Purinergic receptor P2RY12-dependent microglialclosure of the injured blood–brain barrierNanhong Loua,1, Takahiro Takanoa,1, Yong Peia, Anna L. Xaviera, Steven A. Goldmana,b, and Maiken Nedergaarda,b,2

aCenter for Translational Neuromedicine, University of Rochester Medical School, Rochester, NY 14642; and bCenter for Translational Neuromedicine,Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark

Edited by Ben A. Barres, Stanford University School of Medicine, Stanford, CA, and approved December 9, 2015 (received for review October 16, 2015)

Microglia are integral functional elements of the central nervoussystem, but the contribution of these cells to the structural integrityof the neurovascular unit has not hitherto been assessed. We showhere that following blood–brain barrier (BBB) breakdown, P2RY12(purinergic receptor P2Y, G-protein coupled, 12)-mediated chemotaxisof microglia processes is required for the rapid closure of the BBB.Mice treated with the P2RY12 inhibitor clopidogrel, as well as thosein which P2RY12 was genetically ablated, exhibited significantlydiminishedmovement of juxtavascular microglial processes and failedto close laser-induced openings of the BBB. Thus, microglial cells playa previously unrecognized protective role in the maintenance of BBBintegrity following cerebrovascular damage. Because clopidogrel an-tagonizes the platelet P2Y12 receptor, it is widely prescribed for pa-tients with coronary artery and cerebrovascular disease. As such,these observations suggest the need for caution in the postincidentcontinuation of P2RY12-targeted platelet inhibition.

purinergic receptors | microglia | blood–brain barrier | stroke | clopidogrel

The resident immune cells of the central nervous system(CNS) play a variety of roles in both CNS development and

homeostatic maintenance (1). During development, microgliaengulf apoptotic immature neurons and prune synapses toeliminate redundant or inappropriate synaptic connections (2–5). In addition, microglia release a variety of both neurotrophicand gliotrophic factors (6), whereas antiinflammatory M2-stagemicroglia have been shown to accelerate oligodendrocytic dif-ferentiation during remyelination (7). In addition, a number ofstudies have highlighted microglial activation as a hallmark ofneurodegenerative disease (8, 9), as well as in the tissue response toacute ischemic and traumatic brain injury (10, 11). However, de-spite this multiplicity of roles, microglia have not hitherto beenviewed as significant contributors to maintenance of the blood–brain barrier (BBB), despite the fact that a large fraction of juxta-vascular microglial cells are localized in the perivascular space (12).Recent observations show that a coordinated microglial response

to cortical compression injury can reinforce the glial limitans. Inresponse to meningeal cell death, microglia extended processesthrough the compromised glial limitans into the meninges, forminga stable contiguous network resembling a “honeycomb” structureconcurrently with their other retracted processes. The honeycombnetwork formed quickly, within an hour of injury. It was dependentupon purinergic signaling, because transcranial application ofP2RY12 (purinergic receptor P2Y, G-protein coupled, 12) orP2RX4 (purinergic receptor P2X, ligand gated ion channel, 4)inhibitors before compression injury prevented formation of thehoneycomb barrier (13). Microglial cells express high levels ofP2RY12 (14, 15), which serve as chemotactic receptors, directingmovement of microglial cell processes toward local sites of CNSinjury (16–19). We here assessed whether microglial cells viaactivation of P2RY12 receptors contribute to closures of thesmall opening of the BBB. Microscopic opening of the BBB mayoccur during the normal replacement of brain endothelial cellsas part of their life cycle or as a consequence of localizedischemic events.

The purinergic receptor P2RY12 is a clinical target in bothcardiovascular and cerebrovascular disease in that inhibition ofplatelet P2RY12 prevents ADP-induced platelet aggregation andthereby reduces the risk of thrombosis (14, 20). Since its approval in1997 by the Food and Drug Administration, clopidogrel has beenprescribed to over 52 million patients worldwide (21). Clopidogrel is athienopyridine prodrug, whose active metabolite acts as an irreversibleinhibitor of P2RY12 (22). Systemic expression of P2RY12 is limitedto platelets, so that clopidogrel exhibits few side effects in addition tothe prolongation of bleeding time and hemorrhagic risk ascribable toits anti-platelet actions. Other organ systems are essentially devoid ofP2RY12, with the exception of CNS microglial cells.Although clopidogrel reduces the risk of stroke, a large number

of treated patients nonetheless experience cerebral ischemicevents; in one study, 9% of patients taking clopidogrel suffered anischemic event on clopidogrel over a 2.4-year observation period(23). Because BBB compromise is a hallmark of stroke, clopi-dogrel and its active metabolite may thereby gain entry into theaffected CNS, resulting in the local suppression of P2RY12-mediated microglial activation within ischemic tissue.

