Welfare Impact of Carbon Dioxide Euthanasia on Laboratory Mice and Rats : A Systematic Review

Background: There has been increased concern about the suitability of CO as a method for euthanasia of laboratory mice and rats, including the potential discomfort, pain or distress that animals may experience prior to loss of consciousness; time to loss of consciousness; best methods for use of CO; and the availability of better alternatives. These discussions have been useful in providing new information, but have resulted in significant confusion regarding the acceptability of CO for rodent euthanasia. In some cases, researchers and veterinarians have become uncertain as to which techniques to recommend or use for euthanasia of laboratory mice and rats. Methods: The International Association of Colleges of Laboratory Animal Medicine (IACLAM) convened a taskforce to examine the evidence for adverse welfare indicators in laboratory rats and mice undergoing CO euthanasia using a SYRCLE-registered systematic review protocol. Of 3,772 papers identified through a database search (PubMed, Web of Science, CAB Direct, Agricola, and grey literature) from 1900 to 2017, 37 studies were identified for detailed review (some including more than one species or age group), including 15 in adult mice, 21 in adult rats, and 5 in neonates of both species. Experiments or reports were excluded if they only assessed parameters other than those directly affecting animal welfare during CO induction and/or euthanasia. Results: Study design and outcome measures were highly variable and there was an unclear to high risk of bias in many of the published studies. Changes in the outcome measures evaluated were inconsistent or poorly differentiated. It is likely that repeated exposures to carbon dioxide inhalation are aversive to adult rats and mice, based on avoidance behavior studies; however, this effect is largely indistinguishable from aversion induced by repeated exposures to other inhalant anesthetic gasses. Conclusion: There is insufficient evidence to permit an unbiased assessment of the effect of CO inhalation during euthanasia on welfare indicators in laboratory mice and rats. Additional well-designed, unbiased, and adequately powered studies are needed to accurately assess the welfare of laboratory mice and rats undergoing euthanasia via CO gas

Genetic Background Strongly Modifies the Severity of Symptoms of Hirschsprung Disease, but Not Hearing Loss in Rats Carrying Ednrbsl Mutations

Hirschsprung disease (HSCR) is thought to result as a consequence of multiple gene interactions that modulate the ability of enteric neural crest cells to populate the developing gut. However, it remains unknown whether the single complete deletion of important HSCR-associated genes is sufficient to result in HSCR disease. In this study, we found that the null mutation of the Ednrb gene, thought indispensable for enteric neuron development, is insufficient to result in HSCR disease when bred onto a different genetic background in rats carrying Ednrbsl mutations. Moreover, we found that this mutation results in serious congenital sensorineural deafness, and these strains may be used as ideal models of Waardenburg Syndrome Type 4 (WS4). Furthermore, we evaluated how the same changed genetic background modifies three features of WS4 syndrome, aganglionosis, hearing loss, and pigment disorder in these congenic strains. We found that the same genetic background markedly changed the aganglionosis, but resulted in only slight changes to hearing loss and pigment disorder. This provided the important evidence, in support of previous studies, that different lineages of neural crest-derived cells migrating along with various pathways are regulated by different signal molecules. This study will help us to better understand complicated diseases such as HSCR and WS4 syndrome

QTL Analysis Identifies a Modifier Locus of Aganglionosis in the Rat Model of Hirschsprung Disease Carrying Ednrb^[sl] Mutations

Hirschsprung disease (HSCR) exhibits complex genetics with incomplete penetrance and variable severity thought to result as a consequence of multiple gene interactions that modulate the ability of enteric neural crest cells to populate the developing gut. As reported previously, when the same null mutation of the Ednrb gene, Ednrb^[sl], was introgressed into the F344 strain, almost 60% of F344-Ednrb^[sl/sl] pups did not show any symptoms of aganglionosis, appearing healthy and normally fertile. These findings strongly suggested that the severity of HSCR was affected by strain-specific genetic factor (s). In this study, the genetic basis of such large strain differences in the severity of aganglionosis in the rat model was studied by whole-genome scanning for quantitative trait loci (QTLs) using an intercross of (AGH-Ednrb^[sl] x F344-Ednrb^[sl]) F1 with the varying severity of aganglionosis. Genome linkage analysis identified one significant QTL on chromosome 2 for the severity of aganglionosis. Our QTL analyses using rat models of HSCR revealed that multiple genetic factors regulated the severity of aganglionosis. Moreover, a known HSCR susceptibility gene, Gdnf, was found in QTL that suggested a novel non-coding sequence mutation in GDNF that modifies the penetrance and severity of the aganglionosis phenotype in EDNRB-deficient rats. A further identification and analysis of responsible genes located on the identified QTL could lead to the richer understanding of the genetic basis of HSCR development

Identification of antigenic peptides derived from B-cell epitopes of nucleocapsid protein of mouse hepatitis virus for serological diagnosis

