Premature Death In Mice Research Articles

Seizures and premature death in mice with targeted Kv1.1 deficiency in corticolimbic circuits.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related death, likely stemming from seizure activity disrupting vital brain centres controlling heart and breathing function. However, understanding of SUDEP's anatomical basis and mechanisms remains limited, hampering risk evaluation and prevention strategies. Prior studies using a neuron-specific Kcna1 conditional knockout mouse model of SUDEP identified the primary importance of brain-driven mechanisms contributing to sudden death and cardiorespiratory dysregulation; yet, the underlying neurocircuits have not been identified. Using the Emx1-Cre driver, we generated a new conditional knockout mouse model lacking Kcna1 in excitatory neurons of the cortex, hippocampus, amygdala and select vagal afferents. To test whether the absence of Kv1.1 in forebrain corticolimbic circuits is sufficient to induce spontaneous seizures, premature mortality and cardiorespiratory dysfunction, we performed survival studies and EEG, ECG, and plethysmography (EEG-ECG-Pleth) recordings. We demonstrate premature death and epilepsy in corticolimbic conditional knockout mice. During monitoring, we fortuitously captured one SUDEP event, which showed a generalized tonic-clonic seizure that initiated respiratory dysfunction culminating in cardiorespiratory failure. In addition, we observed that cardiorespiratory abnormalities are common during non-fatal seizures in conditional knockout mice, but mostly absent during interictal periods, implying ictal, not interictal, cardiorespiratory impairment as a more reliable indicator of SUDEP risk. These results point to corticolimbic excitatory neurons as critical neural substrates in SUDEP and affirm seizure-related respiratory and cardiac failure as a likely cause of death.

AMPK deficiency in smooth muscles causes persistent pulmonary hypertension of the new-born and premature death

AMPK has been reported to facilitate hypoxic pulmonary vasoconstriction but, paradoxically, its deficiency precipitates pulmonary hypertension. Here we show that AMPK-α1/α2 deficiency in smooth muscles promotes persistent pulmonary hypertension of the new-born. Accordingly, dual AMPK-α1/α2 deletion in smooth muscles causes premature death of mice after birth, associated with increased muscularisation and remodeling throughout the pulmonary arterial tree, reduced alveolar numbers and alveolar membrane thickening, but with no oedema. Spectral Doppler ultrasound indicates pulmonary hypertension and attenuated hypoxic pulmonary vasoconstriction. Age-dependent right ventricular pressure elevation, dilation and reduced cardiac output was also evident. KV1.5 potassium currents of pulmonary arterial myocytes were markedly smaller under normoxia, which is known to facilitate pulmonary hypertension. Mitochondrial fragmentation and reactive oxygen species accumulation was also evident. Importantly, there was no evidence of systemic vasculopathy or hypertension in these mice. Moreover, hypoxic pulmonary vasoconstriction was attenuated by AMPK-α1 or AMPK-α2 deletion without triggering pulmonary hypertension.

Hypoglycemic effect and toxicity of the dry extract of Eugenia biflora (L.) DC. leaves

