Effects of Mild Hypothermia on Superoxide Anion Production, Superoxide Dismutase Expression, and Activity Following Transient Focal Cerebral Ischemia

Neurobiology of Disease 11, 28 – 42 (2002) doi:10.1006/nbdi.2002.0513

Effects of Mild Hypothermia on Superoxide Anion Production, Superoxide Dismutase Expression, and Activity Following Transient Focal Cerebral Ischemia Carolina M. Maier,* ,† Guo Hua Sun,* ,† Danye Cheng,* ,† Midori A. Yenari,* ,†,‡ Pak H. Chan,* ,†,‡ and Gary K. Steinberg* ,†,‡ *Department of Neurosurgery, ‡Department of Neurology and †Stanford Stroke Center, Stanford University, Stanford, California 94305-5487 Received January 4, 2002; revised April 19, 2002; accepted for publication April 30, 2002

Following a transient ischemic insult there is a marked increase in free radical (FR) production within the first 10 –15 min of reperfusion and again at the peak of the inflammatory process. Hypothermia decreases lipid peroxidation following global ischemia, raising the possibility that it may act by reducing FR production early on and by maintaining or increasing endogenous antioxidant systems. By means of FR fluorescence, Western blot, immunohistochemistry, and enzymatic assay, we studied the effects of mild hypothermia on superoxide (O 2ⴚ•) anion production, superoxide dismutase SOD expression, and activity following focal cerebral ischemia in rats. Mild hypothermia significantly reduced O 2ⴚ• generation in the ischemic penumbra and corresponding contralateral region, but did not alter the bilateral SOD expression. SOD enzymatic activity in the ischemic core was slightly reduced in hypothermia-treated animals compared with normothermic controls. Our results suggest that the neuroprotective effect of mild hypothermia may be due, in part, to a reduction in neuronal and endothelial O 2ⴚ• production during early reperfusion. © 2002 Elsevier Science (USA) Key Words: hypothermia; cerebral ischemia; superoxide anion; superoxide dismutase; hydroethidine; free radicals.

INTRODUCTION

(Murakami et al., 1998). Several reports have documented a decrease in free radical generation following reperfusion with lower brain temperature (Globus et al., 1995; Kil et al., 1996; Wenisch et al., 1996; Lei et al., 1997) and a reduction in infiltrating brain neutrophils (Toyoda et al., 1996; Maier et al., 1998). Consistent with this, is the observation by some clinicians that hypothermia may be related to an increase in the incidence of infections, especially with cooling periods longer than 24 h. However, detailed neuropathological studies are needed to support the hypothesis that mild hypothermia protects the ischemic brain by decreasing ROS production. While many histopathological studies on neuroprotection by hypothermia have been reported (Busto et al., 1987; Minamisawa et al., 1990; Dietrich et al., 1993; Nurse & Corbett, 1994), little is known about the effects of mild hypothermia on the

Among the various biochemical events associated with ischemic brain injury, emerging evidence suggests the formation of oxygen-derived free radicals as a common denominator. There are many sources of reactive oxygen species (ROS), including the mitochondrial electron transport chain and activated leukocytes (Phillis, 1994). ROS, which are generated soon after hypoxia/ischemia (Imaizumi et al., 1984) as well as in the later stages during postischemic reperfusion (Kirsch et al., 1992), can attack the major cellular components and alter membrane functions. Superoxide anion (O 2•⫺) in particular, appears to play a key role in the oxidative chain reaction, and may induce lipid peroxidation, protein oxidation, and DNA damage that results in both acute and chronic neuronal injury

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cellular sources of ROS. In a previous study of focal cerebral ischemia, it was demonstrated that the neuroprotective effect of mild intraischemic hypothermia (2-h duration) was sustained over 2 months (Maier et al., 2001). The current study, using identical animal protocols and endpoints, was designed to examine the effects of mild hypothermia on the cellular and molecular events associated with the production of reactive oxygen species implicated in ischemia-induced neuronal damage. We first examined the effects of mild intraischemic hypothermia on the in situ generation of superoxide anion using the cell-permeant probe, hydroethidine (HEt), which is oxidized by O 2•⫺ to a fluorescent dye, ethidium (Et). Since Et is retained intracellularly, this method allows for a semiquantitative assessment of cellular O 2•⫺ production. Under normal physiological conditions, electron transport within mitochondria is responsible for a small percentage of O 2•⫺ production. Scavenging superoxide with SOD is the first step in the cerebral antioxidant system which also includes catalase, glutathione peroxidase (GSPX), and reduced glutathione (GSH). High levels of SOD1 are associated with smaller infarcts following middle cerebral artery occlusion (MCAO) (Imaizumi et al., 1990; Yang et al., 1994; Chan et al., 1998) and overexpression of SOD2 can prevent accumulation of peroxynitrite (ONOO ⫺ ) with a concomitant increase in GSPX activity after focal transient ischemia. Following an ischemic insult, there is a reduction of tissue concentrations of GSH which can be attenuated by mild intraischemic hypothermia (Karibe et al., 1994). Furthermore, recovery of total GSH levels has been shown to precede recuperation of neuronal tissue from oxidative stress (Shivakumar et al., 1995). Therefore, the next step in this study was to determine whether endogenous antioxidants play a role in mediating hypothermic neuroprotection. This was done by examining the temporal expression and anatomical distribution of two isoforms of superoxide dismutase (Cu/Zn-SOD also known as SOD1, and Mn-SOD also known as SOD2), starting at 72 h postischemia and up to 2 months.

