Assessment of exercise-induced alterations in neutrophil function in horses

OBJECTIVE: To evaluate the effects of a standardized exercise test to exhaustion in horses on leukocyte function ex vivo. ANIMALS: 6 Thoroughbred geldings. PROCEDURES: Blood samples were obtained from each horse before exercise; at exhaustion (termed failure); and at 2, 6, 24, 48, and 72 hours after exercise to evaluate hematologic changes, rate of leukocyte apoptosis, and leukocyte production of reactive oxygen species (ROS) ex vivo. To assess leukocyte function, leukocyte ROS production in response to stimulation with lipopolysaccharide, peptidoglycan, zymosan, and phorbol myristate acetate was evaluated. Apoptosis was evaluated via assessment of caspase activity in leukocyte lysates. RESULTS: In response to lipopolysaccharide, production of ROS by leukocytes was significantly increased at 2 hours and remained increased (albeit not significantly) at 6 hours after exercise, compared with the preexercise value. In the absence of any stimulus, leukocyte ROS production was significantly increased at 6 and 24 hours after exercise. In contrast, ROS production in response to phorbol myristate acetate was significantly decreased at 6, 24, and 72 hours after exercise. Leukocyte ROS production induced by zymosan or peptidoglycan was not altered by exercise. Leukocytosis was evident for 24 hours after exercise, and neutrophilia was detected during the first 6 hours. A significant increase in the rate of leukocyte apoptosis was detected at failure and 72 hours after exercise. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that strenuous exercise undertaken by horses causes alterations in innate immune system functions, some of which persist for as long as 72 hours after exercise.     

Heat hypersensitivity at a site proximal to a surgically induced osteochondral fracture in horses

ObjectiveTo evaluate thermal nociceptive thresholds (TNTs) before and after inducing a standardized radiocarpal bone osteochondral fracture (OCF) in horses.Study designProspective, controlled, randomized, masked study.AnimalsA group of 10 Thoroughbred fillies aged 2 years.MethodsSkin temperature and TNTs were measured on the skin over the triceps brachii muscle in both the thoracic limbs before (week 0) and weekly (weeks 1–8) after unilateral arthroscopic induction of a radiocarpal OCF (n = 4) or sham surgery (n = 6) followed by a standardized exercise programme. The contralateral, non-operated thoracic limb was used as a control within each horse. Percentage thermal excursion (%TE) defined as %TE = 100 1 (TNT – skin temperature)/(cut-off temperature – skin temperature) was calculated. Data were analysed with a mixed-effects model followed by Dunnett’s and Tukey’s tests for within and between-limbs comparisons, respectively; p < 0.05 was considered significant.ResultsSkin temperature in the control limb of OCF horses was significantly higher at week 7 than at week 0 (p = 0.0125). At week 1, TNTs and %TE values in operated limbs of OCF horses were significantly reduced compared with their baseline values at week 0 (p ≤ 0.0153) and their values in contralateral control limbs (p ≤ 0.0024) and operated limbs of sham-operated horses (p ≤ 0.0162). At week 2, TNTs and %TE values in operated limbs of OCF horses remained significantly reduced compared with values in operated limbs of sham-operated horses (p ≤ 0.0248).Conclusions and clinical relevanceCreation of an OCF in a radiocarpal bone induced transitory (<2 weeks) ipsilateral heat hypersensitivity proximal to the surgery site (skin over the triceps brachii muscle) in horses. Surgically induced OCF may cause somatosensory abnormalities consistent with secondary thermal hyperalgesia.     

Injection site eosinophilic granulomas and collagenolysis in 3 horses

Three horses were presented with a history of having developed raised cutaneous nodules, within 24-48 hours, in areas of previous injections using standard silicone-coated hypodermic needles. Skin biopsies were taken from a selected cutaneous nodule from all horses for histopathologic evaluation. Histologically, the nodules were consistent with a diagnosis of equine eosinophilic granuloma. A hypersensitivity reaction to the silicone, or another component of the coating formulation, was hypothesized to be responsible for these lesions. Two horses were experimentally injected using both coated and noncoated stainless steel hypodermic needles and skin biopsies were obtained 14 days after injection. The sites of the coated needle injections were characterized by severe eosinophilic granulomatous inflammation with and without collagenolysis. The eosinophilic granulomas with and without collagenolysis observed in these horses are proposed to represent a complex immunologic response to the silicone-based coating of most hypodermic needles.     

