Jackson Laboratory hdac2 flox flox

(a) Schematic representation of the experimental plan. Tamoxifen (TM)(75 mg/kg body weight) was administered to <t>Hdac2</t> flox/flox : C dh5CreERT2 or Ezh 2 flox/flox : Cdh5 CreERT2 8 weeks old mice for generating Hdac2 ECKO and Ezh2 ECKO respectively and brains were collected for phenotypic analysis after 8 weeks of final TM injection. (b) Representative phase microscopy images of the dorsal surface Hdca 2 ECKO mice showing a significant increase (**p ≤0 .01 vs. WT. N=3, one hemisphere from each mouse brain was selected for quantification.) in pial vessel density and cortical vessels stained with laminin showing significantly increased vascular density in Hdac2 ECKO compared to the WT (***p ≤ 0.001 vs. WT. N=6/group and Hdac2 ECKO N=7/group). Scale bar: 500µm. (c) Staining for vessels using laminin and proliferative marker EdU showing proliferating cells in Hdac2 ECKO mice cortical vessels as indicated by white arrows. Scale bar 500µm. Quantification of EdU + cells in vessels showed significantly increased proliferating cells in Hdac2 ECKO mice compared to WT (**p ≤ 0.01 vs WT. N=3/group: quantifications were performed on matching sections from independent brains). (d) BBB permeability assay using 3kDa-FITC dextran showed no leakage of tracer into brain parenchyma in Hdac2 ECKO mice (N=4/group: quantifications were performed on matching sections from independent brains ). Scale bar: 500µm. (e) Laminin staining displayed a reduction in cortical vessel density in Ezh2 ECKO mice compared to WT (*p ≤ 0.05 vs WT. N=3/group: quantifications were performed on matching sections from independent brains). Scale bar 500µm. The vessel tracing in the 20X image using AngioTool showed a significantly reduced vessel length in the Ezh2 ECKO cortex. (**p ≤ 0.01 vs WT. N=3/group quantifications were performed on matching areas of the cortex from independent brains). Scale bar: 5µm . (f) BBB permeability assay using 3kDa tracer shows significant leakage into the brain parenchyma of Ezh2 ECKO mice (***p ≤ 0.001 vs WT. N=5/group: quantifications were performed on matching sections from independent brains). Scale bar: 500 µm. 10x images are acquired and merged using tile scanning. All data are presented as mean ± SD.

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(a) Schematic representation of the experimental plan. Tamoxifen (TM)(75 mg/kg body weight) was administered to Hdac2 flox/flox : C dh5CreERT2 or Ezh 2 flox/flox : Cdh5 CreERT2 8 weeks old mice for generating Hdac2 ECKO and Ezh2 ECKO respectively and brains were collected for phenotypic analysis after 8 weeks of final TM injection. (b) Representative phase microscopy images of the dorsal surface Hdca 2 ECKO mice showing a significant increase (**p ≤0 .01 vs. WT. N=3, one hemisphere from each mouse brain was selected for quantification.) in pial vessel density and cortical vessels stained with laminin showing significantly increased vascular density in Hdac2 ECKO compared to the WT (***p ≤ 0.001 vs. WT. N=6/group and Hdac2 ECKO N=7/group). Scale bar: 500µm. (c) Staining for vessels using laminin and proliferative marker EdU showing proliferating cells in Hdac2 ECKO mice cortical vessels as indicated by white arrows. Scale bar 500µm. Quantification of EdU + cells in vessels showed significantly increased proliferating cells in Hdac2 ECKO mice compared to WT (**p ≤ 0.01 vs WT. N=3/group: quantifications were performed on matching sections from independent brains). (d) BBB permeability assay using 3kDa-FITC dextran showed no leakage of tracer into brain parenchyma in Hdac2 ECKO mice (N=4/group: quantifications were performed on matching sections from independent brains ). Scale bar: 500µm. (e) Laminin staining displayed a reduction in cortical vessel density in Ezh2 ECKO mice compared to WT (*p ≤ 0.05 vs WT. N=3/group: quantifications were performed on matching sections from independent brains). Scale bar 500µm. The vessel tracing in the 20X image using AngioTool showed a significantly reduced vessel length in the Ezh2 ECKO cortex. (**p ≤ 0.01 vs WT. N=3/group quantifications were performed on matching areas of the cortex from independent brains). Scale bar: 5µm . (f) BBB permeability assay using 3kDa tracer shows significant leakage into the brain parenchyma of Ezh2 ECKO mice (***p ≤ 0.001 vs WT. N=5/group: quantifications were performed on matching sections from independent brains). Scale bar: 500 µm. 10x images are acquired and merged using tile scanning. All data are presented as mean ± SD.

Journal: bioRxiv

Article Title: Epigenetic Reactivation of CNS Endothelial Developmental Programs Triggers Adult Brain Angiogenesis, Promotes Post-Stroke Revascularization and Neuronal Regeneration

doi: 10.1101/2025.08.15.670554

Figure Lengend Snippet: (a) Schematic representation of the experimental plan. Tamoxifen (TM)(75 mg/kg body weight) was administered to Hdac2 flox/flox : C dh5CreERT2 or Ezh 2 flox/flox : Cdh5 CreERT2 8 weeks old mice for generating Hdac2 ECKO and Ezh2 ECKO respectively and brains were collected for phenotypic analysis after 8 weeks of final TM injection. (b) Representative phase microscopy images of the dorsal surface Hdca 2 ECKO mice showing a significant increase (**p ≤0 .01 vs. WT. N=3, one hemisphere from each mouse brain was selected for quantification.) in pial vessel density and cortical vessels stained with laminin showing significantly increased vascular density in Hdac2 ECKO compared to the WT (***p ≤ 0.001 vs. WT. N=6/group and Hdac2 ECKO N=7/group). Scale bar: 500µm. (c) Staining for vessels using laminin and proliferative marker EdU showing proliferating cells in Hdac2 ECKO mice cortical vessels as indicated by white arrows. Scale bar 500µm. Quantification of EdU + cells in vessels showed significantly increased proliferating cells in Hdac2 ECKO mice compared to WT (**p ≤ 0.01 vs WT. N=3/group: quantifications were performed on matching sections from independent brains). (d) BBB permeability assay using 3kDa-FITC dextran showed no leakage of tracer into brain parenchyma in Hdac2 ECKO mice (N=4/group: quantifications were performed on matching sections from independent brains ). Scale bar: 500µm. (e) Laminin staining displayed a reduction in cortical vessel density in Ezh2 ECKO mice compared to WT (*p ≤ 0.05 vs WT. N=3/group: quantifications were performed on matching sections from independent brains). Scale bar 500µm. The vessel tracing in the 20X image using AngioTool showed a significantly reduced vessel length in the Ezh2 ECKO cortex. (**p ≤ 0.01 vs WT. N=3/group quantifications were performed on matching areas of the cortex from independent brains). Scale bar: 5µm . (f) BBB permeability assay using 3kDa tracer shows significant leakage into the brain parenchyma of Ezh2 ECKO mice (***p ≤ 0.001 vs WT. N=5/group: quantifications were performed on matching sections from independent brains). Scale bar: 500 µm. 10x images are acquired and merged using tile scanning. All data are presented as mean ± SD.

Article Snippet: Hdac2 flox/flox (Strain #:022625) , Ezh flox/flox (Strain #:022616), mice of both sexes were purchased from Jackson Laboratories.

