No adverse effects on periarticular tissue by intra-articular vitamin D analogue calcipotriol in a reduced-dose zymosan-induced arthritis model in rats.

Calcipotriol, a vitamin D analogue, is an antiproliferative and anti-inflammatory drug currently used in psoriasis. Here, our aim was to analyse the safety of calcipotriol for cartilage and bone in alleviated-dose (0.1 mg instead of usual ≥1mg dose) zymosan-induced arthritis in rats. Theoretically, high doses of vitamin D or analogues could have detrimental effects on bone or cartilage. The rats were divided into four groups: vehicle (n = 9), dexamethasone 0.1 mg/kg (n = 9), calcipotriol 0.1 mg/kg (n = 8) and negative control (n = 10) with no injections. Arthritic rats were given phosphate-buffered saline (PBS) injections to left knees as a control. After euthanasia on day 8, all knees were imaged with micro-computed tomography for surface lesions and decalcified for histological analyses. Contrary to our expectations, no significant changes could be observed in the tomography data and histological scores among the three treatment groups or between the vehicle-treated and non-arthritic group. Calcipotriol did not cause adverse effects on cartilage or subchondral bone within a week, suggesting that it could be safely used in local treatment of arthritis. The alleviated model caused synovitis with local and systemic inflammatory response without cartilage erosions, which might be useful in studying self-limiting synovitis where cartilage or bone effects are not of primary interest.

Central nervous system distribution of buprenorphine in pregnant sheep, fetuses and newborn lambs after continuous transdermal and single subcutaneous extended-release dosing.

Buprenorphine is used during pregnancy for the treatment of opioid use disorder. Limited data exist on the central nervous system (CNS) permeation and distribution, and on the fetal exposure to buprenorphine. The aim of our study was to determine the extent of buprenorphine distribution to CNS in the pregnant sheep, and their fetus at steady-state, and their newborn lambs postdelivery, using three different dosing regimens. Twenty-eight pregnant ewes in late gestation received buprenorphine via 7-day transdermal patch releasing buprenorphine 20 µg/h (n=9) or 40 µg/h (n=11), or an extended-release 8 mg/week subcutaneous injection (n=8). Plasma, cerebrospinal fluid, and CNS tissue samples were collected at steady-state from ewes and fetuses, and from lambs 0.33 - 45 hours after delivery. High accumulation of buprenorphine was observed in all CNS tissues. The median CNS/plasma concentration -ratios of buprenorphine in different CNS areas ranged between 13 and 50 in the ewes, and between 26 and 198 in the fetuses. In the ewes the CNS/plasma -ratios were similar after the three dosing regimens, but higher in the fetuses in the 40 µg/h dosing group, medians 65 - 122, than in the 20 µg/h group, medians 26 - 54. The subcutaneous injection (theoretical release rate 47.6 µg/h) produced higher concentrations than observed after 40 µg/h transdermal patch dosing. The median fetal/maternal concentration -ratios in different dosing groups ranged between 0.21 and 0.54 in plasma, and between 0.38 and 1.3 in CNS tissues, respectively, with the highest ratios observed in the spinal cord. Buprenorphine concentrations in the cerebrospinal fluid were 8 - 13 % of the concurrent plasma concentration in the ewes and 28 % in the fetuses. Buprenorphine was quantifiable in the newborn lambs' plasma and CNS tissues two days postdelivery. Norbuprenorphine was analyzed from all plasma, cerebrospinal fluid, and CNS tissue samples but was nondetectable or below the LLOQ in most. The current study demonstrates that buprenorphine accumulates into CNS tissues at much higher concentrations than in plasma in pregnant sheep, fetuses, and their newborn lambs even 45 hours after delivery.

MATERNAL AND FETAL BUPRENORPHINE PHARMACOKINETICS IN PREGNANT SHEEP DURING TRANSDERMAL PATCH DOSING: Buprenorphine pharmacokinetics in pregnant sheep.

