Dietary nitrate supplementation and cognitive health: the nitric oxide-dependent neurovascular coupling hypothesis.

Diet is currently recognized as a major modifiable agent of human health. In particular, dietary nitrate has been increasingly explored as a strategy to modulate different physiological mechanisms with demonstrated benefits in multiple organs, including gastrointestinal, cardiovascular, metabolic, and endocrine systems. An intriguing exception in this scenario has been the brain, for which the evidence of the nitrate benefits remains controversial. Upon consumption, nitrate can undergo sequential reduction reactions in vivo to produce nitric oxide (•NO), a ubiquitous paracrine messenger that supports multiple physiological events such as vasodilation and neuromodulation. In the brain, •NO plays a key role in neurovascular coupling, a fine process associated with the dynamic regulation of cerebral blood flow matching the metabolic needs of neurons and crucial for sustaining brain function. Neurovascular coupling dysregulation has been associated with neurodegeneration and cognitive dysfunction during different pathological conditions and aging. We discuss the potential biological action of nitrate on brain health, concerning the molecular mechanisms underpinning this association, particularly via modulation of •NO-dependent neurovascular coupling. The impact of nitrate supplementation on cognitive performance was scrutinized through preclinical and clinical data, suggesting that intervention length and the health condition of the participants are determinants of the outcome. Also, it stresses the need for multimodal quantitative studies relating cellular and mechanistic approaches to function coupled with behavior clinical outputs to understand whether a mechanistic relationship between dietary nitrate and cognitive health is operative in the brain. If proven, it supports the exciting hypothesis of cognitive enhancement via diet.

In Utero Treatment of Obstructive Ureterocele.

Lower urinary tract obstruction consists of a heterogeneous group of conditions in which the normal urethral egress of urine from the fetal bladder is impaired. The most frequent diagnoses are posterior urethral valves, urethral atresia, and less common obstructive ureterocele. We report a case of a fetus with prenatal diagnosis of obstructive ureterocele who presented progressive bilateral hydronephrosis. A fetal cystoscopy with laser ablation was performed.

Impairment of neurovascular coupling in the hippocampus due to decreased nitric oxide bioavailability supports early cognitive dysfunction in type 2 diabetic rats.

Numerous epidemiological and preclinical studies have established a strong correlation between type 2 diabetes (T2DM) and cognitive impairment and T2DM is now established as an undisputable risk factor in different forms of dementia. However, the mechanisms underlying cognitive impairment in T2DM are still not fully understood. The temporal and spatial coupling between neuronal activity and cerebral blood flow (CBF) - neurovascular coupling (NVC) - is essential for normal brain function. Neuronal-derived nitric oxide (⦁NO) produced through the nNOS-NMDAr pathway, is recognized as a key messenger in NVC, especially in the hippocampus. Of note, impaired hippocampal perfusion in T2DM patients has been closely linked to learning and memory dysfunction. In this study, we aimed to investigate the functionality of NVC, in terms of neuronal-•NO signaling and spatial memory performance, in young Goto-Kakizaki (GK) rats, a non-obese model of T2DM. For that, we performed direct and simultaneous measurements of •NO concentration dynamics and microvascular CBF changes in the hippocampus upon glutamatergic activation. We found that limited •NO bioavailability, connected to shorter and faster •NO transients in response to glutamatergic neuronal activation, is associated with decreased hemodynamic responses and a decline in spatial memory performance. This evidence supports a close mechanistic association between neuronal-triggered •NO concentration dynamics in the hippocampus, local microvascular responses, and cognitive performance in young diabetic animals, establishing the functionality of NVC as a critical early factor to consider in the cascade of events leading to cognitive decline in T2DM. These results suggest that strategies capable to overcome the limited •NO bioavailability in early stages of T2DM and maintaining a functional NVC pathway may configure pertinent therapeutic approaches to mitigate the risk for cognitive impairment in T2DM.

Nitric Oxide Pathways in Neurovascular Coupling Under Normal and Stress Conditions in the Brain: Strategies to Rescue Aberrant Coupling and Improve Cerebral Blood Flow.

