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P2X receptors are ATP-gated cation channels that mediate fast excita-tory transmission in diverse regions of the brain and in spinal cord1.Several P2X receptor subtypes have the unusual property of changingtheir ion selectivity during prolonged exposure to ATP. Brief exposureto ATP induces the opening of channels that are permeable to bothmono- and divalent cations, whereas more sustained exposures resultin progressive dilatation of the channel pore, with the development ofpermeability to moieties as large as 700 Da, including fluorescentindicators such as propidium, YO-PRO-1 and ethidium2. The P2X7Rwas originally described in cells of hematopoietic origin, in which itsactivation has been linked to cell lysis, an apparent consequence ofefflux of essential metabolites and intracellular messengers3.Although the P2X7R has been found in the nervous system4, its func-tional role in the brain and spinal cord has remained relatively unex-plored.

In addition to mediating neurotransmission, ATP has recently beenidentified as a potent transmitter of astrocytic calcium signaling5,6.Astrocytes release ATP through a regulated pathway, resulting inpropagating intercellular waves of cytosolic calcium entry7. Astrocyticcalcium signaling seems to be a general mechanism by which astro-cytes respond to a variety of stimuli, including synaptic activity,transmitter exposure and traumatic injury8. In turn, astrocytic cal-cium signals are transmitted to adjacent neurons, thereby modulatingtheir synaptic strength9. By this means, local astrocytes transmit dis-tant calcium signals to neurons within their own geographicalmicrodomain, thereby transducing regional signals into local changesin synaptic efficacy10. This ATP-dependent process of calcium wave

propagation occurs not only in the brain, where it has been best stud-ied in situ, but also in the parenchyma of the spinal cord11,12, where itmay have a role in extending local injury.

Because calcium wave propagation is mediated through ATPrelease, we asked whether SCI in particular may be associated withexcessive ATP release, and whether ATP per se is deleterious to threat-ened spinal cord neurons. We specifically asked whether excessiveATP release may lead to spinal neuronal death by activation of neu-ronal P2XRs. This idea is based on the observations that (i) ATPrelease and astrocytic Ca2+ signaling are both triggered by traumaticinjury13–15; (ii) traumatic injury is associated with a decrease in extra-cellular Ca2+, which enhances both astrocytic ATP release and Ca2+

signaling5,16–18 and increases the affinity of P2X7Rs1,19; (iii) P2X7Ractivation directly mediates cell death3; and (iv) spinal cord neurons,including motor neurons, express P2X7Rs4.

RESULTSWe modified a new bioluminescence technique for imaging in liverats with acute SCI5,20. Using this approach, we monitored ATPrelease by light emissions resulting from ATP-triggered luciferasebreakdown of luciferin. This process could be observed in real timewith a liquid nitrogen–cooled CCD (charge-coupled device) camera5

(Fig. 1). At baseline, the dorsal spinal cord showed low but detectablelevels of ATP release. An unexpected pattern of ATP release wasobserved in the traumatized spinal cord after a weight drop impact:The peritraumatic area was characterized by zones that showed sus-tained high ATP release (Fig. 1a–c). These zones of high ATP release

1Department of Neurosurgery, Center for Aging and Developmental Biology, University of Rochester Medical Center, Rochester, New York 14642, USA. 2Departmentof Cell Biology and 3Department of Pathology, New York Medical College, Valhalla, New York 10595, USA. 4Department of Neurology, University of Rochester MedicalCenter, Rochester, New York 14642, USA. 5Department of Neurology and Neuroscience, Cornell University Medical College, New York, New York 10021, USA.Correspondence should be addressed to M.N. (Nedergaard@urmc.rochester.edu).

