2009 - Volume 36 - Issue 2

109-113
Technical Report: Gingival Vein Punction: A New Simple Technique for Drug Administration or Blood Sampling in Rats and Mice
by Daniel Teixeira de Oliveira, Eduardo Souza-Silva & Carlos Rogério Tonussi

Blood collection or intravenous injection of substances in rats and mice are necessary for a wide variety of scientific studies. To date, several methods have been developed to access different vessels, according to the different research purpose. However, animal behavioural responses like stress, pain or traumatic injury during some procedures may influence subsequent results. In this technical report we demonstrate the advantages of using the labialis mandibularis vein route that, in addition, seems to prevent unnecessary animal suffering.

117-122
Clinical, Histological and Biochemical Observations in Spontaneous Senile Cataract in Mastomys Species
by Kalidas N. Kohale

Clinical, histological and biochemical examination of spontaneous lens opacity was carried out in Mastomys coucha species. The incidence of the spontaneous lens opacity was noticed in adult animals, in the age range of 48-72 weeks, up to a maximum prevalence of 2.6% and 13.71% in male and female animals respectively. The affected animals exhibited mild lens opacity initially, which progressed with age, and later on became dense and looked like a mature cataract. The opacity was irreversible and showed no signs of disappearing in the affected animals. Both male and female were affected but, as noted above, the incidence of the abnormality was higher in the females. The mode of inheritance was not investigated. The ophthalmoscopic examination of the affected animals revealed total lenticular opacity characterized by a shallow anterior chamber and bulging of the lens towards the anterior side due to the swelling of the lens. The serum glucose values in affected animals were within the normal range. The histological examination of opaque lenses revealed total alteration in the micro architecture of the lens cells. The SDS-PAGE analysis of lens crystallins, revealed distribution of major lens crystallins within range of 20-30 kDa molecular weights. The immunoblotting of SDS-PAGE separated proteins with anti-crystallin alpha-, beta- and gamma-antibodies indicated expression of major lens crystallins in affected animals. We have concluded that this is a case of spontaneous senile cataract, reported for the first time in Mastomys species.

123-130
Clobetasol 17-Propionate Cream as an Effective Preventive Treatment for Drug Induced Superficial Thrombophlebitis
by E Dvir, S Russo, A Meshorer, R Duvdevani & G Rosenberg

Commonly used therapies for thrombophlebitis have a high failure rate. There are scant data on the application of topical corticosteroids to treat thrombophlebitis. The present study investigated if the potent topical corticosteroid clobetasol 17-propionate cream (Dermovate, Glaxo Wellcome) can be an effective treatment for drug-induced thrombophlebitis. DP-b99, a neuroprotective agent currently undergoing development for acute stroke, can cause injectionsite phlebitis. DP-b99 was administered at doses of 1 and 2 mg/kg by a 1 hour intravenous infusion into the lateral ear vein of groups of 6 and 5 rabbits, respectively. Each rabbit served as its own control by injecting both ears with DP-b99, while treating only one ear with clobetasol cream immediately after treatment, with subsequent applications twice daily for 3 days. Phlebitis was evaluated 1, 3, 5, 24, 32, 48, 56 and 72 hours after DP-b99 treatment using a clinical score ranging from 0 (no reaction) to 4. After 3 days the rabbits were sacrificed for histological analysis of the ears. The phlebitis score was highest at 24 hours. Clobetasol treatment reduced the clinical scores at all time points and shortened the course of phlebitis. Maximal effect was observed 24-48 hours after the first application of clobetasol cream. Histologically, there were fewer cases of thrombophlebitis in the clobetasoltreated ears, and those seen were milder and more focal. To the best of the authors’ knowledge this appears to be the only study to report a phlebitis-ameliorating effect of a topical corticosteroid.

133-136
Time Related Fungal Contamination of Animal Cage Beddings
by Ragnar Rylander, Bethan Foden, Birgit Ewaldsson & Morten Reeslev

The purpose of the study was to measure the extent of fungal contamination in laboratory animal cage beddings over time. The material was analysed for the content of fungal enzyme N-acetylhexosaminidase and the fungal cell wall agent 1,3-beta-glucan at 0-7 days after use. In some cages the values were increased above baseline already at 3 days and at 7 days practically all beddings showed a fungal contamination. It is suggested that the fungal enzyme test can be used for bedding quality control purposes and to monitor fungal contamination in animal cages to prevent pulmonary and other pathologies.

