Neurotoxicity of green- synthesized magnetic iron oxide nanoparticles in different brain areas of wistar rats

Neurotoxicology 77 (2020) 80–93

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Neurotoxicity of green- synthesized magnetic iron oxide nanoparticles in different brain areas of wistar rats

T

Heba M. Fahmya,*, Esraa M. Alya, Faten F. Mohamedb, Neveen A. Noorc, Anwar A. Elsayeda a

Biophysics Department, Faculty of Science, Cairo University, 12613, Giza, Egypt Pathology Department, Faculty of Veterinary Medicine, Cairo University, 12613, Giza, Egypt c Zoology Department, Faculty of Science, Cairo University, 12613, Giza, Egypt b

ARTICLE INFO

ABSTRACT

Keywords: Magnetic iron oxide nanoparticles (MIONs) Oxidative stress (OX) Inductively coupled plasma (ICP) Histopathology Toxicity Carob leaf

Aims: The aim of the present study was to evaluate the toxicity of magnetic iron oxide nanoparticles (MIONs) which were synthesized using carob leaf extract on various brain areas of Wistar rats. Main methods: Carob leaf synthesized-MIONs were characterized using different techniques: Dynamic Light Scattering (DLS), Transmission Electron Microscope (TEM), UV–vis spectrophotometer, Fourier Transform infrared (FTIR), X-Ray Diffraction (XRD) and Atomic Force Microscope (AFM). The toxicity of MIONs in vivo was evaluated by: monitoring rat’s body weight, measuring iron content in different brain areas, evaluating some oxidative stress parameters, estimating acetylcholinesterase (AChE) in addition to histopathological investigations. Key findings: The present study demonstrated no body weight changes of MIONs- treated rats. According to the conditions of the present study, the hippocampus and striatum were the most affected areas and demonstrated neuronal degeneration due to MIONs exposure. MIONs treatment of Wistar rats, also affected the iron homeostasis in both striatum and midbrain by decreasing iron content in these areas. The least affected areas were thalamus and cerebellum. The histopathological examination of brain areas demonstrated moderate neuronal degeneration in hippocampus and striatum, mild neuronal degeneration in cortex and slight degeneration in hypothalamus and pons-medulla areas were detected. Significance: The results suggested that MIONs have a toxic impact on different brain areas and the effect varies according to the brain area.

1. Introduction Nowadays, nanotechnology field is widely developing nanoparticles (NPs) with useful properties for biomedical treatment, therapy, and diagnostics including: hyperthermia, cell separation, labeling, drug delivery, magnetic particle imaging, etc. (Mohammed et al., 2017). Among many NPs used, magnetic nanoparticles (MNPs) have attracted great attention due to their unique features (Montazerabadi et al., 2015). One important class of MNPs is magnetic iron oxide nanoparticles (MIONs) which have been widely used because of their reactive surfaces that can be readily modified with biocompatible coatings (Keshtkar et al., 2018). MNPs can be produced by various methods, the co-precipitation method is the simplest and cost-effective method (Rajput and Kirubha, 2016). Other methods include: hydrothermal method (Jiao and Yang, 2008), sonochemical method (Islam et al., 2011), micro-emulsion method (Deng et al., 2003), electrochemical

⁎

route (Franger et al., 2004) and sol-gel technique (Unal et al., 2010). In contrast to the time-consuming chemical and physical methods which involve harsh procedures, green method is much easier, safer to use and eco-friendly. The previous literature survey demonstrated several studies concerning the green synthesis of MIONs (Cai et al., 2010; Lu et al., 2010; Chrysochoou et al., 2012; Venkateswarlu et al., 2013). The in vivo interaction of MNPs and biological systems is quite complicated and active (Schlachter et al., 2011). MNPs can distribute into various organs, tissues, and cells where they may remain in the same structure or become metabolized. The most in vivo MIONs nanotoxicities result from the overproduction of reactive oxygen species (ROS), including free radicals such as the superoxide anion, hydroxyl radicals and the non-radical hydrogen peroxide (Sharifi et al., 2012; Schrand et al., 2012). The condition of imbalance between ROS and antioxidant defenses, resulting in excessive accumulation of ROS, which is called oxidative stress (Dasuri et al., 2013). Brain is particularly

Corresponding author: Biophysics Department, Faculty of Science, Cairo University, Giza, Egypt. E-mail addresses: [email protected], [email protected], [email protected] (H.M. Fahmy).

https://doi.org/10.1016/j.neuro.2019.12.014 Received 10 August 2019; Received in revised form 22 December 2019; Accepted 27 December 2019 Available online 30 December 2019 0161-813X/ © 2019 Elsevier B.V. All rights reserved.

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vulnerable to oxidative stress for several reasons as its high consumption of oxygen (Niedzielska et al., 2016), it has a high level of polyunsaturated fatty acids in its cell membranes and react as substrates for lipid peroxidation. Iron and copper (which are examples of redox-active metals) are found abundantly in the brain and they are actively involved in catalyzing ROS formation, Brain has relatively low levels of glutathione (GSH) (which plays the role of endogenous antioxidant) (Ferreira et al., 2015). The effect of MNPs on the brain has been studied previously. Some studies showed that MNPs could not cross the blood brain barrier (BBB), Hanini et al. (Hanini et al., 2011). Also, there were studies on the ability of MNPs to cross the BBB without deleterious effect, Reddy et al. (Reddy et al., 2017). But, MNPs may have a deleterious effect on the brain, Kumari et al. (Kumari et al., 2013). Carob (Ceratonia siliqua) is considered as one of the important crops that has been commonly cultivated in the Mediterranean countries for many years and was used in the traditional medicine (Ahmed, 2010). Carob tree is a very valuable nutritional source both for humans and for animals and can even have major nutritional and medicinal applications, depending on their ability and safety (Drouliscos and Malefaki, 1980). Carob leaves have been known to be powerful antioxidant agent (Rasheed, 2006). Sassi et al. (Sassi et al., 2016) reported antioxidant properties of carob leaf extracts against cell DNA oxidative stress caused by H2O2, without genotoxicity. El-Sayyad et al. (El-Sayyad et al., 2017) aimed to link the promising function or phytochemical constituents of carob extracts in treating or protecting the brain disorders against neurotoxicity. The authors reported that Carob-supplementation pre or post treatment with monosodium glutamate may have a lowering effect on the accumulation of hydrogen peroxide in the extracellular space of the brain tissues being investigated and had possible therapeutic impacts (Kennedy and Wightman, 2011). In addition, carob phytochemical components such as ferulic acid have been reported its possible neuroprotection effect against monosodium glutamate treatment (Bravo et al., 1998; Yu et al., 2006). Also, the possibility of its ability as a treatment for neurodegenerative diseases as Alzheimer’s and Parkinson diseases which developed from elevated oxidative stress using natural antioxidant property (El-Sayyad et al., 2017; Mori et al., 2013). The promising of using ferulic acid as a treatment for Alzheimer’s disease arising from maintaining cell viability, increased superoxide dismutase, and inhibited the production of tumor necrosis factor-α and interleukin -1β induced amyloid-beta peptide 25- 35(Aβ25-30) formation (Nabavi et al., 2015). The interest in developing and using MIONs in several applications, especially, in biomedicine must be accompanied with a passion for the knowledge of the effect of these MIONs on the biological systems to be applied safely. Thus, in this study, the toxicity of intravenous (i.v.) injection of carob leaf-synthesized MIONs was evaluated through the measurement of free iron contents and some oxidative stress parameters in addition to the histopathological examinations of different brain areas in adult male Wistar rats.

