Alteration in P-glycoprotein at the blood–brain barrier in
Alteration in P-glycoprotein at the blood–brain barrier in the early period of MCAO in rats
Juan Cen a ,Lu Liu b ,Ming-Shan Li c ,Ling He d ,Li-Juan Wang a ,Yan-qing Liu a ,Meng Liu a and Bian-Sheng Ji a
a Key Laboratory of Natural Medicine and Immune Engineering,
b School of Pharmacy,
c School of Medicine,Henan University,Kaifeng an
d d
Department of Pharmacology,China Pharmaceutical University,Nanjing,China
Keywords
blood–brain barrier;middle cerebral artery occlusion;P-glycoprotein
Correspondence
Bian-Sheng Ji,Key Laboratory of Natural Medicine and Immune Engineering,Henan University,Kaifeng 475000,China.E-mail:jibiansheng@https://www.360docs.net/doc/a84238209.html, Received November 20,2012Accepted January 6,2013doi:10.1111/jphp.12033
Abstract
Objectives The aim of this work was to investigate the alteration in P-glycoprotein (P-gp)at the blood–brain barrier (BBB)after middle cerebral artery occlusion (MCAO)in rats.
Methods Permanent MCAO was veri?ed via 2,3,5-triphenyltetrazolium staining and hematoxylin-eosin staining.The expression of P-gp,matrix metalloproteinase-2(MMP-2),MMP-9,claudin-5,tumour necrosis factor-a (TNF-a )and nitric oxide synthase (NOS)at the BBB was evaluated using western blot or immunostaining analysis.The content of ?uorescein sodium (NaF),rhodamine-123and nimodipine in ischaemic brain tissues was determined using high-performance liquid chromatography.
Key ?ndings Elevated expression of P-gp at the BBB and decreased concentration of P-gp substrates in the ischaemic brain tissues were observed within 4h after MCAO.However,at 6h after MCAO,the concentration of P-gp substrates in the ischaemic hemisphere began to rise even though the expression of P-gp was still increased.Moreover,the expression of claudin-5was decreased;contrarily,the expression of MMP-2,MMP-9,TNF-a as well as NOS gradually increased within 6h after MCAO.
Conclusions P-gp plays a crucial role in limiting the entrance of agents into the brain after MCAO and the speci?c regulation of P-gp expression/activity may provide an important approach for the improvement of pharmacotherapy in ischaemic stroke.
Introduction
P-glycoprotein (P-gp)is located on the luminal surface of specialized endothelial cells lining the blood vessels and plays an important role in limiting entrance of wide range of substances across the blood–brain barrier (BBB)by extruding agents from the brain back into the blood.[1]Accordingly,the status of expression and activity of P-gp at the BBB may determine the brain levels of the agents administered in cerebral diseases such as stroke.[2]There-fore,studying the alterations in P-gp after stroke may provide an opportunity for regulating the transporter activ-ity and determining a rational therapeutic window for the disease.Studies in vitro have revealed an increase in P-gp expression in primary rat brain microvessel endothelial cells
(rBMECs)after ischaemia.[3]However,in in-vivo study,Dazer et al .reported that the Mdr1a mRNA level in the peri-infarcted regions of the brain was not different from that in opposite side after stroke.[4]In the present report,focal cerebral ischaemia was performed by middle cerebral artery occlusion (MCAO)in male Sprague–Dawley rats.At different time points within 6h after MCAO,the alterations in P-gp functional activity and expression at the BBB in the infarcted hemisphere were analysed and compared with those in the contralateral side;the changes in TNF-a and nitric oxide synthase (NOS)expression were also deter-mined at the same time,to examine how ischaemic brain damage affected P-gp at the BBB.The results from these
bs_bs_banner
And Pharmacology
Journal of Pharmacy Research Paper
studies will provide precise adjustment of drug administra-tion time and potential targets for speci?c regulation of P-gp in stroke.
