Efecto del precondicionamiento hipóxico en la respuesta a un insúlto hiperóxico en el sistema nervioso central de ratones reciénnacidos

Resumen

Mammal fetal life elapses in a low oxygen environment relative to the extra uterine. Interestingly, in the human being partial pressure of oxygen increases from fetal 3.3 kPa (25–30 mmHg) to 10.5 kPa (75–85 mmHg) in the first minutes after birth in the newly born infant. This rapid and brisk increase in oxygen availability to tissue generates a physiologic oxidative stress. However, both the lungs and the antioxidant defense system do not mature until late in gestation. Therefore, preterm infants often need respiratory support and oxygen supplementation in the delivery room to achieve postnatal stabilization. The use of supplementary oxygen will cause oxidative stress and damage. In experimental studies in newborn sheep it has been shown that brief resuscitation maneuvers performed in the first minutes after birth produce lung damage and oxidative stress that can have long-lasting consequences. In human newborn infants the use of 100% oxygen instead of air for resuscitation enhances oxidative stress and increases mortality. Furthermore, the use of high oxygen inspired fractions in very preterm infants increased specific morbidities such as bronchopulmonary dysplasia. We hypothesized that delaying oxygenation after birth would preserve reducing equivalents, enhance redox adaptation, and protect the brain against oxygen derived free radicals produced by a hyperoxic insult. The objectives of the study were to determine oxidative stress and damage biomarkers in brain of mice pups during fetal-to-neonatal transition in a breathing atmosphere of FiO2 of 0.14 vs. 0.21. We also studied the consequences of a hyperoxic insult with a FiO2 of 1.0 after postnatal stabilization either with FiO2 of 0.14 or 0.21. Moreover, we analyzed the expression of genes involved in the antioxidant defense system, activation of the expression HIF-1a, inflammation, and the expression of different receptors involved in neurotransmission. Finally, we performed a histological study of different layers of the cerebral cortex to determine morphological changes, apoptosis, and inflammatory changes. Furthermore, we opted to study the effects of perinatal hypoxic preconditioning on mitochondrial morphometry. To proof our hypothesis valid, we designed an experimental model in which we placed pregnant mice in an oxy-cage either with a reduced FiO2 (0.14) or room air (FiO2=0.21) at G18 (8 hours before delivery). 8 hours after birth, both groups were switched to room air (Hx14/21/21 and Nx21/21/21 groups) or subjected to a hyperoxic insult (FiO2=1.0) (Hx14/100/21 and Nx21/100/21) and reset to FiO2=0.21 after 1 hour. Pups were further evaluated either at P1 or kept in the oxy-cage with FiO2=0.21 oxygen until 1 week post-partum (P7). The brains were snap-frozen and kept at -80°C until analysis. We determined the following oxidative stress biomarkers: GSH/GSSG, cysteine/cystine, homocysteine/homocystine ratios, and metabolites of oxidative damage and inflammation such as m-tyr/Phe, o-tyr/Phe, 3NO2-tyr/p-tyr, 3Cl-tyr/p-tyr and 8-OHdG/2dG ratios by liquid chromatography coupled to mass spectrometry (UPLC-MS/MS). In addition, we assessed the expression of antioxidant defense genes, response to HIF-1α, and changes in neurotransmitter receptors by qPCR (both at P1 and P7). Finally, we determined morphology, apoptosis, and inflammation of the different layers of the brain cortex by immunohistochemistry. The mitochondrial morphometry was performed using electron microscopy at P1 mice. In general, we found less oxidative stress and damage to proteins and DNA in the preconditioned hypoxic group. A period of hypoxia after birth seemed to better favor the maintenance of a reducing environment thus protecting them against the switch to a hyperoxic environment. The results obtained in this group were still maintained one week thereafter. In the Hx14/100/21, we found downregulation of antioxidant defense enzymes when compared to the Nx21/100/21 at P1 and P7. Besides, we also observed an upregulation of the HIF-1α targets in mice born under hypoxic conditions. At a histological level, we found higher damage, increased apoptosis and a marked tendency towards inflammation in the cortex of the Nx21/100/21 group when compared to the Hx14/100/21 group. Mitochondria showed better morphology and characteristics during adaptation to re-oxygenation when birth occurred in a hypoxic atmosphere (Hx14/100/21) as opposed to normoxic conditions (Nx21/100/21). We conclude that in mice pups performing fetal-to-neonatal transition under hypoxic conditions (hypoxic preconditioning) and smoothly transitioning to normoxia seemed to better withstand a hyperoxic insult after birth as reflected by less oxidative stress, less damage and less inflammation in brain tissue.