Phenotypic characterization of gastric sensory neurons in mice

Recent studies suggest that the capsaicin receptor [transient receptor potential vanilloid (TRPV)1] may play a role in visceral mechanosensation. To address the potential role of TRPV1 in vagal sensory neurons, we developed a new in vitro technique allowing us to determine TRPV1 expression directly in physiologically characterized gastric sensory neurons. Stomach, esophagus, and intact vagus nerve up to the central terminations were carefully dissected and placed in a perfusion chamber. Intracellular recordings were made from the soma of nodose neurons during mechanical stimulation of the stomach. Physiologically characterized neurons were labeled iontophoretically with neurobiotin and processed for immunohistochemical experiments. As shown by action potential responses triggered by stimulation of the upper thoracic vagus with a suction electrode, essentially all abdominal vagal afferents in mice conduct in the C-fiber range. Mechanosensitive gastric afferents encode stimulus intensities over a wide range without apparent saturation when punctate stimuli are used. Nine of 37 mechanosensitive vagal afferents expressed TRPV1 immunoreactivity, with 8 of the TRPV1-positive cells responding to stretch. A small number of mechanosensitive gastric vagal afferents express neurofilament heavy chains and did not respond to stretch. By maintaining the structural and functional integrity of vagal afferents up to the nodose ganglion, physiological and immunohistochemical properties of mechanosensory gastric sensory neurons can be studied in vitro. Using this novel technique, we identified TRPV1 immunoreactivity in only one-fourth of gastric mechanosensitive neurons, arguing against a major role of this ion channel in sensation of mechanical stimuli under physiological conditions.     

Assessment of Brainstem Function with Auricular Branch of Vagus Nerve Stimulation in Parkinson’s Disease

Background

The efferent dorsal motor nucleus of the vagal nuclei complex may degenerate early in the course of Parkinson’s disease (PD), while efferent nucleus ambiguous, the principal source of parasympathetic vagal neurons innervating the heart, and afferent somatosensory nuclei remain intact.

Objective

To obtain neurophysiological evidence related to this pattern, we tested processing of afferent sensory information transmitted via the auricular branch of the vagus nerve (ABVN) which is known to be connected to autonomic regulation of cardiac rhythm.

Methods

In this cross-sectional observational study, we recorded (i) somatosensory evoked potentials (ABVN-SEP) and (ii) cutaneo-cardioautonomic response elicited by stimulation of the ABVN (modulation of heart-rate variability (HRV index; low frequency power, ln(LF), high frequency power, ln(HF); ln(LF/HF) ratio)) in 50 PD patients and 50 age and sex matched healthy controls. Additionally, auditory evoked potentials and trigeminal nerve SEP were assessed.

Results

Neither ABVN-SEP nor any of the other functional brainstem parameters differed between patients and controls. Although HRV index was decreased in PD patients, modulation of ln(LF/HF) by ABVN-stimulation, likely indicating cardiac parasympathetic activation, did not differ between both groups.

Conclusions

Findings do not point to prominent dysfunction of processing afferent information from ABVN and its connected parasympathetic cardiac pathway in PD. They are consistent with the known pattern of degeneration of the vagal nuclei complex of the brainstem.     

Propofol Causes Vasodilation In Vivo via TRPA1 Ion Channels: Role of Nitric Oxide and BKCa Channels

Background

Transient receptor potential (TRP) ion channels of the A1 (TRPA1) and V1 (TRPV1) subtypes are key regulators of vasomotor tone. Propofol is an intravenous anesthetic known to cause vasorelaxation. Our objectives were to examine the extent to which TRPA1 and/or TRPV1 ion channels mediate propofol-induced depressor responses in vivo and to delineate the signaling pathway(s) involved.

Methods

Mice were subjected to surgery under 1.5–2.5% sevoflurane gas with supplemental oxygen. After a stable baseline in mean arterial pressure (MAP) was achieved propofol (2.5, 5.0, 10.0 mg/kg/min) was administered to assess the hemodynamic actions of the intravenous anesthetic. The effect of nitric oxide synthase (NOS) inhibition with L-NAME and/or calcium-gated K⁺ channel (BK_Ca) inhibition with Penetrim A (Pen A), alone and in combination, on propofol-induced decreases in mean arterial pressure were assessed in control C57Bl/6J, TRPA1-/-, TRPV1^-/- and double-knockout mice (TRPAV^-/-).

