Angiostrongylus cantonensis causes cognitive impairments in heavily infected BALB/c and C57BL/6 mice

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In the brain of mice heavily infected with Angiostrongylus cantonensis , severe damages have been observed in the hippocampus. This component has been considered to have associations with spatial learning and memory in human and vertebrates. This study was designed to determine the impairments in behavior, learning, and memory in BALB/c and C57BL/6 mice heavily infected with the parasite. Methods: Each mouse was inoculated with 50 third-stage larvae of A . cantonensis . After infection, daily changes in weight and dietary consumption, worm recoveries and survival rates were determined. The forced swimming test, open field test, and Morris water maze test were employed to evaluate depression- and anxiety-like behavior as well as impairments in spatial learning and memory, respectively. In addition, in the two strains of mice were also determined. Results: The worm recovery rate in the BALB/c mice was significantly lower than that of C57BL/6 mice since day 14 post-infection. The survival rate in infected BALB/c mice decreased to 0% by day 25 whereas those with swim-training survived three more days. On day 42, the C57BL/6 mice had a survival rate of 85.7% in the swimming group and 70% in the non-swimming group. Significant differences were found in weight between infected and non-infected BALB/c and C57BL/6 mice since day 13 and day 12, respectively with corresponding changes in the dietary consumption. Depression-like behavior was found in the infected BALB/c mice but not in C57BL/6 mice. However, anxiety-like behavior was found to occur only in C57BL/6 mice. Impaired spatial learning and memory were also found in the two strains of mice occurred since day 14 post-infection. Conclusions: Results of this study indicate that A . cantonensis causes depression, anxiety, and impairments in spatial learning and memory in heavily infected mice. Moreover, significantly higher severity was observed in the Th-2 dominant BALB/c mice. Parasitology Angiostrongylus cantonensis behavior forced swimming test learning memory Morris water maze test open field test Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Parasitic infections have been reported to have significant effects on behavior, learning, and memory of the hosts. Mice infected with Heligmosomoides polygyrus and Eimeria vermiformis were respectively observed to have poor spatial learning and a reduced avoidance of the cat odor [1, 2]. However, rodents heavily infected with Strongyloides ratti did not show deficits in learning [3]. Although inconsistent findings were obtained in infected rodents with gastrointestinal parasites, neurotropic parasites such as Toxocara canis [4] and Toxoplasma gondii [5-7] have been documented to cause learning impairments in the hosts. Moreover, cysts of T . gondii in specific forebrain areas of rats had been attributed to attenuation of predator odor aversion and changes in anxiety-related behavior [6]. Association between T . gondii and anxiety and cognitive disorders has also been reported [8, 9]. Angiostrongyl us cantonensis is a neurotropic nematode causing eosinophilic meningitis and/or eosinophilic meningoencephalitis in human. The infection is due to ingestion of raw or uncooked intermediate host (snails or slugs), paratenic hosts (crustaceans, frogs, fishes, or planarians), or vegetables contaminated with the infective third-stage larvae (L3) [10, 11]. After ingestion, L3 penetrate the small intestine and reach the central nervous system through bloodstream. In the brain, the worms molt twice and develop into the fifth-stage larvae or young adults. The young adults cause a series of pathological and immunological changes [12]. Severe headaches, fever, nausea, vomiting, neck stiffness, and neurologic abnormalities are the common clinical manifestations persisting for weeks to months. Although most human infections are self-limited, severe infection may lead to irreversible neurological damages and even death [13]. Using the water maze, BALB/c mice have been determined in learning and memory deficits 1-2 weeks post-infection [14]. In our previous studies, we have reported that A. cantonensis causes significant pathological changes in the brain of infected mice especially in the hippocampus [15] and different temporal-spatial expressions of IL-4, IL-10, and IL-13 between of the infected Th-2 dominant BALB/c and Th-1 dominant C57BL/6 mice [16]. Rats with hippocampal damages required a prolong duration to find the test platform in Morris water test [17]. Moreover, the hippocampal gyrus has been related to depression [18, 19]. Daily feeding of Lactobacillus helveticus to rats with ammonium acetate induced chronic hyperammonemia has been reported to elevate cognitive functions [20]. Treatment with the emotional stabilizer LiCl to young rats inoculated with Streptococcus pneumoniae not only prevented apoptosis and reduced damages in hippocampus but also improve spatial memory and learning [21]. In addition, icariin has been to be effective in treating mice with traumatic brain injuries [22]. Since A. cantonensis infection causes damages to the hippocampus, these changes may lead to impairments in the behavior, learning, and memory of the host. In this study, we employed the force swimming test to evaluate level of depression, open field test to assess anxiety, and Morris water maze test to determine changes in learning and memory in heavily infected BALB/c and C57BL/6 mice with A. cantonensis . Methods Parasite and laboratory animals A Taiwan strain of A . cantonensis has been maintained in our laboratory since 1980. Its life cycle is maintained through Biomphalaria glabrata snails and Sprague-Dawley (SD) rats [16]. The rats for life maintenance and 7-8-week-old BALB/c (H-2 d ) and C57BL/6 (H-2 b ) mice for experimental studies were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan). These animals were reared in the Laboratory Animal Center of Chang-Gung University. They were kept in plastic cages and provided with food and water ad libitum . All procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Chang Gung University (IACUC Approval No.: CGU15-193). Experimental infection On day 21 post-infection, tissues of the snail hosts were removed and homogenized after shell crushing. The tissue samples were digested with 0.6% (w/v) pepsin-HCl (pH 2-3) for at 37°C for 1 h [23]. L3 of A . cantonensis were removed from the standing precipitations using a medical dropper and counted under a dissecting microscope. Each mouse was inoculated with 50 L3 by stomach intubation. Determination of weight and dietary consumption Since mice undertaking the tests have to exercise in the training and testing stages, it is required to determine whether exercise affects the status of infection. There were two controls groups (10 BALB/c mice and 10 C57BL/6 mice) and two corresponding infected groups with equal numbers. Throughout the experimental studies, each mouse was weighed daily. In addition, the daily dietary consumption was determined by recording food and drinking water intakes. Forced swimming test The forced swim test is a behavioral test used for the evaluation on the level of depression after A . cantonensis infection. There were 10 mice in each of the control and infected groups. The test was carried out on days 7, 14, and 21 post-infection. The tested mouse is placed in a transparent cylindrical tank (45 cm height × 10 cm radius) and filled with water (25 ± 1°C) at a level of 15 cm from the bottom for 6 min. The duration for the mouse without swimming in the last 4 min is recorded [24]. Open field test The open field test was used to assess anxiety-like behavior in A . cantonensis infected mice. There were 10 mice in each of the control and infected