Biochemical Discrimination of the Down Syndrome-Related Metabolic and Oxidative/Nitrosative Stress Alterations from the Physiologic Age-Related Changes through the Targeted Metabolomic Analysis of Serum.

Down Syndrome (DS) is a neurodevelopmental disorder that is characterized by an accelerated aging process, frequently associated with the development of Alzheimer's disease (AD). Previous studies evidenced that DS patients have various metabolic anomalies, easily measurable in their serum samples, although values that were found in DS patients were compared with those of age-matched non-DS patients, thus hampering to discriminate the physiologic age-related changes of serum metabolites from those that are truly caused by the pathologic processes associated with DS. In the present study we performed a targeted metabolomic evaluation of serum samples from DS patients without dementia of two age classes (Younger DS Patients, YDSP, aging 20-40 years; Aged DS Patients, ADSP, aging 41-60 years), comparing the results with those that were obtained in two age classes of non-DS patients (Younger non-DS Patients, YnonDSP, aging 30-60 years; Aged-nonDS Patients, AnonDSP, aging 75-90 years). Of the 36 compounds assayed, 30 had significantly different concentrations in Pooled non-DS Patients (PnonDSP), compared to Pooled DS Patients (PDSP). Age categorization revealed that 11/30 compounds were significantly different in AnonDSP, compared to YnonDSP, indicating physiologic, age-related changes of their circulating concentrations. A comparison between YDSP and ADSP showed that 19/30 metabolites had significantly different values from those found in the corresponding classes of non-DS patients, strongly suggesting pathologic, DS-associated alterations of their serum levels. Twelve compounds selectively and specifically discriminated PnonDSP from PDSP, whilst only three discriminated YDSP from ADSP. The results allowed to determine, for the first time and to the best of our knowledge, the true, age-independent alterations of metabolism that are measurable in serum and attributable only to DS. These findings may be of high relevance for better strategies (pharmacological, nutritional) aiming to specifically target the dysmetabolism and decreased antioxidant defenses that are associated with DS.

The Crosstalk between GPR81/IGFBP6 Promotes Breast Cancer Progression by Modulating Lactate Metabolism and Oxidative Stress.

Breast cancer is the most frequent tumor and the leading cause of cancer deaths in women. In recent years, lactate metabolism and, in particular, its receptor GPR81 have been shown to play a vital role in cancer biology. GPR81 is upregulated in breast cancer and promotes tumor growth by tumor cell-derived lactate. Therefore, the search for possible crosstalk and the involvement of new molecules capable of generating this pathology is always in continuous development. In this study, the relationship between GPR81 and IGFBP6 protein in tumor growth and oxidative stress in the human breast cancer cell line MDA-MB-231 was studied. Cells were treated with lactate or the GPR81 receptor agonist and antagonist 3,5-DHBA and 3-OBA, respectively. In addition, oxidative stress and proliferation were also evaluated in cells challenged with the recombinant IGFBP6 protein. Our data showed that lactate induced cell proliferation and wound healing of the MDA-231 breast cancer cell through the overexpression of both the lactate receptor GPR81 and IGFBP6. The increase in IGFBP6 was able, in turn, to improve the mitochondrial fitness and redox state, as suggested by the reduced levels of mitochondrial ROS production after IGFBP6 treatment, presumably mediated by the increase in the ROS detoxifying genes HMOX1, GSTK1 and NQO1. In conclusion, our data highlight a novel axis between GPR81 and IGFBP6 in MDA-231 cells able to modulate lactate metabolism and oxidative stress. This complex signaling may represent a new therapeutic target for breast cancer.

ILB® Attenuates Clinical Symptoms and Serum Biomarkers of Oxidative/Nitrosative Stress and Mitochondrial Dysfunction in Patients with Amyotrophic Lateral Sclerosis.

