Review Article
`
`Glucagon-like peptide-1 receptor
`agonists as neuroprotective agents for
`ischemic stroke: a systematic scoping
`review
`
`Journal of Cerebral Blood Flow &
`Metabolism
`2021, Vol. 41(1) 14–30
`! The Author(s) 2020
`Article reuse guidelines:
`sagepub.com/journals-permissions
`DOI: 10.1177/0271678X20952011
`journals.sagepub.com/home/jcbfm
`
`, Christian Holscher3
`Mark P Maskery1,2
`,
`Stephanie P Jones4, Christopher I Price5, W David Strain6,
`Caroline L Watkins4, David J Werring7 and
`Hedley CA Emsley1,2
`
`Abstract
`Stroke mortality and morbidity is expected to rise. Despite considerable recent advances within acute ischemic stroke
`treatment, scope remains for development of widely applicable neuroprotective agents. Glucagon-like peptide-1 recep-
`tor agonists (GLP-1RAs), originally licensed for the management of Type 2 Diabetes Mellitus, have demonstrated pre-
`clinical neuroprotective efficacy in a range of neurodegenerative conditions. This systematic scoping review reports the
`pre-clinical basis of GLP-1RAs as neuroprotective agents in acute ischemic stroke and their translation into clinical trials.
`We included 35 pre-clinical studies, 11 retrospective database studies, 7 cardiovascular outcome trials and 4 prospective
`clinical studies. Pre-clinical neuroprotection was demonstrated in normoglycemic models when administration was
`delayed by up to 24 h following stroke induction. Outcomes included reduced infarct volume, apoptosis, oxidative
`stress and inflammation alongside increased neurogenesis, angiogenesis and cerebral blood flow. Improved neurological
`function and a trend towards increased survival were also reported. Cardiovascular outcomes trials reported a signif-
`icant reduction in stroke incidence with semaglutide and dulaglutide. Retrospective database studies show a trend
`towards neuroprotection. Prospective interventional clinical trials are on-going, but initial
`indicators of safety and
`tolerability are favourable. Ultimately, we propose that repurposing GLP-1RAs is potentially advantageous but appro-
`priately designed trials are needed to determine clinical efficacy and cost-effectiveness.
`
`Keywords
`Acute stroke, neuroprotection, reperfusion, clinical trials, animal models
`
`Received 2 January 2020; Accepted 18 July 2020
`
`Introduction
`
`Stroke accounts for 6.5 million deaths per year globally
`and by 2030 will result in an annual loss of over 200
`million disability-adjusted life years.1,2 With an increas-
`ing number of strokes occurring in younger patients,
`alongside an increased number of stroke survivors, the
`cost of post-stroke care is rising. There is, therefore,
`significant scope to improve upon the current position.
`Considerable advances have been made in acute
`ischemic stroke (AIS) treatment, notably reperfusion
`therapies, but these are limited to 10–20% of total
`stroke patients following careful clinical and radiolog-
`ical selection.3 Even when intravenous thrombolysis
`and/or endovascular thrombectomy are administered,
`
`1Lancaster Medical School, Lancaster University, Lancaster, UK
`2Department of Neurology, Royal Preston Hospital, Preston, UK
`3Research and Experimental Center, Henan University of Chinese
`Medicine, Zhengzhou, Henan Province, China
`4Faculty of Health and Wellbeing, University of Central Lancashire,
`Preston, UK
`5Institute of Neuroscience, Stroke Research Group, Newcastle
`University, Newcastle, UK
`6NIHR Exeter Clinical Research Facility and Institute of Biomedical and
`Clinical Science, University of Exeter Medical School, Royal Devon &
`Exeter NHS Foundation Trust, Exeter, UK
`7Stroke Research Centre, Department of Brain Repair and Rehabilitation,
`UCL Institute of Neurology and The National Hospital for Neurology
`and Neurosurgery, London, UK
`
`Corresponding author:
`Mark Maskery, Department of Neurology, Royal Preston Hospital,
`Lancashire Teaching Hospitals NHS Foundation Trust, Sharoe Green
`Lane, Preston PR2 9HT, UK.
`Email: Mark.Maskery@doctors.org.uk
`
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`

`Maskery et al.
`
`15
`
`reduction in disability is highly time dependent.4,5
`Scope remains for further improvement, especially for
`patients who are unsuitable for reperfusion therapies or
`those within remote environments.
`Using simpler clinical selection processes, neuropro-
`tective therapies could bring benefits to a wider patient
`group. Neuroprotectants could also enhance the bene-
`fits of reperfusion therapies by preservation of the
`ischemic penumbra and reduction in ischemic reperfu-
`sion injury. Despite many demonstrating pre-clinical
`potential, a suitable agent has not yet been identified
`by translational studies.6 There remains a multitude of
`factors affecting the translation from bench-to-bedside.
