Protective effect of CDP-choline on hypotension and tissue injury in septic shock model

Abstract

CDP-choline is an endogen molecule and also a drug that is used in several trauma and ischemic conditions. It has been demonstrated that it improves the hemodynamic parameters in different shock models and prevents tissue damage in rats. The current study tested the effect of CDP-choline on hypotension, inflammation and tissue injury induced by septic shock model in rats. Twenty-four adult, male Spraque-Dawley rats, weighing 250-300 g were used. Septic shock was induced by cecal ligation-incision (CLI). CDP-choline (100 mg/kg) injected intravenously (i.v.) at the 180th minute of the experiment. The animals were observed for 180 minutes after the injection, then blood and tissue samples were obtained for cytokine measurements and histological examinations, respectively. The cecal ligation-incision decreased arterial pressure and increased heart rate. Intravenous injection of CDP-choline reversed hypotension and increased arterial pressure up to control levels within the first 60 minutes without changing the increase in heart rate. The effect lasted for 3 hours. CDP-choline attenuated the increases in TNF-α, IL-1β and IL-6 levels in septic shock. Moreover, the drug exerted protective effects for the injury induced by septic shock in lungs, liver and kidney tissues; whereas this effect was not present on spleen. In conclusion, the present data suggested that intravenous CDP-choline administration can improve the deteriorations in hemodynamic and inflammatory parameters and can prevent the tissue injury in septic shock-induced by CLI in rats. © 2017, Chartered Inst. of Building Services Engineers. All rights reserved.

Full text

Ankara Üniv Vet Fak Derg, 64, 103-110, 2017
Protective effect of CDP-choline on hypotension and tissue injury in
septic shock model
Çiğdem SEVİM1, Burçin ALTINBAŞ2, Murat YALÇIN2, Sevda İNAN3, Musa Özgür ÖZYİĞİT3,
İlker ARICAN4, Mustafa Sertaç YILMAZ1
1Uludag University, Faculty of Medicine, Department of Medical Pharmacology; 2Department of Physiology; 3Department of
Pathology; 4Faculty of Veterinary Medicine, Department of Anatomy, Bursa, Turkey.
Summary: CDP-choline is an endogen molecule and also a drug that is used in several trauma and ischemic conditions. It has
been demonstrated that it improves the hemodynamic parameters in different shock models and prevents tissue damage in rats. The
current study tested the effect of CDP-choline on hypotension, inflammation and tissue injury induced by septic shock model in rats.
Twenty-four adult, male Spraque-Dawley rats, weighing 250-300 g were used. Septic shock was induced by cecal ligation-incision
(CLI). CDP-choline (100 mg/kg) injected intravenously (i.v.) at the 180th minute of the experiment. The animals were observed for
180 minutes after the injection, then blood and tissue samples were obtained for cytokine measurements and histological examinations,
respectively. The cecal ligation-incision decreased arterial pressure and increased heart rate. Intravenous injection of CDP-choline
reversed hypotension and increased arterial pressure up to control levels within the first 60 minutes without changing the increase in
heart rate. The effect lasted for 3 hours. CDP-choline attenuated the increases in TNF-α, IL-1β and IL-6 levels in septic shock.
Moreover, the drug exerted protective effects for the injury induced by septic shock in lungs, liver and kidney tissues; whereas this
effect was not present on spleen. In conclusion, the present data suggested that intravenous CDP-choline administration can improve
the deteriorations in hemodynamic and inflammatory parameters and can prevent the tissue injury in septic shock-induced by CLI in
rats. Keywords: CDP-choline, cytokine, multiple organ failure, septic shock.
CDP-kolin’in septik şok modelinde hipotansiyon ve doku hasarı üzerine koruyucu etkileri
Özet: CDP-kolin, endojen bir molekül olmasının yanısıra travma ve iskemik koşullarda da kullanılan bir ilaçtır. Değişik şok
modellerinde, sıçanlarda hemodinamik parametreleri iyileştirdiği ve doku hasarına karşı koruyucu etki gösterdiği ortaya konmuştur.
