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2015; 58:7185
Doi:10.1111/jpi.12194
Introduction
Aging is a major factor involved in gradual decline of brain
function and has been implicated in progressive memory
loss, dementia, and cognitive disorders [1, 2]. Numerous
studies have shown a key role of mitochondria in aging
process. Reactive oxygen species (ROS) and oxidative
stress are known to be associated with several age-associated neuronal disorders such as Alzheimers disease (AD)
[3, 4]. The over accumulation of ROS and oxidative stress
triggers cellular lipids, proteins, or DNA damage which
disturbed normal cellular activity and deregulates homeostatic system of neuron, ultimately leading to neuronal cell
death [5]. Hence, to prevent oxidative stress-induced neuronal degeneration could be a potential neurotherapeutic
approach to treat the age-associated neurodegenerative
disease such as AD.
It is well known that injection of D-galactose is a model
for brain aging which induces and accelerates senescence
in rodents to develop AD like symptoms [6]. Elevated
Ali et al.
numerous other functions [15, 16]. Melatonin is amphiphilic in nature and acts as a potent free radical scavenger
and possesses antioxidant, anti-inammatory and antiapoptotic properties [1724]. Its role in protecting the
central nervous system from oxidative damage is well documented [25, 26]. Plasma melatonin levels are higher in
young people than that in old individuals [27]. The
reduced level of melatonin in aged person has been proposed one of the important factors in the development of
age-related neurodegenerative disorders such as AD [28
30]. Recently, Corrales et al. [31] showed that chronic melatonin treatment improved learning and memory in a
mouse model of brain deterioration. In this study, we
investigated the underlying neuroprotective mechanism of
melatonin against D-galactose-induced neurotoxicity. Our
results show that chronic melatonin treatment attenuates
D-galactose-induced memory impairment, synaptic dysfunction, ROS, oxidative stress, neuroinammation, and
neurodegeneration possibly through RAGE/NF-KB65/
JNK signaling pathway.
The MWM test is a well parameter task to evaluate memory functions; we performed MWM as described previously
with some modication [8]. The experimental apparatus
consisted of a circular water tank (100 cm in diameter,
40 cm in height), containing water (23 1C) to a depth of
15.5 cm, which was rendered opaque by adding white ink.
A transparent escape platform (10 cm in diameter, 20 cm in
height) was hidden 1 cm below the water surface and placed
at the midpoint of one quadrant. Each mouse received training per day for six consecutive days using a single hidden
platform in one quadrant with three quadrants of rotational
starting. Latency to escape from the water maze (nding the
submerged escape platform) was calculated for each trial.
On day seven, probe test was performed for the evaluation
of memory consolidation. The probe test was carried out by
removing platform and allowing each mouse to swim freely
for 60 s. The mice spent time in the target quadrant (where
the platform was located during hidden platform training)
was measured. Time spent in the target quadrant is considered to represent the degree of memory consolidation, taken
place after learning. All data were recorded using videotracking software (SMART, Panlab Harward Apparatus;
Bioscience Company, Holliston, MA, USA).
Chemicals
D-galactose, Melatonin, and 20 70 -dichlorodihydrouorescein diacetate (DCFH-DA) were purchased from Sigma
Chemical Co. (St. Louis, MO, USA).
Animals
Male wild type C57BL/6N mice (2530 g, 8 weeks old)
were purchased from Samtako Bio (Osan, Korea). The
mice were acclimatized for 1 week in the university animal house under a 12-h/12-h light/dark cycle at 23C
with 60 10% humidity and provided with food and
water ad libitum. All eorts were made to minimize the
number of mice used and their suering. The experimental procedures were approved through the animal ethics
committee of the Division of Applied Life Sciences,
Department of Biology at Gyeongsang National University, South Korea.
Drug treatment protocols
Mice were divided into the following groups: (i) control
(C) mice treated with saline as a vehicle for 2 months, (ii)
mice treated with D-galactose (D-gal) (100 mg/kg) for
2 months, (iii) mice treated with D-galactose (100 mg/kg)
for 2 months and melatonin 10 mg/kg for 30 days
(D-gal+M), and (iv) mice treated with melatonin 10 mg/kg
alone for 30 days (M).
Melatonin was rst dissolved in 0.1% dimethyl sulfoxide (DMSO) and then makes the nal administered volume in saline. Melatonin (10 mg/kg) or saline was
administered (i.p.) for 30 days each 12 hr before evening.
