Chemical preconditioning with 3-nitropropionic acid: Lack of induction of neuronal tolerance in gerbil hippocampus subjected to transient forebrain ischemia

Brain Research Bulletin, Vol. 58, No. 1, pp. 33–39, 2002 Copyright © 2002 Published by Elsevier Science Inc. 0361-9230/02/$–see front matter

PII: S0361-9230(02)00753-0

Chemical preconditioning with 3-nitropropionic acid: Lack of induction of neuronal tolerance in gerbil hippocampus subjected to transient forebrain ischemia P. Garnier,1 N. Bertrand,1∗ C. Demougeot,1,2 A. Prigent-Tessier,1 C. Marie1 and A. Beley1 1 Unité

de Biochimie, Pharmacologie, Toxicologie, Laboratoire de Pharmacodynamie, Dijon, France; and 2 Service de Neurologie, Center Hospitalier Universitaire, Dijon, France

[Received 14 September 2001; Revised 3 December 2001; Accepted 11 December 2001] 3-NP inhibits cellular respiration by irreversible inactivation of succinate dehydrogenase (SDH), a mitochondrial complex II enzyme responsible for the oxidation of succinate to fumarate in the Krebs cycle and the subsequent transport of electrons in oxidative phosphorylation [8]. It has been shown that 3-NP can generate tolerance in in vitro models of cerebral ischemia [1,30,36]. For instance, 3-NP was shown to induce tolerance against oxygen–glucose deprivation in a rat neuronal-cell enriched culture system [37]. Using a gerbil hippocampal slice model, 3-NP administration at the dose of 4 mg/kg to the animals 3 or 24 h prior to slice preparation was also reported to lead to tolerance towards hypoxia, as evidenced electrophysiologically by an improvement of the field excitatory post-synaptic potential recovery in response to the hypoxic exposure [1]. In addition, 3-NP-induced tolerance was reported in vivo after global [24,33] and focal cerebral ischemia [21,38]. However, the underlying mechanisms by which the 3-NP is responsible for the acquisition of the neuronal tolerance remain to be elucidated. It has been postulated that the generation of oxygen free radicals occurring during the preconditioning stimuli may trigger induction of tolerance to ischemia [29,32] through the neosynthesis of neuroprotective proteins, such as heat shock protein 72 (HSP72) [23], anti-apoptotic protein [5,39] and antioxidant enzymes [14,34]. The objective of the present work was to further investigate the mechanisms responsible for the effectiveness of 3-NP in the acquisition of the tolerance phenomenon. To do so, we have compared the extent of the hippocampal neuronal damage as well as the levels of expression of putative protective proteins, HSP72 and manganese superoxide dismutase (MnSOD) between vehicle and 3-NP pretreated gerbils subjected to a transient forebrain ischemia. In addition, the extent of the post-ischemic astroglial and microglial activation was compared between the two groups.

ABSTRACT: Chemical preconditioning using the mitochondrial toxin, 3-nitropropionic acid (3-NP) has been reported to induce neuroprotection against subsequent global ischemia. To investigate the underlying mechanisms, Mongolian gerbils were pretreated with either vehicle or 3-NP at the dose of 3 or 10 mg/kg, intraperitoneal, 3 days prior to a 5-min bilateral carotid artery occlusion followed by either 48 h or 7 days of blood recirculation. Neuronal damage was assessed by a cresyl violet/fuchsin acid staining. Induction of heat shock protein 72 (HSP72) and manganese superoxide dismutase (MnSOD) expression was evaluated by Western blotting. Astroglial and microglial activation was detected by immunohistochemistry (glial fibrillary acid protein) and by histochemistry (isolectin B4 staining), respectively. Present data show that the hippocampal neuronal damage induced by ischemia were of similar extent between the vehicle- and 3-NP-treated gerbils, whatever the dose tested, indicating that 3-NP did not induce tolerance to transient forebrain ischemia under our experimental conditions. The lack of difference in the post-ischemic level of HSP72 and MnSOD protein expression and in the intensity of astroglial and microglial activation represents further indirect indications of the absence of 3-NP preconditioning effect. In conclusion, although chemical preconditioning with 3-NP is a well-established phenomenon at least in vitro and in models of focal ischemia, the relevance of 3-NP as a preconditioning molecule towards global brain ischemia remains an open question. © 2002 Published by Elsevier Science Inc. KEY WORDS: Chemical preconditioning, 3-Nitropropionic acid, Hippocampus, Gerbil, Transient forebrain ischemia, Neuronal tolerance.

