A refined in vitro model to study inflammatory responses in organotypic membrane culture of postnatal rat hippocampal slices.
BACKGROUND
Propagated tissue degeneration, especially during aging, has been shown to be enhanced through potentiation of innate immune responses. Neurodegenerative diseases and a wide variety of inflammatory conditions are linked together and several anti-inflammatory compounds considered as having therapeutic potential for example in Alzheimer's disease (AD). In vitro brain slice techniques have been widely used to unravel the complexity of neuroinflammation, but rarely, has the power of the model itself been reported. Our aim was to gain a more detailed insight and understanding of the behaviour of hippocampus tissue slices in serum-free, interface culture per se and after exposure to different pro- and anti-inflammatory compounds.


METHODS
The responses of the slices to pro- and anti-inflammatory stimuli were monitored at various time points by measuring the leakage of lactate dehydrogenase (LDH) and the release of cytokines interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-alpha) and nitric oxide (NO) from the culture media. Histological methods were applied to reveal the morphological status after exposure to stimuli and during the time course of the culture period. Statistical power analysis were made with nQuery Advisor, version 5.0, (Statistical Solutions, Saugus, MA) computer program for Wilcoxon (Mann-Whitney) rank-sum test.


RESULTS
By using the interface membrane culture technique, the hippocampal slices largely recover from the trauma caused by cutting after 4-5 days in vitro. Furthermore, the cultures remain stable and retain their responsiveness to inflammatory stimuli for at least 3 weeks. During this time period, cultures are susceptible to modification by inflammatory stimuli as assessed by quantitative biochemical assays and morphological characterizations.


CONCLUSIONS
The present report outlines the techniques for studying immune responses using a serum-free slice culture model. Statistically powerful data under controlled culture conditions and with ethically justified use of animals can be obtained as soon as after 4-5 DIV. The model is most probably suitable also for studies of chronic inflammation.
Huuskonen
Jari
J
Department of Neuroscience and Neurology, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland. Jari.Huuskonen@uku.fi
Suuronen
Tiina
T
Miettinen
Riitta
R
van Groen
Thomas
T
Salminen
Antero
A