Липидированные мезофорные наночастицы, нагруженные тимокиноном, в качестве нового метода лечения аутоиммунного энцефаломиелита у крыс.

Introduction

The neuroinflammatory autoimmune illness multiple sclerosis (MS) is a leading cause of disability worldwide, especially among women [ 1 , 2 ]. It undergoes two main stages: inflammation and demyelination, followed by astroglia proliferation and neurodegeneration [ 3 ]. MS sufferers have visual neuritis, physical disorders, and cognitive impairment. Plaque sites and severity determine symptoms [ 4 ].

Central nervous system (CNS) disorders such as MS begin with the degradation of the blood-brain barrier (BBB) [ 5 ]. There is no cure. However, several treatment alternatives are available [ 6 ]. Neuroinflammation produces cytokines and chemokines that alter tight junction (TJ) and adherens junction (AJ) proteins, connecting endothelial cells (ECs) and causing BBB rupture [ 7 , 8 ].

Experimental autoimmune encephalitis (EAE) is widely recognized as the most extensively used model in MS pathology [ 9 ]. In the EAE model, the immune system attacks myelin proteins in the CNS, causing inflammation and oligodendrocyte loss [ 10 ]. Neuroinflammation, oligodendrocyte biology, demyelination, and remyelination have been extensively studied in animal models of EAE [ 11 ]. Myelin-reactive helper T cells (CD4+ T cells) are hypothesized to mediate immunity to myelin-derived antigens, such as myelin oligodendrocyte glycoprotein (MOG) [ 9 , 10 ]. Incomplete Freund’s adjuvant (IFA) emulsifies MOG to limit immune activation outside the induction phase [ 12 ]. Pertussis toxin disrupts the BBB [ 13 ]. Tail atony precedes hind limb paralysis 8–12 days after disease onset [ 11 ].

Free radicals, also known as reactive oxygen species (ROS), are particularly prevalent in CNS cells due to the oxygen consumption associated with normal biological processes [ 14 ]. However, excessive ROS can cause oxidative stress and inflammation. The BBB, neurons, and myelin are all harmed by ROS [ 13 ]. Inflammation and ROS production can be amplified by demyelinating lesions through the secretion of nitrogen and oxygen free radicals, as well as pro-inflammatory cytokines, thereby further enhancing inflammation and ROS [ 15 ]. Increasing cellular antioxidant production in inflammatory MS lesions protects against ROS-induced cellular damage [ 16 ].

Nigella sativa (N. sativa), or the black seed. It treats several conditions naturally [ 17 ]. Thymoquinone (TQ) and N. sativa seed extracts have demonstrated potent curative effects in studies of autoimmune diseases [ 18 ]. TQ is an antioxidant, anti-inflammatory, antimutagenic, and antigenotoxic treatment for several illnesses [ 17 ]. TQ eliminates oxidative free radicals such as O 2 , OH, and H 2 O 2 [ 19 ]. This notion suggests that TQ can help normalize and prevent inflammation [ 20 ]. By blocking NF-κB signaling, TQ significantly increased GSH levels in an EAE model [ 21 ].

Mesoporous silica nanoparticles (MSNs) are promising vehicles or nanocarriers for regulated drug delivery [ 19 ]. They dominate conventional procedures in neurodegenerative diseases [ 22 ] due to their structural versatility, large surface area, and ease of functionalization. Additionally, MSNs exhibit considerable potential due to their substantial porosity, minimal toxicity [ 23 ], biocompatibility, and wide biodistribution [ 24 ]. MSN contains antioxidant properties and can cross the BBB [ 25 ] Ebabe [ 26 ]). MSN is thermally and chemically stable, making it more stable in a treatment medium than other nanoparticles [ 27 ]. TQ’s curative action and MSN’s drug delivery technology support our findings. The BBB permits lipophilic molecules to pass through by passive diffusion, thereby affecting a drug’s rate of entry into the CNS [ 28 ]. Our work aims to enhance TQ-loaded MSN permeability across the BBB using a lipophilic coating composed of a Lipid bilayer (LB) to increase penetration.

Materials and methods

Chemicals

Sigma-Aldrich supplied asolectin lipid (St. Louis, Missouri, United States), ethanol (99%), tetraethyl orthosilicate (TEOS) (99%), chloroform, dimethyl sulfoxide (DMSO), and TQ (2-isopropyl-5-methyl-1,4-benzoquinone). Ammonium hydroxide (NH4OH, 28%) was obtained from Fluka. The phosphate-buffered saline (PBS, pH 7.4) used in this experiment was obtained from Bio Diagnostic Co., Giza, Egypt. C2H5OCH2CH2OH, or 99% pure 2-ethoxyethanol, was provided by Merck. Bio Diagnostic Co. in Giza, Egypt, supplied the materials and kits used to determine oxidative stress parameters. Complete Freund’s adjuvant (CFA) and rat synthetic myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35-55) were used as a standard adjuvant for the induction of experimental autoimmune encephalomyelitis (EAE)and purchased from Sigma-Aldrich, St. Louis, MO, USA. also, Sigma–Aldrich supplied Luxol® fast blue solution, fluorescein isothiocyanate isomer I, 1,2-dioleoyl-sn-glycero-3-phosphocholine, tetraethyl orthosilicate, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), DOPE-PEG(2000), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

Methods

Different nanoformulations’ preparation

The procedure for creating MSN was derived from the work of Elbialy et al. [ 29 ] and Fahmy et al. [ 30 ]. Briefly, 0.5 g of CTAB was added to 70 mL of distilled water using a magnetic stirrer. An additional 0.5 mL of NH4OH and 30 mL of 2-ethoxyethanol were incorporated into the mixture (co-solvent). Afterward, 2.5 ml of TEOS (silicon source) was added after 30 min of stirring at room temperature, and the combination was stirred for another 24 h. To eliminate any traces of CTAB, the samples were calcined at 600 °C for 6 h. The silica was cleaned and ready for use after centrifuging at 6000 rpm for 20 min, then rinsing with ethanol and deionized water.

TQ-loaded mesoporous silica nanoparticles (MSN-TQ) were prepared by diluting MSN-TQ at a 1:9 ratio of DMSO: saline. At a weight-to-weight ratio of 1:1, the produced TQ solution was added to the MSN aqueous suspension (in deionized water) while stirring continuously. Subsequently, the suspensions were combined for 24 hours at room temperature while spinning at 100 rpm in a water bath.

