Glucagon-like peptide-1 receptor activation reduces ischaemic brain damage following stroke in Type 2 diabetic rats.

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by  Darsalia, Vladimer

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ClinicalScience(2012)122,473–483(PrintedinGreatBritain) doi:10.1042/CS20110374 473
Glucagon-like peptide-1 receptor activation
reduces ischaemic brain damage following
stroke in Type 2 diabetic rats
Vladimer DARSALIA∗, Shiva MANSOURI∗, Henrik ORTSA¨TER∗, Anna OLVERLING∗,
Nino NOZADZE∗, Camilla KAPPE∗, Kerstin IVERFELDT†, Linda M. TRACY†,
Nina GRANKVIST∗, A˚ke SJO¨HOLM∗ and Cesare PATRONE∗
∗
Diabetes Research Unit, Department of Clinical Science and Education, So¨dersjukhuset, Karolinska Institutet, Stockholm,
e
Sweden, and †Department of Neurochemistry, Stockholm University, Stockholm, Sweden
c
n
A B S T R A C T e
i
c
Diabetes is a strong risk factor for premature and severe stroke. The GLP-1R (glucagon-like
S
peptide-1receptor)agonistEx-4(exendin-4)isadrugforthetreatmentofT2D(Type2diabetes)
that may also have neuroprotective effects. The aim of the present study was to determine the
l
efficacyofEx-4againststrokeindiabetesbyusingadiabeticanimalmodel,adrugadministration a
paradigmandadosethatmimicsadiabeticpatientonEx-4therapy.Furthermore,weinvestigated
c
inflammation and neurogenesis as potential cellular mechanisms underlying the Ex-4 efficacy. A
i
total of seven 9-month-old Type 2 diabetic Goto–Kakizaki rats were treated peripherally for n
4weeks with Ex-4 at 0.1, 1 or 5μg/kg of body weight before inducing stroke by transient
i
middlecerebralarteryocclusionandfor2–4weeksthereafter.Theseverityofischaemicdamage l
C
was measured by evaluation of stroke volume and by stereological counting of neurons in the
striatum and cortex. We also quantitatively evaluated stroke-induced inflammation, stem cell
proliferationandneurogenesis.Weshowaprofoundanti-strokeefficacyoftheclinicaldoseofEx-
4indiabeticrats,anarrestedmicrogliainfiltrationandanincreaseofstroke-inducedneuralstem
cell proliferation and neuroblast formation, while stroke-induced neurogenesis was not affected
byEx-4.Theresultsshowapronouncedanti-stroke,neuroprotectiveandanti-inflammatoryeffect
of peripheral and chronic Ex-4 treatment in middle-aged diabetic animals in a preclinical setting
thathasthepotentialtomimictheclinicaltreatment.Ourresultsshouldprovidestrongimpetus
tofurtherinvestigateGLP-1Ragonistsfortheirneuroprotectiveactionindiabetes,andfortheir
possibleuseasanti-strokemedicationinnon-diabeticconditions.
INTRODUCTION is one such comorbid disease where premature stroke
represents one of the most common and serious long-
Stroke is the primary cause of disability in adults and termcomplications[3–6].Theriskofstrokeisincreased
the third most common cause of death. Stroke is often 2-to6-foldinpatientswithT2D[6]withgreaterriskfor
associatedwithcomorbidhealthconditionsthatincrease strokerecurrenceandhighermortalityrate[6,7].Finally,
theriskandseverityofstroke[1,2].T2D(Type2diabetes) a pre-diabetic state with impaired glucose tolerance
Keywords:exendin-4(Ex-4),Goto–Kakizaki(GK)rat,middlecerebralarteryocclusion(MCAO),neurogenesis,neuroprotection.
(cid:3)
Abbreviations: BrdU, bromodeoxyuridine; bw, body weight; CNS, central nervous system; DAPI, 4,6-diamidino-2-
phenylindole; DCX, doublecortin; Ex-4, exendin-4; GK, Goto–Kakizaki; GLP-1R, glucagon-like peptide-1 receptor; IHC,
immunohistochemistry;MCA,middlecerebralartery;MCAO,MCAocclusion;SVZ,subventricularzone;T2D,Type2diabetes.
Correspondence:ProfessorA˚keSjo¨holm([email protected])orDrCesarePatrone([email protected]).
© 2012 The Author(s)
The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attributi(cid:4)oCn NThoen-ACuotmhomrserJcoiuarln Lailcecnocmep (ilhatttipo:n//c(cid:4)rCeat2iv0e1c2omBimocohnesm.oicragl/liScoecniesteys/by-nc/2.5/)
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474 V.Darsaliaandothers
is often detected in stroke patients following hospital
admission, and such patients generally exhibit a poor
prognosis[8,9].
GLP-1R (glucagon-like peptide-1 receptor) agonists
are novel treatments against T2D [10], which may
also have direct neuroprotective effects besides their
gluco-regulatory action. These molecules interact with
the G-protein-coupled GLP-1R and enhance glucose- Figure1 Experimental design and drug administration
dependentinsulinsecretion[11].Exenatide[syntheticEx- paradigm
4(exendin-4)]isastableGLP-1analogueisolatedfrom
thesalivaofthe‘Gilamonster’lizard[12].Itisresistant
todegradationandisapprovedinbothEuropeandthe
U.S.A.fortheclinicaltreatmentofT2D[11]. Animals and experimental groups
GLP-1R is expressed throughout the brain [13,14] Atotalof427–9-month-oldmalediabeticGKratswere
and GLP-1 and Ex-4 can cross the blood–brain barrier used. All experiments were conducted according to the
[15]. GLP-1R agonists have been reported to be ‘Guide for the Care and Use of Laboratory Animals’
neuroprotective [16,17], neurogenic [18,19], enhance published by U.S. National Institutes of Health (NIH
synaptic plasticity [20] and improve different forms of publicationno.85-23,revised1985)andapprovedbythe
learning [16,19]. Furthermore, anti-inflammatory CNS regionalethicscommitteeforanimalexperimentation.