groups. The test was carried out on day 9 post-infection. The open field apparatus consisted of a square tank (40 × 40 cm) with walls 40 cm high made of gray polyvinyl chloride plastic board. Mice were transported to the testing room and left in their home cages for 1 h. The animal was placed in the middle of the tank and allowed to move freely. Speed and time of the locomotor activity was monitored for 1 h. Tendency to anxiety was defined as time in the middle less than time on the edge [25]. Morris water maze test The Morris water maze test was used to assess the effect of A . cantonensis on spatial learning and memory. There were 10 mice in each of the control and infected groups. The mice were trained on days 3-6, 10-13, and 17-20 post-infection and tests on days 7, 14, and 21 days post-infection. The custom-made maze was a circular pool 120 cm in diameter and 40 cm in height, filled with water at 25 ± 1°C making opaque with milk. In the acquisition session, each mouse was given four trials per day and four days of training in total to find a hidden platform located 1.5 cm below the water surface. Each mouse was placed into the pool, facing the wall, with a different starting point for each trial that the direct route to the platform differs each time. The time required by the mouse to find and stand on the platform was recorded for up to 90 s. The mouse was allowed to stay on the platform for 30 s. It was then removed from the maze and placed into its cage. For the mouse did find the platform within 90 s, the animal was placed on the platform for 30 s. The inter-trial interval was at least 30 min. In the probe session, on day 5, the platform was removed from the pool. The mouse was tested in a probe trial for 60 s. Mouse swimming tracts were recorded using a TopScan automated tracking system (Clever Sys Inc., Virginia, USA) [26]. Determination of worm recovery The infected mice were sacrificed by inhalation of 3% (v/v) isoflurane (Panion & BF Biotech Inc., Taipei, Taiwan). Brains were removed from the cranial cavity. Worm recovery rates were then determined by counting the number worms under a dissecting microscope. Statistical analyses Statistical analyses were performed using Microsoft Excel 2010 for Windows (Microsoft, Redmond, WA, USA). Data were expressed as mean ± standard deviation. Differences between groups were analyzed by Student’s t -test. P < 0.05 was considered to be statistically significant. Results Worm recovery and survival rate The worm recovery rate in BALB/c mice tested with the Morris water maze significantly decreased with the time of infection ( t ( 4) = 0.000169, P < 0.001) whereas that of C57BL/6 mice decreased only on day 21 post-infection ( t ( 4) = 0.001452, P < 0.01). In addition, the rate in the BALB/c mice was significantly lower than that of C57BL/6 mice on days 14 ( t ( 4) = 0.000331, P < 0.001) and 21 ( t ( 4) = 0.006658, P < 0.01) (Fig. 1). All animals in the control groups remained alive on day 42 post-infection, giving a survival rate of 100% (data not shown). The survival rate in BALB/c without undertaking the water maze test (the non-swimming group) rapidly decreased to 0% by day 25 whereas those undertaking the test (the swimming group) survived three more days. Among the C57BL/6 mice, the survival rate only became 70% on day 42 in the non-swimming group and that in the swimming remained 85.7% on the same day. The mortality rate in the swimming group decreased from 30% to 14% (Fig. 2). Changes in weight In the non-swimming group, there was no significant difference in weight of infected and non-infected BALB/c mice up to day 13 post-infection ( t ( 9) = 0.320876, P > 0.05). From day 14, the weight of the infected group significantly decreased ( t ( 9) = 0.006903, P < 0.01) (Fig. 3a). On day 21, the non-infected mice had a weight gain of 7% and the infected one had a weight loss of 24%. A similar decreasing pattern was observed in the swimming group. Significantly differences in weight of the infected animals were found since day 13 ( t ( 9) = 0.011617, P < 0.05) (Fig. 3b). On day 21, the non-infected mice had a weight gain of 7% and the infected one had a weight loss of 21%. In the non-swimming group, the weights of infected C57BL/6 mice were found to be significantly lower than the controls ( t ( 9) = 0.031012, P < 0.05) (Fig. 3c). The infected mice had a weight loss of 12% on day 21 post-infection whereas the uninfected controls had a weight of 10%. In the swimming group, the C57BL/6 mice had a change pattern similar to the BALB/c counterparts. From days 1-11, no significant difference was found in the infected and uninfected mice ( t ( 9) = 0.326197, P > 0.05) and the weight of the infected ones significantly decreased since day 12 ( t ( 9) = 0.037645, P < 0.05) (Fig. 3d). On day 21, the uninfected mice has also a weight gain of 10% while the infect ones only had a weight loss of 10%. Dietary consumption In BALB/c mice, there was no significant difference in food intake between the uninfected control and infected one in the swimming group up to day 12 post-infection ( t ( 9) = 0.113181, P > 0.05). Since day 13, the food intake in the infected group significantly decreased ( t ( 9) = 0.004678, P 0.05) and that in the infected ones became significantly decreased since day 13 ( t ( 9) = 0.004806, P < 0.01) (Fig. 4b). In BALB/c mice, there was no significant difference in water intake between the uninfected control and infected one in the swimming group up to day 11 post-infection ( t ( 9) = 0.794676, P > 0.05). Since day 12, the water intake in the infected group significantly decreased ( t ( 9) = 0.002576, P 0.05) and that in the infected ones became significantly decreased since day 14 ( t ( 9) = 0.019749, P < 0.05) (Fig. 5b). Depression- and anxiety-like behavior By the force swimming test, the infected BALB/c mice were determined to have longer immobility time than the uninfected ones on day 21, indicating the occurrence of depression ( t ( 9) = 0.018582, P 0.05). Moreover, the immobility time in the infected mice was found to be significantly shorter than the uninfected ones on day 14 ( t ( 9) = 0.013031, P < 0.05) and day 21 ( t ( 9) = 0.00011, P < 0.001), indicating an increase in activities and no depression occurred (Fig. 6b). Although no special findings was observed in the infected BALB/c mice by the open field test, the infected C57BL/6 mice were found to have significantly higher activity ( t (9) = 0.024914, P < 0.05) (Fig. 7a) and longer walking distance ( t (9) = 0.029771, P < 0.05) (Fig. 7b) the uninfected ones on day 9 post-infection, indicating that anxiety-like behavior occurred in these animals (Fig. 7). Changes in spatial learning After infection with A . cantonensis , BALB/c and C57BL/6 mice were continuously trained in water maze for 4 days to observe their spatial learning ability. Before day 7, no significant difference in escape latency to the platform was found in infected and uninfected BALB/c mice ( t (9) = 0.561855, P > 0.05) (Fig. 8a). Since day 12, the latency in the infected mice was found to be significantly higher than that of the uninfected ones ( t ( 9) = 0.000049, P < 0.001) (Fig. 8b and 8c), indicating that the spatial learning ability of infected mice significantly decreased. Although infected and uninfected C57BL/6 mice showed no significant difference in spatial learning ability from day 1 to day 14 ( t ( 9) = 0.803257, P > 0.05) (Fig. 8d), the latency in the infected ones became significantly higher after day 14 ( t ( 9) = 0.013565, P < 0.05) (Fig. 8e and 8f), indicating significant lower spatial learning ability also occurred in the infected C57BL/6 mice, although the occurring time was delayed. Changes in spatial memory After removing water maze platform, the infected BALB/c mice stayed in the target quadrant of the original platform significantly shorter than the uninfected group ( t (9) = 0.002013, P < 0.01) (Fig. 9a) and the time for the infected mice moving to the target quadrant was significantly longer than that of the uninfected ones (Fig. 9b) on days 7 ( t (9) = 0.016921, P < 0.05), 14 ( t (9) = 0.015133, P < 0.05), and 21 ( t (9) = 0.000047, P < 0.001). In the infected C57BL/6 mice, these phenomena only occurred on days 7 ( t (9) = 0.03045, P < 0.05) and 21 ( t (9) = 0.004776, P < 0.01) (Fig. 9c and 9d). Although the spatial memory ability of the two strains of mice significantly decreased, the ability remained intact in the C57BL/6 in the early stage of A . cantonensis infection. Discussion Mice undertaking the water maze test were required to swim for 4 days in the training stage. The tested infected BALB/c mice may survive after day 28 and C57BL/6 mice had a high survival rate of 87% (Fig. 2). Moreover, the weight loss in those undertaking the test also became lower (Fig. 3). It has been reported that moderate physical exercise may promote metabolism, blood circulation, muscle growth, and immunity, thereby preventing or delaying cardiovascular disease [27] and diabetes [28]. In addition to elevation of the proinflammatory cytokines IL1β, TNF-α, and IL-6 [29], exercise may reduce C-reactive proteins in patients with coronary arteriosclerosis [30] and Type II T helper cytokines (IL-5 and IL-13) in asthmatic BALB/c mice [31]. These findings indicate that regular exercise may ease the clinical symptoms of mice infected with A . cantonensis . However, this suggestion requires further experiments. In our previous study, eosinophilic meningitis was found to occur since day 12 post-infection in ICR mice infected with A . cantonensis and pathologic changes were not limited to the meninges but also in the cerebral parenchyma especially in the hippocampus [13]. Hippocampal reticular injury has been associated with depression [18] and the length of depression is proportional to the volume of hippocampal gyrus loss [19]. In addition, the hypoxia-pituitary-adrenal (HPA) axis loss of function [30] or persistent inflammation [34] may also lead to depression. By the forced swimming test, the infected BALB/c mice were showed to have longer immobility time than the infected C57BL/6 mice. These phenomena may be associated with hippocampus and has been reported to be proportional to the mRNA expression of inducible nitric oxide synthase (iNos) [35]. In this study, we demonstrated that the recovery rate of worms in the two strains of mice infected with A . cantonensis decreased on day 21 (Fig. 1) and mortality of the animals also began to occur around this day (Fig. 2). It is possible that most of young adults may be eliminated by the host immune system. The worms survived around day 35 may continue to live. Moreover, the weight of infected mice began to decline since day 14 (Fig. 3). This finding may be associated with the decrease in food (Fig. 4) and water (Fig. 5) intakes. Since the development of L3 to young adults since day 14 [36], these worms may cause inflammation in the brain of host and changes in behavior. These phenomena may explain the results of the water maze test. Although the infected mice are able to find the hidden platform since day 14, the time stay in this location becomes significantly shorter and the spatial learning and memory abilities significantly reduced. By forced swimming test, infected BALB/c mice were demonstrated to develop depression on day 21. However, infected C57BL/6 mice had significant shorter immobility time (Fig. 6). They were demonstrated to became more active on day 9 post-infection had a total moving distance of 46 m more than the non-infected ones by the open field test (Fig. 7). These findings suggest that the young adults of A . cantonensis may cause mechanical damages to the brain of the host. In the water maze test also demonstrated that the infected BALB/c mice required longer time to find the hidden platform and had shorter residence time at this position than their C57BL/6 counterpart (Fig. 9), showing the spatial memory ability of infected BALB/c mice was more severely reduced. Changes in immune responses have been associated with spatial memory. Although SCID BALB/c mice had a lower spatial memory, significant spatial improvement d memory learning was after injection of T cells in nude mice [37]. A water maze test demonstrated that C57BL/6 mice shaved with the IL-4 gene had low spatial memory learning ability [38]. Combined treatment with Am80 and HX630 to the C57BL/6 mice with amyloid-protein precursor 23 (AβPP23) gene (Alzheimer’s disease) may be effective in restoring spatial memory learning ability through the increase of the IL-4 content in the hippocampus [39]. Injection of IL-4 C-4BL/6 mice with cerebral ischemia has been found to be effective in improving the spatial memory and learning abilities [40]. These results suggest that T cells may secrete IL-4 to reduce the loss of spatial memory learning ability in injured brain. Conclusion In conclusion, A . cantonensis is able to cause cognitive impairments in heavily infected mice. The infection may cause depression, anxiety, and impairments in spatial learning and memory in heavily infected mice. Moreover, significantly higher severity was observed in the Th-2 dominant BALB/c mice. Declarations Availability of data and materials Data supporting the conclusions of this article are included within the article. Acknowledgements We would like to thank Chung-Han Li, Yi-Tang Lin for technical support. Authors’ contributions KYC and LCW designed and coordinated this study and write the manuscript. KYC, KYJ, GJL, PKC, RYT and CJC contributed to carrying out the animal experiments. All authors read and approved the final manuscript. Funding This study was supported in part by grants from the Ministry of Science and 353 Technology, Republic of China (Grand No.: MOST 105-2320-B-182-028-MY3), and the Chang Gung Memorial Hospital (Grand No. CMRPD1H0342 and CMRPD1H0442). Ethics approval Laboratory animal application in this study were approved by the Institutional Animal Care and Use Committee, Chang Gung University (IACUC Approval No.: CGU15-193). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. 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Interleukin-4 is essential for microglia/macrophage M2 polarization and long-term recovery after cerebral ischemia. Stroke. 2016;47:498-504. Cite Share Download PDF Status: Published Journal Publication published 10 Aug, 2020 Read the published version in Parasites & Vectors → Version 3 posted Editor assigned by journal 10 Jun, 2020 Submission checks completed at journal 09 Jun, 2020 Editor invited by journal 09 Jun, 2020 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9315","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":659255,"identity":"7cfb4794-aa9b-4488-9c77-1a65953654a7","order_by":0,"name":"Kai-Yuan Jhan","email":"","orcid":"","institution":"Chang Gung University","correspondingAuthor":false,"prefix":"","firstName":"Kai-Yuan","middleName":"","lastName":"Jhan","suffix":""},{"id":659256,"identity":"daae0d02-3f2f-4a0c-8ac8-82ffcd2a7147","order_by":1,"name":"Guan-Jhih Lai","email":"","orcid":"","institution":"Chang Gung University","correspondingAuthor":false,"prefix":"","firstName":"Guan-Jhih","middleName":"","lastName":"Lai","suffix":""},{"id":659257,"identity":"7a8f0d25-cdc2-4c2f-ba8e-ee2e72fa5b7d","order_by":2,"name":"Pi-Kai Chang","email":"","orcid":"","institution":"Chang Gung University","correspondingAuthor":false,"prefix":"","firstName":"Pi-Kai","middleName":"","lastName":"Chang","suffix":""},{"id":659258,"identity":"065ff451-fcdc-4dc5-b5fd-708f6dd5da05","order_by":3,"name":"Ren-Yu Tang","email":"","orcid":"","institution":"Chang Gung University","correspondingAuthor":false,"prefix":"","firstName":"Ren-Yu","middleName":"","lastName":"Tang","suffix":""},{"id":659259,"identity":"c63b99e5-dd66-4f98-bb2a-89de9fbdb728","order_by":4,"name":"Chien-Ju