Materials and Methods
Materials
The antibodies against claudin-1,claudin-5,matrix metalloproteinase-2(MMP-2),MMP-9,P-gp,tumour necrosis factor-a(TNF-a)and NOS were purchased from Wuhan Boster Bio-engineering Ltd Co.(Wuhan,China). Rhodamine-123(Rh123)and2,3,5-triphenyltetrazolium (TTC)are products of Sigma(St Louis,MO,USA).Fluores-cein sodium(NaF)injection was from Guangzhou Baiyun Shan Ming Xing Pharmaceutical Co.,Ltd(Guangzhou, China).Nimodipine was provided by Shandong Xinhua Pharmaceutical Factory(Zibo,China).All other reagents were of analytical grade and commercially available. Animals
Male Sprague–Dawley rats,250–300g,were purchased from the laboratory animal center of Henan Province (Zhengzhou,China)for the current study.Rats were housed four per cage in standard rat cages.Housing condi-tions,including light cycle(12-h light–dark cycle;on 07:00h off19:00h),temperature(~24°C)and humidity (35–40%),were controlled and food and water was freely available.Rats were handled and weighed daily throughout the experiment and were acclimatized to the new environ-ment for at least5days before surgery.All experimental procedures were approved by the institutional guidelines of Henan University(Kaifeng,China).All experiments were approved by Animal Ethics Committee of Henan University.
Focal cerebral ischaemia
Permanent focal cerebral ischaemia was performed by MCAO as previously described.[5]Rats were anaesthetized with10%chloral hydrate(300mg/kg,i.p.).After a median neck incision,the right common,external and internal carotid arteries were exposed.To block the origin of the middle cerebral artery(MCA),a mono?lament nylon suture(diameter about0.26mm)was prepared by round-ing its tip by heating and coating with poly-l-lysine (Sigma).The nylon suture was introduced into the external carotid artery and advanced along the internal carotid artery(ICA),approximately18–20mm from the carotid bifurcation,until it occluded the origin of the MCA in the circle of Willis.At0,0.5,1,2,3,4and6h after MCAO,the rats were killed under deep anaesthesia and the brains were quickly removed for study.
During surgery,rectal temperatures were recorded and the body temperature was maintained at37°C using ther-mostatically regulated feedback-controlled devices.Local cerebral blood?ow(LCBF)of the MCA territory was meas-ured by laser-Doppler?uxmetry(LDF)(MP150starter system;BIOPAC System,Inc,Goleta,CA,USA).Rats were placed in a supine position and immobilized using a stere-otactic frame.LCBF was measured continuously through-out the whole duration of the experiment.Suf?cient occlusion of the MCA was monitored by LDF(decrease of LCBF to below30%of baseline).
Cerebral infarct volume measurement
Brain infarct volume was evaluated by TTC staining.[6] Essentially,brains were quickly removed,frozen at-20°C for10min and coronally sectioned into six2-mm-thick slices from the frontal pole to the occipital pole.The slices were stained with2%TTC diluted in saline at37°C and the infarct size of each hemisphere was determined.The brain infarcted tissue areas remained white while intact mitochondrial-functioned brain tissue was visualized dark red.Images of TTC-stained sections were captured using a digital camera(CanonA650;Oita,Japan)and then quanti-?ed using Image J(ver1.37c,NIH,Bethesda,MD,USA). The degree of brain damage should be corrected by brain oedema.[7]Therefore,the corrected volume was calculated using the following equation:Percentage lesion volume (%)={[total infarct volume-(right hemisphere volume-left hemisphere volume)]/total brain vol-ume}￥100%.Infarct volume measurements were carried out by an investigator blinded to the treatment groups. Histological assay
Rats were deeply anaesthetized and perfused with0.9% sodium chloride followed by4%paraformaldehyde.Follow-ing decapitation,brains were removed and embedded in paraf?n.Coronal sections were taken at1.20mm anterior to the bregma and3.6mm posterior to the bregma.Five micrometres of the coronal sections were cut and stained with hematoxylin-eosin(HE)and prepared for subsequent microscopic mounting.A light microscope(￥400)was used to capture the histological changes after MCAO.
P-glycoprotein immunostaining
After paraformaldehyde?xation,the brains of rats were taken for P-gp immunostaining.Sections(2mm-thick) were cut on a freezing microtome and immunohistochemis-try was conducted as previously described.[4,8]Brie?y,after incubation with blocking solutions containing3%donkey serum,10m m PBS and0.3%Tween20,tissue sections were exposed overnight at4°C to polyclonal goat anti-P-gp
Juan Cen et al.
Crucial role of P-gp at the BBB
(MDR1,1:500),diluted in PBS containing3%normal donkey serum and0.3%Tween20.Sections were washed and donkey anti-goat rhodamine-conjugated secondary antibodies in3%donkey serum were added.After a short ?xation step,the expression of P-gp in the ischaemic hemi-sphere was photographed by?uorescence microscopy (OlympusBX51,Tokyo,Japan)and the results were quanti-tatively assessed as mean?uorescence intensity(MFI)using IPP6.0software(Ver6.0,Media Cybernetics,Inc,Silver Spring,MD,USA).
