Neuroprotective effects of carnosine and cyclosporine

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Toxicology Mechanisms and Methods
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Neuroprotective effect of carnosine and
cyclosporine-A against inflammation, apoptosis,
and oxidative brain damage after closed head
injury in immature rats
Nayira Ahmed Abdel Baky, Laila Fadda, Nouf M. Al-Rasheed, Nawal M. Al-
Rasheed, Azza Mohamed & Hazar Yacoub
To cite this article: Nayira Ahmed Abdel Baky, Laila Fadda, Nouf M. Al-Rasheed, Nawal M.
Al-Rasheed, Azza Mohamed & Hazar Yacoub (2016) Neuroprotective effect of carnosine
and cyclosporine-A against inflammation, apoptosis, and oxidative brain damage after
closed head injury in immature rats, Toxicology Mechanisms and Methods, 26:1, 1-10, DOI:
10.3109/15376516.2015.1070224
To link to this article: http://dx.doi.org/10.3109/15376516.2015.1070224
Published online: 14 Aug 2015. Submit your article to this journal
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ISSN: 1537-6516 (print), 1537-6524 (electronic)
Toxicol Mech Methods, 2016; 26(1): 1–10
! 2015 Taylor & Francis. DOI: 10.3109/15376516.2015.1070224
RESEARCH ARTICLE
Neuroprotective effect of carnosine and cyclosporine-A against
inflammation, apoptosis, and oxidative brain damage after closed head
injury in immature rats
Nayira Ahmed Abdel Baky1
, Laila Fadda1
, Nouf M. Al-Rasheed1
, Nawal M. Al-Rasheed1
, Azza Mohamed2
, and
Hazar Yacoub1
1
Department of Pharmacology, King Saud University, Riyadh, Saudi Arabia and 2
Biochemistry Department, Faculty of Science for Girls,
King Abdulaziz University, Jeddah, Saudi Arabia
Abstract
Context: Traumatic brain injury in the pediatric population can have a great economic and
emotional impact on both the child’s family and society.
Objective: The present study aimed to compare the effects of carnosine (CAR) and/or
cyclosporine A (CyA) on oxidative brain damage after closed head injury (CHI) in immature rats.
Materials and methods: Thirty-day-old rat pups were divided into five groups: non-traumatic
control group, trauma group underwent CHI, trauma group injected with CAR (200 mg/kg, i.p.)
following CHI for 7 d, trauma group injected with CyA (20 mg/kg, i.p.) given 15 min and 24 h
after CHI, and trauma group treated with CAR and CyA. At the end of the treatment, rats were
sacrificed; blood and brains were collected for assessing different biochemical parameters.
Results: Trauma significantly increased brain level of malondialdehyde, nitric oxide, glucose,
calcium, inflammatory mediators. Brain DNA damage was confirmed by comet assay and the
significant increase in brain caspase-3 activity. Moreover, the serum level of Fas ligand in
traumatized animals was significantly elevated. Concomitant decrease in brain-reduced
glutathione (GSH) and calcium-adenosine triphosphatase activity was observed in the
traumatized-untreated group. Treatment of traumatized animals with CAR and/or CyA
ameliorated all the biochemical changes induced by CHI with marked protective effect in
the combination group.
Discussion and conclusion: CAR and CyA exerted a synergistic neuroprotective effect against CHI
through blocking the induction of lipid peroxidation, reducing inflammatory, and oxidative
stress biomarkers, preserving brain GSH content, and reducing the alterations in brain
apoptotic biomarkers in traumatized animals.
Keywords
Calcium–adenosine triphosphatase, lipid
peroxidation, oxidative damage, sFasL,
traumatic brain injury
History
Received 16 April 2015
Revised 24 June 2015
Accepted 30 June 2015
Published online 14 August 2015
Introduction
Traumatic brain injury (TBI) is a common cause of mortality
and morbidity worldwide, particularly among the young
(Thurman & Guerrero, 1999). The long-term sequel and
consequences after closed head trauma in children are often
more devastating than in adults due to their age and
developmental potential. An extended follow-up of preschool
children after severe TBI revealed learning difficulties,
attention deficits, and memory problems that did not
become manifest until the children entered school (Barlow
et al., 2005).
The significant cell death and subsequent neuronal degen-
eration following TBI is believed to evolve as a consequence
of primary mechanical insult which result in direct mechan-
ical damage to neurons, axons, glia, and blood vessels.
