healthmedicinet

Ethics statement

This research adhered to the tenets of the Declaration of Helsinki. Each participant
provided signed informed consent in accordance with protocols for human subject recruitment
and clinical evaluation approved by the Institutional Review and Ethics Boards of
the National Eye Institute. The animal studies were conducted in compliance with the
ARVO statement for the use of animals, and all animal experiments were performed under
protocols approved by the Institutional Animal Care and Use Committees of National
Eye Institute, National Institutes of Health, USA; and Queen’s University Belfast,
Belfast, UK.

Immunohistochemistry and mRNA expression of human ocular sections

Archived paraffin ocular sections from 5 AMD cases (3 wet, 2 dry) and 5 age-matched
normal eyes were subjected to avidin–biotin-complex immunoperoxidase staining. Endogenous
peroxidase activity was blocked in hydrogen peroxide. Polyclonal antihuman goat IgG
antibodies against total LRP6 (Cell Signaling Technology, Danvers, MA) were applied
at a 1:50 dilution for 60 min at room temperature. Biotinylated rabbit anti-goat IgG
was used as the linker molecule between the LRP6 antibody and avidin–biotin-peroxidase
complex for 60 min before application of the ABC complex (Vector Lab, Mountain View,
CA, USA). Diaminobenzidine-hydrogen peroxide was used as the chromogen. The sections
were counterstained with 1 % methyl green. The staining was evaluated based the intensity
of brown-blackish color on the sections. For ?-catenin immunohistochemistry, the antibody
was antihuman rabbit IgG antibody (sc-7199, Santa Cruz Biotechnology, Inc. Dallas,
TX, USA).

The AMD cases and age-matched normal eyes were used for transcript analysis. Neuroretinal
and RPE cells, Bruch’s membrane and choroidal capillaries in the macular area from
each ocular section were microdissected for RNA extraction using the Paradiseâ„¢ Sample
Quality Assessment Kit (Arcturus Bioscience), followed by cDNA synthesis using Invitrogen
SuperScriptâ„¢ II reverse transcriptase (Invitrogen, Carlsbad, CA). A260/A280 of the
RNA was greater or equal to 1.80, indicating that the RNA was pure. Human Total RNA
(Life Technology, Gaithersburg, MD, USA) was converted to cDNA and used as the control
for RT-PCR reactions. The primers for SYBR Green qPCR were synthesized by SABiosciences
and supplied in the Real-Time RT-PCR Gene Expression Analysis kit (SABiosciences,
Frederick, MD, USA). Following PCR, a thermal melt profile was performed for amplicon
identification. Folder changes (2
^???Ct
analysis method) were calculated by comparing with Ct values obtained from a paralleled
amplification of Human Total RNA.

Human plasma for ELISA quantification of Kallistatin

The study group demographics are summarized in Table 1. Participant selection and clinical evaluation have been previously defined 17]–20]. AMD cases were all in advanced stages and the controls presented either no drusen
or fewer than 5 small drusen (63 µm in diameter) and no signs of other retinal diseases,
including but not limited to high myopia, retinal dystrophies, central serous retinopathy,
vein occlusion, diabetic retinopathy, or uveitis. The patients and controls were all
self-identified as Caucasian and non-Hispanic.

Table 1. Human plasma samples

Kallistatin levels in human plasma were quantified by ELISA (RD Systems, Minneapolis,
MN, USA), as previously described 21]. Briefly, 96 well microplates were coated with mouse anti-human kallistatin capture
antibody overnight at room temperature (RT). Nonspecific binding to capture antibody
was blocking by 1 % BSA in PBS at RT for 1 h. Plasma samples were diluted (1/20,000)
with 1 % BSA in PBS. Samples and standards were added to wells coated with mouse anti-human
kallistatin capture antibody. After incubation at RT for 2 h and the extensive washing
with PBS with Tween-20, the plate was incubated with 100 ?L biotinylated goat anti-human
kallistatin detection antibody for 2 h, followed by incubation with 100 ?L Streptavidin
conjugated to horseradish-peroxidase (RD Systems) for 20 min. After the addition
of H
₂
O
₂
/tetramethylbenzidine for 20 min, 50 ?L 2 N H
₂
SO
₄
was added to stop the reaction. Plate was read immediately at 450 nm (VICTOR3 V™ Multilabel
Counter, PerkinElmer Life And Analytical Sciences, Inc, Waltham, MA, USA). The personnel
testing the samples were blind to the information and samples were identified by numeric
codes. All measurements were performed in triplicate for each sample, and the mean
values were calculated. Inter- and intra-assay variations were 5 and 10 %, respectively.

