Female NMRI Mice Research Articles

The development of response surface pathway design to reduce animal numbers in toxicity studies.

BackgroundThis study describes the development of Response Surface Pathway (RSP) design, assesses its performance and effectiveness in estimating LD50, and compares RSP with Up and Down Procedures (UDPs) and Random Walk (RW) design.MethodsA basic 4-level RSP design was used on 36 male ICR mice given intraperitoneal doses of Yessotoxin. Simulations were performed to optimise the design. A k-adjustment factor was introduced to ensure coverage of the dose window and calculate the dose steps. Instead of using equal numbers of mice on all levels, the number of mice was increased at each design level. Additionally, the binomial outcome variable was changed to multinomial. The performance of the RSP designs and a comparison of UDPs and RW were assessed by simulations. The optimised 4-level RSP design was used on 24 female NMRI mice given Azaspiracid-1 intraperitoneally.ResultsThe in vivo experiment with basic 4-level RSP design estimated the LD50 of Yessotoxin to be 463 μg/kgBW (95% CI: 383–535). By inclusion of the k-adjustment factor with equal or increasing numbers of mice on increasing dose levels, the estimate changed to 481 μg/kgBW (95% CI: 362–566) and 447 μg/kgBW (95% CI: 378–504 μg/kgBW), respectively. The optimised 4-level RSP estimated the LD50 to be 473 μg/kgBW (95% CI: 442–517). A similar increase in power was demonstrated using the optimised RSP design on real Azaspiracid-1 data. The simulations showed that the inclusion of the k-adjustment factor, reduction in sample size by increasing the number of mice on higher design levels and incorporation of a multinomial outcome gave estimates of the LD50 that were as good as those with the basic RSP design. Furthermore, optimised RSP design performed on just three levels reduced the number of animals from 36 to 15 without loss of information, when compared with the 4-level designs. Simulated comparison of the RSP design with UDPs and RW design demonstrated the superiority of RSP.ConclusionOptimised RSP design reduces the number of animals needed. The design converges rapidly on the area of interest and is at least as efficient as both the UDPs and RW design.

A new step toward the artificial ovary: survival and proliferation of isolated murine follicles after autologous transplantation in a fibrin scaffold

To create an artificial ovary to provide an alternative way of restoring fertility in patients who cannot benefit from transplantation of cryopreserved ovarian tissue due to the threat of reintroducing malignant cells. In vivo experimental study. Gynecology research unit in a university hospital. Six-week-old female NMRI mice. Autografting of isolated preantral follicles and ovarian cells (OCs) encapsulated in two fibrin matrices containing low concentrations of fibrinogen (F; mg/mL) and thrombin (T; IU/mL): F12.5/T1 and F25/T4. Follicular density and development, OC survival and proliferation, inflammatory response, and vascularization. After 1 week, the follicle recovery rate ranged from 30.8% (F25/T4) to 31.8% (F12.5/T1). With both fibrin formulations, all follicles were found to be alive or minimally damaged, as demonstrated by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay, and at the growing stage (primary, secondary, and antral follicles), confirmed by Ki67 immunostaining. Isolated OCs also survived and proliferated after grafting, as evidenced by <1% apoptotic cells and a high proportion of Ki67-positive cells. Vessels were found in both fibrin formulations, and the global vascular surface area varied from 1.35% (F25/T4) to 1.88% (F12.5/T1). Numerous CD45-positive cells were also observed in both F25/T4 and F12.5/T1 combinations. The present study is the first to show survival and growth of isolated murine ovarian follicles 1 week after autotransplantation of isolated OCs in a fibrin scaffold. The results indicate that fibrin is a promising candidate as a matrix for the construction of an artificial ovary. Xenotransplantation of isolated human follicles and OCs is the necessary next step to validate these findings.

Pentoxifylline Increases Antiadhesion Effect of Streptokinase on Postoperative Adhesion Formation: Involvement of Fibrinolytic Pathway.

