J Neural Transm DOI 10.1007/s00702-014-1302-3
TRANSLATIONAL NEUROSCIENCES - ORIGINAL ARTICLE
Folic acid prevented cognitive impairment in experimental pneumococcal meningitis Tatiana Barichello • Jaqueline S. Generoso • Lutiana R. Simo˜es • Amanda V. Steckert • Ana Paula Moreira Diogo Dominguini • Paˆmela Ferrari • Carolina Gubert • Fla´vio Kapczinski • Luciano K. Jornada • Lucineia G. Danielski • Fabricia Petronilho • Josiane Budni • Joa˜o Quevedo
•
Received: 21 March 2014 / Accepted: 22 August 2014 Ó Springer-Verlag Wien 2014
Abstract Streptococcus pneumoniae is a common cause of bacterial meningitis, with a high mortality rate and neurological sequelae. In contrast, folic acid plays an important role in neuroplasticity and the preservation of neuronal integrity. In the present study, we evaluated the influence of folic acid on memory, oxidative damage, enzymatic defence, and brain-derived neurotrophic factor (BDNF) expression in experimental pneumococcal meningitis. In animals that received folic acid at a dose of 10 or 50 mg, there was a reduction in both crossing and rearing during an open-field task compared with the training session, demonstrating habituation memory. During a step-down inhibitory avoidance task, there was a difference between the training and the test sessions, demonstrating aversive memory. In the hippocampus, BDNF expression decreased in the meningitis group; however, adjuvant treatment with 10 mg of folic acid increased BDNF expression, decreased lipid peroxidation, protein carbonylation, nitrate/nitrite levels, and
myeloperoxidase activity and increased superoxide dismutase activity. In frontal cortex adjuvant treatment with 10 mg of folic acid decreased lipid peroxidation and protein carbonylation. There is substantial interest in the role of folic acid and related pathways in nervous system function and in folic acid’s potential therapeutic effects. Here, adjuvant treatment with vitamin B9 prevented memory impairment in experimental pneumococcal meningitis.
T. Barichello (&) J. S. Generoso L. R. Simo˜es A. V. Steckert A. P. Moreira Laborato´rio de Microbiologia Experimental, Programa de Po´sGraduac¸a˜o em Cieˆncias da Sau´de (PPGCS), Unidade Acadeˆmica de Cieˆncias da Sau´de (UNASAU), Universidade do Extremo Sul Catarinense, Criciu´ma, SC 88806-000, Brazil e-mail: [email protected]
P. Ferrari C. Gubert F. Kapczinski Laborato´rio de Psiquiatria Molecular, Hospital de Clı´nicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
T. Barichello F. Kapczinski F. Petronilho 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 A. V. Steckert D. Dominguini L. K. Jornada J. Quevedo Laborato´rio de Neurocieˆncias, Programa de Po´s-Graduac¸a˜o em Cieˆncias da Sau´de, Unidade Acadeˆmica de Cieˆncias da Sau´de, Universidade do Extremo Sul Catarinense, Criciu´ma, SC, Brazil
Keywords Meningitis Streptococcus pneumoniae Folic acid Memory Oxidative stress BDNF
Introduction Bacterial meningitis is a life-threatening infectious disease of the central nervous system (CNS) (Brouwer et al. 2010), characterised by acute purulent infection of the
L. G. Danielski F. Petronilho Laborato´rio de Fisiopatologia Clı´nica e Experimental, Programa de Po´s-Graduac¸a˜o em Cieˆncias da Sau´de, Universidade do Sul de Santa Catarina, Tubara˜o, SC, Brazil J. Budni Laborato´rio de Neurodegenerac¸a˜o, Programa de Po´s-Graduac¸a˜o em Cieˆncias da Sau´de, Unidade Acadeˆmica de Cieˆncias da Sau´de, Universidade do Extremo Sul Catarinense, Criciu´ma, SC, Brazil
123
T. Barichello et al.
meninges, affecting the pia, the arachnoid, and the subarachnoid space (van de Beek et al. 2006). The fatal outcome rate of pneumococcal meningitis varies from 16 to 37 %, and approximately 30–52 % of survivors suffer from neurological sequelae, such as seizures, sensory– motor deficits, hearing loss, and learning and memory impairment (van de Beek et al. 2002; Hoogman et al. 2007). Streptococcus pneumoniae is a common causative agent of bacterial meningitis acquired in the community, accounting for two-thirds of cases in Europe and the United States (Brouwer et al. 2010). Pneumococci are highly immunogenic and may be recognised by antigenpresenting cells (APCs) through binding to pattern recognition receptors. Upon activation of these receptors, APCs can release various cytokines, which induce a cascade of inflammatory reactions, including the recruitment of neutrophils (Barichello et al. 2013b). Furthermore, the stimulated immune system may contribute to hyperhomocysteinaemia in certain diseases and in sepsis patients with poor outcomes (Ploder et al. 2010). Folic acid is a cofactor in one-carbon metabolism that promotes the restoration of methionine from homocysteine-mediated preservation of neuronal integrity (Kronenberg et al. 2009). Folic acid has also been shown to prevent memory deficit and the reduction in BDNF levels induced by homocysteine injection (Matte et al. 2009). Another potential way to prevent damage in pneumococcal meningitis is related to the ability of vitamin B to bind to an antigen-presenting protein that stimulates specialised immune cells, which has been suggested as a novel mechanism by which the immune system detects microbial infections (Kjer-Nielsen et al. 2012). We hypothesised that cognitive impairment in adult Wistar rats subjected to pneumococcal meningitis would be reversed or eased by folic acid. In the current study, we evaluated the effects of folic acid on memory, oxidative damage, enzymatic defence and BDNF expression in the brains of Wistar rats subjected to pneumococcal meningitis.
Animal model of meningitis Adult male Wistar rats (250–350 g body weight) from our breeding colony were used in the experiments. All procedures were approved by the Animal Care and Experimentation Committee of UNESC/22/2013, Brazil, and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80–23, revised in 1996). All surgical procedures and bacterial inoculations were performed under anaesthesia, which consisted of intraperitoneal administration of ketamine (6.6 mg/kg), xylazine (0.3 mg/kg), and acepromazine (0.16 mg/kg) (Grandgirard et al. 2007; Barichello et al. 2010b). The rats underwent a cisterna magna tap with a 23-gauge needle. The animals received either 10 lL of artificial cerebral spinal fluid (CSF) as a control or an equivalent volume of S. pneumoniae suspension (meningitis). At the time of inoculation, the animals received fluid replacement and were then returned to their cages (2 mL of sterile saline subcutaneously) (Irazuzta et al. 2008; Barichello et al. 2010b). Eighteen hours later, the animals were assessed clinically using the following scores: 1 = coma; 2 = does not turn upright when positioned on the back; 3 = turn upright within 30 s; 4 = turns upright in \5 s; 5 = normal (Leib et al. 2001). CSF was obtained by puncture of the cisterna magna and 5 lL was cultured on blood agar plate and incubated for 24 h at 35–37 °C in 5 % CO2. At this time point (18 h after pneumococcal meningitis induction), animals were randomly assigned to receive ceftriaxone (100 mg/kg body weight given ip, during 7 days, twice daily, Roche Pharma, RocephinÒ, Brazil) (Barichello et al. 2013c). Ten days after inoculation, the animals were free from infection. All blood cultures that were performed during this period were negative. The animals recovered their weight and grooming habits; their blood counts returned to control levels, and the reactive protein C values were negative (data not shown). Drugs and reagents
Experimental procedures Infecting organism S. pneumoniae (serotype 3) was cultured overnight in 10 mL of Todd Hewitt broth. The cultures were then diluted in fresh medium and grown until the logarithmic phase. These cultures were centrifuged for 10 min at 5,0009g and resuspended in sterile saline at a concentration of 5 9 109 colony-forming units (cfu/mL). The size of the inoculum was confirmed using quantitative cultures (Grandgirard et al. 2007; Barichello et al. 2010a).
123
Folic acid (Sigma Chemical Company, St. Louis, USA) was dissolved in distilled water and administered orally (po) by gavage at a dose of 10 or 50 mg/kg once per day for 7 days, starting 18 h after the induction of pneumococcal meningitis. The folic acid solution was prepared before administration in a volume of 1 mL/kg (Budni et al. 2013). Organisation of the experimental groups The animals were divided into six groups: control, control treated with folic acid at 10 mg/kg, control treated with
Folic acid prevented cognitive impairment
folic acid at 50 mg/kg, meningitis, meningitis treated with folic acid at 10 mg/kg, and meningitis treated with folic acid at 50 mg/kg (10 animals per group). Behavioural tasks Ten days after the induction of meningitis, the animals were randomly assigned to be tested on one of the behavioural tasks: the open field and step-down inhibitory avoidance tasks. After performing the task, the animals were killed and the hippocampus was immediately isolated on dry ice and stored at -80 °C for analysis.
Oxidative stress parameters Malondialdehyde equivalents To determine oxidative damage to lipids, we measured the formation of thiobarbituric acid reactive species (TBARS) during an acid-heating reaction, as previously described by Draper and Hadley (1990). The samples were mixed with 1 mL of 10 % trichloroacetic acid and 1 mL of 0.67 % thiobarbituric acid and then heated in a boiling water bath for 30 min. Malondialdehyde (MDA) equivalents were determined in both the tissue and the submitochondrial particles of the rat brain using a spectrophotometer at an absorbance of 532 nm.