INTRODUCTION It is now well-established that sublethal ischemia leads to increased tolerance against subsequent lethal ischemia in brain [17,18,22]. It has also been reported that such neuronal tolerance can be induced by other sublethal stresses, such as hypoxia [9], hyperthermia [19], oxidative stress [26], spreading depression [20], and, more recently, systemic 3-nitropropionic acid (3-NP) treatment [21,24,33,38].

MATERIALS AND METHODS Animal Experiments Experiments were performed on adult male Mongolian gerbils (60–65 g, Center d’Elevage Janvier, France) in accordance with the

∗ Address for correspondence: N. Bertrand, Unit´ e de Biochimie, Pharmacologie, Toxicologie, Laboratoire de Pharmacodynamie, 7 bld Jeanne d’Arc, BP 87900, 21079 Dijon Cedex, France. Fax: +33-3-80-39-33-00; E-mail: [email protected]

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34 French Department of Agriculture guidelines (license no. 00776). All animals were divided into sham-operated groups treated with either vehicle (n = 11) or 10 mg/kg 3-NP (n = 10); 5-min ischemic control groups treated with vehicle, 3 days before induction of ischemia and reperfused for 48 h (n = 10) or 7 days (n = 11); 5 min-ischemic preconditioned groups treated with either 3 or 10 mg/kg 3-NP, 3 days before induction of ischemia and reperfused for 48 h (n = 8 and 4 at 3 and 10 mg/kg 3-NP, respectively) or 7 days (n = 7 and 16 at 3 and 10 mg/kg 3-NP, respectively). 3-NP was dissolved in saline and pH was adjusted to 7.4 with NaOH and administered to animals intraperitoneally. Transient forebrain ischemia was induced by a 5-min bilateral carotid artery occlusion under anesthesia with 2% halothane in a gas mixture of 30% oxygen/70% nitrous oxide. Body and brain temperatures were monitored with rectal and temporal probes, respectively, and were maintained close to 37–38◦ C by external heating. Sham-operated groups were subjected to anesthesia and all surgical procedures except clamping of the carotid arteries. After 48 h or 7 days of blood recirculation (ischemic groups) and after 3 days of vehicle or 3-NP treatment (sham-operated groups), the animals were anesthetized with pentobarbital and brains were perfused either with isotonic saline solution or further perfused with fixative FAM solution (formol 37%, acetic acid 100%, methanol; 1 v/1 v/8 v) depending on the study. Western blotting experiments were carried out on brains only perfused with saline. Brains perfused with FAM were removed, post-fixed in the same fixative for a week, dehydrated in ethanol and embedded in paraffin. Coronal sections of 10-µm-thick including hippocampus and striatum were used for assessments of the neuronal damage, astroglial and microglial activation. Neuronal Damage Coronal sections were deparaffinized, rehydrated, and stained with fuchsin acid and cresyl violet. Neuronal damage was evaluated by counting surviving neurons in different subdivisions of the CA1 (CA1a , CA1b , CA1c ) and CA3 subfields of the hippocampus (both sides) as described previously [16]. All values obtained in each hippocampal sector for each group were averaged and the data expressed per 5 × 103 µm2 as mean ± SEM. Western Blotting for HSP72 and MnSOD Whole hippocampi were quickly dissected out and homogenized in 10 vol. of cold TES buffer (10 mM, pH 7.5) containing 1 mM EDTA, 250 mM sucrose, 0.1% ethanol, and 200 mM protease inhibitor phenylmethylsulfonyl fluoride. After centrifugation (10,000 × g, 10 min), supernatants were stored at −80◦ C until Western blot analysis. Tissue extract samples were placed in Laemmli solubilized buffer (250 mM Tris, pH 6.8, 4% sodium dodecyl sulfate (SDS), 10% glycerol, 0.006% bromophenol blue, 2% β-mercaptoethanol) and boiled for 3 min. Equal amounts of proteins (80 µg for HSP72, 40 µg for MnSOD per sample) were separated on a SDS–polyacrylamide gel. After electrophotransfer onto a polyvinylidene difluoride (PVDF) membrane (0.2 µm pore size, Pall) the membrane was incubated overnight at 4◦ C in phosphate-buffered saline (PBS) containing 5% non-fat dry milk and 0.1% Tween-20, and then incubated for 3 h with the primary antibody. After three washes, membrane was incubated for 30 min with a 1:5000 dilution of either anti-mouse or anti-rabbit IgG horseradish peroxidase antibody (A-2304, A-0545, respectively, Sigma Chemical Co., St. Louis, MO, USA). Finally, the membrane was washed three times and the bound antibody was visualized with the ECL chemiluminescence system according to the manufacturer’s protocol (Amersham, Orsay, France). The dilutions of the different primary antibodies were as follows: 1:1500 for the mouse monoclonal anti-72 kDa HSP antibody (SPA-810, Stress-

