Introduction
The central nervous system (CNS) can arguably be considered the most important organ system in the body. Hence, the diseases affecting the CNS are a matter of the greatest health concern.[ 1 ] Demyelinating disorders are a group of conditions arising from diseases affecting the myelin sheath or oligodendrocytes and Schwann cells, the cells that form it.[ 2 ] Multiple sclerosis (MS) is the most common demyelinating disease of the CNS, where demyelination occurs alongside axonal and neuronal degeneration.[ 3 ] MS frequently causes young adults to experience neurological difficulties in addition to significant physical or cognitive disablements.[ 4 ] People with MS experience increased anxiety,[ 5 ] locomotive enfeeblement[ 4 ] as well as altered brain chemicals, including decreased serotonin levels.[ 6 ] Oxidative stress, associated with the pathway leading to demyelination in MS, causes altered malondialdehyde (MDA), glutathione (GSH), and catalase level in the brain.[ 7 ]
The treatment for MS mainly focuses on reducing the immune response and they are only effective in slowing the disease progression and attenuation of symptoms. The existing therapies reduce the incidence and severity of new lesions by limiting the activity and availability of immune cells, manifesting clinically through reduction in relapse rates and minimalizing the disability. However, they are unable to cure the disease completely. Finding drugs to treat progression of disease remains greatest unmet need for people with MS. The promotion of regeneration of myelin sheath, through enhancing the progress of endogenous remyelination, will be the most tractable prospects to delay, prevent, or reverse demyelination.[ 8 ] Researches are ongoing to find out such agents which prevent demyelination and enhance the endogenous remyelination process.
Recently, different reports have been published supporting the neuroprotective effects of Flunarizine,[ 9 ] a lipid-soluble piperazine derivative used in the treatment of migraine and vertigo with cerebroselective calcium channel blocking property. Interestingly, it has been demonstrated that preadministration of flunarizine preserves Schwann cell function and myelin sheath structural integrity by avoiding calcium overload, which is a major contributing factor to ischemia-induced nerve degeneration.[ 10 ] Flunarizine may also play a role in regulating neuroinflammation and calcium-mediated neuronal damage, as evidenced by a case report that showed a patient with MS experiencing significant clinical improvement after receiving treatment.[ 11 ] The proposed mechanisms underlying its therapeutic effect may be attributed to its ability to the blockade both voltage-gated N-type calcium channels (Cav2.2) and voltage-gated T-type calcium channels (Cav3.1 and Cav3.3 preferentially), as well as the inhibition mTORC1, producing anti-inflammatory action.[ 11 ] Flunarizine, due to its antioxidant,[ 12 ] calcium channel blocking property, and anti-inflammatory action, can be considered as an excellent neuroprotective candidate.
These reports suggest that flunarizine can be used as a neuroprotective agent, which can also prevent or reverse the demyelination process. In the present study, we evaluated the protective action of Flunarizine in the degeneration of myelin sheath in a cuprizone (CPZ) induced model of demyelination. In this study, the protective effect of the drug in preventing oligodendrocyte apoptosis and myelin destruction is evaluated in mice using various behavioural assays and estimating the concentration of various markers in brain homogenate, and also through histopathological evaluation of the brain.
Materials and Methods
Ethics statement
This experimental study was performed at the College of Pharmaceutical Sciences, Government Medical College, Thiruvananthapuram. All experimental procedures were conducted in compliance with the guidelines of the CCSEA, and the study was approved by the Institutional Animal Ethics Committee of Government Medical College, Thiruvananthapuram (Approval Number: 02/04/IAEC/2022/GMCT). This study was conducted and reported in accordance with the ARRIVE 2.0 guidelines for reporting animal research.
Animals
Thirty-five male Swiss albino mice, weighing 20–25 g, were purchased from the Biomedical Technology wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojapura, Thiruvananthapuram, Kerala, India. These animals were housed in polypropylene cages having stainless steel lids with the provisions for keeping food and drinking water. Husk was used as bedding material, and the animals were housed for 7 days before the start of the experiment. The animals were maintained under standard laboratory conditions, such as temperature: 22°C–25°C, 12 h light/dark cycle, and provided with food and water ad libitum . The entire experimental protocol schedule was performed between 9:00 AM and 4:00 PM to minimize the circadian rhythms effects.
