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The Pathogenesis of MS pathology III: an in vitro model.
By Ilse Smets, PhD
Recent Research
Multiple Sclerosis:
Multiple Sclerosis (MS) is a chronic
inflammatory disease of the central nervous system (CNS). Myelin
breakdown, axonal loss and oligodendrocyte cell death are typical
characteristics of the CNS in MS patients. Recently, Lassmann and
coworkers described 4 different patterns of MS lesions. The typical
markers for MS pattern III lesions are distal 'dying back'
oligodendrogliopathy with preferential loss of MAG and CNPase and
apoptotic nuclear changes. Similar alterations are also found in early
(during the first week of white matter ischemia) stages of white matter
ischemia. Therefore, MS pattern III alterations may reflect hypoxic
white matter damage as a pathogenic component of the lesions.
Picture of oligodendrocytes in culture (with the
courtesy of Marjan Moreels, laboratory of histology UHasselt)
The aim of this research project is to gain
insight in the pathogenic mechanisms of oligodendrocyte injury in MS
pattern III lesions. The first step in this research project is to
develop an in vitro model for induction of the typical MS pattern III
oligodendrocyte characteristics. Primary cultures of oligodendrocytes,
obtained from mature rats, are subjected to hypoxic conditions in an
home-built hypoxic chamber. Cell death will be investigated via flow
cytometry (annexin-propidium iodide) and morphological criteria
(electron microscopy and light microscopy). Immunocytochemistry will be
used to examine changes in expression of myelin proteins e.g. MBP, PLP,
MAG and CNPase.
Former Research
Ischemia in kidney cells:
Since the regeneration of the kidney after an ischemic insult is based on the capacity of surviving cells to recover and proliferate, some of the putative cellular defence mechanisms in sub-lethally injured renal tubular cells are investigated. In view of the high resistance to ischemic injury of distal tubular renal cells, distal tubular cell lines are used as in vitro models: A6 and Madin-Darby canine kidney (MDCK) cell lines. Ischemia is simulated via metabolic inhibition. Different cellular processes are examined during metabolic inhibition: cellular Na+ handling, intracellular pH and volume regulation and the intracellular and intramitochondrial calcium homeostasis.
Mitochondria in MDCK cells were stained with Mito Tracker Green (MTG, left panel) to localize the mitochondria precisely and with Rhod 2 (right panel), a calcium-sensitive probe, to measure intra-mitochondrial calcium concentrations. In order to rule out any contribution of cytosolic and nucleolic Rhod 2 fluorescence, rhod 2 images were analysed only at mitochondrial positions (as determined from the MTG image). (For more details: Smets et al., AJP Renal Physiology, 286, F784-F794, 2004). The images were obtained on a Zeiss LSM 510 Meta laser-scanning confocal microscope attached to an Axiovert 200 frame (Zeiss).
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