Visualization of cell death in mice with focalcerebral ischemia using fluorescent annexin A5,propidium iodide, and TUNEL staining

Peyman Bahmani1, Eyk Schellenberger2, Jan Klohs3, Jens Steinbrink1,4, Ryan Cordell1,Marietta Zille1, Jochen Muller1, Denise Harhausen1, Leo Hofstra5, Chris Reutelingsperger6,Tracy Deanne Farr1, Ulrich Dirnagl1 and Andreas Wunder1

1Department of Experimental Neurology, Center for Stroke Research Berlin (CSB), Small Animal Imaging Center(SAIC), Charite—University Medicine Berlin, Berlin, Germany; 2Department of Radiology, Charite—UniversityMedicine Berlin, Berlin, Germany; 3Institute for Biomedical Engineering, University of Zurich and ETH, Zurich,Switzerland; 4Berlin NeuroImaging Center, Charite—University Medicine Berlin, Berlin, Germany; 5Departmentof Cardiology, Cardiovascular Research Institute, University of Maastricht, Maastricht, The Netherlands;6Department of Biochemistry, University of Maastricht, Maastricht, The Netherlands

To monitor stroke-induced brain damage and assess neuroprotective therapies, specific imaging ofcell death after cerebral ischemia in a noninvasive manner is highly desirable. Annexin A5 has beensuggested as a marker for imaging cell death under various disease conditions including stroke.In this study, C57BL6/N mice received middle cerebral artery occlusion (MCAO) and were injectedintravenously with either active or inactive Cy5.5-annexin A5 48 hours after reperfusion. Some micealso received propidium iodide (PI), a cell integrity marker. Only in mice receiving active Cy5.5-annexinA5 were fluorescence intensities significantly higher over the hemisphere ipsilateral to MCAO than on thecontralateral side. This was detected noninvasively and ex vivo 4 and 8 hours after injection. The majorityof cells positive for fluorescent annexin A5 were also positive for PI and fragmented DNA as detected byterminal deoxynucleotidyl transferase-mediated 20-deoxyuridine 50-triphosphate-biotin nick end labeling(TUNEL) staining. This study demonstrates the high specificity of annexin A5 for visualization of celldeath in a mouse model of stroke. To our knowledge, this is the first study to compare the distribution ofinjected active and inactive annexin A5, PI, and TUNEL staining. It provides important information on theexperimental and potential clinical applications of annexin A5-based imaging agents in stroke.Journal of Cerebral Blood Flow & Metabolism (2011) 31, 1311–1320; doi:10.1038/jcbfm.2010.233; published online 19 January 2011

Keywords: annexin A5; cell death; fluorescence imaging; propidium iodide; stroke; TUNEL staining

Introduction

Specific visualization of cell death with noninvasiveimaging techniques is highly relevant in basic

research and there is great hope that it will translatefor the use in humans. It has been shown thatimaging agents specifically targeting dead cells canbe used to monitor treatment response in cancer(Blankenberg, 2008b), to localize tissue damage inmyocardial infarction (Wolters et al, 2007), andto identify vulnerable plaques in atherosclerosis(Laufer et al, 2009). In cerebral ischemia, a decreaseor arrest of regional blood flow leads to damage ofneurons and other brain cells. Cells in the core of theinfarcted area might not be salvageable, but cells inthe periinfarct region, the so-called ischemic penum-bra, which can only be approximated by usingpositron emission tomography (PET) or magneticresonance imaging (MRI) techniques (Heckl, 2007),might be rescued (Dirnagl et al, 1999; Harraz et al,2008). As cell death is a basic pathophysiologicalfeature in cerebral ischemia, specific noninvasiveimaging of this process would be highly desirable for

Received 29 September 2010; revised 3 December 2010; accepted22 December 2010; published online 19 January 2011

Correspondence: Dr A Wunder, Department of ExperimentalNeurology, Center for Stroke Research Berlin (CSB), Small AnimalImaging Center (SAIC), Charite—University Medicine Berlin,Chariteplatz 1, 10117 Berlin, Germany.E-mail: andreas.wunder@charite.de

This work was financed by the TSB Technologiestiftung Berlin–

Zukunftsfonds Berlin, co-financed by the European Union–

European fund for regional development, the Bundesministerium

fur Bildung und Forschung (BMBF), the German Research

Foundation (ExcellenceCluster DFG-EXC 257 NeuroCure), the

Herman and Lilly Schilling Foundation, and the European Union’s

Seventh Framework Programme (FP7/2008-2013) under grant

agreements n1 201024 and n1 202213 (European Stroke Network).

Journal of Cerebral Blood Flow & Metabolism (2011) 31, 1311–1320& 2011 ISCBFM All rights reserved 0271-678X/11 $32.00

www.jcbfm.com

monitoring stroke-induced brain damage and asses-sing neuroprotective therapies. To date, no clinicallyapproved technique enables the specific visualiza-tion of cells in stroke with an imaging agentspecifically targeting dead or lethally damaged cells.

To envisage cell death-related parameters ex vivo,different staining methods can be applied. Mostcommonly, in situ terminal deoxynucleotidyl trans-ferase-mediated 2’-deoxyuridine 5’-triphosphate-biotin nick end labeling (TUNEL staining), whichdetects fragmented DNA, and fluorescent dyes suchas propidium iodide (PI; 668.4 Da, red fluorescent),are used. As PI cannot penetrate the membrane ofviable cells, it only stains cells with compromisedplasma membrane integrity (Loo and Rillema, 1998;Moore et al, 1998). For noninvasive visualization ofcell death, imaging agents based on small moleculecaspase inhibitors or annexin A5 have been devel-oped (Faust et al, 2009). Annexin A5 is an endogen-ous protein with a molecular weight of about 36 kDathat binds tightly and specifically to phosphatidyl-serine (PS) and other anionic phospholipids. Innormal cells, PS is restricted to the inner surface ofthe cell membrane. The exposure of PS occurs duringapoptosis and necrosis (Galluzzi et al, 2009; Kroemeret al, 2009), therefore, making it a suitable target forspecific imaging of cell death with annexin A5(Blankenberg, 2008a).

