Abstract

Introduction

Dementia is an organic brain disorder clinically characterized by the development of multiple cognitive defects that are severe enough to interfere with daily social and professional functioning.[1] Alzheimer's disease (AD) is the most common cause of dementia in the elderly as according to World Health Organization, 5% of men and 6% of women aged above 60 years suffer from dementia of AD worldwide.[1] AD is basically a neurodegenerative disorder characterized by the progressive accumulation of amyloid beta peptide, neurofibrillary tangles, and hyperphosphorylated microtubule-associated tau protein. Today, there is no cure for this devastating disease and therefore it is of great interest for researchers to find novel drugs that can arrest the disease process. Drugs currently available in the market include inhibitors of acetyl cholinesterase (AChE) and N-methyl D-aspartate-receptor antagonists. These drugs improve the function of yet intact neurons, but do not inhibit the ongoing degenerative process leading to neuronal cell death. Scientists are currently exploring various targets/processes/agents which may provide relief and also stop the progression of dementia.

Riluzole, a 2-amino-6-(trifluoromethoxy) benzothiazole, is a well-known inhibitor of glutamatergic neurotransmission[2] and clinically important for its use in Amyotrophic Lateral Sclerosis. Riluzole is also known to modulate TWIK-Related K⁺ (TREK) channels, the two-pore potassium (K⁺) channel.[3] It is reported to exert potent anti-glutamate,[4] anticonvulsant,[5] anxiolytic,[5] anesthetic,[6] and anti-oxidative[7] actions. It has been observed to exert neuroprotective effect and prevent memory loss and hippocampal neuronal damage in ischemic gerbils.[8] It has also been shown to attenuate cognitive and neuromotor dysfunction associated with brain trauma in rats.[9] Although above reports clearly indicate the potential of riluzole in memory deficits associated with ischemic brain injury, little has been done to explore the memory preserving efficacy of riluzole in animal models of dementia. Furthermore, possible mechanism of action of riluzole in memory deficits also remains to be established. Therefore, the present study has been undertaken to investigate the ameliorative effect and possible mechanism of riluzole in memory deficit in mice.

Materials and Methods

Laboratory Models

Collection of Samples

Blood samples were taken by retro orbital puncture, the animals were then sacrificed by cervical dislocation; brains were removed and homogenized in phosphate buffer (pH = 7.4). The homogenates were than centrifuged at 3 000 rpm for 15 minutes. The supernatant of homogenate was used for biochemical estimations (i.e., brain AChE, thiobarbituric acid reactive species [TBARS], and GSH).

Estimation of Brain Acetyl Cholinesterase Activity

The whole brain AChE activity was measured by the method of Ellman et al.[14] with slight modifications. Change in absorbance per min. of the sample was read spectrophotometrically (DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA) at 420 nm.

Estimation of Brain Thiobarbituric Acid Reactive Species Level

The whole brain TBARS level was measured by the method of Ohkawa et al.[15] with slight modifications. The absorbance was measured spectrophotometrically at 532 nm.

Estimation of Brain Glutathione Level

The whole brain GSH level was measured by the method of Beutler et al.[16] with slight modifications. The absorbance was measured spectrophotometrically at 412 nm.

Experimental Protocol

Ten groups of mice were employed in the present study and each group comprised of eight mice. The groups were run individually over the time.

Results

Effect of Vehicles on Escape Latency Time and Time Spent in Target Quadrant Using Morris Water Maze

Control (Distilled water) mice showed a downward trend in their ELT on subsequent exposure to MWM. There was a significant fall in day 4 ELT of control mice as compared with their day 1 ELT, reflecting normal learning ability [Table 1]. Furthermore, these animals showed a significant rise in day 5 TSTQ, when compared with time spent in other quadrants during retrieval trial conducted on day 5, thus reflecting normal retrieval (memory) as well. Administration of vehicles, i.e., CMC and ACSF did not show any significant effect of day 4 ELT [Table 1] and day 5 TSTQ of control animals [Figure 1].

Table 1

Effect on escape latency time using morris water maze

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Figure 1

Effect on time spent in target Quadrant using Morris Water Maze SN=Sodium nitrite, STZ=Streptozotocin, ACSF=Artificial cerebrospinal fluid, Rlz=Riluzole, Low-dose Riluzole=5mg kg-1, Highdose Riluzole=10mg kg-1. Each group (n=8) represents mean±S.E.M. adenotes P<0.05 vs time spent in other quadrants of Control group, bdenotes P<0.05 vs TSTQ of control group, cdenotes P<0.05 vs TSTQ of STZ group, ddenotes P<0.05 vs TSTQ of STZ + Riluzole group

Effect of Streptozotocin (ICV) and Sodium Nitrite (s.c.) on Escape Latency Time and Time Spent in Target Quadrant Using Morris Water Maze

STZ (ICV)- as well as NaNO₂ (s.c.)-treated mice showed a significant increase in day 4 ELT, when compared with day 4 ELT of control, indicating impairment of acquisition [Table 1]. Furthermore, a significant decrease in day 5 TSTQ of these animals was also noted reflecting impairment of memory as well [Figure 1].