GARNIER ET AL. gen, Victoria, BC, Canada) and 1:5000 for the rabbit polyclonal MnSOD antibody (SOD-110, Stressgen). A computer-based imaging system was used to measure the relative optical density of each specific band obtained after Western immunoblotting. Data were expressed as percentage of the control (represented by the value obtained in the vehicle-treated sham-operated animals), which was considered 100%. Histochemical Detection of Microglial Activation Microglial activation was evidenced on the adjacent paraffinembedded sections used for neuronal damage. All sections were deparaffinized, rehydrated, and treated simultaneously. They were first incubated for 15 min in PBS containing 0.1% Triton X-100 and were subsequently incubated overnight at 4◦ C in PBS containing 10 µg/ml of peroxidase-conjugated isolectin B4 derived from Griffonia simplicifolia seeds (L-5391; Sigma) which localizes microglia. The reaction product was visualized by using 0.06% 3,3 -diaminobenzidine tetrachloride as the chromogen in the presence of 0.02% H2 O2 . Then, the sections were lightly counterstained with hematoxylin, dehydrated in graded ethanol and coverslipped with Eukitt medium. Immunohistochemical Detection of Astroglial Activation Astroglial activation was studied on the deparaffinized and further rehydrated sections adjacent to those used for microglia detection. All sections were treated simultaneously. Endogenous peroxidase activity was blocked for 20 min with 3% H2 O2 solution. Non-specific binding was then blocked for 2 h with PBS containing 10% normal goat serum. The sections were incubated for 48 h at 4◦ C with a mouse monoclonal antiglial fibrillary acidic protein (GFAP) antibody (dilution 1:400, G-3893; Sigma), in order to specifically localize activated astrocytes. They were subsequently incubated with biotinylated anti-mouse IgG antibody for 2 h at room temperature, then with peroxidase conjugated streptavidine complex for 1 h at room temperature by using a Biostain Super ABC kit (Biomeda). The reaction product was visualized by using 0.06% 3,3 -diaminobenzidine tetrachloride as the chromogen in the presence of 0.02% H2 O2 . After the final wash, the immunostained sections were dehydrated in graded ethanol and coverslipped with Eukitt medium. Succinate Dehydrogenase Activity Determination Succinate dehydrogenase (SDH) activity measurements were performed on mitochondrial fractions obtained from hippocampi of additional animals treated with either 3-NP (3 or 10 mg/kg) or vehicle (n = 3 per group). One hour and 3 days after treatment, gerbils were decapitated and brains were rapidly removed. The different cerebral structures were dissected out and homogenized in 10 mM cold sodium phosphate buffer (pH 7.4) containing 0.25 M saccharose. Homogenates were then processed for isolation of mitochondrial fractions [4] and measurement of SDH activity was carried out as follows. Five hundred micrograms of proteins diluted in qsp 1.1 ml of distilled water were directly added in cuve used for spectrophotometric measurement. Two minutes after addition in the cuve of 1 ml of 50 mM potassium phosphate buffer (pH 7.5), 100 µl of CaCl2 (50 mM) and 100 µl of KCN (50 mM), 400 µl of succinate (0.25 M), substrate of the enzyme, 200 µl of 2,6-dichlorophenol–indophenol (0.6 mM) as electron donor, and 100 µl of phenazine methosulfate (1%) as chromogen were rapidly added. The extinction of the optical density was measured at wavelength 600 nm over a 2-min period and the hippocampal SDH activity was expressed as OD min−1 mg−1 proteins.