Acid-base status and electrolyte alterations associated with diarrhea in non-suckling horses

A retrospective search of medical records of 56 adult horses with diarrhea of variable duration disclosed a variety of blood gas and electrolyte abnormalities at the time of admission. The acid-base and electrolyte disturbances were analyzed and classified according to the duration of diarrhea. In horses with acute diarrhea (duration <6 days) the most common disorder was combined anion gap metabolic acidosis and metabolic alkalosis (72%)characterized by significant hyponatremia, hypochloridemia, and hyperkalemia. The most severe acidemia was found in horses with diarrheas of intermediate duration (1–2 weeks). In this group hyperchloremic metabolic acidosis was commonly found (43%). In horses with diarrhea of long duration (>4 weeks) blood gas and electrolyte values were usually within normal limits. Although the above findings suggest differing therapeutic approaches might be based on the duration of the diarrhea, many cases differed markedly from the mean with respect to their underlying disturbances and, therefore, optimal fluid therapy should be based on the patient's blood gas and electrolyte status.     

Age-related quantitative alterations in lymphocyte subsets and immunoglobulin isotypes in healthy horses

OBJECTIVE: To characterize age-associated changes in lymphocyte population subsets and immunoglobulin isotypes. ANIMALS: 30 healthy young light-breed horses (5 to 12 years old) and 30 healthy aged light-breed horses (> 20 years old). PROCEDURE: Lymphocyte subset populations were identified, using monoclonal antibodies to cell surface markers CD5, CD4, CD8, and IgG. Subset populations were quantitated by use of flow cytometric analysis of antibody-stained cells. Serum immunoglobulin concentration was determined using single radial immunodiffusion. RESULTS: Absolute cell counts of total lymphocytes, T cells, CD4+ and CD8+ T cells, and B cells were decreased in aged horses, compared with young horses. There was a significant decrease in the percentage of CD8+ cells and an increase in the CD4+-to-CD8+ cell ratio in the aged population, compared with young horses. However, serum concentration of IgG, IgG(T), IgM, or IgA did not differ with age. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, total lymphocyte count and lymphocyte subset cell counts decrease with age. Age-matched control values are necessary for optimal evaluation of hematologic variables in aged horses. The decrease in lymphocyte subset cell counts in healthy aged horses mimics that seen in other species and may contribute to an age-associated decrease in immunocompetency.     

Exercise-induced alterations in pro-inflammatory cytokines and prostaglandin F2alpha in horses

Using an established standardized exercise test on a high-speed treadmill, thirteen Thoroughbred racehorses were exercised to fatigue (failure); blood samples were obtained before exercise, at failure, and at 2, 6, 24, 48, and 72 h after exercise. The exercise test induced a systemic inflammatory response characterized by a mild transient endotoxemia, leukocytosis, increased leukocyte expression of mRNA for tumor necrosis factor-alpha (TNF-alpha), IL-1 beta, and IL-6, and increased circulating concentrations of TNF-alpha and prostaglandin F2 alpha (PGF 2 alpha), with the most pronounced changes being evident at failure and 2h after exercise. Expression of mRNA for IL-6, TNF-alpha, and IL-1 beta was increased by 120-fold, three-fold, and four-fold, respectively, when compared to pre-exercise values. Plasma concentrations of 6-keto-PGF1alpha and PGE2 did not change in response to the exercise test. Collectively, these findings indicate that brief, strenuous exercise induces endotoxemia and a systemic pro-inflammatory response in horses that persists for at least 2h.     

Clinical pathologic alterations in horses during a water deprivation test

A 72-hour water deprivation test was performed in 12 horses to determine clinical pathologic changes. Reference values for electrolyte (X) clearance, expressed as a percentage of creatinine clearance (CLCR; %CLCRX), were also determined. A comparison was made between urine concentration measurement techniques. Results of %CLCRX determination in 12 horses before water deprivation were 0.034 +/- 0.095 %CLCRNa, 42.4 +/- 9.8 %CLCRK, 0.352 +/- 0.190 %CLCRCl, and 0.710 +/- 0.250 %CLCRP. During water deprivation, there was individual variation for electrolyte clearances, but Na excretion increased significantly (P less than 0.01) at 24 and 48 hours. After 48 hours' water deprivation, %CLCRNa decreased significantly, but was still greater than the initial clearance. Plasma protein was a better indicator of water deprivation (dehydration) in the horse than was PCV. Electrolyte concentrations in serum and urine were determined. Little significant (P less than 0.01) change in acid-base values was noticed after 72 hours' water deprivation. Urine osmolality (as determined by osmometry) was compared with sp gr (determined by refractometry) in determining urine concentration. Initially, sp gr correlated well with urine osmolality determinations, but this correlation decreased after 48 hours.     

