Mol Neurobiol DOI 10.1007/s12035-014-8873-8
Intracerebral Administration of BDNF Protects Rat Brain Against Oxidative Stress Induced by Ouabain in an Animal Model of Mania Samira S. Valvassori & Camila O. Arent & Amanda V. Steckert & Roger B. Varela & Luciano K. Jornada & Paula T. Tonin & Josiane Budni & Edemilson Mariot & Flávio Kapczinski & João Quevedo
Received: 24 June 2014 / Accepted: 18 August 2014 # Springer Science+Business Media New York 2014
Abstract Several studies have suggested that alterations in brain-derived neurotrophic factor (BDNF) and increased oxidative stress have a central role in bipolar disorder (BD). Intracerebroventricular (ICV) injection of ouabain (OUA) in rats alters oxidative stress parameters and decreases BDNF levels in the brain. In this context, the present study aims to investigate the effects of BDNF ICV administration on BDNF levels and oxidative stress parameters in brains of rats submitted to animal model of mania induced by OUA. Wistar rats received an ICV injection of OUA, artificial cerebrospinal fluid (ACSF), OUA plus BDNF, or ACSF plus BDNF. Locomotor activity and risk-taking behavior in the rats were measured using the open-field test. In addition, we analyzed the BDNF levels and oxidative stress parameters (TBARS, Carbonyl, CAT, SOD, GR, and GPx) in the frontal cortex and
S. S. Valvassori (*) : C. O. Arent : A. V. Steckert : R. B. Varela : L. K. Jornada : P. T. Tonin : J. Budni : E. Mariot : J. Quevedo Laboratório de Neurociências, Programa de Pós-Graduação em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil e-mail: [email protected] F. Kapczinski : J. Quevedo (*) Center for Experimental Models in Psychiatry, Department of Psychiatry and Behavioral Sciences, The University of Texas Medical School at Houston, Houston, TX, USA e-mail: [email protected] F. Kapczinski Laboratoryof Molecular Psychiatric and National Institute for Translational Medicine (INCT-TM), Centro de Pesquisas, Hospital de Clínicas de Porto Alegre, 90035-003 Porto Alegre, RS, Brazil
hippocampus of rats. The BDNF was unable to reverse the ouabain-induced hyperactivity and risk-taking behavior. Nevertheless, BDNF treatment increased BDNF levels, modulated the antioxidant enzymes, and protected the OUAinduced oxidative damage in the brain of rats. These results suggest that BDNF alteration observed in BD patients may be associated with oxidative damage, both seen in this disorder. Keywords Bipolar disorder . Animal model of mania . Ouabain . BDNF . Oxidative stress
Introduction Bipolar disorder (BD) is one of the most common, severe, and persistent mental illnesses with psychosocial disability and significant medical and psychiatric comorbidity [1–3]. Despite these facts, little is known about the precise pathophysiology of BD [4]. It is postulated that the reduced neuronal and glial density in certain brain regions, such as the frontal cortex and hippocampus, are involved with mood and cognitive impairment, both observed in BD [5–7]. Accumulating data suggest that alterations in brain-derived neurotrophic factor (BDNF) and increased oxidative stress may play a key role in the pathophysiology of BD. BDNF is associated with cell survival by inhibiting cellular apoptosis through regulation of Bcl-2 family members, which are necessary for the survival and function of neurons [8]. BDNF binds to the receptor tyrosine kinase B (TrKB), activating diverse intracellular cascades involved in cellular survival and growth [9]. Studies showing decreased peripheral BDNF levels during manic and depressive episodes suggest that this neurotrophin may be an important target for the understanding of BD pathophysiology [10–13]. Postmortem
Mol Neurobiol
