Cell Mol Neurobiol (2010) 30:683–692 DOI 10.1007/s10571-009-9492-1

ORIGINAL RESEARCH

Effects of Acute Perinatal Asphyxia in the Rat Hippocampus Juliana Karl Frizzo • Michele Petter Cardoso • Adriano Martimbianco de Assis • Marcos Luiz Perry Cinzia Volonte´ • Marcos Emı´lio Frizzo

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Received: 14 July 2009 / Accepted: 27 December 2009 / Published online: 23 January 2010 Ó Springer Science+Business Media, LLC 2010

Abstract In the present work, we have used a rat animal model to study the early effects of intrauterine asphyxia occurring no later than 60 min following the cesareandelivery procedure. Transitory hypertonia accompanied by altered posture was observed in asphyxiated pups, which also showed appreciably increased lactate values in plasma and hippocampal tissues. Despite this, there was no difference in terms of either cell viability or metabolic activities such as oxidation of lactate, glucose, and glycine in the hippocampus of those fetuses submitted to perinatal asphyxia with respect to normoxic animals. Moreover, a J. K. Frizzo
C. Volonte´
M. E. Frizzo (&) Fondazione Santa Lucia, Neurobiology Unit, CNR/Fondazione Santa Lucia, 65 Via del Fosso di Fiorano, 00143 Rome, Italy e-mail: [email protected]

significant decrease in glutamate, but not GABA uptake was observed in the hippocampus of asphyctic pups. Since intense ATP signaling especially through P2X7 purinergic receptors can lead to excitotoxicity, a feature which initiates neurotransmission failure in experimental paradigms relevant to ischemia, here we assessed the expression level of the P2X7 receptor in the paradigm of perinatal asphyxia. A three-fold increase in P2X7 protein was transiently observed in hippocampus immediately following asphyxia. Nevertheless, further studies are needed to delineate whether the P2X7 receptor subtype is involved in the pathogenesis, contributing to ongoing brain injury after intrapartum asphyxia. In that case, new pharmacologic intervention strategies providing neuroprotection during the reperfusion phase of injury might be identified.

J. K. Frizzo e-mail: [email protected]

Keywords Hypoxia
Excitotoxicity
Glutamate uptake
P2 receptors

M. P. Cardoso
M. E. Frizzo Department of Morphological Sciences, UFRGS, Rua Sarmento Leite, 500, CEP 90050-170 Porto Alegre, Brazil

Introduction

M. P. Cardoso e-mail: [email protected] A. M. de Assis
M. L. Perry Department of Biochemistry, UFRGS, Rua Ramiro Barcelos, 2600, CEP 90035-003 Porto Alegre, Brazil A. M. de Assis e-mail: [email protected] M. L. Perry e-mail: [email protected] C. Volonte´ Institute of Neurobiology and Molecular Medicine, CNR, Via del Fosso di Fiorano 64, Localita` Prato Smeraldo, 00143 Rome, Italy e-mail: [email protected]

Asphyxia can be defined as a condition of impaired blood gas exchange leading, if it persists, to progressive hypoxemia (a decreased partial pressure of oxygen in blood) and hypercapnia (an excess of carbon dioxide in the blood) (Bax and Nelson 1993). It can occur in infants around the time of birth for several reasons, including compression of the umbilical cord, placental abruption, abnormal uterine contractions, or failure of the neonate to successfully begin breathing (de Haan et al. 2006). Hypoxia/ischemia at birth induces severe long-term impaired neurodevelopment, resulting in spasticity, epilepsy, and mental retardation when the insult is severe, or attention-deficit hyperactivity syndrome and minimal brain disorder, when it is mild

