Deferiprone Promoted Remyelination and Functional Recovery through Enhancement of Oligodendrogenesis in Experimental Demyelination Animal Model

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Deferiprone Promoted Remyelination and Functional Recovery through Enhancement of Oligodendrogenesis in Experimental Demyelination Animal Model

https://doi.org/10.21203/rs.3.rs-2588949/v1

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Remyelination refers to myelin regeneration, which reestablishes metabolic supports to axons. However, remyelination often fails in multiple sclerosis (MS), leading to chronic demyelination and axonal degeneration. Therefore, pharmacological approaches toward enhanced remyelination are highly demanded. Recently, deferiprone (DFP) was reported to exert neuroprotective effects, besides its iron-chelating ability. Since DFP exerts protective effects through various mechanisms, which share several factors with myelin formation process, we aimed to investigate the effects of DFP treatment on remyelination. Focal demyelination was induced by injection of lysolecithin, into the optic nerve of male C57BL/6J mice. The animals were treated with DFP/vehicle, starting from day 7 and continued during the myelin repair period. Histopathological, electrophysiological and behavioral studies were used to evaluate the outcomes. Results showed that DFP treatment enhanced remyelination, decreased g-ratio and increased myelin thickness. At the mechanistic level, DFP enhanced oligodendrogenesis and ameliorated gliosis during the remyelination period. Furthermore, our results indicated that enhanced remyelination led to functional recovery as evaluated by the electrophysiological and behavioral tests. Even though the exact molecular mechanisms by which DFP enhanced myelin repair remain to be elucidated, these results raise the possibility of using deferiprone as a therapeutic agent for remyelination therapy in MS.

Biological sciences/Drug discovery

Biological sciences/Neuroscience

Biological sciences/Physiology

Biological sciences/Stem cells

Multiple sclerosis

Myelin Repair

Deferiprone

Oligodendrogenesis

Functional recovery

Myelin is a critical component of the central nervous system (CNS) which facilitates the rapid conduction of action potential, provides metabolic and trophic supports to axons, and ensures the axonal survival ¹. The fundamental importance of myelin is highlighted by demyelinating disorders such as multiple sclerosis (MS). MS is a chronic autoimmune and neurodegenerative disease mainly characterized by focal inflammatory demyelinating lesions within CNS. Most MS current treatments modulate the immune system and reduce inflammation and immune cells infiltration into the CNS. In the early stage of the disease, axons are mainly preserved; however, as the disease progresses, axon damage appears to be the main manifestation of the progressive stage ². It is believed that permanent neurological disabilities during the disease progression, is mostly secondary to prolonged demyelination and consequent irreversible axonal degeneration ³. Therefore, one of the most effective approaches to protect the axons is the enhancement of remyelination, which restores metabolic and trophic supports ⁴. However, in most of patients, remyelination gradually fails and leads to chronic demyelination, which finally results in irreversible axon degeneration and clinical deterioration ⁵.

Both developmental myelination and remyelination are highly orchestrated processes, which occur in a stepwise manner. These processes include oligodendrocyte progenitor cells (OPCs) proliferation, migration, differentiation, process extension and interaction with axons they wish to wrap, and myelin compaction ⁵. Since demyelinated axons are highly susceptible to degeneration, early treatments that enhance remyelination may postpone or even prevent the progression phase of MS.

Recently, iron-chelating agents have been used in treating several neurodegenerative diseases. In this regard, deferiprone (DFP) as an FDA-approved iron chelator has showed well-established neuroprotective effects in different animal models of neurodegeneration such as Alzheimer's disease, Huntington's disease, Parkinson’s disease and different models of retinal degeneration ^6–13. DFP exerts its neuroprotective effects through various mechanisms like reducing accumulated iron and subsequent amelioration of lipid peroxidation and oxidative stress, attenuating the gliosis, as well as alteration in the expression of neurotrophic and antioxidant proteins and enzymes in different animal models of neurodegeneration ^14,15.

