Cannabidiol increases survival and promotes rescue of cognitive function in a murine model of cerebral malaria

Accepted Manuscript Cannabidiol increases survival and promotes rescue of cognitive function in a murine model of Cerebral Malaria Alline Cristina de Campos, Fatima Brant, Aline Silva Miranda, Fabiana Simão Machado, Antônio Lucio Teixeira PII: DOI: Reference:

S0306-4522(15)00019-6 http://dx.doi.org/10.1016/j.neuroscience.2014.12.051 NSC 15951

To appear in:

Neuroscience

Accepted Date:

31 December 2014

Please cite this article as: A.C. de Campos, F. Brant, A.S. Miranda, F.S. Machado, A.L. Teixeira, Cannabidiol increases survival and promotes rescue of cognitive function in a murine model of Cerebral Malaria, Neuroscience (2015), doi: http://dx.doi.org/10.1016/j.neuroscience.2014.12.051

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Original article

Cannabidiol increases survival and promotes rescue of cognitive function in a murine model of Cerebral Malaria. Alline Cristina de Campos1,2,3*; Fatima Brant1,2; Aline Silva Miranda1,2 ; Fabiana Simão Machado1,2 and Antônio Lucio Teixeira2

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Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil. 2

Infectious Diseases and Tropical Medicine Graduate Program, School of Medicine, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil. 3

Department of Pharmacology, School of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil.

*corresponding author Alline C Campos Interdisciplinary Laboratory of Medical Investigation, Room 281 Department of Internal Medicine/Infectious Disease and Tropical Medicine Graduate Program School of Medicine - Federal University of Minas Gerais Av. Alfredo Balena, 190, Belo Horizonte 31130-100, Brazil. Tel.: +55 31 34098073. E-mail: [email protected]

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Abstract Cerebral malaria (CM) is a severe complication resulting from Plasmodium falciparum infection that might cause permanent neurological deficits. Cannabidiol (CBD) is a nonpsychotomimetic compound of Cannabis sativa with neuroprotective properties. In the present work, we evaluated the effects of CBD in a murine model of CM. Female mice were infected with Plasmodium berghei ANKA (PbA) and treated with CBD (30mg/Kg/day- 3 or 7 days i.p.) or vehicle. On 5th day-post-infection (dpi), at the peak of the disease), animals were treated with single or repeated doses of Artesunate, an antimalarial drug. All groups were tested for memory impairment (Novel Object Recognition or Morris Water Maze) and anxiety-like behaviors (Open field or elevated plus maze test) in different stages of the disease (at the peak or after the complete clearance of the disease). Th1/Th2 cytokines and neurothrophins (BDNF and NGF) were measured in prefrontal cortex and hippocampus of experimental groups. PbA-infected mice displayed memory deficits and exhibited increase in anxiety-like behaviors on the 5dpi or after the clearance of the parasitemia, effects prevented by CBD treatment. On 5dpi, TNF-α and IL-6 increased in the hippocampus, while only IL-6 increased in prefrontal cortex. CBD treatment resulted in increase in BDNF expression in the hippocampus and decreased levels of proinflammatory cytokines in the hippocampus (TNF-α) and prefrontal cortex (IL-6). Our results indicate that CBD exhibits neuroprotective effects in CM model and might be useful as an adjunctive therapy to prevent neurological symptoms following this disease. Keywords: Cerebral Malaria; Cannabidiol; inflammation; Cytokines; Brain Derived Neurotrophic Factor.

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Introduction Cerebral Malaria (CM) is the most common form of Plasmodium infection complication. CM is defined as a diffuse encephalopathy characterized by reduced level of arousal, seizures, headache and potentially irreversible neurological symptoms, including behavioral changes and cognitive impairment, being responsible for at least 80% of lethal cases (Molyneux et al. 2000). The pathophysiology of CM results from a combination of exacerbated immunological response, mainly mediated by Th1 mechanisms (Clark and Rockett 1994, Lacerda-Queiroz et al. 2010; Miranda et al. 2013), and mechanical obstruction of the blood flow in the brain by erythrocyte sequestration (Berendt et al. 1994). Together, these processes may affect several brain structure functions leading to long lasting cognitive deficits observed in CM survivors (Carter et al. 2005; Crawley et al. 2001). Despite the effectiveness of antimalarial drugs on treating most Malaria related symptoms, their efficacy on promoting survival and preventing neurological damage in CM cases have been contested (Dao et al. 2007, Dondorp et al. 2005). In this scenario, the combination of antimalarial drugs and neuroprotective compounds would be an interesting strategy for the improvement of the neurological outcome in CM patients (Dai et al. 2012; de Souza et al. 2010; de Miranda et al. 2011a). In the last decade, cannabinoids have emerged as putative modulators of the central nervous system (CNS) immune and plastic events as well as behavioral and cognitive functions (Campos et al. 2012b; Hu et al. 2011, Kaplan 2013; Marchalant et al. 2009; Pazos et al. 2013). Phytocannabinoids are a group formed by terpenophenolic substances derived from Cannabis sativa plant. The plant

produces several compounds,

including two

majors: ∆9-

tetrahydrocannabinol, responsible for the main psychoactive effects induced by Cannabis, and Cannabidiol (CBD), the other major bioactive compound (Mechoulam et al. 1965; Pertwee 3

2005). Regarding the latter, a wealth of evidence indicates that CBD exhibits a wide spectrum of pharmacological properties, including neuroprotective (Campos et al. 2012b), and antiinflammatory (Esposito et al. 2011; Mecha et al. 2013; Napimoga et al. 2009; Saito et al., 2012). CBD decreases the production of inflammatory cytokines and the activation of microglial cells (Kozela et al. 2010, Kozela et al. 2011, Napimoga et al. 2009). It also preserves cerebral circulation during ischemic events and attenuates vascular changes in a model of sepsis-related encephalopathy (Alvarez et al. 2008; Mishima et al. 2005; Ruiz-Valpenas et al. 2011). The effects of CBD in CM remain to be evaluated. In the present work, we demonstrated that CBD repeated injections increase the survival and promote the rescue of both the long-term cognitive deficits and anxiety-like behaviors in a murine model of CM. These effects seem to involve the combined anti-inflammatory and neuroprotective effects of CBD, as evidenced by our results involving cytokines and BDNF levels in the hippocampus. Experimental procedures Animals: Female C57BL/6 mice (6-8 weeks old) were obtained from the Animal Care Facility of the Institute of Biological Sciences, Federal University of Minas Gerais. All animals were housed in groups of 5 mice per cage in a room controlled for temperature (22◦C) and light conditions (lights on at 7:00 am; lights off 7:00 pm) with food and water ad libitum. All experimental procedures described here were previously approved by local ethics committee (CETEA; license number 188/11) and were conducted in accordance to EU Directive 2010/63/EU on the protection of animals used for scientific purposes. Experiments were carried out during the light phase of the cycle (9:00 am-15:00 pm).

