Layer-specific differences in reactive oxygen species levels after oxygen–glucose deprivation in acute hippocampal slices

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Free Radical Biology & Medicine 44 (2008) 1010 – 1022 www.elsevier.com/locate/freeradbiomed

Original Contribution

Layer-specific differences in reactive oxygen species levels after oxygen–glucose deprivation in acute hippocampal slices Ádám Fekete a , E. Sylvester Vizi a , Krisztina J. Kovács b , Balázs Lendvai a , Tibor Zelles a,⁎ a

b

Laboratory of Cellular Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, 1083 Budapest, Szigony u. 43., Hungary Laboratory of Molecular Neuroendocrinology, Institute of Experimental Medicine, Hungarian Academy of Sciences, 1083 Budapest, Szigony u. 43., Hungary Received 15 June 2007; revised 19 September 2007; accepted 25 November 2007 Available online 14 December 2007

Abstract The major role of reactive oxygen species (ROS) in the pathomechanism of ischemia have been widely recognized. Still, measurements of the precise time course and regional distribution of ischemia-induced ROS level changes in acute brain slices have been missing. By using acute hippocampal slices and the fluorescent dye CM-H2DCFDA, we showed that reoxygenation after in vitro ischemia (oxygen–glucose deprivation; OGD) increased ROS levels in the hippocampal CA1 layers vulnerable to ischemia but did not have significant effects in the resistant stratum granulosum in the dentate gyrus (DG). Production of ROS started during OGD, but, contrary to reoxygenation, it manifested as a ROS level increase exclusively in the presence of catalase and glutathione peroxidase inhibition. The mechanism of ROS production involves the activation of NMDA receptors and nitric oxide synthases. The inhibition of ROS response by either AP-5 or L-NAME together with the ROS sensitivity profile of the dye suggest that peroxynitrite, the reaction product of superoxide and nitric oxide, plays a role in the response. Direct visualization of layer-specific effects of ROS production and its scavenging, shown for the first time in acute hippocampal slices, suggests that distinct ROS homeostasis may underlie the different ischemic vulnerability of CA1 and DG. © 2007 Elsevier Inc. All rights reserved. Keywords: Selective vulnerability; CA1 vs dentate gyrus; In vitro ischemia; ROS; Peroxynitrite; CM-H2DCFDA

Introduction Ischemia initiates a multicomponent cascade mechanism that ultimately leads to ischemic cell death [1]. Glutamate (Glu) exci-

U

U

Abbreviations: ROS, reactive oxygen species; NO, nitric oxide; O2 −, U superoxide; H2O2, hydrogen peroxide; ONOO−, peroxynitrite anion; HO , hydroxyl radical; CM-H2DCFDA, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; PI, propidium iodide; OGD, oxygen and glucose deprivation; GPx, glutathione peroxidase; NOS, nitric oxide synthases; SOD, superoxide dismutase; Glu, glutamate; NMDA-R, NMDA receptor; DG, dentate gyrus; SP, stratum pyramidale; SR, stratum radiatum; SL, stratum lacunosum; SM, stratum moleculare; SG, stratum granulosum; ACSF, artificial cerebrospinal fluid; ROI, regions of interest; AP-5, DL-2-amino-5-phosphonopentanoic acid; L-NAME, Nω-nitro-Larginine methyl ester; D-NAME, Nω-nitro-D-arginine methyl ester; TRIM, 1-[2(trifluoromethyl)phenyl]imidazole; ATZ, 3-amino-1,2,4-triazole; MS, mercaptosuccinic acid; SNP, sodium nitroprusside; DMSO, dimethyl sulfoxide; Mops, 3-N[morpholino]propanesulfonic acid; IUPAC, International Union of Pure and Applied Chemistry. ⁎ Corresponding author. Fax: +36 1 210 9423. E-mail address: [email protected] (T. Zelles). 0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2007.11.022

totoxicity [2,3], pathological elevation of intracellular Ca2+ and Na+ [4–6], and generation of reactive oxygen species (ROS; [7,8]) are important components of the cascade. ROS serve as both initiators and perpetrators of pathological processes [1]. Excess levels of ROS are generated in many ways during ischemia, including NMDA receptor (NMDA-R) overactivation during excitotoxicity [9]. Inhibition of glutamate- or NMDA-induced ROS generation has a protective effect on neuronal cultures, indicating a substantial role for ROS production in excitotoxicity [10–12]. ROS are also generated under physiological conditions [13], but their low level is maintained by inherent elimination processes that protect the cells from damage. Enhancement of cellular enzymatic (e.g. superoxide dismutase (SOD; EC 1.15.1.1), catalase (EC 1.11.1.6), glutathione peroxidase (GPx; EC 1.11.1.9; [14]) or nonenzymatic (e.g. ascorbic acid, vitamin E, glutathione (GSH); [15,16]) antioxidant capacities are also protective against ischemia/reperfusion injury. Despite the important role of ROS in the pathomechanism of ischemia, their detection and study have been typically performed

