ETD

• blood brain barrier
• Locus Coeruleus
• neuroinflammation
• neuronal loss
• perinatal hypoxia

This study is based on the hypothesis that perinatal hypoxia (PH) exerts significant harmful effects on the brain nucleus, locus coeruleus (LC). The LC is the main noradrenergic nucleus of the brain, with neurons that diffusely innervate the whole cortical mantle and most subcortical areas. It is involved in several physiological processes, including brain plasticity, memory, attention, the sleep–wake cycle, and the regulation of cortical neuronal excitability.  LC exerts neuroprotective effects by inhibiting neuroinflammation and inducing the synthesis of neurotrophic factors; it may also regulate blood–brain barrier (BBB) integrity and function. Many studies show that the LC modulates seizure susceptibility: LC degeneration lowers seizure threshold, while LC stimulation has anticonvulsant effects. Interestingly, post-mortem studies further suggest that LC may be particularly vulnerable to degeneration following PH. Accordingly, the rationale of this project is that, after PH, the occurrence of an early LC degeneration might play a key role in the development of severe neurodevelopmental conditions, including childhood epilepsy. In parallel, one of the aims of this project is to set up a data set of patients (in collaboration with Stella Maris Epilepsy Unit) on which, in the future, LC features will be assessed through MRI. This step is mandatory to set up a clinical study aimed at the non-invasive, in vivo assessment of the LC in children with childhood epilepsy following post-PH.  
The experimental part of the study was aimed to set up the model of PH, the methods of neuronal counting in the LC and hippocampus, and to analyze the effects of PH on these and other morphological parameters at PN12. In particular, the consequences of perinatal exposure to hypoxia were investigated in C57BL/6J mouse pups. The analyzed experimental groups were: (i) controls, (ii) the PH group, in which pups (PN7) were exposed to 5% O₂ for 20 minutes and, (iii) mice treated with DSP-4 (which is known to lesion the LC in adult mice, thus providing positive controls). Animals were sacrificed at PN12 for morphological analyses.  
To quantify post-PH neuronal damage, an unbiased stereological analysis protocol was set up and applied to brain slices using Stereo Investigator software and apparatus (MBF Bioscience), allowing the estimation of the absolute number of neurons in the LC and in hippocampal subregions. In parallel, to characterize PH-induced neuroinflammation, astrocytic and microglial recruitment and activation were assessed by immunofluorescence using the astroglial marker GFAP and the microglial marker IBA-1. Finally, post-perinatal alterations of the neurovascular unit were investigated by TEM. A quantitative analysis was developed, focusing on luminal diameter, basement membrane thickness, endothelial transcytosis, and tight junction distribution. 
For the small clinical part of the study, a database was created to collect and manage data from patients and control subjects within pediatric populations, selected from the Stella Maris Foundation Epilepsy Unit database. Patients fulfilling the inclusion criteria have begun to be identified. Moreover, I acquired basic information on the MRI methods developed by the Neuroradiology group of the SMF to quantify LC integrity in children. 
The experimental part provides a detailed morphological characterization of perinatal hypoxia-induced alterations in the brain, with an emphasis on its effects on LC integrity and hippocampal degeneration, as well as on its effect on BBB integrity. The delineation of the vulnerability of the LC-NE system to early-life hypoxic injury sets the base for the future study in which the effects in terms of behavior and seizure susceptibility will be assessed. The clinical part of the study sets the stage for a non-invasive investigation of the correlation between LC alterations in children with a history of PH and the development of post-hypoxic epilepsy.