Mouse embryo yield and viability after euthanasia by CO2 inhalation or cervical dislocation

Efficient production of transgenic mice requires high yields of viable, healthy embryos. Cervical dislocation (without prior anesthesia) rather than CO2 inhalation as a means of euthanasia has been justified on the basis of the increased yield of viable ova, but controlled studies have not directly supported this contention. The American Veterinary Medical Association (AVMA) and Canadian Council on Animal Care (CCAC) Guides, and respective Institutional Animal Care and Use Committees (IACUC) have supported the use of CO2 as a preferred, humane method. The study reported here was undertaken to determine the relative yields of viable embryos from mice euthanized either by inhalation of 100% CO2 or by cervical dislocation. Inbred and hybrid mouse strains, representative of common strains used in genetic engineering experimentation included C57BL/6, FVB/N, and B6SJLF1. There was no difference in the embryo yields in comparisons using the two methods of euthanasia (P = 0.534). Decisions regarding the method of euthanasia can be made on the basis of criteria other than those associated with embryo yield and viability.     

Structural teratogenicity evaluation of methyl chloride in rats and mice after inhalation exposure

One hundred bred Fischer-344 female rats were exposed daily for 6 hours to atmospheres containing 0, 100, 500, or 1,500 ppm methyl chloride, 25 females per exposure concentration, from gestation day (gd) 7 through gd 19. On gd 20, the females were sacrificed for evaluation of maternal reproductive and fetal parameters. Maternal and fetal toxicity was apparent at the highest exposure concentration. There were no methyl chloride-induced external, skeletal, or visceral abnormalities seen in the fetuses. One hundred thirty-two C57BL/6 female mice bred to C3H males to produce B6C3F1 offspring were exposed daily for 6 hours to atmospheres containing 0, 100, 500, or 1,500 ppm methyl chloride, 33 females per exposure concentration, from gd 6 through gd 17. Exposure to the entire 1,500-ppm group was terminated on gd 10-14, with the animals killed in extremis. Selective necrosis of neurons in the internal granular layer of the cerebellum, ranging from individual cell involvement to focal areas comprising large numbers of neurons, was found in all females. On gd 18, the females from the other treatment groups, all of which survived, were killed for evaluation of maternal reproductive and fetal parameters. No evidence was seen of maternal or fetal toxicity in these exposure groups. There were no significant alterations in external appearance in fetuses from any of the exposure groups. Visceral examination of mouse fetuses revealed a small, but statistically significant, incidence of heart defects in litters of the 500-ppm group. The anomaly, a reduction or absence of the atrioventricular valve, chordae tendineae, and papillary muscle, was observed on the left side (bicuspid valve) in three fetuses and the right side (tricuspid valve) in six fetuses: three males and six females. It is concluded that methyl chloride inhalation exposure in pregnant rats, during critical periods of embryo and fetal development, is not teratogenic at concentrations which elicit maternal and fetal toxicity. In pregnant mice, methyl chloride was severely toxic to dams following 4 days or more of exposure to 1,500 ppm in air. Methyl chloride, at 500, but not 100 ppm, was teratogenic in mice, leading to a malformation in the heart. No embryo-fetal toxicity or teratogenicity was associated with exposure of mice, during critical periods of embryo and fetal development, to 100 ppm of ethyl chloride.     

Distinct expression/function of potassium and chloride channels contributes to the diverse volume regulation in cortical astrocytes of GFAP/EGFP mice