BACKGROUND: Buprenorphine is used in the opioid maintenance treatment for opioid dependent patients, including pregnant women. Despite the wide use, limited data exists on buprenorphine pharmacokinetics and fetal exposure during pregnancy. The aim of our study was to determine the buprenorphine pharmacokinetics during transdermal patch dosing to pregnant sheep and, to determine the extent of transplacental transfer of buprenorphine to the fetus. METHODS: Pregnant sheep in late gestation (n=50) received 20, 25 or 40 µg/h of buprenorphine as a 7-day extended-release transdermal patch. Plasma samples were collected from the ewe and the fetus on days 1 - 6, and buprenorphine and norbuprenorphine concentrations were determined. During the exposure period the sheep had a surgical procedure on the second day, a recovery phase, and an experimental procedure on the sixth day. In the experiment, hypoxia was induced under anesthesia for 18 sheep to investigate if decreased fetal pH would cause ion-trapping of buprenorphine in the fetus. The fetal/maternal plasma concentration ratio was determined on the second and on the sixth exposure day at baseline and during hypoxia. Maternal pharmacokinetics were modelled with a population pharmacokinetic method using the data from this study and our previous intravenous administration study. RESULTS: The transdermal patch provided an extended release of buprenorphine throughout the exposure period, but the release rate declined approximately 20 h after patch placement. The median fetal/maternal plasma concentration ratio was 13 - 27 % throughout the exposure period at baseline. A ratio over 100 % was observed for four sheep on the sixth exposure day (102 - 269 %). A minor increase was seen in the median fetal/maternal-ratios during maternal hypoxia. Norbuprenorphine was undetected in all plasma samples. CONCLUSIONS: The low transplacental passage of less than one fourth of the ewe's exposure supports buprenorphine as an alternative to methadone in opioid maintenance therapy during pregnancy.

A single intra-articular dose of vitamin D analog calcipotriol alleviates synovitis without adverse effects in rats.

1,25-dihydroxyvitamin-D3 and its derivatives have shown anti-arthritic and chondroprotective effects in experimental animal models with prophylactic dosing. The purpose of this preliminary study was to test the efficacy and safety of calcipotriol, vitamin D analog, as a treatment for a fully-developed knee arthritis in Zymosan-induced arthritis (ZIA) model. Forty 5-month-old male Sprague-Dawley rats were randomized into three arthritis groups and a non-arthritic control group with no injections (10 rats/group). A day after Zymosan (0.1 mg) had been administrated into the right knee joints, the same knees were injected with calcipotriol (0.1 mg/kg), dexamethasone (0.1 mg/kg) or vehicle in a 100 µl volume. The left control knees were injected with saline (PBS) on two consecutive days. All injections, blood sampling and measurements were performed under general anesthesia on days 0, 1, 3 and 8. Internal organs and knees were harvested on day 8 and the histology of the whole knees was assessed blinded. Joints treated with calcipotriol showed a milder histological synovitis than those treated with vehicle (p = 0.041), but there was no statistically significant difference between the dexamethasone and vehicle groups. The clinical severity of arthritis did not differ between the arthritis groups measured by body temperature, swelling of the knee, thermal imaging, clinical scoring or cytokine levels on days 1, 3 and 8. Weight loss was bigger in rats treated with dexamethasone, propably due to loss of appetite,compared to other arthritis groups on days 2-3 (p<0.05). Study drugs did not influence serum calcium ion and glucose levels. Taken together, this preliminary study shows that a single intra-articular injection of calcipotriol reduces histological grade of synovitis a week after the local injection, but dexamethasone did not differ from the vehicle. Calcipotriol may have an early disease-modifying effect in the rat ZIA model without obvious side effects.

Pharmacokinetics of buprenorphine in pregnant sheep after intravenous injection.

