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Luis Alarcon-Martinez, Hector Rieiro, Tugba Demirci, Rocio Leal-Campanario, Susana Martinez-Conde, Stephen Macknik; The Role of Abnormal Blood Flow in Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4607. doi: https://doi.org/.
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The Age-Related Macular Degeneration (AMD) is the main cause of blindness in senior citizens. Chemokines play an important role in this disease, though the precise mechanistic pathways are unknown. In addition to their function in recruiting macrophages, chemokines are important growth factors for the smooth muscle cells found in the macrovessels of the circulatory system, and therefore play a role in blood flow regulation. We studied the retinal vascular supply of an AMD model, the Ccl2 knockout mouse, with fiber-optic-coupled laser-scanning confocal fluorescence microscopy. We imaged the mice retina directly and analyzed whether abnormal blood flow is related to AMD pathogenesis.
We used Ccl2 knockout (KO)(n=45) and Wild Type (WT)(n=35) mice with an age between 8-20 months. We anesthetized and placed them in a stereotaxic positioning rig. We injected a green fluorescein dye through the tail-vein and placed the 300μm fiber-optic objective over the retina through a pinhole made in the superior sclera. We used image analysis software to derive an independent measure of each vessel’s fluorescence-over-time (ΔF/F). After each recording we extracted and incubated the retinas with neuronal, apoptotic, and nuclei markers for stereological analysis. In a subset of animals, we orally administered vasodilators(1.25 g/L) for the last 6 months of life in six Ccl2-/- mice, to normalize a possible abnormal blood flow.
KO animals showed a higher percent of vascular alterations in macrovessels (9.4%KO; 2.6%WT)(t-test; P=0.01), but not in capillaries (1.2%KO; 0.7%WT). The average vasospasm duration (t-test;P=0.05) and magnitude (t-test;P=0.04) were higher for macrovessels in KO versus WT mice. The stereology reveals a higher number of apoptotic (AIF+) neurons in KO than in WT (t-test;P=0.02), and a greater distance between blood vessels and dying AIF+ (21.8%+/-3.8% SEM) neurons than for healthy (AIF-) ones (t-test;P=0.04), whereas there was no difference (t-test;P=0.44) in WT. This stroke-like cell death in the microscopic watershed areas between vessels is further evidence that Ccl2-/- mice suffer from ischemia in macrovessels that leads to hypoxia driven neurodegeneration. Vasodilators treatment normalized the retinal blood flow, distance between AIF+ and AIF- neurons, and increased the number of healthy neurons.
Deleting Ccl2 protein produced abnormal retinal blood flow, leading to neuronal apoptosis.
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