Cannabinoids
(CBs), the psychoactive components of Cannabis sativa L. (marijuana) and their
analogues, exert their effects by activating at least two specific receptors (CB1 and CB2) that
belong to the seven transmembrane G-protein-coupled receptor family. CBs are
known as neurosuppressive drugs. At the cellular level, CBs, through
interaction with Gi/o proteins, attenuate cAMP production, reduce neuronal
activity by modulating potassium channels and inhibit voltage-gated calcium
channels (Howlett, 1995; Pertwee, 1997; Howlett et al., 2010). Gi/o proteins also mediate the effect of
CBs on the mitogen-activated protein kinases extracellular signal-regulated
kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 (Bouaboula et al., 1999; Rueda et al., 2000; Derkinderen et al., 2001; Rubovitch et al., 2004). In vivo, CBs inhibit nociception,
suppress motor activity, impair cognitive processes and short-term memory and
reduce body temperature (Ameri, 1999; Chaperon and Thiebot, 1999). Nevertheless, there are
anecdotal reports on opposite, stimulatory effects of CBs: CBs were shown to
induce aggressive behaviour, hyperalgesia, increased motor activity and
elevated body temperature (Davis et al., 1972; Taylor and Fennessy, 1977; Sulcova et al., 1998). At the cellular level, CBs have also been
shown to couple to Gs proteins (Glass and Felder, 1997;Bash et al., 2003), increase cAMP production (Glass and Felder, 1997; Maneuf and Brotchie, 1997; Bash et al., 2003) and elevate intracellular Ca++ levels
(Sugiura et al., 1997; Rubovitch et al., 2002; Bash et al., 2003). The dual (stimulatory and inhibitory)
effects of CBs depended, in some cases, on the concentration of the drugs:
while regular (high) concentrations induced the conventional inhibitory
effects, low concentrations of CBs induced stimulatory effects (Sulcova et al., 1998; Rubovitch et al., 2002).
Many
studies have demonstrated either neuroprotective or neurotoxic effects of CBs in vitro (Guzman et al., 2002; Sarne and Mechoulam, 2005; van der Stelt and Di Marzo, 2005; Galve-Roperh et al., 2008). In vivo, acute administration of CBs
was found to be protective in various models of acute brain injuries (see
below), while chronic exposure to CBs was found in some cases to result in
neurotoxic consequences both in heavy cannabis users (Block, 1996; Pope and Yurgelun-Todd, 1996; Ehrenreich et al., 1999; Solowij et al., 2002;Matochik et al., 2005; Arnone et al., 2008; McHale and Hunt, 2008) and in animals exposed to
repeated administrations of CB drugs (Fehret al., 1976; Stiglick et al., 1984; Landfield et al., 1988; Scallet, 1991; Lawston et al., 2000). These apparently contradictory effects of
CBs are not yet understood, although several possible explanations for these
opposite findings were suggested (e.g., see Guzman, 2003;Sarne and Mechoulam, 2005; Di Marzo, 2008; Fowler et al., 2010). The present review concentrates on one of
the possible reasons for the dual neuroprotective/neurotoxic effects of CBs in vivo, namely, the opposite effects
of low versus high doses of CBs.
Neuroprotective
effects of CBs in acute brain injuries
Neuroprotective
properties of CBs were demonstrated in several models of acute brain injuries.
Acute in
vivo administration
of the synthetic CB agonist WIN-55,212-2 (1–10 mg·kg−1,
i.p.), but not of its inactive enantiomer WIN-55,212-3, was found to protect
against global and focal ischaemic damage in the hippocampus and cortex (Nagayama et al., 1999). Application of the phytocannabinoid Δ9-tetrahydrocannabinol
(THC) (1 mg·kg−1, i.p.) (van der Stelt et al., 2001a), or of the endocannabinoid
anandamide (1–10 mg·kg−1, i.p.) (van der Stelt et al., 2001b), was found to reduce the infarct
volume through a CB1-dependent mechanism in an in vivo model of ouabain-induced
excitotoxicity. The endocannabinoid 2-arachidonylglycerol (2-AG, 5 mg·kg−1, i.p.)
was found to reduce brain oedema and infarct volume following severe closed
head injury (Panikashvili et al., 2001; 2006) and the intravenous infusion of
the CB1/CB2 agonist
BAY 38-7271 protected against traumatic brain injury and focal ischaemia in
rats (Mauler et al., 2003). Since these early reports, numerous studies
examined the involvement of CB drugs and the endocannabinoid system in
neuroprotection (for recent reviews, see Di Marzo, 2008; Galve-Roperh et al., 2008; Fowler et al., 2010; Viscomi et al., 2010).
