Efficacy of different nitric oxide-based strategies in preventing experimental cerebral malaria by Plasmodium berghei ANKA

Background

Low nitric oxide (NO) bioavailability plays a role in the pathogenesis of human as well as of experimental cerebral malaria (ECM) caused by Plasmodium berghei ANKA (PbA). ECM is partially prevented by administration of the NO-donor dipropylenetriamine NONOate (DPTA-NO) at high concentration (1 mg/mouse), which also induces major side effects such as a sharp drop in blood pressure. We asked whether alternative strategies to improve NO bioavailability with minor side effects would also be effective in preventing ECM.

Methodology/Principal Findings

Mice were infected with PbA and prophylactically treated twice a day with bolus injections of L-arginine, Nω-hydroxy-nor-Arginine (nor-NOHA), tetrahydrobiopterin (BH4), separately or combined, sodium nitrite, sildenafil or sildenafil plus DPTA-NO starting on day 0 of infection. L-arginine and BH4 supplementation, with or without arginase inhibition by nor-NOHA, increased plasma nitrite levels but failed to protect against ECM development. Accordingly, prophylactic treatment with continuous delivery of L-arginine using osmotic pumps also did not improve survival. Similar outcomes were observed with sodium nitrite sildenafil (aimed at inhibiting phosphodiesterase-5) or with DPTA-NO. However, sildenafil (0.1 mg/mouse) in combination with a lower dose (0.1 mg/mouse) of DPTA-NO decreased ECM incidence (82±7.4% mortality in the saline group and 38±10.6% in the treated group; p<0.05). The combined prophylactic therapy did not aggravate anemia, had delayed effects in systolic, diastolic and mean arterial blood pressure and induced lower effects in pulse pressure when compared to DPTA-NO 1 mg/mouse.

Conclusions/Significance

These data show that sildenafil lowers the amount of NO-donor needed to prevent ECM, resulting also in lesser side effects. Prophylactic L-arginine when given in bolus or continuous delivery and bolus BH4 supplementation, with or without arginase inhibition, were able to increase NO bioavailability in PbA-infected mice but failed to decrease ECM incidence in the doses and protocol used.     

L-citrulline supplementation reverses the impaired airway relaxation in neonatal rats exposed to hyperoxia

Background

Hyperoxia is shown to impair airway relaxation via limiting L-arginine bioavailability to nitric oxide synthase (NOS) and reducing NO production as a consequence. L-arginine can also be synthesized by L-citrulline recycling. The role of L-citrulline supplementation was investigated in the reversing of hyperoxia-induced impaired relaxation of rat tracheal smooth muscle (TSM).

Methods

Electrical field stimulation (EFS, 2–20 V)-induced relaxation was measured under in vitro conditions in preconstricted tracheal preparations obtained from 12 day old rat pups exposed to room air or hyperoxia (>95% oxygen) for 7 days supplemented with L-citrulline or saline (in vitro or in vivo). The role of the L-citrulline/L-arginine cycle under basal conditions was studied by incubation of preparations in the presence of argininosuccinate synthase (ASS) inhibitor [α-methyl-D, L-aspartate, 1 mM] or argininosuccinate lyase inhibitor (ASL) succinate (1 mM) and/or NOS inhibitor [N^ω-nitro-L-arginine methyl ester; 100 μM] with respect to the presence or absence of L-citrulline (2 mM).

Results

Hyperoxia impaired the EFS-induced relaxation of TSM as compared to room air control (p < 0.001; 0.5 ± 0.1% at 2 V to 50.6 ± 5.7% at 20 V in hyperoxic group: 0.7 ± 0.2 at 2 V to 80.0 ± 5.6% at 20 V in room air group). Inhibition of ASS or ASL, and L-citrulline supplementation did not affect relaxation responses under basal conditions. However, inhibition of NOS significantly reduced relaxation responses (p < 0.001), which were restored to control level by L-citrulline. L-citrulline supplementation in vivo and in vitro also reversed the hyperoxia-impaired relaxation. The differences were significant (p <0.001; 0.8 ± 0.3% at 2 V to 47.1 ± 4.1% at 20 V without L-citrulline; 0.9 ± 0.3% at 2 V to 68.2 ± 4.8% at 20 V with L-citrulline). Inhibition of ASS or ASL prevented this effect of L-citrulline.

