Placental expression of striatin & endothelial nitric oxide synthase in women with & without pre-eclampsia.

Background & objectives: Striatin is a multi-domain scaffolding protein essential for activating endothelial nitric oxide synthase (eNOS). However, its role in pre-eclampsia remains use explored. Hence, this study aimed to investigate the association between striatin and eNOS in regulating nitric oxide (NO) production in the placenta of women with and without pre-eclampsia. Methods: Forty pregnant women each without (controls) and with pre-eclampsia (cases) were enrolled in the study. Blood striatin and NO concentrations were detected by the ELISA. Protein expression of striatin, phosphorylated eNOS (peNOS), inducible NOS (iNOS) and phosphorylated NF-κB were measured in the placental tissues by Western blot. Twenty four hour urinary protein and serum urea, uric acid and creatinine were analyzed as an autoanalyzer. Placental histology was analyzed by haematoxylin and eosin staining. Results: Compared to normotensive pregnant women, the levels of serum NO and striatin were decreased in pre-eclamptic women. The protein expression of striatin and peNOS was significantly reduced (P<0.05) while p65NF-κB and iNOS were upregulated considerably (P<0.05) in the placenta of cases compared to controls. Interpretation & conclusions: Our results show for the first time that decreased striatin expression was associated with decreased peNOS protein expression in the placental tissue of pre-eclamptic women. Interestingly, no significant difference was found in blood striatin or NO levels between controls and cases. Thus, therapies that improve placental striatin expression are attractive possibilities, both for prevention as well as treatment of endothelial dysfunction in pre-eclampsia.

Direct measurement of nitric oxide (NO) production rates from enzymes using ozone-based gas-phase chemiluminescence (CL).

Nitric oxide (NO) chemiluminescence detectors (CLDs) are specialized and sensitive spectroscopic instruments capable of directly measuring NO flux rates. NO CLDs have been instrumental in the characterization of mammalian nitrite-dependent NO synthases. However, no detailed description of NO flux analysis using NO CLD is available. Herein, a detailed review of the NO CL methodology is provided with guidelines for measuring NO-production rates from aqueous samples, such as isolated enzymes or protein homogenates. Detailed description of the types of signals one can encounter, data processing, and potential pitfalls related to NO flux measurements will also be covered.

Distribution of nitric oxide in the rock cavy (Kerodon rupestris) brain II: The brainstem.

In a recent paper, we described the distribution of Nitric oxide (NO) in the diencephalon of the rock cavy (Kerodon rupestris). This present paper follows this work, showing the distribution of NO synthesizing neurons in the rock cavy's brainstem. For this, we used immunohistochemistry against the neuronal form of nitric oxide synthase (NOS) and NADPH diaphorase histochemistry. In contrast to the diencephalon in the rock cavy, where the NOS neurons were seen to be limited to some nuclei in the thalamus and hypothalamus, the distribution of NOS in the brainstem is widespread. Neurons immunoreactive to NOS (NOS-ir) were seen as rostral as the precommissural nuclei and as caudal as the caudal and gelatinous parts of the spinal trigeminal nucleus. Places such as the raphe nuclei, trigeminal complex, superior and inferior colliculus, oculomotor complex, periaqueductal grey matter, solitary tract nucleus, laterodorsal tegmental nucleus, pedunculopontine tegmental, and other nuclei of the reticular formation are among the locations with the most NOS-ir neurons. This distribution is similar, but with some differences, to those described for other rodents, indicating that NO also has an important role in rock cavy's physiology.

Bacterial-like nitric oxide synthase in the haloalkaliphilic archaeon Natronomonas pharaonis.