studies also have demonstrated that BDNF levels and TrkB, its receptor, decreased in brain from bipolar patients [14, 15]. Interestingly, there is evidence showing that oxidative stress may be increased in conditions where BDNF is described to be decreased in BD [16, 17], indicating an association of BDNF and oxidative stress in BD. Furthermore, preclinical studies have shown that mood stabilizers, lithium, and valproate are able to increase BDNF levels and protect the rat brain against oxidative damage [18, 19]. Studies have demonstrated that oxidative stress could be involved in cell death and in many pathological conditions [20, 21]. The oxidative stress may result from increased reactive oxygen species (ROS) generation, a defective antioxidant defense system, or both [22, 23]. The ROS in the mammalian brain are directly responsible for cell and tissue function and dysfunction. The main source of ROS formation is the mitochondria in the course of electron transport in the oxidative phosphorylation chain. During this process is generated mainly superoxide radical (O2−) and hydrogen peroxide (H2O2). These highly unstable molecules have potential to damage cellular proteins, lipids, carbohydrates, and nucleic acids. The brain is more susceptible to ROS because it metabolizes 20 % of total body oxygen and has a limited amount of antioxidant capacity [22]. A considerable amount of studies supports evidence that oxidative stress can be related to the pathophysiology of BD [24–26]. The most important enzymes of the antioxidant system are the following: superoxide dismutase (SOD), which converts the O2− into H2O2, and catalase (CAT) and glutathione peroxidase (GPx), both of which detoxify the hydrogen peroxide [27]. Reduced glutathione (GSH) has an important role in the neuronal antioxidant system; it is a tripeptide, composed of glutamate, cysteine, and glycine. GSH is used as an electron donor for GPx action, leading to the production of oxidized glutathione (GSSG). Another important enzyme of this system is the glutathione reductase (GR) that is required to catalyze the reduction of GSSG to GSH [27, 28]. The effects of stimulants on behavior have been widely used as an animal model of mania because they induce psychomotor agitation, which is commonly observed during mania [29]. Intracerebroventricular (ICV) injection of ouabain induced hyperactivity in rats by inhibiting Na+/K+-ATPase [30], and it has been suggested as a relevant animal model of mania [29, 31]. Studies with this animal model showed that manic-like hyperactivity induced by ouabain is associated with severe brain damage, increasing formation of lipid and protein oxidation products in the frontal cortex and hippocampus of rats [19, 32, 33]. In addition, the ICV ouabain injection decreased BDNF levels in the hippocampus of animals submitted to this model [18, 34]. In this context, several studies have reported Na+/K+-ATPase alterations in BD patients [30, 35, 36].
Because of these facts, we designed the present study to investigate the effects of ICV BDNF administration on behavior and oxidative stress parameters in the frontal cortex and hippocampus of rats subjected to an animal model of mania induced by ouabain.
Methods and Materials Animals We conducted the study using adult male Wistar rats, weighting between 250–300 g, obtained from our breeding colony. Five animals were housed to a cage, on a 12-h light/ dark cycle (lights on at 7:00 am), with free access to food (standard diet for laboratory animals–NUVILAB CR-1®, Brazil) and water. The rats were maintained at approximately 20±1 °C in a humidified atmosphere of 50 %. All experimental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Brazilian Society for Neuroscience and Behaviour (SBNeC). This study was approved by the local ethics committee (Comitê de Ética no Uso de Animais da Universidade do Extremo Sul Catarinense–32/ 2009).
Drugs Ketamine and xylasine were obtained from Chemie Uetikon/ Germany and Bayer/Germany, respectively. Ouabain (Sigma– Aldrich, USA) and human recombinant BDNF (Chemicon International, USA) were dissolved in artificial cerebrospinal fluid [(aCSF) 145 mM NaCly 2.7 mM KCly1.0 mM MgCl2y2.0 mM NaH2PO4, pH 7.4].
