Astroglial and cognitive effects of chronic cerebral hypoperfusion in the rat

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Astroglial and cognitive effects of chronic cerebral hypoperfusion in the rat Évelin Vicente a , Daniel Degerone b , Liana Bohn b , Francisco Scornavaca b , Alexandre Pimentel b , Marina C. Leite b , Alessandra Swarowsky a , Letícia Rodrigues a , Patrícia Nardin b , Lucia Maria Vieira de Almeida b , Carmem Gottfried a,b , Diogo Onofre Souza b , Carlos Alexandre Netto a,b , Carlos Alberto Gonçalves a,b,⁎ a Post-graduation Program of Neurocience, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil b Post-graduation Program of Biochemistry, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

A R T I C LE I N FO

AB S T R A C T

Article history:

The permanent occlusion of common carotid arteries (2VO) causes a significant reduction of

Accepted 8 November 2008

cerebral blood flow (hypoperfusion) in rats and constitutes a well established experimental

Available online 19 November 2008

model to investigate neuronal damage and cognitive impairment that occurs in human

Keywords:

S100B and glial fibrillary acidic protein (GFAP) – in cerebral cortex and hippocampus tissue,

Astrocyte

glutamate uptake and glutamine synthetase activity in hippocampus tissue, as well as

GFAP

S100B in cerebrospinal fluid. Cognition, as assessed by reference and working spatial

Hippocampus

memory protocols, was also investigated. Adult male Wistar rats were submitted to

Hypoperfusion

10 weeks of chronic cerebral hypoperfusion by the 2VO method. A significant increase of

S100B

S100B and GFAP in hippocampus tissue was observed, as well a significant decrease in

2-VO method

glutamate uptake. Interestingly, we observed a decrease in S100B in cerebrospinal fluid. As

ageing and Alzheimer's disease. In the present study, we evaluated two astroglial proteins –

for the cognitive outcome, there was an impairment of both reference and working spatial memory in the water maze; positive correlation between cognitive impairment and glutamate uptake decrease was evidenced in hypoperfused rats. These data support the hypothesis that astrocytes play a crucial role in the mechanisms of experimental neurodegeneration and that hippocampal pathology arising after chronic hypoperfusion gives rise to memory deficits. © 2008 Elsevier B.V. All rights reserved.

1.

Introduction

Reduction of cerebral blood flow, as occurs in cerebral stroke and advanced ageing, is a prominent risk factor of brain dysfunction. In fact, it has been proposed that a state of chronic

cerebral hypoperfusion represents one of the physiopathological mechanisms of neural damage underlying dementia, including Alzheimer's disease (AD) (Farkas et al., 2004; Ni et al., 1995; Shibata et al., 2004). AD is the major neurodegenerative disorder responsible for dementia in the elderly, which is

⁎ Corresponding author. Departamento de Bioquímica, ICBS, Universidade Federal do Rio Grande do Sul, Ramiro Barcelos, 2600-Anexo, 90035-003, Porto Alegre, RS, Brazil. E-mail address: [email protected] (C.A. Gonçalves). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.11.032

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clinically characterized by a progressive loss of memory and higher cognitive function (Koistinaho and Koistinaho, 2005; Liu et al., 2007; Zlokovic et al., 2005). The main neuropathological findings in AD are amyloid plaques, neuronal loss, neurofibrillary tangles, astrogliosis and microgliosis. The exact etiology of AD is not yet established, however, there is evidence that neurovascular dysfunction contributes to the cognitive impairment seen in AD (Zlokovic et al., 2005). The permanent occlusion of the common carotid arteries, by the two-vessel occlusion method (2VO), results in a significant reduction of cerebral blood flow (hypoperfusion) in the rat; it is accepted as a well established experimental model to investigate neuronal damage and cognitive impairment as it occurs in human ageing and AD (Farkas et al., 2004; Ohta et al., 1997; Tohda et al., 2004). This cognitive impairment is accompanied by progressive loss of hippocampal pyramidal neurons, which is also often observed in human ageing and dementia states (Farkas et al., 2007). Astrocytes are in close morphological and functional association with neurons and the importance of these glial cells in brain disorders has been strongly suggested (e.g. Maragakis and Rothstein, 2006). Glial activation in response to injury stimuli commonly involves changes in glial fibrillary acidic protein (GFAP), S100B levels and glutamate metabolism. GFAP is a specific astrocyte marker; currently, tissue GFAP increase is taken as a sign of astrogliosis associated with

