Thermoregulation by an Australian murine rodent, the ash-grey mouse (Pseudomys albocinereus)

We examine here the thermal physiology of the ash-grey mouse, as there is a paucity of data to explain how Australian rodents meet thermoregulatory demands. Most ash-grey mice remained normothermic over a range of ambient temperatures (10°C to 30°C), although they became hyperthermic at high ambient temperatures. One individual entered torpor at ambient temperatures of 20°C and 25°C, with minimal body temperatures of 24.5°C and 28.4°C respectively, before spontaneously arousing. This is the first evidence of torpor use by an Australian murine rodent. Our data suggest that although ash-grey mice have the physiological ability to use torpor, it is used rarely, presumably due to other behavioural and physiological adaptations. Their higher-than-expected basal metabolic rate (1.56±0.25mLO(2)g(-1)h(-1)) indicates that ash-grey mice do not have a frugal approach to energy expenditure. Other standard physiological variables were typical of a generalised rodent. A readily-available omnivorous diet, nocturnal activity, semi-fossorial habit and social behaviour presumably allow a high energy lifestyle. A reluctance to use torpor, despite an apparent physiological ability to do so, supports the idea that the use of torpor reflects a net balance between the costs and benefits of a heterothermic thermoregulatory strategy.     

A quick protocol for the preparation of mouse retinal cryosections for immunohistochemistry

Immunohistochemistry (IHC) using mouse retinal cryosections is widely used to study the expression and intracellular localization of proteins in mouse retinas. Conventionally, the preparation of retinal cryosections from mice involves tissue fixation, cryoprotection, the removal of the cornea and lens, embedding and sectioning. The procedure takes 1–2 days to complete. Recently, we developed a new technique for the preparation of murine retinal cryosections by coating the sclera with a layer of Super Glue. This enables us to remove the cornea and extract the lens from the unfixed murine eye without causing the eyecup to collapse. In the present study, based on this new technique, we move a step forward to modify the conventional protocol. Unlike in the conventional protocol, in this method, we first coat the unfixed mouse eyeball on the sclera with Super Glue and then remove the cornea and lens. The eyecup is then fixed, cryoprotected and sectioned. This new protocol for the preparation of retinal cryosections reduces the time for the procedure to as little as 2 h. Importantly, the new protocol consistently improves the morphology of retinal sections as well as the image quality of IHC. Thus, this new quick protocol will be greatly beneficial to the community of visual sciences by expediting research progress and improving the results of IHC.     

FVIII gene delivery by muscle electroporation corrects murine hemophilia A

BACKGROUND: Hemophilia A treatment relies on costly factor VIII (FVIII) replacement that may transmit iatrogenic viral diseases. Viral vectors and cell implants are being developed as improvements. We investigated in vivo electroporation of naked DNA as a safe and simple method for correcting FVIII deficiency. METHODS: B-domain-deleted murine FVIII cDNA expression plasmids were constructed with CMV and elongation factor 1alpha promoters for characterisation in murine C2C12 myoblasts. The construct conferring highest in vitro FVIII secretion was electroporated into skeletal muscle of FVII null mice in vivo for phenotypic correction using a protocol that minimised tissue injury. RESULTS: B-domain-deleted murine FVIII cDNA plasmids induced FVIII secretion from stably transfected C2C12 myoblasts (0.54+/-0.20 mU/day/10(5) cells). Phenotypic correction of hemophilic mice was more consistently achieved using a protocol for in vivo electroporation of gastrocnemius muscle with FVIII cDNA that reduced tissue injury by the use of plate electrodes, hyaluronidase pre-treatment and lower field strength. This technique was associated with <10% muscle necrosis. Activated partial thromboplastin time decreased from 51.4+/-3.3 to 34.7+/-1.1 (mean+/-s.e.m.) seconds (p=0.0004) following in vivo electroporation (0.1 mg plasmid/limb; 8x20 ms pulses, 175 V/cm, 1 Hz) of hemophilic mice. All hemophilic mice (8/8) survived hemostatic challenge after muscle electroporation with FVIII cDNA, whereas all (9/9) untreated hemophilic mice died. Plasmid DNA was detectable only in electroporated muscle and not in all other organs tested, including gonads. CONCLUSION: In vivo intramuscular electroporation of naked FVIII plasmid successfully corrects murine hemophilia.     

