Peter Lundh von Leithner

Assessment of Rat and Mouse RGC Apoptosis Imaging in Vivo with Different Scanning Laser Ophthalmoscopes

Purpose: We have recently described a novel way of imaging apoptosing retinal ganglion cells in vivo in the rat. This study investigated if this technique could be used in the mouse, and whether the Heidelberg Retina Angiograph II (HRAII) was appropriate. Methods: Retinal ganglion cell (RGC) death was induced by intravitreal injections in rat and mouse eyes using staurosporine. Fluorescent-labeled apoptosing cells were detected by imaging with both the HRAII and a prototype Zeiss confocal scanning laser ophthalmoscope (cSLO). Averaged in vivo images were analyzed and results compared with histologic analysis. Results: Fluorescent points (FPs) used as a measure of RGC apoptosis in vivo were detected in the mouse eye but only with the HRAII and not the Zeiss cSLO. The HRAII was able to detect 62% more FPs in rat than the Zeiss cSLO. Both cSLOs showed peak FP counts at the 5- to 10-μ m range in rat and mouse. Maximal FP counts were detected in the superior and superior temporal regions in the rat, with no obvious pattern of distribution in the mouse. The HRAII was found to have more FP correspondence with histologically identified apoptosing RGCs. Conclusions: To our knowledge, this is the first demonstration of visualized apoptosing RGC in vivo in a mouse. The improved image quality achieved with the HRAII compared with the Zeiss cSLO was validated by histology. This together with its enhanced maneuverability and the fact that it is already commercially available make the HRAII a potential tool for the early detection and diagnosis of glaucomatous disease in patients.

Complement Factor H Is Critical in the Maintenance of Retinal Perfusion

Vascular pathologies are known to be associated with age-related macular degeneration. Recently, age-related macular degeneration was associated with a single-nucleotide substitution of the complement factor H (CFH) gene, part of the alternative pathway of the complement system, a critical element in the innate immune response. Such polymorphisms are found in more than 50% of cases of age-related macular degeneration. Here we show that the absence of CFH causes an autoimmune response that targets the vascular endothelium of both the inner and outer retinal vascular networks. In CFH-knockout (cfh(-/-)) mice, C3 and C3b, key components of the complement system, are progressively deposited on retinal vessels, which subsequently become restricted and wither, resulting in a reduction of retinal blood supply. This result leads to increased oxygen stress. While such effects are not systemic, these structural changes are mirrored in functional changes with a substantial decline in retinal blood flow dynamics. When the system is challenged functionally by laser-induced choroidal neovascularization, fluorescein leakage was significantly smaller in cfh(-/-) mice compared with controls, likely due to reduced retinal perfusion. These data reveal that in both the presence and absence of exogenous challenge to the innate immune system, CFH is required to maintain normal levels of retinal perfusion. It is likely that C3 and C3b accumulation in the aged CFH-deficient retina is associated with complement-mediated retinal endothelium destruction.

Systemic Administration of Abeta mAb Reduces Retinal Deposition of Abeta and Activated Complement C3 in Age-Related Macular Degeneration Mouse Model

Age-related macular degeneration (AMD) is a leading cause of legal blindness in the Western world. There are effective treatments for the vascular complications of neo-vascular AMD, but no effective therapies are available for the dry/atrophic form of the disease. A previously described transgenic CFH-gene deficient mouse model, (cfh−/−), shows hallmarks of early AMD. The ocular phenotype has been further analysed to demonstrate amyloid beta (Aβ) rich basement membrane deposits associated with activated complement C3. Cfh−/− mice were treated systemically in both prophylactic and therapeutic regimes with an anti-Aβ monoclonal antibody (mAb), 6F6, to determine the effect on the cfh−/− retinal phenotype. Prophylactic treatment with 6F6 demonstrated a dose dependent reduction in the accumulation of both Aβ and activated C3 deposition. A similar reduction in the retinal endpoints could be seen after therapeutic treatment. Serum Aβ levels after systemic administration of 6F6 show accumulation of Aβ in the periphery suggestive of a peripheral sink mechanism. In summary, anti-Aβ mAb treatment can partially prevent or reverse ocular phenotypes of the cfh−/− mouse. The data support this therapeutic approach in humans potentially modulating two key elements in the pathogenesis of AMD – Aβ and activated, complement C3.