Roles of the International Council for Laboratory Animal Science (ICLAS) and International Association of Colleges of Laboratory Animal Medicine (IACLAM) in the Global Organization and Support of 3Rs Advances in Laboratory Animal Science.

Practical implementation of the 3Rs at national and regional levels around the world requires long-term commitment, backing, and coordinated efforts by international associations for laboratory animal medicine and science, including the International Association of Colleges of Laboratory Animal Medicine (IACLAM) and the International Council for Laboratory Animal Science (ICLAS). Together these organizations support the efforts of regional organization and communities of laboratory animal science professionals as well as the development of local associations and professional colleges that promote the training and continuing education of research facility personnel and veterinary specialists. The recent formation of a World Organization for Animal Health (OIE) Collaborating Center for Laboratory Animal Science and Welfare emphasizes the need for research into initiatives promoting laboratory animal welfare, particularly in emerging economies and regions with nascent associations of laboratory animal science.

Anesthetic effects of a mixture of medetomidine, midazolam and butorphanol in two strains of mice.

The combination of ketamine and xylazine is a widely used anesthetic for laboratory animals. However, due to an abuse problem in Japan, ketamine has been specified as a narcotic since 2007. Instead of using ketamine, Kawai et al. reported an injectable formula with an equivalent effect to the mixture of ketamine and xylazine [11]. The mixture of 0.3 mg/kg body weight (b.w.) medetomidine (Med.), 4.0 mg/kg b.w. midazoram (Mid.), and 5.0 mg/kg b.w. butorphanol (But.) produced an anesthetic duration of around 40 min in outbred ICR mice. However, the anesthetic effect of the mixture for inbred mice strains remains unknown. Therefore, we examined anesthetic effects of the mixture of Med., Mid., and But. in the BALB/c and C57BL/6J strains. After intraperitoneal injection into mice, right front paw, left hind paw, and tail pinch reflexes as well as corneal and righting reflexes were observed. Every 5 min, we scored each reflex category as 0 for reaction or 1 for no reaction. As long as the total score was at least 4 out of 5, we considered the mixture as putting a mouse in a surgical anesthetic state. The mixture produced an anesthetic duration of more than 45 min in both strains of mice. These results indicate that the mixture of Med., Mid., and But. can be a useful and effective anesthesia for the BALB/c and C57BL/6J strains of inbred mice as well as outbred ICR mice.

N-Glycosylation modulates the membrane sub-domain distribution and activity of glucose transporter 2 in pancreatic beta cells.

The glucose transporter isoform, GLUT2, -mediated glucose sensing is essential for maintaining normal glucose-stimulated insulin secretion in pancreatic beta cells. We previously reported that GnT-IVa glycosyltransferase is required for the production of an N-glycan structure that acts as a ligand for galectins to form the glycan-galectin lattice that maintains the stable cell surface expression of GLUT2, and cellular glucose transport activity, although the functional relevance of the N-glycosylation of GLUT2 to its membrane sub-domain distribution is not fully understood. In the present study, we demonstrated that disruption of the GLUT2 N-glycan-galectin lattice by the genetic inactivation of GnT-IVa, or by treatment of pancreatic beta cells with competitive glycan mimetics, induced the re-distribution of GLUT2 into the lipid-raft microdomain. This subsequently resulted in the binding of Stomatin to GLUT2 and an attenuation of cellular glucose transport activity. Moreover, disruption of the lipid-raft microdomain by treatment with methyl-ß-cyclodextrin caused the GLUT2 to be released from lipid-rafts and reactivation of the cellular glucose transport activity in GnT-IVa deficient beta cells. These results indicate that the membrane sub-domain distribution of GLUT2 is associated with the glucose transport activity of beta cells, in which the GnT-IVa-dependent formation of the N-glycan-galectin lattice plays an important role. This provides a novel pathophysiological insight into the mechanism of beta cell failure in the pathogenesis of type 2 diabetes.