ResultsSystemic Clopidogrel Suppresses Juxtavascular Microglial Cell ActivationAfter BBB Breakdown. The active metabolite of clopidogrel [mo-lecular weight (MW) 353 Da] has low-BBB permeability (24).Clopidogrel would thus not be expected to interfere with themovement of microglia or their ramified processes under controlconditions. To test this supposition, we evoked small focal lesionswithin the cerebral parenchyma using two-photon focused laser

Significance

Brain endothelial cells, pericytes, and astrocytes participate inmaintenance of the blood–brain barrier (BBB). Juxtavascularmicroglial cells are also an integral part of the neurovascular unit.We show here that, in response to capillary injury, microglialprocesses rapidly form a dense plexiform aggregate at the site ofinjury. Photoablation of microglial cells abolished closure of BBBleakage, whereas inhibition of P2RY12 (purinergic receptor P2Y,G-protein coupled, 12) receptors suppressed microglial pro-cess motility and prolonged BBB closure. Thus, microglial cellsmediate rapid resealing of injury-induced leaks in BBB. Theseobservations may have clinical importance as P2RY12 receptorantagonists are widely used as platelet inhibitors in patientswith coronary artery and cerebrovascular disease at risk forstroke and its attendant disruption of the injured BBB.

Author contributions: N.L., T.T., Y.P., and A.L.X. performed research; T.T., Y.P., and M.N.designed research; T.T. and Y.P. analyzed data; and N.L., T.T., Y.P., S.A.G., and M.N. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1N.L. and T.T. contributed equally to this work.2To whom correspondence should be addressed. Email: nedergaard@urmc.rochester.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1520398113/-/DCSupplemental.

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excitation. We observed a rapid chemotaxic response of nearbymicroglial cell processes in CX3CR1+/EGFP mice (Fig. 1 A and Band Movie S1). Earlier studies have shown that P2RY12 drivesmicroglial cell process movement toward focal lesions (18). Weconfirmed that mice with deletion of P2RY12 (P2RY12−/−)exhibited significantly less process accumulation around focal le-sions (Fig. 1 A and B and Movie S2). In contrast, pretreatment ofP2RY12+/+ mice with 20 mg/kg clopidogrel for 3 d before theexperiment did not suppress microglia process motility, sug-gesting that clopidogrel do not inhibit microglial P2RY12 in

the normal mouse brain in the absence of vascular injury (Fig. 1 Aand B and Movie S3). We next asked whether clopidogrel couldinhibit microglial process motility in the setting of vascular injury.The focal laser injury was targeted to induce injury in singlecapillaries, located 80–150 μm below the pial surface. The capil-lary injury was calibrated to cause minimal, nonhemorrhagicdamage, evaluated by the lack of an extravascular leakage of70 kDa of Texas Red-dextran (Fig. 1C). Similarly to brain pa-renchyma, juxtavascular microglial processes in control mice wereimmediately attracted to the site of the capillary lesion; within 20–30 min, they formed a dense sheet of EGFP+ processes plasteredaround the vessel wall (Fig. 1 C and D and Movie S4), which wassignificantly reduced in CX3CR1/P2RY12−/− mice (P < 0.05,Tukey–Kramer test) (Fig. 1 C and D and Movie S5). Moreover,mice pretreated with clopidogrel exhibited a significant suppres-sion of movement of EGFP+ juxtavascular microglial processestoward laser-injured capillaries (P < 0.01, Tukey–Kramer test) (Fig.1 C and D and Movie S6). Of note, we chose a dose of 20 mg/kgclopidogrel, which increased the bleeding time by 84.8% and re-duced platelet aggregation by 35.5% (Fig. 1E); patients receiving75 mg of clopidogrel daily experienced a mean increase in bleedingtime of ∼140% (25) and an increase in platelet aggregation time of35% (26). Clopidogrel’s only targets in the adult CNS were con-firmed to be microglial cells (14, 15, 18), because the immuno-histochemical labeling of P2RY12 colocalized with Cx3CR1-EGFP(Fig. 1F); blood-borne platelets also expressed high levels ofP2RY12 (27, 28). However, suppression of platelet activity in bloodby clopidogrel is unlikely to be the cause of juxtavascular microglialmotility reduction, because non-P2RY12–dependent plateletantagonists—acetylsalicylic acid (10 mg/kg per day for 3 d) andheparin (200 IU/kg i.v.)—did not reduce the motility of juxta-vascular microglial processes (P > 0.05, Tukey–Kramer test)(Fig. 1D), even though both agents completely suppressed he-mostasis of tail bleeding for over 20 min (n = 3–7). In addition,the same laser injury failed to initiate platelet accumulation in-side the capillary at the injured site (P > 0.05 with vs. withoutinjury, Tukey–Kramer), whereas collagen injection induced theaccumulation of platelets in random positions in capillaries (Fig.1 G and H). Taken together, whereas P2RY12 deletion reducedjuxtavascular microglial chemotaxis in response to both vascu-lar and nonvascular injury, clopidogrel suppressed only micro-glial motility when the injury was targeted to the local vascularbed, the presumed entry site of clopidogrel and its metabolites.

