Abstract
Purpose: :
The lack of a suitable animal model has impeded progress in the study and treatment of age–related macular degeneration (AMD). Senescent CCL2 deficient mice have been shown to develop cardinal features of AMD. Variation within CX3CR1 has been associated with human AMD. In this study, we generated CCL2/CX3CR1 double knock–out (DKO) mice in order to observe the features and timeframe of retinal impairment.
Methods: :
CCL2 and CX3CR1 deficient mice carrying the homozygous null mutated gene were back–crossed to obtain animals heterozygous for both alleles. They were subsequently mated to generate homozygous DKO. Genotyping for wild–type and KOs was performed using a PCR assay. A real–time RT–PCR assay (Taqman) and immunostaining were used to confirm absence of the gene products in vivo. Age–matched homozygous DKOs and wild–type controls with a similar ratio of C57BL/6 x 129 inbred backgrounds were used in this study. Mice were examined by funduscopy and histology. Expression of complement was evaluated by immunohistochemistry.
Results: :
Lack of CCL2 and CX3CR1 transcript and protein in the liver and eye was confirmed in these animals. Among the 400 F2 pups analyzed, 12 animals were DKO, indicating an abnormal Mendelian segregation (1 in 16 expected). F1 males and females cross–bred with heterozygous CCL2+/–/CX3CR1+/– mice were within normal size. DKO mice showed a 20% decrease in weight. DKO mice appeared to be less fertile, with an average of 6 pups per litter as compared to 9 in the controls. Forty percent DKOs showed patchy skin depigmentation, mostly on the face and head. At as early as 1.5 month old, DKO spontaneously developed the typical clinical AMD feature of drusen–like lesions. These lesions became confluent and progressed with age. Histology showed RPE vacuolation and degeneration, drusen formation, photoreceptor atrophy, and a few cases of choroidal neovascularization. Complement component deposits were detected within the RPE, Bruch’s membrane and choroid. Electron microscopy illustrated linear deposits within Bruch’s membrane, and decreased melanosomes and increased lipofusions in the RPE. These human AMD–like morphologies progressed with age.
Conclusions: :
CCL2–/–/CX3CR1–/– mice can be used to represent human AMD. The observations made in this study implicate the important roles of certain chemokines and possible macrophage dysfunction in AMD pathogenesis. Due to the early development of AMD features in this potential model, these mice may serve as a better tool for the study of AMD and may help in evaluating various therapies for this blinding disease.
Keywords: age-related macular degeneration • pathology: experimental • cytokines/chemokines