Effect of sustained postnatal systemic inflammation on hippocampal volume and function in mice.

BACKGROUND: Premature infants are at risk for persistent neurodevelopmental impairment. Children born preterm often exhibit reduced hippocampal volumes that correlate with deficits in working memory. Perinatal inflammation is associated with preterm birth and brain abnormalities. Here we examine the effects of postnatal systemic inflammation on the developing hippocampus in mice. METHODS: Pups received daily intraperitoneal injections of lipopolysaccharide (LPS) or saline between days 3 and 13. Ex vivo magnetic resonance imaging (MRI) and microscopic analysis of brain tissue was performed on day 14. Behavioral testing was conducted at 8-9 wk of age. RESULTS: MR and microscopic analysis revealed a 15-20% reduction in hippocampal volume in LPS-treated mice compared with controls. Behavioral testing revealed deficits in hippocampal-related tasks in LPS-treated animals. Adult mice exposed to LPS during the postnatal period were unable to select a novel environment when re-placed within a 1-min delay, were less able to remember a familiar object after a 1-h delay, and had impaired retention of associative fear learning after 24 h. CONCLUSION: Systemic inflammation sustained during the postnatal period contributes to reduced hippocampal volume and deficits in hippocampus-dependent working memory. These findings support the novel and emerging concept that sustained systemic inflammation contributes to neurodevelopmental impairment among preterm infants.

Systemic and cardiac susceptibility of immune compromised mice to doxorubicin.

BACKGROUND: Anthracycline chemotherapy is an effective and widely used treatment for solid tumors and hematological malignancies regardless of its known cardiotoxicity. The mechanisms of the cardiotoxicity are not fully understood and methods to protect the heart during or following anthracycline chemotherapy are currently unclear. In order to examine the efficacy of human cell based therapy in anthracycline-induced injury, we characterized a mouse model using an immune compromised strain of mice capable of accepting human cells. METHODS: Immune compromised mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) were repeatedly exposed to pharmaceutical grade doxorubicin (0.5 mg/kg - 4 mg/kg). Cardiotoxicity was assessed by echocardiography and µCT imaging of the coronary vascular bed as well as by flow cytometry and by histological assessments of anthracycline-induced cardiac tissue damage. RESULTS: The immune compromised mice were highly susceptible to doxorubicin treatment. Doxorubicin induced both systemic and cardiac toxicities. Gastrointestinal and hepatic injury occurred at 4 mg/kg and 1.5 mg/kg dosing while mice receiving 0.5 mg/kg weekly only displayed hepatic damage. Repeated exposure to 0.5 mg/kg anthracyclines resulted in cardiac toxicity. Flow cytometric analysis of hearts indicated a loss in endothelial and cardiac progenitor cells after doxorubicin treatment. This endothelial loss is corroborated by the lack of small vessels detected by µCT in the hearts of mice exposed to doxorubicin. Histological assessment shows no overt cardiomyocyte injury but livers from mice treated with doxorubicin show marked hepatic plate atrophy with intracytoplasmic and canalicular cholestasis, rare pericentral hepatocellular necrosis and significant zone 3 iron accumulation, likely an indication of metabolic injury due to doxorubicin toxicity. CONCLUSIONS: Immune compromised mice are sensitive to doxorubicin therapy resulting in systemic complications in addition to cardiovascular toxicity. Anthracycline-induced cardiotoxicity is observed at very low doses in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice.

Heterozygous caveolin-3 mice show increased susceptibility to palmitate-induced insulin resistance.

Insulin resistance and diabetes are comorbidities of obesity and affect one in 10 adults in the United States. Despite the high prevalence, the mechanisms of cardiac insulin resistance in obesity are still unclear. We test the hypothesis that the insulin receptor localizes to caveolae and is regulated through binding to caveolin-3 (CAV3). We further test whether haploinsufficiency forCAV3 increases the susceptibility to high-fat-induced insulin resistance. We used in vivo and in vitro studies to determine the effect of palmitate exposure on global insulin resistance, contractile performance of the heart in vivo, glucose uptake in the heart, and on cellular signaling downstream of theIR We show that haploinsufficiency forCAV3 increases susceptibility to palmitate-induced global insulin resistance and causes cardiomyopathy. On the basis of fluorescence energy transfer (FRET) experiments, we show thatCAV3 andIRdirectly interact in cardiomyocytes. Palmitate impairs insulin signaling by a decrease in insulin-stimulated phosphorylation of Akt that corresponds to an 87% decrease in insulin-stimulated glucose uptake inHL-1 cardiomyocytes. Despite loss of Akt phosphorylation and lower glucose uptake, palmitate increased insulin-independent serine phosphorylation ofIRS-1 by 35%. In addition, we found lipid induced downregulation ofCD36, the fatty acid transporter associated with caveolae. This may explain the problem the diabetic heart is facing with the simultaneous impairment of glucose uptake and lipid transport. Thus, these findings suggest that loss ofCAV3 interferes with downstream insulin signaling and lipid uptake, implicatingCAV3 as a regulator of theIRand regulator of lipid uptake in the heart.