An improved murine femur fracture device for bone healing studies

Murine models are commonly used to investigate bone healing and test new treatments before human trials. Our objective was to design an improved murine femur fracture device and determine optimal mass and velocity settings for maximal likelihood of transverse fracture. Fracture reproducibility was maximized using an adjustable kinetic energy level, a novel mouse positioning system and an electromagnet striker release assembly. Sixty wild-type mice of 8-12-week-old male and female with a weight of 26.4+/-6.1g were subjected to an experimental postmortem fracture in the left and right femur (n=120) using variable kinetic energy inputs. A best-fit prediction equation for transverse fracture was developed using multivariate linear regression. Transverse fracture was shown to correlate most highly with kinetic energy with a maximum likelihood at mv2=292 where m is mass (g) and v is velocity (m/s). Model validation with a group of 134 anesthetized C57BL/6 mice resulted in a favorable transverse fracture rate of 85.8%. Simple modifications to existing fracture devices can improve accuracy and reproducibility. The results may assist researchers studying the effects of genetic modifications and novel treatments on boney healing in murine femur fracture models. Maintaining kinetic energy parameters within suggested ranges may also aid in ensuring accuracy and reproducibility.     

Trophoblast cell-specific carcinoembryonic antigen cell adhesion molecule 9 is not required for placental development or a positive outcome of allotypic pregnancies

The carcinoembryonic antigen (CEA) family consists of a large group of evolutionarily divergent glycoproteins. The secreted pregnancy-specific glycoproteins constitute a subgroup within the CEA family. They are predominantly expressed in trophoblast cells throughout placental development and are essential for a positive outcome of pregnancy, possibly by protecting the semiallotypic fetus from the maternal immune system. The murine CEA gene family member CEA cell adhesion molecule 9 (Ceacam9) also exhibits a trophoblast-specific expression pattern. However, its mRNA is found only in certain populations of trophoblast giant cells during early stages of placental development. It is exceptionally well conserved in the rat (over 90% identity on the amino acid level) but is absent from humans. To determine its role during murine development, Ceacam9 was inactivated by homologous recombination. Ceacam9(-/-) mice on both BALB/c and 129/Sv backgrounds developed indistinguishably from heterozygous or wild-type littermates with respect to sex ratio, weight gain, and fertility. Furthermore, the placental morphology and the expression pattern of trophoblast marker genes in the placentae of Ceacam9(-/-) females exhibited no differences. Both backcross analyses and transfer of BALB/c Ceacam9(-/-) blastocysts into pseudopregnant C57BL/6 foster mothers indicated that Ceacam9 is not needed for the protection of the embryo in a semiallogeneic or allogeneic situation. Taken together, Ceacam9 is dispensable for murine placental and embryonic development despite being highly conserved within rodents.     

Oxidative damage during chagasic cardiomyopathy development: role of mitochondrial oxidant release and inefficient antioxidant defense