Motility of Juxtavascular Microglial Cells Contributes to the RapidClosure of the BBB. Our data suggest that at sites of vascular in-jury opening of the BBB may lead to influx of low-molecular-weight compounds, including clopidogrel (MW 353 Da), whichin turn suppress the P2RY12-dependent movement of juxtavas-cular microglial processes to sites of vascular injury (Fig. 2 A–D).To establish whether the laser injury indeed triggered opening ofthe BBB, we developed a technique by which BBB permeabilitycould be serially assessed (Fig. 2A). Alexa Fluor 488 (MW640 Da, 10 μL) was injected into the internal carotid artery every10 min after laser injury. Leakage across the BBB was calculatedas the peak fluorescence signal outside the capillary, divided bythe fluorescence signal inside the vessel lumen within the sameframe (Fig. 2B and Movies S7 and S8). Using this approach, wenoted that the efflux of Alexa Fluor 488 gradually decreasedafter laser injury and that the BBB defect was resealed at 39.6 ±8.6 min in P2RY12+/+ mice. Similarly, neither acetylsalicylic acidnor heparin significantly slowed the closure of BBB leakage afterinjury (P > 0.05, Tukey–Kramer test) (Fig. 2 C and D). In con-trast, both P2RY12−/− and clopidogrel-treated mice exhibitedmuch slower rates of BBB resealing (P < 0.01, Tukey–Kramertest) (Fig. 2 C and D). Because microglia are the only cells of theneurovascular unit that express detectable levels of P2RY12 (14,15, 18) (Fig. 1F), these observations suggest that juxtavascular

Fig. 1. Movement of juxtavascular microglia processes toward injured vesselsrequires P2RY12. (A) Representative time-lapse imaging of laser injury tar-geted outside the vasculature in CX3CR1+/EGFP/P2RY12+/+ mice (Upper),CX3CR1+/EGFP/P2RY12−/− mice (Middle), and CX3CR1+/EGFP/P2RY12+/+ mice re-ceiving clopidogrel (Lower). (Scale bars, 25 μm.) (B, Upper) Juxtavascularmicroglial cell process accumulation in response to laser injury outside thevasculature as shown in A. Fluorescence signal of EGFP around the capillarieswas normalized to fluorescence signal in the whole field. (Lower) Comparisonsof the kinetics of process accumulation. n = 4–11 injuries from four animals; ns,P > 0.05; **P < 0.01, Kruskal–Wallis test. (C) Time-lapse images of juxtavascularmicroglial cell activation in response to laser injury in a CX3CR1+/EGFP/P2RY12+/+

mouse. The laser was targeted to the center of a capillary located 120 μmbelow the cortical surface (yellow star). Laser injury in a CX3CR1+/EGFP/P2RY12−/−

mouse and a CX3CR1+/EGFP/P2RY12+/+ mouse treated with clopidogrel(20 mg/kg). (Scale bars, 20 μm.) (D, Upper) Kinetics of juxtavascularmicroglial process accumulation around the injured capillaries shown inC. Fluorescence signal of EGFP around the capillaries was normalized to fluo-rescence signal in the whole field. (Lower) Comparison of juxtavascularmicroglial cell processes around the injured capillary in CX3CR1+/EGFP/P2RY12+/+,CX3CR1+/EGFP/P2RY12−/−, and CX3CR1+/EGFP/P2RY12+/+ mice treated with clopi-dogrel; in CX3CR1+/EGFP/P2RY12+/+ mice treated with acetylsalicylic acid(10 mg/kg i.p.); and in CX3CR1+/EGFP/P2RY12+/+ mice treated with heparin (200IU/kg i.v.). n = 5–9 capillaries from four to eight animals; ns, P > 0.05; *P < 0.05,**P < 0.01, one-way ANOVA with Tukey–Kramer test. (E, Upper) Tail bleedingtime in vehicle control (n = 7), clopidogrel (5, 20, 30, 40, and 100 mg/kg i.p.daily for 3 d; n = 7–9), and acetylsalicylic acid (10 mg/kg, i.p. daily for 3 d, n = 5).(Lower) Platelet aggregation in whole blood from animals treated with vehiclecontrol (n = 9–15), clopidogrel (5, 20, 30, 40, and 100 mg/kg i.p. daily for 3 d;n = 8–18), and acetylsalicylic acid (10 mg/kg, i.p. daily for 3 d; n = 11).(F) P2RY12 is predominantly, if not exclusively, expressed by juxtavascularmicroglial cells. DAPI (blue), CX3CR1-EGFP (green), P2RY12 (red), Laminin(white). Orthogonal views of XYZ stacked images are shown with planes ofsections shown by white dotted lines. (Scale bar, 25 μm.) (G) Laser injury to acapillary did not induce accumulation of platelets at the injury site. Plateletswere labeled with Calcein AM (green) inside capillaries labeled with Texas Red-dextran (red). Systemic administration of collagen (1 mg/mL) caused aggrega-tions of platelets in random positions. (Scale bars, 20 μm.) (H, Upper) Kinetics ofplatelet accumulation inside the injured capillaries shown in G. (Lower) Com-parison of platelet accumulation inside the capillary at 10 min after with orwithout the laser injury or collagen administration. n = 11 capillaries from fouranimals; ns, P > 0.05; **P < 0.01; one-way ANOVA with Tukey–Kramer test.