Bacterial nitric oxide (NO) synthases (bNOS) play diverse and important roles in microbial physiology, stress resistance, and virulence. Although bacterial and mammalian NOS enzymes have been well-characterized, comparatively little is known about the prevalence and function of NOS enzymes in Archaea. Analysis of archaeal genomes revealed that highly conserved bNOS homologs were restricted to members of the Halobacteria. Of these, Natronomonas pharaonis NOS (npNOS) was chosen for further characterization. NO production was confirmed in heterologously expressed His-tagged npNOS by coupling nitrite production from N-hydroxy-L-arginine in an H2O2-supported reaction. Additionally, the nos gene was successfully targeted and disrupted to create a Nmn. pharaonis nos mutant by adapting an established Natrialba magadii transformation protocol. Genome re-sequencing of this mutant revealed an additional frameshift in a putative cation-acetate symporter gene, which could contribute to altered acetate metabolism in the nos mutant. Inactivation of Nmn. pharaonis nos was also associated with several phenotypes congruent with bacterial nos mutants (altered growth, increased oxygen consumption, increased pigment, increased UV susceptibility), suggesting that NOS function may be conserved between bacteria and archaea. These studies are the first to describe genetic inactivation and characterization of a Nmn. pharaonis gene and provides enhanced tools for probing its physiology.

Pro-inflammatory mediators and signaling proteins in the decidua of pre-eclampsia.

OBJECTIVE: To evaluate the role of CD68+ macrophages and inflammatory/signaling proteins in the decidua of singleton pregnancies with late-onset pre-eclampsia. PATIENTS AND METHODS: This study was designed as a prospective case-control study. Decidual tissue samples were obtained from twenty healthy pregnant women as a control group and twenty pregnant women with late-onset pre-eclampsia showing severe symptoms as the study group. We examined the abundance of CD68+ macrophages in both groups using flow cytometry. Protein and mRNA expression levels of inflammatory/signaling proteins, including inducible nitric oxide synthase, nuclear factor-κB inhibitor α, cyclooxygenase-2, and phosphorylated c-Jun N-terminal kinase, in the decidua of both groups were measured using Western blotting and Reverse Transcription-Polymerase Chain Reaction, respectively. Student's t-tests were performed for statistical analysis. RESULTS: The numbers of CD68+ macrophages were similar in the study and control groups (p=0.47). However, the levels of inducible nitric oxide synthase, nuclear factor-κB, cyclooxygenase-2, and phosphorylated c-Jun N-terminal kinase were significantly increased in the study group. Therefore, pro-inflammatory mediators and signaling proteins in the decidua during pre-eclampsia may be related to the pathogenesis of pre-eclampsia. CONCLUSIONS: Pre-eclampsia-induced alterations in the expression of inflammatory/signaling proteins in the decidua during singleton pregnancies may play a critical role in the pathogenesis of pre-eclampsia.

Induction of brain injury and depression in rats by chronic unpredictable stress associated with the augmentation of nitrosative stress and apoptosis / Inducción de lesión cerebral y depresión en ratas por estrés crónico impredecible asociado con el aumento del estrés nitrosativo y la apoptosis

SUMMARY: Repeated stress is a risk factor for memory impairment and neurological abnormalities in both humans and animals. We sought to investigate the extent of (i) brain tissue injury; (ii) nitrosative and oxidative stress in brain tissue homogenates; (iii) apoptotic and survival biomarkers in brain tissue homogenates; and (iv) immobility and climbing abilities, induced over a period of three weeks by chronic unpredictable stress (CUS). Wistar rats were either left untreated (Control group) or exposed to a variety of unpredictable stressors daily before being sacrificed after 3 weeks (model group). Assessment of depression-like behavior was performed and animals were then culled and harvested brain tissues were stained with basic histological staining and examined under light microscopy. In addition, brain tissue homogenates were prepared and assayed for these parameters; inducible nitric oxide synthase (iNOS), malondialdehyde (MDA), superoxide dismutase (SOD), caspase-3, and B-cell lymphoma 2 (Bcl-2). Histology images showed CUS induced profound damage to the cerebral cortex as demonstrated by severe neuronal damage with shrunken cells, disrupted atrophic nuclei, perineuronal vacuolation and swollen glial cells. CUS also significantly (p<0.05) induced iNOS, MDA, and caspase-3, whereas SOD and Bcl-2 brain tissue levels were inhibited by CUS. In addition, data from the depression-like behavior, forced swimming test showed significant (p<0.05) increase in animal immobility and decrease in climbing ability in the model group of rats. Thus, here we demonstrated a reliable rat model of chronic stress-induced brain injury, which can further be used to investigate beneficial drugs or agents used for a period of three weeks to protect against CUS-induced brain damage.