Oxidative/nitrosative stress and mitochondrial dysfunction is a hallmark of amyotrophic lateral sclerosis (ALS), an invariably fatal progressive neurodegenerative disease. Here, as an exploratory arm of a phase II clinical trial (EudraCT Number 2017-005065-47), we used high performance liquid chromatography(HPLC) to investigate changes in the metabolic profiles of serum from ALS patients treated weekly for 4 weeks with a repeated sub-cutaneous dose of 1 mg/kg of a proprietary low molecular weight dextran sulphate, called ILB®. A significant normalization of the serum levels of several key metabolites was observed over the treatment period, including N-acetylaspartate (NAA), oxypurines, biomarkers of oxidative/nitrosative stress and antioxidants. An improved serum metabolic profile was accompanied by significant amelioration of the patients' clinical conditions, indicating a response to ILB® treatment that appears to be mediated by improvement of tissue bioenergetics, decrease of oxidative/nitrosative stress and attenuation of (neuro)inflammatory processes.

Clobetasol promotes neuromuscular plasticity in mice after motoneuronal loss via sonic hedgehog signaling, immunomodulation and metabolic rebalancing.

Motoneuronal loss is the main feature of amyotrophic lateral sclerosis, although pathogenesis is extremely complex involving both neural and muscle cells. In order to translationally engage the sonic hedgehog pathway, which is a promising target for neural regeneration, recent studies have reported on the neuroprotective effects of clobetasol, an FDA-approved glucocorticoid, able to activate this pathway via smoothened. Herein we sought to examine functional, cellular, and metabolic effects of clobetasol in a neurotoxic mouse model of spinal motoneuronal loss. We found that clobetasol reduces muscle denervation and motor impairments in part by restoring sonic hedgehog signaling and supporting spinal plasticity. These effects were coupled with reduced pro-inflammatory microglia and reactive astrogliosis, reduced muscle atrophy, and support of mitochondrial integrity and metabolism. Our results suggest that clobetasol stimulates a series of compensatory processes and therefore represents a translational approach for intractable denervating and neurodegenerative disorders.

Lung Surfactant Decreases Biochemical Alterations and Oxidative Stress Induced by a Sub-Toxic Concentration of Carbon Nanoparticles in Alveolar Epithelial and Microglial Cells.

Carbon-based nanomaterials are nowadays attracting lots of attention, in particular in the biomedical field, where they find a wide spectrum of applications, including, just to name a few, the drug delivery to specific tumor cells and the improvement of non-invasive imaging methods. Nanoparticles inhaled during breathing accumulate in the lung alveoli, where they interact and are covered with lung surfactants. We recently demonstrated that an apparently non-toxic concentration of engineered carbon nanodiamonds (ECNs) is able to induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells. Therefore, the complete understanding of their "real" biosafety, along with their possible combination with other molecules mimicking the in vivo milieu, possibly allowing the modulation of their side effects becomes of utmost importance. Based on the above, the focus of the present work was to investigate whether the cellular alterations induced by an apparently non-toxic concentration of ECNs could be counteracted by their incorporation into a synthetic lung surfactant (DPPC:POPG in 7:3 molar ratio). By using two different cell lines (alveolar (A549) and microglial (BV-2)), we were able to show that the presence of lung surfactant decreased the production of ECNs-induced nitric oxide, total reactive oxygen species, and malondialdehyde, as well as counteracted reduced glutathione depletion (A549 cells only), ameliorated cell energy status (ATP and total pool of nicotinic coenzymes), and improved mitochondrial phosphorylating capacity. Overall, our results on alveolar basal epithelial and microglial cell lines clearly depict the benefits coming from the incorporation of carbon nanoparticles into a lung surfactant (mimicking its in vivo lipid composition), creating the basis for the investigation of this combination in vivo.

Modulation of Pro-Oxidant and Pro-Inflammatory Activities of M1 Macrophages by the Natural Dipeptide Carnosine.

