Relationship between behavioral alterations and activities of adenylate kinase and creatine kinase in brain of rats infected by Trypanosoma evansi

ARTICLE IN PRESS Experimental Parasitology ■■ (2015) ■■–■■

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Experimental Parasitology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x p r

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Relationship between behavioral alterations and activities of adenylate kinase and creatine kinase in brain of rats infected by Trypanosoma evansi

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Matheus D. Baldissera a,b,*, Virginia C. Rech c, Aleksandro S. Da Silva d, Vivian S.K. Nishihira c, Francine R. Ianiski c, Lucas T. Gressler a, Thirssa H. Grando a, Q2 Rodrigo A. Vaucher b, Claiton I. Schwertz e, Ricardo E. Mendes e, Silvia G. Monteiro a,** a

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Department of Microbiology and Parasitology, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil Laboratory of Microbiology, Centro Universitário Franciscano, Santa Maria, RS, Brazil c Laboratory of Nanotechnology, Centro Universitário Franciscano, Santa Maria, RS, Brazil d Department of Animal Science, Universidade do Estado de Santa Catarina (UDESC), Chapecó, SC, Brazil e Section of Veterinary Pathology, Instituto Federal Catarinense, Concórdia, SC, Brazil b

H I G H L I G H T S

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T. evansi infection alters some important parameters of the energy metabolism in rat brain. Changes in adenylate kinase and creatine kinase lead to behavioral changes. Infected animals with T. evansi presented depressive behavior and decreased memory. Decreases in energy production may be implicated in the pathophysiology of the disease.

G R A P H I C A L

A B S T R A C T

The rats infected developed behavioral alterations: memory deficit and depressant activity and Trypanosoma evansi infection in rats

Histological lesions in the brain

T. evansi infection in rats caused:

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- Adenylate kinase and creatine kinase activity in brain were altered. - Increased proinflammatory cytokine levels in serum: TNF and IFN

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A R T I C L E

I N F O

Article history: Received 7 November 2014 Received in revised form 23 January 2015 Accepted 27 January 2015 Available online Keywords: Phosphoryltransfer network Surra Memory Depression

A B S T R A C T

The aim of this study was to investigate the behavioral assessment and activities of important enzymes involved in the phosphoryl transfer network in rat brains that were experimentally infected with Trypanosoma evansi. Behavioral assessment (cognitive performance), pro-inflammatory cytokines in serum and activities of adenylate kinase (AK), pyruvate kinase (PK), and creatine kinase (CK) in brain were evaluated at 5 and 15 days post-infection (PI). Here we demonstrate a cognitive impairment in the rats infected with T. evansi. At 5 and 15 days PI, a memory deficit and a depressant activity were demonstrated by an inhibition avoidance test and increase in the immobility time in a tail suspension test, respectively. On day 5 PI, a decrease in the CK activity and an increase in the AK activity were observed. On day 15 PI, an increase in the CK activity and a decrease in the AK activity were observed. Considering the importance of energy metabolism for brain functioning, it is possible that the changes in the activity of enzymes involved in the cerebral phosphotransfer network and an increase in the proinflammatory cytokines (TNF and IFN) may be involved at least in part in the cognitive impairment in infected rats with T. evansi. © 2015 Published by Elsevier Inc.

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* Corresponding author. E-mail address: [email protected] (M.D. Baldissera). ** Corresponding author. E-mail address: [email protected] (S.G. Monteiro).

Trypanosoma evansi is a flagellate, etiological agent of the disease known as “Mal das cadeiras” or “Surra” in horses. This parasite has a wide geographical distribution, being found parasitizing many

http://dx.doi.org/10.1016/j.exppara.2015.01.015 0014-4894/© 2015 Published by Elsevier Inc.

