Laboratory Animals current issue

The impact of light, noise, cage cleaning and in-house transport on welfare and stress of laboratory rats

Castelhano-Carlos, M J, Baumans, V — 2009-09-18

Human interaction and physical environmental factors are part of the stimuli presented to laboratory animals everyday, influencing their behaviour and physiology and contributing to their welfare. Certain environmental conditions and routine procedures in the animal facility might induce stress responses and when the animal is unable to maintain its homeostasis in the presence of a particular stressor, the animal's wellbeing is threatened. This review article summarizes several published studies on the impact of environmental factors such as light, noise, cage cleaning and in-house transport on welfare and stress of laboratory rats. The behaviour and physiological responses of laboratory rats to different environmental housing conditions and routine procedures are reviewed. Recommendations on the welfare of laboratory rats and refinements in experimental design are discussed and how these can influence and improve the quality of scientific data.

Preclinical testing on insects predicts human haematotoxic potentials

Berger, J. — 2009-09-18

The substitution of insects for laboratory animals in toxicity testing is likely to become a reality in the framework of prescreening. Haematotoxicological studies of newly developed chemicals, such as food components, drugs, etc. performed on insects can offer advantages in, for example, environmental toxicology. Reliable routine predictions should produce an increase in our knowledge of haemocyte physiology. Although the differences between human physiology and morphology and those of insects are great, the basic functions of insect haemocytes and mammalian leukocytes appear not to have changed during evolution. The use of insects in haematotoxicity assays represents a preclinical testing strategy which will lower costs, accelerate screening and offer ethical benefits.

Vascular ultrasound studies for the non-invasive assessment of vascular flow and patency in experimental surgery in the pig

2009-09-18

Vascular ultrasound is a reliable non-invasive tool used for the routine assessment of vascular flow and patency in human recipients. We describe the use at three different time points (immediately, 1 week and 4 weeks postsurgery) of ultrasound studies and its validation by angiographic studies in 37 swine undergoing carotid graft replacement. We calculated predictive values (>92%), sensitivity (>85%) and specificity (>92%) with high results at all time points. Ultrasound appeared as an accessible non-invasive technique, providing rapid, safe, repeatable and reliable results. It is an excellent alternative to angiography, avoiding risks inherent to invasive methods and therefore contributing to animal welfare.

Development of perioperative care for pigs undergoing laryngeal transplantation: a case series

Murison, P J, Jones, A, Mitchard, L, Burt, R, Birchall, M A — 2009-09-18

Pigs are ideal animal models for airway surgical research, facilitating the successful translation of science into clinical practice. Despite their ubiquitous use, there is a paucity of information on the perioperative care of pigs, especially for major procedures. In a series of experiments to investigate laryngeal transplantation, we combined veterinary and medical experience to develop protocols for perioperative management of pigs, including high dependency care. Novel airway management methods were developed. A pain scoring system was used to direct analgesia use. Fluid balance and electrolytes were monitored closely. Recent animals received a central venous line via the femoral vein two days prior to transplantation to facilitate blood sampling and drug delivery. Intensive monitoring and airway management were required to ensure a successful outcome. Methods for optimal perioperative care are proposed. These results will help future groups wishing to use pigs in airway research, will reduce numbers of animals used and improve animal welfare.

Percutaneous external jugular vein catheterization in piglets using a triangulation technique

Flournoy, W S, Mani, S — 2009-09-18

Chronic jugular vein or central venous cannulation is routinely performed in human and animal patients for access to blood circulation. In mature swine, chronic catheter placement techniques have typically involved venous isolation via extensive cut-down, blunt dissection and manipulation of ventral neck tissues prior to catheter placement. More recently, guide-wire-assisted percutaneous techniques have become standard practice in human and veterinary medicine due to the minimization of soft tissue and vessel damages. Laboratory animal piglets are becoming more popular research models because of their immature immunological system, ease of handling and costs. However, external jugular veins are very difficult to catheterize in paediatric animals including freshly weaned piglets. The objective of this study was to develop a simple, safe and efficient method for external jugular vein cannulation in young piglets. In total, 20 piglets were anaesthetized and percutaneously catheterized with a guide-wire technique using palpable anatomical landmarks and triangulation. With this minimally invasive catheterization, it has allowed our veterinarians and veterinary technicians to quickly and easily obtain central venous access in piglets undergoing operative procedures.