Open-field task Protein carbonyl formation Behaviour was assessed in an open-field apparatus to evaluate both locomotor and exploratory activities. This apparatus was a 40 9 60 cm open field surrounded by 50-cm-high walls made of brown plywood with a front glass wall. Black lines divided the floor of the open field into nine rectangles. The animals were gently placed in the left rear quadrant and were allowed to explore the arena for 5 min. The number of crossings (i.e., the number of times that the animals crossed the black lines, which assesses locomotor activity) and rearing movements (i.e., the exploration behaviour observed in rats subjected to a new environment) were measured. The same person, who was blind to the group treatments, performed all behavioural testing by manual analyses (Vianna et al. 2000). Step-down inhibitory avoidance task The apparatus and procedures have been described in previous reports (Quevedo et al. 1997; Roesler et al. 2003). Briefly, the training apparatus was a 50 9 25 9 25 cm acrylic box (Albarsch, Porto Alegre, Brazil) whose floor consisted of parallel calibre stainless steel bars (1 mm diameter) spaced 1 cm apart. A 7-cm-wide, 2.5-cm-high platform was placed on the floor of the box, against the left wall. In the training trial, the animals were placed on the platform, and their latency to step down on the grid with all four paws was measured with an automatic device. Immediately after stepping down on the grid, the animals received a 0.4 mA foot shock for 2.0 s and were returned to their home cage. A retention test trial was performed 24 h after training (long-term memory). The retention test trial was procedurally identical to the training trial, except that no foot shock was presented. The retention test stepdown latency (maximum, 180 s) was used as a measure of inhibitory avoidance retention (Izquierdo et al. 1998; Bevilaqua et al. 2003).
Oxidative damage to proteins was assessed by determining the amount of carbonyl groups based on their reaction with 0.02 g 2,4-dinitrophenylhydrazine (DNPH) dissolved in 10 mL of water, as previously described by Levine et al. (1994) (Levine et al. 1990). Proteins were precipitated by the addition of 20 % trichloroacetic acid and re-dissolved in DNPH. The absorbance was monitored using a spectrophotometer at 370 nm. Superoxide dismutase activity The method used to assay superoxide dismutase (SOD) activity is based on the capacity of pyrogallol to autoxidise, which is a process that is highly dependent on O2-2, a substrate for SOD (Bannister and Calabrese 1987). Inhibition of the autoxidation of this compound occurs when SOD is present, and the enzymatic activity can be indirectly assayed using a temperature-controlled double-beam spectrophotometer at an absorbance of 420 nm. A calibration curve was performed using purified SOD as the standard to calculate the specific activity of SOD present in the samples. A 50 % inhibition of pyrogallol autoxidation was defined as one unit of SOD, and the specific activity was represented as units per mg of protein. Catalase activity Catalase (CAT) activity was assayed using a temperaturecontrolled double-beam spectrophotometer. This method is based on the disappearance of H2O2 at an absorbance of 240 nm in a reaction medium that contains 20 mM H2O2, 0.1 % Triton X-100, 10 mM potassium phosphate buffer (pH 7.0), and 0.1–0.3 mg protein/mL (Laemmli 1970). CAT unit was defined as 1 mol of H2O2 consumed per minute, and the specific activity was reported as units per mg of protein.
123
T. Barichello et al.
Determination of nitrite and nitrate concentrations The concentrations of nitrite and nitrate were measured in other aliquots of tissue using the Griess reaction by adding 100 ll of reagent [0.1 % (w/v) in H2O and 1 % (w/v) sulphanilamide in 5 % (v/v) H3PO4 concentrate volume (1:1)] to 100 ll of sample. The optical density at 550 nm (OD550) was measured using an ELISA microplate reader (Green et al. 1982). Myeloperoxidase activity Tissues were homogenised (50 mg/mL) in 0.5 % hexadecyltrimethylammonium bromide and centrifuged at 15,000 9 g for 40 min. The suspension was then sonicated three times for 30 s. A supernatant aliquot was mixed with a solution of 1.6 mM tetramethylbenzidine and 1 mM H2O2. Myeloperoxidase (MPO) activity was determined colorimetrically with a spectrophotometer by analysing changes in the absorbance at 650 nm at a temperature of 37 °C (De Young et al. 1989).
the dependent variable was not on an interval scale. Data regarding habituation to the open-field task are reported as the mean difference (training trial–test trial) and the 95 % confidence interval of the difference, as analysed by a paired Student’s t test. Data from the inhibitory avoidance task are reported as medians and interquartile ranges. Data within individual groups were analysed by a Friedman test and multiple comparisons by a Wilcoxon test. Comparisons among groups were performed using Mann–Whitney U tests. All data regarding BDNF levels and oxidative stress were reported as the mean ± SEM and were analysed by two-way ANOVA (meningitis and folic acid), followed by a Bonferroni post hoc test. In our study, we used univariate linear model, and not the multivariate hierarchical analysis or study of intercluster correlation being held because each object (rats) generated an observation, with no variation among objects and only variation between objects (Aarts et al. 2014). P values \0.05 were considered statistically significant. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) software version 20.0.
Assessment of BDNF expression Results The measurement of BDNF levels was performed as previously described (Kauer-Sant’Anna et al. 2007). BDNF serum levels were measured with a sandwich ELISA, using a commercial kit according to the manufacturer’s instructions (Millipore, USA). Briefly, microtitre plates (96-well flat bottom) were coated overnight at 4 °C with samples diluted at 1:100 in sample diluent, and the standard curve ranged from 7.8 to 500 pg/mL BDNF. The plates were then washed four times with wash buffer, and biotinylated mouse anti-human BDNF monoclonal antibody (diluted 1:1,000 in sample diluent) was added, followed by incubation for 3 h at room temperature. After washing, a second incubation with streptavidin–horseradish peroxidase conjugate solution (diluted 1:1,000) was performed for 1 h at room temperature. After the addition of substrate and stop solution, the amount of BDNF was determined (absorbance set at 450 nm). The standard curve demonstrated a direct relationship between optical density (OD) and BDNF concentration. Total protein was measured by a Bradford assay (samples diluted at 1:200), using bovine serum albumin (BSA) as a standard.
Statistics The normality of the data was examined by a Shapiro–Wilk test. Because the null hypothesis was that the data were normally distributed, parametric statistical significance tests were used, except for the inhibitory avoidance task because
123
The bacterial concentration in the CSF at 18 h after pneumococcal meningitis induction were 1.35 ± 0.212 9 103 in the meningitis treated with 10 mg/kg and 1.3 ± 0.141 9 103 in meningitis treated with 50 mg/kg of folic acid. Folic acid did not have antimicrobial effect in vitro, data not shown. In the open-field task (Fig. 1), we illustrated the influence of folic acid on habituation memory 10 days after pneumococcal meningitis induction. There were no differences in the number of crossings and rearing movements among groups during habituation to the open-field training session, demonstrating no difference in motor and exploratory activity (data not shown). However, when we examined the difference between the training and the test sessions, we verified a decrease in crossing and rearing in the untreated meningitis group compared with all other groups, demonstrating habituation memory impairment in this group (P \ 0.05). In Fig. 2, we showed the step-down inhibitory avoidance task 10 days after pneumococcal meningitis induction. There was a difference between the training and the test sessions in the untreated control group, control group/ 10 mg, control group/50 mg, meningitis group/10 mg, and meningitis group/50 mg, demonstrating aversive memory in these groups (P \ 0.05). In the untreated meningitis group, there was no difference between the training and the test sessions, demonstrating impairment of aversive memory in this group.
