Neuroprotective mechanisms of cerium oxide nanoparticles in a mouse hippocampal brain slice model of ischemia

Free Radical Biology & Medicine 51 (2011) 1155–1163

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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Original Contribution

Neuroprotective mechanisms of cerium oxide nanoparticles in a mouse hippocampal brain slice model of ischemia A.Y. Estevez a, b,⁎, S. Pritchard a, K. Harper a, J.W. Aston a, A. Lynch a, J.J. Lucky a, J.S. Ludington a, P. Chatani a, W.P. Mosenthal a, J.C. Leiter c, S. Andreescu d, J.S. Erlichman a a

Biology Department, St. Lawrence University, Canton, NY, USA Psychology Department, St. Lawrence University, Canton, NY, USA Medicine and Physiology, Dartmouth Medical School, Lebanon, NH 03756, USA d Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, NY 13676, USA b c

a r t i c l e

i n f o

Article history: Received 12 February 2011 Revised 2 June 2011 Accepted 3 June 2011 Available online 12 June 2011 Keywords: Cerebral ischemia Nitric oxide Superoxide Peroxynitrite Nanoceria Nitrosylation Mouse Free radicals

a b s t r a c t Cerium oxide nanoparticles (nanoceria) are widely used as catalysts in industrial applications because of their potent free radical-scavenging properties. Given that free radicals play a prominent role in the pathology of many neurological diseases, we explored the use of nanoceria as a potential therapeutic agent for stroke. Using a mouse hippocampal brain slice model of cerebral ischemia, we show here that ceria nanoparticles reduce ischemic cell death by approximately 50%. The neuroprotective effects of nanoceria were due to a modest reduction in reactive oxygen species, in general, and ~ 15% reductions in the concentrations of superoxide (O2•−) and nitric oxide, specifically. Moreover, treatment with nanoceria markedly decreased (~ 70% reduction) the levels of ischemia-induced 3-nitrotyrosine, a modification to tyrosine residues in proteins induced by the peroxynitrite radical. These findings suggest that scavenging of peroxynitrite may be an important mechanism by which cerium oxide nanoparticles mitigate ischemic brain injury. Peroxynitrite plays a pivotal role in the dissemination of oxidative injury in biological tissues. Therefore, nanoceria may be useful as a therapeutic intervention to reduce oxidative and nitrosative damage after a stroke. © 2011 Elsevier Inc. All rights reserved.

Cerebral ischemia resulting from stroke is the third leading cause of death and the leading cause of long-term disability in the United States. The reduction of glucose and oxygen delivery to the brain during a stroke causes bioenergetic failure, which leads to excitotoxicity, oxidative stress, inflammation, blood–brain barrier dysfunction, and ultimately, cell death (reviewed in [1]). The production of free radicals is associated with many of the pathways involved in ischemic cell death. Reactive oxygen and nitrogen species, including superoxide anion (O2•−), hydrogen peroxide (H2O2), hydroxyl radical (HO •−), nitric oxide (NO), and peroxynitrite (ONOO−), accumulate during the ischemic period and induce oxidative damage. Oxidative injury worsens when blood flow is restored during the reperfusion period [2,3]. Currently, there are no effective neuroprotective drug treatments for ischemic stroke: many drugs have been tried, but they were insufficiently potent, were directly toxic, had adverse off-target effects, or failed to achieve effective and sustained levels in the central nervous system [4,5]. Cerium oxide (CeO2) nanoparticles, nanoceria, represent a potential new treatment for stroke and other oxidative disorders that overcomes many of the deficiencies of previous therapies for ⁎ Corresponding author at: Biology Department, St. Lawrence University, Canton, NY, USA. Fax: + 1 315 229 7429. E-mail address: [email protected] (A.Y. Estevez). 0891-5849/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2011.06.006

ischemic brain injury [6–8]. Cerium is a rare earth metal belonging to the lanthanide series of the periodic table. When combined with oxygen in a nanoparticle formulation, cerium oxide adopts a fluorite crystalline structure that has profound antioxidant properties [9,10]. The ability of nanoceria to reversibly bind oxygen and shift between Ce 4+ and Ce 3+ states under oxidizing and reducing conditions plays an important role in scavenging a variety of reactive oxygen species [11,12]. Nanoceria appear to possess both superoxide dismutase mimetic activity (most apparent when cerium is oxidized; Ce 4+) and catalase mimetic activity (most apparent when cerium is reduced; Ce 3+). Thus, oxidized Ce 4+ at the surface of nanoceria appears to dismutate the superoxide radical and form hydrogen peroxide. Hydrogen peroxide is, in turn, disproportionated to molecular oxygen and water [10,13]. The presence of oxygen vacancies in the nanoparticle crystal seems to facilitate the disproportionation of hydrogen peroxide as the peroxides dissociate to oxygen ions (either O − or O 2−), which migrate into oxygen vacancies of the crystalline lattice of the cerium oxide nanoparticles [14,15]. Although nanoceria have been widely used in industrial applications such as oxygen sensors [16] and automotive catalytic converters [17], they have only recently been used to mitigate oxidative stress in a variety of biological model systems [7,8,18]. For example, nanoceria protected a hippocampal neuronal cell line from oxidative stress [7] and

