Guanosine enhances glutamate uptake and oxidation, preventing oxidative stress in mouse hippocampal slices submitted to high glutamate levels.

Glutamate (Glu) is the main mammalian brain neurotransmitter. Concerning the glutamatergic neurotransmission, excessive levels of glutamate in the synaptic cleft are extremally harmful. This phenomenon, named as excitotoxicity is involved in various acute and chronic brain diseases. Guanosine (GUO), an endogenous guanine nucleoside, possesses neuroprotective effects in several experimental models of glutamatergic excitotoxicity, an effect accompanied by an increase in astrocytic glutamate uptake. Therefore, the objective of this study was to investigate the involvement of an additional putative parameter, glutamate oxidation to CO2, involved in ex-vivo GUO neuroprotective effects in mouse hippocampal slices submitted to glutamatergic excitotoxicity. Mice were sacrificed by decapitation, the hippocampi were removed and sliced. The slices were incubated for various times and concentrations of Glu and GUO. First, the concentration of Glu that produced an increase in L-[14C(U)]-Glu oxidation to CO2 without cell injury was determined at different time points (between 0 and 90 min); 1000 µM Glu increased Glu oxidation between 30 and 60 min of incubation without cell injury. Under these conditions (Glu concentration and incubation time), 100 µM GUO increased Glu oxidation (35%). Additionally, 100 µM GUO increased L-[3,4-3H]-glutamate uptake (45%) in slices incubated with 1000 µM Glu (0-30 min). Furthermore, 1000  µM Glu increased reactive species levels, SOD activity, and decreased GPx activity, and GSH content after 30 and 60 min; 100 µM GUO prevented these effects. This is the first study demonstrating that GUO simultaneously promoted an increase in the uptake and utilization of Glu in excitotoxicity-like conditions preventing redox imbalance.

Behavioral, Neurochemical and Brain Oscillation Abnormalities in an Experimental Model of Acute Liver Failure.

Hepatic encephalopathy (HE) represents a brain dysfunction caused by both acute and chronic liver failures, and its severity deeply affects the prognosis of patients with impaired liver function. In its pathophysiology, ammonia levels and glutamatergic system hyperactivity seem to play a pivotal role in the disruption of brain homeostasis. Here, we investigate important outcomes involved in behavioral performance, electroencephalographic patterns, and neurochemical parameters to better characterize the well-accepted animal model of acute liver failure (ALF) induced by subtotal hepatectomy (92% removal of tissue) that produces ALF. This study was divided into three cohorts: (1) rats clinically monitored after hepatectomy every 6 h for 96 h or until death; (2) rats tested in an open-field task (OFT) before and after surgery and had blood, cerebrospinal fluid, and brain tissue collected after the last OFT; and (3) rats that had continuous EEGs recorded before and after surgery for 3 days. The hepatectomized rats presented significant motor behavioral changes accompanied by important abnormalities in classical blood laboratory parameters of ALF, and EEG features suggestive of HE and deep disturbances in the brain glutamatergic system. Using an animal model of ALF induced via subtotal hepatectomy, this work provides a comprehensive and reliable experimental model that increases the opportunity for studying the effects of new treatment strategies to be explored in an unprecedented way. It also presents insights into the pathophysiology of HE in a reproducible model of ALF, which correlates important neurochemical and EEG aspects of the syndrome.

Guanosine Exerts Neuroprotective Effect in an Experimental Model of Acute Ammonia Intoxication.

The nucleoside guanosine (GUO) increases glutamate uptake by astrocytes and acts as antioxidant, thereby providing neuroprotection against glutamatergic excitotoxicity, as we have recently demonstrated in an animal model of chronic hepatic encephalopathy. Here, we investigated the neuroprotective effect of GUO in an acute ammonia intoxication model. Adult male Wistar rats received an intraperitoneal (i.p.) injection of vehicle or GUO 60 mg/kg, followed 20 min later by an i.p. injection of vehicle or 550 mg/kg of ammonium acetate. Afterwards, animals were observed for 45 min, being evaluated as normal, coma (i.e., absence of corneal reflex), or death status. In a second cohort of rats, video-electroencephalogram (EEG) recordings were performed. In a third cohort of rats, the following were measured: (i) plasma levels of glucose, transaminases, and urea; (ii) cerebrospinal fluid (CSF) levels of ammonia, glutamine, glutamate, and alanine; (iii) glutamate uptake in brain slices; and (iv) brain redox status and glutamine synthetase activity in cerebral cortex. GUO drastically reduced the lethality rate and the duration of coma. Animals treated with GUO had improved EEG traces, decreased CSF levels of glutamate and alanine, lowered oxidative stress in the cerebral cortex, and increased glutamate uptake by astrocytes in brain slices compared with animals that received vehicle prior to ammonium acetate administration. This study provides new evidence on mechanisms of guanine-derived purines in their potential modulation of glutamatergic system, contributing to GUO neuroprotective effects in a rodent model of by acute ammonia intoxication.