Bu çalışmada, CDP-kolin’in sıçanlarda septik şokun neden olduğu hipotansiyon, inflamasyon ve doku hasarı üzerine olan etkileri
incelendi. Deneylerde, ag
̆ırlıkları 250-300 g arasında deg
̆is
̧en 24 adet Spraque-Dawley ırkı erkek sıc
̧an kullanıldı. Septik şok modeli
oluşturmak için çekal bağlama-kesme yöntemi uygulandı. CDP-kolin (100 mg/kg), deneyin 180. dakikasında intravenöz (i.v.) olarak
uygulandı. Hayvanlar enjeksiyon sonrası 180 dakika daha gözlendikten sonra sitokin ölçümleri ve histolojik incelemeler için sırasıyla
kan ve doku örnekleri toplandı. Çekal bağlama-kesme kan basıncını azaltırken kalp atım sayısını artırdı. İntravenöz CDP-kolin
uygulaması hipotansiyonu düzeltti ve arteryel basıncı ilk 60 dakika içerisinde, kalp hızındaki yüksekliği değiştirmeden, kontrol
düzeylerine kadar artırdı. Etki 3 saat kadar sürdü. CDP-kolin, septik şoktaki TNF-α, IL-1β ve IL-6 düzeylerindeki yükselmeleri de
azalttı. Bu etkilere ek olarak ilaç; akciğer, karaciğer ve böbreklerde (ancak dalakta değil) septik şokun sebep olduğu hasara karşı
koruyucu etki gösterdi. Sonuç olarak mevcut veriler intravenöz CDP-kolin uygulamasının; çekal bağlama-kesme ile oluşturulan septik
şokun neden olduğu hemodinamik ve inflamatuar parametrelerdeki bozukluklarda iyileşmeye ve meydana gelen doku hasarına karşı
koruyucu olduğunu ortaya koymaktadır.
Anahtar sözcükler: CDP-kolin, çoklu organ yetmezliği, septik şok, sitokin.
Introduction
Sepsis is a complicated syndrome ensuing from a
systemic inflammatory reaction to infection and is the
primary reason of death in critically ill patients (19).
Despite the importance of early antibiotic therapy, several
additional therapeutic strategies are still under
investigation in order to alleviate some of the serious
effects of septic shock (42). Cholinergic modalities are
one of the recently investigated approaches based on the
importance of vagal anti-inflammatory cholinergic
signaling in endotoxemia and sepsis (26, 40).
CDP-choline (cytidine-5-diphosphate choline;
citicoline) is an endogenous molecule and a drug which
has many physiological and pharmacological effects in
several conditions (1, 2, 36). Clinical studies mainly
focused on drug’s anti-ischemic and tissue protective

Çiğdem Sevim - Burçin Altınbaş - Murat Yalçın - Sevda İnan - Musa Özgür Özyiğit - İlker Arıcan - Mustafa Sertaç Yılmaz
104
effects, because CDP-choline protects the membrane from
ischemic insult through the preclusion of fatty acids
release (14), stimulation of phosphatidylcholine synthesis
(28), prevention of cardiolipin and sphingomyelin levels
(27). The drug also decreases oxidative stress by
increasing glutathione synthesis (3). CDP-choline can also
have the antiapoptotic activity by reducing the expression
of all procaspases involved in apoptosis, particularly by
inhibiting the caspase-3 activation (17). When
administered exogenously, i.e., orally or intravenously
(i.v.), CDP-choline quickly hydrolyzed to choline and
cytidine (18, 35). These final metabolites increase in the
circulation, cross blood–brain barrier, are taken up by the
cells/neurons and mediate re-synthesis of the molecule in
the cell (18). We have been investigating its cholinergic
nature and usefulness in several shock conditions for
almost a decade. Our studies have shown that CDP-
choline can exert significant hemodynamic and endocrine
effects in normal and stimulated situations as osmotic
stimulation and hemorrhage (9, 10, 31, 32, 33). It increases
blood pressure in normal circumstances (31), restores
hypotension and increases survival in hemorrhagic shock
(32). The activation of central cholinergic receptors
through the increase in brain choline levels mediates these
effects (32). We also reported that CDP-choline is able to
decrease neuronal injury in spinal cord transected rats by
limiting oxidative injury (11). Additionally, we
demonstrated that intravenously given CDP-choline
exposes intense coverage versus arrhythmias and increase
survival rates in short-term ischemia-reperfusion of
myocardium by activating efferent vagal pathways
followed by increased brainstem cholinergic transmission
throughout the initiation of central muscarinic receptors in
rats (45). More recently we reported that CDP-choline has
protective effects on myocardium from long-term
coronary occlusion–reperfusion induced injury in rats
(12). Furthermore, there were reports that demonstrated
that CDP-choline mediated some protective effect in
endotoxic shock. It was reported that CDP-choline
amends serum lipid responses to endotoxin and precludes
hepatic and renal damage during endotoxemia via a
mechanism which is mediated by nicotinic acetylcholine
receptor (21). CDP-choline alleviates the anomalies in the
hemostasis and avoids the development of dissemine
intravascular coagulopathy during experimental
endotoxemia in dogs (46). These reports also showed that
CDP-choline enhanced survival rate in lethal endotoxin
shock (21). Despite these reports, there is still not enough
information on the possible protective effects of CDP-
choline on septic shock induced negative hemodynamics
and tissue injury. Therefore we hypothesized that CDP-
choline might exert beneficial effect in septic shock by
improving hemodynamic parameters and protect tissue
injury due to its anti-inflammatory and antioxidant effect.