The brain has impressive energy requirements and paradoxically, very limited energy reserves, implying its huge dependency on continuous blood supply. Aditionally, cerebral blood flow must be dynamically regulated to the areas of increased neuronal activity and thus, of increased metabolic demands. The coupling between neuronal activity and cerebral blood flow (CBF) is supported by a mechanism called neurovascular coupling (NVC). Among the several vasoactive molecules released by glutamatergic activation, nitric oxide (•NO) is recognized to be a key player in the process and essential for the development of the neurovascular response. Classically, •NO is produced in neurons upon the activation of the glutamatergic N-methyl-D-aspartate (NMDA) receptor by the neuronal isoform of nitric oxide synthase and promotes vasodilation by activating soluble guanylate cyclase in the smooth muscle cells of the adjacent arterioles. This pathway is part of a more complex network in which other molecular and cellular intervenients, as well as other sources of •NO, are involved. The elucidation of these interacting mechanisms is fundamental in understanding how the brain manages its energy requirements and how the failure of this process translates into neuronal dysfunction. Here, we aimed to provide an integrated and updated perspective of the role of •NO in the NVC, incorporating the most recent evidence that reinforces its central role in the process from both viewpoints, as a physiological mediator and a pathological stressor. First, we described the glutamate-NMDA receptor-nNOS axis as a central pathway in NVC, then we reviewed the link between the derailment of the NVC and neuronal dysfunction associated with neurodegeneration (with a focus on Alzheimer's disease). We further discussed the role of oxidative stress in the NVC dysfunction, specifically by decreasing the •NO bioavailability and diverting its bioactivity toward cytotoxicity. Finally, we highlighted some strategies targeting the rescue or maintenance of •NO bioavailability that could be explored to mitigate the NVC dysfunction associated with neurodegenerative conditions. In line with this, the potential modulatory effects of dietary nitrate and polyphenols on •NO-dependent NVC, in association with physical exercise, may be used as effective non-pharmacological strategies to promote the •NO bioavailability and to manage NVC dysfunction in neuropathological conditions.

Microelectrode Sensor for Real-Time Measurements of Nitrite in the Living Brain, in the Presence of Ascorbate.

The impaired blood flow to the brain causes a decrease in the supply of oxygen that can result in cerebral ischemia; if the blood flow is not restored quickly, neuronal injury or death will occur. Under hypoxic conditions, the production of nitric oxide (●NO), via the classical L-arginine-●NO synthase pathway, is reduced, which can compromise ●NO-dependent vasodilation. However, the alternative nitrite (NO2-) reduction to ●NO, under neuronal hypoxia and ischemia conditions, has been viewed as an in vivo storage pool of ●NO, complementing its enzymatic synthesis. Brain research is thus demanding suitable tools to probe nitrite's temporal and spatial dynamics in vivo. In this work, we propose a new method for the real-time measurement of nitrite concentration in the brain extracellular space, using fast-scan cyclic voltammetry (FSCV) and carbon microfiber electrodes as sensing probes. In this way, nitrite was detected anodically and in vitro, in the 5-500 µM range, in the presence of increasing physiological concentrations of ascorbate (100-500 µM). These sensors were then tested for real-time and in vivo recordings in the anesthetized rat hippocampus; using fast electrochemical techniques, local and reproducible transients of nitrite oxidation signals were observed, upon pressure ejection of an exogenous nitrite solution into the brain tissue. Nitrite microsensors are thus a valuable tool for investigating the role of this inorganic anion in brain redox signaling.

A High Fat/Cholesterol Diet Recapitulates Some Alzheimer's Disease-Like Features in Mice: Focus on Hippocampal Mitochondrial Dysfunction.

BACKGROUND: Ample evidence from clinical and pre-clinical studies suggests mid-life hypercholesterolemia as a risk factor for developing Alzheimer's disease (AD) at a later age. Hypercholesterolemia induced by dietary habits can lead to vascular perturbations that increase the risk of developing sporadic AD. OBJECTIVE: To investigate the effects of a high fat/cholesterol diet (HFCD) as a risk factor for AD by using a rodent model of AD and its correspondent control (healthy animals). METHODS: We compared the effect of a HFCD in normal mice (non-transgenic mice, NTg) and the triple transgenic mouse model of AD (3xTgAD). We evaluated cognitive performance in relation to changes in oxidative metabolism and neuron-derived nitric oxide (•NO) concentration dynamics in hippocampal slices as well as histochemical staining of markers of the neurovascular unit. RESULTS: In NTg, the HFCD produced only moderate hypercholesterolemia but significant decline in spatial memory was observed. A tendency for decrease in •NO production was accompanied by compromised mitochondrial function with decrease in spare respiratory capacity. In 3xTgAD mice, a robust increase in plasma cholesterol levels with the HFCD did not worsen cognitive performance but did induce compromise of mitochondrial function and significantly decreased •NO production. We found increased staining of biomarkers for astrocyte endfeet and endothelial cells in 3xTgAD hippocampi, which was further increased by the HFCD. CONCLUSION: A short term (8 weeks) intervention with HFCD can produce an AD-like phenotype even in the absence of overt systemic hypercholesterolemia and highlights mitochondrial dysfunction as a link between hypercholesterolemia and sporadic AD.