3-NITROPROPIONIC ACID PRECONDITIONING

35 TABLE 1

NEURONAL DAMAGE IN CA1 AND CA3 HIPPOCAMPAL SUBFIELDS AFTER TRANSIENT FOREBRAIN ISCHEMIA IN VEHICLE AND 3-NP-TREATED GERBILS

Group of Animals n Hippocampi

Vehicle-treated sham-operated animals (n = 16)

CA1a CA1b CA1c CA3

39.8 44.2 49.8 27.8

± ± ± ±

3-NP-treated (10 mg/kg) sham-operated animals (n = 14)

1.8 1.0 1.2 0.4

36.0 37.8 40.0 24.4

± ± ± ±

0.6 1.0 1.0 0.4

Vehicle-treated ischemic animals (n = 16)

3-NP-treated (3 mg/kg) ischemic animals (n = 14)

± ± ± ±

5.0 ± 0.2* 5.0 ± 0.2* 4.6 ± 0.2* 24.6 ± 0.6

5.8 6.2 5.8 22.6

0.8* 0.4* 0.4* 1.4

3-NP-treated (10 mg/kg) ischemic animals (n = 26) 4.4 4.2 5.8 23.8

± ± ± ±

0.2* 0.2* 0.4* 0.4*

Neuronal damage was assessed by counting surviving neurons in subdivisions of CA1 (CA1a , CA1b , CA1c ) and in CA3 subfields in the right and left hippocampus and expressed per 5 × 103 µm2 . Ischemia was induced by a 5-min bilateral carotid artery occlusion and followed by 7 days of blood recirculation. Gerbils were treated with vehicle or 3-NP 3 days prior to transient forebrain ischemia. Neuronal counts are expressed as mean ± SEM. ∗ p < 0.05, significantly different from vehicle-treated sham-operated animals.

Data Analysis Quantitative analysis of neuronal damage has been done by two persons blinded to the experimental conditions. Statistical significance was analyzed using one-way ANOVA followed by post-hoc Newman–Keuls test. p ≤ 0.05 was considered to be significant. RESULTS Table 1 summarizes the neuronal damage expressed as the number of surviving neurons in the different hippocampal subfields after vehicle or 3-NP treatment followed or not by the ischemia-recirculation sequence. First, gerbils treated with the highest dose of 3-NP (10 mg/kg) did not exhibit neuronal death in the CA1 and CA3 hippocampal regions after 3 days of treatment. These data evidencing the lack of neurotoxicity of the 3-NP at 10 mg/kg confirmed the sublethal feature of 3-NP treatment under our experimental conditions, necessary for investigating a putative preconditioning effect. Second, after 7 days of blood recirculation, a 5-min ischemia caused a widespread neuronal death in the CA1 subfields of vehicle-treated gerbils, whereas it did not induce significant neuronal damage in the more resistant CA3 subfield. Third, as shown in the table, 3-NP treatment 3 days prior to ischemia was not associated with an increase in the number of surviving CA1 neurons either at 3 mg/kg or at 10 mg/kg. Indeed, the neuronal damage in the different CA1 subfields were found to a similar extent in both vehicle- and 3-NP-treated animals. Table 2 indicates that a single injection of 3-NP at the dose of 3 mg/kg did not induce changes in the hippocampal SDH activity