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decreased both NO and peroxynitrite formation in a murine model of ischemic cardiomyopathy [19]. The aim of this study was to test the hypothesis that nanoceria are neuroprotective in a rodent stroke model. Using an in vitro model of brain ischemia, we examined the neuroprotective capacity and cellular localization of nanoceria. We also measured the effects of nanoceria treatment on specific oxidants to determine the free radical-scavenging profile of cerium oxide. Nanoceria significantly reduced ischemic cell death by N50% at doses ranging from 0.2 to 1 μg/ml. Transmission electron microscopy (TEM) micrographs suggest that these protective effects were due to the proximity of ceria to the principle sources of reactive oxygen species (ROS) production (i.e., mitochondria) and to the targets of ROS and nitrosative agents (lipid membranes and proteins). Nanoceria modestly (~ 30%) reduced the concentration of reactive oxygen species in general. More specifically, treatment with nanoceria reduced the concentration of nitric oxide and superoxide by ~ 15%. Interestingly, ceria profoundly reduced the formation of ischemia-induced 3nitrotyrosine, a modification to proteins induced by peroxynitrite, suggesting that the reduction in peroxynitrite may be greater than the reduction in either of the precursors of peroxynitrite (NO and the superoxide) and that reducing the concentration of peroxynitrite may be a critical mechanism by which cerium oxide nanoparticles are neuroprotective after ischemic injury.

the onset of the ischemic insult or at specific times during the postischemic recovery period. Once the nanoparticles were administered, they remained in the medium throughout the remainder of the experiment. The delivery volume of the cerium oxide nanoparticles was 1 μl per 1 ml aCSF or medium and control slices received an equal volume of distilled water alone (vehicle control). The addition of the ceria to salt-based solutions promoted particle agglomeration at higher concentrations, so distilled water was used as the vehicle and resulted in no significant change in solution osmolarity. Study design

Materials and methods

Quantification of fluorescence images is difficult when there is no method of calibration. Therefore, we used an experimental design in which each set of brain slices studied in the test condition was matched with a similar set of brain slices treated identically in every respect except for the specific experimental intervention being studied (Fig. 1). On every study day, we used two sets of anatomically matched brain slices taken from age- and sex-matched littermates, of which one set was subjected to the test condition and the other set acted as the control. During fluorescence measurements, the light intensity, duration of image capture, and timing of image collection in the sequence of treatment conditions were also identical. Results are expressed as the ratio of the fluorescence in the test condition to the fluorescence in the matched control slice imaged at the same time point within the experimental sequence.

Mouse hippocampal brain slice model of ischemia

Fluorescence imaging

All animals used in this study were housed in St. Lawrence University's vivarium, fed ad libitum, and kept under a normal light/dark cycle. All procedures were approved by the St. Lawrence University Animal Care and Use Committee and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Adult (2–5 months of age) CD1 mice were sacrificed via rapid decapitation and their brains quickly removed and placed in a chilled, choline-based slicing solution containing 24 mM choline bicarbonate, 135 mM choline chloride, 1 mM kynurenic acid, 0.5 mM CaCl2, 1.4 mM Na2PO4, 10 mM glucose, 1 mM KCl, 20 mM MgCl2 (315 mOsm) [20]. Transverse hippocampal slices, 400 μm thick, were cut along a rostralto-caudal axis (−1.2 to −2.8 mm Bregma) using a Leica VT1200 Vibratome (Leica Microsystems, Wetzlar, Germany) and allowed to recover for 1 h in control artificial cerebral spinal fluid (aCSF) containing 124 mM NaCl, 3 mM KCl, 2.4 mM CaCl2, 1.3 mM MgSO4, 1.24 mM KPO4−, 26 mM NaHCO3, 10 mM glucose and bubbled with 5% CO2, 95% O2 gas (pH 7.4, 300 mOsm). Ischemia was induced by placing the brain slices in hypoglycemic, acidic, and hypoxic aCSF (glucose and pH were lowered to 2 mM and 6.8, respectively, and the solution was bubbled with 84% N2, 15% CO2, and 1% O2) at 37 °C for 30 min. Sucrose was added to maintain the osmolarity of the solution at ~295 mOsm. Slices not exposed to ischemia were kept in 37 °C control aCSF during this period. Sections that were studied acutely for 1 h or less were placed in control aCSF. Brain slices that were incubated for periods greater than 1 h were maintained in organotypic culture by placing them in 35-mm culture dishes containing culture medium and Millicell inserts (Millipore, Billerica, MA, USA). The culture medium contained 50% minimum essential medium (Hyclone Scientific, Logan, UT, USA), 25% horse serum, 25% Hanks’ balanced salt solution (supplemented with 28 mM glucose, 20 mM Hepes, and 4 mM NaHCO3), 50 U/ml penicillin, and 50 μg/ml streptomycin, pH 7.2 [21]. Solution osmolarity was measured using a vapor pressure osmometer and corrected to 295 mOsm (Wescor, Logan, UT, USA) if necessary. Hippocampal slices were placed in a culture dish and stored in a NuAire humidified incubator (NuAire, Plymouth, MN, USA) at 37 °C with 5% CO2 for up to 48 h. Cerium oxide nanoparticles (Sigma, St. Louis, MO, USA) at doses ranging from 0.1 to 2 μg/ml were dissolved in sterile, distilled water, sonicated, and administered either at

