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Drug Discovery Research Laboratory, Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus, Hyderabad-500078, IndiaDr. Reddy's Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad-500046, India
Dr. Reddy's Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad-500046, IndiaKareus Therapeutics, SA. 40 Rue Fritz-Courvoisier, 2300 La Chaux-de-Fonds, Switzerland
We propose novel adult zebrafish EAE model for multiple sclerosis drugs screening.
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Disease induction in this model is by using myelin oligodendrocyte glycoprotein.
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The model has been validated using fingolimod hydrochloride (Gilenya).
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Further, detailed validation has been conducted with three known EAE modulators.
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It offers to be a quick simple protocol between in vitro and rodent studies.
Abstract
Introduction
Pre-clinical drug discovery for multiple sclerosis (MS) is a labor intensive activity to perform in rodent models. This is owing to the long duration of disease induction and observation of treatment effects in an experimental autoimmune encephalomyelitis (EAE) model. We propose a novel adult zebrafish based model which offers a quick and simple protocol that can used to screen candidates as a step between in vitro experiments and rodent studies. The experiments conducted for this manuscript were to standardize a suitable model of EAE in adult zebrafish and validate it using known modulators.
Methods
The EAE model was developed by disease induction with myelin oligodendrocyte glycoprotein – 35–55 (MOG) and observation of survival, clinical signs and body weight changes. We have validated this model using fingolimod. We have further performed detailed validation using dimethyl fumarate, dexamethasone and SR1001, which are known modulators of rodent EAE.
Results
The immunization dose for the disease induction was observed to be 0.6 mg/ml of MOG in CFA (Complete Freund's adjuvant), injected subcutaneously (s.c.) near spinal vertebrae. In the validation study with fingolimod, we have demonstrated the modulation of disease symptoms, which were also confirmed by histopathological evaluation. Furthermore, detailed validation with three other known drugs showed that our observations concur with those reported in conventional rodent models.
Discussion
We have standardized and validated the adult zebrafish EAE model. This model can help get a quick idea of in vivo activity of drugs in a week using very low quantities of candidate compounds. Further work will be required to define this model particularly as it is found that zebrafish may not express a MOG homologue.
Experimental autoimmune encephalomyelitis (EAE) is the condition in which interaction between neurological and immune pathological pathways result in features such as axon loss, inflammation and demyelination similar to that of multiple sclerosis (MS) (
). Experimental autoimmune encephalomyelitis can be induced by immunization with self antigens derived from central nervous system (CNS) myelin components, such as myelin-associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP) or proteolipid protein (PLP) (
Mouse models of EAE typically develop within 1–3 weeks following induction, but are often monitored for 4–8 weeks to monitor drug responses, which may require significant quantities of test drug following long duration studies (
). The rationale for the experiments conducted in this manuscript was to develop a model that will be useful as a quick whole organism screen for drugs being developed for multiple sclerosis and associated disorders. We believe that a zebrafish based model can act as a preliminary in vivo model which can help in selecting molecules for further in vivo evaluation in conventional mouse models. A step between in vitro and in conventional mammalian models will help reduce late stage attrition of drug candidates and help select better candidates for detailed in vivo experimentation. Thus, we believe that the proposed model will exhibit 3 R benefits.
Myelin oligodendrocyte glycoprotein peptide residues 35–55 is a myelin component that activates T cells in mice and humans (
). Experimental autoimmune encephalomyelitis is known to be a T-cell dependent disorder because adoptive transfer of in vitro activated myelin-reactive CD4+ T cells have shown to induce the disease (
Critical influences of the cytokine orchestration on the outcome of myelin antigen-specific T-cell autoimmunity in experimental autoimmune encephalomyelitis and multiple sclerosis.
). MOG is a commonly used protein to induce EAE in animal experiments.
Fingolimod hydrochloride (Trade Name: Gilenya) is a marketed multiple sclerosis drug used for treatment of relapsing multiple sclerosis to reduce the frequency of relapses. Fingolimod is a sphingosine 1-phosphate receptor modulator, and is a first in class orally administered drug (
In this manuscript, we have standardized the regimen of MOG for disease induction; we have monitored the clinical scores, body weight and histopathology changes in the fish. We have assessed the rescue of the clinical symptoms seen due to induction of the disease by using an approved and marketed drug fingolimod hydrochloride. We have further validated this model by testing a group of drugs marketed or under development for the treatment of MS which have been shown to be efficacious in the EAE model.
