Age-related changes in the morphology, absorption and fluorescence of melanosomes and lipofuscin...

Vision Ref. Vol. 30, No. 9, pp. 1291-1303, 1990 Printed in Great Britain. All rights resewed

0042-6989~90 S3.00 -t 0.00 Copyright 0 1990 Pergamon Press plc

AGE-RELATED CHANGES IN THE MORPHOLOGY, ABSORPTION AND FLUORESCENCE OF MELANOSOM~S

AND LIPOFUSCIN GRANULES OF THE RETINAL PIGMENT EPITHELIUM

MIKE BOULTON,‘*~ FRANCO DOCCHIO,~$ PIERRETTE DAYHAW-BARKER,~ ROBERTA RAMPONI* and RINALDO CUBEDDU~

‘Department of Clinical Ophthalmology, Institute of Ophthalmology, Judd Street, London WClH 9QS, U.K., 2Centro di Elettronica Quantistica e Strumentazione Elettronica de1 CNR, Istituto di Fisica, Politecnico di Milano, P. za L. da Vinci, 32,20133 Miiano, Italy and ‘Pennsylvania College of Optometry,

1200 West Godfrey Avenue, Philadelphia PA 19141, U.S.A.

(Received 10 July 1989; in revised form 11 January 1990)

Abstract-The morphological and spectral characteristics of purified populations of melanosomes and lipofuscin granules from the human retinal pigment epithelium (RPE) were studied with respect to donor age. All melanosome and lipofuscin fractions exhibited the typical ultrastructural appearance associated with these granules. Absorption profiles of both melanin and Iipofuscin granules d~onstrat~ an increased optical density of the granules with increasing age. The former was associated with an overall increase of melanin within the granules. Melanosomes were weakly fluorescent; emission in the blue decreased with increasing age while emission in the red increased. The fluorescent intensity of lipofuscin granules increased with age.

These results provide support for the concept that melanogenesis is occurring within the human RPE throughout life and that pigment granules within the RPE undergo age-related modifications during life.

Melanin Lipofuscin Morphology Absorption Fluorescence Melanogenesis

INTRODUCTION

The retinal pigment epjthefium (RPE) of normal human eyes contains a range of pigmented inclusions all of which are related to two types of pigment granules: melanin and lipofuscin (Feeney-Burns, 1980). Melanin is a high molecular-weight polymer derived from the enzymatic oxidation of tyrosine and dihydroxy- phenylalanine and is located in membrane limiting granules termed melanosomes (Feeney, 1978). In man the pigment granules usually appear in the RPE between 27-30 days of foetal development and production is presumed to cease within a few weeks (Mann, 1969). These granules mature over the first few years of life undergoing a number of morphological changes

*Present address: Departments of Ophthalmology and Cell and Structural Biology, University of Manchester, Oxford Road, Manchester, U.K.

tTo whom all correspondence should be addressed at: Department of Ophthalmology, Manchester Royal Eye Hospital, Oxford Road, Manchester, U.K.

IPresent address: Dipartimento di Automazione Inde.le, Universita di Bresica, Via Valotti, 9, 25100 Brescia, Italy.

until a full complement of “mature” melanosomes is formed which is thereafter thought to be unchanging (Feeney, 1978). This differs from dermal and uveal melanocytes in which melanogenesis continues throughout life (Garcia, Szabo & Fitzpatrick, 1979).

Lipofuscin is a lipid aggregate which is present within the human RPE as intracellular yellow-brown refractile granules which auto- fluoresce under U.V. light excitation (Feeney, 1978). Lipofuscin is thought to represent the life- long accumulation of lysosomal residual bodies containing the end products of the phagocytosis of photoreceptor outer segments (Boulton, McKechnie, Breda, Bayly & Marshall, 1989; Feeney, 1978; Feeney-Burns & Eldred, 1984; Katz, Drea, Eldred, Hess & Robison, 1986) and to a lesser extent autophagy (Feeney, 1978).

Melanosomes and lipofuscin granules are also invovled in age-related modifications resulting in (a) the formation of melanosome-lipofuscin complexes and (b) remodelling of pigment granules by lytic enzymes (Feeney, 1978).

The morphology of both melanosomes and fipofuscin granules has been examined in different age-groups but studies have primarily

1291

1292 MIKE kXXTON et al

focused on the formation of age-related melanolipofuscin complexes (Feeney, 1978). However, the optical characteristics of melanin and lipofuscin granules have not been comprehensively studied, particularly with regard to age-related changes. Melanosomes either whole or dissolved have been studied with regard to their absorption properties (Gabel, Birngruber & Hillenkamp, 1979; Koltias & Bager, 1985, 1986; Menon, Persad, Haberman & Kurian, 1983; Wolbarsht, Walsh & George, 1981). A constant increase in absorption from the visible to the U.V. end of the spectrum is a general feature of both ocular and non-ocular melanin, Although fluorescence has occasionally been reported for dermal melanin under U.V. irradiation (Arico, Barcellona, Gianmarinaro & Micciancio, 1983) no fluorescence data is available for RPE melanosomes.