Cheng","email":"","orcid":"","institution":"Chang Gung University","correspondingAuthor":false,"prefix":"","firstName":"Chien-Ju","middleName":"","lastName":"Cheng","suffix":""},{"id":659260,"identity":"a87dd175-6981-4cbb-878b-d72b344de614","order_by":5,"name":"Kuang-Yao Chen","email":"","orcid":"","institution":"Chang Gung University","correspondingAuthor":false,"prefix":"","firstName":"Kuang-Yao","middleName":"","lastName":"Chen","suffix":""},{"id":659261,"identity":"80619374-c096-4686-85fd-fce65428e555","order_by":6,"name":"Lian-Chen Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYLCCBww2DAzMzA1QbgIRWhIY0oBaGJG0HCCs5TCQJFaLwfGzh18k1JyP5m8Havnx6zADP3uOAfPHNjxazuSlWSQcu5074zBjA2Nv32EGyZ43BgwH8WgxO5BjZpDYcDu3AaiFmbHnMIPBjRyglm14tJx/A9JyLnc+TIs9QS03cowfJDYcyN0A0sLwA2iLBAEt9jfemDEkHEvO3QjUcrC3IZ1H4syzggNn/+HWItmfY/zhQ41d7rzzhw8++PHHWo6/PXnjg4ozuLUAAZsEjHWAsY2BB8zAqwEY7R8Q7D8E1I6CUTAKRsGIBAAHv1z5X1AqSgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-6862-2215","institution":"Chang Gung University","correspondingAuthor":true,"prefix":"","firstName":"Lian-Chen","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2019-12-10 11:31:43","currentVersionCode":3,"declarations":"","doi":"10.21203/rs.2.18697/v3","doiUrl":"https://doi.org/10.21203/rs.2.18697/v3","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-020-04230-y","type":"published","date":"2020-08-10T12:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":1322940,"identity":"676a1677-db39-4e2c-9c8b-94dd563dc00c","added_by":"auto","created_at":"2020-06-13 02:44:36","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":257146,"visible":true,"origin":"","legend":"Comparison of worm recovery rates between BALB/c (■) and C57BL/6 (■) mice infected with Angiostronglus cantonensis and tested with the Morris water maze.","description":"","filename":"Figure1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9315/v3/Figure1.jpeg"},{"id":1322941,"identity":"b368c2ad-ded2-487a-82b6-9aa65d22689c","added_by":"auto","created_at":"2020-06-13 02:44:37","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":347836,"visible":true,"origin":"","legend":"Effects of the Morris water maze test on the survival rates in BALB/c (─: Non-swimming group, ─: Swimming group) and C57BL/6 (─: Non-swimming group, ─: Swimming group) mice infected with Angiostronglus cantonensis.","description":"","filename":"Figure2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9315/v3/Figure2.jpeg"},{"id":1322942,"identity":"9ce60ae9-9ebc-4884-ad67-4f87b7f4854f","added_by":"auto","created_at":"2020-06-13 02:44:37","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":673669,"visible":true,"origin":"","legend":"Effects of the Morris water maze test on the weight in BALB/c and C57BL/6 mice infected with Angiostronglus cantonensis: (a) BALB/c non-swimming group, (b) BALB/c swimming group, (c) C57BL/6 non-swimming group, (d) C57BL/6 swimming group. ─: Non-infected, ─: Infected.","description":"","filename":"Figure3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9315/v3/Figure3.jpeg"},{"id":1322943,"identity":"47aa274b-b614-4cc9-b764-d59e24517a13","added_by":"auto","created_at":"2020-06-13 02:44:37","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":490198,"visible":true,"origin":"","legend":"Effects of the Morris water maze test on the food intake in (a) BALB/c and (b) C57BL/6 mice infected with Angiostronglus cantonensis. ─: Uninfected mice, ─: Infected mice undertaking the test.","description":"","filename":"Figure4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9315/v3/Figure4.jpeg"},{"id":1322944,"identity":"fcce5386-8f07-4ffd-b156-49785e8d1b71","added_by":"auto","created_at":"2020-06-13 02:44:37","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":532894,"visible":true,"origin":"","legend":"Effects of the Morris water maze test on the water intake in (a) BALB/c and (b) C57BL/6 mice infected with Angiostronglus cantonensis. ─: Uninfected mice, ─: Infected mice undertaking the test.","description":"","filename":"Figure5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9315/v3/Figure5.jpeg"},{"id":1322945,"identity":"91adab3d-1ed5-4c87-8697-e43d6da88f73","added_by":"auto","created_at":"2020-06-13 02:44:37","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":370359,"visible":true,"origin":"","legend":"Determination of depression-like behaviors in (a) BALB/c and (b) C57BL/6 mice infected with Angiostronglus cantonensis by the force swimming test. ■: Uninfected mice, ■: Infected mice.","description":"","filename":"Figure6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9315/v3/Figure6.jpeg"},{"id":1322946,"identity":"626d58c6-8bb8-43ee-9ca1-5e2d884f3b1f","added_by":"auto","created_at":"2020-06-13 02:44:37","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":361794,"visible":true,"origin":"","legend":"Anxiety-like behaviors in C57BL/6 mice infected with Angiostronglus cantonensis by determined the open field test on day 9 post-infection. (a) Activity and (b) Walking distance. ─: Uninfected mice, ─: Infected mice.","description":"","filename":"Figure7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9315/v3/Figure7.jpeg"},{"id":1322947,"identity":"9e6a0a96-2de8-4916-98c7-b8d1b4af34f9","added_by":"auto","created_at":"2020-06-13 02:44:37","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":742276,"visible":true,"origin":"","legend":"Comparison of spatial learning ability in mice by infected with Angiostronglus cantonensis by determining escape latency to the platform in the Morris water maze test. (a-c) BALB/c and (d-f) C57BL/6 mice. ─: Uninfected mice, ─: Infected mice.","description":"","filename":"Figure8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9315/v3/Figure8.jpeg"},{"id":1322948,"identity":"c99ca297-5c4e-4b49-b8d6-0a3b5f45341c","added_by":"auto","created_at":"2020-06-13 02:44:37","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":564102,"visible":true,"origin":"","legend":"Comparison of spatial memory in mice infected with Angiostronglus cantonensis by determining time in the target quadrant (a. BALB/c and c. C57BL/6) and escape latency to the target quadrant in the Morris water maze test (b. BALB/c and d. C57BL/6). ■: Uninfected mice, ■: Infected mice.","description":"","filename":"Figure9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9315/v3/Figure9.jpeg"},{"id":15667322,"identity":"2711c7fd-0a12-401d-9544-303b6313be43","added_by":"auto","created_at":"2021-11-18 13:41:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":782738,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9315/v3/baca6ae3-8d60-4920-8e18-9a1d47647986.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003e\u003cem\u003eAngiostrongylus\u003c/em\u003e \u003cem\u003ecantonensis\u003c/em\u003e causes cognitive impairments in heavily infected BALB/c and C57BL/6 mice\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eParasitic infections have been reported to have significant effects on behavior, learning, and memory of the hosts. Mice infected with \u003cem\u003eHeligmosomoides\u003c/em\u003e \u003cem\u003epolygyrus\u003c/em\u003e and \u003cem\u003eEimeria\u003c/em\u003e \u003cem\u003evermiformis\u003c/em\u003e were respectively observed to have poor spatial learning and a reduced avoidance of the cat odor [1, 2]. However, rodents heavily infected with \u003cem\u003eStrongyloides\u003c/em\u003e \u003cem\u003eratti\u003c/em\u003e did not show deficits in learning [3]. Although inconsistent findings were obtained in infected rodents with gastrointestinal parasites, neurotropic parasites such as \u003cem\u003eToxocara\u003c/em\u003e \u003cem\u003ecanis\u003c/em\u003e [4] and \u003cem\u003eToxoplasma\u003c/em\u003e \u003cem\u003egondii\u003c/em\u003e [5-7] have been documented to cause learning impairments in the hosts. Moreover, cysts of \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e in specific forebrain areas of rats had been attributed to attenuation of predator odor aversion and changes in anxiety-related behavior [6]. Association between \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e and anxiety and cognitive disorders has also been reported [8, 9].