The “Dark Side” of Endocannabinoids: A Neurotoxic Role for Anandamide

Endocannabinoids, including 2-arachidonoylglycerol and anandamide (N-arachidonoylethanolamine; AEA), have neuroprotective effects in the brain through actions at CB1 receptors. However, AEA also binds to vanilloid (VR1) receptors and induces cell

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  The “Dark Side” of Endocannabinoids: A Neurotoxic Rolefor Anandamide *Ibolja Cernak, *†Robert Vink, *JoAnne Natale, *Bogdan Stoica, *Paul M. Lea IV,*Vilen Movsesyan, *Farid Ahmed, *Susan M. Knoblach, *Stanley T. Fricke, and *Alan I. Faden *Department of Neuroscience, Georgetown University Medical Center, Washington D.C., U.S.A.; and †Department of Pathology,University of Adelaide, Australia Summary:  Endocannabinoids, including 2-arachidonoylglyc-erol and anandamide (  N  -arachidonoylethanolamine; AEA),have neuroprotective effects in the brain through actions atCB1 receptors. However, AEA also binds to vanilloid (VR1)receptors and induces cell death in several cell lines. Here weshow that anandamide causes neuronal cell death  in vitro  andexacerbates cell loss caused by stretch-induced axonal injury ortrophic withdrawal in rat primary neuronal cultures. Adminis-tered intracerebroventricularly, AEA causes sustained cerebraledema, as reflected by diffusion-weighted magnetic resonanceimaging, regional cell loss, and impairment in long-term cog-nitive function. These effects are mediated, in part, throughVR1 as well as through calpain-dependent mechanisms, but notthrough CB1 receptors or caspases. Central administration of AEA also significantly upregulates genes involved in pro-inflammatory/microglial-related responses. Thus, anandamideproduces neurotoxic effects both  in vitro  and  in vivo  throughmultiple mechanisms independent of the CB1 receptor.  KeyWords:  Endocannabinoids—Anandamide—Cell death—Microarray—Magnetic resonance imaging—Cognitive deficit. It is now known that much of the tissue damagecaused by central nervous system (CNS) insults resultsfrom delayed biochemical mechanisms (Faden, 2001).One of the secondary processes that may contribute todelayed neuronal death is activation of phospholipase-mediated signaling pathways that leads to membranephospholipid degradation (Chan et al., 1989; Homayounet al., 2000).Endocannabinoids are endogenous lipid ligands,which bind to the same cannabinoid receptors (CB1,CB2) that mediate the effects of    9 -tetrahydrocannabi-nol, the active compound of cannabis (Mechoulam,2002). There are three members of the endocannabinoidfamily discovered to date:  N  -arachidonoylethanolamine(anandamide; AEA), 2-arachidonoylglycerol (2-AG),and 2-arachidonoylglyceryl ether (2-AGE; noladin);these have different affinities for CB1 and CB2 receptorsand distinct biologic effects (Di Marzo et al., 2002). En-docannabinoid signaling system occurs in both neuronsand astrocytes; astrocytes may use this system to com-municate with surrounding neurons or other astrocytes(Walter et al., 2002). During the last decade, consider-able experimental work has demonstrated protective bio-logic effects of endocannabinoids after brain injury (vander Stelt et al., 2002). Indeed, neuroprotective effects of cannabinoids have been shown in global and focal is-chemia, as well as in neuronal cultures subjected to is-chemic conditions (Nagayama et al., 1999; Sinor etal., 2000). Both 2-AG and AEA can protect cerebralrat cortical neurons from  in vitro  ischemia (Sinoret al., 2000), whereas 2-AG reduces cerebral brainedema and infarct volume, decreases hippocampal cellloss, and improves clinical outcome after traumaticbrain injury in mice (Panikashvili et al., 2001). How-ever, it should be noted that in contrast to the datasupporting cannabinoid-induced neuroprotection (Marsi-cano et al., 2003; Mechoulam and Lichtman, 2003), sev-eral studies