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O R I G I N A L P A P E R
Citicoline Protects Brain Against Closed Head Injuryin Rats Through Suppressing Oxidative Stress
and Calpain Over-Activation
Ke Qian • Yi Gu • Yumei Zhao • Zhenzong Li •
Ming Sun
Received: 13 November 2013/ Revised: 23 March 2014/ Accepted: 26 March 2014 / Published online: 2 April 2014
Springer Science+Business Media New York 2014
Abstract Citicoline, a natural compound that functions
as an intermediate in the biosynthesis of cell membranephospholipids, is essential for membrane integrity and
repair. It has been reported to protect brain against trauma.
This study was designed to investigate the protective
effects of citicoline on closed head injury (CHI) in rats.
Citicoline (250 mg/kg i.v. 30 min and 4 h after CHI)
lessened body weight loss, and improved neurological
functions significantly at 7 days after CHI. It markedly
lowered brain edema and blood–brain barrier permeability,
enhanced the activities of superoxide dismutase and the
levels of glutathione, reduced the levels of malondialde-
hyde and lactic acid. Moreover, citicoline suppressed the
activities of calpain, and enhanced the levels of calpastatin,
myelin basic protein and aII-spectrin in traumatic tissue
24 h after CHI. Also, it attenuated the axonal and myelin
sheath damage in corpus callosum and the neuronal cell
death in hippocampal CA1 and CA3 subfields 7 days after
CHI. These data demonstrate the protection of citicoline
against white matter and grey matter damage due to CHI
through suppressing oxidative stress and calpain over-
activation, providing additional support to the application
of citicoline for the treatment of traumatic brain injury.
Keywords Citicoline Closed head injury Oxidative
stress Calpain Corpus callosum Hippocampus
Introduction
Trauma to the brain causes tissue damage by primary and
secondary injury to the neural tissue. Primary injury due to
initial mechanical trauma results in physical disruption of
vessels, neurons and their axons. Secondary injury is an
indirect result of the insult triggered by the primary events,
which leads to further damage and disability. The critical
mechanisms of secondary injury after brain trauma include
inflammation, oxidative stress, ionic imbalance, increased
vascular permeability, mitochondrial dysfunction, and
excitotoxic damage [1–4]. The primary damage cannot be
reversed by medical or surgical means. However, the sec-
ondary damage may be influenced, as it occurs over time
after the primary damage.
For investigating the mechanisms of brain injury and
corresponding therapy, various models of traumatic brain
injury (TBI) have been established. Marmarou’s weight
drop model is one of the most frequently used constrained
rodent models of acceleration closed head injury (CHI).
This model has been shown to induce neurological deficits,
brain edema, increased permeability of blood–brain barrier
(BBB), biochemical changes, and widespread damage of
neurons including hippocampal neurons, axons, dendrites,
and microvasculature, but there was no supratentorial focal
brain lesion [5–7]. Taken together, this model successfully
replicates major biochemical and neurological changes of
diffuse clinical TBI.
K. Qian
Department of Neurosurgery, Beijing Tiantan Hospital, Capital
Medical University, 6 Tiantan Xili, Dongcheng District,Beijing 100050, People’s Republic of China
Y. Gu Y. Zhao M. Sun (&)
Department of Neuropharmacology, Beijing Neurosurgical
Institute, Capital Medical University, 6 Tiantan Xili, Dongcheng
District, Beijing 100050, People’s Republic of China
e-mail: [email protected]
Z. Li
Department of Experimental Zoology, Beijing Neurosurgical
Institute, Capital Medical University, 6 Tiantan Xili, Dongcheng
District, Beijing 100050, People’s Republic of China
1 3
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DOI 10.1007/s11064-014-1299-x
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Citicoline is a naturally occurring compound that func-
tions as an intermediate in the biosynthesis of cell mem-
brane phospholipids. It is hydrolyzed into cytidine and
choline in the intestinal tract and liver, which are essential
for membrane integrity and repair. Citicoline is reported to
protect brain against ischemia and trauma in the rodent
models [8–11]. Furthermore, citicoline shows promise of
clinical efficacy in patients with acute stroke, TBI, andother brain disorders [11, 12]. The therapeutic actions of
citicoline are thought to be due to restorative effects on
phospholipid synthesis in the damaged brain. As far as the
mechanisms of secondary brain injury are considered,
oxidative stress is believed to play a crucial role. Therefore,
for further providing the evidences that citicoline protect
brain against TBI, Marmarou’s weight drop model was
used to investigate the effects of citicoline on neurological
deficits, brain edema, BBB permeability, oxidative stress,
calpain activation, corpus callosum damages, and neuronal
death in hippocampal CA1 and CA3 subfield in this study.
Experimental Procedures
Closed Head Injury
The experimental designs and all procedures were in
accordance with both the National Guidelines for Care and
Use of Laboratory Animals and the Animal Care Guidelines
issued by the Animal Experimental Committee of Beijing
Neurosurgical Institute. Male adult Sprague–Dawley rats
(weighing 290–330 g, Beijing Vital River experimental
animals Technology Ltd., Beijing, China) were kept under
controlled light conditions with a 12-h/12-h light/dark cycle.