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in cell cultures [17–20], which are not optimal for this purpose. Cell metabolism in cultures is adopted to the artificial environment and the anatomical structure of the original tissue is lost. In vivo direct ROS measurements with reasonable time and spatial resolution would be desirable, but they have yet to be performed. Acute brain slices seem to be an adequate model [5,6,21,22] because they preserve the three-dimensional organization and characteristic functions of brain tissue and lack the adaptive changes of cultured slices. However, measurements of ischemic ROS level changes in the function of time were performed on slice cultures and exclusively on hippocampal CA1 pyramidal neurons [23,24]. Among brain slices the hippocampal slice preparation has the advantage of having both ischemia-vulnerable (CA1) and -resistant (dentrate gyrus; DG) regions [25] which makes it an excellent model to explore the relevant intracellular factors responsible for the different sensitivities to ischemia. The roles of ROS in the different ischemic vulnerabilities have been suggested, but this conclusion was based on observations of different scavenger capacities of the regions [26] or on different damaging effects of exogenous ROS generators [27]. However, a study that directly follows the spatial and temporal patterns of ischemia-induced ROS level changes, both in a vulnerable and in a resistant brain region in acute slices, has been missing. For the first time we simultaneously measured the temporal changes in fluorescence of the ROS dye CM-H2DCFDA during ischemia and reoxygenation in layers of the CA1 and DG regions of acute hippocampal slices. We showed differences in the homeostasis of ROS, a characteristic that may define the different vulnerabilities of the two regions. We used pharmacological tools (AP-5, L-NAME, ATZ + MS) to investigate the roles of both the production and the scavenging of ROS. Materials and methods Acute hippocampal slices Procedures were performed in accordance with National Institutes of Health guidelines for the care and use of animals and were approved by the Institutional Animal Care and Use Committee. Male 16–21 days old, Wistar rats were decapitated. The brains were removed and placed in ice-cold cutting solution (composition in mM: NaCl, 126; KCl, 2.5; NaHCO3, 26; CaCl2, 0.5; MgCl2, 5; NaH2PO4, 1.25; glucose, 10; pH 7.4) which was continuously bubbled with 95% O2+ 5% CO2. Coronal slices of 300 μm thickness were cut with a vibratome (Vibratome 1000), separated into left and right halves, and transferred into a mesh-bottom holding chamber containing artificial cerebrospinal fluid (ACSF; in mM: NaCl, 126; KCl, 2.5; NaHCO3, 26; CaCl2, 2; MgCl2, 2; NaH2PO4, 1.25; glucose, 10) bubbled with a mixture of 95% O2+ 5% CO2, leaving the final pH at 7.4. After a 25-min incubation at 32.5 °C, the slices were kept at room temperature until the onset of the experiments [5]. We observed higher fluorescence and larger response to OGD in slices mechanically injured or older than 4 h (data not shown). To ensure good-quality slices with low variability in their state of health no experiment lasted longer than 4 h after decapitation.

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Oxygen and glucose deprivation Ischemia was modeled by a 10 min OGD at 36 °C. OGD solution was bubbled with 95% N2 and 5% CO2 and glucose was replaced by sucrose to maintain osmolarity [28]. We monitored the oxygen level in the perfusion chamber using a dissolved oxygen probe (ISO2; Oxygen Meter; World Precision Instruments). The oxygen tension of ACSF at 36 °C was 722 mmHg in the tissue chamber and was reduced to 52 mmHg during OGD. Intracellular ROS measurement ROS production was monitored at 36 °C by using the oxidation-sensitive dye 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) which is sensitive to peroxynitrite1 (ONOO−), hydrogen peroxide (H2O2; Fig. 1B), and hydroxyl radical (HO˙; Molecular Probes). CMH2DCFDA is a nonfluorescent dye until the acetate groups are removed by intracellular esterases and oxidation occurs within the cell. The dye (50 μg) was freshly dissolved each day in 10 μl dimethyl sulfoxide (DMSO). The slices were loaded with 45 μM CM-H2DCFDA in the dark (45 min; 25 °C; 0.005% [w/v] Pluronic F127). After loading, the slices were washed three times and then left in ACSF for at least 30 min to ensure deacetylation. 5 mM probenecid was added to every solution used after loading to inhibit organic anion transporters that remove fluorescent dyes from the cytoplasm in a temperaturedependent way [29]. Slices were submerged and superfused (3.5 ml/min) in an experimental chamber mounted on a Gibraltar BX1 platform (Burleigh) and viewed with a 10X water immersion objective (Olympus; Fig. 1A) on an Olympus BX50WI upright microscope. Slices were excited at 488 ± 5 nm with a T.I.L.L. Polychrome II monochromator. The illumination was attenuated by means of an adjustable diaphragm installed in the light path. The emitted light (535 ± 25 nm) was detected by a cooled charge-coupled device camera (Photometrics Quantix) and the system was controlled with the Axon Imaging Workbench 4.0 software. To protect the dye from irreversible photooxidation we set excitation intensity as low as possible. During visualization by differential interference contrast (DIC) microscopy (Fig. 1A) a red filter (RG665; Schott Filter Glass) was put over the illuminator. The image frame rate was always 6/min and exposure time was held constant for all experiments. This gave a reasonable time resolution with a reduced level of photooxidation. We studied these layers of the hippocampus: CA1 stratum pyramidale (SP), stratum radiatum (SR), and stratum lacunosum (SL) and dentate gyrus stratum moleculare (SM) and stratum granulosum (SG; see in Fig. 1A). Autofluorescence correction in every examined region of every individual hippocampal slice was made by point by point subtraction of the respective average intensity values of identical

1 The term peroxynitrite is used to refer to both peroxynitrite anion and peroxynitrous acid. IUPAC–recommended names are oxoperoxonitrate (1-) and hydrogen oxoperoxonitrate, respectively.