Procurement of drugs
Flunarizine dihydrochloride was purchased from Yarrow Chem Products, Mumbai. CPZ was purchased from Sigma-Aldrich (India). All other chemicals used in the study are of analytical grade. Solutions of the drugs and chemicals were freshly prepared before use. Flunarizine was suspended in 0.1% carboxy methyl cellulose and was administered by oral route ( p. o. )
Experimental protocol schedule
The male Swiss albino mice were randomly divided into 5 groups ( n = 7 per group). Group 1, control (Diet without CPZ for 6 weeks), Group 2, disease group (CPZ 0.3% through diet for 6 weeks), Group 3, vehicle control (CPZ 0.3% through diet + 0.1% CMC (vehicle) P. O for 6 weeks), Group 4, low dose Group (CPZ 0.3% through diet + flunarizine dihydrochloride P. O.(1.2 mg/kg) for 6 weeks), Group 5, high dose group (CPZ 0.3% through diet + flunarizine dihydrochloride P.O. [2.4 mg/kg] for 6 weeks). Dosing was based on drug repurposing strategy, and the human oral therapeutic doses of flunarizine (5–10 mg/day) were then scaled down to equivalent doses in mice using body surface area normalization. Based on this, 1.2 mg/kg and 2.4 mg/kg were chosen as the lower and higher doses of the therapeutic equivalents to assess dose-dependent effects. Various behavioural parameters were assessed on day 0, 3 rd , and 6 th weeks of the experiment.
Histopathological evaluation of the brain
To assess the morphological changes in the brain, we performed a histological examination using haematoxylin and eosin (H and E) staining. At the end of the experimental period, mice were sacrificed. Brain tissues were carefully removed, rinsed with ice-cold saline, dissected into two hemispheres, and immediately fixed in 10% formalin for 24 h. Tissues were then processed for paraffin embedding, and 5 μm-thick sections were prepared and stained with H and E and observed under a light microscope.[ 13 14 ] For Luxol fast blue (LFB) staining, Paraffin slices were prepared, dewaxed, cleared, and stained with LFB overnight at 60°C. Sections were then differentiated by rinsing in 95% ethanol, 0.05% lithium chloride solution, followed by 70% ethanol. Differentiation continued in distilled water until unmyelinated tissue appeared white, and then the tissues were counterstained using Cresyl violet. The slide was rinsed, dehydrated and then dipped in Xylene for 5 changes for 5 min each, and finally mounted the slides with DPX and observed under a light microscope, where the myelin will appear as blue.[ 15 16 ]
Behavioural assays
Evaluation of motor coordination by the rotarod apparatus
The motor coordination was evaluated using an accelerating rotarod where a mouse was placed on the rotating rod (3 cm diameter). One day before the test session, each mouse was trained in the rod for 3 min, which was accelerated gradually from a speed of 4 rpm to 30 rpm. Training improves animals’ skill and avoids fortuitous falling. After 24 h, each mouse was tested in the rod with the same speed of 25 rpm. The procedure was done on the day before treatment and repeated on 3 rd week and 6 th week.[ 17 18 ]
Evaluation of locomotor activity by actophotometer
The Mice were individually placed in a nontransparent cylinder cage (25 cm diameter × 13 cm height) for 10 min, and then noted the basal activity score of all the animals before the drug treatment on day 0. The activity was again recorded on the 3 rd and 6 th week of the treatment. The frequency of activities was recorded by the total number of movements, including both horizontal and vertical movements.[ 17 19 ]
Evaluation of effect on anxiety by elevated plus maze
Mice from each group were individually placed in the centre facing the open arm and then monitored the number of entries in open/closed arms and average time spent in each arm for 5 min. After 5 min all the parameters were compared. The procedure was done on the day before treatment and repeated on the 3 rd and 6 th week.[ 20 ]
Evaluation of effect on cognition by shuttle box avoidance test
The study involved training mice on a shuttle box with two compartments separated by a wooden wall and a stainless-steel grid floor. The animals learned to control the application of unconditioned stimulus (US) by reacting appropriately to a conditioned stimulus (CS). The first stage of avoidance learning was to escape from the US. On day 1 (habituation), mice were given free access to both compartments for 5 min without any stimulation. The acquisition phase (days 2–6) involved one trial per day. Mice were placed in the electrified compartment and allowed to acclimatize for 30 s, followed by a CS (buzzer, 8 s) and an US (foot shock, 0.4 mA, 10 s) delivered via the grid floor. An escape to the safe compartment in response to the CS was recorded as an avoidance response. Stimulation ended when the animal escaped or when the foot shock was completed. Retention tests were performed on days 7 and 14 under the same conditions as acquisition but without foot shock. Each test consisted of 10 trials, and memory performance was evaluated according to the number of avoidance responses. The avoidance response on day 14 was taken as the baseline (pre-treatment) measure, and the retention test was repeated on day 22 (3 rd week) and day 43 (6 th week) of the treatment regimen.[ 21 ]
Craniotomy and processing of tissue
After 6 weeks, the animals were sacrificed using cervical dislocation and then decapitated. The brains were removed by craniotomy of the vertex, weighed precisely, and processed for biochemical analysis. Some brain samples were immersed in 10% neutral buffered formalin for more than 24 h for histopathological examination. After washing with a 0.1 M phosphate buffer, the brains were tissue processed, the blocks were cut in sections of 5 μm of thickness, and these sections were used for hematoxylin and Eosin (H and E) staining and LFB staining.
