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Laboratory Animals http://lan.sagepub.com/ Aggression in cataract-bearing α-1,3-galactosyltransferase knockout mice

Dorte Bratbo Sørensen, Kirsten Dahl, Annette Kjær Ersbøll, Svend Kirkeby, Anthony J F d'Apice and Axel Kornerup Hansen Lab Anim 2008 42: 34 DOI: 10.1258/la.2007.006057 The online version of this article can be found at: http://lan.sagepub.com/content/42/1/34

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Aggression in cataract-bearing a-1,3-galactosyltransferase knockout mice Dorte Bratbo Sørensen*, Kirsten Dahl*, Annette Kjær Ersbøll†, Svend Kirkeby‡, Anthony J F d’Apice§ and Axel Kornerup Hansen* *Centre for Bioethics and Risk Assessment, Division of Laboratory Animal Science and Welfare, Department of Veterinary Pathobiology, Faculty of Life Sciences, University of Copenhagen, Groennegaardsvej 15, DK-1870 Frederiksberg C, Denmark; †Division of Veterinary Epidemiology, Department of Large Animal Sciences, Faculty of Life Sciences, University of Copenhagen, Groennegaardsvej 8, DK-1870 Frederiksberg C, Denmark; ‡Institute of Oral Medicine, Dental School, University of Copenhagen, Nørre Alle´ 20, DK-2200 Copenhagen N, Denmark; §Immunology Research Centre, St Vincents Hospital, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia

Summary The Gala1-3Galb1-4GlcNAc epitope is the key antigen in the hyperacute rejection of pig-toman xenotransplantation. In the a-1,3-galactosyltransferase knockout (a-1,3GT-KO) mouse – a model for xenograft donor pigs – a targeted mutation of the a-1,3 galactosyltransferase gene (Ggta1) has been constructed. These mice are depleted of the carbohydrate antigen and besides the mice are also known to develop cortical cataracts. The present study aimed at evaluating the morphology and the degree of the cataract in a population of a-GT KO mice, its age of onset, its progression and the impact the cataract may have on aggression, anxiety and perception of light. The a-gal epitope could be shown in the lenses with lectin GS1 B4 in all wild-type and none of the a-GT KO mice. Histology showed apparent cataract in all a-GT KO mice from six weeks of age. Apart from a single wild-type mouse with a small degree of microscopically visible cataract without epithelial involvement at the age of 30 weeks none of the wild-type mice showed signs of cataract. Behavioural testing demonstrated significantly more mounting behaviour and a longer duration of attacking in the a-GT KO mice. Apart from this, the agonistic behaviour was not influenced by genotype. Neither did the genotype affect anxiety or perception of light. Keywords Aggression; a-1,3-galactosyltransferase; cataract; mouse Xenotransplantation, i.e. transplanting an organ from one species to another, is a potential solution for the shortage of organs for transplantation. The pig has been preferred as the potential donor (Dahl et al. 2003, Hansen et al. 2004). However, pig-to-man xenografts will undergo hyperacute rejection due to human antibodies towards the Gal-a1-3Galb1-4GlcNAc-R (a-gal) epitope produced by galactose-conjugation of N-acetyl-lactosamine in the trans-Golgi network. This reaction is catalysed by the enzyme a-1,3-galactosyltransferase (a-GT) Correspondence: Associate Professor D B Sørensen. Email: [email protected] Accepted 3 May 2007 # Laboratory Animals Ltd

and knockout (KO) of the a-GT gene will cause deficiency for this specific carbohydrate epitope. The a-gal epitope is expressed on the surface of cells in non-primate mammals, prosimians and New World primates (Vaughan et al. 1994) while it is absent in humans, apes and Old World monkeys due to a mutation in the ancestral a-GT gene 22 to 32 million years ago (Joziasse et al. 1991, Hennet 2002). The a-gal epitope has thus been demonstrated in homogenates from lenses of both normal pigs and mice and is absent in human lenses (Ogiso et al. 1994). The a-GT KO mouse (Tearle et al. 1996), engineered as a model for a potential xeno-pig, does not express the a-gal epitope on cell membranes. Owing to its small size, fertility