Western blot
Western blot was used to analyse the changes in P-gp, MMP-2,MMP-9,claudin-5and TNF-a as well as NOS in ischaemic cortex microvessels.The protocol followed that used in a previous study with some modi?cations.[9]Cortex was obtained from the MCAO rats of different groups and placed in ice-cold phosphate-buffered saline.After the surface vessels and meninges were removed,the cortex grey matter was minced and incubated at37°C for25min in D-Hanks containing0.05%trypsin.After centrifugation at 800g for5min,the samples were?ltered using150m m and 75m m nylon mesh.Microvessels were harvested on the 75m m nylon mesh and lysed in the ice-cold lysis buffer. After homogenization and centrifugation,the supernatant fractions were collected and analysed for protein level by Enhanced BCA Protein Assay Kit(Beyotime Institute of Biotechnology,Nantong,China.).Tissue lysate(50m g)was resolved on corresponding SDS-PAGE gels with different percentages and then transferred on to nitrocellulose mem-branes.After milk block,the membranes were incubated with primary antibodies overnight at4°C.Then washed with tris-buffered saline and tween20(TBST)three times and incubated with horseradish peroxidase-conjugated sec-ondary antibody at room temperature for1h.Washed another three times with TBST,the membranes were devel-oped by the ECL detection Kit(Pierce Biotechnology,Rock-ford,IL,USA).
Analysis of NaF,Rh123and nimodipine
Rats were deeply anaesthetized and perfused with0.9% sodium chloride before being killed.High-performance liquid chromatography(HPLC)was used for NaF,Rh123 and nimodipine analysis.The concentration ratio of NaF, Rh123and nimodipine in ischaemic cortex hemisphere (ischaemic cortex hemisphere/contralateral cortex hemi-sphere)or normal cortex hemisphere(normal cortex hemisphere/contralateral cortex hemisphere)was used to re?ect the BBB integrity and P-gp function,respectively, and was normalized by protein content.[10–12]NaF and Rh123were diluted in saline(5mg/kg).Nimodipine was diluted in dehydrated alcohol and metered volume in saline (2mg/kg).Rats were divided into three groups and received NaF,Rh123and nimodipine,respectively,by slow intrave-nous injection30min before they were killed.At each time point,respective samples were taken from the ischaemic or the normal hemisphere cortex.For NaF analysis,samples were weighed and homogenized in ice with saline buffer (?ve times the weight of the tissue).Brain homogenates were centrifuged(4°C,2700g,15min)and20m l of the supernatant fraction was injected directly into the HPLC system,which consisted of an ODS C18column (150cm￥4.6mm,5m m),LC10A High Performance Liquid Chromatograph and?uorescence detector(RF540; Shimadzu,Kyoto,Japan)set at an excitation and emission wavelength of480and520nm,respectively.The mobile phase was a mixture of75n m of Na2HPO4(pH9),and 10%(v/v)methanol.The?ow rate was0.75ml/min,the temperature was set at30°C,and the retention time was 4.7min.For Rh123analysis,hemispheres of cortex were weighed and a volume of ice-cold Tris-HCl buffer(0.05m, pH7.4)?ve times the weight of the tissue was added and homogenized on ice.The homogenates of the100–500m l volume was extracted by5ml of the thylacetate/n-butanol mixture,and vortexed for1min.After centrifugation(4°C, 1700g,20min)the supernatant fraction was decanted and evaporated to dryness with reduced pressure at37°C.The residue was dissolved in500m l ice-cold methanol and vor-texed for30s.A volume of20m l was injected into the HPLC system,which was set at an excitation and emission wavelength of480and535nm,respectively.The mobile phase consisted of a mixture of0.025m m phosphate solu-tion and methanol(1:1,v/v).The?ow rate was0.5ml/ min,the temperature was set at40°C,and the retention time was8.5min.The concentration of nimodipine in the cortex was determined by HPLC with a UV detector(SPD-10AVP;Shimadzu)at238nm.