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAngiostrongyl\u003c/em\u003e\u003cem\u003eus\u003c/em\u003e \u003cem\u003ecantonensis\u003c/em\u003e is a neurotropic nematode causing eosinophilic meningitis and/or eosinophilic meningoencephalitis in human. The infection is due to ingestion of raw or uncooked intermediate host (snails or slugs), paratenic hosts (crustaceans, frogs, fishes, or planarians), or vegetables contaminated with the infective third-stage larvae (L3) [10, 11]. After ingestion, L3 penetrate the small intestine and reach the central nervous system through bloodstream. In the brain, the worms molt twice and develop into the fifth-stage larvae or young adults. The young adults cause a series of pathological and immunological changes [12]. Severe headaches, fever, nausea, vomiting, neck stiffness, and neurologic abnormalities are the common clinical manifestations persisting for weeks to months. Although most human infections are self-limited, severe infection may lead to irreversible neurological damages and even death [13]. Using the water maze, BALB/c mice have been determined in learning and memory deficits 1-2 weeks post-infection [14].\u003c/p\u003e\n\u003cp\u003eIn our previous studies, we have reported that \u003cem\u003eA.\u003c/em\u003e \u003cem\u003ecantonensis\u003c/em\u003e causes significant pathological changes in the brain of infected mice especially in the hippocampus [15] and different temporal-spatial expressions of IL-4, IL-10, and IL-13 between of the infected Th-2 dominant BALB/c and Th-1 dominant C57BL/6 mice [16]. Rats with hippocampal damages required a prolong duration to find the test platform in Morris water test [17]. Moreover, the hippocampal gyrus has been related to depression [18, 19]. Daily feeding of \u003cem\u003eLactobacillus\u003c/em\u003e \u003cem\u003ehelveticus\u003c/em\u003e to rats with ammonium acetate induced chronic hyperammonemia has been reported to elevate cognitive functions [20]. Treatment with the emotional stabilizer LiCl to young rats inoculated with \u003cem\u003eStreptococcus\u003c/em\u003e \u003cem\u003epneumoniae\u003c/em\u003e not only prevented apoptosis and reduced damages in hippocampus but also improve spatial memory and learning [21]. In addition, icariin has been to be effective in treating mice with traumatic brain injuries [22]. Since \u003cem\u003eA.\u003c/em\u003e \u003cem\u003ecantonensis\u003c/em\u003e infection causes damages to the hippocampus, these changes may lead to impairments in the behavior, learning, and memory of the host. In this study, we employed the force swimming test to evaluate level of depression, open field test to assess anxiety, and Morris water maze test to determine changes in learning and memory in heavily infected BALB/c and C57BL/6 mice with \u003cem\u003eA.\u003c/em\u003e \u003cem\u003ecantonensis\u003c/em\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch3\u003eParasite and laboratory animals\u003c/h3\u003e\n\u003cp\u003eA Taiwan strain of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecantonensis\u003c/em\u003e has been maintained in our laboratory since 1980. Its life cycle is maintained through \u003cem\u003eBiomphalaria\u003c/em\u003e \u003cem\u003eglabrata\u003c/em\u003e snails and Sprague-Dawley (SD) rats [16]. The rats for life maintenance and 7-8-week-old BALB/c (H-2\u003csup\u003ed\u003c/sup\u003e) and C57BL/6 (H-2\u003csup\u003eb\u003c/sup\u003e) mice for experimental studies were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan). These animals were reared in the Laboratory Animal Center of Chang-Gung University. They were kept in plastic cages and provided with food and water \u003cem\u003ead\u003c/em\u003e \u003cem\u003elibitum\u003c/em\u003e. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Chang Gung University (IACUC Approval No.: CGU15-193).\u003c/p\u003e\n\u003ch3\u003eExperimental infection\u003c/h3\u003e\n\u003cp\u003eOn day 21 post-infection, tissues of the snail hosts were removed and homogenized after shell crushing. The tissue samples were digested with 0.6% (w/v) pepsin-HCl (pH 2-3) for at 37\u0026deg;C for 1 h [23]. L3 of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecantonensis\u003c/em\u003e were removed from the standing precipitations using a medical dropper and counted under a dissecting microscope. Each mouse was inoculated with 50 L3 by stomach intubation.\u003c/p\u003e\n\u003ch3\u003eDetermination of weight and dietary consumption\u003c/h3\u003e\n\u003cp\u003eSince mice undertaking the tests have to exercise in the training and testing stages, it is required to determine whether exercise affects the status of infection. There were two controls groups (10 BALB/c mice and 10 C57BL/6 mice) and two corresponding infected groups with equal numbers. Throughout the experimental studies, each mouse was weighed daily. In addition, the daily dietary consumption was determined by recording food and drinking water intakes.\u003c/p\u003e\n\u003ch3\u003eForced swimming test\u003c/h3\u003e\n\u003cp\u003eThe forced swim test is a behavioral test used for the evaluation on the level of depression after \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecantonensis\u003c/em\u003e infection. There were 10 mice in each of the control and infected groups. The test was carried out on days 7, 14, and 21 post-infection. The tested mouse is placed in a transparent cylindrical tank (45 cm height \u0026times; 10 cm radius) and filled with water (25 \u0026plusmn; 1\u0026deg;C) at a level of 15 cm from the bottom for 6 min. The duration for the mouse without swimming in the last 4 min is recorded [24].\u003c/p\u003e\n\u003ch3\u003eOpen field test\u003c/h3\u003e\n\u003cp\u003eThe open field test was used to assess anxiety-like behavior in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecantonensis\u003c/em\u003e infected mice. There were 10 mice in each of the control and infected groups. The test was carried out on day 9 post-infection. The open field apparatus consisted of a square tank (40 \u0026times; 40 cm) with walls 40 cm high made of gray polyvinyl chloride plastic board. Mice were transported to the testing room and left in their home cages for 1 h. The animal was placed in the middle of the tank and allowed to move freely. Speed and time of the locomotor activity was monitored for 1 h. Tendency to anxiety was defined as time in the middle less than time on the edge [25].\u003c/p\u003e\n\u003ch3\u003eMorris water maze test\u003c/h3\u003e\n\u003cp\u003eThe Morris water maze test was used to assess the effect of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecantonensis\u003c/em\u003e on spatial learning and memory. There were 10 mice in each of the control and infected groups. The mice were trained on days 3-6, 10-13, and 17-20 post-infection and tests on days 7, 14, and 21 days post-infection. The custom-made maze was a circular pool 120 cm in diameter and 40 cm in height, filled with water at 25 \u0026plusmn; 1\u0026deg;C making opaque with milk. In the acquisition session, each mouse was given four trials per day and four days of training in total to find a hidden platform located 1.5 cm below the water surface. Each mouse was placed into the pool, facing the wall, with a different starting point for each trial that the direct route to the platform differs each time. The time required by the mouse to find and stand on the platform was recorded for up to 90 s. The mouse was allowed to stay on the platform for 30 s. It was then removed from the maze and placed into its cage. For the mouse did find the platform within 90 s, the animal was placed on the platform for 30 s. The inter-trial interval was at least 30 min. In the probe session, on day 5, the platform was removed from the pool. The mouse was tested in a probe trial for 60 s. Mouse swimming tracts were recorded using a TopScan automated tracking system (Clever Sys Inc., Virginia, USA) [26].