MATERIALS AND METHODS The following animal protocols were approved by the Stanford University Administrative Panel on Laboratory Animal Care.

Stroke Model For the in situ detection of superoxide anion production, the following protocol was used. Male Sprague–Dawley rats weighing 285–305g (Charles Rivers, Wilmington, DE) were anesthetized with 3% halothane delivered by mask and were maintained in surgical plane of anesthesia with 1% halothane in 200 ml/min oxygen and 800 ml/min air without the use of paralytic agents. Depth of anesthesia was assessed every 15 min by hind limb pinch. Rectal temperature was maintained at 36.5–37.5°C prior to ischemia and ECG leads were placed in order to monitor heart rate and respirations. A midline incision was made in the neck to expose the common carotid (CCA), external carotid (ECA), internal carotid (ICA), and pterygopalatine (PPA) arteries. The CCA, ECA, and PPA were ligated using a 6-0 silk suture. The stroke was produced by inserting a 3-0 monofilament suture (with a flamed tip) 19 –23 mm from the bifurcation of the ICA and ECA. The ostium of the middle cerebral artery (MCA) was occluded for 2 h. Animals were kept (intraischemically) either normothermic (37°C, n ⫽ 3) or hypothermic (33°C, n ⫽ 3). Total body cooling was achieved by spraying alcohol onto the animal and cooling it to the desired temperature with a fan. Rewarming was achieved by a heating pad placed under the animal and a lamp positioned over the animal’s body. At the end of the experiment all animals were returned to normothermia and allowed to reperfuse for 1 h. For determination of the anatomical distribution and temporal expression of superoxide dismutase, the above protocol was used with the following modifications. The occlusion period used was 2 h except for animals survived for 7 days or 2 months that underwent a 90-min occlusion. This was done in order to reduce the high mortality rate observed in normothermic animals, particularly with the longer survival endpoints (Maier et al., 2001). Animals were euthanized with a halothane overdose at 2, 6, 24, 3 days, 1 week, or 2 months following ischemic onset. In Situ Detection of Superoxide Anion (O 2•ⴚ) Production The spatial distribution of O 2•⫺ during cerebral ischemia was investigated by the in situ detection of oxidized hydroethidine (HEt). This method has been used in cells and tissues and is specific for superoxide (Rothe & Valet, 1990; Chan et al., 1998; Miller et al., 1998; Murakami et al., 1998). Neither hydroxyl radical, ©

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30 NO, peroxynitrite, H 2O 2• , hypochlorite, nor singlet O 2•⫺ significantly oxidizes HEt (Bindokas et al., 1996), so we interpreted Et fluorescence to selectively indicate O 2•⫺ generation. HEt (Molecular Probes, Eugene, OR) is taken up by live cells and oxidized to a red fluorescent dye, ethidium. HEt was prepared in dimethylsulfoxide (DMSO) to a concentration of 100 mg/ml, then diluted in PBS to a concentration of 1:100 (1 mg/ml). Fifteen minutes prior to ischemic onset, animals were injected intravenously into the jugular vein with 1 ml solution under halothane anesthesia. Following 2 h MCA occlusion (MCAO) and 1 h reperfusion, the animals (37°C, n ⫽ 3; 33°C, n ⫽ 3) were euthanized and perfused transcardially with 60 ml heparinized normal saline and 300 ml 3.7% paraformaldehyde. Two normothermic and two hypothermic shams in which the suture was not advanced were also included in the study in order to determine the effects of temperature alone on superoxide production. Six additional normothermic animals underwent either 1 h MCAO with no reperfusion (n ⫽ 3) or 2 h MCAO with 15 min of reperfusion (n ⫽ 3). Brains were fixed in 3.7% paraformaldehyde for 24 h. After fixation, 50-␮m sections were cut on a vibratome and then mounted and viewed under a fluorescent microscope (HBO 100 W/2, Zeiss). The measurement of ethidium (Et) fluorescence (excitation ⫽ 510 –550 nm and emission ⬎ 580 nm) was done at a different excitation wavelength (red) from the HEt fluorescence (blue), therefore, the issue of HEt sequestration from mitochondria was not essential for the ethidium measurement. Since ethidium binds to DNA or RNA irreversibly, the data represent an accumulative level of superoxide (Bindokas et al., 1996; Benov et al., 1998). Extracellular O 2•⫺ would not be expected to significantly contribute to the observed cellular fluorescence, since Et is impermeable to cell membranes. In order to estimate superoxide generation, we preselected regions of interests (ROIs) within areas of maximal ischemic injury within the cortex (cortical ischemic core) and infarct border (cortical ischemic penumbra), as well as nonischemic regions from the contralateral side. These regions were photographed under high power (63X) and the amount of fluorescence was measured from 10 to 12 cells per field using an optical densitometer (BioRad Multi-Analyst System) by an individual blinded to the treatment groups. Since ethidium fluorescence stains the whole cell, cell type was readily determined from the morphology but was also confirmed by immunofluorescent labeling with specific cell markers on immediately adjacent sections. •