(central nervous system) effects of GLP-1 have been BeforethestartoftheEx-4/PBStreatments,baseline
reported[21–23].Intracerebroventricularadministration fastingbloodglucoseconcentrationsweremeasuredand
ofEx-415minbeforestrokereducedischaemicdamage the animals were assigned to the different treatment
in mice [24]. Intraperitoneal injection of Ex-4 2h groups so that mean blood glucose values were
before ischaemia and 1h after reperfusion protected equalized. Treatment groups thus created were tested
hippocampalCA1neuronsinaglobalischaemiamodel for normality using the D’Agostino and Pearson
ingerbils[25].Finally,acuteintravenousadministration omnibusnormalitytest.Allratsreceivedintraperitoneal
ofEx-4followingischaemiareducedtheinfarctvolume. injections of Ex-4 [0.1, 1 or 5μg/kg of bw (body
However,theneuroprotectiveeffectwaslostifEx-4was weight) (n=10)] or PBS (n=12) for 4weeks twice
administered 3h after stroke [26]. These studies were daily before being subjected to stroke. Ex-4 and PBS
performedinyounghealthynon-diabeticmice. injections were continued for another 2 or 4weeks
AlthoughtheeffectsofGLP-1Ractivationinthebrain following the stroke. To assess neurogenesis, all rats
are promising, more research is needed to understand receiveddailyintraperitonealinjectionsofthethymidine
themechanismsbehinditsactionandwhetherGLP-1R analogue BrdU (bromodeoxyuridine; 50mg/kg of bw)
activationiseffectiveinthediabeticbrain. for 2weeks following stroke. Rats were killed for
Inthepresentstudy,wetestedtheanti-strokeefficacy immunohistochemical analyses at 2 or 4weeks after
of Ex-4 in diabetes by using middle-aged diabetic GK stroke.TheexperimentaldesignisillustratedinFigure1.
(Goto–Kakizaki)rats.TheGKratisanon-obeseWistar
substrain,whichspontaneouslydevelopsT2Dearlyinlife
[27] and where Ex-4 treatment reduces hyperglycaemia
MCAO [MCA (middle cerebral artery)
[28].
occlusion]
MCAOisastrokeanimalmodelthatcloselyresembles
MATERIALS AND METHODS the most common form of human stroke [29]. Rats
were anaesthetized by spontaneous inhalation of 1.5%
Ex-4 bioactivity assay isofluorane through a snout-mask. Body temperature
ToascertainthebioactivityofEx-4(kindlyprovidedby was maintained at 37–38◦Cusing a heating pad. Stroke
Amylin), insulin secretion was measured from islets of was induced by the intraluminal filament technique
Langerhansisolatedfromnon-diabeticmaleWistarrats. [29].Briefly,commonandexternalcarotidarterieswere
Batchesof15isletspergroupweremaintainedfor30min ligated, and the internal carotid artery was temporarily
at 37◦C in a buffer consisting of 3 or 20mM glucose, closed. A monofilament was advanced through the in-
125mM NaCl, 5.9mM KCl, 1.2mM MgCl , 1.3mM ternalcarotidarterytotheoriginoftheMCA,thewound
2
CaCl and25mMHepes,titratedtopH7.4withNaOH wasclosedandtheanimalwasallowedtowakeupandwas
2
andsupplementedwith1mg/mlBSA(fractionV;Roche placedinitscage.After90minofocclusion,theanimals
Diagnostics)with/without10nMEx-4.Afterincubation, wereanaesthetizedagainandthefilamentwaswithdrawn.
aliquots of buffer were frozen pending analyses of Thesurgeonperformingtheoperationwasblindedtothe
releasedinsulinbyELISA(Mercodia). treatmentgroups.
© 2012 The Author(s)
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GLP-1Rforthetreatmentofstrokeindiabetes 475
Immunocytochemistry computermonitorandthestriatumandcortexdelineated
Animals were deeply anaesthetized and perfused at low magnification. Quantifications were performed
transcardially with 4% (w/v) paraformaldehyde. The usinga×100oil-immersionlenswithnumericaperture
brains were extracted and submersed in 20% sucrose of 1.30 or a ×40 dry lens with numeric aperture of
in phosphate buffer overnight. Then 40-μm-thick 0.75. Ten evenly spaced sections in parallel-cut series
coronal sections were cut using sliding microtome and through the entire striatum were included. Random
stained as free-floating sections. The following primary sampling was carried out using the counting frame,
antibodies were used: mouse anti-Ki67 (1:200 dilution; whichsystematicallywasmovedatpredefinedintervals
Novocastra), a marker of cell proliferation; goat anti- so that ∼200 immunoreactive cells were counted. The
DCX(doublecortin)(1:400dilution;SantaCruzBiotech- total number of cells was estimated according to the
nology),amarkerformigratingneuroblasts;mouseanti- opticalfractionatorformula[31,32].
