research article neuroprotective effect of tea polyphenols...

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2013 Article ID 743938 7 pageshttpdxdoiorg1011552013743938

Research ArticleNeuroprotective Effect of Tea Polyphenols on OxyhemoglobinInduced Subarachnoid Hemorrhage in Mice

Haizhen Mo1 Ying Chen2 Liyong Huang3 Hao Zhang1 Juxiang Li3 and Wenke Zhou3

1 Department of Food Science Henan Institute of Science and Technology Xinxiang Henan 453003 China2 School of Life Science and Technology Henan Institute of Science and Technology Xinxiang Henan 453003 China3Department of Neurosurgery The First Affiliated Hospital of Xinxiang Medical University Weihui Henan 453100 China

Correspondence should be addressed to Ying Chen yingchyahoocn

Received 4 May 2013 Accepted 15 May 2013

Academic Editor Renata Santos

Copyright copy 2013 Haizhen Mo et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Tea polyphenols are of great benefit to the treatment of several neurodegenerative diseases In order to explore the neuroprotectiveeffects of tea polyphenols and their potential mechanisms an established in vivo subarachnoid hemorrhage (SAH) model wasused and alterations of mitochondrial function ATP content and cytochrome c (cyt c) in cerebral cortex were detected Thisstudy showed that the alteration of mitochondrial membrane potential was an early event in SAH progression The trend ofATP production was similar to that of mitochondrial membrane potential indicating that the lower the mitochondrial membranepotential lesser the ATP produced Due to mitochondrial dysfunction more cyt cwas released in the SAH group Interestingly thepreadministration of tea polyphenols significantly rescued the mitochondrial membrane potential to basal level as well as the ATPcontent and the cyt c level in the brain cortex 12 h after SAH After pretreatment with tea polyphenols the neurological outcomewasalso improved The results provide strong evidence that tea polyphenols enhance neuroprotective effects by inhibiting polarizationof mitochondrial membrane potential increasing ATP content and blocking cyt c release

1 Introduction

Polyphenols the most abundant components in fruits teawine and vegetables have attracted attention in recentyears due to their healthy effects It has long been knownthat tea polyphenols have neuroprotective effects in variouspathological states of the nervous system for example bylowering the cognitive impairment in most neurodegener-ative diseases and by reducing the risk of mortality afterstroke [1ndash3] Human epidemiological data shows that therisk of a fatal versus nonfatal stroke is significantly reducedby approximately 21 in people that drink 3 cups of teaper day when compared with nontea drinkers In fact highconsumption of tea particularly green tea contributes to thelower rates of cardiovascular disease observed in the Asianpopulation especially in China indicating that tea drinkingmay be a benefiting lifestyle However other authors indicatethat tea consumption has no correlation with hemorrhagicstroke [4]

Stroke is a leading cause of morbidity and mortalityworldwide Subarachnoid hemorrhage (SAH) accounts foronly 5ndash10 of all strokes but is a major devastating subtypeof stroke affecting 30000 people in North America yearly[5] SAH is caused by the rupture of a brain aneurysm andcan be divided in two stages the first 72 h correspond to theearly brain injury (EBI) and the period after 72 h correspondsto the delayed vasospasm About 21 of SAH survivors donot experience delayed vasospasm indicating that EBI maybe an important stage predicting the outcome of SAH [6 7]In fact all the factors related to the pathological mechanismsof EBI after SAHmay eventually induce irreversible neuronaldeath which is associated with neurological deficits and pooroutcome [8ndash10]

A growing body of evidence from animal models andclinical studies indicate that mitochondrial dysfunction is acommon event in brain injury including in SAH [11] Severallines of research suggest that mitochondrial dysfunction

2 Oxidative Medicine and Cellular Longevity

induced by oxidative stress and inflammation results inmito-chondrial membrane potential (Δ120595

119898) reduction cytochrome

c (cyt c) release and then activation of mitochondrial-dependent cell death providing evidence that mitochondrialimpairment is deleterious [12 13] Therefore mitochondrialfunction is a potential therapeutic target [14]

Recent results suggest that inhibition of a signal pathwaymay not be required for the treatment of SAH [15] In sucha scenario tea polyphenols may be of special interest due totheir multiple functions such as antioxidants antimutageniciron chelators and in glutamate release [16] However teapolyphenolsrsquo effects in the time course after SAH are stillunknownThe aimof this studywas to investigatewhether teapolyphenols have neurological and neurobehavioural effectsor not and the pathway involved in these effects in the in vivoSAHmodelThis study contributes to a better understandingof the role of tea polyphenols in SAH prevention which arenontoxic and inexpensive dietary components

2 Materials and Methods

21 PreparationOxyhemoglobin (OxyHb) Arterial blood col-lected from Kunming mouse with heparin was centrifugedat 2500timesg for 15min The supernatant was discarded Ery-throcytes werewashed 3 timeswith saline solution lysedwithmethylbenzene and centrifuged at 15000timesg for 20min thesupernatantwas collectedUsing filtermembrane theOxyHbsolution was collected adjusted to 3 120583molL using measuredoptical density (OD) at 540 nm and 576 nm and stored atminus80∘C [17]

