Fingolimod attenuates harmaline-induced pas­sive avoidance memory and motor impairments in a rat model of es­sential tremor

Summary

Preclinical data suggest that fingolimod (FTY), a sphingosine-1-phosphate receptor modulator, could be beneficial for treat­­ing com­mon neurological disorders, includ­­ing Parkinson‘s dis­ease, MS and epilepsy. In the cur­rent study, the ef­fects of FTY on harmaline-induced motor and cognitive impairments were studied in male Wistar rats. Pas­sive avoidance memory, exploratory, anxiety-related behaviours, tremor, and motor function were as­ses­sed. The memory impairments observed in harmaline-treated rats were somewhat reversed by administration of FTY. The results showed that FTY could recover step width, left and right step length, but failed to recuperate the mobility duration. FTY improved the time spent in the wire grip and rotarod. Results of our study shed light on the beneficial ef­fects of FTY on cognition and motor function in a model of es­sential tremor (ET) and suggest that sphingosine-1-phosphate receptor modulators have a potential neuroprotective profile for the management of ET.

Key words:

essential tremor – fingolimod – memory – motor function

The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.

The Editorial Board declares that the manu­script met the ICMJE “uniform requirements” for biomedical papers.

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Chinese summary - 摘要

芬戈莫德在基本震颤的大鼠模型中减弱了骆驼因诱导的被动回避记忆和运动损伤

临床前数据表明，芬戈莫德（FTY）是一种鞘氨醇-1-磷酸受体调节剂，可用于治疗常见的神经系统疾病，包括帕金森病，MS和癫痫。 在目前的研究中，在雄性Wistar大鼠中研究了FTY对骆驼因诱导的运动和认知障碍的影响。 评估被动回避记忆，探索性，焦虑相关行为，震颤和运动功能。 通过施用FTY，在harmaline处理的大鼠中观察到的记忆障碍有所逆转。 结果表明，FTY可以恢复步长，左和右的步长长度，但无法恢复活动时间。 FTY减少了线夹和旋转杆所花费的时间。 我们的研究结果揭示了FTY对特发性震颤（ET）模型中认知和运动功能的有益作用，并提示鞘氨醇-1-磷酸受体调节剂具有治疗ET的潜在神经保护特征。

关键词：

特发性震颤 - 芬戈莫德 - 记忆 - 运动功能

Introduction

Es­sential tremor (ET) is defined as a slow progress and mono-symp­tomatic move­ment disorder [1]. With a high prevalence, estimated to be 20 times higher than Parkinson’s dis­ease, ET is known as the most com­mon type of movement disorders [2]. However, despite its high incidence, the etiology of ET is not yet ful­ly understood. Some clinical studies are indicative that along with tremor, patients also suf­fer from cognitive deficit [3– 5]. Thus, an as­sociation may exist between ET and cognitive impairment or dementia [5]. Particularly, the basis of cognitive impairment in ET is still unclear. Furthermore, no pharmacological ther­apy for cognitive impairment of ET patients has been developed [5].

Although the pathophysiologic mecha­nisms of tremor are not clear, many previous experimental studies have provided remarkable insight into the pathogenesis of ET [6]. Several animal models of tremor have been developed for evaluation and find­­ing of therapeutic strategies and pathophysiologic basis of the dis­ease. In this regard, isoproterenol (beta adrenoreceptor agonist), the GABA-A receptor alpha-1 knock-out mouse, and most com­monly the harmaline model have been used by several research groups [7,8]. Furthermore, in addition to caus­­ing several CNS disorders, there is evidence that harmaline plays a crucial role in cognitive disturbances [5]. Besides, many therapeutic agents that improve human tremor also improve harmaline-induced tremor. Thus, this model is supposed to be a useful animal model for as­ses­sment and development of prescribed medications.