RESUMEN: El estrés crónico es un factor de riesgo para el deterioro de la memoria y las anomalías neurológicas tanto en humanos como en animales. Intentamos investigar el alcance de lesión del tejido cerebral; (ii) estrés nitrosativo y oxidativo en homogeneizados de tejido cerebral; (iii) biomarcadores apoptóticos y de supervivencia en homogeneizados de tejido cerebral; y (iv) inmovilidad y habilidades de escalada, inducidas durante un período de tres semanas por estrés crónico impredecible (ECI). Se dejaron sin tratamiento (grupo control) ratas Wistar, o se expusieron a una variedad de factores estresantes impredecibles diariamente antes de ser sacrificadas después de 3 semanas (grupo modelo). Se realizó una evaluación del comportamiento similar a la depresión y luego se sacrificaron los animales y se tiñeron los tejidos cerebrales con tinción histológica básica y se examinaron con microscopía óptica. Además, se prepararon homogeneizados de tejido cerebral y se analizaron los siguientes parámetros; óxido nítrico sintasa inducible (iNOS), malondialdehído (MDA), superóxido dismutasa (SOD), caspasa- 3 y linfoma de células B 2 (Bcl-2). Las imágenes histológicas mostraron que el CUS indujo un daño profundo en la corteza cerebral como lo demuestra el daño neuronal severo con células encogidas, núcleos atróficos alterados, vacuolación perineuronal y células gliales inflamadas. ECI también indujo significativamente (p <0,05) iNOS, MDA y caspase-3, mientras que los niveles de tejido cerebral SOD y Bcl-2 fueron inhibidos por ECI. Además, los datos del comportamiento similar a la de- presión, la prueba de natación forzada mostró un aumento significativo (p <0,05) en la inmovilidad animal y una disminución en la capacidad de escalada en el grupo modelo de ratas. Por lo tanto, aquí demostramos un modelo confiable de daño cerebral crónico en rata inducido por el estrés, que se puede utilizar para investigar medicamentos o agentes beneficiosos usados durante un período de tres semanas para proteger el daño cerebral inducido por ECI.

Simulated vehicle exhaust exposure (SVEE) in rats impairs renal mitochondrial function.

PURPOSE:  Vehicle exhaust emissions primarily comprise of nitrogen, oxygen, water, CO2, NO2, CO, hydrocarbons and particulate matter. While adverse effects of hydrocarbon and particulate matter on cardiovascular functions are known, the effect of pro-oxidants CO2, NO2 and CO are not clear. METHODS:  Here, using an animal model of a simulated mixture of pro-oxidants (0.04% CO2, 0.9 ppm NO2 and 3 ppm CO with air as a base), we examined the effect of simulated vehicle exhaust exposure (SVEE) on various cardiovascular parameters. Male Sprague-Dawley rats were exposed to SVEE or ambient air (Control: CON) for 30 min/day for 2 weeks. Thereafter, systolic and diastolic blood pressure, heart rate and glomerular filtration rate were measured. Later, rats were sacrificed, blood plasma and kidneys were collected. RESULTS:  The systolic and diastolic blood pressure, heart rate and glomerular filtration rate remained unchanged. Plasma corticosterone increased in SVEE rats when compared to CON group. Plasma 8-isoprostane, a systemic marker of oxidative stress, increased while total antioxidant capacity decreased in SVEE but not in CON. Kidney cortical tissue homogenates exhibited increase in superoxide, hydrogen peroxide and protein carbonylation in SVEE but not CON, all indicative of heightened oxidative stress. Renal cortical mitochondrial SOD activity was significantly reduced in SVEE than CON. CONCLUSION:  Significant decline in mitochondrial respiration and oxygen consumption was observed, in addition to low ATP, reduced ATP synthase and cytochrome C oxidase levels, as well as accelerated mitochondrial fission, and reduced fusion processes, were observed in SVEE than CON rats, all indicative of renal mitochondrial impairment.