205

conditions of brain injury (Eng et al., 2000; Vicente et al., 2004). S100B is a calcium-binding protein found in brain tissue, predominantly in astrocytes. This protein has putative intraand extracellular functions: intracellular roles include regulation of protein phosphorylation, cytoskeleton components and transcriptional factors (Donato, 2001; Goncalves et al., 2008); extracellular S100B plays a trophic role on neuronal and glial cells, but elevated extracellular levels of this protein could induce apoptosis in neural cells (Van Eldik and Wainwright, 2003). Cerebrospinal fluid and serum S100B levels have been used as markers of brain insult (Andreazza et al., 2007; Vicente et al., 2004). Moreover, astrocytes are key elements in the brain metabolism of glutamate; they are particularly responsible for removing glutamate from the synaptic cleft and for the synthesis of glutamine, which is sent back to the neuron to renew glutamate stocks (Had-Aissouni et al., 2002; Nedergaard et al., 2002). Therefore, glutamate uptake and glutamine synthetase activities have also been used to characterize astroglial function in brain tissue. The aims of this study were to investigate the cognitive impairment and astrocyte biochemical parameters, namely GFAP, S100B, glutamate uptake and glutamine synthetase, in brain tissue, particularly hippocampus and cerebral cortex, in rats submitted to chronic cerebral hypoperfusion (by the 2VO model for 10 weeks). The working hypothesis is that chronic

Fig. 1 – Cognitive performance of rats submitted to chronic cerebral hypoperfusion evaluated in the water maze. (A) Performance in the reference memory protocol. Each line represents the mean ± standard error. * Significant difference were detected from day 3 onwards when compared to control group (N = 8, repeated-measures ANOVA, p < 0.05); (B) Memory in the probe trial of reference memory, as measured by latency (in s) to find the platform position. Values are mean ± standard error. * Significantly different from control group (N = 8, Student's t test, p < 0.05); (C) Memory in the probe trial of reference memory, as measured by the time spent in the target quadrant (in s). Values are mean ± standard error. * Significantly different from control group (N = 8, Student's t test, p < 0.05); (D) Performance in the working memory protocol. Each line represents the mean ± standard error. * Significant differences were detected from trial 2 onwards when comparing control and hypoperfusion groups (N = 8, one-way ANOVA followed by Duncan's multiple range test, p < 0.05).

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cerebral hypoperfusion provokes cognitive impairment and astroglial alterations.

2.

Results

2.1.

Behavioral effects

The Morris water maze was used to evaluate reference and working spatial memory. Since there were no differences between sham and non-operated groups (data not shown), the cognitive performance and biochemical parameters of hypoperfusion group were compared to those of sham group, referred to as control group, in order to facilitate figure reading. In the reference memory, there was a decline of the average time to find the platform from day 3 onwards in the control group (Fig. 1A) (p = 0.01). The latency to find the platform position was shorter in sham, as compared to the hypoperfusion group in the probe trial (Fig. 1B) (t = −2.011, p = 0.05) and 2VO rats spent less time in the target quadrant, as compared to controls (Fig. 1C) (t = 2.291, p = 0.03). In the working memory task, from trial 2 onwards, there was a decline in the mean time to find the platform in sham rats, this effect did not appear in 2VO animals (Fig. 1 D) (p = 0.01).

2.2.

Changes in GFAP and S100B contents

A significant increase (p = 0.028) in GFAP immunocontent of the hippocampus was observed in rats submitted to chronic hypoperfusion by the 2VO model (Fig. 2A, on the left); this effect was not found in cerebral cortex (p = 0.09, Fig. 2A, on the right). Similarly, hippocampal S100B content was higher in rats submitted to hypoperfusion (p = 0.008, Fig. 2B, on the left), however it did not reach statistical significance in cerebral cortex (p = 0.63, Fig. 2B, on the right). Interestingly, a significant decrease in CSF S100B was observed in the hypoperfusion group, as compared to controls (Fig. 2C).

2.3.