Surgical Procedure Animals were intraperitoneally anesthetized with ketamine (80 mg/kg) and xylasine (10 mg/kg). In a stereotaxic apparatus, the skin of the rat skull was removed and a 27 gauge 9mm guide cannula was placed at 0.9 mm posterior to bregma, 1.5 mm right from the midline, and 1.0 mm above the lateral brain ventricle. Through a 2-mm hole made at the cranial bone, a cannula was implanted 2.6 mm ventral to the superior surface of the skull, according to Paxinos and Watson [37]. The cannula was fixed with jeweler acrylic cement, which was also used to cover the small surface of the skull that was exposed by removal of skin. After surgery, rats were maintained in single cages and recovered from surgery for 3 days (see Fig. 1).
Mol Neurobiol Fig. 1 Schematic illustration of experimental protocol performed with the animal model of mania induced by ouabain (OUA). aCSF=artificial cerebrospinal fluid, BDNF=brain-derived neurotrophic factor. All animals were submitted to the behavioral test (n=10 animals per group); however, for oxidative stress, analysis used an n=5 and for evaluation of BDNF levels also used an n=5 animals per group
Treatment We designed this model to reproduce the management of an acute manic episode. Animals [n = 40 (10 animals each group)] received a single ICV injection of 4 μl of ouabain 10−3 M dissolved in artificial cerebrospinal fluid (aCSF) or 4 μL of aCSF alone on the fourth day following surgery [32, 38]. A 30-gauge cannula was placed into the guide cannula and connected by a polyethylene tube to a microsyringe. The tip of the cannula infusion protruded 1.0 mm beyond the cannula guide aiming at the right lateral brain ventricle. Fifteen minutes after ouabain or aCSF infusion, we delivered human recombinant BDNF (0.25 μg/1 μl per side) or aCSF (1 μl) into the lateral ventricle. There were four groups as follows: group 1, aCSF + aCSF; group 2, aCSF + BDNF; group 3, Ouabain + aCSF; and group 4, Ouabain + BDNF. We measured locomotor activity 7 days after the ICV administration of drugs (see Fig. 1). Note, BDNF dose used in this study was based in a previous study from Alonso and colleagues [39].
Behavioral Assessment We used the open-field task to assess locomotor activity. The task was performed in a 40×60 cm open field surrounded by 50-cm-high walls made of brown plywood with a frontal glass wall. The floor of the open field was divided into nine equal rectangles with black lines. A video camera was installed to register the animal’s behavior. Rats were placed individually in the open field for 5 min, and the following parameters were analyzed: latency to first crossing, number of crossings (times that the animal enters another square with the all paws), number of rearing, and number of visits to center of the apparatus [40, 41]. After each trial, the apparatus was cleaned with a 10 % ethanol solution. The rats were killed by decapitation right after the last open-field, and brain regions (frontal cortex and hippocampus) were dissected, rapidly frozen, and stored at −70 °C until assayed.
Measurement of Brain-Derived Neurotrophic Factor (BDNF) Levels For the analysis of BDNF, the frontal cortex and hippocampus were homogenized in phosphate buffer solution (PBS) with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N0N0-tetraacetic acid (EGTA). The homogenates were centrifuged at 10,000g for 20 min, and the supernatants were collected for quantification of BDNF levels. BDNF levels were measured using sandwich enzyme-linked immunosorbent assay, using commercial kits according to the manufacturer’s instructions (BDNF from Chemicon, USA). Microtiter plates (96-well flat bottom) were coated for 24 h with the samples diluted 1:2 in sample diluent, and standard curve ranged from 7.8 to 500 pg of BDNF. Then, plates were washed four times with sample diluents. Monoclonal anti-BDNF rabbit antibody diluted 1:1,000 in sample diluent was incubated for 3 h at room temperature. After washing, a second incubation with antirabbit antibody peroxidase conjugated diluted 1:1,000 for 1 h at room