International Ophthalmology 16: 251-257, 1992. 9 1992 Kluwer Academic Publishers. Printed in the Netherlands.
Spectrometric investigations in ocular hypertension and early stages of primary open angle glaucoma and of low tension glaucoma multisubstance analysis Dietrich Schweitzer, Sabine Guenther, Mateusz Scibor & Martin Hammer
Department of Ophthalmology, University of Jena, Bachstrasse 18, 0-6900 Jena, Germany
Key words: glaucoma, microcirculation, reflectometry, scattering, spectra deconvolution, xanthophyll Abstract
The approximation of logarithmic difference spectra between the reflectance of the normal fundus and the fundus reflectance in different stages of glaucoma is demonstrated by a model. The influences of fundus pigments like oxihemoglobin, melanin, xanthophyll and rhodopsin as well as the intensity and the exponent of the scattered light are optimized. Glaucomatous alterations in the extinction of these pigments and of the scattering parameters are different in the macula, in the papillo-macular bundle and in the parapapillary region temporal to the optic disc. A lack of oxihemoglobin only in the papillo-macular bundle in first relative losses in the visual field function points to a damaged microcirculation in early POAG. In progressive glaucoma the extinction spectrum of xanthophyll is detectable in the papillo-macular bundle. A decreased intensity of the scattered light and an altered scattering exponent are suggestive of a damage in the nerve fiber layer at early stages of glaucoma.
Introduction
Methods for the measurement of glaucomatous alterations in the thickness of the nerve fiber layer were recently demonstrated by several authors [1-4]. The results of such geometric measurements are important in the diagnosis of glaucoma. But they are limited on changes in the number of axons in the nerve fiber bundles. More evidence for the early diagnosis of glaucoma can be obtained if functional parameters for characterizing the metabolism in the fundus are available. The glaucomatous alteration of the optical density in the nerve fiber layer was studied in the redfree light by Eikelboom et al. [5]. The statistical discrimination between normals and glaucoma suspects, based on calculated values for mean deviation of density and pattern standard deviation of density, results in sensitivity and specificity rates of 80% and 100%. The reflec-
tance from the retina is lower than normal in diffuse nerve fiber layer losses. A high value in the pattern of the density indicates localized nerve fiber layer defects. The most important region for this measurement was situated temporal to the optic disc. Spectral measurements of the nerve fiber layer in monkey eyes with artificially induced arcuate defects were presented by Knighton et al. [6]. The absolute reflectance of a damaged nerve fiber layer decreases as its thickness decreases. The separated reflectance of the intact nerve fiber layer shows a scattering influence with a scattering exponent of 2.56 to 3.05. The aim of our study is to find out alterations in the extinction of fundus pigments or of blood and of scattering properties which occur with increasing severity of glaucoma. This investigation might be a first step in the perception of pathological aspects of metabolism in glaucoma.
