Gene Title Gene Symbol Functionsp21/Cdc42/Rac1-activated kinase 1 PAK1 protein kinase activity p21 (CDKN1A)-activated kinase 2 PAK2 protein kinase ;ATP binding ;transferase activityDown syndrome critical region gene 1-like 1 DSCR1L1 calcium-mediated signalingsuperoxide dismutase 2, mitochondrial SOD2 response to oxidative stressdipeptidylpeptidase 4 DPP4 proteolysis and peptidolysis; immune responseneuronal pentraxin II NPTX2 heterophilic cell adhesion;regulation of synapseserine (or cysteine) proteinase inhibitor SERPINB8 serine protease inhibitor activitycyclin D2 CCND2 regulation of cell cycle;cytokinesisribonuclease, RNase A family RNASE1 RNA binding ;endonuclease activitychondroitin sulfate proteoglycan 2 (versican) CSPG2 heterophilic cell adhesion; cell recognitionDEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked DDX3Y DEAD;ATP bindingprostaglandin D2 synthase 21kDa (brain) PTGDS prostaglandin biosynthesis; transport transmembrane 4 superfamily member 3 TM4SF3 protein amino acid glycosylation;pathogenesisalpha-fetoprotein AFP transport;immune responsekeratin 19 KRT19 structural constituent of cytoskeletonATPase, Ca++ transporting, plasma membrane 4 ATP2B4 cation transport;calcium ion transport;metabolismprostaglandin I2 (prostacyclin) synthase PTGIS prostaglandin biosynthesis;electron transport;lipid metabolismtransferrin receptor (p90, CD71) TFRC proteolysis and peptidolysis; iron ion transport;endocytosis

Table I. Genes expressed in pRPE but not detected in aRPE

ubiquitin specific protease 6 (Tre-2 oncogene) USP6 protein modification /// deubiquitination /// oncogenesisBCL2 binding component 3 BBC3 ---calcium/calmodulin-dependent protein kinase I CAMK1 protein amino acid phosphorylation /// signal transductiondeath associated transcription factor 1 DATF1 regulation of transcription, DNA-dependent /// apoptosisbasic leucine zipper nuclear factor 1 (JEM-1) BLZF1 ---nicotinamide nucleotide adenylyltransferase 2 NMNAT2 ---transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha) TFAP2A ---protein kinase C, alpha PRKCA cell cycle; cell proliferation;induction of apoptosis ubiquitin-conjugating enzyme E2M (UBC12 homolog, yeast) UBE2M ubiquitin cycleTNF receptor-associated factor 2 TRAF2 protein complex assembly /// apoptosis /// signal transductioninterleukin 12A (p35) IL12A immune responsephosphate cytidylyltransferase 2, ethanolamine PCYT2 phospholipid biosynthesis /// biosynthesiszinc finger protein 278, short isoform --- ---paraneoplastic antigen HUMPPA ---programmed cell death 11 PDCD11 rRNA processingleucine-rich repeats and immunoglobulin-like domains 1 LRIG1 ---ubiquitin specific protease 52 USP52 UCH;cysteine-type endopeptidase activity;3.3e-07E1A binding protein p400 EP400 SNF2_N;DNA binding;1.4e-58zinc finger protein 205 ZNF205 regulation of transcription, DNA-dependentsmall nuclear RNA activating complex, polypeptide 4, 190kDa SNAPC4 transcription from Pol II promoter zinc finger protein 44 (KOX 7) ZNF44 regulation of transcription, DNA-dependentADP-ribosylation factor related protein 1 ARFRP1 signal transduction Rho guanine nucleotide exchange factor (GEF) 12 ARHGEF12 PDZ;intracellular signaling cascade;1.5e-10phosphatidylserine receptor PTDSR ---dead ringer-like 1 (Drosophila) DRIL1 regulation of transcription, DNA-dependentMHC class I polypeptide-related sequence B MICB response to stress /// cellular defense response SH3 domain binding glutamic acid-rich protein SH3BGR protein complex assemblynuclear factor of kappa light polypeptide gene enhancer NFKBIL1 ---transcription termination factor, RNA polymerase I TTF1 transcription terminationzinc finger protein 282 ZNF282 regulation of transcription, DNA-dependentperoxisomal acyl-CoA thioesterase PTE1 lipid metabolism /// acyl-CoA metabolism protein kinase, cAMP-dependent, regulatory, type II, beta PRKAR2B protein phosphorylation;intracellular signaling cascadeguanidinoacetate N-methyltransferase GAMT creatine biosynthesis /// muscle contractionleucine rich repeat neuronal 4 LRRN4 neurogenesismitogen-activated protein kinase 8 interacting protein 3 MAPK8IP3 vesicle-mediated transport /// regulation of JNK cascademyeloid/lymphoid or mixed-lineage leukemia 4 MLL4 regulation of transcription, DNA-dependent; apoptosisinterleukin 1 receptor accessory protein IL1RAP ---heat shock 70kDa protein 4 HSPA4 ---zinc finger protein 23 (KOX 16) ZNF23 regulation of transcription, DNA-dependentelongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 2 ELOVL2 fatty acid biosynthesispolymerase (DNA directed), gamma 2, accessory subunit POLG2 DNA replication /// DNA repair /// protein biosynthesisteratocarcinoma-derived growth factor 3, pseudogene TDGF3 ---zinc finger, DHHC domain containing 18 ZDHHC18 ---mitogen-activated protein kinase kinase 5 MAP2K5 signal transductionp53-associated parkin-like cytoplasmic protein PARC ---caspase recruitment domain family, member 10 CARD10 apoptosis;activation of NF-kappaB-inducing kinasecell growth regulator with EF hand domain 1 CGREF1 ---adrenergic, beta-2-, receptor, surface ADRB2 activation of MAPK /// receptor mediated endocytosis P450 (cytochrome) oxidoreductase POR electron transport

