Polyinosinic-Polycytidylic Acid Has Therapeutic Effects against Cerebral Ischemia/Reperfusion Injury through the Downregulation of TLR4 Signaling via TLR3 This information is current as of September 30, 2021. Peng-Fei Wang, Huang Fang, Jing Chen, Sen Lin, Yong Liu, Xiao-Yi Xiong, Yan-Chun Wang, Ren-Ping Xiong, Feng-Lin lv, Jian Wang and Qing-Wu Yang J Immunol 2014; 192:4783-4794; Prepublished online 11 April 2014; Downloaded from doi: 10.4049/jimmunol.1303108 http://www.jimmunol.org/content/192/10/4783 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2014/04/11/jimmunol.130310 Material 8.DCSupplemental References This article cites 46 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/192/10/4783.full#ref-list-1

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Polyinosinic-Polycytidylic Acid Has Therapeutic Effects against Cerebral Ischemia/Reperfusion Injury through the Downregulation of TLR4 Signaling via TLR3

Peng-Fei Wang,* Huang Fang,* Jing Chen,* Sen Lin,†,‡ Yong Liu,* Xiao-Yi Xiong,* Yan-Chun Wang,* Ren-Ping Xiong,x Feng-Lin lv,{ Jian Wang,‖ and Qing-Wu Yang*

Recent reports have shown that preconditioning with the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)) protects against cerebral ischemia/reperfusion (I/R) injury. However, it is unclear whether poly(I:C) treatment after cerebral I/R injury is also effective. We used mouse/rat middle cerebral artery occlusion and cell oxygen-glucose deprivation models to evaluate the thera- peutic effects and mechanisms of poly(I:C) treatment. Poly(I:C) was i.p. injected 3 h after ischemia (treatment group). Cerebral

infarct volumes and brain edemas were significantly reduced, and neurologic scores were significantly increased. TNF-a and IL-1b Downloaded from levels were markedly decreased, whereas IFN-b levels were greatly increased, in the ischemic brain tissues, cerebral spinal fluid, and serum. Injuries to hippocampal neurons and mitochondria were greatly reduced. The numbers of TUNEL-positive and Fluoro-Jade B+ cells also decreased significantly in the ischemic brain tissues. Poly(I:C) treatment increased the levels of Hsp27, Hsp70, and Bcl2 and decreased the level of Bax in the ischemic brain tissues. Moreover, poly(I:C) treatment attenuated the levels of TNF-a and IL-1b in serum and cerebral spinal fluid of mice stimulated by LPS. However, the protective effects of 2/2 2/2 poly(I:C) against cerebral ischemia were abolished in TLR3 and TLR4 mice. Poly(I:C) downregulated TLR4 signaling via http://www.jimmunol.org/ TLR3. Poly(I:C) treatment exhibited obvious protective effects 14 d after ischemia and was also effective in the rat permanent middle cerebral artery occlusion model. The results suggest that poly(I:C) exerts therapeutic effects against cerebral I/R injury through the downregulation of TLR4 signaling via TLR3. Poly(I:C) is a promising new drug candidate for the treatment of cerebral infarcts. The Journal of Immunology, 2014, 192: 4783–4794.

troke is the most common cause of severe disability and the fact that the pathophysiological mechanisms of ischemic the second most common cause of death in the world injury have not been clearly elucidated. Recent research has (1). Currently, recombinant tissue plasminogen activator revealed that inflammation plays an important role in secondary