NeuN (1:100 dilution; Millipore), a neuronal marker; For evaluation of SVZ (subventricular zone) cell
rabbit anti-Iba1 (1:1000 dilution, Wako Chemicals), a proliferation, SVZ cells immunoreactive for Ki67 were
marker for microglia; mouse anti-ED1 (1:200 dilution; counted. DCX is a marker for newly produced
Serotec),amarkerofactivatedmicroglia;ratanti-BrdU neuroblasts from neurogenic brain areas and DCX-
(1:200 dilution; Santa Cruz Biotechnology), to assess positive cells have been shown to migrate into the
neurogenesisincombinationwithNeuN;andrabbitanti- ischaemicstriatum[33].Thereforeneuroblastproduction
GLP-1R(1:50dilution;Abcam).Inthesectionsstained was quantified by counting cells positive for DCX
forGLP-1R,DAPI(4(cid:3),6-diamidino-2-phenylindole)was in stroke-damaged striatum. NeuN is a ubiquitous
usedtovisualizeallofthecells.Sectionswereincubated nuclearmarkerformatureneurons.Thus,neurogenesis
withprimaryantibodiesovernightat4◦Cinphosphate was assessed by counting NeuN/BrdU-positive cells
buffer containing 3% appropriate serum and 0.25% instroke-damagedstriatum.Thesequantificationswere
Triton X-100. Primary antibodies were detected by use performedinthreeevenlyspaced(400μM)brainsections
ofappropriatefluorescentCy3(indocarbocyanine)-,Cy2 onthesideipsilateraltostroke,startingat0.7mmanterior
(carbocyanine)- (both from Jackson ImmunoResearch) toBregma.
or biotin-conjugated (Vector) secondary antibodies
(1:200dilution).Sectionswereincubatedwithsecondary Statistics
antibodies for 2h at room temperature (approx. 21◦C) Statistical analyses were performed using one-way
in phosphate buffer containing 3% of the appropriate or two-way ANOVA, followed by Bonferroni’s or
serum and 0.25% Triton X-100. For chromogenic Dunnet’spost-hoctest.Differencesbetweengroupswere
visualization,biotinylatedsecondaryantibodies,avidin– consideredstatisticallysignificantwhenP<0.05.Results
+
boitincomplex(ABCkit;Vector)anddiaminobenzidine arepresentedasmeans−S.E.M.
wereused.
RESULTS
Tissue damage evaluation and cell
quantification Ex-4 reduces hyperglycaemia in GK rats
Tissue damage evaluation and cell counting were The bioactivity of Ex-4 was shown as its efficacy
performedbyaninvestigatorblindedtotheexperimental to augment several fold the glucose-induced insulin
groups.Fortissuedamageevaluation,theNeuN-labelled secretioninisletsofLangerhansisolatedfromadultnon-
tissue sections were displayed live on the computer diabetic rats (results not shown). Treatment with Ex-4
monitor and the area of contralateral hemisphere and slightlyreducedglycaemiainGKrats,althoughthiseffect
theareaoftheintactipsilateraltissueweremeasuredin didnotattainstatisticalsignificanceinthegroupstreated
everysectioncontainingstrokedamageusingNewCast with0.1and1μg/kgofbw.Onlythegrouptreatedwith
(Visiopharm) software. To compensate for the stroke- Ex-4 at 5μg/kg of bw showed a statistically significant
inducedmorphologicaltissuechanges,theinfarctvolume reductioninglycaemiaafter2and4weeksoftreatment
was calculated by subtracting the volume of remaining (Figures 2A–2D). Animals were fasted for 3h before
tissue in the ipsilateral hemisphere from the volume of bloodglucosemeasurementswereperformed.
thecontralateralhemisphere.
GLP-1R is expressed by neurons in the
Immunoreactive cells were counted using either a
computerized non-biased setup for stereology, driven brain of diabetic rats
by NewCast software, or by using an Olympus To assess GLP-1R expression in the brain, we
BX40epifluorescence/lightmicroscope.Thenumberof immunostained brain sections for GLP-1R. The results
neurons (NeuN) and microglia (Iba1 and ED1) cells showed a widely dispersed expression of GLP-1R
was quantified using the optical fractionator method throughout the brain which co-localized with NeuN-
[31,32].Briefly,brainsectionsweredisplayedliveonthe positive-neurons(Figure3).
© 2012 The Author(s)
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476 V.Darsaliaandothers
points were pooled. Results in Figure 4(E) show that,
followingastrokeinducedbyMCAO,theleastamount
of neurons remained in the stroke-damaged striatum
and cortex of the PBS-treated group. Furthermore, in
the two groups of animals treated with Ex-4 at 0.1 and
1μg/kg of bw, the activation of GLP-1R resulted in
a statistically significant effect above control with an
increased number of surviving neurons in the stroke-
damagedstriatumandcortex(Figure4E).Treatmentwith
Ex-4 at 5μg/kg of bw further increased the number of
Figure2 Ex-4effectsonglycaemiainGKrats survivingneuronscomparedwithbothPBSandthetwo
(A–D)FastedbloodglucoselevelsinGKratstreatedwithPBS(n=12),orEX-4
lowerdosesofEx-4(Figure4E).Whenneuronalsurvival
at 0.1 (n=10), 1 (n=10) or 5μg/kg of bw (n=10) for 4weeks before
was evaluated separately in the striatum and cortex,
inducing a stroke. Bars represent means+−S.E.M. ∗P<0.05 and ∗∗P<0.01 the neuronal quantifications showed that a statistically
compared with week 0 using one-way ANOVA, followed by Bonferroni’s post-hoc
significant neuroprotective effect in striatum was only
analysis. achieved with Ex-4 at 5μg/kg of bw, although a dose-
dependenttrendwasalsonoticeablewiththetwolower
Ex-4doses(Figure4F).Thisneuroprotectiveeffectwas
Ex-4 exhibits dose-dependent anti-stroke muchstrongerinthecortex,whereEx-4treatmentat0.1
efficacy and1μg/kgofbwalsoresultedinstatisticallysignificant
neuroprotection(Figure4G).
