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The Journal of Neuroscience, November 15, 2001, 21(22):8765-8771
Exogenous Anandamide Protects Rat Brain against Acute Neuronal
Injury In Vivo
M.
van der Stelt1,
W.
B.
Veldhuis2, 3,
G. W.
van
Haaften1,
F.
Fezza4,
T.
Bisogno4,
P. R.
Bär3,
G. A.
Veldink1,
J. F. G.
Vliegenthart1,
V.
Di
Marzo4, and
K.
Nicolay2
1 Department of Bio-organic Chemistry, Bijvoet Center
for Biomolecular Research, Utrecht University, 3584 CH Utrecht,
The Netherlands, 2 Department of Experimental In Vivo
Nuclear Magnetic Resonance, Image Sciences Institute, Utrecht
University Medical Center, 3584 CJ Utrecht, The Netherlands,
3 Department of Experimental Neurology, Utrecht University
Medical Center, 3584 CX Utrecht, The Netherlands, and
4 Endocannabinoid Research Group, Istituto per la Chimica
di Molecole di Interesse Biologico, Consiglio Nazionale delle Ricerche,
80078 Pozzuoli, Naples, Italy
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ABSTRACT |
The endocannabinoid anandamide
[N-arachidonoylethanolamine (AEA)] is thought to
function as an endogenous protective factor of the brain against acute
neuronal damage. However, this has never been tested in an in
vivo model of acute brain injury. Here, we show in a
longitudinal pharmacological magnetic resonance imaging study
that exogenously administered AEA dose-dependently reduced neuronal
damage in neonatal rats injected intracerebrally with the
Na+/K+-ATPase inhibitor ouabain.
At 15 min after injury, AEA (10 mg/kg) administered 30 min before
ouabain injection reduced the volume of cytotoxic edema by 43 ± 15% in a manner insensitive to the cannabinoid CB1
receptor antagonist SR141716A. At 7 d after ouabain treatment,
64 ± 24% less neuronal damage was observed in
AEA-treated (10 mg/kg) rats compared with control animals.
Coadministration of SR141716A prevented the neuroprotective
actions of AEA at this end point. In addition, (1) no increase in AEA
and 2-arachidonoylglycerol levels was detected at 2, 8, or 24 hr after
ouabain injection; (2) application of SR141716A alone did not increase
the lesion volume at days 0 and 7; and (3) the AEA-uptake inhibitor,
VDM11, did not affect the lesion volume. These data indicate that there was no endogenous endocannabinoid tone controlling the acute neuronal damage induced by ouabain. Although our data seem to question a
possible role of the endogenous cannabinoid system in establishing a
brain defense system in our model, AEA may be used as a structural template to develop neuroprotective agents.
Key words:
anandamide; 2-AG; astrogliosis; cannabinoid; excitotoxicity; ischemia; magnetic resonance imaging; neonatal rat; neuronal injury; neuroprotection; ouabain
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INTRODUCTION |
The CNS is highly vulnerable
to ischemia. Neuronal death caused by ischemia is executed via a
complex array of processes in which excitotoxicity plays a major role.
In excitotoxicity, cell death is triggered by the overstimulation of
excitatory amino acid receptors. This leads to cytotoxic levels of
calcium and to subsequent activation of destructive pathways, involving
among others caspases, calpains, and the generation of reactive oxygen species (Dirnagl et al., 1999 ; Doble, 1999).
Compounds that interfere with excitotoxicity may be used as
neuroprotective therapeutic agents. Interestingly, the brain has various endogenous protection factors at its disposal (e.g., adenosine, melatonin, and estrogens) (Reiter, 1998; Hurn and Macrae, 2000; Picano
and Abbracchio, 2000). Several reports have also revealed a connection
between the endogenous lipid anandamide
[N-arachidonoylethanolamine (AEA)] and neurodegenerative
diseases (H. S. Hansen et al., 1998, 2000; Di Marzo et al.,
2000b; Baker et al., 2001).
AEA mimics in part the actions of
 9-tetrahydrocannabinol (THC), the
psychoactive compound in marijuana. Together with
2-arachidonoylglycerol (2-AG), AEA represents a class of lipids, termed
endocannabinoids because of their ability to activate the
CB1 and CB2 cannabinoid receptors. AEA is rapidly translocated into the cell via a transporter protein and is then immediately inactivated by a fatty acid amide hydrolase (FAAH) (Pertwee, 1997; Di Marzo et al., 1998).
