Antioxidant, antiinflammatory and antiapoptotic ... - Wiley Online Library

Journal of Neuroscience Research 86:3410–3419 (2008)

Antioxidant, Antiinflammatory and Antiapoptotic Effects of Dapsone in a Model of Brain Ischemia/ Reperfusion in Rats Araceli Diaz-Ruiz,1 Carlos Zavala,2 Sergio Montes,1 Alma Ortiz-Plata,3 Hermelinda Salgado-Ceballos,4 Sandra Orozco-Suarez,4 Concepcioń Nava-Ruiz,3 Ivań Pe´rez-Neri,1 Francisca Perez-Severiano,1 and Camilo Rıós1* 1

Departamento de Neuroquı´mica, Instituto Nacional de Neurologıá y Neurocirugıá Manuel Velasco Suarez S.S.A., D.F. Me´xico, Me´xico 2 Neurocirugıá, Hospital Regional de Alta Especialidad del Bajıó, Leoń Guanajuato, Me´xico 3 Laboratorio de Neuropatologıá, Instituto Nacional de Neurologıá y Neurocirugıá Manuel Velasco Suarez S.S.A., D.F. Me´xico, Me´xico 4 Unidad de Investigacioń Me´dica en Enfermedades Neurolo´gicas, Hospital de Especialidades, Centro Me´dico Nacional Siglo XXI, D.F. Me´xico, Me´xico

Although dapsone (4,40 -diaminodiphenylsulfone) has been described as a neuroprotective agent in occlusive focal ischemia in rats, its mechanism of action is still unknown. To explore this mechanism, oxidative, inflammatory and apoptotic processes were evaluated in the striatum of adult rats using a model of ischemia-reperfusion (I/R), either with or without dapsone treatment. Male Wistar rats were submitted to transient middle cerebral artery occlusion for 2 hr, followed by reperfusion. Rats were dosed either with dapsone (12.5 mg/kg i.p.) or vehicle 30 min before or 30 min after the ischemia onset. Lipid peroxidation (LP) and nitrotyrosine contents were measured 22 hr after reperfusion, and myeloperoxidase activity was evaluated 46 hr after I/R. Different markers for apoptosis and necrosis were also evaluated both at 24 and 72 hr after I/R experimental procedure. LP increased by 37% in ischemic animals vs controls, and this effect was reversed by dapsone treatments. A similar effect was observed regarding nitrotyrosine striatal contents. Myeloperoxidase activity, a marker of inflammatory response, increased 3.7-fold in ischemic animals vs. control rats, and dapsone treatment antagonized that effect. Although apoptosis was increased by the effect of ischemia at both evaluation times, dapsone antagonized that effect only at 72 hr after surgery. Dapsone antagonized all of the I/R end points measured, showing a remarkable ability to decrease markers of damage through antioxidant, antiinflammatory, and antiapoptotic effects. VC 2008 Wiley-Liss, Inc. Key words: dapsone; ischemia/reperfusion; lipid peroxidation; myeloperoxidase; nitrotyrosine; apoptosis

Stroke and other cerebrovascular events represent the third cause of morbidity/mortality around the world, ' 2008 Wiley-Liss, Inc.

with 4.7 million new cases per year, and they represent the first cause of disability (Dirnagl et al., 1999). Thus, cerebrovascular diseases are considered Public Health problems with high social impact, as a result of the high costs derived from stroke treatment and rehabilitation (Porsdal and Boysen, 1999; Diringer et al., 1999; Yoneda et al., 2003; Spieler et al., 2004; Dodel et al., 2004). Brain ischemia is the result of diverse alterations diminishing the blood flow, oxygen, and glucose supply to the brain. Under those conditions, cells with a blood perfusion less than 12 ml/100 g/min in the ischemic center die within a few minutes, whereas those surrounding the ischemic core that are partially perfused might survive for several hours. The tissue surrounding the ischemic core is known as the penumbra ischemic zone (Aronowsky et al., 1999; Leker and Shohami, 2002; Bramlett and Dietrich, 2004). In this area, a series of events initiates a prolonged period of secondary neurodegeneration, expanding the zone of damage, to lead finally to severe neurological dysfunction (Ginsberg, 2003). Several lines of evidence suggest that the secondary neuronal cell death is due to a combination of multiple destructive events triggered by ischemia (Bramlett and Dietrich, 2004), such as vascular changes, edema, Contract grant sponsor: CONACyT; Contract grant number: 47425. *Correspondence to: Camilo Rıós, PhD, Departamento de Neuroquı´mica, Instituto Nacional de Neurologıá y Neurocirugıá, Av. Insurgentes Sur 3877, D.F. Me´xico 14269, Me´xico. E-mail: [email protected] Received 28 May 2007; Revised 26 March 2008; Accepted 29 March 2008 Published online 9 July 2008 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.21775

