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Protective actions of quercetin in
manganese-induced neurotoxicity;
behavioral, neurostructural, and
neurochemical evaluations
1,2,3
Ijomone, O.M., 2Okori, S.O., 4Ebokaiwe, A.P.

Website: Abstract
jecajournal.com
Doi: BACKGROUND AND AIM: Excessive exposure to manganese (Mn) alters neuronal structure and
doi.org/10.4314/jeca.v20i2.7 functions. This leads to manganism, a neurological syndrome similar but not identical to Parkinson’s
disease. Therapeutic interventions for this Parkinsonian syndrome have been largely unsuccessful due
to little knowledge on mechanism of manganese induced brain damage. Oxidative stress is implicated
as one mechanism of Mn induced neurotoxicity. Here, we investigated the effects of quercetin, a potent
antioxidant obtained from fruits and vegetables on Mn induced neurotoxicity.
METHODOLOGY: Adult male rats were exposed to either Mn only or Mn co-administered with 5 or
10 mg/kg of quercetin, while Control rats were treated with normal saline. All treatments were via
intraperitoneal injections for 5 weeks. After treatments, behavioral assessments were performed.
Following which, rats were sacrificed, brain excised for microanatomical analysis and biochemical
quantification of oxidative stress markers.
Submitted: 4th December, 2023 RESULTS: Cognitive and locomotor associated behaviors were significantly impaired in Mn only
Revised: 16th January, 2024 treatment, however quercetin co-administration attenuated only cognitive behavior. Mn treatment
Accepted: 18th January, 2024 induced degenerative changes in brain neurons, accompanied by astrogliosis (abnormal increase in
Published: 15th April, 2024 astrocyte population). Co-administration with quercetin reduced these microstructural deficits.
Additionally, quercetin co-treatment reduced oxidative stress imposed on brain tissues in Mn only
1Department
treatment.
of Human CONCLUSION: These results suggest that the dietary antioxidant quercetin may attenuate structural
Anatomy, School of Health and and behavioral anomalies associated with Mn overexposure, probably due to its ability to resist oxidative
Health Technology, Federal stress.
University of Technology Akure,
Nigeria; 2Department of Keywords:
Anatomy, Faculty of Basic Manganese; quercetin; brain; behavior; oxidative stress
Medical Sciences, Cross River INTRODUCTION
University of Technology, Okuku Globally, overexposures to heavy metals (PD) and results in a parkinsonian syndrome
Campus, Cross River, Nigeria; such as manganese (Mn) are increasingly of known as manganism. This syndrome is
3Laboratory for Experimental
health concern. Mn, an important trace characterized by variety of psychiatric,
and Translational Neurobiology, metal is regularly found within the cognitive and motor disturbances that are
University of Medical Sciences, environment. Mn exist in all tissues and is akin to those inherent to Parkinson’s disease
Ondo, Nigeria; 4Department of required for the maintenance and regulation (PD) (Crossgrove and Zheng, 2004; Erikson et
Chemistry/Biochemistry and of various biochemical and cellular functions al., 2005; Cersosimo and Koller, 2006;
Molecular Biology, Federal (Sidoryk-Wegrzynowicz and Aschner 2013). Sidoryk-Wegrzynowicz and Aschner 2013).
University Ndufu Alike-Ikwo, Neurons and astrocytes are key targets for
Nonetheless, in humans, too much
Ebonyi, Nigeria. Mn upon entering the brain. Hence
accumulation of Mn in the brain from
overexposure is associated with neurological astrocytes serve as main homeostatic
.
deficits. Overexposure to Mn is an regulator and storage site for Mn in the brain
environmental risk factor for Parkinson’s disease (Milatovic et al., 2009).