For imaging purposes, annexin A5 can be labeledwith different signal molecules including radioactiveisotopes for single photon emission computedtomography (SPECT) or PET (Blankenberg, 2008a),fluorochromes for near-infrared fluorescence (NIRF)(Schellenberger et al, 2004b), nanoparticles for MRI(Schellenberger et al, 2002; van Tilborg et al, 2010) ormagneto-optical particles detectable for both opticaland MR imaging (Schellenberger et al, 2004a;Sosnovik et al, 2005). Imaging agents based onannexin A5 have been applied successfully in avariety of disease models and in patients sufferingfrom different diseases including cancer (Belhocineet al, 2002; Petrovsky et al, 2003), myocardialinfarction and ischemia (Dumont et al, 2001; Hofstraet al, 2000; Thimister et al, 2003), and atherosclerosis(Kietselaer et al, 2004; Kolodgie et al, 2003). Blanken-berg and colleagues were the first to use an imagingagent based on annexin A5 for the noninvasivevisualization of cell death in cerebral ischemia. Theyinjected annexin A5 labeled with 99mTc intrave-nously into patients with acute ischemic stroke.Subsequent SPECT imaging showed foci of increasedtracer uptake at sites of ischemic injury. In the samestudy, the authors demonstrated that annexin A5imaging can assess the effects of therapy for ischemiccerebral injury in rats. They concluded that radi-olabeled annexin A5 and SPECT imaging might beused as a noninvasive indicator of drug efficacy inclinical trials (Blankenberg et al, 2006). Similarresults in patients with acute stroke were observedby another group (Lorberboym et al, 2006). Tang et al(2007) demonstrated that 99mTc-labeled annexin A5

and SPECT imaging can be used to monitor theantiapoptotic effects of minocycline therapy in amouse model of focal cerebral ischemia.

The use of targeted imaging agents may involveseveral confounders such as unspecific binding orissues of compartmentalization, particularly whenthe blood–brain barrier (BBB) breaks down in thedisease process. To our knowledge, there are no datathat compare the distribution of systemically admi-nistered active annexin A5 with that of inactiveannexin A5. The latter has no binding properties toPS and can therefore be used as a control forunspecific distribution. Furthermore, there are nodata at the histological level on how annexin A5binding compares to other markers of cell death suchas uptake of PI and TUNEL staining on a single celllevel in focal cerebral ischemia. To address this, weintravenously injected active or inactive annexin A5,both labeled with the NIRF-dye Cy5.5, together withPI into mice after transient middle cerebral arteryocclusion (MCAO). Noninvasive in vivo and ex vivoNIRF imaging were performed and tissue sectionswere prepared. After fluorescence inspection ofCy5.5 and PI, we stained the same sections withTUNEL and finally with hematoxylin and eosin (HE).In this study, we present important information onthe usefulness of annexin A5 imaging in cerebralischemia, especially its specificity in imaging leth-ally damaged or dead cells.

Materials and methods

Preparation of Near-Infrared Fluorescence-LabeledAnnexin A5

Recombinant human annexin A5 (from C Reutelingsperger)was labeled with Cy5.5 (Cy5.5-N-hydroxysuccinimideester, PA 15601, Amersham Biosciences, Buckinghamshire,UK) as described (Schellenberger et al, 2004b). Briefly,active annexin A5 with retained affinity for PS wasprepared by labeling annexin A5 with about one mole ofdye per mole of protein. Inactive annexin A5 with acomplete loss of affinity for PS was prepared with twomoles or more of Cy5.5 per mole of protein (Schellenbergeret al, 2004b).

Animals and Model of Focal Cerebral Ischemia

Twenty-one adult male C57BL6/N mice (Bundesinstitutfuer Risikoforschung, Berlin, Germany) weighing between18 and 20 g were housed under standard conditions withfree access to food and water. All procedures were inaccordance with the German animal welfare laws andapproved under the animal research license G0229/05.Focal cerebral ischemia was induced by transient occlu-sion of the MCA with the intraluminal filament techniqueas previously described (Hara et al, 1996). Mice wereanesthetized with isoflurane in 70% N2O and 30% O2. Theleft common and external carotid arteries were isolated andligated. A microvascular clip was placed on the internal

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carotid artery. An 8-0 nylon monofilament (Suprama,Berlin, Germany) coated with silicon resin (HeraeusKulzer, Hanau, Germany) was introduced through a smallincision in the common carotid artery and advanced to aposition 9 mm distal from the carotid bifurcation forocclusion of the MCA. Rectal temperature was controlledand kept constant at 36.51C±0.51C by means of a feedbacktemperature control unit. After 1 hour, the monofilamentwas removed under isoflurane anesthesia to allow reperfu-sion of the MCA. The internal carotid artery was thenligated and the skin incision was closed.

Application of Fluorescent Annexin A5 and PropidiumIodide

Both annexin A5 preparations were diluted to a volume of150mL in saline and 7 nmol of Cy5.5/mouse (inactiveannexin A5: about 0.1 mg of protein; active annexin A5:about 0.3 mg of protein) were injected intravenously intothe tail vein 48 hours after MCAO. Eight mice were injectedwith inactive annexin A5 and NIRF imaging was per-formed 4 hours (n = 5) or 8 hours (n = 3) postinjection.Three of the mice imaged 4 hours after injection of inactiveannexin A5 were also injected with PI (Molecular Probes,Eugene, OR, USA; 20 mg/kg body weight, diluted in 150 mLsaline). Thirteen mice received active annexin A5 andNIRF imaging was performed 4 hours (n = 8) or 8 hours(n = 5) postinjection. Three of the mice imaged 4 hours afteractive annexin A5 injection were also injected with PI.

