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Objectives: To evaluate the effects of willughbela cochinchinessis (WC) to locomotor disorders in an Alzheimer''s disease (AD) model of mice. Subjects and methods: 50 Swiss mice were separated randomly into 5 experimental groups, 10 mice for each group.
Journal of military pharmaco-medicine No7-2017 WILLUGHBEIA COCHINCHINENSIS AMELIORATES LOCOMOTOR DISORDERS IN MODEL OF ALZHEIMER’S DISEASE MICE Can Van Mao*; Tran Hai Anh*; Le Van Quan* SUMMARY Objectives: To evaluate the effects of willughbela cochinchinessis (WC) to locomotor disorders in an Alzheimer's disease (AD) model of mice Subjects and methods: 50 Swiss mice were separated randomly into experimental groups, 10 mice for each group Group 1: Mice were intraperitoneally injected (i.p) and orally administered (p.o) saline at dose 0.1 mL/10 g; group 2: mice were injected i.p scopolamin 1.5 mg/kg and p.o saline 0.1 mL/10 g; group 3, group and group 5: mice were injected i.p scopolamine 1.5 mg/kg and p.o WC 100 mg/kg, 150 mg/kg and 200 mg/kg, respectively WC and saline were orally administered at 60 minutes and scopolamin and saline were injected i.p at 30 minutes before the behavioral task 60 minutes after WC injections, mice were placed in an open field for minutes Behaviors of mice were observed by a camera and analyzed by Anymaze software Results: WC at doses 150 mg/kg and 200 mg/kg reversed scopolamin-induced hyperactivities in mice Conclusion: These results provided a basic for developing a new drug to treat patients with AD * Keywords: Alzheimer's disease; Willughbela cochinchinessis; Scopolamin; Locomotor behaviors; Mice INTRODUCTION Alzheimer’s deasease is one form of dementia in older humans Mechanism of this kind disease has been suggested to be involved in neurodegeneration and formation of plaques and neurofibrillary tangles [1] These changes in the brain cause behavioral disorders such as cognitive and memory impairments and locomotor hyperactivities In patients with AD, it has been shown that patients with AD expressed hyperactivities in the late afternoon and the evening These hyperactivities are termed as sundown syndrome or sundowing [2, 3] In animal models of AD, previous studies have been demonstrated that animals also exhibit increased locomotor activities including hyperactivity, stereotypic behaviors, and home cage activity disturbances [4] It has been suggested that disorders of neurotransmitter systems, especially cholinergic system and glutamate systems are associated with abnormal behaviors of patients with AD [5] Furthermore, abnormal activity of the cholinergic system affects glutamatergic systems [6] Thus, it has developed a animal model of AD by injecting intraperitoneally scopolamin, a form of anticholiergic drug and used this model to study effects of new drugs or natural plants in animal models of AD * Military Medical University Corresponding author: Cao Van Mao (caovanmao2011@gmail.com) Date received: 15/06/2017 Date accepted: 10/08/2017 16 Journal of military pharmaco-medicine no7-2017 Recently, we applied this model to evaluate effects of WC to scopolamin-induced deficits in cognition and memory in mice However, patients and scopolamin induced animals with AD also showed disorders in locomotion [2, 3, 7, 8] Thus, effects of WC to locomotor disorders should be evaluated We conducted the present study with the aim: To investigate effects of WC to ameliorate locomotor disorders in an animal model of AD SUBJECTS AND METHODS Subjects 50 Swiss mice (150 - 250 g body weight) were used in the present study Animals were housed in individual cages, maintained in controlled temperature and 12h light/dark cycles with free access to water and food The present study was conducted at Department of Physiology, Vietnam Military Medical University All procedures were performed in accordance with the Animal Center Guidelines for the Care and Use of Laboratory Animals at the Vietnam Military Medical University 1.5 mg/kg and p.o treated saline at 0.1 mL/10 g; group 3, group and group (WC groups): mice were i.p injected scopolamin 1.5 mg/kg and p.o WC 100 mg/kg, 150 mg/kg and 200 mg/kg, respectively WC and saline were orally administered at 60 minutes and scopolamin and saline were i.p injected at 30 minutes before the behavioral task * Open field test: 60 minutes after drug treatments, mice were placed in the center of a open field box Open field box was a square box (40 × 40 × 60 cm), covered with polypropylene sheets inside the wooden box (figure 1) Animals were allowed to free explore inside open field box for minutes Behaviors of animals were recorded using a digital video system Data was analyzed offline by ANY-maze software (Stoelting Co., Wood Dale, IL, USA) Materials WC was isolated by Department of Pharmacy, Hochiminh City University of Medicine and Pharmacy and was supplied in power form WC power was dissolved in saline using a magnetic stirrer Methods * Animal grouping and drug treatments: Animals were separated randomly into experimental groups, 10 mice for each group: group (control group): mice were ip and p.o treated saline; group (scopolamin group): mice were i.p treated scopolamin Figure 1: Open field box * Research indicators: In the present study, we analyzed some research indicators as followed: - Travel distances (m) 17 Journal of military pharmaco-medicine No7-2017 - Average speeds (m/s) - Ratios of mobile time/immobile time * Data analysis: of Travel distance, travel speed and ratio mobile time/immobile time were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey’s