Ferric chloride-induced murine carotid arterial injury: A model of redox pathology☆.

1.3 MB·English

by  Li, Wei

RedoxBiology1(2013)50–55
ContentslistsavailableatSciVerseScienceDirect
Redox Biology
journal homepage: www.elsevier.com/locate/redox
Method
Ferric chloride-induced murine carotid arterial injury: A model
$
of redox pathology
Wei Lia,b,n, Thomas M. McIntyrea,b, Roy L. Silversteinc
aDepartmentofCellularandMolecularMedicine,LernerResearchInstitute,ClevelandClinic,9500EuclidAvenue,Cleveland,OH44195,USA
bDepartmentofMolecularMedicine,ClevelandClinicLernerCollegeofMedicineofCase,WesternReserveUniversity,Cleveland,OH,USA
cDepartmentofMedicine,MedicalCollegeofWisconsin,Milwaukee,WI,USA
a r t i c l e i n f o a b s t r a c t
Articlehistory: Ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that
Received1November2012 reportsplateletactivationinthecontextofanasepticclosedvascularsystem.Thismodelisbasedon
Receivedinrevisedform redox-induced endothelial cell injury, which is simpleand sensitive to both anticoagulant and anti-
13November2012 plateletsdrugs.Thetimerequiredforplateletaggregationtooccludebloodflowgivesaquantitative
Accepted14November2012
measureofvasculardamagethatispathologicallyrelevanttothromboticdisease.Wehaverefinedthe
traditional FeCl -induced carotid artery model making the data highly reproducible with lower
3
Keywords: variation. This paper will describe our artifices and report the role of varying the oxidative damage
Ferricchloride byvaryingFeCl concentrationsandexposure.Toexploreamaximumdifferencebetweenexperimental
3
Thrombosis
groups,adjustmentoftheselectedFeCl doseandexposuredurationmaybenecessary.
Carotidartery 3
&2013TheAuthors.PublishedbyElsevierB.V.Allrightsreserved.
Animalmodel
Introduction guineapigsandrabbits[12–19].InthismodelFeCl isappliedtothe
3
topicalsurfaceofanintactvessel,triggeringvascularwallinjuryand
Thromboticcardiovasculardiseasesarethemostcommoncause denudation of the endothelium via a mechanism involving the
of death and disability in the developed world [1–4]. The vessel generation of reactive oxygen species [4,14]. A recent study sug-
wall is a complex system that includes multiple cell types and is gested that FeCl -induced vascular injury was erythrocyte-depen-
3
influenced by multiple extrinsic factors including shear stress, dent,requiringhemolysisandhemoglobinoxidationforendothelial
circulating blood cells, hormones and cytokines, as well as the denudation[20,21].Onemeasurableparameterinthismodelisthe
antioxidant status of the vessel wall [5,6]. Therefore, in vitro elapsedtimefrominjurytocompletevesselocclusion,measuredas
experiments cannot replicate this complex environment, so bloodflowcessationbyDopplerflowmeterorunderdirectobserva-
invivostudiesofanimalmodelsarecriticaltoallowbetterunder- tionwithintravitalmicroscopy[13–15].Arangeoftimesbetween5
standing of mechanisms involved in thrombotic disorders [7–9]. and 30min has been reported in different studies in C57Bl6 mice
Suchstudiesmimichumanpathologyandmayprovideimportant [12–15,22],suggestingthatFeCl concentrations,typesofanesthe-
3
mechanisticinformationrelatedtodiscoveryofnewpathwaysand sia, surgical techniques, mouse age and genomic background,
drugs,aswellasimportantdetailscharacterizingdrugeffects. methodofmeasuringbloodflow,andotherenvironmentalvariables
Ferric chloride (FeCl ) induced vascular injury is a widely used havesignificanteffectsinthismodel.Thiswidevariabilitymaybe
3
modelofthrombosis[10,11].Thisagent,appliedtotheouteraspect acceptable for a single study, but it makes it very difficult to
ofarteriesinducesoxidativedamagetovascularcells[12,13],with comparestudiesfromdifferentgroupsandmaymakedetectionof
loss of endothelial cell protection from circulating platelets and subtledifferencesdifficult.Inourrecentserialstudiesoftheroleof
components of the coagulation cascade. This model is simple and thetype2scavengerreceptorCD36inthrombosisassociatedwith
sensitive to both anticoagulant and anti-platelets drugs, and has diet-inducedatherosclerosiswe have refined the traditionalFeCl -
3
beenperformedoncarotidandfemoralarteries,jugularveins,and inducedcarotid arterymodelmakingthedatahighlyreproducible
mesenteric and cremasteric arterioles and venules in mice, rats, with lower variation [12–15,22,23]. This paper will describe and
shareourskillsandreportdifferentFeCl concentrationordifferent
3
treatingtimeinduceddifferentredoxinjuryonthrombosis.
