traumatic brain injury ( tbi ) is a complex neurodegenerative disease , involving many cellular and molecular pathways , including inflammation ( 1 ) . paradoxically , the inflammatory response can lead to both aggravation and amelioration of the resulting tissue damage , depending on the tissue involved and the type of injury evoked ( 2 ) . after traumatic brain injury ( tbi ) , disruption of blood - brain - barrier ( bbb ) will led to the release of neutrophils , lymphocytes and monocytes from blood to central nervous system and cause inflammatory responses ( 3 ) . estrogen ( e2 ) is a cholesterol - derived hormone that is often known as a reproduction regulator and can play a protective role in different kinds of brain injuries ; it has been revealed that the treatment by estradiol following ischemia , will decrease cortical injuries ( 4 ) . e2 regulates the selective permeability ( 5 ) of the bbb in several models of injury , including systemic inflammation , neurotrauma , and ischemia ( 6 ) . previous study in our laboratory showed that the administration of estrogen and progesterone , alone and in combination after tbi decreases brain edema , blood - brain - barrier permeability , and intracranial pressure ( 7 ) . many of e2 functions are carried via the classic receptors , estrogen receptor alpha and beta . estrogen receptors ( ers ) are involved in signaling pathways of classical and non - classical pathways ( 4 ) . both receptors express throughout the cns , including microglia , astrocytes , and neurons , and also in many immune system cells such as macrophages / monocytes , t and b cells and dendrites are expressed ( 8) . it has been reported that er expression in ischemic brain after stroke greatly increases ( 9 ) . beta receptor agonist in animal models of arthritis and inflammatory bowel disease , has anti - inflammatory effects and suppress gene expression of pro - inflammation . both receptors er and er are involved in cell proliferation induced by estradiol after ischemia ( 10 ) . after brain ischemia , the expression of er in injured cortex increases rapidly ( 11 ) . which of these two receptors alpha and beta - estradiol - mediated control inflammation , depends on the inflamed tissue ( 12 ) . in agreement with previous studies , it was shown that estrogen reduces brain edema and bbb permeability after trauma ( 7 , 13 ) , although the roles of estrogen receptors er and er in the neuroprotective actions of estrogen on tbi remain poorly understood . the goal of the present study was to use these er - selective and non - selective antagonists ( mpp , phtpp and ici 182780 ) to evaluate the relative necessity of er- and er-activation in mediating the neuroprotective effect of estrogen following diffuse tbi in ovx rats . this study was conducted in accordance with the guidelines for the animal experimental protocols of kerman university of medical sciences . knrc/9040 ) of this university , in accordance with the internationally accepted principles for laboratory animal use and care , as found in the european community guidelines ( eec directive of 1986 ; 86/609/eec ) or us guidelines ( nih publication # 8523 , revised in 1985 ) . adult female albino n mary rats ( weighing 200250 g ) were housed in an air - conditioned room at 2225c , with a 12 hr light : 12 hr dark cycle and free access to food and water . in the first step , animals were anesthetized by injection of 60 mg / kg thiopental ( ip ) , sub abdominal area was shaved and an incision of 1 cm was made . both fallopian tubes and vascular base of the ovaries were legated in the proximal parts and then cut from the distal parts . the incision was closed with a 20 silk surgical suture . to avoid interference due to the estrus cycle , all experimental animals were ovariectomized ( ovx ) 2 weeks before the experiments ( 14 ) . before induction of tbi , ovariectomized rats were randomly divided into 10 groups , as follows : sham ( animals that underwent ovariectomy 2 weeks before the start of the experiment and underwent false brain trauma under anesthesia but did not receive hormones or vehicle ) , tbi ( brain injury was induced 2 weeks after ovariectomy ) , e2 , mpp+e2 , phtpp+e2 , mpp+phtpp+e2 , mpp , ici+e2 , oil ( sesame oil , which was used as estrogen solvent ) and dmso ( dimethyl sulfoxide , which was used as antagonists solvent ) . e2(33.3 g / kg ) or oil were administered ( ip ) 30 min after tbi ( 15 , 16 ) . mpp(1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1h - pyrazole hydrochloride ) , er antagonist , and phtpp(4-[2-phenyl-5 , 7-bis ( trifluoromethyl ) pyrazolo [ 1,5-a ] pyrimidine-3- yl ] phenol ) , er antagonist , or dmso were used as follows:1 dose ( 150 g / kg ) of each of mpp and , phtpp was injected two times , 24 hr apart , before tbi and oestrogen treatment ( 17 ) . then treated with e2 ( 33.3 g / kg ) 30 min after tbi ( 13 ) . one group received only an injection of mpp(150 g / kg ) 30 min after tbi ( 17 ) . 