Morris water maze (MWM) test
The behavioral study was performed on mice (n = 15/
group) using MWM and Y-maze test.
72
Y-maze test
The Y-maze was made of black-painted wood. The each
arm of the maze was 50 cm long, 20 cm high and 10 cm
wide at the bottom and 10 cm wide at the top. Each
mouse was placed at the center of the apparatus and
allowed to move freely through the maze for three 8-min
sessions. The series of arm entries was visually observed.
Spontaneous alteration was dened as the successive entry
of the mice into the three arms in overlapping triplet sets.
Alteration behavior (%) was calculated as [successive triplet sets (entries into three dierent arms consecutively)/
total number of arm entries-2] 9 100.
Protein extraction from mouse brain
After behavioral analysis, the mice were killed. The brains
were immediately removed, and hippocampus and cortex
tissue were dissected carefully, frozen on dry ice, and
stored at 80C. The hippocampus tissue was homogenized in 0.2 M PBS with phosphatase inhibitor and protease inhibitor cocktail. The samples were then centrifuged
at 10,000 g at 4C for 25 min. The supernatants were collected and stored at 80C.
Western blot analysis
The protein concentration was measured (Bio-Rad protein
assay kit, Bio-Rad Laboratories, CA, USA). Equal
amounts of protein (1530 lg) were electrophoresis using
412% BoltTM Mini Gels (Novex; Life Technologies, Kiryat Shmona, Israel). The membranes were blocked in 5%
(w/v) skim milk to reduce nonspecic binding and incubated with primary antibodies overnight at 4C at a
1:1000 dilution. After reaction with a horseradish peroxidase-conjugated secondary antibody, as appropriate, the
proteins were detected using an ECL detection reagent
Ali et al.
Inc.). The density values were expressed as the
means SEM. One-way analysis of variance (ANOVA)
followed by a two-tailed independent Students t-test was
used for comparisons among the treated groups and the
control. The ImageJ software was used for immunohistological quantitative analysis. P values <0.05 (P < 0.05)
were considered to be statistically signicant.
Results
To investigate the eect of D-galactose and melatonin on
mice behavior and memory function, Morris water maze
(MWM) and Y-maze tasks were performed. MWM training tests were performed for six consecutive days and the
escape latencies (time to reach the hidden platform) was
recorded. Mice treated with D-galactose showed more
escape latency as compared to the vehicle-treated mice.
Melatonin treatment (10 mg/kg, i.p. for 30 days) to
D-galactose-treated mice showed less latency time than
that of D-galactose-treated mice (Fig. 1A).
After trial session on day 7, the hidden platform
removed and probe test was performed. The number of
platform crossings was signicantly increased by melatonin treatment in the D-galactose-treated mice compared to
the D-galactose-treated-alone mice (Fig. 1B). In addition,
melatonin-treated mice spent more time in the target
quadrant than that of D-galactose alone (Fig. 1C), showing that melatonin reduced D-galactose-induced memory
impairment.
Following the MWM test, we performed a Y-maze task to
analyze the spatial working memory using spontaneous
alteration behavior percentage (%). A higher percentage of
spontaneous alteration behavior was considered to be
enhanced cognitive performance. Measurements of the spontaneous alternation rate from the center of the maze, which
is the percentage of the total number of arm entries that can
(A)
(C)
74
(B)
(D)
Ali et al.
Inc.). The density values were expressed as the
means SEM. One-way analysis of variance (ANOVA)
followed by a two-tailed independent Students t-test was
used for comparisons among the treated groups and the
control. The ImageJ software was used for immunohistological quantitative analysis. P values <0.05 (P < 0.05)
were considered to be statistically signicant.
Results
To investigate the eect of D-galactose and melatonin on
mice behavior and memory function, Morris water maze
(MWM) and Y-maze tasks were performed. MWM training tests were performed for six consecutive days and the
escape latencies (time to reach the hidden platform) was
recorded. Mice treated with D-galactose showed more
escape latency as compared to the vehicle-treated mice.
Melatonin treatment (10 mg/kg, i.p. for 30 days) to
D-galactose-treated mice showed less latency time than
that of D-galactose-treated mice (Fig. 1A).
