Neonatal hypoxia–ischemia reduces ganglioside, phospholipid and cholesterol contents in the rat hippocampus
Introduction
Hypoxia is one of the major pathological events causing neuronal cell injury, neurodegeneration and cell death; it frequently happens in association with ischemia. This combination of hypoxia and ischemia increases the severity of cellular responses to oxygen and nutrient deprivation. If a hypoxic-ischemic insult occurs during the critical cellular or tissue differentiation periods, that episode might have a serious impact on brain maturation; the hippocampus being the most vulnerable structure (Vannucci, 1990, Schmidt-Kastner and Freund, 1991, Nyakas et al., 1996, Northington et al., 2001). Injury resulting from neonatal hypoxia–ischemia in humans contributes to long-term neurological disabilities, including cerebral palsy, epilepsy and mental retardation (Perlman, 1997, Nelson and Grether, 1998, Vannucci, 2000).
The mechanisms for brain injury after hypoxia–ischemia are thought to include energy failure, free radical damage, cytokine and excitatory amino acid excytotoxicity and intracellular calcium increase with activation of endonucleases, proteases and lipases (Delivoria-Papadopoulus and Mishra, 2000). Consequently, hypoxia–ischemia damage comprise several aspects of brain structure and function, including biochemical, histological, anatomical and psychological aspects (Calvert et al., 2002).
The Levine's procedure (Levine, 1960) for neonatal hypoxia–ischemia, as refined by Rice et al. (1981), provides a valuable rat model that replicates much of the neuropathology seen in human neonates. This model is widely accepted and reproducibly creates hippocampal, striatal and cortical damage (Tuor et al., 1996, Hagberg et al., 1997, Vannucci and Vannucci, 1997). In this report, we studied membrane lipid contents of the rat hippocampus following experimental hypoxia–ischemia.
Gangliosides are a family of sialic-containing glycosphingolipid present in high concentration in neuronal membranes. They play important roles in cell–cell interaction, cellular growth and differentiation, signal transduction, adaptation of plasma membrane to environmental variations and may be involved in neuronal development (Ando, 1983, Zeller and Marchase, 1992, Nagai, 1995). On the other hand, GM1 ganglioside prevents in vitro neurotoxicity of glutamate (Favaron et al., 1988), plays a scavenger role (Avrova et al., 1994, Avrova et al., 1998, Mahadik et al., 1993), partially correct the hypoxia-induced neurotransmitter deficits in neonatal rats (Hadjiconstantinou et al., 1990) and reduces the vulnerability of fetal sheep brain to subsequent injuries (Tan et al., 1994). The intraperitonial administration of mixed gangliosides decreases the accumulation of intracellular Ca2+ and stabilizes protein kinase activities (Xie et al., 2000).
Phospholipids make up a heterogeneous group of compounds whose basic structure presents a phosphate radical that is linked to different alcohol's such as the glycerol, sphingosinol and inositol, and may hold specific substituting molecule groups such as choline, serine and ethanolamine (Voet et al., 1999). These membrane compounds carry out structural functions (phospatidylcholine, phosphatidylserine and phosphatidylethanolamine (PE)) and take part in cellular signaling (phosphatidylinositol and sphingomyelin (SM)) (Ohvo-Rekilä et al., 2002).
Cholesterol, chemically derived from cyclopentanoperydrophenanthrene, as well as glycosphingolipids and phospholipids, is an essential component of cellular membranes and is required for viability and cellular proliferation. One of its important functions is its ability to modulate physicochemical properties of cellular membranes. In addition, cholesterol changes behavior and function of proteins residing in membranes, what is necessary for membrane rafts formation (Harder and Simons, 1997, Ohvo-Rekilä et al., 2002, Suzuki, 2002).
Some authors have shown that hypoxia reduces rat and human infant brain ganglioside levels (Rastogi et al., 1968, Domanska-Janik et al., 1982, Qi and Xue, 1991) and alters phospholipid and sterol contents (Ternovoi et al., 1993, Dorszewska and Adamczewska-Goncerzewicz, 2000). In order to study the permanent and irreversible biochemical consequences of neonatal hypoxia–ischemia we have evaluated the pattern and contents of gangliosides and phospholipids, as well as the concentration of cholesterol in rat hippocampus immediately (30 min) and 7, 14, 21, 30, 60 and 90 days after the brain injury.
Section snippets
Animals
A number of 224 7-day-old albino Wistar rats (male and female) weighting 13–19 g, were submitted to hypoxia–ischemia procedure. They were obtained from Departamento de Bioquı́mica, Instituto de Ciências Básicas da Saúde, UFRGS, fed ad libitum and maintained at room temperature on a 12-h light:12-h dark cycle. All animal procedures were approved by the University Animal Care Committee (UFRGS).
Experimental hypoxia–ischemia
Rats were subjected to Levine protocol (Levine, 1960), with minor modifications (Rice et al., 1981
Total ganglioside, phospholipid and cholesterol contents
Fig. 1 shows data relative to the total ganglioside (A), phospholipid (B) and cholesterol (C) contents in hippocampi of rats at different times of recovery after hypoxic-ischemic episode. All lipid parameters evaluated (gangliosides, phospholipids and cholesterol) are significantly lower in HI hippocampi than in respective controls, from 7 to 90 days after the injury; this decrease was not detected immediately after the episode. Fig. 1A–C also reveal lower contents of all three parameters in
Discussion
Brain damage following hypoxia–ischemia in the newborn could result from mechanisms directly related to the hypoxic-ischemic insult per se (energy failure and acute cellular necrosis) and from secondary mechanisms that occur over an extended time. These mechanisms might include disruption of ionic homeostasis, activation of degrading enzymes, increase on cellular oxidation stress that could damage DNA, cell proteins and lipids, thus leading to delayed cellular death (Northington et al., 2001).
Acknowledgements
This work was supported by grants from, CNPq, PIBIC-CNPQ/UFRGS, FAPERGS and PROPESQ/UFRGS. We are grateful to Dr Regina M.C.V. Guaragna for providing the phospholipid standards and to Luciene P. Vianna for WordArt assistance.
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