Carnosine is a natural endogenous dipeptide widely distributed in mammalian tissues, existing at particularly high concentrations in the muscles and brain and possesses well-characterized antioxidant and anti-inflammatory activities. In an in vitro model of macrophage activation, induced by lipopolysaccharide + interferon-gamma (LPS + IFN-γ), we here report the ability of carnosine to modulate pro-oxidant and pro-inflammatory activities of macrophages, representing the primary cell type that is activated as a part of the immune response. An ample set of parameters aimed to evaluate cytotoxicity (MTT assay), energy metabolism (HPLC), gene expressions (high-throughput real-time PCR (qRT-PCR)), protein expressions (western blot) and nitric oxide production (qRT-PCR and HPLC), was used to assess the effects of carnosine on activated macrophages challenged with a non cytotoxic LPS (100 ng/mL) + IFN-γ (600 U/mL) concentration. In our experimental model, main carnosine beneficial effects were: (1) the modulation of nitric oxide production and metabolism; (2) the amelioration of the macrophage energy state; (3) the decrease of the expressions of pro-oxidant enzymes (Nox-2, Cox-2) and of the lipid peroxidation product malondialdehyde; (4) the restoration and/or increase of the expressions of antioxidant enzymes (Gpx1, SOD-2 and Cat); (5) the increase of the transforming growth factor-ß1 (TGF-ß1) and the down-regulation of the expressions of interleukins 1ß and 6 (IL-1ß and IL-6) and 6) the increase of the expressions of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1). According to these results carnosine is worth being tested in the treatment of diseases characterized by elevated levels of oxidative stress and inflammation (atherosclerosis, cancer, depression, metabolic syndrome, and neurodegenerative diseases).

Carnosine Decreases PMA-Induced Oxidative Stress and Inflammation in Murine Macrophages.

Carnosine is an endogenous dipeptide composed of ß-alanine and L-histidine. This naturally occurring molecule is present at high concentrations in several mammalian excitable tissues such as muscles and brain, while it can be found at low concentrations in a few invertebrates. Carnosine has been shown to be involved in different cellular defense mechanisms including the inhibition of protein cross-linking, reactive oxygen and nitrogen species detoxification as well as the counteraction of inflammation. As a part of the immune response, macrophages are the primary cell type that is activated. These cells play a crucial role in many diseases associated with oxidative stress and inflammation, including atherosclerosis, diabetes, and neurodegenerative diseases. In the present study, carnosine was first tested for its ability to counteract oxidative stress. In our experimental model, represented by RAW 264.7 macrophages challenged with phorbol 12-myristate 13-acetate (PMA) and superoxide dismutase (SOD) inhibitors, carnosine was able to decrease the intracellular concentration of superoxide anions (O2-•) as well as the expression of Nox1 and Nox2 enzyme genes. This carnosine antioxidant activity was accompanied by the attenuation of the PMA-induced Akt phosphorylation, the down-regulation of TNF-α and IL-6 mRNAs, and the up-regulation of the expression of the anti-inflammatory mediators IL-4, IL-10, and TGF-ß1. Additionally, when carnosine was used at the highest dose (20 mM), there was a generalized amelioration of the macrophage energy state, evaluated through the increase both in the total nucleoside triphosphate concentrations and the sum of the pool of intracellular nicotinic coenzymes. Finally, carnosine was able to decrease the oxidized (NADP+)/reduced (NADPH) ratio of nicotinamide adenine dinucleotide phosphate in a concentration dependent manner, indicating a strong inhibitory effect of this molecule towards the main source of reactive oxygen species in macrophages. Our data suggest a multimodal mechanism of action of carnosine underlying its beneficial effects on macrophage cells under oxidative stress and inflammation conditions.

Fructose-1,6-Bisphosphate Protects Hippocampal Rat Slices from NMDA Excitotoxicity.