Recently, we have identified two astrocytic subpopulations in the cortex of GFAP-EGFP mice, in which the astrocytes are visualized by the enhanced green–fluorescent protein (EGFP) under the control of the human glial fibrillary acidic protein (GFAP) promotor. These astrocytic subpopulations, termed high response- (HR-) and low response- (LR-) astrocytes, differed in the extent of their swelling during oxygen-glucose deprivation (OGD). In the present study we focused on identifying the ion channels or transporters that might underlie the different capabilities of these two astrocytic subpopulations to regulate their volume during OGD. Using three-dimensional confocal morphometry, which enables quantification of the total astrocytic volume, the effects of selected inhibitors of K⁺ and Cl⁻ channels/transporters or glutamate transporters on astrocyte volume changes were determined during 20 minute-OGD in situ. The inhibition of volume regulated anion channels (VRACs) and two-pore domain potassium channels (K_2P) highlighted their distinct contributions to volume regulation in HR-/LR-astrocytes. While the inhibition of VRACs or K_2P channels revealed their contribution to the swelling of HR-astrocytes, in LR-astrocytes they were both involved in anion/K⁺ effluxes. Additionally, the inhibition of Na⁺-K⁺-Cl⁻ co-transporters in HR-astrocytes led to a reduction of cell swelling, but it had no effect on LR-astrocyte volume. Moreover, employing real-time single-cell quantitative polymerase chain reaction (PCR), we characterized the expression profiles of EGFP-positive astrocytes with a focus on those ion channels and transporters participating in astrocyte swelling and volume regulation. The PCR data revealed the existence of two astrocytic subpopulations markedly differing in their gene expression levels for inwardly rectifying K⁺ channels (Kir4.1), K_2P channels (TREK-1 and TWIK-1) and Cl⁻ channels (ClC2). Thus, we propose that the diverse volume changes displayed by cortical astrocytes during OGD mainly result from their distinct expression patterns of ClC2 and K_2P channels.     

打顶对烟草生长、钾素吸收及其分配的影响

与不打顶相比,打顶并未增加烟株最终的干物质及K累积量,但由于消除了顶端对有机物质及K的强烈竞争,使无谓消耗于顶中的1／3的同化物及K运向其它器官,从而使叶片中累积的同化物及K由不打顶时的1／3左右提高到1／2左右,有利于同化产物及K在叶内的累积;此外打顶不了根对同化产物的竞争能力,因而要系发达、烟叶产量及其含K量显著增加,打顶后用NAA处理茎断口并不能提高烟叶产量,但烟株有烟叶内K的最终累积量增加,因此与公打顶相比可明显提高生育后期叶片K含量。     

Lung mechanics and neuromuscular output during CO2 inhalation after airway anesthesia

Airway anesthesia with aerosolized lidocaine has been associated with an increase in minute ventilation (VE) during CO2 inhalation. The increase in VE may be due to increased neuromuscular output or decreased mechanical load on breathing. To evaluate this we measured VE, breathing pattern, mouth occlusion pressure, and lung mechanics in 20 normal subjects during room-air breathing and then inhalation of 6% CO2-94% O2, before and after airway anesthesia. Measurements of lung mechanics included whole-lung resistance, dynamic and static compliance, and functional residual capacity. Airway anesthesia had no detectable effect on any measurements during room-air breathing. During CO2 inhalation, airway anesthesia produced increases in VE and mean inspiratory flow rate (VT/TI) and more negative inspiratory pleural pressure but had no detectable effect on lung mechanics or mouth occlusion pressure. Pleural pressure was more negative during the latter 25% of inspiration. We concluded that airway receptors accessible to airway anesthesia play a role in determining neuromuscular output during CO2 inhalation.     

Sodium chloride, potassium chloride, and virulence in Listeria monocytogenes.

Virulence, as determined in a mouse model, and the virulence factor activities of catalase, superoxide dismutase, and listeriolysin O were examined in a parental strain (10403S) and in a nonhemolytic mutant strain (DP-L224) of Listeria monocytogenes. The cells were propagated in media containing various concentrations of sodium chloride or potassium chloride. Strains 10403S and DP-L224 exhibited significant increases in catalase activity and listeriolysin O activity when grown in medium containing either salt at 428 mM. The superoxide dismutase activities for both strains increased when they were grown in medium containing either salt. The superoxide dismutase activity was significantly increased only when cells were propagated in medium containing no salt compared with that when they were propagated in medium containing either salt at 1,112 mM. In addition, the listeriolysin O activity was highest for cells propagated in medium containing KCl at 428 mM, while the activity was significantly less for cells propagated in medium containing NaCl at an equal concentration. Virulence was examined in mouse livers and spleens after intravenous infection, and approximate 50% lethal doses were determined after intragastric and intraperitoneal infection. Each method of infection indicated that listeriolysin O is required for virulence, while growth in salt-containing medium or the production of higher levels of catalase, superoxide dismutase, and listeriolysin O do not appear to enhance the virulence of L. monocytogenes.     