Hippocampal histology

In order to evaluate and confirm the hippocampal GFAP change observed in rats submitted to 2VO model, a GFAP immunohistochemistry study was carried out (Fig. 3 shows a representative image). The photomicrographs of astrocyte GFAP immunoreactive (ir) clearly show astrogliosis in the 2VO animals. Astrocytes (black arrows) are present in radiatum and lacunosum-moleculare layers (white arrows) in the hippocampus of control animals (Fig. 3A), while intense marking of astrocytes by GFAP-ir (black arrow) shows up in the 2VO group. There is more astrocyte prolongation (white arrows) in the 2VO animals, as compared to controls (Fig. 3B). We also observed neuron nuclei in the pyramidal layer of hippocampus, by the use of hematoxylin and eosin staining (Figs. 3C and 3D). There is a clear decrease in marked nucleus density (asterisk) in the 2VO rats (panel D), as compared to the control group (panel C), indicating neuronal loss provoked by chronic cerebral hypoperfusion. Bidimensional cell counting revealed a density (±SE) of 1804 ± 75 and 891 ± 49 putative neurons per mm2 in control and 2VO rats, respectively (t = 6.750, p < 0.01).

Fig. 2 – GFAP and S100B content in hippocampus and cerebral cortex of rats submitted to chronic cerebral hypoperfusion. Adult rats were submitted to hypoperfusion for 10 weeks. Hippocampi and cerebral cortex were dissected out and the contents of GFAP (panel A) and S100B (panel B) were measured by ELISA. Values are mean ± standard error of 8 rats in each group. C, Cerebrospinal fluid (CSF) was collected by cisterna magna puncture and the content of S100B was measured by ELISA. Values are mean ± standard error of 6 (2VO) or 8 (control) rats. * Significantly different from control (Student's t test, p < 0.05).

2.4.

Glutamate metabolism in the hippocampus

Glutamate uptake and glutamine synthetase activity were measured in hippocampal slices of control and 2VO rats. A significant decrease in the glutamate uptake (p = 0.03) was observed in 2VO rats (Fig. 4A), however there was no difference in glutamine synthetase activity between groups (p = 0.10, Fig. 4B). A positive correlation was found between cognitive impairment and glutamate uptake decrease in 2VO rats (Fig. 4C; Pearson's correlation, R = 0.899, p = 0.015), but not in control

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Fig. 3 – Immunohistochemistry for GFAP and histological analysis of the hippocampi from rats submitted to chronic cerebral hypoperfusion. Photomicrographs showing GFAP immunoreactive cells (ir) in the hippocampus of control (panel A) and hypoperfusion (panel B) groups. Notice the difference in GFAP-ir in the lacunosum moleculare (white arrows) and radiatum layers of CA1 region (black arrow-heads) between groups. Hippocampal sections show a higher density of hematoxylin–eosin stained nuclei of pyramidal layers (indicated by an asterisk) in control rats (panel C) than in rats submitted to chronic hypoperfusion (panel D). Magnification 100×, scale bar = 150 μm.

animals (Person's correlation, R = 0.163, p = 0.699). No other correlations between cognitive impairment and astroglial changes (hippocampal GFAP increase, hippocampal S100B increase and CSF S100B decrease) were found in the two studied groups assuming p < 0.05.

3.

Discussion

Several types of animal models have been developed to investigate the consequences of chronic reduction of cerebral blood flow (Farkas et al., 2007; Ni et al., 1995; Ohta et al., 1997; Schmidt-Kastner et al., 2005). Progressive cognitive impairment, neuronal loss, cholinergic dysfunction and astrogliosis in the hippocampus have been associated with the 2VO hypoperfusion (Bennett et al., 1998; Schmidt-Kastner et al., 2005). In agreement with that, data presented in Fig. 1 clearly show reference and working spatial memory impairments 10 weeks after 2VO, as evaluated in the Morris water maze task. It was also demonstrated that chronic cerebral hypoperfusion causes significant neuronal loss, an increase of GFAP and S100B and a decrease in glutamate uptake in hippocampal tissue. Moreover, a decrease of S100B in the CSF was also shown. Chronic hypoperfusion by 2VO in the rat has also been associated with visual impairment due to resulting retina damage (Davidson et al., 2000; Stevens et al., 2002). However, a

