Neuroprotection and Recovery

Recent Data at the Bench on Citicoline

ngeles Moro, PhD
Olivia Hurtado, PhD; Ignacio Lizasoain, MD, PhD; Mara A
Background and PurposeIn this work, we review recent data on the actions of citicoline, citicoline is a drug with
demonstrated neuroprotective properties in both animals and humans.
Summary of ReviewFor neuroprotection, mechanisms involved are the improvement of cellular functions aimed to
control excitotoxicity and to maintain cellular adenosine 5!-triphosphate levels by preserving membrane function and
integrity at different levels. Importantly, these actions are theoretically achieved without interfering with possible
underlying mechanisms for neurorepair. Furthermore, citicoline stimulates neuronal plasticity and improves sensorimotor recovery in the chronic phase of experimental stroke.
ConclusionsAlthough the mechanisms of some of these actions remain to be elucidated, so far citicoline appears as a
drug with the ability to promote safe neuroprotection capable of enhancing endogenous protective pathways at the
same time as preparing the scenario for plasticity. (Stroke. 2011;42[suppl 1]:S33-S35.)
Key Words: functional recovery ! glutamate ! neuroprotection ! neuroregeneration

n acute stroke management, current evidence demonstrates

that the most important declines in stroke incidence and
mortality in developed countries have evolved from primary
and secondary prevention measures aimed to better control of
risk factors, including either pharmacological treatments or
other procedures that decrease blood pressure, prevent consequences of atrial fibrillation, and reduce hyperglycemia and
hyperlipidemia, among others.
Although less successful, there have also been interventions with proven benefit for acute stroke treatment. One of
the most significant advances has been the management of
patients in stroke units, which has been shown to reduce
mortality and to improve functional outcome by approximately 20%.1 In addition, recanalization of the occluded
blood vessel with thrombolytics, mainly recombinant tissue
plasminogen activator and, more recently, by mechanical clot
removal or disruption, appears to be 1 of the most effective
treatments for acute ischemic stroke.
However, there have been major disappointments in the
area of pharmacological neuroprotection, where many clinical trials have so far failed. Some of these failures might be
due to deficiencies in trial design rather than absence of
efficacy of the agents tested. In this sense, many current
efforts are now devoted to develop methods for better patient
selection and for analysis of outcomes, a way in which recent
trials have begun to provide some hope. Still, acute stroke
treatment guidelines until now do not encourage treatment
with any neuroprotectant.
Lastly, there is a field for which there are hopes that some
interventions could be useful for patients with stroke, which

is the phase of recovery. Indeed, in this setting, strategies

including rehabilitation programs or pharmacological/cell
therapies may serve to promote repair in late phases and to
decrease stroke-associated disability.
A way to embark on the search for new drugs for the
chronic phase of stroke is by boosting endogenous plasticity
mechanisms. Interestingly, many of these endogenous active
recovery processes initiate very early, in the acute phase of
ischemia. In this line, a very elegant perspective by Lo2 has
recently proposed that, because most molecular targets for
therapy have biphasic roles in stroke pathophysiology, failure
in neuroprotection may partly be due to the fact that many
neuroprotectants inhibit not only mechanisms of damage, but
also those mechanisms needed for repair. This interesting
hypothesis leads to propose that new drugs for acute stroke
treatment should be able to promote a safe neuroprotection
with the ability to preserve those mediators required for
neural repair.

Citicoline: A Drug for Safe Neuroprotection

Toward Repair
Cytidine-5-diphosphocholine (citicoline or CDP-choline) is a
compound normally present in all cells and an intermediate in
the biosynthesis of phosphatidylcholine. Citicoline has been
shown to produce neuroprotective effects in a variety of
central nervous system injury models, including cerebral
ischemia. At the experimental level, it has been reported to
decrease infarct volume and edema and/or to improve neurological deficits either alone or in combination with other
agents (for review, see Adibhatla and Hatcher3).

Received July 19, 2010; accepted September 30, 2010.

