Guanosine Protects Against Cortical Focal Ischemia. Involvementof Inflammatory Response

Gisele Hansel & André Comiran Tonon & Felipe Lhywinskh Guella & Letícia Ferreira Pettenuzzo &

Thiago Duarte & Marta Maria Medeiros Frescura Duarte & Jean Pierre Oses & Matilde Achaval &Diogo Onofre Souza

Received: 1 July 2014 /Accepted: 30 October 2014# Springer Science+Business Media New York 2014

Abstract Stroke is the major cause of death and the mostfrequent cause of disability in the adult population worldwide.Guanosine plays an important neuroprotective role in severalcerebral ischemic models and is involved in the modulation ofoxidative responses and glutamatergic parameters. Becausethe excessive reactive oxygen species produced during anischemic event can trigger an inflammatory response, thepurpose of this study was to evaluate the hypothesis thatguanosine is neuroprotective against focal cerebral ischemia,inhibits microglia/macrophages activation, and mediates aninflammatory response ameliorating the neural damage.Permanent focal cerebral ischemia was induced in adult rats,and guanosine was administered immediately, 1, 3, and 6 hafter surgery. Twenty-four hours after ischemia, the

asymmetry scores were evaluated by the cylinder test; neuro-nal damage was evaluated by Fluoro-Jade C (FJC) stainingand propidium iodide (PI) incorporation; microglia and im-mune cells were evaluated by anti-Iba-1 antibody; and inflam-matory parameters such as interleukins (IL): IL-1, IL-6, IL-10;tumor necrosis factors alpha (TNF-α); and interferon-gamma(INF-γ) were evaluated in the brain tissue and cerebrospinalfluid. The ischemic event increased the levels of Iba-1-positive cells and pro-inflammatory cytokines and decreasedIL-10 levels (an anti-inflammatory cytokine) in the lesionperiphery. The guanosine treatment attenuated the changesin these inflammatory parameters and also reduced the infarctvolume, PI incorporation, and number of FJC-positive cells,improving the functional recovery. Thus, guanosine may havebeen a promising therapeutic agent for the treatment of ische-mic brain injury by reduction of inflammatory process trig-gered in an ischemic event.

Keywords Neuroprotection . Neuroinflammation .

Microglia . Cytokines . Cerebral ischemia . Guanosine

Introduction

Stroke is currently a critical public health problem. Stroke is amajor cause of death and the most frequent cause of long-termdisability in the adult population worldwide. Ischemic strokeis more prevalent than that of hemorrhagic, representing ap-proximately 87 % of all strokes [1–3]. Acute ischemic strokecauses a sudden impairment of blood circulation within abrain area, which results in insufficient substrate delivery tosupport cellular homeostasis. This insufficient substrate deliv-ery results in cellular bioenergetics failure and consequently acomplex, multiple pathway event cascade, which results incellular damage and loss of neurological functions [3–6].Neuroinflammation plays an important role in the

G. Hansel (*) :A. C. Tonon : F. L. Guella : L. F. Pettenuzzo :D. O. SouzaDepartment of Biochemistry, Postgraduate Program in Biochemistry,Institute of Basic Health Sciences, Federal University of Rio Grandedo Sul (UFRGS), Ramiro Barcelos, 2600 – Anexo, Santa Cecília,90035-003 Porto Alegre, RS, Brazile-mail: gihansel@gmail.com

T. DuartePostgraduate Program in Pharmacology, Federal University of SantaMaria (UFSM), Santa Maria, RS, Brazil

M. M. M. F. DuarteHealth Sciences Center, Lutheran University of Brazil (ULBRA) -Campus Santa Maria, Santa Maria, RS, Brazil

J. P. OsesPostgraduate Program in Health and Behavior, Health and BiologicalSciences Center, Catholic University of Pelotas (UCPel), Pelotas,RS, Brazil

M. AchavalDepartment of Compared Histophysiologies and MorphologicScience, Institute of Basic Health Sciences, Federal University of RioGrande do Sul (UFRGS), Porto Alegre, RS, Brazil

Mol NeurobiolDOI 10.1007/s12035-014-8978-0

pathogenesis of ischemic stroke, inducing rapid neuronal deathin the ischemic core, which gradually expands toward the pen-umbra area, and is crucial to brain damage [3]. Microglia, themajor immune cell in the central nervous system (CNS), arerapidly activated in response to ischemia and are typically con-verted from the resting cell type to an activated form [7, 8].During ischemic inflammation, cytokines are released and mod-ulate the extent of damage in animal models. Studies demon-strated that peripheral cytokine levels are increased in ischemicpatients [7]. Tumor necrosis factors alpha (TNF-α) and interleu-kin 1 (IL-1) are produced bymicroglia, intrathecal macrophages,and infiltrating, monocyte-derived macrophages. Interleukin 6(IL-6) is produced by microglia and neurons, and interferon-gamma (INF-γ) is produced mainly by CD4+ and CD8+ T cells[9]. The cytokines produced by immune and injured brain cellsincrease brain edema and/or promote the death of brain cells inthe penumbra area, resulting in a secondary progression of theinfarct lesion [10]. Interleukin 10 (IL-10) is an anti-inflammatorycytokine that is reduced during ischemic events. IL-10 may beneuroprotective against ischemic brain injury [11]. Although theactivation of microglia is a natural process and is necessary fordefense against ischemia, microglial over-activation results in theprogression of neuronal loss and brain injury following ischemicstroke. Thus, important therapeutic strategies for cerebral ische-mia are microglial activation reduction, peripheral immune cellsinfiltration reduction, and releasing pro-inflammatory cytokinesinhibition.