Statistical analysis
All data are expressed as the mean?SD.Signi?cant differ-ence between groups was determined by one-way analysis of variance followed by Tukey’s test.The0.5,1,2,3,4and 6h groups were compared with the0h group(time after MCAO).Results were considered to be statistically signi?-cant when P<0.05.
Results
Time course of ischaemic damage in
brain tissue
As shown in Figure1,the infarct size increased in a time-dependent manner from0.5h to6h after MCAO.In ischaemic brain tissue,pathological changes such as oedema,nuclei shrinkage and even neuronal cell loss were
Juan Cen et al.Crucial role of P-gp at the BBB
observed from 1h to 6h after occlusion (shown in Figure 2).
Time course of BBB permeability and breakdown
The concentration of NaF in brain tissue is positively corre-lated with the BBB permeability.[13]For better comparison,the concentration of NaF in the ischaemic cortex hemi-sphere was divided by that in the contralateral cortex hemi-sphere of the same rat.As shown in Figure 3,the ratio was gradually elevated along with the prolongation of the ischaemia,indicating a time-dependent increase in the BBB permeability;the value of the ratio was about 1.65?0.31(P <0.05)at 6h after MCAO.Moreover,tight junction molecule claudin-5,an important factor responsible for endothelial barrier function,exhibited down-regulated in ischaemic condition in our results (shown in Figure 4).Our results also revealed a time-dependent increase in MMP-2and MMP-9expression (Figure 4).
Time course of P-gp expression and functional activity at the BBB
As shown in Figure 5,the red ?uorescence,which represents P-gp content,was increased both in brain microvessels and in ischaemic brain tissues in a time-dependent way after MCAO.The elevated P-gp expression was also observed via western blot analysis (shown in Figure 4).
Rh123,a typical substrate of P-gp,was used to investigate the P-gp activity at the BBB.As shown in Figure 3,a decreased concentration ratio of Rh123was observed
within 4h and an elevated ratio was observed at 6h after MCAO.In addition,nimodipine,a therapeutic agent and also a substrate of P-gp,exhibited a similar trend to that shown by Rh123(Figure 3).These results indicated that although the P-gp expression was continuously increased within 6h after MCAO (Figures 4and 5),the elevated P-gp functional activity was not strong enough to prevent the agents from penetrating through the broken BBB after 4h.
Time course of TNF-a and NOS expression in brain microvessels
Results from western blot analysis (Figure 4)revealed that TNF-a and NOS expression was promoted after MCAO,synchronously accompanied by enhanced P-gp,indicating their possible regulatory effect on P-gp.
Discussion
Ischaemic stroke is a major cause of death and disability worldwide.Intravenous thrombolysis with recombitant tissue type plasminogen activator is still the only available therapy for patients within 3h of stroke onset.[14]The major reason for the failure of drug therapy is considered to be the weak permeability of the drugs across the BBB.As we know,the BBB mainly consist of capillary endothelial cells with tight junctions,which impede the entrance of toxic com-pounds as well as hydrophilic therapeutic drugs into the brain.[3,15]As a hydrophobic drug ef?ux pump,P-gp is highly expressed at the BBB and is suggested to be the most in?uential factor in the treatment of central nervous system disease.[1]Indeed,up to 50%of the therapeutic agents used are substrates of P-gp.[16]Although the BBB is disturbed after ischaemic stroke,P-gp is overexpressed contrarily.[17]Therefore,to investigate how stroke affects the BBB and P-gp may provide strategy for the improvement of drug therapy.Due to the narrow therapeutic window,only a small proportion (approximately 17%)of individuals are promptly treated with thrombolytic therapy within the ?rst 6h after stroke.[18]Animal models with permanent arterial occlusion,although less commonly used than those with temporary arterial occlusion,are likely to be more relevant to the majority of human ischaemic stroke.Therefore,per-manent MCAO rat model was used in this report.
Given the time-dependent enhancement of BBB perme-ability,?rstly,we should ?nd the time at which the BBB has not been completely broken while P-gp can exert marked action.This study showed that the concentration ratio of hydrophilic tracer NaF in the brain was signi?cantly increased (by 65%,P <0.05)at 6h after MCAO.TTC and HE staining also validated the time-dependent damage of the brain tissue after stroke.Abnormal upregulation of MMP-2and MMP-9and downregulation of claudin-5were reported to be associated with BBB breakdown in
(a)(b)
0.5 h
1 h
2 h
3 h
4 h
6 h
Control
302520151050
0.5
12346Time after MCAO (h)
I n f a r c t s i z e (%)
Figure 1Infarcted brain in MCAO rats.(a)Representative sections showing infarct areas stained by TTC after MCAO from control and at indicated times after occlusion.(b)Infarct volume of the brain in MCAO rats.Each value represents the mean ?SD,n =10,measured as described in Materials and Methods.*P <0.05vs 0.5h;**P <0.01vs 0.5h.
