POTENTIAL NEUROPROTECTIVE EFFECT OF ERYTHROPOIETIN ON ROTENONE-INDUCED PARKINSONISM IN ADULT MALE RATS VIA MODULATION OF MIRNA34A-3-P EXPRESSION

Potential Neuroprotective Effect of Erythropoietin on Rotenone-induced Parkinsonism in Adult Male Rats via Modulation of miRNA34a-3-p Expression

By

Noha Abdel-Aziz Nassef, MD¹, Dalia Mahmoud Eid, MD³, Howaida Saber Salama, MD^4,5, Salwa Fares Ahmed, MD^6,7, Heba Mohamed Tawfik, MD⁸ and Wessam Ezzat Morsy, MD^1,2

1. Associate Professor of Physiology, Faculty of Medicine- Ain-Shams University, Cairo, Egypt.
2. Associate Professor of Physiology, Armed Forces college of Medicine, Cairo, Egypt.
3. Lecturer of Biochemistry and Molecular Biology, Faculty of Medicine-Ain-Shams University, Cairo, Egypt.
4. Associate Professor of Pharmacology, Faculty of Medicine University of Tabuk
5. Associate Professor of Pharmacology, Faculty of Medicine-Assiut University
6. Associate Professor of Histology, Faculty of Medicine-Assiut University
7. Associate Professor, anatomy Department, Faculty of Medicine- University of Tabuk
8. Associate Professor of Geriatrics and Gerontology, Faculty of Medicine- Ain-Shams University, Cairo, Egypt

Corresponding author: Wessam Ezzat Morsy

Email: Dr.wessamezzat@med.asu.edu.eg ,  doctorapioneera@yahoo.com

ABSTRACT

Background: Parkinson’s disease is a devastating neurodegenerative disease with increasing prevalence worldwide.

Objective: To evaluate the possible neuroprotective effect of erythropoietin administration on motor dysfunction in experimentally induced model of Parkinson’s disease.

Materials and Methods: Twenty-six adult male albino rats were divided into 3 groups: Control group (C) including six rats, rotenone-induced Parkinsonian group (PD) including ten rats, and erythropoietin (EPO)-treated parkinsonian group (EPO+PD), ten rats were injected with both rotenone and erythropoietin for two weeks. Motor functions were assessed and then brain extraction and processing were performed for studying the substantia nigra.

Results: Erythropoietin ameliorated the motor dysfunction induced by rotenone as EPO+PD rats demonstrated a significant shortening in the beam traversal time and rotation, descent times of pole test compared to non-treated parkinsonian rats. This was accompanied by significant reduction in the oxidant, inflammatory and apoptotic biomarkers. Also, miR34a-3-p showed marked upregulation with rotenone, and this significantly decreased with erythropoietin treatment. Histological studies revealed that the EPO-treated parkinsonian group showed reduction in neuronal degeneration and gliosis. Immunohistochemical evaluation revealed that the immunoreactivity of tyrosine hydroxylase and Vimentin changed to be almost similar to the control group.

Conclusion: EPO could have a promising neuroprotective effect in Parkinson’s disease. EPO decreased oxidative stress, decreased the inflammatory markers, and had antiapoptotic role evidenced by decrease in caspase 3. A unique finding of our study is upregulation of miR34a-3-p in the rotenone induced parkinsonism group and its downregulation after EPO injection.

Keywords: Parkinson’s disease - TNF-α - erythropoietin - miR34a-3-p - rats.

INTRODUCTION

Parkinson’s disease (PD) is a progressive disabling neurodegenerative disease (Kouli et al.,2018) with a global increase in prevalence during the period between 1990 and 2019 (Safiri et al., 2023). PD is a multifactorial disease, with risk factors including age, genetics, central nervous system infections, and environmental factors (Lees et al., 2009). PD is characterized by loss of nigrostriatal dopaminergic neurons. Neurodegeneration is not only limited to dopaminergic neurons but also involves aggregation of α-synuclein in other cells in the neural network. Thus, PD is a very heterogeneous, widespread disorder that is diagnosed clinically, with no reliable diagnostic test available (Kouli et al.,2018). Erythropoietin (EPO) is a type I cytokine required for the formation and maturation of erythroid cells (Rey et al., 2021). It is synthesized in the liver throughout embryonic development. Renal peritubular interstitial cells become the predominant EPO production site after birth (Pedroso et al., 2018). EPO is produced by various CNS cell types, including neurons, glial cells, astrocytes, oligodendrocytes, and endothelial cells, and plays a role in brain maturation and vascular system development by stimulating neurogenesis (Lombardero et al., 2011). Also, EPO prevents cell injury, suppresses the generation of reactive oxygen species (ROS), inhibits apoptosis, reduces glutamate toxicity, and modulates inflammation (Elliot-Portal et al., 2018). Due to its neuroprotective role, it was suggested that EPO could be effective in the treatment of various cerebrovascular and neurodegenerative disorders, including Alzheimer’s disease (AD), stroke, hypoxic–ischemic encephalopathy, and PD (Pedroso et al., 2012 and Sun et al., 2019). EPO's impact on transcriptional and post-transcriptional regulation may be essential for its neuroprotective effects. Previous studies (Anderson et al., 2013) using mRNA microarrays evaluated genome-wide expression changes induced by EPO.

MicroRNAs (miRNAs) are a family of single-stranded, small, non-coding RNA molecules that play a key role in a variety of biological processes. They bind to the 3՝ untranslated regions of target mRNAs, causing mRNA degradation and translational inhibition, thus playing a role in post-transcriptional gene regulation (O'Brien et al., 2018). Neurodegenerative disorders such as AD were found to have dysregulated miRNA expression (Putteeraj et al., 2017). Furthermore, abnormal expression of specific miRNAs has been confirmed in models of PD as well as in brain samples affected by PD (Hoss et al., 2016). miR-34a is a tumor suppressor transcript, and its absence has been strongly associated with different types of human cancers, particularly those affecting the brain. Notably, miR-34a-3p is widely expressed in the brain and affects various neurodevelopmental and neuropathological processes. Studies have highlighted the connection between miR-34a and neurodegenerative conditions (Chua & Tang, 2019 and Grossi et al., 2021).

