Thioredoxins family proteins immunolocalization in the rat central nervous system

AON-BERTOLINO, MARÍA LAURA; ROMERO, JUAN; HOLUBIEC, MARIANA; BADORREY, SOLEDAD; GALEANO, PABLO; SARACENO, GUSTAVO EZEQUIEL; CODIGNOTTO, EVA; LILLIG, CHRISTOPHER HORST; CAPANI, FRANCISCO

Rosario

Congreso; 10th Inter-American Congress of Electron Microscopy (CIASEM 2009) and 1st Congress of the Argentine Society of Microscopy (SAMIC 2009); 2009

Comité Interamericano de Sociedades de Microscopía Electrónica (CIASEM) y Sociedad Argentina de Microscopía (SAMIC)

The neuronal damage and glial reaction induced by hypoxic ischemic episode has been related to glutamate excitotoxicity, oxidative stress and mitochondrial dysfunction [1] [2]. The Trx (thioredoxin) and Grx (glutaredoxin) systems control the cellular redox potential, keeping a reducing thiol-rich intracellular state, and protecting the cells from the action of reactive oxygen species through thiol redox mechanisms and control signals. Here we examine the CNS distribution of this important family proteins in control rat tissue (the animal model most widely used to study the mechanisms involved in neuronal cell damage during the hypoxic-ischemic event), and performed a Brain Atlas with the most sensitive areas to hypoxia: Neostriatum, Hippocampus, Cerebellum, Substantia Nigra, Cortex, Spinal Cord, and Retina, using the following proteins: Trx-1, Trx-2, TrxR-1, TrxR-2, Txnip, Grx-1, Grx-2, Grx-3, Grx-5, gGCS, Prx-1, Prx-2, Prx-3, Prx-4, Prx-5 and Prx-6 [3]. Adult male rats (N=8) were anaesthetized with 28% (w/v) chloral hydrate, 0.1 ml/100 gr of body weight, and perfused with paraformaldehyde 4% in phosphate buffer 0.1 M, pH 7.4 through the abdominal aorta. Brains were dissected and post-fixed in the same solution during 2 hours, and then immersed overnight in phosphate buffer 0.1 M, pH 7.4 containing 5% of sucrose. Coronal brain sections from the different areas (40μm thick) were cut on an Oxford vibratome and then recovered for light microscopic study. For retina study, after enucleating the eye, the retina was diseccted and included in paraffin. Then sections of 4 microns were cut and mounted in sylanized slices. Brain sections were incubated first with the primary antibodies at 4ºC overnight [3]. Then, the tissue was incubated with the proper secondary antibody [3] and followed by incubation with the Biotin Streptavidin Complex and developed with AEC substrate kit until staining was optimal and examined by light microscopy using a Leica Microscope [3]. Previous studies have suggested that these proteins are distributed in most of the cell types and regions of the CNS; we have observed several differences in intensity between neurons and neuroglia, besides the regional distribution of these proteins on the light microscope analysis. We expect this Atlas will contribute to reveal new insights about the Thioredoxins family proteins distribution to understand, latter, the role of the oxidative pathway in the pathogenesis of the hypoxic ischemic brain damage. References[1]Lillig, CH, and Holmgren, A., Antioxid. Redox Signal; (2007), 9: 35-47.[2]Lillig, CH, Berndt, C., and Holmgren, A. Biochim. Biocphys. Acta, 2008, 1340:1304-1317.[3] María Laura Aón, Bertolino, Juan Ignacio Romero, Pablo Galeano, Mariana Holubiec, Sol Badorrey, Gustavo Ezequiel Saraceno, Christopher Lillig, Francisco Capani. BBA and General Subjects (2009).[4] F. Capani, G.E. Saraceno, V. Boti, L. Aón Bertolino, D. Madureira de Oliveria, G. Barreto, P. Galeano, L.D. Giraldez-Alvarez and H. Coirini, Exp. Neurol. (2009).