Conclusion

The results indicate the presence of an L-citrulline/L-arginine cycle in the airways of rat pups. L-citrulline recycling does not play a major role under basal conditions in airways, but it has an important role under conditions of substrate limitations to NOS as a source of L-arginine, and L-citrulline supplementation reverses the impaired relaxation of airways under hyperoxic conditions.     

Arginase Inhibition Ameliorates Hepatic Metabolic Abnormalities in Obese Mice

Objectives

We examined whether arginase inhibition influences hepatic metabolic pathways and whole body adiposity in diet-induced obesity.

Methods and Results

After obesity induction by a high fat diet (HFD), mice were fed either the HFD or the HFD with an arginase inhibitor, N^ω-hydroxy-nor-L-arginine (nor-NOHA). Nor-NOHA significantly prevented HFD-induced increases in body, liver, and visceral fat tissue weight, and ameliorated abnormal lipid profiles. Furthermore, nor-NOHA treatment reduced lipid accumulation in oleic acid-induced hepatic steatosis in vitro. Arginase inhibition increased hepatic nitric oxide (NO) in HFD-fed mice and HepG2 cells, and reversed the elevated mRNA expression of hepatic genes in lipid metabolism. Expression of phosphorylated 5′ AMPK-activated protein kinase α was increased by arginase inhibition in the mouse livers and HepG2 cells.

Conclusions

Arginase inhibition ameliorated obesity-induced hepatic lipid abnormalities and whole body adiposity, possibly as a result of increased hepatic NO production and subsequent activation of metabolic pathways involved in hepatic triglyceride metabolism and mitochondrial function.     

Effect of Arginase Inhibition on Ischemia-Reperfusion Injury in Patients with Coronary Artery Disease with and without Diabetes Mellitus

Background

Arginase competes with nitric oxide synthase for their common substrate L-arginine. Up-regulation of arginase in coronary artery disease (CAD) and diabetes mellitus may reduce nitric oxide bioavailability contributing to endothelial dysfunction and ischemia-reperfusion injury. Arginase inhibition reduces infarct size in animal models. Therefore the aim of the current study was to investigate if arginase inhibition protects from endothelial dysfunction induced by ischemia-reperfusion in patients with CAD with or without type 2 diabetes (Clinical trial registration number: {"type":"clinical-trial","attrs":{"text":"NCT02009527","term_id":"NCT02009527"}}NCT02009527).

Methods

Male patients with CAD (n = 12) or CAD + type 2 diabetes (n = 12), were included in this cross-over study with blinded evaluation. Endothelium-dependent vasodilatation was assessed by flow-mediated dilatation (FMD) of the radial artery before and after 20 min ischemia-reperfusion during intra-arterial infusion of the arginase inhibitor (N^ω-hydroxy-nor-L-arginine, 0.1 mg/min) or saline.

Results

The forearm ischemia-reperfusion was well tolerated. Endothelium-independent vasodilatation was assessed by sublingual nitroglycerin. Ischemia-reperfusion decreased FMD in patients with CAD from 12.7±5.2% to 7.9±4.0% during saline administration (P<0.05). N^ω-hydroxy-nor-L-arginine administration prevented the decrease in FMD in the CAD group (10.3±4.3% at baseline vs. 11.5±3.6% at reperfusion). Ischemia-reperfusion did not significantly reduce FMD in patients with CAD + type 2 diabetes. However, FMD at reperfusion was higher following nor-NOHA than following saline administration in both groups (P<0.01). Endothelium-independent vasodilatation did not differ between the occasions.

Conclusions

Inhibition of arginase protects against endothelial dysfunction caused by ischemia-reperfusion in patients with CAD. Arginase inhibition may thereby be a promising therapeutic strategy in the treatment of ischemia-reperfusion injury.     

Breast Feeding Increases Vasoconstriction Induced by Electrical Field Stimulation in Rat Mesenteric Artery. Role of Neuronal Nitric Oxide and ATP

Objectives

The aim of this study was to investigate in rat mesenteric artery whether breast feeding (BF) affects the vasomotor response induced by electrical field stimulation (EFS), participation by different innervations in the EFS-induced response and the mechanism/s underlying these possible modifications.