Characterization of a rodent model for the study of arterial microanastomoses with size discrepancy (small-to-large)

Rickard, R F, Wilson, J, Hudson, D A — 2009-09-18

Microsurgical autotransplantation of tissues is employed clinically to reconstruct defects following burns, trauma and surgical cancer ablation, and to correct congenital abnormalities. Transplant vessels of <3 mm are anastomosed by hand under the microscope. Experimentally, anastomotic patency rates decrease with increasing vessel diameter mismatch, and clinically, ratios of 3:1 or greater lead to unacceptably low arterial patency rates. A number of surgical techniques for dealing with size mismatch are described, but no one method has found favour, and few controlled studies of technique are reported. In this report, a rodent superficial caudal epigastric artery (SCEA)/femoral artery (FA) model for the study of these techniques is described in detail. The diameter ratio between these vessels lies in the clinically relevant range of 1:1.5–1:2.5. In the male Wistar rat, external vessel diameters were not found to increase markedly in size between animal weights of 300 and 500 g. The length of FA distal to the origin of the SCEA, which is important in allowing undisturbed distal run-off, was found to be negatively associated with animal body weight, implying that a smaller animal would be better for this model. Mean femoral arterial flow rate, measured by transit-time ultrasound, was noted to be statistically and physiologically significantly higher in the right artery when compared with the left. This model has advantages over interposition vein graft models in that it minimizes vessel compliance mismatch, and avoids the need for a second anastomosis.

The analgesic effects of oral paracetamol in two strains of mice undergoing vasectomy

Dickinson, A. L, Leach, M. C, Flecknell, P. A — 2009-09-18

As the production of transgenic mice increases, the need for vasectomized mice also increases. Currently, there is no accurate method of identifying pain in these mice which can be used routinely and therefore no acceptable analgesic regimens can be established. Sixteen male CBA/CaCrl and 16 male DBA/2JCrl mice were randomly allocated to one of four treatment groups (saline, low, medium and high dose oral paracetamol) and then underwent abdominal vasectomy. Their behaviour was videotaped (filmed) preoperatively and at one hour postoperatively and the data were analysed using an automated system – HomeCageScan. HomeCageScan detected significant changes in 16 behaviours following vasectomy. Such behaviours included twitching, rearing and grooming with varying levels of significance between the strains. No significant effects of drug treatment in any of the behaviours analysed by HomeCageScan were detected in the postoperative observation period. Strain-specific changes do occur in behaviour following abdominal vasectomy and HomeCageScan is capable of detecting these changes. The use of orally administered paracetamol was deemed to be an ineffective analgesic for CBA/CaCrl and DBA/2JCrl mice undergoing vasectomy.

Charles River altered Schaedler flora (CRASF(R)) remained stable for four years in a mouse colony housed in individually ventilated cages

2009-09-18

As recommendations for specific pathogen-free housing change, mouse facilities need to re-derive their colonies repeatedly in order to eliminate specified bacteria or viruses. This paper describes the establishment of a new mouse facility using as starting point a small colony of CD-1 mice colonized with the Charles River altered Schaedler flora (CRASF^®) housed in individually ventilated cages (IVCs). The import of new strains was performed exclusively via embryo transfer using CD-1 mice as recipients. The integrity of the CRASF^® in caecum samples of the original CD-1 colony and of three inbred mouse lines imported into the colony was proven by a quantitative realtime polymerase chain reaction approach. Furthermore, we searched for bacterial contaminants in the gut flora using non-specific 16S rRNA primers. The bacterial sequences found were closely related to but not exclusively sequences of altered Schaedler flora (ASF) members, suggesting that the ASF is heterogeneous rather than restricted to the eight defined bacteria. Moreover, no pathogens were found, neither using the non-specific 16S rRNA primers nor in routine quarterly health monitoring. As one effect of this defined gut flora, interleukin-10 knockout mice are devoid of colitis in our facility. In conclusion, our approach building up a mouse facility using foster mothers and embryo transfer as well as a strict barrier system and IVCs is suitable to maintain a colony free from contaminating bacteria over the long term. CRASF^® remained stable for seven mouse generations and was efficiently transferred to the imported mouse strains.