`Namely, animal models are not perfect in their repre-
`sentation of the heterogeneity of clinical stroke.7 Stroke
`in humans occurs
`in the
`context of
`ageing,
`co-morbidity (hypertension, diabetes mellitus, atrial
`fibrillation, pre-existing cerebrovascular disease) and
`concomitant medication use.8 Furthermore,
`factors
`such as gender, cerebral blood flow, body temperature
`and glycemic status may influence stroke mechanism
`and outcomes associated with therapy.6,9–11
`Glucagon-Like Peptide-1 (GLP-1) receptor agonists
`are gaining increasing momentum as possible neuro-
`protective agents in AIS. GLP-1 is an incretin hor-
`mone. Alongside its role in insulin secretion from the
`pancreas and glucagon suppression, it also crosses the
`blood-brain barrier (BBB) and promotes synaptic func-
`tion, enhances neurogenesis, reduces apoptosis and
`protects neurons from oxidative stress.12 GLP-1 is pro-
`duced in the brain and receptors are distributed
`system.13 GLP-1
`throughout
`the central nervous
`Receptor Agonists (GLP-1RAs), licensed for Type 2
`Diabetes Mellitus (T2DM) have already demonstrated
`pre-clinical neuroprotective efficacy in Alzheimer’s
`Disease and clinical trials in neurodegenerative condi-
`tions are ongoing.12,14
`The aim of this systematic scoping review is to
`report the pre-clinical basis of GLP-1RAs as neuropro-
`tective agents in AIS and their translation into clinical
`trials. In addition to describing the characteristics and
`quality of studies, the objectives are to specifically con-
`sider timing of administration, association with glyce-
`mic status, neuroprotective outcomes and application
`to clinical care.
`
`Materials and methods
`
`Eligibility criteria
`
`in vivo studies
`Pre-clinical: We included pre-clinical
`which administered naturally occurring GLP-1, a
`mimetic or analogue, before, during or after stroke
`induction. Normoglycemic, hyperglycemic and induced
`T2DM models were included.
`
`Studies were excluded if their only focus was hem-
`orrhagic stroke as this does not reflect the proposed
`mechanism for how GLP-1 is involved in ischemic
`tissue injury. Those studies which reported incidence
`of hemorrhagic transformation as a complication of
`AIS were
`included as
`these
`reflect post-stroke
`complications.
`Clinical: We included all prospective clinical trials
`which administered GLP-1RAs before, during or
`after stroke onset with outcome measures defined to
`identify neuroprotective efficacy by way of stroke
`volume reduction or improvement in post-stroke func-
`tion or mortality. We also included any potential fea-
`sibility or safety-based studies in this area.
`Our scoping searches identified that very few pro-
`spective clinical trials measuring stroke outcomes were
`available. Pragmatically, we therefore also included all
`retrospective database analyses of stroke incidence or
`composite cardiovascular outcomes in patients treated
`with GLP-1RAs. Furthermore, we included cardiovas-
`cular outcome trials (CVOTs) of GLP-1RAs to evalu-
`ate the incidence of stroke in this relatively higher risk
`cohort.
`Studies were excluded if their full-text was not avail-
`able or not published in English. Efforts were made to
`contact authors directly to obtain any missing articles
`or data.
`
`Database search strategy
`initial scoping searches,15 we accessed
`After several
`Web of Science on 19 March 2020 to search
`MEDLINE, Web of Science core collection, BIOSIS
`and SciELO from 1 January 2000. Keywords were
`EITHER ‘GLP(-)1, glucagon like peptide(-)1, exena-
`tide, liraglutide, lixisenatide, albiglutide, dulaglutide,
`semaglutide’ AND EITHER ‘stroke, CVA, cerebrovas-
`cular, h(a)emorrhage, small vessel disease’. Articles
`were cross-referenced and references were searched to
`identify further studies of interest.
`All articles/studies were screened independently in
`an unblinded, standardised manner by MM and HE
`by way of title and abstract to identify those suitable
`for full-text review. Queries and disagreements were
`resolved by discussion. Preferred Reporting Items for
`Systematic Reviews and Meta-Analyses (PRISMA)
`guidelines were applied. Pre-clinical
`studies were
`appraised according to Animal Research: Reporting
`of In Vivo Experiments (ARRIVE) guidelines,16 and
`the updated Stroke Therapy Academic
`Industry
`Roundtable Preclinical Recommendations (STAIR)
`guidelines.7 Data supporting the findings of
`this
`review are available from the corresponding author
`upon reasonable request.
`
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`16
`
`Results
`
`Study selection
`
`The literature search identified 797 results (see Figure 1)
`alongside 10 from other sources. After removal of dupli-
`cates, this left 794 for screening. We excluded 593
`articles based upon title and review of abstract leaving
`201 full text articles to review. In total, 35 preclinical
`studies, 11 retrospective database studies, 7 cardiovascu-
`lar outcome trials and 4 prospective clinical studies met
`the inclusion criteria.
`
`Pre-clinical studies
`included studies. As shown in
`Characteristics of
`Table 1, 35 pre-clinical studies were included within
`this review. Studies were completed between 2009 and
`2020. Studies were predominantly based upon mouse
`and rat models of stroke; however, one study utilised a
`gerbil model.17 Stroke induction was either via tran-
`sient (range 30–120 min) or permanent common carot-
`id (CCAO) or middle
`cerebral artery occlusion
`(MCAO). Most studies induced unilateral occlusion
`in keeping with spontaneously occurring stroke onset
`in humans, but six studies utilised a bilateral occlusion
`model. Cerebral ischemia was induced by either liga-
`tion, filament occlusion or ablation of the relevant
`artery.
