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A model for the easy assessment of pressure-dependent damage to retinal ganglion cells using cyan fluorescent protein-expressing transgenic mice.

Tsuruga H, Murata H, Araie M, Aihara M - Mol. Vis. (2012)

Bottom Line: Laser-treated eyes showed a significantly higher IOP than control eyes from 1 to 7 weeks (p<0.01).The pressure insult and the RGC density showed a significant negative correlation (y=-0.070x+97.2, r=0.75, p=0.0008).Moreover, the central, middle, and peripheral areas measured from the optic disc and each of four retinal quadrant areas also showed significant negative correlations.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Tokyo School of Medicine, Tokyo, Japan.

ABSTRACT

Purpose: To establish an animal model for the easy assessment of pressure-dependent damage to retinal ganglion cells (RGCs) using the B6.Cg-TgN(Thy1-CFP)23Jrs/J transgenic mouse strain (CFP mouse), which expresses cyan fluorescent protein (CFP) in RGCs, and to evaluate pressure-dependent RGC death.

Methods: In 20 CFP mice, right eyes were selected to receive laser-induced ocular hypertension eye and the contralateral eyes remained untouched to serve as controls. Intraocular pressure (IOP) was measured each week in both eyes using the microneedle method up to 8 weeks. Based on the line plot of time (x) and IOP (y) in laser-treated and control eyes, the area surrounded by both lines (∫ΔIOP(y) dx) was calculated as a surrogate value of the pressure insult. At 9 weeks, eyes were enucleated and RGCs expressing CFP were evaluated histologically in retinal whole mounts. The correlation between pressure insult and RGC density was evaluated in the whole eye, three concentric regions, and four quadrants.

Results: Laser-treated eyes showed a significantly higher IOP than control eyes from 1 to 7 weeks (p<0.01). The pressure insult and the RGC density showed a significant negative correlation (y=-0.070x+97.2, r=0.75, p=0.0008). Moreover, the central, middle, and peripheral areas measured from the optic disc and each of four retinal quadrant areas also showed significant negative correlations. Our data demonstrate that each retinal area was almost evenly damaged by IOP elevation.

Conclusions: Laser photocoagulation causes a chronic elevation of IOP in CFP mice. The use of CFP mice enabled us to easily evaluate pressure-dependent RGC damage. This glaucomatous CFP mouse model may contribute to the molecular analysis of neurodegeneration and the development of neuroprotective drugs for glaucoma with a great increase in experimental efficiency.

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Related in: MedlinePlus

Counted areas in the whole mount retina. Squares of 200×200 µm were counted at 600 μm (central), 1200 μm (middle), and 1800 μm (peripheral) from the optic disc in each of four retinal quadrants. Scale bar indicates 2 mm.
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f3: Counted areas in the whole mount retina. Squares of 200×200 µm were counted at 600 μm (central), 1200 μm (middle), and 1800 μm (peripheral) from the optic disc in each of four retinal quadrants. Scale bar indicates 2 mm.

Mentions: Nine weeks after laser photocoagulation, the mice were sacrificed. The eyes were enucleated and immediately fixed in 4% paraformaldehyde in 0.1M phosphate buffered saline (PBS, 0.1M, pH 7.4, Muto Pure Chemicals Co. Ltd. Tokyo, Japan) for 1 h at 4 °C. Four radial relaxing incisions were made and the retina was prepared as a flattened whole mount on a glass slide with a coverslip. Images were obtained using a fluorescence microscope (BX50: Olympus, Tokyo, Japan) with a CFP filter set. The number of RGCs expressing CFP was manually counted using image-processing software (ImageJ) in 12 separate areas of 40,000 μm2. Areas that were 600 μm (central), 1200 μm (middle), and 1800 μm (peripheral) away from the optic disc in each of four retinal quadrants were sampled (Figure 3). The average density of RGCs/mm2 for each retinal area was obtained. The identity of the digitized images was masked before analysis. The RGC survival rate in the laser-treated eye was calculated as a percentage of RGC density in the contralateral control eye. The correlation between pressure insult and RGC survival rate was assessed by regression analysis.