After trial session on day 7, the hidden platform
removed and probe test was performed. The number of
platform crossings was signicantly increased by melatonin treatment in the D-galactose-treated mice compared to
the D-galactose-treated-alone mice (Fig. 1B). In addition,
melatonin-treated mice spent more time in the target
quadrant than that of D-galactose alone (Fig. 1C), showing that melatonin reduced D-galactose-induced memory
impairment.
Following the MWM test, we performed a Y-maze task to
analyze the spatial working memory using spontaneous
alteration behavior percentage (%). A higher percentage of
spontaneous alteration behavior was considered to be
enhanced cognitive performance. Measurements of the spontaneous alternation rate from the center of the maze, which
is the percentage of the total number of arm entries that can
(A)
(C)
74
(B)
(D)
Ali et al.
(A)
(B)
IL-1b and TNFa analysis also determined that the immunouorescence reactivity of IL-1b (Fig. 6B) and TNFa
(Fig. 6C) signicantly increased in cortex and hippocampus of D-galactose-treated mice as compared to vehicletreated group. Melatonin treatment (10 mg/kg, i.p. for
30 days) signicantly reduced the immunouorescence
reactivity of the IL-1b and TNFa in cortex, CA1 and CA3
region of hippocampus in the D-galactose-treated mice
compared to that of D-galactose-treated-alone mice
(Fig. 6B and C).
Diverse research data demonstrated that phospho-c-Jun
N-terminal Kinase 1 [p-JNK1] (T183/Y185) known as
stress-activated protein kinase (SAPK) overexpressed in
the oxidative stress condition and involved in the mediation of apoptotic signaling [41, 42]. Therefore, we analyzed
the p-JNK level through Western blot and immunouorescence analysis. Earlier study reported activated p-JNK in
the D-galactose mouse model [43]. Our Western blot
results showed that D-galactose-treated mice also indicated overexpressed p-JNK, while melatonin treatment
(10 mg/kg, i.p. for 30 days) signicantly reduces p-JNK
level compared to the D-galactose-treated mice (Fig. 7A).
76
(B)
Fig. 4. Melatonin
reduces
the
microgliosis, astrocytosis, and RAGE
expression in the D-galactose-treated
mice. (A) The Western blot analysis of
RAGE, GFAP, and Iba-1 in the
hippocampus of mice. The bands were
quantied using Sigma Gel software, and
the dierences are represented by a
histogram. b-Actin was used as a loading
control. The density values are expressed
in arbitrary units (A.U) as the
means SEM
for
the
respective
indicated protein (N = 10 mice/group).
(B) Representative images showing
immunouorescence analysis of the
GFAP.
The
D-galactose-treated
increased the GFAP indicating activated
astrocytes in CA1, DG, and CA3 regions
of
hippocampus.
Treatment
with
melatonin ameliorated the D-galactose
eects and signicantly decreased the
immunoreactivity
of
GFAP.
Magnication 409, Scale bar = 50 lm.
(C) Representative images showing
immunouorescence analysis of the
GFAP in the cortex. Magnication 40 X,
Scale bar = 50 lm. * signicantly
dierent from the vehicle treated; #
signicantly dierent from D-galactose
treated.
Signicance = ***P < 0.01,
###P < 0.001.
(C)
region of the hippocampus as compared to the vehicletreated group. Melatonin treatment signicantly reduced
the immunoreactivity of caspase-3 in the DG, CA1, and
CA3 region of the hippocampus, showing a decreased
number of active caspase-3-positive cells in the melatonin
plus D-galactose-treated group as compared to the
D-galactose-treated-alone group (Fig. 8B). Next, we examined the level of cleaved poly (ADP-ribose) polymerase-1
(PARP-1). Cleaved PARP-1 is responsible for DNA
damage which leads to neurodegeneration [45]. Our Western blot results indicated increased cleaved PARP-1 level
in the D-galactose-treated mice while treatment with melatonin signicantly reduced cleaved PARP-1 level in the
D-galactose-treated mice as compared to D-galactose-treated-alone mice (Fig. 8A). FJB is one of the key markers
for the indication of neurodegeneration [46]. In the
D-galactose-treated group, FJB-positive neuronal cells
were signicantly increased in the DG and CA3 region of
77
Ali et al.
(A)
(B)
IL-1b and TNFa analysis also determined that the immunouorescence reactivity of IL-1b (Fig. 6B) and TNFa
(Fig. 6C) signicantly increased in cortex and hippocampus of D-galactose-treated mice as compared to vehicletreated group. Melatonin treatment (10 mg/kg, i.p. for
30 days) signicantly reduced the immunouorescence
reactivity of the IL-1b and TNFa in cortex, CA1 and CA3
region of hippocampus in the D-galactose-treated mice
compared to that of D-galactose-treated-alone mice
(Fig. 6B and C).
