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Annals of Anatomy 193 (2011) 205–210
Contents lists available at ScienceDirect
Annals of Anatomy
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . d e / a a n a t
Research article
Localization of 4-hydroxy 2-nonenal immunoreactivity in aging
human retinal Müller cells
Tapas C. Nag a,∗, Shashi Wadhwa a, Phalguni Anand Alladi b, Tania Sanyal a
a Department of Anatomy, Neurobiology Laboratory, All India Institute of Medical Sciences, New Delhi 110029, Indiab Department of Neurophysiology, National Institute of Mental Health and Neurosciences, Bangalore 560029, Karnataka, India
a r t i c l e i n f o
Article history:
Received 25 June 2010
Received in revised form 31 January 2011
Accepted 15 February 2011
Keywords:
Human retina
Aging
Müller cells
Lipid peroxidation
4-hydroxy 2-nonenal
Glutamine synthetase
s u m m a r y
Müller cells play a pivotal role in maintaining retinal homeostasis of theextracellular fluid environment.
Information on whether human retinal Müller cells suffer from oxidative stress with normal aging is
lacking.We examinedpost mortem human retinas for thelocalizationof a biomarkerof lipidperoxidation
(4-hydroxy 2-nonenal, 4-HNE) by immunohistochemistry. We procured human eyesfrom donors(N =11;
age: 45–91 years; post mortem delay: 1–3 h), who had no history of ocular diseases. They were fixed
in 4% paraformaldehyde and the retinas cryosectioned and labeled against anti-4-HNE employing the
immunoperoxidase method. Compared to the lower agegroup (45–56 years), in the advanced age group
(67–91 years), immunoreactivity (IR) to 4-HNE was prominent in peripheral Müller cell end-feet, select
cellsin theinner nuclear layerand inouterfibers located inthe macularfiberlayerof Henle.Colocalization
withglutamine synthetase revealedthat the4-HNE positiveprofilesin theinner nuclear layerwere Müller
cells. Quantitative analysisrevealedthat thepercentage of immunopositivecells in theinner nuclear layer
aswell asthe grey levelsof theimmunoreactionproductsin theparafoveal andperipheral retinal regions
significantly increasedin theadvancedage group.The findings indicate that Müllercells of human retina
suffer from lipid peroxidation and are susceptible to damage in the course of normal, advanced aging.
© 2011 Elsevier GmbH. All rights reserved.
1. Introduction
Müller cells are the predominant type of glia of the vertebrate
retina. Their somata extend processes that intimately ensheath
almost every retinal neurons. They perform a number of impor-
tant functions through a metabolic symbiosis between the retinal
neurons and themselves (Bringmann and Reichenbach, 2001;
Bringmann et al., 2006). They maintain homeostasis of the reti-
nal extracellular environment and protect neurons via uptake of
glutamate and secretion of glutathione (Bringmann et al., 2006).In
diseased condition, they react in support of the survival of neu-rons via gliosis, which on the one hand, may provoke neuronal
degeneration.
Information on age-related changes in structure and neuro-
chemistry of human retinal Müller cells is rather limited. These
glial cells show an age dependent decrease of their K+ conductance
(Bringmann et al., 2003); this should cause a disturbance of the
retinal K+ homeostasis, contributing to retinal complications, like
diabetes in elderly patients. The retina suffers from oxidative stress
and this contributes to the progression of gliosis (Asnaghi et al.,
∗ Corresponding author. Tel.: +91 11 26594875; fax: +91 11 26588663.
E-mail address: tapas [email protected] (T.C. Nag).
2003; Baydaset al., 2004). Lipid peroxidation, a type of free radical-
mediated oxidative stress that attack membrane lipids, is reported
to occur in diabetic rat retina, involving reactive changes in Müller
cells (Baydas et al., 2004). It remains unknown whether Müller
cells suffer from oxidative stress in aging and in what manner they
respond to it.