DFP prevented ferroptosis, an iron dependent oxidative cell death in retinal ganglion cell in glaucoma through chelating accumulated iron ¹⁶. In lysolecithin induced demyelination model, DFP treatment led to decrease in the expression of transferrin receptor-1, as a specific marker of ferroptosis, and iron deposition. These effects led to decreased inflammation, microgliosis and astrogliosis in demyelinated optic nerve and preserved myelin and axons in lysolecithin model of demyelination ¹⁷. Furthermore, DFP attenuated clinical score of EAE mice through inhibition of T cells proliferation and infiltration ¹⁸. Besides protective effects, DFP has been shown to reduce apoptosis in OPCs and enhance their survival and promote the cells toward myelin formation in animal model of Pelizaeus-Merzbacher disease (PMD) ¹⁹. However, its effectiveness during myelin repair in animal models of MS has not been reported.

Since DFP reported effects were carried out through mechanisms that may trigger or enhance myelin formation, and considering its anti-inflammatory effects and more robust remyelination in areas with fewer inflammation and gliosis; we hypothesized that deferiprone may also accelerate the remyelination process. Our results showed that DFP treatment enhanced myelin repair through increasing the number of newly formed oligodendrocytes and reducing of gliosis. These effects led to improvement in functional recovery in optic nerve as evaluated by the electrophysiological and behavioral tests.

DFP enhanced remyelination

Following LPC injection, inflammation and demyelination mainly happen during first 3 and 7 days, respectively. Prominent remyelination starts from day 7 in this experimental setting ²⁰. Therefore, to address whether deferiprone enhances myelin repair, we induced focal demyelination in the optic nerve of mice. Afterward, the animals were treated with DFP or vehicle starting from 7 dpi (Fig. 1A). Next, FluoroMyelin staining showed changes in the intensity of myelination at 14 and 21 dpi (Fig. 1B). Quantitative data analysis showed that FluoroMyelin intensity was significantly higher in DFP-treated groups at both 14 (p<0.01) and 21 dpi (p<0.05) (Fig. 1C). For more confirmation, we also checked immunoreactivity against MBP, as a specific marker of myelinating cells (Fig. 1D). Data quantification revealed that MBP intensity in DFP-treated groups was significantly higher than the Control, suggesting enhanced remyelination at 14 (p<0.01) and 21 dpi (p<0.05, Fig. 1E).

[Figure 1]

The myelin sheaths generated during remyelination are usually thinner than those produced during the development. The g-ratio analysis is generally used as a functional and structural index of optimal axonal remyelination ²¹. In order to study remyelination in more details, semi-thin sections were used for toluidine blue staining. In control sections, the axons were almost myelinated. In Vehicle, the axons were mainly demyelinated; however, DFP treatment increased the number of axons with visible myelin sheaths (Fig. 2A). Next, the g-ratio was plotted against axon diameter. The slop of the best-fitted line was calculated (0.3806±0.01381 for control, 0.1818±0.01589 for LPC and 0.3082±0.01135 for DFP-treated groups, Fig. 2B). The comparison of the means of g-ratio showed a significant increase in Vehicle (p<0.001), while DFP-treated group showed a significant reduction toward Control (Fig. 2C, p<0.001). Furthermore, the myelin thickness decreased significantly in vehicle group (p<0.001), whereas in DFP treatment it showed a significant increase toward control group (Fig. 2D, p<0.001). Taken together, these data indicates that DFP promoted remyelination of the demyelinated axons.

[Figure 2]

DFP increased the newly generated oligodendrocytes

To see the cellular basis of observed histological changes, BrdU was injected to label proliferative cells. At 14 dpi, we quantified the number of oligodendrocytes lineage cells as well as proliferating cells among this population (Fig. 3A). Data quantification showed that the total number of olig2⁺ cells was significantly increased in DFP-treated group compared to Vehicle (p<0.05, Fig. 3B). Furthermore, the number of newly generated oligodendrocytes (BrdU⁺/olig2⁺ cells) was significantly increased in DFP group (p<0.05, Fig. 3C). These data suggests that DFP enhances remyelination through inducing the proliferation of OPCs and increasing the number of oligodendrocyte lineage cells.

