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DIFFERENCES IN GENE EXPRESSION PROFILE OF CULTURED ADULT VERSUS
IMMORTALIZED HUMAN RPE
Lee Geng, Hui Cai and Lucian V. Del Priore
Department of Ophthalmology, Columbia University, New York, New York
Genes expressed in ARPE
cells but not detected in adult RPE cells
Table I. Genes expressed in pRPE but not detected in aRPE
Purpose: Immortalized human RPE (cell line ARPE-19) are used widely to draw A.
inferences about the behavior of adult RPE. We
usedGeneDNA
microarray
analysis to compare the
Gene have
Title
Symbol
Functions
p21/Cdc42/Rac1-activated kinase 1
p21 (CDKN1A)-activated kinase 2
Down syndrome critical region gene 1-like 1
superoxide dismutase 2, mitochondrial
dipeptidylpeptidase 4
neuronal pentraxin II
serine (or cysteine) proteinase inhibitor
cyclin D2
ribonuclease, RNase A family
chondroitin sulfate proteoglycan 2 (versican)
DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked
prostaglandin D2 synthase 21kDa (brain)
transmembrane 4 superfamily member 3
alpha-fetoprotein
keratin 19
ATPase, Ca++ transporting, plasma membrane 4
prostaglandin I2 (prostacyclin) synthase
transferrin receptor (p90, CD71)
PAK1
PAK2
DSCR1L1
SOD2
DPP4
NPTX2
SERPINB8
CCND2
RNASE1
CSPG2
DDX3Y
PTGDS
TM4SF3
AFP
KRT19
ATP2B4
PTGIS
TFRC
protein kinase activity
protein kinase ;ATP binding ;transferase activity
calcium-mediated signaling
response to oxidative stress
proteolysis and peptidolysis; immune response
heterophilic cell adhesion;regulation of synapse
serine protease inhibitor activity
regulation of cell cycle;cytokinesis
RNA binding ;endonuclease activity
heterophilic cell adhesion; cell recognition
DEAD;ATP binding
prostaglandin biosynthesis; transport
protein amino acid glycosylation;pathogenesis
transport;immune response
structural constituent of cytoskeleton
cation transport;calcium ion transport;metabolism
prostaglandin biosynthesis;electron transport;lipid metabolism
proteolysis and peptidolysis; iron ion transport;endocytosis
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
B.
Genes expressed in Table
adult
RPE cells but not detected in aRPE-19 cells
II. Selected Genes expressed in aRPE but not in pRPE
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
Numbers of Gene Expressed in RPE cells
7000
6000
Number of Genes
Results:
5000
4000
3000
2000
ARPE
1000
0
pRPE
primary RPE
A.
B.
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.
ARPE-19
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
ubiquitin specific protease 6 (Tre-2 oncogene)
BCL2 binding component 3
calcium/calmodulin-dependent protein kinase I
death associated transcription factor 1
basic leucine zipper nuclear factor 1 (JEM-1)
nicotinamide nucleotide adenylyltransferase 2
transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha)
protein kinase C, alpha
ubiquitin-conjugating enzyme E2M (UBC12 homolog, yeast)
TNF receptor-associated factor 2
interleukin 12A (p35)
phosphate cytidylyltransferase 2, ethanolamine
zinc finger protein 278, short isoform
paraneoplastic antigen
programmed cell death 11
leucine-rich repeats and immunoglobulin-like domains 1
ubiquitin specific protease 52
E1A binding protein p400
zinc finger protein 205
small nuclear RNA activating complex, polypeptide 4, 190kDa
zinc finger protein 44 (KOX 7)
ADP-ribosylation factor related protein 1
Rho guanine nucleotide exchange factor (GEF) 12
phosphatidylserine receptor
dead ringer-like 1 (Drosophila)
MHC class I polypeptide-related sequence B
SH3 domain binding glutamic acid-rich protein
nuclear factor of kappa light polypeptide gene enhancer
transcription termination factor, RNA polymerase I
zinc finger protein 282
peroxisomal acyl-CoA thioesterase
protein kinase, cAMP-dependent, regulatory, type II, beta
guanidinoacetate N-methyltransferase
leucine rich repeat neuronal 4
mitogen-activated protein kinase 8 interacting protein 3
myeloid/lymphoid or mixed-lineage leukemia 4
interleukin 1 receptor accessory protein
heat shock 70kDa protein 4
zinc finger protein 23 (KOX 16)
elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 2
polymerase (DNA directed), gamma 2, accessory subunit
teratocarcinoma-derived growth factor 3, pseudogene
zinc finger, DHHC domain containing 18
