacaine, LP, mepivacaine, and ropivacaine were more toxic to FLSs than preservative-free lidocaine. Several LAs may negatively affect chondrocyte and FLS viability.

Effect of exercise on serum neutrophil gelatinase-associated lipocalin concentration in racehorses.

Serum neutrophil gelatinase-associated lipocalin (sNGAL) is a marker of renal injury, and its concentrations are affected by inflammation. Therefore, it could serve as a useful biomarker of disease or fitness in high-level competition. However, it has not yet been determined if sNGAL concentrations are affected by exercise. The aim of this study was to determine whether concentrations of equine sNGAL were affected by 1000 m galloping as the form of exercise used in the study. Pre- and post-gallop sNGAL, serum amyloid A, and creatinine concentrations were evaluated in 14 healthy Thoroughbred racehorses. The results showed that short, high-intensity exercise did not significantly affect sNGAL concentrations in healthy horses (P = .42), and no significant difference was found in either creatinine or serum amyloid A before and after galloping (P > .05). Therefore, it was determined that sNGAL was not influenced by the type of exercise used in the study and could have the potential to be used as a routine laboratory screening tool in horses even after strenuous exercise. Future research should clarify its use in a larger population and a broader range of equine sport disciplines, including endurance-related exercise.

Cellular Proliferation of Equine Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells Decline With Increasing Donor Age.

Background: Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stem cells (MSCs) are used increasingly for autologous cell therapy in equine practice to treat musculoskeletal and other injuries. Current recommendations often call for 10-100 million MSCs per treatment, necessitating the expansion of primary cells in culture prior to therapeutic use. Of concern, human and rodent studies have shown a decline of both MSC recovery from sampled tissue and in vitro proliferative capacity with increasing donor age. This may be problematic for applications of autologous cell-based therapies in the important equine demographic of older patients. Objectives: To investigate the effect of donor age on the cellular proliferation of equine BM- and AT-MSCs. Study Design: In vitro study. Methods: BM- and AT-MSCs and dermal fibroblasts (biological control) were harvested from horses in five different age groups (n = 4, N = 60); newborn (0 days), yearling (15-17 months), adult (5-8 years), middle-aged (12-18 years), and geriatric (≥22 years). Proliferation of the cells was tested using an EdU incorporation assay and steady state mRNA levels measured for targeted proliferation, aging, and senescence biomarkers. Results: The cellular proliferation of equine BM- and AT-MSCs declined significantly in the geriatric cohort relative to the younger age groups. Proliferation levels in the two MSC types were equally affected by donor age. Analysis of steady state mRNA levels showed an up-regulation in tumor suppressors, apoptotic genes, and multiple growth factors in MSCs from old horses, and a down-regulation of some pro-cycling genes with a few differences between cell types. Main Limitations: Potential age-dependent differences in cell function parameters relevant to cell-therapy application were not investigated. Conclusions: The cellular proliferation of equine BM- and AT-MSCs declined at advanced donor ages. High levels of in vitro proliferation were observed in both MSC types from horses in the age groups below 18 years of age.

Histologic changes and gene expression patterns in biopsy specimens from bacteria-inoculated and noninoculated excisional body and limb wounds in horses healing by second intention.

OBJECTIVE: To evaluate histologic changes and gene expression patterns in body and limb wounds in horses in response to bacterial inoculation. SAMPLE: Wound biopsy specimens from 6 horses collected on days 7, 14, 21, and 27 after excisional wounds (20 wounds/horse) were created over the metacarpal and metatarsal region and lateral thoracic region (body) and then inoculated or not inoculated on day 4 with Staphylococcus aureus and Pseudomonas aeruginosa. PROCEDURES: Specimens were histologically scored for the amount of inflammation, edema, angiogenesis, fibrosis organization, and epithelialization. Quantitative PCR assays were performed to quantify gene expression of 10 inflammatory, proteolytic, fibrotic, and hypoxia-related markers involved in wound healing. RESULTS: Except for gene expression of interleukin-6 on day 27 and tumor necrosis factor-α on day 14, bacterial inoculation had no significant effect on histologic scores and gene expression. Gene expression of interleukin-1ß and -6, serum amyloid A, and matrix metalloproteinase-9 was higher in limb wounds versus body wounds by day 27. Gene expression of cellular communication network factor 1 was higher in limb wounds versus body wounds throughout the observation period. CONCLUSIONS AND CLINICAL RELEVANCE: The lack of clear markers of wound infection in this study reflected well-known difficulties in detecting wound infections in horses. Changes consistent with protracted inflammation were evident in limb wounds, and gene expression patterns of limb wounds shared similarities with those of chronic wounds in humans. Cellular communication network factor warrants further investigation and may be useful in elucidating the mechanisms underlying poor limb wound healing in horses.

