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The Journal of Neuroscience, April 15, 1999, 19(8):2987-2995
Cannabinoids and Neuroprotection in Global and Focal Cerebral
Ischemia and in Neuronal Cultures
Tetsuya
Nagayama1,
Amy
D.
Sinor1, 2,
Roger P.
Simon1,
Jun
Chen1,
Steven H.
Graham1,
Kunlin
Jin1, and
David A.
Greenberg1, 2
Departments of 1 Neurology and
2 Neurobiology, University of Pittsburgh School of
Medicine, Pittsburgh, Pennsylvania 15213
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ABSTRACT |
Marijuana and related drugs (cannabinoids) have been
proposed as treatments for a widening spectrum of medical disorders. R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate (R(+)-WIN 55212-2), a synthetic
cannabinoid agonist, decreased hippocampal neuronal loss
after transient global cerebral ischemia and reduced infarct volume
after permanent focal cerebral ischemia induced by middle cerebral
artery occlusion in rats. The less active enantiomer
S( )-WIN 55212-3 was ineffective,
and the protective effect of R(+)-WIN
55212-2 was blocked by the specific central cannabinoid
(CB1) cannabinoid receptor antagonist
N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide-hydrochloride. R(+)-WIN 55212-2 also protected cultured
cerebral cortical neurons from in vitro hypoxia and
glucose deprivation, but in contrast to the receptor-mediated
neuroprotection observed in vivo, this in
vitro effect was not stereoselective and was insensitive to CB1 and CB2 receptor antagonists. Cannabinoids
may have therapeutic potential in disorders resulting from cerebral
ischemia, including stroke, and may protect neurons from injury through
a variety of mechanisms.
Key words:
cannabinoid; ischemia; stroke; glutamate; excitotoxicity; infarct; neuronal culture
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INTRODUCTION |
Cannabis, the marijuana
plant, has been used since antiquity for its medicinal and psychoactive
properties (Snyder, 1971 ). Both its principal active ingredient,
 9-tetrahydrocannabinol (THC), and synthetic analogs
thereof (cannabinoids) have been proposed as therapy for a variety of
medical conditions, including glaucoma, cancer chemotherapy-induced
nausea and vomiting, acquired immunodeficiency syndrome,
inflammatory disorders, and epilepsy (Jack, 1997). This has contributed
to efforts to legalize marijuana use for therapeutic purposes (Annas,
1997; Kassirer, 1997). However, concern exists about the safety of
cannabinoids, including their possible role in infertility (Schmid et
al., 1997) and the extent to which they share effects with narcotics
(Rodríguez de Fonseca et al., 1997; Tanda et al., 1997). This
controversy persists despite major advances regarding the basic
molecular and cellular mechanisms of cannabinoid action (for review,
see Felder and Glass, 1998), including the discovery of endogenous cannabinoids (Devane et al., 1992; Stella et al., 1997; Randall and
Kendall, 1998), mechanisms for their synthesis and termination of
action (Di Marzo et al., 1994; Beltramo et al., 1997), cannabinoid receptors (Matsuda et al., 1990; Kuster et al., 1993; Howlett, 1995),
receptor-effector coupling pathways (Mackie and Hille, 1992;
Derkinderen et al., 1996), and synthetic cannabinoid agonist and
antagonist drugs (Compton et al., 1992; Rinaldi-Carmona et al.,
1994).
Central cannabinoid (CB1) receptors are coupled to
several signal transduction pathways, including G-proteins that inhibit N-type voltage-gated calcium channels involved in the release of
neurotransmitters (Mackie and Hille, 1992). These channels participate
in release of the excitatory transmitter L-glutamate, which
has been implicated in the death of neurons from stroke, hypoxia,
hypoglycemia, and epilepsy. In hippocampal cultures, cannabinoid
agonists acting through CB1 receptors and G-proteins inhibit glutamate release (Shen et al., 1996), suggesting that they
might reduce glutamate-mediated neuronal injury. Therefore, we examined
the effect of cannabinoids on neuronal death in the selectively
vulnerable CA1 region of rat hippocampus after transient global
cerebral ischemia induced by four-vessel occlusion, on infarct volume
after permanent occlusion of the middle cerebral artery (MCA) and on
the viability of cultured cerebral cortical neurons deprived of oxygen
and glucose in vitro.