Published online 18 July 2004; doi: 10.1038/nm1082

P2X7 receptor inhibition improves recovery after spinalcord injuryXiaohai Wang1, Gregory Arcuino2, Takahiro Takano1, Jane Lin3, Wei Guo Peng1,2, Pinglan Wan2, Pingjia Li1,Qiwu Xu2, Qing Song Liu2, Steven A Goldman4,5 & Maiken Nedergaard1,2

Secondary injury exacerbates the extent of spinal cord insults, yet the mechanistic basis of this phenomenon has largely beenunexplored. Here we report that broad regions of the peritraumatic zone are characterized by a sustained process of pathologic,high ATP release. Spinal cord neurons expressed P2X7 purine receptors (P2X7R), and exposure to ATP led to high-frequencyspiking, irreversible increases in cytosolic calcium and cell death. To assess the potential effect of P2X7R blockade inameliorating acute spinal cord injury (SCI), we delivered P2X7R antagonists OxATP or PPADS to rats after acute impact injury.We found that both OxATP and PPADS significantly improved functional recovery and diminished cell death in the peritraumaticzone. These observations demonstrate that SCI is associated with prolonged purinergic receptor activation, which results inexcitotoxicity-based neuronal degeneration. P2X7R antagonists inhibit this process, reducing both the histological extent andfunctional sequelae of acute SCI.

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were observed in all 24 rats studied and in most rats were restricted toone to three sharply demarcated regions, the areas of which variedinversely as a function of time after injury. At 2 h after injury, the totalarea of high ATP release averaged 1.39 ± 0.18 mm2 (Fig. 1a,e). Agroup of rats studied 6 h after impact also had elevated ATP release,but the mean area of ATP release was smaller in this group, 0.73 ±0.15 mm2 (n = 6 per group; Fig. 1c,e). At 24 h, no rats had areas ofincreased ATP release (Fig. 1d,e). Linear regression of the area of highATP release against the number of hours after injury showed a signif-icant decline (P < 0.05).

ATP release required ongoing metabolism by viable cells, becausecardiac arrest resulted in the rapid cessation of increased ATP release(Fig. 1c). Areas of high ATP release disappeared 4.2 ± 0.5 min (n = 4)after cardiac arrest induced by intravenous injection of 2 M KCl. Incontrast, ATP release from the traumatic lesion itself was depressed,probably as a result of severe cellular damage and cessation of localATP production. The traumatic lesion with low ATP release showedsteady growth. The lesion averaged 0.68 ± 0.06 mm2 after 2 h, 0.88 ±0.04 mm2 after 6 h and 1.01 ± 0.07 mm2 after 24 h (Fig. 1e). Theseobservations indicate that ATP release after acute SCI is characterized

by depressed release from the traumatizedtissue itself, but this traumatized tissue is sur-rounded by areas of increased and sustainedATP release.

Cell death in areas with high ATP releaseWe next asked whether high levels of ATP aredirectly neurotoxic. To assess the effects ofhigh ATP release after cell injury, we usedTUNEL staining to analyze the extent of celldeath in the peritraumatic zone21 (Fig. 2). Wecompared areas of high versus low ATPrelease at matched distances from the lesionsite. Guided by bioluminescence imaging, weinjected DiIC18 (2 µl, 10 µM) at the center ofhigh ATP release (Fig. 2a; n = 10). The ratswere perfusion-fixed 24 h later, and theDiIC18 labeling was clearly recognizable oncryosections (Fig. 2b). Fast blue stainingindicated that a considerable amount ofmyelin had been lost in the areas of high ATPrelease, but this was less so on the oppositeside of the lesion (Fig. 2c). Quantification ofTUNEL-positive cells showed that a signifi-cantly higher number of cells died in areas ofhigh ATP release as compared with tissuewith low ATP release, despite their equal dis-tances from the lesion (Fig. 2d–f). Doublestaining with cell-specific markers demon-strated that MAP-2-positive neurons andO4-positive oligodendrocytes, but not GFAP-positive astrocytes, were TUNEL positive inareas of high ATP release (Fig. 2e). Together,these observations indicate that both neu-ronal and oligodendrocytic injury is exacer-bated in areas of sustained ATP release.

