Deferiprone ameliorates memory impairment in Scopolamine-treated rats: The impact of its iron-chelating effect on β-amyloid disposition

Journal Pre-proof Deferiprone ameliorates memory impairment in Scopolamine-treated rats: the impact of its iron-chelating effect on ␤-amyloid disposition Sylvia F. Fawzi, Esther T. Menze, Mariane G. Tadros

PII:

S0166-4328(19)31031-9

DOI:

https://doi.org/10.1016/j.bbr.2019.112314

Reference:

BBR 112314

To appear in:

Behavioural Brain Research

Received Date:

9 July 2019

Revised Date:

17 October 2019

Accepted Date:

17 October 2019

Please cite this article as: Fawzi SF, Menze ET, Tadros MG, Deferiprone ameliorates memory impairment in Scopolamine-treated rats: the impact of its iron-chelating effect on ␤-amyloid disposition, Behavioural Brain Research (2019), doi: https://doi.org/10.1016/j.bbr.2019.112314

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Sylvia F. Fawzia, Esther T. Menzeb, Mariane G. Tadrosc a

Teaching assistant, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Badr University in Cairo (BUC), Cairo, Egypt – Corresponding author e-mail: [email protected]; [email protected]; [email protected] . b

Lecturer, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt e-mail: [email protected] c

Associate Professor, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt

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e-mail: [email protected]; [email protected]

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Deferiprone ameliorates memory impairment in Scopolamine-treated rats: the impact of its iron-chelating effect on β-amyloid disposition Sylvia F. Fawzia, Esther T. Menzeb, Mariane G. Tadrosb Highlights 

Deferiprone attenuates SCOP-induced behavioral and biochemical changes.

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It reverses increased AChE activity. Thus, it improves cholinergic transmission.

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Prussian blue staining shows improvement in iron deposition.

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Congo red staining shows negative β-amyloid plaques in drug-treated rats.

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Abstract

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by cognitive and memory problems. Scopolamine (SCOP) is a natural anticholinergic drug that was proven

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to cause memory impairment in rats. Chelating agents are potential neuroprotective and

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memory enhancing agents as they can trap iron that enters in pathological deposition of βamyloid (Aβ) which is a hallmark in AD and memory disorders. This study investigated the

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potential neuroprotective and memory enhancing effects of the iron chelating drug, Deferiprone. Three doses (5, 10, and 20 mg/kg) were administered to rats treated with SCOP

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(1.14 mg/kg/day). Systemic administration of SCOP for seven days caused memory impairment which manifested as decreased time spent in platform quadrant in Morris water maze test, decreased retention latencies in passive avoidance test, and increased acetylcholinesterase (AChE) activity, Aβ, and free iron deposition. It was observed that

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pretreatment with Deferiprone increased platform quadrant time in Morris water maze and increased retention latencies in the passive avoidance test. It also attenuated the increase in AChE activity and decreased Aβ and iron deposition. Overall, Deferiprone (10 mg/kg) was determined as the most effective dose. Therefore, this study suggests neuroprotective and memory enhancing effects for Deferiprone in SCOP-treated rats which might be attributed to its iron chelating action and anti-oxidative effect. 2

Key words

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Deferiprone; Chelation therapy; Memory impairment; Scopolamine; β-amyloid

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1. Introduction Alzheimer’s disease (AD) is a dynamic neurodegenerative brain disease that represents approximately 60% of dementia cases [1]. It is characterized by the presence of neuritic plaques containing amyloid-β (Aβ) protein and hyperphosphorylated tau protein filaments as neurofibrillary tangles. Loss of cholinergic cells, especially in the basal forebrain, is accompanied by loss of the neurotransmitter acetylcholine (ACh) which is responsible for memory and cognitive function [2]. As neuro-inflammation and death of brain cells are hallmarks of the disease, it is classified as a neurodegenerative disorder. All these brain

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changes lead to memory loss and changes in thinking and other brain functions [3].

The plaques primarily consist of human Aβ, a 40-mer whose neurotoxicity is due to its pathological aggregation. High concentrations of specific metals such as iron may play a role

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in the pathogenesis of AD by promoting aggregation of Aβ protein [4].

Aβ harmfulness may result from the induction of its pathological aggregation by iron.

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In addition, the iron may also create free radicals. For example, iron binding to Aβ causes

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hydrogen peroxide generation [5]. Iron toxicity is known to result from free radicals’ formation through the Fenton reaction. In that reaction, ferrous iron reacts with hydrogen peroxide to produce reactive oxygen species (ROS) and ferric iron. The subsequent ferric iron can be

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reduced back to Fe2+ using various reducing agents and accordingly recover the beginning reagents [6].

Scopolamine (SCOP), a muscarinic cholinergic receptor blocker, has been generally

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adopted to induce memory deficits in experimental animals. In 1998, three research teams reported decreased activity of the enzyme responsible for ACh synthesis; choline acetyltransferase in the cortex of AD patients. It was noted that this decrease was related to brain lesions and clinical status. It was soon found that neuronal loss occurs in the forebrain basal nucleus of neocortical and hippocampal cholinergic afferent fibres. It was a type of ‘black box’ model in which an unknown pathophysiological process induces deficiency in

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various neurotransmission pathways that were believed to be responsible for the cognitive and behavioural aspects of aging and dementia. Later, the cholinergic hypothesis justified the development of the cholinergic drugs that are prescribed today and the administration of SCOP as a model of investigation for AD research. The SCOP model was used in cognitive research to study the clinical correlations of ACh deficiency. It was applied to AD patients as a marker of cholinergic sensitivity for improving the diagnosis and staging of the disease [7]. Acetylcholinesterase (AChE) inhibitors are the most common drugs used for Dementia of the Alzheimer type [8]. the

intra-peritoneal

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injection

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SCOP,

the

cholinergic

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neurotransmission was blocked which caused cholinergic deficit and impaired memory in rats [9]. This is how rats with SCOP-induced memory deficits were utilized as experimental models

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for screening anti-dementia drugs and sometimes AD drugs [10]. It has been recently reported that memory and cognitive impairment prompted by SCOP in rats is related to changes in brain

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oxidative stress [11].

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Deferiprone is an oral bi-dentate iron chelator which binds to iron in a 3:1 ratio. It reduces body iron content in iron-overloaded animals and humans [12]. Depending on the

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concentration, Deferiprone has been reported to promote, at low concentrations, in-vitro, and conversely to protect against, at high concentrations, oxidative damage caused by oxygen-free radicals [13].