Several lines of evidence indicate that AEA can serve to protect the
brain against neuronal injury (H. S. Hansen et al., 1998, 2000):
(1) AEA and its precursor
N-arachidonoylphosphatidylethanolamine are normally
found in low concentrations in the brain, but their levels increase in
a calcium-dependent manner postmortem and with severe neuronal injury
(Schmid et al., 1990; Kempe et al. 1996; Hansen et al., 1999; H. H. Hansen et al., 2000, 2001; Sugiura et al., 2000); (2)
exogenous AEA protects cerebral neurons from in vitro
ischemia (Sinor et al., 2000); (3) CB1-mediated
closing of N- and P/Q-type calcium channels protects neurons
against in vitro secondary excitotoxicity (Shen et al.,
1996; Shen and Thayer, 1998); (4) we have demonstrated recently that
THC can reduce neuronal damage via the CB1
receptor in an in vivo model of excitotoxicity (Van der
Stelt et al., 2001); (5) WIN55.212, a synthetic cannabinoid, protected
rat brains against focal and global ischemia (Nagayama et al., 1999);
and (6) CB1 expression is enhanced in the
cortical mantle zone in rats after ischemia (Jin et al., 2000). As yet, in vivo neuroprotection by AEA has never been reported.
To date no effective drugs are available to treat brain injury after
transient (global) or permanent focal cerebral ischemia. Insights into
how the brain defends itself may lead to novel strategies to develop
new therapeutic agents. Therefore, it was our goal to test whether the
endogenous cannabinoid system affords the brain protection in an
in vivo model of neuronal injury. In this study we combined
longitudinal pharmacological magnetic resonance imaging (MRI) and
isotope dilution gas chromatography/mass spectrometry (GC/MS)
techniques. Our data indicate that there is no endogenous endocannabinoid tone controlling the acute neuronal damage induced by
ouabain, a
Na+/K+-ATPase
inhibitor, although exogenous AEA can effectively reduce toxin-induced
injury in the neonatal rat brain.
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MATERIALS AND METHODS |
Animal model. The neonatal rat brain was used to test
the neuroprotective effects of endocannabinoids because it has been widely studied in various models of cerebral ischemia and
excitotoxicity (Vannucci, 1990; van Lookeren Campagne et al., 1994;
Dijkhuizen et al., 1996; Renolleau et al., 1998; Hansen et al., 2001).
Neonatal Wistar rats (U:Wu/Cpb; 7-8 d of age) were anesthetized with
ether and immobilized in a stereotaxic frame. A small burr hole was drilled in the cranium over the left hemisphere, 2.5 mm lateral to bregma. A 1 µl syringe was lowered into the left striatum
to a depth of 4.0 mm (Dijkhuizen et al., 1996). Ouabain (0.5 µl; 1 mM, n = 61; Sigma Aldrich,
Zwijndrecht, The Netherlands) or vehicle (0.5 µl, 40 mM Tris-HCl buffer, pH 7.4; n = 8) was injected at a rate of 0.125 µl/min using a microdrive. After
injection, the needle was left in situ for 2 min to avoid
leakage of injection fluid from the needle tract. Body temperature was
maintained at 37°C using a water-filled heating pad and an infrared
heating lamp to compensate for AEA-induced hypothermia. Animals needed for MRI study were then positioned in the magnet and anesthesia was
continued using a mixture of halothane (0.4-1%) in
N2O/O2.
Pharmacological treatments. Animals used for the MRI study
were treated intraperitoneally with anandamide (1 or 10 mg/kg; n = 5 and 6, respectively; Biomol, Heerhugowaard, The
Netherlands), anandamide plus SR141716A (10 and 3 mg/kg;
n = 4; Sanofi Recherche, Montpellier, France),
SR141716A alone (3 mg/kg; n = 5), the selective AEA
membrane transporter inhibitor VDM11 (10 mg/kg; n = 5),
synthesized as described previously (De Petrocellis et al., 2000), or
vehicle alone (n = 12) (all drugs in 1 ml/kg body
weight 18:1:1 v/v PBS/Tween 80/ethanol) 30 min before toxin
injection. There was no difference in body weight and growth rate
between any of the groups. Utrecht University's Animal
Experimentation Committee approved all protocols.
