Early history

Carl Ludwig Wilhelm Bruch first isolated the “lamina vitrea” that we now know as Bruch’s membrane, and described it in his 1844 doctoral thesis,^3,4 where he also first described the tapetum found in many mammals. By light microscopy, Bruch’s membrane appeared transparent with little internal structure. Later studies by Smirnow⁵ divided this membrane into an outer elastic layer (first described by Sattler in 1877) and an inner cuticular layer, separated by a dense plexus of very fine elastic fibers.^6,7

Development of Bruch’s membrane

The bipartite character of Bruch’s membrane arises from the embryology of its tissue. When the optic cup invaginates and folds, its inner layer forms the neural retina, and its outer layer forms the RPE. The RPE lies in contact with mesenchyme. At this apposition, Bruch’s membrane forms by 6–7 weeks’ gestation. Thus, its inner layer is composed of ectodermal tissue and its outer layer is composed of mesodermal tissue. At the border of two layers, the elastic layer forms last, becoming histologically visible by 11–12 weeks.^8–10

The collagen that fills the extracellular space and the later-appearing elastin appear to be made by invading fibroblasts and the filopodia of endothelial cells lining the adjacent choriocapillaris. The two basal laminas are produced by their associated cell layers.¹¹ In addition to collagen IV subunits specific to specialized basal lamina, RPE expresses genes for structural collagen III and angiostatic collagen XVIII in a developmentally regulated manner linked to photoreceptor maturation.¹²

By week 13, fenestrations are apparent in the endothelium facing Bruch’s membrane,¹⁰ indicating that, at this stage, transport across this tissue may be functional. Choroidal endothelial cells originate from paraocular mesenchyme. Development of the choroidal vasculature, and Bruch’s as part of it, depends on differentiated RPE and its production of inductive signals, including basic fibroblast growth factor and vascular endothelial growth factor (VEGF).¹³

RPE basal lamina (RPE-BL)

This ∼0.15-µm-thick layer is a meshwork of fine fibers like other basal laminas in the body.^16,17 The RPE-BL resembles that of the choriocapillaris endothelium but does not contain collagen VI. The RPE-BL contains collagen IV α3–5,¹⁸ like that of kidney glomerulus, another organ with specialized filtration and transport functions. The RPE synthesizes specific laminins that preferentially adhere Bruch’s membrane to the RPE through interaction with integrins.¹⁹

Inner collagenous layer (ICL)

The ICL is ∼1.4 µm thick and contains 70-nm-diameter fibers of collagens I, III, and V in a multilayered criss-cross, parallel to the plane of Bruch’s membrane.¹ The collagen grid is associated with interacting molecules, particularly the negatively charged proteoglycans chondroitin sulfate and dermatan sulfate.^20,21

Elastic layer (EL)

The EL consists of stacked layers of linear elastin fibers, crisscrossing to form a 0.8-µm-thick sheet with interfibrillary spaces of ∼1 µm. This sheet extends from the edge of the optic nerve to the ciliary body pars plana.¹ In addition to elastin fibers, the EL contains collagen VI, fibronectin, and other proteins, and collagen fibers from the ICL and outer collagenous layer (OCL) can cross the EL. Some EL elastin fibers are said to cross the tissue space between the choriocapillaris and join bundles of choroidal elastic tissue.²² The EL confers biomechanical properties, vascular compliance, and antiangiogenic barrier functions. It is more discontinuous in the macula, perhaps explaining why choroidal neovascularization (CNV) is more prominent there.²³ This concept is supported by the extensive laser-induced neovascularization in mice deficient in lysyl oxidase-like 1, an enzyme required for elastin polymerization.²⁴

Outer collagenous layer

The OCL contains many of the same molecular components as the ICL, and the collagen fibrils running parallel to the choriocapillaris additionally form prominent bundles. This layer, unlike the ICL, has periodic outward extensions between individual choriocapillary lumens called intercapillary pillars, where thickness cannot be determined due to the lack of a boundary. Between pillars, OCL thickness can range from 1 to 5 µm.²⁵

Choriocapillaris basal lamina (ChC-BL)

This 0.07-µm-thick layer is discontinuous with respect to Bruch’s membrane due to the interruptions of the intercapillary pillars of the choroid. It is continuous with respect to the complex network of spaces defined by the choriocapillary lumens because the basal lamina envelops the complete circumference of the endothelium. A remarkable structural feature of the adjacent choriocapillary endothelium is fenestrations that are permeable to macromolecules (Fig. 20.2).²⁶ This basal lamina may inhibit endothelial cell migration into Bruch’s membrane, as do basal laminas associated with retinal capillaries.²⁷

Fig. 20.2 Surface of the endothelium of the choriocapillaris showing fenestrations with a bicycle-spoke pattern (yellow arrow) and presumed artifactual openings arising from tissue preparation (cyan arrow); quick-freeze/deep-etch, 64-year-old eye, macula. Bar = 100 nm.