Diverse research data demonstrated that phospho-c-Jun
N-terminal Kinase 1 [p-JNK1] (T183/Y185) known as
stress-activated protein kinase (SAPK) overexpressed in
the oxidative stress condition and involved in the mediation of apoptotic signaling [41, 42]. Therefore, we analyzed
the p-JNK level through Western blot and immunouorescence analysis. Earlier study reported activated p-JNK in
the D-galactose mouse model [43]. Our Western blot
results showed that D-galactose-treated mice also indicated overexpressed p-JNK, while melatonin treatment
(10 mg/kg, i.p. for 30 days) signicantly reduces p-JNK
level compared to the D-galactose-treated mice (Fig. 7A).
76
hippocampus as compared to vehicle-treated group. Melatonin treatment signicantly reduced the neurodegeneration in the DG and CA3 region of hippocampus, showing
a decreased FJB-positive neurons number in the melatonin-treated group as compared to the D-galactosetreatedalone group (Fig. 8C). Next, Nissl staining was used to
observe the extent of neuronal cell death induced by Dgalactose treatment and to examine the neuroprotection
produced through melatonin treatment in the cortex and
hippocampus of D-galactose-treated mice. The number of
survival neurons in the cortex and CA1 and CA3 regions
of hippocampus was reduced in D-galactose-treated mice
compared with vehicle-treated mice. After melatonin treatment, the number of survival neurons signicantly
increased in the cortex and CA1 and CA3 regions of hippocampus in the D-galactose plus melatonin-treated mice
as compared with the D-galactose treatment alone
(Fig. 8D). These Western blot and immunohistochemical
results indicated that melatonin is eective in preventing
apoptosis and neurodegeneration in D-galactose-treated
mice (Figs 7AC and 8AD).
Discussion
Here in, we investigated the ability of melatonin to prevent
the D-galactose-induced memory impairment, synaptic
dysfunction, ROS, oxidative stress, neuroinammation,
and neurodegeneration through RAGE/NF-KB/JNK
pathway. Excessive formation and accumulation of ROS
has been considered a key mediator to induce neuroinammation and neuronal degeneration in various ageassociated neurodegenerative diseases such as AD and
Parkinson disease [3, 4, 47].
Chronic D-galactose administration activates and formation of ROS and has strong anity for free amines of
amino acid in proteins and peptides; and triggers AGEs
accumulation which bind with their receptor RAGE in
vivo, ultimately leading to oxidative stress, resulting neuroinammation, neurodegeneration, and memory impairment [79, 11, 12, 48].
Overexpressed RAGE activates NF-KB, results in the
activation of other inammatory mediators and glial activation, which results in progressive age-related diseases such
as type II diabetes, atherosclerosis, and other chronic neurodegenerative diseases including AD and Parkinson disease
[49, 50]. Elevated NF-KB has been reported in old age [51].
Similarly Terai et al. [52] reported that NF-KB has been
found in neurons, neurobrillary tangles in the brain of
patient with AD after postmortem. Hence, RAGE/NF-KB
signaling pathway is an emerging pathway to study the
progression and treatment of various age-related disease.
Previously it has been established that chronic administration of D-galactose increased ROS level and oxidative
Ali et al.
(A)
(B)
(C)
80
Ali et al.
(A)
(B)
78
Ali et al.
