An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Posterior approach transcleral subretinal injections were performed on 31 healthy eyes from 16 wild type mice with injections of 0.3 µl (n = 18), 0.5 µl (n = 8) and 1.0 µl (n = 5) of 0.01% fluorescein. One eye was excluded from injection due to a pre-existing corneal opacity that prevented structural and functional analysis. Every injected eye is included in this report. No unintended retinal detachments, punctures of the neurosensory retina, or leakage into the vitreous were detected nor was any evidence of lens nicking, inflammatory responses, uveitis, or post-surgical infections in any eyes observed.

Retinal structure was assessed pre-injection, 10 min post-injection and 4 weeks post-injection using OCT imaging (Figures 2 and 3). A 9-point grid, with the center point overlying the center of maximal detachment, was placed on the 10-min post-injection en face scan (Figure 2B). Using vascular landmarks, pre-injection and 4-week post-injection scans were rotated to be in registration with the 10-minute post-injection scan. This allowed identification of exactly the same locations in pre- and post- injection retinal scans (Figure 2A, C).

Subretinal injections resulted in bleb formation that was centered away from the injection site where the needle entered the subretinal space (Figure 2B arrow), and were either flat (Figure 3A) with a shallow detachment stretching over an extended area or domed (Figure 3B – D) with deep detachment in a constrained area (Table 1). A domed bleb was defined as any detachment that exceeded 50 µm orthogonal to the RPE. No rosettes, a wavy pattern produced by stretching of the outer retinal layers, were observed. The extent of injection deposition was visualized with en face OCT (Figure 2D, dashed line), fluorescein fundus imaging (Figure 2E) and OCT B-Scans (Figure 3). The majority of blebs (29 of 31) extended beyond the field of view of the OCT scans, which encompass approximately 10% of the mouse retina. There was no control over bleb shape other than increased frequency of domed blebs with greater injection volumes (Table 1). All blebs resolved by 2 weeks (data not shown).

Total retinal thickness (Bruch's membrane to the nerve fiber layer) was quantitatively assessed 4 weeks after injection for 0.3 µl (Figure 3A), 0.5 µl (Figure 3B) and 1.0 µl (Figure 3C) injection volumes at each point on the grid present in corresponding b-Scans (Table 1). The percent difference (%Δ) was calculated as the difference relative to the pre-injection measurements for both structural and functional metrics. Not all grid points were acquired during all imaging sessions. For example, 3 eyes had 1 or 2 points, 4 eyes had 3 or 4 points, and 23 eyes had 5 to 9 points in all scans. Domed and flat blebs, and injection volumes were collapsed since total retinal thickness was not significantly different for pre- and post-injections, although an overall trend towards retinal thinning was observed for both (Table 2).

Further analysis showed small but statistically significant thinning of 6.5% ± 1.9 overall (pre = 196 ± 1, n = 31; post = 183 ± 4, n = 31, T₆₀ = 3.4, p = 0.001) (Table 3). To determine where retinal thinning occurred, a subset of 10 eyes that contained thickness measurements for all 9 grid points was analyzed. Similar to the analysis for all eyes, the subset of eyes also showed small but significant overall retinal thinning (10.3% ± 3.5, pre = 199 ± 1, n = 10; post = 179 ± 8, n =10, T₁₈ = 2.4, p = 0.02). No retinal thinning was observed at sites distal to the injection site (7.2% ± 4.0, pre = 199 ± 2, n = 10; post = 185 ± 8, n = 10, T₁₈ = 1.7, p = 11). Retinal thinning was observed at the site of maximal retinal detachment (11.2% ± 5.0, pre = 201 ± 2, n = 10; post = 179 ± 10, n = 10, T₁₈ = 2.1, p = 0.04), the injection site without damage (13.8 ± 6.0, pre = 201 ± 1, n = 4; post = 173 ± 10, n = 4, T₆ = 2.8, p = 0.03), injection sites with retinal incarceration when a small region of the retina is pulled through the sclera upon needle withdrawal (14.1% ± 1.0, pre = 199 ± 1, n = 3; post = 171 ± 3, n = 3, T₄ = 8.9, p = 0.001) and injection sites with scarring (26.5% ± 1.6, pre = 200 ± 3, n = 3; post = 147 ± 10, n = 3, T₄ = 5.1, p = 0.007). In total, incarcerations occurred in 14 out of 31 injections, 3 of which resulted in scar formation of the choroid with loss of up to half of the outer retinal layers (Figure 3E). In each instance, however, these were highly localized effects.

