Young Wild-type Mice Research Articles

Altered DNA repair; an early pathogenic pathway in Alzheimer\u2019s disease and obesity

Unrepaired DNA double-strand breaks (DSBs) are lethal. The present study compared the extent of DSBs, neuronal apoptosis, and status of two major DSB repair pathways - homologous combinational repair (HR) and nonhomologous end-joining (NHEJ) - in hippocampus of 5–6 month and 16–18 month-old wild-type and APP/PSEN1 mice fed control diet or high fat diet (60% fat from lard). We performed immunohistochemical staining and quantification for nuclear foci formation of γ-H2AX for DSBs, RAD51, and 53BP1, which represent the functional status of HR and NHEJ, respectively. Increased γ-H2AX and caspase-3 staining indicated greater DSBs and associated neuronal apoptosis in APP/PSEN1 mice at both ages studied. RAD51-positive foci were fewer in APP/PSEN1 indicating that HR processes may be diminished in these mice, although NHEJ (53BP1 staining) appeared unchanged. High fat diet in young wild-type mice led to similar changes to those observed in APP/PSEN1 mice (γ-H2AX and caspase-3 staining, and fewer RAD51-positive foci). Overall, these data suggest that APP/PSEN1- and high fat diet-associated early accumulation of DSBs and neuronal cell death, resulted at least in part, from inhibition of HR, one of the major DSB repair pathways.

AD-Related N-Terminal Truncated Tau Is Sufficient to Recapitulate In Vivo the Early Perturbations of Human Neuropathology: Implications for Immunotherapy.

The NH2tau 26-44 aa (i.e., NH2htau) is the minimal biologically active moiety of longer 20-22-kDa NH2-truncated form of human tau-a neurotoxic fragment mapping between 26 and 230 amino acids of full-length protein (htau40)-which is detectable in presynaptic terminals and peripheral CSF from patients suffering from AD and other non-AD neurodegenerative diseases. Nevertheless, whether its exogenous administration in healthy nontransgenic mice is able to elicit a neuropathological phenotype resembling human tauopathies has not been yet investigated. We explored the in vivo effects evoked by subchronic intracerebroventricular (i.c.v.) infusion of NH2htau or its reverse counterpart into two lines of young (2-month-old) wild-type mice (C57BL/6 and B6SJL). Six days after its accumulation into hippocampal parenchyma, significant impairment in memory/learning performance was detected in NH2htau-treated group in association with reduced synaptic connectivity and neuroinflammatory response. Compromised short-term plasticity in paired-pulse facilitation paradigm (PPF) was detected in the CA3/CA1 synapses from NH2htau-impaired animals along with downregulation in calcineurin (CaN)-stimulated pCREB/c-Fos pathway(s). Importantly, these behavioral, synaptotoxic, and neuropathological effects were independent from the genetic background, occurred prior to frank neuronal loss, and were specific because no alterations were detected in the control group infused with its reverse counterpart. Finally, a 2.0-kDa peptide which biochemically and immunologically resembles the injected NH2htau was endogenously detected in vivo, being present in hippocampal synaptosomal preparations from AD subjects. Given that the identification of the neurotoxic tau species is mandatory to develop a more effective tau-based immunological approach, our evidence can have important translational implications for cure of human tauopathies.

Intrathymic Deletion of IL-7 Reveals a Contribution of the Bone Marrow to Thymic Rebound Induced by Androgen Blockade.