Buprenorphine is a semi-synthetic opioid, widely used in the maintenance treatment for opioid-dependent pregnant women. Limited data exist on the pharmacokinetics of buprenorphine in pregnancy. We conducted a pharmacokinetic study to determine the pharmacokinetics of intravenous buprenorphine in pregnant sheep. Fourteen pregnant sheep in late gestation received 10 µg/kg of buprenorphine as an intravenous bolus injection. Plasma samples were collected up to 48 h after administration. Buprenorphine and its metabolite, norbuprenorphine, were quantified from plasma using a LC/MS/MS method, with lower limits of quantification of 0.01 µg/L and 0.04 µg/L for buprenorphine and norbuprenorphine, respectively. The pharmacokinetic parameters were calculated using noncompartmental analysis. The pharmacokinetic parameters, median (minimum-maximum), were Cmax 4.31 µg/L (1.93-15.5), AUCinf 2.89 h*µg/L (1.72-40.2), CL 3.39 L/h/kg (0.25-6.02), terminal t½ 1.75 h (1.07-31.0), Vss 8.04 L/kg (1.05-49.3). Norbuprenorphine was undetected in all plasma samples. The median clearance in pregnant sheep was higher than previously reported for nonpregnant sheep and human (male) subjects. Our sensitive analytical method was able to detect long terminal half-lives for six subjects, and a wide between-subject variability in the study population. Significance statement: Buprenorphine is widely used for the treatment of opioid use disorder in pregnancy. However, limited data exist on the pharmacokinetics of buprenorphine during pregnancy. As this type of study cannot be done in humans due to ethical reasons, we conducted a study in pregnant sheep. This study provides pharmacokinetic data on buprenorphine in pregnant sheep and helps us to understand the pharmacokinetics of the drug in humans.

Effects of Estrogen Level, Dietary Loading, and Aging on Types I, II, and X Collagen Expression and Structure of Rat Mandibular Condylar Cartilage.

AIMS: To investigate how estrogen level, dietary loading, and aging affect cartilage structure and the expression of major collagens (types I, II, and X) in rat mandibular condylar cartilage (MCC). METHODS: A total of 96 outbred Sprague Dawley female rats were randomly divided into two groups by ovariectomy (OVX) at 7 weeks old. One week later, the rats in each group were further divided into three subgroups on the basis of food hardness: hard food (diet board), normal food (pellet), and soft food (powder). The rats were sacrificed at the age of 5 or 14 months. The thickness of the fibrous, proliferative, and chondroblastic layers of the mandibular condylar cartilage were measured after toluidine blue staining. Immunohistochemical analysis was performed to evaluate the expression levels of types I, II, and X collagen. A linear regression model was used to investigate the main factors affecting changes in thickness and collagen expression. RESULTS: The expression levels of types II and X collagen were decreased by ovarian estrogen deficiency and increased by dietary loading. Increased dietary loading was the main factor affecting an increase in thickness of the cartilage layers, while aging was the main factor affecting a decrease in thickness of the fibrous layer. A significant age-related increase was found in the expression of type I collagen. There was some degree of interaction between aging and dietary loading that affected the thickness of the chondroblastic layer and the expression of type X collagen. CONCLUSION: The physiologic level of estrogen plays a role in MCC development by promoting the expression of types II and X collagen. Dietary loading is essential to increase the expression of types II and X collagen, as well as the thickness of cellular layers, to maintain the integrity of the MCC. Aging seems to reduce the ability of the MCC to withstand occlusal loading.

Oxycodone concentrations in the central nervous system and cerebrospinal fluid after epidural administration to the pregnant ewe.

The main sites of the analgesic action of oxycodone are the brain and spinal cord. The present study describes the concentrations of oxycodone and its metabolites in the brain and spinal cord after epidural administration to the ewe. Twenty pregnant ewes undergoing laparotomy were randomized into two groups to receive epidural oxycodone: infusion group (n = 10, 0.1 mg·kg-1 bolus followed by continuous infusion of 0.05 mg·kg-1 ·h-1 for five days) or repeated boluses group (n = 10, 0.2 + 2x0.1 mg·kg-1 bolus followed by a 0.2 mg·kg-1 bolus every 12 hours for five days). After five days of oxycodone administration, arterial blood samples were collected, the sheep were killed, and a CSF sample and tissue samples from the cortex, thalamus, cerebellum and spinal cord were obtained for the quantification of oxycodone and its main metabolites. The median plasma and CSF concentrations of oxycodone were 9.0 and 14.2 ng·mL-1 after infusion and 0.4 and 1.1 ng·mL-1 after repeated boluses. In the infusion group, the cortex, thalamus and cerebellum oxycodone concentrations were 4-8 times higher and in the spinal cord 1310 times higher than in plasma. In the repeated boluses group, brain tissue concentrations were similar in the three areas, and in the spinal cord were 720 times higher than in plasma. Oxymorphone was the main metabolite detected, which accumulated in the brain and spinal cord tissue. In conclusion, first, accumulation of oxycodone and oxymorphone in the CNS was observed, and second, high spinal cord concentrations suggest that epidural oxycodone may provide segmental analgesia.

