Identification of neurogenic areas in the central nervous system of guinea pigs (Cavia porcellus, Linnaeus 1758): culture, characterization and differentiation of neural precursors

Author(s)

Erika Toledo da Fonseca

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 2012-12-18

Examining board members

Maria Angélica Miglino; Érika Renata Branco; Graciela Conceição Pignatari; Marimelia Aparecida Porcionatto; Rose Elí Grassi Rici

• Advisor: Maria Angélica Miglino

Abstract

The present study concerns the identification of neurogenic areas, culture, identification and characterization of neural precursors obtained from the brain of guinea pigs. Potentially neurogenic areas in the brain of neonates were identified by immunohistochemistry for bromodeoxyuridine (BrdU), following administration of the drug. BrdU incorporation was detected mainly in cells underlying the lateral ventricle, in hippocampus and olfactory bulbs, indicating cell proliferation and suggesting a neurogenic potential for these areas. Subsequently, neural precursors were cultured from cells obtained from the subventricular zone (SVZ) of the lateral ventricles. After approximately one week culture, SVZ cells in active multiplication originated abundant cellular masses (neurospheres, NSFs). Cells dissociated from primary NSFs were capable of originating new NSFs after a few days of culture. NSFs proliferated in number and size, allowing 5-6 weekly subculturings and maintaining growth and viability for up to 60 days. In the meantime, NSFs were frozen and thawed, maintaining the viability and proliferative ability. Viability assays by the colorimetric MTT revealed viability differences among NSFs originated from SVZs from animals of different ages (fetuses, 7, 30 and 180 days of age). Dissociated NSFs underwent approximately 2.3% cell death by apoptosis and 3.1% death by necrosis. Intact and dissociated NSFs were submitted to immunofluorescence (IF), using antibodies for cell markers of stem cells (nestin), neurons (beta-III tubulin), oligodendrocytes (mGalC) and astrocytes (GFAP). A diffuse staining of variable intensity was observed in the cytoplasm of NSFS and dissociated cells, yet the rate of cells expressing each individual marker could not be determined. Upon culture in medium containing B27, without NSFs-specific growth factors, NSFs displayed evidence of neural differentiation, originating cells with morphology distinct from that of NSFs, suggesting differentiation. Flow cytometry analysis of NSFs cells after differentiation revealed approximately 13.3% positive for nestin, around 5.5% positive for beta-III-tubulin, 9% GFAP positive and approximately 7.8% positive for mGalC. Differentiated cells were then submitted to a functional test, by measuring calcium influx upon gamma butiric amino acid (GABA) and glutamate stimuli. GABA and glutamate stimulated some differentiated cells. Thus, cells cultured and differentiated from guinea pig SVZ present physiological indicators of neuronal physiology. Thus, the ability of guinea pig SVZ cells to originate functional neurons in vitro is promising towards further studies and potential therapeutic use of neural stem cells in disorders of the nervous system. (AU)

• FAPESP's process: 10/11008-4 - Identification of central nervous system neurogenics areas of guinea pigs (Cavia Porcellus): culture, characterization and differentiation of neural precursors
• Grantee: Erika Toledo da Fonseca
• Support Opportunities: Scholarships in Brazil - Doctorate