Age of Rats Seriously Affects the Degree of Retinal Damage Induced by Acute High Intraocular Pressure

Abstract

Purpose:
To investigate whether retinal impairment was affected by age of rats in acute glaucoma model.

Methods:
Young adult and aged rats were randomly divided into normal control, 45 mmHg, 60 mmHg and 90 mmHg groups. Intraocular pressures (IOP) of rats were acutely elevated to 45 mmHg, 60 mmHg and 90 mmHg, respectively. Neuron loss in ganglion cell layer (GCL) and activation of retinal macrolgia and microglia 3 days after high IOP treatment were detected by immunofluorescence and further quantitatively analyzed.

Results:
Compared with normal control, significant loss of neurons at GCL of young adult retina wasn't detected until IOP treatment of 90 mmHg. In contrast, obvious loss of neurons at GCL of aged retina was detected at IOP of 45 mmHg (p = 0.002 for central; p = 0.001 for peripheral). The loss level of neurons of aged retina was significantly higher than that of young adult retina at different IOP treatments. Compared with the young adult retina, high IOP induced more significant increase at area percentage of microglia and microglia number in inner part of aged retina. Activation of microglia and macroglia was either in parallel to or earlier than neuron loss of GCL of aged and young adult retina.

Conclusion:
Our data suggest there exists an age-related susceptibility of rat retina to the increased IOP. Therefore, the effect of ages should be considered at glaucoma study of rat models.

Full text

Current Eye Research, Early Online, 1–7, 2014
!Informa Healthcare USA, Inc.
ISSN: 0271-3683 print / 1460-2202 online
DOI: 10.3109/02713683.2014.922194
ORIGINAL ARTICLE
Age of Rats Seriously Affects the Degree of Retinal
Damage Induced by Acute High Intraocular Pressure
Chang Tan
1
,TuHu
2
, Ming-chao Peng
1
, Shu-li Liu
1
,
Jian-bin Tong
1
, Wen Ouyang
1
and Yuan Le
1
1
Department of Anesthesiology, The Third Xiangya Hospital of Central South University, Changsha, P.R. China,
2
Department of Anatomy and Neurobiology, Xiangya School of Medicine, Central South University,
Changsha, P.R. China
ABSTRACT
Purpose: To investigate whether retinal impairment was affected by age of rats in acute glaucoma model.
Methods: Young adult and aged rats were randomly divided into normal control, 45 mmHg, 60 mmHg and
90 mmHg groups. Intraocular pressures (IOP) of rats were acutely elevated to 45 mmHg, 60 mmHg
and 90 mmHg, respectively. Neuron loss in ganglion cell layer (GCL) and activation of retinal macrolgia
and microglia 3 days after high IOP treatment were detected by immunofluorescence and further quantitatively
analyzed.
Results: Compared with normal control, significant loss of neurons at GCL of young adult retina wasn’t detected
until IOP treatment of 90 mmHg. In contrast, obvious loss of neurons at GCL of aged retina was detected at
IOP of 45 mmHg (p= 0.002 for central; p= 0.001 for peripheral). The loss level of neurons of aged retina was
significantly higher than that of young adult retina at different IOP treatments. Compared with the young adult
retina, high IOP induced more significant increase at area percentage of microglia and microglia number
in inner part of aged retina. Activation of microglia and macroglia was either in parallel to or earlier than
neuron loss of GCL of aged and young adult retina.
Conclusion: Our data suggest there exists an age–related susceptibility of rat retina to the increased IOP.
Therefore, the effect of ages should be considered at glaucoma study of rat models.
Keywords: Aging, glaucoma, intraocular pressures, retina, rat
INTRODUCTION
Glaucoma is a common age-related eye disease that
affects 3% of the worldwide population over the age
of 40, making it the second-leading cause of blind-
ness.
1–3
It is characterized by the visual field loss,
the death of a substantial number of retinal ganglion
cells (RGCs) and the loss of their axons in the optic
nerve.
4
Lots of clinical studies have shown that age
4
and elevated intraocular pressures (IOP)
4,5
are the
most extensive document risk factors of glaucoma.
By now, IOP reduction is the only treatment strategy
for all types of glaucoma.
