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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

Laser procedure to increase intraocular pressure. A: Green spots indicate the scheme of laser photocoagulation performed to increase intraocular pressure (IOP) around the whole circumference of the limbus after flattening the anterior chamber by aspiration of aqueous humor. B: The laser-treated eye of one animal at 8 weeks after laser photocoagulation indicated high IOP. Arrow indicates laser spots.
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f1: Laser procedure to increase intraocular pressure. A: Green spots indicate the scheme of laser photocoagulation performed to increase intraocular pressure (IOP) around the whole circumference of the limbus after flattening the anterior chamber by aspiration of aqueous humor. B: The laser-treated eye of one animal at 8 weeks after laser photocoagulation indicated high IOP. Arrow indicates laser spots.

Mentions: Aqueous outflow in the right eye was obstructed by laser photocoagulation at the corneal limbus, as described previously [19]. Briefly, the right eyes received laser-induced ocular hypertension and the contralateral eyes remained untouched to serve as controls. After anesthesia was administered with an intraperitoneal injection of a solution containing ketamine (100 mg/kg) and xylazine (9 mg/kg) and pupils were dilated by instillation of 5 μl of 0.5% tropicamide and 0.5% phenylephrine hydrochloride (Mydrin-P; Santen Pharmaceutical, Osaka, Japan), laser photocoagulation (532 nm wavelength, 200 mW power, 0.05 s duration, 200 μm spot size) was performed. After flattening the anterior chamber by aspiration of aqueous humor, 50–63 laser shots over a 270° circumference of the limbus were applied (Figure 1). To prevent potential infection, an antibiotic ointment was applied topically after the procedure. Additionally, a drop of 0.01% betamethasone was applied topically for 7 days to prevent inflammation.


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)

Laser procedure to increase intraocular pressure. A: Green spots indicate the scheme of laser photocoagulation performed to increase intraocular pressure (IOP) around the whole circumference of the limbus after flattening the anterior chamber by aspiration of aqueous humor. B: The laser-treated eye of one animal at 8 weeks after laser photocoagulation indicated high IOP. Arrow indicates laser spots.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Laser procedure to increase intraocular pressure. A: Green spots indicate the scheme of laser photocoagulation performed to increase intraocular pressure (IOP) around the whole circumference of the limbus after flattening the anterior chamber by aspiration of aqueous humor. B: The laser-treated eye of one animal at 8 weeks after laser photocoagulation indicated high IOP. Arrow indicates laser spots.
Mentions: Aqueous outflow in the right eye was obstructed by laser photocoagulation at the corneal limbus, as described previously [19]. Briefly, the right eyes received laser-induced ocular hypertension and the contralateral eyes remained untouched to serve as controls. After anesthesia was administered with an intraperitoneal injection of a solution containing ketamine (100 mg/kg) and xylazine (9 mg/kg) and pupils were dilated by instillation of 5 μl of 0.5% tropicamide and 0.5% phenylephrine hydrochloride (Mydrin-P; Santen Pharmaceutical, Osaka, Japan), laser photocoagulation (532 nm wavelength, 200 mW power, 0.05 s duration, 200 μm spot size) was performed. After flattening the anterior chamber by aspiration of aqueous humor, 50–63 laser shots over a 270° circumference of the limbus were applied (Figure 1). To prevent potential infection, an antibiotic ointment was applied topically after the procedure. Additionally, a drop of 0.01% betamethasone was applied topically for 7 days to prevent inflammation.

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