In the present study, we used immunohistochemistry to exam-
ine the expression of a biomarker of lipid peroxidation, namely
4-hydroxy 2-nonenal (4-HNE), in human retinas at various ages.
2. Materials and methods
2.1. Eyeballs and fixation
The eyes used in this study were from normal donors (N =11)
who had no history of ocular diseases. They were procured from
The National Eye Bank, Dr Rajendra Prasad Center for Ophthalmic
Sciences, AIIMS, New Delhi. Table 1 shows the age, cause of death
of the donors, and time elapsed between death and fixation of
eyes. The donors were grouped into two categories: the lower age
group (45–56 years; N = 4, eyes employed= 8) and advanced age
group (67–91 years; N = 7, eyes employed= 10). The corneas were
excised and stored by the eye bank authority for transplantation
in the future. The protocols followed here adhered to the tenets of
0940-9602/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.
doi:10.1016/j.aanat.2011.02.004
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Table 1
Information about the donors (N = 11) whose eyes were used.
Agea Sex Cause of deathb Delay in fixationc
45 M Heart attack 3
50 M Haemorrhage 2
52 M Heart attack 2
56 M Myocardial infarction 2
67 M Heart attack 1
75 M Heart attack 2
78 M Cardiac arrest 2
81 M Heart attack 2
83 M Myocardial infarction 2
86 F Cardiac attack 2
91 F Cardio-respiratory attack 1
a In years.b Information obtained from case registry.c In hours; M, male; F, female.
Helsinki declaration for research on human tissues. Written con-
sent from relatives of the donors was obtained for procurement
of the eyes and their use in research. The Institute human Ethics
Committee approved the present study (As-207/2008).
The eyes were fixed in 4% paraformaldehyde in 0.1M phosphatebuffer (pH 7.4) for 48h at 4 ◦C. After washing in buffer, the retina
was cut naso-temporally using the optic disc as a reference point.
The retina was cut for a length of 3–5mm nasal from the optic
disc and 9 mm along the temporal axis, leaving the macula and
peripheral retina (eccentricity: 5–6 mm from the macular border)
intact. The width of the tissue samples was approximately 3 mm.
The samples were cryoprotected in 15–30% sucrose overnight, and
frozen sections (thickness: 14m) cut. They were mounted onto
gelatin-coated slides and stored at −20 ◦C until use.
2.2. Immunohistochemistry
Retinal sections were immunoreacted with an antibody against
4-HNE (rabbit polyclonal, Alpha Diagnostic International, SanAntonio, Texas, Dilution: 5g/ml). Sections were quenched of
endogenous peroxidase activity by treating in 0.3% hydrogen per-
oxide in methanol for 30min and washed. Sections were incubated
in 10% goat normal serum (diluent: 0.01M phosphate-buffered
saline containing 0.5% triton X-100) for 3 h and then in the pri-
mary antibody for 48h at 4 ◦C. After washing, sections were
incubated in the secondary antibody (biotinylated anti-rabbit IgG;
1:200; Vector Laboratories, Burlingame, California, USA) for 6 h at
4 ◦C. The antigen-antibody binding sites in sections were visual-
ized by employing the avidin-biotin immunoperoxidase method
(Vectastain Elite Kit, Vector Laboratories, CA, USA) using 0.06%
diaminobenzidine tetrahydrochloride (DAB) as a chromogen. In
control experiments, incubation of sections in the primary anti-
body wassubstitutedwith thesecondaryantibody. The slidesweredehydrated in ethanol and coverslipped with DPX. Adjacent reti-
nal sections were stained with hematoxylin and eosin to see the
integrity of the cellular layers and identification of macular sub-
regions (parafoveal and perifoveal), using ganglion cell layer and
inner nuclear layer thickness as references. Photographs of the sec-
tions were taken under a Leica microscope and images acquired
with a Leica DFC 420 C digital camera, using software [(Leica
Application Suite, Version 3.4.1; Leica Microsystem (Switzerland)
Limited)].