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Volume change measurement

Fig. 1. Method of measurement of ROS levels in different layers of acute rat hippocampal slices by the fluorescent dye CM-H2DCFDA, which is sensitive to U ONOO−, H2O2, and HO . (A) DIC (upper) and the respective fluorescent (lower) images (10X water immersion objective) of a slice show the hippocampal layers, CM-H2DCFDA loading, and the regions of interest (ROIs) used in the off-line analysis of fluorescence intensities (see Materials and methods). (B) Colored average traces (n = 3) show the responses of different hippocampal layers to the consecutive application of 200 μM H2O2 and 200 μM SNP (left traces; SNP alone has the same response, data not shown) or 10 mM H2O2 alone (right traces). The SEs of the traces are omitted for clarity. Drug perfusions were started when regular experiments finished (after 40-min measurement period). The responses for the exogenous H2O2 and SNP show the appropriate loading of each layer and the sensitivity difference in favor of ONOO−, produced by SNP.

experiments performed on unloaded slices (OGD, n = 6; OGD + ATZ +MS, n = 5). Autofluorescence was 16.5–17.4% of the total signal intensity in the beginning of the experiments and did not differ significantly between the layers. We administered H2O2 and sodium nitroprusside (SNP) to test the sufficiency of loading and sensitivity of CM-H2DCFDA in our preparation at 36 °C (Fig. 1B). Both drugs increased fluorescence in all layers (Fig. 1B) which showed dye loading. The effect of SNP was much larger; 10 mM H2O2 was needed to produce a response comparable with that of 200 μM SNP (Fig. 1B). H2O2 oxidizes U the dye directly. SNP, considered to be an NO donor, also U − generates O2 in biological systems [30,31] and forms ONOO−, which makes CM-H2DCFDA fluorescent more efficiently than U U H2O2. The dye is not sensitive to NO and O2 − (Molecular Probes; [32–35]).

Ischemia induces cell swelling which dilutes dye concentration and decreases the intensity of fluorescence signals. We tried to measure volume changes by calcein AM but this method was not feasible because of the oxidation sensitivity of calcein AM (data not shown; [36]). To determine volume changes of the hippocampal layers directly we have developed a method to measure it in the same slices that were used for ROS experiments. Enlargement of layers in the x (direction of apical dendrites of pyramidal cells) and y (parallel to the pyramidal layer) axes on the transversal hippocampal slices (see Fig. 3A inset) were measured directly on the fluorescent CM-H2DCFDA images off-line. We drew rectangular regions of interest (ROIs) over the layers (in orientation similar to that of the rectangular ROIs on the fluorescent image in Fig. 1A) and enlarged them synchronously with swelling. Side lengths of the rectangular ROIs were expressed in image pixel numbers. Based on the anatomical structures of CA1 and DG, i.e., shape and orientation of principal cells, the third axis (z) of the space was assumed to be the same as the y axis. Volumes were calculated from the three parameters. Dividing the volume measured at the peak of the reoxygenation response (30th min of the traces; x30 min y30 min z30 min = V30 min) by the volume before OGD (10th min of the traces; x10 min y10 min z10 min = V10 min) gave the OGD- and reoxygenation-evoked relative swelling between these time points in each layer (V30 min/V10 min; Fig. 3B). Multiplication of the actual normalized fluorescence values (see Analysis and statistics) by the average relative swelling of the same layer was used to correct the swelling-evoked dilution of CM-H2DCFDA (Fig. 3C). The rectangular ROIs were drawn as follows. Two reference points were picked as diagonal corner points of the ROI rectangle. A reference point was defined as the center of an anatomically not defined but well recognizable and traceable spot which had significantly higher or lower fluorescence intensity than its surrounding. To help precisely identify the center of a spot, we always drew an elliptic ROI around the spot on the actual image and considered the intersection of its diagonals to be the reference point. To decrease the error in measurements of ROI side lengths, reference points were set as far from each other as possible. The x side of a ROI was at least 60% of the whole width of the layer while the y side was not less than 10 pyramidal cell diameters. If there were no reference points meeting all of the above-mentioned criteria the volume enlargement was not determined. Enlarging and positioning the ROIs and counting the pixel numbers on their sides are time consuming and because exploring the fine timedependent details of swelling was not the aim of this work we performed this analysis only at certain defined time points (except in two slices where we followed the swelling with a 0.5-Hz resolution to obtain a more precise picture of the kinetics; Fig. 3B right inset). pH sensitivity measurement of CM-H2DCFDA fluorescence The magnitude of pH-dependent quenching of the CMH2DCFDA dye was measured in different buffers of known pH (7.1, 6.7, 6.4 for MOPS, 4.8 for acetate) in the same imaging

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system. The composition of the buffers were as follows (in mM): NaCl, 142; KCl, 2.5; CaCl2, 2; MgCl2, 2; NaH2PO4, 1.25; glucose, 10; MOPS or acetate, 10. Diacetate groups of CMH2DCFDA were hydrolyzed by addition of NaOH to the stock solution. The fluorescent CM-DCF was formed from CMH2DCF by spontaneous oxidation.

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DL-2-Amino-5-phosphonopentanoic

acid, Nω-nitro-L-arginine methyl ester, Nω-nitro-D-arginine methyl ester, 1-[2-(trifluoromethyl)phenyl]imidazole, 3-amino-1,2,4-triazole, mercaptosuccinic acid, probenecid, and 3-N-[morpholino]propanesulfonic acid were obtained from Sigma–Aldrich Inc. (St. Louis, MO, USA). All other chemicals were obtained from Merck (Darmstadt, Germany).