Pitfalls in mouse norovirus (MNV) detection in fecal samples using RT-PCR, and construction of new MNV-specific primers.

The murine norovirus (MNV), which belongs to the Caliciviridae family, is prevalent in laboratory mice. Since this virus affects macrophages and dendritic cells, infected mice are not suitable for immunological investigations, making it important to detect MNV infections accurately. When we tested RNA extracts derived from mouse feces for MNV detection using nested RT-PCR with a set of MNV-specific primers reported by Goto et al. (Exp. Anim. 58: 135-140, 2009), we found that these primers amplified not only an MNV-specific signal but also amplified a relatively weak signal with a size almost identical to that of the specific signal. Analysis of the nucleotide sequence of this amplified signal revealed that it was at least 98% identical to the exophosphatase gene of a commensal bacterium, Bacteroides vulgatus. Subsequent analysis showed that the signal amplified with a pair of nested primers was from DNA derived from B. vulgatus, which is sometimes present in SPF laboratory mouse feces, and the nested primers used were both partly homologous with the B. vulgatus nucleotide sequence. We thus designed a new set of nested RT-PCR primers that was not cross-reactive with the B. vulgatus genome. PCR products amplified by the newly designed primers were at least 89.3% identical to the MNV RNA polymerase gene in all cases. Our findings demonstrated that the primer set we designed was suitable for detecting an MNV-specific signal without cross-reacting with B. vulgatus DNA in mouse feces.

Effect of three types of mixed anesthetic agents alternate to ketamine in mice.

Ketamine is usually used for murine anesthesia in animal experiments with other anesthetics for its sedation and analgesic effects. However, ketamine was categorized as a narcotic drug in Japan on January 1, 2007. After this act came into effect, a narcotic handling license became necessary for using and possessing ketamine. Pentobarbital sodium, which is also used for laboratory animal experiments as Nembutal, is no longer being manufactured. For these reasons, other anesthetic agents that can be used without a license are needed. In this paper, we examined the use of anesthetics other than ketamine and pentobarbital sodium. A combination anesthetic (M/M/B: 0.3/4/5) was prepared with 0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol. The anesthetics were administered to male ICR mice by intraperitoneal injection. In order to assess anesthetic depth and duration, we stimulated the mice directly after loss of righting reflexes to recovery of these same reflexes and then recorded four parameters--a tail pinch reflex, a pedal withdrawal reflex in the forelimbs, a pedal withdrawal reflex in the hindlimbs, and corneal reflex. Each parameter was scored, and the anesthetic depth, expressed by the total score, was summed. The surgical anesthesia duration of M/M/B: 0.3/4/5 mg/kg was almost identical to the surgical anesthetic duration with a ketamine and xylazine mixture (80-8 mg/kg). These data suggested that mice can be anesthetized by M/M/B: 0.3/4/5 as an alternate to ketamine. We thus can recommend M/M/B: 0.3/4/5 for murine surgical anesthesia.

A diagnostic method for pneumocystis carinii a causative agent of pneumonia in immunodeficient rats.

Immunodeficient animals are in demand in current biomedical research, and they contribute to medical progress. In Pneumocystis infections, a specific histological diagnostic tool may be immunochemistry (IC). However, it was recently reported that the antibody (3F6) was not suitable for detecting Pneumocystis in rats. We purchased another antibody [PNC007] from a commercial source for IC. We could detect positive signals at identical locations with IC and Toluidine blue O in lungs of infected rats. These results corresponded to the results obtained with PCR. We should study the relationship between unexpected positive signals seen in IC and trophic forms in lungs of infected rats. We could clinically diagnose pneumonia caused by Pneumocystis carinii with the diagnostic method we developed.

Implementation of the Three Rs in biomedical research - has the turn of the century turned the tide?

