Validation of a commercially available photometric analytical system for assessment of plasma oxidative status in healthy horses.

Reactive oxygen species (ROS) are the end-products of physiologic functions in health. Oxidative stress occurs when endogenous antioxidants are insufficient to neutralize ROS in the system. As a result, ROS can damage DNA, RNA, proteins, lipids, and cell organelles. To obtain accurate measurements of plasma oxidative stress, levels of both oxidants and antioxidants must be measured. This study validates a commercially available, semi-quantitative, photometric analytical system that measures systemic determinants of reactive oxygen metabolites (dROM) and plasma antioxidant capacity (PAC) in stored equine plasma. The objectives of this work were: 1) to validate a photometric analytical system to quantify dROM and PAC in equine plasma; and 2) to determine expected results for these tests in healthy adult horses. We hypothesized that this system would reliably and reproducibly assess dROM and PAC in equine plasma. We observed expected, dose-dependent increases in dROM generated by adding increasing concentrations of H2O2 or ascorbic acid to equine plasma to provide samples containing a known quantity of oxidants or antioxidants respectively. Mean dROM value in healthy horses was 103.3 ±20.7 U. Carr and mean PAC was 2881.0 ± 313.9 U. Cor. This system reliably and reproducibly quantified dROM and PAC in equine plasma samples.

Circulating Hypothalamic-Pituitary-Adrenal Axis Hormones and Insulin Concentrations in Horses and Ponies.

Mechanisms resulting in breed predispositions to insulin dysregulation (ID) are poorly characterized. Cortisol antagonizes insulin, and free, biologically active cortisol can be increased in ID. Breed-related differences in serum free cortisol fraction (FCF) could contribute to ID, but FCF has not been quantified in equidae predisposed to ID, such as ponies. To compare FCF and other hypothalamic-pituitary-adrenal (HPA) axis hormones between horses and ponies during health and ID. We hypothesized: (1) FCF is higher in ponies than horses in health, and is higher still in ponies with ID and obesity; and (2) FCF is positively correlated with insulin in horses and ponies during health and ID. Thirty-three horses and 24 ponies were sampled before morning feeding in their normal routine. Plasma ACTH and insulin and serum total cortisol concentrations and FCF were measured. ID was defined as evidence of hyperinsulinemia at rest or after oral sugar administration. Data were compared with Mann-Whitney tests and Spearman correlation analysis (P < 0.05). Total cortisol, free cortisol, insulin concentrations, and FCF were comparable in healthy horses (n = 24) and ponies (n = 12), but ACTH concentrations were 29% higher in ponies than in horses (P = 0.016). In animals with ID, total cortisol, free cortisol, and insulin concentrations were similar between horses and ponies, but FCF was increased 40% in ponies (n = 12) compared to horses (n = 9). These data demonstrate differences in insulin, ACTH, and free cortisol during health and ID between ponies and horses.

Variability in peripheral blood enrichment techniques can alter equine leukocyte cellularity, viability and function.

Peripheral blood is commonly sampled to assess the health status of human and veterinary patients. Venous blood collection is a minimally invasive procedure, and in the horse, the common collection site is the jugular vein. Post blood collection, sample processing for leukocyte enrichment can vary by research laboratory with the potential to yield different effects on the enriched cells and their function. The focus of the present study was to compare a common blood dilution-leukocyte enrichment technique using a Histopaque gradient medium (His) to a modified leukocyte buffy coat syringe-lymphocyte separation medium technique (Syr- LSM) with peripheral blood from 12 healthy horses. The endpoints examined included cell recovery/mL of blood, cell viability, leukocyte enrichment purity, leukocyte cell marker subset phenotype, leukocyte spontaneous and mitogen-induced proliferation and secretory TNFα concentrations. Leukocyte cell recovery/mL of whole blood and cell viability was significantly increased in enriched leukocytes from the Syr-LSM technique. Interestingly, the percentage of CD8+ and CD21+ were significantly increased with the His technique as was Con A-induced proliferation. Still, leukocyte cell purity and TNFα concentrations from the 72 h cell culture supernatants were comparable across the two enrichment techniques. To summarize, the type of whole blood leukocyte enrichment technique employed can affect the results of immunologic assay endpoints possibly altering data interpretation.

