C57BL6J Mice Research Articles

5-Fluorouracil disrupts ovarian preantral follicles in young C57BL6J mice.

5-Fluorouracil (5-FU), an anti-cancer drug, has been used for hepatoblastoma (HB) chemotherapy in children, who mayhave impaired ovarian follicle pool reserve with lasting effectsto reproduction. Therefore, this study aimed to investigate 5-FU effects on survival, growth, and morphology of ovarian preantral follicles from C57BL6J young mice. Experiments were carried-out both in vivo and in vitro. Mice were treated with 5-FU injection (450mg/kg i.p) or saline and sacrificed 3days after to obtain ovaries for histology and molecular biology. Ovaries for in vitro studies were obtained from unchallenged mice and cultured under basic culture medium (BCM) or BCM plus 5-FU (9.2, 46.1, 92.2mM). Preantral follicles were classified according to developmental stages, and as normal or degenerated. To assess cell viability, caspase-3 immunostaining was performed. Transcriptional levels for apoptosis (Bax, Bcl2, p53, Bax/Bcl2) and Wnt pathway genes (Wnt2 and Wnt4) were also analyzed. Ultrastructural analyses were carried-out on non-cultured ovaries. In addition, β-catenin immunofluorescence was assessed in mouse ovaries. The percentage of all-types normal follicles was significantly lower after 5-FU challenge. A total loss of secondary normal follicles was found in the 5-FU group. The highest 5-FU concentrations reduced the percentage of cultured normal primordial follicles. Large vacuoles were seen in granulosa cells and ooplasm of preantral follicles by electron microscopy. A significantly higher gene expression for Bax and Bax/Bcl2 ratio was seen after 5-FU treatment. A marked reduction in β-catenin immunolabeling was seen in 5-FU-challenged preantral follicles. In the in vitro experiments, apoptotic and Wnt gene transcriptions were significantly altered. Altogether, our findings suggest that 5-FU can deleteriously affect the ovarian follicle reserve by reducing preantral follicles survival.

Rspo1-LGR4 axis in BMSCs protects bone against radiation-induced injury through the mTOR-dependent autophagy pathway.

While mesenchymal stem cells (MSCs) have been widely used to repair radiation-induced bone damage, the molecular mechanism underlying the effects of MSCs in the maintenance of bone homeostasis under radiation stress remains largely unknown. In this study, the role and mechanisms of R-spondin 1 (Rspo1)-leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) axis on the initiation of self-defense of bone mesenchymal stem cells (BMSCs) and maintenance of bone homeostasis under radiation stress were investigated. Interestingly, radiation increased levels of Rspo1 and LGR4 in BMSCs. siRNA knockdown of Rspo1 or LGR4 aggravated radiation-induced impairment of self-renewal ability and osteogenic differentiation potential of BMSCs. However, exogenous Rspo1 significantly attenuated radiation-induced depletion of BMSCs, and promoted the lineage shift towards osteoblasts. This alteration was associated with the reversal of mammalian target of rapamycin (mTOR) activation and autophagy decrement. Pharmacological and genetic blockade of autophagy attenuated the radio-protective effects of Rspo1, rendering BMSCs more vulnerable to radiation-induced injury. Then bone radiation injury was induced in C57BL6J mice to further determine the radio-protective effects of Rspo1. In mice, administration of Rspo1 recombinant protein alleviated radiation-induced bone loss. Our results uncover that Rspo1-LGR4-mTOR-autophagy axis are key mechanisms by which BMSCs initiate self-defense against radiation and maintain bone homeostasis. Targeting Rspo1-LGR4 may provide a novel strategy for the intervention of radiation-induced bone damage.

Tissue-Specific 1H-NMR Metabolomic Profiling in Mice with Adenine-Induced Chronic Kidney Disease.

