A humanized version of Foxp2 affects cortico-basal ganglia circuits in mice.

It has been proposed that two amino acid substitutions in the transcription factor FOXP2 have been positively selected during human evolution due to effects on aspects of speech and language. Here, we introduce these substitutions into the endogenous Foxp2 gene of mice. Although these mice are generally healthy, they have qualitatively different ultrasonic vocalizations, decreased exploratory behavior and decreased dopamine concentrations in the brain suggesting that the humanized Foxp2 allele affects basal ganglia. In the striatum, a part of the basal ganglia affected in humans with a speech deficit due to a nonfunctional FOXP2 allele, we find that medium spiny neurons have increased dendrite lengths and increased synaptic plasticity. Since mice carrying one nonfunctional Foxp2 allele show opposite effects, this suggests that alterations in cortico-basal ganglia circuits might have been important for the evolution of speech and language in humans.

Standardized, systemic phenotypic analysis of Slc12a1I299F mutant mice.

BACKGROUND: Type I Bartter syndrome is a recessive human nephropathy caused by loss-of-function mutations in the SLC12A1 gene coding for the Na+-K+-2Cl- cotransporter NKCC2. We recently established the mutant mouse line Slc12a1I299F exhibiting kidney defects highly similar to the late-onset manifestation of this hereditary human disease. Besides the kidney defects, low blood pressure and osteopenia were revealed in the homozygous mutant mice which were also described in humans. Beside its strong expression in the kidney, NKCC2 has been also shown to be expressed in other tissues in rodents i.e. the gastrointestinal tract, pancreatic beta cells, and specific compartments of the ear, nasal tissue and eye. RESULTS: To examine if, besides kidney defects, further organ systems and/or metabolic pathways are affected by the Slc12a1I299F mutation as primary or secondary effects, we describe a standardized, systemic phenotypic analysis of the mutant mouse line Slc12a1I299F in the German Mouse Clinic. Slc12a1I299F homozygous mutant mice and Slc12a1I299F heterozygous mutant littermates as controls were tested at the age of 4-6 months. Beside the already published changes in blood pressure and bone metabolism, a significantly lower body weight and fat content were found as new phenotypes for Slc12a1I299F homozygous mutant mice. Small additional effects included a mild erythropenic anemia in homozygous mutant males as well as a slight hyperalgesia in homozygous mutant females. For other functions, such as immunology, lung function and neurology, no distinct alterations were observed. CONCLUSIONS: In this systemic analysis no clear primary effects of the Slc12a1I299F mutation appeared for the organs other than the kidneys where Slc12a1 expression has been described. On the other hand, long-term effects additional and/or secondary to the kidney lesions might also appear in humans harboring SLC12A1 mutations.

Biokinetics of nanoparticles and susceptibility to particulate exposure in a murine model of cystic fibrosis.

BACKGROUND: Persons with cystic fibrosis (CF) are at-risk for health effects from ambient air pollution but little is known about the interaction of nanoparticles (NP) with CF lungs. Here we study the distribution of inhaled NP in a murine CF model and aim to reveal mechanisms contributing to adverse effects of inhaled particles in susceptible populations. METHODS: Chloride channel defective CftrTgH (neoim) Hgu mice were used to analyze lung function, lung distribution and whole body biokinetics of inhaled NP, and inflammatory responses after intratracheal administration of NP. Distribution of 20-nm titanium dioxide NP in lungs was assessed on ultrathin sections immediately and 24 h after a one-hour NP inhalation. NP biokinetics was deduced from total and regional lung deposition and from whole body translocation of inhaled 30-nm iridium NP within 24 h after aerosol inhalation. Inflammatory responses were assessed within 7 days after carbon NP instillation. RESULTS: Cftr mutant females had moderately reduced lung compliance and slightly increased airway resistance compared to wild type mice. We found no genotype dependent differences in total, regional and head deposition or in secondary-organ translocation of inhaled iridium NP. Titanium dioxide inhalation resulted in higher NP uptake by alveolar epithelial cells in Cftr mutants. Instillation of carbon NP induced a comparable acute and transient inflammatory response in both genotypes. The twofold increase of bronchoalveolar lavage (BAL) neutrophils in Cftr mutant compared to wild type mice at day 3 but not at days 1 and 7, indicated an impaired capacity in inflammation resolution in Cftr mutants. Concomitant to the delayed decline of neutrophils, BAL granulocyte-colony stimulating factor was augmented in Cftr mutant mice. Anti-inflammatory 15-hydroxyeicosatetraenoic acid was generally significantly lower in BAL of Cftr mutant than in wild type mice. CONCLUSIONS: Despite lacking alterations in lung deposition and biokinetics of inhaled NP, and absence of significant differences in lung function, higher uptake of NP by alveolar epithelial cells and prolonged, acute inflammatory responses to NP exposure indicate a moderately increased susceptibility of lungs to adverse effects of inhaled NP in Cftr mutant mice and provides potential mechanisms for the increased susceptibility of CF patients to air pollution.