This study aimed to characterize the neuroprotective effect of EPO in the rotenone model of rats with PD via modulation of miRNA34a-3-p expression. Thus, it may provide preliminary evidence for the potential neuroprotective effects of EPO in the context of pharmacological management of PD to halt the disease progression.

MATERIALS AND METHODS

Experimental animals

The current study was initially carried out on 30 rats (4 rats in the rotenone-induced Parkinsonian group died after injection of rotenone), so the study was performed on 26 adult male Wistar rats weighing 200–250 g. Rats were purchased from an animal farm in Helwan (Egypt) and kept in an animal house at the Medical Ain Shams Research Institute (MASRI), Faculty of Medicine, Ain Shams University, under standard conditions of feeding and boarding with free water access throughout the whole study period. Animals were housed in animal cages (50cm width x 50cm length x 30 cm height, 5 rats/cage) with appropriate ventilation, maintained at 22-25 °C, and subjected to normal light-dark cycles.

All animal procedures were performed in compliance with the National Institute of Health guide for the care and use of laboratory animals (NIH Publication 8^th edition, revised 2011) and were approved by the Institutional Animal Ethics Committee for Ain Shams University, Faculty of Medicine (FWA 000017585). The application approval number was FMASU R307/2023.

The study period was 2 weeks. Rats were randomly assigned into 3 groups, as follows:

Group I: Control group (C) [6 rats]: Rats in this group received the vehicle dimethyl sulfoxide and polyethylene glycol (1:1, DMSO/PEG-400 in a volume of 1 ml/kg) by subcutaneous (SC) injection in a similar schedule to that adapted for rotenone injections (every 48 hours for 6 doses).

Group II: Rotenone-induced Parkinsonian group (PD) [10 rats]: Rotenone was given to induce an experimental model of Parkinsonian disease at a dose of 1.5 mg/kg by subcutaneous (SC) injection every 48 hours for 6 doses as described by (Zaitone et al., 2012). They were 14 rats, as mentioned previously, but 4 died.

Group III: Erythropoietin-treated parkinsonian group (EPO+PD) [10 rats]: Rats were injected with 6 doses of rotenone as in group 2 and were treated with subcutaneous injections of erythropoietin at a dose of 1000 IU/kg, 3 times per week for 2 weeks (total 6 doses), as described by El Mahdy et al., (2023).

Experimental procedures

Assessment of motor functions:

At the end of the experimental period (24 hours after the last injection), all rats were monitored for their motor function using a battery of behavioral tests that was done at the same time of the day for all groups (between 9 a.m. and 1 p.m.) to limit circadian influence on animal behavior. These tests were:

1. Challenging beam traversal procedure: A beam of 150 cm length wood of 3 sections, from the widest to the narrowest, was placed so that the narrowest end leads right into the home cage, which was placed on its side. Each rat was placed at the wide end of the beam, and let its tail go. The trial ended when the rat placed one of its forelimbs into the home cage. This was the first "assisted" trial. Several trials were made. Rats were trained for 2 days. On the third day (test day), a video camera was used to record the time needed by each rat to traverse the beam in seconds (beam traversal time) (Fleming et al., 2013).
2. Pole test performance: It was employed to evaluate motor dysfunction because of striatal dopamine depletion (Matsuura et al., 1997). Each rat was positioned facing upward on the top of a pole standing vertically (50 cm long and 1 cm in diameter, made of wood), with the base of the pole placed inside the home cage. Rats were placed with their heads facing upwards on the upmost part and were forced to attempt to descend the pole to enter the home cage. Rats rotated before they descended, so that their heads became directed downward. The time that rats spent attempting to rotate (rotation time in seconds), and the time they spent descending the pole (descending time in seconds) to the home cage were recorded. The best performance among the 3 trials was utilized for comparison (Fleming et al.,2004)a.
3. Spontaneous activity (cylinder test): Rats were placed in a transparent cylinder (60 cm x 25 cm). Spontaneous activity was assessed for 3 minutes, and the number of rears and grooming time were measured. A rear was counted when a rat performs a vertical movement with both forelimbs removed from the ground (Fleming et al.,2004)b.
4. Adhesive removal test: Each rat was scuffed to restrain it, and one adhesive label was placed onto its snout. The rat was released, and a stopwatch was started. When the rat attempted to remove the label with its forepaws, the time was recorded (attempt time in seconds), and when the rat removed it, time was recorded (removal time in seconds) (Fleming et al., 2013).

Brain tissue sampling:

After assessing their motor function, rats were sacrificed. On the day of scarification, the overnight fasted rats were weighed and anesthetized with an intraperitoneal injection of thiopental sodium (EIPICO, Egypt), given at a dose of 40 mg/kg (Cherksey and Altszuler, 1974).

The followings were done: 

1. Brain extraction and dissection:

Brains were extracted and put on ice-cold plates. The brain was cut into two equal hemispheres; a cut was made at the anterior tip of the fornix; the caudate nucleus was collected; and both right and left striatal tissues were dissected and stored at -80 ºC for later determination of the biochemical parameters.

2. Processing of the brain tissue:

Brain tissue samples were probably washed and rinsed with ice before being homogenized in 0.1 M phosphate buffer (pH 7.4) (10% w/v) using a polytron homogenizer at 4 °C. The homogenates were centrifuged at 10.000 rpm for 20 min. Aliquots of supernatants were separated (brain homogenate) and used for the following biochemical assays:

1. Determination of brain levels of oxidative stress markers

Brain malondialdehyde (MDA) was estimated following the procedure outlined by (Mihara and Uchiyama,1978) using the OxiSelect™ Thiobarbituric acid reactive substance (TBARS) Assay Kit (cat# STA-330; Cell Biolabs, San Diego, CA, USA) following the instructions provided by the manufacturer.

