The effect of memantine, an antagonist of the NMDA glutamate receptor, in in vitro and in vivo infections by Trypanosoma cruzi

Chagas disease, caused by Trypanosoma cruzi, is a neglected tropical disease that affects 5–6 million people in endemic areas of the Americas. Presently, chemotherapy relies on two compounds that were proposed as trypanocidal drugs four decades ago: nifurtimox and benznidazole. Both drugs are able to eliminate parasitemia and to avoid seroconversion in infected people when used in the acute phase; however, their use in the chronic phase (the time when the majority of cases are diagnosed) is limited due to their serious side effects. Memantine is a glutamate receptor antagonist in the central nervous system of mammals that has been used for the treatment of Alzheimer’s disease. Our group previously reported memantine as a trypanocidal drug that is able to induce apoptosis-like death in T. cruzi. In the present work, we further investigated the effects of memantine on the infection of RAW 264.7 macrophages and in vivo (in BALB/c mice). Here, we showed that memantine is able to diminish NO and Ca2+ entry in both LPS-activated and non-activated cells. These results, together with the fact that memantine was also able to reduce the infection of macrophages, led us to propose that this drug is able to activate a pro-oxidant non-NO-dependent cell defense mechanism. Finally, infected mice that were treated with memantine had diminished parasitemia, cardiac parasitic load, and inflammatory infiltrates. In addition, the treated mice had an increased survival rate. Taken together, these results indicate memantine to be a candidate drug for the treatment of Chagas disease.


Introduction
Chagas disease is caused by the protozoan Trypanosoma cruzi and affects 5-6 million people in the Americas [1]. Mammals (including humans) become infected when an infected triatomine insect defecates on the skin and expels metacyclic trypomastigotes with the feces, one of the nonproliferative, infective forms of the parasite. These forms are able to internalize into mammalian hosts through the mucosa and small wounds caused by scratching. Once inside the mammalian host, the metacyclic trypomastigotes invade the host cells to reach the cytoplasm, where they initiate their proliferation as amastigotes. After a variable number of cellular divisions, amastigotes undergo a complex differentiation process, yielding a new generation of infective, nonproliferative forms called trypomastigotes. These trypomastigotes burst from the infected cells into the extracellular environment and are able to infect the neighboring cells or to reach the bloodstream, allowing them to extend the infection to other tissues. Eventually, the bloodstream trypomastigotes can be taken by the blood to a new, noninfected triatomine insect during its blood-meal, can infect the insect, and can convert this newly infected insect into a new transmitter of the infection [2].
Chagas disease can be divided into two phases: acute and chronic. The acute phase is mainly asymptomatic with evident parasitemia and undetectable levels of IgG antibodies. The chronic phase is characterized by a robust humoral response with high titers of IgG antibodies and subpatent parasitemia. The chronic phase persists for the host's lifespan. Most patients in the chronic phase (60-70%) are asymptomatic. However, the remaining 30-40% of chronic patients develop recognizable clinical symptoms. The most frequent symptoms are heart hypertrophy and dilatation, esophagus and large intestine dilatations (megavisceras), or a combination of both (reviewed by [3,4]). The treatment of the chagasic infection is largely unsatisfactory [5]. Presently, two drugs discovered approximately 50 years ago are available nifurtimox (Nf) and benznidazole (Bz). Both drugs are highly effective in the acute phase [6]. However, their efficacy in treating the chronic phase, when most patients are diagnosed, is limited due to the serious side effects that occur from the toxicity of the drugs and the long-term treatment required in this phase. Importantly, the emergence of resistant parasites was reported [7]. In view of these facts, there is an urgent need to look for new drugs to treat T. cruzi infections [3,8].
Our group has been exploring drug reposition strategies, consisting of the identification of new uses for drugs already approved for the treatment of any disease in humans [9,10]. In a previous work, Paveto et al. suggested the existence of an L-glutamate receptor N-methyl-Daspartate (NMDA) type in T. cruzi. The facts that: i. excitatory amino acids involved in the NO production in neural cells are able to influence the T. cruzi NO synthase [11]; and ii. epimastigotes are responsive to NMDA (by increasing their motility), and the observed effect is abolished by the non-competitive inhibitor MK-801 [12] reinforces this idea. Additionally, our group characterized a T. cruzi glutamate transporter [13] that could behave as a glutamate receptor. More recently, we showed the sensitivity of T. cruzi to memantine (1,2,3,5,6,7-hexahydro-1,5:3,7-dimethano-4-benzoxonin-3-yl) amines, a tricyclic amine with a low-to-moderate affinity for the N-methyl-D-aspartate (NMDA) receptor [14], which has been indicated for the treatment of Alzheimer's disease [15]. More specifically, we showed that memantine presented an apoptotic-like activity in T. cruzi epimastigotes as well as a trypanocidal effect in infected mammalian cells [14]. In the present work, we show that memantine affects the infection of macrophages by T. cruzi, diminishing the number of infected cells. We also report that, in addition to its effect on the parasite, memantine modifies macrophage activation by slightly diminishing both NO production and intracellular Ca 2+ levels in activated and non-activated macrophages. Finally, infected mice treated with memantine presented a diminished parasitemia peak, heart parasitic load, inflammatory infiltrates, and mortality. As a whole, this work proposes memantine to be an interesting drug to be further explored for the treatment of Chagas disease.

