Impact of surgical severity and analgesic treatment on plasma corticosterone in rats during surgery.

Tissue injury and anaesthesia during surgery induce a stress response associated with increased glucocorticoid secretion from the adrenal glands. This response alters the normal physiology and may cause postoperative morbidity, as well as affect the results during acute experiments. The aim of the present investigation was to study the effect of surgical severity and analgesic treatment on circulating corticosterone in male Sprague-Dawley rats. Male rats were treated with either lidocaine infiltrated during surgery, buprenorphine (0.05 or 0.1 mg/kg subcutaneously) or saline subcutaneously. Each treatment group was subjected to either arterial catheterisation or arterial catheterisation and laparotomy. A catheter was inserted in the common carotid artery and blood was collected during surgery and during anaesthesia 6 h after surgery. Lidocaine treatment reduced the corticosterone levels compared to saline treatment after catheterisation but not after laparotomy. Buprenorphine treatment reduced the corticosterone levels during the first hour after surgery after both catheterisation and laparotomy. The higher buprenorphine dose led to an earlier and more pronounced reduction, especially after laparotomy. In the present study, the corticosterone response during surgery in laboratory rats is correlated with the severity of the procedure, and buprenorphine reduces the surgical stress response more effectively than lidocaine treatment.

Reversal of ketamine/xylazine combination anesthesia by atipamezole in olive baboons (Papio anubis).

BACKGROUND: The potential of Atipamezole (ATI) to reverse Ketamine/Xylazine (KET/XYL) anesthesia in the Olive baboon (Papio anubis) was studied. METHODS: Anesthesia was induced with 10 mg/kg KET and 0.5 mg/kg XYL intramuscularly. Mean arousal time (MAT), heart rate (HR), systolic arterial blood pressure (SAP), rectal temperature, respiratory rate (RR), and hemoglobin oxygen saturation (SpO(2)) were monitored. Baboons were treated with: KET/XYL only, KET/XYL followed by 100 microg/kg ATI or by 200 microg/kg ATI administered 25 minutes after KET/XYL. RESULTS: Atipamezole rapidly reversed depressed HR and SAP (10 +/- 5.2 minutes), RR (5 +/- 2 minutes) and SpO(2) (3 +/- 6 minutes) and significantly decreased MAT (13 +/- 2.2 minutes) vs. KET/XYL alone (35 +/- 5 minutes). Emesis was absent and salivation was observed after administration of 200 microg/kg ATI only. CONCLUSIONS: Atipamezole at 100 microg/kg is sufficient for rapid and smooth reversal of KET/XYL anesthesia in the Olive baboon with minimal side effects.

Prevalence of antibodies and cell mediated immune response against Leishmania major in feral nonhuman primates from Kenya.

In Kenya, Leishmania major is responsible for human cutaneous leishmaniasis (CL). Natural infection with L. major of a vervet monkey and experimental susceptibility of some nonhuman primates (NHPs) from Kenya has been established. However, there has been no comprehensive study of the prevalence of zoonotic CL in Kenya. And also, no investigation has been done to assess whether NHPs could be potential reservoir hosts of L. major even when the involvement of reservoir animals is obligatory in transmission of this parasite. To achieve this, wild caught Chlorocebus aethiops (Vervet monkeys n=213), Papio cynocephalus anubis (olive baboons n=101) and Cercopithecus mitis (Syke's monkeys n=64) from five geographical locations in Kenya were screened for antibodies against L. major using enzyme linked immunosorbent assay (ELISA) and Western blot (WB) analysis. From the population of C. aethiops (n=213) captured, 57 were used in lymphocyte proliferation assay. ELISA revealed a high prevalence of leishmaniasis sero conversion in olive baboons 78/101 (77.2%), vervet monkeys 129/213 (60.6%) and Sykes' monkeys 43/64 (67.2%). WB detected anti-L. major antibodies in 48.5% (49/101) of the baboons, 48% (102/213) of vervet monkeys and 37.5% (24/64) of Sykes' monkey sera. Specific proliferation of peripheral blood mononuclear cells to L. major antigen was demonstrated in 17 of the 57 (29.8%) vervet monkeys. In conclusion, the results of serological assays provide strong circumstantial evidence that CL is prevalent in five Provinces of Kenya and that Kenyan NHPs could be could be a potential reservoir hosts of L. major.