DOI: 10.1258/la.2007.006057. Laboratory Animals (2008) 42, 34 –44

Aggression in cataract-bearing a-GT KO mice

rate and the extensive knowledge on housing and management, the mouse is an attractive tool in xenotransplantation research. Two strains of a-GT KO mice have been generated and in both strains cataract develops at a young age (Tearle et al. 1996, Thall 1999). Why such depletion of a-gal leads to cataracts is unknown. The cataractous a-GT KO mice may give an indication on how impaired vision affects mice. Traditionally, mice are not considered to be essentially dependent on their vision to cope with their surroundings, but being a nocturnal animal, mice may need a greater sensitivity to light than diurnal animals (Pinto & Enroth-Cugell 2000), and in darkness cataractous mice might be unable to register environmental characteristics, which cannot be perceived by e.g. touch or smell. Moreover, gradual impairment of vision may reduce the ability to recognize visual signals communicating dominance and submission, which may result in increased fighting behaviour. It has been demonstrated that FVB/N mice suffering from retinal degeneration show a higher frequency of attacking as well as a longer duration of attack in the intruder test (Pugh et al. 2004). Furthermore, if the same condition were to develop in xeno-donor pigs, which now have been generated through cloning and nuclear transfer (Dai et al. 2002, Lai et al. 2002), it may impose a welfare problem (Dahl et al. 2003), as it has been shown that gradual visual impairment causes aggression towards humans in pigs (Andrea & George 1999). The present study aimed at evaluating the morphology and the degree of the cataract and the impact it may have on aggressive behaviour in the a-GT KO mouse.

Materials Animals C57BL/6.129/CimlKvl–Ggta1tm1Tea (a-GT KO) (Tearle et al. 1996) and C57BL/6JBomTac (wild-type, control animals also called B6), barrier bred and health monitored according to FELASA guidelines (Nicklas et al. 2002), were housed in a transparent standard Makrolon type III cages measuring 425

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266 155 mm (Tecniplast, Buggugiate, Italy) with aspen bedding and aspen nesting material (Tapvei, Kortteinen, Finland). The mice were fed Altromin 1324 standard diet (Brogaarden, Gentofte, Denmark) and given water ad libitum. In both the housing room and the test room, the temperature was maintained at 208C with a relative humidity of 55–80%. The light/dark cycle was reversed to lights on from 22:00 to 10:00 h with no natural light to ensure that the mice were active when performing the behavioural tests during the working day. During the dark period, a red light was on.

Methods Three experiments were done. In the first experiment (experiment 1), a total of 18 male a-GT KO mice and 18 male wild-type mice were used for examining the development of cataract from three weeks of age and up to 30 weeks of age. In the second experiment (experiment 2) on lectin visualization of the a-gal epitope in mouse lenses, a total of 13 female a-GT mice and 13 female wild-type mice of 42–60 weeks of age were used. In experiment 3, body weight and behaviour were evaluated. A total of 18 male and 17 female a-GT KO mice, as well as 19 male and 17 female wild-type mice were used for experiment 3. Experiment 1. Histopathology of the lens Eighteen male a-GT KO mice (Tearle et al. 1996) and 18 male wild-type mice housed in cages of three, four or six animals, respectively, were sampled for histopathology of the lens at the ages of 3, 6, 9, 13, 23 and 30 weeks (Table 1). All animals were anaesthetized by a mixture of fentanyl, fluanisone and midazolam (Flecknell 1996) and a blood sample was obtained from the right retro-orbital Table 1 Numbers of a-GT knockout (KO) mice and wild-type (WT) animals evaluated for lens pathology at different ages Age in weeks KO WT