Please cite this article in press as: Matheus D. Baldissera, Virginia C. Rech, Aleksandro S. Da Silva, Vivian S.K. Nishihira, Francine R. Ianiski, Lucas T. Gressler, Thirssa H. Grando, Rodrigo A. Vaucher, Claiton I. Schwertz, Ricardo E. Mendes, Silvia G. Monteiro, Relationship between behavioral alterations and activities of adenylate kinase and creatine kinase in brain of rats infected by Trypanosoma evansi, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.01.015

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species of domestic and wild animals (Silva et al., 2002). The trypomastigotes present in the blood vessels of vertebrate hosts are transmitted by blood-sucking insects during feeding. The insect vectors are most commonly the tabanid species (Tabanus sp., Chrysops sp., and Hematopota sp.) (Hoare, 1972). The disease causes various clinical signs as well as neurological disorders, such as necrotizing panencephalitis or meningoencephalitis, motor incoordination, necrosis, and demyelination in horses (Aquino et al., 2002; Berlin et al., 2009; Dargantes et al., 2005; Rodrigues et al., 2009). The cattle that were infected presented nervous symptoms, such as excitation, aggressive behavior, and convulsion (Tuntasuvan et al., 1997). In the first report of T. evansi infection in humans, sensory deficit, disorientation, agitation, and aggression were reported (Joshi et al., 2005). It was related, in recent study, that rats infected with T. evansi develop behavioral changes associated with changes in the levels of neurotransmitters such as adenosine triphosphate (ATP), adenosine, and acetylcholine, among others (Wolkmer et al., 2013). Alterations in the levels of ATP were associated with the activity of ectoenzymes as NTPDase (Oliveira et al., 2011), that makes us believe that other enzymes could be involved in metabolism of ATP, as investigated in this study. Adenylate kinase (AK) catalyzes reversible phosphotransfer between ATP and ADP in the presence of AMP and has been implicated in the processing of cellular signals associated with ATP utilization (Dzeja and Terzic, 1998). Impaired AK activity leads to disturbances in cellular functions, and the deficiency of AK is associated with hemolytic anemia (Toren et al., 1994), the main characteristic of the trypanosomosis (Wolkmer et al., 2009). Pyruvate kinase (PK) is a key enzyme of the glycolysis pathway present in all tissues. PK catalyzes the irreversible transphosphorylation between phosphoenolpyruvate and ADP to yield ATP and pyruvate (Valentini et al., 2000). Creatine kinase (CK) catalyzes the reversible transfer of a phosphoryl group from ATP to ADP and creatine to produce phosphocreatine (PCr). The main function of the CK, besides its buffering action, is the replacement of ATP in areas with a high energy demand, such as the brain (Rech et al., 2008). CK-B, the isoform of CK in the brain, is connected to spatial memory acquisition and behavior, development of the hippocampus and phagocytosis (Chang et al., 2008; Jost et al., 2002; Kuiper et al., 2008). ATP is a relevant neuromodulator and neurotransmitter and has played an important role in neural plasticity (Mochel et al., 2012). Research has shown that the alteration of brain energy metabolism is associated with neurodegenerative disorders (De Franceschi et al., 2013). Changes in the concentration of ATP as well as the hydrolysis of this molecule can influence the purinergic cascade and adenosine levels, a nucleoside feature neuromodulator as previously described (Da Silva et al., 2012). These alterations may be related to the pathogenesis of the disease, as well as neurological and nervous system damage in T. evansi infection (Berlin et al., 2009; Rodrigues et al., 2009). Tumor necrosis factor alpha (TNF-α) is a proinflammatory cytokine showing biological effects in the central nervous system (Baune et al., 2012). High levels of TNF-α also been linked to major depressive disorder (Dean et al., 2010), and increased expression of TNF-α were observed in murine models of depressive like behavior and chronic mild stress (Kaster et al., 2012). Therefore, the main objective of this study was to evaluate the relationships between behavior and AK, PK, and CK activities in the brain of rats by T. evansi in two moments of infection. 2. Materials and methods 2.1. Trypanosoma evansi isolate The T. evansi strain was originally isolated from a naturally infected dog (Colpo et al., 2005), which was maintained cryopreserved under laboratory conditions. Initially, 1 rat (R 1 ) was infected