Effect of subcutaneous injection and oral voluntary ingestion of buprenorphine on post-operative serum corticosterone levels in male rats.

BACKGROUND: Adequate peri-operative analgesia may reduce post-operative stress response and improve recovery in laboratory animals. We have established a method involving repeated automated blood sampling, allowing quantification of serum corticosterone levels in rats for stress assessment without stress-inducing handling or restraint. In the present study, the effects of the commonly used route of buprenorphine administration (0.05 mg/kg injected subcutaneously) were compared with oral administration (0.4 mg/kg mixed with Nutella and orally administered by voluntary ingestion) in male Sprague-Dawley rats. METHODS: A catheter was placed in the jugular vein and attached to an Accusampler for automated blood sampling. During 96 h after surgery, blood was collected at specified time points. Pre- and post-operative body weights and water consumption were registered. RESULTS: Buprenorphine significantly suppressed levels of circulating corticosterone after the oral but not after the subcutaneous treatment. Both buprenorphine treatments had a positive impact on maintenance of body weight and water consumption, compared to the control group that received no buprenorphine. CONCLUSION: The present investigation suggests that oral voluntary ingestion ad libitum is an efficacious, convenient and non-invasive way of administering peri-operative buprenorphine to rats, as judged by corticosteroid response and effects on body weight and water consumption.

Stress in cynomolgus monkeys (Macaca fascicularis) subjected to long-distance transport and simulated transport housing conditions.

The stress associated with transportation of non-human primates used in scientific research is an important but almost unexplored part of laboratory animal husbandry. The procedures and routines concerning transport are not only important for the animals' physical health but also for their mental health as well. The transport stress in cynomolgus monkeys (Macaca fascicularis) was studied in two experiments. In Experiment 1, 25 adult female cynomolgus monkeys were divided into five groups of five animals each that received different diets during the transport phase of the experiment. All animals were transported in conventional single animal transport cages with no visual or tactile contact with conspecifics. The animals were transported by lorry for 24 h at ambient temperatures ranging between 20 degrees C and 35 degrees C. Urine produced before, during and after transport was collected and analysed for cortisol by enzyme-linked immunosorbent assay (ELISA). All monkeys exhibited a significant increase in cortisol excretion per time unit during the transport and on the first day following transport.Although anecdotal reports concerning diet during transport, including the provision of fruits and/or a tranquiliser, was thought likely to influence stress responses, these were not corrobated by the present study. In Experiment 2, behavioural data were collected from 18 cynomolgus macaques before and after transfer from group cages to either single or pair housing, and also before and after a simulated transport, in which the animals were housed in transport cages. The single housed monkeys were confined to single transport cages and the pair housed monkeys were kept in their pairs in double size cages. Both pair housed and singly housed monkeys showed clear behavioural signs of stress soon after their transfer out of their group cages.However, stress-associated behaviours were more prevalent in singly housed animals than in pair housed animals, and these behaviours persisted for a longer time after the simulated transport housing event than in the pair housed monkeys. Our data confirm that the transport of cynomolgus monkeys is stressful and suggest that it would be beneficial for the cynomolgus monkeys to be housed and transported in compatible pairs from the time they leave their group cages at the source country breeding facility until they arrive at their final laboratory destination in the country of use.

On-line strategies for determining trace levels of nitroaromatic explosives and related compounds in water.

