Annals of Anatomy 2011

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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




206 T.C. Nag et al. / Annals of Anatomy 193 (2011) 205–210

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




T.C. Nag et al. / Annals of Anatomy 193 (2011) 205–210 207

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





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




T.C. Nag et al. / Annals of Anatomy 193 (2011) 205–210 209

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,





National eye bank, Dr Rajendra Prasad center for Ophthalmic

Sciences, AIIMS, New Delhi, for permitting us to collect the

eyeballs.

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Annals of Anatomy 2011