Food and water were provided ad libitum. With the rat under
chloral hydrate anesthesia (400 mg/kg, i.p.), experimental
CHI was induced using a weight drop device described
previously [5, 13], and modified in our laboratory. Briefly,
the skull of the rat was exposed by a longitudinal incision of
the skin. A metal disc 0.45 cm in diameter and 2 mm in
thickness wasfirmly fixed by quick adhesive to the right skull
vault of the rat. The center of the disc was located 1 mm from
the midline and 2.5 mm posterior to bregma. The rat was
placed on a foam bed in the prone position right under a
25-cm-tall Plexiglas tube. A 200-g weight inside the tube at
25 cm height was allowed to precisely strike the disc
cemented to the skull face. The foambed together with the rat
was then moved away from underneath the tube immediately
after the impact to insure a single hit. The rat was placed on
the operating table for close observation to determine if the
skull vault was fractured. The scalp was then sutured and the
rat was allowed to recover from anesthesia. Rats that died on
impact and those with skull fractures were excluded. In
sham-operated rat, the surgical procedure was prepared for
impact in the same way as above, but the animals were not
subjected to the head trauma. Rectal temperature was con-
tinuously monitored and maintained at 37 ± 0.5 C by a
negative-feedback-controlled heating pad during the whole
experiment. Thebody weights were measured before surgery
and at 7 days after surgery in all animals, and the change of
body weight was expressed as the body weight at 7 daysafter
surgery minus that before surgery (Dbody weight).
Experimental Protocols
Referring to the doses of citicoline used in experimental
TBI and stroke, 250 mg/kg of citicoline was used in this
study [8, 14]. Rats were randomly allocated to 3 groups
treated with citicoline or vehicle: (1) sham group; (2)
vehicle group: CHI ? vehicle, and (3) citicoline group:
CHI ? citicoline. The rats in sham group were given 2 ml/kg
of normal saline intravenously twice, 30 min and again 4 h
after operation, and the rats in vehicle and citicoline groups
were received 2 ml/kg of normal saline and 250 mg/kg of citicoline (Shandong Xinhua Pharmaceutical Co., Ltd.)
intravenously twice, 30 min and again 4 h after induction
of CHI, respectively. Neurological severity score (NSS)
was evaluated 24 h, 48 h, and 7 days after CHI (n = 14
per group). Water content (n = 17 per group), BBB
integrity (n = 14 per group), the levels of malondialdehyde
(MDA), glutathione (GSH), and lactic acid, and the
activities of superoxide dismutase (SOD) in injured tissue
(n = 14 per group) were assayed 24 h after CHI, and the
levels of myelin basic protein (MBP) and aII-spectrin, and
the activities of calpain were determined 24 h after CHI
(n = 7 per group). The histopathology was observed
7 days after CHI (n = 10 per group).
Neurobehavioral Evaluation
In all animals, a neurobehavioral test battery was per-
formed before CHI and at 24 h, 48 h, and 7 days after CHI
by an investigator who was blinded to the experimental
groups. Neurological function was measured in terms of
the NSS, an 18-point scale that assesses functional neuro-
logical status based on the presence of certain reflexes and
the ability to perform motor and behavioral tasks such as
beam walking, beam balance, and spontaneous locomotion.
Table 1 shows a set of modified NSS [15].
Measurement of Water Content in Traumatic Brain
Tissue
Water content in traumatic hemisphere was measured by
the wet–dry weight method as described previously [16].
Briefly, rats were killed 24 h after CHI under 10 % chloral
hydrate anesthesia. The right hemisphere was dissected and
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the surface of sample was gently blotted with tissue paper
to remove small quantities of adsorbent cerebrospinal fluid.
The sample was weighed as wet weight, and then dried in a
120 C incubator for 24 h. The dried tissue was weighed as
dry weight after cooling. Tissue water content (%) was
calculated as (wet weight–dry weight)/wet weight 9 100.
Evaluation of BBB Permeability
The integrity of the BBB was investigated by assessing
extravasation of Evans blue dye (EBD) as previously
described [17, 18]. Briefly, EBD (2 % in saline) was
injected intravenously (4 mg/kg) 24 h after CHI and
allowed to circulate 2 h. To remove the intravascular dye,
we perfused the animals with saline through the left ven-
tricle at 100 cm of water pressure until clear perfusion fluid
was obtained from the right atrium. After the animals were
decapitated, the brains were removed. The right hemi-
sphere was dissected, and incubated in 5 ml of formamide
in room temperature for 3 days. The resulting solution was
centrifuged at 14,0009g for 10 min. The supernatant was
collected and tissue levels of EBD were assessed using amultifunctional microplate reader (Tecan Trading AG,
Salzburg, Austria) at an excitation wavelength of 620 nm
and an emission wavelength of 680 nm. Sample values
were compared with those of EBD standards mixed with
the solvent (0.625–20 lg/ml). The hemisphere was dried in
a 120 C incubator for 24 h and then weighed as dry
weight after cooling. The levels of EBD in each hemi-
sphere were expressed as lg/g dry weight.
Measurement of the Levels of MDA, GSH and Lactic
Acid, and the Activities of SOD
The rat was deeply anesthetized with 10 % chloral hydrate
at 24 h after CHI, and the brain was removed. The right
hemisphere was collected, frozen with liquid nitrogen, and
kept under -70 C until analysis. The samples frozen at
-70 C were irrigated well with a solution of NaCl (0.9 %),
and homogenization at a ratio of 1:10 was achieved. The
homogenate was centrifuged (3,5009g, 20 min, 4 C), and
the supernatant was used to determine the levels of MDA,
GSH and lactic acid, and the activities of SOD by kits
(Nanjing Jiancheng Bioengineering Institute, Nanjing,
China). The protein concentration of the supernatant was
determined by the method of Bradford [19].