252
D. Schweitzer et al.
Methods and materials
Spectral measurements were made with the Jena ophthalmospectrometer whose arrangement has been described in [7]. The spectral dispersed light of a 100 W Xenon lamp illuminated a spot in the fundus. The spot diameter was 0.13 mm. Spectral measurements were done serially in steps of 10 nm within the wavelength range from 430nm to 700 nm. The measured light was related to the intensity, measured in a white standard fundus of a model eye. Such measurements result in reflectance spectra which are different at diverse fundus sites. These reflectance spectra are sum spectra. They are influenced by the absorption of substances in the fundus and the reflexion or scattering of intermediate layers. According to a previous study [8], the locations macula (M), papillo-macular bundle (PMB) and parapapillary region (PP) temporal to the optic disc were investigated. As shown in Table 1, the reflectance spectra at these locations were measured in normals, in ocular hypertension (OH), primary open angle glaucoma (POAG) and low tension glaucoma (LTG), The inclusion criteria for the selection of subjects were determined by a corrected visual acuity better than 0.6 and a refractive error lower than + - 6Dsph and + - 3Dcy I. No clinically relevant pathological alterations could be detected at the anterior segment (e.g. cataract) or in the fundus (e.g. diabetic retinopathy). All subjects had no history of relevant ocular or systemic
diseases. One randomly selected eye was measured in each proband. Only when the eyes of the same proband had different stages of glaucoma, both eyes were included in the study. The ocular hypertension was characterized by a maximal intraocular pressure higher than 21.5 mmHg at several times. The optic disc and the visual field must be normal. For the classification of POAG and of LTG, the increasing severity of the visual field losses was used. The distinctive features mainly correspond to the classification of Aulhorn and Karmeyer [9]. Stage 4 and 5 of that classification are drawn together. A relative defect was considered to be present when at least two adjacent or three non-adjacent test points had a reduction of the sensitivity of 5-9 db or a single test point had a deviation of 10 db in comparison with the normal age-corrected sensitivity [10]. The investigation of the visual field was carried out with the 'OCTOPUS 500 EZ' perimeter in program G1. As shown in [8] and in [11], the statistical comparison of reflectance spectra leads especially in the papillo-macular bundle to a good discrimination between normals and POAG patients suffering from first relative losses in the visual field (sensitivity 77.3%, specificity 79.2%). In case of first absolute defects the separation between normals and POAG patients leads to a sensitivity and a specificity of 88,9% and 86,4%. Based on the Lambert-Beer's law, an estimation of alterations in the tissue structure and in the extinction of pigments in the glaucomatous fundus
Table 1. Survey of the subjects.
Subjects
No.
Normals OH POAG 1 POAG 2 POAG 3 POAG 4 LTG1 LTG2
Papillo-macular bundle PMB
Macula MAC
33 14 35 27 7 5 7 6
No.
Age (years) Mean
Range
41.0 44.2 51.2 57.2 51.4 60.2 47.1 47.0
(23-64) (14-64) (19-72) (26-75) (26-62) (55-65) (16-66) (16-69)
22 11 24 18 5 6 7 5
Age (years)
Parapapillary (halo)-region PP No.
Mean
Range
38.5 43.8 50.5 54.1 54.6 61.8 47.1 42.6
(23-54) (14-64) (19-68) (26-72) (49~52) (55-68) (16-66) (16-53)
17 13 27 20 7 6 6 5
Age (years) Mean
Range
43.8 41.0 49.4 55.1 51.4 61.3 44.0 42.6
(23-64) (14-54) (19-72) (26-72) (26-62) (55--68) (16-58) (16-53)
Spectrometric investigations in ocular hypertension can be obtained. At an appointed location of the healthy fundus the reflectance Ro(1) can be described by a simple model as Ro(1 ) = Rg * 10 2~'E(l)*c*d * Ko,[lo/liln0
Results
In the case of glaucoma the reflectance RI(1) at the same location is (2)
In these equations are: Ro.l(1) - sum reflectance (normal, pathologic), Rg - underground reflectance (sclera), e(1) - spectral extinction coefficient, c - concentration of a pigment, d - thickness of a layer, Do,1 - optical double density (normal, pathologic), K*[lo/l~] - scattering, K - scattering intensity, 1- wavelength, n - scattering exponent. If one forms the logarithms of equations (1) and (2), and, after that, the difference of both, only glaucomatous alterations in the extinctions and in the scattering are documented in equation (3): log(Ro/R0 = + (D1-Do) + log[Ko/K0 + (no-nl)*log[lo/l~]
(3)
The parts of substance-specific extinction alterations and of a changed scattering are estimable quantitatively by means of the multisubstance analysis [7]. The multisubstance analysis is the approximation of a measured spectrum or a calculated logarithmic difference spectrum by a model function. Equation (4) shows a model function which leads to good fits of the logarithmic difference spectra between normal and glaucomatously altered reflectance spectra of the same location: log(RflR 0 = PA[0] + PA[1]*log[lo/li] - PAl2]* EHBO2 + PA[3]*t~Mel + PA[4]*Exar, t - PA[5]*ep, n
ing intensities, PA[1] is the alteration of the scattering exponent and PA[2-5] are the alterations of the substance-specific extinctions.