S by guest on September 30, 2021 (rt-PA) thrombolytic therapy is the only effective treatment for brain insult following cerebral ischemia (3, 4). ischemic stroke (2); this paucity of treatment options is related to TLRs have been demonstrated to play critical roles in the induction of immune and inflammatory responses. Patten-associated molecu- lar patterns and damage-associated molecular patterns (DAMPs) ac- *Department of Neurology, Xinqiao Hospital, Third Military Medical University, tivate TLRs and induce the expression of NF-kB–dependent † Chongqing 40037, China; Department of Development, Regeneration Key Labora- proinflammatory and/or type I IFN, and these molecules tory of Sichuan Province, Chengdu Medical College, Chengdu 610083, China; ‡Department of Histoembryology and Neurobiology, Chengdu Medical College, participate in pathophysiological processes (5). DAMPs released x Chengdu 610083, China; Department 7, Institute of Surgery, State Key Laboratory from ischemic neurons can activate the TLR4/MyD88/NF-kBsig- of Trauma, Burns and Combined Injury, Third Military Medical University, Chongq- ing 400042, China; {Biomedical Engineering College, Chongqing University, Chongq- naling pathway in the neighboring microglia and induce the release ing 400044, China; and ‖Department of Anesthesiology/Critical Care Medicine, School of proinflammatory cytokines, which gives rise to the degeneration of Medicine, Johns Hopkins University, Baltimore, MD 21205 and of neurons (6–8). Recent studies have shown that Received for publication November 19, 2013. Accepted for publication March 7, modification of the TLR-mediated NF-kB signaling pathway sig- 2014. nificantly attenuates ischemic injuries to the organs. Our group and This work was supported by National Natural Science Foundation of China Grants others have demonstrated that cerebral tissue injury is alleviated 81070932 and 81271283. in TLR4-deficient mice after cerebral ischemia/reperfusion (I/R) (9, Address correspondence and reprint requests to Prof. Qing-Wu Yang, Department of Neurology, Xinqiao Hospital and Second Affiliated Hospital, Third Military Medical 10), and preconditioning with TLR 2, 4, 7, and 9 ligands can increase University, 183 Xinqiao Main Street, Shapingba District, Chongqing 400037, China. tolerance to subsequent ischemia; furthermore, significant reductions E-mail address: [email protected] in neurologic impairments have also been reported (11–14). The online version of this article contains supplemental material. Upon activation by dsRNA , TLR3 recruits the regulatory Abbreviations used in this article: CBF, cerebral blood flow; CCA, common carotid Toll/IL-1R domain–containing adapter inducing IFN-b artery; CSF, cerebral spinal fluid; DAMP, damage-associated molecular pattern; k ECA, external carotid artery; FJB, Fluoro-Jade B; ICA, internal carotid artery; I/R, () to activate IFN regulatory factors (IRFs) and NF- B via ischemia/reperfusion; IRF, IFN regulatory factor; MABP, mean arterial blood pres- a TRIF-dependent signaling pathway (15). TLR3-deficient mice sure; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; OGD, do not exhibit reduced cerebral infarct volumes after cerebral I/R oxygen-glucose deprivation; poly(I:C), polyinosinic-polycytidylic acid; rmIFN-b, recombinant mouse IFN-b; rt-PA, recombinant tissue plasminogen activator; TRIF, compared with wild-type mice (16). However, recent reports have Toll/IL-1R domain–containing adapter inducing IFN-b; TTC, triphenyl tetrazolium shown that polyinosinic-polycytidylic acid (poly(I:C)) precon- chloride. ditioning protects against cerebral I/R injury (17, 18). However, it This article is distributed under The American Association of Immunologists, Inc., is unclear whether poly(I:C) treatment after cerebral I/R injury is Reuse Terms and Conditions for Author Choice articles. also effective. Additionally, if treatment is effective, the protec- Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 tive mechanisms remain unknown. It has been demonstrated that www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303108 4784 POLY(I:C) PROTECTS THE BRAIN AGAINST ISCHEMIC INJURY poly(I:C) attenuates LPS-induced liver injury by downregulating laid down so that the body made a 135˚ angle with the fixed head. In this TLR4 expression in macrophages and reducing the levels of TNF-a angle, the dura and spinal medulla were visible, had a characteristic glis- (19). Therefore, we proposed that postischemia treatment with the tening and clear appearance, and the circulatory pulsation of the medulla (i.e., a blood vessel) and adjacent CSF space could be seen. The dura was TLR3 ligand poly(I:C) would also have protective effects then penetrated with a 6-cm-long glass capillary that had a tapered tip with against cerebral I/R injury, which could be mediated by activation an outer diameter of 0.5 mm. Following a noticeable change in resistance of TLR3 and subsequent downregulation of the TLR4 signaling to the capillary insertion, the CSF flows into the capillary. Any CSF samples pathway. contaminated with the slightest trace of blood were discarded. All samples were stored in polypropylene tubes at 270˚C for subsequent ELISA. In this study, we used the mouse/rat middle cerebral artery oc- clusion (MCAO) and cell oxygen-glucose deprivation (OGD) models Poly(I:C) to evaluate the therapeutic effects and mechanisms of postischemia Poly(I:C) (InvivoGen, San Diego, CA) was i.p. injected 3 h after ischemia poly(I:C) treatment. The results suggested that poly(I:C) treatment (treatment group) and the vehicle group was injected i.p. with 0.9% saline. exerts therapeutic effects against cerebral I/R injury through the Poly(I:C) was dissolved in sterile saline to an injection concentration of downregulation of TLR4 signaling via TLR3. Poly(I:C) is a prom- 1 mg/ml. Determination of the content of endotoxin by the limulus assay ising new drug candidate for the treatment of cerebral infarcts. demonstrated that poly(I:C) preparations used in the study were free of any detectable LPS. We tested the effects of different doses of poly(I:C) (0.5, 1.25, or 2.5 mg/g), and found that the 1.25 mg/g dose produced the greatest Materials and Methods effects (data not shown). Therefore, we chose the 1.25 mg/g dose of poly(I:C) Animals for subsequent experiments. In vitro, 20 mg/ml poly(I:C) was added after 3 h of OGD, and the cells were collected 24 h later for use in different assays. Age- and weight-matched male C57BL/6 mice (8–12 wk old, 20–23 g), neonatal C57BL/6 mice born within 24 h, and male SD rats (8–12 wk old, Intracerebral ventricular injection of IFN-b after MCAO Downloaded from 220–250 g) were obtained from the laboratory animal center of the Third b b Military Medical University (Chongqing, China). The TLR32/2 mice (8– Recombinant mouse IFN- (rmIFN- ; Cell Sciences, Canton, MA) or 2/2 vehicle (artificial CSF) was injected into the mice right lateral ventricle as 12 wk old, 21–23 g) and TLR4 mice (8–12 wk old, 20–23 g) were m b purchased from The Jackson Laboratory (Bar Harbor, ME). The TLR32/2 described previously (23). Injections (1 l) of either rmIFN- (200 U) or and TLR42/2 mice have been backcrossed to C57BL/6 mice for more than artificial CSF were administered at 3 h after MCAO. Infarct volume was eight generations. All protocols involving the use of animals were ap- measured 48 h after stroke. proved by the Institutional Animal Care and Use Committee of the Third Assessment of cerebral infarct volume http://www.jimmunol.org/ Military Medical University. The cerebral infarct volume was determined as previously described (9). MCAO model Forty-eight hours after MCAO, animals were sacrificed and perfused with The MCAO model was employed as previously described (20). Briefly, the ice-cold PBS via the ascending aorta. The brains were removed and sec- mice were first anesthetized using 5.0% isoflurane. Then an incision in the tioned coronally (2-mm-thick slices for mice, 5-mm-thick slices for rats). skin was made, and the right common carotid artery (CCA), external ca- The slices were stained with 2% triphenyl tetrazolium chloride (TTC) rotid artery (ECA), and internal carotid artery (ICA) were carefully ex- solution (Sigma-Aldrich, St. Louis, MO) at 37˚C for 15 min. Normal brain posed. Microvascular aneurysm clips were applied to the right CCA and tissue was stained brightly, whereas the infarcted areas were pale white. the ICA. A coated 6-0 filament (Doccol, Redlands, CA) was introduced We measured the entire area of the prosencephalon and the cerebral infarct into an arteriotomy hole, fed distally into the ICA, and advanced a pre- with Image-Pro Plus 5.0 image processing software (Media Cybernetics, by guest on September 30, 2021 determined distance 8 mm from the carotid bifurcation toward the MCA. Rockville, MD). These areas were calculated using the formula V = t(A1 + ... After a 60-min period of focal cerebral ischemia, the filament was gently A2 + An), where V is the volume of the infarct or prosencephalon, t is removed (onset of reperfusion). The collar suture at the base of the ECA the thickness of the slice, and A is the infarct size and the volume ratio of stump was then tightened. The skin incision was closed and anesthesia was the cerebral infarct (cerebral infarction volume/prosencephalon volume). discontinued. Sham mice underwent a neck dissection and coagulation of the ECA, but the MCA was not occluded. Laser Doppler flowmetry Evaluation of neurologic score (PeriFlux system 5000; Perimed, Stockholm, Sweden) was used as a Animals were scored as previously described (20). Briefly, the mouse quality control to ensure that MCAO was induced successfully and to scoring system included performance measurements in five principal tasks: inspect the cerebral blood flow (CBF). Regional CBF decreased by 80% in spontaneous activity during a 3-min period (0–3), symmetry of movement mice after MCAO, and CBF recovered completely after the occlusion was (0–3), open-field path linearity (0–3), beam walking on a 3 3 1-cm beam removed (Fig. 1B). Rectal temperature was monitored and maintained at (0–3), and response to vibrissae touch (0–3). The total scores obtained 6 37.0 0.5˚C during the procedure. The pO2, pCO2, and pH of umbilical using this system thus ranged from 0 to 15, where 15 represents a perfect blood and the mean arterial blood pressure (MABP) were measured score and 0 represents death due to cerebral I/R injury. (details are provided in Supplemental Table I). For the rats, we performed the postural reflex test, which assesses the Permanent MCAO in rats was induced as previously described (21). The upper body posture while the animal is suspended by the tail, and the rats were first anesthetized using 5.0% isoflurane, and the right CCA and forelimb-placing test, which examines sensorimotor integration during ICA were temporarily clamped using a microvascular clip. An incision was forelimb-placing responses to visual, tactile, and proprioceptive stimuli. made between the two sutures around the ECA, through which a silicone- Neurological function was graded on a scale ranging from 0 to 12, with coated 4-0 filament (Doccol) was advanced 18 mm to the origin of the higher scores indicating greater neurologic deficit. MCA. Successful MCAO was confirmed with a decrease in laser Doppler Scoring was performed by two trained investigators who were blinded blood flow (data not shown). Rectal temperature was monitored and to the animal group designation, and the mean score of the subscales was 6 maintained at 37.0 0.5˚C during the procedure. The pO2, pCO2, and pH used as the final score for each animal. of umbilical blood and the MABP were measured (data not shown). The procedures were performed by a blinded operator. The experiment Assessment of brain edema was conducted in agreement with the ARRIVE (Animal Research: Report- ing In Vivo Experiments) guidelines. The success rate of the MCAO was Brain water content was measured as previously described (24). At 48 h ∼90%. The animals were randomly divided into groups by computer- after cerebral ischemia, the right hemispheres were dissected, immediately generated randomization schedules, and blinding to group allocation was weighed (wet weight), dried at 110˚C for 48 h, and reweighed (dry ensured. Dead animals and MCAO model failures were excluded from the weight). The brain water content was calculated with the formula [(wet experiments. weight 2 dry weight)/wet weight] 3 100%. Cerebral spinal fluid sample Nissl staining The sampling of cerebral spinal fluid (CSF) was employed as previously Nissl staining was performed as previously described (20). Paraffin sections described (22). Briefly, the mice were anesthetized and placed prone on the werecutatathicknessof5mm. The sections were deparaffinized and sub- stereotaxic instrument. A sagittal incision of the skin was made inferior sequently stained with 0.1% cresyl violet (Beyotime, Beijing, China) for 2 min. to the occiput. Under the dissection microscope, the s.c. tissue and neck Shrinkage of cell bodies and the nuclear condensation of neurons were muscles through the midline were bluntly separated. Then the mouse was evaluated using microscopy (Olympus BX60; Olympus, Tokyo, Japan). The Journal of Immunology 4785