139-145
Histological and Biomechanical Evaluation of the Preserved Degenerative Dermis in Rat Autologous Skin Transplant Models after a Deep Second Degree Burn
by Jincun Wang, Dequan Li, Feng Xia, Junliang Han, YanYang Tu & Xiao Yuan Huang

To describe the histological and biomechanical changes of the preserved degenerative dermis in rat autologous skin transplant models after a deep second-degree burn. 50 SD rats were divided into 5 groups randomly of 10 rats of each: 7-days group, 9-days group, 14-days group, 21-days group, and 60-days group. Deep second-degree burn wounds were prepared on the back of rats sized 3.5cm×3.5cm. Super tangential excision was performed on the burn wound to preserve the degenerative dermis. Then, autologous epidermis was grafted on the wound. After that, the histological changes of the preserved degenerative dermis tissues and the graft areas were observed by macroscopic, light microscope and electron microscope in the 7, 9, 14, 21, 60 days after the operation. Moreover, the tensile properties of healing deeply burned rat skin were also tested for each group at the same time points mentioned above.
Results: (1) According to the macroscopic observation, 7 days after the operation, the grafted skin was fused with the area of burn wound; A few hairs were growing out on the skin at the 14th day; the injured skin recovered to normality by the 60th day. (2) Hyaline change occurred in the preserved degenerative dermis tissues based on the observation by light microscope. At the 7th day after operation, the dermis papillae and reticular layer could be discerned; by the 21st day, the thickness, structures and morphology of grafted skin were similar to the normal tissues. (3) 7 days after operation, ballooning changes were observed by the electron microscope in the mitochondria and endoplasmic reticulum of damaged cells and the number of the ribosomes was obviously reduced. The subcellular wound improved continuously and approached normality by the 21st day. (4) 9 days after the operation, the tensible strength and maximal strain of the grafting rat skin approached 70% and 90% of natural skin, respectively. (5) 60 days after the operation, the tensile performance of the healing rat skin recovered to the natural level.
Conclusion: The histological and biomechanical changes of the denatured dermis of a deep second degree burn wound may gradually recover to normality after being covered by autologous skin.

147-153
Incidence of Chirodiscoides caviae in Laboratory Rats-Screening, Identification and Treatment
by V.S. Harikrishnan, V.R. Ranaraj & A.C. Fernandez

This is a report on the incidence and treatment of the guinea pig fur mite Chirodiscoides caviae, which was so far considered as host specific, in a conventional colony of laboratory rats. Chirodiscoides caviae infestation in laboratory rats was accidentally observed during the screening of Syphacia obvelata by the peri-anal cellophane tape test (CTT). The organism was identified by comparing the morphology described by various researchers and was differentially diagnosed from other common mites of rat, Radfordia ensifera and Notoedres muris. The adult male mites (n=15) were of 330.2±13.3 μm long and the females (n=15) 495.5±25.2 μm. Later on, the entire rat colony consisting of Wistar, Sprague Dawley and Spontaneously Hypertensive Rats (SHR) and the mice colony of Balb/c and Swiss Albino were randomly sampled and screened for the presence of the mite by the cellophane tape technique. All the rat strains were found positive for C. caviae infestation, which was more concentrated towards the posterior region of the body and, collectively, the screening results of C. caviae revealed that the posterio-dorsal and peri-anal regions are most suitable for sampling-suggesting that, the infestation pattern of C. caviae in rats has similarities to that of guinea pigs. Interestingly the mice colony was found free from the infestation. The Cellophane tape test was found to be an easier method than fur examination by hair plucking and equally accurate for screening of fur mite in a colony of laboratory rats. No clinical symptoms were observed in any of the animals in the colony, which possessed infestation. The facility strictly practised physical separation of animals by species, which pointed to the only possibility of cross infestation being through indirect contact between guinea pigs and laboratory rats and thereby questioning previous reports on the mode of transmission of C. caviae. The entire colony was effectively treated with 0.2% Ivermectin spray followed by 1% spray in an interval of 2 weeks. This report is the first one, which demonstrates the guinea pig fur mite in laboratory rats. It also questions the so far documented “host specificity” and “direct contact” mode of transmission and demonstrates indirect contact as a possible mode of transmission.

155-162
Effects of Sex, Pregnancy and Season on some Haematological and Biochemical Blood Values in Angora Rabbits
by N. Çetin, T. Bekyürek, E. Çetin