Techniques: Injection, Microscopy, Staining, Marker, Permeability

(a) IB4 staining displayed significantly increased vascular density in Hdac2 ECKO mice cortex compared to WT (**p ≤ 0.01vs WT. WT N=6 and Hdac2 ECKO N=5 quantifications were performed on matching sections from independent brains). Scale bar: 500µm. (b) Laminin staining displayed a significant increase in hippocampal vessel density in Hdac2 ECKO mice compared to WT (*p ≤ 0.05 vs WT. WT N=4 and Hdac2 ECKO N=5 quantifications were performed on matching sections from independent brains). 10 x images are acquired and merged using tile scanning. Scale bar 500 µm. (c) Co-staining of Laminin and ZO1 displaying significantly increased ZO1 stained vessels in Hdac2 ECKO mice (**p ≤ 0.01vs WT N=3/group, three similar regions of interest in cortex from matching sections were selected from each mice brains). Scalebar: 500 µm. (d) Tight junction protein CLDN5 and EdU co-staining in Hdac2 ECKO mice vessels indicate newly formed vessels express CLDN5. Scale bar: 50µm. (e) Co-staining of laminin for vessel and PDGFRβ for pericytes displaying pericyte covering on vessels of both WT and Hdac2 ECKO mice. Scale bar: 50µm. (f) Representative images of brain sections from WT and Hdac2 ECKO mice stained for the astrocyte markers ALDH1 and GFAP, co-stained with laminin, and the astrocyte endfeet marker AQP4. Quantification revealed a significant increase in the density of ALDH1 (*p ≤ 0.05 vs. WT; WT: N=4, Hdac2 ECKO: N=6), GFAP (**p ≤ 0.01 vs. WT; WT: N=4, Hdac2 ECKO: N=5), and AQP4 (**p ≤ 0.01 vs. WT; WT: N=3, Hdac2 ECKO: N=3) in Hdac2 ECKO mice. Images were acquired at 10× magnification and merged using tile scanning. Scale bar: 500 µm. Quantifications were performed on matching sections from independent brains. (g) Representative images of laminin staining in liver, lung, and kidney tissues from WT and Hdac2 ECKO mice. Scale bar: 500 µm. (h) Analysis of mice behavior using open field test, Barnes maze test, and Y maze test showed no significant difference in Hdac2 ECKO mice behavior compared to WT (WT N=8 and Hdac2 ECKO N=7). All data are presented as mean ± SD.

Journal: bioRxiv

Article Title: Epigenetic Reactivation of CNS Endothelial Developmental Programs Triggers Adult Brain Angiogenesis, Promotes Post-Stroke Revascularization and Neuronal Regeneration

doi: 10.1101/2025.08.15.670554

Figure Lengend Snippet: (a) IB4 staining displayed significantly increased vascular density in Hdac2 ECKO mice cortex compared to WT (**p ≤ 0.01vs WT. WT N=6 and Hdac2 ECKO N=5 quantifications were performed on matching sections from independent brains). Scale bar: 500µm. (b) Laminin staining displayed a significant increase in hippocampal vessel density in Hdac2 ECKO mice compared to WT (*p ≤ 0.05 vs WT. WT N=4 and Hdac2 ECKO N=5 quantifications were performed on matching sections from independent brains). 10 x images are acquired and merged using tile scanning. Scale bar 500 µm. (c) Co-staining of Laminin and ZO1 displaying significantly increased ZO1 stained vessels in Hdac2 ECKO mice (**p ≤ 0.01vs WT N=3/group, three similar regions of interest in cortex from matching sections were selected from each mice brains). Scalebar: 500 µm. (d) Tight junction protein CLDN5 and EdU co-staining in Hdac2 ECKO mice vessels indicate newly formed vessels express CLDN5. Scale bar: 50µm. (e) Co-staining of laminin for vessel and PDGFRβ for pericytes displaying pericyte covering on vessels of both WT and Hdac2 ECKO mice. Scale bar: 50µm. (f) Representative images of brain sections from WT and Hdac2 ECKO mice stained for the astrocyte markers ALDH1 and GFAP, co-stained with laminin, and the astrocyte endfeet marker AQP4. Quantification revealed a significant increase in the density of ALDH1 (*p ≤ 0.05 vs. WT; WT: N=4, Hdac2 ECKO: N=6), GFAP (**p ≤ 0.01 vs. WT; WT: N=4, Hdac2 ECKO: N=5), and AQP4 (**p ≤ 0.01 vs. WT; WT: N=3, Hdac2 ECKO: N=3) in Hdac2 ECKO mice. Images were acquired at 10× magnification and merged using tile scanning. Scale bar: 500 µm. Quantifications were performed on matching sections from independent brains. (g) Representative images of laminin staining in liver, lung, and kidney tissues from WT and Hdac2 ECKO mice. Scale bar: 500 µm. (h) Analysis of mice behavior using open field test, Barnes maze test, and Y maze test showed no significant difference in Hdac2 ECKO mice behavior compared to WT (WT N=8 and Hdac2 ECKO N=7). All data are presented as mean ± SD.

Article Snippet: Hdac2 flox/flox (Strain #:022625) , Ezh flox/flox (Strain #:022616), mice of both sexes were purchased from Jackson Laboratories.

Techniques: Staining, Marker

( a ) Schematic representation of the experimental design. Tamoxifen (75 mg/kg body weight) was administered to Hdac2 flox/flox : Cdh5CreERT2 or Ezh2 flox/flox : Cdh5CreERT2 mice at 8 weeks of age. After 8 weeks, live CD31+/CD41-/CD45-brain ECs were isolated by FACS sorting and subjected to ultra-low mRNA sequencing. ( b, f ) Differential gene expression analysis showing the number of significantly upregulated and downregulated genes (p ≤ 0.05 N=3/group) in cortical ECs of Hdac2 ECKO (b) and Ezh2 ECKO (f) mice compared to WT controls. ( c, g ) Classification of differentially expressed genes (DEGs) into vascular development-related gene ontology (GO) terms associated with the observed phenotypes in Hdac2 ECKO (c) and Ezh2 ECKO (g) mice. ( d, h ) Subclassification of DEGs based on BBB-related functional categories in Hdac2 ECKO (d) and Ezh2 ECKO (h) mice. ( e, i ) Heatmap showing selected key DEGs categorized into genes related to angiogenesis, cell-cell junctions, transporters, transcription factors, and the Wnt signaling pathway in Hdac2 ECKO (e) and Ezh2 ECKO (i) mice. ( j ) Comparative analysis of the number of DEGs (upregulated and downregulated) that are unique or shared between Hdac2 ECKO and Ezh2 ECKO ECs. ( k ) Classification of unique DEGs in Hdac2 ECKO and Ezh2 ECKO ECs based on GO terms related to angiogenesis, BBB function, and Wnt signaling. ( l ) Top 5 biological processes and diseases associated with the unique upregulated and downregulated genes in Hdac2 ECKO and Ezh2 ECKO mice. For Hdac2 ECKO, additional relevant terms outside the top 5 are also displayed. No significant disease associations were found among the uniquely upregulated genes in Ezh2 ECKO mice.