Comparative study of the distribution of NOS-positive neurons in pigeon intestine.

Nitric oxide (NO) plays an important role in regulating gut motility, mucosal barrier function and secretions in the enteric nervous system. Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) staining has been used to identify nitrergic neurons of the enteric nervous system in different species. However, NADPH-d staining lacks specificity because it also reflects the presence of enzymes other than nitric oxide synthase (NOS). Therefore, NOS immunohistochemistry techniques are needed to test for nitrergic neurons in the avian gut. In the present work, the morphology, density and size of NOS-positive neurons in the duodenum, jejunum, ileum, caecum and rectum myenteric plexus of adult pigeons were investigated using NOS immunohistochemistry and whole-mount preparations techniques. The density of NOS-positive ganglion was highest in the ileum, similar to the caecum and rectum, and the lowest staining levels were observed in the duodenum. The staining intensity of NOS-positive neurons in the duodenum, jejunum and ileum was dark, followed by the rectal regions, with weak staining in the caecum. These results suggested that NOS immunohistochemistry and whole-mount preparation techniques provide an effective assessment method of the ganglia in the pigeon intestinal myenteric nerve plexus and are more accurate for cell counting compared with conventional sections.

Dihydrotanshinone I Alleviates Spinal Cord Injury via Suppressing Inflammatory Response, Oxidative Stress and Apoptosis in Rats.

BACKGROUND Spinal cord injury (SCI) is a serious nervous system injury, causing extremely low quality of life and immensurable economic losses. However, there is few therapies that can effectively cure the injury. The goal of the present study was to explore the potential therapeutic effects of dihydrotanshinone I (DI) for SCI and the involving mechanism. MATERIAL AND METHODS A SCI rat model was structured to investigate the effects of DI on recovery of SCI. Tarlov's scale was employed to assess the neuronal function and histopathological examination was carried out by hematoxylin and eosin staining. In addition, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-1ß, inducible nitric oxide synthase (iNOS), total oxidant status (TOS) and total antioxidant status (TAS) levels were detected. Tunel assay and western blot analysis were performed to evaluate cell apoptosis. Furthermore, western blot assay was used to measure the protein expressions. RESULTS The results demonstrated that the treatment of DI alleviated the pathological damage induced by SCI and promoted the neuronal functional recovery. DI suppressed TNF-alpha, IL-1ß, IL-6, iNOS, and TOS levels while improved the TAS level. Moreover, increased cell apoptosis in SCI rats was inhibited by administration of DI. Most importantly, DI reserved the soaring of TLR4, MyD88, HMGB1, and NOX4 level after induction of SCI. Thus, the observation revealed that the HMGB1/TLR4/NOX4 pathway may be involved in the protective effects of DI on SCI. CONCLUSIONS In conclusion, the findings suggest that DI alleviates SCI by restraining secretion of inflammatory factors, and occurrence of oxidative stress and apoptosis in vivo. DI may be developed into an effective alternative therapy for SCI in clinic.

Comparative study on the efficacy of non-steroidal, steroid and non-use of anti-inflammatory in the treatment of acute epidemic conjunctivitis.

