cerebral infarction , or cerebral ischemia , is a major cause of death and disability in adults , causing a heavy burden for the health system and patients families . studies have pointed out various possible mechanisms in the occurrence of cerebral infarction [ 14 ] . this is supported in animal models , as inflammation aggravated brain tissues even after the primary focal cerebral infarction [ 57 ] . detailed observation revealed that a series of t cell - induced inflammatory responses occurred in the peripheral region of ischemia tissues , and the severity of inflammatory response is positively related with neurological damage . meantime , t cell - secreted cytokines also play a role in the inflammatory injury after cerebral ischemia . numerous in vitro and in vivo studies have indicated tumor necrosis factor ( tnf)- , interleukin ( il)-1 , and il-17 as important factors in the pathogenesis of infraction - induced cerebral tissue damage , as their expression levels are elevated in both ipsilateral brain tissues and peripheral blood serum [ 1014 ] . as a classical co - stimulatory signal molecule , inducible co - stimulatory molecule ( icos ) plays a critical role in the activation of t cell immune efficacy . specifically , t cells can acquire certain co - stimulatory signals by the interaction between icos and its ligands , during the antigen - specific interaction between t cells and dendritic cells or antigen specific b cells . studies have proved the role of co - stimulatory signals induced by the binding between icos and its ligands in regulating various cellular events such as t cell activation / proliferation and secretion of cytokines , including tnf- , il-1 , and il-17 . recent studies have demonstrated the connection between icos - ligand signal pathways and autoimmune disease or inflammatory response . further studies revealed an important role of icos in the differentiation and maintenance of th cells [ 1719 ] . data on the role of icos in cerebral infarction , however , is still lacking . thus , we designed icos - specific sirna to inhibit the in vitro gene expression , and then applied sirna in cerebral infarction rat models . mortality rates and neurological scores , along with serum cytokine levels , were observed to reflect the role of icos in the pathogenesis of cerebral ischemia - related tissue damages . the spleen was removed , ground , filtered , and rinsed by hank s solution . the collected spleen cell suspension 30 ml of erythrocyte lysate was added for 5-min incubation . after the complete lysis of erythrocytes , the mixture was re - centrifuged at 2000 rpm for 3 min to discard erythrocyte debris . after washing by hank s solution , lymphocytes were re - suspended in rpmi-1640 medium ( yubo biotech , china ) with 10% fetal bovine serum ( fbs ) . cells were cultured using dmem culture medium ( yubo biotech , shanghai , china ) containing 5 mm d - glucose at 37c with 5% co2 . rats were used for all experiments , and all procedures were approved by the animal ethics committee of the second xiangya hospital of central south university . one day before the transfection , cultured lymphocytes were inoculated into petri dishes without antibiotics . cells with 30% confluence were transfected with sirna of icos or non - specific ( ns ) controlled sirna ( santa cruz , usa ) using lipofectamine 2000 ( invitrogen , usa ) according to the manufacturer s instructions . in brief , diluted liposome solutions were mixed with sirna , followed by 5-min incubation at room temperature . the mixture was then added into the cultured dish , which was filled with 5 ml of serum - free dmem culture medium . four - eight hours after transfection , cells ( 510 ) in all groups were collected and extracted for total rna using trisol kits ( invitrogen , usa ) following the manufacturer s instructions . icos - specific primers were designed by primer 5.0 software with the following sequences : icos - f , 5-gug cac gac uca aua ta-3 ; icos - r , 5-ttc acg ugc uga cgc ag-3 ; -actin - f , 5-ggt gtg atg gtg ggt atg ggt-3 ; -actin - r , 5-ctg ggt cat ctt ttc acg gt-3. quantitative rt - pcr was carried out using sybr pcr kits ( invitrogen , usa ) following the manufacturer s instructions with the following conditions : pre - denature for 5 min at 94c ; 30 cycles of amplification , each containing 1-min denature at 94c , 1-min annealing at 60c , and 3-min elongation at 72c ; ending with 5-min elongation at 72c . relative expression level of mrna was calculated using 2 method based on the standard curve . thirty sd rats ( 15 males and 15 females ; body weight= 280~300 g ) were obtained from the experimental animal center of the chinese