(Reproduced with permission from Johnson M, Huang J-D, Presley JB, et al. Comparison of morphology of human macular and peripheral Bruch’s membrane in older eyes. Curr Eye Res 2007;32:791–9.)

Lipid accumulation: Bruch’s membrane lipoproteins

Early electron microscopists described aged Bruch’s membrane as being filled with debris, including amorphous electron-dense material, membrane fragments, vesicles, and calcification.^1,25 Debris deposition in ICL and OCL begins in the second decade in the macula and is delayed in equatorial regions, a regional lag also reported for individual components.³⁰ Identifying this material has been a fruitful approach to understanding antecedents of disease.

Most prominent among the changes in Bruch’s membrane is a profound accumulation of lipids. Clinical observations on fluid-filled RPE detachments in older adults led to Bird and Marshall’s hypothesis that a lipophilic barrier in Bruch’s blocked a normal, outwardly directed fluid efflux from the RPE³¹ (as opposed to leakage from CNV). This hypothesis motivated a seminal histochemical study by Pauleikhoff et al.³² that demonstrated oil red O-binding material (esterified cholesterol (EC), triglyceride (TG), fatty acid) localized exclusively to Bruch’s membrane, unlike other stains. This lipid was absent <30 years, variably present at 31–60 years, and abundant at ≥61 years.^33,34 A specific fluorescent marker, filipin, which binds the 3β-hydroxy group of sterols to reveal unesterified (free) cholesterol (UC) or EC depending on tissue pretreatment,³⁵ indicated that EC is a prominent component of the oil red O-binding deposition.³⁵ Macular EC rose linearly from near zero at age 22 years to reach high and variable levels in aged donors. EC was detectable in periphery at ∼1/7 macular levels and increased significantly with age. Hot-stage polarizing microscopy³⁴ similarly demonstrated prominent age-related increases in EC in Bruch’s membrane, manifest as liquid crystals (“Maltese crosses”) when examined through a polarizing filter. Few birefringent crystals signifying the neutral lipid TG were found.

Histochemical, ultrastructural, biochemical, gene expression, and cell biological evidence now indicate that the EC-rich material accumulating with age in Bruch’s membrane is a lipoprotein-containing apolipoprotein B, assembled by the RPE.³⁶ This process, ongoing throughout life yet first revealed by aging, has implications for the formation of AMD-specific lesions, intraocular transport, RPE physiology, nutrition of outer retina, and maintenance of photoreceptor health. In 1926, Verhoeff and Sisson speculated that lipid deposition might precede Bruch’s membrane basophilia and fragmentation, common in older eyes due to “lime salts [calcification] in the elastic layer.”³⁷

Ultrastructural studies described in Bruch’s membrane of older eyes³⁶ numerous small (<100 nm), round, electron-lucent vesicular profiles, implying aqueous interiors. Lipid-preserving preparation techniques together with extraction studies show that these so-called vesicles are actually solid, lipid-containing particles, now considered lipoproteins (Fig. 20.3B). These methods include postfixation in osmium paraphenylenediamine (OTAP)³⁵ and, most strikingly, quick-freeze/deep-etch (QFDE), a freeze fracture method with an etching step to remove frozen water.^38–40 Particles vary in size from 60 to 100 nm but could be as large as 300 nm, occasionally appearing to coalesce (Fig. 20.3).

Fig. 20.3 Lipid wall, a layer of lipoproteins on the inner surface of Bruch’s membrane. (A) Lipoproteins (spherical vesicles of uniform diameter) accumulate 3–4 deep between the retinal pigment epithelium (RPE), basal lamina (black arrowheads), and Bruch’s membrane (BrM), inner collagenous layer (white arrowheads). Thin-section transmission electron micrograph following osmium postfixation. L, lipofuscin. Sectioning plane is vertical; bar = 1 µm. (B) Quick-freeze/deep-etch shows tightly packed Bruch’s membrane lipoproteins in the lipid wall, and that lipoproteins have classic core and surface morphology. Fracture plane is oblique; bar = 200 nm.