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Drugs: Cannabidiol (30mg/Kg- Campos et al., 2013a), kindly supplied by THC Pharm, (Frankfurt/Main, Germany) was dissolved in a mixture of 2% of Tween 80 and saline; Artesunate (64 or 32mg/Kg- Clemmer el at. 2011), purchased from Sigma (St. Louis, MO) was dissolved in 5% sodium bicarbonate in saline. All control groups received vehicle solution as a control for CBD and Artesunate injections. Plamodium berghei ANKA infection and parasitemia assessment: Mice were infected intraperitoneally (i.p.) with 106 red blood cells infected with stages of P. berghei ANKA (PbA) constitutively expressing green fluorescent protein (P. berghei ANKA-GFP- 15cy1 clone) suspended in 0.2 mL PBS (Janse et al. 2006). Non-infected animals received the same volume of vehicle. Parasitemia was determined by flow cytometry. Briefly, a drop of blood was collected from the tail of each animal and dissolved in 2 ml of PBS. Samples were ran on a FACScalibur (Becton Dickinson, San Jose, CA) flow cytometer and read at a 488 nm argon laser by DIVA software (Becton Dickinson, San Jose, CA). Erythrocytes were identified on the basis of their specific forward (FSC) and side (SSC) light-scattering properties, and a total of 100,000 events were counted for each sample. A non-infected blood sample was used as a normalization sample. All groups of mice were observed daily for parasitemia, survival, and neurological signs of CM, as previously described (Miranda et al. 2013). Behavioral analysis: Rapid Murine Coma and Behavior Scale (RMCBS): The progression of CM related symptoms was analyzed by the RMCBS protocol (Carrol et al. 2010). This method intends to simulate the situation found in the clinical practice, and attempts to bring the animal model closer to the human disease. The RMCBS consists of ten parameters based on the components of the SHIRPA 5

score (Gait, balance, motor performance, body position, limb strength, touch scape, pinna reflex, toe pinch, aggression and grooming), each scored from 0 as the lowest, to 2 as the highest, with a maximum possible score of 20. In the present study, the behavioral assessment was carried out from day 3 until day 12 post-infection (p.i.). New Object Recognition Test: The new object recognition (NOR) test was performed on day 17 p.i. Briefly, animals were allowed to explore a square arena (open field - 50 cm x 50cm x 30cm) during 5 min (habituation session). Twenty-four hours later, a training session was conducted. All animals were again allowed to explore the open field for 10 min, however at this time they were exposed to two identical objects (object A1 and A2; Double Lego Toys) positioned in two adjacent corners (at 8 cm distant from the walls). The long-term retention memory (LTM) test took place 24 h after the training session. During the test session, mice explored the open field for 5 min but now in the presence of the familiar (A) and a novel (B) objects. Objects (new and old) were distinct in shape and color but were made by the same material (plastic). The exploratory preference was defined according to Bevins and Besheer (2006) as the percentage of the total time that animal spent investigating the new object. It was calculated by the ratio TN / (TO+TN) * 100 (TN = time spent exploring the new object; TB = time spent exploring the familiar object). The test took place in the light phase of the cycle (10am -2pm) The distance traveled in the apparatus was also measured as a locomotor activity index by the Anymaze software (Stoelting Co., Wood Dale, IL). An investigator blinded to the animal treatments analyzed all tests. Elevated Plus Maze test: The elevated plus-maze (EPM) test was conducted on day 18 p.i. The apparatus was composed by two open arms (30 cm x cm 0,25cm) opposed to two enclosed arms (30cm x 7cm x 15cm), and elevated 60 cm from the floor (made of dark grey plastic). In the 6

beginning of the test, each mouse was placed in the central area of the apparatus with its head facing to a closed arm. The test session duration was 5 min and it was conducted in a soundattenuated temperature-controlled (25±1°C) room, illuminated by a 40W fluorescent bulb placed 3 m above the apparatus. The Anymaze software (Stoelting Co., Wood Dale, USA) was employed for behavioral analysis. It detects the position of the animal in the maze and calculates the number of entries and the time spent in open and enclosed arms. Enclosed-arm entries were considered as an indicator of locomotor activity, whereas percentage of time spent in open arms and percentage open-arm entries were used as measures of anxiety index as previously described (Campos et al., 2014; Lee and Rodgers, 1991). Open field: this test was performed in order to detect anxiety and exploratory indexes of infected animals on day 5 p.i. Animals were placed in a circular arena with transparent walls (30 cm diameter and 50 cm height) during a 5 minute session. The test took place in light phase of the cycle (10am-2pm) and it was recorded and the total crossing and anxiety indexes analyzed by the software Anymaze (Stoelting Co., Wood Dale, USA). Morris Water Maze hidden platform test: independent groups of animals were trained with 6 trials per day (from 10 am- 3pm) over 3 days (from 2nd day p.i. to 4th day p.i.).The test took place in a round blue swimming pool (diameter 900 mm x 500 mm high) filled with water (25.5 cm of depth) located in a sound-attenuated temperature-controlled (25±1°C) room. The temperature of the water was controlled (22±2ºC). During the training session, the platform (made by transparent acrylic- 24.5 cm high x 13cm diameter) was hidden 1 cm below the surface of water that was made opaque with white nontoxic paint. The starting points were changed every trial. The time necessary for each animal to reach the platform was recorded during the trials. Each trial lasted either until the mouse find the platform or for a cut-off time of 60s. All mice were 7

allowed to rest on the platform for 10s. The platform location was the same during the entire training session and animals were allowed to use clues fixed in the walls of the pool. On the day 5 p.i., the platform was removed and all group of animals were tested for the retention of the spatial memory during a 60s session. Time and length of swim path, and swim speed in all quadrants were recorded and analyzed by an automatic video tracking system (AnyMaze, Stoelting). Animals were considered to “remember” the location of the platform when, after training, they showed preference to swim in the target quadrant (represented by the % of time spent in the target quadrant). Experimental design: Experiment 1- Lethality curve and RMCBS analysis: A schematic figure of the experimental design can be seen in the figure 1. From the 3rd day p.i. until the end of the experiments, all groups were checked daily for parasitemia (until the complete parasite clearance) and survival. On the 3rd day p.i. animals started to receive the first of the seven injections of Cannabidiol or vehicle (from the 3rd day p.i to 10th day p.i. 30mg/Kg daily i.p.- protocol based in Dai et al., 2012). On day 5 p.i. animals also received an attack dose of Artesunate (64mg/Kg i.p.) and, subsequently, infected groups received daily doses of Artesunate (32mg/Kg/day for 4 days) until the 10th day p.i. Non-infected group received vehicle injections. The followed experimental groups were obtained: a) Non infected groups: i) vehicle treated or ii) CBD treated; b) PbA infected groups: i) PbA-vehicle; ii) PbA-CBD; iii) PbAvehicle-Artesunate; iv) PbA-CBD-Artesunate. RMCBS assessments were performed from the 3rd to the 12th day p.i only in infected groups. All groups of animals were followed up until 30th day p.i. when they were euthanized (Figure 1A). 8