Materials and Methods
Animal preparation and experimental design: 24
adult (3-4 months old) male Sprague-Dawley rats
weighing 250-300 g (Uludag University Experimental
Animals Breeding and Research Center, Bursa, Turkey)
were used in the current study. Rats were housed three per
cage (Euro Type 3 rat cage, dimensions 425 x 265 mm x
185 mm, floor area: 800 cm2) under a 12-h light/dark cycle
in a temperature-controlled environment (20 oC to 22 oC)
with food pellets (standard diet of rat chow) and tap water
available ad libitum. Experiments were performed on
animals after an acclimation period of one week. Uludag
University Animal Care and Use Ethical Committee
approved both surgical and experimental protocols of the
current study (Approval number: 2010-08/07 and date:
26.11.2010). Animal experiments were performed in
accordance with the Guide for the Care and Use of
Laboratory Animals which is issued by the National
Institutes of Health.
Twenty-four rats were randomly assigned to 3
groups (8 rats/group): group 1 (sham), group 2 (CLI +
saline 1 ml/kg), group 3 (CLI + CDP-choline 100 mg/kg).
All animals’ blood pressure and heart rate recorded during
the experiment as described below. Animals in the second
and third group treated by saline or CDP-choline 3 hours
after undergoing CLI. The rats in each group were killed
at 6 hours after undergoing CLI, blood and tissue
specimens were collected for histological analysis and
cytokine measurements.
Concisely, in sevoflurane anesthetized rats (inducted
by 4% and maintained by 1.5% sevoflurane); left arteria
femoralis and vena jugularis were cannulated for
recording blood pressure and drug injections. Blood
pressure and heart rate were monitored and recorded using
Power Lab data acquisition system and Lab Chart software
(AD Instruments, New Zealand) at 1 min intervals for 6 h.
Mean arterial pressure (MAP) was reported as mmHg and
heart rate (HR) was expressed as beats per minute (bpm).
Cecal ligation-incision procedure: For CLI, midline
laparotomy was accomplished in animals. The cecum was
exteriorized in these animals by cotton sticks, which had
been located in saline (0.9% NaCl) solution. In sham
operated animals, the cecum was slotted back into the
abdomen following a kindly operation. In the CLI-groups,
for avoiding the bowel obstruction; the cecum and the
mesenteric artery and vein were ligated just below the
ileocecal valve. Consequently, the cecum was opened
through a 1.5 cm incision. The cecum was then placed
back into the abdomen. In all groups, saline (2 ml/kg) was
administered intraperitoneally for fluid resuscitation just
before the abdomen was closed. CDP-choline (100 mg/kg)
or saline (1 ml/kg) was intravenously injected 3 hours after
CLI induction. Sham group did not receive any injection.

Ankara Üniv Vet Fak Derg, 64, 2017
105
CDP-choline dose was chosen from dose-response studies
that published previously (45).
Cytokine measurements: For the measurement of
plasma cytokine (IL-1β, IL-6,TNF-) levels, blood
samples (200 µl) were obtained from rats into EDTA
containing cold tubes and centrifuged at +4 °C, 1800 rpm,
for 20 min. All the samples stored at −80 °C until the
measurement. The plasma levels of cytokines were
determined by enzyme immunoassay (ELISA) as per
company's guide (Signosis Inc., CA, USA) using a
microplate reader (BioTek Instruments, Inc., VT, USA)
where the absorbances were read at 450 nm.
Histopathological staining: Animals were perfused
with a fixative that contains paraformaldehyde (4%) in 0.1
mol/L phosphate buffer (pH 7.6) under profound
anaesthesia, at the end of the study. Following the
perfusion, tissue samples (liver, lung, kidney, spleen) were
removed. They fixed for 24–48 h at 37 °C in the same
solution. After consecutive dehydration in 70% ethanol,
80% ethanol, 96% ethanol, and absolute ethanol, the
specimens were fixed in paraffin, and sections were cut 5
µm thick. Finally, they stained with hematoxylin-eosin.
Each slide was examined for congestion, hyperemia,
hemorrhage, degeneration, necrosis, infiltration of
polymorphonuclear leukocytes, necrosis on x40 power
magnification.
Drugs: CDP-choline (Sigma Aldrich Co. Deisenhofen,
Germany) was dissolved in saline (100 mg CDP-choline/1
ml saline).
Statistical analysis: All data are expressed as mean ±
standard error of the mean (S.E.M.) and statistical
analyses were performed by repeated measure one- and
two-way analysis of variance (ANOVA) followed by
Bonferroni’s multiple-comparisons test using SigmaStat
3.0 (Chicago, IL, USA). P<0.05 was considered
statistically significant.
Results
Effect of CDP-choline on blood pressure and heart
rate in septic shock-induced by CLI: Baseline mean
arterial pressure (MAP) values of rats were 119 ± 2 mm
Hg (n=24). These values did not change significantly in
sham group (p>0.05) (Figure 1A). Cecal ligation incision
(CLI) procedure caused the decrease in MAP within 60
min (p<0.05) (Figure 1A). Saline injection 3 h after CLI
did not change the arterial blood pressure in CLI-induced
rats and arterial blood pressure continued to decrease
throughout 3 hours (p>0.05) (Figure 1A). Intravenous
injection of CDP-choline at 3 hours after CLI ameliorated
hypotension in 1 hour (p<0.05) (Figure 1A). Arterial
blood pressure of rats maintained at these levels until the
end of the study in CDP-choline group (Figure 1A).
