Superior cervical gangliectomy induces non-exudative age-related macular degeneration in mice

Abstract

Non-exudative age-related macular degeneration, a prevalent cause of blindness, is a progressive and degenerative disease characterized by alterations in Bruch's membrane, retinal pigment epithelium, and photoreceptors exclusively localized in the macula. Although experimental murine models exist, the vast majority take a long time to develop retinal alterations and, in general, these alterations are ubiquitous, with many resulting from non-eye-specific genetic manipulations; additionally, most do not always reproduce the hallmarks of human age-related macular degeneration. Choroid vessels receive sympathetic innervation from the superior cervical ganglion, which, together with the parasympathetic system, regulates blood flow into the choroid. Choroid blood flow changes have been involved in age-related macular degeneration development and progression. At present, no experimental models take this factor into account. The aim of this work was to analyze the effect of superior cervical gangliectomy (also known as ganglionectomy) on the choroid, Bruch's membrane, retinal pigment epithelium and retina. Adult male C57BL/6J mice underwent unilateral superior cervical gangliectomy and a contralateral sham procedure. Although superior cervical gangliectomy induced ubiquitous choroid and choriocapillaris changes, it induced Bruch's membrane thickening, loss of retinal pigment epithelium melanin content and retinoid isomerohydrolase, the appearance of drusen-like deposits, and retinal pigment epithelium and photoreceptor atrophy, exclusively localized in the temporal side. Moreover, superior cervical gangliectomy provoked a localized increase in retinal pigment epithelium and photoreceptor apoptosis, and a decline in photoreceptor electroretinographic function. Therefore, superior cervical gangliectomy recapitulated the main features of human non-exudative age-related macular degeneration, and could become a new experimental model of dry age-related macular degeneration, and a useful platform for developing new therapies.

Keywords: Age-related macular degeneration; Choroid; Experimental model; Photoreceptors; Retinal pigment epithelium; Superior cervical ganglion.

Fig. 1.

Effect of SCGx on the choroid. (A) Transverse ultrathin sections of the choroid from control and SCGx eyes at 4, 6 and 10 weeks post-surgery. SCGx induced an increase in choroidal thickness (black bars), tissue spreading, and vessels enlargement. (B) Choriocapillaris details from control and SCGx eyes at 4, 6 and 10 weeks post-surgery. A significant increase in nasal and temporal choriocapillaris thickness (black bars) was found in eyes submitted to SCGx at all time points. (C) Quantification of choriocapillaris thickness from control and SCGx eyes at 4, 6 and 10 weeks post-surgery. SCGx induced a significant increase in the nasal and temporal choriocapillaris thickness at 4 and 10 weeks post-surgery. Shown are representative photomicrographs from five animals/group. BrM, Bruch's membrane; ChC, choriocapillaris; Ch, choroid; Sc, sclera; C, control; SCGx, superior cervical gangliectomy. Scale bars: 5 µm. Data are means±s.e.m. (n=5 animals per group), **P<0.01 vs control eyes at 10 weeks post-SCGx, by Student's t-test.

Fig. 2.

Ultrastructural analysis of Bruch's membrane (BrM). (A) Transverse ultrathin sections from control and SCGx eyes at 4, 6 and 10 weeks post-surgery. At 4 weeks post-SCGx, BrM thickness and structure were preserved, whereas, at 6 weeks post-surgery, SCGx induced a significant thickening and a clear loss of its pentalaminar structure, followed by fibrillar collagen accumulation (white arrow), reticular middle electron-dense deposits under the RPE basal membrane (asterisk) and absence of endothelial cells (black arrow) at 10 weeks post-SCGx. Shown are representative photomicrographs at 800 µm, nasally and temporally, from the ONH, from five animals/group. (B) Quantification of nasal and temporal BrM thickness in control eyes and eyes submitted to SCGx at 4, 6 and 10 weeks post-surgery. SCGx induced a significant increase in the thickness of the temporal (but not nasal) BrM after 6 and 10 weeks post-surgery. BI, RPE basal infoldings; BM, basal membrane; IC, internal collagenous layer; EL, elastic layer; OC, outer collagenous layer; EC, endothelial cell; Ch, choroid. Scale bar: 500 nm. Data are means±s.e.m. (n=5 animals per group), **P<0.01 vs control eyes, by Tukey's test.

Fig. 3.