From the Unidad de Investigacion Neurovascular, Departamento de Farmacologa, Facultad de Medicina, Universidad Complutense, Madrid, Spain.
ngeles Moro, PhD, Unidad de Investigacion Neurovascular, Departamento de Farmacologa, Facultad de Medicina,
Correspondence to Mara A
Universidad Complutense, Avda Complutense s/n, 28040, Madrid, Spain. E-mail neurona@med.ucm.es
2010 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org

DOI: 10.1161/STROKEAHA.110.597435

S33
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S34

Stroke

January 2011

In humans, citicoline is the only neuroprotectant that has

shown positive results in all randomized, double-blind
trials and has demonstrated efficacy in a meta-analysis
with an overall safety similar to placebo.4 A large
(n"2600) trial (ICTUS: International Citicoline Trial
on Acute Stroke; Clinicaltrials.gov; October 31, 2006:
www.clinicaltrials.gov/ct/show/NCT00331890ICTUS) is
now enrolling patients with acute ischemic stroke within 24
hours of symptom onset in an effort to lend support to the
results of the meta-analysis.
The effects proposed to explain some of the neuroprotective actions of citicoline have been thoroughly reviewed3,5,6
and include prevention of fatty acid release, stimulation of
PtdCho synthesis, preservation of cardiolipin and sphingomyelin levels, increase of glutathione synthesis and glutathione
reductase activity, restoration of Na#/K#-ATPase activity,
and antiapoptotic effects. This wide range of actions suggests
that its precise mechanism of action is not fully known yet.

Citicoline in Neuroprotection
Data from our laboratory have demonstrated that antiexcitotoxic actions are involved in the neuroprotective actions of
citicoline.7 Indeed, we found that citicoline is able to reduce
infarct volume in parallel to a decrease in ischemia-induced
elevation in brain glutamate levels measured by microdialysis. High extracellular glutamate concentrations in the brain
may result from increased release and/or from decreased
uptake. Ischemia-induced glutamate release has been shown
to be largely due to reversed operation of neuronal glutamate
transporters, a fact that results from a severe depletion in
adenosine 5!-triphosphate levels caused by ischemia.8 Of
note, our results also showed that ischemia-induced adenosine 5!-triphosphate loss was decreased both in vivo and in
vitro by previous administration of citicoline. These results
support the hypothesis that citicoline-induced effect on neuronal adenosine 5!-triphosphate levels accounts, at least in
part, for the decrease in extracellular glutamate and the
subsequent neuroprotection after ischemia very likely by
keeping the normal function of neuronal transporters.
Interestingly, astrocytes play an important role in the
maintenance of extracellular glutamate concentrations by
regulating its uptake; glutamate transporters remove this
neurotransmitter/excitotoxin from extracellular space thereby
helping to terminate glutamatergic synaptic transmission and
to prevent the extracellular glutamate concentration from
rising to neurotoxic values.8 Using cultured rat astrocytes, we
found that citicoline enhances glutamate uptake in these cells.
Of the 5 high-affinity, sodium-dependent glutamate transporters currently known, 2 of them, EAAT1 and EAAT2, are
localized primarily in astrocytes. In our experiments, citicoline remarkably increased astrocytic membrane levels of the
EAAT2 transporter by inducing its translocation from the
cytosol to the membrane, where it is functional and helps to
decrease extracellular glutamate concentrations.
Then, the neuroprotective, antiexcitotoxic effect of citicoline results from a dual action on both neurons and astrocytes:
increased adenosine 5!-triphosphate levels in neurons, either
from an increased production or from decreased consumption, may delay the reversal of neuronal glutamate transport-