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The accumulation of metals in AD brains and the presence of a metal-binding site on Aβ represent promising pharmacological targets. Therefore, compounds with chelation properties, such as Deferiprone, can help treat AD. This study is designed to investigate the potential effects of Deferiprone as a chelating agent in SCOP-induced memory impairment, such as AD, in rats as well as the possible mechanisms underlying these effects.

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2. Materials and methods: 2.1. Animals Male albino rats (initially weighing 160–220 g) were purchased from the National Institute of Research, Cairo, Egypt. Plastic cages were used for housing them at a constant temperature (21 ± 2°C), with alternating 12 h light/dark cycle. Animal chow and water were provided ad libitum. All animal treatments adhered strictly to institutional and international ethical guidelines of the care and use of laboratory animals. The experimental protocol was

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approved by Ain Shams University Faculty of Pharmacy Ethical Committee for the use of animal subjects (Permit no. PhD, 151). 2.2. Drugs and chemicals

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Scopolamine hydrobromide trihydrate, acetylthiocholine iodide, and 5, 5´-Dithiobis (2nitrobenzoic-acid) (DTNB) were purchased from Sigma-Aldrich (MO, USA). Deferiprone was

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received as a free sample for research purpose from ApoPharma Pharmaceuticals (Toronto,

2.3. Experimental design

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Ontario, Canada). The other reagents were of the highest pure grade commercially available.

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The animals were divided into six groups of 10 rats each. The adopted study design is shown in (fig. 1) and chosen according to [14]. Rats were given SCOP which was dissolved in saline and administered intraperitoneally (i.p) at a dose of 1.14 mg/kg body weight for seven consecutive days. Deferiprone (5, 10, and 20 mg/kg body weight) was dissolved in distilled

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water and administered p.o, for 14 days, beginning 7 days before and continued for 7 days one hour before administering SCOP injections. Doses of Deferiprone were selected according to [15] [16]. On day 14, 3.5 hours after the last SCOP injection, the animals were decapitated and their skulls were split on iced phosphate buffer saline. To fix half of their brains for histopathological examination, 10 % formalin was used. The other halves were dissected out

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and the prefrontal cortices and hippocampi were separated, after which 10% (w/v) homogenates in 0.1 M phosphate buffer (pH 7.4) were prepared. As per the results of the behavioural and biochemical examinations, the effective dose of Deferiprone was selected for completing this study. 2.4. Behavioural experiments 2.4.1. Morris water maze test. [14] The effect of Deferiprone on memory was evaluated using Morris water maze. Morris

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water maze is a circular pool which is filled with water and conceptually divided into four quadrants, and a hidden escape platform is placed in one of the pool quadrants and submerged 2 cm below the water surface so that it is not visible at water level. During the five subsequent

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days, the rats underwent three trials per day with the platform in place. For each training trial, the rats were placed in the water facing the pool wall in different pool quadrants, with a

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variable order each day. When a rat located the platform, it was permitted to remain on the platform for 30 s, and if it did not locate the platform within 90 s, it was placed on the platform

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for 30 s. During each trial, the time taken to reach the hidden platform (latency) was recorded. Immediately after the last training trial session, the rats were subjected to a probe trial session

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in which the platform was removed from the pool and the rats were placed in the quadrant facing the platform and were allowed to swim for 90 s to search for it. The time spent by the rats swimming in the platform quadrant was recorded.

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2.4.2. Step-through passive avoidance paradigm [14]. A step-through passive avoidance device was utilized (Ugo Basile, Italy). It is a

Plexiglas cage that encloses two compartments. One compartment is white and is enlightened by a light apparatus, including a 24-V, 10-W knob, affixed to the compartment cover, while the second compartment is dim and made of dark Perspex boards. The two compartments are separated by an automatically operated sliding door. The apparatus incorporated a steel-bar 7

network floor made of 40 parallel bars (0.3 cm in diameter, set 1.2 cm apart). The bars of the dim compartment floor are wired to a constant current high-accuracy eight-shaft scrambling circuit situated in the controller. On day 12, training session was conducted before Deferiprone and SCOP administration. In this session, each rodent was prepared by delicately setting it in the light compartment, and when it ventured through the dim compartment, its four paws were placed on the lattice floor, the door automatically shut, and an electric stun of 0.5 mA was delivered for 2s. The rats which failed to step through within the 90s cut-off time were excluded. On day

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13, the test session involved each rat being introduced to the light compartment and recording the latency time to step-through to the dark compartment which indicated memory acquisition, with an upper cut-off time of 300s. No electric shock was delivered during the test sessions.

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The rats demonstrating immobility from the training test were rejected from the test sessions.

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2.5. Biomarkers of oxidative stress Catalase activity.

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Catalase activity was determined according to kit instructions (Biodiagnostics, Egypt). The homogenates were centrifuged at 4000 g at 4°C for 15 min and then 0.05 ml of

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supernatant was added to a known quantity of H2O2. A catalase inhibitor was used to stop the reaction after exactly one minute. In the presence of peroxidase, the remaining H2O2 reacts with 3, 5-dichloro-2 hydroxybenzene sulfonic acid and 4-aminophenazone to form a chromophore of pink colour that is detected at 510 nm. The intensity of the pink colour is

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inversely proportional to the amount of catalase in the sample [17]. Catalase activities were expressed as U/g tissue. 2.5.2.

Reduced glutathione (GSH) assay.

According to kit instructions (Biodiagnostics, Egypt), 0.5 ml of both hippocampus and cortex tissue homogenate (10% (w/v) in 0.1 M phosphate buffer, pH 7.4) was added to a tube

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with 0.5 ml of 10% trichloroacetic acid. The tubes were shaken gently and intermittently for 15 min, followed by centrifugation at 3000 g for 10 min. An aliquot of the resultant supernatant (0.2 ml) was added to a tube containing 1.7 ml phosphate buffer and 0.1 ml Ellman’s reagent, and then the absorbance was read at 412 nm within 5 min [18]. GSH levels were expressed as mmol/g tissue. 2.5.3. Lipid Peroxidation Products / Malondialdehyde (MDA) assay. According to kit instructions (Biodiagnostics, Egypt), 0.02 ml of both hippocampus and cortex tissue homogenate (10% (w/v) in 0.1 M phosphate buffer, pH 7.4) was added to 1 ml of

534 nm. MDA levels were expressed as µmol/g tissue. [19]

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2.6. AChE activity

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the chromogen (Thiobarbituric acid), were mixed well, and its absorbance was measured at

AChE activity was measured according to the method described by [20] with few

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modifications. The assay was made by adding 2.9 mL of 0.1 mM sodium phosphate buffer (pH 8.0) to 50 μl of the tissue homogenate of cortices and hippocampi (10% (w/v) in 0.1 M

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phosphate buffer (pH 7.4), and the mixture was incubated at 37°C for 5 min. After incubation, 40 μl of acetylthiocholine iodide (154.38 mM) and 10 μl of DTNB (10 mM) were added to the

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reaction mixture, and the formation of thionitrobenzoic acid was recorded at 412 nm for 150 s at 30 s intervals using UV spectrophotometer. The AChE activity was assessed by measuring the change in concentration of thionitrobenzoic acid by time (extinction coefficient 1.36 × 104/molar/cm). AChE activities were expressed as μM/min/mg tissue.

