Figure 1.
Eyes of mice with chronic MCMV infection show more severe CNV as determined by flatmount preparations.
Groups of adult C57BL/6 mice were inoculated intraperitoneally with either MCMV or UV-inactivated virus. At 6 days, 6 weeks, or 12 weeks after inoculation, all mice were subjected to laser treatment to induce CNV, and, four weeks later, flatmount preparations of the posterior pole of mouse eyes were stained with propidium iodide. Flatmount preparations of representative individual mouse eyes showing areas of CNV (white outlines). (D = Optic Disc) (Magnification = 50×) (Bar = 100 um)) (A) Mouse inoculated with UV-inactivated MCMV (control) for 12 weeks prior to laser treatment. (B) Mouse infected with MCMV for 6 days prior to laser treatment. (C) Mouse infected with MCMV for 6 weeks prior to laser treatment. (D) Mouse infected with MCMV for 12 weeks prior to laser treatment.
Figure 2.
Comparative quantitative analysis of flatmount preparations of eyes of all mice of each group confirm that chronic MCMV infection results in more severe CNV.
Groups of adult C57BL/6 mice were inoculated intraperitoneally with either MCMV or UV-inactivated virus (control). At 6 days, 6 weeks, or 12 weeks after inoculation, all mice were subjected to laser treatment to induce CNV, and. four weeks. later flatmount preparations of the posterior pole of mouse eyes were stained with propidium iodide and assessed for different parameters of CNV severity. Asterisks indicate statistical significance when compared with control (p = <0.0001). (A) Comparison of cellular margins (lesion size). (B) Comparison of frequency of large lesions (>2.2 disc areas). (C) Comparison of vascular area (disc areas). (D) Comparison of cellular density index.
Figure 3.
Eyes of mice with chronic MCMV infection show more severe CNV as determined by histopathologic analysis.
Groups of adult C57BL/6 mice were inoculated intraperitoneally with either MCMV or UV-inactivated virus. At 6 days, 6 weeks, or 12 weeks after inoculation, all mice were subjected to laser treatment to induce CNV, and, four weeks later, retinal sections were subjected to histopathologic analysis. (A) Hematoxylin and eosin-stained histopathologic sections of representative individual mouse retinas four weeks after laser treatment showing areas of CNV (white outlines and arrows). (Magnification = 100×) Mouse inoculated with UV-inactivated MCMV (control); mouse infected with MCMV for 6 days prior to laser treatment; mouse infected with MCMV for 6 weeks prior to laser treatment; mouse infected with MCMV for 12 weeks prior to laser treatment. (B) Comparative quantitative analysis of all animals of each group for size of CNV areas as determined by histopathologic analysis. Asterisk indicates statistical significance when compared with control (p = <0.0004).
Figure 4.
Lung tissues collected from chronically infected mice are positive for MCMV-specific IE1 DNA sequences.
Representative 1% agarose gel showing positive signal for PCR assay for MCMV-specific IE1 gene sequences performed on three samples of lung tissue collected from three mice infected with MCMV for 12 weeks. (M = Molecular weight standards).
Table 1.
PCR assay analysis for MCMV-specific IE1 and gH DNA in various cells and tissues collected from mice infected with MCMV for 6 days (acute infection) or 12 weeks (chronic infection).
Table 2.
Macrophage mRNA levels for various cytokines and mediators relevant to CNV formation following acute or chronic MCMV infection.
Figure 5.
MCMV infection stimulates production of VEGF mRNA and VEGF protein in monolayers of IC-21 mouse macrophages.
Monolayers of IC-21 mouse macrophages were either mock infected or infected with MCMV (2.5 PFU/cell) and analyzed for amounts of VEGF mRNA and VEGF protein at 24 hour and/or 48 hour postinfection. Asterisks indicate statistical significance when compared with mock-infected monolayers. (A) Quantitative RT-PCR assay for VEGF mRNA (black bars) and TNF-α mRNA (gray bars) in monolayers of IC-21 macrophages either mock-infected, treated with LPS, inoculated with UV-inactivated virus, or infected with MCMV at 24 hr or 48 hr after infection and compared with mock-infected monolayers. (p = ≤0.04) (B) ELISA for VEGF protein (black bars) in monolayers of IC-21 macrophages infected with MCMV at 48 hr postinfection and compared with mock-infected monolayers (p = 0.01).
Figure 6.
MCMV infection of monolayers of IC-21 mouse macrophages results in VEGF production by virus-infected and bystander uninfected cells.
Monolayers of IC-21 mouse macrophages were either mock infected or infected with MCMV (2.5 PFU/cell). At 24 hour postinfection (MCMV 24 hr pi) or 48 hour postinfection (MCMV 48 hr pi), all mock-infected and MCMV-infected cells were harvested, fixed, and reacted with either control rabbit IgG or anti-VEGF rabbit IgG as primary antibody for visualization of VEGF production. All cells were counterstained with DAPI to visualize cell nuclei. VEGF = Red; DAPI = Blue (Magnification = 200×) White circle emphasizes focus of cells at 48 hr postinfection undergoing cytopathology and plaque formation. Quantification of cells exhibiting positive staining for VEGF was accomplished by using mean values of the number of cells showing obvious red cytoplasmic staining within five random microscopic fields of view.
Figure 7.
Ganciclvir treatment reduces levels of VEGF mRNA and TNF-α mRNA production by splenic macrophages collected from chronically infected mice.
Groups of adult C57BL/6 mice were inoculated intraperitoneally with either MCMV or mock-infected with maintenance medium. At 12 weeks after inoculation, groups of MCMV-infected or mock-infected mice were treated intraperitoneally with ganciclovir for 7 days (40 mg/kg/day). Parallel groups of untreated control MCMV-infected or mock-infected mice received daily intraperitoneal injections of phosphate-buffered saline for 7 days. Following the 7-day regimen of ganciclovir or phosphate-buffered saline treatment, splenic macrophages were collected from treated and untreated MCMV-infected animal groups and compared with splenic macrophages collected from treated and untreated mock-infected animal groups by quantitative real-time RT-PCR assay for comparison of levels of TNF-α mRNA and VEGF mRNA production. Asterisks indicate statistical significance when compared with parallel treated or untreated mock-infected animals. (p = ≤0.009). Gray bars = TNF-α mRNA; Black bars = VEGF mRNA.
Figure 8.
Ganciclvir treatment reduces levels of VEGF mRNA production by MCMV-infected IC-21 macrophages.
Monolayers of IC-21 mouse macrophages were either mock infected or infected with MCMV (2.5 PFU/cell), treated at 1 hour postinfection with 0, 15, 30, or 60 uM of ganciclovir, and harvested at 24 hour postinfection. (A) Quantitative real-time PCR assay for comparison of levels of VEGF mRNA production. Three independent experiments were performed. (B) Immunostaining for detection of cytoplasmic VEGF production. VEGF = Red; DAPI = Blue (Magnification = 200×).