One potential neuroprotective agent is the nucleoside gua-nosine. Several neurodegenerative and neurotoxic modelshave demonstrated that guanosine plays an important role inthe CNS [12–18], exerting trophic effects on neural cells [19,20], and protective modulatory effects on the glutamatergicsystem [18, 20–23]. Studies have shown that guanosine pro-tects against several ischemic models, including oxygen andglucose deprivation [24–26], hypoxia-ischemia models [27],and in vivo permanent and transient ischemic stroke [24, 28,29]. Researchers have claimed that guanosine is neuroprotec-tive against oxidative and inflammatory processes induced byischemia via MAPK/ERK pathway activation [22, 25, 26].Our laboratory has demonstrated that guanosine, by modulat-ing the oxidative response and glutamatergic parameters, pro-tects against damage provoked by permanent focal ischemiainduced by thermocoagulation [30]. Excessive reactive oxy-gen species production during an ischemic event can triggeran inflammatory response through the activation of the tran-scriptional nuclear factor kappa B (NF-κB) [31], which pro-motes the expression of a large number of genes related to thepathology of cerebral ischemia, including those involved inthe inflammatory response, such as IL-1 and TNF-α [32].

Previous experimental studies have demonstrated that gua-nosine modulates the oxidative response during an ischemicevent [30], and this effect is linked to neuroinflammatorymechanisms [31]. Thus, in this study, we tested the hypothesis

that guanosine attenuates cerebral ischemic injury byinhibiting the inflammatory response and modulating thelevels of anti- and pro-inflammatory cytokines in a model ofpe rmanent foca l ce rebra l i schemia induced bythermocoagulation.

Materials and Methods

Animals

Wistar male adult rats (90–100 days old, weighing 300–350 g)were maintained under controlled light and environmentalconditions (12 h light/12 h dark cycle at a constant tempera-ture of 22±2 °C) with water and commercial food pelletsavailable ad libitum. All experimental procedures were per-formed according to the NIH Guide for Care and Use ofLaboratory Animals and the Brazilian Society forNeuroscience and Behavior (SBNeC) recommendations forAnimal Care.

Induction of Permanent Focal Ischemia

The ischemic lesion was induced by thermocoagulation of theblood in the pial vessels of the motor and sensorimotor corti-ces [33]. Briefly, the animals were anesthetized with ketaminehydrochloride (70 mg/kg, i.p.) and xylazine hydrochloride(10 mg/kg, i.p.) and placed in a stereotaxic apparatus. Theskull was surgically exposed, and a craniotomy was per-formed by exposing the left frontoparietal cortex (+2 to!6 mm A.P. and !2 to !4 mm M.L. from the bregma) [34].Blood in the pial vessels was thermocoagulated transdurallyby the approximation of a hot probe close to the dura mater.The rostrocaudal extent of the surface of the frontal andparietal cortices was lesioned. After the procedure, the skinwas sutured, and the body temperature was maintained at37 °C using a heating pad until recovery from the anesthesia.

Drug Treatment

The animals were divided into four groups: sham saline, shamguanosine, ischemia saline, and ischemia guanosine. Theguanosine dose was chosen based on dose–response curvepublished in a previous study [30]. Guanosine (60 mg/kg inNaCl 0.9 %) was purchased from Sigma (St. Louis, MO,USA). All groups received 1 mL/kg i.p. administration ofthe drugs immediately, 1, 3, and 6 h after surgery.

Cylinder Test

This test is based on the spontaneous exploratory behavior ofrodents and reveals forelimb preference when the animal rearsto explore its environment by making forelimb contact with

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the cylinder walls [35, 36]. Animals were subjected to onetrial, 1 day before ischemia (pre-ischemic day) and 24 h afterischemia (post-ischemic day). To prevent habituation to thecylinder, the number of movements recorded was limited to20. The occurrences of ipsilateral (to the lesion) forelimb use,contralateral forelimb use, or simultaneous forelimb use werecounted. The asymmetry score for each animal was calculatedon each day by the formula previously described [37].

Measurement of Infarct Volume

Twenty-four hours after surgery, the animals were sacrificed, andthe brains were rapidly removed from the skull and sectioned inthe coronal plane at 2 mm thick using a rat brain matrix. Theslices were immersed for 30 min in 2 % of 2,3,5-triphenyltetra-zolium chloride (TTC) (Sigma, St. Louis, MO, USA) solution at37 °C followed by overnight fixation in 4 % paraformaldehyde(Sigma, St. Louis,MO,USA). The infarct volumewas calculatedby the formula: Infarct volume=[measured infarct area!slicethickness (2 mm)]+[area of contralateral corresponding struc-ture!slice thickness]![area of ipsilateral corresponding struc-ture!slice thickness] [38, 39]. The brain slices were analyzedwith Image J software (NIH, Bethesda, MD, USA). The resultsare expressed as cubic millimeter.

