Macular Mitochondrial

Flavoprotein Autofluorescence

in Eyes with

Primary Open Angle Glaucoma

Alexander Pinhas1, 2, Moataz Razeen MD1, Theodora Danias1,3, Nikhil

Menon1,2, Nadim Choudhury1,2, Matthew Field4, Joseph Panarelli MD1, Richard B

Rosen MD1

1. New York Eye and Ear Infirmary of Mount Sinai, New York, NY, USA 2. Icahn School of Medicine at Mount Sinai, New York, NY, USA3. Staten Island Technical High School, Staten Island, NY, USA

4. University of Michigan, Ann Arbor, MI, USA

2

Marrus Family Foundation, Bendheim‐Lowenstein Family Foundation, Wise Family Foundation, RD & Linda Peters Foundation, Edith C. Blum Foundation, Chairman's Research Fund of the New York Eye and Ear Infirmary, and an Unrestricted Grant from Research to Prevent Blindness

Acknowledgments

Commercial Relationship DisclosureA Pinhas: NoneM Razeen: NoneT Danias: NoneN Menon: NoneN Choudhury: NoneM Field: OcuSciences: Code E (Employment)J Panarelli: NoneR Rosen: Clarity: Code C (Consultant); Opticology: Code I (Personal Financial Interest); OD-OS: Code C (Consultant); Carl Zeiss Meditech: Code C (Consultant); Optovue: Code C (Consultant); Advanced Cellular Technologies: Code C (Consultant)

Primary Open Angle Glaucoma (POAG): Screening and Assessment

In POAG, intra-ocular pressure (IOP) and structural and functional indices of disease1 are often not sensitive enough to detect its earliest onset, or short-term response to treatment.2

3

1. Preferred Practice Pattern, Primary Open-Angle Glaucoma, American Academy of Ophthalmology 2010.2. Bettin & Matteo. Ophthalmic Res. 2013;50(4):197-208.

Thus, the search for more sensitiveassessment tools continues!

Mitochondrial Metabolic Dysfunction1-3

▶ In retinal ganglion cell (RGC) somas and axons, reactive oxygen species (ROS) cause damage to the mitochondrial oxidative phosphorylation machinery (OxPhos) over time

4

1. Osborne. Exp Eye Res. 2010.2. Munemasa et al. J Neurochem. 2010.3. Zhang et al. J Biol Chem. 1990.

Image borrowed from the Medical Research Council<www.mrc-mbu.cam.ac.uk>

▶ Accumulated damage causes a hypo-metabolic state with decreased ATP production, and with secondary insults leads to RGC dysfunction and eventual death

5

Can We Non-Invasively Measure Mitochondrial Metabolic Dysfunction?

Flavoprotein Fluorescence (FPF)

6

Image borrowed from <www.pixgood.com>

Oxidized (e-1 poor)

Reduced (e-1 rich)

▶ Flavoproteins are redox cofactors serving as temporary place holders for electrons

Flavoprotein Fluorescence (FPF)

7

Image borrowed from <www.pixgood.com>

Oxidized (e-1 poor)

Reduced (e-1 rich)

▶ Exhibit autofluorescence, but only in the oxidized state

▶ Flavoproteins are redox cofactors serving as temporary place holders for electrons

Flavoprotein Fluorescence (FPF)

8

Image borrowed from <www.pixgood.com>

▶ Thus, in POAG, FPF should be observed to increase

▶ Oxidized:reduced ratio of metabolically-active flavoproteins increases with decreased OxPhos activity1

Oxidized (e-1 poor)

Reduced (e-1 rich)

1. Field et al. Exp Eye Res. 2011.

OcuSciencesTM OcuMet BeaconTM:

9

RMA is coupled with a standard slit lamp chin rest

▶ Retinal Metabolic Analysis (RMA) Machine Measures Retinal FPF in Vivo

10

▶ 5 consecutive images using blue excitation light

▶ Shone onto ~15°-circular region of the retina

11

▶ Flavoproteins fluoresce in the green after excitation

▶ Narrow bandpass filter (± 2-3nm) to maximize and optimize FPF signal and minimize other fluorescent signals

12

Intensity (Gray Scale Units)

Num

ber

of P

ixel

s

▶ RMA output is number of pixels versus intensity

▶ FPF score is the mean of the histogram

▶ FPF scores from 5 consecutive images are averaged

The RMA has shown that macular FPF increases with…

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▶ Oxidative stress and apoptosis1

▶ Age2

▶ Diseases such as diabetes mellitus and age-related macular degeneration2-6

1. Field et al. Exp Eye Res. 2011.2. Elner et al. Trans Am Ophthalmol Soc. 2008.3. Elner et al. Arch Ophthalmol. 2008.4. Field et al. Arch Ophthalmol. 2008.5. Field et al. Retina. 2009 Sep. 6. Field et al. Ophthalmic Surg Lasers Imaging.

2012.

Purpose of This Pilot Study

▶ To measure macular and optic disc FPF in eyes with POAG

▶ To compare macular FPF to healthy age-matched controls

▶ To correlate POAG FPF with clinical indicators of disease

14

Methods: Recruitment Criteria

15

▶ Inclusion criteria– Diagnosis of POAG*– BCVA of 20/60 or better, with good central fixation– Pupil dilation of at least 5 mm– Clear phakic lens and clear media

▶ Exclusion criteria– Pre-existing retinal or optic nerve disease other than

POAG– POAG surgery

*All POAG eyes imaged were using IOP-lowering medications.