Ethnopharmacological relevanceThe leaves of Eugenia biflora (Myrtaceae) are traditionally used by Amazonian populations for the control of diabetes. However, their chemical composition has not yet been described and pharmacological evidence has not been reported. ObjectiveThis study aimed to identify the chemical constituents and evaluate the hypoglycemic and toxic effect of the dry extract of the E. biflora leaves (DEEB). Materials and methodsDEEB, obtained by infusion, was analyzed using LC-HRMS and NMR, whose the catechin flavonoid was quantified using NMR. The antidiabetic effect of DEEB was evaluated according to its inhibition of the enzymes α-amylase and α-glucosidase, as well as the content of total phenols, free radical scavengingand antiglycation activities, and its in vitro cell viability. Oral maltose tolerance and chronic multiple dose tests (28 days) in streptozotocin-induced diabetic mice (STZ) were performed. The hypoglycemic effect and toxicity of this extract were evaluated in the multiple dose assay. Biochemical parameters, hemolysis, and levels of the thiobarbituric acid reactive species in the liver were investigated and histopathological analyses of the kidneys and liver were performed. ResultsEight phenolic compounds were identified, with catechin (15.5 ± 1.7 mg g−1) being the majority compound and a possible chemical marker of DEEB. The extract showed inhibition activity of the enzyme α-glucosidase. Chronic administration of DEEB (50 mg/kg of body weight) reduced glucose levels in diabetic animals, similar to acarbose; however, DEEB (100 and 200 mg/kg bw) caused premature death of mice by D22 of the treatment. Our data indicate that one of the mechanisms of toxicity in DEEB may be related to the aggravation of oxidative stress in the liver. This histopathological study indicated that DEEB failed to minimize the progression of the toxicity of diabetes caused by STZ. ConclusionsThis study demonstrated the hypoglycemic potential of E. biflora leaves. However, the prolonged use of this tea can be harmful to its users due to its considerable toxicity, which needs to be better investigated.

PANoptosis is a prominent feature of desmoplakin cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is hereditary cardiomyopathy caused by pathogenic variants (mutations) in genes encoding the intercalated disc (ID), particularly desmosome proteins. ACM caused by mutations in the DSP gene encoding desmoplakin (DSP) is characterized by the prominence of cell death, myocardial fibrosis, and inflammation, and is referred to as desmoplakin cardiomyopathy. The aim of this article was to gain insight into the pathogenesis of DSP cardiomyopathy. The Dsp gene was exclusively deleted in cardiac myocytes using tamoxifen-inducible MerCreMer (Myh6-Mcm Tam) and floxed Dsp (Dsp F/F) mice (Myh6-Mcm Tam:Dsp F/F). Recombination was induced upon subcutaneous injection of tamoxifen (30 mg/kg/d) for 5 days starting post-natal day 14. Survival was analyzed by Kaplan-Meier plots, cardiac function by echocardiography, arrhythmias by rhythm monitoring, and gene expression by RNA-Seq, immunoblotting, and immunofluorescence techniques. Cell death was analyzed by the TUNEL assay and the expression levels of specific markers were by RT-PCR and immunoblotting. Myocardial fibrosis was assessed by picrosirius red staining of the myocardial sections, RT-PCR, and immunoblotting. The Myh6-Mcm Tam: Dsp F/F mice showed extensive molecular remodeling of the IDs and the differential expression of ~10,000 genes, which predicted activation of KDM5A, IRFs, and NFκB and suppression of PPARGC1A and RB1, among others in the DSP-deficient myocytes. Gene set enrichment analysis predicted activation of the TNFα/NFκB pathway, inflammation, cell death programs, and fibrosis. Analysis of cell death markers indicated PANoptosis, comprised of apoptosis (increased CASP3, CASP8, BAD and reduced BCL2), necroptosis (increased RIPK1, RIPK3, and MLKL), and pyroptosis (increased GSDMD and ASC or PYCARD) in the DSP-deficient myocytes. Transcript levels of the pro-inflammatory and pro-fibrotic genes were increased and myocardial fibrosis comprised ~25% of the myocardium in the DSP-deficient hearts. The Myh6-Mcm Tam:Dsp F/F mice showed severe cardiac systolic dysfunction and ventricular arrhythmias, and died prematurely with a median survival rate of ~2 months. The findings identify PANoptosis as a prominent phenotypic feature of DSP cardiomyopathy and set the stage for delineating the specific molecular mechanisms involved in its pathogenesis. The model also provides the opportunity to test the effects of pharmacological and genetic interventions on myocardial fibrosis and cell death.

