The electrophysiological response of medial preoptic neurons to hypoxia and development of a system for patch-clamp measurement with full oxygen control

• A stroke is caused by interruption of the blood supply to the brain. Yearly 15 million people around the globe endure a stroke and the costs and suffering for the people involved and the society are immense. The aim of this thesis was to investigate the response to oxygen deprivation in neurons from the medial preoptic nucleus (MPN) that have a high abundance of neuroglobin. The long term goal is to investigate the neuroprotective role of the protein in relation to stroke. Initially, the electrophysiological response of neurons to hypoxic exposure in an open system was assessed with a conventional patch-clamp setup. The first aim was to see how well the conventional system worked and if it needed improvement. Secondly, the MPN had never been investigated regarding oxygen, deprivation; hence the electrophysiological response under hypoxia needed to be investigated. The conventional patch-clamp system only allowed a reduction of the oxygen content to a level of 3-6% but not total control of the cell environment. The medial preoptic neurons showed mainly an increase of their resting membrane potential at hypoxia. The voltage activated Ca2+ and K+ currents displayed a clear attenuation when cells were subjected to hypoxia. Non-L-type Ca2+ channels were affected by hypoxic exposure and one cell indicated participation of Ca2+ activated K+ channels. However, a response could only be seen in approximately fifty percent of the neurons in the open system. This may have been due to the fact that full control of the oxygen around the neurons at hypoxia could not be achieved. A new system with full control of the ambient oxygen had to be developed in order to investigate this. After the conclusions of the first experiments, a system was developed were a labon- a-chip system was combined with the patch-clamp technique. A microfluidic system with a patch-clamp micropipette integrated was combined with optical tweezers for 3D maneuvering of the neurons. The development of patch-clamp in combination with a microfluidic system and optical tweezers allowed for full oxygen control. The experiments showed that the electrophysiological measurements were not affected by the laser when an infrared laser was used. The microfluidic system allowed very good oxygen control reaching levels of 0.5-1.5 % compared to 3-6 % in the open system. In summary, this work suggests that high voltage activated Ca2+ channels, and K+ channels are involved in the hypoxic depolarization of medial preoptic neurons. Full control of ambient oxygen in cell vicinity could be achieved by the combination of microfluidics, patch-clamp and optical tweezers. The results can be used in future studies to better understand the reaction of the brain to oxygen deprivation caused by stroke.

Ämnesord

TEKNIK OCH TEKNOLOGIER  -- Medicinteknik -- Annan medicinteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Medical Engineering -- Other Medical Engineering (hsv//eng)

Nyckelord

Medical Engineering for Healthcare

Medicinsk teknik för hälsovård

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