Prenatal Diagnosis of Aberrant Right Subclavian Artery: Association with Genetic Abnormalities.

OBJECTIVE: The objective of the present study was to determine the frequency of malformations and chromosomal abnormalities in a population of fetuses with an aberrant right subclavian artery (ARSA). METHODS: This is a 6-year retrospective study of fetuses with a prenatal diagnosis of ARSA conducted during the period between September 2013 and June 2019 at a fetal medicine unit. Data were collected from ultrasound, fetal echocardiograms, genetic studies, and neonatal records. RESULTS: An ARSA was diagnosed in 22 fetuses. An ARSA was an isolated finding in 18 out of 22 cases (82%). Associated abnormal sonographic findings were found in 4 cases. All cases underwent invasive testing. In 1 of the cases, a chromosomal abnormality was detected (mos 45,X [13]/46,X,e(X) (p22.1q22.1)). No cases of congenital heart disease were found in any of these fetuses. There were two cases in which the postnatal evaluation revealed a malformation: one case of hypospadias and 1 case of cleft palate. CONCLUSION: The presence of an isolated ARSA is benign and is not associated with chromosomal abnormalities. The finding of ARSA, however, warrants a detailed fetal ultrasound in order to exclude major fetal abnormalities and other soft markers.

OBJETIVO: O objetivo do presente estudo foi determinar a frequência de malformaçães e anomalias cromossômicas em uma população de fetos com artéria subclávia direita aberrante (ARSA). MéTODOS: Este é um estudo retrospectivo de 6 anos de fetos com diagnóstico pré-natal de ARSA realizado durante o período de setembro de 2013 a junho de 2019 em uma unidade de medicina fetal. Os dados foram coletados de ultrassom, ecocardiograma fetal, estudos genéticos e registros neonatais. RESULTADOS: Um ARSA foi diagnosticado em 22 fetos. Um ARSA foi um achado isolado em 18 dos 22 casos (82%). Achados ultrassonográficos anormais associados foram encontrados em 4 casos. Todos os casos foram submetidos a testes invasivos. Em um dos casos, foi detectada uma anormalidade cromossômica (mos 45, X [13] / 46, X, e (X) (p22.1q22.1)). Nenhum caso de doença cardíaca congênita foi encontrado em qualquer um desses fetos. Houve dois casos em que a avaliação pós-natal revelou a malformação: um caso de hipospádia e 1 caso de fenda palatina. CONCLUSãO: A presença de ARSA isolado é benigna e não está associada a anormalidades cromossômicas. O achado de ARSA, no entanto, justifica uma ultrassonografia fetal detalhada para excluir anormalidades fetais importantes e outros marcadores leves.

Prenatal Diagnosis of Aberrant Right Subclavian Artery: Association with Genetic Abnormalities / Diagnóstico pré-natal de artéria subclávia direita anômala: associação com anormalidades genéticas

Abstract Objective The objective of the present study was to determine the frequency of malformations and chromosomal abnormalities in a population of fetuses with an aberrant right subclavian artery (ARSA). Methods This is a 6-year retrospective study of fetuses with a prenatal diagnosis of ARSA conducted during the period between September 2013 and June 2019 at a fetal medicine unit. Data were collected from ultrasound, fetal echocardiograms, genetic studies, and neonatal records. Results An ARSA was diagnosed in 22 fetuses. An ARSA was an isolated finding in 18 out of 22 cases (82%). Associated abnormal sonographic findings were found in 4 cases. All cases underwent invasive testing. In 1 of the cases, a chromosomal abnormality was detected (mos 45,X [13]/46,X,e(X) (p22.1q22.1)). No cases of congenital heart disease were found in any of these fetuses. There were two cases in which the postnatal evaluation revealed amalformation: one case of hypospadias and 1 case of cleft palate. Conclusion The presence of an isolated ARSA is benign and is not associated with chromosomal abnormalities. The finding of ARSA, however, warrants a detailed fetal ultrasound in order to exclude major fetal abnormalities and other soft markers.

