Determination of the intracellular Ca²⁺ concentration in the N1E-115 neuronal cell line in perspective of its use for peripheric nerve regeneration

Article type: Research Article

Authors: Rodrigues, J.M.^; | Luís, A.L.^; | Lobato, J.V.^{; ;} | Pinto, M.V. | Lopes, M.A. | Freitas, M. | Geuna, S. | Santos, J.D. | Maurício, A.C.^{; ;}

Affiliations: Centro de Estudos de Ciência Animal (CECA), Instituto de Ciências e Tecnologias Agrárias e Agro-Alimentares (ICETA) da Universidade do Porto, Campus Agrário de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, Portugal | Instituto de Ciências Biomédicas de Abel Salazar (ICBAS) da Universidade do Porto, Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal | Departamento de Estomatologia, Centro Hospitalar de Vila Nova de Gaia (CHVNG), Rua Conceição Fernandes, 4434-502 Vila Nova de Gaia, Portugal | Faculdade de Engenharia da Universidade do Porto (FEUP), Rua Dr. Roberto Frias, 4200-465 Porto, Portugal | Departamento de Saúde Pública, UOSP Braga, Portugal | Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, c/o Ospedale San Luigi, Regione Gonzole 10, Orbassano (TO), 10043 Torino, Italy

Note: [] Corresponding author. E-mail: ana.colette@mail.icav.up.pt.

Abstract: Entubulation repair of peripheral nerve injuries has a lengthy history. Several experimental and clinical studies have explored the effectiveness of many biodegradable and non-degradable tubes with or without addition of molecules and cells. The main objective of the present study was to develop an economical and also an easy way for culturing a neural cell line which was capable of growing, differentiating and producing locally nerve growth factors that are otherwise extremely expensive, inside 90 PLA/10 PLG nerve guides. For this purpose the authors have chosen the N1E-115 cell line, a clone of cells derived from mouse neuroblastoma C-1300 with the perspective of using this differentiated cellular system to cover the inside of 90 PLA/10 PLG nerve guides placed to bridge a gap in the rat sciatic nerve experimental model. The N1E-115 cells proliferate in normal culture medium but undergo neuronal differentiation in response to DMSO. Upon induction of differentiation, proliferation of N1E-115 cells ceases, extensive neurite outgrowth is observed and the membranes become highly excitable. While it is known that Ca2+ serves as an important intracellular signal for various cellular processes, such as growth and differentiation. It is also known that can be toxic to cells and is involved in the triggering of events leading to excitotoxic cell death in neurons. The [Ca\[$^{2+}]\tsub{i}$ in non-differentiated N1E-115 cells and after distinct periods of differentiation, have been determined by the epifluorescence technique using the Fura-2-AM probe. The results of this quantitative assessment revealed that N1E-115 cells which undergo neuronal differentiation for 48 hours in the presence of 1.5% DMSO are best qualified to be used to cover the interior of the nerve guides since the [Ca\[$^{2+}]\tsub{i}$ was not found to be elevated indicating thus that the onset the cell death processes was not occurred.

Keywords: N1E-115 cells, nerve regeneration, PLGA, intracellular calcium concentration, epifluorescence technique

Journal: Bio-Medical Materials and Engineering, vol. 15, no. 6, pp. 455-465, 2005