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(Boksa and El-Khodor 2003). There is clinical (Van Erp et al. 2002) and experimental (Pulsinelli et al. 1982) evidences indicating that circuitries within the hippocampus are also extremely vulnerable to hypoxic/ischemic insults (Pulsinelli et al. 1982; Vannucci 1990). Several studies of infants asphyxiated at term report injury to the hippocampus, and this site of damage has been implicated in different long-term outcomes, such as cognitive memory impairment and the psychiatric disorder schizophrenia (de Haan et al. 2006; Wakuda et al. 2008). The vulnerability of the hippocampus has been linked to its dense and highly active glutamatergic neurons that contribute to an excitotoxic cascade (Vannucci 1990). Also extracellular ATP binding to purinergic receptors is among the signals responsible for the hippocampal balance between cell death/survival after the insurgence and progression of hypoxic/ischemic insults (Burnstock 2008; Apolloni et al. 2009). The cell surface purine/pyrimidine nucleotide receptors, termed P2 receptors, are subdivided into two major groups: G protein-coupled P2Y subunits [P2Y1,2,4,6,11–14], and P2X ligand-gated ion channels [P2X1–7] (Burnstock 2007). These receptors have been described as being responsible for varying, even opposite, biological effects (Volonte´ et al. 2006, 2008). Additionally, several studies have suggested a possible cross talk between glutamatergic and purinergic mechanisms, especially in the course of ischemia (Cavaliere et al. 2004a; Frizzo et al. 2007). It is noteworthy that the P2X7 receptor-subtype appears to be causally related to ischemic injury in the brain (Wirkner et al. 2005; Cavaliere et al. 2004b; Milius et al. 2008). Deficiency of oxygen and glucose indeed causes P2X7 receptor hypersensitivity (Wirkner et al. 2005) and up-regulation of the corresponding immunoreactivity, under both in vitro and in vivo conditions (Cavaliere et al. 2004b; Franke et al. 2004; Milius et al. 2008). Additionally, oxygen/glucose deprivation may alter the trafficking properties of P2X7 receptor, thus increasing its integration into the plasma membrane (Milius et al. 2007). Moreover, P2X7 receptor activation has also been reported to promote the release of glutamate and GABA in the hippocampus (Fellin et al. 2006; Papp et al. 2004) as well as under ischemic-like conditions (Sperla´gh et al. 2007). Given that the hippocampus seems to be especially affected by perinatal asphyxia (PA), the aim of this study is to evaluate neurochemical and functional changes in fetuses exposed to asphyxia followed by normoxic reoxygenation, and to study particularly the potential modulation of expression of the purinergic P2X7 receptor subtype.

Methods Pregnant female Wistar rats were housed individually (21 ± 1°C, humidity 60 ± 10%, 12 h light–dark cycle)

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with water and food available ad libitum. In the last day of gestation they were euthanized by neck dislocation and the uterus horns were isolated through an abdominal incision (Bjelke et al. 1991). One horn still containing the fetuses was transferred to a saline bath at 37°C for 15 min to mimics the asphyxia condition, while the other was immediately hysterectomized, providing cesarean-delivered controls. The control pups were removed, cleaned, the umbilical cord was ligated and the animals were allowed to recover in a hood with normal atmospheric conditions at 37°C. The uterine horn that was maintained for 15 min in a saline bath at 37°C was incised and the pups rapidly removed and stimulated to breathe. In order to study the early effects of intrauterine asphyxia the period considered in our work was no longer than 60 min following the cesarean-delivery procedure. We avoided the use of surrogate dams for nursing, so, pups were immediately decapitated or, when necessary, maintained in an incubator (30 ± 1°C) for different times until 60 min. Cesareandelivered control and asphyctic pups were obtained from the same mother, since each rat delivered approximately 10 pups. All animal procedures were approved by the Institutional Ethical Committee (Ethical Committee, UFRGS). Lactate Determination Pups were decapitated and all the blood was immediately drained into an eppendorf coated with sodium fluoride and centrifuged (4,000 rpm to 5 min). Simultaneously, each brain was rapidly dissected and both hippocampi were transferred to wells containing 500 ll of Hank’s balanced salt solution (HBSS) supplemented with 4.6-mM glucose (glucose-HBSS), maintained for 60 min in a humidified atmosphere of 95% air/5% CO2 at 36.5°C. Plasma lactate and lactate present in the incubation medium was determined by an enzymatic colorimetric assay (LOD-PAP, Katal, Brazil). In detail, 10 ll of plasma or incubation medium were incubated for 5 min at 37°C with 1 ml of reagent solution containing lactate oxidase, peroxidase, 4-aminoantipyrine, and N-ethyl-N-(2-hydroxy-3-sulphopropyl)-m-toluidine. Absorbance was read at 540 nm. Assessment of Cell Viability Hippocampal viability and metabolic activity were examined with a mitochondrial assay using MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Whole hippocampi (two per well) were incubated for 60 min with MTT (0.5 mg ml-1) in 500 ll of glucoseHBSS, maintained in a humidified atmosphere of 95% air/ 5% CO2 at 36.5°C. Subsequently, formazan was dissolved with 1 ml DMSO, and the optical density of the solution