This study was undertaken to investigate the effect of sex, season and pregnancy on some blood parameters in Angora rabbits. A total of 45 Angora rabbits (15 males, 15 females and 15 pregnant rabbits), weighing 3.6- 5.3 kg were used in this study. Blood samples were collected from female rabbits in January, April, July and October, and male and pregnant rabbits in January. Erythrocyte counts and haemoglobin concentrations of pregnant rabbits were lower (p<0.05) than those of normal non-pregnant rabbits, while mean corpuscular volume in pregnant rabbits was higher (p<0.05) than those of non-pregnant rabbits. Total leukocyte counts and lymphocyte ratios in pregnant females were lower (p<0.05) than those of normal non-pregnant rabbits. There were no significant differences (p>0.05) in the other haematological values between pregnant and non-pregnant rabbits. Total protein, albumin triglyceride, cholesterol, calcium, and (Inorganic) phosphorus were significantly lower (p<0.05) in pregnant rabbits than the other females, while the mean glucose level was significantly higher (p<0.05). It was determined that male rabbits had higher erythrocyte, haemoglobin and glucose values and lower total cholesterol level than females (p<0.05). Erythrocyte count, haemoglobin concentration, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration of female rabbits were lower (p<0.05) in July, whereas hematocrit level and mean corpuscular volume were higher (p<0.05) in the same month. Leukocyte count and lymphocyte ratio were significantly lower (p<0.05) in July and October. Albumin level was significantly higher (p<0.05) in July. Sex and pregnancy as well as season variously affected most haematology and serum biochemistry variables.

163-165
Comment and answer to the paper entitled Probiotic Biotherapy for Eradication of a Potential Pathogen in a Commercial Rat Breeding Colony
Comment by Ron Boot

With interest I read the paper by Collinder E, Bergsted J, Persson A-K, Norin E and Midtvedt T: Probiotic biotherapy for eradication of a potential pathogen in a commercial rat breeding colony. Scand. J. Lab. Animal Sci. 2008, 35, 199-201.
The paper describes a study in which Lactobacillus reuteri was dosed to rats to clear them from (unspecified) Lancefield group G ß-haemolytic streptococci that were detected in vaginal samples.

167-176
Nine-banded Armadillo Dasypus novemcinctus Animal Model for Leprosy (Hansen’s Disease)
by R. Vijayaraghavan

Leprosy (Hansen’s disease) is a chronic infectious disease of mankind caused by Mycobacterium leprae. Despite several serious scientific attempts, M.leprae has never been cultivated in artificial bacteriological media. When M.leprae was identified as the causative germ of leprosy, Hansen (who discovered the causative germ of leprosy in 1873) attempted to cultivate the germ in animal models, but he failed. Nearly 85 years after the discovery of M. leprae successful transmission and multiplication of M.leprae in the footpads of laboratory mice was achieved. The mouse footpad model, although widely used in experimental leprosy, has its own limitations (Sheppard, 1960; Rojas and Lovik, 2001). In 1971, significant advancement in experimental leprosy research was made when nine-banded armadillos (Dasypus novemcinctus) were successfully infected with M.leprae and developed generalised lepromatous leprosy over a period of 18 to 24 months post inoculation. Since then, the nine-banded armadillo has become a favoured animal model for experimental leprosy (Krichheimer et al., 1972; Job et al., 2003). They remain as the only immunologically intact species which regularly develops lepromatous leprosy.

179-183
The Use of the Techniplast Mouse House® in Four Strains of Mice
by D.B. Soerensen, M. R. Moeller & L. R. Larsen

To combine the good qualities of a dark shelter with the possibility of the staff to monitor the animals without disturbing them, a transparent plastic shelter with a red tint has been designed for mice. As the mice do not see red colours, but perceive them as being grey or dark, this design is thought to provide a dark shelter for mice. As humans do see red colours, the red transparent material allows for inspection of the animals while they are inside the shelter. However, recent studies and anecdotes seem to indicate a preference in some strains for not using the red houses. Four strains of mice, C57BL/6/Bkl, Balb/c/Bkl, CBA/BomTac and Bkl:NMRI, were housed individually with access to a red house as the only shelter. The result showed that there were significant differences in the time spent in the red house and the number of entries between the strains with NMRI mice – and to some extent the Balb/c - spending less time and demonstrating a higher number of entries in the red house than the pigmented strains.

185-191
Single-gene Effects on Body Weight in Selected and Unselected Mouse Lines Detected by Bayesian Marker-free Segregation Analysis
by Anna Wolc, Ewa Skotarczak, Werner Schlote & Tomasz Szwaczkowski

For the purpose of this paper, large animal species are taken to be those animals that are commonly used as farm livestock animals namely: cows, pigs, goats, sheep, horses, camelids and deer. The numbers of procedures in the UK in 2006 involving such animals amounted to around 56,000 out of a total of around 3 million. It may be that human perception of these animals as livestock animals impairs our consideration of their needs, compared to say those of common pet animals, dogs or cats. As the perception of their environment, and the potential to suffer, of livestock animals is likely to be similar however, we should not neglect their needs. The use of large animals in scientific procedures has advantages in some respects – the animals
are in the main domesticated, and are therefore comparatively docile and have been bred to cope with captivity. Nevertheless they can display aggressive behaviour and are capable of causing significant injury, so an understanding of their behaviour can reduce risks to staff caring for and working with these animals. This presentation considers the behaviour of these animals, their needs, signs of discomfort and pain, and means to ameliorate both their welfare and the safety of staff engaged in their use.