(Reproduced with permission from Huang J-D, Presley JB, Chimento MF, et al. Age-related changes in human macular Bruch’s membrane as seen by quick-freeze/deep-etch. Exp Eye Res 2007;85:202–18.)

Lipoprotein particles are first seen among fibrils of the elastic layer in early adulthood, extending inward ultimately to fill most of the open space of the ICL by the seventh decade of life.⁴⁰ Most fatefully, a new layer, the lipid wall,³⁸ then forms with solid particles stacked 3–4 deep occupying nearly 100% of a space between RPE basal lamina and OCL of many older eyes. The lipid wall displaces ICL collagen fibrils that anchor the RPE basal lamina (Fig. 20.3). It is considered a precursor to basal linear deposits, a specific lesion of AMD (see below).

Lipoprotein composition can provide clues to sources of its components.⁴¹ When isolated (Fig. 20.4A), Bruch’s membrane lipoproteins are found to be EC-enriched (EC/total cholesterol = 0.56; EC/TG = 4–11; Fig. 20.4B). For comparison, hepatic very-low-density lipoprotein (VLDL), of similar diameter, is TG-rich. An early report of TG-enriched Bruch’s membrane neutral lipid⁴² was not replicated. Abundant EC points to the only mechanism by which neutral lipids are released directly from cells, an apoB-containing lipoprotein, like hepatic VLDL or intestinal chylomicrons. Significantly, RPE expresses the apoB gene and protein, along with microsomal triglyceride transfer protein (MTP), required for apoB lipidation and secretion. Lack of functional MTP is the basis of abetalipoproteinemia, a rare inherited disorder that includes a pigmentary retinopathy.^43,44 The combination of apoB and MTP within native RPE marks these cells as constitutive lipoprotein secretors.⁴⁵ Secretion of full-length apoB has been demonstrated in rat-derived and human-derived RPE cell lines.^46,47 Consistent with an RPE origin, particles first appear in the elastic layer of Bruch’s membrane and fill in towards the RPE.³⁹

Fig. 20.4 Bruch’s membrane lipoprotein composition. (A) Lipoprotein particles isolated from Bruch’s membrane are large and spherical; negative stain.¹⁵³ (Source: Li C-M, Chung BH, Presley JB, et al. Lipoprotein-like particles and cholesteryl esters in human Bruch’s membrane: initial characterization. Invest Ophthalmol Vis Sci 2005;46:2576–86). Bar = 50 nm. (B) Bruch’s membrane lipoprotein composition inferred from direct assay,^50,153 (Sources: Wang L, Li C-M, Rudolf M, et al. Lipoprotein particles of intra-ocular origin in human Bruch membrane: an unusual lipid profile. Invest Ophthalmol Vis Sci 2009;50:870–7) (Li C-M, Chung BH, Presley JB, et al. Lipoprotein-like particles and cholesteryl esters in human Bruch’s membrane: initial characterization. Invest Ophthalmol Vis Sci 2005;46:2576–86), druse composition, and retinal pigment epithelium gene expression.^139,154 (Sources: Malek G, Li C-M, Guidry C, et al. Apolipoprotein B in cholesterol-containing drusen and basal deposits in eyes with age-related maculopathy. Am J Pathoi 2003;162:413–25) and (TG, Li C-M, Clark ME, Chimento MF, et al. Apolipoprotein localization in isolated drusen and retinal apolipoprotein gene expression. Invest Ophthalmol Vis Sci 2006;47:3119–28) TG, triglyceride; EC, esterified cholesterol; UC, unesterified cholesterol; PL, phospholipid; Apo, apolipoproteins. The question mark signifies that not all apolipoproteins are known.

Indirect evidence that Bruch’s membrane lipoproteins are of intraocular origin also emerges from the epidemiologic literature. If the EC deposition in Bruch’s membrane and AMD-associated lesions were a manifestation of systemic perifibrous lipid and atherosclerosis, then a strong positive correlation between disease status and plasma lipoprotein levels, like that documented for coronary artery disease,⁴⁸ might be expected but has not emerged.⁴⁹

Identifying the upstream sources of Bruch’s membrane lipoprotein constituents is essential for understanding the biological purpose of this pathway and the prospects for eventual clinical exploitation. Studies using isolated lipoproteins from Bruch’s membrane⁵⁰ and Bruch’s membrane choroid EC⁵¹ report a high mole percentage of linoleate (>40%) and low docosahexaenoate (<1%) for all lipid classes.⁵² This composition strongly points away from photoreceptor outer segments (35% docosahexaenoate in membrane phospholipids) as an upstream source, as long postulated,^53,54 and towards plasma lipoproteins (45–55% linoleate in all lipid classes). These data have been interpreted to signify that plasma lipoproteins are major contributors upstream to an apoB lipoprotein of RPE origin. In contrast, the sources of UC in Bruch’s lipoproteins are not yet known and could be outer segments, plasma lipoproteins, endogenous synthesis, or a combination.