Cerebrospinal Fluid and Brain Tissue Processing

Cerebrospinal fluid (CSF) and tissue samples were collected24 h after the ischemic insult and were frozen (!80 °C) untilanalysis. The animals were anesthetized with ketamine hydro-chloride (70mg/kg, i.p.) and xylazine hydrochloride (10mg/kg,i.p.) and placed in a stereotaxic apparatus. CSF was collectedfrom the cisterna magna (about 80 μL) using an insulin syringe(27 gauge 9 1/200 length). The CSF was then centrifuged at3000g for 10 min at 4 °C to obtain a CSF cell-free supernatant.The animal was then submitted to transcardial perfusion with0.9 % saline to eliminate blood from the cerebral tissue. For theflow cytometry assay, the brain was removed from the skull,maintained at 4 °C, and the cortical tissue was collected. Inischemic animals, the brain tissue adjacent to the ischemiclesion (located between the lesion and the cerebral longitudinalfissure, measuring approximately 8 mm!2 mm) was consid-ered the lesion periphery (Fig. 1). This region was chosenbecause previous studies [37] of this model have determinedthat this region has characteristics that are similar to a penumbraarea. The same region was dissected in sham brains and on thecontralateral side of ischemic brains. For the immunohisto-chemistry assay, the animals were transcardially perfused with0.9 % saline followed by 4 % paraformaldehyde in 0.1 Mphosphate buffer (pH 7.4). The brains were removed andpost-fixed in the same solution at room temperature for 4 h.After, the brains were incubated in a 30 % sucrose solution for2 days. Coronal sections (50 μm) were obtained using a

Vibratome (Leica Biosystems, Mannheim, Wetzlar,Germany). Six sections through the rostrocaudal axis wereselected per animal. Stereotaxic positions of the selected sec-tions were standardized for all animals (the first slice wascollected approximately in +1 mm A.P., following one slicefor each 1 mm). The images were captured, and a square regionof interest was created.

Immunohistochemistry Assay

For the immunohistochemistry assay, the sections werepermeabilized and blocked for 1 h with Tris-buffered saline(TBS), pH 7.4, containing 0.1 % of Triton X-100 and 10 % offetal bovine serum. After blocking, the sections were incubat-ed for 24 h at 4 °C with polyclonal rabbit anti-Iba-1 (Wako,Tokyo, Japan, 1:500) in TBS. After being washed four timesfor 10 min with TBS, the sections were incubated with 594Alexa Fluor conjugated goat anti-rabbit (JacksonImmunoResearch Laboratories, Inc., PA, USA) for 2 h atroom temperature. After washing, the sections were mountedon slides coated with 5 % gelatin with chromium and potas-sium sulfate using Vectashield mounting medium (VectorLaboratories, São Paulo, Brazil). All sections werephotographed with confocal microscopy (Olympus, Tokyo,JP). For immunohistochemical analyses, all lighting condi-tions and magnifications were kept constant.

Fluoro-Jade C (FJC) Staining

FJC stain was used to investigate neurodegeneration. Sectionswere subjected to FJC staining in accordance with the manu-facturer’s instructions [40]. Briefly, the sections were immersedin a solution of 1 % sodium hydroxide in 80 % alcohol for5 min, 70 % alcohol for 2 min, distilled water for 2 min, and0.06 % potassium permanganate for 15 min. The sections wereimmersed in a solution of 0.0005 % FJC (MilliporeCorporation, Billerica, MA, USA) in 0.1 % acetic acid vehiclefor 30min. The sections were then rinsed three times in distilledwater and allowed to dry at 45 °C for 20 min before mountingwith DPX medium (Electron Microscopy Sciences, Hatfield,PA, USA). FJC-positive cell counting was performed as previ-ously described [41] with modifications. Six sections locatedinside in the lesion periphery were analyzed. FJC-positive cellswere counted in each image (6 images per animal), and the areawhere the cells were included was measured using the ImageJsoftware. The final value for each animal was: Σ (cells countedper image)/Σ (area containing labeled cells per image, in μm2).