Near-Infrared Fluorescence Imaging Protocol

Before NIRF imaging, the mice were anesthetized byintraperitoneal injection of a 5% chloralhydrate solution(200 mg/kg; Merck, Darmstadt, Germany) and the skinoverlying the parietal cortex was depilated. For NIRFimaging, we used a system developed by our group, whichwe recently described in detail (Klohs et al, 2006). Thesystem enables noninvasive detection of amounts of Cy5.5dye as low as 10�12 mol located deep in the brain of adultpigmented mice. For NIRF imaging of Cy5.5 (absorptionmaximum at 675 nm, emission maximum at 694 nm), weapplied the fluorescence reflectance imaging mode withthe light source for illumination and the detector both onthe same side of the object. The animal’s head wasilluminated with a laser (applied power: 40 mW, wave-length: 682 nm). Fluorescence emission was captured witha charge-coupled device camera equipped with two 710 nminterference filters to block excitation light. Data acquisi-tion time was 60 seconds for noninvasive in vivo NIRFimaging and between 30 and 60 seconds for ex vivo NIRFimaging of the brains removed from the skull and the 1 mmbrain slices, respectively.

Data Processing and Analysis of the Near-InfraredFluorescence Images

Data were processed and analyzed as described previously(Klohs et al, 2006). This included normalization, correctionfor illumination inhomogeneities, and subtraction of

superficial fluorescence from dust or photons from cosmicrays. Region of interest (ROI) analysis was performed withthe normalized noninvasive NIRF images by manuallyselecting anatomically defined ROIs over the right and lefthemispheres of the mice (outlined in Figure 1A). Theaverage fluorescence intensity of all pixels within the ROIwas calculated. Target-to-background ratios (TBRs) werecalculated by dividing the ROI values from the lefthemisphere by the ROI values from the right hemisphere.For statistical comparison of the TBR, an unpairedStudent’s t-test was used. P-values below 0.05 wereconsidered significant.

Infarct Staining with Triphenyl Tetrazolium Chloride

The brains of the mice that did not receive PI underwentinfarct staining with triphenyl tetrazolium chloride (TTC).After noninvasive and ex vivo NIRF imaging, 1 mm coronalbrain slices were cut in a brain matrix using a razor blade.After NIRF imaging, the slices were then incubated in a 2%TTC solution (Sigma-Aldrich, Hamburg, Germany) at 371Cfor 30 minutes. In viable brain tissue, TTC is converted bymitochondria to appear red in color, while the ischemicarea remains colorless. Digital photographs of the TTC-stained slices were taken for documentation.

Histology and Microscopy

The brains of mice that received PI underwent histologyand microscopy. After noninvasive and ex vivo NIRFimaging, the brains were snap-frozen in 2-methylbutaneat �401C and stored at �801C until sectioning. The braintissue was cut in 20 mm coronal cryostat sections takenbetween bregma 1.6 to �1.3 mm. The sections were firstexamined by fluorescence microscopy using a Leica DMRAmicroscope (Leica Microsystems, Heidelberg, Germany)equipped with a nitrogen-cooled charge-coupled devicecamera (Vers Array 512B, Roper Scientific Inc., Duluth,GA, USA) and appropriate filters for Cy5.5, PI, and FITCdetection. The sections were then stained using a TUNELKit (TUNEL stain, ApopTag Fluorescein In Situ ApoptosisDetection Kit, Chemicon International, Temecula, CA,USA). A second fluorescence microscopy was performed,and finally, the sections were stained with HE andexamined with the microscope equipped with a digitalcamera (DC200, Leica Microsystems). The percentage ofcells in the ischemic area (striatum and cortex) thatshowed no labeling/staining and the number of cellspositive for annexin A5 and/or PI, and/or TUNEL stainingwere calculated by examining three sections from each mouse.

Results

Noninvasive Near-Infrared Fluorescence Imaging andEx Vivo Near-Infrared Fluorescence Imaging of theBrains

NIRF imaging was performed 4 and 8 hours afterintravenous injection of either Cy5.5-labeled active

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or inactive annexin A5. Injections were performed inMCAO mice 48 hours after reperfusion. In Figure 1,noninvasive NIRF images of the heads of MCAOmice and respective ex vivo NIRF images of thebrains removed from the skull 4 hours postinjectionof either fluorescent labeled inactive or activeannexin A5 are shown. At both time points, nodifferences were seen between the hemispheres onthe noninvasive NIRF images in mice injected withinactive annexin A5, but mice injected with activeannexin A5 showed higher fluorescence intensitiesover the ischemic hemisphere than over the contral-ateral side. The images of the brains removed fromthe skull showed slightly higher fluorescence in-tensities over the ipsilateral compared with thecontralateral hemisphere after injection of inactiveannexin A5. However, only after injection of activeannexin A5 was the fluorescence intensity consider-ably higher over the ischemic hemisphere than overthe contralateral side. Figure 2 shows the TBRscalculated from noninvasive NIRF images for inac-tive and active annexin A5 at both time points.MCAO mice that received active annexin A5 showedsignificantly higher TBR at 4 and 8 hours postinjec-

tion than the mice that received inactive annexin A5,respectively (1.16±0.07 versus 1.02±0.02 at 4 hours,P = 0.002; 1.18±0.08 versus 1.01±0.01 at 8 hours,P = 0.008).

