cerebral inflammation plays an 
 important role in the pathogenesis of secondary brain injury following 
 traumatic brain injury ( tbi ) [ 1 , 2 ] . proinflammatory nuclear factor kappa b ( nf-b ) 
 signaling pathway has been well documented in previous studies of our 
 laboratory [ 3 , 4 ] . increased levels of inflammatory agents with the injured 
 brain , including tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , 
 interleukin-6 ( il-6 ) , and intercellular adhesion molecule 1 ( icam-1 ) , are 
 believed to contribute to the cerebral damage .
their mediator nf-b activation enhances the transcription of proinflammatory 
 cytokines , and the cytokines are known to in turn activate nf-b .
the 
 positive feedback is believed to serve to amplify inflammatory signals and exacerbate 
 brain injury after tbi .
recent researches have demonstrated that nuclear factor 
 erythroid 2-related factor 2 ( nrf2 ) , a key transcription factor that regulates 
 the cellular antioxidant response , plays a broader role in modulating acute 
 inflammatory response [ 8 , 9 ] . under basal conditions ,
nrf2 is sequestered in 
 the cytoplasm by the cytosolic regulatory protein keap1 . in conditions of 
 oxidative or xenobiotic stress , nrf2 translocates from the cytoplasm to the 
 nucleus , and
sequentially binds to a promoter sequence called the antioxidant 
 response element ( are ) , resulting in a cytoprotective response which is 
 characterized by upregulation of a group of antioxidant enzymes and decreased 
 sensitivity to oxidative damage [ 1012 ] .
these antioxidant enzymes have also 
 been shown to protect cells against acute inflammatory response .
numerous studies have reported that nrf2 plays a critical 
 role in counteracting inflammation in a variety of experimental models .
nrf2 
 protects against allergen - mediated airway inflammation , cigarette 
 smoke - induced emphysema , dextran sulfate sodium ( dss)-mediated colitis 
 , inflammation - mediated colonic tumorigenesis , and inflammatory 
 responses during skin wound healing .
furthermore , nrf2 has also been 
 reported as a crucial regulator of the innate immune response and survival 
 during experimental sepsis . in one of our previous studies
therefore , it may be reasonable to postulate that nrf2 plays 
 an important role in limiting the cerebral inflammatory response after tbi . in 
 our study
, we evaluated the influence of nrf2 genotype on the cerebral upregulation 
 of nf-b activity , proinflammatory cytokine , and icam-1 after tbi .
our experiments were conformed to 
 guide for the care and use of laboratory animals from national institutes of 
 health and approved by the animal care and use committee of nanjing university . 

thomas w. 
 kensler ( johns hopkins university , baltimore , md , usa ) .
homozygous wild - type nrf2 ( + /+ ) 
 and nrf2 ( /)-deficient mice were generated from inbred heterozygous nrf2 ( + / ) 
 mice .
genotypes of nrf2 ( + /+ ) and nrf2 ( / ) mice were confirmed by pcr 
 amplification of genomic dna isolated from the blood .
pcr amplification was 
 carried out by using three different primers , 5-tggacgggactattgaaggctg-3 ( sense for both genotypes ) , 5-cgccttttcagtagatggagg-3 ( antisense for wild - type ) , and 5-gcggattgaccgtaatgggatagg-3 ( antisense for lacz ) . 