In order to determine the potential anti-stroke efficacy
mediated by Ex-4, the measurement of infarct volume
Ex-4 treatment significantly reduces
was assessed at both 2 and 4weeks after stroke. Since
microglia infiltration after stroke, but has
nodifferencewasrecordedbetween2and4weeksafter
strokebetweenthetreatmentgroups,resultsfrom2-and a marginal effect on microglia activation
4-week time points were pooled. The measurement of To evaluate the magnitude of microglia infiltration,
theinfarctvolumerevealed thattreatmentwithEx-4at reflecting inflammatory levels, Iba1-immunoreactive
5μg/kgofbwresultedinasignificantreductionoftissue cellswerecountedusingtheopticalfractionatormethod
damageafterstroke(Figures4A,4C,and4D).Although (see the Materials and methods section). At 2weeks
notstatisticallysignificant,thetreatmentwithEx-4at0.1 after stroke, the number of Iba1-positive microglial
and1μg/kgofbwshowedanoticeabletrendtowardsa cellswithinthestroke-damagedhemisphere(cortexplus
reductionofischaemictissuedamage(Figure4D). striatum;Figure5A)andincortexandstriatumseparately
In order to assess the neuroprotective effect of (Figures 5C and 5E) was similar in the PBS-treated
Ex-4 with greater accuracy than infarct volume and Ex-4-treated groups. However, at 4weeks after
measurement, NeuN-positive neurons were quantified stroke, the number of Iba1-positive cells in stroke-
in both stroke-damaged striatum and cortex using damagedhemispherewassignificantlydifferentbetween
the optical fractionator method (see the Materials and the groups treated with Ex-4 and PBS (Figure 5B).
methods section). As for the volume measurements, When we evaluated microglia infiltration at 4weeks in
no difference was recorded in the number of surviving the ipsilateral striatum and cortex separately, it became
neuronsbetween2and4weeksafterstrokebetweenthe apparent that the Ex-4 treatment had no effect on
treatmentgroups.Thusresultsfrom2-and4-weektime microgliainfiltrationinthestriatum(Figure5D),whilea
Figure3 GLP-1RexpressioninthecortexoftheGKratbrain
(A) DAPI staining (general cell marker); (B) neurons positive for NeuN (neuro-specific marker); (C) GLP-1R-positive cells; (D) merged channels for NeuN and GLP-1R.
ArrowheadsindicatecellspositiveforDAPI,NeuNandGLP-1Rrespectively.Imagesweretakenusinga×40drylenswith0.75numericapertureandColorViewIIFW
digitalcameradrivenbytheCellA imagingsoftware.Brightness/contrastwasadjustedandfinalimagesresizedusingAdobePhotoshopsoftware.(D)Scalebar,50μm.
© 2012 The Author(s)
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GLP-1Rforthetreatmentofstrokeindiabetes 477
Figure4 NeuroprotectiveeffectofEx-4
(A–C)PhotomicrographsofrepresentativeischaemicdamageaftertreatmentwithPBS,orEx-4at0.1or5μg/kgofbw.Dottedlinesoutlinetheischaemicdamage.
(C)Scalebar,2mm.(D)Volumeoftheinfarct.(E–G)StereologicalquantificationofNeuN-positivecellsinstroke-damagedstriatumandcortex.Groups,PBS(n=12),
or EX-4 at 0.1 (n=10), 1 (n=10) or 5 (n=10)μg/kg of bw. Values are means+−S.E.M. ∗P<0.05 and ∗∗P<0.01 between groups using one-way ANOVA,
followedbyBonferroni’spost-hoctest.
statisticallysignificanteffectwasrecordedinthecortex Ex-4 treatment increases stem cell
between each of the Ex-4 treated groups and PBS proliferation and neuroblast formation
(Figure5F). without affecting stroke-induced
Toassessmicrogliaactivation,ED1-positivecellswere
neurogenesis
quantified in a similar manner by using the optical
fractionator method. As for Iba1, at 2weeks after The SVZ of the lateral ventricle and the hippocampal
stroke,thenumberofED1-positivemicroglialcellswas dentate gyrus are the two brain areas in which adult
similar in all experimental groups, both in striatum neurogenesis occurs [34]. To study the effect of Ex-4
and cortex separately or pooled together (Figures 5I, treatment on stroke-induced neurogenesis, all animals
5K and 5M). At 4weeks after stroke, the number of were given daily injections of the thymidine analogue
ED1-positive cells also did not decrease significantly BrdU for 2weeks following MCAO. Brains were
(Figures5J,5Land5N).However,astrongtrendtowards analysed2or4weeksafterstroke(Figure1).
a reduction of the number of ED1-positive microglial Ki67-expressing cells were counted to evaluate
cells in the group treated with Ex-4 at 5μg/kg of stem/progenitor cell proliferation in the SVZ of the
bw was observed in striatum and cortex both pooled brain. The results show that, at 2weeks after stroke,
together or when analysed separately (Figures 5J, 5L Ex-4-treated animals showed 2-fold greater number of
and5N). proliferating cells in the SVZ as compared with the
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478 V.Darsaliaandothers
Figure5 MagnitudeofbraininflammationfollowingEx-4treatmentat2and4weeksafterstroke
(A, B) Stereological quantification of Iba-positive microglial cells in the stroke-damaged hemisphere at 2 (A) and 4 (B) weeks after MCAO. (C, D) Stereological
quantificationofIba1-positivemicroglialcellsin thestroke-damagedstriatumat2(C)and4(D)weeksafterstroke.(E,F)StereologicalquantificationofIba-positive
microglialcellsinthestroke-damagedcortexat2(E)and4(F)weeksafterMCAO.(G,H)RepresentativeimagesofIba1immunoreactivityinthegroupsreceivingPBS
(G)orEx-4at5μg/kgofbw(H)at4weeksafterstroke.(I,J)StereologicalquantificationofED1-positivemicroglialcellsinthestroke-damagedhemisphereat2(I)
and 4 (J) weeks after stroke. (K, L) Stereological quantification of ED1-positive microglial cells in the stroke-damaged striatum at 2 (K) and 4 (L) weeks after MCAO.