Resumo Objetivo O objetivo do presente estudo foi determinar a frequência demalformaçães e anomalias cromossômicas em uma população de fetos com artéria subclávia direita aberrante (ARSA). Métodos Este é um estudo retrospectivo de 6 anos de fetos com diagnóstico prénatal de ARSA realizado durante o período de setembro de 2013 a junho de 2019 em uma unidade de medicina fetal. Os dados foram coletados de ultrassom, ecocardiograma fetal, estudos genéticos e registros neonatais. Resultados Um ARSA foi diagnosticado em 22 fetos. Um ARSA foi um achado isolado em 18 dos 22 casos (82%). Achados ultrassonográficos anormais associados foram encontrados em 4 casos. Todos os casos foram submetidos a testes invasivos. Em um dos casos, foi detectada uma anormalidade cromossômica (mos 45, X [13] / 46, X, e (X) (p22.1q22.1)). Nenhum caso de doença cardíaca congênita foi encontrado em qualquer um desses fetos. Houve dois casos em que a avaliação pós-natal revelou a malformação: um caso de hipospádia e 1 caso de fenda palatina. Conclusão A presença de ARSA isolado é benigna e não está associada a anormalidades cromossômicas. O achado de ARSA, no entanto, justifica uma ultrassonografia fetal detalhada para excluir anormalidades fetais importantes e outros marcadores leves.

Prenatal diagnosis of fibular aplasia-tibial campomelia-oligosyndactyly syndrome: Two case reports and review of the literature.

Fibular aplasia-tibial campomelia-oligosyndactyly also known as FATCO syndrome is a rare condition characterized by fibular aplasia, shortening and anterior bowing of the lower limb at the tibia with overlying soft tissue dimpling and oligosyndactyly. Its etiology is currently unknown, but there is a male predominance. There are less than 30 cases reported in the literature but only three with prenatal diagnosis. We report two cases of FATCO syndrome with prenatal lower limb malformation diagnosis. Identification of the ultrasound findings of this condition in the prenatal stages allows an adequate parental counselling regarding the clinical features, prognosis, and potential treatments.

The Peculiar Facets of Nitric Oxide as a Cellular Messenger: From Disease-Associated Signaling to the Regulation of Brain Bioenergetics and Neurovascular Coupling.

In this review, we address the regulatory and toxic role of ·NO along several pathways, from the gut to the brain. Initially, we address the role on ·NO in the regulation of mitochondrial respiration with emphasis on the possible contribution to Parkinson's disease via mechanisms that involve its interaction with a major dopamine metabolite, DOPAC. In parallel with initial discoveries of the inhibition of mitochondrial respiration by ·NO, it became clear the potential for toxic ·NO-mediated mechanisms involving the production of more reactive species and the post-translational modification of mitochondrial proteins. Accordingly, we have proposed a novel mechanism potentially leading to dopaminergic cell death, providing evidence that NO synergistically interact with DOPAC in promoting cell death via mechanisms that involve GSH depletion. The modulatory role of NO will be then briefly discussed as a master regulator on brain energy metabolism. The energy metabolism in the brain is central to the understanding of brain function and disease. The core role of ·NO in the regulation of brain metabolism and vascular responses is further substantiated by discussing its role as a mediator of neurovascular coupling, the increase in local microvessels blood flow in response to spatially restricted increase of neuronal activity. The many facets of NO as intracellular and intercellular messenger, conveying information associated with its spatial and temporal concentration dynamics, involve not only the discussion of its reactions and potential targets on a defined biological environment but also the regulation of its synthesis by the family of nitric oxide synthases. More recently, a novel pathway, out of control of NOS, has been the subject of a great deal of controversy, the nitrate:nitrite:NO pathway, adding new perspectives to ·NO biology. Thus, finally, this novel pathway will be addressed in connection with nitrate consumption in the diet and the beneficial effects of protein nitration by reactive nitrogen species.