Electron microscopic examination 1:200; anti–IFN-b, 1:200; anti-Hsp27, 1:200; anti-Hsp70, 1:200; anti–IRF-3, 1:200 [Santa Cruz Biotechnology, Santa Cruz, CA]; anti-TLR4, 1:200; Forty-eight hours after MCAO, mice were perfused with 2% glutaraldehyde 3 anti-TRIF, 1:1000; anti-MyD88, 1:1000 [Abcam]; and anti-TLR3, 1:200 through the ascending aorta. Blocks (1 mm ) of ischemic cerebral tissue [eBioscience, San Diego, CA]) and subsequently incubated with peroxidase- were fixed and stored in 0.25% glutaraldehyde. The mitochondrial struc- conjugated secondary Abs (Cell Signaling Technology, Danvers, MA). The ture was observed using a transmission electron microscope (Hitachi, signals were detected using an ECL system, and the membranes were Tokyo, Japan). Normal mitochondria were classified by compacted matrix reprobed with anti-GAPDH (Santa Cruz Biotechnology). The signals were cristae and a continuous outer membrane. In contrast, damaged mito- quantified with scanning densitometry using a bioimaging analysis system chondria were classified into mild and severe injury depending on (LabWorks analysis software; UVP, Upland, CA). mitochondrial shape, matrix density, crystal structure, disrupted outer membrane, and appearance of any abnormal structures (25). Randomly Real-time RT-PCR selected noncontiguous, nonoverlapping, digitized images of each cerebral Ischemic brain tissue was harvested quickly at 24 and 48 h after cerebral tissue pellet were captured, and these images were evaluated by an ob- I/R. Total RNA was extracted with TRIzol (Invitrogen, Gaithersburg, MD) server blinded to the source of mitochondria. according to the manufacturer’s instructions. cDNA was synthesized using TUNEL and Fluoro-Jade B staining the iScript cDNA synthesis (Bio-Rad, Hercules, CA) and real-time RT-PCR was carried out on a Bio-Rad iCycler with the iQ SYBR Green TUNEL and Fluoro-Jade B (FJB) staining were performed according to Supermix (Bio-Rad) in 96-well plates. Primers were purchased from the manufacturers’ protocols. The animals were deeply anesthetized and Shanghai Sangon Biological Engineering (Shanghai, China). The primer perfused through the ascending aorta with cold PBS followed by 4% para- sequences were as follows: TLR2, forward, 59-GAA AGA TGC GCT TCC formaldehyde, and the brains were sectioned coronally into 20-mm-thick TGA AC-39,reverse,59-CGC CTA AGA GCA GGA TCA AC-39.To frozen slices. TUNEL staining was performed using an in situ cell death quantify measurements of expression, a threshold cycle value (CT) detection kit (Roche, Indianapolis, IN). Cell degeneration was detected was calculated using the DDCT method as previously described (27). using an FJB staining kit (Millipore, Billerica, MA). Both assays were Downloaded from performed according to the manufacturers’ protocols. The stained sections EMSA were photographed with a confocal fluorescence microscope (Leica, Wetzlar, EMSA was performed as previously described (28). At 48 h after cerebral Germany). The nuclei were stained with DAPI (blue), and the apoptotic cells + + ischemia, the mice were sacrificed and the brains were removed. Nucle- were TUNEL (green). We calculated the number of DAPI cells (blue) and oprotein was extracted from ischemic brain tissue. Ten micrograms nu- TUNEL-positive cells (green). The percentage of TUNEL+ cells was calcu- 3 cleoprotein of each sample was incubated with the reaction buffer at room lated using the formula green/blue 100%. Four quadrants were selected temperature for 15 min and then [32P]-labeled double-stranded oligonu- from each section, and the number of positive cells in each quadrant was cleotide containing the binding motif of NF-kB(59-AGTTGAGGGGACTTT- http://www.jimmunol.org/ counted. The average value was then calculated. CCCAGGC-39; Promega, Madison, WI) or a [32P]-labeled double-stranded Cell cultures and OGD oligonucleotide corresponding to the IFN-stimulated response element of the IFN-inducible Isg15 gene (59-GATCGGAAAGGGAAACCGAAACTGAA- Primary hippocampal neuron cultures, microglial cell cultures, mixed neuronal- GCC-39, Cell Signaling Technology, Boston, MA) for IRF3 binding was glial cell cultures, and OGD were performed as previously described (14, 26). added to the reaction buffer and incubated for 15 min. After incubation for In brief, the cell cultures were prepared from neonatal mice, and the hippo- 20 min at 25˚C, the reaction mixture was subjected to 6% nondenaturing campi and cortices were dissected and dissociated with 0.25% trypsin-EDTA PAGE. Autoradiography was performed at room temperature. Finally, the (Life Technologies, Birmingham, MI). Primary hippocampal neurons were images were analyzed using a Bio-Rad image analyzer and the results were cultured in neurobasal media (containing 4.5 g/l glucose, supplemented with expressed as OD. GlutaMAX and B27; Life Technologies) for 7 d, microglia were cultured in 10% FBS (Sigma-Aldrich) for 14 d, and the mixed neuronal-glial cell cultures Flow cytometry by guest on September 30, 2021 were cultured in 5% FBS (Sigma-Aldrich) and 5% horse serum (Sigma- Flow cytometry was employed to detect TLR3+ and TLR4+ microglial Aldrich) supplemented with 15 mM glucose for 14 d. OGD was performed cells. In brief, following 3 h of microglial OGD, 20 mg/ml poly(I:C) or by removing the culture medium and replacing it with glucose-free DMEM vehicle was added. The cells were collected 24 h after stimulation, and (Life Technologies); the cultures were then incubated in an anaerobic atmo- mAbs, including allophycocyanin-conjugated anti-TLR4 (1:200; eBioscience), sphere of 85% N2, 10% CO2,and5%H2 at 37˚C for 3 h. OGD was terminated PerCP-Cy–conjugated anti-CD11b (1:200; eBioscience), and FITC-conjugated by replacing the glucose-free DMEM with normal culture medium and anti-TLR3 (1:200; Novus Biologicals, Littleton, CO) were added. The cells returning the cells to a normoxic incubator. Control plates were kept in were incubated in the dark at 4˚C for 30 min and washed twice. Anti-mouse the normoxic incubator during the OGD interval. For in vitro experiments, cells TLR3 (1:100; Abcam) was used to block the TLR3 signal. A BD FACSVerse were treated with either LPS (, 0111:B4; Sigma-Aldrich; flow cytometer (Becton Dickinson, Franklin Lakes, NJ) was used for detec- m b 100 ng/ml), poly I:C (20 g/ml), rmIFN- (500 U/ml), or saline. tion. The percentage of positive cells and the average fluorescence intensities Transwell coculture experiment were analyzed using BD FACSuite software. ELISA Cocultures of neurons and microglial cells were established using a trans- well system (Millipore). Microglia were plated onto the tops of transwell Samples of the supernatants from the ischemic brain tissues, the cultured 3 inserts at a cell density of 1 3 10 /well. Hippocampal neurons were plated cells, the CSF, and serum were obtained, and the inflammatory factors 4 onto the cell plate at a density of 1 3 10 cells/well and were cultured with TNF-a, IL-1b, and IFN-b were quantified with an ELISA kit (Dakewe, 10% FBS in an atmosphere of 5% CO2 at 37˚C for 48 h. Following 3 h of Beijing, China) according to the manufacturer’s protocol. OGD coculture, 20 mg/ml poly(I:C) or vehicle was added to the transwell insert, and immunofluorescence staining of the hippocampal neurons was Statistical analysis performed 24 h after stimulation. The hippocampal neurons were stained 6 b All data are expressed as the means SEM. Differences between multiple with a class III -tubulin Ab (green; 1:200; Abcam, Cambridge, MA), and groups were examined using one-way ANOVA and post hoc Bonferroni the nuclei were stained with DAPI (blue). We determined the number of tests, and independent sample t tests were used for comparisons of two DAPI+ cells (blue) and b-tubulin+ cells (green). The hippocampal neuron 3 groups. When the data were not normally distributed, the nonparametric survival rate was calculated with the formula green/blue 100%. Four Kruskal–Wallis and Mann–Whitney U tests were used. A p value ,0.05 quadrants were selected from each section. The numbers of positive cells was considered statistically significant. in each quadrant were counted, and the average values were calculated. Western blotting Results Poly(I:C) reduced focal cerebral I/R injury Western blotting was performed as previously described (20). At 48 h after cerebral ischemia, the mice were sacrificed and the brains were removed. To evaluate the protective effects of poly(I:C) against cerebral I/R Following 3 h of cellular OGD, 20 mg/ml poly(I:C) or vehicle was added injury, mice were i.p. injected with poly(I:C) (1.25 mg/g) 3 h after and the cells were collected after an additional 24 h. The were cerebral ischemia. Poly(I:C) treatment significantly reduced the electrophoresed through an SDS–polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (Amersham Pharmacia, Piscataway, cerebral infarct volume compared with the I/R group (Fig. 1A; NJ). The polyvinylidene difluoride membranes were first incubated with p , 0.01). However, poly(I:C) treatment 6 h after cerebral is- primary Abs (anti–Bcl-2, 1:200; anti-Bax, 1:200; anti–NF-kB [p65], chemia did not reduce cerebral infarct volume (data not shown). 4786 POLY(I:C) PROTECTS THE BRAIN AGAINST ISCHEMIC INJURY Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 1. Poly(I:C) reduced focal cerebral I/R injury. Poly(I:C) (1.25 mg/g) or vehicle was injected i.p. into mice 3 h after cerebral ischemia. Mice were subjected to cerebral ischemia (60 min), followed by reperfusion (48 h). (A) TTC staining and quantification of the infarct volumes (n = 9). Original mag- nification 31.2. (B) There were no differences in changes in CBF (n = 8). (C) Brain water contents and neurologic scores (15 is a perfect score, and 0 is death due to cerebral I/R injury; n = 8). ELISA of TNF-a,IL-1b,andIFN-b in the ischemic brain tissue (D), CSF (E), and serum (F)(n = 3–5). Nissl staining (G)and transmission electron microscopy analysis (H) (arrow points to mitochondrion; n = 8). Scale bars, 200 mm(left column)and50mm(middle and right columns) in (G), 0.4 mmin(H). ELISA data are representative of three independent experiments. *p , 0.05, **p , 0.01. CA1, cornu ammonis 1; DG, dentate gyrus.