Effects of fructose 1,6-bisphosphate (F-1,6-P2) towards N-methyl-d-aspartate NMDA excitotoxicity were evaluated in rat organotypic hippocampal brain slice cultures (OHSC) challenged for 3 h with 30 µM NMDA, followed by incubations (24, 48, and 72 h) without (controls) and with F-1,6-P2 (0.5, 1 or 1.5 mM). At each time, cell necrosis was determined by measuring LDH in the medium. Energy metabolism was evaluated by measuring ATP, GTP, ADP, AMP, and ATP catabolites (nucleosides and oxypurines) in deproteinized OHSC extracts. Gene expressions of phosphofructokinase, aldolase, and glyceraldehyde-3-phosphate dehydrogenase were also measured. F-1,6-P2 dose-dependently decreased NMDA excitotoxicity, abolishing cell necrosis at the highest concentration tested (1.5 mM). Additionally, F-1,6-P2 attenuated cell energy imbalance caused by NMDA, ameliorating the mitochondrial phosphorylating capacity (increase in ATP/ADP ratio) Metabolism normalization occurred when using 1.5 mM F-1,6-P2. Remarkable increase in expressions of phosphofructokinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase (up to 25 times over the values of controls) was also observed. Since this phenomenon was recorded even in OHSC treated with F-1,6-P2 with no prior challenge with NMDA, it is highly conceivable that F-1,6-P2 can enter into intact cerebral cells producing significant benefits on energy metabolism. These effects are possibly mediated by changes occurring at the gene level, thus opening new perspectives for F-1,6-P2 application as a useful adjuvant to rescue mitochondrial metabolism of cerebral cells under stressing conditions.

Non-toxic engineered carbon nanodiamond concentrations induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells.

Engineered nanoparticles are finding a wide spectrum of biomedical applications, including drug delivery and capacity to trigger cytotoxic phenomena, potentially useful against tumor cells. The full understanding of their biosafety and interactions with cell processes is mandatory. Using microglial (BV-2) and alveolar basal epithelial (A549) cells, in this study we determined the effects of engineered carbon nanodiamonds (ECNs) on cell viability, nitric oxide (NO) and reactive oxygen species (ROS) production, as well as on energy metabolism. Particularly, we initially measured decrease in cell viability as a function of increasing ECNs doses, finding similar cytotoxic ECN effects in the two cell lines. Subsequently, using apparently non-cytotoxic ECN concentrations (2 µg/mL causing decrease in cell number < 5%) we determined NO and ROS production, and measured the concentrations of compounds related to energy metabolism, mitochondrial functions, oxido-reductive reactions, and antioxidant defences. We found that in both cell lines non-cytotoxic ECN concentrations increased NO and ROS production with sustained oxidative/nitrosative stress, and caused energy metabolism imbalance (decrease in high energy phosphates and nicotinic coenzymes) and mitochondrial malfunctioning (decrease in ATP/ADP ratio).These results underline the importance to deeply investigate the molecular and biochemical changes occurring upon the interaction of ECNs (and nanoparticles in general) with living cells, even at apparently non-toxic concentration. Since the use of ECNs in biomedical field is attracting increasing attention the complete evaluation of their biosafety, toxicity and/or possible side effects both in vitro and in vivo is mandatory before these highly promising tools might find the correct application.

Serum Compounds of Energy Metabolism Impairment Are Related to Disability, Disease Course and Neuroimaging in Multiple Sclerosis.