Data from several studies suggest that an interaction between the central and peripheral nervous system may lead to tremor [5,9]. However, it is becom­­ing extremely dif­ficult to ignore the existence of a relationship between ET and the abnormality in the Guil­lain-Mol­laret triangle (rubral nucleus, inferior olive nucleus [ION] and cerebel­lum) [10]. Exposure to harmaline has been shown to induce ET via activation of the olive-cerebel­lar system [8]. It is therefore likely that such con­nections may exist between ION dysfunction and ET [11]. Moreover, it has previously been observed that harmaline increases the ION neuron excitability [12]. Thus, decrement of neuronal excitability of ION by any treatment modality can be a therapeutic target in the treatment of ET.

Fingolimod (FTY) is a fungus metabolite derived from myriocin and known as sphingosine-1-phosphate (S1P) receptor agonist [13]. In recent years, we and other researchers have investigated a variety of approaches for FTY and have revealed beneficial ef­fects on treatment of tremor, MS, acute kidney injury, Alzheimer’s dis­ease, and stroke [14– 16]. Evidence suggests that FTY easily cros­ses the blood brain bar­rier and modulation of its receptors expres­sed on the neural cel­ls may exert neuroprotective functions within the CNS [17].

Furthermore, it has been shown that FTY regulates the plasticity and cell survival in the brain through increase in the brain-derived neurotrophic factor and may be useful in the treatment of cognitive deficits related to neuronal disorders [18]. Therefore, the aim of the cur­rent study was to analyze the pos­sible neuroprotective ef­fects of FTY on motor and cognitive deficits induced by harmaline in the experimental model of ET.

Materials and methods

Animals

Male Wistar rats (40– 60- g, one month old) were used in this study. The rats were divided into five groups of saline, dimethyl sulfoxide (DMSO), harmaline, harmaline + DMSO and FTY + harmaline (N = 10 for each group). All the procedures in this experiment were car­ried out accord­­ing to the Neuroscience Research Center of Kerman Medical University (Ethics Code: KNRC/ REC/ 96/ 1456). All of the animals were maintained on a 12-h light-dark cycle with food and water ad libitum.

Design of the study

Harmaline hydrochloride dihydrate and FTY were purchased from Sigma-Aldrich (Munich, Germany) and dis­solved in nor­mal saline and DMSO, resp. The FTY was administered (1 mg/ kg, intraperitoneal­ly [i. p.]) 24 h before harmaline injection (30 mg/ kg, i. p.). Saline was considered as control (vehicle) for harmaline and DMSO for FTY. Thirty min after administration of harmaline, behavioural tests, includ­­ing open field, wire grip (three trials 180-s with 5-min inter-trial interval), rotarod (three trials 300-s cut off and inter-trial intervals of 5 min) and foot print were car­ried out with adequate amount of rest between each trial and lasted for 3 h in total. Three hours after the end of these behavioural tests, shuttle box test was performed.

Behavioural studies

Observation

An observer who was blinded to the groups under the study rated the tremors. Thirty min after administration of harmaline, dur­­ing the open field test, tremor data were quantitatively scored us­­ing a four-point scale and grad­­ing neurological deficit score test. The normal motor function or no observable tremor was represented by score of 0, occasional tremor af­fect­­ing only the head and neck was considered as a score of 1, intermittent (occasional tremor af­fect­­ing all body parts) was represented by score of 2, persistent (persistent tremor af­fect­­ing all body parts and tail) was scored 3, and severe (persistent tremor render­­ing the animal unable to stand and/ or walk) was considered as a score of 4 [19].

Open-field test

The apparatus of open field consisted of an arena made of opaque Plexiglas (90 ×90 × 45 [H] cm). The arena floor was divided into 16 small squares to define the central and peripheral sections. The rats were placed in the centre of the arena and their behaviour was recorded dur­­ing a 5-min interval by an automated video track­­ing system (Ethovision [Noldus Technology, Wageningen, the Netherlands]). The total time spent in the centre or periphery and the total distance moved were recorded. Between the ses­sions, the chamber was cleaned with 70% ethanol [5].

Rotarod test

An accelerat­­ing rotarod was used to eva­luate the motor coordination and balance performance. Prior to experiment, all animals were trained for the test. The experiment started with a speed of 10 rpm and the rod linearly accelerated to 60 rpm. Each rat undertook three trials with inter-trial interval of 30 min. The maximum cut off time of each trial was 300 s. The total duration of each rat remain­­ing on the rod (fall latency) was measured for evaluation of balance performance [20,21].