Table II. Selected Genes expressed in aRPE but not in pRPE

Conclusions: There are some similarities but significant differences in the gene expression profile of cultured adult and immortalized ARPE cells, and it is important to note that some specific genes are only expressed in one of these two groups. These studies suggest caution should be exercised when generalizing results obtained from ARPE-19 to results that would be obtained with adult RPE.

DIFFERENCES IN GENE EXPRESSION PROFILE OF CULTURED ADULT VERSUS IMMORTALIZED HUMAN RPE

Lee Geng, Hui Cai and Lucian V. Del PrioreDepartment of Ophthalmology, Columbia University, New York, New York

Purpose: Immortalized human RPE (cell line ARPE-19) are used widely to draw inferences about the behavior of adult RPE. We have used DNA microarray analysis to compare the gene expression profiles of these two cell types.

Methods: Cultured primary RPE from five human donors (age: 48 - 80 years) and ARPE-19 cultured to confluence in five dishes were used for DNA microarray study. Total RNA was isolated using a Qiagen RNeasy Mini Kit. First and second strand cDNA were synthesized with a T7-(dT)24 oligomer for priming and double-stranded cDNA was cleaned with Phase Lock Gels-Phenol/Chloroform extraction and ethanol precipitation. Biotin-labeled antisense cRNA was produced by an in vitro transcription reaction (ENZO BioArray High Yield RNA Transcript Labeling Kit) and incubated with fragmentation buffer (Tris-acetate, KOAc and MgOAcat; 94oC for 35 minutes). Target hybridization, washing, staining and scanning probe arrays were done following an Affymetrix GeneChip Expression Analysis Manual. Microarray data were treated with normalization, log transformation, statistic determination for “presence” or “absence” for RPE gene expression profile. Clustering analysis, including PCA was used with Affymetrix Microarray Suite 5.0, Genesis 1.30 software.

Supported by Research to Prevent Blindness, Robert L. Burch III Fund, and the Foundation Fighting Blindness

ARPE

primary RPE

Figure 1. Principle Component Analysis (A) and hierarchic clustering analysis (B) demonstrate that the gene expression profile of the adult RPE (shown in blue color group) and ARPE-19 ( shown in red color group) cluster into two distinct groups with no discernable overlap.

