title: Neonatal anoxia in rats: hippocampal cellular and subcellular changes related to cell death and spatial memory.
abstract: Neonatal anoxia in rodents has been used to understand brain changes and cognitive dysfunction following asphyxia. This study investigated the time-course of cellular and subcellular changes and hippocampal cell death in a non-invasive model of anoxia in neonatal rats, using Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) to reveal DNA fragmentation, Fluoro-Jade® B (FJB) to show degenerating neurons, cleaved caspase-3 immunohistochemistry (IHC) to detect cells undergoing apoptosis, and transmission electron microscopy (TEM) to reveal fine ultrastructural changes related to cell death. Anoxia was induced by exposing postnatal day 1 (P1) pups to a flow of 100% gaseous nitrogen for 25 min in a chamber maintained at 37 °C. Control rats were similarly exposed to this chamber but with air flow instead of nitrogen. Brain changes following anoxia were evaluated at postnatal days 2, 14, 21 and 60 (P2, P14, P21 and P60). In addition, spatial reference memory following anoxia and control treatments was evaluated in the Morris water maze, starting at P60. Compared to their respective controls, P2 anoxic rats exhibited (1) higher TUNEL labeling in cornus ammonis (CA) 1 and the dentate gyrus (DG), (2) higher FJB-positive cells in the CA2-3, and (3) somato-dendritic swelling, mitochondrial injury and chromatin condensation in irregular bodies, as well as other subcellular features indicating apoptosis, necrosis, autophagy and excitotoxicity in the CA1, CA2-3 and DG, as revealed by TEM. At P14, P21 and P60, both groups showed small numbers of TUNEL-positive and FJB-positive cells. Stereological analysis at P2, P14, P21 and P60 revealed a lack of significant differences in cleaved caspase-3 IHC between anoxic and control subjects. These results suggest that the type of hippocampal cell death following neonatal anoxia is likely independent of caspase-3 activation. Neonatal anoxia induced deficits in acquisition and performance of spatial reference memory in the Morris water maze task. Compared to control subjects, anoxic animals exhibited increased latencies and path lengths to reach the platform, as well as decreased searching specifically for the platform location. In contrast, no significant differences were observed for swimming speeds and frequency within the target quadrant. Together, these behavioral results indicate that the poorer performance by anoxic subjects is related to spatial memory deficits and not to sensory or motor deficits. Therefore, this model of neonatal anoxia in rats induces hippocampal changes that result in cell losses and impaired hippocampal function, and these changes are likely related to spatial memory deficits in adulthood.
authors.affiliation: Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil; Núcleo de Cognição e Sistemas Complexos, Centro de Matemática, Computação e Cognição, Universidade Federal do ABC, Santo André, Brazil. Electronic address: silviahonda@usp.br.
authors.forename: S H
authors.initials: SH
authors.lastname: Takada
authors.affiliation: Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: C A
authors.initials: CA
authors.lastname: Dos Santos Haemmerle
authors.affiliation: Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: L C
authors.initials: LC
authors.lastname: Motta-Teixeira
authors.affiliation: Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: A V
authors.initials: AV
authors.lastname: Machado-Nils
authors.affiliation: Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: V Y
authors.initials: VY
authors.lastname: Lee
authors.affiliation: Departamento de Morfologia e Patologia, Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos, São Carlos, Brazil.
authors.forename: L F
authors.initials: LF
authors.lastname: Takase
authors.affiliation: Departamento de Ciências Básicas, Campus de Araçatuba, Universidade Estadual Paulista, Araçatuba, Brazil.
authors.forename: R J
authors.initials: RJ
authors.lastname: Cruz-Rizzolo
authors.affiliation: Núcleo de Cognição e Sistemas Complexos, Centro de Matemática, Computação e Cognição, Universidade Federal do ABC, Santo André, Brazil.
authors.forename: A H
authors.initials: AH
authors.lastname: Kihara
authors.affiliation: Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: G F
authors.initials: GF
authors.lastname: Xavier
authors.affiliation: Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: I-S
authors.initials: IS
authors.lastname: Watanabe
authors.affiliation: Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: M I
authors.initials: MI
authors.lastname: Nogueira
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001238:Asphyxia Neonatorum; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000502:physiology*; D004195:Disease Models, Animal; D006624:Hippocampus / Q000473:pathology / Q000503:physiopathology*; D000860:Hypoxia / Q000473:pathology / Q000503:physiopathology*; D008297:Male; D018782:Maze Learning / Q000502:physiology; D017208:Rats, Wistar; D065852:Spatial Memory / Q000502:physiology*
chemical_list: C505410:Casp3 protein, rat; D053148:Caspase 3
keywords: apoptosis; asphyxia; hippocampal cell death; neonatal hypoxia; spatial memory; transmission electron microscopy