Multiple sclerosis (MS) is characterized by primary inflammation, demyelination, and progressive neurodegeneration. A biochemical MS feature is neuronal mitochondrial dysfunction, compensated by anaerobic metabolism increase, likely aggravating progression of neurodegeneration. Here, we characterized a pragmatic serum profile of compounds related to mitochondrial energy metabolism of potential clinical use. Blood samples of 518 well characterized (disability, disease course) MS patients and 167 healthy controls were analyzed for serum purines, pyrimidines, creatinine, and lactate. Nine of the 15 compounds assayed, hypoxanthine, xanthine, uric acid, inosine, uracil, ß-pseudouridine, uridine, creatinine, and lactate, differed significantly between MS patients and controls (p < 0.0001). Using these nine compounds, a unifying Biomarker Score was calculated. Controls and MS patients had mean Biomarker Scores of 0.4 ± 0.7 and 4.4 ± 1.9, respectively (p < 0.00001). The Biomarker Score was higher in patients with progressive (6.0 ± 1.8 than with relapsing remitting disease course (3.6 ± 1.5, p < 0.00001). High association between the Biomarker Score and increase in disability (EDSS) was also observed. Additionally, in 50 patients who underwent magnetic resonance imaging (MRI), increase in the Biomarker Score correlated to neuroanatomical alterations. These results, obtained in a large cohort of MS patients evaluated for serum metabolic compounds connected to energy metabolism, demonstrated that the Biomarker Score might represent a pragmatic, resource saving, easy to obtain, laboratory tool useful to monitor MS patients and predict at an early stage who will switch from an RR to a progressive disease course. For the first time, it was also clearly shown a link between mitochondrial dysfunction and MRI lesions characteristic of MS.

3-Nitropropionic acid-induced ischemia tolerance in the rat brain is mediated by reduced metabolic activity and cerebral blood flow.

Tissue tolerance to ischemia can be achieved by noxious stimuli that are below a threshold to cause irreversible damage ('preconditioning'). Understanding the mechanisms underlying preconditioning may lead to the identification of novel therapeutic targets for diseases such as stroke. We here used the oxidative chain inhibitor 3-nitropropionic acid (NPA) to induce ischemia tolerance in a rat middle cerebral artery occlusion (MCAO) stroke model. Cerebral blood flow (CBF) and structural integrity were characterized by longitudinal magnetic resonance imaging (MRI) in combination with behavioral, histologic, and biochemical assessment of NPA-preconditioned animals and controls. Using this approach we show that the ischemia-tolerant state is characterized by a lower energy charge potential and lower CBF, indicating a reduced baseline metabolic demand, and therefore a cellular mechanism of neural protection. Blood vessel density and structural integrity were not altered by NPA treatment. When subjected to MCAO, preconditioned animals had a characteristic MRI signature consisting of enhanced CBF maintenance within the ischemic territory and intraischemic reversal of the initial cytotoxic edema, resulting in reduced infarct volumes. Thus, our data show that tissue protection through preconditioning occurs early during ischemia and indicate that a reduced cellular metabolism is associated with tissue tolerance to ischemia.

Serum lactate as a novel potential biomarker in multiple sclerosis.

Multiple sclerosis (MS) is a primary inflammatory demyelinating disease associated with a probably secondary progressive neurodegenerative component. Impaired mitochondrial functioning has been hypothesized to drive neurodegeneration and to cause increased anaerobic metabolism in MS. The aim of our multicentre study was to determine whether MS patients had values of circulating lactate different from those of controls. Patients (n=613) were recruited, assessed for disability and clinically classified (relapsing-remitting, secondary progressive, primary progressive) at the Catholic University of Rome, Italy (n=281), at the MS Centre Amsterdam, The Netherlands (n=158) and at the S. Camillo Forlanini Hospital, Rome, Italy (n=174). Serum lactate levels were quantified spectrophotometrically with the analyst being blinded to all clinical information. In patients with MS serum lactate was three times higher (3.04±1.26mmol/l) than that of healthy controls (1.09±0.25mmol/l, p<0.0001) and increased across clinical groups, with higher levels in cases with a progressive than with a relapsing-remitting disease course. In addition, there was a linear correlation between serum lactate levels and the expanded disability scale (EDSS) (R(2)=0.419; p<0.001). These data support the hypothesis that mitochondrial dysfunction is an important feature in MS and of particular relevance to the neurodegenerative phase of the disease. Measurement of serum lactate in MS might be a relative inexpensive test for longitudinal monitoring of "virtual hypoxia" in MS and also a secondary outcome for treatment trials aimed to improve mitochondrial function in patients with MS.