(A)
(B)
(C)
(D)
Fig. 8. Melatonin decreases apoptotic markers and neurodegeneration in the D-galactose-treated mice. (A) Western blot analysis of
mouse hippocampus using Cyt.C, caspase-9, activated caspase-3, and cleaved PARP-1 antibodies. The bands were quantied using Sigma
Gel software, and the dierences are represented by a histogram. Anti-b-actin was used as a loading control. The density values are
expressed in arbitrary units (A.U) as the means SEM for the respective indicated hippocampus proteins (N = 10 mice/group). (B)
Immunoreactive cells of activated caspase-3 antibody were examined in the DG, CA3, and CA1 regions of the hippocampus of the
D-galactose-treated group. Caspase-3-positive cells were increased in the D-galactose-treated mice compared with the control. Treatment
with melatonin signicantly decreased the D-galactose-induced number of caspase-3-positive cells. Scale bar = 200 lm. (C) Representative
images of FJB staining show that neurodegeneration are induced in the D-galactose-treated mice. FJB-stained apoptotic neurons are
shown in the CA3 and DG regions of the hippocampus. The decreased FJB-positive cells indicate that melatonin treatment (10 mg/kg,
i.p. for 30 days) eectively reduced apoptosis in the hippocampus of D-galactose-treated mice. The images are representative of the staining observed in each section (N = 5 animals/group). Magnication 409. Scale bar = 100 lm. FJB-positive cells in the dierent regions of
each section were analyzed using the computer-based Image J program. (D) Representative photomicrograph of Nissl staining in the DG,
CA3, and CA1 regions of the hippocampus and cortex of the D-galactose-treated group. Neuronal cell death increased after injection of
D-galactose in all regions. Melatonin treatment signicantly decreased neuronal cell death. The results were analyzed using the computerbased Image J program Scale bar = 200 lm.* signicantly dierent from the vehicle treated; # signicantly dierent from D-galactose.
Signicance = **P < 0.01, ***P < 0.001, ##P < 0.01, ###P < 0.001.
82
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Ali et al.
Prof. Myeong Ok Kim for her kind and expert supervision, as she is the corresponding author and holds all the
responsibilities related to this manuscript.
References
1. RAZ N, GHISLETTA P, RODRIGUE KM et al. Trajectories of
brain aging in middle-aged and older adults: regional and
individual dierences. NeuroImage 2010; 51:501511.
2. MORRISON JH, HOF PR. Life and death of neurons in the
aging brain. Science 1997; 278:412419.
3. OLANOW CW. A radical hypothesis for neurodegeneration.
Trends Neurosci 1993; 16:439444.
4. CASTEGNA A, AKSENOV M, AKSENOVA M et al. Proteomic
identication of oxidatively modied proteins in Alzheimers
disease brain. Part I: creatine kinase BB, glutamine synthase,
and ubiquitin carboxy-terminal hydrolase L-1. Free Radic
Biol Med 2002; 33:562571.
5. PARADIES G, PETROSILLO G, PARADIES V et al. Mitochondrial
dysfunction in brain aging: role of oxidative stress and cardiolipin. Neurochem Int 2011; 58:447457.
6. WEI HF, LI L, SONG QJ et al. Behavioral study of the dgalactose induced aging model in C57BL/6J mice. Behav
Brain Res 2005; 157:245251.
7. LU J, ZHENG YL, WU DM et al. Ursolic acid ameliorates cognition decits and attenuates oxidative damage in the brain of
senescent mice induced by d-galactose. Biochem Pharmacol
2007; 74:10781090.
8. LU J, WU D, ZHENG Y et al. Purple sweet potato color alleviates D-galactose-induced brain aging in old mice by promoting
survival of neurons via PI3K pathway and inhibiting cytochrome C-mediated apoptosis. Brain Pathol 2009; 20:598612.
9. LU J, ZHENG YL, LUO L et al. Quercetin reverses d-galactose
induced neurotoxicity in mouse brain. Behav Brain Res 2006;
171:251260.
10. TIAN J, ISHIBASHI K, REISER K et al. Advanced glycation endproduct-induced aging of the retinal pigment epithelium and
choroid: a comprehensive transcriptional response. Proc Natl
Acad Sci USA 2005; 102:1184611851.
11. LEI M, HUA X, XIAO M et al. Impairments of astrocytes are
involved in the d-galactose-induced brain aging. Biochem
Biophys Res Commun 2008; 369:10821087.
12. WU DM, LU J, ZHENG YL et al. Purple sweet potato color
repairs d-galactose-induced spatial learning and memory
impairment by regulating the expression of synaptic proteins.
Neurobiol Learn Mem 2008; 90:1927.
13. LU J, WU DM, ZHENG YL et al. Ursolic acid attenuates DGalactose-induced inammatory response in mouse prefrontal cortex through inhibiting AGEs/RAGE/NF-kB pathway
activation. Cereb Cortex 2010; 20:25402548.
14. MALLIDIS C, AGBAJE I, ROGERS D et al. Distribution of the
receptor for advanced glycation end products in the human
male reproductive tract: prevalence in men with diabetes mellitus. Hum Reprod 2007; 22:21692177.