We next asked whether high levels of extra-cellular ATP per se were sufficient to inducecell death in the uninjured spinal cord. ATPand a series of its analogs, each at 10 mM, wereinjected in 2-µl aliquots into the spinal cord,

just lateral to the dorsal horn. A large number of TUNEL-positive cellsbecame apparent within 24 h, in both gray and white matter (Fig. 2g).The greatest degree of cell death was observed in response to the non-degradable analog ATPγS, but ATP itself also induced significant celldeath (Fig. 2h). Moreover, the specific P2X7R agonist 2′-3′-O-(4-ben-zoylbenzoyl)adenosine 5′-triphosphate (BzATP) induced cell deathwith a potency equivalent to ATP. In contrast, 2-(methylthio)adenosine5′-triphosphate (2MeSATP; an agonist for P2X2, P2X4, P2X5 andP2Y1 receptors) and α,β-methyleneadenosine 5′-triphosphate(αβmeATP; an agonist for P2X1 and P2X3 receptors) injections werenot associated with significant injury, suggesting that other members ofthe P2X family may have a minor role in ATP-induced apoptosis.Together, these observations indicate that high concentrations of extra-cellular ATP, acting through spinal P2X7Rs, can directly trigger celldeath in the uninjured adult spinal cord.

Spinal cord neurons express P2X7RsP2X7Rs are expressed throughout the CNS4, although their functionhas principally been studied in immune cells, in which P2X7R activa-tion can lead to the death of the activated cell3,22. On that basis, we

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Figure 1 Bioluminescence detection of ATP release after acute SCI. (a) Bright-field image of theexposed spinal cord (T12) 2 h after a weight drop impact (left panel). Bioluminescence image of ATPrelease from the same region (right panel). The traumatic lesion shows low ATP release but issurrounded by a peritraumatic zone with high ATP release. The red dotted line indicates area of directimpact. (b) Comparison of relative luminescence signal from the peritraumatic zone (Peri) with highATP release, the traumatic lesion and the opposite side (No lesion) 2 h after impact (n = 10).Brightness scale is luminescence signal in arbitrary units. (c) Bright-field image of post-traumaticspinal cord 6 h after impact (left panel). Persistent high ATP release is evident in peritraumatic zone(middle panel). ATP release in the peritraumatic zone has returned to background amounts 10 minafter cardiac arrest (right panel). (d) Bright-field image (left panel) and bioluminescence image (right panel) 24 h after impact (n = 6). (e) Comparison of the area of the lesion and of the area of theperitraumatic zone with high ATP release at 2–3, 6 and 24 h. Error bars indicate s.e.m. Scale bar isthe same for a, c and d.

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asked if spinal cord neurons might directly express the P2X7R. Wefirst confirmed that spinal cord neurons express numerous mem-brane-localized P2X7R-immunoreactive plaques (Fig. 3a)4. The highexpression of P2X7Rs in spinal cord differs from the weak P2X7Rimmunoreactivity in cortical neurons (Fig. 3b) and the reported lackof protection against ischemic injury in animals with deletion ofP2X7Rs23. To evaluate the extent to which the P2X7Rs in motor neu-rons were functional, we used two-photon laser scanning microscopyof freshly prepared spinal cord slices (postnatal day (P) 10–14) toimage neuronal cytosolic calcium. Bath application of 100 µM ATP orBzATP induced a sustained increase in cytosolic calcium (Fig. 3c).The increase in cytosolic calcium remained high during 20 min ofagonist exposure and failed to normalize thereafter, such that 15 minafter agonist washout, fluo-4 emissions remained two- to threefoldhigher than baseline (Fig. 3d). Thus, prolonged exposure to ATP orBzATP was associated with irreversible increases in neuronal cytosoliccalcium.