We report the development and tests of several systems for the simultaneous determination of 18 energetic compounds and related congeners in untreated water samples. In these systems a Restricted Access Material trap or liquid-chromatography precolumn (with a C(18) or porous graphitic carbon, PGC, stationary phase) followed by a PGC analytical column are used for sample clean-up, enrichment and separation of the trace level analytes, which are then analyzed by mass spectrometry (MS). The relative merits of two MS ionization interfaces (atmospheric pressure chemical ionization, APCI, and atmospheric pressure photoionization, APPI) were also compared for the MS identification and quantification of these analytes. APCI was found to be superior in cases where both alternatives are applicable. A major drawback when applying APPI is that no signal is obtained for the cyclic nitramines and nitrate esters. Using APCI, a wide spectrum of unstable compounds can be determined in a single analysis, and the feasibility of using large volume samples (up to 100 mL) in combination with the sensitivity of the MS detection system provide method detection limits ranging from 2.5 pg/mL (for 2,4-dinitrotoluene and 2,6-diamino-6-nitrotoluene) to 563 pg/mL (for pentaerythritol tetranitrate, PETN), with repeatability ranging from 2 to 7%. Other chemometric parameters such as robustness, selectivity, repeatability, and intermediate precision were also evaluated in the validation of the extraction methods for use in water analysis. Tests with untreated groundwater and drinking water samples, spiked with 20 ng of the analytes, yielded results similar to those obtained with high purity water samples.

Cluster analysis of S100 gene expression and genes correlating to psoriasin (S100A7) expression at different stages of breast cancer development.

Gene expression patterns in ductal carcinoma in situ (DCIS) and invasive and metastatic breast tumors have been determined using serial analysis of gene expression (SAGE). The purpose of this approach was to identify biologically and clinically meaningful subgroups of DCIS with a high risk of progression to invasive disease. The analyses have led to the identification of several differentially expressed genes, such as HIN-1, dermcidin and S100A7 (psoriasin). The aim of the present study was further to delineate the expression profile of S100 genes using information from 22 breast epithelial SAGE libraries. We demonstrated the down-regulation of S100A6 and S100A10 in breast cancer, irrespective of pathological stage. S100P and S100Z were both up-regulated in cancer; whereas S100A7, S100A8 and S100A9 were strongly up-regulated only in DCIS. The hierarchical clustering of S100 gene expression in these 22 libraries revealed two major groups with distinguishable S100 gene expression profiles. One of them was characterized by the high concomitant expression of S100A7, S100A8 and S100A9. Using SAGE informatics, we found 21 genes with a high positive correlation to S100A7 expression in libraries representing different categories of tissues archived at SAGE Genie, suggesting a function of psoriasin that is not tissue specific. Like S100A7, several of these genes displayed cation-binding properties. We also report the strong correlation in the breast epithelial SAGE libraries between the expression of S100A7 and genes reported as being up-regulated in DCIS, as well as in the inflammatory skin disorder, psoriasis; including RGS5, UPK1A, TMPRSS3, S100A9, p53, SCCA1, SCCA2 and KRT17.

Supercritical fluid extraction of energetic nitroaromatic compounds and their degradation products in soil samples.

This paper explores the use of supercritical fluid extraction (SFE), in combination with various analyte collection strategies, for extracting energetic nitroaromatic compounds and their degradation products from soil samples. The required selectivity has been achieved by a combination of an SFE program and active trapping. Several different collection strategies were tested, using a selection of liquids (methanol, toluene, methyl tert-butyl ether, acetonitrile), inert and solid-phase extraction materials (Nexus, Oasis, LiChrolut), and 1-cm liquid chromatography precolumns (porous graphitic carbon, PGC). The best results were obtained using SFE in combination with a PGC precolumn. This setup allows on-line cleanup of the extract, and comparable results were obtained using either GC-ECD or GC-chemical ionization-MS for confirmatory analysis. The time required for a complete analysis was less than 60 min, and only 1 mL of toluene was needed for a 0.5-g representative sample. In contrast, the EPA standard method 8330 required 18-h sonication and 20 mL of acetonitrile for a 4.0-g sample and further time for sample cleanup and HPLC analysis. The method presented here provides method detection limits in the low-nanogram range, with relative standard deviations lower than 7%. The optimized method has been compared and validated with EPA method 8330 in terms of efficiency parameters such as robustness, accuracy (trueness and precision), and capability of detection. The validation demonstrated that the two analytical methodologies give comparable performance for the determination of nitroaromatic compounds, but SFE is superior for analyzing amine degradation products.