$This is an open-access article distributed under the terms of the Creative
CommonsAttributionLicense,whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Abbreviation:CA,carotidartery Materialsandmethods
nCorresponding author at: Department of Cellular and Molecular Medicine,
LernerResearchInstitute,ClevelandClinic,9500EuclidAvenue,Cleveland,
OH44195,USA.Tel.:þ12164455492;fax:þ12164449404. All procedures and manipulations of animals have been
E-mailaddress:[email protected](W.Li). approved by Institutional animal care and use committees
2213-2317/$-seefrontmatter&2013TheAuthors.PublishedbyElsevierB.V.Allrightsreserved.
http://dx.doi.org/10.1016/j.redox.2012.11.001
W.Lietal./RedoxBiology1(2013)50–55 51
(IACUC) of The Cleveland Clinic in accordance with the United vein to label platelets using a 1cc syringe with a fresh 30G
StatesPublicHealthServicePolicyontheHumaneCareandUseof needle.Itisimportanttoflushtheneedlewiththesolutionto
Animals, and the NIH Guide for the Care and Use of Laboratory make sure that no air bubbles remain. To facilitate injection
Animals. theneedlecanbebent2–3mmfromtiptoa901angle,which
will make the tip parallel to the long axis of jugular vein
Equipmentandreagents (Fig. 1D). To stabilize the syringe and keep the needle in
positionthesyringeisheldinonehandandtheneedlewitha
ToolsformousesurgeryareavailablefromFineScienceTools forceps in the other during the injection.After injection, the
(FST). Minimum necessary tools include: dissection scissor site is held with the forceps, the vessel is clamped with a
(14502-14), forceps (11018-12) for cutting skin, scissor (14519- dissection forceps, and the vein is ligated with a 6-0 suture.
14)andforceps(11050-10)fordissectingsofttissue,hemostatic It is important to perform the rhodamine injection before
forceps (13021-12), fine needle holders (12061-10), fine tip exposingtheleftcarotidartery(CA)topreventadventitialCA
forceps [Dumont #5 (11cm) 11254-20 and Dumont #5XL stainingbythefluorescentdyeifleakageorbleedingoccurred
(15cm) 11253-10], 3/8 curve needle, 4-0 silk suture, 6-0 silk duringorafterinjection.
suture, and a hook (10062-12). A black plastic coffee stir tube, 9. Thesofttissueandfasciaaroundtheleftsubmaxillarygland
Whatman#1filterpapercutinto1(cid:2)2mmpieces,15-cmtissue is then dully dissected and pulled toward the 7 o’clock
culturelids,surgicaltape,adissectingmicroscope(orloupeswith position (Fig. 1E) to expose the left sternocleidomastoid
at least 4X magnifications), heating pad withrectal temperature muscle(bluearrow).
monitor probe, and hair cutter were also used. For real time 10. The fascia (Fig. 1E, dotted line) between the left sternoclei-
observation of thrombosis we used a Leica DMLFS fluorescent domastoid muscle and the omohyoid muscle or the sterno-
microscope with an attached Gibraltar Platform (EXFO, Quebec, hyoidmuscle(Fig.1E,greenarrow)locatedtotheleftofthe
Canada) and water immersion objectives at 10x, 20x and 60x tracheaisthendullydissectedwithahemostaticforceps.
