Acute and Chronic Administration of the Branched-Chain Amino Acids Decreases Nerve Growth Factor in Rat Hippocampus

Maple syrup urine disease (MSUD) is a neurometabolic disorder caused by deficiency of the activity of the mitochondrial enzyme complex branched-chain α-keto acid dehydrogenase leading to accumulation of the branched-chain amino acids (BCAA) and their corresponding branched-chain α-keto acids. In this study, we examined the effects of acute and chronic administration of BCAA on protein levels and mRNA expression of nerve growth factor (NGF) considering that patients with MSUD present neurological dysfunction and cognitive impairment. Considering previous observations, it is suggested that oxidative stress may be involved in the pathophysiology of the neurological dysfunction of MSUD. We also investigated the influence of antioxidant treatment (N-acetylcysteine and deferoxamine) in order to verify the influence of oxidative stress in the modulation of NGF levels. Our results demonstrated decreased protein levels of NGF in the hippocampus after acute and chronic administration of BCAA. In addition, we showed a significant decrease in the expression of ngf in the hippocampus only following acute administration in 10-day-old rats. Interestingly, antioxidant treatment was able to prevent the decrease in NGF levels by increasing ngf expression. In conclusion, the results suggest that BCAA is involved in the regulation of NGF in the developing rat. Thus, it is possible that alteration of neurotrophin levels during brain maturation could be of pivotal importance in the impairment of cognition provoked by BCAA. Moreover, the decrease in NGF levels was prevented by antioxidant treatment, reinforcing that the hypothesis of oxidative stress can be an important pathophysiological mechanism underlying the brain damage observed in MSUD.     

Effect of hyperprolinemia on acetylcholinesterase and butyrylcholinesterase activities in rat

Summary. We observed here that acute proline (Pro) administration provoked a decrease (32%) of acetylcholinesterase (AChE) activity in cerebral cortex and an increase (22%) of butyrylcholinesterase (BuChE) activity in the serum of 29-day-old rats. In contrast, chronic administration of Pro did not alter AChE or BuChE activities. Furthermore, pretreatment of rats with vitamins E and C combined or alone, N-nitro-L-arginine methyl ester or melatonin prevented the reduction of AChE activity caused by acute Pro administration, suggesting the participation of oxidative stress in such effects.     

Inhibition of Mitochondrial Respiratory Chain in the Brain of Adult Rats After Acute and Chronic Administration of Methylphenidate

Methylphenidate (MPH) is frequently prescribed for the treatment of attention deficit/hyperactivity disorder. It was previously demonstrated that MPH altered brain metabolic activity. Most cell energy is obtained through oxidative phosphorylation, in the mitochondrial respiratory chain. However, there are still few studies about MPH effects on the brain of adult rats. Thus, in the present study we evaluated the effect of acute or chronic administration of MPH on the activities of mitochondrial respiratory chain complexes I–IV in the brain of adult rats. For acute administration, a single injection of MPH was given to 60-day-old rats. For chronic administration, MPH injections were given to 60-day-old rats once daily for 28 days. Our results showed that complexes I, II, III and IV were inhibited after acute or chronic MPH administration in the hippocampus, prefrontal cortex, striatum and cerebral cortex. On the other hand, cerebellum was not affected.     

Hypermethioninemia Increases Cerebral Acetylcholinesterase Activity and Impairs Memory in Rats

In the present study we investigated the effect of chronic hypermethioninemia on rat performance in the Morris water maze task, as well as on acetylcholinesterase (AChE) activity in rat cerebral cortex. For chronic treatment, rats received subcutaneous injections of methionine (1.34–2.68 μmol/g of body weight), twice a day, from the 6th to the 28th day of age; control rats received the same volume of saline solution. Groups of rats were killed 3 h, 12 h or 30 days after the last injection of methionine to AChE assay and another group was left to recover until the 60th day of life to assess the effect of early methionine administration on reference and working spatial memory of rats. AChE activity was also determined after behavioral task. Results showed that chronic treatment with methionine did not alter reference memory when compared to saline-treated animals. In the working memory task, we observed a significant days effect with significant differences between control and methionine-treated animals. Chronic hypermethioninemia significantly increased AChE activity at 3 h, 12 h or 30 days after the last injection of methionine, as well as before or after behavioral test. The effect of acute hypermethioninemia on AChE was also evaluated. For acute treatment, 29-day-old rats received one single injection of methionine (2.68 μmol/g of body weight) or saline and were killed 1, 3 or 12 h later. Results showed that acute administration of methionine did not alter cerebral cortex AChE activity. Our findings suggest that chronic experimental hypermethioninemia caused cognitive dysfunction and an increase of AChE activity that might be related, at least in part, to the neurological problems presented by hypermethioninemic patients.     

