Optimizing Subretinal Bleb Formation for Visual Streak Involvement in a Porcine Model for Retinal Gene Therapy

Abstract

Purpose: Subretinal (SR) injection in porcine models is a promising avenue for preclinical evaluation of cell and gene therapies. Targeting of the subretinal fluid compartment (bleb) is critical to the procedure, especially if treatment of the cone-rich area centralis is required (i.e., visual streak [VS] in pigs). To our knowledge, this study is the first to investigate the influence of injection site placement on VS involvement in the pig eye.

Methods: We performed 23-gauge pars plana vitrectomy followed by SR injection in 41 eyes of 21 animals (Sus scrofa domesticus). In 27 eyes (65.9%), the injection site was placed superior to the VS, and in 14 eyes (34.1%) it was placed inferior to it. Using intraoperative imaging, blebs were classified based on their propagation behavior relative to the VS.

Results: In 79% of cases, blebs from inferior injection sites developed away from the VS, exhibiting a mean ± SEM vertical anisotropy (AP) of 0.67 ± 0.11. In contrast, blebs from superior injection sites tended to develop toward the VS with an AP of 1.27 ± 0.18 (P = 0.0070). Blebs developed away from the VS in only 41% of injections (P = 0.0212). Inferior blebs were orientated close to 0° (horizontal), whereas superior blebs displayed varied orientations with a mean angle of 56° (P = 0.0008).

Conclusions: Bleb propagation was anisotropic (i.e., directionally biased) and dependent on injection site placement. Superior injection sites led to superior VS detachment. Morphological analysis suggested increased adhesion forces at the VS and superior vascular arcades. This study will aid the planning of surgeries for targeted subretinal delivery in pig models.

Figure 1.

Anatomy of the central pig retina and area centralis. (A) Schematic drawing of the central pig retina. The VS (blue area), the cone-rich area centralis, is located just superior to the optic nerve head (yellow oval) and framed by the superior and inferior vascular arcades. The common trunk of the superior vascular arcades crosses the visual streak. (B) Corresponding intraoperative image showcasing the area centralis anatomy. Note the 41-gauge cannula primed for SR injection at a superior injection site.

Figure 2.

Injection site placement and bleb quantification. (A) Retinotomy site placement immediately superior and inferior to the visual streak. (B) Bleb quantification: The bleb base is approximated as an ellipse (continuous line). X denotes the retinotomy site. The distance between the retinotomy site and distal (P_dist) and proximal (P_prox) bleb margins is illustrated using colored arrows (P_prox, blue; P_dist, orange). For the depicted bleb, P_prox = P_dist. The vertical propagation anisotropy (A_V) is P_prox/P_dist = 1; that is, there is no preference for either distal or proximal propagation of the subretinal fluid that is being injected. As the long (A) and short (B) axes of the ellipse (not marked) are sufficiently different in length (i.e., a/b > 1.1), the long-axis tilt (dashed line) can be measured. The long-axis tilt of the depicted bleb is 0° (i.e., temporal–nasal, or horizontal). (C) Image of a bleb post injection. (D) Bleb quantification measurements for the bleb depicted in C.

Figure 3.

Sample bleb propagation over the course of injection by injection site. (A–D) Inferior injection site. The distance between the injection site and the distal and proximal bleb margins is marked with dashed lines. Distal bleb margin are indicated in orange; proximal bleb margin in blue. Early in the injection (A), immediately after the SR space was opened, the bleb was relatively balanced. Formally, the bleb was biased proximally (i.e., toward the visual streak). After injection of the first ∼50 µL (B), the bleb propagation balanced out. As more volume was injected (C, D), the bleb propagated increasingly away from the VS (final A_V = 0.35). (E, F) Superior injection site. Over the course of the injection, the bleb displayed a more balanced propagation. In the end, the bleb encompassed most of the VS (final A_V = 1.23).

Figure 4.

Average bleb morphology and vertical anisotropy by injection site. (A, B) Subretinal bleb propagation was anisotropic for both vertical anisotropy (A) and bleb area proximal versus distal of the VS (B). Placement of the injection site superior to the VS resulted in development of more balanced blebs, with a tendency to develop toward the VS. In contrast, placement inferior to the VS resulted in development of blebs away from the VS. This difference was highly statistically significant (A_V, P = 0.0070; area, P = 0.0112). Lines and error bars represent median ± interquartile ranges. Curves represent lognormal fit. All values were distributed normally (D'Agostino–Pearson, P > 0.05). ns, not significant). (C) The average bleb morphology for both injection sites. Superior injection sites showed superior VS detachment (mean A_V = 1.27) compared to inferior injection sites (mean A_V = 0.67). The bleb long-axis tilt was in line with the visual streak and the common trunk of the superior vascular arcades, acting as detachment barriers (superior 55° vs. inferior 0°).