3 3 3

6

3 3

9

3 3

13

23

30

2 3

3 3

3 3

Groups with cataract

Laboratory Animals (2008) 42

36

sinus. In all animals, the left eye was inspected for cataract and removed. Hereafter, the animals were euthanized. Sutures were placed in the medial adnexa of the eye for orientation of the tissue at mounting, and the eye was fixed in neutral buffered formaldehyde (4%) for 24 h, processed and embedded in paraffin. All eyes were mounted and cut in a sagittal plane from the posterior surface using cold spray to prevent the lens from tearing. The sections were stained with haematoxylin and eosin and evaluated without knowledge of the genotypes.

Experiment 2: Lectin visualization of the lens Lectin visualization of the a-gal epitope in mouse lenses was done in 13 female a-GT mice and 13 female wild-type mice at 42–60 weeks of age. After cervical dislocation, the eyes were rapidly removed from a-GT KO mice and wild-type mice and immersed in Carnoys fixative (3 volumes of absolute ethanol and 1 volume of galcial acetic acid) for 24 h. After fixation, the eyes were dehydrated through grades of ethanols, cleared in xylene and imbedded in paraffin at 528C. Sections were then cut at 68C, deparaffinized with xylene, rehydrated with graded ethanols and used for lectin histochemistry. The Griffonia simplicifolia 1 isolectin B4 lectin (GS1 B4, Sigma, St Louis, MO, USA) was biotinylated as follows: 1 mg lectin was dissolved in 1 mL NaHCO3 (0.1 mmol/L, pH 8.2) containing 0.1 mmol/L lactose to protect the active site and mixed with Biotin-NHS dissolved in 0.2 mL dimethylformamide. Before incubation with the lectin some sections were treated with 0.1% Triton X-100 solution in tris buffered saline (TBS) for 5 min in order to block possible non-specific binding. The sections were incubated for 24 h at 48C with GS1 B4 1:400 in TBS. After a 3 5 rinse in TBS the sections were immersed in Alexa Fluor 594w streptavidin conjugate (Molecular Probes, Eugene, OR, USA) for 30 min. After a rinse in TBS, the sections were mounted in a fluorescence-mounting medium S3023 (DAKO-Cytomation, Glostrup, Denmark). Controls were carried out to ensure that the Laboratory Animals (2008) 42

D B Sørensen et al.

staining obtained after incubation with lectin indeed reflects the presence of carbohydrate moieties in the sections and is not the result of non-specific lectin-protein interaction. Thus, some sections were immersed for 10 min at room temperature in a 1% aqueous solution of periodic acid (HIO4, 2H2O) before incubation. Other sections were incubated without lectin to exclude probe-independent staining. Genotyping of the mice Genotypes were confirmed by polymerase chain reaction (PCR) on DNA extracted from potassium-EDTA stabilized blood using Dynabeadsw (Dynal, Oslo, Norway). PCR protocol was kindly provided by Evelyn Salvaris (Immunolgy Research Center, St Vincent’s Hospital, Melbourne, Australia). Three primers were used: E9F: (50 -TCA GCA TGA TGC GCA TGA AGA c-30 ), NeoF: (50 -TCT TGA CGA GTT CTT CTG AG-30 ) and E9R: (50 -TGG CCG CGT GGT AGT AAA AA-30 ). In wild-type mice, primers E9R and E9F will produce a 255 bp product. In a-GT KO mice, the same primers will produce small and unreliable amounts of a 1596 bp product, but the primers E9R and NeoF will produce a 364 bp product. HotStarTaq Master Mix (Qiagen, Australia) was used for DNA polymerization. PCR conditions were: for starting: denaturizing (948C) for 15 min, annealing (558C) for 1 min, extension (728C) for 1 min and 30 s. Then nine cycles of denaturizing 1 min, annealing 1 min and extension 1 min and 30 s, and then 25 cycles of denaturizing 30 s, annealing 10 s and extension 1 min and 30 s. Finishing was by denaturizing for 5 min, after which the product was kept cold. Concentration per PCR tube was 2.5 U HotStart Master Mix, 0.5 mmol/L E9F, 1.0 mmol/L E9R, 0.5 mmol/L NeoF, 40 ng DNA. PCR products were visualized in agarose gel with ethidiumbromide. Experiment 3: Body weight and behavioural tests Eighteen male and 17 female a-GT KO mice, as well as 19 male and 17 female wild-type mice were weaned at four weeks of age,