Despite the well-documented effect of castration in thymic regeneration, the singular contribution of the bone marrow (BM) versus the thymus to this process remains unclear. The chief role of IL-7 in pre- and intrathymic stages of T lymphopoiesis led us to investigate the impact of disrupting this cytokine during thymic rebound induced by androgen blockade. We found that castration promoted thymopoiesis in young and aged wild-type mice. In contrast, only young germline IL-7-deficient (Il7-/- ) mice consistently augmented thymopoiesis after castration. The increase in T cell production was accompanied by the expansion of the sparse medullary thymic epithelial cell and the peripheral T cell compartment in young Il7-/- mice. In contrast to young Il7-/- and wild-type mice, the poor thymic response of aged Il7-/- mice after castration was associated with a defect in the expansion of BM hematopoietic progenitors. These findings suggest that BM-derived T cell precursors contribute to thymic rebound driven by androgen blockade. To assess the role of IL-7 within the thymus, we generated mice with conditional deletion of IL-7 (Il7 conditional knockout [cKO]) in thymic epithelial cells. As expected, Il7cKO mice presented a profound defect in T cell development while maintaining an intact BM hematopoietic compartment across life. Unlike Il7-/- mice, castration promoted the expansion of BM precursors and enhanced thymic activity in Il7cKO mice independently of age. Our findings suggest that the mobilization of BM precursors acts as a prime catalyst of castration-driven thymopoiesis.

Oxidation Products of 5-Methylcytosine are Decreased in Senescent Cells and Tissues of Progeroid Mice.

5-Hydroxymethylcytosine and 5-formylcytosine are stable DNA base modifications generated from 5-methylcytosine by the ten-eleven translocation protein family that function as epigenetic markers. 5-Hydroxymethyluracil may also be generated from thymine by ten-eleven translocation enzymes. Here, we asked if these epigenetic changes accumulate in senescent cells, since they are thought to be inversely correlated with proliferation. Testing this in ERCC1-XPF-deficient cells and mice also enabled discovery if these DNA base changes are repaired by nucleotide excision repair. Epigenetic marks were measured in proliferating, quiescent and senescent wild-type (WT) and Ercc1-/- primary mouse embryonic fibroblasts. The pattern of epigenetic marks depended more on the proliferation status of the cells than their DNA repair capacity. The cytosine modifications were all decreased in senescent cells compared to quiescent or proliferating cells, whereas 5-(hydroxymethyl)-2'-deoxyuridine was increased. In vivo, both 5-(hydroxymethyl)-2'-deoxyuridine and 5-(hydroxymethyl)-2'-deoxycytidine were significantly increased in liver tissues of aged WT mice compared to young adult WT mice. Livers of Ercc1-deficient mice with premature senescence and aging had reduced level of 5-(hydroxymethyl)-2'-deoxycytidine and 5-formyl-2'-deoxycytidine compared to aged-matched WT controls. Taken together, we demonstrate for the first time, that 5-(hydroxymethyl)-2'-deoxycytidine is significantly reduced in senescent cells and tissue, potentially yielding a novel marker of senescence.

CD73-A2a adenosine receptor axis promotes innate B cell antibody responses to pneumococcal polysaccharide vaccination.

Many individuals at risk of streptococcal infection respond poorly to the pneumococcal polysaccharide vaccine Pneumovax 23. Identification of actionable pathways able to enhance Pneumovax responsiveness is highly relevant. We investigated the contribution of the extracellular adenosine pathway regulated by the ecto-nucleotidase CD73 in Pneumovax-induced antibody responses. Using gene-targeted mice, we demonstrated that CD73-or A2a adenosine receptor deficiency significantly delayed isotype switching. Nevertheless, CD73- or A2aR- deficient adult mice ultimately produced antigen-specific IgG3 and controlled Streptococcus pneumoniae infection as efficiently as wild type (WT) mice. Compared to adults, young WT mice failed to control S. pneumoniae infection after vaccination and this was associated with lower levels of CD73 on innate B cells. We hypothesized that pharmacological activation of A2a receptor may improve Pneumovax 23 immunization in young WT mice. Remarkably, administration of the A2a adenosine receptor agonist CGS 21680 significantly increased IgG3 responses and significantly enhanced survival after S. pneumoniae challenge. Our study thus suggests that pharmacological activation of the A2a adenosine receptor could improve the efficacy of Pneumovax 23 vaccination in individuals at risk of streptococcal infection.