Prenatal diagnosis of isolated frontonasal dysplasia: A case report.

We report a case of mild frontonasal dysplasia, a complex and rare malformation affecting the central portion of the face, especially the eyes, nose, and forehead, which was diagnosed at 20 weeks of gestation. The diagnosis was made by two- and four-dimensional ultrasound and confirmed at autopsy after pregnancy termination. A review of the literature is presented.

Schaaf-Yang Syndrome: A Real Challenge for Prenatal Diagnosis.

Schaaf-Yang syndrome (SYS) is a rare neurodevelopmental disorder caused by pathogenic variants in the MAGEL2 gene. It is usually a postnatal diagnosis in infants with muscular hypotonia and feeding difficulties. There are no cases diagnosed antenatally. During pregnancy, the most common findings reported are polyhydramnios and decreased fetal movements, which are relatively common and unspecific.We present one case of fetal clubfoot and clinodactyly in a fetus postnatally diagnosed with SYS, as well as a brief review of the prenatal findings associated with this syndrome.

Prenatal diagnosis of frontal encephalocele.

Encephalocele is a rare congenital form of neural tube defect characterized by a protrusion of the meninges and cerebral tissue through a skull defect. These defects are classified according to their location: frontal, parietal and occipital, the last one being the most common form of presentation. The prognosis is related to the anatomical site, the volume of the neural contents and the presence of coexisting abnormalities. Most pregnancies are terminated, since the prognosis is poor. We report a case of an isolated fetal frontal encephalocele diagnosed at 21 weeks of gestation.

Physicochemical stability of contact lenses materials for biomedical applications / Estabilidad fisicoquímica de los materiales de las lentes de contacto, para aplicaciones biomédicas

PURPOSE: The physicochemical stability, thermal and water plasticizing effect on transport properties of contact lenses (CL) were analyzed to verify its capacity to maintain the original properties after being dehydrated and rehydrated. METHODS: Two daily disposable (nesofilcon A and delefilcon A) and two monthly CL (comfilcon A and lotrafilcon B) were used. Measurements of refractive index (RI), water content (WC), chemical structure and thermal properties were taken: new (N), after dehydration (D) and rehydrated (R). RI and WC were accessed using a digital automated refractometer (CLR-12-70). Chemical structure was evaluated by a Fourier Transformed Infrared Spectroscopy (FTIR-ATR) and Differential Scanning Calorimetry (DSC) with a calorimeter (Mettler Toledo DSC-821). RESULTS: The FTIR spectrum of N, D and R was quite similar for all CL, with higher differences observed in the transmission between 3500-3000cm−1 due to the hydroxyl group (OH). After dehydration and rehydration, there were no significant changes in the chemical structure. RI and WC of the CL rehydrated did not vary significantly from the initial CL (p > 0.05) and thermal properties also confirm that the behavior did not change. It was observed that the glass-transition temperature decrease with increased WC. CONCLUSIÓN: No significant alterations were observed in the physicochemical structure of the materials after dehydration and rehydration showing a good stability of their components. The strong water plasticizing effect in the silicone hydrogel lens materials improves flexibility and chain mobility and may should be considered for other biomedical applications

OBJETIVO: Se analizaron la estabilidad fisicoquímica, las propiedades térmicas y el efecto plastificante en las propiedades de transporte de las lentes de contacto (LC), para verificar su capacidad de mantener sus propiedades originales tras la deshidratación y rehidratación. MÉTODOS: Se utilizaron dos lentes de contacto desechables diarias (nesofilcon A y delefilcon A) y dos mensuales (comfilcon A y lotrafilcon B). Se realizaron medidas del índice de refracción (IR), contenido de agua (CA), estructura química y propiedades térmicas: nuevas (N), tras la deshidratación (D) y tras la rehidratación (R). Se accedió a IR y CA utilizando un refractómetro digital automatizado (CLR-12-70). La estructura química se evaluó mediante Espectroscopía de infrarrojos con transformada de Fourier (FTIR-ATR) y Calorimetría diferencial de barrido (DSC) con un calorímetro (Mettler Toledo DSC-821). RESULTADOS: El espectro FTIR de N, D y R fue bastante similar para todas las LC, observándose mayores diferencias en la transmisión entre 3500-3000cm−1 debido al grupo hidroxilo (OH). Tras la deshidratación y rehidratación no se produjeron cambios significativos en la estructura química. Los valores IR y CA de las LC rehidratadas no variaron significativamente con respecto a la LC inicial (p > 0,05), y las propiedades térmicas confirmaron también que el comportamiento no experimentó cambio alguno. Se observó que la temperatura vidrio-transición disminuía al incrementarse WC. CONCLUSIÓN: No se observaron alteraciones significativas en la estructura fisicoquímica de los materiales tras la deshidratación y rehidratación, reflejando una buena estabilidad de sus componentes. El fuerte efecto plastificante del agua en los materiales de las lentes de hidrogel de silicona mejora la flexibilidad y la movilidad de la cadena, debiendo considerarse esta circunstancia para otras aplicaciones biomédicas