(1)
= Rg * 10 -D~ * Ko*[lo/li] n~
Rl(1) = Rg * 10 - m * K,*[lo/li] nl
253
(4)
In this equation, EHBO2is the extinction coefficient of oxihemoglobin [12], ~Me~is the extinction coefficient of melanin [13], eXantis the extinction coefficient of xanthophyll [14], eRh is the extinction coefficient of rhodopsin [15], PA[0] is the logarithm of the wavelength independent relation of the scatter-
The reproducibility of spectra measurements at very low light levels depends on subject specific influences, on the adjustment between subject and measuring device and on the photon noise of the measured light. The photon noise is proportional to the square root of the intensity of the measured light. This error acts as a natural limitation in measurements of the optical density in the fundus [16]. In the shortwave range (450 rim) the reproducibility is in the order of 10-15% and has values of 5-10% at 700 nm. So the reproducibility of spectral measurements in the same subject depends on the wavelength and on the fundus pigmentation. In the investigated groups of normals and patients the variation coefficient is in the order of 40% at 450nm and 25% at 700nm. The multisubstance analysis in this study is applied to the logarithmic difference spectra which were obtained from the mean reflectance spectra of all normals and of all patients suffering from the considered stage of glaucoma. These compared spectra are measured at the same fundus site. As an example, Fig. 1 shows the fit of the logarithmic difference spectrum between normals and patients in the POAG stage 1 measured in the papillomacular bundle. The model function is formed by equation 4. It is assumed that a parameter has an essential influence in fitting if its uncertainty is at least lower than 50% after optimization. The parameters PA[2-5] correspond to the pathologically altered extinction of the substances used in the model function, related to their normal values. The results of the multisubstance analysis are shown in Table 2. In this table, the first value of a parameter (extinction or scattering) is followed by the uncertainty of the corresponding parameter. The correlation coefficient is a measure of the quality which is reached by the approximation.
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D. Schweitzer et al.
Table 2. Alterations in extinctions and in scattering.
Extinction
Scattering
HBO2
Melanin
Xanthophyll
0
-0,111 0,01 - 0,475 0,116 0,351 0,077 - 0,37 0,059 0,23 0,03 0,679 0,07 - 0,69 0,19 0
0
- 0,17 0,054 - 0,09 0,042 0 - 0,21 0,02 0 - 0,09 0,08 - 0,21 0,02 0 0,18 0,05 - 0,17 0,019 - 0,36 0,06 0 -
- 0,23 0,058 - 0,36 0,06 0 0 - 0,36 0,02 0 0 0
0,32 0,089 0 0 1,05 0,1 - 0,459 0,09 0,32 0,08 0,79 0,1 0 - 0,446 0,01 0 - 0,52 0,016 0 - 0,08 0,02
0,105 0,074 0 0,21 0,046 0,12 0,03 0 0,21 0,11 0,25 0,03 0
Rhodopsin
Intensity 1,106 0,01 1,156 0,026 1,122 0,026 - 1,096 0,021 -
- 0,859 0,141 0
- 0,3 0,04 0,93 0,16
-
-
-
0 0,93 0,19
0,098 0,07 0,174 0,027 0
-
0 0,26 0,087 0 0 0,24 0,06 0
0 0
- 0,42 0,16 0
0 0,26 0,046 0
1 , 0 9 6
0,007 1,069 0,027 0
- 0,37 0,11 0
-
1,101 0,015 0 1,148 0,026 1,38 0,032 0
0 - 1,089 0,027 0,186 0,02 - 0,349 0,174 - 0,256 0,029 - 0,219 0,01
Correl. Coeff.
