Animals for the study were procured from Biogen Laboratory Animal Facility, Bangalore, India. The animals were maintained in an air-conditioned animal room with a 12 hour light and dark cycle and diet and water were provided ad libitum throughout the experimental period. The study was conducted between September to October 2020, after receiving proper Institutional Animal Ethical Clearance [SU/CLAR/RD/010/12/2020]. Male Wistar rats with weights ranging from 150 - 200 gm, were chosen for the study.
0.2% Cup was procured from Sigma Aldrich Chemical Company, St.Louis, Missouri, USA, and hydroxypropyl cellulose(HPC) was procured from Sisco Research Laboratories, Mumbai, India. The catalogue numbers were 69586[10099-74-8], 607995[7446-14-2], and 68015[10099-74-8] for cuprizone, MP and HPC respectively.
Experimental procedure and design:
After acclimatization for a period of 1 week, the experiment was carried out for 8 weeks for groups I to V and 11 weeks for group VI, animals were divided into six groups with six rats in each group. Group I (Control) rats were administered 1.5.mL of 1% hydroxypropyl cellulose (HPC)/kg b.w, p.o 35 days. Group II (Cup) rats were administered Cup 450 mg /kg b.w dissolved in 1.5 mL of 1% HPC, p.o for 5 weeks (Basoglu et al. 2013). Group III (Cup+MP group) rats were administered Cup 450 mg/kg b.w, p.o for 5 weeks and from 3rd week, in addition to Cup administration, MP, 20 mg/kg b.w, i.p (intraperitoneal) was included for 3 weeks. Group IV (Cup+LIE) rats were administered Cup 450 mg/kg b.w, p.o for 5 weeks, and from the 3rd week of Cup administration, free swimming with no resistance for 40 min for 5 weeks was included. Group V (Cup + HIE group) rats were administered Cup 450 mg/kg b.w, p.o for 5 weeks, and from the 3rd week of Cup administration, swimming with an added resistance of 9% body weight for 5 weeks was included. Group VI (Cn+Cup+HIE) rats were started with free swimming 40min a day, for 3 weeks. After 3 weeks, the rats were administered Cup 450 mg/kg b.w, p.o for 5 weeks and from the 3rd week of Cup administration, an exercise program same as group V was included. The Cup solution was prepared according to dosage for each day and given through oral gavage.
The rats in group VI were preconditioned with free swimming, in a circular tank with a depth of more than 50cm with a water temperature of 30±5°C, for 40 min, 5 times a week, before administration of Cup. After 3 weeks of Cup administration, rats in the Cup+LIE group were made to swim with no additional resistance for 40 min, 5 days a week, for 5 weeks (Klaren et al. 2014). For Cup+ HIE and Cn+Cup+HIE groups a resistance of 9% of body weight, in the form of a metal ring was tied around the tail of the rat and made to swim for 40 minutes, 5 days a week for 5 weeks (Almeida et al. 2009) In case of slippage of the weight from the tail, it was again reinforced and time monitored. The intensity grading based on the load was adapted from a study done by Gobatto et al. 2001
Induction of demyelination:
Cuprizone, a primary copper chelator, induces selective oligodendrocyte apoptosis by 3 weeks of administration which is followed by activation of innate neuroglial and immune cells in the brain, by astrocytes and microglial proliferation, whereby it finally leads to demyelination of distinct white and grey matter areas (Kipp et al. 2009). There is very minimal involvement of the blood-brain barrier and cells of the immune system is believed to play a role in demyelination induced by Cup (Wolf et al. 2018).
Demyelination induction was achieved by administration of 450 mg /kg b.w Cuprizone dissolved in 1.5mL of 1% HPC (Abe et al. 2015). The control group received 1.5mL of 1% of HPC.