4 mg / kg ici182780 was injected similar to the other two antagonists ( 18 ) . the tbi was induced by the marmarou method ( 16 ) , using a tbi induction device made by the department of physiology , kerman university of medical sciences . the protocol was as follows : a weight of 300 g was dropped from a 2 m height onto the head of the anesthetized rat while a metal disc ( stainless steel , 10 mm in diameter , 3 mm thick ) was attached to the animal s skull . after induction of the trauma , the rats were immediately connected to a respiratory pump untill spontaneous breathing had been restored . the brain edema of each animal was assessed by measuring brain water content 24 hr after tbi . anesthetized animals were sacrificed by cervical dislocation ; the brain was removed , and brain samples were placed in pre - weighed glass vials and weighed ( wet weight ) . the lids were removed and the vials placed in an incubator ( memmert , germany ) at 60c for 72 hr , and then reweighed ( dry weight ) . the percentage of water in each sample was then calculated using a formula published previously ( 16 , 19 ) : ( 100 * [ ( wet weight  dry weight)/wet weight ] ) . the degree of bbb disruption was assessed by measuring evans blue dye leakage ( 16 ) . briefly , evans blue dye was dissolved in 0.01 mol / l pbs at a concentration of 2% , then the dye ( 2 ml / kg iv ) was injected into the tail vein 4 hr after tbi , as a bbb permeability tracer . the rats were then deeply anesthetized and transcardiac perfused with 200 ml heparinized saline through the left ventricle to remove the intravascular dye . the brains were removed , then dissected , weighed , and stored at 80c for quantitative measurement . brain samples were homogenized in 1 ml of 0.1 mol / l pbs , and 0.7 ml of 100% ( w / v ) trichloroacetic acid was added , and centrifuged . after centrifugation for 30 min at 1000 g , the absorbance of evans blue in supernatant was measured at 610 nm using a spectrophotometer ( uv / vis , spectrometer , uk ) . the amount of extravasated evans blue dye was quantified as g / g brain tissue . the data were analyzed by parametric analysis of variance ( anova ) or independent t test . this study was conducted in accordance with the guidelines for the animal experimental protocols of kerman university of medical sciences . knrc/9040 ) of this university , in accordance with the internationally accepted principles for laboratory animal use and care , as found in the european community guidelines ( eec directive of 1986 ; 86/609/eec ) or us guidelines ( nih publication # 8523 , revised in 1985 ) . adult female albino n mary rats ( weighing 200250 g ) were housed in an air - conditioned room at 2225c , with a 12 hr light : 12 hr dark cycle and free access to food and water . in the first step , animals were anesthetized by injection of 60 mg / kg thiopental ( ip ) , sub abdominal area was shaved and an incision of 1 cm was made . both fallopian tubes and vascular base of the ovaries were legated in the proximal parts and then cut from the distal parts . the incision was closed with a 20 silk surgical suture . to avoid interference due to the estrus cycle , all experimental animals were ovariectomized ( ovx ) 2 weeks before the experiments ( 14 ) . before induction of tbi , ovariectomized rats were randomly divided into 10 groups , as follows : sham ( animals that underwent ovariectomy 2 weeks before the start of the experiment and underwent false brain trauma under anesthesia but did not receive hormones or vehicle ) , tbi ( brain injury was induced 2 weeks after ovariectomy ) , e2 , mpp+e2 , phtpp+e2 , mpp+phtpp+e2 , mpp , ici+e2 , oil ( sesame oil , which was used as estrogen solvent ) and dmso ( dimethyl sulfoxide , which was used as antagonists solvent ) . e2(33.3 g / kg ) or oil were administered ( ip ) 30 min after tbi ( 15 , 16 ) . mpp(1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1h - pyrazole hydrochloride ) , er antagonist , and phtpp(4-[2-phenyl-5 , 7-bis ( trifluoromethyl ) pyrazolo [ 1,5-a ] pyrimidine-3- yl ] phenol ) , er antagonist , or dmso were used as follows:1 dose ( 150 g / kg ) of each of mpp and , phtpp was injected two times , 24 hr apart , before tbi and oestrogen treatment ( 17 ) . then treated with e2 ( 33.3 g / kg ) 30 min after tbi ( 13 ) . one group received only an injection of mpp(150 g / kg ) 30 min after tbi ( 17 ) . 