• intermittent hypoxia
• OSAS
• priming
• neuroinflammation
• microglia

Obstructive sleep apnoea syndrome (OSAS) is the most common sleep-related breathing disorder, mainly prevalent in the adult population (5-25% of general population), especially in men (24% compared to the 5% in women). OSAS occurs primarily in the REM phase of sleep, and it consists in recurrent episodes of pharyngeal collapses, leading to repetitive cycles of oxygen desaturation/reoxygenation, which results in intermittent hypoxia at tissues levels, hallmark of this syndrome. A person affected by OSAS experiences more than 5 apnoea episodes per hour and the polysomnography is the gold standard for diagnosis. Evidence suggests that OSA is a risk factor for cognitive impairment. Intermittent hypoxia (IH) causes the expression of specific cytokines in mice, like interleukin 1-β (IL-1β), leading to both systemic and tissue level inflammation. IL-1β is also produced in the CNS by glial cells and neurons, resulting in an early and transient neuroinflammation and microglial changes that could contribute to Cognitive Impairment.
Microglia are normally kept in a resting state but thanks to a series of receptors on cell membranes microglia can detect the slightest changes in the CNS environment and activate themselves: this process is known as “microglial priming”. The activation consists in the changing of specific membrane receptors levels, such as CD86, HLA-DRα and CX3CR1. Whenever damage occurs in the CNS, the microglia activate themselves and start to increase production of these receptors. Under systemic inflammation, the primed microglia cause a higher production of cytokines and inflammatory mediators that can cause neurotoxicity.
The aim of this thesis project was to use a new in vitro model to reproduce IH caused by OSAS to better understand molecular mechanisms that could lead to cognitive impairment, with an insight on microglia priming and inflammatory response. To these aims, C20 human microglia cell line was utilized in the hypoxic in vitro model.
The model is composed by three gas tanks (oxygen, nitrogen, and carbon dioxide) connected to a gas mixer, useful to obtain a mixture with desired gas rates. The gas mixture reaches custom-made resin boxes, realized to maintain cells, cultured in gas-permeable dishes or multiplates, in the defined gas mixture. Cells were exposed to two different protocols of IH; both protocols present 1 initial hour of “pre-conditioning” (at 5% O2, at 37°C) and 6 hours of IH to mimic the OSA patient night-time (at 5%/2% O2, at 37°C), while the second one includes an additional final hour of reoxygenation (at 5% O2, at 37°C) to mimic the awakening condition.
Firstly, a hypoxia detection kit was used to analyse cells hypoxic or normoxic state and to validate the model. The compound EF5 bearing a nitro group under hypoxia condition forms adducts capable of binding macromolecules; an antibody (ELK3-51) specifically binds EF5 adducts. The antibody is conjugated to a fluorophore (Alexa fluor 488®) that allows to measure the intensity of fluorescence, directly correlated with cells hypoxic state.
Then, H2DCF-DA probe was used to quantify the ROS production and to detect the oxidative stress. H2DCF-DA is oxidized by intracellular ROS into a fluorescent compound much more retainable by cells; fluorescence measured is proportional to ROS production.
To characterize microglia response after IH treatments, the mRNA expression of CX3CR1, HLA-DRα and CD86 was analysed by Real Time PCR. Finally, to see if IH could induce microglia priming and an amplified response to inflammation, real time PCR were performed to study the mRNA expression of NF-kB, IL-6 and IL-10 in cells only treated with IH compared to cells treated both with IH and IL-1β. The obtained data by fluorescence measurements confirm successful cells exposure to IH and ROS production after treatments. The mRNA expression analysis in IH exposed microglia showed that IH could modulate specific membrane receptors, and that IH followed by additional inflammatory stimulus may exacerbate inflammatory microglia response.