[Figure 3]

DFP reduced astrogliosis but not microgliosis

LPC induced demyelination is accompanied with profound activation of astrocytes and microglia/macrophages, which leads to glia scar formation surrounding the lesion area. Previously it was shown that astrogliosis and microgliosis were reduced following DFP treatment in LPC-induced demyelination model ¹⁷. Therefore, we asked whether DFP treatment in remyelination phase might reduce astrogliosis and microgliosis, as well. The immunostaining against Iba1 showed a moderate trend of microgliosis reduction by DFP treatment at both time points, although, the means difference was not statistically significant (Fig. 4A, C). GFAP immunostaining indicated that DFP treatment attenuated astrogliosis at both time points (Fig. 4B, D, p<0.05).

[Figure 4]

DFP enhanced functional recovery of visual pathway following demyelination

In order to address weather enhanced remyelination might lead to functional recovery of visual pathway, VEP was recorded before induction of the model, at 7 and 14 dpi (Fig. 5A). Record samples for different groups are shown in figure 5B. The P1 latency was measured and reported as an index of visual pathway conductivity. As shown in figure 5C, compared to the baseline, the P1 latency was increased significantly at 7 dpi in vehicle and DFP groups (p<0.05, p<0.01, respectively). However, a remarkable decrease in P1 latency was observed in mice treated with DFP during the remyelination phase (Fig. 5C, p<0.01 compared to Vehicle). These data support our histological results.

[Figure 5]

DFP recovered visual acuity

To evaluate weather histological and electrophysiological improvements of visual pathway deficits led to the functional improvement in visual acuity, visual cliff and placing tests were performed.. The percentage of the time each animal spend on the pattern area was calculated (Fig. 6A). Before induction of demyelination, animals showed accurate depth perception by approaching the border and retracting toward the safe area (Supp. Video 1A). Whereas, the animals in vehicle group crossed frequently between pattern and cliff areas without stop at the border at 7 and 14 dpi (Supp. Video 1B and C). The quantitative data showed that at baseline all animals spent approximately >60% of the time in the pattern area (59.46± 1.82, 61.09±2.91 for vehicle and DFP-treated groups, respectively), while at 7 dpi this percentage significantly decreased to about 47% (47.37±2.75, p<0.01, 48.37±2.53, P<0.05 in vehicle and DFP groups, respectively, Fig. 6A). DFP treatment at remyelination phase significantly recovered the visual acuity at 14 dpi compared to vehicle group (Supp. Video 1D, p<0.05).

The functional recovery was also evaluated by assessing animal’s response in visual placing test. Intact mice extended the forelimbs toward the bar (Supp. Video 2A), while demyelinated animals showed weak placing response (Supp. Video 2B-C). The quantitative data indicated a significant decline in animal’s response; however, DFP treatment improved the acuity score (p<0.05) which was comparable to the Control (Fig. 6B, Supp. Video 2D). There was a negative significant correlation between P1 latency and the time in pattern area (Fig. 6C, p=0.0395) and placing score (Fig. 6D, p=0.0001).

[Figure 6]

Remyelination is a spontaneous phenomenon by which new myelin sheaths are produced and wrap around demyelinated axons following diseases such as multiple sclerosis. It is believed that this process is finally associated with the restoration of neurological function and prevention of axonal degeneration. Remyelination occurs in some MS lesions; however, in most lesions remyelination usually fails or remains incomplete, which eventually leaves axons vulnerable to degeneration ²². Hence, enhanced remyelination with therapeutic agents may postpone the progression of MS.

In the present study, we investigated the effects of DFP treatment on remyelination process in LPC-induced demyelinated optic nerve. Our results showed that DFP treatment improved myelin repair, attenuated gliosis, enhanced oligodendrogenesis, and restored functional performance of optic nerve pathway and mice visual acuity.