Heart rate of rats in CLI + saline and CLI + CDP-
choline groups increased significantly (p<0.05) (Figure
1B). CDP-choline administration did not affect the rise in
heart rate significantly in septic shock conditions (p>0.05)
(Figure 1B). Measured MAP and HR values during the
experimental protocol are presented in Table 1.
Effect of CDP-choline on IL-1β, IL-6 and TNF-α
levels in septic shock-induced by CLI: Plasma IL-1β, IL-6
and TNF-α levels were 266 ± 54, 1201 ± 125 and 27 ± 9
pg/ml respectively at the end of experiments in sham
group (Figure 2). Cecal ligation incision procedure caused
significant increases in the levels of IL-1β (p<0.05)
(Figure 2A), IL-6 (p<0.05) (Figure 2B) and TNF-α
(p<0.05) (Figure 2C) as observed in saline injected control
group. CDP-choline injection abolished the increases in
IL-1β (p<0.05) and TNF-α (p<0.05), attenuated the
increase in IL-6 levels (p<0.05) (Figure 2) induced by CLI
procedure.
Table 1. Comparison of the values of mean arterial pressure (MAP) and heart rate (HR) in sham, saline and CDP-choline groups before
and after cecal ligation-incision (CLI). Data are means ± S.E.M. (n = 8 rats in each group). *p<0.05, vs sham group. +p < 0.05 vs CLI
+ saline group.
Tablo 1. Çekal ligasyon-insizyon öncesi/sonrasında sham, tuzlu su ve CDP-kolin gruplarında ortalama arter basıncı (MAP) ve kalp
hızı (HR) değerlerinin karşılaştırılması. Veriler ortalama ± ortalamanın standart hatası olarak verilmiştir. *p<0.05, sham grubuna göre.
+p < 0.05 tuzlu su tedavisi uygulanan gruba göre.
MAP (mmHg) and HR (bpm) values
Before CLI
After CLI/Before treatment
After treatment
Sham
(n=8)
118.4 ± 2.7 mmHg
314 ± 15 bpm
120.6 ± 2.6 mmHg
349 ± 15 bpm
111.4 ± 4.3 mmHg
353 ± 18 bpm
CLI + Saline
(n =8)
121.6 ± 2.3 mmHg
327 ± 16 bpm
82.1 ± 3.7 mmHg*
393 ± 17 bpm*
75.3 ± 4.4 mmHg*
462 ± 17 bpm*
CLI + CDP-choline
(n =8)
117.9 ± 1.4 mmHg
325 ± 17 bpm
86.7 ± 3.6 mmHg*
400 ± 18 bpm*
121.4 ± 3.9 mmHg+
431 ± 14 bpm*

Çiğdem Sevim - Burçin Altınbaş - Murat Yalçın - Sevda İnan - Musa Özgür Özyiğit - İlker Arıcan - Mustafa Sertaç Yılmaz
106
Figure 1. Cardiovascular effect of intravenously injected CDP-
choline in CLI-induced septic shock. 3 hours after the CLI
induction, rats were treated with CDP-choline (100 mg/kg; i.v.)
or saline (1 ml/kg; i.v.) and then mean arterial pressure (A) and
heart rate (B) were recorded for the next 3 hours. ‘‘0’’ shows CLI
induction time point. Data are means ± S.E.M. (n = 8 rats in each
group). *p<0.05, vs sham group. +p < 0.05 vs CLI + saline group.
Şekil 1. İntravenöz olarak uygulanan CDP-kolin’in çekal
bağlama-kesme yöntemi ile oluşturulan septik şoktaki
kardiyovasküler etkileri. Çekal bağlama ve kesme işleminin
başlangıcından 3 saat sonra, sıçanların CDP-kolin (100 mg/kg,
i.v.) veya tuzlu su (1 ml/kg; i.v.) ile tedavi edilmelerini takiben
ortalama arter basıncı (A) ve kalp hızı (B) sonraki 3 saat boyunca
kaydedildi. ‘‘0’’ zaman noktası çekal bağlama ve kesme
işleminin uygulandığı anın verisini göstermektedir. Veriler
ortalama ± ortalamanın standart hatası olarak verilmiştir.
*p<0.05, sham grubuna göre. +p < 0.05 tuzlu su tedavisi
uygulanan gruba göre.
Figure 2. The effect of CDP-choline on CLI induced alterations
in plasma cytokine levels. Rats were treated with CDP-choline
(100 mg/kg; i.v.) or saline (1 ml/kg; i.v.) 3 hours after the CLI
induction. At the end of the study (3h after treatment), blood
samples (200 µl) were collected for IL-1β (A), IL-6 (B), and
TNF (C) levels. Data are means ± S.E.M. (n = 8 rats in each
group). *p<0.05, vs sham group. +p < 0.05 vs CLI + saline group.