Long-term proteasomal inhibition in transgenic mice by UBB(+1) expression results in dysfunction of central respiration control reminiscent of brainstem neuropathology in Alzheimer patients.

Aging and neurodegeneration are often accompanied by a functionally impaired ubiquitin-proteasome system (UPS). In tauopathies and polyglutamine diseases, a mutant form of ubiquitin B (UBB(+1)) accumulates in disease-specific aggregates. UBB(+1) mRNA is generated at low levels in vivo during transcription from the ubiquitin B locus by molecular misreading. The resulting mutant protein has been shown to inhibit proteasome function. To elucidate causative effects and neuropathological consequences of UBB(+1) accumulation, we used a UBB(+1) expressing transgenic mouse line that models UPS inhibition in neurons and exhibits behavioral phenotypes reminiscent of Alzheimer's disease (AD). In order to reveal affected organs and functions, young and aged UBB(+1) transgenic mice were comprehensively phenotyped for more than 240 parameters. This revealed unexpected changes in spontaneous breathing patterns and an altered response to hypoxic conditions. Our findings point to a central dysfunction of respiratory regulation in transgenic mice in comparison to wild-type littermate mice. Accordingly, UBB(+1) was strongly expressed in brainstem regions of transgenic mice controlling respiration. These regions included, e.g., the medial part of the nucleus of the tractus solitarius and the lateral subdivisions of the parabrachial nucleus. In addition, UBB(+1) was also strongly expressed in these anatomical structures of AD patients (Braak stage #6) and was not expressed in non-demented controls. We conclude that long-term UPS inhibition due to UBB(+1) expression causes central breathing dysfunction in a transgenic mouse model of AD. The UBB(+1) expression pattern in humans is consistent with the contribution of bronchopneumonia as a cause of death in AD patients.

Microphthalmia, parkinsonism, and enhanced nociception in Pitx3 ( 416insG ) mice.

A new spontaneous mouse mutant was characterized by closed eyelids at weaning and without apparent eyes (provisional gene name, eyeless; provisional gene symbol, eyl). The mutation follows a recessive pattern of inheritance and was mapped to the region of chromosome 19 containing Pitx3. Genetic complementation tests using Pitx3 ( ak/+ ) mice confirmed eyl as a new allele of Pitx3 (Pitx3 ( eyl )). Sequencing of the Pitx3 gene in eyl mutants identified an inserted G after cDNA position 416 (416insG; exon 4). The shifted open reading frame is predicted to result in a hybrid protein still containing the Pitx3 homeobox, but followed by 121 new amino acids. The novel Pitx3 ( eyl/eyl ) mutants expressed ophthalmological and brain defects similar to Pitx3 ( ak/ak ) mice: microphthalmia or anophthalmia and loss of dopamine neurons of the substantia nigra. In addition, we observed in the homozygous eyeless mutants increased extramedullary hematopoiesis in the spleen, frequently liver steatosis, and reduced body weight. There were also several behavioral changes in the homozygous mutants, including reduced forelimb grip strength and increased nociception. In addition to these alterations in both sexes, we observed in female Pitx3 ( eyl/eyl ) mice increased anxiety-related behavior, reduced locomotor activity, reduced object exploration, and increased social contacts; however, we observed decreased anxiety-related behavior and increased arousal in males. Most of these defects identified in the new Pitx3 mutation are observed in Parkinson patients, making the Pitx3 ( eyl ) mutant a valuable new model. It is the first mouse mutant carrying a point mutation within the coding region of Pitx3.

Systemic first-line phenotyping.

With the completion of the mouse genome sequence an essential task for biomedical sciences in the twenty-first century will be the generation and functional analysis of mouse models for every gene in the mammalian genome. More than 30,000 mutations in ES cells will be engineered and thousands of mouse disease models will become available over the coming years by the collaborative effort of the International Mouse Knockout Consortium. In order to realize the full value of the mouse models proper characterization, archiving and dissemination of mouse disease models to the research community have to be performed. Phenotyping centers (mouse clinics) provide the necessary capacity, broad expertise, equipment, and infrastructure to carry out large-scale systemic first-line phenotyping. Using the example of the German Mouse Clinic (GMC) we will introduce the reader to the different aspects of the organization of a mouse clinic and present selected methods used in first-line phenotyping.