Biochemical investigations
Brain homogenate preparation
After washing with ice-cold isotonic saline solution, the brain tissue was homogenized with 10 times (w/v) ice-cold 0.1 M phosphate buffer (pH 7.4). The homogenate was centrifuged at 10,000 rpm for 15 min, the supernatant was separated, and aliquots were used for biochemical estimation.[ 22 23 ]
Estimation of malondialdehyde level
The level of MDA was determined by the method of Ohkawa. This method is based on the reaction of MDA with thiobarbituric acid at low temperature and high pH to form a coloured complex with absorption maxima at 532 nm.[ 24 ]
Estimation of serotonin levels
The serotonin content was estimated using the method of Schulmpf. Reaction between serotonin and o-phthaldialdehyde results in the formation of a fluorophore and is determined spectrofluorimetrically.[ 24 25 ]
Estimation of glutathione level
The assay involves an optimized enzymatic recycling method using GSH reductase and Ellman’s reagent; sulfhydryl reagent 5, 5′-dithio-bis (2-nitrobenzoic acid) (DTNB). GSH reductase reduces oxidized GSH to reduced GSH. DTNB reacts with GSH to form yellow colored chromophore, 5-thionitrobenzoic acid, which has an absorbance maxima at 412 nm.[ 26 ]
Estimation of catalase levels
Catalase activity is measured by ultraviolet (UV) spectroscopic method. In the UV range, H2O2 show a continual increase in absorption with decreasing wavelength. The decomposition of H2O2 can be followed directly by the decrease in the absorbance at 240 nm. The difference in absorbance per unit time is a measure of the catalase activity.[ 27 ]
Results
The neurological motor coordination and balance were evaluated by the latency to fall off by the rotarod test. The fall off time of animals in 5 different groups during the 0 th , 3 rd , and 6 th week of drug treatment has been depicted in Figure 1 . The fall off time in the control group is significantly ( P < 0.001) higher compared with the disease and vehicle control group, whereas the fall off time in the low dose group is significantly ( P < 0.5) higher compared with that of the disease group. By the 6 th week, the fall off time in the control group is significantly higher ( P < 0.001) compared with the disease and vehicle control group, whereas the fall off time in the low dose group is significantly ( P < 0.0001) higher compared with that of the disease group.
Locomotor activity for each group was evaluated by an actophotometer during 0 th , 3 rd , and 6 th week of drug treatment. The locomotor score of animals in 5 different groups following 10 min exploration period in the actophotometer during the 0 th , 3 rd , and 6 th weeks of drug treatment has been depicted in Figure 2 . By the 6 th week, it is evident that CPZ can cause locomotive defects. The disease group showed a statistically significant ( P < 0.001) decrease in the locomotive scores when compared to that of the control group, which is an indication of CPZ-induced locomotive defects. Whereas the treatment groups showed significantly higher ( P < 0.05) locomotor activity compared with that of the disease group.
