the blood - brain barrier ( bbb ) , a barrier between the central nervous system ( cns ) and the systemic circulation , maintains homeostasis within the brain microenvironment . the anatomical substrate of the bbb is the cerebral microvascular endothelium , which , together with astrocytes , pericytes , neurons , and the extracellular matrix , constitutes a  neurovascular unit  that is essential for the health and function of the cns . failure of the bbb is a critical event in the progression of several diseases , such as alzheimer 's disease ( ad ) [ 14 ] . bbb breakdown is accompanied by an increase in the trans - endothelial permeability to substances , which can damage the microenvironment of the brain and affect the structure and function of the cns . therefore , it is not surprising that strategies have been developed to  repair  the bbb in order to restore normal brain homeostasis and prevent the infiltration of pharmacologically active ( noxious ) substances into the brain . cholesterol is one of the most notorious natural risk factors for arteriosclerotic cerebrovascular disease [ 5 , 6 ] . mounting evidence suggests that cholesterol also plays a critical role in the early stage of ad [ 79 ] . hypercholesterolemia leads to increased bbb leakage , an effect that may contribute to ad pathogenesis [ 10 , 11 ] . several epidemiological studies have revealed that cholesterol - lowering statins , which are used for the treatment of coronary arterial disease , are associated with a decreased risk of developing ad . statins have been shown to ameliorate bbb dysfunction resulting from a number of conditions , including diabetes , transient focal cerebral ischemia , and hiv-1 [ 1315 ] . however , the effects of statins on bbb disruptions induced by hypercholesterolemia have not been reported . to address this question , the permeability of the bbb was studied in vivo using rabbits fed a cholesterol - enriched diet and treated with simvastatin , a widely used natural statin derived from fermentation . the effect of simvastatin on bbb was also performed using rat brain microvascular endothelial cells ( rbmecs ) cultured under high - cholesterol conditions in vitro . simvastatin capsules used in vivo were purchased from nantong hua pharmaceutical co. , ltd ( jiangsu , china ) . the simvastatin used in vitro and evans blue dye were obtained from sigma ( st . louis , mo , usa ) . dulbecco 's modified eagle 's medium ( dmem ) , fetal bovine serum ( fbs ) , streptomycin , penicillin , dnase i , collagenase , collagenase / dispase , and fluorescein - isothiocyanate - conjugated bovine serum albumin ( fitc - bsa ) were obtained from invitrogen ( carlsbad , ca , usa ) . antibodies to zo-1 and occludin were purchased from zymed ( san francisco , ca , usa ) . twenty - four adult male new zealand white rabbits ( 2356  212  g ) were used in this study . rabbits were housed individually in the rabbit facility and were kept on a 12  h light/12  h dark cycle at 28  2c and 3748% humidity . the control diet group ( n = 8) consisted of rabbits that received normal rabbit chow for 10 weeks . the other rabbits were fed chow supplemented with 2% cholesterol for 6 weeks and were then randomly assigned to either the cholesterol group ( n = 8) , which was fed chow supplemented with 2% cholesterol or the cholesterol + simvastatin group ( n = 8) , which was fed chow supplemented with 2% cholesterol and 5  mg / kg / d simvastatin for an additional 4 weeks . the olfactory bulbs , brain regions with an intact bbb under normal physiological conditions , have been implicated in alzheimer 's disease by studies reporting that ad patients experience olfactory dysfunction . other research has shown that the hippocampus is characterized by a selective fragility of the bbb and is often affected by ad pathology early in the disease process . accordingly , the olfactory bulbs and hippocampus were two primary brain areas examined in this study . all experiments were approved by the committee for animal care and use at tianjin university of traditional chinese medicine . to assay bbb permeability , evans blue dye ( 4% ; 25  mg / kg ) was injected into rabbits through the ear vein and allowed to circulate for 3  h before the animals were anesthetized with pentobarbital sodium salt ( 30  mg / kg ) . while deeply anesthetized , animals were perfused with 37c saline via the left cardiac ventricle to wash out any vascular evans blue . following perfusion , brains were quickly removed , and the olfactory bulbs and hippocampi were isolated , weighed , and incubated for 72  h with formamide in the dark at room temperature ( 25c ) . after incubation , samples were centrifuged at 10,000  g for 10  min , supernatants were collected , and the absorbance