`
`Journal of Cerebral Blood Flow & Metabolism 41(1)
`
`Twelve studies administered exendin-4,17–28 nine
`used liraglutide,29–37 three used rhGLP-1 (recombinant
`human GLP-1),38–40 three used lixisenatide41–43 and
`one study each reported the utility of semaglutide,43
`poly-microspheres),44
`PEx-4
`(exendin-4
`loaded
`proGLP-1 (long acting GLP-1RA),45 DMB (GLP-1R
`agonist/modulator),46 dual GLP-1/Glucose-dependent
`Insulinotropic Peptide (GIP) agonist
`(GLP-1/GIP
`DA),47 oxyntomodulin (co-activates GLP-1R and glu-
`cagon receptor),48 P7C3 (aminopropyl carbazole com-
`pound)49 and one study directly compared exendin-4
`with liraglutide.50 In eight studies, GLP-1R antago-
`nists, such as Ex-9-39, were administered to study the
`role of
`the GLP-1R in neuroprotective mecha-
`nisms.19,21–23,41,45,46,49
`Two studies investigated multiple doses of GLP-
`1RAs to compare neuroprotective efficacy and con-
`cluded that neuroprotection was dose-dependent.20,51
`Most studies administered GLP-1RAs via intraper-
`itoneal, subcutaneous or transvenous routes. However,
`Zhang et al. reported neuroprotection with both oral
`DMB46 and intranasal exendin-4.22
`Some 14 studies administered GLP-1RAs chroni-
`cally prior to the onset of stroke. Clinically, this
`would represent those patients who receive GLP-
`1RAs as part of
`routine T2DM management
`and then go on to experience AIS.18,19,22,23,29,31,38,
`39,42,44–46,48,51 Chronic
`pre-treatment
`occurred
`
`Figure 1. PRISMA flow chart demonstrating the selection of studies.
`
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`Maskery et al.
`
`17
`
`(continued)
`
`byshRNAsuggestingNPisGLP-1Rmediated.
`insulinandglucoselevelsandNPwasblocked
`tionthanintraperitonealNoimpactonplasma
`
`3),"EAAT2,
`"SOD),#apoptosis("Bcl-2,#Bax,#caspase-
`#oxidativestressparameters(#MDA,"GSH,
`NP–#infarctvolume,#neurologicaldeficit,
`a).LixisenatidemoreNPthanglimepiride.
`#inflammation/apoptosis(#caspase-3,#TNF-
`#oxidativestress(#MDA,"GSH,"catalase),
`
`NP–#infarctvolume,#neurologicaldeficit,
`NP–#infarctvolume,"locomotiveactivity
`inflammation(#COX-2,#PE2)
`#neurologicaldeficit,#oxidativedamage/
`
`NP–#infarctvolume(byupto75%),
`
`byEx-9-39.
`ferenttoEx-4.NPblockedbyshRNAbutnot
`diabeticmice.DMBactivationofGLP-1Rdif-
`onplasmainsulinandglucoselevelsinnon-
`
`Intranasalrouteproduced"CNSconcentra-
`#neurologicaldeficit,#apoptosis(#caspase-3)
`
`NPgivenintranasally–#infarctvolume,
`
`deficit,#apoptosis("Bcl-2,#Bax).Noimpact
`NPgivenorally–#infarctvolume,#neurological
`markers(#S100B,#NSE,#MBP)
`group),#neurologicaldeficit,#braininjury
`NPinDM–#infarctvolume(morethaninsulin
`
`GLP-1RmediatedNP)
`dependent.NPblockedbyshRNA(suggesting
`
`#apoptosis("Bcl-2,#Bax).NPnotglucose
`
`NP–#infarctvolume,#neurologicaldeficit,
`
`moreNPthanEx-4inDM.
`
`#oxidativestress,"cerebralbloodflow.PEx-4
`
`NP–#brainoedema,#cognitivedeficit,
`
`reduceinfarctvolume.
`volumereduction,insulintreatmentdidnot
`diabetic&DMmodelswithsimilarinfarct
`
`#apoptosis("Bcl-2,#Bax).NPinbothnon-
`stressparameters(#MDA,"GSH,"SOD),
`NP–#infarctvolume,#motordeficit,#oxidative
`
`endothelinreceptor.
`
`via#oxidativestressandindependentof
`stressparameters(#MDA,"GSH,"SOD)NP
`NP–#infarctvolume,#motordeficit,#oxidative
`NP–#infarctvolumeandimprovesfunctional
`
`outcome
`
`Mainoutcomes(NP–Neuroprotective)
`
`TDS–14days
`
`u.l.tMCAO(90)
`
`nimodipine
`
`OD–14days
`15
`
`b.l.tCCAO(30)
`u.l.tMCAO(60)
`
`glimepiride
`
`30
`
`u.l.MCAO(60)
`
`Ex-9-39
`
`OD–7days
`
`u.l.tMCAO(90)
`
`IntraperitonealEx-4,shRNA
`
`30
`
`u.l.tMCAO(60)
`
`Ex-4,Ex-9-39,shRNA
`
`TDS–14days
`
`u.l.tMCAO(90)
`
`nimodipine,insulin
`
`OD–7days
`
`u.l.tMCAO(90)
`
`&hypotension
`
`OD–14days
`
`b.ltCCAO(10)
`
`OD–14days
`
`u.l.pMCAO
`
`Non-diabetic,insulin
`
`BD–7days
`
`u.l.pMCAO
`
`15
`
`u.ltMCAO(60)
`
`ischemia
`Post-
`
`ischemia
`Pre-
`
`Administrationtime(min)
`
`(time/min)
`induction
`Stroke
`
`i.p.