Research-Lab Fine Chem Industries, India. Phosphate saline buffer (PSB) pH 7.4 (100 mM / l) was purchased from Bio diagnostic Co., Giza, Egypt. N-1-naphthylene diamine dihydro chloride was purchased from CARLO ERBA reagents, Spain. 85% Phosphoric Acid and Dipotassium hydrogen phosphate (K2HPO4) (99%) were purchased from ElGomhoria Co For Pharmaceutical, Egypt. 5,5' Dithiobis-(2-nitrobenzoic acid) (DTNB) (99%) was purchased from Acros Organics, US. Potassium dihydrogen (KH2PO4) (99%) was purchased from NICE chemicals, India. 96% Ethyl Alcohol (Ethanol) was acquired from Diachem chemicals, US. Carob Leaves were obtained from a local supplier (Giza, Egypt). 2.2. Methods 2.2.1. Preparation of carob leaf-synthesized MIONs MIONs were synthesized according to the method of Awwad & Salem, (Awwad and Salem, 2012). Briefly, 0.53 g of FeCl2.4H2O and 1.11 g of FeCl3.6H2O were dissolved in 100 ml of sterile deionized water. The mixture was heated at 80 °C under mild stirring using magnetic stirrer and under atmospheric pressure for 10 min. Then 5 ml of the aqueous solution of carob leaf extract was added to the mixture. 20 ml of NaOH aqueous solution (1 M) was added to the mixture after 5 min with a rate 3 ml / minute. After cooling, MIONs were obtained by decantation, dilution using sterile distilled water and centrifugation three times to remove heavy biomaterials. Washing for MIONs was done three times with distilled water at 9000 rpm for 30 min. 2.2.2. Physical characterization of the prepared MIONs DLS gives information on the mean particle diameter and the polydispersity index (PDI). The average particle size of MIONs and zeta potential (ZP) were measured using the Zetasizer Nano Series (Nano ZS, Malvern Instruments, UK). Zetasizer measurement was set at 25 °C. DLS technique is based on laser diffraction and can make detection for particles ranging from 0.6–6000 nm. While ZP value was obtained by measuring the direction and velocity of MIONs in the applied electric field. ZP is used to determine the effective electric charge on the NPs surface and the magnitude of the ZP makes prediction of the particle stability. TEM provides images showing each individual particle giving information about the size and shape of the prepared NPs. The size and morphology of MIONs were determined using TEM (JEM 1230 electron microscope Jeol, Tokyo, Japan). Briefly, a very small amount of solution of MIONs in deionized water (14 mg/ml) was dropped on a copper coated with carbon grid and the extra solution was removed using filter paper. The grid was allowed to dry at room temperature prior the examination of samples. The MIONs absorption spectrum was measured using Ultraviolet-visible (UV–Vis) spectrophotometer (Jenway UV6420; Barloworld scientific, Essex, UK). The absorption spectra data were collected in a wavelength range (200−800 nm). The functional groups of the active components of the MIONs were identified using FTIR spectroscopy based on the peak value in the region of infrared radiation. The transmitted peaks were obtained in the range (449.33 4000.6 cm−1) with a FTIR spectroscope (FTIR Edwards High Vaccum, Craeley Sussex, England). XRD was used for phase identification and to determine the crystalline structures of NPs. MIONs were examined using XPERT – PRO – PANalytical – Netherland, equipped with Cu Kα radiation source (λ = 1.54 Å), using Cu anode material at an accelerating voltage and applied current (45 kV - 30 mA) and scanning at 25 °C in the angular range 10°≤ 2θ ≤ 79.99°. The morphology and roughness of MIONs were determined using AFM (Wet – SPM9600 -Scanning Probe microscope -Shimadzu - made in Japan) in non-contact mode. The AFM uses a tip, which is installed at the end of the cantilever, for scanning the specimen surface. When the tip is brought close to the specimen surface, the force between the tip and sample leads to a deflection of the cantilever according to Hooke’s law. The cantilever deflection is measured by using a laser reflected from the top surface of the cantilever in an array of photodiodes (Wang and Chu, 2013). Non-

2. Materials and methods 2.1. Materials Ferric chloride hexahydrate (FeCl3. 6H2O, ≥ 98%), Ferrous chloride tetra hydrate (FeCl2. 4H2O, 99.99%) and Acetyl thiocholine iodide (CH3COSCH2CH2N(CH3)3I) (≥ 99%) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Thiobarbituric Acid, AR (TBA) (99%), 70% Perchloric Acid, AR and Sulfanilamide (C6H8N2O2S), AR (99%) were purchased from Central Drug House (CDH), India. Disodium Edetate (Na2EDTA), Sodium dihydrogen phosphate (Na. H2PO4) (98%) and Disodium phosphate (Na2HPO4) were purchased from EL Nasr Pharmaceutical Chemicals Co (ADWIC), Egypt. Sodium hydroxide (NaOH) was purchased from the Alamia Company for chemicals, Egypt. Trichloro Acetic Acid (TCA) (98%) was purchased from 81

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contact mode of AFM means that the tip does not have contact with sample surface, the cantilever is oscillated near the sample surface, and keeping a distance of the order of tens to hundreds of angstroms away from it (Wang and Chu, 2013; Demircioglu, 2017). In this mode, the instrument measure surface topography by using attractive forces (inter atomic force) between the tip and the sample surface. AFM in this mode, use piezoelectric modulator to keep a minimum distance by Van der Waals forces between the tip and the sample surface and at the same level to the sample surface all the time by a feedback loop control system.

2.2.3.4.1. Determination of lipid peroxidation levels. MDA, a measure of lipid peroxidation, was assayed by measuring the thiobarbituric acid reactive substances according to the method of Ruiz- Larrea et al. (Ruiz-Larrea et al., 1994). In this method, the thiobarbituric acid reactive substances interact with thiobarbituric acid to form a pink colored complex having absorbance at 532 nm which was detected by the spectrophotometer (Jenway UV-6420; Barloworld scientific, Essex, UK). 2.2.3.4.2. Determination of reduced glutathione (GSH) levels. GSH was determined by Ellman,s method (Ellman, 1959). The method is based on the reduction of Ellman’s reagent by –SH groups of GSH to form 2-Nitro-s-mercaptobenzoic acid which has a yellow color. The absorbance of the yellow color is measured spectrophotometrically (Jenway UV-6420; Barloworld scientific, Essex, UK) at 412 nm and GSH concentration was determined by comparison with a standard curve (range from 1 to 6 mmol). 2.2.3.4.3. Determination of nitric oxide (NO) levels. According to the method of Moshage et al. (Moshage et al., 1995), NO levels, measured as nitrite, were determined using Griess reagent. Nitrite, which is a stable product of the NO radical is primarily used as an indicator for the production of NO·. In brief, Griess reagent was added to the tissue supernatant, so, nitrite changed to a deep purple azo compound. The purple/magenta color developed was read at 450 nm using a spectrophotometer (Jenway UV-6420; Barloworld scientific, Essex, UK). 2.2.3.4.4. Determination of acetylcholinesterase (AChE) activity. AChE activity was determined as described by Gorun et al. (Gorun et al., 1978). The principle of this method is based on thiocholine production from the hydrolysis of acetylthiocholine iodide (substrate) by AChE. Thiocholine reacts with the –SH group in reagent DTNB, which is reduced to thionitrobenzoic acid. The absorption of yellow-colored anion thionitrobenzoic acid was read at 412 nm. 2.2.3.4.5. Determination of iron content in different brain areas. After excision and dissection, brain samples were digested using a 1:3 volumetric mix of hydrochloric acid to nitric acid. Digestions were incubated at 70 °C for up to 12 h until all tissues had gone into solution. Then, the total iron content in brain areas was quantified using ICP-OES (uetima Ⅱ- HORIBA France SAS).