age- and weight - matched adult male mice ( 68 weeks , 2832 g ) were 
 separated into four groups ( n = 10 per group ) : group i , sham 
 wild - type ( nrf2 + /+ ) ; group ii , injured 
 wild - type ( nrf2 + /+ ) ; group iii , sham - deficient 
 ( nrf2 / ) ; group iv , injured - deficient 
 ( nrf2 / ) .
the mice of sham and injured groups were subjected to identical anesthetic alone or experimental tbi , respectively .
five mice 
 in each group were sacrificed for electrophoretic 
 mobility shift assay ( emsa ) and enzyme - linked immunosorbent assay ( elisa ) 
 analysis and the others were for immunohistochemistry study .
the mouse 
 model of tbi was employed as described   with recent minor modification .
the mice were anesthetized by intraperitoneal injection 
 with sodium pentobarbital ( 50 mg / kg ) .
a round , flat , and 6 mm diameter teflon impounder was centered between the ears and eyes .
tbi was 
 induced by a 100 g weight 
 dropped from a 12 cm height 
 along a stainless steel string , which translated into 1200 g / cm .
brain injury - induced apnea was then treated 
 for 3 minutes with 100% oxygen administration and chest compression to 
 stimulate the respiration .
this model is generally associated with 20% of 
 mortality within the first 5 minutes postinjury and no delayed mortality was 
 observed thereafter .
heart rate , arterial blood pressure , and rectal temperature were 
 monitored , and the rectal temperature was kept at 37  0.5c ( physical cooling if required ) throughout experiments . at the 24 hours following sham or 
 injury ,
cortex tissue was rapidly taken from the fresh brain at the site of 
 lesion ( figure 1 ) , and stored in liquid nitrogen immediately .
for 
 immunohistochemistry , mice were perfused with cold saline ( 4c ) , 
 followed by 4% neutral - buffered formalin .
the cortex tissue was taken , stored 
 overnight in 4% neutral - buffered formalin , and then embedded in paraffin .
briefly , 
 frozen brain samples were homogenized in 0.8 ml ice - cold buffer a composed of 10 mmol / l hepes ph 7.9 , 10 mmol / l kcl ,   2 mmol / l 
 mgcl2 , 0.1 mmol / l edta , 1 mmol / l dithiothreitol ( dtt ) , and 0.5 mmol / l phenylmethylsulfonyl 
 fluoride ( pmsf ) ( all from sigma chemical co. , st . louis , mo , usa ) .
the homogenates 
 were incubated on ice for 30 minutes and vortexed for 30 seconds after addition 
 of 50 l 10% np-40 ( sigma chemical co. , mo , usa ) .
the mixture was then 
 centrifuged for 10 minutes ( 5000  g , 4c ) .
the pellet was suspended in 100 l ice - cold buffer b composed of 50 mmol / l 
 hepes ph 7.9 , 50 mmol / l kcl , 300 mm nacl , 0.1 mmol / l edta , 1 mmol / ldtt , and 0.5 mmol / l pmsf , and 10% ( v / v ) glycerol 
 and incubated on ice 30 minutes with frequent mixing .
after centrifugation 
 ( 12000  g , 4c ) for 15 minutes , the supernatants were collected as nuclear extracts and 
 stored at 70c for further use .
protein concentration was determined using a 
 bicinchoninic acid assay kit with bovine serum albumin as the standard ( pierce 
 biochemicals , rockford , ill , usa ) .
emsa was performed using a 
 commercial kit ( gel shift assay system ; promega , madison , wis , usa ) 
 following the methods in our laboratory .
consensus oligonucleotide probe ( 5-agttga ggggactttcccaggc-3 ) was end - labeled with [ -p]atp 
 ( free biotech . ,
nuclear protein ( 10 g ) was preincubated in a total volume of 9 l in a 
 binding buffer , consisting of 10 mmol / l tris - hcl ( ph 7.5 ) , 4% glycerol , 1 mmol / l 
 mgcl2 , 0.5 mmol / l m edta , 0.5 mmol
g / l poly-(deoxyinosinic - deoxycytidylic 
 acid ) for 15 minutes at room temperature .
after addition of the 1 l p - labled 
 oligonucleotide probe , the incubation was continued for 20 minutes at room temperature . 

reaction was stopped by adding 1 l of gel loading buffer and the mixture was 
 subjected to nondenaturing 4% polyacrylamide gel electrophoresis in 0.5  tbe 
 buffer ( tris - borate - edta ) .
after electrophoresis was conducted at 390 v for 1 hour , 
 the gel was vacuum - dried and exposed to x - ray film ( fuji hyperfilm , tokyo , japan ) 
 at 70c with an intensifying screed .
levels of nf-b dna binding activity were 
 quantified by computer - assisted densitometric analysis .
frozen brain samples were homogenized in 1 ml of buffer containing 1 mmol / l of pmsf , 1 mg / l of pepstatin a , 1 mg /
l of aprotinin , and 1 mg / l of leupeptin in pbs 
 solution ( ph 7.2 ) with a glass homogenizer and then centrifuged at 12000 g for 20 
 minutes at 4c .
the supernatant was then collected and total 
 protein was determined by the bradford method . 

the levels of inflammatory cytokines were quantified using enzyme - linked 
 immunosorbent assay ( elisa ) kits specific for mouse according to the 
 manufacturers ' instructions ( tnf- from diaclone 
 research , france ; il-1 , il-6 from biosource europe sa , belgium ) and previous study of our laboratory . 

