Ординатура / Офтальмология / Английские материалы / Age-Related Changes of the Human Eye_Cavallotti, Cerulli_2008

Fig. 14.1 b The ultrahigh resolution OCT (UHR-OCT) image, according to Drexler W et al., shows that the ERM is attached to the foveal center and that no signs of imminent hole formation are present. Distortion of the retinal architecture is visible, with intact photoreceptor layer

Fig. 14.1 c The three-dimensional UHR-OCT (3D UHR-OCT) system, according to Glittenberg C. et al., allows a comprehensive analysis of focal and diffuse diseases and provides 3-D images of topographic dynamics from the retinal surface down to the level of photoreceptor segments

Treatment options and prognosis

The principal indication for ERM surgery is a significant disturbance in vision resulting from a decreased visual acuity (VA) with or without metamorphopsia. Usually, patients have a baseline VA of less than 0.6. Since the 1970s, a standard three-port pars plana vitrectomy has been used for the removal of ERMs in patients with symptomatic visual disturbances.13 Vitreoretinal surgery has been found to be

effective in removing ERMs from the macula, improving VA and decreasing metamorphopsia. Favorable visual outcome can be achieved postoperatively in the majority of cases. A significant improvement in VA after surgery has been reported in more than 80 percent of the cases, with more than 90 percent having a subjective improvement in reduction of distortion.14 The surgical complications described include intraoperative detection of retinal breaks (5%), a small amount of intraretinal bleeding after removal of the ERM and postoperative progressive nuclear sclerosis (12%-68%), retinal detachment (5%), macular edema, retinal pigment epitheliopathy, and recurrence of ERM (10%).15

Histological evaluations of tissue removed during vitrectomy have shown that the ILM, as a potential scaffold for cellular proliferation, seems to be associated with recurrence of ERMs. Therefore, the application of dyes such as indocyanine green (ICG), trypan blue (TB) and triamcinolone acetonide (TA) have been introduced to assist the removal of ERMs, the ILM, or both. The use of these dyes improves the visualization of the ILM and ERMs, and consecutively allows a more controllable, easier, and less traumatic membrane peeling. Investigations of functional results after ERM surgery suggest a higher percentage of improved or stable VA and a lower recurrence rate in eyes in which an additional ILM peeling has been performed. Staining of the ERM and/or the ILM has proven to reduce the recurrence rate, but no significant difference was found for VA, reduction of macular edema, postoperative Amsler grid test, or subjective improvement.16 While the use of TB and TA during vitreoretinal surgery seems to be safe, and no adverse effects have been described, the use of ICG to assist the ILM peeling is controversial, and the potential retinal toxicity of ICG—par- ticularly the presence of postoperative visual field defects—is currently under discussion.17,18,19,20 For refining the intraoperative conditions for the surgeon and improving the patients’ outcome, new dyes for intraocular surgery have been developed and investigated in animal studies. Brophenol blue (BPB) or lightgreen SF yellowish (LGSF) produced no significantly detectable toxic effects on the retina in vivo, but the safety and benefits of these novel dyes must be established in preclinical studies.21

Most patients with symptomatic ERM formation are older than 50 years and present different forms of coexisting lens opacities. As vitrectomy generally increases cataract formation, the vitreoretinal procedure is often combined with phacoemulsification and intraocular lens (IOL) implantation. Several studies have reported that a combined vitreoretinal procedure is a safe and effective way to manage cases with vitreoretinal diseases and cataracts, with the functional outcome comparable to those of sequential surgery.22 New types of blue-light filter IOLs have been designed for macular protection, and the implantation of these yellow-colored IOLs has become increasingly common in cataract surgery. Initially, there were concerns about adverse effects on the surgeon’s ability to perform specific vitreoretinal procedures when using these yellowtinted lenses in combined surgery. Nevertheless, a randomized, controlled trial indicates that there is no significant influence on the intraoperative conditions for the surgeon or on the patients’ outcome, and suggests that the routine use of the yellow-tinted IOL in vitrectomy combined with cataract surgery can be recommended.23

For improvement of the surgical technique, investigations covering the minimal invasive methods of vitreoretinal surgery have been conducted. Sutureless 25gauge and 23-gauge vitrectomy systems have been introduced and compared to standard 20-gauge systems for pars plana vitrectomy. According to recent reports, the 25-gauge system has proven to be safe and efficient for ERM surgery. The duration of surgery was comparable to the 20-gauge system, because the shorter time for wound opening and closure is equalized by longer vitrectomy duration. Advantages of the 25-gauge vitrectomy include the minimized surgery-induced trauma, and the reduction of postoperative inflammation and of patients’ discomfort. The 25-gauge vitrectomy seems to allow earlier postoperative visual rehabilitation than conventional 20-gauge vitrectomy for patients presenting ERMs, and it may be preferable to 20-gauge vitrectomy in these cases.24,25 The 23-gauge vitrectomy system is still under investigation.