intraperitoneally with blood cryopreserved containing 106 parasites. This procedure was performed to obtain a large number of parasites for the study. 2.2. Animal model Thirty-six (female) 60-day-old rats weighing an average of 200 ± 10 g were used in this study. They were kept in cages, housed on a light/dark cycle of 12 h in an experimental room with temperature and humidity controlled (23 ± 1 °C; 70% respectively). Animals were fed with commercial rations and water ad libitum. All the animals were subject to a period of 15 days for adaptation. 2.3. Experimental design and parasitemia estimation The animals were divided into two groups (A (n = 12) and B (n = 24)), and these groups were divided into four subgroups (A1 and A2, 6 animals/each group; and B1 and B2, 12 animals/each group). The group A rats were used as control (uninfected). Animals in groups B1 and B2 were inoculated subcutaneously with 0.06 mL of blood from a rat (R1) containing 6.0 × 106 trypanosomes (day 0). Rats were observed and parasitemia was monitored daily through a blood smear. Each slide was prepared with fresh blood collected from the tail vein, stained by the panoptic method, and visualized at a magnification of 1000× according to the method described by Da Silva et al. (2006). 2.4. Behavioral tests 2.4.1. Inhibitory avoidance task Non-spatial long-term memory was investigated using an inhibitory avoidance task according to the method of Sakaguchi et al. (2006), with modifications in the intensity of electric shock and in the exposure time. During the training session (1 day before euthanasia), each rat was placed on the platform. When it stepped down and placed its four paws on the grid floor, an electric shock (0.5 mA) was delivered for 2 s. The acquisition test (on the day of euthanasia) was performed 24 h after training in a similar manner. Each rat was placed again on the platform and the step-down transfer time was recorded. For inhibitory avoidance, a separate group of animals was used. A step-down latency test was taken as a measure of acquisition, and a cut-off time of 300 s was established. 2.4.2. Open field test The open-field test was made of plywood and surrounded by 30 cm height walls. The floor of the open-field, 40 cm length and 40 cm width, was divided by masking tape markers into 9 squares (3 rows of 3). Each animal was placed individually at the center of the apparatus and observed for 4 min to record the locomotor (number of segments crossed with the four paws) and exploratory activities (expressed by the number of time rearing on the hind limbs) (Walsh and Cummins, 1976). The open-field test was per- Q3 formed on the day of euthanasia. 2.4.3. Tail suspension test (TST) The TST is a frequently used test for antidepressant activity (Porsolt et al., 1987). The total duration of immobility induced by tail suspension was measured according to the methods described by Steru et al. (1985). Briefly, animals both acoustically and visually isolated were suspended 50 cm above the floor by adhesive tape placed approximately 1 cm from the tip of the tail. Immobility time was recorded during a 6-min period. Mice were considered immobile only when they hung inhibitory and were completely motionless. This test is a reliable and rapid screening method for antidepressants including those involving the serotonergic system.

Please cite this article in press as: Matheus D. Baldissera, Virginia C. Rech, Aleksandro S. Da Silva, Vivian S.K. Nishihira, Francine R. Ianiski, Lucas T. Gressler, Thirssa H. Grando, Rodrigo A. Vaucher, Claiton I. Schwertz, Ricardo E. Mendes, Silvia G. Monteiro, Relationship between behavioral alterations and activities of adenylate kinase and creatine kinase in brain of rats infected by Trypanosoma evansi, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.01.015