Near-Infrared Fluorescence Imaging of the Brain Slicesand Triphenyl Tetrazolium Chloride Staining

After noninvasive and ex vivo NIRF imaging, 1 mmcoronal brain sections were obtained and NIRFimaging of the sections was performed. Immediatelyafterwards, the sections were incubated with TTC fordemarcation of the lesion. Figure 3 shows NIRFimages of brain sections before and after TTCstaining 4 hours postinjection of either fluorescentinactive or active annexin A5. At both time points,areas with high fluorescence intensities closelycorresponding with the TTC staining were observedin mice that had received active and inactiveannexin A5. Lower fluorescence intensities over theinfarct region were seen in animals injected withinactive annexin A5 compared with the mice thatreceived active annexin A5.

Figure 1 Noninvasive near-infrared fluorescence (NIRF) images of the heads of middle cerebral artery occlusion (MCAO) mice (A, C)and the corresponding ex vivo NIRF images of the brains removed from the skull (B, D) 4 hours after intravenous injection of eitherfluorescent labeled inactive or active annexin A5. The images have the same scaling indicated by the color bar. An example ofanatomically defined regions of interest (ROIs) selected over the right and left hemisphere for calculation of target-to-backgroundratios (TBRs) is shown in A. After injection of both compounds, the noninvasive images of the heads of the mice show strongfluorescence intensities from vessels, especially from the sinus confluens as indicated by the asterisk (C). Strong fluorescence signalsover the stroke area were only seen after injection of active annexin A5 (see dotted line in C).

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Fluorescence Microscopy

After examining the 1-mm brain sections with NIRFimaging, we realized that a strong signal in the areaof the ventricles was observed at both time pointsin all mice. The same was observed in the 20-mmsections. Figure 4A depicts a 20 mm brain section4 hours after injection of active annexin A5, withhigh fluorescent intensities in the ischemic regionand even stronger fluorescence signals in theventricles. Figures 4B–4E show microscopic imagesof a tissue section, also 4 hours after injection ofactive annexin A5 and from the same area demar-cated on the NIRF image in Figure 4A. In theinfarcted area (upper left), strong fluorescencesignals from injected active annexin A5 and injectedPI were detected and the majority of the cells werepositive for TUNEL staining. In contrast, the non-infarcted regions showed no fluorescence frominjected annexin A5 and PI and a negligible numberof cells were TUNEL-positive. The highest fluores-cence intensities from active annexin A5 and PI werelocated in the choroid plexus in the ventricles,whereas only a few cells in this tissue wereTUNEL-positive.

Figure 5 shows microscopic images of 20mm tissuesections 4 hours after injection of either inactiveannexin A5 or active annexin A5 and PI. Thesections were subsequently stained with TUNELand then with HE. Larger vessels showed highfluorescence intensities after injection of inactiveand active annexin A5. Only in mice that receivedactive annexin A5, but not in those injected withinactive annexin A5, were high fluorescence signalsdetected in cells of the ischemic area. Most cellspositive for active annexin A5 were also PI- andTUNEL-positive. Also of note, annexin A5, PI as wellas TUNEL staining were found in the whole infarctarea and delineated the infarct area similar to whatcan be seen on the HE-stained section.

Single Cell Analysis

The percentage of cells showing a distinct labelingpattern was calculated from three sections from eachmouse (Table 1). In the striatum, B59% of the cellswere positive for fluorescent annexin A5, in thecortex about 53%. In both regions, the majority ofcells positive for fluorescent annexin A5 were alsopositive for PI and TUNEL staining (striatum: 84%,cortex: 89%). Most of the remaining cells positive forannexin A5 were at least PI positive. Only a few cellspositive for annexin A5 showed no other label. It hasto be noted that a considerable fraction of the totalnumber of cells showed no annexin A5 signal butwere positive for PI and TUNEL staining or onlypositive for PI (striatum: 18%, cortex: 16%). Thenumber of cells only positive for TUNEL wasnegligible.

Discussion

Our study demonstrates the high specificity ofannexin A5 for the in vivo visualization of lethallydamaged or dead cells in a mouse model of cerebralischemia. This is supported, for the first time, by datathat include both an active PS-binding annexin A5and an appropriate control compound, inactiveannexin A5 without binding properties for PS. Alsofor the first time, the specificity of annexin A5 fordamaged or dead cells is demonstrated on themicroscopic level by comparing the in vivo distribu-tion of active and inactive annexin A5 with other celldamage or cell death markers, namely PI and TUNELstaining. However, we also observed unspecificaccumulation of both annexin A5 forms in thechoroid plexus. Taken together, we conclude thatactive annexin A5, which can be easily labeled withdifferent signal molecules detectable with differentimaging modalities (SPECT, PET, or MRI), is asuitable marker for the visualization of tissue damagein mouse models of cerebral ischemia and it mightalso be useful in patients with stroke.

Figure 2 Target-to-background ratios (TBRs) calculated fromnoninvasive near-infrared fluorescence (NIRF) images 4 and8 hours after intravenous injection of either fluorescent labeledinactive or active annexin A5. TBR’s were calculated by dividingthe average fluorescence intensity of all pixels within the regionof interest (ROI) of the left hemisphere by the values from theright hemisphere. The TBR for each individual mouse and themeans±s.d. are given. Middle cerebral artery occlusion (MCAO)mice that received active annexin A5 showed significantly higherTBR 4 and 8 hours postinjection compared with mice thatreceived inactive annexin A5 (Student’s t-test, *P = 0.002,**P = 0.008).

