optimizing image guided laser-induced choroidal neovascularization in mice

Optimizing Image-Guided Laser-Induced Choroidal Neovascularization in Mice

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Presented by: Dr. Yan Gong, PhDHarvard Medical School

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Micron IV CNV System

Image-Guided Laser

Image-Guided OCT

Optimizing Image-Guided Laser-Induced Choroidal Neovascularization in Mice

Dr. Yan Gong, PhD

The Laboratory of Dr. Lois. E. H. SmithBoston Children’s Hospital

Harvard Medical Schoolyan.gong@childrens.harvard.edu

OR yangong_005@yahoo.com

Copyright 2015 Yan Gong, InsideScientific & Phoenix Research Labs. All Rights Reserved.

Age-related Macular Degeneration

AMD affects over 16 million people in the United States and Europe in 2011. The annual incidence is expected to increase with an aging population, and prevalence in Western countries is anticipated to

reach 25 million by 2020. (Nat Rev Drug Discov. 2012 Nov;11(11):827)

macutene.com

Normal Vision Vision with AMD

Wet AMD - Neovascularization

• Around 10% of AMD patients exhibit wet AMD; however, it accounts for over 90% of the serious lossof vision.

• Wet AMD is characterized by choroidal neovascularization (CNV) with blood vessels from thechoriocapillaris penetrating through Bruch’s membrane into the normal avascular subretinal space.

Laser-induced CNV Model in Mice

• First described using monkey in 1979 (Ryan, 1979), and then adapted to rodents (Yi, 1997).

• Advantages:

Mimics neovessels arising from the choroid through Bruch’s membrane;

Transgenic mice available for mechanism study.

• Disadvantages:

No macula in mice;

Not initiated with aging changes;

Wound healing model involving inflammation.

Experimental Flow Chart

• Only intact eyes without structural abnormalities can be used.

• OCT after laser burn can be used to make sure the photocoagulation is successful.

• Lesion leakage and area can be assessed for statistical analysis.

Lambert V, Lecomte J, Hansen S, Blacher S, et al. (2013) Laser-induced choroidal neovascularization model to study age-related macular degeneration in mice. Nat Protoc 8:2197-2211. doi: 10.1038/nprot.2013.135

Slit Lamp vs Micron

Comparison Items Slit Lamp Micron IV

Cover Glass Needed Not Needed

Size of Laser Spot Adjustable Not Adjustable

Movement of Laser Spot Manual Mechanical

OCT/FFA Component Incompatible Compatible

Slit Lamp vs Micron

• Aiming before lens touching to cornea

• Gently touching of lens to cornea

• Re-aiming after lens touching to cornea

• Aligning the axis of both the mouse eye and the lens

Even Focus is Essential for Consistent Laser Photocoagulation

• Aiming before lens touching to cornea

• Gently touching of lens to cornea

• Re-aiming after lens touching to cornea

• Aligning the axis of both the mouse eye and the lens

Even Focus is Essential for Consistent Laser Photocoagulation

• Aiming before lens touching to cornea

• Gently touching of lens to cornea

• Re-aiming after lens touching to cornea

• Aligning the axis of both the mouse eye and the lens

Even Focus is Essential for Consistent Laser Photocoagulation

• Aiming before lens touching to cornea

• Gently touching of lens to cornea

• Re-aiming after lens touching to cornea

• Aligning the axis of both the mouse eye and the lens

Even Focus is Essential for Consistent Laser Photocoagulation

Laser Burns

• 4 laser burns per eye

• ~2 times of the diameter of the optic nerve disc (OND) away from the center of OND

• >2 times of the diameter of the OND between laser burns

• Avoid major retinal and choroidal vessels

Gong Y, Li J, Sun Y, Fu Z, Liu C-H, et al. (2015) Optimization of an Image-Guided Laser-Induced Choroidal Neovascularization Model in Mice. PLoS ONE 10(7): e0132643. doi:10.1371/journal.pone.0132643

Vaporization Bubble

• The formation of a vaporization bubble immediately after laser photocoagulation indicates the success of a laser burn, which correlates with a rupture of Bruch’s membrane.

FFA & Choroid Flat-Mounts

Ctrl Exp Ctrl Exp

Li J, Liu C-H, Sun Y, Gong Y, Fu Z, et al. Endothelial TWIST1 promotes pathological ocular angiogenesis. Invest Ophthalmol Vis Sci. 2014;55:8267–8277. DOI:10.1167/iovs.14-15623

Data or Mouse Loss

• Cataract and corneal epithelial edema before laser photocoagulation;

• Unsuccessful laser burn without Bruch’s membrane rupture;

• Odd lesion shape due to mouse movement during laser photocoagulation;

• Death of mice after laser photocoagulation;

• Damage of the CNV lesions during tissue dissection and processing.

Data or Mouse Loss

• Cataract and corneal epithelial edema before laser photocoagulation

Data or Mouse Loss

• Cataract and corneal epithelial edema before laser photocoagulation;

• Unsuccessful laser burn without Bruch’s membrane rupture;

• Odd lesion shape due to mouse movement during laser photocoagulation;

• Death of mice after laser photocoagulation;

• Damage of the CNV lesions during tissue dissection and processing.