have revealed that activation of CB1 recep-tors can induce cytotoxic effects in a number of culturedcell systems (Downer et al., 2003) including culturedhippocampal (Chan et al., 1998) and cortical neurons(Downer et al., 2001). Among suggested mechanisms of  Received August 14, 2003; final version received November 5,2003; accepted December 16, 2003.Address and reprint requests to Dr. Ibolja Cernak, Department of Neuroscience, Georgetown University Medical Center, 3970 ReservoirRoad, NW, Research Building, Room EP04, Washington, DC 20057,U.S.A.; e-mail:  Abbreviations used:  ADC, apparent diffusion coefficient; AEA, an-andamide; 2-AG, 2-arachidonoylglycerol; 2-AGE, 2-arachidonoylglyc-eryl ether; BDNF, brain-derived growth factor; CGC, cerebellar gran-ule cell; CPZ, capsazepine; ESTs, expression sequence tags; NAE,  N  -acylethanolamine; NAPE, N-acylated species of phosphatidyletha-nolamine; NM, neurobasal medium; TBI, traumatic brain injury;TIMP, tissue inhibitors of metalloproteinase.  Journal of Cerebral Blood Flow & Metabolism 24: 564–578 © 2004 The International Society for Cerebral Blood Flow and MetabolismPublished by Lippincott Williams & Wilkins, Baltimore 564  DOI: 10.1097/01.WCB.0000117813.35136.12  cannabinoid-induced neurotoxicity are activation of caspase-3-dependent apoptosis (Campbell, 2001; Downeret al., 2001), generation of reactive oxygen species (Chanet al., 1998), sustained ceramide accumulation (Galve-Roperh et al., 2002), activation of the JNK cascade (Sarkerand Maruyama, 2003), and sphingomyelin hydrolysis(Sanchez et al., 1998). It is highly possible that CB1activation may lead to both neurotoxicity and neuropro-tection, depending on a variety of influences such asnature and intensity of the toxic insult, as well as the celltype under study (Downer et al., 2003; Guzman, 2003).Anandamide as an  N  -acylethanolamine (NAE) can besynthesized as a hydrolytic product of N-acylated speciesof phosphatidylethanolamine (NAPE) through a processcatalyzed by phospholipase D (Devane et al., 1992;Schmid, 2000). Under normal conditions, the levels of NAPE and related NAE are very low, with their synthe-sis and metabolism strictly controlled (Schmid, 2000).However, accumulation of NAPE and NAE occurs incells undergoing degeneration and phospholipid degen-eration (Gray, 1976), as well as in conditions associatedwith membrane degradation (Epps et al., 1980). In-creased levels of NAPE and NAE may occur with neu-ronal death induced by glutamate or the mitochondrialrespiratory chain inhibitor, sodium azide (Hansen et al.,1997). Similarly, significant increases in both NAPE andNAE concentrations are found after glutamate-inducedneurotoxicity  in vivo  (Hansen et al., 2001) as well as inpost-decapitative ischemia (Natarajan et al., 1986).These lipid compounds, including AEA, may be formedin response to the high intracellular calcium concentra-tion that occurs in injured cells (Hampson et al., 1998).Although AEA srcinally was identified as an endog-enous ligand for cannabinoid receptors (Devane et al.,1992), more recent data suggest that it might also interactdirectly with other molecular targets, including non-CB1, non-CB2 G-protein coupled receptors (Di Marzo etal., 2000; Sagan et al., 1999), gap junctions (Venance etal., 1995), various ion channels (Hampson et al., 1998;Maingret et al., 2001; Szoke et al., 2000), and vanilloid(VR1) receptors (Zygmunt et al., 1999). Although a sub-stantial body of evidence demonstrates that activation of CB1 receptors by endocannabinoids (Nagayama et al.,1999; Panikashvili et al., 2001), including AEA (van derStelt et al., 2002), has neuroprotective effects, stimula-tion of VR1 receptors has been found to increase intra-cellular Ca 2+ and lead to subsequent cytotoxicity (Olah etal., 2001).VR1 is a nonselective ligand-gated cation channelwith six-transmembrane domains and belongs to