New T530C mutation in the aspartoacylase gene caused Canavan disease with no correlation between severity and N-acetylaspartate excretion.

OBJECTIVE: Canavan disease (OMIM 271900) is a severe autosomal recessive neurodegenerative disorder characterized by spongy degeneration of the brain and caused by mutations in the gene encoding for aspartoacylase (ASPA). The enzyme is responsible for the catalyses of the brain-specific compound N-acetylaspartate (NAA). DESIGN AND METHODS: We report the case of two Egyptian sibling patients suspected of Canavan disease (CD) showing clinical deterioration, white matter degeneration, megalencephaly and severe intellectual impairment. The patients underwent magnetic resonance imaging (MRI) and biochemical analysis of NAA in biological fluid samples (serum and urine). Subsequently, in order to determine the mutation responsible for CD in these two sibs, a molecular biological examination was performed. RESULTS: MRI findings and quantification of high NAA excretion (1378.5 and 680.1µmolNAA/mmolcreatinine in urine of 4months and 4years old patients, respectively) confirmed the diagnosis of CD and prompted a search for the responsible mutation. The molecular biological analysis revealed homozygosity for the substitution T530C (Ile177Thr) in the exon 4 of the ASPA gene in both sibs. A total loss of enzymatic activity was also recorded. CONCLUSIONS: The substitution T530C (Ile177Thr) results in a novel missense mutation causing a CD phenotype with severe clinical characteristics. This mutation was not previously described in the literature. In these two sibs, urinary concentration of NAA appears to correlate inversely to symptom severity and CD progression.

Glucose ameliorates the metabolic profile and mitochondrial function of platelet concentrates during storage in autologous plasma.

BACKGROUND: It is essential that the quality of platelet metabolism and function remains high during storage in order to ensure the clinical effectiveness of a platelet transfusion. New storage conditions and additives are constantly evaluated in order to achieve this. Using glucose as a substrate is controversial because of its potential connection with increased lactate production and decreased pH, both parameters triggering the platelet lesion during storage. MATERIALS AND METHODS: In this study, we analysed the morphological status and metabolic profile of platelets stored for various periods in autologous plasma enriched with increasing glucose concentrations (13.75, 27.5 and 55 mM). After 0, 2, 4, 6 and 8 days, high energy phosphates (ATP, GTP, ADP, AMP), oxypurines (hypoxanthine, xanthine, uric acid), lactate, pH, mitochondrial function, cell lysis and morphology, were evaluated. RESULTS: The data showed a significant dose-dependent improvement of the different parameters in platelets stored with increasing glucose, compared to what detected in controls. Interestingly, this phenomenon was more marked at the highest level of glucose tested and in the period of time generally used for platelet transfusion (0-6 days). CONCLUSION: These results indicate that the addition of glucose during platelet storage ameliorates, in a dose-dependent manner, the biochemical parameters related to energy metabolism and mitochondrial function. Since there was no correspondence between glucose addition, lactate increase and pH decrease in our experiments, it is conceivable that platelet derangement during storage is not directly caused by glucose through an increase of anaerobic glycolysis, but rather to a loss of mitochondrial functions caused by reduced substrate availability.

Decrease in N-acetylaspartate following concussion may be coupled to decrease in creatine.

