Barrier lipid composition and response to plasma lipids: A direct comparison of mouse dorsal back and ear skin.

The skin of the ear and the back are frequently selected sites in skin research using mouse models. However, distinct responses to treatment have been described between these two sites in several studies. Despite the crucial role of the stratum corneum (SC) in the skin barrier function of both dorsal back and ear skin, it remains unclear whether differences in lipid composition might underlie altered responses. Here, we compared the skin morphology and the barrier lipid composition of the ear with the back skin of wild-type mice. The ear contained more corneocyte layers in the SC and its barrier lipid composition was enriched with sphingosine ceramide subclasses, especially the short ones with a total chain length of 33-34 carbons. The free fatty acid (FFA) profile in the ear skin shifted towards shorter chains, significantly reducing the mean chain length to 23.3 vs 24.7 carbons in the back skin. In line, FFA species in the ear displayed a twofold increase in unsaturation index (P < .001). Gene expression in the ear skin revealed low expression of genes involved in lipid synthesis and uptake, indicating a reduced metabolic activity. Finally, the effects of hypercholesterolaemia on SC FFA composition was compared in ear and back skin of apolipoprotein E knockout (APOE-/- ) mice. Interestingly, the FFA profile in APOE-/- ear skin was minimally affected, while the FFA composition in the back skin was markedly changed in response to hypercholesterolaemia. In conclusion, ear and back skin have distinct barrier lipids and respond differently to elevated plasma cholesterol.

Long-term continuous allopregnanolone elevation causes memory decline and hippocampus shrinkage, in female wild-type B6 mice.

Chronic stress in various forms increases the risk for cognitive dysfunction, dementia and Alzheimer's disease. While the pathogenesis behind these findings is unknown, growing evidence suggests that chronic increase in neurosteroid levels, such as allopregnanolone, is part of the mechanism. We treated wild-type C57BL/6J mice with allopregnanolone for 5months, using osmotic pumps. This treatment led to moderately increased levels of allopregnanolone, equivalent to that of mild chronic stress. After an interval of no treatment for 1month, female mice showed impaired learning and memory function in the Morris water maze (MWM) in combination with diminished hippocampus weight and increased cerebellum weight, both correlating to MWM performance. Male mice showed a minor reduction in memory function and no differences in brain structure. We conclude that chronic allopregnanolone elevation can lead to cognitive dysfunction and negative brain alterations. We suggest that allopregnanolone could play a key role in the pathogenesis of stress-induced cognitive disturbances and perhaps dementia.

Ganglioside accumulation in activated glia in the developing brain: comparison between WT and GalNAcT KO mice.

Our previous studies have shown accumulation of GM2 ganglioside during ethanol-induced neurodegeneration in the developing brain, and GM2 elevation has also been reported in other brain injuries and neurodegenerative diseases. Using GM2/GD2 synthase KO mice lacking GM2/GD2 and downstream gangliosides, the current study explored the significance of GM2 elevation in WT mice. Immunohistochemical studies indicated that ethanol-induced acute neurodegeneration in postnatal day 7 (P7) WT mice was associated with GM2 accumulation in the late endosomes/lysosomes of both phagocytic microglia and increased glial fibrillary acidic protein (GFAP)-positive astrocytes. However, in KO mice, although ethanol induced robust neurodegeneration and accumulation of GD3 and GM3 in the late endosomes/lysosomes of phagocytic microglia, it did not increase the number of GFAP-positive astrocytes, and the accumulation of GD3/GM3 in astrocytes was minimal. Not only ethanol, but also DMSO, induced GM2 elevation in activated microglia and astrocytes along with neurodegeneration in P7 WT mice, while lipopolysaccharide, which did not induce significant neurodegeneration, caused GM2 accumulation mainly in lysosomes of activated astrocytes. Thus, GM2 elevation is associated with activation of microglia and astrocytes in the injured developing brain, and GM2, GD2, or other downstream gangliosides may regulate astroglial responses in ethanol-induced neurodegeneration.

Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes.

BACKGROUND: The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics). METHODS: Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin-luciferase based real-time luminometry. RESULTS: Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%. Erythrocytes pre-exposure to 10µM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux. Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers. CONCLUSIONS: MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers. GENERAL SIGNIFICANCE: Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.