22 Animals The animal use and care protocols wereapproved by Institutional Animal Care and Use Committee(IACUC) of Xinxiang Medical University One hundredand eighty adult male Kunming mice weighing from 18 to20 g were purchased from Xinxiang Medical University Allanimals were required to undergo institutional quarantine for7 days prior to use The environment for animal housing wasequipped with controlled temperature (22 plusmn 3∘C) humidity(40ndash70) and a 12 h lightdark alternation The mice weredivided into three groups sham group (119899 = 60) SAH group(119899 = 60) and preadministration tea polyphenols SAH group(119899 = 60) Subsequently each group was subdivided into 3 h6 h 12 h 24 h and 72 h subgroup (119899 = 12) respectivelySAH group was injected with OxyHb and sham group wasgiven isotonic saline for the same period Pretreatment groupwas administered intragastrically with water containing teapolyphenols at a dose of 450mgkgd for 7 days beforeOxyHbinjection [18 19]

23Mouse SAHModel SAHwas performed using themodelreported by Shi et al [20] Under general anesthesia with1 pentobarbital (50mgkg intraperitoneally) animals wereplaced in prone positionThe posterior cervical muscles weredissected through a suboccipital midline skin incision andretracted laterally The exposed transparent atlantooccipitalmembrane was penetrated by a 30 gauge needle Underspontaneous breathing a 23-gauge needle without point was

inserted percutaneously into the skull at a controlling depthof 15mm in the cross position at the sagittal suture 2mmand sutura coronaria 1mm Then 50120583L (150120583molL) ofOxyHb was injected through this hole into subarachnoidspace After the neurological assessment the animals weredecapitated at different time points after SAH Prior todecapitation one part (119899 = 6) was perfused through theleft cardiac ventricle with isotonic saline followed by 4paraformaldehyde in phosphate-buffered saline (PBS) Thebrain tissue was removed and fixed in 4 paraformaldehydefor 48 h and then embedded in paraffin Sections of 4mmthickness were cut using a microtome for histologic studiesThe others (119899 = 6) were sacrificed with 09 saline solutionperfusion through the left cardiac ventricle The fresh brainwas immediately removed and cortex sections selected andthen stored at minus80∘C

24 Mortality and Neurological Functions AssessmentRecently the Garcia scoring system has been developed toevaluate the animal neurological behavior and function ina blinded fashion [21] The results showed that the higherthe neurological score the better the outcome Briefly theneurobehavioral examination was performed at 6 h 12 h24 h and 72 h An 18-point scoring system was used based on(1) spontaneous activity (2) symmetry of limb movement(3) climbing (4) body proprioception (5) movement offorelimbs and (6) response to vibrissae touch (score scale0ndash3 each)

25 Lactate Dehydrogenase (LDH) Assay from Brain Cor-tex The supernatant of all the samples was collected afterhomogenate and the LDH content was determined usingan LDH assay kit according to the manufacturerrsquos instruc-tions (Nanjing Institute of Jiancheng Biological EngineeringChina) [22] LDH cytotoxicity was calculated using ODas LDH cytotoxicity (Ug protein) = (OD sample minus ODblank)(OD standard solutionminusODblank standard solution)times standard solution concentrationsample protein concentra-tion

26 Isolation of Mitochondria from Cerebral Cortex Intactmitochondria were isolated from fresh brain cortex layerusing a tissue mitochondria isolation kit (Beyotime Instituteof Biotechnology China) In brief after homogenization of05 g cortical tissue in ice-cold MSH buffer (10mM HEPESpH75 containing 200mMmannitol 70mMsucrose 10mMEGTA and 20mgmL serum albumin) the homogenatewas centrifuged at 1000timesg at 4∘C for 10min The collectedsupernatantwas then centrifuged at 3500timesg at 4∘C for 10minto obtain a mitochondrial pellet [23]

27 Assay of Mitochondrial Membrane Potential Changes inmitochondrial membrane potential (Δ120595

119898) were measured

using a JC-1 (51015840661015840-tetrachloro-111015840331015840-tetraethylbenzimid-azolylcarbocyanide iodide) staining (mitochondrial mem-brane potential assay kit Beyotime Institute of Biotechnol-ogy China) according to the manufacturerrsquos instructionsBriefly isolated mitochondria were suspended in 05mL

Oxidative Medicine and Cellular Longevity 3

medium containing 5mM JC-1 Samples were analyzedusing an automatic microplate reader (Thermo ScientificUSA) at time scan method The intensities of green (exci-tationemission wavelength = 485538 nm) and red (excita-tionemission wavelength = 485590 nm) fluorescence wereanalyzed in each sample and represented a surrogate markerof loss of mitochondrial Δ120595

119898[24]

28 Assay for Cellular ATP Levels from Brain Cortex Afterbeing thawed samples were homogenized in boiling doubledistilled water in order to denature endogenous ATPasepresent in the tissue The supernatant fraction of thehomogenate was collected after centrifugation at 10000timesgfor 10min ATP levels weremeasured using ATP colorimetricassay kit according to manufacturerrsquos instructions (NanjingInstitute of Jiancheng Biological Engineering China) [25]

29 Immunohistochemistry for Cyt c Antigen Deparaffinizedsections were treated with 03 hydrogen peroxide inmethanol for 15min at room temperature to block endoge-nous peroxidase activity The sections were incubated in001M pH 65 sodium citrate buffer for 10min at 121∘C andcooled to room temperature After being blocked with 10normal goat serum for 1 h at room temperature the slideswere subsequently incubated overnight with anti-cyt c anti-body (BA0781 Boster Bio-Engineering Limited CompanyChina) at a dilution of 1 100 After being extensively washedwith PBS the slides were incubated with Histostain-Pluskit (SP-9001 Zymed USA) The sections were then coun-terstained with DAB (ZLI-9032 Zhongshan Golden BridgeBiotechnology Co LTD China) Quantitative evaluationwas measured using IDA-2000 software (Beijing KonghaiTechnology Company China) At least 10 visual fields werecaptured and more than 500 cells were counted [26 27]