Various
mechanisms can account for the receptor-mediated neuroprotection that is induced
by CBs. The main factor that induces neuronal cell death is the elevation in
intracellular calcium ions during brain insult (ischaemia, epileptic seizure or
mechanical trauma). This elevation of intracellular calcium concentration
initiates a complex cascade of intracellular events such as the stimulation of
numerous enzymes (including proteases like calpains and caspases that
participate in apoptotic cell death) or other calcium-dependent protein–protein
interactions, which affect cell homeostasis and lead to neuronal death. Another
consequence of the rise in intracellular free calcium concentration is the
production of free radicals that attack DNA, mitochondria and the cell membrane
and are considered as major contributors for cell death. In addition, the
elevation in intracellular calcium concentration induces an increased release
of glutamate and the activation of postsynaptic NMDA receptors that stimulates
calcium entry into adjacent cells. Thus, calcium ions have a major role in
spreading the damage to additional brain regions (for review, see Doble, 1999). Hence, the inhibitory effect of CBs on
voltage-gated calcium channels (Caulfield and Brown, 1992; Mackie and Hille, 1992; Twitchell et al., 1997), which attenuates the elevation in
intracellular Ca++ and consequently also the
release of glutamate (Shen et al., 1996; Shen and Thayer, 1998), was suggested as a possible
mechanism for the neuroprotective effects of CBs against excitotoxicity. The
modulation by CBs of other calcium-dependent mechanisms, such as the inhibition
of NO synthesis (Hillard et al., 1999) and the inhibition of the release of the
pro-inflammatory cytokine tumour necrosis factor α (TNFα) (Facchinetti et al., 2003), were also suggested. Other CB actions that
may contribute to their neuroprotective effects include the induction of
hypothermia (Leker et al., 2003), vasodilatation (Wagner et al., 2001), anti-inflammatory effects (e.g. Mareszet al., 2007; Zhang et al., 2007; Fernandez-Ruiz et al., 2008) and neurogenesis (Galve-Roperh et al., 2007). It was also suggested that CB1 receptors
activate intracellular mechanisms such as the phosphatidylinositol 3-kinase
(PI3K/Akt) (Gomez Del Pulgar et al., 2002;Molina-Holgado et al., 2005; Ozaita et al., 2007) and ERK (Valjent et al., 2001; Derkinderen et al., 2003; Tonini et al., 2006; Moranta et al., 2007) pathways that are considered as survival
signals and may contribute to the protective effects of CBs. Other
neuroprotective mechanisms of CBs in either healthy or pathologic conditions
were also suggested (for recent reviews, see Pacher et al., 2006; Di Marzo, 2008; Fowler et al., 2010; Viscomi et al., 2010).
The
various studies that showed protective effects of CBs in acute brain injuries
presented two common experimental features: (a) the doses that were
administered ranged between 1 and 10 mg·kg−1 (the
doses that also induce the conventional acute effects of CBs), and (b) the
drugs were administered immediately before or after the insult. For example,
the CB agonist WIN-55,212-2 (1–10 mg·kg−1, i.p.)
was administered between 40 min before to 120 min after the induction
of global or focal ischaemia in rats (Nagayama et al., 1999); the CB agonist CP 55 940 (4 mg·kg−1, i.p.)
was administered 5 min after the induction of transient global ischaemia
in gerbils (Braida et al., 2000); THC (1 mg·kg−1, i.p.)
(van der Stelt et al., 2001a) or anandamide (1–10 mg·kg−1, i.p.)
(van der Stelt et al., 2001b) were applied 30 min before the
induction of ouabain-induced excitotoxicity in rats; THC (10 mg·kg−1, i.p.)
was administered immediately before or 3.5 h after the induction of
ischaemia in mice (Hayakawa et al., 2007); the endocannabinoid 2-AG (5 mg·kg−1, i.p.)
was found to reduce brain oedema and infarct volume when applied 15 min
after but not when applied 60 min after severe closed head injury in mice
(Panikashviliet al., 2001) and anandamide (10 mg·kg−1, i.p.)
protected the new born mouse brain against AMPA and kainate receptor–mediated
excitotoxicity when injected within the first 4 h following the insult but
not when injected 8–24 h after the insult (Shouman et al., 2006).
As
described above, the neuroprotective properties of CBs are attributed, among
other factors, to their ability to suppress voltage-gated calcium channels (Mackie and Hille, 1992) and consequently to attenuate
the release of glutamate (Shen et al., 1996). In vitro findings, however, have shown
that very low concentrations of CBs can potentiate, rather than suppress,
calcium entry into cells (Okada et al., 1992;Rubovitch et al., 2002). These findings suggest that very low doses
of CB drugs may elevate, rather than decrease, intracellular calcium levels in
the brain, and consequently the release of glutamate, and thus may be
neurotoxic in vivo. We therefore conducted studies in order to
explore the possibility that very low doses of THC may have in vivo neurotoxic effects.