Ammonia detoxification by accelerated oxidation of branched chain amino acids in brains of acute hepatic failure rats

BCAA aminotransferase and BCKA dehydrogenase activities are increased in the mitochondrial fractions from the brains of hepatic failure rats treated with two-thirds removal of CCl4-injured liver. Cerebral leucine decarboxylation was accelerated, and it well correlated with arterial blood ammonia levels. Elevation of brain ammonia content following an intraperitoneal injection of ammonium acetate to hepatic failure rats could be prevented by intravenous infusion of BCAA. Significantly increased brain glutamic acid, glutamine, and alanine contents were noted. These results suggested that accelerated brain BCAA catabolism in acute hepatic failure rats reduce the neurotoxicity of ammonia by promoting the synthesis of glutamic acid and glutamine from BCAA.     

Effect of ACTH and its fragment on brain catecholamines in intact and adrenalectomized rats.

Brain catecholamine metabolism was monitored by distribution of labelled noradrenaline (3H-NA) after intraventricular injection to intact and adrenalectomized rats. The adrenalectomy produced an increased disappearance rate of the labelled pool in the hypothalamus, hippocampus and neocortex. These changes could be prevented by hydrocortisone pretreatment. Painful stimuli resulted in an increased disappearance of the labelled pool in both intact and adrenalectomized rats. The implantation of hydrocortisone into the tuberoinfundibular region prevented the stress-induced changes of the catecholamine metabolism. Intraventricular administration of ACTH1-24 and ACTH4-10 produced a significant increase of the disappearance rate in different brain regions of adrenalectomized rats. The blocking of catecholamine synthesis by intraventricular injection of alpha-methyl-m-tyrosine resulted in a marked decrease of the labelled pool but did not prevent the ACTH-induced decrease of the tracer pool. On the other hand, the blocking of monoamine-oxydase activity by Pargyline led to a marked increase of the labelled pool but intraventricular administration of ACTH led to an increase of the disappearance rate. The mechanism of ACTH action on brain catecholamine metabolism is still obscure, however, an increased release of the NA to ACTH peptides is very likely in the light of the present observations.     

Oxidative damage and antioxidant enzyme activities in experimental hypothyroidism

Free radicals are now well known to damage cellular components. To investigate whether age and thyroid level affect peroxidation speed, we examined the levels of malondialdehyde and antioxidant enzyme activities in different age groups of hypothyroid rats. Hypothyroidism was induced in 30- and 60-day-old Wistar Albino rats by the i.p. administration of propylthiouracil (10 mg kg(-1) body weight) for 15 days. While malondialdehyde levels of 30- or 60-day-old hypothyroid rats were increased in liver, they were decreased in the tissues of the heart and thyroid. While glucose-6-phosphate dehydrogenase activity levels did not change in heart, brain and liver tissues of 30-day-old rats, they increased in brain and heart tissues of 60-day-old experimental groups, but decreased in the liver. Catalase activities decreased in the liver and heart of rats with hypothyroidism, but increased in erythrocytes. In control groups while malondialdehyde levels increased in brain, heart and thymus with regard to age, they decreased in plasma. Glucose-6-phosphate dehydrogenase and catalase activities were not affected by age in tissues of the thymus, thyroid and brain, but they were decreased in the heart tissue. The changes in the levels of lipid peroxidation and antioxidant enzyme activities which were determined in different tissues of hypothyroid rats indicate a cause for functional disorder of these tissues. Moreover, there may be changes depending on age at lipid peroxidation and antioxidant enzyme activity levels.     