1. Determination of catalase activity in brain tissue:

Catalase activity was estimated in the brain tissue homogenate according to the colorimetric method described by Aebi (1984) using catalase “colorimetric method” assay kit (cat# CA 2516, Bio Diagnostic, Giza, Egypt).

1. Determination of brain caspase-3 activity and TNF-α level:

Caspase-3 activity was assessed in brain tissue homogenates using a caspase 3 ELISA kit (cat# HEA626Ra; Cloud-Clone Corp., Katy, Tx, USA) and tumor necrosis factor-α (TNF-α) levels were determined using Rat TNF-α ELISA kit, (cat# CSB-E11987r, CUSABIO), according to the manufacturer’s instructions.   

1. Determination of right and left brain dopamine levels:

Dopamine concentration was determined in brain tissue using rat dopamine (DA) ELISA kit (cat# MBS725908 Mybiosource) according to the manufacturer’s instructions.

1. miRNA isolation from brain tissue:

 miRNA was isolated from the brain tissue samples using the miRNEasy RNA isolation kit (Qiagen, USA) following the manufacturer's protocols. The quality and concentration of miRNA were determined by using NanoDrop 1000 spectrophotometer (NanoDrop Technologies,Inc. Wilmington, USA). Samples with a 260/280 nm absorbance ratio close to 2.0 were considered “pure” for RNA (William et al., 1997). Later, miRNA was stored at -70 °C until it was utilized in the reverse transcription polymerase chain reaction.

1. qRT-PCR analysis of miRNA 34a-3-p expression:

1μg of the extracted miRNA was reverse transcribed to cDNA using the miScript II RT kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Real-time qPCR amplification and analysis were performed using StepOne Plus™ System (Applied Biosystems Inc., Foster City, CA). For the real-time PCR reaction, SYBER green master mix (Qiagen, Valencia, CA, USA) was utilized along with the specific primers of miR-34a-3P and SnU6RNA (as an internal control). The miRNA-specific primer was selected based on the miRNA sequences retrieved from the miRbase database available at (http://microrna.sanger.ac.uk/). The relative expression of miR-34a-3-p was normalized to that of the internal control (SnU6RNA) using the 2^–ΔΔCt cycle threshold method (Livak and Schmittgen, 2001).

Histopathological studies and immunohistochemical analysis:

A 10% formaldehyde solution was injected intracardially into three rats from each group. The substantia nigra (SN) was dissected under a microscope. After that, the specimens underwent processing and were stained using hematoxylin and eosin (H&E) (Bancroft and Gamble,2008). The main antibody, tyrosine hydroxylase (Abcam Cambridge, UK) (TH; 1:100), was incubated on the sections for a whole night at 4 °C. Ab-1, mouse monoclonal antibody, glycophorin A (GA) at a dilution of (1/100), vimentin polyclonal antibody, and anti-glial fibrillary acid protein (anti-GFAP) were all used for an hour at room temperature. The sections were then processed utilizing the universal kit, HRP polymer, Ultra Vision LP system, diaminobenzidine (DAB) plus chromogen, and General Biotechnologies Cambridge, MA, USA (Wang et al.,2010), in accordance with the manufacturer's instructions (Faherty et al., 1999).

Electron microscopic study:

The samples were preserved in cacodylate buffer (pH 7.4), processed for semi-thin sections (0.5-1.0 μm), and stained with 1% Toluidine Blue (pH 7.3). Sections as thin as 50–80 nm were cut from specific blocks, arranged on copper grids, and compared with lead citrate and uranyl acetate. A transmission electron microscope (JEOL-100 CXII; Tokyo, Japan) was used to analyze the slices, and 80 kV photos were taken (Gupta .1983).

Morphometry:

All the histological quantification was done automatically and blindly. Using an Olympus BX53 microscope with a 40-x objective and a connection to an image analysis system equipped for stereological application, the number of tyrosine hydroxylase positive neurons in the substantia nigra was counted. The average cell density (number of positive neurons/mm² of the structure) was used to express the results for stereological evaluation. Using Image-Pro Plus image analysis software, the density of positive cells in the histochemical analysis was computed (Bredesen et al.,2006).

Statistical analysis: Statistics were done using statistical package for the social sciences (SPSS) program (SPSS Inc., version 24). All data were expressed as mean ± SD. Data of behavioral tests were analyzed using the Kruskal Wallis test. Then, Mann-Whitney U-test was conducted between the groups (inter-group analyses). Kruskal-Wallis and Mann-Whitney tests were utilized because our groups were composed of a small number of observations, in which we could not assume for normal distribution All other data were analyzed using One-way analysis of variance (ANOVA) to determine the differences between groups and post hoc Tukey’s test was carried out to find significant intergroup differences. Pearson correlation coefficients were calculated by linear regression analysis using the Least Square Method and a correlation of P < 0.05 considered significant (Armitage and Berry, 1987).

RESULTS

Effects of erythropoietin on motor impairment in rotenone-induced Parkinsonian rats (Table 1):

To determine the extent of the rotenone injury, assessment of motor performance was performed using the challenging beam traversal procedure, pole test, spontaneous activity in the cylinder, and response to sensory stimuli (adhesive removal). Compared to the control group (C), the rotenone-induced parkinsonian group (PD) exhibited a significant impairment in motor performance and coordination deficit, as evidenced by the significant increase in beam traversal time (P1<0.01).

In the pole test, PD rats showed significant prolongation in rotation time and descent time compared to the control group (P1<0.01 and P1<0.05 respectively).

Moreover, the PD group displayed significantly reduced spontaneous motor activity (rear counts) compared to the control group (P1<0.05) but did not significantly alter grooming time. Rotenone injection did not cause a difference in the measured parameters of the adhesive removal test compared to the control group.