PLEASE INSERT THE FIGURE 1 ABOUT HERE Experiment 2- Effects of CBD on behavior, cytokines and neurotrophins at day 5 p.i. without anti-malarial treatment: In the experiment 2, animals were divided in 4 groups: a) Non-infected groups: i) control-vehicle ii) control-CBD; b) PbA infected groups: i) PbA-vehicle ii) PbA-CBD. This experiment was designed to test the possible effects of CBD treatment in the course of CM. On the 3rd day p.i. animals started to receive the first of the seven injections of CBD or vehicle (from the 3rd day p.i to 5th day p.i., 30mg/Kg daily i.p. PbA-infected groups or controls were submitted to the NOR test in order to evaluate cognitive impairment on day 5 p.i. at the peak of CM symptoms (Miranda et al., 2013). As an internal control and to avoid interference of locomotor behavior on NOR results, the distance travelled by animals during the task was also measured. Additional groups were submitted to the Open field test for the detection of anxiety-like behaviors. Two hours after the behavioral tests, animals were euthanized and their brains removed for the assessment of cytokines and neurotrophin levels (Figure 1C). Experiment 3- Behavioral analysis, cytokine and neurotrophins determination on day 5th day post infection in the presence of a single dose of Artesunate. In the experiment 3, animals were divided in 6 experimental groups: a) non-infected groups: i) control-vehicle ii) control-CBD; b) PbA infected groups: i) PbA-vehicle ii) PbA-CBD iii) PbAvehicle + single dose of Artesunate iv) PbA-CBD + single dose of Artesunate (Figure 1C). At day 2 p.i., animals started to be trained in the Morris Water Maze. The learning protocol took place in the morning at least 2 hours after drugs administration. The protocol of drug administration followed the same schematic schedule of the experiment 2, except on day 5 p.i., 9

when two groups of animals (PbA-vehicle and PbA-CBD) received an i.p. injection of Artesunate 2 hours before the Morris Water Maze memory retention test (non-infected groups received vehicle). Two hours after the behavioral test, animals were euthanized under deep anesthesia, their brains removed, hippocampus and prefrontal cortex isolated for determination of cytokines and neurotrophins. PbA infected groups that did not receive Artesunate treatment (PbA-vehicle and PbA-CBD) were not tested in the Morris Water Maze memory retention test as they were not able to swim due to disease severity. They were removed from the behavioral analysis but kept in the biochemical measurements. Experiment 4- Behavioral analysis, cytokine and neurotrophins determination after parasite clearance (17-18pi): Similar to the design of the experiment 1, except by the absence of PbA-infected groups that did not receive Artesunate (as without antimalarial treatment animals died by days 5-7 p.i.). Animals were submitted on day 17 p.i. to the NOR and, on day 18 p.i., to the EPM. PbA groups (PbAvehicle + Artesunate and PbA- CBD + Artesunate) were tested after the complete clearance of the parasite and in the absence of any malaria symptom (Artesunate treatment from 5th to 10th p.i.). Control groups were also tested. Two hours after the last behavioral test, animals were euthanized and their brains removed for the assessment of cytokines and neurotrophins (figure 1C). Sample preparation and analysis: After behavioral analysis (5th or 18th days p.i.) all animals were euthanized under deep anesthesia (a mix of Ketamine-80mg/Kg and Xylazine-60mg/Kg; Rhobifarma; Hortolandia; Brazil), and hippocampal (dorsal and ventral portions) and prefrontal cortex tissues were 10

carefully removed. Samples were then homogenized in lysis buffer. Lysates were centrifuged at 10,000 rpm for 10 min at 4°C, supernatants were collected and stored at -80ºC until their use for determination of cytokines and neurotrophins levels. Th1/Th2 cytokines determination by Cytometric Bead Array (CBA): Analyses of prefrontal cortex and hippocampal cytokines levels were performed using a mouse Th1/Th2 CBA kit (BD Biosciences, San Diego, CA). The cytokines (IL-2, IL-4, IL-6, IL10, IFN-γ and TNF-α) were measured according to the instructions provided by the manufacturer, and analyzed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). Results were normalized by the total protein concentration present in each sample as determined by Bradford assay (Bradford, 1976). Brain-Derived Neurotrophic Factor and Nerve Growth Factor analysis by Enzyme-linked Immunosorbent Assay (ELISA): The concentrations of the neurotrophins BDNF and NGF were determined by ELISA in accordance to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). Results were normalized by the total concentration of protein in each sample determined by Bradford assay. Statistical analysis All obtained data were tested for normality (Kolmorov-Smirnov’s test) and homogeneity of variance (Levene’s test). Differences were detected using analysis of variance (ANOVA) followed, when appropriate, by Duncan’s post-hoc test. Lethality curves were analyzed by Log rank test. Results are expressed as mean ± Standard Error of the Mean (SEM). Learning task of the Morris Water Maze were analyzed by ANOVA of repeated measures. Significance levels

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were set as p<0.05. All statistical analyses were performed with the statistical package SPSS version 22.0 (IBM, New York, NY). Results Cannabidiol treatment increases survival in PbA-infected mice. PbA-infected mice treated with CBD presented a significant improvement in survival rates (χ2= 18.2, df= 3 p< 0.001; Figure 2) with no impact on parasitemia when compared with vehicle-treated infected animals (Table 1). PbA-infected mice that received Artesunate + CBD treatment exhibited higher rates of survival and sustained clearance of the parasite than PbAinfected mice only treated with Artesunate (Figure 2). PbA-infected mice that exhibited sustained clearance of parasitemia (Artesunate and Artesunate+CBD treated mice) had a complete rescue of the clinical signs of CM as evaluated by the RMCBS (Table 2). Combined treatment with Cannabidiol and Artesunate reversed the long-last cognitive deficits and anxiogenic-like responses induced by CM. Figure 3A shows the effect of PbA infection in the NOR performed on day 5 p.i. PbAinfected mice (vehicle and CBD-treated) presented an impairment of recognition memory when compared to the control group (vehicle treated non-infected mice). As indicated by the measures obtained in the NOR, a significant reduction in the percentage of time spent exploring the novel object was observed in PbA-infected mice on day 5 p.i. (fig. 3A; F(2,18)= 2.48; p<0.05). Moreover, there was also a significant difference in the distance travelled between PbA-infected and control groups on day 5 p.i., (fig 3B; F(2,18)= 3.06; p<0.05) that could be related to the sickness behavior also evidenced by the RMCBS scores on day 5 p.i. (Table 2).