Modifications of diagnosis and treatment of ERMs have resulted in a steady improvement of the patients’ outcome. Most cases show very satisfying functional results with a mean improvement of VA of two or more lines in approximately 72 percent of the cases. A less favorable outcome is found in cases with underlying or accompanying diagnoses, like trauma, retinal vascular disorders, or retinal detachment. The clinical implication provided by the UHR-OCT and three-dimensional UHR-OCT has yet to be established. New techniques, like sutureless vitrectomy or novel dyes, may further improve the patients’ outcome.

References

1.Fraser-Bell S, Guzowski M, Rochtchina E, et al. (2003) Five-year cumulative incidence and progression of epiretinal membranes. The Blue Mountains Eye Study. Ophthalmology 110:34-40

2.Hirokawa H, Jalkh AE, Takahashi M, et al. (1986) Role of the vitreous in idiopathic preretinal macular fibrosis. Am J Ophthalmol 101:166-169

3.Appiah AP, Hirose T (1989) Secondary causes of premacular fibrosis. Ophthalmology 96:389-392

4.Cheng L, Freeman WR, Ozerdem U, et al. (2000) Prevalence, correlates, and natural history of epiretinal membranes surrounding idiopathic macular holes. Ophthalmology 107:853-859

5.Gandorfer A, Rohleder M, Kampik A (2002) Epiretinal pathology of vitreomacular traction syndrome. Br J of Ophthalmol 86:902-909

6.Smiddy WE, Maguire AM, Green WR, et al. (1989) Idiopathic epiretinal membranes. Ultrastructural characteristics and clinicopathologic correlation. Ophthalmology 96:811-820

7.Gass JDM. (1987) Stereoscopic Atlas of Macular Diseases. Mosby, St Louis, 671-726

8.Klein R, Klein BEK, Wang Q, et al. (1994) The epidemiology of epiretinal membranes. Trans Am Ophthalmol Soc 92:403-430

9.Wise GN (1975) Clinical features of idiopathic preretinal macular fibrosis. Am J Ophthalmol 79:349-357

10.Massin P, Allouch C, Haouchine B, et al. (2000) Optical coherence tomography of idiopathic macular epiretinal membranes before and after surgery. Am J Ophthalmol 130:732-739

11.Drexler W, Sattmann H, Hermann B (2003) Enhanced visualization of macular pathology using ultrahigh resolution optical coherence tomography. Arch Ophthalmol 121:695-706

12.Glittenberg C, Hermann B, Povazay B, et al. (2006) Creating a steroegraphic three dimensional ultra high resolution optical coherence tomography display system using high-end raytracing software algorithms. Invest Ophthalmol Vis Sci. ARVO Abstract 4054

13.Marchemer R (1974) A new concept for vitreous surgery.7. Two instrument techniques in pars plana vitrectomy. Arch Ophthalmol 92:407-412

14.Wong JG, Sachdev N, Beaumont PE (2005) Visual outcomes following vitrectomy and peeling of epiretinal membrane. Clin Experiment Ophthalmol 33:373-378

15.Pournaras CJ, Donati G, Brazitikos PD, et al. (2000) Macular epiretinal membranes. Semin Ophthalmol 15:100-107

16.Hillenkamp J, Saikia P, Gora F, et al. (2005) Macular function and morphology after peeling of idiopathic epiretinal membrane with and without the assistance of indoyanine green. Br J Ophthalmol 89:437-443

17.Haritoglou C, Kampik A (2006) Staining techniques in macular surgery. Ophthalmologe 103:927-934

18.Hillenkamp J, Saika P, Herrmann WA, et al. (2007) Surgical removal of epiretinal membranes with or without the assistance of indocyanine green: a randomised controlled clinical trial. Graefes Arch Clin Exp Ophthamol 245:973–979

19.Haritoglou C, Gandorfer A, Schaumberger M, et al. (2004) Trypan blue in macular pucker surgery: an evaluation of histology and functional outcome. Retina 24:582-590

20.Tognetto D, Zenoni S, Sanguinetti G, et al. (2005) Staining of the internal limiting membrane with intravitreal triamcinolone acetonide. Retina 25:462-467

21.Schuettauf F, Haritoglou C, May CA, et al. (2006) Administration of novel dyes for intraocular surgery: an in vivo toxicity animal study. Invest Ophthalmol Vis Sci; 47:3573-3578

22.Ling R, Simcock P, McCoombes J, Shaw S (2003) Presbyopic phacovitrectomy. Br J Ophthalmol 87(11):1333-1333

23.Falkner CI, Binder S (2006) UV-filter IOL versus blue light-filter IOL in combined cataract surgery with vitrectomy: a prospective randomized clinical trial. Invest Ophthalmol Vis Sci. ARVO Abstract 1484