However, EPO administration in the EPO+PD group significantly shortened the beam traversal time (P2< 0.01) compared to the PD group and was non-significantly different from the control group.

Regarding the rotation time and the time to descend to home cage in the pole test, a significant decrease in these times was observed in the EPO+PD group compared to the PD group (P2 <0.01 for both), which was not significantly different compared to the control group.

Moreover, the number of rears recorded in the cylinder test was significantly increased (P2 <0.05), while the grooming time was not significantly changed in the EPO+PD group compared to the PD group. No significant differences were detected in the adhesive removal test parameters with erythropoietin treatment.

Table (1): Mean ±SD of results of motor function tests as the challenging beam traversal procedure, pole test, spontaneous activity in the cylinder, and adhesive removal test in the 3 studied groups.

Number in parentheses is the number of observations.

P1: Significance of difference from the control group calculated by Kruskal Wallis test followed by Mann-Whitney U test at P<0.05.

P2: Significance of difference from the rotenone induced Parkinsonian group calculated by Kruskal Wallis test followed by Mann-Whitney U test at P<0.05.

NS: non-significant

Effect of erythropoietin on brain biochemical and molecular changes in rotenone-induced Parkinsonian rats:

As shown in graphs (1 a and b), there was a significant lower level in brain catalase activity and a remarkable higher MDA level in the PD group compared to the control group (all P < 0.001). However, these changes were notably reversed by EPO treatment in the EPO+PD group, with brain catalase activity increasing and MDA levels decreasing significantly (all P < 0.001).

As shown in graphs (1 c and d), the PD group exhibited a significant higher brain TNF-α and caspase-3 levels compared to the control group (all P < 0.001). Conversely, the EPO+PD group demonstrated a significant decrease in these levels (all P < 0.001) compared to the PD group.

The present study revealed variations in brain expression of miR-34a-3-p among the three studied groups. As illustrated in graph (1 e), brain miR-34a-3p expression was significantly upregulated in the PD group compared to the control group (P < 0.001). On the contrary, miR-34a-3-p expression was notably decreased after EPO administration in the EPO+PD group (P <0.001). In graph (1 f), it is illustrated that striatal dopamine levels on both the right and left striatal sides were markedly decreased in the PD group compared to the control group. However, treatment with EPO significantly increased dopamine levels (P<0.01). Additionally, the left side striatal dopamine level was significantly higher in the PD group compared to the corresponding level on the right side (P< 0.01). Regression line studies were shown in graphs 2,3, where is there significant negative correlation between beam traversal time, catalase (CAT) and dopamine and positive correlation between time, caspase, MDA, and miR34a-3-p. Also, descent time significantly decreased with dopamine and increased with TNF, caspase, MDA, and miR34a-3-p.

Graph 1: Comparison of the brain content of: MDA (A), catalase (B), TNF-α (C), caspase-3 (D), miR34a-3-p (E) and right and left striatal dopamine (F) among the studied groups. Values are presented as mean ± SD, a: Significance of difference from the control group calculated by LSD of one-way ANOVA test at P<0.01, b: Significance of difference from the PD group calculated by LSD of one-way ANOVA test at P<0.01, #: Significance of difference from right striatal side at P<0.01.

Correlation studies

There was a highly significant positive correlation between total dopamine and catalase levels and negative correlation between dopamine, MDA, caspase, TNF-α and miR34a-3-p levels as shown in Table 2, (P<0.001) for all.

 Also, in Table 3, there was a significant positive correlation between miR34a-3-p and TNF-α and a negative correlation between the former and catalase, with (P< 0.01) for both.

Table (2): Correlation between total brain dopamine and other biochemical parameters:

R: Coefficients of determination.

P: Significance at P<0.01.

Table (3): Correlation between brain miR34a-3-p expression and TNF-α and catalase levels:

R: Coefficients of determination.

P:  Significance at P<0.01.

Graph (2): Graphs showing significant correlation coefficient (P), Regression lines between beam traversal time and striatal a) MDA b) Catalase c) Caspase-3 d) miR34a-3-p e) Dopamine.

Graph (3): Graphs showing significant correlation coefficient (P), Regression lines between decent time of pole test and striatal a) MDA b) Catalase c) Caspase-3 d) miR34a-3-p e) TNF-α f) Dopamine.

Histological results:

1. Light microscopic results:

Among substantia nigra (SN) sections stained with H&E, the control group showed a normal histological structure of the neurons. SN showed a band of lightly stained neurons along with a few dark ones (Fig. 1a). A higher magnification revealed that the light neurons possessed vesicular nuclei with prominent nucleoli, the dark ones showing nucleoli and dark cytoplasm, numerous glial cells, and average blood vessels (Fig. 1b).

SN sections of the PD group showed a few small-sized degenerated neurons having small pyknotic nuclei and homogenous basophilic cytoplasm with Lewy bodies and an irregular cell membrane, scattered apoptotic neurons with small pyknotic nuclei and homogenous eosinophilic cytoplasm, excess glial cells, and markedly congested blood vessels with marked areas of hemorrhage (Fig. 1c, 1d), while in the EPO+PD group, substantia nigra showed neurons with vesicular nuclei and prominent nucleoli and dark granular cytoplasm with Lewy bodies, clear glial cells, and average blood vessels with small areas of hemorrhage (Fig. 1e, 1f).

In the same line, semi-thin Toluidine Blue-stained sections of the SNc of the control group, neurons appeared variable in size and shape. Their perikarya had large pale nuclei, prominent nucleoli, lightly stained cytoplasm, and average blood vessels (Fig. 2a). The PD group showed small-sized degenerated neurons with small pyknotic nuclei, homogenous basophilic cytoplasm with Lewy bodies, and excess glial cells (Fig. 2b). The EPO+PD group demonstrated average-sized neurons with vesicular nuclei, nucleoli, dark granular cytoplasm with Lewy bodies, numerous glial cells, and average blood vessels (Fig. 2c).