Cell viability measurement Results Following 10 min OGD and 10 min reoxygenation, slices were fixed with 4% paraformaldehyde in 0.1 M phosphatebuffered saline and stained with propidium iodide (PI, 1.5 μM; Molecular Probes) for 30 min to monitor cell viability. Since PI is a membrane-impermeable dye which becomes fluorescent after binding to DNA, cells will not be fluorescent unless their membranes are damaged. Slices were excited with a high-pressure mercury lamp using an excitation filter (535 ± 25 nm). Images of PI-loaded and OGD-treated slices were acquired with a SPOT RT Color Camera (Diagnostic Instruments) viewed with a 2X objective (Nikon) on a Nikon Eclipse E600 microscope. Following image acquisition, the slices were permeabilized by 5 min incubation in 99.8% methanol and reloaded with PI to determine the maximal intensity when all of the cells were damaged. The rate of cell death was calculated as the ratio of fluorescence intensity before and after methanol treatment. Control slices were kept at room temperature, fixed, and loaded with PI in parallel with OGD-treated slices.

The oxygen–glucose-deprivation-evoked increase in ROS level is layer specific in acute hippocampal slices We measured the fluorescence of CM-H2DCFDA-loaded rat hippocampal slices over the CA1 and DG regions with a 10 s time resolution (Fig. 2). We applied 10 min OGD as an in vitro model of ischemia. The OGD insult (OGD phase) was always preceded by a 10 min baseline period (Fig. 2). After about 4 min of OGD, the fluorescence started to decrease in all layers of both

Analysis and statistics Following autofluorescence correction (see above) we fitted an exponential to the pre-OGD period of every individual trace. The actual ROS curve was normalized to this fitted curve. Experiments on loaded control slices confirmed the exponential nature of fluorescence change in the absence of OGD: there is a continuous background production of ROS which oxidizes the dye and increases fluorescence intensity continuously. Traces on figures show the average of these normalized curves with ± SE (standard error of the mean). Number of experiments (n) shows the number of individual slices, each from a different animal. All statistics were performed in the 7th min of OGD (OGD phase; highest section of ROS production during OGD manifested in the presence of catalase and GPx inhibition) and in the 10th min of reoxygenation (reoxygenation phase; highest section of reoxygenic ROS production). Peak amplitudes are expressed as the average of normalized fluorescence intensities of six consecutive points, always taken exactly at the same time of each individual trace. For statistical tests of significance oneway ANOVA followed by Dunnett post-hoc test was used unless specified otherwise. Differences were considered significant at p b 0.05. Chemicals and reagents 5-(and-6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, propidium iodide, calcein AM, and pluronic F127 were purchased from Molecular Probes (Eugene, OR, USA).

Fig. 2. Oxygen and glucose deprivation (OGD, 10 min) evokes layer-specific ROS level elevation in acute hippocampal slices during reoxygenation. The colored traces are averages of ROS responses of the three CA1 and the two DG layers in nine slices. The schematic drawing of the hippocampus with the field of view of the camera (rectangular frame) shows the position of the regions of interest (ROIs; colored areas) over the hippocampal layers and decodes the color of the traces. The gray traces show the average responses of the five layers in control (no OGD) experiments (n = 7). The SE of the traces is omitted for clarity. During OGD, no significant differences in ROS level of the different layers were detected (ANOVA, p = 0.065; pSP-SR = 0.970, pSP-SL = 0.908, pSP-SM = 0.135, pSP-SG = 0.128, pSR-SL = 0.999, pSR-SM = 0.402, pSR-SG = 0.386, pSL-SM = 0.544, pSL-SG = 0.526, pSM-SG = 0.999, power = 0.699, n = 9). However, during reoxygenation, the ROS level in all CA1 layers and SM of the DG increased and significantly differed from the ROS level of the SG, DG ( pSP-SG = 0.0001, pSR-SG = 0.0001, pSL-SG = 0.0001, pSM-SG = 0.0001, n = 9); SP, SR, SL of CA1 and SM of DG were not significantly different from each other ( pSP-SR = 0.999, pSP-SL = 0.913, pSP-SM = 0.996, pSR-SL = 0.948, pSR-SM = 0.987, pSL-SM = 0.737, power = 0.999, n = 9). One-way ANOVA was followed by Tukey post-hoc test.

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regions. When oxygen and glucose supplies were restored (reoxygenation phase), the fluorescence signal started to increase in all CA1 and the DG, SM layers. In contrast, signal intensity over DG, SG continued to decline (Fig. 2). In the 10th min of reoxygenation the difference between the fluorescence intensities of DG, SG vs that of any other layers was significant ( p b 0.001, n = 9). When no OGD treatment was applied (control; n = 7), fluorescence remained unchanged in all layers during the entire length of experiments (Figs. 2 and 3C). Since ischemic insults cause swelling of cells [1,37,38], which results in the dilution of intracellular dye concentration and a decline in fluorescence signal intensity, we investigated the effect of OGD on swelling to exclude false interpretation of the region-