comparison between common carotid ligature (2VO) and internal carotid arteries occlusion (which does not affect the retina) shows that both groups bear spatial memory deficits in the 8-arm radial maze, suggesting that cerebral hypoperfusion itself compromises the learning process, but damage to the visual system aggravates the test results that depend on visual cues. Moreover, auditory and tactile stimuli also guide rats in spatial learning; this is especially true for albino rats with limited vision. Thus, 2VO appears to induce learning deficits, due to non-visual learning, as characterized in the elevated Tmaze and the object recognition test (Farkas et al., 2007). GFAP is commonly used as a marker for changes in astroglial cells during brain development and injury (Eng et al., 2000). In fact, injury of the central nervous system, either as a consequence of trauma, disease, genetic disorders, or chemical insult causes astrocytes to become reactive, a condition characterized by an increase in GFAP (O'Callaghan and Sriram, 2005). It is important to mention that astrocyte reaction is not necessarily accompanied by neuronal death and that astrocytes themselves react to reduction of cerebral blood flow through mechanisms mediated by changes in energetic metabolism, oxygen radical formation and/or cytokine production (Vagnozzi et al., 1997; Zimmer et al., 1991). Glial changes, as evaluated by immunohistochemistry for GFAP, have been observed in 2VO rats already in the first week after reduction of cerebral blood flow (Schmidt-Kastner et al., 2005); there was a was diffuse and transient increment in

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Fig. 4 – Glutamate uptake and glutamine synthetase activity in the hippocampus of rats submitted to chronic cerebral hypoperfusion. Adult rats were submitted to hypoperfusion for 10 weeks. Hippocampi were dissected out and chopped into 0.3 mm slices for measurement of glutamate uptake (panel A) or homogenized for measurement of glutamine synthetase activity (in panel B). Values are mean ± standard error of 6 (2VO) or 8 (control) rats. * Significantly different from controls (Student's t test, p < 0.05). (C) linear regression curve of glutamate uptake versus cognitive performance (as evaluated by latency difference between the 5th and 1st day-Δ latency) in chronic hyperfusion and control groups. Square of the Pearson's correlation coefficients are indicated; p < 0.05 in hyperfusion group.

neocortex, with no observed effects in the hippocampus. However, GFAP increase showed up later (6 months) in the hippocampus, apparently without signals of neuronal damage. Variable hippocampal damage has been reported in 2VO model, possibly caused by differences in rat strain, age, anaesthesia and time of occlusion (Farkas et al., 2004; Pappas et al., 1996; Schmidt-Kastner et al., 2005). In our study, 10 weeks of chronic hypoperfusion caused a significant GFAP increase in the hippocampus as measured by ELISA. Immunohistochemistry of hippocampus confirmed the astrogliosis.

S100B is a calcium binding protein predominantly expressed and secreted by astrocytes in vertebrate brain (Marenholz et al., 2004). Intracellularly, S100B binds to many protein targets, possibly modulating cytoskeleton plasticity, cell proliferation and astrocyte energy metabolism (Donato, 2001; Van Eldik and Wainwright, 2003); however high levels of brain tissue S100B have been observed in neurodegenerative disorders, including Alzheimer's disease (Rothermundt et al., 2003). Interestingly, S100B is able to stimulate the protein phosphatase calcineurin (Leal et al., 2004), which appears to be involved in the inflammatory activation of astrocytes in transgenic Alzheimer's models (Norris et al., 2005). Brain trauma and acute ischemia is associated with peripheral (CSF and/or serum) S100B elevation, probably due to astrocyte protein secretion or release from damaged cells (Rothermundt et al., 2003; Tramontina et al., 2006a). It has been proposed that acute increments of S100B may improve neurogenesis, particularly in the hippocampus (Kleindienst and Ross Bullock, 2006). Moreover, several cell culture studies suggest that, from pico to nanomolar concentrations, S100B stimulates glial proliferation, neuronal survival and protects neurons against glutamate excitotoxicity (Tramontina et al., 2006b; Van Eldik and Wainwright, 2003). However, there is no information about the content of this protein in the cerebrospinal fluid or brain tissue in rats submitted to chronic reduction of cerebral blood flow. The present study shows that S100B, like GFAP, is increased in brain tissue exposed to hypoperfusion, particularly in the hippocampus, as observed in neurodegenerative disorders. Moreover, we found low levels of CSF S100B, what could somehow indicate a lower trophic activity of this protein in rats submitted to chronic reduction of cerebral blood flow. Other astroglial parameters, namely glutamate uptake and glutamine synthetase activities, were investigated in the hippocampus of 2VO rats. A decrease of hippocampal glutamate uptake was revealed, indicating a higher susceptibility of these animals to excitotoxicity. In fact, an altered glutamate uptake has been proposed as one pathophysiological mechanism in Alzheimer's and other neurodegenerative diseases (Maragakis and Rothstein, 2004); resultant hippocampal dysfunction could contribute to the cognitive deficit observed in these animals. In agreement, an increase in glutamate transport was recently associated with cognitive improvement in Aplysia (Collado et al., 2007). Glutamate uptake has also been implicated in a multitude of neuropsychiatric diseases, in addition to its role in the physiology of learning and memory (e.g. Lauriat and McInnes, 2007). The positive correlation between glutamate uptake and latency difference in the reference memory task, in hypoperfused rats, suggests that a possible chronic elevation of extracellular levels of glutamate could be associated with the cognitive deficit reported: animals with low uptake show poor memory performance. This is the first report, to our knowledge, about this correlation in 2VO rats. In support, it has been reported that thiamine deficiency decreases cortical glutamate uptake in the prefrontal cortex and impairs cognitive performance in the water maze task (Carvalho et al., 2006). Further studies in 2VO rats will determine if this alteration involves a decrease in the content of glutamate