(M, N) Stereological quantification of ED1-positive microglial cells in the stroke-damaged cortex at 2 (M) and 4 (N) weeks after stroke. (O, P) Representative images
of ED1 staining in the group treated with PBS (O) and Ex-4 at 5μg/kg of bw (P) at 4weeks after stroke. (P) Scale bar, 2mm. Groups at 2weeks, PBS (n=6)
or Ex-4 at 0.1 (n=5), 1 (n=5) and 5 (n=5)μg/kg of bw. Groups at 4weeks, PBS (n=6) or Ex-4 at 0.1 (n=5), 1 (n=5) and 5 (n=5)μg/kg of
bw.Valuesaremeans+−S.E.M.∗P<0.05betweengroupsusingone-wayANOVA,followedbyDunnet’spost-hoctest.
PBS group (Figures 6A, 6C and 6E), with no statistical led to a 1.5-fold increase in neuroblast production
differencesbetweenthedifferentEx-4doses(Figure6A). compared with the PBS-treated group (Figures 6B, 6G
However, 4weeks after stroke, no difference in the and6I).However,at4weeksafterstroke,nodifference
number of Ki67-positive cells between PBS- and Ex-4- inneuroblastproductionbetweenPBS-andEx-4-treated
treatedgroupswasrecorded(Figures6A,6Dand6F). animalswasrecorded(Figures6B,6Hand6J).
Stroke-induced neuroblast production was evaluated DoublelabellingfortheneuronalmarkerNeuNand
byquantifyingthenumberofDCX-positivecellsinthe the proliferation marker BrdU was used to identify
striatum. The DCX-positive neuroblast quantification mature neurons that were generated after stroke in
revealed that, at 2weeks after stroke, Ex-4 treatment striatum. At 2weeks after stroke, the number of
© 2012 The Author(s)
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GLP-1Rforthetreatmentofstrokeindiabetes 479
Figure6 Stroke-inducedneuralstem/progenitorcellproliferationandneurogenesisinEx-4-treatedGKratSVZandstriatum
(A) Number of Ki67-positive cells in SVZ ipsilateral to stroke at 2 and 4weeks after stroke. (B) Number of DCX+ neuroblasts at 2 and 4weeks after stroke in the
stroke-damagedstriatum.Whiteandblackbarsrepresentanimalskilledat2and4weeksrespectively.(C,D)RepresentativeimagesofKi67immunoreactivityin the
ipsilateral SVZ of PBS-treated rats at 2 and 4weeks after stroke respectively. (E, F) Representative images of Ki67 immunoreactivity in the ipsilateral SVZ of rats
treatedwithEx-4at5μg/kgofbwat2and4weeksafterstrokerespectively.(F)Scalebar,200μm.(G,H)RepresentativeimagesofDCXimmunoreactivityinthe
ipsilateral striatum of PBS-treated rats at 2 and 4weeks after stroke respectively. (I, J) Representative images of DCX immunoreactivity in the ipsilateral striatum of
ratstreatedwithEx-4at5μg/kgofbwat2and4weeksafterstrokerespectively.(J)Scalebar,100μm.(K)NumberofNeuN/BrdU+ neuronsinstroke-damaged
striatumat2(whitebars)and4(blackbars)weeksafterstroke.(L–N)PhotomicrographsofaNeuN/BrdU+ neuroninstroke-damagedstriatum.(L)NeuN,(M)BrdU
and(N)mergedchannelsshowingdoublestaining.(N)Scalebar,20μm.Groupsat2weeks,PBS(n=6)orEx-4at0.1(n=5),1(n=5)and5(n=5)μg/kg
of bw. Groups at 4weeks, PBS (n=6) or Ex-4 at 0.1 (n=5), 1 (n=5) and 5 (n=5)μg/kg of bw. Values are means+−S.E.M. ∗P<0.05 between groups
usingtwo-wayANOVA,followedbyBonferroni’spost-hoctest.w,weeks.
NeuN/BrdU-positive neurons was similar in both DISCUSSION
PBS- and Ex-4-treated groups (Figure 6K). At 4weeks
after stroke, the number of NeuN/BrdU-positive In the present study, we show the anti-stroke efficacy
neurons increased 2-fold in all groups without any of Ex-4 in a preclinical setting that mimics the clinical
differencesbetweenthePBS-andtheEx-4-treatedgroups situation. Although successful in animal models, anti-
(Figure6K). stroke treatment strategies based on early intervention
Similar quantifications were performed in the with neuroprotective drugs have not yet proven their
subgranular zone and granule cell layer of the dentate efficacy in the clinic [35,36]. On the other hand,
gyrus of the hippocampus. In this case, Ex-4 treatment therapeutic strategies during the later and chronic
had no detectable effect on either cell proliferation or phase of stroke have been largely unsuccessful both in
neurogenesis(resultsnotshown). preclinical experiments and clinical trials [36–38]. This
© 2012 The Author(s)
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480 V.Darsaliaandothers
failureislikelyduetotwomainfactors.First,principal neuronaldensityand‘patchy’celllossintheseanimalsas
differences between the research animal models and opposedtothemoreseverecelllossinthecontrolgroup.
typical stroke patients. The majority of stroke patients ThequantificationofsurvivingNeuN-positiveneurons
are elderly [39], where comorbidities such as diabetes usingtheopticalfractionatormethodrevealedthatwhen
and hypertension are common [2,35]. By contrast, in countsfromstriatumandcortexwerecombined,thetwo
most of the preclinical experiments the efficacy of lowerdosesofEx-4significantlyincreasedtheneuronal
drugs is tested on animals that are both young and survival(indicatedbyincreasedneuronalcounts).Since
healthy. It has been calculated that to date, for six infarct volume measurement does not account for
drugsreviewedsystematically,only10%ofpublications neuronal loss in tissue surrounding the infarct, we
which studied the efficacy of these drugs had used suggest that this type of assessment is an accurate
animals with hypertension or diabetes, and none of and precise addition for evaluating stroke damage and
the experiments reported efficacy in aged animals [35]. neuronalsurvival.Unlikethemeasurementsoftheinfarct
Secondly, preclinical drugadministrationparadigms are volume, which dramatically changes depending on the
oftennotrelevantfortheclinicalsituation.Forexample, time after stroke and may lead to misinterpretation
in many cases the preclinical efficacious doses of these oftheresults[42,43],thestereologicalquantificationof
drugs vastly (several orders of magnitude) differed survivingneuronstheusingopticalfractionatormethod
from doses used in clinical practice [40]. Therefore, the providesaccurateinformationandhelpsinavoidingsuch
successful implementation of preclinical achievements pitfalls.
