Alex Pinhas' presentation on flavoprotein fluorescence at the 2015 Association for Research in Vision and Ophthalmology

1. 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, USA
3. 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
2
Commercial Relationship Disclosure
A Pinhas: None
M Razeen: None
T Danias: None
N Menon: None
N Choudhury: None
M Field: OcuSciences: Code E (Employment)
J Panarelli: None
R 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)

3. 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 sensitive
assessment tools continues!

4. 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. 5
Can We Non-Invasively Measure
Mitochondrial Metabolic Dysfunction?

6. 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

7. 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

8. 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.

9. 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. 10
▶ 5 consecutive images using blue
excitation light
▶ Shone onto ~15°-circular region
of the retina

11. 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. 12
Intensity (Gray Scale Units)
NumberofPixels
▶ RMA output is number of pixels
versus intensity
▶ FPF score is the mean of the
histogram
▶ FPF scores from 5 consecutive
images are averaged

13. The RMA has shown that macular
FPF increases with…
13
▶ 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.

14. 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

15. 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.

16. Subjects Imaged
16
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

17. Clinical Indices of Disease
17
▶ 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

18. Results: Macular FPF in POAG
vs. Controls
18
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.
0
100
200
300
400
500
600
Healthy Controls (n=45) POAG (n=15)
MacularFPF
480±134
403±94
t(58)=2.46, p=0.0170

19. 0
100
200
300
400
500
600
700
800
40-49 50-59 60-69
MacularFPF
Decade of Life (yrs)
Healthy Controls
POAG
Macular FPF By Decade of Life
19
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).
*

20. Macular FPF Between Fellow Eyes
20
There is greater variability in macular FPF signal
asymmetry between fellow eyes of 10 POAG patients
compared to 45 healthy controls.
0
5
10
15
20
25
Healthy Controls (n=45) POAG (n=15)
%Difference 13.1±10.6%
6.0±5.8%
t(53)=2.96, p=0.0046

21. Macular versus Optic Disc FPF
21
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
0
100
200
300
400
500
600
700
800
Macular FPF (n=15) Optic disc FPF (n=14)
FPF
536±151
480±134
t(27)=1.06, p=0.299

22. Correlating FPF With
Clinical Indices of Disease
22
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).

23. Case: A 61yo Male with POAG
23
#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: -4.74
PSD: 5.64
MD: -30.8
PSD: 3.37
325µm
OD OS
Macular
Thickness
: 295µm
Macular
Thickness
:

24. Case: A 61yo Male with POAG
24
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: -4.74
PSD: 5.64
MD: -30.8
PSD: 3.37

25. Case: A 61yo Male with POAG
25
OD OS
288µm
Optic Disc
Thickness
: 190µm
Optic Disc
Thickness
:
#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: -4.74
PSD: 5.64
MD: -30.8
PSD: 3.37

26. Case: A 61yo Male with POAG
26
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: -4.74
PSD: 5.64
MD: -30.8
PSD: 3.37

27. 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

28. Future Studies
28
▶ 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

29. Thank You From The Team!
29
Richard B Rosen MDJoseph Panarelli MD
Moataz Razeen MD
Matthew Field
Theodora Danias Nadim
Choudhury
Nikhil Menon
John Choi