- In total, 1076 genes had an increased expression and 879 genes a decreased expression in the occluded MCAs as compared with the control MCAs from female rats. - An enrichment of genes related to apoptosis, regulation of transcription, protein autophosphorylation, inflammation, oxidative stress, and tissue repair and recovery were seen in the occluded MCA. - The high expression genes chosen for qPCR verification (Adamts4, Olr1, JunB, Fosl1, Serpine1, S1pr3, Ccl2 and Socs3) were all shown to be upregulated in the same manner in both females and males after tMCAO (p <. - Conclusions: The cascades of processes initiated in the vasculature following reperfusion are complex. - Dynamic gene expression alterations were observed in the occluded MCAs, and to a less pronounced degree in the non-occluded MCAs. - 1% of the differentially expressed genes) validated for this microarray did not reveal any sex differences. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - Full list of author information is available at the end of the article. - Ischemic stroke is one of the leading causes of death and disability in the world [1]. - In the case of thrombolysis, the risk of these adverse effects does not outweigh the benefits past 4.5 h post stroke, which limits the use in clinical practice [6].. - Despite intense research efforts during several decades with more than 1000 compounds tested and numerous interventions that have shown promise in pre-clinical studies, all failed in the clinical studies [7]. - Some of the main reasons proposed for this “translational roadblock”. - Developing effective treatment strategies for both men and women requires a deeper understanding of sex dif- ferences in the underlying mechanisms of ischemic in- jury. - We have demonstrated that there are differences in the cerebrovascular receptor expression in males versus females both in human brain vessels and in rats after transient middle cerebral artery occlusion (tMCAO, a standard method for this type of experi- mental stroke) [12, 13].. - of the brain against hemorrhage, inflammation and edema by targeting the cerebral arteries is the first step towards successful stroke treatment [14]. - The BBB con- sists of endothelial cells with continuous tight junctions, which offer protection against the pathogens, toxins and reduce the influence of the peripheral immune system in the brain. - The present study was conducted to examine the early cerebrovascular processes of vascular damage after stroke in females and subsequently examine if sex differ- ences and similarities in these responses occurring in the cerebral vessel wall exist. - In order to examine which genes were activated in the early stroke stages (at 3 h), we performed whole-transcriptome expression profiling on middle cerebral arteries (MCAs) of female rats after tMCAO-induced ischemia. - This was also done to iden- tify activated biological processes and pathways locally in the MCAs which potentially could be targeted for vascular protection after stroke. - 05), although they were of the same age. - Insertion of the intraluminal filament resulted in a mean. - Whole transcriptome expression profiling in females In the microarray analysis, bilateral MCA segments (oc- cluded (n sample = 5) and non-occluded (n sample = 6)) from 6 female rats subjected to tMCAO were analyzed. - In total, 1076 genes showed an increased expression and 879 genes had a decreased expression in the occluded MCAs as compared with the control MCAs. - In the contralateral non-occluded MCAs, 111 genes had an increased ex- pression and 92 genes had a decreased expression. - The expressions of 80 of the differentially expressed genes were increased in both the occluded and non-occluded MCAs while the expressions of 67 of the differentially expressed genes were decreased in both the occluded and non-occluded MCAs (Fig. - With the significantly differentially expressed genes in the microarray, a gene ontology (GO) enrichment ana- lysis was performed to identify activated biological. - The genes differentially expressed in the occluded MCAs in relation to control MCAs were overrepresented for 17 PANTHER protein classes, 9 PANTHER pathways and 9 Reactome path- ways (Fig. - 1 Differentially expressed gene count in the occluded and non-occluded middle cerebral arteries (MCAs) from female rats. - Venn diagrams illustrating the number of upregulated and downregulated genes in the occluded and non-occluded MCAs both compared with control MCAs as well as gene overlap between the experimental groups. - 2 Differentially expressed gene distributions in the occluded and non-occluded middle cerebral arteries (MCAs) from female rats. - Volcano plots illustrating distributions of the differentially expressed genes in the occluded and non-occluded MCAs both compared with control MCAs, and in the occluded MCAs compared with the non-occluded MCAs from female rats. - Similar Volcano plots for male stroke rats can be found in the publication by Grell et al. - The number of differentially expressed genes after ex- perimental stroke was considerable higher in the oc- cluded MCAs (1955 differentially expressed genes) than in the non-occluded MCAs where only 203 differentially expressed genes were identified (Fig. - To