traumatic brain injury ( tbi ) is the leading cause of death and disability among the youth in developed countries . annually , 1.5 million people lose their lives due to tbi worldwide ( 1 ) . based on an epidemiological study , accidents are the second leading cause of mortality and morbidity in iran ( 2 ) . tbi following motorcycle accidents is the most common cause of permanent disability of individuals in iran ( 3 ) . tbi is known to cause primary mechanical injury of neurons ; however , the secondary damage following injury is complex with a broad spectrum of symptoms neural inflammation and oxidative stress are the main pathological mechanisms of neuronal cell apoptosis after tbi ( 5 , 6 ) . the role of inflammatory cascade activated after tbi is very important in brain tissue homeostasis , mediated by the release of pro- and anti - inflammatory cytokines and chemokine ( interleukin , tnf- , fas ligand ) , which are normally scarcely detectable in healthy brain tissue , but quickly upregulated in response to stressful or pathological conditions ( 7 ) . different strategies such as melatonin treatment can be adopted to reduce the complications of tbi , using varieties of antioxidant compounds ( 8) . melatonin ( n - acetyl 5-methoxytryptamine ) , originally identified by lerner in 1958 , is a tryptophan derivatives that is produced mainly in the pineal gland . control of circadian rhythm and sleep induction are the main physiological functions of melatonin ( 9 , 10 ) . there is some evidence showing the reduction of melatonin secretion after tbi ( 11 ) ; hence , some scientists tried to use the exogenous treatment of melatonin to evaluate its neuroprotective effects on different neural cell injuries . for instance , administration of melatonin after tbi in an animal contusion model facilitates neurobehavioral recovery ( 3 , 4 ) . besides , experimental and clinical data confirm that melatonin has an important role in the reduction of adhesion molecules and pro - inflammatory cytokines . moreover , experimental evidence supports its function as a direct and indirect antioxidant , free radicals scavenger and antioxidant enzymes stimulating agent ( 12 ) . most tbi research studies have focused on injured neurons , while little attention has been paid to glial cells . glial cells , particularly astrocytes become activated after tbi and have an important role in additional injury ( 13 - 15 ) . activated astrocytes are one of the main sources of pro - inflammatory cytokines , which are the main cause of prominent histological brain injury ( 16 ) . nevertheless , not enough attention has been paid to the effects of melatonin on astrocytes function and activation . since brain tissue is susceptible to inflammation and free radicals ( 17 ) , we conducted a study to examine the neuroprotective effects of melatonin on the prevention of neuronal cells apoptosis and astrocytes activation in male rats after tbi . all experiments of this study were performed in accordance with the animal experimental protocols approved by the ethics committee of kerman university of medical sciences ( ec / knrc/90 - 2 ) . animals ( nmri adult male rats weighing 250300 g ) maintained in a climate controlled room ( 2225 c ) , with a 12-hr light / dark cycle and free access to laboratory chow and water . a total of 40 adult male rats were randomly divided into five groups before tbi induction ; sham ( intact group ) ; a vehicle - treated tbi group that received an injection of vehicle ( 1% ethanol in saline ) after tbi at the corresponding points in time ; and melatonin - treated tbi groups ( tbi+m5 , tbi m10 , tbi+m20 ) that were exposed to brain trauma and received 5 mg / kg , 10 mg / kg , and 20 mg / kg melatonin ip , respectively ( sigma , st . louis , mo ) at 1 , 24 , 48 and 72 hr post - tbi ( 18 ) . eventually , the brain tissues of animals from each group were processed for histological examinations . as previously described by marmarou ( 19 ) , in this method a 250 g weight is dropped onto the head of the anesthetized male rat ( from free - falling tube with a height of 2 m ) , while a 3 mm thick steel s disc with a diameter of 10 mm was attached to the animal s skull . after induction of brain trauma , the animals were connected to a respiratory pump ( germany , tsa animal respiratory compact ) , and following spontaneous breathing and recovery , they were returned to the individual cages ( 20 ) . for the histological assessment of brain tissue , animals were deeply anesthetized with chloral hydrate ( 400 mg / kg , ip , merck ) 72 hr after tbi , and sacrificed by cardiac perfusion with 140180 ml of 0.9% heparinized normal saline followed by 140 to 180 ml of cold 4% paraformaldehyde in phosphate buffered saline ( pbs , ph of 7.5 ) . the perfusion lasted for 12 to 20 min ( until the lungs and liver were clear of blood ) . then , the brains were carefully removed and immediately immersed in an aldehyde fixative ( 10% formaldehyde in 0.1 m sodium phosphate - buffered ) and kept overnight at 4 c . they were then dehydrated in alcohol solution and eventually embedded in paraffin ( 21 , 22 ) . coronal serial sections ( 5 m ) of the brain tissue including the cortex and underlying white matter were cut on a microtome ( using stereotaxic coordinates of the bregma ap + 3.22 to  + 4.78 mm , with 1-mm interval ) , mounted on poly - l - lysine - coated slides ( st louis , mo , sigma chemical co ) , and dried at room temperature overnight ( 23 ) . one set of the sections was stained with hemotoxylin and eosin for morphologic analysis , and the other two sets were subjected to immunohistochemistry ( ihc ) by using glial fibrillary acidic protein ( gfap ) antibody and tunel staining . mouse monoclonal anti - gfap antibody ( 1:500 dilutions , dako a / s denmark ) was used to assess astrocytic reactivity . for this purpose , sections were deparaffinized in a microwave oven ( 65 c ) for 15 min and then in xylene ( 5 min ) . after rinsing in pbs , the sections were put in the citrate buffer ( 10 mm , ph of 6 and temperature of 90c ) for 1 hr and then kept in hydrogen peroxide ( 0.3% h2o2 in 60% methanol / pbs ) for 15 min to inhibit endogenous peroxidase activity . the sections were incubated with primary antibody and stored overnight at 4 c having been rinsed three times in pbs , and were incubated for 1.5 hr at 25 c with a 1:400 diluted secondary antibody ( rabbit anti - mouse antibody ) . to detect antibody - antigen complex , 0.1% diaminobenzidine ( 24 ) the tunel assay was performed on the cerebral sections to detect the cell death ( using the kit pod ; indianapolis , roche , in ) . after incubation with diamino benzidine dab and hydrogen peroxide , a dark brown color revealed damage to neuronal perikarya ( 26 ) . five coronal sections were chosen for cell counting ( between the bregma ap 0.32 and 0.68 mm ) . the number of surviving and degenerating neurons was counted in five random and non - overlapping regions ( at a magnification of x200 ) . percent of neuronal damage was calculated based on the ratio of the number of degenerated neurons to that of both surviving and degenerated neurons . in addition , the number of astrocytes were counted in five random and separated regions of the cortex ( at the level between bregma ap- 0.12 and + 0.28 ) ( 23 ) . one way anova and tukey - kramer multiple post hoc test were used to evaluate the differences between the groups , and p<0.05 was designated for statistical significance . all experiments of this study were performed in accordance with the animal experimental protocols approved by the ethics committee of kerman university of medical sciences ( ec / knrc/90 - 2 ) . animals ( nmri adult male rats weighing 250300 g ) maintained in a climate controlled room ( 2225 c ) , with a 12-hr light / dark cycle and free access to laboratory chow and water . a total of 40 adult male rats were randomly divided into five groups before tbi induction ; sham ( intact group ) ; a vehicle - treated tbi group that received an injection of vehicle ( 1% ethanol in saline ) after tbi at the corresponding points in time ; and melatonin - treated tbi groups ( tbi+m5 , tbi m10 , tbi+m20 ) that were exposed to brain trauma and received 5 mg / kg , 10 mg / kg , and 20 mg / kg melatonin ip , respectively ( sigma , st . louis , mo ) at 1 , 24 , 48 and 72 hr post - tbi ( 18 ) . eventually , the brain tissues of animals from each group were processed for histological examinations . as previously described by marmarou ( 19 ) , in this method a 250 g weight is dropped onto the head of the anesthetized male rat ( from free - falling tube with a height of 2 m ) , while a 3 mm thick steel s disc with a diameter of 10 mm was attached to the animal s skull . after induction of brain trauma , the animals were connected to a respiratory pump ( germany , tsa animal respiratory compact ) , and following spontaneous breathing and recovery , they were returned to the individual cages ( 20 ) . for the histological assessment of brain tissue , animals were deeply anesthetized with chloral hydrate ( 400 mg / kg , ip , merck ) 72 hr after tbi , and sacrificed by cardiac perfusion