4 mg / kg ici182780 was injected similar to the other two antagonists ( 18 ) . the tbi was induced by the marmarou method ( 16 ) , using a tbi induction device made by the department of physiology , kerman university of medical sciences . the protocol was as follows : a weight of 300 g was dropped from a 2 m height onto the head of the anesthetized rat while a metal disc ( stainless steel , 10 mm in diameter , 3 mm thick ) was attached to the animal s skull . after induction of the trauma , the rats were immediately connected to a respiratory pump untill spontaneous breathing had been restored . the brain edema of each animal was assessed by measuring brain water content 24 hr after tbi . anesthetized animals were sacrificed by cervical dislocation ; the brain was removed , and brain samples were placed in pre - weighed glass vials and weighed ( wet weight ) . the lids were removed and the vials placed in an incubator ( memmert , germany ) at 60c for 72 hr , and then reweighed ( dry weight ) . the percentage of water in each sample was then calculated using a formula published previously ( 16 , 19 ) : ( 100 * [ ( wet weight the degree of bbb disruption was assessed by measuring evans blue dye leakage ( 16 ) . briefly , evans blue dye was dissolved in 0.01 mol / l pbs at a concentration of 2% , then the dye ( 2 ml / kg iv ) was injected into the tail vein 4 hr after tbi , as a bbb permeability tracer . the rats were then deeply anesthetized and transcardiac perfused with 200 ml heparinized saline through the left ventricle to remove the intravascular dye . the brains were removed , then dissected , weighed , and stored at 80c for quantitative measurement . brain samples were homogenized in 1 ml of 0.1 mol / l pbs , and 0.7 ml of 100% ( w / v ) trichloroacetic acid was added , and centrifuged . after centrifugation for 30 min at 1000 g , the absorbance of evans blue in supernatant was measured at 610 nm using a spectrophotometer ( uv / vis , spectrometer , uk ) . the amount of extravasated evans blue dye was quantified as g / g brain tissue . the data were analyzed by parametric analysis of variance ( anova ) or independent t test . changes in the brain water content of the ovariectomized rats are shown in figure 1 . figure 1a shows that the brain water content in the tbi ( 78.60.33% ) group was significantly higher than in sham ( 75.650.001% , p<0.001 ) group and brain water content in the group treated with e2 ( 76.760.17% ) was significantly lower than those in the oil ( 78.320.15% ) and dmso ( 78.40.22% ) groups , ( p<0.01 ) . figure 1b shows the effects of administration of estrogen receptors antagonists . in the presence of mpp and phtpp , brain water content decreased and was 74.860.54% in mpp+e2 group and 75.410.88% in phtpp+e2 group . figure 1c shows that the brain water content in the group treated with mpp ( 78.060.32% ) has no significant difference with the water content in dmso group . ( a ) comparison of brain water content ( % ) after traumatic brain injury in ovariectomized ( ovx ) rats ( n = 6 in each group ) . the data are represented as meansem . * * * : p < 0.001 , tbi vs. sham . ( b ) comparison of brain water content in ovx rats treated with ers antagonist . there were no significant differences in mpp+e2 and phtpp+e2 compared to e2 ( c ) brain water content ( % ) after traumatic brain injury in ovx rats treated with mpp and dmso . mpp ( er- antagonist ) , phtpp(er- antagonist ) , ici182780 ( non selective estrogen receptors antagonist ) , dmso ( dimethyl sulfoxide , antagonists solvent ) , oil ( sesame oil , estrogen solvent ) the effects of estrogen and ers antagonists on bbb disruption after tbi are shown in figure 2 . figure 2a shows that the evans blue content in the tbi ( 37.180.4 g / g tissue ) group was significantly higher than in the sham ( 33.250.05 g / g tissue , p<0.05 ) group and the evans blue content in e2 ( 33.50.65 g / g tissue ) was lower than oil ( 37.670.44 g / g tissue ) , tbi , and dmso ( 37.340.22 g / g tissue ) groups . figure 2b shows the effect of administration of estrogen receptors antagonist . in the presence of mpp and phtpp , evans blue content decreased and was 30.780.31 g / g tissue