Folic acid prevented cognitive impairment
Training
50
*
*
*
140
*
20
10
Saline
10 mg/Kg
50 mg/kg
Saline
Control
Rearings (Difference between training and test session)
*
Test
160
30
0
b
*
180
40
Latency (sec)
Crossings (Difference between training and test session)
a
10 mg/Kg
50 mg/kg
120 100 80 60 40 20
Meningitis
0
30
Saline
10 mg/Kg 50 mg/kg Saline Control
Meningitis
Fig. 2 Latency to step down in the inhibitory avoidance task 10 days after pneumococcal meningitis induction. The data from the inhibitory avoidance task are reported as medians and interquartile ranges. Data within individual groups were analysed by a Friedman test and multiple comparisons by a Wilcoxon test. Comparisons among groups were performed using Mann–Whitney U tests, with n = 10 animals per group. *p \ 0.05 indicates differences between the training and the test sessions for the same group
20
*
10
10 mg/Kg 50 mg/kg
0 Saline
10 mg/Kg Control
50 mg/kg
Saline
10 mg/Kg
50 mg/kg
Meningitis
Fig. 1 Habituation to the open-field task 10 days after pneumococcal meningitis induction. The numbers of crossings (a) and rearing (b) movements are reported as the mean difference (training trial–test trial) and the 95 % confidence interval of the difference. These results were analysed by a paired Student’s t test, with n = 10 animals per group. The symbols indicate statistical significance when the training and test sessions were compared (*p \ 0.05)
In Figs. 3 and 4, we illustrated the effects of folic acid on oxidative damage and enzymatic defence in the hippocampus and frontal cortex 10 days after pneumococcal meningitis induction. TBARS levels decreased in the hippocampus and frontal cortex of animals that received adjuvant treatment with 10 mg of folic acid (P \ 0.05), whereas in the meningitis group that received 50 mg, TBARS levels increased compared with levels in the meningitis/10 mg group (P \ 0.05) (Figs. 3a, 4a). Carbonyl levels decreased in the hippocampus and frontal cortex of animals that received adjuvant treatment with 10 mg of folic acid (P \ 0.05). In contrast, in the meningitis group that received 50 mg of folic acid, carbonyl levels increased in the frontal cortex compared with levels in the meningitis/10 mg group (P \ 0.05) (Figs. 3b, 4b). Nitrite/nitrate concentrations decreased in the hippocampus of animals that received adjuvant treatment with 10 mg of folic acid (P \ 0.05, Fig. 3c). MPO concentrations
increased in the hippocampus of the control group that received adjuvant treatment with 50 mg of acid folic (P \ 0.05), whereas adjuvant treatment decreased MPO levels in the hippocampus of animals that received adjuvant treatment with 10 mg of folic acid (P \ 0.05, Fig. 3d). SOD activity increased only in the hippocampus of the control group that received 10 mg of folic acid compared with activity in the untreated control group (P \ 0.05, Fig. 3e). The levels of CAT did not change in either group (Figs. 3f, 4f). In Fig. 5, we showed BDNF expression in the hippocampus and frontal cortex 10 days after pneumococcal meningitis induction. BDNF levels increased in the hippocampus of the meningitis group that received adjuvant treatment with 10 mg of folic acid (P \ 0.05, Fig. 5a). In the frontal cortex, there was a decrease in BDNF levels in the control group/50 mg, meningitis group/10 mg, and meningitis group/50 mg (P \ 0.05, Fig. 5b).
Discussion In the present study, we demonstrated the influence of vitamin B9 on memory, oxidative damage, enzymatic defence, and BDNF expression in experimental pneumococcal meningitis. S. pneumoniae multiplies within the subarachnoid space, initiating a complex immune response (Barichello et al. 2013b). Consequently,
123
a
Saline
0.03
10 mg/kg
#
50 mg/kg
0.02
*
0.01
0.00 Control
Meningitis
Protein carbonyls (nmol/mg protein)
MDA equivalents (nmol/mg protein)
T. Barichello et al.
b 0.025
10 mg/kg
0.020
0.005 0.000
δ
50 mg/kg
0.2
*
0.1
0.0 Control
Meningitis
MPO activity (mU/mg protein)
Nitrite/nitrate concentration (nmol/mg protein)
10 mg/kg
Control
0.015
10 mg/kg 50 mg/kg
*
0.010
0.005
0.000 Control
Meningitis
Meningitis
Saline
0.03
10 mg/kg
δ
50 mg/kg
* *
0.02
0.01
0.00 Control
f CAT activity (U/mg protein)
SOD activity (U/mg protein)
e Saline
*
0.010
d 0.3
δ
50 mg/kg
0.015
c Saline
Saline
Meningitis
Saline
0.6
10 mg/kg 50 mg/kg
0.4
0.2
0.0 Control
Meningitis
Fig. 3 Oxidative damage and enzymatic defence in the hippocampus 10 days after pneumococcal meningitis induction. TBARS levels (a), protein carbonyl levels (b), nitrite/nitrate concentrations (c), MPO activity (d), SOD activity (e), and CAT activity (f). The data are reported as the mean ± SEM. These results were analysed by twoway ANOVA (meningitis and folic acid), followed by a Bonferroni
post hoc test, with n = 10 animals per group. The symbols *p \ 0.05 indicate statistical significance when with compared the untreated meningitis group, #p \ 0.05 indicate statistical significance when compared with meningitis group treated with 10 mg/kg; dp \ 0.05 indicate statistical significance when compared with untreated meningitis vs control saline group
polymorphonuclear cells are attracted from the bloodstream and then activated, releasing large amounts of NO, O2, and H2O2. This formation generates oxidative and nitrosative stress and subsequent lipid peroxidation, mitochondrial damage, and BBB breakdown, which contribute to cell injury during pneumococcal meningitis (Barichello et al. 2013a, b). In the present study, we showed that adjuvant treatment with folic acid at a concentration of 10 mg decreased oxidative stress, increased BDNF levels, however, both concentrations of 10 and 50 mg prevented cognitive impairment in experimental pneumococcal meningitis. Folic acid plays an important
role in neuroplasticity and the preservation of neuronal integrity (Kronenberg et al. 2009). Folic acid supplementation enhances repairs of the adult CNS (Iskandar et al. 2004) and also promotes hippocampal neurogenesis and preserves cognitive functions after stroke in the adult brain (Zhang et al. 2012). Folate is a cofactor in onecarbon metabolism that promotes the restoration of methionine from homocysteine (Kronenberg et al. 2009). Stimulation of the immune system may contribute to hyperhomocysteinaemia in certain diseases and in sepsis patients with poor outcomes (Ploder et al. 2010). Supplementation with folic acid leads to a decrease in the
123
a 0.04
Saline
δ
#
10 mg/kg
0.03
50 mg/kg
0.02
*
0.01
0.00 Control
Meningitis
Protein carbonyls (nmol/mg protein)
MDA equivalents (nmol/mg protein)
Folic acid prevented cognitive impairment
b 0.025
0.015
#
*
*
0.010 0.005 0.000
MPO activity (mU/mg protein)
Nitrite/nitrate concentration (nmol/mg protein)
50 mg/kg
0.10
0.05
0.00 Control
Control
0.03
Meningitis
Saline
δ
10 mg/kg 50 mg/kg
0.02
0.01
0.00
Meningitis
Control
Meningitis
f Saline
0.008
10 mg/kg 50 mg/kg
0.006
0.004
δ
0.002
0.000 Control
Meningitis
CAT activity (U/mg protein)
e SOD activity (U/mg protein)
50 mg/kg
d Saline 10 mg/kg
0.15
δ
10 mg/kg
0.020
c 0.20
Saline
Saline
0.8
10 mg/kg 50 mg/kg
0.6
δ
0.4
0.2
0.0 Control
Meninigitis
Fig. 4 Oxidative damage and enzymatic defence in the frontal cortex 10 days after pneumococcal meningitis induction. TBARS levels (a), protein carbonyl levels (b), nitrite/nitrate concentrations (c), MPO activity (d), SOD activity (e), and CAT activity (f). The data are reported as the mean ± SEM. These results were analysed by twoway ANOVA (meningitis and folic acid), followed by a Bonferroni
post hoc test, with n = 10 animals per group. The symbols *p \ 0.05 indicate statistical significance when compared with the untreated meningitis group, #p \ 0.05 indicate statistical significance when compared with meningitis group treated with 10 mg/kg; dp \ 0.05 indicate statistical significance when compared with untreated meningitis vs control saline group
homocysteine level and rescues pathogenic and epigenetic modifications, showing protective efficacy against homocysteine-induced neurotoxicity (Kalani et al. 2013). Furthermore, treatment with folic acid prevents memory deficit and the reduction in BDNF levels induced by homocysteine injection (Matte et al. 2009). Additionally, alteration of maternal micronutrients, such as folic acid, vitamin B12, and omega-3 fatty acids, reduces BDNF levels in preterm pregnancy (Dhobale and Joshi 2012). In our study, BDNF levels increased in the hippocampus of animals that received adjuvant treatment with 10 mg of folic acid. This upregulated expression of BDNF in experimental pneumococcal meningitis could have an
important role in cognitive protection. The free radicalscavenging properties and possible antioxidant activity of folic acid have been reported. Folic acid inhibits microsomal lipid peroxidation and can efficiently scavenge oxidising free radicals (Joshi et al. 2001). During pneumococcal meningitis, large amounts of oxidative stress are produced (Klein et al. 2006). Accordingly, in our study, folic acid decreased lipid peroxidation, protein carbonylation, nitrate/nitrite levels, and MPO activity and increased SOD activity in the hippocampus. There is substantial interest in the role of folic acid and related pathways in nervous system function (Reynolds 2006) and the potential therapeutic effects of folic acid.
123
T. Barichello et al.
50
BDNF (pg/ug of protein)
References
Saline
a
*
10 mg/kg 50 mg/kg
40 30 20
δ 10 0 Control
Meningitis
b
Saline
30
BDNF (pg/ug of protein)
10 mg/kg 50 mg/kg 20
*
*
*
10
0 Control
Meningitis
Fig. 5 BDNF expression in the hippocampus and frontal cortex 10 days after pneumococcal meningitis induction. The results are shown for the hippocampus (a) and the frontal cortex (b). BDNF levels were assessed by ELISA, and the results are presented in pg per 100 mg of tissue, with n = 10 per group. All data regarding BDNF levels and oxidative stress are reported as the mean ± SEM. These results were analysed by two-way ANOVA (meningitis and folic acid), followed by a Bonferroni post hoc test. The symbols *p \ 0.05 indicate statistical significance compared with the untreated control group; dp \ 0.05 indicate statistical significance when compared with untreated meningitis vs control saline group
In conclusion, only adjuvant treatment with folic acid at 10 mg prevented memory impairment increasing BDNF expression in the hippocampus, decreasing oxidative stress. In addition both doses of 10 and 50 mg prevented memory deficit in animals subjected to pneumococcal meningitis. Acknowledgments Laboratory of Experimental Microbiology (Brazil) is one of the centres of the National Institute for Translational Medicine (INCT-TM) and one of the members of the Center of Excellence in Applied Neurosciences of Santa Catarina (NENASC). This research was supported by grants from CNPq (JQ, FK and TB), FAPESC (JQ and TB), and UNESC (JQ and TB). JQ and TB are CNPq Research Fellows. JSG and LRS is holder of a CAPES studentship. Conflict of interest All authors have read the journal’s policy on the disclosure of potential conflicts of interest. The authors declare that they have no conflict of interest.
