Protective effect of crocin on cuprizone-induced model of multiple sclerosis in mice

Crocin is the main bioactive components of the saffron which has positive role in the nervous system; however, its neuroprotective activity is not fully elicited. So, the aim of the current study was to determine effects of the crocin on reflexive motor and anti-depressive behaviors as well as serum and brain tissue antioxidant activities in cuprizone-induced (CPZ) model of multiple sclerosis (MS) mice. Forty male C57BL/6 mice were randomly assigned into 4 groups. Mice in the control group were received normal diet. In group 2, mice received normal diet and orally received crocin (100 mg/kg) 3 times per week for 5 weeks. In group 3, CPZ-induced demyelination was done by chew palate containing 0.2% (w/w) CPZ for 5 weeks. In group 4, mice feed CPZ containing diet and orally received crocin (100 mg/kg) three times per for 5 weeks. After determination of the MS signs, reflexive motor behavior and depressive tests were done. Also, serum and brain tissue antioxidant activity was determined. According to the data, CPZ had negative effects on hind-limb foot angle, hind- and front-limb suspension, surface righting, grip strength, and negative geotaxis while crocin improved it. Co-administration of the CPZ + crocin reversed effect of the CPZ on the reflexive motor behaviors. CPZ increased immobility time in the forced swimming test (FST) and tail suspension test (TST), while co-administration of the CPZ + crocin reversed effect of the CPZ on immobility time. CPZ decreased number of cross in open field test (OFT) and spending time on rotarod, while co-administration of the CPZ + crocin reversed effect of the CPZ. Malondialdehyde (MDA) production increased, and glutathione peroxidase (GPx), superoxide dismutase (SOD), and total antioxidant status (TAS) levels decreased in serum and brain tissue of the mice treated with CPZ. Pretreatment with crocin decreased adverse effect of the CPZ on serum and brain tissue antioxidants. These results suggested crocin has protective effect against CPZ-induced MS in mice.


Introduction
Multiple sclerosis (MS) is a chronic autoimmune disease which leads to demyelination in central nervous system (CNS) (Fathimoghadam et al. 2019). The mechanisms of demyelinating are not fully determined but mitochondrial dysfunction and inflammatory disorders lead to loss of myelin sheath and nerve conduction deficits (Stadelmann et al. 2019). This condition affects physical, mental, cognitive, and motor activity (Haider et al. 2016). Inflammatory factors and the oxidative stress caused by these free radicals, such as reactive oxygen species (ROS), play a significant role in pathophysiology of the MS. The imbalance between production and distribution of the antioxidant enzymes such as SOD, GPx, CAT, and glutathione or disrupts their function are important due to high sensitivity of the CNS to oxidative stress (Hemmati et al. 2019). Cells, by employing antioxidant machinery, protect themselves against ROS-induced damage (Yánez-Ortiz et al. 2021). In recent years, many natural and synthetic drugs are used to decrease oxidative stress and neurodegenerative mechanisms underlying MS. Even though these medications are effective in preventing immune cell-driven inflammation and decreasing the relapse rate, they are ineffective in subsequent neurodegenerative processes (Zabihi et al. 2017).
Crocus sativus L. (Iridaceae) is commonly known as saffron and widely cultivated in Iran. Saffron is a water-soluble β-carotene with high antioxidant activity (Shokrpour 2019). It has anticancer, anticonvulsant, anti-inflammatory, and antitumor properties (Fernández-Albarral et al. 2020). Crocin has a neural protective role and prescribed for CNS impairments (Shokrpour 2019). Crocin improves memory deficit induced by restraint stress (Dastgerdi et al. 2017), and crocin (60 mg/kg) has protective effect against spatial and cognitive memory impairments (Khani et al. 2018). There are available documents on antioxidant role of crocin in brain following neurodamaging and inflammation, but only a handful of studies are in the case of MS. Beneficial effects of the crocin on cellular antioxidant status in MS were reported. Fathimoghadam et al. (2019) reported orally gavage of crocin (100 mg/ kg) improved MDA, SOD, and GPx levels in rat model of demyelination with ethidium bromide. Peripheral administration of crocin (30 mg/kg) for 21 days improved MDA, SOD, and GPx levels following chronic stress-induced oxidative stress in rat brain (Bandegi et al. 2014). Saffron extracts have protective effects against genotoxins-induced oxidative stress in mice (Bandegi et al. 2014).
Aqueous extract of saffron increased time spends on the open arms of the maze without anxiolytic, hypnotic, or myorelaxation effects; however, adverse results was observed at higher levels (300-550 mg/kg) (Hosseinzadeh and Noraei 2009). Pre-treatment with crocin decreased MDA and nitric oxide levels and increased SOD and nuclear factor-kB in hippocampus of the mice with Parkinson's disease (Rajaei et al. 2016;Mazumder et al. 2017). CPZ models were very useful for pathophysiology of the MS (Torre-Fuentes et al. 2020). Battery reflexes are useful technique to assess neurodevelopmental or neurodegenerative disorders. This method included limb grasping and placing, cliff avoidance, righting, accelerated righting, gait, auditory startle, and posture (Nguyen et al. 2017). Effects of the crocin on locomotion are dose dependent which at low levels (100-200 mg/kg) improves locomotor function and mechanical behavior but higher dose (400 mg/kg) suppressed locomotor activity in the rat model of contused spinal cord injury (Karami et al. 2013).
Several methods were introduced for a model of demyelination in which CPZ-containing diet for 4-6 weeks is preferred by many researchers (Torre-Fuentes et al. 2020). This leads to oligodendrocytes damage, followed by microglia and astroglia activation, which disrupts energy metabolism in the mitochondria. For this reason, C57BL/6 mice are suggested (Burrows et al. 2019). Due to the lake of clear way to treat this disorder, and regarding to positive effects of crocin on reflexive motor behavior in MS, we investigated the protective effect of crocin on CPZ-induced model of MS in C57BL/6 mice.