Figure 2. A. a scatter plot of expression levels of about 6,000 genes in pRPE vs. aRPE-19 shows an incomplete overlap in the gene expression profiles of these two cells types.B. Figure shows the distribution of differentially (1.5-fold) expressed genes in pRPE and aRPE-19 cell types

Figure 3. The expression of 5,932 genes (out of 12,600 genes on microarray Human 95UA chip) was detected in ARPE-19 cells, in comparison to expression of only 4,849 genes in adult RPE cells from all 5 human donor eyes aRPE-19 express

Numbers of Gene Expressed in RPE cells

pRPE ARPE-190

1000

2000

3000

4000

5000

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7000

Nu

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er o

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enes

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Genes expressed in ARPE cells but not detected in adult RPE cells

Results:

Genes expressed in adult RPE cells but not detected in aRPE-19 cells

Bruch's plastic0

100

200

300

400

young old0

100

200

300

400

age of Bruch's membrane

young older0

10

20

30

40

age of Bruch's membrane

Conclusions: This study suggests that Bruch’s membrane promote RPE phagocytosis compared to bare plastic tissue culture wells, and aging of Bruch’s membrane reduces the ability of RPE to ingest beads. To our knowledge, this is the first demonstration that aging of Bruch’s membrane can modulate RPE phagocytosis ability. Further study is required to determine the implications of this age-dependent decrease in RPE phagocytosis in the pathogenesis of AMD.

AGING OF BRUCH’S MEMBRANE DECREASES RETINAL PIGMENT EPITHELIUM (RPE) PHAGOCYTOSIS

Reiko Koyama, Hui Cai and Lucian V. Del PrioreDepartment of Ophthalmology, Columbia University, NY, New York

Purpose: The earliest changes in age-related macular degeneration occur within Bruch’s membrane. Bruch’s membrane aging affects the attachment and survival of the overlying RPE.1-3 Herein we determine the effects of Bruch’s membrane aging on RPE phagocytosis ability.

Methods: Explants of human Bruch’s membrane were prepared as previously described. Donor ages(young eyes: ages 33 - 44; older group: 73 -94). 1-3 Native RPE were removed by bathing the choroid-BM-RPE complex with 0.02 N ammonium hydroxide followed by washing in PBS. The choroid-BM complex was set on polytetrafluoroethylene membrane with the basal lamina of the RPE facing the membrane. 4% agarose was poured onto the choroid -BM- complex from choroidal side and the tissue was kept at 4oC to solidify the agarose. The polytetrafluoroethylene membrane was peeled away and 6 mm circular buttons were trephined from choroid-BM-gel complex. Buttons were placed on 4% agarose at 37oC in non-treated polystyrene wells of a 96 well plate (Figure1). 50,000 immortalized ARPE-19 were seeded onto wells containing Bruch’s membrane explants and bare control wells (plastic only) for 72 hours(5 wells for each age group). 1ul of fluorescent latex beads (3.6x105beads/ul) were added to each well for another 24 hours. ARPE-19 were passaged by trypsinization. Ingested beads were counted using a FACS Flow Cytometer. Data were generated with at least three independent experiments.

References:1. Tezel TH, Del Priore LV, Kaplan HJ. Fate of Human Retinal Pigment Epithelial Cells Seeded onto Layers of Human Bruch’s Membrane. Invest Ophthalmol Vis Sci 1999;40:467-476.2. Tezel TH, Del Priore LV. Repopulation of Different Layers of Host Human Bruch’s Membrane by Retinal Pigment Epithelial Cell Grafts. Invest Ophthalmol Vis Sci 1999;40:767-774.

3. Del Priore LV, Tezel TH. Reattachment Rate of Human Retinal Pigment Epithelium to Layers of Human Bruch’s Membrane. Arch Ophthalmol 116;335-341, 1998.