Pentoxifylline reduces peritoneal adhesions and increases peritoneal fibrinolysis in rodents. Furthermore, the activation of the fibrinolytic system by streptokinase leading to degradation of fibrin is effective in the prevention of adhesion formation. We have investigated the effects of pentoxifylline and streptokinase alone and/or coadministration on postoperative intra-abdominal adhesion formation in adult female NMRI mice. Drugs were administered from the day of surgery until 10days after surgery. At relaparotomy 11days after surgery, the abdomen was opened, and the adhesions were graded in a blinded fashion utilizing the classification system described. Oral gavage administration of lower doses of pentoxifylline (3.125, 6.25, and 12.5mg/kg) had no significant effect on postsurgical adhesion formation, while the higher doses of pentoxifylline (25 and 50mg/kg) significantly decreased postsurgical adhesion formation. Moreover, intraperitoneal injection of lower doses of streptokinase (9.375, 18.75, and 37.5unit/kg, i.p.) had no significant effect on postsurgical adhesion formation, while the higher doses of streptokinase (75 and 150unit/kg) significantly decreased postsurgical adhesion formation. In other series of experiments, coadministration of lower doses of pentoxifylline and streptokinase doses, which were ineffective when given alone, significantly decreased postsurgical intra-abdominal adhesion formation compared with streptokinase control group. The results suggest that pentoxifylline may interfere with streptokinase in the reduction of postoperative intra-abdominal adhesion formation by enhancing local fibrinolytic activity.

Comparing systemic metabolic responses in mice to single or dual infection with Plasmodium berghei and Heligmosomoides bakeri.

Concomitant infections with Plasmodium and gastrointestinal nematodes are frequently observed in humans. At the metabolic level, the cross-talk between the host and multiple coexisting pathogens is poorly characterized. The purpose of this study was to give a comprehensive insight into the systemic metabolic phenotype of mice with a single or dual infection with Plasmodium berghei and Heligmosomoides bakeri. Four groups of eight NMRI female mice were infected with P. berghei or H. bakeri, or with both species concurrently. An additional group remained uninfected, and served as control. Mice were sacrificed at day 19 of the experiment. We collected samples from the liver, spleen, kidney, three intestinal regions, and four brain regions. All biological samples were subjected to (1)H nuclear magnetic resonance spectroscopy, combined with multivariate data analysis, to establish metabolic fingerprints of each tissue from the various infection groups. Compared to uninfected mice, single and dual species infection models showed unique metabolic profiles. P. berghei exerted major effects on glycolysis, tricarboxylic acid cycle, and nucleotide and amino acid metabolism in all studied tissues with the exception of the gut. H. bakeri was characterized by a dysregulation of choline and lipid metabolism in most tissues examined with a particularly strong imprint in the jejunum. Simultaneous co-infection with P. berghei and H. bakeri induced the strongest and most diverse effects in the liver and spleen but led to only minor changes in the intestinal and cerebral parts assessed. Infection with P. berghei showed more pronounced and systemic alterations in the mice metabolic profile than H. bakeri infection. The metabolic fingerprints in the co-infection models were driven by P. berghei infection, whilst the presence of H. bakeri in co-infections had little effect. However, simultaneous co-infection showed indeed the least metabolic disruptions in the peripheral tissues, namely the gut and brain.

Toxoplasmosis: Experimental Vaginal Infection in NMRI Mice and Its Effect on Uterin, Placenta and Fetus Tissues

BackgroundToxoplasma gondii is an important zoonotic pathogen. Vertical transmission of the parasite occurs when females were infected primarily during gestation. This parasite is transmitted to the fetus through the placenta and may cause miscarriage, permanent neurological damage, premature birth and visual impairment. It has been found that mouse is susceptible to Toxoplasma and is particularly an interesting model to the study of congenital infection but whether the entry of T. gondii through vagina route is involved in transmission of the parasite to the placenta and fetus or not.ObjectivesThe current study aimed to find a route of infection which perhaps carried the parasite under natural conditions in human.Materials and MethodsIn the current experimental study, two 6-8 week NMRI female mice were crossed with one male. The pregnant mice were divided into 2 groups: experimental group that was infected by parasite via intra-vaginal (IV) and control group that received the same volume of normal saline via IV. One mouse from each group was killed on the fifth day after infection. The peritoneal fluid, ovary and uterus of mouse samples were taken and divided into two parts. One part used for DNA extraction and the other was kept in formalin and sent for histological study. These steps were repeated seven times and at least 10 mice in each group (case and control) were studied by molecular and histological methods.ResultsPCR using DNA extracted from the experimental group showed that the parasite existed in tissues of the uterus and placenta but not in the embryos and peritoneal fluid. PCR using DNA extracted from the control group was negative.ConclusionsTachyzoite of Toxoplasma and DNA of this parasite were observed in sub mucosa and muscles of the uterus and in the villis of placenta, but not in histological sections of the fetus. Therefore, histological and molecular results were consistent.

