animals and pre - operative care : all animal procedures were approved by the 
 ethics committee for animal care and use at inje university ( approval no . 201229 ) . a total 
 of 47 healthy adult male rats , weighing 240260 g , and all animals were individually housed in plastic cages 
 at controlled temperature ( 22  1c ) , relative humidity ( 55  10% ) and photoperiod 
 ( light / dark conditions 12/12 hr lights on 7:00 a.m. ) . food and water were available 
 ad libitum . preparations of middle cerebral artery occlusion microsurgery : mcao 
 surgery required 40 and 60 nylon threads ( ailee co. , busan , korea ) in rats and mice , 
 respectively . the nylon thread was cut into 4 cm ( for rats ) or 2 cm ( for mice ) pieces , and 
 its tips were blunted to form a bulb shape by heating ( outer diameter of bulb shape or corn 
 dog form : 0.40.45 mm for rats and 0.150.18 mm for mice ) . microsurgical procedures of middle cerebral artery occlusion : all animals 
 were anesthetized with 40 mg / kg tiletamine / zolazepam cocktail ( zoletil ) and 10 mg / kg 
 xylazine ( rompun ) via intraperitoneal injection . after anesthesia , the animals body 
 temperature was maintained at 36.5c to 37.0c using a heating pad on the surgical table . the animals were placed in the supine position and fixed to the surgical table using 
 adhesive tape . the 
 midline neck skin was incised , and the common carotid artery ( cca ) , external carotid artery 
 ( eca ) and internal carotid artery ( ica ) were carefully separated from the vagus nerve . the 
 ica bifurcates into the middle cerebral artery ( mca ) and the pterygopalatine artery ( ppa ) 
 from the cca . the occipital artery ( oa ) originates from the bifurcation point of the eca and 
 the ica . a transient knot was placed on the distal portion of the cca , near the manubrium of 
 the sternum . a microvascular clamp was transiently placed on the ica proximal to the cca 
 junction . two closely spaced permanent knots were then placed on the distal portion of the 
 eca , below the suprathyroid artery , to prevent the backflow of blood . the tied section of 
 the eca was dissected using microscissors to insert the probe , which reached the ica through 
 the cca junction . the microvascular clamp that had been placed on the ica was then removed 
 to allow probe insertion . the probe was carefully inserted into the mca from the cca 
 junction ( up to 1820 mm in rats and 911 mm in mice ) . after confirmation of mca blockage 
 ( mild bending of the ica ) , the rat model exhibited blood supply from the cca , but the mouse 
 model only exhibited blood supply after the occlusion period ( dual occlusion by cca 
 ligation ) . after 60 to 90 min , the probe was carefully withdrawn until the tip was near the 
 arteriotomy for reperfusion . after confirmation of blood flow reperfusion , topical lidocaine 
 gel was applied onto the surgical region to relieve pain and discomfort in the postoperative 
 period . each animal also received 1.0 ml of normal saline subcutaneously 
 for volume replenishment after the surgery . all surgical procedures were finished within 15 
 min , excluding the occlusion and reperfusion periods ( fig . 1.structural diagram of the mcao surgery region and histological results followed by an 
 occlusion period in rats and mice . h&e- and nissl - stained brains of ( b ) mcao mice and ( c ) rats . brain slices 
 were cut at 1-mm ( mice ) and 2-mm ( rats ) intervals rostral and caudal to the bregma 
 ( 0.00 mm ) . the yellow ( h&e staining ) and black ( nissl staining ) dotted lines 
 indicate the marginal penumbra lesion . the black arrow indicates the pale violet color 
 of the ipsilateral lesion of the corpus callosum . the 90 min mcao models not only 
 showed severe infarct lesion but also failure to confirmed corpus callosum . cca : 
 common carotid artery ; eca : external carotid artery ; ica : internal carotid artery ; oa : 
 occipital artery ; ppa : pterygopalatine artery ; mca : middle cerebral artery . ) . structural diagram of the mcao surgery region and histological results followed by an 
 occlusion period in rats and mice . h&e- and nissl - stained brains of ( b ) mcao mice and ( c ) rats . brain slices 
 were cut at 1-mm ( mice ) and 2-mm ( rats ) intervals rostral and caudal to the bregma 
 ( 0.00 mm ) . the yellow ( h&e staining ) and black ( nissl staining ) dotted lines 
