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European Journal of Pharmacology 723 (2014) 330–338
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Behavioural pharmacology
Pyrrolidine dithiocarbamate protects against scopolamine-induced
cognitive impairment in rats
Mai A. Abd-El-Fattah, Noha F. Abdelakader, Hala F. Zaki n
Cairo University, Pharmacology & Toxicology Department, Faculty of Pharmacy, Egypt
art ic l e i nf o
a b s t r a c t
Article history:
Received 10 July 2013
Received in revised form
12 November 2013
Accepted 15 November 2013
Available online 3 December 2013
Alzheimer's disease (AD) is a chronic neurodegenerative disorder that leads to disturbances of cognitive
functions. Although the primary cause of AD remains unclear, brain acetylcholine deﬁciency, oxidative
stress and neuroinﬂammation may be considered the principal pathogenic factors. The present study was
constructed to investigate the anti-amnestic effect of pyrrolidine dithiocarbamate (PDTC) on
scopolamine-induced behavioral, neurochemical and biochemical changes in rats. PDTC (50 and
100 mg/kg) and donepezil (2.5 mg/kg) were orally administered for 14 successive days. Dementia was
induced at the end of the treatment period by a single injection of scopolamine (20 mg/kg; i.p.), and
Y-maze test was conducted 30 min thereafter. Rats were then sacriﬁced and homogenates of cortical and
hippocampal tissues were used for the estimation of noradrenaline, dopamine, serotonin and heat shock
protein 70 contents along with acetylcholinesterase activity. In addition, certain oxidative stress markers,
pro-inﬂammatory and anti-inﬂammatory cytokines were assessed. Histological examination of cortical
and hippocampal tissues was also performed. Scopolamine resulted in memory impairment that was
coupled by alterations in the estimated neurotransmitters, heat shock protein 70, acetylcholinesterase
activity, oxidative stress as well as inﬂammatory biomarkers. Histological analysis revealed serious
damaging effects of scopolamine on the structure of cerebral cortex and hippocampus. Pretreatment of
rats with PDTC in both doses mitigated scopolamine-induced behavioral, biochemical, neurochemical
and histological changes in a manner comparable to donepezil. The observed anti-amnestic effect of
PDTC makes it a promising candidate for clinical trials in patients with cognitive impairment.
& 2013 Elsevier B.V. All rights reserved.
Keywords:
Dementia
Pyrrolidine dithiocarbamate
Acetylcholineesterase
Oxidative stress
Neuroinﬂammation
Heat shock protein 70
1. Introduction
Incidence of dementia has alarmingly increased nowadays
(McCarty, 2006). Alzheimer's disease (AD), the most common
form of senile dementia, is characterized by memory loss accompanied by degeneration of basal forebrain cortical cholinergic
neurons (Heo et al., 2004; Korczyn and Vakhapova, 2007). The
pathogenesis of AD is largely unknown but a number of hypotheses have been proposed including abnormal phosphorylation of
the protein tau, altered calcium homeostasis, oxidative stress,
production of inﬂammatory cytokines, deﬁcits in energy metabolism, and altered protein processing resulting in abnormal
β-amyloid peptide (Aβ) accumulation (Butterﬁeld et al., 2002;
Hardy and Selkoe, 2002; Huang et al., 2003; Zhu et al., 2004;
Ferreira et al., 2006).
n
Correspondence to: Cairo University, Pharmacology & Toxicology Department,
Faculty of Pharmacy, Kasr El-Eini Street, Cairo 11562, Egypt. Tel.: þ2 22 450 3383;
fax: þ20 224 073 411.
E-mail addresses: [email protected], [email protected] (H.F. Zaki).
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejphar.2013.11.008
The cholinergic system plays an important role in learning and
memory (Ellis, 2005). Scopolamine, an anticholinergic drug causes
amnesia in humans and animals (Ogura et al., 2000). Loss of
cholinergic neurons and subsequent deﬁcits in cholinergic neurotransmission in the hippocampus and cerebral cortex are strongly
correlated with clinical signs of cognitive impairment and dementia in AD patients (Mesulam, 2004). Hence, scopolamine-induced
amnesia is widely cited as a model simulating human dementia in
general and AD in particular (Joshi and Parle, 2006).