Journal: bioRxiv

Article Title: Epigenetic Reactivation of CNS Endothelial Developmental Programs Triggers Adult Brain Angiogenesis, Promotes Post-Stroke Revascularization and Neuronal Regeneration

doi: 10.1101/2025.08.15.670554

Figure Lengend Snippet: ( a ) Schematic representation of the experimental design. Tamoxifen (75 mg/kg body weight) was administered to Hdac2 flox/flox : Cdh5CreERT2 or Ezh2 flox/flox : Cdh5CreERT2 mice at 8 weeks of age. After 8 weeks, live CD31+/CD41-/CD45-brain ECs were isolated by FACS sorting and subjected to ultra-low mRNA sequencing. ( b, f ) Differential gene expression analysis showing the number of significantly upregulated and downregulated genes (p ≤ 0.05 N=3/group) in cortical ECs of Hdac2 ECKO (b) and Ezh2 ECKO (f) mice compared to WT controls. ( c, g ) Classification of differentially expressed genes (DEGs) into vascular development-related gene ontology (GO) terms associated with the observed phenotypes in Hdac2 ECKO (c) and Ezh2 ECKO (g) mice. ( d, h ) Subclassification of DEGs based on BBB-related functional categories in Hdac2 ECKO (d) and Ezh2 ECKO (h) mice. ( e, i ) Heatmap showing selected key DEGs categorized into genes related to angiogenesis, cell-cell junctions, transporters, transcription factors, and the Wnt signaling pathway in Hdac2 ECKO (e) and Ezh2 ECKO (i) mice. ( j ) Comparative analysis of the number of DEGs (upregulated and downregulated) that are unique or shared between Hdac2 ECKO and Ezh2 ECKO ECs. ( k ) Classification of unique DEGs in Hdac2 ECKO and Ezh2 ECKO ECs based on GO terms related to angiogenesis, BBB function, and Wnt signaling. ( l ) Top 5 biological processes and diseases associated with the unique upregulated and downregulated genes in Hdac2 ECKO and Ezh2 ECKO mice. For Hdac2 ECKO, additional relevant terms outside the top 5 are also displayed. No significant disease associations were found among the uniquely upregulated genes in Ezh2 ECKO mice.

Article Snippet: Hdac2 flox/flox (Strain #:022625) , Ezh flox/flox (Strain #:022616), mice of both sexes were purchased from Jackson Laboratories.

Techniques: Isolation, Sequencing, Gene Expression, Functional Assay

(a) Diagram depicting Top 20 DEGs (upregulated and downregulated) in Hdac2 ECKO ECs based on fold change (padj ≤ 0.05) and associated top 10 biological processes (upregulated and downregulated). (b) Representative images of brain sections from WT and Hdac2 ECKO mice co-stained for Vegfr2, an angiogenic marker and laminin. Quantification revealed a significant increase in the density of vegfr2 (*p ≤ 0.05 vs. WT; WT: N=3, Hdac2 ECKO: N=3) in Hdac2 ECKO mice. Images were acquired at 10× magnification and merged using tile scanning. Scale bar: 100 µm. Quantifications were performed on matching sections from independent brains. Data is presented as mean ± SD. (c) Image showing Top 20 DEGs (upregulated and downregulated) in Ezh2 ECKO ECs based on fold change (padj ≤ 0.05) and associated top 10 biological processes (upregulated and downregulated).

Journal: bioRxiv

Article Title: Epigenetic Reactivation of CNS Endothelial Developmental Programs Triggers Adult Brain Angiogenesis, Promotes Post-Stroke Revascularization and Neuronal Regeneration

doi: 10.1101/2025.08.15.670554

Figure Lengend Snippet: (a) Diagram depicting Top 20 DEGs (upregulated and downregulated) in Hdac2 ECKO ECs based on fold change (padj ≤ 0.05) and associated top 10 biological processes (upregulated and downregulated). (b) Representative images of brain sections from WT and Hdac2 ECKO mice co-stained for Vegfr2, an angiogenic marker and laminin. Quantification revealed a significant increase in the density of vegfr2 (*p ≤ 0.05 vs. WT; WT: N=3, Hdac2 ECKO: N=3) in Hdac2 ECKO mice. Images were acquired at 10× magnification and merged using tile scanning. Scale bar: 100 µm. Quantifications were performed on matching sections from independent brains. Data is presented as mean ± SD. (c) Image showing Top 20 DEGs (upregulated and downregulated) in Ezh2 ECKO ECs based on fold change (padj ≤ 0.05) and associated top 10 biological processes (upregulated and downregulated).

Article Snippet: Hdac2 flox/flox (Strain #:022625) , Ezh flox/flox (Strain #:022616), mice of both sexes were purchased from Jackson Laboratories.

Techniques: Staining, Marker

( a ) Schematic representation of the top 20 unique genes and the top 10 biological terms associated with upregulated and downregulated genes in Hdac2 ECKO ECs, based on fold change and a significance threshold of padj ≤ 0.05. ( b ) Schematic representation of the top 20 unique genes and the top 10 biological terms associated with upregulated and downregulated genes in Ezh2 ECKO ECs, based on fold change and a significance threshold of padj ≤ 0.05.

Journal: bioRxiv

Article Title: Epigenetic Reactivation of CNS Endothelial Developmental Programs Triggers Adult Brain Angiogenesis, Promotes Post-Stroke Revascularization and Neuronal Regeneration

doi: 10.1101/2025.08.15.670554

Figure Lengend Snippet: ( a ) Schematic representation of the top 20 unique genes and the top 10 biological terms associated with upregulated and downregulated genes in Hdac2 ECKO ECs, based on fold change and a significance threshold of padj ≤ 0.05. ( b ) Schematic representation of the top 20 unique genes and the top 10 biological terms associated with upregulated and downregulated genes in Ezh2 ECKO ECs, based on fold change and a significance threshold of padj ≤ 0.05.

Article Snippet: Hdac2 flox/flox (Strain #:022625) , Ezh flox/flox (Strain #:022616), mice of both sexes were purchased from Jackson Laboratories.

Techniques:

(a) Illustration of experimental strategy. MCAO was induced in 8-weeks-old Hdac2 flox/flox : Cdhh5CreERT2 and Ezh2 flox/flox : Cdh5CreERT2 mice and TM was administered one hour after the completion of surgery (75mg/Kg body weight) for five days and mice were sacrificed on 7 th day for analysis. (b) Behavioral assessments using the Open Field Test (OFT), Barnes Maze Test (BMT), and Y Maze Test (YMT) revealed significant behavioral deficits in MCAO mice, with improvements observed following EC-specific deletion of Hdac2 or Ezh2 . In the OFT, MCAO mice displayed reduced total distance traveled, time spent in the center, and frequency of center entries compared to sham controls. Conversely, MCAO- Hdac2 ECKO and MCAO- Ezh2 ECKO mice exhibited significant improvements in these parameters, approaching sham levels. In the YMT, MCAO mice showed decreased total distance traveled, mean velocity, and zone alternation frequency relative to controls, while MCAO- Hdac2 ECKO and MCAO- Ezh2 ECKO mice demonstrated significant recovery in distance and velocity; MCAO- Hdac2 ECKO mice additionally showed enhanced zone alternation compared to controls. In the BMT, MCAO mice exhibited increased latency to locate the target hole compared to sham controls, whereas MCAO- Hdac2 ECKO and MCAO- Ezh2 ECKO mice showed no significant differences from sham or MCAO groups. Additionally, MCAO mice had reduced mean velocity at the center point, while MCAO- Hdac2 ECKO and MCAO- Ezh2 ECKO mice displayed significant increases compared to MCAO alone. (*p ≤ 0.05, **p ≤ 0.01 vs *** p≤ 0.001 vs sham, #p ≤ 0.05, ##p ≤ 0.01, ###p ≤ 0.001 vs MCAO. N=6/group). (c) Representative Nissl-stained brain sections from mice subjected to sham surgery, MCAO, MCAO- Hdac2 ECKO, and MCAO- Ezh2 ECKO, with infarct areas outlined by a black dotted line. Images were acquired at 10× magnification and reconstructed using tile-scanning microscopy. Scale bar 500µm. Quantification of the infarct area shows a significantly larger infarct in MCAO mice compared to sham, with significantly smaller infarcts in MCAO-Hdac2 ECKO and MCAO- Ezh2 ECKO mice relative to MCAO. (*p ≤ 0.05, ****p ≤ 0.0001 vs sham, ####p ≤ 0.0001 vs MCAO. N=6/group and matching sections were compared between 6 independent mice brains from each group) (d) BBB permeability assay using a 3 kDa FITC-dextran tracer demonstrates leakage into the brain parenchyma in MCAO brain compared to sham. In contrast, MCAO- Hdac2 ECKO and MCAO- Ezh2 ECKO mice exhibit no detectable leakage of the 3 kDa FITC-dextran tracer into the brain parenchyma. 10 x images are acquired and merged using tile scanning. Scale bar 500 µm. (****p ≤ 0.0001 vs sham, ####p ≤ 0.0001 vs MCAO N=6/group and N=6, matching sections were compared between 6 independent mice brains from each group). All data are presented as mean ± SD.