Effects of high rosuvastatin doses on hepatocyte mitochondria of hypercholesterolemic mice

Statins are the cornerstone of therapy for individuals with hyperlipidemia. The aim of this study was to analyze the undesirable effects of mild, moderate and high doses of rosuvastatin in CD-1 male mice who received a cholesterol-rich diet, focusing on the morphological and functional changes on hepatocyte mitochondria. In a mouse model we studied the combined administration of a cholesterol-rich diet along with mild and moderate doses of rosuvastatin (1, 2.5 or 5 mg/kg/day) during several days. After the animals were sacrificed, liver mitochondria were isolated for microscopic studies and to analyze the respiratory function. The respiratory control (state-3/state-4) was evaluated in mice who received high doses of rosuvastatin. Rosuvastatin doses higher than 20 mg/kg/day induced premature death in mice with a hypercholesterolemic diet, but not in mice with a cholesterol-free diet. Doses from 2.5 to 5 mg/kg/day also induced morphological and functional alterations in mitochondria but these hypercholesterolemic animals survived longer. Giving 1 mg/kg/day, which is close to the maximal therapeutic dose for humans, did not affect mitochondrial architecture or respiratory function after two months of treatment. We analyzed the effect of rosuvastatin on hepatic tissue because it is where statins are mainly accumulated and it is the main site of endogenous cholesterol synthesis. Our results contribute to understand the side effects of rosuvastatin in hypercholesterolemic mice, effects that could also affect humans who are intolerant to statins.

Protein Kinase C Downregulation upon Rapamycin Treatment Attenuates Neuroinflammation and Mitochondrial Disease

Mitochondrial dysfunction causes many poorly understood diseases, such as Leigh Syndrome, that are often caused by dysfunctions in proteins involved in the electron transport chain. My lab previously reported mTOR is pathologically involved in the neurodegenerative phenotype and premature death of mice missing the Complex I subunit Ndufs4 (Ndufs4-/- mice). We discovered treatment with rapamycin extends lifespan, reduces neuroinflammation, and attenuates the neurodegenerative phenotype in these mice, although the mechanisms remain unclear. Rapamycin-treated Ndufs4-/- mice exhibited decreased activation of the mTORC1 pathway. It also deactivated the mTORC2 pathway. We observed that phosphorylation of the canonical protein kinase C (PKC) isoforms (PKC-α, -β, and -γ) decreased more than any other kinases, leading us to hypothesize its deactivation contributes to the observed lifespan extension. To test this, we treated Ndufs4-/- mice with three different PKC inhibitors: the pan-PKC inhibitors GO6983 and GF109203X, and the PKC-β specific inhibitor ruboxistaurin. Similar to rapamycin, all three drugs were able to significantly delay the onset of neurological symptoms (i.e. clasping) and increase survival. We also observed that PKC-β inhibition reduced skin inflammation to suppress the hair loss phenotype displayed by Ndufs4-/- mice at weaning. We further discovered PKC-β inhibition reduces neuroinflammation by deactivating the NF-kB inflammatory pathway. These results suggest that mTORC2 may play a critical role in the etiology of mitochondrial diseases such as Leigh Syndrome.

Abolishing UCHL1's hydrolase activity exacerbates TBI-induced axonal injury and neuronal death in mice

Ubiquitin (Ub) C-terminal hydrolase L1 (UCHL1) is a multifunctional protein that is expressed in neurons throughout brain at high levels. UCHL1 deletion is associated with axonal degeneration, progressive sensory motor ataxia, and premature death in mice. UCHL1 has been hypothesized to play a role in the pathogenesis of neurodegenerative diseases and recovery after neuronal injury. UCHL1 hydrolyzes Ub from polyubiquitinated (poly-Ub) proteins, but also may ligate Ub to select neuronal proteins, and interact with cytoskeletal proteins. These and other mechanisms have been hypothesized to underlie UCHL1's role in neurodegeneration and response to brain injury. A UCHL1 knockin mouse containing a C90A mutation (C90A) devoid of hydrolase activity was constructed. The C90A mouse did not develop the sensory and motor deficits, degeneration of the gracile nucleus and tract, or premature death as seen in UCHL1 deficient mice. C90A and wild type (WT) mice were subjected to the controlled cortical impact (CCI) model of traumatic brain injury (TBI), and cell death, axonal injury and behavioral outcome were assessed. C90A mice exhibited decreased spared tissue volume, greater loss of CA1 hippocampal neurons and greater axonal injury as detected using anti-amyloid precursor protein (APP) antibody and anti- non-phosphorylated neurofilament H (SMI-32) antibody immunohistochemistry after CCI compared to WT controls. Poly-Ub proteins and Beclin-1 were elevated after CCI in C90A mice compared to WT controls. Vestibular motor deficits assessed using the beam balance test resolved by day 5 after CCI in WT mice but not in C90A mice. These results suggest that the hydrolase activity of UCHL1 does not account for the progressive neurodegeneration and premature death seen in mice that do not express full length UCHL1. The hydrolase activity of UCHL1 contributes to the function of the ubiquitin proteasome pathway (UPP), ameliorates activation of autophagy, and improves motor recovery after CCI. Thus, UCHL1 hydrolase activity plays an important role in acute injury response after TBI.