The
neurotoxic properties of extremely low doses of THC
We
first conducted some acute experiments in order to determine the doses of THC
that will induce stimulatory effects in mice. Based on our in vitro experiments on the dual effect
of CBs on calcium (Rubovitch et al., 2002), we predicted a 3–4 orders of magnitude
difference between the in vivo inhibitory and stimulatory doses of THC. Our
experiments showed, as expected, that an i.p. injection of 10 mg·kg−1THC to
mice induced acute hypothermia, analgesia and decreased locomotion. On the
other hand, an i.p. injection of 0.001–0.002 mg·kg−1 THC
produced the opposite effects, that is elevation in body temperature,
potentiation of the response to noxious stimuli and increased locomotor
activity (Tselnicker, 2005). The doses of
0.001–0.002 mg·kg−1 THC were hence chosen for
studies on the long-term effects of a low concentration of THC on cognitive
functions in mice.
In
order to test our assumption that ultra-low doses of THC may induce neuronal
damage and impair cognitive functions, we first tested the long-term effect of
THC (0.001 mg·kg−1, i.p.) in two behavioural
assays that assess spatial learning: the Morris water maze (in ICR mice) and
the water T-maze (in C57B1 mice). THC significantly deteriorated the
performance of the mice in both assays 3–7 weeks following the injection (Tselnicker et al., 2007; Senn et al., 2008). The effect of the ultra-low dose of THC was
mild but reproducible and statistically significant and could be overcome by
additional training (see, e.g., figure 1b in Tselnicker et al., 2007 and figure 2 in Sennet al., 2008). The
ability of a single low dose of THC to induce cognitive deficits was blocked by
the CB1 receptor antagonist SR141716A,
indicating the involvement of CB1 cannabinoid
receptors in this effect (Senn et al., 2008). Since swimming can be stressful to mice,
and since mice were shown to perform spatial tasks poorly in a swimming pool
compared with dry land (Whishaw and Tomie, 1996), we next tested the effect
of the low dose of THC in the oasis maze, a land-based spatial learning assay
that was designed to approximate the spatial learning demands required by the
Morris water maze (Clark et al., 2005). Similar to our findings in the water maze
assays, the mice that were injected 3 weeks earlier with a single low dose of
THC exhibited a deficit in the acquisition of spatial learning in the oasis dry
maze (Amal et al., 2010). Moreover, in a modification of the test,
that could assess the ability of the mice to acquire the strategy of actively
looking for a well that contained water, the THC-injected mice performed poorly
compared to vehicle-injected control mice (see figure 4 inAmal et al., 2010). Thus, the administration of a single low
dose of THC to mice had long-term effects on their learning of strategy as
well. We then tested the long-term effect of the low dose of THC on the
performance of the mice in two recognition tests that are believed to be less
stressful and arousing to mice as they do not depend on reward or negative
reinforcement but rather on their natural tendency to explore novel objects (Dere et al., 2007). These tests examine spatial (‘place
recognition’) or non-spatial (‘object recognition’) visual memory without a
requirement for the learning of strategy. Long-term, statistically significant
deteriorating influence of a single low dose of THC was observed in both tests
and persisted for at least 5 months (Amal et al., 2010). Our findings thus demonstrated that the
administration of a single ultra-low dose of THC to mice resulted in a poor
performance in a variety of behavioural tests that examined various aspects of
cognitive functioning. These various long-term cognitive deficits may have
resulted either from several distinct impairments that were induced by THC and
affected different aspects of learning and memory, or from a more general
deficit that affected the performance of the mice in all the assays that were
employed. Our experimental observations supported the latter possibility since
we have noticed a common behavioural characteristic in the mice that were
injected few weeks earlier with THC, who seemed less curious and less prone to
investigate their surroundings, compared with their controls. In the open-field
test, no difference in motor activity was found between the THC- and the
vehicle-injected mice when the test was performed in familiar surroundings
(following habituation of the mice to the arena, as was required for measuring
true ‘motor activity’). However, during the first session of the test
(‘habituation’), when the mice were not familiar yet with their surroundings,
the control group appeared more active than the THC-injected group. Similar behaviour
of the THC-injected mice was noticed in the two recognition tests. In the first
session, when the mice were introduced to two unfamiliar objects in the arena,
control mice spent more time than THC-treated mice in investigating the two
objects. On the second day, however, their activity was reduced and was similar
to that of THC-treated mice (Amal et al., 2010). This lack of curiosity of THC-treated mice
that can also be described as lack of motivation or reduced awareness or
attention may, in our opinion, be the cause for the poor performance of the
mice in all the behavioural tests that were employed. It is interesting to note
that deficits of a similar nature were described following chronic use of
cannabis: repeated administration of high doses of THC to rats resulted in
reduced social interactions 2 weeks following the cessation of the treatment (Quinn et al., 2008); repeated administration of THC to rats
caused an attentional deficit that was detected 7 days later in a test of
visuospatial divided attention (Verrico et al., 2004) and monkey infants that were born to mothers
treated chronically with THC showed an alteration in visual attention (Golub et al., 1982). Furthermore, attentional dysfunction has
been reported repeatedly in human heavy cannabis users (Solowij et al., 1991; Fletcher et al., 1996; Pope and Yurgelun-Todd, 1996; Ehrenreichet al., 1999; McHale and Hunt, 2008).
