Henry S. Adair

Plasma concentrations of endotoxin and platelet activation in the developmental stage of oligofructose-induced laminitis

The link between the fermentation of carbohydrate in the equine large intestine and the development of acute laminitis is poorly understood. Absorption of endotoxin (lipopolysaccharide; LPS) into the plasma has been observed in one experimental model of laminitis, but does not cause laminitis when administered alone. Thus, the potential role of endotoxin is unclear. Platelet activation has previously been demonstrated in the developmental stage of laminitis. Equine platelets are more sensitive than leukocytes to activation by endotoxin, and can be activated directly by LPS in the low pg/ml range, activating p38 MAP kinase and releasing serotonin (5-HT) and thromboxane. The objectives of this study were firstly to determine whether endotoxin and platelet activation could be measured in the plasma of horses in the developmental phase of laminitis induced with oligofructose. Secondly, the time course of events involving platelet activation and platelet-derived vasoactive mediator production was investigated. Laminitis was induced in six Standardbred horses by the administration of 10 g/kg bwt of oligofructose. Plasma samples were obtained every 4h, and platelet pellets were obtained by centrifugation. LPS was measured using a kinetic limulus amebocyte lysate assay, and platelet activation was assessed by Western blotting for the phosphorylated form of p38 MAP kinase. Plasma 5-HT was assayed by HPLC with electrochemical detection and thromboxane B(2) was measured by radioimmunoassay. Clinical signs of laminitis and histopathologic changes were observed in lamellar sections from five of the six horses. Onset of lameness was between 20 and 30 h after the administration of oligofructose. LPS increased above the limit of detection (0.6 pg/ml) to reach a peak of 2.4+/-1.0 pg/ml at 8 h. TNFalpha was also detectable in the plasma from 12 to 24 h. There was a time-dependent increase in platelet p38 MAPK phosphorylation, which peaked at approximately 12 h (3.8+/-1.3 fold increase); plasma 5-HT and thromboxane increased steadily after this time (2.9+/-0.6 and 11.3+/-5.0 fold increases, respectively). These data indicate that small quantities of endotoxin may move into the circulation from the large intestine after the sharp decrease in pH that occurs as a result of carbohydrate fermentation. Correlating these findings with in vitro studies suggests that LPS may primarily activate platelets, leading indirectly to the activation of leukocytes. Therefore, endotoxin may contribute in the initiation of the early inflammatory changes observed in experimental models of acute laminitis.

Equine peripheral blood-derived mesenchymal stem cells: isolation, identification, trilineage differentiation and effect of hyperbaric oxygen treatment

REASONS FOR PERFORMING STUDY Two studies report variability in proliferation and limited adipocyte differentiation of equine peripheral blood-derived adult mesenchymal stem cells, thus casting doubt on their adipogenic potential. Peripheral blood can be a valuable source of adult mesenchymal stem cells if cell culture conditions permissive for their adherence, proliferation and differentiation are defined. Hyperbaric oxygen treatment has been reported to mobilise haematopoietic progenitor stem cells into the peripheral blood in humans and mice, but similar experiments have not been done in horses. OBJECTIVES To optimise cell culture conditions for isolation, propagation and differentiation of adult stem cells from peripheral blood and to assess the effect of hyperbaric oxygen treatment on adult stem cell concentrations. METHODS Peripheral blood was collected from the jugular vein of 6 research mares, and mononuclear cells were isolated. They were subjected to cell culture conditions that promote the adherence and proliferation of adult stem cells. The cells were characterised by their adherence, expression of cellular antigen markers, and trans-differentiation. Each horse was subjected to 3 hyperbaric oxygen treatments, and stem cells were compared before and after treatments. Stem cells derived from adipose tissue were used as controls. RESULTS One-third of the horses yielded viable stem cells from peripheral blood, positive for CD51, CD90 and CD105, and demonstrated osteocyte, chondrocyte and adipocyte differentiation. Hyperbaric oxygen treatment resulted in a significant increase in CD90-positive cells. Horses that did not yield any cells pretreatment did so only after 3 hyperbaric oxygen treatments. CONCLUSIONS AND POTENTIAL RELEVANCE Peripheral blood can be a valuable source of adult stem cells, if one can identify reliable equine-specific markers, provide methods to increase the number of circulating progenitor cells and optimise cell culture conditions for growth and viability. Our findings are important for further studies towards technological advances in basic and clinical equine regenerative medicine.

