RETINAL DEGENERATION IN CCL2/DAF1 DOUBLE-DEFICIENT MICE

Abstract

Purpose: Chemoattractant chemokine ligand 2 (CCL2) can recruit choroidal macrophages to clean up retinal debris, and reduce the inflammation response related to complement activation. Complement decay-accelerating factor (DAF) is a protein which is encoded by the Daf1 gene. DAF is a membrane regulator of the alternative pathway of complement that protects self cells from autologous complement attack through blocking the assembly the C3-convertase (C3bBb complex) of the alternative pathway, accelerating the disassembly of existing C3-convertase, and finally preventing the formation of the membrane attack complex. In view of the link between complement genes and retinal disease, we evaluated the retina of mice lacking Ccl2 and/or Daf1.

Methods: C57BL/6J, Ccl2^−/−, Daf1^−/− and Ccl2^-/-/ Daf1^-/- (DKO) mice were studied. Electroretinography, histological analysis by light microscopy, c3d staining in situ, GRP78 staining in situ, detection of apoptotic cells in situ by TUNEL and fundus imaging by SLO were performed.

Results: At 1 year of age, the a- and b-wave amplitudes of Ccl2^-/-Daf1^-/- , Ccl2^-/- and Daf1^-/- mice were reduced compared to those of WT mice, and the reduction in DKO mice was more prominent than in either single knockout line. TUNEL staining showed no apoptotic cells in WT retina, but many in the ONL and INL in the DKO retina, and a reduced density in these layers of Ccl2^-/- or Daf1^-/- mice. H&E staining of retinal sections showed that cell loss was more pronounced in the inner and outer retina of DKO than in Ccl2^-/- or Daf1^-/- mice. Immunohistochemical analysis showed that c3d deposition along the RPE is elevated in the DKO mice, and to a lesser degree in Ccl2^-/- retina, compared to WT mice. In Daf1^-/- mice, c3d deposition is comparable to WT mice. SLO-AF488nm fundus imaging showed increased number of autofluorescent foci, suggestive of inflammatory cell infiltration, in the three mutant lines with the greatest number in the DKO retina compared to WT. Background autofluorescence emanating from lipofuscin in RPE of DKO appears elevated relative to C57BL/6J, Ccl2^-/-, and Daf1^-/- mice. Immunostaining showed reduced levels of GRP78 in the DKO retina and modest decrease in the inner retina of ccl2^-/- mice.

Conclusions: Mouse mutants for Ccl2^-/-, and/or Daf1^-/- mice have a mild retinal degeneration while Ccl2^-/-/Daf1^-/- DKO mice have a more severe loss of retinal cells. Cell loss involves both the inner and outer nuclear layers.

Keywords: 510 electroretinography: non-clinical • 695 retinal degenerations: cell biology • 554 immunohistochemistry