Supported by Research to Prevent Blindness, Robert L. Burch III Fund, and the Foundation Fighting Blindness

Fig.3 Percentage of RPE cells that ingest beads was higher for RPE cultured onto Bruch’s membrane explant than on bare plastic wells (22.22±11.59% vs. 10.04±3.52%).

% o

f R

PE

in

ges

tin

g b

ead

s

Fig.2. Flow cytometry histogram demonstrating the distribution of fluorescent intensity. RPE cell population which ingest fluorescent beads (green zone) in young BM group (A) is larger than older BM group (B).

Fig 1. Photo shows 6-mm circular buttons which were trephined from BM and placed into 96 well plate

Fig.4 Percentage of RPE cells which have the ability to ingest beads on younger Bruch’s membrane versus older Bruch’s membrane were similar (25.74±15.94% vs. 22.22±11.59%).

p>0.05

% o

f R

PE

in

ges

tin

g b

ead

s

p<0.008

Fig. 5. Comparison of the phagocytosis ability of RPE cells cultured on young Bruch’s membrane explant versus older BM. Average fluorescent intensity per cell (measure of capacity of phagocytosis per cell) on Bruch’s membrane was higher than on plastic (291.61±80.91 vs. 167.50±35.01).

Flo

ure

scen

t in

ten

sity

/cel

l

p<0.0003

Fig. 6 Observation of the population of cells that had ingested beads. The average fluorescent intensity per cell, which is a measure of capacity of phagocytosis per cell in the younger Bruch’s membrane group was higher than on older Bruch’s membrane (312.60±83.80 vs. 267±71.08)

Flu

ore

scen

t in

ten

sity

/cel

l

B. on older Bruch’s membrane

cells ingesting beadscells not ingesting beads

fluorescent intensity

cell

cou

nts

A. on younger Bruch’s membrane

cells ingesting beadscells not ingesting beads

fluorescent intensity

cell

cou

nts

Bruch's plastic0

10

20

30

Results:

p<0.02

Supported by Research to Prevent Blindness, Robert L. Burch III Fund, and the Foundation Fighting Blindness.

Conclusions: Age-related changes within BM alone induce significant spreading of the gene expression profile of proliferation, apoptosis and cell migration genes, with no change in angiogenesis genes. These observations suggest some of cellular changes that develop within the RPE as a function of age, such as occur in age-related macular degeneration, may be the result of substrate-induced alterations in the behavior of the overlying RPE.

BRUCH'S MEMBRANE AGING ALTERS THE RPE EXPRESSION PROFILE OF PROLIFERATION, MIGRATION AND APOPTOSIS BUT NOT ANGIOGENESIS GENES

Hui Cai, Lucian V. Del PrioreDepartment of Ophthalmology, Columbia University, New York, New York

Purpose: Principal component analysis (PCA) is a technique used to determine global changes in gene expression in response to changing cellular conditions. We have used PCA to determine the gene expression pattern of the retinal pigment epithelium (RPE) in response to age-related changes within Bruch’s membrane (BM).

Methods: Immortalized human ARPE-19 cells were seeded onto human BM (five young samples: donor age = 31- 47 yr and five older samples: donor age = 71 – 81 years) harvested from human eye bank eyes. ARPE-19 were harvested 72 hours after seeding onto human BM and total RNA was isolated using a Qiagen RNeasy Mini Kit. First and second strand cDNAs synthesis, Biotin-labeled antisense cRNA Target hybridization, washing, staining and scanning probe arrays were done following an Affymetrix GeneChip Expression Analysis Manual. RPE gene expression profile was analyzed with Affymetrix Miroarray Suite 5.0, SAM and Genesis 1.3 software.

Results:

Figure 1. The expression of approximately 6,000 genes (out of 12,600 genes on microarray Human 95UA chip) was detected. Scatter plot of gene expression within RPE cultured onto 31 year-old vs 38 year-old Bruch’s membrane. More than 96% of genes are expressed consistently among all samples tested within the young age group (data not shown). The correlation co-efficient is 0.989 suggesting limited variation between these individuals.