specific ROS results. In the 10th min of reoxygenation of four slices (out of the nine shown in Fig. 2) we measured the effect of OGD on the enlargement of volume (V) of the imaged layers (Fig. 3A and B). Magnitude of swelling, i.e., relative volume enlargement between the 10th min of reoxygenation and the beginning of OGD treatment (V30 min/V10 min; see Materials and methods) was in the 110–117% range in the SR and SL layers of CA1 and DG layers and 130% in the CA1, SP layer (Fig. 3B). Multiplying the normalized fluorescence values in the 10th min of reoxygenation of each layer by the average relative volume enlargement of the corresponding layer clearly showed that reoxygenation induced a significant increase in ROS levels in the CA1 and DG, SM layers while in DG, SG there was no change compared to the control conditions (Fig. 3C). In two slices we performed the swelling correction in every second minute (Fig. 3C inset). Average trace of the DG, SG layer on this inset followed the baseline while CA1, SP showed the most pronounced increase in ROS levels. Swelling-corrected ROS levels during reoxygenation and their comparisons to control experiments revealed the actual ROS productions devoid of swelling artifacts (Fig. 3C) and confirmed the regional differences in ROS level after OGD (see Fig. 2). Higher-resolution temporal dynamics of swelling (Fig. 3B right inset) showed the coincidence between the onset of swelling and the start of the decline in CM-H2DCFDA fluorescence (Fig. 2). Because the effect of drugs on OGD-evoked ROS production was always compared to the OGD effect alone in the same hippocampal layer and none of the drugs influenced swelling Fig. 3. Correction for the dilution of intracellular CM-H2DCFDA concentration attributable to OGD-evoked cell swelling. Swelling and consequent dilution of CM-H2DCFDA were responsible for the fluorescence decrease caused by 10 min OGD but not for the fluorescence difference between DG, SG and the other layers during reoxygenation (Fig. 2). (A) The extent of 10 min OGD-induced swelling into x (gray column) and y (blank column) dimensions was measured (for details see Materials and methods) in every layer of four slices (n= 4) at the 10th min of reoxygenation, i.e., 30th min of the experiment. The bar graph shows the change of length (ΔL) in %. The inset drawing shows the relation of x and y dimensions to the slice. (B) The changes in volume (V) of the layers were calculated by assuming a similar extent of swelling vertically (z dimension) as that into the y dimension based on the anatomical structure of CA1 and DG. The relation of the three dimensions (x, y, z) to the slice is shown in the left inset. Swelling at the peak of the reoxygenation response (30th min of the experiments) was expressed in relation to the corresponding volume at the beginning of OGD (V30 min/V10 min). The bar graph shows the change of volume (ΔV) in %. The swelling of the layers after OGD was not significantly different from each other (one-way ANOVA, p = 0.521, n = 4). The volume changes were determined separately in each layer of the CM-H2DCFDAloaded slices (CA1, SP: black column; CA1, SR: dark gray column; CA1, SL: middle gray column; DG, SM: light gray column; DG, SG: blank column). In two slices the volume changes were followed with 0.5 Hz resolution (right inset) to demonstrate the kinetic of swelling. Their average traces are shown in the right inset. The colors of the traces correspond to the column bars. (C) Swelling-corrected CMH2DCFDA fluorescence values show significant increase in ROS levels during reoxygenation in all layers of the hippocampus except in DG, SG (right bars in every layer, n =9 vs control experiments, n = 7, left bars in every layer; pSP = 0.0001, pSR = 0.0001, pSL = 0.0027, pSM =0.0031, pSG =0.5802, unpaired t test with Welch correction). The volume-enlargement-evoked decrease of fluorescence in the 10th min of reoxygenation was corrected by multiplying the actual normalized CMH2DCFDA fluorescence with the average of the V30 min/V10 min values (see in B) of the same layer. The inset figure shows the swelling-corrected average ROS traces of the two experiments the swelling of which were followed with 0.5 Hz resolution (see in B right inset). The colors of the curves correspond to the column bars. Error bars represent ±SE; ⁎⁎ pb 0.01; ⁎⁎⁎ pb 0.001.

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significantly during the length of the experiments ( p N 0.05, n = 2– 5; data not shown), correction of fluorescence with swelling was not performed for the rest of the experiments. Another theoretical possibility for the decrease in fluorescence signal is the quenching of the fluorescent dye by acidification after OGD. We measured the pH-dependent fluorescence changes of CM-DCF in different buffers. Fluorescence intensities were normalized to the average intensity value at pH 7.1 (intracellular pH in healthy cells): pH 7.1, 100 ± 1.7%, n = 12; pH 6.7, 96 ± 4.4%, n = 4; pH 6.4, 97 ± 1.8%, n = 4; pH 4.8, 69 ± 1.4%, n = 4. This clearly shows that there is no significant quenching in the 7.1–6.4 pH range which is the range of pHi changes in response to OGD in acute hippocampal slices [39–41]. OGD does not cause detectable cell death during the experiments To test for potential changes in cell viability attributable to OGD during the experiments, we fixed the slices in the reoxygenation phase and stained them with propidium iodide. This showed that the 10 min of OGD did not cause any detectable cell damage compared to control in either the CA1 or the DG regions (Fig. 4). Elevation of ROS in the OGD phase is revealed by inhibition of ROS-defensive scavenger enzymes CM-H2DCFDA fluorescence indicates the net level of ROS but does not directly measure its production. Cells have an efficient antioxidant enzyme system [13,42,43], which can hide ROS production. To test the significance of this defense enzyme system and explore the production of ROS attributable to OGD more directly we preperfused the slices with 3-amino-1,2,4triazole (10 mM) and mercaptosuccinic acid (10 mM), which are inhibitors of the main ROS-scavenger enzymes catalase and GPx, respectively. In the presence of enzyme inhibitors the fluorescence started to rise right after the onset of OGD in every layer (Fig. 5A). However, the presence of the enzyme inhibitors did not enhance the effect of OGD during the reoxygenation phase (Fig. 5A). OGD-evoked ROS production involves the action of NMDA-Rs and NOS Excitotoxicity is one of the main ischemic mechanisms that initiates the cascade of pathological events ultimately leading to cell death [1,2,37]. The excess release of Glu is known to generate ROS [10,11,23,44–46]. We tested the roles of NMDARs and the tightly coupled NOS activity [47,48] in the generation of ROS during OGD and reoxygenation in different hippocampal layers. Ischemia also perturbs mitochondrial function, resulting in the overproduction of superoxide [14,45,49,50], U which is able to react with NO and form the reactive ROS peroxynitrite [51]. CM-H2DCFDA is highly sensitive to oxidation by ONOO− (Molecular Probes; [32,33]). We started to perfuse the slices with the NMDA-R antagonist DL-2-amino-5phosphonopentanoic acid (50 μM) and the nonspecific NOS inhibitor Nω-nitro-L-arginine methyl ester (100 μM) 15 min before OGD. Both AP-5 and L-NAME caused significant