with 140180 ml of 0.9% heparinized normal saline followed by 140 to 180 ml of cold 4% paraformaldehyde in phosphate buffered saline ( pbs , ph of 7.5 ) . the perfusion lasted for 12 to 20 min ( until the lungs and liver were clear of blood ) . then , the brains were carefully removed and immediately immersed in an aldehyde fixative ( 10% formaldehyde in 0.1 m sodium phosphate - buffered ) and kept overnight at 4 c . they were then dehydrated in alcohol solution and eventually embedded in paraffin ( 21 , 22 ) . coronal serial sections ( 5 m ) of the brain tissue including the cortex and underlying white matter were cut on a microtome ( using stereotaxic coordinates of the bregma ap + 3.22 to  + 4.78 mm , with 1-mm interval ) , mounted on poly - l - lysine - coated slides ( st louis , mo , sigma chemical co ) , and dried at room temperature overnight ( 23 ) . one set of the sections was stained with hemotoxylin and eosin for morphologic analysis , and the other two sets were subjected to immunohistochemistry ( ihc ) by using glial fibrillary acidic protein ( gfap ) antibody and tunel staining . mouse monoclonal anti - gfap antibody ( 1:500 dilutions , dako a / s denmark ) was used to assess astrocytic reactivity . for this purpose , sections were deparaffinized in a microwave oven ( 65 c ) for 15 min and then in xylene ( 5 min ) . after rinsing in pbs , the sections were put in the citrate buffer ( 10 mm , ph of 6 and temperature of 90c ) for 1 hr and then kept in hydrogen peroxide ( 0.3% h2o2 in 60% methanol / pbs ) for 15 min to inhibit endogenous peroxidase activity . the sections were incubated with primary antibody and stored overnight at 4 c having been rinsed three times in pbs , and were incubated for 1.5 hr at 25 c with a 1:400 diluted secondary antibody ( rabbit anti - mouse antibody ) . to detect antibody - antigen complex , 0.1% diaminobenzidine ( 24 ) was used in the presence of 0.03% h2o2 ( 25 ) . the tunel assay was performed on the cerebral sections to detect the cell death ( using the kit pod ; indianapolis , roche , in ) . after incubation with diamino benzidine dab and hydrogen peroxide , a dark brown color revealed damage to neuronal perikarya ( 26 ) . five coronal sections were chosen for cell counting ( between the bregma ap 0.32 and 0.68 mm ) . the number of surviving and degenerating neurons was counted in five random and non - overlapping regions ( at a magnification of x200 ) . percent of neuronal damage was calculated based on the ratio of the number of degenerated neurons to that of both surviving and degenerated neurons . in addition , the number of astrocytes were counted in five random and separated regions of the cortex ( at the level between bregma ap- 0.12 and + 0.28 ) ( 23 ) . the data were expressed as meansem . one way anova and tukey - kramer multiple post hoc test were used to evaluate the differences between the groups , and p<0.05 was designated for statistical significance . histological studies in the vehicle group animals showed severe morphological changes including extensively dark pyknotic nuclei and shrunken cytoplasm in the brain cortex following tbi . however , in melatonin treatment groups , the severity of degenerative changes in the nucleus and cytoplasm was lower than those in the vehicle and sham groups ( p<0.05 ) ( table 1 ) . in addition , there were few tunel positive cells of brain cortex in the sham group , while many tunel positive cells were detected in the vehicle group ( figure 1 ) . the effect of melatonin on cortical neuronal death following tbi average number of the degenerated cells ( dc ) average percentage of dead cells p<0.05 for melatonin ( 5 , 10 and 20 mg / kg ) p<0.01(sham vs vehicle ) melatonin treatment reduced apoptosis of neurons induced by tbi . results are expressed as meansem and data were analyzed by one - way anova followed by tukey kramer multiple comparisons test * significantly different from the vehicle group ( p<0.05 )   significantly different from the vehicle group ( p<0.01 ) the results of neuronal cell counting showed significant difference between tbi+ 5 mg / kg melatonin ( p<0.05 ) , tbi+ 10 mg / kg melatonin ( p<0.05 ) , and tbi+20 mg / kg melatonin ( p<0.05 ) groups , compared to the vehicle group ( figure 2 ) . arrows show the apoptotic cells . bar=10 m our findings showed that tbi causes a dramatic increase in the number of activated astrocytes in brain cortex . but , melatonin treatment significantly decreased the number of gfap positive astrocytes ( p<0.05 ) that were not dose dependent ( figure 3 and 4 ) . melatonin treatment