Experimental infection of inbred BALB/c and A/J mice with Massachusetts and Brazilian strains of infectious bronchitis virus (IBV)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Plasma corticosterone levels in mouse models of pain

Background: Pain markedly activates the hypothalamic-pituitary-adrenal (HPA) axis and increases plasma corticosterone release interfering significantly with nociceptive behaviour as well as the mechanism of action of analgesic drugs. Aims/Methods: In the present study, we monitored the time course of circulating corticosterone in two mouse strains (C57Bl/6 and Balb/C) under different pain models. In addition, the stress response was investigated following animal handling, intrathecal (i.t.) manipulation and habituation to environmental conditions commonly used in nociceptive experimental assays. We also examined the influence of within-cage order of testing on plasma corticosterone. Results: Subcutaneous injection of capsaicin precipitated a prompt stress response whereas carrageenan and complete Freund's adjuvant induced an increased corticosterone release around the third hour post-injection. However, carrageenan induced a longer increased corticosterone in C57Bl/6 mice. In partial sciatic nerve ligation, neuropathic pain model corticosterone increased only in the first days whereas mechanical hypersensitivity remained much longer. Animal handling also represents an important stressor whereas the i.t. injection per se does not exacerbate the handling-induced stress response. Moreover, the order of testing animals from the same cage does not interfere with plasma corticosterone levels in the intrathecal procedure. Animal habituation to the testing apparatus also does not reduce the immediate corticosterone increase as compared with non-habituated mice. Conclusion: Our data indicate that HPA axis activation in acute and chronic pain models is time dependent and may be dissociated from evoked hyperalgesia. Therefore, HPA-axis activation represents an important variable to be considered when designing experimental assays of persistent pain as well as for interpretation of data.

Equid herpesvirus type-1 exhibits neurotropism and neurovirulence in a mouse model

Intranasal inoculation of equid herpesvirus type-1 (EHV-1) Brazilian strains A4/72 and A9/92 induced an acute and lethal infection in four different inbred mouse strains. Clinical and neurological signs appeared between the 2nd and 3rd day post inoculation (dpi) and included weight loss, ruffled fur, a hunched posture, crouching in corners, nasal and ocular discharges, dyspnoea, dehydration and increased salivation. These signs were followed by increased reactivity to external stimulation, seizures, recumbency and death. The virus was recovered consistently from the brain and viscera of all mice with neurological signs. Histopathological changes consisted of leptomeningitis, focal haemorrhage, ventriculitis, neuronal degeneration and necrosis, neuronophagia, non-suppurative inflammation, multifocal gliosis and perivascular infiltration of polymorphonuclear and mononuclear cells. Immunohistochemical examination demonstrated that EHV-1 strains A4/72 and A9/92 replicated in neurons of the olfactory bulb, the cortex and the hippocampus. In contrast, mice inoculated with the EHV-1 Brazilian strain A3/97 showed neither weight loss nor apparent clinical or neurological signs; however, the virus was recovered consistently from their lungs at 3 dpi. These three EHV-1 strains showed distinct degrees of virulence and tissue tropism in mice. EHV-1 strains A4/72 and A9/92 exhibited a high degree of central nervous system tropism with neuroinvasion and neurovirulence. EHV-1 strain A3/97 was not neurovirulent despite being detected in the brains of infected BALB/c nude mice. These findings indicate that several inbred mouse strains are susceptible to neuropathogenic EHV-1 strains and should be useful models for studying the pathogenesis and mechanisms contributing to EHV-induced myeloencephalopathy in horses. (C) 2011 Elsevier Ltd. All rights reserved.

Novel insights into mechanisms of food allergy and allergic airway inflammation using experimental mouse models

Over the last decades, considerable efforts have been undertaken in the development of animal models mimicking the pathogenesis of allergic diseases occurring in humans. The mouse has rapidly emerged as the animal model of choice, due to considerations of handling and costs and, importantly, due to the availability of a large and increasing arsenal of genetically modified mouse strains and molecular tools facilitating the analysis of complex disease models. Here, we review latest developments in allergy research that have arisen from in vivo experimentation in the mouse, with a focus on models of food allergy and allergic asthma, which constitute major health problems with increasing incidence in industrialized countries. We highlight recent novel findings and controversies in the field, most of which were obtained through the use of gene-deficient or germ-free mice, and discuss new potential therapeutic approaches that have emerged from animal studies and that aim at attenuating allergic reactions in human patients.