2.3. Image analysis
To assess 4-HNE immunoreactivity levels between lower- and
advanced age groups, image analysis of the DAB reaction productwas done. For this, retinal sections from lower age group (N = 4;
45M, 50M, 52M and 56M) and advance aged group (N =4; 67M,
75M, 81M and86F) were processed simultaneously using the same
antibody dilutions and treatment protocol. Retinal sections were
viewed at 20X magnification, the images acquired using the dig-
ital camera (Leica DFC 420C) and transferred to a video monitor.
The grey level in sections was detected using Leica Q Win software
equipped with the Leicamicroscope. The lightintensity(0.996) was
kept constant throughout the imaging process. Using the standardgrey detection mode, from a 0 to 255 scale, a threshold adjustment
of the staining was calibrated between 0 and 150. The fixation of
theuppergrey value at 150discretely masked only thestained cells
andfiberswith thebinary color,without overlapping theunstained
background tissues. For quantification of grey levels, the retinal
boundary was delimited from the inner limiting membrane to the
outer limiting membrane insidea fixed measuring frame.Four reti-
nal sections, showing consistent staining were selected from each
donorretina andgreylevels measuredat theparafovealand periph-
eral retinal regions. The mean grey values were calculated for both
retinal regions from individual donor retinas.
2.4. Quantification of 4-HNE positive cells
For this,immunolabeledslides were lightly counterstained with
hematoxylin, dehydrated in ethanol and mounted with DPX. For
counting, the same donor retinas were utilized as employed for
image analysis. Under the microscope, at 40× magnification, the
numberof immunopositive cells (appearedin brown)as well as the
immunonegative cells (appeared in blue) in the inner nuclear layer
of four consecutive sections was counted at a length of 285m
in the parafoveal and peripheral retinal regions. From the mean
values, the percentage of immunoreactive cells in the inner nuclear
layer out of total cells present was counted for each donor retina.
2.5. Colocalization of 4-HNE and glutamine synthetase by
confocal laser scanning microscopy
In our study, Müller cell endfeet and select cells of the inner
nuclear layer were found to be 4-HNE immunopositive. To identify
and confirm whether immunopositive profiles of the inner nuclear
layer belonged to Müller cells, we performed colocalization of 4-
HNE with glutamine synthetase, which is a marker of Müller cells
(Linser and Moscona, 1979). For this, peripheral retinal sections
from 75-year, 81-year and 86-year-old donors were selected. For
colocalization of both markers, we used a sequential staining pro-
cedure. The sections were first equilibrated in 0.1M phosphate
buffer (pH 7.4) for 10 min and then blocked with 3% bovine serum
albumin (BSA, Vector Laboratories, Burlingame, CA, USA) for 1 h at
room temperature. This was followed by incubation of the sections
in the rabbit polyclonal glutamine synthetase antibody (dilution:
1:10,000; Sigma Chemicals Company, St. Louis, MO, USA) for 24 h
at 4 ◦C. Thereafter, the sections were incubatedwith theanti-rabbitIgG conjugated to fluorescein isothiocynate (FITC, dilution: 1:500;
Sigma–Aldrich, CA,USA) for4 h at room temperature.Beforebegin-
ning the labeling with the second antibody, the sections were
washed in 0.01 M Phosphate buffer saline-Triton X-100 and then
blocked with 3% BSA for 1 h at room temperature. Thereafter the
sections were incubated with anti-rabbit 4-HNE primary antibody
(dilution 1:1000; Alpha Diagnostic International, San Antonio, TX,
USA), as used in light microscope immunohistochemistry, for 24 h
at 4 ◦C. Anti-rabbit secondary antibody tagged to Cy3 was used
to detect the binding (dilution: 1:500; Sigma–Aldrich, CA, USA).