This mechanical injury is coupled with progressive secondary
necrotic changes that occur from minutes to weeks after the
insult (Faden, 1993; Siesjo et al., 1995). These changes are
sequence of cellular, neurochemical, and metabolic alter-
ations that eventually generate large amounts of toxic and
proinflammatory molecules such as nitric oxide (NO),
prostaglandins, free radicals, and inflammatory cytokines,
which lead to breakdown of the blood–brain barrier (BBB)
and the development of edema. The associated increase in
intracranial pressure (ICP) may then cause local hypoxia and
ischemia, with subsequent neuronal cell death via necrosis
and apoptosis (McIntosh et al., 1996). Since there are no
approved specific pharmacological agents that block the
Address for correspondence: Nayira Ahmed Abdel Baky, PhD,
Department of Pharmacology, King Saud University, Riyadh,
Saudi Arabia. E-mail: nayiraabdelbaky@yahoo.com
Downloadedby[KingSaudUniversity]at01:1318April2016

progression of the secondary injury, the current management
of TBI is mainly supportive and aims at minimize both
secondary insults and the evolution of secondary damage in
the acute phase (Bullock et al., 2002; Kochanek, 2006). For
that pharmacological interventions that could prevent, attenu-
ate, or at least delay the resultant neurological injury are
needed to overcome the cascades of secondary insults which
manifest over minutes to days following the initial trauma. As
oxidative stress is an important contributor to the pathogen-
esis of TBI, therefore, new therapeutic antioxidant strategies
are urgently required (Bayir et al., 2003).
Carnosine (CAR) is one of the most abundant antioxidants
in the brain that is synthesized by the carnosine synthetase
from b-alanine and L-histidine. Carnosine can easily cross the
BBB from the periphery (Matsukura & Tanaka, 2000), and it
is known to quench singlet oxygen radicals, chelate metal
ions, and bind hydroperoxides (Aldini et al., 2002; Boldyrev
et al., 2004; Tang et al., 2007). It also plays many other
prominent roles such as anti-inflammatory agent (Boldyrev
et al., 1999; Zhang et al., 2014). Consequently, CAR could
have a dual action to diminish brain tissue damage after TBI.
On the contrary, cyclosporine-A (CyA) is a multifactorial
neuroprotective agent that putatively exerts neuroprotective
and neurotrophic effects in TBI, sciatic nerve injury, and focal
and global ischemia (Kaminska et al., 2004). It was
demonstrated that CyA administration after TBI reduce
brain damage (Scheff & Sullivan, 1999), and improves not
only motor function (Riess et al., 2001) but also cognitive
performance (Alessandri et al., 2002) in experimental
animals. This effect was correlated to an improvement in
brain oxygen consumption after trauma which reflect
improvement in mitochondrial function, inhibition of mito-
chondrial permeability transition (Hansson et al., 2004;
Sullivan et al., 1999) and improvement in energy recovery
(Nakai et al., 2004). Such effects would also inhibit calcium
accumulation, free radical release, and apoptosis (Mirzayan
et al., 2008; Panickar et al., 2002).
In light of all of the above, this study was aimed to
investigate the neuroprotective effects of carnosine and CyA
on the bases oxidative stress occurring in the brain tissue
following TBI in rats.
Materials and methods
Drugs and chemicals
All drugs and chemicals used in this study were of
analytically pure product of Sigma-Aldrich Chemical Co.,
St. Louis, MO.
Animals
In this study, 30-day-old male Wistar albino rats weighing
50–70 g were used. The rats were obtained from Experimental
Animal Care Center, College of Pharmacy, King Saud
University. Animals have been kept in special cages, and
maintained on a constant 12-h light/12-h dark cycle with air
conditioning and temperature ranging 20–22
C and humidity
(60%). Rats were fed with standard rat pellet chow with free
access to tap water ad libitum for 1 week before the
experiment. Animal utilization protocols were performed in
accordance with the guidelines provided by the Experimental
Animal Laboratory and approved by the Animal Care and Use
Committee of the King Saud University, College of
Pharmacy. All efforts were made to reduce the number of
animals used, as well as to minimize their suffering.
TBI model
We used a modification of a well-described closed head
trauma model in rats for the present study (Awasthi et al.,
1997). To avoid neuroprotective effects of anesthetic agents
(such as halothane or barbiturates), the rats were anesthetized
with ether. Rats were laid on a warmed blanket, and heat
monitoring was performed with a rectal probe to keep the
body temperature at 37.5
C. The contusing device consisted
of a hollow plastic tube 60 cm long, 5 mm wide, and
perforated at 1-cm intervals to prevent air compression. The
device was kept vertical to the surface of the intact skull and
guided a falling weight onto the parietal convexity (3 mm
anterior and 2 mm lateral to the lambda) (Awasthi et al.,
1997). TBI was induced via dropping an object weighing 40 g
through the previously mentioned contusing device to
produce brain contusion. Following closed head injury
(CHI), all rats received 100% oxygen until arousal to decrease
mortality in experimental groups.