Cell viability At given time points postischemia, paired (control and test) slices were incubated for 20 min in culture medium containing 0.81 μM vital exclusion dye Sytox blue (Invitrogen, Carlsbad, CA, USA) and subsequently washed for 15–20 min in culture medium to remove unincorporated dye. Sytox blue is a fluorescent dye that binds DNA and RNA. However, it is excluded from the nucleus by the cell membrane in intact, viable cells. Therefore, it acts as a vital dye and stains only those dead and dying cells in which the cell membrane has become permeable so that the dye has access to the cell interior. After staining and washing, slices were transferred to the stage of a Nikon TE 2000-U (Nikon Instruments, Melville, NY, USA) microscope equipped with epifluorescence attachments and a 150-W xenon light source (Optiquip, Highland Mills, NY, USA). Control aCSF solution was loaded into 60-ml syringes, equilibrated with the 95% O2/5% CO2, and heated to 37 °C using a servo-controlled syringe heater block, stage heater, and in-line perfusion heater (Warner Instruments, Hamden, CT, USA). The sections were continuously perfused with warmed, 95% O2/5% CO2 equilibrated aCSF at a rate of 1 ml per minute [20]. After 5 min, images of the hippocampal formation of each control and test brain slice were collected using a 4× Plan Fluor objective (Nikon Instruments) under identical conditions (i.e., light intensity, exposure time, camera acquisition parameters). Sytox blue fluorescence was measured by briefly (620 ms) exciting the tissue at 480±40 nm and the emitted fluorescence (535±50 nm) from the probe was filtered using a 505 nm, long-pass, dichroic mirror (Chroma Technology, Bennington, VT, USA), intensified, and measured with a cooled CCD gain EM camera (Hamamatsu CCD EM C9100; Bridgewater, NJ, USA). The digital images were acquired and processed with Compix SimplePCI 6.5 software (C Imaging Systems, Cranberry Township, PA, USA). During post hoc image analysis, the detection threshold was set at 65% of maximum, which allowed separate light intensity and area measurements for the pyramidal and dentate granular hippocampal cell layers under control conditions but also prevented signal saturation under ischemic test conditions. The light intensity resulting from the Sytox loading reflected the number of dead or dying cells within the calculated area at or above the detection threshold. The area and light-intensity measurements were performed automatically

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using the Compix SimplePCI 6.5 software, thereby eliminating experimenter bias in selecting the regions of interest (Figs. 1 and 2). The pyramidal layers include the cornu ammonis layers 1–3, oriens layer, stratum radiatum, and lacunosum moleculare, whereas the dentate gyrus layers include the upper and lower blade of the granular layer, the polymorph layer, and the molecular layer. The hippocampal fissure was used to demarcate the transition between cornu ammonis and dentate gyrus layers.

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Measurement of specific reactive oxygen/nitrogen species Relative changes in intracellular ROS, NO, and O2•− production were measured at selected time points after the ischemic insult in brain sections loaded with the appropriate fluorescent probe (Fig. 1) for 20–30 min at 37 °C and then washed for 10 min. For a general estimate of intracellular ROS accumulation, 5-chloromethyl-2′,7′dichlorodihydrofluorescein diacetate (CM-H2DCFDA) was dissolved in dimethyl sulfoxide and used at a final concentration of 20 μM. Given

Sections prepared from same-sex, adult littermates

Experimental Paradigm Control

Test

Place in culture for indicated time

Fluorescence Imaging of anatomically paired sections ROS (CM-H2DCFDA), NO (DAF-FM), Viability (Sytox Blue)

Fig. 1. A schematic representation of the experimental paradigm for cell death assessment or measurement of specific reactive oxygen species. Hippocampal slices from age- and gender-matched littermates were used in each control–test comparison. After an appropriate period of organotypic culture, both test and control slices were studied to determine cell viability or the concentrations of combinations of ROS or the concentrations of specific oxidizing entities. CA fields, cornu ammonis fields. Anatomical brain diagrams reprinted with permission from ‘The Rat Brain In Stereotaxic Coordinates’ by George Paxinos and Charles Watson (2005).