Osteocalcin in the brain: from embryonic development to age-related decline in cognition.

A remarkable, unexpected aspect of the bone-derived hormone osteocalcin is that it is necessary for both brain development and brain function in the mouse, as its absence results in a profound deficit in spatial learning and memory and an exacerbation of anxiety-like behaviour. The regulation of cognitive function by osteocalcin, together with the fact that its circulating levels decrease in midlife compared with adolescence in all species tested, raised the prospect that osteocalcin might be an anti-geronic hormone that could prevent age-related cognitive decline. As presented in this Review, recent data indicate that this is indeed the case and that osteocalcin is necessary for the anti-geronic activity recently ascribed to the plasma of young wild-type mice. The diversity and amplitude of the functions of osteocalcin in the brain, during development and postnatally, had long called for the identification of its receptor in the brain, which was also recently achieved. This Review presents our current understanding of the biology of osteocalcin in the brain, highlighting the bony vertebrate specificity of the regulation of cognitive function and pointing toward where therapeutic opportunities might exist.

Vitamin D Does Not Play a Functional Role in Adipose Tissue Development in Rodent Models.

Several studies have proposed a role of vitamin D in adipogenesis. Here, we sought to study the impact of the vitamin D receptor (Vdr) on adipocyte size in young and old mice and the effect of maternal vitamin D deficiency on fetal adipogenesis. Histological analysis of adipose tissues shows that Vdr knockout (KO) mice have smaller adipocytes than wild-type (WT) mice. Next, we compare young and old Vdr-KO and WT mice and find no differences in adipocyte sizes between weaned Vdr-KO and WT mice. However, 1-year-old Vdr-KO mice, suffering from alopecia, have smaller-sized adipocytes than WT mice, although they consume more food. To elucidate whether vitamin D can directly impact adipocyte development at a critical stage of adipogenesis, we feed rat dams a vitamin D deficient (0 IU kg-1 ) or vitamin D adequate (1000 IU kg-1 ) diet. Neither DNA microarray analysis of the adipose tissues of the newborn rats nor the adipocyte sizes of 21-day-old offspring show significant differences between the two groups. Data indicate that vitamin D does not play a fundamental role in adipogenesis because vitamin D does not affect fetal adipogenesis. Moreover, the smaller adipocytes observed in adult Vdr-KO mice are presumably caused by an increased energy expenditure due to alopecia.

Sestrin2 prevents age-related intolerance to post myocardial infarction via AMPK/PGC-1α pathway

We have revealed that a novel stress-inducible protein, Sestrin2, declines in the heart with aging. Moreover, there is an interaction between Sestrin2 and energy sensor AMPK in the heart in response to ischemic stress. The objective of this study is to determine whether Sestrin2-AMPK complex modulates PGC-1α in the heart and protects the heart from ischemic insults. In order to characterize the role of cardiac Sestrin2-AMPK signaling cascade in aging, C57BL/6 wild type young mice (3–4months), aged mice (24–26months) and young Sestrin2 KO mice were subjected to left anterior descending coronary artery occlusion for in vivo regional ischemia. Intriguingly, ischemic AMPK activation was blunted in aged WT and young Sesn2 KO hearts as compared with young WT hearts. In addition, the AMPK downstream PGC-1α was down-regulated in the aged and Sestrin2 KO hearts during post myocardial infarction. To further determine the regulation of AMPK on mitochondrial functions in aging, the downstream of mitochondrial biogenesis PGC-1α transcriptional factor were measured. The results demonstrated that the PGC-1α downstream effectors TFAM and UCP2 were impaired in the aged and Sestrin2 KO post-MI hearts as compared to the young hearts. While the apoptotic flux markers such as AIF, Bax/Bcl-2 were up-regulated in both aged and Sestrin2 KO hearts versus young hearts. Furthermore, both Sestrin2 KO and aged hearts demonstrated more susceptible to ischemic insults as compared to young hearts. Additionally, the adeno-associated virus (AAV9)-Sestrin2 delivered to the aged hearts via a coronary delivery approach significantly rescued the ischemic tolerance of aged hearts. Taken together, the decreased Sestrin2 levels in aging lead to an impaired AMPK/PGC-1α signaling cascade and an increased sensitivity to ischemic insults.