Flow Cytometry Assay

The tissue samples (30mg) were mechanically dissociated with1 mL of phosphate-buffered saline (PBS), pH 7.4, containing1 mg/mL of collagenase IV; were filtered with a 40-μm nylon

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mesh to remove large clumps of cells and debris; and wereincubated with PBS/collagenase containing 10 μg/mLpropidium iodide (PI). The cells were incubated at room tem-perature in the dark for 30 min, washed twice with PBS, andcentrifuged at 1000g for 10 min at 4 °C to remove the super-natant containing free PI. Afterwards, the cells were perme-abilized with 0.001 % PBS Triton X-100 and blocked for15 min with bovine serum albumin 1 %. After blocking, thecells were incubated for 1 h in blocking solution containing themonoclonal antibodies anti-NeuN diluted 1:100 (MilliporeCorporation, Billerica, MA, USA), anti-GFAP diluted 1:100(Dako, CA, USA), or anti-ionized calcium-binding adaptormolecule 1 (Iba-1) diluted 1:100 (Wako, Tokyo, Japan). Thecells were washed twicewith PBS andwere incubated for 1 h inblocking solution containing Alexa 488 anti-mouse IgG diluted1:200 or Alexa 488-anti-rabbit IgG diluted 1:200 (JacksonImmunoResearch Laboratories, Inc, PA, USA). The level ofPI incorporation and number of positive NeuN, GFAP, or Iba-1-positive cells were determined by flow cytometry(FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA).Alexa Fluor 488 and PI dyes were excited at 488 nm using anair-cooled argon laser. Negative controls (samples with thesecondary antibody) were included for the machine voltagesetup. The emission of fluorochromes was recorded throughspecific band-pass fluorescence filters: green (FL-1; 530 nm/30) and red (FL-3; 670 nm long pass) using a CellQuest Prosoftware (Becton Dickinson, Franklin Lakes, NJ, USA).Fluorescence emissions were collected by using logarithmicamplification. In brief, data from 20,000 events (intact cells)were acquired, and the mean relative fluorescence intensity wasdetermined after the exclusion of debris events from the dataset. All flow cytometric analyses were performed using Flow Josoftware 7.6.3 (Treestar, Ashland, OR). Flow cytometry datawere analyzed and plotted by density as dot plots, which showthe relative FL1 fluorescence on the x-axis and the relative FL3fluorescence on the y-axis. The negative and positive quadrants

were determined by using unstained samples. The number ofcells in each quadrant was computed, and the proportion ofcells stained with PI, NeuN, GFAP, and Iba-1 were expressed aspercentage of control [42].

Inflammatory Cytokine Measurements

The tissue samples (30 mg) were homogenized with PBS/Tris-HCl pH 7.4 and were centrifuged at 5000g for 10 min4 °C to exclude debris. The supernatant was collected toanalyze and measure protein content.

The inflammatory cytokine concentrations were measuredby enzyme-linked immunosorbent assay (ELISA) using com-mercial kits for rat TNF-α, IFN-γ, IL-1, IL-6, and IL-10(eBIOSCIENCE, San Diego, CA, USA) according to themanufacturer’s instructions. Briefly, 96-well microplates wereincubated with the primary antibody at 4 °C overnight,washed, and blocked at room temperature for 1 h. The cyto-kine standards, calibrators, and samples (CSF or tissue) wereadded in the plate in triplicate and incubated at room temper-ature for 2 h. After washing, the secondary antibody conju-gated with peroxidase was added and incubated at roomtemperature for 1 h. After washing, a TMB chromogen(tetramethylbenzidine) was added, and the reaction wasallowed to proceed for 15 min. The enzyme reaction wasstopped by adding Stop solution. The absorbance was mea-sured at 450 nm. The presence and concentration of thecytokines were determined by the color intensity measuredby spectrometry in a micro ELISA reader. The results areexpressed as picogram per milliliter for CSF samples and aspicogram per milligram for tissue samples.

Protein Assay

Protein content was measured using Pierce BCA protein kit(Thermo Scientific, Waltham, MA, USA) with bovine serum

Fig. 1 Schematic illustration ofthe ischemic lesion. Illustrationsshowing the region of inducedlesion (dark gray) and theanalyzed region (gray) that weredissected for the experiments. Theleft (ipsilateral side) and right(contralateral side) frontoparietalcortices were used

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albumin as a standard. Results are expressed as milligram ofprotein.

Statistical Analysis

The results are presented as the mean±S.E.M. The cylindertest was analyzed with a repeated measures analysis of vari-ance (ANOVA) followed by Tukey’s post hoc test. FJC wasanalyzed using Student’s t test. Cytometry assays, cytokines,and cytokine ratios were statistically analyzed using two-wayANOVA followed by Bonferroni’s post hoc test. Probabilityvalues less than 0.05 were considered to be significant. Allanalyses were performed using the Statistical Package forSocial Sciences (SPSS) software version 18.0.

Results

Behavioral Test and Infarct Volume

To evaluate whether the administration of guanosine aftercortical ischemia leads to functional recovery, ischemic ani-mals were treated with guanosine, and their sensorimotorperformance was measured. Both day and group variables,visualized by the “group x day” interaction (F(3,65)=23.9;P<0.0001), were significantly different. The symmetry scoredecreased 24 h after ischemia. The guanosine treatment pro-moted the significant recovery of the contralateral forelimbperformance in support during vertical exploration (Fig. 2a).

In agreement with a previous study [30], brain infarctvolume analysis by TTC staining 24 h after ischemia demon-strated a large amount of tissue damage (visualized by palestaining). Guanosine significantly decreased in 40 % the ex-tent of tissue damage (Fig. 2b, c). The sham groups inducedno damage (Fig. 2c).