Journal: bioRxiv

Article Title: Epigenetic Reactivation of CNS Endothelial Developmental Programs Triggers Adult Brain Angiogenesis, Promotes Post-Stroke Revascularization and Neuronal Regeneration

doi: 10.1101/2025.08.15.670554

Figure Lengend Snippet: (a) Illustration of experimental strategy. MCAO was induced in 8-weeks-old Hdac2 flox/flox : Cdhh5CreERT2 and Ezh2 flox/flox : Cdh5CreERT2 mice and TM was administered one hour after the completion of surgery (75mg/Kg body weight) for five days and mice were sacrificed on 7 th day for analysis. (b) Behavioral assessments using the Open Field Test (OFT), Barnes Maze Test (BMT), and Y Maze Test (YMT) revealed significant behavioral deficits in MCAO mice, with improvements observed following EC-specific deletion of Hdac2 or Ezh2 . In the OFT, MCAO mice displayed reduced total distance traveled, time spent in the center, and frequency of center entries compared to sham controls. Conversely, MCAO- Hdac2 ECKO and MCAO- Ezh2 ECKO mice exhibited significant improvements in these parameters, approaching sham levels. In the YMT, MCAO mice showed decreased total distance traveled, mean velocity, and zone alternation frequency relative to controls, while MCAO- Hdac2 ECKO and MCAO- Ezh2 ECKO mice demonstrated significant recovery in distance and velocity; MCAO- Hdac2 ECKO mice additionally showed enhanced zone alternation compared to controls. In the BMT, MCAO mice exhibited increased latency to locate the target hole compared to sham controls, whereas MCAO- Hdac2 ECKO and MCAO- Ezh2 ECKO mice showed no significant differences from sham or MCAO groups. Additionally, MCAO mice had reduced mean velocity at the center point, while MCAO- Hdac2 ECKO and MCAO- Ezh2 ECKO mice displayed significant increases compared to MCAO alone. (*p ≤ 0.05, **p ≤ 0.01 vs *** p≤ 0.001 vs sham, #p ≤ 0.05, ##p ≤ 0.01, ###p ≤ 0.001 vs MCAO. N=6/group). (c) Representative Nissl-stained brain sections from mice subjected to sham surgery, MCAO, MCAO- Hdac2 ECKO, and MCAO- Ezh2 ECKO, with infarct areas outlined by a black dotted line. Images were acquired at 10× magnification and reconstructed using tile-scanning microscopy. Scale bar 500µm. Quantification of the infarct area shows a significantly larger infarct in MCAO mice compared to sham, with significantly smaller infarcts in MCAO-Hdac2 ECKO and MCAO- Ezh2 ECKO mice relative to MCAO. (*p ≤ 0.05, ****p ≤ 0.0001 vs sham, ####p ≤ 0.0001 vs MCAO. N=6/group and matching sections were compared between 6 independent mice brains from each group) (d) BBB permeability assay using a 3 kDa FITC-dextran tracer demonstrates leakage into the brain parenchyma in MCAO brain compared to sham. In contrast, MCAO- Hdac2 ECKO and MCAO- Ezh2 ECKO mice exhibit no detectable leakage of the 3 kDa FITC-dextran tracer into the brain parenchyma. 10 x images are acquired and merged using tile scanning. Scale bar 500 µm. (****p ≤ 0.0001 vs sham, ####p ≤ 0.0001 vs MCAO N=6/group and N=6, matching sections were compared between 6 independent mice brains from each group). All data are presented as mean ± SD.

Article Snippet: Hdac2 flox/flox (Strain #:022625) , Ezh flox/flox (Strain #:022616), mice of both sexes were purchased from Jackson Laboratories.

Techniques: Staining, Microscopy, Permeability

(a) Experimental strategy schematic. MCAO was induced in 8-week-old WT, Hdac2 flox/flox : Cdh5CreERT2 , and Ezh2 flox/flox : Cdh5CreERT2 mice. Tamoxifen (75 mg/kg) was given one hour post-surgery for 5 days. Mice were sacrificed on day 7 post-MCAO. Ipsilateral cortical ECs were isolated and FACS sorted (CD31 + /CD41 − /CD45 − ) for ultra-low-input mRNA sequencing. (b) RNA sequencing results showing the number of upregulated and downregulated genes (padj ≤ 0.05 N=3/group) in MCAO, MCAO- Hdac2 ECKO, and MCAO- Ezh2 ECKO mice relative to sham mice. (c) Enrichment analysis of upregulated and downregulated genes in MCAO mice, highlighting the top 10 associated biological processes. ( d ) Heatmap of the top 20 DEGs in MCAO mice, ranked by fold change (padj ≤ 0.05 N=3/group) relative to sham, with corresponding expression levels in MCAO-Hdac2 ECKO and MCAO- Ezh2 ECKO mice compared to sham. (e) Heatmap of DEGs in MCAO vs sham categorized by selected GO terms (angiogenesis, cell-cell junctions, transporters, transcription factors, Wnt signaling), with corresponding expression levels in MCAO- Hdac2 ECKO and MCAO- Ezh2 ECKO. All data are presented as mean ± SD.

Journal: bioRxiv

Article Title: Epigenetic Reactivation of CNS Endothelial Developmental Programs Triggers Adult Brain Angiogenesis, Promotes Post-Stroke Revascularization and Neuronal Regeneration

doi: 10.1101/2025.08.15.670554

Figure Lengend Snippet: (a) Experimental strategy schematic. MCAO was induced in 8-week-old WT, Hdac2 flox/flox : Cdh5CreERT2 , and Ezh2 flox/flox : Cdh5CreERT2 mice. Tamoxifen (75 mg/kg) was given one hour post-surgery for 5 days. Mice were sacrificed on day 7 post-MCAO. Ipsilateral cortical ECs were isolated and FACS sorted (CD31 + /CD41 − /CD45 − ) for ultra-low-input mRNA sequencing. (b) RNA sequencing results showing the number of upregulated and downregulated genes (padj ≤ 0.05 N=3/group) in MCAO, MCAO- Hdac2 ECKO, and MCAO- Ezh2 ECKO mice relative to sham mice. (c) Enrichment analysis of upregulated and downregulated genes in MCAO mice, highlighting the top 10 associated biological processes. ( d ) Heatmap of the top 20 DEGs in MCAO mice, ranked by fold change (padj ≤ 0.05 N=3/group) relative to sham, with corresponding expression levels in MCAO-Hdac2 ECKO and MCAO- Ezh2 ECKO mice compared to sham. (e) Heatmap of DEGs in MCAO vs sham categorized by selected GO terms (angiogenesis, cell-cell junctions, transporters, transcription factors, Wnt signaling), with corresponding expression levels in MCAO- Hdac2 ECKO and MCAO- Ezh2 ECKO. All data are presented as mean ± SD.