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After obtaining the previous results, the best effective dose of Deferiprone was chosen

for the rest of the study 2.7. Histopathological examination First, 24 hours fixation of brain samples was conducted by 10% formalin. It was then washed with tap water and serial dilutions of alcohol (methyl, ethyl and absolute ethyl) for

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dehydration. Specimens were cleared in xylene and embedded in paraffin at 56°C in hot air oven for 24 hours. Paraffin bees wax tissue blocks were prepared for coronal sectioning at 4μm thickness by a revolving microtome. The obtained tissue sections were gathered on glass slides, deparaffinized, and recoloured by hematoxylin (H) and eosin (E) stain for routine examination by the light electric microscope [21]. 2.8. Iron deposition detection by Prussian blue staining of brain tissue Brain iron deposition was determined using the Perl’s Prussian blue staining techniques

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which was modified from a previous study [22]. Paraffin blocks were cut using a sliding microtome (5-μm thickness) and stained with Perl’s super vital dye solution. The Perl’s stained tissue slides were examined under a light microscope [23].

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2.9. Amyloid-β deposition by histochemical analysis

Paraffin blocks were cut at 25 μm using a sliding microtome and stored floating in

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phosphate-buffered saline at 4ºC. They were de-waxed and rehydrated by incubating them in

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distilled water for 30 s. Then, they were incubated in alkaline saturated Sodium Chloride solution for 20 m and then in Congo red filtered solution for 30 m. Sections of these blocks

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were then rinsed by dipping them in 95% ethanol and in 100% ethanol eight times each, without too much rinsing. This step was followed by incubation in xylene, and coverslips were put using a xylene-based mountant and allowed to dry overnight for examination under light microscope [24].

Statistical analysis

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2.10.

All parametric tests were analysed by one-way ANOVA, while Morris water maze training data were analysed by two-way ANOVA followed by Bonferroni post-hoc test. All data were expressed as mean ± standard error of the mean (SEM). Passive avoidance test data were analysed by Kruskal Wallis followed by Dunn’s test as a post-hoc test, and its data were

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expressed as medians and interquartile range. All statistical analyses were performed using the GraphPad Prism software (version 5.01, Inc., 2007, San Diego California USA). Probability values of less than 0.05 were considered statistically significant. 3. Results: 3.1. Behavioural experiments 3.1.1. Morris water maze test. Two-way ANOVA test was performed to analyse transfer latency during training days for the treated rats in each group and statistical significance was found (F (5,176) = 15.38, P <

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0.0001, F (4,176) = 67.38, P < 0.0001, F (20,176) = 0.78, P > 0.05) for significance between treatments, days and interaction between them, respectively. Therefore, no significant interaction was found between treatment groups and time (fig. 2-A). Further statistical analysis

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with Bonferroni test showed that SCOP (1.14 mg/kg) resulted in a longer transfer latency time

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compared to the control group on the third, fourth, and fifth days, while pretreatment with Deferiprone (10 & 20 mg/kg) significantly attenuated SCOP-induced increased transfer latency

SCOP-treated group.

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on the same days. The effect of Deferiprone (5 mg/kg) was not statistically significant from the

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One-way ANOVA test showed significant effect on the time spent in platform quadrant (F (5, 30) = 6.838, P < 0.001) (fig. 2-B). Further statistical analysis with Tukey test showed that SCOP resulted in a shorter time in platform quadrant compared to the control group, while pretreatment with Deferiprone (10 & 20 mg/kg) significantly attenuated SCOP-induced effect.

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The effect of the other Deferiprone dose (5 mg/kg) was not statistically significant from the SCOP-treated group. 3.1.2. Step-through passive avoidance test The Kruskal–Wallis test showed significant effect on the step-through latency (H (5, 54) = 17.08, P < 0.01) (fig. 2-C). Further statistical analysis with Dunn’s test indicated that

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SCOP led to a shorter latency to step through the dark compartment compared to the control group, while pretreatment with Deferiprone (10 mg/kg) significantly attenuated this short latency time. Moreover, Deferiprone alone did not cause any significant changes in stepthrough latency compared to the control group. 3.2. Effects of Deferiprone on hippocampal and cortical oxidative status 3.2.1. Hippocampal and cortical catalase activity in SCOP-treated rats. One-way ANOVA showed significant differences among groups on both hippocampal and cortical catalase activities (F (5,30) = 10.82, P < 0.0001, F (5,30) = 5.505, P = 0.001,

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respectively) followed by Tukey post-hoc test. Results showed that administration of SCOP significantly reduced catalase enzyme activity in both hippocampal and cortical tissues compared to the control groups. However, pretreatment with Deferiprone (10 and 20 mg/kg)

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attenuated the decrease in catalase activity compared to the SCOP-treated group (fig. 3-A, B).

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3.2.2. Hippocampal and cortical GSH levels in SCOP-treated rats.

One-way ANOVA showed significant difference between groups in both hippocampal

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and cortical GSH levels (F (5,30) = 6.286, P < 0.001, F (5,30) = 7.091, P<0.001, respectively) (fig. 4-A, B). It was found that administrating SCOP significantly reduced GSH levels

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compared to the control groups, while pretreatment with Deferiprone (10 and 20 mg/kg) significantly attenuated this decrease in the hippocampus and Deferiprone (10 mg/kg) also attenuated this decrease in the cortex.

3.2.3. Hippocampal and cortical MDA levels in SCOP-treated rats.

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One-way ANOVA showed significant difference in both hippocampal and cortical

MDA levels (F (5,30) = 10.06, P < 0.0001, F (5,30) = 4.931, P<0.01, respectively) (fig. 5-A, B). SCOP-treated rats had significantly increased hippocampal and cortical MDA levels compared to the control group, while this effect was reversed in Deferiprone-treated (5, 10, 20 mg/kg) rats in the hippocampus and Deferiprone-treated (10 mg/kg) rats in the cortex.