In situ regeneration of retinal pigment epithelium by gene transfer of E2F2: a potential strategy for treatment of macular degenerations.

The retinal pigment epithelium (RPE) interacts closely with photoreceptors to maintain visual function. In degenerative diseases such as Stargardt disease and age-related macular degeneration, the leading cause of blindness in the developed world, RPE cell loss is followed by photoreceptor cell death. RPE cells can proliferate under certain conditions, suggesting an intrinsic regenerative potential, but so far this has not been utilised therapeutically. Here, we used E2F2 to induce RPE cell replication and thereby regeneration. In both young and old (2 and 18 month) wildtype mice, subretinal injection of non-integrating lentiviral vector expressing E2F2 resulted in 47% of examined RPE cells becoming BrdU positive. E2F2 induced an increase in RPE cell density of 17% compared with control vector-treated and 14% compared with untreated eyes. We also tested this approach in an inducible transgenic mouse model of RPE loss, generated through activation of diphtheria toxin-A gene. E2F2 expression resulted in a 10-fold increase in BrdU uptake and a 34% increase in central RPE cell density. Although in mice this localised rescue is insufficiently large to be demonstrable by electroretinography, a measure of massed retinal function, these results provide proof-of-concept for a strategy to induce in situ regeneration of RPE for the treatment of RPE degeneration.

Abstract 21054: Hematopoietic Factors Are Sufficient to Increase AF in Aged, Atherosclerotic Mice

Rationale: Aging is a strong risk factor for atrial fibrillation (AF), the most common sustained arrhythmia in the clinical practice. Atrial inflammation correlates with increased AF susceptibility but whether chronic age-related inflammation that occurs with atherosclerosis contributes to AF is unknown. Hypothesis: Hematopoietic factors contribute to AF inducibility in aged atherosclerotic mice. Methods: We used burst electrical stimulation (tachypacing) of the right atrium to test susceptibility to AF in-vivo in young and aged atherosclerotic, low density lipoprotein receptor knockout (C57BL/6 Ldlr -/- ) mice, and young and old wildtype (C57BL/6 WT) mice. We also determined the role of the stroma vs. the hematopoietic system, by creating bone marrow chimeras in which aged or young Ldlr -/- mice were transplanted with age-matched or mismatched bone Ldlr -/- bone marrow. We used whole-cell patch-clamping investigate atrial sodium currents. Confocal microscopy and patch-clamping in current clamp mode was used to simultaneously record action potentials and Ca 2+ transients. Results: Upon atrial tachypacing, aged atherosclerotic mice underwent longer and more frequent AF episodes than young (incidence, 0.47±0.09 vs 0.12±0.02 episodes/attempts; p<0.05 ) and aged WT mice (0.47±0.09 vs 0.16±0.07 episodes/attempts; p<0.05 ). Importantly, young-to-old bone marrow chimeric mice exhibited reduced AF incidence (0.23 ± 0.05 episodes/attempts), whereas old-to-young chimeric mice exhibited increased AF incidence (0.45 ± 0.08 episodes/attempts). Atrial myocytes from aged atherosclerotic mice presented a 50% reduction in the cardiac inward sodium current (I Na ). Moreover, compared to young, aged atherosclerotic mice showed prolonged action potential duration (APD90: 34.0±5.0 ms vs 48.1±7.0ms; p<0.05), post-pacing spontaneous calcium releases (25% vs 81.8%; p<0.05) and triggered activity (0% vs 27.2 %; p<0.05). Conclusions: Aged atherosclerotic prone mice are susceptible to AF induction. Hematopoietic factors are sufficient to increase AF propensity with aging in the atherosclerotic host possibly by causing electrophysiological remodeling and intracellular Ca 2+ handling abnormalities.