reveal if the biological processes activated in the occluded MCAs are similar to those activated in the non-occluded MCAs, we looked for overlapping overrepresented GO terms.. - were overrepresented in the list of differentially expressed genes for both the occluded and non-occluded MCAs when compared with control MCAs (Fig. - Focusing on the gene-specific transcriptional regula- tors, 108 and 15 differentially expressed genes in the oc- cluded and non-occluded MCAs were annotated to this GO term, respectively. - Fo- cusing on the occluded MCAs, 94 of the gene-specific transcriptional regulators were DNA-binding Table 1 Gene enrichment analysis for the occluded middle cerebral arteries (MCAs) from female rats. - Overview of the top 15 overrepresented gene ontology (GO) biological process terms with the highest fold enrichment and top 15 overrepresented GO biological process terms with lowest p-value identified for the differentially expressed genes in the occluded MCAs compared with control MCAs. - qPCR for validation of target genes for both sexes To validate the results from the microarray, eight high- expression genes of interest were chosen for quantitative real-time polymerase chain reaction (qPCR) analysis in MCAs from both female and male rats (a new set of ani- mals were operated on in both sexes, 5 stroke females and 6 stroke males were included in the analysis as well. - The expression of the target genes in the occluded MCAs, non-occluded MCAs and control MCAs from the microarray analysis can be found in Fig. - All of the 8 analyzed genes (Ccl2, oxidized low- density lipoprotein receptor 1 (Olr1), a disintegrin and metalloproteinase with thrombospondin type 1 motif, 4 (Adamts4), serine protease inhibitor, clade E, member 1 (Serpine1), sphingosine 1 phosphate. - 3 Overrepresented protein classes and pathways in the occluded middle cerebral artery (MCA) from female rats. - Seventeen PANTHER protein classes and 18 PANTHER and Reactome pathways were overrepresented amongst the differentially expressed genes in the occluded MCAs from female rats compared with control MCAs. - In the non-occluded MCAs, only 5 overrepresented pathways and protein classes were identified all of which were also identified for the occluded MCAs. - receptor 3 (S1pr3), Socs3, JunB and Fosl1) were sig- nificantly upregulated in the occluded MCAs com- pared with control MCAs (Figs. - In addition, Ccl2, Socs3, Fosl1, JunB and Serpine1 were also upreg- ulated in the non-occluded MCAs as compared to control MCAs. - Sex did not have a significant effect on the expression of any of the 8 analyzed genes (p Figs. - differentially expressed in each of the experimental groups (Fig. - Within this study, whole transcriptome ex- pression profiling revealed a considerable number of dif- ferentially expressed genes (more than 2000) in the MCAs from female rats after tMCAO. - From the microarray analysis, we found a markedly increased expression of Adamts4, Socs3, Fosl1, JunB, Serpine1, Olr1, Ccl2 and S1pr3 in the ischemic artery compared to non-ischemic artery at 3-h post reperfusion in female rats. - In a further validation of the genes we compared both sexes and found a similar degree of increase in expression. - Thus, the results of this part of the study confirmed an increased expression of genes in the vasculature that contributes to inflamma- tion, endothelial dysfunction and breakdown of the BBB.. - Adamts4 is of interest since it for the first time is shown to be upregulated in the cerebral arteries in re- sponse to stroke. - Other metalloproteinases were also found to be overex- pressed in the microarray analysis of the occluded MCA (matrix metallopeptidase 19 (Mmp19), Adamts1, Adamts5, Adamts9, ADAM metallopeptidase domain 8 (Adam8) and Adam9), but not to the same degree as Adamts4. - It is highly expressed in VSMCs in macrophage-rich areas in the thoracic aorta of patients with thoracic aortic dissection and increased serum levels have been associated with carotid. - Venn diagram visualizing differentially expressed genes in the occluded middle cerebral artery compared with control MCAs that overlaps between the overrepresented Reactome pathway, cytokine signaling in immune system (Fig. - Table 2 Gene-specific transcriptional regulators and kinase modulators in the occluded middle cerebral arteries (MCAs) from female rats. - Overview of the differentially expressed genes, annotated to a subontology of the overrepresented gene-specific transcriptional regulator (PC00264) and kinase modulator (PC00140) PANTHER protein classes, in the occluded MCAs compared with control MCAs. - The time course of the Mmp8, Mmp9 and Mmp13 were carefully monitored in the MCAs immedi- ately after the SAH up until 48 h in male rats [16].. - Although this process has been well characterized in the settings of developing atheroscler- osis, it has not yet been established what role Olr1 plays. - 6 Further analysis of the upregulated gene targets in the female rat middle cerebral arteries (MCAs). - Heatmap and dendrograms illustrating hierarchical clustering of the expression of the gene targets for the experimental groups, occluded MCAs (R), non-occluded MCAs (L) and control MCAs (C), based on outcomes from the Affymetrix whole-transcriptome