Methods

Experiments were performed in female Sprague-Dawley rats (3 months old), divided into three groups: Control (in oestrous phase), mothers after 21 days of BF, and mothers that had recovered their oestral cycle (After BF, in oestrous phase). Vasomotor response to EFS, noradrenaline (NA) and nitric oxide (NO) donor DEA-NO were studied. Neuronal NO synthase (nNOS) and phosphorylated nNOS (P-nNOS) protein expression were analysed and NO, superoxide anion (O₂ ^.–), NA and ATP releases were also determined.

Results

EFS-induced contraction was higher in the BF group, and was recovered after BF. 1 µmol/L phentolamine decreased the response to EFS similarly in control and BF rats. NA vasoconstriction and release were similar in both experimental groups. ATP release was higher in segments from BF rats. 0.1 mmol/L L-NAME increased the response to EFS in both control and BF rats, but more so in control animals. BF decreased NO release and did not modify O₂ ^.– production. Vasodilator response to DEA-NO was similar in both groups, while nNOS and P-nNOS expressions were decreased in segments from BF animals.

Conclusion

Breast feeding increases EFS-induced contraction in mesenteric arteries, mainly through the decrease of neuronal NO release mediated by decreased nNOS and P-nNOS expression. Sympathetic function is increased through the increased ATP release in BF rats.     

Is arginase a potential drug target in tobacco-induced pulmonary endothelial dysfunction?

Background

Tobacco-induced pulmonary vascular disease is partly driven by endothelial dysfunction. The bioavailability of the potent vasodilator nitric oxide (NO) depends on competition between NO synthase-3 (NOS3) and arginases for their common substrate (L-arginine). We tested the hypothesis whereby tobacco smoking impairs pulmonary endothelial function via upregulation of the arginase pathway.

Methods

Endothelium-dependent vasodilation in response to acetylcholine (Ach) was compared ex vivo for pulmonary vascular rings from 29 smokers and 10 never-smokers. The results were expressed as a percentage of the contraction with phenylephrine. We tested the effects of L-arginine supplementation, arginase inhibition (by N(omega)-hydroxy-nor-l-arginine, NorNOHA) and NOS3 induction (by genistein) on vasodilation. Protein levels of NOS3 and arginases I and II in the pulmonary arteries were quantified by Western blotting.

Results

Overall, vasodilation was impaired in smokers (relative to controls; p < 0.01). Eleven of the 29 smokers (the ED⁺ subgroup) displayed endothelial dysfunction (defined as the absence of a relaxant response to Ach), whereas 18 (the ED⁻ subgroup) had normal vasodilation. The mean responses to 10⁻⁴ M Ach were −23 ± 10% and 31 ± 4% in the ED⁺ and ED⁻ subgroups, respectively (p < 0.01). Supplementation with L- arginine improved endothelial function in the ED⁺ subgroup (−4 ± 10% vs. -32 ± 10% in the presence and absence of L- arginine, respectively; p = 0.006), as did arginase inhibition (18 ± 9% vs. -1 ± 9%, respectively; p = 0.0002). Arginase I protein was overexpressed in ED⁺ samples, whereas ED⁺ and ED⁻ samples did not differ significantly in terms of NOS3 expression. Treatment with genistein did not significantly improve endothelial function in ED⁺ samples.

Conclusion

Overexpression and elevated activity of arginase I are involved in tobacco-induced pulmonary endothelial dysfunction.     

Local Arginase Inhibition during Early Reperfusion Mediates Cardioprotection via Increased Nitric Oxide Production

Consumption of L-arginine contributes to reduced bioavailability of nitric oxide (NO) that is critical for the development of ischemia-reperfusion injury. The aim of the study was to determine myocardial arginase expression and activity in ischemic-reperfusion myocardium and whether local inhibition of arginase within the ischemic myocardium results in increased NO production and protection against myocardial ischemia-reperfusion. Anesthetized pigs were subjected to coronary artery occlusion for 40 min followed by 4 h reperfusion. The pigs were randomized to intracoronary infusion of vehicle (n = 7), the arginase inhibitor N-hydroxy-nor-L-arginine (nor-NOHA, 2 mg/min, n = 7), the combination of nor-NOHA and the NO synthase inhibitor N^G-monomethyl-L-arginine (L-NMMA, 0.35 mg/min, n = 6) into the jeopardized myocardial area or systemic intravenous infusion of nor-NOHA (2 mg/min, n = 5) at the end of ischemia and start of reperfusion. The infarct size of the vehicle group was 80±4% of the area at risk. Intracoronary nor-NOHA reduced infarct size to 46±5% (P<0.01). Co-administration of L-NMMA abrogated the cardioprotective effect mediated by nor-NOHA (infarct size 72±6%). Intravenous nor-NOHA did not reduce infarct size. Arginase I and II were expressed in cardiomyocytes, endothelial, smooth muscle and poylmorphonuclear cells. There was no difference in cytosolic arginase I or mitochondrial arginase II expression between ischemic-reperfused and non-ischemic myocardium. Arginase activity increased 2-fold in the ischemic-reperfused myocardium in comparison with non-ischemic myocardium. In conclusion, ischemia-reperfusion increases arginase activity without affecting cytosolic arginase I or mitochondrial arginase II expression. Local arginase inhibition during early reperfusion reduces infarct size via a mechanism that is dependent on increased bioavailability of NO.     