Wire grip test

We used the wire grip test to check the muscle strength and balance. Each rat suspended with the forepaws on a hori­zontal steel wire (80 cm long, diameter 6 m­m) and held in vertical position while it grasped the wire. Each rat was given three trials with a 30-min inter-trial rest interval. For each animal, the latency to fall was recorded with a stopwatch [22].

Gait analysis test (Footprint)

This test as­ses­ses the animal’s walk­­ing patterns and gait kinematics. To perform this experiment, the hind paws of each rat were stained with non-toxic inks and the animals were al­lowed to transverse freely through a plexiglass tun­nel (100 [length] × 10 [width] × 10 [height] cm) end­­ing in a darkened cage. The floor of the tun­nel was lined with a fresh sheet of white absorbent paper at each final test. The hind paw stride lengths were measured by the distance (cm) between the respective paw prints to the centre of the ipsilateral print. The hind paw stride widths were measured through the distance between the centres of the respective paw prints to the cor­respond­­ing contralateral stride length measurements at a right angle. Footprints at the begin­n­­ing and at the end of each run were not measured [23].

Pas­sive avoidance test

This test, as a fear-aggravated test, is used for evaluat­­ing learn­­ing and memory in rodents. The shuttle box device with dimensions of 100 [length] × 25 [width] × 25 [height] cm and consist­­ing of two compartments (light and dark) separated by a door was used. In this experiment, the animal learns to avoid an environment in which a prior aversive stimulus has been delivered. In the learn­­ing phase of the test, the animals were familiarized with the test by putt­­ing them in the light chamber (door closed) for 5 min. The next day, each rat was returned to the light compartment, the door opened and the animal was al­lowed to move to the dark chamber before the door was closed. This phase was repeated once and if an animal failed to move into the dark compartment, it was removed from the study. One hour later, each animal was placed in the light compartment, the door opened, and on enter­­ing the dark compartment, was given an electric shock (0.5 mA, 2 s; via wires embedded in the dark chamber floor) [24,25]. This part of the process was repeated up to 5× at 1-h intervals until the animal learned to avoid the dark compartment (remain­­ing in light compartment for at least 300 s) and the number of shocks required for learn­­ing was recorded. Twenty-four hours after the learn­­ing phase, the as­ses­sment phase of the test was undertaken. The animal was placed in the light chamber (door opened) and the time until the animal entered the dark compartment recorded as the step-through latency (STL). The total time spent in the dark compartment (TDC) dur­­ing a period of 5 min was also recorded [5].

Statistical analysis

Graph Pad Prism 6 (Graph Pad Software, La Jol­la, CA, USA) was used for statistical analysis of data and figure production. All data were first as­ses­sed for normality us­­ing a Kolmogorov--Smirnov test. The normal­ly distributed data were then analyzed us­­ing one-way ANOVA test. Where a main ef­fect was seen in ANOVA tests, pairwise comparisons between groups were then made us­­ing Tukey’s post hoc analysis. Data that were not normal­ly distributed were analyzed us­­ing Kruskal-Wal­lis test. All data were expres­sed as median and interquartile range and p < 0.05 was considered sta­tistical­ly significant.

Results

The ef­fect of FTY on tremor and gait disorder

The results obtained from the tremor scale score showed an increase in tremor in the harmaline group compared to the saline group (p < 0.05). Although the score of FTY group showed a significant increment compared to the saline group, it is apparent that FTY has improved the tremor scale score compared to the harmaline group (p < 0.05) (Fig. 1A). Moreover, the hind paw stride width, an indication for gait, was also higher in the harmaline group, as compared to the saline group (p < 0.05) (Fig. 1B). In addition, the FTY recovered the gait in this group compared to saline group (p < 0.05). However, FTY did not completely improve and recover the gait back to the control levels (Fig. 1B). The saline and DMSO groups showed the same levels for tremor scale score and gait.