In this study, we evaluated the oxidant status and antioxidant defense capabilities of the heart during the course of Trypanosoma cruzi infection and disease development in a murine model system. Our data show that the extent of protein carbonylation and lipid peroxidation is increased in the heart, but not the skeletal muscle, of infected mice. The level of oxidative injury biomarkers in the myocardium consistently increased with chronic disease severity. The antioxidant defense constituted by catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GSR), and reduced glutathione was increased in murine heart and skeletal tissue in response to the stress of T. cruzi infection. After the initial burst, CAT, GPx, and GSR remained unresponsive to the severity of chronic tissue damage in chagasic hearts. The cardiac level of Mn(2+) superoxide dismutase (MnSOD) was diminished in chagasic mice. Our data suggest that the host responds to acute injuries by activating antioxidant defenses that are of sufficient magnitude to scavenge the reactive oxidants in skeletal tissue. The myocardia of infected mice, however, sustain increased oxidative injuries with disease progression. We surmise that MnSOD deficiencies, resulting in the increased release of mitochondrial free radicals, lead to sustained oxidative stress that exceeds the cardiac antioxidant defense capacity and contribute to persistent oxidative damage in chagasic myocardium.     

Validation of imaging-based quantification of glenohumeral joint kinematics using an unmodified clinical biplane fluoroscopy system

Model-based tracking, using CT and biplane fluoroscopy, allows highly accurate quantification of glenohumeral motion and changes in the subacromial space. Previous investigators have used custom-built biplane fluoroscopes designed specifically for kinematic applications, which are available at few institutions and require FDA approval prior to clinical use. The aim of this study was to demonstrate the utility of an off-the-shelf clinical biplane fluoroscope for kinematic applications by validating model-based tracking for measurement of glenohumeral motion using an unmodified clinical system. Biplane images of each shoulder of a cadaver torso were acquired at various joint positions and during simulated movements along anatomical planes of motion. The pose of each humerus and scapula was determined using model-based tracking and compared to a bead-based gold standard. Error due to a temporal-offset between corresponding biplane images, characteristic of clinical biplane systems, was determined by comparison of measured and known relative position of 2 bead clusters of a phantom that was imaged while moved throughout the fluoroscopy image volume. Model-based tracking had global kinematic mean absolute errors of 0.27 mm and 0.29° (static), and 0.22–0.32 mm and 0.12–0.45° (dynamic). Glenohumeral mean absolute errors were 0.39 mm and 0.45° (static), and 0.36–0.42 mm and 0.41–0.48° (dynamic). The temporal-offset was predicted to add errors of 0.06–0.85 mm and 0.05–0.28° for cadaveric trials for the speeds examined. For defined speeds, sub-millimeter and sub-degree kinematic accuracy and precision were achieved using an unmodified clinical biplane fluoroscope for quantification of glenohumeral motion.     

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of chronic CNS disorders, there is an increasing demand for murine models with high validity at the behavioral level. However, multiple pathogenic mechanisms and complex functional deficits often impose challenges to reliably measure and interpret behavior of chronically sick mice. Therefore, the assessment of peripheral pathology and a behavioral profile at several time points using a battery of tests are required. Video-tracking, behavioral spectroscopy, and remote acquisition of physiological measures are emerging technologies that allow for comprehensive, accurate, and unbiased behavioral analysis in a home-base-like setting. This report describes a refined phenotyping protocol, which includes a custom-made monitoring apparatus (Integrated Behavioral Station, INBEST) that focuses on prolonged measurements of basic functional outputs, such as spontaneous activity, food/water intake and motivated behavior in a relatively stress-free environment. Technical and conceptual improvements in INBEST design may further promote reproducibility and standardization of behavioral studies.     