Neuroprotective Mechanisms of Dapsone

overproduction of reactive oxygen species, and lipid peroxidation (LP; Leker and Shohami, 2002). In addition, during the ischemic period, ATP depletion causes a decrease in sodium-potassium ATPase pump activity, which in turn generates a disruption in the plasma membrane potential, leading to release of glutamate and other neurotransmitters (Globus et al., 1988; Santos et al., 1996; Melani et al., 1999). This anomalous increase in extracellular excitatory neurotransmitters produces excitotoxicity, accompanied by a rise in the cytosolic calcium concentrations, in turn associated with activation of calcium-dependent enzymes, such as calpain, calcineurin, and phospholipases, with a subsequent proteolysis of calpain substrates, activation of constitutive nitric oxide synthase (cNOS) enzyme, and release of arachidonic acid (White et al., 2000; Pisani et al., 2004; Shirakura et al., 2005). These processes are particularly important after ischemia insofar as they initiate a cascade of inflammatory responses, overproduction of free radicals, and activation of apoptosis (Zheng and Yenari, 2004; Chan, 2004; Margaill et al., 2005). Recent reviews propose that a combined strategy, acting simultaneously on the main tissue damage-generating mechanisms, could be an optimal treatment for patients with stroke (Leker and Shohami; 2002). On the other hand, dapsone (4,40 -diamino-diphenyl-sulfone), has been a well-known drug since its discovery in the 1940s, when it was used as chemotherapy for leprosy and more recently for the treatment of Pneumocystis carinii infections. It is also used as an adjunctive therapy in the treatment of various skin diseases, taking advantage of its antiinflammatory actions (Leoung et al., 1986). Also, it has been shown that dapsone is able to antagonize the neurotoxic effects of kainic and quinolinic acids, both agonists of excitatory amino acid receptors (Santamaria et al., 1997). More recently, our group showed that dapsone has neuroprotective effects in a model of acute ischemia in rats, leading to a better functional recovery and preservation of the nervous tissue in the animals treated with dapsone 30 min after ischemia (Rios et al., 2004). However, because the mechanism of neuroprotection of dapsone is still unknown, we tested here its possible antioxidant, antiexcitotoxic, and antiinflammatory properties with the aim of explaining how dapsone protects neurons from ischemia/reperfusion (I/R)-induced damage using the middle-cerebral artery (MCA) occlusion model in rats. MATERIALS AND METHODS Animals Male Wistar rats weighing 250–300 g were maintained under standard laboratory conditions and had free access to food and water. The protocols for animal use were approved by the Animals Ethics Committee of the National Institute of Neurology and Neurosurgery of Mexico. Surgery We used the MCA occlusion experimental model of focal cerebral ischemia reported by Longa et al. (1989), modiJournal of Neuroscience Research

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fied to achieve reperfusion after 2 hr of ischemia. Animals were anesthetized with 3% halothane using a facemask. Body temperature was maintained at 378C with a warm pad during the surgical procedure and afterward until the recovery of rats from anesthesia. A longitudinal incision was made in the middle of the ventral cervical skin. The right common carotid artery, right internal carotid artery, and right external carotid artery were exposed. The distal portion of the right external carotid artery was then ligated and cut. A nylon suture (3-0) was introduced into the lumen of the right external carotid toward the direction of the internal carotid. The suture was advanced for 17 mm into the right internal carotid and left there. Finally, the incision was closed and left under controlled conditions for 2 hr. After this time, the reperfusion started by opening the wound to retract the filament, which was pulled out completely. Immediately after reperfusion, rats were evaluated for neurological deficits using the scale described by Longa et al. (1989). This scale rates the presence of neurological signs in rats as follows: 0 5 no observable deficit, 1 5 forelimb flexion, 2 5 unidirectional circling, 3 5 falling to the contralateral side, 4 5 decreased level or lack of consciousness, 5 5 death after surgery. Only those animals showing a value >2 were included in the study. All animals were allocated in individual acrylic cages with sterile sawdust and received food and water ad libitum. Pharmacological Treatments Rats were randomly allocated into four groups, as follows: group 1, sham operation plus vehicle; group 2, 2 hr of ischemia and reperfusion plus vehicle: groups 3 and 4: 2 hr of ischemia and reperfusion plus dapsone at a dose of 12.5 mg/ kg every 24 hr (Rios et al., 2004) starting either 30 min before or 30 min after ischemia, respectively. Lipid Peroxidation Assay Lipid fluorescence product formation was measured after ischemia and reperfusion using the technique described by Triggs and Willmore (1984), as modified by Santamaria and Rıós (1993). All animals were sacrificed 24 hr after ischemia, the time of peak levels of LP reported by Thiyagarajan and Shrama (2004). Rats were killed by decapitation, and their corpora striata were dissected out, according to Iversen and Glowinski (1966). Striatal tissue was weighed and homogenized in 3 ml of cold 0.9% NaCl solution. One-milliliter aliquots from the homogenate were added to 4 ml of a chloroform-methanol mixture (2:1 v/v). After stirring, the mixture was ice-cooled for 30 min to allow phase separation and the fluorescence of the chloroform layer was measured in a Perkin-Elmer LS50B Luminescence spectrophotometer at 370 nm of excitation and 430 nm of emission wavelengths. The sensitivity of the spectrophotometer was adjusted to 150 units of fluorescence with a quinine standard solution (0.1 lg/ml). Results were expressed as fluorescence units/g of wet tissue. Myeloperoxidase Activity Assay Myeloperoxidase activity was measured according to Weaver et al. (2002). Briefly, striatal tissue was homogenized in 20 volumes of ice-cooled 5 mM phosphate buffer, pH 6.0.