Emphasis has been given to the fluorescence properties of lipofuscin as a means to (a) identify these granules in situ and (b) understand the mechanisms of lipofuscin formation. Using this knowledge it has been possible to monitor the topographical distribution, as well as age- and pathology related changes within the RPE (Eldred & Katz, 1988; Feeney-Bums, Hilder- brand & Eldridge, 1984; Weiter, Delori, Wing & Fitch, 1986; Wing, Blanchard & Weiter, 1978). Attempts at elucidating the absorption characteristics of lipofuscin are lacking, probably due to the complexity of this lipid aggregate. Eldred and Katz (1988) however, have demonstrated that chloroform extracts show an increase in absorbance from the visible to the U.V.

In an attempt to extend our understanding of RPE melanosomes and lipofuscin we have compared the morphology and spectral properties of purified populations of these granules with respect to age.

MATERIALS AND METHODS

Melanin and lipofuscin were isolated from human RPE cells as previously described (Boul- ton & Marshall, 1985). In brief, RPE cells were isolated from four groups of donor eyes: 20 foetal eyes between 12 and 22 weeks of gestation, 33 eyes between S and 29 yr of age, 31 eyes between 30 and 49 yr of age, and 44 eyes > SO yr of age. The mean age of each group was 18 weeks of gestation, 16, 41 and 68 yr of age respectively. These subdivisions were chosen in order to provide sufficient material for each

group. For RPE cell isolation the eyes were hemisected at the equator and the anterior portion discarded. The vitreous and neural retina were gently removed and the RPE cells gently brushed off Bruch’s membrane using a camel hair brush. When freed, the cells were washed out of the eye cup with Dulbecco’s phosphate-buffered saline without calcium and magnesium (PBSA). Cell suspensions were stored at -20°C and approp~ate age groups pooled until a suitable cell volume and been isolated. The cells were disrupted by sonication for 60 set at 4°C using a Soniprep 1 SO (MSE). Cellular debris was removed by centrifugation at 60g for 7 min. The resultant low speed supernatant was centrifuged at 6OOOg for IOmin to sediment the pigment granules. This sediment was resuspended in 0.3 M sucrose and layered onto a discontinuous sucrose gradient consisting of eight different con~ntrations of sucrose solution (2, 1.8, 1.6, 1.55, 1.5, 1.4, 1.2 and 1 .O M) and centrifuged on a MSE super- speed centrifuge at 103,OOOg for f hr. The prin- cipal band, lipofuscin (Ll), was located between the 1.2 and 1.4 M interfaces, while melanosomes (M) formed the pellet. There was no Ll band present for the foetal eyes. In all groups, 5 yr and above, there were intermediate bands located at each interface between Ll and M. Only at the 1.8 and 2 M interface in the > SO yr old group was there sufficient material for ex~~mentation. This fraction was termed as L2. Each fraction was isotated and further purified by running it on a second identical gradient and the fractions were washed four times with PBSA in order to remove the sucrose.

Melanosomes from bovine RPE were isolated as described above.

Microscopy

Samples of the Ll and M fractions from the different age groups and the L2 fraction from the > SO yr age group were examined as follows:

(a) using a Zeiss IN2 bright field and fluorescence microscope (fluorescence filters included a BP-450-490 exciter and an LP 520 barrier);

(b) fixed with 2.5% glutaraldehyde and pro- cessed for electron microscopy as previously described (Bouiton & Marshall. 1985) with the exception that the sections were examined unstained.

Preparation of fractions In an attempt to standard&e the analysis we

adjusted the concentration of ail the fractions,

Fig. I. Electron micrographs of unstained sections of melanin samples from different age groups (a) foetal. (b) 5-29 yr, (c) 30-49 yr and (d) > 50 yr. The scale bar is 2 jtm.

Fig. 2. Electron micrographs of unstained sections of lipofuscin (a-c), melanolipofuscin (d) and bovine

melanin (e) samples from different age groups. (a) and (d) > 50 yr, (b) 30-49 yr, (c) 5-29 yr and (d) I yr.

The scale bar is 2 orn.

1293

Spectral properties of RPE pigment granules 1295

suspended in buffered-saline, to an equal optical density of 0.2 at the wavelength of 514 nm using a Perkin-Eimer 554 spectrophotometer. The number of granules in each fraction was determined by mixing the granules with a known number of 12 pm latex microspheres (Sigma) and spraying the suspension onto a plastic coverslip. The two types of particles were counted on an Hitachi S 510 scanning electron microscope and the total number of pigment granules in each sample were determined. In some instances equal optical densities of equivalent volumes of the M fractions were dissolved by boiling in 1 M KOH for IOmin. The KOH treated samples were subsequently cent~fuged at 21 ,OOOg, since no pellet was obtained it was concluded that the granules had been completely dissolved. For fluorescence spectroscopy studies at the microscopic level melanin and lipofuscin granules were dried onto quartz glass slides.