The regional CBF decreased by 80% in mice after MCAO, and in pH, pO2,pCO2, MABP, or rectal temperature between the CBF recovered completely after the occlusion was removed poly(I:C)-treated and vehicle groups (Supplemental Table I). (Fig. 1B). Additionally, no remarkable differences were found Compared with the I/R group, the brain water content of the right The Journal of Immunology 4787 hemisphere was significantly decreased and the neurologic membranes were severely damaged, resulting in irregularly shaped scores were increased markedly in the poly(I:C) group (Fig. 1C; organelles with poor membrane integrity in the I/R group; in contrast, p , 0.05). Moreover, the IFN-b content was increased mark- in the poly(I:C) group, mitochondria demonstrated evidence of edema edly, and the levels of the inflammatory factors TNF-a and IL-1b with matrix, but with intact surrounding membranes (Fig. 1H). were decreased significantly in the ischemic brain tissue (Fig. 1D; p , 0.01), CSF (Fig. 1E; p , 0.01), and serum (Fig. 1F; p , 0.01). Poly(I:C) increased the expression of Bcl2, Hsp27, and Hsp70, Similar results were observed in mixed neuronal-glial cell cultures decreased Bax expression, and reduced cellular degeneration after OGD treated with poly(I:C) (Supplemental Fig. 1A). Nissl and apoptosis staining showed neuronal damage in the cornu ammonis 1 and Neuron apoptosis plays an important role in cerebral ischemia dentate gyrus fields of hippocampal formation characterized by injury. Bcl-2 is important for cell survival owing to its antiapoptotic shrunken cell bodies accompanied by shrunken and pyknotic nuclei effects, whereas Bax can promote apoptosis. We examined the effect in the I/R mice for 48 h (Fig. 1G). Hippocampal neuron damage was of poly(I:C) treatment on the levels of Bcl-2 and Bax after cerebral significantly reduced in the poly(I:C) treatment group (Fig. 1G). I/R. The results showed that poly(I:C) treatment significantly in- Transmission electron microscopy demonstrated that mitochondrial creased Bcl-2 expression (Fig. 2A; p , 0.01) and significantly Downloaded from http://www.jimmunol.org/

