Espinosa-Heidmann Diego G, Suner Ivan J, Hernandez Eleut P, Monroy Dagoberto, Csaky Karl G, Cousins Scott W
Bascom Palmer Eye Institute, Department of Ophthalmology, The University of Miami School of Medicine, Miami, Florida, USA.
Invest Ophthalmol Vis Sci. 2003 Aug;44(8):3586-92. doi: 10.1167/iovs.03-0038.
Macrophage recruitment to the choroid has been proposed to contribute to the pathogenesis of choroidal neovascularization (CNV) in AMD. The study was conducted to determine whether treatment with clodronate liposomes (CL(2)MDP-lip), which cause depletion of blood monocytes and lymph node macrophages, diminishes the severity of neovascularization in a mouse model of laser-induced CNV.
Laser-induced CNV was performed in female 16-month-old C57BL/6 mice. Macrophages were depleted by use of CL(2)MDP-lip intraperitoneally and subcutaneously 72 and 24 hours before and every 2 to 3 days after laser injury. Control mice received injections of either PBS alone or PBS liposomes. Blood monocyte and choroidal macrophage depletion were documented by flow cytometry and choroidal flatmount preparation analysis, respectively. Two weeks after laser injury, mice were injected intravenously with fluoresceinated dextran. The right eyes were removed and prepared for flatmount analysis of CNV surface area (in relative disc areas or DA), vascularity (relative fluorescence), and cellularity (propidium iodide stain). The mice were then perfused with 10% formaldehyde, and the left eyes were removed for histopathology. The means of the various parameters for four CNV lesions per eye were calculated. Fluorescein angiography was also performed.
Flow cytometry of circulating monocytes and immunohistochemical analysis of choroidal macrophage density confirmed the effective depletion of blood monocytes and choroidal macrophages respectively in CL(2)MDP-lip-treated mice. Compared with the control, flatmount analysis of macrophage depleted mice demonstrated a significant reduction in size of the CNV area (2.8 +/- 0.5 DA vs. 1.4 +/- 0.1 DA; P < 0.043). The treated group also revealed less vascularity (1.6 +/- 0.1 units vs. 1.1 +/- 0.0 units; P < 0.0092) and cellularity of CNV lesions (3.3 +/- 0.6 DA vs. 1.7 +/- 0.1 DA, P < 0.04). Histopathology revealed that, in the macrophage-depleted group, CNV was smaller in diameter (1270 +/- 73 pixels vs. 770 +/- 82 pixels, P < 0.0006) and thickness (120 +/- 7 pixels vs. 96 +/- 7 pixels, P < 0.019).
Macrophage depletion using CL(2)MDP-lip reduces size, cellularity, and vascularity of CNV. This observation supports the hypothesis that macrophages contribute to the severity of CNV lesions.
有研究提出巨噬细胞募集至脉络膜会促进年龄相关性黄斑变性（AMD）中脉络膜新生血管（CNV）的发病机制。本研究旨在确定用氯膦酸二钠脂质体（CL(2)MDP-lip）进行治疗是否会减轻激光诱导的CNV小鼠模型中新生血管形成的严重程度，氯膦酸二钠脂质体会导致血液单核细胞和淋巴结巨噬细胞减少。
对16月龄雌性C57BL/6小鼠进行激光诱导的CNV实验。在激光损伤前72小时和24小时以及损伤后每2至3天，通过腹腔和皮下注射CL(2)MDP-lip来消耗巨噬细胞。对照小鼠分别注射单独的PBS或PBS脂质体。通过流式细胞术和脉络膜铺片制备分析分别记录血液单核细胞和脉络膜巨噬细胞的消耗情况。激光损伤两周后，给小鼠静脉注射荧光葡聚糖。取出右眼并制备用于对CNV表面积（以相对视盘面积或DA表示）、血管化程度（相对荧光）和细胞密度（碘化丙啶染色）进行铺片分析。然后用10%甲醛灌注小鼠，取出左眼进行组织病理学检查。计算每只眼睛四个CNV病变的各种参数的平均值。还进行了荧光素血管造影。
循环单核细胞的流式细胞术和脉络膜巨噬细胞密度的免疫组织化学分析分别证实了CL(2)MDP-lip处理的小鼠中血液单核细胞和脉络膜巨噬细胞的有效消耗。与对照组相比，对巨噬细胞消耗的小鼠进行铺片分析显示CNV区域大小显著减小（2.8±0.5 DA对1.4±0.1 DA；P<0.043）。治疗组还显示CNV病变的血管化程度较低（1.6±0.1单位对1.1±0.0单位；P<0.0092）和细胞密度较低（3.3±0.6 DA对1.7±0.1 DA，P<0.04）。组织病理学显示，在巨噬细胞消耗组中，CNV的直径（1270±73像素对770±82像素，P<0.0006）和厚度（120±7像素对96±7像素，P<0.019）较小。
使用CL(2)MDP-lip消耗巨噬细胞可减小CNV的大小、细胞密度和血管化程度。这一观察结果支持了巨噬细胞会影响CNV病变严重程度的假说。
