Review Article Aneurysmal Subarachnoid Hemorrhage Models: Do They Need a Fix? - Hindawi

14 Pages·2015·1.28 MB·English

HindawiPublishingCorporation
StrokeResearchandTreatment
Volume2013,ArticleID615154,13pages
http://dx.doi.org/10.1155/2013/615154
Review Article
Aneurysmal Subarachnoid Hemorrhage Models: Do They
Need a Fix?
FatimaA.Sehba1andRyszardM.Pluta2
1DepartmentsofNeurosurgeryandNeuroscience,MountSinaiSchoolofMedicine,NewYork,NY10029,USA
2SurgicalNeurologyBranch,NationalInstituteofNeurologicalDisordersandStroke,NationalInstitutesofHealth,Bethesda,
MD20892,USA
CorrespondenceshouldbeaddressedtoFatimaA.Sehba;[email protected]
Received15April2013;Revised31May2013;Accepted4June2013
AcademicEditor:R.LochMacdonald
Copyright©2013F.A.SehbaandR.M.Pluta.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
Thediscoveryoftissueplasminogenactivatortotreatacutestrokeisasuccessstoryofresearchonpreventingbraininjuryfollowing
transientcerebralischemia(TGI).Thatthisdiscoverydependedupondevelopmentofembolicanimalmodelreiteratesthatproper
strokemodelingisthekeytodevelopnewtreatments.IncontrasttoTGI,despiteextensiveresearch,preventionortreatmentof
braininjuryfollowinganeurysmalsubarachnoidhemorrhage(aSAH)hasnotbeenachieved.AlackofadequateaSAHdisease
modelmayhavecontributedtothisfailure.TGIisanimportantcomponentofaSAHandsharesmechanismofinjurywithit.We
hypothesizedthatmodifyingaSAHmodelusingexperienceacquiredfromTGImodelingmayfacilitatedevelopmentoftreatment
foraSAHanditscomplications.ThisreviewfocusesonsimilaritiesanddissimilaritiesbetweenTGIandaSAH,discussestheexisting
TGIandaSAHanimalmodels,andpresentsamodifiedaSAHmodelwhicheffectivelymimicsthediseaseandhasapotentialof
becomingabetterresourceforstudyingthebraininjurymechanismsanddevelopingatreatment.
1.Introduction 2.IschemicandHemorrhagicBrainInjury
Stroke is the second major cause of death worldwide. Ischemic stroke occurs when blood supply to the brain is
Accordingto theWorldStrokeOrganizationapproximately reducedtoalevelthatcerebralfunctionandmetabolismare
15 million people suffer from stroke each year. Five million no longer maintained. Cerebral ischemia could be focal or
people die from it, and another 5 million are left perma- global and transient or permanent. A mix of any of them
nentlydisabled[1].Ischemicstrokeconstitutesthemostand isalsopossible;forinstance,afteraneurysmalsubarachnoid
hemorrhagic stroke 15 to 30% of the total annual stroke hemorrhage,apatientcandevelopatransientglobalischemia
cases [2]. The 21-day to 1-month case fatality ranges from (evokedbytemporaryincreasedICP)followedbypermanent
13 to 23% for ischemic stroke, as compared to 25–35% for focalischemiabecauseofathrombosisordelayedvasospasm.
hemorrhage stroke [3]. The cost of survivor care and lost Transientfocalischemia(TFI)affectsaspecificbrainregion,
productivity (conservatively estimated to be more than 54 and transient global ischemia (TGI) affects the whole brain
billiondollarsannually)necessitatesresearchtoreducestroke for a limited time; both are followed by reperfusion and/or
mortalityanddisability.Anessentialstepinthisdirectionis hyperperfusion. In contrast to transient ischemia, in per-
developinganexperimentalmodelthatreplicatesthehuman manent ischemia blood flow is never reestablished to the
condition.Numerousanimalmodelsaddressingcausesand part(local)orthewhole(global)brain.Hemorrhagicstroke
pathophysiology of ischemic and hemorrhagic stroke have occurs when blood flow in the brain is reduced due to
been developed. Whereas research using these models has theintracranialbleeding.Aneurysmalsubarachnoidhemor-
clearly influenced the treatment of global ischemic stroke, rhage(aSAH),anontraumatictypeoftheintraduralbleeding,
it has made relatively small impact on treatment of hemor- constitutes5%ofallstrokesandoccurswhenanintracranial
rhagicstroke. aneurysmburstsandspewsbloodunderhighpressureinto
2 StrokeResearchandTreatment
the subarachnoid space. Such a violent flow of blood into Variationsthatallowinvestigatortocontrolinjuryintensity
a narrow, CSF-filled space results in a dramatic increase in areavailable.Amild-to-moderateinjuryiscreatedbykeeping
intracranialpressureanddecreaseinthecerebralperfusion arterial blood pressure normal during carotid occlusion
pressure (CPP) and cerebral blood flow (CBF) [4–6]. The [40, 51]. A severe injury is achieved by reducing arterial
ICP-dependentreductioninCBFafterSAHisbeneficialbut blood pressure to 40–50mmHg during carotid occlusion.
alsoharmful,beneficialasitsavesapatient’slifebyallowinga Blood pressure reduction is achieved by phlebotomy or by
bloodclottosealthedomeoftherupturedaneurysmandstop pharmacologicalmanipulation[52].