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After homogenization, samples were centrifuged at 30,000g for 30 min at 48C (Sorvall OTD55B Ultracentrifuge). The pellets were washed out with phosphate buffer and centrifuged again as described above. Pellets were resuspended in 0.5% hexadecyltrimethylammonium bromide, 50 mM phosphate buffer, pH 6.0. Then, samples underwent three freeze-thawing cycles, followed by 30 sec of ultrasonic disruption (ultrasonic processor Daigger GEX130 PD). After 20 min of incubation at 48C, samples were centrifuged again at 12,000g for 15 min at 48C, and supernatants were collected to assay myeloperoxidase activity. Reaction was started by mixing 0.1-ml aliquots of the sample with 2.9 ml of 50 mM phosphate buffer containing 0.167/ml of o-dianisidine dihydrochloride (Sigma, St. Louis, MO) and 0.0005% H2O2. Changes in absorbance at 460 nm in periods of 15 sec over 2 min were monitored with a UV-visible spectrophotometer (Lambda 20; Perkin Elmer). One unit of myeloperoxidase activity was calculated as the amount of sample needed to metabolize 1 lmol of hydrogen peroxide per minute at 258C. Results were expressed as international units of myeloperoxidase per gram of wet tissue.

3-Nitrotyrosine Assay Animals were sacrificed 24 hr after ischemia, according to Takizawa et al. (1999), the peak time for nitrotyrosine formation. The samples were processed as reported by Kaur et al. (1998). Striata from animals were dissected out from both the ipsilateral and the contralateral hemispheres and homogenized in 500 ll phosphate-buffer saline (PBS; pH 7.4), and an equal volume of 10% trichloroacetic acid was added to the homogenate. After stirring, samples were centrifuged at 18,000g for 15 min at 58C, the supernatants discarded, and the pellets suspended in 500 ll ethanol/ethyl acetate (1:1), stirred for 10 sec, and centrifuged at the same conditions described above. This step was repeated twice. Supernatants were then discarded, and pellets were resuspended in 600 ll of 6 M hydrochloric acid in glass tubes that were hermetically sealed. Samples were hydrolyzed at 110 6 58C. After 22 hr of hydrolysis, tubes were opened, and samples were evaporated to dryness. Residues were resuspended in 300 ll deionized water, vortexed, and filtered through Millipore membranes (0.45 lm pore diameter of nitrocellulose).

Chromatographic Analysis of 3-Nitrotyrosine Samples were analyzed by reversed-phase high-performance liquid chromatography with an isocratic LC 250 pump (Perkin Elmer). The mobil phase consisted of a 50 mM sodium acetate buffer solution (pH 4.7) with 1% HPLC-grade absolute methanol at a flow rate of 0.8 ml/min. Samples were injected into an Adsorbosphere Catecholamine column (4.6 3 100 mm, 3 mm; Alltech) using a 20-ll loop. Signals were recorded with an electrochemical detector (Metrohm 656) at 950 mV of applied voltage and 1 nA of sensitivity. Peaks were integrated using Turbochrom 4.0 software (Perkin Elmer). The ratios of 3-nitrotyrosine to tyrosine concentrations were determined for each sample and expressed as a percentage.