temperature was carried out. After the addition of streptavidin enzyme, substrate, and stop solution, the amount of BDNF was determined for absorbance in 450 nm. In order to calculate the concentration of BDNF in the sample, Microsoft Excel 2010 was used and the known concentrations of BDNF were plotted on the X-axis and corresponding OD on the Y-axis. The standard curve of BDNF analysis resulted in a graph that shows a direct relationship between BDNF concentrations (pg/mL) and corresponding ODs (absorbance). Thiobarbituric Acid Reactive Species (TBARS) Content in the Rat Brain The rat frontal cortex and hippocampus for TBARS were homogenized in Na2PO4 KCl (30 mM Na2PO4, 14 mM KCl, pH=7.4). As a marker of lipid peroxidation, we measured the formation of TBARS during an acid-heating reaction, as previously described [42]. Briefly, the samples were mixed with 1 ml of trichloroacetic acid 10 % and 1 ml of
Mol Neurobiol
thiobarbituric acid 0.67 % and then heated in a boiling water bath for 15 min. TBARS were determined by the absorbance at 535 nm. Protein Carbonyl Content The rat frontal cortex and hippocampus for carbonyl protein were homogenized in KCl KH2PO4 (12 mM KCl, 0.038 mM KH2PO4, pH=7.4). Oxidative damage to proteins was measured by the quantification of carbonyl groups based on the reaction with dinitrophenylhydrazine (DNPH), as previously described [43]. Proteins were precipitated by the addition of 20 % trichloroacetic acid and were dissolved in DNPH; the absorbance was read at 370 nm.
decreased between T1 and T2 (in nmol). T1 is the time of first reading (A1) (in min). T2 is the time of second reading (A2) (in min). V is the pretreated sample volume added into the reaction well (in ml). The GR kit measures the rate of oxidation of NADPH to NADP+, which is accompanied by a decrease in absorbance at 340 nM. One unit of GR is defined as the amount of enzyme that will cause the oxidation of 1.0 nmol of NADPH to NADP+ per minute at 25 °C. Protein Determination All biochemical measures were normalized to the protein content with bovine albumin as standard [44]. Statistical Analysis
Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GPx), and Glutathione Reductase (GR) Activities Measurement The rat frontal cortex and hippocampus for SOD, CAT, GPx, and GR activities were homogenized in Na2PO4 KCl (30 mM Na2PO4, 14 mM KCl, pH=7.4). We measured SOD, CAT, GPx, and GR activities using the corresponding assay kits by Cayman Chemical. The method utilized to evaluated SOD activity employs xanthine and xanthine oxidase to generate superoxide radicals that react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT) to form a formazan dye that is assayed spectrophotometrically at 492 nm at 37 °C. The inhibition in the production of the chromogen is proportional to the activity of SOD present in the sample; one unit of SOD causes 50 % inhibition of the rate of the reduction of INT under the conditions of the assay. CAT is an enzyme able to degrade peroxides, including hydrogen peroxide (H2O2), and its activity assessment is based upon establishing the rate of H2O2 degradation spectrophotometrically at 240 nm at 25 °C. CAT activity was calculated in terms of micromoles of H2O2 consumed per minute per milligram of protein, using a molar extinction coefficient of 43.6 M−1 cm−1. The GPx kit utilizes an indirect measure of GPx activity. Oxidized glutathione is produced via reduction of hydrogen peroxide by GPx and is recycled to its reduced state by GR and nicotinamide adenine dinucleotide phosphate oxidized (NADP+). The oxidation of nicotinamide adenine dinucleotide phosphate reduced (NADPH) to NADP+ is accompanied by decreased absorbance of light at 340 nM. One unit of GPx is defined as the amount of enzyme that will cause the oxidation of 1.0 nmol of NADPH to NADP+ per minute at 25 °C. The standard curve of GPx analysis resulted in a graph that shows a direct relationship between NADPH concentrations (nmol) and corresponding ODs (absorbance). To calculate the GPx activity, MasterPlex ReaderFit was used and the following formula was used: GPx=B/T1–T2*V=nmol/min/ ml=mU/mL. Where B is the NADPH amount that was
