Suitability of rat brain microtissues for the investigation of neuroinflammation and single stranded oligonucleotide induced neurotoxicity Stefan Kustermann, Tanja Minz, Christine Zihlmann, Christian Elsässer, Sabine Sewing Pharmaceutical Sciences, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd
In order to investigate molecular and cellular mechanism of neurotoxicity of drug candidates, simple 2D-neuronal models are mostly not sufficient as they lack multiple cell types and do not represent the physiological cellular microenvironment. To enable interaction of multiple cell types and the establishment of appropriate environmental cues we have investigated a rat 3-dimensional brain micro tissue model. The model comprises of different cell types of the adult rat brain including neurons and glia cells, which maintain homeostasis and support and protect neurons, rendering the model suitable to study neuronal glia interactions. Characterization of the 3D-micro tissues with specific markers for neuronal and glial cells revealed presence of neurons, astrocytes, oligodendrocytes and microglia after 3 and 11 days in culture. Treatment of microtissues with LPS and IFNγ resulted in a strong upregulation of cellular TNFα and iNOS mRNA as well as in Iba1 mRNA and protein upregulation as demonstrated by qPCR and ICC, respectively. In addition slight TNFα release into the medium was observed after 24 and 72h of treatment. We finally investigated the suitability of this model for the assessment of single stranded oligonucleotide induced effects in the brain. After 3 and 11 days of oligonucleotide exposure an almost 100% knock-down of target mRNA was observed, indicating good penetration and even distribution of the oligonucleotide molecules within the 3D structure. Taken together the presented rat brain microtissue is a relevant model to study mechanisms of neuroinflammation and is amenable also for the investigation of oligonucleotide driven effects in the brain.
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