`
`rhGLP-1
`
`DM
`
`2018Rat
`
`Fangetal.39
`
`i.p.
`i.c.v.
`
`i.c.v
`
`Lixisenatide
`OXM
`
`DM
`
`Abdel-latifetal.422018Rat
`Lietal.48
`2017Rat
`
`Ex-4
`
`2017Rat
`
`Kimetal.23
`
`i.n.
`
`Ex-4
`
`2016Mouse
`
`Zhangetal.22
`
`p.o.
`
`DMB
`
`2016Mouse
`
`Zhangetal.46
`
`i.p.
`
`rhGLP-1
`
`DM
`
`2016Rat
`
`Jiangetal.38
`
`shRNA
`
`i.p.
`
`Pro-GLP-1
`
`2015Mouse
`
`Zhangetal.45
`
`Non-diabetic,Ex-4
`
`s.c.
`
`PEx-4
`
`DM
`
`2015Rat
`
`Chienetal.44
`
`s.c.
`
`Liraglutide
`
`DM
`
`2014Rat
`
`Briyaletal.29
`
`BQ123
`
`i.p.
`
`Ex-4
`
`2012Rat
`
`Briyaletal.19
`
`Lietal.18
`GLP-1RAadministeredpriortostrokeonset(includingchronicpre-treatment)
`
`2009Rat
`
`i.c.v.
`
`Ex-4
`
`Sub-studies
`
`route
`Admin
`
`modelCo-morb.Drug
`Animal
`
`Year
`
`Author
`
`Table1.Overviewofthepre-clinicalstudies.
`
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`

`18
`
`Journal of Cerebral Blood Flow & Metabolism 41(1)
`
`(continued)
`
`youngandoldanimalmodels
`tered4.5hrsafterstrokeonset.NPinboth
`administration.NotNPwhenEx-4adminis-
`kg.StrokevolumenotaffectedbyEx-4
`50mg/kgat90/180minandat90minfor5mg/
`inflammatorymarkers.NPinnon-DM/DMat
`Nonstatisticallysignificantreductioninpro-
`
`NP–"neuronalsurvival,"M2microglialmarkers
`#oxidativestress,"VEGFincortexbutnotin
`
`raisedintracellularcAMPlevels
`GLP-1Ractivation).NPprobablymediatedvia
`
`NP–#infarctvolume,#neurologicaldeficit,
`
`thestriatum.
`
`activation),"intracellularcAMPlevels(dueto
`#oxidativestress,#inflammation(#microglial
`
`NP–#infarctvolume,#neurologicaldeficit,
`
`NPbetweenDM&non-DM
`signallingpathway)Nosignificantdifferencein
`
`(#MPO),"Nrf2,"HO-1(antioxidativestress
`#oxidativestress("SOD),#inflammation
`NP-#infarctvolume,#neurologicaldeficit,
`
`dosedependent.
`proliferation&neuroblastformation.NPis
`
`NP–#infarctvolume,#neurologicaldeficit,
`
`andnaloxone
`blockedbyEx-9-39,b-endorphinanti-serum
`"hippocampalb-endorphinexpression,NP
`
`0
`
`u.l.tMCAO(120)
`
`nimodipine
`
`thenOD1week
`
`90,180or270
`
`14monthobese/DMmice
`
`u.l.tMCAO(30)
`
`60
`
`u.l.tMCAO(90)
`
`0,60,180
`
`u.ltMCAO(60)
`
`BD–7days
`
`BD–7days
`
`u.l.pMCAO
`
`Non-DM
`
`0
`
`15
`
`u.l.tMCAO(60)
`
`butmarginaleffectonactivation,"stemcell
`4weekspostMCAO),#microglialinfiltration
`NP–#infarctvolume(nodifferencebetween2&
`activity,#microglialactivation
`NP–#neurologicaldeficit,"GLP-1Rimmunore-
`
`BD–4weeksBD–2–4weeks
`
`u.l.tMCAO(90)
`
`60
`
`120
`
`b.l.tCCAO(5)
`
`cemiccontrolamelioration(MetforminnotNP)
`non-DMmodel,NPnotassociatedwithgly-
`
`NP–#oxidativestressparameters(#MDA,
`
`Lixisenatide"NP
`#IL-1b,#TNF-a),"viablehippocampalneu-
`#caspase-3),#inflammatorymarkers(#MPO,
`"GSH,"SOD),#apoptosis("Bcl-2,#Bax,
`
`ronsonhistologicalstaining.Higherdose
`
`NP–#infarctvolume,#neurologicaldeficitonlyin
`
`Mainoutcomes(NP–Neuroprotective)
`
`OD–14days
`
`OD–7days
`
`u.l.tMCAO(30)
`
`Non-diabetic,metformin
`
`ischemia
`Post-
`
`ischemia
`Pre-
`
`Administrationtime(min)
`
`(time/min)
`induction
`Stroke
`
`i.p.