sorted by gender and allocated randomly to home cages consisting of both a-GT KO mice and wild-type mice, either males or females. Seven cages (high-density cages) housed six mice (3 of each genotype) per cage. Eight cages (low-density cages) housed four mice (2 of each genotype) per cage. Females and males were not housed together. Three males were euthanized during the study period due to fighting or for other reasons. All mice were euthanized at 23 weeks of age and clinical examinations as well as histopathology of the lenses were performed as described above. Body weight All mice in experiment 3 were weighed once a week from 4 to 23 weeks of age.

Behavioural tests Assessment of social interaction and aggression All male mice (18 a-GT KO mice and 19 wild-type mice) were tested in the social interaction test with young, group-housed DBA/2J males as opponents (Votava et al. 2001). The test mouse was allowed 30 min of habituation to a new transparent standard Makrolon type III cage with aspen bedding and no lid before a young, group-housed DBA/2J male was introduced as an opponent for 4 min. To avoid inducing single-housing related high levels of aggression, both test animals and opponents were housed in groups in their home cages prior to testing and no more than 30 min of habituation was allowed to avoid that test animal would perceive the test cage as its territory. The interaction was videotaped for later analysis, but the experimenter stayed in the room to intervene, if any fighting exceeded 10 s, which, however, did not occur. Videotapes were analysed using Observer 3.0 (Noldus Information Technology, Wageningen, The Netherlands). Assessment of emotionality and sensitivity to light All mice in experiment 3 were tested for emotionality and sensitivity to light. A

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two-compartment test box was constructed. The test box was divided into two equally sized compartments (both measuring 370 240 210 mm), one of which was dark and sheltered and the other was open and illuminated (light intensity 600 lux). The two compartments were of equal size and apart from the closed compartment being roofed and dark, there were no differences. This design was used to avoid that perception of the dark compartment as a small shelter would obscure possible effects of different light intensities in the open compartment. At test-start the mouse was placed in the open compartment, facing the wall opposite to the entrance hole to the dark compartment. To assess the sensitivity to light all animals were tested with both 8 and 800 lux light in the open compartment in a crossover design, testing the first half of the animals in each group in bright and then in dim light, and the other half in the reverse order. Test time was 10 min during which behaviour was videotaped for analysis by Observer 3.0 (Noldus Information Technology). Number of transitions between compartments and time spent in the open compartment as indicators of anxiety-related behaviour (McIlwain et al. 2001), and latency to enter dark compartment as an indicator of initial activity was recorded (McIlwain et al. 2001). By comparing the time spent in the open compartment under different light conditions (bright or dim), an assessment of the aversiveness of the light resulted. Statistical analysis The statistical analysis system (SAS) was used for all statistical analyses (SAS Institute Inc, version 8.2) using a 5% significance level. When assessing social interaction and aggression, the effect of cage density (number of animals in the home cage) and genotype (a-GT KO or wild type) were tested on a number of outcome variables. As none of the outcome variables followed a Gaussian distribution, a non-parametric analysis of variance was performed as an analysis of variance of the rank transformed outcome variable (PROC RANK and PROC MIXED in SAS). Cage density and genotype were Laboratory Animals (2008) 42