2.2.3. In vivo experiments 2.2.3.1. Experimental animals. Adult male Wistar rats were used in the present work of age (6–8 weeks) and mean weight of (146 ± 1.80 gm). The animals were kept in standard cages in groups of five animals in each cage under fixed housing conditions (12 h light/dark cycles) and temperature (25 ± 1◦C). The water and chow were provided ad libitum. Experimental protocols and procedures used in this study were approved by the Cairo University, Faculty of Science Institutional Animal Care and Use Committee (IACUC) (Egypt) with approval number (CUIF 6 18). 2.2.3.2. Experimental design. Thirty rats were randomly divided into two main groups: control group and MIONs-treated group (15 rats in each group, 6 rats of each group were used for iron content by inductively coupled plasma - optical emission spectrometry (ICP-OES) measurement, while 9 rats of each group were used for biochemical analysis and histopathology examination; the right half of each brain sample was used for biochemical analysis while the left half of each brain sample was used for histopathology examination. The rats of the MIONs-treated group received single i.v. injection with MIONs with a dose of 10 mg/ kg (Jain et al., 2008). Animals of the control group received i.v. injection with saline by the same volume as the treated group. Animals were sacrificed by sudden decapitation on the fourth day following the injection. 2.2.3.3. Handling of tissue samples. After decapitation, the brain of each animal was removed and rapidly dissected on an ice-cold petri dish. After dissection, different brain areas were obtained (cortex, hippocampus, thalamus, hypothalamus, striatum, midbrain, cerebellum and Pons-medulla). The right half of each brain area was weighed then frozen at −20 °C to be used for biochemical analysis. The left half of each brain sample, that was specified for histopathology examination was fixed at 10% buffered formalin for twentyfour hours. Washing was done with tap water, and then serial dilutions of alcohol (methyl, ethyl and absolute ethyl) were used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56 °C hot air oven for 24 h. Paraffin bees wax tissue blocks were prepared for sectioning at 8 microns by slide microtome. The obtained tissue sections were collected on glass slides, deparaffinized and stained with Hematoxylin and Eosin (H & E) stain. Slides were then examined through the light microscope (Zeiss, Germany) as described by (Bancroft and Gamble, 2008).

2.2.4. Statistical analysis Results were expressed as mean ± S.E.M (Standard Error of Mean) and were analyzed using student’s t-test with significance level (p < 0.05). The standard error is used here to provide an estimation of the accuracy of a parameter (mean) and is used to deduce information from a sample to some relevant population (Westmoreland and Standard Errors, 2006). Statistical Package for Social Sciences origin software (version 94E) was used for all data. The percent of variation in the value of data for treated group with respect to the control was also calculated and presented as percentage difference.

%Difference (%D) =

Treated value Control value × 100% Control value

3. Results

2.2.3.4. Determination of biochemical parameters. An automatic Homogenizer (Heidolph DIAX 900, Germany) was used for homogenization of brain samples before analysis. All samples were homogenized in 4 ml ice cold phosphate saline buffer (PSB) (50 mM, pH = 7.4). Except the cortex, it was homogenized in 5 ml ice cold phosphate saline buffer. The homogenate for all samples was centrifuged at 8000 rpm for 15 min, at 4 °C using a high-speed cooling centrifuge (VS-18000 M small size high speed refrigerated centrifuge, Korea) and clear supernatant was used for the analysis of the levels of malondialdehyde (MDA) as a measure of lipid peroxidation, reduced GSH, nitric oxide (NO) and acetyl choline esterase (AChE) activity.

3.1. Characterization of MIONs 3.1.1. Particle size, shape, Zeta potential and roughness surface The average particle size of MIONs using DLS was: 80.18 ± 23.01 nm with PDI distribution = 0.41, as shown in Fig. 1(a) and Table 1. TEM images Fig. 2(a, b) and Table 1 show the presence of regular and relatively homogeneous MIONs. The morphology of MIONs was nearly spherical in shape. The size obtained from TEM measurements was found to be 15.63 ± 2.38 nm. The mean zeta potential for MIONs (Fig. 1(b) and Table 1) was found to be -26.0 ± 2.55 mV. The topographic image by AFM for MIONs is demonstrated in Fig. 2(c). The 82

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corresponded to Fe3O4 (JCPDS 04-006-06550). 3.2. Effect of i.v. Injection of MIONs in adult male Wistar rats on 3.2.1. Body weight As shown in Fig. 6(a), the change in weight along the whole experimental time was nonsignificant in the treated group as compared to the control group. 3.2.2. Iron content in brain areas Fig. 6(b) illustrates the iron content in the different brain areas. The iron content of the striatum and midbrain were significantly decreased in the treated group by 29.58% and 52.97%, respectively as compared to the control group. Meanwhile, nonsignificant changes in the iron content levels were detected in the cortex, hippocampus, thalamus, hypothalamus, cerebellum and Pons-medulla in the treated group as compared to the control group. 3.2.3. Oxidative stress parameters in brain areas Data concerning the effectiveness of i.v. injection of MIONs on the MDA contents in the different brain areas of adult male rats and the percentage differences in comparison to the control values is demonstrated in Table 2 and Fig. 7(a). As seen in Table 2 and Fig. 7(a), MDA levels showed significant decreases in the cortex, hippocampus, hypothalamus and cerebellum of treated groups as compared to the control groups, being -24.12%, -25.88 %, -51.42 % and -30.91 % below the control values, respectively. Meanwhile, there were non-significant decreases in MDA levels in the striatum, thalamus, midbrain and pons-medulla of the treated group relative to the control group. Table 3 and Fig. 7(b) represent the effectiveness of i.v. injection of MIONs on the GSH contents in the different brain areas of adult male rats and the percentage differences in comparison to the control values. As shown in Table 3 and Fig. 7(b), there were non-significant changes in GSH levels in the brain areas of the treated group in comparison to the control values except for a significant increase in the hippocampus (16.40 %) and significant decrease in the thalamus (-25.66 %) with respect to control levels. Data representing the effectiveness of i.v. injection of MIONs on the NO levels in the different brain areas of adult male rats and the percentage differences in comparison to the control values are presented in Table 4 and demonstrated in Fig. 7(c). The NO levels recorded significant decreases in the cerebellum and Pons-medulla (Table 4 and Fig. 7(c)) of the treated groups, being -30.19 % and -26.14 % below the control values, respectively. However, significant increase was recorded in the NO level of the thalamus with respect to control value (52.65 %). Non-significant changes were observed in the NO levels in the cortex, hippocampus, striatum, hypothalamus and midbrain relative to control values. The effectiveness of i.v. injection of MIONs on the AChE activities in the different brain areas of adult male rats and the percentage differences in comparison to the control values are presented in Table 5 and demonstrated in Fig. 7(d). As demonstrated in Table 5 and illustrated in Fig. 7(d), there were significant decreases in the AChE activities in the hippocampus, hypothalamus, midbrain, cerebellum and Pons-medulla (-10.86 %, -33.81 %, -14.99 %, -17.58 % and -13.37 %, respectively) of the treated group compared to the control group. However, the cortical, striatal and