Neonatal NMDA receptor blockade alters anxiety- and depression-related behaviors in a sex-dependent manner in mice

There is increasing evidence that N-methyl-d-aspartate (NMDA) receptor blockade in the neonatal period has a long-lasting influence on brain and behavior development and has been linked to an increased risk for neuropsychiatric disorders in later life. We sought to determine whether postnatal NMDA receptor blockade can affect normal development of body weight, corticosterone levels, anxiety- and depression-related behaviors in male and female mice in adulthood. For this purpose, male and female NMRI mice were treated with either saline or phencyclidine (PCP; 5 and 10 mg/kg, s.c.) on postnatal days (PND) 7, 9, and 11, and then subjected to different behavioral tests, including open field, elevated plus-maze, elevated zero-maze, light-dark box, tail suspension test and forced swimming test in adulthood. The results indicated that neonatal PCP treatment reduced body weight during neonatal and adulthood periods, and did not alter baseline corticosterone levels in both male and female mice. Moreover, this study obtained some experimental evidence showing the PCP at dose of 10 mg/kg increases stress-induced corticosterone levels, anxiety- and depression-related behaviors in males, while decreasing levels of anxiety without any significant effect on depression in female mice in adulthood. These data support the argument that neonatal NMDA receptor blockade can lead to behavioral abnormalities and psychiatric diseases in adulthood. Collectively, our findings suggest that neonatal exposure to PCP may have profound effects on the development of anxiety- and depression-related behaviors in a sex- and dose-dependent manner in mice.

Metabonomic investigations of age- and batch-related variations in female NMRI mice using proton nuclear magnetic resonance spectroscopy

The NMRI outbred mouse model is widely used for studying metabolic disease, toxicity, and infection, yet information regarding baseline metabolism of this murine strain is relatively sparse. Using different batches of female NMRI mice, we assessed the stability of the metabolic phenotype with increasing age and weight, and determined the influence of acclimatization on the metabolic profile of biofluids (urine, plasma, and faecal water). Differences in urinary concentrations of 3-ureidopropionate, 2-oxoisocaproate, trimethylamine, and glycine were detected between three batches of 9-week-old female NMRI mice using proton nuclear magnetic resonance ((1)H NMR) spectroscopy coupled with multivariate statistical analysis. An acclimatization period of 2 weeks was imposed after the mice entered the laboratory environment. Strong differences in the faecal metabolome pre- and post-acclimatization were found (reduction in amino acid concentrations), whilst the urine metabolome showed increased levels of trimethylamine-N-oxide, phenylacetyl glycine, and hippurate with decreased excretion of formate and betaine post-acclimatization. Temporal variation in the metabolite profiles over a 16-week study stabilized around 7-week-old animals. The results from this study strongly argue for inclusion of an acclimatization period prior to starting an investigative procedure, and suggest that the metabolic phenotypes of female NMRI mice are more stable at around 7 weeks of age. We have also identified a set of metabolites that are more susceptible to variation in concentration. This information can serve as a benchmark in order to establish confidence in systematic variation attributable to pathology or therapeutic intervention above the background metabolic variation in the NMRI mouse.

