INTRODUCTION:

Depression and cognitive disturbances commonly coexist in the elderly. Prevalence of cognitive disturbance in the elderly is about 10-15%¹, and that of depression is around 10-20%.² Prescription of antidepressants is increasing throughout the world^3,4,5 and the prevalence of depression in elderly population is increasing.^6,7,8 Dementia along with depression in an elderly person can further worsen their quality of life.^9,10 Among antidepressants, tricyclic antidepressants tend to produce memory disturbances mainly through their anticholinergic property.¹¹

The other group of antidepressants which is frequently prescribed is selective serotonin reuptake inhibitors (SSRI) which selectively target reuptake of serotonin.^12,13Compared to tricyclic antidepressants, SSRIs are well tolerated¹⁴. Many studies have shown that neurons transmitting serotonin are involved in the formation of memory.^15,16,17However, a few studies have shown that drugs with serotonergic property disturb memory formations in animals.^18,19,20 Some authors proposed that neurons releasing acetylcholine and serotonin at their nerve ending interact with each other, and this interaction modulates the process of memory formation²¹. The nature of this complex interaction is still elusive.

Escitalopram is a commonly used antidepressant that belongs to selective serotonin reuptake inhibitors group. Acute escitalopram has shown to improve performance in a spatial reversal learning task.²² Escitalopram has also shown to improve memory in the novel object recognition task, facilitation of fear conditioning.

Scopolamine, an anticholinergic drug interferes with the consolidation of memory by blocking cholinergic signals in the hippocampus and neocortex^23. Scopolamine-induced amnesia model is a well-established model mimicking pathophysiology of Alzheimer’s disease.

There is no study in the accessible literature evaluating the effect of chronic escitalopram treatment on scopolamine-induced memory impairment, hence this study was planned to evaluate the chronic effect of escitalopram on scopolamine-induced amnesia in Wistar albino rats

METHODS:

Twenty-four adult male albino Wistar rats (150-200g) used in the current study were acquired from the central animal house facility of our institute. The animals were assigned to four groups and were marked with different colors for identification. Different treatment groups were: Group 1 (control) 2% gum acacia orally, Group 2 (disease model) scopolamine 20mg/kg IP, Group 3 (E 10 +S) escitalopram 10mg/kg, Group 4 (E5+S) escitalopram 5mg/kg. Three rats were housed in a single cage. The bedding was changed on alternative day. They were provided with standard animal feed and water ad libitum.

The protocol was approved by the Institutional Animal Ethics Committee before the start of the study. ( IAEC number: IAEC/KMC/86/2016 dated 13.09.2016) The experiments were carried out strictly in accordance with CPCSEA guidelines.

High and low doses of escitalopram were used in the current study. The dose of escitalopram was taken from a previous study²⁴. Scopolamine was used at a dose of 20 mg/kg to induce memory impairment²⁵. Escitalopram was powdered and dissolved in 2% gum acacia and given orally for 28 days. Scopolamine was dissolved in distilled water administered single dose intraperitoneally thirty minutes before the start of the behavioural test. In water maze, scopolamine was injected thirty minutes before the start of the acquisition trial.

Test procedure:

Two behavioural paradigms were used to assess the effect of test and scopolamine on learning and memory – Morris water maze and T maze. One-month washout was given between the two behavioural paradigms. Once behavioural procedures are completed, the animals were killed humanly by cervical dislocation. The brain was carefully extracted for biochemical and histopathological examination.

Water maze:

Water maze experiment was carried out described by Vorhees et.al.²⁶ The apparatus consists of a round tank. (165cm X 35) The tank was filled with water and maintained at 25⁰C. Milk was added to water to remove transparency. The tank was divided into four equal zones. (NE, NW, SE, and SW). A platform (10cm²) was kept in one of the zones just submerged in water. Black and white symbol board was placed as a cue. The position of extra-maze cue and platform were kept constant throughout the learning sessions. The water maze test consists of two phases. The animals were familiarised to water maze for two days by allowing them to swim freely.

a) Spatial task acquisition phase:

Four trials of 2 min each are done for every animal for four days continuously during which the study animal was trained to escape from cold water by reaching the hidden platform and staying there for 20 sec. The location of the platform was fixed in all the trials. (Target quadrant) Four start positions were used (North, South, East, and West). Each day animals were given a series of daily trials with random start positions. In case of inability of the animal to locate the platform even after 90sec, it was guided to the platform. Time to reach platform was counted.

b) Retrieval trial:

On the last day of the experiment, the platform was taken away. The animal was kept in a new place in the maze, directed towards the tank wall in opposite quadrant as that of the original target quadrant. The animal was removed after the 30s. Percentage of distance traveled and the percentage of time spent in target quadrant was noted.

