The Complement System Is Critical in Maintaining Retinal Integrity during Aging

Abstract

The complement system is a key component of innate immunity comprised of soluble components that form a proteolytic cascade leading to the generation of effector molecules involved in cellular clearance. This system is highly activated not only under general inflammatory conditions such as infections, collagen diseases, nephritis, and liver diseases, but also in focal ocular diseases. However, little is known about the role of the complement system in retinal homeostasis during aging. Using young (6-week-old) and adult (6-month-old) mice in wild type (C57BL/6) and complement knockout strains (C1q^-/-, Mbl a/c^-/-, Fb^-/-, C3^-/-, and C5^-/-), we compared amplitudes of electroretinograms (ERG) and thicknesses of retinal layers in spectral domain optical coherence tomography between young and adult mice. The ERG amplitudes in adult mice were significantly decreased (p < 0.001, p < 0.0001) compared to that of young mice in all complement knockout strains, and there were significant decreases in the inner nuclear layer (INL) thickness in adult mice compared to young mice in all complement knockout strains (p < 0.0001). There were no significant differences in ERG amplitude or thickness of the INL between young and adult control mice. These data suggest that the complement system plays an important role in maintaining normal retinal integrity over time.

Keywords: EM; ERG; OCT; aging; complement system; retina.

Figure 1

Representative examples of ERG a- and b-wave responses in young (6 weeks) and adult (6 months) mice in each experimental group. In C57BL/6 mice, the amplitudes of a- and b-waves between young and adult mice were almost identical. On the other hand, both amplitudes were impaired in aged C1q^−/−, Mbl^−/−, Fb^−/−, C3^−/−, and C5^−/− mice. A scale was shown at the bottom right.

Figure 2

Quantification of ERG amplitudes in a- and b-waves of young (6 weeks) and adult (6 months) mice in each experimental group. In C57BL/6 mice (A,B), there was no significant difference in the amplitude of a- and b-waves between the young (n = 5) and old (n = 5) mice. By contrast, both amplitudes were significantly decreased in old compared to in young C1q^−/− mice (a-wave: p < 0.001, b-wave: p < 0.0001; young n = 3, adult n = 3), Mbl^−/− mice (a-wave: p < 0.0001, b-wave: p < 0.0001; young n = 6, adult n = 5), Fb^−/− mice (a-wave: p < 0.001, b-wave: p < 0.0001; young n = 4, adult n = 8), C3^−/− mice (a-wave: p < 0.001, b-wave: p < 0.0001; young n = 6, adult n = 4), and C5^−/− mice (a-wave: p < 0.001, b-wave: p < 0.0001; young n = 3, adult n = 3) (C–L).

Figure 3

Comparison of ERG a- and b-wave amplitudes among experimental mouse strains at 6 weeks (A,B) and 6 months (C,D) of age. The amplitude of a- and b-waves in complement knockout strains in C1q^−/−, Mbl^−/−, Fb^−/−, and C3^−/− mice were lower than those of C57BL/6 mice at each age, except for C5^−/− mice at 6 weeks of age.

Figure 4

Mean thickness of retinal sub-layers measured by Spectral Domain Optical Coherence Tomography in young (6 weeks) and adult (6 months) mice in each experimental group. (A) There was no significant thinning in any retinal layer between 6-week-old and 6-month-old C57BL/6 mice. (B–F) Contrastingly, INL thinning was identified in all complement knockout strains at the age of 6 months. Values and statistical analysis are summarized in Table 1. RNFL, retinal nerve fiber layer; IPL/GC, inner plexiform layer/ganglion cell; INL, inner nuclear layer; ONL, outer nuclear layer. Bar = 100 μm. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^†p < 0.0001.

Figure 5

Representative light microscopic and electron microscopic images comparing the retinal layer thickness of C57BL/6 (A–D) and C1q^−/− (E–H) mice at 6 weeks (A,C,E,G) and 6 months (B,D,F,H) of age. (A–D) No thinning in any retinal layer were observed between 6-week-old and 6-month-old C57BL/6 mice. (E–H) In C1q^−/− mice at 6 months of age, the thickness of inner nuclear layer seemed to be thinner than that in C1q^−/− mice at 6 weeks of age. Bar = 100 μm, (F) Bar = 10 μm (H).

Figure 6

Representative light microscopic (A,B) and electron microscopic images (C,D) comparing the retinal layer thickness of C3^−/− mice at 6 weeks (A,C) and 6 months of age (B,D). (A–D) The thickness of the inner nuclear layer in C3^−/− mice at 6 months of age (B,D) appears thinner than that in C3^−/− mice at 6 weeks of age (A,C). (A,B) Bar = 100 μm. (C,D) Bar = 10 μm.

Figure 7

Representative comparison of retinal layer thickness between 6-week-old and 6-month-old Fb^−/− (A,B), C5^−/− (C,D), and Mbl^−/− (E,F) mice. (A–F) There was no apparent difference in retinal thickness of each layer between mice at 6 weeks of age and those at 6 months of age. (A–F) Bar = 100 μm.

Figure 8

Phagocytized microglial cells in the inner nuclear layer of C1q^−/− mice at 6 months of age. (A–D, white arrows) (A) A microglial cell phagocytizing dead cells, with dense particles observed in the microglial cell body at the border between the inner nuclear layer and inner plexiform layer (magnification: x1,400). (B) High magnification image of (A) (magnification: x9,300). (C,D) Microglial cells containing dense particles were detected in other parts of the inner nuclear layer. (C: x13,000, D: x9,300). (A) Bar = 10 μm, (B) Bar = 2 μm, (C) Bar = 500 nm, (D) Bar = 2 μm.

Figure 9

Assessment of dense inclusions in the outer plexiform layer (OPL) (A,B, white arrows). Dense inclusions in the OPL have been reported previously in microglia-depleted retinas, and were due the destruction of pre-synaptic termini in the OPL (Wang et al., 2016). The presence of dense inclusions was identified in alternative complement pathway deficient mice and corresponded to the border between the tip of photoreceptors and neighboring bipolar cells. (A) Fb^−/− mice at 6 weeks of age. Bar = 500 nm. (B) Fb^−/− mice at 6 months of age. Bar = 500 nm.