PURPOSE: To evaluate the effects of prednisolone against sodium diclofenac both with ciprofloxacin compared to artificial tears on the symptoms and signs of acute viral conjunctivitis. METHODS: Study included 37 patients diagnosed with acute conjunctivitis and distributed by three groups: A (1% prednisolone acetate + ciprofloxacin (0.3%); B (Sodium diclofenac (0.1%) + ciprofloxacin (0.3%) and C (artificial tears + ciprofloxacin (0.3%). Patients received medication 6/6 hours daily. Signs and symptoms (e.g. lacrimation, burning, photophobia, etc.) were scored at baseline and on the first, third, fifth and seventh days and in the end of treatment using a standardized questionnaire and slit lamp anterior segment examination. RESULTS: All three groups demonstrated an improvement in the signs and symptoms of conjunctivitis in their follow-up visits. There was no significant difference in symptom and sign scores between Group A and B and B and C in the study visits ( p >0.05). However, the comparison between groups A and C showed a clinical trend (p=0.05) on third evaluation suggesting better clinical action using the corticosteroids. CONCLUSION: The prednisolone acetate was not superior to the use of sodium diclofenac or artificial tears in relieving the signs and symptoms of viral conjunctivitis.

Changes in nitric oxide synthase levels are associated with impaired cardiac function and tolerance to ischemia-reperfusion injury in male rats with transient congenital hypothyroidism.

Transient congenital hypothyroidism (TCH) has long-lasting consequences on the cardiovascular system during adulthood. The aim of this study was to determine whether nitric oxide (NO) and NO-producing enzymes are involved in impaired cardiac function as well as decreased tolerance to ischemia-reperfusion (IR) injury in adult male rats with TCH. Pregnant rats were divided into control and hypothyroid groups. Male offspring rats were categorized in control and hypothyroid (TCH) groups at week 16. Levels of NOx (nitrate+nitrite) and neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) were measured in hearts of rats and isolated perfused hearts from both groups were subjected to IR. Levels of NOx and NOSs were also measured in both groups after ischemia. Compared with controls, heart NOx levels were higher at baseline (48.0 ± 4.9 vs. 35.0 ± 2.6 µmol/L; P = 0.034) and following IR (103.6 ± 4.2 vs. 70.2 ± 2.7 µmol/L; P < 0.001) in rat with TCH. At baseline, compared with controls, heart iNOS and nNOS levels were significantly higher in rats with TCH (6.12 ± 0.34 vs. 4.78 ± 0.27 ng/mg protein; P = 0.008 for iNOS and 4.87 ± 0.28 vs. 3.55 ± 0.23 ng/mg protein; P = 0.003 for nNOS). Following IR, in rats with TCH, heart iNOS levels increased (11.75 ± 2.02 vs. 6.12 ± 0.34, ng/mg protein; P = 0.015) whereas nNOS level decreased (4.10 ± 0.25 vs. 4.87 ± 0.28 ng/mg protein; P = 0.063). Adverse effects of TCH on cardiac function are associated with increased ratio of iNOS/eNOS; in addition, increased heart nNOS levels are involved in impaired cardiac function while its decrease is associated with decreased tolerance to IR injury.

The Autonomic Innervation and Uterine Telocyte Interplay in Leiomyoma Formation.

The autonomic innervation of the uterus is involved in multiple pathophysiological processes in both humans and animals. Pathological conditions such as adenomyosis or inflammatory pelvic disease are usually accompanied by significant alterations in uterine innervation. In the current study, we focused on autonomic innervation of uterine fibroids, the identification of recently described interstitial cells, telocytes, and the possible interplay between these structures. In this work, uterine telocytes were identified by immunopositivity for c-kit, CD34, and PDGFRα. Nerves were revealed by immunolabeling for neuronal markers: protein gene product PGP 9.5, inducible nitric oxide synthase (iNOS), choline acetyltransferase (ChAT), and tyrosine hydroxylase (TH). The gross organization of myometrial tissue has been analyzed by routine histology. The results demonstrated that the density of iNOS and ChAT-immunopositive neurons in the uterine fibroids was higher than that in the control samples. The density of telocytes in the fibrosis foci was lower than that in the normal myometrium. Our results suggest that autonomic innervation and telocytes are involved in the microenvironment imbalance characteristic of uterine leiomyoma. Since NOS-positive nerves play an important role in oxidative stress modulation, they might lead to a decrease in the number of telocytes, which are crucial components in the pathogenesis of leiomyoma formation.