academy of science ( shanghai ) . rats were anaesthetized by 3% pentobarbital ( 1 ml/100 g ) and fixed on the table . a middle anterior neck incision was made to expose the left common carotid artery , followed by the separation of internal / external carotid artery . a metal clip was used to temporally block the common carotid artery and a 0.5-ml thrombus mixture was then injected via a catheter inserted into the left external carotid artery , which was clipped immediately after the infusion . the common carotid artery was opened to let the thrombus enter the cerebral artery , causing multifocal cerebral infarction . a parallel sham group was also prepared with the same surgical procedure and saline injection . one day after the surgery , all rats were examined for neurological behaviors and were graded as : grade 0 , normal activity ; grade i ( mild ) , able to walk but with decreased body resistance ( animals can be pushed back for 10~15 cm ) ; grade ii ( moderate ) , can walk but with cycled gait pattern ; grade iii ( severe ) , can stand but not walk . all rats meeting these criteria were randomly divided into model group , icos sirna treatment group and sirna control group . three days after the surgery , 0.2 ml saline , icos sirna , or ns controlled sirna were applied via intravenous injection . mortality rates of all animals were continuously monitored and neurological behaviors were observed 2 weeks after the injection . venous blood samples were collected and extracted for lymphocytes , which were cultured in dmem medium containing 10% fbs . after 48 h , supernatants of cell culture were collected and tested for tnf- , il-1 , and il-17 levels using elisa kits ( bd corp . the spss 17.0 software package was used to analyze all collected data , which are presented as mean  standard deviation ( sd ) . the spleen was removed , ground , filtered , and rinsed by hank s solution . the collected spleen cell suspension 30 ml of erythrocyte lysate was added for 5-min incubation . after the complete lysis of erythrocytes , the mixture was re - centrifuged at 2000 rpm for 3 min to discard erythrocyte debris . after washing by hank s solution , lymphocytes were re - suspended in rpmi-1640 medium ( yubo biotech , china ) with 10% fetal bovine serum ( fbs ) . cells were cultured using dmem culture medium ( yubo biotech , shanghai , china ) containing 5 mm d - glucose at 37c with 5% co2 . rats were used for all experiments , and all procedures were approved by the animal ethics committee of the second xiangya hospital of central south university . one day before the transfection , cultured lymphocytes were inoculated into petri dishes without antibiotics . cells with 30% confluence were transfected with sirna of icos or non - specific ( ns ) controlled sirna ( santa cruz , usa ) using lipofectamine 2000 ( invitrogen , usa ) according to the manufacturer s instructions . in brief , diluted liposome solutions were mixed with sirna , followed by 5-min incubation at room temperature . the mixture was then added into the cultured dish , which was filled with 5 ml of serum - free dmem culture medium . four - eight hours after transfection , cells ( 510 ) in all groups were collected and extracted for total rna using trisol kits ( invitrogen , usa ) following the manufacturer s instructions . icos - specific primers were designed by primer 5.0 software with the following sequences : icos - f , 5-gug cac gac uca aua ta-3 ; icos - r , 5-ttc acg ugc uga cgc ag-3 ; -actin - f , 5-ggt gtg atg gtg ggt atg ggt-3 ; -actin - r , 5-ctg ggt cat ctt ttc acg gt-3. quantitative rt - pcr was carried out using sybr pcr kits ( invitrogen , usa ) following the manufacturer s instructions with the following conditions : pre - denature for 5 min at 94c ; 30 cycles of amplification , each containing 1-min denature at 94c , 1-min annealing at 60c , and 3-min elongation at 72c ; ending with 5-min elongation at 72c . relative expression level of mrna was calculated using 2 method based on the standard curve . thirty sd rats ( 15 males and 15 females ; body weight= 280~300 g ) were obtained from the experimental animal center of the chinese academy of science ( shanghai ) . rats were anaesthetized by 3% pentobarbital ( 1 ml/100 g ) and fixed on the table . a middle anterior neck incision was made to expose the left common carotid artery , followed by the separation of internal / external carotid artery . a metal clip was used to temporally block the common carotid artery and a 0.5-ml thrombus mixture was then injected via a catheter inserted into the left external carotid artery , which was clipped immediately after the infusion . the common carotid artery was