These data showed that treatment with harmaline induced severe tremor as­sociated with significant functional deficits. Furthermore, a main ef­fect for treatment with harmaline and/ or FTY upon left (Fig. 1C) and right (Fig. 1D) step lengths was not detected.

The ef­fect of FTY on explorative and anxiety-like behaviours

Harmaline and harmaline + DMSO groups showed a significant decrease in total distance moved as compared to the control group (p < 0.05) (Fig. 2A). No significant dif­ference was observed among FTY, saline and DMSO groups (p > 0.05) (Fig. 2A). In Fig. 2A, B, there was a clear and significant trend of decrease in velocity (Fig. 2B) and mobility (Fig. 2C) upon treatment with harmaline and/ or FTY. The results indicated that the rats in FTY+ harmaline and harmaline plus DMSO groups showed a decrease in velocity (Fig. 2B), mobility (Fig. 2C) compared to the saline and DMSO groups (p < 0.05) indicat­­ing that FTY treatment did not have any ef­fects on harmaline-induced disturbances in velocity and mobility (p > 0.05, Fig. 2B, C). No significant dif­ference in duration of stay­­ing in the centre and peripheral zones was observed which is an indirect indicator of anxiolytic-like ef­fects and implies that there is no impaired anxiety-like behaviour at least in the open field test (data have not shown).

The ef­fect of FTY on muscle strength and balance function

In order to as­sess the muscle strength and balance function, we performed a wire grip test. In three consecutive trials, the animals of the harmaline and harmaline + DMSO groups showed a decrease in fal­l­­ing time and the time taken on the rod as compared to the control group (p < 0.05) (Fig. 3A). However, FTY treatment did not show any recovery impact for either parameters. Rats of the FTY and harmaline groups spent a shorter duration on the wire grip as compared to the saline and DMSO groups (p < 0.05). Moreover, no significant dif­ferences were observed among FTY, harmaline, and harmaline + DMSO groups (p > 0.05) (Fig. 3A).

In addition, the balance function was evaluated by averag­­ing the three repeated trials of the time stayed on rod; the result indicated that harmaline significantly (p < 0.05) decreased the time on the rod when compared with the saline group, but FTY failed to increase the time that animals spent on the rod. No significant dif­ference was observed between the FTY and harmaline and harmaline + DMSO groups (Fig. 3B).

The ef­fect of harmaline and FTY on pas­sive avoidance

In this test, the number of shocks that animals received for the learn­­ing phase evaluated the ef­fects of treatment with FTY. In Fig. 4A, it can be seen that the harmaline and harmaline + DMSO groups required significantly more shocks to learn the task (p < 0.05 vs. saline). However, further analysis showed that FTY treatment improved the learn­­ing impairment induced by harmaline (p < 0.05) (Fig. 4A). In addition, 24 h after learning, the ef­fect of treatment on the step through latency, an index for memory retrieval, was evaluated. The result revealed that this measure was significantly decreased in both harmaline and harmaline + DMSO groups (p < 0.05) (Fig. 4B) compared with the saline groups. Treatment with FTY not only improved learning, but also partial­ly improved the STL time to the level of saline group. However, no significant dif­ference was observed between the harmaline + FTY and harmaline and harmaline + DMSO groups. This result indicates that FTY treatment was able to partial­ly improve the harmaline-induced reduction in the step through latency. Moreover, we evaluated the total time spent in the dark compartment. The rats in the harmaline group spent more time in the dark chamber compared with the saline group, while pretreatment with FTY non-significantly decreased this parameter compared with the harmaline group.

Discus­sion

The present study demonstrated that pretreatment with FTY improved learning, memory deficit, and tremor status in the rat’s model of tremor induced by harmaline, as evaluated by several behavioural tasks. However, there were no significant improvements in muscle strength, balance function, explorative, and anxiety-like behaviour.