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Immunohistochemistry Animals whose brain tissue was used for immunohistochemical detection of SOD1 and SOD2 were euthanized with a halothane overdose. The brains were quickly removed and sliced into 3-mm-thick coronal sections. The brain slices were then incubated in 2% triphenyltetrazolium chloride (TTC) at 37°C for 15 min and fixed in 10% buffered formalin (pH 7.4) for 1 week. Animals survived for 2 months were anesthetized with 3% halothane and transcardially perfused with heparinized saline (600 cc) followed by perfusion with 600 cc of 10% buffered formalin (pH 7.4). The brains were kept in the skull in formalin overnight, removed, and fixed for an additional 7 days. Following paraffin embedding, 6-␮m-thick sections were obtained. Sections were washed with 0.2% Triton X-100, treated for endogenous peroxidases with hydrogen peroxide, then blocked with 1.5% normal horse serum in phosphate buffered solution (PBS). Sections were then incubated with primary antibody followed by secondary antibody and the avidin-biotin-horseradish peroxidase method was used to detect antibody using the Vector ABC kit (Elite Vectastain ABC Kit, Vector Labs, Burlingame, CA) and colorized with DAB (Vector DAB Kit, Vector Labs). Antibodies to SOD1 and SOD2 were generously provided by Dr. Kanefusa Kato (Department of Biochemistry, Institute of Developmental Research, Kasugai, Japan). Negative controls were run in parallel using adjacent sections without primary antibody. The sections were then counterstained with H & E, cleared, and mounted. For determination of cell type in the HEt study, fluorescence labeling was performed using a fluorescein conjugated secondary antibody (1:20, Vector Laboratories), while peroxidase-conjugated secondary antibodies were used in paraffin-embedded tissue (1:100, Vector Laboratories). MAP2 was used to identify neurons, glial fibrillary acid protein (GFAP) to identify astrocytes (Amersham, Arlington Heights, IL), 2⬘,3⬘cyclic nucleotide 3⬘phosphohydrolase (CNP) to identify oligodendrocytes (Promega, Madison, WI), and isolectin B4 (IB4) to identify microglia/monocytes (Sigma Chemical Co, St. Louis, MO).

Western Blot Analysis A separate set of animals (n ⫽ 3 per group) was subjected to transient MCAO as described above. Sham controls were similarly prepared from animals that received no ischemia or surgery. Animals were anesthetized and transcardially perfused with 600 cc

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of heparinized saline. The brains were quickly removed and placed on dry ice. Samples from predefined brain regions (striatum, cortical core, and cortical penumbra) within a 2- to 3-mm-thick section at the level of the anterior commissure (region of maximal injury) were isolated and homogenized and then stored at ⫺70°C in Laemmli’s solubilizing buffer with protease inhibitors (1 nM PMSF, 1 ␮M leupeptin, 1 ␮M pepstatin) until further use. Thirty microliters of buffer were added per milligram of tissue. Protein concentration was determined using a BCA Protein Assay kit (Pierce Laboratories, Rockford, IL) with BSA as a standard. Aliquots containing 15 ␮g (SOD1 and SOD2) of protein in lysis buffer with 2-mercaptoethanol and 5% bromophenol blue were subjected to 12.5% SDS–polyacrylamide gel electrophoresis. Protein bands were transferred from the gel to polyvinylidinene fluoride (PVDF) membranes (Millipore, Bedford, MA), and blocked with 5% milk in PBS. Membranes were probed for the protein of interest by incubating in the primary antibody of interest for 1 h followed by a horseradish peroxidase (HRP)-conjugated secondary antibody (1:200 dilution). Membranes were washed between steps with 1% BSA in 0.01 M PBS. Blots were visualized with the ECL system (Amersham) and exposed to X-ray film for 1 s or up to 20 min. Densitometric measurements were made from the film using a GS-700 imaging densitometer (Bio-Rad, Hercules, CA), then quantified using the Multi-Analyst System (Bio-Rad). For quantification of relative protein expression, the optical density of the protein band of interest was normalized to the optical density of actin. The actin bands were detected by re-probing the same membrane with the appropriate antibody after stripping. Since it was possible that actin levels were also affected by the ischemic insult, in some instances total protein was measured by staining the membranes with Ponceau Red and measuring the optical density of the entire lane. Thus the optical density of the protein band of interest (SOD1 or SOD2) was also normalized to total protein levels in the sample being studied. SOD Activity Assay Following MCAO, brain samples from additional animals (n ⫽ 3 per group at 2 and 24 h postinsult) were assayed for SOD activity using the xanthine/ xanthine oxidase (XO) and ferrycytochrome c method (Crapo et al., 1978). Briefly, tissue samples were homogenized, centrifuged and the supernatant collected. To determine the concentration of XO (Sigma-Aldrich,