38


included as fixed effects and cage as a random effect. In the test for emotionality and sensitivity to light, the effect of cage density (number of animals in the home cage), gender, genotype, light intensity, order of light intensity tested (either dark condition first and then the lit condition or reversed) and run-through (first or second run-through the test) were tested on number of transitions, latency to enter the dark compartment and time spent in open compartment. As the three outcome variables did not follow a Gaussian distribution, a non-parametric analysis of variance was performed for each rank transformed outcome variable (PROC RANK and PROC MIXED in SAS vs. 8.2, SAS Institute Inc. A 5% significance level was used). Cage density, gender, genotype, light intensity, order of light tested and round were included as fixed effects. Cage nested within gender and cage density and mouse nested within cage density, genotype and order of light tested were included as random effects.

Results Body weight At weaning, no differences in body weight were found, but at 23 weeks of age, the a-GT KO mice were significantly smaller than controls (Table 2). Histopathology of the lens; experiment 1 Macroscopic inspection revealed that a-GT KO mice aged from six weeks and up had bilateral cataracts recognizable as an opaque area in the centre of the eye (Table 1). At 23 weeks of age bilateral cataract was evident in all a-GT KO mice, both at inspection prior to euthanasia and by histopathology of the lens. None of the mice in the control group Table 2

showed any abnormalities of the lens at the macroscopic evaluation at any time. Histological evaluation of lenses from a-GT KO mice older than three weeks revealed that lens epithelium had become hyperplastic with numerous displaced and swollen epithelial cells, known as bladder cells (Figure 1). The lens cortex was severely damaged by necrosis and lens nucleus was affected in most, but not all, incidences (Figure 2b). Also nucleated cells are present below the level of the epithelia in the anterior cortex indicating abnormalities in the maturation process. In the equatorial regions this necrosis had given rise to numerous lakes of proteinaceous material. Occasionally, migratory epithelial cells were seen in the posterior region of the lens where the cuboidal epithelium is normally not present. Also at the posterior region of the lens proteinaceous lakes were present consisting of material from the lyzed fibres. In the lens cortex, degeneration appeared as morgagnian globules together with fragmented fibres with rounded ends distinctive from artificial fragmentation that would appear as fragments with sharp endings. The lens nucleus was also affected in all cataractous lenses except from three of the a-GT KO mice, one mouse aged nine and two aged 23 weeks, respectively, in a similar way with loss of normal fibre structure and release of cell contents due to necrosis (Figure 2b). The above described picture was seen in a-GT KO mice older than three weeks. In all eight a-GT KO mice aged 13 weeks or more dystrophic calcification of the lens cortex was present. Histopathology of the lens; experiment 3 Histological examination of the behaviourally examined mice at the age of 23 weeks confirmed that all a-GT KO mice had

Body weight (mean + SD) in a-GT knockout (KO) and wild-type (WT) mice at 4 and 23 weeks of age Male

Age (weeks) KO WT

4 12.39 13.08

Female 23 29.43 §§§ 32.82 §§§

4 11.4 12.21

23 21.51 §§§ 25.44 §§§

P values comparing males and females at respectively 4 and 23 weeks of age and the two genotypes a-GT KO and wild-type mice at 4 and 23 weeks of age. Asterisks indicate that the body weight is significantly different between genotypes ( P , 0.0001, P , 0.001). Section marks indicate that body weight is significantly different between the sexes (§§§P , 0.0001)



39

Figure 1 Anterior region of lens from a-GT KO mouse. Lens fibres have undergone liquefaction necrosis and proteins have been released to the extracellular space to form morgagnian globules. The lens epithelium is hyperplastic and swollen epithelial cells known as ‘bladder cells’ appear in the cortex near the capsule