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transporters or in their activity. It is worth pointing out that increased GFAP is commonly associated with astroglial activation. Herein we found, in addition to GFAP increment, a decrease in glutamate uptake. This is in agreement with the idea that increased GFAP alone does not allow us to draw conclusions about the type of astroglial response, be it beneficial or deleterious (e.g. Liberto et al., 2004). Moreover, it is important to mention that, due to astroglial heterogeneity, we cannot rule out, at this moment, which opposite changes in GFAP and glutamate uptake might be occurring in different cell populations. In summary, the present study showed specific astroglial changes in rats after 10 weeks of 2VO, namely hippocampal gliosis (based on GFAP and S100B contents), reduced CSF S100B and reduced hippocampal glutamate uptake. These changes have been put forward in patients with neurodegenerative disorders, including Alzheimer's disease, and so reinforce the importance of this model for the investigation of astrocytes as targets of new therapeutic strategies. Such changes, particularly decreased glutamate uptake, could also be associated to the cognitive impairment observed in rats submitted to chronic hypoperfusion.

4.

Experimental procedures

4.1.

Chemicals

Antibody anti-S100B (clone SH-B1), glutamate, γ-glutamylhydroxamate and fast OPD were purchased from Sigma-Aldrich. 3 L-[2,3- H] glutamic acid and conjugated-peroxidase anti-rabbit IgG were purchased from Amersham; Rabbit polyclonal antiS100 and anti-GFAP were obtained from DAKO.

4.2.

Animals

Fifty adult (120-day old) male Wistar rats, weighing 280– 300 g, obtained from the Department of Biochemistry colony of the Federal University of Rio Grande do Sul, were housed with food and water ad libitum under a 12 h light/dark cycle at a temperature of 25 °C. All procedures were in accordance with the international guiding principles for biomedical research involving animals from CIOMS/WHO and were approved by the local authorities at the Federal University of Rio Grande do Sul. Rats were divided into three groups: non-operated (N = 16), sham surgery (N = 16), and 2VO (N = 18). Two set of experiments were carried out, using half of the rats of each group in each set. After behavioral tasks, rats were anaesthetized, as subsequently described, for CSF puncture, immunohistochemistry and hematoxylin–eosin staining (first set of experiments) or for brain slice preparation (second set of experiments), aiming to evaluate S100B and GFAP contents, glutamate uptake and glutamine synthetase activity. Since there were no behavioral differences between sham and non-operated groups (data not shown), the biochemical parameters of the 2VO group were compared to those of the sham group, referred to as the control group in text and Figure legends. Notice that Figs. 1, 2 (A–B) and 4 correspond to a set of experiments, whereas Figs. 2C and 3 correspond to another set.

4.3.

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Surgery procedure

Rats were anesthetized with Ketamine and Xylazine (75 and 10 mg/Kg, respectively), common carotid arteries of the animals were exposed and carefully separated from carotid sheath, cervical sympathetic and vagal nerves. Both common carotid arteries were ligated with surgical silk in 2VO rats; sham-operated animals suffered no artery ligature. After the surgical procedure, rats were put on a heating pad to maintain body temperature at 37.5 ±0.5 °C and were kept on it until recovery from anesthesia (Lavinsky et al., 2006). Two rats submitted to 2VO died after surgery. Animals were submitted to behavioral tasks and biochemical analysis after 10 weeks of chronic hypoperfusion.