Use and Reuse of Animal Behavioral, Molecular, and Biochemical Data in Alzheimer's Disease Research: Focus on 3Rs and Saving People's Tax Dollars.

Several decades of research on cell and animal models contributed tremendously to understanding human diseases. Particularly, research on rodents and non-human primates revealed that animal research is a major and important component in biomedical research in learning complex pathophysiological processes. Further, animal research helped us to understand human diseases, such as Alzheimer's disease. In addition, animal research has also helped us to test hundreds of drugs and develop treatments for human use. Researchers can gain a better understanding of key biological and physiological processes in humans by comparing them to laboratory animals. Based on their relevance and resemblance to people, or even usual living conditions, scientists rationalize the use of particular animal models in their studies. It is suggested that in the National Institutes of Health and other agencies-funded research, animal models should be carefully selected to study the biology and pathophysiology of human health and diseases such as Alzheimer's disease and other dementias. However, it is critical to use a minimum number of animals for human research. Further, it is also noted that the use and reuse of behavioral,  molecular, and biochemical data from wild-type (WT) control mice with mutant lines of disease models, as long as the genetic background is the same in both WT and disease mice. On the other hand, anonymous readers have challenged the use and reuse of WT mice data for comparison. In the current article, we discuss the minimum utility of animals, covering the 3Rs, Replacement, Reduction, and Refinement, and also discuss the use and reuse of behavioral, molecular, and biochemical data.

Cyclic nucleotide phosphodiesterase 3A1 protects the heart against ischemia-reperfusion injury.

Phosphodiesterase 3A (PDE3A) is a major regulator of cAMP in cardiomyocytes. PDE3 inhibitors are used for acute treatment of congestive heart failure, but are associated with increased incidence of arrhythmias and sudden death with long-term use. We previously reported that chronic PDE3A downregulation or inhibition induced myocyte apoptosis in vitro. However, the cardiac protective effect of PDE3A has not been demonstrated in vivo in disease models. In this study, we examined the role of PDE3A in regulating myocardial function and survival in vivo using genetically engineered transgenic mice with myocardial overexpression of the PDE3A1 isozyme (TG). TG mice have reduced cardiac function characterized by reduced heart rate and ejection fraction (52.5±7.8% vs. 83.9±4.7%) as well as compensatory expansion of left ventricular diameter (4.19±0.19mm vs. 3.10±0.18mm). However, there was no maladaptive increase of fibrosis and apoptosis in TG hearts compared to wild type (WT) hearts, and the survival rates also remained the same. The diminution of cardiac contractile function is very likely attributed to a decrease in beta-adrenergic receptor (ß-AR) response in TG mice. Importantly, the myocardial infarct size (4.0±1.8% vs. 24.6±3.8%) and apoptotic cell number (1.3±1.0% vs. 5.6±1.5%) induced by ischemia/reperfusion (I/R) injury were significantly attenuated in TG mice. This was associated with decreased expression of inducible cAMP early repressor (ICER) and increased expression of anti-apoptotic protein BCL-2. To further verify the anti-apoptotic effects of PDE3A1, we performed in vitro apoptosis study in isolated adult TG and WT cardiomyocytes. We found that the apoptotic rates stimulated by hypoxia/reoxygenation or H2O2 were indeed significantly reduced in TG myocytes, and the differences between TG and WT myocytes were completely reversed in the presence of the PDE3 inhibitor milrinone. These together indicate that PDE3A1 negatively regulates ß-AR signaling and protects against I/R injury by inhibiting cardiomyocyte apoptosis.

Proposed mechanistic description of dose-dependent BDE-47 urinary elimination in mice using a physiologically based pharmacokinetic model.