ATP acts as an excitatory neurotransmitter in spinal cordTo analyze neuronal responses to ATP in detail, we next patchedspinal cord neurons and recorded in current clamp configuration.Exposure to 100 µM ATP or BzATP consistently induced high-fre-quency spiking, with little tendency to inactivate (Fig. 4a). Theincreased spiking activity was attenuated by pretreatment with theP2X7R antagonist adenosine 5′-triphos-phate-2′ ,3′-dialdehyde (OxATP; 100 µM;Fig. 4b) but not by a combined treatmentwith NBQX and CPP (AMPA and NMDAreceptor antagonists, respectively; Fig. 4c).These data indicate that ATP-evoked action

potentials were driven by P2X7R-linked channels, rather than byactivation of NMDA or AMPA receptors. NBQX and CPP alsofailed to reduce BzATP-induced Ca2+ increases (data not shown).The affinity for either BzATP or ATP was low, with a Kd of 87.1 µM(Fig. 4d) and 59.5 µM (Fig. 4e), respectively. The high agonist con-centrations that were required for activation are similar to previousreports, but a direct comparison with activity in the posttraumaticspinal cord was complicated by the fact that the affinity of P2X7Rsfor ATP increases as an inverse function of the extracellular con-centration of divalent cations1,19. The extracellular concentrationof Ca2+ falls to 0.1 mM immediately after SCI and remainsdepressed for several hours, recovering to only one-third (0.44 mM) of the normal value (1.1 mM) 3 h after injury16,17. Theartificial cerebrospinal fluid (ACSF) used during our recordingscontained 2 mM Ca2+ and 1 mM Mg2+, and it is therefore likely thatthe P2X7Rs in the post-traumatic spinal cord are activated by lowerATP concentrations than those observed here.

P2X7R antagonists improve functional recovery after SCIWe next asked whether the P2X7R antagonist OxATP protectedagainst secondary injury after SCI. Hindlimb motor function wasassessed using the Basso-Beattie-Bresnahan (BBB) locomotor ratingscale to evaluate the recovery of rats exposed to a weight dropimpact. OxATP (2 nM) or vehicle was injected 2.5 mm rostral and

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Figure 2 Areas of high ATP release are associatedwith increased apoptosis. (a) Bioluminescenceimage of ATP release 2 h after weight drop impact.DiIC18 was injected 200 µm into the spinal cord atposition a. Position b indicates a symmetricallyopposed site at an equal distance from the lesionsite. (b) Fluorescence detection of DiIC18 injectionsites. (c) Luxol fast blue shows degree ofmyelination in areas a–a and b–b. In b, c and d,italicized a and b refer to positions in a. (d) TUNELstaining shows apoptotic cells (arrowheads) inareas a–a and b–b. Scale bar is the same for c andd. (e) TUNEL staining (red) of MAP2-positiveneurons (white, left panel) and O4-positiveoligodendrocytes (white, middle panel), but notGFAP-positive astrocytes (white, right panel). (f) Comparison of number of TUNEL-positive cellsin rats undergoing bioluminescence imaging, dyeinjection and positive identification of areas a–aand b–b (P < 0.004, Student’s t-test; n = 6). Twoof these rats had regions of high ATP release bothrostral and caudal to the point of impact, whereasin the others, the zone of high ATP release waseither lateral (n = 1) or medial (n = 1) to or on bothsides of (n = 2) the point of impact. (g) Injection ofATP or ATP agonists induces apoptosis in theuninjured spinal cord. TUNEL-positive cells 24 hafter injection of ATP. Inset shows TUNEL-positivecells in larger magnification. (h) Comparison ofnumber of apoptotic cells after injection of ATPγS,ATP, BzATP, 2MesATP, αβmeATP and vehicle(analysis of variance (ANOVA) with Fisher’sprotected least significant difference (PLSD)). n = 4–8 animals in each group.

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caudal from the epicenter of injury immediately before impact. Ratstreated with OxATP had a mean BBB score of 2.9 ± 0.7, which wastwo points higher than matched vehicle-treated controls (P = 0.0006,unpaired t-test) 1 d after injury. OxATP-treated rats thereafter had amore rapid recovery, yielding five points or higher improvement rel-ative to their controls during the remainder of the observationperiod (P = 0.002; Fig. 5a). Thus, OxATP effectively improved func-tional recovery after SCI.