`
`rhGLP-1
`
`DM
`
`2015Rat
`
`Zhaoetal.40
`
`2monthold&
`
`i.p.
`
`Ex-4
`
`2014MouseDM
`
`Darsaliaetal.25
`
`i.p.
`
`Liraglutide
`
`2013Rat
`
`Satoetal.32
`
`Teramotoetal.242011Mouse
`GLP-1RAadministeredfollowingstrokeonset(includingdelayedadministration)
`
`Ex-4
`
`t.v.
`
`i.p.
`
`Liraglutide
`
`DM
`
`2018Rat
`
`Dengetal.30
`
`Ex-9-39
`
`i.c.v.
`
`Ex-4/Catapol
`
`2015Rat
`
`Jiaetal.21
`
`0.1,2or5mg/kgofEx-4
`
`i.p.
`
`i.p.
`
`Ex-4
`
`DM
`
`2012Rat
`
`Darsaliaetal.20
`
`HyunLeeetal.172011Gerbil
`GLP-1RAadministeredpriortoandfollowingstrokeonset
`
`Ex-4
`
`0.7and7nmol/kglixisenatideb.l.tCCAO(60)
`
`i.p.
`
`Lixisenatide
`
`2020Rat
`
`Gadetal.51
`
`s.c.
`
`Liraglutide
`
`DM
`
`2018Rat
`
`Filchenkoetal.31
`
`Sub-studies
`
`route
`Admin
`
`modelCo-morb.Drug
`Animal
`
`Year
`
`Author
`
`Table1.Continued.
`
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`19
`
`(continued)
`
`ingGLP-1Rindependentpathway.
`inhibitedbyGLP-1RantagonistEx-39suggest-
`
`#inflammation(TNF-a)–theseeffectsnot
`(#GSH,#MDA,#catalase,#NO),
`#apoptosis(caspase-3),#oxidativestress
`NP–#infarctvolume,#neurologicaldeficit,
`
`delayedby1day
`
`("NeuN,"GFAP,"vWF).NPwhenfirstdose
`"neurovascularremodelling
`"glucosemetabolisminischemicpenumbra
`NP–#neurologicaldeficit,"GLP-1Rexpression,
`#stressrelatedhyperglycemiawithoutcausing
`"Bcl-2,#Bax),#DNAdamage(#TUNEL),
`#apoptosis(#ROS,#caspase-3,-8,-9,#PARP,
`
`(18-FDG-PET)
`
`hypoglycemia.
`
`#inflammation(#ICAM-1,NF-2jB,p50,p65).
`#TUNEL,#Bax,#PARP,"Bcl-2),
`#ROS,"SOD),#apoptosis(#caspase-3,
`#cerebraloedema,#oxidativestress(#DHE,
`dysfunction,"cerebralmicrocirculation.
`
`notNP.
`microglia,NPinhyperglycemicmodel.Insulin
`
`NP–#neurologicaldeficitincluding#bladder
`
`NP–#infarctvolume,#neurologicaldeficit,
`NP–#DNAdamage("APE1,#TUNEL)
`
`1&24hrs
`
`b.l.tCCAO(30)
`
`Ex-9-39,pentoxyphylline
`
`i.p.
`
`Lixisenatide
`
`Abdel-latifetal.412018Rat
`
`for4weeks
`24hrs,thenOD
`
`1,3and7days
`1hr,thenODfor
`
`0&24hrs
`
`u.l.MCAO(90)
`
`s.c.
`
`Liraglutide
`
`2017Rat
`
`Dongetal.34
`
`u.lpMCAO
`
`b.l.tCCAO(60)
`
`u.l.tMCAO&
`
`s.c.
`
`t.v.
`
`Liraglutide
`
`2016Rat
`
`Zhuetal.33
`
`Ex-4
`
`2016Rat
`
`Yangetal.28
`
`0,3,6or12hrs
`
`u.l.tMCAO(60)
`
`Ex-4orliraglutidei.p.
`
`2016MouseDM
`
`Lietal.50
`
`Maskery et al.
`
`(#TNF-a),#MMP-9activation,#Iba-1positive
`oedema,#oxidativestress,#inflammation
`tion.Ex-4NP–#infarctvolume,#cerebral
`"cerebraloedema,"hemorrhagictransforma-
`Hyperglycemiaassociatedwith"infarctvolume,
`
`hemorrhagictransformation.
`inhibitor.Amelioratedwarfarinassociated
`
`HHE),#inflammation,NPblockedbyP13K
`integrity,#oxidativestress(#8-OHdG,#
`NP–#infarctvolume,#neurologicaldeficit,"BBB
`
`damage(iNOS).DAmoreNPthanSA.
`
`coseinhighdosegroup(nohypoglycemia)
`Statisticallysignificantreductioninbloodglu-
`
`#apoptosis("Bcl-2,#Bax),#nitricoxide
`NP–#infarctvolume,#neurologicaldeficit,
`
`#nitricoxidedamage(iNOS,eNOS)
`#oxidativestress(#MDA,"GSH,"SOD),
`
`NP–#infarctvolume,"neuronalsurvival,
`
`Mainoutcomes(NP–Neuroprotective)
`
`60
`
`0
`
`60
`
`u.l.tMCAO(60)
`
`insulin
`
`i.p.
