The FASEB Journal Research Communication

Nanoparticle-mediated delivery of superoxide dismutase to the brain: an effective strategy to reduce ischemia-reperfusion injury
Maram K. Reddy* and Vinod Labhasetwar*,,1
*Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA; and Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska, USA
ABSTRACT

Excessive production of reactive oxygen species (ROS) after cerebral ischemia and reperfusion is implicated in brain damage through different cellular and molecular mechanisms, and it is further aggravated by impaired cellular antioxidant defense systems under ischemic conditions. Therapeutic strategies based on exogenous delivery of the native form of superoxide dismutase (SOD), a free radical scavenger, are limited because of its short half-life (6 min) in vivo and poor permeability across the blood-brain-barrier (BBB). We encapsulated SOD in biodegradable poly(D,L-lactide co-glycolide) nanoparticles (SOD-NPs) and tested their efcacy in a rat focal cerebral ischemia-reperfusion injury model. We hypothesized that localized brain delivery of SOD-NPs would sustain the protective effect of SOD by neutralizing the deleterious effects of ROS formed following ischemia-reperfusion. SOD-NPs were administered at the time of reperfusion via the intracarotid route to maximize their localization in the brain. Animals receiving SOD-NPs (10,000 U of SOD/ kg) demonstrated a 65% reduction in infarct volume, whereas an equivalent dose of SOD in solution (SODSol) increased it by 25% over saline control (P<0.001; data at 6 h following reperfusion). Control NPs alone or mixed with SOD-Sol were ineffective in reducing infract volume, with results similar to saline control, indicating the protective effect of the encapsulated enzyme. SOD-NPs maintained BBB integrity, thereby preventing edema, reduced the level of ROS formed following reperfusion, and protected neurons from undergoing apoptosis. Animals treated with SOD-NPs demonstrated greater survival than those with saline control (75% vs. 0% at 28 days) and later regained most vital neurological functions. SOD-NPs may be an effective treatment option in conjunction with a thrombolytic agent for stroke patients.Reddy, M. K., Labhasetwar, V. Nanoparticle-mediated delivery of superoxide dismutase to the brain: an effective strategy to reduce ischemia-reperfusion injury. FASEB J. 23, 1384 1395 (2009) Key Words: antioxidant enzymes blood-brain barrier biodegradable polymers sustained release nanomedicine

Thromboembolic stroke is common among acute ischemic strokes and is the major cause of death and disability in the elderly (1). In the management of such strokes, the rst line of treatment is to limit the ischemic insult by promoting early reperfusion (2). Although reperfusion of ischemic tissue improves clinical outcome in some patients, in others, it may exacerbate brain damage, and is attributed to cerebral ischemiareperfusion injury (3). Reperfusion with thrombolytic agents like tissue-type plasminogen activator (t-PA) is effective within 3 h of the onset of ischemic symptoms, but t-PA cannot be administered to most stroke patients because of the risk of brain hemorrhage and edema formation due to the breakdown of the blood-brain barrier (BBB) (4). An alternative strategy is needed, one that can overcome the limitations of current therapy, particularly by extending the window of intervention and minimizing the damage caused by reperfusion injury. Reactive oxygen species (ROS) are generated soon after a vessel occlusion is cleared, as well as at later stages of ischemic reperfusion due to the inammatory process, and are the main mediators of reperfusion injury (3). ROS are usually scavenged by antioxidant enzymes, primarily by superoxide dismutase (SOD), by catalyzing the dismutation reaction of the superoxide anion to hydrogen peroxide. Catalase and glutathione peroxidase, on the other hand, protect cells from the toxic effects of hydrogen peroxide by catalyzing its decomposition into water (5). Under normal conditions, there is a balance between free-radical generation and free-radical scavenging; however, under ischemic conditions, the imbalance between the two processes results in oxidative stress and contributes to several pathophysiological conditions. During reperfusion, oxidative stress causes damage as a result of the excess production of ROS and insufciency of the free-radical scavenging process. This insufciency occurs from overconsumption of antioxidant enzymes
Correspondence: Department of Biomedical Engineering/ND-20, Cleveland Clinic, 9500 Euclid Ave., Cleveland, OH 44195, USA. E-mail: labhasv@ccf.org doi: 10.1096/fj.08-116947
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and their rapid degradation, as well as failure to adequately replenish antioxidants in ischemic brain tissue (6). It is known that, during reperfusion, activated leukocytes adhere to endothelial cells lining the brains blood vessels, then extravasate across the BBB and inltrate brain parenchyma. These inltrating leukocytes contribute to further generation of ROS in addition to triggering the cascade of inammatory events, which eventually results in deterioration of the salvageable penumbra (3). The strategy of exogenous delivery of the native form of SOD to neutralize the deleterious effect of ROS has been ineffective because SOD has a short half-life (6 min) in circulation and poor permeability across the BBB (7). Different alternatives have been investigated to address these issues, for instance, pegylation and lecithinization of SOD to improve its stability in circulation (8) and fusion of SOD with cell membranepenetrating peptides like transactivator of transcription-peptide or tetanus toxin fragment to increase its ability to cross the BBB (9). However, there are limitations to these modications. For example, although pegylated SOD (PEG-SOD) increases the enzymes stability in circulation from 6 min to 36 h, it limits the permeability of SOD across cerebral cell membranes (10). Similarly, fusion of different cell-penetrating or cell-specic peptides to target proteins requires a crosslinker via a chemical process that could potentially cause denaturation and loss of activity of the target protein (11). There is also a concern regarding possible immune-mediated anaphylactic responses to hybrid proteins given to patients (12). Intravenous delivery of SOD loaded into liposomes has shown partial inhibition of infarct volume, but the instability of liposomes in vivo (half-life 4.2 h) limits the duration of SOD activity and hence its efcacy (13, 14). In the present study, we formulated SOD encapsulated in sustained-release biodegradable poly(d,l-lactide co-glycolide) (PLGA) nanoparticles (SOD-NPs) and examined their ability to inhibit reperfusion injury in a rat transient focal cerebral ischemia-reperfusion injury model. We hypothesized that encapsulating SOD in NPs would protect it from degradation and that localized brain delivery of SOD-NPs would provide the sustained protective effect of the enzyme by neutralizing the deleterious effect of ROS. Animals treated with SOD-NPs demonstrated greater survival than saline control and regained most neurological functions on recovery.