OBJECTIVES: To assess the time course changes in N-acetylaspartate (NAA) and creatine (Cr) levels in the brain of athletes who suffered a sport-related concussion. PARTICIPANTS: Eleven nonconsecutive athletes with concussive head injury and 11 sex- and age-matched control volunteers MAIN OUTCOME MEASURES: : At 3, 15, 30, and 45 days postinjury, athletes were examined by proton magnetic resonance spectroscopy for the determination of NAA, Cr, and choline (Cho) levels. Proton magnetic resonance spectroscopic data recorded for the control group were used for comparison. RESULTS: Compared with controls (2.18 ± 0.19), athletes showed an increase in the NAA/Cr ratio at 3 (2.71 ± 0.16; P < .01) and 15 (2.54 ± 0.21; P < .01) days postconcussion, followed by a decrease and subsequent normalization at 30 (1.95 ± 0.16, P < .05) and 45 (2.17 ± 0.20; P < .05) days postconcussion. The NAA/Cho ratio decreased at 3, 15, and 30 days postinjury (P < .01 compared with controls), with no differences observed in controls at 45 days postconcussion. Compared with controls, significant increase in the Cho/Cr ratio after 3 (+33%, P < .01) and 15 (+31.5%, P < .01) days postinjury was observed whereas no differences were recorded at 30 and 45 days postinjury. CONCLUSIONS: This cohort of athletes indicates that concussion may cause concomitant decrease in cerebral NAA and Cr levels. This provokes longer time for normalization of metabolism, as well as longer time for resolution of concussion-associated clinical symptoms.

Potentially neuroprotective gene modulation in an in vitro model of mild traumatic brain injury.

In this study, we investigated the hypothesis that mild traumatic brain injury (mTBI) triggers a controlled gene program as an adaptive response finalized to neuroprotection, similar to that found in hibernators and in ischemic preconditioning. A stretch injury device was used to produce an equi-biaxial strain field in rat organotypic hippocampal slice cultures at a specified Lagrangian strain of 10 % and a constant strain rate of 20 s(-1). After 24 h from injury, propidium iodide staining, HPLC analysis of metabolites and microarray analysis of cDNA were performed to evaluate cell viability, cell energy state and gene expression, respectively. Compared to control cultures, 10 % stretch injured cultures showed no change in viability, but demonstrated a hypometabolic state (decreased ATP, ATP/ADP, and nicotinic coenzymes) and a peculiar pattern of gene modulation. The latter was characterized by downregulation of genes encoding for proteins of complexes I, III, and IV of the mitochondrial electron transport chain and of ATP synthase; downregulation of transcriptional and translational genes; downregulation and upregulation of genes controlling the synthesis of glutamate and GABA receptors, upregulation of calmodulin and calmodulin-binding proteins; proper modulation of genes encoding for proapoptotic and antiapoptotic proteins. These results support the hypothesis that, following mTBI, a hibernation-type response is activated in non-hibernating species. Unlike in hibernators and ischemic preconditioning, this adaptive gene programme, aimed at achieving maximal neuroprotection, is not triggered by decrease in oxygen availability. It seems rather activated to avoid increase in oxidative/nitrosative stress and apoptosis during a transient period of mitochondrial malfunctioning.

Metabolic profile of amniotic fluid as a biochemical tool to screen for inborn errors of metabolism and fetal anomalies.

Physiologic concentration in amniotic fluid (AF) of several metabolites has not been established with certainty. In this study, we initially assayed purines, pyrimidines, and amino compounds in 1,257 AF withdrawn between the 15th and the 20th week of gestation from actually normal pregnancies (normal gestations, normal offspring). Results allowed to determine physiologic reference intervals for 45 compounds. In these AF, not all purines and pyrimidines were detectable and uric acid (238.35±76.31 µmol/l) had the highest concentration. All amino compounds were measurable, with alanine having the highest concentration (401.10±88.47 µmol/l). In the second part of the study, we performed a blind metabolic screening of AF to evaluate the utility of this biochemical analysis as an additional test in amniocenteses. In 1,295 additional AF from normal pregnancies, all metabolites fell within the confidence intervals determined in the first part of the study. In 24 additional AF from women carrying Down's syndrome-affected fetuses, glutamate, glutamine, glycine, taurine, valine, isoleucine, leucine, ornithine, and lysine were different from physiologic reference values. One AF sample showed phenylalanine level of 375.54 µmol/l (mean value in normal AF=65.07 µmol/l) and was from a woman with unreported phenylketonuria with mild hyperphenylalaninemia (serum phenylalanine=360.88 µmol/l), carrying the IVS 4+5 G-T and D394A mutations. The fetus was heterozygote for the maternal D394A mutation. An appropriate diet maintained the mother phenylalanine in the range of normality during pregnancy, avoiding serious damage in fetal and neonatal development. These results suggest that the metabolic screening of AF might be considered as an additional biochemical test in amniocenteses useful to highlight anomalies potentially related to IEM.