Effects of acepromazine maleate, isoxsuprine hydrochloride and prazosin hydrochloride on laminar microcirculatory blood flow in healthy horses

Summary Laser Doppler flowmetry was used to determine the effects of acepromazine maleate, isoxsuprine hydrochloride, and prazosin hydrochloride on laminar microcirculatory blood flow. Five adult horses, weighing 450–500 kg and free of evidence of laminitis or other inflammatory conditions of the lower limb were used. Holes were drilled on the dorsum of each front hoof to the depth of the junction of the sensitive and insensitive laminae. Temperature, pulse rate and respiratory rate were obtained before begining the measurements. Pulse rate was measured at 15-minute intervals throughout the measurement period. A laser Doppler probe was placed in each hole. Baseline Doppler flow measurements were obtained. Following baseline determinations either acepromazine maleate (0.066 mg/kg), isoxsuprine hydrochloride (0.6 mg/kg) or prazosin hydrochloride (0.025 mg/kg) were administered intravenously. Drugs were randomly assigned and repeated 3 times in each horse. Laminar microcirculatory blood flow was then measured continuously for 180 minutes. These data were analyzed using univariate (mixed model) repeated measures analysis. None of the drugs evaluated had any significant effect on laminar microcirculatory blood flow. Acepromazine maleate, isoxsuprine hydrochloride and prazosin hydrochloride have no laminar microcirculatory blood flow effect in standing, healthy horses at dosages used. Efficacy in the use of these 3 drugs for the treatment of laminitis is questionable. However, this study was performed in healthy horses and the results may not be applicable in horses with laminitis.

Effects of domperidone on digital laminar microvascular blood flow in clinically normal adult horses

OBJECTIVE To determine effects of domperidone and acepromazine maleate on microvascular blood flow in digital laminae of clinically normal adult horses. ANIMALS 8 clinically normal adult horses (4 mares and 4 geldings). PROCEDURES In a 4-period crossover study, domperidone was administered PO at 1.1 mg/ kg and 5.5 mg/kg and IV at 0.2 mg/kg; acepromazine was administered IV at 0.04 mg/kg. The washout period between treatments was 1 week. A 3-minute measurement of laminar microvascular blood flow (LMBF) was obtained with laser Doppler flowmetry. Baseline measurements were obtained at -2, -1, and 0 hours prior to administration of drugs. Post-treatment measurements were obtained at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, and 12 hours. Percentage change from baseline values in LMBF for each treatment was subsequently calculated. RESULTS Oral administration of domperidone at 1.1 mg/kg and 5.5 mg/kg significantly increased LMBF, compared with baseline values, beginning 4 hours after administration, and this effect persisted for at least 8 hours. Intravenous administration of domperidone at 0.2 mg/kg significantly increased LMBF, compared with baseline values, at 10 and 12 hours after administration. Administration of acepromazine (0.04 mg/kg, IV) significantly increased LMBF, compared with baseline values, at 3, 5, 8, and 10 hours after administration. No adverse effects of drugs were detected in any horse. CONCLUSIONS AND CLINICAL RELEVANCE Domperidone may be useful for preventing vasoconstriction and reduction in LMBF believed to occur in horses with laminitis, but additional research of the drugs effects in horses with laminitis is required.

Effects of hyperbaric oxygen treatment on horses with experimentally induced endotoxemia

OBJECTIVE To determine the effectiveness of preinduction hyperbaric oxygen treatment (HBOT) in ameliorating signs of experimentally induced endotoxemia in horses. ANIMALS 18 healthy adult horses. PROCEDURES Horses were randomly assigned to 1 of 3 equal-sized treatment groups to receive normobaric ambient air and lipopolysaccharide (LPS), HBOT and LPS, or HBOT and physiologic saline (0.9% NaCl) solution. Horses were physically examined, and blood was obtained for a CBC and to determine concentration or activity of plasma tissue necrosis factor-α, blood lactate, and blood glucose before the horses were treated with HBOT and then intermittently for 6 hours after administration of LPS or physiologic saline solution. RESULTS All LPS-treated horses developed signs and biochemical and hematologic changes consistent with endotoxemia. Treatment with HBOT significantly ameliorated the effect of LPS on clinical endotoxemia score but did not significantly improve other abnormalities associated with endotoxemia. CONCLUSIONS AND CLINICAL RELEVANCE The protective effect of HBOT was minimal, and results did not support its use as a treatment for horses prior to development of endotoxemia.