Systemic overexpression of C-C motif chemokine ligand 2 promotes metabolic dysregulation and premature death in mice with accelerated aging.

Injection of tissues with senescent cells induces changes that mimic aging, and this process is delayed in mice engineered to eliminate senescent cells, which secrete proinflammatory cytokines, including C-C motif chemokine ligand 2 (Ccl2). Circulating levels of Ccl2 correlate with age, but the impact of Ccl2 on tissue homeostasis has not been established. We generated an experimental model by crossbreeding mice overexpressing Ccl2 with progeroid mice bearing a mutation in the lamin A (Lmna) gene. Wild-type animals and progeroid mice that do not overexpress Ccl2 were used as controls. Ccl2 overexpression decreased the lifespan of the progeroid mice and induced the dysregulation of glycolysis, the citric acid cycle and one-carbon metabolism in skeletal muscle, driving dynamic changes in energy metabolism and DNA methylation. This impact on cellular bioenergetics was associated with mitochondrial alterations and affected cellular metabolism, autophagy and protein synthesis through AMPK/mTOR pathways. The data revealed the ability of Ccl2 to promote death in mice with accelerated aging, which supports its putative use as a biomarker of an increased senescent cell burden and for the assessment of the efficacy of interventions aimed at extending healthy aging.

Splicing Factor SRSF1 Is Essential for Satellite Cell Proliferation and Postnatal Maturation of Neuromuscular Junctions in Mice.

SummarySatellite cells are main muscle stem cells that could provide myonuclei for myofiber growth and synaptic-specific gene expression during the early postnatal development. Here, we observed that splicing factor SRSF1 is highly expressed in myoblasts and its expression is closely related with satellite cell activation and proliferation. By genetic deletion of SRSF1 in myogenic progenitors, we found that SRSF1 is critical for satellite cell proliferation in vitro and in vivo. Most notably we also observed that SRSF1 is required for the functional neuromuscular junction (NMJ) formation, as SRSF1-deficient mice fail to form mature pretzel-like NMJs, which leads to muscle weakness and premature death in mice. Finally, we demonstrated that SRSF1 contributes to muscle innervation and muscle development likely by regulating a restricted set of tissue-specific alternative splicing events. Thus, our data define a unique role for SRSF1 in postnatal skeletal muscle growth and function in mice.

A Gut Feeling about C9ORF72

C9ORF72 mutations are the most common genetic cause of ALS and FTD, leading to neurodegeneration via complex mechanisms. Mutations also lead to loss of C9ORF72 function and inflammatory diseases in patients and knockout mice. Burberry et al. now show that C9orf72-associated inflammation and premature death in mice are directly modified by the gut microbiome.

Biallelic variants in ADARB1, encoding a dsRNA-specific adenosine deaminase, cause a severe developmental and epileptic encephalopathy

BackgroundAdenosine-to-inosine RNA editing is a co-transcriptional/post-transcriptional modification of double-stranded RNA, catalysed by one of two active adenosine deaminases acting on RNA (ADARs), ADAR1 and ADAR2. ADARB1 encodes the enzyme ADAR2...

Lipid Hydrolase Enzymes: Pragmatic Prolongevity Targets for Improved Human Healthspan?