in mpp+e2and 31.40.38 g / g tissue in phtpp+e2 groups ( p<0.001 ) . figure 2c shows that the evans blue content in the groups treated with mpp ( 36.460.68 ) has no significant difference with the evans blue in the e2 group . ( a ) comparison of evans blue content ( g / g tissue ) after traumatic brain injury in ovariectomized ( ovx ) rats ( n = 6 in each group ) . the data are represented as meansem . * * : p < 0.01 , tbi vs. sham . # # : p < 0.01 , e2 vs. tbi , oil and dmso groups . ( b ) comparison of brain water content in ovx rats treated with ers antagonist . there were no significant differences in mpp+e2 and phtpp+e2 compared to e2 ( c ) evans blue content ( g / g tissue ) after traumatic brain injury in ovx rats treated with mpp and dmso . mpp(er- antagonist ) , phtpp(er- antagonist ) , ici182780 ( non selective estrogen receptors antagonist ) . dmso ( dimethyl sulfoxide , antagonists solvent ) , oil ( sesame oil , estrogen solvent ) changes in the brain water content of the ovariectomized rats are shown in figure 1 . figure 1a shows that the brain water content in the tbi ( 78.60.33% ) group was significantly higher than in sham ( 75.650.001% , p<0.001 ) group and brain water content in the group treated with e2 ( 76.760.17% ) was significantly lower than those in the oil ( 78.320.15% ) and dmso ( 78.40.22% ) groups , ( p<0.01 ) . figure 1b shows the effects of administration of estrogen receptors antagonists . in the presence of mpp and phtpp , brain water content decreased and was 74.860.54% in mpp+e2 group and 75.410.88% in phtpp+e2 group . figure 1c shows that the brain water content in the group treated with mpp ( 78.060.32% ) has no significant difference with the water content in dmso group . ( a ) comparison of brain water content ( % ) after traumatic brain injury in ovariectomized ( ovx ) rats ( n = 6 in each group ) . the data are represented as meansem . * * * : p < 0.001 , tbi vs. sham . ( b ) comparison of brain water content in ovx rats treated with ers antagonist . there were no significant differences in mpp+e2 and phtpp+e2 compared to e2 ( c ) brain water content ( % ) after traumatic brain injury in ovx rats treated with mpp and dmso . mpp ( er- antagonist ) , phtpp(er- antagonist ) , ici182780 ( non selective estrogen receptors antagonist ) , dmso ( dimethyl sulfoxide , antagonists solvent ) , oil ( sesame oil , estrogen solvent ) the effects of estrogen and ers antagonists on bbb disruption after tbi are shown in figure 2 . figure 2a shows that the evans blue content in the tbi ( 37.180.4 g / g tissue ) group was significantly higher than in the sham ( 33.250.05 g / g tissue , p<0.05 ) group and the evans blue content in e2 ( 33.50.65 g / g tissue ) was lower than oil ( 37.670.44 g / g tissue ) , tbi , and dmso ( 37.340.22 g / g tissue ) groups . figure 2b shows the effect of administration of estrogen receptors antagonist . in the presence of mpp and phtpp , evans blue content decreased and was 30.780.31 g / g tissue in mpp+e2and 31.40.38 g / g tissue in phtpp+e2 groups ( p<0.001 ) . figure 2c shows that the evans blue content in the groups treated with mpp ( 36.460.68 ) has no significant difference with the evans blue in the e2 group . ( a ) comparison of evans blue content ( g / g tissue ) after traumatic brain injury in ovariectomized ( ovx ) rats ( n = 6 in each group ) . the data are represented as meansem . * * : p < 0.01 , tbi vs. sham . # # : p < 0.01 , e2 vs. tbi , oil and dmso groups . ( b ) comparison of brain water content in ovx rats treated with ers antagonist . there were no significant differences in mpp+e2 and phtpp+e2 compared to e2 ( c ) evans blue content ( g / g tissue ) after traumatic brain injury in ovx rats treated with mpp and dmso . mpp(er- antagonist ) , phtpp(er- antagonist ) , ici182780 ( non selective estrogen receptors antagonist ) . dmso ( dimethyl sulfoxide , antagonists solvent ) , oil ( sesame oil , estrogen solvent ) previous studies have reported , the neuroprotective effects of estrogen on brain edema and blood - brain barrier permeability ( 7 , 13 ) , in the present study , the roles of alpha and beta estrogen receptors in the development of these effects were investigated . the main findings of this research are as follows : 1-estrogen exerts its anti - edema effects through both receptors , so that any differences between the results of combined use of selective antagonists and a non - selective antagonist do not exist . 