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3.3. Hippocampal and cortical AChE activity One-way ANOVA showed statistical significance between the groups on hippocampal as well as cortical AChE activities (F (5,30) = 7.12, P<0.001, F (5,30) = 13.21, P<0.0001, respectively). Further, SCOP-treated rats showed significant increase in hippocampal and cortical AChE activity compared to the control group. Pretreatment with Deferiprone (10 and 20 mg/kg) significantly attenuated this increase (fig. 6-A, B). 3.4. Histopathological examination of the hippocampal and cortical tissues by H&E staining

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H&E staining of CA1, CA3, and dentate gyrus regions of the hippocampi revealed hypo-cellularity of pyramidal cells layer with scattered shrunk cells that had pyknotic nuclei, glial cells infiltration, and mild oedema was observed in the SCOP-treated group. In addition,

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Deferiprone-treated (10 mg/kg) group showed increased number of pyramidal cells layer with few degenerating cells, pyknotic nuclei, and mild glial cells infiltration in CA1, and intact

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neurons associated with glial cells infiltration were found in the in CA3 and dentate gyrus

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regions, while the Deferiprone only-treated group showed apparent intact neurons and organized hippocampal layers (fig. 7, 8, 9). Furthermore, H&E staining of outer cortical layers

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showed diffused neuronal degeneration with several shrunken dark pyknotic nuclei and moderate intercellular oedema as well as perineuronal oedema in the SCOP-treated group and Deferiprone-treated (10 mg/kg) groups. In addition, the SCOP-treated group samples showed more pronounced glial cells infiltration which is relevant to [25] which found that glial cells

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participate in the pathogenesis of AD. Slightly protected cortical region with few numbers of apparent intact neurons having vesicular nuclei was shown in the Deferiprone only-treated group. However, several pyknotic darkly stained neurons were demonstrated as along with mild intercellular oedema (fig. 10).

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3.5. Histopathological examination of iron deposition in different areas of the brain by Prussian blue staining Several focal areas of ferric iron showed positive reaction with Prussian blue staining in the cerebral cortex and hippocampus of the SCOP-treated rats. However, fewer scattered ones were noticed in both brain areas in the Deferiprone-treated (10 mg/kg) rats and negative reaction was observed towards Prussian blue staining in cortical and hippocampal regions in the drug-only-treated rats (fig. 11).

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3.6. Histopathological examination of Aβ deposition in different areas of the brain by Congo red staining Multiple positive Aβ plaques were noticed in Congo red-stained sections of the cerebral cortex of the SCOP and Deferiprone-treated groups. This plaques deposition was reversed in

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the Deferiprone-treated (10 mg/kg) group which showed negative reaction to Congo red

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staining (fig. 12).

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4. Discussion AD is the most common form of senile dementia. It is characterized by memory loss accompanied by degeneration of cortical and hippocampal cholinergic neurons [26]. The pathogenesis of AD is multifactorial and includes degeneration of cholinergic neurons, abnormal phosphorylation of the protein tau, oxidative stress, and altered protein processing resulting in abnormal Aβ accumulation [27]. These changes are eventually accompanied by the damage and death of neurons [28]. This hallmark is evident in certain brain areas including the hippocampus [29] that consists of three regions which are the dentate gyrus region, CA1 and

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CA3 fields [30], and the cerebral cortex.

In this study, SCOP was selected because of its effect on memory impairment caused by the imbalance of oxidative stress in brain [31].These effects are somehow similar to those

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occurring in older people and result in age-related dementia [32] [33]. Thus, SCOP-induced memory impairment in an animal model imitates some of the dysfunction of the CNS, such as

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AD, which includes hippocampal-dependent learning and memory deficits, oxidative stress,

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and behavioural disorder [34]. In addition, SCOP increases the brain iron in accordance with [35], Aβ accumulation by making imbalance between α, β secretases, and increases oxidative

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stress and glial cell infiltration [36]. It was also found that Muscarinic antagonism increases neuronal dysfunction and apoptosis by increasing oxidative stress. As activation of muscarinic receptor causes protection from oxidative stress-induced apoptosis, muscarinic receptorcoupled signalling activity becoming impaired with AD’s progression results in neurons losing

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the ability to counteract the harmful effects of oxidative stress [37]. Moreover, cholinergic signals by the vagal nerve were found to attenuate inflammatory cytokine production [38], and their blocking by SCOP was expected to trigger inflammation. Furthermore, activating ACh receptors increases cellular proliferation and neurogenesis throughout development and vice versa. Any interference with this function by muscarinic blockade may provoke apoptosis [39].

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Regarding iron and chelation, iron binds to Aβ and promotes its aggregation [4] [40]. This altered conformational status of the peptide promotes an increased neurotoxicity of Aβ [41]. Previous studies have shown that Aβ does not spontaneously aggregate but that there is an age-dependent reaction with excess brain metals such as iron which induces the protein to precipitate into metal-enriched masses called plaques [42]. Thus, an iron chelating agent such as Deferiprone is expected to reduce the iron-mediated pathological disposition of Aβ as well as reduce the induced oxidative stress caused by iron. Further, this expectation is in accordance with the current study findings in histopathology and Congo red staining of Aβ as mentioned

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and later discussed.

It was found that the Deferiprone drug, which has an iron chelating activity, can pass the blood–brain barrier and can thus be present at effective concentration at the site of action in

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brain [43]. Previous studies on Deferiprone have shown that the drug was efficacious in Parkinson’s disease (PD) both in in-vitro [44] and in-vivo experiments [15]. Moreover, short-

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term Deferiprone therapy in PD subjects has been observed to be safe and associated with

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decrease in iron in specific brain regions, thus supporting future longer-term clinical trials in PD where the neuroprotective effects of Deferiprone can be fully assessed [45].