Fig. 4. The 10 min OGD treatment did not produce cell damage during the experiments as shown by propidium iodide (PI) staining of hippocampal slices. Slices exposed to oxygen–glucose deprivation (OGD, n = 4, gray bars) for 10 min or perfused exclusively with normal ACSF (control, n =4, white bars) were fixed and then stained with PI 10 min after OGD at the time point when all the reoxygenation statistics were calculated. Rates of cell death were expressed as the percentage of total cell loss induced by methanol (see Material and methods). Fluorescent images show control and OGD-treated slices prior to and after methanol exposition. The effects of OGD were compared to controls, which showed a basic cell loss resulting from the preparation and maintenance of the slices in an artificial environment. The bar graph summarizes the data from the two principal cell layers (SP of CA1 and SG of DG). Error bars represent ±SE, pSP = 0.8257, pSG =0.4896; unpaired t test.

decreases in the OGD-evoked fluorescence rise during the reoxygenation phase in all CA1 layers and DG, SM, while they did not have significant effects in the DG, SG (Fig. 6). Increase in ROS production during OGD, revealed by ATZ + MS (Fig. 5A), was also inhibited by AP-5 and L-NAME in the principal cell layer of the CA1 but not of the DG (Fig. 5B top). Although the effects of the inhibitors were not significant in DG, there seemed to be a trend of inhibition. During reoxygenation AP-5 and L-NAME similarly showed an inhibitory action in CA1, SP and had no significant effect in DG, SG in the presence of ATZ + MS (Fig. 5B bottom). Perfusion of D-NAME was ineffective ( p N 0.05, n = 4; data not shown). U In ischemia models, NO production can be either detrimental or beneficial to the brain [52]. The contradictory effects observed with different NOS inhibitors raise the possibility that different U NOS isoenzymes could be responsible for NO production in

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Fig. 5. Production of ROS started right at the beginning of OGD as was revealed by the inhibition of the ROS-defensive enzymes catalase and glutathione peroxidase (GPx). The ROS level increase during OGD was NMDA-R and NOS-dependent. (A) As compared to the OGD experiments (10 min OGD; n = 9; gray curves), 15 min pretreatments with the catalase and GPx inhibitors ATZ (10 mM) and MS (10 mM) revealed significant increases in ROS levels in every imaged hippocampal layer during OGD ( pSP = 0.00001, pSR = 0.00002, pSL = 0.00003, pSM = 0.00001, pSG = 0.00005, power = 0.98–1.00, n = 6; black curves) but not during reoxygenation ( pSP = 0.320, pSR = 0.934, pSL = 0.904, pSM = 0.927, power = 0.96–0.99; pSG = 0.945, power = 0.70; n = 6; black curves). Dotted gray lines represent ±SE. (B) NMDA-R (AP-5, 50 μM) and NOS (L-NAME, 100 μM) inhibition were able to prevent significantly the OGD-evoked ROS increase during both the OGD and the reoxygenation phase in CA1, SP but not in DG, SG (OGD in CA1, SP: p(ogd)-(ogd + atz + ms) = 0.00001, p(ogd + atz + ms)-(ogd + atz + ms + ap-5) = 0.015, p(ogd + atz + ms)-(ogd + atz + ms + l-name) = 0.006, power = 0.96; OGD in DG, SG: p(ogd)-(ogd + atz + ms) = 0.00005, p(ogd + atz + ms)-(ogd + atz + ms + ap-5) = 0.467, p(ogd + atz + ms)-(ogd + atz + ms + l-name) = 0.075, power = 0.42; reoxygenation in CA1, SP: p(ogd)-(ogd + atz + ms) = 0.320, p(ogd + atz + ms)-(ogd + atz + ms + ap-5) = 0.002, p(ogd + atz + ms)-(ogd + atz + ms + l-name) = 0.005, power = 0.95; reoxygenation in DG, SG: p(ogd)-(ogd + atz + ms) = 0.945, p(ogd + atz + ms)-(ogd + atz + ms + ap-5) = 0.726, p(ogd + atz + ms)-(ogd + atz + ms + l-name) = 0.992, power = 0.12). See Materials and methods for data analysis and statistics. ⁎ p b 0.05; ⁎⁎ p b 0.01; ⁎⁎⁎ p b 0.001; OGD, n = 9; ATZ + MS, n = 6; ATZ + MS + AP-5, n = 5; ATZ + MS + L-NAME, n = 5.

different cell types or regions. For selective inhibition of neuronal NOS (nNOS), we administered 1-[2-(trifluoromethyl)phenyl] imidazole (TRIM, 100 μM), which inhibits nNOS with an IC50 of 28.2 μM and endothelial NOS (eNOS) with an IC50 of 1057.5 μM [53]. Contrary to L-NAME, TRIM had a significant inhibitory effect only in CA1, SR and SL, although there appears to be a trend of inhibition in DG, SM and CA1, SP (Fig. 6B). Discussion Harmful elevation of ROS levels is prevented by a complex antioxidant system of the cells. Ischemic challenges shift this

oxidoreduction balance to the direction of oxidation. We explored the effect of in vitro ischemia on the oxidoreduction equilibrium of acute rat hippocampal slices by means of CM-H2DCFDA. Imaging of fluorescent ROS-sensitive dyes has the advantage of directly measuring the kinetics of ROS with spatial and temporal resolution. Other ROS dyes, such as dihydroethidium or dihydrorhodamine 123, bind to helical polynucleotides (DNA, RNA) with a concomitant quantum yield increase [54] or accumulate in mitochondria with changing fluorescence depending on mitochondrial potential [55]. The dichlorofluorescein derivative CMH2DCFDA is free of these limitations in the quantification of ROS level or in localization to the actual oxidation site. To