Juan Cen et al .
Crucial role of P-gp at the BBB
stroke.[15,19]The results of this study showed that both the decrease in claudin-5(the most important component of tight junctions)and the increase in MMP-2and MMP-9(which could degrade the extracellular matrix,basal lamina proteins and tight junctions after stroke)occurred in a time-dependent manner.[20–22]In contrast to the two peaks of BBB permeability appearing at 3h and 72h during reperfusion in temporary MCAO rats,[23]the present report showed that BBB permeability had a continual increasing trend from 0.5h to 6h in permanent ischaemic stroke.The content of Rh123in the brain tissue trended to decrease within 4h and began to increase at 6h after MCAO.Con-
sidering the above results,the investigation on the changes in P-gp was carried out within 6h after permanent MCAO.Recently,researchers have reported changes in P-gp after cerebral ischaemia.Samoto et al .observed a loss in P-gp expression (day 3)followed by a recovery (day 5)in the post-ischaemic period in a rat model of focal ischaemia.[24]
(a)(e)(f)(g)
(b)(c)(d)
Figure 2Morphological changes in ischaemic brain cortex in MCAO rats.a,Control.b,0.5h after occlusion.c,1h after occlusion.d,2h after occlusion.e,3h after occlusion.f,4h after occlusion.g,6h after occlusion.
NaF
Rh123NMD
2.5
21.510.50
0.51234
6
Time after MCAO (h)
D r u g c o n c n r a t i o (i s c h a e m i c /c o n t r a l a t e r a l c o r t e x h e m i s p h e r e )
Figure 3Concentration ratio of NaF,Rh123and nimodipine (NMD)in brain tissue in MCAO rats.Each value represents the mean ?SD,n =10and was measured as described in Materials and Methods.*P <0.05vs 0.5h;**P <0.01vs 0.5h.
a b c d e f g
P-gp
MMP-2MMP-9
Claudin-5
NOS β-actin
TNF-αFigure 4Western blot analysis of P-gp,MMP-2,MMP-9,claudin-5and TNF-a as well as NOS expression at BBB after MCAO in rats.a,Control.b,0.5h after occlusion.c,1h after occlusion.d,2h after occlusion.e,3h after occlusion.f,4h after occlusion.g,6h after occlusion.
Juan Cen et al .Crucial role of P-gp at the BBB
However,Dazert et al .reported that P-gp expression in the peri-infarcted region was not signi?cantly different from that in the contralateral site in temporal ischaemic rats.[4,25]These varied results may be attributed to the differences in the models chosen,the stress applied and the brain regions assayed.The present study revealed that P-gp expression was continuously elevated within 6h after permanent MCAO in rats.However,the concentration ratio of Rh123and nimodipine was only decreased within 4h and began to increase at 6h after ischaemia,which seemed not to be in accordance with the changes in P-gp expression.In fact,the accumulation of the P-gp substrate in the brain is the outcome of integration between the P-gp ef?ux activity and the BBB permeability.The more the BBB permeability
increases,the less the agent delivery is affected by P-gp.When the in?uence of BBB permeability overwhelmingly exceeds the effect of P-gp,the agent will be free to enter the brain.Accordingly,our result suggested that P-gp played a crucial role in limiting the entrance of the agents into the brain in the early period of stroke,especially within 4h after ischaemia.Therefore,speci?c regulation of P-gp activ-ity and reasonable choice of drug administration time may provide reference for the improvement of clinical pharma-cotherapy.Our results may also contribute to a better understanding of the drug–drug interactions with regard to P-gp in stroke.In addition,the upregulated expression of P-gp is reported to occur not only in brain endothelial cells but also in astrocytes and neurons after ischaemic
brain
(a)(b)
P-gp-associated MPI of brain microvessels P-gp-associated MFI of brain parenchyma
8007006005004003002001000
Control 0.5 h 1 h 2 h 3 h 4 h 6 h
Figure 5Immunostaining analysis of P-gp expression (red ?uorescence)in brain microvessels and brain tissues in ischaemic hemisphere cortex after MCAO in rats.a.Representative photographs of P-glycoprotein staining from each group by OlympusBX51?uorescence microscopy (￥400).b.Quantitative assessment of the mean ?uorescence intensity (MFI)of P-glycoprotein using IPP 6.0software.*P <0.05vs control;#P <0.05vs control.