St. Louis, MO) necessary to obtain a variation of absorbance of approximately 0.02 untis per minute at 550 nm without SOD, dilutions of XO stock (suspension in 2.3 M (NH4)SO4 at a concentration of 50 U/ml) were made. A 96 multiple-well plate was loaded with 50 ␮l of the different XO concentrations along with 100 ␮l of SOD buffer and 150 ␮l of reactive solution (0.1 mM cytochrome c, 0.5 mM xanthine, 1 mM KCN, SOD buffer). Optical density measurements were recorded for 2 min (one reading/10 seconds) using Spectra MAX 340 (Molecular Devices Corporation, Sunnyvale, CA) to determine the appropriate XO concentration needed to perform the SOD assay. Samples were serially diluted in SOD buffer and loaded in a 96 well plate followed by addition of 150 ␮l of reactive solution and 50 ␮l of diluted XO. The variation of optical density was measured as stated above. SOD activity was determined in triplicate. One unit of SOD was defined as the quantity of enzyme necessary to produce 50% inhibition of the rate of reduction of ferrycytochrome c. Superoxide dismutase activity was expressed in U/mg soluble protein (Copin et al., 2000). Protein content in the samples was determined using the BCA Protein Assay kit (Pierce, Rockford, IL).

RESULTS Production of O 2•ⴚ Following Focal Cerebral Ischemia As previously observed by Kondo, et al (Kondo et al., 1997), 2 h of MCA occlusion followed by 1 h reperfusion demonstrated Et fluorescence which was particularly intense within subcellular organelles (dot like regions within the cells), suggestive of mitochondrial production of O 2•⫺ (Figs. 1A and 1C). Furthermore, Et-positive cells appeared to have neuronal morphology, similar to that observed on adjacent sections stained with the neuronal marker, MAP2 (Figs. 1A–1C). Astrocytes, while easily discernible by GFAP, were not ethidium-positive (Fig. 1D). Very prominent ethidium fluorescence was observed in endothelial cells, particularly in the ischemic cortex, of both normothermic and hypothermic animals (Fig. 1E). Occasionally, oligodendrocytes also displayed Et-fluorescence (Fig. 1F). The effects of mild intraischemic hypothermia on the generation of O 2•⫺ can bee seen on Fig. 2. Optical density measurements from each region of interest (ROI) showed that mild intraischemic hypothermia significantly reduced the generation of O 2•⫺ in the ischemic penumbra (P ⫽ 0.049) but had no effect ©

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the 2 h MCAO/15 group compared with the animals in the 1 h MCAO/0 reperfusion group (P ⬍ 0.0001) SOD1 and SOD2 Expression

FIG. 2. Effect of mild intraischemic hypothermia on oxygen radical generation. Optical density measurements from 10 –12 cells per region of interest in three animals per temperature group. Hypothermic animals showed significantly reduced ethidium fluorescence in the peri-infarct area as well as in the corresponding contralateral region compared with normothermic controls by ANOVA and Student t test (*P ⬍ 0.05). Data are expressed as mean ⫾ SEM. IC, ischemic cortex; IP, ischemic penumbra; N-IC, nonischemic cortex; N-IP, nonischemic penumbra.

on O 2•⫺ production in the infarct core. There were no significant differences in optical density measurements (i.e., superoxide production) between normothermic and hypothermic shams (data not shown). Following 1 h MCAO without reperfusion (1 h MCAO/0), normothermic animals showed a slight increase in ethidium fluorescence in the ischemic cortex compared with the corresponding region in the contralateral side, while animals undergoing 2 h MCAO with 15 min of reperfusion (2 h MCAO/15) showed a 94% increase in the ischemic penumbra compared with the corresponding contralateral region. There were no differences in OD measurements from the nonischemic hemisphere of the 1 h MCAO/0 group compared with animals in the 2 h MCAO/15 group. There was, however, a 58% increase in superoxide anion production in the ischemic penumbra of