is largely dependent on how closely the experimental TheEx-4-mediatedefficacywasdose-dependent,with
paradigmsmatchthelikelyclinicalscenario. 5μg/kgofbwbeingthemosteffectivedoseinthestudy.
In the present study, we have evaluated the anti- Ex-4hasbeenreportedtocrosstheblood–brainbarrier
stroke efficacy of the GLP-1R analogue Ex-4. The innormalmice[15]andGLP-1RisexpressedonNeuN-
neuroprotectiveeffectofEx-4administeredimmediately positiveneurons.
or within an hour after ischaemia in non-diabetic adult Althoughtheanti-strokeeffectmediatedbyEx-4was
micehasbeenpreviouslydemonstrated[25,26].However, foundtobedose-dependent,itwasnotlinearbetweenthe
theneuroprotectiveeffectwas notseenwhenEx-4was threeadministereddoses.Onepossibilitytoexplainthis
administered after a 3h delay [26]. These results are non-linearityisthatEx-4atthetwolowerdosesmayact
clearly interesting and demonstrate GLP-1R-mediated viatheknownGLP-1R,whiletheneuroprotectiveeffect
acute neuroprotection. However, their applicability to mediatedbythehighestdoseofEx-4isalsooccurringvia
the clinical setting is remote due to the relatively short aGLP-1R-independentpathwayrecentlydescribed[44].
effective intervention time-window (up to 1h) and the Linearitymayalsonotbeexpectedconsideringthatthe
Ex-4 doses employed that were up to 100times higher incretineffectbyEx-4occursontopofalreadyexisting
(10μg/kgofbw)whencomparedwiththeclinicaldose GLP-1 levels in the blood. Finally, although Ex-4 can
[26]. pass the blood–brain barrier [15], it is hard to predict
To closely simulate the way Ex-4 is used in clinical whatamountofEx-4canreachbothstriatumandcortex.
practice,wehaveusedmiddle-agedType2diabeticGK The neuroprotective effect of Ex-4 was stronger in
rats, among which one group was treated with Ex-4 the cortex than in the striatum. To investigate whether
at 0.1μg/kg of bw, which corresponds to the clinical thiseffectisduetodifferencesinGLP-1Rexpressionin
dose that is used in Type 2 diabetic patients. Since Ex- cortexandstriatum,quantitativestudieswillhavetobe
4 is a marketed drug for the treatment of T2D, our performedatearliertimepointsthantheonesusedinour
rationale was to mimic the clinical setting of Type 2 studythusbettertargetingtheneuroprotectivewindow
diabeticpatientsonEx-4treatment,wheretheperipheral (hourstofewdaysafterMCAO).Whilethepossibility
and chronic administration of Ex-4 will likely occur that the cortical and striatal neurons react differently
bothbeforeandafterstroke.Therefore,wehavetreated to Ex-4 treatment cannot be ruled out, this difference
GKratsinasimilarmannerandadministeredclinically is most likely due to the characteristics of the stroke
relevantdosesofEx-4forseveralweeksbothbeforeand modelthatwehaveemployed.Inthismodel,ischaemic
afterstroke.Wedemonstratehere,forthefirsttime,anti- damageoriginatesinthestriatumandthenspreadsacross
strokeefficacyofEx-4inapreclinicalsettingthatmimics the overlaying cortex depending on the duration of the
theclinicalsituation. MCAO. Thus, as cortex contains more ischaemic
Whenweevaluatedthetissuedamagebymeasuringthe penumbralarea,theneuroprotectiveinterventioncanbe
infarctvolume,onlythehighestdoseofEx-4(5μg/kgof moreeffective.
bw)showedastatisticallysignificanteffectincomparison Ex-4stimulatesinsulinsecretion[45]andanumberof
withthecontrolgroup,whereasthetwolowerEx-4doses previousstudieshavereporteddecreasedischaemicbrain
(0.1 and 1μg/kg of bw) produced a noticeable but not injury when hyperglycaemic levels of diabetic animals
statistically significant trend towards reduction of the were normalized using insulin [46,47]. In our study,
infarctvolume.Closerexaminationrevealed‘thinning’of however,thetreatmentwithEx-4at0.1and1μg/kgof
© 2012 The Author(s)
The author(s) has (cid:4)pCaidT hfoer Athuitsh aorrtsicJleou tron bale cfroemeplyil aatviaonila(cid:4)bCle u2n0d1e2r Bthioec theermmisca olf Stohcei eCtyreative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/)
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GLP-1Rforthetreatmentofstrokeindiabetes 481
bwsignificantlyreducedstrokeinjurywithoutshowing work has shown that Ex-4 enhances neurogenesis in
asignificantimpactonhyperglycaemia(Figures2Band non-diabetic rodents [18,19], in this work we studied
2C). Thus we suggest that, in our case, the reduction whether Ex-4 had the potential to enhance stroke-
in neuronal damage mediated by Ex-4 at 0.1–1μg/kg induced neurogenesis and/or to promote survival of
ofbwislargelyindependentofglycaemiceffects.Only the newly generated neurons in the diabetic brain.