2. Animal ethics statement
All zebrafish experiments were performed following animal ethics guidelines of the institution as per the animal ethics laws of India. A licensed veterinarian supervised all the experimentation.
3. Materials and Methods
3.1 Animal care and maintenance
Wild type zebrafish (Danio rerio) were procured from local vendor (Vikrant Aquaculture, Mumbai, India) and maintained in re-circulatory system with controlled environment conditions with a temperature of 28 °C, and a light/dark cycle of ~14/10 h. They were fed thrice with live hatched brine shrimp and dry food (supplied by Vikrant Aquaculture, Mumbai, India) and were maintained as previously described (
). Four to six months old fish were used for these experiments.
3.2 Chemicals, drugs and drug administration
All drugs were purchased from Sigma Aldrich, USA. All other routine chemicals were purchased from Sisco Research Laboratories, Hyderabad, India. Complete Freund's adjuvant (CFA) was also procured from Sigma Aldrich, USA (Cat. No. F5881). MOG (Sequence: MEVGWYRSPFSRVVHLYRNGK) was purchased from GenScript HK Limited, Hong Kong. The drugs were administered using either oral (
) routes. These methods ensured the delivery of exact doses of the drugs in terms of milligrams per kilograms (mg/kg) of body weight.
3.3 Optimization of immunization dose
Experimental autoimmune encephalomyelitis (EAE) was induced by immunization with myelin oligodendrocyte glycoprotein – 35–55 (MOG). MOG in CFA was injected subcutaneously (s.c.) in the mid spine regions (near the end of the precaudal vertebrae as depicted in the Graphical Abstract) using 10 µl bevel-tipped Hamilton syringe with a volume of 5 µl/fish. Three concentrations of MOG: 0.3, 0.6 and 1 mg/ml were tested to standardize the dose that showed maximum efficiency for induction of clinical symptoms, body weight reduction with low mortality. The vehicle control was injected CFA at the same site as MOG injection. The different groups were the following: vehicle control (CFA s.c.), MOG in CFA 0.3 mg/ml s.c., MOG in CFA 0.6 mg/ml s.c and MOG in CFA 1 mg/ml s.c. The clinical signs were assigned scores to the surviving fish in the following order: 1: Normal, 2: Loss of Gait, 3: Mild Paralysis, 4: Total Paralysis. Each of the clinical signs can be seen in Video 1. All fish were observed for 7 days post treatment for clinical scores, body weight and mortality.
The following is the Supplementary material related to this article Video 1.
The video shows each of the clinical signs that have been assigned a score.
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3.4 Validation with the fingolimod hydrochloride (hereafter referred as fingolimod)
In this study immunization of zebrafish was done on the 1st day with standardized concentration of MOG. Fingolimod treatment was performed using two regimens (I) progressing regimen wherein the treatment started immediately after immunization; and, (II) therapeutic regimen wherein treatment started after disease development on day 3. The doses of 0.1, 0.3 and 1 mg/kg of fingolimod were administered orally (p.o.) for 7 days in both the regimens (Dose volume used: MOG – 10 µl s.c.; fingolimod – 5 µl p.o.). Control group was injected with CFA s.c. and water p.o.. All fish were observed daily for 7 days of treatment for clinical symptoms, mortality and body weights were recorded on days 1 and 7 of treatment. Qualitative scoring of EAE signs were done in blinded fashion. To ensure that the scoring is conducted in blinded fashion, video recordings (3–7 min) were performed each day by the personnel performing drug administration, the videos was coded by the supervisor, and the qualitative scoring was performed by trained personnel not involved in experimental design or immunization or drug administration. In the therapeutic regimen, the treatment started on day 3, hence all observations that were made on day 7 of treatment were day 9 post immunization, however, the data reported has been with respect to the days of treatment. Statistical analysis for clinical scores was performed using GraphPad Prism® software using Kruskal-Wallis analysis followed by Dunn's multiple comparison test. Statistical analysis for body weight loss was performed using One-way ANOVA followed by Dunnet's Post-hoc test.