Apoptosis and Necrosis Assays Cellular death was estimated in tissue sections of striatal tissue by two different techniques: annexin V plus 4,6-diamidino-2-phenylindole (DAPI) stain and TUNNEL plus NeuN and propidium iodide immunohistochemistry. Groups of animals were killed either at 24 or 72 hr after the ischemic event with the aim of analyzing the early and late cellular death, according to Chen et al. (1998) and Endres et al. (1998). Annexin V Immunostaining For the immunohistochemical detection and quantification of apoptotic processes, the analysis was based on cell membrane alterations as revealed by annexin V assay, which is used as a marker of early apoptotic cellular death. Once the apoptotic process is activated in a cell, the phosphatidylserine is distributed asymmetrically in the cellular membrane, and a phospholipid-binding protein that binds phosphatidylserine in a calcium-dependent manner can be used as a marker for quantitative measurement of apoptotic cell death (Koopman et al., 1994). Sections were incubated for 2 hr in normal horse serum (1:200) in PBS, to block nonspecific binding sites. They were incubated for 2 hr with polyclonal rabbit antiannexin V (200 lg/ml; Santa Cruz Biotechnology, Santa Cruz, CA). After repeated washes (3 3 15 min) in 0.1 M PB 1% Triton, sections were exposed for 2 hr at room temperature to a secondary antiserum (fluorescein goat anti-rabbit IgG; 1:250; Zymed, South San Francisco, CA). Sections were rinsed three times with PBS. DAPI Nuclei Staining Morphological changes in the nuclear chromatin of cells undergoing apoptosis were detected by staining them with the DNA binding fluorochrome DAPI, previously described by Culmsee et al. (2005) and Culmsee and Krieglstein (2005). Sections were incubated with DAPI (1 lg/ml) for 15 min. Coverslips were then washed in PBS, mounted with Vectashield (Vector Laboratories, Burlingame, CA), and subjected to fluorescence microscopy. When evaluating the staining, background and baseline immunofluorescence was minimized by fluorescence gain adjustment. TUNEL Staining (In Situ DNA Nick-End Labeling) In situ DNA nick-end labeling (TUNEL staining) was performed as described elsewhere (Fujikawa et al., 1999, 2000). In brief, brain transversal sections were washed in PBS, incubated for 15 min at room temperature with 20 lg/ml of proteinase K in Tris/HCl, and washed in PBS; the sections were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 5 min on ice (2–88C); and the slides were rinse twice with PBS. After the slides were incubated in a humidified chamber for 60 min at 378C with 50 ll TUNEL reaction mixture on the sample, the sections were rinsed three times with PBS. Samples were analyzed under a fluorescence microscope in this state. Positive control sections were made by exposing sections to DNAase I solution prior to the TdT step, and negative control sections were made by substituting PBS for TdT. Both controls were included each time sections were stained. Double staining was performed by incubating Journal of Neuroscience Research


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the slides with the Neun-antimouse antibody (1:500) during 24 hr (Chemicon, Temecula, CA), followed by incubation with a secondary antibody anti-mouse Alexa 647 (Molecular Probes, Eugene, OR). Then the slides were stained with propidium iodide by 1 min and finally were mounted with Vectashield (Vector Laboratories, Burlingame, CA). All samples were analyzed under a fluorescence microscope (Zeiss HBO50) and later with a confocal microscope (Leica model DMIRE 2, connected to a TCSSP2AOBS scanner), by a pathologist blind to the treatment that each group had received. Caspase-3 and Caspase-9 Activities The activities of caspase-9 and caspase-3 were evaluated 24 and 72 hr after ischemia to assess events related to both early and late apoptic death (Chen et al., 1998). Caspase-9 activity was measured by using a caspase-9 fluorometric protease ELISA Kit that recognizes the sequence LEHD (Chemicon MCH6 kit), following the manufacturer’s instructions. The assay detects the cleavage of LEHD-AFC (7-amino-4-trifluoromethyl coumarin), as a substrate. LEHD-AFC emits blue light that is detected at 400 nm; upon cleavage of the substrate by MCH-6 or related caspases, free AFC emits a yellow-green fluorescence at 505 nm, which was quantified by using a fluorescence plate reader (BioTek model FLX 800TB). Comparison of the fluorescence of AFC from an apoptotic sample with an uninduced control allowed us to determine the increase in caspase-9 activity. Caspase-3 activity was evaluated by using a Calbiochem Caspase-3 Activity Assay Kit. The enzyme cleaves specifically after aspartate residues in a different peptide sequence (DEVD). The DEVD substrate is also labeled with the fluorescent molecule AFC, so the reaction can be monitored by a blue to green shift in fluorescence upon cleavage of the AFC fluorophore. The results were expressed in arbitrary fluorescence units per milligram of protein 6 SE of three animals per groups. Statistical Analysis All results are expressed as mean 6 SEM. Statistical significance between groups was determined by analysis of variance, followed by the Dunnett’s test, after testing for homogeneity of variances. Statistical significance between contralateral and ipsilateral striata values was determined using paired-samples t-test. Statistical analysis was performed in the SPSS 13.0 software.

