Short and Long Term alterations in the rat CNS induced by Perinatal Asphyxia

F. CAPANI, G. E. SARACENO, P. GALEANO, M. L. AÓN BERTOLINO, J.I ROMERO1, R. KÖLLIKER-FRERS, M. HOLUBIEC, S. BADORREY , M.V. AYALA. A. MORENO, L. D. GIRALDEZ ÁLVAREZ

Congreso; XI Interamerican Congress of Electron Microscopy (CIASEM 2011); 2011

CIASEM

Perinatal asphyxia (PA) induced short and long term synaptic and cytoskeletal alterations that has been associated with neuronal cell death following hypoxia. The lack of knowledge on the mechanisms underlying this dysfunction prompted us to investigate the morphological changes in the synapse and neuronal cytoskeleton using light and electron microscopy techniques combined with western blot and immunocytochemical analysis. For this study we used a well established murine model of (PA). Full-term pregnant rats on gestational day 22, were anesthetized, rapidly decapitated and the uterus horns were isolated and placed in a water bath at 37 °C for different time of asphyxia and intra-asphyctic hypothermia. When their physiological conditions improved, they were given to surrogate mothers. One month, four month, 6 month and 18 month after PA, rats were subjected to an intracardiac perfusion using a solution of paraformaldehyde and glutaraldehyde in different concentrations. One month changes of F-actin cytoskeleton in the synapses in neostriatum were studied using photooxidation with phalloidin-eosin as is described in [1]. Four and 18 months were studied using conventional EM and inmunocytochemistry [4]. For 6 months changes we combined ethanolic phosphotungstic acid (E-PTA) staining, immunolectron microscopy for ubiquitin, electron tomography with 3-D reconstruction techniques [2]. After one month of the PA insult, an increase in the F-actin staining in neostriatum and hippocampus synapses was observed at confocal and electron microscopic level using correlative fluorescent electron microscopy for phalloidin-eosin. Mushroom-shaped spines showed the most consistent staining. Four month of asphyxia showed strong alterations in the dendrite and astroglial cytoskeleton [4] After six months of PA, PSDs of the rat neostriatum are highly modified. We observed an increment of PSD thickness related with the duration and severity of the hypoxic insult. In addition PSDS showed and increase in the ubiquitination level. Using 3-d reconstruction and electron tomography we observed showed clear signs of damage in the asphyctic PSD (Fig.1). These changes are correlated with intense staining for ubiquitin. Overall these results demonstrate that synaptic dysfunction following PA might be produced by early changes in the actin organization and long-term misfolding and aggregation of proteins in the PSDs. Finally after 18 month clear reduction in the number of synapses was observed in the PA animals. Therefore we hypothesize that the synaptic and neuronal cytoskeleton changes in rat CNS could lead to the cellular dysfunction and death. For this study we used a well established murine model of (PA). Full-term pregnant rats on gestational day 22, were anesthetized, rapidly decapitated and the uterus horns were isolated and placed in a water bath at 37 °C for different time of asphyxia and intra-asphyctic hypothermia. When their physiological conditions improved, they were given to surrogate mothers. One month, four month, 6 month and 18 month after PA, rats were subjected to an intracardiac perfusion using a solution of paraformaldehyde and glutaraldehyde in different concentrations. One month changes of F-actin cytoskeleton in the synapses in neostriatum were studied using photooxidation with phalloidin-eosin as is described in [1]. Four and 18 months were studied using conventional EM and inmunocytochemistry [4]. For 6 months changes we combined ethanolic phosphotungstic acid (E-PTA) staining, immunolectron microscopy for ubiquitin, electron tomography with 3-D reconstruction techniques [2]. After one month of the PA insult, an increase in the F-actin staining in neostriatum and hippocampus synapses was observed at confocal and electron microscopic level using correlative fluorescent electron microscopy for phalloidin-eosin. Mushroom-shaped spines showed the most consistent staining. Four month of asphyxia showed strong alterations in the dendrite and astroglial cytoskeleton [4] After six months of PA, PSDs of the rat neostriatum are highly modified. We observed an increment of PSD thickness related with the duration and severity of the hypoxic insult. In addition PSDS showed and increase in the ubiquitination level. Using 3-d reconstruction and electron tomography we observed showed clear signs of damage in the asphyctic PSD (Fig.1). These changes are correlated with intense staining for ubiquitin. Overall these results demonstrate that synaptic dysfunction following PA might be produced by early changes in the actin organization and long-term misfolding and aggregation of proteins in the PSDs. Finally after 18 month clear reduction in the number of synapses was observed in the PA animals. Therefore we hypothesize that the synaptic and neuronal cytoskeleton changes in rat CNS could lead to the cellular dysfunction and death.