Innate immune responses of equine monocytes cultured in equine platelet lysate.

Platelet lysate (PL) has been extensively used for the laboratory expansion of human mesenchymal stem cells (MSC) in order to avoid fetal bovine serum (FBS) which has been associated with immune-mediated host reactions and transmission of bovine-origin microbial contaminants. Before suggesting the routine use of PL for MSC culture, we wanted to further investigate whether PL alone might trigger inflammatory responses when exposed to reactive white blood cells such as monocytes. Our objectives were to evaluate the inflammatory profile of equine monocytes cultured with equine PL (ePL) and to determine if ePL can modulate the expression of inflammatory cytokines in lipopolysaccharide (LPS)-stimulated monocytes. In a first experiment, equine monocytes were isolated and incubated with donor horse serum (DHS), FBS, six individual donors ePL or pooled ePL from all horses. In a second experiment, monocytes were stimulated with E. coli LPS in the presence of 1, 5 or 10% DHS and/or pooled ePL. After 6h of incubation, cell culture supernatants were assayed via ELISA for production of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and Interleukin 1ß (IL-1ß) as well as for the anti-inflammatory Interleukin 10 (IL-10). Equine monocytes incubated with pooled ePL produced significantly less TNF-α and significantly more IL-10 than monocytes incubated in FBS. A statistically significant difference was not identified for the production of IL-1ß. The second experiment showed that pooled ePL added to LPS-stimulated equine monocytes resulted in a significant reduction in TNF-α and IL-1ß production. IL-10 production was not significantly upregulated by the addition of ePL to LPS-stimulated monocytes. Finally, the addition of ePL to LPS-stimulated monocytes in the presence of various concentrations of DHS resulted to statistically significant decrease of TNF-α and IL-1ß compared to the control groups. This is the first study to demonstrate that ePL suppresses the release of pro-inflammatory cytokines from stimulated equine monocytes. These results encourage further exploration of PL as a homologous media substitute for FBS but also opens the possibility of investigating its use as means to suppress cell-mediated inflammation.

The effect of free and carrier-bound cortisol on equine neutrophil function.

Cortisol is a key anti-inflammatory hormone that increases in bacterial sepsis and circulates predominantly bound to cortisol binding globulin (CBG). Only unbound cortisol was believed to be biologically active, but recent evidence suggests that CBG-bound cortisol also regulates inflammation. The objective of this study was to evaluate the effects of free and CBG-bound cortisol on equine neutrophil function ex vivo. We hypothesized that CBG would enhance cortisol-mediated suppression of neutrophil pro-inflammatory responses. Neutrophils isolated from 8 foals and 6 adult horses were exposed to Staphylococcus aureus antigen (SAA) alone and with hydrocortisone (HC), CBG, or both (CBG+HC). Inflammatory cytokine (TNF-α, IL-8) and reactive oxygen species (ROS) production were measured and compared among stimulants and between ages with linear mixed-effects models. CBG and CBG+HC inhibited ROS production induced by SAA in both foal and horse neutrophils, maintaining it at levels comparable to baseline production (P≤0.060-0.907). TNF-α production was not significantly different among stimulants (P=0.284). CBG+HC significantly (P≤0.016) increased IL-8 production by neutrophils in response to SAA in both foals and adults, although the response of foals was significantly greater than that of adults (P<0.001). These findings suggest that CBG directly modulates equine neutrophil responses, but the effects are cytokine- and age-specific.

Measurement of cortisol concentration in the tears of horses and ponies with pituitary pars intermedia dysfunction.