expression profiling (microarray analysis). - in the acute phase after ischemia-reperfusion and in par- ticular in the affected MCAs.. - Previous studies have shown that the expression of Ccl2 is highly increased in the brain after stroke, and that si- lencing the Ccl2 gene is protective in stroke models [32]. - We have previously reported on the increased presence of the chemokine (C-X-C motif) ligand 2. - Again, this clearly shows that the process is ongoing in the cerebral vessel walls at least 48 h after experimental SAH.. - Differential gene expression of the selected upregulated gene targets was validated by qPCR. - Inflamma- tion is crucial for repair and recovery after stroke, but in the initial phase, reduction of inflammation has been shown to be beneficial. - Our findings revealed that following tMCAO there are dynamic alterations in gene expres- sion in the immediate MCA, but also on the contralat- eral side, albeit to a less pronounced degree. - The sample size was set based on experience from earlier studies conducted in the lab.. - 1 where both upregulation and downregulation of the genes were considered. - The reason for the discrepancy is due to (1) Tetratricopeptide repeat domain 39B (Ttc39b) is downregulated in the non-occluded MCAs and upregulated in the occluded MCAs when compared with control MCAs, and (2) FK506 binding protein 3 (Fkbp3) is upregulated in the non-occluded MCAs and downregulated in the occluded MCAs when compared with control MCAs. - environmental enriched cages with a 12 h light/dark cycle (lights on 7 am-7 pm), climate- and humidity- con- trolled environment and with free access to food and water in the University of Lunds animal facility. - All experimental Table 4 Overlapping differentially expressed genes found for each of the experimental tMCAO stroke groups (Fig. - Thirty-two differentially expressed genes found for each of the following experimental stroke groups (tMCAO [transient middle cerebral artery occlusion] stroke models): Occluded versus non-occluded middle cerebral arteries (MCAs) from Wistar-Kyoto (WKY) male rats (results from Grell et al. - Female rats that were in the high estrogen phase, proestrus, were not included to re- duce variation due to hormone fluctuations. - A laser Doppler probe (Perimed, Järfälla, Sweden) was fixed to the skull 6 mm laterally of the midline and 1 mm posterior to bregma. - An incision was made in midline of the neck, exposing the carotid artery. - Animals with a minimum flow reduction of 50% were included in the study. - Only animals showing a score of 3–4 in the test were re-anesthetized after 120 min and the filament removed, which resulted in an in- crease in cerebral blood flow (as measured with Laser Doppler flowmetry). - Animals with a minimum flow in- crease of 30% were included in the study. - A ratio of sample absorbance at 260 nm and 280 nm in the range of 1.7 to 2.1 was accepted. - The concentration and quality of the concentrated RNA was determined with a NanoDrop ND1000 spectropho- tometer (ThermoFisher Scientific, MA, USA).. - The integrity of the RNA was measured with the Agilent Bioanalyzer (Agilent Technologies, CA, USA). - Subsequently, overlap between overrepresented GO terms for the differentially expressed genes in the occluded MCAs and non- occluded MCAs both compared with control MCAs were identified.. - Ccl2, S1pr3, Socs3, Serpine1, JunB and Fosl1 were selected due to oc- currence in biological processes that were significantly enriched in the GO analysis in combination with analysis of fold change and review of literature. - Adamts4 was se- lected due to a very high relative expression, and Olr1 was selected due to the availability of previous data in the same model in hypertensive and normotensive males. - In the PCR-validation, the mean computed cycle thresh- old (CT) for the triplicates of each sample was calcu- lated. - Since a higher dCT value corresponds with a lower expression, the axes in the figures were re- versed to offer a more intuitive reading of the figures.. - Here, they investigated differential expression of genes in the occluded MCAs compared with the non-occluded MCAs in WKY male rats. - The funding bodies were not involved in the design, analysis and reporting of the study. - The datasets generated or analysed during the current study are available in the Gene Expression Omnibus (GEO) repository, GSE162072, and in the published article [and its supplementary information file. - However, this did not affect the outcomes of the study. - Estrogens may reduce mortality and ischemic damage caused by middle cerebral artery occlusion in the female rat. - Gene expression profiling in the human middle cerebral artery after cerebral ischemia. - Inhibition of mitogen-activated protein kinase 1/2 in the acute phase of stroke improves long-term neurological outcome and promotes recovery processes in rats.. - Stretch-induced activation of the transcription factor activator protein-1 controls monocyte chemoattractant protein-1 expression during arteriogenesis. - Ischemic penumbra in a model of reversible middle cerebral artery occlusion in the rat. - Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination
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