24.Kellner L, Wimopissinger B, Stolba U, et al. (2007) 25-gauge versus 20-gauge system for pars plana vitrectomy: a prospective randomized clinical trial. Br J Ophthalmol 91:945–948

25.Kadonosono K, Yamakava T, Uchio E, et al. (2006) Comparison of visual function after epiretinal removal by 20-gauge and 25-gauge vitrectomy. Am J Ophthalmol 142:513-515

Introduction

Age-related macular degeneration (AMD) is a progressive neurodegenerative disease of the central retina. and represents the most common cause of legal blindness in industrialized countries.1 Current pathophysiologic concepts suggest that this multifactorial disease affects primarily the RPE and the thin connective tissue layer (Bruch’s membrane) interposed between the RPE and choriocapillaries. Electron microscopy revealed accumulation of lipofuscin granules in the cytoplasm of RPE, and deposition of randomly distributed amorphous or granular material in the Bruch’s membrane.2 Histo-chemically, these letters contain mainly extracellular matrix-associated lipids and glycoproteins.3 Frequently, similar materials build up clinically detectable focal deposits—so-called soft Drusen—located just beneath the RPE.5 The subsequent impairment of metabolic exchange between choriocapillaries and RPE leads to secondary degeneration of photo-receptor cells resulting in further deposition of abnormal metabolites in the Bruch’s membrane, and finally, permanent loss of the visual functions at the affected macular area.5,6 Biochemical studies have shown increasing lipid deposits in Bruch’s membrane exponentially correlated with age. The macular area was preferentially affected.7 Further studies on normal donor eyes showed that the Bruch’s membrane contains significant amount of lipids derived from long-chain polyunsaturated fatty acids normally found in the photoreceptor outer segment, providing support for the cellular, but not plasma origin of these lipids.8 These findings suggested that incomplete catabolism of the photoreceptors results in intraand extracellular accumulation of undigested materials—first of all lipid peroxides. Recent studies showed that Bruch’s membrane is also enriched in cholesterol, particularly in esterified cholesterol, which increases significantly with age. Photoreceptors are poor in unesterified cholesterol, and RPE can not esterify cholesterol. Thus, cholesterol deposition in human Bruch’s membranes may rather have plasma than photoreceptor origin.9 All these findings justify an assumption that both catabolism and uptake of lipids by RPE are compromised in AMD. Although the critical role of mitochondria and peroxisomes in the abnormal lipid metabolism of RPE was suggested several years ago, this concept was never confirmed in humans.5,10,11 Bruch’s membrane is intriguingly similar to the arterial intima because both of them contain extracellular matrix molecules that potentially can interact and bind lipids. Furthermore, Bruch’s membrane alterations in AMD and atherosclerotic vascular lesions possess several common features.9 These observations are in accordance with epidemiologic studies that showed an association between atherosclerosis and the presence of early AMD and their risk factors.12,13

Ultrastructural and histo-chemical studies showed that alterations of the Bruch’s membrane may also include focal or diffuse thickening of the basement membrane of the RPE and choriocapillaries.2,14 Similar thickening of capillary basement membrane is a well known feature of ageand diabetes-related microangiopathy. Although electron microscopy shows homogeneous appearance of basement membrane, histochemically it contains type IV collagen, heparan sulfate proteoglycans, and

laminin. Polarization microscopy revealed that collagen filaments are present in axiparallel linear order in the basement membranes, and the carbohydrate components of proteoglycan macromolecules are also in linear order, which is correlated with that of the collageneous framework.15 A lipid layer formed by transversally oriented hydrocarbon chains of lipids has also been described in the basement membranes.16,17

Although extracellular matrix (ECM) alterations in aging and in several diseases (neurodegenerative diseases, metabolic diseases, and atherosclerosis) have been widely studied, neither the origin of these structural lipids nor their pathologic significance were explored. Currently, the role of peroxisomes in the intracellular accumulation of lipids is under exploration in several laboratories, but its contribution to extracellular lipid deposition is unknown. The central role of mitrochondria in aging and in the pathogenesis of the above mentioned diseases is well-established.18 In spite of that, these mitochondrial diseases are usually associated with marked changes in content and composition of ECM. The correlation between mitochondrial pathology and ECM alterations have not yet been studied. For the present studies, all of our specimens were obtained from surgically removed human eyes. The time between enucleation of eyeball and fixation was extremely short, thus post-mortem changes of membrane lipids were insignificant. We also used these specimens for polarization microscopy for the study of ECM alterations. This is a very sensitive technique for revealing macromolecular organization of proteoglycans and lipids. It may also give quantitative information on ageor disease-related changes of each basement membrane component.19 This unique human material and special microscopic techniques permitted an excellent insight into the pathology of a common eye disease, which has neither animal model nor treatment, and which may be considered as a paradigm for neurodegenerative diseases. Furthermore, these studies also added new information to the pathology of ECM—apparently suitable for learning more on aging, atherosclerosis, diabetic microangiopathy, and infiltrative neoplastic diseases.