magnifications. Video imaging was performed using a QImaging 11. A needle with 6-0 silk suture (about 15cm long) is passed
Retigo Exi 12-bit mono digital camera (Surrey, Canada) and under the sternocleidomastoid muscle at the level indicated
Streampix version 3.17.2 software (Norpix, Montreal, Canada). by blue arrow in Fig. 1E and pulled laterally toward the
Ketamine(Ketaset100mg/ml,FortDodgeAnimalHealth,IA,USA) 10o’clockpositiontoexposethecommonCAalongorunderthe
andxylazine(100mg/ml,Hospira,IL,USA)wereusedforanesthe- sternohyoid muscle and/or omohyoid muscle. This procedure
sia and rhodamine 6G (Sigma 252433-1G, 0.5mg/ml in saline, mustbeperformedwithcaretoavoidinjuringtheleftjugular
0.2mm filtered) was injected intravenously used to label blood vein, which is located outside of the sternocleidomastoid
components. A stock solution of 30% FeCl (Anhydrous, Sigma muscle.
3
12321) in water was prepared and diluted prior to use at final 12. The CA is seen after pulling away the sternocleidomastoid
concentrationsof2.5%,5%,7.5%,10%and12.5%. muscle (Fig. 1F arrow), which is accompanied by the vagus
nerve, the white structure seen in Fig. 1G (green arrow).
Arterialinjuryprotocol The very thin omohyoid muscle seen between the yellow
dottedlinesinFig.1FisremovedifitcoverstheCA.
1. EighttotwelveweeksoldC57Bl6miceareanesthetizedwith 13. The hemostatic forceps are used to dissect the soft tissue
ketamine (100mg/kg)/xylazine (10mg/kg) via intraperito- around the CA without touching the CA, and then fine tip
neal injection. This anesthetic method facilitates manipula- forceps are used to pick up the lateral fascia around the CA
tion of the animal during surgery and during transfer from while avoiding the vagus nerve and CA. Another fine tip
surgicalbenchtothemicroscopestage. forcepisthenusedtopunchaholebetweentheCAandvagus
2. Fur on the neck and upper chest is removed with a small nerve. A hook is passed through the hole and the CA gently
animalelectricclipper.Dipilatorycreamisnotused. lifted so that a fine tip forcep can be placed under the CA.
3. Mice are secured in the supine position on the lid of 15cm The hook and forceps are then in opposite directions along
tissue culture plate using tape (about 10cm) to secure hind theCAtostripadventitialsofttissue.
limbsandlowerbody,andtwopiecesoftape(2cm(cid:2)0.5cm) 14. To block background fluorescence, a black plastic coffee
to secure the forelegs. A 4-0 suture is wrapped around the stirreriscutintoa3mmpiece,pressedandcutlongitudinally
incisorstokeeptheneckstraight. toyieldtwopiecesof‘‘U’’shapeplastic.Onepieceisinserted
4. The mouse is placed with head towardsthe operator under a withforcepsundertheCAwhilegentlyliftingtheCAwiththe
surgical light source. A midline incision is made through the hook(Fig.1H).
skinfromsternumtochinasshowninthelinedrawninFig.1A. 15. ToavoiddryingofCA,2–3dropsofsalineareappliedtothe
5. The structures visualized first under the skin are the sub- surgical field and the mouse and plate lid are transferred
maxillaryglands(Fig.1BandD,bluearrows)whicharelinked together to the microscope stage and the 10x water lens is
byathinfascia(dottedline).Thisfasciacanbedullydissected placedintheappropriateposition.
with a hemostatic forceps exposing the manubrium (black 16. Sincemechanicaltraumacandirectlyinjuretheendothelium
arrowinFig.1C)andthetrachea(TinFig.1D). and lead to thrombus formation [24] it is necessary to
6. ThesofttissueseentotherightoftheblackarrowinFig.1C observeandrecordvesselimagestoconfirmthatnosurgical
andtheskinareheldtogetherwithaforcepsandadissection injuryhappenstothevesselpriortotheFeCl injury.Itisalso
3
forcepsistheninsertedunderthefasciatowardthe2o’clock necessarytoconfirmthatthereisnoremainingtissuearound
position(yellowdottedlineinFig.1C)whichisthepositionof theCA,whichmayformabarrierandattenuatetheeffectof
theclavicle.Thefasciaisthenfreedfromthejugularveinby FeCl -inducedinjury.