Wnt pathway component mRNA array

Total RNA from mouse retina/RPE was extracted using Trizol (Invitrogen, Carlsbad,
CA, USA). cDNA was synthesized using reverse transcriptase (Invitrogen). The array
to identify possible differences in the ocular expression of Wnt-related genes between
Ccl2^?/?/Cx3cr1^?/?/rd8 and wild type B6 N mice was performed using Mouse WNT Signaling Pathway RT
²
Profiler™ PCR Arrays according to the manufacturer’s protocols (Qiagen/SABiosciences,
Gaithersburg, MD, USA). The plate contains assays for 92 Wnt pathway associated genes
and 4 assays to candidate endogenous control genes (Additional file 2: Table S1). The reaction was run on an ABI 7500 System (Applied Biosystems, CA, USA).
Expression values were determined with DataAssistâ„¢ v2.0 Software (Applied Biosystems).

Animals and treatment

The animal study was conducted in compliance with the ARVO statement for the use of
animals, and all animal experiments were performed under protocols approved by the
Institutional Animal Care and Use Committees of National Eye Institute, National Institutes
of Health, USA and Queen’s University, Belfast, UK. Mice were originated and bred
from National Eye Institute, National Institutes of Health, USA (Ccl2^?/?/Cx3cr1^?/?/rd8) or Queen’s University, Belfast, UK (Ccl2^?/?/Cx3cr1 ^gfp/gfp
).

The generation of Ccl2^?/?/Cx3cr1^?/?
mice on rd8 background (Ccl2^?/?/Cx3cr1^?/?/rd8) and Ccl2^?/?/Cx3cr1^?/?
without rd8 background (Ccl2^?/?/Cx3cr1 ^gfp/gfp
) and their pathological features have been reported previously 10], 16]. Specifically, both mouse strains were generated as a model of progressive, focal
retinal degeneration that mimics certain features of human AMD lesions, predominant
dry AMD phenotype 10], 16]. Ccl2^?/?/Cx3cr1^?/?/rd8 develops retinal lesions spontaneously while the retinal lesions in Ccl2^?/?/Cx3cr1 ^gfp/gfp
can be induced by exposing of 3-month old mice to blue light (465 nm) for 8 h/day
for 6 months. The monoclonal anti-LRP6 antibody was obtained using a recombinant peptide
comprising the E1E2 domain of murine LRP6 as the antigen 7]. Nonspecific mouse IgG (mIgG) purchased from Vector Laboratories (Burlingame, CA,
USA) was used as a control for anti-LRP6.

Single injections of 500 ng of anti-LRP6 in 1 ?L volume were intravitreally administered
to the right eyes of 6-week-old Ccl2^?/?/Cx3cr1^?/?/rd8; the contralateral eye was injected with 500 ng of mIgG in the same volume as a control
(n = 35). The same amount of anti-LRP6 was intravitreally injected once per month
into one eye of 6-month-old Ccl2^?/?/Cx3cr1 ^gfp/gfp
(i.e. after 3 months light exposure, n = 20, 10 for treatment and 10 for control).
The control eyes were intravitreally given 500 ng (1 ?L) mIgG once every month. Eyes
were harvested at 2 months (Ccl2^?/?/Cx3cr1^?/?/rd8) or 3 months (Ccl2^?/?/Cx3cr1 ^gfp/gfp
) after the injection for various measurements. The eyes of C57BL/6N with rd8 mutations were used for an assay control.

Fundus photography and scoring

We evaluated the lesion changes by comparing sequential fundus photographs taken in
the same fundus area of each eye before the injection and every month after the injection
using a Karl Storz veterinary otoendoscope 22]. Progression was defined as a 10 % increase in the number of retinal lesions (Score
+1), a 50 % increase in the lesion size in at least one out of three of the lesions
(Score +2), more than five fused lesions or the appearance of more than two chorioretinal
scars (Score +3), and diffuse chorioretinal scars (Score +4) compared to baseline.
Regression was defined as a 10 % decrease in the number of retinal lesions (Score
?1), a 50 % decrease in lesion size in at least one out of three of the lesions (Score
?2), a 50 % disappearance of retinal lesions (Score ?3) or a total disappearance
of retinal lesions (Score ?4). To avoid bias, a masked observer evaluated all fundus
photographs. The lesion scores of each eye were estimated by cross-comparison of the
same area based on above criteria. Fundus photography taken from the mice tested in
Belfast site used the topic endoscopic fundus imaging (TEFI) system described previously
22], 23].