the treatment with Ex-4 at 5μg/kg of bw resulted in a Our results show that Ex-4 treatment significantly
significantreductioninglycaemia(Figure2D),although increased stem/progenitor cell proliferation in the SVZ
theseanimalsstillremainedhyperglycaemic.Thisdoseof andneuroblastproductioninthestriatum.However,this
Ex-4wasalsothemosteffectiveinreducingtheischaemic effectwasonlynoticeableat2weeksafterstroke,whereas
brain injury, and therefore a partial contribution of the no differences between the groups were observed after
Ex-4-mediated glucose lowering to the reduction of 4weeksofEx-4treatment.Thekineticsofstroke-induced
the ischaemic damage cannot be ruled out. To entirely neuralstem/progenitorcellproliferationinGKratshave
excludethepossibilitythatEx-4inducesneuroprotection not been yet characterized. However, it is conceivable
by lowering glucose, future studies will be done using that at this later time point, any proliferative effect of
blood glucose-lowering drugs, titrated to achieve the Ex-4treatmentmaybemaskedbytheproliferativeeffect
same glycaemic effects as Ex-4, in head-to-head com- inducedbystroke.
parisons. Interestingly, and in support of our findings, Although at 2weeks after stroke, Ex-4 treatment
Teramoto et al. [26] recently reported that the anti- increased stem cell and neuroblast proliferation, this
stroke efficacy by GLP-1R activation in non-diabetic treatmentdidnotresultinanyincreaseinthegeneration
miceoccurredwithunchangedinsulinlevels,suggesting of mature neurons. Both at 2 and 4weeks after stroke
thatitsCNSeffectsarenotduetochangesinglycaemia. all groups showed similar numbers of NeuN/BrdU-
At 2weeks after stroke, all groups showed similar positiveneurons,althoughthenumberofNeuN/BrdU-
numbers of Iba-positive microglial cells in the injured positiveneuronsat4weekswas2-foldhighercompared
brainhemisphere.However,at4weeksfollowingstroke, with 2weeks in both PBS and Ex-4 treatment groups.
significantly fewer Iba1-positive microglial cells were This is most likely due to neuroblast differentiation
detected in the cerebral cortex of Ex-4-treated groups. [33]. Thus, although a greater number of proliferating
Theinflammationprocessinthebrainafterstrokemay cells and neuroblasts at 2weeks after stroke in the
last for several weeks and is believed to have both Ex-4 treated groups did not result in an increase in
beneficial and detrimental effects that largely depend neuronal differentiation, our results provide evidence
on the time after the stroke onset [48,49]. As evident thatEx-4indeedstimulatestheformationofnewbrain
from Figures 5(A), 5(B), 5(E) and 5(F), the difference cells following stroke. Whether these Ex-4-induced
inIba1-positivenumbersat4weeksismainlyattributed proliferating cells can contribute to the reduction
to the increase of microglia infiltration from 2weeks of the ischaemic damage remains to be investigated.
and onwards in the PBS group, whereas it remained However, future therapeutic strategies based on the
largelyunchangedinEx-4-treatedgroups.Weinterpret combinationofEx-4withneuro-survivingfactors,might
our results as the Ex-4 treatment after 4weeks had havethepotentialtoincreasethesurvivalandneuronal
halted the microglia infiltration, but had not reduced differentiationofthesenewlygeneratedcells.
its magnitude compared with the 2weeks time point. In conclusion, we report here a pronounced anti-
Furthermore, since the change in microglia infiltration stroke, neuroprotective and anti-inflammatory effect of
after Ex-4 treatment did not correlate either with the peripheral and chronic Ex-4 treatment in middle-aged
size of the ischaemic lesion or the stroke-induced stem diabetic animals in a preclinical setting that has the
cellproliferationandneurogenesis,itisunclearwhether potentialtomimictheclinicaltreatmentconditions.Our
the modulation of microglia infiltration by Ex-4 has resultsarestrongmotivatingfactorstofurtherinvestigate
any significant impact on the ischaemic damage and GLP-1R agonists for their neuroprotective action in
stroke-induced neurogenesis. Although it is possible diabetes, as well as for their possible use as anti-stroke
thatthedecreasedmicrogliainfiltrationcouldinfluence therapiesinnon-diabeticconditions.
neurogenesisatlatertime-points,newexperimentsusing
these later-time points will be needed to answer this
question. AUTHOR CONTRIBUTION
Obesity and diabetes impair adult neurogenesis and
animal studies have shown decreased neurogenesis VladimerDarsaliaperformedanddesignedthestrokeex-
in diabetic animal models [50–54]. Stroke induces periments,performedpartoftheimmunohistochemical
neurogenesis in both rodents and humans [33,55–57]. studies, contributed to the discussion, wrote the paper,
However,itisknownthatthegreatmajorityofthenew- performed stereology analysis, and acquired and pro-
born neuroblasts induced by stroke do not reach the cessedtheimagesandFigures.ShivaMansouriperformed
maturestateduetopoorsurvival[58].Sinceourprevious the immunohistochemical and screening of ischaemic
© 2012 The Author(s)
The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attributi(cid:4)oCn NThoen-ACuotmhomrserJcoiuarln Lailcecnocmep (ilhatttipo:n//c(cid:4)rCeat2iv0e1c2omBimocohnesm.oicragl/liScoecniesteys/by-nc/2.5/)
which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
482 V.Darsaliaandothers
damage, and contributed to the discussion. Henrik 7 Harmsen,P.,Lappas,G.,Rosengren,A.andWilhelmsen,
Ortsa¨ter provided expertise in diabetes, contributed to L.(2006)Long-termriskfactorsforstroke:twenty-eight
yearsoffollow-upof7457middle-agedmeninGoteborg,
the discussion and performed the bioactivity studies of Sweden.Stroke37,1663–1667
Ex-4.AnnaOlverlingperformedtheimmunohistochem- 8 Kruyt,N.D.,Biessels,G.J.,Devries,J.H.andRoos,Y.B.