3
openingtheforceps. 17. Apapertowelisfoldedtomakeathinandfinetipandusedto
7. Thefascia,softtissueandskinalongtheyellowdottedlinein carefullyblotthesalinearoundtheCA(avoidtouchingCA)as
Fig. 1C are cut to expose the right jugular vein (Fig. 1D, well as in other places in the surgical field. This is very
arrow). importanttoavoiddilutionoftheusedFeCl solution.
3
8. 100ml of rhodamine 6G solution (or other drugs or fluores- 18. Using a fine tip forceps to pick up a piece of filter paper
centfree radicalindicators) is injectedinto theright jugular (1(cid:2)2mm) saturated with FeCl solution and place directly
3
52 W.Lietal./RedoxBiology1(2013)50–55
Fig.1. Procedurestoexposethejugularvein,injectionofrhodamine6Gflurescencedyeintothecirculationsystemandexposureofleftcarotidartery.Tindicatestrachea.
Seetextfordetails.
ontheCAfortimedpoints.Thenthefilterpaperisremoved segmentitbecomesdifficulttoidentifybloodflowinthethrombi.
fromtheCAandrinsedwithsaline.Toaidvisualizationofthe To solve this issue the imaging is moved to the proximal un-
bloodstreamthefilterpaperisplacedtowardthedistalend injuredsite(Fig.1I,greenarrow).Inthissite,bloodflowshouldbe
of the exposed CA (Fig. 1I) making sure to leave a short stillclearlyidentified.Theendpointsoftheexperimentare:when
segment of the proximal site (green arrow in Fig. 1I) for bloodflowhasceasedfor 430s,orifocclusionisnotseenafter
observinglatephasebloodflow. 30min,thetimeisrecordedas30minforstatisticalcomparisons.
Observationofthrombiformationandtheimagerecord Statisticalanalysis
Thrombus formation is identified by accumulation of the One-way ANOVA (Bonferroni/Dunn) is used to determine the
fluorescent platelets, which is observed in real-time recording differences among groups. Unpaired t test is used to determine
videoimagesorundermicroscope.Typically,werecordvideofor differencesbetweengroups.Dataarerepresentedasmean7SEM,
10s everyminutefor thefirst 10min and then10severy other andPo0.05isconsideredsignificant.
minute.Makesuretonotethevideoframenumberateachtime
point for later analysis. After injury the vessel is scanned from Resultsanddiscussion
distal to proximal sites of the exposed segments to provide a
completeviewoftheinitialinjury,andthenfocusontheinterest Animal models of thrombosis not only provide valuable
areaforfurtherimaging.Whenthethrombusoccupiestheentire informationaboutthemechanismsofthrombosis,butalsoarea
W.Lietal./RedoxBiology1(2013)50–55 53
Before 1 4 8 12 16 20 24 28 30
wt
cd36-/-
Before 1 2 4 6 8 10 12 14 30
wt
stopped
cd36-/-
no stop
Before 1 2 4 6 8 10 11 24 28’30”
wt
stopped
cd36-/-
stopped
Before 1 2 3 4 5 6 6’45”
stopped
wt
cd36-/-
stopped
Fig. 2. Effect of different concentration of FeCl3 on thrombosis. Representative video images of thrombi formation in carotid artery (CA) treated with different
concentrationofFeCl3,2.5%(A),5%(B),7.5%(C),and10%(D)areshown.Bloodflowwasfromrighttoleft.‘‘Before’’indicatesimagestakenimmediatelypriortoFeCl3
treatment.Numbersindicatetimeinminutesafterremovingthefilterpaper.ArrowsinAindicatethetinythrombiwhichwerefirstobserved.