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The cognitive deficits evidenced in the NOR persisted after the clearance of the parasitemia by Artesunate treatment (Figure 3C), indicating that the clearance of the parasite was not enough to prevent MC-induced neurological deficits. These data are in agreement with previous reports on other antimalarial drug in which authors have shown persistent cognitive and motor deficits after successful chloroquine treatment (Dai et al., 2010). Interestingly, the combined treatment with Artesunate + CBD completely rescues the cognitive performance of the infected animals in the NOR (3C; F(3,29)= 4.2; p<0.05). It is worth mentioning that no locomotor deficits were observed after complete clearance of the parasite by Artesunate, (figure 3D). Also, as evidenced by results of the Morris Water Maze test (figure 4A-B), single injection of Artesunate at the peak of the disease did not prevent the development of memory impairment induced by CM on the day 5 after infection (Figure 4B. F(3.17)= 7.03; p< 0.01). In contrast, in combination with CBD, Artesunate was effective in preventing cognitive deficits induced by CM. Although a trend of CBD treatment in improving learning performance in PbA infected mice was observed, no statistical significance was achieved in the learning task performed (Figure 4A). Our group has previously shown that PbA-infected mice exhibited an anxiogenic-like behavior in the EPM on day 5 p.i. (de Miranda et al. 2011a). This finding was replicated in the current work using a different parading, i.e. the open field test. On day 5 p.i, PbA mice treated with vehicle exhibited a decrease in the distance travelled in the center of the arena when compared to the control group. This anxiogenic-like effect was prevented by CBD treatment (Figure 5A). No effects in the number of crossing where detected (Figure 5B).

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Moreover, long-lasting analysis of anxiety-like behavior in animals that were completed free from parasites (18th day post infection) showed that PbA-Artesunate group displayed a decrease in number of entries into the open arms of the EPM (5A; F(3,27)= 3.6; p<0.05), and the time spent in the open arms (6A; F(3,27)= 7.0; p<0.01). Similarly to the results obtained in the NOR, the combined treatment with CBD + Artesunate was able to prevent PbA-induced anxietylike behavior, and turned PbA-infected animals less anxious (Figure 6A). No effect on enclosed arm entries was detected (Figure 6B).

Combined Cannabidiol and Artesunate treatment increases BDNF expression on hippocampal formation Results from the neurothrophin determination can be seen in the figure 7 (A-D). On the day 5 pi, PbA-infection tended to decrease the expression of BDNF in the prefrontal cortex (F(7,31)= 2.1; p=0.056; Figure 5A). This effect was attenuated by CBD treatment. In the hippocampal formation, CBD + Artesunate treatment significantly increased BDNF expression when compared to all other groups (F(7,31)= 13.8; p<0.001; Figure 5C). No effect on NGF expression was found in the brain areas studied (Figure 7B and 7D), although a trend in increase NGF in both prefrontal cortex and hippocampus can be observed in CBD + Artesunate group. Single dose of Artesunate, in combination or not with CBD, does not seem to have effects on BDNF or NGF production on day 5 p.i. (data not shown). Plamodium berghei ANKA infection-induced up regulation of brain cytokines is reversed by Artesunate and CBD treatment.

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On the 5th day p.i., PbA-infected mice presented increased level of TNF-α (F(7,31)= 2.7; p<0.05) in the hippocampus (Table 3). This up-regulation on TNF-α was prevented by Artesunate treatment in combination or not with CBD. An up-regulation of IL-6 was also found in the hippocampus (F(7,31)= 4.99; p<0.01) and prefrontal cortex (F(7,31)= 4.27; p<0.05) of infected mice on the 5th day p.i. Of note, CBD treatment in combination with artesunate remarkably reduced IL-6 levels in prefrontal cortex (Table 2). No effects on other analyzed cytokines were observed (Table 3). Analysis of cytokine expression on day 5 p.i. revealed increase in the production of IL-6 and IFN-γ in the hippocampus while up-regulation of TNF-α, IFN-γ and IL-17 in the prefrontal cortex in PbA infected groups (treated or not with CBD) were detected (Table 4). A single dose of Artesunate, in combination or not with CBD prevented the up-regulation of those cytokines in both brain areas (Table 4). Discussion The results showed herein reinforce the emerging view that CBD produces neuroprotective effects. Although CBD per se did not impact on parasitemia on the 5th day p.i., CBD was able to increase significantly PbA-infected mice survival. Despite the antiinflammatory effects of Artesunate at the peak of CM, they were not able to prevent the associated cognitive deficits. CBD treatment, in association with Artesunate, prevents these cognitive deficits observed in the murine model of CM. After the complete clearance of parasitemia, CBD, in combination with Artesunate, prevents the long-term cognitive deficits of CM. Moreover, our results suggested, for the first time, that the anxiogenic effects induced by CM are long lasting, and, interestingly, this behavior was attenuated by CBD. Although the 15

specific mechanism underlying CBD neuroprotective effects in the murine model of CM remains to be determined, it seems to be related with its anti-inflammatory and/or its capacity to up regulate BDNF expression in the hippocampal formation. Anti-inflammatory properties of CBD and Artesunate contribute to the neuroprotective effects on CM neurological outcome. A poor cognitive outcome is a common feature in human and experimental CM. Although the mechanisms underlying this phenomenon remain largely unknown (Desruisseaux et al. 2008; John et al. 2008), cognitive and behavioral dysfunctions in clinical and experimental CM have been associated with inflammatory processes in the CNS, including increase in the production of pro-inflammatory cytokines (Desruisseaux et al. 2008; John et al. 2008; de Miranda et al. 2011b, Miranda et al. 2013). In patients with severe Malaria, serum levels of TNFα and IL-6 correlate with indices of severity of the disease (Mendonca et al., 2014; Sinha et al., 2008; Urquhart et al., 1994; Wenisch et al., 1999). Moreover, cognitive deficits in children with CM were associated with increased concentration of TNF-α in the cerebrospinal fluid (John et al., 2008). In the murine model of CM, it has demonstrated that the main neurological symptoms in the acute phase of the disease were associated with the CNS increase in IL-1β and TNF-α levels (Lacerda-Queiroz et al. 2010, Miranda et al. 2013). Recently, our group demonstrated that the memory deficits exhibited by animals infected with PbA in the late phase of CM was associated with elevated levels of pro-inflammatory cytokines in the hippocampus and prefrontal cortex (Miranda at al. 2013). It has also been suggested that the cognitive impairment promoted by CM may be associated with microglial cell activation in the cerebral cortex and hippocampus (Desruisseaux et al. 2008).