Pattern of nitrergic cells and fibers organization in the central nervous system of the Australian lungfish, Neoceratodus forsteri (Sarcopterygii: Dipnoi).

The Australian lungfish Neoceratodus forsteri is the only extant species of the order Ceratodontiformes, which retained most of the primitive features of ancient lobe finned-fishes. Lungfishes are the closest living relatives of land vertebrates and their study is important for deducing the neural traits that were conserved, modified, or lost with the transition from fishes to land vertebrates. We have investigated the nitrergic system with neural nitric oxide synthase (NOS) immunohistochemistry and NADPH-diaphorase (NADPH-d) histochemistry, which yielded almost identical results except for the primary olfactory projections and the terminal and preoptic nerve fibers labeled only for NADPH-d. Combined immunohistochemistry was used for simultaneous detection of NOS with catecholaminergic, cholinergic, and serotonergic structures, aiming to establish accurately the localization of the nitrergic elements and to assess possible interactions between these neurotransmitter systems. The results demonstrated abundant nitrergic cells in the basal ganglia, amygdaloid complex, preoptic area, basal hypothalamus, mesencephalic tectum and tegmentum, laterodorsal tegmental nucleus, reticular formation, spinal cord, and retina. In addition, low numbers of nitrergic cells were observed in the olfactory bulb, all pallial divisions, lateral septum, suprachiasmatic nucleus, prethalamic and thalamic areas, posterior tubercle, pretectum, torus semicircularis, cerebellar nucleus, interpeduncular nucleus, the medial octavolateral nucleus, nucleus of the solitary tract, and the dorsal column nucleus. Colocalization of NOS and tyrosine hydroxylase was observed in numerous cells of the ventral tegmental area/substantia nigra complex. Comparison with other vertebrates, using a neuromeric analysis, reveals that the nitrergic system of Neoceratodus shares many neuroanatomical features with tetrapods and particularly with amphibians.

Local anaesthetics upregulate nitric oxide generation in cord blood and adult human neutrophils.

Nitric oxide (NO) generation by systemic neonatal neutrophils is not clarified. It is also not known whether local anaesthetics (LAs) transferred to the fetal systemic circulation following maternal epidural blockade may affect this process. In the present study, NO generation was evaluated in neutrophils from cord blood (CB, n = 11) and adult blood (n = 10) following exposure to bupivacaine (0.0005, 0.005, 1 mM), lidocaine (0.002, 0.02, 4 mM) and ropivacaine (0.0007, 0.007, 1.4 mM) using flow cytometry, as well as indirectly by determining nitrite concentrations in cell incubation media. To determine the role of NO synthase (NOS) isoforms in NO generation following exposure to LAs, experiments were repeated in the presence of the NOS inhibitors, NG-nitro-L-arginine methyl ester and aminoguanidine; in addition, the expression of NOS isoforms was analysed. CB neutrophils produced less NO than adult neutrophils. LAs, especially ropivacaine and lidocaine, stimulated neutrophil NO generation, but in CB neutrophils this effect was negligible at clinically relevant drug concentrations. A mechanism involving NOS activity was responsible for the observed phenomena. In conclusion, LAs are able to upregulate neutrophil NO production, but in neonates this effect is likely to be clinically insignificant.