at 1 h and 3 days post-injection. On the other hand, the highest dose of 3-NP (10 mg/kg) was associated with a significant decrease in SDH activity (approximately −22%) after both 1 h and 3 days of treatment. As shown in Figs. 1 and 2, 3-NP treatment at the dose of 10 mg/kg did not induce expression of HSP72 and MnSOD proteins at 3 days post-injection. In vehicle-treated group, a 5-min transient ischemia followed by 48 h of blood recirculation led to a significant induction of both HSP72 and MnSOD as evidenced by a 25 and 100% increase, respectively, as compared to sham-operated group. After 7 days of recirculation, HSP72 expression returned then to control values while MnSOD expression remained not significantly enhanced. Finally, our data show that 3-NP pretreatment did not change the level of HSP72 and MnSOD expression in ischemic hippocampus. Indeed, the protein induction profile observed in 3-NP-treated groups was found closely similar to that of vehicle-treated groups at both times of blood recirculation. Fig. 3 shows microphotographs of representative hippocampal sections stained for studying astroglial and microglial activation. Astrocytes immunostained for GFAP were scarcely present in vehicle- and 3-NP-treated sham-operated animals (Fig. 3A and B). Our data shows that ischemia led to an astrocytic activation after 7 days of reperfusion, which was found to a similar extent in the hippocampus of vehicle- and 3-NP-treated gerbils (Fig. 3C and D). Likewise, 3-NP treatment prior to ischemia did not induce significant difference in the microglial activation during the reperfusion period as compared to the vehicle-treated group (Fig. 3G and H).

TABLE 2 HIPPOCAMPAL SUCCINATE DEHYDROGENASE (SDH) ACTIVITY FOLLOWING 3-NP TREATMENT

3-NP treatment 3 mg/kg Vehicle treatment SDH activity (OD min−1 mg−1 proteins)

141 ± 3

10 mg/kg

1h

3 Days

1h

3 Days

134 ± 2

151 ± 4

112 ± 2*

111 ± 3*

SDH activity was performed on mitochondrial fractions obtained from gerbil hippocampus after 1 h and 3 days of 3-NP treatment. SDH activity was expressed as mean ± SEM from n = 6 hippocampi in each group. ∗ p < 0.05, significantly different from vehicle-treated sham-operated animals.

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GARNIER ET AL.

FIG. 1. Western immunoblot analysis of gerbil hippocampus heat shock protein 72 (HSP72). Samples containing 80 µg of proteins were analyzed by SDS–polyacrylamide gel electrophoresis and immunoblotting by using a mouse monoclonal anti-HSP72 antibody. (A) Representative immunoblot of HSP72 expression. Lane 1: Vehicle-treated sham-operated animal (control); Lane 2: 3-NP-treated sham-operated animal; Lane 3: Vehicle-treated ischemic animal after 48 h of blood recirculation; Lane 4: 3-NP-treated ischemic animal after 48 h of blood recirculation; Lane 5: Vehicle-treated ischemic animal after 7 days of blood recirculation; Lane 6: 3-NP-treated ischemic animal after 7 days of blood recirculation. (B) Densitometric analysis from three independent experiments with three ischemic animals and three sham-operated animals of each subgroup (mean±SEM of values expressed as percentage of the control, which was considered 100%). The dose of 3-NP used in this study was of 10 mg/kg. Significant differences were assessed by analysis of variance followed by post-hoc Newman–Keuls test (p < 0.05). (*) Significantly different compared with vehicle-treated sham-operated animals.

