2008 - Volume 35 - Issue 4

221-229
The Use of HiSPECT to Investigate Dopaminergic Involvement in the Development of Stereotypic Behaviour
by Christel Palmyre Henri Moons, Kathelijne Peremans, Simon Vermeire, Eva Vandermeulen, André Dobbeleir, Katleen Hermans, Frank Olof Ödberg & Kurt Audenaert

Functional molecular imaging is becoming increasingly popular for in vivo research on small animals, because it has a number of scientific advantages over ex vivo methods. The molecular parameters themselves can be used in other areas of investigation also, such as monitoring of the dopaminergic and serotonergic involvement in the development of stereotypic behaviour. In Single Photon Emission Computed Tomography (SPECT), a radioactive substance with specific affinity for a certain molecular target is injected intravenously and after a period of time the radioactivity that is not washed out from the region of interest, is measured. A relative measure of quantification, i.e., the binding index (BI), can then be calculated. This paper aims to introduce a broad readership to one possible application of SPECT by presenting preliminary data about the dopamine transporter (DAT) status in the Mongolian gerbil (Meriones unguiculatus). 99mTechnetium-labelled Ethylcysteinate Dimer tracer (24.42 ± 5.92 MBq) was injected in the femoral vein of four gerbils to provide brain perfusion images that allowanatomical identification of DAT-rich regions that were imaged in another four gerbils using 123I-labelled FP-CIT tracer (44.33 ± 11.66 MBq). Furthermore, the optimal scan time for FP-CIT was established in one gerbil. The study was successful in obtaining brain perfusion images as well as demonstrating regional binding of FP-CIT to the basal ganglia, DAT-rich areas in the brain. The optimal scan time for DAT-imaging was 4½ hours. Our preliminary data suggest the Mongolian gerbil is a suitable model for combining SPECT and behavioural observations in the investigation of stereotypic behaviour.

231-236
Evaluation of Corncob as Bedding for Rodents
by Thomas C. Krohn & Axel Kornerup Hansen

As an alternative to aspen bedding, corncob may be used for rodents. Previous studies have shown that the ammonia level in cages using corncob bedding is reduced compared to cages with aspen bedding. The reduced level of ammonia prolongs the interval between cage changing, and it may therefore be beneficial for the facility to use corncob. The aim of the present study was to measure and evaluate the animal preferences for corncob compared to aspen bedding and also analyse the properties of corncob compared to aspen bedding. When analysing the bedding’s ability to absorb water, corncob showed lower water absorption compared to aspen bedding; the more corncob in the mixture, the less water is absorbed. Both mice and rats rejected cages with pure corncob during the day (sleeping time), and none of the animals preferred corncob mixed with aspen, only equally accepted it. In conclusion, neither rats nor mice prefer corncob, even not in mixtures with aspen bedding and enrichment. In the light of the common standard for bedding being wood chips, and the lack of preference for corncob mixture, corncob seems to be a poor alternative to wood based bedding.

239-248
Investigating the Effect of Withania somnifera, Selenium and Hydrocortisone on Blood Count and Bronchial Lavage of Experimental Asthmatic BALB/c Mice
by HM Oberholzer, E Pretorius, E Smit, OE Ekpo, P Humphries, RE Auer & MJ Bester

The murine asthmatic Balb/c mouse model was used to investigate the effect of W. somnifera L., the antioxidant selenium, W. somnifera and selenium in combination and hydrocortisone as positive control on the number of white blood cells in blood smears and bronchial lavage smears as well as the platelet distribution of asthmatic Balb/c mice. The mice were sensitised, nebulized and treated over a period of 43 days and blood smears were made of each individual animal and bronchial lavage was performed by injecting 0.3ml of saline into the trachea of the mice, both on the day of termination. Light microscopy analysis of the bronchial lavage revealed a significant decrease in the number of eosinophils counted in the asthmatic and the different treatment groups. In the asthmatic group, numerous platelet clumps were found distributed between white blood cells. Platelets were also found in the other treatment groups but are not as prevalent as in the asthmatic group. Results from the blood smears showed the same trends, where cell counts in control and hydrocortisone blood smears were decreased compared to that of the asthma group. It is concluded that W. somnifera and W. somnifera combined with selenium significantly decreased the white blood cells in both bronchial lavage as well as blood smears, suggesting that W. somnifera indeed has an anti-inflammatory potential and it, in combination with an anti-oxidant like selenium, might successfully be used in the treatment of asthma.