Harmaline induces action dependent tremor, and is wel­l-noted as an easy animal model for seek­­ing and evaluat­­ing new ther­apies for ET [26– 28]. After injection of harmaline, tremor developed im­mediately. Consistent with this notion, we showed that harmaline induced tremor, which was seen as marked deficits in the performance of all behavioural tasks employed. One pos­sible explanation for harmaline induced tremor might be that it synchronizes the electrical activity in the ION [29]. Moreover, evidence shows that the harmaline causes an increase in excitability by open­­ing calcium chan­nels and subsequently bring­­ing about tremor [30]. Many studies have shown that the use of T-type calcium chan­nel blockers in treat­­ing neurological dis­eases in animal models is promising [31]. Recent studies have demonstrated that pulses of T-type calcium in the ION increased by harmaline and T-type calcium chan­nel blockers might be of therapeutic potential for the treatment of tremor [32].

In the cur­rent study, we examined the ef­fect of FTY pretreatment upon harmaline-induced tremor and its symp­toms. FTY al­leviated some symp­toms as­sociated with ET in a potential­ly relevant animal model. The findings of our previous study provided the pharmacological evidence for the specific involvement of S1P receptors in mediat­­ing motor deficits induced by harmaline and demonstrated that pretreatment with FTY one hour before harmaline injection could exert protective impacts on impaired locomotion and balance in harmaline--exposed animals [33]. In line with these findings, in the cur­rent experiment, FTY significantly decreased the harmaline--induced tremor score, show­­ing helpful ef­fects on the behavioural disturbance exhibited. There are no precise putative mechanisms to explain how FTY exhibits such ameliorative ef­fects, but it may partly be explained by the fact that FTY directly inhibits the atrioventricular conduction [34]. It is also pos­sible that FTY inhibits fir­­ing activity at ION and modulates tremor and further research should be ensued to investigate the direct ef­fects of FTY on fir­­ing properties of the neurons of the ION.

In addition, we and other researchers have shown that harmaline impaired the muscle strength and balance function [26,35]. Although FTY showed a beneficial ef­fect on tremor, it failed to improve the muscle strength and balance function in the harmaline treated rats. A find­­ing that could be explained by the fact that impairment of muscle strength and balance function may be related to peripheral deficit in muscles rather than the CNS activity. This claim can also be supported by the results of explorative and anxiety-like behaviours where FTY failed to recover these responses. In addition, since harmaline acts as weak or partial agonist of benzodiazepine receptors [36], it has been suggested that its actions may in part be mediated by the benzodiazepine receptors which have a crucial role in the regulation of anxiety [37]. However, our results delineate that FTY might not interact with benzodiazepine receptors to les­sen anxious-related behaviours.

In the pas­sive avoidance test, it was shown that harmaline impaired the learn­­ing and memory performance as indicated by increased number of received shocks and STL time. It has been suggested that harmaline inactivates gastrointestinal monoamine oxidase and al­lows the first-pass metabolism of dimethyltryptamine access to systemic circulation and the CNS system where dimethyltryptamine acts as an injurious insult [38]. However, FTY improved the STL and memory performance in harmaline treated animals. It has been suggested that administration of FTY improves learn­­ing and memory in the rat’s model of Alzheimer’s dis­ease [39]. The FTY may also act directly via S1P receptors and thereafter activates endogenous neuroprotective mechanisms [40]. Furthermore, it has been shown that FTY ameliorates neural deficit and synaptic plasticity impairment in stroke, independent from its im­munosuppres­sive ef­fects [16]. Moreover, recent evidence has demonstrated that FTY increases the production of neurotrophic factors such as brain-derived neurotrophic factor and mediates neuroprotective functions [41]. In conclusion, FTY appears to act as a neuroprotective factor to improve learn­­ing and memory impairment induced by harmaline and more investigations are needed to evaluate the mechanism of therapeutic ef­fects of FTY on motor and cognitive impairments.

Fund­­ing for this study was provided by Kerman University of Medical Sciences as a grant for the PhD research of Narjes Dahmardeh.

Accepted for review: 10. 5. 2018

Accepted for print: 31. 10. 2018

Mohammad Shabani, PhD

Kerman Neuroscience Research Center

Neuropharmacology Institute

Kerman University of Medical Sciences

Jehad Blvd

Ebn Sina Avenue

76198-13159 Kerman, Iran

e-mail: shabani@kmu.ac.ir