Address for Correspondence: This is an open access journal, and articles are distributed How to cite this article: Ijomone, O.M., Okori S.O.,
Ijomone, O.M. under the terms of the Creative Commons Attribution‑Non Ebokaiwe A.P. Protective actions of quercetin in
Laboratory for Experimental Commercial‑Share Alike 4.0 License, which allows others to manganese-induced neurotoxicity; behavioral,
remix, tweak, and build upon the work non‑commercially, as neurostructural, and neurochemical evaluations J Exp
and Translational long as appropriate credit is given and the new creations are Clin Anat 2023; 20(2):39-49.
Neurobiology, University of licensed under the identical terms.
doi.org/10.4314/jeca.v20i2.7
Medical Sciences, Ondo, For reprints contact: jecajournal@gmail.com
Nigeria.
omijomone@unimed.edu.ng

Journal Of Experimental and Clinical Anatomy – Volume 20, Issue 2, December, 2023 39
 Ijomone et al.: Quercetin protects against Mn neurotoxicity

However, amplified accumulation of Mn in astrocytes may Group 4: received MnCl2 and 10 mg/kg of quercetin.
modify glutamate release and elicit excitatory neurotoxicity
(Erikson and Aschner, 2003). Mn favorably accumulates in All administrations were via daily intraperitoneal injections
mitochondria, where it upsets oxidative phosphorylation and for 5 weeks. Doses used for rat models of Mn toxicity were
intensifies the generation of reactive oxygen species (ROS). generated based on previous studies that have shown
Increased striatal concentrations of ascorbic acid and significant increase in Mn accumulation in brain tissues and
glutathione (GSH), antioxidants that when increased, signal biochemical alterations in rats (Bouabid et al., 2014; Cordova
the presence of elevated burden from ROS, as well as other et al., 2012; Marreilha dos Santos et al., 2011). Additionally,
markers of oxidative stress, have been previously reported the selection of the dose, route and duration was based on
(Milatovic et al., 2009). Oxidative stress is implicated as one previous studies (Chan et al., 2014; Dong et al., 2014; Nassiri-
of the mechanisms of neurotoxicity by Mn (Karki et al., 2013; Asl et al., 2013; Bhutada et al., 2010), which have shown the
Avila et al., 2012). effectiveness of intraperitoneally administered quercetin in
protecting against damage in major organs.
Quercetin is a classic flavones-type flavonoid; ubiquitously
distributed in fruits and vegetables and comprises the Neurobehavioral studies
dominant part of flavonoids from daily foods. Quercetin, is a Rats were kept over-night in behavior testing room for
potent antioxidant, known to prevent oxidative injury and cell acclimatization before administration. Behavioral studies
death by chelating metal ions, scavenging oxygen radicals and were carried out 1 hour after last administration in quiet
defending against lipid peroxidation (Ebokaiwe et al., 2016). room between the hours of 10 am and 3 pm. The apparatuses
Previous study has shown the protective effect of dietary were cleaned with 5% ethanol before testing a new animal to
quercetin against oxidative toxicity induced by lead, eliminate possible bias due to odours left by previous animal.
cadmium, atrazine on several organs in rat, including liver, The tests were recorded using a digital camera and later
renal, testis (Ebokaiwe and Farombi, 2015). scored by trained blind observers.
Given that oxidative stress is one of the major mechanisms Y-maze test: The tests were performed as previously
characterizing Mn induced neurotoxicity, it is hypothesized described (Ijomone et al., 2015; Kim et al., 2008) using
that quercetin may attenuate toxic effects of Mn on brain protocols adapted from Mori et al. (2001). Y-maze task
structures and functions. In view of the foregoing, the present assesses short-term spatial memory as a measure of cognitive
study investigated the effects of quercetin on behavior and functions using spontaneous alternation behaviors of rats. In
neuronal histology following Mn-induced neurotoxicity in rat this test, rats are placed on a start arm of a Y-shaped maze
models. and allowed to move freely for 8 min. Hind paws of the rats
have to be completely within an arm to be considered as rats
Materials and Methods
having entered the arm. Entering all 3 arms in the overlapping
Animal care and treatments triplet sets is defined as spontaneous alternation. The
percentage of spontaneous alternation is calculated as
Twenty-four male adult albino strain Wistar rats (150-200 g) {spontaneous alternation / (total number of arm entries – 2)}
were used for this study. They were bred at the Animal × 100.
Holdings of Faculty of Basic Medical Sciences of the
University. Animals were housed in clean plastic cages in a Open-field test (OFT): The tests were carried out as previously
clean environment of 12 hours day/light cycle, at room described (Ijomone et al., 2014; Brown et al., 1999) in an
temperature. Animals in all groups were allowed access to apparatus consisting of a box (72x72x36 cm) with the floor
standard laboratory rat pellets and water ad libitum. All divided into 18x18 square units. In this test, animals are
experimental protocols were in strict accordance with the placed in the centre of the box and allowed to move freely for
guidelines for animal research, as detailed in the NIH 5 min. In the present study, we obtained the following
Guidelines for the Care and Use of Laboratory Animals (NIH, parameters; locomotion frequency (number of crossings
2011) and conformed to local institutional guidelines for the from one square to the other), rearing frequency (number of
protection of animal welfare during the experiments. times the animals stood on their hind paws), rearing against
the wall (no of times the animals stood on their hind paws
The rats were randomly assigned into 4 groups of 6 animals against the wall), hinding (calculated by adding the rearing
and treated as follows; frequency to rearing against the wall).
Group 1: Control rats received normal saline Histology and histomorphometry
Group 2: received 10 mg/kg manganese chloride (MnCl2) as Following behavioural tests, rats were sacrificed and brains
MnCl2 tetrahydrate (MnCl2.4H2O, Sigma-Aldrich, US). were harvested and fixed using neutral buffered formalin.
The tissues were processed with the routine tissue processing
Group 3: received MnCl2 and 5 mg/kg of quercetin (Sigma-
procedure and stained with haematoxylin and eosin using
Aldrich, US).
established protocols (Bancroft and Gamble 2008).