Results

Propofol decreased MAP in control mice and this effect was markedly attenuated in TRPA1^-/- and TRPAV^-/- mice but unaffected in TRPV1^-/-mice. Moreover, pretreatment with L-NAME or Pen A attenuated the decrease in MAP in control and TRPV1^-/- mice, and combined inhibition abolished the depressor response. In contrast, the markedly attenuated propofol-induced depressor response observed in TRPA1^-/- and TRPAV^-/- mice was unaffected by pre-treatment with Pen A or L-NAME when used either alone or in combination.

Conclusion

These data demonstrate for the first time that propofol-induced depressor responses in vivo are predominantly mediated by TRPA1 ion channels with no involvement of TRPV1 ion channels and includes activation of both NOS and BK_Ca channels.     

Myocardial Structural and Biological Anomalies Induced by High Fat Diet in Psammomys obesus Gerbils

Background

Psammomys obesus gerbils are particularly prone to develop diabetes and obesity after brief period of abundant food intake. A hypercaloric high fat diet has been shown to affect cardiac function. Here, we sought to determine whether a short period of high fat feeding might alter myocardial structure and expression of calcium handling proteins in this particular strain of gerbils.

Methods

Twenty Psammomys obesus gerbils were randomly assigned to receive a normal plant diet (controls) or a high fat diet. At baseline and 16-week later, body weight, plasma biochemical parameters (including lipid and carbohydrate levels) were evaluated. Myocardial samples were collected for pathobiological evaluation.

Results

Sixteen-week high fat dieting resulted in body weight gain and hyperlipidemia, while levels of carbohydrates remained unchanged. At myocardial level, high fat diet induced structural disorganization, including cardiomyocyte hypertrophy, lipid accumulation, interstitial and perivascular fibrosis and increased number of infiltrating neutrophils. Myocardial expressions of pro-apoptotic Bax-to-Bcl-2 ratio, pro-inflammatory cytokines [interleukin (IL)-1β and tumor necrosis factor (TNF)-α], intercellular (ICAM1) and vascular adhesion molecules (VCAM1) increased, while gene encoding cardiac muscle protein, the alpha myosin heavy polypeptide (MYH6), was downregulated. Myocardial expressions of sarco(endo)plasmic calcium-ATPase (SERCA2) and voltage-dependent calcium channel (Cacna1c) decreased, while protein kinase A (PKA) and calcium-calmodulin-dependent protein kinase (CaMK2D) expressions increased. Myocardial expressions of ryanodine receptor, phospholamban and sodium/calcium exchanger (Slc8a1) did not change.

Conclusions

We conclude that a relative short period of high fat diet in Psammomys obesus results in severe alterations of cardiac structure, activation of inflammatory and apoptotic processes, and altered expression of calcium-cycling determinants.     

Suppressive Role of PPARγ-Regulated Endothelial Nitric Oxide Synthase in Adipocyte Lipolysis

Introduction

Metabolic syndrome causes insulin resistance and is associated with risk factor clustering, thereby increasing the risk of atherosclerosis. Recently, endothelial nitric oxide synthase deficient (eNOS-/-) mice have been reported to show metabolic disorders. Interestingly, eNOS has also been reported to be expressed in non-endothelial cells including adipocytes, but the functions of eNOS in adipocytes remain unclear.

Methods and Results

The eNOS expression was induced with adipocyte differentiation and inhibition of eNOS/NO enhanced lipolysis in vitro and in vivo. Furthermore, the administration of a high fat diet (HFD) was able to induce non-alcoholic steatohepatitis (NASH) in eNOS-/- mice but not in wild type mice. A PPARγ antagonist increased eNOS expression in adipocytes and suppressed HFD-induced fatty liver changes.

Conclusions

eNOS-/- mice induce NASH development, and these findings provide new insights into the therapeutic approach for fatty liver disease and related disorders.     

Effects of Incretin-Based Therapies on Neuro-Cardiovascular Dynamic Changes Induced by High Fat Diet in Rats

Background and Aims

Obesity promotes cardiac and cerebral microcirculatory dysfunction that could be improved by incretin-based therapies. However, the effects of this class of compounds on neuro-cardiovascular system damage induced by high fat diet remain unclear. The aim of this study was to investigate the effects of incretin-based therapies on neuro-cardiovascular dysfunction induced by high fat diet in Wistar rats.