There has been increasing pressure from the public against animal experimentation for testing and research purposes. The Three Rs (replacement, reduction, and refinement) principle is thought to be a key foundation concept in optimising the welfare of animals used in experiments. This retrospective study attempts to investigate the transition of the Three Rs in biomedical research through a review of articles published in Nature Medicine. We categorised all of the articles published in Nature Medicine from 1998 to 2003, on the basis of the pain and distress of the animals used in the experiments featured in the analysed article. We found there were no large fluctuations in the distribution of these categories over this time period. We also examined each article for the presence of a statement relating to the humane use of laboratory animals, and found that the number of articles which included such a statement dramatically increased in 2002. Over the years studied, there was a decreasing trend in the total number of animal types used for the experiments in the articles. Our results suggest that: a) more encouragement by journal editors might improve the attitude of scientists in terms of animal welfare; and b) the progress of replacement appears to be a more long-term effort in the field of biomedical research.

Proteinuria-induced chronic kidney disease in the ICGN/Oa mice with a mutation of Tensin2 gene.

ICGN/Oa mice are used to study the pathophysiological mechanisms underlying proteinuria-induced chronic kidney disease (CKD). Recently, a mutation of tensin2 gene (Tns2) was suggested to be responsible for proteinuria in the inbred ICGN mice. We identified the wild-type (+/+), heterozygous (+/nep), and homozygous (nep/nep) ICGN/Oa mice by PCR assay. The homozygotes developed proteinuria, resulting in nephrotic syndrome (NS) as early as 5 weeks and CKD by 15 weeks. However, the heterozygotes did not show the symptoms of these renal failures. These results indicate that the homozygous tensin2 mutation is necessary for the ICGN/Oa mice to develop proteinuria-induced CKD. Furthermore, we examined the time course of tubulointerstitial fibrosis and the kinetics of tubular epithelial cells (TECs) in the ICGN/Oa mice using immunohistochemical and TUNEL assays. In the renal parenchyma of the five-week-old homozygotes, the expression of alpha-SMA and type I collagen were higher than those in the age-matched wild-type. Additionally, increased TEC proliferation was found at 5 weeks, and increased TEC apoptosis was by 15 weeks in the homozygotes. Tubulointerstitial fibrosis precedes TEC apoptosis in the proteinuria-induced CKD model mice, and that tubulointerstitial fibrosis may be the triggering event of the disease.

Serological evidence for hepatitis e virus infection in laboratory monkeys and pigs in animal facilities in Japan.

In laboratory animal facilities, monkeys and pigs are used for animal experiments, but the details of hepatitis E virus (HEV) infection in these animals are unknown. The risk of infection from laboratory animals to humans has become a concern; therefore, much attention should be paid to the handling of these animals during their care and use, including surgical procedures performed on infected animals. In this connection, serum samples collected from 916 monkeys and 77 pigs kept in 23 animal facilities belonging to the Japanese Association of Laboratory Animal Facilities of National University Corporations (JALAN) and the Japanese Association of Laboratory Animal Facilities of Public and Private Universities (JALAP) in Japan were examined for the purpose of detecting antibodies to HEV and HEV RNA by using ELISA and RT-PCR, respectively. One hundred and seven serum samples of 916 (11.7%) monkeys were positive for anti-HEV IgG, and 7 and 17 serum samples of 916 (0.8% and 5.3%) monkeys were positive for anti-HEV IgM and IgA, respectively. Thirty-six samples from 62 (58.1%) farm pigs were positive for anti-HEV IgG, whereas all samples tested from miniature pigs were negative (0/15, 0%). Seven samples from 62 (9.1%) farm pigs and 7 samples from 916 (0.8%) monkeys were positive for IgM antibody, but these HEV-IgM antibody positive serum samples were HEV-RNA negative by RT-PCR. The IgM antibody positive rate (9.1%) of farm pigs was much higher than that of monkeys (0.8%). These results suggest the relative levels of risk of HEV infection from these animals to animal handlers and researchers who work with them in laboratory animal facilities.