Fig. 1. (a) Particle size distribution of MIONs using dynamic light scattering technique, (b) The zeta potential distribution of MIONs.

image and Table 1 showed that the morphology of MIONs is nearly spherical. The MIONs are rough with roughness surface = 1.54 ± 0.61. 3.1.2. Ultraviolet-visible (UV–vis) spectrum of MIONs The UV–vis absorption spectrum for MIONs was used to confirm the formation and the stability of the synthesized NPs in aqueous solution (deionized water). Fig. 3 shows the UV–vis absorption spectrum of MIONs with the characteristic absorption band observed at 235 nm. 3.1.3. Fourier transform infrared (FTIR) analysis The FTIR spectrum of MIONs was recorded as shown in Fig. 4. The spectrum revealed some characteristic peaks: the peak at 3429.78 cm−1 corresponds to OeH stretching, the peak at 2922.59 cm−1 was due to (CH3 and CH2) functional groups, the peak at 2187.85 cm−1 assigned to (C^C alkyne group. In addition, 2 peaks at 1634.38 cm−1 and 875.52 cm−1 corresponding to C]C aromatic ring and alkyne, respectively were evident. The peak recorded at 1437.67 cm−1 was due to –C-H bending. The peaks that appeared at 1114.65 cm−1 and 1057.76 cm−1 corresponded to CeO stretching and finally, a peak at 565.04 cm−1 was due to (Fe-O stretching). 3.1.4. X-ray diffraction (XRD) analysis The XRD pattern of IONPs is shown in Fig. 5. The diffraction peaks Table 1 Fe3O4 NPs mean size, shape, zeta potential and surface roughness. Nanoparticles

Mean particle size (nm)

Fe3O4

DLS 80.18 ± 23.0 and PDI = 0.41

Shape TEM 15.63 ± 2.38

TEM nearly spherical

83

AFM nearly spherical

Mean zeta potential (mv)

Surface roughness (AFM)

−26.0 ± 2.55

1.54 ± 0.61

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Fig. 2. (a, b) Transmission electron microscope (TEM) images of MIONs, (c) Topographic image by AFM for MIONs.

thalamic AChE activity showed non- significant changes.

neuronal degeneration starting from the external granular layer of cerebral cortex gray matter (Fig. 8(a)) and becomes more pronounced in the inner pyramidal layer that showed red pyramidal neurons. These red neurons are characterized by intense eosinophilic cytoplasm with

3.2.4. Histopathological investigations The microscopic examination of the treated group revealed

Fig. 3. UV–vis absorption spectrum of MIONs. 84

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Fig. 4. Fourier transform infrared (FTIR) spectrum of MIONs.

pyknotic nuclei (Fig. 8(b)). The same reaction was detected in a multiform layer of the cerebral cortex (Fig. 8(c)). Moreover, the cerebral cortex white matter of the treated group showed glial cell proliferation (Fig. 8(d)). The hippocampus of the treated group demonstrated a reduction of cellular density and loss of pyramidal neurons, the lesions appeared as a focal area involving CA2 area (Fig. 9(a)) and become more severe in the CA3 area (Fig. 9(b)). The dentate gyrus showed individual necrosis of granular cells with necrosis of pyramidal neurons in the dentate gyrus hilus (Fig. 9(c)). The striatum of the treated group showed neuronal degeneration and neuronophagia of neurons with gliosis and capillary endothelial proliferation, these alterations were detected in caudate putamen (Fig. 10(a, b, c)) and associated with malacia (Fig. 10(d)) and with focal gliosis. The neuronal degeneration with neuronophagia was more pronounced in Globus pallidus (Fig. 11(a) and (b)). The medulla of the treated group showed individual necrosis of ganglionic neurons with neuronophagia (Fig. 12(a) and (b)). The hypothalamus of the treated group exhibited individual necrosis of neurons with neuronophagia in the gray matter of hypothalamus associated with mild gliosis (Fig. 13). There were no alterations detected in the thalamus, midbrain and cerebellum of the treated group.

Fig. 6. (a) Effect of i.v. injection of MIONs on the body weight of adult male Wistar rats, (b) Effect of i.v. injection of MIONs on the iron content in the different brain areas of adult male Wistar rats.

sizes (Zhu et al., 2011). In addition, Brain is the body's most sensitive organ to study oxidative damage (Afifi et al., 2016). So, comprehensive studies were carried out to investigate safety problems associated with iron oxide nanoparticles (IONPs)exposure as they demonstrated damage to the CNS, which can lead to neurodegenerative disorders (Wang

4. Discussion MIONs can produce a variety of tissue reactions, including cell activation, ROS production, and cellular death, due to their ultra-fine

Fig. 5. XRD spectrum of MIONs. 85

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Table 2 Effect of i.v. injection of MIONs on MDA levels in different brain areas of adult male Wistar rats.

Table 3 Effect of i.v. injection of MIONs on GSH levels in different brain areas of adult male Wistar rats.

Brain areas

Control group Mean ± S.E.M. (nmole/ ml)

Treated group Mean ± S.E.M. (nmole/ml)

%D

t test

Brain areas

Control group Mean ± S.E.M. (mmol/ ml)

Treated group Mean ± S.E.M. (mmol/ml)

%D

t test

Cortex

16.84 ± 0.95 (6) 8.99 ± 0.55 (6) 16.21 ± 0.60 (7) 7.66 ± 0.83 (6) 14.40 ± 1.04 (6) 14.60 ± 0.87 (6) 59.54 ± 2.93 (6) 4.13 ± 0.23 (7)

12.78 ± 1.02 (6) 6.66 ± 0.71 (6) 14.28 ± 1.22 (6) 6.67 ± 0.73 (6) 6.99 ± 0.95 (6) 13.62 ± 1.05 (8) 41.14 ± 2.64 (6) 3.93 ± 0.26 (7)

−24.12 %

*

Cortex

n.s.

*

Hippocampus

16.40 %

*

−11.92 %

n.s.

Striatum

6.02 %

n.s.

−12.89 %

n.s.

Thalamus

−25.66 %

*

−51.42 %

*

Hypothalamus

11.67 %

n.s.

−6.71 %

n.s.

Midbrain

−12.57 %

n.s.

−30.91 %

*

Cerebellum

5.81 %

n.s.

−4.82 %

n.s.

Pons-Medulla

1.09 ± 0.08 (7) 30.74 ± 1.56 (6) 25.49 ± 1.45 (7) 47.59 ± 4.84 (8) 105.16 ± 4.62 (5) 34.35 ± 2.63 (7) 14.29 ± 0.96 (6) 14.51 ± 0.79 (6)

−4.89 %

−25.88 %

1.15 ± 0.14 (6) 26.41 ± 1.03 (8) 24.04 ± 1.34 (6) 64.01 ± 3.68 (6) 94.17 ± 4.55 (6) 39.29 ± 2.23 (7) 13.51 ± 0.69 (6) 15.86 ± 1.04 (6)

−8.51 %

n.s.

Hippocampus Striatum Thalamus Hypothalamus Midbrain Cerebellum Pons-Medulla

Values represent the mean ± S.E.M. The number of animals is shown between parentheses. % D: percentage difference in comparison to control group. *p < 0.05 significant. n.s.: non-significant.