thebleedingandharmfulasitlimitsbloodflowtothewhole Theadvantagesof2-VOmodelincludeone-stagesurgical
brainforunpredictabletimeandmayresult,inthebest-case preparation, production of high-grade forebrain ischemia,
scenario,inatransientlocalorglobalischemicbraininjury abilitytocontrolventilationtoensurenormoxiaandnormo-
or,intheworstcase,braindeath.Thus,tobestofourknowl- carbia,easeofreestablishingcerebralcirculation,suitability
edge most of aSAH bleeding is associated with transient for chronic studies, and a relatively low failure rate. The
globalhypoperfusionand/orischemia.ThougharoleofTGI disadvantageisthatpharmacologicallyinducedhypotension
inaSAHoutcomehasbeensuspectedsinceearly19thcentury, maycomplicatetheinterpretationofresults[78].
its true nature remains poorly defined and its importance
largelyunappreciated.Asaconsequence,thedifferencesand
3.1.2. Four-Vessel Occlusion Model (4-VO). Ischemia in this
similaritiesbetweenTGIandaSAHarenotdetermined,and
model is created by almost simultaneous occlusion of four
knowledge that TGI researchers have accumulated over the
majorcerebralvessels:bilateralbothvertebralandcommon
yearsisnotusedtofurtherunderstandingoftheSAH-related
carotid arteries [43]. Usually, first, the vertebral arteries are
injury to the brain. Three reasons of this oversight are: (1)
electrocoagulated, and then the common carotid arteries
eventsleadingtoa“spontaneous”TGIandTGIevokedbyan
are occluded by tightening the ligatures around them [78].
aneurysmalSAHaredifferent,(2)thesudden/abruptnature
This model has been extensively studied to assure a high
ofaSAHeventmakesassociationwithTGIdifficulttostudy
incidence of successful ischemia with acceptable mortality
[5, 7], and (3) until recently, most research on improving
rate.Nevertheless,eveninthebesthands,animalsurvivalrate
patient outcome has been on delayed cerebral vasospasm
following4-VOisonly50%[43,79].Amodificationof4-VO
(AKA delayed neurological deficits) and not on events that
whichcombinesamildsystemichypotension(80–90mmHg)
occur early after aSAH [8]. Lately, limited improvement in
with bilateral carotid occlusion creates less morbidity and
patient outcome after more than half a century of research
moreuniformbraininjury[80,81].
convincedmanyresearcherstoreevaluatethesignificanceof
Both 2- and 4-VO models are frequently used to study
earlyeventsandmoreimportantlytheinfluenceofearlyTGI
TGI (see Table 1). 2-VO model is often preferred over
afteraSAHontheoutcome[9].
4-VOmodelasitrequireslesssurgicalmanipulations;4-VO
Inrecognitionofthisnewtrendwereviewanimalmodels
requires two state surgical preparation and rarely achieves
of TGI and aSAH, discuss why ischemic, but not aSAH
completereversalofglobalischemia[82].
models have proven successful in reducing the death and
disabilityafterstroke,andproposeamodifiedaSAHmodel
3.1.3.BihemisphericForebrainCompressionIschemia(BFCI).
thatincorporatesfeaturesofTGImodelandcouldbeabetter
ThismodelwasdevelopedbyKramerandTuynmanin1967
resourceforstudyingtheinjurymechanismsandtreatment
to define the duration of ischemia tolerated by the brain
ofaSAH.
[28]. Ischemia here is induced by increasing intracranial
pressuretothelevelofsystolicbloodpressuresothatcerebral
3.AnimalModelsofTGIandSAH perfusionisdisrupted.Theincreaseinintracranialpressure
isachievedbyinfusionofartificialcerebrospinalfluid(CSF)
3.1. TGI Models (Table 1). Animal models of TGI induce intothecisternamagna.Cushing’sreflexevokedbyincreased
eithercompleteorincompleteglobalischemia.Incomplete ICP can be reduced by administration of the ganglion-
global ischemia blood flow is ceased completely, and in the blockingdrug[83].
incompleteglobalischemiabloodflowdecreasestoadegree TGI produced by BFCI is consistent, reproducible and
thatcellularmetabolismandfunctioncannolongerbemain- successfullycreatedinseveralanimalspecies.ThoughBFCI
tained [49, 50]. The injury and survival are proportional to modelisnotasextensivelyusedasthe2-VOor4-VOmodel,
thedurationofglobalischemicinsult:greaterwhentheinsult it provides an excellent foundation for the modified aSAH
isashortandresolvable;lasting10to30minutesandlower modelthatwelaterproposeinthisreview(seebelow).
whentheinsultislongerorpermanent.Thus,permanentTGI
modelsworkbestforstudyingthemechanismsofinjury,and
3.2. SAH Models (Table 2). Brain injury evoked by aSAH
theresolvableTGImodelsworkbestforstudyingtherapeutic
consistsofearlyanddelayedevents.Earlyeventsincluderise
interventions. Below we describe the most extensively used
in ICP, fall in CBF and CPP at the time of aSAH, and the
TGImodels.SeeTable1foralistofTGImodelsandanimal
delayed eventsarearterialvasospasmanddelayed ischemic
speciesused.
deficits that develop 3–7 days after the initial bleed. Due to
unpredictablenature(noteveryaneurysmruptures)ofaSAH
3.1.1. Two-Vessel Occlusion (2-VO) Model. Ischemia in this [5,7],theinformationonultra-earlyeventsisavailableonly
model is created by a transient bilateral carotid occlusion. as the patient is admitted and monitored after the initial
StrokeResearchandTreatment 3
Table1:Experimentalmodelsoftransientglobalischemia.
