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. 2023 Aug:233:109543.
doi: 10.1016/j.exer.2023.109543. Epub 2023 Jun 28.

Ex vivo ocular perfusion model to study vascular physiology in the mouse eye

Ahmed M Eltanahy  1 , Cristian Franco  1 , Priscilla Jeyaraj  1 , Shipra Goswami  1 , Elena Hughes  1 , Albert L Gonzales  2
Affiliations

Ex vivo ocular perfusion model to study vascular physiology in the mouse eye

Ahmed M Eltanahy et al. Exp Eye Res. 2023 Aug.

Abstract

Several hypotheses have been tested to understand whole organ regulation in other organs such as the brain and kidney, but no such hypothesis has yet been proposed for ocular circulations. To some extent resolve this deficit our ex vivo mouse eye perfusion model takes the first step in elucidating the mechanisms controlling the individual components of the ocular circulation. Various isolated ocular vascular preparations have been utilized in studies of ocular vascular biology, physiology, and pharmacology, including studies on both normal and pathological conditions. However, there is still significant potential for further studies to improve our understanding of ocular circulation and its regulation. The choroid specifically is inaccessible to direct visualization due to the retina's high metabolic requirement with a transparency that cannot be compromised by an overly rich vascular network on the inner retinal side hindering the visualization of the choroid. In this technical paper, we provide a detailed description of all the steps to be followed from the enucleation of mouse eyes to cannulation of the ophthalmic artery and perfusion and ex vivo confocal microscopy imaging of the dynamic nature of the choroid circulation.

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Figures

Figure 1
Figure 1. Microdissection steps of the Ophthalmic Artery.
After euthanasia, the skin is dissected from the skull (A) a longitudinal incision is made along the sagittal suture to yield a hemi-section of the skull (B) The orbital bone is carefully cut along the dashed line to separate the eyeball and its surround soft tissue (Video S1) (C). (D&E) Be careful not to dissect the double nerve bundle running along the medial border of the harderian gland (CN3) and the nervous tissue should be dissected with care to yield the isolated eye with its vascular supply intact starting from the ophthalmic artery. The dashed red lines in (D) represent the anatomical localization of the ophthalmic artery beneath the cranial nerves. (E) Shows the ligated axillary branches of the ophthalmic artery, The Extraocular Muscles (EOMs) are left intact. CN2 = 2nd cranial nerve, CN3 = 3rd cranial nerve, CN5= 5th cranial nerve. OA= Ophthalmic artery. Scale Bars: A & B= 2 mm, C= 1 mm, D&E= 500 uM.
Figure 2
Figure 2. Cannulation of the Ophthalmic Artery and experimental setup.
Anatomical illustration of the isolated mouse eye with its blood supply and cannulation point (A&B). (C) 10x imaging of an arterially perfused NG2-dsRed albino eye via a transscleral view, refer to video S6 for z stack rendering and intravascular lectin perfusion. (D&E) Experimental setup for ex-vivo confocal microscopy imaging setup showing an enucleated and arterially cannulated eye superfused with oxygenated and 37° C Physiological Ocular Perfusate buffer (Refer to table S1&S2 for supporting information). ON, Optic Nerve; CRA, Central Retinal Artery; EOM, Extraocular Muscles; OA, Ophthalmic artery; LPCA, Long Posterior Ciliary Artery; PCA, Posterior Ciliary Arteries; ACA, Anterior Ciliary Artery.
Figure 3
Figure 3. Structural and functional imaging of the choroid circulation.
(A) Isolectin perfused ex vivo eye preparation using 10x magnification (A) 60x magnification (i&ii). (B-D) Representative images (of 10 images for each group) classifying the structural heterogeneity of the choriocapillaris; finger-like pattern (B), maze-like pattern (C) and honeycomb like pattern (D) in the lectin perfused ex-vivo eye. Scale Bars: A= 200 μm, i&ii= 25 μm. (E) Ex-vivo functional Ca2+ imaging of the choriocapillaris showing spontaneous Ca2+ events (yellow arrowheads) using cdh5-Gcamp6f in our ex-vivo preparation (refer to Video S7). Scale Bar: B-D= 25 μm, E= 15 μm. IB4, Isolectin B4, SMCs= Smooth Muscle Cells.
Figure 4
Figure 4. Flow dynamics of the choriocapillaris.
(A) Representative images of the perfusion of 150 KD FITC-dextran in choriocapillaris in the arterially perfused ex vivo preparation. Flow of FITC-dextran increased at increasing perfusion pressures. Scale Bar: 20 μm. (B) Representative time-lapse images and bead tracking path summary (1, yellow) of a flowing 1μm fluorescent bead (yellow arrowhead and dashed circle) through the choriocapillaris. When tracking three separate beads entering the region of interests, we observed that the beads entered the capillary network from different vessels (1, 2, and 3) but exited through the same venule (v) suggesting that the presence of inter-capillary anastomosis. Scale Bar: 25 μm. (Refer to Video S4&S5).
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