spinal cord injury ( sci ) initiates a series of cellular and molecular cascade events and that a combination of secondary injury factors leads to a progressive neuronal injury     . it is generally accepted that , among the secondary injury factors , inflammation is the major one and plays a central role in regulating the pathogenesis of sci     [ 2 , 3 ] . attenuation of the early inflammatory response after sci may limit the extent of tissue injury and the consequent disability     [ 46 ] . the proinflammatory cytokine tnf- , which is upregulated immediately after sci , can enhance vascular permeability     . hence , tnf- is considered as a proinflammatory cytotoxic cytokine . and tnf- is primarily responsible for initiating the cascade of other cytokines in the classic immune response     [ 6 , 8 ] . inflammatory cells can release matrix metalloproteinases ( mmps ) , especially mmp-9 , which can penetrate the bbb     . mmp-9 is upregulated in leucocytes entering the spinal cord , directly facilitating their extravasation , and promoting the tissue damage that they caused     [ 9 , 10 ] . it might be a vascular permeabilizing factor and is a key determinant in secondary tissue damage after sci . nuclear factor erythroid 2-related factor 2 ( nrf2 ) , a leucine zipper redox - sensitive transcription factor , is a pleiotropic regulator of cell survival mechanisms . under the normal condition , nrf2 is sequestered in the cytoplasm by a cytosolic regulatory protein named keap1 . under condition 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 the expression of antioxidant and cytoprotective genes that attenuate tissue injury     [ 1214 ] . recent studies have demonstrated that nrf2 plays a broad role in modulating acute inflammatory response . nrf2 plays a protective role in cigarette smoke - induced emphysema     , dextran sulfate sodium- ( dss- ) mediated colitis     , inflammation - mediated colonic tumorigenesis     , and allergen - mediated airway inflammation     . our previous studies have shown that nrf2 played an important role in protecting traumatic brain injury- ( tbi- ) induced secondary brain injury by regulating inflammatory cytokines and attenuating the pulmonary inflammatory response and nf-b activation after tbi     [ 12 , 19 ] . we have found that sci could induce the activation of nrf2-are pathway in spinal cord tissue ( data not shown ) , just as pomeshchik et al . showed . therefore , it is reasonable to postulate that nrf2 plays an important role in limiting the spinal cord inflammatory response after sci . in the present study , we evaluated the effect of nrf2 genotype on the activation of nf-b , tnf- , and mmp-9 in spinal cord after sci . our experiments were conformed to the guide for the care and use of laboratory animals by national institutes of health ( nih publications no . 80 - 23 ) and approved by the animal care and use committee of nanjing university . male icr mice ( 2832  g ) were housed at 23  1c in humidity - controlled animal quarters with 12 hours light / dark cycle , and with ad libitum access to food and water throughout the study . 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 = 42 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 sci , respectively . the mouse compression model of sci established as described previously used to study spinal cord injury in mice     . briefly , the animals were anesthetized with sodium pentobarbital ( 50  mg / kg , ip ) . a longitudinal skin incision was made to expose the spine between t8 and t10 vertebral body levels , and the paravertebral muscles of the thoracic - level ( t8t10 ) vertebrae were removed . laminectomy was performed at the t8t10 level , using an operating microscope ( m500-n , laica , germany ) . in sham - operated mice , the muscles and skin wound was then closed . in order to cause an acute - compression injury , we used a vascular clip ( with 10  g force , kent scientific corporation , ins 14120 , usa ) to injure the cord at t9 by bilateral compression for 1 minute . after surgery , the animals were given a subcutaneous saline injection ( 1 - 2  ml ) immediately . they were left to recover on a warm pad until thermoregulation and the righting reflex was re - established . the rectal temperature was monitored and was kept at 37  0.5c ( with physical cooling if required ) throughout the experiment . as described , animals received manual bladder expression twice daily until urinary retention was relieved     . at 24 hours following sham operation or sci , thirty mice in each group were sacrificed for spinal cord segments collection to assay nf-b activity , tnf- , and mmp-9 . briefly , mice were deeply anaesthetized with pentobarbital sodium ( 80  mg / kg ) and were transcardially perfused with cold saline ( 4c ) , and then the tissue segments containing the lesion ( 1  cm on each side of the lesion ) were rapidly removed and stored at liquid nitrogen immediately . six mice of each group were sacrificed at 48 hours after operation for assays of spinal cord water content of spinal cord segments . the last six mice in each group were evaluated by histopathology at 24 hours after sci . they were perfused with cold saline ( 4c ) , followed by 4% neutral - buffered formalin . spinal cord segments containing the lesion ( 0.5  cm on each side of the lesion ) were taken , stored overnight in 4% neutral - buffered formalin , then were paraffin embedded , and cut into 5  m - thick sections . tissue sections ( thickness 5  m ) were deparaffinized with xylene , stained with hematoxylin / eosin ( h&e ) , and studied using light microscopy ( eclipse e100 , nikon , japan ) . the spinal cord water content was measured as previously described . at 48 hours after sci , spinal cords were removed from t7 to t11 vertebral body levels , weighed , heated at 98c for 48 hours , and reweighed . damaged neurons were counted and the histopathologic changes of the gray matter were scored on a 6-point scale     [ 23 , 24 ] : ( 0 ) , no lesion observed ; ( 1 ) , gray matter contained 1 to 5 eosinophilic neurons ; ( 2 ) , gray matter contained 5 to 10 eosinophilic neurons ; ( 3 ) , gray matter contained more than 10 eosinophilic neurons ; ( 4 ) , small infarction ( less than one - third of the gray matter area ) ; ( 5 ) , moderate infarction ( one - third to one - half of the gray matter area ) ; ( 6 ) , large infarction ( more than half of the gray matter area ) . the scores of all the sections from each spinal cord were averaged to give a final score for an individual mouse . nuclear protein extraction followed the method described by jin et al . in our laboratory     . protein concentration was determined using a bicinchoninic acid assay kit with boving 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 described in detail elsewhere     . consensus oligonucleotide probe ( 5-agttgaggggactttcccaggg-3 ) was end labeled with [ -]-atp ( free biotech . , nuclear protein ( 20  g ) was preincubated in a total volume of 20  l in a binding buffer , consisting of 10  mmol / l tris - hcl ( ph 7.5 ) , 1  mmol / l mgcl2 , 0.5  mmol / l nacl , 4% glycerol , 0.5  mmol / l edta , 0.5  mmol / l dtt , and 2  g poly di - dc for 20 minutes at room temperature . after addition of the 1  l p - labled oligonucleotide probe , the incubation was continued for 20 minutes at room temperature . after adding 1  l of gel loading buffer , the reaction was stopped , and the mixture was resolved by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5tbe buffer ( tris - borate - edta ) at 390  v for 1 hour at 4c . the gel was dried and exposed to x - ray film ( fuji hyperfilm , tokyo , japan ) at 70c with an intensifying screed . total rna was extracted from mouse spinal cord segments with ezgene tissue rna miniprep kit ( biomiga , inc . , san diego , ca , usa ) according to the manufacturer 's instructions . the cdna was synthesized using reverse transcription system ( promega corporation , madison , wi , usa ) and oligo dt from 2  g of total rna . the cdna was then amplified by pcr using various primer sets as follows : tnf- , 5-acggcatggatctcaaagac-3 , and 5-ggtcactgtcccagcatctt-3 ; mmp-9 , 5-ctactctgaagacttgccg-3 , and 5-ccatacagtttatcctggtc-3 ; -actin , 5-agtgtgacgttgacatccgta-3 , and 5-gccagagcagtaatctccttct-3     [ 19 , 27 ] . pcr products were detected by agarose gel electrophoresis in 2% nusieve agarose gels ( fmc , usa ) and visualized by ethidium bromide staining . the intensity of the bands was quantified using imagej program , and the ratios of each gene product to -actin product were used as indices of tnf- and mmp-9 mrna expression . portions of spinal cord tissues were homogenized as previously described in pbs containing 2  mmol / l of phenylmethylsulfonyl fluoride ( pmsf , sigma chemical co. ) , 1  mg / l pepstatin a , 1  mg / l aprotinin , 1  mg / l leupeptin , and phosphate - buffered saline solution ( ph 7.2 ) and centrifuged at 12,000 g for 20 minutes at 4c . the supernatant was then collected and total protein was determined by using the bradford method . tnf- protein was quantified using elisa kits specific for mouse according to the manufacturers ' instructions ( diaclone research , france ) and a previous study of our laboratory     . the cytokine tnf- content in the spinal cord samples was expressed as pg per milligram total protein . spinal cord tissues were homogenized in ripa buffer ( 1% np40 , 0.5% sodium deoxycholate , 0.1% sds , 1  mm edta , 1  mm egta , 1  mm na3vo4 , 20  mm naf , 0.5  mm dtt , 1  mm pmsf , and protease inhibitor cocktail in pbs ph 7.4 ) . the homogenate was centrifuged at 1050 g ( 4c ) for 10 min , and microsomal fraction was subsequently centrifuged at 17,000 g ( 4c ) for 20 minutes     . fifty micrograms of the resulting cytosolic protein extracts were heat denatured in laemmli sample loading buffer , separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis , and electrotransferred onto a nitrocellulose membrane . for immunoblotting , membranes were blocked with 5% nonfat dry milk in saline buffer overnight , at 4c , and then incubated with primary anti - mmp-9 antibody ( 1  :  500 , calbiochem , darmstadt , germany ) for 2 hours at room temperature in 1pbs , 5% w / v nonfat dry milk , and 0.1% tween-20 ( pmt ) . the membranes were washed three times for 5 minutes in pmt and the secondary incubations were performed with horseradish peroxidase - linked antirabbit igg ( 1  :  5000 , jackson immunoresearch , west grove , pa , usa ) . positive signals were visualized using enhanced chemiluminescence ( ecl ; amersham biosciences , bucks , united kingdom ) , and serial exposures were made to radiographic film ( fuji hyperfilm , tokyo , japan ) . densitometric analysis of the blots was performed with the image analysis program imagej . to ascertain that blots were loaded with equal amounts of proteic lysates 2% sds , 62.5  mm tris - hcl , ph 6.7 ) at 50c for 30 minutes , then washed , blocked , and reprobed overnight at 4c with mouse anti--actin monoclonal antibody ( 1  :  5000 ; sigma ) . we used gelatin zymography analysis to show mmp-9 activity of spinal cord tissue at 24 hours after sci . samples of spinal cord , prepared from the epicenter , were quickly frozen at 80c . each sample was weighed and homogenized ( 1  :  4 w / v ) in lysis buffer containing 50  mm tris - hcl , ph 7.4 , 150  mm nacl , 5  mm cacl2 , 0.2  mm nan3 , and 0.01% triton . equal amounts of the supernatant were analyzed by gel zymography as described previously on 10% sds - polyacrylamide gels copolymerized with substrate ( 1  mg / ml gelatin ) . the proteins were renatured by incubation in 2.5% triton x-100 and then incubated in substrate buffer ( 50  mm tris - hcl , ph 8.5 , 5  mm cacl2 ) for 2436 hours at 37c to enable the mmp-9 to cleave the gelatin . after rinsing in water , each gel was stained with coomassie blue for 4 hours and destained in 50% methanol . the results were analyzed by one - way anova followed by a student - newman - keuls test for multiple comparisons . similar water content of spinal cord tissue was detected in both sham - operated nrf2 ( + /+ ) and nrf2 ( / ) mice . at 48 hours after sci , water content of spinal cord significantly increased in both injured nrf2 ( + /+ ) and nrf2 ( / ) mice compared with the sham - operated mice , respectively . however , the pathological change was severer in nrf2 ( / ) mice than that in nrf2 ( + /+ ) mice ( 0.85  0.03 versus 0.78  0.02 , p < .05 , figure 1 ) . the degree of the spinal cord injury at the perilesional area was evaluated at 24 hours after sci . significant damage to the spinal cord was observed in the sci mice than that in the sham - operated mice in both nrf2 ( + /+ ) and nrf2 ( / ) mice . notably , the histological injury was severer in nrf2 ( / ) mice than that in nrf2 ( + /+ ) mice at 24 hours after sci ( 5.3  0.08 versus 4.2  0.13 , p < .01 , figure 2 ) . as shown in figure 3 , low nf-b banding activity was detected in sham - operated mice of both genotypes . sci induced activation of nf-b in the spinal cord in both nrf2 ( + /+ ) and nrf2 ( / ) mice . nrf2 ( / ) mice showed an increased susceptibility