The stress kinase JNK regulates gap junction Cx43 gene expression and promotes atrial fibrillation in the aged heart

BackgroundThe stress kinase c-jun N-terminal kinase (JNK) is critical in the pathogenesis of cardiac diseases associated with an increased incidence of atrial fibrillation (AF), the most common arrhythmia in the elderly. We recently discovered that JNK activation is linked to the loss of gap junction connexin43 (Cx43) and enhanced atrial arrhythmogenicity. However, direct evidence for JNK-mediated impairment of intercellular coupling (cell-cell communication) in the intact aged atrium is lacking, as is evidence for whether and how JNK suppresses Cx43 in the aged human atrium. Methods and resultsJNK activity in human atrial samples is correlated with both reduced Cx43 expression and increasing age. Using a unique technique of optical mapping space constant measurement, we found that impaired intercellular coupling and reduced Cx43 were linked to enhanced activation of JNK in intact aged rabbit atria. These JNK-associated alterations were further confirmed in naturally JNK activated aged mice and in cardiac-specific inducible MKK7D (JNK upstream activator) young mice. Moreover, JNK inhibition, using either JNK specific inhibitors in aged wild-type (WT) mice and JNK activator anisomycin-treated young WT mice or JNK1/2 dominant-negative mice with genetically inhibited cardiac JNK activity, completely eliminated these functional abnormalities. Furthermore, we discovered for the first time that long-term JNK activation downregulates Cx43 expression via c-jun suppressed transcriptional activity of the Cx43 gene promoter. ConclusionOur results demonstrate that JNK is a critical regulator of Cx43 expression, and that augmented JNK activation in aged atria downregulates Cx43 to impair cell-cell communication and promote the development of AF. JNK inhibition may represent a promising therapeutic approach to prevent or treat AF in the elderly.

Aging-Induced Modulation of Pituitary Adenylate Cyclase-Activating Peptide- and Vasoactive Intestinal Peptide-Induced Vasomotor Responses in the Arteries of Mice

Pituitary adenylate cyclase-activating peptide (PACAP; 1-38 and 1-27) and vasoactive intestinal peptide (VIP) are related neuropeptides of the secretin/glucagon family. Overlapping signaling through G-protein-coupled receptors mediates their vasomotor activity. We previously showed that PACAP deficiency (PACAP-KO) shifts the mechanisms of vascular response and maintains arterial relaxation through the VIP backup mechanism and (mainly) its VPAC1R, but their age-dependent modulation is still unknown. We hypothesized that backup mechanisms exist, which maintain the vasomotor activity of these peptides also in older age. Thus, we investigated the effects of exogenous VIP and PACAP peptides in isolated carotid arteries of 2- and 15-month-old wild-type (WT) and PACAP-KO mice. All peptides induced relaxation in the arteries of young WT mice, whereas in young PACAP-KO mice PACAP1-27 and VIP, but not PACAP1-38, induced relaxation. Unlike VIP, PACAP-induced vasomotor responses were reduced in aging WT mice. However, in the arteries of aging PACAP-KO mice, PACAP1-27- and VIP-induced responses were reduced, but PACAP1-38 showed a greater vasomotor response compared to that of young PACAP-KO animals. There were no significant differences between the vasomotor responses of aging WT and PACAP-KO mice. Our data suggest that, in the absence of PACAP both in young and old ages, the vascular response is mediated through backup mechanisms, most likely VIP, maintaining proper vascular relaxation in aging-induced PACAP insufficiency.