210 Statistical Analysis The statistical analysis was per-formed using the Statistical Package for the Social Sci-ences (SPSS Inc Chicago IL USA) program All datawere reported as means plusmn SD of three independent exper-iments The physiological variables were analyzed by one-way ANOVA followed by LSDmultiple comparison post hocanalysis The neurological scores were compared by Kruskal-Wallis nonparametric test followed by multiple comparisonprocedures by Duncanrsquos method For all comparisons 119875 lt005 was considered statistically significant

3 Results

31 Mortality and Neurological Scores Of a total of 180mice 8 (44) died over the course of the experiment 5(83) in the SAH group 3 (50) in the tea polyphenolspretreated animals and 0 in the sham group This suggeststhat tea polyphenols pretreatment reduced the mortality inconsequence of SAH

The neurological scores obtained for sham SAH andtea polyphenols + SAH groups are depicted in Figure 1The neurological score observed for mice with SAH wassignificantly lower than that of the sham group from 6 h

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

201816141210

86420

6 12 24 72Time (h)

Neu

rolo

gica

l sco

res

ShamSAHSAH + tea polyphenols

998779998779lowastlowast998779998779

lowastlowast998779998779

Figure 1 Neurological deficits in OxyHb-induced SAH miceValues are expressed as mean plusmn SD of triplicate samples lowastlowast119875 lt 001versus sham 998779998779119875 lt 001 versus SAH

to 72 h after SAH (119875 lt 001 versus sham) whereas theimproved neurological scores were observed after 12 h ofSAH in animals pretreatedwith tea polyphenols pretreatmentgroup (119875 lt 001 versus SAH) indicating that tea polyphenolsrescued neuronal injury

32 The Neuroprotective Role of Tea Polyphenols in EarlyBrain Injury after SAH LDH activity is the most widely usedmarker in cytotoxic studies Using this assay we detected aneuroprotective role for tea polyphenols in the EBI stage afterSAH LDHactivitywas stable in the cortical tissue of the shamgroup (Figure 2) After SAH LDH levels were significantlyincreased in the cortex along with the SAH progress (119875 lt001 versus sham) but the peak level was observed 12 hafter SAH indicating that LDH activity might be timedependent However pretreatment with tea polyphenols ledto a significant decrease in LDHactivity compared to the SAHgroup (119875 lt 001 versus SAH) A significant difference wasobserved between groups pretreated with tea polyphenolsand sham (119875 lt 001 versus sham) except at 12 h LDHlevels after pretreatment with tea polyphenols were variablecompared to those detected in the sham group

33 The Preventive Effect of Tea Polyphenols on Mitochon-drial Depolarization in the Cortex after SAH Mitochondrialmembrane potential (Δ120595

119898) a widely recognized biomarker

of mitochondrial function can be measured using a cationiclipophilic dye JC-1 OxyHb caused significant mitochondrialmembrane depolarization in the SAH group which wasexpressed as an increase in JC-1 greenred fluorescenceratios (119875 lt 001 versus sham) (Figure 3) However thepreadministration of tea polyphenols prevented the lossof mitochondrial membrane potential (119875 lt 005 versussham) 12 h after SAH no significant difference was observedcompared with the sham group (119875 gt 005 versus sham) The



lowastlowast


16000

14000

12000

10000

8000

6000

4000

2000

03 6 12 24 72

Time (h)

LDH

activ

ity (U

gpr

ot)






998779998779

Figure 2 Effect of tea polyphenols on LDH activity in OxyHb-induced SAH Data are expressed as the mean plusmn SD of threeindependent experiments lowastlowast119875 lt 001 versus sham 998779998779119875 lt 001versus SAH

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowast998779

910

876543210

3 6 12 24 72Time (h)

JC-1

(rel

ativ

e gre

enr

ed fl

uore

scen

ce ra

tios)


998779998779998779998779998779998779

998779998779

Figure 3 Effect of tea polyphenols on themitochondrial membranepotential Data are expressed as themean plusmn SD of three independentexperiments lowast119875 lt 005 versus sham lowastlowast119875 lt 001 versus sham 998779119875 lt005 versus SAH 998779998779119875 lt 001 versus SAH

decrease ofmitochondrialmembrane potential in SAHgroupwas greatly alleviated by tea polyphenols pretreatment with atime-dependent effect (119875 lt 005 versus SAH)

34 Tea Polyphenols Increasing ATP Content in the Devel-opment of SAH After SAH ATP content was determinedusing a validatedATPdetection assay in the cortex (Figure 4)Within the cortical region the steady-state ATP level wasstable in the sham group at different time points However

lowastlowast

lowastlowast lowastlowastlowastlowast lowastlowast

lowastlowast

3500

3000

2500

2000

1500

1000

500

03 h 6 h 12 h 24 h

Time

ATP

cont

ent (120583

moL

gpr

ot)


3 d

998779998779

998779998779



lowastlowast998779998779lowastlowast998779998779

Figure 4 Alteration of ATP content after SAH All the experimentswere performed as described in Section 2 Data are expressed as themean plusmn SD of three independent experiments lowastlowast119875 lt 001 versussham 998779998779119875 lt 001 versus SAH

