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. 2024 Jul 23;25(15):8008.
doi: 10.3390/ijms25158008.

Optimization of an Ischemic Retinopathy Mouse Model and the Consequences of Hypoxia in a Time-Dependent Manner

Inez Bosnyak  1 , Nelli Farkas  2 , Dorottya Molitor  1 , Balazs Meresz  1 , Evelin Patko  1 , Tamas Atlasz  1   3 , Alexandra Vaczy  1 , Dora Reglodi  1
Affiliations

Optimization of an Ischemic Retinopathy Mouse Model and the Consequences of Hypoxia in a Time-Dependent Manner

Inez Bosnyak et al. Int J Mol Sci. .

Abstract

The retina is one of the highest metabolically active tissues with a high oxygen consumption, so insufficient blood supply leads to visual impairment. The incidence of related conditions is increasing; however, no effective treatment without side effects is available. Furthermore, the pathomechanism of these diseases is not fully understood. Our aim was to develop an optimal ischemic retinopathy mouse model to investigate the retinal damage in a time-dependent manner. Retinal ischemia was induced by bilateral common carotid artery occlusion (BCCAO) for 10, 13, 15 or 20 min, or by right permanent unilateral common carotid artery occlusion (UCCAO). Optical coherence tomography was used to follow the changes in retinal thickness 3, 7, 14, 21 and 28 days after surgery. The number of ganglion cells was evaluated in the central and peripheral regions on whole-mount retina preparations. Expression of glial fibrillary acidic protein (GFAP) was analyzed with immunohistochemistry and Western blot. Retinal degeneration and ganglion cell loss was observed in multiple groups. Our results suggest that the 20 min BCCAO is a good model to investigate the consequences of ischemia and reperfusion in the retina in a time-dependent manner, while the UCCAO causes more severe damage in a short time, so it can be used for testing new drugs.

Keywords: hypoxia; ischemia; retina.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Optical coherence tomography (OCT) results and layers of the retina. The dots show the individual values, while the lines show the averages. Statistical analysis was performed with the linear random effect mixed model, and multiple comparisons were performed with Satterthwaite’s method. ˙ p < 0.1, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. change in the control group. UCCAO: unilateral common carotid artery occlusion. Control: n = 10; 10-min BCCAO: n = 12; 13-min BCCAO: n = 14; 15-min BCCAO: n = 14; 20-min BCCAO: n = 17; UCCAO: n = 25. (A) OCT results of inner retinal layers. The upper bracket shows the inner layers of the retina. (B) OCT results of middle retinal layers. The middle bracket indicates the middle layers of the retina. (C) OCT results of the outer part of the retina. The bottom bracket shows the outer retinal layers. (D) OCT results of total retinal thickness. (E) A representative OCT image. Scale bar: 202 µm. RNFL: retinal nerve fiber layer. IPL: inner plexiform layer. INL: inner nuclear layer. OPL: outer plexiform layer. ONL: outer nuclear layer. IS/OS: inner and outer segments of photoreceptor cells. RPE: retinal pigment epithelium.
Figure 2
Figure 2
Results of optical coherence tomography (OCT) measurements of the different retinal layers. The dots show the individual values, while the lines show the averages. Statistical analysis was performed with the linear random effect mixed model, and multiple comparisons were performed with Satterthwaite’s method. ˙ p < 0.1, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. change in control group. Control: n = 10; 10 min BCCAO: n = 12; 13 min BCCAO: n = 14; 15 min BCCAO: n = 14; 20 min BCCAO: n = 17; UCCAO: n = 25. The thickness of (A) nerve fiber layer; (B) retinal pigment epithelium; (C) whole photoreceptor cells; (D) outer segment of photoreceptor cells; (E) inner segment of photoreceptor cells.
Figure 3
Figure 3
Brn3a labeling of retinal ganglion cells. Statistical analysis was performed with Kruskal–Wallis ANOVA and uncorrected Dunn’s post hoc test. n = 3/group. (A) Representative images of ganglion cells from the different groups in the central region of the retina under 20× magnification. Scale bar: 50 µm. (B) Representative images of ganglion cells from the different groups in the peripheral region of the retina under 20× magnification. Scale bar: 50 µm. (C) Representative image of a whole mount retina preparation. Rectangle I. indicates the location of the images of the central region, while the rectangle II. shows the location of the images of the peripheral region. Scale bar: 100 µm. (D) Ganglion cell count in the peripheral regions of the retina. * p < 0.05 vs. control. Area = 0.344 mm2. (E) Number of ganglion cells in the central regions of the retina. * p < 0.05 vs. control. Area = 0.344 mm2.
Figure 4
Figure 4
(A) Representative images of GFAP staining on retinal whole mounts from the peripheral regions of the retina. UCCAO: unilateral common carotid artery occlusion. Scale bar: 10 µm (B) Representative images of GFAP labeling with DAPI counterstaining on cross sections from the same groups as above. INL: inner nuclear layer. ONL: outer nuclear layer. Scale bar: 10 µm (C) Results of Western blot measurements. Statistical analysis: one-way ANOVA, Dunnet’s post hoc test. * p < 0.05 vs. control. n = 4/group × 3 runs (D) A representative blot of GFAP expression with the same groups as above. GFAP: glial fibrillary acidic protein. GAPDH: internal control.
Figure 5
Figure 5
Illustration of the surgical procedure. Schematic drawing of the isolated right common carotid artery, with the vagus nerve next to it. (A) Representative picture of the bilateral common carotid artery occlusion (BCCAO). Both common carotid arteries are isolated and occluded with surgical microclips. (B) The right common carotid artery is occluded with two sutures and cut between the two sutures to avoid reperfusion.
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Grants and funding

This research work was conducted with the support of the National Academy of Scientist Education Program of the National Biomedical Foundation under the sponsorship of the Hungarian Ministry of Culture and Innovation (FEIF/646-4/2021- ITM_SZERZ). National Research, Development and Innovation Fund FK129190, K135457; PTE AOK-TANDEM, National Brain Research Program NAP3. HUN-REN TKI14016; and Higher Education Institutional Excellence Programme of the Ministry of Human Capacities in Hungary: TKP2021-EGA-16.

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