Abstract
Congenital toxoplasmosis (TC) from Toxoplasma gondii positive mother to child transmission results in fetal death, abortion, or infantile neurologic and neurocognitive deficits as well as chorioretinitis. This study aims to analyze the morphological changes in brain and skeletal muscle cells of Swiss mouse embryos during experimental congenital toxoplasmosis. Swiss mice, before mating, were gavage inoculation infected with approximately 25 or 50 cysts of ME‐49 strain T. gondii. Eighteen day postcoitus maternal and embryonic muscle and brain samples were collected and processed for histopathological analysis. The muscle tissue from embryos of infected mothers, in comparison with healthy muscle myofibers, exhibited discontinuous and shorter myofibrils, more interfibrillar space and immature cells with fewer stained and poorly defined striated profiles. These in vivo findings might be related to an adhesion protein decrease, observed in vitro, where myogenesis was completely affected during Toxoplasma infection. The neurogenesis was severely affected with irregularly arranged cells, reduced cell density, and a significant intercellular space increase. The brain tissue presented ischemia, cell death, necrosis, and thrombi, increasing according to the degree of the acute infection, which compromised the neurogenesis, thereby justifying brain size decrease in these embryos.
Keywords
- Toxoplasma gondii
- experimental toxoplasmosis
- myogenesis
- neurogenesis
1. Introduction
The ability to transmigrate through the placenta to replicate in various fetal tissues, evading the immune system of the fetus, makes Toxoplasma a major cause of prenatal complications. This infection can seriously interfere with the development of the fetus, possibly leading to abortion or serious pathologies after birth with severe consequences in childhood, adolescence, or adulthood [3–7]. Among the most common brain afflictions are encephalitis, altered mental status, seizures, weakness, cranial nerve disorders, sensory abnormalities, movement disorders, psychomotor or mental retardation, and behavioral alterations. Acute retinocorioidite is a severe condition, which may develop, involving vitreous hypervascularization and retinal necrosis with a total loss of vision [6, 8–12]. With regard to muscle tissue, the
2. Experimental design
Three experimental protocols were developed in an attempt to establish trans‐chromosome (TC) in mice, premating infection, imminently postmating, and during pregnancy. Swiss mice during the mating period (five females and one male per experiment,
For the diagnosis of possible morphological changes in muscular and cerebral tissue caused by parasite presence in embryos from
3. Results
From three experimental protocols for
The severity of the
In addition, by macroscopic analysis, besides the swelling of some organs, there was malformation of the anterior and posterior limbs as well as significant decrease in skull size characterizing microcefalia in these animals (Figure 1B). These embryos from infected mothers were extremely swollen, both brain and muscle tissues retaining so much fluid that it was impossible to obtain samples for histological analysis (Figure 1B). Some of these embryos exhibited a very committed development such as malformation of the trunk (Figure 1C) and even total absence of hindlimb formation (Figure 1C and D).
Group 2 females inoculated with 50 cysts, although fertilized, did not terminate pregnancy, dying in few days. Many embryos were reabsorbed. In this experimental condition, there was nearly a total loss of females and their embryos.
Longitudinal sections of pregnant mouse muscle tissue infected with 25 cysts of
Brain samples of control embryos and infected mothers were examined. All control embryo brain tissues did not show any changes, the brain tissue normal and cell density characteristics of healthy tissue. Tissue section showed arranged cells in a pink color with the cytoplasm unaltered in purple, the nuclei with no visible changes in the chromatin arrangement and almost no intercellular space (Figure 4A). In contrast, the brains of embryos from infected mothers exhibited irregularly arranged cells with higher concentrations at apparently less injured points, reduced cell density, and a significant increase of intercellular space, possibly due to the decrease of the adherent junctions (Figure 4B–D). In addition, there was weak cytoplasmic labeling and nuclear marking disparity, some larger nuclei almost transparent, others much smaller, marked. The presence of mononuclear cell inflammatory infiltrates was noted, such as macrophages, lymphocytes, mast cells, and eosinophils. There were several points with massive presence of red blood cells, implying recent thrombi in all analyzed sections (Figure 4B and C). In addition, different levels of brain tissue impairment were apparent (Figure 4D), such as presence of cells similar to astrocytes and neurons, which possibly suffered karyolysis (weak nuclear staining) and eosinophilia (Figure 5B and C). Some of these cells presented nearly transparent cytoplasm resulting from the loss of basophilia and others an apoptotic process characteristic (Figure 5C). No analyzed sections revealed parasites.
4. Discussion
This study investigated the role of
Our first experimental strategy was gavage infection of the female with cysts aiming to mimic
Our experimental model with Swiss mice involving infection of the female prior to mating demonstrated that during the first week of pregnancy, there was significant fertility reduction, embryos with low birth weight, fetal reabsorption, miscarriages, and stillbirths, presenting a loss rate of over 50% for Group 1 (inoculated with 25 cysts) and almost 100% in Group 2 (inoculated with 50 cysts). However, since each cyst can vary in bradyzoite numbers (which may die or survive), it was not possible to define the parasite concentration that generated a certain vertical transmission rate. This would explain the loss of more than 70% of the embryos, considering that bradyzoites differentiate to tachyzoites that can damage the placenta, leading to loss of the embryo by spontaneous abortion or absorption, as proposed by Vargas‐Villavicencio et al. [20]. These data confirm that the severity of Toxoplasma infection in females and embryos depends upon the concentration of parasites [18–20, 31]. TC is one of the most serious consequences of acute Toxoplasma infection, the greatest severity observed in early pregnancy as demonstrated experimentally in this study.
Embryos from infected mothers presented brain tissue with possible levels of ischemia and morphological aspects compatible with cell death mechanisms. Inflammatory reactions were apparent suggesting the focal presence of mononuclear cell infiltrates, necrosis, and thrombi, which increased according to the degree of infection development, compromising the neurogenesis. It is known that during apoptosis, the cells lose their adherent junctions [32], and lesions, due to ischemia or hypoxia together with other stimuli, also triggering cell death (necrosis and apoptosis) [33, 34]. Some cells, similar to astrocytes and neurons, underwent karyolysis (weak nuclear staining) and eosinophilia with the nearly transparent cytoplasm resulting from the loss of basophilia [35, 36]. The results described here during the cerebral histological analysis point to neuropathogenesis induced by the
The systemic inflammatory response induced by the parasite (toxoplasmosis sepsis) witnessed in our experiments may have caused multiple organ failure of these embryos, as proposed earlier [17]. Despite the already known involvement of muscle tissue in the development of the toxoplasmosis chronic phase [42], no studies involving embryos from
Acknowledgments
The authors thank Sandra Maria Oliveira Souza for technical support. English review and revision by Mitchell Raymond Lishon, native of Chicago, Illinois, USA‐UCLA 1969.
Our work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Fundação Oswaldo Cruz (Programa Estratégico de Apoio à Pesquisa em Saúde—PAPES IV e VI), Pronex—Programa de Apoio a Núcleos de Excelência—CNPq/FAPERJ and Instituto Oswaldo Cruz/Fiocruz.
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