Increase of nitrosative stress in patients with eosinophilic pneumonia

Background

Exhaled nitric oxide (NO) production is increased in asthma and reflects the degree of airway inflammation. The alveolar NO concentration (Calv) in interstitial pneumonia is reported to be increased. However, it remains unknown whether NO production is increased and nitrosative stress occurs in eosinophilic pneumonia (EP). We hypothesized that nitrosative stress markers including Calv, inducible type of NO synthase (iNOS), and 3-nitrotyrosine (3-NT), are upregulated in EP.

Methods

Exhaled NO including fractional exhaled NO (FE_NO) and Calv was measured in ten healthy subjects, 13 patients with idiopathic pulmonary fibrosis (IPF), and 13 patients with EP. iNOS expression and 3-NT formation were assessed by immunocytochemistory in BALf cells. The exhaled NO, lung function, and systemic inflammatory markers of the EP patients were investigated after corticosteroid treatment for 4 weeks.

Results

The Calv levels in the EP group (14.4 ± 2.0 ppb) were significantly higher than those in the healthy subjects (5.1 ± 0.6 ppb, p < 0.01) and the IPF groups (6.3 ± 0.6 ppb, p < 0.01) as well as the FE_NO and the corrected Calv levels (all p < 0.01). More iNOS and 3-NT positive cells were observed in the EP group compared to the healthy subject and IPF patient. The Calv levels had significant positive correlations with both iNOS (r = 0.858, p < 0.05) and 3-NT positive cells (r = 0.924, p < 0.01). Corticosteroid treatment significantly reduced both the FE_NO (p < 0.05) and the Calv levels (p < 0.01). The magnitude of reduction in the Calv levels had a significant positive correlation with the peripheral blood eosinophil counts (r = 0.802, p < 0.05).

Conclusions

These results suggested that excessive nitrosative stress occurred in EP and that Calv could be a marker of the disease activity.     

A Promising Approach to Effectively Reduce Cramp Susceptibility in Human Muscles: A Randomized,Controlled Clinical Trial

Background

To investigate if the cramp threshold frequency (CTF) can be altered by electrical muscle stimulation in a shortened position.

Methods

A total of 15 healthy male sport students were randomly allocated to an intervention (IG, n = 10) and a non-treatment control group (CG, n = 5). Calf muscles of both legs in the IG were stimulated equally twice a week over 6 weeks. The protocol was 3×5 s on, 10 s off, 150 µs impulse width, 30 Hz above the individual CTF, and was at 85% of the maximal tolerated stimulation energy. One leg was stimulated in a shortened position, inducing muscle cramps (CT), while the opposite leg was fixated in a neutral position at the ankle, hindering muscle cramps (nCT). CTF tests were performed prior to the first and 96 h after the 6^th (3 w) and 12^th (6 w) training session.

Results

After 3 w, the CTF had significantly (p<0.001) increased in CT calves from 23.3±5.7 Hz to 33.3±6.9 Hz, while it remained unchanged in nCT (pre: 23.6±5.7 Hz, mid: 22.3±3.5 Hz) and in both legs of the CG (pre: 21.8±3.2 Hz, mid: 22.0±2.7 Hz). Only CT saw further insignificant increases in the CTF. The applied stimulation energy (mA² • µs) positively correlated with the effect on the CTF (r = 0.92; p<0.001).

Conclusions

The present study may be useful for developing new non-pharmacological strategies to reduce cramp susceptibility.