Results are presented as the means±standard deviations. Differences among experimental groups were determined by two-way ANOVA followed by Tukey’s post hoc test. A value of p
Intracerebral Administration of BDNF Protects Rat Brain Against Oxidative Stress Induced by Ouabain in an Animal Model of Mania Samira S. Valvassori & Camila O. Arent & Amanda V. Steckert & Roger B. Varela & Luciano K. Jornada & Paula T. Tonin & Josiane Budni & Edemilson Mariot & Flávio Kapczinski & João Quevedo
Received: 24 June 2014 / Accepted: 18 August 2014 # Springer Science+Business Media New York 2014
Abstract Several studies have suggested that alterations in brain-derived neurotrophic factor (BDNF) and increased oxidative stress have a central role in bipolar disorder (BD). Intracerebroventricular (ICV) injection of ouabain (OUA) in rats alters oxidative stress parameters and decreases BDNF levels in the brain. In this context, the present study aims to investigate the effects of BDNF ICV administration on BDNF levels and oxidative stress parameters in brains of rats submitted to animal model of mania induced by OUA. Wistar rats received an ICV injection of OUA, artificial cerebrospinal fluid (ACSF), OUA plus BDNF, or ACSF plus BDNF. Locomotor activity and risk-taking behavior in the rats were measured using the open-field test. In addition, we analyzed the BDNF levels and oxidative stress parameters (TBARS, Carbonyl, CAT, SOD, GR, and GPx) in the frontal cortex and
S. S. Valvassori (*) : C. O. Arent : A. V. Steckert : R. B. Varela : L. K. Jornada : P. T. Tonin : J. Budni : E. Mariot : J. Quevedo Laboratório de Neurociências, Programa de Pós-Graduação em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil e-mail: [email protected] F. Kapczinski : J. Quevedo (*) Center for Experimental Models in Psychiatry, Department of Psychiatry and Behavioral Sciences, The University of Texas Medical School at Houston, Houston, TX, USA e-mail: [email protected] F. Kapczinski Laboratoryof Molecular Psychiatric and National Institute for Translational Medicine (INCT-TM), Centro de Pesquisas, Hospital de Clínicas de Porto Alegre, 90035-003 Porto Alegre, RS, Brazil
hippocampus of rats. The BDNF was unable to reverse the ouabain-induced hyperactivity and risk-taking behavior. Nevertheless, BDNF treatment increased BDNF levels, modulated the antioxidant enzymes, and protected the OUAinduced oxidative damage in the brain of rats. These results suggest that BDNF alteration observed in BD patients may be associated with oxidative damage, both seen in this disorder. Keywords Bipolar disorder . Animal model of mania . Ouabain . BDNF . Oxidative stress
Introduction Bipolar disorder (BD) is one of the most common, severe, and persistent mental illnesses with psychosocial disability and significant medical and psychiatric comorbidity [1–3]. Despite these facts, little is known about the precise pathophysiology of BD [4]. It is postulated that the reduced neuronal and glial density in certain brain regions, such as the frontal cortex and hippocampus, are involved with mood and cognitive impairment, both observed in BD [5–7]. Accumulating data suggest that alterations in brain-derived neurotrophic factor (BDNF) and increased oxidative stress may play a key role in the pathophysiology of BD. BDNF is associated with cell survival by inhibiting cellular apoptosis through regulation of Bcl-2 family members, which are necessary for the survival and function of neurons [8]. BDNF binds to the receptor tyrosine kinase B (TrKB), activating diverse intracellular cascades involved in cellular survival and growth [9]. Studies showing decreased peripheral BDNF levels during manic and depressive episodes suggest that this neurotrophin may be an important target for the understanding of BD pathophysiology [10–13]. Postmortem
Mol Neurobiol
