It is now known that much of the tissue damage caused by central nervous system (CNS) insults results from delayed biochemical mechanisms (Faden, 2001). One of the secondary processes that may contribute to delayed neuronal death is activation of phospholipase-mediated signaling pathways that leads to membrane phospholipid degradation (Chan et al., 1989; Homayoun et al., 2000).

Endocannabinoids are endogenous lipid ligands, which bind to the same cannabinoid receptors (CB1, CB2) that mediate the effects of Δ9-tetrahydrocannabinol, the active compound of cannabis (Mechoulam, 2002). There are three members of the endocannabinoid family 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 receptors and distinct biologic effects (Di Marzo et al., 2002). Endocannabinoid signaling system occurs in both neurons and astrocytes; astrocytes may use this system to communicate with surrounding neurons or other astrocytes (Walter et al., 2002). During the last decade, considerable experimental work has demonstrated protective biologic effects of endocannabinoids after brain injury (van der Stelt et al., 2002). Indeed, neuroprotective effects of cannabinoids have been shown in global and focal ischemia, as well as in neuronal cultures subjected to ischemic conditions (Nagayama et al., 1999; Sinor et al., 2000). Both 2-AG and AEA can protect cerebral rat cortical neurons from in vitro ischemia (Sinor et al., 2000), whereas 2-AG reduces cerebral brain edema and infarct volume, decreases hippocampal cell loss, and improves clinical outcome after traumatic brain injury in mice (Panikashvili et al., 2001). However, it should be noted that in contrast to the data supporting cannabinoid-induced neuroprotection (Marsicano et al., 2003; Mechoulam and Lichtman, 2003), several studies have revealed that activation of CB1 receptors can induce cytotoxic effects in a number of cultured cell systems (Downer et al., 2003) including cultured hippocampal (Chan et al., 1998) and cortical neurons (Downer et al., 2001). Among suggested mechanisms of cannabinoid-induced neurotoxicity are activation of caspase-3-dependent apoptosis (Campbell, 2001; Downer et al., 2001), generation of reactive oxygen species (Chan et al., 1998), sustained ceramide accumulation (Galve-Roperh et al., 2002), activation of the JNK cascade (Sarker and Maruyama, 2003), and sphingomyelin hydrolysis (Sanchez et al., 1998). It is highly possible that CB1 activation may lead to both neurotoxicity and neuroprotection, depending on a variety of influences such as nature and intensity of the toxic insult, as well as the cell type under study (Downer et al., 2003; Guzman, 2003).

Anandamide as an N-acylethanolamine (NAE) can be synthesized as a hydrolytic product of N-acylated species of phosphatidylethanolamine (NAPE) through a process catalyzed by phospholipase D (Devane et al., 1992; Schmid, 2000). Under normal conditions, the levels of NAPE and related NAE are very low, with their synthesis and metabolism strictly controlled (Schmid, 2000). However, accumulation of NAPE and NAE occurs in cells undergoing degeneration and phospholipid degeneration (Gray, 1976), as well as in conditions associated with membrane degradation (Epps et al., 1980). Increased levels of NAPE and NAE may occur with neuronal death induced by glutamate or the mitochondrial respiratory chain inhibitor, sodium azide (Hansen et al., 1997). Similarly, significant increases in both NAPE and NAE concentrations are found after glutamate-induced neurotoxicity in vivo (Hansen et al., 2001) as well as in post-decapitative ischemia (Natarajan et al., 1986). These lipid compounds, including AEA, may be formed in response to the high intracellular calcium concentration that occurs in injured cells (Hampson et al., 1998). Although AEA originally was identified as an endogenous ligand for cannabinoid receptors (Devane et al., 1992), more recent data suggest that it might also interact directly with other molecular targets, including non-CB1, non-CB2 G-protein coupled receptors (Di Marzo et al., 2000; Sagan et al., 1999), gap junctions (Venance et al., 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 substantial body of evidence demonstrates that activation of CB1 receptors by endocannabinoids (Nagayama et al., 1999; Panikashvili et al., 2001), including AEA (van der Stelt et al., 2002), has neuroprotective effects, stimulation of VR1 receptors has been found to increase intracellular Ca2+ and lead to subsequent cytotoxicity (Olah et al., 2001).