Şekil 2. CDP-kolin’in çekal bağlama-kesmenin neden olduğu
plazma sitokin seviyeleri değişiklikleri üzerine etkileri. Çekal
bağlama ve kesme işleminin başlangıcından 3 saat sonra,
sıçanlar CDP-kolin (100 mg/kg, i.v.) veya tuzlu su (1 ml/kg; i.v.)
ile tedavi edildi. Çalışmanın sonunda (Tedaviden sonra 3 saat)
IL-1β (A), IL-6 (B) ve TNF (C) seviyelerinin ölçümü için kan
örnekleri (200 µl) toplandı. Veriler ortalama ± ortalamanın
standart hatası olarak verilmiştir. *p<0.05, sham grubuna göre.
+p < 0.05 tuzlu su tedavisi uygulanan gruba göre.

Ankara Üniv Vet Fak Derg, 64, 2017
107
Figure 3. The effect of CDP-choline on CLI induced organ failure. Rats were treated with CDP-choline (100 mg/kg; i.v.) or saline (1
ml/kg; i.v.) 3 hours after the CLI induction. Histopathological comparison of liver [sham (A), saline (B), CDP-choline (C) groups],
lung [sham (D), saline (E), CDP-choline (F) groups], kidney [sham (G), saline (H), CDP-choline (I) groups] and spleen [sham (J),
saline (K), CDP-choline (L) groups] tissues. “arrow” shows vacuolar degeneration; “star” shows hemorrhage; “triangle” shows
necrosis.
Şekil 3. CDP-kolin’in çekal bağlama-kesmenin neden olduğu doku hasarı üzerine etkileri. Çekal bağlama ve kesme işleminin
başlangıcından 3 saat sonra, sıçanlar CDP-kolin (100 mg/kg, i.v.) veya tuzlu su (1 ml/kg; i.v.) ile tedavi edildi. Karaciğer, [sham (A),
tuzlu su (B), CDP-kolin (C) grupları], akciğer [sham (D), tuzlu su (E), CDP-kolin (F) grupları], böbrek [sham (G), tuzlu su (H), CDP-
kolin (I) grupları] ve dalak [sham (J), tuzlu su (K), CDP-kolin (L) grupları] dokularının histopatolojik karşılaştırılması. “ok”, vasküler
dejenerasyonu; “yıldız”, hemorajiyi; “üçgen”, nekrozu göstermektedir.
Effect of CDP-choline on tissue injury induced by
septic shock model in rats: Since septic shock leads to the
multiple organ failure due to the tissue injury, we
investigated whether CDP-choline can exert tissue
protective effect in septic shock condition. Therefore
histopathological examination was performed on lung,
liver, kidney and spleen tissue. In CLI + saline group,
vacuolar degeneration, necrosis and inflammatory
reaction, increasing inflammatory cells in portal area were
seen in liver widely to moderate; while these changes were
observed in CLI + CDP- choline group slightly to
moderate. In lung; severe inflammatory response
(including bronchopneumonia and increased BALT
activity) and severe hemorrhage were observed in CLI +
saline group, while inflammatory response and
hemorrhage were just moderate in CLI + CDP- choline
group. In kidney, degeneration in tubules and necrosis
were seen slightly in CLI + saline group. The severity of
lesions in CLI + CDP- choline group was moderate extent.
In spleen, there were remarkable siderosis, necrosis and
depletion of lymphoid tissue in saline treated CLI-induced
sepsis group. However this changes decreased in CDP-
choline treated CLI sepsis (Figure 3).
Discussion and Conclusion
The present data shows that intravenously injected
CDP-choline restores blood pressure, attenuates the
increase in plasma IL-6, IL-1, TNF-a levels and prevents
lung, liver and kidney injury in septic shock induced by
cecal ligation and incision model.
Cecal ligation and incision model decreased arterial
blood pressure and caused hypotension within 3 hours
(Figure 1). CDP-choline that was injected at 3rd hours after
CLI increased blood pressure and reversed hypotension
without affecting the heart rate changes. The dose of CDP-
choline was chosen from our earlier experiments in which
the drug was shown to exert both cardiovascular and tissue
protective effects in several conditions (11, 31, 32, 45).
Our previous reports repeatedly demonstrated that CDP-
choline, administered either intracerebroventricularly or
intravenously, can affect blood pressure and exert pressor
response in normal and hypotensive conditions (31, 32).
Therefore the present finding showing that CDP-choline
restores hypotension induced by CLI is in aggreement
with those previous reports. Besides, the observation that
CDP-choline did not alter the increase in heart rate caused
by CLI is in good accordance with the previous data

Çiğdem Sevim - Burçin Altınbaş - Murat Yalçın - Sevda İnan - Musa Özgür Özyiğit - İlker Arıcan - Mustafa Sertaç Yılmaz
108
demonstrating that CDP-choline did not influence heart
rate changes while producing pressor effect in normal and
haemorrhaged conditions (31, 32).