the paraffin - embedded sections ( 4 m ) were used for immunohistochemical 
 assay , which was performed with a goat antimouse icam-1(cd54 ) antibody ( diluted 
 1:200 , r&d systems , inc .
, 
 minn , usa ) , 
 according to previous studies of our laboratory .
the sections were 
 incubated with the diluted antibody overnight at 4c in a humid chamber , washed , and blocked with 1.6%   h2o2 in phosphate - buffered saline 
 ( pbs ) for 10 minutes .
after washing with pbs again , sections were then 
 incubated with biotinylated second antibodies for 1 hour at room temperature . 

diaminobenzidine ( dab ) was used as chromogen and counterstaining was done with 
 hematoxylin .
the number of positive microvessels in each section was counted in 
 10 microscopic fields ( at 100  magnifications ) and averaged for the positively 
 immunostained vessel number of per visual field .
all data were 
 expressed as mean  sem , student 's t - test was used to analyze the differences between the sham and tbi groups within a 
 single genotype as well as between genotypes .
nf-b activation in the nuclear 
 extracts was assessed by emsa . as shown in 
 figure 2 , low nf-b banding activity 
 ( weak emsa autoradiography )
tbi induced activation of nf-b in the cortex 
 of both nrf2 ( + /+ ) and nrf2 ( / ) mice .
nrf2 ( / ) mice showed an increased 
 susceptibility to tbi - induced activation of nf-b than their wild - type nrf2 ( + /+ ) counterparts .
concentrations of tnf- , il-1 , and il-6 in the brain samples were measured by 
 elisa . as shown in figure 3 , low concentrations of tnf- , il-1 , and il-6
tbi 
 induced upregulation of tnf- , il-1 , and il-6 in the cortex of both nrf2 ( + /+ ) and nrf2 
 ( / ) mice .
nrf2 ( / ) mice showed larger increase in cortical levels of tnf- , il-1 , and il-6 than their wild - type 
 nrf2 ( + /+ ) littermates after tbi . for assessment of the expression of icam - a in the brain after tbi , 
 immunohistochemical study for icam-1 was performed .
as shown in figure 4 , few 
 icam-1-immunostained cerebral microvessels were observed in sham - operated mice 
 of both genotypes . at the 24 hours after tbi
, the number of icam-1 positive 
 vessels was significantly increased in the cortex of both nrf2 ( + /+ ) and nrf2 
 ( / ) mice .
nrf2 ( / ) mice showed larger increase in the number of icam-1 
 positive vessels than their wild - type 
 nrf2 ( + /+ ) littermates after tbi .
the most important finding of this 
 study is that nrf2 ( / ) mice had more inflammatory cytokines tnf- , il-1 and il-6 
 production , icam-1 expression , and their mediator nf-b activation in brain after tbi compared 
 with their wild - type nrf2 ( + /+ ) counterparts .
these findings reported here 
 suggest for the first time that nrf2 may play an important role in limiting the 
 cerebral inflammatory response after tbi through modulating the proinflammatory 
 nuclear factor kappa b ( nf-b ) signaling pathway .
activation of nf-b signaling pathway has been shown to be 
 central to the pathophysiology of cerebral inflammatory response induced by tbi 
 [ 6 , 7 ] .
nf-b can be activated by lesion - induced oxidative stress , bacterial 
 endotoxin , and cytokines .
the functional importance of nf-b in 
 inflammation is based on its ability to regulate the promoters of multiple inflammatory 
 genes , including tnf- , il-1 , il-6 , and icam-1 .
tnf- 
 is reported to be a major initiator of inflammation and is released early after 
 an inflammatory stimulus .
il-6 
 is increased after tnf- and is considered to be an important proinflammatory 
 cytokine in contribution to both morbidity and mortality in condition of 
  uncontrolled  inflammation .
icam-1 , a member of the immunoglobulin 
 superfamily which can be profound induced after cytokine challenge , is 
 important in the recruitment of leukocytes during the inflammatory process . 