ATP levels were dramatically altered both in the SAH and thetea polyphenols pretreatment groups with a distinct patternInterestingly the ATP content increased rapidly showing thehighest values 3 h after SAH both in the SAH and the teapolyphenols pretreatment groups (119875 lt 001 versus sham)After 3 h of SAH ATP levels dramatically declined and thelowest levels were observed at 3 days after SAH suggestinga time-dependent ATP depletion (119875 lt 001 versus sham)In the tea polyphenols treatment group a fluctuation in theATP level was observed ATP levels were gradually reducedfrom6 h to 24 hwhereas a significant increase inATP contentwas observed 3 days after SAH (119875 gt 005 versus sham 119875 lt001 versus SAH) In conclusion tea polyphenols significantlyincreased ATP content after SAH

35 Effect of Tea Polyphenols on the Cyt c in EBI after SAHConsidering that cytoplastic cyt c level is increased alongwith depolarization of mitochondrial membrane potentialcyt c content was measured in the different experimentalmodels using immunohistology As shown in Figure 5(a) amoderate and stable immunoreactivity was observed in thesham group whereas robust cyt c levels were observed atall time points after SAH However after pre-administrationof tea polyphenols cyt c levels were gradually decreased tobasal level The cyt c levels were significantly increased inthe SAH group compared to the sham group (119875 lt 001versus sham) (Figure 5(b)) Interestingly cyt c values afterpre-administration of tea polyphenols were higher than thatof sham but lower than that of SAH group Significantdifference was observed from 3 h to 24 h of sham (119875 lt 001versus sham) as well as 3 h 24 h and 3 days of SAH (119875 lt 005versus SAH) indicating that tea polyphenols can block cyt crelease after SAH


3 h

6 h

12 h

24 h

Sham SAH Tea polyphenols

3 d

(a)

lowastlowast


lowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

9

8

7

6

5

4

3

2

1

03 6 12 24 72

Time (h)Re

lativ

e cyt

c in

tens

ityShamSAHSAH + tea polyphenols

998779998779

(b)

Figure 5 Tea polyphenols prevented cyt c release (a) Identification of cyt c by immunohistochemical assay (times400) (b) Quantification of cytc level by densitometry At least 10 visual fields were captured and more than 500 cells were counted The quantification represents meansand standard deviations of results from three independent experiments lowastlowast119875 lt 001 versus sham 998779119875 lt 005 versus SAH 998779998779119875 lt 001 versusSAH

4 Discussion

Mitochondria contribute to many cellular events involv-ing intracellular calcium homeostasis reduction-oxidationpotential cell cycle regulation and synaptic plasticity [28]There is a current awareness that mitochondria are highlylikely subjected to insults therefore mitochondrial dys-function may act as one of the main trigger events incentral nervous system disease such as Parkinsonrsquos diseaseAlzheimerrsquos disease and ischemic stroke leading to celldeath and ultimately diseased brain [29ndash31] The depolar-ization of the mitochondrial membrane potential (Δ120595

119898) is a

common event in mitochondrial dysfunction through whichapoptosis necrosis and autophagy can be driven [32 33]Ourresults show a significant decrease in Δ120595

119898in the SAH group

compared with sham group Several studies have shownthat tea polyphenols significantly prevent cell swelling andthe decline in the Δ120595

119898[34] In line with these reports

we observed that in the pretreated tea polyphenols groupthe Δ120595

119898gradually increased to basal level 12 h after SAH

indicating that one mechanism by which tea polyphenolsexert their protective effects is possibly by inhibition of thedepolarization of the inner membrane potential Due to lackof glycolytic capacity more mitochondria are required toproduce the necessary energy in the brain than that in otherorgans [35] In pathological conditions loss of Δ120595

119898leads

to the mitochondrial permeability transition (MPT) poreopening and to osmotic swelling of the mitochondrial matrixand to defective oxidative phosphorylation thus impairing

ATP synthesis After SAH the ATP levels were decreasedcompared to sham controls but could be restored by teapolyphenols pretreatment indicating that tea polyphenolsmay block mitochondrial dysfunction followed by increasedATP content eventually leading to neuronal cell survival

Since the brain is highly sensitive to changes inmitochon-drial respiration due to its higher consumption of oxygenand fewer free radicals scavenger ability changes inΔ120595

119898may

also ultimately lead to apoptosis through downregulation ofantiapoptotic proteins as well as activation of proapoptoticpathways [36] More specifically mitochondrial dysfunctionwould inhibit cytochrome c oxidase activity and lead torelease of cyt c into the cytosol which initiates the caspasecascade There is no doubt that mitochondrial respiratorydysfunctions leading to lowΔ120595

119898might result in the release of

pro-apoptotic proteins such as the apoptosis inducing factor(AIF) and cyt c [37] The latter activates caspase-9 thus acti-vating caspase-3-dependent apoptotic pathway In additionin low Δ120595

119898conditions PINK1 accumulates on the surface

of the mitochondria and recruits Parkin an ubiquitin ligasewhich ubiquitinylates Bcl-2 further inducing cyt c relatedand apoptosis [38 39] It has been shown that increased cyt cmediates DNA fragmentation and apoptosis in mouse brainsin subarachnoid hemolysate [40] In other words with SAHprogression neuronal cells enter the apoptotic pathway in thecortex However tea polyphenols can significantly inhibit cytc release by blocking mitochondrial dysfunction at the EBIstage after SAHThis finding is supported by previous resultsthat showed that the number of apoptotic cells was reduced


after using green tea extract for pre-treatment of ischemia ingerbils [41]