Although the mechanism of the CDP-choline’s
pressor effect was not investigated in this study, we may
imply that the activation of central cholinergic nicotinic
and/or muscarinic receptors followed by the enhancement
of central cholinergic transmission is the leading part of its
pressor effect. Because we have previously shown that
intravenously injected CDP-choline increases choline
levels in the plasma and the brain, improves neuronal
acetylcholine synthesis and release; the increase in
neuronal Ach release activates central nicotinic receptors
which results in the pressor response through the
stimulation of the peripheral catecholamine and
vasopressin release (32, 33). On the other hand, it has been
shown that, choline is a selective agonist for 7 nicotinic
acetylcholine receptors (6). It has also been reported that
choline exerts pressor effect by directly activating 7-
nicotinic acetylcholine receptors (23). Hence, we may also
suggest that choline metabolite may mediate the pressor
effect of CDP-choline observed in this study.
Septic shock induced by cecal ligation insicion
model caused an increase in plasma TNF-, IL-6 and IL-
1 levels. CDP-choline administration significantly
attenuated the increase of these cytokine levels (Figure 2).
The excessive increase of these proinflammatory
molecules indicates the progressive and deleterious
inflammatory response to infection in septic shock.
Therefore, treatment approaches aimed at reducing the
release of these molecules in endotoxemia or sepsis are
very important (29). One of those treatment strategies is to
activate vagal anti-inflammatory cholinergic signaling
because the stimulation of efferent vagus nerve inhibits the
release of proinflammatory molecules and attenuates the
progression of shock in endotoxemia (8). CDP-choline
can be considered one of those cholinergic modalities
since it has been shown to activate efferent vagal
cholinergic pathway by stimulating central muscarinic
receptors throughout the enhanced cholinergic transmission
in short term myocardial ischemia-reperfusion injury (45).
Moreover, 7-nicotinic acetylcholine receptors have a
unique role in mediating vagal anti-inflammatory pathway
(25, 39). Several reports revealed that the activation of 7-
nicotinic acetylcholine receptors by nicotine (a
nonselective nicotinic agonist) or GTS-21 (a selective 7
nicotinic acetylcholine receptor agonist) blocked the TNF-
 release and improved survival in experimental septic
conditions (26, 40). Considering that choline is the main
functional mediator of CDP-choline and is a selective
agonist for 7 nicotinic acetylcholine receptors together
with the above information, we may suggest that the
stimulation of vagal cholinergic anti-inflammatory
signaling through the initiation of central muscarinic
cholinergic receptors or peripheral 7-nicotinic acetylcholine
receptors may mediate the CDP-choline induced inhibiton
of the proinflammatory cytokine release in septic shock
conditions.
In the present study, histological data demonstrated
that CLI model caused multiple organ injury including
liver, kidney, lung and spleen. Reduced tissue perfusion
during the hypotensive phase, inflammation through the
excessive release of proinflammatory cytokins and
oxidative stress are the main reasons of these tissue
damages in septic shock conditions (5). CDP-choline
administration attenuated tissue injuries and decreased
vacuolar degeneration, necrosis and hemorrhage in liver,
kidney and lung tissue. CDP-choline-induced tissue
protection may have been through i) the improved tissue
perfusion due to drug’s pressor effect, ii) the alleviation of
inflammatory conditions due to the drug-induced decrease
in plasma proinflammatory cytokins. CDP-choline also
can reduce oxidative stress in several pathological
conditions (3, 11). Therefore the antioxidant effect of
CDP-choline may have another explanation of its tissue
protective effect.
In summary, the outcomes of the current study show
that CDP-choline is able to restore hypotension, to
decrease the plasma levels of proinflammatory cytokins
and to protect tissue injury induced by septic shock model.
Acknowledgements
This work was supported by a grant from the Uludag
University Commission of Scientific Research Projects
(UAP (T) – 2011/13) awarded to Dr. M. Sertaç Yilmaz.
This study was presented in FASEB meeting in 2013
where abstract only is published in the meeting abstract
supplement (47).
References
1. Adibhatla RM, Hatcher JF (2002): Citicoline mechanisms
and clinical efficacy in cerebral ischemia. J Neurosci Res,
70, 133-139.
2. Adibhatla RM, Hatcher JF (2005): Cytidine 5’-
diphosphocholine (CDP-choline) in stroke and other CNS
disorders. Neurochem Res, 30, 15-23.
3. Adibhatla RM, Hatcher JF, Dempsey RJ (2001): Effects
of citicoline on phospholipid and glutathione levels in
transient cerebral ischemia. Stroke, 32, 2376-2381.
4. Adibhatla RM, Hatcher JF, Dempsey RJ (2004):
Cytidine-5'-diphosphocholine (CDP-choline) affects CTP:
Phosphocholine cytidylyltransferase and lyso-
phosphatidylcholine after transient brain ischemia. J
Neurosci Res, 76, 390-396.