MRI experiments. MRI was performed on a 4.7 T Varian
horizontal bore spectrometer. Excitation and signal detection were
accomplished by means of a Helmholtz volume coil (9 cm) and an
inductively coupled surface coil (2 cm), respectively. A
single-scan diffusion-trace MRI sequence [four b
values = 100-1300 sec/mm2;
repetition time (TR) = 3 sec; echo time (TE) = 100 msec] was used to generate quantified images of the tissue water
trace apparent diffusion coefficient (ADC). Diffusion-trace and
T2-weighted imaging (TE = 18, 40, 62, and 84 msec; TR = 2 sec; number of transients = 2) were performed
in all animals (2.2 × 2.2 cm field of view, 64 × 64 datamatrix), starting at t = 15 min after injection on day 0; both types of imaging were repeated 1 week later. As expected, no changes in T2-weighted MRI were detected at
this early time point. Both the T2-weighted and
the diffusion-weighted datasets consisted of seven consecutive,
1.5-mm-thick slices with a 0 mm slice gap. To minimize interference at
the slice boundaries, slices were acquired in alternating order (1, 3, 5, 7, 2, 4, 6), thus maximizing the time between excitation of two
neighboring slices. For the diffusion-weighted imaging we used a double
spin-echo pulse sequence with four pairs of bipolar gradients with
specific predetermined signs in each of the three orthogonal
directions, as described recently (de Graaf et al., 2001). The
combination of gradient directions leads to cancellation of all
off-diagonal tensor elements, thus effectively measuring the trace of
the diffusion tensor. This provides unambiguous and rotationally
invariant ADC values in one experiment, circumventing the need for
three separate experiments. For each b value, two
scans were averaged. The total scan time for acquisition of seven
slices with four b values and two averages was 17 min.
Data analysis. ADC and T2 maps were
generated by monoexponential fitting using the Interactive Data
Language software package (Research Systems Inc., Boulder, CO).
Parametric images were analyzed in anatomic regions of interest using
the same software package. Pixels in the ipsilateral hemisphere were
considered pathological if their ADC value or T2
value differed more than twice the SD of the mean value in the
contralateral hemisphere. The ventricles were segmented out in the
average ADC and T2 measurements. The lesion
volume per slice was calculated by multiplying the lesion area
(equaling the number of pathological pixels × field-of-view in
cm2/number of points acquired per image)
by the slice thickness. The total lesion volume was obtained by
summation of the lesion volumes for all slices. The absence of a slice
gap makes interpolation of lesion areas between slices unnecessary,
reducing systematic errors to within-slice "averaging" of signal
intensity. Statistical analysis was performed using SPSS 9.0 (SPSS
Inc., Chicago, IL). Differences between groups were analyzed
using Student's t test; reported p values
correspond to two-tailed significance.
Histology. After the last MRI measurements, animals needed
for histology were transcardially perfused with 4% paraformaldehyde in
0.1 M PBS. Dissected brains were post-fixed
overnight by immersion in the same fixative, cryoprotected in 10%
sucrose in PBS for 24 hr followed by 25% sucrose in PBS for 72 hr, and
quickly frozen in liquid nitrogen-cooled isopentane. Coronal sections
(10 µm) were cut and stained for glial fibrillary acidic protein
(GFAP), for Nissl substance, or with hematoxylin-eosin using standard procedures. The position of the histological slices was matched to the
position of the MRI images by known position relative to bregma, after
which a gross correlation was done.
Protein analysis. Mouse melanoma cells (B16-G4F) were
transiently transfected with 6-10 µg of recombinant cDNA (pcDNA3
vector) encoding the CB2 receptor (a kind gift
from Dr. R. Delwel, Institute of Hematology, Erasmus University,
Rotterdam, The Netherlands). Rat tissue (brain and spleen) and cells
were homogenized and sonicated for 10 sec three times in buffer
containing (in mM): 50 Tris, 1 EDTA, and 3 MgCl2, pH 7.4. Membrane fractions were prepared by centrifugation at 5000 × g for 5 min. Samples were
stored at  20°C until further use.