Pleiotropic effects in Eya3 knockout mice.

BACKGROUND: In Drosophila, mutations in the gene eyes absent (eya) lead to severe defects in eye development. The functions of its mammalian orthologs Eya1-4 are only partially understood and no mouse model exists for Eya3. Therefore, we characterized the phenotype of a new Eya3 knockout mouse mutant. RESULTS: Expression analysis of Eya3 by in-situ hybridizations and beta-Gal-staining of Eya3 mutant mice revealed abundant expression of the gene throughout development, e.g. in brain, eyes, heart, somites and limbs suggesting pleiotropic effects of the mutated gene. A similar complex expression pattern was observed also in zebrafish embryos. The phenotype of young adult Eya3 mouse mutants was systematically analyzed within the German Mouse Clinic. There was no obvious defect in the eyes, ears and kidneys of Eya3 mutant mice. Homozygous mutants displayed decreased bone mineral content and shorter body length. In the lung, the tidal volume at rest was decreased, and electrocardiography showed increased JT- and PQ intervals as well as decreased QRS amplitude. Behavioral analysis of the mutants demonstrated a mild increase in exploratory behavior, but decreased locomotor activity and reduced muscle strength. Analysis of differential gene expression revealed 110 regulated genes in heart and brain. Using real-time PCR, we confirmed Nup155 being down regulated in both organs. CONCLUSION: The loss of Eya3 in the mouse has no apparent effect on eye development. The wide-spread expression of Eya3 in mouse and zebrafish embryos is in contrast to the restricted expression pattern in Xenopus embryos. The loss of Eya3 in mice leads to a broad spectrum of minor physiological changes. Among them, the mutant mice move less than the wild-type mice and, together with the effects on respiratory, muscle and heart function, the mutation might lead to more severe effects when the mice become older. Therefore, future investigations of Eya3 function should focus on aging mice.

Postnatal lung function in the developing rat.

Little is known about lung function during early stages of postnatal maturation, although the complex structural changes associated with developing rat lung are well studied. We therefore analyzed corresponding functional (lung volume, respiratory mechanics, intrapulmonary gas mixing, and gas exchange) and structural (alveolar surface area, mean linear intercept length, and alveolar septal thickness) changes of the developing rat lung at 7-90 days. Total lung capacity (TLC) increased from 1.54 +/- 0.07 to 16.7 +/- 2.46 (SD) ml in proportion to body weight, but an increase in body weight exceeded an increase in lung volume by almost twofold. Series dead space volume increased from 0.21 +/- 0.03 to 1.38 +/- 0.08 ml but decreased relative to TLC from 14% to 8%, indicating that parenchymal growth exceeded growth of conducting airways. Diffusing capacity of CO (D(CO)) increased from 8.1 +/- 0.8 to 214.1 +/- 23.5 micromol min(-1) hPa(-1), corresponding to a substantial increase in surface area from 744 +/- 20 to 6,536 +/- 488 cm(2). D(CO) per unit of lung volume is considerably lower in the immature lung, inasmuch as D(CO)/TLC in 7-day-old rats was only 42% of that in adult (90 day-old) rats. In humans, however, infants and adults show comparable specific D(CO). Our functional and structural analysis shows that gas exchange is limited in the immature rat lung. The pivotal step for improvement of gas exchange occurs with the transition from bulk alveolarization to the phase of expansion of air spaces with septal reconstruction and microvascular maturation.

Candidate genes controlling pulmonary function in mice: transcript profiling and predicted protein structure.