Physicochemical stability of contact lenses materials for biomedical applications.

PURPOSE: The physicochemical stability, thermal and water plasticizing effect on transport properties of contact lenses (CL) were analyzed to verify its capacity to maintain the original properties after being dehydrated and rehydrated. METHODS: Two daily disposable (nesofilcon A and delefilcon A) and two monthly CL (comfilcon A and lotrafilcon B) were used. Measurements of refractive index (RI), water content (WC), chemical structure and thermal properties were taken: new (N), after dehydration (D) and rehydrated (R). RI and WC were accessed using a digital automated refractometer (CLR-12-70). Chemical structure was evaluated by a Fourier Transformed Infrared Spectroscopy (FTIR-ATR) and Differential Scanning Calorimetry (DSC) with a calorimeter (Mettler Toledo DSC-821). RESULTS: The FTIR spectrum of N, D and R was quite similar for all CL, with higher differences observed in the transmission between 3500-3000cm-1 due to the hydroxyl group (OH). After dehydration and rehydration, there were no significant changes in the chemical structure. RI and WC of the CL rehydrated did not vary significantly from the initial CL (p>0.05) and thermal properties also confirm that the behavior did not change. It was observed that the glass-transition temperature decrease with increased WC. CONCLUSION: No significant alterations were observed in the physicochemical structure of the materials after dehydration and rehydration showing a good stability of their components. The strong water plasticizing effect in the silicone hydrogel lens materials improves flexibility and chain mobility and may should be considered for other biomedical applications.

Platinized carbon fiber-based glucose microbiosensor designed for metabolic studies in brain slices.

In order to understand how energy metabolism adapts to changes in neuronal activity it is imperative to perform direct measurements of the flux of glucose (and other metabolites) in brain tissue. Metabolic studies using brain slice preparations are attractive due to the controllability of recording conditions, absence of anesthetic interference and refined animal experimental protocols. In this work, taking advantage of the small size and versatility of carbon fiber microelectrodes (CFMs), we aimed to develop an amperometric glucose microbiosensor suitable for glucose measurement in brain slices. Potentiostatic- or galvanostatic-driven platinum electrodeposition was used to improve the analytical properties of CFMs towards detection of hydrogen peroxide. The platinized CFMs served as platform for the development of glucose microbiosensors through the immobilization of glucose-oxidase (GOx) by cross-linking with glutaraldehyde in the presence of BSA. Selective glucose measurements were attained by modifying the electrode with a permselective layer of meta-phenylenediamine and by integrating a null sensor. The in vitro characterization studies support the good analytical features of the CFM/Pt-based microbiosensors to reliably measure glucose in brain tissue. The ex vivo experiments in rodent hippocampal slices validated their suitability to measure evoked changes in extracellular glucose. This approach, encompassing the use of null sensor to cross-check the selectivity on a moment-to-moment basis, allowed us to provide the temporal and quantitative profile of extracellular glucose changes in hippocampal slices following a spreading depolarization event. Overall, these results support the potential of these microbiosensors to be used as a valuable tool to investigate the complex nature of glucose utilization in brain tissue linked to neuronal activation both in physiological and pathological conditions.

Age-Dependent Impairment of Neurovascular and Neurometabolic Coupling in the Hippocampus.

