the heart consists of the cardiac muscle , vasculature , and to a lesser extent interstitial infiltrating cells .
it is considered that the gene expressions are separately regulated in these tissue components under physiological and diseased conditions [ 1 , 2 ] .
although the mrna expression analysis has been established in the whole myocardium , it is difficult to investigate the expression level of each tissue component .
recently , the laser microdissection ( lmd ) method has been developed to isolate specific microscopic regions from tissue samples and separately collect the specimens of interest , which enables us to selectively evaluate the mrna expression levels in targeted cell clusters in the tissues , especially in malignant tissues .
the regions of interest are marked on the monitor of a vertical microscope and cut out by the laser beam under computer control .
the isolated samples fall down into collecting tubes , which are subjected to quantitative real - time reverse - transcribed polymerase chain reaction ( rt - pcr ) or gene - chip / microarray assay .
thus , we sought to establish a method to selectively collect the muscular region and arterial region in myocardial sections using the lmd method .
the mrna expression levels of maker genes specific for cardiac myocytes or vascular smooth muscle cells ( vsmcs ) were analyzed in the muscular and vascular samples , respectively , obtained from rat heart sections .
the study protocol was reviewed and approved by the animal care and treatment committee of kurume university .
male wistar - kyoto rats ( wky ) and stroke - prone spontaneously hypertensive rats ( shr - sp ) were purchased from slc ( shizuoka , japan ) and housed under standard conditions of humidity , room temperature , and a 12  :  12-hour dark - light cycles .
they were provided with free access to tap water and chow .   at 24 weeks
, blood pressure was measured using a tail - cuff sphygmomanometer ( mk-2000st , muromachi , tokyo , japan ) , as described previously .
thereafter , rats were anesthetized with intraperitoneal ketamine ( 50  mg / kg ) and xylazine ( 10  mg / kg ) .
percentage of left ventricular fractional shortening was measured using an echocardiography equipped with a 10  mhz transducer ( aloka , tokyo , japan ) [ 58 ] . the next day
, rats were euthanized with an overdose of pentobarbital ( 100  mg / kg , intraperitoneally ) . after the rats were perfused with ice - cold saline ( 4c ) at 100  mmhg , the heart was removed .
the left ventricle was snap - frozen in isopentane / dry ice , embedded in oct compound , and sectioned with cryostat .
the cryosections ( 7  m in thickness ) were mounted on ice - cold pen - slides ( leica microsystems , wetzlar , germany ) .
fresh cryosections were fixed in rnase - free - ethyl acetate ( acetic acid  :  ethanol = 1  :  19 ) and stained with 0.05% toluidine blue dissolved in rnase - free distilled water .
the regions of the cardiac muscle and intramyocardial arteries were identified based on microscopic observation and were separately isolated from the section using lmd6000 system ( leica microsystems ) . to collect vascular samples
, we consistently placed the laser cut line on the outside border of the medial vsmc layer ( figure 1(a ) ) .
the isolated fragments were collected in the cap of an eppendorf tube ( eppendorf japan , tokyo , japan ) containing trizol reagent ( life technologies japan , tokyo , japan ) .
muscular samples were dissected from the myocardium area without microscopically visible vasculatures and infiltrating cells ( figure 1(b ) ) .
the vascular sample included 40 cross - sections of the intramyocardial arteries for each animal .
the muscular fragments with a total of 6  10  m were collected in each animal .
total rna was purified using rneasy micro ( qiagen , valencia , ca ) according to the manufacturer 's instructions .
electropherogram exhibited clear peaks for 18s and 28s ribosomal rnas in the purified rna samples ( figure 2(a ) ) .
rna was reverse transcribed using a high capacity rna - to - cdna kit ( ge health care , waukesha , wi ) .
equal amount of the resulting cdna was subjected to real - time pcr using the taqman universal pcr master mix and a sequence detection system model 7700 ( life technologies japan ) [ 5 , 9 , 10 ] .
primer pairs and taqman probes for rat type b - natriuretic peptide ( bnp ) , -smooth muscle actin ( -sma ) , and -actin were obtained from ge health care .
good amplification plots for target genes were shown in the muscular and vascular samples ( figure 2(b ) ) .