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Fig. 6. ROS response during reoxygenation was NMDA-R and NOS mediated. Reoxygenation ROS levels were significantly inhibited by preperfusion of AP-5 (50 μM, n = 6; black line in A) and L-NAME (100 μM, n = 7; black line in B) in the CA1 and DG, SM (AP-5: pSP = 0.009, pSR = 0.0009, pSL = 0.00005, pSM = 0.014; L-NAME: pSP = 0.008, pSR = 0.026, pSL = 0.0008, PSM = 0.014, power = 0.96–0.99) but not in the DG, SG (AP-5: pSG = 0.999; L-NAME: pSG = 0.996, power = 0.7) compared to OGD curves (n = 9; gray line in both A and B). Note that OGD did not have any effects on the ROS levels in DG, SG (see also Figs. 2 and 3C), so the lack of effect of the inhibitors is not surprising in this layer. The other NOS inhibitor, TRIM (100 μM, n = 4), which has a selectivity against nNOS, had a significant inhibitory effect in stratum radiatum and lacunosum of the CA1 ( pSP = 0.339, pSR = 0.034, pSL = 0.042, pSM = 0.113, pSG = 0.999; see the appropriate power values above; black dotted line in B). Dotted gray lines represent ±SE. See Materials and methods for data analysis and statistics.

attenuate its disadvantages, such as poor retention in cells, photoconversion, and photobleaching, we used the organic anion transporter inhibitor probenecid and reduced unnecessary illumination. Measurement of ROS levels in vivo after ischemic injury offers the best opportunity to investigate the real pathological processes; however, these experiments have the disadvantages of poor spatial and temporal resolution, indirect test tube determination of free radicals, and difficulties of administering pharmacological compounds [56,57]. Culture preparations (either monolayers or slices), which are free of these limitations, have the major disadvantages of metabolic and structural changes. We used acute rat brain slice preparations [5,6,22] which offer the

potential for good spatial and temporal resolution measurements of intracellular ROS level changes with low levels of metabolic adaptation and disturbance of in vivo anatomical structure. Spatial and temporal pattern of OGD-evoked ROS production in acute hippocampal slices Each layer of the vulnerable CA1 region and DG, SM showed an increase in CM-H2DCFDA fluorescence during reoxygenation while SG, the principal cell layer of the resistant DG region [25], continued the decline in fluorescence, which started during OGD in every layer.

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OGD in acute hippocampal slices, similar to in vivo ischemia, results in cell swelling [38,58], which causes intracellular dye dilution and an apparent loss in fluorescence. We have developed a method to measure OGD-evoked volume changes of CA1 and DG layers in the same CM-H2DCFDA-loaded acute slices that were used for ROS measurements. Another advantage of this method is that it measures the swelling directly and is not distorted by fluorescence intensity changes caused by the oxidation of socalled “inert” fluorescein-based dyes, such as calcein-AM [36], which is typically used for volume determination. The simultaneous onset of swelling and drop in CM-H2DCFDA fluorescence and the disappearance of this fluorescence decrease on swelling-corrected traces showed that there was no detectable change in ROS levels in any layers during OGD. However, during reoxygenation the levels of ROS increased in every layer except in the resistant SG, DG. The most dynamic increase in ROS levels after OGD was in CA1, SP, but this was hidden on the non-volumecorrected traces by the most pronounced swelling of the CA1, SP. Theoretically the OGD-evoked acidification could also be responsible for the fluorescence signal decrease by quenching the fluorescein-based ROS dye. However, CM-H2DCFDA is a chlorinated form with a pKa value of ∼ 4.8 and the decrease of apparent pHi in murine hippocampal slices under conditions identical to ours was not higher than ∼0.15 pH unit during either OGD or reoxygenation [39]. Other in vitro brain slice studies show similar small rate of acidifications after OGD (e.g., [40,41]). This two order of magnitude difference between the pKa of CM-H2DCFDA and the pHi during or after OGD ensures that our results are not influenced significantly by pHi changes caused by 10 min OGD in acute hippocampal slices. Our measurements on the pH dependency of CM-H2DCFDA fluorescence also supported that CM-DCF does not significantly change its fluorescence, at least in the pH range 7.1–6.4. The identical ROS responses of the different layers to the perfusion of exogenous H2O2 or SNP at the end of the experiments demonstrated that all layers were loaded properly. Absence of membrane damage during the experiments, shown by the PI experiments, do not support significant dye leakage also. We can conclude that the increase in CM-H2DCFDA fluorescence and its layer specificity in response to 10 min OGD represent a real layer-specific ROS level increase during reoxygenation which is not attributable to artifacts resulting from swelling, acidification, or insufficient dye content of the cells. To test the influence of the enzymatic scavenger system on shaping the ROS response to OGD, we inhibited both catalase and GPx, which degrade H2O2 and play a decisive role in the maintenance of the intracellular oxidoreduction balance. Inhibition of the two enzymes revealed that the production of ROS starts right after the onset of OGD. In vivo experiments also suggested that free radical production starts during ischemia and that it peaks at the beginning of reperfusion [56,57]. How can ROS be formed during OGD if oxygen is deprived and how can the change in O2 supply during OGD be related to in vivo ischemia? The amount of oxygen in our tissue chamber was not zero during OGD and that could be the source of O2 for ROS