The study for each regimen was conducted separately wherein fish were assigned in six treatment groups with twelve fish per group at the beginning of treatment. The different groups were the following: vehicle control (CFA s.c. + water p.o.), MOG control (MOG 0.6 mg/ml s.c. + water p.o.), Fingolimod 0.1 mg/kg (MOG 0.6 mg/ml s.c.+ Fingolimod 0.1 mg/kg p.o.), Fingolimod 0.3 mg/kg (MOG 0.6 mg/ml s.c.+ Fingolimod 0.3 mg/kg p.o.), Fingolimod 1 mg/kg (MOG 0.6 mg/ml s.c.+ Fingolimod 1 mg/kg p.o.).
The spinal sections from four groups of zebrafish: control, MOG, Fingolimod 1 mg/kg (from prophylactic regimen) and Fingolimod 1 mg/kg (from therapeutic regimen) were analyzed for histopathological assessment at the end of the study to know the extent of inflammation, neurodegeneration and demyelination and also the effect of fingolimod treatment. The histological evaluation was performed on representative sample of spinal tissue taken from fixed tissue. Tissue processing and staining was performed as per standard sequential staining protocols. Infiltration cells were counted by using ImageJ Analysis Software in the hematoxylin and eosin stained sections. The region of interest was converted to 8 bit type to clearly visualize infiltrated cells as intense dark spots and the cells in each section were counted accordingly (
). Glial cell count per section was also performed using ImageJ Analysis Software, in the sections stained using crystal violet stain. In this case, background subtraction of the selected area was performed and the cyton and nucleus in neuron which appear as dark purple spots were counted (
)). For myelination, the intensity of blue stained region of spinal cord section, in the luxol fast stained sections, was measured using RGB Plug-in in ImageJ Analysis Software (
). Statistical analysis was performed using GraphPad Prism® software using One-way ANOVA followed by Dunnet's Post-hoc test.
3.5 Detailed validation with additional drugs
Further validation of model was conducted in the prophylactic regimen of this model. Three drugs: dimethyl fumarate, dexamethasone and SR1001; were selected for the validation. Dimethyl fumarate is a drug approved and marketed for MS and related disorders (
); it was administered orally at doses of 15, 30 and 60 mg/kg and effects were evaluated for a period of seven days. Dexamethasone is a corticosteroid and has been shown to be effective in prevention and treatment of EAE in rodents (
); it was tested after intra-peritoneal administration at 0.3, 1 and 3 mg/kg doses. SR1001 is a ligand of RAR-related orphan receptors (ROR), which has been demonstrated to suppress Th17 cell differentiation and cytokine expressions. It is being developed by The Scripps Research Institute, and has been proven efficacious in rat model of EAE (
). It was tested in the zebrafish EAE model through intra-peritoneal administration at 25, 50 and 75 mg/kg doses.
4. Results
4.1 Optimization of immunization dose
Acute onset of EAE in zebrafish after immunization with mylein oligodendrocyte glycopeptide (MOG), known to develop progressive paralysis, began to show clinical signs 3 – 4 days from the day of immunization. The dose of MOG in CFA was standardized as 0.6 mg/kg, based upon efficiency to induce clinical symptoms, a significant reduction in percent body weight reduction with low incidence of mortality (See Fig. 1). The lower mean clinical score to 1 mg/kg dose of MOG could be attributed to local accumulation or spillage at higher concentration; however, we did not probe it further as 0.6 mg/kg dose satisfied the conditions for reasonable disease induction. The vehicle control group (CFA s.c.) did not show any clinical signs. From the animal ethics perspective, disease induction in zebrafish resulted in mortality numbers similar to those observed in conventional mouse models of EAE published in recent studies (
Fig. 1Standardization of immunization dose with myelin oligodendrocyte glycoprotein (MOG): (a) Effects of MOG (0.3 mg/kg, 0.6 mg/kg and 1 mg/kg) as mean clinical score of paralysis like activity seen every day from day 1 to day 7. (b) Mean clinical score on Day 7. Statistical analysis for clinical scores was performed using GraphPad Prism® software using Kruskal-Wallis analysis followed by Dunn's multiple comparison test comparing all other groups with Vehicle Control. Data are represented using mean and standard error of the mean (±S.E.M.) clinical score on day 7 post immunization (*p<0.05, **p<0.01 and ***p<0.001) (n=10 at the beginning of treatment). (c) Kaplan-Meier survival analysis performed to know survival probability after administration of MOG 0.6 mg/kg (n=36 at the beginning of treatment). (d) Effects of MOG on survival. Data are represented as Percentage Survival on day 7 post immunization (n=10).