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MATERIALS AND METHODS |
Animals. Animal experiments were approved by local
committee review and were conducted according to policies on the use of animals of the Society for Neuroscience. Male Sprague Dawley rats weighing 300-330 gm (global ischemia studies) or 280-310 gm (focal ischemia studies) were used. Anesthesia was induced with 4%
isoflurane, 66% N2O, and 30% O2 and, after
intubation, maintained with 1.5% isoflurane, 68.5% N2O,
and 30% O2. The left femoral artery was cannulated to
monitor arterial blood pressure, blood gases, and blood glucose
concentration. Rectal temperature was monitored continuously and
maintained at 37.0-37.5°C using a heating pad. In global ischemia
studies, brain temperature was monitored with a 29 gauge thermocouple
implanted in the right striatum and was maintained at 36-37°C with a
temperature-regulated heating lamp. In focal ischemia studies, the
temperature of the temporalis muscle contralateral to MCA occlusion was
monitored and maintained at 37.0-37.5°C in the same manner.
Global cerebral ischemia. Global cerebral ischemia lasting
15 min, followed by reperfusion, was induced by four-vessel occlusion in anesthetized rats (Pulsinelli et al., 1982). Animals were placed in
a Kopf stereotactic frame, and the vertebral arteries were coagulated
and transected at the junction of the C1 and C2 vertebrae under
microscopic guidance. The common carotid arteries (CCAs) were then
exposed, the external carotid arteries (ECAs) were ligated, and
administration of isoflurane was discontinued. Three minutes later, the
CCAs were occluded reversibly for 15 min with microvascular clips, and
perfusion was then restored. The electroencephalogram was monitored to
ensure isoelectricity during the period of ischemia. Temperature was
monitored from the time of intubation until ~30 min after the onset
of reperfusion, for a total of ~50-70 min.
Focal cerebral ischemia. Permanent focal ischemia was
induced by intraluminal occlusion of the MCA with a suture (Longa et al., 1989). Under a microscope, the left ECA was ligated with a 6-0 silk suture and dissected distally, and the left internal carotid
artery (ICA) was isolated and separated from the vagus nerve. The
extracranial branch of the left ICA was ligated close to its origin
with a 6-0 silk suture. A 3-0 surgical monofilament nylon suture with a
rounded tip was introduced into the left ICA lumen through the stump of
the left ECA and advanced 20-21 mm past the CCA bifurcation. The
suture was left in place until rats were killed 24 hr after the
onset of ischemia. Temperature was monitored from the time of
intubation until ~30 min after the onset of ischemia, for a total of
~30-45 min.
Drug administration.
R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate (R(+)-WIN 55212-2 mesylate) and
S()-WIN 55212-3 mesylate were purchased from
Research Biochemicals (Natick, MA) and
N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A) was obtained from the National Institute on Drug Abuse. Drugs were dissolved in dimethylsulfoxide (DMSO) and given as 300 µl
injections by the intraperitoneal route, 40 min before occlusion of the
CCAs (global ischemia) or 30 min before or 30, 60, or 120 min after MCA
occlusion (focal ischemia). R(+)-WIN 55212-2 produced
dose-dependent behavioral effects consisting of slight drowsiness (1 mg/kg), more marked drowsiness with limb rigidity and hypokinesia in
~50% of animals (3 mg/kg), and more severe rigidity and hypokinesia
in 100% of animals (10 mg/kg).
Quantification of neuronal loss. Three days after exposure
to global ischemia, animals were perfused transcardially with 200 ml of
saline and then 300 ml of 4% paraformaldehyde in 0.1 M
phosphate buffer, pH 7.4, and killed by decapitation. The brains were
removed and post-fixed in the same paraformaldehyde solution for 5 d and then embedded in paraffin, and 6 µm sections through the dorsal hippocampus (anteroposterior coordinate, bregma 3.0 mm) were cut on a microtome and processed for staining with cresyl violet. Neuronal counts in a predesignated region of CA1 were obtained from six
to eight animals per condition.
Quantification of infarct volume. Rats subjected to focal
ischemia were anesthetized with an overdose of chloral hydrate and decapitated. The brains were removed and sectioned coronally at 2 mm
intervals. Sections were immersed in 2% 2,3,5-triphenyltetrazolium hydrochloride (TTC) in saline for 20 min at 37°C and then fixed for
30 min in 4% paraformaldehyde (Isayama et al., 1991). Six sections per
brain were analyzed for infarct size using a computerized image
analysis system (MCID, St. Catharine's, Ontario, Canada). Infarct area in each section was calculated by subtracting the residual
uninfarcted, TTC-stained area of the ischemic (left) hemisphere from
the total area of the nonischemic (right) hemisphere (Swanson et al.,
1990). Infarct volume at 24 hr measured in this manner is equivalent to
infarct volume determined from hematoxylin- and eosin-stained sections
(Isayama et al., 1991).