Treatment of dementia of Alzheimer type is based upon the
neurotransmitter replacement approach. Current medications
approved by Food and Drug Administration include acetylcholinesterase (AchE) inhibitors as donepezil, for mild to moderate
cases, and memantine, an N-methyl-D-aspartate (NMDA) receptor
antagonist, for the treatment of moderate to severe Alzheimer
dementia. All of these drugs seem to be able to produce modest
symptomatic improvements in some of the patients (Scarpini
et al., 2003; Cummings, 2004).
The dithiocarbamates are a class of metal chelating antioxidants reported to be potent inhibitors of nuclear factor kappa B
(NF-κB), a transcription factor regulating expression of pro-
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inﬂammatory and pro-apoptotic genes, both in vivo and in vitro
(Schreck et al., 1992; Cuzzocrea et al., 2002). The most potent
member of this class is pyrrolidine dithiocarbamate (PDTC) which
enhances cellular defense mechanisms via upregulation of cytoprotective genes, including heat shock protein 70 (HSP70) and
endogenous antioxidants such as superoxide dismutase and
reduced glutathione (GSH) (Borrello and Demple, 1997; Wild and
Mulcahy, 1999; Kim et al., 2001). Moreover, PDTC possesses metal
chelating activity (Iseki et al., 2000) and reduces the transcription
of numerous pro-inﬂammatory mediators, such as tumor necrosis
factor-alpha (TNF-α), interleukin-1beta (IL-1β) and inducible nitric
oxide synthase (Ziegler-Heitbrock et al., 1993; Satriano and
Schlondorff, 1994) via inhibition of NF-κB (Schreck et al., 1992; Li
et al., 1999).
Although PDTC has been shown to improve learning and
memory functions of the transgenic APP/PS1 mice model for AD
(Malm et al., 2007), yet its effects on the role of oxidative stress
and neuroinﬂammation in AD has not been fully elucidated. The
present study was performed to investigate the possible modulatory effect of PDTC in scopolamine-induced dementia and to
compare such effects with donepezil, a commonly used agent in
dementia and AD. The study design included evaluated effects of
PDTC on some of the underlying mechanisms involved in AD
progression.
2. Material and methods
2.1. Animals
Adult male albino Wistar rats, weighing 150–200 g each, were
used in the present study. Animals were allocated in groups and
were allowed to accommodate for one week in the animal house
at Faculty of Pharmacy, Cairo University, before subjecting them to
experimentation. They were provided with a standard pellet diet
and were given water ad libitum. The animals were kept at
a temperature of 22 73 1C and a 12 h light/dark cycle as well as
a constant relative humidity throughout the experimental period.
The study was approved by the Ethics Committee for Animal
Experimentation at Faculty of Pharmacy, Cairo University, Egypt.
2.2. Drugs and chemicals
Scopolamine hydrobromide was purchased from Sigma-Aldrich
(MO, USA). It was dissolved in saline and i.p. administered in a
dose of 20 mg/kg (Casas et al., 1999). PDTC was purchased from
Sigma-Aldrich (MO, USA). It was suspended in 1% tween 80 and
administered p.o. in doses of 50 and 100 mg/kg (Pfeilschifter et al.,
2010). Donepezil hydrochloride was purchased from Pﬁzer (Cairo,
Egypt). It was dissolved in saline and administered p.o. in a dose of
2.5 mg/kg (Kosasa et al., 1999). Reagent kits for IL-1β, interleukin10 (IL-10) and HSP70 were purchased from Invitrogen (California,
USA), Bender MedSystems (Vienna, Austria), and R&D Systems
Chemical (MN, USA), respectively. Kit for AchE was purchased
from Quimica Clinica Aplicada (S.A., Spain).