MSN loaded with TQ and coated with LB (LB-MSNs) were synthesized with certain modifications from the method stated by Tu et al. [ 31 ]. Asolectin (a mixture of phospholipids), which consists of 24% saturated, 62% polyunsaturated, and 14% monounsaturated fatty acids, was dissolved in chloroform (organic solvent) and kept under a nitrogen flow with a steady evaporation rate to create a thin film of lipid (under the vacuum). Following a 10-minute vortex in PBS (2 mL, 1 mM, pH 7.4), the lipid film was rehydrated, resulting in a cloudy lipid suspension. Subsequently, the product was sonicated for 10 minutes in a water bath maintained at 50 °C. Liposomes were prepared as previously described and stored at 40 °C.To prepare the LB-MSNs, 0.5 mL of TQ-MSN was mixed with the exact amount of liposomes previously prepared, and then agitated at 400 rpm for approximately 90 min.

Characterization of nanoformulations

TEM

TEM was used to examine the morphological features of the LB-MSNs and MSNs-TQ formulations using a JEM-1230 electron microscope (JEOL, Tokyo, Japan). TEM was used to determine the actual size of the nanoparticles produced. Each sample was placed in a beaker of deionized water and suspended on a carbon-coated copper grid. The grid was allowed to air-dry at room temperature for approximately 5 minutes before testing.

Particle size (PS) applying DLS

The Zeta sizer examined the LB-MSNs and MSNs-TQ formulations for hydrodynamic diameter and size distribution (Nano ZS, Malvern Instruments, UK). Laser diffraction measures the scattering of laser light from diluted samples at 90° and at room temperature. The polydispersity of the sample can be determined using this method, as indicated by the PDI.

2.3.3. ZP

The zeta sizer measured the nanoparticle surface charge. Samples were measured at room temperature using Nano ZS (Malvern Instruments, Malvern, UK). The nanoparticles’ electrophoretic mobility was measured as they moved in an applied electrical field, enabling calculation of their zeta potentials.

In vitro drug release

Using a dialysis bag, the diffusion technique was employed to investigate the sustained release of TQ from LB-MSNs in vitro. The release experiment was carried out using a dialyzer, which required sterile dialysis bags with a maximum molecular weight cutoff of 12,000 daltons. Dialysis bags controlled the release of chemicals in distilled water for a specific time. 10 mL of LB-MSNs was placed in the dialysis bags. After placing the sealed dialysis bag into the containers, 30 ml of the releasing medium was added. The containers were shaken at 100 rpm at a temperature of 37 °C. At regular intervals, 2 mL of the medium surrounding the beaker’s interior was withdrawn and substituted with a new medium (distilled water). A UV-Vis Spectrophotometer was used to quantify free TQ concentrations at 253 nm, using a calibration curve (Jenway UV-6420; Barloworld Scientific, Essex, UK).

Encapsulation efficiency

The encapsulation efficiency of LB-MSNs was determined from the TQ’s calibration curve based on Eq. 1 .

In vivo experiments

Experimental animals

Male Wistar rats aged 6 to 8 weeks were used in this study. The animals were procured from a local supplier, not from a privately owned institution/individual/or farm. The animals used in the experiment were housed and handled humanely and provided with food and water as needed. The procedure for the in vivo study (CU-I-F-28–21) was approved by the Institutional Animal Ethics Committee. No informed consent from the owner(s) was required as the animals were not privately owned.

Toxicity test

A pilot trial was conducted to choose a suitable dose for our experiment. Two rats per group were administered doses (600, 300, 100, and 10 mg/kg) of DMSO, TQ, MSNs, and LB-MSN over two days. All the rats under investigation showed no toxicological symptoms. Therefore, our original experimental groups used one-tenth of the maximum tolerated dose (MTD) (60 mg/kg).

EAE induction:

With some modifications, the EAE model was performed as described by Aharoni et al. [ 32 ]. 200 µl of MOG35-55 dissolved in 100 µl of CFA, comprising 3.3 mg/ml of heat-inactivated Mycobacterium tuberculosis, was administered subcutaneously once at the base of the tail to induce EAE. After MOG vaccination, each rat received an intraperitoneal administration of 150 ng of Pertussis toxin. All rats were anesthetized with light halothane. A successful EAE model manifested 14 days later, with daily scores showing loss of tail toxicity and hind-limb and forelimb weakening.

Experimental design:

Wistar rats were investigated, with an average weight of 110 ± 10 g. The animals were kept under consistent conditions, including a constant temperature (27 degrees Celsius) and a light/dark cycle of 12 hours. Random assignment was made for 35 rats to one of five groups (each with seven rats) and treated orally once daily for 14 days following the onset of clinical symptoms with the following: (1) saline and DMSO (10% DMSO: 90% saline) injections were given to the animals in the control group, and (2) DMSO injections were given to the rats in the EAE group. Animals with EAE exposed to treatment formulas were divided into three groups: (3) MSNs, where MSNs (60 mg/kg) were injected; (4) TQ, where free TQ (60 mg/kg) was administered; and (5) LB-MSN, wherein a mixture of TQ and MSNs (60 mg of TQ/kg to 60 mg of MSNs/kg) was injected. The dosage was determined based on the results of the toxicology study. All animals were euthanized by sudden decapitation on the 28 th day after induction. It is important to note that no anesthesia was used during this process. The rationale behind this decision is that anesthesia could potentially interfere with brain measurements, a critical aspect of our study. Our research focuses on augmenting Thymoquinone’s curative and remyelinating effects by using a novel formula of MSN coated with LB. The goal is to enhance the compound’s entry into the BBB. Therefore, to ensure the accuracy of our results, we opted not to use anesthesia during the euthanasia process.

Clinical scoring:

Every day from the starting day of induction to the day of decapitation (day 28), all rats were weighed and examined to monitor the progression of their illness. Rats in the control groups were subjected to the same stress levels as those in the other EAE-induced groups. Loss of tail toxicity was scored as 1, the hind limbs’ weakness or partial paralysis as 2, the hind limbs and the rest of the body’s complete paralysis as 3, the hind limbs and the rest of the body’s paralysis as 4, the hind and forelimbs’ paralysis as 5, and death as 5 [ 33 ].