Following LPC injection, the maximum extent of demyelination occurs at 7 dpi. This process is continued by remyelination in the following days ²³. Furthermore, our previous report showed prolonged demyelination period when LPC was injected into the optic nerve behind the globe. In this model, effective remyelination was observed at 28 dpi ²⁰. Accordingly, we evaluated the possibility of remyelination enhancement by DFP treatment starting from day 7. Comparing the extent of demyelination in optic nerve at 14 and 21 dpi showed that DFP accelerated the myelin repair process. We further confirmed enhanced remyelination by DFP, using toluidine blue staining on semi-thin sections. Our findings showed that LPC administration led to increasing in the g-ratio, which was reversed toward the control level in DFP-treated mice. Furthermore, we observed a significant reduction in myelin thickness in vehicle group, whereas DFP treatment restored myelin thickness. This data is in consistence with the previously reports that show myelin restoring therapies decrease the g-ratio toward the control level ²⁴.

DFP has been reported to carry out neuroprotective effects through various mechanisms. In an animal model of memory impairment, DFP rescued memory deficit through the elevation of hippocampal BDNF, antioxidant enzyme catalase (CAT) and quinone oxidoreductase 1 (NQO1) ¹⁴. Furthermore, DFP attenuated neuropathology and improved the outcomes following traumatic brain injury by reducing lipid peroxidation, oxidative stress, and gliosis as well as an alteration in BDNF/TrkB expression ¹⁵. Additionally, Nrf2 and erythropoietin were reported as two key factors in neuroprotection provided by DFP ²⁵. Although the exact molecular mechanism by which DFP can accelerate myelin repair remains to be clarified, some of the mentioned mechanisms share signaling molecules with remyelination pathways that could be involved in DFP-mediated enhanced remyelination.

Successful remyelination requires OPCs proliferation and migration into the demyelinated area, which subsequently differentiate into the myelinating oligodendrocytes and wrap around demyelinated axons ²⁶. This process is termed oligodendrogenesis. In progressive MS, although OPCs are present in the CNS, new oligodendrogenesis and remyelination capacity seem to be limited ²⁷. Thus, pharmacological intervention with the ability to enhance oligodendrogenesis has received much attention in remyelination studies.

In order to evaluate possible changes in oligodendrogenesis we used BrdU labeling to mark proliferating cells during DFP treatment. We observed increase in the total number of olig2⁺ cells in DFP group, which is a general marker for the oligodendrocyte lineage cell including early progenitors to mature cells. Increasing the total number of oligodendrocytes in lesion site could be the result of increasing the migration or the enhancement of proliferation of these cells. Additionally, double staining against BrdU/Olig2 revealed that DFP treatment increased the number of newly formed oligodendrocytes. Although, the effect of DFP treatment on remyelination has not been reported before, enhancing OPC survival and promoting the cells toward myelin formation by DFP treatment has been evaluated in vitro as well as in in vivo experimental setting in an animal model of Pelizaeus-Merzbacher disease ¹⁹.

Previously, we observed the protective effect of DFP treatment on demyelination extent, inflammation, and gliosis during the course of myelin breakdown ¹⁷. We then asked whether DFP treatment attenuates the remained gliosis in remyelination phase. Our findings showed a significant decrease in astrogliosis; however, we observed a moderated reduction in the number of Iba1⁺ cells in DFP-treated mice. Following CNS insults gliosis surrounded the damaged area. Reactive astrocytes and activated microglia are the major cellular components of gliosis. Dense glial scar formation occurs during chronic stages of demyelination in MS and increases with lesion development as well ²⁸. This gliosis response not only prevents or impairs the recruitment of OPCs toward MS lesions but also inhibits OPC differentiation, maturation and final myelin sheath formation ²⁹. Although some studies argue about the detrimental or supportive effects of reactive astrocytes on myelin repair, there is sufficient evidence that remyelination is more robust in areas with fewer reactive astrocytes ³⁰. In agreement with our results, DFP treatment has been previously reported to attenuate the astrogliosis and microgliosis in an animal model of traumatic brain injury, with improving the functional outcomes ¹⁵. Collectively, these data suggest that reduced glial activation in DFP-treated animals may lead to enhanced remyelination.

