Perfusion system for studying dynamic metabolomics in rat brain slices exposed to oxygen–glucose deprivation using proton and phosphorus nuclear magnetic resonance

The aim of the current study was to establish a controlled and reproducible model to study metabolic changes during oxygen–glucose deprivation (OGD) in rat brain using a nuclear magnetic resonance (NMR)-compatible perfusion system. Rat brains were cut into 400-μm thick slices and perfused with artificial cerebrospinal fluid (aCSF) in a 10-mm NMR tube inside a 600-MHz NMR spectrometer. Four experimental conditions were tested: (1) continuous perfusion with aCSF with glucose and normoxia, and (2) 30-, (3) 60-, or (4) 120-min periods of OGD followed by reperfusion of aCSF containing glucose and normoxia. The energetic state of perfused brain slices was measured using phosphorus (31P) NMR and metabolite changes were measured using proton (1H) NMR. aCSF samples were collected every 30 min and analyzed using 1H NMR. The sample temperature was maintained at 36.7 ± 0.1°C and was checked periodically throughout the experiments. Brain slice histology was compared before and after OGD in the perfusion system using hematoxylin–eosin–saffron staining. NMR data clearly distinguished three severity groups (mild, moderate, and severe) after 30, 60, and 120 min of OGD, respectively, compared with the control group. 31P NMR spectra obtained from controls showed that phosphocreatine levels were stable for 5 h inside the perfusion system. Control 1H NMR spectra showed that lactate, N-acetylaspartic acid, glutamate, γ-aminobutyric acid, and creatine metabolite levels were stable over time, with lactate levels having a tendency to gradually increase due to the recirculation of the aCSF in the perfusion system. A controlled and reproducible perfusion system was established to study the energetic and metabolic changes in rat brain slices during and after OGD of varying severity.publishedVersio

Single-centre, triple-blinded, randomised, 1-year, parallel-group, superiority study to compare the effects of Roux-en-Y gastric bypass and sleeve gastrectomy on remission of type 2 diabetes and β-cell function in subjects with morbid obesity: a protocol for the Obesity surgery in Tønsberg (Oseberg) study

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The effect of melatonin on brain metabolism and development after hypoxic-ischemic brain injury in the newborn rat evaluated by multimodal MR, 2014

Reduced supply of blood and oxygen to the brain around birth, known as perinatal hypoxic-ischemic (HI), may cause brain injury in newborns. In this project we investigated the effects of melatonin as a possible treatment that can reduce the damage. A ratmodel for HI injury (Vannucci-model) was used to compare the brain injury development of rats treated with melatonin (injections 0, 6 and 25 hours after injury) with rats without treatment (given injections with 5% DMSO or saline which were used as disolvants for melatonin). The rats were scanned with magnetic resonance 1, 7, 20 and 43 days after the HI injury with T2-weighted images, diffusion weighted images and diffusion tensor imaging. Spin-Echo RARE T2-weighted images (day 1, 7, 20 and 43): RARE factor 8, effective TE 70 ms, TR 3500 ms, 4 or 8 averages, Field of View 20 x 20 mm2, acquisition matrix 160 x 160, 15 slices á 1 mm. Diffusion weighted images (day 1/P08): echo planar imaging (EPI) sequence with Stejskal-Tanner diffusion weighting using 6 b-values (100/200/400/600/800/1000 s/mm2) in 3 orthogonal directions, TE 30 ms, TR 3000 ms, 6 averages, Field of View 17.2 x 15 mm2, acquisition matrix 110 x 96, 9 slices á 1 mm. Diffusion tensor images (day 7, 20 and 43) using an EPI sequence with Stejskal-Tanner diffusion preparation with a b-value of 750 s/mm2 in 81 directions. 10 b0-images. TE 27.50 ms, TR 3000 ms, Field of View 19.25 x 13.65 mm2, acquisition matrix 110 x 78 reconstructed to 220 x 156, 12 axial slices of 0.75 mm thickness

Transient effect of melatonin treatment after neonatal hypoxic-ischemic brain injury in rats