FIGURE 2. Poly(I:C) increased the expression of Bcl2, Hsp27, and Hsp70, decreased Bax expression, and reduced cellular apoptosis and by guest on September 30, 2021 degeneration. Poly(I:C) (1.25 mg/g) or vehicle was injected i.p. into mice 3 h after cerebral ischemia. Mice were subjected to cerebral ischemia (60 min), followed by reperfusion (48 h). (A) Expression of Bcl-2 and Bax were examined by Western blot- ting (n =3).(B)Hsp27andHsp70 expression were examined by West- ern blotting (n = 3). TUNEL+ (C) and FJB+ (D) cell counts were re- markably reduced in ischemic brain tissues after 48 h of MCAO with poly(I:C) treatment (n = 8). *p , 0.05, **p , 0.01. Western blotting data are representative of three in- dependent experiments. 4788 POLY(I:C) PROTECTS THE BRAIN AGAINST ISCHEMIC INJURY decreased Bax expression (Fig. 2A; p , 0.05) in the ischemic brain Neuronal degeneration was detected with FJB staining. In this tissues compared with the I/R group; similar results were obtained study, compared with I/R group, the numbers of TUNEL+ and in the mixed neuronal-glial cell OGD model (Supplemental Fig. FJB+ cells in ischemic brain tissues were markedly decreased in 1B). Recent studies have shown that Hsp27 and Hsp70 have pro- the poly(I:C) group (Fig. 2C, 2D; p , 0.01). In the transwell co- tective effects against cerebral I/R injury (29, 30). Our results also culture model, the hippocampal neuronal survival rate was mark- showed that poly(I:C) significantly increased the levels of Hsp27 edly increased with poly(I:C) treatment (Supplemental Fig. 1C). (Fig. 2B; p , 0.05) and Hsp70 (Fig. 2B; p , 0.01) in the ischemic brain tissues compared with the I/R group. Poly(I:C) protected against cerebral ischemia via TLR3 TUNEL staining is a molecular biological-histochemical system We investigated whether the protective effects of poly(I:C) are for sensitive and specific staining of DNA fragmentation (31). exerted via TLR3. First, Western blotting showed that the ex- Downloaded from http://www.jimmunol.org/