Animals and study protocol
Male C57BL/6 mice (4-6 weeks old; weighing 19 ± 2 g) were purchased from Pasture Institute (Tehran, Iran). The mice were kept in standard plastic cages at laboratory conditions (temperature of 22 ± 2 °C and 12/h light/dark cycle). Food and tap water were available ad libitum. One week after acclimatization, mice randomly allocated into 4 experimental groups (n = 10). The mice in the control group were treated with normal diet. In group 2, normal diet was provided and mice orally gavage with crocin (100 mg/kg) (> 98.0% purity, Sigma Chemical Co., St Louis, MO, USA) 3 times per week for 5 weeks. In group 3, acute demyelination was induced by feeding mice with 0.2% (w/w) CPZ (Sigma Aldrich, St. Louis, MO, USA) mixed with ground chow for 5 weeks (Zhu et al. 2021). In group 4, mice received diet containing CPZ (0.2% w/w) for 5 weeks and during this time orally received crocin (100 mg/kg) three times per week. MS corresponding animal model, experimental autoimmune encephalomyelitis (EAE), is widely used to understand disease pathogenesis and test novel therapeutic agents. These defects are quantified using a standard EAE scoring system on a 0-5 disease severity scale: 0, no disease; 1, loss of tail tone; 2, hind limb weakness; 3, hind limb paralysis; 4, hind limb paralysis and forelimb paralysis or weakness; and 5, moribund/death (Shahi et al. 2019). At the end of the study, reflexive motor behavior and depressive tests were done (Fig. 1).

Ambulation
Crawling behavior is used to test the walking ability of the mice following MS signs (Williams and Scott 1954). Mice were placed in a transparent enclosure, where they were visible from the top and all 4 sides. To motivate mice to walk, we used a gentle tail prod. The ambulation score was 0 = no movement, 1 = crawling with asymmetric limb movement, 2 = slow crawling but symmetric limb movement, and 3 = fast crawling/ walking. To eliminate alterations in test due to learning, the test was performed in triplicate within 3 min (Feather-Schussler and Ferguson 2016).

Hind-limb foot angle
Following the signs of the MS, hind limb posture changes wherein walking the hind limbs is positioned under the body. Therefore, angle among the hind-limbs in walking position was less than crawling position (Feather-Schussler and Ferguson 2016). A plain open field box equipped with a camera above on was used to record the mice movements around the box. The foot angle was determined by recorded videos. In recorded videos, a line was drawn from the end of the heel/shin to the tip of the toe. The measurements recorded only in the mice that were performing a full stride in a straight line, and their feet were flat on the ground. To minimize experimental errors, average of the 3-5 sets of the foot angles was calculated for 1 3 each animal. No repeat related learning was observed in this test (Feather-Schussler and Ferguson 2016).