Article Snippet: Hdac2 flox/flox (Strain #:022625) , Ezh flox/flox (Strain #:022616), mice of both sexes were purchased from Jackson Laboratories.

Techniques: Isolation, Sequencing, RNA Sequencing, Expressing

(a) Graph depicting significantly upregulated and downregulated genes (padj ≤ 0.05) in contralateral cortical ECs from sham and WT MCAO mice. (b) Image highlighting top upregulated and downregulated DEGs (minimum 2-fold change, padj ≤ 0.05) in contralateral cortical ECs of MCAO mice. (c) List of the top 20 DEGs (upregulated and downregulated) in ipsilateral cortical ECs of MCAO- Hdac2 ECKO mice versus sham, ranked by fold change (pad ≤ 0.05). (d) List of the top 20 DEGs (upregulated and downregulated) in ipsilateral cortical ECs of MCAO- Ezh2 ECKO mice versus sham, ranked by fold change (padj ≤ 0.05). (e) Graph depicting significantly upregulated and downregulated genes (p≤ 0.05) in contralateral cortical ECs from sham and MCAO- Hdac2 ECKO mice and classification of differentially expressed genes (DEGs) into vascular development-related and BBB gene ontology (GO) terms. (f) Graph showing the number of significantly upregulated and downregulated genes (p ≤ 0.05) in cortical ECs of in contralateral cortical ECs from sham and MCAO- Ezh2 ECKO mice mice and classification of differentially expressed genes (DEGs) into vascular development-related and BBB gene ontology (GO) terms.

Journal: bioRxiv

Article Title: Epigenetic Reactivation of CNS Endothelial Developmental Programs Triggers Adult Brain Angiogenesis, Promotes Post-Stroke Revascularization and Neuronal Regeneration

doi: 10.1101/2025.08.15.670554

Figure Lengend Snippet: (a) Graph depicting significantly upregulated and downregulated genes (padj ≤ 0.05) in contralateral cortical ECs from sham and WT MCAO mice. (b) Image highlighting top upregulated and downregulated DEGs (minimum 2-fold change, padj ≤ 0.05) in contralateral cortical ECs of MCAO mice. (c) List of the top 20 DEGs (upregulated and downregulated) in ipsilateral cortical ECs of MCAO- Hdac2 ECKO mice versus sham, ranked by fold change (pad ≤ 0.05). (d) List of the top 20 DEGs (upregulated and downregulated) in ipsilateral cortical ECs of MCAO- Ezh2 ECKO mice versus sham, ranked by fold change (padj ≤ 0.05). (e) Graph depicting significantly upregulated and downregulated genes (p≤ 0.05) in contralateral cortical ECs from sham and MCAO- Hdac2 ECKO mice and classification of differentially expressed genes (DEGs) into vascular development-related and BBB gene ontology (GO) terms. (f) Graph showing the number of significantly upregulated and downregulated genes (p ≤ 0.05) in cortical ECs of in contralateral cortical ECs from sham and MCAO- Ezh2 ECKO mice mice and classification of differentially expressed genes (DEGs) into vascular development-related and BBB gene ontology (GO) terms.

Article Snippet: Hdac2 flox/flox (Strain #:022625) , Ezh flox/flox (Strain #:022616), mice of both sexes were purchased from Jackson Laboratories.

Techniques:

(a) Diagrammatic representation of experimental approach to generate EC-specific β-catenin gain of function (β-cat GOF) mice. TM was injected into 8-week-old adult Ctnnb1 (ex3)flox/flox : Cdh5CreERT2 mice for 5 days, and the brain was collected after 8 weeks for analysis. (b) Cortical vessels stained with laminin reveal comparable vascular density in β-catenin GOF and WT mice. BBB permeability assay with a 3 kDa FITC-dextran tracer shows no leakage into the brain parenchyma, similar to WT mice. Images were acquired at 10× magnification and merged via tile scanning. Scale bar: 500 µm. (N =4/group and matching sections were compared between 4 independent mice brains from each group) (c) Graph depicting DEGs (p ≤ 0.05 N=3/group) in cortical ECs of β-catenin GOF mice versus WT mice, with upregulated and downregulated genes classified across five key EC functions (angiogenesis, cell-cell junctions, transporters, transcription factors, Wnt pathway). (d) Heatmap of selected differentially expressed genes across five key EC functions in β-cat GOF mice versus WT mice. (e) Graph showing the number of Wnt-related genes in Hdac2 ECKO and β-cat GOF mice, along with the overlap in Wnt-related gene expression between the two groups. (f) Representative Nissl-stained brain sections from mice subjected to sham surgery, MCAO, and MCAO with β-cat GOF, with infarct areas outlined by black dotted lines. Quantification reveals significantly larger infarct areas in MCAO and MCAO-β-catenin GOF mice compared to sham (****p ≤ 0.0001 vs. sham; #p ≤ 0.05 vs. MCAO; N=6/group, matching sections were compared between 6 independent mice brains from each group). Images were acquired at 10× magnification and merged via tile scanning. Scale bar 500 µm. (g) BBB permeability assay using 3kDa tracer showing leakage into ipsilateral brain parenchyma in MCAO and MCAO with β-catenin GOF mice and the fluorescence intensity in brain parenchyma of WT-MCAO and MCAO with MCAO with β-cat GOF mice didn’t show any significant difference. 10 x images are acquired and merged using tile scanning. Scale bar= 500 µm. (****p ≤ 0.0001 vs sham, **p ≤ 0.01 vs sham. N=6/group and matching sections were compared between 6 independent mice brains from each group). All data are presented as mean ± SD.

Journal: bioRxiv

Article Title: Epigenetic Reactivation of CNS Endothelial Developmental Programs Triggers Adult Brain Angiogenesis, Promotes Post-Stroke Revascularization and Neuronal Regeneration

doi: 10.1101/2025.08.15.670554

Figure Lengend Snippet: (a) Diagrammatic representation of experimental approach to generate EC-specific β-catenin gain of function (β-cat GOF) mice. TM was injected into 8-week-old adult Ctnnb1 (ex3)flox/flox : Cdh5CreERT2 mice for 5 days, and the brain was collected after 8 weeks for analysis. (b) Cortical vessels stained with laminin reveal comparable vascular density in β-catenin GOF and WT mice. BBB permeability assay with a 3 kDa FITC-dextran tracer shows no leakage into the brain parenchyma, similar to WT mice. Images were acquired at 10× magnification and merged via tile scanning. Scale bar: 500 µm. (N =4/group and matching sections were compared between 4 independent mice brains from each group) (c) Graph depicting DEGs (p ≤ 0.05 N=3/group) in cortical ECs of β-catenin GOF mice versus WT mice, with upregulated and downregulated genes classified across five key EC functions (angiogenesis, cell-cell junctions, transporters, transcription factors, Wnt pathway). (d) Heatmap of selected differentially expressed genes across five key EC functions in β-cat GOF mice versus WT mice. (e) Graph showing the number of Wnt-related genes in Hdac2 ECKO and β-cat GOF mice, along with the overlap in Wnt-related gene expression between the two groups. (f) Representative Nissl-stained brain sections from mice subjected to sham surgery, MCAO, and MCAO with β-cat GOF, with infarct areas outlined by black dotted lines. Quantification reveals significantly larger infarct areas in MCAO and MCAO-β-catenin GOF mice compared to sham (****p ≤ 0.0001 vs. sham; #p ≤ 0.05 vs. MCAO; N=6/group, matching sections were compared between 6 independent mice brains from each group). Images were acquired at 10× magnification and merged via tile scanning. Scale bar 500 µm. (g) BBB permeability assay using 3kDa tracer showing leakage into ipsilateral brain parenchyma in MCAO and MCAO with β-catenin GOF mice and the fluorescence intensity in brain parenchyma of WT-MCAO and MCAO with MCAO with β-cat GOF mice didn’t show any significant difference. 10 x images are acquired and merged using tile scanning. Scale bar= 500 µm. (****p ≤ 0.0001 vs sham, **p ≤ 0.01 vs sham. N=6/group and matching sections were compared between 6 independent mice brains from each group). All data are presented as mean ± SD.