Neurovascular and neurometabolic coupling are critical and complex processes underlying brain function. Perturbations in the regulation of these processes are, likely, early dysfunctional alterations in pathological brain aging and age-related neurodegeneration. Evidences support the role of nitric oxide (•NO) as a key messenger both in neurovascular coupling, by signaling from neurons to blood vessels, and in neurometabolic coupling, by modulating O2 utilization by mitochondria. In the present study, we investigated the functionality of neurovascular and neurometabolic coupling in connection to •NO signaling and in association to cognitive performance during aging. For this, we performed in vivo simultaneous measurements of •NO, O2 and cerebral blood flow (CBF) in the hippocampus of F344 rats along chronological age in response to glutamatergic activation and in correlation with cognitive performance. Firstly, it is evidenced the temporal sequence of events upon glutamate stimulation of hippocampal dentate gyrus, encompassing the local and transitory increase of •NO followed by transitory local changes of CBF and pO2. Specifically, the transient increase of •NO is followed by an increase of CBF and biphasic changes of the local pO2. We observed that, although the glutamate-induced •NO dynamics were not significantly affected by aging, the correspondent hemodynamic was progressively diminished accompanying a decline in learning and memory. Noteworthy, in spite of a compromised blood supply, in aged rats we observed an increased ΔpO2 associated to the hemodynamic response, suggestive of a decrease in the global metabolic rate of O2. Furthermore, the impairment in the neurovascular coupling observed along aging in F344 rats was mimicked in young rats by promoting an unbalance in redox status toward oxidation via intracellular generation of superoxide radical. This observation strengthens the idea that oxidative stress may have a critical role in the neurovascular uncoupling underlying brain aging and dysfunction. Overall, data supports an impairment of neurovascular response in connection with cognition decline due to oxidative environment-dependent compromised •NO signaling from neurons to vessels during aging.

Analysis of respiratory capacity in brain tissue preparations: high-resolution respirometry for intact hippocampal slices.

The evaluation of mitochondrial function provides the basis for the study of brain bioenergetics. However, analysis of brain mitochondrial respiration has been hindered by the low yield associated with mitochondria isolation procedures. Furthermore, isolating mitochondria or cells results in loss of the inherent complexity of the central nervous system. High-resolution respirometry (HRR), is a valuable tool to study mitochondrial function and has been used in diverse biological preparations ranging from isolated mitochondria to tissue homogenates and permeabilized tissue biopsies. Here we describe a novel methodology for evaluation of mitochondrial respiration using tissue preparations from the central nervous system, namely acute hippocampal slices from rodents, with HRR. By using acute intact hippocampal slices, tissue cytoarchitecture, intercellular communication and connectivity are preserved. Mitochondrial respiration was evaluated by using an adapted substrate-uncoupler-inhibitor titration (SUIT) protocol and the expected responses were observed. This methodology can be used to detect differences in mitochondrial function at the oxidative phosphorylation level and for studies with different brain oxidative substrates in physiological and neuropathological settings, by using a system that better represents the in vivo conditions than isolated mitochondria and/or cells.

Combined in Vivo Amperometric Oximetry and Electrophysiology in a Single Sensor: A Tool for Epilepsy Research.

Seizures are paroxysmal events in which increased neuronal activity is accompanied by an increase in localized energetic demand. The ability to simultaneously record electrical and chemical events using a single sensor poses a promising approach to identify seizure onset zones in the brain. In the present work, we used ceramic-based platinum microelectrode arrays (MEAs) to perform high-frequency amperometric recording of local pO2 and local field potential (LFP)-related currents during seizures in the hippocampus of chronically implanted freely moving rats. Resting levels of O2 in the rodent brain varied between 6.6 ± 0.7 µM in the dentate gyrus (DG) region of the hippocampus and 22.1 ± 4.9 µM in the cerebral cortex. We also observed an expected increase in hippocampal pO2 (15 ± 4% from baseline) in response to tail pinch stress paradigm. Finally, induction of status epilepticus by intrahippocampal injection of pilocarpine induced biphasic changes in pO2 in the hippocampus. The initial dip at seizure onset (ΔO2 = -4.5 ± 0.7 µM) was followed by a prolonged hyperoxygenation phase (ΔO2 = +10.4 ± 2.9 µM). By acquiring the amperometry signal with a high sampling rate of 100 Hz we decomposed the raw signal in an oximetry recording (<1 Hz) and LFP recording (>1 Hz), demonstrating that each individual Pt site can simultaneously report changes in local pO2 and LFP-related currents during pilocarpine-induced seizure activity. This has high potential for translation into the clinical setting supported on intracranial grid or strip electrodes.