Purchase this article with an account.
Shuichen Zhang, David A. Leske, Jonathan M. Holmes; Neovascularization Grading Methods in a Rat Model of Retinopathy of Prematurity. Invest. Ophthalmol. Vis. Sci. 2000;41(3):887-891.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
purpose. The method of counting cell nuclei above the internal limiting membrane
in histologic sections is considered the standard when quantifying
neovascularization (NV) in rodent oxygen-induced retinopathy (OIR). An
alternative, more rapid method of counting clock hours in flatmounted
adenosine diphosphatase (ADPase)–stained rat retinas is analogous to
clinically scoring retinopathy of prematurity (ROP). In the present
study, the validity of counting clock hours was evaluated by a direct
comparison of these techniques. The intereye correlation of NV score
and retinal vascular area were also studied.
methods. Newborn Sprague–Dawley rats were exposed to cycles of O2 (80–10%) for 7 days, followed by 5 days of room air recovery.
Preretinal NV was quantified by three masked observers counting clock
hours in flatmounted ADPase-stained retinas of both eyes. Retinal
vascular and total retinal areas were calculated using
computer-assisted analysis. Representative retinas that had been scored
positive (n = 10) and negative (n = 3)
for NV and room air control retinas (n = 3) were
embedded in paraffin. Each entire peripheral retinal quadrant was
serially sectioned at 6 μm and stained with hematoxylin and eosin.
Nuclei above the internal limiting membrane were then counted in a
masked manner. The total number of nuclei counted per retina was
defined as the nucleus count (704–938 sections per retina; 12,900
sections). Correlations were evaluated using Spearman rank
results. The nucleus count was 0 to 44 in room air control retinas, 0 to 40 in
negative OIR retinas, and 250 to 5634 in positive OIR retinas. The
nucleus count was highly correlated with the clock hour score
(r s = 0.95, P =
0.0001). For the paired retinas, there was a significant correlation
between right and left eyes in the severity of NV (clock hours; r s = 0.76, P =
0.0001) and the ratio of retinal vascular area to total retinal area
(r s = 0.81, P =
conclusions. The more rapid method of counting clock hours in flatmounted
ADPase-stained retinas is valid for quantifying NV in rat models of
ROP. Incidence and severity of NV and vascularized areas were similar
between left and right eyes, which permits the use of paired retinas
for complementary research techniques.
This PDF is available to Subscribers Only