opened to let the thrombus enter the cerebral artery , causing multifocal cerebral infarction . a parallel sham group was also prepared with the same surgical procedure and saline injection . one day after the surgery , all rats were examined for neurological behaviors and were graded as : grade 0 , normal activity ; grade i ( mild ) , able to walk but with decreased body resistance ( animals can be pushed back for 10~15 cm ) ; grade ii ( moderate ) , can walk but with cycled gait pattern ; grade iii ( severe ) , can stand but not walk . all rats meeting these criteria were randomly divided into model group , icos sirna treatment group and sirna control group . three days after the surgery , 0.2 ml saline , icos sirna , or ns controlled sirna were applied via intravenous injection . mortality rates of all animals were continuously monitored and neurological behaviors were observed 2 weeks after the injection . venous blood samples were collected and extracted for lymphocytes , which were cultured in dmem medium containing 10% fbs . after 48 h , supernatants of cell culture were collected and tested for tnf- , il-1 , and il-17 levels using elisa kits ( bd corp . the spss 17.0 software package was used to analyze all collected data , which are presented as mean  standard deviation ( sd ) . the transfection of cultured lymphocytes by icos sirna significantly suppressed the expression of icos , as suggested by the rt - pcr study in which icos - sirna transfection decrease the expression of the target gene by more than 40% ( figure 1 ) . as shown in table 1 , 6 out of 20 ( 30% ) rats died 2 weeks after the application of saline , 4 out of 20 ( 20% ) rats that received ns sirna died , and only 1 out of 20 ( 5% ) rats died that received icos sirna . between - group comparison showed statistical significance ( p<0.05 , 1-way anova ) . these results suggest the potential effect of icos sirna in decreasing the mortality rate of cerebral infarction . two weeks after the injection , there were 4 , 10 , and 6 rats in the model group being classified into grade i , ii , and iii , respectively . in the ns sirna group , when rats received icos sirna , however , the number of grade iii rats decreased to zero , and the number of grade i animals increased to 13 . all these differences were of statistical significance ( 1-way anova , p<0.05 ) , showing that sirna can improve the neurological behavior and ameliorate motor deficits of cerebral infarction . as shown in figure 2 and table 2 , the application of icos sirna can significantly decreased the level of tnf- , il-1 , and il-17 in peripheral blood by more than 50% ( p<0.05 , t test ) , suggesting the role of icos in the induction of secondary inflammation after cerebral ischemia . the transfection of cultured lymphocytes by icos sirna significantly suppressed the expression of icos , as suggested by the rt - pcr study in which icos - sirna transfection decrease the expression of the target gene by more than 40% ( figure 1 ) . as shown in table 1 , 6 out of 20 ( 30% ) rats died 2 weeks after the application of saline , 4 out of 20 ( 20% ) rats that received ns sirna died , and only 1 out of 20 ( 5% ) rats died that received icos sirna . between - group comparison showed statistical significance ( p<0.05 , 1-way anova ) . these results suggest the potential effect of icos sirna in decreasing the mortality rate of cerebral infarction . two weeks after the injection , there were 4 , 10 , and 6 rats in the model group being classified into grade i , ii , and iii , respectively . in the ns sirna group , when rats received icos sirna , however , the number of grade iii rats decreased to zero , and the number of grade i animals increased to 13 . all these differences were of statistical significance ( 1-way anova , p<0.05 ) , showing that sirna can improve the neurological behavior and ameliorate motor deficits of cerebral infarction . as shown in figure 2 and table 2 , the application of icos sirna can significantly decreased the level of tnf- , il-1 , and il-17 in peripheral blood by more than 50% ( p<0.05 , t test ) , suggesting the role of icos in the induction of secondary inflammation after cerebral ischemia . a reliable and consistent methodology to induce cerebral ischemia is therefore of critical importance for elucidating the pathology of cerebral ischemia . our animal models showed limb paralysis on the contralateral side with various severities , consistent with multi - focal cerebral infarction in clinics [ 59 ] . all animals that received the thrombosis infusion reached neurological deficit grade i or above ( table 3 ) , suggesting the efficacy of the surgery . stress response can be