2 -bbb protection after tbi by estrogen is mediated by both receptors , which is similar to the results of the anti - edema effect of estrogen . 3- the estrogen receptor alpha antagonist ( mpp ) has no agonistic effect on its receptor . tbi caused an increase in brain water content and bbb permeability , these increases were inhibited by estrogen compared to vehicle ( oil ) . there have been a number of reports supporting a neuroprotective role for estrogen after various insults to the cns . ovariectomy increases ischemic brain damage in females , and estradiol exerts neuroprotective and anti - inflammatory action against the ischemic brain when administered immediately upon ovariectomy ( 20 ) . e2 regulates the selective permeability of the bbb in several models of injury ( 5 ) . recently , our research showed that estrogen loss facilitates , whereas administration of a different dose of estradiol could prevent edema formation ( 7 ) and destruction of the bbb after tbi ( 13 ) . a significant reduction in the brain water content after tbi was induced by estrogen this suggested that neither selective er antagonist ( mpp ) nor er antagonist ( phtpp ) can abolish these effects of estrogen . on the other hand , administration of the two antagonists together ( mpp + phtpp ) or the use of non - selective estrogen receptor antagonist ( ici182780 ) eliminates the inhibitory effect of estrogen on brain edema and bbb permeability , this means that , the effects of estrogen were not inhibited by the administration of antagonists alone , but when these antagonists were administered in combination , they have an inhibitory effect . it is suggested that , probably both receptors are involved in the development of neuroprotective effects of estrogen , we found that more than one receptor is activated by estrogen at the same time , perhaps activation of both receptors at the same time has reciprocal modulatory effects . whenever the effect of estrogen was observed during receptor inhibition , it was caused by activation of the other receptor at the same time . to the best of our knowledge , the roles of neither er antagonist nor er antagonist in tbi have been reported thus far . because this study is the first to use a selective er and er antagonist to investigate the effects on tbi . however , the effects of these receptors or antagonists in other non - tbi studies , and other estrogen - mediated effects have been demonstrated , including : the receptors er and er are essential for e2-mediated regulation of bbb permeability , anti - inflammatory properties , and regulation of neutrophil recruitment into the brain ( 4 ) . both er receptors are involved in the control of inflammation by estradiol ( 21 ) , neuromodulation , and neuroprotection processes after brain injury ( 22 ) . the affinity of mpp to er is only 11% of that of e2 ( 23 ) . er antagonists ( mpp and er antagonist ( r , r)-thc ) treatments alone did not significantly change npy release levels ( 24 ) . mpp failed to attenuate the anorexigenic effects of estradiol and an er agonist in ovx rats ( 25 ) . mpp did attenuate the estrous - related decrease in food intake in cycling rats ( 25 ) . 17estradiol protects dopamine neurons from injury , an effect that was blocked by ici 182,780 ( 26 ) . estrogen neuroprotection was related to microglial activation of estrogen receptors ( ers ) , this protective effect was overridden by pretreatment with ici 182,780 , the specific era , only partially blocked the effects of estrogen ( 6 ) . an alternate mechanism which could explain the differential effects of ici and mpp , is an involvement of non - genomic / non - classical signaling mechanisms activated by anti - estrogen - occupied er . indeed , ici - mediated erk1/2(extracellular signal - regulated kinases ) activation appears to be critical for modulation in immature cerebellar neurons ( 28 ) . some studies suggest that mpp acts as an er antagonist following in vitro applications , but exerts mixed er agonist / antagonist actions following in vivo applications ( 29 ) . it is possible that the mpp concentration in this experiment was too low to completely compete with e2 for binding to er  , because it is reported that mpp is needed in high excess to fully antagonize e2 ( 30 ) . there are several probable mechanisms by which er and er attenuate bbb permeability and brain edema after tbi that result from the inflammatory response ; up regulation of defense - related genes , il-6 , mmp-9 , and some of phagocytic receptors ( 31 ) , protection of cortical cells against oxidative glutamate