Pharmacokinetics of tilmicosin in chicken joints based on a novel microdialysis sampling method.

Microdialysis is a continuous direct sampling technique used in live animals to study pharmacokinetic (PK) characteristics of drugs directly in target organs. The antibiotic tilmicosin used to treat arthritis in chickens caused by Mycoplasma synoviae. However, the PK study of tilmicosin in chicken joint has not been reported. The aim of this study was to explore the PK characteristics and penetration of tilmicosin by microdialysis incorporated with High-Performance Liquid Chromatography Mass Spectrometry (HPLC-MS/MS). An articular cavity microdialysis sampling model was established by determining in vivo and in vitro recovery results. Tilmicosin was orally administered to chickens and flow rate testing combined with retro-dialysis were used to determine tilmicosin concentration in the target synovial space. HPLC-MS/MS quantification of tilmicosin from plasma and joint dialysate indicated that recovery was negatively correlated with flow rate and the optimal perfusion rate was determined to be 1.0 µL/min. The AUC, Cmax , MRT and t1/2 in plasma were 4.6, 3.0, 2.2 and 1.6 times higher than in the joint dialysate, respectively, but Tmax did not significantly differ. The penetration of tilmicosin from plasma to joint (AUCdialysate /AUCplasma ) was 0.24 and indicated tilmicosin concentration in joints was much lower than that of plasma. Microdialysis technology provides a novel technique to study pharmacokinetics directly in target tissues and our study provides a reference for the clinical use of tilmicosin for treatment of M. synoviae infections in articular cavities.

Lung microdialysis study of florfenicol in pigs after single intramuscular administration.

For most bacterial lung infections, the concentration of unbound antimicrobial agent in lung interstitial fluid has been considered as the gold standard for estimating the antibacterial efficacy. In this study, the pharmacokinetics of florfenicol (FF) in porcine lung interstitial fluid was investigated after single intramuscular administration at two different doses (20 and 50 mg/kg). Twelve pigs underwent thoracotomy under general anesthesia. Then, the CMA/30 probe was implanted into the lung and perfused at 1 µL/min. The microdialysis (MD) samples were collected on a preset schedule and analyzed by high-performance liquid chromatography (HPLC). Noncompartmental pharmacokinetic analysis was performed. FF exhibited rapid distribution and slow elimination in porcine lung interstitial fluid. The main pharmacokinetic parameters at 20 and 50 mg/kg were 4.88 ± 0.54 and 10.36 ± 2.52 µg/mL for the maximum concentration (Cmax ), 3.25 ± 0.32 and 3.50 ± 0.27 h for the time to Cmax (Tmax ), 9.47 ± 6.84 and 7.75 ± 3.23 h for the half-life (t1/2 ), 0.10 ± 0.06 and 0.10 ± 0.04 1/h for the terminal elimination rate constant (λz ), 13.85 ± 7.97 and 11.42 ± 2.79 h for the mean residence time (MRT), 37.77 ± 8.13 and 71.15 ± 16.99 h·µg/mL for the area under the curve from time 0 to 18.25 h (AUC0-18.25 ), and 51.18 ± 20.11 and 88.78 ± 27.58 h·µg/mL for the area under the curve from time 0 to infinity (AUC0-∞ ), respectively.

Microdialysis measurements of lamellar perfusion and energy metabolism during the development of laminitis in the oligofructose model.