Trial Registry

German Clinical Trials Register DRKS00005312     

Metabolic Imaging of Human Kidney Triglyceride Content: Reproducibility of Proton Magnetic Resonance Spectroscopy

Objective

To assess the feasibility of renal proton magnetic resonance spectroscopy for quantification of triglyceride content and to compare spectral quality and reproducibility without and with respiratory motion compensation in vivo.

Materials and Methods

The Institutional Review Board of our institution approved the study protocol, and written informed consent was obtained. After technical optimization, a total of 20 healthy volunteers underwent renal proton magnetic resonance spectroscopy of the renal cortex both without and with respiratory motion compensation and volume tracking. After the first session the subjects were repositioned and the protocol was repeated to assess reproducibility. Spectral quality (linewidth of the water signal) and triglyceride content were quantified. Bland-Altman analyses and a test by Pitman were performed.

Results

Linewidth changed from 11.5±0.4 Hz to 10.7±0.4 Hz (all data pooled, p<0.05), without and with respiratory motion compensation respectively. Mean % triglyceride content in the first and second session without respiratory motion compensation were respectively 0.58±0.12% and 0.51±0.14% (P = NS). Mean % triglyceride content in the first and second session with respiratory motion compensation were respectively 0.44±0.10% and 0.43±0.10% (P = NS between sessions and P = NS compared to measurements with respiratory motion compensation). Bland-Altman analyses showed narrower limits of agreement and a significant difference in the correlated variances (correlation of −0.59, P<0.05).

Conclusion

Metabolic imaging of the human kidney using renal proton magnetic resonance spectroscopy is a feasible tool to assess cortical triglyceride content in humans in vivo and the use of respiratory motion compensation significantly improves spectral quality and reproducibility. Therefore, respiratory motion compensation seems a necessity for metabolic imaging of renal triglyceride content in vivo.     

Assessment of Choroidal Thickness in Healthy and Glaucomatous Eyes Using Swept Source Optical Coherence Tomography

Purpose

To evaluate choroidal thickness (CT) in healthy and glaucomatous eyes using Swept Source Optical Coherence Tomography (SS-OCT).

Methods

A cross-sectional observational study of 216 eyes of 140 subjects with glaucoma and 106 eyes of 67 healthy subjects enrolled in the Diagnostic Innovations in Glaucoma Study. CT was assessed from wide-field (12×9 mm) SS-OCT scans. The association between CT and potential confounding variables including age, gender, axial length, intraocular pressure, central corneal thickness and ocular perfusion pressure was examined using univariable and multivariable regression analyses.

Results

Overall CT was thinner in glaucomatous eyes with a mean (± standard deviation) of 157.7±48.5 µm in glaucoma compared to 179.9±36.1 µm in healthy eyes (P<0.001). The choroid was thinner in both the peripapillary and macular regions in glaucoma compared to controls. Mean peripapillary CT was 154.1±44.1 µm and 134.0±56.9 µm (P<0.001) and macular CT 199.3±46.1 µm and 176.2±57.5 µm (P<0.001) for healthy and glaucomatous eyes respectively. However, older age (P<0.001) and longer axial length (P<0.001) were also associated with thinner choroid and when differences in age and axial length between glaucomatous and healthy subjects were accounted for, glaucoma was not significantly associated with CT. There was also no association between glaucoma severity and CT.

Conclusions

Glaucoma was not associated with CT measured using SS-OCT; however, older age and longer axial length were associated with thinner choroid so should be considered when interpreting CT measurements.     

iNOS-Dependent Increase in Colonic Mucus Thickness in DSS-Colitic Rats

Aim

To investigate colonic mucus thickness in vivo in health and during experimental inflammatory bowel disease.

Methods

Colitis was induced with 5% DSS in drinking water for 8 days prior to experiment, when the descending colonic mucosa of anesthetized rats was studied using intravital microscopy. Mucus thickness was measured with micropipettes attached to a micromanipulator. To assess the contributions of NOS and prostaglandins in the regulation of colonic mucus thickness, the non-selective NOS-inhibitor L-NNA (10 mg/kg bolus followed by 3 mg/kg/h), the selective iNOS-inhibitor L-NIL (10 mg/kg bolus followed by 3 mg/kg/h) and the non-selective COX-inhibitor diclofenac (5 mg/kg) were administered intravenously prior to experiment. To further investigate the role of iNOS in the regulation of colonic mucus thickness, iNOS −/− mice were used.