Calpain Spectrophotometric Assay
The tissue of right hemisphere was dissected according to the
experimental protocols at 4 C, and sample was prepared.
Briefly, the tissue was homogenized in 5 volumes of
homogenization buffer (20 mM N -2-hydroxyethylpiperazine-
N ’-20-ethanesulfonic acid (HEPES), 1.5 mM MgCl2, 10 mM
KCl, 1 mM EDTA, 1 mM ethyleneglycol bis(2-aminoethyl
ether)tetraacetic acid (EGTA), 250 mM sucrose, 1 mM
dithiothreitol (DTT), and 10 lg/ml of each of aprotinin, and
leupeptin, pH 7.5). Sample was centrifuged at 1,0009g at
4 C for 15 min to separate the sample into supernatant A and
pellet A. Pellet A was discarded, and supernatant A was
further centrifuged at 16,0009g for 20 min at 4 C, and the
supernatant B was used as the cytosolic fraction. The protein
concentrations in cytosolic fractions were determined by the
method of Bradford [19].
Calpain activity was estimated by a spectrophotometric
assay that uses azocasein as a substrate for endog-
enous calpain. The endogenous calpain activity in tissue
Table 1 Neurological severity score of rats after neurotrauma
Motor tests Score
Raising rat by the tail
Flexion of forelimb 1
Flexion of hindlimb 1
Head moved .10 to vertical axis within 30 s 1
Placing rat on the floor (normal = 0; maximum = 3)
Normal walk 0
Inability to walk straight 1
Circling toward the paretic side 2
Fall down to the paretic side 3
Sensory tests
Placing test (visual and tactile test) 1
Proprioceptive test (deep sensation, pushing the paw against
the table edge to stimulate limb muscles)
1
Beam balance tests (normal = 0; maximum = 6)
Balances with steady posture 0
Grasps side of beam 1
Hugs the beam and one limb falls down from the beam 2
Hugs the beam and two limbs fall down from the beam, or
spins on beam([60 s)
3
Attempts to balance on the beam but falls off ([40 s) 4
Attempts to balance on the beam but falls off ([20 s) 5
Falls off: No attempt to balance or hang on to the beam
(\20 s)
6
Reflexes absent and abnormal movements
Pinna reflex (head shake when touching the auditory
meatus)
1
Corneal reflex (eye blink when lightly touching the cornea
with cotton)
1
Startle reflex (motor response to a brief noise from snappinga clipboard paper) 1
Seizures, myoclonus, myodystony 1
Maximum points 18
One point is awarded for the inability to perform the tasks or for the
lack of a tested reflex; 13–18 indicates severe injury; 7–12, moderate
injury; 1–6, mild injury
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homogenates was obtained by measuring the amount of azo
chromophore released into solution after the addition of
azocasein and calcium [20, 21]. Briefly, a 20 ll aliquot of
the cytosolic fraction was added to 200 ll of assay buffer
(100 mM HEPES, 0.02 % b-mercaptoethanol, 10 mM
KCl, pH 7.5). Next, 30 ll of an azocasein (Sigma-Aldrich
Co., St Louis, MO, USA) stock solution (20 mg/ml) was
added, and the assay was initiated by adding 30 ll of 10 mM CaCl2. The samples were incubated at 37 C for
2 h and were placed on ice before adding 130 ll of 20 %
trichloroacetic acid. The samples were maintained at -
20 C for 5 min and then at 4 C for 15 min. The samples
were centrifuged for 10 min at 16, 0009g, and the 130 ll
supernatant was placed in a separate tube to which 130 ll
of 1 N NaOH was added to maximize absorbance of the
azo chromophore. The absorbance of the supernatant at
440 nm was determined and compared with that of
supernatants from homogenates that were not incubated
with calcium. The result was expressed as absorption
value/h/mg protein.
Western Blot Analysis
The tissues were collected and the cytosolic fractions were
prepared by the methods used in the section of calpain
assay. Calpastatin, MBP and aII-spectrin were determined
from cytosolic fraction separated by sodium dodecyl sul-
fate–polyacrylamide gel electrophoresis (SDS-PAGE).
Forty-microgram proteins were separated by SDS-PAGE,
and the proteins on the gel were transferred onto a nitro-
cellulose membrane. The membrane was then probed withantibody reactive with calpastatin (1:100; Santa Cruz
Biotechnology, CA, USA), MBP (1:500; Sigma-Aldrich,
St. Louis, MO, USA), or aII-spectrin (1:500; Chemicon
International Inc., Temecula, CA, USA) at 4 C overnight,
and subsequently incubated with alkaline phosphatase-
conjugated secondary antibody for 2 h at room tempera-
ture. The color reaction was observed by incubation of
membrane with Nitroblue tetrazolium/5-Bromo-4-chloro-
3-inoloyl-phosphate (NBT/BCIP) (Chemicon International
Inc, Temecula, CA, USA), and the integrated optical den-
sities(IODs) of the protein bands detected by Western blot
analysis were analyzed by gel image analyzer (Alpha-ImagerTM 2200, Aalpha Innotech Co., USA). b-actin
(1:2,000; Abcam Inc., Cambridge, MA, UK) was used as
an internal control, and the IODs of the protein bands were
normalized to b-actin immunoreactivity.