123
Aarts E, Verhage M, Veenvliet JV, Dolan CV, van der Sluis S (2014) A solution to dependency: using multilevel analysis to accommodate nested data. Nat Neurosci 17(4):491–496 Bannister JV, Calabrese L (1987) Assays for superoxide dismutase. Methods Biochem Anal 32:279–312 Barichello T, Belarmino E Jr, Comim CM, Cipriano AL, Generoso JS, Savi GD, Stertz L, Kapczinski F, Quevedo J (2010a) Correlation between behavioral deficits and decreased brainderived neurotrophic (correction of neurotrofic) factor in neonatal meningitis. J Neuroimmunol 223(1–2):73–76 Barichello T, Savi GD, Silva GZ, Generoso JS, Bellettini G, Vuolo F, Petronilho F, Feier G, Comim CM, Quevedo J, Dal-Pizzol F (2010b) Antibiotic therapy prevents, in part, the oxidative stress in the rat brain after meningitis induced by Streptococcus pneumoniae. Neurosci Lett 478(2):93–96 Barichello T, Generoso JS, Milioli G, Elias SG, Teixeira AL (2013a) Pathophysiology of bacterial infection of the central nervous system and its putative role in the pathogenesis of behavioral changes. Rev Bras Psiquiatr 35(1):81–87 Barichello T, Generoso JS, Simoes LR, Elias SG, Quevedo J (2013b) Role of oxidative stress in the pathophysiology of pneumococcal meningitis. Oxidative Med Cell Longev 2013:371465 Barichello T, Simoes LR, Generoso JS, Sangiogo G, Danielski LG, Florentino D, Dominguini D, Comim CM, Petronilho F, Quevedo J (2013c) Erythropoietin prevents cognitive impairment and oxidative parameters in Wistar rats subjected to pneumococcal meningitis. Transl Res 163(5):503–513 Bevilaqua LR, Kerr DS, Medina JH, Izquierdo I, Cammarota M (2003) Inhibition of hippocampal Jun N-terminal kinase enhances short-term memory but blocks long-term memory formation and retrieval of an inhibitory avoidance task. Eur J Neurosci 17(4):897–902 Brouwer MC, Tunkel AR, van de Beek D (2010) Epidemiology, diagnosis, and antimicrobial treatment of acute bacterial meningitis. Clin Microbiol Rev 23(3):467–492 Budni J, Zomkowski AD, Engel D, Santos DB, dos Santos AA, Moretti M, Valvassori SS, Ornell F, Quevedo J, Farina M, Rodrigues AL (2013) Folic acid prevents depressive-like behavior and hippocampal antioxidant imbalance induced by restraint stress in mice. Exp Neurol 240:112–121 De Young LM, Kheifets JB, Ballaron SJ, Young JM (1989) Edema and cell infiltration in the phorbol ester-treated mouse ear are temporally separate and can be differentially modulated by pharmacologic agents. Agents Actions 26(3–4):335–341 Dhobale M, Joshi S (2012) Altered maternal micronutrients (folic acid, vitamin B(12)) and omega 3 fatty acids through oxidative stress may reduce neurotrophic factors in preterm pregnancy. J Maternal Fetal Neonatal Med 25(4):317–323 Draper HH, Hadley M (1990) Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 186:421–431 Grandgirard D, Steiner O, Tauber MG, Leib SL (2007) An infant mouse model of brain damage in pneumococcal meningitis. Acta Neuropathol 114(6):609–617 Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR (1982) Analysis of nitrate, nitrite, and (15 N) nitrate in biological fluids. Anal Biochem 126(1):131–138 Hoogman M, van de Beek D, Weisfelt M, de Gans J, Schmand B (2007) Cognitive outcome in adults after bacterial meningitis. J Neurol Neurosurg Psychiatry 78(10):1092–1096 Irazuzta J, Pretzlaff RK, Zingarelli B (2008) Caspases inhibition decreases neurological sequelae in meningitis. Crit Care Med 36(5):1603–1606
Folic acid prevented cognitive impairment Iskandar BJ, Nelson A, Resnick D, Skene JH, Gao P, Johnson C, Cook TD, Hariharan N (2004) Folic acid supplementation enhances repair of the adult central nervous system. Ann Neurol 56(2):221–227 Izquierdo I, Barros DM, Mello e Souza T, de Souza MM, Izquierdo LA, Barros DM, Medina JH (1998) Mechanisms for memory types differ. Nature 393(6686):635–636 Joshi R, Adhikari S, Patro BS, Chattopadhyay S, Mukherjee T (2001) Free radical scavenging behavior of folic acid: evidence for possible antioxidant activity. Free Radic Biol Med 30(12): 1390–1399 Kalani A, Kamat PK, Givvimani S, Brown K, Metreveli N, Tyagi SC, Tyagi N (2013) Nutri-epigenetics ameliorates blood-Brain barrier damage and neurodegeneration in hyperhomocysteinemia: role of folic acid. J Mol Neurosci 13:13 Kauer-Sant’Anna M, Andreazza AC, Valvassori SS, Martins MR, Barbosa LM, Schwartsmann G, Roesler R, Quevedo J, Kapczinski F (2007) A gastrin-releasing peptide receptor antagonist blocks D-amphetamine-induced hyperlocomotion and increases hippocampal NGF and BDNF levels in rats. Peptides 28(7): 1447–1452 Kjer-Nielsen L, Patel O, Corbett AJ, Le Nours J, Meehan B, Liu L, Bhati M, Chen Z, Kostenko L, Reantragoon R, Williamson NA, Purcell AW, Dudek NL, McConville MJ, O’Hair RA, Khairallah GN, Godfrey DI, Fairlie DP, Rossjohn J, McCluskey J (2012) MR1 presents microbial vitamin B metabolites to MAIT cells. Nature 491(7426):717–723 Klein M, Koedel U, Pfister HW (2006) Oxidative stress in pneumococcal meningitis: a future target for adjunctive therapy? Prog Neurobiol 80(6):269–280 Kronenberg G, Colla M, Endres M (2009) Folic acid, neurodegenerative and neuropsychiatric disease. Curr Mol Med 9(3): 315–323 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680–685 Leib SL, Clements JM, Lindberg RL, Heimgartner C, Loeffler JM, Pfister LA, Tauber MG, Leppert D (2001) Inhibition of matrix metalloproteinases and tumour necrosis factor alpha converting enzyme as adjuvant therapy in pneumococcal meningitis. Brain 124(Pt 9):1734–1742
Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, Ahn BW, Shaltiel S, Stadtman ER (1990) Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 186:464–478 Matte C, Pereira LO, Dos Santos TM, Mackedanz V, Cunha AA, Netto CA, Wyse AT (2009) Acute homocysteine administration impairs memory consolidation on inhibitory avoidance task and decreases hippocampal brain-derived neurotrophic factor immunocontent: prevention by folic acid treatment. Neuroscience 163(4):1039–1045 Ploder M, Kurz K, Spittler A, Neurauter G, Roth E, Fuchs D (2010) Early increase of plasma homocysteine in sepsis patients with poor outcome. Mol Med 16(11–12):498–504 Quevedo J, Vianna M, Zanatta MS, Roesler R, Izquierdo I, Jerusalinsky D, Quillfeldt JA (1997) Involvement of mechanisms dependent on NMDA receptors, nitric oxide and protein kinase A in the hippocampus but not in the caudate nucleus in memory. Behav Pharmacol 8(8):713–717 Reynolds E (2006) Vitamin B12, folic acid, and the nervous system. Lancet Neurol 5(11):949–960 Roesler R, Schroder N, Vianna MR, Quevedo J, Bromberg E, Kapczinski F, Ferreira MB (2003) Differential involvement of hippocampal and amygdalar NMDA receptors in contextual and aversive aspects of inhibitory avoidance memory in rats. Brain Res 975(1–2):207–213 van de Beek D, Schmand B, de Gans J, Weisfelt M, Vaessen H, Dankert J, Vermeulen M (2002) Cognitive impairment in adults with good recovery after bacterial meningitis. J Infect Dis 186(7):1047–1052 van de Beek D, de Gans J, Tunkel AR, Wijdicks EF (2006) Community-acquired bacterial meningitis in adults. N Engl J Med 354(1):44–53 Vianna MR, Alonso M, Viola H, Quevedo J, de Paris F, Furman M, de Stein ML, Medina JH, Izquierdo I (2000) Role of hippocampal signaling pathways in long-term memory formation of a nonassociative learning task in the rat. Learn Mem 7(5):333–340 Zhang X, Huang G, Liu H, Chang H, Wilson JX (2012) Folic acid enhances Notch signaling, hippocampal neurogenesis, and cognitive function in a rat model of cerebral ischemia. Nutr Neurosci 15(2):55–61
123
TRANSLATIONAL NEUROSCIENCES - ORIGINAL ARTICLE
Folic acid prevented cognitive impairment in experimental pneumococcal meningitis Tatiana Barichello • Jaqueline S. Generoso • Lutiana R. Simo˜es • Amanda V. Steckert • Ana Paula Moreira Diogo Dominguini • Paˆmela Ferrari • Carolina Gubert • Fla´vio Kapczinski • Luciano K. Jornada • Lucineia G. Danielski • Fabricia Petronilho • Josiane Budni • Joa˜o Quevedo
•
Received: 21 March 2014 / Accepted: 22 August 2014 Ó Springer-Verlag Wien 2014
Abstract Streptococcus pneumoniae is a common cause of bacterial meningitis, with a high mortality rate and neurological sequelae. In contrast, folic acid plays an important role in neuroplasticity and the preservation of neuronal integrity. In the present study, we evaluated the influence of folic acid on memory, oxidative damage, enzymatic defence, and brain-derived neurotrophic factor (BDNF) expression in experimental pneumococcal meningitis. In animals that received folic acid at a dose of 10 or 50 mg, there was a reduction in both crossing and rearing during an open-field task compared with the training session, demonstrating habituation memory. During a step-down inhibitory avoidance task, there was a difference between the training and the test sessions, demonstrating aversive memory. In the hippocampus, BDNF expression decreased in the meningitis group; however, adjuvant treatment with 10 mg of folic acid increased BDNF expression, decreased lipid peroxidation, protein carbonylation, nitrate/nitrite levels, and
myeloperoxidase activity and increased superoxide dismutase activity. In frontal cortex adjuvant treatment with 10 mg of folic acid decreased lipid peroxidation and protein carbonylation. There is substantial interest in the role of folic acid and related pathways in nervous system function and in folic acid’s potential therapeutic effects. Here, adjuvant treatment with vitamin B9 prevented memory impairment in experimental pneumococcal meningitis.
