the toll - like receptors ( tlrs ) are a recently discovered family of innate immune recognition receptors ( prrs ) . innate immune system is rapidly activated by recognition of pathogenic microorganisms and special structure of cell wall that is a highly conserved pathogen - associated molecular patterns ( pamps ) . there are at least 13 human tlrs identified to date , in which toll - like receptor 4 ( tlr4 ) is the earliest discovered and the most studied . the main function of tlr4 is to identify the lipopolysaccharide ( lps ) of gram - negative bacterial cell wall and activates innate immune system . chen et al . recently   reported that tlr4 was highly expressed in the surface of iris - ciliary macrophages in the rats with uveitis induced by footpad injection of vibrio cholera lipopolysaccharide . it suggested that tlr4 may be involved in the pathogenesis of acute anterior uveitis ( aau ) . in our study , changes in the expression of tlr4 , downstream transduction molecules myd88 , and nf-b were investigated in iris - ciliary during eiu by the same research method . the role of tlr4 transduction pathway was further explored in in the pathogenesis of aau . adult male pathogen - free wistar rats ( 810 weeks old , weighing 150200  g ) were obtained from vital river laboratory animal technology co. ltd ( beijing , china ) . animals were randomly divided into five groups ( n = 10 per group ) for the following time points : before lps injection ( 0  h ; control group ) and 6  h , 12  h , 24  h , and 48  h after lps injection . lipopolysaccharide ( v. cholera , classical biotype , serotype ogawa ) was kindly provided by lanzhou institute of biologic products ( lanzhou , china ) . rabbit polyclonal antibody tlr4 and myd88 , mouse monoclonal antibody nf-b p65 was purchased from santa cruz biotechnology ( santa cruz , ca ) . affinity purified normal rabbit igg was purchased from boster biotechnology ( wuhan , china ) . mouse igg1 was purchased from serotec ( oxford , uk ) , dab kit was purchased from zhongshan goldbridge biotechnology ( beijing , china ) . removal of the iris was under a stereo microscope ( leica - m165 c ; leica , wetzlar , germany ) . slides were examined under an optical microscope ( leica - dm-4000b ; leica , wetzlar , germany ) . images were captured by a slit lamp with an anterior segment camera system ( topcon sl - d7 ; topcon , japan ) , and analysis using image - management software ( adobe photoshop cs3 . mice received a single injection of 200  g [ 4 , 5 ] lps dissolved in 100  l sterile saline ( ns ) in one rear footpad . the eyes were examined by slit microscope before the injection and at different time after the injection , and the intensity of anterior segment inflammation was evaluated by slit lamp . the intensity of anterior segment inflammation was examined by slit microscope before the injection and at 2  h intervals after the injection . inflammatory signs were recorded in detail , and photographs were taken . the severity of uveitis was graded from 0 to 4 by a investigator blinded to study protocol   as follows : 0 = no  inflammation ; 1 = discrete vasodilatation of the iris and the conjunctiva vessels ; 2 = moderate dilatation of the iris and the conjunctival vessels with moderate flare in the anterior chamber ; 3 = intense iridal hyperemia with intense flare in the anterior chamber ; and 4 = the  same clinical signs as 3 with fibrinous exudates in the pupillary area . intracardiac perfusion of rats was performed at different time points in order to eliminate the effect of blood to immunohistochemical staining . animals were deeply anesthetized using 17.5% chloral hydrate ( 2  ml / kg ) by intraperitoneal injection . through an abdominal incision in the midsagittal plane to expose the chest wall , the rat was flushed through the left ventricle with 250300  ml of phosphate - buffered saline ( pbs ) , 1  iu heparin per ml of pbs until the outflow becomes colorless , then 4% paraformaldehyde(250  ml ) was flushed through the heart . movements in the limbs and tail ( fixation has reached the extremities ) indicated adequate perfusion . after being fixed in 4% paraformaldehyde for additional 1 - 2 the iris - ciliary body complex was gently dissected into 3~4 segments under the stereomicroscope and stored in eppendorf tube with pbs at 80c . rats were killed by overdose of pentobarbital ( 100  mg / kg ) 24  hours after being immunized with lps . the eyes of rats were enucleated and placed in 10% neutral buffered formalin solution for 24  h. after stationary liquid was washed out , tissue sample was immersed in 50% , 75% , 80% , 90% , and 100% alcohol for 1  h , respectively , to dehydrate . then , the tissue was put into paraffin for 1  h  3 for embedding after being treated with xylene for 30 minutes . sagittal sections ( 4  m thick ) were cut near the optic nerve head and stained with hematoxylin and eosin . after three rinses with pbs ( 5  min each ) , 0.3% triton - x 100 was used to perforate the cell membrane for 30  min . then , endogenous peroxidase activity in the whole mounts was blocked by 0.3% h2o2 for 30  min at room temperature . after another three rinses with pbs , the tissues were blocked with 5% goat serum for 30  min , and incubated with primary antibody against tlr4 , myd88 , and nf-b p65 ( dilution , 1  :  50 ) overnight at 4c . after several washes in pbs , the tissues were incubated with biotinylated antirabbit and antimouse secondary antibody ( 1  :  200 ) for 2  h at room temperature . after three further washes in pbs , the tissues were incubated with mixture of a and b ( 1  :  100 ) for 30  min at 37c . staining was visualized by the biotin - avidin - peroxidase method using diaminobenzidine as chromogen . negative controls were performed by replacing the primary antibody with species - matched and isotype - matched antibodies with the same concentration of the primary antibody . the method employed in our study involved counting the total number of immunopositive cells per iris segment ( a strip 0.29  mm or 1 graticule in width from the pupil margin to iris base chosen randomly around the circumference ) . the mean length of the irides ( base - pupil margin ) in the present study was approximately 1  mm ( 0.97  mm or 3.4 graticule lengths ) ; therefore , the mean area of a segment was 0.281  mm . a minimum of two and up to five segments were counted per iris from one eye , and an overall mean density / mm per animal was obtained . cells were counted by a blinded investigator ( one of the authors , who was unaware of the treatment ) . different layers of stained cells could be distinguished in separate focal planes of the whole mounts . all cells of different layers in one field were counted under a microscope with 20x or 40x objective lens . quantitative data were expressed as means  standard deviation ( sd ) and were analyzed with one - way analysis of variance ( anova ) followed by significant difference procedure ( lsd ) test for multiple comparisons among experimental groups with control groups . in addition to the control group , ocular inflammatory signs were observed in the each rat of remaining four groups after lps injection . conjunctival edema , ciliary congestion , and blood vessels dilatation in the iris began to appearance within 4~6  h after the lps injection . at 12~16  h , aqueous flaring and fibrinous pupillary membrane was seen , reaching a maximum at 22~24  h. occlusion of pupil even was found by slit microscope ( figure 1(a ) ) . generally , inflammation subsided gradually after 24  h , and the exudation had decreased 48  h after lps injection ( figure 1(b ) ) . at 72~76  h , the score of eiu in wistar rat at different time was described in figure 2(a ) and table 1 . h - e staining results were consistent with inflammatory manifestations in wistar rats at 24  h after lps immunization . a large number of infiltration of inflammatory cells and fibrin exudations could be seen in the anterior and posterior chamber ( figure 3(a ) ) ; massive neutrophil adhered behind corneal endothelial cell ( figure 3(b ) ) ; thickened iris stroma with vasodilatation and a majority of inflammatory cell infiltration in the vitreous were observed ( figure 3(c ) ) . tlr4 could not be detected in the iris - ciliary body complex in 0  h group ( figure 4(a ) ) ; at 12  h , much tlr4 positive cells were found around blood vessels ( figure 4(b ) ) ; the number of tlr4 positive cells significantly increased in the iris and ciliary body of all rats at 24  h ( figure 4(c ) ) and reached the peak at 48  h ( figure 4(d ) ) . the number of positive cells had reduced at 72  h ( figure 4(e ) ) . there was statistical significance to positive cells overall among these groups ( f = 46.79 , p < .05 anova ) . however , no positive cells could be detected in negative control with a nuclear counterstain ( figure 4(f ) ) . the trend of changes in myd88 expression was consistent with the tlr4 ( figure 2(b ) ) . myd88 could not be detected at 0  h , but positive cells were observed in iris at 12  h ( figure 5(a ) ) and reached the maximum during 24~48  h ( figures 5(b ) and 5(c ) ) . the number of positive cells had reduced at 72  h ( figure 5(d ) ) . there was statistical significance to positive cells among these groups ( f = 54.37 , p < .05 anova ) . nf-b p65 could not be found at 0  h group , but nf-b p65 positive cells were located in the cytoplasm or nucleus at other groups and its expression was gradually increased from 12  h to 48  h. immunopositive cells were predominantly round - ovoid cells mainly scattering in iris stroma . the number of positive cells had reduced at 72  h. there