Methods and Results

We have evaluated fasting glucose levels and insulin resistance, heart rate variability quantified on time and frequency domains, cerebral microcirculation by intravital microscopy, mean arterial blood pressure, ventricular function and mitochondrial swelling. High fat diet worsened biometric and metabolic parameters and promoted deleterious effects on autonomic, myocardial and haemodynamic parameters, decreased capillary diameters and increased functional capillary density in the brain. Biometric and metabolic parameters were better improved by glucagon like peptide-1 (GLP-1) compared with dipeptdyl peptidase-4 (DPP-4) inhibitor. On the other hand, both GLP-1 agonist and DPP-4 inhibitor reversed the deleterious effects of high fat diet on autonomic, myocardial, haemodynamic and cerebral microvascular parameters. GLP-1 agonist and DPP-4 inhibitor therapy also increased mitochondrial permeability transition pore resistance in brain and heart tissues of rats subjected to high fat diet.

Conclusion

Incretin-based therapies improve deleterious cardiovascular effects induced by high fat diet and may have important contributions on the interplay between neuro-cardiovascular dynamic controls through mitochondrial dysfunction associated to metabolic disorders.     

Dietary Intervention for Overweight and Obese Adults: Comparison of Low-Carbohydrate and Low-Fat Diets. A Meta-Analysis

Background

Reduced calorie, low fat diet is currently recommended diet for overweight and obese adults. Prior data suggest that low carbohydrate diets may also be a viable option for those who are overweight and obese.

Purpose

Compare the effects of low carbohydrate versus low fats diet on weight and atherosclerotic cardiovascular disease risk in overweight and obese patients.

Data Sources

Systematic literature review via PubMed (1966–2014).

Study Selection

Randomized controlled trials with ≥8 weeks follow up, comparing low carbohydrate (≤120gm carbohydrates/day) and low fat diet (≤30% energy from fat/day).

Data Extraction

Data were extracted and prepared for analysis using double data entry. Prior to identification of candidate publications, the outcomes of change in weight and metabolic factors were selected as defined by Cochrane Collaboration. Assessment of the effects of diets on predicted risk of atherosclerotic cardiovascular disease risk was added during the data collection phase.

Data Synthesis

1797 patients were included from 17 trials with <1 year follow up in 12. Compared with low fat diet, low carbohydrate was associated with significantly greater reduction in weight (Δ = -2.0 kg, 95% CI: -3.1, -0.9) and significantly lower predicted risk of atherosclerotic cardiovascular disease events (p<0.03). Frequentist and Bayesian results were concordant. The probability of greater weight loss associated with low carbohydrate was >99% while the reduction in predicted risk favoring low carbohydrate was >98%.

Limitations

Lack of patient-level data and heterogeneity in dropout rates and outcomes reported.

Conclusions

This trial-level meta-analysis of randomized controlled trials comparing LoCHO diets with LoFAT diets in strictly adherent populations demonstrates that each diet was associated with significant weight loss and reduction in predicted risk of ASCVD events. However, LoCHO diet was associated with modest but significantly greater improvements in weight loss and predicted ASCVD risk in studies from 8 weeks to 24 months in duration. These results suggest that future evaluations of dietary guidelines should consider low carbohydrate diets as effective and safe intervention for weight management in the overweight and obese, although long-term effects require further investigation.     

Moderate exercise training is more effective than resveratrol supplementation for ameliorating lipid metabolic complication in skeletal muscle of high fat diet-induced obese mice

[Purpose]

The aim of this study was to compare the effectiveness of moderate exercise training or resveratrol supplementation with a low fat diet on lipid metabolism in the skeletal muscle of high fat diet-induced obese mice.

[Methods]

C57BL/6J mice (5 weeks old, n = 30) were fed a high fat diet (45% fat) for 8 weeks first to make them obese. Afterward, all the mice were fed a low fat diet during 8 weeks of intervention with moderate exercise training and resveratrol supplementation. Before the intervention, the mice were separated into 3 groups: low-fat diet control (HLC; n = 10), low fat diet with resveratrol (HLR; n = 10) or low fat diet with exercise (HLE n = 10). The exercise group (HLE) performed treadmill running for 30-60 min/day at 10-22 m/min, 0% grade, 5 times/week for 8 weeks, while the resveratrol group (HLR) received a daily dose of resveratrol (10 mg/kg of body weight), 5 days/week for 8 weeks.