Absorption spectroscopy of lipofuscin and melanin granules

Absorption spectroscopy of the M, Ll and L2 fractions was performed using a Perkin-Elmer Model 554 dual beam spectrophotometer. All spectra were performed from 250 to 700nm. Absorption spectra of dissolved M fractions were measured with a 1 M KOH solution as the reference, while spectra of intact granules were determined using saline as the reference. The spectral resolution of the system was 2 nm.

Fluorescence spectroscopy

Fluorescence spectroscopy of the M, Ll and L2 fractions was performed using a fluorescence spectrophotometer (Perkin-Elmer, Model 650- 40, U.S.A.). Emission spectra of each fraction were taken using several excitation wavelengths. Among these, 364 and 476nm were used to comply with the measurements at the microscopic level. Moreover, 364 nm excitation has been used fairly routinely in the literature.

All the excitation and emission spectra were corrected for the Raman peak of the excitation light in saline. The spectra were also corrected for the spectral sensitivity of the detection system using standard procedures (i.e. quantum counters, scattered light). The spectral resolution of the spectrofluorimeter was set at 5 nm by varying the width of the entrance slit of the emission mono~hromator.

Fluorescence spectroscopy at the microscopic

level

Fluorescence emission spectra of granules at the microscopic level were recorded using a laser microspectrofluorimeter (Cubeddu, Docchio, Liu, Ramponi & Taroni, 1990). In brief, the system made use of a coherent tunable source of subnanosecond pulses, a Leitz MPV2 fluorescence microscope, a stepping motor-driven monochromator (Jarrel-Ash Model 82-410) a microchannel plate photomultiplier (Hamamatsu R1564UOl), and a time-correlated single photon counting unit. A 40 x microscope objective was used for the measurements. The beam spotsize with this objective was about 10pm dia. Single granules of each fraction could be irradiated, although in this case the detected fluorescence was very weak. Therefore in the majority of our measurements, the fluorescence was collected from groups of 5-10 granules in order to obtain a significant signal-to-noise ratio. The quartz slides exhibited negligible fluorescence at both excitation wavelengths. The spectral resolution was 1 nm. The spectra were corrected for the overall spectral sensitivity of the system, to compare them with those obtained from the solutions. The calibration curve for the correction was obtained by taking the emission spectrum of an incandescent lamp of known spectral sensitivity.

RESULTS

Morphological chaiacteristics of the diflerent fractions

Brightfield and fluorescence microscopy revealed that there was no contamination of the Ll fractions with melanin and only minimal contamination (< 1%) of the melanin fraction by lipofuscin. The L2 fraction exhibited characteristics of both melanin and lipofuscin.

Electron microscopy demonstrated that each separate fraction consisted of a homogeneous population of granules (Figs 1 and 2). Melanin granules from the >5 yr age groups were 2-3 @rn in length and all exhibited the typical electron dense morphology of mature melanin granules [Fig. l(b-d)]. The foetal melanin fraction showed immature melanosomes in several stages of melanization [Fig. l(a)]. The bovine melanin fraction consisted of typical melanin granules surrounded by amorphous debris. Further attempts at isolating a pure population of bovine melanosomes all resulted in contamination with this amorphous material [Fig. 2(e)].

1296 MINCE bUtTON et al

Table 1. The number of granules in each fraction when adjusted to

an O.D. of 0.2 at 514 nm

Sample Granules/ml x lad (group) (SD)

Ll (5-29) 2.40(0.16) LI (30-49 2.73 (0.23) Ll (50-80) 2.50 (0.20) L2 (50-80) 2.36 (0.14) M (f&al) 2.12 (0.09) M (5-29) 2.30 (0.15) M (30-49) 1.81 (0.11) M (50-80) 2.00 (0.09) M (bovine) I.50 (0.06)

The Ll fractions exhibited a less electron dense and more diffuse morphology typical of lipofuscin [Fig. 2(a)-(c)]. The L2 fractions had the typical characteristics of melanolipofuscin complexes consisting of a varied population of particles with diflering electron densities [Fig. 2(d)].

The number of granules in each fraction when adjusted to an optical density of 0.2 at 514 nm are shown in Table 1.