was determined ( ex 620  nm and em 680  nm ) with a jasco fp-777 spectrofluorimeter ( jasco uk ltd , essex , uk ) . evan 's blue concentrations were calculated from standard curves , and results were expressed as evan 's blue / specimen wet weight . to evaluate the expression of the tight junction proteins , occludin and zonula occluden-1 ( zo-1 ) , cryostat brain sections ( 10  m ) of the olfactory bulbs and hippocampi were fixed with acetone and then rinsed 3 times in 0.01  m pbs for 5  min each . fluorescently labeled primary antibodies against zo-1 ( zymed , clone zo1 - 1a12 , dilution 1  :  200 ) and occludin ( zymed , clone oc-3f10 , dilution 1  :  200 ) were diluted in pbs containing 1% triton x-100 , applied to the brain sections and incubated overnight at 4c in a dark humidified chamber . sections were rinsed 3 times in 0.01  m pbs for 5  min each and were subsequently examined by leica microscopy . total serum cholesterol was measured in venous blood collected from rabbit ear veins using standard enzymatic techniques with a fully automatic biochemistry analyzer . following perfusion , brains were removed quickly and the cortex and hippocampus were removed and weighed . cholesterol levels in the cortex and hippocampus were measured using reverse - phase hplc ( zorbax xdb c18 4.6  250  mm spheri-5  rp c18 column , 5  m , agilent , flow rate , 0.8  ml / min ) with varian prostar 325 ultraviolet detection at 208  nm and lc workstation v6.2 chromatographic data system . primary cultures of rat brain microvessel endothelial cells ( rbmecs ) were isolated from wistar rat ( approximately 100  g ) brains using a combination of enzyme digestion and ultracentrifugation approaches as described previously , with minor modifications . in brief , fresh rat brains were obtained , and the surface vessels and meninges of the brains were removed . the grey matter was cut into 1  mm pieces , digested with 39  u / ml dnase i and 0.7  mg / ml collagenase at 37c for 1  h , and centrifuged at 1000  g for 10  min . after centrifugation , the supernatant was discarded , 20% bsa was added , and the resulting mixture was centrifuged at 1000  g for 20  min . the dark red pellet was collected and subjected to further enzymatic digestion in 39  u / ml dnase i and 1  mg / ml collagenase / dispase for 1  h. after centrifugation , the supernatant was discarded , and brain capillary fragments were seeded onto matrigel - coated transwell filters ( a pore size of 0.4  m transwell , corning life sciences , usa ) in dulbecco 's modified eagle 's medium supplemented with 20% fbs , 100  g / ml heparin , 30  g / ml ecgf , and 2  mm glutamine . the purity of rbmecs was > 95% as determined by factor - viii - related antigen and immunocytochemical staining ( data not shown ) . these cultures , which were used as an in vitro model of the bbb , were maintained for 810 days to obtain confluence prior to starting experiments . when a stable transendothelial electrical resistance ( teer ) value was obtained , the cultures were washed twice with serum - free dmem and treated with water - soluble cholesterol ( 10  m ) for 24  h with or without simvastatin ( 5  m ) to evaluate disruption transendothelial permeability . teer was measured using a millicell - ers electrical resistance apparatus ( millipore , eschborn , germany ) 24  h after treatment with simvastatin . to study the effects of simvastatin on teer , the transwell inserts with the rbmecs cultures mentioned above were placed into the chamber electrode filled with hepes - buffered ( 25  mm ) serum - free dmem . the symmetrically apposing electrodes were situated above and beneath the membrane , allowing a uniform current density to flow across the membrane . resistance values of multiple transwell inserts from each experimental group were expressed in common units ( /cm ) after subtracting the value of a blank cell - free filter . transendothelial permeability to macromolecules was assessed by passage of fluorescein isothiocyanate - conjugated bovine serum albumin ( fitc - bsa ) across the monolayer as described previously . in brief , primary rbmecs were treated with cholesterol in the presence / absence of simvastatin as mentioned above . fitc - bsa ( 50  g / ml ) was added to the upper chambers and its diffusion across the model bbb was monitored using an fl600 microplate fluorescent reader ( biotek , winooski , vt ) ( ex 488  nm , em 525  nm ) 3  h after the addition of fitc - bsa . bbb permeability was calculated using the following formula : ( bsa lower chamber )  100/(bsa upper chamber ) . statistical significance for multiple comparisons was determined using one - way anovas and tukey 's post hoc tests with spss software ( spss , chicago , il , usa ) . evans blue