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. 2025 Dec 8;10(50):61159–61168. doi: 10.1021/acsomega.5c00405

Penetration of the Blood–Brain Barrier and Liver for Repairing Motor Disorders in a Rat Model of Parkinson’s Disease Using a Nanocopolymer

Aliaa A Razzak †,‡, Zahraa S Al-Garawi †,*, Adawya J Haider §, Bahaa Hassan ∥
PMCID: PMC12750232  PMID: 41476516

Abstract

Parkinson’s disease (PD) is a neurodegenerative disorder that results in dopamine deficiency and motor symptoms. The blood–brain barrier (BBB) poses a significant challenge in delivering therapeutics to the central nervous system (CNS). In this study, a new nanocopolymer was developed to penetrate the BBB and deliver dopamine to the substantia nigra pars compacta (SNpc) in a Parkinson’s-induced rat model. The dopamine loading was 85%, with a release efficiency of 75%. Subsequently, 70 Swiss rats were induced to have Parkinson’s using 6-OHD. After 3 days of administration, Parkinson’s symptoms emerged and were confirmed by specialists. The induced animals were divided into subgroups based on the dosage of the nanocopolymer (10, 20, or 40 mg for 14 days), after which they were dissected. The brains were sectioned, and the nanocopolymer’s penetration was observed and imaged with fluorescence microscopy. Results showed that the nanocopolymer successfully crossed the BBB and the capillary endothelium in the striatum and substantia nigra. Presence of dopamine in the striatum was confirmed by the presence of yellow–green fluorescence in the substantia nigra. Reduced dopamine autoxidation provides sustained delivery of dopamine to the brain. The results also demonstrated the efficacy of this nanocopolymer in penetrating the rat liver, where it affected liver cells significantly more than it did in the brain. The developed nanocopolymer effectively crossed the BBB and elevated dopamine levels in the brain, alleviating motor symptoms in a rat model as an early-stage treatment for Parkinson’s disease.

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Introduction

The blood–brain barrier (BBB) is a significant challenge in the delivery of diagnostic and therapeutic agents to the central nervous system (CNS), which makes the treatment and diagnosis of the CNS-related diseases complex. − The BBB is composed of various cell types, including unfenestrated endothelial cells (ECs) connected through tight junctions (TJs), pericytes, astrocytes, and microglial cells. , Despite its highly selective nature, the BBB allows specific essential molecules such as nutrients and amino acids (AAs) to cross. − However, it prevents the passage of many drugs, giant biopharmaceutical molecules, into the CNS.

Innovative strategies using dopamine-loaded albumin/chitosan nanocopolymers offered promising solutions for delivering therapeutic agents to the brain. − Unlike direct brain injections, which require complex surgeries and are often unfavorable for patients, nanocopolymers can enhance the BBB penetration properties of therapeutic agents. − Dopamine and albumin, when incorporated or conjugated with chitosan nanoparticles, significantly improved the delivery of treatments for CNS diseases. − The cationic nature of these nanocopolymers enables them to penetrate the BBB through adsorptive-mediated transcytosis pathways facilitated by electrostatic interactions between the cationic peptides and anionic microdomains of the BBB. −

Adsorptive-mediated transcytosis is a nonspecific transport mechanism triggered by electrostatic interactions between positively charged molecules and the negatively charged membranes of BBB endothelial cells. − The BBB-penetrating peptides are particularly used to bind BBB receptors and typically do not interact with other cell types, proteins, and small particles but can interact broadly with various cell types and tissues. , Albumin-assisted nanoparticle passage through the BBB, potentially via endogenous albumin routes including receptor-mediated transcytosis, efficiently delivers dopamine at the site of action, BBB. Chitosan is a well-studied polymer with beneficial biological and chemical characteristics including mucoadhesion and ease of functionalization. Chitosan-based nanocarriers (CsNCr) form ionic connections with endothelial cells, allowing drug crossing over the BBB via adsorptive-mediated transcytosis. The advantage of these nanocopolymers is their ability to deliver therapeutics into neurons in addition to crossing the BBB, thereby targeting intracellular proteins with diverse cargoes. In Parkinson’s disease (PD), using nanocopolymers used to pass the BBB and release therapeutic agents directly into the brain. This targeted delivery system can significantly alleviate PD symptoms and improve the quality of life in PD lesion models. − Specifically, albumin, peptides, and chitosan serve as effective BBB delivery bioagents, carrying dopamine to treat damaged dopaminergic neurons in the striatum and substantia nigra. ,,