0

2000

4000

6000

8000

10000

12000

14000

0 2000 4000 6000 8000 10000 12000 14000

on 31 yo BM

on

38 y

o B

M

PC

#2

18.3

%

PC#1 47.2%

PC

#2

22.

0%

B.

PC#1 33.1%

PC

#2

18.

4%

C. D.

PC#1 35/2%

PC

#2

21

.0%

A.

PC#3 1

2.4%

PC#1 27.4%PC#3

10.

7%

PC#3 1

1.3%

PC#3 15.5%

Figure 2. Principal component analysis of gene expression. (A) The pattern of gene expression of human RPE seeded onto Bruch’s membrane explants from younger donors (blue) shows a tighter clustering than the gene expression profile of RPE seeded onto older donors (red) Bruch’s membrane (B) Cell proliferation and migration genes of RPE seeded onto young Bruch’s membrane (blue) show a tight clustering with spread in the expression profile of proliferation-related genes of human RPE seeded onto older Bruch’s membrane explants (red). (C) There was a similar pattern for apoptosis-related and genes . (D) There is no age-dependent alteration in the spread in the expression profile of angiogenesis genes.

qRT-PCRold/young P value old/young

160025_at transforming growth factor, alpha TGFA 1.7 0.0031 up1933_g_at ATP-binding cassette, sub-family C (CFTR/MRP) ABCC5 0.5 0.0058 n/c31886_at 5'-nucleotidase, ecto (CD73) NT5E 1.7 0.005733377_at vitronectin (serum spreading factor) VTN 0.4 0.0049 down348_at kinesin family member C1 KIFC1 1.6 0.007435016_at CD74 antigen (invariant polypeptide of MHC) CD74 1.5 0.007236197_at catilage GP-39 protein Y08374 0.4 0.0016 n/c36310_at keratin, hair, acidic, 1 KRTHA1 1.5 0.004136916_at sialyltransferase 4C SIAT4C 1.6 0.006636924_r_at secretogranin II (chromogranin C) SCG2 0.4 0.0059 n/c37393_at hairy and enhancer of split 1 HES1 0.5 0.010638489_at heparin-binding growth factor binding protein HBP17 2.4 0.0036 n/c38957_at doublecortin and CaM kinase-like 1 DCAMKL1 0.4 0.0010 down39171_at catenin, beta interacting protein 1 CTNNBIP1 0.5 0.0041 n/c39375_g_at G-2 and S-phase expressed 1 GTSE1 1.5 0.009139771_at Rho-related BTB domain containing 1 RHOBTB1 0.5 0.0002 down40257_at Homo sapiens clone 24649 mRNA sequence Al400011 1.9 0.009340641_at BTAF1 RNA polymerase II BTAF1 1.7 0.010541119_f_at Homo sapiens, clone IMAGE:4310637 W27452 1.7 0.008841479_s_at RAD51 homolog C RAD51C 1.5 0.0091

MicroarrayProbe Set ID Gene Name Symbol

Table I. 20 genes and EST’s with the lowest p-values. Microarray data suggests that aging of Bruch’s membrane increases the expression level of numerous genes. We performed RT-PCR on several genes of interest, including up regulated genes transforming growth factor alpha, CD74 antigen, and heparin-binding growth factor binding protein, and down regulated genes that include the ATP-binding cassette, vitronectin, cartilage GP-39 protein, doublecortin and CaM kinase-like 1, and catenin. RT-PCR confirms the up regulation of TGF alpha and the down regulation of vitronectin, doublecortin and CaM kinase-like 1, and Rho-related BTB domain containing 1.

OVERALL EXPRESSION

MIGRATION GENES

PROLIFERATION GENES

ANGIOGENESIS GENES

YOUNG BM

OLD BM

Supported by Research to Prevent Blindness, Robert L. Burch III Fund, and the Foundation Fighting Blindness

Conclusions: Aging of Bruch’s membrane downregulates the expression profile of vitronectin mRNA and its receptor in human RPE. The vitronectin receptor may play an important role in phagocytosis of photoreceptor outer segments and vitronectin partially mediates RPE attachment to human Bruch’s membrane. These observations suggest some of the changes seen in age-related macular degeneration may be the result of substrate-induced alterations in the behavior of the overlying RPE.