T. Barichello (&) J. S. Generoso L. R. Simo˜es A. V. Steckert A. P. Moreira Laborato´rio de Microbiologia Experimental, Programa de Po´sGraduac¸a˜o em Cieˆncias da Sau´de (PPGCS), Unidade Acadeˆmica de Cieˆncias da Sau´de (UNASAU), Universidade do Extremo Sul Catarinense, Criciu´ma, SC 88806-000, Brazil e-mail: [email protected]
P. Ferrari C. Gubert F. Kapczinski Laborato´rio de Psiquiatria Molecular, Hospital de Clı´nicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
T. Barichello F. Kapczinski F. Petronilho 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 A. V. Steckert D. Dominguini L. K. Jornada J. Quevedo Laborato´rio de Neurocieˆncias, Programa de Po´s-Graduac¸a˜o em Cieˆncias da Sau´de, Unidade Acadeˆmica de Cieˆncias da Sau´de, Universidade do Extremo Sul Catarinense, Criciu´ma, SC, Brazil
Keywords Meningitis Streptococcus pneumoniae Folic acid Memory Oxidative stress BDNF
Introduction Bacterial meningitis is a life-threatening infectious disease of the central nervous system (CNS) (Brouwer et al. 2010), characterised by acute purulent infection of the
L. G. Danielski F. Petronilho Laborato´rio de Fisiopatologia Clı´nica e Experimental, Programa de Po´s-Graduac¸a˜o em Cieˆncias da Sau´de, Universidade do Sul de Santa Catarina, Tubara˜o, SC, Brazil J. Budni Laborato´rio de Neurodegenerac¸a˜o, Programa de Po´s-Graduac¸a˜o em Cieˆncias da Sau´de, Unidade Acadeˆmica de Cieˆncias da Sau´de, Universidade do Extremo Sul Catarinense, Criciu´ma, SC, Brazil
123
T. Barichello et al.
meninges, affecting the pia, the arachnoid, and the subarachnoid space (van de Beek et al. 2006). The fatal outcome rate of pneumococcal meningitis varies from 16 to 37 %, and approximately 30–52 % of survivors suffer from neurological sequelae, such as seizures, sensory– motor deficits, hearing loss, and learning and memory impairment (van de Beek et al. 2002; Hoogman et al. 2007). Streptococcus pneumoniae is a common causative agent of bacterial meningitis acquired in the community, accounting for two-thirds of cases in Europe and the United States (Brouwer et al. 2010). Pneumococci are highly immunogenic and may be recognised by antigenpresenting cells (APCs) through binding to pattern recognition receptors. Upon activation of these receptors, APCs can release various cytokines, which induce a cascade of inflammatory reactions, including the recruitment of neutrophils (Barichello et al. 2013b). Furthermore, the stimulated immune system may contribute to hyperhomocysteinaemia in certain diseases and in sepsis patients with poor outcomes (Ploder et al. 2010). Folic acid is a cofactor in one-carbon metabolism that promotes the restoration of methionine from homocysteine-mediated preservation of neuronal integrity (Kronenberg et al. 2009). Folic acid has also been shown to prevent memory deficit and the reduction in BDNF levels induced by homocysteine injection (Matte et al. 2009). Another potential way to prevent damage in pneumococcal meningitis is related to the ability of vitamin B to bind to an antigen-presenting protein that stimulates specialised immune cells, which has been suggested as a novel mechanism by which the immune system detects microbial infections (Kjer-Nielsen et al. 2012). We hypothesised that cognitive impairment in adult Wistar rats subjected to pneumococcal meningitis would be reversed or eased by folic acid. In the current study, we evaluated the effects of folic acid on memory, oxidative damage, enzymatic defence and BDNF expression in the brains of Wistar rats subjected to pneumococcal meningitis.
Animal model of meningitis Adult male Wistar rats (250–350 g body weight) from our breeding colony were used in the experiments. All procedures were approved by the Animal Care and Experimentation Committee of UNESC/22/2013, Brazil, and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80–23, revised in 1996). All surgical procedures and bacterial inoculations were performed under anaesthesia, which consisted of intraperitoneal administration of ketamine (6.6 mg/kg), xylazine (0.3 mg/kg), and acepromazine (0.16 mg/kg) (Grandgirard et al. 2007; Barichello et al. 2010b). The rats underwent a cisterna magna tap with a 23-gauge needle. The animals received either 10 lL of artificial cerebral spinal fluid (CSF) as a control or an equivalent volume of S. pneumoniae suspension (meningitis). At the time of inoculation, the animals received fluid replacement and were then returned to their cages (2 mL of sterile saline subcutaneously) (Irazuzta et al. 2008; Barichello et al. 2010b). Eighteen hours later, the animals were assessed clinically using the following scores: 1 = coma; 2 = does not turn upright when positioned on the back; 3 = turn upright within 30 s; 4 = turns upright in \5 s; 5 = normal (Leib et al. 2001). CSF was obtained by puncture of the cisterna magna and 5 lL was cultured on blood agar plate and incubated for 24 h at 35–37 °C in 5 % CO2. At this time point (18 h after pneumococcal meningitis induction), animals were randomly assigned to receive ceftriaxone (100 mg/kg body weight given ip, during 7 days, twice daily, Roche Pharma, RocephinÒ, Brazil) (Barichello et al. 2013c). Ten days after inoculation, the animals were free from infection. All blood cultures that were performed during this period were negative. The animals recovered their weight and grooming habits; their blood counts returned to control levels, and the reactive protein C values were negative (data not shown). Drugs and reagents
Experimental procedures Infecting organism S. pneumoniae (serotype 3) was cultured overnight in 10 mL of Todd Hewitt broth. The cultures were then diluted in fresh medium and grown until the logarithmic phase. These cultures were centrifuged for 10 min at 5,0009g and resuspended in sterile saline at a concentration of 5 9 109 colony-forming units (cfu/mL). The size of the inoculum was confirmed using quantitative cultures (Grandgirard et al. 2007; Barichello et al. 2010a).
123
Folic acid (Sigma Chemical Company, St. Louis, USA) was dissolved in distilled water and administered orally (po) by gavage at a dose of 10 or 50 mg/kg once per day for 7 days, starting 18 h after the induction of pneumococcal meningitis. The folic acid solution was prepared before administration in a volume of 1 mL/kg (Budni et al. 2013). Organisation of the experimental groups The animals were divided into six groups: control, control treated with folic acid at 10 mg/kg, control treated with
Folic acid prevented cognitive impairment
folic acid at 50 mg/kg, meningitis, meningitis treated with folic acid at 10 mg/kg, and meningitis treated with folic acid at 50 mg/kg (10 animals per group). Behavioural tasks Ten days after the induction of meningitis, the animals were randomly assigned to be tested on one of the behavioural tasks: the open field and step-down inhibitory avoidance tasks. After performing the task, the animals were killed and the hippocampus was immediately isolated on dry ice and stored at -80 °C for analysis.
Oxidative stress parameters Malondialdehyde equivalents To determine oxidative damage to lipids, we measured the formation of thiobarbituric acid reactive species (TBARS) during an acid-heating reaction, as previously described by Draper and Hadley (1990). The samples were mixed with 1 mL of 10 % trichloroacetic acid and 1 mL of 0.67 % thiobarbituric acid and then heated in a boiling water bath for 30 min. Malondialdehyde (MDA) equivalents were determined in both the tissue and the submitochondrial particles of the rat brain using a spectrophotometer at an absorbance of 532 nm.