RESULTS Dapsone Inhibits Lipid Peroxidation in the Striatum After Ischemia And Reperfusion The lipid fluorescence products measured in the striata of animals 24 hr after ischemia are shown in Figure 1. I/R group showed 37% and 41% increases of LP (P < 0.05) vs. sham and contralateral striata, respectively. In the group treated with dapsone, both before and after I/R, a decrease of LP was observed to values similar to those of sham-operated animals. Journal of Neuroscience Research

Fig. 1. Striatal lipid peroxidaton in rats 24 hr after ischemia/reperfusion (I/R). Sham: rats without I/R; I/R: rats with 2 hr of ischemia and 22 of reperfusion plus vehicle; I/R 1 DDS-before: animals with 2 hr of ischemia and 22 of reperfusion treated with dapsone (12.5 mg/kg) 30 min before ischemia; I/R 1 DDS-after: animals submitted to 2 hr of ischemia and 22 of reperfusion treated with dapsone 30 min after ischemia. The results are expressed as means 6 SEM of six to eight animals per group. *Different from both sham group and contralateral striatum (P < 0.05). One-way ANOVA followed by Dunnett’s test for between-groups comparison and paired-samples t-test for contralateral vs. ipsilateral comparisons.

Dapsone Antagonizes Myeloperoxidase Activity in the Striatum After I/R The presence of neutrophils in the samples after I/ R was indirectly determined by measuring tissue myeloperoxidase activity 48 hr after ischemia. The results are shown in Figure 2. We observed an increase of 3.7-fold of tissue myeloperoxidase activity as a consequence of I/ R, compared with both sham group and ipsilateral striatum (P < 0.05). That effect was fully antagonized by dapsone treatment administered either before or after ischemia. The Treatment With Dapsone Diminishes the Amount of Nitrotyrosine Present in the Striatum After I/R The striatal concentrations of nitrotyrosine analyzed 24 hr after ischemia are shown in Figure 3. A statistically significant increase (P < 0.05) was observed in the I/R group compared with both sham and contralateral striatum values. Again, dapsone antagonized that effect when it was administered before or after I/R. Dapsone Reduces Cellular Death in Striatum After I/R After the I/R experimental procedure, dapsone diminished cellular death in the striatum by both apoptotic and necrotic mechanisms. The effect of dapsone on those processes of cell death is shown in Figure 4. In the sham group, no apparent cell death from apoptosis was observed, and just a few instances by necrosis,

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Fig. 2. Striatal myeloperoxidase activity in rats 48 hr after ischemia/ reperfusion (I/R). Sham: rats without I/R; I/R: rats with 2 hr of ischemia and 46 hr of reperfusion plus vehicle; I/R 1 DDS-before: animals with 2 hr of ischemia and 46 hr of reperfusion treated with dapsone (12.5 mg/kg each 24 hr) 30 min before ischemia; I/R 1 DDS-after: animals submitted at 2 hr of ischemia and 46 hr of reperfusion treated with dapsone 30 min after ischemia. The results are expressed as means 6 SEM of six animals per group. *Different from both sham group and contralateral striatum (P < 0.05). One-way ANOVA followed by Dunnett’s test for between-groups comparison and paired-samples t-test for contralateral vs. ipsilateral comparisons.

Fig. 3. Striatal nitrotyrtosine 24 hr after ischemia/reperfusion (I/R). Sham: rats without I/R; I/R: rats with 2 hr of ischemia and 22 hr of reperfusion plus vehicle; I/R 1 DDS-before: animals with 2 hr of ischemia and 22 hr of reperfusion treated with dapsone (12.5 mg/kg each 24 hr) 30 min before ischemia; I/R 1 DDS-after: animals submitted at 2 hr of ischemia and 22 hr of reperfusion treated with dapsone 30 min after ischemia. The results are expressed as means 6 SEM of three to six animals per group. *Different from both sham group and contralateral striatum (P < 0.05). One-way ANOVA followed by Dunnett’s test for between-groups comparison and pairedsamples t-test for contralateral vs. ipsilateral comparisons.

whereas, in the I/R group, many neurons and nonneuronal cells were positive for apoptotic and necrotic stains. Nevertheless, the main cellular type affected by necrosis was the neurons that were marked with NeuN