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It should be noted that PD and AD share certain pathological features such as protein accumulation and involvement of iron in their pathogenesis [44]. A study noted that pretreatment of Aβ with deferoxamine, an iron chelating agent, also decreased the neuronal degeneration after Aβ was included in cultured neurons [46]. Thus, the present study was

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conducted to determine the effect of Deferiprone as a chelating agent on SCOP-induced memory impairment in rats. Behavioural experiments showed increased latency time in Morris water maze training

days and decreased time spent in platform quadrant during the probe test for the SCOP-treated rats. This is in accordance with [14] where both were reversed in the Deferiprone-treated (10, 20 mg/kg) rats, and thus, Deferiprone was found to improve memory in the SCOP-injected rats

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as they exhibited improved target quadrant preference as shown by time spent in platform quadrant. Passive avoidance test in the SCOP-treated rats had a significantly shorter step-through latency compared to the control rats, which is in accordance with [14]. Administration of Deferiprone (10 mg/kg) before the SCOP treatment significantly lengthened the step-through latency, thus indicating that Deferiprone reversed SCOP-mediated memory impairment. Biochemical assays were performed to investigate the underlying mechanisms involved in the behavioural and pathological alterations caused by SCOP and/or Deferiprone. Rats

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treated with SCOP showed significant decrease in catalase activity and GSH levels in brain tissues, particularly the hippocampal and cortical tissues as per [47]. Pretreatment with Deferiprone (10 and 20 mg/kg) significantly reversed the SCOP effects on hippocampal and

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cortical catalase activity while Deferiprone (10 mg/kg) reversed this effect on hippocampal and cortical levels of GSH. In addition, pretreatment with Deferiprone attenuated SCOP-associated

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oxidative stress as it reduced cortical and hippocampal MDA content by increasing the ROS

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scavenging activity of cortical and hippocampal GSH.

The observed oxidative stress changes may have led to the pathological deposition of

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iron, and therefore, Aβ protein aggregation is as follows. Loss of α7-nicotinic ACh receptors leads to enhanced Aβ oligomer accumulation in mouse model of AD [48]. The present study showed a significant increase in AChE activity in SCOP-treated rats, which is in accordance with previous studies [8] [34] [49] . However,

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pretreatment with Deferiprone significantly attenuated this increase. Therefore, SCOP could have lowered ACh levels in AD brain, suggesting degeneration of cholinergic neurons [50]. Moreover, in the present study, it increased hippocampal and cortical AChE activity compared to that in the control group, which is in accordance with [8]. Dysfunction of cholinergic system in the prefrontal cortex and the hippocampus appears to be crucial in the pathogenesis of cognitive impairment, including that in AD. Both

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muscarinic and nicotinic receptors, as a part of cholinergic system, are important for memory formation and SCOP, which is the antagonist of the first one, significantly impairs memory, thus inducing cognitive impairment. ACh is a brain neurotransmitter that is significant for both learning and memory as well as motivation. Hence, any substances with the ability to decrease cholinergic neurotransmission in the CNS such as SCOP are of great interest from the pharmacological perspective. After testing three doses of Deferiprone, it was observed that the lower Deferiprone dose (5 mg/kg) led to no improvement in the behavioural, biochemical, and AChE activity

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when challenged with SCOP, and the higher Deferiprone dose (20 mg/kg) showed approximately the same effect as Deferiprone (10 mg/kg) in most results. Therefore, the experiment proceeded with the 10 mg/kg dose to further investigate the possible mechanisms

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underlying its protective effects on memory impairment in rats.

Histopathological examination of brain tissues showed inflammation, oedema, and

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degenerated vacuolated neurons in different brain areas in the SCOP-treated group, while

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improvement was noticed in the Deferiprone-treated (10 mg/kg) group in some brain areas. Prussian blue staining was conducted to evaluate iron levels in both prefrontal cortex

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and hippocampus as a defect in homoeostasis of redox-active metals such as iron contributes to the neuropathology of AD. This abnormal homoeostasis plays an important role in the misfolding process associated with the production of Aβ from amyloid precursor protein (APP) [51]. In the present study, Prussian blue staining of iron showed several focal areas of ferric

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iron positivity in the SCOP-treated group, while a reasonable improvement was noticed in the Deferiprone-treated (10 mg/kg) group. Moreover, as iron is crucial in Aβ pathological deposition, staining of Aβ was conducted in both cerebral cortex and hippocampus regions because of Deferiprone’s indirect action on its accumulation by chelation of iron. Positive Aβ plaques were found in the cortex of the SCOP-treated groups, while negative reactivity was observed in the Deferiprone-treated (10 mg/kg) group. In addition, as Aβ fibril accumulation is

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more prone to starting with increased rates in the cortex in early Aβ stage [52], this finding may explain the negative reactivity in hippocampus region of the disease group. Perhaps if the rats were left for a longer time, hippocampal plaques may have appeared. This may need further future investigation. Thus, these findings may elaborate the behavioural and pathological improvements found in Deferiprone-pretreated rats. 5. Conclusion Deferiprone (10 mg/kg) dose improved memory retention and restored the histological

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abnormalities including reduction of Aβ accumulation and iron deposition in SCOP injected rats. This activity could be, at least partly, explained based on its chelating properties and attenuating the increase of cholinesterase activity.

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6. Acknowledgements

We are grateful to ApoPharma Pharmaceuticals (Toronto, Ontario, Canada) for

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7. Funding:

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providing free Deferiprone sample for research purpose.

This research did not receive any specific grant from funding agencies in the public,

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commercial, or not-for-profit sectors. 8. Declarations of interest