mitogen-activated protein kinase kinase 5
p53-associated parkin-like cytoplasmic protein
caspase recruitment domain family, member 10
cell growth regulator with EF hand domain 1
adrenergic, beta-2-, receptor, surface
P450 (cytochrome) oxidoreductase
USP6
BBC3
CAMK1
DATF1
BLZF1
NMNAT2
TFAP2A
PRKCA
UBE2M
TRAF2
IL12A
PCYT2
--HUMPPA
PDCD11
LRIG1
USP52
EP400
ZNF205
SNAPC4
ZNF44
ARFRP1
ARHGEF12
PTDSR
DRIL1
MICB
SH3BGR
NFKBIL1
TTF1
ZNF282
PTE1
PRKAR2B
GAMT
LRRN4
MAPK8IP3
MLL4
IL1RAP
HSPA4
ZNF23
ELOVL2
POLG2
TDGF3
ZDHHC18
MAP2K5
PARC
CARD10
CGREF1
ADRB2
POR
protein modification /// deubiquitination /// oncogenesis
--protein amino acid phosphorylation /// signal transduction
regulation of transcription, DNA-dependent /// apoptosis
------cell cycle; cell proliferation;induction of apoptosis
ubiquitin cycle
protein complex assembly /// apoptosis /// signal transduction
immune response
phospholipid biosynthesis /// biosynthesis
----rRNA processing
--UCH;cysteine-type endopeptidase activity;3.3e-07
SNF2_N;DNA binding;1.4e-58
regulation of transcription, DNA-dependent
transcription from Pol II promoter
regulation of transcription, DNA-dependent
signal transduction
PDZ;intracellular signaling cascade;1.5e-10
--regulation of transcription, DNA-dependent
response to stress /// cellular defense response
protein complex assembly
--transcription termination
regulation of transcription, DNA-dependent
lipid metabolism /// acyl-CoA metabolism
protein phosphorylation;intracellular signaling cascade
creatine biosynthesis /// muscle contraction
neurogenesis
vesicle-mediated transport /// regulation of JNK cascade
regulation of transcription, DNA-dependent; apoptosis
----regulation of transcription, DNA-dependent
fatty acid biosynthesis
DNA replication /// DNA repair /// protein biosynthesis
----signal transduction
--apoptosis;activation of NF-kappaB-inducing kinase
--activation of MAPK /// receptor mediated endocytosis
electron transport
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.
Supported by Research to Prevent Blindness, Robert L. Burch III Fund, and the Foundation Fighting Blindness
AGING OF BRUCH’S MEMBRANE DECREASES RETINAL PIGMENT EPITHELIUM (RPE)
PHAGOCYTOSIS
Reiko Koyama, Hui Cai and Lucian V. Del Priore
Department of Ophthalmology, Columbia University, NY, New York
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.
30
p<0.02
20
10
cells ingesting beads
cells ingesting beads
30
p>0.05
20
10
0
young
400
p<0.0003
300
200
100
older
age of Bruch's membrane
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%).
Flourescent intensity/cell
B. on older Bruch’s membrane
cells not
ingesting
beads
plastic
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%).
Fluorescent intensity/cell
cells not
ingesting
beads
0
Bruch's
Results:
A. on younger Bruch’s membrane
40
% of RPE ingesting beads
% of RPE ingesting beads
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.
400
p<0.008
300
200
100
0
0
Bruch's
plastic
young
old
age of Bruch's membrane
fluorescent intensity
Fig 1. Photo shows 6-mm circular
buttons which were trephined from
BM and placed into 96 well plate
fluorescent intensity
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).
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
Supported by Research to Prevent Blindness, Robert L. Burch III Fund, and the Foundation Fighting Blindness.
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).
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)
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.
BRUCH'S MEMBRANE AGING ALTERS THE RPE EXPRESSION PROFILE OF
PROLIFERATION, MIGRATION AND APOPTOSIS BUT NOT ANGIOGENESIS GENES
Hui Cai, Lucian V. Del Priore
Department of Ophthalmology, Columbia University, New York, New York
PROLIFERATION GENES
A.
B.
OVERALL
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
YOUNG BM
22.0%
OLD BM
PC#2
PC#2 18.3%
EXPRESSION
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 fr
PC#1
27.4%
C.
PC#1
D.
PC#2
Results:
ANGIOGENESIS GENES
PC#2
21.0%
18.4%
MIGRATION GENES
47.2%
14000
PC#1
PC#1 35/2%
33.1%
12000
on 38 yo BM
10000
8000
6000
4000
2000
0
0
2000
4000
6000
8000
10000
12000
14000
on 31 yo BM
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.
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.