[Results]

Body weight was significantly reduced in HLE. Further, the lipogenesis marker SREBP and the inflammatory cytokine TNF-α were significant reduced in HLE. However, there was no significant effect from resveratrol supplementation with a low fat diet. Taken together, exercise training with a low fat diet has the positive effect of ameliorating lipid disturbance in the skeletal muscle of high fat diet-induced obese mice.

[Conclusion]

These findings suggest that exercise training with a low fat diet is most effective to improve lipid metabolism by reducing lipogenesis and inflammation in the skeletal muscle of high fat diet-induced obese mice.     

Isorhamnetin Attenuates Atherosclerosis by Inhibiting Macrophage Apoptosis via PI3K/AKT Activation and HO-1 Induction

Background and Purpose

Isorhamnetin (Iso) is a flavonoid compound extracted from the Chinese herb Hippophae rhamnoides L. Previous studies have revealed its anti-cancer, anti-inflammatory, and anti-oxidant activities. This study investigated the ability of Iso to inhibit oxidized low-density lipoprotein (ox-LDL)-induced cell apoptosis in THP-1-derived macrophages. The effects of Iso on atherosclerosis in vivo were also evaluated in apolipoprotein E knockout (ApoE-/-) mice fed a high fat diet.

Methods and Results

Iso showed significant inhibitory effects on ox-LDL-induced THP-1-derived macrophage injuries via decreasing reactive oxygen species levels, lipid deposition, and caspase-3 activation, restoring mitochondrial membrane potential, reducing the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells, and regulating apoptosis-related proteins. We also determined the protective effects of Iso by PI3K/AKT activation and HO-1 induction. Iso reduced the atherosclerotic plaque size in vivo in ApoE-/- mice as assessed by oil red O, Sudan IV staining, and CD68-positive cells, and reduced macrophage apoptosis as assessed by caspase-3 and TUNEL assays in lesions.

Conclusion

In conclusion, our results show that Iso inhibited atherosclerotic plaque development in ApoE-/- mice by PI3K/AKT activation and HO-1 induction.     

Beneficial Effects of an Alternating High- Fat Dietary Regimen on Systemic Insulin Resistance,Hepatic and Renal Inflammation and Renal Function

Background

An Alternating high- cholesterol dietary regimen has proven to be beneficial when compared to daily high- cholesterol feeding. In the current study we explored whether the same strategy is applicable to a high- fat dietary regimen.

Objective

To investigate whether an alternating high- fat dietary regimen can effectively diminish insulin resistance, hepatic and renal inflammation and renal dysfunction as compared to a continuous high- fat diet.

Design

Four groups of male ApoE*3Leiden mice (n = 15) were exposed to different diet regimens for 20 weeks as follows: Group 1: low- fat diet (10 kcal% fat); Group 2: intermediate- fat diet (25 kcal% fat); Group 3: high- fat diet (45 kcal% fat) and Group 4: alternating- fat diet (10 kcal% fat for 4 days and 45 kcal% fat for 3 days in a week).

Results

Compared to high fat diet feeding, the alternating and intermediate- fat diet groups had reduced body weight gain and did not develop insulin resistance or albuminuria. In addition, in the alternating and intermediate- fat diet groups, parameters of tissue inflammation were markedly reduced compared to high fat diet fed mice.

Conclusion

Both alternating and intermediate- fat feeding were beneficial in terms of reducing body weight gain, insulin resistance, hepatic and renal inflammation and renal dysfunction. Thus beneficial effects of alternating feeding regimens on cardiometabolic risk factors are not only applicable for cholesterol containing diets but can be extended to diets high in fat content.     

Trypanosoma cruzi Infection Is a Potent Risk Factor for Non-alcoholic Steatohepatitis Enhancing Local and Systemic Inflammation Associated with Strong Oxidative Stress and Metabolic Disorders

Background

The immune mechanisms underlying experimental non-alcoholic steatohepatitis (NASH), and more interestingly, the effect of T. cruzi chronic infection on the pathogenesis of this metabolic disorder are not completely understood.

