research on stroke mechanisms reveals secondary injury develops through the inflammatory reaction induced by the leukocytes that adhere to the micro vessels during cerebral ischemia - reperfusion.9 ) this mechanism was verified in animal experiments using middle cerebral artery occlusion and reperfusion ( mcao - r ) models.5)12 ) experiments using the mcao - r model , found reduced leukocyte infiltration and enhanced neurologic functions in mice that were injected with adhesion - molecule - specific monoclonal antibodies ( mabs ) , or in mice whose adhesion molecules were removed through genetic manipulation , compared with wild - type mice.13)21)25 ) studies on adiponectin are active , and many favorable effects of adiponectin on the vascular system reported .
adiponectin activates angiogenesis in ischemic damages,19 ) and inhibits atherosclerosis.15 ) in addition , a negative association between the initial infarction volume and the adiponectin level in ischemic stroke was confirmed.6 ) in serial measurement of adiponectin level in ischemic cerebrovascular disease patients , a gradual decrease was observed.24 ) in this study , the effects of adiponectin on the interaction between leukocytes and endothelia were investigated through experiments based on the hypothesis that adiponectin might inhibit the secondary inflammatory reaction in cerebral ischemia .
all experiments were conducted according to the guidelines issued by the institutional animal care and use committee and complied with the national institutes of health ( nih ) guide for the care and use of laboratory animals . for the experiments of this study , 10 to 12-week - old adiponectin - deficient mice ( apn - ko ) ( n = 8) and wild - type mice ( wt ) ( n = 8) were prepared .
each group was sub - divided into 2 groups by reperfusion time ; 6h apn - ko , 12h apn - ko , 6h wt and 12h wt .
general anesthesia was given to mice using 2% isoflurane and 70% nitrous oxide , and 30% oxygen was supplied during the experiments via a face mask .
the mice 's body temperatures were maintained at 36.5 - 37.5 during the experiments , as measured by a rectal thermometer .
focal cerebral ischemia was induced using the intraluminal filament technique.11 ) after the left common carotid artery was dissected and completely exposed , the external carotid artery was tied by 4 - 0 silk . thereafter ,
silicon - coated 8 - 0 monofilament was headed to the middle cerebral artery through the common carotid artery .
the inserted monofilament was tightened with a 6 - 0 silk suture to prevent bleeding while advancing the monofilament and removing the monofilament during reperfusion .
the mice were placed on a stereotactic frame to perform 46 mm - sized craniotomy in the left parietal area using a dental drill .
extreme caution was required to avoid damage to the dura mater and the tissues beneath it .
after the craniotomy , the dura mater was opened using 26-gauge needles to expose the brain cortex of the mca territory and the artificial cerebrospinal fluid was arranged to flow over the surface of the brain ( fig .
a straight pial venule ( 100 m long and 30 - 40 m in diameter ) was selected . to observe the microcirculation ,
a fluorescence intravital microscope was used at 780 specimen - to - monitor magnification . using 1 ml fluorescein isothiocyanate - albumin that was intravenously injected shortly before data collection ,
a bright contrast was obtained , and the length and diameter of the microvessel were measured . using 1 ml of the 0.1% rhodamine that was intravenously injected 10 minutes before data collection , the leukocytes and platelets were selectively stained .
data were collected at 6 and 12 hours after 1-hour occlusion . at each data selection ,
the target venule was recorded for 30 seconds through a microscope , and the number of rolling leukocytes and leukocytes that adhered to endothelium were counted and documented ( fig .
the statistical significance of differences between the groups of apn - ko and wt mice in the measurements taken 6 and 12 hours after the reperfusion was assessed by the mann - whitney u - test .
all experiments were conducted according to the guidelines issued by the institutional animal care and use committee and complied with the national institutes of health ( nih ) guide for the care and use of laboratory animals . for the experiments of this study , 10 to 12-week - old adiponectin - deficient mice ( apn - ko ) ( n = 8) and wild - type mice ( wt ) ( n = 8) were prepared .