Ovarian Stimulation Affects the Population of Mouse Uterine NK Cells at Early Pregnancy

The aim of this study was to determine the influence of ovarian stimulation on endometrial mouse NK cell population. For superovulation, the female adult NMRI mice were injected i.p. with 10 IU of the pregnant mare serum gonadotropin followed 48 h later by an i.p. injection of 10 IU human chorionic gonadotropin hormone. Ovarian stimulated and nonstimulated mice were mated with fertile male. The presence of vaginal plug proved natural pregnancy, and this day was considered as day one of pregnancy. Tissue samples were prepared from the uterine horn and spleen of both groups of study on 7th day of pregnancy. Serum estradiol-17β and progesterone were measured at the same time. The tissue cryosections were prepared and double stained for CD 161 and CD3 markers, and NK cells population was analyzed. Relative frequency of NK cells was significantly lower in stroma and myometrium in hyperstimulated mice compared with the control group. However, no difference was seen in percentage of NK cells in spleen. The ovarian stimulation influences the proportion of uterine NK cells and may affect the embryo implantation.

A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders

Cobalamin uptake and transport in mammals are mediated by three cobalamin-binding proteins: haptocorrin, intrinsic factor, and transcobalamin. The nature of cobalamin-binding proteins in lower vertebrates remains to be elucidated. The aim of this study was to characterize the cobalamin-binding proteins of the rainbow trout (Oncorhynchus mykiss) and to compare their properties with those of the three human cobalamin-binding proteins. High cobalamin-binding capacity was found in trout stomach (210 pmol/g), roe (400 pmol/g), roe fluid (390 nmol/liter), and plasma (2500 nmol/liter). In all cases, it appeared to be the same protein based on analysis of partial sequences and immunological responses. The trout cobalamin-binding protein was purified from roe fluid, sequenced, and further characterized. Like haptocorrin, the trout cobalamin-binding protein was stable at low pH and had a high binding affinity for the cobalamin analog cobinamide. Like haptocorrin and transcobalamin, the trout cobalamin-binding protein was present in plasma and recognized ligands with altered nucleotide moiety. Like intrinsic factors, the trout cobalamin-binding protein was present in the stomach and resisted degradation by trypsin and chymotrypsin. It also resembled intrinsic factor in the composition of conserved residues in the primary cobalamin-binding site in the C terminus. The trout cobalamin-binding protein was glycosylated and displayed spectral properties comparable with those of haptocorrin and intrinsic factor. In conclusion, only one soluble cobalamin-binding protein was identified in the rainbow trout, a protein that structurally behaves like an intermediate between the three human cobalamin-binding proteins.

89 Identical obligate anaerobic bacteria in sputum of patients with COPD and CF

Research into the immunopathogensis of chronic bacterial colonisation of cystic fibrosis (CF) patients has been hampered by lack of a suitable animal model. Whilst models of chronic infection have been described, they rely on the use of artificial embedding material to prevent rapid clearance of the bacteria. We hypothesised that careful selection of the bacterial isolate, mouse strain and infection regime, will result in a clinically relevant murine model of chronic S. aureus (SA) and P. aeruginosa (PA) infection without the need for embedding materials. Previously published models have largely used PA01 and lab attenuated SA strains. We have screened several hundred clinical isolates of SA and PA and selected 5 strains of each for murine infection experiments, based on genotype and phenotype. The selected SA isolates include haemolytic strains from CF and non-CF sputum. The PA isolates originated from CF or ICU patients and were selected based on phenotype including ability to form biofilm in vitro. Significant differences in the in vivo virulence of these strains have been shown. Equally essential is mouse strain. C57BL6 mice are used almost exclusively, however this strain are inherently resistant to bacterial infection. We have compared the survival and colonisation of C57BL6, A/J, BALBc, Biozzi, FVB/N, NIH, SJL, CD1, MF1 and NMRI female mice with PA and SA. Identifying a combination of mouse and bacterial strain resulting in the prolonged survival of the animal whilst maintaining chronic pulmonary infection, will allow investigation of the intricate immunological and physiological disease processes involved during chronic pulmonary infection of CF patients.