Polybrominated diphenyl ethers (PBDEs) have been used in a wide variety of consumer applications as additive flame retardants. In North America, scientists have noted continuing increases in the levels of PBDE congeners measured in human serum. Some recent studies have found that PBDEs are associated with adverse health effects in humans, in experimental animals, and wildlife. This laboratory previously demonstrated that urinary elimination of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) is saturable at high doses in mice; however, this dose-dependent urinary elimination has not been observed in adult rats or immature mice. Thus, the primary objective of this study was to examine the mechanism of urinary elimination of BDE-47 in adult mice using a physiologically based pharmacokinetic (PBPK) model. To support this objective, additional laboratory data were collected to evaluate the predictions of the PBPK model using novel information from adult multi-drug resistance 1a/b knockout mice. Using the PBPK model, the roles of mouse major urinary protein (a blood protein carrier) and P-glycoprotein (an apical membrane transporter in proximal tubule cells in the kidneys, brain, intestines, and liver) were investigated in BDE-47 elimination. The resulting model and new data supported the major role of m-MUP in excretion of BDE-47 in the urine of adult mice, and a lesser role of P-gp as a transporter of BDE-47 in mice. This work expands the knowledge of BDE-47 kinetics between species and provides information for determining the relevancy of these data for human risk assessment purposes.

The Effect of High Molecular Weight Hyaluronic Acid and Latanoprost Eyedrops on Tear Functions and Ocular Surface Status in C57/BL6 Mice.

Anti-glaucoma eye drop treatment often induces ocular surface problems, including dry eyes, and may be associated with poor medication compliance. This study aimed to investigate the effects of a novel high molecular weight hyaluronic acid and Latanoprost eye drop on intraocular pressure, as well as the tear function and ocular surface alterations in wild type mice, comparing the results with the mice receiving commercially available Latanoprost eye drops and mice receiving no treatment. The mice were divided into three groups: Group I, control group (no treatment group); Group II, commercial Latanoprost eye drop (LP); and Group III, Comfort Shield (CS) + Latanoprost (LP) eye drop (CS + LP). The CS + LP eye drop group had an IOP lowering effect comparable to the commercial LP eye drop group. The mice receiving LP eye drops had significantly worse corneal staining scores, lesser goblet cell density(GCD), higher numbers of CD45+ staining cells, significantly higher tear film concentrations of IL-6 and IL1-b, and a significantly lower expression of corneal ZO-1 mRNA compared with the mice receiving CS + LP 7 days after eye drop instillations (p < 0.05). In conclusion, the new CS + LP formulation appeared to induce less inflammation, less corneal vital staining, and a better barrier status with an IOP lowering effect comparable to the commercial LP eye drops.

An Imbalance in the Pro/mature BDNF Ratio Occurs in Multiple Brain Regions During Normal Ageing in Wild-Type Mice.

The early transition to Alzheimer's disease is characterized by a period of accelerated brain atrophy that exceeds normal ageing. Identifying the molecular basis of this atrophy could facilitate the discovery of novel drug targets. The precursor of brain-derived neurotrophic factor, a well characterized neurotrophin, is increased in the hippocampus of aged rodents, while its mature isoform is relatively stable. This imbalance could increase the risk of Alzheimer's disease by precipitating its pathological hallmarks. However, less is known about how relative levels of these isoforms change in middle-aged mice. In addition, the underlying mechanisms that might cause an imbalance are unknown. The main aim of this study was to determine how precursor brain-derived neurotrophic factor changes relative to its mature isoform with normal brain ageing in wild type mice. A secondary aim was to determine if signaling through the neurotrophin receptor, p75 influences this ratio. An increasing ratio was identified in several brain regions, except the hippocampus, suggesting a neurotrophic imbalance occurs as early as middle age. Some changes in receptors that mediate the isoforms effects were also identified, but these did not correspond with trends in the isoforms. Relative amounts of precursor brain-derived neurotrophic factor were mostly unchanged in mutant p75 mice. The lack of changes suggested that signaling through the receptor had no influence on the ratio.

Tooth Loss Induces Memory Impairment and Glial Activation in Young Wild-Type Mice.