Animals
Six-to eight-week-old BALB/c female mice were obtained from the animal facility of the Department of Parasitology of ICB, USP. The animals were kept under controlled climatic conditions with free access to food and water (ad libitum). All laboratory procedures involving animals were previously authorized by the Ethics Committee on the Use of Animals for Experimentation of the Institute of Biomedical Sciences (ICB)-University of São Paulo (USP) (Protocol 107, Fls 132, Book 02).

Mammalian cells and parasites
The RAW 264.7 (macrophage) cell line was routinely cultivated in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), supplemented with 2 mM sodium pyruvate, 0.15% (w/v) NaCO 3 , 100 units mL -1 penicillin and 100 μg mL -1 streptomycin at 37 C in a humid atmosphere containing 5% CO 2 . Tissue culture-derived trypomastigotes of the T. cruzi Y-strain were obtained from infections of the LLC-MK 2 cell line (multiplicity of infection: 10 trypomastigotes/cell) as previously described [16]. Trypomastigotes were collected from the supernatant of LLC-MK 2 cells at days 6 to 10 postinfection and were transferred to other bottles for new passages and/or used for infection assays. The bloodstream trypomastigote form of the T. cruzi Y-strain was maintained by infecting the BALB/c mice. The recovery of trypomastigotes was performed weekly and was used for the infection assays.

Determination of RAW 264.7 macrophage viability
RAW 264.7 cells (5.0 x 10 5 cells mL -1 ) were cultured in 24-well plates in RPMI medium supplemented with FCS (10%) in the presence of different concentrations of memantine (ranging from 10 to 800 μM) or none (control). Cell viability was evaluated 48 h after the initiation of treatment using an MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) [17]. The inhibitory concentration of 50% of cells (IC 50 ) was determined by fitting the data to a typical dose-response sigmoidal curve using the program OriginPro8.
Effect of memantine on the intracellular amastigote of T. cruzi RAW 264.7 cells (2.5 x 10 4 per well) were cultivated in 24-well plates in RPMI medium (10% FCS) and kept at 37˚C. After 24 h, the cells were infected with 6.25 x 10 5 trypomastigotes per well (moi = 25) for 5 h. After this time, free parasites were removed by washing twice with PBS; RPMI medium (10% FCS) was replaced, and the cells were treated with different concentrations of memantine (range 1 μM to 150 μM) or Benznidazole (range 0.5 μM to 100 μM) for the following 48 h. Then, the cells were washed with PBS, fixed with 4% paraformaldehyde, washed again and stained with Hoechst 33258. After washing out the excess of dye, the cells were observed by fluorescence microscopy. The infected cells were counted from a sample of 400 randomly chosen cells for determination of the infection rate. The number of amastigotes was also counted to determine the rate of amastigote per cell. All experiments were performed in technical and biological triplicates.

Evaluation of the nitric oxide (NO) production from RAW 264.7 macrophage culture
Macrophages require activation by particular quantities of Escherichia coli-derived LPS for NO production detection. Thus, a dose-response curve was produced to determine the ideal concentration of LPS required for activating the RAW 264.7 cells. The macrophages were stimulated with different concentrations of LPS ranging from 1 to 100 μg/ml for 24, 48 and 72 h. The NO evaluation was based on the nitrite released measure from the supernatant of the cultured cells. The RAW 264.7 cells (2.5 x 10 5 cells mL -1 ) were cultured in 96-well plates in RPMI medium (10% FCS) in the presence of different concentrations of memantine (ranging from 1 to 100 μM) or none (control) and were stimulated by 10 μg/ml LPS for 24 h or no stimulation. Over this period, the nitrite concentration of the cell supernatant was quantified using the Griess reaction, as described by [18].