Samples containing 30 µg of protein were separated by 10% SDS-PAGE
(reducing) and transferred to a nitrocellulose membrane. Membranes were
blocked overnight in 1% gelatin in PBS-0.1% Tween (CB1, FAAH) or in PBS-0.1% Tween containing 5%
nonfat milk powder (CB2). Membranes were rinsed
twice and washed once for 15 min and four times for 5 min each with 20 ml of PBS-0.1% T. The membranes were incubated with
polyclonal primary antibodies for 3 hr (1:1000, CB1; Cayman Chemicals, Ann Arbor, MI), 2 hr
(1:1000, CB2; Cayman Chemicals) and 1.5 hr
(1:5000, FAAH; a kind gift from Dr. M. Maccarrone, University
of Rome "Tor Vergata," Rome, Italy) and washed. A donkey anti-rabbit antibody conjugated to horseradish peroxidase (1:5000; Bio-Rad, Richmond, CA) was used as a secondary antibody (1 hr incubation). Membranes were washed and developed with Western-blotting detection reagents (Amersham Pharmacia Biotech, Arlington Heights, IL)
according to the manufacturer's manual.
Lipid extraction. Neonatal rats [postnatal day 7 (P7) and P8] injected intracerebrally with ouabain or vehicle
were killed by decapitation at 0, 2, 8, and 24 hr after induction of
excitotoxicity (n = 6 for each time point). The
ipsilateral and contralateral hemisphere were rapidly removed and
separately homogenized in 5 ml of ice-cold Tris buffer (50 mM, pH 7.4). Lipids were extracted according to
the method of Bligh and Dyer (1959). One nanomole of
d8-AEA and 1 nmol of
d8-2-AG (Cayman Chemicals) were added as internal
standards. The organic phases were dried under nitrogen and purified by
normal-phase HPLC performed as described previously (Bisogno et
al., 1997). Mono-AGs and AEA standards were eluted after 18-23 and
27-28 min, respectively. The mono-AG fraction contained the 1-, (3)-,
and 2-stereoisomers.
To limit postmortem accumulation of endocannabinoids, the time between
decapitation and homogenization in cold organic solvents was kept as
short and constant as possible (<5 min) and the tissues were kept on ice.
GC/MS analysis. HPLC fractions were dried under a flow of
nitrogen and derivatized with 15 µl of
N-methyl-N-trimethylsilyl-trifluoroacetamide containing 1% trimethylchlorosilane for 2 hr at room temperature, thus
yielding the trimethylsilyl derivatives of AEA and 2-AG. The two
derivatized fractions were analyzed by GC/MS performed as described
previously (Bisogno et al., 1997, 1999). The derivatives of both
deuterated and nondeuterated AEA, 2-AG, and 1-(3)AG standards were
eluted after 18, 19, and 19.5 min, respectively. MS detection was run
in the selected ion-monitoring mode to improve sensitivity. Selected
ions for AEA were at a mass to charge ratio (m/z) of 427 and 419 corresponding to the molecular ions for
d8-AEA and nondeuterated AEA, and at a
m/z of 412 and 404, corresponding to the loss of a methyl
group in both compounds. Selected ions for 2-AG were at a
m/z of 530 and 522, corresponding to the molecular ions of
d8-2-AG and nondeuterated 2-AG, and at a
m/z of 515 and 507, corresponding to the loss of a methyl
group in both compounds. The endocannabinoids were identified on the
basis of the same retention time as the deuterated internal standards
of the corresponding MS signals with the appropriate relative
abundance. The amounts of AEA and 2-AG were calculated from the peak
area ratios between the signals at a m/z of 404 and 412 and
at a m/z of 507 and 515, respectively. A linear correlation
between these area ratios and the amounts of standards was observed in
separate studies. In the case of 2-AG, the amount of the 1-(3) isomer,
which is almost exclusively formed during tissue workup and lipid
purification (Stella et al., 1997), was added to the amount of the
2-isomer.
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RESULTS |
The presence of CB1 and
CB2 receptors and FAAH in the neonatal rat brain
was verified by Western blotting; endocannabinoid levels were
determined by isotope dilution GC/MS. Western blot analysis
demonstrated the presence of the CB1 receptor in
7- and 14-d-old rat brains, whereas the CB2
receptor could not be detected (Fig. 1).
FAAH was also detected in 7-d-old rats (Fig. 1). Neonatal rat brains
(P7) contained 32.5 ± 6.5 pmol/gm AEA and 1.17 ± 0.22 nmol/gm 2-AG, which is in the same order of magnitude as reported previously (Sugiura et al., 1995; Kondo et al., 1998; Berrendero et
al., 1999; Bisogno et al., 1999).
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Figure 1.
Western blot of CB1 and
CB2 cannabinoid receptors and FAAH in the neonatal rat (P7
and P14) and adult brain. CB2 receptor was absent in the
rat brain but was detected in the spleen and in the
CB2-transfected cell line.