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PY DG

resulting supernatant, containing the cytosolic fraction, was frozen at −80 °C until use. Total protein concentration for each sample was determined using a Bio-Rad Bradford protein assay kit (Bio-Rad, Hercules, CA, USA) following the 96-well standard microplate procedure as per the manufacturer's protocol (Bio-Rad). Absorbance was determined at 595 nm using a μQuant microplate spectrophotometer (BioTek, Winooski, VT, USA). Nitrotyrosine (3-NT) levels in each sample were quantified in triplicate using the OxiSelect nitrotyrosine ELISA kit (Cell BioLabs, San Diego, CA, USA) according to the manufacturer's protocol.

Statistical analysis

Electron microscopy To determine the distribution and location of the ceria nanoparticles in situ, brain slices were fixed and stained with glutaraldehyde (2%) and osmium tetraoxide (5%), respectively. Dehydration in increasing concentrations of acetone (50, 95, 100%) was followed by resin infiltration to generate a capsule. Nanoceria were imaged using a JEOL 2010 high-resolution scanning transmission electron microscope (JEOL USA, Peabody, MA, USA) coupled with a Gatan camera (Gatan, Warrendale, PA, USA).

SYTOX fluorescence area (% control)

the intense photoxidation associated with this compound, we used light-exposure durations and intensities that did not result in a progressive increase in the CM-H2DCFDA signal intensity after brief, repeated exposures. Diacetate (4-amino-5-methylamino-2′,7′difluorofluorescein diacetate; DAF-FM diacetate; 20 μM) was used to estimate intracellular NO accumulation, MitoSOX red was used to determine O2•− accumulation (5 μM). After the wash period, the sections were mounted into the perfusion chamber, and fluorescence images were collected as described above for Sytox blue. The exposure time was approximately 200–300 ms for DAF-FM and CM-H2DCFDA.

a

500

* 400

*

CA Fields DG

300

200 control

100

0 2

4

24

48

Time post-ischemia (hours)

b SYTOX light intensity (% control)

Fig. 2. Images and method used in the automated quantification of cell death using Sytox blue. After the appropriate treatment, slices were loaded with 0.81 μM Sytox blue (in cell culture medium) for 30 min to load dead or dying cells. After being washed to remove unincorporated dye, the slices were transferred to the stage of a Nikon TE 2000U microscope equipped with epifluorescence attachments and a Hamamatsu EM-CCD C9100 digital camera. Images were collected under identical conditions (i.e., light intensity, camera acquisition variables) with the detection threshold set at 65% of maximum, which allowed separate light-intensity and area measurements for the pyramidal layer (PY) and dentate granular (DG) layer (red outline) with sufficient sensitivity to detect cell death under conditions and still prevent signal saturation under test conditions. The pyramidal layers included the cornu ammonis layers 1–3, oriens layer, stratum radiatum, and lacunosum moleculare. The dentate gyrus layers included the upper and lower blade of the granular layer, the polymorph layer, and molecular layer. The hippocampal fissure was used to demarcate the transition between the dentate gyrus and the cornu ammonis layers. Slices were always imaged in pairs obtained from same-sex littermates and similar anatomical sections were used for comparisons; one served as a control and the other as a treatment (as shown in Fig. 1).

For fluorescence imaging experiments, data are presented as percentage control (this represents the ratio of the test to the control treatments from anatomically matched sections from same-sex, agematched littermate brains sliced and imaged on the same day). For statistical analysis of sections stained with Sytox blue, paired t tests were performed comparing light intensity and area in matched,

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CA Field DG Background

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140 120

control

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24

48

Time post-ischemia (hours) Brain homogenate preparation and enzyme-linked immunosorbent assays (ELISA) At 2 h postischemia, four to eight slices per experiment were diced on a chilled glass plate, homogenized, and fractioned as described by Li et al. [22]. Diced tissue was homogenized with ice-cold homogenization buffer (250 mM sucrose, 10 mM Hepes at pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 1% Halt protease inhibitor cocktail) in a glass homogenizer and centrifuged for 3 min at 2000g and 4 °C using an Allegra 64 ultracentrifuge (Beckman–Coulter, Fullerton, CA, USA). The supernatant was decanted into another ultracentrifuge tube and spun for 12 min at 12,000g, and the

Fig. 3. Time course of ischemia-induced changes in Sytox fluorescence without nanoceria present. We measured the (a) area and (b) intensity of Sytox blue fluorescence in cultured mouse hippocampal slices. All slices were exposed to 30 min of ischemia and imaged at the time points indicated. Ischemia-induced cell death peaked between 4 and 24 h. Data are presented as mean percentage ± SEM of control (paired anatomical sections of same-sex littermates sliced and imaged on the same day). The control slices were not exposed to ischemia, but the test slices were. As described under Materials and methods, cornu ammonis fields (CA fields) refers to the pyramidal and surrounding layers, whereas DG refers to the dentate gyrus and surrounding layers. Background represents all of the fluorescence in the slice outside of the cornu ammonis and dentate gyrus layers. Statistical significance was determined using a paired t test and *p b 0.05 denotes significant difference compared to matched control slices (n = 6–8 pairs).

