1. Introduction
Inflammation is one of the major events that contribute to the pathology of neurodegenerative diseases such as spinal cord injury (SCI), Alzheimer disease, Parkinson disease, multiple sclerosis, and epilepsy (Gao & Hong, 2008). In other words, immune system activation seen in neurodegenerative disease has photogenic and supportive roles (Amor, Puentes, Baker, & Van Der Valk, 2010). Hence, inflammation management is an essential issue in treating neurodegenerative diseases. 
One of the primary drugs administered to manage inflammation following neurodegenerative disorders is methylprednisolone (MP). For instance, an intravenous injection of a high concentration of MP (30 mg/kg of body weight in the first time followed by 4.5 mg/kg/h for 23 hours) is used during the first hours after SCI to reduce the secondary injury (Bracken et al., 1990). In addition, this drug has been reported as a promising approach for treating Alzheimer disease (Alisky, 2008), Parkinson disease (Sato, Asoh, Metoki, & Satoh, 2003), multiple sclerosis (Saidha, Mok, Butler, Fanning, & Harrington, 2010), stroke (Altamentova et al., 2020), and epilepsy (Almaabdi et al., 2014). Although some neurological improvements have been reported following the administration of MP, the systemic administration of a high dose of the drug shows important side effects, including wound infection, pneumonia, gastrointestinal bleeding, and myopathy (Gerndt et al., 1997; Qian et al., 2005). Thus, changing the route of administration from systemic to local might be a promising approach for decreasing these adverse effects of MP. 
On the other hand, the treatment of neurodegenerative diseases goes beyond pharmacotherapy, and new approaches like applying neural stem/progenitor cells (NS/PCs) are in progress (Ronaghi, Erceg, Moreno‐Manzano, & Stojkovic, 2010; Russo, 2020). Several basic investigations indicate that transplanted NS/PCs survive, migrate, and differentiate into neurons, astrocytes, and oligodendrocytes (Aligholi et al., 2016; Cummings et al., 2005). In addition, NS/PCs release trophic factors such as nerve growth factors and brain-derived neurotrophic factors, which can be helpful for neuroregeneration (Lu, Jones, Snyder, & Tuszynski, 2003). Moreover, functional recovery has been reported after transplantation of NS/PCs to the damaged tissue of the spinal cord (Sankavaram et al., 2019). Despite these outstanding properties of NS/PCs, their transplantation must be combined with other agents to modulate their behavior (Garbossa, Boido, Fontanella, Fronda, Ducati, & Vercelli, 2019).
Based on the above statements, the combination of NS/PCs and MP has been considered. In this sense, what is needed to be clear is the effect of MP on the behavior of NS/PCs. One study reported that administration of MP after brain ischemia increased survival and migration of NS/PCs (hong Jing, Ping Hou, Feng Song, & Yin, 2012). Using MP in a brain injury model showed that this drug supported oligodendrocytes but did not affect the survival of neurons (Lee et al., 2008). In addition, the proliferation of NS/PCs in an SCI model decreased by MP (Obermair, Schroter, & Thallmair, 2008). In an in vitro study, a reduction in proliferation of spinal cord-derived NS/PCs after exposure to MP was reported (Wang et al., 2014). Due to these discrepancies, the present study evaluated the effects of different concentrations of MP on survival, proliferation, and migration of rat embryonic NS/PCs using neurosphere assay. 
2. Materials and Methods
All methods were performed in accordance with the institutional guidelines of Shiraz University of Medical Sciences for animal care and use.
Culture of NS/PCs