Article Snippet: Hdac2 flox/flox (Strain #:022625) , Ezh flox/flox (Strain #:022616), mice of both sexes were purchased from Jackson Laboratories.

Techniques: Injection, Staining, Permeability, Gene Expression, Fluorescence

(a) Illustration of study methodology. Hdac2 ECKO;β-catenin GOF double-mutant mice were generated by administering tamoxifen (75 mg/kg body weight) to 8-week-old Hdac2 flox/flox ; Ctnnb1 (ex3)flox/flox ; Cdh5CreERT2 mice daily for 5 days. Brains were harvested 4 weeks later for analysis. (b) isolectin B4 (IB4) staining and quantification demonstrating increased vascular density in Hdac2 ECKO:β-catenin GOF double mutant mice after one month compared to WT mice and one month Hdac2 ECKO mice. Representative images of BBB permeability assay using a 3 kDa-FITC tracer showed no significant BBB leakage in all groups. (**p ≤ 0.01 vs. WT #p ≤ 0.05 vs. Hdac2 ECKO. Matching sections were analyzed from independent mice from each group. WT N=4/group, Hdac2 ECKO N=3/group, and double mutant N=4/group). 10 x images are acquired and merged using tile scanning. Scale bar 500 µm. (c) Schematic of the experimental framework. Tamoxifen was administered to 8-week-old Hdac2 flox/flox ; Cdh5CreERT 2 mice to generate Hdac2 ECKO mice. At 8 weeks post-TM, mice were split into two groups: one injected with AAVBI30 -Flt1-eGFP , the other with AAVBI30 -Flt1- m -Hdac2-eGFP . Mice were sacrificed 8 weeks later, and samples were collected for analysis. Images show brain vessels stained with laminin (vessel marker) and GFP (viral expression), with merged images confirming viral vector expression primarily in brain vessels. Scale bar: 1 µm. (d) Representative images of IB4 staining and BBB permeability assay using 3 kDa-Cy3 tracer. Quantification of cortical brain vessels revealed significantly increased vessel density in Hdac2 ECKO mice with control vector and Hdac2 ECKO mice with Hdac2 re-expression vector compared to WT (*p ≤ 0.05, ****p ≤ 0.0001), though the Hdac2 re-expression group showed significantly reduced density versus the control vector group (##p ≤ 0.01). BBB leakage quantification showed no significant differences between groups. (N= 5/group for IB4; N=6/group for BBB assay. Matching sections were analyszed from independent mice from each group). Images were acquired at 10× magnification and merged via tile scanning. Scale bar: 500 µm. All data are presented as mean ± SD.

Journal: bioRxiv

Article Title: Epigenetic Reactivation of CNS Endothelial Developmental Programs Triggers Adult Brain Angiogenesis, Promotes Post-Stroke Revascularization and Neuronal Regeneration

doi: 10.1101/2025.08.15.670554

Figure Lengend Snippet: (a) Illustration of study methodology. Hdac2 ECKO;β-catenin GOF double-mutant mice were generated by administering tamoxifen (75 mg/kg body weight) to 8-week-old Hdac2 flox/flox ; Ctnnb1 (ex3)flox/flox ; Cdh5CreERT2 mice daily for 5 days. Brains were harvested 4 weeks later for analysis. (b) isolectin B4 (IB4) staining and quantification demonstrating increased vascular density in Hdac2 ECKO:β-catenin GOF double mutant mice after one month compared to WT mice and one month Hdac2 ECKO mice. Representative images of BBB permeability assay using a 3 kDa-FITC tracer showed no significant BBB leakage in all groups. (**p ≤ 0.01 vs. WT #p ≤ 0.05 vs. Hdac2 ECKO. Matching sections were analyzed from independent mice from each group. WT N=4/group, Hdac2 ECKO N=3/group, and double mutant N=4/group). 10 x images are acquired and merged using tile scanning. Scale bar 500 µm. (c) Schematic of the experimental framework. Tamoxifen was administered to 8-week-old Hdac2 flox/flox ; Cdh5CreERT 2 mice to generate Hdac2 ECKO mice. At 8 weeks post-TM, mice were split into two groups: one injected with AAVBI30 -Flt1-eGFP , the other with AAVBI30 -Flt1- m -Hdac2-eGFP . Mice were sacrificed 8 weeks later, and samples were collected for analysis. Images show brain vessels stained with laminin (vessel marker) and GFP (viral expression), with merged images confirming viral vector expression primarily in brain vessels. Scale bar: 1 µm. (d) Representative images of IB4 staining and BBB permeability assay using 3 kDa-Cy3 tracer. Quantification of cortical brain vessels revealed significantly increased vessel density in Hdac2 ECKO mice with control vector and Hdac2 ECKO mice with Hdac2 re-expression vector compared to WT (*p ≤ 0.05, ****p ≤ 0.0001), though the Hdac2 re-expression group showed significantly reduced density versus the control vector group (##p ≤ 0.01). BBB leakage quantification showed no significant differences between groups. (N= 5/group for IB4; N=6/group for BBB assay. Matching sections were analyszed from independent mice from each group). Images were acquired at 10× magnification and merged via tile scanning. Scale bar: 500 µm. All data are presented as mean ± SD.

Article Snippet: Hdac2 flox/flox (Strain #:022625) , Ezh flox/flox (Strain #:022616), mice of both sexes were purchased from Jackson Laboratories.

Techniques: Mutagenesis, Generated, Staining, Permeability, Injection, Marker, Expressing, Plasmid Preparation, Control