Full-field electroretinography was performed prior to injection and repeated 4 weeks after injection with relative changes assessed individually for each eye to assess functional alterations (Table 1). Only dark-adapted and rod-mediated responses were recorded. Intensity response functions were fitted with Michaelis-Menten equations to derive Vmax for both photoreceptors (a-waves) and the middle retina (b-waves). Responses were not affected by bleb shape and were therefore collapsed (Table 4). There was a small but statistically significant decrease in Vmax on a-waves (F(1, 28) = 7.1, p = 0.013) but not b-waves (F(1, 28) = 4.0, p = 0.055) after injection (Table 4). Representative waveforms for pre- and post-injection of 0.3 µl, 0.5 µl and 1.0 µl are shown (Figure 4). There was an effect of injection volume on both a– and b-waves (F(2, 28) = 6.2, p = 0.006 and F(2, 28) = 8.8, p = 0.001, respectively).

Lastly, 3D modeling was used to visualize the structure of both domed and flat blebs. An example of a domed bleb (Figure 5A) shows a domed fluid-filled detachment contained within the scan area. Examples from flat blebs (Figure 5B, C) show shallow fluid-filled areas that extend beyond the region of the scan. When retinal incarceration occurs, a small hole in the reconstruction is seen (Figure 5C). Artificial boundaries are shown at the edge of a scan to allow the reconstruction (Figure 5C). Calculation of bleb volumes from these sample injections indicated that a minimum of 0.15 µl and 0.01 µl was successfully targeted to the subretinal space for 0.3 µl injections resulting in domed and flat blebs, respectively. The calculated volume injected likely underestimates the actual volume due to resolution of imaging and reconstruction, resorption of fluid during the procedure as occurs in human subretinal injections, and for flat or domed blebs particularly, if the entire detachment was not represented in the OCT scans.

Table 1. Functional and Structural Effects of Subretinal Injections by Eye. Metrics of individual eyes and their outcomes from subretinal injections. Retinal responses to light (middle retina b-waves and photoreceptor a-waves) and retinal thickness measurements (Bruch's membrane to nerve fiber layer) are given. Footnotes:
^aBT = Bleb Type, Flat (F) or Domed (D)
^b#gp = number of grid points measured
* Eyes used for reconstruction of injection volume.
** Only 1 grid point available for measurement at scar.
# Eyes with scar formation.

Table 2. Effect of Subretinal Injections on Total Retinal Thickness. Analysis of bleb shape and injection volume on retinal thickness.

Table 3. Effect of Damage on Retinal Structure. Analysis of site-dependent retinal thinning. Footnotes: *Analysis from 10 mice with data from all 9 grid points.

Table 4. Effect of Subretinal Injections on Scotopic Rod-mediated a– and b-waves. Analysis of bleb shape and injection volume on retinal responses to light (middle retina b-waves and photoreceptor a-waves).

Figure 1. Schematic Representation of Subretinal Injection. A top-down schematic of a mouse eye in the socket shows the approach in traditional subretinal injections, using a transcorneal (arrow a) or transcleral (arrow b) injection site near the pars plana. This method uses a transcleral approach near the posterior pole (arrow c), achieved by rotating the eye nasally to expose the posterior pole. Please click here to view a larger version of this figure.

Figure 2. Registration of OCT Images Allows Identification of Retinal Sites for Thickness Analysis. A-C, en face OCT image of mouse 1OS in original orientation. A) Image of retina pre-injection with fused registration grid. B) Image of retina 10 min post-injection. A 9-point grid was positioned with the center point at the site of maximal retinal detachment. Injection site is visible (arrow). C) Image of retina 4 weeks post-injection with fused registration grid. D-E, images of mouse 9OS. D) En face OCT image of retina 4 weeks post-injection showing extend of subretinal detachment. E) Overlay of fundus (green) and en face OCT images of retina 10 min post-injection. Scale bar = 100 µm. Please click here to view a larger version of this figure.

Figure 3. Subretinal Injections Cause Temporary Retinal Detachments with Minimal Retinal Thinning. Representative OCT B-Scans at the site of maximal retinal detachment are shown for pre-injection, 10 min post-injection and 4 weeks post-injection. A) Formation and resolution of a flat bleb from a 0.3 µl injection. B) Formation and resolution of a domed bleb from a 0.5 µl injection. C) Formation and resolution of a domed bleb from a 1.0 µl injection. D) Example of severe choroidal scarring and retinal thinning at injection site. Scale bar = 100 µm. Please click here to view a larger version of this figure.

Figure 4. Retinas Retain Normal Function After Bleb Resolution. Waveforms for scotopic rod-mediated responses are shown for pre-injection (black line) and 4 weeks post-injection (red dashed line) for A) 0.3 µl, B) 0.5 µl and C) 1.0 µl injections at 9 illumination intensities ranging from 4.37 x 10^-6 to 0.51 cd/m². Please click here to view a larger version of this figure.

Figure 5. 3D reconstruction of blebs. A) Software-generated 3D reconstructions of representative A) domed and B, C) flat blebs from 0.3 µl injections. Ribbing is an artifact of reconstruction software. Artificial boundaries were placed in C to allow reconstruction. Scale bar = 150 µm. Please click here to view a larger version of this figure.