REASONS FOR PERFORMING STUDY: Failure of lamellar energy metabolism, with or without ischaemia, may be important in the pathophysiology of sepsis-associated laminitis. OBJECTIVES: To examine lamellar perfusion and energy balance during laminitis development in the oligofructose model using tissue microdialysis. STUDY DESIGN: In vivo experiment. METHODS: Six Standardbred horses underwent laminitis induction using the oligofructose model (OFT group) and 6 horses were untreated controls (CON group). Microdialysis probes were placed in the lamellar tissue of one forelimb (all horses) as well as the skin dermis of the tail in OFT horses. Dialysate and plasma samples were collected every 2 h for 24 h and concentrations of energy metabolites (glucose, lactate, pyruvate) and standard indices of energy metabolism (lactate to glucose ratio [L:G] and lactate to pyruvate ratio [L:P]) determined. Microdialysis urea clearance was used to estimate changes in tissue perfusion. Data were analysed nonparametrically. RESULTS: Median glucose concentration decreased to <30% of baseline by 8 h in OFT lamellar (P = <0.01) and skin (P<0.01) dialysate. Lactate increased mildly in skin dialysate (P = 0.04) and plasma (P = 0.05) but not lamellar dialysate in OFT horses. Median pyruvate concentration decreased to <50% of baseline in OFT lamellar dialysate (P = 0.03). A >5-fold increase in median L:G compared with baseline occurred in OFT lamellar and skin dialysate (P<0.03). From a baseline of <20, median L:P increased to a peak of 80 in OFT skin and 38.7 in OFT lamellar dialysates (P<0.02); however, OFT lamellar dialysate L:P was not significantly different from CON. Urea concentration decreased significantly in OFT lamellar dialysate (increased urea clearance) but not in OFT skin or CON lamellar dialysate. CONCLUSIONS: Increased lamellar perfusion occurred during the development of sepsis-associated laminitis in the oligofructose model. Glucose concentrations in the lamellar interstitium decreased, suggesting increased glucose consumption but there was no definitive evidence of lamellar energy failure.

The effect of weightbearing and limb load cycling on equine lamellar perfusion and energy metabolism measured using tissue microdialysis.

REASONS FOR PERFORMING STUDY: Lamellar perfusion is thought to be affected by weightbearing and limb load cycling; this may be critical in the development of supporting limb laminitis. OBJECTIVES: To document the effects of unilateral weightbearing and altered limb load cycling on lamellar energy metabolism and perfusion. STUDY DESIGN: Randomised, controlled (within subject), experimental trial. METHODS: Nine Standardbred horses were instrumented with microdialysis probes in the foot lamellar tissue and skin (over the tail base). Urea (20 mmol/l) was added to the perfusate. Samples were collected every 15 min for a 1 h control period, then during periods of unilateral weightbearing (opposite limb held off the ground for 1 h); enhanced static limb load cycling (instrumented limb lifted every 10 s for 30 min); reduced limb load cycling activity (i.v. detomidine sedation) and continuous walking (30 min). Dialysate concentrations of glucose, lactate, pyruvate and urea were measured and lactate:glucose (L:G) and lactate:pyruvate (L:P) ratios were calculated. For each intervention, values were compared with baseline using nonparametric statistical testing. RESULTS: Lamellar dialysate glucose increased and L:G decreased significantly during enhanced static limb load cycling. Glucose and pyruvate increased, and L:G, L:P and urea decreased significantly during walking. Simultaneous skin dialysate values did not change significantly. There were no significant dialysate changes during unilateral weightbearing or after detomidine administration, but only the latter resulted in a significant decrease in limb load cycling frequency. CONCLUSIONS: Increases in limb load cycling frequency (particularly walking) caused dialysate changes consistent with increased lamellar perfusion. Unilateral weightbearing (1 h) and a sedation-induced reduction in limb load cycling frequency did not have a detectable effect on lamellar perfusion. More research is needed to confirm the role of hypoperfusion in supporting limb laminitis, but strategies to increase limb load cycling may be important for prevention.

Microdialysis measurements of equine lamellar perfusion and energy metabolism in response to physical and pharmacological manipulations of blood flow.

REASONS FOR PERFORMING STUDY: A suitable method for evaluating lamellar perfusion changes and their metabolic consequences is currently lacking. OBJECTIVES: To examine perfusion changes in lamellar tissue using serial microdialysis measurements of urea clearance and energy metabolites. STUDY DESIGN: Randomised, controlled (within subject) experimental trial. METHODS: Nine Standardbred horses were instrumented with microdialysis probes in the foot lamellar tissue and skin (over the tail base). Urea (20 mmol/l) was added to the perfusate and its clearance was used to estimate local perfusion. Samples were collected every 15 min for a 1 h control period, then during application of a distal limb tourniquet, during periods when norepinephrine or potassium chloride (KCl) were included in both skin and lamellar perfusates, and after systemic (intravenous) acetylpromazine. Dialysate concentrations of glucose, lactate, pyruvate and urea were measured and lactate:glucose (L:G) and lactate:pyruvate (L:P) ratios calculated. Values were compared with pre-intervention baseline and also between simultaneous skin and lamellar samples using nonparametric statistical methods. RESULTS: Lamellar glucose decreased and lactate, urea, L:G and L:P increased significantly with tourniquet application, without significant changes in skin dialysate values. Lamellar and skin glucose decreased and L:G increased significantly during norepinephrine infusion, but mild increases in urea were not significant at either site. KCl caused significant decreases in lamellar and skin L:G, and an increase in skin glucose, but did not affect urea clearance. Acetylpromazine caused profound decreases in lamellar glucose and L:P, with increased L:G and pyruvate, but did not affect urea clearance or any skin dialysate values. CONCLUSIONS: Significant changes in microdialysis urea clearance only occurred with severe lamellar hypoperfusion. However, changes in dialysate metabolite concentrations reflected less profound fluctuations in perfusion. This method may be useful for examining lamellar perfusion and energy balance during laminitis development and for the evaluation of vasoactive therapeutics.