Histopathological Examination
Animals were anesthetized with chloral hydrate, and tran-
scardially perfused with heparinized normal saline followed
by 4 % paraformaldehyde 7 days after CHI. Brains were
removed, fixed, embedded in paraffin, and the 8-lm-thick
coronal sections through the hippocampus were collected.
Hematoxylin eosin (HE) staining was performed following
the procedures described in our previous paper [22]. The
sections were examined with light microscopy and pictures
were taken with a digital camera. Quantification of neurons
in the CA1 and CA3 hippocampus was performed in twoadjacent HE-stained coronal sections of the dorsal hippo-
campus for each animal. All attempts were made to use the
same region of the dorsal hippocampus as was used for
evaluation. Clearly defined pyramidal neurons (cell body
and nucleus) in CA1 and CA3 hippocampus were counted
in two high power fields, and hippocampal neuronal sur-
vival in CA1 and CA3 subfields was expressed as neurons
per high power field.
For the study of the white matter injury, the coronal
sections (8 lm thickness) through the hippocampus were
stained with Luxol fast blue–periodic acid Schiff (LFB–
PAS) and Bielschowsky’s silver impregnation [23, 24].The LFB–PAS and Bielschowsky’s silver stains were used
to measure optical densities (ODs) of myelin (LFB–PAS)
and axons (Bielschowsky’s stain) in the corpus callosum.
The measured OD values reflect the stainability of white
matter, and a decreased OD value indirectly reflects
destruction of white matter because of loss of stainability
[25]. For Bielschowsky’s silver stain, slices were rinsed in
distilled water after deparaffination, and then transferred to
a 20 % solution of silver nitrate for 30 min at 37 C. The
slices were washed with distilled water, differentiated in
10 % formaldehyde. After rinsing in distilled water, slices
were stained by ammoniacal silver solution for 30 s. After
washing in distilled water, slices were incubated with
0.1 % gold chloride for 3 min and immersed in 5 %
sodium thiosulphate in distilled water. Finally, slices were
rinsed, dehydrated, cleared, mounted, and stored in 4 C
for a few days until evaluation and photo acquiring. For
LFB–PAS stain, paraffin-embedded 8-lm thick slices were
rinsed in distilled water after deparaffination and then
transferred through 95 % ethanol to a 0.1 % solution of
luxol fast blue (LFB; Sigma-Aldrich, St. Louis, MO, USA)
in 95 % ethanol and 0.05 % acetic acid. After staining for
16 h at 60 C, slices were washed with distilled water,
differentiated in 0.05 % aqueous lithium carbonate fol-
lowed by 70 % ethanol. After rinsing in distilled water,
slices were oxidized in 0.5 % periodic acid and then
stained in 0.5 % Schiff’s solution for 15 min. Slices were
finally counterstained with hematoxylin. The slices stained
with Bielschowsky’s silver and LFB–PAS–Hematoxylin
were examined with light microscopy and pictures were
taken with a digital camera, and the average ODs of corpus
callosum were measured using an image analysis program
(Beijing Konghai Co., China).
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Statistical Analysis
Data were presented as mean ± SE. Comparisons between
groups were statistically evaluated by one-way ANOVA with
a post hoc LSD test (body weight loss, brain edema, BBB
permeability, the levels of MDA, GSH, lactic acid, calpasta-
tin, MBP and aII-spectrin, and the activities of SOD and
calpain, the neuronal numbers in CA1 and CA3 subfields, theaverage optical density of Bielschowsky’s silver stain and
LFB–PAS–Hematoxylin stain, and the calpain activity). NSS
was analyzed with a nonparametric Mann–Whitney U test. A
probability of \0.05 was considered statistically significant.
Results
Effects of Citicoline on Body Weight Loss After CHI
Before surgery, there was no significant difference among
body weights in all groups (Table 2). CHI rats treated withvehicle solution have higher amounts of weight loss com-
pared to those of sham-operated rats (P\ 0.01). Treatment
with citicoline markedly reduced the weight loss after CHI
(P\ 0.05 vs. vehicle-treated rats).
Effects of Citicoline on Neurobehavior After CHI
Before induction of CHI, all animals showed no significant
neurological deficits. The vehicle-treated rats showed signif-
icant neurological deficits at after 24 h, 48 h and 7 days of
CHI versus sham-operated rats (all P\0.001). Treatment
with citicoline markedly reducedthe NSS after 24 h, 48 h and
7 days of CHI (Fig. 1; all P\0.01 vs. vehicle-treated rats).
Effects of Citicoline on Water Contents and BBB
Integrity After CHI
CHI induced a significant increase in the percentage of water
content in the injured hemisphere (Fig. 2 a. P\ 0.01 vs.
sham-operated rats). Compared with vehicle-treated rats,
treatment with citicoline reduced the percentage of water
content significantly (P\ 0.01 vs. vehicle-treated rats).