Juan Cen et al .
Crucial role of P-gp at the BBB
damage.[6]The results of the P-gp immuno?uorescence assay in this report also showed that the elevated P-gp expression occurred both in brain microvessels and in the ischaemic brain tissues.It is worth further elucidating the alterations in P-gp in different brain regions and in different cells.
Given the enhanced P-gp expression,speci?c inhibition appears to be a promising approach for promoting thera-peutic ef?cacy of drugs.P-gp is expressed not only in the central nervous system but also in the liver,kidneys,pan-creas and other organs.[26]Therefore,non-speci?c inhibition of P-gp may lead to enhanced drug penetration into the brain,delayed metabolism of drugs or even elevated long-term risk for various diseases.[17]Identifying factors which speci?cally modulate P-gp in ischaemic brain may provide an alternative strategy.Pro-in?ammatory cytokine TNF-a has been reported to be involved in the regulation of P-gp. In isolated rat brain capillaries and renal proximal tubules, TNF-a could cause a loss in P-gp activity and expression via toll-like receptor4,tumour necrosis factor receptor1, endothelin-B and endothelin-B receptors as well as NOS, protein kinase C and nuclear factor-k B.[8,27,28]However,it is not clear whether this factor has the same cascade relation-ship in stroke.A previous report showed that TNF-a is largely produced by microglia and macrophages in mice with ischaemic stroke.[29]The present study revealed that the gradually increased TNF-a and NOS expression was also found in brain microvessels and synchronously accompa-nied by the enhanced P-gp,suggesting that this pro-in?ammatory pathway may exert an important role in the regulation of P-gp in stroke.It is of speci?c interest to eluci-date the exact mechanisms that drive P-gp expression in response to cerebral ischaemia.
Conclusions
The results of this study suggested that speci?c regulation of P-gp expression/activity and precise adjustment of drug administration time may provide an important approach for the improvement of pharmacotherapy in the early period of ischaemic stroke.
Declarations
Con?ict of interest
The Author(s)declare(s)that they have no con?icts of interest to disclose.
Funding
This work was supported by National Natural Science Foundation of China(No81072647);Nature Science Foun-dation of Science and Technology Of?ce of Henan Province, China(No102300410091);National12th Five-year Plan ‘Major Scienti?c and Technological Special Project for Sig-ni?cant New Drugs Creation’project of‘Novel G protein-coupled receptor targeted drug screening system and key technology research’(NO.2012ZX09504001-001);Program for New Century Excellent Talents in University(No.NCET-10-0817).
References
1.Cordon-Cardo C et al.Expression of
the multidrug resistance gene product (P-glycoprotein)in human normal and tumor tissues.J Histochem Cyto-chem1990;38:1277–1287.
2.Spudich A et al.Inhibition of multid-
rug resistance transporter-1facilitates neuroprotective therapies after focal cerebral ischemia.Nat Neurosci2006;
9:487–488.
3.Hermann DM et al.Role of drug
ef?ux carriers in the healthy and dis-eased brain.Ann Neurol2006;60:489–498.
4.Dazert P et al.Differential regulation
of transport proteins in the periinfarct region following reversible middle cer-ebral artery occlusion in rats.Neuro-science2006;142:1071–1079.
5.Longa EZ et al.Reversible middle
cerebral artery occlusion without
craniectomy in rats.Stroke1989;20:
84–91.
https://www.360docs.net/doc/a84238209.html,zarowski A et al.Neuronal mdr-1
gene expression after experimental
focal hypoxia:a new obstacle for neu-
roprotection?J Neurol Sci2007;258:
84–92.
7.Shimakura A et al.Neutrophil elastase
inhibition reduces cerebral ischemic
damage in the middle cerebral artery
occlusion.Brain Res2000;858:55–
60.
8.Bauer B et al.Tumor necrosis factor
alpha and endothelin-1increase
P-glycoprotein expression and trans-
port activity at the blood-brain
barrier.Mol Pharmacol2007;71:667–
675.