each group was sub - divided into 2 groups by reperfusion time ; 6h apn - ko , 12h apn - ko , 6h wt and 12h wt .
general anesthesia was given to mice using 2% isoflurane and 70% nitrous oxide , and 30% oxygen was supplied during the experiments via a face mask .
the mice 's body temperatures were maintained at 36.5 - 37.5 during the experiments , as measured by a rectal thermometer .
focal cerebral ischemia was induced using the intraluminal filament technique.11 ) after the left common carotid artery was dissected and completely exposed , the external carotid artery was tied by 4 - 0 silk .
thereafter , silicon - coated 8 - 0 monofilament was headed to the middle cerebral artery through the common carotid artery .
the inserted monofilament was tightened with a 6 - 0 silk suture to prevent bleeding while advancing the monofilament and removing the monofilament during reperfusion .
after preparation of the focal cerebral ischemia and reperfusion model , the mice were placed on a stereotactic frame to perform 46 mm - sized craniotomy in the left parietal area using a dental drill .
extreme caution was required to avoid damage to the dura mater and the tissues beneath it .
after the craniotomy , the dura mater was opened using 26-gauge needles to expose the brain cortex of the mca territory and the artificial cerebrospinal fluid was arranged to flow over the surface of the brain ( fig .
as a target vessel , a straight pial venule ( 100 m long and 30 - 40 m in diameter ) was selected . to observe the microcirculation ,
a fluorescence intravital microscope was used at 780 specimen - to - monitor magnification . using 1 ml fluorescein isothiocyanate - albumin that was intravenously injected shortly before data collection ,
a bright contrast was obtained , and the length and diameter of the microvessel were measured . using 1 ml of the 0.1% rhodamine that was intravenously injected 10 minutes before data collection , the leukocytes and platelets were selectively stained .
data were collected at 6 and 12 hours after 1-hour occlusion . at each data selection ,
the target venule was recorded for 30 seconds through a microscope , and the number of rolling leukocytes and leukocytes that adhered to endothelium were counted and documented ( fig .
the statistical significance of differences between the groups of apn - ko and wt mice in the measurements taken 6 and 12 hours after the reperfusion was assessed by the mann - whitney u - test .
the behaviors of the leukocytes in the target vessel of apn - ko and wt mice was observed and recorded for 30 seconds at 6 and 12 hours after the occlusion , using an intravital fluorescence microscope .
the number of rolling and adhesion leukocytes of each experimental group was counted and recorded and converted to per mm  sec ( table 1 ) . in 6hwt mice ,
a mean of 55.48 per mm  sec of rolling leukocytes were observed while a mean of 653.82 per mm  sec of rolling leukocytes were observed in 6hapn - ko mice . in 12hwt mice ,
a mean of 405.92 per mm  sec of rolling leukocytes was observed in all 4 groups . in the 12hapn - ko mice ,
rolling leukocytes were observed in all 4 groups , and in particular , leukocyte adhesion was observed in experimental group iii , with a mean of 1364.78 per mm  sec of total leukocytes .
mann - whitney u - test was used to test for statistical significance of the outcomes .
leukocyte numbers between the apn - ko and wt mice 6 hours after the occlusion differed significantly with p = 0.029 .
the difference at 12 hours after the occlusion was also statistically significant with p = 0.029 ( fig .
adiponectin is an adipocyte - derived bioactive protein that initially attracted attention in the treatment of metabolic syndrome.14 ) clinically , the plasma adiponectin level is negatively associated with dyslipidemia , hypertension , and the c - reactive protein level,17 ) and hypoadiponectinemia ( defined as plasma adiponectin level < 4 g / ml ) increases the risk of type 2 dm , hypertension , and coronary heart disease.16)17 ) studies of the role of adiponectin in the cardiovascular system have been conducted using apn - ko mice .
these studies found that apn - ko mice showed exaggerated myocardial remodeling under pressure - overload conditions , and severe cardiac injuries were reported during ischemia - reperfusion.20)22 ) by contrast , when adiponectin was injected into the apn - ko mice , pathological cardiac hypertrophy and ischemia - induced myocardial damages were inhibited.18 ) these protective effects have driven research on the effects of adiponectin on the cerebrovascular system .