MMP-3 and MMP-8 in rat mandibular condylar cartilage associated with dietary loading, estrogen level, and aging.

OBJECTIVES: The structure of the mandibular condylar cartilage (MCC) is regulated by dynamic and multifactorial processes. The aim of this study was to examine the effects of altered dietary loading, estrogen level, and aging on the structure of the condylar cartilage and the expressions of matrix metalloproteinase (MMP) -3 and MMP-8 of rat MCC. METHODS: In this study, Crl:CD (SD) female rats were randomly divided into 3 groups according to dietary hardness: hard diet (diet board), normal diet (pellet), and soft diet (powder). In each group, the rats were further divided into 2 subgroups by ovariectomy at the age of 7 weeks. The rats were sacrificed at 5- and 14-month-old. Histomorphometric analysis of the MCC thickness was performed after toluidine blue staining. Immunochemical staining was done for MMP-3 and MMP-8. A linear mixed model was used to assess the effects of dietary loading, estrogen level, and aging. RESULTS: Increased dietary loading was the main factor to increase the MMP-3 expression and the anterior and central thickness of the MCC. Lack of estrogen was the main factor associated with decreased MMP-8. Aging was associated with the thickness changes of the whole condylar cartilage and the reduced expression of MMP-8. CONCLUSION: The condylar cartilage structure and metabolism of the female rats are sensitive to dietary loading changes, estrogen level as well as aging. The proper balance of these factors seems to be essential for the maintenance of the condylar cartilage.

Foramen ovale blood flow and cardiac function after main pulmonary artery occlusion in fetal sheep.

NEW FINDINGS: What is the central question of this study? At near-term gestation, foramen ovale blood flow accounts for a significant proportion of fetal left ventricular output. Can the foramen ovale increase its volume blood flow when right ventricular afterload is increased by main pulmonary artery occlusion? What is the main finding and its importance? Foramen ovale volume blood flow increased during main pulmonary artery occlusion. However, this increase was attributable to an increase in fetal heart rate, because left ventricular stroke volume remained unchanged. These findings suggest that the foramen ovale has a limited capacity to increase its volume blood flow. ABSTRACT: The foramen ovale (FO) accounts for the majority of fetal left ventricular (LV) output. Increased right ventricular afterload can cause a redistribution of combined cardiac output between the ventricles. To understand the capability of the FO to increase its volume blood flow and thus LV output, we mechanically occluded the main pulmonary artery in seven chronically instrumented near-term sheep fetuses. We hypothesized that FO volume blood flow and LV output would increase during main pulmonary artery occlusion. Fetal cardiac function and haemodynamics were assessed by pulsed and tissue Doppler at baseline, 15 and 60 min after occlusion of the main pulmonary artery and 15 min after occlusion was released. Fetal ascending aorta and central venous pressures and blood gas values were monitored. Main pulmonary artery occlusion initially increased fetal heart rate (P < 0.05) from [mean (SD)] 158 (7) to 188 (23) beats min-1 and LV cardiac output (P < 0.0001) from 629 (198) to 776 (283) ml min-1 . Combined cardiac output fell (P < 0.0001) from 1524 (341) to 720 (273) ml min-1 . During main pulmonary artery occlusion, FO volume blood flow increased (P < 0.001) from 507 (181) to 776 (283) ml min-1 . This increase was related to fetal tachycardia, because LV stroke volume did not change. Fetal ascending aortic blood pressure remained stable. Central venous pressure was higher (P < 0.05) during the occlusion than after it was released. During the occlusion, fetal pH decreased and P C O 2 increased. Left ventricular systolic dysfunction developed while LV diastolic function was preserved. Right ventricular systolic and diastolic function deteriorated after the occlusion. In conclusion, the FO has a limited capacity to increase its volume blood flow at near-term gestation.

Pharmacokinetics of intra-articular vitamin D analogue calcipotriol in sheep and metabolism in human synovial and mesenchymal stromal cells.