Values represent the mean ± S.E.M. Number between parentheses shows the number of animals used. %D: percentage difference in comparison to control group. p < 0.05 significant. n.s.: non-significant changes.

et al., 2007, 2009). MIONs can be synthesized via chemical, physical or biological methods. Several researches used biological methods as they are eco- friendly and economic (Rastogi et al., 2018). Based on these facts, the aim of the present work was to evaluate the toxicity of MIONs which were synthesized using carob leaf extract (as a green route synthesis) on the brain of male Wistar rats. In the present work, MIONs were synthesized using Awwad & Salem [33] method. Before the evaluation of the toxicity of MIONs in the rats, physical and chemical characterizations of MIONs were carried out, as the physiological response is highly dependent on the physicochemical characteristics of NPs (Oberdörster et al., 2005). UV- visible spectrum, FTIR analysis and XRD verified the successful synthesis of

MIONS. TEM, DLS and AFM techniques were used to obtain information about the morphology and size of the synthesized particles. The images of TEM and AFM confirmed that MIONs were nearly spherical in shape. In addition, the AFM was used to determine the roughness of particles that may lead to the elevated surface activity of the brain-deposited NPs and can contribute to cell interaction and free radical manufacturing; which may cause brain damage and increase the danger of neurodegenerative disorders (Sun et al., 2011). The size of the particles was 15.63 ± 2.38 nm using TEM technique while the average particle size using DLS was found to be 80.18 ± 23.01 nm. The difference in the size values obtained through the two techniques was because DLS determines the size of particles, including their solvation shell while TEM Fig. 7. (a) Effect of i.v. injection of MIONs on MDA levels in different brain areas of adult male Wistar rats, (b) Effect of i.v. injection of MIONs on GSH levels in different brain areas of adult male Wistar rats, (c) Effect of i.v. injection of MIONs on NO levels in different brain areas of adult male Wistar rats, (d) Effect of i.v. injection of MIONs on AChE activity in different brain areas of adult male Wistar rats.

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They found no significant changes in GSH in the cortex similar to the present study. However, they reported non-significant decrease in thiobarbituric acid reactive substances (TBARS (levels in the cortex which was dissimilar to the present obtained results. Also, Chahinez et al. (Chahinez et al., 2016) tested two different doses of Fe3O4 NPs that were administrated orally in male rabbits for 14 days, and they reported significant elevations in stromal MDA levels of cerebral cortex which was not in line with the present study. In addition, the results of the present study were contrary to Dhakshinamoorthy et al. (Dhakshinamoorthy et al., 2017), who reported different findings after intraperitoneal injection (IP) injection of two different doses of Fe2O3 NPs in Male Swiss Albino mice for 4 weeks. As the cerebral cortex, for the two different doses exhibited significant increases in MDA levels. The difference in the findings in the cerebral cortex was observed also, in the significant increase in NO levels, the iron content and AChE activity. IONPs may cause several cytotoxic impacts, including oxidative harm, cell cycle alterations and apoptosis (Kiliç et al., 2017). In the present study, a significant decrease in the MDA level was evident in the cortex of MIONs-treated group, a finding which could be due to the neuronal degeneration in the cerebral cortex. This interpretation was further supported by the histopathological examination results of the cerebral cortex, which demonstrated the toxic effect of MIONs in this brain area emphasized as neuronal degeneration. Also, Wang et al. (Wang et al., 2011) reported neuropathological alterations observed using Nissl staining in cerebral cortex of the exposed CD-ICR male mice to two types of Fe2O3 NPs (ɣ-Fe2O3 and α-Fe2O3), NPs were intranasally instilled day after day for 40 days. In contrary to present findings, Estevanato et al. (Estevanato et al., 2011) reported that when Female Swiss mice were injected IP with magnetic albumin nanoparticles (MAN) at different time intervals, the NPs did not cause histopathological changes in the cerebral cortex area although the clusters of NPs in this area of the brain were evident. Also, Kumari et al. (Kumari et al., 2012) reported no pathological changes in the brains exposed to different sizes of Fe2O3, when the NPs with different doses were orally administered in female Wistar rats for 28 days. Moreover, in another study that used an ethanolic extract of Centella Asiatica (CA) plant to synthesize iron oxide nanoparticles (CAIONPs) and evaluated their toxicity in Swiss albino mice of either sex through oral administration of the NPs for 14 days (Pravallika et al., 2019). The authors reported that CAIONPs didn’t induce any histopathological changes on the cerebral cortex. In the present work, glial cell proliferation was observed in the cortex, hippocampus, striatum, hypothalamus and medulla of MIONs-treated group. The activation of glial cells is involved in most brain injuries and neurodegenerative diseases resulting from inflammation (Wu and Tang, 2018). In the present study, there were significant increases in GSH levels in the hippocampus accompanied with significant decreases in MDA levels and a significant decrease in AChE activity. The present results contradict with those of Wang et al. (Wang et al., 2009). Their study exhibited declines in GSH levels in the hippocampus for two different tested sizes of Fe2O3 NPs. The significant decrease of GSH levels was associated with a significant elevation of total NOS activities in the hippocampus region and non-significant decreases in TBARS levels. Also, Wu et al. (Wu et al., 2013) used radiolabeled (125I-Fe3O4-NPs) in male Sprague Dawley (SD) rats through intranasal instillation of them at different times. The authors reported non- significant changes in the MDA levels in the hippocampus region at different times. Dhakshinamoorthy et al. (Dhakshinamoorthy et al., 2017) reported different results as the hippocampus exhibited a significant increase in the iron content of the hippocampus region, in addition to significant increases in: MDA levels, NO levels and AChE activity in Male Swiss Albino mice for two different doses of Fe2O3 NPs. GSH is known as an important antioxidant and acts as redox buffer in living cells (González-Fraguela et al., 2018). Also, higher levels of GSH denote enhanced defense mechanism of the body (Cantin and Bégin, 1991; Martensson et al., 1991). So, the high levels of GSH in the

Table 4 Effect of i.v. injection of MIONs on NO levels in different brain areas of adult male Wistar rats. Brain areas

Control group Mean ± S.E.M. (μmol/L)

Treated group Mean ± S.E.M. (μmol/L)

%D

t test

Cortex

2.64 (8) 1.36 (6) 1.21 (6) 1.66 (6) 2.46 (7) 2.75 (7) 3.96 (6) 9.64 (8)

2.55 (8) 1.35 (8) 0.85 (6) 2.53 (8) 2.00 (6) 2.63 (7) 2.76 (7) 7.12 (7)

± 0.13

−3.18 %

n.s.

± 0.10

−1.39 %

n.s.

± 0.10

−29.73 %

n.s.

± 0.20

52.65 %

*

± 0.25

−18.54 %

n.s.

± 0.16

−4.33 %

n.s.

± 0.12

−30.19 %

*

± 0.19

−26.14 %

*

Hippocampus Striatum Thalamus Hypothalamus Midbrain Cerebellum Pons-Medulla

± 0.29 ± 0.11 ± 0.18 ± 0.16 ± 0.20 ± 0.15 ± 0.10 ± 0.43

Values represent the mean ± S.E.M. Number between parentheses shows the number of animals used. %D: percentage difference in comparison to control group. *p < 0.05 significant. n.s.: non-significant changes. Table 5 Effect of i.v. injection of MIONs on AChE activity in different brain areas of adult male Wistar rats. Brain areas

Control group Mean ± S.E.M. (μmol/min/g tissue)

Treated group Mean ± S.E.M. (μmol/min/g tissue)

%D

t test

Cortex

20.60 ± 0.90 (7) 30.92 ± 1.38 (6) 144.26 ± 5.53 (7) 73.09 ± 3.91 (7) 52.68 ± 1.71 (7) 46.91 ± 1.68 (6) 23.79 ± 0.78 (7) 43.48 ± 1.69 (6)

21.44 ± 0.73 (8) 27.56 ± 0.83 (8) 150.68 ± 8.80 (6) 63.57 ± 2.75 (6) 34.87 ± 2.28 (6) 39.88 ± 1.65 (6) 19.61 ± 0.60 (6)

4.07 %

n.s.