was statistical significance to positive cells among these groups ( f = 85.32 , p < .05 , figures 6(a)6(d ) . uveitis is a common inflammatory disease that was a potential threaten to visual loss , which mainly affects iris , ciliary body , and choroid . at present , the pathogenic mechanisms of uveitis is not clear . the majority of uveitis may be caused by nonimmune factors , only a small part of the infectious uveitis is due to pathogen invasion . acute anterior uveitis , especially hla - b27-associated aau is a common noninfectious uveitis , but clinical and laboratory research have proven gram negative bacteria such as klebsiella , salmonella , yersinia , and shigella species can trigger it . tlr-4 is a main receptor that recognizes lipopolysaccharide of gram - negative bacterial cell wall . in our study , tlr4 was not expressed in the normal wistar rat iris whereas chang et al .   and brito et al .   found that tlr4 positive cells expressed in the normal hunman iris - ciliary body . this may be due to the different subjects and methods applied since tlr4 expression is too low in normal wistar rat to be detected by iris stretched preparation technology . in our study , we found that tlr4 was expressed in iris - ciliary body after lps administration . the inflammatory response reached the maximum at 24  h after lps administration , then the degree of inflammatory response was gradually reduced , but tlr4 positive cells continued to increase until 48  h in the iris . tlr4 expression significantly decreased at 72  h comparing with it at 48  h ( p < .001 ) . in our study , we had observed that the changes of tlr4 was relative to the degree of anterior segment inflammation , which suggested that lps - related gram negative bacteria could excessively activate tlr4-mediated innate immunity and adaptive immunity that may resulted in incidence of aau . upon lps recognition , tlr4 undergoes oligomerization and recruits its downstream adaptors through interactions with the tir ( toll - interleukin-1 receptor ) domains , resulting in inflammatory reaction finally . tlr4 signaling has been divided into myd88-dependent and myd88-independent ( trif - dependent ) pathways . our study found that many myd88 positive cells were expressed in the iris at 24  h after lps administration , peaked at 48  h , and then gradually decreased . it showed that tlr4 activated its downstream signaling molecules through a myd88-dependent pathway conduction in the pathogenesis of aau . reported that myd88-deficient mice were completely resistant to experimental autoimmune uveitis ( eau ) in th1 mediated autoimmunity response . taken together , these findings suggest myd88-dependent pathway plays an essential role in lps / tlr4 signaling . after myd88 activation , another adaptor protein traf6 ( tnf receptor - associated factor 6 ) is critical for the myd88-dependent pathway . it leads to the phosphorylation of ib proteins which makes nf-b / i-b trimer complex degradation . subsequencely , nf-b is activated and transfered into nucleus . in our study , nf-b p65 positive cells could not be observed in normal wistar rat iris . at 12  h after lps immunization , nf-b p65 positive cells were detected in the cytoplasm and nucleus of iris , reaching the maximum at 48  h , and then had decreased at 72  h. compared to tlr4 and myd88 , nf-b p65 reduction is more obvious from 48  h to 72  h , which can better reflect attenuation of inflammation directly . report in that nf-b p65 was activated in iris - ciliary body after a footpad injection of lps in the rats . under the normal circumstances , the p65 subunit of nf-b binds with its inhibitor i-b to form i-b - nf-bp50/p65 trimeric complex , which is in the nonactivation state . when the i-b inhibitory protein degradates , nf-b antibody recognizes and activates p65 . in addition , todaro et al .   found that nf-b was highly expressed in behcet 's peripheral blood t cells , which contributes to the regulation of the apoptosis - related factors and death receptors leading to apoptosis resistance in bd t cell subsets . in the research , the reduction of nf-b p65 may be induced in the down regulation of the peripheral lymphocyte apoptosis by this transduction pathway , which results in regression of inflammation reaction . we found more positive tlr4 and myd88 cells than nf-b p65 cells in the same field at 72  h , which may be due to feedback in the pathway . further studies , including functional studies , are required to identify the roles of nuclear factor in the network of uveitis . our study revealed that the expression of tlr4 , myd88 , and nf-b p65 in the iris changed during eiu . these findings suggest the important role of tlr4 and its associated factors in the pathogenesis of uveitis and will provide some insightful ideas of the mechanism of uveitis .