Front-limb suspension
This test was conducted on mice to hang onto the wire with both forepaws. After grasping the wire, mice were released and the time needed to fall was recorded in seconds. To minimize alterations in testing due to learning, the test was performed in triplicate within 3 min (Feather-Schussler and Ferguson 2016).

Hind-limb suspension
The hind-limb suspension test was performed to determine mice strength and neuromuscular function. They were placed face down into the standard 50-mL conical laboratory tube. The mice's hind legs hung over the wire and were released, and hind-limb posture score was recorded as score 0: constant clasping of the hind-limb by holding onto the tube; score 1: weakness was apparent and the hind-limb were almost in a clasped position with the tail raised; score 2: hind-limb were close to each other and often touching; score 3: weakness was apparent, closer together and rarely touched each other; score: 4 normal hind limb separation with tail raised (Feather-Schussler and Ferguson 2016). To minimize alterations in testing due to learning, the test was carried out in triplicate within 3 min (Feather-Schussler and Ferguson 2016).

Surface righting
The surface righting reflex is a motor ability of the mice to flip onto their feet from their supine position (Heyser 2004). This can be measured by surface righting test. This test was performed on a cotton sheet and kept in position for 5 s. Then, they were released, and time needed to return to their prone position was recorded. As no repeat related learning reported in this test, triplicate within 3 min was done (Feather-Schussler and Ferguson 2016).

Grip strength
The test was conducted to determine the grip strength in which animals can grab onto a screen and generate a reading of the grip force. In summary, a 16 × 18 fiberglass screen was used in which the surface is rotated slowly from a horizontal to the vertical position, to challenge the grasping of all four limbs (Corti 2017). The hanging impulse which indicated the force needed to resist gravity was calculated per below formula: (Venerosi et al. 2009).

Negative geotaxis
Mice were placed downward on a 45° wooden surface which is used as a slope. Then, they were released and time needed by weight(g) × latencytofall(s) Fig. 1 Flow chart of study procedure mice to face the slope upward due to vestibular cues of gravity was documented (Feather-Schussler & Ferguson 2016).

FST
The forced swimming test (FST) was done according to a previously reported method (Nasehi et al. 2019). Mice were individually plunged in ht: 25 cm; diameter: 15 cm) containing 10 cm of water to a glass cylindrical container at 25 °C. Each mouse was left in the cylinder for 6 min. When mouse ceased struggling and remained floating motionless in the water, the total duration of immobility during the last 4 min of the 6 min testing period was measured.

TST
The tail suspension test (TST) is known as common techniques for assessing antidepressant-like activity in mice (Cryan et al. 2005). The TST was performed based on the method described by Steru et al. (1985) and Alimohammadi et al. (2019). Briefly, the mice were away from nearest objects and were both acoustically and visually isolated from observing or interacting each other. Then, mice were suspended 50 cm above the floor by adhesive tape placed approximately 1 cm from the extremity of the tail. Immobility time was monitored during a 6-min period.

OFT
The open field test (OFT) was used to determine the possible effects of crocin on the locomotor and exploratory activities in mice. The test was done using a 45-× 45-× 30-cm 3 wooden box. The floor of the open field box was divided by masking tape markers into 9 squares. Each animal was placed individually at the center of the apparatus. Then, the number of segments crossed with the four paws was recorded for a period of 6 min (Donato et al. 2014).

Rotarod test
The accelerated rotarod test is a standard sensory-motor test to investigate animals' motor coordination and learning skills through measuring the ability of the mice to stay and run on the accelerated rod. The test was done for 8 min with an acceleration of 0-20 rpm. When mice were fell off the rod or started to rotate with the rotarod without running, the time was recorded. After an initial training trial, mice were tested for 5 trials over 2 days. The recovery phase between trials was 10 min (Eltokhi et al. 2021).

Antioxidant activity
At the end of the tests, blood samples were collected via cardiac puncture and serums were obtained and stored at − 80 °C for further analysis. Also, animals were sacrificed and brain cortex was removed and homogenized in a solution of 0.32 mol/l sucrose, 1 mmol/l EDTA, 10 nmol/l Tris-HCl, pH 7.4, respectively. Subsequently, the tissues were centrifuged at 13,600 g for 30 min. The supernatants were collected and stored at − 80 °C for further analysis. Protein concentration in various samples was determined based on the method of Bradford (1976) with bovine serum albumin as a standard. The assessed oxidative stress biomarkers included MDA, SOD, GPx, and TAS were determined using Zell Bio GmbH (Germany) assay kits.