\u003c/p\u003e\n\u003ch3\u003eDetermination of worm recovery\u003c/h3\u003e\n\u003cp\u003eThe infected mice were sacrificed by inhalation of 3% (v/v) isoflurane (Panion \u0026amp; BF Biotech Inc., Taipei, Taiwan). Brains were removed from the cranial cavity. Worm recovery rates were then determined by counting the number worms under a dissecting microscope.\u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eStatistical analyses were performed using Microsoft Excel 2010 for Windows (Microsoft, Redmond, WA, USA). Data were expressed as mean \u0026plusmn; standard deviation. Differences between groups were analyzed by Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered to be statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003ch3\u003eWorm recovery and survival rate\u003c/h3\u003e\n\u003cp\u003eThe worm recovery rate in BALB/c mice tested with the Morris water maze significantly decreased with the time of infection (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e4)\u003c/sub\u003e = 0.000169, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) whereas that of C57BL/6 mice decreased only on day 21 post-infection (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e4)\u003c/sub\u003e = 0.001452, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). In addition, the rate in the BALB/c mice was significantly lower than that of C57BL/6 mice on days 14 (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e4)\u003c/sub\u003e = 0.000331, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) and 21 (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e4)\u003c/sub\u003e = 0.006658, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) (Fig. 1).\u003c/p\u003e\n\u003cp\u003eAll animals in the control groups remained alive on day 42 post-infection, giving a survival rate of 100% (data not shown). The survival rate in BALB/c without undertaking the water maze test (the non-swimming group) rapidly decreased to 0% by day 25 whereas those undertaking the test (the swimming group) survived three more days. Among the C57BL/6 mice, the survival rate only became 70% on day 42 in the non-swimming group and that in the swimming remained 85.7% on the same day. The mortality rate in the swimming group decreased from 30% to 14% (Fig. 2).\u003c/p\u003e\n\u003ch3\u003eChanges in weight\u003c/h3\u003e\n\u003cp\u003eIn the non-swimming group, there was no significant difference in weight of infected and non-infected BALB/c mice up to day 13 post-infection (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.320876, \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). From day 14, the weight of the infected group significantly decreased (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.006903, P \u0026lt; 0.01) (Fig. 3a). On day 21, the non-infected mice had a weight gain of 7% and the infected one had a weight loss of 24%. A similar decreasing pattern was observed in the swimming group. Significantly differences in weight of the infected animals were found since day 13 (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.011617, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig. 3b). On day 21, the non-infected mice had a weight gain of 7% and the infected one had a weight loss of 21%.\u003c/p\u003e\n\u003cp\u003eIn the non-swimming group, the weights of infected C57BL/6 mice were found to be significantly lower than the controls (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.031012, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig. 3c). The infected mice had a weight loss of 12% on day 21 post-infection whereas the uninfected controls had a weight of 10%. In the swimming group, the C57BL/6 mice had a change pattern similar to the BALB/c counterparts. From days 1-11, no significant difference was found in the infected and uninfected mice (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.326197, \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) and the weight of the infected ones significantly decreased since day 12 (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.037645, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig. 3d). On day 21, the uninfected mice has also a weight gain of 10% while the infect ones only had a weight loss of 10%.\u003c/p\u003e\n\u003ch3\u003eDietary consumption\u003c/h3\u003e\n\u003cp\u003eIn BALB/c mice, there was no significant difference in food intake between the uninfected control and infected one in the swimming group up to day 12 post-infection (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.113181, \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). Since day 13, the food intake in the infected group significantly decreased (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.004678, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) (Fig. 4a). There was also no difference in food intake in C57BL/6 infected mice in the swimming group and the uninfected controls up to day 12 (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.692658, \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) and that in the infected ones became significantly decreased since day 13 (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.004806, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) (Fig. 4b).\u003c/p\u003e\n\u003cp\u003eIn BALB/c mice, there was no significant difference in water intake between the uninfected control and infected one in the swimming group up to day 11 post-infection (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.794676, \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). Since day 12, the water intake in the infected group significantly decreased (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.002576, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) (Fig. 5a). There was also no difference in food intake in C57BL/6 infected mice in the swimming group and the uninfected controls up to day 13 (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.204049, \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) and that in the infected ones became significantly decreased since day 14 (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.019749, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig. 5b).\u003c/p\u003e\n\u003ch3\u003eDepression- and anxiety-like behavior\u003c/h3\u003e\n\u003cp\u003eBy the force swimming test, the infected BALB/c mice were determined to have longer immobility time than the uninfected ones on day 21, indicating the occurrence of depression (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.018582, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig. 6a). No significant difference was found in the immobility time in the infected and uninfected C57BL/6 mice on day 7 (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.158094, \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). Moreover, the immobility time in the infected mice was found to be significantly shorter than the uninfected ones on day 14 (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.013031, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) and day 21 (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.00011, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001), indicating an increase in activities and no depression occurred (Fig. 6b). Although no special findings was observed in the infected BALB/c mice by the open field test, the infected C57BL/6 mice were found to have significantly higher activity (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(9)\u003c/sub\u003e = 0.024914, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig. 7a) and longer walking distance (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(9)\u003c/sub\u003e = 0.029771, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig. 7b) the uninfected ones on day 9 post-infection, indicating that anxiety-like behavior occurred in these animals (Fig. 7).