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To claim ‘specificity’ of an imaging agent, anappropriate control experiment is mandatory. Underpathological conditions, the perfusion of the tissueand the permeability of vessels that can lead toextravasation of molecules can change dramaticallycompared with normal tissue. In many brain dis-eases, the BBB is impaired, potentially leading toextravasation of compounds into the parenchymaand thus to unspecific probe accumulation. This isparticularly relevant for imaging in brain tumors,multiple sclerosis, and in stroke (Ballabh et al, 2004;Zlokovic, 2008). Thus, the signal measured afterinjection of an imaging agent might be, at leastpartially, due to unspecific extravasation. This effectcan be very prominent and is the underlyingprinciple of contrast generation in Gd-enhancedMRI of BBB disruption. In validation studies, controlexperiments can be performed with knockout micelacking the target molecule, as we have shown inprevious studies (Klohs et al, 2008, 2009a). In thecurrent study, we used inactive annexin A5 as acontrol (Schellenberger et al, 2004b). At both 4 and8 hours after injection, mice with both active andinactive annexin A5 showed higher fluorescenceintensities in the ischemic hemisphere comparedwith the contralateral side. However, the TBR ofinactive annexin A5 was considerably lower thanthat of the mice injected with active annexin A5(Figures 1–3). Therefore, the signal detected in the

ischemic hemisphere from inactive annexin A5 canbe attributed to extravasation. Accordingly, a part ofthe signal detected after injection of the activecompound is also caused by BBB impairment.However, we cannot define the exact percentageattributable to leakage, since absolute quantificationof the NIRF signals is not possible. This can beaddressed in future studies using radioactive annex-in A5 preparations labeled with different radio-isotopes, which can be detected separately.

To our knowledge, the literature on the pharma-cokinetics of annexin A5 is surprisingly sparse. ThePK of annexin A5 labeled with 99mTc and 111In hasbeen studied in rats (Greupink et al, 2009) and mice(Wen et al, 2003), respectively. A two-compartmentmodel has been postulated with a rapid eliminationof annexin A5 in the first phase ( < 10 minutes) and aslower one in the second phase ( > 60 minutes).Furthermore, there are studies on the PK of 99mTc-labeled annexin A5 in rabbit and swine models(Stratton et al, 1995; Tait et al, 1994). Owing to itssize and water solubility, annexin A5 is rapidlycleared by the kidneys with a short half-life in bloodof about an hour or less. Among the organs studied,the kidneys showed by far the highest uptake. Inhumans, > 90% of the blood activity was clearedwith a half-life of 24 minutes (Kemerink et al, 2003).In our study, we found no significant differencesbetween the TBRs at 4 and 8 hours after injection of

Figure 3 Ex vivo near-infrared fluorescence (NIRF) images of coronal brain slices (A, C) and white light images of the same sectionsafter triphenyl tetrazolium chloride (TTC) staining (B, D) taken from middle cerebral artery occlusion (MCAO) mice 4 hours afterintravenous injection of either fluorescent labeled inactive or active annexin A5. The fluorescence images have the same scalingindicated by the color bar. After injection of inactive annexin A5 only slightly higher fluorescence intensities were detected, whereasafter injection of active annexin A5 fluorescence was considerably higher over the ischemic hemisphere than over the contralateralside. Areas of higher fluorescence intensities compared with the contralateral side corresponded closely with the areas of theinfarction appearing as pale areas in the TTC stain. The infarct extends through striatal and cortical areas.

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active annexin A5. This indicates that annexin A5 isonly washed out slowly from the lesion and thatimaging might be performed at comparably late timepoints after injection.

The TBR after injection of active NIRF-annexin A5is clearly lower ( < 1.2) than that found in similarstudies with Cy5.5-labeled compounds. For example,TBRs above 1.5 are typically produced when usingan antibody directed against the proinflammatoryCD40 receptor (Klohs et al, 2008) or with a matrixmetalloproteinases-cleavable macromolecular ima-ging agent (Klohs et al, 2009a) or fluorescentlylabeled albumin (Klohs et al, 2009b). The low TBRin our study is, in part, attributable to unspecificsignals from the ventricles (Figure 4), which werenot present in the earlier studies, and lead toa comparably low TBR. However, the analysis ofuptake ratios in ischemic compared with normalbrain areas in animal models using tomographicimaging systems such as SPECT are, due to theirbetter spatial resolution, expected to be less influ-enced by the background signal in the ventricles.

To further evaluate the specificity of systemicallyadministered annexin A5, we compared its distribu-tion with that of intravenously injected PI andTUNEL staining. The choroid plexus showed fluor-escence signals from both PI and active annexin A5that were even stronger than that of the ischemicarea, whereas only a few cells were positive forTUNEL (Figure 4). The choroid plexus separates thecerebrospinal fluid containing ventricles and spinalcanal from the blood. The blood–cerebrospinal fluidbarrier, like the BBB, consists of tight junctions(Nabeshima et al, 1975), but is more permeable thanthe BBB (Johanson et al, 2008). It remains unclearwhy annexin A5 and PI bind to the choroid plexus,while other molecules such as antibodies or albumindo not. Radiolabeled annexin A5 has already beenused in animal and human studies of stroke withoutsuch a characterization (Blankenberg et al, 2006).Some of the images of these studies are indicative ofunspecific signal from the choroid plexus, especiallyin the paper by Blankenberg et al (2006). The strongsignal in the area of the ventricles detected withSPECT can be blood pool, as discussed in the paperor accumulation of the compound in the choroidplexus or both. From the images presented in thatpaper, this cannot be ruled out, but it is neithermentioned nor further investigated.

The specificity of active Cy5.5-annexin A5 fordead or lethally damaged cells is also supported byour single cell analysis. On the microscopic level,cells in the ischemic area with high fluorescenceintensity were only detected for active annexin A5but not for inactive annexin A5. The majority ofthese cells were also PI- and TUNEL-positive. Somecells were also only PI- and TUNEL-positive or onlyPI-positive (see Table 1). This can be explained bylimited delivery of fluorescent annexin A5 ordifferent detection sensitivities. Our results corre-spond to the findings by Unal Cevik and Dalkara

Figure 4 Ex vivo near-infrared fluorescence (NIRF) image of abrain slice showing the distribution of active annexin A5 (A) andmicroscopic images of a tissue section from the area markedon the NIRF image (B–E) taken from a mouse 4 hours afterintravenous injection of active annexin A5 (B) and propidiumiodide (PI) (C), which was subsequently stained with terminaldeoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-tripho-sphate-biotin nick end labeling (TUNEL) (D), and finally withhematoxylin and eosin (HE) (E). High fluorescence intensitiesfrom injected annexin A5 and PI were not only detected in theischemic region (upper left), but also in the choroid plexus in theventricles (see red arrows), while only a few cells in this tissuewere TUNEL-positive.