Open field test (OFT)

The apparatus used for the OFT test was a square with a surface area of 66 cm 2 , a height of 30 cm, and walls surrounding it. Thirty-six individual squares made up the open field base. Almost a quarter of the testing area was devoted to the middle third. The test lasted 10 minutes. OF characteristics included traversed square numbers, the period passed in the middle zone, the frequency with which the animal reared, groomed, passed fecal boli, and urinated, and the size of urine patches. The OF test was repeated twice during the experiment: the first on day 13 after vaccination and the second on day 26 after therapy.

Manipulation of tissue specimens:

At the end of day 14 of the therapy duration, all the animals, including those given the treatment and those that served as the control, were sacrificed by rapid decapitation. The animals’ brains were quickly extracted, put on a Petri dish filled with ice, and then dissected to remove the brain areas (Hippocampus, cortex, medulla, and cerebellum). Before studying any section of the brain, the sections were weighed and then frozen. After preparing a 4 ml solution of cold PBS, brain sections were homogenized using an automated homogenizer at 50 mM and pH 7.4. Following a 15-minute centrifugation at 8000 RPM and 4 °C utilizing a high-speed cooling centrifuge, the homogenate was separated into a transparent supernatant, which was subsequently used for additional measurements.

TQ distribution in the brain regions

The presence of TQ was detected chromatographically at 25 °C. Isocratically, at a flow rate of 1 ml/min, methanol, potassium dihydrogen phosphate buffer, and acetonitrile (20 mM, pH 4.5) were administered as the mobile phase into the HPLC instrument’s C18 column (25 cm, 4.6 mm) at a volumetric ratio of 50:20:30 (v/v/v). (USA YL 9100 HPLC).

Oxidative biomarkers:

Nitric oxide (NO)

We used the bio diagnostic kit no. 25 33 to evaluate NO levels. Using the Griess reagent and a known nitrite content, NO levels were determined using the method described by Moshage et al. [ 34 ]. When the Griess reagent was added to the tissue lysate, the nitrite became an azo compound with a rich purple color. A spectrophotometer (Jenway UV-6420; Barloworld Scientific, Essex, UK) was used to measure the absorbance at 450 nm.

Lipid peroxidation (MDA)

Malondialdehyde (MDA) is a biomarker of lipid peroxidation. The method developed by Begoña Ruiz-Larrea et al. [ 35 ] to measure MDA involved detecting thiobarbituric acid-reactive molecules. Thiobarbituric acid forms a pink complex with the reactive chemicals shown above. The absorbance of the newly synthesized chemical was evaluated photometrically at 532 nm.

Reduced glutathione (GSH)

A biodiagnostic kit (No. GR 25 11) was used to measure GSH concentrations by the Ellman method, in which the sulfhydryl group of GST reacts with Ellman’s reagent, 5,5’-dithiobis(2-nitrobenzoic acid) [ 36 ]. A spectrophotometer was set at 412 nm to detect yellow nitro-mercaptobenzoic acid.

Catalase

Catalase activity (measured by spectrophotometry) was assayed using a Biodiagnostic Kit (No. CA 25 1) according to Aebi’s method [ 37 ]. After 1 min, the reaction between catalase and H 2 O 2 stops when catalase is inhibited. Observing the interaction of hydrogen peroxide with 3,5-dichloro-2-hydroxybenzene sulfonic acid (DHBS) and 4-aminophenazone helps evaluate the catalase action in a sample. The absorbance of the chromophore was recorded at 510 nm.

Histopathology and immunohistochemistry

The selected nervous tissue specimens were fixed in 10% neutral buffered formalin. Specimens were routinely processed, sectioned, and stained with Hematoxylin and Eosin (H&E), as described by Suvarna et al. [ 38 ]. The paraffin-embedded sections from the spinal cord and sciatic nerves were further stained with Luxol Fast Blue, as described by Suvarna et al. [ 38 ]. Tissue sections were examined using an Olympus BX43 light microscope and captured with an Olympus DP27 camera linked to CellSens Dimensions software (Olympus). Small-sized Spinal cord and sciatic nerve specimens were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer and then post-fixed in 1% osmium tetroxide for 1 h. Subsequently, the 1-μm-thick semi-thin sections were stained with toluidine blue for further evaluation by transmission electron microscope (TEM).

For immunohistochemistry, Paraffin-embedded sections were used. Protein-blocking steps were followed by rehydration, and endogenous peroxidase was blocked with hydrogen peroxide and bovine serum albumin (BSA). The tissue sections were rinsed several times with Tris buffer, then the primary antibodies were added (anti-GFAP, Santa Cruz Biotechnology, Inc., Heidelberg, Germany) at a dilution of 1:150 for 12 h at 4 °C. After that, washing was performed 2 hours at room temperature with secondary horseradish peroxidase (HRP)-labeled antibody (goat anti-mouse HRP-labeled secondary antibody, Abcam, UK) at a dilution of 1:1000. The color was developed using the DAB Substrate Kit. The expression intensity was measured using ImageJ software for color deconvolution of the captured images.

Brain tissue morphology using transmission electron microscopy (TEM)

After the hippocampus and cortex were extracted from the brains of the various experimental groups, they were fixed at 4 °C in 0.1 M phosphate buffer (pH 7.2), 1.5% paraformaldehyde, and 1% glutaraldehyde. The specimens were cleaned in a pH 7.2; 0.1 M cacodylate buffer and then post-fixed in 1% osmium tetroxide for 1.5 h at room temperature. After a brief rinse in deionized water, the samples underwent dehydration in a succession of more concentrated ethanol baths, followed by infiltration with propylene oxide, and were then embedded in EPON epoxy resin. The specimens were polymerized at 60 °C for 24 h after being mixed with epoxy resin and then placed in capsules. RMC MT-X ultramicrotomes cut thin slices on copper grids. A JEOL JEM-1400 transmission electron microscope was used to take 80-kV images (JEOL, Ltd., Tokyo, Japan).

Statistical analysis

The Statistical Package for the Social Sciences (SPSS) 19 was used to analyze all the data. Non-parametric statistics (the Kruskal-Wallis test combined with the Mann-Whitney U statistics) were utilized to determine the highest clinical score, the number of recurrences, and the number of remissions. A p -value of 0.05 was considered statistically significant. Similarly, analysis of variance (ANOVA) was used to examine body mass, open-field behavior, and oxidative stress. When the F-test was significant ( p > 0.05), Duncan’s post hoc test was performed. Additionally, a percentage was provided, indicating the change in value compared to the control.