Application of in vivo microdialysis for investigation of unbound drug concentrations of intravenously administered sulfadimidine in the paranasal sinus mucosa of horses.

OBJECTIVE: To monitor concentrations of sulfadimidine in the paranasal sinus mucosa (PSM) of unsedated horses following IV administration of trimethoprim-sulfadimidine via in vivo microdialysis. ANIMALS: 10 healthy adult horses. PROCEDURES: Concentric microdialysis probes were implanted into the subepithelial layers of the frontal sinus mucosa of standing sedated horses. Four hours after implantation, trimethoprim-sulfadimidine (30 mg/kg) was administered IV every 24 hours for 2 days; dialysate and plasma samples were collected at intervals during that 48-hour period and analyzed for concentrations of sulfadimidine. The dialysate concentration and relative loss of sulfadimidine from the perfusate were used to calculate the PSM concentration. RESULTS: Microdialysis probe implantation and subsequent in vivo microdialysis were successfully performed for all 10 horses. Following the first and second administration of trimethoprim-sulfadimidine, mean ± SD peak concentrations of sulfadimidine were 55.3 ± 10.3 µg/mL and 51.5 ± 8.7 µg/mL, respectively, in plasma and 9.6 ± 4.5 µg/mL and 7.0 ± 3.3 µg/mL, respectively, in the PSM. Peak sulfadimidine concentrations in the PSM were detected at 5.9 ± 2.7 hours and 5.4 ± 2.3 hours following the first and second drug administrations, respectively. For 12 hours, mean PSM sulfadimidine concentration remained greater than the minimum inhibitory concentration indicative of sulfonamide susceptibility of equine bacterial isolates (4.75 µg/mL). CONCLUSIONS AND CLINICAL RELEVANCE: In vivo microdialysis for continuous monitoring of PSM sulfadimidine concentrations in unsedated horses was feasible. Intravenous administration of trimethoprim (5 mg/kg) and sulfadimidine (25 mg/kg) proved likely to be efficient for treating sinusitis caused by highly susceptible pathogens, providing that the dosing interval is 12 hours.

Equine lamellar energy metabolism studied using tissue microdialysis.

Failure of lamellar energy metabolism may contribute to the pathophysiology of equine laminitis. Tissue microdialysis has the potential to dynamically monitor lamellar energy balance over time. The objectives of this study were to develop a minimally invasive lamellar microdialysis technique and use it to measure normal lamellar energy metabolite concentrations over 24 h. Microdialysis probes were placed (through the white line) into either the lamellar dermis (LAM) (n = 6) or the sublamellar dermis (SUBLAM) (n = 6) and perfused continuously over a 24 h study period. Probes were placed in the skin dermis (SKIN) for simultaneous comparison to LAM (n = 6). Samples were collected every 2 h and analysed for glucose, lactate, pyruvate, urea and glycerol concentrations. LAM was further compared with SUBLAM by simultaneous placement and sampling in four feet from two horses over 4 h. Horses were monitored for lameness, and either clinically evaluated for 1 month after probe removal (n = 4) or subjected to histological evaluation of the probe site (n = 10). There were no deleterious clinical effects of probe placement and the histological response was mild. Sample fluid recovery and metabolite concentrations were stable for 24 h. Glucose was lower (and lactate:glucose ratio higher) in LAM compared with SUBLAM and SKIN (P < 0.05). Pyruvate was lower in SUBLAM than SKIN and urea was lower in LAM than SKIN (P < 0.05). These differences suggest lower perfusion and increased glucose consumption in LAM compared with SUBLAM and SKIN. In conclusion, lamellar tissue microdialysis was well tolerated and may be useful for determining the contribution of energy failure in laminitis pathogenesis.