Noninvasive determination of intraocular pressure (IOP) in nonsedated mice of 5 different inbred strains

BACKGROUND: Noninvasive intraocular pressure (IOP) measurement in mice is critically important for understanding the pathophysiology of glaucoma. Rebound tonometry is one of the methods that can be used for obtaining such measurements. We evaluated the ability of the rebound tonometer (RT) to determine IOP differences among various mouse strains and whether differences in corneal thickness may affect IOP measurements in these animals. MATERIALS AND METHODS: Five different commonly used mouse strains (BALB/C, CBA/CAHN, AKR/J, CBA/J, and 129P3/J) were used. IOP was measured in eyes from 12 nonsedated animals (6 male and 6 female) from each strain at 2 to 3 months of age using the RT. IOPs were measured in all animals, on 2 different days between 10 AM and 12 PM. Subsequently, a number of eyes from each strain were cannulated to provide a calibration curve specific for that strain. Tonometer readings for all strains were converted to apparent IOP values using the calibration data obtained from the calibration curve of the respective strain. For comparison purposes, IOP values were also obtained using the C57BL/6 calibration data previously reported. IOP for the 5 strains, male and female animals, and the different occasion of measurement were compared using repeat measures analysis of variance. The central corneal thickness (CCT) of another group of 8 male animals from each of the 5 strains was also measured using an optical low coherence reflectometry (OLCR) pachymeter modified for use with mice. CCT values were correlated to mean IOPs of male animals and to the slopes and intercept of individual strain calibration curves. RESULTS: Noninvasive IOP measurements confirm that the BALB/C strain has lower and the CBA/CAHN has higher relative IOPs than other mouse strains while the AKR/J, the CBA/J, and the 129P3/J strains have intermediate IOPs. There is a very good correlation of apparent IOP values obtained by RT with previously reported true IOPs obtained by cannulation. There was a small but statistically significant difference in IOP between male and female animals in 2 strains (129P3/J and AKR/J) with female mice having higher relative IOPs. No correlation between CCT and IOP was detected. CCT did not correlate with any of the constants describing the calibration curves in the various strains. CONCLUSIONS: Noninvasive IOP measurement in mice using the RT can be used to help elucidate IOP phenotype, after prior calibration of the tonometer. CCT has no effect on mouse IOP measurements using the RT.

Genetically determined heterogeneity of lung disease in a mouse model of airway mucus obstruction

Mucus clearance is an important airway innate defense mechanism. Airway-targeted overexpression of the epithelial Na(+) channel β-subunit [encoded by sodium channel nonvoltage gated 1, beta subunit (Scnn1b)] in mice [Scnn1b-transgenic (Tg) mice] increases transepithelial Na(+) absorption and dehydrates the airway surface, which produces key features of human obstructive lung diseases, including mucus obstruction, inflammation, and air-space enlargement. Because the first Scnn1b-Tg mice were generated on a mixed background, the impact of genetic background on disease phenotype in Scnn1b-Tg mice is unknown. To explore this issue, congenic Scnn1b-Tg mice strains were generated on C57BL/6N, C3H/HeN, BALB/cJ, and FVB/NJ backgrounds. All strains exhibited a two- to threefold increase in tracheal epithelial Na(+) absorption, and all developed airway mucus obstruction, inflammation, and air-space enlargement. However, there were striking differences in neonatal survival, ranging from 5 to 80% (FVB/NJstrains also exhibited variable Clara cell necrotic degeneration in neonatal intrapulmonary airways and a variable incidence of pulmonary hemorrhage and lung atelectasis. The spontaneous occurrence of a high surviving BALB/cJ line, which exhibited delayed onset of Na(+) hyperabsorption, provided evidence that: 1) air-space enlargement and postnatal death were only present when Na(+) hyperabsorption occurred early, and 2) inflammation and mucus obstruction developed whenever Na(+) hyperabsorption was expressed. In summary, the genetic context and timing of airway innate immune dysfunction critically determines lung disease phenotype. These mouse strains may be useful to identify key modifier genes and pathways.