5 Conclusion

In the present study we found that neuroprotective effectsof tea polyphenols may rely on their mitochondrial pro-tection behavior Our studies showed that tea polyphenolspretreatment reduces mitochondrial dysfunction markersand increases neurological scores after SAH [42 43] Thissuggests that dietary supplementation with tea polyphenolscould be a potential candidate for prevention of SAH As teapolyphenols represent a class of natural dietary componentsfurther research will be necessary to better identify whichpolyphenols play the roles in SAH and which signalingpathways are involved in these neuroprotective effects

Conflict of Interests

The authors have declared no conflict of interests

Acknowledgments

This research was supported by the National Nature Sci-ence Foundation of China (no 31200519) the Program forNew Century Excellent Talents in University (NCET) theProgram for Science and Technology Innovation Talentsin Universities of Henan Province (HASTIT) and the Sci-ence and Technology Key Projects of Henan Province (no122102110116)

References

[1] O Weinreb S Mandel T Amit and M B H Youdim ldquoNeu-rological mechanisms of green tea polyphenols in Alzheimerrsquosand Parkinsonrsquos diseasesrdquo Journal of Nutritional Biochemistryvol 15 no 9 pp 506ndash516 2004

[2] B A Sutherland R M A Rahman and I Appleton ldquoMech-anisms of action of green tea catechins with a focus onischemia-induced neurodegenerationrdquo Journal of NutritionalBiochemistry vol 17 no 5 pp 291ndash306 2006

[3] S Kuriyama A Hozawa K Ohmori et al ldquoGreen tea consump-tion and cognitive function a cross-sectional study from theTsurugaya Projectrdquo American Journal of Clinical Nutrition vol83 no 2 pp 355ndash361 2006

[4] B E Sumpio A C Cordova D W Berke-Schlessel F Qin andQH Chen ldquoGreen tea the ldquoAsian Paradoxrdquo and cardiovasculardiseaserdquo Journal of the American College of Surgeons vol 202no 5 pp 813ndash825 2006

[5] J B Bederson E S Connolly Jr H H Batjer et al ldquoGuidelinesfor the management of aneurismal subarachnoid hemorrhagea statement for healthcare professionals from a special writinggroup of the stroke council American Heart AssociationrdquoStroke vol 40 no 3 pp 994ndash1025 2009

[6] A Alaraj F T Charbel and S Amin-Hanjani ldquoPeri-operativemeasures for treatment and prevention of cerebral vasospasmfollowing subarachnoid hemorrhagerdquo Neurological Researchvol 31 no 6 pp 651ndash659 2009

[7] W J Cahill J H Calvert and J H Zhang ldquoMechanisms ofearly brain injury after subarachnoid hemorrhagerdquo Journal of

Cerebral Blood Flow and Metabolism vol 26 no 11 pp 1341ndash1353 2006

[8] H Yatsushige R P Ostrowski T Tsubokawa A Colohan andJ H Zhang ldquoRole of c-Jun N-terminal kinase in early braininjury after subarachnoid hemorrhagerdquo Journal of NeuroscienceResearch vol 85 no 7 pp 1436ndash1448 2007

[9] S Park M Yamaguchi C Zhou J W Calvert J Tang and JH Zhang ldquoNeurovascular protection reduces early brain injuryafter subarachnoid hemorrhagerdquo Stroke vol 35 no 10 pp 2412ndash2417 2004

[10] J Cahill J W Calvert S Marcantonio and J H Zhang ldquop53may play an orchestrating role in apoptotic cell death afterexperimental subarachnoid hemorrhagerdquoNeurosurgery vol 60no 3 pp 531ndash545 2007

[11] G Fiskum R E Rosenthal V Vereczki et al ldquoProtectionagainst ischemic brain injury by inhibition of mitochondrialoxidative stressrdquo Journal of Bioenergetics and Biomembranesvol 36 no 4 pp 347ndash352 2004

[12] S Marchi C Giorgi and J M Suski ldquoMitochondria-roscrosstalk in the control of cell death and agingrdquo Journal of SignalTransduction vol 2012 Article ID 329635 17 pages 2012

[13] C Mammucari and R Rizzuto ldquoSignaling pathways in mito-chondrial dysfunction and agingrdquo Mechanisms of Ageing andDevelopment vol 131 no 7-8 pp 536ndash543 2010

[14] P H Reddy and T P Reddy ldquoMitochondria as a therapeu-tic target for aging and neurodegenerative diseasesrdquo CurrentAlzheimer Research vol 8 no 4 pp 393ndash409 2011

[15] F A Sehba R M Pluta and J H Zhang ldquoMetamorphosis ofsubarachnoid hemorrhage research fromdelayed vasospasm toearly brain injuryrdquoMolecular Neurobiology vol 43 no 1 pp 27ndash40 2011

[16] J V Higdon and B Frei ldquoTea catechins and polyphenols healtheffectsmetabolism and antioxidant functionsrdquoCritical Reviewsin Food Science and Nutrition vol 43 no 1 pp 89ndash143 2003

[17] T Sasaki H Kasuya H Onda et al ldquoRole of p38 mitogen-activated protein kinase on cerebral vasospasm after subarach-noid hemorrhagerdquo Stroke vol 35 no 6 pp 1466ndash1470 2004