to sci - induced activation of nf-b than their wild - type nrf2 ( + /+ ) counterparts ( 3.80  0.07 versus 2.30  0.05 , p < .01 , figures 3(a ) and 3(b ) ) . the mrna levels of two inflammatory gene products , tnf- and mmp-9 , were measured by rt - pcr . similar mrna expression levels of them were detected in the spinal cord samples of both sham - operated groups . at 24 hours after sci , increased mrna expression levels in spinal cord , including tnf- and mmp-9 , were detected in both injured nrf2 ( + /+ ) and nrf2 ( / ) mice compared with the sham - operated mice , respectively . higher mrna expression levels of tnf- were found in nrf2 ( / ) mice than in nrf2 ( + /+ ) mice ( 2.01  0.05 versus 0.97  0.05 , p < .01 , figures 4(a ) and 4(b ) ) . similarly , significant difference of mmp-9 mrna level was also observed between nrf2 ( / ) mice and nrf2 ( + /+ ) mice ( 0.99  0.07 versus 0.78  0.04 , p < .01 , figures 4(c ) and 4(d ) ) . the protein expression level of tnf- in the spinal cord samples of both sham - operated nrf2 ( + /+ ) and nrf2 ( / ) mice was also similar . at 24 h after sci , higher spinal cord tissue protein expression levels of tnf- were detected in both injured nrf2 ( + /+ ) and nrf2 ( / ) mice compared with their respective sham - operated mice . similarly , higher protein expression levels of tnf- were found in nrf2 ( / ) mice than in nrf2 ( + /+ ) mice ( 3966.60  122.67 versus 3424.04  79.66 , p < .05 , figure 5 ) . there is no difference on mmp-9 expression in the spinal cord samples between sham - operated nrf2 ( + /+ ) and nrf2 ( / ) mice , as shown by the immunoreactive band migrating at ~92  kda . at 24 hours after sci , significant upregulation of mmp-9 expression levels was detected in both injured nrf2 ( + /+ ) and nrf2 ( / ) mice compared with their respective sham - operated mice . higher mmp-9 expression levels were found in nrf2 ( / ) mice than in nrf2 ( + /+ ) mice ( 0.98  0.03 versus 0.88  0.02 , p < .01 , figures 6(a ) and 6(b ) ) . the activity of mmp-9 in spinal cord tissue was measured by gelatin zymography analysis . in the spinal cord samples of both sham - operated nrf2 ( + /+ ) and nrf2 ( / ) mice , the activity of mmp-9 was similar . at 24 h after sci , higher activity of mmp-9 was detected in both injured nrf2 ( + /+ ) and nrf2 ( / ) mice compared with their respective sham - operated mice , while the activity change was severer in nrf2 ( / ) mice than in nrf2 ( + /+ ) mice ( 27922.79  1185.68 versus 22666.08  1316.41 , p in this study , we found that the mice lacking nrf2 function have significantly enhanced secondary spinal cord injury characterized by increased spinal cord edema and severer inflammatory response . increased sci - induced nf-b activation and expression of the inflammatory cytokines , tnf- and mmp-9 , were observed in the spinal cord of nrf2 ( / ) mice compared to nrf2 ( + /+ ) mice . in addition , higher level of mmp-9 activity was found in the spinal cord of nrf2 ( / ) mice than that in nrf2 ( + /+ ) mice after sci . to our knowledge , the findings in this study suggest for the first time that nrf2 plays a protective role against sci - induced second spinal cord injury , possibly by limiting the inflammatory response in the spinal cord after sci . activation of nf-b signaling pathway has been shown to be central to the pathophysiology of spinal cord inflammatory response induced by sci , and nf-b can be activated by lesion - induced oxidative stress and cytokines     [ 6 , 30 ] . the functional importance of nf-b in inflammation is based on its ability to regulate the promoters of multiple inflammatory genes . animal sci models have shown to have infiltration of inflammatory cells , endothelial activation and injury leading to increased vascular permeability , edema formation , and accumulation of inflammatory mediators     . mmp-9 is produced by neutrophils and endothelia , facilitating leukocytes diapedesis which may be a vascular permeabilizing factor . in spinal cord injury , mmps , including mmp-9 , contribute to early secondary pathogenesis by disrupting the blood spinal cord barrier and promoting inflammation     . tnf- is a major initiator of inflammation and is released early after an inflammatory stimulus . there is evidence that tnf- also plays an important role in the recruitment of inflammatory and immune cells to the injured site . moreover , the infiltration of immune and inflammatory cell to sci sites is a major contributor to secondary degeneration . consistent with other studies , we show that tnf- and mmp-9 were also upregulated during sci , spinal cord water content at the injury site increased at 2 days after sci , and significant damage to the spinal cord was observed in the tissue from sci mice     [ 9 , 22 , 24 ] . nrf2 has been reported to be the key regulator of the expression of a group of antioxidant and detoxification enzymes and is considered to play an important role in modulating inflammation in a variety of experimental models     [ 38 , 39 ] . also , the nrf2 signaling pathway has been shown to modulate nf-b activation and expression of nf-b - dependent genes including mmp-9 . in our past studies , the results suggested that nrf2 plays an important role in protecting tbi - induced secondary brain injury , possibly by limiting the cerebral upregulation of nf-b activity , proinflammatory cytokine after tbi     . recently , pomeshchik et al . showed that sci could induce nrf2-are pathway activation in spinal cord , just as our laboratory has found ( data not shown ) . also in vitro , phase ii enzyme inducers can promote the expression of antioxidative enzymes through nrf2 and show neuroprotective potential on motor neuron survival in traumatic spinal cord injury . in the present study , nrf2 ( / ) mice were shown to have severer spinal cord edema , more nf-b activity , more inflammatory cytokine tnf- , and mmp-9 expression in spinal cord after sci compared with their wild - type nrf2 ( + /+ ) counterparts . taken together , these studies showed that nrf2 may play an important protective role in limiting the spinal cord inflammatory response after sci .   although numerous in vivo studies have reported that nrf2 plays a critical role in counteracting the inflammation in a variety of experimental models     [ 15 , 16 , 25 ] , the precise mechanism underlying this network is still unclear . several lines of evidence indicate that nrf2 interferes with inflammatory signaling pathways by inhibiting nf-b activation through the maintenance of cellular redox status . oxidative stress from reactive oxygen species ( ros ) was considered as a major pathway of secondary injury in sci . 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 has been shown to play an important role in limiting ros levels and thereby affects the redox - sensitive nf-b signaling pathway involved in inflammation     [ 14 , 40 ] . augmentation of cellular antioxidative or detoxification systems via activation of nrf2-regulated enzymes results in decreased inflammatory cytokine production via inactivation of nf-b . this constitutes a possible anti - inflammatory mechanism for the attenuated inflammatory response and tissue damage seen in spinal cord from nrf2 ( + /+ ) mice but not nrf2 ( / ) mice after sci . in conclusion , the present study showed that nrf2 ( / ) mice are more susceptible to sci - induced spinal cord edema , nf-b activity , inflammatory tnf- production , and mmp-9 expression , which then contributed to exacerbated spinal cord injury after sci . these findings raise the possibility that nrf2 might be a new therapeutic target for the treatment of sci .
matrix metalloproteinase-9 ( mmp-9 ) plays an important role in the acute periods of spinal cord injury ( sci ) , and its expression is related to the inflammation which could cause the disruption of the blood - spinal barrier ( bbb ) . 
 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 upregulating of nuclear factor kappa b ( nf-b ) activity , tumor necrosis factor- ( tnf- ) , and mmp-9 after sci . 
 wild - type nrf2 ( + /+ ) and nrf2-deficient ( nrf ( / ) ) mice were subjected to an sci model induced by the application of vascular clips ( force of 10  g ) to the dura after a three - level t8-t10 laminectomy . 
 we detected the wet / dry weight ratio of impaired spinal cord tissue , the activation of nf-b , the mrna and protein levels of tnf- and mmp-9 , and the enzyme activity of mmp-9 . 
 nrf2 ( / ) mice were demonstrated to have more spinal cord edema , nf-b activation , tnf- production , and mmp-9 expression after sci compared with the wild - type controls . 
 the results suggest that nrf2 may play an important role in limiting the upregulation of nf-b activity , tnf- , and mmp-9 in spinal cord after sci .