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(2003). They compared systemically administered PI(0.5 mg injected into the ipsilateral ventricle or 20 mg/kg injected intravenously) with subsequent TUNELstaining at 6 and 72 hours in two mice models offocal cerebral ischemia (30 minutes of transientMCAO with either intravenous or intracerebroven-tricular injection and permanent MCAO injectedintracerebroventricularly). The authors showed thatonly discrete populations of cells positive for eitherPI or TUNEL were detected in areas with mild

ischemic injury in the 30-minute model. In regions ofmore severe ischemic damage, most of the cells had amixed phenotype of both markers, in addition tocells labeled with only one of the markers, which issimilar to our study. However, the fraction of onlyPI-positive cells in our study was larger, and wedetected only a few exclusively TUNEL-positivecells. These differences might be attributed todifferences in the animal models used. For example,Unal Cevik and Dalkara found that only a few cellswere positive for PI and TUNEL at 6 hours afterischemia. As we have not studied early time pointsafter ischemia, it is not clear whether the stainingpattern (annexin A5, PI, and TUNEL) is different atearly and late time points after reperfusion. Thisremains to be determined in future studies usingfluorescent annexin A5.

Cell death can be classified according to morpho-logical, enzymological, functional, or immunologicalcharacteristics (Galluzzi et al, 2009; Kroemer et al,2009). Neither TUNEL staining, annexin A5 binding,nor PI uptake is specific for a certain type of celldeath. Further complexity ensues with the differentsensitivities and specificities of the methods used toevaluate cell death. In addition, some of the ‘celldeath markers’ may bind to cells that are not yet fullycommitted to cell death, and may be ‘rescued’ undercertain conditions. For instance, Yang et al (2002)

Figure 5 Microscopic images of tissue sections taken from middle cerebral artery occlusion (MCAO) mice 4 hours after intravenousinjection of propidium iodide (PI) and either inactive annexin A5 (A) or active annexin A5 (B, C; C shows the area boxed in B at ahigher magnification). Sections were subsequently stained with terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate-biotin nick end labeling (TUNEL) and hematoxylin and eosin (HE). Fluorescence from inactive and active annexin A5was associated with larger vessels. In contrast to mice that received inactive annexin A5, additional signal was detected in cells of theischemic area of mice injected with active annexin A5. The majority of these signals corresponded closely with the signals from PIand the TUNEL stain.

Table 1 Percentage of cells in the striatum and the cortex of thebrain showing certain labeling patterns

% Cells showing Striatum Cortex

Mean±s.d. Mean±s.d.

No label 23.4±7.0 31.0±9.0A+PI+TU 49.6±9.0 47.0±8.4A+PI 8.1±3.7 4.4±1.3A 1.4±0.7 1.7±0.5PI+TU 6.6±4.0 7.0±2.6PI 11.2±3.3 8.5±2.8

PI, propidium iodide; TUNEL, terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate-biotin nick end labeling.In both regions, the majority of cells positive for fluorescent annexin A5 werealso positive for PI and TUNEL staining (n = 3 mice each).

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described reversible PS expression on the cellsurface of granulocytes and monocytes. Kenis et al(2010) demonstrated reversible PS expression incardiomyocytes subjected to brief episodes of ische-mia. Although we are not aware of any data onreversible PS expression in the brain after stroke, wecannot exclude it. Taken together, we believe thatannexin A5 can be used as a marker for ‘dead cells’ or‘lethally damaged cells’, without further qualifica-tion of the type of cell death.

In summary, we have shown that cell deathinduced by cerebral ischemia can be specificallyvisualized using NIRF imaging of annexin A5 in amouse model of stroke. Imaging of annexin A5 maybe applied to further study cell death in animalmodels of stroke and other diseases. As annexin A5can be labeled for SPECT, PET, or MRI, it might alsoprove useful in the diagnosis and therapy monitoringof stroke and other diseases in humans, providedthat the resolution of the imaging approach allowsfor the discrimination of unspecific signal enhance-ment in the ventricles.

Acknowledgements

The authors thank Ingo Przesdzing for his support inthe histological parts of the study.

Disclosure/conflict of interest

The authors declare no conflict of interest.

References

Ballabh P, Braun A, Nedergaard M (2004) The blood-brainbarrier: an overview: structure, regulation, and clinicalimplications. Neurobiol Dis 16:1–13

Belhocine T, Steinmetz N, Hustinx R, Bartsch P, JerusalemG, Seidel L, Rigo P, Green A (2002) Increased uptakeof the apoptosis-imaging agent (99m)Tc recombinanthuman Annexin V in human tumors after one courseof chemotherapy as a predictor of tumor response andpatient prognosis. Clin Cancer Res 8:2766–74

Blankenberg FG (2008a) Monitoring of treatment-inducedapoptosis in oncology with PET and SPECT. Curr PharmDes 14:2974–82

Blankenberg FG (2008b) In vivo imaging of apoptosis.Cancer Biol Ther 7:1525–32

Blankenberg FG, Kalinyak J, Liu L, Koike M, Cheng D,Goris ML, Green A, Vanderheyden JL, Tong DC, YenariMA (2006) 99mTc-HYNIC-annexin V SPECT imaging ofacute stroke and its response to neuroprotective therapywith anti-Fas ligand antibody. Eur J Nucl Med MolImaging 33:566–74