Fig. 4. Representative photomicrographs of slides from the striatum, analyzed with a confocal microscope either 24 hr or 72 hr after ischemia/reperfusion (I/R). Slides stained with annexin V in green and a nuclear staining in blue using DAPI are shown in photomicrographs on the left side and slides stained with TUNEL in green, propidium iodine in red and NeuN in blue, are shown in photomicrographs in the right side. Sham (rat without I/R): no annexin-positive cells are shown in A and just a few TUNEL-positive cells in B (yellow arrows), with a good preservation of neurons and nonneuronal cells. I/R (rat with ischemia/reperfusion plus vehicle): many annexin- and TUNELpositive cells are shown in C (white arrows) and in D, respectively, with a poor preservation of neurons and nonneuronal cells. I/R 1 DDS-before (rat with ischemia/reperfusion treated with dapsone at 12.5 mg/kg each 24 hr, 30 min before ischemia): some annexin- and TUNEL-positive cells can be seen in E and F, respectively, along with a better preservation of the nervous tissue. I/R 1 DDP-after (rat with ischemia/reperfusion treated with dapsone (12.5 mg/kg each 24 hr, 30 min after ischemia): few annexin- and TUNEL-positive cells can be seen in G and H, respectively, with a much better preservation of the nervous tissue. Scale bars 5 20 lm.

antibody and propidium iodide stain. When dapsone treatment was used, both types of cellular death were decreased, especially in the I/R 1 DDS-after group, showing a preservation of the nervous tissue (Fig. 4). In Figure 5 it can be observed that the fluorescent intensity in the striatum 24 hr after ischemia in the I/R group was 2.5 6 0.3 FU, whereas the groups treated Journal of Neuroscience Research


Fig. 5. Apoptotic fluorescence intensity measurements. Results are expressed as means 6 SEM of four animals per group. A: Apoptosis evaluated 24 hr after ischemia and reperfusion. B: Apoptosis measured 72 hr after damage. Sham: rats without I/R; I/R: rats with ischemia/ reperfusion plus vehicle; I/R 1 DDS-before: animals with ischemia/ reperfusion treated with dapsone (12.5 mg/kg each 24 hr) 30 min before ischemia; I/R 1 DDS-after: animals subjected to ischemia/reperfusion treated with dapsone 30 min after ischemia. Different from all other groups, *P < 0.05, ANOVA followed by Dunnett’s test.

with dapsone before or after ischemia showed values of 1.5 6 0.5 and 1.75 6 0.7 FU, respectively, showing a tendency to diminish without reaching statistical significance. The fluorescent intensity evaluated in the striatum 72 hr after I/R was 3.5-fold that for the sham group. Dapsone treatments diminished those values significantly (P < 0.05) in the groups that received the treatment with dapsone both before and after ischemia (1.8 6 0.1 and 1.7 6 0.7, respectively). Dapsone Diminishes Both Caspase-9 and Caspase-3 Activities After I/R in Striatum The results of caspase-9 activity evaluated 24 and 72 hr after ischemia in striatum are shown in Figure 6. Journal of Neuroscience Research

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Fig. 6. Striatal caspase-9 activity measured 24 hr (A) and 72 hr (B) after ischemia and reperfusion. I/R: rats with 2 hr of ischemia and 22 or 70 hr of reperfusion plus vehicle; I/R 1 DDS-before: animals with 2 hr of ischemia and 22 or 70 hr of reperfusion treated with dapsone (12.5 mg/kg every 24 hr) 30 min before ischemia; I/R 1 DDS-after: animals submitted at 2 hr of ischemia and 22 or 70 hr of reperfusion treated with dapsone 30 min after ischemia. The results are expressed as means 6 SEM of three animals per group. *Different from I/R group (P < 0.05). One-way ANOVA followed by Dunnett’s test.

Twenty-four hours after I/R, caspase-9 activity increased significantly compared with sham-group values (5.61 6 0.52). The activity values for the I/R group were 8.97 6 2.2, and the values for the groups treated with dapsone showed a diminution to 3.94 6 2.43 and 5.97 6 0.30 for the dapsone before and after I/R groups, respectively. However, those values did not reach statistical significance (Fig. 6A). A similar pattern of caspase-9 activity was observed after 72 hr of I/R, with values of 11.21 6 1.04 for the I/R group and diminished values for the groups treated with dapsone before (7.71 6 0.94) and after (3.55 6 1.97) I/R. In

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I/R group (10.59 6 2.05) and in the groups treated with dapsone 30 min before (9.41 6 3.82) and after (9.79 6 0.21) I/R. No statistically significant changes were observed at this time point. Figure 7B shows caspase-3 activity values 72 hr after ischemia. We observed a significant diminution of caspase-3 activity only in the group that received dapsone 30 min after ischemia (P < 0.05).