 indicate the marginal penumbra lesion . the black arrow indicates the pale violet color 
 of the ipsilateral lesion of the corpus callosum . the 90 min mcao models not only 
 showed severe infarct lesion but also failure to confirmed corpus callosum . cca : 
 common carotid artery ; eca : external carotid artery ; ica : internal carotid artery ; oa : 
 occipital artery ; ppa : pterygopalatine artery ; mca : middle cerebral artery . measurements of neurological dysfunction in rats and mice followed by 
 mcao : the battery of rat mcao model tests consisted of evaluation of the modified 
 neurological severity score ( mnss ) . table 1table 1.modified neurological severity score points ( mnss)motor functionsscoreraising rat by tail3flexion of forelimb1flexion of hindlimb1head moved > 10 to vertical axis within 30 
 sec1placing rat on floor ( normal=0 ; 
 maximum=3)3normal walk0inability to walk straight1circles toward paretic side2falls down to paretic side3sensory tests2placing test ( visual and tactile test)1proprioceptive test ( deep sensation , pushing paw 
 against table edge to stimulate limb muscles)1beam balance tests ( normal=0 ; 
 maximum=6)6balances with steady posture0grasps side of beam1hugs beam and 1 limb falls down from beam2hugs beam and 2 limbs fall down from beam , or spins on 
 beam ( > 60 sec)3attempts to balance on beam but falls off ( > 40 
 sec)4attempts to balance on beam but falls off ( > 20 
 sec)5falls off ; no attempt to balance or hang on to beam 
 ( < 20 sec)6reflex absence and abnormal movements4pinna reflex ( head shake when auditory meatus is 
 touched)1corneal reflex ( eye blink when cornea is lightly 
 touched with cotton)1startle reflex ( motor responses to a brief noise from 
 snapping a clipboard paper)1seizures , myoclonus , myodystony1maximum points18one point is awarded for inability to perform the tasks or for lack of a tested 
 reflex : 1318 , severe injury ; 712 , moderate injury ; 16 , mild injury . neurological function was graded on a scale of 0 to 
 18 ( normal score : 0 , maximal deficit score : 18 ) . the mnss evaluation involves a composite of 
 motor , sensory , reflex and balance tests . in the severity scores of injury , 1 point is 
 awarded for the inability to perform the test or for the lack of a tested reflex ; thus , the 
 higher the score , the more severe the injury . likewise , mouse mcao models have been 
 suggested to have an association with a neurological disability status score ( ndss ) ( table 2table 2.neurological disability status scaledegree of deficitneurobehavioral alterations0none2hypomobility ( slight)passivity4hypomobility ( moderate)flattened posturehunched backataxic gaitpiloerectiondecreased body tonedecreased muscular strengthmotor incoordination ( slight)6circlingtremor / twitches / convulsionsforelimb flexionmotor incoordination ( moderate)8hypomobility ( severe)motor incoordination ( severe)respiratory distress10death ) . one point is awarded for inability to perform the tasks or for lack of a tested 
 reflex : 1318 , severe injury ; 712 , moderate injury ; 16 , mild injury . evaluation of infarct lesion between rats and mice followed by mcao : the 
 subjects were anesthetized with an intraperitoneal injection of zoletil ( 40 mg / kg ) and 
 rompun ( 10 mg / kg ) . subsequently , they underwent transcardial perfusion with 0.1 m pbs 
 ( phosphate - buffered saline ) ( ph 7.4 ) , followed by fix with 4% nbp ( neutrally buffered 
 paraformaldehyde ) . the brain tissues were rapidly removed and stored in the same solution 
 ( 4% nbp ) for 24 hr at 4c and then passed through a gradient ( 10 , 20 , and 30% ) of sucrose 
 solutions for cryoprotection . next , tissue samples were embedded in tissue - tek oct compound 
 ( sakura finetek , torrance , ca , u.s.a . ) . brain tissues were dissected at 10 and 20 
 m using a cryostat ( hm525 ; microm international gmbh , walldorf , 
 germany ) . they were then stained with either h&e ( 10-m thick sections ) 
 or cresyl violet acetate ( nissl ; 20-m thick sections ) , serially dehydrated 
 in etoh solutions , cleared with xylene and mounted with permount ( sp15 - 500 ; fisher 
 scientific , fair lawn , nj , u.s.a . ) . statistical analysis : data were collected from repeated experiments and 
 are presented as means  sd . student`s all analyses were performed using the spss software ( spss ver . 
