Subretinal administration of SNP induced controllable, focal retinal degeneration in cynomolgus monkeys
To overcome the lethal side effect of systemic delivery of retinotoxic reagents or random or uneven distribution of the retinal lesion after their intravitreal delivery[13], and to facilitate the study of potential therapeutic interventions, such as stem cell grafts and retinal prosthesis, we subretinally injected 100 μL of SNP solutions at three doses (0.05 mM, 0.1 mM and 0.2 mM) or NS into the posterior pole of the monkey retina next to the fovea under a surgical microscope (Figure 1A). Within 1 h after SI, SD-OCT images showed that SNP solutions or NS caused a hypo-reflective retinal bleb of about 9 mm in diameter, indicating the successful delivery (Figure 1B). In 14 cynomolgus monkeys (28 eyes) studied, 6 eyes received 0.05 mM SNP, 14 eyes 0.1 mM SNP, 5 eyes 0.2 mM SNP and 3 eyes NS, respectively. During the observation period after administration, with SD-OCT and fundus photographs, the focal lesions with various degree of severity were clearly noticed and consistently located in the posterior pole of retina in all animals including NS group. The other area of retina in the monkeys was not noticeably affected by the treatment (Figure 1C-D). All monkeys were in good health without systemic side effect or death. However, due to the unexpected injury to lens and retinal vessels in operation, complications in a few eyes were observed, including cataract (2 eyes), vitreous hemorrhage (1), retinal tear and detachment (1), and endophthalmities (1).
SNP caused acute retinal degeneration of cynomolgus monkeys in a dose-dependent manner
With the advantage of the noninvasive and time-saving in vivo measurements of retinal layers over histological test, SD-OCT was employed to dynamically evaluate the damage severity of retinal structures after the SNP treatment in the monkeys. The SI of NS or SNP solutions in three doses led to acute local retinal injury with various severities from slight, mild, moderate to severe alterations within 28 days after administration (Figure 2 A-D). In the vehicle control group, NS (3/3, 100 %) caused a slight injury with slight hyper-reflection of retinal pigment epithelium (RPE) and photoreceptor segments, sign of the cell swelling. In 0.05 mM SNP group, the majority of eyes (5/6, 83 %) presented mild injury of retina lesions with swelling, disorganization or loss of RPE, segments and part of outer nuclear layer (ONL), and 1 eye (1/6, 17 %) had severe retinal lesions with disruption of most retina and choroid. In 0.1 mM SNP group, 11 out of 14 eyes (11/14, 79 %) presented moderate injury of retina lesions with evident swelling and/or loss of RPE and outer neural retina layer, 2 out of 14 eyes (2/14, 14 %) mild and 1 out of 14 eyes (1/14, 7 %) severe. In 0.2 mM SNP group, 4 out of 5 eyes (4/5, 80 %) presented severe injury of retina lesions with the loss of most of the neural retina and RPE accompanied by the involvement of choroid, and 1 out of 5 eyes (1/5, 20 %) moderate. The time course images of SD-OCT revealed the pathological changes of cynomolgus monkey retinas over the 28-day observation period (Figure 3). The tissue swelling indicated by the hyper-reflection of retinas was marked in all SNP groups on day 7 (D7) after administration, then gradually disappeared till D28. The cell loss and disorganization of part or all of outer retinal layers were seen on D7 and thereafter in SNP treatment groups. Compared to the NS group, all of three doses of SNP caused significant reduction of the retinal thickness (from OPL to BM, and from ILM to BM) of cynomolgus monkeys over time, with the 0.2 mM SNP being the most significant (Figure 4).
Functional examination with mfERG was performed in cynomolgus monkey eyes with NS and 0.1 mM SNP SI (Figure 5 and see Additional file 1). NS SI did not cause noticeable changes in the amplitudes of P1 (Amp. P1) between pretreatment (amplitude 36.8 ± 8.1 nV/deg²) and D7 after treatment (amplitude 31.8 ± 8.2 nV/deg², P = 0.49; Figure 5A, C), implying the SI approach itself did not cause evident functional change of the retina. However, 0.1 mM SNP significantly reduced the responses since D7 after treatment (amplitude 13.6 ± 5.6 nV/deg², P = 0.01), compared to the pretreatment (amplitude 33.1 ± 5.0 nV/deg², Figure 5B-D).
SNP-induced stable and long-lasting retinal degenerations in cynomolgus monkeys
To determine the long-term effect of SNP SI on retina, some monkeys were followed up for more than 5 months after the treatment. Multimodal imaging (BAF, IR, FA and ICGA) performed in the 5th month disclosed focal lesions surrounded by relatively normal retina in the SNP groups of three doses, but no obvious damages in the NS control group. The size and shape of these damaged lesions were similar to those described above (Figure 6). Both FA and ICGA showed that fluorescence leakage and tissue staining were obvious in the damaged area with the fluorescence intensity related to the degree of retinal damage in a concentration-dependent manner, while no leakage and tissue staining found in the control group (Figure 6).
In the 7th month after treatment, mfERG was also performed in the cynomolgus monkey eyes with 0.1 mM SNP SI. Compared to the results of the pretreatment and on the 14th days after treatment, the Amp. P1 in the 7th month were significantly reduced (see Additional file 2), indicating the SNP administration caused permanent dysfunction of retina.
Histological examination in the 7th month after SNP treatment confirmed that SNP SI caused focal retinal degeneration of cynomolgus monkeys in a dose-dependent manner, which was consistent with the results of SD-OCT described above (Figure 7). 0.05 mM SNP did not cause remarkable structural changes in retina and choroid; 0.1 mM SNP caused the depletion of outer neural layer including RPE, ONL and outer plexiform layer (OPL); while 0.2 mM SNP destroyed the entire retina and choroid. Immunostaining with the antibody recoverin revealed that photoreceptors were clearly eliminated by 0.1 mM or 0.2 mM SNP, but were almost not affected by 0.05 mM SNP.