RPE EXPRESSION OF VITRONECTIN AND ITS RECEPTOR ARE DOWNREGULATED WITH AGING OF HUMAN BRUCH’S MEMBRANE

L. V. Del Priore, H. Cai, and T. H. Tezel Department of Ophthalmology, Columbia University, New York, New York

Purpose: Previous studies shows that vitronectin is a major constituent of ocular drusen and vitronectin mRNA is synthesized in RPE cells. The purpose of this study is to determine if Bruch’s membrane aging alters the level of vitronectin mRNA and its receptor in the overlying RPE.

Methods: DNA microarray and semi-quantitative RTPCR method were used for this study. Immortalized human ARPE-19 cells were seeded onto human Bruch’s membrane (five samples from donors age < 50 yr and five samples from donors age > 70 yr) harvested from eye bank eyes. ARPE-19 cells were harvested 72 hours after seeding onto human Bruch’s membrane and total RNA was isolated using a Qiagen RNeasy Mini Kit. First and second strand cDNAs synthesis, Biotin-labeled antisense cRNA target hybridization (on Affymetrix U95A chip), washing, staining and scanning probe arrays were done following an Affymetrix GeneChip Expression Analysis Manual. Real-time quantitative polymerase chain reaction (Roche Lightcycler) using samples generated from different experiments with different donor tissues were used to confirm and further study the genes expression patterns. Oligo primers were determined with LC Primer Design and RTPCR data were analyzed with Lightcycler3 Data Analysis software.

Supported by Research to Prevent Blindness, Robert L. Burch III Fund, and the Foundation Fighting Blindness.

Figure 3. Real time semi-quantitative RT-PCR. The Bruch’s membrane samples were from different batches of donors than those shown above. (A) Quantitative RT-PCR was performed , after establishing standard curves with GAPDH house-keeping gene using serial dilutions of total RNA. (B) Vitronectin expression level is decreased in RPE cells seeded onto aged Bruch’s membrane (dashed lines in duplicates). (C) vitronectin receptor alphaV subunit mRNA in RPE cells is also decreased upon culturing on aged BM (dashed lines).

B.

C.

A. GADPH VN

VN RECEPTOR

Figure 1. A. Plot of individual DNA microarray data on vitronectin (in red color) and its receptor subunit alphaV (in blue color) transcript expression levels. Data show a general decreased expression level trends for both VN and its receptor mRNA in RPE cells seeded onto aged Bruch’s membrane. B. Heat map shows VN and its receptor expression levels (high level in red color and low level in green) in RPE cells cultured on BM explant from different donor ages.

Figure 2. DNA microarray data show vitronectin (VN) and its receptor alphaV transcript expression patterns. ARPE –19 cells were cultured on different aged Bruch’s membrane explants for 72 hours. Data show VN and its receptor subunit alphaV expression level decreases in RPE cells overlaying on aged Bruch’s membrane.

young older young older

BM donor ages

VN and its receptor expression level inRPE cultured on different aged BM

0

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400

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600

VN receptorVN

Rela

tive V

N e

xp

ressio

n l

evel

VN and its receptor expression level inRPE cultured on different ages of BM

31 35 38 47 71 76 810

1

2

3

4

5

6

VNVN receptor

BM donor age

Rela

tive V

N e

xp

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evel

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B.

Results:

ABSTRACT

Purpose: To determine whether the observed anatomy of macular holes can be explained by a hydrodynamic model in which fluid flow through the hole is balanced by fluid pumping across the RPE. We use this model to draw conclusions about the possible role of vitreomacular traction in determining the morphology of macular holes and their resolution after vitreous surgery.Design: Cross sectional.

Methods: Retrospective study in a clinical practice. The study included 42 eyes of 42 patients, each with a stage 3 or 4 macular hole (Gass classification). Macular holes were staged on the basis of clinical exam, Optical Coherence Tomography, and intraoperative findings. We measured the radius of the macular hole and the radius of the surrounding cuff of subretinal fluid from color or red free fundus photographs, and determined the relationship between these 2 variables.