 19.0 , ibm , chicago , il , u.s.a . ) . alteration of body weight in rodent mcao animal models : mcao surgery was 
 applied to 8-week - old rats and mice , as well as equalization of the body weight from 240260 
 g ( rats ; 60 min : 253.08  5.62 g and 90 min : 256.89  2.26 g ) and 2025 g ( mice ; 60 min : 
 23.30  0.79 g and 90 min : 23.50  1.19 g ) . the subjects showed significant reductions of 
 body weight compared with those before onset ( rats ; 60 min : 23.70  7.43 g and 90 min : 36.29 
  9.31 g ) ( mice ; 60 min : 2.03  0.54 g and 90 min : 3.17  1.11 g ) 
 ( p<0.01 ) ( fig . ( a ) 
 alteration of body weight of rat mcao models followed by an occlusion period . ( b ) 
 alteration of body weight of mouse mcao models followed by an occlusion period . ( c ) 
 neurological severity score of rat mcao models at 24 hr after transient mcao surgery 
 by occlusion period . ( d ) neurological disability status of mouse mcao models at 24 hr 
 after transient mcao surgery by occlusion period . the 90 min occlusion period was clearly 
 associated with a severe score , which affected survival ratio and model identity . mnss : modified neurological severity score ; ndss : neurological disability status 
 score . * p<0.05 , * * p<0.01 compared with that at 
 onset , student s t - test . ) . in 60 min occlusion models , there were reductions of approximately 10% compared 
 with initial body weight in rats and mice . however , 90 min occlusion models showed 
 approximately 15% reductions compared with initial body weight 
 ( p<0.01 ) . ( a ) 
 alteration of body weight of rat mcao models followed by an occlusion period . ( b ) 
 alteration of body weight of mouse mcao models followed by an occlusion period . ( c ) 
 neurological severity score of rat mcao models at 24 hr after transient mcao surgery 
 by occlusion period . ( d ) neurological disability status of mouse mcao models at 24 hr 
 after transient mcao surgery by occlusion period . occlusion period did not 
 significantly affect neurological score . the 90 min occlusion period was clearly 
 associated with a severe score , which affected survival ratio and model identity . mnss : modified neurological severity score ; ndss : neurological disability status 
 score . * p<0.05 , * * p<0.01 compared with that at 
 onset , student s t - test . survival ratio of mcao rodent models : the 60 min occlusion rat model 
 animals showed a survival ratio of 80.95% . a total of 6 rats were excluded in the 
 moderate - severity mcao model . specifically , 3 rats were lost due to respiratory failure 
 after reperfusion , 1 rat died from bleeding from the jugular vein , and 2 rats were excluded 
 due to mild symptoms . therefore , a total of 21 rats underwent mcao surgery , and 15 of them 
 showed moderate symptoms ( model engineering success ratio : 72.43% ) . in the mice , a total of 16 mice also showed moderate - severity symptoms , 
 as well as 3 mice that died due to respiratory failure . in terms of comparison of the 90 min 
 occlusion mcao models , rat was lost 
 due to cardiac arrest at approximately 80 min during occlusion , 1 rat died from bleeding 
 from the occipital artery , and 3 rats were lost due to cardiac arrest with respiratory 
 failure . a total of 8 rats were also lost due to respiratory failure after reperfusion . therefore , a total of 26 rats underwent mcao surgery , and 11 rats showed moderate to severe 
 symptoms . in the mouse mcao model animals , 10 mice were also lost due to respiratory 
 failure , as well as 2 mice that died from bleeding upon occipital artery rupture . likewise , 
 18 mice were subjected to mcao surgery , and 6 mice also showed high severity symptoms ( table 3table 3.results of neurological behavior tests and alteration of body weightocclusion periodratsmicemnssbody weight ( g)ndssbody weight ( g)onset24 hronset24 hr60 min(n=19)11.93  0.96253.08  5.62229.38  6.51(23.70  7.43)6.81  0.83 ( n=16)23.30  0.7921.28  0.68(2.03  0.54)90 min(n=26)13.18 0.98256.89  2.26220.60  7.92(36.29  9.31)8.50  0.55(n=18)23.50  1.1920.18  0.33(3.17  1.11 ) ) . measurement of neurological dysfunction : all subjects were evaluated for 
 neurological dysfunction following mnss ( for rats ) and ndss ( for mice ) . a period of occlusion of 60 min occlusion might be 
 appropriate for moderate severity . a total of 19 rats showed moderate - severity symptoms 
 ( 11.93  0.96 ) ( fig . 2c ) . however , 2 rats showed 
 mild symptoms , excluding circling gait , spasticity of paretic limbs or falling down on the 
 beam . in the mice , in contrast , 90 min occlusion more often 
 showed higher severity than 60 min . a total of 11 rats showed high - severity symptoms ( 13.18 
  0.98 ) , including falling down on the paretic side in open field , falling down in 20 sec on 
 the beam and the absence of a proprioceptive response ( fig . 2c ) . likewise , 6 mice presented high - severity symptoms , including severe 
 hypomobility , respiratory distress and motor in coordination ( fig . comparison of infarct lesions of rodent mcao animal model : transient mcao 
 models were affected by the occlusion period . therefore , the occlusion period was divided 
 into 60 and 90 min . the animals were 
 employed for the following histological studies : ttc staining ( 60 min occlusion : 3 rats and 
 3 mice ; 90 min occlusion : 3 rats and 2 mice ) , h&e and nissl staining ( 60 min occlusion : 
 12 rats and 13 mice ; 90 min occlusion : 8 rats and 4 mice ) . we previously found that the 
 infarct volume is affected by the occlusion period using the ttc staining method . therefore , h&e and nissl staining was performed according to the 
 protocol described in the methods section . in the comparison of the infarct volume using 
 h&e staining , the bregma 0.00 mm ( slice # 3 ) region showed the largest infarct volume at 
 60 min of occlusion in both rat and mouse models ( rats , 58.43  0.38% ; mice , 46.70  0.81% ) . 