−10.86 %

*

4.45 %

n.s.

−13.03 %

n.s.

−33.81 %

*

−14.99 %

*

−17.58 %

*

37.67 ± 0.71 (7)

−13.37 %

*

Hippocampus Striatum Thalamus Hypothalamus Midbrain Cerebellum Pons-Medulla

Values represent the mean ± S.E.M. Number between parentheses shows the number of animals used. %D: percentage difference in comparison to control group. *p < 0.05 significant. n.s.: non-significant changes.

shows the size of particles individually (without solvation shell) (Uskoković, 2012). This difference in size values showed by TEM and DLS agrees with the results obtained by Arsalani et al. (Arsalani et al., 2019). ZP is used to predict the stability of the particle. The particles tend to aggregate in the range 0–5 mV and are minimally stable in the range 5–20 mV. They are moderately stable in the range 20–40 mV and highly stable in the range ≥ 40 mV (Titus et al., 2019). The present study revealed moderate stability of the synthesized NPs as ZP was -26.0 ± 2.55 mV. In the present work, the effect of MIONs was evaluated on the brain cortex. MDA levels demonstrated significant decreases in the MIONstreated group as compared to the control group. Wang et al. (Wang et al., 2009) studied the effect of intranasal exposure of repeatedly lowdose of two different sizes of Fe2O3 NPs in different mice brain regions. 87

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Fig. 8. (a) Histological section of the cerebral cortex gray matter external granular layer of treated rat showing individual neuronal degeneration of small pyramidal neurons with deeply eosinophilic cytoplasm and basophilic nuclei (arrow) (H&E, X400), (b) Histological section of the cerebral cortex gray matter inner pyramidal layer of treated rat showing red necrotic pyramidal neurons with dark eosinophilic cytoplasm and pyknotic nuclei (arrow) (H&E, X400), (c) Histological section of the cerebral cortex gray matter multiform layer of treated rat showing individual neuronal degeneration of the pyramidal (long arrow) and granular neurons (short arrow) (H&E, X400), (d) Histological section of the cerebral cortex white matter internal capsule of treated rat showing small focal glia cell proliferation (H& E, X400). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

hippocampus area in the present study may be a normal response of brain owing to free radicals’ production from MIONs treatment in the rats. The action of GSH was observed in the significant decrease of MDA levels, which is considered a biomarker of lipid peroxidation. AChE, a fundamental enzyme among the crucial enzymes for neurotransmission in higher living organisms, was found in the extracellular surface of neurons in the brain (Bugata et al., 2019). The AChE activity is a very effective biochemical assay for neurotoxic effect [22]. It is considered a vital constituent of the cholinergic system which plays an important role in the cognitional function (Dhakshinamoorthy et al., 2017). AChE hydrolyze acetylcholine (ACh) to choline and acetyl-CoA (Meena et al., 2015). From our results MIONs inhibited AChE enzyme that leads to accumulation of ACh in hippocampus. The elevated Ach continuously triggered post synaptical cholinergic stimulation and consequently led to neurotoxicity signs (Kumari et al., 2012). The signs of neurotoxicity in the hippocampus area were obvious in the histopathological examination results that illustrated loss and lesions of neurons in CA2 area and became more severe in the CA3 area in addition to necrosis of neurons in the dentate gyrus. Wang et al. (Wang et al., 2007) reported similar findings after using fine Fe2O3 NPs in CD-ICR male mice administrated by intranasal instillation. The authors reported heavy fatty degeneration of neurons in the CA3 area in the hippocampus on the 14th day after intranasal instillation of fine Fe2O3 particles in mice. Also, Wang et al. (Wang et al., 2011) observed a significant reduction in the number of intact neuronal cells in hippocampus CA1 area of the treated mice and the pyramidal neurons organized loosely with clear thinner layers than those in normal mice. However, the authors Kumari et al. (Kumari et al., 2012), Pravallika et al. (Pravallika et al., 2019) and Wu et al. (Wu et al., 2013) reported no histopathological changes in the hippocampus after oral administration of Fe2O3 NPs, CAIONPs and intranasal instillation of 125I-Fe3O4-NPs, respectively. In the present study, significant decreases in iron content levels in the striatum were exhibited. In addition, histopathological changes in this area were detected as neuronal degeneration and neuronphagia of neurons with gliosis and capillary endothelial proliferation. In line with our results, Wu et al. (Wu et al., 2013) reported non- significant changes in the MDA levels in the striatum at different times. For instance, iron plays a key role in the ordinary function of the brain, such as synthesis and packaging of neurotransmitters; uptake and degradation of the neurotransmitters into other iron-containing proteins (Grantham-McGregor and Ani, 2001). Iron is essential for neuronal function and activity, development of dendritic connection, the myelination processes of nerve fibers, and proper functioning of enzyme systems that regulate cellular energy (Beard, 2007; Sheida et al., 2017).

Iron is a cofactor for many iron-containing enzymes that are important to produce monoamine neurotransmitters (Youdim and Green, 1978). So, iron decrease in the brain may lead to low activities of these enzymes, which would affect the contents of biogenic amines (Hu et al., 2010). A previous study showed significant decreases in the iron levels in the brain of CD-1 (ICR) female mice, this was observed after the intragastric administration of mice with anatase TiO2 NPs for 60 consecutive days (Hu et al., 2010). The study exhibited histopathology changes in the brain after the treatment with NPs and the authors suggested that these alterations may indirectly or directly disturb the homeostasis of trace elements in the brain. The previous study may be similar to ours as the striatum exhibited histopathological changes accompanied with iron decrease. Similar to the present study, neuropathological alterations detected in the striatum of mice treated with IONPs (Wang et al., 2011). The alterations presented as cellular swelling, vacuolar degeneration, nuclear chromatin condensation and fragmentation. Our findings are not in line with those of Kumari et al. (Kumari et al., 2012) and Pravallika et al. (Pravallika et al., 2019) who reported no pathological changes in the brains. Also, Wu et al. (Wu et al., 2013) showed no histopathological changes in the striatum after their treatment with Fe3O4 NPs. The effect of MIONs on the thalamus was exhibited as a significant increase in NO level that was accompanied by a significant decrease in GSH level without any histopathological lesions. Kumari et al. (Kumari et al., 2012) and Pravallika et al. (Pravallika et al., 2019) reported similar findings as there were no histopathological changes in the thalamus. In addition, Gorman et al. (Gorman et al., 2018) reported no histopathological changes in the thalamus of male Sprague–Dawley rats after i.v. injection with two different ultra-small paramagnetic iron oxide particles. GSH is an antioxidant and its function is the detoxification of endogenous and exogenous toxic compounds, the depletion in GSH levels may be an indicator of its consumption in the protection of cells from the generated free radicals (Mazzetti et al., 2015). NO exhibited several functions in the nervous system: in the process of endothelium-dependent vasodilatation (Duncan and Heales, 2005; Michell et al., 2004), in neurotransmission (Yamamoto et al., 2015), and in host-defense mechanisms (Akyol et al., 2004). NO is known to be a two-edged sword. It has beneficial effects, mediates and protects neuronal activity in the lower constitutive mode. Secondly, it is an indiscriminate damaging molecule in high, unregulated fashion (Džoljić et al., 2015). NO can react quickly with superoxide anion resulting in the formation of the peroxynitrite anion (ONOO–) that is known with its high cytotoxic (Lipton et al., 1993). The decrease in GSH levels leads to increased NOS 88