251-257
Refinement Recommendations for Intravenous Injection and Body Heat Conservation in Mongolian Gerbils used in SPECT Studies
by Christel Palmyre Henri Moons, Kathelijne Peremans, Philippe Büscher, Simon Vermeire, Eva Vandermeulen, Kurt Audenaert, Katleen Hermans & Frank Olof Ödberg

This paper describes two aspects of Single Photon Emission Computed Tomography (SPECT) which have been refined for use in small animals, as demonstrated by a study on Mongolian gerbils: intravenous injection of radiotracers in the femoral vein and body heat conservation in animals anaesthetized at the time of scanning. Elaborating on recommendations made by other authors, we have combined chemical and physical restraint to optimize injection efficiency as well as to reduce handling time of the animals. Finally, we provide a detailed description of a SPECT-compatible waterheated scanning bed, the application of which we believe is not limited to scintigraphic studies.

259-263
3-methylhistidine as an Indicator for Protein Beakdown: An Experimental Model in Male Capra hircu
by A.M. Almeida, L.M. Schwalbach, H.O. de Waal, J.P.C. Greyling & L.A. Cardoso

The role of the amino acid 3-methylhistidine as an indicator of protein breakdown and weight loss is often suggested. Despite existing information for other animal species, little is known about the actual levels of 3-methyhistidine in the serum of less studied domestic species such as the goat. We have evaluated the 3-methyhistidine serum concentrations in young Boer goat bucks subjected to two distinct feeding regimens: winter-grass hay with or without supplementation. Non-supplemented animals had a negative nitrogen balance and experienced weight loss throughout the experiment and significantly higher concentrations of 3-methyhistidine than supplemented animals that had a slight increase in live weight. This amino acid can be considered a valid indicator of protein breakdown and weight decrease in male goats. Serum 3-methylhistidine concentrations in adequately fed male goats were similar throughout the assay (20-40 μmol/l) whereas in weight-losing animals, concentrations of up to 170μmol/l can be expected.

265-280
Using The BALB/c Asthmatic Mouse Model to Investigate the Effects of Hydrocortisone and a Herbal Asthma Medicine on Animal Weight
by OE Ekpo & E Pretorius

Euphorbia hirta is an anti-inflammatory phytomedicine used to treat asthma. It contains, amongst other components, Quercitrin, which is converted to Quercetin in the alimentary canal and is thought to be responsible for its therapeutic potential. In this study, the BALB/c asthmatic mouse mode was used to investigate the short and long term effects of Euphorbia hirta on asthma and weight change. Hydrocortisone was used as positive control. Data showed that animals in both the short and long term control groups experienced slow but steady progressive weight increments. Both immunization and nebulization had positive weight gain effects on the animals but the effects were more pronounced following immunization but were only minimal following nebulization. Prolonged treatment with hydrocortisone remarkably reduced the cumulative weight gained following prior experimental procedures (immunization and nebulization), followed by a slow and sustained increase in the rate of weight gain. On the other hand, prolonged administration of the plant causes only a minimal reduction in weight gained due to induced asthmatic conditions. It is concluded that E. hirta, besides reducing asthma symptoms similarly to that of hydrocortisone (as seen with white blood cell counts), does not impact on weight gain as severely as hydrocortisone.

283-296
Factors influencing the Yield of Transgenic Mouse Embryos for Cryopreservation
by Anna Schwab & Johannes Schenkel

Cryopreservation of pre-implantation embryos is a powerful tool to save and handle the increasing number of transgenic mouse lines, often consisting of very small populations. However, the outcome of embryos following superovulation and mating varies and is one of the limiting factors for cryopreservation. Using the data received together with the cryopreservation of more than 100,000 mouse embryos, the role of embryo yield limiting and of yield raising factors was analyzed: annual rhythms, seasonal changes (possibly due to environmental effects, such as extreme weather conditions), infections of a mouse colony, or the mating frequency of males. Then again to increase embryo yields two pheromonal effects were investigated: Especially the housing of female donors in small groups (Lee-Boot effect), but also the exposure to the future males’ bedding two days before mating (Whitten effect) resulted in remarkably higher embryo yields.

299-301
Accidental Ear Burns Following Anaesthesia in Mice
by L Antunes, ML Pinto & AL Silva & HC Alves

Homoeothermic heating systems are frequently used to prevent the occurrence of hypothermia during mice anaesthesia. The burn hazard is a relatively frequent but not well document post-operative situation in small animal practice. This paper reports a case of ear burns following anaesthesia in ICR (CD-1) mice.