Experimental groups and treatment
Rats were randomly allocated into five groups (each having
seven rats): a control group: in which rats were not undergone
closed head trauma; CHI group: in which rats underwent
closed head trauma; CHI/CAR group: in which rats under-
went CHI and treated i.p. with carnosine; CHI/CyA group: in
which rats underwent CHI and treated i.p. with cyclosporine
A; and CHI/CAR + CyA group: in which rats underwent CHI
and treated i.p. with carnosine and cyclosporine A. Control
and trauma groups received no medication but only received
physiological saline as a vehicle control. The CAR group
received 200 mg/kg, i.p. carnosine (Boldyrev et al., 1999),
immediately after truma, and for a period of 7 d following
CHI. CyA (20 mg/kg, i.p.) was administrated 15 min and 24 h
after CHI (Sullivan et al., 2000). All rats were sacrificed at
day 7 after CHI, blood was collected, and serum was
separated, and stored at À80
C. After removal of the skull
bones, the brain lobes were separated immediately, rinsed in
cold isotonic saline, homogenized, and freezed at À80
C for
different biochemical estimations.
Biochemical analyses
Determination of lipid peroxides (MDA) and glutathione
(GSH) level in brain tissue
The degree of lipid peroxidation in brain tissues was
determined by measuring thiobarbituric acid reactive sub-
stances (TBARS) in the supernatant tissue from brain
homogenate (Uchiyama Mihara, 1978). The absorbance
was measured spectrophotometrically at 532 nm and quanti-
fied as nanomoles of malondialdehyde (MDA)/g wet tissue.
Brain tissue levels of acid-soluble thiols, mainly reduced
glutathione (GSH), were determined calorimetrically at
412 nm (Ellman, 1959). Homogenates were precipitated
2 N. A. A. Baky et al. Toxicol Mech Methods, 2016; 26(1): 1–10

with trichloroacetic acid, and after centrifugation, pellets were
used for the estimation of protein thiols (Protein-SH)
expressed as mmol/gm wet tissue.
Determination of brain tissue total nitrate/nitrite
concentrations
Total tissue nitrate/nitrite, an indirect measure for NO
synthesis, was estimated according to the method described
by Green et al. (1982) using the Griess reagent (sulfanilamide
and N-1-naphthylethenediamine dihydrochloride) in the acid
medium.
Determination of xanthine oxidase (XO) activity
in brain tissue
The XO activity was assessed according to the method of
Prajda Weber’s (1975). The enzyme activity was measured
spectrophotometrically by the formation of uric acid from
xanthine with increase in absorbance at 293 nm. This method
is based on the amount of uric acid produced by XO from the
xanthine, added to the medium. Tissue homogenate was
incubated (50 mL) for 30 min at 37
C in 2.85 mL of medium
containing phosphate buffer (pH 7.5, 50 mM) and xanthine
(0.067 mmol final concentration in each tube). The reaction
was stopped by addition of 0.1 mL 100% (w/v) TCA and the
mixture was centrifuged at 5000Âg for 15 min. The absorp-
tion at 293 nm of the resultant clear supernatant was measured
against blank. One unit of activity was defined as 1 mmol of
uric acid formed per minute at 37
C, pH 7.5. The activity was
expressed in units per gram protein (U/g protein).
Determination of Na/K-ATPase in brain tissue
Na,K-ATPase was measured in brain tissue by means of an
enzyme-coupled kinetic assay with pyruvate kinase and
lactate dehydrogenase, assuming ouabain (2.5 mM) inhibition
measured the Na,K-ATPase activity (Simon et al., 1996).
Determination of calcium ions concentration
Calcium concentration in the supernatant of brain tissue
homogenates was assessed by atomic absorption spectropho-
tometry with a modification of the method of Willis (1961).
Standard calcium solutions were prepared from dried calcium
carbonate in the range 2.0–20.0 mg/100 mL. Standards and
supernatants were diluted 1:25 with 0.5 lanthanum chloride
(with respect to lanthanum ion), and the percentage of
absorbance was recorded with the Perkin-Elmer Model 303
atomic absorption spectrophotometer (Perkin-Elmer Inc.,
Shelton, CT). From a standard curve of the standards against
percentage of transmission, the mg calcium/100 mL for each
specimen were calculated. Each estimation was performed in
duplicate.