The volume effect in irradiated mouse colorectum

Damage of the colorectum is the dose-limiting normal tissue complication following radiotherapy of prostate and cervical cancers. One approach for decreasing complications is to physically reduce the treatment volume. Mathematical models have been previously developed to describe the change in associated toxicity with a change in irradiated volume, i.e. the "volume effect", for serial-type normal tissues including the colorectum. The first goal of this thesis was to test the hypothesis that there would not be a threshold length in the development of obstruction after irradiation of mouse colorectum, as predicted by the Probability model of the volume effect. The second goal was to examine if there were differences in the threshold and in the incidence of colorectal obstruction after irradiation of two mouse strains, C57B1/6 (C57) and C3Hf/Kam (C3H), previously found to be fibrosis-prone and-resistant, respectively, after lung irradiation due, in part, to genetic differences. The hypothesis examined was that differences in incidence between strains were due to the differential expression of the fibrogenic cytokines $\rm TGF\beta$ and $\rm TNF\alpha.$ Various lengths of C57 and C3H mouse colorectum were irradiated and the incidence of colorectal obstruction was followed up to 15 months. A threshold length was observed for both mouse strains, in contradiction of model predictions. The mechanism of the threshold was epithelial regeneration after irradiation. C57 mice had significantly higher incidence of colorectal obstruction compared to C3H mice, especially at smaller irradiated lengths. Colorectal tissue was obtained at various times after irradiation and prepared for histology, immunohistochemistry and RNase protection assay for measurement of $\rm TGF\beta 1,$ 2, 3 and $\rm TNF\alpha$ mRNA. Distinct strain differences in the histological time of appearance and spatial locations of fibrosis were observed. However, there were no consistent strain difference in mRNA levels or immunolocalization for any of the cytokines examined. The data indicate the need for volume effect models that account for biologically important processes, such as the effect of epithelial regeneration after irradiation. As well, changes in fibrogenic cytokines at the mRNA level do not contribute to the strain difference in radiation-induced colorectal obstruction. ^

Mouse models and quantitative trait loci of susceptibility to bleomycin- and radiation-induced pulmonary fibrosis

Radiotherapy involving the thoracic cavity and chemotherapy with the drug bleomycin are both dose limited by the development of pulmonary fibrosis. From evidence that there is variation in the population in susceptibility to pulmonary fibrosis, and animal data, it was hypothesized that individual variation in susceptibility to bleomycin-induced, or radiation-induced, pulmonary fibrosis is, in part, genetically controlled. In this thesis a three generation mouse genetic model of C57BL/6J (fibrosis prone) and C3Hf/Kam (fibrosis resistant) mouse strains and F1 and F2 (F1 intercross) progeny derived from the parental strains was developed to investigate the genetic basis of susceptibility to fibrosis. In the bleomycin studies the mice received 100 mg/kg (125 for females) of bleomycin, via mini osmotic pump. The animals were sacrificed at eight weeks following treatment or when their breathing rate indicated respiratory distress. In the radiation studies the mice were given a single dose of 14 or 16 Gy (Co$\sp{60})$ to the whole thorax and were sacrificed when moribund. The phenotype was defined as the percent of fibrosis area in the left lung as quantified with image analysis of histological sections. Quantitative trait loci (QTL) mapping was used to identify the chromosomal location of genes which contribute to susceptibility to bleomycin-induced pulmonary fibrosis in C57BL/6J mice compared to C3Hf/Kam mice and to determine if the QTL's which influence susceptibility to bleomycin-induced lung fibrosis in these progenitor strains could be implicated in susceptibility to radiation-induced lung fibrosis. For bleomycin, a genome wide scan revealed QTL's on chromosome 17, at the MHC, (LOD = 11.7 for males and 7.2 for females) accounting for approximately 21% of the phenotypic variance, and on chromosome 11 (LOD = 4.9), in male mice only, adding 8% of phenotypic variance. The bleomycin QTL on chromosome 17 was also implicated for susceptibility to radiation-induced fibrosis (LOD = 5.0) and contributes 7% of the phenotypic variance in the radiation study. In conclusion, susceptibility to both bleomycin-induced and radiation-induced pulmonary fibrosis are heritable traits, and are influenced by a genetic factor which maps to a genomic region containing the MHC. ^

Quantitative Analysis of Mouse Retinal Layers Using Automated Segmentation of Spectral Domain Optical Coherence Tomography Images.

