2012 - Volume 39 - Issue 1


Modified Tapvei OÜ Stairs Induce an Anxiolytic Effect in Female C57BL/6 Mice in the Elevated Plus-Maze Test
by K. Őkva, T. Nevalainen, K. Mauranen & P. Pokk

We evaluated the effect of modified Tapvei OÜ stairs (stairs) on the behaviour of female C57BL/6 and BALB/c mice in the elevated plus-maze (EPM) test. The mice were kept under standard conditions for 4 weeks (control) or exposed to stairs for 3 or 4 weeks, and were assessed thereafter with the EPM. The C57BL/6 mice displayed less anxiety, when compared with the BALB/c mice. Exposure to stairs had an anxiolytic effect in C57BL/6 mice, but not in BALB/c. The strain-dependent effects of stairs should be considered in the design of housing refinements and behavioural experiments.

The Suspended Formalin Test: A Method Designed for Studying Formalin-Induced Behaviour in the Speke’s Hingeback Tortoise (Kinixys spekii)
by Joakim Dahlin, Titus I. Kanui, Stanley N. Wambugu & Klas S.P. Abelson,

The present study aimed to develop a method for testing pain-related behaviour induced by formalin in the Speke’s hingeback tortoise (Kinixys spekii). These animals retract their head and limbs into their shell when approached, making behavioural testing almost impossible. It was found that suspending the animals in the air, facing away from the observer, made the animals keep their limbs out of the shell. Subcutaneous injection of formalin induced easily identifiable and quantifiable behaviours that lasted for 20 minutes. Contrary to the biphasic effect of formalin observed in rats and mice, the response in tortoises was monophasic. The suspended formalin test may be useful for studying nociceptive mechanisms in tortoises, which in turn will be important for a further understanding of the nociceptive system in reptiles as well as in mammals.

Effective Decontamination of Laboratory Animal Rooms with Vapour-phase (“Vaporized”) Hydrogen Peroxide and Peracetic Acid
by Tohru Kimura

The purpose of this study was to investigate the decontaminant effects of vapour-phase hydrogen peroxide(VHP) and peracetic acid (PAA) in laboratory animal rooms. Methodologically and microbiologically, both methods were evaluated as an alternative to traditional methods. In the VHP decontamination process, the cycle consisted of 4 phases (dehumidification, conditioning, decontamination and aeration). The residual vapour was catalytically decomposed into water and oxygen. The complete process of room decontamination with VHP took 14-15 hours.
In PAA decontamination, the dry fog system produced very fine droplets of disinfectant that were dispersed throughout the laboratory animal rooms. The overall decontamination process by PAA mist took 3 hours, which was much faster than VHP decontamination. In both methods, no corrosion appeared on the material surfaces. The results of chemical and biological indicators showed complete decontamination after exposure to VHP and PAA fumigation. In the airborne microbiological examinations, total colony counts for the surface and environmental microorganisms were minimal. There were no significant differences between VHP and PAA fumigation. In laboratory facilities, conventional decontaminating methods will be gradually converted into VHP or PAA decontamination. This experiment confirmed that the VHP method is suitable for the decontamination of a relatively limited space whereas the PAA method is applicable to the rapid decontamination of spacious laboratory animal rooms. In conclusion, decontamination with VHP and PAA holds great promise as an effective alternative to currently used formaldehyde fumigation.

PET Neuroimaging in Pigs
by Aage Kristian Olsen Alstrup, & Donald F. Smith

Current interest in studying molecular processes as they occur in the living brain has accelerated the use of laboratory animals for neuroimaging of novel radiolabelled compounds. In particular, positron emission tomography (PET) has contributed to the development of radiolabelled compounds for assessing molecular processes in the living brain. The dynamics of PET typically require a relatively large organ size and blood supply in order to properly evaluate radioligand binding kinetics. To fulfil these requirements, pigs have often been used in such studies. Today, much is known about the metabolism, neurotransmission and molecular binding properties of the living porcine brain, and most findings support similarities between neuronal mechanisms in pigs and humans. Here, we review 10-years of PET findings on neuromolecular processes in the living porcine brain and, whenever possible, we relate PET findings in pigs to those obtained in humans.