For negative controls, the primary antibody was replaced with the
dilution buffer. The fluorescent images were captured using laser
scanning confocal microscope (DMIRE-TCS Leica, Germany) using
laser excitation at 488nm for FITC and 514 nm for Cy3. Emissionband widths of 495–540 nm for FITC and 550–620 nm for Cy3 were
maintained to avoid non-specific overlap of emission frequencies
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Fig. 1. Immunohistochemicallocalization of 4-HNE in a 56-year-old donorretina. (A) Fromthe periphery, IR is apparent in manyMüller cell end-feet (arrowheads) and cells
(in deep brown color) of the inner nuclear layer (inl, arrow). (B) From the macular perifoveal region, showing IR in many cells of the i nner nuclear layer (inl, arrow), but little
or no IR in Müller cell end-feet. (C) From the parafoveal region, Müller cell outer fibers located within the fiber layer of Henle (asterisk) are strongly immunoreactive, as are
the cells of the inner nuclear layer (inl, arrow). All sections are counterstained with hematoxylin (blue color). gcl, ganglion cell layer; Ipl, inner plexiform layer; onl, outer
nuclear layer. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
(Alladiet al., 2010). Allimages were captured using 20×magnifica-tion at a constant photomultiplier tube voltage of 537. Further, the
software controls and microscope settings such as optical zoom,
scan speed, pinhole aperture and image resolution were kept uni-
form.
2.6. Statistical analysis
The grey levels of the DAB immunoreaction product as well as
the percentage of 4-HNE immunopositive cells in the inner nuclear
layer in the parafovea and periphery of both donor group (young
vs advanced aged groups) retinas were statistically analyzed using
non-parametric Mann–Whitney U test.
3. Results
Histologically, all donor retinas appeared well-organized with
minimal autolytic changes. In retinas from lower age-groupdonors
(45–56 years; N = 4), the expression of 4-HNE was observed in
few Müller cell end-feet located at the periphery and cells of the
inner nuclear layer (Fig. 1A). At the macular region, very little
immunoreactivity (IR) could be detected in Müller cell end-feet
(not shown), but relative to the periphery, many cells of the inner
nuclear layerwas 4-HNE immunopositivein 45-year to 56-year-old
donor retinas (Fig. 1B, 56-year-old). Additionally, the outer fibers
of Müller cells located within the fiber layer of Henle were strongly
immunopositive (Fig. 1C). From the seventh decade of life onward
(donor age > 67 years), IR was found extensively in many cells of
the inner nuclear layer of the macula (Fig. 2A, 81-year-old) andin Müller cell end-feet located in the peripheral retina (Fig. 2B,
81-year-old; Fig. 2C, 83-year-old and Fig. 2D, 86-year-old). Such
a pattern of 4-HNE IR (widespread in peripheral Müller cell end-
feet and macular inner nuclear layer cells) was uniformly noted in
allother advanced aged retinas (donorages: 75–91 years), with the
exception that in three retinas (81-, 83- and 86-year-old donors),
photoreceptor outer segments were also labeled (Fig. 2A, 81-year-old).
Quantitative analysis of retinal sections immunolabeled with 4-
HNE and counterstained with hematoxylin revealed that in both
parafovea and periphery, the percentage of immunopositive cells
of the inner nuclear layer increased in the retinas of the advanced
age group, when compared with the lower age group ((Fig. 3;
p≤0.02). In the latter, the mean values (with standard deviations)
at the parafovea and periphery were 27.28±2.78 and 23.97±5.09,
respectively; the corresponding values in the advanced age group
were 41.82±0.71 and 46.63±1.11, respectively. This increase
in the advanced age group was statistically significant at both
parafovea( p≤0.02) andperiphery ( p≤0.02).This wasalso thesitu-
ation when the grey levels were determined at both retinal regions
in young vs advanced aged group ( p≤0.02; Table 2).Colocalization of 4-HNE immunofluorescence with glutamine
synthetase (a marker for Müller cells;) by confocal laser scanning
microscopy revealed that many 4-HNE positive cells of the inner
nuclear layer were indeed Müller cells (Fig. 4). In some cases, IR in
the processes originating from the Müller cell bodies andterminat-
ing into end-feet was evident (Fig. 4A–C, 75-year-old).