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Increase in pro-inflammatory cytokines signaling is involved in cognitive impairment described in other conditions than CM. For instance, psychological stress increases IL-6 in the brain and it is associated with memory deficits in rats (Patki et al. 2013). In humans, elevated circulating levels of IL-6 have been associated with poor memory performance in the elderly (Mooijaart et al. 2013) and in patients with major depressive disorder (Grassi-Oliveira et al. 2011). Thus, our results are in agreement with these previous experimental and clinical findings. Moreover they reinforce the view that increased levels of the pro-inflammatory cytokines TNF-α and IL-6 in specific brain areas, such as hippocampus and prefrontal cortex, are related with memory and behavior impairment (Yuan and Raz 2014, Yonelinas 2013). TNF-α is also involved in the control of anxiety-like behaviors and brain excitability. Genetic modulation of TNF-α reduced anxiety-like behavior in autoimmune encephalitis-induced anxiety in mice (Haji et al, 2012). TNF-α antagonist also reduces anxiety in chronic pain models in mice (Chen et al, 2013). In the hippocampus, increase in TNF-α levels is associated with cellular death (Liu et al., 2014) and dramatic reduction of plastic events, such as LTP and the number of dendritic spines (Strehl et al., 2014), mechanisms important for cognitive function and stress-related disorders. In this line, our results suggested that the reduction in levels of TNF-α in the hippocampus could contribute to CBD ability to prevent long-term cognitive and anxiogenic symptoms in CM. IL-6 is involved in the pathogenesis of several neuropsychiatric conditions, including epilepsy, schizophrenia, anxiety disorders (Atzori et al., 2012). For instance, IL-6 gene is upregulated in patients with higher levels of anxiety when compared with asymptomatic controls (Murphy et al., 2014). IL-6 signaling in the PFC has been related to the control of hyperexcitability states observed in disorders like epilepsy (Atzori et al., 2012, Garcia-Oscos et 17

al., 2014). Importantly, seizure is one of the symptoms observed in course of CM that could lead to death. Garcia-Oscos et al. (2014) demonstrated that IL-6 signaling in the PFC is an important mechanism for the control of CNS excitability induced by periphery inflammatory stimuli such as LPS. Our results suggested that CBD, after the successful treatment of malaria, prevents the increase of IL-6 in the PFC, an effect that is associated with better cognitive and behavioral parameters when compared with animals that received only Artesunate. Although CBD does not seem to produce a clear addictive anti-inflammatory effect to Artesunate in CM, its anti-inflammatory properties have been reported in the periphery and in the CNS in different experimental models (Barrichello et al. 2012; Magen et al. 2010; Mecha et al. 2013; Napimoga et al. 2009; Ruiz-Valdepenas et al. 2011). For instance, in a model of hepatic encephalopathy, CBD chronic treatment ameliorated cognitive and locomotor activities by restoring BDNF levels and decreasing the mRNA expression of the type-1 TNF-α receptors in the brain (Magen et al. 2010). An intravital microscopy study showed that CBD reduced vascular changes, CNS leucocyte migration and TNF-α expression induced by the administration of LPS in rodents (Ruiz-Valdepenas et al. 2011). Of note, we have recently shown that a single dose of Artesunate decreases inflammatory parameters in the brain and in the circulation of PbA-infected mice (Miranda et al. 2013). Clemmer et al. (2011) also found that a single dose of Artemisinin was highly effective in clearing PbA parasitemia at day 5 p.i., rescuing mice from the late-stages of CM (Clemmer et al. 2011). These authors suggested that the decrease in the brain inflammatory response could result from a faster parasitemia clearance associated with an intrinsic anti-inflammatory property presented by Artemisinin derivatives (Clemmer et al. 2011). However, our results clearly indicated that the anti-inflammatory activity of Artesunate does not prevent all neurological 18

impairment observed in the murine model of CM at the peak of disease or in the long-term (Miranda et al. 2013). Although treatment with Artesunate promoted the complete rescue of the neurological symptoms measured by RMCBS, it failed to prevent the long-lasting cognitive and anxiety-like behaviors observed in the course of CM. Complete reversion of neurological and behavioral outcomes of CM only occurred when Artesunate was administered in combination with CBD. Artesunate may act as a facilitator of CBD neuroprotective effects due to its antimalarial effects or, alternatively, as a synergic agent when in combination with CBD. Regarding the later, one possible mechanism, which is connected with inflammatory pathways and would be beneficial in the course of CM, is the modulation of the expression of adhesion molecules that controls, for instance, blood brain barrier permeability. ICAM-1 and VCAM-1 haves been systematically described to be up-regulated in the pathogenesis of CM (Armah et al. 2005; Erdman et al. 2011;Lacerda-Queiroz et al. 2010; Miranda et al. 2011). The effects of CBD, in combination or not with Artesunate, on the expression of those molecules in the course of CM remain to be determined. The rescue of cognitive function by CBD may also be mediated by the up regulation of BDNF signaling. CBD treatment promoted an increase in the hippocampal levels of BDNF after the clearance of the parasitemia. The expression of BDNF in the hippocampus has been associated with the improvement of cognitive performance in several inflammatory diseases (Barrichelo et al. 2012; Bechara et al. 2013; Ickes et al. 2000). For instance, decreased memory performance was associated with low brain levels of BDNF in rats submitted to experimental meningitis induced by Streptococcus pneumoniae (Barichello et al. 2010). Comim et al. (2012) reported that low BDNF levels in the hippocampus were associated with long-term cognitive deficits in mice 19

submitted to the murine model of CM even after Chloroquine treatment. Although CBD does not promote changes in BDNF levels in the hippocampus or prefrontal cortex in rodent models of anxiety and depression (Campos et al. 2012a; Zanelati et al. 2010), this seems to be different in other models. For instance, Magen and colleagues (2010) demonstrated that CBD treatment prevented the decrease of BDNF hippocampal expression induced by a model of hepatic encephalopathy-induced by bile duct ligation. CBD also increased BDNF levels in the hippocampus of rats submitted to a model of amphetamine-induced oxidative stress (Valvassori et al. 2011). Given the number of studies that have associated increase in BDNF levels and better cognitive performance (Carlino et al. 2013; Dincheva et al. 2012), it is reasonable to assume that the increased BDNF levels in the hippocampus found in CBD and Artesunate treated mice explain the rescue of cognitive deficits. The reason why CBD only increased the levels of BDNF in the hippocampus but not in the PFC is unknown. In our study, the effects of CBD on BDNF levels in the hippocampus of animals submitted to a memory task would result in a better performance in the short term, being the effects on PFC evidenced only in a later time-point. BDNF is a molecule that can be expressed either constitutively or in an inducible-manner (Sakata et al., 2013). It has been shown that disruption of BDNF signaling in the PFC has no impact on working memory process (Sakata et al., 2013). Recently, Rosas-Vidal et al. (2014) showed that after a memory related task, BDNF levels increased in the hippocampus but not in the PFC. They suggested that during the process of learning, BDNF starts being produced early in the hippocampus, followed by later production in areas connected with the hippocampus, such as the PFC (Rosas-Vidal et al., 2014). A trend of increase of NGF in the PFC and hippocampus could be observed following CBD treatment and after the complete remission of malaria symptoms (Figure 7B and 7D). Given the importance of NGF in neuroplastic events (Levi20

