Human NGF “Painless” Ocular Delivery for Retinitis Pigmentosa: An In Vivo Study

Intravitreal delivery of hNGFp has a moderate effect on cone survival in RP mice. A, Experimental design used for the single intravitreal injection. The animals were divided into two groups as for the previous experiments, control (ctr) and hNGFp-treated (treat) and once intravitreal injected with PBS (ctr) or hNGFp (treat) at age P50; hNGFp dosage, 0.54 ng/gr. B, Whole-mount retinas of control (left) and treated mice (right) stained with cone arrestin (red signal). The dotted lines define the degenerated area. C, qPCR quantification of genes involved in photoreceptors survival and microglial activation. Gnat2 (ctr = 8, treat = 9; p = 0.050) and Arr3 (ctr = 5, treat = 6; p = 0.184) are genes expressed in rods and cones, ntrk1 (ctr = 5, treat = 6; val = 0.003) is the gene for trkA expression while ngfr (ctr = 8, treat = 9; p = 0.327) is the one for p75NTR receptor expression. Gfap (ctr = 5, treat = 6; p = 0.749) refers to macroglial activation while tmem119 (ctr = 5, treat = 6; p = 0.007) represent the activation of microglia. Arg1 (ctr = 8, treat = 9; p = 0.552) and nos2 (ctr = 8, treat = 9; p = 0.916) are, respectively, genes expressed by anti-/inflammatory and pro-/inflammatory microglia. Gene expression is reported as the ratio between the ctr and treat animals. Normalization was done on actin expression for each sample. D, Degenerated area quantification expressed as mm2 (ctr = 3, treat = 3; p = 0.153) and percentage (ctr = 3, treat = 3; p = 0.146) with respect to the total retinal area. E, Quantification of cone arrestin signal. Absolute cone number per retina (ctr = 7, treat = 8; p = 0.835); cone density (ctr = 7, treat = 8; p = 0.723); and retinal area (ctr = 7, treat = 8; p = 0.729). Error bars are ±SEM. t test was used to compare the ctr versus treat mean for each experiment.

Retinal thickness is not rescued by intravitreal delivery of hNGFp in RP mice. A, B, Representative images of the ctr (left) and treated (right) retinal section used to measure the thickness of the ONL. C, D, zoom of retinal sections. E, Statistical analysis of ONL measurements shows no changes in thickness upon hNGFp administration (ctr = 4, treat = 5: p = 0.495). Error bars are ±SEM.

Intravitreal delivery of hNGFp shows partial immunomodulation of retinal inflammation in RP mice. A, Whole-mount retinas of the control (left) and treated mice (right) stained with CD11b (green signal). The dotted lines define the degenerated area. A and B are zoom images of retinal microglia in the peripheral and central area (B) qPCR quantification of genes involved in inflammatory responses. Myd88 (ctr = 8, treat = 9; p = 0.019) and csf1 (ctr = 5, treat = 6; p = 0.782) are transcriptional factors involved in the activation of pro- and anti-inflammatory response. Lamp2 (ctr = 5, treat = 6; p = 0.0005) is a protein involved in the autophagy process. Nr3c1 (ctr = 5, treat = 6; p = 0.002) codifies for glucocorticoid receptor expression. Ccl12 ctr = 5, treat = 6; p = 0.250), ccl2 (ctr = 5, treat = 6; p = 0.928) and cxcl5 (ctr = 5, treat = 6; p = 0.861) are chemokines and cytokines.

Increased dosage of hNGFp intravitreal delivered has no beneficial effects on cone survival and retinal inflammation of RP mice. A, Whole-mount retinas of control (left) and treated mice (right) stained with cone arrestin (red signal). The dotted lines define the degenerated area. B, qPCR quantification of genes involved in photoreceptors survival and microglial activation. Gnat2 (ctr = 7, treat = 8; p = 0.042) and Arr3 (ctr = 7, treat = 8; p = 0.486) are genes expressed in rods and cones, ntrk1 (ctr = 7, treat = 8; p = 0.050) is the gene for trkA expression while ngfr (ctr = 7, treat = 8; p = 0.092) is the one for p75NTR receptor expression. Gfap (ctr = 7, treat = 8; p = 0.500) refers to macroglial activation while tmem119 (ctr = 7, treat = 8; p = 0.270) represent the activation of microglia. Arg1 (ctr = 7, treat = 8; p = 0.831) and nos2 (ctr = 7, treat = 8; p = 0.415) are genes expressed by microglial cells. C, Degenerated area quantification expressed as mm2 (ctr = 6, treat = 7; p = 0.698) and percentage (ctr = 6, treat = 7; p = 0.858) with respect to the total retinal area. D, Quantification of cone arrestin–positive cells. Absolute cone number per retina (ctr = 7, treat = 8; p = 0.221); cone density (ctr = 7, treat = 8; p = 0.316); and retinal area (ctr = 7, treat = 8; p = 0.519). Error bars are ±SEM. t test was used to compare the ctr versus treat mean for each experiment.

wtNGF and hNGFp treatments show no differences in cone survival. Cone counts in retinal preparations from rd10 mice injected intravitreally with wtNGF (n = 4), hNGFp (n = 6), and control solution (n = 6). Error bars are ±SEM. One-way ANOVA test. p = 0.7087.

Intranasal delivery of hNGFp has no effect on cone rescue in RP mice. A, Graphical representation of the experimental protocol adopted for the treatment. The animals were divided into two groups, control (ctr) and hNGFp treated (treat), and manipulated for 1 week before the start of the experimental protocol. Finally, they were intranasally treated with drops of PBS (ctr) or hNGFp (treat) 3 d/week from age P25 to P45 (left panel) or P60 (right panel). hNGFp dosage, 0.54 ng/gr. B, C, Volcano plots of qPCR array show no changes of gene expression related to photoreceptor survival and the inflammatory pathway. The dots represent each tested gene with its mean fold change and the statistical value (-log10pval). The red and green dots are, respectively, the downregulated and upregulated genes in the treated animals with respect to the control mice. The dotted line represents the significance threshold [-log10(0.05) = 1.33.]. Volcano plot in B, N:(ctr = 7, treat = 7), and in C, N:(ctr = 6, treat = 6). D, Whole-mount image (left side) of a rd10 retina (aged P45) stained with cone arrestin (red signal) showing how the acquisition for cone counting was done. The 16 dotted squares represent the investigated retinal areas for each animal while the white squares in A and B show zoom images of central and peripheral cone arrestin signals. E, Quantification of cone arrestin signal. Absolute cone number per retina (ctr = 4, treat = 4; p = 0.399); cone density (ctr = 4, treat = 4; p = 0.582); and retinal area (ctr = 4, treat = 4; p = 0.781). Each dot represents a different animal. Error bars are ±SEM.

RPE analysis after intranasal delivery of hNGFp does not demonstrate structural amelioration. The administration failed to elicit visible effects on the morphology of the RPE, here shown upon immunolabeling with antibodies against ZO-1, a protein of the RPE tight junctional complexes. The number of discontinuities in the ZO-1 array (arrows), which is high in rd10 mutants as a secondary effect of photoreceptor loss (A), was not rescued by hNGFp administration (B), as demonstrated by a quantitative analysis shown in C, where the number of intersections between ZO-1 profiles and a reference grid has been assessed systematically and compared statistically (t test; p = 0.62). Error bars are ±SEM. t test was used to compare the ctr versus treat mean for each experiment.