Evaluation of the gene expression of inducible NO synthase in RAW 264.7 macrophages
To evaluate the gene expression of inducible nitric oxide synthase, 2 x 10 6 cells per well were cultured for 18 h in 6-well plates in RPMI medium (10% FCS) in the presence of different concentrations of memantine (ranging from 1 to 100 μM) or none (control) and were stimulated by 10 μg/ml LPS for 18 h. After the incubation time, the supernatant was discarded, and the adhered cells were homogenized with Trizol for RNA extraction (Thermo Fisher Scientific). cDNA was synthetized using the Reverse Transcription Kit SuperScriptII (Thermo Fisher Scientific). qPCR was performed with SYBR Green (Fermentas) for detecting the gene expression levels of iNOS. All reactions were run in triplicate on an Eppendorf RealPlex Real Time PCR System (Eppendorf) with the standard thermal cycling conditions. The runs were normalized with the ACT-β gene. The threshold cycle (2 -ΔΔCt ) method of comparative PCR was used for the data analysis.

Analysis of intracellular Ca 2+ levels in RAW 264.7 macrophages
Cells (2.5 x 10 4 cells/well) were cultivated in 96-well plates in RPMI medium (10% FCS), stimulated or not stimulated (control) with LPS, and treated or not treated (control) with different concentrations of memantine (ranging between 1 and 100 μM) for 24 h. Then, 5 μM fluo-4 AM (Invitrogen) was added to the cultures for 1 h. After the incubation, the cells were washed twice with HEPES-glucose (50 mM HEPES, 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO 4 , 5.5 mM glucose and 2 mM CaCl 2 , pH 7.4). The reading was performed on a Spectra Max M3 fluorometer, Molecular Devices, using excitation λ 490 nm and emission λ 518 nm [14].

Evaluation of parasitemia and survival of BALB/c mice
Mice were separated into the two groups with ten mice each: Untreated mice and infected; treated mice and infected. The animals were intraperitoneally infected with 1x10 3 trypomastigotes (Y strain) per mice. From the start of the infection, mice were intraperitoneally treated with MEM (10 mg/kg/day) or PBS (vehicle) for 10 consecutive days. The effect of the treatment was evaluated by following-up parasitemia. The parasitemia was registered between days 7 and 12 and the number of trypomastigotes was quantified in 25 microscope fields at 400x magnification with 10 μL of blood (Nikon Eclipse E200) [19]. The survival of the mice was also monitored for 40 days postinfection, when mice in this model becomes chronically infected [20]. After this time, all mice were humanely euthanized according to the guides of the Brazilian Society for Animal Welfare (SBCAL), and the protocols approved by the Ethics Committee on the use of Animals for Experimentation of ICB-USP (Protocol 107, Fls 132, Book 02). All experiments were repeated four times with a minimum of 10 animals/group. For the survival curves, log-rank and Wilcoxon-Gehan-Breslow tests were used. All data were analyzed in GraphPad Prism 5.0. p < 0.05 was considered significant.

Quantification of the tissue parasite load
On the 15 th day postinfection, samples of lung, spleen, bladder, heart, intestine, and skeletal muscle were obtained from the infected BALB/c mice for the quantification of the parasite load. The fragments were transferred to formaldehyde (10%) and were then processed by gradual dehydration in ethanol solutions, followed by immersion in xylene, and subsequently embedded in paraffin. Tissue sections 5 μm thick were obtained and stained with hematoxylin and eosin (H&E) and analyzed by light microscopy. The number of amastigote nets was counted in 20 random microscope fields using a 400x magnification. In parallel, tissue fragments were submitted for DNA extraction using the DNAeasy Blood and Tissue Kit as recommended by the manufacturer. The tissue parasitic load was also performed using quantitative PCR as previously described [21]. The cycle threshold values obtained by the Eppendorf Real-Plex software were converted to the number of parasites per 5 ng of tissue DNA. Their averages were normalized according to the TNF-α gene.