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In addition, the decline in AChE levels leads to the accumulation of Ach and this accumulation may lead to brain neurotoxicity. A significant decrease in the iron content of in the midbrain area was observed in our study due to MIONs administration. This deficiency of iron content in the midbrain was associated with deficiency in AChE activity. Abdel-Wahhab et al. (Abdel-Wahhab et al., 2017) reported similar results in the midbrain of male Sprague-Dawley rats after 3 weeks from their IP administration with Eudragit® nanoparticles (EUDNPs). The study exhibited a significant decrease in AChE activity. But, a significant increase in AChE activity was obtained in the midbrain region of male Sprague-Dawley rats after 4 h, 48 h and 1 week from IP injection. The increment was also detected after 1 week and 3 weeks of their oral treatment. Our findings are consistent with Kumari et al. (Kumari et al., 2012) and Pravallika et al. (Pravallika et al., 2019) studies as they reported no histopathological changes were in the midbrain area like ours. Conflicting with Gorman et al. (Gorman et al., 2018) study, who reported vacuolation of midbrain parenchyma in all animals treated groups with USPIO. In addition, in contrary to our findings, Wang et al. (Wang et al., 2008) reported non-significant changes in iron contents in the midbrain of CD–ICR male mice after their intranasal instillation with two different sizes of ferric oxide particles for different times. In addition, Wang et al. (Wang et al., 2011) reported significant increases in the iron content in the midbrain of mice treated with Fe2O3 NPs. Cellular IONPs can liberate iron in the cells, which may have a harmful effect, mainly by iron-catalyzed ROS formation (Lévy et al., 2010). Alternatively, increased cellular iron levels following IONPs loading can account for observed upregulation of cellular ferritin (Raschzok et al., 2011), altered macrophage immune responses (Yeh et al., 2010) and delayed cytotoxicity induced by IONPs (Lunov et al., 2010). Hohnholt et al. (Hohnholt et al., 2011) reported in an in vitro study, that a strong iron accumulation was observed after exposure of oligodendroglial cell line OLN-93 to dimercaptosuccinate (DMSA)coated IONPs and this was accompanied with a strong increase in the amount of the cellular iron storage protein ferritin. The large amounts of accumulated iron in OLN-93 cells did not cause ROS production or an alteration in the cellular thiol reduction potential. Authors refer these findings to the fact of the the strong ferritin upregulation, the cells could safely store excess iron released from the IONPs that was not needed for proliferation and hence prevent the formation of ROS by iron-mediating Fenton chemistry. Also, when DMSA coated Fe3O4 NPs degraded in in mouse macrophage RAW264.7, cells manifested that this NPs could degrade into iron ion in lysosome after intracellular internalization and caused significant increase of intracellular iron ion (Liu and Wang, 2012). The released iron ion altered the iron and osmosis homeostasis of cell and led to compensatory cell responses to transcriptional gene regulation that maintain intracellular iron and osmosis homeostasis. The compensatory responses appeared as downregulation of transcription of several major genes like Transferrin receptor (Tfrc) and Transferrin (Trf) that are responsible for transferring iron ion into cells (Richardson and Ponka, 1997) and simultaneous upregulation of transcription of major genes responsible for transferring intracellular iron ion out cells, like Slc40a1 (encoding ferroportin) and Lipocalin-2 (Lcn2). Cultured brain astrocytes cells when exposed to IONPs, also exhibited up-regulation of ferritin synthesis and transient appearance of ROS due to iron that is released from them (Geppert et al., 2012). Such studies suggest that IONPs can influence cell iron pool. CNS-trace metal imbalance could be linked to many pathological conditions of the brain, such as neuronal injury, neurodegenerative diseases and brain ischemia (Konoha et al., 2006). Restless legs syndrome (RLS) is one of a neurological disease and is characterized by reducing iron function (Ward et al., 2014). Autopsy and MRI studies demonstrated decreased iron levels in the substantial nigra (located in the midbrain) in patients with restless leg syndrome and the degree of deficiency in iron levels in this region are associated with severity of disease (Allen and Earley, 2007). As mentioned previously, iron is one

Fig. 9. (a) Histological section of the hippocampus CA2 of treated rat showing reduction and loss of pyramidal neurons (square) with individual necrosis of pyramidal neurons (arrow) (H&E, X400), (b) Histological section of the hippocampus CA3 of treated rat showing marked cellular reduction and loss of pyramidal neurons with necrosis of pyramidal neurons (arrow) and glia cell proliferation (H&E, X400), (c) Histological section of the hippocampus dentate gyrus of treated rat showing pyknosis of granular cells with neuronal degeneration of pyramidal neurons with dark eosinophilic cytoplasm (H&E, X400).

which is another biological molecule related to inflammation that is followed with a large amount of genotoxic reactive nitrogen species, such as NO (Wu and Tang, 2018; Heales et al., 1996). From the previous literature, we can say that the significant decrease in GSH levels may be due to its consumption in scavenging the liberated ROS as NO while the effect of these ROS production was not highly toxic enough to exhibit changes in the histopathology of this brain area. In the present study, the hypothalamus of the brain demonstrated significant decreases in MDA levels, significant decreases in AChE levels concomitant with slight histopathologic changes. Kumari et al. (Kumari et al., 2012) and Pravallika et al. (Pravallika et al., 2019) reported contradictory findings to ours. As, they reported that there were no histopathological changes in the brain after treatment with NPs. Also, in Gorman et al. (Gorman et al., 2018) study, the authors found no histopathological changes in the hypothalamus of male Sprague–Dawley rats. As previously mentioned, the decline in MDA levels here may arise from the degeneration that occurred in this area. 89

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Fig. 10. (a) Histological section of the striatum caudate putamen of treated rat showing individual necrosis of neurons with neuronophagia (arrow) (H&E, X400), (b) Histological section of the striatum caudate putamen of treated rat showing neuronal degeneration (arrow) with gliosis (H&E, X400), (c) Histological section of the striatum caudate putamen of treated rat showing proliferation of glia cell and endothelial capillary (arrow) (H& E, X400), (d) Histological section of the striatum caudate putamen of treated rat showing focal malacia with glia cell proliferation (circle) (H&E, X400).

