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Immune system involvement in sex-specific vulnerability to prenatal stress

Periodic Reporting for period 1 - ISSVS (Immune system involvement in sex-specific vulnerability to prenatal stress)

Reporting period: 2015-07-01 to 2017-06-30

Substantial psychological or immunological stress in pregnant women can have consequences for the unborn offspring. Animal models have shown that fetal males are more vulnerable to stress than fetal females. And research in humans corroborate the finding that prenatal stress has different effects in males than in females.
Furthermore, evidence is accumulating that prenatal stress may play an important role in the development of neurodevelopmental disorders with a complex multifactorial etiology, which often show a male bias in incidence.
The mechanisms underlying the sex-specific effects of prenatal stress are largely unknown, but recent evidence implicates a crucial role for microglia, the resident immune cells of the brain.
Although in the adult brain microglia are activated only after infection or injury, in the developing neonate microglia show a structurally activated pattern, especially in males. This time period of male specific microglia activation coincides with the critical period for hormonal action that causes sexual differentiation of the brain. And these observations are not merely a coincidence; recently microglia were found to play a pivotal role in the process of sexual differentiation of the male brain.
In this project we investigated whether this male-specific neonatal activation of microglial cells makes males more vulnerable to the effects of stress than females, since we know that overactivation of microglia is detrimental and associated with a wide range of neurological disorders.
These experiments contribute to important novel insights into the sex-specific sensitivity to the prenatal environment, and help understand the male predominance in many neurodevelopmental disorders.
Mice were prenatally exposed to lipopolysaccharide (LPS) or a control solution (saline) administered to the pregnant female. LPS is a molecule present on Gram negative bacteria that elicits a strong immune response in mammals. Once born, the pups within a litter were appointed to different experimental groups: control females, control males, females of which we manipulated the microglia to resemble the male typical state, males of which we manipulated the microglia to resemble the female typical state.
Brains were collected of three batches of mice. (1) 4 days after birth (sex specific microglia activation is expected), (2) 21 days after birth (in typical mice the sex specific microglia activation has disappeared) and (3) 80 days after birth (when the mice reached maturity). Immunohistochemistry was performed to investigate a variety of proteins in 4 main brain areas of interest: the preoptic area (where the sex differences in neonatal microglia activation were first observed by the McCarthy lab), and three areas because of their relevance to neurodevelopmental disorders: amygdala, medial prefrontal cortex, hippocampus. Also, blood was collected to investigate corticosterone levels, and spleen and thyroid were weighed.
Brain tissue for epigenetic analyses was collected, but this tissue has not been processed yet due to time restraints.
A separate batch of brains of mice aged 4 days was used to investigate the morphology, quantity and distribution of the microglia in the different brain areas in more detail using three dimensional imaging of solvent-cleared organs (3DISCO) and lightsheet microscopy.
Finally, a batch of mice was tested on behavioral tasks that were designed to test the functionality of a subset of the brain areas investigated in the molecular assays: a hippocampal-dependent object recognition task including an open field tests, amygdala dependent fear conditioning test, and a social recognition task.
The project contributes to the understanding of the mechanisms underlying the sex bias that is present in most neurodevelopmental disorders, and in Autism Spectrum Disorders in specific. This understanding is imperative to potential prevention, treatment, identification of risk factors, and early intervention.
The Center for Disease Control estimated the incidence of Autism Spectrum Disorders is 1 in 68 children in 2016. Early intervention is proven to substantially increase the quality and productivity of the lives of patients and their families. Besides improving quality of life, the economic implication are also substantial since the lifetime costs of 1 ASD patient is estimated to be $2.5 million in the US and £1.5 million in the UK.
Furthermore, the core of this project is the sex-specific mechanisms that are at work in mammals, a feature that is often entirely neglected, but of which awareness of its importance is steadily rising. Many drugs are tested for safety and efficacy on the male population solely and likewise, many preclinical studies are performed only in males even when the development of neurodevelopmental disorders is investigated. The latter is especially remarkable since the sex difference is often one of the best-known features of the disorder. In this project the sex specific workings of the brain is its main focus point.