(a) CompleteTGImodels
TGImethod Keyfeatures Species References
Cardiacarrest Epinephrineinjection,defibrillation,and
Mouse,ratandmonkey [10–12]
(i)KClinjection CPRareusedforresuscitation
CanbeusedwithCPRtostudy
(ii)Ventricularfibrillation Cat,dog,pigandmonkey [13–16]
resuscitation
Aorticocclusion Inhibitsflowthroughoutthebody Rat,rabbit,catanddog [17–19]
Neckcuff/tourniquetwithhypotension Inhibitionofbloodflowtothehead Rat,cat,dogandmonkey [20–24]
Extracranialarteryocclusion
(i)Innominateandsubclavianarteries Cat [25,26]
Inhibitionofbloodflowtothehead
(ii)Brachiocephalicandsubclaviannearaortic
Monkey [27]
origin
(b) IncompleteTGImodels
TGImethod Keyfeatures Species References
Intracranialhypertension
Rabbit,cat,dogand
(i)Fluidinfusionincerebralcistern [28–30]
Abraincompressioninjury monkey
(ii)Ballooninflation Rat,cat,dogandmonkey [31–34]
Immediateischemiaandreperfusion
Extracranialarteryocclusion allowspossibilityofpermanent
occlusion
Bilateralcommoncarotid(2-VO)
Mouse,rat,gerbil,sheep
(i)Withouthypotension [35–39]
Createsmild-to-moderateinjury andmonkey
(ii)Withhypotension Rat,rabbit,catandmonkey [40–42]
Bilateralcommoncarotid+vertebral Rat,rabbit,cat,dogand [43–48]
Createssevereinjury
arteries(4-VO) monkey
Table2:ExperimentalmodelsofaSAHand/orvasospasm.
SAHmethod Phasestudied
Species Bloodinjection Reference
Arterypuncture Clot EBI Vasospasm
Single Double
Mouse + + + − + ? [53–55]
Rat + + + − + +/− [56–60]
Rabbit + + + − + +/− [61–63]
Cat + + + + [5,64–66]
Pig + + + + + [67,68]
Dog + + + + + + [69–73]
Nonhumanprimate + + + + +/− + [74–77]
aneurysm rupture. Consequently, information obtained is 3.2.1. Blood or Hemolysate Injection or Infusion. Injection
alreadydelayed,unlessrebleedoccurs,usuallywithinhours model involves introduction of autologous fresh blood
after the initial bleed. However, because of the lingering [56,67,74,87–89] into the cisterna magna, prechiasmatic
effects ofthe initialbleed, the data obtainedduringrebleed cistern[90],ornexttoanintracranial[91,92]oranextracra-
cannotbedirectlyextrapolatedasamimicofthefirstaSAH nialartery[84,93–97].Thismodelisquiteextensivelyusedto
[5,7].Nevertheless,informationobtainedduringtherebleed studyearlyanddelayedinjuryafteraSAH.Inseveralspecies
has been used to develop animal models of aSAH. These (mouse, rat, and dog), a second blood injection 24 to 48
modelsarewidelyusedtostudyearlyanddelayedbraininjury hoursafterthefirstisnecessaryfordevelopmentofdelayed
afteraSAHandareacceptedasmimicsofclinicalaSAH(see vasospasm. Advantages of this model are that it produces
Table 2 for details) [84–86]. aSAH models can be broadly reproducible injury and allows use of saline injected sham
dividedintothreecategories. control.Disadvantageisafailuretoreproducethemechanical
4 StrokeResearchandTreatment
trauma,thefirstinsultfeltbythecerebralvasculatureupon other hand, a spectacular success was the development of
aneurysmrupture(forreviewsee[98]andreferenceswithin). thrombolytic therapy with recombinant tissue plasminogen
activator (rtPA) against acute transient focal and global
ischemicstrokebasedontheresultsofstudiesusingarabbit
3.2.2. Blood Clot Placement. In this model arterial blood
model of embolic stroke [106, 107]. The success of a rabbit
is withdrawn and allowed to clot ex vivo and then surgi-
embolic model versus failure of favored ischemic primate
cally placed on the adventitial surface of an artery. Both
modelindevelopmentofsuccessfultreatmentmayindicate
intracranial(themiddlecerebralartery[75])andextracranial
thatanaccuratemodelofadiseaseshouldprovideresultsthat
(femoral [96]) arteries have been used for clot placement.
arereproducedacrossspeciesandsuccessfullytranslatedto
Thismodelstudiesdelayedvasospasmandnotearlyinjury.
clinic.
Advantages of this technique are the well-defined course of
vasospasmandlowanimalmortalitythatpermitspharmaco-
logical intervention. Disadvantages are lack of reproducing 5.aSAHModelsandComponentsofInjury
mechanical trauma (see above) and the high cost of exper-
iment; this model is predominantly used in larger animals: Mortality, neurological deficits, and diminished quality of
dog,pig,andmonkey. life are the most important end points of a brain injury
evoked by TGI and aSAH. However, some and not all of
the mechanisms that TGI and aSAH evoke are shared (see
3.2.3.ArterialPuncture. ThisaSAHmodelinvolvespuncture
Table3).ForexamplefallinCBFcreatingtemporaryglobal
of the intracranial artery adjacent to the skull base by an
perfusiondeficitsoccursbothinTGIandaSAH,butinjury
endovascular filament. The model is considered the best
byaprolongedpresenceofbloodinthesubarachnoidspace
mimicofhumanaSAHasitreplicatesthemechanicaltrauma
characterizesSAHonly.Thus,inanewdesiredaSAHmodel
felt by cerebral vasculature upon aneurysm rupture, as well
all components of injury, the presence of controllable TGI,
as the events observed during rebleed in aSAH patients:
and an intracranial bleeding need to occur simultaneously.