305-310
Protocol for Providing Additional Pseudo-Pregnant Recipient Mice for Embryo Transfer and Intra-Uterine Insemination by Plugging in the Middle of the Day
by Åse Roos, Maria Liljander, Anders Forslid & Ragnar Mattsson

The fact that 10% of female mice enter oestrus and allow mating in the middle of the day is an old observation that has been more or less forgotten. We here show that this old knowledge can be used to improve the efficacy of both embryo transfer and insemination protocols. The present technical paper shows that rapid re-arrangements of mating cages, to achieve pseudo-pregnant recipients in the middle of the day, can be of great advantage in emergency situations. Such emergency situations occur repeatedly, i.e. when a scientist has forgotten to re-arrange her/his mating cages, and the last important male suddenly has become ill and may die within a few hours. A rapid technique for uterine artificial insemination in mice in such situations is extremely valuable. An artificial intra-uterine insemination requires only a minimum of planning, a minimum of instrumentation and a minimum of surgical training. The artificial insemination must be performed shortly after mating due to rapid constriction of the utero-tubual junction (UTJ). This means that the timing of the insemination is very important. We here show that the success rate for embryo transfers, when using recipients plugged in the middle of the day, was the same as for ordinary overnight mating protocols. In addition, it should be noted that the success rate (frequency of pregnancies) for uterine inseminations was 55% if using F1 recipients of C57BL/6J (considerable lower if using recipients of inbred C57BL/6J), which is amazingly high, since inseminations in mice is known to be tricky to perform in a reproducible manner.

313-320
A Research Model of Measuring the Tensile Strength of Colonic Anastomosis in Wistar Rats
by K. A. Ekmektzoglou, T. Xanthos, I. A. Dontas, G. C. Zografos, P. Giannopoulos, A. Pantopoulou, S. A. Papanicolopulos, S. K. Kourkoulis & D. N. Perrea

The present experimental study investigates the mechanical behavior of intestinal anastomoses in Wistar rats. More specifically the response of the anastomoses to a properly applied uniaxial direct tensile load is studied and the respective tensile strength is determined. The surgical procedure of large bowel anastomoses is described in detail. In addition the authors provide a thorough description of the experimental apparatus, designed especially for measuring the tensile strength of the specimens under study, with special consideration in gripping of the intestine, the load application and the data acquisition and storage systems. This experimental model provides an excellent method for measuring the anastomotic strength and therefore a flexible tool for the comparative evaluation of various anastomotic techniques.

321-328
Effects of Diets Containing Different Types of Carbohydrates on Hepatic Metabolism
by Gabriela Salim Ferreira de Castro, Lillian Pimenta de Almeida, Helio Vannucchi, Guilherme Vannucchi Portari& Alceu Afonso Jordão

The study of diets rich in different quantities of simple and complex carbohydrates is essential as an aid in the prevention of several types of organic damage. Objectives: To assess the effects of hyperglycidic, isocaloric diets with variations in carbohydrate type (simple or complex) on the metabolism of rats. Methodology: Forty Wistar rats were divided into 4 groups: control group (57.96% starch and 12.04% saccharose - CG), carbohydrate mixture group (35% saccharose, 35% starch - MG), simple-carbohydrate-group (70% saccharose - SG), and complex-carbohydrate-group (70% starch - CCG). The animals were allowed to habituate to the diets and then received them for 28 days, with free access to water. Results: Ration weight and consumption did not differ between groups. The amount of hepatic fat was found to be greater in SG compared to CG. Hepatic malondialdehyde (MDA) determination revealed that SG presented the lowest value, although this result was accompanied by the lowest vitamin E value, demonstrating that the consumption of this antioxidant was higher in SG. Reduced glutathione values did not differ between groups, raising the hypothesis that in this case vitamin E was the first antioxidant barrier to be utilized. Blood glucose levels differed between CG, MG and SG in relation to CCG only on day 14; however, the oral glucose tolerance test performed at the end of the experiment did not demonstrate a difference between groups. Serum fructosamine differed significantly between MG and CG and CCG and also between SG and CG and CCG, with the values for MG and SG being higher than those for the other groups. Conclusion: The consumption of simple carbohydrates led to hepatic steatosis and altered the antioxidant system even within a short period of time, in addition to modifying the fructosamine values, revealing an increase in glycated serum proteins that may cause damage over a longer period of time.