Age-related changes in human macular Bruch's membrane as seen by quick-freeze/deep-etch

https://doi.org/10.1016/j.exer.2007.03.011Get rights and content

Abstract

Lipid-containing inclusions have been observed in human Bruch's membrane (BrM) and are postulated to be associated with age-related maculopathy (ARM), a major cause of legal blindness in developed countries. The dehydration associated with specimen preparation for thin-section transmission electron microscopy causes loss of these inclusions. Better preservation of the ultrastructure of the inclusions and tissue is achieved by using a quick-freeze/deep-etch preparation. We use this technique to examine normal human macular BrM in order to characterize the deposition of the lipid-rich inclusions and their age-related accumulation within different layers of the tissue. We find that various inclusions mentioned in other studies can be formed by combinations of three basic structures: lipoprotein-like particles (LLPs), small granules (SGs) and membrane-like structures. These inclusions are associated with collagen and elastic fibrils by fine filaments. In younger eyes, these inclusions are found mostly in the elastic (EL) and outer collageneous layer (OCL) and occupy a small fraction of the interfibrillar spacing. As age increases, LLPs and SGs gradually fill the interfibrillar spacing of the EL and inner collageneous layer (ICL) of the tissue, and later form a new sublayer, the lipid wall, within the boundary region between the basal lamina of retinal pigment epithelium (RPE) and ICL. Because the formation of the lipid wall only occurs after these inclusions fill the ICL, and it seems unlikely that the LLPs can pass through the packed layer, this result suggests a possible RPE origin of the LLPs that make up the lipid wall.

Introduction

Bruch's membrane (BrM) is a thin layer of connective tissue located between the retinal pigment epithelium (RPE) and the choriocapillaris. Due to its specific location and properties, this tissue is thought to be a vital limiting layer for metabolic transport between the RPE cells and the choriocapillaris (Marshall et al., 1998). The RPE in turn plays an important role in the maintenance of photoreceptor metabolism (Kornzweig, 1977, Anderson et al., 1978, Matsumoto et al., 1987, Korte et al., 1989, Giusto et al., 1997). Compromise of the nutrient and waste transport through this region would eventually affect the photoreceptor's normal function and thus has been suggested as a possible cause of age-related maculopathy (ARM) (Sarks, 1976, Grindle and Marshall, 1978, Bird and Marshall, 1986, Chen et al., 1992, Starita et al., 1996), a leading cause of blindness in developed countries (Foster and Resnikoff, 2005). Age-related changes and accumulation of apparent “debris” in BrM are thought to compromise this transport (Killingsworth, 1987, Chen et al., 1992, Moore et al., 1995, Starita et al., 1996).

With advanced age, both the thickness and complexity of BrM increase primarily due to extracellular matrix remodeling and accumulation of inclusions in this region (Ramrattan et al., 1994). These inclusions include advanced glycation endproducts (AGEs), coated vesicle-like bodies, spiked coated vesicle-like bodies, electron-lucent droplets, membranous debris, coated membrane-bound bodies (CMBBs), long spacing collagen and lipoprotein-like particles (LLPs) (Hogan and Alvarado, 1967, Grindle and Marshall, 1978, Feeney-Burns and Ellersieck, 1985, Killingsworth, 1987, van der Schaft et al., 1991, Handa et al., 1999, Ruberti et al., 2003, Li et al., 2005a, Yamada et al., 2006). In the later stage of human life, these inclusions presumably fuse or aggregate to form drusen and the basal deposits that are associated with age-related macular degeneration (Sarks, 1976, Pauleikhoff et al., 1990, van der Schaft et al., 1992, Green and Enger, 1993, Curcio and Millican, 1999). The accumulation of these inclusions may impede physiologic transport through this region (Hogan and Alvarado, 1967, Killingsworth, 1987, Curcio and Millican, 1999). The hydraulic conductivity, the permeability to macromolecular transport, and the elasticity of BrM are all found to decrease with advancing age (Moore et al., 1995, Starita et al., 1996, Hussain et al., 2002, Ugarte et al., 2006), and these events may disturb the metabolism of the RPE and photoreceptor cells.

In order to characterize these ultrastructural changes in BrM, studies using conventional thin-sectioning transmission electron microscopy (TEM) and histochemical methods have identified various inclusions and the age-related changes of the tissue (Nakaizumi et al., 1964, Feeney-Burns and Ellersieck, 1985, Killingsworth, 1987, Curcio et al., 2001). However, preparative steps for these procedures, such as the dehydration necessary for conventional TEM, result in ultrastructural loss and removal of lipids from the tissue (Guyton and Klemp, 1988, Overby et al., 2001, Ruberti et al., 2003). A better characterization of the accumulation of the lipid-rich inclusions, along with ultrastructural features and the consequent age-related changes of BrM, can provide valuable information related to the progression of accumulation of these inclusions and their influence on transport through this region.

Ruberti et al. (2003) used the quick-freeze/deep-etch (QFDE) technique to preserve lipids and to visualize the ultrastructural features within BrM. Their images of a small number of older eyes not only demonstrated lipid-rich particles accumulating in BrM, but also revealed the “lipid wall” that is formed by these particles in the inner ICL in some older eyes. In this study, we use this technique to examine normal human BrM in a larger series of eyes in order to characterize the progression of inclusion deposition and age-related ultrastructural changes of the extracellular compartment.

Section snippets

Eye tissues

Human eyes were obtained from Alabama Eye Bank within 4 h post-mortem. Eyes with drusen larger than 63 μm or any other grossly visible chorioretinal pathologic disturbance in the macula were excluded. Also, eyes from donors with diabetes or receiving artificial respiration longer than 5 days were excluded. Sixteen normal eyes were examined in this study, including two previously described eyes (Ruberti et al., 2003) (Table 1).