Absorption specfroscopy of the M fractions

Absorption spectra of dissolved M fractions are shown in Fig. 3. All samples exhibited an absorbance profile which decreased from 250 to 700 nm. The M fraction from > 50 yr age group showed an absorbance 8 times greater at 250 nm than at 700 nm. This ratio decreased with younger age groups, down to a factor of 4 in the case of the foetal M fraction. The shape of the absorption curves were also related to age groups. The foetal M fraction demonstrated a marked change in the slope of the absorption

curve at about 3OOnm, while this change became less apparent in the M samples from the older age groups. Bovine melanin presented an absorption curve which was intermediate between foetal M fractions and the >5-29 yr age group. Additional shoulders in the range 280-340 nm were present in this spectrum.

Absorption spectra of intact M granules in suspension are shown in Fig. 4. The curves from the > 5 yr age groups were ail characterised by an almost equal wavelength dependence, with a rise in absorbance form 250 up to 5OOnm and a subsequent decrease towards longer wavelengths. The absorption curve of foetal M and bovine M fractions exhibited a wavelength dependence similar to that of dissolved granules, with a decrease in absorbance towards longer wavelengths. Light scattering was not a significant problem since the shape of the spectra did not depend on the concentration of the granules in a range of two orders of magnitude.

Absorption spectroscopy of intact LI and L2 fractions in suspension

Since there was no direct procedure to dis- solve the Ll and L2 fractions, all absorption and fluorescence studies were conducted on intact granules. All the Ll fractions exhibited similar broad absorption spectra [Fig. 5(a)]. A peak at about 400nm was apparent for the 30-49yr and the ?50yr age groups, although the peak was less pronounced in the former. The absorption of L2 granules from the > 50 yr age group [Fig. 5(b)] exhibited a wavelength dependence similar to that of the Ll granules from the 30-49yr group. Light scattering was not a

Wavelength (nm)

Fig. 3. Absorption spectra of dissolved mcbmia f&ns from diScrent age groups. (-): foctol, ( -. -1: 5-29 yr, (- .. -): M-49 yr, (. . . . .): z= 50 yr old m&nin (- - -): bovine m&mia. Absorption (O.D.) is

expressed in arbitrary units (A.U.).


0 I I 1 I I

300 400 500 600 700

Wavelength (nmf

Fig. 4. Absorption spectra of intact melanin granules from different age groups. The number of granules in the samples is the same as that used to perform the spectra in solution. (-): foetal, (-, -): 5-29 p,

(-..-): 30-49yr, (.... .): > 50yr old melanin and (- - -): bovine melanin. Absorption (O.D.) is expressed in arbitrary units (A.U.).

significant problem sin& the shape of the spectra did not depend on the concentration of the granules in a range of two orders of magnitude.

Fluorescence spectroscopy of the iU samples

The excitation and emission spectra of intact M fractions from different age groups are shown in Fig. 6. A summary of the excitation and emission characteristics of these granules is presented in Table 2. All M fractions exhibited a rather weak emission when excited at 364nm. Foetal melanin demonstrated the highest emission intensity, with a single emission peak around 450 nm [Fig. 6(a)]. The corresponding excitation peak was located at 350nm. The

emission peak at 450 nm decreased dramatically in the 5-29yr group, with a corresponding decrease in the 350 nm excitation peak. This component remained, thereafter, almost unchanged in the older age groups. A secondary peak at about 570 nm, extending from 500 to 700 nm, was observed starting from the 5-29 yr age group. Correspondingly, there was an enhancement of the excitation spectrum in the region 330-370 nm. This secondary emission increased with age: the > 50 yr old group exhibited the highest emission intensity in this region. Concomitantly, an enhan~ment of the red component in the excitation spectrum was observed [Fig. 6(d)]. Bovine melanin [Fig. 6(e)] showed

0’ I I I I I

300 400 300 600 700

Wavelength (nmf

Fig. 5(a). Caption overleaf:

1298 MX.E BOULTON et al.

0.5 (bf

Wavelsngth (nm)

Fig. S(b)

Fig. 5. Absorption spectra of intact L1 (a) and L2 (b) granttics in suspwion. (-): 5-29 yr Ll &actions, (- - -): 30-49 yr fractions and (- r -): > SO yr. LI fractions. Absorpkm (O.D.) is qwcssed in arbitrwy

units (AN.).

excitation and emission spectra which were intermediate between foetal M and M from the 5-29 yr age group. Here, the red emission peak was still nearly absent, but the red component in the excitation spectrum w8s 8ecn to bc h&r than the blue component. Excitation spectra did not show any appreciabk difference for concentrations of granules up to 10 times than that reported here.

Emission spectra of groups of M granules were performed at the microscopic level for comparison with the spectra from suspe&d granules. As 8 typicai ex8mple, Fig. 7 shows 8 comparison between the emission spectrum of suspended M granules from the >50 yr age group, and of dried granules under the microscope. In all the cases the spectra obtained on individual granules were similar to the spectra of granules in suspension.