Fig. 1: A photomicrograph of haematoxylin and eosin-stained section in substantia nigra; control group a) neurons with vesicular nuclei (↑) and dark cytoplasm (short arrow), and a glial cell (arrowhead) (X 400). b) higher power showing average neurons (long arrow), and glial cells (short arrow), and blood vessels (arrowhead) (X1000). PD c) pyknotic nuclei (long arrow), homogenous basophilic cytoplasm (curved arrow) and irregular cell membrane (arrowhead), glial cells (short arrow), and hemorrhage (irregular arrow) (X 400). d) Higher magnification showing small-sized neurons (long arrow) and homogenous basophilic cytoplasm with Lewy bodies (curved arrow) and congested blood vessels (short arrow) (X 1000). EPO+PD e): Showing average nuclei (long arrow) and dark granular cytoplasm (short arrow), and glial cells (arrowhead) (X 400). f) neurons with average nuclei showing nucleoli (curved arrow) and dark granular cytoplasm (long arrow), and glial cells (arrowhead) (X 1000).

Fig. 2:  Photo micrograph of semithin section stained with toluidine blue in substantia nigra: control group 2a) showing neurons with vesicular nuclei (long arrow) and dark cytoplasm, glial cells (arrowhead), and blood vessels (curved arrow) (X 1000). PD 2b) substantia nigra showing pyknotic nuclei, homogenous basophilic cytoplasm with Lewy bodies (arrowhead) and irregular cell membrane (curved arrow), and excess glial cells (long arrow) (X 1000). EPO+PD 2c) substantia nigra showing neurons with vesicular nuclei (long arrow), dark granular cytoplasm with Lewy bodies (arrowhead), average glial cells (curve arrow) and average blood vessels (short arrow) (X 1000).

1. Immuno-histochemical results:

Tyrosine hydroxylase expression in the substantia nigra:

Substantia nigra of the control group revealed many neurons with marked cytoplasmic reactivity for tyrosine hydroxylase (Fig. 3a, 3b), while in the PD group, substantia nigra showed few neurons with weak cytoplasmic reactivity for tyrosine hydroxylase (Fig. 3c, 3d). Substantia nigra of the EPO+PD group demonstrated many neurons with moderate cytoplasmic reactivity for tyrosine hydroxylase (Fig. 3e, 3f).

Fig. 3: Immuno-stained sections of substantia nigra tyrosine hydroxylase expression in glial cells and neurons showing; control group 3a): Neurons with marked cytoplasmic reactivity for tyrosine hydroxylase (arrow) (X 400)3b): Higher magnification (arrow (X 1000) PD group: 3c): Showing neurons with negative cytoplasmic reactivity for tyrosine hydroxylase (arrow) (X 400). 3d): High power view (arrow) (X 1000). EPO+PD group: 3e): Substantia nigra showing many neurons with moderate cytoplasmic reactivity for Tyrosine hydroxylase (arrow) (X 400) 3f): high power view (arrow) (X 1000).

GFAP expression in the substantia nigra:

Substantia nigra of the control group showed negative reactivity for GFAP in neurons, and evenly distributed glial cells with average processes (Fig. 4a, 4b), meanwhile, PD group showed negative reactivity (for GFAP in neurons and excess densely stained glial cells with excess processes (gliosis) (Fig. 4c, 4d). The EPO+PD group demonstrated negative reactivity for GFAP and evenly distributed glial cells with average processes (Fig. 4e, 4f).

Fig. 4: Immuno-stained sections of substantia nigra GFAP expression showing; Control group:4a): Negative reactivity for GFAP in neurons (arrowhead), and evenly distributed glial cells with average processes (long arrow) (X 400). 4b): High power view (X 1000). PD group:  4c): Substantia nigra showing negative reactivity for GFAP in neurons (arrow head), and excess densely-stained glial cells with excess processes (gliosis) (long arrow) (X 400).4d): High power view (X 1000) EPO+PD: 4e): Substantia nigra showing negative reactivity for GFAP in neurons (arrow head), and evenly distributed glial cells with average processes (long arrow) (X 400). 4f): High power view (X 1000).

Vimentin expression in the substantia nigra:

Substantia nigra showed negative reactivity for vimentin in neurons and positive reactivity in few background cells and in blood vessel walls (Fig. 5a, 5b). The PD group substantia nigra showed negative reactivity for vimentin in neurons and positive reactivity in many background cells and in vessel walls (Fig. 5c, 5d). The EPO+PD group substantia nigra showed negative reactivity for vimentin in neurons and positive reactivity in few background cells and in vessel walls (Fig. 5e, 5f).

Fig. 5: Immuno-stained sections of substantia nigra vimentin expression showing; Control group:  5a): negative reactivity for vimentin in neurons (arrowhead), and few positive background cells (arrow) (X 400) 5b): high power view showing negative reactivity in blood vessel walls (short arrow) (vim X 1000). PD group: substantia nigra 4c): showing negative reactivity for vimentin in neurons (arrowhead), and positive reactivity in many background cells (long arrow) and in vessel walls (short arrow) (X 400).4d): high power view (X 1000). EPO+PD: 5e): substantia nigra showing negative reactivity for vimentin in neurons (arrowhead), and few positive background cells (long arrow) (X 400). 5f): high power (X 1000).

1. Electron microscopic results:

Neuronal cells of SN of the control group revealed nuclei with dispersed chromatin and prominent nucleoli, average ovoid mitochondria, microtubules, dendrites, and myelinated axons with intra-cytoplasmic inclusions (Fig. 6a, 6b). The glial cells exhibit euchromatic nuclei, numerous microtubules, and heterochromatic nuclei surrounded by a thin rim of dark cytoplasm and distinguished thick processes (Fig. 7a).

