Production of digoxigenin-labelled RNA probes and the detection of cytokine mRNA in rat spleen and brain by in situ hybridization

Abstract

Non-radioactive in situ hybridization is a sensitive method for determining the site of production for secretory molecules such as cytokines. We report here on the central and peripheral induction of proinflammatory cytokines by endotoxin, and outline procedures for the generation and application of rat-specific digoxigenin (Dig)-labelled RNA probes for the localization of mRNA by in situ hybridization. Rats were injected either intravenously (i.v.) or intracerebroventricularly (i.c.v.) with vehicle or lipopolysaccharide (LPS) and sacrificed at various time intervals post-injection. Rats were then perfused with 4% paraformaldehyde and the spleens and brains were removed and cryoprotected in 30% sucrose. Dig-labelled, rat-specific, antisense and sense RNA probes were generated by in vitro transcription from PCR-derived templates. Positive staining with all the antisense probes was cytoplasmic, whereas the sense probes showed no staining. Numerous tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) mRNA positive cells were observed in the marginal zone and in the red pulp of the spleen after iv LPS injections, whereas sections from saline-treated animals showed minimal cytokine mRNA expression. Cells positive for TNF-alpha and IL-1beta mRNA were detectable in the brain after i.c.v. injections of LPS, but not after icv injection of vehicle. An antisense probe for c-fos was utilized in these studies as a positive control for our procedure due to its anatomically specific expression in the rat brain after LPS. In conclusion we have demonstrated that in situ hybridization with Dig-labelled RNA probes is an efficient, sensitive and reliable tool to localize cytokine mRNA production in rat tissue.