studies also have demonstrated that BDNF levels and TrkB, its receptor, decreased in brain from bipolar patients [14, 15]. Interestingly, there is evidence showing that oxidative stress may be increased in conditions where BDNF is described to be decreased in BD [16, 17], indicating an association of BDNF and oxidative stress in BD. Furthermore, preclinical studies have shown that mood stabilizers, lithium, and valproate are able to increase BDNF levels and protect the rat brain against oxidative damage [18, 19]. Studies have demonstrated that oxidative stress could be involved in cell death and in many pathological conditions [20, 21]. The oxidative stress may result from increased reactive oxygen species (ROS) generation, a defective antioxidant defense system, or both [22, 23]. The ROS in the mammalian brain are directly responsible for cell and tissue function and dysfunction. The main source of ROS formation is the mitochondria in the course of electron transport in the oxidative phosphorylation chain. During this process is generated mainly superoxide radical (O2−) and hydrogen peroxide (H2O2). These highly unstable molecules have potential to damage cellular proteins, lipids, carbohydrates, and nucleic acids. The brain is more susceptible to ROS because it metabolizes 20 % of total body oxygen and has a limited amount of antioxidant capacity [22]. A considerable amount of studies supports evidence that oxidative stress can be related to the pathophysiology of BD [24–26]. The most important enzymes of the antioxidant system are the following: superoxide dismutase (SOD), which converts the O2− into H2O2, and catalase (CAT) and glutathione peroxidase (GPx), both of which detoxify the hydrogen peroxide [27]. Reduced glutathione (GSH) has an important role in the neuronal antioxidant system; it is a tripeptide, composed of glutamate, cysteine, and glycine. GSH is used as an electron donor for GPx action, leading to the production of oxidized glutathione (GSSG). Another important enzyme of this system is the glutathione reductase (GR) that is required to catalyze the reduction of GSSG to GSH [27, 28]. The effects of stimulants on behavior have been widely used as an animal model of mania because they induce psychomotor agitation, which is commonly observed during mania [29]. Intracerebroventricular (ICV) injection of ouabain induced hyperactivity in rats by inhibiting Na+/K+-ATPase [30], and it has been suggested as a relevant animal model of mania [29, 31]. Studies with this animal model showed that manic-like hyperactivity induced by ouabain is associated with severe brain damage, increasing formation of lipid and protein oxidation products in the frontal cortex and hippocampus of rats [19, 32, 33]. In addition, the ICV ouabain injection decreased BDNF levels in the hippocampus of animals submitted to this model [18, 34]. In this context, several studies have reported Na+/K+-ATPase alterations in BD patients [30, 35, 36].
Because of these facts, we designed the present study to investigate the effects of ICV BDNF administration on behavior and oxidative stress parameters in the frontal cortex and hippocampus of rats subjected to an animal model of mania induced by ouabain.
Methods and Materials Animals We conducted the study using adult male Wistar rats, weighting between 250–300 g, obtained from our breeding colony. Five animals were housed to a cage, on a 12-h light/ dark cycle (lights on at 7:00 am), with free access to food (standard diet for laboratory animals–NUVILAB CR-1®, Brazil) and water. The rats were maintained at approximately 20±1 °C in a humidified atmosphere of 50 %. All experimental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Brazilian Society for Neuroscience and Behaviour (SBNeC). This study was approved by the local ethics committee (Comitê de Ética no Uso de Animais da Universidade do Extremo Sul Catarinense–32/ 2009).
Drugs Ketamine and xylasine were obtained from Chemie Uetikon/ Germany and Bayer/Germany, respectively. Ouabain (Sigma– Aldrich, USA) and human recombinant BDNF (Chemicon International, USA) were dissolved in artificial cerebrospinal fluid [(aCSF) 145 mM NaCly 2.7 mM KCly1.0 mM MgCl2y2.0 mM NaH2PO4, pH 7.4].