Results

Determination of the nanoformulation characteristics

The present study involved TEM observation of formulations comprising spherical MSNs and LB-MSN. As shown in Fig. 1 (A), the silica nanoparticles had minor aggregates with identical pore arrangement and an average diameter of 144.52 ± 0.06 nm. While the LB thickness of the prepared LB-MSNs was approximately 5.53 nm, the particles exhibited a spherical morphology, with an average diameter of 171.21 ± 14.59 nm (Fig. 1 B).

DLS findings are shown in Fig. 1 (C), and they demonstrate the average hydrodynamic diameters of three developed nanoparticles (MSNs: 291.9 ± 64.3 nm, MSN-TQ: 383 ± 113.5 nm, and LB-MSNs: 504 ± 73.6 nm). Moreover, the polydispersity index (PDI) indicates a uniform composition across all formulations (values are MSNs: 0.234, MSN-TQ: 0.251, and LB-MSNs: 0.474 nm). The MSNs had a negative net charge (−28.2 ± 2.48 mV). The net charge after TQ loading elevated to −56.1±6.74 mV. The ZP charge decreased slightly to −50.2 ± 6.3 mV upon application of the LB.

In vitro release experiment

This section describes the in vitro release profile of TQ from MSNs (Fig. 2 ). The release profile exhibited an initial prompt release of TQ from the outermost layer (surface) of MSNs, followed by a more gradual and prolonged release, which can be attributed to the release of TQ via the MSNs’ internal pores. The restriction and regulation of the gradual discharge of TQ out of the internal pores are possibly linked to the intermolecular affinity between the silica present in MSNs and the oxygen atoms in TQ itself.

In vivo experiments

Daily clinical scores

In our study, relapsing-remitting encephalomyelitis (RRE) was indicated (Table 1 ) by alternating relapse and remission intervals, which agrees with Mir et al. [ 39 ], who used the same induction procedures. LB-MSN was the only treatment among all groups that demonstrated a significant reduction in relapse days compared with the EAE group.

Impact of EAE induction and different treatments on rat body weight during the experimental period

The rats’ body weight was recorded during the experimental period, as shown in Fig. 3 . The clinical manifestations of EAE include considerable weight loss, anorexia, and decreased desire for sweet solutions. The EAE group showed a significant reduction in body weight compared with the control group. TQ and LB-MSN failed to restore the control body weight, resulting in a decrease in body weight that was even more pronounced than in the EAE group.

Open field test (OFT) parameters

The OFT can be used to assess locomotion, exploratory behavior, and rats’ general performance, which can be influenced by sickness [ 40 ]. On the 13 th day post-immunization, the EAE group consumed the lowest time in the center in the open field test, whereas the TQ, in addition to the LB-MSN groups, spent more time in the center. Rearing and the number of squares crossed were most remarkable in the two groups and lowest in the EAE group on the same day. On the 26 th day post-immunization, grooming behavior was significantly lower in the LB-MSN and TQ groups.

Distribution of TQ in different brain regions

Table 2 presents the quantitative distribution of TQ across various brain regions for the two rat groups. The first group, the TQ group, received TQ, while the second group, the LB-MSN-treated group, received TQ-loaded MSNs coated with LB. The findings indicate that the incorporation of TQ into MSNs covered with LB has led to improvements in drug targeting toward the cerebellum, cortex, hippocampus, and medulla by 11.64%, −32.09%, −22.56%, and 16.09%, respectively, compared with the TQ group. Nonetheless, no noteworthy differences in TQ levels were observed between the two cohorts under investigation.

Antioxidant and oxidant stress biomarker assessment

Nitric oxide (NO) levels

No results in the EAE model showed a significant change compared to the control group (Table 3 ). In contrast, the TQ group showed a significant elevation in the medulla (163.54%) and a significant reduction in the cerebellum and cortex (40.02% and 20.92%). For MSNs, NO demonstrated a substantial elevation in the medulla (27.56%) and significant decreases in the cerebellum and cortex (23.84% and 16.95%, respectively). Compared with the control group, LB-MSNs showed a significant elevation in the cortex and medulla (13.13% and 78.94%, respectively) and a significant decrease in the cerebellum (16.31%). The four mentioned groups showed no significant change in the hippocampus in the control group.

Malondialdehyde levels (MDA)

Compared with the control group, the EAE model showed a significant increase in MDA levels in the cortex (17.4%), hippocampus (120.86%), and medulla (201.21%), and a significant reduction in the cerebellum (96.51%). MDA exhibited a significant increase in all tested areas in the TQ group. In MSNs, MDA indicated a significant increase in the following areas: cortex (62.57%), hippocampus (75.22%), and medulla (435.54%). However, MSNs showed no significant change in the cerebellum. Compared with the control group, the LB-MSNs group showed significantly higher levels in all tested areas (Table 4 ).

Glutathione-reduced (GSH) levels

GSH levels indicated a significant elevation in the EAE group compared with the control group only in the cerebellum area (34.17%) (Table 5 ). Still, no significant alterations were indicated for the cortex, hippocampus, or medulla. In the TQ group, significant reductions were observed in the cerebellum and medulla (25.64% and 56.13%, respectively); however, there was no significant alteration in the cortex or hippocampus. The MSNs group demonstrated a significant elevation in the hippocampus (45.79%) and a significant decline in the cerebellum (7.34%), with no significant change in the cortex and medulla. Regarding the control, the LB-MSNs group showed a significant elevation (26.53%, 22.82%) in both the cortex and the hippocampus. In comparison, significant decreases of 25.29% and 41.41% in GSH were observed in the cerebellum and medulla, respectively.

Catalase activity

Catalase results revealed a significant elevation in the hippocampus (41.53%) compared with the control group. In contrast, catalase indicated a significant reduction in the cortex, cerebellum, and medulla (45.48%, 32.64%, and 45.31%, respectively). TQ catalase revealed a significant elevation in the cerebellum (122.4%) and a significant reduction in the cortex (84.75%), but no significant alterations in the hippocampus and medulla. For MSNs, catalase showed a significant elevation in the cerebellum (55.86%), but a significant reduction in the following tested areas: cortex, hippocampus, and medulla (55.89%, 25.25%, and 40.05%, respectively). Compared with the control group, LB-MSN results showed a significant increase in the medulla (7.9%) and a significant decrease in the cortex (91.37%). However, no significant changes were observed in the cerebellum and hippocampus (Table 6 ).