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None

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References

Jo

ur na

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[1] C. Holmes, J. Amin, Dementia, Medicine 44(11) (2016) 687-690. [2] D.J. Selkoe, Alzheimer's disease: a central role for amyloid, Journal of Neuropathology & Experimental Neurology 53(5) (1994) 438-447. [3] L. Rizzi, I. Rosset, M. Roriz-Cruz, Global epidemiology of dementia: Alzheimer’s and vascular types, BioMed research international 2014 (2014). [4] P.W. Mantyh, J.R. Ghilardi, S. Rogers, E. DeMaster, C.J. Allen, E.R. Stimson, J.E. Maggio, Aluminum, iron, and zinc ions promote aggregation of physiological concentrations of β amyloid peptide, Journal of neurochemistry 61(3) (1993) 1171-1174. [5] X. Huang, C.S. Atwood, M.A. Hartshorn, G. Multhaup, L.E. Goldstein, R.C. Scarpa, M.P. Cuajungco, D.N. Gray, J. Lim, R.D. Moir, The Aβ peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction, Biochemistry 38(24) (1999) 76097616. [6] J.A. Gaasch, P.R. Lockman, W.J. Geldenhuys, D.D. Allen, C.J. Van der Schyf, Brain iron toxicity: differential responses of astrocytes, neurons, and endothelial cells, Neurochemical research 32(7) (2007) 1196-1208. [7] C. Gilles, S. Ertlé, Pharmacological models in Alzheimer's disease research, Dialogues in clinical neuroscience 2(3) (2000) 247. [8] S.A. El-Marasy, R.M. Abd-Elsalam, O.A. Ahmed-Farid, Ameliorative effect of silymarin on scopolamine-induced dementia in rats, Open access Macedonian journal of medical sciences 6(7) (2018) 1215. [9] J.H. Oh, B.J. Choi, M.S. Chang, S.K. Park, Nelumbo nucifera semen extract improves memory in rats with scopolamine-induced amnesia through the induction of choline acetyltransferase expression, Neuroscience Letters 461(1) (2009) 41-44. [10] J. Chen, Y. Long, M. Han, T. Wang, Q. Chen, R. Wang, Water-soluble derivative of propolis mitigates scopolamine-induced learning and memory impairment in mice, Pharmacology Biochemistry and Behavior 90(3) (2008) 441-446. [11] Y. Fan, J. Hu, J. Li, Z. Yang, X. Xin, J. Wang, J. Ding, M. Geng, Effect of acidic oligosaccharide sugar chain on scopolamine-induced memory impairment in rats and its related mechanisms, Neuroscience letters 374(3) (2005) 222-226. [12] A.N. Saliba, A.R. Harb, A.T. Taher, Iron chelation therapy in transfusion-dependent thalassemia patients: current strategies and future directions, Journal of blood medicine 6 (2015) 197. [13] J.A.B. Balfour, R.H. Foster, Deferiprone, Drugs 58(3) (1999) 553-578. [14] Y.J. Jang, J. Kim, J. Shim, C.-Y. Kim, J.-H. Jang, K.W. Lee, H.J. Lee, Decaffeinated coffee prevents scopolamine-induced memory impairment in rats, Behavioural brain research 245 (2013) 113-119. [15] R.B. Mounsey, P. Teismann, Chelators in the treatment of iron accumulation in Parkinson's disease, International journal of cell biology 2012 (2012). [16] G. Abbruzzese, G. Cossu, M. Balocco, R. Marchese, D. Murgia, M. Melis, R. Galanello, S. Barella, G. Matta, U. Ruffinengo, A pilot trial of deferiprone for neurodegeneration with brain iron accumulation, Haematologica (2011) haematol. 2011.043018. [17] H. Aebi, [13] Catalase in vitro, Methods in enzymology, Elsevier1984, pp. 121-126. [18] G.L. Ellman, Tissue sulfhydryl groups, Archives of biochemistry and biophysics 82(1) (1959) 70-77. [19] M. Uchiyama, M. Mihara, Determination of malonaldehyde precursor in tissues by thiobarbituric acid test, Analytical biochemistry 86(1) (1978) 271-278. [20] G.L. Ellman, K.D. Courtney, V. Andres Jr, R.M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochemical pharmacology 7(2) (1961) 88-95.

20

Jo

ur na

lP

re

-p

ro of

[21] D. Hopwood, J. Bancroft, A. Stevens, Theory and practice of histological techniques, Theory and practice of histological techniques Google Scholar (1996). [22] S. Kumfu, S. Chattipakorn, K. Chinda, S. Fucharoen, N. Chattipakorn, T type calcium channel blockade improves survival and cardiovascular function in thalassemic mice, European journal of haematology 88(6) (2012) 535-548. [23] J. Sripetchwandee, S. Wongjaikam, W. Krintratun, N. Chattipakorn, S.C. Chattipakorn, A combination of an iron chelator with an antioxidant effectively diminishes the dendritic loss, tau-hyperphosphorylation, amyloids-β accumulation and brain mitochondrial dynamic disruption in rats with chronic iron-overload, Neuroscience 332 (2016) 191-202. [24] D.M. Wilcock, M.N. Gordon, D. Morgan, Quantification of cerebral amyloid angiopathy and parenchymal amyloid plaques with Congo red histochemical stain, Nature protocols 1(3) (2006) 1591. [25] D. Dzamba, L. Harantova, O. Butenko, M. Anderova, Glial Cells–The Key Elements of Alzheimer´ s Disease, Current Alzheimer Research 13(8) (2016) 894-911. [26] A.D. Korczyn, V. Vakhapova, The prevention of the dementia epidemic, Journal of the neurological sciences 257(1-2) (2007) 2-4. [27] A. Ferreira, C. Proença, M. Serralheiro, M. Araujo, The in vitro screening for acetylcholinesterase inhibition and antioxidant activity of medicinal plants from Portugal, Journal of ethnopharmacology 108(1) (2006) 31-37. [28] A.s. Association, 2016 Alzheimer's disease facts and figures, Alzheimer's & Dementia 12(4) (2016) 459-509. [29] H. Braak, E. Braak, J. Bohl, Staging of Alzheimer-related cortical destruction, European neurology 33(6) (1993) 403-408. [30] J.F. Guzowski, J.J. Knierim, E.I. Moser, Ensemble dynamics of hippocampal regions CA3 and CA1, Neuron 44(4) (2004) 581-584. [31] P. Goverdhan, A. Sravanthi, T. Mamatha, Neuroprotective effects of meloxicam and selegiline in scopolamine-induced cognitive impairment and oxidative stress, International Journal of Alzheimer’s Disease 2012 (2012). [32] U. Ebert, W. Kirch, Scopolamine model of dementia: electroencephalogram findings and cognitive performance, European journal of clinical investigation 28(11) (1998) 944-949. [33] Y. Zhang, X. Yang, G. Jin, X. Yang, Y. Zhang, Polysaccharides from Pleurotus ostreatus alleviate cognitive impairment in a rat model of Alzheimer’s disease, International journal of biological macromolecules 92 (2016) 935-941. [34] K. Skalicka-Wozniak, B. Budzynska, G. Biala, A. Boguszewska-Czubara, Scopolamineinduced memory impairment is alleviated by xanthotoxin: role of acetylcholinesterase and oxidative stress processes, ACS chemical neuroscience 9(5) (2018) 1184-1194. [35] B. Wang, Y. Zhong, C. Gao, J. Li, Myricetin ameliorates scopolamine-induced memory impairment in mice via inhibiting acetylcholinesterase and down-regulating brain iron, Biochemical and biophysical research communications 490(2) (2017) 336-342. [36] R. Kaur, S. Parveen, S. Mehan, D. Khanna, S. Kalra, Neuroprotective effect of ellagic acid against chronically scopolamine induced Alzheimer’s type memory and cognitive dysfunctions: possible behavioural and biochemical evidences, Int. J. Preven. Med. Res 1 (2015) 45-64. [37] P. De Sarno, S.A. Shestopal, T.D. King, A. Zmijewska, L. Song, R.S. Jope, Muscarinic receptor activation protects cells from apoptotic effects of DNA damage, oxidative stress, and mitochondrial inhibition, Journal of Biological Chemistry 278(13) (2003) 11086-11093. [38] M. Rosas Ballina, K. Tracey, Cholinergic control of inflammation, Journal of internal medicine 265(6) (2009) 663-679. [39] R.R. Resende, A. Adhikari, Cholinergic receptor pathways involved in apoptosis, cell proliferation and neuronal differentiation, Cell Communication and Signaling 7(1) (2009) 20.