Results: The mean age of the patients was 68.0 7 years old (range 51-80). 25 patients had stage 3 macular holes and 17 patients with stage 4 macular holes. The radius of the neurosensory detachment radius was related to the square of the macular hole radius for stage 3 and stage 4 holes, with no significant difference between the stage 3 and stage 4 linear trend lines (p=0.999). There was no correlation between patient age and the area of the macular hole (r = 0.0645) or neurosensory detachment plus hole (r=0.156) over the range of age in this study (51-80 years). However, the area of the doughnut-shaped cuff of subretinal fluid increased with increasing patient age (p = 0.0493), thus suggesting an age-dependent decline in the pumping ability of the RPE.

Conclusions: Our data is consistent with a hydrodynamic model in which macular hole anatomy is determined by a balance between fluid flow through the hole and fluid outflow across the RPE. Since Stage 3 and 4 macular holes exhibit a similar relationship between the size of the macular hole and the size of the cuff of subretinal fluid around the hole, simple relief of vitreomacular traction would not lead to resolution of the subretinal fluid cuff unless it is accompanied by a reduction in hole diameter due to approximation of wound edges.

ANATOMICAL SIMILARITIES BETWEEN STAGE 3 AND STAGE 4 MACULAR HOLES: IMPLICATIONS FOR TREATMENT

Jon Wender, Tomohiro Iida, M.D., Lucian V. Del Priore, M.D., Ph.D.

Department of Ophthalmology, Columbia University, New York, NY

ConclusionsOur data is consistent with a hydrodynamic model of macular hole anatomy in which fluid flow through the hole is balanced by the outflow of fluid across the RPE. Since Stage 3 and 4 macular holes exhibit a similar relationship between the size of the macular hole and the size of the cuff of subretinal fluid around the hole, simple relief of vitreomacular traction would not lead to resolution of the subretinal fluid cuff unless it is accompanied by a reduction in hole diameter due to approximation of wound edges.

r1

r2

a1

a2

F in

F out

Mathematical Model of Macular Holes

Our model relies on the fact that there is flow of fluid from the vitreous cavity, across the intact retina and RPE, into the choroid in the normal human eye. The development of a full thickness defect in the neurosensory retina will allow fluid from the vitreous cavity to flow through the hole and detach the retina from the RPE (Fig. 1). Fluid flow through the hole will create an enlarging neurosensory detachment. The neurosensory detachment around the hole will increase in size and ultimately be limited by the pumping ability of the underlying RPE. In equilibrium (i.e., when there is no further enlargement of the hole), the fluid flow into the hole (Fin) and fluid flow out

of the hole through the RPE (Fout) will be equal (Fig. 1).

For a Newtonian fluid, the rate of fluid flow into the hole is limited by the size of the macular hole itself. Mathematically, fluid flow through the macular hole (Fin) is inversely proportional to the resistance (R) to

fluid flow through the hole. Thus, we write:

(Eq. 1) Fin 1/R

The resistance is proportional to the square of the area of the macular hole, where the area of the macular hole is given by r1

2. Thus,

(Eq. 2) Fin = / 1/2r14 = 2r1

4, where is an arbitrary constant.

The flow out (Fout) of the subretinal space is due to active pumping of fluid by the underlying RPE. If we

assume that the pumping ability of the RPE is homogeneous (i.e., does not vary across the area of the neurosensory detachment), then outward flow will be directly proportional to the area of the RPE under the macular hole and the surrounding neurosensory detachment. Thus, we write:

(Eq. 3) Fout = Kr22

where r2 is the radius of the surrounding neurosensory detachment (Fig. 1). A priori, it is not known if K is a

constant or varies with patient age. We note this explicitly by writing:

(Eq. 4) Fout = K(age) r22

In this model, the subretinal fluid cuff will increase in size until enough RPE is exposed to allow the flow out to balance the fluid inflow through the hole. In equilibrium,

(Eq. 5) Fin = Fout

(Eq. 6) 2 r14 = Kr2

2

(Eq. 7) r14 = (K/) r2

2

(Eq. 8) r12 = (K/) r2

Thus, the hydrodynamic model predicts that r12 would be proportional to r2; i.e., as the radius of the macular

hole doubles, the radius of the neurosensory detachment would quadruple.