Background: Tooth loss is closely associated with Alzheimer's disease (AD). Previously, we reported that tooth loss induced memory impairment in amyloid precursor protein knock-in mice by decreasing neuronal activity and synaptic protein levels and increasing glial activation, neuroinflammation, and pyramidal neuronal cell loss without altering amyloid-ß levels in the hippocampus. However, the effects of tooth loss in young wild-type mice have not been explored yet. Objective: We investigated the effects of tooth loss on memory impairment, neuronal activity, synaptic protein levels, glial activation, and pyramidal neuronal cell loss in young wild-type mice. Methods: Two-month-old wild-type mice were randomly divided into control and tooth loss groups. In the tooth loss group, maxillary molar teeth on both sides were extracted, whereas no teeth were extracted in the control group. Two months after tooth extraction, we performed a novel object recognition test to evaluate memory function. Glial activation, neuronal activity, synaptic protein levels, and the number of pyramidal neurons were evaluated using immunofluorescence staining, immunohistochemistry, and western blotting. Results: The tooth loss group exhibited memory impairment and decreased neuronal activity and the levels of synaptic proteins in both the hippocampus and cortex. Moreover, tooth loss increased the activation of phosphorylated c-Jun N-terminal kinase (JNK), heat shock protein 90 (HSP90), and glial activation and reduced the number of pyramidal neurons in the hippocampus. Conclusion: Tooth loss in the young wild-type mice will attenuate neuronal activity, decrease synaptic protein levels, and induce pyramidal neuronal loss, and eventually lead to memory impairment.

Alpha-NETA, as a CMKLR1 Small Molecule Antagonist, Protects against Renal Ischemia Reperfusion Injury in Mice.

BACKGROUND: Ischemia-reperfusion (IR) injury is one of the major causes of acute kidney injury (AKI). Chemerin chemokine-like receptor 1 (CMKLR1) has been reported to be involved in the progression of IR injury. Here, we investigated the protective role of CMKLR1 antagonist, α-NETA, in IR mouse model, and dissected the underlying regulatory mechanism. METHODS: IR injury mouse model was established to evaluate the protective effects of α-NETA on IR injury. Kidney injury-associated parameters and functions were examined to evaluate the renal function of Sham, IR, and IR+ α-NETA mice. Renal morphological changes and apoptosis were determined by PAS and TUNEL staining in IR and α-NETA treated mice. ELISA, RT-qPCR, and western blot were performed to examine the inflammatory responses and expression of CMKLR1. RESULTS: α-NETA administration attenuated IR-induced renal tubular injury and epithelial cell apoptosis in IR injury mice. Kidney injury-related cystatin C, kidney injury molecule-1, neutrophil gelatinaseassociated lipocalin, and renal morphology were significantly improved. Mechanistically, α-NETA suppressed the inflammatory responses by inhibiting the expression of CMKLR1, and then protected the IR-induced renal damage and restored renal function. CONCLUSION: CMKLR1 plays an important role in renal ischemia-reperfusion injury, targeting CMKLR1 by using the small molecule inhibitor α-NETA is a potential treatment strategy for AKI.

PlexinA1-deficient mice exhibit decreased cell density and augmented oxidative stress in parvalbumin-expressing interneurons in the medial prefrontal cortex.

PlexinA1 (PlxnA1) is a transmembrane receptor for semaphorins (Semas), a large family of axonal guidance cues vital during neural development. PlxnA1 is expressed in embryonic interneurons, and PlxnA1 deletion in mice leads to less interneurons in the developing cortex. In addition, PlxnA1 has been identified as a schizophrenia susceptibility gene. In our previous study, PlxnA1 knockout (KO) mice under a BALB/cAJ genetic background exhibited significantly increased self-grooming and reduced prepulse inhibition, a reliable phenotype for investigating the neurobiology of schizophrenia. However, the mechanism underlying the abnormal behavior of PlxnA1 KO mice remains unclear. We first confirmed PlxnA1 mRNA expression in parvalbumin-expressing interneurons (PV cells) in the medial prefrontal cortex (mPFC) of adult mice. Immunohistochemical analysis (IHC) showed significantly decreased densities of both GABAergic neurons and PV cells in the mPFC of PlxnA1 KO mice compared with wild type mice (WT). PV cells were found to express molecule interacting with CasL 1 (MICAL1), an effector involved in Sema-Plxn signaling for axon guidance, suggesting MICAL1 and PlxnA1 co-expression in PV cells. Furthermore, IHC analysis of 8-oxo-dG, an oxidative stress marker, revealed significantly increased oxidative stress in PlxnA1-deficient PV cells compared with WT. Thus, increased oxidative stress and decreased PV cell density in the mPFC may determine the onset of PlxnA1 KO mice's abnormal behavior. Accordingly, deficient PlxnA1-mediated signaling may increase oxidative stress in PV cells, thereby disrupting PV-cell networks in the mPFC and causing abnormal behavior related to neuropsychiatric diseases.