Cathepsin L deficiency results in reactive oxygen species (ROS) accumulation and vascular cells activation

Recent evidence suggests a link between cathepsin L (CTSL) and vascular diseases. However, its contribution to reactive oxygen species (ROS) homeostasis in the vasculature remains unknown. p66shc is a redox enzyme implicated in mitochondrial ROS generation and translation of oxidative signals. In this study, we explored the relationship between CTSL and oxidative damage in vasculature and whether the oxidative damage is mediated by p66shc.Carotid arteries from aged mice (24 months old) showed a reduction in CTSL expression compared with young wild-type mice (4 months old). Local knockdown of CTSL in carotid arteries of young mice by adenoviral vector encoding the short hairpin RNA targeting CTSL leading to premature vascular aging, as shown by mitochondrial disruption, increased β-galactosidase–positive cells, reduced telomerase activity, and up-regulation of p66shc. Knockdown of CTSL decreased the expression of mitochondrial oxidative phosphorylation (OXPHOS) complexes I, III, and IV, leading to increased mitochondrial ROS and hyperpolarization of the mitochondrial membrane in vitro. Furthermore, knockdown of CTSL also stimulated ROS production and senescence in vascular cells, accompanied by the up-regulation of p66shc.However, p66shc knockdown blunted the alteration in ROS production, and senescence in CTSL knockdown vascular cells. This study suggests that CTSL knockdown partially induces vascular cells damage via increased ROS production and up-regulation of p66shc.

Deletion of T-type calcium channels Cav3.1 or Cav3.2 attenuates endothelial dysfunction in aging mice.

Impairment of endothelial function with aging is accompanied by reduced nitric oxide (NO) production. T-type Cav3.1 channels augment nitric oxide and co-localize with eNOS. Therefore, the hypothesis was that T-type channels contribute to the endothelial dysfunction of aging. Endothelial function was determined in mesenteric arteries (perfusion) and aortae (isometric contraction) of young and old wild-type (WT), Cav3.1, and Cav3.2 knockout mice. NO production was measured by fluorescence imaging in mesenteric arteries. With age, endothelium-dependent subsequent dilatation (following depolarization with KCl) of mesenteric arteries was diminished in the arteries of WT mice, unchanged in Cav3.2-/- preparations but increased in those of Cav3.1-/- mice. NO synthase inhibition abolished the subsequent dilatation in mesenteric arteries and acetylcholine-induced relaxations in aortae. NO levels were significantly reduced in mesenteric arteries of old compared to young WT mice. In Cav3.1-/- and Cav3.2-/- preparations, NO levels increased significantly with age. Relaxations to acetylcholine were significantly smaller in the aortae of old compared to young WT mice, while such responses were comparable in preparations of young and old Cav3.1-/- and Cav3.2-/- mice. The expression of Cav3.1 was significantly reduced in aortae from aged compared to young WT mice. The level of phosphorylated eNOS was significantly increased in aortae from aged Cav3.1-/- mice. In conclusion, T-type calcium channel-deficient mice develop less age-dependent endothelial dysfunction. Changes in NO levels are involved in this phenomenon in WT and Cav3.1-/- mice. These findings suggest that T-type channels play an important role in age-induced endothelial dysfunction.

Brain Interleukin-1 Facilitates Learning of a Water Maze Spatial Memory Task in Young Mice

The proinflammatory cytokine interleukin-1 (IL-1) is produced by many types of cells, including immune cells in the periphery and glia and neurons in the brain. The type I IL-1 receptor (IL-1r1) is primarily responsible for transmitting the inflammatory effects of IL-1 and mediates several biological functions by binding to either IL-1α or IL-1β. IL-1β activation is associated with hippocampus-dependent memory tasks. Although IL-1β impairs spatial memory under certain pathophysiological conditions, IL-1β may be required for the normal physiological regulation of hippocampal plasticity and memory. In addition, brain IL-1β levels are thought to change in the hippocampus in an age-dependent manner. These findings suggest that IL-1β may have a beneficial, temporary effect on learning and memory in young mice, but the matter remains unclear. Therefore, we hypothesized that hippocampal IL-1β has a beneficial effect on spatial learning and memory in young mice via IL-1r1, which is diminished in adults. We investigated the performance of young (3-month-old) and adult (6-month-old) wild-type mice, IL-1β knockout mice (IL-1βko) and IL-1r1 knockout mice (IL-1r1ko) in learning a spatial memory task with a fixed platform in a water maze (WM) and measured the levels of IL-1β and IL-1α in the hippocampus and cortex of adult and young mice by using homogeneous time-resolved fluorescence (HTRF). Learning was significantly impaired in the training trials of the WM spatial memory task in young IL-1βko and IL-1r1ko mice but not in adult IL-1βko and IL-1r1ko mice. Moreover, young IL-1r1ko mice but not IL-1βko mice showed an impairment in long-term memory extinction, suggesting that IL-1α might facilitate memory extinction. In this study, the cytokine assay using HTRF did not indicate a higher expression of hippocampal IL-1 in young mice but cortical IL-1β and IL-1α were significantly increased in adult mice. We need to investigate the role of cortical IL-1 and the local IL-1 expression in the hippocampal neurons in the future.