Cochlear function in mice following inhalation of brevetoxin-3

Brevetoxin-3 was shown previously to adversely affect central auditory function in goldfish. The present study evaluated the effects of exposure to this agent on cochlear function in mice using the 2f₁-f₂ distortion-product otoacoustic emission (DPOAE). Towards this end, inbred CBA/CaJ mice were exposed to a relatively high concentration of brevetoxin-3 (~400 g/m³) by nose-only inhalation for a 2-h period. Further, a subset of these mice received a second exposure a day later that lasted for an additional 4 h. Mice exposed only once for 2 h did not exhibit any notable cochlear effects. Similarly, mice exposed two times, for a cumulative dose of 6 h, exhibited essentially no change in DPOAE levels.     

Euthanasia via CO2 inhalation causes premature cortical granule exocytosis in mouse oocytes and influences in vitro fertilization and embryo development

Generation of high quality mouse metaphase II oocytes is an integral part for efficient in vitro fertilization (IVF), and subsequent embryo production for reproductive studies and genome banking. The main objectives of this study were to investigate the impact of various euthanasia methods on IVF, embryo development, and subcellular structures of MII mouse oocytes. Following superovulation regimen, female mice were euthanized by high flow CO₂ (H CO₂), low flow CO₂ (L CO₂), or cervical dislocation (CD). The MII oocytes obtained from these mice were evaluated for subcellular integrity by assessing their cortical granules and F‐actin. Furthermore, fertilization and subsequent embryonic development competence up to blastocyst stage were also evaluated in vitro. The oocytes collected from females euthanized by CD resulted in significantly higher two‐cell development rates (p = 0.028) and subsequently lead to in higher embryo development rates (p = 0.027) compared with oocytes from females euthanized by L CO₂. The cortical granule integrity analysis revealed significantly higher rate of premature cortical granules exocytosis (PCGE) for L CO₂ group compared with CD and H CO₂ groups (p < 0.001). These data collectively suggest that CO₂ associated PCGE during euthanasia procedure is the main cause of decreased IVF rates and CD is the optimal euthanasia method for the purpose of obtaining good quality MII oocytes for mouse IVF and other reproductive studies.     

Ferrokinetics and erythropoiesis in mice after long-term inhalation of benzene

Ferrokinetics and erythropoiesis were examined in mice exposed for 6 or 7 weeks to an airborne concentration of 300 ppm of benzene, for 6 h per day, and 5 days per week. Ferrokinetic indicators showed only a slightly enhanced production of haeme and erythrocytes in the spleen (133% +/- 18% and 122% +/- 17%, respectively). Production did not change in the femoral marrow; a decline of CFU-C, BFU-E and especially CFU-E (34% +/- 8%) took place there and a shift of cellularity into less mature developmental classes in the erythroblast compartment, without this compartment as a whole being damaged. The erythrocytes produced have an enhanced MCV (109% +/- 0%) and MCH (109% +/- 1%) with an unchanged MCHC; their concentration in blood sank to 87% +/- 1%. The absolute reticulocyte count rose to 160% +/- 16%. 59Fe incorporation into the liver declined far below the level attributable to decreased accessibility of the tracer (84% +/- 4%). A shortening of the life span of late erythroblasts and circulating erythrocytes was deduced from these findings and methodological problems related to some of the seemingly controversial findings are discussed.     

Loss of SPEF2 function in mice results in spermatogenesis defects and primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) results from defects in motile cilia function. Mice homozygous for the mutation big giant head (bgh) have several abnormalities commonly associated with PCD, including hydrocephalus, male infertility, and sinusitis. In the present study, we use a variety of histopathological and cell biological techniques to characterize the bgh phenotype, and we identify the bgh mutation using a positional cloning approach. Histopathological, immunofluorescence, and electron microscopic analyses demonstrate that the male infertility results from shortened flagella and disorganized axonemal and accessory structures in elongating spermatids and mature sperm. In addition, there is a reduced number of elongating spermatids during spermatogenesis and mature sperm in the epididymis. Histological analyses show that the hydrocephalus is characterized by severe dilatation of the lateral ventricles and that bgh sinuses have an accumulation of mucus infiltrated by neutrophils. In contrast to the sperm phenotype, electron microscopy demonstrates that mutant respiratory epithelial cilia are ultrastructurally normal, but video microscopic analysis shows that their beat frequency is lower than that of wild-type cilia. Through a positional cloning approach, we identified two sequence variants in the gene encoding sperm flagellar protein 2 (SPEF2), which has been postulated to play an important role in spermatogenesis and flagellar assembly. A causative nonsense mutation was validated by Western blot analysis, strongly suggesting that the bgh phenotype results from the loss of SPEF2 function. Taken together, the data in this study demonstrate that SPEF2 is required for cilia function and identify a new genetic cause of PCD in mice.     