5. Ahmad A, Druzhyna N, Szabo C (2016): Delayed
treatment with sodium hydrosulfide improves regional
blood flow and alleviates cecal ligation and puncture
(CLP)-induced septic shock. Shock, Epub ahead of print
(doi: 10.1097/SHK.0000000000000589)

Ankara Üniv Vet Fak Derg, 64, 2017
109
6. Alkondon M, Pereira EF, Barbosa CT, et al. (1997):
Neuronal nicotinic acetylcholine receptor activation
modulates gamma-aminobutyric acid release from CA1
neurons of rat hippocampal slices. J Pharmacol Exp Ther,
283, 1396-1411.
7. Blusztajn JK, Wurtman RJ (1983): Choline and
cholinergic neurons. Science, 22, 614-620.
8. Borovikova LV, Ivanova S, Zhang M, et al. (2000): Vagus
nerve stimulation attenuates the systemic inflammatory
response to endotoxin. Nature, 405, 458-462.
9. Cavun S, Savci V (2004): CDP-choline increases plasma
ACTH and potentiates the stimulated release of GH, TSH
and LH: The cholinergic involvement. Fundam Clin
Pharmacol, 18, 513-523.
10. Cavun S, Savci V, Ulus IH (2004): Centrally injected
CDP-choline increases plasma vasopressin levels by
central cholinergic activation. Fundam Clin Pharmacol, 18,
71-77.
11. Coskun C, Avci B, Ocak N, et al. (2010): Effect of
repeatedly given CDP-choline on cardiovascular and tissue
injury in spinal shock conditions: Investigation of the acute
phase. J Pharm Pharmacol, 62, 497-506.
12. Coskun C, Avci B, Yalcin M, et al. (2014): Protective
effect of CDP-choline on ischemia-reperfusion-induced
myocardial tissue injury in rats. Ir J Med Sci, 183, 539-548.
13. Deitch EA (1998): Animal models of sepsis and shock: A
review and lessons learned. Shock, 9, 1-11.
14. Dorman RV, Dabrowiecki Z, Horrocks LA (1983):
Effects of CDP-choline and CDP-ethanolamine on the
alterations in rat brain lipid metabolism induced by global
ischemia. J Neurochem, 40, 276-279.
15. Downing JEG, Miyan JA (2000): Neural
immunoregulation: Emerging roles for nerves in immune
homeostasis and disease. Immunol Today, 21, 281-289.
16. Esmon CT (2004): Why do animal models (sometimes) fail
to mimic human sepsis? Crit Care Med, 32, 219-222.
17. Fiedorowicz M, Makarewicz D, Stan´czak-Mrozek KI,
et al. (2008): CDP-choline (citicoline) attenuates brain
damage in a rat model of birth asphyxia. Acta Neurobiol
Exp, 68, 389-397.
18. G-Coviella IL, Wurtman RJ (1992): Enhancement by
cytidine of membrane phospholipids synthesis. J
Neurochem, 59, 338-343.
19. Hotchkiss RS, Karl IE (2003): The pathophysiology and
treatment of sepsis. N Engl J Med, 348, 138.
20. Ilcol YO, Gurun MS, Taga Y, et al. (2003): Choline
increases serum insulin in rat when injected
intraperitoneally and augments basal and stimulated
acetylcholine release from the rat minced pancreas in vitro.
Eur J Biochem, 270, 991-999.
21. Ilcol YO, Yilmaz Z, Cansev M, et al. (2009): Choline or
CDP-choline alters serum lipid responses to endotoxin in
dogs and rats: Involvement of the peripheral nicotinic
acetylcholine receptors. Shock, 32, 286-294.
22. Klein J (2000): Membrane breakdown in acute and chronic
neurodegeneration: Focus on choline-containing
phospholipids. J Neural Transm, 107, 1027-1063.
23. Li XD, Buccafusco JJ (2004): Role of alpha7 nicotinic
acetylcholine receptors in the pressor response to
intracerebroventricular injection of choline: Blockade by
amyloid peptide Abeta1-42. J Pharmacol Exp Ther, 309,
1206-1212.
24. Marshall JC, Vincent JL, Fink MP, et al. (2003):
Measures, markers, and mediators: toward a staging system
for clinical sepsis. A report of the Fifth Toronto Sepsis
Roundtable, Toronto, Ontario, Canada, October 25-26,
2000. Crit Care Med, 31, 1560-1567.
25. Parrish WR, Rosas-Ballina M, Gallowitsch-Puerta M, et
al. (2008): Modulation of TNF release by choline requires
alpha7 subunit nicotinic acetylcholine receptor-mediated
signaling. Mol Med, 14, 567-574.
26. Pavlov VA, Ochani M, Yang LH, et al. (2007): Selective
alpha7-nicotinic acetylcholine receptor agonist GTS-21
improves survival in murine endotoxemia and severe sepsis.
Crit Care Med, 35, 1139-1144.