2.3. Experimental design
Animals were classiﬁed into two sets, each of ﬁve groups (10
rats each). The treatment period for animals was 14 days, and at
the end of the treatment period (1 h after the last dose of test
agents), all animals were i.p. injected with scopolamine hydrobromide (20 mg/kg) except the ﬁrst group of each set, which
served as a normal group. Group II animals served as control
dementia. Animals in groups I and II received 1% tween 80 (p.o.).
Group III was daily treated with donepezil hydrochloride (2.5 mg/
331
kg, p.o.), while rats of groups IV and V were treated daily with
PDTC ammonium salt at two dose levels (50 and 100 mg/kg, p.o.).
The Y-maze spontaneous alternation test was conducted 30 min
after scopolamine injection. Immediately after performing the
behavioral test, rats were sacriﬁced by decapitation; thereafter,
in the ﬁrst set, both cortical and hippocampal tissues were
homogenized in 75% (v/v) aqueous methanol (HPLC grade) for
the evaluation of monoamines. In the second set of experimental
animals, cortical and hippocampal tissues were homogenized in
ice-cold 50 mM phosphate buffer (pH ¼ 7.4) for the estimation of
thiobarbituric acid reactive substances (TBARS), GSH, IL-1β, IL-10
and HSP70 contents as well as AchE activity. Finally, brains of 2–3
rats from each group were preserved in 10% formalin and kept for
histopathologic examination.
2.4. Y-maze spontaneous alternation test
The Y-maze test was performed as described by Wall and
Messier (2002). The maze was made of 3 identical arms, 40 cm
long, 35 cm high and 12 cm wide, positioned at equal angles and
labeled A, B, and C. Rats were placed at the end of one arm and
allowed to move freely through the maze during a 5 min session.
Spontaneous alternation was examined by visually recording the
pattern of entrance into each arm in the maze for each rat. Arm
entry was considered to be complete when the hind paws of the
rat were completely placed in the arm. Alternation was deﬁned as
successive entries into the three arms on overlapping triplet set
(i.e., ABC, BCA…). The spontaneous alternation performance score
(SAP), spontaneous alternation percentage (SAP%) and total arm
entries (TAE) were calculated.
2.5. Estimation of brain dopamine, noradrenaline and serotonin
contents
Dopamine, noradrenaline and serotonin contents were estimated in cortex and hippocampus according to the method of
Pagel et al. (2000) using a fully automated high pressure liquid
chromatography system (HPLC; Perkin-Elmer, MA, USA). In brief,
homogenates were spun at 4000 g, for 20 min and supernatants
were immediately extracted from trace elements and lipids by the
use of solid phase extraction Chromabond column NH2 phase
(Chromabond, USA). Monoamine standards or samples (20 μl)
were injected directly into the AQUA column (150 4.6 mm 5 μ
C18) from Phenomenex [CA, USA] and a mobile phase composed of
97:3 mixture (v) of methanol: acetonitrile was used. A constant
ﬂow rate of 1.5 ml/min was maintained throughout the experiment that lasted for 12 min and the absorbance of monoamines
was measured at 270 nm.
2.6. Estimation of brain lipid peroxides content
The thiobarbituric acid reaction of Mihara and Uchiyama (1978)
was adopted for estimation of lipid peroxides level. To cortical and
hippocampal homogenates orthophosphoric acid (1%) and thiobarbituric acid (0.6%) were added, mixtures were boiled for 45 min
at 100 1C, then cooled. The colored product was extracted by
n-butanol, vortexed and centrifuged at 3000 g for 15 min. The
absorbance of the organic layer was read at 535 and 520 nm and
the difference in absorbance was calculated as lipid peroxides level
expressed as TBARS (nmol/g wet tissue).
2.7. Estimation of brain reduced glutathione content
The method for the assessment of GSH (mg/g wet tissue) in the
cortex and hippocampus was based on that of Beutler et al. (1963).