Methodology/Principal Findings

We evaluated immunological parameters in male C57BL/6 wild type and TLR4 deficient mice fed with a standard, low fat diet, LFD (3% fat) as control group, or a medium fat diet, MFD (14% fat) in order to induce NASH, or mice infected intraperitoneally with 100 blood-derived trypomastigotes of Tulahuen strain and also fed with LFD (I+LFD) or MFD (I+MFD) for 24 weeks. We demonstrated that MFD by itself was able to induce NASH in WT mice and that parasitic infection induced marked metabolic changes with reduction of body weight and steatosis revealed by histological studies. The I+MFD group also improved insulin resistance, demonstrated by homeostasis model assessment of insulin resistance (HOMA-IR) analysis; although parasitic infection increased the triglycerides and cholesterol plasma levels. In addition, hepatic M1 inflammatory macrophages and cytotoxic T cells showed intracellular inflammatory cytokines which were associated with high levels of IL6, IFNγ and IL17 plasmatic cytokines and CCL2 chemokine. These findings correlated with an increase in hepatic parasite load in I+MFD group demonstrated by qPCR assays. The recruitment of hepatic B lymphocytes, NK and dendritic cells was enhanced by MFD, and it was intensified by parasitic infection. These results were TLR4 signaling dependent. Flow cytometry and confocal microscopy analysis demonstrated that the reactive oxygen species and peroxinitrites produced by liver inflammatory leukocytes of MFD group were also exacerbated by parasitic infection in our NASH model.

Conclusions

We highlight that a medium fat diet by itself is able to induce steatohepatitis. Our results also suggest a synergic effect between damage associated with molecular patterns generated during NASH and parasitic infection, revealing an intense cross-talk between metabolically active tissues, such as the liver, and the immune system. Thus, T. cruzi infection must be considered as an additional risk factor since exacerbates the inflammation and accelerates the development of hepatic injury.     

Exercise training is more effective than resveratrol supplementation on alleviation of inflammation in peritoneal macrophages of high fat diet mice

[Purpose]

This study investigated the effect of exercise training and resveratrol supplementation with low fat diet on proinflammatory profiles by Lipopolysaccharide (LPS)-stimulation in peritoneal macrophage of high fat diet mice.

[Methods]

To accomplish the purpose of this study, C57BL/6 male mice were fed high fat diet (45% fat diet) for 8 weeks. Then these mice were divided into 3 groups; HLC (high fat diet and low fat diet for 8 weeks as the control, n=10), HLR (high fat diet and low fat diet for 8 weeks with resveratrol supplementation, n=10). HLE (high fat diet and low fat diet for 8 weeks with moderate exercise training, n=10). Resveratrol (10 mg/kg) was administrated once a day, 5 days/week for 8 weeks. Exercise training was performed for 8 weeks on a treadmill running for 30-60 min/day at 10-22 m/min, 0% grade, 5 days/week. After exercise training, all the peritoneal macrophage was collected and LPS (0, 0.5, 1.0 μg/ml) were used to stimulate the cells. Then peritoneal macrophage TNF-α, IL-6, MCP-1, IL12p70, IFN-γ, IL-10 were measured by BD cytometric bead array mouse inflammation kit.

[Results]

As a result, body weight and total cholesterol were significantly reduced in HLE compared with HLC (p<.05). Also, TNF-α and MCP-1 were decreased in HLE compared with HLC (p<.05) by LPS-stimulation (0, 0.5, 1.0 μg/ml) and IL-6, IL-12p70 and IFN-r were decreased in HLE compared with HLC (p<.05) by LPS-stimulation (1.0 μg/ml). But resveratrol supplementation did not affect the result.

[Conclusion]

These findings suggest that exercise training has beneficial effects on body weight, total cholesterol, peritoneal macrophage and proinflammatory cytokine in high fat diet mice.     

Sucrose counteracts the anti-inflammatory effect of fish oil in adipose tissue and increases obesity development in mice

Background

Polyunsaturated n-3 fatty acids (n-3 PUFAs) are reported to protect against high fat diet-induced obesity and inflammation in adipose tissue. Here we aimed to investigate if the amount of sucrose in the background diet influences the ability of n-3 PUFAs to protect against diet-induced obesity, adipose tissue inflammation and glucose intolerance.