(2010)Hyperglycemiainacuteischemicstroke:
icalneurogenesisexperiments.CamillaKappeperformed
pathophysiologyandclinicalmanagement.Nat.Rev.
GLP-1R immunohistochemical experiments. Kerstin Neurol.6,145–155
Iverfeldt and Linda M. Tracy designed and performed 9 Haratz,S.andTanne,D.(2011)Diabetes,hyperglycemia
andthemanagementofcerebrovasculardisease.Curr.
part of the inflammation studies. Nino Nozadze Opin.Neurol.24,81–88
performed immunohistochemical inflammation studies. 10 Perry,T.andGreig,N.H.(2003)Theglucagon-like
peptides:adouble-edgedtherapeuticsword?Trends
Nina Grankvist performed bioactivity studies of Ex-4.
Pharmacol.Sci.24,377–383
A˚keSjo¨holmprovidedexpertiseindiabetes,theGKrats, 11 Drucker,D.J.andNauck,M.A.(2006)Theincretin
conceivedtheresearchplan,contributedtothediscussion system:glucagon-likepeptide-1receptoragonistsand
dipeptidylpeptidase-4inhibitorsintype2diabetes.Lancet
andeditedthepaperpriortosubmission.CesarePatrone 368,1696–1705
contributed to the discussion, conceived, designed and 12 Eng,J.,Kleinman,W.A.,Singh,L.,Singh,G.andRaufman,
J.P.(1992)Isolationandcharacterizationofexendin-4,an
co-ordinated the research plan, the stroke experiments
exendin-3analogue,fromHelodermasuspectumvenom.
andwrote/editedthepaperpriortosubmission. Furtherevidenceforanexendinreceptorondispersedacini
fromguineapigpancreas.J.Biol.Chem.267,7402–7405
13 Goke,R.,Larsen,P.J.,Mikkelsen,J.D.andSheikh,S.P.
ACKNOWLEDGEMENTS (1995)DistributionofGLP-1bindingsitesintheratbrain:
evidencethatexendin-4isaligandofbrainGLP-1binding
sites.Eur.J.Neurosci.7,2294–2300
WethankPetraWolbert(KarolinskaInstitutet),Richelle
14 Hamilton,A.andHolscher,C.(2009)Receptorsforthe
Fall, Diana Rydholm (So¨dersjukhuset AB) for skilled incretinglucagon-likepeptide-1areexpressedonneurons
inthecentralnervoussystem.Neuroreport20,1161–1166
animaltechnicalassistance,DrHansPettersson(Karol-
15 Kastin,A.J.andAkerstrom,V.(2003)Entryofexendin-4
inska Institutet) for help with statistical analyses, and intobrainisrapidbutmaybelimitedathighdoses.Int.J.
JeannetteLundbladMagnusson(So¨dersjukhusetAB)for Obes.Relat.Metab.Disord.27,313–318
16 During,M.J.,Cao,L.,Zuzga,D.S.,Francis,J.S.,
laboratorytechnicalassistance.
Fitzsimons,H.L.,Jiao,X.,Bland,R.J.,Klugmann,M.,
Banks,W.A.,Drucker,D.J.andHaile,C.N.(2003)
Glucagon-likepeptide-1receptorisinvolvedinlearning
FUNDING
andneuroprotection.Nat.Med.9,1173–1179
17 Holst,J.J.,Burcelin,R.andNathanson,E.(2011)
This work was supported by the Regional Agreement NeuroprotectivepropertiesofGLP-1:theoreticaland
practicalapplications.Curr.Med.Res.Opin.27,547–558
on Medical Training and Clinical Research (ALF) 18 Bertilsson,G.,Patrone,C.,Zachrisson,O.,Andersson,A.,
betweenStockholmCountyCouncilandtheKarolinska Dannaeus,K.,Heidrich,J.,Kortesmaa,J.,Mercer,A.,
Nielsen,E.,Ronnholm,H.andWikstrom,L.(2008)
InstitutetandtheAFAInsurance[grantnumber110067],
Peptidehormoneexendin-4stimulatessubventricularzone
Diabetes Research and Wellness Foundation [grant neurogenesisintheadultrodentbrainandinduces
number 0245/2010W], A˚hle´n-stiftelsen [grant number recoveryinananimalmodelofParkinson’sdisease.
J.Neurosci.Res.86,326–338
mG8/09], Stroke-Riksfo¨rbundet, European Foundation 19 Isacson,R.,Nielsen,E.,Dannaeus,K.,Bertilsson,G.,
for the Study of Diabetes (EFSD)/sanofi–aventis, the Patrone,C.,Zachrisson,O.andWikstrom,L.(2011)The
glucagon-likepeptide1receptoragonistexendin-4
FredrikandIngridThuring’sFoundation,theAxeland
improvesreferencememoryperformanceanddecreases
Signe Lagermans Donation Foundation, the Loo and immobilityintheforcedswimtest.Eur.J.Pharmacol.650,
Hans Ostermans Foundation, Stohnes stiftelse and the 249–255
20 McClean,P.L.,Gault,V.A.,Harriott,P.andHolscher,C.
KarolinskaInstitutet. (2010)Glucagon-likepeptide-1analoguesenhance
synapticplasticityinthebrain:alinkbetweendiabetesand
Alzheimer’sdisease.Eur.J.Pharmacol.630,158–162
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© 2012 The Author(s)
The author(s) has (cid:4)pCaidT hfoer Athuitsh aorrtsicJleou tron bale cfroemeplyil aatviaonila(cid:4)bCle u2n0d1e2r Bthioec theermmisca olf Stohcei eCtyreative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/)
which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.