6-Coumarin was obtained from Polysciences, Inc. (Warrington, PA, USA). Formulation of nanoparticles SOD-NPs were formulated using a multiple emulsion solvent evaporation technique, as described in our previous study (15). In a typical formulation procedure, 81 mg PLGA polymer and 9 mg DMT were dissolved in 3 ml chloroform; separately, an aqueous solution of proteins was prepared by mixing 12 mg SOD (50,000 U) and 18 mg RSA in 300 l double-distilled water. NPs with a higher dose of SOD were prepared by using 24 mg SOD and 6 mg RSA. The aqueous protein solution was emulsied into the polymer solution by vortexing for 1 min, followed by sonication using a microtip probe sonicator (XL 2015 Sonicator Ultrasonic Processor; Misonix Inc., Farmingdale, NY, USA) for 2 min in an ice bath at 55 W of energy output to form an oil-in-water emulsion. RSA was used in the present formulation to stabilize the encapsulated enzyme from interfacial inactivation, and DMT was used to facilitate the release of encapsulated enzyme from NPs (16). The oil-in-water emulsion was further emulsied into an aqueous solution of 2% w/v PVA by rst vortexing followed by sonication, as described above. The emulsion was stirred overnight on a magnetic stir plate at room temperature, followed by stirring for 1 h in a vacuum desiccator placed on a magnetic stir plate to allow for complete removal of the organic solvent. NPs thus formed were recovered by ultracentrifugation at 30,000 rpm for 20 min at 4C (50 Ti Rotor, Beckman Optima LE-80K; Beckman Instruments, Palo Alto, CA, USA) and washed twice with water to remove PVA and unencapsulated proteins by resuspending NPs each time in water followed by ultracentrifugation. The supernatant and the washings were collected and analyzed for the amount of SOD that was not encapsulated in NPs using the enzyme activity assay described below. The nal suspension of NPs was prepared in 10 ml of water, and aliquots were lyophilized in sterile tubes for 2 days (Freeze Dryer; VirTis Company, Gardiner, NY, USA). An identical protocol was used to prepare control NPs with RSA alone (weight30 mg). To prepare NPs loaded with HRP protein, the procedure was identical to that used for SOD-NPs except that SOD was replaced with the same weight of HRP protein (12 mg). Dye-loaded NPs were prepared similarly to control NPs (with RSA only) except that 50 g of 6-coumarin dye (0.5 mg/ml in chloroform), which acts as a marker for NPs, was added to the polymer solution prior to emulsication (17). Characterization of SOD-NPs NPs were characterized for size using both transmission electron microscopy (TEM) and dynamic light scattering (DLS), zeta potential, and in vitro release of the encapsulated SOD (15). The samples collected from the release study were assayed for SOD enzyme activity using a kit from Dojindo Molecular Technologies, Inc. (Gaithersburg, MD, USA) and as described in our previous study (15). In brief, A 20-l aliquot of each sample (n3) was added to a 96-well plate, followed by the assay reagents, which was then incubated at 37C for 20 min, and the absorbance was measured at 450 nm using a microplate reader (BT 2000 Microkinetics, Bio-Tek Instruments, Inc., Winooski, VT, USA). The standard plot (0.1 to 200 U/ml SOD) was prepared by diluting SOD in dilution buffer provided by the manufacturer, and the linear concentration range was determined. If necessary, the samples were diluted so that the readings were within the linear range of the standard curve. The supernatant and washings from the NP formulation protocol were also assayed for the
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MATERIALS AND METHODS