Cellular Degeneration

As previously shown [41], the procedure of thermocoagulationinduced a large amount of neurodegeneration as revealed byFJC staining. The number of FJC-positive cells observed in thelesion periphery 24 h after ischemia was significantly lower inischemic animals treated with guanosine (138.6±8.9 to 97.0±4.1 number of FJC+ cells, compared ischemic animals to ische-mic animals treated with guanosine, respectively; P<0.05)(Fig. 3a–c).

For assessing cell types and cell viability, flow cytometryanalysis was carried out. There was no difference amonggroups in the total number of neurons and astrocytes markedby anti-NeuN and anti-GFAP antibodies, in the contralateraland ipsilateral sites (Fig. 4a, c). In addition, the anti-NeuN andanti-GFAP antibodies co-stained with PI identified neuronal

and astrocytic damage. PI incorporation in neurons in theischemic group (in the ipsilateral side) significantly increasedmore than threefold compared to that in the sham group.Guanosine treatment was able to abolish the increase in PIincorporation 24 h after ischemia (Fig. 4b). In the ischemicgroup (in the ipsilateral side), 24 h after ischemia, the numberof anti-GFAP cells co-stained with PI increased twofold com-pared to that in the sham group. The ischemic group treatedwith guanosine (131.8±18.5 %) had a partial decrease in PIincorporation compared to that in the ischemic group (213.8±22.8 %) (Fig. 4d). Together, these findings indicate that ische-mia increased neuronal and astrocytic damage in the lesionperiphery, and guanosine treatment provided neuronal andastrocytic neuroprotection 24 h after ischemia.

Guanosine Decreased Microglia/Macrophages and Reducedthe Activated Microglia Cell in the Lesion Periphery

The Iba-1 antibody detects microglia as well as peripheralimmune cells, such as monocytes/macrophages. To eliminateperipheral intravascular cells, the tissue was perfused with0.9 % saline. Using flow cytometry analysis, the number ofIba-1-positive cells increased 2.5-fold comparing the ischemicgroup to the sham group, 24 h after ischemia. The increase ofIba-1-positive cells was abolished by guanosine treatment(Fig. 5).

Immunohistochemistry was carried out using Iba-1 formicroglia labeling. The Iba-1 antibody allows visualizationof microglia and their processes. No difference was detectedon the contralateral side across all groups. In both shamgroups, the microglia present in the lesion periphery had atypical resting morphology with cellular processes branchingfrom the small soma with further distal arborization (Fig. 6).Twenty-four hours after insult, the ischemic group had severalcells with enlarged somata and fewer and shorter processes,which is characteristic of the activated state (Fig. 6). Theischemic group treated with guanosine had an intermediarymorphology. These groups had a smaller number of cells withactivated characteristics and cells with intermediary processesbranching from the somata in the lesion periphery (Fig. 6).

Guanosine-Attenuated Changes in the Levelsof Pro-inflammatory and Anti-inflammatory Cytokines

In this study, the levels of pro-inflammatory cytokines weremeasured in the CSF (Fig. 7) and in the lesion periphery(Fig. 8) 24 h after ischemia. The IL-1, IL-6, TNF-α, andIFN-γ levels were significantly augmented in both the CSFand lesion periphery in the ischemic animals. Guanosinetreatment significantly returned the levels of all these cyto-kines to sham levels. IL-10 is an anti-inflammatory cytokine.The levels of IL-10 decreased in the ischemic animals com-pared to the sham group, and guanosine treatment inhibited

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this reduction both in the CSF and lesion periphery. The IL-1/IL-10, IL-6/IL-10, TNF-α/IL-10, and IFN-γ/ IL-10 ratioswere calculated to verify the relationship between the pro-and anti-inflammatory cytokines. These ratios were increasedin the ischemic animals in the CSF and lesion periphery, andthis effect was minimized with guanosine treatment (Table 1).

Discussion

The neuroprotective effects of guanosine in ischemic strokehave been studied in several stroke models. The data fromin vitro models suggest that guanosine protects against oxygenand glucose deprivation [22–26], increases glutamate uptakein hypoxia-ischemia models [27, 43], and protects againstglucose deprivation [17]. In vivo guanosine is neuroprotectiveagainst permanent and transient ischemic stroke, promotesfunctional improvement, and reduces infarct volume [24, 28,29]. Our group recently demonstrated that guanosine

neuroprotection in the permanent focal ischemia model hascaused a significant and lasting recovery in the function ofimpaired forelimb, maintained for at least 15 days. This neu-roprotection was related to the modulation and maintenance ofcellular redox environment and glutamatergic system 24 hafter ischemia [30]. During ischemic events, the excessivereactive oxygen species production can trigger an inflamma-tory response and contribute to the expansion of brain injuryand delayed neuronal death. Thus, the novel finding of thisstudy is that treatment with guanosine early after permanentfocal ischemia suppressed the activation of microglia cells, anassumption based upon the cell morphology by immunohis-tochemistry. The treatment with guanosine also reduced thenumber of Iba-1-positive cells (which could be due to a resultof microglia migration and/or by the infiltration of peripheralimmune cells) in the lesion periphery, reduced the levels ofpro-inflammatory cytokines, and increased the levels of anti-inflammatory cytokines in the brain and CSF. These effectslead to a diminished neural damage and consequent functionalrecovery.