Changes in mineralised tissue at the site of origin of condylar fracture are present before athletic training in Thoroughbred horses

Abstract

AIM: To show that changes are present at the site of origin of metacarpal condylar fracture in young Thoroughbred horses before they enter race training.

METHOD: Bone slices, 2 mm thick, in three mediolateral planes through the centre of rotation of the metacarpo-phalangeal joint (MCPJ) of both distal third metacarpal bones (Mc3) of 12 Thoroughbred horses aged 17 months, were imaged using point-projection digital X-ray imaging (µXR).

RESULTS: In some horses, linear or ovoid radiolucency was found in articular calcified cartilage (ACC) and subchondral bone of the palmaro-distal aspect of the sagittal groove, exactly at the site of more advanced stages of condylar fatigue fracture. An incidental finding was ovoid radiolucency in the apex of the dorso-distal aspect of the sagittal ridge, with or without fragmentation or disturbance of the subchondral mineralised tissue line, resembling equine osteochondrosis.

CONCLUSIONS AND CLINICAL RELEVANCE: The findings imply that the aetiology of condylar fatigue fracture in young Thoroughbred horses includes abnormality in development of the bone and joint that is present before athletic activity occurs.     

Effect of the injection site on the pharmacokinetics of procaine penicillin G in horses

The plasma penicillin concentrations were determined in 5 horses given an IV injection of sodium penicillin G; plasma penicillin concentrations were also determined in a crossover experiment, where animals were given procaine penicillin G subcutaneously at 1 site and IM at 4 sites. The mean penicillin plasma peak concentration and bioavailability were highest after the drug was injected in the neck and biceps musculature. Injections in the gluteal muscle and in the subcutaneous sites resulted in similar, but lower, more persistent penicillin plasma concentrations and a lower bioavailability than were obtained with injection in the neck and biceps musculature. The pharmacokinetic data obtained after penicillin was administered via the pectoral muscle route exhibited an intermediate position. Therapeutic implications of the routes of administration with respect to hemolytic streptococcal infections are discussed.     

Intravenous and sublingual buprenorphine in horses: pharmacokinetics and influence of sampling site

ObjectiveTo describe the pharmacokinetics and adverse effects of intravenous (IV) and sublingual (SL) buprenorphine in horses, and to determine the effect of sampling site on plasma concentrations after SL administration.Study designRandomized crossover experiment; prospective study.AnimalsEleven healthy adult horses between 6 and 20 years of age and weighing 487–592 kg.MethodsIn the first phase; buprenorphine was administered as a single IV or SL dose (0.006 mg kg^?1) and pharmacokinetic parameters were determined for each route of administration using a noncompartmental model. In the second phase; the jugular and lateral thoracic veins were catheterized for simultaneous venous blood sampling, following a dose of 0.006 mg kg^?1 SL buprenorphine. For both phases, plasma buprenorphine concentrations were measured using ultra-performance liquid chromatography with mass spectrometry. At each sampling period, horses were assessed for behavioral excitement and gastrointestinal motility.ResultsFollowing IV administration, buprenorphine mean ± SD half-life was 5.79 ± 1.09 hours. Systemic clearance (Cl) following IV administration was 6.13 ± 0.86 mL kg^?1 minute^?1 and volume of distribution at steady-state was 3.16 ± 0.65 L kg^?1. Following IV administration, horses showed signs of excitement. Gastrointestinal sounds were decreased following both routes of administration; however, none of the horses exhibited signs of colic. There was a significant discrepancy between plasma buprenorphine concentrations measured in the jugular vein versus the lateral thoracic vein following phase 2, thus pharmacokinetic parameters following SL buprenorphine are not reported.Conclusions and clinical relevanceBuprenorphine has a long plasma half-life and results in plasma concentrations that are consistent with analgesia in other species for up to 4 hours following IV administration of this dose in horses. While buprenorphine is absorbed into the circulation following SL administration, jugular venous sampling gave a false measurement of the quantity absorbed and should not be used to study the uptake from SL administration.     