The ganglionic eminence of a 13.5-day-old rat embryo was harvested using a stereomicroscope. After mechanical cutting by surgical knife, the specimens were dissociated using 0.05% trypsin/EDTA (Invitrogen, USA) for 5 min at 37°C. Then, a soybean trypsin inhibitor was added (Sigma, USA). After centrifugation and discarding the supernatant, the single cells were plated in Dulbecco’s modified Eagle’s medium/F12 (Invitrogen, USA) containing 1% N2 supplement (Invitrogen, USA), 2% B27 supplement (Invitrogen, USA), 1% penicillin/streptomycin, 1% glutaMax (Invitrogen, USA), 20 ng/mL epidermal growth factor (EGF; Miltenybiotech, Germany), and incubated at 37°C and 5% CO2. During the following days, NS/PCs proliferated as free-floating clusters (neurospheres). When the diameter of the spheres became about 200 µm, subculturing was done, and the cells were replated into a fresh growth medium. Following the second passage, the obtained NS/PCs were used for the study.
Study design
The cells obtained from the second passage (3×104 cells per well) were cultured in 96-well plates and used for investigation. A range of 0.25 μg/mL to 1000 μg/mL of MP was used in MP-based previous in vitro studies (Kuppermann, Zacharias, & Kenney, 2014; Mealey, Chen, & Schanz, 1971; Wang et al., 2014). In the present investigation, we selected concentrations of 0, 5, 10, 15, or 20 μg/mL of MP (Sigma, USA) due to the toxicity of this drug in higher concentrations for NS/PCs (according to our pilot study). Cell viability, proliferation, and migration of NS/PCs were evaluated using the following methods (4 well/group).
Cell viability assessment
The NS/PCs were exposed to different concentrations of MP for 7 days. Then, an MTT assay was done to evaluate cell viability. Briefly, the cells were incubated with MTT solution for 4 hours; then, the reaction was ceased by dimethyl sulfoxide (DMSO). Absorbance was measured by an ELISA microplate reader at 570 nm.
Proliferation assay 
Two parameters of the number and the diameter of neurospheres were considered proliferation index. The NS/PCs were cultured in 96-well plates containing the neurosphere medium (3 wells per group). The number of neurospheres in each well was calculated on days 3, 5, and 7 under an inverted microscope (Optika, Italy). Moreover, the diameter of neurospheres was measured in 5 photos taken from the corners and center of each well, using Infinity software on days 3, 5, and 7. The average diameter of two diagonals perpendicular to each other was reported as the neurosphere diameter. 
Cell migration assay
To evaluate the migration of the cells, the neurospheres were cultured in poly-L-ornithine-treated plates, and then the migration was monitored by taking photos on days 1, 3, 5, and 7. The average distance passed by the three cells located at the farthest distance to the margin of the neurosphere was measured by Infinity software as an index of cell migration.
Statistical analysis
The study data are presented as Mean±SD. The normal distribution of data was tested prior to statistical analysis. One-way analysis of variance (ANOVA) was used if data were distributed normally and the Kruskal-Wallis ANOVA test (KWT) for nonparametric data. The least significant difference was used as a post hoc test. P<0.05 was considered statistically significant.
3. Results
Primary culture
The isolated ganglionic eminence was cultured in a serum-free medium. As indicated in Figure 1, the NS/PCs were proliferated as free-floating neurospheres. One week after primary culturing, the diameter of the neurospheres was more than 100 µm; thus, passaging was done. The single cells obtained from the second passage were used for the rest of the study.