Probe Set ID
160025_at
1933_g_at
31886_at
33377_at
348_at
35016_at
36197_at
36310_at
36916_at
36924_r_at
37393_at
38489_at
38957_at
39171_at
39375_g_at
39771_at
40257_at
40641_at
41119_f_at
41479_s_at
Gene Name
transforming growth factor, alpha
ATP-binding cassette, sub-family C (CFTR/MRP)
5'-nucleotidase, ecto (CD73)
vitronectin (serum spreading factor)
kinesin family member C1
CD74 antigen (invariant polypeptide of MHC)
catilage GP-39 protein
keratin, hair, acidic, 1
sialyltransferase 4C
secretogranin II (chromogranin C)
hairy and enhancer of split 1
heparin-binding growth factor binding protein
doublecortin and CaM kinase-like 1
catenin, beta interacting protein 1
G-2 and S-phase expressed 1
Rho-related BTB domain containing 1
Homo sapiens clone 24649 mRNA sequence
BTAF1 RNA polymerase II
Homo sapiens, clone IMAGE:4310637
RAD51 homolog C
Microarray
old/young
P value
TGFA
1.7
0.0031
ABCC5
0.5
0.0058
NT5E
1.7
0.0057
VTN
0.4
0.0049
KIFC1
1.6
0.0074
CD74
1.5
0.0072
Y08374
0.4
0.0016
KRTHA1
1.5
0.0041
SIAT4C
1.6
0.0066
SCG2
0.4
0.0059
HES1
0.5
0.0106
HBP17
2.4
0.0036
DCAMKL1
0.4
0.0010
CTNNBIP1
0.5
0.0041
GTSE1
1.5
0.0091
RHOBTB1
0.5
0.0002
Al400011
1.9
0.0093
BTAF1
1.7
0.0105
W27452
1.7
0.0088
0.0091
RAD51C
1.5
Symbol
qRT-PCR
old/young
up
n/c
down
n/c
n/c
n/c
down
n/c
down
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 Rhorelated BTB domain containing 1.
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
Supported by Research to Prevent Blindness, Robert L. Burch III Fund, and the Foundation Fighting Blindness
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
VN and its receptor expression level in
RPE cultured on different aged BM
Figure 2. DNA microarray data show
vitronectin (VN) and its receptor alphaV
500
transcript expression patterns. ARPE –19 cells
400
were cultured on different aged Bruch’s
VN receptor
VNseeded onto
Methods: DNA microarray and semi-quantitative RTPCR method were used for this study. Immortalized
human ARPE-19 cells were
human
Bruch’s
(five samples from don
membrane
explants
for 72membrane
hours. Data show
300
VN
and
its
receptor
subunit
alphaV
expression
200
level decreases in RPE cells overlaying on aged
100
Bruch’s membrane.
Relative VN expression level
600
0
young
older
young
older
BM donor ages
GADPH
A.
Results:
C.
A.
VN and its receptor expression level in
RPE cultured on different ages of BM
Relative VN expression level
6
5
VN
VN receptor
4
3
2
1
0
31 35 38
47
BM donor age
B.
71
76
81
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.
VN
RECEPTOR
B.
VN
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).
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.
Supported by Research to Prevent Blindness, Robert L. Burch III Fund, and the Foundation Fighting Blindness.
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
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.
Mathematical Model of Macular Holes
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/2r14 = 2r14, 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 = Kr2
2
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)
a1
r2
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 = Kr22
(Eq. 7)
r14 = (K/) r22
(Eq. 8)
r12 = (K/) r2
2000000
800
1800000
700
1600000
600
Stage III
Stage IV
500
Stage III
Stage IV
400
1400000
1200000
1000000
Stage III
800000
Stage IV
s
Stage III
600000
Stage IV
400000
300
200000
200
a2
0
20000
40000
60000
80000
100000
120000
0
30000
140000
80000
macular hole radius 2 (um 2)
F in
180000
230000
280000
330000
380000
430000
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.
2
Table 1. Patient age (years) vs. a1, a2, a2-a1, and a2 /a1
3000000
F out
130000
m acular hole are a (um 2)
Figure 2. Relationship between neurosensory detachment radius (r2) and
macular hole radius squared (r12). 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).
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 r12. Thus,
(Eq. 2)
r1
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.
2500000
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.
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.
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.
neurosensory detachment area (um 2)
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).
Results
subretinal fluid cuff area (um 2)
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.
neurosensory detachment radius (um)
ABSTRACT
2000000
Stage III
<66
≥66
p-value
180,000 + 117,000
695,000 + 475,000
514,000 + 383,000
2,970,000 + 2,600,000
227,000 + 144,000
1,090,000 + 839,000
858,000 + 715,000
5,760,000 + 5,420,000
0.267
0.0617
0.0493
0.0296
Stage IV
1500000
Stage III
Stage IV
1000000
500000
0
0
50000000000
1E+11
1.5E+11
2
a1 (um )
2
a2 (um )
2
a2-a1 (um )
2
2
a2 /a1 (um )
2E+11
macular hole area2 (um 2)
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).
Conclusions
Our 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.