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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

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Neurochem Res (2014) 39:1206–1218

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


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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



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


Neurochem Res (2014) 39:1206–1218 1213

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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].


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


1214 Neurochem Res (2014) 39:1206–1218

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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

Neurochem Res (2014) 39:1206–1218 1215

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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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http://dx.doi.org/10.1111/j.1471-4159.2006.03882.xhttp://dx.doi.org/10.1111/j.1471-4159.2006.03882.xhttp://dx.doi.org/10.1016/S0378-4274(02)00427-7http://dx.doi.org/10.1038/sj.jcbfm.9600595http://dx.doi.org/10.1111/j.1471-4159.2010.06749.xhttp://dx.doi.org/10.1111/j.1471-4159.2011.07228.xhttp://dx.doi.org/10.1111/j.1471-4159.2011.07228.xhttp://dx.doi.org/10.1016/j.braindev.2008.06.010http://dx.doi.org/10.1089/089771503321532842http://dx.doi.org/10.1089/neu.2006.0224http://dx.doi.org/10.1002/jnr.10672http://dx.doi.org/10.1016/s0304-3940(97)00239-5http://dx.doi.org/10.1016/0197-0186(87)90049-0http://dx.doi.org/10.1016/0197-0186(87)90049-0http://dx.doi.org/10.1001/jama.2012.13256http://dx.doi.org/10.1016/S0140-6736(12)60813-7http://dx.doi.org/10.1023/A:1018999225435http://dx.doi.org/10.1111/j.2042-7158.1994.tb03252.xhttp://dx.doi.org/10.1016/j.brainres.2005.07.067http://dx.doi.org/10.1016/j.brainres.2005.07.067http://dx.doi.org/10.1038/nrneurol.2012.166-c1http://dx.doi.org/10.1038/nrneurol.2012.166-c1http://dx.doi.org/10.1016/j.brainres.2005.07.067http://dx.doi.org/10.1016/j.brainres.2005.07.067http://dx.doi.org/10.1111/j.2042-7158.1994.tb03252.xhttp://dx.doi.org/10.1023/A:1018999225435http://dx.doi.org/10.1016/S0140-6736(12)60813-7http://dx.doi.org/10.1001/jama.2012.13256http://dx.doi.org/10.1016/0197-0186(87)90049-0http://dx.doi.org/10.1016/0197-0186(87)90049-0http://dx.doi.org/10.1016/s0304-3940(97)00239-5http://dx.doi.org/10.1002/jnr.10672http://dx.doi.org/10.1089/neu.2006.0224http://dx.doi.org/10.1089/089771503321532842http://dx.doi.org/10.1016/j.braindev.2008.06.010http://dx.doi.org/10.1111/j.1471-4159.2011.07228.xhttp://dx.doi.org/10.1111/j.1471-4159.2011.07228.xhttp://dx.doi.org/10.1111/j.1471-4159.2010.06749.xhttp://dx.doi.org/10.1038/sj.jcbfm.9600595http://dx.doi.org/10.1016/S0378-4274(02)00427-7http://dx.doi.org/10.1111/j.1471-4159.2006.03882.xhttp://dx.doi.org/10.1111/j.1471-4159.2006.03882.x


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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 .

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