Although, the alterations that appeared in the midbrain region as a result of i.v injection of MIONs are signs of neurotoxicity, they may be not toxic enough to make histopathological alterations. The impact of MIONs on the cerebellum of the brain was evident in the significant decrease recorded in the MDA levels, NO levels and AChE. Dhakshinamoorthy et al. (Dhakshinamoorthy et al., 2017) reported opposed results than ours, the authors detected significant increases in MDA, NO levels and AChE activity in the cerebellum of the brain in male Swiss Albino mice that are IP- treated with Fe2O3 NPs, this was accompanied by significant increases in the iron content. The present results demonstrated a non-significant alteration in the iron content in the cerebellum. Like our findings, Kumari et al. (Kumari et al., 2012), Pravallika et al. (Pravallika et al., 2019) and Estevanato et al. (Estevanato et al., 2011) reported no histopathological changes in the cerebellum. The decrease in the MDA levels detected in the present study may be due to the decrease in NO levels. As, it was previously reported that the reduction in NO levels decreases peroxynitite production, which causes the peroxidation in tissue to decrease and quenches the formation of the MDA (Fahmy et al., 2019). As discussed previously, the significant decrease in AChE levels may be a sign of neurotoxicity. In addition, the histopathological examination revealed no lesions in the cerebellum. From the present findings recorded from the cerebellum, it may be concluded that the MIONs were non-toxic on the cerebellum further they may enhance the antioxidant system in this brain region. However, the reported decrease in the AChE activity shown in the present study still need more investigations. MIONs caused significant decreases in both NO levels and AChE levels in the Pons-Medulla in the present study. These alterations were accompanied with slight histopathological changes in this region. Wang et al. (Wang et al., 2009) found similar findings, they reported non- significant changes in both GSH and TBARS levels in the brain stem of CD-ICR male mice. In contrary to our findings, Wang et al. (Wang et al., 2007), Kumari et al. (Kumari et al., 2012) and Pravallika et al. (Pravallika et al., 2019) reported no histopathological changes in the Pons-medullary regions of the brain. It's worth to mention that although, the accumulation of the MAN NPs in the medulla of female Swiss mice, Estevanato et al. (Estevanato et al., 2011) reported no histopathological changes in this area at different time points. The significant decreases in both NO levels and AChE levels in the Pons-Medulla region of the brain here may be related to each other. As it was reported in previous studies, NO has a modulatory effect on ACh release in the medial pontine reticular formation (a nucleus in the Pons) when NOS inhibitor NG-nitro-L-arginine (NLA) decreases ACh release in

Fig. 11. (a) Histological section of the striatum Globus pallidus of treated rat showing necrosis of neurons with neuronophagia (arrow) and gliosis (H&E, X400), (b) Histological section of the striatum globus pallidus of treated rat showing neuronophagia (arrow) (H&E, X400).

of the most important biometals that has a role in the CNS and the deficiency in iron may affect brain functions and leads to a reduction in dopamine receptors D2 number and sensitivity leading to decreasing the amount of pulses that pass-through nerve cells (Yehuda and Youdim, 1989). As previously mentioned, the deficiency of iron in the striatum in the present study may be due to the histopathological changes detected in this brain area as the degeneration may indirectly or directly disturbs the iron homeostasis. But the midbrain did not exhibit any histopathological lesions, so the decline in the iron content in the striatum and midbrain areas may be because of the effect of MIONs on the iron regulatory proteins and MIONs may have a harmful effect on genetic levels. Further investigations are still needed to confirm whether MIONs have harmful effects on the genetic levels or not. 90

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safety of silica-coated IONPs-containing rhodamine B isothiocyanate on the brain, after their intraperitoneal administration for 4 weeks into mice, and by Jain et al. (Jain et al., 2008) who intravenously administered oleic acid–coated IONPs. However, Kumari et al. (Kumari et al., 2012) and Kumari et al. (Kumari et al., 2013) when used Fe2O330 NPs orally in Wistar rats, NPs caused significant decrease in AChE activity and total, Na+, K+, Mg2+, and Ca2+-ATPases in a dose-dependent manner in the brain. Also, Kim et al. (Kim et al., 2013) used the intraneural injection of four types of IONPs with different surface and core chemistries, namely DMSA-coated IONPs (both maghemite and magnetite), PEG-coated magnetite NPs, and PEG-Au-coated magnetite NPs into an intact sciatic nerve of SD rats. The authors detected that 48 h after IONPs injection, the levels of mitogen-activated protein kinase/extracellular signal-regulated kinase and caspase 3 (as indicators of inflammation and apoptosis, respectively), were significantly increased in nerves of all IONPs-treated animals. In addition, de Oliveira et al. (de Oliveira et al., 2014) observed apparent toxicity of IONPs coated with cross-linked aminated dextran and intraperitoneally injected to zebrafish. The toxicity observed in the downregulation in brain AChE activity, impaired swimming performance parameters, significant increase in iron levels in the brains and triggered of apoptosis. In general, this discrepancy in the findings and potential neurotoxicity of IONPs in the present study and other studies may be due to various factors related to the NPs as their biocompatibility, iron release capacity, presence and chemical features of coating or due to setting of experiments as exposure conditions or pathway, type of tissue or cell affected and IONPs (Wang et al., 2011). So, the data extracted from such studies would help in defining the safety limits and conditions of use of such NPs in order to avoid or reduce hazards, especially those associated with their exponentially growing biomedical applications. Up to the author’s knowledge, this is the first study to evaluate the toxicity of green synthesized MIONs (carob leaf-synthesized) in different brain areas of adult male Wistar rats.

Fig. 12. (a) Histological section of the medulla of treated rat glia cell proliferation (H&E, X400), (b) Histological section of the medulla of treated rat showing necrosis of neurons with neuronophagia (arrow) and gliosis (H&E, X600).

5. Conclusion The present study evaluated the toxicity of carob leaf-synthesized MIONs on different brain areas of adult male Wistar rats. MIONs, which were 15.63 ± 2.38 nm in size, spherical in shape, with moderate stability and rough surface, demonstrated toxic impacts on various brain areas and the effect varies from one area to another. According to the conditions of the present study, the hippocampus and striatum were the most affected areas and demonstrated severe damage due to MIONs exposure. MIONs injection in Wistar rats affected the iron homeostasis in both striatum and midbrain by decreasing iron content in these areas. This study was the first step to evaluate the toxicity of carob-synthesized MIONs and further investigations are needed to understand the mechanism of MIONs toxicity of the brain. It would be useful to understand MIONs interactions with biological system for further use in biomedical applications.

Fig. 13. Histological section of the hypothalamus of treated rat showing individual necrosis of neurons with neuronophagia in the gray matter of hypothalamus associated with mild gliosis (H&E, X200).

this region of the brain (Leonard and Lydic, 1997). Hence, it may be suggested that the significant decreases in the AChE levels recorded in the present study may be a compensatory response to acetylcholine decrease. It was previously reported that relative NO depletion occurs in early phases of cerebral injury e.g. after traumatic brain injury, subarachnoid hemorrhage, post-cardiac arrest and ischemic stroke (Ahn et al., 2004; Tuzgen et al., 2003). This happens at the same time as the blood flow decreases (Hlatky et al., 2003). Combining these suggestions together, we can say the slight histopathological changes in our study in the Pons-medulla may be related to the impact of MIONs on the blood flow to this area. A number of studies have examined the potential neurotoxic effects of IONPs in different animal models. Though, findings are often conflicting. Muldoon et al. (Muldoon et al., 2005) reported that intracerebral inoculated or intraarterially administered iron oxide-based MRI contrast agents in the normal rat brains didn’t produce apparent toxicity in the brain. Also, Kim et al. (Kim et al., 2005) reported the

CRediT authorship contribution statement Heba M. Fahmy: Conceptualization, Methodology, Software. Esraa M. Aly: Data curation, Formal analysis, Methodology, Writing - original draft. Faten F. Mohamed: Visualization, Investigation. Neveen A. Noor: Investigation, Supervision, Validation. Anwar A. Elsayed: Supervision. Declaration of Competing Interest The authors declare no conflict of interest 91

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