4.4.

Behavioral procedure

After 10 weeks of chronic cerebral hypoperfusion, rats were submitted to behavioral testing for the study of reference and working memory in the Morris water maze. The maze consisted of a black circular pool (180 cm of diameter and 60 cm high) filled with water (temperature around 24 ± 1 °C, depth 30 cm), situated in a room that was rich in consistently located spatial cues. The pool was conceptually divided into four quadrants and had four points designed as starting positions (N, S, W or E). An escape platform (10 cm diameter) was placed in the middle of one of the quadrants, 1.5 cm below the water surface (Morris, 1984). Two behavioral protocols, for reference and working memory, were utilized.

4.5.

Reference memory protocol

In this task the rats received five training days (sessions) and a probe trial in the 6th day. Each session consisted of four trials with a 15 min intertrial interval. A trial began when the rat was placed in the water at one of the four starting positions, chosen at random, facing the wall. The order of starting position varied in every trial and any given sequence was not repeated on acquisition phase days. The rats were given 60 s to located the platform; if the animal did not succeed it was gently guided to the platform and left on it for 10 s. Rats were dried and returned to their home cages after each trial. The latency to find the platform was measured in each trial and the mean latency for every training day was calculated. The probe trial consisted of a single trial, as described before, with the platform removed. Here, the latency to reach the original platform position, as well as the time spent in the target quadrant, were measured (Pereira et al., 2007; Vicente et al., 2004). Sessions were recorded by a video system to allow for post-hoc analysis. After a oneweek interval, rats were submitted to the working memory task.

4.6.

Working memory protocol

This protocol consisted of four trials/day, during four consecutive days, with the platform position changed daily. Each trial was conducted as described in the reference memory protocol, with an intertrial interval of 5 min. Latency to find the platform was measured in each trial and the average latency for each trial was calculated, allowing to observe the ability of the animals in locating the novel position of the platform in the day (Pereira et al., 2007).

210 4.7.

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Quantification of S100B and GFAP

S100B content in the hippocampus and cerebrospinal fluid (CSF) was measured by ELISA (Leite et al., 2008). Briefly, 50 μL of sample plus 50 μL of Tris buffer were incubated for 2 h on a microtiter plate previously coated with monoclonal antiS100B (SH-B1). Polyclonal anti-S100B was incubated for 30 min and then peroxidase-conjugated anti-rabbit antibody was added for a further 30 min. A colorimetric reaction with ophenylenediamine was measured at 492 nm. The standard S100B curve ranged from 0.025 to 2.5 ng/mL. ELISA for GFAP was carried out by coating the microtiter plate with 100 μL samples containing 30 μg of protein for 48 h at 4 °C. Incubation with a polyclonal anti-GFAP from rabbit for 2 h was followed by incubation with a secondary antibody conjugated with peroxidase for 1 h, at room temperature; the standard GFAP curve ranged from 0.1 to 10 ng/mL (Tramontina et al., 2007).

4.8.

Immunohistochemistry for GFAP

Rats were anesthetized using ketamine/xylazine and were perfused through the left cardiac ventricle with 20 mL of saline solution, followed by 20 mL of 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4. The brains were removed and left for post-fixation in the same fixative solution at 4 °C for 2 h. After this, the material was cryoprotected by immersing the brain in 30% sucrose in phosphate buffer at 4 °C. The brains were sectioned (60 μm) on a cryostat (Leitz) and sections were treated in 10% methanol and 3% H2O2 for 30 min. Sections were then preincubated in 2% bovine serum albumin (BSA) in phosphatebuffered saline (PBS) containing 0.3% Triton X-100 for 30 min and incubated with polyclonal anti-GFAP from rabbit diluted 1:200 in 2% BSA in PBS-Triton X-100 for 48 h at 4 °C. After washing several times, tissue sections were incubated in a rabbit PAP-conjugated anti-rabbit IgG diluted 1:50 in PBS at room temperature for 2 h. The immunohistochemical reaction was revealed by incubating the sections in a histochemical medium that contained 0.06% 3,3-diaminobenzidine (DAB) dissolved in PBS for 10 min and then, in the same solution containing 1 μM of 3% H2O2 per mL of DAB medium for approximately 10 min. Afterwards, the sections were rinsed in PBS, dehydrated in ethanol, cleared with xylene and covered with Entellan and coverslips. Images were viewed with a Nikon inverted microscope and images transferred to computer with a digital camera (Sound Vision Inc. Wayland, MA) (Feoli et al., 2008).