Figure 2b depicted the concentration of EBD (lg/g dry
weight) extracted from injured hemisphere 24 h after CHI
in sham-, vehicle-, and citicoline-treated rats. The con-
centration of EBD increased significantly after CHI
(P\ 0.01 vs. sham-operated rats). Treatment with citico-
line markedly reduced the concentration of EBD in the
injured hemisphere compared with vehicle-treated rats
(P\ 0.01).
Table 2 Effects of citicoline on the body weight loss after 7 days of
CHI (mean ± SE)
Groups Body weight before surgery (g) Dbody weight (g)
Sham 317.5 ± 5.3 8.9 ± 5.9
Vehicle 315.3 ± 2.9 -18.3 ± 7.4#
Citicoline 326.5 ± 3.4 3.1 ± 5.8*
n = 14 per group. CHI closed head injury. Vehicle or citicoline was
administered by intravenous injection over 1 min, twice 30 min and
again 4 h after induction of closed head injury. The changes of body
weight (Dbody weight) were expressed as the body weight at 7 days
after surgery minus that before surgery
* P\ 0.05 versus vehicle-treated rats#
P\ 0.01 versus sham-operated rats
Fig. 1 Effects of citicoline on the neurological severity score (NSS)
after closed head injury. Vehicle or citicoline was administered by
intravenous injection over 1 min, twice 30 min and again 4 h after
induction of closed head injury. Data were presented as mean ± SE.
n = 14. #
P\0.001 versus sham-operated rats. *P\ 0.01 versus
vehicle-treated rats
Fig. 2 Effects of citicoline on water content and Evan’s blue dye
(EBD) in traumatic tissue 24 h after closed head injury. Vehicle or
citicoline was administered by intravenous injection over 1 min,
twice 30 min and again 4 h after induction of closed head injury.
a water content (n = 17). b EBD content (n = 14). Data are
mean ± SE. #
P\0.01 versus sham-operated rats. *P\0.01 versus
vehicle-treated rats
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Effects of Citicoline on the Levels of MDA, GSH,
and Lactic Acid, and the Activities of SOD After CHI
Figure 3 showed the levels of MDA, GSH, and lactic acid,
and the activities of SOD in all groups. CHI produced a
significant reduction in the activity of SOD and the level of
GSH (Fig. 3a, b; P\ 0.05, 0.01, respectively), and an
elevation in the levels of MDA and lactic acid in injured
hemisphere (Fig. 3c, d; both P\ 0.01). Compared with
vehicle-treated rats, treatment with citicoline markedly
enhanced the activity of SOD and the level of GSH
(P\ 0.05, 0.01 vs. vehicle-treated rats, respectively), andreduced the levels of MDA and lactic acid (both P\ 0.01
vs. vehicle-treated rats).
Effects of Citicoline on the Degradation of aII-Spectrin
Endogenous aII-spectrin (240 kDa), a well-characterized
calpain substrate, can be cleaved into 150 and 145-kDa
fragment [26]. As illustrated in Fig. 4, the levels of aII-
spectrin in traumatic brain tissue following CHI were
decreased significantly (P\ 0.01 vs. sham-operated rats),
and the levels of 145 kDa fragment of aII-spectrin were
increased (P\ 0.01). Treatment with citicoline markedlyenhanced the levels of aII-spectrin (P\ 0.05 vs. vehicle-
treated rats), and reduced the levels of 145 kDa fragment of
aII-spectrin in traumatic brain tissue (P\ 0.05 vs. vehicle-
treated rats).
Effects of Citicoline on the Levels of MBP
MBP is a major constituent of the myelin sheath in nervous
system, which is a marker of demyelination due to
neurological diseases [27, 28]. We analyzed the effects of
citicoline on the levels of MBP in traumatic brain tissue
after CHI, and the results were shown in Fig. 5. The levels
of MBP in traumatic brain tissue following CHI was
lessened significantly (P\0.01 vs. sham-operated rats).
Citicoline treatment markedly enhanced the levels of MBP
compared with vehicle-treated rats (P\ 0.05).
Effects of Citicoline on the Levels of Calpastatin
Calpastatin is well-known as an endogenous calpain
inhibitor. The effects of citicoline on the protein levels of calpastatin in traumatic brain tissue were illustrated in
Fig. 6. The protein levels of calpastatin in traumatic brain
tissue 24 h after CHI in vehicle-treated rats decreased
significantly (P\ 0.01 vs. sham-operated rats). Treatment
with citicoline markedly enhanced the calpastatin protein
levels in traumatic brain tissue 24 h after CHI (P\ 0.05
vs. vehicle-treated rats).
Effects of Citicoline on Calpain Activities After CHI
Results were shown in Fig. 7. The calpain activities in
traumatic brain tissue in vehicle-treated rats increasedsignificantly (P\ 0.05 vs. sham-operated rats). Treatment
with citicoline markedly reduced the activities of calpain in
traumatic brain tissue (P\ 0.05 vs. vehicle-treated rats).
Effects of Citicoline on Corpus Callosum Damage
After CHI
LFB–PAS–hematoxylin and Bielschowsky’s silver stain
were used to investigate the morphology of corpus
Fig. 3 Effects of citicoline on
the activity of SOD, and the
levels of GSH, MDA and lactic
acid in traumatic tissue 24 h
after closed head injury. Vehicle
or citicoline was administered
by intravenous injection over
1 min, twice 30 min and again
4 h after induction of closed
head injury. a SOD activity.
b GSH level. c MDA level.
d lactic acid level. pro protein.