Methodology/Principal Findings

We fed C57BL/6J mice a protein- (casein) or sucrose-based high fat diet supplemented with fish oil or corn oil for 9 weeks. Irrespective of the fatty acid source, mice fed diets rich in sucrose became obese whereas mice fed high protein diets remained lean. Inclusion of sucrose in the diet also counteracted the well-known anti-inflammatory effect of fish oil in adipose tissue, but did not impair the ability of fish oil to prevent accumulation of fat in the liver. Calculation of HOMA-IR indicated that mice fed high levels of proteins remained insulin sensitive, whereas insulin sensitivity was reduced in the obese mice fed sucrose irrespectively of the fat source. We show that a high fat diet decreased glucose tolerance in the mice independently of both obesity and dietary levels of n-3 PUFAs and sucrose. Of note, increasing the protein∶sucrose ratio in high fat diets decreased energy efficiency irrespective of fat source. This was accompanied by increased expression of Ppargc1a (peroxisome proliferator-activated receptor, gamma, coactivator 1 alpha) and increased gluconeogenesis in the fed state.

Conclusions/Significance

The background diet influence the ability of n-3 PUFAs to protect against development of obesity, glucose intolerance and adipose tissue inflammation. High levels of dietary sucrose counteract the anti-inflammatory effect of fish oil in adipose tissue and increases obesity development in mice.     

Replacement of Dietary Saturated Fat by PUFA-Rich Pumpkin Seed Oil Attenuates Non-Alcoholic Fatty Liver Disease and Atherosclerosis Development,with Additional Health Effects of Virgin over Refined Oil

Background and Aims

As dietary saturated fatty acids are associated with metabolic and cardiovascular disease, a potentially interesting strategy to reduce disease risk is modification of the quality of fat consumed. Vegetable oils represent an attractive target for intervention, as they largely determine the intake of dietary fats. Furthermore, besides potential health effects conferred by the type of fatty acids in a vegetable oil, other minor components (e.g. phytochemicals) may also have health benefits. Here, we investigated the potential long-term health effects of isocaloric substitution of dietary fat (i.e. partial replacement of saturated by unsaturated fats), as well as putative additional effects of phytochemicals present in unrefined (virgin) oil on development of non-alcoholic fatty liver disease (NAFLD) and associated atherosclerosis. For this, we used pumpkin seed oil, because it is high in unsaturated fatty acids and a rich source of phytochemicals.

Methods

ApoE*3Leiden mice were fed a Western-type diet (CON) containing cocoa butter (15% w/w) and cholesterol (1% w/w) for 20 weeks to induce risk factors and disease endpoints. In separate groups, cocoa butter was replaced by refined (REF) or virgin (VIR) pumpkin seed oil (comparable in fatty acid composition, but different in phytochemical content).

Results

Both oils improved dyslipidaemia, with decreased (V)LDL-cholesterol and triglyceride levels in comparison with CON, and additional cholesterol-lowering effects of VIR over REF. While REF did not affect plasma inflammatory markers, VIR reduced circulating serum amyloid A and soluble vascular adhesion molecule-1. NAFLD and atherosclerosis development was modestly reduced in REF, and VIR strongly decreased liver steatosis and inflammation as well as atherosclerotic lesion area and severity.

Conclusions

Overall, we show that an isocaloric switch from a diet rich in saturated fat to a diet rich in unsaturated fat can attenuate NAFLD and atherosclerosis development. Phytochemical-rich virgin pumpkin seed oil exerts additional anti-inflammatory effects resulting in more pronounced health effects.     

Inhibition of Neuroblastoma Tumor Growth by Ketogenic Diet and/or Calorie Restriction in a CD1-Nu Mouse Model

Introduction

Neuroblastoma is a malignant pediatric cancer derived from neural crest cells. It is characterized by a generalized reduction of mitochondrial oxidative phosphorylation. The goal of the present study was to investigate the effects of calorie restriction and ketogenic diet on neuroblastoma tumor growth and monitor potential adaptive mechanisms of the cancer’s oxidative phosphorylation system.

Methods

Xenografts were established in CD-1 nude mice by subcutaneous injection of two neuroblastoma cell lines having distinct genetic characteristics and therapeutic sensitivity [SH-SY5Y and SK-N-BE(2)]. Mice were randomized to four treatment groups receiving standard diet, calorie-restricted standard diet, long chain fatty acid based ketogenic diet or calorie-restricted ketogenic diet. Tumor growth, survival, metabolic parameters and weight of the mice were monitored. Cancer tissue was evaluated for diet-induced changes of proliferation indices and multiple oxidative phosphorylation system parameters (respiratory chain enzyme activities, western blot analysis, immunohistochemistry and mitochondrial DNA content).