Homogenates were deproteinized with 5-sulfuosalicylic acid (10%) for
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30 min at 4 1C then centrifuged at 3000 g for 15 min at 4 1C. An
aliquot of the acid soluble supernatant was diluted with phosphate
buffer (0.3 M, pH¼7.7) and 5,5′-dithiobis-2-nitrobenzoic acid (1 mM)
was added to the samples, where its optical density was determined at
412 nm.
non-parametric one way ANOVA followed by Dunn's multiple
comparisons test. A probability level of less than 0.05 was
accepted as being signiﬁcant in all types of statistical tests.
3. Results
2.8. Estimation of brain contents of interleukin-1beta, interleukin-10,
and heat shock protein 70 as well as acetylcholinesterase activity
Cortical and hippocampal IL-1β, IL-10 and HSP 70 were
assessed using ELISA kits and results were expressed as pg/g wet
tissue. AchE activity (U/g wet tissue) was measured according to
the method of den Blaauwen et al. (1983) using a colorimetric
reagent kit. All the procedures of the used kits were performed
following the manufacturer's instruction manual.
2.9. Histological examination of cortical and hippocampal tissues
Histological assessment was performed on the brains of 2–3
rats randomly selected from each group. Brains were immediately
ﬁxed in 10% phosphate buffered formaldehyde, subsequently
embedded in parafﬁn, and 5 mm longitudinal sections were performed. The sections were stained with hematoxylin and eosin (H
and E) and examined microscopically.
2.10. Statistical analysis
Data were expressed as means 7 SEM. Comparisons between
means were carried out using one way analysis of variance
(ANOVA) followed by least signiﬁcant difference (LSD) test. Results
of behavioral experiments were analyzed using Kruskal–Wallis
Administration of scopolamine (20 mg/kg) as a single i.p.
injection resulted in a signiﬁcant reduction in SAP% by 38.11% as
compared to normal group. On the other hand, no signiﬁcant
changes were noted on TAE and SAP (Fig. 1A and B); accordingly
scopolamine did not affect locomotor activity of rats. Pretreatments with donepezil and PDTC (50 and 100 mg/kg) increased
SAP%, while no signiﬁcant change was observed in any of the other
tested parameters as compared to control dementia, except for
PDTC (50 mg/kg) which resulted in a signiﬁcant increase in TAE as
compared to normal group (Fig. 1A–C).
Rats injected with scopolamine displayed a signiﬁcant decrease
in the cortical dopamine content by 53.64% as compared with the
normal group (Table 1). Treatment with donepezil and PDTC (50
and 100 mg/kg) for 14 days signiﬁcantly increased cortical dopamine content as compared with scopolamine-induced dementia
group. On the other hand, no signiﬁcant changes were elicited in
both noradrenaline and serotonin cortical contents and in hippocampal catecholamine contents in all treated groups compared to
normal group (Tables 1 and 2).
Administration of scopolamine signiﬁcantly increased both
cortical and hippocampal TBARS content by 86.39% and 19%,
respectively as compared to that of the normal group. Administration
of donepezil and PDTC (50 and 100 mg/kg) protected against
scopolamine-induced increase in lipid peroxidation (Fig. 2A and B).
Rats that received scopolamine showed signiﬁcant increase in both
Fig. 1. Effect of pyrrolidine dithiocarbamate (PDTC) on Y-maze behavioral test in scopolamine-induced demented rats. Each bar with vertical line represents the mean
percentage of (A) spontaneous alternation percentage (SAP%), (B) total arm entries (TAE) and (C) spontaneous alteration performance (SAP) for each group (8 rats)7 SEM.
n
P o0.05 vs. normal, @P o 0.05 vs. scopolamine; using One-Way ANOVA followed by Dunn's multiple comparisons test.
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cortical and hippocampal GSH content by 35.6% and 23.1%, respectively as compared to that of the normal group. Pretreatment of rats
with donepezil and PDTC in the dose of 50 mg/kg protected against
scopolamine-induced elevation of GSH content. On the other hand,
pretreatment with PDTC (100 mg/kg) resulted in a signiﬁcant
decrease in cortical GSH content, while failed to produce any
signiﬁcant change in hippocampal GSH content (Fig. 3A and B).