88 A novel approach to the development of a murine model of cystic fibrosis associated chronic pulmonary bacterial infection

Research into the immunopathogensis of chronic bacterial colonisation of cystic fibrosis (CF) patients has been hampered by lack of a suitable animal model. Whilst models of chronic infection have been described, they rely on the use of artificial embedding material to prevent rapid clearance of the bacteria. We hypothesised that careful selection of the bacterial isolate, mouse strain and infection regime, will result in a clinically relevant murine model of chronic S. aureus (SA) and P. aeruginosa (PA) infection without the need for embedding materials. Previously published models have largely used PA01 and lab attenuated SA strains. We have screened several hundred clinical isolates of SA and PA and selected 5 strains of each for murine infection experiments, based on genotype and phenotype. The selected SA isolates include haemolytic strains from CF and non-CF sputum. The PA isolates originated from CF or ICU patients and were selected based on phenotype including ability to form biofilm in vitro. Significant differences in the in vivo virulence of these strains have been shown. Equally essential is mouse strain. C57BL6 mice are used almost exclusively, however this strain are inherently resistant to bacterial infection. We have compared the survival and colonisation of C57BL6, A/J, BALBc, Biozzi, FVB/N, NIH, SJL, CD1, MF1 and NMRI female mice with PA and SA. Identifying a combination of mouse and bacterial strain resulting in the prolonged survival of the animal whilst maintaining chronic pulmonary infection, will allow investigation of the intricate immunological and physiological disease processes involved during chronic pulmonary infection of CF patients.

Analysis of DNA fragmentation in mouse embryos exposed to an extremely low-frequency electromagnetic field

Effects of extremely low-frequency electromagnetic fields (ELF-EMFs) on DNA damage in biological systems are still a matter of dispute. The aim of the present study was to investigate the possible effect of electromagnetic field exposure on DNA fragmentation in cells (blastomers) of mouse blastocysts.Eighty female NMRI mice were randomly divided into 2 groups of 40 animals each. The control group was left unexposed whereas the animals in the EMF-group were exposed to a 50-Hz EMF at 0.5 mT 4 h per day, 6 days a week for a duration of 2 weeks. After the 8th day of exposure, the female mice in both groups were superovulated (with injections of pregnant mare serum gonadotropin and human chorionic gonadotropin) and then mated overnight. At approximately 4 days after mating (102 h after the human chorionic gonadotropin treatment), blastocysts were obtained by flushing the uterus horns. The mean numbers of pregnant mice, blastocysts after flushing, blastomers within the blastocysts, and the DNA fragmentation index following staining in both groups were compared using statistical methods (SPSS, the Chi-square test, the Student's t-test and the Mann-Whitney U-test, P < 0.05). The results showed that the mean number of blastocysts after flushing was significantly decreased in the EMF-group compared to that of the control group (P < 0.03). The DNA fragmentation index was significantly increased in the EMF-group compared to control (10.53% vs. 7.14%; P < 0.001). However, there was no significant difference in the mean numbers of blastomers and numbers of pregnant mice between the EMF-exposed and control group. Our findings indicate that the EMF exposure in preimplantation stage could have detrimental effects on female mouse fertility and embryo development by decreasing the number of blastocysts and increasing the blastocysts DNA fragmentation.

Chemometric analysis of biofluids from mice experimentally infected with Schistosoma mansoni

BackgroundThe urinary metabolic fingerprint of a patent Schistosoma mansoni infection in the mouse has been characterized using spectroscopic methods. However, the temporal dynamics of metabolic alterations have not been studied at the systems level. Here, we investigated the systems metabolic changes in the mouse upon S. mansoni infection by modeling the sequence of metabolic events in urine, plasma and faecal water.MethodsTen female NMRI mice, aged 5 weeks, were infected with 80 S. mansoni cercariae each. Ten age- and sex-matched mice remained uninfected and served as a control group. Urine, plasma and faecal samples were collected 1 day before, and on eight time points until day 73 post-infection. Biofluid samples were subjected to 1H nuclear magnetic resonance (NMR) spectroscopy and multivariate statistical analyses.ResultsDifferences between S. mansoni-infected and uninfected control mice were found from day 41 onwards. One of the key metabolic signatures in urine and faecal extracts was an alteration in several gut bacteria-related metabolites, whereas the plasma reflected S. mansoni infection by changes in metabolites related to energy homeostasis, such as relatively higher levels of lipids and decreased levels of glucose. We identified 12 urinary biomarkers of S. mansoni infection, among which hippurate, phenylacetylglycine (PAG) and 2-oxoadipate were particularly robust with regard to disease progression. Thirteen plasma metabolites were found to differentiate infected from control mice, with the lipid components, D-3-hydroxybutyrate and glycerophosphorylcholine showing greatest consistency. Faecal extracts were highly variable in chemical composition and therefore only five metabolites were found discriminatory of infected mice, of which 5-aminovalerate was the most stable and showed a positive correlation with urinary PAG.ConclusionsThe composite metabolic signature of S. mansoni in the mouse derived from perturbations in urinary, faecal and plasma composition showed a coherent response in altered energy metabolism and in gut microbial activity. Our findings provide new mechanistic insight into host-parasite interactions across different compartments and identified a set of temporally robust biomarkers of S. mansoni infection, which might assist in derivation of diagnostic assays or metrics for monitoring therapeutic response.