Data are mean ± SE. n = 14.#
P\ 0.05 and ##
P\0.01
versus sham-operated rats.
*P\ 0.05 and **P\0.01
versus vehicle-treated rats
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callosum after CHI. Representative photomicrographs
chosen from right corpus callosum were shown in Fig. 8.
The myelin sheaths and axons were moderately damaged.Myelin sheaths lost their LFB–PAS stainability and
appeared as empty spaces (vacuoles) separating myelin
sheaths in the lesion areas of white matter (Fig. 8b2).
Axons appeared as irregular, twisted profiles and showed
segmental fragmentation with Bielschowsky’s stain
(Fig. 8c2). Moreover, increased cellular reactions occurred
in the injured corpus callosum. Citicoline treatment
decreased the damage of myelin sheaths and axons after
Fig. 4 Effects of citicoline on the levels of 240 kDa aII-spectrin and
145 kDa aII-spectrin fragment in traumatic brain region 24 h after
closed head injury. Vehicle or citicoline was injected intravenously
30 min after closed head injury. S sham, V vehicle, c Citicoline.
a Western blot analysis using aII-spectrin antibody. b, c The bar
graphs reflected the densitometric data of 240 kDa aII-spectrin and
145 kDa aII-spectrin fragment from the experiment of aII-spectrin
Western blot respectively. Data are mean ± SE. n = 7. #
P\ 0.01
versus sham-operated rats. *P\0.05 versus vehicle-treated rats
Fig. 5 Effects of citicoline on the levels of MBP in traumatic brainregion 24 h after closed head injury. Vehicle or citicoline was
injected intravenously 30 min after closed head injury. S sham,
V vehicle, C citicoline. a Western blot analysis using MBP antibody.
b The bar graphs reflected the densitometric data of MBP from
Western blot. Data are mean ± SE. n = 7. #
P\ 0.01 versus sham-
operated rats. *P\0.05 versus vehicle-treated rats
Fig. 6 Effects of citicoline on the levels of calpastatin in traumatic
brain region 24 h after closed head injury. Vehicle or citicoline was
injected intravenously 30 min after closed head injury. S sham,
V vehicle, C citicoline. a Western blot analysis using calpastatin
antibody. b The bar graphs reflected the densitometric data of
calpastatin from Western blot. Data are mean ± SE. n = 7.#
P\ 0.01 versus sham-operated rats. *P\ 0.05 versus vehicle-
treated rats
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CHI. Overall statistical analysis demonstrated that the
axonal injury and damage to the myelin sheath in corpus
callosum in vehicle-treated rats (P\0.001, 0.01 vs. sham-
operated rats, respectively), and citicoline markedly
reduced the axonal injury and damage to the myelin sheath
in corpus callosum (P\ 0.01, 0.05 vs. vehicle-treated rats,
respectively).
Effects of Citicoline on the Neuronal Cell Death
in Hippocampal CA1 and CA3 Subfields
HE staining was used to investigate the morphology of
dead cells in hippocampal CA1 and CA3 subfields after
CHI; the results were illustrated in Fig. 9. CA1 and CA3
neurons in sham-operated rats were normal, with a clearly
Fig. 7 Effects of citicoline on the calpain activity in traumatic brain
region 24 h after closed head injury. Vehicle or citicoline was
injected intravenously 30 min after closed head injury. Data are
mean ± SE. n = 7. #
P\ 0.05 versus sham-operated rats. *P\ 0.05
versus vehicle-treated rats
Fig. 8 Effects of citicoline on
corpus callosum damage after
7 days of closed head injury.
Vehicle or citicoline was
administered by intravenous
injection over 1 min, twice
30 min and again 4 h after
induction of closed head injury.
a1–a3, b1–b3, and c1–c3
Representative photographs of
corpus callosum stained by HE,
LFB–PAS–hematoxylin, and
Bielschowsky’s silver method
in sham-, vehicle- and
citicoline-treated rats,
respectively (original
magnification 1009). d, e Ba r
graphs show the average optical
density of corpus callosum
stained by LFB–PAS–
hematoxylin and
Bielschowsky’s silver method
in each group, respectively.
Data are mean ± SE. n = 10.#
P\ 0.01 and ##
P\0.001
versus sham-operated rats.
*P\ 0.05 and **P\0.01
versus vehicle-treated rats
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rounded appearance and intact well-defined membranes, a
clear nucleus, distinct nucleoli, and a clear cytoplasm
(Fig. 9a). In vehicle-treated rats, some neurons showed
shrunken and distorted shape, pyknosis and dark staining,
and the number of normal neurons in the hippocampal CA1
and CA3 subfields were significantly decreased versus that in
sham-operated rats (Fig. 9; both P\ 0.001 vs. sham-oper-ated rats). Treatment with citicoline markedly enhanced the
numbers of normal neurons in CA1 and CA3 subfields
(Fig. 9; both P\ 0.01 vs. vehicle-treated rats).