Neuropathology in different areas of the CNS and sciatic nerves

Olfactory bulb

Microscopic examination of the control group revealed typical histological structures of the olfactory bulb’s cellular layers. The EAE model group showed diffuse gliosis with perivascular (PV) mononuclear cell infiltration in the olfactory nerve fibrillar layer (ONL) associated with extensive demyelination; there was glial infiltration in between periglomerular cells in the glomerular layer (GL) in addition to individual necrosis of mitral cells comprising the mitral layer (ML) (Fig. 4 a and b). The external plexiform layer showed few infiltrations of PV mononuclear cells. The inflammatory reaction and demyelination were slightly reduced in the TQ-treated group (Fig. 4 c and d) and markedly alleviated in LB-MSN (Fig. 4 e and f).

Cerebral cortex and striatum

The EAE model group showed PV aggregation of lymphocytes admixed with macrophages around the meningeal blood vessel (bv) (Fig. 5 a). Intense PV lymphocytic aggregation in the deeper cerebral cortex was observed (Fig. 5 b), associated with neuronal necrosis. PV lymphocytic aggregation was reduced in the TQ-treated group (Fig. 5 c). At the same time, a moderate inflammatory reaction in the PV area comprising the meningeal vasculature was noted in the MSNs-treated group. In contrast, few lymphocytes were detected in the LB-MSN group.

The EAE model exhibited diffuse gliosis and neuronal necrosis in the internal capsule (Fig. 5 d), caudate putamen (Fig. 5 e), and globus pallidus (Fig. 5 f). The glial reaction was reduced in all treated groups, and marked improvement was observed in the LB-MSN-treated group (Fig. 5 g- i).

The hippocampus

The microscopic examination of the hippocampus revealed histopathological alterations affecting all layers, but more pronounced in the CA3 region and the dentate gyrus. There was a marked reduction in neuronal cellular density in CA3 cells with massive necrosis of pyramidal cells associated with gliosis (Fig. 6 a). The dentate gyrus showed necrosis of pyramidal neuronal cells, astrogliosis, and microgliosis in the dentate hilus (Fig. 6 b). Histopathological alterations were markedly reduced in all treated groups, with the reduction more pronounced in the mixed treatment group (Fig. 6 c–h).

Mid-brain and medulla

The substantia nigra exhibited microgliosis with chromatolysis of neuronal cells (Fig. 7 a & b). The glia reaction was slight in the TQ (Fig. 7 c) and MSNs treated groups, whereas no apparent histopathological alterations were noted in the LB-MSN treated group (Fig. 7 d).

Histopathological alterations in the medulla were detected only in the EAE model, characterized by lesions with reactive astrocytes (gemistocytes) and microgliosis, admixed with mononuclear cells (Fig. 7 e & f).

Spinal cord

The EAE model showed extensive demyelination of the white matter with Wallerian degeneration of axons comprising the white matter (Fig. 8 a) associated with microgliosis and astrogliosis (Fig. 8 b). Hypercellularity of grey and white matter was evident in the EAE group. Neuronal necrosis is characterized by grey-matter, with perivascular mononuclear cell aggregation extending into the neuropil, associated with astrogliosis and microgliosis, and accompanied by the formation of axonal spheroids (Fig. 8 c). The TQ-treated group showed reduced demyelination and a glial reaction in the white matter. In contrast, the grey matter showed lymphocytic aggregation (Fig. 8 d), microgliosis, and perivascular aggregation of a few lymphocytes (Fig. 8 e) associated with necrosis of individual neurons (Fig. 8 f). The MSNs-treated group showed demyelination and Hypercellularity of white matter that was slightly reduced in comparison with the EAE untreated group. Intense PV lymphocytic aggregation extending slightly into the neuropil of the grey matter was detected (Fig. 8 g). The neurons showed necrosis. The neuropathology of white and grey matter lesions was significantly reduced in the LB-MSN treated group (Fig. 8 h).

Sciatic nerve

The EAE model group showed marked lymphocytic infiltration of the neurovascular stroma, which extended into the perineurium and epineurium (Fig.s. 9 a & b). The nerve fascicles showed loss of Schwann cells, demyelination, and axonal degeneration (Fig. 9 c). The developed histopathological alterations were partially reduced in the TQ (Fig. 9 d & e) and MSNs treated groups (Fig. 9 f & g) and markedly reduced in LB-MSN treated groups (Fig. 9 h & i).

GFAP expression in the spinal cord:

Microscopic examination of the spinal cord from the EAE group demonstrated a significant elevation in GFAP+ cells in spinal cords with an increase in the number, dendrites, and size of immune-positive astrocytes (Fig. 10 a). TQ, MSNs, and LB-MSN treated groups demonstrated a significant decline in GFAP expression compared with the EAE group (Fig. 10 b, c, d). At the same time, no significant difference was observed between the TQ- and MSN-medicated groups. Treatment with both resulted in a significant decline in GFAP expression compared with the other treatment groups alone.

Luxol fast staining for demyelination of the spinal cord and sciatic nerve

The EAE group exhibited extensive demyelination of the white matter in the sciatic nerve and spinal cord (Figs. 11 , 12 a). Mild, moderate, and minimal demyelination were evident in the sciatic nerve and spinal cord in the TQ-treated group (Figs. 11 , 12 b), MSNs-treated group (Figs. 11 , 12 c), and LB-MSN-treated group (Figs. 11 , 12 d).

Ultrastructure of myelin sheath employing TEM in Wistar rats

Spinal cords

The results of toluidine-stained semi-thin sections demonstrated the normal distribution of myelin and axons in the spinal cord (Fig. 13 a), while splitting of myelin (Fig. 13 b) and chromatolysis comprising neurons (Fig. 13 c) were detected in the spinal cord of the EAE untreated group. At the same time, no significant difference in axonal or myelin structure and distribution was detected in the other EAE-treated groups (Fig. 13 e–f).

TEM revealed that in controls, normal axon and myelin distributions were observed in the spinal cord structure (Fig. 14 a). The EAE group showed perineuronal edema, necrotic neurons, marked demyelination, and mitochondrial swelling associated with axonal disruption (Fig. 14 c-d) whereas, in the TQ treated model, the results revealed marked alleviation in demyelination of axons with the typical structure on neurons of the spinal cord tissue (Fig. 14 e-f). The MSNs treated model showed focal myelin splitting and necrosis of neurons with axonal inclusion (Fig. 14 g). On the other hand, mild perineuronal edema, focal axonal distortion, and swelling of SER and mitochondria with standard myelin structure were detected in the LB-MSN treated model (Fig. 4 h).