Neuronal cell culture. Neuronal cultures were prepared from
16-17 d Sprague Dawley rat embryos (Yu et al., 1986). Cerebral hemispheres were removed aseptically, freed of meninges, olfactory bulbs, basal ganglia, and hippocampi, and incubated at 37°C in Ca2+- and Mg2+-free Earle's
balanced salt solution containing 0.01% trypsin (1:250). After 30 min,
10% horse serum (HS) was added. Cells were placed in 2 ml of fresh
MEM, triturated, and resuspended in Eagle's MEM prepared without
glutamine and with twice the usual concentration of other amino acids
and four times the usual concentration of vitamins (MEM-Pak; Cell
Culture Facility, University of California, San Francisco, CA); this
medium had been supplemented on the day of plating with glucose (final
concentration, 30 mM), 2 mM glutamine, and 15 mM HEPES, pH 7.4. Cell suspensions were filtered through a
70 µm Falcon nylon cell strainer, supplemented with 10% HS and 10%
fetal bovine serum (FBS), and seeded at 3 × 105 cells per well on 24-well Corning (Corning, NY)
tissue culture dishes coated with 100 µg/ml
poly-D-lysine. Cultures were incubated for 20 min at 37°C
in humidified 95% air and 5% CO2, and one-half of
the medium was replaced with medium containing 5% HS and 5% FBS.
Cytosine arabinoside (AraC, 10 µM) was added on the sixth day in vitro (DIV). At 7 DIV, two-thirds of the medium was
replaced with medium lacking AraC; thereafter, one-half of the medium
was replaced with fresh medium twice weekly. Experiments were conducted at 18 DIV, when cultures consisted primarily of neurons (92 ± 1%
MAP2-immunoreactive cells, 6 ± 1% GFAP-immunoreactive cells; n = 12).
In vitro model of ischemia. To model neuronal
ischemia in vitro, cultures were exposed to combined hypoxia
and glucose deprivation (Koretz et al., 1994). Two-thirds of the medium
was replaced three times with serum-free medium, with or without 30 mM glucose. Glucose-containing cultures were then incubated
for 24 hr at 37°C in humidified 95% air and 5% CO2.
Glucose-deprived cultures were placed in a Billups-Rothenberg (Del Mar,
CA) modular incubator chamber, which was flushed for 5 min with 95%
N2 and 5% CO2, sealed for 15 min,
flushed again for 30 sec with 5% N2 and 5%
CO2, and resealed. The chamber was placed in a
water-jacketed incubator at 37°C for 8 hr and then returned to 95%
air and 5% CO2 and glucose-containing medium for 16 hr. In
some experiments, glucose-containing, normoxic cultures were treated
for 8 hr with and then 16 hr without 1 mM NMDA or 1 mM AMPA in the presence or absence of the NMDA
antagonist
(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-benzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) or the AMPA antagonist
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (all from Research
Biochemicals, Natick, MA). Stock solutions of cannabinoid receptor
agonists and antagonists were prepared in DMSO and diluted to a final
concentration not exceeding 7.36 µM DMSO in the
drug-treated cultures. At concentrations at least 10-fold higher than
this, DMSO had no effect on neuronal viability in either normoxic or
hypoxic cultures.
Cytotoxicity assays. Fluorescence of Alamar blue (Accumed
International, Westlake, OH), an indicator that changes color from blue
to red and fluoresces when reduced by cellular metabolic activity, was
used to measure the viability of cultured neurons. One-half of the
culture medium was replaced with MEM-Pak containing 10% (v/v) Alamar
blue, and cultures were incubated for 3 hr at 37°C in humidified 95%
air and 5% CO2. Fluorescence was determined in a Millipore
(Bedford, MA) CytoFluor 2300 automated plate-reading fluorometer, with
excitation at 530 nm and emission at 590 nm. As reported previously,
Alamar blue fluorescence in these cultures varies linearly with cell
number, decreases with exposure to hypoxia or excitotoxins, and
correlates with the extent of cellular injury determined by lactate
dehydrogenase release (White et al., 1996).
Detection of DNA damage. DNA polymerase I-mediated
biotin-dATP nick translation (PANT) labeling was used to detect DNA
single-strand breaks (Didier et al., 1996). Cultures were fixed for 15 min at room temperature by adding 20% paraformaldehyde in PBS, pH 7.4, to a final concentration of 4%, washed three times with PBS, and permeabilized for 10 min with 0.5% Triton X-100 in PBS.
H2O2 (1%) was added for 15 min at room
temperature to neutralize endogenous peroxidase, after which cultures
were again washed three times with PBS. Cells were then incubated for
60 min at 37°C in PBS, pH 7.6, containing 5 mM
MgCl2, 10 mM 2-mercaptoethanol, 50 µg/ml bovine serum albumin (BSA), 20 µM dCTP, dGTP,
dTTP, and biotinylated-dATP, and 50 U/ml Escherichia coli
DNA polymerase I (Sigma, St. Louis, MO). Cells were washed three times
with ice-cold PBS and once with 5 mg/ml BSA in PBS (PBS-BSA), incubated
for 1 hr at room temperature with streptavidin-horseradish peroxidase
(HRP) in PBS-BSA (Vector Laboratories, Burlingame, CA), and washed four times in PBS. The HRP complex was detected after incubation for 5 min
at room temperature in PBS containing 0.5 mg/ml diaminobenzidine, 0.03% H2O2, and nickel (added according
to the manufacturer's instructions). Cells with dense nuclear staining
were considered PANT-positive and were counted at 200× magnification
in at least three randomly selected fields per well. Data were
expressed as a percentage of all cells in the same fields, with
reactions conducted in the absence of DNA polymerase I serving as
controls for nonspecific labeling.