Neuroprotective effects of guanosine administration on behavioral, brain activity, neurochemical and redox parameters in a rat model of chronic hepatic encephalopathy.

It is well known that glutamatergic excitotoxicity and oxidative stress are implicated in the pathogenesis of hepatic encephalopathy (HE). The nucleoside guanosine exerts neuroprotective effects through the antagonism against glutamate neurotoxicity and antioxidant properties. In this study, we evaluated the neuroprotective effect of guanosine in an animal model of chronic HE. Rats underwent bile duct ligation (BDL) and 2 weeks later they were treated with i.p. injection of guanosine 7.5 mg/kg once a day for 1-week. We evaluated the effects of guanosine in HE studying several aspects: a) animal behavior using open field and Y-maze tasks; b) brain rhythm changes in electroencephalogram (EEG) recordings; c) purines and glutamate levels in the cerebral spinal fluid (CSF); and d) oxidative stress parameters in the brain. BDL rats presented increased levels of glutamate, purines and metabolites in the CSF, as well as increased oxidative damage. Guanosine was able not only to prevent these effects but also to attenuate the behavioral and EEG impairment induced by BDL. Our study shows the neuroprotective effects of systemic administration of guanosine in a rat model of HE and highlights the involvement of purinergic system in the physiopathology of this disease.

Chronic sulforaphane oral treatment accentuates blood glucose impairment and may affect GLUT3 expression in the cerebral cortex and hypothalamus of rats fed with a highly palatable diet.

Obesity and insulin resistance are the key factors underlying the etiology of major health problems such as hypertension, diabetes and stroke. These important health issues lead researchers to investigate new approaches to prevent and treat obesity and insulin resistance. Good candidates are the phytochemical compounds that have been extensively studied in the field. Therefore, the aim of this study was to test whether sulforaphane (SFN, 1 mg kg⁻¹, 4 months treatment), a potent inducer of antioxidant enzymes present in cruciferous vegetables, had some beneficial effects on obesity and insulin resistance induced by a highly palatable (HP) diet in male Wistar rats. Glucose tolerance, serum and hepatic lipid levels, lipid profile, ALT, AST, urea and creatinine, GLUT1 and GLUT3 levels in the cerebral cortex, hippocampus and hypothalamus were analyzed. Glucose tolerance was lower in the HP diet groups, especially in the HP group treated with SFN. Except for the liver triacylglycerols, no differences were found in serum lipids, hepatic and kidney markers of the HP diet groups. Although expression of GLUT1 was similar between groups for all three brain structures analyzed, expression of GLUT3 in the cortex and hypothalamus had a tendency to decrease in the HP diet group treated with SFN. In conclusion, SFN at the specific dose was able to accentuate glucose intolerance and may affect GLUT3 expression in the cerebral cortex and hypothalamus.

Evidences that maternal swimming exercise improves antioxidant defenses and induces mitochondrial biogenesis in the brain of young Wistar rats.

Physical exercise during pregnancy has been considered beneficial to mother and child. Recent studies showed that maternal swimming improves memory in the offspring, increases hippocampal neurogenesis and levels of neurotrophic factors. The objective of this work was to investigate the effect of maternal swimming during pregnancy on redox status and mitochondrial parameters in brain structures from the offspring. Adult female Wistar rats were submitted to five swimming sessions (30 min/day) prior to mating with adult male Wistar rats, and then trained during the pregnancy (five sessions of 30-min swimming/week). The litter was sacrificed when 7 days old, when cerebellum, parietal cortex, hippocampus, and striatum were dissected. We evaluated the production of reactive species and antioxidant status, measuring the activities of superoxide-dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GPx), as well as non-enzymatic antioxidants. We also investigated a potential mitochondrial biogenesis regarding mitochondrion mass and membrane potential, through cytometric approaches. Our results showed that maternal swimming exercise promoted an increase in reactive species levels in cerebellum, parietal cortex, and hippocampus, demonstrated by an increase in dichlorofluorescein oxidation. Mitochondrial superoxide was reduced in cerebellum and parietal cortex, while nitrite levels were increased in cerebellum, parietal cortex, hippocampus, and striatum. Antioxidant status was improved in cerebellum, parietal cortex, and hippocampus. SOD activity was increased in parietal cortex, and was not altered in the remaining brain structures. CAT and GPx activities, as well as non-enzymatic antioxidant potential, were increased in cerebellum, parietal cortex, and hippocampus of rats whose mothers were exercised. Finally, we observed an increased mitochondrial mass and membrane potential, suggesting mitochondriogenesis, in cerebellum and parietal cortex of pups subjected to maternal swimming. In conclusion, maternal swimming exercise induced neurometabolic programing in the offspring that could be of benefit to the rats against future cerebral insults.

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome.

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.