Surgical Procedure Animals were intraperitoneally anesthetized with ketamine (80 mg/kg) and xylasine (10 mg/kg). In a stereotaxic apparatus, the skin of the rat skull was removed and a 27 gauge 9mm guide cannula was placed at 0.9 mm posterior to bregma, 1.5 mm right from the midline, and 1.0 mm above the lateral brain ventricle. Through a 2-mm hole made at the cranial bone, a cannula was implanted 2.6 mm ventral to the superior surface of the skull, according to Paxinos and Watson [37]. The cannula was fixed with jeweler acrylic cement, which was also used to cover the small surface of the skull that was exposed by removal of skin. After surgery, rats were maintained in single cages and recovered from surgery for 3 days (see Fig. 1).
Mol Neurobiol Fig. 1 Schematic illustration of experimental protocol performed with the animal model of mania induced by ouabain (OUA). aCSF=artificial cerebrospinal fluid, BDNF=brain-derived neurotrophic factor. All animals were submitted to the behavioral test (n=10 animals per group); however, for oxidative stress, analysis used an n=5 and for evaluation of BDNF levels also used an n=5 animals per group
Treatment We designed this model to reproduce the management of an acute manic episode. Animals [n = 40 (10 animals each group)] received a single ICV injection of 4 μl of ouabain 10−3 M dissolved in artificial cerebrospinal fluid (aCSF) or 4 μL of aCSF alone on the fourth day following surgery [32, 38]. A 30-gauge cannula was placed into the guide cannula and connected by a polyethylene tube to a microsyringe. The tip of the cannula infusion protruded 1.0 mm beyond the cannula guide aiming at the right lateral brain ventricle. Fifteen minutes after ouabain or aCSF infusion, we delivered human recombinant BDNF (0.25 μg/1 μl per side) or aCSF (1 μl) into the lateral ventricle. There were four groups as follows: group 1, aCSF + aCSF; group 2, aCSF + BDNF; group 3, Ouabain + aCSF; and group 4, Ouabain + BDNF. We measured locomotor activity 7 days after the ICV administration of drugs (see Fig. 1). Note, BDNF dose used in this study was based in a previous study from Alonso and colleagues [39].
Behavioral Assessment We used the open-field task to assess locomotor activity. The task was performed in a 40×60 cm open field surrounded by 50-cm-high walls made of brown plywood with a frontal glass wall. The floor of the open field was divided into nine equal rectangles with black lines. A video camera was installed to register the animal’s behavior. Rats were placed individually in the open field for 5 min, and the following parameters were analyzed: latency to first crossing, number of crossings (times that the animal enters another square with the all paws), number of rearing, and number of visits to center of the apparatus [40, 41]. After each trial, the apparatus was cleaned with a 10 % ethanol solution. The rats were killed by decapitation right after the last open-field, and brain regions (frontal cortex and hippocampus) were dissected, rapidly frozen, and stored at −70 °C until assayed.
Measurement of Brain-Derived Neurotrophic Factor (BDNF) Levels For the analysis of BDNF, the frontal cortex and hippocampus were homogenized in phosphate buffer solution (PBS) with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N0N0-tetraacetic acid (EGTA). The homogenates were centrifuged at 10,000g for 20 min, and the supernatants were collected for quantification of BDNF levels. BDNF levels were measured using sandwich enzyme-linked immunosorbent assay, using commercial kits according to the manufacturer’s instructions (BDNF from Chemicon, USA). Microtiter plates (96-well flat bottom) were coated for 24 h with the samples diluted 1:2 in sample diluent, and standard curve ranged from 7.8 to 500 pg of BDNF. Then, plates were washed four times with sample diluents. Monoclonal anti-BDNF rabbit antibody diluted 1:1,000 in sample diluent was incubated for 3 h at room temperature. After washing, a second incubation with antirabbit antibody peroxidase conjugated diluted 1:1,000 for 1 h at room temperature was carried out. After the addition of streptavidin enzyme, substrate, and stop solution, the amount of BDNF was determined