Dirnagl U, Iadecola C, Moskowitz MA (1999) Pathobiologyof ischaemic stroke: an integrated view. Trends Neurosci22:391–7

Dumont EA, Reutelingsperger CP, Smits JF, Daemen MJ,Doevendans PA, Wellens HJ, Hofstra L (2001) Real-timeimaging of apoptotic cell-membrane changes at the single-cell level in the beating murine heart. Nat Med 7:1352–5

Faust A, Hermann S, Wagner S, Haufe G, Schober O,Schafers M, Kopka K (2009) Molecular imaging of apoptosisin vivo with scintigraphic and optical biomarkers–a statusreport. Anticancer Agents Med Chem 9:968–85

Galluzzi L, Aaronson SA, Abrams J, Alnemri ES, AndrewsDW, Baehrecke EH, Bazan NG, Blagosklonny MV,Blomgren K, Borner C, Bredesen DE, Brenner C, CastedoM, Cidlowski JA, Ciechanover A, Cohen GM, DeLaurenzi V, De Maria R, Deshmukh M, Dynlacht BD,El-Deiry WS, Flavell RA, Fulda S, Garrido C, Golstein P,Gougeon ML, Green DR, Gronemeyer H, Hajnoczky G,Hardwick JM, Hengartner MO, Ichijo H, Jaattela M,Kepp O, Kimchi A, Klionsky DJ, Knight RA, KornbluthS, Kumar S, Levine B, Lipton SA, Lugli E, Madeo F,Malomi W, Marine JC, Martin SJ, Medema JP, Mehlen P,Melino G, Moll UM, Morselli E, Nagata S, NicholsonDW, Nicotera P, Nunez G, Oren M, Penninger J, PervaizS, Peter ME, Piacentini M, Prehn JH, Puthalakath H,Rabinovich GA, Rizzuto R, Rodrigues CM, RubinszteinDC, Rudel T, Scorrano L, Simon HU, Steller H, TschoppJ, Tsujimoto Y, Vandenabeele P, Vitale I, Vousden KH,Youle RJ, Yuan J, Zhivotovsky B, Kroemer G (2009)Guidelines for the use and interpretation of assays formonitoring cell death in higher eukaryotes. Cell DeathDiffer 16:1093–107

Greupink R, Sio CF, Ederveen A, Orsel J (2009) Evaluationof a 99mTc-labeled AnnexinA5 variant for non-invasiveSPECT imaging of cell death in liver, spleen andprostate. Pharm Res 26:2647–56

Hara H, Huang PL, Panahian N, Fishman MC, MoskowitzMA (1996) Reduced brain edema and infarction volumein mice lacking the neuronal isoform of nitric oxidesynthase after transient MCA occlusion. J Cereb BloodFlow Metab 16:605–11

Harraz MM, Dawson TM, Dawson VL (2008) Advances inneuronal cell death 2007. Stroke 39:286–8

Heckl S (2007) Future contrast agents for molecularimaging in stroke. Curr Med Chem 14:1713–28

Hofstra L, Liem IH, Dumont EA, Boersma HH, van HeerdeWL, Doevendans PA, De Muinck E, Wellens HJ,Kemerink GJ, Reutelingsperger CP, Heidendal GA(2000) Visualisation of cell death in vivo in patientswith acute myocardial infarction. Lancet 356:209–12

Johanson CE, Duncan III JA, Klinge PM, Brinker T, StopaEG, Silverberg GD (2008) Multiplicity of cerebrospinalfluid functions: new challenges in health and disease.Cerebrospinal Fluid Res 5:10

Kemerink GJ, Liu X, Kieffer D, Ceyssens S, Mortelmans L,Verbruggen AM, Steinmetz ND, Vanderheyden JL, GreenAM, Verbeke K (2003) Safety, biodistribution, anddosimetry of 99mTc-HYNIC-annexin V, a novel humanrecombinant annexin V for human application. J NuclMed 44:947–52

Kenis H, Zandbergen HR, Hofstra L, Petrov AD, DumontEA, Blankenberg FD, Haider N, Bitsch N, Gijbels M,Verjans JW, Narula N, Narula J, Reutelingsperger CP(2010) Annexin A5 uptake in ischemic myocardium:demonstration of reversible phosphatidylserine exter-nalization and feasibility of radionuclide imaging.J Nucl Med 51:259–67

Kietselaer BL, Reutelingsperger CP, Heidendal GA, DaemenMJ, Mess WH, Hofstra L, Narula J (2004) Noninvasivedetection of plaque instability with use of radiolabeledannexin A5 in patients with carotid-artery atherosclero-sis. N Engl J Med 350:1472–3

Klohs J, Baeva N, Steinbrink J, Bourayou R, Boettcher C,Royl G, Megow D, Dirnagl U, Priller J, Wunder A (2009a)

Visualization of cell death in mice with focal cerebral ischemiaP Bahmani et al

1319

Journal of Cerebral Blood Flow & Metabolism (2011) 31, 1311–1320

In vivo near-infrared fluorescence imaging of matrixmetalloproteinase activity after cerebral ischemia.J Cereb Blood Flow Metab 29:1284–92

Klohs J, Grafe M, Graf K, Steinbrink J, Dietrich T, Stibenz D,Bahmani P, Kronenberg G, Harms C, Endres M, LindauerU, Greger K, Stelzer EH, Dirnagl U, Wunder A (2008) Invivo imaging of the inflammatory receptor CD40 aftercerebral ischemia using a fluorescent antibody. Stroke39:2845–52

Klohs J, Steinbrink J, Bourayou R, Mueller S, Cordell R,Licha K, Schirner M, Dirnagl U, Lindauer U, Wunder A(2009b) Near-infrared fluorescence imaging with fluor-escently labeled albumin: a novel method for non-invasive optical imaging of blood-brain barrier impair-ment after focal cerebral ischemia in mice. J NeurosciMethods 180:126–32