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In our model, excitotoxicity was triggered by the unilateral
intrastriatal injection of 0.5 µl of ouabain (1 mM) in 7- to 8-d-old rats. Ouabain, a cardiac glycoside, inhibits
Na+/K+-ATPases
and induces cellular swelling, eventually leading to pancellular
necrosis and infarction (Lees et al., 1990; Lees, 1991; Lees and Leong,
1994, 1995; Stelmashook et al., 1999). The acute cellular swelling is
conveniently monitored by diffusion-weighted MRI. ADC maps of brain
tissue water, calculated from diffusion-weighted MRIs acquired 15 min
after ouabain injection, showed hypointensity regions with reduced ADC
values (~0.65 × 10 3
mm2/sec1)
in the ipsilateral hemisphere in all animals (Fig.
2). Normal ADC values (~1.11 × 103
mm2/sec1)
were measured in the contralateral hemisphere of the ouabain-injected rats (Fig. 2) and in the brains of the control animals, which received
only vehicle (0.5 µl of Tris-HCl, 40 mM, pH 7.4). The reduction in ADC values in the ipsilateral hemisphere after ouabain injection is thought to reflect neuronal swelling (i.e., cytotoxic edema) because of a relocation of part of the extracellular water into
depolarized cells (van Lookeren Campagne et al., 1994; Dijkhuizen et
al., 1996). The same brain regions, including the caudate putamen, cortex, and hippocampus, were affected in all animals (Fig. 2). In this
acute phase, AEA reduced the volume of brain tissue with cytotoxic
edema dose-dependently. The volume of tissue at risk to go into
infarction was reduced by 43 ± 15% (p < 0.05) at 10 mg/kg AEA (Figs. 2 and
3A). This effect was observed
at the borders of the affected tissue, namely the cortex and striatum.
Coinjection of the CB1 receptor antagonist
SR141716A with AEA did not reverse AEA action (Figs. 2 and
3A). Application of SR141716A or VDM11 (an endocannabinoid
uptake inhibitor) alone did not change lesion volume at day 0 compared
with vehicle-treated animals (Figs. 2 and 3A).
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Figure 2.
Coronal ADC maps of neonatal rat brains 15 min
after ouabain injection. Hypointensities correlate to cytotoxic edema.
Treatments: A, Vehicle; B, AEA (1 mg/kg);
C, AEA (10 mg/kg); D, AEA plus SR141716A
(10 and 3 mg/kg, respectively); E, SR141716A (3 mg/kg);
F, VDM11 (10 mg/kg).
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Figure 3.
Mean lesion volumes (±SE) of ouabain-injected
rats on day 0 (A) and day 7 (B) based on ADC and T2 maps
(hyperintensities), respectively. An asterisk denotes a
p value of <0.05 compared with vehicle-treated rats.
The numbers at the bottom indicate the dose of
compound in milligrams per kilogram. See Materials and Methods for
lesion volume calculation.
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After 7 d, the effect of AEA treatment on neuronal damage was
assessed using T2-weighted imaging and verified
by standard histology. Normal T2 values
(T2 = 73 ± 1 msec) were observed in contralateral hemispheres and in the brains of control animals (Fig.
4). The T2 maps of
ouabain-injected animals demonstrated both hyperintensities and
hypointensities (Fig. 4). Both types of T2
abnormalities indicate pathological changes. Hyperintense areas
correspond to vasogenic edema, tissue loss, and ventricle dilation,
whereas hypointensities can correlate to astrogliosis [i.e.,
phenotypic changes (hypertrophy)] and proliferation of astroglial
cells in response to neuronal injury (Fig. 4) (Feuerstein et al., 1994;
van Lookeren Campagne et al., 1994). Infarct size based on
T2 hyperintense abnormalities was
dose-dependently reduced in the AEA-treated group compared with the
control animals (Figs. 3B and 4). The infarct volume was
64 ± 24% (p < 0.05) at 10 mg/kg AEA,
smaller than in the vehicle-treated animals. Protection was primarily
observed in the caudate putamen, cortex, and hippocampus. This effect
was blocked by the CB1 antagonist
(p < 0.05) (Figs. 3B and 4). The
infarct was ~2.5-fold larger than that in vehicle-treated animals
(p < 0.05) and primarily involved the
hippocampus and caudate putamen. Application of SR141716A and VDM11
alone did not affect lesion size. Conventional histology (Nissl and
hematoxylin-eosin staining) showed a similar lesion pattern on brain
sections and confirmed the assessment made by T2
map analysis (data not shown).
