Graefe's Arch Clin Exp Ophthalmol (1986)224:265-270 Graefe's Archive for Clinical and Experimental

Ophthalmology © Springer-Verlag 1986

Correlation of Scheimpflug photography of the anterior eye segment with biochemical analysis of the lens Application of a frozen-sectioning technique to investigate differences in protein distribution of single lens layers

O. Hockwin, M.H.J. Ahrend, and J. Bours Department of Experimental Ophthalmology, University of Bonn, D-5300 Bonn, Federal Republic of Germany

Abstract. Normal and cataractous lenses were separated mechanically into lens equator and inner cylinder and the latter then sectioned in a freezing microtome. Fractions with 120-140 sections each were collected representing sin- gle lens layers, and the content of water-soluble and insolu- ble proteins was determined. Protein profiles for each lens layer were obtained by means of isoelectric focusing in spe- cial agarose gels. Using this microsectioning technique, it was possible to demonstrate differences in the protein distri- bution in single layers of both normal and cataractous hu- man lenses. Comparison of the protein profiles of the nor- mal lens and the lenses of different cataract morphology used in this study demonstrates the potential usefulness of this methodology for future research with cataract lenses.

Introduction

In older patients suffering from cataracts, Scheimpfiug pho- tographs of anterior segments of the eye demonstrate a great degree of variability in terms of cataract morphology. These opacities, which are generally characterized by the clinician as senile cataracts, are now of increasing interest to the lens researcher. Analysis of whole human lenses con- tributes little to the understanding of age-related changes or processes leading to lens opacification, because such analysis yields only average values for the biochemical com- ponents of interest and not their lenticular distributions. Some insight into the mechanisms causing or leading to cataract formation could be gained by layer-by-layer analy- sis of lens regions. Such an analysis can be achieved in vivo by utilizing Scheimpflug photography to monitor changes in lens transparency during opacification.

Hockwin and Kleifeld (1965) and Hockwin et al. (1966) have proposed facilitating regional biochemical analysis of the lens by separating it into equatorial ring, anterior and posterior cortices, and nucleus. This separation was based on the assumption that these lens parts contain different protein species. This separation method has been applied to the study of age-related changes in lenticular metabolism (Hockwin and Ohrloff 1981, 1984) and of single protein species (Bours 1984).

A major contribution to research with human catarac-

Offprint requests to: Prof. Dr, O. Hockwin, Abt. fiir experimentelle Ophthalmologie, Sigmund-Freud-Strasse 25, D-5300 Bonn-1 (Venusberg), FRG

tous lenses has been made by Horwitz et al. (1981), who utilized a regional approach by analyzing an opaque part of a cataractous lens and comparing it with a transparent part of the same lens. Further, Takemoto et al. (1982/1983) and Horwitz et al. (1983) have analyzed microdissected nor- mal and cataractous human lenses. Only the nuclear part of the lens was analyzed. However, these procedures have provided incomplete information about the overall lens dis- tribution of various biochemical parameters and how these may be affected during cataractogenesis. Using the whole lens, a microsectioning technique was developed in the pres- ent study, which allows for the localization and subsequent biochemical analysis of proteins in cataractous lens layers, while simultaneously determining their distributions in the same lens (Ahrend et al. 1985).

Materials and methods

Three adult human lenses, which were received from the University of Bonn Eye Clinic after intracapsular extrac- tion, were included in this study. The first was a normal lens from a 62-year-old male who had, 9 days prior to sur- gery, sustained a traumatic lenticular luxation; the lens wet weight (LWW) was 256.1 mg. The second was a lens with a posterior subcapsular cataract, discrete opacity at the pos- terior subcapsular region, waterclefts, and yellowed cortex and nucleus; it was from a 74-year-old female and the LWW was 262.5 rag. The third was a lens with a hyperma- ture cataract from a 77-year-old diabetic female who was also suffering from diabetic retinopathy; the LWW was 257.1 mg, These lenses were photographed with the Scheimpflug camera (TOPCON SL 45) 1 day prior to sur- gery according to the method of Hockwin and Dragomi- rescu (1981).