Fig. 7. Striatal caspase-3 activity measured 24 hr (A) and 72 hr (B) after ischemia and reperfusion. I/R: rats with 2 hr of ischemia and 22 or 70 hr of reperfusion plus vehicle; I/R 1 DDS-before: animals with 2 hr of ischemia and 22 or 70 hr of reperfusion treated with dapsone (12.5 mg/kg each 24 hr) 30 min before ischemia; I/R 1 DDS-after: animals submitted at 2 hr of ischemia and 22 or 70 hr of reperfusion treated with dapsone 30 min after ischemia. The results are expressed as means 6 SEM of three animals per group. *Different from I/R group (P < 0.05). One-way ANOVA followed by Dunnett’s test.

this case, the latter group reached statistical significance, compared with I/R values (Fig. 6B). The results of caspase-3 activity are shown in Figure 7 and are similar to the pattern displayed by caspase9. In our hands, the sham group showed values below the detection limit of the technique, so the bar for this group is depicted as zero. Caspase-3 activity was, however, detected 24 hr after ischemia (Fig. 7A), both in the

DISCUSSION In the present study, we evaluated the effect of dapsone, both as an antioxidant and as an antiinflammatory drug, in a model of I/R in rats, by measuring several end points in the striatal tissue from adult rats. Results showed that I/R induced a significant increase in all of the parameters assessed: LP; content of nitrotyrosine; caspase-9 and caspase-3 activities; and, remarkably, myeloperoxidase activity, as has been shown previously in the literature (Nakazawa et al., 2000; Horikawa et al., 2001; Kato et al., 2003). A decrease in the constitutive NOS activity was also observed 48 hr after I/R (data not shown), as previously reported by others (Parmentier et al., 1999). Dapsone, administered 30 min after I/R, antagonized all those effects at the tested dose. The ability of dapsone to antagonize the damaging events after I/R measured here is probably the explanation for the reported neuroprotective properties of this sulfone both in experimental models (Santamaria et al., 1997) and recently, in human patients with acute stroke (Nader-Kawachi et al., 2006). It has been proposed that a neuroprotective therapy should include simultaneous actions to prevent further oxidative stress, exacerbated inflammatory response, and excitotoxicity, as the optimal alternative to reduce tissue damage after I/R (Laker et al., 2004; Bramlett and Dietrich, 2004). The antiexcitotoxic effects of dapsone have been previously demonstrated by our group (Rodriguez et al., 1999) and may be related to an antagonism of dapsone on voltage-gated calcium channels, as with the structurally related drug Zonisamide (Leppik, 2004). The antioxidant effects of dapsone shown here could also be related to an antagonism on calcium-dependent processes, insofar as dapsone was able to decrease N-methyl-D-aspartate (NMDA) receptor-activated striatal LP (Rodriguez et al., 1999). In this context, Suda et al. (2005) tested in vitro the antioxidant effect of dapsone, in neutrophils isolated from venous blood obtained from healthy human donors. In that study, dapsone was able to suppress the production of superoxide anions, through a calcium-dependent mechanism, suggesting that antioxidant and antiexcitotoxic effects associated with dapsone are both calcium dependent. Oxidative stress generated after I/R turns on mechanisms of cellular death, as thoroughly documented in the literature (Chistophe and Nicholas, 2006); among these, many processes are calcium dependent (Kristian and Siesjo, 1998) and susceptible to attenuation by dapsone. Journal of Neuroscience Research