Subjects Imaged

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n # of eyes Mean Age (yrs)

Age Range (yrs)

Healthy Controls 45 90 54 40-66

POAG 15 25 59 42-68

▶ One eye per subject was used in data analysis▶ Both eyes were used only in determining signal

asymmetry between fellow eyes– Both eyes were imaged in 10/15 POAG patients and

in all 45 healthy controls

Clinical Indices of Disease

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▶ From patient chart– Number of medications (#meds)– BCVA– IOP– Humphrey 24-2 visual fields (VF)

• mean deviation (MD)• pattern standard deviation (PSD)

▶ Spectralis SD-OCT before RMA imaging– Cup-to-disc ratio (CDR)– Circumferential peripapillary RNFL thickness– Macular thickness

Results: Macular FPF in POAGvs. Controls

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Macular FPF of 15 POAG eyes was significantly higher than that of 45 age-matched healthy control eyes.Results are reported as group averages ± SD.

Healthy Controls (n=45) POAG (n=15)0

100

200

300

400

500

600M

acu

lar

FP

F480±134

403±94

t(58)=2.46, p=0.0170

40-49 50-59 60-690

100

200

300

400

500

600

700

Healthy ControlsPOAG

Decade of Life (yrs)

Mac

ula

r F

PF

Macular FPF By Decade of Life

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When stratified by decade of life, significant difference* in FPF between POAG and healthy control eyes was observed only in the 60-69yo group (611±62.3 versus 489±70.8, t(19)=3.86, p=0.0010).

*

Macular FPF Between Fellow Eyes

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There is greater variability in macular FPF signal asymmetry between fellow eyes of 10 POAG patients compared to 45 healthy controls.

Healthy Controls (n=45)

POAG (n=15)0

2

4

6

8

10

12

14%

Dif

fere

nce

13.1±10.6%

6.0±5.8%

t(53)=2.96, p=0.0046

Macular versus Optic Disc FPF

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In POAG eyes, FPF was often greater at the optic disc compared to the macula, but not significantly. We do not have optic disc FPF data for healthy

age-matched controls for comparison

Macular FPF (n=15) Optic disc FPF (n=14)0

100

200

300

400

500

600F

PF

536±151480±134

t(27)=1.06, p=0.299

Correlating FPF WithClinical Indices of Disease

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So far, we have not found statistically-significant correlation between clinical indices of disease and FPF signal in POAG (for either macular or optic disc FPF).

Case: A 61yo Male with POAG

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#Meds :BCVA :

IOP :CDR :

Peripapillary RNFL :Macular FPF :

Optic Disc FPF :

2

20/40 15 mmHg 0.55 107 µm 550±7.69 569±21.1

#Meds :BCVA :

IOP :CDR :

Peripapillary RNFL :Macular FPF :

Optic Disc FPF :

2

20/100 13 mmHg 0.99 38.8 µm 695±15.1 828±3.21

MD: -4.74PSD: 5.64

MD: -30.8PSD: 3.37

325µm

OD OS

MacularThickness

: 295µmMacularThickness

:

Case: A 61yo Male with POAG

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Macular FPF:550±7.69

Macular FPF:695±15.1

OD OS

#Meds :BCVA :

IOP :CDR :

Peripapillary RNFL :Macular FPF :

Optic Disc FPF :

2

20/40 15 mmHg 0.55 107 µm 550±7.69 569±21.1

#Meds :BCVA :

IOP :CDR :

Peripapillary RNFL :Macular FPF :

Optic Disc FPF :

2

20/100 13 mmHg 0.99 38.8 µm 695±15.1 828±3.21

MD: -4.74PSD: 5.64

MD: -30.8PSD: 3.37

Case: A 61yo Male with POAG

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OD OS

288µmOptic DiscThickness

: 190µmOptic DiscThickness

:

#Meds :BCVA :

IOP :CDR :

Peripapillary RNFL :Macular FPF :

Optic Disc FPF :

2

20/40 15 mmHg 0.55 107 µm 550±7.69 569±21.1

#Meds :BCVA :

IOP :CDR :

Peripapillary RNFL :Macular FPF :

Optic Disc FPF :

2

20/100 13 mmHg 0.99 38.8 µm 695±15.1 828±3.21

MD: -4.74PSD: 5.64

MD: -30.8PSD: 3.37

Case: A 61yo Male with POAG

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Optic Disc FPF:569±21.1

Optic Disc FPF:828±3.21

OD OS

#Meds :BCVA :

IOP :CDR :

Peripapillary RNFL :Macular FPF :

Optic Disc FPF :

2

20/40 15 mmHg 0.55 107 µm 550±7.69 569±21.1

#Meds :BCVA :

IOP :CDR :

Peripapillary RNFL :Macular FPF :

Optic Disc FPF :

2

20/100 13 mmHg 0.99 38.8 µm 695±15.1 828±3.21

MD: -4.74PSD: 5.64

MD: -30.8PSD: 3.37

Conclusions

27

▶ FPF measured by the RMA appears sensitive to the increased mitochondrial dysfunction in POAG.

▶ Search continues for indices of disease that FPF may correlate with– Although lack of correlation should not discount FPF

as useful

Future Studies

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▶ Expanding current POAG FPF database

▶ Creating an age-matched normative FPF database at the optic disc

▶ Exploring the use of RMA for earlier POAG diagnosis

▶ Longitudinal studies using RMA for assessing adequacy of therapy in POAG, including substances that enhance mitochondrial function

Thank You From The Team!

29Richard B Rosen MDJoseph Panarelli MD

Moataz Razeen MD

Matthew Field

Theodora Danias Nadim Choudhury

Nikhil Menon

John Choi