Lipoproteins may thus be assembled from several sources, including outer segments, remnant components from the photoreceptor nutrient supply system, and endogenous synthesis. According to this model,⁵² plasma lipoproteins serve as vehicles for delivery of lipophilic nutrients (carotenoids,⁵⁵ vitamin E, and cholesterol⁵⁶) to photoreceptors by RPE, which has functional receptors for low-density lipoprotein (LDL) and high-density lipoprotein.^57,58 Nutrients are stripped from these lipoproteins by the RPE for delivery to the photoreceptors, and the remnants are repackaged for secretion into Bruch’s membrane as part of apoB-containing lipoproteins, where they begin to accumulate during age and become toxically modified to instigate inflammation in AMD.

Other aging changes

Bruch’s membrane thickens throughout adulthood (20–100 years) two- to threefold under the macula and becoming more variable between individuals at older ages.^25,59,60 Equatorial Bruch’s membrane changes little while Bruch’s membrane near the ora serrata increases twofold during this time.²⁵ In the macula, the OCL thickens more prominently than the ICL.⁶¹ A large ultrastructural study of 121 human donor eyes demonstrated that the macular EL is 3–6 times thinner than peripheral EL²³ at all ages.

Unbalanced regulation of extracellular matrix molecules and their modulator matrix are thought to result in Bruch’s membrane thickening. Increased histochemical reactivity for glycoconjugates, glycosaminoglycans (GAGs), collagen, and elastin is seen in the macula relative to equator and near the ora serrata.²⁵ Collagen solubility declines with age.⁶² Matrix metalloproteinases MMP-2 and MMP-3 increase with age, as does a potent tissue inhibitor of metalloproteinases, TIMP-3. TIMP-3 immunoreactivity reaches adult levels at 30 years of age near vasculature in lung, kidney, and in Bruch’s membrane, signifying the end of developmental organogenesis.⁶³ The reduction or absence of TIMP-3 is proangiogenic, as this protein not only regulates metalloproteinases during the normal turnover of Bruch’s membrane matrix components, but it also binds to VEGF.^64,65

The EL thickens with age but decreases relative to overall thickening of Bruch’s membrane.²³ Thus elastin referenced to other Bruch’s constituents, as detected by Raman spectroscopy, decreases with age.⁶⁶ Similar arguments can be made for collagen III and IV. A prominent age change,⁶⁷ noted early,³⁷ is calcification and ensuing brittleness. This process involves fine deposition of electron-dense particulate matter,¹⁴ confirmed as calcium phosphate⁶⁸ on individual elastin fibrils.

Long-lived proteins like collagens are modified in vivo by nonenzymatic Maillard and free radical reactions to yield advanced glycation end products (AGEs) and the formation of lipid-derived reactive carbonyl species like 4-hydroxyhexenal and linoleate hydroperoxide, collectively called age-related lipoperoxidation end products (ALEs). Accumulation of AGEs and ALEs, characteristic of diabetes and atherosclerosis, also occurs in aging Bruch’s membrane (Table 20.1). Finally, other components more prominent in aged eyes include complement components C3d, C5b-9, and pentraxin-3, a homolog of the acute-phase respondent C-reactive protein. Thus, at the molecular level, aging Bruch’s membrane contains evidence of many biological activities, including remodeling, oxidative damage, and inflammation, in addition to lipoprotein accumulation.