Journal Of Experimental and Clinical Anatomy – Volume 20, Issue 2, December, 2023 40
 Ijomone et al.: Quercetin protects against Mn neurotoxicity

Furthermore, separate sections were stained with tetramethylbenzidine and 100mM H2O2 in 43mM NaH2PO4
phosphotungstic acid hematoxylin (PTAH) – a specific (pH 5.4) according to Eiserich at al., (1998).
technique for demonstrating astrocytes– as described by
Drury and Wallington, (1980). Briefly, Mallory’s PTAH Determination of Nitric oxide (NO) level (nitrite): Nitric oxide
prepared with 0.1 g hematoxylin and 2 g phosphotungstic measured as nitrite was determined by using Griess reagent,
acid was dissolved in 100 ml of distilled water and allowed to according to the method of Moshage et al. (1995) where
naturally ripen. Deparaffinized sections were treated with 4% nitrite, stable end product of NO radical, is mostly used as an
iron alum for about 60 minutes, rinsed in water, and indicator for the production of NO.
transferred to Mallory PTAH for about 18 hours. Sections Statistical analysis
were blot dry with filter paper, differentiated rapidly in
absolute alcohol, cleaned, air dried and mounted. Data were expressed as mean±SEM. Data comparisons were
performed using One-way ANOVA, followed by Student
A digital microscope (OMAX microscopes, Irvine, CA, USA) Newman-Keuls (SNK) for post hoc. GraphPad Prism (Version
was utilized to obtain photomicrographs of the region of the 5.03, GraphPad Software, USA.) was the statistical package
hippocampus and striatum were obtained using landmarks used for data analysis. Statistical significance was set at
from the rat brain atlas (Paxinos and Watson 2007). Normal P<0.05.
appearance (intact) and degenerating neuronal cells were
identified and counted at x400 magnification using the cell RESULTS
counter tool on Image Analysis and Processing for Java (Image
J) program. The percentage of intact neurons was calculated Y-maze and Open field tests
as number of intact neurons divided by total number of
Percentage of spontaneous alternation on the Y-maze was
neurons and then multiplied by 100. Astrocytes were also
significantly decreased following MnCl2 administration
identified and counted in PTAH stained sections.
compared to the Control. Co-administration with quercetin at
Biochemical assays 10 mg/kg significantly attenuated MnCl2 effects on Y-maze
spontaneous alternation but not at 5 mg/kg [F3,16=6.06;
Brains samples from the Control and treated rats were P=0.0059] (Fig. 1). Locomotion frequency on the OFT was
separately homogenized in eight volumes of 50 mM of Tris- significantly reduced following MnCl2 administration
HCl buffer (pH 7.4) containing 1.15% potassium chloride, and compared to the Control, an effect that was not improved
the homogenates were centrifuged at 10,000×g for 15 significantly with quercetin administration [F3,20=5.16;
minutes at 4 ºC. Supernatant; post mitochondria fraction was P=0.0084]. On the other hand, hinding was significantly
collected for enzyme assays. lowered following MnCl2 treatment compared to the Control,
however no effect was observed following quercetin co-
Determination of catalase (CAT) activity: CAT activity was
treatment [F3,20=3.57; P=0.0323] (Fig. 2).
assayed by the method of Claiborne (1995).

