Elevated IOP was induced in RAG
−/− and control mice by injection of polystyrene microbeads into the anterior chamber as described previously by others.
31–33 In our hands, the injections resulted in increased IOP that developed within 1 week in approximately 70% of treated eyes, although the magnitude of the observed IOP changes varied between individuals. Overall, individuals in the RAG
−/− group tended to respond to this treatment with slightly higher elevation of IOP than normal mice. In the interest of yielding comparable data, eyes were selected from each strain to yield groups with similar IOP prior to subsequent analyses of the induced glaucomatous changes (
N = 12 per genotype and for each time point, 72 eyes total). The observed IOP increased from 9.9 and 10.1 mm Hg prior to microbead injection, to 14.7 and 14.8 mm Hg after 4 days, and continued to increase to 19.5 and 20.6 mm Hg by the end of the experiment in the normal and the RAG
−/− mice, respectively (
Fig. 1). The IOP differences between the groups are statistically not significant (
P = 0.81, 0.24, and 0.45 on days 10, 16, and 28, respectively).
To confirm the development of glaucomatous damage and to assess any potential differences between normal and RAG
−/− experimental glaucomatous change, RGC density was determined following immunohistochemical identification of γ-synuclein positive RGC (
Fig. 2A). As expected, continued exposure to elevated IOP resulted in progressive RGC loss and optic nerve damage (
Fig. 2A). While eyes obtained from both RAG
−/− and control mice 14 days after induction of elevated IOP display essentially normal RGC densities (
P = 0.46 and 0.76, respectively), retinas harvested 28 days after induction of elevated IOP exhibit 21.5% (RAG
−/−) and 29.6% (control) loss of RGC (
P = 3.66 × 10
−06 and 2.06 × 10
−08; respectively). The differences between the RAG
−/− and control mice in the number of surviving RGC are not statistically significant (
P = 0.42 and 0.07 after 14 and 28 days of elevated IOP, respectively).
The degree of axonal damage in the optic nerve was determined using a grading scheme as previously described.
6,17 Increasing axonal damage is indicated by a rise in the number of PPD stained axons and increasing amounts of gliosis. Optic nerves evaluated are assigned to one of five damage categories ranging from 1 (no damage) to 5 (severe damage) as illustrated in the examples in
Figure 2C. Our data demonstrate that 2 weeks after induction of elevated IOP damage to the optic nerve was not significant in either group (
P = 0.66 in control mice and 0.24 in RAG1
−/−;
Fig. 2B). However, 28 days after IOP elevation, evidence of mild optic nerve damage was frequently evident and a fraction of samples displayed signs of advanced damage as indicated by PPD staining of numerous axons and clear signs of gliosis (
P = 0.03 in control mice and 0.0005 in RAG1
−/− compared with baseline values). Congruent with our data indicating a similar degree of RGC loss in RAG
−/− and normal mice, we did not observe a statistically significant difference in the extent of optic nerve damage between the two groups (
P = 0.73 after 14 days and 0.78 after 28 days).
The development of RGC damage is accompanied by increased immunohistochemical labeling of C1q and the C5b-9 complex (
Fig. 3). Four weeks after microbead injection, immunohistochemical detection of C1q and C5b-9 revealed increased labeling in the ganglion cell layer (GCL) in both normal and RAG
−/− mice in animals with elevated IOP. Other layers of the retina are not complement-immunoreactive, although occasionally weak C1q binding was observed in the outer plexiform layer (OPL). Immunoreactivity to complement components can also frequently be observed within the retinal vasculature (arrowhead in
Fig. 3F) presumably due to the presence of complement components in the serum. In contrast, retinal labeling is absent in both mouse strains at normal IOP. Eyes that received microbead injections, but failed to develop elevated IOP, did not display C1q or C5b-9 immunoreactivity (
Figs. 3C,
3F).
In order to demonstrate that the complement components detected are fixed on the cell membranes, we separated soluble retinal protein from the cell membrane fraction and analyzed the latter by Western blot analyses. As described in the “Methods” section, our experimental approach yields fractions of insoluble cellular components (P1) as well as a fraction of cellular constituents that remain soluble after centrifugation at 10,000
g (S1). This fraction S1 can further be divided by ultracentrifugation into soluble components (S2) and a pellet enriched in cell membranes and cell-membrane associated proteins (P2).
Figures 4A and
4B demonstrate the effectiveness of our separation method. Glucose Transporter 1 (
Glut1), a membrane spanning protein, is retained in the membrane fraction P2 (
Fig. 4A). Conversely, β-tubulin, a biomarker for soluble protein, is detected in the soluble protein fraction S2, but is removed from the membrane enriched fraction P2 (
Fig. 4B).
Retinal membrane preparations (P2) were then obtained from RAG
−/− and normal mice with and without elevated IOP and evaluated for the presence of C1q and
C3 by Western blot analyses (
Figs. 4C,
4D). Membrane associated dimers of C1q (66 kDa) are readily detectable in the membrane enriched retinal fraction obtained from mice with elevated IOP (
Fig. 4C). Low levels of C1q are also occasionally observed in control mice with normal (low) IOP. Importantly, membrane-associated C1q can also be demonstrated in mice lacking RAG1, suggesting antibody independent fixation of C1q in the glaucomatous retina.
Western blot analysis of the same protein fractions for the presence of C3 also revealed an accumulation of this protein in retinas obtained from mice with elevated IOP but not those with normal IOP (
Fig. 4D). A major band with an apparent molecular weight of 63 kDa, as well as minor bands at 75 kDa and 101 kDa representing iC3bα, iC3bβ, and the uncleaved C3α' chains, respectively, are present. Congruent with our findings for C1q, membrane-associated
C3 was observed in control and RAG
−/− mice, indicating that C1q fixation led to activation of the complement cascade in both mouse strains.
Finally, C1q fixation on the surface of RGC in the absence of antibodies can also be demonstrated in a primary cell culture system of mouse retinal cell suspensions. RGC maintained in cell culture exhibit considerable cell stress and typically die within days or weeks. During this period, the accumulation of complement components can be observed. Here we maintained RGC in cell culture medium without serum to rule out an exogenous source of immunoglobulins. Adult mouse retinae were enzymatically dissociated and maintained in serum free cell culture media. After 6 days in culture, numerous RGC were identified immunohistochemically by the presence of neurofilaments heavy chain (NFH). Many of these cells are also immunoreactive with antibodies directed against C1q (
Fig. 5). These findings not only further indicate that immunoglobulins are not required for C1q fixation on RGC, but also indicate that both C1q and the ligand fixing it to these cells are not serum derived and are consequently synthesized by retinal cells, possibly RGC themselves.