for absorbance in 450 nm. In order to calculate the concentration of BDNF in the sample, Microsoft Excel 2010 was used and the known concentrations of BDNF were plotted on the X-axis and corresponding OD on the Y-axis. The standard curve of BDNF analysis resulted in a graph that shows a direct relationship between BDNF concentrations (pg/mL) and corresponding ODs (absorbance). Thiobarbituric Acid Reactive Species (TBARS) Content in the Rat Brain The rat frontal cortex and hippocampus for TBARS were homogenized in Na2PO4 KCl (30 mM Na2PO4, 14 mM KCl, pH=7.4). As a marker of lipid peroxidation, we measured the formation of TBARS during an acid-heating reaction, as previously described [42]. Briefly, the samples were mixed with 1 ml of trichloroacetic acid 10 % and 1 ml of
Mol Neurobiol
thiobarbituric acid 0.67 % and then heated in a boiling water bath for 15 min. TBARS were determined by the absorbance at 535 nm. Protein Carbonyl Content The rat frontal cortex and hippocampus for carbonyl protein were homogenized in KCl KH2PO4 (12 mM KCl, 0.038 mM KH2PO4, pH=7.4). Oxidative damage to proteins was measured by the quantification of carbonyl groups based on the reaction with dinitrophenylhydrazine (DNPH), as previously described [43]. Proteins were precipitated by the addition of 20 % trichloroacetic acid and were dissolved in DNPH; the absorbance was read at 370 nm.
decreased between T1 and T2 (in nmol). T1 is the time of first reading (A1) (in min). T2 is the time of second reading (A2) (in min). V is the pretreated sample volume added into the reaction well (in ml). The GR kit measures the rate of oxidation of NADPH to NADP+, which is accompanied by a decrease in absorbance at 340 nM. One unit of GR is defined as the amount of enzyme that will cause the oxidation of 1.0 nmol of NADPH to NADP+ per minute at 25 °C. Protein Determination All biochemical measures were normalized to the protein content with bovine albumin as standard [44]. Statistical Analysis
Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GPx), and Glutathione Reductase (GR) Activities Measurement The rat frontal cortex and hippocampus for SOD, CAT, GPx, and GR activities were homogenized in Na2PO4 KCl (30 mM Na2PO4, 14 mM KCl, pH=7.4). We measured SOD, CAT, GPx, and GR activities using the corresponding assay kits by Cayman Chemical. The method utilized to evaluated SOD activity employs xanthine and xanthine oxidase to generate superoxide radicals that react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT) to form a formazan dye that is assayed spectrophotometrically at 492 nm at 37 °C. The inhibition in the production of the chromogen is proportional to the activity of SOD present in the sample; one unit of SOD causes 50 % inhibition of the rate of the reduction of INT under the conditions of the assay. CAT is an enzyme able to degrade peroxides, including hydrogen peroxide (H2O2), and its activity assessment is based upon establishing the rate of H2O2 degradation spectrophotometrically at 240 nm at 25 °C. CAT activity was calculated in terms of micromoles of H2O2 consumed per minute per milligram of protein, using a molar extinction coefficient of 43.6 M−1 cm−1. The GPx kit utilizes an indirect measure of GPx activity. Oxidized glutathione is produced via reduction of hydrogen peroxide by GPx and is recycled to its reduced state by GR and nicotinamide adenine dinucleotide phosphate oxidized (NADP+). The oxidation of nicotinamide adenine dinucleotide phosphate reduced (NADPH) to NADP+ is accompanied by decreased absorbance of light at 340 nM. One unit of GPx is defined as the amount of enzyme that will cause the oxidation of 1.0 nmol of NADPH to NADP+ per minute at 25 °C. The standard curve of GPx analysis resulted in a graph that shows a direct relationship between NADPH concentrations (nmol) and corresponding ODs (absorbance). To calculate the GPx activity, MasterPlex ReaderFit was used and the following formula was used: GPx=B/T1–T2*V=nmol/min/ ml=mU/mL. Where B is the NADPH amount that was
Results are presented as the means±standard deviations. Differences among experimental groups were determined by two-way ANOVA followed by Tukey’s post hoc test. A value of p