Calcipotriol (MC903) is a side chain analogue of the biologically active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Due to its anti-inflammatory and anti-proliferative effects on stromal cells, calcipotriol is a promising candidate for the local treatment of arthritis. In this preliminary work, we studied the pharmacokinetics and safety of calcipotriol after an IV (0.1 mg/kg given to one sheep) and intra-articular dose (0.054 mg/kg, 0.216 mg/kg and 0.560 mg/kg given to three sheep). The terminal half-life of calcipotriol was approximately 1 h after an IV dose. After intra-articular dosing, the systemic absorption was between 1 and 13% during the observed 24 h. Hypercalcemia or other clinical adverse effects did not occur in any animal during the study, and no macroscopic or microscopic alterations were seen in the synovium of the calcipotriol-injected knees compared to the vehicle knees. The in vitro metabolism of calcipotriol was analyzed with LC-MS from human synovial and mesenchymal stromal cell cultures. Both cell types were able to metabolize calcipotriol with MC1080 and MC1046 as the main metabolites. CYP24A1 transcripts were strongly induced by a 48-hour calcipotriol exposure in mesenchymal stromal cells, but not consistently in synovial stromal cells, as determined by RT-qPCR. Calcipotriol proved to be safe after a single intra-articular dose with applied concentrations, and it is metabolized by the cells of the joint. Slow dissolution of calcipotriol crystals in the joint can extend the pharmaceutical impact on the synovium, cartilage and subcortical bone.

Oxycodone pharmacokinetics and fetal exposure after intravenous or epidural administration to the ewe.

INTRODUCTION: There are limited data on oxycodone pharmacokinetics during pregnancy and on fetal exposure after maternal administration. The present study describes the pharmacokinetics of intravenous (i.v.) oxycodone in pregnant sheep and fetal exposure after intravenous and epidural administration. MATERIAL AND METHODS: Ten pregnant sheep received 0.1 mg·kg-1 oxycodone intravenously, and blood samples were collected up to 24 hours. Seven days later, the ewes were randomized to receive 0.5 mg·kg-1 oxycodone intravenously (n = 5) or epidurally (n = 5) as a single bolus, before laparotomy for placement of catheters into the fetal superior vena cava and carotid artery. Paired maternal and fetal blood samples were taken when the fetal arterial catheter was in place and at the end of surgery. Maternal blood samples were taken up to 24 hours. RESULTS: After 0.1 mg·kg-1 oxycodone intravenously, the median clearance was 5.2 L·h-1 ·kg-1 (range 4.6-6.2), but the volume of distribution varied between 1.5 and 4.7 L·kg-1 . The area under the curve was 17 h·ng·mL-1 (range 14-19) and the plasma concentration at 2 minutes 60 ng·mL-1 (range 50-74). Following administration of 0.5 mg·kg-1 intravenously or epidurally, oxycodone concentrations were similar in the maternal and the fetal plasma. Accumulation of oxymorphone in the fetus occurred; fetal-to-maternal ratios were 1.3-3.5 (median 2.1) in the i.v.-group and 0.9-3.0 (1.3) in the Epidural-group. CONCLUSIONS: We determined the pharmacokinetics of oxycodone in pregnant sheep. We showed accumulation of oxymorphone, which an active metabolite of oxycodone, in the fetus. Further studies in human pregnancies are required to evaluate the safety of oxycodone.

Changes in Type I and Type II Collagen Expression in Rat Mandibular Condylar Cartilage Associated with Aging and Dietary Loading.

AIMS: To evaluate the usefulness of diet board feeding as a model for temporomandibular joint (TMJ) research, characterize dietary loading-related morphometric changes in the mandibular condylar cartilage of aging rats, and investigate changes in type I and type II collagen expression in different age, sex, and diet groups. METHODS: Material was collected from a study that examined the effects of 1-year and 2-year diet board feeding on rats. In diet board feeding, rats must gnaw wood to reach their food, leading to a higher masticatory workload. The material analyzed was comprised of 150 TMJ samples from 75 Hsd:Sprague Dawley rats grouped according to feeding method (diet board [experimental group] or ad libitum [control group]), sex, and experiment length (1 or 2 years). The rats were sacrificed at the age of 15 or 26 months (15-M rats or 26-M rats). From the TMJ samples, 5-µm-thick sections were cut parallel to the sagittal plane of the mandibular condyle. Histomorphometric analysis of the thickness of the condylar cartilage and the number of cartilage cells was performed after toluidine blue staining. Immunohistochemical staining included type I and type II collagen antigens. Differences in the thickness of the cellular layer and the number of cells in the condylar cartilage were analyzed by means of a repeated-measures analysis of variance (ANOVA) model, and differences in the type of collagen with a one-way random-effects ANOVA model. RESULTS: Condylar cartilage was significantly thicker in the 15-M diet board-fed rats than in the 15-M control rats and in the 26-M rats than in the 15-M rats. The number of cells was larger in the 26-M female rats than in the 26-M male rats. Type I collagen expression was significantly higher in the 15-M diet board-fed female rats than in the 15-M controls. Type II collagen showed increased expression in older rats compared to younger rats. CONCLUSION: Condylar cartilage is sensitive to the interplay between loading, aging, and sex of middle-aged and older rats. High loading of condylar cartilage increased the thickness of cartilage in younger rats.