rapidfallincerebralbloodflowandbloodaccumulationinto
Unfortunately, the current animal models dissociate TGI
subarachnoidspace[4–6,98].However,duetoanumberof
fromaSAH,replicatesubarachnoidbleedbutnotaperfusion
reasonsexplainedelsewherethismodelprovidesapoorcon-
deficitthatcreatesTGI,andthusthesemodelsonlypartially
trolofbleedingandhighmortality(forreviewsee[98–100]).
imitate injury produced by aSAH. This shortcoming may
Otherdisadvantagesincludecomplicatedsurgicalprocedure
havecontributedtoalackofclinicaltranslationoftherapies
that requires a trained person and difficultly in adaptation
found successful in animals. A more inclusive model that
to other, larger species. Nevertheless, arterial puncture is
incorporates all components of brain injury after aSAH is
frequentlyusedtostudyearlyinjuryafteraSAHespeciallyin
requiredtoacceleratethedevelopmentofadequatetreatment
rodents.
forimprovingthepatient’soutcome.
4.SuccessofEmbolicIschemiaModel and
6.AModifiedaSAHModel
LessonLearntaboutaSAH
AnumberofdifferentaSAHmodelsareavailableforstudying
The research focused on treatment of cerebral ischemia
injurymechanismandtreatment.Eachcarriesitsownadvan-
has been successful. It has provided us with recombinant
tagesanddisadvantages.Oneshortcomingcommonatallis
tissueplasminogenactivator(r-tPA)that,whenusedwithin
thelackofrequirementofCPPfallatSAHinductiontoalevel
4.5 hours after ischemic episode, reduces brain injury and
thatensuresTGI.Asaresultthesemodelsreplicatesomebut
improves the outcome [101]. In contrast, despite extensive
notallofthecomponentsofinjurythatarepresentinhuman
research, a therapy that could be translated to clinical SAH
aSAH(discussedabove).WehereproposeamodifiedaSAH
has not been found. Though several compounds have been
modelthatreproducesallofthecomponentsofinjuryafter
found promising against SAH in animals, none succeed in
aSAHandinadditionrequireslimitedsurgicalmanipulation,
clinicaltrials[98].
carries low mortality, can be easily adapted to a number of
Aproperdiseasemodelingmayhavecontributedtothe
species, and makes comparisons and interpretation of data
success of TGI research. That varying degree and duration
fromdifferentlaboratoriespossible.
of CBF reduction produce varying effects on the neurovas-
After reviewing the existing aSAH models (above) we
cular unit has been realized [102], and animal models that
haveformedanopinionthatperhapsanadaptationofKramer
addressaspecificproblemareaccordinglydeveloped.Focal
and Tuynman’s TGI model (explained above), that uses
ischemiamodelsstudyinjuryfollowingathromboticevent,
autologous arterial blood instead of artificial CSF, provides
and global ischemia models study injury following cardiac
thebestfoundationforthemodifiedaSAHmodel[28].
arrest. Both models focus on developing a time-dependent
Belowwedetailthreefeaturesessentialtothismodified
intervention.Animalspeciesusedrangefromrodentstothe
aSAH model. We discuss the reason we consider them
AHArecommendedprimates[103,104].However,eventhis
essentialandthetechniquesthatcanbeusedtoattainthem.
meticulous approach has not always worked. An example
of failure is the free radical-trapping agent NXY-059 that
showed promise as a neuroprotectant in rat and primate 6.1. Blood Injection. As blood upon aneurysm rupture is
ischemicmodelsbutwasineffectiveinpatients[105].Onthe released under high pressure and pools into subarachnoid
StrokeResearchandTreatment 5
Table3:RiskfactorsofTGIversusaneurysmalSAH.
Factor TGI aSAH
Highbloodpressure Shared Shared
Smoking Shared Shared
Alcoholabuse Shared Shared
Stress Shared Shared
Cardiacarrestorshockthatcreatesprolongedhypoxiaorhypoglycemia Strokeonly
Pathologicallyelevatedcerebralmetabolicrate Strokeonly
Decreasedcerebralperfusionpressure Strokeonly
Age(years) ≥65 ≤56
Gender Menprevalence Womenprevalence
Intracranialaneurysm −/+ +
cisterns, a proper replication requires the same to occur in detailed description check references in Table 2). In short,
the animal model. The location, speed, and volume blood ananimalisanesthetizedandplacedinapronepositioning
injectionallareimportantconsiderationsforconsistentrepli- withtheheadtiltedforward.Theatlantooccipitalmembrane
cationofaSAHinjuryinanimalsusedwithinanexperiment isexposedviaaskinincisionfromthemidlineontheback
andacrosslaboratories. of the neck and a delicate dissection of muscles from the
occipitalboneandC1-2vertebralbodies.Theatlantooccipital
membraneisthenpuncturedwitha27-gaugeneedleorPE-
6.1.1.BloodDeliveryRoute. Technicaldetailsofeachproce- 10 catheter that is attached to a syringe filled with fresh
durecanbefoundinadequatereference(s)inTable2. autologousblood.Theinjectionfollowsthesameparameters
Several routes have been successfully used for intracra- as the percutaneous infusion. The muscle is reapproached
nial, subarachnoid injection/infusion of blood. A brief with sutures and wound closed and covered with antibiotic
descriptionoftheseroutesandtechniquesispresentedbelow. creamstospeedthehealingandpreventinfection.Advantage
DetailscanbefoundinthereferencesinTable2. ofthisapproachisapossibilityofsealingtheholebytissue
glueastheneedleisremoved.