The proposed mechanisms behind the functional improvements include restoring brain dopamine concentrations, preventing neuronal damage from oxidative stress, and protecting dopamine from degradation during transport to ensure it reaches its target effectively. , Additionally, these nanocopolymers are stable, biodegradable, low-cost materials and can be modified to evade macrophage detection and enhance their therapeutic potential. ,,

Therefore, this study was designed to examine the potency of a new nanocopolymer composed of chitosan/albumin/dopamine to penetrate the BBB and make changes in the brain tissues of a rat model over a course of 14 days. We hypothesized that this nanocopolymer could reduce PD symptoms and improve the quality of life in PD lesion rats through stable and targeted dopamine delivery.

Results and Discussion

The in vivo study aimed to confirm the ability of the manufactured nanocopolymer (chitosan/albumin/dopamine) to penetrate the BBB and deliver dopamine effectively to the striatum within the nigrostriatal pathway of rat models induced with PD. Histological and fluorescence analyses revealed significant dopaminergic cell loss in the substantia nigra pars compacta (SNpc) of animals following 6-OHDA injection; see Figure .

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Fluorescence micrographs of substantial nigra­(SN)­coronal section of brain tissue for (1) flourescence and microscopic images of control-ve (healthy rats) as a non-lesion with 6-OHDA, (2) flourescence and microscopic images of control+ ve (lesion rats) with 6- OHDA without nanocopolymer (fluoresces represent the red dots of toxin) black region present the defect cell, (2A) flourescence and microscopic images of lesion rats with 6- OHDA with nanocopolymer (10mg/animal) red triangle (yellow-green dots ) showed the treatment and red arrows show defect cell, (2B) flourescence and microscopic images of lesion rats with 6- OHDA with nanocopolymer (20mg/animal) red arrow shows death neuron red triangle (yellow-green dots ) shows the treatment, (2C) flourescence and microscopic images of substantial nigra (SN) coronal section of brain tissue for lesion rats with 6- OHDA with nanocopolymer (40 mg/animal) ) red triangle (red arrows show the low level of defect in cell nerves). Magnification: 20*20, (2D) fluorescence and microscopic images of lesion rats with 6-OHDA with nanocopolymer (40 mg/animal). (Yellow-green dots indicate the treatment.) The magnification of all images was 20*20. E) The histogram shows the statistics of images 1, 2, 2A, AB, and 2D based on the mean and SD of the red intensity area.

Neurobehavioral Observations

The rats were divided into five groups, as shown in Table . Over 15 to 21 days, we observed abnormal neurobehavior with clinical signs of PD. Loss of dopaminergic neurons in the substantia nigra, which project to the striatum, was the pathological hallmark most closely associated with the motor symptoms of PD.

1. Classification of Rat Groups.

group no. of rat experimental setup
1 5 control −ve: healthy rats were orally administered a saline solution until the end of the experiment
2 65 control +ve: lesioned rats were orally administrated with 1000 μL 6-OHDA, every 2 days for 3 weeks
after 3 weeks, the neurobehavioral symptoms appeared; group 2 was divided into 4 groups
2A 5 control + ve: lesioned rats with 6-OHDA (without nanocopolymer)
2B 20 lesioned rats with 6-OHDA received an intraperitoneal injection (i.p) and 100 μL nanocopolymer 10 mg/animal body weight, every day until the autopsy. This group was subdivided into 4 classes according to time of anatomy: I, II, III, and IV (5 rats per class)
2C 20 lesioned rats with 6-OHDA received intraperitoneal injection (i.p) and 100 μL nanocopolymer 20 mg/animal body weight every day until the autopsy. This group was subdivided into 4 classes according to time of anatomy: I, II, III, and IV (5 rats per class)
2D 20 lesioned rats with 6-OHDA received an intraperitoneal injection (i.p) and 100 μL nanocopolymer 40 mg/animal body weight every day until the autopsy; this group was subdivided into 4 classes according to time of anatomy: I, II, III, and IV (5 rats per class)
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Class I: Autopsy 2 days after nanocopolymer injection, class II: autopsy 5 days after nanocopolymer injection, class III: autopsy 7 days after nanocopolymer injection, and class IV: autopsy 14 days after nanocopolymer injection.