reduced the number of glial fibrillary acidic protein ( gfap ) positive astrocytes . results are expressed as meansem , and data were analyzed by one - way anova followed by tukey - kramer multiple comparisons test * significantly different from the vehicle group ( p<0.05 )   significantly different from the vehicle group ( p<0.01 ) immunohistochemical analysis of glial fibrillary acidic protein ( gfap ) positive cells in brain cortex of rat . histological studies in the vehicle group animals showed severe morphological changes including extensively dark pyknotic nuclei and shrunken cytoplasm in the brain cortex following tbi . however , in melatonin treatment groups , the severity of degenerative changes in the nucleus and cytoplasm was lower than those in the vehicle and sham groups ( p<0.05 ) ( table 1 ) . in addition , there were few tunel positive cells of brain cortex in the sham group , while many tunel positive cells were detected in the vehicle group ( figure 1 ) . the effect of melatonin on cortical neuronal death following tbi average number of the degenerated cells ( dc ) average percentage of dead cells p<0.05 for melatonin ( 5 , 10 and 20 mg / kg ) p<0.01(sham vs vehicle ) melatonin treatment reduced apoptosis of neurons induced by tbi . results are expressed as meansem and data were analyzed by one - way anova followed by tukey kramer multiple comparisons test * significantly different from the vehicle group ( p<0.05 )   significantly different from the vehicle group ( p<0.01 ) the results of neuronal cell counting showed significant difference between tbi+ 5 mg / kg melatonin ( p<0.05 ) , tbi+ 10 mg / kg melatonin ( p<0.05 ) , and tbi+20 mg / kg melatonin ( p<0.05 ) groups , compared to the vehicle group ( figure 2 ) . our findings showed that tbi causes a dramatic increase in the number of activated astrocytes in brain cortex . but , melatonin treatment significantly decreased the number of gfap positive astrocytes ( p<0.05 ) that were not dose dependent ( figure 3 and 4 ) . melatonin treatment reduced the number of glial fibrillary acidic protein ( gfap ) positive astrocytes . results are expressed as meansem , and data were analyzed by one - way anova followed by tukey - kramer multiple comparisons test * significantly different from the vehicle group ( p<0.05 )   significantly different from the vehicle group ( p<0.01 ) immunohistochemical analysis of glial fibrillary acidic protein ( gfap ) positive cells in brain cortex of rat . the results of this study have shown the neuroprotective effect of melatonin after tbi in male rat . our findings indicate that tbi causes a dramatic increase in neuronal cell death in brain tissue , and based on tunel assay , the percentage of apoptotic cells significantly decreased in the melatonin treatment groups . it seems that melatonin plays a great role in antiapoptotic activities via the inhibition of intrinsic apoptotic pathways , as well as , the activation of several associated signal molecules in various brain regions in tbi model ( 27 ) . in an experimental study , it was evaluated that treatment with melatonin ( 5 and 10mg / kg , ip ) improved the survival rate in a stroke model of mice ( 28 ) . melatonin has been evaluated as effective in tbi , through increasing superoxide dismutase ( sod ) and glutathione peroxidase ( gpx ) activities ( 29 ) . the result of the present study does not show any significant difference between three different doses of melatonin in our animal groups . a previous study has also shown that melatonin significantly attenuated neuronal cell death in hippocampal ca1 and ca3 regions and dentate gyrus of immature rats after head trauma , which was equally effective at doses of 5 and 20 mg / kg ( 30 ) . in another experimental animal model , melatonin treatment led to glial cell death reduction in white matter in mid - gestation fetal sheep following umbilical cord occlusion ( 31 ) . the anti - inflammatory effect of melatonin has been introduced as a chief protective mechanism against brain injury ( 32 , 33 ) . however , the modulation behaviors of melatonin to suppress activated astrocytes have not been particularly investigated in a research with respect to tbi . astrocytes activation occurs in response to many cns pathologies , such as trauma , tumor formation , and neurodegenerative disease . the process of astrocyte activation results in so - called  reactive astrogliosis  , which is a reaction with specific structural and functional characteristics including hypertrophy of cellular process of astrocytes ( 34 ) . up - regulation of