Surveys on attitudes towards legalisation of euthanasia: importance of question phrasing.

AIM: To explore whether the phrasing of the questions and the response alternatives would influence the answers to questions about legalisation of euthanasia. METHODS: Results were compared from two different surveys in populations with similar characteristics. The alternatives "positive", "negative", and "don't know" (first questionnaire) were replaced with an explanatory text, "no legal sanction", four types of legal sanctions, and no possibility to answer "don't know" (second questionnaire). Four undergraduate student groups (engineering, law, medicine, and nursing) answered. RESULTS: In the first questionnaire (n = 684) 43% accepted euthanasia (range 28-50%), 14% (8-33%) did not, and 43% (39-59%) answered "don't know". Two per cent of the respondents declined to answer. In comparison with previous surveys on attitudes to euthanasia the proportion of "don't know" was large. The results of the second questionnaire (n = 639), showed that 38% favoured "no legal prosecution" (26-50%). However, 62% (50-74%) opted for different kinds of legal sanctions, and two of four groups expressed significantly different views in the two surveys. A proportion of 10% declined to answer the second questionnaire. CONCLUSION: An introduction of an explanatory text and a wider range of response alternatives produced differences between the results of the two surveys conducted.

Implementation of the 'three Rs' in biomedical research.

A systematic sample of 2800 articles published between 1970 and 2000 in 14 major biomedical journals was analysed to assess the implementation of the 'Three Rs' (replacement, reduction and refinement) in biomedical research. During this period the total number of articles published annually by the journals more than doubled, but the proportion of studies using animals decreased by 30 per cent. There was a significant increase, from 21 per cent to 35 per cent, in the proportion of animal studies which made use of untreated euthanased animals as donors of biological materials, a gradual decrease in the number of chronic studies on animals, and a 50 per cent decrease in the average number of animals used per published paper. There was an improvement in the reporting of the specification of the animals' husbandry, conditions of care and environment. Parameters of importance for the evaluation of the scientific quality of the research and the welfare of the animals were generally poorly reported, but the proportion of papers with adequate information on most of the parameters analysed increased between 1970 and 2000.

Impact of chronic catheterization and automated blood sampling (Accusampler) on serum corticosterone and fecal immunoreactive corticosterone metabolites and immunoglobulin A in male rats.

Jugular catheters were inserted in nine male rats under general isofluorane anesthesia and the catheters were connected to a commercially available computerized blood sampling device (Accusampler). Blood samples (150 microl) were collected every 4 h during the first 24 h after surgery and every 12 h during the following 72 h until 94 h after surgery, when the animals were killed. All fecal pellets were collected at blood sampling. Serum corticosterone and fecal concentrations of immunoreactive corticosterone metabolites and immunoglobulin A (IgA) were quantified by ELISAs. In blood, high corticosterone concentrations (>200 ng/ml) were recorded in the first samples obtained after surgery, but the concentrations decreased steadily during the day and became cyclical, showing a diurnal variation with high levels during evenings and low levels in the mornings. The automatic blood sampling itself did not result in recordable increases in serum corticosterone concentrations. The time delay between the presence of elevated corticosterone levels in blood and in feces was approximately 12 h. Fecal immunoreactive corticosterone metabolite levels remained elevated during the 94 h study period after surgery. The fecal concentrations of IgA showed substantial between-animal variation and decreased non-significantly after the surgery. Like serum corticosterone, fecal IgA showed a diurnal variation in amounts excreted, in this case with high values in the morning and low values in the evening. The concentrations of fecal corticosterone and IgA were negatively correlated in samples obtained before surgery but no correlation existed after surgery. This indicates that fecal immunoreactive corticosterone metabolites, but not IgA, constitute a good marker of acute stress. For immunoreactive corticosterone metabolites as well as for IgA, the concentration in feces correlated well with total excretion, making single fecal samplings usable as a measure of total secretion.