In the present experiment, scopolamine administration increased
cortical and hippocampal cholinesterase enzyme activity by 48.39%
Table 1
Effect of pyrrolidine dithiocarbamate administration on cortical monoamines
concentrations.
Groups
Normal
Scopolamine
Donepezil
PDTC 50
PDTC 100
Parameters
Dopamine
(μg/g tissue)
Noradrenaline
(μg/g tissue)
Serotonin
(μg/g tissue)
0.4667 0.017
0.2167 0.018a
0.5357 0.045b
0.6977 0.038a,b,c
0.698 7 0.055a,b,c
1.187 0.02
1.067 0.03
1.187 0.10
1.09 7 0.04
1.017 0.01
0.094 7 0.004
0.085 7 0.004
0.1007 0.012
0.087 7 0.004
0.098 7 0.003
Data are expressed as mean7 SEM of 8 animals.
a
P o0.05 vs. normal, using One-Way ANOVA followed by LSD multiple
comparisons test.
b
Po 0.05 vs. scopolamine, using One-Way ANOVA followed by LSD multiple
comparisons test.
c
Po 0.05 vs. donepezil, using One-Way ANOVA followed by LSD multiple
comparisons test.
Table 2
Effect of pyrrolidine dithiocarbamate administration on hippocampal monoamines
concentrations.
Groups
Normal
Scopolamine
Donepezil
PDTC 50
PDTC 100
Parameters
Dopamine
(μg/g tissue)
Noradrenaline
(μg/g tissue)
Serotonin
(μg/g tissue)
0.3367 0.009
0.3337 0.020
0.3197 0.017
0.303 7 0.041
0.3197 0.011
1.25 70.08
1.29 70.08
1.16 70.07
1.11 70.07
1.22 70.06
0.084 7 0.007
0. 085 7 0.006
0.0777 0.007
0.0697 0.005
0.0697 0.005
Data are expressed as mean 7 SEM of 8 animals. Statistical analysis were performed
using One-Way ANOVA followed by LSD multiple comparisons test.
333
and 70.73%, respectively as compared to normal group (Fig. 4A and B).
Elevation of cortical and hippocampal cholinesterase activity was
normalized by all of the used agents (Fig. 4A and B).
In the current investigation, administration of scopolamine
resulted in 36.2% and 30.68% decrease in both cortical and hippocampal IL-10 content, respectively as compared to the normal group
(Fig. 5A and B). Treatments with donepezil and PDTC (50 mg/kg)
provided signiﬁcant protection against scopolamine-induced IL-10
depletion. However, treatment with PDTC (100 mg/kg) increased
cortical IL-10 as compared to control dementia and reduced
hippocampal IL-10 content as compared with the normal group
(Fig. 5A and B). Scopolamine resulted in a signiﬁcant increase in
both cortical and hippocampal IL-1β content by 98.72% and 54.83%,
respectively as compared with the normal group. Treatments with
donepezil and PDTC (50 and 100 mg/kg) prevented such increase in
cortical and hippocampal IL-1β content (Fig. 6A and B).
Administration of scopolamine signiﬁcantly increased both cortical and hippocampal HSP 70 content by 20.86% and 37%, respectively as compared with the normal group. Treatment of rats with
donepezil signiﬁcantly decreased scopolamine-induced elevation in
cortical HSP 70 content as compared with control dementia.
Treatment with PDTC (50 mg/kg) prevented scopolamine-induced
increase in cortical and hippocampal HSP 70 content by 27.3% and
34.71%, respectively as compared to control dementia, while treatment with 100 mg/kg decreased cortical HSP 70 by 26.88% and did
not signiﬁcantly affect hippocampal HSP 70 content, when compared to control dementia (Fig. 7A and B).