Comet assay
Comet assay was used to analyze the level of DNA damage in
brain tissues after TBI. Brain tissues were pressed through a
screen in homogenization buffer (0.075 M NaCl and 0.024 M
EDTA, pH 7.5), at a ratio of 1 g of tissue to 1 mL of buffer, and
then cooled to 4
C. A Potter-type homogenizer was used
(Sasaki et al., 1997). All collected samples were prepared for
analysis by using a modification of the method of Singh et al.
(1988): 6 mL of brain homogenate were placed onto precleaned
microscope slides, previously precoated with 300 mL of 0.6%
NMP agarose. After solidification on ice for 10 min, the slides
were covered with 0.5% LMP agarose. After the agarose gel
has solidified, slides were immersed for 1 h in ice-cold lysis
solution, consisting of 100 mM Na2EDTA, 2.5 M NaCl,
10 mM Tris-HCl, and 1% sodium sarcosinate, adjusted to pH
10 with 1% Triton X-100 and 10% DMSO, added just prior to
use. Before electrophoresis, slides were removed from the
lysing solution and placed for 20 min in a horizontal electro-
phoresis unit (near the anode), filled with an alkaline buffer, in
order to allow the unwinding of DNA and to express alkali-
labile damage. The electrophoresis alkaline solution consisted
of 1 mM Na2EDTA and 300 mM NaOH, pH 13. After the
unwinding of DNA, electrophoresis was carried out in the
freshly prepared alkaline solution for 20 min at 25 V (300 mA).
Alkali unwinding and electrophoresis were performed at 4
C.
Electrophoresis at high pH results in structures resembling
comets, as observed by fluorescence microscopy; the intensity
of the comet tail relative to the head reflects the number of
DNA breaks. The slides were then neutralized by adding Tris
buffer (pH 7.5), stained with ethidium bromide (Sigma,
St. Louis, MO), covered and stored in sealed boxes at 4
C
for analysis. All preparation steps were performed under
dimmed light to prevent additional DNA damage. Images of
100 randomly selected cells (50 counts on each duplicate slide)
were analyzed for each sample. A total of 500 cells from each
group were analyzed under a Leitz Orthoplan epifluorescence
microscope (Leitz, Wetzlar, Germany) (magnification 250Â)
equipped with an excitation filter of 515–560 nm and a barrier
filter of 590 nm. The microscope was connected through a
camera to a computer-based image analysis system (Comet
Assay IV software, Perspective Instruments, Haverhill Suffolk,
UK). Comets were randomly captured at a constant depth of
the gel, avoiding the edges of the gel, occasional dead cells, and
superimposed comets. DNA damage was measured as tail
length (TL ¼ distance of DNA migration from the centre of the
body of the nuclear core), and tail intensity DNA (TI ¼ % of
genomic DNA that migrated during the electrophoresis from
the nuclear core to the tail) (Singh et al., 1988).
Determination of brain glucose level
Glucose was measured according to method adopted previ-
ously by Miwa et al. (1972) using a glucose kit (enzymatic
method) (Wako, Osaka, Japan).
Assessment of brain inflammatory cytokine concentration
The concentration of inflammatory cytokines (TNF-a and
IL-6) in brain tissue was determined using commercially
available ELISA assays following the instructions supplied by
the manufacturer (DuoSet kits, RD Systems; Minneapolis,
MN). The results are shown as pg/100 mg tissue.
Determination of brain caspase 3 level
Caspase 3-like protease was assayed according to the method
described by Vaculova Zhivotovsky (2008).
DOI: 10.3109/15376516.2015.1070224 Neuroprotective effect of carnosine and cyclosporine-A 3

Determination of serum Fas ligand
sFasL was determined using commercial enzyme-linked
immunosorbent assay (ELISA) kits (Diaclone, Besancon,
France).
Statistical analysis
The experimental data were statistically analyzed using one-
way analysis of variance (ANOVA) followed by the Tukey–
Kramer as a post-ANOVA test for multiple comparisons. Data
were expressed as mean ± SD. Differences were considered
significant at p value of less than 0.05.
Results
Oxidative stress biomarkers in brain tissue
Oxidative stress biomarkers (XO, NO, and MDA) as well as
non-enzymatic antioxidant marker (GSH) in brain tissue of
normal and different experimental TBI rat groups are shown
in Table 1. TBI induced pronounced increases in oxidative
stress biomarkers with concomitant decrease in tissue
antioxidant level compared with normal animals (p50.05).