Microdialysis in equine research: a review of clinical and experimental findings.

Microdialysis is a method for sampling compounds from extracellular fluid with minimal tissue trauma. Small hollow probes that are 0.2-0.5mm in diameter are inserted into the tissue and slowly perfused. The probe membrane is semi-permeable and a flux of the solutes occurs exclusively according to the concentration gradients. The recovered dialysate reflects changes in the composition of the extracellular water phase with a minor time delay. Because microdialysis is a continuous sampling method, it differs from point sample methods, such as blood sampling. The ability to obtain local measurements in the tissues has led to important discoveries in the detection of tissue changes within the areas of pharmacokinetics, pharmacodynamics, pathology and pathophysiology. New technological solutions, such as transportable pumps, fluid collectors and bedside analysers, have made microdialysis an indispensable tool for the surveillance of critically ill human patients, such as after brain injuries and reconstructive surgeries. The use of microdialysis in equine medicine has been sparingly described with only 14 published studies within muscle, pulmonary and hoof lamellar tissue, nasal mucosa, intestinal wall, uterine, allantoic and cerebrospinal fluid and blood. Only a few papers have been published within each area, indicating that few equine researchers are aware of the unique opportunities provided by the technique. This review discusses the theory and applications of microdialysis with a special emphasis on clinical and experimental equine studies, which may be useful to veterinary experimental and clinical researchers.

Synovial distribution of "systemically" administered acetylsalicylic acid in the isolated perfused equine distal limb.

BACKGROUND: This study investigated synovial concentrations of acetylsalicylic acid (ASA) and its metabolite salicylic acid (SA) in the equine fetlock joint following systemic administration of ASA. Salicylates were chosen because SA is the only nonsteroidal anti-inflammatory drug for which threshold levels exist for plasma and urine in equine sports. To avoid animal experiments, the study was conducted using an ex vivo model of the isolated perfused equine distal limb in combination with plasma concentrations obtained from literature.Salicylate concentrations in the joint were determined using microdialysis and high performance liquid chromatography (HPLC). Any anti-inflammatory effect of synovial ASA concentrations was assessed using an ASA EC50 (half maximal effective concentration) determined in equine whole blood. RESULTS: The ASA concentration in the synovial fluid (n=6) reached a maximum of 4 µg/mL, the mean concentration over the entire perfusion period was 2 µg/mL. Maximum SA concentration was 17 µg/mL, the average was 14 µg/mL. ASA and SA concentration in the synovial fluid exceeded systemic concentrations 2 h and 3.5 h after "systemic" administration, respectively. CONCLUSIONS: ASA and SA accumulated in the in the synovial fluid of the ex vivo model despite decreasing systemic concentrations. This suggests a prolonged anti-inflammatory effect within the joint that remains to be further elucidated.

Oral administration of lysine restores food intake and ventromedial hypothalamic dopamine in chicken on a lysine-free diet.

1. This study was conducted to examine whether oral administration of lysine solution affect food intake and the ventromedial hypothalamic (VMH) monoamines in chickens fed on a lysine-free diet. 2. Chickens were assigned to four treatment groups. Two groups of chickens were given two different doses of lysine solution (0.1 g and 0.07 g in 1 ml of saline) exogenously (orally) while being fed on a lysine-free diet, and these results were compared with a control diet plus saline group. Another group of chickens was fed on a lysine-free diet without lysine supplementation, and their results were compared with the lysine treated groups. The extracellular dopamine (DA), norepinephrine (NE) and serotonin (5-HT) in the VMH of freely moving chicken were measured by in vivo microdialysis. 3. There was no significant difference in food intake between the control diet and the lysine supplemented groups during the time-course of the experiments. Food intake significantly decreased at 4, 5 and 6 h in the lysine-free diet plus saline group compared with the lysine supplemented groups. Of the VMH monoamines, the DA concentration remained close to the baseline in the lysine supplemented groups. This DA concentration was significantly lower than the baseline in the lysine-free diet plus saline group at 3.5 h and thereafter. 4. No significant difference from the baseline was observed for NE in the lysine-free diet plus saline group. The 5-HT concentrations were close to the baseline for all groups throughout the experiments. 5. The findings suggest that oral administration of lysine solution to chickens fed on a lysine-free diet restored food intake which was associated with the variations of VMH DA concentration.