Delivery of the Nanocopolymer to the BBB

The fluorescence image in Figure shows the penetration of the nanocopolymer into the BBB based on its concentration. The images indicated a reduction in damaged cells as the concentration increased up to 20 mg. Over 20 mg, fibrosis of the liver cells was expected and appeared. The nanocopolymer (20 mg and 40 mg) showed a reduction of neuronal cell damage. This result displayed the antioxidant properties of the nanocopolymer and its ability to prevent autoxidation induced by free radicals produced from the toxic pathway. Also, Figure shows the presence of nanoparticles within neuronal cells and channels. The presence of yellow–green fluorescence within the substantia nigra suggests successful crossing into the BBB and delivery of dopamine to the target regions. This penetration is crucial for mitigating the effects of dopamine depletion in PD.

Figure (1) shows the healthy rat tissues by fluorescence analysis of the substantia nigra (SN) coronal section of brain tissue for the negative control group without receiving 6-OHDA and nanocopolymer treatment.

Figure (2) shows images of SN for the control group that received 6-OHDA without nanocopolymer treatment. Fluorescence microscopy revealed significant neuronal damage and a lack of fluorescence in the substantia nigra, indicating that no nanocopolymer was present to mitigate the effects of dopamine loss. The black regions in the images represent areas of severe cellular degradation, which correlated to the absence of motor function improvements in these rats.

Figure (2A) shows images of SN for a group treated with 10 mg (1.33 mg/mL) of the nanocopolymer. The fluorescence analysis showed a moderate penetration of nanoparticles, with a noticeable but limited presence of yellow–green fluorescence. This suggests partial BBB crossing and a moderate level of dopamine delivery. While some neuroprotection was observed, the extent of neuronal recovery was not as pronounced as in the higher-dose groups.

Figure (2B) shows images of SN for a group treated with 20 mg (2.67 mg/mL) of the nanocopolymer. The images showed a significant increase in the fluorescence intensity, which indicated a higher concentration of nanoparticles within the substantia nigra. This group exhibited substantial neuroprotection, with reduced areas of cell damage and an improved neuronal structure. The relation between the fluorescence intensity and motor function improvement suggests that this dose effectively delivered dopamine to the affected brain regions.

Figure (C and D) shows images of SN for a group treated with a 40 mg (5.33 mg/mL) nanopolymer. The fluorescence intensity in these images was the highest among all groups, indicating the maximal penetration of the BBB and extensive distribution of the nanocopolymer within the brain. Images showed minimal areas of cell damage and well-preserved neuronal structures. The doses (20,40) mg significantly improved motor coordination and overall function, confirming the therapeutic potential of the nanocopolymer at this concentration.

Figure illustrates the fluorescence micrographs of the nanocopolymer and highlights its potential to mitigate cell damage and neuronal death caused by oxidative stress. This was particularly examined by 10, 20, and 40 mg/animal of the nanocopolymer for 2 (I), 5 (II), 7 (III), and 14 (IV) days, a study course. Results indicated a gradual recovery of damaged tissues after the demonstration of the nanocopolymer. The best penetration of the BBB appeared after 14 days (Figure ) when the damaged cells decreased and dopamine levels increased. These results also indicated a lack of dopamine in the substantia nigra (SN) in the brain, which is responsible for the nervous signaling of movement in the body and thus treats PD. Therefore, penetration by the nanocopolymer could help restore the nervous signaling of the SN and facilitate the treatment.

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Fluorescence micrographs of the substantia nigra (SN) coronal section of brain tissue for three groups of rats with Parkinson’s disease (2B, 2C, and 2D) dissected after a specified period following the administration of the nanocopolymer (I after 2 days, II after 5 days, III after 7 days, and IV after 14 days). The degenerative changes in the dopaminergic pathway appeared in red (yellow arrows). The tissue improvement increases after 7 days of injection until 14 days (red triangles). The histograms at the right-hand side show the statistics of all images in 2B, 2C, and 2D channels based on the weighted intensity area.

The yellow–green dots appearing in Figures (2A–D) and indicated the presence of nanodopamine in the brain tissue compared to that observed in the healthy −ve and + ve positive control. All images were interpreted by a specialist physician and then statistically analyzed. Additional fluorescence images are attached in Supporting Information file S1.