P2X7R antagonists improve neuronal survival after SCITo evaluate the effect of OxATP on tissue injury, we next analyzed his-tological alterations in rats sacrificed 24 h after impact. In accordancewith previous reports, diverse signs of irreversible injury were evidentin the peritraumatic zone (Fig. 5b)24,25. Numerous TUNEL-positivecells were present in the peritraumatic zone, and many of these cellsshowed a nuclear morphology consistent with apoptosis, includingchromatin condensation and fragmentation. Cresyl violet counter-staining showed another large fraction of cells with enlarged nucleiand loss of Nissl staining, which is suggestive of necrosis (Fig. 5b).Not all cells, however, died according to the classical pathways ofapoptosis or necrosis. Caspase-3 activation is the key step in the

process of apoptosis, but only 11 ± 4% ofTUNEL-positive cells were positive for caspase-3 activation (Fig. 5c). The lack ofcaspase-3 activation in TUNEL-positive cellsmay indicate that not all TUNEL-positive

cells died by apoptosis, but it could also indicate that caspase-3immunoreactivity was lost in the late stages of apoptosis. BecauseDNA fragmentation is an objective indicator of cell death, TUNELstaining was used to quantify the effect of OxATP on cell injury.OxATP-treated rats showed a highly significant (>75%) reduction in

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c Figure 3 Motor neurons express functionalP2X7Rs. (a) Two-photon laser scanning microscopyshows P2X7Rs (red) localized to the membrane ofMAP-2-positive (white) spinal cord neurons. Cellswere counterstained with a nuclear marker Sytox(green). (b) Immunoreactivity against P2X7Rs incortex of the same rats. Scale bar is the same for aand b. (c) BzATP induces an irreversible increasein neuronal cytosolic calcium in acutely preparedspinal cord slices. The neuron was patched incurrent clamp at its resting membrane potential(baseline, –57 mV) with a pipette containing fluo-4and Alexa Fluor-594 (to visualize fine processes).Bath application of 100 µM BzATP induces acalcium elevation detected by an increase in fluo-4emission in the cell body as well as in theprocesses. The calcium elevation fails to recoverafter washout (20-min exposure to BzATP followedby 20-min washout). (d) Summary of calciumimaging experiments. Histogram shows peakincreases in fluo-4 signal during BzATP (100 µM)and ATP (100 µM) and after a 15- to 20-minwashout (recovery).

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c d eFigure 4 P2X7R activation induces high-frequency firing in spinal cordneurons. (a) Action potentials triggered by bath application of 100 µM ATPfollowed by bath application of 100 µM BzATP. Recording is from the sameneurons with a recovery period of 20 min between exposures to agonists.(b) OxATP (100 µM) prevents BzATP-induced action potential firing (sameneuron shown on left and right panel). (c) Average spiking rate undervarious experimental conditions. Both ATP (100 µM; n = 6) and BzATP(100 µM; n = 9) increase spiking rate. Combined application of NBQX (10 µM) and CPP (5 µM) has no significant effect on BzATP-inducedincrease in the firing rate (P > 0.05; n = 6). OxATP (100 µM) preventsBzATP-induced increase in spike rate (n = 7). (d) The spiking rate increasesin a dose-dependent manner after addition of BzATP (n = 5–9). (e) ATP also causes a dose-dependent increase in the spike rate (n = 5).

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the number of TUNEL-positive cells in both gray and white matter,relative to vehicle-treated controls (Fig. 5d), strengthening the conclusion that the P2X7R antagonist limited secondary injury.