Age-related decline of the acute local inflammation response: a mitigating role for the adenosine A2A receptor.

Aging is accompanied by an increase in markers of innate immunity. How aging affects neutrophil functions remains of debate. The adenosine A2A receptor (A2AR), essential to the resolution of inflammation, modulates neutrophil functions. We sought to determine whether or not A2AR protects against the effects of aging. We monitored neutrophil influx, viability, and activation as well as cytokine accumulation in wild-type (WT) and A2AR-knockout mice (KO) at three different ages.Several readouts decreased with aging: neutrophil counts in dorsal air pouches (by up to 55%), neutrophil viability (by up to 56%), elastase and total protein in exudates (by up to 80%), and local levels of cytokines (by up to 90%). Each of these parameters was significantly more affected in A2AR-KO mice. CXCL1-3 levels were largely unaffected. The effects of aging were not observed systemically. Preventing neutrophil influx into the air pouch caused a comparable cytokine pattern in young WT mice. Gene expression (mRNA) in leukocytes was affected, with CXCL1 and CCL4 increasing and with TNF and IL-1∝ decreasing.Conclusion: Aging has deleterious effects on the acute inflammatory response and neutrophil-related activities, and defective migration appears as an important factor. A functional A2AR signaling pathway delays some of these.

Pancreatic neuro-insular network in young mice revealed by 3D panoramic histology.

It has been proposed that the neuro-insular network enables rapid, synchronised insulin secretion. However, to date, acquiring the pancreatic tissue map to study the neural network remains a challenging task as there is a lack of feasible approaches for large-scale tissue analysis at the organ level. Here, we have developed 3-dimensional (3D) panoramic histology to characterise the pancreatic neuro-insular network in young mice. Pancreases harvested from young wild-type B6 mice (3 and 8weeks old) and db/db mice (3weeks old; db/db vs db/+) were used to develop 3D panoramic histology. Transparent pancreases were prepared by optical clearing to enable deep-tissue, tile-scanning microscopy for qualitative and quantitative analyses of islets and the pancreatic tissue network in space. 3D panoramic histology reveals the pancreatic neurovascular network and the coupling of ganglionic and islet populations via the network. This integration is identified in both 3- and 8-week-old mice, featuring the peri-arteriolar neuro-insular network and islet-ganglionic aggregation. In weaning hyperphagic db/db mice, the 3D image data identifies the associated increases in weight, adipose tissue attached to the pancreas, density of large islets (major axis >150μm) and pancreatic sympathetic innervation compared with db/+ mice. Our work provides insight into the neuro-insular integration at the organ level and demonstrates a new approach for investigating previously unknown details of the pancreatic tissue network in health and disease.