Sciatic nerve

The results of toluidine-stained semi-thin sections demonstrated a normal distribution of myelin and axons in the peripheral sciatic nerve (Fig. 14 a). The EAE untreated model showed extensive demyelination and myelin splitting (Fig. 14 b). A Marked reduction in demyelination of axons was detected in other treated groups, with more effect in the LB-MSN treated model (Fig. 14 c–e).

TEM revealed normal axon, myelin distribution, and Schwann cell structures (Fig. 16 a). The EAE group showed splitting and separation of the myelin sheath, perivascular edema, and axonal disruption with vacuolization of Schwann cells that showed swelling of SER, in addition to the presence of macrophages (Fig. 16 b-d). The sciatic nerve of TQ or MSNs treated models showed focal splitting of the myelin with remyelination (Figs. 16 e-g). Treatment with TQ and MSNs resulted in normal myelinated axons, accompanied by focal, mild vacuolization of Schwann cells (Fig. 16 h).

Discussion

EAE is the most employed model for MS, a human inflammatory demyelinating illness of the CNS. Strong T-cell responses elicited by immunization with myelin antigens initiate the pathogenesis of EAE, an antigen-driven autoimmune disease characterized by the degeneration of CNS myelin. Immunization with myelin peptides or CNS tissue causes EAE. Various disease characteristics arise from different immunization regimes. [ 41 , 42 ]. Crossing the BBB is the primary challenge researchers face when testing promising new formulations to treat neurological disorders, as the BBB prevents these drugs from entering the brain [ 43 ]. MSN has been confirmed to cross the BBB [ 27 ].

The present study demonstrates the development of an MS animal model using the EAE model. In our research, the development of the EAE model was corroborated by the clinical manifestation of EAE, including considerable weight loss, anorexia, and decreased desire for sweet solutions. This behavior is proven to be induced in acute phases and restored in recovery phases [ 44 ]. According to reports, pro-inflammatory cytokines involving TNF-α and IL-1β are activated and produced in the brain and peripheral tissues, mediating such behavioral reactions [ 44 , 45 ]. In the existing study, we demonstrated that the EAE model exhibits anxiety-like behavior before the onset of clinical symptoms and that there are neurophysiological changes in the nucleus striatum that are linked to intense microglial activation. All of these events require an excess of TNF-α. Thus, the current decline in the OFT’s parameters (number of crossed squares, grooming, and rearing) after 13 days of daily EAE therapy supports the rat model of EAE development.

The physical characterization of the synthesized nanoformulations suggests their potential for use in drug delivery applications. Spherical MSNs with identical pore arrangements have an average width of 144.52 ± 0.06 nm, whereas LB-MSNs have a larger width of 171.21 ± 14.59 nm, including approximately 5.53 nm from the LB. The DLS results show that the average hydrodynamic diameters of the three formulated nanoparticles are within the optimal range for drug delivery, with the LB-MSNs having the largest diameter of 504 ± 73.6 nm. The uniform composition of the formulations, as indicated by the low PDI values, demonstrated that the generated MSNs had a negative net charge (−28.2 ± 2.48 mV). However, the net charge after TQ loading improved to −56.1 ± 6.74 mV.

Furthermore, upon application of the LB, the charge decreased slightly to −50.2 ± 6.3 mV, suggesting that the particles are stable and do not aggregate. Furthermore, the higher charge indicates enhanced stability and a potential to bypass the BBB and the immune system, allowing it to reach multiple areas at varying concentrations. The high encapsulation efficiency (93% ± 2.5) of TQ in the MSNs is attributed to the enormous pore surface area, which can accommodate substantial amounts of TQ. These results suggest that the synthesized nanoformulations have potential for use in drug delivery applications. They could be further explored for their therapeutic effects in treating neuroinflammatory disorders such as EAE.

The release profile of TQ from the LB-MSNs was analyzed in vitro, revealing that TQ release from the formulation is rapid within the first 5 h and then slows. The progressive release of TQ through the MSNs’ internal pores is most likely what causes the sluggish discharge, whereas the quick release of TQ is most likely the result of TQ being released from the MSNs’ outer surface. However, the release of TQ from the MSNs’ interior pores may be restricted because of the binding forces between the silica of MSNs and the oxygen of TQ compounds [ 46 ]. These results suggest that the formulation of LB-MSNs could be a promising strategy for controlling the release of TQ from the nanoparticles’ surfaces and interior pores, enabling the delivery of targeted TQ doses to different brain regions. Further studies are necessary to investigate the in vivo release profile of TQ from the LB-MSNs formulation and its therapeutic effects in treating neuroinflammatory disorders, such as EAE.

These study findings reveal that rats with EAE experienced significant weight loss, consistent with previous studies on the clinical manifestation of this disease. However, TQ and LB-MSN did not restore control of body weight or even decrease it. Although studies [ 47 , 48 ] on N. sativa have reported significant decreases in waist circumference and body weight, as well as reduced body fat mass, feeling of hunger, and appetite, the effect of TQ and LB-MSNs on body weight may be complex and depend on various factors. The findings suggest that additional studies are crucial to fully understand the effect of TQ and LB-MSNs on body weight in the EAE model while also considering the potential impact of the RRE model of MS and the limitations of the OFT. Despite these limitations, the LB-MSN formulation improved the rats’ behavior in the first and second OFT, suggesting a potential therapeutic application for this formulation in managing EAE.

Regarding the OFT, on the 13 th day post-immunization, the EAE group recorded the least time spent in the center (Table 7 ), which is consistent with Haji et al. [ 49 ], who showed that EAE-induced mice tend to remain outside the center of the open field with a preference to be closer to walls, a phenomenon known as thigmotaxis. The results also show that the TQ group consumed more time in the center than both the control and EAE groups, an observation that aligns with the reported anxiolytic effects [ 50 ]; moreover, the rats of the LB-MSN group spent almost three times the time consumed by the EAE group and around two times the time spent by the control, suggesting that LB-MSN treatment was associated with increased the exploratory behavior and lowered the anxiety of the test individuals. However, on the 26 th day post-immunization, all groups showed no significant difference in time spent in the center, which could be attributed to the test being performed during a relapsing phase.