The elevated plus maze measures parameters such as total time spent in the open arm (sec) and number of entries in the open arm. Total time spent in open arm is evaluated for 5 groups at the 0 th , 3 rd , and 6 th week of treatment. The anxiety of animals expressed as total time spent in open arm in 5 different groups following 5 min exploration period in the elevated plus maze during 0 th , 3 rd , and 6 th week of drug treatment has been depicted in Figure 3a . From Figure 3a , by 6 th week, there was a significant decrease ( P < 0.0001) in total time spent in open arm in the disease group compared with that of the control group, indicating increased anxiety levels in the disease group. But the treatment group, especially the low dose group showed significantly higher ( P < 0.001) amount of total time spent in open arm compared with that of disease group, indicating decreased anxiety levels compared with that of the disease group. The number of entries to the open arm of the elevated plus maze was evaluated during the 0 th , 3 rd , and 6 th week of drug treatments. The anxiety of animals expressed as number of entries into open arm in 5 different groups following 5 min exploration period in the elevated plus maze during the 0 th , 3 rd , and 6 th week of drug treatment has been depicted in Figure 3b . The total number of entries into the open arm was evaluated. By the 6 th week, there was a significant decrease ( P < 0.0001) in the total number of entries into open arm in the disease group compared with that of the control group, indicating increased anxiety levels in the disease group. But the treatment group, especially low dose group showed significantly higher ( P < 0.0001) total number of entries into in open arm compared with that of the disease group, indicating decreased anxiety levels compared with that of disease group.
The cognitive effect of the drug was evaluated using the shuttle box avoidance test in mice. Shuttle box avoidance test apparatus mainly evaluates the number of avoidance responses by mice in 10 trials. The number of avoidance responses by mice is evaluated for 5 groups at the 0 th , 3 rd , and 6 th week of treatment. The memory of animals is expressed as the number of avoidance responses by mice in 5 different groups following 10 trials in the shuttle box apparatus during 0 th , 3 rd , and 6 th week of drug treatment has been depicted in Figure 4 . The number of avoidance responses by mice in 10 trials in shuttle box apparatus was recorded. By 3 rd week, the number of avoidance responses by mice in the disease group is significantly reduced ( P < 0.001) compared with that of control group, whereas the number of avoidance responses by the animals in the low dose and high dose group is significantly increased ( P < 0.05) compared with that of the disease group. Similarly, by 6 th week, the number of avoidances responses by the animals in the disease and vehicle control groups is significantly reduced ( P < 0.0001) compared with that of control group whereas the number of avoidances responses by the animals in the high dose group and low dose group is significantly increased ( P < 0.001 and P < 0.001, respectively) compared with that of disease group indicating retaining of memory.
The serotonin level of animals is expressed as μg/g protein in five different groups and has been depicted in Figure 5 . From the Figure 5 , the brain serotonin level was significantly lower in the CPZ group when compared to control ( P < 0.0001) and it indicates the depressive behavior and cognitive impairment induced by CPZ. The drug high-dose and low-dose treatment considerably increased serotonin level when compared to the CPZ group, which was statistically significant ( P < 0.001 and P < 0.0001, respectively).
Catalase level in mice brain homogenate is evaluated using UV spectrophotometric analysis. The catalase level of animals is expressed as as micromoles of hydrogen peroxide decomposed per minute per milligram protein in 5 different groups and has been depicted in Figure 6 . From Figure 6 , the brain catalase level was significantly lower in the CPZ group when compared to the control ( P < 0.0001) and it indicates the possible oxidative stress in the brain induced by CPZ. The drug high dose and low-dose treatment considerably increased ( P < 0.0001) catalase level when compared to the CPZ group.
GSH level in mice brain homogenate is evaluated using colorimetric analysis. The GSH level of animals is expressed as μmol/g tissue and has been depicted in Figure 7 . From Figure 7 , the brain GSH level was significantly lower in the CPZ group when compared to control ( P < 0.0001) and it indicates the possible oxidative stress in the brain induced by CPZ. The drug high dose and low dose treatment considerably increased ( P < 0.0001) GSH level when compared to the CPZ group.
MDA level in mice brain homogenate is evaluated using colorimetric analysis. The MDA level of animals is expressed as nmol/g/weight of brain and has been depicted in Figure 8 . From Figure 8 , the brain MDA level was significantly higher in the CPZ group when compared to the control ( P < 0.0001) and it indicates the possible oxidative stress in the brain induced by CPZ. The drug high-dose and low-dose treatment considerably decreased ( P < 0.05 and P < 0.001, respectively) MDA level when compared to the CPZ group.