Chronic kidney disease (CKD) results in the impaired filtration of metabolites, which may be toxic or harmful to organs/tissues. The objective of this study was to perform unbiased 1H nuclear magnetic resonance (NMR)-based metabolomics profiling of tissues from mice with CKD. Five-month-old male C57BL6J mice were placed on either a casein control diet or adenine-supplemented diet to induce CKD for 24 weeks. CKD was confirmed by significant increases in blood urea nitrogen (24.1 ± 7.7 vs. 105.3 ± 18.3 mg/dL, p < 0.0001) in adenine-fed mice. Following this chronic adenine diet, the kidney, heart, liver, and quadriceps muscles were rapidly dissected; snap-frozen in liquid nitrogen; and the metabolites were extracted. Metabolomic profiling coupled with multivariate analyses confirm clear separation in both aqueous and organic phases between control and CKD mice. Severe energetic stress and apparent impaired mitochondrial metabolism were observed in CKD kidneys evidenced by the depletion of ATP and NAD+, along with significant alterations in tricarboxylic acid (TCA) cycle intermediates. Altered amino acid metabolism was observed in all tissues, although significant differences in specific amino acids varied across tissue types. Taken together, this study provides a metabolomics fingerprint of multiple tissues from mice with and without severe CKD induced by chronic adenine feeding.

Unique Subtype of Microglia in Degenerative Thalamus After Cortical Stroke.

Stroke disrupts neuronal functions in both local and remotely connected regions, leading to network-wide deficits that can hinder recovery. The thalamus is particularly affected, with progressive development of neurodegeneration accompanied by inflammatory responses. However, the complexity of the involved inflammatory responses is poorly understood. Herein we investigated the spatiotemporal changes in the secondary degenerative thalamus after cortical stroke, using targeted transcriptome approach in conjunction with histology and flow cytometry. Cortical ischemic stroke was generated by permanent occlusion of the left middle cerebral artery in male C57BL6J mice. Neurodegeneration, neuroinflammatory responses, and microglial activation were examined in naive and stroke mice at from poststroke days (PD) 1 to 84, in both ipsilesional somatosensory cortex and ipsilesional thalamus. NanoString neuropathology panel (780 genes) was used to examine transcriptome changes at PD7 and PD28. Fluorescence activated cell sorting was used to collect CD11c+ microglia from ipsilesional thalamus, and gene expressions were validated by quantitative real-time polymerase chain reaction. Neurodegeneration in the thalamus was detected at PD7 and progressively worsened by PD28. This was accompanied by rapid microglial activation detected as early as PD1, which preceded the neurodegenerative changes. Transcriptome analysis showed higher number of differentially expressed genes in ipsilesional thalamus at PD28. Notably, neuroinflammation was the top activated pathway, and microglia was the most enriched cell type. Itgax (CD11c) was the most significantly increased gene, and its expression was highly detected in microglia. Flow-sorted CD11c+ microglia from degenerative thalamus indicated molecular signatures similar to neurodegenerative disease-associated microglia; these included downregulated Tmem119 and CX3CR1 and upregulated ApoE, Axl, LpL, CSF1, and Cst7. Our findings demonstrate the dynamic changes of microglia after stroke and highlight the importance of investigating stroke network-wide deficits. Importantly, we report the existence of a unique subtype of microglia (CD11c+) with neurodegenerative disease-associated microglia features in the degenerative thalamus after stroke.

Geometric mapping of the eye lens and its epithelium reveals changes that accompany emmetropia

PurposeTo investigate changes in both lens shape and the epithelial distribution as emmetropia is established in C57Bl6J mice between postnatal weeks 4 to 6.MethodsWe used a computational approach to produce geometrical maps of the mouse lens and its epithelium. Conveniently our approach ulitises data collected from intact lenses imaged using a conventional laser scanning confocal microscope. It delivers cartesian coordinates for every epithelial cell in the lens epithelium. We chose to investigate lenses during postnatal weeks 4 to 6, a time during lens development when emmetropia is established in this inbred strain of mice (Tkatchenko et al., 2010).ResultsGeometric mapping standardises the measurement and analysis of lens epithelial cell densities. Emmetropia is established in C57Bl6J inbred mice, but many other strains exhibit considerable refractive error (Tkatchenko et al., 2019). The eye lens plays its part in the development and establishment of emmetropia in mice (Tkatchenko et al., 2010; Pardue et al., 2013). We record a change in the shape of the eye lens from a spheroid to a lentoid shape between postnatal weeks 4 and 6. The distribution of the epithelial cells in the lens epithelium also changes from a non‐zonal to a zonal distribution, with characteristics that typify the epithelia of adult animals. We correlate these observations regarding lens geometry with genetic mutations in lens cytoskeletal proteins found in those strains that present with refractive error, but which are absent from C57Bl6J (Sandilands et al., 2004). We discuss the potential significance of these observations in the context of the mechanistic basis of the lens contribution to emmetropia.ConclusionsThe development of emmetropia in the C57Bl6J inbred strain of mice correlates with changes in epithelial cell organisation and changes in lens geometry, which when combined with strain‐specific genetic differences offer a mechanistic understanding involving the lintermediate filament cytoskeleton.Pardue, M. T., et al., (2013) Exp Eye Res, 114, 96–105.Sandilands, A., et al (2004). Eye Res. 78, 109–123.Tkatchenko, T. V. et al. (2019). BMC Medical Genomics, 12, 1–24.Tkatchenko, T. V., Shen, Y. and Tkatchenko, A. V. (2010) Invest Ophth Vis Sci, 51, 21–27. https://doi.org/10.1167/iovs.08‐2767