Statistical analysis. Results are reported as mean
values ± SE. The significance of differences between means was
assessed by Student's t test (single comparisons) or by
ANOVA and post hoc t tests (multiple comparisons),
with p < 0.05 considered statistically significant.
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RESULTS |
Global cerebral ischemia
In control animals that underwent sham CCA occlusions, ~300
neurons could be counted in the designated region of CA1, whereas only
zero to five neurons were seen after global ischemia (Figs. 1, 2).
R(+)-WIN 55212-2, a synthetic aminoalkylindole
cannabimimetic compound, competes with THC for binding to cannabinoid
receptor sites in brain (Kuster et al., 1993) and reproduces
pharmacological and behavioral effects of THC (Compton et al., 1992).
When animals were given R(+)-WIN 55212-2 mesylate
intraperitoneally 40 min before the induction of global cerebral
ischemia, there was a dose-dependent increase in neuronal survival that
reached maximal levels (56% of sham-operated controls) at 1 mg/kg. At
higher doses, neuronal survival declined. The doses that protected
against ischemic neuronal death were similar to those associated with
behavioral actions such as locomotor inhibition, antinociception, and
THC-like discriminative effects (Compton et al., 1992). Neuroprotection by R(+)-WIN 55212-2 (1 mg/kg) was not associated with
significant changes before, during, or after the induction of ischemia
in mean arterial blood pressure, PaO2,
PaCO2, pH, or blood glucose concentration
compared with values in animals that received DMSO vehicle alone (Table
1).
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Figure 1.
Histological evidence for cannabinoid-mediated
protection of CA1 hippocampal neurons in global cerebral ischemia. Rats
underwent sham surgery or 15 min of cerebral ischemia induced by
four-vessel occlusion, with or without the administration of drugs 40 min before the onset of ischemia. Animals were killed 3 d later
and paraffin-embedded brain sections were stained with cresyl violet.
Sections are shown at low (left, 25×) and high
(right, 400×) power, after sham surgery
(Sham) or after the induction of ischemia without
(Isch) or with previous administration of
R(+)-WIN 55212-2 [R(+)],
S()-WIN 55212-3 [S()], or SR
141716A (SR) at the indicated doses (milligrams per
kilogram, i.p.). Asterisks at left
indicate the region within CA1 from which the fields shown at
right were taken.
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Figure 2.
Neuron counts in CA1 after global cerebral
ischemia. Animals were treated as described in the legend to Figure 1,
undergoing sham surgery (Sham) or global cerebral
ischemia without drugs (Isch) or with previous
administration of R(+)-WIN 55212-2 [R(+)], S()-WIN 55212-3 [S()], or SR 141716A (SR) at the
indicated doses (milligrams per kilogram, i.p.). Neurons were counted
in cresyl violet-stained sections. Data shown are mean values ± SEs from six to eight animals per condition. *p < 0.05 (ANOVA with post hoc t tests) relative to neuron
counts in animals subjected to ischemia without drug treatment
(Isch).
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R(+)-WIN 55212-2 is one of a pair of enantiomers that
exhibit stereoselectivity in cannabinoid receptor radioligand binding assays and behavioral studies. R(+)-WIN 55212-2 has
~2500-fold greater affinity than its enantiomer, S()-WIN
55212-3, for CB1 receptor binding sites on rat cerebellar
membranes (Kuster et al., 1993), and the S() enantiomer is
inactive in behavioral assays of cannabimimetic activity that show
potent effects of R(+)-WIN 55212-2 (Compton et al., 1992).
When animals were treated with S()-WIN 55212-3 before
ischemia, neuronal counts in CA1 were not significantly different from
counts obtained after ischemia alone and were significantly lower than
counts in animals given R(+)-WIN 55212-2.
SR141716A is a selective, competitive antagonist that inhibits
radioligand binding to CB1 cannabinoid receptors in rat
brain synaptosomal membranes, cannabinoid-induced inhibition of smooth muscle contraction in mouse vas deferens, and behavioral effects of
R(+)-WIN 55212-2, including locomotor inhibition and
antinociception (Rinaldi-Carmona et al., 1994). When administered alone
before ischemia, SR141716A failed to alter neuronal counts. However, when 1 mg/kg SR141716A was given together with 1 mg/kg
R(+)-WIN 55212-2, the neuroprotective effect of the latter
was reduced by 80%.