Statistical analysis
Obtained data were analyzed by one-way analysis of variance (ANOVA) and presented as the mean ± standard error (SE). For treatments having differences, mean values were compared with the Tukey HSD test. P < 0.05 were considered to indicate significant differences among the treatments.

Discussion
To our knowledge, this is the first report on effects of crocin on reflexive motor behavior following excremental MS mice. As seen, crocin had positive effects on hind-limb foot angle, hind-and front-limb suspension, surface righting, grip strength, and negative geotaxis, while CPZ had adverse effect. Feeding of the CPZ for 4-6 weeks leads to oligondendrocytes damage followed by microglia and astroglia activation (Torre-Fuentes et al. 2020). CPZ-induced oligondendroglia death acts via disrupting the energy metabolism in the mitochondria. It is reported that solid lipid nanoparticles loaded with dimethyl fumarate improved deficit score, grasping ability, forelimb strength, and motor function in CPZ model of rats (Ojha 2018) and our results were in agreement to this report. Mice exposed to 0.2% CPZ for 4 to 6 weeks had impaired sensorimotor gating and less social interaction CPZ-exposed mice spent more time in the open arms of an elevated plus maze and exhibited spatial working memory impairment (Xu et al. 2009). Although myelin damage frequently occurs in the white matter of the MS patients, evidence suggest that demyelination is also taking place in grey matter. However, the pathophysiology of increasing anxiety in MS patients is still unclear. Previous studies reported that anxiety behavior in MS patients is associated with demyelination and inflammation in CNS structures such as spinal cord and hippocampus (Zhu et al. 2021). (Burrows et al. 2019).
In this study, co-administration of the CPZ + crocin significantly decreased adverse effect of the CPZ on the reflexive motor behavior tests. CPZ increased immobility time in the FST, TST, and crocin reversed it. Co-administration of the CPZ + crocin reversed effect of the CPZ on immobility time. CPZ decreased number of cross in OFT and spending time on rotarod and CPZ + crocin reversed effect of the CPZ. Crocin (12.5, 25, and 50 mg/kg) decreased immobility time in FST and TST, and our finding was in agreement to this report. Sohrabi Asadabad et al. (2017) reported crocin treatment improved motor activity in open field and rotarod tests in doxorubicin-induced memory impairment. Crocin with bioactive properties has both anti-inflammatory and anti-oxidant activities. It attenuates lipopolysaccharideinduced anxiety and depressive-like behaviors via suppressing NF-kB and NLRP3 signaling pathway (Zhang et al. 2018). It is reported that acute treatment with crocin (40 mg/ kg) produced antidepressant-like effect in FST without affecting the baseline locomotion in mice (Amin et al. 2015).
Immobility time in FST resembles a state of despair and mental depression and was decreased by intraperitoneal crocin in acute study. It has been reported that neurochemical pathways involved in mediating performance in FST and TST are not exactly the same (Chatterjee et al. 2012). It has been suggested that stress may provoke a modification in dopamine release in different brain areas and that the FST, in its own accord as a stressor, may be responsible for this modification. Monoamine metabolism changes following the mouse forced swimming test but not the tail suspension test (Renard et al. 2003).
Employing a battery of tests allows us to explore the effects of the drug on different behavioral tasks to achieve complementary and/or converging information on the antidepressant activities of drugs (Amin et al. 2015). The FST and TST do not reproduce the pathophysiology of depression but they are useful in that they induce changes that are sensitive to therapeutic agents in a manner predictive of their effects in humans. The FST and TST have been used extensively for this purpose, but the selectivity of these tests for monoamine-based mechanisms may limit their ability to detect novel mechanisms (Chatterjee et al. 2012). C57BL/6 mice treated with CPZ-containing diet had lower monoamine oxidase and dopamine β-hydroxylase activities in the hippocampus and prefrontal cortex (Xu et al. 2009). CPZ exposure C57BL/6 mice for 3 weeks had greater demyelination and oligodendrocyte loss, and after 6 weeks, activated astrocytes and microglia were detected in the demyelinated area due to monoamine oxidase activity as an oxidative enzyme (Xu et al. 2009).