Continuous enriched environment improves learning and memory in adult NMRI mice through theta burst-related-LTP independent mechanisms but is not efficient in advanced aged animals

IntroductionEffects of 3-month continuous environmental enrichment (EE) on cognitive abilities and on theta burst-related synaptic plasticity of CA1 hippocampal neuronal networks have been assessed in 6- and 20-month old NMRI female mice. ResultsEE decreased anxiety-like behavior and improved learning and memory performances in adult but not in aged mice. Electrophysiological results in CA1 hippocampal slices showed that basal synaptic transmission was not affected by EE in adult mice whereas it was partially improved in aged animals, even though not sufficient to rescue the decrease related to aging. Besides, no effect of EE on N-methyl-d-aspartate receptor activation and theta-burst-induced long-term potentiation was found in adult or aged animals. DiscussionThese results indicate that continuous EE is able to improve cognitive abilities in adult NMRI female mice, that does not correlate with changes in theta burst-related synaptic plasticity within neuronal networks. In addition, the lack of effects in aged animals suggests the existence of a critical delay for the beneficial effects of EE on cognitive aging.

Combined oral toxicity of azaspiracid-1 and yessotoxin in female NMRI mice

For many years, the presence of yessotoxins (YTXs) in shellfish has contributed to the outcome of the traditional mouse bioassay and has on many occasions caused closure of shellfisheries. Since YTXs do not appear to cause diarrhoea in man and exert low oral toxicity in animal experiments, it has been suggested that they should be removed from regulation. Before doing so, it is important to determine whether the oral toxicity of YTXs is enhanced when present together with shellfish toxins known to cause damage to the gastrointestinal tract. Consequently, mice were given high doses of YTX, at 1 or 5 mg/kg body weight, either alone or together with azaspiracid-1 (AZA1) at 200 μg/kg. The latter has been shown to induce damage to the small intestine at this level. The combined exposure caused no clinical effects, and no pathological changes were observed in internal organs. These results correspond well with the very low levels of YTX detected in internal organs by means of LC-MS/MS and ELISA after dosing. Indeed, the very low absorption of YTX when given alone remained largely unchanged when YTX was administered in combination with AZA1. Thus, the oral toxicity of YTX is not enhanced in the presence of sub-lethal levels of AZA1.

Local and systemic effects of angiotensin receptor blockade in an emphysema mouse model