Immunoreactivity against Cu/Zn-SOD (SOD1) showed that this enzyme was uniformly expressed throughout the brain at basal levels and did not appear to be upregulated by the ischemic insult or by temperature manipulations at any of the time points studied (Fig. 3). SOD1 was observed primarily in neurons and was not readily detected in glial or endothelial cells. However, strong SOD1 immunoreactivity was observed in cells from the choroid plexus at 6 h postischemic onset, but was greatly diminished by 3 days and returned to basal levels by 7 days postinsult. Strong SOD1-positive cells were also evident in the septum at 2 months. Immunoreactivity to SOD2 (Fig. 3) was observed throughout the brain and intensity of the immunoreaction was variable, depending on the brain area and the cell-type being examined. Following 2 h MCAO there was an increase in SOD2 immunoreactivity that was very prominent bilaterally at 6 h postischemic onset. At this time point SOD2 immunoreactivity was seen mostly in neurons in both the striatum (ischemic core) and throughout the cortex (ischemic core and penumbra), as well as in endothelial cells and choroid plexus. SOD2-positive cells were found throughout the neocortex, particularly in the ischemic penumbra, and were also prominent in the contralateral side. SOD2 immunoreactivity was greatly diminished in the ischemic core by 3 days postinsult and was virtually absent in the core by 7 days. By 3 days postinsult, SOD2 immunoreactivity was detected not only in neurons but also in reactive astrocytes, oligodendrocytes, and microglia. Intense SOD2 immunoreactivity was particularly evident in the peri-infarct regions of the cortex. Figures 4A– 4C shows SOD2 immunoreactivity and cellular localization of the enzyme in coronal sections from a representative normothermic animal 1 week following the stroke. At 2 months postinsult

FIG. 1. (A) Low magnification image (10X) from a coronal section of a normothermic animal at 15 min of reperfusion indicating ethidium fluorescence; striatum (St), corpus callosum (CC), cortex (Ctx). (B) Photomicrograph (20X) of the peri-infarct border of the same animal where N indicates normal, non-ischemic tissue, P is penumbra and C is the infarct core. (C) Ethidium fluorescence is observed within neurons in the peri-infarct regions after 2 h MCAO and 1 h reperfusion (arrows). At higher magnification it is possible to observe the intense fluorescence within subcellular organelles (dot like regions within the cells), suggestive of mitochondrial production of superoxide (E). Morphology outlined by Et fluorescence is neuronal, similar to that observed on an adjacent section stained with MAP2 (D). Astrocytes are easily discerned with GFAP staining, however, ethidium fluorescence is absent in these cells (F). Prominent Et-fluorescence is observed in endothelial cells of the ischemic cortex (G) and occasionally in oligodendrocytes (H).

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there was still prominent SOD2 immunostaining, but this was restricted mostly to the peri-infarct area and the corresponding homotopic region on the contralateral side. Figure 5 shows the progression of the stroke at 2 months. At this time point reactive astrocytes were very evident in the rim of the lesion due to gliosis and scar formation, in both the normothermic and hypothermic animals. Note, however, that normothermic animals essentially lost all the tissue in the infarcted region, while hypothermia-treated animals retained it. Sham animals showed very faint SOD2 immunoreactivity throughout the brain at all time points studied and negative controls did not display any immunoreactivity. As seen on Fig. 6, SOD2 is a dimeric protein of approximately 80 kDa; similar blots were obtained for SOD1. Optical density measurements from immunoblots did not show any obvious or significant differences in the levels of SOD2 expression between normothermic controls and hypothermia-treated animals. Immunohistological examination of coronal sections adjacent to the tissue sampled for Western blotting showed that there was a good correlation between immunoreactivity to SOD2 and optical density measurements from immunoblots within the same animal. While measuring the level of actin in immunoblots has been an accepted method for verifying equal protein loading of all lanes in a gel, we often found that, particularly in animals that survived for 2 months, actin levels were also variable. Total protein levels obtained from optical density measurements following Ponceau red staining of membranes indicated that there was equal protein loading of all lanes in a gel. SOD Activity Results from the enzyme activity assays can be seen in Fig. 7. At 2 h postinsult, there was a trend toward increased SOD activity in the ischemic cortex of both normothermic and hypothermic animals compared with the contralateral cortex. This increase in SOD activity became significant by 24 h: 1.4-fold increase in the ischemic tissue compared with the nonischemic side. Compared with the 2-hour time point, SOD ac-

tivity at 24 hours increased 3-fold and 3.4-fold in the ischemic cortex of normothermic and hypothermic animals, respectively. In the ischemic striatum there was a 2.3-fold and 2-fold increase in SOD activity in normothermic and hypothermic animals, respectively. There was a small reduction in enzymatic activity in the infarcted striatum of hypothermia-treated animals compared with normothermic animals (9.86 ⫾ 0.5 and 11.8 ⫾ 0.8 U/mg protein, respectively; P ⫽ 0.05) and a similar trend at 24 h (19.7 ⫾ 1.9 and 26.6 ⫾ 2.9 U/mg protein, respectively; P ⫽ 0.07).