Histological examination of stained brain sections revealed
serious damaging effects of scopolamine on brain tissues
(Figs. 8B and 9B) compared to normal brain sections (Figs. 8A
and 9A). Scopolamine-induced brain damage consisted of focal
gliosis in cerebral cortex; in addition, the hippocampus showed
spongiosis associated with edema and congestion in the blood
vessels at the brain ﬁssure (Figs. 8B and 9B). Spongiosis was
diagnosed based on the appearance of vacuoles in the brain
matrix. Gliosis results from activation of glial cells following
induction of dementia. The perivascular edema in scopolamineinduced dementia group appears to be associated with thickening
in the endothelium resulting in increased ﬂuid permeability
(inﬁltration) outside the vascular wall. Administration of donepezil
provided partial protection against scopolamine-induced brain
injury by preventing blood vessels congestion, while showing only
edema in the hippocampus and focal gliosis in cerebral cortex
(Figs. 8C and 9C). Pretreatment with PDTC (50 mg/kg) showed
edema and congestion in the blood vessels associated with focal
spongiosis in the hippocampus, while restored the normal
Fig. 2. Effect of PDTC on cortical (A) and hippocampal (B) thiobarbituric acid reactive substances (TBARS) content in scopolamine-induced demented rats. Each bar with
vertical line represents the mean of 8 rats7 SEM. nPo 0.05 vs. normal, @Po 0.05 vs. scopolamine, #Po 0.05 vs. donepezil; using One-Way ANOVA followed by LSD multiple
comparisons test.
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Fig. 3. Effect of PDTC on cortical (A) and hippocampal (B) reduced glutathione (GSH) content in scopolamine-induced demented rats. Each bar with vertical line represents
the mean of 8 rats7SEM. nPo 0.05 vs. normal, @Po 0.05 vs. scopolamine, #Po 0.05 vs. donepezil; using One-Way ANOVA followed by LSD multiple comparisons test.
Fig. 4. Effect of PDTC on cortical (A) and hippocampal (B) cholinesterase activity in scopolamine-induced demented rats. Each bar with vertical line represents the mean of
8 rats7 SEM. nPo 0.05 vs. normal, @Po 0.05 vs. scopolamine, #Po 0.05 vs. donepezil; using One-Way ANOVA followed by LSD multiple comparisons test.
structure of cerebral cortex tissues (Figs. 8D and 9D). On the other
hand, pretreatment with PDTC (100 mg/kg) provided marked
protection against scopolamine-induced brain injury revealed by
near restoration of the normal structure of brain tissues and
showing only edema in the hippocampus (Figs. 8E and 9E).
4. Discussion
Scopolamine-induced amnesia has been used extensively to
evaluate potential therapeutic agents for treating AD (Blokland
et al., 2006; Kwon et al., 2009). Scopolamine is a muscarinic
receptor antagonist that inhibits central cholinergic neuronal
activity and inﬂuences the expression of a broad spectrum of
genes associated with muscarinic receptor signaling pathways,
apoptosis, and cell differentiation in rat brain (Hsieh et al., 2003).
Hence, it causes profound memory impairment in animals and
humans as degeneration and dysfunction of cortical cholinergic
neurons is closely associated with cognitive deﬁcits in AD
(Giacobini, 1998; Lieben and Blokland, 2005).
Indeed in the current study, single i.p. injection of scopolamine
was coupled by signiﬁcant decrease in spontaneous alternation
percentage of rats in Y-maze test coupled with increased activity
of AchE activity in cortex and hippocampus together with marked
histopathologic changes in both regions. Scopolamine-induced
changes were attenuated by pretreatment of rats with donepezil
and PDTC in both doses used.
Similar results regarding the effects of scopolamine (Jung et al.,
2012; Kim et al., 2013; Oh et al., 2013) and donepezil (Hu et al., 2012;
Jung et al., 2012; Kim et al., 2013) on the aforementioned parameters
were reported.
Donepezil is a piperidine class AchE inhibitor, designed especially for AD (Sugimoto et al., 1995). It was proven to improve
cognitive function of mild to moderate AD patients and showed
excellent tolerability without hepatotoxicity (Mihara et al., 1993;
Rogers et al., 1998).
PDTC was not previously investigated in scopolamine-induced
amnesia model. However, in a study performed by Cheng et al.