APercutaneousDeliveryofBlood.Thisrouteisoftenfavored
insmallandlargeanimals(rabbit,cat,dog,pig,andmonkey). A Direct Anterior Intracisternal (Prechiasmatic) Blood Injec-
This technique requires good anatomical knowledge and tion.Thisroutewastraditionallyusedinlargeanimals(dog,
reasonablebutbasicsurgicalskills.Briefly,afterproperanes- monkey)[90,108]andhasbeenrecentlyadaptedtorodents
thesiaandskinpreparation(includesshavingthebackofthe [109–111]. The technique used requires advanced surgical
head,betweentheears,andtheflexorsurfaceoftheneck),a skills,stereotacticapparatus,andaccesstoradiologicalequip-
shortbevel25-/27-gaugeneedleattachedtoaninsulinsyringe ment. It can be achieved via intra(peri)orbital approach
(size depends on the species used, can range from insulin [110,111]withorwithoutenucleation[90]orthroughtrans-
to 10cc) is introduced in the midline directly below the parenchymal approach (Table 2) [109]. Approach used to
palpable edge of the cranium after significant head flexion. accessprechiasmaticcisterndiffersamongspecies.Inrodents
The needle is slowly advanced until an access to cisterna a prechiasmatic cistern is usually approached by placing
magnaisconfirmedwithCSFpresenceinthesyringe.Atthis the animal in prone position and advancing a 27-gauge
momentasyringeisexchangedfortheonethatisfilledwith needle attached to a 1mL syringe with nonheparin blood
fresh,arterial,andpreferablynonheparinizedblood.Bloodis stereotacticallyuntilthetipreachesthebaseoftheskulland
thenquickly(inlessthan1min)injectedintosubarachnoid a proper placement in a prechiasmatic cistern is confirmed
space.Thevolume,time,andspeedofinjectionareguidedby byflowofCSFintothesyringe[109].Theorbitandtheoptic
theriseinICPtomeanarterialbloodpressurerenderingCPP foramenhavealsobeenusedtoaccessperichiasmaticcistern.
zerooradropinCBFbelow10mL/100g/min.Atthismoment
the needle is quickly removed, and steady compression is
appliedtotheneck.Theanimal’sheadistheneitherreturned 6.1.2.BloodInjectionParameters:Volume,Length,andSpeed.
to neutral or slightly extended position with the body of The volume, length, and speed of blood infusion dictate
animal(ifpossible)tilteddownforabout5–10mintoallow the degree of ICP rise and CPP reduction and thus the
blood to flow toward the anterior cisterns. Monitoring of intensityofSAHbeingcreated.ToensuresimilarityofSAH
ICP,CPP,andCBFcontinuesunderanesthesiaoranimalis intensity within an experimental group these parameters
awakedafterremovalofmonitoringdevices. needtobestandardizedandcloselymonitored.Thishowever
is not a simple task, as intracranial volume differs within
A Direct Infusion into the Cisterna Magna. This is another andbetweenspeciesmakinganinvestigator’scontrolonthe
frequently used route for blood delivery. It requires signifi- injury intensity difficult. Consequently, in current practice
cantlymoresurgicalexperiencebutisstillrelativelyeasy(for a consensus on the injection parameters that work the best
6 StrokeResearchandTreatment
in a particular species does not exist, and a wide array of mechanisms and test therapies, animal research
volume,length,andspeedoptionsareavailableandusedfor remains to be the cornerstone of scientific research
injectingbloodinasinglespecies.Adownfallofthisisthat and drug development. However, respect for lives
sincetheintensityofSAHdependsuponthevolume,length, of all creatures is essential and is an important
andspeedofinjection,variationsintheseparametersmakes consideration in animal research. Reducing distress
a comparison and interpretation of results from different and suffering in animals is a crucial consideration
laboratories difficult, if not impossible. For instance, SAH in development of an animal model. A number of
in rat has been induced by injecting 100microL autologous steps can be taken to prevent unnecessary animal
bloodoverseconds[110],0.2mLofbloodformorethan1min sufferingduringexperimentation.Thesestepsinclude
[109]or0.3mLbloodfor1min[111]. (1) use of perioperative and postoperative analgesia
and anesthesia; (2) use of proper life support; (3)
OntheModel.InamodifiedaSAHmodelinjectionparam- aseptic surgical technique; and (4) little amount of
etersshouldbeguidedbythechangesofICPandCPP.The surgicalmanipulationetc.
parameters that evoke dramatic but transient reduction in
CPP to near zero should be selected and used for creating
SAH.Theseparameterswillofcoursedifferbetweenspecies
Use of perioperative and postoperative analgesia and
andevenwithinaspecies,buttheselectioncriteria(transient
anesthesia during surgery reduce distress caused by the
reductionintheCPPtonearzero)willremainsame.Thiswill
surgical manipulations for inducing SAH. The type and
facilitate comparisondata obtained in different laboratories
dose of anesthetic and analgesic depends upon the animal
andamongdifferentspecies.