\u003c/p\u003e\n\u003ch3\u003eChanges in spatial learning\u003c/h3\u003e\n\u003cp\u003eAfter infection with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecantonensis\u003c/em\u003e, BALB/c and C57BL/6 mice were continuously trained in water maze for 4 days to observe their spatial learning ability. Before day 7, no significant difference in escape latency to the platform was found in infected and uninfected BALB/c mice (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(9)\u003c/sub\u003e = 0.561855, \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) (Fig. 8a). Since day 12, the latency in the infected mice was found to be significantly higher than that of the uninfected ones (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.000049, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) (Fig. 8b and 8c), indicating that the spatial learning ability of infected mice significantly decreased. Although infected and uninfected C57BL/6 mice showed no significant difference in spatial learning ability from day 1 to day 14 (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.803257, \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) (Fig. 8d), the latency in the infected ones became significantly higher after day 14 (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(\u003c/sub\u003e\u003csub\u003e9)\u003c/sub\u003e = 0.013565, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig. 8e and 8f), indicating significant lower spatial learning ability also occurred in the infected C57BL/6 mice, although the occurring time was delayed.\u003c/p\u003e\n\u003ch3\u003eChanges in spatial memory\u003c/h3\u003e\n\u003cp\u003eAfter removing water maze platform, the infected BALB/c mice stayed in the target quadrant of the original platform significantly shorter than the uninfected group (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(9)\u003c/sub\u003e = 0.002013, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) (Fig. 9a) and the time for the infected mice moving to the target quadrant was significantly longer than that of the uninfected ones (Fig. 9b) on days 7 (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(9)\u003c/sub\u003e = 0.016921, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), 14 (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(9)\u003c/sub\u003e = 0.015133, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), and 21 (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(9)\u003c/sub\u003e = 0.000047, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). In the infected C57BL/6 mice, these phenomena only occurred on days 7 (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(9)\u003c/sub\u003e = 0.03045, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) and 21 (\u003cem\u003et\u003c/em\u003e\u003csub\u003e(9)\u003c/sub\u003e = 0.004776, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) (Fig. 9c and 9d). Although the spatial memory ability of the two strains of mice significantly decreased, the ability remained intact in the C57BL/6 in the early stage of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecantonensis\u003c/em\u003e infection.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMice undertaking the water maze test were required to swim for 4 days in the training stage. The tested infected BALB/c mice may survive after day 28 and C57BL/6 mice had a high survival rate of 87% (Fig. 2). Moreover, the weight loss in those undertaking the test also became lower (Fig. 3). It has been reported that moderate physical exercise may promote metabolism, blood circulation, muscle growth, and immunity, thereby preventing or delaying cardiovascular disease [27] and diabetes [28]. In addition to elevation of the proinflammatory cytokines IL1\u0026beta;, TNF-\u0026alpha;, and IL-6 [29], exercise may reduce C-reactive proteins in patients with coronary arteriosclerosis [30] and Type II T helper cytokines (IL-5 and IL-13) in asthmatic BALB/c mice [31]. These findings indicate that regular exercise may ease the clinical symptoms of mice infected with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecantonensis\u003c/em\u003e. However, this suggestion requires further experiments.\u003c/p\u003e\n\u003cp\u003eIn our previous study, eosinophilic meningitis was found to occur since day 12 post-infection in ICR mice infected with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecantonensis\u003c/em\u003e and pathologic changes were not limited to the meninges but also in the cerebral parenchyma especially in the hippocampus [13]. Hippocampal reticular injury has been associated with depression [18] and the length of depression is proportional to the volume of hippocampal gyrus loss [19]. In addition, the hypoxia-pituitary-adrenal (HPA) axis loss of function [30] or persistent inflammation [34] may also lead to depression. By the forced swimming test, the infected BALB/c mice were showed to have longer immobility time than the infected C57BL/6 mice. These phenomena may be associated with hippocampus and has been reported to be proportional to the mRNA expression of inducible nitric oxide synthase (iNos) [35].\u003c/p\u003e\n\u003cp\u003eIn this study, we demonstrated that the recovery rate of worms in the two strains of mice infected with \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecantonensis\u003c/em\u003e decreased on day 21 (Fig. 1) and mortality of the animals also began to occur around this day (Fig. 2). It is possible that most of young adults may be eliminated by the host immune system. The worms survived around day 35 may continue to live. Moreover, the weight of infected mice began to decline since day 14 (Fig. 3). This finding may be associated with the decrease in food (Fig. 4) and water (Fig. 5) intakes. Since the development of L3 to young adults since day 14 [36], these worms may cause inflammation in the brain of host and changes in behavior. These phenomena may explain the results of the water maze test. Although the infected mice are able to find the hidden platform since day 14, the time stay in this location becomes significantly shorter and the spatial learning and memory abilities significantly reduced.\u003c/p\u003e\n\u003cp\u003eBy forced swimming test, infected BALB/c mice were demonstrated to develop depression on day 21. However, infected C57BL/6 mice had significant shorter immobility time (Fig. 6). They were demonstrated to became more active on day 9 post-infection had a total moving distance of 46 m more than the non-infected ones by the open field test (Fig. 7). These findings suggest that the young adults of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecantonensis\u003c/em\u003e may cause mechanical damages to the brain of the host. In the water maze test also demonstrated that the infected BALB/c mice required longer time to find the hidden platform and had shorter residence time at this position than their C57BL/6 counterpart (Fig. 9), showing the spatial memory ability of infected BALB/c mice was more severely reduced.\u003c/p\u003e\n\u003cp\u003eChanges in immune responses have been associated with spatial memory. Although SCID BALB/c mice had a lower spatial memory, significant spatial improvement d memory learning was after injection of T cells in nude mice [37]. A water maze test demonstrated that C57BL/6 mice shaved with the IL-4 gene had low spatial memory learning ability [38]. Combined treatment with Am80 and HX630 to the C57BL/6 mice with amyloid-protein precursor 23 (A\u0026beta;PP23) gene (Alzheimer\u0026rsquo;s disease) may be effective in restoring spatial memory learning ability through the increase of the IL-4 content in the hippocampus [39]. Injection of IL-4 C-4BL/6 mice with cerebral ischemia has been found to be effective in improving the spatial memory and learning abilities [40]. These results suggest that T cells may secrete IL-4 to reduce the loss of spatial memory learning ability in injured brain.