Dextran sulphate-induced tau assemblies cause endogenous tau aggregation and propagation in wild-type mice.

Accumulation of assembled tau protein in the central nervous system is characteristic of Alzheimer's disease and several other neurodegenerative diseases, called tauopathies. Recent studies have revealed that propagation of assembled tau is key to understanding the pathological mechanisms of these diseases. Mouse models of tau propagation are established by injecting human-derived tau seeds intracerebrally; nevertheless, these have a limitation in terms of regulation of availability. To date, no study has shown that synthetic assembled tau induce tau propagation in non-transgenic mice. Here we confirm that dextran sulphate, a sulphated glycosaminoglycan, induces the assembly of recombinant tau protein into filaments in vitro. As compared to tau filaments induced by heparin, those induced by dextran sulphate showed higher thioflavin T fluorescence and lower resistance to guanidine hydrochloride, which suggests that the two types of filaments have distinct conformational features. Unlike other synthetic filament seeds, intracerebral injection of dextran sulphate-induced assemblies of recombinant tau caused aggregation of endogenous murine tau in wild-type mice. AT8-positive tau was present at the injection site 1 month after injection, from where it spread to anatomically connected regions. Induced tau assemblies were also stained by anti-tau antibodies AT100, AT180, 12E8, PHF1, anti-pS396 and anti-pS422. They were thioflavin- and Gallyas-Braak silver-positive, indicative of amyloid. In biochemical analyses, accumulated sarkosyl-insoluble and hyperphosphorylated tau was observed in the injected mice. In conclusion, we revealed that intracerebral injection of synthetic full-length wild-type tau seeds prepared in the presence of dextran sulphate caused tau propagation in non-transgenic mice. These findings establish that propagation of tau assemblies does not require tau to be either mutant and/or overexpressed.

Ongoing Electroencephalographic Rhythms Related to Exploratory Movements in Transgenic TASTPM Mice.

BACKGROUND: The European PharmaCog study (http://www.pharmacog.org) has reported a reduction in delta (1-6 Hz) electroencephalographic (EEG) power (density) during cage exploration (active condition) compared with quiet wakefulness (passive condition) in PDAPP mice (hAPP Indiana V717F mutation) modeling Alzheimer's disease (AD) amyloidosis and cognitive deficits. OBJECTIVE: Here, we tested the reproducibility of that evidence in TASTPM mice (double mutation in APP KM670/671NL and PSEN1 M146V), which develop brain amyloidosis and cognitive deficits over aging. The reliability of that evidence was examined in four research centers of the PharmaCog study. METHODS: Ongoing EEG rhythms were recorded from a frontoparietal bipolar channel in 29 TASTPM and 58 matched "wild type" C57 mice (range of age: 12-24 months). Normalized EEG power was calculated. Frequency and amplitude of individual delta and theta frequency (IDF and ITF) peaks were considered during the passive and active conditions. RESULTS: Compared with the "wild type" group, the TASTPM group showed a significantly lower reduction in IDF power during the active over the passive condition (p < 0.05). This effect was observed in 3 out of 4 EEG recording units. CONCLUSION: TASTPM mice were characterized by "poor reactivity" of delta EEG rhythms during the cage exploration in line with previous evidence in PDAPP mice. The reliability of that result across the centers was moderate, thus unveiling pros and cons of multicenter preclinical EEG trials in TASTPM mice useful for planning future studies.

Reduction of BMP6-induced bone formation by calcium phosphate in wild-type compared with nude mice.

