Dose-Dependent Neurobehavioral and Neurochemical Effects of Oral Donepezil in Male Albino Rats

1. INTRODUCTION

Progressive loss of cholinergic neurons has been found in Alzheimer’s Disease (AD) and related dementias from the basal forebrain which leads to a marked loss of acetylcholine in the cortex and hippocampus and is responsible for many of the cognitive deficits seen in these patients [1]. Prior studies proposed that donepezil, a reversible acetylcholinesterase (AChE) inhibitor, is one of the most commonly used drugs in mild to severe symptomatic AD. Due to the inhibitory action of donepezil on the enzymatic degradation of acetylcholine, acetylcholine has increased activity in the synapse which are also thought to include nicotinic receptor upregulation and the reduction of glutamate receptor-mediated excitotoxicity [2]. Shiekh and Ammar [3] elaborated that clinically donepezil exhibited the dose-dependent effect with measurable improvement in cognition and global function at the standard dosing (mg/kg/day) and higher doses in more advanced disease [3].

Previous literature reported that donepezil has, however, not been limited to its precognitive activity [4]. Since acetylcholine has high affinity and binding to the monoaminergic systems which are known to regulate mood (dopamine, serotonin and noradrenaline) the classics, cholinesterase inhibitors are candidates for mood modulation, with seemingly contradictory results. The pro-depressant effect of high-dose cholinergic stimulation has been well-established, while several recent studies have reported that AchE inhibitors, such as donepezil, have antidepressant-like properties at low doses [4]. Notably, low and medium doses of donepezil decreased immobility time in the rats Forced Swim Test (FST), while a higher dose tended to induce depressive-like behavior, showing a U-shaped dose-response relationship between cholinergic modulation of despair-like behavior [4]. A similar finding has been reported in rats, in a chronic mild stress model, where sub-clinical doses of donepezil were found to have antidepressant, anxiolytic and precognitive effects [5]. Such observations suggest that there is a biphasic effect of cholinergic augmentation, depending on how much acetylcholine is raised in the synapses of the brain.

A researcher reported that Morris Water Maze (MWM) is the most widely used behavioral assessment test of learning and memory in rodents and is still one of the most common readouts for pharmacology studies investigating the hippocampal-dependent cognition [6]. In the medial prefrontal cortex and hippocampus, donepezil increases extracellular acetylcholine, which in turn has been shown to cause enhanced dopaminergic release in these regions, as well as to affect the monoamine tone (elevated, in the case of cholinergic augmentation). Cholinergic augmentation is expected to increase escape latency in the MWM, but also to change monoamine tone, which is increased after cholinergic augmentation. At the same time, oxidative stress is becoming a recognized mechanism in the pathogenesis of neurodegenerative diseases and excessive levels of ROS, including lipid peroxidation markers, Malondialdehyde (MDA) are associated with cognitive impairment as well as the development of depressive pathology. In cognitive impairment rodent models, donepezil has been demonstrated to increase brain SOD activity and decrease MDA content in addition to increasing water-maze performance indicating that some of the neurobehavioral effects of donepezil may be an antioxidant effect, which does not occur with cholinergic action alone [7].

Previous literature shows little understanding about behavioral despair, spatial long-term memory, oxidative status and monoamine levels at the same time in the same group of healthy rats, but with very different doses of donepezil. It is crucial to consider such an integrated evaluation as donepezil is often used orally in clinical practice and the decision on dose in practice involves the trade-off between the precognitive effect and possible adverse effects of a widening or reversal of benefit at higher doses [8]. The present study was, therefore, planned to determine the effects of oral administration of donepezil (5, 10 and 20 mg/kg) on neurobehavioral parameters including depressive-like behavior, long-term memory and oxidative stress markers with neurochemicals contents in male albino rats. Study will predict a dose-dependent and possibly non-monotonic enhancement of these behavior and biochemical parameters with donepezil.

2. MATERIALS AND METHODS

2.1. Animals and Experimental Groups

Twenty-four male albino rats of weighing 150-200 gm, were locally bred, were fed ad libitum with standard rat chow feed and provided water and kept under standard laboratory conditions (12-hour light/dark cycle, controlled temperature and humidity). All experiments were performed using the institutional guidelines for the care and use of laboratory animals. The animals were randomly divided into four groups (six animals in each group): a vehicle (water)-treated control group and three donepezil-treated groups receiving 5 mg/kg, 10 mg/kg and 20 mg/kg daily through the treatment period of 28 days respectively. Doses were selected on the basis of literature review [9].

2.2. Behavioral Assessment

2.2.1. Forced Swimming Test (FST)

Forced swimming test described by Porsolt et al., [10] and standardized for rats was used as an assessment of depressive-like behavior [11]. Animals were briefly pretested in a cylinder mentioned by Sajid et al., [12] with no opportunity to escape from the water, for 15 min a day. After twenty hours a test swim was performed for 5 min in which the total time spent without active escape-oriented movement was measured.

2.2.2. Morris Water Maze Test

The Morris water maze test was used to assess spatial Long-Term Memory (LTM): a round pool of opaque water with a hidden, submerged escape platform in a fixed quadrant [6]. Extra maze visual cues were provided during repeated training acquisition trials, and the delayed probe/retention trial either involved removal of the platform or a change in platform position, with latency to find the platform/quadrant measured as an index of long-term memory, a shorter latency represents good long-term memory consolidation and retrieval [13].

2.2.3. Biochemical and Neurochemical Estimation

After behavioral testing, all animals were sacrificed and whole brain was quickly dissected and homogenized for biochemical analysis. The endogenous antioxidant capacity measured by the level of the enzyme Superoxide Dismutase (SOD) and the level of Malondialdehyde (MDA) that reflects occurrence of lipid peroxidation and oxidative damage were estimated in the brain homogenates by standard spectrophotometric assays as described by [12].

Standard chromatographic quantification methods were used to measure the levels of brain Dopamine (DA) and serotonin (5-HT) while using standard curve of DA and 5-HT by HPLC. Following the protocol by Haider et al., [14].

All the chemicals and reagents have been purchased from Merck, Darmstadt, Germany and Sigma-Aldrich, St. Louis, MO, USA.

2.3. Statistical Analysis

Data are presented as mean ± SEM (Standard Error of the Mean) for all the groups. Individual differences were compared by one way analysis of variance (ANOVA) and an appropriate post-hoc multiple comparison test. Immobility was slightly reduced in 5 mg/kg dose, significantly reduced in 10 mg/kg dose and 20 mg/kg dose slightly increased again by 10 mg/kg whereas remain lessen as compared to control 5 mg/kg dose.

3. RESULTS

3.1. Forced Swim Test (FST)

Fig. (1) shows the effect on 28 days oral administration of donepezil on depression-like symptoms in rats. Data was analyzed through one way ANOVA shows significant effect (F = 5.640, df = 3,20, ** ++p < 0.01). The graph shows a non-significant effect at 5mg/kg as compared to control, while the 10 mg/kg dose significantly reduced depression-like response and minimizing immobility time. However, 20 mg/kg dose of Donepezil shows non-significant effect as compared to 10mg/kg which showing a biphasic U-shaped or bi-phasic response in forced swim/tail suspension test.