DISCUSSION Reactive oxygen species have been implicated in the pathogenesis of cerebral infarction following both global and focal ischemia (Malinski et al., 1993; Dirnagl et al., 1995; Forman et al., 1998; Peters et al., 1998). Superoxide, which is directly toxic to neurons (Patel et al., 1996) appears to be a key factor (Beckman et al., 1990; Kondo et al., 1997). One of the major approaches to ameliorate ischemia-induced brain damage is to minimize the destructive actions of ROS using various therapeutic strategies including administration of ROS-scavenging drugs, upregulation of endogenous ROS-scavenging mechanisms, and prevention of leukocyte infiltration into the affected brain tissue (Juurlink, 1997). Hypothermia has been reported to decrease lipid peroxidation following global ischemia (Lei et al., 1994), suggesting that decreased production of ROS may be partly responsible for its neuroprotective effect. Studies in rodents using microdialysis measurements of NO synthesis, measurements of nitric oxide synthase (NOS) activity or of plasma nitrite plus nitrate levels have shown that intraischemic mild hypothermia decreases nitric oxide (NO) and hydroxyl radical (OH •⫺) levels following global and focal cerebral ischemia (Kader et al., 1994; Kil et al., 1996; Kumura et al., 1996). Extending mild hypothermia into the reperfusion period has also been shown to significantly reduce oxygen radicals as measured by spectroscopy with spin trapping.

FIG. 3. Representative photomicrographs of immunoreactivity against SOD1 and SOD2 in the rat cortex (ipsilateral to the infarct unless otherwise indicated) following transient focal cerebral ischemia. SOD2 immunoreactivity was very prominent bilaterally starting at 6 h post-ischemic onset. By 3 days postinsult SOD2 immunostaining was very prominent in the cortex, particularly in the peri-infarct area, as well as in the contralateral homotopic region. At 2 months postinsult, SOD2 immunoreactivity was restricted mostly to the peri-infarct regions of the cortex.

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In the present study we show that mild intraischemic hypothermia significantly reduces the generation of superoxide in peri-infarct areas and corresponding contralateral regions as measured by ethidium fluorescence. The burst-like pattern of ROS production following reversible MCAO has been shown to occur in the first 10 –15 min of reperfusion (Peters et al., 1998). In this study we also observed an early increase of ROS production in the ischemic hemisphere (94% compared with the nonischemic hemisphere) in normothermic animals undergoing 2 h MCAO followed by 15 min of reperfusion. This increase in ROS production also coincides with increases in excitatory amino acids (EAAs), particularly glutamate (Busto et al., 1989). Mild hypothermia appears to blunt the peak in glutamate that occurs within 60 min of ischemia onset and in some instances delays it by 20 min (Graham et al., 1990; Huang et al., 1998). A few groups, including ours, have shown that mild hypothermia is still effective even when applied after glutamate peaks (delayed by 60 to 120 min) (Baker et al., 1992; Maier et al., 2001). Therefore, there is reason to believe that mild hypothermia may exert its protective effects by directly altering processes such as generation of ROS. In this study we looked at 1 h into normothermic reperfusion; however, we still cannot rule out the possibility that mild hypothermia may have simply delayed the generation of ROS. This still suggests that hypothermia may extend the therapeutic window for stroke by reducing FR production, a key component of

FIG. 4. (A) Representative coronal section of a normothermic animal at 7 days postinsult. The entire ipsilateral hemisphere shows reactive astrocytes expressing both GFAP (blue) and SOD2 (brown), particularly in the peri-infarct zone. A and B were taken from the area marked by a yellow square on the coronal section and show colocalization of GFAP and SOD2 in reactive astrocytes, while neurons show only SOD2 staining. Panel C shows a hypertrophied astrocyte (arrow) expressing both GFAP and SOD2 taken from the border of the infarct (area marked by a yellow circle). (B) Representative coronal section of a normothermic animal at 7 days postinsult (same animal as 4A). The ipsilateral hemisphere shows increased CNP staining (blue) in the peri-infarct zone. A and B were taken from the area marked by a yellow square on the coronal section and show colocalization of CNP and SOD2 (brown) in oligodendrocytes (arrow). (C) Same animal as in A and B. The ipsilateral hemisphere shows a large number of IB4- positive cells (microglia and monocytes) in the peri-infarct region. A (taken from the area marked by a yellow square on the coronal section) shows the general distribution of IB4-positive cells (brown) in the infarct border. B shows colocalization of IB4 and SOD2 (blue) in microglia associated with neurons expressing high levels of SOD2 (arrow). However, the majority of IB-4-positive cells did not show expression of SOD2 (arrowhead).