Simultaneous percutaneous implantation of a microdialysis probe for monitoring perineural concentrations of local anaesthetics during peripheral nerve block in rabbits.

OBJECTIVE: To develop a technique that allows simultaneous percutaneous implantation of both a microdialysis probe and injection catheter in order to monitor the perineural pharmacokinetics of local anaesthetics (LA) after a femoral block. STUDY DESIGN: Prospective experimental study. ANIMALS: Five anaesthetized male New Zealand rabbits with a mean ± SD weight of 3.2 ± 0.2 kg. METHODS: After femoral nerve localization by electrostimulation, an injection catheter and a microdialysis probe were slowly and simultaneously inserted into a cannula left into place in the perineural region. Both were then secured into place, after removal of the cannula. At the end of the experiment, methylene blue was injected to confirm the distance from the femoral nerve during subsequent postmortem anatomical dissection of the injection site. RESULTS: Staining was adequate and the catheter found to be located within 4 mm of the femoral nerve in three out of five rabbits. CONCLUSIONS AND CLINICAL RELEVANCE: This procedure allows direct implantation of a microdialysis probe near the injection site of LA during a femoral nerve block without loosing nerve localization accuracy. This procedure has been used successfully to monitor the regional pharmacokinetics of LA after a peripheral nerve block.

Effect of surgery for cannula implantation into the hypothalamus on the behaviour and neural activity of broiler chicks.

1. The effects of a surgical operation, to implant a guide cannula in the chick hypothalamus for microdialysis, on behavioural responses and neural activity in broiler chicks are described. 2. General behavioural activities (feeding, preening, sitting, drinking, cage pecking and beak wiping), open field and locomotor activity tests were conducted to evaluate the effects related to surgery in the immediate 4 d following this procedure. Perfusion of Ringer solution with high K(+) after 4 d of guide cannula implantation was used to estimate the neural activity resulting from surgery through stimulation of monoamine release by in vivo brain dialysis. 3. The results of direct behavioral observations indicated that the stress provoked by surgical guide cannula insertion caused behavioural alterations that are particularly evident in the immediate days following this procedure. Open-field tests on day 4 after surgery showed that, compared to the intact control chickens, the treated chicks had a shorter latency to ambulate and defecate, with more vocalisation. Locomotor activity was less in the treated chicks than in the controls. 4. After 4 d of guide cannula implantation, the serotonin concentration started to increase 30 min after the onset of perfusing high-K(+) Ringer solution. It reached its highest value at one hour, suggesting that the 4 d after surgery is enough to alleviate some neurochemical dysfunction resulting from surgery. The results of behavioural observations, open-field and locomotor activity tests indicate that the surgical operation caused stress and fear in chicks which persisted up to 4 d.

Development of intraosseous infusion of the distal phalanx to access the foot lamellar circulation in the standing, conscious horse.

Intraosseous (i.o.) infusion of the distal phalanx (IOIDP) as a delivery route targeting hoof lamellar tissue of standing, conscious horses was evaluated. Following sedation and regional nerve blockade in six Standardbred horses, a microdialysis (MD) probe was implanted into the hoof lamellar tissue of one forelimb. A purpose designed cannulated bone screw was introduced into the body of the distal phalanx, approximately 6 cm from the MD probe. Gentamicin solution (25 mg/mL) was infused at 20 microL/min through the bone screw for 2 h without the application of a tourniquet. MD and blood samples were collected at regular intervals and analysed for gentamicin concentrations. Gentamicin was present in lamellar tissue at much higher concentrations than peripheral serum. The mean concentration of gentamicin was 24.4, 20.5 and 4.4 microg/mL in extracellular fluid (ECF) and 0.28, 0.5 and 0.32 microg/mL in serum samples collected 60, 120 and 150 min after IOIDP was started, respectively. A clinically safe and efficacious i.o. drug delivery to the hoof lamellar tissue of standing, conscious horse was developed.