DISCUSSION Sugino et al. [33] have first reported the neuroprotective effect of 3-NP preconditioning in a model of transient global ischemia. These authors have shown that the highest effectiveness of 3-NP to induce neuronal tolerance to transient forebrain ischemia in gerbils was obtained at a dose of 3 mg/kg, 3 days after intraperitoneal administration. Indeed, they have observed that gerbils exhibited neuronal damage of similar extent to those obtained in vehicle-treated animals when gerbils were pretreated with 3-NP at lower dose (1 mg/kg) and higher dose (10 mg/kg) and when the interval between the injection of 3-NP and the ischemic episode was shortened (1 day) or elongated (4 and 7 days). As indicated in the Introduction, the present study was aimed to elucidate by which mechanisms the chemical preconditioning with 3-NP treatment induced neuronal tolerance to cerebral ischemia. To do so, our study was initially based exactly on the experimental conditions of Sugino et al. [33] in terms of 3-NP dose (3 mg/kg), duration of treatment (3 days), administration route (intraperitoneal), animal model (gerbil of similar age, gender, and strain), control of

FIG. 2. Western immunoblot analysis of gerbil hippocampus manganese superoxide dismutase (MnSOD) protein. Samples containing 40 µg of proteins were analyzed by SDS–polyacrylamide gel electrophoresis and immunoblotting by using a rabbit polyclonal anti-MnSOD antibody. (A) Representative immunoblot of MnSOD expression. Lane 1: Vehicle-treated sham-operated animal (control); Lane 2: 3-NP-treated sham-operated animal; Lane 3: Vehicle-treated ischemic animal after 48 h of blood recirculation; Lane 4: 3-NP-treated ischemic animal after 48 h of blood recirculation; Lane 5: Vehicle-treated ischemic animal after 7 days of blood recirculation; Lane 6: 3-NP-treated ischemic animal after 7 days of blood recirculation. (B) Densitometric analysis from three independent experiments with three ischemic animals and three sham-operated animals of each subgroup (mean±SEM of values expressed as percentage of the control, which was considered 100%). The dose of 3-NP used in this study was of 10 mg/kg. Significant differences were assessed by analysis of variance followed by post-hoc Newman–Keuls test (p < 0.05). (*) Significantly different compared with vehicle-treated sham-operated animals.

the body and brain temperatures, anesthesia, and duration of transient global ischemia (5 min of bilateral carotid artery occlusion). Unlike the data of Sugino et al. [33], our findings do not show a 3-NP-induced neuronal tolerance to transient global ischemia in gerbils. The reasons for the discrepancies between our data and those of Sugino et al. [33] are unclear. Our results show that 3-NP at the dose of 3 mg/kg did not induce SDH inhibition. Assuming that the 3-NP-induced tolerance is due to SDH inhibition, we postulated that the lack of protection has to be related to this absence of SDH inhibition. However, the dose 10 mg/kg presented in our study led to sustained inhibition of the enzyme whereas it was also ineffective in term of preconditioning. Importantly, we also tested a higher dose of 3-NP (20 mg/kg) but, given the SDH inhibition was found to be similar to that obtained with the dose 10 mg/kg (data not shown), we have chosen the latter dose to ensure the absence of neurotoxic effect. A higher sensitivity of our gerbils to ischemia cannot explain either the lack of neuroprotection, because comparable number of hippocampal neurons survived to ischemia after 7 days of blood recirculation in both studies. Finally, the tolerance was not an earlier onset phenomenon in our study because the number of surviving hippocampal neurons after 48 h of blood recirculation did not differ between vehicle- and 3-NP-treated gerbils (data not shown).

3-NITROPROPIONIC ACID PRECONDITIONING

FIG. 3. Photographs of representative astroglial and microglial activation in gerbil hippocampus. Astroglial (A–D) and microglial (E–H) activation was evidenced by using a mouse anti-glial fibrillary acidic protein antibody and a peroxidase conjugated isolectin B4, respectively. Staining was performed on paraffin-embedded sections of vehicle-treated sham-operated gerbil (A, E), of 3-nitropropionic acid (3-NP)-treated sham-operated gerbil (B, F), of vehicle-treated ischemic animal after 7 days of blood recirculation (C, G), and of 3-NP-treated ischemic animal after 7 days of blood recirculation (D, H). The dose of 3-NP used in this study was of 10 mg/kg. The sections were lightly counterstained with hematoxylin. Scale bar: 200 µm.