Open-field task Protein carbonyl formation Behaviour was assessed in an open-field apparatus to evaluate both locomotor and exploratory activities. This apparatus was a 40 9 60 cm open field surrounded by 50-cm-high walls made of brown plywood with a front glass wall. Black lines divided the floor of the open field into nine rectangles. The animals were gently placed in the left rear quadrant and were allowed to explore the arena for 5 min. The number of crossings (i.e., the number of times that the animals crossed the black lines, which assesses locomotor activity) and rearing movements (i.e., the exploration behaviour observed in rats subjected to a new environment) were measured. The same person, who was blind to the group treatments, performed all behavioural testing by manual analyses (Vianna et al. 2000). Step-down inhibitory avoidance task The apparatus and procedures have been described in previous reports (Quevedo et al. 1997; Roesler et al. 2003). Briefly, the training apparatus was a 50 9 25 9 25 cm acrylic box (Albarsch, Porto Alegre, Brazil) whose floor consisted of parallel calibre stainless steel bars (1 mm diameter) spaced 1 cm apart. A 7-cm-wide, 2.5-cm-high platform was placed on the floor of the box, against the left wall. In the training trial, the animals were placed on the platform, and their latency to step down on the grid with all four paws was measured with an automatic device. Immediately after stepping down on the grid, the animals received a 0.4 mA foot shock for 2.0 s and were returned to their home cage. A retention test trial was performed 24 h after training (long-term memory). The retention test trial was procedurally identical to the training trial, except that no foot shock was presented. The retention test stepdown latency (maximum, 180 s) was used as a measure of inhibitory avoidance retention (Izquierdo et al. 1998; Bevilaqua et al. 2003).
Oxidative damage to proteins was assessed by determining the amount of carbonyl groups based on their reaction with 0.02 g 2,4-dinitrophenylhydrazine (DNPH) dissolved in 10 mL of water, as previously described by Levine et al. (1994) (Levine et al. 1990). Proteins were precipitated by the addition of 20 % trichloroacetic acid and re-dissolved in DNPH. The absorbance was monitored using a spectrophotometer at 370 nm. Superoxide dismutase activity The method used to assay superoxide dismutase (SOD) activity is based on the capacity of pyrogallol to autoxidise, which is a process that is highly dependent on O2-2, a substrate for SOD (Bannister and Calabrese 1987). Inhibition of the autoxidation of this compound occurs when SOD is present, and the enzymatic activity can be indirectly assayed using a temperature-controlled double-beam spectrophotometer at an absorbance of 420 nm. A calibration curve was performed using purified SOD as the standard to calculate the specific activity of SOD present in the samples. A 50 % inhibition of pyrogallol autoxidation was defined as one unit of SOD, and the specific activity was represented as units per mg of protein. Catalase activity Catalase (CAT) activity was assayed using a temperaturecontrolled double-beam spectrophotometer. This method is based on the disappearance of H2O2 at an absorbance of 240 nm in a reaction medium that contains 20 mM H2O2, 0.1 % Triton X-100, 10 mM potassium phosphate buffer (pH 7.0), and 0.1–0.3 mg protein/mL (Laemmli 1970). CAT unit was defined as 1 mol of H2O2 consumed per minute, and the specific activity was reported as units per mg of protein.
123
T. Barichello et al.
Determination of nitrite and nitrate concentrations The concentrations of nitrite and nitrate were measured in other aliquots of tissue using the Griess reaction by adding 100 ll of reagent [0.1 % (w/v) in H2O and 1 % (w/v) sulphanilamide in 5 % (v/v) H3PO4 concentrate volume (1:1)] to 100 ll of sample. The optical density at 550 nm (OD550) was measured using an ELISA microplate reader (Green et al. 1982). Myeloperoxidase activity Tissues were homogenised (50 mg/mL) in 0.5 % hexadecyltrimethylammonium bromide and centrifuged at 15,000 9 g for 40 min. The suspension was then sonicated three times for 30 s. A supernatant aliquot was mixed with a solution of 1.6 mM tetramethylbenzidine and 1 mM H2O2. Myeloperoxidase (MPO) activity was determined colorimetrically with a spectrophotometer by analysing changes in the absorbance at 650 nm at a temperature of 37 °C (De Young et al. 1989).
the dependent variable was not on an interval scale. Data regarding habituation to the open-field task are reported as the mean difference (training trial–test trial) and the 95 % confidence interval of the difference, as analysed by a paired Student’s t test. Data from the inhibitory avoidance task are reported as medians and interquartile ranges. Data within individual groups were analysed by a Friedman test and multiple comparisons by a Wilcoxon test. Comparisons among groups were performed using Mann–Whitney U tests. All data regarding BDNF levels and oxidative stress were reported as the mean ± SEM and were analysed by two-way ANOVA (meningitis and folic acid), followed by a Bonferroni post hoc test. In our study, we used univariate linear model, and not the multivariate hierarchical analysis or study of intercluster correlation being held because each object (rats) generated an observation, with no variation among objects and only variation between objects (Aarts et al. 2014). P values \0.05 were considered statistically significant. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) software version 20.0.
Assessment of BDNF expression Results The measurement of BDNF levels was performed as previously described (Kauer-Sant’Anna et al. 2007). BDNF serum levels were measured with a sandwich ELISA, using a commercial kit according to the manufacturer’s instructions (Millipore, USA). Briefly, microtitre plates (96-well flat bottom) were coated overnight at 4 °C with samples diluted at 1:100 in sample diluent, and the standard curve ranged from 7.8 to 500 pg/mL BDNF. The plates were then washed four times with wash buffer, and biotinylated mouse anti-human BDNF monoclonal antibody (diluted 1:1,000 in sample diluent) was added, followed by incubation for 3 h at room temperature. After washing, a second incubation with streptavidin–horseradish peroxidase conjugate solution (diluted 1:1,000) was performed for 1 h at room temperature. After the addition of substrate and stop solution, the amount of BDNF was determined (absorbance set at 450 nm). The standard curve demonstrated a direct relationship between optical density (OD) and BDNF concentration. Total protein was measured by a Bradford assay (samples diluted at 1:200), using bovine serum albumin (BSA) as a standard.
Statistics The normality of the data was examined by a Shapiro–Wilk test. Because the null hypothesis was that the data were normally distributed, parametric statistical significance tests were used, except for the inhibitory avoidance task because
123
The bacterial concentration in the CSF at 18 h after pneumococcal meningitis induction were 1.35 ± 0.212 9 103 in the meningitis treated with 10 mg/kg and 1.3 ± 0.141 9 103 in meningitis treated with 50 mg/kg of folic acid. Folic acid did not have antimicrobial effect in vitro, data not shown. In the open-field task (Fig. 1), we illustrated the influence of folic acid on habituation memory 10 days after pneumococcal meningitis induction. There were no differences in the number of crossings and rearing movements among groups during habituation to the open-field training session, demonstrating no difference in motor and exploratory activity (data not shown). However, when we examined the difference between the training and the test sessions, we verified a decrease in crossing and rearing in the untreated meningitis group compared with all other groups, demonstrating habituation memory impairment in this group (P \ 0.05). In Fig. 2, we showed the step-down inhibitory avoidance task 10 days after pneumococcal meningitis induction. There was a difference between the training and the test sessions in the untreated control group, control group/ 10 mg, control group/50 mg, meningitis group/10 mg, and meningitis group/50 mg, demonstrating aversive memory in these groups (P \ 0.05). In the untreated meningitis group, there was no difference between the training and the test sessions, demonstrating impairment of aversive memory in this group.