[Alternatives to animal experimentation v.s. animal rights terrorism].

Systematic modern animal experimentation was established by Bernard Claude who wrote "An Introduction to the Study of Experimental Medicine" in 1865. At this point, the public was already asking that the pain and distress of experimental animals be reduced. For this, scientists, William Russell and Rex Burch in 1959 proposed the principles of alternatives to animal experimentation, the "3Rs". Since that time, animal welfare advocates have promoted the 3Rs concept in biomedical research communities. However, cruel animal experiments have continued and there are reports of radical extremists showing their opposition by invasion, arson, theft and even bombing of institutions involved, resulting in killing of the animals. SHAC, one extremist group believed to be animal welfare activitists was recognized as a terrorist group after the 9.11 tragedy in USA and the government viewed their activities very seriously. In 2001, British animal extremists invaded Japanese universities and stole laboratory resources; one individual was arrested and sentenced to prison for three years; Japanese who assisted in the incident were arrested and one was sentenced for one year. In 2006, SHAC USA members were prosecuted and sentenced for up to 6 years for their terrorism activities including arson. We need to consider the background of these activities which are financially supported by animal welfare advocates. The way we, as scientists who conduct such experiments can respond is by promoting alternatives to this experimentation. In Japan, the animal welfare law was revised in 2005 stressing the importance of 3Rs in scientific activities with animals. The promotion of 3Rs should be strengthened in the pharmaceutical community.

The possible involvement of tensin2 in the expression and extension of nephrin by glomerular podocytes in mice.

The prevalence of chronic kidney disease (CKD) is increasing worldwide and proteinuria is a critical prognostic indicator of CKD. Nephrin is produced by podocytes and functions as a slit barrier for inhibition of proteinuria. Nephrin expression is frequently decreased in CKD patients. Nevertheless, the mechanism by which nephrin declines during CKD-related pathological states remains to be determined. Using tensin2-deficient mice (ICGN/Oa strain), we provide evidence that tensin2 is important for glomerular nephrin expression in vivo. In heterozygous mice with a single mutated tensin2 allele, nephrin expression was maintained, while albuminuria was not observed. In contrast, nephrin expression was impaired, especially in the central zones of glomeruli of homozygous mice (with double mutated tensin2 alleles), even at one week after birth. In homozygous mice, extension of synaptopodin, a key actin-associated protein, was also suppressed in the central zone of glomerular tufts. Consistent with the loss of nephrin and synaptopodin expression, severe albuminuria was detected in homozygous ICGN/Oa mice. Therefore, we suggested that tensin2 is involved in expression and extension of nephrin, while tensin2 deficiency may result in proteinuria, associated with the loss of slit integrity.

Hepatocyte growth factor (HGF) promotes oligodendrocyte progenitor cell proliferation and inhibits its differentiation during postnatal development in the rat.

Hepatocyte growth factor (HGF) was initially cloned as a mitogen for hepatocytes and has been identified as a neurotrophic factor for a variety of neurons. However, few attempts have assessed the role of HGF in cells of oligodendrocyte lineage. The purpose of this study was to elucidate the role of HGF in such cells during development. Double immunostaining for either c-Met/HGF receptor or phospho-c-Met with either NG2 or RIP in rat striatum at postnatal day 3 (P3), P7, and P14 revealed that c-Met was phosphorylated on tyrosine residues and thereby activated in NG2(+) oligodendrocyte progenitor cells (OPCs) at P3-P14 and in RIP(+) oligodendrocytes at P14. Intrastriatal injections of recombinant human HGF at both P7 and P10 revealed that the relative ratio of BrdU(+)/NG2(+) cells per total number of NG2(+) cells increased, while BrdU(+)/MBP(+) oligodendrocyte numbers decreased. Western blot analysis showed a down-regulation of myelin basic protein (MBP) after HGF injection. Electron microscopy revealed that the numbers of myelinated nerve fibers decreased after HGF treatment. Furthermore, administration of anti-HGF IgG into the striatum increased the number of BrdU(+)/MBP(+) oligodendrocytes. These findings demonstrated that HGF increases proliferation of OPCs and attenuates their differentiation into myelinating oligodendrocytes, presumably by favoring neurite outgrowth that may be inhibited by the myelin inhibitory molecules on oligodendrocytes. Down-regulation of HGF mRNA in the striatum from P7 to P14, as revealed by quantitative real-time RT-PCR, may be favorable for OPC differentiation into myelinating oligodendrocytes. Our findings suggest that c-Met signaling, together with HGF regulation, plays an important role in developmental oligodendrogenesis.