4.9. Hematoxylin–eosin staining and pyramidal cell counting Slices from the same brains used for GPAP immunohistochemistry were also stained with hematoxylin–eosin. A light microscope was used for analysis. The digitized images obtained at 40× from the pyramidal layer of the CA1 dorsal hippocampal region were converted to an 8-bit gray scale (0– 255 gray levels). All lighting conditions and magnifications were kept constant. The number of pyramidal neuron per mm2 (bidimensional counting) was estimated using a Nikon Eclipse E-600 microscope (500×), coupled to a digital camera. A region of interest measuring 0.023 mm2 was overlaid at the analyzed region. The nucleus located inside this square or

intersected by the upper and/or right edges of the square were counted. Cells that were intersected by the lower and/ or left edges of the square were not counted. At least 2 sections were analyzed in each brain and counted manually. Neurons considered as necrotic, i.e. when they exhibited pyknosis, karyorrhexis, karyolysis, cytoplasmic eosinophilia (“red neuron”), or loss of affinity for hematoxylin (“ghost neuron”) were excluded from counting (Davidson et al., 2000; Henninger et al., 2007).

4.10.

Glutamate uptake

The animals were sacrificed by decapitation, the brains were removed and placed in cold saline medium with the following composition (in mM): 120 NaCl; 2 KCl; 1 CaCl2; 1 MgSO4; 25 HEPES; 1 KH2PO4 and 10 glucose, adjusted to pH 7.4 and previously aerated with O2. The hippocampi were dissected and transverse slices of 0.3 mm were obtained using a McIlwain Tissue Chopper. Slices were then immediately transferred into 24-well culture plates, each well containing 0.3 mL of physiological medium and only one slice. Glutamate uptake was performed as previously described (Feoli et al., 2006). Briefly, media were replaced by Hank's balanced salt solution (HBSS) containing (in mM): 137 NaCl; 0.63 Na2HPO4; 4.17 NaHCO3; 5.36 KCl; 0.44 KH2PO4; 1.26 CaCl2; 0.41 MgSO4; 0.49 MgCl2 and 1.11 glucose, in pH 7.2. The assay was started by the addition of 0.1 mM L-glutamate and 0.66 μCi/mL L-[2,3-3H] glutamate. Incubation was stopped after 5 min by removal of the medium and rinsing the slices twice with ice-cold HBSS. Slices were then lysed in a solution containing 0.1 M NaOH and 0.01% SDS. Sodium-independent uptake was determined using N-methyl-D-glucamine instead of sodium chloride. Sodium-dependent glutamate uptake was obtained by subtracting the non-specific uptake from the specific uptake. Radioactivity was measured with a scintillation counter.

4.11.

Glutamine synthetase (GS) activity

The enzymatic assay was performed, as previously described (Feoli et al., 2006). Briefly, homogenized tissue samples were added to a reaction mixture containing (in mM): 10 MgCl2; 50 Lglutamate; 100 imidazole–HCl buffer (pH 7.4); 10 2-mercaptoethanol; 50 hydroxylamine–HCl; 10 ATP and incubated for 15 min at 37 °C. The reaction was stopped by the addition of 0.4 mL of a solution containing: 370 mM ferric chloride; 670 mM HCl; 200 mM trichloroacetic acid. After centrifugation, the supernatant was measured at 530 nm and compared to the absorbance generated by standard quantities of γ-glutamylhydroxamate treated with ferric chloride reagent.

4.12.

Protein measurement

Protein was measured by Lowry's method (Peterson, 1977) using bovine serum albumin as a standard.

4.13.

Statistical analysis

Parametric data are reported as mean ± SEM and were analyzed by Student's t test (when two groups were considered) or by one-way analysis of variance (ANOVA), followed by

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Duncan's multiple range test when indicated, in the SPSS-16.0. Values of p < 0.05 were considered significant.

Acknowledgments Brazilian funds from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal do Ensino Superior (CAPES), FINEP/Rede IBN 01.06.0842-00.

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