The lenses were frozen and divided into equatorial ring and inner cylinder by means of a trephine with a bore hole corresponding to 80% of the equatorial lens diameter (Rink et al. 1982). The frozen lens was placed on a petri dish with the anterior surface upwards. The trephine was placed into the center of this surface and pressed through the lens towards the petri dish. In this way, the equator, as the youngest part of the lens, could effectively be separated from the lens inner cylinder, representing the anterior cor- tex, nucleus, and posterior cortex. This cylinder was fixed with Tissue-tek II (Lab-Tek Products, Division of Miles Laboratories, USA) on a microtome sample holder and frozen with CO2. Sections of about 10 g were made at

266

PC AC 10 5 1 PC AC

Fig. l a-c. Scheimpflug photographs of: a a normal human lens, from a 63-year-old female, b a posterior subcapsular cataractous lens from a 74-year-old female, and e a hypermature cataractous lens from a 77-year-old female. A grid is superimposed on the photographs to show how cryostat microtome sections were made and collected into ten fractions (arrow). AC, anterior cortex; PC, posterior cortex

100- % WS-CRYSTALLINS

75- : ~ \ o o / °9 ~

50- \ . . . . , , / , • / \ /

25 . . . . "/ '"

0 i w r i i i j i i i i Layer No. ; 2 3 z, 5 6 7 8 9 10

I Region: EQAC N PC

100- % WI-PROTEINS

A

75- [ " ,-.. mf ~ •

/ \. • \

50- "-o~°--o~o" \

0 [ l l l l l i ~ , , I

Layer No. 1 2 3 4 5 6 7 8 910 i

Region: EQAC N PC

Fig. 2. Display of the percentage of water-soluble crystallins (WS) and water-insoluble proteins (WI) as a function of lens region. Layers 1-10 were collected from sections through anterior cortex (AC), nucleus (N) and posterior cortex (PC). o . . . . o Normal luxated lens, 62-year-old male. • . . . . • Posterior subcapsular cataractous lens, 74-year-old female. • . . . . • Hypermature cataractous lens, 77-year-old female. EQ, equatorial ring

--20 ° C with a cryostat microtome (W. Dittes, Heidelberg, F R G ) and grouped into ten lens samples with always about 130-140sections. Each sample corresponded to about 0.5 mm lens layer thickness. Care was taken that each sam- ple contained nearly the same wet weight. For each lens layer the LWW, the water-soluble (WS) protein content, and the water-insoluble (WI) protein content were mea- sured by weight. The dry weight (DW = WS + WI) and the ratio R of water-soluble to water-insoluble proteins ( R = WS/WI) were then calculated after weight determination of WS and WI. Crystallin profiles were obtained using agar- ose/0.5% linearly polymerized acrylamide gel isoelectric fo- cusing according to the method of McLachlan and Cornell

20- T R:W /w,

15-

10-

!\ / 0 I ~ ~ , r ~ ~ , , ~

Layer No. 1 2 3 /, 5 6 7 8 910 t

Region: EQ AC N PC

Fig. 3. The ratio R of the water-soluble crystallins (WS) to water- insoluble proteins (WI) as a function of lens region, calculated from Fig. 2

(1983), upgraded by riboflavin polymerization of the acryl- amide. Determination of crystallin moieties of WS crystallin (in %) was performed in a Joyce-Loebl densitometer (Bours and Hockwin 1983).