Effect of Riluzole on Streptozotocin and Sodium Nitrite-induced Impairment of Learning and Memory Using Morris Water Maze

Treatment with riluzole prevented STZ as well as NaNO₂ -induced rise in day 4 ELT [Table 1] and fall in day 5 TSTQ [Figure 1] in a dose-dependent manner. However, administration of riluzole did not show any significant per se effect on day 4 ELT [Table 1] and day 5 TSTQ [Figure 1].

Effect of Riluzole on Streptozotocin and Sodium Nitrite-induced Changes in Brain Acetyl Cholinesterase Activity

Administration of ICV STZ as well as NaNO₂ (s.c.) showed a significant increase in brain AChE activity in mice as compared with control [Figure 2]. While treatment with riluzole significantly prevented ICV STZ and NaNO₂ induced rise in brain AChE activity in a dose-dependent manner [Figure 2]. However, administration of riluzole did not show any significant per se effect on brain AChE activity [Figure 2].

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Figure 2

Effect on brain acetylcholinesterase (AChE) activity SN=Sodium nitrite, STZ=Streptozotocin, ACSF=Artificial cerebrospinal fluid, Rlz=Riluzole, Low-dose Riluzole=5 mg/kg, High-dose Riluzole=10 mg/kg. Each group (n=8) represents mean±S.E.M. ^adenotes P<0.05 vs control group, ^bdenotes P<0.05 vs SN group, ^cdenotes P<0.05 vs STZ group

Effect of Riluzole on Streptozotocin and Sodium Nitrite-induced Changes in Oxidative Stress Levels of Brain

STZ as well as NaNO₂ treatment showed a significant increase in brain oxidative stress levels manifested in terms of increased TBARS level [Figure 3] and decreased reduced form of GSH level [Figure 4], when compared with control. Treatment with riluzole significantly and dose dependently reduced the STZ and NaNO₂ -induced rise in brain oxidative stress levels [Figures ​[Figures33 and ​and4].4]. However, administration of riluzole did not show any significant per se effect on brain oxidative stress levels [Figures ​[Figures33 and ​and44].

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Figure 3

Effect on brain thiobarbituric acid reactive species (TBARS) levels SN=Sodium nitrite, STZ=Streptozotocin, ACSF=Artificial cerebrospinal fluid, Rlz=Riluzole, Low-dose Riluzole=5 mg/kg, Highdose Riluzole=10 mg/kg. Each group (n=8) represents mean±S.E.M. ^adenotes P<0.05 vs control group, ^bdenotes P<0.05 vs SN group, ^cdenotes P<0.05 vs STZ group

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Figure 4

Effect on brain reduced glutathione (GSH) levels SN=Sodium nitrite, STZ=Streptozotocin, ACSF=Artificial cerebrospinal fluid, Rlz=Riluzole, Low-dose Riluzole=5 mg/kg, High-dose Riluzole=10 mg/ kg. Each group (n=8) represents mean±S.E.M. ^adenotes P<0.05 vs control group, ^bdenotes P<0.05 vs SN group, ^cdenotes P<0.05 vs STZ group

Discussion

MWM test employed in the present study is one of the most widely accepted models to evaluate learning and memory of the animals.[11] A significant decrease in day 4 ELT of control animals during ongoing acquisition trials denoted normal acquisition of memory and an increase in TSTQ, in search of missing platform during retrieval trial indicated, retrieval of memory. Animals of both sexes have been employed in this study; the idea was to observe the effect of drug not only in males but in animals of both sexes. The fact that estrogen is known to improve memory is taken care by equally distributing the male and female mice in all groups, including that of control.