Impaired development and reduced lung capacity are risk factors of asthma and chronic obstructive pulmonary disease. Previously, our genomewide linkage analysis of C3H/HeJ (C3H) and JF1/Msf (JF1) mouse strains identified quantitative trait loci (QTLs) associated with the complex traits of dead space volume (Vd), total lung capacity (TLC), lung compliance (CL), and diffusing capacity for CO (D(CO)). We assessed positional candidate genes by comparing C3H with JF1 lung transcript levels by microarray and by comparing C3H, BALB/cByJ, C57BL/6J, A/J, PWD/PhJ, and JF1 strains, using exon sequencing to predict protein structure. Microarray identified >900 transcripts differing in C3H and JF1 lungs related to lung development, function, and remodeling. Of these, three genes localized to QTLs associated with differences in lung function. C3H and JF1 strains differed in transcript and protein levels of superoxide dismutase 3, extracellular [SOD3; mouse chromosome (mCh) 5: VD] and transcript of trefoil factor 2 (TFF2; mCh 17: TLC and D(CO)), and ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2; mCh 15: TLC and CL). Nucleotide sequencing of Sod3, Tff2, and previously identified Relaxin 1 (Rln1; mCh 19: CL) uncovered polymorphisms that could lead to nonsynonymous amino acid changes and altered predicted protein structure. Gene-targeted Sod3(-/-) mice had increased conducting airway volume (Vd/TLC) compared with strain-matched control Sod3(+/+) mice, consistent with the QTL on mCh 5. Two novel genes (Tff2 and Enpp2) have been identified and two suspected genes (Sod3 and Rln1) have been supported as determinants of lung function in mice. Findings with gene-targeted mice suggest that SOD3 is a contributing factor defining the complex trait of conducting airway volume.

Visualization of the visual cortex in minipigs using fMRI.

Minipigs are widely used to examine physiologic mechanisms under experimental settings. The purpose of our study was to localize the visual cortex of minipigs using visual stimulation in functional magnetic resonance imaging (fMRI). Five male Göttinger minipigs were studied with fMRI during visual stimulation with series of light impulses conducted into the MR cabin via fibre optical wave guides (EPI sequences, block design, 1.5 T clinical routine MR-scanner). Statistics were performed using SPM'99. All minipigs showed activation of the visual cortex due to light stimulation. The activation could be superimposed to T2-weighted structural images with good spatial resolution revealing the exact location of the visual cortex. We were able to localize the visual cortex in minipigs for the first time utilizing fMRI on a routine scanner. However, this may be useful for precise positioning of electrodes or implants or be used as a further uncomplicated non-invasive method in physiologic experiments.

Intravenous infusion anesthesia with Propofol-Midazolam-fentanyl for experimental surgery in swine.

There is a need for prolonged anesthesia procedures in experimental surgery. Animals in this study received fentanyl, Midazolam, and Propofol administered by continuous intravenous infusion for anesthesia along with controlled ventilation. Time of anesthesia was 413 +/- 95 min. Animals could be extubated 20 +/- 12 min after operation. Animals recovered completely from anesthesia by day 1 after surgery with almost normal physical activity. This study clearly shows that intravenous infusion anesthesia is safe and easy to handle in prolonged anesthesia for experimental surgery in swine. This anesthetic protocol can also be used for intraoperative transportation.

Micron-sized intrapulmonary particle deposition in the developing rat lung.

Little is known about the effects of postnatal developmental changes in lung architecture and breathing patterns on intrapulmonary particle deposition. We measured deposition in the developing Wistar-Kyoto rat, whose lung development largely parallels that of humans. Deposition of 2-µm sebacate particles was determined in anesthetized, intubated, spontaneously breathing rats on postnatal days (P) 7 to 90 by aerosol photometry (Karrasch S, Eder G, Bolle I, Tsuda A, Schulz H. J Appl Physiol 107: 1293-1299, 2009). Respiratory parameters were determined by body plethysmography. Tidal volume increased substantially from P7 (0.19 ml) to P90 (2.1 ml) while respiratory rate declined from 182 to 107/min. Breath-specific deposition was lowest (9%) at P7 and P90 and markedly higher at P35 (almost 16%). Structural changes of the alveolar region include a ninefold increase in surface area (Bolle I, Eder G, Takenaka S, Ganguly K, Karrasch S, Zeller C, Neuner M, Kreyling WG, Tsuda A, Schulz H. J Appl Physiol 104: 1167-1176, 2008). Particle deposition per unit of time and surface area peaked at P35 and showed a minimum at P90. At an inhaled particle number concentration of 10(5)/cm(3), there was an estimated 450, 690, and 330 particles/(min × cm(2)) at P7, P35, and P90, respectively. Multiple regression models showed that deposition depends on the mean linear intercept as structural component and the breathing parameters, tidal volume, and respiratory rate (r(2) > 0.9). In conclusion, micron-sized particle deposition was dependent on the stage of postnatal lung development. A maximum was observed during late alveolarization (P35), which corresponds to human lungs of about eight years of age. Children at this age may therefore be more susceptible to micron-sized airborne environmental health hazards.

Model for the deposition of aerosol particles in the respiratory tract of the rat. I. Nonhygroscopic particle deposition.