posthoc test for multiple comparison, using SPSS 19.0 Results were considered to be statistically significant at p < 0.05 All results were expressed as mean ± SEM RESULTS Changes in travel distance Figure 2: Travel distance of mice Figure showed travel distance of mice in the open field test There was a significant difference in travel distance of mice in the experimental groups [F(5.49) = 3.82, p = 0.016] Post hoc test indicated that mean travel distance of mice in the scopolamin group was significantly longer than this in the control group (Tukey test, p < 0.05) After WC treatment, travel distance of mice decreased gradually from WC 100 mg/kg to WC 200 mg/kg However, a significant decrease in mean travel distance of mice was observed in only WC 200 mg/kg group (Tukey test, p < 0.05) Changes in average speed Figure 3: Average speed of mice 18 Journal of military pharmaco-medicine no7-2017 Figure showed changes in average speed of mice in the open field test One way ANOVA indicated that there was a significant difference in average speed of mice in experimental groups [F(4.49) = 4.12, p = 0.011] Post hoc test indicated that there was a significant increase in average speed of mice in scopolamin group, compared to this in the control group (Tukey test, p < 0.05) After WC treatments, average speed also reduced gradually from WC 100 mg/kg to WC 200 mg/kg However, compared to average speed of mice in control group, a significant decrease in average speed was also observed in the WC 200 mg/kg group only (Tukey’s test, p < 0.05) Changes in ratio of mobile time/immobile time Figure 4: Ratio of mobile time/immobile time of mice Figure showed changes in ratio of mobile time/immobile time of mice in the experimental groups One way ANOVA indicated that there was a significant difference in ratio of mobile time/immobile time of mice between experimental groups [F(4.49) = 6.11, p = 0.01] Post hoc test indicated that mean ratio of mobile time/immobile time of mice in the scopolamin group was significantly higher than this in the control group (Tukey test, p < 0.05) When mice were treated by WC, these ratios decreased gradually Ratios of mobile time/immobile time of mice in WC 150 mg/kg group and WC 200 mg/kg group were significantly lower than those in the scopolamin group (Tukey test, p < 0.05) Ratios of mobile time/immobile time of mice expressed mobile or immobile tendencies of animals If this ratio is higher than 1, it indicates that animals might tend to be mobile On the contrary, it indicates that animals might tend to be immobile Results in figure indicated that WC at doses 150 mg/kg and 200 mg/kg reversed scopolamin induced increasing mobile tendencies of mice DISCUSSION Hyperactivity is one of behavioral disorders in both patients with AD and 19 Journal of military pharmaco-medicine No7-2017 animal models of AD In patients with AD, it was demonstrated that they display sleeping and locomotion disorders These disorders are more serious in the late afternoon and evening Thus, these disorders were called as sundown syndrome or sundowning [2, 3] Locomotor disorders are observed in animal models of AD, including the scopolamin-induced Alzheimer model [7, 8] In these models, mice exhibit hyperactivity tendencies during the dark phase (the active phase of mice) [4] Thus, in developments of new drugs or natural plants to treat for AD, study on locomotor functions of experimental animals is necessary Open field test used widely to investigate locomotor functions in small animals such as rats and mice This test allows to evaluate many research indicators Thus, this test will help us to study more particularly and exactly the effects of new drugs to locomotor functions of experimental animals [9] In the present study, the open field test was used to assess effects of WC to scopolamine- induced hyperactivities of animals with Alzheimer-like symptoms Results showed changes in some research indicators to indicate that WC ameliorated disorders in locomotor functions of animals These are: WC has effects to decrease travel distance, average speed and ratio of mobile time/immobile time of animals with scopolamin-induced hyperactivities These results along with our previous study’s results [6] provided a important basic to apply new plants for treating AD in humans 20 CONCLUSION In the present study, we used open field test to investigate effects of WC on scopolamin induced locomotor disorders in animals with Alzheimer-like symptoms Our results indicated that WC at doses 150 mg/kg and 200 mg/kg reduced scoplamin-induced hyperactivities in experimental animals The reduction of hyperactivities was expressed by significant decreases in travel distance, average speed and ratio of mobile time/immobile timey These results provided important basic for next researches to use WC for treatment of AD in humans ACKNOWLEDGEMENTS This work was supported by Grant 106YS.05-2013.24 from Vietnam’s National Foundation for Science and Technology Development (NAFOSTED) REFERENCE Crews L, Masliah E Molecular mechanisms of neurodegeneration in Alzheimer's disease Hum Mol Genet 2010, 19(R1):R12-20 Vitiello M.V1, Bliwise D.L, Prinz P.N Sleep in Alzheimer's disease and the sundown syndrome Neurology 1992, 42 (7 Suppl 6), pp.83-93 Bliwise D L What is sundowning? 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Changes in travel distance Figure 2: Travel distance of mice Figure showed travel distance of mice in the open field test There was a significant difference in travel distance of mice in the experimental