Mechanical ventilation and volutrauma: study in vivo of a healthy pig model

Mechanical ventilation is essential in intensive care units. However, it may itself induce lung injury. Current studies are based on rodents, using exceptionally large tidal volumes for very short periods, often after a "priming" pulmonary insult. Our study deepens a clinically relevant large animal model, closely resembling human physiology and the ventilator setting used in clinic settings. Our aim was to evaluate the pathophysiological mechanisms involved in alveolo/capillary barrier damage due to mechanical stress in healthy subjects. We randomly divided 18 pigs (sedated with medetomidine/tiletamine-zolazepam and anesthetised with thiopental sodium) into three groups (n=6): two were mechanically ventilated (tidal volume of 8 or 20 ml/kg), the third breathed spontaneously for 4 hours, then animals were sacrificed (thiopental overdose). We analyzed every 30' hemogasanalysis and the main circulatory and respiratory parameters. Matrix gelatinase expression was evaluated on bronchoalveolar lavage fluid after surgery and before euthanasia. On autoptic samples we performed zymographic analysis of lung, kidney and liver tissues and histological examination of lung. Results evidenced that high Vt evoked profound alterations of lung mechanics and structure, although low Vt strategy was not devoid of side effects, too. Unexpectedly, also animals that were spontaneously breathing showed a worsening of the respiratory functions.     

Rapid immunomagnetic negative enrichment of neutrophil granulocytes from murine bone marrow for functional studies in vitro and in vivo

Polymorphonuclear neutrophils (PMN) mediate early immunity to infection but can also cause host damage if their effector functions are not controlled. Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue. One prerequisite for PMN analysis is the availability of purified cells from primary organs. While human PMN are easily isolated from peripheral blood, this approach is less suitable for mice due to limited availability of blood. Instead, bone marrow (BM) is an easily available reservoir of murine PMN, but methods to obtain pure cells from BM are limited. We have developed a novel protocol allowing the isolation of highly pure untouched PMN from murine BM by negative immunomagnetic isolation using a complex antibody cocktail. The protocol is simple and fast (∼1 h), has a high yield (5–10*10⁶ PMN per animal) and provides a purity of cells equivalent to positive selection (>80%). Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals. This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo.     

Burrowing in rodents: a sensitive method for detecting behavioral dysfunction

Virtually all rodents display burrowing behavior, yet measurement of this behavior has not yet been standardized or formalized. Previously, parameters such as the latency to burrow and the complexity of the burrow systems in substrate-filled boxes in the laboratory or naturalistic outdoor environments have been assessed. We describe here a simple protocol that can quantitatively measure burrowing in laboratory rodents, using a simple apparatus that can be placed in the home cage. The test is very cheap to run and requires minimal experimenter training, yet seems sensitive to a variety of treatments, such as the early stages of prion disease in mice, mouse strain differences, lesions of the hippocampus and prefrontal cortex in mice, also effects of lipopolysaccharide and IL-1beta in rats. Other species such as hamsters, gerbils and Egyptian spiny mice also burrow in this apparatus, and with suitable size modification probably almost any burrowing animal could be tested in it. The simplicity, sensitivity and robustness of burrowing make it ideal for assessing genetically modified animals, which in most cases would be mice. The test is run from late afternoon until the next morning, but only two measurements need to be taken.     

Optimization of the touchscreen paired-associate learning (PAL) task for mice and its dorsal hippocampal dependency

Two versions of the touchscreen paired-associate learning (PAL) task have been developed for rodents: same PAL (sPAL) and different PAL (dPAL). These tasks are very important in studying murine models of Alzheimer’s disease and schizophrenia, and have also been used to test object-location memory in various studies. However, the relatively long time needed for the tasks (approx. 50 days for mice) limits their widespread use. By giving training that was more intensive with a higher number of trials, we shortened the time required for learning saturation in sPAL and dPAL to about one-third of the time required for the generally used protocol. Furthermore, by applying a reduced number of objects and trial types for sPAL, we developed a simplified version of sPAL, termed 2-object sPAL, in which mice could reach the fully learned level in 6 days. Our pharmacological experiments indicate that the dorsal hippocampal CA1 region is crucial for the performance of the two PAL tasks with the new protocols and the new 2-object sPAL. This work has significantly enhanced the usefulness of the touchscreen PAL tasks to increase the speed of learning, but they remain highly hippocampus-dependent object-location memory tasks.     

