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Updated: Jul 27, 2026
Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area
Published on: February 2, 2017
Circ-camk4 involved in cerebral ischemia/reperfusion induced neuronal injury
Zhao-Huan Zhang1,2, Yue-Rong Wang3, Fei Li2
1School of Preclinical Medicine, Wannan Medical College, Wuhu 241001, China. School of Life Sciences, Shanghai University, Shanghai, 200444, China.
Abstract:
Stroke and subsequent cerebral ischemia/reperfusion (I/R) injury is a frequently occurring disease that can have serious consequences in the absence of timely intervention. Circular RNAs (circRNAs) in association with microRNAs (miRNAs) and RNA-binding proteins (RBPs) can influence gene expression. However, whether circRNAs have a role in cerebral I/R injury pathogenesis, especially soon after onset, is unclear. In this study, we used the SD rat middle cerebral artery occlusion (MCAO) model of stroke to examine the role of circRNAs in cerebral I/R injury. We used high-throughput sequencing (HTS) to compare the expression levels of circRNAs in cerebral cortex tissue from MCAO rats during the occlusion-reperfusion latency period 3 hours after I/R injury with those in control cerebral cortices. Our sequencing results revealed that expression levels of 44 circRNAs were significantly altered after I/R, with 16 and 28 circRNAs showing significant up- and down-regulation, respectively, relative to levels in control cortex. We extended these results in vitro in primary cultured neuron cells exposed to oxygen-glucose deprivation/reperfusion (OGD/R) using qRT-PCR to show that levels of circ-camk4 were increased in OGD/R neurons relative to control neurons. Bioinformatics analyses predicted that several miRNAs could be associated with circ-camk4 and this prediction was confirmed in a RNA pull-down assay. KEGG analysis to predict pathways that involve circ-camk4 included the glutamatergic synapse pathway, MAPK signaling pathway, and apoptosis signaling pathways, all of which are known to be involved in brain injury after I/R. Our results also demonstrate that levels of the human homolog to circ-camk4 (hsa-circ-camk4) are elevated in SH-SY5Y cells exposed to OGD/R treatment. Overexpression of hsa-circ-camk4 in SH-SY5Y cells significantly increased the rate of cell death after OGD/R, suggesting that circ-camk4 may play a key role in progression of cerebral I/R injury.
Insights
Circular RNAs (circRNAs) are altered after stroke-induced brain injury. Circ-camk4 levels increase, promoting cell death and potentially contributing to cerebral ischemia/reperfusion injury.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Stroke and cerebral ischemia/reperfusion (I/R) injury pose significant health risks.
- Circular RNAs (circRNAs), microRNAs (miRNAs), and RNA-binding proteins (RBPs) regulate gene expression.
- The role of circRNAs in early cerebral I/R injury remains largely unknown.
Purpose of the Study:
- To investigate the role of circRNAs in the early stages of cerebral I/R injury.
- To identify specific circRNAs involved in the pathogenesis of I/R injury.
Main Methods:
- Middle cerebral artery occlusion (MCAO) rat model for stroke and I/R injury.
- High-throughput sequencing (HTS) to analyze circRNA expression in brain tissue.
- In vitro studies using oxygen-glucose deprivation/reperfusion (OGD/R) in primary neurons and SH-SY5Y cells.
- Quantitative reverse transcription PCR (qRT-PCR), RNA pull-down assays, and bioinformatics analyses.
Main Results:
- HTS revealed 44 significantly altered circRNAs in rat cerebral cortex 3 hours post-I/R.
- 16 circRNAs were upregulated and 28 were downregulated.
- Circ-camk4 expression increased in OGD/R-treated neurons and SH-SY5Y cells.
- Bioinformatics predicted and RNA pull-down confirmed miRNA interactions with circ-camk4.
- KEGG analysis implicated circ-camk4 in glutamatergic synapse, MAPK signaling, and apoptosis pathways.
- Overexpression of hsa-circ-camk4 exacerbated OGD/R-induced cell death in SH-SY5Y cells.
Conclusions:
- CircRNAs are dynamically regulated following cerebral I/R injury.
- Circ-camk4 is upregulated in response to I/R and promotes neuronal cell death.
- Circ-camk4 may be a key player in the progression of cerebral I/R injury.