Current and future regenerative medicine - principles, concepts, and therapeutic use of stem cell therapy and tissue engineering in equine medicine.

This paper provides a bird's-eye perspective of the general principles of stem-cell therapy and tissue engineering; it relates comparative knowledge in this area to the current and future status of equine regenerative medicine.The understanding of equine stem cell biology, biofactors, and scaffolds, and their potential therapeutic use in horses are rudimentary at present. Mesenchymal stem cell isolation has been proclaimed from several equine tissues in the past few years. Based on the criteria of the International Society for Cellular Therapy, most of these cells are more correctly referred to as multipotent mesenchymal stromal cells, unless there is proof that they exhibit the fundamental in vivo characteristics of pluripotency and the ability to self-renew. That said, these cells from various tissues hold great promise for therapeutic use in horses. The 3 components of tissue engineering - cells, biological factors, and biomaterials - are increasingly being applied in equine medicine, fuelled by better scaffolds and increased understanding of individual biofactors and cell sources.The effectiveness of stem cell-based therapies and most tissue engineering concepts has not been demonstrated sufficiently in controlled clinical trials in equine patients to be regarded as evidence-based medicine. In the meantime, the medical mantra "do no harm" should prevail, and the application of stem cell-based therapies in the horse should be done critically and cautiously, and treatment outcomes (good and bad) should be recorded and reported.Stem cell and tissue engineering research in the horse has exciting comparative and equine specific perspectives that most likely will benefit the health of horses and humans. Controlled, well-designed studies are needed to move this new equine research field forward.

Molecular characterization and chromosomal assignment of equine cartilage derived retinoic acid sensitive protein (CD-RAP)/melanoma inhibitory activity (MIA).

Cartilage-derived retinoic acid sensitive protein (CD-RAP) also known as melanoma inhibitory activity (MIA) has already been established as a marker for chondrocyte differentiation and a number of cancerous conditions in humans. Studies have also shown that CD-RAP/MIA is a potential marker of joint disease. The objective of this study was to characterize the equine CD-RAP/MIA gene and thus make it available as a marker in cartilage research and clinical studies. Gene analysis revealed that the equine gene (GenBank accession no. EF679787) consists of four exons and three introns, and the homology to the human gene is 90% for the translated region. The upstream sequence includes regulatory elements and putative transcription factor binding sites previously described in the human and murine promoter regions. The deduced amino acid sequence consists of 130 aa including a signal peptide of 23 aa, and has a 91% identity to the human protein. Using radiation hybrid mapping, the CD-RAP/MIA gene was localized to the p arm of equine chromosome 10 (ECA10p), which is in accordance with prediction based on the current human-equine comparative map. Gene expression studies showed expression of CD-RAP/MIA mRNA in articular cartilage and chondrocytes from horses with no signs of joint disease. The expression decreased as the cells dedifferentiated in monolayer culture. We also identified an equine CD-RAP/MIA splice variant similar to that reported in humans. The CD-RAP/MIA protein was detected in equine synovial fluid, serum and culture medium from chondrocyte cultures. In conclusion, CD-RAP/MIA is expressed in equine cartilage and chondrocytes, and the protein can be detected in equine serum, synovial fluid and in culture medium from chondrocyte cultures. The equine gene and resulting protein share great homology with the human gene, making future studies on CD-RAP/MIAs potential as a marker in joint disease possible using the equine joint as a model.

Concepts for the clinical use of stem cells in equine medicine.

Stem cells from various tissues hold great promise for their therapeutic use in horses, but so far efficacy or proof-of-principle has not been established. The basic characteristics and properties of various equine stem cells remain largely unknown, despite their increasingly widespread experimental and empirical commercial use. A better understanding of equine stem cell biology and concepts is needed in order to develop and evaluate rational clinical applications in the horse. Controlled, well-designed studies of the basic biologic characteristics and properties of these cells are needed to move this new equine research field forward. Stem cell research in the horse has exciting equine specific and comparative perspectives that will most likely benefit the health of horses and, potentially, humans.

Validation of an ELISA for detection of neutrophil gelatinase-associated lipocalin (NGAL) in equine serum.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) has been shown to be a useful marker of kidney injury in people and dogs, but has not been described in horses. OBJECTIVES: The aim of the study was to validate a commercially available porcine-specific ELISA to measure serum concentrations of equine NGAL. METHODS: Intra- and interassay imprecisions were evaluated by multiple measurements on equine serum pools. Assay inaccuracy was determined by the linearity under dilution. Overlapping performance was assessed by measuring NGAL concentrations in horses with normal and elevated serum creatinine levels. RESULTS: Intra- and interassay imprecision (coefficient of variation) ranged from 5.35% to 28.39%. The ELISA showed no signs of inaccuracy. Overlapping performance was acceptable, as the assay was able to detect the expected differences of NGAL levels in horses with normal and elevated serum creatinine concentrations. CONCLUSIONS: Equine serum NGAL concentrations could be quantified reliably using the porcine-specific ELISA.