ers that leads to glutamate release and subsequent excitotoxicity; in addition, an increased capacity of astrocytes to
remove excess extracellular glutamate would contribute to
decrease ischemic damage.
In a subsequent study we demonstrated that citicoline
induces a specific relocalization of EAAT2 into rat brain lipid
raft microdomains,9 specialized membrane microdomains
enriched in cholesterol and glycosphingolipids (for review,
see Simons and Toomre10) that serve as platforms for a
variety of cellular functions such as vesicular trafficking and
signal transduction, transmembrane signaling, lipid and protein sorting, viral uptake, and regulated proteolysis. The
relocalization of EAAT2 into lipid rafts caused by citicoline
may represent a new and efficient mechanism of regulation of
glutamate transporter activity and subsequent management of
extracellular glutamate levels because (1) EAAT2 is more
strongly associated with lipid rafts than other glutamate
transporters expressed in the brain; and (2) this association is
important for the trafficking of EAAT2 to the lipid rafts in
which glutamate uptake is more efficient.11 Importantly,
increased EAAT2 association to lipid rafts by citicoline is
associated with an increased function, as shown by improved
glutamate uptake by lipid raft-associated plasma membrane
vesicles. Interestingly, this phenomenon also takes place in
vivo, where late administration of citicoline, 4 hours after the
onset of the ischemic injury, is able to increase the EAAT2
association to lipid raft fractions, concomitantly to a reduction in infarct size, strongly supporting this mechanism as a
mediator of neuroprotection in ischemic stroke.
Several studies have shown that EAAT2 deletion is related
to larger increases in extracellular glutamate, neuronal damage, and brain edema after experimental brain ischemia.1215
Interestingly, we reported that a polymorphism in the EAAT2
glutamate transporter promoter associates with higher and
sustained plasma glutamate concentrations and increased
frequency of neurological deterioration in patients with
stroke,16 underscoring the importance of EAAT2 as a pharmacological target in the ischemic scenario. Although only
experimentally a few drugs (estrogen, !-lactam antibiotics,
glucocorticoid, and peroxisome proliferator-activated
receptor-" agonists) are known to increase EAAT2 expression,1720 our present findings support the use of citicoline
with this purpose not only in stroke, but also in other
neurological disorders in which an altered function or expression of EAAT2 has been described such as epilepsy, Alzheimer disease, Huntington disease, and amyotrophic lateral
sclerosis, among others.8

Citicoline in Neurorepair
Stroke is a leading cause of death and long-lasting disability
with a high socioeconomic burden; among 30-day survivors
of first-ever stroke, approximately half survive for 5 years; of
survivors, one third remain disabled, and 1 in 7 are in
permanent institutional care.21 Taking into account that hemiparesis is the most common cause of disability after stroke, it
seems essential to develop effective therapies to improve the
motor recovery of these patients.
Due to its pleiotropic actions, we investigated whether
chronic treatment with citicoline starting 24 hours after

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Hurtado et al
ischemia onset might affect stroke recovery and neuronal
plasticity after focal ischemic injury in the rat.22 Our results
showed that citicoline improves both sensorimotor integration and asymmetrical motor behavior after experimental
stroke as determined by using the staircase skilled reaching
test and the elevated body swing test. Interestingly, this was
found in concordance with an increase in both dendritic
complexity and spine density of pyramidal neurons of Layer
V in the sensorimotor cortex contralateral to the insult in the
group of animals treated with citicoline. The neurons studied
include those that project to the forelimb region of striatum
among Layer V pyramidal cells, $40% project into dorsoventral striatumand others into the rat spinal cord. Therefore, citicoline is likely to contribute to forelimb movement
recovery acting on its different components. In addition, it
has been suggested that changes in dendritic complexity are
implicated in sensorimotor recovery induced by drugs or by
rehabilitative training23 after experimental stroke. Our results
are in agreement with previous evidence in animals and
humans in which large infarcts affecting multiple structures
involve reorganization and compensatory recruitment of the
undamaged contralesional hemisphere.23
In summary, citicoline increases neuronal plasticity and
contributes to sensorimotor function recovery after experimental stroke. In the present collection of reviews, other
actions of citicoline in different aspects of stroke pathophysiology are described that further support the potential of this
drug for recovery. Taking into account that citicoline has
been extensively studied in volunteers and patients demonstrating that is a well-tolerated and safe drug,4 as opposed to
other therapies assayed for recovery, all these results may
possess important implications by enhancing the therapeutic
options not only for neuroprotection, but also for rehabilitation and overcoming suffering of patients with stroke.

Sources of Funding
This work was supported by grants from the Spanish Ministry of
Education and Science SAF2009-08145 and the Spanish Ministry of
Health RENEVAS RD06/0026/0005 (M.A.M.) and SAF200803122, CIDEM-TNR (I.L.), and Ferrer Internacional (I.L.).

Disclosures
I.L. has received grants from Ferrer Internacional. The funding
sources had no role in study design or conduct; collection, management, analysis, or interpretation of the data; or preparation, review,
or final manuscript approval.

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Neuroprotection and Recovery: Recent Data at the Bench on Citicoline

Olivia Hurtado, Ignacio Lizasoain and Mara ngeles Moro
Stroke. 2011;42:S33-S35; originally published online December 16, 2010;
doi: 10.1161/STROKEAHA.110.597435

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