Results

200

300

400

500

600

700

800

0 20000 40000 60000 80000 100000 120000 140000

macular hole radius2 (um2)

ne

uro

se

ns

ory

de

tac

hm

en

t ra

diu

s (

um

)

Stage III

Stage IV

Stage III

Stage IV

Figure 2. Relationship between neurosensory detachment radius (r2) and macular hole radius squared (r1

2). Data fit to a linear regression model, with no significant difference between the stage 3 (y = 0.0042x + 220.04, R2 = 0.8098) and stage 4 (y = 0.0042x + 243.61, R2 = 0.8046) linear trend lines (p=0.999).

0

500000

1000000

1500000

2000000

2500000

3000000

0 50000000000 1E+11 1.5E+11 2E+11

macular hole area2 (um2)

neu

rosen

so

ry d

eta

ch

men

t are

a (

um

2 )

Stage III

Stage IV

Stage III

Stage IV

Figure 3. Relationship between neurosensory detachment area and macular hole area squared. Note that the data now fits a linear regression model with no significant difference noted between stage 3 (y = 10-05 + 275658, R2 = 0.7677) and stage 4 (y = 10-05x + 337788, R2 = 0.8691) linear trend lines (p=0.904).

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

30000 80000 130000 180000 230000 280000 330000 380000 430000

macular hole area (um 2)

su

bre

tin

al fl

uid

cu

ff a

rea (

um

2)

Stage III

Stage IV

Stage III

Stage IV

s

Figure 4. Subretinal fluid cuff area (a2-a1) vs. macular hole area (a1) for stage 3 and stage 4 macular holes. There appears to be no significant difference between the 2nd order polynomial trend lines for stage 3 (y = 10-05x2 - 0.7656x + 258038, R2 = 0.6752) vs. stage 4 (y = 10-05x2 - 1.8772x + 426462, R2 = 0.8225) macular holes.

Table 1. Patient age (years) vs. a1, a2, a2-a1, and a22/a1

<66

≥66 p-value

a1 (um2) 180,000 + 117,000 227,000 + 144,000 0.267 a2 (um2) 695,000 + 475,000 1,090,000 + 839,000 0.0617 a2-a1 (um2) 514,000 + 383,000 858,000 + 715,000 0.0493 a2

2/a1 (um2) 2,970,000 + 2,600,000 5,760,000 + 5,420,000 0.0296

Mean age: 68.0 7 years (range 51-80)• 25 patients with stage 3 macular holes• 17 patients with stage 4 macular holes• The neurosensory detachment radius was related to the square of the macular hole radius for stage 3 and stage 4 holes, with no significant difference between the stage 3 and stage 4 linear trend lines (p=0.999). • Similarly, the neurosensory detachment area was related to the square of the macular hole area for stage 3 and stage 4 holes, with no significant difference between the stage 3 and stage 4 linear trend lines.

FIGURE 1. (Top) Schematic diagram of fluid dynamics of macular hole with surrounding neurosensory detachment. r1 = radius of macular hole, r2 = radius of subretinal fluid cuff. (Bottom) Diagram illustrating fluid dynamics for macular holes. Fin and Fout represent the fluid flow into and out of the subretinal space, respectively. For a Newtonian fluid, Fin is inversely proportional to the resistance. Fout is proportional to the area of the underlying RPE.

Methods Retrospective study in a clinical practice. The study included 42 eyes of 42 patients, each with a stage 3 or 4 macular hole (Gass classification). Macular holes were staged on the basis of clinical exam, Optical Coherence Tomography, and intraoperative findings. We measured the radius of the macular hole and the radius of the surrounding cuff of subretinal fluid from color or red free fundus photographs, and determined the relationship between these 2 variables.