Fundus site
Glaucoma stage
0
0,903
MAC
OH
2,087 0,319 1,34 0,27 0,546 0,02 0
0,914
PMB
0,715
PP
0,87
MAC
0,98
PMB
2,69 0,32 2,6 0,36 1,15 0,07 0,81 0,35 0,4 0,17 1,07 0,09 1,9 0,3 2,16 0,37 0
0,92
PP
0,89
MAC
0,98
PMB
0,89
PP
0,67
MAC
0,98
PMB
0,93
PP
0,77
MAC
0,972
PMB
0,43 0,3 0 - 1,44 0,39 0,44 0,11 0
0,95
PP
0 0,67
MAC PMB
0,977
PP
0,987
MAC
0,818
PMB
0,98
PP
Exponent
0,62 0,15 0,36 0,086
POAG1
POAG2
POAG3
POAG4
LTG1
LTG2
Glaucomatous alterations in the extinction of oxihemoglobin (HBO2), melanin, xanthophyU and rhodopsin, as well as in the scattering intensity and in the scattering exponent in the fundus. The values of the extinction and of the scattering intensity are relative alterations to the healthy status. In each field the value of the optimized parameter is followed by its uncertainty. The scattering exponent in this table is the difference between the scattering exponents under normal and under pathologic conditions. In the multisubstance analysis were used the logarithmic difference spectra of the mean reflectance spectra of normals and of single pathologic stages at the locations macula, papillo-macular bundle and parapapillary region temporal to the optic disc.
Spectrometric investigations in ocular hypertension (I,~
D0-D)"
g.2tl
0.15
0 .IO
0,0"5 -
0,00 450
500
550
600
650
700 nm
Fig. 1. Approximation of the logarithmic difference spectrum of normals and P O A G 1 patients of the papillo-macular bundle (PMB) by the model according equation 4 .. + . . + . logarithmic difference spectrum, ... model function.
Discussion
In Fig. 2 the change in the extinction of the oxigensaturated blood is demonstrated depending on progressive glaucoma. A reduced extinction of blood in early stage P O A G 1 is most distinctive in the PMB. From stage P O A G 3 a strong reduction of 3 6 + - 6 % in the extinction of blood is also detectable in the PP. That means, in P O A G the retinal microcirculation seems to be earlier damaged than the ciliary system. In LTG 1 the largest reduction in the extinction of blood is detectable in the PP. As the parapapillary region is mainly supplied by the short posterior ciliary vessels, this behaviour points to a damage in the ciliary vessel system in LTG. Ciliary vasospasms could be the reason for this pathologic stage, as supposed by Flamer [17[. From the spectrometric measurements it can be concluded that already in O H the microcirculation is reduced in both the retina and the choroid. As demonstrated in Fig. 3, the pigmentation in PP shows an increasing tendency in POAG, whereas the relative extinction of melanin is decreased in the macula. The pigmentation in PP is lower in P O A G 2 than in P O A G 1. This alteration in the pigmentation is in accordance with the observations of Jonas [18]. In the PMB the pigmentation is faintly altered with progressive POAG. It is an interesting fact, that xanthophyll appears in the papillo-macular bundle in both P O A G and LTG. As shown in Fig. 4, the extinction of xanthophyll in the macula is
255
particularly enlarged in POAG 1 and P O A G 2. According to Snodderly et al. [19], who investigated the isolated retina by microspectrophotometry, xanthophyll is detectable also outside the fovea. This pigment is located mostly in the receptor axon layer and in the inner plexiform layer. In a healthy eye the nerve fiber layer acts as a scattering screen and hinders the penetration of the measuring light in these layers which contain xanthophyll. Probably in glaucoma the nerve fiber layer is thin and the reflected light is diminished also by the absorption of xanthophyll. As demonstrated in Fig. 5, the glaucomatous alteration of the scattering intensity shows different courses at the investigated fundus sites. In OH the scattering intensity is decreased at all fundus sites. In P O A G 1, the scattering intensity is reduced in both the papillo-macular bundle and the macula, but it is increased in the parapapillary region. With increasing severity of glaucoma the scattering intensity shows an increased course in the macula, whereas it has a decreased tendency in the PMB. A reduction in the wavelength-independent scattering intensity arises from a lower concentration of scattering particles in the measured volume. If the scattering is mainly caused by particles in the nerve fiber layer, a reduced wavelength-independent scattering corresponds to a thinning of this layer. This thinning is easily detectable in the papillo-macular bundle, because the retina thickness has a maximum in the middle of the distance between optic disc and fovea in normals [20]. In this area also the concentration of the ganglion cell axons has a maximum [21]. Alterations in the size of the scattering particles correspond to the difference of the scattering exponents in normal subjects and glaucoma patients. As demonstrated in Fig. 6, the scattering exponent is reduced in both the PMB and the macula. In the parapapillary region the scattering exponent is enlarged. The weakly decreased scattering exponent as measured at the PMB suggests to a loss of large scattering particles in POAG 2 and P O A G 3. The strongly reduced scattering exponent in the macula indicates a loss of small particles in the stages POAG 2 and POAG 4. The increased scattering exponent in PP is caused by the appearance of scattering particles. In POAG 1 a large number of
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D. Schweitzer et al.