Results

Scheimpflug photographs taken with a T O P C O N SL 45 slit camera are illustrated in Fig. 1. With this technique it is possible to correlate in vivo observations of the patients' eyes before operation with biochemical data obtained from the lens after intracapsular extraction. The location of opa- cities can be accurately measured with microdensitometer image analysis of the film negatives (Hockwin et al. 1983; Shibata et al. 1984). The Scheimpflug photo o f the normal lens (Fig. 1 a) showed a hazy density in the anterior cortex and the nucleus, which is the result of normal aging. In Fig. 1 b, besides a characteristic posterior subcapsular cata- ract, a high density area was also located in the anterior cortex and the nucleus without influencing normal age-re- lated transparency. In Fig. I c, a hypermature cataractous

267

P

o[ H M [ Llr. Nlr.... R E G I O N : AC N PC

i l l

9.4

7.4

6.'7

Fig. 4. Crystallin profiles of a posterior subcapsular cataractous lens (case 2). Layers 1-10 were composed of mean 130 sections each. Mean fraction wet weight (FWW)= 18.2 mg, ~ W S = 50.7 mg, ~WI=20 .2 mg, ~DW=70 .9 mg, R=2.50. AC, anterior cortex; PC, posterior cortex; N, nucleus; ~, ~-crystallins;/?, fl-crystallins, fls, fl-slow crystallin; 7, 7-crystallins; HM, high-molecular weight crystallin material. Reference pH = isoelectric points of a reference mixture of calibration proteins (Broad pI Calibration Kit, pH 3-10, Pharmacia Fine Chemicals)

L r. N o. 1

R E G I O N : E Q AC

2 3 4 5 6 7 8 9 10 R e f . p H

N P C

Fig. 5. Protein profiles of a hypermature cataractous lens (case 3). Layers 1 10 were composed of a mean 140 sections each. Mean FWW = 15.2 rag, ~ W S = 22.8 rag, ~ W I = 25.4 mg, ~ D W = 48.2 mg, R = 0.90. For further explanation, see Figs. 4 and 5

lens is shown, in which large areas of the lens were opac]- fled.

The amounts of WS and W I proteins in lens layers 1-10 were measured by weight and calculated as the % WS and % W I proteins (Fig. 2a, b). The highest amounts of WS proteins were in the normal lens and the lowest amounts in the hypermature cataractous lens (HMC). The poster ior subcapsular cataract (PSC) showed an intermediate posi- t ion (Fig. 2a). Conversely, the amount of W I proteins (% W I - - 1 0 0 - % WS) was greatest in hypermature cata- ract and least in the normal lens (Fig. 2b). In all three lenses, the largest amounts of WS crystallins were in both cortices, in layers 1-3 and 8-10. The smallest amounts of

W I proteins were si tuated in these same layers, because this was the youngest tissue in the lens.

When the rat io R between WS and W I was calculated from Fig. 2, then the crystallins from normal lens, going from the anterior cortex (AC) to the poster ior cortex (PC), demonstra ted a U-shaped dis tr ibut ion for this ratio. F o r the hypermature cataractous lens, the ratio R remained lower th roughout the whole lens than in PSC (Fig. 3). In normal lens as well in cataractous lenses, the R at the PC was lower than at the AC. This means that in natura l aging, the PC contains more W I than the A C (Bours et al. 1978; Bouts 1980a). The PC side of the PSC lens contained much more W I than the AC. Values for the nucleus (N) were

268

P O S T E R I O R S U B C ' A P S U k A R

C A T A R A C T , 7 4 Y E A R S

a

i ~ w = mF . . . . .

H V P E R M A T U R E C A T A R A C T `

Fig. 6a and b. Densitometric tracings of isoelectric focusing gel patterns of: a 10 layers (numbered 1 to 10) from a posterior cataractous lens; b 10 layers (1 to 10) and equatorial ring (EQ) from a hypermature cataractous lens. These tracings were made directly from the blue-stained gels, shown in Figs. 4 and 5

always situated at the minimum of the U-shaped curves. In this way, small differences between the single lens layers could be detected. In all three layers, the protein content of the equatorial rings (EQ) were given for comparison. The protein content of EQ was similar to that of AC and PC (Figs. 2 and 3).

