1. INTRODUCTION
Evaluation of Alternative Methods to Quantify Ocular Angiogenesis in Mice
K.Paes (KPaes@lexpharma.com); E.Wang; R.Wudali; D.S.Rice
Lexicon Pharmaceuticals, Inc., The Woodlands, TX, United States 77381
Presented at the Association for Research in Vision and Ophthalmology, 2010 Annual Meeting, May 2
nd
- 6
th
– Fort Lauderdale, Florida
Classical techniques to evaluate the effects of angiostatic
compounds in rodent models of ocular angiogenesis are
laborious and time consuming. We sought to identify novel
approaches to generate high quality results in a rapid fashion.
We chose to characterize these approaches in three
conventional mouse models of ocular angiogenesis:
developmental retinal angiogenesis (DRA), retinopathy of
prematurity (OIR) and laser-induced choroidal
neovascularization (CNV) using a known anti-angiogenic
compound Rapamycin.
Disclosure: The authors of this poster are employees of, and have received stock options from,
Lexicon Pharmaceuticals, Inc
References: Dejneka, et.al. Molecular Vision 2004.
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RESULTS RESULTS (continued)
CONCLUSIONS
METHODS
Rapamycin Treatment Impairs Retinal and Vascular
Development in Neonatal Mice.
Rapamycin Inhibits Angiogenesis in the Oxygen-Induced
Retinopathy Model
VEHICLE RAPAMYCIN
VEHICLE-RAW IMAGE RAPAMYCIN-RAW IMAGE
THRESHOLDED IMAGE THRESHOLDED IMAGE
SKELETON AND JUNCTIONS SKELETON AND JUNCTIONS
VEHICLE RAPAMYCIN
ISOLECTIN NG2
THRESHOLDED
NG2 IMAGE
Neovascularization is assessed by NG2 immunohistochemical staining of
OIR retinas and morphometric area analysis.
Neovascular nuclei can be quantified from digital histological slides using
semi-automated nuclear algorithms.
Morphometric analysis of the retinal vasculature from wholemount retinas
using AngioSys (TCS Cellworks).
Abbreviated Materials and Methods. Please contact Kim Paes (KPaes@lexpharma.com) for complete and detailed methods.
Developmental Retinal Angiogenesis Model (DRA):
C57BL/6J neonatal mice from several litters were randomized at the time of birth and redistributed to nursing mothers. Rapamycin
(R-5000, LC Laboratories) was formulated in a DMSO/PBS vehicle control. Rapamycin and vehicle control were administered
once daily by subcutaneous delivery in the nape of the neck at 4mg/Kg from postnatal day (PD) 0 to PD4. On PD5, eyes were
enucleated and fixed in 4% paraformaldehyde overnight at 4°C. Wholemount retinas were prepared and gently agitated overnight
at 4°C in a solution of isolectin IB4 Alexa 488. Retinas were then removed from the staining solution and washed in 1XTBS-T
several times at 4°C. Radial incisions were made in each retina towards the optic nerve head and mounted on histological slides
using Flouromount G. Images of each retina were taken and analyzed using Adobe PhotoShop or AngioSys (TCS Cellworks) for
the effects of Rapamycin on retinal and vascular development respectively.
Oxygen-induced proliferative retinopathy (OIR):
C57BL/6 litters were housed in a hyperoxic environment from PD7 to PD12, and then returned to room air (relative hypoxia) for an
additional five days as described by L.E. Smith 1994. Rapamycin or DMSO/PBS vehicle control were formulated and administered
as described in procedures related to the developmental retinal angiogenesis model, except that administration occurred once
daily from PD12 (return to room air) to PD16 by intraperitoneal delivery. Neonatal mice were euthanized on PD17, and eyes from
each animal were enucleated and placed in 4% paraformaldehyde or Davidson’s fixative (Poly Scientific) for wholemount retinal
immunohistochemical staining or paraffin embedding and sectioning examination, respectively. Retinal wholemounts were
examined in Adobe PhotoShop to measure the central avascular and neovascular areas. Histological sections were imaged and
analyzed using an Aperio ScanScope and nuclear algorithm suite.
Laser-induced Choroidal Neovascularization (CNV):
Disruption of Bruch’s membrane to induce CNV was achieved by activation of a 532nm solid-state LASER (Lumenis Novus
Spectra) adjusted to 130mW power, 0.05s duration, single-pulse, and 50um spot size in C57BL/6 adult mice following pupillary
dilation. Four LASER spots were placed approximately 2-4 disc diameters away from the optic nerve head of each eye and
spaced equally from one another. Animals were dosed with 4mg/Kg rapamycin suspension in DMSO/PBS vehicle or DMSO/PBS
vehicle control. Dosing was initiated one day prior to LASER-induced choroidal neovascularization (CNV) induction and continued
once daily throughout the course of the experiments. Enucleation was performed eight days following the LASER procedure and
eyes were place in a 4% paraformaldehyde fixative.
Fundus images were obtained at two and seven days following LASER treatment using a retinal fundus imaging system (KOWA).
At the seven day time point, angiograms were performed in all subjects by injecting 60uL of 100mg/mL 10% fluorescein (Akorn)
intraperitoneally. Angiogram images were captured using the yellow-free filter of the retinal camera at one-minute and six-minute
post-injection time points. Digital angiogram images were analyzed in Adobe Photoshop. Data are represented for individual
lesion area differences and also for the average of all lesion area differences within one animal.
Fixed eyes were processed for RPE/choroidal wholemounts and stained with isolectin IB4. Flatmounts were prepared after
several days of staining and Z-stack images were taken using a confocal microscope to quantify the volume of the lesions using
Imaris software (Bitplane). The volume (um
3
) within the three-dimensional skin was calculated by the software and is represented
as individual lesion volumes and average of all lesion volumes within one animal
Statistical analysis: The statistical significance between experimental and control groups was determined by an unpaired
Student’s t-test and considered significant if two tailed p values were <0.05.
RESULTS (continued)
Neovascular leakage and lesion volume is reduced in CNV
mice treated with Rapamycin
RAPAMYCIN: 1 MINUTE ANGIOGRAM
RAPAMYCIN: 6 MINUTE ANGIOGRAM
VEHICLE: 1 MINUTE ANGIOGRAM
VEHICLE: 6 MINUTE ANGIOGRAM
We have developed alternative methods that aide in the
quantification of ocular angiogenesis in three widely used
models.
These analysis methods allow for swift quantification
compared to traditional manual techniques while also
minimizing observer bias.
We continue to improve our methods by utilizing tools that
increase the quality and efficiency of our studies and data.