Rats are used to test the toxicological and pharmacological effects of aerosol particles on the organism. For estimates of the delivered aerosol dose, lung deposition models provide a valuable tool. Here a previously developed deposition model for nonhygroscopic and hygroscopic aerosol particles in the lungs of man (Ferron et al., J. Aerosol Sci. 1988, 19:611) is adapted to the rat by implementing a lung structure for the rat combined with empirical equations for particle deposition due to impaction/sedimentation in the extrathoracic region and in bifurcations. To account for the effect of body weight (BW) on the physiological parameters (lung size, respiration frequency) we present BW-scaling laws with an estimated accuracy of about 16%. The present model shows good agreement with the measured total deposition (per breath) and other models from the literature to within the variability of the experimental data (20% absolute). Our calculations show that the variability of the experimental data is consistent with the combined effects from realistic variations in particle properties (mainly density) and physiological parameters (mainly activity level). For the alveolar region, which is of particular significance for pharmacological and health studies, we show that although the activity level may change the deposited dose by up to a factor of 2.2 for particles between 0.05 and 2.0 microm in diameter, the alveolar dose is almost independent (to within 10%) of activity level for particles between 0.5 and 1 microm, which makes this size range advantageous for pharmacological and toxicological experiments. The present model allows estimates of the total and regional particle dose deposited in the lungs of rats, which are consistent with experimental data. The advantage of the present model is that hygroscopic growth can be included in the calculations.

Chronically implanted epidural electrodes in Göttinger minipigs allow function tests of epiretinal implants.

BACKGROUND: To test the function of implantable devices for electrical stimulation of the retina, long-term registration tests of cortical-evoked potentials are required. Skin electrodes are not appropriate to provide representative recordings, due to the voluminous pneumatic frontal sinus of minipigs. Therefore, epidural electrodes were permanently implanted in minipigs and tested with visual and electrical retinal stimulation. The present study describes long-term recordings of electrically evoked cortical potentials in minipigs. METHODS: Three-channel silver-silver chloride electrodes were fixed on the epidura dorsal to the primary visual cortex of one hemisphere in Göttinger minipigs. Repeated light stimulation was performed and platinum-polyimide film multielectrode arrays were implanted on the retina for electrical stimulation and were later removed. Cortical potentials were recorded after stimulation with short biphasic charge-balanced currents. RESULTS: For up to 18 months, the implanted epidural electrodes allowed recording of visual cortex potentials evoked by visual or electrical retina stimulation. Small changes of response amplitudes in subsequent experiments indicated a stable location and recording properties of the cortex electrodes. Visual stimulation often yielded stronger responses for the contralateral eye. Superthreshold electrical retina stimulation evoked cortical responses with less delay compared to visual stimulation. We found threshold currents of 50 microA for charge-balanced biphasic trains of current impulses. Postoperative examination showed an attached and unchanged retina. CONCLUSIONS: The minipig model is appropriate for the implantation of epiretinal stimulation electrodes and for the long-term tests of retinal implants by recording of cortical responses with chronically implanted epidural electrodes.

TNF-alpha antibodies are as effective as ischemic preconditioning in reducing infarct size in rabbits.

Pretreatment with tumor necrosis factor-alpha (TNF-alpha) antibodies abolishes myocardial infarct size reduction by late ischemic preconditioning (IP). Whether or not TNF-alpha is also important for myocardial infarct size reduction by classic IP is unknown. Anesthetized rabbits were untreated (group 1, n = 7), classically preconditioned by 5 min left coronary artery occlusion/10 min reperfusion (group 2, n = 6), or pretreated with TNF-alpha antibodies without (group 3, n = 6) or with IP (group 4, n = 6) before undergoing 30 min of occlusion and 180 min of reperfusion. Infarct size in group 1 was 44 +/- 11 (means +/- SD)% of the area at risk. With a comparable area at risk, infarct size was reduced to 13 +/- 7%, 23 +/- 8%, and 19 +/- 12% (all P < 0.05) in groups 2, 3, and 4, respectively. The circulating TNF-alpha concentration was increased during ischemia in group 1 from 752 +/- 403 to 1,542 +/- 482 U/ml (P < 0.05) but remained unchanged in all other groups. Circulating TNF-alpha concentration during ischemia and infarct size correlated in all groups (r = 0.76). IP, TNF-alpha antibodies, and the combined approach reduced infarct size to a comparable extent. Therefore, the question of whether or not TNF-alpha is causally involved in the infarct size reduction by IP in rabbits could not be answered.