The
finding that a single extremely low dose of THC causes cognitive deficits
similar to those caused by repeated treatments with high doses of the drug are
in agreement with our previously published hypothesis on the deteriorating
effects of chronic exposure to cannabis (Sarne and Keren, 2004). According to this hypothesis,
the detrimental outcomes of intermittent applications of CBs result from the
exposure of the organism to the very low concentration of the drug that is
present in the body for a prolonged time following each application, due to the
slow washout of the lipophilic drug.
The
surprising finding that a single administration of such an ultra-low dose of
THC causes cognitive deficits led us to test whether similar doses of THC would
induce biochemical effects in the brain. Indeed, we found that the injection of
0.001–0.002 mg·kg−1 of THC triggered a biochemical
pathway that led to a delayed activation of ERK1/2 in the cerebellum that
peaked at 24 h and then declined (Senn et al., 2008; Amal et al., 2010). This finding was in contrast to the
reported rapid activation of ERK following the injection of high doses of THC
(1–10 mg·kg−1, doses that induce the
conventional acute effects of the drug) that peaked at 10–30 min
post-injection and then declined (Derkinderen et al., 2003; Rubino et al., 2004). We have also searched for long-term (weeks)
neurochemical changes that develop in parallel to the long-term behavioural
effects that were induced by the ultra-low dose of THC. We have therefore
recently tested the amounts of total and phosphorylated (active) ERK in the
cerebellum 7 weeks following the injection of 0.002 mg·kg−1 THC to
mice. A consistent significant decrease in phosphorylated ERK was found in the
cerebella of THC-injected mice compared to vehicle-injected mice, while there
was no difference in the amount of total ERK between the two groups of mice.
Interestingly, in other brain regions (hippocampus and frontal cortex), a
sustained activation of ERK (namely, elevation in phosphorylated ERK with no
change in total ERK) was observed 7 weeks after the injection of THC. These
findings suggested that a single injection of an ultra-low dose of THC to mice
can induce both short-term (days) and long-term (weeks) modifications in the
brain. Further studies are needed in order to understand the relevance of the
long-term decline in ERK activity in the cerebellum to our findings in the
behavioural assays, since ERK has been shown to have an important role in
regulating many processes of cellular homeostasis, including both cell survival
and cell death (reviewed in Agell et al., 2002).
To
conclude, we have shown that a single administration of an extremely low dose
of THC to mice induced long-term cognitive deficits that were detected by
several tests that evaluated different aspects of memory and learning. The
deficits were usually mild and were not accompanied by any apparent
neurological (motor or sensory) damage. We have also shown that a single
injection of the ultra-low dose of THC evoked long-term neurochemical processes
that may affect brain plasticity. These findings led us to examine whether this
low dose of THC, which induces minor damage to the brain, may activate
preconditioning and/or postconditioning mechanisms and thus will protect the
brain from more severe insults.
Pre-
and postconditioning
The
discovery of the phenomena of preconditioning, where a minor noxious stimulus
protects various organs, including the brain, from a subsequent more severe
insult (Murry et al., 1986; Kitagawa et al., 1991) and of postconditioning, where the
protective intervention is applied following the insult (Zhao and Vinten-Johansen, 2006; Pignataro et al., 2008), prompted studies that aimed to find ways to
utilize therapeutically pre- and postconditioning mechanisms (Hausenloy and Yellon, 2009).
Preconditioning
research was initially conducted in cardiology, following the finding of Murry
and coworkers, who have shown that there was a considerable reduction in
myocardial infarct size resulting from prolonged ischaemia, in dogs that had
been submitted earlier to four cycles of brief coronary occlusion followed by
reperfusion (Murry et al., 1986). Since then, the molecular mechanisms of
both early and delayed cardiac preconditioning have been extensively studied (Das and Das, 2008), and large clinical trials have
confirmed the existence of myocardial preconditioning in humans (Dirnagl et al., 2003). The phenomenon of preconditioning was later
described in other organs, including the brain. The interest in ischaemic
preconditioning (‘tolerance’) in the brain started with the finding that a
brief bilateral carotid occlusion 2 days before global ischaemia protected
neurons from death in several brain areas of the gerbil (Kitagawa et al., 1991). Similar to the findings in the heart,
preconditioning can protect the brain almost immediately (‘early
preconditioning’) or after a delay of 1–7 days (‘delayed preconditioning’). The
molecular signalling cascades that are involved in both types of
preconditioning were extensively studied (reviewed in Gidday, 2006; Dirnagl et al., 2009). These cascades include, for example, the
activation of ERK, Akt, nitric oxide synthase and various neurotrophins (Gidday, 2006; Dirnagl et al., 2009). Preconditioning can be induced by different
harmful stimuli like ischaemia, hypoxia, trauma, hyperthermia or by chemical
substances and is not specific to the type of injury. The preconditioning
stimulus can be different from the insult (‘cross preconditioning’) or even
remote, since preconditioning of one organ can protect a different organ (Hausenloy and Yellon, 2008).