Materials PLGA (inherent viscosity, 1.32 dl/g, copolymer ratio 50:50) was purchased from Lactel (Cupertino, CA, USA). Polyvinyl alcohol (PVA; average MW 30,000 70,000), CuZn-SOD from bovine erythrocytes, rat serum albumin (RSA), horseradish peroxidase (HRP), Evans blue dye, poly-l-lysine (PLL), trichloroacetic acid, and dimethyl tartaric acid (DMT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
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SOD activity using the same protocol. The encapsulation efciency of SOD in NPs was determined from the difference in the total SOD added in the formulation and that which did not get encapsulated. In our previous study, we have demonstrated that the protein loading in NPs determined using the indirect method as described above correlates well with the amount of the protein released from NPs in vitro than that determined by direct extraction of protein in an aqueous phase from the NPs dissolved in an organic solvent. This occurs as NPs do not completely dissolve in organic solvents due to the PVA associated with them at the interface, which acts as a barrier in dissolution of NPs in an organic solvent (18). To determine the binding of SOD to control NPs, a suspension of control NPs mixed with SOD-Sol was centrifuged at 30,000 rpm for 20 min at 4C (50 Ti Rotor, Beckman Optima LE-80K), and the supernatant was again centrifuged at 14,000 rpm using a microcentrifuge (Eppendorf Microcentrifuge, model 5417; Fisher Scientic, Pittsburgh, PA, USA) in Eppendorf tubes at 4C for 20 min to ensure complete removal of NPs. The supernatant was analyzed for the protein content using Pierce 660-nm Protein Assay Kit (Thermo Fisher Scientic Inc., Waltham, MA, USA) with supernatant from the suspension of control NPs (without SOD) as blank (n5). The color intensities were measured at 660 nm using a Vmax Kinetic Microplate Reader (Molecular Devices Corp., Sunnyvale, CA, USA). Focal cerebral ischemia-reperfusion injury model Male Sprague-Dawley rats (250 300 g) were purchased from Charles River Laboratories (Wilmington, MA, USA). All animal procedures were approved by the Institutional Animal Care and Use Committees of the Cleveland Clinic and the University of Nebraska Medical Center. Animals were anesthetized via intraperitoneal administration of ketamine (80 mg/kg) and xylazine (10 mg/kg) followed by maintenance of anesthesia with 1.5% isourane in 70% nitrous oxide and 30% oxygen. The left femoral artery was cannulated using polyethylene tubing (PE-50) to monitor blood pressure and measure arterial blood gases. Transient focal cerebral ischemia was accomplished by middle cerebral artery occlusion, as reported previously (19). Under a surgical microscope (Leica MZ6; Diagnostic Instruments, Sterling Heights, MI, USA), a midline incision was made in the neck, and the left common carotid and external carotid arteries were carefully exposed and dissected from adhering tissue. A 4-0 monolament nylon suture (Ethicon, Johnson & Johnson, Somerville, NJ, USA) was inserted at the bifurcation of the external into the internal carotid artery. The suture tip was rounded by heating and then coated with a 1% PLL solution in water before insertion. PLL coating facilitates adhesion of the suture to the vessels endothelial lining, resulting in improved, more consistent occlusion of the blood vessel. The suture was advanced until light resistance was felt; it was then secured in place with a ligature. Blood vessel occlusion was further conrmed by measuring cerebral blood ow using a Doppler blood owmeter (ADInstruments Inc., Colorado Springs, CO, USA). To determine cerebral blood ow, animals were placed in the supine position, with the head rmly immobilized in a stereotactic frame (Model 900 Small Animal Stereotactic; David Kopf Instruments, Tujunga, CA, USA). A burr hole (1.5 mm diameter) was drilled into the skull using a surgical bone drill system (Microtorque II; Harvard Apparatus, Holliston, MA, USA) at 5 6 mm lateral and 12 mm posterior to the bregma, without injury to the dura mater. The laser Doppler ow probe (Standard Pencil Probe, MNP 100XP; ADInstruments, Inc.) was carefully positioned at the craniectomy site using a three-way micromanipulator (Narishige International,
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Inc., East Meadow, NY, USA). The above procedure was carried out prior to blood vessel occlusion to induce ischemia. Cerebral blood ow was continuously monitored (2-Hz sampling rate) from before the onset of ischemia until 5 min after reperfusion. Middle cerebral artery occlusion was conrmed by reduction in the local cerebral blood ow from the baseline value. One hour after ischemia, the intraluminal suture was withdrawn to allow for reperfusion, which was conrmed by restoration of the local cerebral blood ow to baseline. The incision was sutured, and animals were allowed to recover, during which time their bodies were kept warm with a heating lamp. Blood pressure was monitored before, during, and after the release of occlusion (ADInstruments, Inc.). Arterial blood samples were collected from the femoral arterial catheter before, during, and immediately after the release of occlusion to determine levels of blood gases (arterial PO2, arterial PCO2 and pH) (ABL-500; Radiometer, Copenhagen, Denmark) and glucose (Accu-Chek, Roche Diagnostics, Indianapolis, IN, USA). Animals were euthanized using an overdose of pentobarbital (120 mg/kg). In some procedures, this dose of pentobarbital was used to induce deep anesthesia prior to transcardial perfusion with 150 ml of heparinized saline (10 U/ml). Lastly, in some of the remaining procedures, a xative solution (as specied in each protocol) was passed following perfusion with heparinized saline to x the brain tissue. The perfusion was carried out at a rate of 5 ml/min. Efciency of uptake of NPs by the brain via different routes of administration Because the efciency of NP uptake by the brain is critical to achieving a therapeutic dose of SOD, we rst determined the brain uptake of NPs via intravenous, intrajugular, and internal carotid arterial routes. The rationale behind using intrajugular and internal carotid arterial routes of administration was to minimize the exposure of NPs to the reticulo-endothelial system and to increase the circulating concentration of NPs in the cerebral vasculature, with the assumption that this method would result in greater accumulation of NPs in the brain than that could have been achieved via intravenous administration of the same dose of NPs. The general procedure for preparation of a suspension of NPs prior to its administration to animals involved reconstituting a desired dose of lyophilized NPs in 0.5 ml sterile saline, vortexing the sample briey, then sonicating it in a water bath sonicator (S140; L&R Ultrasonic, Kearny, NJ, USA) for 10 min. For experiments with control NPs mixed with SOD-Sol, a suspension of NPs was prepared as above, then mixed with the desired dose of SOD-Sol. Usually, a 50% extra dose was prepared to account for the dose retained in the catheter. Each suspension of NPs was prepared just prior to its administration. The respective blood vessel was cannulated with PE-10 tubing, and the suspension was infused at a rate of 100 l/min using an infusion pump (11 Plus Infusion Pump, Harvard Apparatus) at the time of reperfusion. An identical protocol was used for administration of SOD-Sol or saline in control groups. To determine the efciency of uptake of NPs into the brain, NPs loaded with 6-coumarin dye (35 mg/kg) were infused through the right jugular vein, the left external carotid artery, or the tail vein. For intravenous administration, the tail vein was rst cannulated with a 27-gauge needle, which was then connected to PE-10 tubing. Following 1 h of reperfusion, animals were transcardially perfused with heparinized saline. Immediately after death, the rat brains were collected, then homogenized using a Tissue-Tearor (Model 985370; Biospec Products, Inc., Bartlesville, OK, USA). Each
REDDY AND LABHASETWAR