On the other hand, dapsone is a well-known antiinflammatory drug acting through several mechanisms, including inhibition of neutrophil recruitment (Zhu and Stiller, 2001). Recent evidence indicates that therapies that down-regulate or interfere with mechanisms of inflammation may diminish the damage after ischemia and reperfusion (Shen et al., 2007). Dapsone is a direct and irreversible inhibitor of myeloperoxidase, so a diminished production of hypochlorous acid from this enzyme is expected, with a consequent reduction in oxidizing cytotoxic molecules in the tissue interstitial space (Bozeman et al., 1992). Dapsone also inhibits the association of leukotriene B4 with its specific receptor sites (Maloff et al., 1988), to prevent the generation of 5-lipoxygenase metabolites from polymorphonuclear leukocytes (Wozel et al., 1997). The synthesis of prostaglandin and leukotriene by macrophages is also reduced by dapsone (Bonney and Humes, 1984). The antagonistic effect of dapsone on the I/Rinduced rise in nitrotyrosine content enhancement is probably related to an inhibition on both NMDA and non-NMDA receptors by the sulfone, as was previously shown by our group (Santamaria et al., 1997). Nitrotyrosine is produced mainly by the action of peroxynitrite on tyrosine residues of proteins. Very low peroxynitrite concentrations are commonly found in brain tissue, but the enhanced production of nitric oxide by NOS enzymes and the superoxide anion overproduction coming from xantine oxidase activity (Warner et al., 2004) after I/R lead to substantial peroxynitrite formation (Eliasson et al., 1999). Because constitutive NOS and xantine oxidase are calcium-dependent enzymes, dapsone is probably reducing their activity to render less peroxynitrite formed and decreased nitrotyrosine content as a consequence. Thus, nitric oxide overproduction may play a role in secondary damage after I/R by combining with superoxide anion to form peroxynitrite that, in turn, reacts readily with amino acids, proteins, carbohydrates, lipids, and DNA (Willmot et al., 2005) Another mechanism to explain the dapsoneinduced reduction in nitrotyrosine formation (Lau and Baldus, 2006) is related to its effect on myeloperoxidase activity. Nitric oxide is formed during myeloperoxidasecatalyzed oxidation of nitrites, and the secondary oxidation of nitrites by myeloperoxidase has been reported as a mechanism for myeloperoxidase- and neutrophil-mediated damage during inflammation (Hazen et al., 1999). The inhibitory action of dapsone on myeloperoxidase activity may then diminish oxidative stress by two different routes: by reducing the nitrergic damage and by reducing the damage exerted by HClO, the direct product of myeloperoxidase activity. Finally, the treatment with dapsone diminished the cellular death either by apoptosis or by necrosis. Previous observations of delayed neuronal death after ischemia have suggested the possibility of an ‘‘apoptosis–necrosis’’ continuum (Portera-Cailliau et al., 1997). Thus, depending on the degree and duration of ischemia, brain cells may die by an ionic cascade, rapidly (necrotic cell Journal of Neuroscience Research

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death); or by molecular cascade, slowly (apoptotic cell death; Lee et al., 1999; Kuschinsky and Gillardon, 2000; MacManus and Buchan, 2000). In this context, when ischemia is mild and short, the resulting cell death may be apoptotic; i.e., neurons appear to be spared initially but later go on to die slowly (Petito et al., 1997). Although excitotoxic necrosis is considered the predominant mechanism of cell death by ischemia, in most cases, as in the present work, apoptosis may occur in penumbral neurons that escape excitotoxic death (Petito et al., 1997). In the present study, both forms of cellular death were seen, mainly in neurons marked with TUNEL and propidium iodide stains, in high proportion at 24 hr, indicating that dapsone protects only some neurons from late molecular death. On the other hand, because apoptosis is the result of dysregulation of calcium, increased glutamatergic signaling, oxidative stress, and inflammatory responses (Mehta et al., 2007), both ischemia and inflammatory responses have been suggested as factors that might mediate apoptosis (Padosch and Bottiger, 2003; Huang et al., 2006). Because only the late cellular death was significantly reduced by dapsone, it may indicate that the predominant neuron-protective mechanism of dapsone works in the poststroke recovery period as a late event; i.e., inflammation. This is further supported by the dapsone-induced decrease in caspase-9 and caspase-3 activities found only at the late (72 hr) times after I/R. Thus, the reductions of inflammation and cellular death by dapsone might be suggested as the main neuroprotective mechanisms associated with dapsone. CONCLUSIONS Dapsone treatment antagonized all of the end points measured: LP, myeloperoxidase and constitutive nitric oxide activities, nitrotyrosine content, and late apoptosis, in the striata of rats, showing a remarkable ability to decrease important processes of I/R-induced damage through antioxidant, antiinflammatory, and antiapoptotic effects. As a whole, results show a good candidate profile for dapsone as a neuroprotective drug in stroke. REFERENCES Aronowski J, Cho KH, Strong R, Grotta JC. 1999. Neurofilament proteolysis after focal ischemia; when do cells die after experimental stroke? J Cereb Blood Flow Metab 19:652–660. Bonney RJ, Humes JL. 1984. Physiological and pharmacological regulation of prostaglandin and leukotriene production by macrophages. J Leukoc Biol 35:1–10. Bozeman PM, Learn DB, Thomas EL. 1992. Inhibition of the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase by dapsone. Biochem Pharmacol 44:553–563. Bramlett HM, Dietrich WD. 2004. Pathophysiology of cerebral ischemia and brain trauma: similarities and differences. J Cereb Blood Flow Metab 24:133–150. Chan PH. 2004. Mitochondria and neuronal death/survival signaling pathways in cerebral ischemia. Neurochem Res 29:1943–1949. Chen J, Nagayama T, Jin K. Stetler RA, Zhu RL, Graham SH, Simon RP. 1998. Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia. J Neurosci 18:4914–4928.

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