4
In order to investigate
the neuropathologic mechanism of glaucoma and
develop effective therapeutic interventions, many
kinds of experimental glaucoma models were
formed by elevating IOP or changing gene MYOC or
a1 subunit of collagen type 1 in rats,
6,7
mice,
8,9
monkeys,
10,11
dogs,
12
cats,
12
and several other spe-
cies.
12
Studies based on these animal models have
shown that neuropathology is related with depriv-
ation of target neurotrophic factor,
13,14
oxidative
stress,
15,16
mitochondrial dysfunction,
17
excitotoxic
damage,
18–20
and inflammation,
21
activation of apop-
totic signals,
22,23
ischemia.
24,25
Unfortunately, the
pathophysiologic mechanisms underlying glaucoma
are not understood, though researches in the field
of glaucoma are substantial. In addition, in these
Correspondence: Yuan Le, Department of Anesthesiology, The Third Xiangya Hospital of Central South University, Changsha, P.R. China.
E-mail: leyuanxy@csu.edu.cn
Received 18 December 2013; revised 21 April 2014; accepted 1 May 2014; published online 25 August 2014
1
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animal models of glaucoma, only young adult ani-
mals were used, neglecting the fact that glaucoma
is an age-related eye disorder and that age is an
important risk factor of glaucoma. Interestingly,
Samuel et al. found that RGCs density and synapse
density of inner plexiform layer and the areas of
dendritic and axonal arbors of RGCs all decreased at
the aged retina, compared to that of the young adult
retina.
26
Moreover, Wang et al. found that the loss
of RGCs after optic nerve crush in old mice was faster
than that in young mice.
27
Bakalash et al. found that
the protection of chondroitin sulfate-derived disac-
charide on retinal cells from IOP-induced death was
age-dependent in rats.
28
These data indicate that age
significantly impacts on the retinal injury and recov-
ery. On the other hand, high IOP is the most extensive
document risk factors of glaucoma. Kawai et al. also
detected that acute high IOP treatment induced
significant loss of RGCs at aged retinae that were
pretreated with chronic high IOP, compared to that
of age-matched rats.
29
However, in this study, the rats
were pretreated with chronic high IOP, therefore it
remains unknown whether aged and young adult rats
with same IOP treatment would yield the same retinal
injury.
Rat is a commonly-used model animal of glau-
coma. Rat shares similar anatomical
30,31
and develop-
mental
32,33
features of the anterior chamber, and
similar aqueous outflow pathway with the human.
Rat and human genomes are highly conservative.
34
These provide the basis for the use of rats as
a glaucoma model animal. In addition, rats are
inexpensive and easy to house and handle.
35
Their
eyes are easy to obtain, and the sample number for
studies can be large.
35
Moreover, elevating IOP of
rats reproduced the core phenotypes of glaucoma,
including RGC loss and damage of the optic nerve.
36
Thus, rat is a good model animal of glaucoma. Here
we constructed the rat glaucoma model by elevating
the IOP with cannulation of the anterior chamber,
and compared the retinal difference between young
adult and aged rats 3 d after IOP treatment of
45 mmHg, 60 mmHg or 90 mmHg. We found that
increased IOP induced more serious retinal damage
at aged retina than at young adult retina. At the same
time, increased IOP also activated more the astrocytes
and microglia at the aged retina.
MATERIALS AND METHODS
Animals and Grouping
Twelve young adult (aged 2 months, 200–250 g) and
twelve aged (aged 18 months, 500–550 g) female
Sprague-Dawley rats were purchased from Central
South University (P.R. China). All rats were raised
under controlled environmental conditions on a 12 h
light/dark cycle with ad libitum access to food and
water. All experimental protocols were approved by
the local animal ethics committee, and the guidelines
for animal experiments of Central South University
Young adult and aged rats were randomly divided
into normal control (n= 3, 6 eyes), 45 mmHg (n=3,
6 eyes), 60 mmHg (n= 3, 6 eyes), 90 mmHg (n=3,
6 eyes) groups. Intraocular pressures of rats in
45 mmHg, 60 mmHg, 90 mmHg (n= 3, 6 eyes) groups
were increased acutely to 45 mmHg, 60 mmHg and
90 mmHg, respectively. All the rats were killed at
3 days after high intraocular pressures.
Acute IOP Model
The animal model was prepared following reported
method.