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Figure 4.
Coronal T2 maps of neonatal rat brains
7 d after ouabain injection. Hyperintensities correlate to
vasogenic edema, tissue loss, and ventricle dilatation. Treatment:
A, Vehicle; B, AEA (1 mg/kg);
C, AEA (10 mg/kg); D, AEA plus SR141716A
(10 and 3 mg/kg, respectively); E, SR141716A (3 mg/kg);
F, VDM11 (10 mg/kg).
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The hypointense regions on the T2 maps
corresponded to regions exhibiting increased staining for GFAP on brain
sections of ouabain-treated rats, which is typical of astrogliosis
(Fig. 5A-D) (Van der Stelt et
al., 2001). No indications for hemorrhage were found. Astrogliotic
tissue constituted ~44% of the lesion on T2 maps of nontreated animals and usually surrounded the edematous tissue
and the dilated ventricles (Fig. 5B). The volume of
astrogliotic tissue in AEA-treated rats was not affected compared with
nontreated rats (p > 0.05), which is in
accordance with our previous observation that THC reduces astrogliosis
via a CB1- and
CB2-independent mode of action in our model (Van
der Stelt et al., 2001) (our unpublished results). This
reinforces the notion that classical cannabinoids also have other modes
of action in addition to their interaction with the
CB1 and CB2 receptors
(Burstein, 1999).
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Figure 5.
Transversal T2 map
(A) of an ouabain-injected animal showing that
vasogenic edema is surrounded by astrogliosis (hypointense area)
(B). Nissl-staining (C) and
GFAP-staining (D) of a brain section of an
ouabain-injected rat demonstrate a sharp line between affected and
healthy tissue.
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Finally, rat brain endocannabinoid levels were measured 2, 8, and 24 hr
after ouabain or vehicle injection. No rises in concentrations of AEA
and 2-AG were observed after inducing acute neuronal damage in the
ipsilateral hemisphere (Fig. 6). There
were no significant differences in endocannabinoid levels between
ipsilateral and contralateral hemispheres and between vehicle- and
ouabain-injected animals (data not shown).
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DISCUSSION |
The (patho)physiological role of the
endogenous cannabinoid system is beginning to be unraveled (Giuffrida
et al., 1999; Galve-Roperh et al., 2000; Di Marzo et al., 2001). It has
been postulated that the endocannabinoid system may serve to establish
a defense system for the brain during neurotoxicity and ischemia
(H. S. Hansen et al., 2000; Jin et al., 2000; Van der Stelt et
al., 2001). Yet the therapeutic effects of classical and synthetic
cannabinoids were contradictory in models in which
transient (20-120 min) or permanent cerebral ischemia was induced
(Nagayama et al., 1999; Louw et al., 2000; C. J. Hillard, personal communication). Because the cannabinoid
system has complex (cerebro)vascular effects (Randall and
Kendall, 1998; Kunos et al., 2000), this might explain the difference
in therapeutic outcome in the various models of stroke.
We investigated the presumed neuroprotective properties of the most
studied endocannabinoid, AEA, in an in vivo model of
secondary excitotoxicity, in which neuronal injury was induced by
unilateral intrastriatal injection of the
Na+/K+-ATPase
inhibitor ouabain without direct cerebrovascular intervention. Ouabain
rapidly perturbs ion homeostasis and induces cellular swelling and
glutamate-dependent damage of cells, which can be prevented in part by
blockade of the NMDA receptor (Lees et al., 1990; Cousin et
al., 1995; Greene and Greenamyre, 1996; Basarsky et al., 1999).
Diffusion-weighted MRI data acquired 15 min after the injection of
ouabain showed that exogenous AEA dose-dependently reduces in
vivo cellular swelling in the early phase after the induction of
excitotoxicity. We have shown previously that THC was able to reduce
in vivo cellular swelling in a
CB1-mediated manner in the same model (Van der
Stelt et al., 2001). The reduction in cellular swelling was not
attenuated by the CB1 antagonist SR141716A. Because there are no CB2 receptors detected in
the brain, the early in vivo neuroprotective action of AEA
does not seem to be mediated via the CB1 or
CB2 receptors.