A novel
protective approach that was recently described was ischaemic postconditioning,
where the protective intervention is applied following the insult.
Postconditioning was found effective both in cardiac ischaemia (Zhao and Vinten-Johansen, 2006) and in the brain (Pignataro et al., 2008), and recently the existence of remote
postconditioning was also suggested (Hausenloy and Yellon, 2009). Similar to
preconditioning, postconditioning can be produced by various chemical agents
(reviewed in Gross and Gross, 2006). Moreover, it has been
suggested that pre- and postconditioning share common signalling pathways (Hausenloy and Yellon, 2009).
Pre-
and postconditioning treatments with an ultra-low dose of THC provide long-term
neuroprotection
We have
recently conducted experiments in order to examine whether a single ultra-low
dose of THC, which induces minor cognitive deficits in mice, may activate
preconditioning and/or postconditioning mechanisms and thus will protect the
brain from more severe insults (a detailed account of our findings was recently
published: Assaf et al., 2011). Two different brain insults that cause
cognitive damage and mimic different clinical situations were used as
experimental models: (i) the injection of pentylenetetrazole (PTZ) that induces
seizures that correlate to the petit mal type of epilepsy and (ii) repeated
short sessions of exposure to carbon monoxide (CO) that induce partial anoxia
and correlate to various hypoxic pathological conditions.
The
epileptogenic drug PTZ was previously shown to impair cognitive functions (Lamberty and Klitgaard, 2000; Wang et al., 2008). Epileptic seizures can cause neuronal
damage via the release of glutamate that triggers the excitotoxic cascade (Charriaut-Marlangueet al., 1996) and were shown to induce neuronal
death (Sankar et al., 1998; Huang et al., 2002; Troy et al., 2002) and memory impairment (Huang et al., 2002; Rutten et al., 2002). Moreover, various insults to the brain such
as stroke or trauma are known to induce seizures that augment the risk of
damage (Bladin et al., 2000; De Reuck et al., 2006; Christensen et al., 2009; Pitkanen et al., 2009). The CO intoxication model was shown to
induce excitotoxicity accompanied by synaptic and cellular loss in the
hippocampus and the development of learning deficits in mice (Ishimaru et al., 1991; Maurice et al., 1999; Meunier et al., 2006). CO exposure induces anoxia, which is the
main damage-inducing factor in brain insults such as stroke, cardiac arrest or
suffocation. Thus, these two experimental models (PTZ and CO) represent a wide
spectrum of pathological conditions.
In our
experiments, a single injection of PTZ (60 mg·kg−1) to
mice caused acute clonic–tonic seizures (stage 5 according to Clement et al., 2003) that lasted for 2–10 min. This
treatment induced cognitive deficits that were detected 3 weeks later by the
oasis maze. The potential of a single ultra-low dose of THC to protect the mice
and prevent the development of PTZ-induced cognitive deficits was then studied.
THC (0.002 mg·kg−1) was initially injected to
mice 3 days before the administration of PTZ (60 mg·kg−1), and
its effect on the performance of the mice three weeks later in the oasis maze
was examined. The time point of 3 days before the insult was chosen since at 3
days the protection induced by ischaemic preconditioning had been reported to
be maximal (Obrenovitch, 2008). We found that the mice that were
injected 3 weeks earlier with PTZ needed a significantly longer time than the
control mice to find the well that was filled with water in the oasis maze. The
mice that had been pretreated with THC 3 days before the administration of PTZ
needed a significantly shorter time than the PTZ-injected mice to find the
water-filled well, indicating that pretreatment with THC protected the mice
from the cognitive deficits that were induced when PTZ alone was injected.
Moreover, no significant difference between the performance of the mice that
were pretreated with THC before the injection of PTZ and the control group was
found, suggesting the absence of cognitive deficits in the PTZ-injected
THC-pretreated mice. Similar results were obtained when THC was injected either
1 day or 7 days before the administration of PTZ. Our findings thus suggested
that a preconditioning treatment of mice with an ultra-low dose of THC, 1–7
days before the administration of PTZ, can protect the mice from PTZ-induced
cognitive deficits.