this inflammatory agent network is believed to be important in the generation 
 of acute inflammatory response .
nf-b activation enhances the transcription of 
 proinflammatory cytokines , and the cytokines are known to in turn activate 
 nf-b .
the positive feedback is believed to serve to amplify inflammatory 
 signals and exacerbate brain injury after tbi . in the present study ,
we 
 evaluated the influence of nrf2 genotype in the tbi - induced activation of proinflammatory 
 nf-b signaling pathway in the brain .
the results showed that disruption of nrf2 in mice caused a greater activation of 
 nf-b signaling pathway which played a critical role in the pathophysiology of 
 cerebral inflammatory response induced by tbi .
the observed interplay between 
 nrf2 and nf-b signaling corresponds well to the results of study on experimental sepsis , 
 which have demonstrated that nrf2-deficient mice displayed increased nf-b 
 activation in response to lipopolysaccharide ( lps ) .
although numerous in vivo studies 
 have reported that nrf2 plays a critical role in counteracting the inflammation 
 in a variety of experimental models [ 1419 ] , the findings 
 which we have confirmed and extended in the model of tbi in the present study , the 
 precise mechanism underlying this network is still unclear .
several lines of 
 evidence suggest that nrf2 regulates the inflammatory response by inhibiting 
 proinflammatory nf-b activation through maintenance of redox homeostasis . 

oxidative stress from reactive oxygen species ( ros ) is believed to be involved 
 in the progression of secondary brain injury following tbi .
activation of 
 the nf-b signaling pathway has been shown to be responsive to excess ros and 
 is important in the generation of inflammation .
nrf2 , as a key antioxidant 
 transcription factor involved in the intracellular antioxidant defense systems , 
 has been shown to play an important role in limiting ros levels and thereby 
 affect redox - sensitive nf-b signaling pathway involved in the inflammation [ 8 , 1012 ] .
the 
 protective function of nrf2 is mainly mediated by a group of nrf2-regulated 
 antioxidant and detoxifying enzymes .
therefore , the augmentation of cellular 
 antioxidative or detoxification systems via activation of nrf2-regulated 
 enzymes resulting in decreased proinflammatory cytokines production and 
 adhesion molecules expression via inactivation of nf-b represents a possible 
 anti - inflammatory mechanism for the attenuated inflammatory response seen in 
 brains from nrf2 ( + /+ ) mice but not nrf2 ( / ) mice after tbi .
we then in this 
 study postulated that nrf2 regulates the tbi - induced cerebral inflammatory 
 response may at least in part through modulating the cerebral redox status and proinflammatory 
 nf-b signaling pathway .
additional work is necessary to 
 elucidate the whole mechanisms involved in these complicated 
 networks . in summary , this present study showed that nrf2 plays a 
 protective role in tbi - induced cerebral upregulation of inflammatory agents in 
 mice .
we found that nrf2 ( / ) mice are more susceptible to tbi - induced 
 cerebral nf-b activation , inflammatory cytokine 
 tnf- , il-1 and il-6 production , and icam-1 expression , which then contributed to exacerbated brain injury after tbi . to the 
 best of our knowledge , this is the first study that elucidates the interplay between nrf2 and proinflammatory nf-b signaling pathway in the brain following tbi .
these 
 findings raise the possibility that nrf2 will be a new therapeutic target for 
 the treatment tbi .
 inflammatory response plays an important role in the pathogenesis of secondary brain injury after traumatic brain injury ( tbi ) . 
 nuclear factor erythroid 2-related factor 2 ( nrf2 ) is a key transcription factor that plays a crucial role in cytoprotection against inflammation . 
 the present study investigated the role of nrf2 in the cerebral upregulation of nf-b activity , proinflammatory cytokine , and icam-1 after tbi . 
 wild - type nrf2 ( + /+ ) and nrf2 ( /)-deficient mice were subjected to a moderately severe weight - drop impact head injury . 
 electrophoretic mobility shift assays ( emsas ) were performed to analyze the activation of nuclear factor kappa b ( nf-b ) . 
 enzyme - linked immunosorbent assays were performed to quantify the production of tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , and interleukin-6 ( il-6 ) . 
 immunohistochemistry staining experiments were performed to detect the expression of intercellular adhesion molecule-1 ( icam-1 ) . 
 nrf2 ( / ) mice were shown to have more nf-b activation , inflammatory cytokines tnf- , il-1 and il-6 production , and icam-1 expression in brain after tbi compared with their wild - type nrf2 ( + /+ ) counterparts . the results suggest that nrf2 plays an important protective role in limiting the cerebral upregulation of nf-b activity , proinflammatory cytokine , and icam-1 after tbi . 