FIGURE 3. Poly(I:C) protected against cerebral ischemia via TLR3. Poly(I:C) (1.25 mg/g) or vehicle was injected i.p. into mice 3 h after ce- rebral ischemia. Mice were sub- jected to cerebral ischemia (60 min), followed by reperfusion (48 h). (A) Expression of TLR3 in the ischemic braintissueswasexaminedbyWest- by guest on September 30, 2021 ern blotting (n =3).(B) Infarction volumes and neurologic scores in TLR32/2 mice (n =8).Original magnification 31.1. The numbers of TUNEL+ cells (C)andFJB+ cells in ischemic cerebral tissues (D)werenot significantly different between the two groups after 48 h of MCAO (n =9). Following 3 h of cell OGD, 20 mg/ml poly(I:C) was added and the cells were detected 24 h later. (E)The hippocampal neuronal survival rates were not different in the OGD plus poly(I:C) group compared with the OGD plus vehicle group after 24 h of OGD (n =3).**p , 0.01. Data are representative of at least three inde- pendent experiments. The Journal of Immunology 4789 pression of TLR3 in the poly(I:C) group was increased significantly poly(I:C), we detected the proinflammatory levels of mice (Fig. 3A; p , 0.01), and similar results were observed in the mi- responses to subsequent LPS treatment following poly(I:C) pre- croglia OGD model (Supplemental Fig. 2A, 2B). Second, in the treatment. The results showed that poly(I:C) pretreatment indeed TLR32/2 mice, the cerebral infarct volumes and neurologic scores attenuated the levels of TNF-a and IL-1b in serum and CSF of mice did not differ in the poly(I:C) group compared with the I/R group stimulated by the LPS (Fig. 5F, 5G; p , 0.01), and similar results (Fig.3B).TheTUNEL+ and FJB+ cell counts in the ischemic were also obtained in vitro model (Fig. 5H, p , 0.01). Because cerebral tissues with poly(I:C) treatment were not decreased (Fig. TLR2 plays important roles in cerebral I/R injury (32), we assessed 3C, 3D), and the hippocampal neuron survival rate was not in- whether TLR2 had been downregurlated in ischemic brain tissues creased in the poly(I:C) treatment group (Fig. 3E). after administration of poly(I:C) with the real-time RT-PCR. The results showed that the TLR2 mRNA was not downregu- Poly(I:C) conferred protection against cerebral I/R injury lated in poly(I:C) group at 24 and 48 h after mice MCAO com- through the downregulation of TLR4 signaling via TLR3 pared with the I/R group (Supplemental Fig. 2C). Next, we It has been reported that pretreatment with the TLR3 ligand explored whether poly(I:C) could alleviate cerebral I/R injury in poly(I:C) downregulates the expression of TLR4 on macrophages the TLR42/2 MCAO model. The results showed that poly(I:C) did and decreases TNF-a levels to attenuate LPS-induced liver injury not reduce cerebral infarction volumes or brain water content and (19). We hypothesized that the induction of cerebral ischemic did not improve neurologic scores in the TLR42/2 MCAO mice protective effects by poly(I:C) might also be mediated by down- compared with the I/R group (Fig. 6A). We further investigated regulation of TLR4 signaling. In the MCAO model, Western blot the effect of TLR3 on TLR4 signaling in the OGD model treated 2 2 analysis showed that the expression levels of TRIF, IRF3, and with poly(I:C). In the TLR3 / microglia OGD model, the poly(I:C)- Downloaded from IFN-b (TLR3 downstream signaling molecule) in the ischemic brain induced downregulation of TLR4 signaling and NF-kBactivitywere tissues were significantly increased in the poly(I:C) group (Fig. 4A; abolished (Fig. 6B, 6C). Moreover, treatment with the anti-mouse p , 0.01). EMSA showed that poly(I:C) significantly increased TLR3 Ab yielded similar results (Supplemental Fig. 2D, 2E). IRF3 activity (Fig. 4B; p , 0.01). Similar results were obtained in Flow cytometry revealed that poly(I:C) significantly reduced the the microglia OGD model (Fig. 4C, 4D; p , 0.01). In contrast, the TLR4+ microglia, whereas treatment with TLR3 Abs diminished

expression levels of TLR4, MyD88, and NF-kB p65 were decreased the downregulation of TLR4 (Fig. 6D). Finally, in the microglia http://www.jimmunol.org/ significantly in the poly(I:C) group (Fig. 5A; p , 0.05). NF-kB OGD model, IFN-b significantly downregulated the expression activity also was decreased in the poly(I:C) group (Fig. 5B; p , levels of TLR4, MyD88, NF-kB p65, and NF-kB activity (Fig. 0.01), and similar results were also obtained in the microglia OGD 7A–C). Moreover, we tested the effect of exogenous IFN-b ad- model (Fig. 5C, 5D). Flow cytometry further verified that poly(I:C) ministration in the right lateral at 3 h after MCAO. Mice treated reduced the proportion of TLR4+ microglia (Fig. 5E; p , 0.05). To with intracerebral ventricular injection of rmIFN-b showed a sig- verify that TLR4 is indeed downregulated via TLR3 treated with nificant reduction in infarct volume (Fig. 7D; p , 0.01), the by guest on September 30, 2021