Oxidative Stress in a Model of Toxic Demyelination in Rat Brain: The Effect of Piracetam and Vinpocetine

We studied the role of oxidative stress and the effect of vinpocetine (1.5, 3 or 6 mg/kg) and piracetam (150 or 300 mg/kg) in acute demyelination of the rat brain following intracerebral injection of ethidium bromide (10 μl of 0.1%). Results: ethidium bromide caused (1) increased malondialdehyde (MDA) in cortex, hippocampus and striatum; (2) decreased total antioxidant capacity (TAC) in cortex, hippocampus and striatum; (3) decreased reduced glutathione (GSH) in cortex and hippocampus (4); increased serum nitric oxide and (5) increased striatal (but not cortical or hippocampal) acetylcholinesterase (AChE) activity. MDA decreased in striatum and cortex by the lower doses of vinpocetine or piracetam but increased in cortex and hippocampus and in cortex, hypothalamus and striatum by the higher dose of vinpocetine or piracetam, respectively along with decreased TAC. GSH increased by the higher dose of piracetam and by vinpocetine which also decreased serum nitric oxide. Vinpocetine and piracetam displayed variable effects on regional AChE activity.     

Effect of Acute Administration of 3-Butyl-1-Phenyl-2-(Phenyltelluro)Oct-En-1-One on Oxidative Stress in Cerebral Cortex,Hippocampus, and Cerebellum of Rats

Organotellurium compounds have been synthesized since 1840, but pharmacological and toxicological studies about them are still incipient. Therefore, the objective of this study was to verify the effect of acute administration of the organochalcogen 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one on some parameters of oxidative stress in the brain of 30-day-old rats. Animals were treated intraperitoneally with a single dose of the organotellurium (125, 250, or 500 μg/kg body weight) and sacrificed 60 min after the injection. The cerebral cortex, the hippocampus, and the cerebellum were dissected and homogenized in KCl. Afterward, thiobarbituric acid reactive substances (TBARS), carbonyl, sulfhydryl, catalase (CAT), superoxide dismutase (SOD), nitric oxide (NO) formation, and hydroxyl radical production were measured in the brain. The organotellurium enhanced TBARS in the cerebral cortex and the hippocampus, and increased protein damage (carbonyl) in the cerebral cortex and the cerebellum. In contrast, the compound provoked a reduced loss of thiol groups measured by the sulfhydryl assay in all the tissues studied. Furthermore, the activity of the antioxidant enzyme CAT was reduced by the organochalcogen in the cerebral cortex and the cerebellum, and the activity of SOD was inhibited in all the brain tissues. Moreover, NO production was increased in the cerebral cortex and the cerebellum by this organochalcogen, and hydroxyl radical formation was also enhanced in the cerebral cortex. Our findings indicate that this organotellurium compound induces oxidative stress in the brain of rats, corroborating that this tissue is a potential target for organochalcogen action.     

Cognition Enhancing and Neuromodulatory Propensity of <Emphasis Type="Italic">Bacopa monniera</Emphasis> Extract Against Scopolamine Induced Cognitive Impairments in Rat Hippocampus

Cognition-enhancing activity of Bacopa monniera extract (BME) was evaluated against scopolamine-induced amnesic rats by novel object recognition test (NOR), elevated plus maze (EPM) and Morris water maze (MWM) tests. Scopolamine (2 mg/kg body wt, i.p.) was used to induce amnesia in rats. Piracetam (200 mg/kg body wt, i.p.) was used as positive control. BME at three different dosages (i.e., 10, 20 and 40 mg/kg body wt.) improved the impairment induced by scopolamine by increasing the discrimination index of NOR and by decreasing the transfer latency of EPM and escape latency of MWM tests. Our results further elucidate that BME administration has normalized the neurotransmitters (acetylcholine, glutamate, 5-hydroxytryptamine, dopamine, 3,4 dihydroxyphenylacetic acid, norepinephrine) levels that were altered by scopolamine administration in hippocampus of rat brain. BME administration also ameliorated scopolamine effect by down-regulating AChE and up-regulating BDNF, muscarinic M1 receptor and CREB expression in brain hippocampus confirms the potent neuroprotective role and these results are in corroboration with the earlier in vitro studies. BME administration showed significant protection against scopolamine-induced toxicity by restoring the levels of antioxidant and lipid peroxidation. These results indicate that, cognition-enhancing and neuromodulatory propensity of BME is through modulating the expression of AChE, BDNF, MUS-1, CREB and also by altering the levels of neurotransmitters in hippocampus of rat brain.     