Classification and reporting of severity experienced by animals used in scientific procedures: FELASA/ECLAM/ESLAV Working Group report.

Directive 2010/63/EU introduced requirements for the classification of the severity of procedures to be applied during the project authorisation process to use animals in scientific procedures and also to report actual severity experienced by each animal used in such procedures. These requirements offer opportunities during the design, conduct and reporting of procedures to consider the adverse effects of procedures and how these can be reduced to minimize the welfare consequences for the animals. Better recording and reporting of adverse effects should also help in highlighting priorities for refinement of future similar procedures and benchmarking good practice. Reporting of actual severity should help inform the public of the relative severity of different areas of scientific research and, over time, may show trends regarding refinement. Consistency of assignment of severity categories across Member States is a key requirement, particularly if re-use is considered, or the safeguard clause is to be invoked. The examples of severity classification given in Annex VIII are limited in number, and have little descriptive power to aid assignment. Additionally, the examples given often relate to the procedure and do not attempt to assess the outcome, such as adverse effects that may occur. The aim of this report is to deliver guidance on the assignment of severity, both prospectively and at the end of a procedure. A number of animal models, in current use, have been used to illustrate the severity assessment process from inception of the project, through monitoring during the course of the procedure to the final assessment of actual severity at the end of the procedure (Appendix 1).

Diurnal rhythms of blood glucose, serum ghrelin, faecal IgA and faecal corticosterone in rats subjected to restricted feeding using the diet board.

Laboratory rats are generally fed ad libitum, although this method is associated with obesity and an increased frequency of spontaneous tumours. It has been challenging looking for ways to limit feed consumption in group-housed rats without any setbacks to animal welfare and scientific results. The diet board, as a method of dietary restriction, was used in the present study. Diet board feeding allows group housing and should result in enhanced welfare compared with traditional methods of dietary restriction. With respect to animal model robustness and translatability of results it is important that the feeding regime does not affect diurnal rhythmicity of biological parameters. In the present study the effects of diet board feeding on diurnal rhythms of blood glucose, serum ghrelin, faecal immunoglobulin A (IgA) and faecal corticosterone were assessed. The diet board did not alter diurnal rhythms, and adds weight to the use of this method for dietary restriction which should benefit animal health and the validity of scientific results generated from the animals.

Foetal Fentanyl Exposure and Ion Trapping after Intravenous and Transdermal Administration to the Ewe.

Opioids given to pregnant and parturient women are relatively freely transferred across the placenta. Spinal, epidural and intravenous fentanyl has been studied in pregnant women and neonates, but foetal safety of fentanyl dosing with transdermal patch during pregnancy and labour is not sufficiently studied. Foetal pH is physiologically lower than maternal pH, and thus, opioids, which are weak bases, are ionized and may cumulate to foetus. Foetal asphyxia may further worsen acidosis, and ion trapping induced by low pH is assumed to increase the foetal exposure to opioids. Here, we show that no correlation between foetal acidosis and ion trapping of fentanyl could be found. In three experiments, 29 pregnant sheep were administered fentanyl with 2 µg/kg/h patch supplemented with IV boluses/infusion. Foetal exposure to fentanyl was extensive, median 0.34 ng/ml (quartiles 0.21, 0.42), yet drug accumulation to foetus was not observed, and median of foetal/maternal concentration (F/M) ratio was 0.63 (0.43, 0.75) during the first hours after the fentanyl administration. Low foetal pH and pH difference between ewe and the foetus did not correlate with fentanyl concentration in the foetus or F/M ratio. At steady-state during the second patch worn, foetal plasma fentanyl was low, 0.13 ng/ml, and the median of F/M ratio was 0.69. Our results demonstrate that drug accumulation to foetus caused by ion trapping seen with some weak base opioids may not be that significant with fentanyl. These results have a clinical relevance when fentanyl is dosed to pregnant woman and the foetus is acidemic.