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was measured spectrophotometrically using the difference between the 570 and 630 nm wavelengths. Lactate dehydrogenase (LDH) is a stable cytosolic enzyme and its activity into the incubation medium was used as another marker of cell damage. Hippocampi from control and asphyxiated animals were transferred to multiwell dishes, two hippocampi from each animal per well. Afterward, each well was washed with 1 ml of HBSS, which was replaced by 500 ll glucose-HBSS. The multiwell dish was incubated in a humidified atmosphere of 95% air/5% CO2 at 36.5°C. LDH-activity in the samples of incubation medium was determined using an LDH Liquiform kit (Labtest, Brazil). After 2 h, 25 ll of the incubation medium were added to 1 ml of freshly made sodium pyruvate and NADH. This solution was immediately measured at 340 nm. Data are shown as percentages of the whole hippocampus lysis. Glutamate and GABA Uptake Following asphyxia, hippocampi were quickly obtained and transferred to multiwell dishes containing 0.3 ml HBSS at 36.5°C. Uptake was assessed by adding 0.66 lCi mL-1 3 -1 L-[2,3- H] of glutamate (33 Ci mmol , Amersham) with 100 lM unlabeled glutamate or 0.67 lCi mL-1 [2,3-3H] GABA (89 Ci mmol-1, Amersham) with 25 lM unlabeled GABA. To evaluate the optimal time for assaying L-[3H] glutamate or [3H] GABA uptake, we carried out incubation time courses using hippocampus (data not shown), and the time chosen was 4 and 10 min for glutamate and GABA, respectively. Glutamate and GABA uptake were performed after 15 min of asphyxia at different times after the injury (0, 15, 30, or 60 min). Until 15, 30, or 60 min after asphyxia, the hippocampi were maintained in a humidified atmosphere of 95% air/5% CO2 at 36.5°C before being incubated with glutamate or GABA. Incubation was stopped by two ice-cold washes with 1 ml of HBSS immediately followed by addition of 0.5 M NaOH. Aliquots of lysates were taken in order to determine the intracellular content of L-[3H] glutamate or [3H] GABA through scintillation counting. To determine the actual uptake, parallel experiments were done on ice, using N-methyl-D-glucamine instead of sodium chloride in the incubation medium. Values obtained in these conditions were subtracted from the uptake at 36.5°C. Protein determination was assessed using the method described by Bradford (1976). Substrate Oxidation and Incorporation into Lipids and Proteins The lactate, glucose, and glycine metabolisms were studied immediately after hysterectomy in non-asphyctic and