Comparative study on the efficacy of non-steroidal, steroid and non-use of anti-inflammatory in the treatment of acute epidemic conjunctivitis

Abstract Purpose To evaluate the effects of prednisolone against sodium diclofenac both with ciprofloxacin compared to artificial tears on the symptoms and signs of acute viral conjunctivitis. Methods Study included 37 patients diagnosed with acute conjunctivitis and distributed by three groups: A (1% prednisolone acetate + ciprofloxacin (0.3%); B (Sodium diclofenac (0.1%) + ciprofloxacin (0.3%) and C (artificial tears + ciprofloxacin (0.3%). Patients received medication 6/6 hours daily. Signs and symptoms (e.g. lacrimation, burning, photophobia, etc.) were scored at baseline and on the first, third, fifth and seventh days and in the end of treatment using a standardized questionnaire and slit lamp anterior segment examination. Results All three groups demonstrated an improvement in the signs and symptoms of conjunctivitis in their follow-up visits. There was no significant difference in symptom and sign scores between Group A and B and B and C in the study visits ( p >0.05). However, the comparison between groups A and C showed a clinical trend (p=0.05) on third evaluation suggesting better clinical action using the corticosteroids. Conclusion The prednisolone acetate was not superior to the use of sodium diclofenac or artificial tears in relieving the signs and symptoms of viral conjunctivitis.

Ketogenic diet enhances neurovascular function with altered gut microbiome in young healthy mice.

Neurovascular integrity, including cerebral blood flow (CBF) and blood-brain barrier (BBB) function, plays a major role in determining cognitive capability. Recent studies suggest that neurovascular integrity could be regulated by the gut microbiome. The purpose of the study was to identify if ketogenic diet (KD) intervention would alter gut microbiome and enhance neurovascular functions, and thus reduce risk for neurodegeneration in young healthy mice (12-14 weeks old). Here we show that with 16 weeks of KD, mice had significant increases in CBF and P-glycoprotein transports on BBB to facilitate clearance of amyloid-beta, a hallmark of Alzheimer's disease (AD). These neurovascular enhancements were associated with reduced mechanistic target of rapamycin (mTOR) and increased endothelial nitric oxide synthase (eNOS) protein expressions. KD also increased the relative abundance of putatively beneficial gut microbiota (Akkermansia muciniphila and Lactobacillus), and reduced that of putatively pro-inflammatory taxa (Desulfovibrio and Turicibacter). We also observed that KD reduced blood glucose levels and body weight, and increased blood ketone levels, which might be associated with gut microbiome alteration. Our findings suggest that KD intervention started in the early stage may enhance brain vascular function, increase beneficial gut microbiota, improve metabolic profile, and reduce risk for AD.

Detection of Nitric Oxide by Membrane Inlet Mass Spectrometry.

Membrane inlet mass spectrometry (MIMS) is a reproducible and reliable method for the measurement of nitric oxide in aqueous solution with a lower limit of detection of 10 nM and a linear response to 50 µM. MIMS utilizes a semipermeable membrane to partition analytes based on physicochemical properties from the bulk sample into the mass spectrometer. Silastic tubing allows the introduction of small gaseous molecules including nitric oxide (NO) into the high vacuum of a mass spectrometer. We describe the measurement of NO generated chemically from nitrite and MAHMA NONOate as well as enzymatically by nitric oxide synthase (NOS).

Spectrophotometric assays for measuring redox biomarkers in blood and tissues: the NADPH network.

Nicotinamide adenine dinucleotide (NAD+/NADH) along with its phosphorylated form (NADP+/NADPH) are two molecules ubiquitously present in all organisms, and they play key roles as cofactors in fundamental catabolic and anabolic processes, respectively. The oxidation of NADPH to NADP+ initiates a cascade of reactions, where a network of molecules is implicated. The molecules of this cascade form a network with eminent translational potential in redox metabolism. A special point of interest is that spectrophotometric assays have been developed both for NADH/NADPH and the molecules directly regulated by them. Therefore, crucial molecules of the NADPH-dependent redox network can be measured, and the results can be used to assess the bioenergetic and/or oxidative stress status. The main aim of this review is to collectively present the NADPH-related molecules, namely NADPH, NADH, NAD+ kinase, NADPH oxidase, peroxiredoxin, thioredoxin, thioredoxin reductase, and nitric oxide synthase, that can be measured in blood and tissues with the use of a spectrophotometer, which is probably the most simple, inexpensive and widely used tool in biochemistry. We are providing the researchers with reliable and valid spectrophotometric assays for the measurement of the most important biomarkers of the NADPH network in blood and other tissues, thus allowing the opportunity to follow the redox changes in response to a stimulus.

