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. 2021 Oct 5;16(10):e0258199.
doi: 10.1371/journal.pone.0258199. eCollection 2021.

Behavioral alterations in long-term Toxoplasma gondii infection of C57BL/6 mice are associated with neuroinflammation and disruption of the blood brain barrier

Affiliations

Behavioral alterations in long-term Toxoplasma gondii infection of C57BL/6 mice are associated with neuroinflammation and disruption of the blood brain barrier

Leda Castaño Barrios et al. PLoS One. .

Abstract

The Apicomplexa protozoan Toxoplasma gondii is a mandatory intracellular parasite and the causative agent of toxoplasmosis. This illness is of medical importance due to its high prevalence worldwide and may cause neurological alterations in immunocompromised persons. In chronically infected immunocompetent individuals, this parasite forms tissue cysts mainly in the brain. In addition, T. gondii infection has been related to mental illnesses such as schizophrenia, bipolar disorder, depression, obsessive-compulsive disorder, as well as mood, personality, and other behavioral changes. In the present study, we evaluated the kinetics of behavioral alterations in a model of chronic infection, assessing anxiety, depression and exploratory behavior, and their relationship with neuroinflammation and parasite cysts in brain tissue areas, blood-brain-barrier (BBB) integrity, and cytokine status in the brain and serum. Adult female C57BL/6 mice were infected by gavage with 5 cysts of the ME-49 type II T. gondii strain, and analyzed as independent groups at 30, 60 and 90 days postinfection (dpi). Anxiety, depressive-like behavior, and hyperactivity were detected in the early (30 dpi) and long-term (60 and 90 dpi) chronic T. gondii infection, in a direct association with the presence of parasite cysts and neuroinflammation, independently of the brain tissue areas, and linked to BBB disruption. These behavioral alterations paralleled the upregulation of expression of tumor necrosis factor (TNF) and CC-chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β and CCL5/RANTES) in the brain tissue. In addition, increased levels of interferon-gamma (IFNγ), TNF and CCL2/MCP-1 were detected in the peripheral blood, at 30 and 60 dpi. Our data suggest that the persistence of parasite cysts induces sustained neuroinflammation, and BBB disruption, thus allowing leakage of cytokines of circulating plasma into the brain tissue. Therefore, all these factors may contribute to behavioral changes (anxiety, depressive-like behavior, and hyperactivity) in chronic T. gondii infection.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Flow chart showing the experimental protocol with the number of animals used.
Death registered, and mice included in 2 (endpoint registered at "30 and 60 dpi") or 1 (endpoint registered at "90 dpi") independent experiments.
Fig 2
Fig 2. C57BL/6 mice chronically infected with the ME-49 Toxoplasma gondii strain survive and show weight and muscle strength loss.
(A) Mice were infected with 5 cysts of the ME-49 T. gondii strain, the clinical follow-up and the mortality were assessed and recorded weekly, and the kinetics of infection was evaluated at 30, 60 and 90 dpi. (B) The survival curve shows a survival of 100% at 30 and 60 dpi, and 83% (19/24) at 90 dpi, compared to 100% in the age- and sex-matched NI control group. (C) Infected mice showed body weight loss during the acute phase of the infection (up to 15 dpi), after this period, the weight loss ceased. (D) Muscle strength was compromised in infected mice; values of muscle strength are shown as gram force (gf) / body weight (g). Each experimental group consisted of 4–6 NI mice and 10–19 T. gondii-infected mice. Each circle represents an individual mouse. Data are expressed as means ± SEM, and were analyzed using Welch’s test (C), and ordinary one-way ANOVA (D). **, p<0.01. ***, p<0.001, comparing T. gondii-infected and NI mice.
Fig 3
Fig 3. Mice with long-term chronic Toxoplasma gondii infection show preserved locomotor capacity.
(A) The gait analysis was performed using the footprint test and were evaluated as such: the spread of the fingers and overlapping distance between the anterior and posterior limbs, right and left, and the stride length of limbs, width of the front and rear base. (B) The toe spread was normal in the right and left limb. (C) |No alteration was observed in the right and left step overlap of infected mice. (D) The forelimb and hindlimb stride were increased as the infection progresses. (E) The width of the front base was enlarged in infected mice, but the hind base was preserved. Each experimental group consisted of 4–6 NI mice and 8–12 T. gondii-infected mice. Each circle represents an individual mouse. Data are expressed as means ± SEM, and were analyzed using ordinary one-way ANOVA. *, p<0,05, **, p<0.01. ***, p<0.001, comparing T. gondii-infected and NI mice.
Fig 4
Fig 4. Anxiety and depressive-like behavior are detected in long-term chronically Toxoplasma gondii-infected C57BL/6 mice.
(A) Chronically infected mice showed reduced time in the central zone in OFT, and (B) increased time exploring the peripheral area of the open field. (C) Depressive-like behavior was revealed as enhanced time of immobility in TST. Each experimental group consisted of 4–6 NI mice and 10–15 T. gondii-infected mice. Each circle represents an individual mouse. Data are expressed as means ± SEM, and were analyzed using t-Student test. *, p<0,05, **, p<0.01. ***, p<0.001, comparing T. gondii-infected and NI mice.
Fig 5
Fig 5. Hyperactive behavior is observed in chronically Toxoplasma gondii-infected C57BL/6 mice.