developed severe cataract as described above. One wild-type mouse at the age of 30 weeks showed a small degree of cataract at the microscopic evaluation as the lens cortex had a minimal area of liquefaction necrosis, the epithelium however being unaffected. Apart from this, all wild-type mice had normal lenses (Figure 2a). Lectin visualization of the lens After staining with GS1 B4 all wild-type mice showed clear presentations of the a-gal epitope in the lens epithelia (Figure 3a), while none of the a-GT KO mice showed such positive reactions (Figure 3b). Behavioural observations In the social interaction test, a-GT KO mice showed significantly longer duration of attacks, they showed significantly more mounting behaviour and they tended to attack more (Table 3). None of the other behavioural patterns tested in the social interaction design were significantly affected by genotype. The cage density in the home cage significantly influenced several behavioural patterns (Table 3). There was no significant interaction between genotype and cage density on social behaviour. There was no effect of genotype in the combined emotionality and sensitivity-to-light test when

Figure 2 Normal lens from a wild-type mouse (a) and eye from an a-GT KO mouse (b) showing subcapsular cataract comprising both cortex and nucleus. In lens cortex and nucleus fibres are necrotic. The released fibre content from lyzed fibres is visible as lakes of proteinaceous material in the lens. In an abortive attempt to generate new fibres the lens epithelium becomes hyperplastic

corrected for gender, cage density, lighting condition and number of run-through. However, within each mouse genotype, significant differences were found in time spent in the open compartment both in relation to light conditions and in relation to cage Laboratory Animals (2008) 42

40

Figure 3 GS1 B4 lectin staining of lenses from wild-type mouse (a) and a-GT KO mice (b)

density in home cages (Table 4). As expected mice from both genotypes preferred the closed and darkest compartment, but no significant difference between the genotypes in the degree of preference of compartment in matched light conditions could be established.

Discussion Deficiency of the a-gal epitope in mice results in severe cataract in mice between three and six weeks of age in 100% of a-GT KO mice. The impaired visual abilities of the cataractous mice do not seem to affect their perception of light or their ability to distinguish between a closed or an a open compartment. However, increased aggressive behaviour was seen as a-GT KO mice showed longer duration of attacks and more Laboratory Animals (2008) 42


mounting behaviour compared with wildtype mice in the social interaction test. The manifestation of cataract in a-GT KO mice was homogeneous in appearance and the onset of the cataract was comparable with the observations done by Eyssens (1999), who found that macroscopic cataract appears around day 36. The rapid progression from no lesions in 3-week-old a-GT KO mice to fully developed cataract, in most instances comprising the whole lens including nucleus, in 6-week-old a-GT KO mice also correlated with the findings of Eyssens (1999). Cataract in a-GT KO mice was not present at birth, but developed rapidly hereafter and before the eye was fully developed. This finding point towards a role for an a-gal epitope bearing molecule involved in the maintenance of lens fibres or involved in the differentiation of epithelial cells to lens fibres. This hypothesis is supported by the fact that the wild-type mice clearly expressed the a-gal epitope in their lenses (Figure 3a) and furthermore by the findings of Ogiso et al., who found that the a-gal epitope was associated with differentiation, elongation and interaction of the lens fibres in the embryonic rat (Ogiso et al. 1997). The biochemical aetiology of the present cataract is not clear. However, a role for the a-gal epitope in development of the lens is consistent with the presence of nucleated cells beneath the epithelial surface that normally does not contain nucleated cells. Occasional cataracts has previously been described in C57BL/6 (Pierro & Spiggle 1967, Smith et al. 1994, Wolf et al. 2000), and we did find one wild-type mouse with minor cataract. Moreover, the development of cataracts in KO mice deficient for certain connexins has been shown to be influenced by unidentified factors dependent on the genetic background (West & Fisher 1985, Gong et al. 1999, Gerido et al. 2003). Thus, an effect attributable to the background can therefore not be ruled out. In this study, the a-GT KO mice had a significantly higher total duration of attacks and they showed more mounting behaviour, indicating a slightly higher level of aggression in a-GT KO mice. The gradual impairment of vision resulting from the cataract