(2006), PDTC prevented Aβ-induced neuropathological and neuroinﬂammatory responses, as well as the learning and spatial
memory deﬁcits. Moreover, PDTC has been shown to improve
learning and memory functions of the transgenic APP/PS1 mice
model for AD (Malm et al., 2007). The reported protective effects
of PDTC in models of cerebral ischemia (Nurmi et al., 2006;
Pfeilschifter et al., 2010) were attributed to inhibition of NF-κB
(Schneider et al., 1999; Zhao et al., 2012). Suppression of NF-κB
pathway has been shown to attenuate cognitive deﬁcits induced in
rats by diabetes (Kuhad et al., 2009).
The observed neuroprotective effects of PDTC may also be related
to its ability to inhibit expression of matrix metalloproteinase-9
(MMP-9) (Zhao et al., 2012); as increased expression of MMP-9 has
been linked to cognitive impairment in elderly patients (Gaudet et al.,
2010). Moreover, MMP- 9 inhibition has also been reported to improve
Aβ-mediated cognitive impairment and neurotoxicity (Mizoguchi
et al., 2009).
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335
Fig. 5. Effect of PDTC on cortical (A) and hippocampal (B) interleukin-10 (IL-10) in scopolamine-induced demented rats. Each bar with vertical line represents the mean of
8 rats7 SEM. nPo 0.05 vs. normal, @Po 0.05 vs. scopolamine, #Po 0.05 vs. donepezil; using One-Way ANOVA followed by LSD multiple comparisons test.
Fig. 6. Effect of PDTC on cortical (A) and hippocampal (B) interleukin-1β (IL-1β) in scopolamine-induced demented rats. Each bar with vertical line represents the mean of
8 rats7 SEM. nPo 0.05 vs. normal, @Po 0.05 vs. scopolamine, #Po 0.05 vs. donepezil; using One-Way ANOVA followed by LSD multiple comparisons test.
Fig. 7. Effect of PDTC on cortical (A) and hippocampal (B) heat shock protein (HSP) in scopolamine-induced demented rats. Each bar with vertical line represents the mean of
8 rats7 SEM. nPo 0.05 vs. normal, @Po 0.05 vs. scopolamine, #Po 0.05 vs. donepezil; using One-Way ANOVA followed by LSD multiple comparisons test.
In the current experiments, scopolamine-induced pathological
and behavioral changes were coupled by decrease in cortical
dopamine content that was prevented by pretreatment with donepezil or PDTC. Scopolamine-induced dementia was shown to be
associated with reduced brain dopamine content (Zhou et al., 2009).
Neurotransmitters play a critical role in the brain circuits
involved in various aspects of memory. The importance of acetylcholine is illustrated by the psychopathology of AD (Giacobini,
1998; Lieben and Blokland, 2005). Dopamine in the prefrontal
cortex also contributes to information storage, particularly working memory (Iversen, 1998). In fact dopamine agonists improve
scopolamine-induced deﬁcits by increasing the release of acetylcholine (Steele et al., 1997).
Increased free radical activity appears to fuel Alzheimer's
pathology acting simultaneously as a mediator, product and trigger
for this "clogging" process and its related neural damage (JimenezJimenez et al., 1997; McIntosh et al., 1997). Lipid peroxidation
produced by the attack of reactive oxygen metabolites (ROM) on
cell membranes result in membrane ﬂuidity and permeability,
which eventually leads to oxidative destruction of cellular membranes and cell lysis (Halliwell and Gutteridge, 1990). Free radicals
are thought to cause behavioral deﬁcits in experimental animals
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Fig. 8. Photomicrograph of sections of brain hippocampus tissue of (A) normal rat showing the normal histological structure of hippocampus (hp); (B) scopolamine-treated
rat showing spongiosis in hippocampal matrix (S) with edema (O) and congestion in blood vessels (V); (C) demented rat treated with donepezil (5 mg/kg) showing edema
(O); (D) demented rat treated with PDTC (50 mg/kg) showing congestion of blood vessels (V), edema (O) and spongiosis (S) and (E) demented rat treated with PDTC (100 mg/
kg) showing edema in hippocampus (O) (H and E X 40, 60).