The results of our previous study showed that some degrees of iron accumulation is still observed at 7 dpi, which is the peak of demyelination ¹⁷, Therefore, some part of the observed effects of DFP on remyelination might also be as the result of the removal of accumulated iron at the beginning of the remyelination phase and providing a more permissive environment for OPCs to remyelinate.

It is believed that enhancing remyelination in the setting of acute demyelination, potentially, re-establishes trophic and metabolic supports to axons and restore fast impulse conduction ⁴. In the current study, we used mouse model of optic nerve demyelination. This model gives the opportunity to evaluate visual pathway integrity and functional recovery by both electrophysiological and behavioral studies of visual acuity. In order to address weather enhanced-remyelination led to functional integrity of visual pathway, VEP was recorded before LPC and at 7 and 14 dpi. A delay in the conductivity of visual impulse corresponds to damage along the visual pathway. We observed a significant increase in P1 latency at 7 dpi, which was reveres toward baseline in DFP-treated mice compared to the Vehicle at 14 dpi. Furthermore, behavioral evaluation supported histological and electrophysiological outcomes. Following LPC-induced demyelination, a decrease in the visual acuity was observed. Our findings showed an improved visual acuity at 14 dpi in DFP-treated group, which implies myelin repair at a functional level. The improvement in behavioral tests was negatively correlated with P1 latency in VEP records. Efficient remyelination may increase the efficiency of impulse conduction and improve the visual placing and cliff perception.

In summary, deferiprone showed great potential to enhance oligodendrogenesis, and generate functional myelin sheaths (Fig. 7). These results raise the possibilities for using deferiprone as a therapeutic agent in remyelination therapy in MS. However, further studies are needed to determine whether DFP treatment does not lead to brain iron depletion, since iron is required for remyelination and myelin maintenance. Even though the safety of DFP is well established in clinical trials of neurodegenerative disease ^31–35, its efficacy and safety must be evaluated in MS patients.

Animals

Fifty-five male C57BL/6J mice (8–10-week-old, 21-25 g) were purchased from Pasteur Institute (Karaj, Iran) and were kept in standard cages under 12h light/dark cycle and controlled temperature 22±2 °C. The animals had free access to food and water. All procedures were conducted in accordance with the principals described in international guidelines for research involving laboratory animals and were approved by the Ethics Committee for Biomedical Research at Tarbiat Modares University (Ethical approved ID: IR.MODARES.REC.1398.005). All animal procedures were in accordance with ARRIVE guidelines

Demyelination induction

Focal demyelination was induced by direct injection of lysolecithin (lysophosphatidylcholine (LPC), Sigma-Aldrich, L1381), into the optic nerve, as previously reported ²⁰ Briefly, animals were deeply anesthetized using intraperitoneal (i.p.) injection of ketamine 70 mg/kg (Bremer Pharma GmbH, Germany) and xylazine hydrochloride 10 mg/kg (Hoogstraten, Belgium); a well working anesthesia methods in out setting. The conjunctiva was dissected along the glob and the optic nerve was exposed. LPC (1 µl, 1%) was injected into the optic nerve behind the globe. Topical administration of tetracycline was used to prevent infection ^20,36. It should be noted that animals used for VEP recording, visual cliff and placing, received LPC, bilaterally.

Experimental design and treatments

In order to determine the effects of DFP treatment on remyelination, after randomized assignment to animal groups, animals received daily intraperitoneal injection (i.p.) of DFP (Avicenna Laboratories Inc., Iran, 10 mg/kg), twice a day at 8 a.m. and 7 p.m., starting at 7 days post injection (7 dpi). Vehicle group received same volume of saline. The animals were sacrificed at 14 or 21 dpi, during the remyelination phase. In order to label newly generated oligodendrocytes, animals received BrdU (5-bromo-2-deoxyuridine, B5002, Sigma–Aldrich, Germany, 100 mg/kg. i.p.), for four consequent days starting at 7 dpi (Fig. 1A).