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microglia are critical to the rapid closure of BBB defects. Ul-trastructural analysis based on electron microscopy (EM) of thelaser injury revealed, as expected, aggregation of densely packedprocesses, which completely ensheathed the site of injury. Adjacentprocesses exhibited closely apposed membrane (Fig. 3A). Immu-nolabeling revealed that the juxtavascular microglial cell processesextending toward the site of laser injury exhibited very high P2RY12expression (Fig. 3B), as well as polarized expression of the adherensjunction molecule E-cadherin. In contrast, a tight junction proteinoccludin was not detected (Fig. 3B). These observations showed thatmicroglial cell processes were in direct contact with each other afterthey aggregated around the site of injury. It is possible that theE-cadherin–positive membrane appositions will restrict diffusionbetween the lesion and surrounding tissue and thus reseal acuteBBB openings. An alternative possibility—that juxtavascular micro-glial process-mediated restriction of the injured capillary wall reducedcapillary perfusion—was not supported because capillary diameter,erythrocyte flow velocity, and flux did not differ among P2RY12+/+,P2RY12− /−, and P2RY12+/+ mice treated with clopidogrel(P > 0.05, ANOVA) (Fig. 3 C–F).To assess the role of juxtavascular microglial cells in BBB

resealing using an alternative approach, we next used laser injury toablate juxtavascular microglial cells. Pulsed two-photon laser abla-tion of EGFP+ cells yields a higher degree of localized injury thancontinuous lasers, and has been successfully used to ablate organ-elles in single cells (29), as well as to sever individual dendrites ofsensory neurons (30), and to functionally inactivate individual in-terneurons (31). The femtosecond pulsed laser was tuned for highabsorbance by EGFP (910 nm) and focused on the center of jux-tavascular microglial soma. Constant laser exposure (60–120 s)resulted in the irreversible loss of fluorescence signal in the targetedmicroglial cells (Fig. 4 A and B). Repeated collection of 3D Z-stacksconfirmed that EGFP did not recover after laser ablation, and thatablated juxtavascular microglial cells exhibited nuclear staining withpropidium iodide, a marker of irreversible membrane damage (32).Remarkably, BBB leaks failed to reseal during a 70-min observationperiod when all juxtavascular microglia located within a radius of 40μm from the capillary (six to nine cells) were ablated (Fig. 4 C and

D). Of note, no dye leak was observed after ablation of juxtavascularmicroglial cells in the absence of laser injury to the capillary (Fig.4D). Thus, the congregation of activated juxtavascular microglialprocesses at sites of capillary injury appears to contribute signifi-cantly to the closure of BBB defects following capillary injury.Clopidogrel inhibits juxtavascular microglial process movement,thereby impairs resealing of the BBB, and by so doing prolongsinjury-associated transudation.

DiscussionThe experiments in this study show that P2RY12-mediated ac-tivation of juxtavascular microglial cells contributes to the rapidclosure of small openings in the BBB, and that the movement ofmicroglial cell processes toward the site of vascular injury is a keydeterminant of how rapidly those leaks are closed (Figs. 1 and 2).Our in vivo analysis suggested that juxtavascular microglial cellprocesses aggregate to form a physical barrier, consisting ofE-cadherin–expressing membrane appositions, and that the micro-glial cuff around the vessel wall temporally assumes the functionsof the BBB lost in the setting of acute vascular injury (Fig. 3). Incontrast, laser-targeted elimination of juxtavascular microglialcells (Fig. 4), as well as both pharmacological inhibition and ge-netic deletion of P2RY12 receptors, delayed resealing of smallopenings in the BBB. Although our EM analysis suggests thatjuxtavascular microglial processes formed a physical barrier thattemporarily sealed the BBB, our data also permit the possibilitythat these microglial processes released trophic factors thataccelerated endothelial cell closure of the BBB opening. As such,this study, to our knowledge, is the first to identify a key role formicroglial cells as integral to the structural integrity of the neu-rovascular unit, and paramount in the acute closure of injury-associated BBB leaks. Juxtavascular microglia thus join pericytesand astrocytes as critical contributors to the unique barrier func-tions of brain endothelial cells (33, 34).Although most relevant studies have highlighted the relative

specificity of brain P2RY12 expression to microglial cells, somecontroversy on the point exists, in that studies have variablyreported that P2RY12 might also be expressed in brain endothelial