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there was an increasing trend of Sytox fluorescence intensity within the cornu ammonis and dentate gyrus fields at 2 and 4 h postischemia, it was not statistically significant (Fig. 3b). The fluorescence intensity of the slice background (that part of the image other than the dentate gyrus and cornu ammonis areas) did, however, increase significantly at both 4 and 24 h postischemia. These changes in percentage fluorescence intensity in the cornu ammonis and dentate gyrus fields and in the background probably represent the fluorescence contribution of dead cells due to slice trauma (which caused high fluorescence intensity in control slices as well as ischemic slices). The intensity reflects the leakiness of individual cell membranes, but it is a less robust measure of the extent of cell death (because it better reflects the unique features of Sytox binding to individual cells rather than the population of cells). Based on these results, we concluded that area measurement was a more sensitive indicator of ischemia-induced hippocampal cell death than intensity. Thus, Sytox results indicating cell death are hereafter presented as the area measurements only. The decline in cell death by 48 h postischemia suggests that dead and dying cells were removed from the remaining living tissue in culture, and previous work has shown that this process can continue up to 2 weeks [23]. Peak tissue death after ischemia was greater in the cornu ammonis fields compared to the dentate gyrus, which is consistent with previous in vitro findings [24–26]. These results show

cerium-treated or untreated sections. In contrast, estimates of the intracellular accumulation of reactive oxygen species were made by measuring the average gray level from the entire visual field of the hippocampal formation. For most sections, this required two serial images from which the light intensities and areas were summed. The resultant values were averaged with the data obtained from the contralateral side to get an overall intensity and area value for each slice (Fig. 1). Statistical comparisons between treatment conditions were made using paired t tests and p b 0.05 was taken to denote significant differences. Results We evaluated the extent of tissue death in the cornu ammonis pyramidal fields, dentate gyrus, and surrounding cell layers after 30 min of transient, global ischemia in hippocampal brain slices using the vital exclusion dye Sytox blue. Using identical image-capture protocols and detection thresholds, we measured the area and intensity of Sytox fluorescence at various time points after the ischemic insult. The area of cell death increased significantly in the cornu ammonis fields 4 h postischemia, peaked at 24 h, and returned toward control values by 48 h postischemia compared to the same time points in control, nonischemic anatomically matched brain slices (Fig. 3a). Although

a

Control

Ischemia (30 min) + 1 µg/mL CeO2

Ischemia (30 min)

b CA DG

SYTOX Fluorescence Area (% Control)

180 160 140 120 100

Control

80

*

*

*

60

*

*

*

40 20

*

0 0.1

0.2

0.5

1.0

2.0

[CeO2] (µg/mL) Fig. 4. Neuroprotective effect of nanoceria treatment after ischemia. Nanoceria significantly decreased the area of ischemia-induced cell death in hippocampal slices. (a) Pseudocolor images of brain slices loaded with Sytox blue. (b) Quantification of the area of Sytox fluorescence in mouse hippocampal slices after 30 min of ischemia and treatment with varying doses of nanoceria (added to the solution at the onset of the ischemic insult). All Sytox measurements were made 1 h postischemia. Data are presented as mean percentage ± SEM of control (percentages defined by the ratio of anatomically matched test and control slices from same-sex littermates sliced and imaged on the same day). Statistical significance was determined using paired t tests and *p b 0.01 denotes significant differences compared to vehicle controls (n = 3–6 pairs).

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SYTOX fluorescence area (% control)

1160

120 control

100 80 60

*

40 20

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0 0

2

4

Post-ischemia timepoint of CeO2 (1 µg/mL) addition (hours) Fig. 5. The impact of the timing of nanoceria administration after ischemia on cell death. The times indicate how long after ischemia nanoceria were added to the hippocampal brain slices. Nanoceria (1 μg/ml) treatment was neuroprotective when given within 4 h of the onset of ischemia. The nanoceria treatment was more effective when given sooner after ischemia. Data are presented as mean percentage ± SEM of control. Statistical significance was determined using paired t tests and *p b 0.005 and **p b 0.001 denote significant differences compared to vehicle controls (n = 6 or 7 pairs).

that significant ischemic injury occurs after relatively brief exposure to ischemic solutions in slices prepared from adult mice. Nanoceria have potent free-radical scavenging properties and display both superoxide dismutase- and catalase-mimetic activities [10,13]. Because reactive oxygen species play a pivotal role in ischemic cell death, we tested whether nanoceria were neuroprotective and decreased cell death in our stroke model. We used commercially available particles (Sigma–Aldrich) with an average size of 10 nm and a negative ζ potential at physiological pH (Supplementary Fig. 1). The addition of nanoceria (0.1–2 μg/ml) to slices at the onset of ischemia resulted in a dose-dependent decrease in cell death measured 24 h postischemia (Fig. 4). The optimum dose under organotypic culture conditions seemed to be 1 μg/ml. Higher concentrations (2 μg/ml) resulted, however, in visible particle precipitation into the solution, thereby decreasing biological effectiveness. For this reason, studies of the 2 μg/ml dose were not pursued further. The neuroprotective effects of nanoceria were observed as long as the particles were added before ischemia or within 4 h postischemia (Fig. 5), results that are comparable to those of some of the best drugs currently being developed to treat stroke [27]. Because some studies have demonstrated toxic effects of nanoceria on various cell lines [28,29], we exposed nonischemic brain