Fig. 2 Recovery of the impaired forelimb in the cylinder tests 24 h beforeand after ischemia and infarct volume of the lesion 24 h after ischemia. aGraph showing the performance of sham saline (n=8), sham guanosine(n=8), ischemia saline (n=10), and ischemia guanosine (n=10) groups.Ischemia and ischemia guanosine groups were significantly differentfrom the sham group 24 h after ischemia. The ischemia guanosine grouppartially recovered the use of the impaired forelimb. Data are expressed aspercentage of symmetry score. *P<0.001 comparing sham group vs.

ischemia and ischemia guanosine groups; #P<0.01 comparing ischemiaand ischemia guanosine. b Analysis of cortex infarct volume 24 h aftercerebral ischemia. Ischemia guanosine group had a significantly reducedinfarct volume compared to that of the ischemia group. Data areexpressed as cubic millimeter. *P=0.014, n=8 per group. c The repre-sentative TTC staining was evidenced in both ischemic and ischemictreated with guanosine groups. In sham animals, the TTC staining wasanalyzed, but no lesion was observed

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Neurons are extremely susceptible to changes in bloodflow, and clinical studies have shown impaired neuronal func-tion after only 10 min of ischemia [44]. In animal models ofischemic stroke, neural cells in the core rapidly become com-mitted to die. Therefore, therapeutic strategies aimed at rescu-ing these neurons have failed. As a consequence, attention hasincreasingly become focused on the penumbra, where neuro-nal death can be extended for days to weeks [3, 6]. FJCstaining has a good affinity for entirely degenerating neurons[40]. Here, FJC staining was used to study the effect ofguanosine treatment on ischemia-induced neurodegenerationin the lesion periphery. The number of degenerating neuronsin the lesion periphery 24 h after ischemia significantly de-creased in the ischemic group treated with guanosine. Inaddition, the flow cytometry analysis 24 h after ischemicinsult indicated that although the total number of neuronsand astrocytes were not decreased in the ipsilateral site in theischemic animals, the number of PI positive in neurons andastrocytes was increased. The guanosine treatment totallyreversed the increase in PI incorporation in neurons andpartially restored this effect in astrocytes. These findings

indicate that the beneficial effects of guanosine may be dueto neuroprotection in the lesion periphery, the main presump-tive site of restorative processes. Guanosine treatment dimin-ishes the neurodegeneration and consequently promotes in-creased functional recovery.

Accumulating evidence demonstrates the relationship be-tween inflammation and neuronal cell damage in cerebralischemia [45]. Thus, the mechanisms of neuroprotective strat-egies against cerebral ischemia may target inflammatory al-terations involved in cellular damage. Neuroinflammation isone of the key pathological events contributing to the progres-sion of damage caused by ischemia [46]. Post-ischemic in-flammatory responses are characterized by the activation ofastrocytes and microglia, as well as the infiltration of poly-morphonuclear granulocytes and monocytes/macrophages in-to the ischemic brain region [47]. In this study, the number ofmicroglia/macrophages, in the lesion periphery, had substan-tially increased 24 h after ischemia, and the guanosine treat-ment abolished this effect. Analyzing the morphology byimmunohistochemistry, the sham group had the characteristicsof resting microglia with small somata and long, distal

Fig. 3 Neuronalneurodegeneration evaluation(FJC staining) 24 h after ischemia.Images of the lesion peripherywere analyzed. The images weretaken from representative coronalsections of ischemic animals (a)and ischemic animals treated withguanosine (b). Note that thenumber of FJC-positive(degenerating) neurons wasgreater in ischemic animalscompared to ischemic guanosine-treated animals. c Quantificationof degenerating neurons.Ischemic guanosine group had asignificant reduction in thenumber of FJC-positive neuronsin the lesion periphery. Data areexpressed as number of FJC-positive cells. *P=0.007, n=5 pergroup

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arborized processes. The ischemic group had characteristics ofactivated microglia. The microglia cells had reduced the

complexity of their shape by retracting the branches of theirprocesses (so that they are resorbed into the cell body), whichincreases the intensity stained near the soma. The ischemicgroup treated with guanosine had an intermediary morpholo-gy with some microglia having activated characteristics butstill with branched processes. Resident microglia are the majorcells involved in inflammatory processes in the brain and arethe first cells to respond to brain injury. They are activatedwithin minutes after the onset of focal cerebral ischemia. Thisactivation may last for several weeks and plays a critical roleduring ischemia mainly in the penumbra area [7, 48, 49].Besides the activation of microglia, there was an increase ofthe infiltration of polymorphonuclear granulocytes andmonocytes/macrophages into the ischemic brain region bythe breakdown of the protective blood–brain barrier [7, 47].Despite reactive microglia are morphologically and function-ally similar to blood-derived monocytes/macrophages [50,51], studies have shown that blood-derived macrophages arerecruited into the ischemic brain tissue, most abundantly atdays 3–7 after stroke [52, 53]. Moreover, studies have shownthat infiltrating macrophages remain much lower than that ofactivated resident microglia [7, 50, 52]. Thus, it is possible to