Results

Colitic rats had a thicker firmly adherent mucus layer following 8 days of DSS treatment than untreated rats (88±2 µm vs 76±1 µm). During induction of colitis, the thickness of the colonic mucus layer initially decreased but was from day 3 significantly thicker than in untreated rats. Diclofenac reduced the mucus thickness similarly in colitic and untreated rats (−16±5 µm vs −14±2 µm). While L-NNA had no effect on colonic mucus thickness in DSS or untreated controls (+3±2 µm vs +3±1 µm), L-NIL reduced the mucus thickness significantly more in colitic rats than in controls (−33±4 µm vs −10±3 µm). The importance of iNOS in regulating the colonic mucus thickness was confirmed in iNOS−/− mice, which had thinner colonic mucus than wild-type mice (35±3 µm vs 50±2 µm, respectively). Furthermore, immunohistochemistry revealed increased levels of iNOS in the colonic surface epithelium following DSS treatment.

Conclusion

Both prostaglandins and nitric oxide regulate basal colonic mucus thickness. During onset of colitis, the thickness of the mucus layer is initially reduced followed by an iNOS mediated increase.     

Opposite Effect of Mast Cell Stabilizers Ketotifen and Tranilast on the Vasoconstrictor Response to Electrical Field Stimulation in Rat Mesenteric Artery

Objectives

We analyzed whether mast cell stabilization by either ketotifen or tranilast could alter either sympathetic or nitrergic innervation function in rat mesenteric arteries.

Methods

Electrical field stimulation (EFS)-induced contraction was analyzed in mesenteric segments from 6-month-old Wistar rats in three experimental groups: control, 3-hour ketotifen incubated (0.1 αmol/L), and 3-hour tranilast incubated (0.1 mmol/L). To assess the possible participation of nitrergic or sympathetic innervation, EFS contraction was analyzed in the presence of non-selective nitric oxide synthase (NOS) inhibitor L-NAME (0.1 mmol/L), α-adrenergic receptor antagonist phentolamine (0.1 µmol/L), or the neurotoxin 6-hydroxydopamine (6-OHDA, 1.46 mmol/L). Nitric oxide (NO) and superoxide anion (O₂ ^.-) levels were measured, as were vasomotor responses to noradrenaline (NA) and to NO donor DEA-NO, in the presence and absence of 0.1 mmol/L tempol. Phosphorylated neuronal NOS (P-nNOS) expression was also analyzed.

Results

EFS-induced contraction was increased by ketotifen and decreased by tranilast. L-NAME increased the vasoconstrictor response to EFS only in control segments. The vasodilator response to DEA-NO was higher in ketotifen- and tranilast-incubated segments, while tempol increased vasodilator response to DEA-NO only in control segments. Both NO and O₂ ^- release, and P-nNOS expression were diminished by ketotifen and by tranilast treatment. The decrease in EFS-induced contraction produced by phentolamine was lower in tranilast-incubated segments. NA vasomotor response was decreased only by tranilast. The remnant vasoconstriction observed in control and ketotifen-incubated segments was abolished by 6-OHDA.

Conclusion

While both ketotifen and tranilast diminish nitrergic innervation function, only tranilast diminishes sympathetic innnervation function, thus they alter the vasoconstrictor response to EFS in opposing manners.     

“Immunonutrition” Has Failed to Improve Peritonitis-Induced Septic Shock in Rodents

Background

Immunonutrition in sepsis, including n-3 poly-unsaturated fatty acids (PUFAs) or L-arginine supplementation, is a controversial issue that has yielded a great number of studies for the last thirty-five years, and the conclusions regarding the quantity and quality of this support in patients are deceiving. The aim of the present experimental study is to investigate the effects of a pretreatment with enteral nutrition enriched with n-3 PUFAs or L-arginine on vascular dysfunctions, inflammation and oxidative stress during septic shock in rats.

Design

Rats were fed with enteral Peptamen^® HN (HN group), Peptamen^® AF containing n-3 PUFAs (AF group) or Peptamen^® AF enriched with L-arginine (AFA group). On day 4, peritonitis by cecal ligation and puncture (CLP) was performed. Rats were resuscitated (H18) once septic shock was established. After a 4-hour resuscitation, vessels and organs were harvested to assess inflammation, superoxide anion, nitric oxide and prostacyclin levels. Ex-vivo vascular reactivity was also performed.