valuable tool for pre-clinical evaluation of compounds that may which are unable to bind and respond to oxidized lipoprotein
be used for treatment and prevention of the atherothrombotic particlesandapoptoticcellparticles,astoolstocomparedifferent
diseases [10,16,21,25]. Although the FeCl induced vessel injury concentrationofFeCl onthrombosis.Nothrombiwereobserved
3 3
model is a well-accepted tool for studying thrombosis, the in the absence of FeCl application, showing that the surgical
3
mechanismofFeCl inducedvascularlesionandthedeterminants procedures did not induce vascular injury (Fig. 2). Treatment of
3
initiatingthrombusformationremainsincompletelyunderstood. CA with 2.5% FeCl for 1min immediately induced formation of
3
OneestablishedconceptisthatFeCl mediatedoxidationinduces scatteredtinythrombiinwtmice(Fig.2A,column1min,arrows).
3
endothelial cell denudation and subsequent exposure of the Withtime,thethrombibecamelarger,butat30minthosewere
subendothelial matrix to the blood stream, thus activating the not large enough to impact blood flow. There were no thrombi
coagulatingsystem[4,21]. formedin2ofthe3cd36nullmiceandonly4tinythrombiwere
We made several refinements to the FeCl model to enhance observed(startingat8min)inthethirdmouse.
3
itsutilityandreproducibility.Theseinclude:(1)exposingtheCA Treatment with 5% FeCl for 1min immediately induced
3
sufficientlytoprovideenoughlength((cid:3)5mm)toallowapplica- thrombi formation in wt and cd36 null mice, and the border of
tion of the filter paper while leaving space to clearly observe the injured area was easy to identify (not shown). The thrombi
bloodflowbyintravitalmicroscopy;(2)strippingtheadventitial formed initially were smaller in cd36 null mice than in wt mice
soft tissue cleanly around the CA to allow the filter paper (Fig. 2B). These initial thrombi were washed away by the blood
to contact the vessel wall directly and produce an even injury; stream,sofewerthrombiwereseenat2min.Thrombistartedto
(3)underlyingtheCAwitha‘‘U’’shapedpieceofblackplasticto enlarge 3–4min after removing the filter paper and these later
separate it from surroundingtissue, preventFeCl diffusion, and forming thrombi were stable and usually did not wash away.
3
block background fluorescence. This strategy is very important Thethrombiinwtmiceformedfasterandwerelargerthanthose
when using this model to examine vessel wall reactive oxygen in cd36 null mice; cessation of blood flow was seen in 3 of the
species formation by injection of free radical indicator into the 5wtmicewithanaveragevesselocclusiontimeof23minwhile
blood system [26]; (4) directly injecting a fluorescence dye into none of the cd36 null mice showed flow cessation within the
the jugular vein to label platelets in vivo. This does not affect 30minofobservation.
