male sprague - dawley rats ( 210 - 220 g , n=30 ) were obtained from samtako co. ( animal breeding center , osan , korea ) and were divided into two groups , sham - operated and middle cerebral artery occlusion ( mcao ) animals ( n=15 per group ) . rats were allowed free access to food and water , and were kept in a temperature and light - controlled environment . all experimental procedures for animal use were approved by the institutional animal care and use committee of gyeongsang national university ( gnu - la-015 ) . to induce focal cerebral ischemia , rats were anesthetized with zolretil ( 50 mg / kg , virbac , carros , france ) before the mcao operation . the right common carotid artery , external carotid artery , and internal carotid artery were exposed through a neck midline incision . a 4/0 nylon monofilament with a heated round tip was introduced into the right external carotid artery and advanced into the internal carotid artery until the tip blocked the origin of the middle cerebral artery . sham - operated rats underwent the same surgical procedure without the insertion of the nylon monofilament . twenty - four hours after blocking the middle cerebral artery , the brains were removed and cut into 2 mm thick coronal slices . the brain slices were stained in 2% triphenyltetrazolium chloride ( ttc ; sigma , st . louis , mo , usa ) at 37 for 20 min and fixed in 10% formalin . for the histopathological study , the brain slices were embedded with paraffin and the paraffin block were cut into 4 m coronal section . the paraffin sections were deparaffined in xylene , rehydrated in gradient ethanol from 100% to 70% , and stained with hematoxylin and eosin solution ( sigma ) . the dehydrated tissue sections were mounted with permount ( sigma ) and observed under a light microscope . twenty - four hours after mcao , the right cerebral cortices were isolated , homogenized in lysis buffer ( 8 m urea , 4% chaps , ampholytes , and 40 mm tris - hcl ) , and centrifuged at 16,000 g for 20 min at 4. after centrifugation , the pellets were dissolved in lysis buffer and the protein concentration was measured using the bradford method ( bio - rad , hercules , ca , usa ) according to the manufacturer 's protocol . the ettan ipgphor 3 system ( ge healthcare , uppsala , sweden ) was used for first - dimensional isoelectric focusing ( ief ) . immobilized ph gradient ( ipg ) gel strips ( 17 cm , ph 4 - 7 and ph 6 - 9 ; bio - rad ) were rehydrated in buffer ( 8 m urea , 2% chaps , 20 mm dtt , 0.5% ipg buffer , and bromophenol blue ) for 13 hours . the protein samples ( 100 g ) were loaded onto the ipg strips using a sample cup and ief was performed as previously described . the strips were subjected to gradient gels ( 7.5 - 17.5% ) for sodium dodecyl sulfate ( sds ) gel electrophoresis . the gels were loaded on protein - ii xi electrophoresis equipment ( bio - rad ) and subjected to a 5 ma for 2 hours , followed by 10 ma for 10 hours at 10. the gels were fixed in 12% acetic acid and 50% methanol for 2 hours and washed with 50% ethanol for 20 min . the gels were stained with silver solution ( 0.2% silver nitrate ) for 20 min and developed in a solution of 0.2% sodium carbonate . the gel images were scanned with an agfa arcus 1200 ( agfa - gevaert , mortsel , belgium ) . pdquest 2-d analysis software ( bio - rad ) was used for analysis of the protein spots . protein spots were excised from the gels and processed for matrix assisted laser desorption ionizationtime of flight ( maldi - tof ) . peptides were analyzed with a voyager system de - str biospectrometry workstation ( applied biosystem , foster city , ca , usa ) . ms - fit and profound programs were used to detect proteins and the databases swiss - prot and ncbi were used to identify protein sequences . the right cerebral cortices were homogenized in lysis buffer [ 1% triton x-100 , 1 mm edta in pbs ( ph 7.4 ) ] and centrifuged at 15,000 g for 20 min at 4. after centrifugation , the supernatants were harvested for further processing and the protein concentration was measured according to the manufacturer 's protocol . from each sample , 30 g of total protein was loaded on 10% sds polyacrylamide gel and electrophoresis was performed . the electrophoresed proteins were transferred onto polyvinylidene fluoride membranes ( millipore , billerica , ma , usa ) and the membranes were washed in tris - buffered saline containing 0.1% tween-20 ( tbst ) . the membranes were blocked with 5% milk solution for 1 hour to minimize non - specific antibody binding and incubated with antibodies against anti - dlp-1 ( diluted 1:1,000 , bd biosciences , san jose , ca , usa ) and antiactin ( diluted 1:1,000 , santa cruz biotechnology , santa cruz , ca , usa ) as the primary antibodies . the membranes were washed with tbst and reacted with horseradish perxoxidase - conjugated goat anti - rabbit igg secondary antibody ( 1:5,000 ; santa cruz biotechnology ) . the enhanced chemiluminescence western blot analysis system ( amersham pharmacia biotech , piscataway , nj , usa ) was used for the detection of immunoreactive bands . the cell culture system was performed used as previously described . a mouse hippocampal cell line ( ht22 ) was grown in dulbecco 's modified eagle 's medium ( without l - glutamine ) with 10% fetal bovine serum . the cell density was maintained at a confluence of 70% or less based on a previously described . the cells were treated with 5 mm glutamate ( sigma ) or vehicle for 24 hours . the cell viability was investigated by measuring the metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) , as previously described . mtt solution ( 5 mg / ml ) was added to each well of a 96-well plate and incubated for 4 hour at 37. after removal of the mtt solution , a solubilization solution containing 50% dimethylformamide and 20% sds ( ph 4.8 ) was added and incubated overnight . the absorbance was then measured at a wavelength of 570 nm using a microplate reader . the cell viability was represented as the percentage of neuroprotection vs. control set at 100% . the intensity analysis was carried out using sigma gel 1.0 ( jandel scientific , san rafael , ca , usa ) and sigma plot 4.0 ( spss inc . , the ratio of intensity is described as spots intensity of mcao - operated animal to spots intensity of sham - operated animal . the results in each group were compared by one - way analysis of variance ( anova ) followed by student 's t - test . male sprague - dawley rats ( 210 - 220 g , n=30 ) were obtained from samtako co. ( animal breeding center , osan , korea ) and were divided into two groups , sham - operated and middle cerebral artery occlusion ( mcao ) animals ( n=15 per group ) . rats were allowed free access to food and water , and were kept in a temperature and light - controlled environment . all experimental procedures for animal use were approved by the institutional animal care and use committee of gyeongsang national university ( gnu - la-015 ) . to induce focal cerebral ischemia , rats were anesthetized with zolretil ( 50 mg / kg , virbac , carros , france ) before the mcao operation . the right common carotid artery , external carotid artery , and internal carotid artery were exposed through a neck midline incision . a 4/0 nylon monofilament with a heated round tip was introduced into the right external carotid artery and advanced into the internal carotid artery until the tip blocked the origin of the middle cerebral artery . sham - operated rats underwent the same surgical procedure without the insertion of the nylon monofilament . twenty - four hours after blocking the middle cerebral artery , the brains were removed and cut into 2 mm thick coronal slices . the brain slices were stained in 2% triphenyltetrazolium chloride ( ttc ; sigma , st . louis , mo , usa ) at 37 for 20 min and fixed in 10% formalin . for the histopathological study , the brain slices were embedded with paraffin and the paraffin block were cut into 4 m coronal section . the paraffin sections were deparaffined in xylene , rehydrated in gradient ethanol from 100% to 70% , and stained with hematoxylin and eosin solution ( sigma ) . the dehydrated tissue sections were mounted with permount ( sigma ) and observed under a light microscope . twenty - four hours after mcao , the right cerebral cortices were isolated , homogenized in lysis buffer ( 8 m urea , 4% chaps , ampholytes , and 40 mm tris - hcl ) , and centrifuged at 16,000 g for 20 min at 4. after centrifugation , the pellets were dissolved in lysis buffer and the protein concentration was measured using the bradford method ( bio - rad , hercules , ca , usa ) according to the manufacturer 's protocol . the ettan ipgphor 3 system ( ge healthcare , uppsala , sweden ) was used for first - dimensional isoelectric focusing ( ief ) . immobilized ph gradient ( ipg ) gel strips ( 17 cm , ph 4 - 7 and ph 6 - 9 ; bio - rad ) were rehydrated in buffer ( 8 m urea , 2% chaps , 20 mm dtt , 0.5% ipg buffer , and bromophenol blue ) for 13 hours . the protein samples ( 100 g ) were loaded onto the ipg strips using a sample cup and ief was performed as previously described . the strips were subjected to gradient gels ( 7.5 - 17.5% ) for sodium dodecyl sulfate ( sds ) gel electrophoresis . the gels were loaded on protein - ii xi electrophoresis equipment ( bio - rad ) and subjected to a 5 ma for 2 hours , followed by 10 ma for 10 hours at 10. the gels were fixed in 12% acetic acid and 50% methanol for 2 hours and washed with 50% ethanol for 20 min . the gels were stained with silver solution ( 0.2% silver nitrate ) for 20 min and developed in a solution of 0.2% sodium carbonate . the gel images were scanned with an agfa arcus 1200 ( agfa - gevaert , mortsel , belgium ) . pdquest 2-d analysis software ( bio - rad ) was used for analysis of the protein spots . protein spots were excised from the gels and processed for matrix assisted laser desorption ionizationtime of flight ( maldi - tof ) . peptides were analyzed with a voyager system de - str biospectrometry workstation ( applied biosystem , foster city , ca , usa ) . ms - fit and profound programs were used to detect proteins and the databases swiss - prot and ncbi were used to identify protein sequences . the right cerebral cortices were homogenized in lysis buffer [ 1% triton x-100 , 1 mm edta in pbs ( ph 7.4 ) ] and centrifuged at 15,000 g for 20 min at 4. after centrifugation , the supernatants were harvested for further processing and the protein concentration was measured according to the manufacturer 's protocol . from each sample , 30 g of total protein was loaded on 10% sds polyacrylamide gel and electrophoresis was performed . the electrophoresed proteins were transferred onto polyvinylidene fluoride membranes ( millipore , billerica , ma , usa ) and the membranes were washed in tris - buffered saline containing 0.1% tween-20 ( tbst ) . the membranes were blocked with 5% milk solution for 1 hour to minimize non - specific antibody binding and incubated with antibodies against anti - dlp-1 ( diluted 1:1,000 , bd biosciences , san jose , ca , usa ) and antiactin ( diluted 1:1,000 , santa cruz biotechnology , santa cruz , ca , usa ) as the primary antibodies . the membranes were washed with tbst and reacted with horseradish perxoxidase - conjugated goat anti - rabbit igg secondary antibody ( 1:5,000 ; santa cruz biotechnology ) . the enhanced chemiluminescence western blot analysis system ( amersham pharmacia biotech , piscataway , nj , usa ) was used for the detection of immunoreactive bands . the cell culture system was performed used as previously described . a mouse hippocampal cell line ( ht22 ) was grown in dulbecco 's modified eagle 's medium ( without l - glutamine ) with 10% fetal bovine serum . the cell density was maintained at a confluence of 70% or less based on a previously described . the cells were treated with 5 mm glutamate ( sigma ) or vehicle for 24 hours . the cell viability was investigated by measuring the metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) , as previously described . mtt solution ( 5 mg / ml ) was added to each well of a 96-well plate and incubated for 4 hour at 37. after removal of the mtt solution , a solubilization solution containing 50% dimethylformamide and 20% sds ( ph 4.8 ) was added and incubated overnight . the absorbance was then measured at a wavelength of 570 nm using a microplate reader . the cell viability was represented as the percentage of neuroprotection vs. control set at 100% . the intensity analysis was carried out using sigma gel 1.0 ( jandel scientific , san rafael , ca , usa ) and sigma plot 4.0 ( spss inc . , the ratio of intensity is described as spots intensity of mcao - operated animal to spots intensity of sham - operated animal . the results in each group were compared by one - way analysis of variance ( anova ) followed by student 's t - test . the difference for comparison we confirmed that the mcao operation led to brain damage and neuronal cell death using ttc staining ( figure 1 ) . the infarct regions were 2.20.5 and 24.84.18% in sham - operated and mcao - induced animals , respectively . neuron in mcao - induced animals typically had a scalloped and shrunken morphology and apoptotic bodies containing intensive