Proline Reduces Creatine Kinase Activity in the Brain Cortex of Rats

Type II hyperprolinemia is an inherited disorder caused by a deficiency of ¹-pyrroline-5-carboxilic acid dehydrogenase, whose biochemical hallmark is proline accumulation in plasma and tissues. Although neurological symptoms occur in most patients, the neurotoxicity of proline is still controversial. The main objective of the present study was to investigate the effect of acute and chronic administration of proline on creatine kinase activity of brain cortex of Wistar rats. Acute treatment was performed by subcutaneous administration of one injection of proline to 22-day-old rats. For chronic treatment, proline was administered twice a day from the 6th to the 21st postpartum day. The results showed that creatine kinase activity was significantly inhibited in the brain cortex of rats subjected to acute proline administration. In contrast, this activity was increased in animals subjected to chronic administration. We also measured the in vitro effect of proline on creatine kinase activity in cerebral cortex of 22-day-old nontreated rats. Proline significantly inhibited creatine kinase activity. Considering the importance of creatine kinase forthe maintenance of energy homeostasis in the brain, it is conceivable that an alteration of this enzyme activity in the brain may be one of the mechanisms by which proline might be neurotoxic.     

Proline Alters Antioxidant Enzyme Defenses and Lipoperoxidation in the Erythrocytes and Plasma of Rats: In Vitro and In Vivo Studies

In the present study, we investigated, in vivo (acute and chronic) and in vitro, the effects of proline on the activities of antioxidant enzymes such as catalase (CAT), glutathione peroxidase and superoxide dismutase (SOD) in erythrocytes and also investigated the effect on thiobarbituric acid-reactive substances (TBARS) in the plasma of rats. For the experiments, the number of animals per group ranged from eight to ten. For acute administration, 29-day-old rats received one subcutaneous injection of proline (18.2 μmol/g body weight) or an equivalent volume of 0.9% saline solution (control) and were killed 1 h later. For chronic treatment, buffered proline was injected subcutaneously into rats twice a day at 10 h intervals from the 6th to the 28th day of age. Rats were killed 12 h after the last injection. For in vitro studies, proline (30.0 μM to 1.0 mM) was added to the incubation medium. Results showed that acute administration of proline reduced CAT and increased SOD activities, while chronic treatment increased the activities of CAT and SOD in erythrocytes and TBARS in the plasma of rats. Furthermore, in vitro studies showed that proline increased TBARS in the plasma (0.5 and 1.0 mM) and CAT activity (1.0 mM) in the erythrocytes of rats. The influence of the antioxidants (α-tocopherol plus ascorbic acid) on the effects elicited by proline was also studied. Treatment with antioxidants for 1 week or from the 6th to the 28th day of age prevented the alterations caused by acute and chronic, respectively, proline administration on the oxidative parameters evaluated. Data indicate that proline alters antioxidant defenses and induces lipid peroxidation in the blood of rats.     

The effects of some porphyrinogenic drugs on the brain cholinergic system.