 however , the infarct volume was slightly smaller in the caudal slices . infarct lesion of 
 mcao animal models with occlusion for 90 min was significantly extended to retrograde at # 3 , 
 # 4 and # 5 compared with those of mcao animal models with occlusion for 60 min in rats and 
 mice ( * p<0.05 , * * p<0.01 ) ( table 4table 4.results of infarct volume in rats and mice following occlusion periodslice no.60 min occlusion#1#2#3#4#5rats30.68  4.5041.01  2.8858.43  0.3855.09  0.1845.74  3.00mice20.94  1.1145.78  1.5346.70  0.8137.88  1.5431.86 1.08slice no.90 min occlusion#1#2#3#4#5rats36.75  2.0945.72  1.1860.63  1.2058.47  0.6858.79  1.00mice41.98  2.4250.84  1.9655.53  1.5352.49  1.6750.57  2.36*p<0.05 , * * p<0.01 : 60 min vs. 90 min in rats 
 and mice . ) . 1b and c , black arrow ) without a clear division 
 between superficial and profundus layer in cerebrum ( fig . * p<0.05 , * * p<0.01 : 60 min vs. 90 min in rats 
 and mice . the intraluminal mcao model mimics one of the most common types of ischemic stroke in 
 human . however , mouse stroke models have 
 insteadoften involved genetically or molecularly manipulated mice in research using knockout 
 and knockdown techniques [ 5 , 6 , 17 ] . knockout mice have been 
 informative in the discovery of unexpected biological functions of specific molecules . in particular , targeted molecules were identified as 
 having roles in pathophysiological mechanisms . in stroke - related studies , knockout mice have 
 been used to identify the inhibitory mechanism of cell death signaling , as well as applied 
 in the pharmacological developmental industry through elucidation of the effects of 
 candidate molecules that exhibit therapeutic efficiency [ 9 , 17 ] . genetically mutated mouse models 
 are essential for exploring genetic diseases or undisclosed disease mechanisms . numerous studies have described mcao surgery procedures [ 3 , 4 , 10,11,12 , 14 , 19 , 20 ] . however , there are different 
 procedures , such as in terms of filament size and tip diameter , insert length , occlusion 
 period and surgical techniques . the aim of the present study was to establish a standard 
 mouse mcao model surgery procedure , as well as to compare it with rat mcao , which is 
 extensively applied in stroke - related animal research . in mcao surgery , when all procedures have been completed , recovery of the body temperature 
 must be achieved , since numerous studies have proposed a body temperature range of 37.0  
 0.5c , as well as indicating that the operation period can also affect hypothermia . body 
 temperature is one of the essential factors affecting the extent of infarction , hypothermia 
 decreases and hyperthermia increases the infarct lesion size . consequently , we exclude animals that have undergone 
 operation for longer than 15 min . the rat mcao model used a 40 monofilament nylon , which is 
 made of a 0.40.45-mm outer - diameter thread by heating . numerous studies have suggested that 
 the optimal thread requires 60 monofilament nylon for occlusion , which is made with a 
 0.170.2-mm outer - diameter by heating [ 6 , 13 ] . mouse mcao models showed similar infarct volume to the rat mcao models upon 60 min 
 occlusion . in particular , the bregma 0.00 mm region showed the largest infarct volume in 
 both rat and mouse brains , as well as presenting a pattern of infarct lesion from rostral to 
 caudal in 60 min occlusion models . conversely , infarct volume was shown to be slightly reduced from bregma 
 0.00 to caudal ( fig . we previously suggested that the occlusion period by mcao surgery affects the infarct 
 volume in rat and mouse models of focal cerebral ischemia ; rat and mouse 90 min mcao models 
 exhibited significantly augmented infarct volume from bregma 0.00 to 4.00 mm and from 2.00 
 to 2.00 mm , respectively . brain infarct volume 
 might be associated with the survival rate . in terms of the survival ratio , the 60 min occlusion rat model showed an approximately 80% 
 survival ratio , as well as a 72.43% ischemic model engineering success ratio . likewise , 
 mouse model animals showed approximately 85% survival and 85% ischemic model engineering 
