O. Dössel and W.C. Schlegel (Eds.): WC 2009, IFMBE Proceedings 25/VIII, pp. 176–179, 2009. www.springerlink.com Development of a Multifunctional Microfluidic System for Studies of Nerve Cell Activity during Hypoxic and Anoxic Conditions Nazanin Bitaraf 1 , Ahmed Ahmed 1 , Michael Druzin 2 , and Kerstin Ramser 1 1. Department of Computer Science and Electrical Engineering, Luleå University of Technology, 971 87 Luleå, Sweden 2. Department of Integrative Medical Biology, Section for Physiology, Umeå University, 901 87 Umeå, Sweden Abstract—Hemoproteins usually supply cells and tissue with oxygen. A new hemoprotein mainly present in nerve cells called Neuroglobin was recently discovered. Enhanced expres- sion of the protein has been shown to reduce hypoxic neural injury but the mechanism behind this function remains un- known. Methods enabling investigation of the protein in single functional neurons need to be developed. Here, we have stud- ied how the electrical signaling capacity of a neuron was af- fected by hypoxic environments. Preliminary results show a trend of higher noise-level when a neuron is exposed to hy- poxic compared to normoxic surroundings, which implies increased ion-channel activity. The setup used today shows shortages such as reduced control over the oxygen content due to leakage. Therefore, a gas-tight, multifunctional microfluidic system is under development which enables us to study influ- ences of Neuroglobin concentrations on neuronal activity dur- ing hypoxia and anoxia. For electrophysiological recordings a patch-clamp micro pipette will be molded into the walls of the microfluidic system. A single biological cell is steered towards the pipette and attached there by means of optical tweezers. The Neuroglobin oxygen binding state will be studied using optical spectroscopy and the neuron environment will be ma- nipulated by applying flows of varying oxygen content through the microfluidic system. This system will constitute a powerful tool in the investigation of the Neuroglobin mechanism of action. Keywords—Neuroglobin, hypoxia, multifunctional microfluidic system, patch clamp, optical tweezers, optical spectroscopy I. INTRODUCTION The nerve system depends on oxygen for its survival and if the oxygen flow to a cell is restrained the cell may suffer irreparable damage or die. The oxygen supply to tissue and cells is usually provided by hemoproteins. In 2000, a new hemoprotein was discovered and since this hemoprotein is mainly present in nerve cells it has been named Neuroglobin (Ngb) [1]. Much research has been conducted to learn more about the protein. Its 3D structure and abundance have been studied extensively. The function, on the other hand, is still a matter of debate [2]. Our main interest, i.e. the research performed to unravel Ngb’s possi- ble function as a neuroprotectant against hypoxic or ischemic injury, has been examined previously [3]. Evi- dence was found that neuronal hypoxia induces Ngb expres- sion, and enhanced Ngb expression reduces hypoxic neu- ronal injury. The challenge that still remains is to investigate the mechanism by which Ngb functions during oxygen deprivation. Previous research has mainly been implemented on the protein in its purified form and there is a need of better methods to investigate Ngb activity in a functional biologi- cal cell. Our research group aims to develop a multifunc- tional microfluidic system enabling supervision of influ- ences of Ngb concentrations on neuronal activity during hypoxia and anoxia. II. METHODS A. Preparation of neurons and solutions Ethical approval of the procedures described was given by the regional ethics committees for animal research (“Stockholms södra djuförsöksetiska nämnd”, approval No. S201/04 and “Umeå djurförsöksetiska nämnd”, approval No. A17-05). Cell preparation Neurons were prepared from the brains of male Sprague Dawley rats who were decapitated without anes- thetics, the brains were rapidly removed and placed in pre- oxygenated ice-cold (4 °C) incubation solution (150mM NaCl, 5mM KCl, 2mM CaCl 2 , 10mM HEPES, 10mM glu- cose, 4.93mM Tris-base, pH 7.4) which was also used throughout the entire slicing procedure. Slicing procedure A vibratome (Vibratome 100plus, Ted Pella, Red- ding, CA, USA, or Vibroslicer 752 M, Campden Instru- ments, Leicestershire, UK) was used to cut 200 - 300 mm thick coronal slices from the part of the block of brain tissue containing the anterior hypothalamus. Slices were then