Ticagrelor alleviates sepsis-induced myocardial injury via an adenosine-dependent pathway in a mouse sepsis model.

The purpose of this study was to determine whether ticagrelor, a classic anti-platelet drug, has a therapeutic effect on sepsis-induced myocardial injury. The C57BL6J mice received oral ticagrelor (10, 25 and 50 mg/kg) for seven days after which cecum ligation and puncture (CLP) were performed. An adenosine-receptor antagonist (CGS15943) was administered two hours before CLP. After 24 h, cardiac function was measured using cardiac echocardiography, then the heart and blood were collected. Hematoxylin and eosin (HE) staining and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL staining) were used to observe pathological changes and cardiomyocyte apoptosis. Plasma concentration of TNF-α, IL-6 and adenosine and myocardial tissue levels of TNF-α and IL-6 were determined. Survival analysis was performed. Western blot was used to determine the expression of a signalling protein in the myocardial tissue. The HE and TUNEL staining showed less inflammatory cell infiltration and less cardiomyocyte apoptosis in the ticagrelor group. Cardiac echocardiography showed preserved heart function in the ticagrelor group. Plasma TNF-α, IL-6 and relative expression of TNF-α and IL-6 in myocardial tissue were significantly lower in the ticagrelor group. Plasma adenosine levels were significantly higher in the ticagrelor group. Adenosine-receptor antagonists significantly blocked the protective effect of ticagrelor. Ticagrelor reduced the mortality of sepsis mice, and this reduction was blocked by the adenosine-receptor antagonist. Western blot showed that ticagrelor activated the phosphorylation of AKT and mTOR. Adenosine-receptor antagonists inhibited the activation of AKT and mTOR. The protective effect of ticagrelor was dependent on adenosine-receptor activation, with downstream upregulation of phosphorylation of AKT and mTOR.

Pharmacologic inhibition of serotonin htr2b ameliorates hyperglycemia and the altered expression of hepatic FGF21, Sdf2l1, and htr2a in db/db mice and KKAy mice

Plasma fibroblast growth factor 21 (FGF21) levels and hepatic FGF21, serotonin 2a receptor (htr2a), and stromal cell-derived factor 2 like 1 (Sdf2l1) expression are increased in insulin-resistant C57BL6J mice fed a high-fat diet. Here we show that plasma FGF21 levels and hepatic FGF21, Sdf2l1, and htr2a expression were decreased in 6-week-old db/db mice compared with C57BL6J mice, whereas they were increased in 6-week-old KKAy mice compared with KK mice. Expression of hepatic htr2b was increased in db/db mice and KKAy mice compared with controls. Treatment with the selective htr2b antagonist SB204741 suppressed the hyperglycemia in either db/db mice or KKAy mice. Treatment with SB20471 reversed the decreases in plasma FGF21 levels and hepatic FGF21, Sdf2l1, and htr2a expression in db/db mice, whereas it suppressed the increases in plasma FGF21 levels and hepatic FGF21, Sdf2l1, and htr2a expression in KKAy mice. Moreover, treatment with SB204741 increased plasma FGF21 levels and expression of hepatic FGF21, htr2a, and Sdf2l1 in C57BL6J mice, whereas it decreased plasma FGF21 levels and hepatic FGF21 expression in KK mice. These findings suggest that pharmacologic inhibition of htr2b ameliorates the hyperglycemia and altered expression of hepatic FGF21, Sdf2l1 and htr2a in obese and diabetic db/db and KKAy mice.

Endurance exercise plus overload induces fatigue resistance and similar hypertrophy in mice irrespective of muscle mass.