The posterior segment of the normal eyes was preserved by immersion in

Results

The five layers of BrM lying between the RPE and the fenestrated choriocapillaris, when examined using QFDE, could be distinguished by their morphological features (Fig. 1). The layers are not always continuous across the images. On both aspects of the tissue resided two thin layers of tight meshwork, the basal lamina of the RPE (BL-RPE) (Fig. 2A) and the basal lamina of the choriocapillaris (BL-CC) (Fig. 2D). The smooth and wide band-like material seen in the center of BrM were elastic fibrils

Discussion

In this study, by utilizing both the QFDE and OTAP techniques for sample preparation, we were able to demonstrate ultrastructural features of BrM not easily appreciated using conventional techniques. The limited usage of chemical treatments and ultra-fast slam freezing of the sample tissue of QFDE should have minimized artifacts in our preparations. As diseased eyes were excluded from this study, the results represent a plausible progression of age-related change within BrM.

Our findings are in

Conclusions

Grindle and Marshall (1978) proposed that a hydrophobic barrier in BrM could compromise the transport between RPE cells and choroids and this could lead to RPE detachments. In the current study, the QFDE and OTAP techniques applied to the examination of the age-related changes in BrM provided a detailed characterization of age-related changes in the ultrastructure of the components of the tissue. Our findings not only confirmed several types of inclusions demonstrated by other morphological

Acknowledgements

We would like to thank Dr Jeffery W. Ruberti for technical assistance. This study is supported by NIH EY014662, NIH EY06109, and The American Health Assistance Foundation.

References (65)

  • G. Malek et al.

    Apolipoprotein B in cholesterol-containing drusen and basal deposits of human eyes with age-related maculopathy

    Am. J. Pathol.

    (2003)
  • K.M. Meek et al.

    The staining pattern of collagen fibrils. Improved correlation with sequence data

    J. Biol. Chem.

    (1979)
  • D. Pauleikhoff et al.

    Drusen as risk factors in age-related macular disease

    Am. J. Ophthalmol.

    (1990)
  • S.M. Sabesin et al.

    Electron microscopic studies of the assembly, intracellular transport, and secretion of chylomicrons by rat intestine

    J. Lipid Res.

    (1977)
  • C. Starita et al.

    Hydrodynamics of aging Bruch's membrane: implications for macular disease

    Exp. Eye Res.

    (1996)
  • T.L. van der Schaft et al.

    Histologic features of the early stages of age-related macular degeneration. A statistical analysis

    Ophthalmology

    (1992)
  • P. Vijayagopal et al.

    Varied low density lipoprotein binding property of proteoglycans synthesized by vascular smooth muscle cells cultured on extracellular matrix

    Atherosclerosis

    (2005)
  • Y. Yamada et al.

    The expression of advanced glycation endproduct receptors in rpe cells associated with basal deposits in human maculas

    Exp. Eye Res.

    (2006)
  • D.H. Anderson et al.

    Mammalian cones: disc shedding, phagocytosis, and renewal

    Invest. Ophthalmol. Vis. Sci.

    (1978)
  • A.C. Bird et al.

    Retinal pigment epithelial detachments in the elderly

    Trans. Ophthalmol. Soc. UK

    (1986)
  • Y.V. Bobryshev et al.

    Accumulation of co-localised unesterified cholesterol and neutral lipids within vacuolised elastin fibres in athero-prone areas of the human aorta

    Atherosclerosis

    (1999)
  • T.M. Bocan et al.

    Human aortic fibrolipid lesions. Immunochemical localization of apolipoprotein B and apolipoprotein A

    Arteriosclerosis

    (1988)
  • J.C. Chen et al.

    Functional loss in age-related Bruch's membrane change with choroidal perfusion defect

    Invest. Ophthalmol. Vis. Sci.

    (1992)
  • C.A. Curcio et al.

    Basal linear deposit and large drusen are specific for early age-related maculopathy

    Arch. Ophthalmol.

    (1999)
  • C.A. Curcio et al.

    Accumulation of cholesterol with age in human Bruch's membrane

    Invest. Ophthalmol. Vis. Sci.

    (2001)
  • A. Foster et al.

    The impact of Vision 2020 on global blindness

    Eye

    (2005)
  • N.M. Giusto et al.

    Lipid metabolism in photoreceptor membranes: regulation and mechanisms

    Neurochem. Res.

    (1997)
  • N. Gordiyenko et al.

    RPE cells internalize low-density lipoprotein (LDL) and oxidized LDL (oxLDL) in large quantities in vitro and in vivo

    Invest. Ophthalmol. Vis. Sci.

    (2004)
  • C.F. Grindle et al.

    Ageing changes in Bruch's membrane and their functional implications

    Trans. Ophthalmol. Soc. UK

    (1978)
  • J.R. Guyton et al.

    Ultrastructural discrimination of lipid droplets and vesicles in atherosclerosis: value of osmium-thiocarbohydrazide-osmium and tannic acid-paraphenylenediamine techniques

    J. Histochem. Cytochem.

    (1988)
  • J.R. Guyton et al.

    Quantitative ultrastructural analysis of perifibrous lipid and its association with elastin in nonatherosclerotic human aorta

    Arteriosclerosis

    (1985)
  • G.S. Hageman et al.

    Molecular composition of drusen as related to substructural phenotype

    Mol. Vis.

    (1999)
  • Cited by (0)

    View full text