thefamily of transient release-potential ion-channels (Ben-ham et al., 2002). It may be activated by exogenouscompounds such as capsaicin, the pungent component of chili peppers, and resiniferatoxin, a plant toxin (Caterinaet al., 1997; Szallasi and Blumberg, 1990; Szallasi,2002). In the rat, VR1-positive neurons are locatedthroughout the neuroaxis (Szallasi and Di Marzo, 2000),and the distribution of AEA is mainly overlapping withthe localization of VR1 receptors (Szallasi and Di Marzo,2000). There is ample evidence now (Ross, 2003) thatthe interaction of AEA with VR1 receptors is specific,whereas the efficacy of AEA as a VR1 agonist dependson numerous factors including receptor reserve, phos-phorylation, CB1 receptor activation, voltage, tempera-ture, and pH, among others.Although a decade has passed since the discovery of AEA, conflicting data on its biologic effects are stillemerging, and the mechanisms of AEA actions remainunclear. The root of the controversy resides in the factthat AEA is an endogenous ligand for both cannabinoidand vanilloid receptors, which often manifest opposingeffects. For example, the neuroprotective effects of theendocannabinoids through CB1 have been demonstratedin numerous  in vitro  and  in vivo  models (Gomez DelPulgar et al., 2002; Maccarrone et al., 2000; Mechoulamet al., 2002b), whereas the activation of VR1 receptorsseems to be involved in various forms of neuronal celldeath (Hail and Lotan, 2002; Hail, 2003; Jambrina et al.,2003). Indeed, in recent studies, AEA was shown to in-duce apoptotic cell death in human neuroblastomaCHP100, lymphoma U937, and PC-12 cells (Maccarroneet al., 2000; Sarker et al., 2000); the formation of apo-ptotic bodies induced by AEA corresponds to increasesin intracellular calcium, mitochondrial uncoupling, andcytochrome  c  release (Maccarrone et al., 2000). Thesepro-apoptotic effects of AEA were mediated via VR1receptors (Maccarrone et al., 2000). However, it shouldbe noted that recent results (Veldhuis et al., 2003) dem-onstrated that arvanil, a ligand for both VR1 and CB1receptors, leads to neuroprotective effects acting at bothCB1 and VR1. Moreover, it has been shown that the  invivo  neuroprotective effects of AEA are mediated byCB1 but not by VR1 or by lipoxygenase metabolites(Veldhuis et al., 2003). Taken together, it is possible thatduring pathologic conditions such as inflammation orcell damage when pH is decreased, the PKC-dependentsignaling system is activated, and NAPE is increasinglysynthesized and released by cells; under these conditions,AEA may become more active at VR1 than CB1 recep-tors. We hypothesize that AEA may induce either neu-roprotection or neurotoxicity, depending on the balanceof its action on CB1 receptors on the one hand, and VR1receptors or calcium-mediated signal transduction path-ways on the other. We have addressed these questionsusing both  in vitro  and  in vivo  model systems. METHODSAnimals All protocols involving the use of animals were in com-pliance with the Guide for the Care and Use of Laboratory  ANANDAMIDE NEUROTOXICITY 565  J Cereb Blood Flow Metab, Vol. 24, No. 5, 2004  Animals published by NIH (DHEW publication NIH 85-23-2985), and were approved by the Georgetown University Ani-mal Use Committee. Male Sprague-Dawley rats (340 to 380 g)for  in vivo  studies and female pregnant rats used to prepareneuronal and glial cultures were purchased from Harlan (India-napolis, IN, U.S.A.). Drugs Anandamide (AEA) and capsazepine (CPZ) were purchasedfrom Tocris (Tocris Cookson, Ellisville, MO, U.S.A.), and dis-solved in a minimum of ethyl alcohol and diluted with saline(the final concentration of ethanol was 2% (v/v). Five micro-liters of AEA and CPZ contained 20 nmol/L and 35 nmol/L,respectively. AM251, a specific CB1 antagonist, (  N  -(piperidin-1-yl)-5-(4-iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1  