Table 2. Summary of the fluorevxnct cbaractitica of Melanin and lipofuacin fractions

Sampie (SroUPf

LI (S-29) LI (30-49) Ll (so-so) L2 (SO-SO) M (foctalf hi (5-29) M (30-49) M (50-80) M (bovine)

EXCitati0n

peaks t=d

370-405-470 370-470 310-470 400-460

3stzO 350-430 350-450 350-460

. , 470-570-610 470-570-680

470-570 46Q-550-680

440

$:E 440-560 440-560

Rdative

a 1.00 1.71 1.95 0.71 1.1% 0.27 0.15 0.27 0.28

The CXtit8tiOn and emission spectra of the Ll fractions from di&ent age group 8re shown in Fig. 8. A summary of the excitation and emission characteri&s of the Ll and L2 granules is shown in Table 2. All Ll fractions exhiited a broad emission spectrum with a peak at 600 nm when excited at 364nm. This emission peak rem8incd unchanged as the cxoitation wavelength was set at 476 mn. A comparison of the emission spectra from the various age groups showed four main spectral regions of interest: the main peak located at 600-61Onm, a blue-green shoulder located at 470nm, a green-yellow shoulder at 55Onm, and a far red shoulder 8t 68onm. with respect to the main peak at 600-610 run, the blue-green component was evident in the 5-29yr old and in the 30- 49 yr old age group, but it was not present in the > 50 yr old age group. The same b&&our was exhibited by the green-yellow peak. By contrast, the far red 5uorescence component ~8s shown cbnsistcntly to increase with increasing age.

With the exception of the 5-29 yr group, the excitation spectra with emission monitored at 610 nm were identical to those with emission at 570 nm. In the 5-29 yr group the excitation qectrum at 61Onm was appreciably different from that at 550 nm, with the main peak being shifted to 480 nm, and a second peak located at 405 nm. Excitation sbcctra did not show any ‘Normalised to chat of LI (S-29). .


60

40

20

100

80

60

40

20

0

100 ? ?

I 80

Q * ti

60

j 40

5 20

100

I 80

60

40

20

o- 100

80

60

40

20

I

300 400 500 600

II

~

(a)

(bl

II

I”“\. (c)

I I I I

(dl

(e)

400 500 600 700

Wavelength (nm)

Fig. 6. Excitation and emission spectra of intact melanin granules from different age groups. Left curves (I): excitation spectra with emission monitored at 570nm. Right curves (II): emission spectra with excitation performed at 364nm. (a) Foetal, (b) 5-29 yr, (c) 30-49 yr, (d) >5Oyr and (e) 1 yr

bovine melanin. Excitation and emission spectra are expressed in arbitrary units (A.U.).

appreciable difference for concentrations of granules up to 10 times than that reported here.

The fluorescence intensity increased with increasing age (Table 2). The spectra showed a marked increase in the emission and excitation peaks being about 40°/ higher in the older group than in the younger.

The L2 fraction demonstrated a spectrum which was intermediate between those of Ll and of melanin [Fig. 8(d)], and a markedly reduced fluorescence intensity with respect to the Ll fractions of the corresponding age group.

As for the case of melanin, emission spectra of groups of Ll and L2 granules were performed at the microscopic level for comparison with the spectra from suspended granules. As a typical exampIe, Fig. 9 shows a comparison between the emission spectrum of suspended Ll granules from the >50 yr age group, and of dried granules under the microscope. As for the M fraction, in none of the cases did the spectra obtained on individual granules show appreciable differences with respect to the spectra of granufes in suspension.

“R 3CV9-D

1300 MIKE BOGLTON~SI al

I I I 400 500 600 700

Wavotength fnml

Fig. 7. Comparison between emission spectra of melanin samples from the 50-80 age group, suspended in saline (a) and at the microscopic level (b), excited at 364 nm. Excita- tion and emission spectra are expressed in arbitrary units

(A.U.).

DISCUSSiON

The interpretation of the photobiological events in the RPE has become dependent on a better understanding of the photophysical and photochemical properties of melanosomes and lipofuscin granules. Melanin is a well- established amorphous semiconductor and acts as a converter of excited-state energy into heat as well as a sink for free radicals (McGuinness & Proctor, 1973; McGuinness, Corry 8r Proctor, 1974). Thus the inclusion of melanin within cells constantly exposed to optical radiation provides a protection factor. Although the primary biological role of melanin remains difficult to estab- lish, it has been suggested that abnormalities in melanosomes are associated with certain diseases (Proctor, 1976). Lipofuscin is an age- related pigment whose precise function remains unknown but its presence within the RPE has been implicated in a number of age related diseases of the outer retina (Eagle, 1984; Foos & Trese, 1982; Gass, 1973; Sarks, 1976).