Tissue processing and sectioning

Animals were deeply anesthetized using ketamine and xylazine, followed by transcardial perfusion with phosphate buffer solution (PBS) and 4% paraformaldehyde (PFA). Optic nerve was immediately harvested and post fixed in 4% PFA for 12 h, then preserved in 30% sucrose for 24 h, followed by embedding in optimal cutting medium (OCT; Bio-Optica, Italy). Ten-micrometer transvers sections were obtained using a cryostat microtome (Histo-Line Laboratories, Italy) and mounted on super-frost plus slides.

Myelin staining and Immunofluorescence studies

Myelin staining was done using FluoroMyelin. Following rehydration, the sections were incubated with FluoroMyelin (1:300, f34652, ThermoFisher, Oregon, USA) for 30 min at room temperature. Data quantification was done by measuring the fluorescent intensity using ImageJ software.

For immunostaining, briefly, frozen sections were rehydrated in PBS, followed by incubation in triton-X100, blocking solution (NGS 10%) and the relevant primary antibodies. For nuclear antigens, the sections were pre-treated with 2 N HCl for 30 min at 37 °C followed by incubation in trypsin for 20 min before blocking. After 24 h, the sections were incubated by corresponding secondary antibody coupled to a fluorescent dye for 2 h. After washing in PBS, DAPI (4′,6-diamidino-2-phenylindole, Sigma-Aldrich; D-9542) was used to counterstain the cell nuclei. Myelin basic protein (MBP) and Glial fibrillary acidic protein (GFAP) staining intensities were quantified by ImageJ software. The number of total ionized calcium binding adaptor molecule 1 (Iba1) positive cells, oligodendrocyte transcription factor (Olig2) positive cells and Olig2⁺/BrdU⁺ cells were presented as the number of cells per section. All photos were captured under BX-51 fluorescent microscope (Olympus Optical Co, Ltd., Tokyo, Japan). Each data point for staining studies represents 3-5 sections per mouse and 5 mice per group. The list of antibodies used in this study is provided in table 1.

Table 1. List of primary and secondary antibodies used in this study

Primary antibody	Label	Species isotype	Supplier	Dilution
MBP	-	Chicken polyclonal	Aves MBP88837983	1:1000
Iba1	-	Rabbit polyclonal	FUJIFILM Wako Pure Chemical	1:1000
GFAP	-	Rabbit polyclonal	Dako Z0334	1:500
Olig2	-	Rabbit polyclonal	Proteintech 13999-1-AP	1:500
BrdU	-	Mouse monoclonal	Abcam, ab8039	1:100
anti-chicken IgY	Texas Red	Rabbit	Abcam, ab6751	1:500
anti-rabbit IgG	Alexa Fluor® 488	Goat	ThermoFisher, A11008	1:1000
anti-rabbit IgG	Alexa Fluor® 568	Goat	ThermoFisher, A11036	1:1000
anti-mouse IgG	Alexa Fluor® 568	Goat	ThermoFisher, A-11004	1:1000

Toluidine blue staining on semi-thin sections and g-ratio analysis were used to assess the profile of demyelinated/remyelinated axons. Sample preparation and staining were done as previously reported ³⁷. Briefly, after perfusion with PBS, optic nerves were harvested and post fixed in 2.5% glutaraldehyde in PBS, pH 7.2, for 3 h at 4 °C. Afterward, the samples were incubated in 1% osmium tetroxide for 3 h followed by dehydration in graded series of alcohol and embedded in resin. Semi-thin transverse sections (500 nm) were prepared and stained with toluidine blue. Using light microscope, the images were captured. The g-ratio was measured as the ratio of axon to fiber diameter. A 10-μm × 10 μm grid was placed on each section using ImageJ. Three squares area (100 μm²) were selected on a radial oriented line for evaluation. The minimum number of axons evaluated in this experiment was 370 per animal. Three sections were averaged for each individual animals and each group included 3 mice.