`
`Ex-4
`
`2016MouseDM
`
`Kurokietal.27
`
`u.l.tMCAO(45)
`
`P13Kinhibitor
`
`t.v.
`
`Warfarin&Ex-4
`
`2016Mouse
`
`Chenetal.26
`
`u.l.tMCAO(60)
`
`GLP-1RA(SA)
`
`i.p.
`
`GLP-1/GIP(DA)
`
`2016Rat
`
`Hanetal.47
`
`ischemia
`Post-
`
`ischemia
`Pre-
`
`Administrationtime(min)
`
`(time/min)
`induction
`Stroke
`
`Sub-studies
`
`route
`Admin
`
`modelCo-morb.Drug
`Animal
`
`Year
`
`Author
`
`Table1.Continued.
`
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`

`

`20
`
`Journal of Cerebral Blood Flow & Metabolism 41(1)
`
`fibrillaryacidicprotein(glialmarker);vWF:vonWillebrandfactor(endothelialmarker);b-tub3:betatubulinIII;GSK-3:glycogensynthasekinase3;IL:interleukin.
`NOS:nitricoxidesynthase;BBB:bloodbrainbarrier;MMP-9:matrixmetalloproteinase-9;ICAM-1:intracellularadhesionmolecule-1;NeuN:neuronalnuclearprotein(neuronalmarker);GFAP:Glial
`myelinbasicprotein;COX-2:cyclo-oxygenase-2;PE2:ProstaglandinE2;EAAT2:Excitatoryaminoacidtransporter-2;MPO:myeloperoxidase;HO-1:hemeoxygenase-1;Nrf2:nuclearfactorerythroid-2;
`protein4;Caspase-3;PARP:poly-ADPribosepolymerase;TUNEL:terminaldeoxynucleotidyltransferasedUTPnickendlabeling;S100b:s100-calciumbindingproteinB;NSE:neuronspecificenolase;MBP:
`MDA:malondialdehyde;GSH:glutathione;8-OHdG:8-hydroxy-2-deoxyguanosine;HHE:4-hydroxyhexenal;DHE:dihydroethidium;ROS:reactiveoxygenspecies;Bcl-2:Bcelllymphoma2;Bax:Bcl-2-like
`diabetesmellitus(healthy);u.l.:unilateral;b.l.:bilateral;MCAO:middlecerebralarteryocclusion;CCAO:commoncarotidarteryocclusion;(t/p)MCAO:transient/permanent;SOD:superoxidedismutase;
`compound;i.p.:intraperitoneal;s.c.:subcutaneous;p.o.:oral;i.n.:intranasal;t.v.:transvenous;i.c.v.:intracerebroventricular;OD/BD/TDS:once/twice/threetimesdaily;DM:diabetesmellitus;Non-DM:non
`6,7-dichloro-2-methyl-sulfonyl-3-N-tert-butylaminoquinoxaline;OXM:oxyntomodulin;shRNA:smallhairpinRNA(targetsGLP-1R);BQ123-endothelinreceptorantagonist;P7C3-aminopropylcarbazole
`SA:singleagonist;DA:dualagonist;Ex-4-exendin-4;PEx-4:Exendin-4loadedpoly-microspheres;Ex-9-39:GLP-1Rantagonist;pro-GLP-1:longactingGLP-1RA;rhGLP-1:recombinanthumanGLP-1;DMB:
`
`effects.
`COX-2)P13kinhibitorpartiallyreversedNP
`
`#inflammation(#TNF-a,IL-18,IL-1b,IL-6,
`#apoptosis("Bcl-2,#caspase-3,#TUNEL),
`
`NP–#infarctvolume,#cerebraloedema,
`NP–#neurologicaldeficit,#oxidativestress
`
`glycemiainnormoglycemicmodel.
`
`factorsignalling("ERK1,"ERS-1),Nohypo-
`#caspase-3),"neurogenesis,improvedgrowth
`#neuronalloss,#apoptosis("Bcl-2,#Bax,
`
`NP–#infarctvolume,#neurologicaldeficit,
`
`dependent.
`
`manner).NPwhenfirstdosedelayedby1day
`releaseisinablood-glucosedependent
`andcontrols(asexpected,stimulatedinsulin
`enceinbloodglucoselevelsbetweenliraglutide
`
`"adamts20,#GSK-3)NPisGLP-1R
`("doublecortin,"b-tub3,"adam11,
`NP–#neurologicaldeficit,"neurogenesis
`
`0thenOD
`ODfor7days
`
`u.lpMCAO
`u.l.pCCAO
`
`P13kinhibitor
`
`i.p.
`i.p.
`
`Liraglutide
`Liraglutide
`
`2020Rat
`2019Mice
`
`Zengetal.37
`Heetal.36
`
`days1,7,14or21days
`
`2hrs,thenalternate
`
`u.l.pMCAO
`
`Bloodglucosemonitoring
`
`i.p.
`
`Semaglutide
`
`2019Rat
`
`Yangetal.43
`
`120
`
`u.l.tMCAO(40)
`
`Ex-9-39
`
`t.v.