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2.5. Sample collection

Uninfected 5 days post-infection

On day 5 post infection (PI) (subgroups A1 and B1) and day 15 PI (subgroups A2 and B2), the sample collections were performed. The animals were sacrificed by decapitation without use of anesthetic. Whole blood was collected after the decapitation of the animals, and stored in tubes without anticoagulant. The samples were centrifuged at 3000 rpm for 15 minutes to obtain serum for the measurement levels of proinflammatory cytokines. Then, the brain was removed for biochemical and histological analyzes, as will be described later. The serum was stored at −20 °C for 1 week, and the brain was stored at −80 °C for 2 weeks to determine the enzyme activity of energy metabolism. 2.6. AK, PK, and CK activities The brains were removed and dissected on a glass dish over ice. For the assay of the enzymes, brains were washed in SET buffer (0.32 M sucrose, 1 mM EGTA, 10 mM Tris–HCl, pH 7.4) and homogenized (1:10 w/v) in the same SET buffer with a Potter glass homogenizer. The homogenate was centrifuged at 800 × g for 10 min at 4 °C. Part of the supernatant was centrifuged and used for the determination of AK activity; the pellet was discarded and the rest of the supernatant was centrifuged at 10,000 × g for 15 min at 4 °C. The supernatant of this second centrifugation was collected for the determination of PK and CK activities. AK activity was measured with a coupled enzyme assay with hexokinase (HK) and glucose 6-phosphate dehydrogenase (G6PD), according to Dzeja et al. (1999). The reaction mixture contained 100 mM KCl, 20 mM HEPES, 20 mM glucose, 4 mM MgCl2, NADP+, 1 mM EDTA, 4.5 U/mL of HK, 2 U/mL of G6PD, and 20 μL of homogenate. The reaction was initiated by the addition of 2 mM ADP and the reduction of NADP+ was followed at 340 nm for 3 min in a spectrophotometer. Reagent concentration and assay time (3 min) were chosen to assure the linearity of the reaction. Results were expressed in μmol of ATP formed per min per mg of protein. PK activity was assayed essentially as described by Leong et al. (1981). The incubation medium consisted of 0.1 M Tris/HCl buffer, pH 7.5, 10 mM MgCl2, 0.16 mM NADH, 75 mM KCl, 5.0 mM ADP, 7 U of lactate dehydrogenase, 0.1% (v/v) Triton X-100, and 10 μL to homogenize. After 10 min of preincubation at 37 °C, the reaction was started by the addition of 1 mM phosphoenol pyruvate. All assays were performed in duplicate at 25 °C. The results were expressed as μmol of pyruvate formed per min per mg of protein. CK activity was assayed as a method described by Hughes (1962). The reaction mixture contained the following final concentrations: 65 mM Tris–HCl buffer, pH 7.5, 7 mM phosphocreatine, 9 mM MgSO4 and 20 μL of homogenize. The reaction was started by the addition of 0.3 μmol of ADP after 10 min of pre-incubation at 37 °C. The color was developed by the addition of 0.1 mL 2 2% α-naphtol and 0.1 mL 0.05% diacetyl in a final volume of 1 mL. Reading was performed after 20 min of the wavelength at 540 nm. The results were expressed as μmol of creatine formed per min per mg of protein. The protein contents of brain homogenates were determined by the method of Lowry et al. (1951), using serum bovine album as the standard. 2.7. Proinflammatory cytokines Proinflammatory cytokine quantification (tumor necrosis factor (TNF-α) and interferon gamma (IFN-γ)) was assessed by ELISA assay using commercial Quantikine Immunoassay kits (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. The concentrations of the TNF-α and IFN-γ were determined by the intensity of the color measured spectrophotometrically using a microplate reader.

Number of Trypanosomes/field (1000x)

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Days post-infection Fig. 1. Average parasitemia of infected animals with Trypanosoma evansi, corresponding 0–15 days post-infection.

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2.8. Histology After euthanasia, brain fragments of uninfected rats and infected rats with T. evansi were collected and stored in a 10% buffered formalin solution. For histopathology, sagittal sections were obtained with an interval of 3 mm between the regions and stained with hematoxylin and eosin. A method for staining nervous tissue with Giemsa dye was used (Iñiguez et al., 1985). After 2 weeks storage, histopathology technique was performed.