Melatonin has potential neuroprotective capabilities after neonatal hypoxia-ischemia (HI), but long-term effects have not been investigated. We hypothesized that melatonin treatment directly after HI could protect against early and delayed brain injury. Unilateral HI brain injury was induced in postnatal day 7 rats. An intraperitoneal injection of either melatonin or vehicle was given at 0, 6 and 25 hours after hypoxia. In-vivo MRI was performed 1, 7, 20 and 43 days after HI, followed by histological analysis. Forelimb asymmetry and memory were assessed at 12–15 and at 36–43 days after HI. More melatonin treated than vehicle treated animals (54.5% vs 15.8%) developed a mild injury characterized by diffusion tensor values, brain volumes, histological scores and behavioral parameters closer to sham. However, on average, melatonin treatment resulted only in a tendency towards milder injury on T2-weighted MRI and apparent diffusion coefficient maps day 1 after HI, and not improved long-term outcome. These results indicate that the melatonin treatment regimen of 3 injections of 10 mg/kg within the first 25 hours only gave a transient and subtle neuroprotective effect, and may not have been sufficient to mitigate long-term brain injury development following HI

Preconditioning Triggered by Carbon Monoxide (CO) Provides Neuronal Protection Following Perinatal Hypoxia-Ischemia

Perinatal hypoxia-ischemia is a major cause of acute mortality in newborns and cognitive and motor impairments in children. Cerebral hypoxia-ischemia leads to excitotoxicity and necrotic and apoptotic cell death, in which mitochondria play a major role. Increased resistance against major damage can be achieved by preconditioning triggered by subtle insults. CO, a toxic molecule that is also generated endogenously, may have a role in preconditioning as low doses can protect against inflammation and apoptosis. In this study, the role of CO-induced preconditioning on neurons was addressed in vitro and in vivo. The effect of 1 h of CO treatment on neuronal death (plasmatic membrane permeabilization and chromatin condensation) and bcl-2 expression was studied in cerebellar granule cells undergoing to glutamate-induced apoptosis. CO's role was studied in vivo in the Rice-Vannucci model of neonatal hypoxia-ischemia (common carotid artery ligature +75 min at 8% oxygen). Apoptotic cells, assessed by Nissl staining were counted with a stereological approach and cleaved caspase 3-positive profiles in the hippocampus were assessed. Apoptotic hallmarks were analyzed in hippocampal extracts by Western Blot. CO inhibited excitotoxicity-induced cell death and increased Bcl-2 mRNA in primary cultures of neurons. In vivo, CO prevented hypoxia-ischemia induced apoptosis in the hippocampus, limited cytochrome c released from mitochondria and reduced activation of caspase-3. Still, Bcl-2 protein levels were higher in hippocampus of CO pre-treated rat pups. Our results show that CO preconditioning elicits a molecular cascade that limits neuronal apoptosis. This could represent an innovative therapeutic strategy for high-risk cerebral hypoxia-ischemia patients, in particular neonates

Neuron-astrocyte interactions, pyruvate carboxylation and the pentose phosphate pathway in the neonatal rat brain

Glucose and acetate metabolism and the synthesis of amino acid neurotransmitters, anaplerosis, glutamate-glutamine cycling and the pentose phosphate pathway (PPP) have been extensively investigated in the adult, but not the neonatal rat brain. To do this, 7 day postnatal (P7) rats were injected with [1-(13)C]glucose and [1,2-(13)C]acetate and sacrificed 5, 10, 15, 30 and 45 min later. Adult rats were injected and sacrificed after 15 min. To analyse pyruvate carboxylation and PPP activity during development, P7 rats received [1,2-(13)C]glucose and were sacrificed 30 min later. Brain extracts were analysed using (1)H- and (13)C-NMR spectroscopy. Numerous differences in metabolism were found between the neonatal and adult brain. The neonatal brain contained lower levels of glutamate, aspartate and N-acetylaspartate but similar levels of GABA and glutamine per mg tissue. Metabolism of [1-(13)C]glucose at the acetyl CoA stage was reduced much more than that of [1,2-(13)C]acetate. The transfer of glutamate from neurons to astrocytes was much lower while transfer of glutamine from astrocytes to glutamatergic neurons was relatively higher. However, transport of glutamine from astrocytes to GABAergic neurons was lower. Using [1,2-(13)C]glucose it could be shown that despite much lower pyruvate carboxylation, relatively more pyruvate from glycolysis was directed towards anaplerosis than pyruvate dehydrogenation in astrocytes. Moreover, the ratio of PPP/glucose-metabolism was higher. These findings indicate that only the part of the glutamate-glutamine cycle that transfers glutamine from astrocytes to neurons is operating in the neonatal brain and that compared to adults, relatively more glucose is prioritised to PPP and pyruvate carboxylation. Our results may have implications for the capacity to protect the neonatal brain against excitotoxicity and oxidative stress