Nude mice have been extensively used to investigate the potency of tissue engineering strategies for bone repair. However, the contribution of pro-inflammatory and proregenerative stimuli of the host for the process of new bone formation and integration remains poorly understood. In this study, ectopic bone formation was investigated in nude (Nu) versus wild-type (WT) mice. Calcium phosphate (CaP) scaffolds (CopiOs [Zimmer] and Bio-Oss [Geistlich]) were loaded with different concentrations of rhBMP6 (40, 120, and 240 ng/mm3 rhBMP6) and implanted subcutaneously in Nu (BALB/c and NMR1) and WT (BALB/c and c57BL/6) mice. CaP scaffolds loaded with rhBMP6 did not form bone in WT mice. However, in Nu mice, 40 ng/mm3 rhBMP6 was sufficient to generate relevant volumes of new bone at 6 weeks after implantation. Looking into potential underlying mechanisms, TNF-α blocking antibodies were injected intraperitoneally but could not restore bone formation. Also, mouse periosteal cells (mPDCs) seeded in CopiOs loaded with rhBMP6 did not significantly improve the outcome. Abrogation of bone formation was associated with dense cellular infiltration, in particular with the presence of CD3+ T-lymphocytes. To probe a correlation between calcium ions and impaired bone formation in WT mice, type 1 collagen gels were loaded with rhBMP6 and calcium chloride and injected subcutaneously. These gels generated new bone in WT mice despite the increased percentage of CD3+ cells at Day 3 after implantation as compared with control gels. Overall, this study illustrated the negative effect of the inflammatory host response on the bone-forming capacity of rhBMP6 coated on bioceramic scaffolds.

Congenital Zika Virus Infection in Immunocompetent Mice Causes Postnatal Growth Impediment and Neurobehavioral Deficits.

A small percentage of babies born to Zika virus (ZIKV)-infected mothers manifest severe defects at birth, including microcephaly. Among those who appeared healthy at birth, there are increasing reports of postnatal growth or developmental defects. However, the impact of congenital ZIKV infection in postnatal development is poorly understood. Here, we report that a mild congenital ZIKV-infection in pups born to immunocompetent pregnant mice did not display apparent defects at birth, but manifested postnatal growth impediments and neurobehavioral deficits, which include reduced locomotor and cognitive deficits that persisted into adulthood. We found that the brains of these pups were smaller, had a thinner cortical layer 1, displayed increased astrogliosis, decreased expression of microcephaly- and neuron development- related genes, and increased pathology as compared to mock-infected controls. In summary, our results showed that even a mild congenital ZIKV infection in immunocompetent mice could lead to postnatal deficits, providing definitive experimental evidence for a necessity to closely monitor postnatal growth and development of presumably healthy human infants, whose mothers were exposed to ZIKV infection during pregnancy.

Time Courses of Cortical Glucose Metabolism and Microglial Activity Across the Life Span of Wild-Type Mice: A PET Study.

Contrary to findings in the human brain, 18F-FDG PET shows cerebral hypermetabolism of aged wild-type (WT) mice relative to younger animals, supposedly due to microglial activation. Therefore, we used dual-tracer small-animal PET to examine directly the link between neuroinflammation and hypermetabolism in aged mice. Methods: WT mice (5-20 mo) were investigated in a cross-sectional design using 18F-FDG (n = 43) and translocator protein (TSPO) (18F-GE180; n = 58) small-animal PET, with volume-of-interest and voxelwise analyses. Biochemical analysis of plasma cytokine levels and immunohistochemical confirmation of microglial activity were also performed. Results: Age-dependent cortical hypermetabolism in WT mice relative to young animals aged 5 mo peaked at 14.5 mo (+16%, P < 0.001) and declined to baseline at 20 mo. Similarly, cortical TSPO binding increased to a maximum at 14.5 mo (+15%, P < 0.001) and remained high to 20 mo, resulting in an overall correlation between 18F-FDG uptake and TSPO binding (R = 0.69, P < 0.005). Biochemical and immunohistochemical analyses confirmed the TSPO small-animal PET findings. Conclusion: Age-dependent neuroinflammation is associated with the controversial observation of cerebral hypermetabolism in aging WT mice.

The influence of different calorie restriction protocols on serum pro-inflammatory cytokines, adipokines and IGF-I levels in female C57BL6 mice: short term and long term diet effects.