After 3 weeks of Cup administration, 20 mg/kg b.w of MP was administered through intraperitoneal route for 3 weeks (Cammer et al. 1999)
After the assigned experimental period, the rats were euthanized by an overdose of 1% isofluorane. After which, the rats were perfused intracardially with 50mM phosphate-buffered saline. Brain tissue was carefully dissected and washed in saline and transferred to formalin containers for histopathology and wrapped in aluminum foil and kept in refrigeration at - 80°C for immunohistochemistry, immunofluorescence, and Quantitative real-time - Polymerase chain reaction (qRT PCR)
Luxol fast blue (LFB) staining:
.The coronal sections of brain issue was stained with Luxol fast blue (LFB). Brain tissue slides were incubated in Luxol Fast Blue Solution (0.1%) for 24 h at room temperature. The sections were rinsed thoroughly in distilled water and then dipped in Lithium Carbonate Solution (0.05%) several times. Differentiation for the sections were then continued by repeatedly dipping in alcohol reagent (70%) until the gray matter became colorless and the white matter remained blue. Then, the sections were rinsed in distilled water, followed by incubation with Cresyl Echt Violet (0.1%) for 2–5 min. The sections were rinsed quickly in distilled water, dehydrated quickly in three changes of absolute alcohol, cleared in three changes of xylene, and mounted with mounting medium. The slides were viewed under the Olympus binocular Bright field Microscope at 60x magnification.
The tissues were immersed in ice-cold PBS followed by freshly prepared filtered 4% Paraformaldehyde (PFA) in PBS. After embedding, the tissues were cut using the Leica cryostat CM1850 and stored in cryoprotective solution (25% glycerol, 25% ethylene glycol in PBS) at -20°C. The sections were incubated with polyclonal rabbit anti-MBP (1:1,000, Abcam, Cambridge) overnight at 25°C, then treated with streptavidin-peroxidase complex (1:200). Sections were visualized by the reaction with 3,3-diaminobenzidine tetrachloride (Sigma Aldrich) in 0.1 M Tris-HCl buffer (pH 7.2). The sections were then dehydrated, mounted on a slide, and visualized under a bright field microscope (Labomed).
Chronic demyelination usually leads to astrocyte proliferation. The corpus callosum frozen brain sections (10 μ m) were dried for 2 h at room temperature and then fixed with 4% PFA in PBS for 20 min. After blocking non-specific antibody binding with 5% non-fat dried milk for 20 min, sections were incubated overnight with rabbit anti-GFAP (1:4000, Enzo Life Sci) diluted in blocking buffer at 4 °C. Subsequently, sections were washed in TBS and incubated in a dark, humid chamber with the appropriate fluorescently labelled IgG’s-Alexa Fluor 594 (1:400) combined with donkey anti-rabbit IgG Alexa Fluor 488 (1:400) diluted in TBS-T for 2 h at room temperature. After several washes in TBS, sections were mounted with polyvinyl alcohol mounting medium. Immunofluorescence was examined using a Leica confocal laser scanning microscope (Leica Microsystems).
Fgene expression level of BDNF in the hippocampus by qRT-PCR:
The total RNA was prepared from the brain hippocampus tissue using a TRIzol reagent purchased from and RNeasy® Mini kits (Qiagen, Germany). QPCR Master Mix Kit were purchased from Invitrogen, Biomedica. Complementary DNA was first synthesized from total RNA using reverse transcriptase. PCR was performed using a (Applied Biosystems, USA). The operating conditions were as follows: for glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 30 cycles of denaturation at 95∘C for 30sec, annealing at 58∘ C for 30sec, and extension at 72∘C for 30 sec; for BDNF, 27 cycles of denaturation at 95∘C for 30sec, annealing at 57∘C for 30sec, and extension at 72∘C for 30 sec. The PCR products were separated on 1.2% agarose gels and stained with ethidium bromide. The density of each band was quantified using an image-analyzing system. The expression levels were compared with each other by calculating the relative density of the target band, such as BDNF, to that of GAPDH.
The primer sequence was as in Table 1.
Table 1: showing the primer sequence for qRT-PCR BDNF in the hippocampus region
DATA AVAILABILITY STATEMENT
All data generated or analysed during this study are included in this article
All the data were represented by mean ±SD. Comparison between groups was made using one-way ANOVA with Bonferonni's t-test with Sigma plot 13 software. P-value <0.001 was taken as statistically significant. Immunostained slides were quantified using Image J software. The expression of the BDNF gene was measured using GAPDH as an internal control.