Macrophage depletion diminishes lesion size and severity in experimental choroidal neovascularization

Diego G. Espinosa-Heidmann, Ivan J. Suner, Eleut P. Hernandez, Dagoberto Monroy, Karl G. Csaky, Scott W. Cousins

Research output: Contribution to journalArticle

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Abstract

PURPOSE. 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 (CL2MDP-lip), which cause depletion of blood monocytes and lymph node macrophages, diminishes the severity of neovascularization in a mouse model of laser-induced CNV. METHODS. Laser-induced CNV was performed in female 16-month-old C57BL/6 mice. Macrophages were depleted by use of CL2MDP-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. RESULTS. Flow cytometry of circulating monocytes and immunohistochemical analysis of choroidal macrophage density confirmed the effective depletion of blood monocytes and choroidal macrophages respectively in CL2MDP-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). CONCLUSIONS. Macrophage depletion using CL2MDP-lip reduces size, cellularity, and vascularity of CNV. This observation supports the hypothesis that macrophages contribute to the severity of CNV lesions.

Original languageEnglish (US)
Pages (from-to)3586-3592
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume44
Issue number8
DOIs
StatePublished - Aug 1 2003

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Choroidal Neovascularization
Macrophages
Lip
Monocytes
Lasers
Liposomes
Flow Cytometry
Clodronic Acid
Choroid
Propidium
Fluorescein Angiography
Wounds and Injuries
Dextrans
Inbred C57BL Mouse
Formaldehyde
Coloring Agents
Fluorescence
Lymph Nodes

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Macrophage depletion diminishes lesion size and severity in experimental choroidal neovascularization. / Espinosa-Heidmann, Diego G.; Suner, Ivan J.; Hernandez, Eleut P.; Monroy, Dagoberto; Csaky, Karl G.; Cousins, Scott W.

In: Investigative Ophthalmology and Visual Science, Vol. 44, No. 8, 01.08.2003, p. 3586-3592.

Research output: Contribution to journalArticle

Espinosa-Heidmann, Diego G. ; Suner, Ivan J. ; Hernandez, Eleut P. ; Monroy, Dagoberto ; Csaky, Karl G. ; Cousins, Scott W. / Macrophage depletion diminishes lesion size and severity in experimental choroidal neovascularization. In: Investigative Ophthalmology and Visual Science. 2003 ; Vol. 44, No. 8. pp. 3586-3592.
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abstract = "PURPOSE. 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 (CL2MDP-lip), which cause depletion of blood monocytes and lymph node macrophages, diminishes the severity of neovascularization in a mouse model of laser-induced CNV. METHODS. Laser-induced CNV was performed in female 16-month-old C57BL/6 mice. Macrophages were depleted by use of CL2MDP-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. RESULTS. Flow cytometry of circulating monocytes and immunohistochemical analysis of choroidal macrophage density confirmed the effective depletion of blood monocytes and choroidal macrophages respectively in CL2MDP-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). CONCLUSIONS. Macrophage depletion using CL2MDP-lip reduces size, cellularity, and vascularity of CNV. This observation supports the hypothesis that macrophages contribute to the severity of CNV lesions.",
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T1 - Macrophage depletion diminishes lesion size and severity in experimental choroidal neovascularization

AU - Espinosa-Heidmann, Diego G.

AU - Suner, Ivan J.

AU - Hernandez, Eleut P.

AU - Monroy, Dagoberto

AU - Csaky, Karl G.

AU - Cousins, Scott W.

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N2 - PURPOSE. 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 (CL2MDP-lip), which cause depletion of blood monocytes and lymph node macrophages, diminishes the severity of neovascularization in a mouse model of laser-induced CNV. METHODS. Laser-induced CNV was performed in female 16-month-old C57BL/6 mice. Macrophages were depleted by use of CL2MDP-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. RESULTS. Flow cytometry of circulating monocytes and immunohistochemical analysis of choroidal macrophage density confirmed the effective depletion of blood monocytes and choroidal macrophages respectively in CL2MDP-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). CONCLUSIONS. Macrophage depletion using CL2MDP-lip reduces size, cellularity, and vascularity of CNV. This observation supports the hypothesis that macrophages contribute to the severity of CNV lesions.

AB - PURPOSE. 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 (CL2MDP-lip), which cause depletion of blood monocytes and lymph node macrophages, diminishes the severity of neovascularization in a mouse model of laser-induced CNV. METHODS. Laser-induced CNV was performed in female 16-month-old C57BL/6 mice. Macrophages were depleted by use of CL2MDP-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. RESULTS. Flow cytometry of circulating monocytes and immunohistochemical analysis of choroidal macrophage density confirmed the effective depletion of blood monocytes and choroidal macrophages respectively in CL2MDP-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). CONCLUSIONS. Macrophage depletion using CL2MDP-lip reduces size, cellularity, and vascularity of CNV. This observation supports the hypothesis that macrophages contribute to the severity of CNV lesions.

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