The hematoxylin and eosin (H and E) stain is the standard reference stain used in the study of histochemical tissue pathology. It can provide a comprehensive picture of the microanatomy of tissues. Figure 9 represents the brain sections of Swiss albino mice after different types of treatment for 6 weeks. The green arrows indicate vacuole formation and black arrow indicates macrophages. The control group showed both neural cells and glial cells, whereas the disease group and the vehicle control group showed a decrease in neural cells and also exhibited the presence of inflammatory cells and macrophages, indicating the presence of neuroinflammation and degeneration. However, low dose group and high dose group showed a decrease in the inflammatory cells and an increase in the neural cells, indicating the capability of flunarizine to provide neuroprotection in albino mice.
The LFB staining is specific for myelin in the CNS. It is a commonly used stain to observe myelin under light microscopy. Figure 10 represents the brain sections of Swiss albino mice after different types of treatment for 6 weeks. Compared to the normal group, demyelinated axons are seen in the disease group and vehicle control group, as indicated by lesser blue staining. At the same time, myelinated axons are seen in the low dose group and high dose group as indicated by more blue staining, thus indicating the capacity of Flunarizine to protect the myelin sheath.
Discussion
A chronic autoimmune demyelinating disease of the CNS MS is typified by progressive neurological deficits, axonal damage, and neuroinflammation.[ 8 ] Using behavioral, biochemical and histopathological evaluation to replicate MS-like effects, the current study assessed the neuroprotective potential of flunarizine dihydrochloride against CPZ-induced demyelination in Swiss albino mice.
The CPZ model is the common model for examining the demyelination process, specifically in the corpus callosum, caused by oligodendrocyte apoptosis and subsequent myelin loss. Although the CPZ model does not have an autoimmune component and does not reflect the heterogeneity of MS, it is nevertheless an established model for studying toxin-induced oligodendrocyte death and demyelination. Through histopathological analysis employing H and E and LFB staining, significant differences between the groups were discovered. The disease and vehicle control groups showed vacuole formation, inflammatory cell infiltration, and demyelination, whereas the flunarizine-treated groups showed partial preservation of myelin, reduced inflammatory markers, and increased neuronal density. The histopathological improvements observed in the flunarizine-treated group, such as preservation of myelin sheath integrity and reduced vacuolation, align with the functional outcomes noted in the study.
The motor deficits observed in the disease and vehicle control groups are likely due to the demyelination of the corpus callosum, leading to impaired nerve conduction. Flunarizine treatment significantly mitigated these deficits, as evidenced by increased fall-off time and improved locomotor scores. These behavioral improvements may be the result of flunarizine’s calcium channel blocking properties preventing oligodendrocyte apoptosis and this might have preserved axonal conduction and neuromuscular signaling,[ 28 ] thereby enhancing muscle grip strength and coordination.
Approximately, half of people who has MS experience anxiety. Researchers suspect that anxiety in MS results from both the disease process, causing changes within the brain, in combination with social consequences and uncertainties associated with living with MS. The heightened anxiety-like behaviors compared exhibited by disease group animals compared to controls might be a consequence of demyelination and neuroinflammation on prefrontal cortex, amygdala, and hippocampus, areas of the brain involved in emotional regulation.[ 29 ] Flunarizine treatment effectively attenuated these anxiety-like behaviors, indicating its potential anxiolytic action in the context of MS. This could be attributed to its calcium channel blocking activity, which modulates neurotransmitter release and synaptic plasticity,[ 30 ] thus stabilizing mood and behaviour.