`
`P7C3
`
`2018Mouse
`
`Wangetal.49
`
`"angiogenesis("VEGF)Nosignificantdiffer-
`
`24hrs,thenODfor14daysNP–#infarctvolume,#neurologicaldeficit,
`
`Mainoutcomes(NP–Neuroprotective)
`
`ischemia
`Post-
`
`ischemia
`Pre-
`
`Administrationtime(min)
`
`u.l.pMCAO
`
`(time/min)
`induction
`Stroke
`
`i.p.
`
`Liraglutide
`
`2018Mouse
`
`Chenetal.35
`
`Sub-studies
`
`route
`Admin
`
`modelCo-morb.Drug
`Animal
`
`Year
`
`Author
`
`Table1.Continued.
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`

`Maskery et al.
`
`21
`
`between 14 days prior and 15 minutes prior to stroke
`onset, for one to three times daily.
`A further four studies administered GLP-1RAs
`prior to and following stroke onset for between 0 and
`four weeks – with dosing schedules of up to twice
`daily.17,20,21,30
`Most closely aligned with the proposed clinical
`application of administering treatment to GLP-1RA
`naı¨ ve patients in the hyper-acute AIS setting, 17 studies
`only administered the medication following stroke
`onset.24–28,32–37,40,41,43,47,49,50 First dose was delayed
`between 0 min and 24 h post-onset and continued for
`up to four weeks.
`Alterations in physiological parameters such as
`body temperature have the potential to impact the out-
`come of stroke. Li et al. reported no significant change
`in body temperature when measured before and after
`treatment with exendin-4.18 We did not identify any
`GLP-1RA study of AIS which varied temperature
`between groups. Most studies regulated body temper-
`ature within normal physiological parameters during
`MCAO surgery and animals were housed within a
`temperature-controlled environment.
`
`Quality of included studies. Pre-clinical study meth-
`odology was appraised according to the STAIR 2009
`criteria, with a maximum available score of 7. Median
`score was 3 (range 2–7). Inclusion/exclusion criteria
`and reporting of potential conflicts of interest/funding
`were consistently reported in 100% and 94% of studies,
`respectively. Fewer studies commented on randomisa-
`tion (54%), allocation concealment (17%) and blinded
`assessment of outcome (43%). Only four studies (11%)
`reported performing a sample size calculation.
`Reporting of pre-clinical studies was also assessed
`using the ARRIVE guidelines, with a maximum score
`of 36. Median score was 22 (range 14–29). Methods,
`statistical analysis, outcomes and confidence intervals
`were well reported, as were ethical and funding state-
`ments, but the justification for animal models, transla-
`tion to human biology, limitations and adverse events
`were frequently not reported.
`
`Impact of hyperglycemia in stroke models. Twelve
`studies were based on hyperglycemic rodents, with or
`without a normoglycemic control group.
`Kuroki et al. demonstrated that hyperglycemia was
`associated with an increase in infarct volume, cerebral
`edema and hemorrhagic transformation in a mouse
`model that underwent transient, unilateral occlusion
`of the MCAO for 60 min.27 They reported that intra-
`peritoneal administration of exendin-4 60 min after
`stroke onset was associated with a reduction in these
`parameters which was not
`replicated by insulin
`monotherapy.
`
`Briyal et al. reported that liraglutide reduced infarct
`volume by a similar amount in both diabetic and nor-
`moglyemic models, but this neuroprotection was again
`not reproduced in the insulin treatment arm despite
`resolution of hyperglycemia.29 Deng et al. further con-
`firmed neuroprotection was independent of glycemic
`status prior to stroke onset.30 Jiang et al. reported
`that stroke infarct volume in the rhGLP-1 group was
`significantly reduced when compared to insulin treat-
`ment.38 Metformin was also shown to ameliorate
`hyperglycemia but did not confer the additional neuro-
`protective outcomes associated with liraglutide.31
`One study demonstrated similar neuroprotective
`outcomes between 2-month-old healthy mice and 14-
`month-old overweight, diabetic mice treated with exen-
`din-4.25
`Whilst GLP-1RAs were associated with a reduction
`in blood glucose in hyperglycemic models,40 no study
`reported hypoglycemia when GLP-1RAs were admin-
`istered to normoglycemic models. This is to be expected
`as GLP-1RA-associated insulin secretion from the pan-
`creas is glucose dependent.35 Indeed, Zhang et al. con-
`cluded
`that
`neuroprotection was
`not
`glucose
`dependent.45
`Yang et al. monitored blood glucose in rats which
`underwent intraperitoneal administration of semaglu-
`tide starting 2 h following unilateral permanent MCAO
`occlusion, further demonstrating neuroprotection in
`the absence of hypoglycemic episodes.43
`
`Infarct volume and neuronal survival. Administration
`of GLP-1RAs prior to, at the point of, or delayed fol-
`lowing stroke onset were associated with reduction in
`infarct volume. In total, nine studies demonstrated a
`reduction in infarct volume with exendin-4,18–24,26,27
`eight with liraglutide,29–33,35–37 three with rhGLP-1,
`three with lixisenatide and one with each of PEx-4,
`proGLP-1, DMB, OXM, GLP-1/GIP DA and sema-
`glutide. Liraglutide was associated with a reduction in
`infarct volume when the first dose was delayed by up to
`1 day.35 Kim et al. reported a reduction in infarct
`volume by up to 75% in a rat model of transient
`MCAO. Darsalia et al. reported that exendin-4 admin-
`istration was associated with a reduction in stroke
`volume when administered for four weeks prior to,
`following stroke onset.20
`and two to four weeks
`However, in a later study, whereby they only adminis-
`tered exendin-4 following stroke onset, it did not sig-
`nificantly reduce infarct volume but did reduce overall
`neuronal loss.25 Reduction in neuronal loss was also
`rhGLP-140
`semaglutide,43
`and
`reported with
`lixisenatide.51
`
`Cellular function. Apoptosis represents a chain of
`enzymatic
`events
`resulting
`in programmed cell
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`

`22
`
`Journal of Cerebral Blood Flow & Metabolism 41(1)
`
`death.52 Whilst controlled apoptosis is essential for the
`maintenance of homeostasis, dysregulated apoptosis,
`for example in within the ischemic penumbra, can
`in increased cell death.53 Bcl-2 is an anti-
`result
`apoptotic protein which functions at least in part by
`reducing cytochrome C release from the mitochondria.