Focal cerebral ischemia
MCA occlusion produced ipsilateral cerebral infarcts averaging
~210 mm3 in volume, which could be detected after
24 hr by the loss of TTC staining (Figs.
3, 4).
Pretreatment with 1 mg/kg R(+)-WIN 55212-2 given 30 min
before the onset of ischemia reduced infarct size by ~30%. The drug
was similarly effective when given 30 min after the onset of ischemia,
but its effect was lost when administration was delayed by 60-120 min.
Cerebral salvage occurred primarily in the periphery of the ischemic
region, leading to enhanced survival of penumbral cortical tissue,
without affecting the ischemic core in the striatum. Reduction of
infarct size by R(+)-WIN 55212-2 (1 mg/kg) was not
accompanied by alterations in mean arterial blood pressure,
PaO2, PaCO2, pH, or
blood glucose concentration, measured 10 min after the onset of
ischemia, compared with values in control animals given DMSO vehicle
alone (Table 2).
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Figure 3.
Histological evidence for cannabinoid-mediated
reduction in infarct size after permanent MCA occlusion. The left MCA
(on the viewer's right) was occluded with a nylon
suture as described in Materials and Methods. Animals were given DMSO
vehicle 30 min before occlusion (Veh); 1 mg/kg
R(+)-WIN 55212-2 30 min before [R(+)
30], or 30 [R(+) 30], 60 [R(+) 60], or 120 [R(+) 120] min
after occlusion; or 1mg/kg R(+)-WIN 55212-2 and 1 mg/kg
SR141716A [R(+) & SR], 1 mg/kg SR141716A alone
(SR), or 1 mg/kg S()-WIN 55212-3 [S()], all 30 min before occlusion. Drugs were administered intraperitoneally.
Animals were killed 24 hr after the onset of occlusion, and brain
sections were treated with TTC (red), which stains
viable tissue red but does not stain infarcted tissue. The brains shown
are representative of six animals per condition. S()-WIN 55212-3 [S()],
or 10 µM THC. After an additional 16 hr under control
conditions, cell viability was measured by Alamar blue fluorescence and
expressed as a percentage of fluorescence above background in control
cultures. Data shown are mean values ± SEs from 4-12 cultures
per condition. *p < 0.05 (ANOVA with post
hoc t tests) relative to fluorescence in cultures exposed to
hypoxia and glucose deprivation in the presence of 10 nM
R(+)-WIN 55212-2. C, Concentration
dependence of neuroprotection induced by R(+)-WIN
55212-2. Experiments were performed as described in B
above, except that different concentrations of R(+)-WIN
55212-2 were used, and results were expressed as a percentage of the
maximal drug-induced increase in viability above that in cultures
exposed to hypoxia and glucose deprivation without drugs. Data shown
are mean values ± SEs from 4-12 cultures per condition.
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Figure 4.
Infarct volumes after focal cerebral ischemia.
Animals were treated as described in the legend to Figure 3, undergoing
permanent MCA occlusion without drugs (Veh) or with
intraperitoneal administration of 1 mg/kg R(+)-WIN
55212-2 given 30 min before (30) or 30, 60, or 120 min
after the onset of ischemia; 1 mg/kg R(+)-WIN 55212-2 and 1 mg/kg SR141716A, both given 30 min before ischemia; 1 mg/kg
SR141716A alone given 30 min before ischemia; or 1 mg/kg
S()-WIN 55212-3. Data shown are mean values ± SEs from six animals per condition. *p < 0.05 (ANOVA with post hoc t tests) relative to infarct
volumes in animals subjected to ischemia and treated with vehicle only
(Veh).
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Table 2.
Blood pressure, arterial blood gases, and serum glucose 10 min after the onset of permanent focal cerebral ischemia
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The CB1 cannabinoid receptor antagonist SR141716A had no
effect on infarct size when administered alone but reversed the
protective effect of R(+)-WIN 55212-2 given 30 min before
the onset of ischemia. The less active agonist isomer,
S()-WIN 55212-3, failed to alter infarct volume.
In vitro hypoxia and glucose deprivation
Both normoxic and hypoxic cortical cultures expressed
CB1 receptor immunoreactivity on Western blots probed with
a polyclonal rabbit IgG (Calbiochem, La Jolla, CA) raised against the
N-terminal domain of the human CB1 receptor (Fig.