Genetic uniformity of Echinococcus multilocularis collected from different intermediate host species in Hokkaido, Japan.

DNA from several isolates of Taenia taeniaeformis and Echinococcus multilocularis were digested with restriction enzymes and hybridized with digoxigenated oligonucleotide probe (CAC)5. Within the six wild isolates of Taenia taeniaeformis from Norway rats in Hokkaido, although several bands were common among isolates, fingerprinting patterns were specific to each isolate. In the case of E. multilocularis, regardless of hosts from which each isolate has been isolated, the five isolates collected from Hokkaido, showed the same fingerprinting pattern. These results indicate that there was very little genetic difference among these isolates. Although the fingerprinting pattern of E. multilocularis from St. Lawrence Is. was similar to that of the Hokkaido isolates, some bands were different from those in the Hokkaido isolates. Echinococcus multilocularis in Hokkaido seems to be closely-related genetically to that from St. Lawrence Is.

Deficiency of the tensin2 gene in the ICGN mouse: an animal model for congenital nephrotic syndrome.

The ICGN mouse is a model for nephrotic syndrome (NS) which presents with proteinuria, hyperlipidemia, and edema. In this study we attempted to identify the gene(s) responsible for NS. By analyzing albuminuria in 160 (ICGN x MSM)F(1) x ICGN backcross progenies, we found that NS in the ICGN mouse is caused by more than one gene. We then performed a quantitative trait locus (QTL) analysis and detected a QTL with a very high LOD score peak in the telomeric region of Chr 15. By analyzing the nucleotide sequence of 22 genes located close to the QTL, we found that the tensin2 gene of the ICGN mouse possessed an 8-nucleotide deletion mutation in exon 18, leading to a frameshift and giving rise to a terminal codon at a premature position. Analyses of in situ hybridization and immunohistochemistry revealed that tensin2 was expressed in podocytes and tubular epithelial cells in normal mice but not in the ICGN mouse. These data raise the possibility that a mutation of the tensin2 gene is responsible for NS of the ICGN mouse and tensin2 is a prerequisite for the normal kidney function.

JSAAE activities in alternatives to animal experimentation for undergraduate schools: forum for citizens in primary and secondary schools.

The aim of this paper is to introduce some educational activities by the Japanese Society of Alternatives to Animal Experiments (JSAAE). The JSAAE is an academic society to promote the Three Rs in Japan and also the international biomedical community. Our activities include the education of not only scientists but also the public. In particular, activities should be focused on the education in schools regarding alternatives. The JSAAE organised a forum for citizens in alternatives education in primary and secondary schools during their 14th annual meeting.

Activities in Japan and other Asian countries & summary of the satellite session.

In Japan, the main alternative activities are led by the Japanese Society of Alternatives to Animal Experiments (JSAAE). Therefore, the activities of the JSAAE are discussed as representative of Japanese activities. The JSAAE was established in 1982, as an academic society. Since then, the JSAAE has developed gradually, and it now has 338 memberships and 13 supporting organisations. The JSAAE annually publishes Alternatives to Animal Testing and Experimentation (AATEX) as its official journal, in English. For communication among members and the distribution of information, we regularly publish the newsletter of the JSAAE in Japanese.