Transcriptional responses in a mouse model of silicone wire embolization induced acute retinal artery ischemia and reperfusion
- Department of Ophthalmology, Renmin Hospital of Wuhan University, China
- Department of Clinical Laboratory, Institute of Translational Medicine, Renmin Hospital of Wuhan University, China
Figures
Figure 1
Modeling procedure, validation, and cervical artery anatomy.
(A1–G1) Schematic illustration of unilateral pterygopalatine ophthalmic artery occlusion (UPOAO). (A2–G2) Practical operation of UPOAO. (A1, A2) Blunt separation and exposure of the left cervical …
Figure 2
Staining and quantification of retinal ganglion cells (RGCs) at different ischemia and reperfusion times.
Flat-mounted retina RGCs were labeled with Brn3a staining. (A, B) Representative pictures of the peripheral field (A), and quantification of surviving RGCs in all fields (B) in the 30 min ischemia …
Figure 3 with 1 supplement
Comparison of electroretinographic (ERG) dark-adapted responses at different ischemia and reperfusion times.
Following the evaluation of surviving retinal ganglion cells (RGCs), visual function in sham and unilateral pterygopalatine ophthalmic artery occlusion (UPOAO) experimental eyes at various ischemia …
Figure 3—figure supplement 1
Response times of a-waves and b-waves in electroretinographic (ERG) at different light intensities.
(A, B) Response times of a-waves and b-waves were measured under different light intensities at 3 days and 7 days post-unilateral pterygopalatine ophthalmic artery occlusion (UPOAO) in the 30 min …
Figure 4 with 1 supplement
Changes in retina morphology in the unilateral pterygopalatine ophthalmic artery occlusion (UPOAO) model.
(A) Representative optical coherence tomography (OCT) images of the mouse retina at 3 days. The Green lines indicate the OCT scan area starting from the optic disc. Local magnification and layering …
Figure 4—figure supplement 1
Quantification of inner nuclear layer (INL) + outer plexiform layer (OPL) thickness and outer nuclear layer (ONL) + inner segment/outer segment (IS/OS) + retinal pigment epithelium (RPE) thickness in optical coherence tomography (OCT) during 3 days and 7 days Reperfusion of unilateral pterygopalatine ophthalmic artery occlusion (UPOAO) Animals.
The INL + OPL thickness and the ONL + IS/OS + RPE thickness were measured and compared at distances of 1.5 papillary diameters (PD), 3.0 PD, and 4.5 PD from the optic disc using OCT. (A, B) …
Figure 5 with 1 supplement
Changes in bipolar cells, horizontal cells, and cholinergic amacrine cells in unilateral pterygopalatine ophthalmic artery occlusion (UPOAO) mice.
(A) Representative images of mouse retina co-stained with DAPI and PKCα at 3 days (B) Representative images of mouse retina co-stained with DAPI and PKCα at 7 days. (C) Quantification of PKCα …
Figure 5—figure supplement 1
Changes in photoreceptor cells in unilateral pterygopalatine ophthalmic artery occlusion (UPOAO).
(A) Representative images of mouse retina co-stained with DAPI and Recoverin at 3 days. (B) Representative images of mouse retina co-stained with DAPI and Recoverin at 7 days. n=3. Data were …
Figure 6 with 5 supplements
Transcriptomic features at different times of reperfusion.
RNA-seq evaluation was performed at 0 day, 3 days, and 7 days reperfusion periods in unilateral pterygopalatine ophthalmic artery occlusion (UPOAO) revealing enrichment in pathways related to immune …
Figure 6—figure supplement 1
Hub genes in protein-protein interaction (PPI) analysis and gene set enrichment analysis (GSEA) during the non-reperfusion stage in unilateral pterygopalatine ophthalmic artery occlusion (UPOAO).
(A) Hub genes identified through PPI analysis of differential expression genes (DEGs) in the non-reperfusion group. (B–D) GSEA analysis of the pathway of the cell killing (B), lymphocyte …
Figure 6—figure supplement 2
Hub genes, gene set enrichment analysis (GSEA) analysis at 3 days reperfusion in unilateral pterygopalatine ophthalmic artery occlusion (UPOAO), and relation with mitochondrial genes.
(A) Hub genes were identified through protein-protein interaction (PPI) analysis of differential expression genes (DEGs) in the 3 days reperfusion group. (B) GSEA analysis of the pathways related to …
Figure 6—figure supplement 3
Hub Genes, gene set enrichment analysis (GSEA) analysis at 7 days reperfusion in unilateral pterygopalatine ophthalmic artery occlusion (UPOAO), and relation with immune genes.