H  -pyrazole-3-carboxamide) was obtained from Tocris (TocrisCookson, Ellisville, MO, U.S.A.), first dissolved in dimethylsulfoxide (DMSO) and then diluted in saline (the final concen-tration of DMSO was 10%), whereas 5   L contained 35nmol/L. z-DEVD-fmk (  N  -benzyl-oxycarbonyl-Asp(OMe)-Val-Asp(OMe)-fluoromethylketone) was purchased from EnzymeSystems (Livermore, CA, U.S.A.), whereas SJA6017 (  N  -(4-fluorophenylsulfonyl)- L -valyl- L -leucinal), the calpain inhibitorVI, was obtained from Calbiochem (San Diego, CA, U.S.A.).Both drugs were dissolved in DMSO and diluted in saline toaccommodate a dosing volume of 5   L containing 160 ng and1   g, respectively. The vehicle for the AEA- and CPZ-treatedanimals was saline containing 2% of ethyl alcohol, whereas thevehicle for AM251-, z-DEVD-fmk-, and SJA6017-treatedgroups consisted of saline with 10% DMSO. Injection of CPZ(35 nmol/L), AM251 (35 nmol/L), z-DEVD-fmk (160 ng), andSJA6017 (1  g) did not induce significant changes in cognitiveoutcome or alteration in blood pressure, compared to vehiclecontrol (results not shown). Moreover, administration of thesedrugs did not alter the apparent diffusion coefficient (ADC),measured by diffusion-weighted magnetic resonance imaging(MRI) (results not shown); ADC significantly correlates withthe changes of extracellular water and as such reflects brainedema (Albensi et al., 2000). Because there were no significantdifferences between cognitive performances, blood pressure,ADC values, and gene profile in animals treated withsaline/ethyl alcohol or saline/DMSO vehicles, these animalswere pooled into one group. Cell cultures Glia were prepared from 1- to 3-day-old rat cortices, andneurons were prepared from 17- to 18-day-old rat embryoniccortices, as described previously in detail (Lea et al., 2002).Cortical neuronal cultures were used to examine AEA dose – response curves between 0.5 and 100   M, and cytotoxicitymeasured by lactate dehydrogenase (LDH) release. Mixed neu-ronal – glial cultures were used to test the effects of AEA onmechanical (stretch) injury-induced LDH release. Cerebellargranule cell (CGC) cultures were prepared as previously de-scribed (Toman et al., 2002). In experiments using trophic sup-port withdrawal, CGCs cultured in neurobasal medium (NM)with 2% B27 supplement and 25 mmol/L KCl were washedonce in NM and placed in B27-free NM containing 5 mmol/LKCl. Stretch injury Using the srcinal method described by Ellis et al. (Ellis etal., 1995), cells cultured on a deformable membrane werestretched with compressed gas. Stretch (7.5-mm deformationsof the membrane; 50-millisecond duration) was applied to thecells using a cell injury controller (Biomedical EngineeringFacility, Medical College of Virginia, VA, U.S.A.). Assay for  in vitro  cell death Lactate dehydrogenase activity cell culture growth mediawas quantified as an index of cell death (Mukhin et al., 1997).Injury- and/or anandamide-induced release of LDH was mea-sured using a CytoTox-96 nonradioactive cytotoxicity assay kit(Promega, Madison, WI, U.S.A.) according to the manufactur-er ’ s protocol 24 hours after injury or application of AEA togrowth media. Relative absorbance was measured at 490 nmusing a Multiskan Ascent microplate reader (Labsystem Oy,Helsinki, Finland). LDH levels in injured or treated cultureswere expressed as a percentage of a mean value (100%) inuninjured cultures. Intracerebroventricular injections Surgical anesthesia was induced and maintained with 4%and 2% isoflurane, respectively, using a flow rate of 1.0 to 1.5L/min oxygen. A guide cannula for microinjection of drugs wasimplanted into right lateral cerebroventricle. The drugs wereadministered to rats in a volume of 5   L for 2 minutes using amicroliter syringe. Administration of CPZ, AM251, SJA6017,or z-DEVD-fmk was performed 5 minutes after AEA injection. Nuclear magnetic resonance