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homogenate was lyophilized in a 20-ml scintillation vial for 2 days at 60C and 7-m Hg vacuum. After noting the tissue weight of the lyophilized samples, dye from the NPs was extracted by adding 10 ml methanol to each lyophilized sample of the entire brain tissue. Samples were kept on an orbital shaker rotating at 150 rpm (Environ Orbital Shaker; Barnstead International, Dubuque, IA, USA) and maintained at room temperature. After 48 h of shaking, 1.5 ml of methanol extract from each sample was collected into 1.5 ml Eppendorf tubes. Each sample was centrifuged at 14,000 rpm for 10 min in an Eppendorf microcentrifuge to remove tissue debris. The supernatant was analyzed for dye content using high-performance liquid chromatography, as described previously (17). A standard plot was prepared with known concentrations of the dye-loaded NPs (0 100 g) added to the brain homogenates of control (saline-injected) animals and processed as above. In a separate set of experiments to visualize localization of NPs, the procured brains were snap-frozen in dry ice and stored at 80C until taken for sectioning. Coronal brain sections (8-m thickness from 2 mm posterior of the frontal pole) were examined under a confocal microscope (Zeiss LSM 510; Carl Zeiss, Jena, Germany) using a uorescein isothiocyanate lter (Ex 495 nm, Em 520 nm). Although the set of experiments described above provided evidence for uptake of NPs into the brain, it was difcult to identify the distribution of NPs in different regions (e.g., cortex vs. striatum) against the dark background. Therefore, we carried out other experiments in which HRP-loaded NPs were injected in the same way as detailed above. The rationale for using HRP-NPs was that particles in the brain sections may be better visualized by treating the sections with a peroxidasereducing agent, diaminobenzidine (DAB), which gives a dark brown color. Brain sections were treated with DAB substrate (Peroxidase Substrate Kit; Vector Laboratories, Burlingame, CA) and viewed under a light microscope (Leica DMR; Leica Microsystems Inc. Bannockburn, IL, USA). Prior to the experiments with SOD-NPs, we determined the feasibility of delivering NPs via the intracarotid route and tested for signs of side effects. We tested two doses of control NPs (10 mg/kg and 35 mg/kg, n3 each) on normal animals (without induction of ischemia) and monitored them for 1 wk. We saw no signs of toxic effects from NPs, animals showed no apparent behavioral changes or mortality. Evaluation of the efcacy of SOD-NPs in inhibiting reperfusion injury Five groups were used to assess inhibition of reperfusion injury: SOD-NPs and controls, which were saline, control NPs, SOD-Sol (10,000 U/kg), and SOD-Sol mixed with control NPs. The dose of control NPs (dose35 mg/kg) was same as the dose of SOD-loaded NPs. Two doses of SOD (10,000 and 20,000 U/kg) encapsulated in NPs were tested. SOD-NPs prepared with low-dose SOD (1 mg SOD-NPs400 U SOD) and high-dose SOD (1 mg SOD-NPs800 U SOD) were used. These doses of SOD were based on a previous study using SOD-loaded liposomes (SOD dose25,000 U/kg) that demonstrated a partial inhibition of infarction in a rat ischemiareperfusion model (18 33% inhibition at 24 h following reperfusion) (13). Doses of SOD-NPs or controls were administered via the intracarotid route at the time of reperfusion following 1 h of ischemia, as described above. In an acute study, animals were euthanized at 6 h following reperfusion, whereas in chronic studies, animals were euthanized at different times (7, 14, or 28 days) following reperfusion. Animals that did not show reduction in cerebral blood ow by 70% from baseline during surgical occlusion were excluded from the study. Animals that showed a clot deposit
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at the base of the brain, arising from hemorrhage caused by inadvertent vessel perforation during intraluminal suture advancement, were also excluded from the study because this error would have compromised the pathophysiological relevance of the ischemia-reperfusion injury model (20). Typically, animals with subarachnoid hemorrhage died within 24 h following reperfusion. Quantication of infarct lesions The brains harvested at different times after reperfusion with heparinized saline were immersed in cold saline for 10 min, then sectioned coronally into six 2-mm-thick slices (from rostral to caudal, rst to sixth slice) using a Brain Matrix (Electron Microscopy Sciences, Hateld, PA, USA). Brain slices were incubated in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma) in PBS, pH 7.4. The sections in TTC solution were incubated in a water bath maintained at 37C for 1520 min, then transferred to a 10% phosphatebuffered formalin solution (Sigma). TTC-stained brain sections were scanned using a atbed scanner (HP Scanjet 3970; Hewlett Packard, Palo Alto, CA, USA) at 600 dpi resolution. Areas of infarct lesion were measured using ImageJ Analysis Software (http://rsb.info.nih.gov/ij/). Measurements were performed by manually outlining the margins of the infarcted areas. Unstained areas of brain sections were dened as infarcted. To minimize artifacts associated with postischemic edema caused by infarction, infarct area and volume were indirectly calculated. Briey, the infarcted area on the ipsilateral (ischemic) side was indirectly measured by subtracting the noninfarcted area in the ipsilateral hemisphere from the total nonischemic area of the contralateral (nonischemic) hemisphere. Hemispheric infarcted areas were calculated separately on each coronal slice from 1 to 6; each such area was dened as a percentage of the affected hemisphere (21). Neurological assessment Neurological behavior was evaluated using a modied neurological severity score at different times after initiation of reperfusion (6 h or 7, 14, or 28 days). The behavior score was derived by evaluating each animal for the following tasks: motor activity like exion response of forelimb and hind limb and ability to hold the head in vertical axis, while raising the rat by tail. Rats were also evaluated for the ability to walk straight, ability to bear weight on the paretic side, circling movements, shaking, and seizures. Similarly sensory responses like visual and tactile response, deep sensory response by pinching toes, and other reex responses like pinna reex by touching the auditory meatus, eye blink in response to touching the cornea with a cotton applicator, and sensitivity to a brief and sudden loud noise by knocking rat cage (22). To assess the extent of injury, one point was counted for inability to correctly perform any single motor task or if animals exhibit abnormal behavior like circling movement, shaking and seizures or show lack of any reex response. Severe injury was reected by scores of 10 14, moderate 59, and mild 1 4. Evans blue leakage Breakdown of the BBB was assessed by the Evans blue exudation technique. The dye solution (4% w/v in saline) was injected (2.5 ml/kg) through the tail vein before reperfusion. After 6 h of reperfusion, animals were transcardially perfused with heparinized saline before harvesting their brains. Digital photographs (Nikon D200, 7.1 megapixels; Nikon, Osaka,
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Japan) of the brains were taken prior to dividing them into right and left hemispheres. Each hemisphere was homogenized in 1 ml of 50% trichloroacetic acid in distilled water, then centrifuged at 14,000 rpm (Eppendorf Microcentrifuge, model 5417R) for 20 min at 4C (23). Absorbance of the supernatant from each homogenate was measured at 610 nm using a spectrophotometry (UV-1601PC, UV-Visible Spectrophotometer; Shimadzu Scientic Instruments, Inc., Columbia, MD, USA). The tissue content of Evans blue was quantied from a standard curve derived from known amounts (0 5 g/ml) of the dye prepared in the same solvent. Cerebral edema Following 6 h of reperfusion, animals were transcardially perfused with heparinized saline, and the brains were quickly removed and dissected through the interhemispheric ssure into ischemic and nonischemic hemispheres; their weights were noted and then immediately dried in an oven at 100C for 48 h. The percentage of hemispheric water content was calculated using the formula [(wet weight dry weight)/wet weight] 100 (24). Apoptosis After 6 h of reperfusion, animals were transcardially perfused with heparinized saline followed by 4% paraformaldehyde solution in PBS (100 ml). The harvested brains were stored in the same xative for 24 h, and then transferred to a 20% aqueous sucrose solution. After overnight storage in that solution, the brains were removed from the solution and stored at 80C until taken for sectioning. Sections of 5-m thickness were prepared and TUNEL reactivity was measured using the Apop Tag Plus Fluorescein in situ Apoptosis Detection Kit (Chemicon International, Temecula, CA, USA). TUNEL-positive cells per eld were counted and expressed as the average from six randomly selected areas in the ischemic penumbra at the level of the bregma. For detection of apoptosis of neurons using electron microscopy, animals were transcardially perfused with heparinized saline followed with 4% paraformaldehyde solution (100 ml), then 1% glutaraldehyde solution in PBS (100 ml). Brains were isolated and cut into 2-mm-thick coronal sections. These sections were incubated in the same xative solution overnight at room temperature. Under a dissecting microscope (EMZ 8TR, Meizo Techno; Sheerin Scientic, Shawnee, KS, USA), brain penumbral regions were identied, and tissue blocks were prepared using Unicryl embedding resin (Ted Pella, Redding, CA, USA). These tissue blocks were postxed in 0.5% osmium tetroxide (Electron Microscopy Sciences) for 2 h at 4C. Ultrathin sections (80 100 nm in thickness) were made from the above-processed tissue blocks on a Reichert microtome (Leica) using a diamond knife. Completed sections were stained with 2% uranyl acetate and imaged with a Philips 201 transmission electron microscope (Philips, Amsterdam, The Netherlands), as described above. Detection of ROS ROS in brain sections were determined using 6-carboxy-2,7dichlorodihydrouorescein diacetate (DCDHF-DA; Molecular Probes, Invitrogen Corp., Carlsbad, CA, USA). DCDHF-DA is an ester that is freely permeable across the cell membrane. On entering the cells, it loses its diacetate group by esterase action, thereby becoming a substrate for oxidation by ROS and producing the highly uorescent intracellular product DCF. Generation of ROS was visualized in nonischemic vs. ischemic brain hemispheres of controls and animals treated
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with SOD-NPs. For this experiment, following 6 h of reperfusion, animals were perfused with heparinized saline followed by 4% paraformaldehyde. Brain sections of 15-m thickness were prepared, washed 3 times with PBS, and incubated with DCDHF-DA solution for 30 min at 37C, then washed twice with PBS before viewing under a uorescence microscope (Ex 495 nm, Em520 nm; Leica DMR). Statistical analysis Statistical analysis was performed using Minitab 5 software (Minitab Inc., State College, PA, USA). All values were expressed as means se. Differences in infarct areas among different groups were analyzed by using one-way analysis of variance followed by Tukeys post hoc test. All other parameters among the groups were analyzed using an unpaired Students t test. Differences with a probability value of P 0.05 were considered to be signicant.