On the other hand, liver tissue analysis of Parkinsoǹs rat models indicated health status following nanocopolymer administration of 20 and 40 mg doses. Figure (A–C) shows the construction of nanocopolymer in the liver cells and a decrease in the damaged tissue. The optimal dose of the nanocopolymer for recovering from severe stages of PD was 20 mg. This result was in line with previous studies, ,, while a dose of 40 mg was considered an overdose due to the increase of damaged cells. , Figure D shows the defect % of cells in the brain and liver after delivery of the nanocopolymer in rats. The defective cells reduced as the concentration increased; however, at a concentration of 20 mg nanocopolymer, the rats restored balanced performance to the level of the nonlesion animals, more than at the other concentrations. This work demonstrates the potential of nanocopolymers to deliver drugs across the BBB and improve the treatment of Parkinson’s in animal models by significantly reducing neuronal cell damage. A previous study used albumin/PLGA nanoparticles loaded with dopamine (ALNP-DA) in a 6-OHDA mouse model of PD. These nanoparticles had a particle size of 353 nm and a zeta potential of −27. The ALNP-DA effectively crossed the BBB, replenishing dopamine in the nigrostriatal pathway and significantly improving motor symptoms compared to that observed in untreated lesioned animals and those treated with l-dopa.

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Fluorescence and micrographs of the coronal section of liver tissue: (A) fluorescence and microscopic images of lesioned rats induced with 6-OHDA with nanocopolymer (10 mg/animal), (B) fluorescence and microscopic images of lesioned rats induced with 6-OHDA with nanocopolymer (20 mg/animal), (C) fluorescence and microscopic images of liver tissue for lesioned rats induced with 6-OHDA with nanocopolymer (40 mg/animal). Magnification:20*20. The histograms at the right-hand side show the statistics of all images based on the weighted intensity area. (D) Effect of nanocopolymer on the brain and liver cells of rats induced with 6-OHDA without nanocopolymer; lesioned rats treated with nanocopolymer 10 mg, 20 mg, and 40 mg/body weight. All groups were related to the mean of the defect cells. Gray bars refer to the standard deviation of the mean.

At a 20 mg dose, ALNP-DA restored motor coordination to the level of control animals. Another study evaluated ropinirole-loaded lipid nanoparticles in a 6-OHDA rat model of PD with a particle size >250 nm and a zeta potential of 30 mV. The nanoparticles showed improved pharmacokinetic and pharmacodynamic properties compared to the drug alone, with enhanced neuroprotective effects. Polymeric nanoparticles alone may not be sufficient for effective BBB permeation; incorporating appropriate delivery systems that signal BBB opening while preserving the drug’s pharmacological activity is crucial. The excellent properties of our nanosystem chitosan/albumin/dopamine, such as particle size (38–190 nm), zeta potential (+73 mV), encapsulation efficiency (74.31 ± 0.29), and dopamine release over 96 h in both acidic media (pH 5.4) and physiological conditions (pH 7.4), facilitated efficient BBB crossing through an adsorption mechanism in the cell membrane’s negative charges.

Scheme shows a simple representation of passing of the nanocopolymer through the BBB from the blood into the brain.

1. Penetration of the BBB by the Nanocopolymer .

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a Reproduced from a free domain (https://commons.wikimedia.org/wiki/File:Blood_vessels_brain_english.jpg.)

In conclusion, the nanocopolymer (chitosan/albumin/dopamine) successfully circumvented the BBB and was detected within brain tissue, specifically in the substantia nigra pars compacta (SNpc) with a deficiency of DA in the SNpc of a rat model of PD. The best concentration of the nanocopolymer that showed excellent penetration was 20 mg/body weight of the animal, and PD symptoms were reduced after 14 days of administration; see Scheme . Fluorescence analysis revealed yellow dots scattered throughout the tissue, indicating the presence of nanocluster agglomerates emitting fluorescence. As a mechanism of penetration, there are two main possibilities: (1) adsorptive mediator and (2) receptor mediator. In the adsorptive mechanism, the penetration relies upon charge–charge interactions with the endothelial membranes, which lead to absorbing proteins or nanocopolymers directly and passing them to the CNS (Scheme A). The second mechanism is the receptor mediator mechanism, which requires a specific binding between the nanocopolymer and the endothelial cell and acts as a receptor (Scheme B).

2. Two Possible Mechanisms of Penetration of the Nanocopolymer into the BBB: (A) Transcellular Route and (B) Receptor-Mediator Route.

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These results underscore the potential of this nanocopolymer as a therapeutic strategy for delivering dopamine to the brain, offering a promising approach to treating Parkinson’s disease.

Materials and Methods

Materials

6-OHDA hydrochloride was purchased from Sigma-Aldrich, St. Louis, MO, USA. The fluorescence microscope (Fluorescence VANOX-OLYMPUS, Tokyo, Japan), 0.9% sodium chloride injection, was purchased from MOH, USA. Acridine Orange (Polysciences or Sigma, USA). The source of ascorbic acid, formaldehyde, glycine, glycerine, ethylene glycol, sucrose, phosphate-buffered saline (PBS), chloroform, and Xylazine was from Sigma, USA. The preparation process was conducted at room temperature (RT) using double deionized water (DDW) of 99.99% purity.