A model for the easy assessment of pressure-dependent damage to retinal ganglion cells using cyan fluorescent protein-expressing transgenic mice.

Tsuruga H, Murata H, Araie M, Aihara M - Mol. Vis. (2012)

Counted areas in the whole mount retina. Squares of 200×200 µm were counted at 600 μm (central), 1200 μm (middle), and 1800 μm (peripheral) from the optic disc in each of four retinal quadrants. Scale bar indicates 2 mm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472928&req=5

f3: Counted areas in the whole mount retina. Squares of 200×200 µm were counted at 600 μm (central), 1200 μm (middle), and 1800 μm (peripheral) from the optic disc in each of four retinal quadrants. Scale bar indicates 2 mm.
Mentions: Nine weeks after laser photocoagulation, the mice were sacrificed. The eyes were enucleated and immediately fixed in 4% paraformaldehyde in 0.1M phosphate buffered saline (PBS, 0.1M, pH 7.4, Muto Pure Chemicals Co. Ltd. Tokyo, Japan) for 1 h at 4 °C. Four radial relaxing incisions were made and the retina was prepared as a flattened whole mount on a glass slide with a coverslip. Images were obtained using a fluorescence microscope (BX50: Olympus, Tokyo, Japan) with a CFP filter set. The number of RGCs expressing CFP was manually counted using image-processing software (ImageJ) in 12 separate areas of 40,000 μm2. Areas that were 600 μm (central), 1200 μm (middle), and 1800 μm (peripheral) away from the optic disc in each of four retinal quadrants were sampled (Figure 3). The average density of RGCs/mm2 for each retinal area was obtained. The identity of the digitized images was masked before analysis. The RGC survival rate in the laser-treated eye was calculated as a percentage of RGC density in the contralateral control eye. The correlation between pressure insult and RGC survival rate was assessed by regression analysis.

Bottom Line: Laser-treated eyes showed a significantly higher IOP than control eyes from 1 to 7 weeks (p<0.01).The pressure insult and the RGC density showed a significant negative correlation (y=-0.070x+97.2, r=0.75, p=0.0008).Moreover, the central, middle, and peripheral areas measured from the optic disc and each of four retinal quadrant areas also showed significant negative correlations.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Tokyo School of Medicine, Tokyo, Japan.

ABSTRACT

Purpose: To establish an animal model for the easy assessment of pressure-dependent damage to retinal ganglion cells (RGCs) using the B6.Cg-TgN(Thy1-CFP)23Jrs/J transgenic mouse strain (CFP mouse), which expresses cyan fluorescent protein (CFP) in RGCs, and to evaluate pressure-dependent RGC death.

Methods: In 20 CFP mice, right eyes were selected to receive laser-induced ocular hypertension eye and the contralateral eyes remained untouched to serve as controls. Intraocular pressure (IOP) was measured each week in both eyes using the microneedle method up to 8 weeks. Based on the line plot of time (x) and IOP (y) in laser-treated and control eyes, the area surrounded by both lines (∫ΔIOP(y) dx) was calculated as a surrogate value of the pressure insult. At 9 weeks, eyes were enucleated and RGCs expressing CFP were evaluated histologically in retinal whole mounts. The correlation between pressure insult and RGC density was evaluated in the whole eye, three concentric regions, and four quadrants.

Results: Laser-treated eyes showed a significantly higher IOP than control eyes from 1 to 7 weeks (p<0.01). The pressure insult and the RGC density showed a significant negative correlation (y=-0.070x+97.2, r=0.75, p=0.0008). Moreover, the central, middle, and peripheral areas measured from the optic disc and each of four retinal quadrant areas also showed significant negative correlations. Our data demonstrate that each retinal area was almost evenly damaged by IOP elevation.

Conclusions: Laser photocoagulation causes a chronic elevation of IOP in CFP mice. The use of CFP mice enabled us to easily evaluate pressure-dependent RGC damage. This glaucomatous CFP mouse model may contribute to the molecular analysis of neurodegeneration and the development of neuroprotective drugs for glaucoma with a great increase in experimental efficiency.

Show MeSH
Related in: MedlinePlus