The failure of SR141716A to block a reduction in cytotoxic edema by AEA
in the early phase does not seem to be a matter of dose and
pharmacokinetics. The antagonist (3 mg/kg) did block the late effects
(after 7 d) of AEA (10 mg/kg). In addition, a lower dose of
SR141716A (1 mg/kg) was more than sufficient to block the
neuroprotective actions of THC (1 mg/kg) at days 0 and 7 (Van der Stelt
et al., 2001). It is noteworthy that THC is a more potent agonist of
the CB1 receptor than AEA. In addition, SR141716A
is effective in blocking the behavioral effects of THC in the mouse
"tetrad" with AD50s of ~0.1 mg/kg, whereas some of the
actions of AEA were insensitive to SR141716A (Adams et al., 1998).
Moreover, some of the behavioral actions of AEA in mice were still
observed in the CB1 knock-out mice (Di Marzo et
al., 2000a). Others (Sinor et al., 2000) have shown that
neuroprotection of AEA in in vitro experiments was also
independent of CB1 and CB2 receptors.
Recent data demonstrate that AEA is capable of interacting with other
molecular targets, such as 5-hydroxytryptamine receptors, NMDA
receptors, vanilloid receptors, L-type calcium channels, Shaker-related
K+ channels, TASK-1 channels, a
non-CB1 G-protein-coupled AEA receptor in
astrocytes, and a non-CB1,
non-CB2 G-protein-coupled receptor for AEA and
WIN55.212-2 in the mouse brain (Howlett and Mukhopadhyay, 2000;
Breivogel et al., 2001), some of which may contribute to a reduction in
cellular swelling. For example, the inhibition of gap junctions and
intracellular calcium signaling in striatal astrocytes by the
non-CB1 G-protein-coupled AEA receptor (Venance et al., 1995) or the inhibition of L-type calcium channels (Johnson et
al., 1993) may prevent glutamate exocytosis and the spreading of
excitotoxicity. We cannot rule out the possibility that metabolites of
AEA may account for some of the observed effects. Additional studies
are necessary to understand the molecular mechanism of AEA-induced
reduction of cytotoxic edema.
T2-weighted MRI data recorded 1 week after
ouabain injection showed that exogenous AEA reduced neuronal damage by
64 ± 24% (p < 0.05). Compared with the
early phase, AEA-induced neuroprotection was blocked by the
CB1 receptor antagonist after 7 d. This can be explained by the different stages in the cascade of events induced
by excitotoxicity. Calcium entry is held responsible for delayed
neurodegenerative events, which can occur even if the initial cellular
swelling is reversed or prevented (Choi, 1987; Doble, 1999). We have
suggested previously that THC protected rat brains in the late phase
via the CB1-mediated closing of N- and
P/Q-type calcium channels and inhibition of glutamatergic transmission
in the same model (Huang et al., 2001; Van der Stelt et al., 2001). It
is reasonable to assume that this CB1-mediated process of closing voltage-sensitive calcium channels also contributes to the observed neuroprotection of AEA after 7 d. As noted before for THC, AEA-induced neuroprotection was observed in brain regions such
as the cortex, striatum, and hippocampus (Van der Stelt et al.,
2001).
Strikingly, the combination of AEA and the antagonist produced an
infarct that was 2.5 times larger than that seen for the control group.
This observation is not likely to be explained by (1) blockade of the
effect of endogenously released cannabinoids, (2) inverse agonism of
SR141716A, or (3) toxic effects of SR141716A. In fact, treatment of
rats with SR141716A at 1 and 3 mg/kg or with SR141716A plus THC (1 mg/kg) did not increase infarct size significantly (Van der Stelt et
al., 2001). In addition, endocannabinoid levels do not appear to
increase after ouabain injection.
Hampson et al. (1998) have shown that AEA can enhance calcium influx
presumably via direct activation of NMDA receptors. This enhancement
could only be observed when CB1 receptors were
blocked by SR141716A. This might explain the extra deleterious effect of the combination AEA and SR141716A. Interestingly, the site and size
of the infarcted region in these animals were similar to those observed
in animals that received a unilateral intrastriatal injection with NMDA
(our unpublished results).