Comparison of polymerase chain reaction primer sets for amplification of rodent Pasteurellaceae

Boot, R, Vlemminx, M J, Reubsaet, F A G — 2009-09-18

Monitoring of rodents for Pasteurellaceae infection may be carried out by the polymerase chain reaction (PCR). We tested which of 17 rodent Pasteurellaceae strains were detected by three PCR primer sets. By phylogenetic analysis, 12 strains were assigned to the Rodent cluster and five strains to other clusters, namely the Somnus cluster, Pasteurella sensu stricto, Actinobacillus sensu stricto, the Mannheimia and Rossii cluster. A primer set developed to detect biotype Heyl [Pasteurella] pneumotropica produced amplicons from three strains and appeared specific for this taxon. A primer set developed to detect biotype Jawetz [P.] pneumotropica produced amplicons from the [P.] pneumotropica type strain and two other strains within the Rodent cluster. A primer set as described by Bootz and his co-workers (Bootz F, Kirschnek S, Nicklas W, Wyss SK, Homberger FR. Detection of Pasteurellaceae in rodents by polymerase chain reaction analysis. Lab Anim Sci 1998;48:542–6) for the detection of all Pasteurellaceae indeed detected all bacterial strains examined. Bootz's primer set should be used to monitor rodents for Pasteurellaceae infection by PCR as FELASA recommends the monitoring of rodents for all Pasteurellaceae taxa. Health monitoring reports should specify the primer set(s) used for PCR testing rodents for Pasteurellaceae infection.

Morphological study on dental caries induced in WBN/KobSlc rats (Rattus norvegicus) fed a standard laboratory diet

2009-09-18

In our previous studies, WBN/KobSlc was characterized as a rat strain in which only males began to develop pancreatitis, and then presented with diabetic symptoms. In the course of studying their pancreatic inflammation, we detected molar caries in prediabetic males feeding on a standard diet (CRF-1) widely used for experimental animals. The purpose of this study is to confirm whether the WBN/KobSlc strain is caries-susceptible to the diet reported to be non-cariogenic, and to examine the effect of a prediabetic condition on their dental caries. For a morphological study, 25 male WBN/KobSlc rats aged 3.2–7.8 months and 24 females of the same strain aged 3.3–6.6 months were used, along with 10 males and 10 females of 8.2-month-old F344 rats. Marked dental caries were detected in the mandibular molars of male and female WBN/KobSlc rats regardless of pancreatitis, although no similar changes were observed in any teeth of the F344 strain fed the same diet. Soft X-ray examination revealed that the caries began in the crown and progressed horizontally and vertically, and that a severe radiolucent lesion extensively expanded to the entire crown, corresponding to a macroscopically deleted molar. The caries had gradually developed mainly in the second mandibular molar from more than 3.5 months of age, while none were seen in any rats before that time. The WBN/KobSlc rats were caries-susceptible even to the standard laboratory diet, and pancreatitis was not directly associated with the onset of dental caries in this strain.

Design and evaluation of a low-cost respiratory monitoring device for use with anaesthetized animals

Devonshire, I M, Preston, M J, Dommett, E J, Murphy, K L, Greenfield, S A — 2009-09-18

This report describes a simple, non-invasive electronic device that employs a compact accelerometer integrated circuit to transduce movements in the chest wall of an anaesthetized animal into an analogue signal that can be used to calculate the rate and relative depth of respiration. The device requires amplification by signal processing hardware/software which are common to most experimental laboratories. We assessed the sensitivity of the device by its ability to detect changes in respiratory patterns produced by modulating the depth of anaesthesia in isoflurane-anaesthetized Wistar rats. It is widely accepted that many anaesthetic agents affect respiratory patterns, especially respiratory rate (RR), which is often used as an important index of anaesthetic depth. Respiratory parameters obtained with the device were compared with concurrently recorded electroencephalographic and cardiac measures. Different concentrations of anaesthetic agent produced four depths of anaesthesia, identified using established electroencephalographic criteria. The accelerometer was attached easily and securely to the location of maximal chest wall movement and produced a strong respiratory signal that was detectable in all four anaesthetic stages. Deepening the anaesthesia produced a gradual decrease in RR, a decrease in dominant spectral frequency of the electroencephalogram (EEG) but no change in the heart rate. There was a significant correlation between RR and the dominant spectral frequency of the EEG, indicating that one useful application of the monitor could be to identify anaesthetic stages. The results demonstrate that respiratory parameters can be recorded using a simply constructed, low-cost device and suggest an application in the monitoring of anaesthetic depth.