Discussion
It is well-known thatoxidative stress plays an important rolein
thepathogenesis of TBI [2, 3]. Reactiveoxygenspecies(ROS)
are highly reactive molecules, which are formed during nor-
mal cellular processes, but the production is tightly controlled
by scavenging system, including SOD, GSH-peroxidase and
catalase, as well as lowmolecular weight antioxidants such as
ascorbic acid, a-tocopherols, GSH, melatonin, etc. The brain
is particularly vulnerable to oxidative injury because of its
high rate of oxygen consumption, intense production of ROS,and high levels of transition metals and polyunsaturated fatty
acids. Neuronal membranes are rich in polyunsaturated fatty
acids, which are prime targets for ROS attack, and MDA is a
main breakdown product of lipid peroxidation in brain. After
TBI, the ROS levels increase markedly, and the anti-oxidative
defense mechanisms are depleted. Decreases in the activities
of SOD and GSH peroxidase, reduction in GSH, and increases
in thelevelof MDA aredemonstrated in rodent modelsof TBI
[2, 29–31].
Fig. 9 Effects of citicoline on
neuronal cell survival in CA1
and CA3 subfields in
hippocampus after 7 days of
closed head injury. Vehicle or
citicoline was administered by
intravenous injection over
1 min, twice 30 min and again
4 h after induction of closed
head injury. a1–a3
Representative photographs of
hippocampus stained by HE in
each group (original
magnification 409). b1–b3 and
c1–c3 Representative
photographs of CA1 and CA3
subfields in hippocampus in
each group, respectively
(original magnification 2009).
d Bar graphs reflected the
survival neurons in CA1 and
CA3 subfields in each group.
HPF high power field. Data
were presented as
mean ± SEM. n = 10.#
P\ 0.001 versus sham-
operated rats. *P\ 0.01 versus
vehicle-treated rats
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It is reported that TBI elicits the hydrolysis of phos-
pholipids enriched in neuronal membranes, in which,
phospholipase A2 (PLA2) plays an important role [32, 33].
PLA2 activation following TBI is harmful to neurons
through degrading phospholipids, enhancing calcium
influx, and increasing the release of free fatty acids. Ara-
chidonic acid (AA), one of free fatty acids, is metabolized
by cyclooxygenase/lipoxygenase, and oxidative metabo-lism of AA is considered to be a major source of ROS
during brain trauma [34, 35].
Citicoline is a key intermediary in the biosynthesis of
phosphatidylcholine, sphingomyelin, and other neuronal
membrane phospholipid components. When administered
exogenously, citicoline is hydrolyzed to form choline and
cytidine. These two components are incorporated into the
phospholipid fraction of the membrane and microsomes,
but also contributed to metabolic functions such as the
formation of nucleic acids, proteins, and acetylcholine [11].
As an intermediate of synthesis of membrane phospholip-
ids and inhibitor of PLA2, citicoline can restore membraneintegrity and normal functions by stimulating phospholipid
synthesis, suppressing phospholipid degradation, and
reducing the release of AA during brain ischemia [36–38].
Moreover, choline can be metabolized to GSH, increasing
the GSH levels and GSH reductase activity after transient
cerebral ischemia [39]. Iincreased GSH may contribute to
neuroprotection by removing hydrogen peroxide and
attenuating lipid peroxidation [40]. Therefore, it is rea-
sonable that citicoline could increase the synthesis of
phospholipids, reduce the activation of PLA2, and enhan-
ces the levels of GSH, subsequently, lessening the degra-
dation of membrane phospholipids and the release of AA,
suppressing the production of ROS and lipid peroxidation,
terminally, restoring membrane integrity and protecting the
brain against CHI, as shown in this study that citicoline
lessens the brain edema and BBB breakdown, enhances the
level of GSH and the activity of SOD, reduces the levels of
lactic acid and MDA, subsequently, improving neurologi-
cal functions, reducing the weight loss, and attenuating the
damage of corpus callosum and the neuronal cell death in
CA1 and CA3 subfields in hippocampus in the rat model of
CHI.
Contusions due to CHI are commonly associated with
hemodynamic changes including focal reductions in cere-
bral blood flow. This ‘ischemia-like’ pattern leads to
accumulation of lactic acid due to anaerobic glycolysis,
increased membrane permeability, and subsequent edema
formation [1]. In vitro the involvement of lactic acidosis in
the generation of ROS and lipid peroxidation has been
demonstrated [41, 42]. Therefore, it is reasonable that the
accumulation of lactic acid during CHI could contribute to
oxidative stress. The present study shows that the levels of
lactic acid in injured tissue are increased. Treatment with
citicoline reduced the levels of lactic acid in injured tissue
in a rat model of CHI, suggesting that citicoline could not
only improve energy metabolism, but suppress lactic acid-
induced ROS production due to mitochondrial dysfunction,
as citicoline can help stabilize cellular membranes and
restore mitochondrial function under brain ischemia [43,
44].
After determining the protective effects of citicolineagainst oxidative stress-mediated damage in the rat model
of CHI, we primarily investigate whether citicoline exerts
neuroprotection through suppressing the over-activation of
calpain, as calpain plays a key role in neuropathologic
events following TBI [4].
Calpains, one family of cysteine proteases, are activated
by calcium and autolytic processing, and regulated
reversibly by calcium and calpastatin, an endogenous cal-
pain inhibitor [45]. Intact aII-spectrin is a major structural
component of the membrane cytoskeleton located in axons,
presynaptic terminals and cell bodies [46, 47] The degra-
dation of aII-spectrin mediated by calpain leads to theformation of 150 and 145 kDa fragments [26], which are
reported to be increased in cortex, subcortical white matter,
and hippocampus during experimental diffuse axonal
injury [48, 49]. MBP is the major protein component in
myelin sheath which encases axons [27, 28], and it is
reported to be degraded by calpain in cortex and hippo-
campus after TBI [50].
