Materials and Methods
Twenty-four male Sprague Dawley rats (age: 4-5 weeks, 120-170 g) were provided from the National Laboratory Animal Center, Mahidol University, Salaya, Nakornprathom. The experimental method was accepted by the Khon Kaen University Ethics Committee in Animal Research (ACUC-KKU-57/62). The rats were adjusted to the experimental condition for one week before the experiment. Four rats were housed in a cage and maintained under standard laboratory conditions (12 h light-dark cycle, 23±2 °C temperature). They were fed with food and water ad libitum.
VPA (Sigma-Aldrich, Inc., St. Louis, USA) and Hsd (ChemFaces Biochemical, Wuhan, China) were freshly prepared every day before the drug administration. VPA at a dose of 300 mg/kg was dissolved in 0.9% saline solution by intraperitoneal (i.p.) injection two times every day at 10 a.m. and 3 p.m. for 14 days. Hsd at a dose of 100 mg/kg/day was dissolved in propylene glycol (Ajax Finechem Pty Ltd., Auckland, New Zealand) and given by oral gavage for 21 days. Rats were randomly divided into 4 groups. The vehicle group received 0.9% saline solution (i.p.) and propylene glycol by oral gavage. The VPA group received 300 mg/kg of VPA (i.p.) at 10 a.m. and 3 p.m.4,17. The Hsd group received 100 mg/kg of Hsd by oral gavage. The VPA+Hsd group administered with VPA and Hsd at the same dose and time as in the VPA and Hsd groups.
Figure 1 Timeline of drug administration and behavioral tests.
The NOL and NOR tests were adapted from Dix and Aggleton18, which are spatial and recognition memory tasks. Both tests were divided into three parts including habituation, familiarization and choice trials. One day before the NOL and NOR testing, all rats were habituated to the environment of an arena (open field black acrylic 50 x 50 x 50 cm.) without objects for 30 minutes. The objects used in these tests composed of the identical objects (plastic bottles and triangles). The behavioral tests were recorded using EthoVision® XT (EthoVision®, XT version 12, Noldus, Wageningen, Netherlands)
Novel object location (NOL) test
On the testing day, all rats were habituated to the arena without objects for 3 minutes. In the familiarization trial, they were allowed to survey two similar objects put in different positions for 3 minutes and then returned to their cage for 15 minutes. In the choice trial, they were enabled to explore two similar objects, one of which was placed in the same location and the other one was placed in a new location for 3 minutes.
Novel object recognition (NOR) test
On the testing day, all rats were habituated to the arena without objects for 3 minutes. In the familiarization trial, rats were placed at the center of the arena to explore two similar objects placed in different locations for 3 minutes and then moved to home cage for 15 minutes. During the choice trial, rats were put back to the arena and enabled to explore one familiar object and a novel object in the same locations for 3 minutes.
The exploratory activity of rats was measured when their noses directed to the object from a distance within 2 cm.5,18. The exploration time was used to calculate discrimination index (DI) that is defined as the ability to discriminate between novel and familiar locations or objects. A positive DI value means the rats can discriminate the objects in the novel location or novel object from the objects in the familiar location or familiar object. By contrast, a negative DI values reflect the rats cannot discriminate the objects in the novel location or novel object from the objects in the familiar location or familiar object. If the DI value is not significantly higher than 0, indicating that rats cannot discriminate between two objects by showing equal exploration4,8,16.
The data were expressed as mean ± standard error of mean (SEM) using GraphPad Prism 8.0 software (GraphPad software Inc., San Diego, CA, USA). Two-way repeated measure analysis of variance (ANOVA) was used to analyze body weight. One-way ANOVA was used to determine total exploration time. The DIs were compared using one sample t-test. A significance was considered as p<0.05
1. Body weight
In this study, body weight of rats in all groups were not significantly different throughout the experiment (p>0.05, Fig 2). The result indicates that Hsd and VPA did not have a negative effect on the body weight of all rats.
Figure 2 Body weight of rats throughout the drugs administration.
2. Behavioral tests
During the behavioral tests, total exploration times were recorded before and after three days of drugs administration to evaluate locomotor activity of rats. The DIs were calculated in the choice trial in both of the behavioral tests.
2.1 NOL test
The total exploration times in the NOL test were not significant differences among groups before (p>0.05, one-way ANOVA, Fig 3A) and after treatment (p>0.05, one-way ANOVA, Fig 3B). This result indicates that the rats in all groups had similar locomotor activity. Additionally, the DIs of all groups before treatment were higher than 0 (p<0.05, one sample t-test, Fig 4A). The results indicate that rats in all groups could discriminate between two identical objects in different locations. After treatment, the DIs of the vehicle, Hsd and VPA+Hsd groups were significantly different from zero (p<0.05, one sample t-test, Fig 4B). Whereas, the DI of the VPA group was not significantly different from zero (p>0.05, one sample t-test, Fig 4B). These results indicate that rats in the vehicle, Hsd and VPA+Hsd groups could discriminate between two identical objects in different locations but rats in the VPA group could not discriminate. These indicate that Hsd could counteract VPA-induced spatial memory deficits.