VR1 is a nonselective ligand-gated cation channel with six-transmembrane domains and belongs to the family of transient release-potential ion-channels (Benham et al., 2002). It may be activated by exogenous compounds such as capsaicin, the pungent component of chili peppers, and resiniferatoxin, a plant toxin (Caterina et al., 1997; Szallasi and Blumberg, 1990; Szallasi, 2002). In the rat, VR1-positive neurons are located throughout the neuroaxis (Szallasi and Di Marzo, 2000), and the distribution of AEA is mainly overlapping with the localization of VR1 receptors (Szallasi and Di Marzo, 2000). There is ample evidence now (Ross, 2003) that the interaction of AEA with VR1 receptors is specific, whereas the efficacy of AEA as a VR1 agonist depends on numerous factors including receptor reserve, phosphorylation, CB1 receptor activation, voltage, temperature, and pH, among others.

Although a decade has passed since the discovery of AEA, conflicting data on its biologic effects are still emerging, and the mechanisms of AEA actions remain unclear. The root of the controversy resides in the fact that AEA is an endogenous ligand for both cannabinoid and vanilloid receptors, which often manifest opposing effects. For example, the neuroprotective effects of the endocannabinoids through CB1 have been demonstrated in numerous in vitro and in vivo models (Gomez Del Pulgar et al., 2002; Maccarrone et al., 2000; Mechoulam et al., 2002b), whereas the activation of VR1 receptors seems to be involved in various forms of neuronal cell death (Hail and Lotan, 2002; Hail, 2003; Jambrina et al., 2003). Indeed, in recent studies, AEA was shown to induce apoptotic cell death in human neuroblastoma CHP100, lymphoma U937, and PC-12 cells (Maccarrone et al., 2000; Sarker et al., 2000); the formation of apoptotic bodies induced by AEA corresponds to increases in intracellular calcium, mitochondrial uncoupling, and cytochrome c release (Maccarrone et al., 2000). These pro-apoptotic effects of AEA were mediated via VR1 receptors (Maccarrone et al., 2000). However, it should be noted that recent results (Veldhuis et al., 2003) demonstrated that arvanil, a ligand for both VR1 and CB1 receptors, leads to neuroprotective effects acting at both CB1 and VR1. Moreover, it has been shown that the in vivo neuroprotective effects of AEA are mediated by CB1 but not by VR1 or by lipoxygenase metabolites (Veldhuis et al., 2003). Taken together, it is possible that during pathologic conditions such as inflammation or cell damage when pH is decreased, the PKC-dependent signaling system is activated, and NAPE is increasingly synthesized and released by cells; under these conditions, AEA may become more active at VR1 than CB1 receptors. We hypothesize that AEA may induce either neuroprotection or neurotoxicity, depending on the balance of its action on CB1 receptors on the one hand, and VR1 receptors or calcium-mediated signal transduction pathways on the other. We have addressed these questions using both in vitro and in vivo model systems.

METHODS

Animals All protocols involving the use of animals were in compliance with the Guide for the Care and Use of Laboratory Animals published by NIH (DHEW publication NIH 85-23-2985), and were approved by the Georgetown University Animal Use Committee. Male Sprague-Dawley rats (340 to 380 g) for in vivo studies and female pregnant rats used to prepare neuronal and glial cultures were purchased from Harlan (Indianapolis, IN, U.S.A.).