Histopathological analysis in the cardiac tissue
Cardiac tissue sections 5 μm thick were obtained on the 15 th day postinfection, stained with H & E and analyzed by light microscopy. Six nonconsecutive slides from the heart of each mouse were analyzed in a blinded fashion. Areas of inflammatory infiltrates were quantified by an image analysis system (Bioscan Optimas; Bioscan Inc., Edmonds, Wash). The sum of the infiltrated areas from the six slides was calculated for each mouse. The final individual score was expressed in square micrometers of inflammatory infiltrates per square millimeter of area examined.

Statistical analysis
The experimental data were input into GraphPad Prism version 4.0 software for construction of the graphs. In addition, t-tests or one-way ANOVA analyses were performed, followed by Tukey's and logrank tests for statistical analysis. Differences with a p value <0.05 were considered statistically significant.

In vitro
Memantine affects the intracellular cycle of T. cruzi. The effect of memantine on the intracellular cycle of T. cruzi was evaluated. First, we evaluated the toxicity of memantine in macrophages of the RAW 264.7 lineage. The cells were treated with different concentrations of memantine (10-800 μM) for 24, 48 and 72 h; we observed that the macrophages tolerated memantine at concentrations up to 100 μM, showing an IC 50 of 580 ± 22 μM, 279 ± 2 μM and 257 ± 4.7 μM, respectively (Fig 1A-1F).
Once the cytotoxic effect of memantine was evaluated, the RAW 264.7 macrophages were subjugated to infection. For this, the cells were incubated with trypomastigotes for 5 h, and then the trypomastigotes remaining in the supernatants were washed out. The infected cells were incubated for 12 h at 37˚C. Then, the culture medium was replaced with culture medium containing different concentrations of memantine (1-150 μM) or Benznidazole (0.5-100 μM). These treatments were maintained for 48 h. The treatment with memantine reduced the number of infected cells at concentrations tested, showing an IC 50 of approximately 22 μM (approximately five times the IC 50 obtained for Benznidazole) when compared to the untreated group (Fig 2). The number of intracellular amastigotes as a function of the memantine or benznidazole concentrations were counted. None of the treatments cured the infected cells. However, it is worth mentioning that both, memantine and benznidazole had similar effects, diminishing approximately by half the number of intracellular amastigotes at a concentration of 25 μM.

Memantine decreases NO production at intermediate concentrations in RAW 264.7 macrophages in vitro.
Due to the ability of memantine to reduce the number of infected cells in a dose-dependent manner, we were interested in checking whether memantine was acting as a trypanocidal compound by inducing macrophage activation. To evaluate the possible effect of memantine on the activation of RAW 264.7 macrophages, we first evaluated their sensitivity to LPS, a well-known macrophage activator (control). For this, the cells were incubated with different concentrations of LPS (1-100 μg/ml) for 24 h, and we considered the ability of the cells to produce NO as a measurement of activation. The NO production increased linearly with the LPS concentration; thus, among the concentrations tested, we chose 10 μg/ml (the maximum concentration tested) for further experiments (S1A Fig). Next, we performed a time-course experiment to follow the NO production for up to 72 h. We observed a significant increase in the period of 24 h, followed by a plateau that was maintained at 48 and 72 h (S1B Fig). We then evaluated the effect of memantine treatment (1-100 μM) on nitric oxide (NO) production and iNOS gene expression after 24 h of LPS stimulation. Unexpectedly, memantine treatment showed a reduction in nitrite production at concentrations of 10 and 50 μM, as well as in the expression of iNOS mRNA. Interestingly, the concentration of 100 μM did not interfere with NO production, which suggests a dose-dependent anti-inflammatory effect (Fig  3A and 3B).
Memantine reduces intracellular Ca 2+ levels in RAW 264.7 macrophages. It is known that the pathological activation of NMDA receptors, either by direct or indirect mechanisms, possibly results in an increase of intracellular calcium [22]. Based on these observations, we  (1-100 μM). Exposure of the LPS-activated cells to memantine resulted in a dosedependent decrease in intracellular calcium levels (Fig 3C). Remarkably, this effect was not dependent on macrophage activation since no differences in the intracellular calcium levels were observed in the non-activated cells.