In Fig. 4, the protein profiles are shown of the fractions taken from the lens with a posterior subcapsular cataract by isoelectric focusing (IEF). The results for WS crystallins from the normal luxated lens were similar to those of Fig. 4, and are, therefore, not shown. In the 74-year-old posterior subcapsular cataractous lens, the only affected area was the PC, whereas the rest of the lens was transparent and normal in appearance (Fig. 1 b). The natural aging process in this lens was already advanced, as can be observed by

the progressive density areas seen by Scheimpflug photOgra- phy at the anterior cortex and nucleus (Fig. 1 b). Even with cataractogenic processes, age changes can be shown by the sharp decrease in 7-crystallins, the increase of fl-crystallins of lower IEP (fl0, especially at the posterior cortical side, and the increase in high-molecular-weight material due to polymerization of a-crystallins of mainly lower molecular weight (Bours and Hockwin 1983). In all lens layers, fls- crystallin remained in the WS fraction (Fig. 4). The highest amounts of fl-crystallins of high molecular weight (fix) were detected in layers 6-8 from the nucleus to the posterior cortex. The same results were observed for the fl-crystallins in the normal luxated lens (results not shown).

In the hypermature cataractous lens, the 7-crystallins were entirely absent. The fl-crystallins were significantly di- minished due to insolubilization offls-crystallin, other low- molecular-weight fl-crystallins (IEPs 6.8 to 7.00), and fl- crystallins with IEPs between 6.0 and 6.8 (tim) (Fig. 5). In the nucleus, the loss of fl-crystallins (tim) was even more prominent (layers 5 and 6) than at the AC and PC. The presence of fl-crystallins of high molecular weight (ill) was obvious in the nucleus, in layers 4 to 7.

The ratios R = WS/WI of the normal whole luxated lens, the whole subcapsular cataractous lens, and the whole hy- permature cataractous lens were 2.60, 2.50 and 0.90, respec- tively. This indicated that WS decreases and WI increases in the subcapsular catara~ztous lens, but considerably more in the hypermature cataractous lens.

Densitometry of the patterns from Figs. 4 and 5 was carried out and compared, as shown in Fig. 6. The densito- metric profiles in Fig. 6 a were taken to represent the nor- mal situation. In the hypermature cataract, the fl~i~ crystal- lins were very much diminished in concentration, especially in layers 3-10 (Fig. 6b).

The distributions of the WI crystallins as percentage of dry weight are shown in Fig. 7. The curves have a U- shaped distribution similar to that in Fig. 3. Compared to the posterior subcapsular cataract, the decrease in the fl~- and 7-crystallin moieties in the hypermature cataract was balanced by an increase in e-crystallins, especially in layers 1, 2, and 6-10 (Fig. 7a). The fl-crystallins demon- strated a substantial decline in all fl-crystallin groups, but most clearly in the tim crystallins (Fig. 7c, d). It appeared that all fl-crystallins were primarily involved in the insolubi- lization process during cataractogenesis. They were reduced by a factor of about 2 (Fig. 7 c, d).

From the low-molecular weight moiety, the fls-crystal- lins diminished drastically in the hypermature cataract (Fig. 7b) and nearly disappeared completely from layer 3- 10 (Figs. 5, 7b). Here, the comparison with the Scheimpflug made in vivo photograph is shown. In the hypermature cataractous lens (Fig. ] c), the first layer at the AC is fully transparent. In this layer the fls-crystallin is still present, but diminishes in layer 2 and disappears finally in opaque layers 4-10. The 7-crystallins, which were entirely absent in Fig. 5, were still present with about 0.6% in Fig. 4 but could not be traced by the densitometer (Fig. 6 a).

For comparison, the crystallin content of the equatorial rings was also analyzed and is represented in Figs. 5 and 7a-d. The ~- and fl-crystallin content of the equator was similar to that of the posterior cortex (Fig. 7a-c), but in the hypermature cataract the fl-crystallin content of the equator was higher for flin crystallins.