Drugs Anandamide (AEA) and capsazepine (CPZ) were purchased from Tocris (Tocris Cookson, Ellisville, MO, U.S.A.), and dissolved in a minimum of ethyl alcohol and diluted with saline (the final concentration of ethanol was 2% (v/v). Five microliters 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-1H-pyrazole-3-carboxamide) was obtained from Tocris (Tocris Cookson, Ellisville, MO, U.S.A.), first dissolved in dimethyl sulfoxide (DMSO) and then diluted in saline (the final concentration of DMSO was 10%), whereas 5 μL contained 35 nmol/L. z-DEVD-fmk (N-benzyl-oxycarbonyl-Asp(OMe)-Val-Asp(OMe)-fluoromethylketone) was purchased from Enzyme Systems (Livermore, CA, U.S.A.), whereas SJA6017 (N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal), the calpain inhibitor VI, was obtained from Calbiochem (San Diego, CA, U.S.A.). Both drugs were dissolved in DMSO and diluted in saline to accommodate a dosing volume of 5 μL containing 160 ng and 1 μg, respectively. The vehicle for the AEA- and CPZ-treated animals was saline containing 2% of ethyl alcohol, whereas the vehicle for AM251-, z-DEVD-fmk-, and SJA6017-treated groups consisted of saline with 10% DMSO. Injection of CPZ (35 nmol/L), AM251 (35 nmol/L), z-DEVD-fmk (160 ng), and SJA6017 (1 μg) did not induce significant changes in cognitive outcome or alteration in blood pressure, compared to vehicle control (results not shown). Moreover, administration of these drugs did not alter the apparent diffusion coefficient (ADC), measured by diffusion-weighted magnetic resonance imaging (MRI) (results not shown); ADC significantly correlates with the changes of extracellular water and as such reflects brain edema (Albensi et al., 2000). Because there were no significant differences between cognitive performances, blood pressure, ADC values, and gene profile in animals treated with saline/ethyl alcohol or saline/DMSO vehicles, these animals were pooled into one group.

Cell cultures Glia were prepared from 1- to 3-day-old rat cortices, and neurons were prepared from 17- to 18-day-old rat embryonic cortices, 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 μM, and cytotoxicity measured by lactate dehydrogenase (LDH) release. Mixed neuronal–glial cultures were used to test the effects of AEA on mechanical (stretch) injury-induced LDH release. Cerebellar granule cell (CGC) cultures were prepared as previously described (Toman et al., 2002). In experiments using trophic support withdrawal, CGCs cultured in neurobasal medium (NM) with 2% B27 supplement and 25 mmol/L KCl were washed once in NM and placed in B27-free NM containing 5 mmol/L KCl.

Stretch injury Using the original method described by Ellis et al. (Ellis et al., 1995), cells cultured on a deformable membrane were stretched with compressed gas. Stretch (7.5-mm deformations of the membrane; 50-millisecond duration) was applied to the cells using a cell injury controller (Biomedical Engineering Facility, Medical College of Virginia, VA, U.S.A.).

Assay for in vitro cell death Lactate dehydrogenase activity cell culture growth media was quantified as an index of cell death (Mukhin et al., 1997). Injury- and/or anandamide-induced release of LDH was measured using a CytoTox-96 nonradioactive cytotoxicity assay kit (Promega, Madison, WI, U.S.A.) according to the manufacturer's protocol 24 hours after injury or application of AEA to growth media. Relative absorbance was measured at 490 nm using a Multiskan Ascent microplate reader (Labsystem Oy, Helsinki, Finland). LDH levels in injured or treated cultures were expressed as a percentage of a mean value (100%) in uninjured cultures.