Montalcini et al., 1996), the participation of this neurotrophin on CBD neuroprotective effects cannot be ruled out. Regarding the effects of CBD on preventing anxiogenic-like behaviors in PbA-infected mice (either at the peak of the disease or after the complete remission of the malaria symptoms), the involvement of 5-HT1A receptors activation is likely. Several pieces of evidence have suggested that CBD induces anti-anxiety effects after acute, repeated or intracerebral administration by facilitating 5-HT1A receptors activation (Campos and Guimarães, 2008; Campos et al., 2012a; Campos et al., 2013b; Fogaça et al., 2014). CBD neuroprotective effects could be also be explained by several mechanisms (Campos et al. 2012b; Izzo et al. 2009). CBD is proposed to act as partial, full or yet inverse agonist at several receptors in the CNS, including CB1, CB2, GPR55, TRPV1 and 5-HT1A receptors. CBD inhibits the anandamide hydrolysing enzyme fatty acid amide hydrolase (FAAH) and the adenosine transporter (Campos and Guimaraes 2008; Campos and Guimaraes 2009; Campos et al. 2012b; Izzo et al. 2009), indirectly increasing the levels of these neurotransmitters. Moreover CBD has a potent action in inhibiting oxidative and nitrosative stress, a mechanism that has been related to its neuroprotective effects with implications for the treatment of neurodegenerative diseases such as Alzheimer’s, Huntington’s and Parkinson’s diseases (Esposito et al. 2006; Esposito et al. 2011; Fernandez-Ruiz et al. 2013; Garcia-Arencibia et al. 2009; Sagredo et al. 2007). This putative anti-oxidant effect of CBD is of particular interest for CM. Oxidative stress is believed to play a role in tissue damage during the course of Malaria. In fact, an increase in the concentration of reactive oxygen species (ROS, e.g. superoxide anion and the hydroxyl radical) produced by activated neutrophils is observed in peripheral tissues and in the brain of PbA-infected mice (Pino et al, 2013; Reis et al., 2010). Also, the treatment of PbA21

infected mice with antioxidants (e.g: N-acetylcysteine) together with Chloroquine at the first signs of CM prevented the development of late cognitive impairment (Reis et al. 2010). Although the antioxidant properties of CBD is likely to be responsible for its neuroprotective actions in the murine model of CM, the relevance of this particular mechanism or other involved in the beneficial effects of this phytocannabinoid on CM model remains to be determined. Conclusion Taken together, our results suggested that CBD may be a promising candidate as an adjuvant (in combination with antimarial drugs) to prevent brain damage and neurological outcomes of CM in humans. This assumption is reinforced by several reports suggest the use of CBD in humans (Bergamaschi et al. 2011; Demiracka et al. 2010, Zuardi et al. 1993). Nevertheless new studies are needed to determine the specific mechanisms involved in CBD effects.

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Conflict of interest The authors declare no conflict of interest. Acknowledgments This work was supported by Conselho Nacional de Desenvolvimento Científico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (Fapemig), Brazilian government funding agencies. ACC was CNPq fellowship recipient. References Alvarez, F.J., Lafuente, H., Rey-Santano, M.C., Mielgo, V.E., Gastiasoro, E., Rueda, M., Pertwee, R.G., Castillo, A.I., Romero, J. and Martinez-Orgado, J. (2008) Cannabidiol reduces brain damage and improves functional recovery after acute hypoxia-ischemia in newborn pigs. Pediatr Res., 64, 653-658. Armah, H., Wired, E.K., Dodoo, A.K., Adjei, A.A., Tettey, Y., Gyasi, R. (2005) Cytokines and adhesion molecules expression in the brain in human cerebral malaria. Int J Environ Res Public Health., 2, 123-131. Atzori, M., Garcia-Oscos F. and Mendez J.A. (2012) Role of IL-6 in the etiology of hyperexcitable neuropsychiatric conditions: experimental evidence and therapeutic implications. Future Med Chem, 4, 2177-2192. Barichello, T., Belarmino, E. Jr., Comim, C.M., Cipriano, A.L., Generoso, J.S., Savi, G.D., Stertz, L., Kapczinski, F. and Quevedo, J. (2010) Correlation between behavioral deficits and decreased brain-derived neurotrophic factor in neonatal meningitis. J Neuroimmunol., 223, 7376. Barichello, T., Ceretta, R.A., Generoso, J.S., Moreira, A.P., Simoes, L.R., Comim, C.M., Quevedo, J., Vilela, M.C., Zuardi, A.W., Crippa, J.A., Teixeira, A.L. (2012) Cannabidiol reduces host immune response and prevents cognitive impairments in Wistar rats submitted to pneumococcal meningitis. Eur J Pharmacol. 697, 158-164. Bechara, R.G. and Kelly, A.M. (2013). Exercise improves object recognition memory and induces BDNF expression and cell proliferation in cognitively enriched rats. Behav Brain Res., 245, 96-100. Berendt, A.R., Ferguson, D.J., Gardner, J., Turner, G., Rowe, A., McCormick, C., Roberts, D., Craig, A., Pinches, R., Elford, B.C. et al (1994) Molecular mechanisms of sequestration in malaria. Parasitology., 108 Suppl, S19-28. 23