Retrospective analysis of local injection site adverse reactions associated with 230 allogenic administrations of bone marrow-derived mesenchymal stem cells in 164 horses

BACKGROUND Bone marrow-derived mesenchymal stem cells (BM-MSCs) are frequently used in the treatment of musculoskeletal injuries. Fully characterised cells that are readily available for use is optimum. Allogenic BM-MSCs can satisfy the need for rapid treatment, however, their safety has been questioned. OBJECTIVES Objectives were to characterise BM-MSCs from an adult donor horse, in vitro, and to identify and describe adverse reactions that occurred following their injection into other horses. We hypothesised that BM-MSCs capable of proliferation, differentiation and lacking MHC II from one donor could be implanted into another individual without significant adverse reactions and the frequency of adverse reactions in clinical cases would be similar to that previously reported for autologous BM-MSCs. STUDY DESIGN Retrospective clinical study. METHODS BM-MSCs were proliferated and characterised from one donor and cryopreserved for clinical use. Medical records for horses injected with allogenic BM-MSCs from this donor at a single hospital were used. After routine lameness exam, lesions were identified using diagnostic ultrasound or MRI. Post injection reaction was defined as increased pain, swelling, or heat at or near injection site, or increased lameness. Treatments required for each reaction were noted. RESULTS BM-MSCs proliferated and underwent differentiation. Cells were found to be negative for MHC-II (<2%) and were viable after cryopreservation and shipping. Ten of 230 (4.35%) injections were noted to be associated with an adverse reaction. Adverse reactions occurred in synovial structures (n = 3) and in soft tissues (n = 7). MAIN LIMITATIONS This investigation could underestimate the number and severity of reactions. Mild reactions, such as synovitis, may have been missed. Also, anti-inflammatory drugs could overshadow mild reactions, making them less likely to be detected. CONCLUSIONS Fully characterised allogenic BM-MSCs originating from a single donor horse can be administered to horses with soft tissue injuries with a low rate of adverse reaction. The Summary is available in Portuguese - see Supporting Information.

Validity and repeatability of goniometry in normal horses

PURPOSE To assess validity and inter- and intra-tester reliability of equine goniometry and to establish values for carpal, metacarpophalangeal, tarsal, and metatarsophalangeal flexion and extension in horses. SUBJECTS Seventeen healthy equine subjects of varied breeds were used. METHODS Three investigators blindly and independently measured in triplicate the extension and flexion of carpal, metacarpophalangeal, tarsal, and metatarsophalangeal joints of 17 horses after sedation. Radiographs of these joints in flexion and extension were acquired while under sedation. Goniometric and radiographic measurements were compared statistically and were correlated. A Bland-Altman plot was constructed. Inter- and intra-tester repeatability of goniometry were evaluated by calculating intraclass correlation coefficients (ICC). Mean flexion and extension of carpal, metacarpophalangeal, tarsal, and metatarsophalangeal joints were calculated. RESULTS Goniometric and radiographic measurements did not differ statistically and were significantly correlated (correlation coefficients ranged from 0.59 - 0.89). The mean difference between goniometric and radiographic measurements was 0.4°. Triplicate measurements collected by the three raters did not differ significantly within raters (ICC ranging from 0.950 - 0.995) and between raters (ICC ranging from 0.942 - 0.989). CONCLUSION Goniometry is a valid and repeatable tool for evaluation of the range of motion of carpal, metacarpophalangeal, tarsal, and metatarsophalangeal joints in standing, sedated healthy horses.

Laser Doppler flowmetry for measurement of laminar capillary blood flow in the horse

Current methods for in vivo evaluation of digital hemodynamics in the horse include angiography, scintigraphy, Doppler ultrasound, electromagnetic flow and isolated extracorporeal pump perfused digit preparations. These techniques are either non-quantifiable, do not allow for continuous measurement, require destruction of the horse orare invasive, inducing non- physiologic variables. In vitro techniques have also been reported for the evaluation of the effects of vasoactive agents on the digital vessels. The in vitro techniques are non-physiologic and have evaluated the vasculature proximal to the coronary band. Lastly, many of these techniques require general anesthesia or euthanasia of the animal. Laser Doppler flowmetry is a non-invasive, continuous measure of capillary blood flow. Laser Doppler flowmetry has been used to measure capillary blood flow in many tissues. The principle of this method is to measure the Doppler shift, that is, the frequency change that light undergoes when reflected by moving objects, such as red blood cells. Laser Doppler flowmetry records a continuous measurement of the red cell motion in the outer layer of the tissue under study, with little or no influence on physiologic blood flow. This output value constitutes the flux of red cells and is reported as capillary perfusion units. No direct information concerning oxygen, nutrient or waste metabolite exchange in the surrounding tissue is obtained. The relationship between the flowmeter output signal and the flux of red blood cells is linear. The principles of laser Doppler flowmetry will be discussed and the technique for laminar capillary blood flow measurements will be presented.