According to our findings, CPZ increased MDA and decreased GPx, SOD, and TAS and these effects reversed by crocin. The oxidative stress plays important role in the pathogenesis of MS. The lowest antioxidant activity and highest neuro-inflammation is the main aspects in MS (Ladan Moghadam 2016). Free radicals can damage many types of nerve cells and have the strongest effect on oligodendrocytes and neurons. In addition, the sites with the most extensive oxidative damage in MS are observed in the myelin sheath and oligodendrocytes due to their weak antioxidant defense systems, high endogenous metabolic activity rates, and low intrinsic antioxidant enzyme levels (Zha et al. 2022). Microglia and astrocytes are vulnerable to the ROS due to their low antioxidant capacity. So, increased ROS leads to demyelination. SOD is first line indicator for demyelination in the MS disease (Vodjgani et al. 2021). CPZ by inhibiting SOD activity leads to mega mitochondria formation, perikaryon, and myelin sheath degeneration (Khalilian et al. 2021). Also, GPx acts as a donor to eliminate ROS and inhibiting oxidative damage to neurons and oligodendrocytes (Vodjgani et al. 2021). Therefore, antioxidants and anti-inflammatory agents have valuable in the prevention and treatment of the MS (Asadi et al. 2020). As observed, co-administration of the CPZ + crocin reversed adverse effect of the CPZ on serum antioxidants. Asadi et al. (2015) reported crocin administration inhibits β amyloid induced apoptosis by antioxidant properties which acts as a potential neuroprotective pharmaceutical agent for management of Alzheimer's disease. Crocin decrease nitric oxide release from microglia triggered by interferon gamma and β amyloid (Azari et al. 2018). Saffron, with its active compounds, may help with neurodegenerative diseases such as MS through attenuation of oxidative damage and inflammation (Ghiasian et al. 2019). Oral administration of the crocin increases levels of the crocin in the plasma and brain of the rat. Also, crocin inhibits ROS-induced oxidative stress in stroke-prone spontaneously hypertensive rats (Yoshino et al. 2011). Crocin has higher anti-inflammatory and antioxidant activity compared to other bioactive components of the saffron (Amin et al. 2015). Ethanoic extract of saffron enhances antioxidant status in the hippocampus of experimental models of MS (Ghaffari et al. 2015). Crocin (30 mg/day) decreases lipid peroxidation and increases total thiol groups and total antioxidant capacity in patients with MS (Ahmadi et al. 2020). Crocin decreases release of proinflammatory cytokines, microglial activation, and cellular apoptosis in mice with traumatic brain injury (Ghiasian et al. 2019). Thus, administration of the natural products improves serum and brain antioxidant levels which inhibits free radical and CPZ-induced nerve injury (Zha et al. 2022). It is important to note that despite CPZ-induced demyelination model has been widely tested and exhibits demyelination behaviors similar to those observed in patients with MS, but MS and the artificial model of MS are not the same. Thus, it needs to be pointed out that findings in mice do not always translate in to the same findings in MS. Despite the direct mechanism for how crocin improves antioxidant activity during MS, but it seems crocin increases the expression of brainderived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) in the rat hippocampus (Lu et al. 2020). Crocin activates extracellular regulated protein kinase signaling pathways via upregulating endogenous pituitary adenylate cyclase-activating polypeptide, then enhances synaptic plasticity and improves neuron survival which plays an antidepressant role in the mice (Lu et al. 2020). Pretreatment with crocin impresses neuroprotective effects by decreased microglial activation, pro-inflammatory cytokine release, and apoptosis in traumatic brain damage (Wang et al. 2015).
In conclusion, these results suggested crocin has protective effect against on CPZ-induced MS in mice. Crocin ameliorates adverse effect of the CPZ on antioxidant activity in the brain tissue of the mice. Based on limitations of the current study, however, we were not able to determine immunohistochemically staining of the brain section to demonstrate demyelination and re-myelination and pro-inflammatory cytokines level. Also, we were not able to determine cellular mechanism for observed results. Perhaps, some portions of the observed positive effects of the crocin on reflexive motor behavior and depressives test are related to its antioxidant properties. Further researches suggested to determine possible cellular and molecular pathways for observed results.