FIGURE 4. Poly(I:C) activated TLR3 signaling. (A) Expression of TRIF, IRF3, and IFN-b were examined by Western blotting in the brain tissues after 48 h of MCAO (n = 3). (B) IRF3 activity was determined by EMSA (n = 3). (C) Expression of TRIF, IRF3, and IFN-b were examined by Western blotting in microglia after 24 h of OGD (n = 3). (D) IRF3 activity was determined by EMSA in microglia (n = 3). Western blotting and EMSA data are representative of three independent experiments. **p , 0.01. CI, competitive inhibition; NC, negative control; SS, supershift. 4790 POLY(I:C) PROTECTS THE BRAIN AGAINST ISCHEMIC INJURY Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 5. Poly(I:C) downregulated TLR4 signaling. (A) Expression of TLR4, MyD88, and NF-kB p65 were examined by Western blotting in the ischermic brain tissues after 48 h of MCAO (n = 3). (B) NF-kB activity was determined by EMSA (n = 3). (C) Expression of TLR4, MyD88, and NF-kB p65 were examined by Western blotting in microglia after 24 h of OGD (n = 3). (D) NF-kB activity was determined by EMSA in microglia (n = 3). (E) TLR4 expression in microglia after 24 h of OGD as determined by flow cytometry (red, OGD plus vehicle; green, OGD plus poly(I:C); n = 4). Poly(I:C) was i.p. injected into mice 3 h before LPS (2 mg/g) i.p. injection. Serum and CSF samples were collected after 24 h. The levels of TNF-a and IL-1b in the serum (F) and CSF (G) were detected with ELISA (n = 3–5). Microglia were treated with either LPS (100 ng/ml) or vehicle for 3 h, and then poly(I:C) was added for 24 h. The levels of TNF-a and IL-1b (H) were detected with ELISA. Data are representative of at least three independent experiments. *p , 0.05, **p , 0.01. CI, competitive inhibition; NC, negative control; SS, supershift. expressions of TLR4, MyD88, and NF-kB p65 (Fig. 7E; p , 0.05), chose to administer poly(I:C) at 3, 24, and 48 h after cerebral and NF-kB activity (Fig. 7F; p , 0.01). These results suggest that ischemia because the inflammatory response mediated by the poly(I:C) confers protection against cerebral I/R injury through TLR4/MyD88 pathway occurs ∼24–48 h following cerebral I/R; the downregulation of TLR4 signaling via TLR3. Ref. 33). The neurologic scores of the poly(I:C) treatment group were significantly higher than those of the I/R group at 1, 3, 10, Poly(I:C) exerted protective effects after cerebral I/R injury in and 14 d after ischemia (Fig. 8A; p , 0.05), and the cerebral infarct mice and rats volumes were significantly reduced at 14 d (Fig. 8B; p , 0.01). The long-term protective effects of poly(I:C) after cerebral I/R Based on the suggestions of the Stroke Therapy Academic In- injury were evaluated by i.p. injection of poly(I:C) (1.25 mg/g) dustry Roundtable (34), the protective effects of poly(I:C) against at 3 and 24 h and once again 48 h after cerebral ischemia (we cerebral I/R injury were evaluated in a rat model of permanent The Journal of Immunology 4791 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 6. Poly(I:C) exerted therapeutic effects against cerebral I/R injury through the downregulation of TLR4 signaling via TLR3. (A) Infarction volumes, neurologic scores, and brain water contents after 48 h of MCAO (n = 8). Original magnification 31.1. (B) The expression levels of TLR4, MyD88, and NF-kB p65 were examined by Western blotting in TLR32/2 microglia after 24 h of OGD (n = 3). (C) NF-kB activity was determined by EMSA in TLR32/2 microglia (n = 3). (D) TLR4 expression in microglia after 24 h of OGD as determined by flow cytometry (red, OGD plus vehicle; green, OGD plus poly(I:C); n = 4). *p , 0.05. Data are representative of at least three independent experiments. cerebral ischemia. Poly(I:C) (1.25 mg/g) or vehicle was injected 14 d after ischemia, indicating that poly(I:C) may have long-term i.p. into rats 3 h after cerebral ischemia. Rats were subjected to protective effects. To our knowledge, this study is the first to cerebral ischemia (48 h). Cerebral infarct volumes (Fig. 8C; p , provide evidence suggesting that poly(I:C) has therapeutic effects 0.01) and the severity of neurologic deficits (Fig. 8D; p , 0.05) against cerebral I/R injury. Moreover, the protective effects of were significantly reduced in the poly(I:C) treatment group at 48 h poly(I:C) were abolished in the TLR32/2 mouse MCAO model, after cerebral ischemia. suggesting that the protective effects of poly(I:C) are mediated via TLR3. Discussion It has been reported that poly(I:C) preconditioning could reduce Ischemia preconditioning can reduce cerebral I/R injury (35), and I/R injury (17, 18), and our results demonstrated that poly(I:C) recent reports have shown that poly(I:C) preconditioning protects treatment 3 h after ischemia had therapeutic effects on cerebral I/R against cerebral I/R injury (17, 18). However, it has not been injury. However, a recent study has reported that TRL3 activation determined whether postischemia poly(I:C) treatment would also increased the susceptibility of neonatal brain to hypoxia and is- be effective in protecting against cerebral I/R injury. Additionally, chemia (36). The precise reasons of the observed differences of the mechanism of this potentially protective effect remains un- these results were not completely clear, which may be related known. The results showed that poly(I:C) treatment 3 h after to different animals, models, and dose of poly(I:C). We used ischemia had therapeutic effects on cerebral I/R injury. Most 1.25 mg/g poly(I:C) by i.p. injection, whereas Stridh et al. (36) used importantly, postischemia poly(I:C) treatment ameliorated neu- 10 mg/kg. Moreover, we adopted the MCAO model, whereas the rologic deficits and reduced the cerebral infarct volume up to hypoxic-ischemia model was used by Stridh et al. Moreover, 4792 POLY(I:C) PROTECTS THE BRAIN AGAINST ISCHEMIC INJURY Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 7. IFN-b downregulated TLR4 signaling and attenuated cerebral infarction volume. Following 3 h of microglia OGD, 500 U/ml rmIFN-b was added, and the microglia were detected 24 h later. (A) IFN-b treatment downregulated the expression levels of TLR4, MyD88, and NF-kB p65. (B) NF-kB activity was determined by EMSA in microglia (n = 3). (C) TLR4+ microglia after 24 h of OGD (n = 3). (D) Infarction volume was decreased in mice treated with intracerebral ventricular injection of rmIFN-b after 48 h of MCAO (n = 8). Original magnification 31.1. (E) Intracerebral ventricular injection of rmIFN-b downregulated the expression levels of TLR4, MyD88, and NF-kB p65 in ischemic brain tissues. (F) NF-kB activity was determined by EMSA (n = 3). *p , 0.05, **p , 0.01. Data are representative of at least three independent experiments.

Puyal et al. (37) showed that the adult mice pathophysiological involved in ischemic neuronal injury and aggravate neurologic mechanism of cerebral I/R injury were different from the neonatal deficits (3). Furthermore, I/R can also injure the blood–brain mice, and whose early autophagic death was prominent in the barrier and cause increases in the expression levels of adhesion ischemic penumbra of neonatal mice; however, necrotic and ap- molecules and the infiltration of inflammatory cells from the cir- optotic cell death in the early ischemic neurons play an important culation, both of which can aggravate inflammatory injury (35, role in the adult mice (38). Therefore, we speculated that the 40). Our data demonstrated that poly(I:C) administered 3 h after different pathophysiological mechanisms may also be an impor- ischemia can inhibit inflammatory responses and reduce cerebral tant reason of the differing results. I/R injury. These findings suggest that the application of rt-PA Currently, rt-PA thrombolytic therapy administered ∼3–4.5 h combined with poly(I:C) could attenuate I/R inflammation inju- following ischemic stroke is the only effective treatment (2, 39); ries and improve neurologic deficits. Research in this area is on- however, revascularization contributes to I/R injury and can ag- going. gravate neurologic deficits. It is well known that inflammation It is generally acknowledged that neuronal apoptosis is involved plays an important role in cerebral I/R injury. Proinflammatory in the response of the brain to cerebral I/R injury (41). Bcl-2 is factors produced by microglia, such as TNF-a and IL-1b, are important for cell survival and antiapoptotic effects, whereas Bax The Journal of Immunology 4793