Memantine treatment reduces parasitemia and increases the survival of T. cruziinfected BALB/c mice.
Since memantine showed a reduction in the number of infected cells in vitro, we considered it relevant to evaluate memantine's effect in vivo. The available clinical data shows the concentration administered in the treatment of patients with [EXSCINDED]The in vivo trypanocidal activity of memantine neurodegenerative diseases and the reported side effects for the use of memantine in other animal models (ataxia, muscle relaxation, and amnesia); the side effects were only observed in relatively high doses related to the concentration considered to be of therapeutic importance [23], and we chose 10 mg/kg of body weight as the ideal concentration for our trial. BALB/c mice infected with 1x10 3 bloodstream trypomastigotes were treated for 10 consecutive days. The treated animals showed decreased parasitemia on the days corresponding to the parasitemic peak (7 to 10 d.p.i) by approximately 40% compared to the control group (Fig 4A). The mouse survival rate was 12.5% compared to the control group survival rate of 7.5% (p = 0.0347) (Fig 4B). In summary, memantine treatment decreased parasitemia and extended the survival of infected BALB/c mice.
The memantine treatment reduces the parasitic load and increases the inflammatory infiltrate in the heart of infected BALB/c mice. To evaluate the effect of memantine on the parasitic load in different tissues from treated mice, we performed real-time PCR to quantify the number of parasites. For this, we obtained DNA, equivalent to 5 ng of tissue DNA (P E /5 ng DNA), from the heart, bladder, intestine, skeletal muscle and liver. Among the evaluated tissues, the heart showed the highest parasitic load (equivalent to 3,427 ± 451 P E /5 ng DNA). The memantine-treated mice showed a significant reduction in the parasitic load in the heart   Parasitemia in BALB/c mice infected by the intraperitoneal route with 1x10 3 forms of sanguine trypomastigotes of strain Y of T. cruzi. The number of blood trypomastigotes on days equivalent to the parasitemic peak was evaluated in infected mice, treated or not treated with memantine (10 mg/kg per day). Comparison between the groups treated and not treated with memantine (p <0.05). Data are expressed as the mean ± standard deviation. (B) Infected animals treated or not treated with memantine (10 mg/kg per day). The experiments were repeated four times with 10 animals/ group. Comparison between the groups treated and not treated with memantine (log-rank test) (p <0.05). https://doi.org/10.1371/journal.pntd.0007226.g004 [EXSCINDED]The in vivo trypanocidal activity of memantine (35.3%) compared to the parasitic load in the values obtained from the control mice (p<0.05) (Fig 5A; S2A-S2E Fig). Remarkably, the quantification of the number of amastigote nests per mm 2 , evaluated by microscopy observation, confirmed these data: the hearts from the control mice demonstrated a mean of 2.3 ± 0.35 nests/mm 2 , while the memantine-treated mouse hearts showed a mean of 1.2 ± 0.15 nests/mm 2 (a reduction of approximately 45%, Fig 5B). Moreover, we also observed that the area of the inflammatory infiltrates (normalized per mm 2 ) was significantly reduced in the treated heart tissues: those from the treated animals showed a mean value of 8.66 ± 4.15 inflammatory infiltrate/mm 2 , while those from the control group presented a mean value of 80.53 ± 31.73 inflammatory infiltrate/mm 2 (Fig 5C, as an illustrative example of the amastigote nests and infiltrates in treated mice vs control mice see Fig 5D).
All the obtained data for the parasitic load, amastigote nest and inflammatory infiltrate quantification demonstrate that treatment with memantine reduces the risk of tissue parasiticassociated damage, both by diminishing the parasitic load and by diminishing the inflammatory response. [EXSCINDED]The in vivo trypanocidal activity of memantine

