As the first step to get historical background data for physiological examinations in juvenile dogs, hematology and blood chemistry data obtained from juvenile beagle dogs (less than 3 months of age) used in the control group of toxicity studies conducted in our laboratory were summarized and compared with those obtained from adult beagle dogs (6 months of age). In the hematological examination, growth of beagle dogs was shown to be associated with increases in erythrocyte parameters and with decreases in reticulocyte and leukocyte counts. In the blood chemical examination, growth of beagle dogs was shown to be associated with increases in aspartate aminotransferase, alanine aminotransferase, and creatinine and with decreases in creatine phosphokinase, glucose, total cholesterol, and calcium. The differential leukocyte ratio showed no age relation, but the actual count showed a tendency toward decrease. Alkaline phosphatase showed a tendency to increase from 0 months of age to 3 months of age, but it decreased at 6 months of age. The present results were roughly similar to those previously reported.
Oxidative stress and the production of reactive oxygen species are known to play a major role in neuronal cell damage, but the exact mechanisms responsible for neuronal injury and death remain uncertain. In the present study, we examined the effects of oxidative stress on spontaneous spike activity and depolarizing outward potassium current by exposing the Retzius neurons of the leech to cumene hydroperoxide (CHP) and hydrogen peroxide (H2O2), the oxidants commonly used to examine oxidative mechanisms mediating cell death. We observed that relatively low concentrations of CHP (0.25, 1, and 1.5 mM) led to a marked prolongation of spontaneous repetitive activity. The prolonged action potentials showed an initial, spike-like depolarization followed by a plateau phase. In contrast, H2O2 at the same and much higher concentrations (0.25 to 5 mM) did not significantly change the duration of spontaneous spike potentials of leech Retzius nerve cells (LRNCs). In the voltage clamp experiments, calcium-activated outward potassium currents, needed for the repolarization of the action potential, were suppressed with CHP, but not with H2O2. The present findings indicate that CHP is a more potent oxidant and neurotoxin than H2O2 and that the effect of CHP on the electrophysiological properties of LRNCs may be due to the inhibition of the potassium channels.
The isolated perfused kidney is commonly utilized as a screening tool for renal clearance and metabolism, and to correlate renal drug deposition to renal function. Here, we report on several aspects of this procedure that will facilitate a higher experimental success rate and lead to a reduction in animal use. First, we investigated the utility of inulin and creatinine as commonly used markers to measure glomerular filtration rate. For inulin, in the presence of either 20 mM glucose or 4.5% dextran in the buffer, significant interference was observed using an anthrone-based colorimetric assay. These findings suggest that caution needs to be exercised when using glucose or dextran and when inulin is quantitated using this method. Under these circumstances the use of alternative methods of inulin quantitation such as fluorescently tagged inulin is preferred. Second, we optimized bovine serum albumin (BSA) and BSA/dextran compositions that are routinely recommended as oncotic agents in the perfusion buffer and found that a 4% BSA/1.67% dextran composition had the best viability of kidney biomarkers in accordance with recommended threshold parameters. These considerations will be of particular relevance to researchers utilizing the isolated perfused kidney as a screening tool to measure renal biology and drug metabolism as well as applications to investigate diabetic nephropathy.
There are many coat colors in the laboratory mouse, Mus musculus. On the basis of traditional genetics, there are four loci, A-D, related to coat color expressions. As shown by previous studies, Japanese wild mice have gray backs and white bellies and are assumed to carry the Aw allele at the A (agouti) locus, which is dominant over any other alleles. However, we collected Japanese wild mice from central Honshu with black coats. To understand this black coat expression, we performed cross experiments concerning the four loci using wild-caught mice and DBA/2 laboratory mice from the standpoint of traditional genetics. The offspring of the current crosses showed the wild type, the blackish type, and the intermediate type from some combinations of parents. Considering the coat colors of the offspring, we did not obtain any evidence that the Japanese wild mice always carry the Aw allele at the A locus. Furthermore, we were not able to explain the current coat color expressions using the traditional logic with regard to the A-D loci and concluded that it is possible for another locus (loci) to be related to the coat color expressions. On the other hand, skull characteristics and external body measurements of the captured wild mice were fundamentally different from those of DBA/2 and offspring from captured wild mice and DBA/2 combinations. Thus, we concluded that the Japanese wild mice had original criteria from a morphological viewpoint.