What is the central question of this study? Does combining endurance and hypertrophic stimuli blunt the adaptations to both modalities and is this effect greater in muscles with larger baseline fibre cross sectional area? What is the main finding and its importance? Endurance exercise and hypertrophic stimuli can be combined to increase fatigue resistance and fibre size without blunting either adaptation regardless of baseline fibre size. Previous studies have demonstrated that fibre cross-sectional area (FCSA) is inversely related to oxidative capacity, which is thought to be determined by diffusion limitations of oxygen, ADP and ATP. Consequently, it is hypothesised that (1) when endurance training is combined with a hypertrophic stimulus the response to each will be blunted, and (2) muscles with a smaller FCSA will show a larger hypertrophic response than those with a large FCSA. To investigate this, we combined overload with endurance exercise in 12-month-old male mice from three different strains with different FCSA: Berlin High (BEH) (large fibres), C57BL/6J (C57) (normal-sized fibres) and Berlin Low (BEL) (small fibres). The right plantaris muscle was subjected to overload through denervation of synergists with the left muscle acting as an internal control. Half the animals trained 30min per day for 6weeks. The overload-induced hypertrophy was not blunted by endurance exercise, and the exercise-induced increase in fatigue resistance was not impaired by overload. All strains demonstrated similar absolute increases in FCSA, although the BEH mice with more fibres than the C57 mice demonstrated the largest increase in muscle mass and BEL mice with fewer fibres the smallest increase in muscle mass. This study suggests that endurance exercise and hypertrophic stimuli can be combined without attenuating adaptations to either modality, and that increases in FCSA are independent of baseline fibre size.

Moderate Aerobic Exercise Training Prevents the Augmented Hepatic Glucocorticoid Response Induced by High-Fat Diet in Mice.

Glucocorticoids (GCs) are critical regulators of energy balance. Their deregulation is associated with the development of obesity and metabolic syndrome. However, it is not understood if obesity alters the tissue glucocorticoid receptor (GR) response, and moreover whether a moderate aerobic exercise prevents the alteration in GR response induced by obesity. Methods: To evaluate the GR response in obese mice, we fed C57BL6J mice with a high-fat diet (HFD) for 12 weeks. Before mice were sacrificed, we injected them with dexamethasone. To assess the exercise role in GR response, we fed mice an HFD and subjected them to moderate aerobic exercise three times a week. Results: We found that mice fed a high-fat diet for 12 weeks developed hepatic GC hypersensitivity without changes in the gastrocnemius or epididymal fat GR response. Therefore, moderate aerobic exercise improved glucose tolerance, increased the corticosterone plasma levels, and prevented hepatic GR hypersensitivity with an increase in epididymal fat GR response. Conclusion: Collectively, our results suggest that mice with HFD-induced obesity develop hepatic GR sensitivity, which could enhance the metabolic effects of HFD in the liver. Moreover, exercise was found to be a feasible non-pharmacological strategy to prevent the deregulation of GR response in obesity.

Spontaneous Mouse Behavior in Presence of Dissonance and Acoustic Roughness.

According to a novel hypothesis (Arnal et al., 2015, Current Biology 25:2051–2056), auditory roughness, or temporal envelope modulations between 30 and 150 Hz, are present in both natural and artificial human alarm signals, which boosts the detection of these alarms in various tasks. These results also shed new light on the unpleasantness of dissonant sounds to humans, which builds upon the high level of roughness present in such sounds. However, it is not clear whether this hypothesis also applies to other species, such as rodents. In particular, whether consonant/dissonant chords, and particularly whether auditory roughness, can trigger unpleasant sensations in mice remains unknown. Using an autonomous behavioral system, which allows the monitoring of mouse behavior over a period of weeks, we observed that C57Bl6J mice did not show any preference for consonant chords. In addition, we found that mice showed a preference for rough sounds over sounds having amplitude modulations in their temporal envelope outside the “rough” range. These results suggest that some emotional features carried by the acoustic temporal envelope are likely to be species-specific.