Discussion
In the present work, we provide evidence of the therapeutic potential of memantine in the in vitro and experimental infection by T. cruzi. As memantine is currently used in patients with moderate to severe stages of Alzheimer's disease [15], we propose to further study its repurposing to treat the infection by T. cruzi.
Under pathological conditions, memantine is used as a noncompetitive antagonist drug of the voltage-dependent N-methyl-D-aspartate (NMDA) receptor to block the effects of elevated glutamate levels [24]. The NMDA receptor belongs to the family of ionotropic glutamate receptors and is involved in a variety of central nervous system (CNS) functions and processes [25]. In mammals, these receptors play important physiological roles, and despite their predominance in the CNS, NMDA receptors have also been identified in peripheral and visceral sites located on the postsynaptic dendrite membranes [25,26]. In T. cruzi, there are no reports in the literature on the presence of a canonical NMDA-type glutamate receptor. However, as previously mentioned, it was showed that T. cruzi epimastigotes are responsive to NMDA [11,12]. This information is compatible with our previous finding that the CL-14 strain of T. cruzi is susceptible to memantine and that amastigotes infecting CHO-K 1 cells are the most susceptible forms in vitro [14]. Here, we show that treatment with memantine significantly reduces the infection rate (infected/noninfected cells) in RAW 264.7 macrophages. However, as memantine diminishes NO production in infected macrophages, it is unlikely to attribute its effect of an increase in the host cell natural defense mechanism against the parasite. Therefore, other possible mechanisms altering the viability of the intracellular amastigotes were explored. Our data show that memantine induced an increase in mitochondrial function in relation to the control cells. Similarly, Prado demonstrated that Neuro-2A neural cells increased their mitochondrial reducing power when pretreated with 0.5-50 μM memantine. However, when the same cells were treated with lower memantine concentrations, the calcium influx was decreased with the concomitant increase in the mitochondrial reducing power [27]. Additionally, Chen and colleagues observed that memantine at low doses may play an antiinflammatory and neuroprotective role, although the anti-inflammatory effects are still uncertain [28]. These findings corroborate our data, where memantine induced a decrease in NO production in LPS-stimulated RAW 264.7 macrophages (10 μg/mL) at concentrations of 10 and 50 μM memantine. However, the concentration of 100 μM memantine suggests a pro-oxidant effect in our assays. Additionally, memantine was shown to have potential effects as a neurotransmitter and a neuroprotective compound [29], to inhibit the ATP-sensitive potassium channels (K + /ATP) in substantia nigra (dopaminergic) neurons [30] and to suppress the internal currents induced by electroporation [31]. In fact, Tsai and colleagues demonstrated that the concentrations used to inhibit at 50% the internal rectifying potassium channels (IK (IR)) is similar to the memantine IC 50 (12 μM) in RAW 264.7 macrophages. These channels act as metabolic sensors and are sensitive to ATP, that is, when calcium levels are high, closure of the channel occurs [32]. We previously demonstrated that memantine affects the energetic metabolism of the parasite, inducing decreased levels of ATP and triggering mechanisms that lead to apoptosis in epimastigotes of T. cruzi (CL strain, clone 14) [14]. In the present work, we showed that 100 μM memantine induces a decrease in the intracellular calcium levels in both LPS-stimulated and nonstimulated cells. This supports the hypothesized macrophage NMDA receptor [33], suggesting that memantine would be able to block it. This is consistent with the previous observations that, in lymphocytes, NMDA receptors are involved in the regulation of intracellular calcium levels [34] as well as the levels of ROS [35].
Since memantine decreased T. cruzi infection in macrophages in vitro, we evaluated the effect of memantine treatment on infection in vivo. The treatment of BALB/c mice infected with Y strain bloodstream trypomastigotes with memantine for 10 consecutive days reduced the parasitemia by 40% during the parasitemic peak when compared to the control group. Importantly, the dose used in our work (10 mg/kg per day) can be considered safe: doses of 10 mg or 20 mg per day were shown to be beneficial for mice in terms of improving the functional capacity of daily activities and the behavioral disorders characteristic of Alzheimer´s disease [36].
During the acute phase of Chagas disease, it is known that the parasites are present in many tissues of the host. Our data showed a high parasitic load in the heart with 3427 ± 451 parasites equivalent to 5 ng tissue DNA (PE/5 ng DNA). As observed, memantine, at the dose used, was able to significantly reduce the tissue parasitic load in this tissue by approximately 35.3%. It was reported that animals inoculated with the trypomastigote form of the Y strain show an extremely high parasitic load on the 7th and 8th d.p.i. in the spleen and liver, among others [37]. This might explain why we did not observe significant differences or a consistent parasitic load in these tissues. When we evaluated the number of amastigote nests in the cardiac tissue, our results indicate that the control group presented a mean of 2.3 ± 0.35 nests/mm 2 , while the animals treated with memantine (10 mg/kg per day) presented an average of 1.2 ± 0.15 nests/ mm 2 in cardiac tissue, which is consistent with the data obtained by using real-time PCR. Remarkably, this reduction was consistent with that observed when the inflammatory infiltrates in the cardiac tissue were analyzed. These datasets are consistent with previously published results that show the susceptibility of amastigote forms to memantine [14]. This is of extreme relevance since the amastigotes are responsible for maintaining chronic infection in patients. It was shown that amastigotes can enter a dormant state, which makes them resistant to benznidazole [38]. Further studies should be conducted to evaluate the possible trypanocidal effect of memantine on dormant amastigotes. Taken together, all these findings point memantine as an interesting starting point for the development of an optimized alternative therapy for Chagas disease.