Compelling evidence suggests that lipid metabolism, which plays critical roles in fat storage, cell membrane maintenance, and cell signaling, is intricately linked to aging. Lipid hydrolases are important enzymes that catalyze the hydrolysis of more complex lipids into simpler lipids. Diverse interventions targeting lipid hydrolases can prolong or shorten life in model organisms. For example, the genetic removal of or RNAi knockdown against a phospholipase can reduce lifespan in Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus. The removal of lysosomal acid lipase results in premature death in mice, while its overexpression in nematodes generates lean, long-lived individuals. The overexpression or inhibition of diacylglycerol lipase leads to enhanced or reduced longevity, respectively, in both worms and flies. Lifespan can also be extended by knocking down triacylglycerol lipases in yeast, overexpressing fatty acid amide hydrolase in worms, or removing hepatic lipase in a mouse model of coronary disease. Conversely, flies lacking the triacylglycerol lipase Brummer are obese and short lived. Linking sphingolipids and aging, removing the sphingomyelinase inositol phosphosphingolipid phospholipase shortens chronological lifespan in Saccharomyces cerevisiae, while inhibiting an acid sphingomyelinase in worms or inactivating alkaline ceramidase in flies extends lifespan. The clinical potential of manipulating these enzymes is highlighted by the FDA-approved obesity drug orlistat, which is an inhibitor of pancreatic and hepatic lipases that induces weight loss and improves insulin/glucose homeostasis. Additional research is warranted to better understand how these lipid hydrolases impact aging and to determine if clinical interventions targeting them are capable of improving human healthspan.

Essential Role of Endothelial MCPIP in Vascular Integrity and Post-Ischemic Remodeling.

MCP-1-induced protein (MCPIP, also known as Zc3h12a or Regnase-1), a newly identified suppressor of cytokine signaling, is expressed in endothelial cells (ECs). To investigate the role of endothelial MCPIP in vascular homeostasis and function, we deleted the MCPIP gene specifically in ECs using the Cre-LoxP system. EC-specific MCPIP deletion resulted in systemic inflammation, increased vessel permeability, edema, thrombus formation, and premature death in mice. Serum levels of cytokines, chemokines, and biomarkers of EC dysfunction were significantly elevated in these mice. Upon lipopolysaccharide (LPS) challenge, mice with EC-specific MCPIP depletion were highly susceptible to LPS-induced death. When subjected to ischemia, these mice showed defective post-ischemic angiogenesis and impaired blood flow recovery in hind limb ischemia. In aortic ring cultures, the MCPIP-deficient ECs displayed significantly impaired vessel sprouting and tube elongation. Mechanistically, silencing of MCPIP by small interfering RNAs in cultured ECs enhanced NF-κΒ activity and dysregulated synthesis of microRNAs linked with elevated cytokines and biomarkers of EC dysfunction. Collectively, these results establish that constitutive expression of MCPIP in ECs is essential to maintaining endothelial homeostasis and function by serving as a key negative feedback regulator that keeps the inflammatory signaling suppressed.

Cardiospecific deletion of αE-catenin leads to heart failure and lethality in mice.

αE-catenin is a component of adherens junctions that link the cadherin-catenin complex to the actin cytoskeleton. The signaling function of this protein was recently revealed. In the present study, we investigated the role of αE-catenin in the pathogenesis of heart failure. We mated αE-catenin conditional knockout mice with αMHC-Cre mice and evaluated their mutant offspring. We found that αE-catenin knockout caused enlargement of the heart and atria, fibrosis, the upregulation of hypertrophic genes, and the dysregulation of fatty acid metabolism via the transcriptional activity of Yap and β-catenin. The activation of canonical Wnt and Yap decreased the activity of main regulators of energy metabolism (i.e., adenosine monophosphate-activated protein kinase and peroxisome proliferator-activated receptor α) and dysregulated hypertrophic pathway activity (i.e., phosphatidylinositide 3-kinase/Akt, cyclic adenosine monophosphate/protein kinase A, and MEK1/extracellular signal regulated kinase 1/2). The loss of αE-catenin also negatively affected cardio-hemodynamic function via the protein kinase A pathway. Overall, we found that the embryonic heart-specific ablation of αE-catenin leads to the development of heart failure with age and premature death in mice. Thus, αE-catenin appears to have a crucial signaling function in the postnatal heart, and the dysfunction of this gene causes heart failure through canonical Wnt and Yap activation.

Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice

Manganese is an essential metal that becomes toxic at elevated levels. Loss-of-function mutations in SLC30A10, a cell-surface-localized manganese efflux transporter, cause a heritable manganese metabolism disorder resulting in elevated manganese levels and parkinsonian-like movement deficits. The underlying disease mechanisms are unclear; therefore, treatment is challenging. To understand the consequences of loss of SLC30A10 function at the organism level, we generated Slc30a10 knock-out mice. During early development, knock-outs were indistinguishable from controls. Surprisingly, however, after weaning and compared with controls, knock-out mice failed to gain weight, were smaller, and died prematurely (by ∼6-8 weeks of age). At 6 weeks, manganese levels in the brain, blood, and liver of the knock-outs were ∼20-60-fold higher than controls. Unexpectedly, histological analyses revealed that the brain and liver of the knock-outs were largely unaffected, but their thyroid exhibited extensive alterations. Because hypothyroidism leads to growth defects and premature death in mice, we assayed for changes in thyroid and pituitary hormones. At 6 weeks and compared with controls, the knock-outs had markedly reduced thyroxine levels (∼50-80%) and profoundly increased thyroid-stimulating hormone levels (∼800-1000-fold), indicating that Slc30a10 knock-out mice develop hypothyroidism. Importantly, a low-manganese diet produced lower tissue manganese levels in the knock-outs and rescued the phenotype, suggesting that manganese toxicity was the underlying cause. Our unanticipated discovery highlights the importance of determining the role of thyroid dysfunction in the onset and progression of manganese-induced disease and identifies Slc30a10 knock-out mice as a new model for studying thyroid biology.

Effect of a novel oncolytic herpes simplex virus type II on lung adenocarcinoma

Objective To establish the subcutaneous transplantation tumor models with Lewis lung adenocarcinoma in C57BL/6 mice, and to observe the influence of oHSV2, DDP and drug combination on tumor volume, median survival time and weight of tumor-burdened mice. Methods Subcutaneous transplantation tumor models were established with Lewis lung adenocarcinoma in tumor-burdened mice. Tumor-burdened mice were randomly divided into the control group, oHSV2 group, DDP group, oHSV2/DDP sequential group, DDP/oHSV2 sequential group and oHSV2+ DDP combination group with 12 rats in each group using the random number table method. The tumor size and weight of mice were measured every 3 days. Results On the 21st day, the tumor size of tumor-burdened mice in every group was as follows: control group (1.82±0.06)cm3, oHSV2 group (0.63±0.05)cm3, DDP group (0.58±0.03)cm3, oHSV2/DDP sequential group (0.49±0.05)cm3, DDP/oHSV2 sequential group (0.42±0.04)cm3, and the difference was statistically significant (F=1 359.01, P=0.000). The data in oHSV2+ DDP group were put away because of premature death in mice. The differences were statistically significant between control group and oHSV2 group (P=0.000), control group and DDP group (P=0.000), control group and oHSV2/DDP sequential group (P=0.000), control group and DDP/oHSV2 sequential group (P=0.000), oHSV2 group and DDP group (P=0.017), DDP group and DDP/oHSV2 sequential group (P=0.000), oHSV2/DDP sequential group and DDP/oHSV2 sequential group (P=0.001). The weight of tumor-burdened mice in every group was listed as follows: control group (21.64±0.40)g, oHSV2 group (21.34±0.37)g, DDP group (15.96±0.43)g, oHSV2/DDP sequential group (19.04±0.31)g, DDP/oHSV2 sequential group (16.34±0.30)g, and the difference was statistically significant (F=588.67, P=0.000). The difference was not statistically significant between control group and oHSV2 group (P=0.076). However, the differences were statistically significant between control group and DDP group (P=0.000), control group and oHSV2/DDP sequential group (P=0.000), control group and DDP/oHSV2 sequential group (P=0.000), oHSV2 group and DDP group (P=0.000), oHSV2 group and oHSV2/DDP sequential group (P=0.000), DDP group and DDP/oHSV2 group (P=0.013), oHSV2/DDP sequential group and DDP/oHSV2 sequential group (P=0.000). The median survival time of tumor-burdened mice in every group was displayed as follows: control group 23 d , oHSV2 group 32 d, DDP group 30 d, oHSV2/DDP sequential group 37 d, DDP/oHSV2 sequential group 39 d, oHSV2+ DDP combination group 16 d, and the difference was statistically significant (χ2=120.81, P=0.000). The differences were statistically significant between control group and oHSV2 group (χ2=10.88, P=0.001), control group and DDP group (χ2=10.69, P=0.001), oHSV2 group and DDP/oHSV2 sequential group (χ2=10.09, P=0.001), DDP group and DDP/oHSV2 sequential group (χ2=9.67, P=0.002). However, the differences were not statistically significant between oHSV2 group and DDP group (χ2=0.00, P=0.996), oHSV2/DDP sequential group and DDP/oHSV2 sequential group (χ2=2.70, P=0.100). Conclusion On the premise of that the weight of mice is no affected, oHSV2 can inhibit the tumor size and prolong the median survival time of tumor-burdened mice effectively, and the effect of DDP/oHSV2 sequential group is the most significant. This article provides an experimental basis for exploring therapeutic methods of lung adenocarcinoma. Key words: Oncolytic virus; Cisplatin; Lung neoplasms

