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Optimizing Image-Guided
Laser-Induced Choroidal
Neovascularization in Mice
Sponsored by:
Presented by: Dr. Yan Gong, PhD
Harvard Medical School
InsideScientific is an online educational environment
designed for life science researchers. Our goal is to aid in
the sharing and distribution of scientific information
regarding innovative technologies, protocols, research
tools and laboratory services.
Thank you to our webinar sponsor
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. Smith
Boston Children’s Hospital
Harvard Medical School
yan.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 loss
of vision.
• Wet AMD is characterized by choroidal neovascularization (CNV) with blood vessels from the
choriocapillaris 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 Power
Lesion 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 choroidal
neovascularization 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/

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Optimizing image guided laser-induced choroidal neovascularization in mice

  • 1. Optimizing Image-Guided Laser-Induced Choroidal Neovascularization in Mice Sponsored by: Presented by: Dr. Yan Gong, PhD Harvard Medical School
  • 2. InsideScientific is an online educational environment designed for life science researchers. Our goal is to aid in the sharing and distribution of scientific information regarding innovative technologies, protocols, research tools and laboratory services.
  • 3. Thank you to our webinar sponsor
  • 4. Micron IV CNV System
  • 7. Optimizing Image-Guided Laser-Induced Choroidal Neovascularization in Mice Dr. Yan Gong, PhD The Laboratory of Dr. Lois. E. H. Smith Boston Children’s Hospital Harvard Medical School yan.gong@childrens.harvard.edu OR yangong_005@yahoo.com Copyright 2015 Yan Gong, InsideScientific & Phoenix Research Labs. All Rights Reserved.
  • 8. 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
  • 9. Wet AMD - Neovascularization • Around 10% of AMD patients exhibit wet AMD; however, it accounts for over 90% of the serious loss of vision. • Wet AMD is characterized by choroidal neovascularization (CNV) with blood vessels from the choriocapillaris penetrating through Bruch’s membrane into the normal avascular subretinal space.
  • 10. 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.
  • 11. 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
  • 12. Slit Lamp vs Micron
  • 13. 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
  • 14. • 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
  • 15. • 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
  • 16. • 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
  • 17. • 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
  • 18. 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
  • 19. 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.
  • 20.
  • 21. 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
  • 22. 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.
  • 23. Data or Mouse Loss • Cataract and corneal epithelial edema before laser photocoagulation
  • 24. 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.
  • 25. Data or Mouse Loss • Odd lesion shape due to mouse movement during laser photocoagulation
  • 26. 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.
  • 27. • 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
  • 28. • 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 Power Lesion 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
  • 29. • 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
  • 30. 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
  • 31. 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.
  • 32. 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 choroidal neovascularization 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
  • 34. Disclosure • No Financial Disclosure. • Dr. Gong, Dr. Smith, Dr. Chen and all other lab members have no financial interest in the Phoenix Research Labs.
  • 35. Thank You! For additional information on Phoenix Research Labs equipment specially designed for vision research in rodents please visit: http://www.phoenixreslabs.com/