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Updated: Aug 24, 2026
Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique
Published on: December 15, 2023
Translation-state analysis of gene expression in mouse brain after focal ischemia
John P MacManus1, Tyson Graber, Christian Luebbert
1Experimental Stroke Group, Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada. John.MacManus@nrc-cnrc.gc.ca
Abstract:
Confounding any genome-scale analysis of gene expression after cerebral ischemia is massive suppression of protein synthesis. This inefficient translation questions the utility of examining profiles of total transcripts. Our approach to such postischemic gene profiling in the mouse by microarray analysis was to concentrate on those mRNAs bound to polyribosomes. In our proof-of-principle study, polysomally bound and unbound mRNAs were subjected to microarray analysis: of the 1,161 transcripts that we found to increase after ischemia, only 36% were bound to polyribosomes. In addition to the expected increases in heat-shock proteins and metallothioneins, increases in several other bound transcripts involved in the promotion of cell survival or antiinflammatory behavior were noted, such as CD63 (Lamp3), Lcn2 (lipocalin-2), Msn (moesin), and UCP2 (uncoupling protein 2), all of which showed increases in cognate protein by Western blotting. The list of heretofore nonfunctionally annotated transcripts (RIKEN clones/ESTs) that increased appeared to be novel. How some transcripts are selected in ischemic brain for translation into protein, while others are rejected, is not clear. The length of the 5'-UTR in the ischemically induced transcripts that occur in the NCBI RefSeq database did not indicate any general tendency to be more than 200 nt, nor to be longer than the 5'-UTRs of the unbound transcripts. Thus, the presence of a complex 5'-UTR region with internal ribosome entry sites (IRES) or polypyrimidine tracts (TOP) does not appear to be the basis of selection for translation in ischemic brain.
Insights
Investigating gene expression after cerebral ischemia requires focusing on polyribosome-bound messenger RNAs (mRNAs). This study identified specific mRNAs translated into proteins, revealing novel targets for cell survival and anti-inflammatory responses post-ischemia.
Area of Science:
- Neuroscience
- Molecular Biology
- Genomics
Background:
- Cerebral ischemia causes massive protein synthesis suppression, complicating genome-scale gene expression analysis.
- Examining total transcript profiles after ischemia may be misleading due to inefficient translation.
Purpose of the Study:
- To develop a method for analyzing gene expression after cerebral ischemia by focusing on actively translated mRNAs.
- To identify specific transcripts that are translated into proteins following ischemic events in the brain.
Main Methods:
- Microarray analysis of polyribosome-bound and unbound mRNAs in a mouse model of cerebral ischemia.
- Validation of increased transcript levels by Western blotting for cognate proteins.
Main Results:
- Of 1,161 transcripts increased after ischemia, only 36% were polyribosome-bound.
- Increased polyribosome-bound transcripts included those involved in cell survival (e.g., CD63, Lcn2, Msn, UCP2) and anti-inflammatory responses.
- Novel, unannotated transcripts were also identified among the increased, translated mRNAs.
- Transcript 5'-UTR length did not appear to be a selection factor for translation.
Conclusions:
- Focusing on polyribosome-bound mRNAs provides a more accurate profile of functional gene expression post-cerebral ischemia.
- Several novel transcripts are translated into proteins promoting cell survival and anti-inflammatory actions after ischemia.
- The mechanism for selective mRNA translation in the ischemic brain remains unclear and is not explained by 5'-UTR length.