Calorie restriction (CR) is an effective intervention to prevent chronic diseases including cancer. Although many factors, i.e., sex hormones, IGF-I and mTOR have been studied in response to CR, the molecular mechanisms of CR remain to be identified. Our objective was to determine the short and long-term effects of different CR protocols on pro-inflammatory cytokines. Our hypothesis was that Intermittent CR (ICR) would result in greater inhibition of pro-inflammatory serum cytokines compared to Chronic CR (CCR) as we previously found ICR to be more protective in the prevention of mammary tumor development. From ten weeks of age female C57BL6 mice were maintained on either ad libitum (AL) fed, ICR or CCR protocols (overall CR of ~75% of AL) for up to 74 weeks of age. Blood samples were collected for measurements of serum interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), adiponectin, leptin, IGF-I and insulin at specified ages. For ICR mice samples were collected following 3 weeks of restriction (ICR-R) and after one week of refeeding (ICR-RF). In general, both modes of CR significantly reduced serum IL-6, TNF-α, IGF-I and leptin levels compared to AL with IL-6 levels 24 and 3.5 fold and TNF-α levels t 11 and 1.5 fold lower in ICR and CCR groups, respectively at study termination. There was a trend for adiponectin and insulin to be highest in ICR-RF mice. Body weights were positively correlated with IL-6, TNF-α, insulin and leptin but negatively correlated with adiponectin-to-leptin ratio. Moreover, there was a positive correlation between IL-6 and TNF-α. Beneficial effects of ICR may function through pro-inflammatory cytokine pathways.

Distribution of ASIC4 transcripts in the adult wild-type mouse brain.

Acid-sensing ion channel 4 (ASIC4) belongs to the ASIC gene family of neuronal proton-gated cation channels, and is the least understood subtype among the members. Previous studies of ASIC4 expression in the mammalian central nervous system have shown that ASIC4 is abundantly expressed in the spinal cord and in various brain regions, such as the cerebral cortex, the hippocampus, and the cerebellum. However, the detailed distribution of ASIC4 transcripts in mammalian brains still remains to be elucidated. In the present study, radioactive in situ hybridization histochemistry with an ASIC4-specific cRNA probe was performed on wild-type mouse brains, followed by X-gal staining experiments with Asic4-lacZ reporter mice Asic4tm1a(KOMP)Mbp. It was found that ASIC4 mRNAs were widely expressed throughout the wild-type brain, but preferentially concentrated in the olfactory bulb, the piriform cortex, the caudate putamen, the preoptic area, the paraventricular nucleus, the medial habenular nucleus, the pretectal area, the lateral geniculate nucleus, the amygdaloid complex, the superior colliculus, the interpeduncular nucleus, and the granule cell layer of the ventral hippocampus, and these results were in agreement with the X-gal-positive reactions observed in the mutant brain. In addition, X-gal staining combined with immunohistochemistry identified intense signals for ASIC4 transcriptional activity in most of the choline acetyltransferase (ChAT)-positive principal neurons located in the basal forebrain cholinergic nuclei. Our data provide useful information to speculate possible roles of ASIC4 in diverse brain functions.

Interaction of (12)C ions with the mouse retinal response to light.

Astronauts in orbit reported phosphenes varying in shape and orientation across the visual field; incidence was correlated with the radiation flux. Patients with skull tumors treated by (12)C ions and volunteers whose posterior portion of the eye was exposed to highly ionizing particles in early studies reported comparable percepts. An origin in radiation activating the visual system is suggested. Bursts (∼ 4 ms) of (12)C ions evoked electrophysiological mass responses comparable to those to light in the retina of anesthetized wild-type mice at threshold flux intensities consistent with the incidence observed in humans. The retinal response amplitude increased in mice with ion intensity to a maximum at ∼ 2000 ions/burst, to decline at higher intensities; the inverted-U relationship suggests complex effects on retinal structures. Here, we show that bursts of (12)C ions presented simultaneously to white light stimuli reduced the presynaptic mass response to light in the mouse retina, while increasing the postsynaptic retinal and cortical responses amplitude and the phase-locking to stimulus of cortical low frequency and gamma (∼ 25-45 Hz) responses. These findings suggest (12)C ions to interfere with, rather than mimicking the light action on photoreceptors; a parallel action on other retinal structures/mechanisms resulting in cortical activation is conceivable. Electrophysiological visual testing appears applicable to monitor the radiation effects and in designing countermeasures to prevent functional visual impairment during operations in space.