Cardiovascular and Haemostatic Changes in a Rabbit Microsphere Model of Pulmonary Thrombosis
by Line Olrik Berthelsen, Henrik Duelund Pedersen, Annemarie Thuri Kristensen & Mikael Tranholm.

In order to further understand the pathogenesis of disseminated intravascular coagulation (DIC), to diagnosethe condition in the early stage and to provide effective therapeutic intervention, valid and clinical relevant animal models are needed. Microvascular pulmonary thrombosis is one of the hallmarks in DIC. In the search for markers of microvascular pulmonary thrombosis in DIC, the present study evaluated local pulmonary effects of microvascular pulmonary thrombosis caused by polystyrene microspheres, generally assumed to be inert. Simultaneously the haemostatic system was evaluated to investigate whether microspheres are truly inert in a rabbit model of microvascular pulmonary thrombosis. Cardiovascular and haematological parameters were measured at three time points over a 30 minute period from rabbits administered 45 µm microspheres as a single bolus dose of 0.3, 6, 16 or 32 µl microspheres/ml blood. Injection of 0.3 µl microspheres/ml blood resulted in no changes. Injection of 6 µl microspheres/ml blood resulted in a minor increase in cardiovascular parameters whereas injection of 16 µl microspheres/ml blood resulted in significant changes of cardiovascular parameters without impact on haemostatic parameters. A lethal effect was shown after injection of 32 µl microspheres/ml blood. With i.v. injection of microspheres we succeeded in establishing an inert mechanical model of fixed size microvascular emboli in the lung vasculature of the rabbit. We showed that selected cardiovascular parameters rapidly and dose dependently reflect obstructions in the pulmonary vasculature and therefore could be very valuable and reliable parameters in experimental models of disseminated intravascular coagulation for early detection of microvascular pulmonary thrombosis.

Rare Spontaneous Proliferating Tumour in Rat: Fibrosarcoma
by Madhav Mugale, & Vikash Singh,

A 1 year old, male Wistar rat presented to clinic having a subcutaneous greyish-white mass extending to the supraorbital area of the left eye. The stained impression-smear in the microscope small field showed bunches of round cells in diffused masses, which were present in various stages of cell division. Large numbers of cells were present in the mitosis phase. Large populations of neoplastic spindle cells, rare pleomorphic multinucleated cells, and rare leukocytes were observed. The primary tumour was characterized by fusiform spindle cells producing various amounts of interlacing bundles of collagen. The spindleshaped cells contained moderate amounts of eosinophilic, fibrillar cytoplasm with an oval nucleus, coarsely clumped chromatin, and one or more nucleoli. Some of the round cells were present in the nucleolar remoulding stage and the presence of immature proliferating cells had previously been identified as spindle cell fibrosarcomas. During progression, the tumour acquires self-dependence and the ability to invade other tissues and metastasize. The primary tumour was characterized by fusiform spindle cells producing various amounts of interlacing bundles of collagen. The cells formed a characteristic herringbone pattern and mitotic figures were frequent. The relationship to human fibrosarcoma is noted.

Spontaneous Congenital Hydrocephalus in Sprague Dawley Rat
by Mohmad Sadik A. Mulla, Vinod Kumar Goyal, Santanu Jana & Ramakrishna Nirogi

Hydrocephalus is a neurological disorder that results from the accumulation of excess cerebrospinal fluid in the ventricles of the brain culminating in an enlarged cranium. This sporadic disorder may occur as a congenital malformation in many mammalian species including inbred rodent colonies at an early juvenile stage of life. Under conventional husbandry practice of breeding, 5 pups showing some clinical signs of neurological dysfunction at the age of 20 days were examined thoroughly. Detailed macroscopic examination demonstrated dome shaped head, thinned and deformed parietal bone, open/closed suture depending upon the severity of ventricular dilatation. Microscopic examination revealed dilated lateral ventricles, compressed and attenuated cortical mantle, spongy appearance of the sub-ventricular zone, stretched ventricular ependyma, flattened ependymal cell lining and infiltration of mononuclear cells in the ventricular lining.