The morphological and spectral data presented confirm that the Ll fraction is hpoftin, the L2 fraction is melanolipofuscin and that M is melanin. As predicted no lipofuscin or melanolipofuscin granules were present in the foetal eyes. The intermediate bands observed on the discontinuous sucrose gradient were assumed to be melanolipofuscin complexes in varying stages of formation. Thus the fraction

termed L2 represented only it part of the mefanohpofuscin content of RPE cells. Since sufficient melanolipofuscin could only be isolated from the > 50 yr age group it was impos-

sible to conduct an age-related comparison. Absorption profiles of dissolved melanin

granules were similar to those reported in the literature for eumelanin (Gabel et al., 1979; Kollias & Bager, 1985; Menon et al.. 1983). However, differences were apparent between melanosomes from different age groups. Given that equivalent volumes of granules at an optical density of 514 nm contained approximately the same numbers of granules it must be concluded that the age-dependent increase in absorbance at the shorter wavelengths reflects an increase in the melanin content of the mefanosomes. The absorption peak in the blue- near U.V. region with bovine melanosomes was almost certainly due to the amorphous debris associated with this fraction. Absorption spectra of intact melanosomes in suspension demonstrated a masking effect due to the high individual optical density of each granule: this effect results in a distortion, or flattening, of the spectrum with respect to the spectrum exhibited by the dissolved material (Latimer, 1983; Duysens, 1956). Younger melanosomes exhibit a less distorted spectrnm, and therefore a fess marked masking effect than older granules. This finding correlates well with the differences in the optical density observed from the spectra of dissolved granules.

Scattering of light by the granules might, in principle, explain the change in profiles of the spectra of suspended granules with respect to those of dissolved granules. The involvement of both Rayleigh and/or Mie scattering on the absorption spectra is, however, ruled out due to the similarity of absorption spectra of suspended granules two orders of magnitude of optical density greater than those reported in this study.

It has always been assumed that RPE melanosomes are non-fluorescent since only lipofuscin and melanoIipofu~in granules are observed to fluoresce when examined by fluorescence microscopy. The weak fluorescence intensity exhibited by intact melanosomes and the use of barrier filters with a cut off at 520 nm may explain why melanosomes have not been found to ffuoresce by standard fluorescence microscopy techniques. A weak fluorescence in the blue has also been reported by Eldred and Katz (1988) studying fluorophores in the RPE

Spectral properties of RPE pigment granules I301

100

80

60

40

20

0

80

60

* E Y 100 I5 I

80

60

40

20

0- 300 400 500 600 400 500 500 700

(a)

(b)

Id)

Wavelength (nm)

Fig. 8. Excitation and emission spectra of intact LI and L2 granules in suspension. Left curves (I): excitation spectra with emission monitored at 570 nm. Right curves (II): emission spectra with excitation performed at 364 nm. (a) 5-29yr old Li fraction, (b) 30-49yr oId Ll fraction, (c) >50yr Ll fraction and (d) >50yr L2 fractions. Excitation and emission spectra are expressed in arbitrary units (A.U.).

but the source and nature of this fluorescence were not characterized. The decrease in the blue part of the spectrum with age may be related to the increase in density of melanosomes with age; the fluorescence yield of a granule can decrease due to autoabsorption in a granule with a higher packing ratio. The shift towards the red in the fluorescence spectra of melanosomes from older donors could be explained by either a wavelength-~l~tive intragranule absorption or the incorporation of fluorescent material in the older melanosomes. Contamination of the older melanosome fraction by lipofuscin or melanolipofuscin granules is not a consideration since the 20% increase in fluorescent density in the older group greatly exceeds the < I % contamination by lipofuscin granules. The fluorescence spectra of dissolved melanosomes are not reported since the fluorescence profile and

quantum yield of a granule is dependent on its structure, density and dimensions.

Owing to the inability to completely solubilize lipofuscin granules, absorption spectra could only be obtained from intact granules. These spectra exhibited the previously mentioned masking effect and indicated an age-dependent increase in the optical density of lipofuscin granules, as was found to be the case for melanin. Eldred and Katz (1988) have obtained absorption spectra from chloroform extracts of lipofuscin and shown a wavelength dependence with a decrease in absorbance towards longer wavelengths. The fluorescence emission spectra of lipofuscin either as suspended granules or at the microscopic level compared well with those reported by other authors (Eldred, Mueller, Stark & Feeney-Bums, 1982; Weiter et al., 1986). However, a significant increase (about

1302 MIKE &XJLTON et al

?OO

80

60

40

(b)

Y 0- 400 700

Wavelength tnmi Fig. 9. Comparison bctwccn emission spectra of Li samples from the > 50 yr age group, suspended in saline (a) and at the microscopic level (b) excited at 364 nm. Excitation and emission spectra are expressed in arbitrary units (A.U.).