platelet count or function, and obviates the need to sacrifice Treatment of the vessels with 7.5% FeCl induced more rapid
3
additionalanimalsasplateletdonors. increase in the size of thrombi in wt mice, but had no obvious
differenceincd36nullmice(Fig.2C).Asreportedpreviously[27],
DosedependenceofFeCl -inducedvesselocclusiontime thisconcentrationofFeCl inducedvesselocclusioninbothtypes
3 3
of mice; however, the vessel occlusion time was significantly
We previously have reported that deletion of cd36 prolonged elongated in cd36 null mice. When the FeCl concentration was
3
vesselocclusiontimes[13].Wenowusedwtandcd36nullmice, 10% or greater the vessel wall oxidation injury was presumably
54 W.Lietal./RedoxBiology1(2013)50–55
maximal(Fig.2D)andnodifferencesinocclusiontimewereseen When thrombi become larger and cover the entire area of
betweenthetwotypesofmice.Fig.3showsvesselocclusiontime injury,itbecomesdifficulttoobserveflow(e.g.Fig.2B,8mintime
plottedasafunctionofFeCl anddemonstratesashifttotheright pointinwt,andonlinevideo).Thismaybethereasonwhysome
3
oftheresponsecurveincd36nullmicecomparedtowt,suggest- researchersthinkthatthismethodislesssensitivethanmeasure-
ing a pro-thrombotic effect of CD36. These data are also consis- mentofbloodflowbytransonicflowmeter.Bysurgicallyexposing
tent with our recent data that cd36 deletion induced an theCAoverasufficientlengthandplacingthefilterpapercloseto
antioxidanteffectonthevesselwall[14]. the distal site (Fig. 1I) we were able to observe and record the
normal area proximal to the site of injury as shown in Fig. 1I
(arrow) and Fig. 2C (e.g. 6–11min in 7.5% FeCl treated wt
3
32.5 * mouse), and blood flow can be identified clearly. When blood
flowceases,numerouslargercells(leukocytes)starttoadhereto
30
* the vessel wall at the proximal site of the thrombi; therefore,
27.5
p = 0.02 p = 0.04 large cell accumulation can be used as a secondary marker for
min) 25 oxidatvevasculardamage.Flowusuallystoppedwithin2minof
m ( 22.5 theappearanceoflargecells(seeonlinevideoformoreinforma-
n ti 20 tion). In our studies we recorded the time when blood flow
sio 17.5 completely stopped. This may differ from some studies using a
clu 15 p<0.0001 p<0.0001 transonicflowprobewherebackgroundnoisemakesitdifficultto
c
o determinetheflowprecisely,especiallywhenitfallstolessthan
sel 12.5 10%ofbaseline[28].
Ves 10 Supplementary material related to this article can be found
7.5 onlineathttp://dx.doi.org/10.1016/j.redox.2012.11.001.
wt
5
2.5 cd36-/- ThedurationofFeCl3treatmentcorrelateswithtimetovessel
0 occlusion
FeCl (%) 2.5 5 7.5 10 12.5
3
PublishedstudieshaveusedarangeofexposuretimesofFeCl
3
Fig.3. VesselocclusiontimesafterdifferentconcentrationsofFeCl3treatment. treatment,makingcomparisonsamongstudiesdifficult.Todeter-
MiceweretreatedasinFig.2andocclusiontimeassessedbyintravitalmicro- mine the effect of varying exposure times in this model system
scopy. *po0.01 wt vs. cd36 null at the same concentration of FeCl3. One-way we treated cd36 null mice with 5% FeCl for 1, 3 and 5min.
ANOVABonferroni/Dunnwasusedtodeterminethedifferencesamonggroups. 3
Dataarerepresentedasmean7SEM.N¼3for2.5%FeCl3treatedwtandcd36null As shown in Fig. 4, increasing the duration of FeCl3 exposure
mice;n¼4for5%FeCl3treatedcd36nullmice;andn¼5inallotherconditions. dramatically increased the rate of thrombus enlargement and
Before 1 2 4 8 12 16 20 24 30
1 min
no stop
3 min
5 min
8’30” stopped
p = 0.006
p = 0.2
35
p = 0.02
30
n)
mi
m ( 25
n ti 20
o
si
u
cl 15
c
o
sel 10
s
Ve
5
0
1 3 5
FeCl 5% (min)
3
Fig.4. DurationofFeCl3treatmentonthrombosis.(A)RepresentativevideoimagesofthrombiinCAexposedto5%FeCl3for1,3or5min.‘‘Before’’indicatesbeforeFeCl3
treatment.Numbersindicatetimeinminutesafterremovingthefilterpaper.(B)Statisticalanalysisofvesselocclusiontime.N¼3for1minandn¼5for3and5min
treatments.One-wayANOVA(Bonferroni/Dunn)wasusedtodeterminethedifferencesamonggroups.Dataarerepresentedasmean7SEM.
W.Lietal./RedoxBiology1(2013)50–55 55
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