Successful Treatment for Idiopathic Thrombocytopenic Purpura in a Japanese Monkey
by Tohru Kimura

In human beings and dogs, idiopathic thrombocytopenic purpura (ITP) is a well-known disease in which antibodies bound to the surface platelets result in premature platelet destruction by macrophages. However, there is a paucity of information dealing with ITP in non-human primates, especially the Japanese monkey (Macaca fuscata). The case is described of a female Japanese monkey suffering from ITP in the Center for Experimental Animals. Physical examinations revealed characteristic findings such as mucosal and cutaneous petechiae, ecchymoses and purpura, epistaxis and mucous membrane pallor. The monkey had severe thrombocytopenia (10,000/µl) on initial hematological examination. Immunosuppressive glucocorticoid therapy had remarkable effects on this condition, with the platelet count rapidly reaching the normal range. On tapering the dose of predonisolone, the number of platelets decreased and the monkey suffered a relapse of ITP. Although immunosuppressive therapy was resumed with the initial dose of predonisolone, the monkey was relatively slow to respond. The initial treatment revealed an apparently faster increase in platelet count than the second treatment following the recurrence of ITP. The monkey remained in complete remission for more than one year after cessation of predonisolone treatment. In blood coagulation profiles and serum biochemical findings, there were no marked changes throughout this investigation. Neither endoscopy nor the stool antigen test provided evidence that natural infection with Helicobacter pylori caused ITP in this monkey. This is the first case of successful treatment for idiopathic thrombocytopenic purpura in a Japanese monkey.

Dose Optimization for Using the Contrast Agent Gadofosveset in Magnetic Resonance Imaging (MRI) of Domestic Pig Brain
by Erik S. Poulsen, Aage K.O. Alstrup, Dora G. Zeidler, Niels Hjort & Leif Østergaard

Pigs are useful models in stroke research, and Magnetic Resonance Imaging (MRI) is a useful tool for measurements of brain pathophysiology. Perfusion Weighed Imaging (PWI) with standard Gd-based chelates (i.e. gadobutrol) provides crucial information about breakdown of the Blood-Brain-Barrier (BBB) in patients. Gadofosveset is also a Gd-based contrast agent, but with a higher binding to serum albumin. The prolonged plasma-half life of gadofosveset allows the acquisition of steady state angiographies, which may increase the sensitivity for detection of BBB leakage. We hypothesize that the contrast dosage with gadofosveset can be optimized for PWI and subsequent steady-state Magnetic Resonance Angiography (MRA) in pigs. Anesthetized domestic pigs (females; N=6) were MRI scanned four times in one day: they were initially imaged during a standard gadobutrol bolus injection (0.1 mmol/kg). Then they received three successive gadofosveset bolus injections of varying dosages (0.015-0.09 mmol/kg). Based on projection from our data,we suggest that a bolus injection of 0.0916 mmol/kg gadofosveset would yield contrast similar to that of a standard dose of 0.1 mmol/kg gadobutrol in dynamic susceptibility contrast MRI at 3 T. In conclusion, our results demonstrate the feasibility of gadofosveset based PWI in pig brain research. The relaxation and plasma half-life properties allow detailed steady-state MRA angiographies and may prove useful in detecting subtle BBB disruption of significance in stroke models and human patients.