Fig. 2. P2RY12 is required for rapid closure of the BBB, and juxtavascular microglia processes may temporarily seal BBB openings. (A) Experimental setup. Thelarge MWweight tracer, Texas Red-dextran, was injected i.v. to outline the vasculature, and the small molecular Alexa Fluor 488 (10 μL, 80 μM) was repeatedlydelivered by a catheter inserted into the internal carotid artery every 10 min to map the duration of closure of BBB openings induced by laser injury of singlecapillaries. (B, Upper panels) Time lapse of Alexa Fluor 488 (green) passage through a control, noninjured capillary. (Lower panels) Similar time lapse of AlexaFluor 488 (green) passage through a capillary exposed to laser injury. The capillary is outlined by Texas Red-dextran (red). The dotted white square indicatesthe region used for quantification of Alexa Fluor 488 leakage. Alexa Fluor 488 leakage was defined as “peak fluorescence signal intensity outside the vesseldivided by fluorescence signal intensity inside the vessel.” (Scale bar, 10 μm.) (C) Scatter histograms of Alexa Fluor 488 leakage in P2RY12+/+ mice (black),P2RY12−/−mice (orange), P2RY12+/+ mice receiving clopidogrel (20 mg/kg, red), acetylsalicylic acid (10 mg/kg, blue), or heparin (200 IU/kg, turquoise). Differentcolor gradients indicate an individual set of capillaries. The lines indicate the average of linear regression curves, obtained by averaging the slopes andY-intercept of each regression line from a single capillary. The average regression lines were used to obtain BBB closure time (X-intercept). (D) Summaryhistogram of BBB closure time. n = 4–7 capillaries from four to seven animals; ns, P > 0.05; **P < 0.01; one-way ANOVA with Tukey–Kramer test.

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cells (35), human smooth muscle vascular cells (36), and astrocytes(37). However, these reports have without exception been based onanalysis of cultured cells or ex vivo preparations. In contrast, a re-cent transcriptome analysis showed that microglial cells are the onlycell type in brain that express P2RY12 (38), confirming earlierstudies (15, 18) as well as our own analysis (Fig. 1F). Anotherquestion is how selective clopidogrel inhibits P2RY12 receptors.Our analysis suggested that clopidogrel is a highly specific P2RY12inhibitor in accordance with the literature (39, 40) because clopi-dogrel had no effect on juxtavascular microglia process motility orthe duration of closure of small BBB openings in mice with deletionof P2RY12 (P2RY12−/− KO) (Figs. 1 B and D and 2C).Clopidogrel is widely prescribed to patients with cardio- and

cerebrovascular disease on the basis of its platelet-targeted anti-thrombotic actions, which are affected through inhibition of theplatelet P2RY12. Large prospective studies have supportedclopidogrel’s use for the prevention of both heart attack andstroke, principally as an adjunct to acetylsalicylic acid, and re-cently as prophylaxis for stent restenosis following angioplasty,in a patient population similarly at risk for coronary, carotid,and intracerebral thrombotic events; these studies have includedclopidogrel in unstable angina to prevent recurrent events (CURE)(41), clopidogrel for the reduction of events during observation(CREDO) (42), atrial fibrillation clopidogrel trail with irbesartan for

prevention of vascular events (ACTIVE) (43), and clopidogrel forhigh atherothrombotic risk and ischemic stabilization, management,and avoidance (CHARISMA) (44–46), among others. However, asignificant number of patients proceed to develop stroke de-spite optimal medical management and prophylactic clopidogrel(23). Because a hallmark of ischemic brain injury is BBB break-down (47, 48), which in turn can allow the influx of both clopi-dogrel and its active metabolite into the ischemic tissue, we askedwhether clopidogrel might the affect microglial P2RY12 re-ceptors, essentially as unintended targets. Specifically, we askedwhether clopidogrel—and its active metabolite, already present inthe bloodstream at the time of an acute stroke—might reducelocal P2RY12-dependent microglial activation, and therebyworsen stroke outcome. Our analysis showed that clopidogrel in-deed suppressed the movement of juxtavascular microglial pro-cesses toward photolytic lesions in the vessel wall, and by so doingsuppressed the closure of BBB leaks. If microglial P2RY12 plays asimilar role in humans, then these observations might have sig-nificant implications for current platelet-directed strategies for theprevention of thrombosis, which call for P2RY12 inhibition.However, two prior studies have shown that P2RY12 antagonistadministration after ischemia is beneficial: The first study inducedglobal stroke in mice and reported that clopidogrel administered5 min to 4 h after stroke reduced delayed neuronal loss aftertransient occlusion of the common carotid arteries (49). The sec-ond study exposed rats to occlusion of the middle cerebral artery