Folic acid prevented cognitive impairment
Training
50
*
*
*
140
*
20
10
Saline
10 mg/Kg
50 mg/kg
Saline
Control
Rearings (Difference between training and test session)
*
Test
160
30
0
b
*
180
40
Latency (sec)
Crossings (Difference between training and test session)
a
10 mg/Kg
50 mg/kg
120 100 80 60 40 20
Meningitis
0
30
Saline
10 mg/Kg 50 mg/kg Saline Control
Meningitis
Fig. 2 Latency to step down in the inhibitory avoidance task 10 days after pneumococcal meningitis induction. The data from the inhibitory avoidance task are reported as medians and interquartile ranges. Data within individual groups were analysed by a Friedman test and multiple comparisons by a Wilcoxon test. Comparisons among groups were performed using Mann–Whitney U tests, with n = 10 animals per group. *p \ 0.05 indicates differences between the training and the test sessions for the same group
20
*
10
10 mg/Kg 50 mg/kg
0 Saline
10 mg/Kg Control
50 mg/kg
Saline
10 mg/Kg
50 mg/kg
Meningitis
Fig. 1 Habituation to the open-field task 10 days after pneumococcal meningitis induction. The numbers of crossings (a) and rearing (b) movements are reported as the mean difference (training trial–test trial) and the 95 % confidence interval of the difference. These results were analysed by a paired Student’s t test, with n = 10 animals per group. The symbols indicate statistical significance when the training and test sessions were compared (*p \ 0.05)
In Figs. 3 and 4, we illustrated the effects of folic acid on oxidative damage and enzymatic defence in the hippocampus and frontal cortex 10 days after pneumococcal meningitis induction. TBARS levels decreased in the hippocampus and frontal cortex of animals that received adjuvant treatment with 10 mg of folic acid (P \ 0.05), whereas in the meningitis group that received 50 mg, TBARS levels increased compared with levels in the meningitis/10 mg group (P \ 0.05) (Figs. 3a, 4a). Carbonyl levels decreased in the hippocampus and frontal cortex of animals that received adjuvant treatment with 10 mg of folic acid (P \ 0.05). In contrast, in the meningitis group that received 50 mg of folic acid, carbonyl levels increased in the frontal cortex compared with levels in the meningitis/10 mg group (P \ 0.05) (Figs. 3b, 4b). Nitrite/nitrate concentrations decreased in the hippocampus of animals that received adjuvant treatment with 10 mg of folic acid (P \ 0.05, Fig. 3c). MPO concentrations
increased in the hippocampus of the control group that received adjuvant treatment with 50 mg of acid folic (P \ 0.05), whereas adjuvant treatment decreased MPO levels in the hippocampus of animals that received adjuvant treatment with 10 mg of folic acid (P \ 0.05, Fig. 3d). SOD activity increased only in the hippocampus of the control group that received 10 mg of folic acid compared with activity in the untreated control group (P \ 0.05, Fig. 3e). The levels of CAT did not change in either group (Figs. 3f, 4f). In Fig. 5, we showed BDNF expression in the hippocampus and frontal cortex 10 days after pneumococcal meningitis induction. BDNF levels increased in the hippocampus of the meningitis group that received adjuvant treatment with 10 mg of folic acid (P \ 0.05, Fig. 5a). In the frontal cortex, there was a decrease in BDNF levels in the control group/50 mg, meningitis group/10 mg, and meningitis group/50 mg (P \ 0.05, Fig. 5b).
Discussion In the present study, we demonstrated the influence of vitamin B9 on memory, oxidative damage, enzymatic defence, and BDNF expression in experimental pneumococcal meningitis. S. pneumoniae multiplies within the subarachnoid space, initiating a complex immune response (Barichello et al. 2013b). Consequently,
123
a
Saline
0.03
10 mg/kg
#
50 mg/kg
0.02
*
0.01
0.00 Control
Meningitis
Protein carbonyls (nmol/mg protein)
MDA equivalents (nmol/mg protein)
T. Barichello et al.
b 0.025
10 mg/kg
0.020
0.005 0.000
δ
50 mg/kg
0.2
*
0.1
0.0 Control
Meningitis
MPO activity (mU/mg protein)
Nitrite/nitrate concentration (nmol/mg protein)
10 mg/kg
Control
0.015
10 mg/kg 50 mg/kg
*
0.010
0.005
0.000 Control
Meningitis
Meningitis
Saline
0.03
10 mg/kg
δ
50 mg/kg
* *
0.02
0.01
0.00 Control
f CAT activity (U/mg protein)
SOD activity (U/mg protein)
e Saline
*
0.010
d 0.3
δ
50 mg/kg
0.015
c Saline
Saline
Meningitis
Saline
0.6
10 mg/kg 50 mg/kg
0.4
0.2
0.0 Control
Meningitis
Fig. 3 Oxidative damage and enzymatic defence in the hippocampus 10 days after pneumococcal meningitis induction. TBARS levels (a), protein carbonyl levels (b), nitrite/nitrate concentrations (c), MPO activity (d), SOD activity (e), and CAT activity (f). The data are reported as the mean ± SEM. These results were analysed by twoway ANOVA (meningitis and folic acid), followed by a Bonferroni
post hoc test, with n = 10 animals per group. The symbols *p \ 0.05 indicate statistical significance when with compared the untreated meningitis group, #p \ 0.05 indicate statistical significance when compared with meningitis group treated with 10 mg/kg; dp \ 0.05 indicate statistical significance when compared with untreated meningitis vs control saline group
polymorphonuclear cells are attracted from the bloodstream and then activated, releasing large amounts of NO, O2, and H2O2. This formation generates oxidative and nitrosative stress and subsequent lipid peroxidation, mitochondrial damage, and BBB breakdown, which contribute to cell injury during pneumococcal meningitis (Barichello et al. 2013a, b). In the present study, we showed that adjuvant treatment with folic acid at a concentration of 10 mg decreased oxidative stress, increased BDNF levels, however, both concentrations of 10 and 50 mg prevented cognitive impairment in experimental pneumococcal meningitis. Folic acid plays an important
role in neuroplasticity and the preservation of neuronal integrity (Kronenberg et al. 2009). Folic acid supplementation enhances repairs of the adult CNS (Iskandar et al. 2004) and also promotes hippocampal neurogenesis and preserves cognitive functions after stroke in the adult brain (Zhang et al. 2012). Folate is a cofactor in onecarbon metabolism that promotes the restoration of methionine from homocysteine (Kronenberg et al. 2009). Stimulation of the immune system may contribute to hyperhomocysteinaemia in certain diseases and in sepsis patients with poor outcomes (Ploder et al. 2010). Supplementation with folic acid leads to a decrease in the
123
a 0.04
Saline
δ
#
10 mg/kg
0.03
50 mg/kg
0.02
*
0.01
0.00 Control
Meningitis
Protein carbonyls (nmol/mg protein)
MDA equivalents (nmol/mg protein)
Folic acid prevented cognitive impairment
b 0.025
0.015
#
*
*
0.010 0.005 0.000
MPO activity (mU/mg protein)
Nitrite/nitrate concentration (nmol/mg protein)
50 mg/kg
0.10
0.05
0.00 Control
Control
0.03
Meningitis
Saline
δ
10 mg/kg 50 mg/kg
0.02
0.01
0.00
Meningitis
Control
Meningitis
f Saline
0.008
10 mg/kg 50 mg/kg
0.006
0.004
δ
0.002
0.000 Control
Meningitis
CAT activity (U/mg protein)
e SOD activity (U/mg protein)
50 mg/kg
d Saline 10 mg/kg
0.15
δ
10 mg/kg
0.020
c 0.20
Saline
Saline
0.8
10 mg/kg 50 mg/kg
0.6
δ
0.4
0.2
0.0 Control
Meninigitis
Fig. 4 Oxidative damage and enzymatic defence in the frontal cortex 10 days after pneumococcal meningitis induction. TBARS levels (a), protein carbonyl levels (b), nitrite/nitrate concentrations (c), MPO activity (d), SOD activity (e), and CAT activity (f). The data are reported as the mean ± SEM. These results were analysed by twoway ANOVA (meningitis and folic acid), followed by a Bonferroni
post hoc test, with n = 10 animals per group. The symbols *p \ 0.05 indicate statistical significance when compared with the untreated meningitis group, #p \ 0.05 indicate statistical significance when compared with meningitis group treated with 10 mg/kg; dp \ 0.05 indicate statistical significance when compared with untreated meningitis vs control saline group
homocysteine level and rescues pathogenic and epigenetic modifications, showing protective efficacy against homocysteine-induced neurotoxicity (Kalani et al. 2013). Furthermore, treatment with folic acid prevents memory deficit and the reduction in BDNF levels induced by homocysteine injection (Matte et al. 2009). Additionally, alteration of maternal micronutrients, such as folic acid, vitamin B12, and omega-3 fatty acids, reduces BDNF levels in preterm pregnancy (Dhobale and Joshi 2012). In our study, BDNF levels increased in the hippocampus of animals that received adjuvant treatment with 10 mg of folic acid. This upregulated expression of BDNF in experimental pneumococcal meningitis could have an
important role in cognitive protection. The free radicalscavenging properties and possible antioxidant activity of folic acid have been reported. Folic acid inhibits microsomal lipid peroxidation and can efficiently scavenge oxidising free radicals (Joshi et al. 2001). During pneumococcal meningitis, large amounts of oxidative stress are produced (Klein et al. 2006). Accordingly, in our study, folic acid decreased lipid peroxidation, protein carbonylation, nitrate/nitrite levels, and MPO activity and increased SOD activity in the hippocampus. There is substantial interest in the role of folic acid and related pathways in nervous system function (Reynolds 2006) and the potential therapeutic effects of folic acid.
123
T. Barichello et al.
50
BDNF (pg/ug of protein)
References
Saline
a
*
10 mg/kg 50 mg/kg
40 30 20
δ 10 0 Control
Meningitis
b
Saline
30
BDNF (pg/ug of protein)
10 mg/kg 50 mg/kg 20
*
*
*
10
0 Control
Meningitis
Fig. 5 BDNF expression in the hippocampus and frontal cortex 10 days after pneumococcal meningitis induction. The results are shown for the hippocampus (a) and the frontal cortex (b). BDNF levels were assessed by ELISA, and the results are presented in pg per 100 mg of tissue, with n = 10 per group. All data regarding BDNF levels and oxidative stress are reported as the mean ± SEM. These results were analysed by two-way ANOVA (meningitis and folic acid), followed by a Bonferroni post hoc test. The symbols *p \ 0.05 indicate statistical significance compared with the untreated control group; dp \ 0.05 indicate statistical significance when compared with untreated meningitis vs control saline group
In conclusion, only adjuvant treatment with folic acid at 10 mg prevented memory impairment increasing BDNF expression in the hippocampus, decreasing oxidative stress. In addition both doses of 10 and 50 mg prevented memory deficit in animals subjected to pneumococcal meningitis. Acknowledgments Laboratory of Experimental Microbiology (Brazil) is one of the centres of the National Institute for Translational Medicine (INCT-TM) and one of the members of the Center of Excellence in Applied Neurosciences of Santa Catarina (NENASC). This research was supported by grants from CNPq (JQ, FK and TB), FAPESC (JQ and TB), and UNESC (JQ and TB). JQ and TB are CNPq Research Fellows. JSG and LRS is holder of a CAPES studentship. Conflict of interest All authors have read the journal’s policy on the disclosure of potential conflicts of interest. The authors declare that they have no conflict of interest.