Development of an intra-lamellar microdialysis method for laminitis investigations in horses.

Microdialysis (MD) was used for continuous monitoring of the lamellar extracellular fluid (ECF) in six mature healthy Standardbred horses. MD probes were introduced into the lamellar tissue under local anaesthesia. Following intravenous injection of gentamicin (5mg/kg), MD and serum samples were collected for 24h and analysed using a sensitive ELISA test for gentamicin and fluorescence polarization immunoassay for urea concentrations. Calibration of probes was performed through in vivo urea recovery and in vitro gentamicin and urea recovery. Data obtained from different body compartments were compared statistically. The concentration of gentamicin was significantly higher in serum than lamellar ECF for the first 8h (P<0.05), but remained above the minimum inhibitory concentration (MIC) in the ECF for a further 4h. The method will be useful to monitor changes in the lamellar ECF during laminitis and the local kinetics of drugs used therapeutically for this condition.

A pilot study to evaluate a novel subcutaneous continuous glucose monitoring system in healthy Beagle dogs.

Serial blood glucose measurements are currently regarded as the 'gold standard' for evaluating glycaemic control of canine diabetic patients. The aim of this study was to investigate a subcutaneous continuous glucose monitoring system based on microdialysis. Analyses were performed by taking interstitial glucose samples from two different anatomical regions (interscapular region [IR], thoracic region [TR]) in six healthy Beagle dogs during induced hypoglycaemia and hyperglycaemia, the feeding period and a period of stable glucose values. For comparison, plasma glucose concentration was measured simultaneously by a hexokinase-based automated chemistry analyser. The mean absolute differences ranged from 11.7 mg/dL (SD 23.5 mg/dL, TR) to 5.3 mg/dL (SD 19.2 mg/dL, IR) with a correlation of r=0.43-0.92 (P<0.01). Sensitivity for the detection of hypoglycaemia was 85.0% TR and 93.3% IR, and the specificity was 99.5% TR and 99.7% IR, respectively. In a modified Clarke error grid analysis, 99.3% TR and 99.7% IR of the values fell into zone A and B. Collapse of the microdialysis fibre was a recurrent problem although this was easily detectable by fluctuations in system pressure. The microdialysis system reflects physiological as well as induced variations in blood glucose in a valid manner.

Metabolism during anaesthesia and recovery in colic and healthy horses: a microdialysis study.

BACKGROUND: Muscle metabolism in horses has been studied mainly by analysis of substances in blood or plasma and muscle biopsy specimens. By using microdialysis, real-time monitoring of the metabolic events in local tissue with a minimum of trauma is possible. There is limited information about muscle metabolism in the early recovery period after anaesthesia in horses and especially in the colic horse. The aims were to evaluate the microdialysis technique as a complement to plasma analysis and to study the concentration changes in lactate, pyruvate, glucose, glycerol, and urea during anaesthesia and in the recovery period in colic horses undergoing abdominal surgery and in healthy horses not subjected to surgery. METHODS: Ten healthy university-owned horses given anaesthesia alone and ten client-owned colic horses subjected to emergency abdominal surgery were anaesthetised for a mean (range) of 230 min (193-273) and 208 min (145-300) respectively. Venous blood samples were taken before anaesthesia. Venous blood sampling and microdialysis in the gluteal muscle were performed during anaesthesia and until 24 h after anaesthesia. Temporal changes and differences between groups were analysed with an ANOVA for repeated measures followed by Tukey Post Hoc test or Planned Comparisons. RESULTS: Lactate, glucose and urea, in both dialysate and plasma, were higher in the colic horses than in the healthy horses for several hours after recovery to standing. In the colic horses, lactate, glucose, and urea in dialysate, and lactate in plasma increased during the attempts to stand. The lactate-to-pyruvate ratio was initially high in sampled colic horses but decreased over time. In the colic horses, dialysate glycerol concentrations varied considerably whereas in the healthy horses, dialysate glycerol was elevated during anaesthesia but decreased after standing. In both groups, lactate concentration was higher in dialysate than in plasma. The correspondence between dialysate and plasma concentrations of glucose, urea and glycerol varied. CONCLUSION: Microdialysis proved to be suitable in the clinical setting for monitoring of the metabolic events during anaesthesia and recovery. It was possible with this technique to show greater muscle metabolic alterations in the colic horses compared to the healthy horses in response to regaining the standing position.