4. Discussion
In this study, we have noted 4-HNE expression in Müller cells,
with prominent IR that was localized in their end-feet located in
peripheral retina. The little IR found in the macular counterparts
is due to their lower density as well as smaller size in this special-ized retinal region than at the periphery (Nishikawa and Tamai,
2001). Examinations of our materials have shown that with an
advancement of age, the IR became stronger in peripheral Müller
cells end-feet. Besides, in the macula, fibers located in the outer
retina showed clear immunopositivity. Since no cone photorecep-
tors were labeled, the immunopositive fibers that were located in
Table 2
Grey values of the DAB reaction products determined in the donor retinas. The lower grey values indicate the relative increase in the level of immunoreactions, and vice
versa.
Regions Young Mean±SD Aged Mean±SD
45M 50M 52M 56M 67M 75M 81M 86F
Grey values Grey values
Parafovea 186 183 181 179 182.25 ± 2.99 180 173 175 158 171.50 ± 9.46
Periphery 160 157 142 131 147.5 ± 13.52 149 152 138 122 140.25 ± 13.57
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Fig. 2. Immunohistochemical localization of 4-HNE in advanced age donor retinas. (A) Macular region from 81-year-old donor, showing IR in cells of the inner nuclear layer
(inl) and in outer fibers located within the fiber layer of Henle (asterisk). Note IR in outer segments in the photoreceptor layer (prl). (B) Peripheral region from 81-year-
old donor, showing IR in Müller cell end-feet (arrows) and cells of the inner nuclear layer (inl). (C and D) Peripheral region from 83-year- and 91-year-old donor retinas,
respectively. IR is intense in Müller cell end-feet (arrows). Cells of the inner nuclear layer (inl) are also immunopositive. All sections are counterstained with hematoxylin
(blue color). gcl, ganglion cell layer; Ipl, inner plexiform layer; onl, outer nuclear layer; prl, photoreceptor layer. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of the article.)
the fiber layer of Henle of the macula were perhaps the outer fibers
of Müller cells. These fibers, along with the perikarya in the inner
nuclear layerand vitreal end-feet indicate sitesof lipidperoxidation
in Müller cells of human retina in normal aging process.
The murine retina suffers from oxidative stress under differ-
ent experimental conditions (e.g., upon exposure to light and
intra-vitreal iron load) and these contribute to appearance of lipid
peroxidation markers in photoreceptors (De La Paz and Anderson,
1992; Wiegand et al., 1983; Tanito et al., 2006; Rogers et al., 2007).
4-HNE is a major end product of lipid peroxidation and has been
widely accepted as an inducer of oxidative stress (Uchida, 2003). It
Fig. 3. Histogram showing percentage of 4-HNE immunopositive cells in the inner
nuclear layer of parafoveal and peripheral retinas in young vs aged group. Note the
significant increasein percentage of cellsin the advancedaged group in bothretinal
regions (* p <0.02).
hasbeen found that retinal damagecaused by light exposure canbe
reduced by various types of synthetic antioxidants, and so oxidative
stress was considered to be involved in the pathogenesis of light-
induced retinal damage. Several authors (Uchida and Stadtman,
1992; Tanitoet al., 2005) reported an increase in theretinal protein
modification by 4-HNE accumulation. Because in our study promi-
nent IR was seen in aging Müller cell end-feet (indicating increased
level of 4-HNE), the proteins located in those compartments (e.g.,
potassium channel, aquaporins) may get modified by 4-HNE. This
may equally be their cell bodies located in the inner nuclear layer.