`Conversely, Bax is a pro-apoptotic protein and
`increases cytochrome C levels.52 Increased Bcl-2 and
`reduced Bax levels, contributing to an increased
`Bcl-2:Bax ratio and representing reduced levels of apo-
`ptosis, have been reported in three studies of liraglu-
`tide29,33,37 and one study for each pro-GLP-1,45
`rhGLP-1,39 DMB,22 exendin-4,50 lixisenatide,51 GLP-
`1/GIP DA47 and semaglutide.43 Critically, this reduc-
`tion of apoptosis was reproduced in normoglycemic
`models when administered after stroke onset.33,37,43,47
`Caspase proteins are also pro-apoptotic and
`increased levels are associated with higher rates of
`cell death.54 Liraglutide,
`exendin-4,
`lixisenatide,
`rhGLP-1 and semaglutide have been shown to reduce
`levels
`of
`caspase-3
`in
`pre-clinical models
`of
`stroke.33,37,39,41–43,46,50,51 Zhu et al. have also demon-
`strated reduced levels of caspase 8 and 9 in models
`administered liraglutide.33
`Cleavage of PARP by caspases is a signal of apo-
`ptosis and has been implicated in cerebral ischemia.53
`Reduction in PARP has been demonstrated with both
`liraglutide and exendin-4 treatment.33,50
`TUNEL assays detect DNA degradation during the
`later
`stages of apoptosis and have demonstrated
`reduced apoptotic activity with liraglutide and exen-
`din-4.37,50,55
`GLP-1RA administration following stroke induc-
`tion has been associated with an anti-inflammatory
`effect.26 Tumor necrosis factor alpha (TNF-a) is a cyto-
`kine synthesised by many cell lines, but particularly by
`macrophages and microglia.56 TNF-a is involved with
`the inflammatory response following stroke onset.56
`Five studies reported a reduction in TNF-a when com-
`pared to controls, three with lixisenatide41,42,51 and one
`with each of liraglutide37 and exendin-4.27 Indeed, two
`reported reduced microglial activation17,24
`studies
`whilst a further study by Darsalia et al. reported
`reduced microglial infiltration, but a marginal effect
`on activation.20 Reduced levels of other markers of
`inflammation, such as myeloperoxidase and interleu-
`kin, have also been reported.30,37,51
`One study reported a non-statistically significant
`reduction in pro-inflammatory markers.25
`There is also deterioration in markers of oxidative
`stress following AIS. Twelve studies reported an
`improvement in oxidative stress parameters following
`GLP-1RA administration in AIS – five studies of exen-
`din-4,19,24,26,27,50 four studies of liraglutide,29,30,32,36
`lixisenatide41,42
`rhGLP.40
`two of
`and one of
`
`reported reduced levels of
`Predominantly, studies
`malondialdehyde and increased concentrations of glu-
`tathione and superoxide dismutase.
`Dong et al. performed 18-FDG PET imaging in a
`rat model of unilateral transient MCAO. In the ani-
`mals treated with subcutaneous liraglutide, there was
`radiological evidence of increased glucose metabolism
`within the ischemic penumbra.34
`
`function. The neurovascular unit
`Neurovascular
`incorporates both cellular and extracellular compo-
`nents involved in the regulation of cerebral blood
`flow and blood-brain barrier function – it is involved
`with the maintenance of cerebral homeostasis and con-
`trol of cerebral blood flow.57
`GLP-1RAs have been shown to increase cerebral
`blood flow following AIS when compared with con-
`trols.44 Li et al. reported that cerebral microcirculation
`is reduced following AIS, but improved to a similar
`degree within 4–12 h after MCAO in diabetic mice
`treated with either liraglutide or exendin-4 after stroke
`induction.50 Blood–brain barrier integrity has also been
`shown to improve with GLP-1RA treatment.26
`is
`Vascular endothelial growth factor
`(VEGF)
`known to promote angiogenesis and protect ischemic
`neurons from injury, demonstrating a crucial role in the
`neurovascular remodelling post-AIS.58 Chen et al.
`demonstrated that intraperitoneal liraglutide therapy,
`administere