21

Jo

ur na

lP

re

-p

ro of

[40] Y. Kuroda, M. Kawahara, Aggregation of amyloid β-protein and its neurotoxicity: enhancement by aluminum and other metals, The Tohoku journal of experimental medicine 174(3) (1994) 263-268. [41] G.M. Bishop, S.R. Robinson, Q. Liu, G. Perry, C.S. Atwood, M.A. Smith, Iron: a pathological mediator of Alzheimer disease?, Developmental neuroscience 24(2-3) (2002) 184187. [42] A.I. Bush, Metal complexing agents as therapies for Alzheimer’s disease, Neurobiology of aging 23(6) (2002) 1031-1038. [43] A.M. Fredenburg, R.K. Sethi, D.D. Allen, R.A. Yokel, The pharmacokinetics and bloodbrain barrier permeation of the chelators 1, 2 dimethyl-, 1, 2 diethyl-, and 1-[ethan-1′ ol]-2methyl-3-hydroxypyridin-4-one in the rat, Toxicology 108(3) (1996) 191-199. [44] F. Molina Holgado, A. Gaeta, P.T. Francis, R.J. Williams, R.C. Hider, Neuroprotective actions of deferiprone in cultured cortical neurones and SHSY 5Y cells, Journal of neurochemistry 105(6) (2008) 2466-2476. [45] A. Martin-Bastida, R.J. Ward, R. Newbould, P. Piccini, D. Sharp, C. Kabba, M.C. Patel, M. Spino, J. Connelly, F. Tricta, R.R. Crichton, D.T. Dexter, Brain iron chelation by deferiprone in a phase 2 randomised double-blinded placebo controlled clinical trial in Parkinson’s disease, Scientific Reports 7(1) (2017) 1398. [46] C.A. Rottkamp, A.K. Raina, X. Zhu, E. Gaier, A.I. Bush, C.S. Atwood, M. Chevion, G. Perry, M.A. Smith, Redox-active iron mediates amyloid-β toxicity, Free Radical Biology and Medicine 30(4) (2001) 447-450. [47] A. Nigam, M. Kulshreshtha, D. Panjwani, Pharmacological evaluation of Hibiscus abelmoschus against scopolamine-induced amnesia and cognitive impairment in mice, Advances in Human Biology 9(2) (2019) 116. [48] C.M. Hernandez, R. Kayed, H. Zheng, J.D. Sweatt, K.T. Dineley, Loss of α7 nicotinic receptors enhances β-amyloid oligomer accumulation, exacerbating early-stage cognitive decline and septohippocampal pathology in a mouse model of Alzheimer's disease, Journal of Neuroscience 30(7) (2010) 2442-2453. [49] D.-Y. Choi, Y.-J. Lee, S.Y. Lee, Y.M. Lee, H.H. Lee, I.S. Choi, K.-W. Oh, S.B. Han, S.Y. Nam, J.T. Hong, Attenuation of scopolamine-induced cognitive dysfunction by obovatol, Archives of pharmacal research 35(7) (2012) 1279-1286. [50] M. Kristensen, Neurotransmitters in Alzheimer's disease, Ugeskrift for laeger 152(30) (1990) 2165-2168. [51] R.J. Ward, F.A. Zucca, J.H. Duyn, R.R. Crichton, L. Zecca, The role of iron in brain ageing and neurodegenerative disorders, The Lancet Neurology 13(10) (2014) 1045-1060. [52] S. Palmqvist, M. Schöll, O. Strandberg, N. Mattsson, E. Stomrud, H. Zetterberg, K. Blennow, S. Landau, W. Jagust, O. Hansson, Earliest accumulation of β-amyloid occurs within the default-mode network and concurrently affects brain connectivity, Nature communications 8(1) (2017) 1214.

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Figure legends

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Figure 1: Experimental design

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Control SCOP Deferiprone (5 mg/kg) + SCOP Deferiprone (10 mg/kg) + SCOP Deferiprone (20 mg/kg) + SCOP Deferiprone (20 mg/kg)

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a

a

40

b b

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a

Step-Through latency time (sec)

5 ay

4

D

ay D

D

ay

ay D

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10

b

3

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2

1 ay D

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Time Spent in Platform quadrant (sec)

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b

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0

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a

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Time to reach platform in training days (Mean) sec

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400

b

300

a 200

100

0

Figure 2: The effect of Deferiprone on (A) 5-days Morris water maze latency time, (B) probe test of water maze and (C) step through latency of passive avoidance task of SCOP-treated rats. Data were analyzed by two-way ANOVA followed by Bonferroni post-hoc test, one-way ANOVA followed by Tukey post-hoc test and Kruskal Wallis followed by Dunn’s post-hoc test, respectively. SCOP was

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bb

700

b

600

400

a

200

b 600

b 500

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Control SCOP Deferiprone Deferiprone Deferiprone Deferiprone

(5 mg/kg) + SCOP (10 mg/kg) + SCOP (20 mg/kg) + SCOP (20 mg/kg)

a

300

0

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b

(U /gtissue)

C ortical C atalaseA ctivtiy

800

(U/gtissue)

Hippocam pal CatalaseActivtiy

administered at a dose of 1.14 mg/kg for 7 days starting on day 7 one hour after Deferiprone administration and Deferiprone was administered at doses of 5, 10, 20 mg/kg for 14 days. Morris water maze latency time was measured for 5 days starting from day 7 to day 11, probe test was performed on day 11 and passive avoidance acquisition training was performed on day 12, while the test on day 13. a, b, are considered statistically significant compared to control group and SCOP-treated group, respectively, at P<0.05. Data are represented as mean ± SEM (n = 6,7) for (A), (n = 6) for (B), and as medians and interquartile range (n = 10) for (C).