Klohs J, Steinbrink J, Nierhaus T, Bourayou R, Lindauer U,Bahmani P, Dirnagl U, Wunder A (2006) Noninvasivenear-infrared imaging of fluorochromes within the brainof live mice: an in vivo phantom study. Mol Imaging5:180–7

Kolodgie FD, Petrov A, Virmani R, Narula N, Verjans JW,Weber DK, Hartung D, Steinmetz N, Vanderheyden JL,Vannan MA, Gold HK, Reutelingsperger CP, Hofstra L,Narula J (2003) Targeting of apoptotic macrophages andexperimental atheroma with radiolabeled annexin V: atechnique with potential for noninvasive imaging ofvulnerable plaque. Circulation 108:3134–9

Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, AlnemriES, Baehrecke EH, Blagosklonny MV, El-Deiry WS,Golstein P, Green DR, Hengartner M, Knight RA, KumarS, Lipton SA, Malorni W, Nunez G, Peter ME, Tschopp J,Yuan J, Piacentini M, Zhivotovsky B, Melino G (2009)Classification of cell death: recommendations of theNomenclature Committee on Cell Death 2009. CellDeath Differ 16:3–11

Laufer EM, Winkens HM, Corsten MF, Reutelingsperger CP,Narula J, Hofstra L (2009) PET and SPECT imaging ofapoptosis in vulnerable atherosclerotic plaques withradiolabeled Annexin A5. Q J Nucl Med Mol Imaging53:26–34

Loo DT, Rillema JR (1998) Measurement of cell death.Methods Cell Biol 57:251–64

Lorberboym M, Blankenberg FG, Sadeh M, Lampl Y (2006)In vivo imaging of apoptosis in patients with acutestroke: correlation with blood-brain barrier permeabil-ity. Brain Res 1103:13–9

Moore A, Donahue CJ, Bauer KD, Mather JP (1998)Simultaneous measurement of cell cycle and apoptoticcell death. Methods Cell Biol 57:265–78

Nabeshima S, Reese TS, Landis DM, Brightman MW (1975)Junctions in the meninges and marginal glia. J CompNeurol 164:127–69

Petrovsky A, Schellenberger E, Josephson L, Weissleder R,Bogdanov Jr A (2003) Near-infrared fluorescent imagingof tumor apoptosis. Cancer Res 63:1936–42

Schellenberger EA, Bogdanov Jr A, Hogemann D, Tait J,Weissleder R, Josephson L (2002) Annexin V-CLIO: ananoparticle for detecting apoptosis by MRI. MolImaging 1:102–7

Schellenberger EA, Sosnovik D, Weissleder R, Josephson L(2004a) Magneto/optical annexin V, a multimodalprotein. Bioconjug Chem 15:1062–7

Schellenberger EA, Weissleder R, Josephson L (2004b)Optimal modification of annexin V with fluorescentdyes. Chembiochem 5:271–4

Sosnovik DE, Schellenberger EA, Nahrendorf M, NovikovMS, Matsui T, Dai G, Reynolds F, Grazette L, Rosenz-weig A, Weissleder R, Josephson L (2005) Magneticresonance imaging of cardiomyocyte apoptosis with anovel magneto-optical nanoparticle. Magn Reson Med54:718–24

Stratton JR, Dewhurst TA, Kasina S, Reno JM, CerqueiraMD, Baskin DG, Tait JF (1995) Selective uptake ofradiolabeled annexin V on acute porcine left atrialthrombi. Circulation 92:3113–21

Tait JF, Cerqueira MD, Dewhurst TA, Fujikawa K, RitchieJL, Stratton JR (1994) Evaluation of annexin V as aplatelet-directed thrombus targeting agent. Thromb Res75:491–501

Tang XN, Wang Q, Koike MA, Cheng D, Goris ML,Blankenberg FG, Yenari MA (2007) Monitoring theprotective effects of minocycline treatment with radi-olabeled annexin V in an experimental model of focalcerebral ischemia. J Nucl Med 48:1822–8

Thimister PW, Hofstra L, Liem IH, Boersma HH, KemerinkG, Reutelingsperger CP, Heidendal GA (2003) In vivodetection of cell death in the area at risk in acutemyocardial infarction. J Nucl Med 44:391–6

Unal Cevik I, Dalkara T (2003) Intravenously administeredpropidium iodide labels necrotic cells in the intactmouse brain after injury. Cell Death Differ 10:928–9

van Tilborg GA, Vucic E, Strijkers GJ, Cormode DP, Mani V,Skajaa T, Reutelingsperger CP, Fayad ZA, Mulder WJ,Nicolay K (2010) Annexin A5-functionalized bimodalnanoparticles for MRI and fluorescence imaging ofatherosclerotic plaques. Bioconjug Chem 21:1794–803

Wen X, Wu QP, Ke S, Wallace S, Charnsangavej C, Huang P,Liang D, Chow D, Li C (2003) Improved radiolabeling ofPEGylated protein: PEGylated annexin V for noninva-sive imaging of tumor apoptosis. Cancer Biother Radio-pharm 18:819–27

Wolters SL, Corsten MF, Reutelingsperger CP, Narula J,Hofstra L (2007) Cardiovascular molecular imaging ofapoptosis. Eur J Nucl Med Mol Imaging 34(Suppl1):S86–98

Yang MY, Chuang H, Chen RF, Yang KD (2002) Reversiblephosphatidylserine expression on blood granulocytesrelated to membrane perturbation but not DNA strandbreaks. J Leukoc Biol 71:231–7

Zlokovic BV (2008) The blood-brain barrier in health andchronic neurodegenerative disorders. Neuron 57:178–201

Visualization of cell death in mice with focal cerebral ischemiaP Bahmani et al

1320

Journal of Cerebral Blood Flow & Metabolism (2011) 31, 1311–1320