Structural role of Bruch’s membrane

Bruch’s membrane encircles more than half the eye and stretches with the corneoscleral envelope as intraocular pressure (IOP) increases. It therefore withstands this stretch and returns to its original shape when IOP decreases. This tissue also stretches to accommodate changes in choroidal blood volume. Finally, the choroid (and Bruch’s membrane with it) may act as a spring that pulls the lens during accommodation.^73,74 For these reasons, then, Bruch’s membrane requires elasticity. Marshall and Hussain’s group estimated the modulus of elasticity in Bruch’s membrane choroid preparations to be 7–19 MPa.⁷⁵ These values are similar to those of sclera (although sclera is much thicker and thus can support more load), consistent with the notion that Bruch’s membrane contributes to load bearing. After early adulthood, the modulus of elasticity of human Bruch’s membrane–choroid complex increases (P < 0.001) at a rate of ∼1% per year. Bruch’s membrane stiffness in AMD eyes does not differ from age-matched normals.⁷⁶

Transport role of Bruch’s membrane

The choroid services the metabolic needs of the outer retina, facilitated in part by fenestrated endothelium. Oxygen, electrolytes, nutrients, and cytokines destined for the RPE and photoreceptors pass from the choriocapillaris and through Bruch’s membrane, and waste products travel back in the opposite direction for elimination. Vitamins, signaling molecules, and other factors needed for photoreceptor function are carried to the RPE by lipoprotein particles passing through Bruch’s membrane, as do the RPE-produced lipoproteins that are eliminated in the opposite direction. The RPE pumps water from the subretinal space to counter the swelling of the interphotoreceptor matrix GAGs. This fluid also flows across Bruch’s membrane to reach the circulation. Thus, many transport processes involve Bruch’s membrane, as reviewed here.

Age-related changes in hydraulic conductivity and disease

Fisher was the first to measure L_p of human Bruch’s membrane,⁹¹ finding that L_p decreased significantly with age. However, his values for L_p of Bruch’s membrane and other tissues are much lower than those found by later investigators.^85,92,93 Marshall and Hussain’s group carefully revisited these measurements using Bruch’s membrane/choroid with RPE removed, a preparation that was simpler to create. They showed using laser ablation that the flow resistance of these preparations was entirely due to Bruch’s membrane.⁸⁹ They also found that flow rate increased linearly with driving pressure, indicating that L_p of Bruch’s membrane is relatively insensitive to pressure up to 25 mmHg.

They reported that L_p of macular Bruch’s membrane exhibited a dramatic, exponential decline throughout life (Fig. 20.5), dropping from 130 × 10⁻¹⁰ m/s/Pa in young children to 0.52 × 10⁻¹⁰ m/s/Pa in old age. L_p of macular Bruch’s membrane dropped more rapidly with age than did that of the periphery, consistent with an accelerated process occurring in the macula.^1,84,94,95 Note that the lowest value measured for L_p of Bruch’s membrane in normal eyes is similar to the calculated minimum value of L_p that allows complete fluid resorption (0.4 × 10⁻¹⁰ m/s/Pa; see above). Marshall and Hussain’s group reached similar conclusions regarding this process.⁹⁴

Fig. 20.5 Hydraulic conductivity (L_p) of Bruch’s membrane as a function of age. Dotted lines are exponential fits to data from macular and peripheral regions, respectively. Note that all of the data from eyes with age-related macular degeneration (AMD) (taken only in peripheral region) have lower values of L_p than the best fit to data taken from peripheral Bruch’s membrane of nondiseased eyes.

(Reproduced with permission from Hussain AA, Starita C, Marshall J. Transport characteristics of ageing human Bruch’s membrane: implications for age-related macular degeneration (AMD). In: Ioseliani OR, editor. Focus on macular degeneration research. Hauppauge, New York: Nova Biomedical Books; 2004.)

Determining L_p of Bruch’s membrane in isolated macular samples of AMD eyes is difficult due to scar formation and other changes.⁹⁴ However, Marshall and Hussein’s group showed that, in the periphery, L_p of Bruch’s membrane is decreased in AMD eyes as compared to age-matched normal eyes (Fig. 20.5).⁹⁴ Assuming that similar processes occur in macular Bruch’s membrane due to the profound lipid accumulation in this region, then in diseased eyes, the RPE must generate higher pressures at its basal surface to drive fluid into the choriocapillaris, with further pathological consequences.³¹ Above an unknown threshold level, higher pressure will cause the RPE-BL to separate from the ICL, leading to RPE detachment and fluid accumulation, as seen in 12–20% of AMD patients.⁹⁴

What causes the dramatic age-related decrease in L_p of Bruch’s membrane? It is natural to suspect the age-related lipid accumulation. In fact, McCarty et al.⁹⁶ showed that lipid particles trapped in an extracellular matrix can generate very significant flow resistance, more than would be expected based simply on their size and number. However, Marshall and Hussain’s group observed that most of the marked change in L_p occurred before age 40 (Fig. 20.6A) while the increase in Bruch’s membrane lipid content occurred largely after this age. They thus concluded that other age-related changes must be responsible for changes in L_p.^1,84

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