40%) in the fiuorescent intensity of lipofuscin was observed with increasing age. The small variations between the spectra from suspended granules and those under the microspectrofluorimeter may well be explained by the photo- bleaching that occurs during the short ptdse excitation under the microscope. The fluorescence spectra observed were deemed not to be influenced by light scatter since, (1) the emission spectra were consistently the same under different irradiation conditions (i.e. large volume i~adiation, single granule irradiation and small cross section irradiation; and (2) the shape of the spectra did not depend on the concentration of granules. Roth excitation and emission spectra of all lipofuscin fractions may be thought as combinations of fractions termed “green-yellow” and “orange-red” emitters by Eldred and Katz (19gg), ahhough in our study the peak of the excitation spectra are shifted from 330 to 36Onm.

There are a number of important biological irnp~~tio~ from this study. First, since the melanin content of meianosomcs increases with age, it must be conduded that melanogencsis is occurring throughout life in human RPE. This is contrary to the commonly held view that RPE meianogenesis occurs during a brief win- dow of embryogenesis and is therea& switched off. This view has been derived from electron microscopy studies and the inability to demon- strate tyrosinase activity in mature melano-

somes (Hogan, Alvarado & Weddeil, 1971; Zinn & Benjamin-Henkind. 1979). However, the concept of melanin productton in the RPE throughout life (albeit at a slow rate) has been introduced by previous authors. Spitznas (1971) described morphological evidence consistent with active melanin synthesis in adult human RPE and Dryja, O’Neil-Dryja. Pawelek and Albert (1978) reported low levels of tyrosinase activity within adult bovine RPE. Second, the increase in melanin content of individual granules may be essential to compensate for the overall decrease in melanosomes in the RPE with age (Feeney-Burns et al., 1984; Wing et al., 1978). Third. the change in slope of the dissolved foetal melanin absorption curve compared to the older human melanin suggests that either, the granules are undergoing a change in configuration; there is a decrease in the number of ionic sites during polymer reconstruction; or a combination of the two. This correlates with the morphological changes melanosomes are undergoing during their ‘“maturation” (Feeney, 1978). Given that the spectral characte~sti~s of bovine melanosomes isolated from 1 yr old animals were intermediate between human foetal melanin and young human melanosomes, care should be taken in equating studies on bovine melanin with the human situation. Fourth, there are significant age related changes in both the optical density and fluorescent intensities of individual lipofuscin granules. It is possible that increases in fluorescence are due to one of the following: (I) an increase in one or a few of the ten partialiy purified fluoropho~s identified by Eldred and Katz { 1988); (2) equal increase in all fractions with age; (3) the presence of fluorophores in the granules heretofore not identified due to the complex structure of the granules. Thus, at this point, the biological inte~retation of the age-related changes in the spectral characteristics of melanin and lipofuscin granules observed in this study and their relevance to visual function awaits further investigations.

~ckn5wledge~~fs-~is research was supported by grants from the British Council, CNR (Italy), FDA and Fight for Sight. The authors wish to thank M.R.C. Capon for provid- ing the foctal material and M. Bayly. S. Rothcry, W. Q. Liu and S. Tebaldini for their technical assistance.

REFERENCES

Arico. M., Barccllona, M., Gianmarinaro, M. S. & Micciancio, S. (1983). Time-resolved spectrofluorimetry


of melanin in human melanoma. In Martelluci, S. & Chester, A. N. (Eds.), Laser photobiology and pholo- medicine (pp. 101-106). New York: Plenum Press.

Boulton, M. & Marshall, J. (1985). Repigmentation of human retinal pigment epithelial cells in vitro. Experimental Eye Research, 41, 209-218.

Gass, J. D. (1973). Drusen and disciform macular detach- ment and degeneration. Archives of Ophthalmology, 90,

206-217. Hogan, M. J., Alvarado, J. A. & Weddell. J. E. (1971).

Histology of Ihe human eye (Chap. 9. pp. 393-522). Philadelphia: Saunders.

Boulton, M., McKechnie, N. M., Breda, J., Bayly, M. & Marshall, J. (1989). The formation of autofluorescent granules in cultured human RPE. Invesrigarive Ophrhal- mology and Visual Science, 30, 82-89.

Cubeddu, R., Docchio, F., Liu, W. Q., Ramponi, R. & Taroni, P. (I 990). A system for time-resolved fluorescence spectroscopy with multiple picosecond gateing. Reviews in Scientific Instrumentarion. In press.

Dryja, P. P., O’Neil-Dryja, M., Pawelek, J. M. & Albert, D. M. (1978). Demonstration of tyrosinase activity in the adult bovine uveal tract and retinal pigment epithelium. Investigative Ophlhalmology and Visual Science, 17, 5 1 I - 514.

Katz, M. L., Drea, C., Eldred, G., Hess, H. & Robinson, W. G. (1986). Influence of early photoreceptor degeneration on lipofuscin in the retinal pigment epithelium. Experimental Eye Research, 43, 56 1- 573.

Kollias, N. & Bager, A. (1985). On the assessment of human melanin in vivo. Journal of Investigative Dermatology, 85, 38-42.