Oxidative stress is reported to induce the activation of
calpain through enhancing intracellular Ca2? concentration
[51, 52]. Inhibition of lipid peroxidation improved main-
tenance of mouse cortical mitochondrial bioenergetics and
calcium buffering following severe TBI, and reduced the
calpain-mediated cytoskeletal damage [53, 54]. In addition,
Ascorbic acid, potent antioxidant, significantly suppressed
150/145 kDa subunits of a-spectrin breakdown products in
brain after hypoxic-ischemic injury in the immature rat
brain, indicating the inhibition of calpain activation by
ascorbic acid [55]. In multiple rodent models of TBI,
neuronal and axonal calpain are activated and involved in
the damage of neurons and axons, which have been dem-
onstrated through investigating the proteolysis of substrates
and the protection of calpain inhibitors [4, 50, 56, 57].
These data suggest that oxidative stress would disrupt
intracellular calcium homeostasis, subsequently, resulting
in the activating calpain and the degradation of structural
proteins, leading to the axonal damage and neuronal cell
death in the rat model of CHI. Citicoline is reported to
decrease the production hydroxyl radical after transient
forebrain ischemia of gerbil [58]. Our results showed that
citicoline enhanced the level of GSH and the activity of
SOD, reduced the levels of lactic acid and MDA, sup-
pressed the over-activation of calpain, and the degradation
of aII-spectrin and MBP after CHI. These data suggest that
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citicoline would suppress oxidative stress-induced the
over-activation of calpain and the degradation of aII-
spectrin and MBP due to CHI, by which, reducing the
damage of corpus callosum and the neuronal death in
hippocampus, and promoting the recovery of neurological
function and protects brain against CHI.
As a neuroprotectvie agent, citicoline must pass through
BBB and enter into brain under neuropathological condi-tions. It is reported that the radioactivity increases in brain
after radio-labeled citicoline is administrated [59, 60].
Moreover, citicoline has been used with varying degrees of
success in the experimental and clinical therapy of stroke
and TBI [8–12]. In addition, BBB is opened after TBI [61].
These researches provide convincing evidences that citic-
oline may cross BBB and reach the traumatic area to exert
its cytoprotection when it is administrated after TBI. It is
shown that the levels of choline and cytidine in plasma are
increased 2 h following the administration of single oral
dose of 2 g citicoline in healthy volunteers. And in healthy
individuals receiving a citicoline infusion of 3 g in 500 mlphysiological saline over 30 min, citicoline levels are vir-
tually detectable immediately after the end of the infusion
period, and plasma levels of choline and cytidine reach
peak at that time [62]. These data suggest that injection of
citicoline should exert its actions more quickly than oral
administration, although the bioavailability and metabo-
lism of citicoline are believed to be same between oral and
intravenous route [11]. Secades reviews the studies con-
ducted in the treatment of patients with head injuries, and
concludes that citicoline accelerates recovery from post-
traumatic coma and improves gait, achieving an improved
final functional outcome and shortening hospital stays in
these patients. Citicoline also improves the amnesic and
cognitive disorders seen after head trauma of minor
severity that constitute the so called post-concussional
syndrome [11]. However, in a phase 3, double-blind ran-
domized clinical trial of Citicoline Brain Injury Treatment
Trial (COBRIT) and an international, randomized. Multi-
centre, placebo-controlled study (ICTUS trial), citicoline is
reported to be no efficacious in treating TBI and ischemic
stroke, respectively [63, 64]. In COBRIT and ICTUS trials,
citicoline is administered within 24 h after onset of TBI or
ischemic stroke. In addition, liposome encapsulated citic-
oline can prolong the vesicle circulation time, increase
brain uptake of the drug, and protect brain against brain
ischemia at low doses [65–67]. Therefore, further clinical
trial of citicoline should consider the route of administra-
tion, liposomal formulation, and the therapeutic window of
citicoline against TBI or ischemic stroke in patients [68].
In conclusions, this study provides the evidences that
citicoline administered intravenously protects brain against
white matter and grey matter damage due to CHI, and
suppressing oxidative stress and calpain over-activation
may be one mechanism of citicoline against CHI. Our data
provide additional support to the application of citicoline
for the treatment of TBI.
Acknowledgments All authors have read the manuscript and
approved the final version of the manuscript. We thank Miss Jingjing
Yang for excellent technical assistance.
Conflict of interest All authors have read the manuscript and the journal’s policy on the disclosure of potential conflicts of interest, and
all authors have none to declare.
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C o p y r i g h t o f N e u r o c h e m i c a l R e s e a r c h i s t h e p r o p e r t y o f S p r i n g e r S c i e n c e & B u s i n e s s M e d i a
B . V . a n d i t s c o n t e n t m a y n o t b e c o p i e d o r e m a i l e d t o m u l t i p l e s i t e s o r p o s t e d t o a l i s t s e r v
w i t h o u t t h e c o p y r i g h t h o l d e r ' s e x p r e s s w r i t t e n p e r m i s s i o n . H o w e v e r , u s e r s m a y p r i n t ,
d o w n l o a d , o r e m a i l a r t i c l e s f o r i n d i v i d u a l u s e .