DNA analysis from stool samples: a fast and reliable method avoiding invasive sampling methods in mouse models of bleeding disorders

Kalippke, K., Werwitzke, S., von Hornung, M., Mischke, R., Ganser, A., Tiede, A. — 2009-09-18

Mouse models with targeted disruptions of coagulation factor genes are used to study disorders of haemostasis such as haemophilia. Standard protocols to obtain biopsies for genotyping in breeding programmes are based on invasive sampling methods such as tail clipping. These procedures imply a high risk of fatal bleeding, especially in haemophilic mouse models. Here we used a non-invasive sampling method obtaining stool samples for DNA isolation in a breeding programme, aiming to introduce targeted disruptions of Fc receptor genes (Fc gamma receptor IIB and III) into the haemophilia A mouse model (factor VIII deficiency). Faecal pellets were reliably obtained from individual mice and high-quality DNA was extracted with a mean yield of 7.1 µg/pellet. Polymerase chain reaction amplification of wild-type and knockout alleles for Fc receptor and factor VIII genes was similar, comparing stool and peripheral blood as the source of genomic DNA. Definite genotype identification was achieved in a first attempt in 336 of 352 analyses (95%). Repeated analysis of homozygous knockout animals confirmed the first result in all cases. No animal was lost due to bleeding from the procedure. In conclusion, DNA isolation from stool is a preferable method for genotyping in laboratory animals, especially in models of bleeding disorders. Avoiding loss of animals due to bleeding implies a substantial improvement in animal welfare by reducing the number of animals used and may also advance the effectiveness of breeding programmes in these disease models.

Modified PAXgeneTM method allows for isolation of high-integrity total RNA from microlitre volumes of mouse whole blood

Krawiec, J A, Chen, H, Alom-Ruiz, S, Jaye, M — 2009-09-18

Analysis of gene expression is often used to evaluate the effects of experimental manipulations in laboratory animals. Blood is a rich source of potential biomarkers, including gene expression information, which may be obtained from whole blood. When compared with the end of a study, when whole blood samples can be easily obtained for gene expression measurements, the limiting volumes of whole blood obtainable from animals during the course of an experiment requires a method for RNA isolation from a minimal volume of whole blood. The PAXgene^TM Blood RNA Extraction System originally designed for isolation of total RNA from 2.5 mL of human whole blood, was modified and successfully used to isolate high-integrity total RNA from as little as 50 µL of mouse whole blood. Fifty microlitres of mouse whole blood yielded an average of 2.3 µg highly intact total RNA, of sufficient quality and quantity allowing for multiple gene expression determinations. The utility of this method was demonstrated by confirming the time- and dose-dependent upregulation of haem oxygenase-1 (Hmox1) mRNA in response to a single injection of cobalt protoporphyrin. The successful isolation of total RNA from small volumes of mouse whole blood can allow for serial sampling on the same animals, thereby reducing the number of animals required for experimentation.

A simple method for confirming correct endotracheal intubation in mice

Watanabe, A, Hashimoto, Y, Ochiai, E, Sato, A, Kamei, K — 2009-09-18

Endotracheal intubation in mice is both a common and important technique. However, it is a difficult procedure because of the small orotracheal size and the success rate is variable. There have been many reports of refined techniques that facilitate intubation but only a few reports have proposed how to verify the proper placement of the endotracheal tube. We describe a novel, safe and reliable method to confirm endotracheal intubation in mice using an extension tube for intravenous infusion. One drop of water was instilled in the extension tube and connected to the end of the catheter used as an endotracheal tube. When the catheter was inserted correctly into the trachea, the water in the extension tube oscillated in synchrony with the movement of the mouse's thorax, indicating correct placement of the catheter. This method was simple, reliable and use materials that are routinely available. This method is helpful for experimental mouse models that require airway access.

Spontaneous thymic lymphomas in the non-obese diabetic/Shi-scid, IL-2R{gamma}null mouse

2009-09-18

The NOD/Shi-scid, IL-2R^null (NOG) mouse is a severely immunodeficient mouse used for the engraftment of human tissues and cells. In this study, 2406 mice (8–62 weeks old, 503 males and 1903 females) were subcutaneously engrafted with human tissues. In 16 mice (12–26 weeks old, 1 male and 15 females), a mass was seen in the anteroventralis of the thorax on gross examination with an incidence of 0.7%. Histologically, the masses were composed of sheets of lymphoblastic cells. A ‘starry sky’ pattern was observed with numerous mitoses. Immunohistochemically the lymphoblastic cells were positive for Thy 1. The lymphoblastic cells were also seen in the spleen, lung, liver, kidney and heart. The gross and histopathological findings led to the diagnosis of spontaneous thymic lymphoma in NOG mice.

Laboratory Animal Science and Technology (Vol. I/II)

Wilkinson, M. — 2009-09-18