Electroretinography (ERG)

Light-adapted ERG responses for Ccl2^?/?/Cx3cr1^?/?/rd8 were used to evaluate cone-pathway function in the retina following a published procedure
24]. Briefly, mice adapted to room light were anesthetized with i.p. injection of a mixture
containing ketamine and xylazine. Pupils were dilated with 2.5 % phenylephrine (Alcon
Inc, Fort Worth, TX, USA) and 1 % tropicamide (Alcon Inc), and cornea was anesthetized
with 0.5 % proparacaine (Alcon Inc). Animals were placed on a heating pad to maintain
body temperature. ERG responses were recorded with gold-wire electrodes placed on
the center of the cornea and measured using a commercial Espion E2 system (Diagnosys
LLC, Lowell, MA, USA). Light emitting diodes with built in ultraviolet (UV) colordome
provide ganzfeld UV (365 nm) or green (514 nm) light stimulus. Photopic ERG responses
were recorded in the presence of a rod-saturating background of 20 sc cd/m
²
. The dark-adapted ERG was recorded for Ccl2^?/?/Cx3cr1 ^gfp/gfp
(LKC Technologies, Gaithersburg, MD, USA) 25].

Histology

Whole mouse eyes were fixed in 4 % glutaraldehyde-10 % formalin and then embedded
in methacrylate. The eyes were serially sectioned in the vertical pupillary-optic
nerve plane. Each eye was cut into 6 sections. All sections were stained with hematoxylin
and eosin. If an ocular lesion was observed, another 6-12 sections were cut through
the lesion. These slides were also stained with Periodic Acid Schiff (PAS) to highlight
the Bruch’s membrane and the basement membrane of small neovascular vessels.

Retinal lipofuscin extraction and quantification

A2E ([2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetra-enyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-trimethyl-1-cyclohexen-1-yl)
1E,3E,5E,7E-hexatrienyl]-pyridinium) is the major component of lipofuscin fluorophores
generated from the visual cycle flux of all-trans-retinol. The molecule is particularly
correlated with retinal aging and AMD pathogenesis 26]. A2E was extracted via chloroform/methanol as previously described 27]. The extracts dissolved in methanol were separated by HPLC (Agilent 1100 LC, Wilmington,
DE, USA) and detected by an ultraviolet detector at a wavelength of 435 nm. A2E was
quantified using external A2E standards 28].

Western blot analysis

Western blot analysis detected the phosphorylated level of LRP6. Mouse retina was
dissected and homogenized for protein extraction. Equal amounts (50 ?g) of total protein
from each sample were resolved by SDS polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred onto a nitrocellulose membrane. The membrane was blocked with 5 %
nonfat milk and separately blotted with primary antibodies (anti-Non-phosphorylated
?-Catenin, Cell Signaling Technology; anti-phosphorylated LRP6, Cell Signaling Technology;
and anti-LRP6, self-made). After thorough washes, the membrane was incubated with
the peroxidase-conjugated horse anti-rabbit or horse anti-mouse antibody (Vector Lab).
The signal was developed with the enhanced chemiluminescence (ECL) system (Pierce,
Rockford, IL, USA). Membranes were stripped and re-blotted with anti-?-actin (Sigma-Aldrich,
St. Louis, MO, USA) as the loading control. Densitometry of the signal bands on digital
images was quantified using FluorChem Q software (ProteinSimple, Santa Clara, CA,
USA).

Quantification of mRNA expression in mouse eyes by RT-PCR

cDNA synthesis was described as above. The primers/probes were purchased from Applied
Biosystems as inventoried TaqMan gene expression reagents. Relative quantitative real
time PCR was performed according to ABI’s instructions. To determine the Ct values,
the threshold level of fluorescence was set manually in the early phase of PCR amplification.
ABI SDS 1.3.1 software and the 2
^???Ct
analysis method were used to determine relative amounts of product using Gapdh as an endogenous control. Each sample was analyzed in triplicate.

Statistical analysis

Multiple means were compared by paired T-test. Differences were considered significant
when p 0.05. The collective ERG amplitudes were calculated by the area under curve (AUC)
and compared by T-test between the groups. Comparison of plasma kallistatin levels
between AMD patients were performed by independent-samples T test.