T maze:

T maze was a wooden apparatus with three arms, two-goal arms (50cm X 10cm) and a start arm (50cm X 20 cm) as shown in Figure-7. There were two guillotine doors to separate goal arm from start arm. Walls of the arms were raised to a height of 30cm. A circular food well of diameter 2cm was placed in one of the goal arms. The rewarded alteration was performed as described by Deacon et.al.²⁷

Habituation and training:

One day before the actual experiment, the animals were habituated by putting a hand into the cage for five mins. The animals were fed with the reward in their home cage for two days before the start of the trial to decrease hyponeophagia. The day before the start of training, the animals were food rationed overnight. During training, the animals were trained to reach the goal arm to get a reward. The animals which do not reach even after the 90s were repeated. The animals were trained for four days.

Rewarded alteration:

After four days of habituation, the animals were tested for rewarded alteration. Sample run and choice run was performed in a trial. In the sample run, one of the goal arms was blocked by raising the door (choice arm). In another arm, the reward was placed (sample arm). The animal was allowed to move from start arm to goal arm and consume the reward. In the choice run, both the goal arms were open. The animal was free to choose between the arms. If the animal moved to the arm opposite to the sample arm, then it was regarded as a correct response. Percentage of correct response was calculated by the formula: Percentage of correct response = a Total number of correct response/total number of trials X100.

Biochemical estimation:

Acetylcholinesterase (AChE) assay:

The level of acetylcholinesterase was estimated as described in the previous studies^28,29,30. The whole brain was used for this procedure. After weighing, the brain was homogenised with 0.9% NaCl. It was then centrifuged at 3000 rpm. Supernatant liquid (0.5ml) was added to a flask (25ml) and was filled up to volume with DTNB solution, 4ml of this solution is added to two test tubes. 1ml of a solution containing 75mg of acetylcholine iodide in 50ml of distilled water was added to them. To one of the test tubes, eserine was added. Test tube with eserine was utilized for zeroing. Change in colour was measured using a spectrophotometer.

Histopathological examination:

The whole brain was sectioned and stained with haematoxylin and eosin stain for histopathological examination. The brain was cut and dehydrated in ascending concentrations of alcohol. It was kept in xylene for 1 to 2 hours. It was further immersed in paraffin wax and embedded. Five-micron thick sections were cut and mounted on glass slides.

Staining procedure:

They were deparaffinized in xylol. Then the sections were hydrated through descending grades of alcohol and washed thoroughly in 70% alcohol to remove the yellow color of picric acid. They were washed in tap water for bluing followed by staining with 1% eosin for 30 seconds and haematoxylin for 10 minutes. Sections were dehydrated with ascending grades of alcohol, cleared and mounted.

Statistical analysis:

SPSS V .22 (IBM Co., Armonk, NY, USA) was used for analyzing the data. The results were expressed as mean ± SD (Standard Deviation). ANOVA was used to compare the difference between different groups, and it was followed by Tukey’s post hoc test. A p-value of < 0.05 was considered to be statistically significant.

RESULTS:

Morris water maze -Acquisition Trials:

Escape latency was calculated during acquisition trials. It was the time taken by the animal to reach hidden the platform. Animals with impaired memory take a longer time to reach the platform. As shown in figure 1, on day 1 and 2, there was no significant difference between the groups. (p > 0.05) There was a significant decrease in escape latency in groups treated with escitalopram 10 mg/kg compared to control on day 3 and 4 of acquisition trials (p < 0.05). Escape latency in lower dose group (escitalopram 5mg/kg) was comparable to control, and there was no statistically significant difference.

Figure -1: Escape latency assessed by water maze during four days of acquisition trials. * denotes p-value of less than 0.05 compared “to the control group.”

Probe Trial:

Percentage of time spent and distance traveled in the target quadrant was measured during the probe trial. Animals with impaired memory tend to spend less time in the target quadrant. Animals treated with scopolamine showed a statistically significant reduction in time spent and distance traveled in the target quadrant (p < 0.05) compared to control group animals which are suggestive of scopolamine-induced impaired memory.

Both percentages of time and distance were comparable to control group animals in a higher dose of escitalopram pre-treated animals when co-administered with scopolamine. The animals pre-treated with a lower dose of escitalopram when co-administered with scopolamine showed the statistically significant reduction in both time and distance compared to control group animals. (p < 0.05) This finding suggests that pre-treatment with a higher dose of escitalopram (10mg/kg) was able to reverse scopolamine-induced memory impairment. The data is presented in Figure-2.