Methods

Manufacture of the Nanocopolymer (Chitosan/Albumin/Dopamine)

The nanocopolymer was prepared and well-characterized in our previous work. Its cytotoxicity is really limited. Briefly, nanochitosan (4.5 g) was dissolved in 150 mL of 1% acetic acid and ultrasonicated (Ultrasonic Cell disrupter, UCD-150, P/V150W/220 V50 Hz, biobased, Shandong, China, CO, LTD) for 2 min. Then, albumin (1.12 g) was added to 100 mL of phosphate-buffered saline (PBS). The mixture of chitosan and albumin was syndicated and stirred at 1500 rpm at room temperature under an argon gas atmosphere. After 2 min, the mixture was incubated in a 40 °C water bath, dopamine (2.25 g in 50 mL of distilled water) was added, and the mixture was stirred for 30 min. Finally, a supersaturated solution of TPP (dissolved in 25 mL of distilled water) was added to the mixture and centrifuged (Sigma 3–16 KL, Germany) at 11500 rpm for 30 min. The resulting gel was stored at −8 °C for further studies.

In Vivo Study

In vivo studies were conducted using 70 Swiss adult males (albino rats), aged 8–10 weeks and weighing 200–250 g. The rats were housed under controlled conditions, with a 12 h light/dark cycle, at 25 °C, and allowed limited access to water and standard pellet food. All animal handling was conducted with proper humane care. All procedures followed the NIH guide for the care and use of laboratory animals, and the Local Ethics Committee and Animal Research Committee approved the experimental protocol (ethics committee approval code: BCSMU/0524/0005C).

6-Hydroxy Dopamine (6-OHDA)–Lesioned Rat Model of Parkinson’s Disease

8 μg of 6-OHDA was dissolved in 2 μL of a 0.2% ascorbic acid saline solution for a final concentration of 4 mg/μL. After 3 weeks of acclimatization, rats received the neurotoxin 6-OHDA orally (1 cm3 every 2 days for 3 weeks), until the appearance of neurobehavioral symptoms indicative of PD. Rats were under the care and recorded observation until their behaviors were closely monitored to assess muscular rigidity-like symptoms. Some rats were excluded because they did not show symptoms even after 3 weeks. The net number of rats enrolled in the study was 70.

Assessment of Muscular Rigidity-like Symptoms

  • 1

    The catalepsy bar test

This evaluated muscular rigidity-like symptoms in the rats. This test measured the inability to correct a constrained posture, which indicates muscular rigidity. Rats were gently placed with their forelimbs on a 10 cm high stainless-steel bar, while their hind limbs remained on the floor. Then, the time consumed by the rats to remove their paws from the bar was recorded. The maximum descent latency was set at 180 s. Interpretation for a longer time to remove the paws from the bar indicated increased muscular rigidity and stiffness in the rats. This method provided a quantitative measurement of the degree of muscular rigidity, a characteristic symptom of PD models.

  • 2

    Paucity of movement test

Paucity of movement is a specific test for PD. It was used to assess the bradykinesia and akinesia in rats from the paucity of movement. It was assessed by a series of behavioral tests. Behavioral tests included a general assessment of locomotor activity, such as measuring movement grip or strength of the front paws. In addition, the stepping test was to measure akinesia, and the pole test was to measure bradykinesia.

Preparation of the Nanocopolymer

Nanocopolymer (10, 20, and 40 mg) was dissolved in 7.5 mL of normal saline solution (0.9% NaCl) and vortexed for final concentrations of 1.33 2.67, and 5.33 mg/mL. 100 μL of the solution was administered intraperitoneally (i.p) daily to test the appropriate concentration.