[18] B Scolaro D Delwing-De Lima J G P Da Cruz and DDelwing-Dal Magro ldquoMate tea prevents oxidative stress in theblood and hippocampus of rats with acute or chronic ethanoladministrationrdquo Oxidative Medicine and Cellular LongevityArticle ID 314758 2012

[19] S Guo J Q Yan T B Yang X Q Yang E Bezard and BL Zhao ldquoProtective effects of green tea polyphenols in the 6-OHDA rat model of Parkinsonrsquos disease through inhibition ofROS-NO pathwayrdquo Biological Psychiatry vol 62 no 12 pp1353ndash1362 2007

[20] W Shi L Y Huang R Z Wang et al ldquoTime course ofoxyhemoglobin induces apoptosis in mice brain cells in vivordquoActa Neurochirurgica no 104 pp 23ndash26 2008

[21] X Zhu C Chen D Ye et al ldquoDiammonium glycyrrhizinateupregulates PGC-1120572 and protects against A1205731-42-induced neu-rotoxicityrdquo PLoS ONE vol 7 no 4 Article ID e35823 2012

[22] B K Singh M Tripathi B P Chaudhari P K Pandey andP Kakkar ldquoNatural terpenes prevent mitochondrial dysfunc-tion oxidative stress and release of apoptotic proteins duringnimesulide-hepatotoxicity in ratsrdquo PLoS ONE vol 7 no 4Article ID e34200 2012

[23] KChenQ Zhang JWang et al ldquoTaurine protects transformedrat retinal ganglion cells from hypoxia-induced apoptosis bypreventing mitochondrial dysfunctionrdquo Brain Research vol1279 pp 131ndash138 2009


[24] D Xinmin D Yunyou P Chaosheng et al ldquoDexamethasonetreatment attenuates early seawater instillation-induced acutelung injury in rabbitsrdquo Pharmacological Research vol 53 no 4pp 372ndash379 2006

[25] Y Liu D B Kintner G Begum et al ldquoEndoplasmic reticulumCa2+ signaling and mitochondrial Cyt c release in astrocytesfollowing oxygen and glucose deprivationrdquo Journal of Neuro-chemistry vol 114 no 5 pp 1436ndash1446 2010

[26] Y Chen and Q Kong ldquoEvaluation of centrosome abnormalitiesand p53 inactivation in chemical induced hepatocellular car-cinogenesisrdquo Neoplasma vol 56 no 2 pp 169ndash176 2009

[27] J H Garcia S Wagner K F Liu X J Hu and J P Mohr ldquoNeu-rological deficit and extent of neuronal necrosis attributable tomiddle cerebral artery occlusion in rats statistical validationrdquoStroke vol 26 no 4 pp 627ndash635 1995

[28] M P Mattson M Gleichmann and A Cheng ldquoMitochondriainNeuroplasticity andNeurological DisordersrdquoNeuron vol 60no 5 pp 748ndash766 2008

[29] R X Santos S C Correia X Wang et al ldquoAlzheimerrsquos diseasediverse aspects ofmitochondrial malfunctioningrdquo InternationalJournal of Clinical and Experimental Pathology vol 3 no 6 pp570ndash581 2010

[30] R A Vaishnav I N Singh D M Miller and E D Hall ldquoLipidperoxidation-derived reactive aldehydes directly and differen-tially impair spinal cord and brain mitochondrial functionrdquoJournal of Neurotrauma vol 27 no 7 pp 1311ndash1320 2010

[31] H Chen and D C Chan ldquoMitochondrial dynamicsmdashfusionfission movement and mitophagymdashin neurodegenerative dis-easesrdquo Human molecular genetics vol 18 no 2 pp R169ndashR1762009

[32] C H Jing L Wang P P Liu C Wu D Ruan and GChen ldquoAutophagy activation is associatedwith neuroprotectionagainst apoptosis via a mitochondrial pathway in a rat model ofsubarachnoid hemorrhagerdquo Neuroscience vol 213 pp 144ndash1532012

[33] N Hail and R Lotan ldquoCancer chemoprevention andmitochon-dria targeting apoptosis in transformed cells via the disruptionof mitochondrial bioenergeticsredox staterdquo Molecular Nutri-tion and Food Research vol 53 no 1 pp 49ndash67 2009

[34] F Gomez-Pinilla and T T Nguyen ldquoNatural mood foodsthe actions of polyphenols against psychiatric and cognitivedisordersrdquo Nutritional Neuroscience vol 15 no 3 pp 127ndash1332012

[35] P H Reddy ldquoMitochondrial dysfunction in aging andAlzheimerrsquos disease strategies to protect neuronsrdquoAntioxidantsand Redox Signaling vol 9 no 10 pp 1647ndash1658 2007

[36] D R Green and G Kroemer ldquoThe pathophysiology of mito-chondrial cell deathrdquo Science vol 305 no 5684 pp 626ndash6292004

[37] G Cheng R H Kong L M Zhang and J N ZhangldquoMitochondria in traumatic brain injury and mitochondrial-targetedmultipotential therapeutic strategiesrdquo British Journal ofPharmacology vol 167 no 4 pp 699ndash719 2012

[38] D Chen F Gao B Li et al ldquoParkin mono-ubiquitinates Bcl-2and regulates autophagyrdquo Journal of Biological Chemistry vol285 no 49 pp 38214ndash38223 2010