Where trauma significantly increased brain TBARS, NO
content and, XO activity, and this was accompanied by a
reduction in brain-reduced GSH level of traumatized animals
compared with control rats (p50.001). Treatment of
traumatized animals with any form of single treatment
protocol significantly down-modulated the induced oxidative
stress markers level (lipid peroxides, nitric oxide, and XO
activity), and significantly increased GSH levels, when
compared with levels of such parameters in the untreated
group (p50.05). Interestingly, combined treatment with CAR
and CyA exerted a synergistic antioxidant effect against
oxidative brain damage as compared with traumatized non-
treated animals (p50.001) or those treated with either drug
alone (p50.05).
Inflammatory cytokine level in brain tissue
The levels of immunologic proinflammatory biomarkers,
including TNF-a and IL-6, in the normal and traumatized rat
groups are illustrated in Figure 1. These biomarkers were
dramatically elevated in the brain tissue of traumatized rats
compared with the normal group. CAR and CyA treatment
significantly reduced the elevation in inflammatory cytokines
level as compared with traumatized non-treated pups
(p50.05).
Brain ATPase activity and glucose concentration
Figure 2 shows the level of brain Na/K–ATPase in normal and
different experimental TBI rat groups. A significant decrease
in the enzyme activity was noticed in the brain tissue of
traumatized rats compared with normal animals. TBI rats that
underwent injection of the studied agents, each alone or in
combination showed marked attenuation of trauma induced
Figure 1. Effects of carnosine and/or cyclosporine A treatment on brain inflammatory biomarkers: (a) IL-6, (b) TNF-a after CHI in rat pups. Values are
means ± SD (n¼7). a
p50.001, b
p50.01, and c
p50.05 compared with the normal control group, ***p50.001, **p50.01 compared with the
traumatized non-treated group, respectively, using ANOVA followed by the Tukey–Kramer as the post-ANOVA test.
Table 1. Effects of carnosine and/or cyclosporine A treatment on malonaldehyde, glutathione, nitric oxide levels, and xanthene oxidase activity in
traumatized brain tissues of rat pups.
Groups MDA (nmol/g tissue) GSH (mmol/g tissue) XO (nmol/mg protein) NO (mmol/g tissue)
Control 1.50 ± 0.077 3.422 ± 0.165 13.045 ± 0.501 0.726 ± 0.04
CHI 3.82 ± 0.237a
1.248 ± 0.095a
53.63 ± 3.11a
3.42 ± 0.25a
CHI/CAR 2.19 ± 0.119a
*** 3.070 ± 0.188c
*** 14.56 ± 0.322*** 0.819 ± 0.02***
CHI/CyA 2.34 ± 0.082a
*** 2.485 ± 0.143a
*** 21.09 ± 1.28a
*** 0.973 ± 0.06***
CHI/CAR+CyA 1.58 ± 0.042***###$$$ 3.332 ± 0.171***### 13.817 ± 0.324***### 0.743 ± 0.05***
Values are means of 7 ± SD. a
p50.001 and c
p50.05 compared with the normal control group, ***p50.001 compared with the traumatized non-
treated group, ###p50.001 compared with CyA-treated group, and $$$p50.001 compared with CAR-treated group, using ANOVA followed by the
Tukey–Kramer as the post-ANOVA test.

depletion in brain Na/K–ATPase compared with untreated
rats, with maximal improvement in the combination group
(p50.05).
Meanwhile, brain glucose level was significantly increased
in traumatized rats compared with normal animals (Figure 3).
Administration of CAR and/or CyA, markedly reduced the
dramatic increase in the brain glucose level of traumatized
rats compared with untreated animals.
Assessment of apoptosis and DNA damage
The effect of TBI on apoptotic brain DNA damage was
assessed through the determination of different apoptotic
biomarkers, as well as utilizing comet assay. Figure 4 shows
the effect of truma on apoptotic biomarkers (FasL in serum,
caspase-3 activity, and Ca+2
in brain tissue). These bio-
markers were significantly up-regulated in traumatized rats.
Administration of CAR and/or CyA to rats beneficially down-
modulated the increases in these biomarkers (p50.05).
Meanwhile, Figures 5 and 6 show a significant increase in
the tail length and DNA% (tail DNA content) in the brain
tissue of traumatized rats compared with normal healthy ones.
All the observed changes in the level of DNA damage after
CAR or CyA treatment were not statistically proved to be
significant from traumatized non-treated pups. However, the
combination treatment reduced the level of DNA damage
more significantly than either agent alone compared with the
control group.