9.Cen J et al.HZ08,a great regulator to
reverse multidrug resistance via cycle
arrest and apoptosis sensitization in
MCF-7/ADM.Eur J Pharmacol2010;
647:21–30.
10.He L,Ji BS.In vitro and in vivo study
of CJY,an iso?avone,on
p-glycoprotein function in rats.
J Chemother2008;20:361–367.
11.Meng F et al.Pinocembrin attenuates
blood-brain barrier injury induced
by global cerebral ischemia-
reperfusion in rats.Brain Res2011;
1391:93–101.
12.Zhang L et al.P-glycoprotein
restricted transport of nimodipine
across blood-brain barrier.Acta Phar-
macol Sin2003;24:903–906.
13.van Vliet EA et al.COX-2inhibition
controls P-glycoprotein expression
and promotes brain delivery of pheny-
toin in chronic epileptic rats.Neurop-
harmacology2010;58:404–412.
14.Zaleska MM et al.The development of
stroke therapeutics:promising mecha-
nisms and translational challenges.
Neuropharmacology2009;56:329–
341.
Juan Cen et al.Crucial role of P-gp at the BBB
15.Loscher W,Potschka H.Drug resist-
ance in brain diseases and the role of drug ef?ux transporters.Nat Rev Neu-rosci2005;6:591–602.
16.Wang Z et al.Valproic acid attenuates
blood-brain barrier disruption in a rat model of transient focal cerebral ischemia:the roles of HDAC and MMP-9inhibition.J Cereb Blood Flow Metab2011;31:52–57.
17.Lee G,Bendayan R.Functional expres-
sion and localization of P-glycoprotein in the central nervous system:rel-evance to the pathogenesis and treat-ment of neurological disorders.Pharm Res2004;21:1313–1330.
18.Sims NR,Muyderman H.Mitochon-
dria,oxidative metabolism and cell death in stroke.Biochim Biophys Acta 2010;1802:80–91.
19.Koto T et al.Hypoxia disrupts the
barrier function of neural blood vessels through changes in the expres-sion of claudin-5in endothelial cells.
Am J Pathol2007;170:1389–1397. 20.Shimizu F et al.Pericyte-derived glial
cell line-derived neurotrophic factor
increase the expression of claudin-5
in the blood-brain barrier and the
blood-nerve barrier.Neurochem Res
2012;37:401–409.
21.Nitta T et al.Size-selective loosening
of the blood-brain barrier in claudin-
5-de?cient mice.J Cell Biol2003;161:
653–660.
22.Yang Y et al.Matrix
metalloproteinase-mediated disrup-
tion of tight junction proteins in cer-
ebral vessels is reversed by synthetic
matrix metalloproteinase inhibitor in
focal ischemia in rat.J Cereb Blood
Flow Metab2007;27:697–709.
23.Jiao H et al.Speci?c role of tight junc-
tion proteins claudin-5,occludin,and
ZO-1of the blood-brain barrier in a
focal cerebral ischemic insult.J Mol
Neurosci2011;44:130–139.
24.Samoto K et al.P-glycoprotein expres-
sion in brain capillary endothelial
cells after focal ischemia in rat.Acta
Neurochir Suppl(Wien)1994;60:257–
260.
https://www.360docs.net/doc/a84238209.html,ngmade SJ et al.Pregnane X recep-
tor(PXR)activation:a mechanism for
neuroprotection in a mouse model of
Niemann-Pick C disease.Proc Natl
Acad Sci U S A2006;103:13807–
13812.
26.Linnet K,Ejsing TB.A review on the
impact of P-glycoprotein on the pen-
etration of drugs into the brain.Focus
on psychotropic drugs.Eur Neuropsy-
chopharmacol2008;18:157–169.
https://www.360docs.net/doc/a84238209.html,ler DS et al.Modulation of
P-glycoprotein at the blood-brain
barrier:opportunities to improve
central nervous system pharmaco-
therapy.Pharmacol Rev2008;60:196–
209.
28.Roberts DJ,Goralski KB.A critical
overview of the in?uence of
in?ammation and infection on
P-glycoprotein expression and activity
in the brain.Expert Opin Drug Metab
Toxicol2008;4:1245–1264.
29.Clausen BH et al.Interleukin-1beta
and tumor necrosis factor-alpha are
expressed by different subsets of
microglia and macrophages after
ischemic stroke in mice.J Neuroin-
?ammation2008;5:46.
Juan Cen et al.
Crucial role of P-gp at the BBB