Visual evoked potential (VEP) recording

To assess the functional integrity of mouse visual pathway as an index of demyelination and remyelination processes, we recoded visual evoked potential (VEP) prior to demyelination induction, at the peak of demyelination at 7 dpi and after 7 days of treatment at 14 dpi, as mentioned earlier ^23,38,39. Briefly, animals were anesthetized and fixed in the stereotaxic frame. A monopolar electrode was implanted on the visual cortex contralateral to the stimulated eye (AP: −3.6 mm, L: +2.3) ⁴⁰. The reference electrode was implanted on the pre-frontal cortex with the coordination of AP: +2 mm, L: +0.5 mm. The electrodes were fixed in the skull using dental cement. For VEP recording, the re-anesthetized animals were placed in dark and electrically grounded Faraday cage (60 × 60 × 60 cm) and allowed for darkness adaptation for 5 min. All recordings were performed at morning 8-12 a.m. The contralateral eye was closed to prevent any interfering light stimulation. The flashlight was used with the frequency of 0.5 Hz for 300 trials. Signal averaging was used to reveal the evoked potential wave and the P1 latency was calculated for each time point. Each animal group included 9-12 mice.

Behavioral tests

To assess visual acuity, visual cliff and visual placing tests were done. Briefly, animals were placed in an open-top box with glass base, on the 90 cm height frame with a high-contrast checkered pattern paper attached to the one half of the box underneath. The clear side gives the impression of a cliff at the edge of the pattern paper. The vibrissae were cut to eliminate the tactile placing responses, which could interfere with the test. The animals were placed on the border of the cliff area and allowed to choose for 5 min. The percentage of the time each animal spend on the pattern area was calculated ³⁷.

For visual placing test, mice were suspended by tail, lowered toward a bar. Healthy mice extend the forelimbs toward the bar when observed. The response was scored as follow. 0 reflects no placing response, 1 represents weak placing response and 2 represents a clear placing response ⁴¹. Each assessment was the average of 3 replicates. A camera recorded the animal’s responses during the test and someone blinded to the animal grouping scored the responses.

Statistical analysis

The normality of data was assessed using Shapiro-Wilk test or The Kolmogorov–Smirnov test. In order to compare DFP-treated animals with the vehicle, two-tailed unpaired Student's t-test for parametric data and Mann-Whitney test for nonparametric data were used. To compare more than two groups, one way ANOVA with Tukey’s multiple comparisons test or Kruskal-Wallis test were used. In order to compare the groups with the factor of time, two-way repeated measured ANOVA with Bonferroni’s post-hoc test was used. In order to compare two variables among the groups, two-way ANOVA with Bonferroni’s multiple comparisons test was used. To explore correlation between variables Pearson’s correlation (r) for parametric data and Spearman’s correlation (r) for nonparametric were used. All data are presented as means ± SEM. Significance is considered when p- value <0.05. No blinding procedure was considered for data analysis. Sample size was considered based on data variability observed in previous works.

Acknowledgment

This study was supported by grants INSF4004518 from Iran National Science Foundation, Med79865, and IG-39803 from Tarbiat Modares University.

Author’s contribution

MJ: Conceptualization; AR, FF, MJ: Data curation, AR, FF, MJ: Methodology; AR, FF, MJ: Formal analysis and investigation; AR: Writing - original draft preparation; MJ: Writing - review and editing; MJ: Funding acquisition; MJ: Resources; MJ: Supervision. All authors confirmed the final version of manuscript.

Data availability

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of interest

The authors declared no conflict of interests.

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No competing interests reported.

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Deferiprone Promoted Remyelination and Functional Recovery through Enhancement of Oligodendrogenesis in Experimental Demyelination Animal Model

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