Fig. 3. Laser injury induces accumulatation of juxtavascular microglia pro-cesses and does not affect capillary perfusion. (A) Electron microscopic imageof laser injury in cerebral cortex. Yellow dotted line with a yellow star in-dicates the site of the focal injury. Green dotted line indicates the accumu-lated juxtavascular microglia processes, with arrows indicating the closeapposition of adjacent microglia processes extended toward the injury site.(B) Immunohistochemical analysis of focal laser injury site in cerebral cortexof CX3CR1+/EGFP animals. P2RY12 and E-cadherin (red) colocalized with EGFP(green) and are highly expressed in microglial cell processes encircling theinjury site. In contrast, occludin (red) was detected only in vascular endo-thelial cells, but not in microglial cells. (Scale bars, 20 μm.) (C) Time lapse of alaser-injured capillary (red) with microglia (gray) in CX3CR1+/EGFP/P2RY12+/+,CX3CR1+/EGFP/P2RY12−/−, and CX3CR1+/EGFP/P2RY12+/+ mice treated with20 mg/kg clopidogrel. (Scale bars, 10 μm.) (D) Plots of capillary diameter atthe site of laser injury plotted as a function of time in CX3CR1+/EGFP/P2RY12+/+,CX3CR1+/EGFP/P2RY12− /−, and CX3CR1+/EGFP/P2RY12+/+ mice treated with20 mg/kg clopidogrel. n = 3–5 capillaries from three to five animals. (E, Left)Strategy for collecting time series of XT line-scan images in capillaries filledwith Texas Red-dextran (red). (Right) Line scans were collected at 0–64 minafter laser injury. (F) Plots of RBC velocity and flux of capillary exposed to laserinjury in P2RY12+/+, P2RY12−/−, and P2RY12+/+ mice treated with 20 mg/kgclopidogrel. n = 5–12 capillaries from three animals.

Fig. 4. Juxtavascular microglia ablation attenuates vascular closure. (A) Ab-lation of juxtavascular microglial cell. Propidium iodide (30 μM) was appliedafter a juxtavascular microglia was ablated with focused laser radiation.Only the ablated microglial cell (blue circle) that lost EGFP fluorescence wasstained with a cell death marker propidium iodide (red). (Scale bar, 20 μm.)(B) Projection images (55 μm in z direction) and orthogonal views (XZ and YZplanes at yellow dotted lines) of a field before (Left) and after (Right) laserablation of six juxtavascular microglial cells (white with blue circles) locatedwithin a radius of 40 μm around the target capillary (red, at the crosshair) ina CX3CR1+/EGFP/P2RY12+/+ mouse. (Scale bars, 20 μm.) (C) Time series of ex-periment with ablation of juxtavascular microglial cells shows that the re-gion around the injured capillary remained free of microglial cell processesfor the duration of the experiment. No juxtavascular microglial cellprocesses were in contact with the injured capillary at 70 min. (Scale bar,20 μm.) (D) Scatter histogram of Alexa Fluor 488 leakage in microglia-ablatedCX3CR1+/EGFP/P2RY12+/+ mice. Different color gradients indicate an individ-ual set of capillaries. The line indicates the average of linear regressioncurves (y = 0.0144x–1125.84), obtained by averaging slopes and Y-intercept ofeach regression line from each capillary. Rate of BBB closure was 1.44± 0.87%/min(n = 5 capillaries), indicating that the leak worsened over time rather thangradually closing. (Inset) Scatter histogram of Alexa Fluor 488 leakage followingjuxtavascular microglial cell ablation but without laser injury to the capillary.The line indicates the average of linear regression curves (y = 0.00021x–0.01092;n = 5 capillaries).

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followed by injection of ticagrelor (a reversible P2RY12 antago-nist) 10 min to 36 h later (50). Both studies ascribed the apparentneuroprotective effect of P2RY12 inhibition to a suppression ofthe inflammatory response to ischemic cellular injury.How then can these observations be reconciled with our

findings that clopidogrel potently inhibited the repair of vascularinjury, thus potentially aggravating edema and ischemic injury?Clinically, clopidogrel must be administered for several consec-utive days to achieve a therapeutic effect, as the active metab-olite of clopidogrel is produced in the liver (51). The typicalpatient takes clopidogrel for months or years before an ischemicevent. By administering clopidogrel after the ischemic event, thetwo aforementioned studies (49, 50) would not have beenexpected to achieve functional P2RY12 inhibition until severaldays after injury, long after the acute role of microglia in rean-nealing the BBB would have been accomplished. As such, thedesigns of these studies would have effectively eliminated anydeleterious effects of clopidogrel at the time of acute injury. Incontrast, our study was designed to model a more clinically rel-evant scenario, by administering clopidogrel over 3 consecutivedays before the ischemic event, so that functional P2RY12 in-hibition would be in effect at the time of vascular occlusion, asmight be expected of a patient on clopidogrel prophylaxis whoproceeds to nonetheless have a cerebral ischemic event. Also, weselected a dose of clopidogrel that induced a clinically relevantprolongation of bleeding time and platelet aggregation, whichwas verified to be so at the time of the ischemic event. Using thisdesign, we found that clopigogrel indeed prolongs the opening ofthe BBB at the time of experimental vaso-injury and thereby mayaggravate ischemic injury in those patients who proceed to havean ischemic event while on clopidogrel as a prophylactic. To-gether, these data suggest the hitherto unappreciated impor-tance of juxtavascular microglial cells in the structural integrityand functional maintenance of the gliovascular unit and BBB,while highlighting the need for further studies modeling thepotential risks of inadvertent microglial inhibition when targetingP2RY12 for purposes of platelet inhibition.