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modulation

of

Sakata, K., Martinowich K., Woo N.H., et al. (2013) Role of activity-dependent BDNF expression in hippocampal-prefrontal cortical regulation of behavioral perseverance. Proc Natl Acad Sci U S A, 110, 15103-15108. Sinha, S., Mishra, S.K., Sharma, S., et al. (2008) Polymorphisms of TNF-enhancer and gene for FcgammaRIIa correlate with the severity of falciparum malaria in the ethnically diverse Indian population. Malar J., 7, 13. Strehl, A., Lenz M., Itsekson-Hayosh Z., et al. (2014) Systemic inflammation is associated with a reduction in Synaptopodin expression in the mouse hippocampus. Exp Neurol, 261, 230-235. Urquhart, A.D. (1994) Putative pathophysiological interactions of cytokines and phagocytic cells in severe human falciparum malaria. Clin Infect Dis., 19, 117-131. Valvassori, S.S., Elias, G., de Souza, B., Petronilho, F., Dal-Pizzol, F., Kapczinski, F., Trzesniak, C., Tumas, V., Dursun, S., Chagas, M.H., Hallak, J.E., Zuardi, A.W., Quevedo, J., Crippa, J.A. (2011) Effects of cannabidiol on amphetamine-induced oxidative stress generation in an animal model of mania. J Psychopharmacol., 25, 274-280. Wenisch, C., Linnau, K.F., Looaresuwan, S., Rumpold, H. (1999) Plasma levels of the interleukin-6 cytokine family in persons with severe Plasmodium falciparum malaria. J Infect Dis., 179, 747-750. Yonelinas, A.P. (2013) The hippocampus supports high-resolution binding in the service of perception, working memory and long-term memory. Behav Brain Res., 254, 34-44. Yuan, P. & Raz, N. (2014) Prefrontal cortex and executive functions in healthy adults: A metaanalysis of structural neuroimaging studies. Neurosci Biobehav Rev., 42C, 180-192. Zanelati, T.V., Biojone, C., Moreira, F.A., Guimaraes, F.S., Joca, S.R. (2010) Antidepressantlike effects of cannabidiol in mice: possible involvement of 5-HT1A receptors. Br J Pharmacol., 159, 122-128. Zuardi, A.W., Cosme, R.A., Graeff, F.G., Guimaraes FS (1993) Effects of ipsapirone and cannabidiol on human experimental anxiety. J Psychopharmacol., 7, 82-88.

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Legend for figures Figure 1- Schematic design of the experiments A- Experiment 1. PbA (Plamodium berghei ANKA); RMCBS Rapid Murine Coma and Behavior Scale); dpi (days post-infection); BSchematic design of the experiment 2 and 4. C- Experiment 3. Figure 2- Survival curve of mice infected with Plasmodium berghei ANKA (PbA) (n = 6/group) treated or not with Cannabidiol (CBD) and Artesunate (Art).

Figure 3- Cannabidiol treatment rescues cognitive performance of Plasmodium berghei ANKA (PbA) infected mice treated with Artesunate as assessed by the Novel Object Recognition test. Animals were tested after the clearance of the parasitemia on days 5 or 17 post infection. (A) 5th day post infection Long-term memory; (B) 5th day post infection distance travelled 24 hours after training session- (n= 4, 8, 9 respectively) (C) 17th day post infection Long-term memory; (B) 17th day post infection distance travelled 24 hours after training session- n= 9, 9, 4, 9 respectively. Results are expressed as mean ± SEM. Asterisk indicates statistical differences from all groups. Figure 4- Effects of single doses of Artesunate in combination with Cannabidiol in the cognitive performance of PbA-infected mice in the Morris Water Maze. A- Cued learning performance of animals trained from de day 2 to day 4 after PbA infection. B- Memory retention task after a single injection of Artesunate (32mg/Kg) or vehicle on the 5 day post infection. Results are expressed as mean ± SEM. * indicates statistical differences from all groups (n=5/group; p< 0.01). Figure 5- Anxiety-like index and motor behavior measured in the open field test on the 5th day post infection of PbA. Animals were treated with Vehicle or Cannabidiol from the day 3-5 after infection with PbA. (A) Distance travelled in the center of the arena; (B) number of crossing. 30

Results are expressed as mean ± SEM. * indicates statistical differences from all other groups/ (n= 5 animals/group). Figure 6- Anxiety-like behaviors in the course of cerebral malaria in mice treated or not with Cannabidiol after the complete remission of malaria symptoms. Animals were tested on day 18 post-infection after completely clearance of parasitemia by Artesunate treatment. In the upper panel (A) open columns represent the percent of entries onto the open arms while the hatched columns represent the percent of the time spent in the open arms. In the lower panel (B) the open columns represent the number of enclosed arms entries. Results are expressed as mean ± SEM. * indicates statistical differences from all other groups; # indicates difference from veh/veh group. (n= 9, 9, 4, 9 animals/group, respectively). Figure 7- Prefrontal cortex and hippocampus protein levels of the neurotrophins BDNF and NGF of mice infected with Plasmodium berghei ANKA (PbA) and controls treated or not with Cannabidiol and Artesunate (n= 4-5 per group). Prefrontal cortex and hippocampus were harvest on 5th or 18th days post-infection Neurotrophic factors were determined by ELISA. Upper panel: BDNF and NGF levels in the prefontal cortex; lower panel: hippocampus. Results are expressed as mean ± SEM. *indicates differences from all other groups. Legend for Supplementary figure S1- BDNF and NGF levels of animals infected with PbA treated or not with a single dose of Artesunate (32mg/Kg) combined or not with CBD on the peak of the CM symptoms (5dpi). Prefrontal cortex and hippocampus were harvest on 5th day post-infection. Further specifications please see Figure 7 (n= 5/group).

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Table 1- Representative data of Parasitemia of the survival curve Days post infection Treatment

4th

5th

10th

20th

30th

Vehicle+ PbA + Art

6.7±0.5

2.7±0.1*

0.03±0.01

nd

nd

CBD + PbA+ Art

7.4±0.9

2.5±0.2*

1.0±0.3

nd

-

Vehicle+ PbA

7.7±0.3

18.8±2.8

-

-

-

CBD + PbA

8.1±0.6

15.9±2.8

-

-

-

CBD- Cannabidiol (30mg/Kg); Art- Artesunate (64mg/Kg on 5th dpi, 32mg/Kg 6-10 dpi). Results expressed as mean ± SEM. (-) indicate that the measures was not performed because the animals died. (nd) parasite not detected. n= 6 per group * represent difference from all groups, p< 0.05.

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Table 2- RMCBS score measured in Plasmodium berghei-ANKA (PbA) infected mice. Days post-infection Treatment

3rd

4th

5th

6th

7th

8th

9th

10th

11th

12th

Vehicle+ Pba

20±0

19±0.2

8.4±1.6*

0±0*

-

-

-

-

-

-

CBD + Pba

20±0

20±0

12.6± 1

9.8±1*

1±1

-

-

-

-

-

Vehicle+ Pba + Art

20±0

20±0

15.8±1

20±0

20±0

20±0

20±0

20±0

20±0

20±0

CBD + Pba + Art

20±0

20±0

18.8±1

20±0

20±0

20±0

20±0

20±0

20±0

20±0

CBD- Cannabidiol (30mg/Kg); Art- Artesunate (64mg/Kg on 5th dpi, 32mg/Kg 6-10 dpi). RMCBS- Rapid Murine Coma and Behavior Scale. Results expressed as mean ± SEM. (-) indicate that the test was not performed because the animals died. * differences from all groups, p<0.05.