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2.9. Statistical analyses The data were tested for normality and transformed when necessary. AK and CK data were not normally distributed and were ranktransformed before statistical analysis. Then, the statistical analysis to results from AK, PK, and CK activities, and proinflammatory cytokines were performed by Student’s t-test and the data from behavioral tests were analyzed by Duncan test. All analyses were performed through the Statistical Package for the Social Sciences (SPSS) software. A probability <0.05 was accepted as significant.

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3. Results 3.1. Disease course The data of the course of the disease are shown in Fig. 1. Infected animals showed on average 14 trypanosomes/field (1000×) at 5 days PI. On day 15 PI, the infected animals are on average 20 trypanosomes/fields (1000×).

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3.2. Behavioral changes In days 5 and 15 PI in inhibitory avoidance task, the infected animals reduced (p < 0.05) the latency time when compared to uninfected (Fig. 2A). Open field tests no significant differences in number of crossings and rearings revealed between the groups. Therefore, the animals do not exhibit locomotor problems, and this validates the other behavioral tests. A time-course analysis of the antidepressant profile of infection in the TST was accomplished. The immobility time in the TST of animals infected is shown in Fig. 2B. The infected animals (5 and 15 days PI) increased the immobility time in the TST test compared to their respective uninfected controls (p < 0.05).

Please cite this article in press as: Matheus D. Baldissera, Virginia C. Rech, Aleksandro S. Da Silva, Vivian S.K. Nishihira, Francine R. Ianiski, Lucas T. Gressler, Thirssa H. Grando, Rodrigo A. Vaucher, Claiton I. Schwertz, Ricardo E. Mendes, Silvia G. Monteiro, Relationship between behavioral alterations and activities of adenylate kinase and creatine kinase in brain of rats infected by Trypanosoma evansi, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.01.015

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Fig. 2. Behavioral assessment of rats experimentally infected with T. evansi compared to the controls 5 and 15 days post-infection. Inhibitory avoidance task (A) and tail suspension test (B). The data are expressed as average and standard deviation (in seconds) in session latency and immobility from 6 animals for each group (uninfected), and 12 animals for each group (infected). Statistical analysis was performed by a Duncan test. *p < 0.05; **p < 0.01; ***p < 0.001.

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3.3. AK, PK and CK activities The AK, PK, and CK activities in the brain are shown in Fig. 3. The AK activity increased (p < 0.05) in the infected group on day 5 PI, and decreased (p < 0.05) in the infected group on day 15 PI when compared to the control group. The CK activity decreased (p < 0.05) in the infected group on day 5 PI, and showed an increase (p < 0.05) activity on day 15 PI compared to the control group. There were no differences between the groups regarding PK activity (p > 0.05). 3.4. Proinflammatory cytokines The levels of the proinflammatory cytokines data are shown in Fig. 4. The cytokines were quantified in serum 5 and 15 days postinfection. Increased serum levels of TNF-α and INF-γ in T. evansi infected animals compared to uninfected animals were observed. A gradual increase in the cytokines levels according to the disease progression was observed.

Fig. 3. Average and standard error of adenylate kinase (AK), pyruvate kinase (PK), and creatine kinase (CK) activity in rats experimentally infected with T. evansi compared to the controls 5 and 15 days post infection. The data are expressed as average and standard error from 6 animals for each group (uninfected), and 12 animals for each group (infected). Statistical analysis was performed by Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001.

38 extensive associated with mild congestion in the meninges was observed on day 5 PI (Fig. 5A) and using Giemsa staining trypomastigotes of T. evansi within a thrombus in the meningeal vessels were observed (Fig. 5B). On day 15, a diffuse moderate lymphocytic inflammatory infiltrate, associated with multiple intravascular fibrin thrombi in the meninges, was observed (Fig. 6A) as well as the necrosis of neurons (polioencephalomalacia) moderate multifocal associated with satellitosis in the cortex (Fig. 6B). 4. Discussion

3.5. Histology The uninfected rats showed no brain injuries. In contrast, infected animals had different lesions in the brain, according to the time of infection. Mild lymphocytic inflammatory infiltrate focally