species being used. An investigator can refer to species-
specific guideline on anesthesia and analgesia provided by
6.1.3. Factors Influencing Choice of Technique Used for SAH their institution for agents that work best in the species
Induction. Anumberoffactorsneedtobeconsideredbefore used. The depth of anesthesia ensures that animal does
atechniquecanbeselectedforcreatingaSAH.Someofthese not feel pain during surgery. A frequent check of corneal
factorsareasfollows: reflex and limb pinch as well as monitoring of heart rate
is commonly employed to confirm anesthesia depth. Such
(i)Simplicity and Reproducibility. A technique that is
as for rat Ketamine-Xylazine combination (50mg/5mg/Kg;
simpleandreproducibleisincreasinglyattractiveand
intraperitoneal administration) is often used for reducing
has greater chances of becoming a favored method
perioperativepainandbuprenorphine(0.05mg/Kg,subcuta-
forstudyingaproblem.Asimpletechniqueallowsfor
neousadministration)twicedailyforreducingpostoperative
a short training period and reduces the chances of
pain. In addition, inspired isoflurane (1% to 2% in oxygen-
surgeon’smistake.Reproducibilityofinjurydecreases
supplementedroomair)isfrequentlyusedduringsurgeryto
the cost of a project by reducing the number of
maintaindeepsedationinrats.
animal required for an experiment. Above, we have
Properlifesupportduringsurgeryreducesanimalmor-
examined simplicity and reproducibility of available
tality. This support includes monitoring and regulation of
aSAHtechniques.
breathing,bodytemperature,andafluidintake.Theincrease
(ii)EaseofAdaptation.Atechniqueallowingforadapta-
in ICP upon blood infusion may increase pressure at the
tioninseveralanimalspeciespermitscomparisonof
respiratorycenterstothepointthatanimalstopsbreathing.A
results.Severalanimalspecieshavebeenusedtostudy
respiratorysupportthatensuresbreathingsuchasintubation
SAH. These range from smaller animals: mice, rats,
or placement of a nose cone ensures that animal does
rabbits,andgerbils,tolargeranimals:cats,dogs,pigs,
not expire. Similarly, unless a project is studying the effect
andnonhumanprimates.Foranimalspeciesusedfor
of temperature on injury, body temperature of animal is
aparticularSAHtechniqueseeTable2andassociated ∘
maintained at 37 C (such as by a thermoblanket) from the
references. Primates, due to their higher ranking in
start of anesthesia until the animal recovers. For proper
the evolution ladder, are considered the best choice
hydrationringerlactateisadministeredasrequired.
for replication of human conditions. However, not
Aseptic surgical technique protects against infection.
every investigatorand laboratory is equipped to use
As a minimum requirement, this includes sterilization of
primates.Fortunately,thesuccessofarabbitembolic
surgical equipments, applying antiseptics such as iodine to
modelversusfailureoffavoredprimatemodelproves
thewoundsuponclosingandiftheprojectpermits,adminis-
thatitisthediseasemodelingandnotthecloseness
trationofantibioticstopreventinfectionfromoccurringand
of species to human that translates into a successful
speedhealing.
treatment.Replicationandcross-validationofresults
The amount of surgical manipulation can result into
in more than one animal species are perhaps a
animal death. In general, the more the surgical steps, the
strongerindicationoffuturesuccessfultranslationin
moreinvasivetheprocedurebecomes.Incontrast,asimple
clinicaltrial.Suchoptionwillonlybeavailableifthe
procedurereducesunnecessarypainandsuffering.
technique used to create SAH is applicable in other
specieswithnooronlyminimalmodifications.
OntheModel.ThetechniqueusedforSAHinductionshould
(iii)Low Mortality with Ethically Acceptable Morbidity. be simple, reproducible and allow adaption into different
Since computer simulation cannot be used to study species.
StrokeResearchandTreatment 7
6.2.MonitoringofSAHPhysiology. Physiologicalmonitoring intraparenchymalprobemaynotmatchtheintraventricular
isanessentialfeatureofmodifiedaSAHmodelasitconfirms pressure[113].
theintensityofSAH.Thisinformationcanbeusedtoensure
that all animals within and across an experimental group On the Model. The intraventricular measurement, despite
receive similar intensity and to interpret the results from beingtechnicallydemanding,seemstobeamethodofchoice
differentlaboratories. forthenewaSAHmodel.
6.2.2. Blood Pressure and Heart Rate Changes (“Cushing’s
6.2.1.ICPandCPPChanges. Equilibriumbetweenbrain,and
Reflex”). Cerebralperfusionpressure(CPP)isanimportant,
cranial vault volume via controlled intracranial blood and
if not crucial, clinical tool that provides information on
CSF flow is essential for maintenance of normal ICP. This
perfusion of brain [113]. CPP falls as ICP increases. An
equilibrium is disturbed by blood released upon aneurysm
ICP rise that is near or above systolic blood pressure leads
rupture. An ICP rise that occurs at aSAH reflects sub-
to complete perfusion arrest; a reduction of CPP to zero.
arachnoidbloodvolume,statusofbrainandcerebrovascular
RecoveryofCPPbeginsasICPdeclinesafterreachingapeak.
disturbances. Furthermore, peak ICP value and the pattern
CPP is estimated as the difference between ICP and mean
ofitsdeclineassociatewiththeintensityofinjuryafterSAH
arterialbloodpressure:CPP=MABP−ICP.
[7,112].Hence,continuousandreliableICPmonitoringvia
Furthermore,anincreaseinICPatSAHevokesCushing’s
a simple and easy technique is desired to determine and
reflex, a hypothalamic response to ischemia. During this
control the injury intensity and understand the underlying
reflex systolic blood pressure rises, heart rate decreases,
pathophysiologiceventsafteraSAH.
andrespirationbecomesirregular(sympatheticstimulation);
each either directly or indirectly influences CPP and CBF.