Portacaval shunting causes differential mitochondrial superoxide production in brain regions.

The portacaval shunting (PCS) prevents portal hypertension and recurrent bleeding of esophageal varices. On the other hand, it can induce chronic hyperammonemia and is considered to be the best model of mild hepatic encephalopathy (HE). Pathogenic mechanisms of HE and dysfunction of the brain in hyperammonemia are not fully elucidated, but it was originally suggested that the pathogenetic defect causes destruction of antioxidant defense which leads to an increase in the production of reactive oxygen species (ROS) and the occurrence of oxidative stress. In order to gain insight into the pathogenic mechanisms of HE in the brain tissue, we investigated the effects of PCS in rats on free radicals production and activity levels of antioxidant and prooxidant enzymes in mitochondria isolated from different brain areas. We found that O2·- production, activities of Mn-superoxide dismutase (Mn-SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione transferase (GT), nitric oxide synthase (NOS), and levels of carbonylated proteins differed between the four brain regions both in the amount and response to PCS. In PCS rats, Mn-SOD activity in the cerebellum was significantly decreased, and remained unchanged in the neocortex, hippocampus and striatum compared with that in sham-operated animals. Among the four brain regions in control rats, the levels of the carbonyl groups in mitochondrial proteins were maximal in the cerebellum. 4 weeks after PCS, the content of carbonylated proteins were higher only in mitochondria of this brain region. Under control conditions, O2·- production by submitochondrial particles in the cerebellum was significantly higher than in other brain regions, but was significantly increased in each brain region from PCS animals. Indeed, the production of O2·- by submitochondrial particles correlated with mitochondrial ammonia levels in the four brain regions of control and PCS-animals. These findings are the first to suggest that in vivo levels of ammonia in the brain directly affect the rate of mitochondrial O2·- production.

NADPH-diaphorase-positive neurons in the human inferior colliculus: morphology, distribution and clinical implications.

Using the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reaction with nitroblue tetrazolium, we provided a detailed investigation of the distribution, dimensional characteristics and morphology of NADPH-d-positive neurons in the three main subdivisions of the human inferior colliculus (IC): central nucleus, pericentral nucleus, and external nucleus. In accordance with their perikaryal diameter, dendritic and axonal morphology, these neurons were categorized as large (averaging up to 45 µm in diameter), medium (20-30 µm), small (13-16 µm) and very small (7-10 µm). Their morphological differences could contribute to varying functionality and processing capacity. Our results support the hypothesis that large and medium NADPH-d-positive cells represent projection neurons, while the small cells correspond to interneurons. Heretofore, the very small NADPH-d-positive neurons have not been described in any species. Their functions-and if they are, indeed, the smallest neurons in the IC of humans-remain to be clarified. Owing to their location, we posit that they are interneurons that connect the large NADPH-d-positive neurons and thereby serve as an anatomical substrate for information exchange and processing before feeding forward to higher brain centers. Our results also suggest that the broad distribution of nitric oxide (NO) synthesis in the human IC is closely tied to the neuromodulatory action of NO on collicular neurotransmitters such as GABA and glutamate, and to calcium-binding proteins such as parvalbumin. A deeper understanding of the relationship between NADPH-d-positive fibers in all IC connections and their co-localization with other neurotransmitters and calcium-binding proteins will assist in better defining the function of NO in the context of its interplay with the cerebral cortex, the sequelae of the aging process and neurodegenerative disorders.