imaging At 24 hours, 48 hours, and 7 days after drug administration,all animals (n  6/group) were reanesthetized with isofluraneand subjected to magnetic resonance imaging examination us-ing a Bruker 7T/21 cm Biospec-Avance system (Bruker, Karls-ruhe, Germany) as previously described (Albensi et al., 2000).Briefly, animals were placed in the heated Plexiglas holder, anda respiratory motion detector was positioned over the thorax tofacilitate respiratory gating. The animal bed was positionedwith the head in the center of the magnet within a 72-mm  1 Hbirdcage resonator (Bruker). Field homogeneity across thebrain was optimized and a sagittal scout image was acquired(RARE [rapid acquisition relaxation enhancement pulse se-quence] image, field of vision, 4  ×  4 cm; 128  ×  128 resolution;repetition time [TR] to echo time [TE], 1,500/10 millisecondswith a RARE factor of 8, making the effective TE 40 millisec-onds). Multislice T 2 -weighted images were then acquired toobtain eight contiguous slices commencing at the end of theolfactory bulb and working caudally (field of vision, 3  ×  3 cm;slice thickness 2 mm; 256  ×  256 resolution; TR/TE 2,000/20ms; four echo images and two averages). Diffusion weighted(DWI) images were then acquired with a spin-echo pulse se-quence that had diffusion gradients added before and after therefocusing pulse. Gradient strength was varied in six steps us-ing sensitization values ranging from 20 to 1,000 s/mm 2 . A 256 ×  256 matrix was used with a 3-cm field of view, TR 2.0seconds, TE 502 milliseconds, slice thickness of 2 mm, and 4echoes. Diffusion maps were generated by applying the Stej-skal-Tanner equation in association with a Marquart algorithmusing a commercially available Paravision software (Bruker,Billerica, MA, U.S.A.). ADCs were calculated for four regions:left cortex, right cortex, left subcortex, and right subcortex (Fig.1). ADCs were expressed as 10 −5 mm 2  /s ± SD. Nuclear magnetic resonance spectroscopy Anandamide-treated animals used in MRI experiments at 24hours were also subject to phosphorus magnetic resonancespectroscopy using a Bruker 7T/21 cm Biospec-Avance system(Bruker, Karlsruhe, Germany) as previously described in detailelsewhere (Vink and McIntosh, 1990). Additionally, naïve orvehicle-treated animals were used as spectroscopy controls.  I. CERNAK ET AL.566   J Cereb Blood Flow Metab, Vol. 24, No. 5, 2004  Because there were no differences in vehicle-treated or na  ï  veanimals (results not shown), they were pooled for statisticalanalysis (n  6). Briefly, animals were placed in a speciallyconstructed, temperature-controlled Plexiglas holder and a5-mm  ×  9-mm surface coil was placed centrally over the ex-posed skull. Skin and muscle were retracted well clear of thecoil to prevent contributions from these tissues. The animalswere then inserted into the center of a 7.0-Tesla magnet inter-faced with a Bruker spectrometer and field homogeneity opti-mized on the water signal before acquisition of phosphorusspectra. Phosphorus spectra were obtained in 20-minute blocksusing a 90 °  pulse calibrated for a 2-mm cortical depth, a 700-millisecond delay time, and a 5,000-Hz spectral width contain-ing 2,048 data points. Rectal temperature and respiration weremonitored at all times. The anesthesia was maintained usingisoflurane. At the conclusion of the acquisition period, animalswere removed from the magnet, their wounds were closed, andthe animals were returned to their cages.Phosphorus magnetic resonance spectra were analyzed usingthe resident Bruker computer software program. After convo-lution difference (400/20 Hz), chemical shifts and integrals of the individual peaks were determined following line fitting.Intracellular pH, brain free magnesium concentration, and cy-tosolic phosphorylation potential were then determined as de-scribed in detail elsewhere (Vink et al., 