Figure 3 Total exploration time of the NOL test before (A) and after treatment (B).
Figure 4 The discrimination index of the NOL test before (A) and after treatment (B). (*p<0.05, **p<0.01 significant difference compared to zero)
2.2 NOR test
The total exploration times in the NOR test were not significant differences among groups before and after treatment (p>0.05, one-way ANOVA, Fig 5A-B). This result indicates that rats in all groups had a similar locomotor activity. In addition, the results of the DIs before treatment were significant differences among groups (p<0.05, one sample t-test, Fig 6A). These indicate that all rats were able to discriminate between the novel and familiar objects. For the DI analysis after treatment, there were significant differences in the vehicle, Hsd and VPA+Hsd groups (p<0.05, one sample t-test, Fig 6B) but it was not found in the VPA group (p>0.05, one sample t-test, Fig 6B). The results indicate that rats in the vehicle, Hsd and VPA+Hsd groups had the ability to discriminate between the novel and familiar objects. However, rats in the VPA group could not discriminate between the novel and familiar objects. These findings reveal that Hsd could improve VPA-impaired the recognition memory in rats.
Figure 5 Total exploration time of the NOR test before (A) and after treatment (B).
Figure 6 The discrimination index of the NOR test before (A) and after treatment (B). (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 significant difference compared to zero)
The present study demonstrates the effect of Hsd on memory impairment after receiving VPA treatment. During this study, we found that Hsd could improve VPA-induced memory deficits in rats. However, both Hsd and VPA did not affect body weight and locomotor activity.
The results after administration show that the body weight of rats in all groups were not significantly different, indicating that Hsd and VPA did not affect body weight. This finding suggests that Hsd did not affect body weight which is consistent with previous studies19,20. Besides the effect of Hsd on body weight, VPA treatment for 14 days was not significantly different in other groups. Similarly, the body weight of rodent pups exposed to VPA in utero was not significantly different when compared to the controls21.
In this study, the rats administrated with both Hsd and VPA had no effect on locomotor activity that was evaluated using total exploration time. This indicating that all rats had normal movement and could perform the behavioral tests. Accordingly, previous studies have found that Hsd and VPA did not have negative effect on locomotor activity using distance moved, speed and number of crossing analysis6,8,16.
Both the NOL and NOR tests were used to evaluate memory impairments, which are dependent on hippocampus. The NOL test is a test for spatial memory that involved in hippocampus-dependent memory related to spatial locations, configurations or routes22. The NOR test is a test for recognition memory that is associated with the hippocampus and perirhinal cortex. The perirhinal cortex is an area that receives the information including visual, olfactory and somatosensory stimuli before entering into the hippocampus23. The potential of spatial and recognition memory is associated with neurogenesis in the SGZ of the hippocampal DG2426. The hippocampus plays a role in learning and memory27. The results in this study demonstrates that VPA induced memory impairments, which are consistent with previous study4,8,28. VPA treatment can induce memory impairments, which are associated with decreasing in hippocampal neurogenesis including cell proliferation, survival and differentiation4,6,8,29. In the NOL test, the DIs show that rats treated with VPA could not discriminate between two identical objects in the familiar and novel locations, indicating that VPA impaired spatial memory. In the NOR test, similarly, rats in the VPA group showed an inability to discriminate between the familiar object and novel object, indicating an impairment in recognition memory. However, impairments of both spatial and recognition memory were improved by Hsd co-administration. This was confirmed by the DIs of NOL and NOR tests. Likewise, the effect of Hsd (100 mg/kg) improved learning and memory impairment in a mouse model of Alzheimers disease30. Hsd promotes cell survival by increasing the number of new neurons derived from hippocampal neurogenesis16. The ability of learning and memory is linked to the number of generated neurons in the SGZ of the DG in the hippocampus31. Therefore, further study of neuroprotective effects of Hsd should focus on the relationship of hippocampal neurogenesis and cognition.
This study confirmed that VPA treatment could induce both spatial and recognition memory impairments however Hsd could improve these memory impairments. Hsd, a flavanone glycoside, is one of the most important antioxidants, and also functions as a neuroprotective agent. A previous study has reported that Hsd is a mediated of improvement of neural growth factors and endogenous antioxidants defense, alleviating neuro-inflammatory and apoptotic pathways15. Similarly, a recent report has shown that effects of Hsd reduces the negative effects of methotrexate (MTX) on neurogenesis, oxidative stress, and antioxidant enzymes24. Moreover, Tamilselvam et al.32 reported that Hsd protects against rotenone-induced oxidative stress and apoptosis in a cellular model for Parkinsons disease. These studies support our findings that Hsd can improve memory impairments in rats induced by VPA.
This study reveals that Hsd significantly improved memory impairments induced by VPA in adult rats. This finding postulates the ability of Hsd to improve memory impairments caused by the deterioration of the brain functions.
The author would like to thank invitation research from Faculty of Medicine (IN63242), Khon Kaen University for financial support.
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