PURPOSE Quantification of retinal layers using automated segmentation of optical coherence tomography (OCT) images allows for longitudinal studies of retinal and neurological disorders in mice. The purpose of this study was to compare the performance of automated retinal layer segmentation algorithms with data from manual segmentation in mice using the Spectralis OCT. METHODS Spectral domain OCT images from 55 mice from three different mouse strains were analyzed in total. The OCT scans from 22 C57Bl/6, 22 BALBc, and 11 C3A.Cg-Pde6b(+)Prph2(Rd2) /J mice were automatically segmented using three commercially available automated retinal segmentation algorithms and compared to manual segmentation. RESULTS Fully automated segmentation performed well in mice and showed coefficients of variation (CV) of below 5% for the total retinal volume. However, all three automated segmentation algorithms yielded much thicker total retinal thickness values compared to manual segmentation data (P < 0.0001) due to segmentation errors in the basement membrane. CONCLUSIONS Whereas the automated retinal segmentation algorithms performed well for the inner layers, the retinal pigmentation epithelium (RPE) was delineated within the sclera, leading to consistently thicker measurements of the photoreceptor layer and the total retina. TRANSLATIONAL RELEVANCE The introduction of spectral domain OCT allows for accurate imaging of the mouse retina. Exact quantification of retinal layer thicknesses in mice is important to study layers of interest under various pathological conditions.

Tumor burden and clonality in multiple intestinal neoplasia mouse/normal mouse aggregation chimeras

Aggregation chimeras were formed between C57BL/6 mice heterozygous for the Apcmin (Min) mutation and wild-type SWR mice, that differ in their Pla2g2a status, a modifier of Apcmin, and also in their resistance to intestinal polyp formation. Variation in the dolichos biflorus agglutinin-staining patterns of the intestines of these mouse strains was used to determine the chimeric composition of the intestine in individual mice and to examine the clonal composition of adenomas. Macroscopic adenoma numbers in chimeric mice were compared with the expected adenoma numbers based on the percentage of C57BL/6J-Apcmin/+ epithelium in individual mice. These results unexpectedly show that there was no apparent inhibitory effect of the SWR-derived (Pla2g2a wild-type) tissue on adenoma formation in the C57BL/6J-Apcmin/+ epithelium. This suggests that the main genetic modifiers of the Min phenotype act at a cellular or crypt-restricted level with no discernable systemic effect. All adenomas were seen to contain C57BL/6J-Apcmin/+-derived epithelium, confirming that the germ-line mutation of the mApc gene is necessary to initiate tumorigenesis in this model system, and that the mApc gene acts in a cell autonomous fashion.

Screening for imprinted genes by allelic message display: Identification of a paternally expressed gene Impact on mouse chromosome 18

A systematic screen termed the allelic message display (AMD) was developed for the hunting of imprinted genes. In AMD, differential display PCR is adopted to image allelic expression status of multiple polymorphic transcripts in two parental mouse strains, reciprocal F1 hybrids and pooled backcross progenies. From the displayed patterns, paternally and maternally expressed transcripts can be unequivocally identified. The effectiveness of AMD screening was clearly demonstrated by the identification of a paternally expressed gene Impact on mouse chromosome 18, the predicted product of which belongs to the YCR59c/yigZ hypothetical protein family composed of yeast and bacterial proteins with currently unknown function. In contrast with previous screening methods necessitating positional cloning efforts or generation of parthenogenetic embryos, this approach requires nothing particular but appropriately crossed mice and can be readily applied to any tissues at various developmental stages. Hence, AMD would considerably accelerate the identification of imprinted genes playing pivotal roles in mammalian development and the pathogenesis of various diseases.