Max

0.0 0.0 1.0 1.7 0.0 14.1

0.0 0.0 0.0 0.0 0.0 1.5

0.0 0.0 2.0 2.8 0.0 9.0

0.0 0.0 3.0 6.1 0.0 21.10

0.0 0.0 0.0 0.0 0.0 5.4

0.0 0.0 1.0 0.95 0.0 8.85

Q1

0.0 0.0 2.50 3.70 0.0 11.50

Med

0.0 0.0 5.5 6.75 0.0 18.9

5.0 5.1 10.0 33.2 3.0 26.8

0.0 0.0 0.0 0.0 0.0 1.5

0.0 0.0 3.0 5.3 0.0 21.1

Q1

0.0 0.0 7.0 15.7 0.0 26.6

Med

Min

Max

5.0 5.1 23.0 58.6 3.0 52.8

Min

Q3

1.0 1.7 9.0 20.2 1.0 26.5

Six males/cage (n ¼ 21)

0.0 0.0 5.0 6.75 0.0 21.95

Med

Four males/cage (n ¼ 16)

Cage density

0.0 0.00 0.0 0.0 0.0 7.2

Q1

Min

P value (non-parametric ANOVA of rank transformed outcome variables)

b (Observed behavioural pattern in mice housed at different cage densities) Number of attacks Duration of attacks Number of chases Duration of chasing Mounting behaviour Duration of anogenital sniffing

a (Observed behavioural pattern in the two genotypes) Number of attacks Duration of attacks Number of chases Duration of chasing Mounting behaviour Duration of anogenital sniffing

Q3

Wild type (n ¼ 19) Med

Min

Q1

a-GT KO (n ¼ 18)

Genotype

1.0 0.5 14.0 32.9 0.0 35.0

Q3

0.0 0.0 13.0 21.6 0.0 32.4

Q3

3.0 2.8 24.0 58.6 3.0 69.3

Max

1.0 0.8 24.0 48.0 1.0 69.3

Max

0.393 0.468 0.009 0.007 0.746 0.0001

P

0.051 0.045 0.819 0.885 0.049 0.703

P

Table 3 Behavioural patterns in the social interaction test for a-GT knockout (KO) and wild-type mice (wild type). (a) Observed behavioural pattern in the two genotypes. (b) Observed behavioural pattern in mice housed at different cage densities

Aggression in cataract-bearing a-GT KO mice 41


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Table 4


The following observed behaviours were significantly influence by a number of variables as listed

Observation behaviour

Significant variable

Level

Median

Min– max

Latency to enter dark compartment (min)

Cage density in home cage

4 animals/cage 6 animals/cage 1st run-through 2nd run-through Females Males 4 animals/cage 6 animals/cage Bright light (800 lux) Dim light (8 lux) 1st run-through 2nd run-through 4 animals/cage 6 animals/cage Bright light (800 lux) Dim light (8 lux) 1st run-through 2nd run-through

0.07 0.15 0.23 0.05 25 19 17 23 15

0.02– 3.1 0.02– 10.00 0.03– 10.00 0.02– 10.00 0– 93 0– 46 3– 68 0– 93 0– 42

27.5 23 20 1.14 3.13 1.53

0– 93 0– 68 0– 93 0.06– 6.34 0.21– 10.00 0.13– 10.00

2.51 2.50 1.53

0.6– 10.00 0.42– 10.00 0.06– 10.00

Number of run-through Number of transitions

Gender Cage density in home cage Light condition in open compartment No of run-through

Time spent in open compartment (min)

Cage density in home cage Light condition in open compartment No of run-through

Asterisks denoted the level of significance: P , 0.001;