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Table 3- Cytokine profile of Prefrontal cortex and hippocampus of controls and Plasmodium berghei-ANKA (PbA) infected mice. Experimental Groups 5dpi Veh

CBD

PbAVeh

PbACBD

Veh

CBD

PbAArt/Veh

PbAArt/CBD

PFC

2.23±0.4

1.84±0.4

2.74±0.3

3.1±0.4

3.1±0.2

2.4±0.3

2.5±0.2

2.5±0.2

Hippocampus

2.1±0.1

2.0±0.1

2.1±0.5

2.3±0.1

2.1±0.1

2.5±0.1

0.6±0.4

0.5±0.3

PFC

0.2±0.01

0.7±0.3

0.9±0.7

1.0±0.8

3.1±0.2

2.4±0.3

2.5±0.2

2.5±0.2

Hippocampus

0.7±0.5

0.2±0.01

1.0±0.3

1.1±0.9

0.2±0.04

0.2±0.01

0.2±0.03

0.2±0.04

PFC

0.02±0.01

0.02±0.3

12.5±2.5

13.0±0.8

1.5±1.4

3.9±1.8

14.8.±4.0

1.9±0.8

Hippocampus

0.1±0.01

0.2±0.3

5.1±0.6*

4.4±0.7*

0.4±0.4

1.0±0.5

3.1±0.6

2.3±0.8

PFC

17.7±2.4

17.8±2.4

30.0±5.7

28±8.4

9.0.±4.1

14.8±4.3

14.6±5.8

15.5±2.8

Hippocampus

16.5±3.6

18.9±2.0

27.7±0.9

24.3±5

17.4±6.4

18.7±5.6

25.8±4.3

18.6±4.3

PFC

1.6±0.2

1.4±0.2

0.8±0.3

1.2±0.3

0.7±0.3

0.5±0.3

0.5±0.3

07±0.4

Hippocampus

0.9±0.4

1.1±0.2

0.3±0.1

1.3±0.3

0.5±0.2

0.8±0.3

1.7±0.1

0.8±0.5

PFC

1.2±0.6

1.2±0.5

5.5±1.2

7.5±3.6

2.5±0.9

3.6±1.1

3.0±0.7

3.5±1.3

Hippocampus

1.2±0.2

0.5±0.3

3.4±0.7*

4.2±0.8*

1.2±0.2

1.0±0.3

3.7±0.2*

2.2±0.3

PFC

1.4±0.09

1.0±0.3

4.3±1.6

4.3±1.5

1.8±0.5

3.2±0.7

2.8±0.8

3.4±1.8

Hippocampus

1.0±0.5

1.2±0.3

2.7±0.8

3.7±1.5

1.5±0.5

2.4±0.3

2.1±0.3

3.2±1.3

Cytokine

IL-2

IL-4

IL-6

IL-10

IL-17

TNF-α

IFN-γ

18dpi

PFC- prefrontal cortex. Veh- Vehicle; CBD- Cannabidiol (30mg/Kg); Art- Artesunate (64mg/Kg on 5th dpi, 32mg/Kg 6-10th dpi). Results expressed as mean ± SEM (pg/µg of protein). * indicates difference from all groups; p<0.05. (n= 4, 6, 5, 5, 5, 5, 4, 4 per group respectively)

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Table 4 - Cytokine profile of prefrontal cortex and hippocampus of controls and Plasmodium berghei-ANKA (PbA) infected mice treated or not with a single dose of Artesunate (5th day after infection). Experimental Groups Vehicle

Control

Pba

Pba+Art

Control

Pba

Pba+Art

PFC

0.9±0.05

0.7±0.05

0.9±0.04

0.8±0.02

0.8±0.05

1.2±0.08

Hippocampus

1.3±0.1

0.9±0.04

1.3±0.7

1.2±0.05

0.9±0.05

1.2±0.08

PFC

1.1±0.04

1.1±0.4

1.2±0.04

1.2±0.04

1.1±0.01

1.2±0.02

Hippocampus

1.2±0.04

1.2±0.05

1.3±0.07

1.2±0.05

1.2±0.05

1.33±0.03

PFC

0.9±0.2

0.7±0.3

1.1±0.1

0.5±0.09

1.3±0.3

1.1.±0.4

Hippocampus

1.0±0.3

3.4±0.5*

1.1±0.3

0.8±0.06

4.1±0.5*

3.1±0.6

PFC

2.6±0.7

0.8±0.4

2.2±0.4

1.4.±0.7

0.6±0.2

2.9±1.3

Hippocampus

7.9±2.7

12.9±5.5

7.7±2.6

2.3±0.7#

3.0±0.8

8.2±1..3

PFC

0.09±0.02

0.03±0.01*

0.09±0.3

0.06±0.01

0.01±0.001*

0.09±0.03

Hippocampus

0.2±0.06

0.2±0.04

0.3±0.07

0.1±0.02

0.1±0.01

0.2±0.07

PFC

0.5±0.2

1.6±0.03*

1.0±0.4

0.8±0.2

1.4±0.2*

0.7±0.2

Hippocampus

1.5±0.1

1.9±0.1

1.6±0.2

1.4±0.3

2.0±0.4

1.6±0.2

PFC

1.6±0.09

3.5±0.3*

1.1±0.1

1.1±0.1

7.0±0.8*

4.8±1.1

Hippocampus

1.5±0.05

6.8±0.4*

2.4±0.2

1.5±0.1

5.8±0.8*

4.8±1.1

Cytokine

IL-2

IL-4

IL-6

IL-10

IL-17

TNF-α

IFN-γ

CBD

PFC- prefrontal cortex. Veh- Vehicle; CBD- Cannabidiol (30mg/Kg); Art- Artesunate (64mg/Kg on 5th dpi, 32mg/Kg 6-10th dpi). Results expressed as mean ± SEM (pg/µg of protein). * indicates difference from all groups; # indicates difference from vehicle treated groups and CBD+ artesunate group; p<0.05. n=5/group.

35

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Highlights > Late anxiogenic effects are observed in mice infected with Plasmodium berghei ANKA. > Artesunate does not prevent late anxiogenic effect or cognitive impairment of CM. > Cannabidiol adjuvant treatment increases survival in the murine model of CM. > Cannabidiol adjuvant treatment promotes rescue of behavioral and cognitive function. > Cannabidiol effects might involve up regulation of hippocampal levels of BDNF.

36