RESULTS Characterization of NPs The mean size of SOD-NPs as measured using TEM was 81 4 nm, and the particles were mostly spherical in shape (Fig. 1A). However, the mean hydrodynamic diameter of NPs as measured by DLS was 291 nm (polydispersity index0.12) (Fig. 1B). Hydration of the multilayered PVA, estimated to be 10,000 molecules/NP (25), that remains associated with NPs at the interface despite repeated washing has been determined in our previous study to be responsible for the discrepancies in size between the TEM and DLS measurements (26). The zeta potential of SOD-NPs was 24.5 1.8 mV. The other formulations of NPs (dye-loaded, control, or HRPloaded) demonstrated almost identical physical properties as SOD-NPs. The encapsulation efciency of SOD in NPs was 75 3.6% (n3); i.e., 75% of the added protein was entrapped into NPs. Thus, each milligram of NPs contained 90 g SOD (equivalent to 400 U). Assuming the same encapsulation efciency, the dose of SOD present in each milligram of high-dose SODNPs was estimated to be 800 U. The release of SOD from NPs was sustained, with 8.2 1.6% of release in 24 h, 26.8 1.5% in 1 wk, 41.5 3.2% in 4 wk, and 78 9.3% in 3 mo (Fig. 1C). In the experiment in which control NPs were mixed with SOD solution, it was found that only 1.6% of the added SOD binds to NPs. Localization of NPs in the brain via different routes of administration Administration of NPs via the internal carotid artery resulted in a 13-fold greater uptake of NPs in the brain compared with that via either tail or intrajugular veins (Fig. 2A). The uptake was 0.18 mg NP/brain via carotid artery, which was 0.013 and 0.011 mg, respectively, via intrajugular and intravenous routes. The greater efcacy of uptake of NPs via intracarotid arterial adminisREDDY AND LABHASETWAR

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B
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Figure 1. Characterization of SOD-NPs. A) Transmission electron micrograph of SOD-NPs. Scale bar 100 nm. B) Particle size analysis by dynamic light scattering (DLS). C) Release of SOD from NPs under in vitro conditions in double diffusion chambers. SOD released from NPs was quantitated by measuring the enzyme activity. Data are means se; n 3.

tration was further conrmed by uorescence microscopic pictures of the brain sections. More uorescent particles were found within the brain parenchyma of the ischemic (ipsilateral) vs. nonischemic (contralateral) hemisphere (Fig. 2B). Studies with HRP-loaded NPs conrmed that NPs had localized primarily in the cortex and striatal regions of the ischemic hemisphere (Fig. 2C).