P2X7R antagonists are effective treatments after injuryOxATP is an irreversible P2X7R antagonist, which reacts covalentlywith unprotonated lysine residues present at the nucleotide bindingsite26. As a result, its maximal inhibitory effect is obtained only30–120 min after exposure in cultured cells26,27. On this basis, we pre-treated rats with OxATP in the initial experiments described above.Given the efficacy observed, we next chose to model a more likelytherapeutic use, by administering the antagonist 1 h after weight dropimpact in a model of mild SCI (the experimental height was reducedfrom 10 to 6.25 mm). Again, OxATP (2 nM) significantly improvedfunctional recovery and decreased the number of TUNEL-positivecells (Fig. 5e,f). Similarly, another P2X7R antagonist, pyridoxal-5′-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), improvedfunctional recovery and reduced the number of TUNEL-positive cellswhen administered 1 h after injury (Fig. 5e,f). The protective effect ofPPADS was encouraging because its inhibitory action is also slow andis first detectable after a 15-min incubation at 37 °C in culture27.Thus, two P2X7R antagonists, which differ with regard to both struc-ture and mechanism of action, decreased histological injury andimproved recovery of motor function after SCI. These data supportthe notion that P2X7R antagonists that are administered after injuryhave potential as new therapeutic agents in acute SCI.

DISCUSSIONP2XRs mediate fast excitatory postsynaptic currents by openingcation channels that exhibit a remarkably high permeability to Ca2+

and that are expressed in multiple types of neurons28. P2XR expres-sion is region specific. For example, P2X2Rs are highly expressed bymyenteric neurons, whereas P2X3R expression seems restricted tonociceptive neurons. P2X7Rs were first described in immune cellsand microglial cells29, where they have been implicated in the releaseof cytokines and in the induction of cell death. Recent studies havedocumented that P2X7Rs also are expressed by mossy fibers in theCA3 area of hippocampus30, by cultured astrocytes and Schwanncells31,32 and by spinal cord neurons4.

ATP released in the setting of trauma has been linked to an efflux ofcytosolic ATP from mechanically traumatized cells33. Our study failedto support this concept, because ATP release was below control valuesat the site of impact. That being said, technical issues prevented usfrom imaging immediately after injury, and it is possible that wemissed an initial peak release. Imaging was initiated in most rats

within 10 min after impact, and ATP release was consistentlydepressed in the center of the injury. It is conceivable that ATPreleased on impact may be hydrolyzed within minutes and that thesevere cellular disintegration is incompatible with further ATP pro-duction. Rather, the unexpected finding of this study is that broadperitraumatic zones showed high ATP release for hours after SCI. Thecellular events that lead to these prolonged episodes of increased ATPrelease remain to be defined, but they seem to reflect a combination ofcontinued production of energy metabolites and a persistent inabilityto regulate efflux pathways. Notably, ATP has been identified as theprimary transmitter of astrocytic Ca2+ signaling5. It has not yet beendetermined whether astrocytic Ca2+ signaling is activated in the set-ting of SCI, but mechanical stimulation has for the last decade beenthe preferred technique for induction of astrocytic Ca2+ signal-ing10,34.

Notably, the high ATP release was restricted to two or three sharplydefined zones, whereas other areas of the peritraumatic tissue showednormal or depressed ATP release. These observations are in line withprevious work, which has documented a heterogeneous pattern ofalterations in the post-traumatic spinal cord with regard to bothblood flow and extracellular concentration of glutamate35,36. BecauseATP is a potent stimulator of astrocytic glutamate release37,38, it islikely that glutamate excitoxicity contributes to neuronal death inareas of high ATP release39,40.