 success ratio . however , 90 min occlusion models showed a poor model engineering success 
 ratio followed by cardiac arrest , respiratory failure and severe ischemic damage . as a 
 result , 90 min was associated with 42.03% and 33.34% model engineering success ratios in 
 rats and mice , respectively . we reported that transient mcao models exhibited a moderate severity score by the mnss test 
 [ 12 , 19 , 
 20 ] . in terms of a comparison of neurological 
 dysfunction , 60 min occlusion rat model animals revealed optimal moderate - severity symptoms , 
 including flexion pattern of forelimb , circling gait toward the paretic side and poor 
 balance or falling down on the beam . however , 90 min occlusion rat model animals exhibited 
 severe neurological symptoms , such as falling down on the paretic side during gait , not 
 attempting to balance on the beam , and myodystony . mouse mcao model animals also showed moderate neurological 
 disability status , as well as presenting with moderate - severity symptoms , including circling 
 gait , moderate hypomobility , excessive urination , forelimb flexion and body rotation by tail 
 suspension . likewise , 90 min mouse mcao model animals showed a severe neurological 
 disability status , such as severe motor in coordination namely no attempt to climb to the 
 top of the edge on the inclination board , severe hypomobility namely no movement during 30 
 sec and respiratory distress . in conclusion , we have elucidated that the mouse mcao model procedure could induce a 
 moderate - severity mcao model , as well as that our method might obtain a higher survival 
 ratio and stable model engineering using mice . to produce results relevant for a stroke study , standardized and high - quality rodent models 
 are very important in experimental stroke research . in addition , rodent mcao model 
 engineering has to consider monofilament standard for a hand - made probe , tip outer diameter 
 ( unrelated to silicone coating or rounding by heating ) , probe insert length ( is needed 
 regarding hemorrhage or aca infarction ) and maintenance of body conditions during operation 
 ( body temperature control ) and occlusion time ( moderate versus severe score models and 
 identity of model ) . the following is a summary of rodent mcao models : 
 ( 1 ) 0.400.45 mm probe of 40 nylon ( for rats ) and 0.150.18 mm probe of 60 nylon ( for 
 mice ) by heating(2)inserted probe length from bifurcated ica and eca : 1820 mm ( for rats ) and 911 mm ( for 
 mice)(3)operation time : a maximum of 15 min(4)occlusion time : 60 min(5)mca occlusion allows cca reperfusion ( for rats ) or cca binary occlusion ( for mice ) 
   0.400.45 mm probe of 40 nylon ( for rats ) and 0.150.18 mm probe of 60 nylon ( for 
 mice ) by heating inserted probe length from bifurcated ica and eca : 1820 mm ( for rats ) and 911 mm ( for 
 mice ) operation time : a maximum of 15 min occlusion time : 60 min mca occlusion allows cca reperfusion ( for rats ) or cca binary occlusion ( for mice )
rodent models of focal cerebral ischemia that do not require craniotomy have been 
 developed by intraluminal suture middle cerebral artery occlusion ( mcao ) . 
 mouse mcao 
 models have been widely used and extended to genetic studies of cell death or recovery 
 mechanisms . 
 therefore , we compared surgery - related parameters and techniques between such 
 rats and mice . in rodent mcao models , has to be considered body temperature during the 
 operative period , as well as the need for the use of a standardized tip in terms of the 
 outer diameter of probes . 
 induction of focal cerebral ischemia was measured by 
 neurological dysfunction parameters . 
 our methods could induce stable moderate - severity 
 ischemic brain injury models and histological alteration at 24 hr after mcao surgery . 
 moreover approximately 80% ( rats ) and 85% ( mice ) survival ratios were shown indicating 
 with model engineering success . 
 finally , we described and compared major parameters 
 between rats and mice , including probe size , thread insert length , operation and occlusion 
 periods , and differences in the procedures .