Figure-2: “Percentage of time spent and distance traveled in the target quadrant during the probe trial of the water maze task. * p < 0.05 vs control, vs # p < 0.05 vs scopolamine.”

T MAZE:

Scopolamine treated animals showed a significant decline in the percentage of correct response implying that scopolamine produced memory impairment. However, it was reversed by higher doses of escitalopram, but lower dose failed to reverse scopolamine-induced memory impairment as shown in figure 3.

Figure-3: Percentage of correct response in T maze. E10+S denotes escitalopram 10 mg/kg co-administered with scopolamine. * “ p < 0.05 vs control; vs # p < 0.05 vs scopolamine.”

Brain Acetylcholinesterase (AchE) levels:

A significant increase in AchE was noted in scopolamine treated animals compared to control group animals indicating inhibition of cholinergic activity by scopolamine. Higher doses of escitalopram were able to reverse the scopolamine-induced inhibition of cholinergic activity. The result is represented in Table-1.

Table- 1: Effect of various drugs on brain acetyl cholinesterase (AchE) levels (micromole/L/g tissue). Results are in Mean ± SD. ^a p<0.05 vs control; ^abp<0.05 vs scopolamine.

Treatment group	Brain AchE levels (micromole/L/g tissue).
Control	1.82 ±0.15
Scopolamine	4.5 ±1^a
Escitalopram 10 mg/kg + scopolamine	1.5 ±0.7^ab
Escitalopram 5 mg/kg + scopolamine	3.4 ±0.65^a

Histopathological examination:

Control group rats showed normal architecture of rat brain hippocampus (figure 4a). On the other hand, scopolamine treated rats showed perivascular edema, pyknotic cells with damaged cell membrane in the cortex, a loss of brain tissue (arrow), edema and neuronal degeneration in the hippocampus ( figure 4b).

In the rats pre-treated with a higher dose of escitalopram 10 mg/kg, the brain section showed minimal damage when co-administered with scopolamine in the hippocampus (figure 4c). Damaged structure was observed with a lower dose of escitalopram similar to scopolamine group (figure 4d).

Figure- 4: Sample microscopic photographs of effects of various drugs on rat hippocampus. A. Control; B. Scopolamine; C. Escitalopram 10mg/kg + scopolamine; Escitalopram 5 mg/kg + scopolamine.

DISCUSSION:

Results of our study revealed that chronic administration of escitalopram could reverse scopolamine-induced memory impairment in a dose-dependent manner in our study scopolamine impaired memory in both water maze and T maze coupled with a significant increase in acetylcholinesterase activity and marked histopathological changes. Our results are consistent with the previous study results which showed that scopolamine administration impairs spatial memory formation^31,32,33,34.

Various clinical studies have shown that escitalopram ameliorates cognitive deficits in depressive patients.^35,36 In animal studies, escitalopram improved memory in various models such as memory deficits induced by maternal separation³⁰ and chronic stress-induced deficits in extradimensional set shifting³⁷. SSRIs have shown even neuroprotective action in the hippocampus of rats with depressive behaviour^38,39.

0.3 and 1mg/kg of escitalopram have been shown to enhance memory in spatial reversal task⁴⁰. One more study done by Schilstrom et al. showed that escitalopram (5mg/kg) improved memory.⁴¹ The probable mechanism underlying cognitive enhancing property of escitalopram may be due to their direct action on hippocampal neuronal remodelling⁴².

Consolo et al. have shown that citalopram, an SSRI, by increasing serotonin enhances the acetylcholine release in hippocampal tissue. This facilitation is mediated by 5HT₃ receptors⁴³. In our study higher dose of escitalopram reversed scopolamine-induced amnesia through increasing cholinergic activity. The mechanism could be similar to that of citalopram. The exact cause for its dose-dependent action observed in our study is yet to be elucidated.

This study has some limitations. Firstly, the study was done only in one species and sex. Only male rats were used for the current study to avoid behavioural changes during estrus cycles. Secondly, other mechanisms that might have been involved was not studied.

In conclusion, chronic treatment with escitalopram reversed scopolamine-induced behavioural, biochemical and histopathological changes in a dose-dependent manner. Further clinical studies are required to confirm the same. If proved it suggests that escitalopram may be the preferred drug in elderly patients in depression.

ACKNOWLEDGEMENT:

The authors are grateful to Manipal Academy of Higher Education for the research facilities.

CONFLICT OF INTEREST:

The authors declare no conflict of interest

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Received on 27.12.2019 Modified on 28.05.2020

Research J. Pharm. and Tech. 2021; 14(4):1887-1892.

DOI: 10.52711/0974-360X.2021.00333