Together, these results indicate that P2X7-mediated neurodegener-ation contributes to secondary injury after SCI. Bioluminescenceimaging of ATP release from rats subjected to experimental cord con-tusion showed that areas surrounding the traumatic lesion have anabnormally high and sustained pattern of ATP release. Spinal cord

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bFigure 5 P2X7R antagonists reduce apoptosis and improve functionalrecovery after SCI. (a) Functional recovery evaluated by the BBB scale for 6 weeks after SCI. OxATP was administered immediately before impact (n = 19 for controls and n = 15 for OxATP-treated rats; *P < 0.002;unpaired t-test). Data represent mean ± s.e.m. (b) Histological evaluation ofcell death in the peritraumatic zone in a rat sacrificed 24 h after injury.Sections were processed for the TUNEL assay and were counterstained withcresyl violet. Cellular alterations typical of both necrosis (arrow) andapoptosis (arrowhead) are evident. (c) Caspase-3 activation in TUNEL-positive cells in the peritraumatic zone of a rat sacrificed 24 h after SCI. (d) Comparison of number of TUNEL-positive cells in rats treated beforeinjury with OxATP or vehicle and sacrificed 24 h after injury. (Student’s t-test). (e) Functional recovery in rats treated after injury with OxATP,PPADS or vehicle. (*P < 0.05; ANOVA with Fisher’s PLSD). (f) Histogramcomparing number of TUNEL-positive cells in rats treated after injury withOxATP, PPADS or vehicle and sacrificed 24 h after impact (ANOVA withFisher’s PLSD).

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neurons expressed an abundance of P2X7Rs, and exposure of freshlyprepared spinal cord slices to ATP or BzATP evoked high-frequencyfiring and irreversible increases in cytosolic Ca2+. Areas with highATP release surrounding the epicenter of impact were characterizedby the appearance of many dying cells 24 h after injury. When injectedlocally in the peritraumatic zone, OxATP (a P2X7R antagonist)strongly reduced both the number of TUNEL-positive cells and theseverity of histological injury. This was associated with significantlyimproved behavioral recovery of the OxATP-treated rats relative totheir controls, which was sustained throughout the postinjury obser-vation. OxATP as well as PPADS also reduced the severity of injurywhen injected 1 h after injury. Of note, P2X7R is expressed by bothleukocytes and macrophages, including microglia, and its activation isa strong inflammatory stimulus41. Accordingly, P2X7R-deficient micehave significantly attenuated inflammatory responses42, reflecting adiminution of interleukin-1-mediated leukocyte mobilization43.Acute SCIs cause highly inflammatory environments, and P2X7 acti-vation of local microglial cells may have adverse effects. Additionalstudies are required to establish whether OxATP-mediated P2X7Rinhibition after SCI may derive in part from its potent anti-inflamma-tory effects, over and above its protection of vulnerable neurons andoligodendrocytes from purinergic excitotoxicity44,45. The beneficialeffects of P2X7R inhibition on SCI may be a twin function of both thehigh density of P2X7-receptive cells in the spinal cord and the strongcontribution of inflammatory damage to SCI. The systemic adminis-tration of P2X7R antagonists may thus constitute a new and much-needed approach for the treatment of acute SCI46,47.

METHODSTraumatic model and agonist injections. Adult female Sprague-Dawley rats(220–250 g) were anesthetized with pentobarbital (50 mg/kg i.p.). When unre-sponsive, a laminectomy was carried out on T12 vertebrae. Dura was leftintact, and injury was produced by the weight drop device developed at NewYork University. The dorsal surface of the spinal cord was subjected to a weightdrop impact using a 10-g weight (2.5-mm tip diameter) dropped at an experi-mental height of either 6.25 or 10 mm (ref. 48). ATP and agonists (10 mM,2 µl) were stereotaxically injected into T12. All agents were purchased fromSigma. Experiments were approved by the Institutional Animal Care and UseCommittee of University of Rochester and New York Medical College.

Bioluminescence imaging of ATP. ATP release from the exposed spinal cordwas imaged by chemiluminescence in real time as described7. Luciferase (0.132mg/ml) and luciferin (0.332 mg/ml) were added to an oxygenated ACSF solu-tion49. Dura was kept intact, and the enzyme mixture was delivered by a silastictube inserted under the dura at a rate of approximately 1 ml/h (Minipump RT-202, VWR). Light production from the luciferin-luciferase reaction wasimaged by a liquid nitrogen–cooled CCD camera (VersArray 1300B, PrincetonInstruments) using a 50-mm camera lens (Olympus), 4 × 4 binning and 30-sintegration per frame7. In bioluminescence imaging experiments, we reducedthe tip of the rod to 1 mm and the weight to 5 g (dropped at 10 mm) to avoidrupture of the dorsal venous complex, because the high content of ATP inblood interfered with imaging. The lower limit for detection of ATP release is50 nM (ref. 7).