15. CHEN LD, MANCHESTER LC, POEGGELER B et al. Melatonin: a
potent endogenous hydroxyl radical scavenger. Endocr J
1993; 1:5760.
16. HARDELAND R. Melatonin and the theories of aging: a critical
appraisal of melatonins role in antiaging mechanisms. J
Pineal Res 2013; 55:325356.
17. RODRIGUEZ C, MAYO JC, SAINZ RM et al. Regulation of antioxidant enzymes: a signicant role for melatonin. J Pineal
Res 2004; 36:19.
84
18. GALANO A, TAN DX, REITER RJ. On the free radical scavenging activities of melatonins metabolites, AFMK and AMK. J
Pineal Res 2013; 54:2452557.
19. ZHANG HM, ZHANG Y. Melatonin: a well-documented antioxidant with conditional pro-oxidant actions. J Pineal Res 2014;
57:131146.
20. MAURIZ JL, CALLADO PS, VENEROSO C et al. A review of the
molecular aspects of melatonins anti-inammatory actions:
recent insights and new perspectives. J Pineal Res 2013; 54:1
14.
21. JOU MJ, PENG TI, HSU LF et al. Visualization of melatonins
multiple mitochondrial levels of protection against mitochondrial Ca+-mediated permeability transition and beyond in rat
brain astrocytes. J Pineal Res 2010; 48:2038.
22. DAS A, MCDOWELL M, PAVA MJ et al. The inhibition of
apoptosis by melatonin in VSC4.1 motoneurons exposed to
oxidative stress, glutamate excitotoxicity, or TNF-a toxicity
involves membrane melatonin receptors. J Pineal Res 2010;
48:157169.
23. WANG Z, WU L, YOU W et al. Melatonin alleviates secondary
brain damage and neurobehavioral dysfunction after experimental subarachnoid hemorrhage: possible involvement of
TLR4-mediated inammatory pathway. J Pineal Res 2013;
55:399408.
24. MARTIN V, SANCHEZ-SANCHEZ AM, PUENTE-MONCADA N et al.
Involvement of autophagy in melatonin-induced cytotoxicity
in glioma-initiating cells. J Pineal Res 2014; 57:308316.
25. GARCIA JJ, LOPEZ-PINGARRON L, ALMEIDA-SOUZA P et al. Protective eects of melatonin in reducing oxidative stress and in
preserving the uidity of biological membranes: a review. J
Pineal Res 2014; 56:225237.
26. REITETER RJ, TAN DX, LEON J et al. When melatonin gets on
your nerves: its benecial actions in experimental models of
stroke. Exp Biol Med (Maywood) 2005; 230:104117.
27. PARADIES G, PETROSILLO G, PARADIES V et al. Melatonin, cardiolipin and mitochondrial bioenergetics in health and disease. J Pineal Res 2010; 48:297310.
28. WIECHMANN AF, SHERRY DM. Role of melatonin and its
receptors in the in the vertebrate retina. Int Rev Cell Mol
Biol 2013; 300:211242.
29. REITER RJ. The pineal gland and melatonin in relation to
aging: a summary of the theories and of the data. Exp Gerontol 1995; 30:199212.
30. ROSALES-CORRAL SA, ACUNA-CASTROVIEJO D, COTO-MONTES A
et al. Alzheimers disease: pathological mechanisms and the benecial actions of melatonin. J Pineal Res 2012; 52:167202.
31. CORRALES A, MARTINES P, GARCIA S et al. Long-term oral
administration of melatonin improves spatial learning and
memory and protects against cholinergic degeneration in middle-aged Ts65Dn mice, a model of Down syndrome. J Pineal
Res 2013; 54:346358.
32. SHAH SA, LEE HY, BRESSAN RA et al. Novel osmotin attenuates glutamate-induced synaptic dysfunction and neurodegeneration via the JNK/PI3K/Akt pathway in postnatal rat
brain. Cell Death Dis 2014; 5:e1026.
33. SHAH SA, YOON GH, KIM MO. Protection of the developing
brain with anthocyanins against ethanol-induced oxidative
stress and neurodegeneration. Mol Neurobiol 2014; doi:10.
1007/s12035-014-8805-7.
34. LIDA T, FURUTA A, NISHIOKA K et al. Expression of 8-oxoguanine DNA glycosylase is reduced and associated with neurobrillary tangles in Alzheimers disease brain. Acta
Neuropathol 2002; 103:2025.
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