ICPMeasurement.Symptomslikeheadache,nausea,vomit-
Thus,monitoringofBPandheartratechangesisnecessary
ing(particularlyprojecting),andthepresenceofpapilledema
toaccessCPPchangesafterSAH.
stronglysuggestanincreasedintracranialpressure;however,
they do not allow for close monitoring of ICP changes.
(1) Blood Pressure Measurement. Mean arterial pressure can
Fortunately, ICP can be assessed by a number of ways;
bemeasuredbyinvasiveandnoninvasivemethods.
howeverallthesemethodsareinvasive.
(i)Invasive Method. This surgical method is based on
(i)IntraventricularCatheter.Inthismethodaburrholeis experimentsconductedbyStephenHalesin1733,that
drilledinthefrontalregion,andundereitherstereo- showed that blood pressure and heart beat can be
tactic or under radiographic guidance a catheter observed by a glass tube inserted into an artery of
is introduced into the frontal horn or the lateral horse who inserted a glass tube in artery of horse
ventricleandsecuredtotheskin.Thismethodallows and observed changes in blood pressure with the
forcontinuousandaccurateassessmentofICPandfor heart beat [114]. Not much has changed since then,
eventual intervention if an ICP increase jeopardizes andtoobtainreliableandlong-lastingmonitoringin
CBF. surgicalsettingsunderanesthesia,asterilecatheteris
placed into radial or femoral artery. This method is
(ii)Intraparenchymal Probe. The placement of an intra- used mostly for acute experiments and/or in bigger
parenchymalprobewithapressuresensororafiber- animalsbuthasbeenusedtomeasurebloodpressure
optic catheter is an alternative to the ventricular insmallanimals:rabbit(ear)androdents(tailartery).
catheter.However,thismethodispronetoareference
(ii)NoninvasiveMethod.Thismethodisfurtherdivided
driftwhilerecalibrationisimpossibleaftertheprobe
intoauscultatoryoroscillometricmethods.
isinplace.Furthermore,thelocalchangesofpressure
evokedbymetabolicchangesrelatedtodiseaseor(a
The auscultatory method is most commonly used for mea-
traumatic probe placement) can dramatically influ-
suring blood pressure in clinics. It is based on Korotkoff’s
encerecordings.
1905discoveryoftheauscultatorysounds[115].Thismethod
usesabloodpressurecuffandstethoscope(ormorerecently
(iii)Subdural Bolt. A burr hole is drilled, and a hollow
amicrophone),whichareappliedonthearm(monkey),leg,
screwisinsertedthroughthedura,andpulsationsof
or tail (rodents) to register animal’s pulse tones. It allows
CSFinasubarachnoidspacearerecordedviaasensor.
for single, serial, or continuous measurements but usually
requiresanesthesia, which may influencethe results. More-
(iv)EpiduralSensor.Aburrholeisdrilled,andanepidural
over,ifthestethoscopeisused,resultscanbeinconsistentand
sensor is inserted between theskulland the dura to
operatordependent.However,themeasurementsofsystolic
registerduraltension(pulsations).
anddiastolicpressuresallowforaneasyandoftenautomated
assessmentofmeanarterialpressure.
Theaccuracyofmeasurementsbysubduralboltorepidural The oscillometric method is widely used for blood pres-
sensorislowerthanthosebyintraventricularcatheter.Addi- suremeasurementintheexperimentalsettings.Itmeasures
tionalcaveatsare(1)ICPisnotuniformlydistributedthrough oscillationscausedbybloodflow(i.e.,pulse)bymeansofa
thebrain,and(2)localpressuremeasurementsmadebyan pressurecuff.Thissimplemethoddoesnotrequireaskilled
8 StrokeResearchandTreatment
operator and hence can be automated for blood pressure aremadefromalimited(local)areaonlyandmaynot
recording.However,itdoeshaveseveral,above-mentioned, representthewholebrain(global)CBFchanges.
limitationsrelatedtotheuseofacuff.
(ii)Transcranial Doppler Method (TCD). This is a non-
invasive method that was introduced by Aaslid et
(2) Heart Rate Monitoring. Sympathetic stimulation during
al. in 1982 [122]. It measures blood flow velocity
Cushingreflexleadstoreductioninheartrate(bradycardia)
and not blood flow. The linear relationship between
and significant increase of BP. The following techniques
CBF and mean flow velocity under most of the
havebeenusedformonitoringheartrateandothercardiac
experimental and many clinical conditions allows
changesfollowingSAH.
for accurate assessment of CBF by TCD method
(i)ECG Monitoring. ECG changes are registered when andpermitsreal-timeCBFmeasurements[121–123].
theICPincreasestowardthesystolicarterialpressure. This method is easy to use, allows for continuous
data collection over a long period of time, can be
(ii)Transesophageal Echocardiography. Can be used in
used repeatedly, and allows comparison with other
largeanimalstoassesswallmotionchangesandaortic
experiments or data sets [113, 123]. The usefulness
andpulmonaryflowvelocitiesatSAH[116].
ofTCDforassessmentofCBFandarterialdiameter
(iii)Serum Markers of Myocardial Injury. An increased has been confirmed by numerous experimental and
serum creatine kinase-MB and cardiac troponin-1 clinical studies of SAH [121, 124–128]. In addition,
(cTn-1)concentrationisoftenusedtodiagnoseacute TCDassessesvascularresistanceandreactivityaswell
myocardialinjuryafterSAH.However,asCK-MBcan asstatusofautoregulationofCBF.Thisisofsignificant
bereleasedfromnon-cardiacmuscledamage,cTn-1is valuesinceCBFisconstantintheCPPrangeofabout
asuperiorindicatorofmyocardialinjury[117]. 50–150mmHg because of autoregulation, which is
frequently disturbed after aSAH. The limitations of
TCD include indirect CBF measurement and inter-
On the Model. In addition to the ICP measurements, BP
operatorvariability.