Objectives COPD with emphysema causes marked neurohumoral activation. Angiotensin II receptors are highly expressed within the lung and interfere with mechanisms involved in the progression of emphysema. This study examined the effects of an angiotensin II receptor blocker (ARB) on pulmonary and systemic manifestations of emphysema in a mouse model. Methods Female NMRI mice received five intratracheal instillations of porcine pancreatic elastase (emphysema; n = 11) or phosphate-buffered saline (PBS; n = 4). Emphysema severity was quantified histologically by mean linear intercept, exercise tolerance by treadmill running distance, and lung biomechanics by compliance. Following emphysema induction, 6 mice were treated with the ARB irbesartan for 8 weeks, while 5 mice receiving standard food served as controls. Results Following emphysema induction, mean linear intercept was higher in elastase-treated than in PBS-treated lungs (103.0 ± 6.2 μm vs. 35.0 ± 0.6 μm; p = 0.043) while running distance was shorter in emphysema mice (418.6 ± 83.5 m vs. 906.6 ± 244.6 m, p = 0.028). Irbesartan-treated emphysema mice showed a lower mean linear intercept (90.8 ± 3.8 μm vs. 121.5 ± 8.1 μm; p = 0.005), improved compliance (163.6 ± 55.9 μl/cmH 2O vs. 354.4 ± 72.5 μl/cmH 2O; p = 0.063) and greater running distance ( p ANOVA = 0.015) compared to emphysema mice receiving standard food. Conclusions The ARB irbesartan elicits encouraging beneficial effects on emphysema severity, lung biomechanics and exercise capacity in an emphysema mouse model. These findings might help to understand the corresponding positive effects of angiotensin II receptor blockade noticed in patients with COPD.

71 WILL DEHYDRATION BY EXPOSURE TO SUCROSE IMPROVE POST-VITRIFICATION SURVIVAL OF MURINE EMBRYOS VITRIFIED BY THE OPEN PULLED STRAW METHOD?

Vitrification of mammalian embryos comprises suspension in highly concentrated solutions of penetrating cryoprotectants. These cryoprotectants are known to be extremely cytotoxic to cells in an unfrozen state. In the present experiment, it was attempted to reduce the amount of cryoprotectants entering the cells to a minimum by at least partially dehydrating the blastomeres before exposing them to vitrification solutions. Sucrose is known to be a non-permeating osmotically active agent and is, therefore, suited to serve this purpose. It has been shown that concentrations of up to 1.1 M sucrose are well tolerated (Krag et al. 1985 Theriogenology 23, 199). Morphologically intact mouse blastocysts collected from superovulated 5- to 8-week-old virgin female NMRI mice were randomly allocated to 4 treatment groups (100 embryos/group). A control group (Group 1) was vitrified according to the slightly modified (El-Gayar et al. 2008 Cryobiology 57, 191–194) protocol of (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58). The protocol involves exposure to 10% dimethyl-sulfoxide (Me2SO) + 10% ethylene glycol (EG) for 1 min; 20% Me2SO + 20% EG for 20 s; loading into straws and plunging directly into liquid nitrogen. In embryos of Group 2, the procedure mentioned above was preceded by exposure to a 1 M sucrose solution for 1 min. In Group 3, the procedure of Group 2 was followed; however, vitrification solution no. 1 (10% Me2SO + 10% EG) was omitted. In Group 4, sucrose was added to both vitrification solutions at a concentration of 0.2 mol with the 10% Me2SO + 10% EG-solution and of 0.4 mol with the 20% Me2SO + 20% EG-solution. After warming, embryos of all groups were cultured in vitro in microdrops of M16 medium for 48 h. Differences between means were tested for significance by the chi-square test. All embryos were recovered after warming. In the control group, 97% of the embryos had continued development to the expanded blastocyst stage and 89% had proceeded to hatch. The corresponding values for Groups 2, 3, and 4 were 81 and 64%; 60 and 29%, and 93 and 88%, respectively. Earlier studies have shown that in vitro hatching is closely correlated with in vivo survival (El-Gayar et al. 2008 Cryobiology 57, 191–194; Cryoletters 2010, in print). The differences between the control group and Groups 2 and 3 were significant (P &lt; 0.01), whereas with Group 4 it was not (P &gt; 0.05). Thus, exposure to sucrose before vitrification (Groups 2 and 3) compromised the viability of murine blastocysts rather than protecting them, whereas addition of sucrose to vitrification solutions did not. Because embryo survival after cryopreservation by the standard OPS procedure was exceptionally high, it was impossible to detect a potential improvement. This study was partly supported by a grant from the Egyptian government.