Results

Compared to CLP-AF or CLP-HN groups, 47.6% of CLP-AFA rats died before the beginning of hemodynamic measurements (vs. 8.0% and 20.0% respectively, p<0.05). AF and AFA rats required significantly increased norepinephrine infusion rates to reach the mean arterial pressure objective, compared to CLP-HN rats. Both CLP-AF and CLP-AFA reduced mesenteric resistance arterial contractility, decreased vascular oxidative stress, but increased NF-κB (0.40±0.15 in CLP-AF and 0.69±0.06 in CLP-AFA vs. 0.09±0.03 in SHAM rats and 0.30±0.06 in CLP-HN, ß-actin ratio, p<0.05) and pIκB expression (0.60±0.03 in CLP-AF and 0.94±0.15 in CLP-AFA vs. 0.04±0.01 in SHAM rats and 0.56±0.07 in CLP-HN, ß-actin ratio, p<0.05), nitric oxide and prostacyclin production in septic rats.

Conclusions

Although n-3 PUFAs or L-arginine supplementation exhibited an antioxidant effect, it worsened the septic shock-induced vascular dysfunction. Furthermore, mortality was higher after L-arginine supplementation.     

Comparison of Retinal Nerve Fiber Layer Thickness In Vivo and Axonal Transport after Chronic Intraocular Pressure Elevation in Young versus Older Rats

Purpose

To compare in young and old rats longitudinal measurements of retinal nerve fiber layer thickness (RNFLT) and axonal transport 3-weeks after chronic IOP elevation.

Method

IOP was elevated unilaterally in 2- and 9.5-month-old Brown-Norway rats by intracameral injections of magnetic microbeads. RNFLT was measured by spectral domain optical coherence tomography. Anterograde axonal transport was assessed from confocal scanning laser ophthalmolscopy of superior colliculi (SC) after bilateral intravitreal injections of cholera toxin-B-488. Optic nerve sections were graded for damage.

Results

Mean IOP was elevated in both groups (young 37, old 38 mmHg, p = 0.95). RNFL in young rats exhibited 10% thickening at 1-week (50.9±8.1 µm, p<0.05) vs. baseline (46.4±2.4 µm), then 7% thinning at 2-weeks (43.0±7.2 µm, p>0.05) and 3-weeks (43.5±4.4 µm, p>0.05), representing 20% loss of dynamic range. RNFLT in old rats showed no significant change at 1-week (44.9±4.1 µm) vs. baseline (49.2±5.3 µm), but progression to 22% thinning at 2-weeks (38.0±3.7 µm, p<0.01) and 3-weeks (40.0±6.6 µm, p<0.05), representing 59% loss of dynamic range. Relative SC fluorescence intensity was reduced in both groups (p<0.001), representing 77–80% loss of dynamic range and a severe transport deficit. Optic nerves showed 75–95% damage (p<0.001). There was greater RNFL thinning in old rats (p<0.05), despite equivalent IOP insult, transport deficit and nerve damage between age groups (all p>0.05).

Conclusion

Chronic IOP elevation resulted in severely disrupted axonal transport and optic nerve axon damage in all rats, associated with mild RNFL loss in young rats but a moderate RNFL loss in old rats despite the similar IOP insult. Hence, the glaucomatous injury response within the RNFL depends on age.     

The Supplementary Motor Area Exerts a Tonic Excitatory Influence on Corticospinal Projections to Phrenic Motoneurons in Awake Humans

Introduction

In humans, cortical mechanisms can interfere with autonomic breathing. Respiratory-related activation of the supplementary motor area (SMA) has been documented during voluntary breathing and in response to inspiratory constraints. The SMA could therefore participate in the increased resting state of the respiratory motor system during wake (i.e. "wakefulness drive to breathe").

Methods

The SMA was conditioned by continuous theta burst magnetic stimulation (cTBS, inhibitory) and 5 Hz conventional rTMS (5 Hz, excitatory). The ensuing effects were described in terms of the diaphragm motor evoked response (DiMEPs) to single-pulse transcranial magnetic stimulation over the motor cortex. DiMEPs were recorded at baseline, and at 3 time-points ("post1", "post2", "post3") up to 15 minutes following conditioning of the SMA.

Results

cTBS reduced the amplitude of DiMEPs from 327.5±159.8 µV at baseline to 243.3±118.7 µV, 217.8±102.9 µV and 240.6±123.9 µV at post 1, post 2 and post 3, respectively (F = 6.341, p = 0.002). 5 Hz conditioning increased the amplitude of DiMEPs from 184.7±96.5 µV at baseline to 270.7±135.4 µV at post 3 (F = 4.844, p = 0.009).