123
Aarts E, Verhage M, Veenvliet JV, Dolan CV, van der Sluis S (2014) A solution to dependency: using multilevel analysis to accommodate nested data. Nat Neurosci 17(4):491–496 Bannister JV, Calabrese L (1987) Assays for superoxide dismutase. Methods Biochem Anal 32:279–312 Barichello T, Belarmino E Jr, Comim CM, Cipriano AL, Generoso JS, Savi GD, Stertz L, Kapczinski F, Quevedo J (2010a) Correlation between behavioral deficits and decreased brainderived neurotrophic (correction of neurotrofic) factor in neonatal meningitis. J Neuroimmunol 223(1–2):73–76 Barichello T, Savi GD, Silva GZ, Generoso JS, Bellettini G, Vuolo F, Petronilho F, Feier G, Comim CM, Quevedo J, Dal-Pizzol F (2010b) Antibiotic therapy prevents, in part, the oxidative stress in the rat brain after meningitis induced by Streptococcus pneumoniae. Neurosci Lett 478(2):93–96 Barichello T, Generoso JS, Milioli G, Elias SG, Teixeira AL (2013a) Pathophysiology of bacterial infection of the central nervous system and its putative role in the pathogenesis of behavioral changes. Rev Bras Psiquiatr 35(1):81–87 Barichello T, Generoso JS, Simoes LR, Elias SG, Quevedo J (2013b) Role of oxidative stress in the pathophysiology of pneumococcal meningitis. Oxidative Med Cell Longev 2013:371465 Barichello T, Simoes LR, Generoso JS, Sangiogo G, Danielski LG, Florentino D, Dominguini D, Comim CM, Petronilho F, Quevedo J (2013c) Erythropoietin prevents cognitive impairment and oxidative parameters in Wistar rats subjected to pneumococcal meningitis. Transl Res 163(5):503–513 Bevilaqua LR, Kerr DS, Medina JH, Izquierdo I, Cammarota M (2003) Inhibition of hippocampal Jun N-terminal kinase enhances short-term memory but blocks long-term memory formation and retrieval of an inhibitory avoidance task. Eur J Neurosci 17(4):897–902 Brouwer MC, Tunkel AR, van de Beek D (2010) Epidemiology, diagnosis, and antimicrobial treatment of acute bacterial meningitis. Clin Microbiol Rev 23(3):467–492 Budni J, Zomkowski AD, Engel D, Santos DB, dos Santos AA, Moretti M, Valvassori SS, Ornell F, Quevedo J, Farina M, Rodrigues AL (2013) Folic acid prevents depressive-like behavior and hippocampal antioxidant imbalance induced by restraint stress in mice. Exp Neurol 240:112–121 De Young LM, Kheifets JB, Ballaron SJ, Young JM (1989) Edema and cell infiltration in the phorbol ester-treated mouse ear are temporally separate and can be differentially modulated by pharmacologic agents. Agents Actions 26(3–4):335–341 Dhobale M, Joshi S (2012) Altered maternal micronutrients (folic acid, vitamin B(12)) and omega 3 fatty acids through oxidative stress may reduce neurotrophic factors in preterm pregnancy. J Maternal Fetal Neonatal Med 25(4):317–323 Draper HH, Hadley M (1990) Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 186:421–431 Grandgirard D, Steiner O, Tauber MG, Leib SL (2007) An infant mouse model of brain damage in pneumococcal meningitis. Acta Neuropathol 114(6):609–617 Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR (1982) Analysis of nitrate, nitrite, and (15 N) nitrate in biological fluids. Anal Biochem 126(1):131–138 Hoogman M, van de Beek D, Weisfelt M, de Gans J, Schmand B (2007) Cognitive outcome in adults after bacterial meningitis. J Neurol Neurosurg Psychiatry 78(10):1092–1096 Irazuzta J, Pretzlaff RK, Zingarelli B (2008) Caspases inhibition decreases neurological sequelae in meningitis. Crit Care Med 36(5):1603–1606
Folic acid prevented cognitive impairment Iskandar BJ, Nelson A, Resnick D, Skene JH, Gao P, Johnson C, Cook TD, Hariharan N (2004) Folic acid supplementation enhances repair of the adult central nervous system. Ann Neurol 56(2):221–227 Izquierdo I, Barros DM, Mello e Souza T, de Souza MM, Izquierdo LA, Barros DM, Medina JH (1998) Mechanisms for memory types differ. Nature 393(6686):635–636 Joshi R, Adhikari S, Patro BS, Chattopadhyay S, Mukherjee T (2001) Free radical scavenging behavior of folic acid: evidence for possible antioxidant activity. Free Radic Biol Med 30(12): 1390–1399 Kalani A, Kamat PK, Givvimani S, Brown K, Metreveli N, Tyagi SC, Tyagi N (2013) Nutri-epigenetics ameliorates blood-Brain barrier damage and neurodegeneration in hyperhomocysteinemia: role of folic acid. J Mol Neurosci 13:13 Kauer-Sant’Anna M, Andreazza AC, Valvassori SS, Martins MR, Barbosa LM, Schwartsmann G, Roesler R, Quevedo J, Kapczinski F (2007) A gastrin-releasing peptide receptor antagonist blocks D-amphetamine-induced hyperlocomotion and increases hippocampal NGF and BDNF levels in rats. Peptides 28(7): 1447–1452 Kjer-Nielsen L, Patel O, Corbett AJ, Le Nours J, Meehan B, Liu L, Bhati M, Chen Z, Kostenko L, Reantragoon R, Williamson NA, Purcell AW, Dudek NL, McConville MJ, O’Hair RA, Khairallah GN, Godfrey DI, Fairlie DP, Rossjohn J, McCluskey J (2012) MR1 presents microbial vitamin B metabolites to MAIT cells. Nature 491(7426):717–723 Klein M, Koedel U, Pfister HW (2006) Oxidative stress in pneumococcal meningitis: a future target for adjunctive therapy? Prog Neurobiol 80(6):269–280 Kronenberg G, Colla M, Endres M (2009) Folic acid, neurodegenerative and neuropsychiatric disease. Curr Mol Med 9(3): 315–323 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680–685 Leib SL, Clements JM, Lindberg RL, Heimgartner C, Loeffler JM, Pfister LA, Tauber MG, Leppert D (2001) Inhibition of matrix metalloproteinases and tumour necrosis factor alpha converting enzyme as adjuvant therapy in pneumococcal meningitis. Brain 124(Pt 9):1734–1742
Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, Ahn BW, Shaltiel S, Stadtman ER (1990) Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 186:464–478 Matte C, Pereira LO, Dos Santos TM, Mackedanz V, Cunha AA, Netto CA, Wyse AT (2009) Acute homocysteine administration impairs memory consolidation on inhibitory avoidance task and decreases hippocampal brain-derived neurotrophic factor immunocontent: prevention by folic acid treatment. Neuroscience 163(4):1039–1045 Ploder M, Kurz K, Spittler A, Neurauter G, Roth E, Fuchs D (2010) Early increase of plasma homocysteine in sepsis patients with poor outcome. Mol Med 16(11–12):498–504 Quevedo J, Vianna M, Zanatta MS, Roesler R, Izquierdo I, Jerusalinsky D, Quillfeldt JA (1997) Involvement of mechanisms dependent on NMDA receptors, nitric oxide and protein kinase A in the hippocampus but not in the caudate nucleus in memory. Behav Pharmacol 8(8):713–717 Reynolds E (2006) Vitamin B12, folic acid, and the nervous system. Lancet Neurol 5(11):949–960 Roesler R, Schroder N, Vianna MR, Quevedo J, Bromberg E, Kapczinski F, Ferreira MB (2003) Differential involvement of hippocampal and amygdalar NMDA receptors in contextual and aversive aspects of inhibitory avoidance memory in rats. Brain Res 975(1–2):207–213 van de Beek D, Schmand B, de Gans J, Weisfelt M, Vaessen H, Dankert J, Vermeulen M (2002) Cognitive impairment in adults with good recovery after bacterial meningitis. J Infect Dis 186(7):1047–1052 van de Beek D, de Gans J, Tunkel AR, Wijdicks EF (2006) Community-acquired bacterial meningitis in adults. N Engl J Med 354(1):44–53 Vianna MR, Alonso M, Viola H, Quevedo J, de Paris F, Furman M, de Stein ML, Medina JH, Izquierdo I (2000) Role of hippocampal signaling pathways in long-term memory formation of a nonassociative learning task in the rat. Learn Mem 7(5):333–340 Zhang X, Huang G, Liu H, Chang H, Wilson JX (2012) Folic acid enhances Notch signaling, hippocampal neurogenesis, and cognitive function in a rat model of cerebral ischemia. Nutr Neurosci 15(2):55–61
123
