Abstract
(Englisch)
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The goals of the study were to:
- determine upregulated 14-3-3 expression in scrapie infected hippocampal slice cultures
- identify specifically up regulated isomers of 14-3-3
- investigate a possible colocalisation of 14-3-3 and PrPSc
PrP overexpressing mice (tg20) and wild type mice (wt) were used. PrP knock out mice served as negative controls (ko). The hippocampus was isolated and cut into slices of 400-450 mm thickness on a tissue chopper. Slices were placed in a Petri dish and transferred to Millipore culture plate inserts. Establishing slice cultures required much effort, since the slice preparation requires very gentle and quick handling. After three to four days, the slice cultures began to exhibit severe astrocytic gliosis and severe neuronal loss. We managed to keep 20% of the cultures in viable condition for 3-4 weeks, seldom for 7-8 weeks.
Cultures were infected with prions. Attempts to detect 14-3-3 and PrPSc in the medium by Western blot revealed severe background problems caused by the culture medium containing horse serum. Only the brain homogenate that served as positive control gave a strong signal at 33 kD. This fact caused us to change to serum free culture medium (Gilbcos neurobasal medium without glutamine, B27, glutamine) resulted in equal survival times of the cultures. However, 14-3-3 in the medium was still below detectability.
Slice culture survival period and incubation time might have been to short for prion pathogenesis. Therefore, we infected CD 1 mice intracerebral with 30 ml RML10-3. Intracerebral mock-infected mice served as controls. 150 days post infection, mice were sacrificed. Hippocampal slice cultures were prepared as described above and kept in culture for one week. Culture medium was collected every second to third day. We still failed to detect 14-3-3 or PrPSc in the medium.
There is no doubt that we have been learning a number of important things about the biology of hippocampal slices, and their suitability (or lack thereof) to addressing questions related to prion diseases. In summary, we can say that although this study has not been as successful as we had been hoping, it has helped us moving forward and establishing the slice technology, which will be used in the future in continued attempts to ask the questions proposed at the beginning of this study (which continue to be highly relevant to prion diseases), as well as a question related to the establishment of an in vitro cell culture model for the replication of disease-associated prion protein.
In addition, Western blot for 14-3-3 in spinal fluid was established for diagnostic purposes on CJD suspected patients. Spinal fluid samples of CJD patients exhibited a strong signal at 33 kD. Some samples showed an additional band of about 32 kD. 14-3-3 turned out not to be very specific. We found 14-3-3 positive also in patients with neurosyphilis (2/2), paraneoplastic limbencephalitis (2/2), suspected Hashimoto encephalitis (1/1) and anaplastic astrocytoma (1/1). How ever, false negative results were rarely obtained; in one case spinal fluid was initially negative for 14-3-3 and became positive in the course of illness.
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