intermediate filament proteins , particularly gfap by reactive astrocytes may be applied to distinguish reactive astrogliosis . hence , the present study has focused on reactive astrogliosis based on gfap immunoreactivity ( 35 ) . in fact , astrocytes , as the largest and the most abundant glial cells in cns , may be the target of melatonin . we have demonstrated that the number of gfap positive cells is reduced in the melatonin treatment groups , which indicates the alleviation of astrogliosis process induced by tbi . melatonin has been shown to modify immunity and the stress response , and has an antioxidant activity ( scavenge free radicals ) ( 31 ) . melatonin has a greater antioxidant effect than vitamin e , and can indirectly increase the expression of other endogenous antioxidant enzymes ( 36 ) . ananth and colleagues demonstrated that domoic acid - induced astrocyte activation is attenuated significantly by the intravenous administration of melatonin in the hippocampus of adult rats ( 37 ) . also , studies have shown that among the pathological changes occurred in the acute post - traumatic period , impairment of the blood brain barrier ( bbb ) has a major role and allows the entry of circulating lymphocytes , monocytes , and neutrophils into the injured site , which directly affects inflammation and glial cell reactivities that may lead to neural cell death ( 38 , 39 ) . in addition , it was shown that melatonin administration significantly up regulated stat1 dna - binding activity , and led to the reduction of pro - inflammatory cytokines such as il-6 , and nitric oxide synthase nos ( 33 ) . however , the modulation behaviors of melatonin to astrocyte , an important source of pro - inflammatory cytokines , have not yet been extensively explored . proliferation of astrocytes and their migration toward the lesion site can be recorded by gfap immunoreactivity ( 40 ) . also , we showed the importance of the therapeutic time - window after the initial insult of tbi . it is critical to start interventions as soon as possible upon tbi ( 41 ) . in our experiment , the treatment started one hr after tbi and continued until 72 hr , which is a critical time period for histological examination . in addition , this study only reveals the neuroprotective effects of melatonin after a short time period following tbi . therefore , further investigations need to be performed to clarify the molecular mechanisem of melatonin in suppression of astrocyte reactivity . findings support that melatonin is highly protective of both neurons and glial cells following tbi in our animal model . in general , the present findings suggest that melatonin treatment of tbi markedly diminish astrocyte reactivity ( astrogliosis ) , as well as the number of apoptotic neurons in brain cortex of traumatic brain injury in rat model .
objective(s):melatonin is known as an anti - inflammatory agent , and it has been proven to exert neuroprotection through inhibition of cell death ( apoptosis ) in several models of brain injury . 
 secondary injury following the primary traumatic brain injury ( tbi ) results in glial cells activation , especially astrocytes . 
 in fact , astrocyte activation causes the production of pro - inflammatory cytokines that may lead to secondary injury . since most tbi research studies have focused on injured neurons and paid little attention to glial cells , the aim of current study was to investigate the effects of melatonin against astrocytes activation ( astrogliosis ) , as well as inhibition of apoptosis in brain tissue of male rats after tbi.materials and methods : the animals were randomly allocated into five groups : sham group , tbi+ vehicle group ( 1% ethanol in saline ) and tbi+ melatonin groups ( 5 mg / kg , 10 mg / kg and 20 mg / kg ) . 
 all rats were intubated and then exposed to diffuse tbi , except for the sham group . 
 immunohistochemical methods were conducted using glial fibrillary acidic protein ( gfap ) marker and tunel assay to evaluate astrocyte reactivity and cell death , respectively.results:the results showed that based on the number of gfap positive astrocytes in brain cortex , astrogliosis was reduced significantly ( p<0.05 ) in melatonin- treated groups ( no dose dependent ) compared to the vehicle group . furthermore , based on tunel results , melatonin treatment considerably reduced the number of apoptotic cells ( p<0.05).conclusion : in total , the present findings suggest that melatonin treatment following tbi diminishes astrocyte reactivity and neuronal cells apoptosis in brain cortex in the rat model .