35
Briefly, under anesthesia of 2% pentobar-
bital (40 mg/kg), 30-gauge needle connected to the
instillation instrument filled with normal saline
were inserted into the anterior chamber of rats. The
intraocular pressures was elevated to 45 mmHg,
60 mmHg or 90 mmHg, and then maintained for
60 min. After maintenance of 60 min, the 90 mmHg
of intraocular pressures was decreased through
80 mmHg for 5 min, 70 mmHg for 5 min, 60 mmHg
for 5 min, 30 mmHg for 5 min. finally, the needle
inserted into the anterior chamber was taken out.
For the condition of 45 mmHg and 60 mmHg of
intraocular pressures, after maintenance of 60 min,
the intraocular pressure was directly decreased
to 30 mmHg. Five minutes later, the needle inserted
into the anterior chamber was taken out.
Retinal Tissue Preparation
Under deep anesthesia, rats were firstly infused with
0.9% saline at 37 C, and then with 4% paraformalde-
hyde. The eyes were enucleated. After removing the
corneas and the lenses, the remainder of the eye
including the retina was post-fixed in 4% paraformal-
dehyde for 4 hr more at 4 C and then immersed in
30% sucrose. Cross sections were cut in parallel to the
equator of eyes through the optic disc of the retinae
at a thickness of 14 mm by a cryostat machine,
mounted on glass slides, dried at room temperature,
and finally stored at 20 C.
Immunofluorescence
Retinal sections stored at 20 C were taken out and
warmed at room temperature. After washing with
0.01 M phosphate buffered saline (PBS) for 10 min,
sections were incubated in blocking solution (5% BSA
and 0.3% Triton X- 100 in 0.1 M PB) for 1 hr at room
temperature. Then the sections were incubated in
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primary antibodies
36–38
(NeuN, 1:1000, Millipore,
Billerica, MA; Iba-1, 1:1000, Wako Chemical,
Richmond, MA; GFAP, 1:2000, Millipore) overnight
at 4 C. On the second day, these sections were
washed with 0.01 M PBS for three times and then
incubated in the secondary antibodies labeled with
fluorescent dyes (1:200, Jackson Immuno research) for
2 hours at room temperature. Through three washes
of PBS, these sections were covered with mounting
medium with DAPI (vector). As negative controls, an
adjacent series of sections were processed using the
same procedures without the primary antibodies.
Analysis
Three sections from each eye for each staining were
chosen. 1/6, 3/6, and 5/6 of the retinal radius away
from the optic nerve head were referred to as central,
middle, and peripheral retina, respectively
39
. Images
of central (about 600 mm to the midpoint of optic nerve
head) and peripheral(about 4000 mm to the midpoint
of optic nerve head) parts of retinal sections were
captured under 40Objective lens (image field:
430 mm320 mm) on a confocal microscopy of Leica.
The neuron of retinal ganglion cell layers was defined
by co-labeling of NeuN (neuron marker) and DAPI
(nuclear marker). The microglia was defined by co-
labeling of Iba1 (microglia marker) and DAPI (nuclear
marker). The number of neuron and microglia in
retinal ganglion cell layers of captured pictures were
blindly counted, respectively. Percentage of Iba1
positive area to area of inner retina (including nerve
fiber layer, ganglion cell layer and inner plexiform
layer) and relative mean gray value of GFAP staining
at inner retina were measured by Image J.
40,41
For
quantitaion of each test, 36 images were used.
All data are presented as mean ± standard devi-
ation (mean ± SD). Two-way analysis of variance
(ANOVA) followed by LSD test were used for
means comparisons. PValues50.05 were considered
statistically significant.