Determination of nitroaromatic compounds in air samples at femtogram level using C18 membrane sampling and on-line extraction with LC-MS.

This paper explores the use of C18 solid-phase extraction membranes for sampling very low concentrations of nitroaromatic compounds in the atmosphere. After sampling, analytes trapped in the membrane are desorbed on-line directly by a chromatographic mobile phase. The analytes are then separated onto a porous graphitic carbon (PGC) HPLC column. Finally, they are analyzed by an LC-MS/MS detector equipped with an atmospheric pressure chemical ionization (APCI) interface. The method was validated by controlled exposure of the membranes to standard gaseous mixtures of 2,4,6-trinitrotoluene (TNT) and 2,4-dinitrotoluene (2,4-DNT). The developed method was fully characterized, and no breakthrough was observed when sampling volumes up to 9.2 m3. Analyses of membranes following medium- and long-term storage demonstrated that samples could be stored on the C18 membranes without degradation or losses. In addition, the results obtained with this technique were compared with those obtained by a gas chromatographic method in which analytes were collected on Tenax TA and thermally desorbed. The developed method allows sampling at flow rates of 15 L/min and has method detection limits in the femtogram/liter range, with a relative standard deviation lower than 10%. An additional advantage of this method is that it separates most of the TNT and DNT isomers, as demonstrated by applying the method to the analysis of headspace over military-grade TNT explosives.

Fetal antigen 1 (FA1) in the adult rat adrenal gland, ovary and pituitary gland.

Fetal antigen 1 (FA1) is a circulating glycoprotein containing six epidermal growth factor (EGF)-like repeats. FA1's larger membrane-bound precursor is defined by the cDNAs referred to as either human delta-like (dlk) or human adrenal specific cDNA, pG2. In rodents FA1 has also been studied under the names of preadipocyte factor 1 (Pref-1), and zona glomerulosa-specific factor (ZOG). FA1 is abundantly expressed in fetal tissues, but in the mature cells of the adult organism the tissue presence of the protein seems to be restricted to neuroendocrine tissues. The present study demonstrates FA1 localisation in endocrine tissues of the adult female rat in which the protein was found present in the medulla and the zona glomerulosa of the cortex of the adrenal glands, in the pars distalis of the adenohypophysis, and in the ovarian granulosa lutein cells. No staining was found in the pancreas, which is in contrast to what has been described in the human.

Religious beliefs and opinions on clinical xenotransplantation--a survey of university students from Kenya, Sweden and Texas.

This study investigated the association between religious beliefs and opinions on xenotransplantation among students from three different countries. A lower proportion of religious students accepted xenotransplantation than did non-religious students. A higher proportion of Protestant students seemed to accept xenotransplantation than did Muslim and Roman Catholic students. A higher proportion of the religious respondents had not formed an opinion on xenotransplantation compared to non-religious students. There was no difference according to gender on views on xenotransplantation, but a higher proportion of older students seemed to accept xenotransplantation than did younger students. A higher proportion of non-vegetarians reported acceptance of xenotransplantation than did vegetarians. Acceptance of xenotransplantation was higher in Sweden compared to the two other regions, and the proportion of students who had formed an opinion was higher as well.

Development and validation of a sensitive ELISA for quantification of secretory IgA in rat saliva and faeces.

Non-invasive measures of immunological markers are an attractive means of stress assessment in laboratory animals. Salivary IgA has been used successfully as a stress marker in the human, and several reports indicate the potential of secretory IgA as a non-invasive measure of stress in animals. The present paper describes the development of an ELISA using commercially available components for the quantification of rat IgA and validation of this assay for the quantification of rat secretory IgA in saliva and faeces. The concentration of IgA in rat saliva varied significantly between duplicate samples obtained from individual rats, and the viscosity and small total volume of rat saliva gave unsatisfactory results for IgA. Faecal IgA was present in high concentrations, and duplicate samples varied by only 2-3%. However, faecal IgA seemed less stable than IgA in other biological compartments, and this finding must be taken into consideration when using quantitative measurements of IgA as a marker of mucous humoral immune status.