In central nervous system, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) hydrolyse acetylcholine. Diminished cholinesterase activity is known to alter several mental and psychomotor functions. The symptoms of cholinergic crisis and those observed during acute attacks of acute intermittent porphyria are very similar. The aim of this study was to investigate if there could be a link between the action of some porphyrinogenic drugs on brain and the alteration of the cholinergic system. To this end, AChE and BuChE activities were assayed in whole and different brain areas. Muscarinic acetylcholine receptor (mAChR) levels were also measured. Results obtained indicate that the porphyrinogenic drugs tested affect central cholinergic transmission. Quantification of mAChR gave quite different levels depending on the xenobiotic. Veronal administration inhibited 50% BuChE activity in whole brain, cortex and hippocampus; concomitantly cortex mAChR was 30% reduced. Acute and chronic isoflurane anaesthesia diminished BuChE activity by 70-90% in whole brain instead cerebellum and hippocampus mAChR levels were only altered by chronic enflurane anaesthesia. Differential inhibition of cholinesterases in the brain regions and their consequent effects may be of importance to the knowledge of the mechanisms of neurotoxicity of porphyrinogenic drugs.     

Glutamyl transferase and acetylcholinesterase activity in various areas of the rat brain in alcoholic intoxication

Albino mongrel rats were used for the determination of the gamma-glutamyl transferase (gamma-GTF) and acetylcholine esterase (AChE) activities in various brain areas (cerebral hemispheres, cerebellum, hippocampus, brain stem) during acute (1.5; 4 and 6 g/kg i. p.) and chronic (15 months) alcoholic intoxication and alcohol withdrawal (24-48 h, 4 and 8 days). An increase or a decrease in the activity of these two enzymes in the various rat brain areas depends on the dose of ethanol and the time of its action. The activity of gamma-GTF grew in all brain areas during chronic ethanol intoxication; the activity of AChE was also enhanced in three brain areas but it was diminished in cerebral hemispheres. Alcohol withdrawal caused diverse changes in the activities of these two enzymes in various areas of the brain. A tendency to normalization of the gamma-GTF and AChE activities is manifested 4-8 days after alcohol withdrawal.     

Biochemical, Histological, and Memory Impairment Effects of Chronic Copper Toxicity: A Model for Non-Wilsonian Brain Copper Toxicosis in Wistar Rat

Animal models of copper toxicosis rarely exhibit neurological impairments and increased brain copper accumulation impeding the development of novel therapeutic approaches to treat neurodegenerative diseases having high brain Cu content. The aim of this study was to investigate the effects of intraperitoneally injected copper lactate (0.15 mg Cu/100 g body weight) daily for 90 days on copper and zinc levels in the liver and hippocampus, on biochemical parameters, and on neurobehavioral functions (by Morris water maze) of male Wistar rats. Copper-administered animals exhibited significantly decreased serum acetylcholinesterase (AChE) activity and impaired neuromuscular coordination and spatial memory compared to control rats. Copper-intoxicated rats showed significant increase in liver and hippocampus copper content (99.1 and 73 % increase, respectively), 40.7 % reduction in hepatic zinc content, and interestingly, 77.1 % increase in hippocampus zinc content with concomitant increase in copper and zinc levels in serum and urine compared to control rats. Massive grade 4 copper depositions and grade 1 copper-associated protein in hepatocytes of copper-intoxicated rats were substantiated by rhodanine and orcein stains, respectively. Copper-intoxicated rats demonstrated swelling and increase in the number of astrocytes and copper deposition in the choroid plexus, with degenerated neurons showing pyknotic nuclei and dense eosinophilic cytoplasm. In conclusion, the present study shows the first evidence in vivo that chronic copper toxicity causes impaired spatial memory and neuromuscular coordination, swelling of astrocytes, decreased serum AChE activity, copper deposition in the choroid plexus, neuronal degeneration, and augmented levels of copper and zinc in the hippocampus of male Wistar rats.     