The PD group illustrated a neuronal cell body with a small pyknotic nucleus with clumped chromatin in the periphery, prominent nucleoli, small, condensed mitochondria, few microtubules, small dendrites, and less myelinated axons (Fig. 6c, 6d). Glial cells have small nuclei, clumped chromatin in the periphery and large intra-nuclear vacuoles, small condensed and distorted mitochondria, few microtubules, large intra-cytoplasmic inclusions (Lewy bodies), few distorted dendrites, and less myelinated axons (Fig. 7b).

Meanwhile, EPO+PD revealed a neuronal cell body with nuclei showing dispersed chromatin and prominent nucleoli (Fig. 6e, 6f), scattered glial cells with nuclei showing clumped chromatin, small mitochondria, average microtubules, scattered intra-cytoplasmic inclusion (Lewy body), and few small dendrites (Fig. 7c).

Fig. 6 : Electron micrograph of a section substantia nigra showing Control group: 6a): showing neuronal cell body with nucleus (N) showing dispersed chromatin and prominent nucleoli (long arrow), average mitochondria (arrow head), average microtubules (short arrow), average dendrites (curved arrow), myelinated axons (irregular arrow), and intra-cytoplasmic inclusions (double arrow).6b): Showing cytoplasm showing mitochondria (arrow head), microtubules (short arrow), dendrites (irregular arrow), myelinated axons (curved arrow), and intra-cytoplasmic inclusions (double arrow). PD: 6c): Showing neuron with small pyknotic nucleus (N) condensed chromatin (long arrow) and nucleoli (double arrow), small condensed mitochondria (arrow head), few microtubules (short arrow), small dendrites (irregular arrow), and less myelinated axons (curved arrow).6d): Showing apoptotic nucleus (N) clumped chromatin (arrow head), large cytoplasmic vacuoles (irregular arrow), intra-cytoplasmic inclusion; Lewy body (short arrow) and few distorted dendrites (curved arrow), and another nucleus of pericyte (double arrow). EPO+PD: 6e): Showing nucleus (N) showing dispersed chromatin with prominent nucleoli (arrow head), small mitochondria (irregular arrow), average microtubules (short arrow), intra-cytoplasmic inclusion; Lewy body (curved arrow), and few dendrites (double arrow) .6f): showing neuronal cell body with nucleus (N) dispersed chromatin with prominent nucleoli (arrow head), small mitochondria (irregular arrow), average microtubules , intra-cytoplasmic inclusion; Lewy body (curved arrow), and few dendrites.

Fig. 7: Electron micrograph of a section substantia nigra showing; Control group: 7a): Showing glial cell with nucleus (N) showing dispersed chromatin (long arrow), average ovoid mitochondria (arrowhead), average microtubules (short arrow), average dendrites (irregular arrow), and myelinated axons (curved arrow). PD group:7b): Showing glial cell with small nucleus (N) and clumped condensed chromatin (long arrow), small, distorted mitochondria (arrowhead), large cytoplasmic vacuoles (irregular arrow), few microtubules (short arrow), and small dendrites (curved arrow). EPO+PD group: 7c) Showing glial cell with nucleus (N) showing dispersed chromatin (arrowhead), small mitochondria (irregular head), microtubules (short arrow), intra-cytoplasmic inclusion; Lewy body (curved arrow), and few dendrites (double arrow).

1. Morphometry results:

There was a significant increase in the mean number of dark neurons in the PD group compared to those in the control group. The number of dark neurons in the EPO+PD group was significantly reduced compared to those in the PD group. The mean count of light neurons showed a non-significant increase in the PD group compared to the control group. On the other hand, they showed a significant increase in the EPO+PD group compared to those in the PD group (Table 4). The number of TH-positive dopaminergic neurons/field in the SNc was significantly decreased in the PD group compared to the control group. Erythropoietin treatment significantly increased the number of TH-positive dopaminergic neurons compared to those in the PD group (Table 5).

Table 4: Count of pale and dark neurons in SNc in the 3 studied groups.

SNc= substantia nigra pars compacta

Values are presented as mean ± SD, a. Significance of difference calculated by LSD independent t-test at P<0.01 between the control group and the PD group, b. Significance of difference calculated by LSD of independent t-test at P<0.01 between the PD and EPO+PD group. c. Significance of difference calculated by LSD independent t- test at P<0.01 between the control and EPO+PD group

Table 5: Number of tyrosine hydroxylase-positive cells/field in the SNc in 3 studied groups.

SNc= substantia nigra pars compacta

Values are presented as mean ± SD, a. Significance of difference calculated by independent t- test at P<0.01 between the control group and the PD group, b. Significance of difference calculated independent t- test at P<0.01 between the PD and EPO+PD group. c. Significance of difference calculated by independent t-test at P<0.00001 between the control and EPO+PD group.

DISCUSSION

Although the precise mechanism of dopaminergic neuron loss in PD is unclear, numerous neurobiochemical evidence may be implicated. Loss of substantia nigra neurons can be attributed to several factors, such as oxidative stress, neuro-inflammation, mitochondrial dysfunction, apoptosis, and excitotoxicity (Burke,2008).

The current study investigated the possible antioxidant, anti-inflammatory, and anti-apoptotic mechanisms underlying EPO-induced neuroprotection in rotenone-induced Parkinsonism in rats. Behavioral and motor coordination deficits were evidenced in the rotenone-induced PD group by a significant increase in beam traversal time, prolonged rotation, and descent time in the pole test, as well as a reduction in spontaneous motor activity compared to the control group. These findings significantly improved after EPO injection, suggesting its promising neuroprotective role. Similar improvement in behavioral alterations after injection of EPO in rats with PD have been previously reported, which was dose-dependent (Abd-Elhalim et al., 2014).