Discussion
TBI is a complex dynamic process that initiates a multitude of
cascades of pathological cellular pathways. Oxidative stress is
the principal factor in traumatic brain injury that initiates
events and results in protracted neuronal dysfunction and
remodeling. The current study documented increase in brain
cellular oxidative stress biomarkers (XO, NO, and MDA),
with concomitant decrease in the non-enzymatic antioxidant
(GSH) as well as in the activity of membrane bound enzyme
Na+
/K+
ATPase in rats following experimental TBI. Our
results were in line with the data presented from previous
experimental studies (Ansari et al., 2008; Hou et al., 2012).
The brain is particularly vulnerable to oxidative injury
because of its high rate of oxygen consumption, intense
production of reactive radicals, and high levels of transition
metals, such as iron, that catalyze the production of reactive
radicals (Popa-Wagner et al., 2013). XO can generate reactive
oxygen species (ROS) and reactive nitrogen species (RNS) as
NO, which can induce oxidative stress and inflect tissue
injury (Yeldandi et al., 2000). Moreover, neuronal membranes
are rich in polyunsaturated peroxidizable fatty acids PUFAs
that represent a source of lipid peroxidation along with high
levels of iron that act as a prooxidant (Badjatia et al., 2012;
Chen et al., 2013; Reiter, 1998). Lipid peroxidation directly
damages neuronal membranes and yields a number of
secondary products responsible for extensive cellular
damage including MDA. Peroxidation of membrane lipids
affects a variety of functions resulting in increased membrane
rigidity, decreased activity of membrane-bound enzymes (as
Na-K ATPase), impairment of membrane receptors, and
altered permeability, thus affecting neuronal homeostasis,
leading to brain dysfunction (Farooqui Horrocks, 1998).
Drugs that reduce oxidative stress status appear to be a
rational choice for the prevention of those neurological
disorders. Treatment of traumatized rats with either CAR or
CyA, significantly reduced the induced oxidative stress
biomarkers (MDA, NO, and XO) and up-modulate the
decreased GSH and Na-K ATPase versus untreated trauma-
tized rats. CAR as well as its combination with CyA was the
most effective against TBI-induced oxidative stress. The
effectiveness of CAR was mainly related to its neuro-
protective and antioxidant activities that it is capable of
counteracting oxidative process through its free radical
Figure 3. Effects of carnosine and/or cyclosporine A treatment on
glucose level in traumatized brain tissues. Values are means ± SD
(n ¼ 7). a
p50.001 compared with the normal control group,
***p50.001 compared with the traumatized non-treated group, and
##p50.01 compared with the CyA-treated group, using ANOVA
followed by the Tukey–Kramer as the post-ANOVA test.
Figure 2. Effects of carnosine and/or cyclosporine A treatment on
Na+
/K+
-ATPase activity in traumatized brain tissues. Values are
means ± SD (n ¼ 7). a
p50.001 compared with the normal control
group, ***p50.001 compared with the traumatized non-treated group,
###p50.001 compared with the CyA-treated group, and $p50.05
compared with the CAR-treated group, using ANOVA followed by the
Tukey–Kramer as the post-ANOVA test.

scavenger (Boldyrev et al., 2004; Murad et al., 2011). CAR
and related compounds have been linked to several antioxi-
dant activities (Guiotto et al., 2005), including the scavenging
of peroxyl and hydroxyl radicals, the chelation of transition
metals (Babizhayev et al., 1994), and protection of cells from
RNS (Fontana et al., 2002). CAR was also found to exhibit a
significant antioxidant protecting effect in case of brain
damaged induced either by ischemic injury or hypobaric
Figure 4. Effect of carnosine and/or cyclosporine A treatment on apoptosis biomarkers; (a) serum sFasL, (b) brain caspase-3 activity, and (c) Ca2+
concentration in brain tissues after CHI in rat pups. Values are mean ± SD of seven rats. a
p50.001, b
p50.01, and c
p50.05 compared with the normal
control group, ***p50.001, **p50.01, *p50.05 compared with the traumatized non-treated group, and $p50.05 compared with the CAR-treated
group, using ANOVA followed by the Tukey–Kramer as the post-ANOVA test.
Figure 5. Effects of carnosine and/or cyclosporine A treatment on brain DNA oxidative damage biomarkers: (a) tail DNA %, (b) tail length after CHT
in rat pups. Values are means ± SD (n ¼ 7). b
p50.01 and c
p50.05 compared with the normal control group using ANOVA followed by the Tukey–
Kramer as the post-ANOVA test.

hypoxia (Dobrota et al., 2005; Stvolinskii et al., 2003).
Amelioration of oxidative stress by CyA may be related to its
direct inhibitory effects on activation of microglia cells,
which have a role in inflammatory cytokines and free radical
production (Hailer, 2008). This effect may be beneficial for
prevention of neuroinflammation and neurodegeneration
induced by TBI. Previous prospective clinical trials also
showed that CyA have a good safety profile when treatments
were initiated within the first 12 h post-injury in patients with
severe head injuries (Mazzeo et al., 2009).
