Edited by: Isabel Varela-Nieto, Consejo Superior de Investigaciones Científicas (CSIC), Spain
Reviewed by: Antje Grosche, Ludwig-Maximilians-Universität München, Germany; Enrica Strettoi, Istituto di Neuroscienze (CNR), Italy
*Correspondence: Kip M. Connor
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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 (
Retinal degeneration is a common form of neurodegenerative disease and a leading cause of vision loss worldwide. In the United States alone, about 3.4 million people are estimated to have vision loss due to retinal degeneration, and its prevalence is predicted to increase in industrialized countries with aging populations (Wert et al.,
The complement system is an essential component of the innate immune system and plays an important role in immune function and disease pathogenesis. The complement system is a hub-like surveillance network of the innate immune system that plays a vital role in the modulation of immune and inflammatory responses (Walport,
The complement system is complex, with numerous complement effectors and a variety of receptors with different effects (McGeer et al.,
The complement system is thought to contribute to general homeostasis by eliminating immune complexes and apoptotic cells, as well as mediating cross-talk with immune cells for adaptive immune functions (Ricklin et al.,
The complement system is active in the retina, RPE, and choroid under endogenous conditions. Immunohistochemistry has revealed that C1q is expressed in the retinal ganglion cell (RGC) layer even during the developmental stage (Stevens et al.,
The Massachusetts Eye and Ear Animal Care Committees approved all animal procedures and experiments prior to beginning, and all animals were treated in accordance with the guidelines set forth by the Association of Research for Vision and Ophthalmology.
In this study, C57BL/6 (stock no. 000664) mice were purchased from Jackson Laboratories (Bar Harbor ME, USA).
Full-field ERGs were recorded simultaneously from both eyes. Animals were dark-adapted overnight (>12 h). Mice were weighed and anesthetized with intraperitoneal injections of a mixture of ketamine (60 mg/kg) and xylazine (9 mg/kg). Mydriasis was achieved with one drop of 0.5% tropicamide with 5% phenylephrine. Corneal anesthesia was performed with a single drop of 0.5% proparacaine hydrochloride ophthalmic solution (Akorn Inc. Illinois). A warm heating pad was used to maintain body temperature (37°C).
ERGs were recorded using HMS's ERG LAB system (OcuScience, Nevada). Stimulus flashes were presented in a Ganzfeld bowl. Stimulus intensities ranging from −1.5 to 1.0 log cd s/m2 in 0.5-log units steps were used under dark-adapted conditions. Light stimuli were presented with a 1-min interval between successive stimuli.
Before optical coherence tomography (OCT) imaging was performed, each animal was anesthetized by intraperitoneal Avertin injection (125 mg/kg), and the pupils were dilated with a drop of 0.5% tropicamide with 5% phenylephrine. Two repeated volumetric images, centered on the optic nerve head, were acquired in both eyes using spectral-domain OCT (SD-OCT; Bioptigen, Inc., Davis, NC). All SD-OCT images consisted of 1,000 A-scans and 100-averaged B scans (each B-scan was the average of three B-scans). These parameters correspond to the area of approximately 1.4 × 1.4 mm.