Chronic cervical compressive myelopathy in horses: clinical correlations with spinal cord alterations

Histological examination was performed on the cervical spinal cord from 13 horses with chronic cervical compressive myelopathy of 4 to 29 months duration. Structural alterations were correlated with clinical features. At the level of compression, the spinal cord was grossly deformed. Histological alterations included nerve fibre swelling and degeneration, occasional spheroids, astrocytic gliosis, increased macrophage activity and increased perivascular collagen. Myelin degeneration or loss at the level of the compressive lesion was greatest in the ventral and lateral funiculi and less consistently present in the dorsal funiculi. Asymmetry of lesions in the dorsal funiculi was associated with asymmetry of clinical signs in 5 horses. Histological alterations in areas of Wallerian degeneration were similar to that at the level of spinal cord compression, except that perivascular collagen was not increased. Wallerian degeneration was present cranial to the compressed site in the superficial portions of the lateral funiculi and in the middle of the dorsal funiculi. Caudal to the compressed site it was present in the ventral funiculi adjacent to the ventral median fissure and in the middle of the lateral funiculi. Deformation of the spinal cord did not correlate with the severity or duration of clinical signs but was positively correlated with the amount of perivascular collagen increase. The amount of nerve fibre swelling was not correlated with the severity of clinical signs but was negatively correlated with their duration. A rapid loss of nerve fibres apparently occurred early in the course of compression, since there was a marked decrease in the amount of nerve fibre swelling and Marchi stained degenerating myelin with increasing clinical duration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Systemic and colonic venous plasma biochemical alterations in horses during low-flow ischemia and reperfusion of the large colon.

The purpose of this study was to determine the effects of low-flow ischemia and reperfusion (I-R) of the large colon on 16 systemic venous (SV) and colonic venous (CV) plasma biochemical variables in horses. Horses (n = 24) were randomly allocated to 3 groups: sham-operated (n = 6), 6 h ischemia (n = 9), and 3 h ischemia followed by 3 h reperfusion (n = 9). SV and CV heparinized blood was collected at 0, 1, 3, 3.25, 4, and 6 h. The SV-CV difference was calculated for each variable. The SV, CV, and SV-CV difference for albumin, total protein, and calcium decreased significantly (P < 0.05) across time in horses of all groups, but there were no differences among groups. SV phosphorous was significantly increased from baseline (BL) at 1 to 6 h in horses of all groups, but there were no differences among groups. CV phosphorous was significantly greater than BL from 1 to 6 h in group-2 horses and from 1 to 3 h in group-3 horses. SV potassium was not different among groups, but was significantly higher at 6 h, compared with BL in horses of all groups. CV potassium was significantly greater than BL from 1 to 6 h in horses of groups 2 and 3. SV glucose was greater at 6 h compared with all previous times in horses of all groups, but there were no difference among groups. CV glucose was significantly lower than BL and group-1 values in horses of groups 2 and 3 during ischemia, but returned to BL during reperfusion in group-3 horses. CV anion gap was significantly greater and SV-CV anion gap was significantly more negative in horses of groups 2 and 3, compared with group-1 horses during ischemia. The biologic relevance of these alterations is unknown, but they may contribute to histopathologic, hemodynamic, and metabolic alterations characteristic of low-flow I-R. Alternatively, these alterations may simply reflect colonic injury sustained during I-R. Results suggest that the colon utilizes glucose as a fuel and generates acid anions during low-flow ischemia. Increased CV phosphorous and potassium during I-R likely occurs as a result of leakage of intracellular stores subsequent to cellular damage.     

Temporal and linear kinematics in elite and riding horses at the trot

Standard videography was used to determine the temporal and linear variables of 32 Spanish Purebreds at trot. Horses were organized into three groups: Group A (nonelite), comprising 13 stallions without any specific training program, which were hand led; Group B1 (elite), formed by 19 horses considered to be well-gaited and dressage trained, and which were also hand led; and Group B2 (ridden elite), comprising the same horses as the latter group but this time ridden. The horses ran at working trot, according to the trainers. Each animal was recorded using a Hi8 video camera. For temporal measurements, a 1/50 s time code was added to video images. Recordings were analyzed to provide times (seconds) at landing, mid-stance position, lift off and at subsequent landing in each limb. These data allowed the stride to be divided into its component phases by analysis of the variables for individual limbs, diagonal pairs and overall stride. The linear variables (meters) analyzed were stride length and overtracking, obtained for each stride from digitized images and calculated on a spreadsheet. Data were then compared between Groups A-B1 and Groups B1–B2.

