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 arteries. (B1, B2) Arterial suture ligation. (C1, C2) Insertion of the silicone wire embolus. The artery was incised to create a hole, and the silicone wire embolus was inserted. (D1, D2) Artery disconnection and movement of the silicone wire embolus. The artery was cut along the incision, and the silicone wire embolus was retracted and reinserted. (E1, E2) Removal of the silicone wire embolus and reperfusion. (F1, F2) Suture removal. The sutures at both ends of the disconnected vessel were knotted, and the other two sutures were removed. (G1, G2) Anatomic reduction and suturing of the skin. (H, I) Canavalin A label vasculature of the UPOAO mouse retina. The silicone wire embolus was inserted into artery before perfusing rhodamine-labeled canavalin A into the heart of UPOAO mouse. The sham eye served as an unpracticed control eye, while the UPOAO lateral eye represented the experimental eye. Retinal vessels in the sham eye (H) exhibited fluorescence filling, while retinal vessels in the UPOAO lateral eye (I) remained unfilled. Scale bar = 1 mm. (J and K) Fluorescein Fundus Angiography (FFA) performed before removing the silicone wire embolus from the UPOAO mouse. The vessels in the sham lateral retina (J) were perfused, while the lateral retinal perfusion in UPOAO (K) was delayed. (L) Schematic illustration of cervical artery anatomy and ocular blood supply. Embolization of the PPA resulted in ocular ischemia. The red arrow indicates the site of the silicone wire embolus occlusion. The silicone wire embolus used a type 602156 wire, extended to 7mm with a diameter of 0.21mm. The blue arrows indicate the modelling locations of the HIOP model and the UCCAO model, respectively. CCA: common carotid artery; ICA: internal carotid artery; ECA: external carotid artery; PPA: pterygopalatine artery; MCA: middle cerebral artery; Infraorbital art.: infraorbital artery; OA: ophthalmic artery; SPCA: short posterior ciliary artery; LPCA: long posterior ciliary artery; CRA: central retinal artery.

Antibodies Used in Staining of Flat-Mounted Retina and Sections

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-minute ischemia and 3-days reperfusion group. n = 3. (C, D) Representative pictures of the peripheral field (C), and quantification of surviving RGCs in all fields (D) in the 30-minute ischemia and 7-days reperfusion group. n = 3. (E, F) Representative pictures of the peripheral field (E), and quantification of surviving RGCs in all fields (F) in the 60-minute ischemia and 3-days reperfusion group. n = 5. (G, H) Representative pictures of the peripheral field (G), and quantification of surviving RGCs in all fields (H) in the 60-minute ischemia and 7-days reperfusion group. n = 5. The results showed a significant loss of RGCs after 60 minutes of ischemia. Data were presented as means ± s.e.m, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, t-test. Scale bar = 100 μm.

Comparison of Electroretinographic (ERG) Dark-Adapted Responses at Different Ischemia and Reperfusion Times.

Following the evaluation of surviving RGCs, visual function in sham and UPOAO experimental eyes at various ischemia and reperfusion times was assessed using ERG. (A) Representative waveforms in the four groups at the stimulus light intensities of 0.1, 1.0, and 10.0 cd.s/m2, respectively. (B) Quantification of a-wave and b-wave amplitudes in the 30-minute ischemia and 3-days reperfusion group. n = 5. (C) Quantification of a-wave and b-wave amplitudes in the 30-minute ischemia and 7-days reperfusion group. n = 5. (D) Quantification of a-wave and b-wave amplitudes in the 60-minute ischemia and 3-days reperfusion group. n = 7. (E) Quantification of a-wave and b-wave amplitudes in the 60-minute ischemia and 7-days reperfusion group. n = 8. Dark-adapted responses showed almost similar a-wave amplitudes but significantly decreased b-wave amplitudes in the 60-minute ischemia groups. The amplitudes of b-waves declined at 3-days and even more prominently at 7-days. (F, G) Representative OPs and quantification of amplitudes in the 60-minute ischemic groups. n = 7 in the 3-days reperfusion group; n = 8 in the 7-days reperfusion group. The amplitudes of OPs decreased significantly at 7-days reperfusion. The decline in b-waves and OPs along with the loss of RGCs, supports the selection of a 60-minute ischemic duration as an appropriate choice. Data were presented as means ± s.e.m, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, two-way ANOVA test for a-waves and b-waves; paired t-test for OPs.

Changes in Retina Morphology in the UPOAO Model.