Data or Mouse Loss

• Odd lesion shape due to mouse movement during laser photocoagulation

Data or Mouse Loss

• Cataract and corneal epithelial edema before laser photocoagulation;

• Unsuccessful laser burn without Bruch’s membrane rupture;

• Odd lesion shape due to mouse movement during laser photocoagulation;

• Death of mice after laser photocoagulation;

• Damage of the CNV lesions during tissue dissection and processing.

• Hemorrhage

• Choroidal damage

• Fused lesions

• Outlier lesions

• Only eligible lesion in an eye

Exclusion Criteria

Poor SH, Qiu Y, Fassbender ES, et al. Reliability of the mouse model of choroidal neovascularization induced by laser photocoagulation. Invest Ophthalmol Vis Sci. 2014;55:6525-6534. DOI:10.1167/ivos.14-15067

• 240 mW is the optimal laser power level for laser photocoagulation in C57BL/6J mice using the Micron IV laser system.

• The CNV lesion area is proportional to the laser power level.

Laser PowerLesion Type (%) 180 mW 240 mW 300 mW 360 mW

Lesion Included 46 (63.9%) 75 (93.8%) 63 (82.9%) 49 (64.5%)

No BM Rupture 20 (27.8%) 2 (2.50%) 1 (1.32%) 0 (0.00%)

Bleeding (G1&G2) 0 (0.00%) 1 (1.25%) 5 (6.58%) 14 (18.4%)

Choroidal Damage 0 (0.00%) 0(0.00%) 2 (2.63%) 5 (6.58%)

Fused Lesion 0 (0.00%) 0 (0.00%) 2 (2.63%) 4 (5.26%)

Outlier Lesion 6 (8.33%) 2 (2.50%) 3 (3.95%) 4 (5.26%)

Total Shots/Mice 72/10 80/10 76/10 76/10

Laser Power 180 mW 240 mW 300 mW 360 mW

Number of CNV Lesions 46 75 63 49

Mean Area CNV (µm2) 17627.4 30433.0 47067.9 76326.2

SEM 12582.2 20368.9 28330.5 31594.9

SD 1855.14 2352.00 3569.30 4513.55

% Relative to 240 mW 57.9 100 155 251

• There are larger lesion area and higher variation in older mice than younger ones, especially female mice.

• C57BL/6J mice at 6-8 weeks old of both genders are most recommended.

Mouse Age/Gender

Mice

6-8 Week

Female 6-8 Week Male

12-16 Week

Female 12-16 Week Male

Number of CNV Lesions 67 58 66 60

Mean Area CNV (µm2) 33202.5 29445.4 71770.9 52104.9

SEM 10835.0 13059.7 35599.2 26895.8

SD 1323.70 1714.82 4381.96 3472.23

% Relative to 6-8 Week

Male 113 100 244 177

Feed ω-6 ω-3

Number of CNV lesions 110 105

Mean area CNV (µm2) 38893.5 28960.5

SEM 18796.5 11896.3

SD 1792.17 1160.96

% inhibition - 25.5

• Dietary intake of omega-3 long-chain polyunsaturated fatty acid reduces laser-induced CNV in mice.

Lesion Area vs ω-6/3 LCPUFA Feed

Summary

• Mice at 6-8 weeks of age are ideal for laser-induced CNV.

• Even focus is essential for producing consistent laser photocoagulation.

• Formation of a vaporization bubble indicates successful laser shot.

• Laser shot at 240 mW for 70 ms is optimal for C57BL/6J mice.

• Exclusion criteria are necessary for evaluation of CNV lesions.

References

• Lambert V, Lecomte J, Hansen S, Blacher S, et al. (2013) Laser-induced choroidal neovascularization model to study age-related macular degeneration in mice. Nat Protoc 8:2197-2211. doi: 10.1038/nprot.2013.135

• Poor SH, Qiu Y, Fassbender ES, et al. (2014) Reliability of the mouse model of choroidalneovascularization induced by laser photocoagulation. Invest Ophthalmol Vis Sci 55:6525-6534. doi:10.1167/ivos.14-15067

• Li J, Liu C-H, Sun Y, Gong Y, Fu Z, et al. (2014) Endothelial TWIST1 promotes pathological ocular angiogenesis. Invest Ophthalmol Vis Sci 55:8267–8277. doi:10.1167/iovs.14-15623

• Gong Y, Li J, Sun Y, Fu Z, Liu C-H, et al. (2015) Optimization of an Image-Guided Laser-Induced Choroidal Neovascularization Model in Mice. PLoS ONE 10(7): e0132643. doi:10.1371/journal.pone.0132643

Thank You

Yan

Lois

Jing

Jie

yan.gong@childrens.harvard.edu

Disclosure

• No Financial Disclosure.

• Dr. Gong, Dr. Smith, Dr. Chen and all other lab members have no financial interest in the Phoenix Research Labs.

Thank You!For additional information on Phoenix Research Labs equipment specially designed for vision research in rodents please visit:

http://www.phoenixreslabs.com/