The RGC layer, inner plexiform layer and ganglion cell complex (IPL/GC), INL, and ONL were measured using SD-OCT B-scan cross-sectional images (Supplementary Figure
Mouse eyes were enucleated and immersed in half strength Karnovsky's fixative (2% formaldehyde + 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4; Electron Microscopy Sciences, Hatfield, Pennsylvania) at room temperature. An eyecup was created with each eye by dissecting away the anterior portion from the posterior portion. Eyecup samples were then placed back into half-strength Karnovsky's fixative for a minimum of 24 h under refrigeration. After fixation, samples were rinsed with 0.1 M sodium cacodylate buffer, post-fixed with 2% osmium tetroxide in 0.1 M sodium cacodylate buffer for 1.5 h, en bloc stained with 2% aqueous uranyl acetate for 30 min, then dehydrated with graded ethyl alcohol solutions, transitioned with propylene oxide, and resin infiltrated in tEPON-812 epoxy resin (Tousimis, Rockville, Maryland) utilizing an automated EMS Lynx 2 EM tissue processor (Electron Microscopy Sciences, Hatfield, Pennsylvania). The whole posterior eye cup was processed and embedded as a single block in transparent epoxy resin to prevent outer segment detachment during subsequent processing and sectioning procedures. The mid optic nerve head plane within the embedded eyecup was targeted for sectioning through the use of a stereoscope observing all directions of the transparent embedded block, then scored for sectioning using the optic nerve as an exterior reference. An initial precision saw cut was made into the embedded block away from the mid-level plane then ground down using a frosted slide. The edges of the eyecup tissue were trimmed using razor blades for ultramicrotomy. Sections were subsequently generated at 1 micrometer thickness and stained with toluidine blue stain. The orientation of photoreceptor outer segments was assessed in addition to the level through optic nerve head. The angle of cut and orientation in the both the x and y directions where made to generate a mid-level plane with regions of retinal photoreceptor outer segments oriented longitudinally prior to thin sectioning at 80 nanometers thickness for electron microscopy. Ultrathin sections (70–90 nm) were cut from the epoxy block using a Leica EM UC7 ultramicrotome (Leica Microsystems, Buffalo Grove, IL) and a diamond knife, collected onto 2 × 1 mm single slot formvar/carbon coated grids, and were stained with aqueous 25% Uranyl Acetate Replacement stain (Electron Microscopy Sciences, Hatfield, Pennsylvania) and Sato's lead citrate using a modified Hiraoka grid staining system. Grids were imaged using a FEI Tecnai G2 Spirit transmission electron microscope (TEM) (FEI, Hillsboro, Oregon) at 80 kV interfaced with an AMT XR41 digital CCD camera (Advanced Microscopy Techniques, Woburn, Massachusetts) for digital TIFF file image acquisition. TEM imaging of all layers of the retina was used to capture representative regions. When analyzing the number of dense inclusions in the outer plexiform layer (OPL), 10 images taken at x11,000 magnification were selected every 50 μm from the optic nerve head in each mouse strain.
All data are expressed as means ± SE. ERG comparisons in a-wave or b-wave signals were made between 6-week-old and 6-month-old mice using two-way analysis of variance (ANOVA), and comparisons of retinal thickness were analyzed using a two-tailed student's
Significant decreases in the ERG amplitude of both a-wave and b-waves were detected in all strains of complement knockout mice that were 6 months old, compared to that of complement knockout mice (strain-matched mice) at 6 weeks of age; however, no such age-related differences were noted in C57BL/6 mice (Figures
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
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
Comparison of ERG a- and b-wave amplitudes among experimental mouse strains at 6 weeks
Inner nuclear layer (INL) thickness was significantly decreased in
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.
Changes in thickness of different retinal layers in 6-week-old or 6-month-old mice from each experimental strain.
C57BL/6 | 63.35(0.38) | 10 | 62.53(0.38) | 6 | 0.18 | |
65.46(0.22) | 4 | 62.25(0.36) | 6 | < |
||
64.73(0.51) | 7 | 64.03(0.51) | 10 | 0.34 | ||
65.50(0.48) | 5 | 61.03(0.32) | 14 | < |
||
62.45(0.34) | 14 | 58.83(0.46) | 8 | < |
||
65.94(0.40) | 6 | 60.76(0.55) | 7 | < |
||
C57BL/6 | 30.38(0.24) | 10 | 30.07(0.27) | 6 | 0.42 | |
30.90(0.34) | 4 | 26.88(0.22) | 6 | < |
||
31.34(0.42) | 7 | 27.88(0.34) | 10 | < |
||
31.43(0.48) | 5 | 23.32(0.46) | 14 | < |
||