OBJECTIVE To compare tear cortisol concentrations between horses and ponies with pituitary pars intermedia dysfunction (PPID) and healthy nonaged (≤ 15 years old) and aged (≥ 20 years old) horses and to determine whether serum and tear cortisol concentrations were correlated. ANIMALS 11 horses and ponies with PPID and 20 healthy control horses and ponies (11 nonaged and 9 aged). PROCEDURES Paired tear and serum samples were obtained from PPID and control animals. All animals were free of active ocular disease. Tear and serum cortisol concentrations were measured with an ELISA and chemiluminescent assay, respectively. Groups were compared with Kruskal-Wallis and Mann-Whitney U tests, and Spearman correlation analysis was used to examine relationships between tear and serum cortisol concentrations within groups. RESULTS Median tear cortisol concentration was significantly higher in PPID animals than in aged control animals, despite comparable serum cortisol concentrations in PPID and aged control animals. Median tear-to-serum cortisol concentration ratios were also significantly higher in PPID animals than in aged control animals. Serum and tear cortisol concentrations were not significantly correlated in PPID or control animals. CONCLUSIONS AND CLINICAL RELEVANCE Some horses and ponies with PPID had increased tear cortisol concentrations, compared with concentrations in healthy aged animals. Localized cortisol production in the tear film or altered cortisol binding dynamics could have contributed to this increase. Further studies are warranted to evaluate these mechanisms and to determine whether increased tear cortisol concentrations are associated with delays in corneal wound healing in horses and ponies with and without PPID.

Feline mesenchymal stem cells and supernatant inhibit reactive oxygen species production in cultured feline neutrophils.

Feline bone marrow-derived MSCs (BMMSCs), adipose-derived MSCs (AMSCs) and fibroblasts (FBs) were isolated and cultured. Tri-lineage differentiation assays and flow cytometry were used to characterize MSCs. Neutrophils (NPs) were isolated from whole blood and the NPs production of reactive oxygen reactive oxygen species (ROS) was measured. NPs were cultured alone, with MSC culture supernatant (SN), BMMSCs or AMSCs. NPs incubated with BMMSCs had significantly lower ROS production than NPs incubated with AMSCs (p=0.0006) or FB (p<0.0001); NPs ROS production significantly decreased with increasing BMMSC cell number (p=0.0023) and significantly increased with NPs were incubated with FB compared to BMMSC (p=0.0003). Both BMMSC SN and AMSC SN had statistically significantly lower ROS production than FB SN when incubated with NPs (both p<0.0001). ROS production was significantly reduced with increased fractions of SN from BMMSCs (p=0.0467) and AMSCs (p=0.0017).

Detection of endogenous cortisol in equine tears and blood at rest and after simulated stress.

OBJECTIVE: To determine whether cortisol is present in equine tears at rest and during simulated stress and compare tear cortisol to serum free and total cortisol. MATERIALS AND METHODS: Fourteen healthy adult horses were included. Paired tear total cortisol and serum total and free cortisol concentrations were measured with ELISA, chemiluminescent immunoassay, and ultrafiltration methodology, respectively, in 10 horses at rest once daily for five consecutive days. In an additional four horses, paired tear and serum samples were collected for cortisol measurement before and after adrenocorticotropic hormone (ACTH) stimulation (cosyntropin, 1 µg/kg IV). RESULTS: Cortisol was detectable in equine tears at rest. Following ACTH stimulation, tear cortisol increased significantly from baseline at 60-120 min (P ≤ 0.001). Serum total and free cortisol also increased significantly at 30-180 min after ACTH stimulation (P ≤ 0.001). Both serum and tear cortisol returned to baseline concentrations by 360 min. Changes in tear cortisol were similarly associated with changes in serum total and free cortisol, although high tear cortisol concentrations suggest a portion of tear cortisol may be protein-bound. DISCUSSION: Cortisol is present in equine tears and increases in concert with serum cortisol following ACTH stimulation. Further study is needed to determine whether endogenous cortisol in tears contributes to ocular pathology.