Maintenance of Seasonal Differences in Reproductive Characteristics of Bank Voles After 30 Years Captive Breeding
by Kapusta J, Kruczek M, Pochro´n E & Olejniczak P

Bank vole, Myodes (Clethrionomys) glareolus, serves as an object of research in many scientific disciplines. Both wild and laboratory-reared animals are used in such studies but the latter, contrary to the wild individuals, reproduce throughout the year. The aim of this study was to investigate the effect of the season outside the breeding chambers on the reproductive characteristics of bank voles kept under constant, optimal breeding conditions for 30 years. The comparison was made between summer, a breeding season, and winter, a non-breeding season, in four consecutive years. Significant differences in reproduction and development were observed. More females gave birth in summer than in winter. The number of pups born were similar in both seasons but more pups survived the first day of life in summer than in winter. Moreover, more young survived to the time of weaning (19 days) and reached higher level of body weight in summer than in winter. Season influenced the rate of morphological development of the reproductive tract of weaned males and those individuals born in summer had significantly heavier testes than those born in winter. Differences in sexual maturation were observed also in 6 week old males. Sperm concentration, as well as the proportion of viable sperm, motile sperm and not swollen sperm were higher in males born in summer. Our results may have important implications for bank vole breeders and scientists working on other captive bred rodents.

Apneic Oxygenation in Diagnosis and Treatment of Lung Tumours in an Experimental Porcine Model
by B. Kjaergaard, P.R. Zepernick, M.S. Nielsen, H.K. Jensen, D.T. Arp, J.B. Thomsen, J. Nielsen, T. McCulloch, J. Carl

Objectives: Respiratory movements may complicate diagnostic and therapeutic procedures such as biopsies and stereotactic irradiation therapy in lung cancer patients. An attempt to avoid respiratory movements, up to 30 minutes, long enough for procedures was performed in an animal study. Methods: Ten anaesthetized minipigs ~30 kg were intubated in the trachea and small NiTi-stents were placed in various parts of the lungs. Using a muscle relaxing drug, the pigs were deprived of the ability to breathe for 30 minutes, a longer time than normally used for positioning and irradiation or for biopsies. No attempt to hyperventilate the animals was made prior to the apneic period. After a lung recruitment manoeuvre, a constant oxygen pressure of 20 cm water was applied to the airways. Using X-ray fluoroscopy, the position of the stents and thereby the movements of the lung, were monitored. Arterial gas analyses were performed every 5 minutes during the apneic period. Results: All animals survived 30 minutes of apneic oxygenation. The median arterial oxygen partial pressure actually rose from 11.8 to 54.3 kPa and there were no changes in oxygen saturation. The median arterial carbon dioxide partial pressure rose from 6.9 to 18.7 kPa and the median pH fell from 7.41 to 7.04 during 30 minutes of apneic oxygenation. Our setup, or our strategy of anaesthesia, did not immobilise the internal parts of the lungs satisfactorily, and must be improved before it can be used in a clinical situation. Conclusion: Physiologically, it is possible to stop respiration using apneic oxygenation for periods long enough to perform biopsies or stereotactic radiation therapy.

Refinement and Reduction Value of Aspen Furniture and Restricted Feeding of Rats in Conventional Cages
by Niina Kemppinen, Anna Meller, Jann Hau, Kari Mauranen, Tarja Kohila & Timo Nevalainen

This study evaluated the impact of aspen furniture on cardiovascular parameters, locomotor activity (LA) and faecal welfare indicators in rats. A total of 12 BN and 12 F344 male rats were group housed (n=3) in conventional cages. In this crossover study, responses of all rats to the following cage furniture items were investigated: two types of simple maze, a rectangular tube and a control with no cage furniture. In one of the two maze groups, the rats had to gnaw through wood in order to obtain food. The mean values of the LA in all groups and differences in mean arterial pressure (MAP) and heart rate of the rats housed in the various furniture item groups were compared to the values of the rats housed in control cages with no furniture, on days two, six, ten and 14 in each period (both light and dark phases). The F344 rats were generally more active than the BN rats during the dark phase, but not during the light phase. Based on the MAP results, the tube appeared to be a poor choice for F344 rats, while for BN rats all furniture items seemed beneficial, with both board types apparently superior to the tube. In general, F344 rats had higher faecal corticosterone levels than BN rats with the reverse being true for secretory IgA values. In conclusion, LA and cardiovascular parameters seemed appropriate ways to evaluate the impact of cage furniture on physiological parameters, and covered structures such as tubes do not seem to provide any enrichment value in these two rat strains.