To further confirm the occurrence of neurodegeneration in substantia nigra pars compacta (SNpc) by rotenone injection, the hallmark of PD, SNc was assessed biochemically and histologically. A significantly reduced dopamine level in SNc of PD group was observed, which is PD's vulnerable region as it is extremely susceptible to neurodegeneration because each of their heavily branching, unmyelinated axons is under extreme pressure (Tagliaferro and Burke, 2016). Moreover, degenerative alterations in this study were confirmed histologically, as the SNc showed degenerated neurons with neuronal cell body having apoptotic nuclei, glial cells with small nuclei, condensed and distorted mitochondria, large intra-cytoplasmic inclusions, which are the Lewy bodies, and distorted dendrites. Our findings concurred with those of previous studies (Fa et al.,2016 and Bloem et al., 2021). PD is primarily characterized by pathological aggregation of insoluble alpha-synuclein (α-syn) in intracellular deposits known as Lewy bodies in midbrain nigral neurons. Alpha-syn plays a physiological role in controlling neuroplasticity, neuronal maturation, and dopamine production (Bendor et al., 2016).

Microglial participation in the pathophysiology of PD has been pointed out (Gao et al., 2003), which runs parallel to the gliosis demonstrated in our histochemical results. In the PD animal group, the glial fibrillary astrocytic protein (GFAP) SN showed negative reactivity for GFAP in neurons, and glial cells with excess gliosis showed negative reactivity for vimentin in neurons. Dopaminergic degeneration changes the size and density of striatal astrocytes, and their expression of GFAP is upregulated by astrocyte hypertrophy (Clairembault et al., 2014).

In addition, glia has a highly expressed vimentin, which declines in the brain with old age, and the immunoreactivity to vimentin processes increased significantly following dopaminergic denervation (Bendor et al., 2016). Vimentin is linked to epithelial-mesenchymal transition enlisted in tissue growth during an inflammatory state of stress. It may contribute to the abnormal angiogenesis, blood vessels congestion, and pericytes alteration demonstrated in this study, which coincides with the findings of other studies (Clairembault et al., 2014).

Considerable experimental data points to reactive oxygen species (ROS) as a major contributor to dopaminergic neuronal loss in the PD brain. ROS are extensively generated both in neurons and glia from dopamine metabolism (Dumont and Beal, 2011). In the current study, rotenone induced oxidative stress in the brains of the PD group, as evidenced by a significant increase in brain MDA levels and a correspondingly significant decrease in brain catalase activity.

Many studies have assessed the crucial antioxidant effect of EPO in animal models with PD. It was reported that restoration of antioxidant activity of glutathione peroxidase in the striatum of Parkinson’s animal model occurred after intra-striatal infusion of EPO (Thompson et al., 2020). Additionally, a previous study highlighted a reduction in MDA levels following EPO treatment in an animal model of rotenone-induced neurodegeneration (Erbaş et al., 2015). Therefore, it was noticeable in the present study that EPO was efficient in counteracting oxidative stress, as evidenced by a significant decrease in brain MDA content and restoration of brain catalase activity, confirming that EPO treatment ameliorated ROS formation and promoted the survival of vulnerable neurons.

The involvement of neuroinflammation in the pathogenesis of PD is also supported in the literature (Chen et al., 2005). Interestingly, the current study revealed a significant overexpression of the potent pro-inflammatory cytokine, TNF-α, secreted mainly by microglia in the brain of rotenone-induced Parkinson’s rats compared to the control group. After EPO treatment, there was a marked decrease in TNF-α overexpression with subsequent improvement of tyrosine hydroxylase (TH) immune-reactivity, which was clearly demonstrated in our histological result, supporting EPO's potent anti-inflammatory and neuroprotective effects. This effect has been previously reported when treatment with EPO provided neuroprotection through attenuation of TNF-α in animal models after traumatic brain injury (Zhou et al., 2017).

Since apoptosis is one of the processes related to neurodegeneration and the pathogenesis of PD, we have investigated the impact of EPO treatment on neural apoptosis in the PD rat model. We evaluated the activity of caspase-3 as a pro-apoptotic protein and a prominent mediator of genomic DNA fragmentation (Lu et al., 2012).

In the present study, the PD group exhibited a significant increase in caspase-3 activity. Notably, this activity was significantly reduced in the EPO+PD group following EPO treatment, suggesting the ability of EPO to modulate components of the apoptotic cascade and prevent the activation of caspases. This was further confirmed by restoration of the normal histological   features to be more or less similar to the control. Previously, it was found that EPO maintains mitochondrial membrane potential, prevents cytochrome c release, and inhibits apoptotic protease activating factor-1 (Apaf-1) activity, thereby blocking the activation of caspase-3 (Shang, et al., 2012).

An intriguing finding years ago was that the parkinsonian brain exhibits abnormal miRNA expression (Hoss et al., 2016). This remarkable finding prompted researchers to consider if such miRNA dysregulation could contribute to the neuropathological alterations associated with PD (Titze-de-Almeida and Titze-de-Almeida, 2018). Our study represents a crucial step in this direction, by comparing the levels of miRNA 34a-3-p expression in the brain of rotenone-induced parkinsonian rats with the levels after EPO treatment.

The selection of miR‑34a in the study was based on its fundamental role in neurogenesis and neural differentiation, as it is abundantly expressed in the adult brain (Rostamian- Delavar et al., 2018). Recently, numerous studies connecting miR-34a to neurodegenerative diseases have been reported. (Ahmadzadeh-Darinsoo et al., 2022). In the present study, miR-34a-3-p was found to be up-regulated after rotenone injection and markedly down regulated with EPO treatment. These results suggest that miR-34a-3-p upregulation is involved in the dopaminergic neuronal death. Based on this information we related the restoration of dopaminergic neurons after EPO treatment in PD to modulation of miRNA expression.

According to the miRDB online database, both TNF-α and catalase are predicted as targets of miR-34a-3p. Consequently, miR34a -3-P overexpression resulted in downregulation of catalase, and treatment with EPO led to downregulation of miR34a-3-P expression, which in turn resulted in the restoration of catalase activity. However, our study revealed that an increase in miR34a -3-p expression was strongly correlated to TNF-α levels, and this observation appears contradictory to the expected outcome. This discrepancy may be explained by the inability of miR-34a-3-p expression to counteract the neuroinflammation induced by rotenone, which led to elevated TNF-α levels and neurological damage.