Determination of superoxide dismutase (SOD) activity:

Superoxide dismutase activity was determined by measuring
the inhibition of autoxidation of epinephrine (at pH 10.2) at
30°C by the method of Misra and Fridovich (1972).

Determination of glutathione peroxidase (GSH-Px) activity:

Activity of GSH-Px was determined by the method of Rotruck
et al. (1973).

Determination of glutathione S-transferase (GST) activity: GST

activity was determined by the method of Habig et al. (1974)
using CDNB as a substrate.

Determination of reduced glutathione (GSH) level: GSH was

determined according to Jollow et al. (1974).

Determination of lipid peroxidation (LPO) level: LPO was

quantified as malondialdehyde (MDA), according to the
Fig. 1. Effect of Mn and Quercetin co-administered with Mn
method described by Farombi et al. (2000).
on short term memory on the Y-maze task. Values are
Determination of Myeloperoxidase (MPO) activity: An expressed as mean + SEM. *P<0.05, **P<0.01. One-way
aliquote of the post-mitochondrial supernatant of the brain ANOVA followed by SNK for post-tests.
homogenate was allowed to react with a solution of 1.2 mM

Journal Of Experimental and Clinical Anatomy – Volume 20, Issue 2, December, 2023 41
 Ijomone et al.: Quercetin protects against Mn neurotoxicity

large nuclei and prominent nucleoli. On the other hand,

striatal neurons are predominantly medium-sized with
prominent nuclei and nucleoli. Treatment with MnCl2 showed
increases in neurons with degenerating features in both
hippocampus and striatum. Neurodegenerating features in
routinely stained brain sections include; prominent
eosinophilic cytoplasm, pyknotic nuclei, neuron swelling
and/or vacuolation within the cytoplasm (Garman, 2011).
Image J cell count analysis revealed significant decrease in
percentage of intact neurons following MnCl2 treatment
compared to the Control, in both hippocampus [F3,12=15.76;
P=0.0002] and striatum [F3,12=205.4; P<0.0001]. However,
neurodegeneration was worse in the striatum with over 50%
reduction in intact neurons. Co-administration with quercetin
significantly improved percentage of intact neurons in both
brain regions, with 10 mg/kg quercetin treatment better
improved compared to 5 mg/kg treatment (Fig. 3).

PTAH staining for astrocytes showed that MnCl2 induced

astrocytosis in hippocampus and striatum. Astrocytosis or
astrogliosis is an abnormal increase in astrocyte population.
MnCl2 treatment significantly increased number of astrocytes
compared to the Control in both the hippocampus
[F3,12=14.29; P=0.0003] and striatum [F3,12=14.48; P=0.0003].
Co-administration with quercetin at 10 mg/kg attenuated the
observed astrocytosis in these brain regions, however 5
mg/kg quercetin co-treatment showed no such attenuating
effect (Fig. 4).

Biochemical assays of markers of oxidative stress

There was significant decrease in activities of SOD

[F3,20=43.99; P<0.0001], CAT [F3,20=131.5; P<0.0001], GST
[F3,20=67.62; P<0.0001], and GSH-Px [F3,20=52.65; P<0.0001]
following MnCl2 administration compared to the Control.
Quercetin co-administration attenuated the effects of MnCl2
on these oxidative stress markers, with 10 mg/kg quercetin
treatment better improving the activities of these markers
Fig. 2. Effect of Mn and Quercetin co-administered with Mn on compared to 5 mg/kg quercetin (Fig. 5).
locomotor and exploratory activities on the OFT. Values are
expressed as mean + SEM. *P<0.05, **P<0.01. One-way ANOVA Levels of GSH [F3,20=19.15; P<0.0001] were significantly
followed by SNK for post-tests. lowered following MnCl2 administration compared to the
Control, with quercetin co-treatment also attenuating this
effect. On the other hand, levels of LPO [F3,20=26.16;
Histology and histomorphometry P<0.0001] and NO [F3,20=38.12; P<0.0001], and MPO
[F3,20=34.18; P<0.0001], activity were significantly increased
Histologically, intact neurons in the hippocampal pyramidal with MnCl2 administration, while quercetin co-treatment at
layer are characterized by their large pyramidal shape, with both 5 and 10 mg/kg reduced these effects (Fig. 6).