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Figure 1

Figure 3: Effects of SCOP and/or Deferiprone on hippocampal catalase activity (A) and

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cortical catalase activity (B). One-way ANOVA showed statistical significance between groups

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(P<0.0001, P<0.01), respectively followed by Tukey post-hoc test for further statistical analyses. a and b are considered statistically significant compared to control group and SCOP-

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120

b

260 240

Control SCOP Deferiprone Deferiprone Deferiprone Deferiprone

(5 mg/kg) + SCOP (10 mg/kg) + SCOP (20 mg/kg) + SCOP (20 mg/kg)

a

220 200 180

Figure 2

Figure 4: Effects of SCOP and/or Deferiprone on hippocampal GSH levels (A) and cortical

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140

m m o l/gtis s u e

a 160

C o rtic a lG S H

180

b

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m m o l/gtis s u e

H ip p o c a m p a lG S H

200

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treated group, respectively, at P<0.05. Data are represented as mean ± SEM (n = 6)

GSH levels (B). One-way ANOVA showed (P< 0.001, P<0.0001) respectively followed by Tukey post-hoc test. a and b are considered statistically significant compared to control group and SCOP-treated group, respectively, at P<0.05. Data are represented as mean ± SEM (n = 6)

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6

a

Control SCOP Deferiprone Deferiprone Deferiprone Deferiprone

a Cortical M DA

b

12

b

10

b

(m ol/gtissue)

(m ol/gtissue)

Hippocam pal M DA

14 5

b

(5 mg/kg) + SCOP (10 mg/kg) + SCOP (20 mg/kg) + SCOP (20 mg/kg)

4

8 6

3

Figure 3

Figure 5: Effects of SCOP and/or Deferiprone on hippocampal MDA levels (A) and cortical MDA levels (B). One-way ANOVA showed (P< 0.0001, P<0.01) respectively followed by Tukey post-hoc test. a and b are considered statistically significant compared to

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control group and SCOP-treated group, respectively, at P<0.05. Data are represented as mean ±

b 16

b

a

b

15

10

14

B

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18

20

b

Control SCOP Deferiprone Deferiprone Deferiprone Deferiprone

(5mg/kg) + SCOP (10mg/kg) + SCOP (20mg/kg) + SCOP (20mg/kg)

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a

(M/min/mg tissue)

A

Cortical AChE Activity

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Figure 4

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Figure 6: Effects of SCOP and/or Deferiprone on hippocampal AChE activity (A) and cortical AChE activity (B). One-way ANOVA showed statistical significance between the groups (P<0.001, P<0.0001), respectively followed by Tukey post-hoc test for further statistical analyses. a, b are considered statistically significant compared to control group and SCOP-treated group, respectively, at P<0.05. Data are represented as mean ± SEM (n = 6).

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(M/min/mg tissue)

Hippocampal AChE Activity

SEM (n = 6)

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Figure 7: H & E staining of CA1 region of the hippocampi of rats belonging to the control group (A), SCOP-treated group (B), Deferiprone (10 mg/kg) and SCOP-treated group

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(C), Deferiprone (20 mg/kg)-treated group (D). Group (A) showed normal histological

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architecture of molecular, pyramidal and polymorphic layers most of neurons are apparent intact having vesicular nuclei, group (B) showed hypo-cellularity of pyramidal cells layer with

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scattered shrunk cells with pyknotic nuclei (arrow), glial cells infiltration (arrow head) and mild edema of neutrophil, group (C) showed increased number of pyramidal cells layer with

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few degenerating cells with pyknotic nuclei (arrow) and mild glial cells infiltration, group (D)

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showed apparent intact neurons and organized hippocampal layers.

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Figure 8: H & E staining of CA3 region of the hippocampi of rats belonging to the

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control group (A), SCOP-treated group (B), Deferiprone (10 mg/kg) and SCOP-treated group (C), Deferiprone (20 mg/kg)-treated group (D). Group (A) showed many apparent intact

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pyramidal cells with vesicular nuclei and mild glial cells infiltration, group (B) showed

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scattered degenerating cells with pyknotic nuclei (arrow), intercellular edema (star) and glial cells infiltration in between, group (C) showed apparent intact neurons associated with

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increased numbers of glial cells (arrow), group (D) showed apparent intact neurons and

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organized hippocampal layers with mild glial cells infiltration.

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Figure 9: H & E staining of dentate gyrus region of the hippocampi of rats belonging to the control group (A), SCOP-treated group (B), Deferiprone (10 mg/kg) and SCOP-treated

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group (C), Deferiprone (20 mg/kg)-treated group (D). Group (A) showed many apparent intact

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granular cells, group (B) showed many degenerated and vacuolated neurons with dark pyknotic nuclei (arrow) and edema, group (C) showed apparent intact neurons, mild edema and glial

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cells infiltration (arrow head), group (D) apparent intact neurons with mild edema.

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Figure 10: H & E staining of outer cortical layers of rats belonging to the control group (A), SCOP-treated group (B), Deferiprone (10 mg/kg) and SCOP-treated group (C),

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Deferiprone (20 mg/kg)-treated group (D). Groups (B) and (C) showed diffuse neuronal

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degeneration with many shrunken dark pyknotic nuclei (arrow) and moderate intercellular edema as well as perineuronal edema. (B) samples also showed more pronounced glial cells

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infiltration (arrow head). Group (D) showed slightly protected cortical region with few

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numbers of apparent intact neurons with vesicular nuclei (red arrow).

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Figure 11: Prussian blue staining of iron in rats belonging to the control group (A), SCOP-treated group (B), Deferiprone (10 mg/kg) and SCOP-treated group (C), Deferipronealone-treated group (D). The control group demonstrated negative reactivity for Prussian blue staining in different brain regions, the cortex (A1) and hippocampus (A2). SCOP-treated rats

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showed many focal areas of ferric iron positivity for Prussian blue staining in the cortex (B1) and hippocampus (B2). SCOP- and Deferiprone (10 mg/kg) - showed negative reactivity for Prussian blue staining and cortical region (C1) with minimally detected focal positive deposits in hippocampus (C2). Deferiprone-alone-treated rats showed negative reactivity in both the cortex (D1) and hippocampus (D2)

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Figure 12: Congo red staining of Aβ in rats belonging to the control group (A), SCOP-

treated group (B), Deferiprone (10 mg/kg) and SCOP-treated group (C), Deferiprone-alonetreated group (D). Groups A, C and D revealed negative reactivity to Congo red stain in different brain regions, Group B showed multiple positive plaques of Aβ deposits in cerebral cortex and negative reactivity in hippocampus.

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