Limitation of the study

The study started with 30 rats, but 4 died after induction of parkinsonism.

Ethics approval: The study was approved by the Institutional Animal Ethics Committee for Ain Shams University, Faculty of Medicine. The application approval number was FMASU R307/2023.

Competing interests: The authors declare no conflicts of interest.

Funding: The study is self-funded.

Data Availability:

The authors declare that all relevant data supporting the findings of this study are available within the article.

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التأثير الوقائي العصبي المحتمل للإريثروبويتين على مرض الشلل الرعاش الناجم عن الروتينون في ذكور الجرذان البالغة عبر تعديل إبانة miRNA34a-3-p

نهى عبد العزيز ناصف - استاذ مساعد بقسم الفسيولوجيا الطبية- كلية الطب – جامعة عين شمس

هويدا صابر سلامه علي-استاذ مساعد الفارماكولوجي الطبية -كلية الطب- جامعة اسيوط

سلوى فارس احمد حسن- استاذ مساعد علم الأنسجة وبيولجيا الخلية- كليه الطب- جامعه اسيوط

داليا محمود عبد الحميد عيد- مدرس الكيمياء الحيوية والبيولوجيا الجزيئية-كلية الطب- جامعة عين شمس

هبة محمد توفيق – استاذ مساعد بقسم طب وصحة المسنين وعلوم الاعمار- كلية الطب- جامعة عين شمس.

وسام عزت مرسي - استاذ مساعد بقسم الفسيولوجيا الطبية- كلية الطب – جامعة عين شمس وكلية طب القوات المسلحة.

خلفية البحث: مرض الشلل الرعاش هو مرض تنكس عصبي مدمر ذو انتشار متزايد في جميع أنحاء العالم.

الهدف من البحث: تقييم التأثير الوقائي العصبي المحتمل للمعالجة بعقار الإريثروبويتين على الخلل الحركي في نموذج مستحث تجريبيًا لمرض الشلل الرعاش والكشف عن الآليات الأساسية وراء هذا التاثير.

مواد وطرق البحث: أجريت الدراسة الحالية على ستة وعشرين جرذاً ذكراً بالغاً تم تقسيمها إلى ثلاث مجموعات: المجموعة الضابطة وتضم 6 جرذان، ومجموعة مرض الشلل الرعاش   المستحث عن طريق حقن الجرذان بمادة الروتينون وتضم 10 جرذان، حيث تم إعطاء الروتينون الي 10 جرذان بجرعة 1.5 ملغم/كغم/48 ساعة، تحت الجلد لمدة 6 مرات ومجموعة الشلل الرعاش المعالجة بالإريثروبويتين، تم حقن 10 جرذان بكل من الروتينون والإريثروبويتين لمدة أسبوعين. تم تقييم الوظائف الحركية، ومن ثم تم إجراء استخراج الدماغ ومعالجته للدراسات البيوكيميائية والنسيجية.

نتائج البحث: أوضحت النتائج أن الإريثروبويتين خفف من الخلل الحركي الناجم عن الروتينون حيث أظهرت جرذان    التي تمت معالجتها بالاريثرويويتن قصرًا ذو دلالة احصائية في وقت اجتياز العارضة الخشبية الموصلة الى قفص الجرذان في اختبار (beam test)، وأوقات دوران الجرذان من اعلي القصب استعدادا للنزول عليه والوقت الذي استغرقته الجرذان للنزول من اعلي القطب الموضوع عموديا في اختبار) (pole test مقارنة بجرذان الشلل الرعاش الغير معالجة. وكان ذلك مصحوبًا بانخفاض ذو دلالة احصائية في المؤشرات الحيوية المؤكسدة والالتهابية وموت الخلايا المبرمج (المالوندايالدهيد ، MDA و معامل نخر الورم  الفا، TNF-α وكاسبيز 3 ، caspase 3  على التوالي). بالإضافة إلى ذلك، أظهر تحليل qRT-PCR لـ miR34a-3-p ارتفاعًا ملحوظًا مع الروتينون، وانخفض انخفاضا ذو دلالة احصائية مع العلاج بالإريثروبويتين.

وقد كشفت الدراسات النسيجية أن مجموعة الشلل الرعاش التي عولجت بالايريثروبويتين أظهرت انخفاضًا في تنكس الخلايا العصبية والدبق. علاوة على ذلك، أظهر التقييم الهيستوكيميائي المناعي أن الفعالية المناعية لإنزيم التيروزين هيدروكسيليز (tyrosine hydroxylase) ، حامض بروتين الدبق الليفي (GFAP) والفيمنتين   (vimentin) قد تغيرت لتصبح مشابهة تقريباً للمجموعة الضابطة.

الاستنتاج: يمكن أن يكون للاريثروبيوتين تأثير وقائي عصبي واعد في مرض الشلل الرعاش ، حيث قلل من التنكس العصبي والدبق. ثبط الايريثروبويتين في هذه الدراسة الإجهاد التأكسدي حيث اتضح ذلك في تقليل المالوندايلدهيد  (MDA) واستعادة نشاط الكاتلايز (catalase)  في الجرذان المصابة بالشلل الرعاش والتي تم معالجتها بالارثروبويتين ، مما أدى أيضًا إلى انخفاض في علامات الالتهابات مثل  معامل نخر الورم الفا ( (TNF-α وكان له دور مضاد للخلايا يتضح من انخفاض كاسبيز 3  (caspase 3)في نماذج الجرذان والنتيجة الفريدة لدراستنا هي تنظيم miR34a-3-p في مجموعة الشلل الرعاش الناجم عن الروتينون وإلغاء تنظيمه بعد حقن الايريثروبيوتين.

الكلمات الافتتاحية: الشلل الرعاش - معامل نخر الورم الفا - الايريثروبويتين - miR34a-3-p - الجرذان.