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In this study, we decided to evaluate a possible relationship between behavioral changes and enzyme activity of energy metabolism and cytokines in rat brain infected by T. evansi during acute phase of the disease. The course of infection has a traditional

Please cite this article in press as: Matheus D. Baldissera, Virginia C. Rech, Aleksandro S. Da Silva, Vivian S.K. Nishihira, Francine R. Ianiski, Lucas T. Gressler, Thirssa H. Grando, Rodrigo A. Vaucher, Claiton I. Schwertz, Ricardo E. Mendes, Silvia G. Monteiro, Relationship between behavioral alterations and activities of adenylate kinase and creatine kinase in brain of rats infected by Trypanosoma evansi, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.01.015

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Fig. 4. Mean and standard deviation of the proinflammatory cytokines of rats experimentally infected with T. evansi compared to the controls 5 and 15 days postinfection. The data are expressed as average and standard deviation from 6 animals for each group (uninfected), and 12 animals for each group (infected). Statistical analysis was performed by Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001.

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variation in animals regarding the response to the degree of parasitemia (França et al., 2011; Paim et al., 2011b; Wolkmer et al., 2013). As expected, the rats infected with T. evansi showed high parasitemia at 5–6 days PI. After this period, a reduction in parasitemia was observed, similar to the chronic disease process. Behavior alterations in infected rats by T. evansi were observed this study. Using the open-field test, neither locomotor abnormalities nor anxiety was detected in infected rats with T. evansi. Conversely, we observed that T. evansi infection increased the immobility time in the TST test, suggesting depressive behavior of these animals in the two time points of infection. According to Dantzer et al. (2008), stressors such as inflammatory cytokines emerged as important factors of depressive behavior, when the increases in the production of the IFN-γ and TNF-α have been associated with depressive behavior. As literature, animals infected with T. cruzi also showed depressive behavior (Vilar-Pereira et al., 2012), i.e. TNF plays a pivotal role as an immunological stressor in depressive behavior during chronic infection. The progressive increases in the serum levels of TNF-α and IFN-γ on infected animals with T. evansi are correlated to the inflammatory response against infection (Baldissera et al., 2014; Gao and Pereira, 2002). Cytokines have been shown to affect many behaviors, including effects on memory and motor activity (Dantzer and Kelley, 2007). In this work, we observed that T. evansi infection decreased latency time in the inhibitory avoidance task compared to uninfected animals, suggesting memory impairment. Some authors have related a relationship between depressed individuals and cognitive deficits, including memory (Bearden et al., 2006; Vasic et al., 2008), which was evidenced in this study.

Fig. 5. Rats experimentally infected with Trypanosoma evansi, after 5 days of infection. (a) Mild lymphocytic inflammatory infiltrate stained with hematoxylin and eosin, and (b) trypomastigotes inside thrombus in meningeal vessels stained with Giemsa.

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A decrease on the CK activity in the brain may be associated with the disruption of neuronal functions, loss of hippocampal mossy fiber bodies, and changes in mitochondrial structure that are typical for pathological conditions, such as encephalomyopathies (Watanabe et al., 2002), which are neurological signs observed in the infection caused by T. evansi (Rodrigues et al., 2009). An increase in the NTPDase activity in the cerebral cortex of rats infected with T. evansi has already been reported, which may be related to the increase of ATP in the brain (Oliveira et al., 2011). A reduction of ecto adenosine desaminase (E-ADA) activity in the brain of infected rats was also observed (Da Silva et al., 2011) corroborating other studies related to the increased level of adenosine (Da Silva et al., 2012), an important neuromodulator. A very high turnover of ATP is necessary to maintain electrical membrane potentials, as well as the signaling activities of the central and peripheral nervous system (Shulman et al., 2004) and considering that the CK system is essential for the maintaining a stable ATP level in living cells, the depletion of CK activity has been implicated in the pathogenesis of a number of diseases, especially in the brain (Schlattner and Wallimann, 2000). Several authors suggested an involvement of CK in learning and memory (Poon et al., 2005). In a study carried out by Wolkmer et al. (2013) trypanosomosis