Single transient intraocular pressure elevations cause prolonged retinal ganglion cell dysfunction and retinal capillary abnormalities in mice

Transient intraocular pressure (IOP) elevations are likely to occur in certain forms of glaucoma and after intravitreal injections to treat various retinal diseases. However, the impact of these transient IOP elevations on the physiology of individual retinal ganglion cells (RGCs) is unknown. In this report, we explore how transient IOP elevations in mice affect RGC physiology, RGC anatomy, and retinal arteriole and capillary structure. Transient IOP elevation was induced in 12-week old wild type C57BL6J mice by injecting sodium hyaluronate into the anterior chamber. IOP was measured immediately after the injection and again 1 and 7 days later. Average peak IOP after injection was ~50 mmHg and subsequent IOPs returned to normal. RGC physiology was assessed with a multielectrode array (MEA) by calculating a spike triggered average (STA) at the same time points. RGC counts and retinal vascular structure were assessed 14 days after injection with immunohistochemistry to label RGCs and blood vessels. Transient IOP elevation caused a marked reduction of scotopic STA presence and delayed center and surround STA peak times that did not recover. Transient IOP elevation also caused a reduced photopic receptive field size and spontaneous firing rate, both of which showed some recovery with time. Transient IOP elevation also induced vascular remodeling: the number of capillary branches was decreased within the superficial and intermediate vascular plexi. RGC counts, retinal arteriole diameter, and deep capillary plexus branching were unaffected. These previously unappreciated findings suggest that transient IOP elevation may cause unrecognized and potentially long-term pathology to RGCs and associated neurovascular units which should be accounted for in clinical practice.

Clostridioides difficile Toxin A Remodels Membranes and Mediates DNA Entry Into Cells to Activate Toll-Like Receptor 9 Signaling

Clostridioides difficile toxin A (TcdA) activates the innate immune response. TcdA co-purifies with DNA. Toll-like receptor 9 (TLR9) recognizes bacterial DNA to initiate inflammation. We investigated whether DNA bound to TcdA activates an inflammatory response in murine models of C difficile infection via activation of TLR9. We performed studies with human colonocytes and monocytes and macrophages from wild-type and TLR9 knockout mice incubated with TcdA or its antagonist (ODN TTAGGG) or transduced with vectors encoding TLR9 or small-interfering RNAs. Cytokine production was measured with enzyme-linked immunosorbent assay. We studied a transduction domain of TcdA (TcdA57-80), which was predicted by machine learning to have cell-penetrating activity and confirmed by synchrotron small-angle X-ray scattering. Intestines of CD1 mice, C57BL6J mice, and mice that express a form of TLR9 that is not activated by CpG DNA were injected with TcdA, TLR9 antagonist, or both. Enterotoxicity was estimated based on loop weight to length ratios. A TLR9 antagonist was tested in mice infected with C difficile. We incubated human colon explants with an antagonist of TLR9 and measured TcdA-induced production of cytokines. The TcdA57-80 protein transduction domain had membrane remodeling activity that allowed TcdA to enter endosomes. TcdA-bound DNA entered human colonocytes. TLR9 was required for production of cytokines by cultured cells and in human colon explants incubated with TcdA. TLR9 was required in TcdA-induced mice intestinal secretions and in the survival of mice infected by C difficile. Even in a protease-rich environment, in which only fragments of TcdA exist, the TcdA57-80 domain organized DNA into a geometrically ordered structure that activated TLR9. TcdA from C difficile can bind and organize bacterial DNA to activate TLR9. TcdA and TcdA fragments remodel membranes, which allows them to access endosomes and present bacterial DNA to and activate TLR9. Rather than inactivating the ability of DNA to bind TLR9, TcdA appears to chaperone and organize DNA into an inflammatory, spatially periodic structure.

Dietary Cyanidin-3-Glucoside Attenuates High-Fat-Diet–Induced Body-Weight Gain and Impairment of Glucose Tolerance in Mice via Effects on the Hepatic Hormone FGF21