(a) Illustration of the research approach. 8-week-old WT, Hdac2 flox/flox ; Cdh5CreERT2 , and Ctnnb1 (ex3)flox/flox ; Cdh5CreERT2 mice underwent MCAO. One week post-MCAO, TM was administered daily for 5 days. 1 month after MCAO animals received 300 µg of EdU per injection over three consecutive days, with each series separated by a 10-day interval. This treatment regimen was repeated three times, and the mice were sacrificed the day after the final injection for analysis. (b) Nissl and NeuN-stained brain sections from sham, MCAO, MCAO- Hdac2 ECKO, and MCAO-β-cat GOF mice, with infarct areas marked with asterisk in Nissl-stained images. Nine weeks post-MCAO, Nissl staining revealed significant ipsilateral hemisphere loss in MCAO and MCAO-β-cat GOF mice compared to sham, as quantified by the ipsilateral/contralateral area ratio. NeuN immunostaining corroborated this loss, showing a reduced ratio of NeuN + cells in the ipsilateral/contralateral hemispheres. In contrast, MCAO- Hdac2 ECKO mice exhibited a significant increase in both the Nissl-stained ipsilateral/contralateral area ratio and the NeuN + cell ratio.(****p ≤ 0.0001 vs sham, ####p ≤ 0.0001 vs MCAO, $$$$p ≤ 0.0001 vs MCAO- Hdac2 -ECKO. N=3/group, matching sections form 3 independent brain) 10 x images are acquired and merged using tile scanning. Scale bar 500 µm. (c) Representative immunofluorescence images of laminin and Edu (green fluorescent dots) stained brain sections from MCAO and MCAO- Hdac2 ECKO mice. A digitally magnified view highlights Edu positive proliferating cells inside vessels within the core infarct region of MCAO- Hdac2 ECKO mice. Quantification of vascular density and Edu positive vessels in the ipsilateral hemisphere revealed significantly increased vessel density and proliferating vessels (*p ≤ 0.05, ****p ≤ 0.0001 vs MCAO; MCAO, N=3/group, and images were quantified from similar areas of matching sections from three independent brains) in MCAO- Hdac2 ECKO mice compared to MCAO controls. (d) BBB permeability assay using a 3 kDa FITC-conjugated tracer in Sham, MCAO, and MCAO- Hdac2 ECKO mice. Sham mice exhibited intact vessels with no detectable leakage, whereas MCAO brains showed significant tracer leakage in the infarct core. In contrast, MCAO- Hdac2 ECKO mice demonstrated no significant leakage. (****p ≤ 0.0001 vs. Sham; ####p ≤ 0.0001 vs. MCAO- Hdac2 ECKO; N=3/group, two matching sections were taken for analysis from 3 independent brains). Images were acquired at 10× magnification and merged using tile scanning. Scale bar = 500 µm. (e) Representative T2-weighted magnetic resonance imaging (MRI) of MCAO and MCAO- Hdac2 mice. Images depict MCAO and MCAO- Hdac2 mice at 7 days and 9 weeks post-surgery. The ratio of infarct volume at 7 days to the change in ipsilateral cortical volume between 7 days and 9 weeks was quantified in MCAO and MCAO- Hdac2 ECKO mice. Compared to MCAO, MCAO- Hdac2 ECKO brains exhibited a significantly higher ratio (*p ≤ 0.01 vs. MCAO; N = 3 per group; three matching sections per mouse were analyzed). All data are presented as mean ± SD.

Journal: bioRxiv

Article Title: Epigenetic Reactivation of CNS Endothelial Developmental Programs Triggers Adult Brain Angiogenesis, Promotes Post-Stroke Revascularization and Neuronal Regeneration

doi: 10.1101/2025.08.15.670554

Figure Lengend Snippet: (a) Illustration of the research approach. 8-week-old WT, Hdac2 flox/flox ; Cdh5CreERT2 , and Ctnnb1 (ex3)flox/flox ; Cdh5CreERT2 mice underwent MCAO. One week post-MCAO, TM was administered daily for 5 days. 1 month after MCAO animals received 300 µg of EdU per injection over three consecutive days, with each series separated by a 10-day interval. This treatment regimen was repeated three times, and the mice were sacrificed the day after the final injection for analysis. (b) Nissl and NeuN-stained brain sections from sham, MCAO, MCAO- Hdac2 ECKO, and MCAO-β-cat GOF mice, with infarct areas marked with asterisk in Nissl-stained images. Nine weeks post-MCAO, Nissl staining revealed significant ipsilateral hemisphere loss in MCAO and MCAO-β-cat GOF mice compared to sham, as quantified by the ipsilateral/contralateral area ratio. NeuN immunostaining corroborated this loss, showing a reduced ratio of NeuN + cells in the ipsilateral/contralateral hemispheres. In contrast, MCAO- Hdac2 ECKO mice exhibited a significant increase in both the Nissl-stained ipsilateral/contralateral area ratio and the NeuN + cell ratio.(****p ≤ 0.0001 vs sham, ####p ≤ 0.0001 vs MCAO, $$$$p ≤ 0.0001 vs MCAO- Hdac2 -ECKO. N=3/group, matching sections form 3 independent brain) 10 x images are acquired and merged using tile scanning. Scale bar 500 µm. (c) Representative immunofluorescence images of laminin and Edu (green fluorescent dots) stained brain sections from MCAO and MCAO- Hdac2 ECKO mice. A digitally magnified view highlights Edu positive proliferating cells inside vessels within the core infarct region of MCAO- Hdac2 ECKO mice. Quantification of vascular density and Edu positive vessels in the ipsilateral hemisphere revealed significantly increased vessel density and proliferating vessels (*p ≤ 0.05, ****p ≤ 0.0001 vs MCAO; MCAO, N=3/group, and images were quantified from similar areas of matching sections from three independent brains) in MCAO- Hdac2 ECKO mice compared to MCAO controls. (d) BBB permeability assay using a 3 kDa FITC-conjugated tracer in Sham, MCAO, and MCAO- Hdac2 ECKO mice. Sham mice exhibited intact vessels with no detectable leakage, whereas MCAO brains showed significant tracer leakage in the infarct core. In contrast, MCAO- Hdac2 ECKO mice demonstrated no significant leakage. (****p ≤ 0.0001 vs. Sham; ####p ≤ 0.0001 vs. MCAO- Hdac2 ECKO; N=3/group, two matching sections were taken for analysis from 3 independent brains). Images were acquired at 10× magnification and merged using tile scanning. Scale bar = 500 µm. (e) Representative T2-weighted magnetic resonance imaging (MRI) of MCAO and MCAO- Hdac2 mice. Images depict MCAO and MCAO- Hdac2 mice at 7 days and 9 weeks post-surgery. The ratio of infarct volume at 7 days to the change in ipsilateral cortical volume between 7 days and 9 weeks was quantified in MCAO and MCAO- Hdac2 ECKO mice. Compared to MCAO, MCAO- Hdac2 ECKO brains exhibited a significantly higher ratio (*p ≤ 0.01 vs. MCAO; N = 3 per group; three matching sections per mouse were analyzed). All data are presented as mean ± SD.

Article Snippet: Hdac2 flox/flox (Strain #:022625) , Ezh flox/flox (Strain #:022616), mice of both sexes were purchased from Jackson Laboratories.

Techniques: Injection, Staining, Immunostaining, Immunofluorescence, Permeability, Magnetic Resonance Imaging

(a) Six serial coronal images, arranged from rostral to caudal, depict representative MCAO and MCAO- Hdac2 mice at 7 days and 9 weeks post-surgery. Infarct regions appear as distinct T2-hyperintense areas in the brains of both groups at 7 days post-surgery.

Journal: bioRxiv

Article Title: Epigenetic Reactivation of CNS Endothelial Developmental Programs Triggers Adult Brain Angiogenesis, Promotes Post-Stroke Revascularization and Neuronal Regeneration

doi: 10.1101/2025.08.15.670554

Figure Lengend Snippet: (a) Six serial coronal images, arranged from rostral to caudal, depict representative MCAO and MCAO- Hdac2 mice at 7 days and 9 weeks post-surgery. Infarct regions appear as distinct T2-hyperintense areas in the brains of both groups at 7 days post-surgery.

Article Snippet: Hdac2 flox/flox (Strain #:022625) , Ezh flox/flox (Strain #:022616), mice of both sexes were purchased from Jackson Laboratories.