Journal Of Experimental and Clinical Anatomy – Volume 20, Issue 2, December, 2023 42
 Ijomone et al.: Quercetin protects against Mn neurotoxicity

Fig. 3. (A) – Photomicrographs of hippocampus (CA3) and striatum (CPu) of control and treated rats. H&E x 400, Scale bars – 25 µm. Arrows
– intact neurons; arrow heads – prominent eosinophilic cytoplasm; dashed arrows – neuron swelling and/or vacuolation within the
cytoplasm. Boxes a, b, c, and d, are enlarged to show details of histological changes in (B). (C) – Image J analysis of H&E stained sections.
Values are expressed as mean + SEM. **P<0.01, ***P<0.001. One-way ANOVA followed by SNK for post-tests.

Fig. 4. (A) – Photomicrographs of PTAH stained sections of the hippocampus (CA3) and striatum (CPu) of control and treated rats. PTAH x
400, Scale bars – 25 µm. PTAH technique particularly marks astrocytes as purplish to bluish cells (dashed arrows) on brain sections. Neurons
in the sections of the hippocampus and striatum are clearly identified (arrows). (B)– Image J analysis of PTAH stained sections. Number of
astrocytes where identified and counted. Values are expressed as mean + SEM. *P<0.05, **P<0.01. One-way ANOVA followed by SNK for
post-tests.

Journal Of Experimental and Clinical Anatomy – Volume 20, Issue 2, December, 2023 43
 Ijomone et al.: Quercetin protects against Mn neurotoxicity

Fig. 5. Effect of Mn and Quercetin co-administered with Mn on activities of SOD (A), CAT (B), GST (C), and GSH-Px (D). Values are expressed
as mean + SEM. **P<0.01, ***P<0.001. One-way ANOVA followed by SNK for post-tests.

Fig. 6. Effect of Mn and Quercetin co-administered with Mn on GSH (A), LPO (B), MPO (C), and NO (D). Values are expressed as mean + SEM.
*P<0.05, ***P<0.001. One-way ANOVA followed by SNK for post-tests.

Journal Of Experimental and Clinical Anatomy – Volume 20, Issue 2, December, 2023 44
 Ijomone et al.: Quercetin protects against Mn neurotoxicity