Luteolytic effect of prostaglandin F 2 alpha on bovine corpus luteum depends on cell composition and contact.

Prostaglandin F(2 alpha) (PGF(2 alpha)) is a main luteolytic factor in vivo; however, its direct luteolytic influence on steroidogenic cells of bovine corpus luteum (CL) is controversial and not fully understood. The aim of the study was to clarify PGF(2 alpha) action on bovine CL in different in vivo and in vitro conditions and to examine whether the contact among all main types of CL cells is necessary for luteolytic PGF(2 alpha) action. In experiment 1, the bovine CL (day 15 of the oestrous cycle) was perfused using in vivo microdialysis system with dinoprost (an analogue of PGF(2 alpha)) for 0.5 h. Dinoprost caused a short-time increase in progesterone (P4), whose concentration decreased thereafter (at 6-, 10-, 12- and 24-h after treatment). In experiment 2, the direct effect of PGF(2 alpha) on P4 accumulation in CL steroidogenic cells cultured in monolayer (day 15 of the cycle) was determined. PGF(2 alpha) after 24 h of incubation increased P4 accumulation in steroidogenic CL cells. In experiment 3 steroidogenic, endothelial CL and immune cells (day 15 of the cycle) were incubated with PGF(2 alpha) in cocultures for 24 h in glass tubes and the levels of P4, stable metabolites of nitric oxide (NO) and leukotriene (LT) C(4) were determined. Although PGF(2 alpha) treatment increased P4 secretion in homogeneous steroidogenic CL cell culture, the decrease in P4 secretion in cocultures of all types of CL cells was observed. The secretion of NO and LTC(4) increased after the treatment of PGF(2 alpha) both in pure cultures of CL cells and in cocultures. The interactions between endothelial and immune cells with steroidogenic CL cells are needed for luteolytic PGF(2 alpha) action within the bovine CL. Our results indicate that the cell coculture model, including the main types of CL cells, is the most approximate to study PGF(2 alpha) role in vitro.

Development of amoxicillin enzyme-linked immunosorbent assay and measurements of tissue amoxicillin concentrations in a pigeon microdialysis model.

A sensitive ELISA was developed for the detection of amoxicillin (AMX) in serum, urine, and milk. The ELISA used an indirect competitive method produced by coating the plate with ovalbumin conjugated with AMX hapten. Antibodies against AMX-BSA were detected by a goat-antirabbit antibody conjugated with peroxidase. Calibration standard curves ranged from 1.28 ng/mL to 20 microg/mL [IC(50) (inhibition concentration 50%) = 100 ng/mL], and the limits of detection were 1.3, 2.7, and 4.8 ng/mL for urine, milk, and serum, respectively. The intra- and interassay variations were less than 4 and 9.6%. The antibody produced against AMX cross-reacted highly with penicillin G (77%); cross-reacted moderately with ampicillin, oxacillin, and cloxacillin (56.9, 51.4, and 48.8%, respectively); but was considered non-cross-reactive with dicloxacillin (7.4%), cefadroxil (<1%), and cefazolin (<1%). Concentrations of AMX were measured simultaneously in venous blood and muscles by using the developed AMX ELISA in an in vivo microdialysis model designed for pigeons. Following i.m. injection (25 mg/kg), AMX attained a peak blood level of 4.74 +/-0.30 mu g/mL and decreased with a half-life of 2.38 +/-0.16 h. In contrast, measurements in pectoral and femoral muscles exhibited delayed appearances, reduced peak concentrations, and prolonged half-lives of 4.07 +/-0.48 (pectoral) and 3.01 +/-0.26 (femoral) that were significantly different from each other and those in the blood (P < 0.05). Blood protein binding was calculated to be 27.9 +/-5.7%. This study demonstrated the semiquantitative application of a selective AMX ELISA in the first microdialysis procedure for continuous monitoring of drug levels in specific tissues of pigeons and maybe useful for related studies in other poultry species.

