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Localization of 4-hydroxy 2-nonenal immunoreactivity in aging human retinal Müller cells
Annals of Anatomy 193 (2011) 205–210
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Annals of Anatomy journal homepage: www.elsevier.de/aanat
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 b
Department of Anatomy, Neurobiology Laboratory, All India Institute of Medical Sciences, New Delhi 110029, India 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 the extracellular fluid environment. Information on whether human retinal Müller cells suffer from oxidative stress with normal aging is lacking. We examined post mortem human retinas for the localization of a biomarker of lipid peroxidation (4-hydroxy 2-nonenal, 4-HNE) by immunohistochemistry. We procured human eyes from 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 age group (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 cells in the inner nuclear layer and in outer fibers located in the macular fiber layer of Henle. Colocalization with glutamine synthetase revealed that the 4-HNE positive profiles in the inner nuclear layer were Müller cells. Quantitative analysis revealed that the percentage of immunopositive cells in the inner nuclear layer as well as the grey levels of the immunoreaction products in the parafoveal and peripheral retinal regions significantly increased in the advanced age group. The findings indicate that Müller cells 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 important functions through a metabolic symbiosis between the retinal neurons and themselves (Bringmann and Reichenbach, 2001; Bringmann et al., 2006). They maintain homeostasis of the retinal 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 neurons via gliosis, which on the one hand, may provoke neuronal degeneration. Information on age-related changes in structure and neurochemistry 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). 0940-9602/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.aanat.2011.02.004
2003; Baydas et al., 2004). Lipid peroxidation, a type of free radicalmediated 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 examine 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
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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 50 52 56 67 75 78 81 83 86 91
M M M M M M M M M F F
Heart attack Haemorrhage Heart attack Myocardial infarction Heart attack Heart attack Cardiac arrest Heart attack Myocardial infarction Cardiac attack Cardio-respiratory attack
3 2 2 2 1 2 2 2 2 2 1
a b c
In years. Information obtained from case registry. In hours; M, male; F, female.
Helsinki declaration for research on human tissues. Written consent 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 0.1 M phosphate buffer (pH 7.4) for 48 h 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 3–5 mm 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: 14 m) 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, San Antonio, Texas, Dilution: 5 g/ml). Sections were quenched of endogenous peroxidase activity by treating in 0.3% hydrogen peroxide in methanol for 30 min and washed. Sections were incubated in 10% goat normal serum (diluent: 0.01 M phosphate-buffered saline containing 0.5% triton X-100) for 3 h and then in the primary antibody for 48 h 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 visualized 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 antibody was substituted with the secondary antibody. The slides were dehydrated in ethanol and coverslipped with DPX. Adjacent retinal sections were stained with hematoxylin and eosin to see the integrity of the cellular layers and identification of macular subregions (parafoveal and perifoveal), using ganglion cell layer and inner nuclear layer thickness as references. Photographs of the sections 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 product was done. For this, retinal sections from lower age group (N = 4; 45M, 50 M, 52M and 56M) and advance aged group (N = 4; 67M,
75M, 81M and 86F) were processed simultaneously using the same antibody dilutions and treatment protocol. Retinal sections were viewed at 20X magnification, the images acquired using the digital camera (Leica DFC 420 C) and transferred to a video monitor. The grey level in sections was detected using Leica Q Win software equipped with the Leica microscope. The light intensity (0.996) was kept constant throughout the imaging process. Using the standard grey detection mode, from a 0 to 255 scale, a threshold adjustment of the staining was calibrated between 0 and 150. The fixation of the upper grey value at 150 discretely masked only the stained cells and fibers with the binary color, without overlapping the unstained background tissues. For quantification of grey levels, the retinal boundary was delimited from the inner limiting membrane to the outer limiting membrane inside a fixed measuring frame. Four retinal sections, showing consistent staining were selected from each donor retina and grey levels measured at the parafoveal and peripheral 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, immunolabeled slides 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 number of immunopositive cells (appeared in 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 285 m 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 4HNE 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 procedure. The sections were first equilibrated in 0.1 M 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 incubated with the anti-rabbit IgG conjugated to fluorescein isothiocynate (FITC, dilution: 1:500; Sigma–Aldrich, CA, USA) for 4 h at room temperature. Before beginning 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 488 nm for FITC and 514 nm for Cy3. Emission band 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. Immunohistochemical localization of 4-HNE in a 56-year-old donor retina. (A) From the periphery, IR is apparent in many Müller 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 inner 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.)