Dietary polyphenols including anthocyanins target multiple organs. We aimed to assess the involvement of glucagon-like peptide 1 (GLP-1), leptin, insulin and fibroblast growth factor 21 (FGF21) in mediating metabolic beneficial effects of purified anthocyanin cyanidin-3-glucoside (Cy3G). Intestinal proglucagon gene (Gcg; encoding GLP-1) and liver Fgf21 expression were assessed in 6-wk-old male C57BL-6J mice fed a low-fat-diet (LFD; 10% of energy from fat), alone or with 1.6mg Cy3G/L in drinking water for 3 wk [experiment (Exp.) 1; n=5/group]. Similar mice were fed the LFD or a high-fat diet (HFD; 60% energy from fat) with or without Cy3G for 20 wk. Half of the mice administered Cy3G also received 4 broad-spectrum antibiotics (ABs) in drinking water between weeks 11 and 14, for a total of 6 groups (n=8/group). Metabolic tolerance tests were conducted between weeks 2 and 16. Relevant hormone gene expression and plasma hormone concentrations were assessed mainly at the end of 20 wk (Exp. 2). In Exp. 1, Cy3G administration increased ileal but not colonic Gcg level by 2-fold (P<0.05). In Exp. 2, Cy3G attenuated HFD-induced body-weight gain (20.3% at week 16), and improved glucose tolerance (26.5% at week 15) but not insulin tolerance. Although Cy3G had no effect on glucose tolerance in LFD mice, LFD/Cy3G/AB mice showed better glucose tolerance than LFD/Cy3G mice (23%). In contrast, HFD/Cy3G/AB mice showed worse glucose tolerance compared with HFD/Cy3G mice (15%). Beneficial effects of Cy3G in HFD mice were not associated with changes in plasma leptin, insulin or GLP-1 concentrations. However, Cy3G increased hepatic Fgf21 expression in mice in Exp. 1 by 4-fold and attenuated Fgf21 overexpression in HFD mice (Exp. 2, 22%), associated with increased expression of genes that encode FGFR1 and β-klotho (>3-fold, P <0.05). Dietary Cy3G may reduce body weight and exert metabolic homeostatic effects in mice via changes in hepatic FGF21.

Modeling a potential SANS countermeasure by experimental manipulation of the translaminar pressure difference in mice

The spaceflight-associated neuro-ocular syndrome (SANS), which may present after prolonged exposure to microgravity, is thought to occur due to elevated intracranial pressure (ICP). Intracranial pressure interacts with intraocular pressure (IOP) to define the translaminar pressure difference (TLPD; IOP−ICP). We combined inducible models of ICP and IOP elevation in mice to interrogate the relationships among ICP, IOP, and TLPD, and to determine if IOP elevation could mitigate the phenotypes typically caused by elevated ICP and thereby serve as a countermeasure for SANS. Ten C57BL6J mice of both genders underwent experimental elevation of ICP via infusion of artificial cerebrospinal fluid into the subarachnoid space. One eye also underwent experimental elevation of IOP using the bead injection model. Intraocular pressure and ICP were monitored for 2 weeks. Optokinetic-based contrast sensitivity was measured at baseline and after 2 weeks, and post-mortem studies of optic nerve and retina anatomy were performed. Photopic contrast sensitivity was reduced more in IOP elevated than control eyes. Scotopic contrast sensitivity was reduced similarly in IOP elevated and control eyes. However, the pattern of scotopic vision loss was not uniform in IOP elevated eyes; there was minimal loss in eyes that most closely approximated the normal TLPD. Optic nerve axon loss, increased optic nerve disorganization, and retinal ganglion cell loss all occurred similarly between IOP elevated and control eyes. Elevation of IOP in eyes with elevated ICP may counterbalance some effects on vision loss but exacerbate others, suggesting complex relationships among IOP, ICP, and TLPD.

Stimulus-Driven Retinal Intrinsic Signal Optical Imaging in Mouse Demonstrates a Dominant Rod-Driven Component.

PurposeThe primary hypotheses tested are that (1) there exist stimulus-driven intrinsic optical signals in the mouse retina similar to those previously observed in other species, and (2) these optical signals require an intact rod photoreceptor phototransduction cascade.MethodsWe used 38 wild-type C57BL6J mice and 18 genetic knockout Gnat1−/− mice to study the light-evoked retinal intrinsic response. A custom mouse fundus camera delivered visual stimuli and collected mouse retinal imaging data of changes in retinal reflectance for further analysis. The retina was stimulated in the high-mesopic range with a 505-nm light-emitting diode while also being illuminated with 780-nm near-infrared light.ResultsWild-type C57BL6J mice yielded retinal imaging signals that typically showed a stimulus-driven decrease in retinal reflectance of ∼0.1%, with a time course of several seconds. The signals exhibit spatial specificity in the retina. Overall, the mouse imaging signals are similar in sign and time course to those reported in other mammalian species but are of lower amplitude. In contrast, functional retinal imaging of Gnat1−/− mice that lack a functional rod transducin yielded no such stimulus-driven signals.ConclusionsPrevious studies have not shown which pathway component is essential for the generation of these imaged signals. The absence of the intrinsic signal responses in Gnat1−/− knockout mice indicates that a functional rod transducin is likely to be necessary for generating the retinal intrinsic signals. These studies, to the best of our knowledge, demonstrate for the first time in vivo mouse retinal functional imaging signals similar to those previously shown in other mammalian species.

