Kinematic Locomotion Changes in C57BL/6 Mice Infected with Toxoplasma Strain ME49

Chronic infection with the intracellular parasite Toxoplasma gondii produces an accumulation of cysts in the brain and muscle, causing tissue damage. The cysts in the brain motor regions affect some kinematic locomotion parameters in the host. To localize the brain cysts from Toxoplasma gondii and study the changes in kinematic locomotion in C57BL/6 mice. Female adult C57BL/6 mice were infected orally with 30 ME-49 Toxoplasma gondii cysts. An uninfected group (n = 7) and two infected groups, examined 15 and 40 days postinfection, were used for this study. To evaluate kinematic locomotion, the mice were marked with indelible ink on the iliac crest, hip, knee, ankle, and phalangeal metatarsus of the left and right hindlimbs. At least three recordings were carried out to obtain videos of the left and right hindlimbs. Mice were video recorded at 90 fps at a resolution of 640 × 480 pixels while walking freely in a transparent Plexiglass tunnel. We measured the hindlimb pendular movement and the hindlimb transfer [linear displacement] curves for each step and evaluated them statistically with Fréchet dissimilarity tests. Afterward, the mice were sacrificed, and the brain, heart, skeletal muscle, lung, liver, and kidney were obtained. The different tissues were stained with hematoxylin and eosin for analysis with optical microscopy. Topographic localization of the cysts was made using bregma coordinates for the mouse brain. The cysts were distributed in several brain regions. In one mouse, cyst accumulation occurred in the hippocampus, coinciding with an alteration in foot displacement. The step length was different among the different studied groups.


Introduction
Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that infects both humans and animals. Toxoplasma is transmitted orally, through a transplacental pathway or blood transfusion or via transplant [1]. A third of the world's population has been affected by this parasite.

Locomotion Studies
All mice from the three groups were marked with nontoxic indelible ink in the (A) iliac crest, (B) the hip, (C) the knee, (D) the ankle, and (E) the phalangeal metatarsus, in both the left and right hindlimbs ( Figure 1A). Mice were video recorded at 90 fps at a resolution of 640 x 480 pixels while walking freely in a transparent Plexiglass tunnel [14,15].
The marks on the mice were manually annotated on each frame of the videos using proprietary software developed by our research group, and marks were used to track their displacement in the horizontal and vertical directions during mouse movements ( Figure 1B). The duration and covered distance of the translation phase for all steps for each group were computed.
Step length was divided and evaluated in pixels. Then, transformed into centimeters and plotted in the ordinate, the different mice groups were plotted on the abscissa.
The translation phase patterns of the marks of all mice were compared by two means: the analysis of the pendulum-like movement [16,17] that is measured as the curve corresponding to the changes in the angle resulting from connecting points A and E with respect to time ( Figure 1C), and the analysis of the joint transitions that was performed by computing curves corresponding to the amplitude of the joints movement in the horizontal and vertical directions across time ( Figure 1B).
Steps length and speed can be different among mice. Therefore, to evaluate the joints, motion patterns were necessary to normalize the curves. We divided the steps using 20 points at equal time bins during the step cycle. The beginning of the translation phase corresponded to the 0% step cycle, and the point was corresponding to the end of the translation phase corresponded to 100% of the step cycle ( Figure 1D).
Statistics for all normalized curves corresponding to each mark of the control group mice were computed for analyses. Then, each bin corresponding to chronic-and acute-infected mice was compared with respect to the statistics of the control curves [15,16]. distributed (p-value of 0.08). Then, we performed a t-test with a 95% confidence level (α = 0.05) to determine if statistically significant differences existed between the control vs chronic and control vs acute groups [15].

Histopathological Samples
Mice infected and remaining alive for 15 (N = 7) days and those surviving for 40 (N = 12) days, as well as mice in the control group (N = 7), were anesthetized with isofluorane and sacrificed. Subsequently, a craniotomy of the mouse was performed to extract the brain, while other organs such as the heart, skeletal muscle, lung, liver, and kidney were also extracted; they were washed with saline solution before formol fixation. Then, the brains were placed in tubes containing PBS at 10% until the sample was covered. Twenty-four hours later, samples were washed with distilled water and then fixed. PBS was replaced with 70% alcohol. Samples were embedded in paraffin to obtain 0.5-micron slices [6].