Fig. 4 Flow cytometry analysis 24 h after ischemia in the lesion periphery(contralateral and ipsilateral sides). a NeuN-positive cells. b NeuN-positivecells co-stainedwith propidium iodide (PI), *P<0.001 comparing sham salineand ischemic guanosine vs. ischemic saline. c GFAP-positive cells. d GFAP-positive cells co-stained with PI, *P<0.001 comparing sham saline vs.ischemic saline; #P<0.01 comparing ischemia guanosine vs. ischemia saline;and &P<0.05 comparing sham saline vs. ischemia guanosine. Cells stained

only with the secondary antibody were used to set the negative region of thedot plot. Cells with fluorescence above the negative region were consideredstained and were counted as neurons (NeuN positive) or astrocytes (GFAPpositive). Data are reported as the means±S.E.M. of 8–10 animals per groupand are expressed as a percent of control. Statistical differences were deter-mined by a two-way ANOVA followed by a Bonferroni post-test

Fig. 5 Flow cytometry analysis by Iba-1 24 h after ischemia in the lesionperiphery (contralateral and ipsilateral sides). Cells stained only with thesecondary antibody were used to set the negative region. Cells with fluores-cence above the negative region were considered stained and were counted asIba-1-positive cells. Data are reported as the means±S.E.M. of 9–13 animalsper group and are expressed as a percent of control. Statistical differences weredetermined by a two-way ANOVA followed by Bonferroni post-tests,*P<0.001 comparing sham saline and ischemia guanosine vs. ischemia saline

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assume that the vast majority of macrophage-like cells in theischemic brain represented activated resident microglia, espe-cially during the first 24 h, diminished effect with guanosinetreatment.

The inflammatory response not only may exacerbate sec-ondary brain injury in the acute stage of ischemia but also maybeneficially contribute to brain recovery after an ischemicevent. Thus, the inhibition of inflammatory responses couldworsen brain repair and long-term functional recovery afterischemic stroke [54, 55]. Although inflammatory response isnecessary for processing reparative mechanisms of tissuedamage after cerebral ischemia, the over-production of pro-inflammatory cytokine mediators contributes to the expansionof brain injury and the delayed neuronal death [46, 7].Cerebral ischemia could break the dynamic balance between

the pro-inflammatory and anti- inflammatory responses; thus,studies indicate that inhibition of inflammatory responsescould decrease brain injury and improve neurological out-come [54].

Here, the microglia/macrophages increase was accompa-nied by an increase of pro-inflammatory cytokines IL-1, IL-6,TNF-α, and INF-γ and a decrease of the anti-inflammatorycytokine IL-10 in both the lesion periphery and CSF in theischemic group. The increased production of pro-inflammatory cytokines has been observed in experimentalmodels of brain ischemia and in patients with acute stroke andmay be associated with large cerebral infarct volume and poorstroke outcome [56]. TNF-α, INF-γ, and IL-1 are rapidlyexpressed in the ischemic brain after the disease onset, andtheir upregulation persists for days. Despite the unclear effects

Fig. 6 Iba-1 immunohistochemistry 24 h after permanent focal ischemia. Representative images (!40 and !60) of 5 animals per group. Six slices peranimal were used. The lesion periphery in the ipsilateral and contralateral sides was analyzed

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of IL-6, due to its anti-inflammatory properties to induce IL-1receptor antagonist synthesis, studies have shown that theupregulation of TNF-α, INF-γ, IL-1, and IL-6 followingischemic events leads to the disruption of the blood–brain

barrier, increases the infarct volume, and causes neuronal celldeath [2, 56, 9]. Some investigators have reported that IL-10 isthe main down regulator of deleterious effects of pro-inflammatory cytokines. Clinical stroke studies have

Fig. 7 CSF inflammatory cytokines levels: a TNF-α, b INF-γ, c IL-1, dIL-6, and e IL-10, 24 h after ischemia. Data are reported as the means±S.E.M. of 8–16 animals per group and are expressed as picogram permilliliter. Statistical differences were determined by a two-way ANOVA

followed by Bonferroni post-tests. *P<0.001 comparing sham saline andischemia guanosine vs. ischemia saline in a, b, c, and e; *P<0.001comparing sham saline and vs. ischemia saline; and #P<0.01 comparingischemia guanosine vs. ischemia saline in d

