Behavioral and Neuropathological Changes After Toxoplasma gondii Ocular Conjunctival Infection in BALB/c Mice

Abstract

Ocular infection with Toxoplasma gondii causes toxoplasmosis in mice. However, following ocular infection with tachyzoites, the cause of the accompanying progressive changes in hippocampal-dependent tasks, and their relationship with the morphology and number of microglia, is less well understood. Here, in 6-month-old, female BALB/c mice, 5 μl of a suspension containing 48.5 × 10⁶ tachyzoites/ml was introduced into the conjunctival sac; control received an equal volume of saline. Before and after instillation, all mice were subject to an olfactory discrimination (OD) test, using predator (cat) feces, and to an open-field (OF) task. After the behavioral tests, the animals were culled at either 22 or 44 days post-instillation (dpi), and the brains and retinas were dissected and processed for immunohistochemistry. The total number of Iba-1-immunolabeled microglia in the molecular layer of the dentate gyrus was estimated, and three-dimensional reconstructions of the cells were evaluated. Immobility was increased in the infected group at 12, 22, and 43 dpi, but the greatest immobility was observed at 22 dpi and was associated with reduced line crossing in the OF and distance traveled. In the OD test, infected animals spent more time in the compartment with feline fecal material at 14 and at 43 dpi. No OD changes were observed in the control group. The number of microglia was increased at 22 dpi but returned to control levels by 44 dpi. These changes were associated with the differentiation of T. gondii tachyzoites into bradyzoite-enclosed cysts within the brain and retina. Thus, infection of mice with T. gondii alters exploratory behavior, gives rise to a loss in predator's odor avoidance from 2 weeks after infection, increased microglia number, and altered their morphology in the molecular layer of the dentate gyrus.

Keywords: Toxoplasma gondii; behavioral tests; hippocampus; microglia response; neuroinfection; ocular conjunctival instillation.

Figure 1

Experimental timeline. 1. Days 1 to 4: behavioral tests performed before conjunctival instillation. 2. Ophthalmic instillation of either an infected suspension (n = 20) or equal volume of saline (n = 9), 65 days after starting the behavioral tests. 3. Behavioral tests at 22 and 44 dpi. 4. Animal euthanasia and histological procedures at 22 and 44 dpi.

Figure 2

Photomicrographs of IBA1-immunolabeled brain sections from control (A–E) and infected mice at 22 and 44 dpi. The molecular layer of dentate gyrus is pink shaded, and granular and polymorphic layers are yellow- and blue-shaded areas in low-power picture. Dotted squares progressively show greater magnifications. Scale bars: (A, F, K) = 250 µm; (B, G, L) = 250 µm; (C, H, M) = 250 µm; (D, I, N) = 125 µm; (E, J, O) = 25 µm.

Figure 3

Graphic representations of T. gondii-induced behavioral and microglial changes as disease progressed. In the open field task, infected groups at 12, 22, and 43 dpi showed a significant increase of immobility with greater mean values at 22 dpi. In general, crossed lines and travelled distances were reduced as immobility increased. Contrast values between the times spent in the periphery and in the center of the open arena increased at 12 and 22 dpi, returning to the pre-instillation levels at 44 dpi. Infected animals increased the time spent in the feline’s odor compartment relatively to the total time test, at 14 dpi, and this altered outcome did return to baseline. The mean number of microglia at 22 dpi was greater than that of the control group. At 44 dpi, this significant difference disappeared. (A–E) Graphic representations display standard error bars. Microglial number graphic representation (F) displays standard deviation.

Figure 4

Brain sections photomicrographs of periventricular and perivascular infiltrates in the lateral septum (A–C), striatum (D–F), cerebral cortex (G–I), and cerebellum (J–L) in T. gondii-infected animals at 22 dpi. Scale bars: (A, D, G, J) = 250 µm; (B, E, H, K) = 125 µm; (C, F, I, L) = 25 µm.

Figure 5

Photomicrographs of hippocampal sections to illustrate perivascular infiltrate (A–C), T. gondii parasite encystment (D–F), and vascular congestion in the hippocampus fissure (G–I) at 22 dpi. Scale bars: (A, D, G) = 200 µm; (B, E, H) = 100 µm; (C, F, I) = 30 µm.

Figure 6

Retinal photomicrographs T. gondii-infected mice at 22 dpi. (A, B) Flat mount of retina counterstained with Giemsa after T. gondii immunolabeling. (C, D) Low- and high-power photomicrographs of retinal sections stained with hematoxylin–eosin at the same time window. (A) High-power pictures of two retinal T. gondii pseudocysts. (B) Interferential contrast microscopic image of a retinal T. gondii pseudocyst. Macrophages and polymorphonuclear cells (neutrophils) in the retinal section of infected mouse is shown in (C, D). Scale bars: (A) = 25 µm; (B–D) = 50 µm.