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FIG. 5. Representative coronal sections of a normothermic and a hypothermic animal at 2 months postinsult. Although there is no tissue left in the infarcted area of the normothermic animal, the peri-infarct zone still shows reactive astrocytes expressing both GFAP (blue) and SOD2 (brown). The hypothermia-treated animal shows a significantly smaller infarct. Top panels show immunoreactivity to SOD2 (brown), GFAP (blue), and CNP (blue). Lower panels show immunoreactivity to SOD2 (blue) and IB4 (brown), indicating that the inflammatory response is still going on 2 months after the stroke.

ischemia/reperfusion (I/R) injury. The deleterious consequences of delayed restoration of blood flow are attributed to reperfusion injury, a process that further damages brain cells, the ischemic arterial wall, and the microvasculature (Hamann et al., 1995; Aronowski et

al., 1997). The cerebral endothelial cells that compose the BBB are a source of ROS and may therefore be important mediators of I/R injury. Upon examining the cellular sources of superoxide during the first hour of reperfusion we have found Et-positive cells to be ©

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FIG. 6. Western blot analysis showed that SOD2 is a dimeric protein of approximately 80 kDa. SOD2 expression is prominent in all brain regions examined irrespective of temperature. Similar results were obtained for SOD1.

Maier et al.

primarily neuronal, although Et-positive endothelial cells were also observed throughout the infarcted region. Et signals have been shown to appear in neurons early during reperfusion (4 h) as well as in endothelial cells (24 h) (Murakami et al., 1998). A second source of ROS in later stages of postischemic reperfusion is activated leukocytes (Phillis, 1994). We have previously shown that mild hypothermia may act by reducing the number of infiltrating leukocytes, thus attenuating the generation of ROS that occurs several days following transient MCA occlusion (Maier et al., 1998). A study by Nakajima and colleagues (Nakajima et al., 1997) showed that while 10 min of global cerebral ischemia at 32°C did not induce

FIG. 7. Effect of mild hypothermia on SOD activity (expressed in U/mg soluble protein) at 2 and 24 h post-MCAO. (A) At 2 h postinsult there is no significant increase in SOD activity in the ischemic cortex compared with the contralateral, nonischemic cortex of either normothermic or hypothermic animals. (B) By 24 h there is 1.4-fold increase in SOD activity in the ischemic cortex compared with the contralateral side in both groups by ANOVA and Student t test (*P ⬍ 0.05). (C) SOD activity increases significantly (ⱖ 3-fold) between 2 h and 24 h in the ischemic cortex of both temperature groups (*P ⬍ 0.0001). (D) A significant increase in enzyme activity is observed at 24 h in the ischemic striatum of both temperature groups (**P ⱕ 0.0002) compared with the respective 2-h group. At 2 h postinsult, hypothermia-treated animals show a reduction in SOD activity compared with normothermic animals at this time point (*P ⬍ 0.05). There is a similar trend at 24 h (P ⫽ 0.07). Data are expressed as mean ⫾ SEMs.

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delayed neuronal damage at 7 days postinsult, 20 min caused extensive hippocampal degeneration. Interestingly, the distribution of activated microglia was restricted mostly to the selectively vulnerable CA1 region. Additional evidence linking hypothermia and highly reactive free radicals comes from studies on endogenous antioxidants in brain tissue. After 3 h of transient focal cerebral ischemia, Karibe and colleagues (Karibe et al., 1994) showed that intraischemic mild hypothermia suppresses the reduction of cortical tissue concentrations of the endogenous antioxidants (ascorbate and glutathione) detected 3 h into reperfusion. Based on the observation that expression of free radical scavengers such as SOD increases following an ischemic insult (Liu et al., 1993; Matsuyama et al., 1993), Fukuhara et al. (1994) examined the induction of the cytosolic isoform of SOD (Cu/Zn-SOD or SOD1) in rats 6 h after cerebral contusion under hypothermia. Results showed an increase in SOD1 mRNA in the periphery of the contusion and decreased brain edema in hypothermic animals. Whether mild hypothermia affects the translation of SOD1 mRNA into protein is not known. The regional and subcellular localization of SOD and the temporal profile of its expression under hypothermia are also unclear. SOD1 is a constitutively expressed cytosolic antioxidant isoenzyme which plays a protective role mainly during reperfusion following ischemia (Chan et al., 1993). SOD2 on the other hand, is primarily located in mitochondria which have been shown to be both the sites of O 2•⫺ production and the target of free radical attacks (Murakami et al., 1998). The present study shows that Cu/ZnSOD is constitutively and uniformly expressed in the brain. MnSOD, on the other hand, is inducible after an ischemic insult and immunoreactivity to SOD2 varies according to brain area and cell-type being examined. SOD2 expression is primarily neuronal and can be observed in the peri-infarct areas as early as 2 h following ischemic onset under conditions of normothermia and hypothermia. By 7 days postinsult, when the inflammatory response is still in progress, we also find SOD2-positive reactive astrocytes and activated microglia. At this point, SOD2 immunoreactivity may be a result of superoxide production not only by the glial cells, but also by macrophages which are present in the infarcted tissue (Maier et al., 2001). Our results are consistent with work by Kato and colleagues (Kato et al., 1995) who showed an accumulation of reactive glial cells with intense SOD2 immunoreactivity in the