Staining with Hematoxylin and Eosin
The slides were placed in a hematoxylin container (Sigma Aldrich,Darmstadt,Germany) for two minutes, and then, the excess was removed with distilled water. The slices were submerged for a three-second period in acid alcohol (1% HCl in 70% alcohol), washed with water, and then immersed in yellow eosin (Sigma) for a minute and a half before being washed with tap water for thirty seconds. For dehydration, passages were made in increasing gradients of alcohol and xylol as follows: 70% alcohol for 3 s, 90% alcohol for 3 s, 96% alcohol for 3 min, twice in 100% alcohol for 5 min, and then

Statistical Analysis
To validate the data normality we used the Kolmogorov-Smirnov Test. Our data are normally distributed (p-value of 0.08). Then, we performed a t-test with a 95% confidence level (α = 0.05) to determine if statistically significant differences existed between the control vs chronic and control vs acute groups [15].

Histopathological Samples
Mice infected and remaining alive for 15 (N = 7) days and those surviving for 40 (N = 12) days, as well as mice in the control group (N = 7), were anesthetized with isofluorane and sacrificed. Subsequently, a craniotomy of the mouse was performed to extract the brain, while other organs such as the heart, skeletal muscle, lung, liver, and kidney were also extracted; they were washed with saline solution before formol fixation. Then, the brains were placed in tubes containing PBS at 10% until the sample was covered. Twenty-four hours later, samples were washed with distilled water and then fixed. PBS was replaced with 70% alcohol. Samples were embedded in paraffin to obtain 0.5-micron slices [6].

Staining with Hematoxylin and Eosin
The slides were placed in a hematoxylin container (Sigma Aldrich, Darmstadt, Germany) for two minutes, and then, the excess was removed with distilled water. The slices were submerged for a three-second period in acid alcohol (1% HCl in 70% alcohol), washed with water, and then immersed in yellow eosin (Sigma) for a minute and a half before being washed with tap water for thirty seconds. For dehydration, passages were made in increasing gradients of alcohol and xylol as follows: 70% alcohol for 3 s, 90% alcohol for 3 s, 96% alcohol for 3 min, twice in 100% alcohol for 5 min, and then twice in xylol for 5 min. Finally, combined with entellan, the obtained cuts were observed under a microscope (Carl-Zeiss Oberkochen, Germany) at 10 X and 40 X.
The Histological analysis was qualitative. We name four categories (Absent, Mild, Moderate, and Severe). The categories were established according to the morphological severity damage and the number of affected cases. The topographical location of cysts in the brains of infected mice was localizated using the bregma coordinates of the mouse brain [18].

Step and Displacement Analysis
There were no significant differences among the studied groups in pendular movement (not shown). In contrast, the results indicated a statistically significant difference in the step length of control vs chronically infected mice, as well as between the control and acute groups in the left and right hindlimbs (Figures 2 and 3).
Microorganisms 2019, 7, x FOR PEER REVIEW 5 of 16 twice in xylol for 5 min. Finally, combined with entellan, the obtained cuts were observed under a microscope (Carl-Zeiss Oberkochen, Germany) at 10 X and 40 X. The Histological analysis was qualitative. We name four categories (Absent, Mild, Moderate, and Severe). The categories were established according to the morphological severity damage and the number of affected cases. The topographical location of cysts in the brains of infected mice was localizated using the bregma coordinates of the mouse brain [18].

Step and Displacement Analysis
There were no significant differences among the studied groups in pendular movement (not shown). In contrast, the results indicated a statistically significant difference in the step length of control vs chronically infected mice, as well as between the control and acute groups in the left and right hindlimbs (Figures 2 and 3).   In the box plot graphics, 75% of the data are placed in each box, and the median and its respective standard deviations are calculated and platted. Statistically significant differences existed between the control vs chronic and control vs acute groups ( Figure 4). In the box plot graphics, 75% of the data are placed in each box, and the median and its respective standard deviations are calculated and platted. Statistically significant differences existed between the control vs chronic and control vs acute groups ( Figure 4).
The curves for linear hindlimb displacement in infected mice were similar among the control and experimental groups. However, there was a relevant difference in the pattern of the curve for the foot displacement of mouse 7 in the chronic group ( Figure 5). Note that the amplitude of the metatarsal joint displacement (lifting up) curves was out of range, more extensive than that of the control group, as depicted in the box plots.  The curves for linear hindlimb displacement in infected mice were similar among the control and experimental groups. However, there was a relevant difference in the pattern of the curve for the foot displacement of mouse 7 in the chronic group ( Figure 5). Note that the amplitude of the metatarsal joint displacement (lifting up) curves was out of range, more extensive than that of the control group, as depicted in the box plots.   The curves for linear hindlimb displacement in infected mice were similar among the control and experimental groups. However, there was a relevant difference in the pattern of the curve for the foot displacement of mouse 7 in the chronic group ( Figure 5). Note that the amplitude of the metatarsal joint displacement (lifting up) curves was out of range, more extensive than that of the control group, as depicted in the box plots.