Fig. 9. Photomicrograph of sections of brain cortex tissue of (A) normal rat showing the normal histological structure of cortex (c); (B) scopolamine-treated rat showing focal
gliosis (g); (C) demented rat treated with donepezil (5 mg/kg) showing the normal histological structure of cortex (c); (D) demented rat treated with PDTC (50 mg/kg)
showing the normal histological structure of cortex (c) and (E) demented rat treated with PDTC (100 mg/kg) showing the normal histological structure of cortex (c) (H and E
X 40, 60).
(Fukui et al., 2001, 2002). Stadtman (1992) reported that balanced
antioxidants are required to control the cognitive and motor
functions of the cerebral cortex and the hippocampus.
Indeed in the present study, scopolamine induced deﬁcits was
accompanied by increased cortical and hippocampal lipid peroxidation
manifested by increased formation of TBARS in both regions. An
Author's personal copy
M.A. Abd-El-Fattah et al. / European Journal of Pharmacology 723 (2014) 330–338
increase in cortical and hippocampal GSH contents by scopolamine
was also observed.
GSH provides major protection in oxidative injury by participating in the cellular defense systems against oxidative damage
(Jefferies et al., 2003). Decreased cellular GSH contents and
capacity for GSH synthesis sensitize cells to certain drugs but
GSH may be induced in cells exposed to oxidative stress as an
adaptive process (Ilbey et al., 2009). This could probably explain
the observed increase in cortical and hippocampal GSH content.
Treatment of rats with donepezil or PDTC reduced scopolamineinduced increase in brain TBARS. Regarding the present effect on
changes induced in brain GSH content by PDTC, only the small dose
level was effective.
PDTC functions as an antioxidant due to two structural features: direct scavenging of ROM by the dithiocarboxy group and
chelating activity for heavy metal ions that may catalyze formation
of ROM (Liu et al., 1999).
ROM has been reported to be a crucial event in the activation of
NF-κB (Schreck et al., 1992). Therefore, it has been proposed that
antioxidants, as PDTC, by quenching ROM, may in turn block NF-κB
activation and ﬁnally inhibit NF-κB mediated increased production
of pro-inﬂammatory cytokines as TNF-α and IL-1β (Nemeth et al.,
1998). Indeed in the current experiments, pretreatment of rats
with PDTC prevented scopolamine-induced increases in cortical
and hippocampal IL-1β content. IL-10, an anti-inﬂammatory cytokine, was suppressed by scopolamine administration and prevented by pretreatment with PDTC in the small dose level. PDTC
bears numbers of beneﬁcial properties plus its antioxidant activity
including anti-inﬂammatory and immunomodulatory effects
(Cuzzocrea et al., 2002), all of which could account for the
observed effects of PDTC on cortical and hippocampal cytokines
contents.
Induction of scopolamine dementia was coupled by increased
cortical and hippocampal contents of HSP 70 that was prevented
by pretreatment with PDTC. Although, HSP 70 mRNA is not
detected in the brain under normal condition (Wagstaff et al.,
1996), HSP 70 is expressed in the brain in response to any stressful
condition and is therefore suggested as cytoprotective protein in
ameliorating tissue damage (Kelly and Yenari, 2002; Yasuda et al.,
2005).
In conclusion, the prevalence of AD and dementia has increased
signiﬁcantly nowadays. As none of the available medications
appear to be able to stop the disease progression, there is
enormous medical need for the development of novel therapeutic
strategies that target the underlying pathogenic mechanisms in
AD. Keeping in mind that PDTC has broad cytoprotective properties as observed in the current study; its use might slow progression of the multifactorial AD. Further studies may be needed,
however, to examine the role of PDTC in scopolamine-induced
dementia after development of symptoms to simulate what
actually happens in patients with AD.
Acknowledgment
The authors are very grateful to Dr. Adel M. Bakeer, Professor of
Pathology, Faculty of Veterinary Medicine, Cairo University, for
examining and interpreting histopathologic aspects of this study.
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