toxicity ( 32 ) , neuroprotection of estrogen against activated microglia ( 33 ) and inhibition of the cytoplasmic transport of nf - kb ( nuclear factor kappa - light - chain - enhancer of activated b cells ) ( 34 ) . mpp and pthpp inhibited dna synthesis , oxygen species ( ros ) formation induced by estrogens according to their receptors affinity ( 36 ) . experiments with phtpp , indicated that e2-mediated neuroprotection against nmda toxicity was mediated via er receptor ( 37 ) . many studies have reported a primary role for er as a regulator of the anti - inflammatory properties of estradiol , including the anti - inflammatory and neuroprotective activity of estradiol ( 38 ) , neuroprotection after ischemia and inhibition of microglial activation ( 39 ) . studies involving transgenic mice with targeted deletion of either er or er indicate that er , but not er , is required for the e2-mediated suppression of bbb permeability ( 8) . in addition , a growing number of studies have reported anti - inflammatory roles for er , such as neuroprotection after ischemia and , inhibition of microglia activity ( 40 ) . in mammals , the actions of mpp vary , depending on the species , physiological conditions , dosing method , and tissue ( 17 , 41 - 43 ) . since currently , there is no evidence that phtpp , exerts the mixed agonist / antagonist properties that are often seen for mpp ( 29 ) , in another part of the present study , mpp was used to replace estrogen after tbi , it was observed this compound did not reduce brain edema , and bbb permeability . in other words , mpp has no agonist effect on er. this finding is consistent with other studies that have reported that mpp counteracted the effects of ppt(estrogen receptor agonist ) but had little effect on its own ( 44 ) . on the other hand , it has also been reported that estrogen receptor/(er ) , even in absence of estrogen ( e2 ) , plays a critical role in lactotroph homeostasis ( 45 ) . in vivo tests of mpp s effects on food intake revealed estrogenic activity , suggesting that mpp functions as a serm(selective estrogen receptor modulator ) , rather than as an er antagonist ( 46 ) . the present study showed that the administration of ppt or phtpp alone will not lead to reduced brain edema , and prevention of bbb disruption following tbi , but a combined administration of mpp and phtpp or ici inhibited the e2-induced decrease in brain edema and bbb disruption , this may suggest that estrogen neuroprotection was mediated via both er and er. both of them can play roles in classic and non - classic pathways . further study is needed and intracellular mechanism activation in the presence of e2 should be evaluated . these data , makes them exciting therapeutic targets for further study , in addition , perhaps it is essential that non - genomic mechanisms are also investigated .
objective(s):estrogen ( e2 ) has neuroprotective effects on blood - brain - barrier ( bbb ) after traumatic brain injury ( tbi ) . in order to investigate the roles of estrogen receptors ( ers ) in these effects , 
 er- antagonist ( mpp ) and , er- antagonist ( phtpp ) , or non - selective estrogen receptors antagonist ( ici 182780 ) were administered.materials and methods : ovariectomized rats were divided into 10 groups , as follows : sham , tbi , e2 , oil , mpp+e2 , phtpp+e2 , mpp+phtpp+e2 , ici+e2 , mpp , and dmso . 
 e2 ( 33.3 g / kg ) or oil were administered 30 min after tbi . 1 dose ( 150 g / kg ) of each of mpp , phtpp , and ( 4 mg / kg ) ici182780 was injected two times , 24 hr apart , before tbi and estrogen treatment . bbb disruption ( evans blue content ) and brain edema ( brain water content ) evaluated 5 hr and 24 hr after the tbi were evaluated , respectively.results:the results showed that e2 reduced brain edema after tbi compared to vehicle ( p<0.01 ) . 
 the brain edema in the mpp+e2 and phtpp+e2 groups decreased compared to the vehicle ( p<0.001 ) . 
 there was no significant difference in mpp+phtpp+e2 and ici+e2 compared to tbi . 
 this parameter in mpp was similar to vehicle . 
 evans blue content in e2 group was lower than vehicle ( p<0.05 ) . 
 the inhibitory effect of e2 on evans blue was not reduced by mpp+e2 and phtpp+e2 groups , but decreased by treatment with mpp+phtpp or ici . 
 mpp had no effect on evans blue content.conclusion:a combined administration of mpp and phtpp or ici inhibited the e2-induced decrease in brain edema and bbb disruption ; this may suggest that these effects were mediated via both receptors .