The actual reasons for oxidative stress in aging human retina
are not clear (Beatty et al., 2000; Shen et al., 2007), but could
be attributed to Sunlight, smoking, nutritional status (lack of carotenoids in diets) and decreased antioxidant defense mecha-
nisms with normal aging. Due to senility, reactive oxygen species
production could be greater in aging retina and they can cause
the peroxidative change of lipids, proteins and nucleic acids in the
absence of the required level of retinal endogenous antioxidants
(e.g.,carotenoids, Vitamin E) and antioxidant enzymes (glutathione
peroxidase, glutathione-S-transferases, catalase and superoxide
dismutase). Here, we show oxidative stress in aging human retina
involving Müller cells. Müller glia support many important physio-
logical functionsthat areperformed byretinal neurons (Bringmann
et al., 2006). They are believed to be resistant to damage/changes
in processes in which neurons show the initial sufferings. The fact
that aging Müller cells suffer from oxidative stress, as is evident
in our study, raises issues about how the general physiologicalwell-being of the retina in aging would be maintained. For exam-
ple, Müller cells protect retinal neurons via uptake of glutamate
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Fig. 4. Colocalization of 4-HNE and glutamine synthetase in retinas from 75-year (A–C), 81-year (D–F) and 86-year (G–I) old donors. Photographs of the left panel (A, D, G)
show glutamine synthetase immunofluorescence, those of the mid panel (B, E, H) show 4-HNE immunofluorescence and the right panel (C, F, I) shows the merge view of
both immunofluorescence. In all three retinas, the glutamine synthetase immunofluorescence (green FITC label) in Müller cell bodies located the inner nuclear layer (inl)
colocalizes with 4-HNE immunofluorescence(red Cy 3 label), as is evident in mergedviews (yellow, right panel, arrows).Asterisks in (A–C) denotelabels in Müller cell outer
fibers located within the fiber layer of Henle and arrowheads denote the vitreal processes terminating in end-feet. Intense labels are seen in Müller cell end-feet (ef) in
86-year-old donor retina (G–I). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
and secretion of glutathione (Bringmann et al., 2006). Decreased
glutamate uptake (due to low expression of the glutamate trans-
porter, GLAST and/or decreased activity of Na, K, ATPase ( Rauen
et al., 1998; MacGregor and Matschinsky, 1986) was shown tocause a decrease of glutathione synthesis in Müller cells (Reichelt
et al., 1997), a condition that must enhance oxidative stress in the
retina. So, one possible reason for oxidative stress in Müller cells
may be related to the level of glutathione in these glia in aging,
and is worthwhile for future study. Even if the glutathione level
remains unaltered, its remarkably high levels in Müller cells ( Pow
and Crook, 1995; Paasche et al., 1998) make them susceptible to
glutathione-depleting agents (Ulyanova et al., 2001). Since 4-HNE
is a strong electrophile, one possibility is that it can significantly
alter cellular redox status by depleting sulfhydryl compounds,such
as glutathione (Uchida, 2003).
In parallel with studies showing lipid peroxidation by 4-HNE,
several other reports have indicated a protective role for 4-HNE in
oxidative stress. 4-HNE accumulation may exert a protective roleby upregulating glutathione S-transferases (Fukuda et al., 1997;
Awasthi et al., 2004), the endogenous antioxidant defense system
predominantly involved in cellular detoxification. Similar is the sit-
uation with the expression of heme-oxygenase-1 in Müller cells
of mouse retina in organ culture in response to oxidative stress
(Ulyanova et al.,2001) andin macrophages,as a protective responseagainst 4-HNE (Iles et al., 2005). Thus, it is important to know how
redox status is affected by oxidative stress in Müller cells of aging
human retina. Glutaredoxins are small redox enzymes, which use
glutathione as a cofactor.Retinal localization of theseenzymes after
oxidative stress is not known. There is need to assess this issue
in depth, especially to know the endogenous antioxidant defense
mechanism of Müller cells against oxidative stress occurring as a
result of aging and other insults.
Acknowledgements
The work was supported by funds from the Department of
Biotechnology, Govt. of India (BT/PR 10195/BRB/10/589/2007)and Institute Research grant (F. 6-1/2009 Acad (Para-Med.) to
TCN. We sincerely thank Prof. Radhika Tandon, Officer-In-Charge,
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National eye bank, Dr Rajendra Prasad center for Ophthalmic
Sciences, AIIMS, New Delhi, for permitting us to collect the
eyeballs.
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