Effect of SCGx on the RPE melanin content and RPE65 immunoreactivity. (A) Representative photomicrographs showing RPE melanin content loss (arrows) at the temporal (but not nasal) RPE at 800 µm from the ONH. Scale bar: 25 µm. (B) SCGx induced a significant decrease in melanin content in the temporal RPE, which, at 4 weeks post-SCGx (black triangles), was evident at 800 µm temporally from the ONH and afterwards spread to almost all the temporal side, except for the periphery (1600-2000 µm from the ONH), at 6 (white triangles) and 10 (black squares) weeks post-SCGx. No differences in the nasal RPE melanin content were observed between control eyes (white circles) and SCGx eyes at all time points examined. Data are means±s.e.m. (n=5 animals per group), **P<0.01 vs control eyes, by Tukey's test. (C) Representative RPE photomicrographs of RPE65 immunostaining at 800 µm nasally and temporally from the ONH. A decrease in RPE65 immunostaining in the temporal (but not nasal) RPE was observed in eyes submitted to SCGx at 4, 6 and 10 weeks post-surgery. Shown are representative photomicrographs from five animals/group. OS, photoreceptor outer segments; RPE, retinal pigment epithelium; Ch, choroid. Scale bar: 50 µm.

Fig. 4.

Retinal histology, sub-retinal deposit analysis, and ONL and RPE apoptosis. (A) Representative retinal semi-thin sections at 800 µm nasally and temporally from the ONH stained with Toluidine Blue from control eyes and eyes submitted to SCGx are shown. Besides the loss of RPE melanin content (white asterisk) at 4 weeks post-SCGx, drusenoid deposits (white arrows) between temporal RPE and PRs were found at 6 weeks post-SCGx, followed by major alterations in the temporal area such as RPE vacuolization (black arrow) and endothelial cell alterations (arrowhead), which were found at 10 weeks post-SCGx. (B) C3 immunoreactivity in representative transverse retinal sections. Linear C3(+) deposits within the temporal BrM were observed at 6 and 10 weeks post-SCGx. Shown are representative photomicrographs from five animals/group. Scale bars: 25 µm (A); 10 µm (B). (C) SCGx induced a significant increase in TUNEL(+) nuclei (arrows) in the temporal ONL and RPE at 800 µm from the ONH at 10 weeks post-surgery. Representative photomicrographs from five animals/group are shown. Scale bars: 25 μm (retina); 50 μm (RPE). RPE, retinal pigment epithelium; Ch, choroid; RPE, retinal pigment epithelium; OS, photoreceptor outer segments; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; GCL, ganglion cell layer; IS, photoreceptor inner segments; C, control; SCGx, superior cervical gangliectomy. Data are means±s.e.m. (n=5 animals per group), **P<0.01 vs control eyes, by Student's t-test.

Fig. 5.

Effect of SCGx on retinal function. (A-C) The average amplitudes of scotopic ERG a-wave, b-wave and OPs are shown. A significant decrease in ERG a-wave amplitude was observed after 4, 6 and 10 weeks post-SCGx (white dots), whereas no alterations in the ERG b-wave and OP amplitudes were found at any time point studied. Gray bars show naïve ERG a- and b-wave and OP amplitudes. No differences were found between naïve and control eyes (black dots) in all parameters and time points studied. Data are means±s.e.m. (n=10 animals per group), *P<0.05 and **P<0.01 vs naïve eyes, by Tukey's test. (D) Representative scotopic ERG and OP traces of control eyes and SCGx eyes.

Fig. 6.

Effect of SCGx on the RPE and PR ultrastructure. Shown are representative transverse ultrathin RPE (A) and PR (B) sections from sham-treated eyes and eyes at 4, 6 and 10 weeks post-SCGx. (A) At 6 weeks post-surgery, SCGx induced the occurrence of vacuoles (black arrow) adjacent to the temporal (at 800 µm from the ONH) RPE basal infoldings. These changes were more evident, and vacuoles appeared more apical and often filled with membranous content (asterisk), at 10 weeks post-SCGx. Moreover, granules of middle-high electron-density were also present at the temporal RPE at 10 weeks post-SCGx (white arrow). No differences were observed at the nasal RPE between control and SCGx eyes at any time point studied. Shown are representative photomicrographs from five animals/group. OS, photoreceptors outer segments; RPE, retinal pigment epithelium; BI, basal infoldings. Scale bar: 200 nm. (B) SCGx induced focal losses of plasmatic membrane (black arrow) and the replacement of discs with an electron-dense material (white asterisk) in the temporal retina at 6 weeks post-surgery. These alterations were more evident at 10 weeks post-SCGx, and focal complete losses of discs and blebs (black asterisk) were observed. There were no alterations in nasal PR outer segment plasmatic membrane (white arrow) and discs (white arrowhead) between control and SCGx eyes at any time point studied. Shown are representative photomicrographs from five animals/group. Scale bar: 200 nm.