Background
Stroke is a major cause for disability and death affecting one in four people during their lifetime [
1]. Stroke survivors have only limited therapeutic options and are often left with considerable disabilities [
2,
3]. The development of new therapeutics for stroke is not trivial as the ischemic cascade involves many pathological processes such as cellular excitotoxicity, oxidative stress, inflammation, blood-brain barrier disruption and scarring [
4]. Therefore, unbiased screening studies decoding the transcriptional response of stroked tissue are important to identify new potential targets for therapy [
5,
6]. Since human stroke brain tissue is only of limited suitability for the analysis (i.e., it can only be accessed in lethal strokes and the processing time may lead to poor RNA quality), the majority of transcriptional profiling of stroked tissue was performed in rodents. Although animal models are designed to generate reproducible infarcts in highly controlled conditions, there is recognized heterogeneity among stroke models and species [
7]. The most commonly used stroke model is the middle cerebral artery occlusion (MCAo) model; in this model the most commonly affected artery in human stroke is surgically obscured. The occlusion can either be transient to produce reperfusion after 30–120 min (tMCAo) or permanent (pMCAo) [
8,
9]. Alternatively, the photothrombotic stroke model is popular for permanent ischemia in defined brain regions that lead to long-term functional deficits [
9‐
11].
In this study, the gene expression omnibus (GEO) RNA sequencing data were obtained from stroke tissue across different stroke models, sex, time points, and species. The gene expression profiles were integrated to identify and compare common and differentially expressed genes (DEG) and enriched pathways in the individual groups.
Methods
data search from GEO microarray data repositories were searched in November 2022. The search terms were “stroke”, “ischemia”, “tMCAo”, “pMCAo”, “MCAo”, “photothrombotic stroke”. Selected organisms were “homo sapiens”, “macaca”, “mus musculus” and “rattus norvegicus”. Datasets were excluded that did not include brain tissue samples (Suppl. Figure
3). (Most human studies 49/51 had to be excluded because they performed RNAseq with blood samples). Further studies were excluded with missing information, duplicates, pooled samples, poor quality controls, and no clear separation between stroked and non-stroked groups. No separation between stroke and non-stroke groups was considered to be a result of either a failed stroke induction or incorrect allocation of datasets, leading to the exclusion of these studies. An overview of the used datasets can be found in Suppl. Table
1. Significant genes for each group were identified using R Studio RankProd [
12]. All datasets were annotated and converted uniformly using genome wide annotation resources.
Study selection
The search initially retrieved 338 articles, of which 34 met the inclusion criteria.
Data processing and annotation
First, all data frames were pre-processed and normalized using the trimmed mean of M-values TMM in edgeR to account for differences in library size, sequencing depth, and gene length and reduce technical variability. Gene annotation and identifiers across species was performed using biomaRt mapIDs package. All molecular identifiers were converted in gene symbols and capitalized (to account for different species). Comparison of different species, time points and stroke model were performed by calculating the changes between stroked and the corresponding control transcriptome using the edgeR package and then comparing the overlap of up- and downregulated genes via Venn diagrams.
Venn Diagram
Overlap between upregulated and downregulated genes were visualized using venn.diagram function in R. Differentially expressed genes with fold change +/- 0.5 were compared from different species, time points and stroke models.
Functional enrichment analysis of DEGs
Analysis of functional enrichment analysis for DEGs was performed using EdgeR [
13] and clusterProfiler 4.0 using the function gseGO [
14] in RStudio.
Discussion
Dissecting the molecular profile after stroke promises to identify new targets for potential therapeutic compounds. However, the multifaceted ischemic cascade and the variety of used animal models complicates the search of promising pathways related to stroke. Here, major differences were identified in publicly available gene expression profiles after stroke that varied depending on the time period, the animal/rodent species, and the stroke model used. These alterations affected primarily upregulated genes and affected both general and stroke-related pathways. The biggest alteration in the gene expression was identified for the different time periods after stroke, supporting the hypothesis that timing of therapy in stroke is of primary importance [
16].
Furthermore, RNA datasets from primates and humans were only comparable to a limited extent with the rodent datasets. The human and primate datasets had considerably fewer analysed genes as they were derived from micro-array studies. Additionally, obtaining high quality RNA from human stroked brain tissue is challenging, mainly because of the time gap between the patient’s death and the collection of tissue, which can contribute to the decline in RNA quality.
The heterogenicity of the stroke pathophysiology is a further limitation in identifying novel targets for therapy. For instance, the molecular signature of the stroke core may considerably vary from the penumbra and different cell types may contribute to gene expression changes. However, there are only a limited number of studies that have thoroughly investigated these differences [
17]. Novel advancements with single cell/nucleus and spatially resolved transcriptomics may provide further insights in near future [
18,
19].
Many detrimental and regenerative processes after stroke occur in parallel over time. The classification of time points after stroke in acute (0-24 h), subacute (1-7d) and long-term (7-28d) was based on previous preclinical studies and studies used in this meta-analysis. The general aim in this classification was to describe gene expression differences in time frames often used to evaluate treatments that either inhibit with the initial stroke progression (acute), reduce inflammatory responses or are neuroprotective (subacute) and regenerative treatments (long-term). For instance, interference with early-response pathways such as CCL2 are often investigated acutely after stroke within the first 24 h [
20]. Anti-inflammatory drugs blocking with subacute post-ischemic inflammation are usually tested in the first 24-7d after stroke [
21] and therapies aimed to improve long-term recovery evaluate the effects later than 7d [
9,
10,
22]. A limitation of the study is that dynamic changes in gene expression in between these time periods are not detectable. A potential alternative could involve employing a data-driven unsupervised approach to establish time frames that are not reliant on previous research, but rather on gene network behavior. This method could enable future investigators to design interventions more effectively.
Species-dependent differences may be attributed to differences in the brain circulation of mice and rats. While the overall function and structure of the brain vasculature are similar in both mice and rats, the Circle of Willis is more developed in rats than that in some strains of mice including C57Bl6 [
23,
24]. These differences may affect the collateral blood perfusion and are known to considerably vary even within mice of different genetic backgrounds [
24,
25].
Many differentially expressed genes were also associated with inflammatory responses after stroke. It has previously been shown that mice and rats exhibit distinct inflammatory reactions after spinal cord injury [
26]. These differences include for instance timing and magnitude of immune cell infiltration [
26], also more recent studies indicate sex-specific differences within the species that could not be assessed in this analysis [
27].
This meta-analysis reveals that considerable gene expression differences exist between mice and rats as well as the used stroke models. This data supports the recommendations to confirm the effect of experimental therapeutic compounds in at least two animal species or different stroke models [
28]. Additionally, the study was designed to examine the effect of sex on gene expression after stroke. However, there is still a lack of datasets for the female sex after stroke as all studies used male rodents or mixed sex. The sex plays an important role in human stroke pathology [
29] and should be further investigated in preclinical gene expression studies in near future.
Conclusion
In sum, this meta-analysis identifies distinct gene expression changes in stroked brain tissue across species, time points and stroke models. These differences affected general and stroke-related pathways and need to be considered when evaluating new potential therapeutic compounds.
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