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecantonensis\u003c/em\u003e is able to cause cognitive impairments in heavily infected mice. The infection may cause depression, anxiety, and impairments in spatial learning and memory in heavily infected mice. Moreover, significantly higher severity was observed in the Th-2 dominant BALB/c mice.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eAvailability of data and materials\u003c/h3\u003e\n\u003cp\u003eData supporting the conclusions of this article are included within the article.\u003c/p\u003e\n\u003ch3\u003eAcknowledgements\u003c/h3\u003e\n\u003cp\u003eWe would like to thank Chung-Han Li, Yi-Tang Lin for technical support.\u003c/p\u003e\n\u003ch3\u003eAuthors\u0026rsquo; contributions\u003c/h3\u003e\n\u003cp\u003eKYC and LCW designed and coordinated this study and write the manuscript. KYC, KYJ, GJL, PKC, RYT and CJC contributed to carrying out the animal experiments. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eThis study was supported in part by grants from the Ministry of Science and 353 Technology, Republic of China (Grand No.: MOST 105-2320-B-182-028-MY3), and the Chang Gung Memorial Hospital (Grand No. CMRPD1H0342 and CMRPD1H0442).\u003c/p\u003e\n\u003ch3\u003eEthics approval\u003c/h3\u003e\n\u003cp\u003eLaboratory animal application in this study were approved by the Institutional Animal Care and Use Committee, Chang Gung University (IACUC Approval No.: CGU15-193).\u003c/p\u003e\n\u003ch3\u003eConsent for publication\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch3\u003eCompeting interests\u003c/h3\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch3\u003eAuthor details\u003c/h3\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Department of Parasitology, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan. \u003csup\u003e2\u003c/sup\u003e Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan. \u003csup\u003e3\u003c/sup\u003e Molecular Infectious Disease Research Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKavaliers M, Colwell DD. 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Anti-apoptotic effects of Sonic hedgehog signalling through oxidative stress reduction in astrocytes co-cultured with excretory-secretory products of larval \u003cem\u003eAngiostrongylus\u003c/em\u003e \u003cem\u003ecantonensis\u003c/em\u003e. Sci Rep. 2017;7:41574.\u003c/li\u003e\n\u003cli\u003e24. Yankelevitch-Yahav R, Franko M, Huly A, Doron R. The forced swim test as a model of depressive-like behavior. J Vis Exp. 2015;97:52587.\u003c/li\u003e\n\u003cli\u003e25. Kuniishi H, Ichisaka S, Yamamoto M, Ikubo N, Matsuda S, Futora E, et al. Early deprivation increases high-leaning behavior, a novel anxiety-like behavior, in the open field test in rats. Neurosci Res. 2017;123:27-35.\u003c/li\u003e\n\u003cli\u003e26. Chang PK, Yu L, Chen JC. Dopamine D3 receptor blockade rescues hyper-dopamine activity-induced deficit in novel object recognition memory. Neuropharmacology. 2018;133:216-23.\u003c/li\u003e\n\u003cli\u003eStampfer MJ, Hu FB, Manson JE, Rimm EB, Willett WC. 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Am J Epidemiol. 1969;89: 331-44.\u003c/li\u003e\n\u003cli\u003eKipnis J, Cohen Ha, Cardon M, Ziv Y, Schwartz M. T cell deficiency leads to cognitive dysfunction: Implications for therapeutic vaccination for schizophrenia and other psychiatric conditions. PNAS. 2004;101: 8180-5.\u003c/li\u003e\n\u003cli\u003eDerecki NC, Cardani AN, Yang CH, Quinnies KM, Crihfield A, Lynch KR, et al. Regulation of learning and memory by meningeal immunity: a key role for IL-4. J Exp Med. 2009;207: 1067-80.\u003c/li\u003e\n\u003cli\u003eKawahara K, Suenobu M, Yoshida A, Koga K, Hyodo A, Ohtsuka H, et al. Intracerebral microinjection of interleukin-4/interleukin-13 reduces \u0026beta;-amyloid accumulation in the ipsilateral side and improves cognitive deficits in young amyloid precursor protein 23 mice. Neuroscience. 2012;207:243-60.\u003c/li\u003e\n\u003cli\u003eLiu X, Liu J, Zhao S, Zhang H, Cai W, Cai M. Interleukin-4 is essential for microglia/macrophage M2 polarization and long-term recovery after cerebral ischemia. Stroke. 2016;47:498-504.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Angiostrongylus cantonensis, behavior, forced swimming test, learning, memory, Morris water maze test, open field test","lastPublishedDoi":"10.21203/rs.2.18697/v3","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.18697/v3","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Parasitic infections may cause significant effects on behavior, learning, and memory of the hosts. In the brain of mice heavily infected with \u003cem\u003eAngiostrongylus\u003c/em\u003e \u003cem\u003ecantonensis\u003c/em\u003e, severe damages have been observed in the hippocampus. This component has been considered to have associations with spatial learning and memory in human and vertebrates. This study was designed to determine the impairments in behavior, learning, and memory in BALB/c and C57BL/6 mice heavily infected with the parasite.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Each mouse was inoculated with 50 third-stage larvae of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecantonensis\u003c/em\u003e. After infection, daily changes in weight and dietary consumption, worm recoveries and survival rates were determined. The forced swimming test, open field test, and Morris water maze test were employed to evaluate depression- and anxiety-like behavior as well as impairments in spatial learning and memory, respectively. In addition, in the two strains of mice were also determined.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The worm recovery rate in the BALB/c mice was significantly lower than that of C57BL/6 mice since day 14 post-infection. The survival rate in infected BALB/c mice decreased to 0% by day 25 whereas those with swim-training survived three more days. On day 42, the C57BL/6 mice had a survival rate of 85.7% in the swimming group and 70% in the non-swimming group. Significant differences were found in weight between infected and non-infected BALB/c and C57BL/6 mice since day 13 and day 12, respectively with corresponding changes in the dietary consumption. Depression-like behavior was found in the infected BALB/c mice but not in C57BL/6 mice. However, anxiety-like behavior was found to occur only in C57BL/6 mice. Impaired spatial learning and memory were also found in the two strains of mice occurred since day 14 post-infection.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Results of this study indicate that \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ecantonensis\u003c/em\u003e causes depression, anxiety, and impairments in spatial learning and memory in heavily infected mice. Moreover, significantly higher severity was observed in the Th-2 dominant BALB/c mice.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","manuscriptTitle":"Angiostrongylus cantonensis causes cognitive impairments in heavily infected BALB/c and C57BL/6 mice","msid":"","msnumber":"","nonDraftVersions":[{"code":3,"date":"2020-06-13 02:44:36","doi":"10.21203/rs.2.18697/v3","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2020-06-10T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-06-09T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-06-09T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 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