activated after acute cerebral infarction for the further induction of the hypothalamus - pituitary - adrenal gland axis , thereby causing the secretin of cortical hormones for modulating t cell distribution and function [ 1216 ] . icos and its ligand , on the other hand , exert critical functions on t cell - related immune response . in this study , icos - specific sirna was for the first time synthesized and utilized in rat cerebral infarction models . the efficacy of sirna can be confirmed by in vitro lymphocytes ( figure 1 ) . further in vivo testing showed that , compared to those that received ns - controlled sirna or saline , cerebral infarction rats after icos sirna injection had significantly lowered mortality rate ( table 1 ) and improved motor behaviors , as shown by neurological grades ( table 3 ) . our results suggest that the suppression of icos gene expression can effectively improve the limb movement and motor coordination of cerebral infarction rats , lower the mortality rate , and ameliorate neural tissue damage . a further investigation revealed a possible mechanism of icos by showing that the application icos sirna decreased the secretion of tnf- , il-1 , and il-17 from th1 lymphocytes ( figure 2 ) . these data suggest that icos is potentially involved in the inflammatory response after primary cerebral ischemia . various studies have shown that an acute inflammatory response occurred after the onset of acute cerebral infarction , resulting in the elevated expression of pro - inflammatory cytokines , including tnf- , il-1 , and il-17 [ 1719 ] . other studies demonstrated the efficacy of multiple neural protective agents such as edaravone in decreasing serum levels of pro - inflammatory factors and treating cerebral ischemia [ 1216 ] . our results revealed a similar role of icos sirna in suppressing pro - inflammatory cytokines and in inhibiting inflammatory response . both in vitro and in vivo studies have shown the critical function of icos and its ligands in both formation and function of t cells , th1 , and th17 cells [ 2023 ] . further studies showed the facilitation of icos signal pathway in the secretion of th1-related cytokines ( ifn- , tnf- , and il-1 ) and th17-related cytokine ( il-17 ) . icos gene knockout mice showed no immunity against bacterial or viral inflammation , accompanying with deficits in th cell - related immune response , thereby attributing the th cell immunity to the icos signal pathway . studies also showed that the absence of icos inhibited the progression of autoimmune disease or graft rejection . in summary , this study for the first time indicated the potential treatment efficacy of icos sirna in cerebral infarction , possibly via the inhibition of th1 and th17 cytokine secretion . in clinical practice , only thrombolytic therapy at the super - acute period , urokinase and streptokinase have been approved for treating cerebral infarction . all these methods , however , only exert satisfactory efficacy within 3~6 hours of the occurrence of cerebral ischemia . our studies showed that the application of icos sirna 3 days after cerebral infarction can still improve motor behavior and decrease mortality rates , thereby providing a novel candidate for brain ischemia treatment .
backgroundt cell - induced inflammatory response and related cytokine secretion at the injury site may participate in the pathogenesis of cerebral infarction . 
 recent studies established inducible co - stimulatory molecule ( icos ) as a novel t cell - related factor for its activation and functions . 
 we thus investigate the role of icos in cerebral infarction.material/methodsthe sirna of icos was first used to suppress the gene expression in cultured lymphocytes . 
 an in vivo study was then performed by intravenous application of icos sirna in cerebral infarction rats . 
 survival rates , neurological scores , serum tumor necrosis factor ( tnf)- , interleukin ( il)-1 , and il-17 levels were observed.resultsthe expression of icos in cultured lymphocytes was significantly suppressed by sirna . in the in vivo study , the application of sirna effectively lowered mortality rates of rats , in addition to the improvement of neurological behaviors and amelioration of cerebral tissue damage . 
 serum levels of tnf- , il-1 and il-17 were all significantly suppressed after sirna injection.conclusionsicos sirna can protect brain tissues from ischemia injuries after cerebral infarction , improve limb movement and coordination , lower the mortality rate of rats , and inhibit t cell - induced cytokines . 
 these results collectively suggest the potential treatment efficacy of icos sirna against cerebral infarction .