Neuro-glia-vascular alterations by common neonatal clinical conditions

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2014

Neonatal common diseases, such as sepsis and jaundice, may damage the normal development of the central nervous system. Still, the cellular interactions behind the origin of the sequelae remain unknown. Thus, we intended to: (1) investigate the in vitro response of brain microvascular endothelial cells (BMEC), either alone or in the presence of other CNS cells, to stimuli that mimic those diseases– lipopolysaccharide (LPS) for sepsis and unconjugated bilirubin (UCB) for jaundice; (2) to evaluate in vivo the neuronal outcome and determine which cellular interactions may be involved, and lastly (3) to dissect if the sequelae depend on the neurodevelopmental stage in which neonatal inflammation occurs. First, we observed that both LPS and UCB induce damage of mitochondria and of rough endoplasmic reticulum, ultimately leading to cell death in monocultures of rat BMEC. In addition, these molecules cause blood-brain barrier (BBB) breakdown through P-glycoprotein inhibition, activation of matrix metalloproteinases (MMPs), loss of intercellular junctions – transient by LPS and lasting by UCB. Increased permeability and junctional disruption were more severe in co-cultures with astrocytes, particularly in LPS-treated cells. Secondly, newborn mice were treated with LPS during the first week of life (equivalent to human preterm birth) to examine acute-to-delayed effects in the developing neurovascular unit. LPS induced acute loss of body and brain weight, in addition to cerebellar hypoplasia. The latter was linked to neuronal loss/shrinkage, as well as to delayed myelination. Moreover, we observed a late pro-inflammatory response with acute astrogliosis in the parenchyma and microvasculature interface, which was lost at later days after a fast and progressive increase of activated microglia near the vessels. Lastly, we performed further in vivo studies to assess the acute effects of LPS-induced neuroinflammation during the second week of life (equivalent to human term birth). Alike in preterm neuroinflammation, we observed neuronal and white matter loss/shrinkage, with increased glial numbers. We further explored the existence of lasting neurodevelopment impairment in young adult age. Interestingly, neuroinflammation during the first week of life led to lasting neuronal loss/shrinkage, whereas neuroinflammation in the second week further induced lasting white matter injury and reduction of astrocytic population. In agreement, when dissecting the effects of priming during the neonatal period, animals with neuroinflammation during the first week had less marked effects compared to an acute young adult response whereas priming during the second week additionally caused neuronal loss and microgliosis. In conclusion, LPS and UCB act directly on BMEC, impairing BBB integrity which may favor the access of blood-borne molecules into the brain. This increases the risk of altered neurodevelopment, as is the case of neuronal atrophy and delayed myelination observed in our second study. The particular susceptibility of astrocytes to LPS-induced inflammation may not only depend on BMEC response to the stimuli but also on an intricate association with activated microglia. Even though young adults exposed to inflammation in the first week of life recover from the initial loss of astrocytes, they still sustain severe neuronal compromise. On the other hand, young adults subjected to inflammatory stimuli in the second week of life have a more marked demyelination and glial response. These findings contribute to a better understanding of the cellular interactions involved in lasting sequelae observed in sepsis survivors and how they depend on the window of neurodevelopmental stage after birth.

Fundação para a Ciência e a Tecnologia (FCT); National Institutes of Health (NIH); National Institute of Neurological Disorders and Stroke (NINDS); Fundação Luso-Americana para o Desenvolvimento (FLAD); European Molecular Biology Organization (EMBO)