Brain Toxoplasma Cyst Distribution
The brain cyst distribution for this particular mouse is illustrated in Figure 6. T. gondii cysts were located in groups of three to four cysts in the hippocampus zone in the cuts of bregma −1.94 to 1.34. However, Toxoplasma was widely distributed in all brain zones.

Brain Toxoplasma Cyst Distribution
The brain cyst distribution for this particular mouse is illustrated in Figure 6. T. gondii cysts were located in groups of three to four cysts in the hippocampus zone in the cuts of bregma −1.94 to 1.34. However, Toxoplasma was widely distributed in all brain zones. In all mice, cysts were widely distributed in the brain. We analyzed several brain coronal cuts to observe the distribution of T. gondii cysts. The cuts were made from bregma −6.0 to 0.38. In general, the cysts were localized randomly in several brain zones, as illustrated in Figure 7. In all mice, cysts were widely distributed in the brain. We analyzed several brain coronal cuts to observe the distribution of T. gondii cysts. The cuts were made from bregma −6.0 to 0.38. In general, the cysts were localized randomly in several brain zones, as illustrated in Figure 7. Toxoplasma cysts were widely distributed in all brain regions, as illustrated in Table 1. However, some brain zones (hf, LH, PoDG, RSA, SIBF, S2, TeA, V1,ZID) were preferred. In these zones there were more than ten cysts per region. Toxoplasma cysts were widely distributed in all brain regions, as illustrated in Table 1. However, some brain zones (hf, LH, PoDG, RSA, SIBF, S2, TeA, V1,ZID) were preferred. In these zones there were more than ten cysts per region.

Cysts in Acute Mice
In acute infected mice we only observed eighteen cysts (most of them in primary motor cortex, (N = 5), three in caudate putamen. Ten cysts were distributed in six brain regions.

Histological Analysis
A microscopic analysis of the mouse tissue was examined and showed a preferential tropism of Toxoplasma gondii in the central nervous system. In the acute infection group, gliosis nodules with remarkable necrosis accompanied by few Toxoplasma cysts either inside or outside of the gliosis were observed. We observed a neuronal hypoxia booth in cortical and hippocampal regions, with a less prominent meningovascular inflammatory component. The cysts were also found in the cerebellum. In the chronically infected mouse group, mild-to moderate-intensity myocarditis was found in most animals, with myopathy in 20% of cases; lymphocytic myopathy was the predominant type. We also noted pulmonary disease characterized by patches of a mixed inflammatory infiltrate with lymphocytes macrophages and polymorphonuclear leukocytes. A mild portal-reactive hepatitis was found in the liver, and in one case, mononuclear focal inflammation was found in the renal parenchyma ( Figure 8). In the chronically infect mouse group, we also observed more brain cysts compared to the acute experimental group. Cyst distribution appeared in several brain regions from the metencephalon to the telencephalon with an accumulation in the hippocampus. Gliosis was variable, with a tendency to be present in the cortical and periventricular regions. Mild to severe meningitis was accompanied by parenchymal perivasculitis and included endothelial proliferation (Figure 9).
In six out of 12 cases, skeletal muscle damage occurred. Damage included mild to focal myositis. There was evidence of cysts in the sarcoplasm in one case. In four out of 12 cases there was dystrophic calcification of muscle fibers that ranged from moderate or severe. Mild to moderate chronic inflammation was observed in the lungs in both the peribronchiolar and parenchymal tissue. Focal pneumonitis with a chronic component was also observed (Figure 9, Table 2). In the chronically infect mouse group, we also observed more brain cysts compared to the acute experimental group. Cyst distribution appeared in several brain regions from the metencephalon to the telencephalon with an accumulation in the hippocampus. Gliosis was variable, with a tendency to be present in the cortical and periventricular regions. Mild to severe meningitis was accompanied by parenchymal perivasculitis and included endothelial proliferation (Figure 9).
In six out of 12 cases, skeletal muscle damage occurred. Damage included mild to focal myositis. There was evidence of cysts in the sarcoplasm in one case. In four out of 12 cases there was dystrophic calcification of muscle fibers that ranged from moderate or severe. Mild to moderate chronic inflammation was observed in the lungs in both the peribronchiolar and parenchymal tissue. Focal pneumonitis with a chronic component was also observed (Figure 9, Table 2).  The intensity of the damage corresponds to: − (none shown), + (slight), ++ (moderate) and +++ (severe).