[39] C Zhu XWang F Xu et al ldquoThe influence of age on apoptoticand other mechanisms of cell death after cerebral hypoxia-ischemiardquo Cell Death and Differentiation vol 12 no 2 pp 162ndash176 2005

[40] P G Matz M Fujimura A Lewen Y Morita-Fujimura andP H Chan ldquoIncreased cytochrome c-mediated DNA frag-mentation and cell death in manganese-superoxide dismutase-deficient mice after exposure to subarachnoid hemolysaterdquoStroke vol 32 no 2 pp 506ndash515 2001

[41] J T Hong S R Ryu H J Kim et al ldquoProtective effect ofgreen tea extract on ischemiareperfusion-induced brain injuryin Mongolian gerbilsrdquo Brain Research vol 888 no 1 pp 11ndash182001

[42] C S Yang J D Lambert and S Sang ldquoAntioxidative andanti-carcinogenic activities of tea polyphenolsrdquo Archives ofToxicology vol 83 no 1 pp 11ndash21 2009

[43] S Bastianetto S Krantic J G Chabot andRQuirion ldquoPossibleinvolvement of programmed cell death pathways in the neuro-protective action of polyphenolsrdquo Current Alzheimer Researchvol 8 no 5 pp 445ndash451 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


induced by oxidative stress and inflammation results inmito-chondrial membrane potential (Δ120595

119898) reduction cytochrome

c (cyt c) release and then activation of mitochondrial-dependent cell death providing evidence that mitochondrialimpairment is deleterious [12 13] Therefore mitochondrialfunction is a potential therapeutic target [14]

Recent results suggest that inhibition of a signal pathwaymay not be required for the treatment of SAH [15] In sucha scenario tea polyphenols may be of special interest due totheir multiple functions such as antioxidants antimutageniciron chelators and in glutamate release [16] However teapolyphenolsrsquo effects in the time course after SAH are stillunknownThe aimof this studywas to investigatewhether teapolyphenols have neurological and neurobehavioural effectsor not and the pathway involved in these effects in the in vivoSAHmodelThis study contributes to a better understandingof the role of tea polyphenols in SAH prevention which arenontoxic and inexpensive dietary components

2 Materials and Methods

21 PreparationOxyhemoglobin (OxyHb) Arterial blood col-lected from Kunming mouse with heparin was centrifugedat 2500timesg for 15min The supernatant was discarded Ery-throcytes werewashed 3 timeswith saline solution lysedwithmethylbenzene and centrifuged at 15000timesg for 20min thesupernatantwas collectedUsing filtermembrane theOxyHbsolution was collected adjusted to 3 120583molL using measuredoptical density (OD) at 540 nm and 576 nm and stored atminus80∘C [17]

22 Animals The animal use and care protocols wereapproved by Institutional Animal Care and Use Committee(IACUC) of Xinxiang Medical University One hundredand eighty adult male Kunming mice weighing from 18 to20 g were purchased from Xinxiang Medical University Allanimals were required to undergo institutional quarantine for7 days prior to use The environment for animal housing wasequipped with controlled temperature (22 plusmn 3∘C) humidity(40ndash70) and a 12 h lightdark alternation The mice weredivided into three groups sham group (119899 = 60) SAH group(119899 = 60) and preadministration tea polyphenols SAH group(119899 = 60) Subsequently each group was subdivided into 3 h6 h 12 h 24 h and 72 h subgroup (119899 = 12) respectivelySAH group was injected with OxyHb and sham group wasgiven isotonic saline for the same period Pretreatment groupwas administered intragastrically with water containing teapolyphenols at a dose of 450mgkgd for 7 days beforeOxyHbinjection [18 19]

23Mouse SAHModel SAHwas performed using themodelreported by Shi et al [20] Under general anesthesia with1 pentobarbital (50mgkg intraperitoneally) animals wereplaced in prone positionThe posterior cervical muscles weredissected through a suboccipital midline skin incision andretracted laterally The exposed transparent atlantooccipitalmembrane was penetrated by a 30 gauge needle Underspontaneous breathing a 23-gauge needle without point was

inserted percutaneously into the skull at a controlling depthof 15mm in the cross position at the sagittal suture 2mmand sutura coronaria 1mm Then 50120583L (150120583molL) ofOxyHb was injected through this hole into subarachnoidspace After the neurological assessment the animals weredecapitated at different time points after SAH Prior todecapitation one part (119899 = 6) was perfused through theleft cardiac ventricle with isotonic saline followed by 4paraformaldehyde in phosphate-buffered saline (PBS) Thebrain tissue was removed and fixed in 4 paraformaldehydefor 48 h and then embedded in paraffin Sections of 4mmthickness were cut using a microtome for histologic studiesThe others (119899 = 6) were sacrificed with 09 saline solutionperfusion through the left cardiac ventricle The fresh brainwas immediately removed and cortex sections selected andthen stored at minus80∘C

24 Mortality and Neurological Functions AssessmentRecently the Garcia scoring system has been developed toevaluate the animal neurological behavior and function ina blinded fashion [21] The results showed that the higherthe neurological score the better the outcome Briefly theneurobehavioral examination was performed at 6 h 12 h24 h and 72 h An 18-point scoring system was used based on(1) spontaneous activity (2) symmetry of limb movement(3) climbing (4) body proprioception (5) movement offorelimbs and (6) response to vibrissae touch (score scale0ndash3 each)

