University of Twente Student Theses


Ischemic core : a method to analyse cell swelling

Lamberti, M. (2019) Ischemic core : a method to analyse cell swelling.

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Abstract:Stroke is one of the most common causes of death. Ischemic stroke occurs due to the reduction of blood flow in the affected area of the brain. The reduction of blood supply causes a lack of oxygen inducing a shortfall of ATP production. In the core of the infarct, center of irreversible damages, the dysfunction of the sodium-potassium pump and, as a result, the influx of water inside the cells lead to cell swelling and necrotic cell death. To understand if there might be a possibility for future treatments in this region, the first step would be to quantify cells swelling. The present study aims to design, validate and apply a new method for quantifying changes in cells size in the ischemic core. To analyse cell swelling, ischemia was induced in in vitro cultures of rat cortical neurons, by incubating the cell with 10mM sodium-azide (NaN3) and 5mM 2-deoxy- D-glucose for 10 minutes. After verifying that this procedure did not enhance apoptosis, cell swelling was assayed by acquiring fluorescent images with an inverted microscope every 30 seconds for 10 minutes. Fluorescence was obtained using a CellTracker green live staining. Two-dimensional images were analysed using an inhouse developed algorithm, with the purpose to identify all individual cells, and to determine their size and size changes during chemical ischemia. The algorithm was validated acquiring fluorescent images of blue microbeads with known diameter. The number of swollen cells under ischemic conditions was significantly larger than under control conditions. Around 21% of ischemic cells showed cell swelling. Their area reached an increase of about 20% of the initial size. These results show that the chosen method of chemical ischemia leads to cell swelling. Furthermore the designed algorithm is able to determine the correct size of cells in the x-y plane, as well as temporal evolution of this area. It is possible that swelling occurred only in a specific subset of cells, e.g. only astrocytes. Moreover, it might be that, in some cells, swelling remained undetected because it occurred predominantly in the vertical direction, perpendicularly to the observed plane.
Item Type:Essay (Master)
Faculty:TNW: Science and Technology
Subject:42 biology, 50 technical science in general
Programme:Biomedical Engineering MSc (66226)
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