Materials and MethodsMouse Strains. CX3CR1+/EGFP mice were purchased from Jackson Labs (strainname B6.129P-CX3CR1tm1Litt/J, stock no. 005582), P2RY12−/− mice wereobtained from the European Mutant Mouse Association (stock no.EM:02301). Both strains are in the C57BL/6J background, and CX3CR1+/EGFP/P2RY12+/+ and CX3CR1+/EGFP/P2RY12−/− littermates were generated by crossingthe two lines. All experiments were performed in accordance with protocolsapproved by Animal Use Committees at the University of Rochester.

Animal Preparation for in Vivo Imaging. The 8- to 12-wk-old male mice wereanesthetized with a mixture of ketamine (70 mg/kg) and xylazine (10 mg/kg) i.p.and artificially ventilated (SAR-830, CWE). A custom-made metal plate was gluedto the skull with dental acrylic cement, and a 1.5-mm cranial window was pre-pared over the parietal cortex (2 mm posterior and 3 mm lateral from bregma).The dura was left intact and artificial cerebrospinal fluid containing 1%agarose was placed between the window and a glass coverslip (52). Si-lastic catheters (PE10) were inserted into the left femoral artery and vein.During two-photon imaging the anesthesia was changed to isoflurane(1.2%), and body temperature was maintained at 37 °C by a circulatingwarm-water blanket (T/Pump, Stryker). Blood samples (40 μL) from the fem-oral artery were analyzed by a blood-gas analyzer (Rapidlab 248, Bayer),and experiments were completed only if physiological variables remainedwithin normal limits. The normal limits for pCO2 were set at 35–45 mm Hg;for pO2, 80–115 mm Hg; and for arterial blood pH, 7.35–7.45 (52).

In Vivo Two-Photon Laser Scanning Microscopy. A custom-built microscopeattached to a Ti:Sapphire laser (Mai Tai, SpectraPhysics), a scanning box(FV300, Olympus) operated by FluoView software (Olympus), and a 20×water-immersion objective lens (0.95 N.A., Olympus) was used for imaging.Excitation wavelength was set to 910 nm, whereas two-channel detection ofemission wavelength was achieved by a 565-nm dichroic mirror (ChromaTechnology) and two external photomultiplier tubes. A 515/50 bandpassfilter (Chroma Technology) detected EGFP and Alexa Fluor 488 (Invitrogen)

emission wavelength, and a 620/60 bandpass filter (Chroma Technology)detected Texas Red-dextran. Texas Red-dextran (MW 70 kDa, 0.1 mL of 1%in saline; Invitrogen) was administered through a femoral-vein catheterbefore imaging. Time-lapse images of CX3CR1+/EGFP microglial cells wererecorded every 10 s at a depth of 80–150 μm from the cortical surface. Thetwo-photon laser power was adjusted daily to 40 mW below the objectivelens. Individual capillaries with an average diameter of 4–6 μm were injuredby repeated line scanning (1 μm) targeted to the center of the capillary lu-men for 20 s. Tissue injury was carried out the same way except by focusingthe laser to a site devoid of blood vessels. Microglial cell ablation was ac-complished by focusing the imaging two-photon laser on the center of thesoma of EGFP+ cells. The duration of laser exposure (typically 60–120 s) wasadjusted to irreversibly ablate the EGFP signal in soma and processes. Pro-pidium iodide (30 μM; Sigma) was locally applied with a glass micropipette(tip: 2–3 μm) after ablation. Accumulation of microglial cell processes inCX3CR1+/EGFP mice was quantified as the increase in EGFP fluorescence signalsurrounding the site of laser injury in a field of ∼10 × 4 μm and normalizedto the EGFP fluorescence signal of the whole field (180 × 180 μm) (18). Forquantification of dye leakage, Alexa Fluor 488 cadaverine (10 μL of 80 μMdissolved in saline) was injected through a catheter (PE10) inserted throughthe external carotid artery into the right internal carotid artery while imagingthe injured capillary at high speed (1–1.2 Hz) for 30 s. An image frame withhighest fluorescence signal intensity outside the capillary was chosen, andAlexa Fluor 488 leakage was quantified as peak fluorescence signal intensity ina field (10 × 4 μm) positioned immediately outside the injured capillary andnormalized to fluorescence signal intensity in the capillary in the same frame.In each experiment, this image sequence of Alexa Fluor 488 diffusion wascollected at 10-min intervals, the percentage of peak dye leak was plotted overtime, and then a linear curve was fitted by the least-square method. The slope(closure rate) and Y-intercept of each regression line were pooled together toobtain the average slope and Y-intercept. BBB closure time was obtained usingaverage slope and Y-intercept fitted to the linear line. Capillary flow velocityand flux were measured using two-photon imaging in line-scan mode (2–3 ms/line, 10 μm length, 2,000 lines per image). Average velocity and flux per imagewere calculated by Matlab software (MathWorks) with custom software bydetecting the movement of red blood cells as black lines devoid of Texas Red-dextran (53). Platelet aggregation in vivo was imaged with Texas Red-dextranand Calcein AM (100 μL of 5 mM; Invitrogen) administered through a femoralvein before the imaging (54, 55). Collagen (10–50 μL of 1 mg/mL dissolved insaline; Gibco) was administered through a catheter inserted into the ex-ternal carotid artery to induce platelet aggregation.