The effect of MP on the viability of the NS/PCs
The results of the MTT assay on day 7 post-treatment showed that following exposure to 10, 15, and 20 μg/mL of MP, the survival of the NS/PCs decreased significantly compared to that of the group without any drug exposure (P<0.05). In contrast, the cell viability did not change by 5 μg/mL of MP (Figure 2).



The effect of MP on the proliferation of the NS/PCs
The number of neurospheres produced from the single cells after 3 days was not significantly different between the MP-treated groups and the non-treated group. Although, the number of neurospheres significantly decreased in the 15 μg/mL MP group compared to that of the 5 and 10 μg/mL of MP groups (P<0.05, Figure 3F). On days 5 and 7 of post-treatment of the NS/PCs with MP, there was no significant difference in the number of neurospheres between the groups. On the other hand, the diameter of neurospheres dramatically reduced following exposure of the NS/PCs to different concentrations of MT compared to the non-treated group. This effect could be observed 3, 5, and 7 days after exposure to MP (P<0.05) except for the concentration of 20 μg/mL of MP on day 5 (Figure 3G).



The effect of MP on the migration of the NS/PCs
As illustrated in Figure 4, an increasing trend could be seen in the migration capacity of the NS/PCs in all groups from day 1 to day 7 following exposure to different concentrations of MP. On days 1 and 3 after exposure, there were no significant differences between the groups in the migration index. Although, the migration of NS/PCs treated with 20 μg/mL of MP was higher than that of the non-treated group on days 5 and 7 following exposure.



4. Discussion
In the present study, we indicated that high concentrations of MP threatened the survival of NS/PCs, and the proliferation capacity of NS/PCs decreased by exposure to different concentrations of MP. However, the highest concentrations of MP (20 μg/mL) enhanced the migration capacity of NS/PCs.
As demonstrated in the present study, previous investigations showed the anti-proliferative effect of MP. Wenhao et al. reported that the inhibitory effect of MP on the proliferation of NS/PCs is related to a decrease in the expression of hypoxia-inducible factor-1α (HIF-1α) and Hes1 (Wang et al., 2014). HIF-1α helps the cells against apoptosis and increases cell survival (Majmundar, Wong, & Simon, 2010). Based on the results of another study that evaluated the expression of various genes associated with neurogenesis, the anti-proliferative effect of methylprednisolone is related to the up-regulation of ferritin heavy chain 1 (Fth1) and insulin-like growth factor-binding protein (IGFBP-3) genes as well as down-regulation of endothelin receptor type B (EndrB) (Li, Wang, Tang, Huang, Wu, & Shen, 2012). Recently, Li et al. reported that MP decreased the survival of fetal neural stem cells. Using an EndrbB agonist, they explored the role of the PI3K/Akt pathway and lncRNA in this adverse effect of MP (Li et al., 2020). Our investigation indicates that all studied concentrations of MP decrease the proliferation of NS/PCs. Moreover, previous studies indicate that MP inhibits the proliferation of the endogenous NS/PCs (Obermair et al., 2008). Accordingly, based on our results and previous investigations, MP inhibits the proliferation of both endogenous and exogenous NS/PCs. 
The present study demonstrates the improved migration of NS/PCs following exposure to high concentrations of MP as a positive finding. The migration capacity is one of the main factors in NS/PCs transplantation (Arocena & Collinson, 2012), reported for MP-treated NS/PCs for the first time in this study. However, further mechanistic investigations in this area are needed. However, considering the anti-survival and anti-proliferative effects of MP, its effect on migration of NS/PCs may not be beneficial combined with therapeutic approaches. 
In conclusion, MP increases the migration capacity of NS/PCs only in a high concentration, but it reduces the survival and proliferation of NS/PCs. These effects of MP on NS/PCs should be considered in future combination therapies, including MP+NS/PCs for neurodegenerative diseases.
Ethical Considerations
Compliance with ethical guidelines
This study was approved by the Ethics Committee of the Shiraz University of Medical Sciences (Code: IR.SUMS.REC.1395.S34).
Funding
This research was supported by the research project (No. 8054), Funded by Shiraz University of Medical Sciences.
Authors' contributions
Conceptualization and Supervision: Hadi Aligholi; Methodology: Ahmad Soltani; Investigation, Writing – original draft, and Writing – review & editing: All authors; Data collection: Fatemeh Shamsi, Zahra Zeraatpisheh, Mina Salmannejad; Data analysis: Zohreh Bagheri; Funding acquisition and Resources: Hadi Aligholi.
Conflict of interest
The authors declared no conflict of interest.
Acknowledgments
The authors gratefully acknowledge the support of Shiraz University of Medical Sciences (grant number 10750-74-01-94).

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