To determine hyperactivity, we evaluated the time spent immobile, the distance and the speed of travel in OFT, and the activity in FST. (A) Infected mice did not show differences in immobility time when in the OFT. (B) At 60 dpi, mice showed increased distance traveled in OFT. At 30 (18%) and 90 (21%) dpi only a reduced percentage of mice showed increase in the distance traveled. Red dot line shows mean distance of NI group + 2 standard deviation. (C) The walk velocity in OFT was increased as infection progressed. (D) In FST, immobility time decreased as infection progressed. Each experimental group consisted of 4–6 NI mice and 8–15 T. gondii-infected mice. Each circle represents an individual mouse. Data are expressed as means ± SEM, and were analyzed using t-Student test (A-C) and the Mann-Whitney test (D). *, p<0.05, **, p<0.01. ***, p<0.001, comparing T. gondii-infected and NI mice.
Fig 6
Fig 6. The numbers of parasite cysts in the brain decrease and the sizes increase as Toxoplasma gondii infection progresses in mice C57BL/6.
(A) The number of cysts decreased as the early (30 dpi) infection coursed to long-term chronic infection (60 and 90 dpi). (B) The size of the cysts increased as infection progressed from early to long-term chronic phase. (C) The graphs show the relative frequencies of cysts in each class at the three timepoints analyzed. Each experimental group consisted of 8–12 T. gondii-infected mice. Each circle represents the number of cysts in the brain of each animal (A), and the diameter of each cyst (B). Data are expressed as means ± SEM, and analyzed using the Mann-Whitney test. #, p <0.05, ##, p <0.01. ###, p <0.001, comparing different timepoints.
Fig 7
Fig 7. Some regions of the brain of C57BL/6 Toxoplasma gondii-infection mice are intensely infected.
(A) The stereotaxic coordinates of the bregma of the mouse ́s brain were used to construct representative maps of the topographic location of the cysts in each brain area: Olfactory areas (OLF), Isocortex (ICTX), Cerebral Nuclei (CNU), Hippocampal formation (HPF), Thalamus (TH), Hypothalamus (HY), Midbrain (MB), Pons (P), Medulla (MY) and Cerebellum (CB). (B) The cysts were localized in all brain regions, but the isocortex, thalamus and midbrain were colonized more intensely in the early (30 dpi) and long-term (60 and 90 dpi) chronic infection (each color represents an individual mice). The histograms show the percentage of cysts per area studied. (C) Representative pictures show individual cysts or multiple cysts per microscopic field in different brain areas, associated or not with inflammatory foci. Bar = 60 μm. Each experimental group consisted of 4–10 T. gondii-infected mice. Data were analyzed using ordinary one-way ANOVA.
Fig 8
Fig 8. Toxoplasma gondii-infected C57BL/6 mice showed generalized neuroinflammation in early and long-term chronic phase of infection.
(A) Infected mice presented inflammatory foci with meningoencephalitis and perivascular inflammatory cuffs in all evaluated areas of the CNS in early (30 dpi) and long-term (60 and 90 dpi) chronic infection. Bar = 330 μm. (B) Cysts devoid of inflammation were firstly detected in the CNS at the acute phase (20 dpi, left panel), and the inflammation is already settled in the chronic phase (45 dpi, right panel). Bar = 100 μm. (C) Increased expression of the pro-inflammatory cytokines IFNγ and TNF was detected in the acute phase, and TNF expression was sustained at the chronic phase of infection. (D) In the acute phase, the expression of MIP1α/CCL3, MIP1β/CCL4, RANTES/CCL5 and MCP-1/CCL2, was increased. The upregulation of the expression of TNF and CC-chemokines were sustained at 45 dpi. Each experimental group consisted of 2–4 NI mice and 2–10 T. gondii-infected mice, in two independent experiments. Data are expressed as means ± SEM, and were analyzed using ordinary one-way ANOVA. *, p<0,05 comparing T. gondii-infected and NI mice, and #, p<0.05 comparing acute and chronic groups of T. gondii infected mice.
Fig 9
Fig 9. Systemic cytokine expression is upregulated in the early and long-term chronic Toxoplasma gondii infection.
(A) The images show representative data plots of the FACS analysis of CBA. (B) Chronically T. gondii-infected mice showed increased levels of pro-inflammatory cytokines at 30 and 60 dpi. At 90 dpi, all cytokine levels showed a tendency to decrease or, even, exhibited cytokines levels like those found in sera of NI controls, except for IFNγ and MCP-1/CCL2 levels. Each experimental group consisted of 2–3 NI mice and 2–5 T. gondii-infected mice, in two independent experiments. Data are expressed as means ± SEM, and were analyzed using ordinary one-way ANOVA. *, p<0,05, **, p<0.01. ***, p<0.001, comparing T. gondii-infected and NI mice.
Fig 10
Fig 10. The increase in BBB permeability is concomitant with edema in Toxoplasma gondii-infected C57BL/6 mice.
(A) Representative images of brains depicting EB extravasation at the timepoints assessed. (B) Representative sagittal sections of the encephala, at 30 and 60 dpi. (C) The concentrations of EB in the brain tissue of infected mice increased at the three studied timepoints, indicating the increase in BBB permeability. (D) Cerebral edema, manifested by a significant increase in the relative brain weight (brain weight in milligram / whole body weight in gram), was present in the timepoints evaluated. Each experimental group consisted of 4–6 NI mice and 8–19 T. gondii-infected mice, in two independent experiments. Each circle represents an individual mouse. The data are expressed as means ± SEM, and were analyzed using Mann-Whitey test (C) and t-Student test (D). **, p<0.01. ***, p<0.001, comparing T. gondii-infected and NI mice.

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