In our study, ICV STZ not only impaired learning and memory of mice but also produced a significant rise in brain AChE activity as well as oxidative stress (increase in TBARS and decrease in GSH) levels. STZ (ICV) model has been described as an appropriate animal model for dementia, typically characterized by progressive impairment of learning abilities and memory capacities.[17] Significant memory loss was seen after two weeks of second dose of ICV STZ. Cerebral glucose and energy metabolism is associated with oxidative stress. After ICV administration, the highest concentration of STZ (3 mg/kg) reaches the fornix and periventricular white matter at the level of third ventricle, which show the greatest damage and ICV STZ-induced dementia is independent of its hyperglycemic effect.[18] Although the mechanism of action of ICV STZ on memory impairment is not yet known, it probably involves the induction of oxidative stress[19] to which myelin is particularly vulnerable. Damage to myelin by oxidative stress is seen in disorders such as AD with cognitive impairment.[20] In addition, reduced energy metabolism and synthesis of acetyl-CoA ultimately results in cholinergic deficiency and thereby memory deficit in ICV STZ rats.

Similar to that of ICV STZ, NaNO₂ (s.c.) treatment in the present investigation also resulted in significant loss of memory along with rise in brain oxidative stress (i.e., increase in TBARS and decrease in GSH) levels and brain AChE activity. NaNO₂ has been used to induce chemical hypoxia and associated memory loss in animals and is a good model of experimental dementia.[13] Basically, by virtue of its chemical nature, NaNO₂ produces methemoglobinemia eventually leading to cerebral hypoxia.[21] Administration of NaNO₂ at dose of 75 mg/kg (s.c.) has been reported to produce significant impairment of learning and memory in animals.[13] However, in contrast to ICV STZ, memory loss induced by NaNO₂ is acute in nature. One of the most serious consequences of hypoxia/ischemia in human beings is a decline of memory and ability to learn and acquire novel experiences. Cerebral hypoxia/ischemia occurring with environmental limitations, insufficient blood flow, respiratory dysfunction, the use of some toxic chemical/substance or during aging have been demonstrated to result in a high incidence of memory deficits.[22] Furthermore, it has been observed that ischemia/hypoxia leads to an increase in extracellular excitatory amino acid concentrations resulting in glutamate receptor-mediated excitotoxic events.[23] During hypoxia, increased synaptic release and impaired cellular re-uptake of glutamate results in large increases in extracellular glutamate eventually leading to neurodegeneration and dementia.[23]

In the present study, riluzole has significantly reversed STZ as well as NaNO₂ -induced memory deficits, manifested in the terms of increase in MWM performance. Riluzole was administered for 14+4=18 days to STZ animals because STZ-treated mice show a persistent memory deficit in MWM test after 14 days of the second dose; hence, a prolonged treatment with riluzole is required. On the other hand, NaNO₂ -treated mice show early memory loss; hence, riluzole was administered for 4 days to these mice. Riluzole treatment also produced a significant decrease in brain AChE activity and oxidative stress (decrease in TBARS and increase in GSH) levels. Riluzole as mentioned earlier possesses potent neuroprotective,[8,9] anti-glutamate, and anti-oxidative actions.[7] Therefore, it looks evident that riluzole in this study alleviated STZ as well as NaNO₂ -induced memory deficits by virtue of its above mentioned effects. Furthermore, observed inhibition of brain AChE activity with riluzole might also have played a significant role. Moreover, at this point, riluzole induced modulation of TREK channels, the two-pore potassium (K ⁺) channel can also not be ruled out.

TREK-1 (also known as KCNK2) is a member of the most recently discovered family of two-pore-domain K⁺ channels, so called because they contain two pore-forming domains in their primary sequence. The class of mammalian two-pore-domain K⁺ channel subunits now includes 15 members, which are thought to dimerize to form functional channels. Human TREK-1 channels are highly expressed in many regions of the central nervous system such as prefrontal cortex, hippocampus, hypothalamus, midbrain serotonergic neurons of the dorsal raphe nucleus, and in sensory neurons of dorsal root ganglia and are generally believed to play a critical role in controlling neuronal excitability.[24] A remarkable feature of the TREK-1 channel is its sensitivity to a wide variety of endogenous and exogenous modulators.[25] It has been well documented that under conditions of hypoxia, TREK-1 becomes insensitive.[25] Neuroprotective riluzole has been demonstrated to activate TREK-1.[26] Recent study has also documented that TREK-1 play an important role in learning and memory processes.[27] Therefore, it may be speculated that hypoxia induced due to administration of NaNO₂ led to insensitivity of TREK-1 channels with subsequent memory loss, this effect was abolished by our pretreatment of riluzole, a neuroprotective and an activator of TREK-1 channels.[26]

Conclusion

It may be concluded that beneficial effect of riluzole in dementia may involve its multiple effects including anti-oxidative and anticholinesterase actions. Nevertheless, further studies are needed to elucidate the full potential and mechanism of riluzole as memory restorative agent with special focus on TREK-1 channels.

Acknowledgments

Footnotes

References