Kv7 (KCNQ) channel openers induce hypothermia in the mouse

Kv7 channels, encoded by corresponding kcnq genes, are expressed both centrally and peripherally where they serve to dampen neuronal activity. While Kv7 channel openers have shown efficacy in neurological and neuropsychiatric disease models, the impact of Kv7 channel activation on physiological endpoint markers have not been addressed in detail. In this study we assessed the effect of a range of Kv7 channel openers with different affinity for neuronal Kv7.2-5 channel subunits on body temperature regulation in mice. Female NMRI mice were acutely exposed to vehicle (10% Tween-80, i.p.), retigabine (3–30mg/kg, i.p., pan-Kv7 channel opener), (S)BMS-204352 (60–240mg/kg, i.p., Kv7.4/5 channel-preferring opener), ICA-27243 (1–10mg/kg, i.p., Kv7.2/3 channel-preferring opener), or S-(1) (10–60mg/kg, i.p., Kv7.2/3 channel-preferring opener), and rectal body temperature was measured 15–120min post-injection. Retigabine (>10mg/kg), ICA-27243 (≥10mg/kg), and S-(1) (≥30mg/kg) dose-dependently lowered rectal body temperature with maximal doses of each Kv7 channel opener inducing a marked drop (>4°C) in rectal temperature. The Kv7 channel openers showed differential temporal pharmacodynamics, which likely reflects their different pharmacokinetic profiles. Pretreatment with the pan-Kv7 channel blocker XE-991 (1.0mg/kg, i.p.) completely reversed the hypothermic effect of the pan-Kv7 opener, retigabine (15mg/kg), whereas ICA-27243-induced hypothermia (10mg/kg) could only be partially prevented by XE-991. Because ICA-27743 and S-(1) are Kv7.2/3 channel subunit-preferring compounds, this suggests that the Kv7.2/3 channel isoform is the predominant substrate for Kv7 channel opener-evoked hypothermia. These data indicate the physiological relevance of Kv7 channel function on body temperature regulation which may potentially reside from central inhibitory Kv7 channel activity.

Schistosoma mansoni Infection but Not Egg Antigen Promotes Recovery from Colitis in Outbred NMRI Mice

The ability of intestinal helminths to manipulate the immune system of their host towards a Th2 response has been proposed to modulate auto-immune and allergic diseases. This initial study investigated the anti-inflammatory potential of S. mansoni and soluble egg antigen (SEA) in a murine model of colitis. Colitis was induced in female NMRI mice by 5% dextran sulfate sodium (DSS) for 7days, either 9weeks post-infection with S. mansoni or during treatment with SEA. In addition to clinical signs of colitis, colon histology, immunohistochemistry, and flow cytometry of leukocytes were performed. Colon cytokines were measured using a quantitative real-time technique. Infection with cercariae of S. mansoni attenuated DSS-induced colitis. Clinical symptoms such as weight loss and shortening of colon length were significantly prevented. Histological scores and cell infiltration were affected and expression of pro-inflammatory cytokines in the colons of infected DSS colitis mice was reduced. In contrast, application of SEA failed to improve colitis, even though some findings like earlier manifestation of inflammation and local induction of Th2 cytokines were similar to the effects of cercarial infection. The results presented here suggest that SEA treatment could not protect mice from acute colitis. However, both infection with S. mansoni and injection of SEA affect mucosal immune responses.

Therapeutic levels of erythropoietin (EPO) achieved after gene electrotransfer to skin in mice

Gene electrotransfer refers to gene transfection by electroporation and is an effective non-viral method for delivering naked DNA into cells and tissues. This study presents data from gene electrotransfer with erythropoietin (EPO) to mouse skin. Nine-week-old female NMRI mice received one, two or three intradermal injections of 50 microg EPO plasmid and were subsequently electroporated. With plate electrodes and 100 microg of EPO, a significant increase in hemoglobin (P<0.01) was observed compared with controls. The level of hemoglobin peaked after 5 weeks but stayed significantly elevated for more than 3 months. Serum EPO was significantly increased (P<0.001) 24 h after the transfection and remained significantly different compared with controls until the maximum level of serum EPO was reached after 2 weeks. Eight weeks after the transfection serum EPO returned to baseline. In this study, we have established that gene electrotransfer to skin of even small amounts of DNA can lead to systemically therapeutic levels of protein. This means that in addition to DNA vaccinations, there is a potential utility for electroporation in alleviating systemic diseases such as cancer and protein deficiency disorders.