dark masses were found in cell nuclei . however , sham - operated animals had intact neurons in the cerebral cortex . figure 2 shows change in dlp-1 expression in the cerebral cortices of sham - operated and mcao - induced animals . the peptide mass of dlp-1 is 14/90 and the sequence of this protein is 21% . dlp-1 protein levels were decreased in mcao - induced animals compared to sham - operated animals . the levels of dlp-1 were 1.190.04 and 0.810.03 in the cerebral cortices of sham - operated and mcao - induced animals , respectively . in the in vitro study , glutamate exposure led to neuronal cell death in cultured ht22 cells ( figure 4a ) . moreover , western blot analysis showed that glutamate toxicity induces decrease in dlp-1 levels in ht22 cells . the levels of dlp-1 were 0.9 30.03 and 0.370.03 in vehicle - treated and glutamate - treated groups , respectively ( figure 4b , 4c ) . cerebral ischemia caused by surgical induction of mcao induces neuronal cell death in cerebral cortex . the results of this study confirmed that mcao - induced injury increases infarct regions and apoptotic cell death in the cerebral cortex . in this study , we focused on dlp-1 expression in the cerebral cortex after mcao and as well as in glutamate - exposed hippocampal neurons . a decrease in dlp-1 protein expression was identified after mcao - induced brain injury using a proteomic approach . moreover , western blot analysis clearly demonstrated that focal cerebral ischemia results in a significant decrease in dlp-1 protein expression . oxidative stress is involved in the development of neurodegenerative diseases including stroke , parkinson 's disease , and alzheimer 's disease . oxidative stress leads to the generation of ros , atp depletion , mitochondrial permeability transition pore opening , and results in apoptotic cell death . mitochondria , a major source of ros , are involved in the integration of cellular responses to stress . moreover , dlp-1 mediates outer mitochondrial membrane fission , modulates mitochondrial morphology , and regulates mitochondria - dependent cell death . dlp-1 depletion induces not only serious brain hypoplasia but also death during embryonic development and after birth in dlp-1 knockout mice . changes in dlp-1 expression during mcao injury induce mitochondrial dysfunction , resulting in neuronal cell death . in cultured hippocampal cells , glutamate neurotoxicity generates ros , increases mitochondrial damage , and consequently leads to cell death . the increase in intracellular ca by ischemic brain injury induces mitochondrial dysfunction and leads to neuronal cell death via mitochondrial permeability transition . this study demonstrated that glutamate treatment leads to ischemic conditions and results in neuronal cell death . dlp-1 is an important mitochondrial fission and fusion protein that plays an important role in mitochondrial dynamic actions . imbalances in mitochondrial fission and fusion lead to structural impairment and dysfunction of mitochondria , consequently disrupting atp generation and causing neuronal cell death . this study revealed decreased dlp-1 levels in rats with mcao - induced injury and in glutamate - exposed ht22 cells . these results suggest that decreased dlp-1 expression leads to the imbalance of mitochondrial fission and fusion , resulting in neuronal cell death .
dynamin - like protein i ( dlp-1 ) is an important mitochondrial fission and fusion protein that is associated with apoptotic cell death in neurodegenerative diseases . in this study 
 , we investigated dlp-1 expression in a focal cerebral ischemia animal model and glutamate - exposed hippocampal - derived cell line . 
 middle cerebral artery occlusion ( mcao ) was surgically induced in adult male rats to induce focal cerebral ischemic injury . 
 brain tissues were collected 24 hours after the onset of mcao . 
 mcao induces an increase in infarct volume and histopathological changes in the cerebral cortex . 
 we identified a decrease in dlp-1 in the cerebral cortices of mcao - injured animals using a proteomic approach and western blot analysis . 
 moreover , glutamate treatment significantly decreased dlp-1 expression in a hippocampal - derived cell line . 
 the decrease in dlp-1 indicates mitochondrial dysfunction . 
 thus , these results suggest that neuronal cell injury induces a decrease in dlp-1 levels and consequently leads to neuronal cell death .