Results

Ketogenic diet and/or calorie restriction significantly reduced tumor growth and prolonged survival in the xenograft model. Neuroblastoma growth reduction correlated with decreased blood glucose concentrations and was characterized by a significant decrease in Ki-67 and phospho-histone H3 levels in the diet groups with low tumor growth. As in human tumor tissue, neuroblastoma xenografts showed distinctly low mitochondrial complex II activity in combination with a generalized low level of mitochondrial oxidative phosphorylation, validating the tumor model. Neuroblastoma showed no ability to adapt its mitochondrial oxidative phosphorylation activity to the change in nutrient supply induced by dietary intervention.

Conclusions

Our data suggest that targeting the metabolic characteristics of neuroblastoma could open a new front in supporting standard therapy regimens. Therefore, we propose that a ketogenic diet and/or calorie restriction should be further evaluated as a possible adjuvant therapy for patients undergoing treatment for neuroblastoma.     

Differential Effects of C1qa Ablation on Glaucomatous Damage in Two Sexes in DBA/2NNia Mice

Purpose

To determine the sex and age-related effects of C1qa ablation on retinal ganglion cell (RGC) and optic nerve (ON) axonal loss in a mouse model of glaucomatous neurodegeneration.

Methods

Congenic C1qa mice were generated in the DBA/2NNia background. Female and male knockout (-/-), heterozygous (+/-), and wild type (+/+) mice were aged up to 14 months and IOPs were recorded in a subset of animals. Retinas of mice from all three groups at 5–6, 9–10 and 11–13 months of age were flat-mounted after retrograde labeling with Fluorogold. Imaged retinas were scored (RGC score) semi-quantitatively on a 10 point scale by two independent observers. A subset of retinas and optic nerves were also used for measurement of total number of RGCs. Semi-thin sections of ON were imaged and graded (ON score) for the amount of axonal damage semi-quantitatively, by two masked observers. Analysis of covariance (ANCOVA) was used for statistical comparisons. Microglial cells in flat-mounted retinas of 5–6 month old C1qa -/- and C1qa +/+ mice were used for assessment of microglial activation utilizing morphological criteria.

Results

Female C1qa -/- mice had significantly higher IOP (p<0.000001, ANOVA) between 8 and 13 months of age compared to C1qa +/+ animals. No differences in IOPs between animals of the three genotypes were observed in males. At 5–6 months of age, there was no difference in RGC or ON scores between the three genotypes in animals of either sex. At 9–10 months of age, female mice didn’t show significant differences in RGC or ON scores between the three genotypes. However, male C1qa -/- and C1qa +/- mice of the same age had better RGC and ON scores (p<0.003 and p<0.05, ANCOVA, for RGC and ON scores, respectively) compared with C1qa +/+ mice. At 11–13 months of age, female C1qa -/- mice had better RGC scores (p<0.006, ANCOVA) compared to C1qa +/+ and C1qa +/- animals. Accordingly, C1qa -/- mice had higher RGC counts (p<0.03, t-test) compared to C1qa +/+ animals. In male mice, there was a tendency for 12 month old C1qa -/- animals to have better RGC scores and higher RGC counts, but this didn''t reach statistical significance. ON scores in 11–13 month old animals of either sex were not different between all three genotype. Microglial activation in male 5–6 month old C1qa -/- mice was decreased compared to C1qa +/+ animals; no such effect was seen in females.

Conclusions

Absence of C1qa ameliorates RGC and ON loss in the DBA/2NNia strain, but this effect differs between the two sexes. C1q-mediated RGC damage seems to be more potent than IOP-mediated RGC loss. In contrast, C1qa absence provides axonal protection early on, but this protection cannot overcome the effects of significant IOP elevation.     

Impaired H-Reflex Gain during Postural Loaded Locomotion in Individuals Post-Stroke

Objective

Successful execution of upright locomotion requires coordinated interaction between controllers for locomotion and posture. Our earlier research supported this model in the non-impaired and found impaired interaction in the post-stroke nervous system during locomotion. In this study, we sought to examine the role of the Ia afferent spinal loop, via the H-reflex response, under postural influence during a locomotor task. We tested the hypothesis that the ability to increase stretch reflex gain in response to postural loads during locomotion would be reduced post-stroke.