Cognitive dysfunction, particularly memory loss is the most common mental change in people with MS. The significant decline in avoidance responses during the shuttle box test is indicative of memory impairment. This is consistent with previous reports demonstrating CPZ-induced demyelination in the hippocampus,[ 31 ] a brain region critically involved in learning and memory processes. The increased number of avoidance responses, in essence, cognitive enhancement in Flunarizine-treated groups, may be attributed to flunarizine’s ability to mitigate oxidative stress and neuroinflammation, both of which are central to MS pathogenesis and cognitive decline.[ 32 ]
These functional improvements produced by flunarizine were further substantiated by the evaluation of biochemical markers in the brain homogenate. Serotonin plays a significant role in learning, memory, mood, and sleep patterns. Recent researches has shown that a decrease in serotonin level may be attributed to neuroinflammation, anxiety, motor impairments and delay in cognitive process.[ 6 ] The significant decline in brain serotonin levels in the animals in the disease group is consistent with previous findings connecting demyelination and neuroinflammation to imbalance in brain serotonin level. This reduction in serotonin likely contributed to the observed anxiety, motor and cognitive impairments in the disease group. Notably, flunarizine treatment significantly restored serotonin levels toward normal, which might be attributed to its antioxidant and anti-inflammatory effects.[ 32 ] This may have protected serotonergic neurons from CPZ-induced oxidative damage, thereby preserving normal serotonin synthesis and release.
Oxidative stress is a key contributor to the pathogenesis of MS and plays a central role in CPZ induced demyelination and neurodegeneration. Elevated markers of oxidative stress in the disease group are likely due to CPZ induced neuronal damage. Treatment with low and high doses of Flunarizine effectively reduced this oxidative stress, as evidenced by a significant reduction in MDA levels and restoration of catalase and GSH levels. This suggests that flunarizine exerts a protective antioxidant effect by scavenging free radicals and likely enhancing the activity of endogenous antioxidant systems.[ 12 ] By preventing oxidative stress, flunarizine may help prevent demyelination and subsequent neuronal loss, further supporting its neuroprotective role in demyelinating conditions such as MS.
Notably, the lower dose of flunarizine caused more evident behavioral improvements than the higher dose. Flunarizine has a long half-life and H₁-histaminergic and dopaminergic (D₂) receptor antagonism, which at higher doses may cause sedation and loss of motor activity. These CNS-depressant actions could counteract the improvement of behavioral performance despite the biochemical or histological neuroprotection, thus explaining the relatively poor functional efficacy of the higher dose.
These findings align with previous reports highlighting flunarizine’s ability to block both N-type (Cav2.2) and T-type (Cav3.1) voltage-gated calcium channels and inhibit mTORC1 signaling, suppressing proinflammatory microglial activation and promoting anti-inflammatory microbial activation, thereby reducing neuroinflammation and promoting remyelination. Flunarizine’s cerebroselective calcium channel blocking, antioxidant, and anti-inflammatory properties collectively contribute to its neuroprotective action in the CPZ-induced demyelination model. These findings suggest its potential as a promising therapeutic candidate in preventing or slowing the progression of demyelinating conditions such as MS.
Conclusions
In the present study, CPZ 0.3% in powdered chow diet for 6 weeks was used to bring about mice model of MS. After 6 weeks, demyelination-associated MS, which was characterized by histopathological, behavioural and biochemical studies.
The CPZ induced motor skill dysfunction, reduction in locomotor activity, elevated anxiety and decline in cognitive abilities were prevented by Flunarizine. It was found that increased levels of serotonin produced by Flunarizine may be the reason for the reduction in the anxiety and memory impairment associated with MS. It was also found that Flunarizine treatment leads to an increase in the catalase and GSH level and decrease in MDA level, indicating the reduction in brain oxidative stress caused by CPZ. Overall, the findings from biochemical estimations suggest the ability of flunarizine dihydrochloride to reverse the neuronal damage caused by CPZ.
Histopathological evaluation of the brain using hematoxylin and eosin stain and LFB stain from different treatment groups showed that flunarizine was capable of attenuating or preserving the demyelination-associated neuronal damages caused by CPZ up to a certain extent.
In summary, flunarizine showed neuroprotective and myelin-preserving properties in a toxin-induced demyelination model. Although these results do not prove the efficacy of flunarizine in MS, they encourage further research into the potential use of this drug as an adjunct therapy for the treatment of demyelination-induced neurodegeneration. As it is evident from the above results, the effect of Flunarizine at high dose (2.4 mg/kg) was found to be less pronounced than low dose (1.2 mg/kg) Flunarizine, presumably due to its long half-life and H1 and D2 blocking property leading to CNS depression. From the present study, Flunarizine dihydrochloride at a low dose was found to be beneficial in ameliorating the symptoms of neurological damage resembling that of MS.
Conflicts of interest
There are no conflicts of interest.
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