Mouse hepatitis virus (MHV) infection is found commonly in laboratory mice and this virus has been known to cause various diseases such as subclinical infection, enteritis, hepatitis, and encephalitis. Serological tests are used commonly to diagnose MHV infection. Complete MHV virions have been used primarily as antigens for serological diagnosis to date. To develop an antigen that is more specific, more sensitive, and easier to prepare for serological diagnosis, the antigenic sites in the MHV-nucleocapsid (N) protein were screened in this study. Sixteen antigenic linear sequences in the N protein were found using antisera obtained from mice infected naturally with MHV and a peptide array containing overlapping 10-mer peptides covering the entire N protein. From these antigenic sequences, two synthesized peptides, ILKKTTWADQTERGL and RFDSTLPGFETIMKVL, which were consistent with positions 24-38 and 357-372 of the N protein respectively, were used as antigens in ELISA. Evaluation of ELISA with these peptides revealed that both peptides were specific to anti-MHV antisera. Furthermore, ELISA performed using these peptides was more sensitive than commercial ELISA used for a screening sera from mice infected accidentally to MHV maintained in cages, suggesting that these peptides are useful for serological diagnosis of MHV infection

Analysis of the cell cycle of fibroblasts derived from the LEC rat after X-irradiation

The LEC rat is reported to exhibit hypersensitivity to X-irradiation, deficiency in DNA double-strand break repair, and radio-resistant DNA synthesis. This character of the LEC rat has been thought to be due to abnormal G１arrest in cells after X-irradiation. In this report, we re-investigated the effect of X-irradiation on the cell cycle in primary-cultured fibroblasts . Primarycultured fibroblasts derived from LEC and BN rats were exposed to４Gy of Xray and their cell cycle analysis was performed with a flow cytometer. Fibroblasts derived from both rats showed normal response of the cell cycle, indicating the arrest at both G１‐and G２／M-phase and no difference in the cell cycle population between fibroblasts derived from both rats. In contrast, when the same analysis was performed using the cell line, L７and W８，which had been established from the lung fibroblasts of LEC and control WKAH rats, respectively, by immortalizing with SV４０T-antigen, L７cells but not W８cells showed impaired G１arrest and abnormal cell cycle. These results suggest that fibroblasts derived from LEC rats possess the normal cell cycle response after Xirradiation, if they are kept naive as not immortalized with SV４０T-antigen

Functional analysis of duck, goose, and ostrich 2 '-5 '-oligoadenylate synthetase

Up-to-date the flavivirus infection in avian taxa is not clearly defined. Several reports have demonstrated that many viruses belonging to Flaviviridae may cause diseases in poultry species; however, the susceptibility of other avian species is variable and still unclear. In human and mice, the 2'-5'-oligoadenylate synthetase (OAS) proteins are associated with resistance to the flavivirus infection as well as other virus infections. However, the avian OAS proteins are rarely studied. In our previous studies, we confirmed that the chicken OAS-like protein (chOASL) expressed OAS-enzymatic activity (the classical OAS/RNase L-dependent pathway) as well as the anti-flavivirus activity (the putative OAS/RNase L-independent pathway). Therefore, the current study aimed at functional analysis of avian OAS proteins from duck, goose, and ostrich. The duOASL, goOASL, and osOAS1 proteins expressed enzymatic activity as well as chOASL, whereas osOASL expressed little enzymatic activity. On the other hand, duOASL, goOASL, and osOASL possessed significant antiviral activity against West Nile virus (WNV)-replicon replication as well as chOASL, whereas osOAS1 did not. In addition, similar to chOASL, their antiviral activity was independent of RNase L activation. These results suggest that OASL is the only OAS protein in the duck and goose as well as chicken and possesses both OAS-enzymatic and anti-flavivirus activities, whereas the ostrich possesses both OAS1 and OASL proteins with sharing the functional activities, OAS-enzymatic and anti-flavivirus activities, respectively. It is of interest that the ostrich undergoes differential process in OAS gene evolution from other poultries and thus possesses different molecular mechanism in antiviral activity

Exon skipping of exonuclease 1 in MRL/MpJ mice is caused by a nucleotide substitution of the branchpoint sequence in intron eight

In MRL/MpJ mice, there is a genetic mutation of exonuclease 1 (Exo1), in which the exon 9 is sometimes deleted. In the presentstudy, to check the generation ofthe spliced exons, exon 8-intron 8-exon 9 (pCX/Ex/EIE/B and pCX/Ex/EIE/M) plasmids were temporally transfected in vitro into BALB 3T3 cells, and RT-PCR using appropriate primer pair was carried out 1 day after transfection. In these constructions, pCX/Ex/EIE/B was derived from genomic sequence of C57BL/6 mice, and pCX/Ex/EIE/M was from MRL/MpJ. A spliced band was detected in pCX/Ex/EIE/B, but was present little or very weakly in pCX/Ex/EIE/M. Next, the same spliced band was demonstrated in the pCX/Ex/EIE/M(T) plasmid, in which the branchpoint sequence (BPS) of pCX/Ex/EIE/M including the exon 9 was changed into that of pCX/Ex/EIE/B. The splicing did not occur in the del1/B mutant, in which 1960 nucleotides of the intron 8 were deleted, whereas it was detected in the del2/B plasmid deleted 1036 nucleotides in its middle region. These results suggest that the nucleotide T to A mutation of the BPS in the intron 8 is at least a sufficient for generation of splice variants (tr-1 and tr-2 Exo1)