Effects of ryanodine on skeletal muscle lactate and pyruvate in malignant hyperthermia-susceptible and normal swine as assessed by microdialysis.

BACKGROUND: The caffeine/halothane contracture test in North America and the in vitro contracture test in Europe are currently the only validated bioassays for diagnosing malignant hyperthermia susceptibility and phenotyping families. Both tests are invasive requiring surgical muscle biopsy. Here, we report first use of the selective ryanodine receptor type I agonist ryanodine in a percutaneous microdialysis protocol designed to test whether microdialysis-induced local metabolic responses of skeletal muscle due to ryanodine receptor activation can differentiate between malignant hyperthermia-sensitive and normal pigs. METHODS: Six microdialysis catheters were implanted percutaneously into the adductor muscles of the right and left thighs of malignant hyperthermia-susceptible (n = 9) and normal (n = 8) anaesthetized (ketamine/propofol) and mechanically ventilated swine. Systemic blood gases, haemodynamic parameters and creatine kinase levels were measured before, during and after microdialysis perfusion of ryanodine. After a post-implantation equilibration period of 30 min, one catheter perfused (2 micro min-1) with 0.9% NaCl (control) and was compared with the remaining five catheters perfused with increasing concentrations of ryanodine (0.2-100 micromol). Lactate and pyruvate levels were measured enzymatically. RESULTS: Continuous perfusion with ryanodine revealed dose-dependent sigmoidal increases in the dialysate lactate and lactate-pyruvate ratio parameters; these effects were greatly augmented in malignant hyperthermia-susceptible pigs compared to normal pigs (two- to threefold): estimated EC50 greatly decreased (>19-fold) while the maximum effect increased (>twofold) in the malignant hyperthermia-susceptible group. CONCLUSION: The in vivo percutaneous microdialysis protocol for skeletal muscle, using ryanodine as the ryanodine receptor type I agonist and dialysed lactate-pyruvate parameters as metabolic index, can reproducibly differentiate between malignant hyperthermia-susceptible and normal swine.

Continuous monitoring of penicillin G and gentamicin in allantoic fluid of pregnant pony mares by in vivo microdialysis.

REASONS FOR PERFORMING STUDY: Most current treatments for placentitis in mares are empirical with few control studies to evaluate their effectiveness. OBJECTIVE: To monitor drug concentrations in allantoic fluid of pregnant pony mares using in vivo microdialysis and establish if this method would be useful for determining allantoic concentrations of drugs in normal mares and those with placentitis. METHODS: Five late gestational pony mares had microdialysis probes inserted into the allantoic fluid using transabdominal ultrasound-guided allantocentesis. Single injections of penicillin G (22,000 u/kg), gentamicin (6.6 mg/kg bwt) and flunixin meglumine (1 mg/kg bwt) were administered i.v. and dialysate samples collected continuously for 24 h. In a separate study, drug concentrations were monitored in allantoic fluid of 2 mares with experimental placentitis induced by intracervical inoculation with Streptococcus equi ssp. zooepidemicus. Drug concentrations were measured by high performance liquid chromatography (penicillin G, flunixin meglumine) or enzyme-linked immunosorbent assay (gentamicin). RESULTS: Penicillin G and gentamicin achieved average peak concentrations of 9.8+/-2.2 and 8.5+/-3.1 microg/ml, respectively, in allantoic fluid of noninfected mares. Pharmacokinetic comparisons indicate that penicillin G persists much longer in allantoic fluid than blood, whereas gentamicin exhibited similar profiles in the 2 compartments. Flunixin meglumine was not detected in allantoic fluid. In infected mares, penicillin G achieved a similar peak concentration in allantoic fluid (11.2 microg/ml) whereas peak gentamicin concentration (3.9 microg/ml) appeared to be reduced relative to drug concentrations in noninfected mares. CONCLUSIONS: Microdialysis is a useful technique for continuous in vivo monitoring of drugs in equine allantoic fluid. Our results indicate that penicillin G and gentamicin undergo effective placental transfer in pregnant mares and in 2 mares that transplacental drug transfer may be altered selectively if active placental infection is present. POTENTIAL RELEVANCE: Further studies are needed to evaluate the feasibility of using increased dose intervals for penicillin G and an increased dose rate of gentamicin to effectively combat placental infections in mares.