Klebsiella pneumoniae, Corynebacterium kutscheri, and Streptococcus pneumoniae are important pathogens that cause respiratory infections in laboratory rodents. In this study, we used species-specific triplex PCR analysis to directly detect three common bacterial pathogens associated with respiratory diseases. Specific targets were amplified with conventional PCR using the tyrB gene from K. pneumoniae, gyrB gene from C. kutscheri, and ply gene from S. pneumoniae. Our primers were tested against purified DNA from another eleven murine bacteria to determine primer specificity. Under optimal PCR conditions, the triplex assay simultaneously yielded a 931 bp product from K. pneumoniae, a 540 bp product from C. kutscheri, and a 354 bp product from S. pneumoniae. The triplex assay detection thresholds for pure cultures were 10 pg for K. pneumoniae and S. pneumoniae, and 100 pg for C. kutscheri. All three bacteria were successfully identified in the trachea and lung of experimentally infected mice at the same time. Our triplex PCR method can be used as a useful method for detecting pathogenic bacterial infections in laboratory rodents.
On the basis of our 2011 microbiological monitoring tests, we report here the current microbiological status of mice and rats housed in experimental facilities in Japan. We tested more than 14,000 mice, 6,000 serum samples, 500 fecal or cecal samples, and 200 lung samples from 3,549 mouse facilities within Japanese universities and institutes (U/I), pharmaceutical companies and contract research organizations (P/C). We also tested more than 1,500 rats, 1,600 serum samples, and 20 fecal or cecal samples from 772 U/I and P/C rat facilities. Bacterial cultures, serology, microscopy, PCR, and DNA analysis using DNA chips were performed. Staphylococcus aureus (18.8% in mouse facilities, 58.6% in rat facilities) was the most prevalent agent in both the mouse and rat facilities. The next most prevalent agents in the mouse facilities were murine norovirus (11.97%), intestinal protozoa (0.05-8.49%, from various species), Pasteurella pneumotropica (5.32%), and Helicobacter hepaticus (3.17%), while intestinal protozoa (0.74-6.84% from various species), Syphacia muris (6.20%), Pseudomonas aeruginosa (3.61%), and Pasteurella pneumotropica (3.05%) were the subsequent most prevalent agents in the rat facilities. These results suggest that the currently prevalent microbes in laboratory mice and rats in Japan are mainly opportunistic pathogens, intestinal protozoa, and microbes with low pathogenicity.
Prolactin (PRL) has numerous physiological functions that are mediated by its receptors in target cells. Expression of the rat PRL receptor (PRLR) gene is regulated in a tissue-specific manner via the transcriptional activation of five distinct first exons, i.e., E11, E12, E13, E14, and E15. In the present study, we investigated the expression profiles of these first exon variants of PRLR mRNA in the rat choroid plexus, which is considered to be a site of receptor-mediated PRL transport from the blood to cerebrospinal fluid. Real-time PCR analysis revealed that E13-, E14-, and E15-PRLR mRNA expression levels increased in the choroid plexus in male and female rats during postnatal development, with markedly higher level of E14-PRLR mRNA. In female rats, the E14-PRLR mRNA expression levels increased markedly during lactation compared with the diestrus state, whereas there was no increase in the E13- and E15-PRLR mRNA levels. The E14-PRLR mRNA expression pattern was similar to that of the total PRLR mRNA. The PRL plasma concentration generally correlated with the E14-PRLR mRNA expression levels in both sexes. These findings suggest that PRLR gene expression in the choroid plexus is upregulated mainly via the transcriptional activation of the E14-first exon.
Micro X-ray computed tomography (micro-CT) is widely used in preclinical studies of small animals. However, due to the low soft tissue contrast, segmentation of soft tissues in the micro-CT image is a challenging problem. To gain a better understanding of the macroscopic anatomy of the mouse embryo, 3 fixation methods and 3 metal stainings were examined for micro-CT using C57BL/6J mouse embryos in the present study. The examination demonstrated that 1% acetic acid/95% ethanol fixative together with zinc staining provided a high contrast micro-CT image, enabling the segmentation of soft tissues. Then, using this condition, the macroscopic embryo structure of the nude mouse was examined, revealing lack of a thymus. It appears that micro-CT with the fixation and staining condition devised in the present study could be a powerful tool in detecting the effects of various mutations at embryonic stages.
A combination of hematoma aspiration and local delivery of chemicals may be more effective than either therapy in intracerebral hemorrhage (ICH). The aim of the present study was to develop a rat model of hematoma aspiration plus intralesional injection after ICH. ICH was induced in adult Sprague-Dawley rats by an intrastriatal injection of bacterial collagenase IV. Hematoma aspiration was performed 3.5 h after ICH onset. Following aspiration, normal saline was injected into the lesion cavity. Hematoma aspiration with or without subsequent saline injection significantly reduced the hematoma volume, lesion volume, and perihematomal neutrophil infiltration. Hematoma aspiration plus subsequent intralesional injection is simple, feasible, and safe. This ICH model can be used to assess the effectiveness of hematoma removal plus local delivery of chemicals.