Brain energy depletion in a rodent model of diffuse traumatic brain injury is not prevented with administration of sodium lactate.

Lactate has been identified as an alternative fuel for the brain in situations of increased energy demand, as following a traumatic brain injury (TBI). This study investigates the effect of treatment with sodium lactate (NaLac) on the changes in brain energy state induced by a severe diffuse TBI. Rats were assigned to one of the eight groups (n=10 per group): 1-sham, normal saline; 2-TBI, normal saline; 3-TBI, hypertonic saline; 4-TBI, 100mM NaLac, 5-TBI, 500 mM NaLac; 6-TBI, 1280 mM NaLac; 7-TBI, 2000 mM NaLac and 8-TBI-500 mM NaLac+magnesium sulfate. Cerebrums were removed 6h after trauma. Metabolites representative of the energy state (ATP, ATP-catabolites), N-acetylaspartate (NAA), antioxidant defenses (ascorbic acid, glutathione), markers of oxidative stress (malondialdehyde, ADP-ribose) and nicotinic coenzymes (NAD(+)) were measured by HPLC. TBI induced a marked decrease in the cerebral levels of ATP, NAA, ascorbic acid, glutathione and NAD(+) and a significant rise in the content of ATP-catabolites, malondialdehyde and ADP-ribose. These alterations were not ameliorated with NaLac infusion. We observed a significant reduction in cerebral NAD(+), an essential co-enzyme for mitochondrial lactate-dehydrogenase that converts lactate into pyruvate and thus replenishes the tricarboxylic acid cycle. These results suggest that the metabolic pathway necessary to consume lactate may be compromised following a severe diffuse TBI in rats.

The protein kinase C activator phorbol myristate acetate decreases brain edema by aquaporin 4 downregulation after middle cerebral artery occlusion in the rat.

The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) is known to interact with aquaporin 4 (AQP 4), a water-selective transporting protein that is abundant in astrocytes, and has experimentally been found to decrease osmotically-induced cell swelling. The purpose of this study was to examine whether PMA reduces brain edema following focal ischemia induced by middle cerebral artery (MCA) occlusion by modulation of AQP4 expression. Male Sprague-Dawley rats were randomly assigned to either sham surgery (n = 6), or a continuous intravenous infusion of vehicle (1% dimethylsulfoxide), followed by MCA occlusion (n = 18), and administration of PMA at 50 microg/kg (n = 6) or at 200 microg/kg (n = 6) starting 60 min before or 30 min (200 microg/kg; n = 6) or 60 min (200 microg/kg; n = 6) after MCA occlusion. Cerebral blood flow was monitored with laser Doppler over the MCA territory, and confirmed a 70% reduction during occlusion. After a 2-h period of ischemia and 2 h of reperfusion, the animals were sacrificed for assessment of brain water content and sodium and potassium concentration. AQP4 expression was assessed by immunoblotting and quantified by densitometry (n = 24). Statistical analysis was performed by ANOVA followed by Tukey's post-hoc test. PMA treatment at 200 microg/kg significantly reduced brain water concentration in the infarcted area when started 60 min before or 30 min after occlusion (p < 0.001 and p = 0.022, respectively), and prevented the subsequent sodium shift (p < 0.05). PMA normalized the AQP4 upregulation in ischemia (p = 0.021). A downregulation of AQP4 in the ischemic area paralleling the reduction in brain edema formation following PMA treatment suggests that the effect was mediated by AQP4 modulation.