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show the joint action of enzymes, noting that when the activity of one of them (CK or AK) is decreased, the activity of the other increases (Dzeja and Terzic, 2003; Janssen et al., 2003), as was also observed in this study. It is possible that the non-altered PK activity is a response to the decrement and increases in the CK and AK in an attempt to preserve energetic homeostasis (Dzeja and Terzic, 2003). The involvement of the central nervous system (CNS) in infection is widely discussed, because brain lesions are observed, due to the presence of the parasite (Bal et al., 2012; Berlin et al., 2009; Rodrigues et al., 2009). According to Bal et al. (2012), since the protozoa were detected in the blood vessels of the brain, the changes in the brain might, therefore, be due to toxic substances released by the parasite. It has been reported that pathological changes in the brain are due to constant irritation caused by the presence of parasites (Poursines and Dardenne, 1943). Researchers observed that a reduction of creatine kinase activity may potentially impair energy homeostasis (Burbaeva et al., 1999), contributing to brain damage, and that reduced functional brain activity may result in cognitive deficits (Sackeim, 2000), such as observed in this study. In summary, in this study we demonstrated for the first time that an infection with T. evansi alters some important parameters of the phosphoryltransfer network in rat brain, as AK and CK. The enzymatic and behavioral alterations are probably consequences of cell and tissue lesions, as well as the presence of the parasite in the brain. Therefore, it is possible to hypothesize that the inhibition in the CK activity may be impair brain energy homeostasis altering brain functioning and so contributing to the behavioral and cognitive deficits. Ethics committee The procedures were approved by the Animal Welfare Committee of Universidade do Estado de Santa Catarina under number 01.27.14. References 1 2 3

Fig. 6. Rats experimentally infected with Trypanosoma evansi, after 15 days of infection. (a) Marked inflammatory infiltrates in the meninges, and (b) neuron necrosis with satellitosis in the region of the cerebral cortex.

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caused by T. evansi impaired memory and increased anxiety behavior in rats that were experimentally infected. These authors reported the infection decreased Na+, K+-ATPase activity in the cortex and hippocampus 5 days post-infection. The inhibition of Na+, K+ATPase activity expression has been associated in rats with learning and memory impairment of different behavioral tasks (Dos Reis et al., 2002). The same authors observed an increase of acetylcholinesterase activity, which led to a rapid degradation of acetylcholine, an important neurotransmitter associated with memory. Substantial evidence supports a direct coupling of CK with growth cone migration, with Na+, K+-ATPase and neurotransmitter release, playing a relevant role in neuronal and synaptic plasticity, as well as an involvement of CK in the maintenance of membrane potentials, Q4 homeostasis, and restoration of ion gradients before and after depolarization (Schlattner and Wallimann, 2000). The infection by T. evansi had been associated with increase on oxidative stress variables in the brain of rats (Paim et al., 2011a), and there is also a relationship between oxidative stress and brain energy metabolism in some pathologies (Amaral et al., 2010; Mescka et al., 2011). The AK, PK, and CK enzymes act on the transfer of phosphoryl grouping of ATP from mitochondria to the cytosol (phosphoryltransfer network), ensuring the flow of energy to places of high energy demand, such as the brain (Ames, 2000). Some studies

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Please cite this article in press as: Matheus D. Baldissera, Virginia C. Rech, Aleksandro S. Da Silva, Vivian S.K. Nishihira, Francine R. Ianiski, Lucas T. Gressler, Thirssa H. Grando, Rodrigo A. Vaucher, Claiton I. Schwertz, Ricardo E. Mendes, Silvia G. Monteiro, Relationship between behavioral alterations and activities of adenylate kinase and creatine kinase in brain of rats infected by Trypanosoma evansi, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.01.015

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