Cerebral infarction and neurological decit There was no change in the blood gases (PO2 and PCO2) or pH level at 10 min before, during, or after reperfusion. The group treated with SOD-NPs (10,000 U/kg) demonstrated a decrease in the infarct lesion compared with the saline control group. The total calculated infarcted volume of the ischemic hemi-

Figure 2. Localization of NPs in the brain via different routes in rat ischemia-reperfusion model. A suspension of NPs was administered after 1 h of ischemia and at the time of reperfusion. NP levels or their localization in the brain were determined 1 h after NP administration. A) Relative efcacy of uptake of NPs via intravenous (IV), intracarotid (IC), and intrajugular (IJ) routes. Data are means se; n 3. *P 0.005. B) Representative uorescence photomicrographs of brain sections (left: nonischemic hemisphere; right: ischemic hemisphere). Ischemic side of the brain shows more uorescence of the 6-coumarin dye-loaded NPs scattered in the brain parenchyma than the nonischemic side. Scale bars 100 m. C) Representative brain sections (left: nonischemic hemisphere; right: ischemic hemisphere) after intra-arterial injection of HRP-loaded NPs. Enlarged portion (20) of ischemic side shows more particles (DAB staining) scattered in the brain parenchyma (indicated by arrows) than in nonischemic hemisphere.
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sphere in animals treated with SOD-NPs measured 6 h after reperfusion was reduced by 65% compared with that in saline control rats (Table 1). Similarly, the infarct lesion area in each coronal slice of brain was signicantly less in the group treated with SOD-NPs than in saline controls (Fig. 3A, B). Interestingly, the animals that received SOD-Sol (10,000 U/kg) showed a 25% higher infarct volume than saline controls (Fig. 3A, B, Table 1). Neurological decit scores in animals treated with SOD-NPs were signicantly lower than those in saline controls or treated with SOD-Sol (Fig. 3C). There was no signicant difference in infarct volume between the two doses of SOD (10,000 and 20,000 U/kg) loaded into NPs (202.5%, n6 vs. 253.5%, n3; P0.26). We tested the effect of control NPs, control NPs mixed with SOD-Sol (10,000 U/kg), and the SOD dose that is released from NPs over 6 h (SOD dose4 U) on the infarct lesion to ensure that the protective effect is, in fact, due to the encapsulated SOD. This dose of SOD released at 6 h was calculated from the efciency of localization of NPs in the brain (1.23%), SOD-loading in NPs, and the amount of SOD released in 6 h. We observed no protective effect of SOD-Sol at this dose. However, with an equivalent dose of control NPs (loaded with RSA alone), we found a marginal reduction in the infarct lesion as compared with saline controls, but it was not signicant. Control NPs mixed with SOD-Sol also did not improve the outcome, and the results were similar to saline control (Table 1). Animals treated with SOD-NPs demonstrated reduction in infarct lesions with time (Fig. 4A) and improvement in neurological behavior as they recovered (Fig. 4B). About 25% of the group treated with SOD-NPs died within 48 h after ischemia-reperfusion (Fig. 4C), but those that survived continued to recover with time, as evident from their improved neurological scores (Fig. 4B). Evans blue leakage and cerebral edema Extravasation of Evans blue dye from the ischemic hemisphere was signicantly reduced in animals treated with SOD-NPs than in saline control or SOD-Sol groups (Fig. 5A, B). The SOD-Sol group showed slightly greater extravasation of Evans blue than saline control, but this difference was statistically insignicant (P0.6). Water content,

or edema, in the ischemic hemisphere was signicantly greater than that in the nonischemic hemisphere in saline control group (801 vs. 731.8%, n3; P0.05) (Fig. 5C). SOD-Sol and Saline control showed no signicant difference in edema formation. There was no signicant change in water content between ischemic and nonischemic brain hemispheres in the group treated with SODNPs (754 vs. 744%, n3). Neuronal apoptosis and ROS levels The number of TUNEL-positive cells in the group treated with SOD-NPs was signicantly fewer than in the saline control group (155 vs. 805 apoptotic cells/0.1 mm2, P0.05) (Fig. 6A). Examination of brain sections by electron microscopy further conrmed these results. Neurons from animals treated with SODNPs showed almost normal neuronal morphology, whereas neurons from the saline control group demonstrated characteristic features of apoptosis such as nuclear shrinkage, chromatin condensation, and loss of nuclear membrane (Fig. 6B). Brains of rats treated with SOD-NPs demonstrated a signicant reduction in the ROS activity compared to that in the brains of saline control animals (Fig. 6C).

DISCUSSION Our study demonstrated signicant reduction in cerebral infarct lesions following intracarotid administration of SOD-NPs in the rat cerebral ischemia-reperfusion model. The study also showed signicant reduction in the leakage of Evans blue to the brain, suggesting the protective effect of SOD-NPs in maintaining the BBB integrity, which also resulted in reduction of edema formation and inhibition of neuronal apoptosis. Further, SOD-NPs signicantly reduced ROS activity following reperfusion. The combined effects of SOD-NPs conferred better survival than controls, as well as signicant neurological recovery, which improved further with time. The efcacy of SOD-NPs could be the result of the protection of the encapsulated enzyme and its sustained release in active form within the brain. Changes in the antioxidant status of brain tissue during ischemia and reperfusion inuence the extent of postischemic brain damage. Endogenous SOD levels

TABLE 1. Efcacy of SOD-NPs in reducing infarct volume and neurological recovery of animals following 1 h of ischemia and 6 h of reperfusion
Group Number Dose of SOD (U/kg) Infarct volume (%) Neurological score

SOD-NPs SOD-NPs Saline control SOD-Sol Control NPs Control NPs SOD-Sol
*P 0.001 vs. saline control values.

6 3 6 3 4 5

10,000 20,000 0 10,000 0 10,000

20.1 2.5* 25 3.5* 56.3 2.5 75.4 1.8* 48.7 3.7 50.4 5.9

3.5 0.5* 3.7 1.2* 10.4 0.4 11 0.5 9.5 0.9 8.6 1.2

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Figure 3. Efcacy of SOD-NPs in reducing infarct volume and promoting neurological recovery of animals following ischemia-reperfusion. At the time of reperfusion following 1 h of ischemia, animals received via carotid artery either saline (control), SOD-Sol, or a suspension of SOD-NPs. Animals were euthanized 6 h following reperfusion. A) Representative coronal brain sections stained with TTC solution from animals treated with saline control, SOD-Sol, or SOD-NPs. Dark colored regions in the TTC-stained sections indicate nonischemic areas; pale-colored regions indicate ischemic portions of the brain. B) Bar graph showing quantication of ischemic lesion area from the above groups. Data are means se. *P 0.001 vs. saline control. C) Bar graph showing neurological severity score. Data are means se. *P 0.05 vs. saline control (saline control, n6; SOD-NPs, n6; SOD-Sol, n3).

