2.7. Assessment of cognitive function

Learning and memory functions were evaluated on days 31–36 for Morris water maze test and day 32–33 for Novel Object Recognition test.

Cognitive function of rats was assessed using Morris Water Maze test as described earlier [20]. The test apparatus was a circular water tank (180 cm in diameter and 60 cm high) made up of dark grey plastic that was partially filled with water. Full cream milk (liquid) was used to render the water opaque. The pool was divided virtually into four equal quadrants, labelled A– B–C–D. A platform (12.5 cm in diameter and 38 cm high) was placed in one of the four maze quadrants (the target quadrant) and submerged 2.0 cm below the water surface. The platform remained in the same quadrant for the entire experimental duration. The rats were required to find the platform using only distal spatial extra-maze cues available in the testing room. The cues were maintained constant throughout the testing period. The rats received four consecutive daily training trials for 6 days following completion of treatment, with each trial having a ceiling time of 60s and a trial interval of approximately 90s. Each rat had to swim until it climbed onto the platform submerged underneath the water. After climbing onto the platform, each rat remained there for 30s before the commencement of the next trial. The escape platform was kept in the same position relative to the distal cues. If any rat fails to reach the escape platform within the maximally allowed time of 60s, it was gently guided to the platform and allowed to remain there for 30s before the next trial. The time to climb the platform (latency in seconds) was measured. A probe trial was conducted on the 6^th day. The platform was removed from the maze and rats were expected to spend more time in the quadrants initially housing the platform. The total time spent in quadrants other than that for the platform was measured and recorded for each rat as a measure of memory decline.

The object recognition test for assessing non spatial memory in rats was deployed. The test environment was an open field (80cm long × 50cm high × 60cm wide). On day 1, each rat was placed in the middle of the open field and allow to explore the field for 10 min. On day 2, 2 non movable identical objects were placed at the northwest and south east pole of the test field while the rats were allowed to explore the test area. On day 3, one of the identical object was replaced with a novel object and day two experiment was repeated for each rat. During the inter trial interval, the objects and open-field apparatus was cleaned with 70% ethanol to avoid a confounding error induced by the influence of odour. The total of time which each rat spent exploring each object on day 3 was recorded and calculated as a novel object ratio (NOR) as follows: [21]

Where T_novel = time spent to explore the novel object and T_familiar = time spent to explore the familiar object.