slices to the same concentration range of nanoceria and measured cell viability. Nanoceria had no adverse effect on tissue viability (Supplementary Fig. 2) and, on the contrary, increased tissue viability at lower concentrations. Because there was no ischemic treatment in this set of experiments, the improved viability was most likely due to the scavenging of reactive oxygen species generated in response to slice trauma. To explore the site of action of ceria, we collected TEM images from ischemic brain slices treated either with or without nanoceria (1 μg/ml). TEM images revealed that nanoceria localized to lipid membranes, mitochondria, and neurofilaments (Fig. 6). The proximity of the nanoceria to mitochondria is interesting and may be of therapeutic relevance as these organelles are known to generate reactive oxygen species and trigger cell death pathways during ischemia [30]. Consistent with a neuroprotective effect of nanoceria, the mitochondria of the slices exposed to ischemia alone appeared swollen, with disorganized cristae, compared to the highly structured mitochondria visible in nanoceria-treated slices. To evaluate the putative free radical-scavenging properties of nanoceria, we first determined the relative ischemia-induced accumulation of ROS in brain slices at 1, 2, 4, and 24 h postischemia. We used

Fig. 6. TEM micrographs demonstrating the location of cerium oxide nanoparticles within hippocampal brain slices. The hippocampal brain slices shown were exposed to 30 min of ischemia and allowed to recover for 24 h. Nanoceria (white arrows) are located in high densities in the mitochondria and associated with neurofilaments (black arrows). Note the relative disorganization of the mitochondrial cristae in the ischemic slice compared to the more normal-appearing mitochondrial cristae in the slice treated with identical ischemia but in the presence of nanoceria.

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a

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Table 1 Effects of nanoceria on the accumulation of ROS, NO, and O2•− measured 1–2 h postischemia using fluorescence indicator dyes. Fluorescence indicator (species)

Ischemia

Ischemia + 1 μg/ml CeO2

CM-H2DCFDA (ROS) DAF-FM (NO) MitoSOX red (O2•−)

7987 ± 949 8330 ± 370 6870 ± 1443

5364 ± 890⁎ 6903 ± 585⁎ 5852 ± 1273⁎

Data are presented as mean ± SEM fluorescence intensity (in arbitrary units) of each indicator. Statistical significance was determined using a paired t test (n = 5–15 pairs). ⁎ p b 0.05 compared to ischemia alone.

b

Fig. 7. Time course of (a) ROS (measured by CM-H2DCFDA fluorescence) and (b) NO accumulation (measured by DAF-FM fluorescence) in hippocampal brain slices after ischemia. Data are presented as mean percentage ± SEM of control fluorescence (fluorescence ratio of age- and gender-matched test and control sections of littermates sliced and imaged on the same day). Statistical significance was determined using paired t tests comparing ischemic slices to nonischemic controls (n = 4–16 pairs); *p b 0.05, **p b 0.01, ***p b 0.001.

CM-H2DCFDA, a cell-permeant indicator that becomes fluorescent when oxidized by a variety of ROS, including peroxyl (ROO•) and HO•− radicals and ONOO −. We found that the CM-H2DCFDA signal peaked 2 h after the ischemic insult compared to control values (Fig. 7a). Because NO can contribute to the production of peroxynitrite, but is not the only radical detected by CM-H2DCFDA, we measured NO production with the NOspecific probe DAF-FM at 1, 2, 4, and 24 h postischemia (Fig. 7b). DAFFM is nonfluorescent until it reacts with a nitrosonium cation (produced by the spontaneous oxidation of nitric oxide) to form a fluorescent adduct that becomes trapped in the cell's cytoplasm. We found that DAF-FM fluorescence peaked at 1 h postischemia and gradually returned toward control levels after 24 h, a finding that is similar to the time course of NO levels in stroke patients [31,32]. Having delineated the time course of ROS production, we next tested the hypothesis that the neuroprotective effects of nanoceria are due to a decrease in ischemia-induced ROS. To do this, we added the nanoparticles to brain slices at the onset and after ischemia and measured ROS levels in the hippocampus 2 h postischemia using the nonspecific indicator CM-H2DCFDA. Nanoceria (1 μg/ml) decreased ischemia-induced ROS production by approximately 30% (Table 1). Because CM-H2DCFDA is a promiscuous indicator with differential affinities for the various free radicals and because the decrease in ROS production observed was less than the observed decrease in cell death (~50%; Fig. 4), we hypothesized that the neuroprotection by nanoceria might be associated with the scavenging of specific species of ROS. To examine this further, we used more specific probes such as DAF-FM