the most recent studies find that plasma adiponectin level is closely related with the cerebrovascular system .
chen at al . described hypoadiponectinemia as an independent and significant risk factor for cerebrovascular disease while efstathiou et al . reported an increased risk of mortality for hypoadiponectinemia patients who suffered ischemic insults.4)6 ) in addition ,
advanced intracranial atherosclerosis patients were observed to have had significantly low plasma adiponectin levels.1 ) in this study , the effects of adiponectin on the early stage of inflammation reaction in cerebral ischemia - reperfusion were investigated . at both 6 and 12 hours after the occlusion
, the apn - ko mice group showed significantly more rolling and adhesion leukocytes than the wt mice group did .
although firm adhesion of the leukocytes to the endothelium was observed only in experimental group iii of the 12hapn - ko mice , the rolling leukocytes would form a firm adhesion on the endothelium through the media of leukocyte adhesion molecule cd11b
/ cd18 ( integrin ) and its endothelial ligand icam-1 . the activated leukocytes that adhered to the endothelium release toxic mediators to damage the surrounding vasculatures or parenchymal cells , or induce a change in blood rheology and accelerated thrombosis by inducing platelet aggregation.7)8)10 ) inhibition of the endothelial adhesion - accumulation of the leukocytes after cerebral ischemia - reperfusion improves electrophysiological and neurological function , and reduces cerebral edema and cerebral infarction size.3)5)23 ) therefore , adiponectin that restrains the leukocyte - endothelium interaction is considered to inhibit the secondary inflammatory reaction and is neuroprotective in cerebral ischemia - reperfusion .
although the leukocyte accumulation in cerebral ischemia - reperfusion steadily increased to reach a peak after 24 - 48 in other studies,2)10 ) more advanced studies with a larger sample size may be needed in the future .
in this study , the effects of adiponectin on the early inflammatory reactions in cerebral ischemia - reperfusion were investigated . as a result
, statistically more rolling and adhesion leukocytes were observed in the apn - ko mice than in the wt mice in the experiments with the mcao - r model . in conclusion ,
adiponectin was observed to inhibit the interaction between the endothelium and leukocytes in cerebral ischemia - reperfusion , and accordingly , adiponectin might prevent the secondary insult caused by the inflammation reaction .
 objectivein this study , we investigate the role of adiponectin in the interaction between leukocytes and endothelium in the secondary inflammatory reaction of cerebral ischemia.methodsadiponectin knock - out mice group ( apn - ko ) ( n = 8) and wild - type mice group ( wt ) ( n = 8) were prepared . 
 each group was sub - divided into 2 groups by reperfusion time . 
 one - hour middle cerebral artery occlusion and reperfusion were induced using the intraluminal filament technique . at 6 and 12 hours after the occlusion 
 , the mice were placed on a stereotactic frame to perform craniotomy in the left parietal area . 
 after craniotomy , a straight pial venule was selected as a target vessel . with the fluorescence intravital microscope , 
 the number of rolling leukocytes and leukocytes that adhered to endothelium were counted and documented at 6 and 12 hours after the reperfusion.resultsat 6 and 12 hours after the reperfusion , more rolling leukocyte and leukocyte adhesion were observed in the apn - ko mice than in the wt mice . 
 the difference in leukocyte numbers between the apn - ko and wt mice was found to be statistically significant ( p = 0.029 ) by mann - whitney u-test.conclusionwe found that adiponectin inhibits the interaction between the endothelium and leukocytes in cerebral ischemia - reperfusion . 
 therefore adiponectin might prevent the secondary insult caused by the inflammation reaction . 
INTRODUCTION
MATERIALS AND METHODS
Animal preparation
Model of middle cerebral artery occlusion and reperfusion
Cranial-window preparation
Intravital microscopy
Statistical analysis
RESULTS
DISCUSSION
CONCLUSION