Memory and Learning Deficits Are Associated With Ca2+ Dyshomeostasis in Normal Aging.

Neuronal intracellular Ca2+ homeostasis is critical to the normal physiological functions of neurons and neuronal Ca2+ dyshomeostasis has been associated with the age-related decline of cognitive functions. Accumulated evidence indicates that the underlying mechanism for this is that abnormal intracellular Ca2+ levels stimulate the dysregulation of intracellular signaling, which subsequently induces neuronal cell death. We examined intracellular Ca2+ homeostasis in cortical (in vivo) and hippocampal (in vitro) neurons from young (3-months), middle-age (12-months), and aged (24-months) wild type C57BL6J mice. We found a progressive age-related elevation of intracellular resting calcium ([Ca2+]r) in cortical (in vivo) and hippocampal (in vitro) neurons associated with increased hippocampal neuronal calpain activity and reduced cell viability. In vitro, removal of extracellular Ca2+ or treatment with SAR7334 or dantrolene reduced [Ca2+]r in all age groups and dantrolene treatment lowered calpain activity and increased cell viability. In vivo, both middle-aged and aged mice showed cognitive deficits compared to young mice, which improved after dantrolene treatment. These findings support the hypothesis that intracellular Ca2+ dyshomeostasis is a major mechanism underlying the cognitive deficits seen in both normal aging and degenerative neurologic diseases.

Estrogen protects dental roots from orthodontic-induced inflammatory resorption

ObjectiveRoot resorption is a side effect of orthodontic tooth movement (OTM). Despite the recognized role of estrogen on bone, there is little information about their effects on orthodontic-induced inflammatory root resorption (OIIRR). We aimed to investigate if estrogen deficiency affects OIIRR in two mice strains. MethodsFemale Balb/C (Balb) and C57BL6/J (C57) mice were ovariectomized (OVX) and replaced with estradiol (E2). Tooth samples subjected or not to OTM were collected and analyzed by microCT, histomorphometry and qPCR. ResultsOVX resulted in decreased root volume (RV/TV) and root mineral density (RMD) in Balb mice without OTM. In contrast, OVX did not modify physiological root structure of C57 mice. OTM and OIIRR were increased after OVX in both mice strains after 30 days. E2 replacement reversed this phenotype in Balb, but not in C57 mice. Due to the significant increase of OIIRR in OVX Balb mice, the expression of key molecules was investigated in periodontium. Accordingly, these mice showed increased expression of receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor alpha, matrix metalloproteinases-2 and -13 and decreased osteoprotegerin (OPG) and interleukin-10 expression after OTM. E2 replacement reversed the changes of these markers. ConclusionThe lack of estrogen in Balb mice without OTM triggered loss of root structure which was positively correlated to RANKL/OPG ratio. Regardless of mouse strain, the absence of estrogen following OTM induced OIIRR. Mechanisms involve the imbalance of RANKL/OPG system, inflammatory and osteoclastic makers.

P0679LOSARTAN REDUCES INFLAMMATION INDUCED BY TERTIARY LYMPHOID TISSUES IN ENSUING CKD AFTER ACUTE KIDNEY INJURY

Abstract Background and Aims Chronic inflammation and renin-angiotensin system (RAS) overactivation have been demonstrated as a central mechanism that results in the development of progressive chronic kidney disease (CKD). Evidences have indicated that tertiary lymphoid tissues (TLT) form after acute kidney injury (AKI), especially in old-aged mice. Heterogeneous fibroblasts which appear and secret proinflammatory cytokines after AKI underlie TLT formation. There is an association between TLT sizes and impaired renal function along with increase of proinflammatory cytokines, suggesting a probable cause of persistent inflammation after AKI. In this study, we explored the relationship between TLT development and RAS activity. The phenotypes of heterogeneous fibroblasts diversified after AKI is also investigated. Method Here we utilized ischemia-reperfusion injury to cause ensuing CKD both in a CD-1 and C57BL6J mice model. We also used staining of CD3 and B220 to label and confirm multiple TLTs which represents T and B lymphocytes respectively. Results Administration of losartan, a kind of RAS inhibitor, offered protection against subsequent CKD development and attenuated number and size of TLT besides blood pressure reduction. Moreover, heterogeneous fibroblasts diminished and proinflammatory cytokines such as CXCL13, CCL21 and CCL19 which trigger the initiation of TLT were downregulated. In experiments of several fibroblast cell lines, angiotensin II (Ang II) administration could induce differentiation of fibroblasts with distinct phenotypes expressing p75 neurotrophin receptor and retinaldehyde dehydrogenase 2 which contribute to TLT formation. These fibroblasts expressing PDGFRβ stimulated production of aforementioned proinflammatory cytokines and expression of pro-fibrotic genes. Conclusion Our data demonstrates that TLT plays an important role in AKI-CKD transition. We also verify that Ang II causes phenotypic change of fibroblasts which constitute TLT. Losartan can ameliorate TLT formation through inhibition of these distinctive fibroblasts. It is a plausible mechanism to reduce ensuing CKD.