Methods

Selection of Materials

Fifty-two human eyes were involved in these histo-pathologic studies (25 female and 27 male, aged 56 to 87 years—mean age 68 years). Twenty-six of them were affected by early AMD, and 26 eyes were used as age matched normal controls. Clinical criteria for AMD was pigment irregularities and/or soft Drusen by ophthaimoscopy, while histological criteria for AMD wasa presence of soft Drusen and/or basement membrane thickening. These eyes were surgically removed because of malignant tumor or severe ocular trauma—neither of them affected the posterior pole of the eyeball.

Transmission Electron Microscopy

Small pieces of the retina and choroid were dissected at the posterior pole, immediately (< 2 minutes) after the removal of the eyeball, and fixed at 40C in 2 percent buffered glutaraldehyde for two hours, and post-fixed in 2 percent osmium tetroxide for another two hours. The post-fixation with osmium tetroxide has been found to be effective for demonstrating lipid peroxides as tetramethylbensidine.20 The specimens were dehydrated, embedded in araldite, sectioned with Reichert ultramicrotome, contrasted with lead-citrate and uranyl-acetate, and studied with Zeiss 109 Electron Microscope.

Light Microscopy

Other small pieces of the retina and choroids were fixed in 10 percent buffered formaldehyde for 48 hours, then dissected, One half of the eyeball was dehydrated, embedded in paraffin, and the 6-micron thick sections were stained with hema- toxyline-eosine and PAS-Hale staining.

Polarization Microscopy

This technique, by the use of histochemical reactions to selectively modify the anisotropy (topo-optical reactions), is a unique microscopic approach for the study of membrane lipids, proteoglycans, and collagen fibrils at macromolecular levels.

For the Study of Lipids

Other small pieces of the retina and choroids after fixation were embedded in gelatin. Ten-micron thick frozen sections were made with cryostat, and the unstained sections were mounted in gum arabic. This simple technique suppresses the birefringence of all structures except lipids, and thus permits the study of lipid structures. For lipid extraction, metanol-chlorophorm 1:3 mixture was used for 24 hours.

For the Study of Proteoglycan Structures

Aldehyde-Bisulfite-Toluidine Blue (ABT) staining reactions were used.15 This technique allows the polarization microscopic studies of vicinal OH groups, based on the aldehyde-bisulfate addition reaction followed by toluidine blue staining at pH1.0. During the procedures, vicinal OH groups are transformed into dialdehydes

by periodic acid, and then transformed into negatively charged groups by the bisulfite addition reaction. In this way, they are rendered capable of binding toluidine blue at low pH, which results in basophilia and anisotropy indicating linear order of the OH groups in the reacting macromolecules. In detail, the deparaffined sections were first treated with 0.5 percent periodic acid for 30 minutes, then for 30 more minutes with a saturated solution of sodium bisulfate. After short rinsing with water, the slices were stained for five minutes with toluidine blue at pH 1.0 (0.1% toluidine blue in 0.1 normal HCl). The controls were stained with toluidine blue at pH 4.5 (0.1% toluidine blue in McIlvain buffer). After staining, the dye solution was blotted off with filter paper and then 1 percent potassium ferricyanide solution was dropped onto the slides. This resulted in stabilization of the dye binding in the oriented state in which the dye has been bound by the structures originally and in maintaining its optical effects—the metachromatic basophilia and anisotropy. Without being rinsed in water, the slices were mounted in gum arabic containing 1 percent potassium ferricyanide. This gum arabic layer was allowed to dry for two to three days.

For the Study of Collagen Fibrils

The deparaffined sections were mounted in 50 percent phenol-containing canada balsam. Addition of phenol specifically inverses and increases the anisotropy of collagen fibers, but not the other protein fibrils. These slices were studied with a Leitz-Ortoplan-Pol microscope. The light retardation (anisotropy) was measured with a Brace-Kohler rotary compensator in 580 nm light.19 Five serial sections for every specimen were used and the light retardation was measured in ten different areas of each section—thus, every date point of patients represented a mean value of 50 measurements.

Statistical Analysis

For the study, the relationship between age and anisotropy of the ECM components was analyzed using a simple linear regression model, and Fisher’s transformation was used for the calculation of correlation coefficients.

Results

Alterations of the RPE

Numerous lipofuscin granules can be seen in the RPE—some of them are fused with melanosomes forming melanolipofuscin granules. Mitochondria in the RPE show focal loss of cristae and increased translucency of matrix. Some of the mitochondria show normal structure. Several microsomes and peroxisomes of various