Conclusions

The corticospinal pathway to the diaphragm can be modulated in both directions by conditioning the SMA. This suggests that the baseline respiratory activity of the SMA represents an equipoise from which it is possible to move in either direction. The resting corticofugal outflow from the SMA to phrenic motoneurones that this study evidences could putatively contribute to the wakefulness drive to breathe.     

Assessment of Clinical Signs of Liver Cirrhosis Using T1 Mapping on Gd-EOB-DTPA-Enhanced 3T MRI

Objectives

To assess the differences between normal and cirrhotic livers by means of T1 mapping of liver parenchyma on gadoxetic acid (Gd-EOB-DTPA)-enhanced 3 Tesla (3T) MR imaging (MRI).

Methods

162 patients with normal (n = 96) and cirrhotic livers (n = 66; Child-Pugh class A, n = 30; B, n = 28; C, n = 8) underwent Gd-EOB-DTPA-enhanced 3T MRI. To obtain T1 maps, two TurboFLASH sequences (TI = 400 ms and 1000 ms) before and 20 min after Gd-EOB-DTPA administration were acquired. T1 relaxation times of the liver and the reduction rate between pre- and post-contrast enhancement images were measured.

Results

The T1 relaxation times for Gd-EOB-DTPA-enhanced MRI showed significant differences between patients with normal liver function and patients with Child-Pugh class A, B, and C (p < 0.001). The T1 relaxation times showed a constant significant increase from Child-Pugh class A up to class C (Child-Pugh class A, 335 ms ± 80 ms; B, 431 ms ± 75 ms; C, 557 ms ± 99 ms; Child-Pugh A to B, p < 0.001; Child-Pugh A to C, p < 0.001; Child-Pugh B to C, p < 0.001) and a constant decrease of the reduction rate of T1 relaxation times (Child-Pugh class A, 57.1% ± 8.8%; B, 44.3% ± 10.2%, C, 29.9% ± 6.9%; Child-Pugh A to B, p < 0.001; Child-Pugh A to C,p < 0.001; Child-Pugh B to C, p < 0.001).

Conclusion

Gd-EOB-DTPA-enhanced T1 mapping of the liver parenchyma may present a useful method for determining severity of liver cirrhosis.     

Magnetic Resonance Imaging Determined Visceral Fat Reduction Associates with Enhanced IL-10 Plasma Levels in Calorie Restricted Obese Subjects

Background

Obesity is characterized by a low grade chronic inflammation state. Indeed circulating pro-inflammatory cytokines, such as TNF-α and IL-6, are elevated in obese subjects, while anti-inflammatory cytokines, such as IL-10, appear to be reduced. Cytokines profile improves after weight loss, but how visceral or subcutaneous fat loss respectively affect pro- or anti-inflammatory cytokines plasma levels has not been precisely assessed. Therefore in the present study we correlated changes in circulating cytokine profile with quantitative changes in visceral and subcutaneous adipose tissue depots measured by an ad hoc Magnetic Resonance Imaging (MRI) protocol before and after weight loss.

Materials and Methods

In 14 obese subjects, MRI determination of visceral and subcutaneous fat and plasma glucose, insulin, TNF-α IL-6, and IL-10 measurements were performed before and after a caloric restriction induced weight loss of at least 5% of the original body weight.

Results

Weight loss improved insulin sensitivity (QUICKI Index: 0.35±0.03 vs 0.37±0.04; P<0.05), increased IL-10 (3.4±1.9 vs 4.6±1.0 pg/mL; P<0.03), and reduced TNF-α and IL-6 plasma levels (2.5±1.3 vs 1.6±1.5 pg/mL, P<0.0015, 2.3±0.4 vs 1.6±0.6 pg/mL, P<0.02 respectively). A significant correlation was observed between the amount of visceral fat loss and the percentage reduction in both TNF-α (r = 0.56, p<0.05) and IL-6 (r = 0.19 p<0.05) plasma levels. In a multiple regression analysis, the amount of visceral fat loss independently correlated with the increase in IL-10 plasma levels.

Conclusion

The reduction in visceral adipose tissue is the main driver of the improved inflammatory profile induced by weight loss.