Functional outcome analysis. The BBB 21-point open-field locomotor ratingscale was used for evaluating hindlimb movement48. The rats were blindlyevaluated every day for 2 weeks after injury and later on a biweekly basis.

Tissue preparation. Transcardiac perfusion was carried out in anesthetizedrats 24 h after SCI by 4% paraformaldehyde in phosphate-buffered saline21.The rats were anesthetized with ketamine (60 mg/kg) and xylazine (10 mg/kg).After 12 h postfixation, the spinal cord was cryoprotected in 30% sucrose andcut in serial 20-µm parasaggital sections. Myelin was visualized with Luxol fastblue48,49. Immunohistochemistry, cresyl violet and TUNEL staining were carried out as described21,50,51. Antibody against activated caspase-3 was

purchased from Cell Signaling. Immunofluorescence was visualized usingconfocal microscopy (BioRad MRC1024), and images were analyzed byMetamorph software (Universal Imaging Corp.).

Electrophysiology. Spinal cord slices were prepared from 10- to 14-d-oldSprague Dawley rats anesthetized with ketamine and xylazine. A section of thelumbar spinal cord (T9–L4) was rapidly dissected out and immersed in an ice-cold solution containing 240 mM sucrose, 2.5 mM KCl, 0.5 mM CaCl2, 10 mMMgCl2, 26 mM NaHCO3, 1.25 mM NaH2PO4 and 10 mM glucose (saturatedwith 95% O2/5% CO2). After isolation, the lumbar cord was immobilized inlow-melting-point agarose, and transverse slices (300–350 µm) were cut usinga vibratome (TPI). After sectioning, slices were transferred to an oxygenatedACSF that contained 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2,26 mM NaHCO3, 1.25 mM NaH2PO4 and 10 mM glucose and were allowed torecover for at least 1 h before recording.

Individual neurons were identified with a 40× objective under infrared dif-ferential interference contrast microscopy (DIC; Olympus BX61WI). The neu-rons were current clamped at their resting membrane potentials, ranging from–53 to –70 mV (ref. 52). The pipette solution contained 10 mM KCl, 130 mMKCH3SO3, 2 mM MgCl2, 20 mM HEPES, 5 mM sodium phosphocreatine,4 mM Mg-ATP and 0.3 mM GTP. Membrane currents were filtered at 1–2 KHzand were digitized at 5–10 KHz using Axopatch 700A amplifier, Clampex8software and DigiData 1332A interface (Axon Instruments Inc.). Series resist-ance (<20 MΩ) was carefully monitored throughout the experiments; experi-ments were terminated if the series resistance changed by more than 15%. Allexperiments were done at room temperature (21–23 °C).

Multiphoton laser scanning imaging. Calcium imaging was carried out with acustom-built two-photon laser scanning microscope linked to a Ti:sapphirelaser (Mai Tai, Spectra-Physics) using Fluoview software (Olympus) and a 60×objective (0.9 n.a., infrared; Olympus). Motor neurons were patched in whole-cell configuration with pipettes containing Alexa Fluor-594 (50 µM) and fluo-4 (100 µM) for a minimum of 20 min before imaging. Both fluorescenceindicators were excited at 810 nm, and the emission signal was separated byemission filters.

ACKNOWLEDGMENTSThis study was supported by the National Institute of Neurological Disorders andStroke and the New York State Spinal Cord Injury Research Program.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 18 May; accepted 25 June 2004Published online at http://www.nature.com/naturemedicine/

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