in brain tissue are reduced during ischemia and remain below the baseline values until several days after reperfusion (27). Therefore, it is suggested that antioxidant supplementation with exogenous SOD over and above the baseline value may be effective in protecting the brain from ischemia-reperfusion injury. The protective effect of SOD-NPs could be due to the direct antioxidant effect of the active enzyme released from the NPs localized in the brain. Since the BBB integrity was maintained, the SOD-NPs localized in the endothelium of the cerebral vasculature could have protected it from ROS-induced damage. Previously, we studied the dynamics of transport of NPs across the brain endothelium and parenchyma in mice. The results demonstrated NP localization in the endothelium (28). NPs also showed localization in the brain parenchyma, as

evident from brain sections of animals in which HRPloaded NPs were infused (Fig. 2C). It is quite possible that increased permeability of the BBB during reperfusion following transient focal brain ischemia might have allowed SOD-NPs to cross the BBB to the brain parenchyma (3). The SOD-NPs localized in the brain may have protected the neurons, which are very susceptible to damage due to ROS because neurons are rich in oxidation-prone, polyunsaturated fatty acids (5). Intracarotid arterial administration of SOD-Sol at the dose of 10,000 U/kg, which is the same total dose of SOD administered in NPs, increased infarct volume by 25% over that of saline control (Table 1). This increase may have been caused by permeation of SOD through the leaky BBB, which could have aggravated the brain damage as a result of overproduction of

Figure 4. Recovery of animals following ischemia-reperfusion treated with SOD-NPs. A) Representative coronal brain sections stained with TTC at different time points following reperfusion (6 h, or 7, 14, or 28 days; n6 for each time point) after treatment with SOD-NPs. B) Bar graph showing neurological severity score. Control is saline control at 6 h following reperfusion. Data are means se. C) Survival of animals treated with SOD-NPs compared with saline control. All animals in saline control group died within 3 days, whereas 75% of animals treated with SOD-NPs not only survived but showed improvement in neurological recovery with time (*P0.06 at 28 days vs. 6 h).
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Figure 5. Protection of the BBB and inhibition of edema formation in animals treated with SOD-NPs in ischemia-reperfusion. A) Representative brain showing Evans blue leakage in saline control- and SOD-NP-treated rats at 6 h after reperfusion. B) Bar graph showing quantitative measurement of Evans blue in rats receiving saline (control) vs. those treated with SOD-Sol and SOD-NPs. Data are means se (n3/group). *P 0.05 vs. saline control. No signicant difference between SOD-Sol and saline control, P 0.6. C) Bar graph showing hemispheric (ischemic vs. nonischemic) water content in rats treated with saline control and rats treated with SOD-Sol and SOD-NPs at 6 h after reperfusion. Data are means se. *P 0.05 vs. nonischemic hemisphere (n3/group).

hydrogen peroxide by scavenging the superoxide radical produced during reperfusion, thus overwhelming the endogenous hydrogen peroxide scavengers (catalase and glutathione peroxidase) and exacerbating cerebral injury (29). This effect was partially inhibited when the same dose of SOD-Sol mixed with control NPs was used. Since we observed insignicant binding of the added SOD to NPs (1.6%), the reduced infarct volume in this treatment group compared to SOD-Sol alone could be due to the NPs immobilized in the leaky endothelium that could have partially inhibited the passage of the unbound SOD to the brain parenchyma, thus minimizing the damaging effect of excess SOD. There was no signicant difference in the extravasation of Evans blue with SOD-Sol as compared to saline control, suggesting that the excess SOD has no signicant effect on the integrity of the BBB and the damaging effect of it is limited to the brain parenchyma. This

difference could also be due to the sensitive nature of neurons to oxidative stress caused by hydrogen peroxide than the endothelial cells forming the BBB. Other investigators have shown that continuous intra-arterial infusion of SOD-Sol at the rate of 400 U/h for 3 h during reperfusion reduced reperfusion injury, although rapid loss of enzymatic activity (90%) was noted only 30 min after the infusion (12). It appears that continuous supplementation of a lower dose of SOD has a protective effect, whereas rapid administration of high doses has a detrimental effect. Therefore, the mechanism of efcacy of SOD-NPs could also be attributed to the sustained delivery of SOD into the brain, which could have neutralized the ROS formed over a period of time following reperfusion. The total localization of NPs via the intracarotid route was 0.18 mg/brain (average dry weight of brain 420 mg), which is equivalent to 1.23% efciency of localiza-