Discussion
We did not find pendulum angular movement changes among the different study groups. Angular changes in the different hindlimb joints could compensate for the absence of change in the pendular movement. Further studies using camera recordings at 250 frames/sec will be done to clarify this point. In contrast, we observed alterations in the stride length as found by other authors [8]. We did not study rear paw drags. The metatarsial joint displacement curve was different in one

Discussion
We did not find pendulum angular movement changes among the different study groups. Angular changes in the different hindlimb joints could compensate for the absence of change in the pendular movement. Further studies using camera recordings at 250 frames/sec will be done to clarify this point. In contrast, we observed alterations in the stride length as found by other authors [8]. We did not study rear paw drags. The metatarsial joint displacement curve was different in one chronically infected mouse compared to the control group. In this mouse, we observed cyst accumulation in the hippocampus, which can alter the locomotion kinematics. In rats, penetrating and damaging the hippocampus produces an alteration in locomotion speed [19].
We did not study whether the hippocampus cysts had enough time to liberate the bradyzoites to produce the kinematic alterations in this particular mouse. Note the clear neuron damage and hippocampal cysts in Figure 9C,D. The method used in these experiments to explore locomotion kinematics is reliable enough to detect changes in a joint through linear displacement curves.Traumatic brain injure in rats and mice hippocampus produce locomotion velocitiy reduction [17,19]. In this study most of cysts were found in a mouse with clearly change in join metatharsal displacement.
A sensorimotor deficit in C57BL/6 mice infected with the ME-49 Toxoplasma strain was also reported previously [8]. Gait deficits, measured as the number of missteps, the mean stride length and the number of rear paw drags, were significantly different between T. gondii-infected and control mice, p < 0.05 [8].
In an open field locomotion study of female C57BL/6 mice infected with transgenic Toxoplasma gondii ME49, it was found that the animals with brain cysts reached higher locomotion speeds in as early as the first second of locomotion and showed an initial acceleration during that first second of movement that was double that of control groups [9]. We did not study mice in open field locomotion to compare to the results of Alfonso et al. [9]. It is our intention to perform further studies on mice in open field locomotion using several recording cameras to study several kinematic locomotion parameters. For the first time, we implemented an analysis of iliac crest, hip, and ankle joint translation and a computational program to evaluate statistical significance among curves generated in different mouse groups using a Fréchet dissimilarity analysis. This method will be used in further experiments to statistical evaluate join displacement [20,21].
Acute infected mice had neuronal gliosis and necrosis damage, as well as an apparent cyst breakdown with consequent bradyzoites liberation, which changed them to chronic status and produced meningeal-encephalitic damage [8]. Neurons, astrocytes, and microglia are damaged by the presence of cysts in the neuronal body and in the dendrites and axons [22,23]. We found cysts in damaged neurons.
The cysts in chronic infected mice were distributed in several brain regions: mainly hippocampus, and the telencephalons.A minor number of cysts appeared in the brain stem and cerebellum. In acute infected mice we observed few cysts than in chronic infected mice, distribuited in several regions, manly in motor cortex. Given the distribution, several brain functions could be affected. It was also suggested that the presence of brain cysts was due to local damage in the hamate-encephalic brain barrier or alterations in metabolic blood flow [6]. It was suggested that the intercellular adhesion molecule 1 (ICAM-1) is upregulated in cellular barriers during Toxoplasma infection. Soluble human ICAM-1 and ICAM-1 antibodies inhibit the transmigration of parasites across cellular barriers, implicating this receptor in the process of transmigration.
Furthermore, an intercellular adhesion molecule (ICAM-1) immunoprecipitated the mature form of the parasite and adhesion microneme protein (MIC2) present on the parasite surface, indicating that this interaction may contribute to cellular migration [24,25] We did not study the intracellular adhesion molecules, but the Toxoplasma was widely distributed in the brain, suggesting that the brain barrier had broken down. The present results also demonstrated a disperse distribution of brain cysts, although there was an apparent predominance in the hippocampus. It is important to mention that in acute-infected mice, there was no histopathological damage in tissues such as the lung, liver, heart, and kidney, but damage was present in the chronically infected mice.
Our results suggest the importance of the possible presence of cysts liberating bradyzoites. The presence of cysts in the neural cell body, bradyzoites liberation, and muscle calcification in chronically infected mice produced skeletal muscle damage as well as mild to moderate focal myositis and dystrophic calcification of muscle fibers. This damage has been reported in Toxoplasma-infected mice and increases between days 12 and 30 postinfection [15,25]; we believe that this damage in the skeletal muscle reduced the locomotion of the host.

Conclusions
This study contributes with relevant information that increases knowledge in relation to the damage produced by Toxoplasma gondii in several tissues as well as a quantitative analysis of kinematic locomotion.