Changes in Antioxidant Defense Enzymes after d-amphetamine Exposure: Implications as an Animal Model of Mania

Studies have demonstrated that oxidative stress is associated with amphetamine-induced neurotoxicity, but little is known about the adaptations of antioxidant enzymes in the brain after amphetamine exposure. We studied the effects of acute and chronic amphetamine administration on superoxide dismutase (SOD) and catalase (CAT) activity, in a rodent model of mania. Male Wistar rats received either a single IP injection of d-amphetamine (1 mg/kg, 2 mg/kg, or 4 mg/kg) or vehicle (acute treatment). In the chronic treatment rats received a daily IP injection of either d-amphetamine (1 mg/kg, 2 mg/kg, or 4 mg/kg) or vehicle for 7 days. Locomotor behavior was assessed using the open field test. SOD and CAT activities were measured in the prefrontal cortex, hippocampus, and striatum. Acute and to a greater extent chronic amphetamine treatment increased locomotor behavior and affected SOD and CAT activities in the prefrontal cortex, hippocampus and striatum. Our findings suggest that amphetamine exposure is associated with an imbalance between SOD and CAT activity in the prefrontal cortex, hippocampus and striatum.     

Proline promotes decrease in glutamate uptake in slices of cerebral cortex and hippocampus of rats

In the present study we first investigated the in vitro and in vivo effects of proline on glutamate uptake in the cerebral cortex and hippocampus slices of rats. The action of alpha-tocopherol and/or ascorbic acid on the effects elicited by administration of proline was also evaluated. For in vitro studies, proline (30.0 microM and 1.0 mM) was added to the incubation medium. For acute administration, 29-day-old rats received one subcutaneous injection of proline (18.2 micromol/g body weight) or saline (control) and were sacrificed 1 h later. Results showed that addition of proline in the assay (in vitro studies) reduces glutamate uptake in both cerebral structures. Administration of proline (in vivo studies) reduces glutamate uptake in the cerebral cortex, but not in the hippocampal slices of rats. In another set of experiments, 22-day-old rats were pretreated for one week with daily administration of alpha-tocopherol (40 mg/kg) or ascorbic acid (100 mg/kg) or with both vitamins. Twelve hours after the last vitamins injection, rats received a single injection of proline or saline and were killed 1 h later. Pretreatment with alpha-tocopherol and/or ascorbic acid did not prevent the effect of proline administration on glutamate uptake. alpha-Tocopherol plus ascorbic acid prevented the inhibitory effect of acute hyperprolinemia on Na(+),K(+) -ATPase activity in the cerebral cortex of 29-day-old rats. The data indicate that the effect of proline on reduction of glutamate uptake and Na(+),K(+) -ATPase activity may be, at least in part, involved in the brain dysfunction observed in hyperprolinemic patients.     

Polyacrylamide gel electrophoresis of rat brain acetylcholinesterase: isoenzymes of normal rat brain

Abstract— Triton-solubilized acetylcholinesterase (EC 3.1.1.7) of rat brain was submitted to vertical flatbed polyacrylamide gel electrophoresis. Three anodally migrating isoenzyme zones with low relative mobilities could be resolved, each of which on quantitative densitometry appeared to consist of more than one subzone. More than 50 per cent of the total AChE activity was exhibited by the isoenzyme zone closest to the origin (isoenzyme zone 3). Regional differences in AChE isoenzyme activity were quantitative only with the caudate-putamen complex, midbrain, pons and medulla oblongata exhibiting relatively high content of the three isoenzymes and the cerebral cortex and olfactory bulb possessing weak isoenzyme activities. Intermediate levels of isoenzyme activities were observed in the cerebellum and hippocampus. In all areas examined, the relative percentage values for each isoenzyme remained constant. AChE isoenzymes from the forebrain, brain stem and cerebellum of 15- and 30-day-old rats appeared to have identical patterns. In brain stem, no quantitative differences could be detected in the isoenzyme activities between 15 and 30 days of age. At both ages, the isoenzymes of male and female rats did not show any qualitative differences. The single cholinesterase (EC 3.1.1.8) isoenzyme which could be identified in brain stem supernatants of 30-day-old rats was weakly reactive and appeared to have the same relative mobility as the major acetylcholinesterase zone, zone 3. Acetylcholinesterase isoenzymes failed to demonstrate any differential response toward varying concentrations of inhibitors and to changes in pH. While there were basic similarities in the acetylcholinesterase and cholinesterase isoenzyme patterns of brain, serum, liver, skeletal muscle and intestine, brain alone exhibited a marked preponderance of the acetylcholinesterase isoenzyme zone 3.