Intracerebroventricular injections Surgical anesthesia was induced and maintained with 4% and 2% isoflurane, respectively, using a flow rate of 1.0 to 1.5 L/min oxygen. A guide cannula for microinjection of drugs was implanted into right lateral cerebroventricle. The drugs were administered to rats in a volume of 5 μL for 2 minutes using a microliter 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 isoflurane and subjected to magnetic resonance imaging examination using a Bruker 7T/21 cm Biospec-Avance system (Bruker, Karlsruhe, Germany) as previously described (Albensi et al., 2000). Briefly, animals were placed in the heated Plexiglas holder, and a respiratory motion detector was positioned over the thorax to facilitate respiratory gating. The animal bed was positioned with the head in the center of the magnet within a 72-mm 1H birdcage resonator (Bruker). Field homogeneity across the brain was optimized and a sagittal scout image was acquired (RARE [rapid acquisition relaxation enhancement pulse sequence] image, field of vision, 4 × 4 cm; 128 × 128 resolution; repetition time [TR] to echo time [TE], 1,500/10 milliseconds with a RARE factor of 8, making the effective TE 40 milliseconds). Multislice T 2 -weighted images were then acquired to obtain eight contiguous slices commencing at the end of the olfactory bulb and working caudally (field of vision, 3 × 3 cm; slice thickness 2 mm; 256 × 256 resolution; TR/TE 2,000/20 ms; four echo images and two averages). Diffusion weighted (DWI) images were then acquired with a spin-echo pulse sequence that had diffusion gradients added before and after the refocusing pulse. Gradient strength was varied in six steps using sensitization values ranging from 20 to 1,000 s/mm2. A 256 × 256 matrix was used with a 3-cm field of view, TR 2.0 seconds, TE 502 milliseconds, slice thickness of 2 mm, and 4 echoes. Diffusion maps were generated by applying the Stejskal-Tanner equation in association with a Marquart algorithm using 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 mm2/s ± SD. Download Open in new tab Download in PowerPoint

Nuclear magnetic resonance spectroscopy Anandamide-treated animals used in MRI experiments at 24 hours were also subject to phosphorus magnetic resonance spectroscopy using a Bruker 7T/21 cm Biospec-Avance system (Bruker, Karlsruhe, Germany) as previously described in detail elsewhere (Vink and McIntosh, 1990). Additionally, naïve or vehicle-treated animals were used as spectroscopy controls. Because there were no differences in vehicle-treated or naïve animals (results not shown), they were pooled for statistical analysis (n = 6). Briefly, animals were placed in a specially constructed, temperature-controlled Plexiglas holder and a 5-mm x 9-mm surface coil was placed centrally over the exposed skull. Skin and muscle were retracted well clear of the coil to prevent contributions from these tissues. The animals were then inserted into the center of a 7.0-Tesla magnet interfaced with a Bruker spectrometer and field homogeneity optimized on the water signal before acquisition of phosphorus spectra. Phosphorus spectra were obtained in 20-minute blocks using a 90° pulse calibrated for a 2-mm cortical depth, a 700-millisecond delay time, and a 5,000-Hz spectral width containing 2,048 data points. Rectal temperature and respiration were monitored at all times. The anesthesia was maintained using isoflurane. At the conclusion of the acquisition period, animals were removed from the magnet, their wounds were closed, and the animals were returned to their cages. Phosphorus magnetic resonance spectra were analyzed using the resident Bruker computer software program. After convolution difference (400/20 Hz), chemical shifts and integrals of the individual peaks were determined following line fitting. Intracellular pH, brain free magnesium concentration, and cytosolic phosphorylation potential were then determined as described in detail elsewhere (Vink et al., 1994). Briefly, intracellular pH was determined from the chemical shift of the inorganic phosphate peak (δPi) relative to phosphocreatine (PCr) in the magnetic resonance spectra using the equation p H = 6.77 + log ( δ P i − 3.29 5.68 − δ P i ) (1) Similarly, free magnesium concentration was determined from the chemical shift difference between the α and β peaks of ATP using the equation [ M g 2 + ] = K d ( 10.82 − δ α − β δ α − β − 8.35 ) (2) where δ α–β is the chemical shift difference between the α and β peaks of ATP. The K d for MgATP was initially assumed to be 50 μmol/L at pH 7.2 and 0.15 mol/L ionic strength and was corrected for pH according to Bock et al. (Bock et al., 1987). Cytosolic phosphorylation potential (PP) was determined according to the equation P P = [ Σ A T P ] [ Σ A D P ] [ Σ P i ] (3) where Σ represents all the ionic forms of the free species. The concentration of ADP was calculated from the creatine kinase equilibrium equation after correcting the equilibrium constant for pH and free magnesium concentration as previously described in detail elsewhere (Vink et al., 1994). Concentrations of the other metabolites were determined from the integrated peak areas of the respective MRS peaks, assuming that preinjury the normal values for PCr and ATP were 4.72 and 2.59 μmol/g, respectively, and that the total creatine pool remained constant at 10.83 μmol/g (Siesjo, 1981; Veech et al., 1979). Brain water content was assumed to be 80%, with the intracellular compartment accounting for 78% of the total water (Siesjo, 1981).