The phenotypic results of validation study with fingolimod in the prophylactic and therapeutic regimens have been depicted in Fig. 2 and can be appreciated in Video 1. We would reiterate here that, the treatment started on day 3 in the therapeutic regimen, thus, observations made on day 7 of treatment were day 9 post immunization, and, data reported has been with respect to the days of treatment. fingolimod at doses of 0.3 and 1 mg/kg showed marked improvement of ~10% and 20% respectively, in percentage survival as compared to MOG immunized group on day 7 post immunization (Fig. 2: (a) & (b)). Mean clinical scores showed daily improvement with fingolimod treatment (Fig. 2: (c) & (d)) and on day 7 (Fig. 2: (e) & (f)) showed statistically significant dose dependent improvement in the clinical score as compared to the MOG immunized group. The body weight loss data (Fig. 2: (g) & (h)) suggests dose dependent and statistically significant improvement in this parameter at doses 0.3 and 1 mg/kg of fingolimod as compared to MOG group. In the fingolimod 1 mg/kg group, on day 7, the mean clinical score and body weight loss data was similar to the vehicle control group suggesting the efficacy of fingolimod upon clinical progression of disease. The phenotypic effects, recorded on Day 7 of treatment in the prophylactic regimen of the model, of Control (untreated), MOG induced EAE and Fingolimod 1 mg/kg body weight treatment in adult zebrafish can be seen in Video 1.
Fig. 2Phenotypic effects seen in validation with fingolimod. Graph labels are: Percent Survival on Day 7, Mean Clinical Score over 7 Days, Mean Clinical Score on Day 7 and Percent Body Weight Loss on Day 7 in Prophylactic Regimen (a, c, e, g) and Therapeutic Regimen (b, d, f, h). Survival data are represented as Percentage Survival. Clinical score and body weight loss are represented using mean and standard error of the mean (±S.E.M.). For Mean Clinical Score on Day 7 statistical analysis for clinical scores was performed using GraphPad Prism® software using Kruskal-Wallis analysis followed by Dunn's multiple comparison test comparing all other groups with Vehicle Control. For Percent Body Weight Loss on Day 7; GraphPad Prism® software was used for conducting One-way ANOVA followed by Dunnet's Post-hoc test comparing all other groups with Vehicle Control (*p<0.05, **p<0.01 and ***p<0.001) (n=12 at the beginning of treatment).
The histopathological evaluation of spinal sections was performed on four groups: control, MOG, Fingolimod 1 mg/kg (from prophylactic regimen) and Fingolimod 1 mg/kg (from therapeutic regimen). The infiltration cell number (Figs. 3(a) and 4(a)) clearly indicates that the extent of inflammation was more and statistically significant in MOG immunized fish when compared to vehicle control and fingolimod treatment at 1 mg/kg (in both prophylactic and therapeutic regimens). The histopathology of spinal cord region of MOG immunized fish also showed statistically significant decrease in glial cell count per section (Figs. 3(b) and 4(b)) when compared to vehicle control and the glial cell density was found to be within normal limit in 1 mg/kg fingolimod treated. Luxol fast staining indicates the extent of loss of myelin based on reduction in intensity of staining (Figs. 3(c) and 4(c)). MOG immunized fish were found to show slightly low intensity, though statistically not significant, of myelination when compared to vehicle control and fingolimod treatments in both regimens. These are aspects of further refinement of this model; however, in our judgment, the phenotypic effects and other histopathological changes satisfy the conditions to consider it as a reasonable and quick screening model for further investigation.