The current study also showed marked increases in
proinflammatory cytokines (TNF-a and IL-6) in rat sera in
response to TBI compared with control animals. The produc-
tion of such inflammatory cytokines following TBI was
documented in a range of biological compartments (blood,
CSF, and brain extracellular space) (Helmy et al., 2011;
Sandhir et al., 2004). Also, elevated TNF-a and IL-6 have
been detected in the brain parenchyma within the early hours
after brain injury in both humans and rodents (Helmy et al.,
2011; Sandhir et al., 2004). It is also documented that TNF-a
and IL-6 play a determinant role in disrupting BBB, causing
capillary leakage and accelerating the formation of cerebral
edema and tissue injury (Luo et al., 2013; Qian et al., 2010;
Wang et al., 2007). Thus inhibiting pro-inflammatory cyto-
kines induced by TBI is a neuro-protective (Shohami et al.,
1996). Treatment of traumatized rats with CAR and/or CyA,
significantly decreased serum levels of TNF-a and IL-1b
compared with untreated traumatized ones. Similar results
were obtained from previous studies that showed reduced IL-
6 and TNF-a level after oral administration of CAR in animal
models of brain damage and diabetes mellitus (Lee et al.,
2005; Qian et al., 2010,). Additionally, CAR has the ability to
inhibit the synthesis of microglial inflammatory and oxidative
stress mediators in LPS-induced brain damage (Fleisher-
Berkovich et al., 2009). On the contrary, CyA can specifically
inhibit the activities of immunocytes and inhibit proinflam-
matory cytokines secreted by activated microglia, T cells, and
mononuclear phagocytes (Qian et al., 2010; Signoretti et al.,
2004).
Induction of DNA fragmentation, which is one of the
major cause of neuro-degeneration following TBI, have been
shown in a number of studies (Kassubek et al., 2012; Morita-
Fujimura et al., 1999a,b). Our data were in accordance with
the previous results. Where TBI induced brain DNA
Figure 6. Effect of carnosine and/or cyclo-
sporine A treatment on the level of DNA
damage after CHI in rat pups. Comet assay
showing the degree of DNA damage in the
brain tissue of normal and different experi-
mental TBI groups, (1) control rats, (2)
traumatized rats, (3) traumatized rats treated
with CAR, (4) traumatized rats treated with
CyA, and (5) traumatized rats treated with
CAR and CyA.

fragmentation as documented by significant increase in the
tail length and DNA% in the tail in the brain tissue of
traumatized rats. Using Comet assay, DNA damage was also
detected in a variety of injury models at both acute and
chronic time points (Dagci et al., 2009; Huang et al., 2007;
Martin Liu, 2002). On the contrary, substantial evidence
suggests oxidative/nitrosative stress associated with second-
ary brain injury can affect DNA integrity (Hall et al., 2010).
Failure to repair DNA lesions may result in blockages of
transcription and replication, mutagenesis, and/or cellular
cytotoxicity (Kasparek Humphrey 2011). Administration of
CAR and/or CyA to traumatized rats significantly alleviated
the brain tissue from DNA damage in traumatized rats
compared with untreated rats. CAR in combination with CyA
was the most effective one. The protective effect of CAR
against DNA damage was previously documented in brain
cortex and medulla of rats induced by propionic acid toxicity
as well as in peripheral blood derived human CD4 + T cell
clones (El-Ansary et al., 2013; Hyland et al., 2000). The
remarkable beneficial effect of CAR reported in the present
study may be attributed to its antioxidant/free radical
scavenging abilities which have the major role in DNA
damage (Boldyrev et al., 1988). Also, CyA was previously
documented to allow spontaneous DNA repair in human
peripheral blood mononuclear cells (Ori et al., 2012).
The current work showed also marked increases in
apoptosis biomarkers (sFasL in serum, caspase-3 activity,
and Ca+2
in brain tissue) level-traumatized rats compared
with normal control animals. These results were in line with
data from previous studies (Niu et al., 2012; Sun et al., 2008).
The Fas ligand (FasL; also called the CD95 or APO-1 ligand),
a member of the growing TNF family, is synthesized as a type
II membrane protein that induces apoptosis by binding to its
receptor, Fas (also called CD95 or APO-1) in Fas-expressing
cells through activating caspases (Nagata, 1997). The mem-
brane-bound FasL (mFasL) can be cleaved by a metallopro-
teinase to become a soluble form (sFasL) as a cytokine
(Tanaka et al., 1996). When the cell surface molecule Fas is
triggered by its agonist Fas ligand, the result is apoptosis of
these cells and tissue destruction (Siegel et al., 2000). Upon
sFasL binding, to Fas expressed on cell surface allows
activation of caspases including caspase 3 leading to apop-
tosis after 48 h of binding (Li et al., 2004; Siegel et al., 2000).