Stride duration (0.639±0.064 vs 0.672±0.037 s) and stride length (247.7±21.9 vs 238.5±18.1 cm) of horses from A and B1 groups were similar; Group B1 was characterized by limb support and overlap values close to those of Group A; however, limb swing (forelimbs: 0.391±0.032 s; hind limbs: 0.374±0.027 s), diagonal swing (0.365±0.027 s) and inter-overlap (0.131±0.035) were of greater duration. Additionally, the proportion of horses with advanced placement was greater in Group B1.

Group B2 displayed greater stride duration (0.718± 0.054 s) and lower stride length (217.3±26.1 cm) than B1 group. The analysis revealed mounted horses to be characterized by limb and diagonal swings similar to those of Group B1; however, limb support and overlap were significantly greater, and inter-overlap and suspension phase were shorter.     

Comparative histomorphological study of endometrium in mares

Uterine acute post‐breeding inflammation is a physiological tissue response to the entry of exogenous elements, with persistent endometritis being the main pathology responsible for subfertility in the mare (Equus ferus caballus; Linnaeus, 1758). Mares can be classified as susceptible or resistant to endometritis according to their ability to remove intrauterine fluid within 48 hr after experimental inoculation. Endometrial biopsy is a technique that is commonly used to establish the degree of lesions that can affect the fertility of the mare. Endometrial histomorphometry is an objective and highly precise diagnostic method. The aim of this study was to compare, during oestrus, the endometrial histomorphometry of mares previously classified as susceptible (SM) or resistant (RM) to endometritis. Endometrial biopsies from 24 mares at the oestrus phase of the cycle were obtained. For the histomorphometric analysis, samples were histologically processed and subjected to routine Haematoxylin–Eosin staining. For the evaluation, the variables were considered as follows: 1‐Height of the lining and glandular epithelia (Lining SM = 15.9 μm vs. RM = 13.3 μm; Glandular SM = 15.0 μm vs. RM = 13.0 μm); 2‐Perpendicular diameters of endometrial glands (SM = 51.3 μm vs. RM = 44.8 μm); 3‐Number of endometrial glands per field (SM = 24.8 glands/field vs. RM = 20.5 glands/field). The results from this study suggest the existence of a relationship between the studied characteristics and the susceptibility/resistance to post‐breeding endometritis in mares. Thus, increased epithelial height, greater glandular density and greater development of the glands during oestrus would be related to a higher susceptibility to endometritis.     

Time-dependent alterations in gene expression of interleukin-8 in the bronchial epithelium of horses with recurrent airway obstruction

OBJECTIVE: To evaluate time-dependent alterations in gene expression of chemokines in bronchial epithelium of recurrent airway obstruction (RAO)-affected horses and whether alterations resulted from increases in gene expression of interleukin (IL)-17 in cells isolated from bronchoalveolar lavage fluid (BALF). ANIMALS: 8 RAO-susceptible horses and 9 control horses. PROCEDURE: In 2 experiments, both groups of horses were evaluated after being maintained on pasture and after being stabled and fed dusty hay for 1, 14, 35, and 49 days (experiment 1) or 14 and 28 days (experiment 2). In experiment 1, gene expression of IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and Toll-like receptor 4 (TLR4) in epithelium and IL-8, IL-17, and TLR4 in BALF cells was measured. In experiment 2, bronchial biopsy specimens were evaluated for IL-8 immunoreactivity. RESULTS: In RAO-susceptible horses after 14 days of challenge exposure, there was a 3- and 10-fold increase in gene expression of IL-8 for epithelial and BALF cells and an increase in IL-8 immunoreactivity in epithelial cells. Challenge exposure failed to alter gene expression of CXCL1, GM-CSF, G-CSF, and TLR4 in epithelial cells of any horses at any time point. During challenge exposure, gene expression of BALF cell IL-17 was downregulated in control horses (day 1) and upregulated in RAO-affected horses (day 35). CONCLUSIONS AND CLINICAL RELEVANCE: Epithelial-derived IL-8 may promote airway neutrophilia, but the inciting stimulus is unlikely to be IL-17 because upregulation of this gene is subsequent to that of IL-8 in epithelial cells.