(A) Hub genes identified through protein-protein interaction (PPI) analysis of differential expression genes (DEGs) in the 7 days reperfusion group. (B) GSEA analysis of the pathways related to the …
Figure 6—figure supplement 4
Upregulation of immune inflammation-related gene expression in the 7 days reperfusion group.
The genes Abca1, Cd86, Cd48, Tlr4, Tlr6, and Tspo were significantly upregulated in the 7 days reperfusion group, while other immune-inflammatory and chemotaxis-related genes showed no significant …
Figure 6—figure supplement 5
Co-expressed genes during 3 days and 7 days reperfusion.
(A) Venn diagram showing the overlap of differential expression genes (DEGs) between the 3 days and 7 days reperfusion groups, along with a list of the 17 overlapping DEGs. (B) Gene ontology (GO) …
Figure 7
Peripheral leukocyte infiltration and retinal resident microglial activation.
Rhodamine-labeled canavalin A was used for immediate cardiac perfusion to visualize blood vessels, followed by CD45 immunofluorescent staining to observe the relationship between blood vessels and …
Figure 8
Transcriptomic results comparison between unilateral pterygopalatine ophthalmic artery occlusion (UPOAO), high intraocular pressure (HIOP), and unilateral common carotid artery occlusion (UCCAO) models.
RNA-seq comparison between UPOAO and extravascular occlusion models: HIOP and UCCAO. (A) Schematic illustration of the HIOP model. (B) Gene ontology (GO) analysis of differential expression genes …
Figure 9
The characteristic features of unilateral pterygopalatine ophthalmic artery occlusion (UPOAO) model during ischemia-reperfusion periods.
Author response image 1
Thickness of OPL, ONL, IS/OS+RPE in HE staining.
n=3; ns: no significance (p>0.05).
Author response image 2
Cited from Ji, K., et al., Resveratrol attenuates retinal ganglion cell loss in a mouse model of retinal ischemia reperfusion injury via multiple pathways.
Experimental Eye Research, 2021. 209: p. 108683.
Author response image 3
Schematic diagram illustrating the selection of regions.
The figure was captured using a fluorescence microscope (BX63; Olympus, Tokyo, Japan) under a 4X objective. Scale bar=500 µm.
Author response image 4
Cited from Feng, L., et al., Ripa-56 protects retinal ganglion cells in glutamate-induced retinal excitotoxic model of glaucoma. Sci Rep, 2024. 14(1): p. 3834.
Videos
Video 1
Blunt dissection of cervical vessels.
The neck was exposed, and the cervical arteries were separated using blunt dissection techniques.
Video 2
Insertion of silicone wire embolus and induction of ischemia.
The silicone wire embolus was inserted into the PPA, initiating the ischemic condition. PPA: the pterygopalatine artery.
Video 3
Extraction of silicone wire embolus and reperfusion.
The silicone wire embolus was removed, allowing reperfusion to occur. The mouse’s wound was sutured, and standard feeding procedures were followed.
Tables
Table 1
Antibodies used in staining of flat-mounted retina and sections.
| Antibodies | Species | Dilution | Company |
|---|---|---|---|
| PKCα | rabbit | 1:1000 | Sigma |
| Recoverin | rabbit | 1:1000 | Millipore |
| Calbindin | rabbit | 1:5000 | SWANT |
| ChAT | goat | 1:200 | Millipore |
| Iba1 | rabbit | 1:500 | Wako |
| CD45 | rabbit | 1:100 | CST |
| Rhodamine-labeled canavalin A | / | 1:125 | MKBio |
| Goat anti-mouse IgG Alexa Fluor 488 | goat | 1:500 | Jackson |
| Donkey anti-rabbit IgG Alexa Fluor 594 | donkey | 1:500 | Jackson |
| Donkey anti-goat IgG Alexa Fluor 488 | donkey | 1:500 | Jackson |
Additional files
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Supplementary file 1
Summary table.
Time course of all the morphological, functional, cellular, and transcriptome changes in the UPOAO model.-: no change; ↑: increase; ↓: decrease; /: not involved in the article; #: supplementary content: also increase after 60 min-ischemia and 1d-reperfusion.
- https://cdn.elifesciences.org/articles/98949/elife-98949-supp1-v1.docx
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Supplementary file 2
Primers used in this study.
- https://cdn.elifesciences.org/articles/98949/elife-98949-supp2-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/98949/elife-98949-mdarchecklist1-v1.docx