monitoringisarequiredfeatureofamodifiedaSAHanimal
model. The technique used for monitoring BP and cardiac (iii)Jugular Oximetry. As TCD, jugular oximetry does
changes in the new aSAH animal model will depend upon not measure CBF directly. Here, CBF is calcu-
nature of experiment and its requirements. If an animal latedfromarteriojugularoxygensaturationdifference
survivalisrequired,thennoninvasiveBPmonitoringshould (AJDO2).ThemeasurementsassessCBFinrelationto
be used. Similarly, if the effect of SAH on heart rate is of metabolic activity but are adequate only if coupling
concern,thenasimpleECGmonitoringwillworkfine. between CBF and metabolism is intact. Another
limitationisthatoximetryassessesoxygencontentin
ajugularbulbthatmaybetterrepresenthemispherical
6.2.3. CBFChangesandPossibilityofRepeatedArteriography
andnotglobalCBF.
or TCD for a Delayed Vasospasm Assessment. CBF mon-
itoring and vasospasm assessment provide useful tools to (iv)CerebralAngiography.Spasminlargecerebralarteries
examinepotentialtherapeuticoptions.Ananimalmodelpro- setsin3–7daysafterSAH.Angiographyisfrequently
vides these assessments and, in addition, can help establish used to assess the presence and severity of delayed
the influence of acute phase on the following subacute and arterialvasospasm.Thoughthistechniqueisinvasive
delayedphasesofbraininjuryafteraSAH. it can be used repeatedly to follow the development
and effects of pharmacological intervention on the
CBF Monitoring. CBF can be assessed quantitatively or
delayedvasospasm[129].
133
qualitatively. xenon method is a method for quantitative
(v)EEG Monitoring. EEG changes are registered when
assessment of CBF, which was described by Kety-Schmidt
the ICP surpassed the systolic arterial pressure and
[118]. CBF is calculated from data obtained from several
the electrical silence results of arrest of the cerebral
detectors placed on the head surface after administration
circulation.
of radioactive xenon gas. This method is widely used in
bothclinicalandexperimentalsettings.However,itmeasures
CBF mostly from cortical and subcortical structures of the
On the Model. CBF measurement is crucial for a modified
middle cerebral artery, and the measurements obtained are
aSAH model and should be performed using a technique
not reproducible by other CBF measurement methods. In
thatisreliable,simple,easy,noninvasive,andallowsrepeated
addition, this method is cumbersome, requires significant
measurements.TCDfulfillsthisselectioncriterion.
investment,knowledge,andexperience.
(i)Thermal Diffusion Method. This method estimates 6.3.OutcomeAssessment. Ananimaloutcomeisanessential
cortical or interstitial blood flow from the temper- endpoint of an aSAH study. It confirms the importance
ature difference between the two gold plates at the of a pathway being studied in aSAH induced injury and
tipoftheprobeplacedonorinthebrainthrougha helps decide whether modification of this pathway would
burrhole[119–121].Thismethodprovidescontinuous be beneficial. It is also essential that outcome assessments
quantitative real-time CBF. However, measurements studiedinanimalsarerelevanttothehumanconditionsothat
StrokeResearchandTreatment 9
treatmentsfoundeffectiveinanimalscanbetranslatedtothe (4)Additional attributes are adaptable to other species
patients[99]. (range from rodents to primates) with little modifi-
In aSAH patient neurological and functional deficits cationandlowmortalityandmorbidity.
developearlyand/orafterseveraldays.Inpatients,statusat
admission and early deficits are assessed by the Hunt and
8.Summary
Hess, the Glasgow coma (GCS), and the World Federation
ofNeurologicalSurgeons(WFNS)gradingscales[130].The Inadequate disease modeling may have contributed to the
long-termoutcomeinSAHpatientsisassessedbyKatzman, failureofimprovingoutcomeinaSAHpatients.Wepresented
Rankin, and/or Barthel scores. In animals, neurological hereaproposalofamodifiedmodelofaSAHthatincorpo-
injury is studied indirectly as diminished response to an ratesallofthecomponentsandelementsofinjuryafteraSAH,
external stimulus or reduced function or directly as death whichmayprovideabetterresourceforstudyingtheinjury
of brain cells by immunostaining or assays for apoptosis, mechanismsanddevelopingatreatment.
autophagy, or neurodegeneration. The methods used for
assessingneurologicalandfunctionaldeficitsinSAHanimals
Acknowledgment
are less than perfect and often erroneously incorporate
procedures intended for assessing focal ischemic injury.
ThisworkwassupportedbytheAmericanHeartOrganiza-
Furthermore, though a battery of exams for a number of
tionGrant[10GRNT4570012(FAS)],theNationalInstitutes
speciesexists,aspecies-specificlimitationforassessmentsis
ofHealthNationalCenterforResearchResources[NS078369
often not recognized (for review see [131, 132]). The review
(FAS)], and Intramural Research Program (RMP) of the
by Jeon and colleagues provides an excellent guide to the
NationalInstitutesofStrokeandNeurologicalDisorders.
techniques used to assess outcome in rodents after aSAH
[99].
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