DISCUSSION (Daoust et al., 2014). Another study on mouse striatum

showed that Mn treatment resulted in neuronal injury that
Overexposure to Mn induces parkinsonian-like syndrome was evident by the presence of condensed, pyknotic neurons
that includes behavioral deficits such as motor un- and presence of dying neurons (Liu et al. 2006a). It was
coordination and cognitive impairment, as well as possible that compromised behaviors observed in the
degenerative changes to certain brain regions (Liu et al., present study following Mn administration, was due to the
2006a). In the present study, the protective effect of increased degeneration of neurons in these brain regions.
quercetin on Mn-induced neuronal dysfunction in rat was The hippocampus is well recognized for its role in cognitive
revealed. processes of memory and learning (Squire, 2009; Moser et
al., 2008). The striatum on the other hand, is associated with
In the present study, behavioral analysis of performance on
central control of overall control of locomotor functions
the Y-maze revealed that quercetin co-administration with
(Voytek and Knight, 2010). It is also likely that the greater
Mn exposure at 10 mg/kg significantly attenuated memory
neuronal damage imposed on the striatum by Mn exposure
deficits caused by Mn exposure. Spontaneous alternation is a
may have resulted in the inability of quercetin co-
classic indicator of short-term memory on the Y-maze
administration to attenuate Mn-impaired locomotor related
(Ijomone et al., 2015). In support of our present observation
deficits. This is in contrast to attenuating effect of quercetin
on short-term memory, several studies have shown cognitive
treatment on Mn-impaired cognitive abilities, probably due
enhancing effects of quercetin in experimental rodent
to the less damage on hippocampal neurons.
models where memory impairment was induced as well as in
rodent models with no induced memory deficits. Quercetin The present study also showed that Mn induced astrocytosis
attenuates detrimental effects on spatial learning and in both the hippocampus and striatum. Several studies have
memory on the Morris water maze following cognitive revealed that astrocytosis is indicative of neurotoxicity and
impairment induced by chronic stress (Mohammadi et al., consequent neuronal injuries (Jahanshahi et al., 2013;
2014), in 6-OHDA rat model of Parkinson disease (Sriraksa et Granado et al., 2010; Granado et al., 2011; Adori et al., 2006;
al., 2012), and in D-galactose-induced aged mice (Liu et al., Aguirre et al., 1999), as we have also observed in the present
2006b). Quercetin enhances memory retrieval of kindled rats study following Mn treatment. Liu et al. (2006a) observed
in the passive avoidance test (Nassiri-Asl et al., 2013), and that increase in astrocyte-derived NO together with
ameliorate cognitive dysfunction in diabetic rats (Bhutada et astrocytosis is involved in neurodegeneration imposed on the
al., 2010). Additionally, quercetin showed cognitive striatum following Mn treatment. Our present study revealed
enhancing effects when administered to normal rats via that quercetin at 10 mg/kg attenuated abnormal astrocyte
intranasal route (Tong-Un et al., 2010). proliferation and this may be a contributing mechanism in
protecting against neuronal damage.
The primary interest in the OFT is movement, which is greatly
influenced by locomotor output and exploratory drive. Previous studies have shown that exposure to Mn
Locomotion frequency and rearing are indications of animals’ significantly induced oxidative stress in the brain, thus
overall locomotor and exploratory activities respectively eliciting various pathological conditions associated with
(Ijomone et al., 2014). Previous studies have shown that neurodegenerative diseases (Milatovic et al., 2009). Our
quercetin improves overall locomotor activities in several findings were consistent; having yielded the same
models of motor deficits. Quercetin was shown to improve observations in the brain of rats exposed to Mn. Oxidative
motor coordination, balance and gait in MPTP-induced stress and formation of free radicals are the major factors of
mouse model of Parkinson’s disease (Lv et al., 2012) and 3- many neurodegenerative disorders (Ebokaiwe and Farombi,
NP-induced model of Huntington’s disease (Sandhir and 2015). Among the prominent antioxidant enzymes; SOD is at
Mehrotra, 2013). However, behavioral analysis on the OFT in the front line, it helps to dismutate superoxide radicals to less
the present study showed that though quercetin harmful product like H2O2 which also can be a potential
administration had no effect on reduced locomotion oxidant. CAT and GSH-Px acts in tandem to convert the
following Mn administration, it prevented reduction in generated H2O2 to water and less harmful products
exploratory activity induced by Mn. (Ebokaiwe et al., 2013) whereas GST, a phase-II xenobiotic
detoxifying enzyme, helps in the excretion of the toxic
Here, we have examined microstructural changes to two
products of oxidative stress from the system (Ebokaiwe and
regions – hippocampus and striatum – of the brain due to
Farombi, 2015). However, when there is a surge in production
their high susceptibility to Mn deposition (Daoust et al., 2014;
of superoxide radicals, the integrity of these enzymes are
Robinson et al., 2013; Liu et al., 2006a; Erikson et al., 2005).
compromised which could be the reason for the significant
Histological analyses in the present study suggest that Mn
decrease in the activities of SOD, CAT, GSH-Px enzymes and
exposure imposes degeneration in the hippocampal and
GST, a phase-II xenobiotic detoxifying enzyme, following Mn
striatal neurons with percentage of neurodegeneration
administration. Induction of oxidative stress by Mn exposure
worse in the striatum. A previous study showed that mouse
was further confirmed by significant elevation in the levels of
primary hippocampal neurons are sensitive to Mn toxicity
LPO; which destroy and compromise the integrity of the

Journal Of Experimental and Clinical Anatomy – Volume 20, Issue 2, December, 2023 45
 Ijomone et al.: Quercetin protects against Mn neurotoxicity

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