Kollias, N. & Bager, A. (1986). On the assessment of melanin in human skin in vivo. Photochemistry and Photo-

biology, 43, 49-54.

Duysens, L. (1956). The flattening of the absorption spectrum of suspensions, as compared to that of solutions. Biochemica Biophysics Acta, 19, I- 12.

Eagle, R. C. (1984). Mechanisms of maculopathy. Ophrhal- mology. 91, 613-626.

Latimer, P. (1983). The deconvolution of absorption spectra of green plant materials. Improved corrections for the sieve effect. Photochemistry and Photobiology, 37(6), 731-734.

Mann, I. (1969). The development of rhe human eye. London: BMA.

Eldred, G. E. & Katz, M. L. (1988). Fluorophores of the human retinal pigment epithelium. Experimental Eye Research, 47, 71-86.

McGuinness, J. & Proctor, P. H. (1973). The importance of the fact melanin is black. Journal of Theoretical Biology, 39, 677-678.

Eldred, G. E., Mueller, G. V., Stark, W. S. & Feeney-Burns, L. (1982). Lipofuscin: Resolution of discrepant fluorescent data. Science, 216, 757-159.

McGuinness, J., Corry, P. M. & Proctor, P. H. (1974). Amorphous semiconductor switching in melanins. Science, 183, 853-854.

Feeney, L. (1978). Lipofuscin and melanin of human retinal pigment epithelium: Fluorescence, enzyme cytochemical and ultrastructural studies. Investigariue Ophthalmology and Visual Science, 17, 583-600.

Menon, I. A., Persad, S., Haberman, H. F. & Kurian, C. J. (I 983). A comparative study of the physical and chemical properties of melanins isolated from human black and red hair. Journal of Investigative Dermatology, 80, 202-206.

Feeney-Burns, L. (1980). The pigments of the RPE. In Zadunaisky, J. A. & Davson, H. (Eds.), Current topics in eye research (pp. I l9- 178). New York: Academic Press.

Proctor, P. H. (1976). The role of melanin in human neurobiological disorders. Pigment Cell Research, 3, 378-383.

Feeney-Burns, L. & Eldred, G. E. (1984). The fate of the phagosome: Conversion to “age pigment” and impact in human retinal pigment epithelium. Transacrions of the Ophrhalmological Society of the United Kingdom, 103, 416-421.

Sarks, S. H. (1976). Ageing and degeneration in the macular region: A clinico-pathological study. British Journal of Ophthalmology, 60, 324-340.

Spitznas, M. (1971). Morphogenesis and nature of the pigment granules in the adult human retinal pigment epithelium. Zeitschrifr fur Zeliforschung Mikroskopie und Anatomic, 122, 378-388.

Feeney-Burns, L., Hilderbrand, E. S. & Eldrige, S. (1984). Ageing human RPE: Morphometric analysis of macular, equatorial and peripheral cells. Investigative Ophrhalmology and Visual Science, 25, 195-200.

Foos, R. Y. & Trese, M. T. (1982). Chorioretinal junction. Vacularisation of Bruch’s membrane in the peripheral fundus. Archives of Ophthalmology, IlW, 1492- 1503.

Gabel, V-P., Birngruber, R. & Hillenkamp, F. (1979). Visible and near infrared light absorption in pigment epithelium and choroid. Excerpfa Medica Inrernational Congress Series, 450, 658-662.

Weiter, J. J., Delori, F. C., Wing, G. L. & Fitch, K. A. (1986). Retinal pigment epithehal lipofuscin and melanin and choroidal melanin in human eyes. Investigative Ophthalmology and Visual Science, 27, 145- 152.

Wing, G. L., Blanchard, G. C. & Weiter, J. J. (1978). The topography and age relationships of lipofuscin concentration in the retinal pigment epithelium. Investigative Ophthalmology and Visual Science, 17, 601-611.

Wolbarsht, M. L., Walsh, A. W. & George, G. (1981). Melanin, a unique biological absorber. Applied Optics, 20, 2184-2186.

Garcia, R. I., Szabo. G. & Fitzpatrick, T. B. (1979). Zinn, K. M. & Benjamin-Henkind, J. V. (1979). Anatomy Molecular and cell biology of melanin. In Zinn, K. M. & of the human retinal pigment epithelium. In Zinn, K. M. Marmor, M. F. (Ed.), The retinalpigment epirhelium (pp. & Marmor, M. F. (Eds.), The retinal pigment epithelium l24- 147). Cambridge, Mass.: Harvard University Press. (pp. 3-31). Cambridge, Mass.: Harvard University Press,

Age-related changes in the morphology, absorption and fluorescence of melanosomes and lipofuscin...

Documents

Transcript of Age-related changes in the morphology, absorption and fluorescence of melanosomes and lipofuscin...