Mouse intragastric infusion (iG) model

Direct intragastric delivery of a diet, nutrient or test substance can be achieved in rodents (mice and rats) on a long-term (2-3 months) basis using a chronically implanted gastrostomy catheter and a flow-through swivel system. This rodent intragastric infusion (iG) model has broad applications in research on food intake, gastrointestinal (GI) physiology, GI neuroendocrinology, drug metabolism and toxicity, obesity and liver disease. It achieves maximal control over the rate and pattern of delivery and it can be combined with normal ad libitum feeding of solid diet if so desired. It may be adopted to achieve infusion at other sites of the GI system to test the role of a bypassed GI segment in neuroendocrine physiology, and its use in genetic mouse models facilitates the genetic analysis of a central question under investigation.     

The regulation of the oxidative processes in the tissues of Muridae like rodents caught in the Chernobyl accident zone

The results of the investigations of the radioactive contamination consequences on the lipid peroxidation (LPO) processes in organs and tissues of wild rodents which were caught in the Chernobyl NPP accident 30-km zone during 1986-1993 are generalized. The behaviors of the technogenic contamination effect on dynamic of changes of the LPO physico-chemical regulatory system parameters and the generalized parameters of the phospholipid composition in organs of the different radioresistance wild rodents are revealed in dependence on the radioactive contamination level and the duration of the radiation factor exposure. Different sensitivity of the LPO regulatory system parameters in wild rodent tissues to the radioactive contamination of their environment and the unequal ability to normalization of the antioxidant status and the energy exchange in tissues result in the change of the scale and character of interrelations between the reciprocal parameters in norm and have an influence on the development of qualitatively new subpopulations of wild rodents due to the transition of the cell regulatory system to the another level of the function.     

Induction of antigen-specific immune responses against malignant brain tumors by intramuscular injection of sindbis DNA encoding gp100 and IL-18

We constructed pSin-SV40-HDV-SV40pA, an improved Sindbis DNA expression vector, and evaluated the potential of this vector system for brain tumor therapy. We investigated whether immunizing mice with xenogeneic DNA encoding human gp100 and mouse IL-18 would enhance the antitumor responses. To study the immune mechanisms involved in tumor regression, we examined tumor growth in B16-gp100-implanted brain tumor models using T-cell subset-depleted and IFN-gamma-neutralized mice. Hugp100/mIL-18 vaccination was also investigated for its antitumor effects against the wild-type murine B16 tumor, which expresses the murine gp100 molecule. Genetic immunization using plasmid pSin 9001 DNA codelivery of human gp100 and mouse IL-18 resulted in enhanced protective and therapeutic effects on the malignant brain tumors. The antitumor and protective effects were mediated by both CD4(+)/CD8(+) T cells and IFN-gamma. Vaccination with hugp100/mIL-18 conferred a significant survival merit to wild-type B16 tumor-harboring mice. Immunogene therapy with the improved Sindbis virus vector expressing xenogeneic gp100 and syngeneic IL-18 may be an excellent approach for developing a new treatment protocol. Thus, the Sindbis DNA system may represent a novel approach for the treatment of malignant brain tumors.     

Passive induction of experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is a widely used animal model of the human demyelinating disease multiple sclerosis. EAE is initiated by immunization with myelin antigens in adjuvant or by adoptive transfer of myelin-specific T cells, resulting in inflammatory infiltrates and demyelination in the central nervous system. Induction of EAE in rodents typically results in ascending flaccid paralysis with inflammation primarily targeting the spinal cord. This protocol describes passive induction of EAE by adoptive transfer of T cells isolated from mice primed with myelin antigens into na?ve mice. The advantages of using this method versus active induction of EAE are discussed.     