(detects NO) and MitoSOX red (detects O2•−) to measure the effects of nanoceria on ischemia-induced peak accumulation of these oxidizing species 1 to 2 h postischemia. MitoSOX red is a cell-permeant indicator that localizes to mitochondria and gets oxidized by O2•− to produce red fluorescence. We observed that the NO and O2•− levels decreased modestly after the addition of nanoceria (~15%; Table 1). Peroxynitrite, generated by the reaction of NO and O2•−[33], is produced under ischemic conditions and acts as both an oxidant and a nitrating agent to cause cellular damage [34,35]. Although peroxynitrite is labile and difficult to measure, it has a long enough half-life to diffuse in tissues, and it can readily react with tissue CO2 to form other reactive intermediates that lead to sustained protein modifications, such as the formation of 3-NT [33,36]. Several studies have shown that 3nitrotyrosine levels are increased after ischemia in human and animal studies [37]. In an effort to determine whether the neuroprotective effects of nanoceria were due to a reduction in peroxynitrite-mediated ischemic damage, we used ELISA to quantify 3-NT formation. Treatment with 1 μg/ml nanoceria significantly (p b 0.05) reduced ischemia-induced tissue nitrosylation at 2 h postischemia (Fig. 8) by approximately 70%. These results suggest that the neuroprotective effects of nanoceria are probably due, in large part, to a reduction in the damaging downstream effects of ONOO−, namely tissue nitrosylation. Discussion The hippocampus is selectively vulnerable to injury and cell death after transient global cerebral ischemia, and multiple mechanisms have been proposed to explain this phenomenon, including pathways of excitotoxicity, apoptosis, oxidative stress, and neuroinflammation [26,27]. In our novel in vitro model of ischemic brain injury, we use both acute and organotypic hippocampal slice cultures (OHSC) to evaluate the extent of cell death using perfusates that mimic ischemia and reperfusion. The solutions that we used reflect the cardinal features of cerebral ischemia observed in vivo: hypoxia, glucose deprivation, and

Fig. 8. Assessment of protein nitrosylation with and without nanoceria treatment. Nanoceria (1 μg/ml) treatment significantly decreased the concentration of ischemiainduced 3-NT measured using ELISA. Data are presented as mean ± SEM concentration of 3-NT normalized to protein levels of five pairs of experiments (four to six brain slices per experiment) done in triplicate. Statistical significance was determined using a paired t test (n = 5 pairs); *p b 0.05.

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acidosis. In contrast to other investigators using OHSC systems, who generally prepare slices from younger animals (~P7–P30), we used adult animals (8–20 weeks), which have a more mature, fully developed central nervous system [25,38]. In our view, this is an important distinction because it reflects the reality that stroke most commonly afflicts older individuals. In addition, animals less than 30 days of age do not have the full complement of neurons and glia compared to adult animals [39]. Thus, our model accounts for the influence of age on neurological outcome after a stroke and incorporates all three essential changes in the brain tissue environment after a stroke (hypoxia, glucose deprivation, and acidosis). Perhaps for these reasons, we found that just 30 min of simulated ischemia induced significant cell death in the cornu ammonis and dentate gyrus regions in our model (Fig. 3), whereas it is often necessary in other models of ischemic injury to expose tissue to hypoxia and/or hypoglycemia for much longer periods of time. Finally, by using neuroanatomically matched test and control sections from ageand sex-matched littermates, we decreased much of the variability associated with separate, independent experiments of fluorescence measurements. Cerium oxide nanoparticles significantly mitigated ischemic cell death (Fig. 4) in hippocampal slices in our study. Although nanoceria possess both superoxide dismutase- and catalase-mimetic properties [10,13], the catalytic interactions with specific free radicals in biological systems have not been fully elucidated. Using highresolution scanning tunneling microscopy, Esch et al. [12] demonstrated that electron shuffling within the lattice structure of nanoceria creates oxygen vacancies that allow redox reactions to occur while ceria cycles between the +3 and the + 4 oxidation states. However, this demonstration occurred at very high temperatures (such as those observed in catalytic converters), so the precise mechanism of interaction and catalysis of nanoceria with specific ROS in biological systems is not clearly defined. To our knowledge, our experiments using fluorescence indicators (Table 1) are the first to evaluate the effects of nanoceria on the accumulation of specific reactive oxygen species in intact, adult neural tissue with functioning circuitry (as opposed to cultured cell lines) and suggest that the ability of the nanoparticles to neutralize radicals in biological systems differs substantially from reports in tissue-free systems [40]. Specifically, we observed moderate effects of nanoceria on ischemia-induced accumulation of ROS, NO, and O2•− (Table 1), but profound reductions in 3-NT formation (Fig. 8). This is not a complete surprise. The superoxide radical is not a particularly strong oxidizing agent in the physiological pH range—it has already acquired an extra electron, which decreases its oxidizing potential. Nitric oxide is a radical with a half-life measured in seconds, and it readily reacts with the superoxide radical to form peroxynitrite. Thus, two less potent oxidizing agents react to form an extremely toxic free radical, peroxynitrite; a reaction that is actually facilitated by the enzyme superoxide dismutase [41]. Thus, the modest reductions in NO and the superoxide radical after treatment with nanoceria may profoundly limit the formation of peroxynitrite. It is also possible that nanoceria may directly react with and detoxify peroxynitrite (although as noted above, the exact interactions of nanoceria with free radicals in biological systems at physiological temperatures are unknown). Regardless of the chemical mechanism, the striking reduction in tyrosine nitrosylation after nanoceria treatment suggests that the effects of reducing peroxynitrite accumulation may play a key role in the neuroprotective properties of nanoceria after ischemic injury. It is possible that although multiple ROS are present, some of these species may not have equal affinity for the surface of nanoceria or access to the oxygen vacancies in the lattice structure proposed to play a role in the antioxidant capability of nanoceria by virtue of their intrinsic chemical reactivity, half-life, or diffusivity [12]. Alternatively, it is possible that the physical location of the nanoceria within the cell may dictate the species scavenged most effectively based on the proximity of ROS production. Thus it may be that the sparing effects of nanoceria are due in part to the intracellular