Hyperactive mTOR signals in the proopiomelanocortin-expressing hippocampal neurons cause age-dependent epilepsy and premature death in mice.

Epilepsy is a frequent comorbidity in patients with focal cortical dysplasia (FCD). Recent studies utilizing massive sequencing data identified subsets of genes that are associated with epilepsy and FCD. AKT and mTOR-related signals have been recently implicated in the pathogenic processes of epilepsy and FCD. To clarify the functional roles of the AKT-mTOR pathway in the hippocampal neurons, we generated conditional knockout mice harboring the deletion of Pten (Pten-cKO) in Proopiomelanocortin-expressing neurons. The Pten-cKO mice developed normally until 8 weeks of age, then presented generalized seizures at 8–10 weeks of age. Video-monitored electroencephalograms detected paroxysmal discharges emerging from the cerebral cortex and hippocampus. These mice showed progressive hypertrophy of the dentate gyrus (DG) with increased expressions of excitatory synaptic markers (Psd95, Shank3 and Homer). In contrast, the expression of inhibitory neurons (Gad67) was decreased at 6–8 weeks of age. Immunofluorescence studies revealed the abnormal sprouting of mossy fibers in the DG of the Pten-cKO mice prior to the onset of seizures. The treatment of these mice with an mTOR inhibitor rapamycin successfully prevented the development of seizures and reversed these molecular phenotypes. These data indicate that the mTOR pathway regulates hippocampal excitability in the postnatal brain.

Liver Cholesterol Overload Aggravates Obstructive Cholestasis by Inducing Oxidative Stress and Premature Death in Mice.

Nonalcoholic steatohepatitis is one of the leading causes of liver disease. Dietary factors determine the clinical presentation of steatohepatitis and can influence the progression of related diseases. Cholesterol has emerged as a critical player in the disease and hence consumption of cholesterol-enriched diets can lead to a progressive form of the disease. The aim was to investigate the impact of liver cholesterol overload on the progression of the obstructive cholestasis in mice subjected to bile duct ligation surgery. Mice were fed with a high cholesterol diet for two days and then were subjected to surgery procedure; histological, biochemical, and molecular analyses were conducted to address the effect of cholesterol in liver damage. Mice under the diet were more susceptible to damage. Results show that cholesterol fed mice exhibited increased apoptosis and oxidative stress as well as reduction in cell proliferation. Mortality following surgery was higher in HC fed mice. Liver cholesterol impairs the repair of liver during obstructive cholestasis and aggravates the disease with early fatal consequences; these effects were strongly associated with oxidative stress.