gerbil hippocampus at 7 days following global ischemia. It is remarkable that SOD2 expression is still prevalent at 2 months postinsult. While the significance of this finding is not clear, one can speculate that SOD2 is being expressed in response to superoxide production by reactive astroglia during the process of gliosis, a characteristic stage of chronic stroke (Jabs et al., 1999). The fact that the SOD2 immunoreactivity is bilateral is not entirely surprising and has also been observed following focal cortical thrombotic lesions in rats (Bidmon et al., 1998). In that study, however, SOD2 immunoreactivity increased in the entire ipsilateral and contralateral cortex up to day 7, but decreased globally thereafter. Our results also suggest that SOD2 expression is more dependent on the degree of ischemic damage than on temperature manipulations. We find no obvious differences in SOD2 expression between the two temperature groups at any of the time points studied. This may simply reflect a technical problem with the detection of very high levels of SOD2 in the tissue. In this case small alterations in SOD2 protein levels would not be detectable. Another possibility is that SOD2 expression remains unaltered by transient temperature manipulations, but enzyme activity is affected. Using a rat brain decapitation model, Chan et al. (1988) showed that SOD1 activities were significantly decreased in cerebral cortex and hippocampus at 30 and 60 min of ischemia, whereas the enzyme activities were decreased at 60 min in cerebellum and caudate areas. The reduction of SOD2 activities followed the same pattern of SOD1 in various brain regions. However, glutathione peroxidase activities in these brain regions were not affected. This suggests that enzymatic activity may be differentially altered in various brain regions and independent of protein expression patterns. In the current study we found that SOD activity was nearly identical in the non-infarcted tissue of both normothermic and hypothermic animals at 2 and 24 h postinsult. There were no significant differences in enzyme activity between temperature groups in the ischemic cortex; however, SOD activity was slightly reduced in the ischemic striatum of hypothermia-treated animals at 2 h with a similar trend at 24 h postinsult. The robust increase in SOD activity at 24 h relative to the 2-h time point suggests that oxidative processes play a very significant role in the development of the infarct. Neutrophils are a significant source of ROS. These cells begin to infiltrate the infarcted tissue 6 –24 h after ischemia, followed by a massive invasion of monocytes at 2–3 days postinsult ©

2002 Elsevier Science (USA) All rights reserved.

40 (Kochanek & Hallenbeck, 1992). A decrease in neutrophil infiltration in hypothermia-treated animals (Maier et al., 2001) may induce a less robust antioxidant response in these animals and could, therefore, explain the small decrease in striatal SOD activity compared with normothermic animals. While it is generally accepted that disturbances in oxidant-antioxidant balance play a significant part in rendering tissue more vulnerable to free radical induced injuries, the exact role of ROS in ischemic injury is still not clear. Recent studies with transgenic (Tg) and knockout (KO) mutant rodents have provided greater insight on the role of specific oxidants and antioxidants in cerebral ischemia. Chan and colleagues have demonstrated that ischemic infarction is significantly reduced at 3 or 24 h in mice overexpressing SOD1 compared with wild-type littermates after transient focal cerebral ischemia (Yang et al., 1994; Kamii et al., 1996). However, these animals are not protected against permanent MCA occlusion, lending further support to the notion that oxidative stress plays a role in reperfusion injury. Transgenic mice overexpressing human SOD2 show reduced membrane lipid peroxidation and neuronal death 24 h after transient focal cerebral ischemia (Keller et al., 1998). In a similar paradigm, KO mice with targeted-disruption of sod1 (Kondo et al., 1997) or sod2 (Murakami et al., 1998) are more susceptible to ischemic injury compared with wild-type animals. Therefore, these animals provide a unique opportunity to test the efficacy of mild hypothermia and at the same time determine whether SOD2 expression is critical for the neuroprotection afforded. Indeed, we have recently shown that mild hypothermia-treated SOD2-KO mice have a significant reduction in infarct size at 3 days post MCAO compared with normothermic SOD2-KO mice (Maier et al., 2001). In conclusion, while mild intraischemic hypothermia does not appear to alter the expression of either SOD1 or SOD2, it does reduce the levels of superoxide produced during the first hour of reperfusion in this model. This work lends further support for the use of mild hypothermia in the treatment and prevention of stroke and to the notion that mild hypothermia exerts its protective effects, in part, by attenuating free radical mediated damage.

ACKNOWLEDGMENTS We thank Beth Houle for histological figures preparation, David Kunis and David Onley for technical assistance, Dr. Ray Sobel and

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Maier et al. Dr. Brent Harris (Department of Pathology, Stanford University) for their expert advice. This research was supported by AHA Western States Affiliate Predoctoral Fellowship 98-405 (C.M.M.), by NIHNINDS Grant RO1 NS27292 and PO1 NS37520 (G.K.S.), by AHA Beginning Grant in Aid 0060081Y (M.A.Y.), by NIH-NINDS Grant RO1 NS25372 (P.H.C.), and by funding provided by Bernard and Ronni Lacroute (G.K.S.).

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