5A). Exposure of cortical cultures to hypoxia and glucose deprivation reduced neuronal viability (Alamar blue fluorescence), with approximately half-maximal toxicity at
8 hr and maximal toxicity by 24 hr. The NMDA antagonist MK-801 reduced
toxicity, as did R(+)-WIN 55212-2 and the endogenous
cannabinoid anandamide (Fig. 5B). Exposure for 8 hr to 1 mM NMDA or AMPA decreased neuronal viability to an extent
similar to that seen with hypoxia and glucose deprivation, but although
MK-801 blocked NMDA toxicity, and the AMPA antagonist CNQX blocked AMPA
toxicity under these conditions, R(+)-WIN 55212-2 was
ineffective (data not shown). PANT staining, indicative of cells with
DNA single-strand breaks (Chen et al., 1997), also increased after
hypoxia and glucose deprivation, and this increase was attenuated by
both MK-801 and R(+)-WIN 55212-2 (Fig.
6).
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Figure 5.
Pharmacological characterization of
cannabinoid-mediated neuroprotection from hypoxia and glucose
deprivation in vitro. Cultured cerebral cortical neurons
were exposed for 24 hr to normoxia and glucose (control), or to 8 hr of
hypoxia and glucose deprivation followed by 16 hr of recovery
(hypoxic). A, Western analysis of control (lane
1) and hypoxic (lane 2) cultures probed with an
antibody against the CB1 cannabinoid receptor
(arrow). B, Pharmacological features of
cannabinoid-mediated neuroprotection. Cultures were exposed to hypoxia
and glucose deprivation in the absence (Hypoxia) or
presence of 10 µM MK-801 (MK), 100 nM anandamide (Anand), 10 nM
R(+)-WIN 55212-2 (alone [R(+)] or
together with the CB1 antagonist SR141617A [R(+) & CB1] or the CB2 antagonist SR144528 [R(+)
& CB2], each at 300 nM-1 µM), 10 nM
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Figure 6.
PANT staining for DNA single-strand breaks after
in vitro hypoxia and glucose deprivation. Cultures were
maintained for 8 hr under control conditions (A)
or exposed for 8 hr to hypoxia and glucose deprivation in the absence
(B) or presence of 10 µM MK-801
(C) or 10 nM R(+)-WIN
55212-2 (D). After an additional 16 hr under
control conditions, cultures were fixed and processed for PANT staining
as described in Materials and Methods. Cells with dense nuclear
staining were considered PANT-positive and were counted at 200×
magnification in at least three randomly selected fields per well.
E, Quantitative data were expressed as a percentage of
all cells in the same fields and are shown are mean values ± SEs
from 6-12 cultures per condition. *p < 0.05 (ANOVA with post hoc t tests) relative to values in
cultures exposed to hypoxia and glucose deprivation in the absence of
drugs (Hypoxia).
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To test whether the same CB1 receptor-mediated mechanism
involved in protection from in vivo ischemia was also
responsible for in vitro neuroprotection by
R(+)-WIN 55212-2, we examined the effects of the less-active
enantiomer S()-WIN 55212-3, the CB1 agonists
anandamide and THC, the CB1 antagonist SR141716A, and the
CB2 antagonist
N-[(1S)-endo-1,3,3-trimethyl
bicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528; obtained from the National Institute on Drug Abuse) (Rinaldi-Carmona et al., 1998). As shown in Figure 5B,
protection from hypoxia and glucose deprivation in our cultures did not
have the pharmacological features of a CB1 or
CB2 receptor-mediated process. Thus, although the
endogenous cannabinoid anandamide reduced hypoxic injury, THC did not.
Moreover, neuroprotection lacked stereoselectivity and was insensitive
to CB1 and CB2 antagonists. As was also
observed in our studies on global ischemia, the protective effect of
R(+)-WIN 55212-2 was lost at higher concentrations (Fig. 5C).
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DISCUSSION |
The major finding we report is that the synthetic cannabinoid
R(+)-WIN 55212-2, an analog of the psychoactive constituents of marijuana, protects brain tissue against ischemic injury. This effect appears to be mediated through CB1 cannabinoid
receptors, because it is stereospecific and is blocked by a selective
CB1 receptor antagonist, SR141716A. We observed
neuroprotection by R(+)-WIN 55212-2 in both global and focal
ischemia and, in the latter case, with drug administration either
before or up to 30 min after the onset of ischemia.
Cannabinoid-mediated protection from global ischemia was seen in the
CA1 region of hippocampus, which is especially vulnerable to ischemic
injury, and inhibition of glutamate release by cannabinoids has been
demonstrated in cells cultured from this region (Shen et al., 1996).
Cannabinoid receptor levels in hippocampus, including CA1, and in
cerebral cortex, which was the major site of cannabinoid-mediated
neuroprotection in our focal ischemia model, are among the highest in
the brain (Matsuda et al., 1990; Kuster et al., 1993; Adams et al.,
1998).