RESULTS
Same High IOP Resulted in More Serious
Loss of Neurons at the Ganglion Cell Layer
of Aged Retinae than at Young Adult Retinae
RGC loss is the main pathological feature of glaucoma
retina. Thus we detected the neuron loss of ganglion
cell layer (GCL) of aged and young adult retina
3 days after IOP treatment of 45 mmHg, 60 mmHg,
and 90 mmHg (Figure 1). Two way ANOVA analysis
showed that aging significantly increased neuron loss
of GCL 3 days after IOP treatment (F(1,40)=138.99,
p50.001, for central; F(1,40)=84.14, p50.001, for
peripheral). Moreover, IOP treatment also
exhibited significant effects on neuron loss of GCL
(F (3,40)=214.13, p50.001, for central; F(3,40)=109.10,
p50.001, for peripheral). Further analysis showed
that at the normal control, the number of neurons
in GCL of peripheral (10.16 ± 0.87) and central
(15.33 ± 1.03) aged retina were not different from that
of young adult retina, respectively (p= 0.220 for
central; p= 0.341 for peripheral) (Figure 1). At IOP
treatment of 45 mmHg, the number of neurons in GCL
of peripheral (6.16 ± 0.97) and central (13.00 ± 1.11)
aged retina were significantly less than that of normal
aged retina, respectively (p= 0.002 for central; p= 0.001
for peripheral) (Figure 1). More obvious neuron loss
of peripheral and central aged retina was detected at
IOP treatment of 60 mmHg and 90 mmHg. However,
significant neuron loss of peripheral (3.00 ± 0.35) and
central (7.16 ± 1.17) retina could be detected at IOP
treatment of 90 mmHg at the young adult retinae,
compared to that of normal young adult retinae
(p50.001 for central; p50.001 for peripheral)
(Figure 1). These data implied that aged retina was
susceptible to IOP, compared to the young adult
retina.
Microglia of Aged Retinae was activated
more easily than that of Young Adult
Retinae under High IOP Treatment
Activated microglia played important roles in damage
of glaucoma by releasing inflammatory factors.
Previous results showed that peripheral retina was
susceptible to changed IOP, compared to the central
retina.
39
Thus we detected the activation of microglia
of peripheral retina by counting the number and the
area percentage of microglia marked by Iba1 in inner
retina (Figure 2). Two-way ANOVA analysis showed
that aging significantly increased the activation of
microglia 3 days after IOP treatment (F (1,40)=116.72,
p50.001, for area percentage; F (1,40)=156.89,
p50.001, for microglia number). Moreover, IOP treat-
ment also significantly affected the activation of
microglia (F (3,40)=250.13, p50.001, for area percent-
age; F(3,40)=190.03,p50.001, for microglia number)
(Figure 2). We found that at the normal control, the
number(1.00 ± 0.58) and the area percentage
(4.72% ± 0.51%) of microglia of aged retinae were not
different from that of young adult retina(p= 0.088
for number; p= 0.051 for area percetage) (Figure 2).
At day 3 after IOP treatments of 45 mmHg, 60 mmHg,
or 90 mmHg, the number and the area percentage of
microglia at aged and young adult retina both were
significantly increased, compared to that of normal
control(p50.05) (Figure 2). The number and the area
percentage of microglia of aged retinae were signifi-
cantly higher than that of young adult retina at each
IOP treatment (p50.05) (Figure 2). These showed that
Age of Rats Affects the Degree of Retinal Damage 3
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increased IOP induced more obvious activation of
microglia at aged retina than at young adult retina.
Macroglia was Activated at the Retinae of
Aged and Young Adult Retina Under High
IOP Treatment
Mu
¨ller glial cells and astrocytes belong to macroglia in
the retina. Activation of macroglia was characterized
by increased processes and enlarged cell body. Here
we detected macroglia activation by measurement of
the relative gray value of GFAP staining, the marker
of macroglia, in the inner retina. Similar to that in
microglia, increased IOP induced stronger GFAP
expression at ganglion cell layer and more obvious
GFAP positive processes in inner plexiform layer at
aged and young adult retina, compared with the
normal retina (Figure 3). It was noticed that at aged
retina, many GFAP positive processes extended into
the inner plexiform layer, even into the inner nuclear
layer after high IOP treatment (Figure 3).
Two-way ANOVA analysis of relative mean gray
value of GFAP staining at the inner retina showed that
age didn’t significantly increased the activation of
macroglia 3 days after IOP treatment (F (1,40)=3.24,
FIGURE 1 Neurons of ganglion cell layer of aged retina were more susceptible to IOP damage than that of young adult retina.
Neurons of ganglion cell layer were marked by NeuN (red). Compared with the age-matched normal control (A, E, I, M), IOP
treatment of 45 mmHg, 60 mmHg, and 90 mmHg induced the loss of neurons at ganglion cell layer of young adult (B–D, J–L) and aged
(F–H, N–P) retinae 3 days after treatment. Quantitative analysis showed that at central (F) and peripheral (N) parts of aged retina, IOP
treatment of 45 mmHg was enough to induce significant loss of neurons of ganglion cell layer, compared to that of normal aged retina
(p= 0.002 for central; p= 0.001 for peripheral) (Q, R). In contrast, significant loss of neurons at ganglion cell layer of central and
peripheral young adult retinae was detected until IOP treatment of 90 mmHg (p50.001 for central; p50.001 for peripheral; Q, R).
GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer. *p50.05 versus control;
#p50.05 versus matched part of young adult retina at the same IOP treatment. Bar = 50 mm.
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p= 0.080). However, IOP treatment significantly
affected the activation of macroglia (F (3,40)=73.22,
p50.001; Figure 3). The relative mean gray value of
GFAP staining at the inner retina was significantly
increased at young adult and aged retinae, compared
with that of the normal control (p50.05; Figure 3).
The same IOP treatment didn’t produce significant
difference of relative mean gray value of GFAP
staining in the retina between aged and young adult
rats (p= 0.236 for 45 mmHg, p= 0.052 for 60 mmHg,
p= 0.561 for 90 mmHg; Figure 3).
DISCUSSION
The aim of this study was to investigate whether
retinal damage was affected by age of rats in
glaucoma model. We found that compared to that of
the young adult retina, (1) neurons at the ganglion cell
layer of aged retina were more susceptible to
increased IOP; (2) microglia of aged retina was more
easily activated by the increased IOP. These results
suggest that age of rats should be taken into account
at studying of experimental glaucoma.
Loss of RGCs was the main pathological feature of
glaucoma retina and the basis of visual defects.
42,43
Thus we first detected the loss of neuron at GCL after
different IOP treatments. We found that at young
adult retina, significant loss of neurons at GCL wasn’t
detected until IOP of 90 mmHg. However significant
loss of neurons at GCL of aged retina was detected
at IOP of 45 mmHg and more as IOP increased.
In addition, the loss of neurons in aged retina was
significantly higher than that of young adult retina at
different IOP treatments. These results suggested
that neurons of GCL of aged retina were more
susceptible to increased IOP, compared to young
adult retina. Our data are consistent with a recent
report in which after induction of prolonged cere-
bral hypoperfusion, 8-month-old mice showed more
severe white matter injury and working memory
dysfunction, compared with that of 2-month mice.
41
Previous researches showed that activation
of glial cells contributed much to the loss
of RGCs of glaucoma retina.
38–42
Inman et al.
found that in chronic glaucoma model of mice,
microglial cell number increased by two–fold.
44
Moreover, minocycline treatment reduced the retina
microglial activation in the DBA/2 J mouse model of
glaucoma, corresponding to the improved optic nerve
integrity.
45
Interestingly, in our study, we detected that
high IOP increased area percentage and number of
FIGURE 2 Activation of microglia at aged retina was more obvious than that of young adult retina 3 days after high IOP treatment.
Area percentage and number of microglia (green) were used to show the activation of microglia. Three days after IOP treatment of
45 mmHg, 60 mmHg, and 90 mmHg, microglia of inner retina of young adult (B–D) and aged (F–H) retinae both were activated,
compared with the age-matched normal control (A, E). Quantitative analysis showed that area percentage (J) and number (I) of
microglia of aged inner retina increased more, relative to that of young adult retina at each IOP treatment (p50.05). GCL, ganglion cell
layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer. *p50.05 versus control; #p50.05 versus young
adult retina at the same IOP treatment. Bar = 50 mm.
Age of Rats Affects the Degree of Retinal Damage 5
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microglia in inner retina at IOP of from 45 mmHg to
90 mmHg. Under the same IOP, the increased degrees
of area percentage of microglia and microglia number
at the inner part of aged retina were much higher than
that at young adult retina. Moreover, these changes of
microglia were in parallel to or earlier than the loss
of neurons at GCL. These suggested that more obvious
activation of microglia possibly contributed to the
easier loss of neurons at aged retinae after high IOP
treatment, compared to young adult retinae. But the
molecular mechanism that microglia differentially
contributed to the age-related susceptibility of neurons
of GCL needs to be further studied. In brief, we found
that age seriously affects the degree of retinal damage
induced by acute high intraocular pressure.
DECLARATION OF INTEREST
There is no conflict of interest. The authors alone are
responsible for the content and writing of the paper.
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