P , 0.01;

may reduce the ability to recognize the visual signals related to agonistic behaviour and thus cataractous mice may not be able to apprehend submissive appeasement behaviour of the opponents. Even though mice aggression are clearly connected to olfactory stimuli (e.g. Gray & Hurst 1995, Van Loo et al. 2000, Latham & Mason 2004), male aggression may also be modulated by other factors (Mucignat-Caretta et al. 2004). Visual deprivation has been shown to result in a higher frequency of attacking as well as a longer duration of attack in the intruder test in FVB/N mice suffering from retinal degeneration (Pugh et al. 2004), which correlates well with the results of this study. In another study, mice were blinded using opaque contact lenses. In these mice, chasing behaviour was not found to be affected; however, the frequency of misdirected attacks was significantly increased and the frequency of successful attacks were reduced. Hence, deprivation of vision seems to impair the attack phase in mouse agonistic behaviour (Strasser & Dixon 1986). The changes observed in this study in agonistic behaviour cannot be ascribed to environmental factors, as all KO mice were housed in cages with B6s, ensuring the same environment for both strains. However, a not Laboratory Animals (2008) 42

P , 0.05

yet defined effect of the genetic modification on agonistic behaviour cannot be ruled out. Aggression in genetically modified mice has been shown to be influenced by several genes (Miczek et al. 2001) and there is no obvious pattern linking certain gene products to either an increase or a decrease in aggression. Hence, aggression in mice is probably under polygenic influence. Moreover, factors like background strain, maternal environment and the characteristic of the intruder males (breeding males, docile strains, aggressive strains or even bulbectomized males) has been demonstrated to influence aggressive behaviour (Miczek et al. 2001). The changes in agonistic behaviour could thus be ascribed to a variety of factors. It is important to realize that an increased level of aggression may severely reduce the welfare of socially housed animals – especially for the subdominant and subordinate individuals. However, the overall effect of the cataract on aggression was rather discrete in a-GT KO mice. Gender and cage density seem to have more pronounced effects. In the modified light/dark test, the number of transitions as well as latency to enter the dark compartment was influenced by several factors (Table 4); but no difference was found between a-GT KO mice and


controls. Both a-GT KO and wild-type mice found the brightly lit compartment aversive and were most likely able to visually discriminate between the open and the closed compartment as both genotypes spent significantly less time in the open compartment under both light conditions. The difference in time spent in the open compartment in dim vs. bright light was not different between the two genotypes. Hence, the a-GT KO mice did not appear to be less sensitive to bright light or less able to perceive the open area as being open and hence more dangerous. However, these behavioural patterns were influenced by cage density as mice housed at high cage density showed a longer latency to enter the dark compartment, had a higher rate of transitions and spent significantly longer time in the open compartment (Table 4), which all indicate a lower level of anxiety (Hascoet et al. 2001, McIlwain et al. 2001). Males housed in groups of higher density also showed significantly more ano-genital sniffing and chasing behaviour, i.e. increased aggression. When the open area was lit, the number of transitions was significantly lower, and mice of both genotypes stayed longer in the dark compartment indicating a higher level of anxiety (McIlwain et al. 2001). The significant smaller body weight of a-GT KO mice compared with wild-type mice cannot be explained by the present data but it may be hypothesized that other physiological changes due to knockout of a-GT may cause yet unidentified phenotypic consequences in a-GT KO mice. In conclusion, the behavioural impact on the a-GT KO mouse is not dramatic, however, there is still evidence of an increase in some specific aggressive behaviours. To further elucidate the true level of aggression in the a-GT KO mouse, a standard intruder test could be performed, in which all elements of the agonistic behaviour of this mouse could be studied in detail. Moreover, testing the visual acuity of these cataractous animals may add information to their visual abilities, which would also be of importance for interpretation of the behavioural data in this study. The result of the present study emphasizes the need for behavioural

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phenotypic characterizations of genetically modified animals, as the genetic modification may have effects on behaviour and welfare, which were not anticipated – both in relation to conspecifics, but also in relation to the staff in the laboratory animal facility. Acknowledgements Lennart Kurland and Preben Lund are kindly thanked for skilful breeding of the transgenic strains and conscientious caring for the animals. Henrik Elvang Jensen is kindly thanked for providing good quality photos of the mouse lenses.

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