(A) Representative 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 are annotated. GCC: ganglion cell complex, including RNFL, GCL and IPL layers. (B and C) Quantification of GCC and total retinal thickness at 3-days. The thickness of the GCC and the entire retina in OCT was measured and compared at distances of 1.5 PD, 3.0 PD and 4.5 PD from the optic disc, respectively. n = 5. (D) Representative OCT images of the mouse retina at 7-days. (E and F) Quantification of GCC and total retinal thickness at 7-days. n = 5. (G) Representative HE images of the mouse retina at 3-days. (H, I and J) Quantification of NFL + GCL, IPL, and INL thickness at 3-days. Retinal thickness in HE was measured near the optic nerve head and compared. n = 3. (K) Representative HE images of the mouse retina at 7-days. (L, M and N) Quantification of NFL + GCL, IPL, and INL thickness at 7-days. n = 3. Data were presented as means ± s.e.m, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, two-way ANOVA test in OCT and paired t-test in HE. PD: papillary diameters; RNFL: retinal nerve fiber layer; GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; IS: inner segment; OS outer segment; RPE: retinal pigment epithelium. Scale bar = 100 μm.

Changes in Bipolar Cells, Horizontal Cells, and Cholinergic Amacrine Cells in 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 fluorescence density at 3-days. (D) Quantification of PKCα fluorescence density at 7-days. n = 4. (E) Representative images of mouse retina co-stained with DAPI and Calbindin at 3-days. (F) Representative images of mouse retina co-stained with DAPI and Calbindin at 7-days. Horizontal cell somata are indicated by white asterisks. (G) Quantification of Calbindin fluorescence density at 3-days. (H) Quantification of Calbindin fluorescence density at 7-days. n = 3. (I) Representative images of mouse retina co-stained with DAPI and ChAT at 3-days. (J) Representative images of mouse retina co-stained with DAPI and ChAT at 7-days. Cholinergic amacrine cell somata are indicated by white asterisks. (K) Quantification of ChAT fluorescence density at 3-days. (L) Quantification of ChAT fluorescence density at 7-days. n = 3. Data were presented as means ± s.e.m, *: p<0.05, **: p<0.01, ***: p<0.001, t-test. Scale bar = 50 μm.

Transcriptomic Features at Different Times of Reperfusion.

RNA-seq evaluation was performed at 0-day, 3-days, and 7-days reperfusion periods in UPOAO revealing enrichment in pathways related to immune cells migration, oxidative stress, and immune inflammation. (A, D, G) Volcano plots display differential expression genes (DEGs) between UPOAO and sham eyes in the no-perfusion group (A), 3-days perfusion group (D), and 7-days perfusion group (G), respectively. Red dots: significant upregulated genes, green and blue dots: significant downregulated genes, grey dots: stable expressed genes, adjusted P < 0.05. log2FC = 1. (B, E, H) Gene ontology (GO) analysis of differential genes in the non-perfusion group (B), 3-days group (E), and 7-days group (H). The DEGs between UPOAO and sham eyes were enriched in pathways associated with immune cells migration (0d), oxidative stress (3d), and immune inflammation (7d), respectively. (C, F, I) Heatmap displaying the top 100 downregulated and top 100 upregulated DEGs between UPOAO and sham at non-perfusion, 3-days, and 7-days reperfusion, respectively. The box represents the genes related to immune cells migration (C), oxidative stress (F), and immune inflammation (I). The ranking was determined by the magnitude of fold change. In each heatmap, the upper box represents the top 10 downregulated genes, while the lower box represents the top 10 upregulated genes.

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 CD45+ cells in sham (A), 1-day perfusion group (B), 3-days perfusion group (C), and 7-days perfusion group (D). The presence of CD45+ cells within blood vessels is indicated by white arrows. Scale bar = 50 μm. CD45+ cell counts were performed in whole retinas of 1-day (E), 3-days (F), 7-days (G). The cellular morphology and distribution of microglial cells in the superficial retina were assessed in 3-days (H) and 7-days (I). Activated microglial cells are indicated by white asterisks. Microglial cell counting was conducted in the superficial retina of 3-days (J) and 7-days (K). Data points for CD45+ cells were derived from four flat-mounted retinas, and data points for microglial cells were from five flat-mounted retinas. Data were presented as means ± s.e.m, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, t-test. Scale bar = 50 μm.

Transcriptomic Results Comparison between UPOAO, HIOP, and UCCAO Models.