FIGURE 8. Poly(I:C) exerted protective effects against cerebral I/R injury in mice and rats. Poly(I:C) (1.25 mg/g) or vehicle was injected i.p. into mice at 3 and 24 h and once again 48 h after cerebral ischemia. (A) The neurologic scores were significantly different between the poly(I:C) group and the vehicle group (p , 0.001, repeated measures ANOVA on ranks; n = 8). When the days were analyzed individually, there were differences between the vehicle and poly(I:C) groups on days 1, 3, 10, and 14 (p , 0.05, n = 8). (B) TTC staining after 14 d of MCAO (n = 6). Poly(I:C) (1.25 mg/g) or vehicle was injected i.p. to rats 3 h after cerebral ischemia. Rats were subjected to cerebral is- chemia (48 h). Original magnification 31.1. (C) Ce- rebral infarct volumes after 48 h of MCAO in the rat model of permanent MCAO (n = 9). (D) Neurological scores after 48 h of MCAO in rats (normal score, 0; maximal score, 12; n = 9). *p , 0.05, **p , 0.01. Downloaded from has been demonstrated to promote apoptosis. It has been reported with the I/R group, suggesting that poly(I:C) protects against ce- that LPS can induce the apoptosis of dental pulp cells and the rebral I/R injury via downregulation of TLR4 signaling. However, expression of Bax (42), whereas TLR4-deficient mice exhibit at- poly(I:C) is a ligand of TLR3, not TLR4; thus, we hypothesized tenuated doxorubicin-induced cardiac apoptosis, increased levels that poly(I:C) provides therapeutic effects against cerebral I/R http://www.jimmunol.org/ of Bcl-2, and decreased levels of Bax (43). Our results are in good injury through the downregulation of TLR4/MyD88 signaling agreement with these results. Poly(I:C) treatment increased Bcl-2 via TLR3. This view was confirmed based on the following evi- expression significantly and decreased Bax expression in both the dence. First, in TLR32/2 mice, poly(I:C) did not downregulate MCAO model and the mixed neuronal-glial cellular OGD model. TLR4/MyD88 signaling, and the protective effect of poly(I:C) The protective effects of poly(I:C) in cerebral I/R injury are ap- against cerebral I/R injury was abolished. Second, after blocking parently related to the increase in Bcl-2 expression, the reduction with the TLR3 mAb, the expression levels of TLR4, MyD88, and in Bax expression, and the attenuation of neuronal apoptosis. NF-kB were not decreased. Third, IFN-b significantly downreg- Recent research has demonstrated that Hsp27 and Hsp70 have ulated the expression of TLR4, MyD88, and NF-kB in the wild- protective effects in cerebral I/R injury (29, 30). In the present type microglial OGD model. Fourth, intraventricular injection of by guest on September 30, 2021 study, we also observed that Hsp27 and Hsp70 levels were sig- rmIFN-b after mice MCAO attenuated infarct volume and down- nificantly increased in the poly(I:C) group. This finding suggests regulated TLR4 signaling and NF-kB activity. Finally, poly(I:C) that the protective effects of poly(I:C) are related to the increased levels pretreatment attenuated the levels of TNF-a and IL-1b in serum of Hsp27 and Hsp70. However, it remains unclear how poly(I:C), and CSF of mice stimulated by the LPS. It has been reported that a TLR3 ligand, increases Hsp27 and Hsp70 levels in ischemic cerebral the inducible production of TRIM30-a (activated by IRF3) tissue. Recent reports have shown that poly(I:C) cannot only in- negatively regulates the TLR4/NF-kB signaling pathway (46). duce Hsp70 expression but also promote the extracellular release Although similar results were observed in the IFN-b group, the of Hsp70 (44). It has also been reported that the signaling mole- exact molecular mechanisms by which IFN-b mediates the cule IFN-b, which is downstream of TLR3, can upregulate the downregulation of the TLR4/MyD88 pathway require further expression of Hsp70 in splenic dendritic cells (45). This finding study. suggests that the protective effects of the poly(I:C)-dependent The Stroke Therapy Academic Industry Roundtable has pro- upregulation of Hsp27 and Hsp70 may be related to the activa- posed some suggestions to facilitate the translation of drugs from tion of the TLR3/TRIF signaling pathway. However, details re- the bench to clinical trials. First, ideal neuroprotective drugs should garding the molecular mechanisms of this process require further have short- and long-term effects. Second, the drugs should be study. effective in at least two animal models. Finally, the drugs should be We and other investigators have demonstrated that TLR4 sig- protective against both transient and permanent focal ischemia naling plays a critical role in the response to cerebral I/R injury (9, (34). Thus, based on our results, poly(I:C) meets the requirements 10). The DAMPs act on TLR4 of microglia to activate the of the above suggestions. Poly(I:C) reduced the cerebral infarct MyD88/NF-kB signaling pathway, which produces inflammatory volume and improved neurologic deficits 14 d after cerebral is- factors that contribute to inflammatory injury and aggravate chemia, and similar results were demonstrated in the rat model of neurologic impairments (7). Thus, inhibition of TLR4 signaling permanent MCAO. Therefore, poly(I:C) is a promising drug for strongly protects against cerebral ischemia (7, 15). It has been the treatment of cerebral I/R injury, and related clinical research reported that pretreatment with the TLR3 ligand poly(I:C) down- trial is ongoing. regulates the expression of TLR4 on macrophages and decreases Collectively, our data show that poly(I:C) exerted therapeutic TNF-a levels to attenuate LPS-induced liver injury (19). Our results effects against cerebral I/R injury through the downregulation of also revealed that the levels of TLR4 and its downstream signaling TLR4/MyD88 signaling via TLR3. We suggest that the application molecules MyD88 and NF-kB p65 were downregulated, and TNF-a of rt-PA in combination with poly(I:C) could ameliorate cerebral and IL-1b levels were decreased significantly after postischemia I/R inflammatory injury and improve neurologic deficits. Therefore, poly(I:C) treatment. Moreover, poly(I:C) did not further alleviate poly(I:C) is a promising new drug candidate for the treatment of cerebral I/R injury in the mouse TLR42/2 MCAO model compared cerebral infarcts. 4794 POLY(I:C) PROTECTS THE BRAIN AGAINST ISCHEMIC INJURY

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