Figure 6. Inhibition of apoptosis in animals treated with SOD-NPs. A) Representative photomicrographs showing TUNEL staining in penumbral region of brain sections from saline control- and SOD-NP-treated rats at 6 h after reperfusion (n3). Scale bars 100 m. B) Representative electron micrographs of penumbral region of brain sections of sham saline control- and SOD-NP-treated rats at 6 h after reperfusion. Scale bars 500 nm. C) Representative photomicrograph of brain section from the area of ischemic penumbra at the level of the bregma, showing ROS levels (detectable as proportional to uorescence intensity) in control and SOD-NP-treated group. Scale bar 10 m.
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tion of NPs. On the basis of this efciency of uptake, the amount of SOD localized in the brain would be 74 U for the 10,000 U/kg dose of SOD administered in NPs. From the release prole of SOD from NPs, it can be estimated that 1 U/day SOD provides the protective effect in this model. We have tested two doses of SOD in NPs (10,000 and 20,000 U/kg), but there was no signicant difference in the infarct volume between the two groups. It would be interesting to study the effect at lower doses in a dose-response study to determine the optimal therapeutic dose of SOD-NPs. Further studies could involve optimizing the release rate and duration of release of the encapsulated SOD from NPs, and combining SOD with catalase in NPs, as catalase could neutralize the hydrogen peroxide formed as a result of the dismutation reaction of the superoxide anion (5). The optimized delivery of antioxidant enzymes in NPs could lead to rapid recovery and better survival than that seen with the current formulation. In our previous study, in vitro treatment of neurons with PEG-SOD (dose100 U/ml) showed no protective effect from hydrogen peroxide-induced oxidative stress, whereas SOD-NPs at an equivalent dose demonstrated complete protection (15). Despite its extended half-life in the circulation, PEG-SOD did not demonstrate a consistent protective effect in cerebral ischemia-reperfusion injury (10). He et al. (30) have reported a marginal reduction in infarct volume with PEG-SOD at the 10,000 U/kg SOD dose but no effect at lower or higher doses. The inefcacy of PEG-SOD in cerebral ischemia has been attributed to its difculty penetrating into cerebral cells (31). Our previous study has shown neuronal uptake of a model protein (uorescein isothiocyanate-labeled bovine serum albumin) encapsulated in NPs as early as 15 min following incubation in culture, and its uptake increased with incubation time. However, cells incubated with this model protein in solution did not exhibit its internalization and remained mostly adhered to the cell membrane (15). Thus, encapsulation of SOD in NPs could have facilitated the intracellular delivery of the enzyme following their localization in the brain and might have neutralized the effect of the ROS generated both in the intracellular and extracellular environments, resulting in signicant reduction in neuronal apoptosis (Fig. 6AC). A series of acute, subacute, and chronic events occur after the incidence of stroke and reperfusion (32), some of which could be the consequence of one leading to the others. For example, ROS-induced damage to cells from irreversible oxidation of cellular macromolecules (i.e., proteins, lipids, nucleic acids, etc.) could trigger cell-death pathways (33), as well as the recruitment of neutrophils and inltration of macrophages at the ischemic site. Neutrophils are detected as early as 5 h after stroke, but the recruitment of macrophages is delayed as late as 7 days after stroke (34), and they have been reported to remain at the lesion site over several months. In addition, several proinammatory cytokines (such as interleukin-1, tumor necrosis factor , and interferon-) display very
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different temporal proles, ranging from 6 h to 72 h after stroke (32). Therefore, it has been suggested that an ideal intervention in stroke should demonstrate a therapeutic effect in all the phases of illness (i.e., acute, subacute, and chronic) to maximize the clinical benet (35). It is possible that SOD-NPs prevented the cascade of events or acted at several stages in the pathophysiology of reperfusion injury. Breakdown of the BBB is an early event arising from free-radical damage, especially from superoxide radicals generated in endothelial cells during ischemia and reperfusion (36). SOD-NPs localized in the lining of the cerebral vasculature protected the BBB from free-radical injury, which then could have prevented the leukocyte adhesion that otherwise is a common event following reperfusion, as activated endothelial cells express intracellular adhesion molecules (37). We observed a signicant reduction in infarct volume at 6 h with SOD-NPs, suggesting that most of the ischemic penumbra, i.e., the hypoperfused tissue, was rescued because of the direct free-radical scavenging effect of SOD released from NPs that had localized in the brain. We also noticed that as the animals recovered, infarct volume was reduced, and it became almost undetectable at 28 days (Fig. 4A). It is possible that the infarcted tissue could have been cleared by phagocytes, an important process of brain repair after stroke (38), which is facilitated in the absence of oxidative stress (39). In addition, the conducive conditions created in the brain as a result of the sustained presence of SOD and its antiapoptotic and anti-inammatory mechanisms (40) could have played a role in facilitating the process of angiogenesis and neurogenesis in the infarcted brain. Although further studies are needed to completely understand the mechanism of their efcacy, we found that SOD-NPs are effective in achieving the overall goal of neurological recovery and increasing survival of animals (Fig. 4B, C). Rats treated with SOD-NPs in our study regained almost all vital neurological functions by 28 days, with the exception of slight impairment in forelimb movement and paresis of the eye muscles on the infarcted side. A marginal reduction in infarct lesion observed with control NPs at 6 h could possibly be an effect of NPs entrapped in the transiently opened endothelial junctions that may have delayed the BBB breakdown (Table 1). The major challenge in the treatment of cerebral ischemia is to extend the therapeutic window of intervention. The benecial effects of thrombolytic treatment are offset by reperfusion-mediated injury resulting from excessive production of ROS and impaired cellular antioxidant defenses. Therefore, it is thought that coadministration of SOD-NPs with a thrombolytic agent may reduce the reperfusion-induced injury associated with thrombolytic therapy because of the protective effect of SOD from ROS-induced damage to the BBB and its anti-inammatory and antiapoptotic actions (40). Many studies have suggested the benecial effect of combining neuroprotective therapy with clot-lysing agents to reduce reperfusion injury, as these agents
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cause additional oxidative stress (41). Plekhanova et al. (42) have shown that urokinase stimulates the production of superoxide radical in vascular smooth muscle cells, and Mu hl et al. (43) have reported augmentation of oxidative stress in patients with acute pulmonary embolism treated with different thrombolytics, including t-PA. These studies suggest that antioxidants such as SOD can play an important protective role in mitigating oxidative stress during thrombolysis. It has also been shown that t-PA promotes disruption of the microvascular basal lamina to cause BBB disruption (44). Because SOD-NPs arguably protect the BBB, their use could prevent the deleterious effects of t-PA by inhibiting its transport to the parenchyma. It would be advantageous if SOD-NPs could be administered prior to t-PA administration, so that the BBB is protected to minimize the passage of t-PA to the brain to prevent its neurotoxic effect. This is feasible with a recanalization technique that could allow a catheter to pass through the clot for NP administration (45). The damaging effects of ischemic-reperfusion injury can extend beyond the ischemic area because of systemic inammatory response in stroke condition (46). Because of the anti-inammatory effect of SOD, the dose of SOD-NPs distributed to other body compartments may provide protection to other tissues. Thus, SOD-NPs may demonstrate their protective efcacy in ischemia-reperfusion injury via different pathways. A similar therapeutic strategy can also be developed to rescue other organs, such as heart, kidney, and liver, from the ischemia-reperfusion-induced damage caused by ROS. Thus, SOD-NPs could be explored for therapeutic applications in several oxidative stress conditions.

2. 3.

4. 5. 6.

7. 8.

9.

10. 11.

12.

CONCLUSIONS Ours is the rst study that demonstrates the protective effect of SOD-encapsulated NPs in reducing cerebral injury and promoting neurological recovery in a rat cerebral ischemia-reperfusion model. The mechanism of efcacy appears to be due to sustained delivery of the encapsulated SOD, thus neutralizing the deleterious effect of ROS on BBB and neurons. The overall outcome was the animals in the treatment group not only demonstrated greater survival than untreated control but regained most vital neurological functions with time.
This work was supported by grants from the Cleveland Clinic and a Technology Advancement grant from the University of Nebraska Medical Center, Omaha, NE (to V.L.). We thank Ms. Melissa Jedlicka for proofreading the manuscript.

13.

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