Cytochrome P450-2E1 is involved in aging-related kidney damage in mice through increased nitroxidative stress

The aim of this study was to investigate the role of cytochrome P450-2E1 (CYP2E1) in aging-dependent kidney damage since it is poorly understood. Young (7 weeks) and aged female (16–17 months old) wild-type (WT) and Cyp2e1-null mice were used. Kidney histology showed that aged WT mice exhibited typical signs of kidney aging such as cell vacuolation, inflammatory cell infiltration, cellular apoptosis, glomerulonephropathy, and fibrosis, along with significantly elevated levels of renal TNF-α and serum creatinine than all other groups. Furthermore, the highest levels of renal hydrogen peroxide, protein carbonylation and nitration were observed in aged WT mice. These increases in the aged WT mice were accompanied by increased levels of iNOS and mitochondrial nitroxidative stress through altered amounts and activities of the mitochondrial complex proteins and significantly reduced levels of the antioxidant glutathione (GSH). In contrast, the aged Cyp2e1-null mice exhibited significantly higher antioxidant capacity with elevated heme oxygenase-1 and catalase activities compared to all other groups, while maintaining normal GSH levels with significantly less mitochondrial nitroxidative stress compared to the aged WT mice. Thus, CYP2E1 is important in causing aging-related kidney damage most likely through increasing nitroxidative stress and that CYP2E1 could be a potential target in preventing aging-related kidney diseases.

RIPK1-RIPK3-MLKL-dependent necrosis promotes the aging of mouse male reproductive system.

A pair of kinases, RIPK1 and RIPK3, as well as the RIPK3 substrate MLKL cause a form of programmed necrotic cell death in mammals termed necroptosis. We report here that male reproductive organs of both Ripk3- and Mlkl-knockout mice retain 'youthful' morphology and function into advanced age, while those of age-matched wild-type mice deteriorate. The RIPK3 phosphorylation of MLKL, the activation marker of necroptosis, is detected in spermatogonial stem cells in the testes of old but not in young wild-type mice. When the testes of young wild-type mice are given a local necroptotic stimulus, their reproductive organs showed accelerated aging. Feeding of wild-type mice with an RIPK1 inhibitor prior to the normal onset of age-related changes in their reproductive organs blocked the appearance of signs of aging. Thus, necroptosis in testes promotes the aging-associated deterioration of the male reproductive system in mice.

BLUNTED RESPONSE TO STRESS IN YOUNG IL-10TM/TM MICE: IMPLICATION FOR THE VULNERABILITY IN FRAILTY

AbstractThe hypothalamic pituitary adrenal axis (HPA axis) is a major neuroendocrine system for stress response and for regulating the immune system. Aged rats as well as older humans demonstrate progressive loss of control of the HPA axis, resulting in impaired diurnal rhythm and hypersecretion of glucocorticoids during times of stress. Higher levels and a blunted diurnal rhythm of cortisol have been observed in frail compared to more robust older adults. Understanding the factors underlying disturbed glucocorticoid secretion that precede age-related diseases and frailty are of considerable importance to prevent vulnerability and disability in late life. In order to study this system in vivo, we utilized a mouse model of chronic inflammation and as frailty and measured changes in plasma corticosterone and pro-inflammatory levels after acute cold stress. The plasma corticosterone level was determined 2 weeks before cold stimulation in 10 young (3 months), 10 old (21 months) C57BL/6J mice, and 10 young IL-10 tm/tm (3 months). 5 mice from each group were exposed to cold stress, the remainder served as controls and were not exposed to cold. The treatment groups were exposed to four degrees Celsius for 5 hours and the controls were kept in room temperature conditions at twenty five degrees Celsius. The results showed higher basal plasma corticosterone (P<0.01) and normal IL-6 and TNFR-1 levels in young IL-10tm/tm, compared to young and old C57BL/6J mice. However, there were unchanged corticosterone level and higher IL-6 and TNFR-1 levels (P=0.04, 0.02, respectively) in young IL-10tm/tm mice after acute cold stress compared to young wild type mice. Lower GRa mRNA expression in hippocampus was also observed in control and treated young IL-10tm/tm (P<0.01), compared the age-matched wild type mice. These findings provide initial evidence for the hypothesis that HPA axis dysfunction is through whole life of frail subjects and even earlier than changes in immune system.