at 24 weeks of age , systolic blood pressure was 120.5  12.2  mmhg in wky ( n = 5 ) and 252.5  16.7  mmhg in shr - sp ( n = 3 ) ( p < 0.001 ) .
percentage of left ventricular fractional shortening was 26.8  4.1% in wky ( n = 5 ) and 29.8  5.2% in shr - sp ( n = 3 ) ( no significance ) .
we present one example of real - time rt - pcr analysis in wky , which showed bnp mrna expression in the muscular samples , but not in the vascular samples ( figure 3 ) .
next , we compared the expression levels of bnp , a molecular marker of cardiac myocyte hypertrophy , between wky and shr - sp ( figure 4 ) .
shr - sp had a significantly greater bnp expression than wky in the muscular samples .
bnp mrna was not expressed in the vascular samples of wky and shr - sp .
the present study demonstrated that the lmd method enabled us to selectively collect myocardium and intramyocardial arteries from the heart section .
selective sampling was verified because the expression of cardiac myocyte - specific gene marker , bnp , was detected exclusively in the muscular samples , whereas the vsmc - specific marker , -sma , was expressed only in the vascular samples . moreover ,
hypertrophic gene upregulation , as assessed by bnp expression , was significantly greater in the muscular samples obtained from shr - sp than from wky .
recently , it has been reported that the lmd method is useful for selective sampling of the arterial component from the surrounding tissues , for example , the isolation of the arterial lesions in the lung specimen of patients with familiar pulmonary hypertension   and the isolation of the collateral vessels in the ischemic hindlimb in mice .
also , the lmd method was used for selective sampling of the intimal plaques in the human atherosclerotic lesions [ 13 , 14 ] .
however , there have been few studies using the lmd method for gene expression analysis of the heart .
thus , we sought to establish the method to selectively collect muscular and vascular samples from the heart sections using the lmd method .
as shown in figure 1(a ) , the intramyocardial arterioles were surrounded by thin loose connective tissue separating from the cardiac muscle tissue .
thus , the microscopic guidance allows us to easily isolate vascular samples . in this study
, we successfully collected and analyzed the intramyocardial arteries with a diameter of approximately 50  m .
this finding was in line with the previous studies demonstrating that relatively small vascular samples , such as intrapulmonary arteries at a size between 50 and 200  m and intrarenal arterioles with a diameter of approximately 100  m , were selectively isolated using the lmd method [ 16 , 17 ] .
in contrast , it was necessary that we consistently took a great care in isolating muscular samples by avoiding microscopically visible vessels and infiltrating cells .
the mrna expressions of bnp and -sma were not detected in the vascular and muscular samples , respectively ( figure 3 ) .
moreover , the bnp mrna upregulation associated with cardiac hypertrophy was documented specifically in the muscular samples in shr - sp ( figure 4 ) .
these findings suggested that the contamination of cardiac myocytes in the vascular samples or vsmcs in the muscular samples was negligible . in conclusion , the lmd method enabled us to separately collect the muscular and vascular samples from the myocardial sections and to selectively evaluate the mrna expression changes in individual tissue component .
 
 background . 
 the heart consists of various kinds of cell components . however , it has not been feasible to separately analyze the gene expression of individual components . 
 the laser microdissection ( lmd ) method , a new technology to collect target cells from the microscopic regions , has been used for malignancies . 
 we sought to establish a method to selectively collect the muscular and vascular regions from the heart sections and to compare the marker gene expressions with this method . 
 methods and results . 
 frozen left ventricle sections were obtained from wistar - kyoto rats ( wky ) and stroke - prone spontaneously hypertensive rats ( shr - sp ) at 24 weeks of age . using the lmd method , 
 the muscular and vascular regions were selectively collected under microscopic guidance . 
 real - time rt - pcr analysis showed that brain - type natriuretic peptide ( bnp ) , a marker of cardiac myocytes , was expressed in the muscular samples , but not in the vascular samples , whereas -smooth muscle actin , a marker of smooth muscle cells , was detected only in the vascular samples . 
 moreover , shr - sp had significantly greater bnp upregulation than wky ( p < 0.05 ) in the muscular samples . conclusions . 
 the lmd method enabled us to separately collect the muscular and vascular samples from myocardial sections and to selectively evaluate mrna expressions of the individual tissue component . 