1994). Briefly, intra-cellular pH was determined from the chemical shift of theinorganic phosphate peak (  Pi) relative to phosphocreatine(PCr) in the magnetic resonance spectra using the equationpH  =  6.77  +  log   P i  −  3.295.68  −   P i   ( 1 ) Similarly, free magnesium concentration was determined fromthe chemical shift difference between the  and  peaks of ATPusing the equation   Mg 2 +   =  K d  10.82  −    −    −   −  8.35    ( 2 ) where    −   is the chemical shift difference between the    and   peaks of ATP. The  K  d   for MgATP was initially assumed tobe 50   mol/L at pH 7.2 and 0.15 mol/L ionic strength and wascorrected for pH according to Bock et al. (Bock et al., 1987).Cytosolic phosphorylation potential (PP) was determined ac-cording to the equationPP  =  ATP  ADP  P i   ( 3 ) where    represents all the ionic forms of the free species. Theconcentration of ADP was calculated from the creatine kinaseequilibrium equation after correcting the equilibrium constantfor pH and free magnesium concentration as previously de-scribed in detail elsewhere (Vink et al., 1994). Concentrationsof the other metabolites were determined from the integratedpeak areas of the respective MRS peaks, assuming that prein- jury the normal values for PCr and ATP were 4.72 and 2.59  mol/g, respectively, and that the total creatine pool remainedconstant at 10.83   mol/g (Siesjo, 1981; Veech et al., 1979).Brain water content was assumed to be 80%, with the intracel-lular compartment accounting for 78% of the total water(Siesjo, 1981). Morris water maze test Cognitive outcome (spatial learning) was determined usingthe hidden platform version of the Morris water maze as pre-viously described (Hamm et al., 1996). Briefly, rats (n   10/group) were trained to locate a hidden, submerged platformusing constant extra-maze visual information, while the moni-toring was performed using a PC-controlled video system (Ac-cuScan Instruments, Columbus, OH, U.S.A.). The apparatusconsists of a large, white circular pool (900-mm diameter, 500mm high, water temperature 24 ± 1 ° C) with a Plexiglas plat-form 76 mm in diameter painted white and submerged 15 mmbelow the surface of the water (225 mm high). The surface of the water is rendered opaque with the addition of dilute, white,nontoxic paint. During training, the platform remained in aconstant location hidden in one quadrant 14 cm from the side-wall. The rat was gently placed in the water facing the wall atone of four randomly chosen locations separated by 90 ° . Thelatency to find the hidden platform within a 90-second criteriontime was recorded by a masked observer. A series of 16 train-ing trials, administered in blocks of four, were conducted ondays 17, 18, 19, and 20 in rats after drug injection. To controlfor visual discriminative ability or motor impairment, the sameanimals were finally required to locate a clearly visible black platform (placed in a different location) raised 5 mm above thewater surface at least 2 hours after the last training trial. Theresults were expressed as a latency to find the platform (sec-onds) ± SD. Western immunoblotting The animals were killed and the brain was immediately re-moved. After dissection, the brain samples were stored at − 80 ° C. For analysis, the brain tissue was resuspended in lysisbuffer (60 mmol/L Tris-HCl, pH 7.8 containing 150 mmol/LNaCl, 5 mmol/L EDTA, 10% glycerol, 2 mmol/L Na 3 VO 4 , 25mmol/L NaF, 10   g/ml leupeptin (Sigma), 10   g/ml aprotinin(Sigma), 1 mmol/L AEBSF (Sigma), 1 mmol/L Pepstatin(Sigma), 1 mmol/L Microcystin LR (Sigma), 0.1% sodium do-decyl sulfate (SDS), 0.5% Na deoxycholate, and 1% TritonX-100 (Calbiochem, La Jolla, CA, U.S.A.). The samples wereincubated on ice for at least 30 minutes and centrifuged at20,000 g for 15 min. The soluble fraction representing total cellextracts was recovered and stored at  − 80 ° C until use. Proteinconcentration in the samples was determined with the BCAassay kit (Pierce, Rockford, IL, U.S.A.). Equal protein aliquots(25 to 50   g) were resolved by SDS-polyacrylamide gel elec-trophoresis and transferred to nitrocellulose membranes (Hy-bond-C super; Amersham, Arlington Heights, IL, U.S.A.). Af-ter transfer, the gels were stained with GelCode blue stainreagent (Pierce) to verify equal protein loading. The mem-branes were probed with specific primary antibodies and the FIG. 1.  