Fig. 3Histopathological effects seen on day 7 of treatment in validation with fingolimod with four groups: Vehicle control, MOG control, Fingolimod 1 mg/kg (from prophylactic regimen abbreviated as P) and Fingolimod 1 mg/kg (from therapeutic regimen abbreviated as T). Graph labels are: (a) Number of Infiltrated Cells (b) Number of Glial cells (c) Intensity of Luxol Fast Staining. Data are represented using mean and standard error of the mean (±S.E.M.). GraphPad Prism® software was used for conducting One-way ANOVA followed by Dunnet's Post-hoc test comparing all other groups with Vehicle Control (*p<0.05, **p<0.01 and ***p<0.001; n=3).
Fig. 4Black and white images (see electronic supplementary data for colored images) of representative spinal cord histopathological sections for vehicle control, MOG control, Fingolimod 1 mg/kg (P) and Fingolimod 1 mg/kg (T) seen on day 7 of treatment in validation study with Fingolimod. The arrows (→) point towards examples of cells counted or blue intensity measured using ImageJ.
). Thus the observations in zebrafish model concur with those reported in conventional rodent models.
4.3 Detailed validation with additional drugs
Three known drugs have been evaluated, as a part of validation of the model, and the data is represented in Table 1. Dimethyl fumerate when administered orally at doses of 5, 15 and 60 mg/kg showed dose dependent improvement in survival rates. Clinical scores and body weight loss parameters also improved when compared to the MOG group, however there was no dose dependence and the data looked like a saturated effect at the doses tested. Dexamethasone was administered intra-peritoneally at doses 0.3, 1 and 3 mg/kg. There was an improvement in survival rates, clinical scores and body weight loss when compared to MOG treated groups; however, at highest dose the clinical score and body weight loss increased, which could be because of immunosuppressant effect of dexamethosone. SR1001, a synthetic ROR ligand, showed improvement in survival rate at one dose i.e. 50 mg/kg when compared to MOG treated group. It showed improvement in clinical score and body weight loss at all doses, however, not in a dose dependent manner. This could be because the highest dose had severe mortality.
Table 1Validation Study with Known Drugs: Phenotypic effects seen on day 7. Data are represented as mean and standard error of the mean (±S.E.M.). Statistical analysis for clinical scores was performed using GraphPad Prism® software using Kruskal-Wallis analysis of variance followed by Dunn's multiple comparison test, whereas, statistical analysis for body weight loss was performed using One-way ANOVA followed by Dunnet's Post-hoc test. Comparison is between all other groups with Vehicle Control (*p<0.05, **p<0.01 and ***p<0.001).
In summary we have tested wide chemical classes of drugs, which have different mechanisms of action, through different routes of administration and at different doses to validate the model.
5. Discussion and conclusions
5.1 Major advantages of the model
We have developed and validated, a novel zebrafish EAE model, that can be used to test drug candidates for MS and related disorders. The model proposed by us has the following advantages: (i) Quick: the efficacy of candidate drugs can be evaluated in a short span of 7 days; (ii) Low Compound Requirement: the efficacy evaluation can be performed using very little amounts of test compound, for example, compound requirement to test at 10 mg/kg dose (sample size of 10 fish weighing approximately 0.5 g each) for 7 days will be 350 µg. This is a minuscule quantity as compared to that required for a rodent EAE study; and; (iii) Inexpensive In vivo Data: the cost of maintaining zebrafish is very low as compared to rodents and in vivo data can be obtained quickly and with low quantities of test compound, further making it inexpensive.
5.2 Relevance in drug discovery
The EAE model has been used for pre-clinical evaluation of candidates being screened for MS and associated disorders for quite some time; however, there has been a question on the translational potential of the animal efficacy data in clinics. Despite this skepticism EAE is the most widely used model for in vivo efficacy evaluation and almost all drugs approved for MS were tested in this model before selecting them as clinical candidates (
). Therefore, we believe that this model will play a significant role in pre-clinical evaluation of drug candidates in the near future as well. The zebrafish model for EAE can be used preceding the use of the rodent EAE models. This model can be used as a filter at lead optimization stage wherein substantial number of compounds selected through in vitro screening can be filtered using this model. The most promising compounds thereafter can be screened in the rodent models. Using all the advantages of the zebrafish models stated above, late stage attrition of compounds and associated costs can be saved, furthermore, the possibility of oral/intra-peritoneal drug administration can indicate the their dose range for rodent testing.