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asphyctic fetuses. For the measurement of protein synthesis, lipid synthesis and CO2 production, two hippocampi per well were incubated in: (1) 1.0 ml of Dulbecco’s phosphate buffered saline (D-PBS), pH 7.4, containing ? 0.2 mM glycine ? 0.3 lCi [U-14C] glycine (101 mCi mmol-1, Amersham); or (2) 1.0-ml D-PBS, pH 7.4, containing ?10 mM lactate ? 0.2 lCi L-[U-14C] lactate (154 mCi mmol-1, Amersham); or (3) 1.0 ml D-PBS, pH 7.4, containing 5.0 mM glucose ? 0.3 lCi L-[U-14C] glucose. The flasks were previously gassed with a 95% O2:5% CO2 mixture for 10 min and incubated for 1 h in a Dubnoff metabolic shaker (60 cycles min-1, 36.5°C). The incubation was stopped by adding 0.25 ml 50% trichloroacetic acid (TCA). Then 0.25 ml of 1 M hyamine hydroxide was injected and the flasks were shaken for further 30 min (36.5°C) to trap CO2. The flask contents were homogenized and centrifuged. The precipitate was washed three times with 10% TCA, and lipids were extracted with chloroform– methanol (2:1). The precipitate resulting after washing with chloroform–methanol was dissolved in concentrated formic acid and the radioactivity was measured. This radioactivity represents protein synthesis from the amino acids. All the results were expressed considering the initial specific activity of the incubation medium. Protein Extraction Hippocampi were homogenized in RIPA buffer [phosphate buffer saline, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethanesulfonyl fluoride, and 2 lM Leupeptin] at pH 7.6. After 30 min incubation on ice, the samples were centrifuged at 10,0009g for 10 min at 7°C and the supernatants collected. Protein determination in the supernatants was assessed using the method described by Bradford (1976). SDS-PAGE electrophoresis buffer (pH 6.8) containing 4% SDS, 5% mercaptoethanol, 2 mM EDTA, and 50 mM Tris–HCl was added to each sample, which was then boiled (3 min) and stored at -20°C. Western Blot Analysis Samples containing equal amounts of protein (50 lg) were subjected to SDS-polyacrylamide gel electrophoresis (10% acrylamide) and transferred to nitrocellulose filters (Hybond C, Amersham Biosciences, MI, Italy). Filters were incubated with a blocking solution containing 10 mM Tris (pH 7.5), 150 mM NaCl, 0.1% Tween-20 (Tris buffered saline with Tween 20, T-TBS), and 5% non-fat powdered milk for 1 h at room temperature, rinsed in T-TBS, and then incubated 3 h with specific antibodies diluted in T-TBS containing 5% non-fat powdered milk [P2X7 (Alomone) was diluted 1:500; monoclonal antibody

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recognizing b-actin (Sigma) was diluted 1:2,000]. After three rinses in T-TBS, the filters were incubated for 1 h at room temperature with horseradish peroxidase-conjugated antirabbit or antimouse (both from Cell Signaling Technology) IgG, diluted 1:5,000 in T-TBS. The immunoreactions were performed using ECL chemiluminescence kit (Amersham Biosciences), and quantified with 1D Image Analysis Software in a Kodak Image Station 440CF. Statistical Analysis Data were analyzed using one-way analysis of variance (ANOVA) for multiple group comparison. Post hoc analysis was carried out using the Duncan multiple range test. Values of P \ 0.05 were considered statistically significant.

Results In the normoxic group, we observed 100% animal survival, while in the group exposed to asphyxia for 15 min, the survival rate was 90 ± 6%. It is important to note that when the length of asphyxia within the model is increased to 20 min (severe PA), the mortality rate is 50 ± 5% (Fig. 1a). Hence, in our study, we analyzed the effects of 15 min of asphyxia. Immediately after being removed from the uterus, the asphyxiated pups exhibited bluish coloring, loss of movement, and sporadic gasping. Around 20 min following asphyxia, the original pink coloring returned, and the breathing became regular. Hypertonic phenotype accompanied by altered posture was observed from 30 ± 1 min until 58 ± 2 min after asphyxia (Fig. 1b). This condition began as lumbar rectification, followed by extension of posterior legs with resistance-to-motion (Fig. 1c). The sharp increase in muscle tone was completely reversed near the end of the observation period. Given that lactate is invariably produced in the event of hypoxia and poor tissue perfusion, we also verified this parameter in our model of intrapartum asphyxia. As expected, the plasma levels of lactate in normoxia (8.09 ± 0.55 mmol/l) were significantly enhanced after 15 min of asphyxia (13.23 ± 0.13 mmol/l), Fig. 2a. Moreover, lactate released from asphyxiated hippocampal tissue was also significantly higher (81%) than in controls (Fig. 2b). This was obtained in the absence of plasma membrane damage up to 60 min after asphyxia, as measured by LDH efflux assay (Fig. 3a) and Fluoro-Jade histochemistry (data not shown). Likewise, metabolic activity determined with the MTT/formazan assay did not differ significantly between controls and asphyxiated animals. Furthermore, for both control and asphyxiated animals, the increase in the metabolic activity determined by the MTT/ formazan assay at 60 min was not significantly different