Diagrammatic representation of the different regions uti-lized for apparent diffusion coefficient determinations. LC, leftcortex; RC, right cortex; LSC, left subcortex; RSC, right subcor-tex. The right side is ipsilateral, whereas the left side is contra-lateral to the anandamide injection.  ANANDAMIDE NEUROTOXICITY 567   J Cereb Blood Flow Metab, Vol. 24, No. 5, 2004  immune complexes were detected using appropriate horserad-ish peroxidase – liked secondary antibodies (Amersham Phar-macia Biotech), chemiluminescence reagents (Super SignalWestDura, Pierce), and Kodak Biomax MR-1 films (Sigma). Calpain activity assay Tissue was homogenized in extraction buffer (K240-100,Biovision), incubated on ice for 20 minutes, and then centri-fuged (10,000 rcf, 4 ° C). Supernatant was removed and centri-fuged again (10,000 rcf, 4 ° C). The final supernatant was ali-quoted and frozen. Later, protein concentration was determinedusing the method of Bradford (Bradford, 1976). Fifty micro-grams of protein was then diluted in reaction buffer (25% Ex-traction Buffer, 5% DMSO, 13.6 mmol/L Tris-HCL [pH 7.5],2.7 mmol/L dithiothreitol, 2.7 mmol/L CaCl 2 , and 500   mol/LSuc-Leu-Tyr-AMC) and activity was measured at excitation of 360 nm and an emission of 530 nm on a microplate fluores-cence reader (Cytofluor4000, PerSeptive Biosystems). Caspase activity assay Assay for caspase-3- and caspase-9-like activity was per-formed as previously described (Yakovlev et al., 1997). Ali-quots of cytosolic extracts (25   g of protein in 100   L of extraction buffer) are preincubated at 37 ° C for 30 minutes, andthen mixed with an equal volume of 40   mol/L fluorescenttetrapeptide substrate (Ac-DEVD-AMC or Ac-LEHD-AMC,respectively; Bachem, Torrance, CA, U.S.A.) in the samebuffer solution. Free aminomethylcoumarin (AMC) accumula-tion, which resulted from cleavage of the aspartate – AMC bond,is monitored continuously in each sample during 30 minutes in96-well microtiter plates, using a CytoFluor II fluorometer(PerSeptive Biosystems, Framingham, MA, U.S.A.) at 360-nmexcitation and 460 emission wavelengths. The emission fromeach well is plotted against time. Linear regression analysis of the initial velocity (slope) of each curve yielded an activity foreach sample. Data are expressed as a percentage of the caspaseactivity in samples from sham-treated control animals. Histology Brains, prefixed in 4% phosphate-saline buffered formalde-hyde, were cut using a cryostat, and serial 6-  m, anterior-to-posterior sections were made. The sections were stained withhematoxylin and eosin. Cell count of neuronal profile was per-formed for the pyramidal cell layer of CA3 hippocampal area,in 10 randomly chosen sections from each brain, as previouslydescribed (Bramlett et al., 1997). All neurons were included,regardless of whether they were normal or with changed struc-tural characteristics. Hippocampal cell count in our vehicle-treated animals (254 ± 4 neurons in CA3 area) was comparableto values from the literature (Bramlett et al., 1997). The neuralprofile of the CA3 hippocampal sector of treated animals wasexpressed as a percentage of the total number of the neuronalcells in the CA3 region in vehicle-treated animals (100%). Cellcount data were analyzed statistically using the  t  -test for inde-pendent samples. Gene profiling by oligonucleotide microarray Cortical and hippocampal