location of the nanoparticles, which may interrupt the oxidant cascade at the source of oxidant generation or cellular targets (e.g., mitochondria, lipid membranes; Fig. 6), and in part to the specific physicochemical characteristics of each radical involved. Whether neuroprotection by nanoceria is due to direct interaction with peroxynitrite or the combined reduction in NO and O2•− or both is unclear. Regardless of the mechanism, our results suggest that reducing the effects of peroxynitrite is an important step in mitigating ischemiainduced cell death. Peroxynitrite is both an oxidizing and a nitrating agent that can induce inflammation and cell death via necrotic or apoptotic mechanisms (reviewed in [33]). In addition, nitration of tyrosine residues can have a profound impact on protein activity and can trigger an immune response [36]. For example, Niu et al. demonstrated that treatment with nanoceria was protective in a mouse model of cardiomyopathy by several mechanisms including the inhibition of protein nitration and a decrease in inflammatory mediators [19]. In addition, nanoceria decrease ROS production and inflammation in a mouse macrophage cell line [42]. It is likely that the time course of generation and accumulation of ROS species may vary depending on the type of pathology. The fact that nanoceria have the capacity, based on ex vitro experiments, to scavenge all biologically relevant ROS suggests that its therapeutic relevance may extend beyond ischemic injury. Indeed, recent work from our lab has shown that nanoceria are effective in decreasing motor deficits in experimental autoimmune encephalitis, a mouse model of relapsing multiple sclerosis [47]. In this study, nanoceria were not toxic. Ceria-based nanoparticles have exhibited toxicity in some model systems, but not in others [42– 44]. These divergent results may be due to cell-specific responses, methodological differences in the viability measurements made, or the physical properties, concentration, size, and synthetic route of the nanoparticles used in the various studies. For example, exposure of human bronchial epithelial cells (BEAS-2B) to nanoceria reduced viability when measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) assay [28]. MTT undergoes reductive cleavage by an incompletely understood cellular enzymatic system to yield a color precipitate in live cells. Because of its reliance on enzymatic processes, any intervention that alters one of the enzymes involved in dye cleavage may lead to misleading viability results (e.g., [45]). In light of this, it is interesting that a study using propidium iodide to measure viability demonstrated no toxic effects of nanoceria on a variety of cell lines, including BEAS-2B cells [46]. Furthermore, no overt pathology has been detected in intact mice exposed long term (9 months) to nanoceria via weekly tail vein injections (W.E. DeCoteau et al., unpublished observations; [42]). Although we have not studied the biodistribution of the commercially available nanoparticles used in this study, we have used mass spectroscopy to analyze the distribution of the nanoceria injected into the whole animal (W.E. DeCoteau et al., unpublished observations). In these preliminary studies, significant concentrations of nanoceria were detected in the spleen and liver, presumably in cells of the reticuloendothelial system. Smaller concentrations accumulated in other tissues, probably in proportion to blood flow. Most relevant to the current work, we measured significant accumulations of nanoceria in brain tissue of normal mice. In summary, we have preliminary evidence of a neuroprotective effect in an animal model of multiple sclerosis; nanoceria do not appear to be toxic in mice, and easily detectible concentrations of nanoceria appear in the brain when administered via tail vein injections. Based on the results presented herein, we propose that the direct or indirect scavenging of peroxynitrite by nanoceria contributes to its neuroprotective effects in our in vitro model of ischemia. This, combined with the regenerative nature of the catalytic activity of nanoceria, prolonged half-life of nanoceria in tissue, and access of nanoparticles to the relatively sequestered intracellular space of biological tissues [10,12,13,40], endows these particles with great potential for use as neuroprotectants. Experiments are currently

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