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Fig. 1 Hypertonic phenotype follows perinatal asphyxia. Panel a represents quantitative data on survival after 15 or 20 min of asphyxia. In panel b, the paradigm used is depicted, where dotted line is intrauterine asphyxia (15 min) followed by postpartum period (60 min). In the postpartum period, asphyxiated pups exhibited altered tonus from 30 ± 1 until 58 ± 2 min. In panel c, the upper image is a control and the lower one an asphyxiated pup presenting hypertonia and altered posture (mean ± SEM, n = 12)

than the values observed immediately after the uterus hysterectomy (Fig. 3b). Lactate, produced in glial cells and other fetal compartments during asphyxia, may become the only utilizable and thus obligatory substrate for the recovery of aerobic energy metabolism upon reoxygenation (Seidl et al. 2000). Accordingly, we studied lactate and additionally glucose and glycine oxidations in the hippocampus immediately after intrauterine asphyxia. Hippocampi from non-asphyctic and asphyctic fetuses were incubated for 1 h in D-PBS previously gassed, in order to mimic reoxygenation. Oxidation of L-[U-14C] lactate by tissue submitted to asphyxia

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Fig. 3 Perinatal asphyxia does not affect cellular viability and metabolic activity in the hippocampus. The analysis of LDH-activity 60 min after asphyxia showed there to be no difference in comparison with control (Panel a). Panel b shows the metabolic activity in the hippocampus immediately (0 min) and after 60 min of asphyxia. In both the cases, significant differences were not observed in comparison with controls. Results are shown as mean ± SEM, n = 5

Fig. 2 Effect of PA on measured lactate concentration. Panel a shows lactate present in the plasma where a significant increase (64%) was observed after 15 min of asphyxia. Panel b shows the levels of lactate released from hippocampus, which were significantly enhanced by asphyxia (81%). Data are represented as mean ± SEM, n = 12, P \ 0.05

for 15 min was not different from the control (Fig. 4a). Similar results were obtained for L-[3-14C] glucose and [U-14C] glycine oxidation (Fig. 4b, c). Given that glutamate is released following hypoxia/ anoxia in the hippocampus (Perlman 2006), glutamate uptake was tested immediately (0 min), or 15, 30, and 60 min after asphyxia (Fig. 5a). Glutamate uptake was significantly decreased at 0 min (80.2 ± 4.3%), it presented another significant reduction at 15 min (54.2 ± 5.2%), and then remained constant at 30 (63.6 ± 5.4%) and 60 min (55.4 ± 7.7%). Since GABA uptake also occurs through a Na-dependent process, we tested whether PA could also affect GABA uptake (Fig. 5b). However, in contrast to

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Fig. 4 Effect of PA on lactate, glucose, and glycine metabolisms. Hippocampi from normoxic and asphyxiated fetuses were weighed and incubated for 60 min in D-PBS, containing L-[U-14C] lactate, 14 14 L-[U- C] glucose, or [U- C] glycine for the measurement of protein synthesis, lipid synthesis, and CO2 production. Metabolism of lactate, glucose, and glycine are represented in panels (a), (b), and (c), respectively (data are mean ± SEM, n = 3)

glutamate, we observed a slight, but not significant, increase in the GABA uptake following asphyxia. The ATP-ionotropic P2X7 receptor, whose expression has been shown to be enhanced following hypoxia/ ischemia (Cavaliere et al. 2004b) was also investigated. A significant enhancement in P2X7 protein expression was observed immediately following asphyxia (296 ± 25%), which decreased to levels near control at 60 min (Fig. 6).

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Fig. 5 Perinatal asphyxia affects glutamate transport activity. L-[3H] glutamate and [3H] GABA uptake were performed at different times following 15 min of asphyxia. Panel a shows that glutamate uptake was significantly reduced (20%) immediately after asphyxia and remained at around 50% from 15 to 60 min following asphyxia (* different from control, ** different from control and all other groups, at P \ 0.05, n = 5). On the other hand, in the same conditions, GABA transport was not affected (Panel b, n = 6). Data are mean ± SEM

Discussion This study was aimed at investigating the early effects of intrauterine asphyxia on a rat animal model. Here, we reported a transitory hypertonia, i.e., abnormally increased resistance to externally imposed movements around a joint (Scher 2008), accompanied by altered posture in