Palmitate diet-induced loss of cardiac caveolin-3: a novel mechanism for lipid-induced contractile dysfunction.

Obesity is associated with an increased risk of cardiomyopathy, and mechanisms linking the underlying risk and dietary factors are not well understood. We tested the hypothesis that dietary intake of saturated fat increases the levels of sphingolipids, namely ceramide and sphingomyelin in cardiac cell membranes that disrupt caveolae, specialized membrane micro-domains and important for cellular signaling. C57BL/6 mice were fed two high-fat diets: palmitate diet (21% total fat, 47% is palmitate), and MCT diet (21% medium-chain triglycerides, no palmitate). We established that high-palmitate feeding for 12 weeks leads to 40% and 50% increases in ceramide and sphingomyelin, respectively, in cellular membranes. Concomitant with sphingolipid accumulation, we observed a 40% reduction in systolic contractile performance. To explore the relationship of increased sphingolipids with caveolins, we analyzed caveolin protein levels and intracellular localization in isolated cardiomyocytes. In normal cardiomyocytes, caveolin-1 and caveolin-3 co-localize at the plasma membrane and the T-tubule system. However, mice maintained on palmitate lost 80% of caveolin-3, mainly from the T-tubule system. Mice maintained on MCT diet had a 90% reduction in caveolin-1. These data show that caveolin isoforms are sensitive to the lipid environment. These data are further supported by similar findings in human cardiac tissue samples from non-obese, obese, non-obese cardiomyopathic, and obese cardiomyopathic patients. To further elucidate the contractile dysfunction associated with the loss of caveolin-3, we determined the localization of the ryanodine receptor and found lower expression and loss of the striated appearance of this protein. We suggest that palmitate-induced loss of caveolin-3 results in cardiac contractile dysfunction via a defect in calcium-induced calcium release.

Interactive 3D Analysis of Blood Vessel Trees and Collateral Vessel Volumes in Magnetic Resonance Angiograms in the Mouse Ischemic Hindlimb Model.

The quantitative analysis of blood vessel volumes from magnetic resonance angiograms (MRA) or µCT images is difficult and time-consuming. This fact, when combined with a study that involves multiple scans of multiple subjects, can represent a significant portion of research time. In order to enhance analysis options and to provide an automated and fast analysis method, we developed a software plugin for the ImageJ and Fiji image processing frameworks that enables the quick and reproducible volume quantification of blood vessel segments. The novel plugin named Volume Calculator (VolCal), accepts any binary (thresholded) image and produces a three-dimensional schematic representation of the vasculature that can be directly manipulated by the investigator. Using MRAs of the mouse hindlimb ischemia model, we demonstrate quick and reproducible blood vessel volume calculations with 95 - 98% accuracy. In clinical settings this software may enhance image interpretation and the speed of data analysis and thus enhance intervention decisions for example in peripheral vascular disease or aneurysms. In summary, we provide a novel, fast and interactive quantification of blood vessel volumes for single blood vessels or sets of vessel segments with particular focus on collateral formation after an ischemic insult.

Palmitate-Induced Translocation of Caveolin-3 and Endothelial Nitric Oxide Synthase in Cardiomyocytes.

PROBLEM STATEMENT: Palmitate is a known cardiac lipotoxin that blunts cardiomyocyte contractile function and induces apoptosis, likely via accumulation of the lipotoxic ceramide. Ceramide is a sphingolipid and localizes to caveolae, which are lined in the inner membrane leaflet by caveolin proteins. In this study, we investigated the effects of palmitate on caveolin proteins and on endothelial Nitric Oxide Synthase (eNOS), a signaling mediator that binds to caveolin-3, the muscle-specific caveolae scaffolding protein. APPROACH AND RESULTS: Mice fed a high palmitate diet for 12 weeks showed pathologically increased coronary flow in the ex vivo Langendorff heart especially at low extracellular calcium concentrations. In these hearts, eNOS Ser1177 phosphorylation was increased compared to standard or high fat control diet hearts. This suggested that eNOS, a potent vasodilator in the heart, is affected by palmitate. In vitro experiments showed that exposure of HL-1 cardiomyocytes to palmitate causes translocation of eNOS from the plasma membrane to a perinuclear location and causes an 80% decrease in Thr495 phosphorylation. This corresponded with a 41% decrease in NO production. To determine the mechanism of the loss of plasma membrane bound eNOS, we investigated the effect of palmitate on caveolin-3 and found decreased caveolin-3 protein levels by 70% compared to control cells. The remaining 30% of caveolin-3 was localized to a perinuclear location. In contrast to previous studies, palmitate did not cause apoptosis in cardiomyocytes. CONCLUSION: Overall, we show for the first time that a high palmitate diet leads to loss of caveolin-3 in cardiomyocytes and to coronary dysfunction of the mouse heart, via uncoupling of eNOS.