We next
tested whether preconditioning with a single low dose of THC will similarly
protect the mice from cognitive deficits that were induced by another insult to
the brain, namely CO intoxication. Mice were exposed to CO (for 12 s)
thrice, with 45 min intervals between exposures (according to Ishimaru et al., 1991; Meunier et al., 2006). This treatment induced a cognitive deficit
that was detected 3–7 weeks following the exposure to CO by the oasis maze. The
injection of a single dose of THC (0.002 mg·kg−1) 1 or
3 days before the exposure to CO significantly protected the mice and prevented
the appearance of this cognitive deficit. These findings corroborate the
results that were obtained with PTZ as the insult and suggest that THC may have
a potential as a preconditioning treatment in a broader spectrum of brain
injuries.
While
the current review has been prepared, another group reported that a high
(conventional) dose of WIN 55,212-2 (1 mg·kg−1)
protected rats from focal cerebral ischaemia when injected 24 h before the
insult (Hu et al., 2010). This report further supports the idea that
CBs may produce conditioning effects, although it is not clear whether the
protective effect in that study resulted from the high concentration of the
injected drug or alternatively, from the low concentration that was present in
the body several hours after the injection, as had been suggested by us before
to explain the deteriorating effects of chronic exposure to cannabis (see above
and Sarne and Keren, 2004).
Our
next goal was to test whether the ultra-low dose of THC will also protect the
mice from PTZ-induced cognitive deficits when injected after the administration
of PTZ. In this set of experiments, THC (0.002 mg·kg−1) was
injected 1 or 3 days following the administration of PTZ (60 mg·kg−1).
Three weeks later, the mice were tested for cognitive deficits in the oasis
maze. The results indicated that a postconditioning treatment with this single
low dose of THC 1 or 3 days following the insult prevented the appearance of
PTZ-induced cognitive deficits, as detected by the oasis maze assay.
To
further establish the pre- and postconditioning potential of THC, the mice were
also tested in the place- and object recognition assays, which assess spatial
and non-spatial visual memory respectively. Similar to the results that were
obtained using the oasis maze, the treatment with a single dose of THC 1–7 days
before the injection of PTZ, or 1–3 days after the injection of PTZ, prevented
the development of PTZ-induced cognitive deficits that were detected 3–7 weeks
later. The performance of the pre- and postconditioned mice in the tests was
significantly better than the performance of the PTZ-injected mice and not
different from the control mice, suggesting the absence of cognitive deficits
that could be detected by the recognition assays.
The
molecular mechanism(s) of THC-induced pre- and postconditioning is not defined
yet. If, indeed, the protective effect of THC is secondary to its deteriorating
effect and the mobilization of an endogenous compensatory mechanism(s)
(‘conditioning’), it is expected to involve CB1 receptors,
since these receptors were previously shown by us to mediate the cognitive
deficits that were induced by THC (Senn et al., 2008). Nevertheless, the introduction of selective
CB1 and CB2 antagonists,
or the use of CB receptor knockout mice, will reveal the involvement of CB
receptors in the THC-induced pre- and postconditioning.
To
conclude, our results suggest that a pre- or a postconditioning treatment with
extremely low doses of THC, several days before or after brain injury, may
provide effective long-term neuroprotection and be used as a therapeutical
treatment in a wide spectrum of brain insults.
Therapeutical
potential
Brain
damage is a leading cause of long-term disability and mortality worldwide.
Brain damage can be induced by stroke (ischaemic or haemorrhagic), by traumatic
brain injury (TBI), by hypoxic or anoxic conditions (e.g. due to suffocation,
cardiac arrest, complications of general anaesthesia or carbon monoxide
poisoning), by epileptic seizures or by various toxins. The consequences of
brain injury depend on the amount of brain tissue that was damaged and the part
of the brain where the injury occurred and can range from transient or
long-term cognitive, emotional or motor deficits in the case of mild or
moderate injury, to coma or even brain death in the case of severe injury. The
initial insult induces multiple processes that lead to a rapid apoptotic and necrotic
cell death in the core area of the injury, including excitotoxicity (excessive
release of glutamate that generates the accumulation of toxic concentrations of
intracellular free calcium and of nitrogen and oxygen free radicals),
acidotoxicity and ionic imbalance (for a detailed review, see Doyle et al., 2008). The cells in the region surrounding the
core area of the injury (penumbra) degenerate more slowly, over a period of
hours or days following the initial insult, by mechanisms such as apoptosis and
inflammation. These cells may be salvaged by therapeutical intervention, but,
as the processes that lead to cellular death are already in progress, the time
window for treatment is limited. Currently, two major therapeutical approaches
for the rescue of penumbral cells are considered: (a) the use of
neuroprotective drugs that suppress biochemical pathways that mediate cellular
death (e.g. NMDA receptor antagonists, calcium channel blockers, antioxidants
or anti-inflammatory drugs) and (b) neurotrophic factors that induce
synaptogenesis, proliferation of dendritic spines and regeneration of neuronal
cells. However, despite two decades of research, clinical trials did not yield
any effective neuroprotectant drugs, and the only treatment available today is
thrombolysis, which restores the interrupted blood flow in the case of stroke,
using, for example, the recombinant tissue plasminogen activator that has a
therapeutical time window of 3 h (Zaleska et al., 2009).