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Fig. 6 Perinatal asphyxia induces P2X7 expression in hippocampus. Western blot analysis of P2X7 expression was done immediately (0 min) or 60 min after asphyxia. Panel a beta-actin antibody was used as internal standard for loading and transferring control. In panel b, densitometric analysis of the respective blots was performed and the quantitative data compared to the control condition, considered 100%, and shown as mean ± SEM (n = 5). Note that PA provoked a significant and transitory increase in P2X7 expression (P \ 0.01)

asphyxiated pups. Despite this phenomenon not be directly related with hippocampus, it was described given that a similar phenotype was not mentioned previously after procedures of intrauterine asphyxia. Although this condition was seen to be temporary, in our experiments, it was also intense and severe. Additionally, asphyxiated pups showed an evident metabolic acidosis, which is without doubt one of the most important clinical indicators of neonatal asphyxia. Actually, studies have demonstrated a correlation between the degree of acidosis and the neonatal neurological outcome (Hanrahan et al. 1998). Values of lactate measurements were compared in the diagnosis and short-term prognosis of intrapartum asphyxia in term neonates. Lactate concentration in the plasma greater than 9 mmol/l has been associated with moderate or severe hypoxic ischemic encephalopathy (HIE) in human neonates (Silva et al. 2000). In another study, a plasma lactate concentration of 11.09 ± 4.6 mmol/l in the first hour after birth was associated with moderate to severe HIE (Shah et al. 2004). In human neonates with severe hypoxia, plasma lactate values above 15 mmol/l predicted death (Deshpande and Platt 1997) and an unfavorable developmental outcome in survivors (Cheung et al. 1996). In our

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paradigm, lactate values were determined immediately after the asphyxia period, and a significant increase in plasma lactate values was observed. Additionally, we observed a significant increase in the lactate released from hippocampal tissue after asphyxia. Despite this, here we found that both cell viability and metabolic activity in the hippocampus of fetuses submitted to PA were not significantly different from normoxic animals. In our model, we observed that oxidation of lactate and glucose by the asphyctic hippocampus was not significantly different from that of control fetuses. These results are in agreement with data from adult animals and humans that indicate that cerebral metabolism is maintained during severe reductions in oxygen delivery, until near oxygen starvation (Cohen et al. 1967; Gjedde et al. 2002). It is noteworthy that, in contrast to the neocortex and striatum, post-ischemic glucose utilization in the hippocampus equaled that of control values at 1 h (Pulsinelli et al. 1982). An additional important energy source for the oxidative metabolism of neonates is the amino acid glycine, which is oxidized exclusively by astrocytes (dos Santos Fagundes et al. 2001). Ischemia elicits the rapid release of various amino acids, one of which is glycine. There is a direct correlation between the severity of HIE and the extracellular glycine concentration (Rolda´n et al. 1999; Oda et al. 2007), although its effects on ischemic injury remain controversial (Oda et al. 2007). Here, we observed that oxidation of glycine in the asphyctic hippocampus was not significantly different from that of the control animals. Taken together these results suggest that the oxidative energetic metabolism of asphyctic pups immediately after asphyxia is not essentially different from normoxic fetuses. The release of the excitatory amino acid glutamate is crucial in the pathogenesis of neuronal death following hypoxic ischemic insult (Perlman 2006; Hagberg et al. 1993). In fact, in asphyxiated infants the concentration of glutamate in the cerebrospinal fluid (CSF) was 387% that of the control values (Hagberg et al., 1993), and Khashaba et al. (2006) demonstrated a significant correlation between its concentration in CSF and severity of encephalopathy after PA. Furthermore, glutamate concentration was significantly increased in more severe grades of HIE, when compared to milder grades (Hagberg et al. 1993; Khashaba et al. 2006). Additionally, its concentration was highest immediately after birth, and Khashaba et al. (2006) concluded that early involvement of this amino acid in the process of encephalopathy was probably triggered and peaked either prenatally or in the immediate postnatal period. Since there is no extracellular conversion of glutamate, brain tissue needs a very high glutamate uptake activity to protect itself against excitotoxicity (Danbolt 2001). Here, we observed a significant decrease in glutamate uptake immediately after PA. The clearance of the