In principle, the neuroprotective action of R(+)-WIN 55212-2 could be exerted in a variety of ways. For example, the ability of
cannabinoids to inhibit ion flux through calcium channels (Mackie and
Hille, 1992) and thereby reduce glutamate release (Shen et al., 1996)
suggests that these effects could be responsible for neuroprotection.
However, cannabinoid receptor activation has been shown to affect a
variety of second mesenger systems, including the cAMP and
phospholipase A2-cyclooxygenase signal transduction pathways (Chan et al., 1998), so reduction of calcium influx may not
necessarily be responsible for cannabinoid-mediated neuroprotection. The vasoactive properties of cannabinoids (Randall and Kendall, 1998)
raise the possibility that they may alter blood flow in the ischemic
brain, but although it would be premature to conclude that such
alterations have no role in the neuroprotective effect of cannabinoids,
a major contribution of altered blood flow seems unlikely. Protection
was seen not only in focal ischemia, in which blood flow might be
redistributed from nonischemic to ischemic brain regions, but also in
global cerebral ischemia, which affects the brain diffusely. Moreover,
isoelectricity of the electroencephalogram, reflecting ischemia severe
enough to interrupt brain function, was present during global ischemia
in both cannabinoid-treated and untreated animals, arguing against
cannabinoid-induced preservation of cerebral blood flow. Cannabinoids
can also promote hypothermia, which is neuroprotective in some
settings. Although rectal and brain temperature were held constant
during the early stages of our global and focal ischemia studies, a
contributory effect of late hypothermia on outcome cannot be excluded.
Finally, cannabinoids exert anti-inflammatory effects, at least in part
by inhibiting the proliferation of lymphocytes and inducing their death
by apoptosis (Schwarz et al., 1994), and inflammation has been
implicated in focal ischemic brain injury (Nogawa et al., 1997).
However, inflammation appears to be a less prominent feature of
transient global cerebral ischemia (Dirnagl et al., 1994), in which we
found neuroprotection by cannabinoids similarly effective.
We also observed neuronal protection in an in vitro cell
culture model of neuronal hypoxia and glucose deprivation, but the mechanism appeared to differ from that observed in vivo. In
particular, neuroprotection in vitro lacked
stereoselectivity for R(+)-WIN 55212-2 over
S()-WIN 55212-3 and was insensitive to inhibition by
cannabinoid receptor antagonists. This resembles in some respects the
effect reported by Hampson et al. (1998b), who found that comparatively
high concentrations of THC reduced excitotoxicity in cultured cortical
neurons by a receptor-independent mechanism. In contrast to those
investigators, however, we found no effect of THC and no protection
against directly applied excitatory amino acids or the nitric oxide
donor sodium nitroprusside (data not shown), arguing against an
antioxidant effect such as that they proposed. Whether the
nonreceptor-mediated neuroprotective effect of cannabinoids that we
observed in vitro also operates in vivo is
unclear, although there is precedent for the coexistence of parallel,
receptor-mediated and non-receptor-mediated effects of cannabinoids on
neurotransmission and signal transduction (Felder et al., 1992; Hampson
et al., 1998a). Therefore, it would not be altogether surprising if
similarly parallel neuroprotective mechanisms were found.
Two other in vitro studies of cannabinoid effects have
appeared recently and require comment. One described a toxic
effect of THC on cultured hippocampal neurons (Chan et al., 1998), and the other showed a protective effect of R(+)-WIN
55212-2 on cultured hippocampal neurons exposed to excitotoxicity
induced by Mg2+ depletion (Shen and Thayer, 1998).
Both effects appeared to be CB1 receptor-mediated. Thus,
neurotoxic and neuroprotective as well as receptor-mediated and
non-receptor-mediatedeffects of cannabinoids can be observed in
vitro. These differences are likely to depend on a variety of
influences, apparently including the nature of the toxic insult, the
source of cells, and the particular cannabinoid used. In light of our
finding that cannabinoids afford receptor-mediated neuroprotection
against global and focal cerebral ischemia in vivo
(occurring in hippocampus and cerebral cortex, respectively), the
discrepant results of our and other in vitro studies accent
the importance of using in vivo models to establish how
potential therapeutic agents are likely to affect intact organisms.
Whether the in vivo protective effect of cannabinoids that
we observed is permanent or only delays ischemic death beyond the time
frames examined remains to be shown, as does the relationship between
histological and functional improvement. Nevertheless, the ability of
cannabinoids to improve histological outcome after both global and
focal cerebral ischemia in rats indicates that further investigation of
its potential therapeutic role in cerebral ischemia, such as occurs in
stroke and after cardiac arrest, may be warranted.
| |
FOOTNOTES |
Received July 13, 1998; revised Jan. 27, 1999; accepted Feb. 1, 1999.
This work was supported by National Institutes of Health Grants NS24728
and NS35965. We thank Wei Pei and Marie Rose for help with biochemical assays.