Aquaporin water channels and lung physiology

Fluid transport across epithelial and endothelial barriers occurs in the neonatal and adult lungs. Biophysical measurements in the intact lung and cell isolates have indicated that osmotic water permeability is exceptionally high across alveolar epithelia and endothelia and moderately high across airway epithelia. This review is focused on the role of membrane water-transporting proteins, the aquaporins (AQPs), in high lung water permeability and lung physiology. The lung expresses several AQPs: AQP1 in microvascular endothelia, AQP3 in large airways, AQP4 in large- and small-airway epithelia, and AQP5 in type I alveolar epithelial cells. Lung phenotype analysis of transgenic mice lacking each of these AQPs has been informative. Osmotically driven water permeability between the air space and capillary compartments is reduced approximately 10-fold by deletion of AQP1 or AQP5 and reduced even more by deletion of AQP1 and AQP4 or AQP1 and AQP5 together. AQP1 deletion greatly reduces osmotically driven water transport across alveolar capillaries but has only a minor effect on hydrostatic lung filtration, which primarily involves paracellular water movement. However, despite the major role of AQPs in lung osmotic water permeabilities, AQP deletion has little or no effect on physiologically important lung functions, such as alveolar fluid clearance in adult and neonatal lung, and edema accumulation after lung injury. Although AQPs play a major role in renal and central nervous system physiology, the data to date on AQP knockout mice do not support an important role of high lung water permeabilities or AQPs in lung physiology. However, there remain unresolved questions about possible non-water-transporting roles of AQPs and about the role of AQPs in airway physiology, pleural fluid dynamics, and edema after lung infection.     

6-MBOA affects testis size, but not delayed-type hypersensitivity, in white-footed mice (Peromyscus leucopus).

Many rodents use day length to time reproduction to occur when resources are abundant, but some species also use supplementary environmental cues. One supplementary cue is the plant-derived compound, 6-methoxy-2-benzoxazolinone (6-MBOA). Most rodents grow their gonads in response to 6-MBOA in their diets, but it is presently unknown whether they also use 6-MBOA to adjust other aspects of physiology, specifically their immune systems. 6-MBOA is structurally similar to melatonin, and seasonal changes in rodent immune activities are often mediated by melatonin. We therefore predicted that white-footed mice (Peromyscus leucopus), which breed seasonally and are reproductively sensitive to melatonin, would adjust their immune systems when fed 6-MBOA. 6-MBOA treated mice in long day lengths regressed their testes to a greater extent than mice fed a standard diet, or mice kept in short day lengths and fed 6-MBOA or a standard diet. One type of immune activity (delayed-type hypersensitivity) was not affected by 6-MBOA, however, although responses were greater in short versus long day mice. In sum, P. leucopus responded reproductively to 6-MBOA, although differently than other species; immune activity was unaffected. Other aspects of the immune system, especially in herbivorous rodents, may be affected by 6-MBOA and thus warrant further study.     

6-MBOA affects testis size, but not delayed-type hypersensitivity, in white-footed mice (Peromyscus leucopus)

Many rodents use day length to time reproduction to occur when resources are abundant, but some species also use supplementary environmental cues. One supplementary cue is the plant-derived compound, 6-methoxy-2-benzoxazolinone (6-MBOA). Most rodents grow their gonads in response to 6-MBOA in their diets, but it is presently unknown whether they also use 6-MBOA to adjust other aspects of physiology, specifically their immune systems. 6-MBOA is structurally similar to melatonin, and seasonal changes in rodent immune activities are often mediated by melatonin. We therefore predicted that white-footed mice (Peromyscus leucopus), which breed seasonally and are reproductively sensitive to melatonin, would adjust their immune systems when fed 6-MBOA. 6-MBOA treated mice in long day lengths regressed their testes to a greater extent than mice fed a standard diet, or mice kept in short day lengths and fed 6-MBOA or a standard diet. One type of immune activity (delayed-type hypersensitivity) was not affected by 6-MBOA, however, although responses were greater in short versus long day mice. In sum, P. leucopus responded reproductively to 6-MBOA, although differently than other species; immune activity was unaffected. Other aspects of the immune system, especially in herbivorous rodents, may be affected by 6-MBOA and thus warrant further study.