The
discovery of the phenomena of pre- and postconditioning presented a different
therapeutical approach, namely, the possible activation of endogenous
mechanisms by which the brain protects itself and recovers from damage. One of
the main advantages of pre- and postconditioning stimuli is that the time
window for their application is long (days) (Zhao, 2009), in contrast to the therapeutical time
window for pharmacological intervention with neuroprotective drugs that is very
short (hours). Thus, conditioning procedures may be used to either protect
patients that are at risk of injury or to treat the insulted brain following
injury.
Our
findings demonstrate the potential of a single treatment with a very low dose
of THC to induce pharmacological pre- and postconditioning and protect the
brain from the development of cognitive deficits due to epileptic seizures and
CO intoxication and probably from other insults that involve excitotoxicity. As
described above, the therapeutical time window for protective intervention with
the conventional doses of CBs is short (±4 h in rodents), while the
preconditioning treatment with the ultra-low dose of THC can be employed 1–7
days before the insult and the postconditioning treatment can be applied for at
least 3 days following the insult. In both cases, the ameliorating consequences
of these treatments last for at least several weeks. Our findings with THC
preconditioning thus render this drug a potential candidate for inducing
neuroprotection in advance in patients that are at risk of cognitive damage
due, for example, to heart or brain surgery or to complications of anaesthesia
or percutaneous coronary intervention. Similarly, the ability of the low dose
of THC to induce postconditioning may prove to be effective in treating the
insulted brain following traumatic injury, stroke, suffocation or cardiac
arrest, insults that are known to induce long-term cognitive decline.
Traditional
preconditioning approaches use sublethal doses of otherwise damaging insults
such as brief episodes of ischaemia, hyperthermia or hypoxia, or low doses of
toxins (reviewed in Gidday, 2006), and therefore, their future
therapeutical benefit is questioned, due to safety, efficacy and ethical
considerations (Dirnagl et al., 2009). THC is already safely used in the clinic
for various pathological conditions (Pertwee, 2009). According to our findings in mice,
the dose of THC that induced pre- and post-insult protection is 3–4 orders of
magnitude lower then the dose that induced the acute conventional effects of
THC. A treatment with such extremely low doses of THC may therefore have a
potential to provide safe, long-term neuroprotection before or after brain
injury, without the undesired psychotropic effects of the conventional dose of
the drug. Moreover, since similar molecular mechanisms are involved in brain
injuries and neurodegenerative diseases (Zemke et al., 2004), it is possible that a chronic treatment
with very low doses of THC may prove beneficial in neurodegenerative diseases
as well.
What
can be learned from in
vitro studies
The
current review focused on the in vivo neuroprotective/neurotoxic profile of CB drugs.
Yet in
vitro studies
may shed more light on the cellular mechanisms that underlie this dual activity
of CBs. As was mentioned above, many studies have demonstrated either
beneficial or deleterious effects of CBs on the survival of various cells in
culture (reviewed in Guzman et al., 2002; Sarne and Mechoulam, 2005; van der Stelt and Di Marzo, 2005; Galve-Roperh et al., 2008). For example, CB agonists acting
through CB1 receptors protected hippocampal
neurons in culture from synaptically mediated excitotoxicity (Shen and Thayer, 1998). In cultures of mouse spinal
cord neurons, application of THC attenuated, via CB1 receptors,
kainate-induced toxicity (Abood et al., 2001). Similarly, the CB agonist CP-55 940
protected cultured cortical neurons from glutamatergic excitotoxicity by CB1 receptor–mediated
inhibition of voltage-dependent calcium channels (Hampson and Grimaldi, 2001). CBs have also shown in vitro neuroprotective potential in
models of neurodegenerative diseases. For example, CBs abrogated
microglia-mediated neurotoxicity after amyloid addition to rat cortical
cocultures through the activation of CB1 and CB2receptors
(Ramirez et al., 2005), and CB1 agonists
were shown to be neuroprotective in an in vitro model of Huntington's disease (Scotteret al., 2010). On the other hand, in vitro treatment of neuronal cell
lines or cultured hippocampal neurons and cortical neurons or hippocampal
slices with THC has been shown to induce neuronal death (Chan et al., 1998; Guzman et al., 2002; Downer et al., 2003; 2007b). It should be noted that some of the in vitro protective effects of CBs were
not mediated by CB receptors (e.g., see Hampson et al., 1998; Nagayama et al., 1999; Sinor et al., 2000; Marsicano et al., 2002), a fact that may correlate to their non-CB
receptor–mediated protective effects in some of the in vivo studies (Shohami and Mechoulam, 2000; Lastres-Becker et al., 2005).