Rearing behavior is a method of gathering information about the new environment in which the rat has been placed [ 51 ]. It is also suggested that rearing and the number of squares crossed are associated with exploratory behavior, locomotion, and anxiety. As they increase, this indicates increased exploration drive and decreased anxiety [ 52 ]. The assessment of locomotor and exploratory behavior revealed a time-dependent effect of the treatments. On day 13 post-immunization, animals in the EAE group showed the most impaired performance, with the lowest scores for both rearing and squares crossed. The TQ and LB-MSN treatments significantly ameliorated these deficits, resulting in higher scores that correlate with improved locomotion, exploratory behavior, and reduced anxiety. The beneficial effect of TQ aligns with its known properties in stress alleviation and motor enhancement [ 53 ]. The association between neuropathological improvement and behavioral recovery provides a strong biological rationale. However, the study did not experimentally establish a direct causal relationship, and behavioral improvements are likely multi-factorial, potentially involving complex interactions between reduced neuroinflammation and systemic physiological factors.

By day 26, the results profile had shifted. Locomotor activity (squares crossed) was highest in the EAE group, while the other groups showed no significant differences from one another. Exploratory behavior (rearing) was lowest in the LB-MSN group at this later time point, with no significant variation among the other groups.

Rodents’ self-grooming can be observed in behaviors such as paw licking, head washing, and face and nose washing. It has been suggested that it is a mechanism for dearousal in rodents [ 54 ]. Grooming in rats is enhanced by exposure to novel environments [ 55 ]. The results for grooming are fascinating, as the grooming behavior of the control and EAE groups is very similar on the 13 th and 26 th days post-immunization. In contrast, the LB-MSN and TQ groups showed significantly lower grooming behavior on the 26th day post-immunization.

Freezing, an unlearned response to specific situations, is a valid characteristic of emotionality [ 56 , 57 ]. The lowest freezing time on the 13 th day for LB-MSN is consistent with TQ’s anxiolytic activity, as reported by Firdaus et al. [ 50 ]. Urination and defecation are indicators of fear and emotion, and they can even be used to study individual differences in emotion [ 58 – 60 ]. Even though the LB-MSN group showed lower defecation than the control and EAE groups, the differences in urination and defecation among the groups were not significant.

In general, the LB-MSN formula was associated with improved behavioral outcomes in the OFT. While these improvements in exploration and locomotion provide a plausible biological rationale when viewed alongside the evidence of remyelination, it is important to clarify that this correlation does not definitively prove a direct causal link. Other factors, such as the formulation’s impact on systemic inflammation or peripheral nerve function, could also contribute to the behavioral shifts. Furthermore, the difference between the first and second OFT is logical, as reperforming the OFT and preexposing rats to the same arena may yield different results [ 40 ]. Another contributing factor to these differences may be the relapsing-remitting disease course observed in the MS model utilized.

In the pathological mechanism of MS, oxidative stress plays a vital role in the demyelination and exacerbation of the disease [ 61 ]. NO, an oxidative stress biomarker, is produced at low levels in the normal CNS and plays a beneficial role in regulating blood flow. However, when inflammation occurs, as in MS animal models, NO is continuously produced by inducible nitric oxide synthase (iNOS), an enzyme not typically present in the brain. A higher NO concentration increases BBB permeability, induces inflammation, demyelination, and oligodendrocyte destruction [ 62 ]. Another oxidative stress biomarker is MDA, a final lipid peroxidation product produced by ROS in cells [ 63 ]. To overcome oxidative stress, cells are equipped with antioxidant machinery such as GSH and catalase to protect them from ROS-induced cellular damage [ 64 ]. Inflammatory lesions in the white matter lead to demyelination in EAE and MS, resulting in clinical impairments. Rats with relapsing EAE exhibit a significant inflammatory response, primary demyelination with modest remyelination, and some axonal loss in the neuropathology of the active CNS lesion. As the disease progresses, these lesions become less severely demyelinated, more gliotic, and axon-depleted [ 10 ]. The measurements of oxidative stress biomarkers in this study present a complex picture that varies significantly across CNS regions. The results reveal that the modulation of oxidative stress by TQ and LB-MSNs is highly region-specific rather than a generalized systemic effect. In the cortex, in vivo experiments conducted by Parveen et al. [ 65 ] and Hozayen et al. [ 66 ] revealed that MSNs suppressed antioxidant enzymes and increased ROS production. These findings align with an in vitro study by Zhou et al. [ 67 ], which showed that MSNs induced elevated ROS, MDA production, and GSH depletion in a rat pheochromocytoma (PC12) cell line. These studies, combined with the biodistribution results showing lower TQ concentrations in the cortex of the LB-MSNs group compared with the free TQ group, suggest that MSNs may have contributed to localized oxidative stress in certain areas, where TQ concentrations were insufficient to alleviate it fully.Therefore, the data suggest that the LB-MSN formulation did not simply “normalize” oxidative stress across the entire brain. However, the lack of a uniform antioxidant profile across the CNS does not preclude histological benefit. Moving on to the EAE model, the TQ-treated group exhibited decreased lymphocytic aggregation, whereas the MSNs-treated group showed a moderate inflammatory response. In contrast, the LB-MSN-treated group demonstrated the most notable improvement, with some lymphocytes observed in the meningeal vasculature. In addition, all treated groups showed a decrease in glial response, with the LB-MSN-treated group exhibiting the most significant improvement in the internal capsule, caudate putamen, and globus pallidus, where diffuse gliosis and neuronal necrosis were observed in the EAE model. These findings suggest that the therapeutic potential of LB-MSNs in reducing inflammation and glial responses may occur through mechanisms—such as the modulation of inflammatory cytokines or physical shielding of the BBB—that are partially independent of the specific oxidative stress pathways measured. Its protective effects are more likely attributable to potent anti-inflammatory mechanisms or direct remyelinating effects.The non-significant change in TQ distribution levels between the TQ and LB-MSN groups in the cerebellum resulted in non-significant differences in $NO$, MDA, and $GSH$ levels. However, the exacerbated results in the TQ and LB-MSN groups for $NO$, MDA, and $GSH$ in certain regions are attributed to the potential pro-oxidant effects of MSNs and TQ. Studies have shown that higher TQ concentrations can induce ROS production, indicating pro-oxidant effects [ 68 , 69 ]. El-Najjar et al. [ 70 ] also explained that semiquinone, a product of TQ reduction by reductases, induces ROS generation. This complexity suggests that while the formulation improves structural outcomes, it may simultaneously trigger localized compensatory oxidative responses.The observed increase in NO and MDA levels in the medulla contradicts the reported antioxidant effects of TQ extracted from N. sativa [ 71 ], suggesting that MSNs may have induced localized oxidative stress in the medulla, as demonstrated by Zhou et al. [ 67 ]. The decrease in $GSH$ levels observed in the medulla may help protect cells from free radicals, as $GSH$ functions as an antioxidant by altering the solubility of harmful chemicals [ 72 ]. Regarding histopathology, microgliosis and neuronal cell chromatolysis were observed in the substantia nigra across all groups, with minimal glial reactivity observed in the groups treated with TQ and MSNs. The LB-MSN group showed no overt histopathological changes, indicating its potential as a therapeutic agent. It is important to note that the histological recovery in the medulla occurred despite the localized increase in oxidative markers, reinforcing the hypothesis that the formulation’s primary benefit may be anti-inflammatory or anti-apoptotic rather than purely antioxidant.In the hippocampus, MDA levels increased significantly across all treated groups compared with the control group. Additionally, GSH levels significantly increased in both the MSN and LB-MSN groups but did not change significantly in the TQ group. The results indicate no statistically significant alterations in catalase activity across all experimental groups compared with the untreated group. The hippocampus is highly susceptible to lipid peroxidation due to its abundance of polyunsaturated fatty acids and iron [ 73 ]. However, unlike Zhou et al. [ 67 ], who confirmed that ROS levels dramatically deplete cellular GSH, the hippocampus’s GSH levels showed an increase, possibly enhanced by the effect of TQ loaded on the MSN, as TQ plays a vital role in producing thiol content and increasing GSH levels [ 74 ]. These data further highlight the regional heterogeneity of the CNS response to nanoformulations. In summary, although the oxidative stress data do not support a generalized alleviation of oxidative stress, the TQ and LB-MSN groups demonstrated potential therapeutic effects in reducing demyelination and axonal disruption in the EAE model. Further research is needed to fully decouple the pro-oxidant potential of MSNs from the protective anti-inflammatory effects of the loaded TQ. Nonetheless, these results suggest that loading TQ onto MSNs coated with LB may enhance its therapeutic potential in reducing demyelination and axonal disruption in neuroinflammatory disorders such as EAE, likely through a multifaceted mechanism that warrants further molecular characterization.