Fentanyl Pharmacokinetics in Pregnant Sheep after Intravenous and Transdermal Administration to the Ewe.

Fentanyl is used for pain treatment during pregnancy in human beings and animals. However, fentanyl pharmacokinetics during pregnancy has not been fully established. The aim of this study was to characterize fentanyl pharmacokinetics in pregnant sheep after intravenous and transdermal dosing during surgical procedure performed to ewe and foetus. Pharmacokinetic parameters reported for non-pregnant sheep and nominal transdermal dose rate were utilized for a priori calculation to achieve analgesic fentanyl concentration (0.5-2 ng/ml) in maternal plasma. A total of 20 Aland landrace ewes at 118-127 gestational days were used. In the first protocol, 1 week before surgery, 10 animals received 2 µg/kg fentanyl intravenous bolus, and on the operation day, transdermal fentanyl patches at nominal dose rate of 2 µg/kg/hr were applied to antebrachium, and ewes were then given a 2 µg/kg intravenous bolus followed by an intra-operative 2.5 µg/kg/hr infusion. In the second protocol, 10 animals received fentanyl only as transdermal patches on the operation day and oxycodone for rescue analgesia. The data were analysed with population pharmacokinetic modelling. Intra- and post-operative fentanyl concentrations were similar and slightly lower than the a priori predictions, and elimination and distribution clearances appeared slower during than before or after the surgery. Transdermal patches provided sustained fentanyl absorption for up to 5 days, but the absorption rate was slower than the nominal dose rate and showed a high interindividual variability. Further research is warranted to evaluate the clinical relevance of the observations made in sheep.

Reducing suffering in experimental autoimmune encephalomyelitis (EAE).

This report is based on discussions and submissions from an expert working group consisting of veterinarians, animal care staff and scientists with expert knowledge relevant to the field. It aims to facilitate the implementation of the Three Rs (replacement, reduction and refinement) in the use of animal models or procedures involving experimental autoimmune encephalomyelitis (EAE), an experimental model used in multiple sclerosis research. The emphasis is on refinement since this has the greatest potential for immediate implementation. Specific welfare issues are identified and discussed, and practical measures are proposed to reduce animal use and suffering. Some general issues for refinement are summarised to help achieve this, with more detail provided on a range of specific measures to reduce suffering.

Reducing suffering in animal models and procedures involving seizures, convulsions and epilepsy.

This report is based on discussions and submissions from an expert working group consisting of veterinarians, animal care staff and scientists with expert knowledge relevant to the field and aims to facilitate the implementation of the Three Rs (replacement, reduction and refinement) in the use of animal models or procedures involving seizures, convulsions and epilepsy. Each of these conditions will be considered, the specific welfare issues discussed, and practical measures to reduce animal use and suffering suggested. The emphasis is on refinement since this has the greatest potential for immediate implementation, and some general issues for refinement are summarised to help achieve this, with more detail provided on a range of specific refinements.

Guidelines for the veterinary care of laboratory animals: report of the FELASA/ECLAM/ESLAV Joint Working Group on Veterinary Care.

Veterinary professionals working in partnership with other competent persons are essential for a successful animal care and use programme. A veterinarian's primary responsibilities are defined by their own professional regulatory bodies, but in this area of work there are further opportunities for contribution, which will assist in safeguarding the health and welfare of animals used in research. These guidelines are aimed not only at veterinarians to explain their duties, and outline the opportunities to improve the health and welfare of animals under their care, but also at employers and regulators to help them meet their responsibilities. They describe the desirability for postgraduate education towards specialization in laboratory animal medicine and detail the many competencies necessary to fulfil the role of the laboratory animal veterinarian. They detail the need for veterinary expertise to promote good health and good welfare of animals used in biomedical research during husbandry as well as when under experimental procedures. Regulatory and ethical aspects are covered as are the involvement of the veterinarian in education and training of others working in the animal care and use programme. Managerial aspects, including occupational health and safety, are also areas where the veterinarian's input can assist in the successful implementation of the programme.