Methods

Fifteen individuals with chronic post-stroke hemiparesis and 13 non-impaired controls pedaled on a motorized cycle ergometer with specialized backboard support system under (1) seated supported, and (2) non-seated postural-loaded conditions, generating matched pedal force outputs of two levels. H-reflexes were elicited at 90°crank angle.

Results

We observed increased H-reflex gain with postural influence in non-impaired individuals, but a lack of increase in individuals post-stroke. Furthermore, we observed decreased H-reflex gain at higher postural loads in the stroke-impaired group.

Conclusion

These findings suggest an impaired Ia afferent pathway potentially underlies the defects in the interaction between postural and locomotor control post-stroke and may explain reduced ability of paretic limb support during locomotor weight-bearing in individuals post-stroke.

Significance

These results support the judicious use of bodyweight support training when first helping individuals post-stroke to regain locomotor pattern generation and weight-bearing capability.     

Prevention of Diet-Induced Obesity Effects on Body Weight and Gut Microbiota in Mice Treated Chronically with Δ9-Tetrahydrocannabinol

Objective

Acute administration of cannabinoid CB₁ receptor agonists, or the ingestion of cannabis, induces short-term hyperphagia. However, the incidence of obesity is lower in frequent cannabis users compared to non-users. Gut microbiota affects host metabolism and altered microbial profiles are observed in obese states. Gut microbiota modifies adipogenesis through actions on the endocannabinoid system. This study investigated the effect of chronic THC administration on body weight and gut microbiota in diet-induced obese (DIO) and lean mice.

Methods

Adult male DIO and lean mice were treated daily with vehicle or THC (2mg/kg for 3 weeks and 4 mg/kg for 1 additional week). Body weight, fat mass, energy intake, locomotor activity, whole gut transit and gut microbiota were measured longitudinally.

Results

THC reduced weight gain, fat mass gain and energy intake in DIO but not lean mice. DIO-induced changes in select gut microbiota were prevented in mice chronically administered THC. THC had no effect on locomotor activity or whole gut transit in either lean or DIO mice.

Conclusions

Chronic THC treatment reduced energy intake and prevented high fat diet-induced increases in body weight and adiposity; effects that were unlikely to be a result of sedation or altered gastrointestinal transit. Changes in gut microbiota potentially contribute to chronic THC-induced actions on body weight in obesity.     

Ghrelin selectively reduces mechanosensitivity of upper gastrointestinal vagal afferents

Ghrelin is a peptide released from gastric endocrine cells that has an orexigenic effect via a vagal pathway. Here we determine the effect of ghrelin on mechanosensitivity of upper-intestinal vagal afferent fibers in ferret and mouse. The responses of gastroesophageal vagal afferents to graded mechanical stimulation were determined in vitro before and during application of ghrelin to their peripheral endings. Three types of vagal afferent were tested: tension receptors responding to circumferential tension, mucosal receptors responding only to mucosal stroking, and tension/mucosal (TM) receptors in ferret esophagus that responded to both stimuli. In the mouse, ghrelin did not significantly affect the response of mucosal receptors to mucosal stroking with calibrated von Frey hairs. However, it significantly reduced responses of tension receptors to circumferential tension (P < 0.005; two-way ANOVA) by up to 40%. This inhibition was reversed by the ghrelin receptor antagonist [d-Lys-3]-growth hormone-releasing peptide (GHRP)-6. In the ferret, ghrelin significantly reduced the response of mucosal and TM receptors to mucosal stroking with calibrated von Frey hairs. Surprisingly, ghrelin did not significantly alter the response to circumferential tension in either tension or TM receptors. RT-PCR analysis indicated that both ghrelin and its receptor are expressed in vagal afferent cell bodies in mouse nodose ganglia. In conclusion, ghrelin selectively inhibits subpopulations of mechanically sensitive gastroesophageal vagal afferents; there is also potential for ghrelin release from vagal afferents. However, the subpopulation of afferents inhibited differs between species. These data have broad implications for ghrelin's role in food intake regulation and reflex control of gastrointestinal function.