There is an obvious question related to the translation of zebrafish data to conventional mammalian models. Zebrafish are being increasingly proposed as screening tools for potential remyelination therapies due to their regenerative abilities, suggesting its relevance as a screening tool for MS and related disorders (
). However, all the models suggested are larval models, which even though act as alternative to animal experimentation, have limitations of (i) not having fully developed organ systems, (b) inability to test poorly soluble drugs, and, (c) inability to evaluate drug kinetics and carrying out pharmacokinetic – pharmacodynamic correlation. All these factors attribute to the questions relating to translation of zebrafish data to other mammals. The use of adult zebrafish and its ability to overcome the limitations of a larval model have been suggested in several publications before (
). Homology searches of the peptide used showed some similarity (~40%) with zebrafish butyrophilin-like molecules (NCBI Sequence ID: NP_001103953.1), involved in lipid metabolism, and cross-reactive antibodies between MOG and butyrophilin have been reported (
). However, whilst this peripheral antigen could account for molecular mimicry triggering MOG-reactive autoimmunity, it is not clear how this would manifest as a CNS restricted disease. Alternatively this peptide could induce an unusual cross reactivity at the T cell level, as it has been reported that T cells specific for MOG 35–55 peptide can also react with neurofilament medium 18–30 and such that the MOG peptide can induce disease in MOG-deficient mice (
). Repetition of these results in this study can confirm the value of the model and further work will be needed to determine the target auto-antigen driving the paralysis in the zebrafish.
Zebrafish express orthologues of glucocorticosteroid receptors (
), which could mediate the effects of the immunomodulatory drugs (dexamethasone, dimethyl fumarate and SR1001 respectively) used here. In addition, although zebrafish have spingosine-1-phosphate receptors (
), it remains to be established whether the mechanism of action of fingolimod in zebrafish is via influences of egress from the spleen or other surrogate lymphoid tissue present in zebrafish (
Sphingosine 1 Phosphate at the Blood Brain Barrier: Can the Modulation of S1P Receptor 1 Influence the Response of Endothelial Cells and Astrocytes to Inflammatory Stimuli?.
An argument can be made regarding the relation of paralysis observed in fish to demyelination as the phenotypes seen in fish might be due to inflammation near the site of injection. This argument can be countered by the following facts of the experiment: (a) the control group was injected with CFA s.c. at the same site as MOG injected groups and did not show the paralytic phenotypes suggesting that the paralysis was due to MOG; (b) MOG induced inflammation is an established method for creating an EAE models; and; (c) the MOG induction has shown that on day 7 post immunization the intensity of myelination was slightly reduced in the luxol fast stained sections. The aspects of detailed pathological assessment, booster injections, larger set of drugs, biomarkers, endpoints other than mortality, culling the animals before death, etc. will need to be further investigated to refine this model for optimal utilization.
We propose this model to be a potential tool for quick assessment of candidate drugs and also to study disease pathobiology. The present manuscript can be inspiring to researchers in the field to explore and refine this model further as larger network of laboratories will be required to make it robust for use of industry and academia.
6. Conclusions
This is the first report, to the best of our knowledge, suggesting an in vivo adult zebrafish EAE model. It is possibly the quickest and most inexpensive in vivo model proposed for drug discovery of MS and related disorders. This model will need to undergo a wider validation by using larger set of drugs, identification of biomarkers and through larger network of zebrafish laboratories. There is scope to further improve this model for drug screening as well as for biological research; however, this will be the starting point of all such efforts.
Conflict of interest declaration
The authors have no conflict of interest with respect to this manuscript. Authors receive monetary compensation from their affiliation; however, the present manuscript does not in part or full disclose any information pertaining to the research of authors’ affiliations.
Funding and Acknowledgements
The authors gratefully acknowledge Dr. Reddy's Institute of Life Sciences, Hyderabad, India, for funding this research and for infrastructural facilities; Birla Institute of Technology and Sciences, Hyderabad for PhD registration and research guidance of the first author.
Supplementary material The video shows the typical phenotypic effects of Control (untreated), MOG Induced EAE and Fingolimod 1 mg/kg body weight treatment in adult zebrafish. The videos are representative and have been recorded on Day 7 of treatment in the prophylactic regimen of the model.
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