Protocols

Rodents are the most common PD model due to their relatively low cost of maintenance and their ability to recapitulate many of PD’s key hallmarks, including motor defects. After being induced with 6-OHDA, the symptoms that appeared were evaluated using the catalepsy bar test, the patience of movement test, the stepping test, and the pole test. A longer time to remove the paws from the bar indicated increased rigidity and stiffness in the rats, according to this common assessment of PD. After 3 weeks of administration, PD symptoms were evaluated, such as balance improvement, increased movement activity, and decreased stiffness and tremor, which indicated a good response to the nanopolymer. Therefore, animals in group 2 were divided into 4 subgroups (2A, 2B, 2C, and 2D); see Table . To determine the optimal concentration of the nanocopolymer that could penetrate the BBB, groups 2B–2D were administered different concentrations of the nanocopolymer (10, 20, and 40 mg/animal body weight). To identify the optimal time for the maximal effect of the nanocopolymer, each group was further divided into four classes: I, II, III, and IV (five rats per class). Each class was dissected after a specified period following the administration of the nanocopolymer (I: after 2 days, II: after 5 days, III: after 7 days, and IV: after 14 days) (Table ). The study workflow is illustrated in Scheme .

3. Workflow of the Study.

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The rats were fasted overnight and subsequently euthanized for dissection. During dissection, the brain and liver were carefully excised. These tissues were functionalized with Acridine Orange diluted in a saline solution for fluorescence and histological analyses.

Brain and Liver Dissection

Xylazine and Ketamine were administered to facilitate anesthesia, blood flow, and the humane handling of the animals during the procedure. Euthanasia was used post blood collection; the animals were placed in a jar containing chloroform to ensure euthanasia before dissection. The animal selected for dissection was secured on a rectangular cork plate using pins to immobilize the front and back limbs. Then, the abdominal incision was done by toothed forceps, and the skin of the lower abdomen was gently lifted and incised with medical scissors toward the chest and along both sides to expose the abdominal and thoracic organs, targeting the liver for removal. The scalp was carefully removed, and then incisions were made along both sides of the skull. The selected portion of the skull and the back of the head were removed. Finally, the brain was gently and precisely lifted to avoid tissue damage.

Tissue Fixation and Processing

Extracted brains and livers were fixed in 4% formaldehyde for 24 h (Fixation). After Fixation, brains were immersed in a 30% sucrose solution (0.1 M phosphate buffer, pH 7.4) for 48 h at 4 °C (Cryopreservation). After Cryopreservation, coronal sections (50 μm thick) of the brain, including the striatal region and substantia nigra (Sectioning), were prepared based on the Paxinos and Franklin atlas.

Histological and Fluorescence Analyses

Brain slices were placed in an antifreezing solution (30% glycerine and 30% ethylene glycol) at 4 °C until further processing. Then slices were rinsed in 0.1 M and pH 7.4 of PBS for 10 min to remove the antifreezing solution. They were incubated for 1 h in 0.3 M glycine solution at room temperature with constant stirring and rinsed again with PBS, mounted on gelatinized slides. It was labeled with Acridine Orange (AO) fluorescent dye, which fluoresces red (>630 nm). Slides were left at room temperature before coverslips were placed and sealed with a colorless enamel (Figure ).

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Steps of the experiment. (A) Healthy rats. (B) Oral administration of 6-OHDA, (C) nanocopolymer preparation, (D) i.p injection, (E) liver anatomy, (F) brain anatomy, (G) conserving brain, (H) disordered cell, and (I) covariation healthy. The image on the right-hand side marks the beginning of PD. The initial symptoms of PD arise from losing neurons in the SNpc and the denervation of their axons in the caudate nucleus and putamen, reproduced from ref no permission was required.

Imaging and Analysis

To determine the nanocopolymer’s efficacy in crossing the BBB and its potential therapeutic impact on the target regions in the brain and liver, slides were imaged and digitally photographed using a fluorescence microscope (20× magnification), as shown in Figure .

Acknowledgments

This study was proposed for A.A.R. as part of her Ph.D. plan. The authors thank Mustansiriyah University, the University of Technology, and Sàad Al-Witry Neuroscience Hospital for their valuable support and help during the study.

All data is available through the manuscript.

A.A.R. Performed the experiments, analyzed the results, wrote the main manuscript text, and edited the final draft; Z.S.A.-G. designed the study, supervised the work, interpreted the results, wrote the main manuscript text, and revised and edited the final draft; A.J.H. supervised the study, wrote the main text, and edited the final draft; B.H. (doctor in medicine) consulted on the neurological part, revised, edited, and approved the final version. All authors have read and agreed to the published version of the manuscript.

No funding received.

Experiments in this study were performed following all national or local guidelines and regulations. Written approval was obtained from the Ethical Approval Committee of The College of Science Research Ethics Committee, Mustansiriyah University, Iraq (REF: BCSMU/0524/0005C on April 1st, 2023). Study data/information was used for research purposes only. Informed consent was obtained from each participant.

The authors declare no competing financial interest.

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