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extracellular glutamate occurs by a special uptake mechanism, which uses the Na?, K? electrochemical gradient as a driving force (Danbolt 2001). In contrast to glutamate, GABA uptake, which is also dependent of the Na?, K? electrochemical gradient, was not significantly affected by 15 min of asphyxia. Taking into account that in a similar PA model, significant differences in ATP decline were only observed after 15 min of asphyxia (Seidl et al. 2000), we could hypothesize the existence of sufficient ATP to maintain a Na?-dependent process. Thus, the maintenance of glutamate uptake under normoxic levels remains unexplained. The huge extracellular glutamate concentrations described after PA (Perlman 2006; Hagberg et al. 1993; Khashaba et al. 2006) could be explained at least partially by the ineffectiveness of its transporters, perhaps not by energy deficiency, but maybe by a kind of specific inhibition. Moreover, recent in vivo experiments showed that the hippocampal tissue levels of glutamate were elevated immediately after the hypoxic–ischemic insult and remained enhanced even 7 days after the asphyctic insult (Papazisis et al. 2008). The maintenance of glutamate uptake under normoxic levels could thus explain, at least partially, the imbalance in glutamate turn-over in the neonatal hippocampus. However, additional experiments are necessary to better understand this feature. P2X7 receptor activation is known to promote glutamate release in the hippocampus (Fellin et al. 2006; Papp et al. 2004; Sperla´gh et al. 2007) and it was recently described as being able to quickly inhibit [3H] glutamate uptake in RBA-2 cells (Lo et al. 2008). In line with these results, we observed a significant increase in P2X7 expression after 15 min of PA, and its immunocontent returned to control levels 60 min after asphyxia. The up-regulation of the P2X7 immunoreactivity elicited by ischemic-like conditions has been described previously (Cavaliere et al. 2004b; Franke et al. 2004). However, its function in the hippocampus subjected to asphyxia remains unclear. Nonetheless, recent findings suggest a mechanism by which glutamate can be released from astrocytes in response to extracellular ATP binding to P2X7 receptors. Although the channel opened by P2X7 ligand binding is not highly selective for glutamate, the strong driving force for glutamate release favors a significant glutamate efflux through the activated channel, corresponding to the release of approximately [30% of the intracellular glutamate pool within the 6-min observation interval (Duan et al. 2003). Thus, the up-regulation in P2X7 protein that we observe after PA could very likely cause the glutamate accumulation reported under our experimental conditions. Since the overall increased expression of the P2X7 receptor is transient and returns to control values within 60 min, an increasing extracellular concentration of glutamate such as that occurring during PA (Perlman 2006; Khashaba et al.

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2006) might even be responsible for a feedback loop and consequent down-regulation of the purinergic receptor itself. This might very well represent a cellular effort to counteract and possibly halt the detrimental outcome of PA. To our knowledge, this is the first study demonstrating an increase in P2X7 receptor expression after intrauterine asphyxia. Further studies are needed to identify whether this receptor is directly involved in the pathogenesis, contributing to ongoing brain injury after perinatal asphyxia. Hypoxia–ischemia (HI) administered on postnatal day (pnd) 7 in rats produces a severe form of injury with high mortality rates and is thought to represent brain damage occurring during birth of term infants (Stadlin et al. 2003). Conversely, HI insults administered to rats at pnd 3 produce only minor brain injury and offer a reliable model for damage expected in a preterm infant (from 24 to 28 weeks of gestation) suffering a nonlethal perinatal insult (Stadlin et al. 2003). In the rat, graded intrauterine asphyxia has been successfully used to mimic an asphyxial insult occurring in the premature human brain (Boksa et al. 1995; Bjelke et al. 1991; Chen et al. 1997), since the level of brain maturation in the rat at birth is comparable to that of the human fetus in the last trimester of gestation (Dobbing and Sands 1979). In effect, some studies have shown that morphological/biochemical outcomes caused by early neonatal hypoxia are time- and age-dependent (e.g., Towfighi and Mauger 1998; Northington et al. 2001). Hence, taking into account that intrauterine versus postnatal insults are very different conditions, their relative contribution to the outcome of the diseased asphyctic CNS, including, e.g., expression levels of P2X7 receptors and lactate levels, is undoubtedly an interesting issue and would deserve further investigation. Acknowledgments This research was supported by Grants from the Brazilian National Research Council (CNPq) and by MIUR project # 2006059022-005 and Grant from Ministero della Salute RF05.105. Juliana K. Frizzo is a recipient of a post-doctoral fellowship from the Brazilian funding agency CNPq.

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