samples were harvested at 24hours, 48 hours, and 7 days after AEA (20 nmol/L, n  3/timepoint) or vehicle administration (n  3/time point). Total RNAfrom homogenized tissue was extracted using TRIzol reagent(Invitrogen Corporation, Carlsbad, CA, U.S.A.), and then con-verted to biotinylated cRNA and hybridized to 22 AffymetrixU34A oligonucleotide microarray containing 8,800 genes andexpression sequence tags (ESTs), as described by us (Di Gio-vanni et al., 2003). Signal intensity values for each oligonucleo-tide probe set were calculated using Affymetrix GeneChipMAS 5.0 software. Any data not meeting stringent quality con-trol criteria were repeated to meet standards. Signal intensityfor each gene on the microarrays from AEA and vehicle-treatedanimals was normalized to levels in na  ï  ve cortex. Statisticalanalysis and hierarchic clustering was performed using Gene-Spring 5.0. Within each region, genes with significant changeswere grouped by the primary function of their gene product. Semiquantitative reverse transcriptase-polymerasechain reaction One   g of total RNA used for gene profiling was also usedfor cDNA synthesis using SuperScript reverse transcriptase(Gibco BRL, Bethesda, MD, U.S.A.) and oligo(dT)-primer.The amount of synthesized cDNA was evaluated by polymer-ase chain reaction (PCR) using primers specific for ribosomalprotein RPL19. PCR reactions were performed in a PTC-225Thermal Cycler (MJ Research, Waltham, MA, U.S.A.) usingAmpliTaq polymerase (Perkin Elmer Life Sciences, Torrance,CA, U.S.A.). Each PCR reaction was repeated at least twice.The thermal cycling parameters were as follows: 1 minute 30seconds at 94 ° C followed by 30 cycles of 30 seconds at 94 ° C,1 minute 30 seconds at 59 ° C, 1 minute at 72 ° C, and finalincubation for 5 minutes at 72 ° C. PCR reaction products wereanalyzed by agarose gel-electrophoresis. Intensity of injuredcortex and hippocampus (n    3) was adjusted to respectivevehicle controls (n  3) using a housekeeping gene RibosomalProtein (Invitrogen Corp., Carlsbad, CA, U.S.A.) L-19 (RPL-19). Normalized cDNA was then used to estimate the relativeabundances of tissue inhibitors of metalloproteinase (TIMP)-1,MHC class II and brain-derived growth factor (BDNF) to con-firm the array hybridization data. Primers for each gene werelocated in different exons. Different dilutions of cDNA sampleswere used for different genes to provide linear range of PCRreactions (Di Giovanni et al., 2003). Data analysis Continuous variables subjected to repeated measurementsduring a period of time (ADC, water maze studies) were ana-lyzed using a repeated-measures analysis of variance followedby Tukey pairwise comparison at each time point. MRS data,activity assays, and cell counts were analyzed with a  t  -test.Analysis of variance followed by  t  -test with Bonferroni correc-tions for multiple comparisons were used for comparisons of anandamide- and vehicle-treated groups;  P  < 0.05 was consid-ered to reflect a statistically significant difference. RESULTS  In vitro  study We initially tested the effect of AEA on cell viabilitymeasured by LDH release in primary cortical neuronalcultures, as previously described (Mukhin et al., 1997).Concentrations of AEA greater than 50   mol/L signifi-cantly increased LDH release, indicating a neurotoxicaction (Fig. 2a). We subsequently examined the effect of anandamide on rat cortical neurons exposed to a moder-ate stretch injury. This trauma model has been used tostudy diffuse axonal injury-induced molecular and bio-chemical responses  in vitro  (Ellis et al., 1995); it causesboth necrotic and apoptotic pathways of cell death (Pikeet al., 2000). Addition of 25   mol/L AEA to cell culturemedia, a dose that did not cause cell death by itself,  I. CERNAK ET AL.568  J Cereb Blood Flow Metab, Vol. 24, No. 5, 2004
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