The intracellular potassium and chloride channels: Properties,pharmacology and function (Review)

Channels selective for potassium or chloride ions are present in membranes of intracellular organelles such as sarcoplasmic (endoplasmic) reticulum, mitochondria, nucleus, synaptic vesicles, and chromaffin, and zymogen granules. They probably play an important role in cellular events such as compensation of electrical charges during transport of Ca²⁺, ΔpH formation in mitochondria or V-ATPase containing membrane granules, and regulation of volume changes, due to potassium and chloride transport into intracellular organelles. Intracellular potassium and chloride channels could also be the target for pharmacologically active compounds. This mini-review describes the basic properties, pharmacology, and current hypotheses concerning the functional role of intracellular potassium and chloride channels.     

Breathing pattern in anesthetized chickens: CO2 inhalation and vagotomy

The breathing patterns of 20 anesthetized chickens were studied during progressive suppression of intrapulmonary chemoreceptors (IPC) by various concentrations of CO2 and following bilateral vagotomy. The vagotomy breathing pattern, characterized by a marked accentuation of expiratory times with prolonged expiratory pauses, was markedly different from that induced by CO2 inhalation. Removal of neural input to the central respiratory centers from IPC does not appear to be solely responsible for the altered breathing pattern following vagotomy in birds.     

Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man

Chloride channels play important roles in the plasma membrane and in intracellular organelles. Mice deficient for the ubiquitously expressed ClC-7 Cl(-) channel show severe osteopetrosis and retinal degeneration. Although osteoclasts are present in normal numbers, they fail to resorb bone because they cannot acidify the extracellular resorption lacuna. ClC-7 resides in late endosomal and lysosomal compartments. In osteoclasts, it is highly expressed in the ruffled membrane, formed by the fusion of H(+)-ATPase-containing vesicles, that secretes protons into the lacuna. We also identified CLCN7 mutations in a patient with human infantile malignant osteopetrosis. We conclude that ClC-7 provides the chloride conductance required for an efficient proton pumping by the H(+)-ATPase of the osteoclast ruffled membrane.     

Female sexual behavior in the male rat: facilitation by cortical application of potassium chloride.

Two groups of male rats castrated in adulthood were injected daily with estradiol benzoate for 7 days. During the period of injections the subjects were tested for feminine responses to mounts by nonexperimental males. Potassium chloride was applied to the cortices of the subjects in one of the groups before each test, whereas the other group was similarly treated with saline. Both of the groups displayed lordosis responses to mounts, but showed no feminine soliciting responses. The potassium chloride-treated rats exhibited significantly higher lordosis rates than the control subjects. The results suggest that the cerebral cortex exerts an inhibitory influence on the execution of lordosis responses by male rats.     

Modulation of brain S2 serotonin receptors by lithium, sodium and potassium chloride

The effect of LiCl, NaCl, and KCl on serotonin competition for 3H-ketanserin binding to S2 serotonin receptors in homogenates of rat prefrontal cortex were investigated. LiCl was the most potent of the ionic modulators in lowering the apparent affinity of serotonin for the S2 serotonin receptor. A threshold effect was noted at 12 mM LiCl (a 60% change in IC50); at 120 mM LiCl a nine-fold shift in the serotonin IC50 was noted. 120 mM NaCl or KCl demonstrated similar effects as 12 mM LiCl in reducing serotonin's apparent affinity. These results indicated that monovalent cations modulate S2 serotonin receptor affinity for serotonin and that lithium ion is more potent than sodium or potassium.