The Hepatoprotective Effect of Taurisolo, a Nutraceutical Enriched in Resveratrol and Polyphenols, Involves Activation of Mitochondrial Metabolism in Mice Liver.

Liver diseases affect millions of people worldwide. In most of the cases, severe hepatic dysfunction and liver cancer stem from mild and common clinical signs including hepatic steatosis, insulin resistance, liver inflammation, and oxidative stress, all together referred to as Nonalcoholic Fatty Liver Disease (NAFLD). Nutraceuticals endowed with antioxidant activity have been shown to reduce NAFLD risk factors and exert hepatoprotective effects. Here, we test the protective effect exerted on liver by the antioxidant Taurisolo, a nutraceutical formulation produced by grape pomace and enriched in Resveratrol and Polyphenols. We analyze the effect of Taurisolo on liver cells by profiling the metabolome of in vitro cultured hepatic HuH7 cells and of C57BL-6J mice fed a High Fat Diet and treated with the nutraceutical. Both in vitro and in vivo, we provide evidence that Taurisolo reduces risk factor markers associated with NAFLD. Taurisolo stimulates glucose uptake and reduces hepatic cholesterol and serum triglycerides. Furthermore, we give new insights into the mechanism of action of Taurisolo. The nutraceutical increases mitochondrial activity and promotes respiration and ATP production, fostering catabolic reactions like fatty acid β-oxidation and amino acid catabolism. On the contrary, Taurisolo reduces anabolic reactions like biosynthesis of cholesterol, bile acids, and plasma membrane lipids.

Nonnutritive sweetener consumption during pregnancy, adiposity, and adipocyte differentiation in offspring: evidence from humans, mice, and cells

Obesity often originates in early life, and is linked to excess sugar intake. Nonnutritive sweeteners (NNS) are widely consumed as "healthier" alternatives to sugar, yet recent evidence suggests NNS may adversely influence weight gain and metabolic health. The impact of NNS during critical periods of early development has rarely been studied. We investigated the effect of prenatal NNS exposure on postnatal adiposity and adipocyte development. In the CHILD birth cohort (N = 2298), we assessed maternal NNS beverage intake during pregnancy and child body composition at 3 years, controlling for maternal BMI and other potential confounders. To investigate causal mechanisms, we fed NNS to pregnant C57BL6J mice at doses relevant to human consumption (42 mg/kg/day aspartame or 6.3 mg/kg/day sucralose), and assessed offspring until 12 weeks of age for: body weight, adiposity, adipose tissue morphology and gene expression, glucose and insulin tolerance. We also studied the effect of sucralose on lipid accumulation and gene expression in cultured 3T3-L1 pre-adipocyte cells. In the CHILD cohort, children born to mothers who regularly consumed NNS beverages had elevated body mass index (mean z-score difference +0.23, 95% CI 0.05-0.42 for daily vs. no consumption, adjusted for maternal BMI). In mice, maternal NNS caused elevated body weight, adiposity, and insulin resistance in offspring, especially in males (e.g., 47% and 15% increase in body fat for aspartame and sucralose vs. controls, p < 0.001). In cultured adipocytes, sucralose exposure at early stages of differentiation caused increased lipid accumulation and expression of adipocyte differentiation genes (e.g., C/EBP-α, FABP4, and FASN). These genes were also upregulated in adipose tissue of male mouse offspring born to sucralose-fed dams. By triangulating evidence from humans, mice, and cultured adipocytes, this study provides new evidence that maternal NNS consumption during pregnancy may program obesity risk in offspring through effects on adiposity and adipocyte differentiation.