RNA-seq comparison between UPOAO and extravascular occlusion models: HIOP and UCCAO. (A) Schematic illustration of the HIOP model. (B) GO analysis of differential expression genes (DEGs) in the HIOP model at 7-days perfusion. (C) Venn diagram indicating the overlapping DEGs (146 genes) between HIOP and UPOAO models, as well as the remaining DEGs specific to each group (593 genes in each group). (D) GO analysis of the overlapping DEGs between HIOP and UPOAO models. (E) GO analysis of the remaining DEGs in the HIOP model at 7-days perfusion, excluding the overlapping DEGs. (F) GO analysis of the remaining DEGs, excluding the overlapping DEGs. (G) Heatmap showing the inter-sample distribution of lipid and steroid-related DEGs from the analysis of the remaining DEGs in the UPOAO model. (H) Schematic illustration of the UCCAO model. (I) GO analysis of DEGs in the UCCAO model. (J) Hub genes identified through protein-protein interaction (PPI) network analysis of the DEGs in the UCCAO model. (K) Venn diagram indicating the overlapping DEGs between UCCAO and UPOAO models (15 genes). (L) Venn diagram indicating the presence of two overlapping DEGs between UCCAO DEGs and m6A-related genes.

The Characteristic Features of UPOAO Model during Ischemia-Reperfusion Periods.

Response Times of a-Waves and b-Waves in 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-UPOAO in the 30-minutes ischemia group. n = 5. (C, D) Response times of a-waves and b-waves were measured under different light intensities at 3-days and 7-days post-UPOAO in the 60-minute ischemia group. n = 7 at 3-days; n = 8 at 7-days. Data were presented as means ± s.e.m, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, two-way ANOVA test.

Quantification of INL+OPL Thickness and ONL+IS/OS+RPE Thickness in OCT during 3-days and 7-days Reperfusion of UPOAO Animals.

The INL+OPL thickness and the ONL+IS/OS+RPE thickness were measured and compared at distances of 1.5 PD, 3.0 PD and 4.5 PD from the optic disc using OCT. (A and B) Quantification of INL+OPL layer thickness (A) and ONL+IS/OS+RPE layer thickness (B) at 3-days. n = 5. No significant differences were observed. (C and D) Quantification of INL+OPL layer thickness (C) and ONL+IS/OS+RPE layer thickness (D) at 7-days. n = 5. No significant differences were observed. Data were presented as means ± s.e.m, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, two-way ANOVA test.

Changes in Photoreceptor Cells in 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 presented as means ± s.e.m, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, t-test. Scale bar = 50 μm.

Hub Genes in PPI Analysis and Gene Set Enrichment Analysis (GSEA) during the Non-Reperfusion Stage in UPOAO.

(A) Hub genes identified through PPI analysis of DEGs in the non-reperfusion group. (B-D) GSEA analysis of the pathway of the cell killing (B), lymphocyte mediated-immunity (C), and leukocyte mediated-immunity (D) in the non-reperfusion group.

Hub Genes, GSEA Analysis at 3-days Reperfusion in UPOAO, and Relation with Mitochondrial Genes.

(A) Hub genes identified through PPI analysis of DEGs in the 3-days reperfusion group.

(B) GSEA analysis of the pathways related to plasma membrane protein complex (top left), bounding membrane of organelle (top right), and cell surface (bottom). (C) Venn diagram showing the overlapping genes between mitochondrial genes and DEGs at 3-days reperfusion (44 genes). (D) GO analysis of the 44 overlapping DEGs.

Hub Genes, GSEA Analysis at 7-days Reperfusion in UPOAO, and Relation with Immune Genes.

(A) Hub genes identified through PPI analysis of DEGs in the 7-days reperfusion group.

(B) GSEA analysis of the pathways related to the reactome innate immune system (top left), reactome immunoregulatory interactions between a lymphoid and a non-lymphoid cell (top right), reactome immune system (bottom left), and reactome adaptive immune system (bottom right). (C) Venn diagram showing the overlapping genes between immune genes and DEGs at 7-days reperfusion (112 genes). (D) GO analysis of the 112 overlapping DEGs.

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 differences. The data points were from the retina of four animals. Data were presented as means ± s.e.m, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, Paired t-test.

Co-Expressed Genes during 3-days and 7-days Reperfusion.

(A) Venn diagram showing the overlap of DEGs between the 3-days and 7-days reperfusion groups, along with a list of the 17 overlapping DEGs. (B) GO analysis of the 17 overlapping DEGs.

Primers Used in This Study.

Summary Table:

Time Course of all the Morphological, Functional, Cellular, and Transcriptome Changes in the UPOAO Model.