269

30-

20-

10-

0 LR, NO. REGION: AC

DW b

p s - O R Y S T A L L I N S

0

o-o_ pSc ..J

e ~ H M C

I 2 4 6 8 10 I

N PC EO

30-

20-

10-

//o DW

o - C R Y S T A L L I N S

\ .::c a

- C R Y S T A L L I N S

0--1 •

Po..-I

~o-~

5o-

4o-

3o-

20-

lo-

o-

p s c

o

\ !,,

o

C "1 7 0 -

60-

504

D W

d 13 - C R Y S T A L LI NS

H M C

40-

30-

20-

10-

o ~ , v ~ u v v v , ~0 i v u , i i ~ i i v io i o n LR N O . 2 4 6 8 1 L R . N O . 2 4 6 8 1 L R N O . 2 4 REC, I O N A C N P C E Q R E G . : A C N P C E ~ R E G : AC

[3 T o O •

./P:

'1' 'l' 1 I I I I T + ,, I N P C E O

Fig. 7a-d. The WS crystallin distribution in percentage of the dry weight (DVV) of ~-, /~- and y-crystallin components in different layers of the human cataractous lens, evaluated by densitometry of agarose/polyacrylamide isoelectric focusing gel patterns, shown in Figs. 4 and 5. PSC, posterior subcapsular cataract; HMC, hypermature cataract; Lr. No., layer number; reg., lens region; AC, anterior cortex; N, nucleus; PC, posterior cortex; EQ, equatorial ring; fir, total fl-crystallins; ill, fin, flHi are fl-crystallin fractions, in this order diminishing in molecular weight; fl+, fl-slow crystallin. [] . . . . [] Total c~-crystallins in PSC; • . . . . • total e-crystallins in HMC; o . . . . o flrcrystallins; fl.-crystallins; • . . . . • fl.rcrystallins ; /Tr-crystallins; o . . . . o flccrystallin in PSC; • . . . . • fl+-crystallin in HMC

D i s c u s s i o n

In the present study, only three human lenses were ana- lyzed. This may well be just an example of the potential usefulness for the application of the frozen-sectioning tech- nique in lens research.

The normal aging process in human lens, as well in bovine and rat lens, may generally be described as a strong increase of WI proteins, as reflected in the decrease of the ratio R = W S / W I (Zigman etal. 1970; Bours 1980b). Dische and Zil (1951) also determined the % WI in human normal and cataractous lenses. When their data were recal- culated, a ratio for human adult normal lens of 2.57 was found, and for total cataractous lenses, 0.83 and 0.65 were found. These values are in good agreement with the data presented here. Of all human lenses studied, nuclear layers 4-8 contained the highest amounts of WI proteins (Fig. 2) and lowest ratio R compared with the AC, PC,

and EQ (Fig. 3). This means that a substantial part of the aging processes occur in the lens nucleus. This supports what was reported by Bessems et al. (1983) and by Bours et al. (1976, 1978).

With this sectioning technique and subsequent isoelec- tric focusing protein analysis, protein profiles for normal or cataractous lenses from humans or experimental animals can be determined. This technique has the potential o f de- tecting minor differences in protein composition between adjacent lens layers. When used in combination with Scheimpflug photography, protein analysis of lens micro- sections facilitates a better understanding of the biochemi- cal and morphologic changes occurring in distinct regions of the lens due to aging or cataractogenesis.

Acknowledgements. The authors thank Professor M. Spitznas, Di- rector of the University of Bonn Eye Clinic, for providing human lenses. Many thanks are due to Dr. D.R. Sisk for review of the

270

manuscript and to V. Dragomirescn, M. Leyendecker, and H. Len- gert for Scheimpflug photographs. This research was supported by the Deutsche Forschungs-Gemeinschaft (Ho 249-13-3/84) and was carried out within the scope of the EEC Concerted Action on Cellular Aging and Diseases (EURAGE).

References

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Received June 28, 1985 / Accepted October 30, 1985