While
the in
vivo protective
and toxic effects of CBs may be the result of complex processes that involve
various systems (e.g. effects on body temperature, inflammation, blood flow,
etc.), the dual effects of CBs on survival in vitro occur at the cellular level, by
the activation of intracellular pro-survival or pro-death signalling
mechanisms. CBs, via CB receptors, were shown to activate different signal
transduction mechanisms in different cell types. This differential activation
may lead to diverse effects on cell survival. For example, two sub-clones of C6
glioma cells exhibited different sensitivity to the toxic, anti-tumoral action
of THC: in C6.9 cells, THC induced cellular death that correlated to sustained
ceramide accumulation and Raf1/ERK activation, while in C6.4 cells, THC did not
induce cellular death, and no sustained accumulation of ceramide and activation
of Raf1/ERK was found (Galve-Roperh et al., 2000). Moreover, while THC increased the
synthesis of ceramide and induced apoptosis in transformed glioma cells, it did
not affect ceramide synthesis and failed to produce apoptosis in native
astrocytes and even protected them from oxidative stress (Carracedo et al., 2004). It is conceivable that CBs may activate
distinct cellular pathways in different cell types in vivo as well. Hence, different
neurons in different brain regions, which regulate different physiological
functions, may be affected differently by CB drugs, leading to different
functional consequences in vivo.
Recent
reports have shown that CBs can mediate distinct signalling mechanisms in a
single neuron, depending on the state of the neuron (Kellogg et al., 2009; Roloff and Thayer, 2009). The physiological state of
the neuron at the time of the application of the CB may therefore affect the
outcome of the CB treatment. Our recent study (Bologov et al., 2011) showed that various CB agonists (CP 55,940,
THC, HU-210 and WIN 55,212-2) significantly reduced the viability of N18TG2
neuroblastoma cells that were grown under stressful conditions (glucose- and
serum-free medium) but not when the same cells were grown under optimal
conditions (normal medium). These in vitro experiments suggest that the
consequences of the administration of CBs in vivo may be affected by the
stressful or pathologic conditions of the organism (see, e.g., Di Marzo, 2008). Furthermore, THC was shown to
activate pro-apoptotic mechanisms in cerebral cortical slices obtained from the
neonatal rat brain but not from adult brain (Downer et al., 2007a), indicating different sensitivity to the
pro-apoptotic effect of THC of immature cortical cells, compared with mature,
differentiated cortical cells. Similarly, we have recently shown that exposure
of differentiating N18TG2 cells, but not of dividing cells, to CB agonists
significantly increased their viability (Bologovet al., 2011). These studies imply that the
neuroprotective/neurotoxic effect of CBs in vivo may be affected by the age and
stage of development of the organism (Downer and Campbell, 2010).
In vitro studies
offer an experimental setup that enables a better analysis of the signalling
pathways that lead to either the survival or the death of the cells, yet the
relevance of the in vitro findings to the in vivo effects of CBs is not always
clear. Nevertheless, a thorough research of the in vitro effects of CBs on the survival
of isolated neurons may direct our future experiments with living organisms, in
order to further elucidate the dual, neuroprotective/neurotoxic profile of CB
drugs.
Special
Issue: Cannabinoids in Biology and Medicine, Part I. Guest Editors: Itai Bab
and Steve Alexander
Abbreviations
2AG
2-arachidonylglycerol
BAY
38-7271
[(–)-(R)-3-(2-hydroxymethylindanyl-4-oxy)phenyl-4,4,4-trifluoro-1-sulfonate]
CBs
cannabinoids, the psychoactive
ingredients of the cannabis plant, their synthetic analogues and the endogenous
ligands that act through CB1 and/or CB2 receptors.
This definition excludes, within the framework of the present review, the
non-psychoactive ingredients of cannabis such as cannabidiol
CO
carbon monoxide
CP
55 940
[(–)-cis-3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-trans-4-(3-hydroxypropyl)cyclohexanol)]
ERK
extracellular signal-regulated
kinase
HU-210
((-)-11-hydroxy-Δ8-tetrahydrocannabinol-dimethylheptyl)
i.p.
intraperitoneal
i.v.
intravenous
JNK
c-Jun N-terminal kinase
NO
nitric oxide
PTZ
pentylenetetrazole
Raf1
murine leukaemia viral oncogene
homolog 1
THC
Δ9-tetrahydrocannabinol
TNFα
tumour necrosis factor α
WIN
55,212-2
[(R)-(+)-(2,3-fihydro-5-methyl-3-[(morphonolinyl)methyl]pyrrolol[1,2,3-del]-1,4-benzoxazin-yl)(1-naphtaleneyl)methanone
mesylate]
WIN
55,212-3
[(3S)-2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone
monomethanesulfonate