193-201
Effect of Strontium Ranelate on Femur Densitometry and Antioxidative/Oxidative Status in Castrated Male Rats
by R P. Radzki, M. Bienko, R. Filip, E. Albera & M. Kankofer

The studies were aimed at determinatning of the effect of strontium ranelate (SR) on the mineralization processes and selected parameters of oxidative stress in orchidectomized rats during the development of osteopenia. Male Wistar rats were sham-operated (SHO) and orchidectomized (ORX). ORX animals were divided into control (ORX-C) and gavaged with SR (ORX-SR), at a dose of 900mg/kg/b.w. After 60 days the animals were scanned for determination of bone mineral density (BMD) of the whole skeleton. Isolated femora were examined by DEXA and pQCT. Tomographic measurements were performed for a total slice and separately for the cortical and trabecular parts of the distal end of the femora. The intensity of lipid peroxidation (ILP) and total antioxidant capacity (TAC) in blood serum were measured. SR treatment increased vBMD and BMC of total, trabecular and cortical bone in ORX rats compared to ORX-C and SHO rats. ORX significantly increased TAC in control animals, and SR limited this increase. ILP in SHO and ORX-C rats which on a similar level. SR increased ILP by 21.3%, as compared to SHO. SR improved densitometric and geometric parameters of femora by orchidectomized rats what prevented degradation of bone tissue. Beneficial effects of SR were also demonstrated in stabilization of TAC in ORX rats at the level noted in SHO rats.

205-213
Body Weight and Faecal Corticosterone Metabolite Excretion in Male Sprague-Dawley Rats Following Short Transportation and Transfer from Group-housing to Single-housing
by Joakim Dahlin, Jenn Lam, Jann Hau, Pudji Astuti, Harry Siswanto & Klas S. P. Abelson

Body weight and faecal excretion of corticosterone metabolites (CM) were recorded daily in 18 young male Sprague-Dawley rats from the day they arrived at the animal facility from the breeder. The animals were group-housed (n=3) and divided in two groups after 7 days. One group (n=9 animals in 3 cages) was moved to another room in the facility and the other group remained in the original holding room. After an additional 7 days the animals which had been moved were separated and single housed for an additional 7 days. The body weights developed normally in all rats during the three-week period. Faecal CM excretion appeared high immediately after the rats arrived from the breeder, and decreased to reach significantly lower levels 6 days after arrival. This was likely related to natural fluctuations in faecal CM output rather than substantial stress. None of the husbandry procedures performed during the study had any effect on faecal corticosterone metabolite secretion compared to control. The results suggest that neither transportation from the breeder, moving within the facility, nor being transferred from group housing to single housing are events stressful enough to be reflected by the parameters analysed in the present study. However, the faecal CM excretions clearly fluctuate over several days, which must be considered when using faecal samples for non-invasive stress assessment.

215-221
Blood Sampling from the Tail Vein, in Comparison with Two Other Techniques, Causes Less Stress to Mice
by J. Madetoja*, M. Madetoja, J. Mäkinen, E. Riuttala & J. Jokinen

It is important to use the optimal method for repeated blood sampling to ensure minimal stress to mice, and also to provide better pharmacokinetic and pharmacodynamic data. The aim of the present study was to compare the impact of blood sampling methods on corticosterone and adrenocorticotropic hormone (ACTH) levels in mice. Hsdwin:NMRI mice were divided into four sampling groups: control group (I), vena facialis (II), tail vein (III) and saphenous vein (IV). The first blood samples, obtained from vena facialis, tail or saphenous vein of conscious mice, were taken at time point 0. The second blood sample was taken by decapitation from groups II-IV with isoflurane anaesthesia at time point 20 min. The control group animals were anesthetized and decapitated at 20 min time point. Corticosterone levels in plasma were analyzed at time point 0 and 20 min, and ACTH at time point 20 min. Saphenous bled mice, in comparison with vena facialis and tail vein sampled mice, indicated statistically significant greater (P < 0.05) level of corticosterone at sampling point (0 min). Rising levels of corticosterone in all groups differed statistically (P < 0.05) from the control group level, indicating that all tested bleeding methods were stressful to the experimental animals. However, the tail vein bleeding method stressed statistically significantly (P < 0.05) less in comparison with vena facialis and saphenous vein bleeding. At time point 20 min, only saphenous vein bled mice showed statistically significant greater (P < 0.05) blood levels of ACTH compared to tail vein bled mice. Conditions in sampling and rising levels of corticosterone and/or ACTH level did not show direct correlation. In conclusion the results suggest that the tail bleeding method accomplished least stress to mice and next less vena facialis bleeding. Blood collection technique from the saphenous vein was the most stressful to the experimental animals.