Flame retardants in indoor air at an electronics recycling plant and at other work environments.

Air samples from a plant engaged in recycling electronics goods, a factory assembling printed circuit boards, a computer repair facility, offices equipped with computers, and outdoor air have been analyzed with respect to their content of brominated hydrocarbon and phosphate ester flame retardants. Polybrominated diphenyl ethers, polybrominated biphenyls, 1,2-bis(2,4,6-tribromophenoxy)-ethane, tetrabromobisphenol A, and organophosphate esters were all detected in the indoor air samples, with the highest concentrations being detected in air from the recycling plant. In air from the dismantling hall at the recycling plant the average concentrations of decabromodiphenyl ether, tetrabromobisphenol A, and triphenyl phosphate were 38, 55, and 58 pmol/m3, respectively. Significantly higher levels of all of these additives were present in air in the vicinity of the shredder at the dismantling plant. This is the first time that 1,2-bis(2,4,6-tribromophenoxy)-ethane and several arylated phosphate esters are reported to be contaminants of air in occupational settings. At all of the other sites investigated, low levels of flame retardants were detected in the indoor air. Flame retardants associated with airborne particles, present at elevated levels, pose a potential health hazard to the exposed workers.

Undergraduate and postgraduate students' responses to mandatory courses (FELASA category C) in laboratory animal science.

The results from the analysis of the course evaluations from FELASA category C compulsory courses in laboratory animal science at Uppsala University showed that the students realize that theoretical knowledge of the subject and practical skills are of great importance to the success of their future research involving animal experiments. All the subjects and elements of the course, in particular the practicals on animal handling and procedures using live anaesthetized animals, were fully appreciated by the students.

Response mechanisms of thermionic detectors with enhanced nitrogen selectivity.

The response mechanisms of a thermionic detector with enhanced nitrogen selectivity operating in an inert gas environment were investigated. According to accepted theory, the analyte has to contain electronegative functional groups in order for negative ions to be formed by the extraction of electrons from the thermionic source. This leads to a selective detector response for compounds containing nitro groups or multiple halogens. However, in the tests described here, polycyclic aromatic nitrogen hydrocarbons (PANHs), acridines, and carbazoles were used as reference substances. These compounds contain no electronegative functional groups. None of the investigated acridines exhibited any response from the detector, but carbazoles generated a strong structure-related detector response. By examining partial charges for all hydrogens of all individual carbazoles and acridine, it was demonstrated that the acidic hydrogen atom attached to the nitrogen heteroatom of the carbazoles has a strong influence on the detector response. Ionization of carbazoles may occur by dissociation of the nitrogen-hydrogen bond during contact with the thermionic surface. Support for this theory was provided by the linear relationship between the relative detector response and the deprotonization energy of the carbazoles (coefficients of determination of 0.90 and 0.98 for linear and quadratic models, respectively, were obtained). Further, there appeared to be no linear relationship between the detector response and electron affinity of the carbazoles, (R2 value, 0.32). Thus, the mechanism involved in ionization of the carbazoles is probably not direct electron transfer from the thermionic surface to the carbazoles. Principal component analysis (PCA) showed that the thermal conductivity of chemically inert detector gases also has an influence on the detector response. The investigated gases were helium, neon, nitrogen, carbon dioxide, and argon. It was found that thermal conductivity can be used to rank the detector response for the carbazoles, and there was no discernible response when helium, which has the highest thermal conductivity, was used as the detector gas.

Attitude of Swedish veterinary and medical students to animal experimentation.

Nearly all veterinary and medical students (94 per cent) found it morally acceptable to use animals in research and believed it to be a necessity in order to treat human diseases. In contrast with the medical students a substantial proportion of veterinary students (40 per cent) considered themselves animal rights activists. Unlike the medical curriculum, the veterinary curriculum contains a two-week course in laboratory animal medicine, and a higher proportion of the students who had not been through this course was opposed to the use of animals in research than of the students who had completed the course. The course modified the views of half the students; more than 26 per cent of them became more positive towards animal use in research after the course, whereas 3 per cent became more negative.