Correspondence should be addressed to Dr. David A. Greenberg,
Department of Neurology, University of Pittsburgh, S-526 Biomedical Science Tower, 3500 Terrace Street, Pittsburgh, PA 15213.
| |
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E. Molina-Holgado, J. M. Vela, A. Arevalo-Martin, G. Almazan, F. Molina-Holgado, J. Borrell, and C. Guaza
Cannabinoids Promote Oligodendrocyte Progenitor Survival: Involvement of Cannabinoid Receptors and Phosphatidylinositol-3 Kinase/Akt Signaling
J. Neurosci.,
November 15, 2002;
22(22):
9742 - 9753.
[Abstract]
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[PDF]
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S. Parmentier-Batteur, K. Jin, X. O. Mao, L. Xie, and D. A. Greenberg
Increased Severity of Stroke in CB1 Cannabinoid Receptor Knock-Out Mice
J. Neurosci.,
November 15, 2002;
22(22):
9771 - 9775.
[Abstract]
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[PDF]
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S. Parmentier-Batteur, K. Jin, L. Xie, X. O. Mao, and D. A. Greenberg
DNA Microarray Analysis of Cannabinoid Signaling in Mouse Brain in Vivo
Mol. Pharmacol.,
October 1, 2002;
62(4):
828 - 835.
[Abstract]
[Full Text]
[PDF]
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T. Gomez del Pulgar, M. L. de Ceballos, M. Guzman, and G. Velasco
Cannabinoids Protect Astrocytes from Ceramide-induced Apoptosis through the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway
J. Biol. Chem.,
September 20, 2002;
277(39):
36527 - 36533.
[Abstract]
[Full Text]
[PDF]
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W.-R. Schabitz, A. Giuffrida, C. Berger, A. Aschoff, M. Schwaninger, S. Schwab, and D. Piomelli
Release of Fatty Acid Amides in a Patient With Hemispheric Stroke: A Microdialysis Study
Stroke,
August 1, 2002;
33(8):
2112 - 2114.
[Abstract]
[Full Text]
[PDF]
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F. Mauler, J. Mittendorf, E. Horvath, and J. de Vry
Characterization of the Diarylether Sulfonylester (-)-(R)-3-(2-Hydroxymethylindanyl-4-oxy)phenyl-4,4,4-trifluoro-1-sulfonate (BAY 38-7271) as a Potent Cannabinoid Receptor Agonist with Neuroprotective Properties
J. Pharmacol. Exp. Ther.,
July 1, 2002;
302(1):
359 - 368.
[Abstract]
[Full Text]
[PDF]
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R. Mechoulam, M. Spatz, and E. Shohami
Endocannabinoids and Neuroprotection
Sci. Signal.,
April 23, 2002;
2002(129):
re5 - re5.
[Abstract]
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M. van der Stelt, W. B. Veldhuis, G. W. van Haaften, F. Fezza, T. Bisogno, P. R. Bar, G. A. Veldink, J. F. G. Vliegenthart, V. Di Marzo, and K. Nicolay
Exogenous Anandamide Protects Rat Brain against Acute Neuronal Injury In Vivo
J. Neurosci.,
November 15, 2001;
21(22):
8765 - 8771.
[Abstract]
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M. van der Stelt, W. B. Veldhuis, P. R. Bar, G. A. Veldink, J. F. G. Vliegenthart, and K. Nicolay
Neuroprotection by {Delta}9-Tetrahydrocannabinol, the Main Active Compound in Marijuana, against Ouabain-Induced In Vivo Excitotoxicity
J. Neurosci.,
September 1, 2001;
21(17):
6475 - 6479.
[Abstract]
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[PDF]
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G. T. Carter and B. S. Rosen
Marijuana in the management of amyotrophic lateral sclerosis
American Journal of Hospice and Palliative Medicine,
July 1, 2001;
18(4):
264 - 270.
[Abstract]
[PDF]
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G. Cao, W. Pei, J. Lan, R. A. Stetler, Y. Luo, T. Nagayama, S. H. Graham, X.-M. Yin, R. P. Simon, and J. Chen
Caspase-Activated DNase/DNA Fragmentation Factor 40 Mediates Apoptotic DNA Fragmentation in Transient Cerebral Ischemia and in Neuronal Cultures
J. Neurosci.,
July 1, 2001;
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4678 - 4690.
[Abstract]
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B. Moesgaard, G. Petersen, J. W. Jaroszewski, and H. S. Hansen
Age dependent accumulation of N-acyl-ethanolamine phospholipids in ischemic rat brain: a 31P NMR and enzyme activity study
J. Lipid Res.,
June 1, 2000;
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[Abstract]
[Full Text]
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TOP
ABSTRACT
INTRODUCTION