(Alladi et al., 2010). All images were captured using 20× magnification 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 uniform. 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-group donors (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 layer was 4-HNE immunopositive in 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) and in 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 endfeet and macular inner nuclear layer cells) was uniformly noted in all other advanced aged retinas (donor ages: 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-yearold). Quantitative analysis of retinal sections immunolabeled with 4HNE 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) and periphery (p ≤ 0.02). This was also the situation 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 and terminating 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 specialized 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 photoreceptors 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
Mean ± SD
Young 45M
50M
52M
56M
183 157
181 142
179 131
67M
Grey values Parafovea Periphery
186 160
Mean ± SD
Aged 75M
81M
86F
173 152
175 138
158 122
Grey values 182.25 ± 2.99 147.5 ± 13.52
180 149
171.50 ± 9.46 140.25 ± 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-yearold 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 layer and vitreal end-feet indicate sites of lipid peroxidation in Müller cells of human retina in normal aging process. The murine retina suffers from oxidative stress under different 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 increase in percentage of cells in the advanced aged group in both retinal regions (*p < 0.02).
has been found that retinal damage caused by light exposure can be reduced by various types of synthetic antioxidants, and so oxidative stress was considered to be involved in the pathogenesis of lightinduced retinal damage. Several authors (Uchida and Stadtman, 1992; Tanito et al., 2005) reported an increase in the retinal protein modification by 4-HNE accumulation. Because in our study prominent 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 mechanisms 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 physiological functions that are performed by retinal 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 physiological well-being of the retina in aging would be maintained. For example, 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 merged views (yellow, right panel, arrows). Asterisks in (A–C) denote labels 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 transporter, GLAST and/or decreased activity of Na, K, ATPase (Rauen et al., 1998; MacGregor and Matschinsky, 1986) was shown to cause 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 role by 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 situation 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) and in macrophages, as a protective response against 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 these enzymes 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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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 b
Department of Anatomy, Neurobiology Laboratory, All India Institute of Medical Sciences, New Delhi 110029, India 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 the extracellular fluid environment. Information on whether human retinal Müller cells suffer from oxidative stress with normal aging is lacking. We examined post mortem human retinas for the localization of a biomarker of lipid peroxidation (4-hydroxy 2-nonenal, 4-HNE) by immunohistochemistry. We procured human eyes from 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 age group (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 cells in the inner nuclear layer and in outer fibers located in the macular fiber layer of Henle. Colocalization with glutamine synthetase revealed that the 4-HNE positive profiles in the inner nuclear layer were Müller cells. Quantitative analysis revealed that the percentage of immunopositive cells in the inner nuclear layer as well as the grey levels of the immunoreaction products in the parafoveal and peripheral retinal regions significantly increased in the advanced age group. The findings indicate that Müller cells 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 important functions through a metabolic symbiosis between the retinal neurons and themselves (Bringmann and Reichenbach, 2001; Bringmann et al., 2006). They maintain homeostasis of the retinal 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 neurons via gliosis, which on the one hand, may provoke neuronal degeneration. Information on age-related changes in structure and neurochemistry 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). 0940-9602/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.aanat.2011.02.004
2003; Baydas et al., 2004). Lipid peroxidation, a type of free radicalmediated 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 examine 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
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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 50 52 56 67 75 78 81 83 86 91
M M M M M M M M M F F
Heart attack Haemorrhage Heart attack Myocardial infarction Heart attack Heart attack Cardiac arrest Heart attack Myocardial infarction Cardiac attack Cardio-respiratory attack
3 2 2 2 1 2 2 2 2 2 1
a b c
In years. Information obtained from case registry. In hours; M, male; F, female.