formation in this phase. Experiments on mitochondrial ROS production show that, although the formation of ROS is stopped during anoxia, under hypoxic conditions it is increased [59–61]. This paradoxical increase in production of ROS at lower O2 concentration is probably attributable to the inhibition of cytochrome oxidase by NO and ONOO−, formed during ischemia. These reactive nitrogen species decrease the affinity of the terminal oxidase to oxygen [62–64], thus making the mitochondrial electron transfer chain more reduced upstream, favoring U O2 − production [60,65,66]. Decrease of pO2 level during ischemia in vivo depends on many factors (e.g., type of occluded vessels, region of the brain, blood pressure, brain edema, or duration and extent of occlusion) and it does not always reach the absolute zero level, even in the core region [67–70]. Global complete ischemia or the center of the core region during a longer focal ischemic insult may form the exceptions [67,71]. OGD in our hippocampal slice preparation may not be relevant to these anoxic cases but can be considered an in vitro model for all the others. The increase in ROS level during OGD in the presence but not in the absence of the enzyme inhibitors means that the scavenger system is able to neutralize the effect of OGD, which is also conceivable during ischemia in vivo. The ineffectiveness of ATZ plus MS in the reoxygenation phase may suggest that the antioxidant capacity becomes exhausted during OGD [60,66,72,73], an idea which is further strengthened by the abrupt burst of ROS formation when the O2 supply is restored [74]. Because differences in the amount of antioxidants between brain regions are reported during normal brain functioning [74–77] and antioxidant enzyme activities may change region specifically during ischemia [19,78], the site-specific differences of the antioxidant capacity may participate in the development of the hippocampal layer-specific ROS levels after OGD. However, indications of the overwhelmed antioxidant defences during reoxygenation rather support the layer specificity in production than in elimination of ROS in our case. Pathological elevation of ROS levels is considered a cell death initiator [1]. The issue whether there are any detectable effects of the layer-selective ROS increase on cell damage emerged. Propidium iodide staining showed significant cell death neither in the vulnerable CA1 nor in the resistant DG regions. Our data suggest that, although the generation of ROS started immediately after the onset of OGD, definite cell death needs either more time to develop or higher ROS levels. Mechanism of ROS generation induced by OGD Mitochondria are considered the main source of ROS in U ischemia [8,55,79,80] during which excess amounts of O2 − are U − produced. Scavenging of O2 is performed by its dismutation to H2O2 by SOD. H2O2 is then neutralized by catalase and GPx U U to form H2O and O2 [81]. However, if NO is present, O2 − reacts with it at a reaction rate about three to six times faster than that with SOD to produce ONOO− the reactive free radical U [82–85]. Production of NO is also enhanced in ischemia [86–88]. nNOS was found in hippocampal neurons [89,90] and eNOS is present in the hippocampus in endothelial cells [89,91].

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Fig. 7. Spatial (hippocampal layers) and temporal (OGD vs reoxygenation) heterogeneity of OGD-evoked ROS response and its modulation by pharmacological tools. As demonstrated in the inset sample curves in the top right corner, red indicates a significant increase while blue indicates a significant decrease in ROS levels. During OGD (in vitro ischemia) ROS levels were not modified and AP-5 (50 μM), L-NAME (100 μM), and TRIM (100 μM) pretreatment did not influence this. However, in the presence of inhibition of catalase and GPx by ATZ + MS (10 mM both), OGD increased ROS levels in all layers of CA1 and DG. During reoxygenation ROS levels were elevated in all imaged layers except in SG of the DG. AP-5 and L-NAME inhibited the OGD-evoked ROS response significantly in the CA1 and DG, SM but were ineffective in the DG, SG. TRIM had a significant inhibitory effect in SR and SL of CA1. The presence of ATZ + MS did not alter reoxygenic ROS response.

By inhibiting NMDA-Rs and NOS we provided evidence for their involvement in ROS production in all CA1 and DG, SM layers during OGD and reperfusion. The finding that inhibition of NOS by L-NAME had an effect on ROS production similar to that of NMDA-R inhibition by AP-5 together with observations of the higher ONOO− and lower H2O2 sensitivities of CM-H2DCFDA U U and its insensitivity to NO and O2 − ([32,33]; Molecular Probes; − Fig. 1) suggest that ONOO is an important component of the ROS response produced by OGD in the hippocampal CA1 region U and DG, SM. Loading acute hippocampal slices with the NOsensitive fluorescent dye DAF-FM DA [88] showed a slightly U higher NO elevation in CA1 than in DG after OGD. This finding U shows that NO is available for ONOO− production and is in line with the regional differences that we found in changes of ROS levels. The less-pronounced effect of the selective nNOS inhibitor TRIM in CA1, SP and DG, SM than in CA1, SR and SL comU pared to L-NAME suggests that NO produced by eNOS may also take part in the generation of ONOO− in CA1, SP and DG,

SM, although partial (∼70%) inhibition of nNOS by 100 μM TRIM [53] as an alternative explanation cannot be excluded. In summary (Fig. 7), we first showed in acute hippocampal slices the ROS-producing effect of OGD with reasonable temporal and spatial resolution. The effect is layer specific. ROS increased in the vulnerable CA1 region and did not change in the ischemiaresistant SG, DG during reoxygenation. Production of ROS started right after OGD onset, i.e., during ischemia, but it was sufficiently quenched by catalase and GPx. The mechanism of ROS production measured in our system involves the activation of NMDA-Rs and NOS, and the type of ROS that we predominantly detected was presumably ONOO−. These findings suggest the causative role of ROS in the layer-specific ischemic vulnerability. Acknowledgments This work was supported by the Hungarian Medical Research Foundation (123/2003; 576/2006), the Hungarian Research

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