Several lines of evidence gained in this study indicate that there is
no endogenous cannabinoid tone controlling the acute neuronal damage
induced by ouabain: (1) No increases in AEA and 2-AG levels were
detected at 2, 8, or 24 hr after ouabain injection. (2) Application of
SR141716A alone (3 mg/kg) did not increase the lesion volume at day 0 or at day 7. This implies that activation of CB1
receptors by constitutive levels of AEA, 2-AG, or any other novel
CB1 ligand, such as noladin ether (Hanus et al.,
2001), does not tonically protect the brain. (3) The AEA uptake
inhibitor VDM11 did not reduce neuronal swelling at day 0, nor did it
reduce the infarct volume after 7 d. This also argues against a
CB1 receptor-independent tonic protective role of
endogenous AEA and 2-AG. Thus, in our in vivo model of acute
neuronal damage, the data do not support the previously proposed role
of the endogenous cannabinoid system in neuroprotection.
However, we cannot exclude the possibility that technical issues have
prevented the detection of a tonic protection by endocannabinoids. The
lesion was considered pathological when ADC or T2
values differed more than twice the SD of the mean value in the
contralateral hemisphere. Thus, the periphery of the infarct with
smaller changes in ADC or T2 was not taken into
account, but may have benefited from a possible endogenous release of
cannabinoids. In addition, endocannabinoid levels were measured in
total hemispheres, and a possible local upregulation of AEA and 2-AG
could have been missed. However, this last possibility appears unlikely
if one considers the massive accumulation of endocannabinoids observed in other models of neuronal injury (Sugiura et al., 2000; Hansen et
al., 2001).
In addition, Hansen et al. (2001) have demonstrated that the increase
in N-acylphosphatidylethanolamines varies in different in vivo models of neuronal damage and is dependent on the
type of cell death. High levels of these lipids, which act as
biosynthetic precursors for AEA and its congeners, were found in
NMDA-injected neonatal rats. However, only moderate and low increases
were observed in a closed head-injury model and in an apoptotic model,
respectively. Although our model does not represent a model of
apoptotic cell death, ouabain-induced injury was not severe enough to
elicit endocannabinoid formation.
In this respect it is interesting to note that Wilson and Nicoll (2001)
have suggested that release of relevant levels of endocannabinoids
probably only occurs in response to particularly intense stimuli. Thus,
endogenous AEA may only be released after the intense stimulus, and,
hence, too late to exert a protective action, whereas exogenous AEA may
inhibit the ouabain-induced glutamatergic transmission, thereby
preventing spreading and reducing the effect of the toxic stimulus.
This explanation is consistent with recent studies in which endogenous
cannabinoids were shown to mediate retrograde signaling from
postsynaptic neurons to presynaptic terminals in hippocampal and
cerebellar synapses (Kreitzer and Regehr, 2001; Ohno-Shosaku et al.,
2001; Wilson and Nicoll, 2001).
In summary, we were able to accumulate data that strongly suggest that
there is no endogenous endocannabinoid tone controlling the acute
neuronal damage induced by ouabain. In contrast, our data, together
with previous reports, indicate that exogenous AEA can protect the
neonatal rat brain via a variety of mechanisms. Although our findings
do question the role of the endogenous cannabinoid system in
establishing a tonic brain defense system in our model, AEA may be used
as a structural template to develop new neuroprotective agents.
| |
FOOTNOTES |
Received June 28, 2001; revised Aug. 21, 2001; accepted Sept. 5, 2001.
W.B.V. is supported financially by the Netherlands Organization for
Scientific Research, Medical Sciences Council. V.D.M. is supported in
part by the Ministero per l'Universita' e Ricerca Scientifica e
Tecnologica 3933. We are indebted to H. Veldman and G. van Vliet for
expert technical assistance. We thank Sanofi Recherche for the gift of
SR141716A, Dr. R. van Sluis for the development of the data-processing
program, and Dr. R. A. H. Adan for the cell culture facilities.
M.v.d.S. and W.B.V. contributed equally to the work.
Correspondence should be addressed to G. A. Veldink, Department of
Bio-organic Chemistry, Bijvoet Center for Biomolecular Research,
Padualaan 8, Utrecht University, 3584 CH Utrecht, The Netherlands,
E-mail: veldink{at}accu.uu.nl; or V. Di Marzo, Endocannabinoid Research
Group, Istituto per la Chimica di Molecole di Interesse Biologico,
Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, Ex
Comprensorio Olivetti, Fabbricato 70, 80078 Pozzuoli, Napoli, Italy,
E-mail: vdimarzo{at}icmib.na.cnr.it.
| |
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TOP
ABSTRACT
INTRODUCTION
Compound via MeSH