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38 It is well known that, on the one hand, inhibition of mitochondrial respiration generates reactive oxygen species (ROS) formation [6,10] and on the other hand, 3-NP induces oxidative stress [3,31,32,38]. Furthermore, the abolition of tolerance by the free radical scavenger, dimethylthiourea, strongly suggests that ROS generation may be a necessary step in the establishment of ischemic tolerance [32]. In addition, there is accumulating evidence indicating that moderate ROS levels can regulate cellular functions including signaling cascades and transcriptional/post-transcriptional control of gene expression [7,25]. Thus, provided the inhibition of SDH by 3-NP is important enough to generate free radicals, ROS generation may in turn induce the expression of proteins capable of protecting the brain from subsequent lethal insult. For instance, Gorman et al. [12] have reported in vitro on HL-60 human myelocytic cells that the antioxidant molecules such as pyrrolidine dithiocarbamate and 1,10-phenanthroline reduced significantly the heat shock-induced HSP72 expression, which was associated with an increased caspase activity, index of apoptosis. Hence, ROS could contribute to the induction of HSP72, which may subsequently lead to cellular protection. Likewise, MnSOD was also shown to be induced by ROS generation [36] and to have protective properties [7,15,35]. Our data showing that pretreatment with 3-NP did not lead to changes in the level of induction of HSP72 and MnSOD following transient ischemic exposure may also be considered as an indirect indication of the lack of 3-NP preconditioning effect observed in our study. Likewise, the absence of significant difference in post-ischemic astroglial and microglial activation between vehicleand 3-NP-treated hippocampus represents another indirect argument for the lack of tolerance induction, because both astroglial and microglial activation can be considered as a marker of the intensity of cerebral stress [11,13]. In addition, a correlation between neuroprotection and microglial and astrocytic activation was found in preconditioned rats that exhibited a decrease in microglial and astrocytic activation compared to control animals at both 3 and 7 days of survival [28]. Thus, none of the direct (neuronal loss) and indirect (HSP72 and MnSOD expression, glial activation) parameters examined in our study revealed cerebral protection. Furthermore, although Nakase et al. [24] have reported an early-onset tolerance induced by 3-NP (20 mg/kg) occurring in CA2 , CA3 , and CA4 hippocampal subfields in rat subjected to global cerebral ischemia, these authors failed to demonstrate neuronal protection in the CA1 subfield. So, it can be pointed out that the study of Sugino et al. [33] represents the only one showing a 3-NP-induced protection of CA1 neurons subjected to subsequent transient global ischemia. One reason might be the wide variability of the effects of 3-NP. Indeed, studies on 3-NP neurotoxic effects have shown both major strain differences [27] and important inter-individual variability [2] in rats subjected to systemic 3-NP injection. On the other hand, no study has been carried out in gerbils yet. Thus, although Mongolian gerbils used for animal experimentation are all Meriones unguiculatus, the source (supplier) may be responsible for the experimental variability. If so, in our minds, 3-NP preconditioning effect against global ischemia in gerbils is at the best very modest. However, our results do not exclude the studies that have shown that 3-NP treatment is capable of inducing tolerance in models of focal ischemia. Indeed, Wiegand et al. [38] have reported that 3-NP can induce profound tolerance to focal cerebral ischemia in the rat when administered in a single dose (20 mg/kg) 3 days before ischemia, even though the extent of SDH inhibition induced by 3-NP (20%) was similar to that obtained in our study. However, the reduction of the infarct volume by 3-NP was of a lesser extent after a transient focal ischemia (−35%) than after a permanent focal ischemia (−70%). Likewise, Kuroiwa et al. [21] have

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