27. Rao AM, Hatcher JF, Dempsey RJ (2000): Lipid
alterations in transient forebrain ischemia: Possible new
mechanisms of CDP-choline neuroprotection. J
Neurochem, 75, 2528-2535.
28. Rao AM, Hatcher JF, Dempsey RJ (2001): Does CDP-
choline modulate phospholipase activities after transient
forebrain ischemia? Brain Res, 893, 268-272.
29. Riedemann NC, Guo RF, Ward PA (2003): Novel
strategies for the treatment of sepsis. Nat Med, 9, 517-524.
30. Rivera CA, Wheeler MD, Enomoto N, et al. (1998): A
choline rich diet improves survival in a rat model of
endotoxin shock. Am J Physiol, 275, 862-867.
31. Savci V, Cavun S, Goktalay G, et al. (2002):
Cardiovascular effects of intracerebroventricularly injected
CDP-choline in normotensive and hypotensive animals:
The involvement of cholinergic system. Naunyn
Schmiedebergs Arch Pharmacol, 365, 388-398.
32. Savci V, Goktalay G, Cansev M, et al. (2003):
Intravenously injected CDP-choline increases blood
pressure and reverses hypotension in haemorrhagic shock:
Effect is mediated by central cholinergic activation. Eur J
Pharmacol, 468, 129-139.
33. Savci V, Goktalay G, Ulus IH (2002):
Intracerebroventricular choline increases plasma
vasopressin and augments plasma vasopressin response to
osmotic stimulation and hemorrhage. Brain Res, 942, 58-
70.
34. Savci V, Ulus IH (1997): Cardiovascular effects of central
choline during endotoxin shock in the rat. J Cardiovasc
Pharmacol, 30, 667-675.
35. Savci V, Wurtman RJ (1995): Effect of cytidine on
membrane phospholipid synthesis in rat striatal slices. J
Neurochem, 64, 378-384.
36. Secades JJ (2011): Citicoline: Pharmacological and
clinical review, 2010 update. Rev Neurol, 52, 1-62.
37. Stoll AL, Renshaw PF, DeMicheli E, et al. (1995):
Choline ingestion increases the resonance of choline
containing compound in human brain: An in vivo proton
magnetic resonance study. Biol Psych, 37, 170-174.
38. Ulus IH, Wurtman RJ, Mauron C, et al. (1995): Choline
increases acetylcholine release and protects against the
stimulation-induced decrease in phosphatide levels within
membranes of rat corpus striatum. Brain Res, 484, 217-227.
39. Wang H, Yu M, Ochani M, et al. (2003): Nicotinic
acetylcholine receptor alpha7 subunit is an essential
regulator of inflammation. Nature, 421, 384-388.

Çiğdem Sevim - Burçin Altınbaş - Murat Yalçın - Sevda İnan - Musa Özgür Özyiğit - İlker Arıcan - Mustafa Sertaç Yılmaz
110
40. Wang H, Liao H, Ochani M, et al. (2004): Cholinergic
agonists inhibit HMGB1 release and improve survival in
experimental sepsis. Nat Med, 10, 1216-1221.
41. Weiss GB (1995): Metabolism and actions of CDP-choline
as an endogenous compound and administered exogenously
as citicoline. Life Sci, 56, 637-660.
42. Wenzel RP, Edmond MB (2012): Septic shock devaluating
another failed treatment. N Engl J Med, 366, 2122.
43. Wijdicks EF, Stevens M (1992): The role of hypotension
in septic encephalopathy following surgical procedures.
Arch Neurol, 49, 653.
44. Wurtman RJ (1992): Choline metabolism as a basis for the
selective vulnerability of cholinergic neurons. Trends
Neurosci, 15, 117-122.
45. Yilmaz MS, Coskun C, Yalcın M, et al. (2008): CDP-
choline prevents cardiac arrhythmias and lethality induced
by short-term myocardial ischemia-reperfusion injury in the
rat: involvement of central muscarinic cholinergic
mechanisms. Naunyn Schmiedebergs Arch Pharmacol, 378,
293-301.
46. Yilmaz Z, Ilcol YO, Torun S, et al. (2006): Intravenous
administration of choline or cdp-choline improves platelet
count and platelet closure times in endotoxin-treated dogs.
Shock, 25, 73-79.
47. Yilmaz MS, Sevim C, Altinbas B, et al. (2013): CDP-
choline protects against sepsis-induced acute tissue injury
in rats. The FASEB J, 27, Supplement 888.8.
Geliş tarihi: 25.12.2015 / Kabul tarihi: 20.07.2016
Address for correspondence:
Mustafa Sertaç Yılmaz, MD, PhD
Uludağ Universitesi Tıp Fakultesi,
Tıbbi Farmakoloji Anabilim Dalı,
Gorukle, 16059, Bursa, Turkey.
Tel: + 90 224 295 3566 Fax: + 90 224 442 8102
e-mail: sertacyilmaz@uludag.edu.tr