Antioxidant supplementation accelerates cachexia development by promoting tumor growth in C26 tumor-bearing mice

More than 50% of patients with advanced stages of colon cancer suffer from progressive loss of skeletal muscle, called cachexia, resulting in reduced quality of life and shortened survival. It is becoming evident that reactive oxygen species (ROS) regulate pathways controlling skeletal muscle atrophy. Herein we tested the hypothesis that antioxidant supplementation could prevent skeletal muscle atrophy in a model of cachectic Colon 26 (C26) tumor-bearing mice. Seven-week-old BALB/c mice were subcutaneously inoculated with colon 26 (C26) cancer cells or PBS. Then C26-mice were daily gavaged during 22 days either with PBS (vehicle) or an antioxidant cocktail whose composition is close to that of commercial dietary antioxidant supplements (rich in catechins, quercetin and vitamin C). We found that antioxidants enhanced weight loss and caused premature death of mice. Antioxidants supplementation failed to prevent (i) the increase in plasma TNF-α levels and systemic oxidative damage, (ii) skeletal muscle atrophy and (iii) activation of the ubiquitin-proteasome system (MuRF-1, MAFbx and polyubiquitinated proteins). Accordingly, immunohistological staining for Ki-67 and the expression of cell cycle inhibitors demonstrated that tumor of supplemented mice developed faster with a concomitant decrease in oxidative damage. Previous studies have shown that the use of catechins and quercetin separately can improve the musculoskeletal function in cachectic animals. However, our results indicate that the combination of these antioxidants reduced survival and enhanced cachexia in C26-mice.

Abstract 216: Genetic Deletion of Cardiomyocyte Mineralocorticoid Receptors Prevents Cardiac Dysfunction and Premature Death in Mice Lacking Glucocorticoid Receptors in the Heart

Heart failure is one of the leading causes of death in the Western world, and stress is increasingly associated with adverse cardiac outcomes. Glucocorticoids are primary stress hormones, but their direct role in heart physiology and pathology is poorly understood. The actions of glucocorticoids are mediated classically by the glucocorticoid receptor (GR); however, in cardiomyocytes glucocorticoid occupancy and activation of the mineralocorticoid receptor (MR) may also contribute to the observed glucocorticoid response. To elucidate the in vivo function of glucocorticoid signaling in the heart, we generated mice with cardiomyocyte-specific deletion of GR (cardioGRKO). The cardioGRKO mice spontaneously develop cardiac hypertrophy by 2 months of age and left ventricular systolic dysfunction and dilatation between 3-6 months of age, and they die prematurely from heart failure. The mean survival age of the cardioGRKO mice was 9.3 months, and 97% (38 of 39) of the mice died prior to reaching 12 months of age. Loss of GR in the heart was not accompanied by an increase in fibrosis. To investigate whether cardiomyocyte MR signaling contributes to the pathology in the cardioGRKO mice, we generated for the first time mice lacking both GR and MR in the heart (cardioGRMRKO). Despite showing increased expression of classic hypertrophic marker genes, the cardioGRMRKO mice were protected from the development of cardiac hypertrophy, and no major alterations were observed in the function and chamber size of the left ventricle. Moreover, the cardioGRMRKO mice were protected from premature death as 94% (15 of 16) of the mice survived past the 13 month study endpoint. Microarray analysis revealed marked differences in gene expression profiles between the cardioGRKO and cardioGRMRKO hearts. These findings reveal that cardiomyocyte MR signaling, when unopposed by GR signaling, plays a major role in the progression of cardiac disease. Moreover, they suggest that combining GR agonists with MR antagonists may represent an improved therapeutic approach for treating heart failure.