25 Lactate Dehydrogenase (LDH) Assay from Brain Cor-tex The supernatant of all the samples was collected afterhomogenate and the LDH content was determined usingan LDH assay kit according to the manufacturerrsquos instruc-tions (Nanjing Institute of Jiancheng Biological EngineeringChina) [22] LDH cytotoxicity was calculated using ODas LDH cytotoxicity (Ug protein) = (OD sample minus ODblank)(OD standard solutionminusODblank standard solution)times standard solution concentrationsample protein concentra-tion

26 Isolation of Mitochondria from Cerebral Cortex Intactmitochondria were isolated from fresh brain cortex layerusing a tissue mitochondria isolation kit (Beyotime Instituteof Biotechnology China) In brief after homogenization of05 g cortical tissue in ice-cold MSH buffer (10mM HEPESpH75 containing 200mMmannitol 70mMsucrose 10mMEGTA and 20mgmL serum albumin) the homogenatewas centrifuged at 1000timesg at 4∘C for 10min The collectedsupernatantwas then centrifuged at 3500timesg at 4∘C for 10minto obtain a mitochondrial pellet [23]

27 Assay of Mitochondrial Membrane Potential Changes inmitochondrial membrane potential (Δ120595

119898) were measured

using a JC-1 (51015840661015840-tetrachloro-111015840331015840-tetraethylbenzimid-azolylcarbocyanide iodide) staining (mitochondrial mem-brane potential assay kit Beyotime Institute of Biotechnol-ogy China) according to the manufacturerrsquos instructionsBriefly isolated mitochondria were suspended in 05mL



119898[24]




3 Results




lowastlowast

lowastlowast

lowastlowast

lowastlowast

201816141210

86420

6 12 24 72Time (h)

Neu

rolo

gica

l sco

res


998779998779lowastlowast998779998779










lowastlowast


16000

14000

12000

10000

8000

6000

4000

2000

03 6 12 24 72

Time (h)

LDH

activ

ity (U

gpr

ot)






998779998779


lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowast998779

910

876543210

3 6 12 24 72Time (h)

JC-1

(rel

ativ

e gre

enr

ed fl

uore

scen

ce ra

tios)


998779998779998779998779998779998779

998779998779




lowastlowast


lowastlowast

3500

3000

2500

2000

1500

1000

500

03 h 6 h 12 h 24 h

Time

ATP

cont

ent (120583

moL

gpr

ot)


3 d

998779998779

998779998779








3 h

6 h

12 h

24 h


3 d

(a)

lowastlowast


lowastlowast


lowastlowast

lowastlowast

lowastlowast

9

8

7

6

5

4

3

2

1

03 6 12 24 72

Time (h)Re

lativ

e cyt

c in

tens


998779998779

(b)


4 Discussion


119898) is a








119898leads




119898may








5 Conclusion




Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















119898[24]




3 Results




lowastlowast

lowastlowast

lowastlowast

lowastlowast

201816141210

86420

6 12 24 72Time (h)

Neu

rolo

gica

l sco

res


998779998779lowastlowast998779998779










lowastlowast


16000

14000

12000

10000

8000

6000

4000

2000

03 6 12 24 72

Time (h)

LDH

activ

ity (U

gpr

ot)






998779998779


lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowast998779

910

876543210

3 6 12 24 72Time (h)

JC-1

(rel

ativ

e gre

enr

ed fl

uore

scen

ce ra

tios)


998779998779998779998779998779998779

998779998779




lowastlowast


lowastlowast

3500

3000

2500

2000

1500

1000

500

03 h 6 h 12 h 24 h

Time

ATP

cont

ent (120583

moL

gpr

ot)


3 d

998779998779

998779998779








3 h

6 h

12 h

24 h


3 d

(a)

lowastlowast


lowastlowast


lowastlowast

lowastlowast

lowastlowast

9

8

7

6

5

4

3

2

1

03 6 12 24 72

Time (h)Re

lativ

e cyt

c in

tens


998779998779

(b)


4 Discussion


119898) is a








119898leads




119898may








5 Conclusion




Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















lowastlowast


16000

14000

12000

10000

8000

6000

4000

2000

03 6 12 24 72

Time (h)

LDH

activ

ity (U

gpr

ot)






998779998779


lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowast998779

910

876543210

3 6 12 24 72Time (h)

JC-1

(rel

ativ

e gre

enr

ed fl

uore

scen

ce ra

tios)


998779998779998779998779998779998779

998779998779




lowastlowast


lowastlowast

3500

3000

2500

2000

1500

1000

500

03 h 6 h 12 h 24 h

Time

ATP

cont

ent (120583

moL

gpr

ot)


3 d

998779998779

998779998779








3 h

6 h

12 h

24 h


3 d

(a)

lowastlowast


lowastlowast


lowastlowast

lowastlowast

lowastlowast

9

8

7

6

5

4

3

2

1

03 6 12 24 72

Time (h)Re

lativ

e cyt

c in

tens


998779998779

(b)


4 Discussion


119898) is a








119898leads




119898may








5 Conclusion




Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















3 h

6 h

12 h

24 h


3 d

(a)

lowastlowast


lowastlowast


lowastlowast

lowastlowast

lowastlowast

9

8

7

6

5

4

3

2

1

03 6 12 24 72

Time (h)Re

lativ

e cyt

c in

tens


998779998779

(b)


4 Discussion


119898) is a








119898leads




119898may








5 Conclusion




Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusion




Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article neuroprotective effect of tea polyphenols...

Documents