Assessments of Bleeding Time and Platelet Aggregation. Mice were fastedovernight before experiments. Clopidogrel (Tocris Bioscience) was preparedas a 100-mM stock solution in DMSO and administered i.p. at a dose of 5, 20,30, 40, and 100 mg/kg animal in 0.2 mL saline for 3 d before the experiment.Acetylsalicylic acid was prepared as 10 mg/mL in saline and administered i.p.(10 mg/kg) for 3 d. For bleeding tests and platelet aggregation assays, themice were anesthetized with ketamine (70 mg/kg) and xylazine (10 mg/kg)i.p. Their tails were transected 2 mm from the tip with a no. 10 scalpel bladeand immersed in a 20-mL scintillation vial filled with normal saline at 37 °C.Bleeding time was determined as time to cessation of bleeding withina 20-min observation period (27). Bleeding was considered stopped if nobleeding was observed for 30 s and was done only once in eachmouse. Whole-blood platelet aggregation was measured by an impedance aggregometer(Chronolog). Blood samples were collected with a 19-gauge needle by directvenipuncture into a syringe containing 1:10 volume of 3.2% sodium citrateand left at room temperature for 15 min. The sample was mixed with saline(1:2 dilution) to make 1 mL in a test vial and allowed to warm to 37 °C for5 min. The electrode was placed and the sample was continuously stirred at1,200 × g. The sample was stimulated with either 0.2 μg/mL collagen (Chro-nolog) or 0.1 mM arachidonic acid (Chronolog) with 0.2 mM CaCl2, and thereaction during the 15-min recording time was recorded.

Immunohistochemistry and Electron Microscopy for Analysis of MicroglialProcesses. Forty-five minutes before the tissue harvest, animals receivedfocal laser injuries as described above. Deeply anesthetized mice were thentranscardially perfused with PBS and 4% (wt/vol; dissolved in PBS solution, pH7.4) paraformaldehyde (PFA) followed by brain isolation and postfixation in4% PFA for 3 h at room temperature. Vibratome coronal sections (50 μm) wereprepared (Vibratome Series 3000) and blocked in PBS containing 0.2% Triton Xand 7% normal donkey serum (Vector Labs). Specific antibodies for P2RY12(1:2,000, kindly provided by David Julius, University of California, SanFrancisco) and Laminin (1:400, Abcam 14055) were applied with 0.1%Triton X and 1% normal donkey serum overnight at 4 °C. Antibodies

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against occludin (1:50, Life Technologies 33–1500) or E-cadherin (1:100,Abcam 76055 and Life Technologies 33–4000) were applied after treating thesections with proteases (0.2 mg/mL, 10 min at 37 °C; Sigma P5147). The an-tibodies were visualized following a 2-h incubation period (at room tem-perature) with appropriated secondary antibodies conjugated to fluorophore(1:250, Jackson ImmunoResearch). DAPI was used for nuclear counterstaining,and slides were mounted with ProLong Antifade (Life Technologies). Immu-nolabeled brain sections were imaged and analyzed using the confocal mi-croscope with a 60× oil-immersion objective lens (1.25 N.A.; Olympus).

For electron microscopy of focal injury, the brains from Cx3CR1+/EGFPmice were perfusion-fixed with 4% PFA, and transverse sections (50 μm)were cut. The sections were examined with the confocal microscope to lo-cate the positions of the focal injuries before being postfixed with 2.5%glutaraldehyde/4% PFA overnight at 4 °C. The brain sections were thenpostfixed in 1.0% osmium tetroxide for 30 min and dehydrated in a gradedseries of ethanol up to 100%, transitioned into proplyene oxide, and

infiltrated with EPON/Arialdite epoxy resin. The brain slices were then po-lymerized for 24 h between two glass slides (pretreated with Sialane toprevent adhesion). Thin sections were cut at 70 nm, and the images werecaptured with a transmission electron microscope (Hitachi 7650) and a Gatan11 megapixel digital camera system.

Statistical Analysis. All histograms are expressed as mean ± SE. Normality ofthe data was examined with the Shapiro–Wilk test. One-way ANOVA, one-way repeated measure ANOVA, two-way ANOVA with the Tukey–Kramerpost hoc test, and t test were used where appropriate. The Mann–Whitneynonparametric test with Bonferroni-corrected alpha level was used wherenormality was not assumed.

ACKNOWLEDGMENTS. This work was supported by National Institutes ofHealth Grants R01DE022743, R01NS075177, and R01AT007945.

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