is normally excluded from the brain parenchyma by the bbb and is only detectable when the integrity of the bbb is compromised . increased bbb permeability was observed in rabbits fed a high - cholesterol diet ( supplemented with 2% cholesterol for 10 weeks ) , as evidenced by the increased evans blue content in their olfactory bulbs and hippocampi when compared to those of rabbits fed a normal diet . oral administration of simvastatin at a dose of 5  mg / kg / day for 4 weeks ( weeks 710 ) attenuated the high cholesterol diet - induced leakage of evan 's blue dye in both the olfactory bulb and the hippocampus . the tight junctions ( tjs ) between the endothelial cells serve to restrict blood - borne substances from entering the brain . zo-1 and occluding were the major interendothelial junctional proteins . thus , to determine if the effects of simvastatin on bbb permeability resulted from alterations in the major interendothelial junctional proteins , the expression of zo-1 and occludin was examined in rabbits fed a high - cholesterol diet treated with simvastatin . the results revealed decreased immunostaining for zo-1 ( figures 2(a ) and 3(a ) ) and occludin ( figures 2(b ) and 3(b ) ) in the olfactory bulbs and hippocampi of rabbits fed a high - cholesterol diet for 10 weeks when compared to rabbits fed a normal diet . however , simvastatin ( 5  mg / kg / day for 4 weeks ) had no effects on either the expression of occludin or zo-1 . therefore , it is of interest to evaluate the effects of high doses of simvastatin on levels of cholesterol in serum and in brain . as expected , rabbits fed a cholesterol - enriched diet for 10 weeks exhibited over a 20-fold increase in total serum cholesterol concentration . treatment of high - cholesterol - diet - fed rabbits with simvastatin ( 5  mg / kg / day for 4 weeks ) significantly reduced plasma cholesterol levels . however , cholesterol levels in the hippocampus and cortex were not affected by a cholesterol - enriched diet in the presence or absence of simvastatin ( table 1 ) . to further verify the observed effects of simvastatin , primary cultures of brain endothelial cells , the most important component of the bbb , were examined . primary rbmecs were stimulated with cholesterol ( 10  m ) in the presence / absence of simvastatin ( 5  m ) for 24  h. potential changes in the integrity of the bbb were assessed by measuring teer and the permeability of an rbmec monolayer to fitc - bsa . figure 4(a ) shows that cholesterol markedly retarded the development of teer of rbmec monolayers during postconfluent growth . a significant effect of simvastatin ( 5  m ) became visible after a 24  h treatment of rbmecs . consistent with the results of teer , cholesterol was shown to induce increased permeability to the large molecular weight tracer fitc - bsa , and this effect was significantly blocked by simvastatin ( figure 4(b ) ) . our results showed that simvastatin significantly reduces leakage of evan 's blue dye across the bbb but does not affect the expressions of the tight junction proteins , occlud , and zo-1 , in rabbits fed a cholesterol - rich diet . in addition , our data indicate that simvastatin alters plasma levels of cholesterol but does not affect levels of cholesterol in the brain . we also observed that cotreatment with simvastatin blocked the cholesterol - induced increase in transendothelial permeability and the cholesterol - induced decrease in teer observed in primary cultured rbmecs . the semipermeable bbb restricts the diffusion of blood - borne substances into the brain parenchyma . disruptions in the bbb can cause chemical imbalances in the neuronal  milieu ,  ultimately resulting in synaptic and neuronal dysfunction . disruption of the bbb is a hallmark of alzheimer 's disease [ 10 , 11 ] . furthermore , elevated plasma cholesterol has been shown to increase bbb permeability and is a possible risk factor for ad [ 2 , 2326 ] . consistent with the above findings , the present study demonstrated that high levels of cholesterol can induce leakage of the bbb in vivo and disrupt the integrity of in vitro models of the bbb . ad , a type of dementia that affects millions of elderly individuals , is the fourth leading cause of death among the elderly in developed countries . epidemiological studies reveal a lower prevalence of ad in patients with hypercholesterolemia who are taking statins , a frequently prescribed class of lipid - lowering drugs [ 28 , 29 ] . several experiments have shown that statins may be potentially beneficial in the treatment of ad via their direct effects on brain cholesterol metabolism [ 30 , 31 ] . however , other studies have failed to demonstrate a reduction in the ratio between 24s - hydroxycholesterol and cholesterol , in response to statin treatment . at the same time , recent retrospective epidemiological studies have reported that the use of statins , but not nonstatin lipid - lowering agents , may reduce the risk of developing ad [ 8 , 12 ] . consistent with previous findings , our results also demonstrate that short - term simvasatin treatment does not affect brain total cholesterol ; however , it has an effect on bbb integrity . this result suggests that the mechanisms by which statins protect against ad may extend beyond their lipid - lowering effects . cholesterol has been mainly considered for dementia through promoting the generation of amyloid . however , immunization trials targeting the removal of amyloid- plaques in alzheimer 's disease have so far failed to stop the progression of dementia . recent data show that to reduce oxidative stress and inflammation - related factors in the brain by interfering with the permeability of the bbb may enhance the therapeutic effect mentioned above . therefore , to maintain the integrity of the bbb maybe more closely associated with the action of simvastatin on preventing ad instead of lipid - lowering effects . the current study provides the first evidence for the protective effects of simvastatin against bbb disruptions both in rabbits with hypercholesterolemia and in rbmecs exposed to cholesterol - enriched media . in rabbits fed a high - cholesterol diet , simvastatin reduced the leakage of evan 's blue dye into the olfactory bulb and hippocampus . to follow up these findings , we subsequently carried out an in vitro study using brain microvascular endothelial cells exposed to 10  m cholesterol as a model of bbb disruption . we found that simvastatin blocks the teer decline and increased transendothelial permeability induced by high cholesterol , a result that corresponded well with our observed results in vivo . because tight junctions between cerebral microvascular endothelial cells form the basis of the bbb , we examined the expression of two tight junction proteins , occludin and zo-1 , after simvastatin treatment . however , we found that simvasatin had no effect on the expression of tight junction proteins . these results suggest that simvastatin blocks bbb leakage induced by elevated cholesterol , independent of changes in the expression of the tight junction molecules , occludin and zonula occluden-1 . this finding is in agreement with at least one previous report in the literature , which showed similar results in an in vitro model of multiple sclerosis . in the present study , we did not explore the detailed molecular mechanisms whereby simvastatin protects against bbb disruption . however , we did demonstrate that simvastatin is effective in ameliorating bbb disruption induced by high - cholesterol diet in rabbits and in reducing cholesterol - induced endothelial permeability in an in vitro model of bbb disruption . to the best of our knowledge , the present study is the first to show that simvastatin improves bbb integrity both in rabbits fed a high - cholesterol diet and in a cholesterol - induced in vitro model of bbb disruption using primary cultured rbmecs . our data provide a new mechanism underlying the neuroprotective activity of simvastatin and suggest that simvastatin may be useful in the treatment of bbb disruptions induced by hypercholesterolemia .
background . hypercholesterolemia and disruptions of the blood brain barrier ( bbb ) have been implicated as underlying mechanisms in the pathogenesis of alzheimer 's disease ( ad ) . 
 simvastatin therapy may be of benefit in treating ad ; however , its mechanism has not been yet fully understood . 
 objective . to explore whether simvastatin could block disruption of bbb induced by cholesterol both in vivo and in vitro 
 . methods . 
 new zealand rabbits were fed cholesterol - enriched diet with or without simvastatin . 
 total cholesterol of serum and brain was measured . 
 bbb dysfunction was evaluated . to further test the results in vivo , rat brain microvascular endothelial cells ( rbmecs ) 
 were stimulated with cholesterol in the presence / absence of simvastatin in vitro . 
 bbb disruption was evaluated . 
 results . 
 simvastatin blocked cholesterol - rich diet induced leakage of evan 's blue dye . 
 cholesterol content in the serum was affected by simvastatin , but not brain cholesterol . 
 simvastatin blocked high - cholesterol medium - induced decrease in teer and increase in transendothelial fitc - labeled bsa passage in rbmecs . 
 conclusions . 
 the present study firstly shows that simvastatin improves disturbed bbb function both in vivo and in vitro . 
 our data provide that simvastatin may be useful for attenuating disturbed bbb mediated by hypercholesterolemia .