Study limitations

While the findings of this study provide promising evidence for the use of LB-coated MSNPs as a delivery vehicle for Thymoquinone, several limitations must be acknowledged. First, the use of a single animal model (EAE) limits the generalizability of these results to the full clinical complexity of human multiple sclerosis. Second, the study utilized a single dose of the formulation, and the lack of dose-response data precludes a definitive determination of the optimal therapeutic window. Third, while indirect evidence suggests improved delivery, the bioavailability of TQ and its specific mechanism of crossing the BBB were not directly quantified in this study.

Furthermore, the observed improvements in locomotor and exploratory behavior, while strongly correlated with histological evidence of remyelination, provide a biological rationale rather than a definitive causal link. The notable disconnect between some oxidative stress data and the observed histological improvement suggests that other unexamined mechanisms, such as potent anti-inflammatory pathways or anti-apoptotic effects, may be involved. Finally, while the initial safety profile of the LB-MSNs appears favorable, further long-term toxicity studies and high-resolution molecular mapping are necessary before these findings can be translated toward clinical applications.

Conclusion

In conclusion, this study evaluated the potential of Thymoquinone (TQ) delivered via lipid-bilayer-coated mesoporous silica nanoparticles (LB-MSNs) as a therapeutic strategy for neuroinflammatory conditions. While our findings revealed notable histological improvements and reduced axonal demyelination in the EAE model, the underlying biochemical mechanisms appear complex and region-specific. The LB-MSN formulation was associated with improved exploratory behavior and localized modulation of oxidative stress markers; however, these effects did not represent a generalized normalization of the oxidative state across all examined CNS regions.

These preliminary results suggest that the protective profile of the LB-MSN formulation may be driven by multifaceted anti-inflammatory or remyelinating pathways that warrant deeper exploration. As a preliminary investigation using a single-animal model and a single dosage, this study provides a biological rationale for the use of lipid-coated nanomaterials. However, it does not yet establish definitive clinical efficacy or complete pharmacokinetic profiles. Future research focusing on dose-response relationships, direct quantification of BBB penetration, and high-resolution molecular mapping of inflammatory cytokines is essential to fully elucidate the therapeutic potential of this approach in the management of multiple sclerosis.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Acknowledgements

This work was supported by the Science & Technology Development Fund (Egypt) (STDF), Project ID: 33500, Young Researcher.

Abbreviations

The blood-brain barrier

Cetyltrimethylammonium bromide

3, 5-dichloro-2-hydroxybenzene sulfonic acid

Dynamic light scattering

Dimethyl sulfoxide

Folic acid

Reduced glutathione

A lipid bilayer was used to cover MSNs

Malondialdehyde lipid peroxidation

Multiple sclerosis

Mesoporous silica nanoparticles

Nitric oxide

Phosphate-buffered saline

Polydispersity index

Particle size

Arginine–glycine–aspartic acid

Transmission electron microscopy

Tetraethyl orthosilicate

Thymoquinone

Zeta potential

Author contributions

H.M.F. designed this study. H.M.F., A.S.E., M.M.A., A.S.M., E.S.E., T.M.A., A.B.M., M.W.A., F.F.M., and M.M. F. wrote the primary manuscript, conducted experiments, and generated Figs. All contributors evaluated the manuscript. All contributors reviewed and approved the manuscript.

Funding

Project ID 33500: Young researcher funded by the Science & Technology Development Fund (Egypt) (STDF).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The Institutional Animal Ethics Committee authorized the in vivo protocol. Number: ( CU-I-F-28–21 ). This study’s animal procedures followed the ARRIVE guidelines and relevant restrictions. Informed consent did not apply. “The Institutional Animal Care and Use Committee (CU-IACUC), Cairo University” accepted all experimental protocols.

Consent for publication

NA.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

Data Availability Statement

No datasets were generated or analysed during the current study.