Techniques:

(a) Schematic representation of the experimental design. MCAO was induced in 8-week-old Hdac2 flox/flox : Cdh5CreERT2 mice, followed by TM injection for five days starting one week post-MCAO to delete Hdac2 . The experimental animals received 300 µg of EdU per injection over three consecutive days, with each series separated by a 10-day interval. This treatment regimen was repeated three times, and the mice were sacrificed the day after the final injection. (b) Representative images of EdU + cells (green fluorescent dots) in the dentate gyrus and subventricular zone, with quantification showing a significant increase in EdU + cells in MCAO- Hdac2 ECKO mice compared to MCAO (*p ≤ 0.05 vs. MCAO; MCAO N=3, MCAO- Hdac2 ECKO N=3). Scale bar: 500 µm. (c) Deletion of CNS EC Hdac2 after MCAO increased the number of proliferating neurons in the cortex. Immunofluorescent image of MCAO- Hdac2 ECKO cortex stained with NeuN and EdU. Areas within white dotted lines are digitally magnified to show the colocalization of EdU and NeuN. The number of EdU + /NeuN + cells in the cortical sections was quantified, showing a significant increase in MCAO- Hdac2 ECKO compared to MCAO (***p ≤ 0.001 vs. MCAO; N =3 /group; four matching sections per mouse were analyzed). 10x images were acquired and merged using tile scanning. Scale bar: 500 µm. (d) 40x images showing EdU + excitatory neurons (TBR1) in the cortex and SVZ (**p ≤ 0.01 vs. MCAO; N = 3/group; four matching sections per mouse were analyzed) and inhibitory neurons (Calbindin) in the cortex and SVZ (**p ≤ 0.01 vs. MCAO; N = 3/group; four matching sections were analyzed) of MCAO- Hdac2 ECKO. EdU + (green), TBR1 + (red), Calbindin + (red), and DAPI + (blue). Scale bar: 5 µm. (e) Representative images of TBR1 and Calbindin stained sections in sham, MCAO, and MCAO- Hdac2 ECKO. The ratio of TBR1 + and Calbindin + cells in the ipsilateral versus contralateral cortex was calculated, revealing a significant decrease in MCAO brains compared to sham, whereas MCAO- Hdac 2 ECKO brains exhibited a significant increase compared to MCAO. Additionally, TBR1 + cells in MCAO- Hdac2 ECKO brains showed a significant increase relative to sham. (*p ≤ 0.05, ****p ≤ 0.0001 vs. sham; ##p ≤ 0.01, ###p ≤ 0.001 vs. MCAO; N=3/group. Matching sections from each group were taken for analysis). 10x images were acquired and merged using tile scanning. Scale bar: 500 µm. All data are presented as mean ± SD.

Journal: bioRxiv

Article Title: Epigenetic Reactivation of CNS Endothelial Developmental Programs Triggers Adult Brain Angiogenesis, Promotes Post-Stroke Revascularization and Neuronal Regeneration

doi: 10.1101/2025.08.15.670554

Figure Lengend Snippet: (a) Schematic representation of the experimental design. MCAO was induced in 8-week-old Hdac2 flox/flox : Cdh5CreERT2 mice, followed by TM injection for five days starting one week post-MCAO to delete Hdac2 . The experimental animals received 300 µg of EdU per injection over three consecutive days, with each series separated by a 10-day interval. This treatment regimen was repeated three times, and the mice were sacrificed the day after the final injection. (b) Representative images of EdU + cells (green fluorescent dots) in the dentate gyrus and subventricular zone, with quantification showing a significant increase in EdU + cells in MCAO- Hdac2 ECKO mice compared to MCAO (*p ≤ 0.05 vs. MCAO; MCAO N=3, MCAO- Hdac2 ECKO N=3). Scale bar: 500 µm. (c) Deletion of CNS EC Hdac2 after MCAO increased the number of proliferating neurons in the cortex. Immunofluorescent image of MCAO- Hdac2 ECKO cortex stained with NeuN and EdU. Areas within white dotted lines are digitally magnified to show the colocalization of EdU and NeuN. The number of EdU + /NeuN + cells in the cortical sections was quantified, showing a significant increase in MCAO- Hdac2 ECKO compared to MCAO (***p ≤ 0.001 vs. MCAO; N =3 /group; four matching sections per mouse were analyzed). 10x images were acquired and merged using tile scanning. Scale bar: 500 µm. (d) 40x images showing EdU + excitatory neurons (TBR1) in the cortex and SVZ (**p ≤ 0.01 vs. MCAO; N = 3/group; four matching sections per mouse were analyzed) and inhibitory neurons (Calbindin) in the cortex and SVZ (**p ≤ 0.01 vs. MCAO; N = 3/group; four matching sections were analyzed) of MCAO- Hdac2 ECKO. EdU + (green), TBR1 + (red), Calbindin + (red), and DAPI + (blue). Scale bar: 5 µm. (e) Representative images of TBR1 and Calbindin stained sections in sham, MCAO, and MCAO- Hdac2 ECKO. The ratio of TBR1 + and Calbindin + cells in the ipsilateral versus contralateral cortex was calculated, revealing a significant decrease in MCAO brains compared to sham, whereas MCAO- Hdac 2 ECKO brains exhibited a significant increase compared to MCAO. Additionally, TBR1 + cells in MCAO- Hdac2 ECKO brains showed a significant increase relative to sham. (*p ≤ 0.05, ****p ≤ 0.0001 vs. sham; ##p ≤ 0.01, ###p ≤ 0.001 vs. MCAO; N=3/group. Matching sections from each group were taken for analysis). 10x images were acquired and merged using tile scanning. Scale bar: 500 µm. All data are presented as mean ± SD.

Article Snippet: Hdac2 flox/flox (Strain #:022625) , Ezh flox/flox (Strain #:022616), mice of both sexes were purchased from Jackson Laboratories.

Techniques: Injection, Staining

(a) Representative images of DCX-stained sections in MCAO and MCAO- Hdac2 ECKO. Areas within white dotted lines are digitally magnified to show DCX + cells in MCAO and MCAO- Hdac2 ECKO. 10x images were acquired and merged using tile scanning. Scale bar: 500 µm. 40x image from MCAO- Hdac2 ECKO mouse displaying the pattern of distribution of DCX + cells in the ipsilateral striatum. Scale bar: 50 µm. Quantification of the number of DCX + cells in the ipsilateral cortex, revealing a significant increase in MCAO- Hdac 2 ECKO brains compared to MCAO. (**p ≤ 0.01 vs. MCAO; N =3 /group; three matching sections per mouse were analyzed). (b) Graph displaying quantification of cortical thickness between ipsilateral and contralateral sides of MCAO- Hdac 2 ECKO brains. Cortical thickness was significantly increased in the ipsilateral sides of MCAO- Hdac 2 ECKO brains. (*p ≤ 0.05 vs. MCAO; N =3 /group; matching sections per mouse were analyzed).

Journal: bioRxiv

Article Title: Epigenetic Reactivation of CNS Endothelial Developmental Programs Triggers Adult Brain Angiogenesis, Promotes Post-Stroke Revascularization and Neuronal Regeneration

doi: 10.1101/2025.08.15.670554

Figure Lengend Snippet: (a) Representative images of DCX-stained sections in MCAO and MCAO- Hdac2 ECKO. Areas within white dotted lines are digitally magnified to show DCX + cells in MCAO and MCAO- Hdac2 ECKO. 10x images were acquired and merged using tile scanning. Scale bar: 500 µm. 40x image from MCAO- Hdac2 ECKO mouse displaying the pattern of distribution of DCX + cells in the ipsilateral striatum. Scale bar: 50 µm. Quantification of the number of DCX + cells in the ipsilateral cortex, revealing a significant increase in MCAO- Hdac 2 ECKO brains compared to MCAO. (**p ≤ 0.01 vs. MCAO; N =3 /group; three matching sections per mouse were analyzed). (b) Graph displaying quantification of cortical thickness between ipsilateral and contralateral sides of MCAO- Hdac 2 ECKO brains. Cortical thickness was significantly increased in the ipsilateral sides of MCAO- Hdac 2 ECKO brains. (*p ≤ 0.05 vs. MCAO; N =3 /group; matching sections per mouse were analyzed).

Article Snippet: Hdac2 flox/flox (Strain #:022625) , Ezh flox/flox (Strain #:022616), mice of both sexes were purchased from Jackson Laboratories.

Techniques: Staining

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