Morris water maze test Cognitive outcome (spatial learning) was determined using the hidden platform version of the Morris water maze as previously described (Hamm et al., 1996). Briefly, rats (n = 10/group) were trained to locate a hidden, submerged platform using constant extra-maze visual information, while the monitoring was performed using a PC-controlled video system (AccuScan Instruments, Columbus, OH, U.S.A.). The apparatus consists of a large, white circular pool (900-mm diameter, 500 mm high, water temperature 24 ± 1°C) with a Plexiglas platform 76 mm in diameter painted white and submerged 15 mm below 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 a constant location hidden in one quadrant 14 cm from the sidewall. The rat was gently placed in the water facing the wall at one of four randomly chosen locations separated by 90°. The latency to find the hidden platform within a 90-second criterion time was recorded by a masked observer. A series of 16 training trials, administered in blocks of four, were conducted on days 17, 18, 19, and 20 in rats after drug injection. To control for visual discriminative ability or motor impairment, the same animals were finally required to locate a clearly visible black platform (placed in a different location) raised 5 mm above the water surface at least 2 hours after the last training trial. The results were expressed as a latency to find the platform (seconds) ± SD.

Western immunoblotting The animals were killed and the brain was immediately removed. After dissection, the brain samples were stored at −80°C. For analysis, the brain tissue was resuspended in lysis buffer (60 mmol/L Tris-HCl, pH 7.8 containing 150 mmol/L NaCl, 5 mmol/L EDTA, 10% glycerol, 2 mmol/L Na 3 VO 4 , 25 mmol/L NaF, 10 μg/ml leupeptin (Sigma), 10 μg/ml aprotinin (Sigma), 1 mmol/L AEBSF (Sigma), 1 mmol/L Pepstatin (Sigma), 1 mmol/L Microcystin LR (Sigma), 0.1% sodium dodecyl sulfate (SDS), 0.5% Na deoxycholate, and 1% Triton X-100 (Calbiochem, La Jolla, CA, U.S.A.). The samples were incubated on ice for at least 30 minutes and centrifuged at 20,000 g for 15 min. The soluble fraction representing total cell extracts was recovered and stored at −80°C until use. Protein concentration in the samples was determined with the BCA assay kit (Pierce, Rockford, IL, U.S.A.). Equal protein aliquots (25 to 50 μg) were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Hybond-C super; Amersham, Arlington Heights, IL, U.S.A.). After transfer, the gels were stained with GelCode blue stain reagent (Pierce) to verify equal protein loading. The membranes were probed with specific primary antibodies and the immune complexes were detected using appropriate horseradish peroxidase–liked secondary antibodies (Amersham Pharmacia Biotech), chemiluminescence reagents (Super Signal WestDura, 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 centrifuged (10,000 rcf, 4°C). Supernatant was removed and centrifuged again (10,000 rcf, 4°C). The final supernatant was aliquoted and frozen. Later, protein concentration was determined using the method of Bradford (Bradford, 1976). Fifty micrograms of protein was then diluted in reaction buffer (25% Extraction 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 μmol/L Suc-Leu-Tyr-AMC) and activity was measured at excitation of 360 nm and an emission of 530 nm on a microplate fluorescence reader (Cytofluor4000, PerSeptive Biosystems).