Fig. 8 Inflammatory cytokines levels: a TNF-α, b INF-γ, c IL-1, d IL-6,and e IL-10 measured in the lesion periphery 24 h after ischemia. Data arereported as the means±S.E.M. of 10 animals per group and are expressedas picogram per milligram of protein. Statistical differences were deter-mined by a two-way ANOVA followed by Bonferroni post-tests.*P<0.001 comparing sham saline and ischemia guanosine vs. ischemia

saline in a, b, and c; *P<0.001 comparing sham saline and vs. ischemiasaline; #P<0.01 comparing ischemia guanosine vs. ischemia saline in d;*P<0.001 comparing sham saline and vs. ischemia saline; #P<0.01comparing ischemia guanosine vs. ischemia saline; &P<0.05 comparingsham saline vs. ischemia guanosine in e

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demonstrated that IL-10 decreases during ischemic events,and increases in IL-10 are associated with better patient out-comes [56, 57]. Interestingly, the guanosine treatment afterischemia decreased the production of pro-inflammatory cyto-kines and restored IL-10 to sham levels.

Studies have described the balance between pro- and anti-inflammatory factors to be a more accurate reflection of theinflammatory environment [58, 59]. Due to significant inter-individual differences in the concentrations of pro- or anti-inflammatory factors, here, we evaluated the ratio of pro- toanti-inflammatory factors, which provided a useful measure ofthe net immunological effect of the circulating cytokines and/or other factors associated with damage. It is important to notethat the ratios of IL-1/IL-10, IL-6/IL-10, TNF-α/IL-10, andIFN-γ/ IL-10 were reduced in the ischemic animals treatedwith guanosine compared to that in the ischemic group 24 hafter ischemia in the lesion periphery and CSF. Taken togeth-er, these findings indicate a favorable effect of guanosinetreatment, which altered the balance of pro- and anti-inflammatory cytokines during an ischemic event.

It has been shown that the suppression of some inflamma-tory components is associated with reduced neuronal celldeath, reduced infarct volume, and improved behavioral out-comes in stroke models [2, 9, 57]. Studies have shown thatguanosine is neuroprotective against oxidative and inflamma-tory processes [22, 26, 30, 60, 61]. D’Alimonte et al. [61]showed the participation of guanosine in inflammatory re-sponse by the inhibition of TNF-α, IL-1β, and NF-κB nucleartranslocation in microglial cells via reducing CD40. Dal-Cimet al. [26] demonstrated that guanosine is capable of decreas-ing nuclear p65 expression induced by an oxidative insult.Our group [60] also showed that guanosine prevented oxida-tive and inflammatory response by azide-induced oxidativedamage through the heme-oxygenase-1 signaling pathway,pointing to the involvement of NF-κB. Moreover, NF-κBhas been related to excessive ROS production and genes such

as IL-6, IL-1, and TNF-α [31, 62]. Thus, guanosine has beenimplicated in the modulation of MAPK/ERK signaling path-ways, which lead to the inhibition of NF-κB and confers aneuroprotective property against oxidative and inflammatoryprocesses [22, 25, 26, 60]. Therefore, here, the decreased pro-inflammatory interleukin levels might be due to the inhibitionof NF-κB. Although the mechanisms by which guanosine actsare not fully known, the present study demonstrated for thefirst time that the administration of guanosine after permanentfocal cerebral ischemia reduced microglial/macrophage cellnumber and pro-inflammatory cytokine levels and increasedanti-inflammatory cytokine levels in the brain. The reductionof the inflammatory state was accompanied by a decrease inneuronal damage and infarct volume, improving behavioraloutcomes.

Acknowledgments This work was supported by the ConselhoNacional de Desenvolvimento Científico e Tecnológico (CNPq),Coordenação de Aperfeiçoamento de Pessoal de Nível Superior(CAPES), Fundação de Amparo à Pesquisa do Estado do Rio Grandedo Sul (FAPERGS), Financiadora de Estudos e Projetos (FINEP),IBN.Net 01.06.0842-00, and Instituto Nacional de Ciência e Tecnologiapara Excitotoxicidade e Neuroproteção (INCTEN).

Conflict of Interest None.

References

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Table 1 Pro-inflammatory/anti-inflammatory ratio

Groups IL-1/IL-10 IL-6/IL-10 TNF-α/IL-10 INF-γ/IL-10

CSF

Sham saline 0.48±002 0.62±0.02 0.90±0.03 1.09±0.05

Sham guanosine 0.59±0.05 0.68±0.08 0.88±0.06 1.09±0.07

Ischemia saline 4.24±0.76* 5.36±1.20* 6.35±1.31* 8.07±1.55*

Ischemia guanosine 1.04±0.08** 1.27±0.13** 1.30±0.12** 1.94±0.15**

Cortical tissue

Sham saline 0.52±0.03 0.75±0.06 1.01±0.03 1.33±0.06

Sham guanosine 0.60±0.06 0.75±0.06 1.06±0.05 1.27±0.07

Ischemia saline 4.13±0.53* 5.00±0.81* 5.76±0.09* 8.12±1.02*

Ischemia guanosine 1.46±0.09** 1.74±0.13** 1.83±0.15** 2.57±0.18**

Data are shown as mean (SEM)

*P<0.001, sham saline vs. ischemia saline; **P<0.001, ischemia saline vs. ischemia guanosine,

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