Daily endogenous cortisol production and hydrocortisone pharmacokinetics in adult horses and neonatal foals.

OBJECTIVE: To compare daily endogenous cortisol production rate and the pharmacokinetics of an i.v. bolus of hydrocortisone between neonatal foals and adult horses. ANIMALS: 10 healthy full-term 2- to 4-day-old foals and 7 healthy adult horses. PROCEDURES: Blood samples were collected from each horse every 15 to 20 minutes for 24 hours for determination of 24-hour mean cortisol concentration. Afterward, dexamethasone (0.08 mg/kg) was administered i.v. to suppress endogenous cortisol production. Twelve hours afterward, hydrocortisone sodium succinate (1.0 mg/kg) was administered as a rapid i.v. bolus and serial blood samples were collected to determine hydrocortisone pharmacokinetics. Cortisol concentrations, daily cortisol production rate, and hydrocortisone pharmacokinetics were determined, and results were compared between adult horses and foals. RESULTS: The mean ± SD 24-hour cortisol concentration was significantly lower in foals (20 ± 4 ng/mL) than in horses (26 ± 6 ng/mL), but the daily cortisol production rate was significantly greater in foals (6,710 ± 320 ng/kg/d) than in horses (2,140 ± 400 ng/kg/d). For hydrocortisone, foals had a significantly greater volume of distribution at steady state (1.92 ± 1.11 L/kg) and total body clearance (1.39 ± 0.108 L/kg/h) and significantly lower peak plasma concentration (1,051 ± 343 ng/mL) than did horses (0.58 ± 0.15 L/kg, 0.349 ± 0.065 L/kg/h, and 8,934 ± 3,843 ng/mL, respectively). CONCLUSIONS AND CLINICAL RELEVANCE: Important differences were detected in cortisol production and metabolism between neonatal foals and adult horses consistent with lower plasma protein binding of cortisol in foals. This decrease may contribute to cortisol insufficiency during prolonged critical illness in neonatal foals.

Antimicrobial-induced endotoxin and cytokine activity in an in vitro model of septicemia in foals.

OBJECTIVE: To determine which antimicrobials that are used to treat neonatal foals with septicemia attributable to Escherichia coli will minimize endotoxin release from bacteria and subsequent activity of inflammatory mediators while maintaining bactericidal efficacy. SAMPLE POPULATION: Blood samples from 10 healthy foals. PROCEDURE: Escherichia coli isolates A and B were isolated from 2 septicemic foals, and minimal inhibitory concentrations (MIC) were determined for 9 antimicrobials. Five of these antimicrobials were tested in vitro at 2 and 20 times their respective MIC. Whole blood or mononuclear cells grown in tissue-culture media were incubated with 105 colony-forming units of E. coli and each antimicrobial or saline (0.9% NaCl) solution. After 6 hours, number of viable bacteria remaining was determined, and supernatant was tested for endotoxin and tumor necrosis activity. RESULTS: Testing in whole blood was compromised by bactericidal effects of the blood itself. In mononuclear cell suspensions, each antimicrobial significantly reduced the number of viable bacteria to low or undetectable amounts. Antimicrobials did not differ significantly in efficacy of bacterial killing. Amikacin used alone or in combination with ampicillin resulted in significantly less endotoxin activity than did ampicillin, imipenem, or ceftiofur alone. There was a correlation between TNF-alpha and endotoxin activity. CONCLUSIONS AND CLINICAL RELEVANCE: Aminoglycosides appear less likely to induce endotoxemia and TNF-alpha synthesis during bactericidal treatment of E. coli septicemia, compared with beta-lactam antimicrobials. Use of ampicillin, imipenem, or ceftiofur in the treatment of septicemic neonatal foals should be accompanied by appropriate treatment for endotoxemia.