Caspase activity assay Assay for caspase-3- and caspase-9-like activity was performed as previously described (Yakovlev et al., 1997). Aliquots of cytosolic extracts (25 μg of protein in 100 μL of extraction buffer) are preincubated at 37°C for 30 minutes, and then mixed with an equal volume of 40 μmol/L fluorescent tetrapeptide substrate (Ac-DEVD-AMC or Ac-LEHD-AMC, respectively; Bachem, Torrance, CA, U.S.A.) in the same buffer solution. Free aminomethylcoumarin (AMC) accumulation, which resulted from cleavage of the aspartate–AMC bond, is monitored continuously in each sample during 30 minutes in 96-well microtiter plates, using a CytoFluor II fluorometer (PerSeptive Biosystems, Framingham, MA, U.S.A.) at 360-nm excitation and 460 emission wavelengths. The emission from each well is plotted against time. Linear regression analysis of the initial velocity (slope) of each curve yielded an activity for each sample. Data are expressed as a percentage of the caspase activity in samples from sham-treated control animals.

Histology Brains, prefixed in 4% phosphate-saline buffered formaldehyde, were cut using a cryostat, and serial 6-μm, anterior-to-posterior sections were made. The sections were stained with hematoxylin and eosin. Cell count of neuronal profile was performed for the pyramidal cell layer of CA3 hippocampal area, in 10 randomly chosen sections from each brain, as previously described (Bramlett et al., 1997). All neurons were included, regardless of whether they were normal or with changed structural characteristics. Hippocampal cell count in our vehicle-treated animals (254 ± 4 neurons in CA3 area) was comparable to values from the literature (Bramlett et al., 1997). The neural profile of the CA3 hippocampal sector of treated animals was expressed as a percentage of the total number of the neuronal cells in the CA3 region in vehicle-treated animals (100%). Cell count data were analyzed statistically using the t-test for independent samples.

Gene profiling by oligonucleotide microarray Cortical and hippocampal samples were harvested at 24 hours, 48 hours, and 7 days after AEA (20 nmol/L, n = 3/time point) or vehicle administration (n = 3/time point). Total RNA from homogenized tissue was extracted using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, U.S.A.), and then converted to biotinylated cRNA and hybridized to 22 Affymetrix U34A oligonucleotide microarray containing 8,800 genes and expression sequence tags (ESTs), as described by us (Di Giovanni et al., 2003). Signal intensity values for each oligonucleotide probe set were calculated using Affymetrix GeneChip MAS 5.0 software. Any data not meeting stringent quality control criteria were repeated to meet standards. Signal intensity for each gene on the microarrays from AEA and vehicle-treated animals was normalized to levels in naïve cortex. Statistical analysis and hierarchic clustering was performed using Gene-Spring 5.0. Within each region, genes with significant changes were grouped by the primary function of their gene product.

Semiquantitative reverse transcriptase-polymerase chain reaction One μg of total RNA used for gene profiling was also used for 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 polymerase chain reaction (PCR) using primers specific for ribosomal protein RPL19. PCR reactions were performed in a PTC-225 Thermal Cycler (MJ Research, Waltham, MA, U.S.A.) using AmpliTaq 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 30 seconds 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 final incubation for 5 minutes at 72°C. PCR reaction products were analyzed by agarose gel-electrophoresis. Intensity of injured cortex and hippocampus (n = 3) was adjusted to respective vehicle controls (n = 3) using a housekeeping gene Ribosomal Protein (Invitrogen Corp., Carlsbad, CA, U.S.A.) L-19 (RPL-19). Normalized cDNA was then used to estimate the relative abundances of tissue inhibitors of metalloproteinase (TIMP)-1, MHC class II and brain-derived growth factor (BDNF) to confirm the array hybridization data. Primers for each gene were located in different exons. Different dilutions of cDNA samples were used for different genes to provide linear range of PCR reactions (Di Giovanni et al., 2003).