29.03(0.31) | 14 | 22.14(0.46) | 8 | < |
||
31.78(0.30) | 6 | 24.68(0.80) | 7 | < |
||
C57BL/6 | 55.47(0.31) | 10 | 54.22(0.61) | 6 | 0.06 | |
51.67(0.84) | 4 | 49.39(0.24) | 6 | < |
||
55.22(0.64) | 7 | 54.53(1.17) | 10 | 0.64 | ||
54.25(0.68) | 5 | 52.32(0.41) | 14 | < |
||
52.32(0.40) | 14 | 50.79(0.57) | 8 | < |
||
56.93(0.56) | 6 | 54.24(0.34) | 7 | < |
||
C57BL/6 | 15.93(0.20) | 10 | 15.51(0.26) | 6 | 0.22 | |
15.75(0.23) | 4 | 15.76(0.15) | 6 | 0.98 | ||
16.72(0.44) | 7 | 15.83(0.31) | 10 | 0.11 | ||
15.08(0.14) | 5 | 15.15(0.48) | 14 | 0.86 | ||
16.91(0.17) | 14 | 14.77(0.16) | 8 | < |
||
15.90(0.25) | 6 | 15.54(0.36) | 7 | 0.70 |
According to retinal single cell recordings, it is thought that the ERG is a mass retinal response in which the a-wave is generated by photoreceptors and the b-wave primarily reflects activation of bipolar cells. Thus, we analyzed the correlation between a-wave amplitude and ONL thickness, the correlation between b-wave amplitude and INL thickness, and the correlation between b-wave amplitude and IPL/GC thickness. A positive correlation between the b-wave amplitude and INL thickness (
Light microscopic and electron microscopic images were obtained in each mouse strain in order to define the morphological changes induced in the retina when there was a deficiency in complement system activity. INL thinning was observed via electron and light microscopy in
Representative light microscopic and electron microscopic images comparing the retinal layer thickness of C57BL/6
Representative light microscopic
Representative comparison of retinal layer thickness between 6-week-old and 6-month-old
Phagocytized microglial cells in the inner nuclear layer of
Assessment of dense inclusions in the outer plexiform layer (OPL) (
The number of dense inclusions in each mouse strain at 6-weeks or 6-months of age.
C57BL/6J | 0 | 0 |
3 | 5 | |
16 | 9 | |
5 | 11 | |
0 | 0 | |
2 | 3 |
Retinal functional abnormalities were detected in
In C57BL/6 mice, a mild decrease in a-wave and b-wave amplitudes was observed in 6-month-old mice compared to in 6-week-old mice; however, the changes were not significant in this study, indicating that retinal function is preserved from 6 weeks to 6 months in C57BL/6 mice. A previous study detected significant reductions in a-wave and b-wave amplitudes in 12-month-old mice, but such reductions were limited in 6-month-old mice, as compared to 2-month-old mice in the C57BL/6 strain (Li et al.,
Decreased amplitudes of both a- and b-waves at 6 months of age were identified not only in alternative pathway knockout mice (
In this study, we discovered that INL thickness in SD-OCT was significantly decreased in
The mechanism for neuronal loss in the INL and ONL in complement knockout strains, remains unclear. Yu et al. demonstrated that decreased PKCα staining in the INL correlated with a reduction in the number of Calbundin D28 positive cells, and found swelling of the outer segment in aged
Hoh therefore concluded that inhibition of C3 for treatment of age-related macular degeneration (AMD) patients might be deleterious (Hoh Kam et al.,
Interestingly, we detected dense inclusions in the OPL via electron microscopy, which may correspond to the degeneration of synapses between photoreceptors and bipolar cells (Somogyi et al.,
When analyzing morphological changes in INL and IPL by electron microscopy, we focused on morphological changes in amacrine cells, bipolar cells and horizontal cells. We recognized the star-like shape of nuclei in amacrine cells as an index. The Kolmer's organelle is highly important in order to identify horizontal cells (Hogan et al.,
Of note, our study has shown the functional (Figure
Recently, evidence has been accumulating regarding the association between genetic alterations in complement factor H (CFH) with the development of age-related macular degeneration (AMD) in clinical studies. CFH has also been a topic of intense investigation in basic research (Anderson et al.,
To the best of our knowledge, this is the first study to suggest that, in addition to C3, C3aR, and C5aR(Yu et al.,
Drafting the article and conception or design of the work: RM and KC. Critical revision of the article, data analysis and interpretation: RM, YO, CK, DH, JL, and KC.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors are grateful to G. Stahl at Brigham and Women's Hospital, who gave us
The Supplementary Material for this article can be found online at:
Electroretinogram
electron microscopy
inner nuclear layer
retinal degeneration
spectral domain optical coherence tomography
inner plexiform layer/ganglion cell layer
outer nuclear layer
optic nerve head.