Lenzlinger et al. (2002) reported that increasing sFas ligand
concentrations in serum of patients with severe TBI may be
correlated significantly with severity of brain injury. The Fas–
Fas ligand system may have a pivotal role in causing edema
and local tissue destruction in the brain after severe head
injury. Meanwhile, intracellular calcium (Ca2+
) is a key
element in maintaining physiological functions of nerve cells
(Verkhratsky et al., 1998). Alteration in cellular Ca2+
homeostasis is likely another key mechanism that contributes
to secondary neuronal damage and cell apoptosis in TBI
(Stoica Faden, 2010). During the process of TBI, neurons
and other brain cells are excited abnormally, following which
Ca2+
channels open, and release of Ca2+
from intracellular
stores, that lead to a sustained cellular Ca2+
overload.
Elevated intracellular calcium initiates also many cellular
pathways including the activation of proteases including
calpains, and caspases, nitric oxide synthase as well as DNA-
degrading endonucleases. The over-activation of these mol-
ecules can lead to mitochondrial dysfunction, oxidative stress
and overproduction of free radicals, diminished ATP produc-
tion, activation of cell death signaling pathways and ultim-
ately cell death. These large amounts of Ca2+
can rapidly
import into mitochondria (Sullivan et al., 2005; Walker
Tesco, 2013; Weber, 2012).
CAR and/or CyA administration in the current study
reduced apoptosis biomarkers level (FasL in serum, caspase-3
activity, and Ca+2
in brain tissue) in traumatized rat. These
results may indicate the antiapoptotic beneficial role of these
agents that may aid in decreasing the secondary traumatic
brain injury and improving neurological outcome.
Considerable studies have showed that CAR provides anti-
apoptotic role in the animal models of hypoxia-ischemia brain
damage and subarachnoid hemorrhage (SAH)-induced early
brain injury (EBI) through lowering expression of caspase-3
protein (Wang et al., 2013; Zhang et al., 2011, 2014). On the
contrary, CyA was reported to have anti-apoptotic activity in
human gingival fibroblasts through down-regulating mito-
chondrial transition pore, decreasing the level of both
cytochrome c and caspase-3 associated with mitochondria-
mediated apoptosis (Jung et al., 2008). CyA also inhibits CNS
mitochondrial dysfunction and prevents calcium efflux by
interfering with calcium release from mitochondria, which
leads to secondary cascade of events that ends with persistent
damage within the CNS (Sullivan et al., 1999).
The current investigation also demonstrated significant
increase in brain glucose level in traumatized rats compared
with control healthy rats. The excessive increase in brain
glucose may be due to its essential need for more energy
production in defending against brain damage. Some reports
stated that early after TBI, cerebral glucose utilization is
increased in response to release of ions and excitatory amino
acids, such as glutamate from injured cells (Faden et al.,
1989; Statler et al., 2003). It is also documented that increased
glucose uptake and its over utilization through hyperglyco-
lysis by brain after traumatic injury (Statler et al., 2003; Yang
et al., 1985). Treatment of traumatized rats with CAR and/or
CyA pronouncedly down-regulated the alteration in brain
glucose level. CAR and its combination with CyA were more
effective in regulating brain glucose level than CyA. The
reducing effect of these agents on brain glucose level may be
attributed to their beneficial effect in ameliorating brain
injury and/or having important role in energy production by
attenuating mitochondrial depression. CAR has been showed
to increase ATP production by activating oxidative phosphor-
ylation (Churchil et al., 1995), and increasing the liberation of
ATP in mammalian muscles during anoxic stress (Millar
et al., 1993). It also normalized adenylate energy charge in the
chronic infection (Soliman et al., 2001).
In conclusion, we demonstrated that administration of
CAR and/or CyA have a protective effect against the
oxidative injury following TBI in experimental rats. This
protective effect may be related to their beneficial effect in
maintaining brain homeostasis through reducing oxidative
stress, inflammatory mediators, oxidative DNA damage, and
apoptosis. These data suggest that carnosine may have
potential value in therapy of TBI especially if combined
with cyclosporine A.

Declaration of interest
This research was supported by a grant from the Research
Center of the Center for Female Scientific and Medical
Colleges, Deanship of Scientific Research, King Saud
University.
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