Production of serum amyloid A in equine articular chondrocytes and fibroblast-like synoviocytes treated with proinflammatory cytokines and its effects on the two cell types in culture.

OBJECTIVE: To investigate the role of the major equine acute phase protein serum amyloid A (SAA) in inflammation of equine intraarticular tissues. SAMPLE: Articular chondrocytes and fibroblast-like synoviocytes (FLSs) from 8 horses (4 horses/cell type). PROCEDURES: Chondrocytes and FLSs were stimulated in vitro for various periods up to 48 hours with cytokines (recombinant interleukin [IL]-1ß, IL-6, tumor necrosis factor-α, or a combination of all 3 [IIT]) or with recombinant SAA. Gene expression of SAA, IL-6, matrix metalloproteinases (MMP)-1 and -3, and cartilage-derived retinoic acid-sensitive protein were assessed by quantitative real-time PCR assay; SAA protein was evaluated by immunoturbidimetry and denaturing isoelectric focusing and western blotting. RESULTS: All cytokine stimulation protocols increased expression of SAA mRNA and resulted in detectable SAA protein production in chondrocytes and FLSs. Isoforms of SAA in lysed chondrocytes and their culture medium corresponded to those previously detected in synovial fluid from horses with joint disease. When exposed to SAA, chondrocytes and FLSs had increased expression of IL-6, SAA, and MMP3, and chondrocytes had increased expression of MMP-1. Chondrocytes had decreased expression of cartilage-derived retinoic acid-sensitive protein. CONCLUSIONS AND CLINICAL RELEVANCE: Upregulation of SAA in chondrocytes and FLSs stimulated with proinflammatory cytokines and the proinflammatory effects of SAA suggested that SAA may be involved in key aspects of pathogenesis of the joint inflammation in horses.

Serum insulin-like growth factor 1 in the aging horse.

BACKGROUND: Insulin-like growth factor 1 (IGF-1) has important roles in anabolic processes in the musculoskeletal system and has been reported to decrease with age in both people and horses. OBJECTIVES: The objective of this study was to determine serum IGF-1 levels in the aging horse from early to late adulthood (age range 5-27 years). METHODS: Healthy horses (n = 72) were used in a cross-sectional study, while 37 paired serum samples were available for a longitudinal study. Serum IGF-1 protein was determined using an ELISA kit validated for use in equine samples. RESULTS: No association was found between serum IGF-1 levels and age in the cross-sectional study. In the longitudinal study, a latent variable model fitted to the data revealed that horses in general experienced a 5.2% increase of serum IGF-1 levels over a 5-year period, but horses crossing a change point around 9 years of age between the 2 samples experienced an 11.0% decrease. CONCLUSIONS: In this study, there was no evidence for aging being a factor in changes of IGF-1 levels in an adult and old horse population.

Validation of the IDS Octeia ELISA for the determination of insulin-like growth factor 1 in equine serum and tendon tissue extracts.

BACKGROUND: Insulin-like growth factor (IGF-1) is an important mediator of tissue repair in horses. OBJECTIVES: The aim of the study was to evaluate whether IGF-1 could be measured reliably in equine serum and tendon tissue extracts, using an IGF-1 ELISA kit developed for human serum and plasma. METHODS: A glycyl-glycine pretreatment protocol of samples was compared with the pretreatment procedure recommended by the manufacturer. Intra- and inter-assay imprecision were evaluated by repeated measurements of equine serum pools. Assay inaccuracy was determined based on the linearity of serially diluted equine serum samples and tendon tissue extracts. The recovery of IGF-1 was evaluated in serum and tendon tissue extracts spiked with known amounts of IGF-1. RESULTS: The range of IGF-1 released using the manufacturer's pretreatment was between 23% and 56% of the amount released using the gly-gly pretreatment in different equine samples. In serum pools with low, intermediate, and high IGF-1 concentrations, intra-assay imprecision was 4.0%, 4.0%, and 3.1%, respectively, and inter-assay imprecision was 13.9%, 7.3%, and 12.8%, respectively. The recovery of serially diluted serum was 96 ± 3% when diluted with serum, and 72 ± 15% when diluted with PBS. The recovery after dilution was 108 ± 17% in tendon tissue extracts. Recovery from serum spiked with a fixed amount of IGF-1 was 101 ± 5% and 99 ± 7% from tendon tissue samples. CONCLUSIONS: The IDS Octeia IGF-1 ELISA kit can be used for measuring IGF-1 in equine serum and tendon tissue extract after pretreatment with glycyl-glycine.