Strain-specific differences in mouse hepatic wound healing are mediated by divergent T helper cytokine responses

Hepatic fibrosis represents the generalized response of the liver to injury and is characterized by excessive deposition of extracellular matrix. The cellular basis of this process is complex and involves interplay of many factors, of which cytokines are prominent. We have identified divergent fibrosing responses to injury among mouse strains and taken advantage of these differences to examine and contrast T helper (Th)-derived cytokines during fibrogenesis. Liver injury was induced with carbon tetrachloride, fibrosis was quantitated, and Th1/Th2 cytokine mRNAs measured. Liver injury in BALB/c mice resulted in severe fibrosis, whereas C57BL/6 mice developed comparatively minimal fibrosis. Fibrogenesis was significantly modified in T and B cell-deficient BALB/c and C57BL/6 severe combined immunodeficient (SCID) mice compared with wild-type counterparts, suggesting a role of Th subsets. Fibrogenic BALB/c mice exhibited a Th2 response during the wounding response, whereas C57BL/6 mice displayed a Th1 response, suggesting that hepatic fibrosis is influenced by different T helper subsets. Moreover, mice lacking interferon γ, which default to the Th2 cytokine pathway, exhibited more pronounced fibrotic lesions than did wild-type animals. Finally, shifting of the Th2 response toward a Th1 response by treatment with neutralizing anti-interleukin 4 or with interferon γ itself ameliorated fibrosis in BALB/c mice. These data support a role for immune modulation of hepatic fibrosis and suggest that Th cytokine subsets can modulate the fibrotic response to injury.

Telomeres in the mouse have large inter-chromosomal variations in the number of T2AG3 repeats

The ultra-long telomeres that have been observed in mice are not in accordance with the concept that critical telomere shortening is related to aging and immortalization. Here, we have used quantitative fluorescence in situ hybridization to estimate (T2AG3)n lengths of individual telomeres in various mouse strains. Telomere lengths were very heterogeneous, but specific chromosomes of bone marrow cells and skin fibroblasts from individual mice had similar telomere lengths. We estimate that the shortest telomeres are around 10 kb in length, indicating that each mouse cell has a few telomeres with (T2AG3)n lengths within the range of human telomeres. These short telomeres may be critical in limiting the replicative potential of murine cells.

Genome scan for teratogen-induced clefting susceptibility loci in the mouse: Evidence of both allelic and locus heterogeneity distinguishing cleft lip and cleft palate

Nonsyndromic clefting of the lip and palate in humans has a highly complex etiology, with both multiple genetic loci and exposure to teratogens influencing susceptibility. Previous studies using mouse models have examined only very small portions of the genome. Here we report the findings of a genome-wide search for susceptibility genes for teratogen-induced clefting in the AXB and BXA set of recombinant inbred mouse strains. We compare results obtained using phenytoin (which induces cleft lip) and 6-aminonicotinamide (which induces cleft palate). We use a new statistical approach based on logistic regression suitable for these categorical data to identify several chromosomal regions as possible locations of clefting susceptibility loci, and we review candidate genes located within each region. Because cleft lip and cleft palate do not frequently co-aggregate in human families and because these structures arise semi-independently during development, these disorders are usually considered to be distinct in etiology. Our data, however, implicate several of the same chromosomal regions for both forms of clefting when teratogen-induced. Furthermore, different parental strain alleles are usually associated with clefting of the lip versus that of the palate (i.e., allelic heterogeneity). Because several other chromosomal regions are associated with only one form of clefting, locus heterogeneity also appears to be involved. Our findings in this mouse model suggest several priority areas for evaluation in human epidemiological studies.

Regional and strain-specific gene expression mapping in the adult mouse brain

To determine the genetic causes and molecular mechanisms responsible for neurobehavioral differences in mice, we used highly parallel gene expression profiling to detect genes that are differentially expressed between the 129SvEv and C57BL/6 mouse strains at baseline and in response to seizure. In addition, we identified genes that are differentially expressed in specific brain regions. We found that approximately 1% of expressed genes are differentially expressed between strains in at least one region of the brain and that the gene expression response to seizure is significantly different between the two inbred strains. The results lead to the identification of differences in gene expression that may account for distinct phenotypes in inbred strains and the unique functions of specific brain regions.