Helsinki declaration for research on human tissues. Written consent 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 0.1 M phosphate buffer (pH 7.4) for 48 h 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 3–5 mm 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: 14 m) 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, San Antonio, Texas, Dilution: 5 g/ml). Sections were quenched of endogenous peroxidase activity by treating in 0.3% hydrogen peroxide in methanol for 30 min and washed. Sections were incubated in 10% goat normal serum (diluent: 0.01 M phosphate-buffered saline containing 0.5% triton X-100) for 3 h and then in the primary antibody for 48 h 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 visualized 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 antibody was substituted with the secondary antibody. The slides were dehydrated in ethanol and coverslipped with DPX. Adjacent retinal sections were stained with hematoxylin and eosin to see the integrity of the cellular layers and identification of macular subregions (parafoveal and perifoveal), using ganglion cell layer and inner nuclear layer thickness as references. Photographs of the sections 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 product was done. For this, retinal sections from lower age group (N = 4; 45M, 50 M, 52M and 56M) and advance aged group (N = 4; 67M,
75M, 81M and 86F) were processed simultaneously using the same antibody dilutions and treatment protocol. Retinal sections were viewed at 20X magnification, the images acquired using the digital camera (Leica DFC 420 C) and transferred to a video monitor. The grey level in sections was detected using Leica Q Win software equipped with the Leica microscope. The light intensity (0.996) was kept constant throughout the imaging process. Using the standard grey detection mode, from a 0 to 255 scale, a threshold adjustment of the staining was calibrated between 0 and 150. The fixation of the upper grey value at 150 discretely masked only the stained cells and fibers with the binary color, without overlapping the unstained background tissues. For quantification of grey levels, the retinal boundary was delimited from the inner limiting membrane to the outer limiting membrane inside a fixed measuring frame. Four retinal sections, showing consistent staining were selected from each donor retina and grey levels measured at the parafoveal and peripheral 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, immunolabeled slides 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 number of immunopositive cells (appeared in 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 285 m 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 4HNE 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 procedure. The sections were first equilibrated in 0.1 M 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 incubated with the anti-rabbit IgG conjugated to fluorescein isothiocynate (FITC, dilution: 1:500; Sigma–Aldrich, CA, USA) for 4 h at room temperature. Before beginning 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 488 nm for FITC and 514 nm for Cy3. Emission band 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. Immunohistochemical localization of 4-HNE in a 56-year-old donor retina. (A) From the periphery, IR is apparent in many Müller 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 inner 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.)
(Alladi et al., 2010). All images were captured using 20× magnification 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 uniform. 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-group donors (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 layer was 4-HNE immunopositive in 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) and in 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 endfeet and macular inner nuclear layer cells) was uniformly noted in all other advanced aged retinas (donor ages: 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-yearold). Quantitative analysis of retinal sections immunolabeled with 4HNE 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) and periphery (p ≤ 0.02). This was also the situation 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 and terminating 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 specialized 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 photoreceptors 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
Mean ± SD
Young 45M
50M
52M
56M
183 157
181 142
179 131
67M
Grey values Parafovea Periphery
186 160
Mean ± SD
Aged 75M
81M
86F
173 152
175 138
158 122
Grey values 182.25 ± 2.99 147.5 ± 13.52
180 149
171.50 ± 9.46 140.25 ± 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-yearold 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 layer and vitreal end-feet indicate sites of lipid peroxidation in Müller cells of human retina in normal aging process. The murine retina suffers from oxidative stress under different 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 increase in percentage of cells in the advanced aged group in both retinal regions (*p < 0.02).
has been found that retinal damage caused by light exposure can be reduced by various types of synthetic antioxidants, and so oxidative stress was considered to be involved in the pathogenesis of lightinduced retinal damage. Several authors (Uchida and Stadtman, 1992; Tanito et al., 2005) reported an increase in the retinal protein modification by 4-HNE accumulation. Because in our study prominent 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 mechanisms 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 physiological functions that are performed by retinal 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 physiological well-being of the retina in aging would be maintained. For example, 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 merged views (yellow, right panel, arrows). Asterisks in (A–C) denote labels 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 transporter, GLAST and/or decreased activity of Na, K, ATPase (Rauen et al., 1998; MacGregor and Matschinsky, 1986) was shown to cause 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 role by 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 situation 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) and in macrophages, as a protective response against 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 these enzymes 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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