Fig. 2. Fig. 3. Fig. 4. Fig. 5.
Change Change Change Change
of the of the of the of the
extinction of oxihemoglobin at different fundus sites in progressive glaucoma. extinction of melanin at different fundus sites in progressive glaucoma. extinction of xanthophyll at different fundus sites in progressive glaucoma. scattering intensity at different fundus sites in progressive glaucoma.
small particles leads to a highly increased scattering exponent in the parapapillary region. The results in this study are in agreement with the investigations of Knighton [6]. It was found by this group that the scattering in artificially induced nerve fiber losses is reduced and the scattering coefficient is in the order of 2.5 to 3.1. In scattering measurements the
influence of a turbid lense is negligible in first order. In aperture diaphragm separation between illuminating and reflection light the common measuring volume is in the fundus. The influence of age-related alterations must be taken into account. The demonstrated glaucomatous alterations in the extinction of fundus pigments and in the light scattering can be considered as a first step in the in vivo investigation of the pathologic metabolism.
References
Fig. 6. Change of the scattering exponent at different fundus sites in progressive glaucoma.
1. Caprioli J, Ortiz-Colberg R, Miller JM, Tressler Ch. Measurements of peripapillary nerve fiber layer contour in glaucoma. Am J Ophthalmol 1989; 108: 404-13. 2. Zeimer RC, Shahidi M, Mori MT, Benhamon E. In vivo evaluation of a noninvasive method to measure the retinal thickness in primates. Arch Ophthalmo11989; 107: 1006-9. 3. Cristini G, Cennamo G, Daponte P. Choroidal thickness in primary glaucoma. Ophthalmologica 1991; 202: 81-5. 4. Weinreb RN, Dreher AW, Coleman A, Quigley H, Shaw
Spectrometric investigations in ocular hypertension
5.
6.
7.
8.
9.
10.
11.
12.
B, Reiter K. Histopathologic validation of fourier-ellipsometry measurements of retinal nerve fiber layer thickness. Arch Ophthalmol 1990; 108: 557-60. Eikelboom RH, Cooper RL, Barry ChJ. A study of variance in densitometry of retinal nerve fiber layer photographs in normals and glaucoma suspects. Invest Ophthalmol Vis Sci 1990; 31: 2373-83. Knighton RW, Jacobson SG, Kemp CM. The spectral reflectance of the nerve fiber layer of the macaque retina. Invest Ophthalmol Vis Sci 1989; 30: 2393~t02. Schweitzer D, Tr6ger G, Koenigsdoerffer E, Klein S. Multisubstanzanalyse - Nachweis yon Substanzen in einzelnen Schichten des Augenhintergrundes. Fortschr Ophthalmol 1991; 88: 554-61. Schweitzer D, Klein S, Stein A, Truckenbrodt C. Glaukomdiagnostik mittels Fundusspektrometrie in Bereichen auBerhalb der Papille. Klin Mbl Augenheilk 1991; 198: 544-9. Aulhorn E, Karmeyer H. Frequency distribution in early glaucomatous visual field defects. 2nd Internat Visual Field Symposium, Tfibingen (1976). In: Doe Ophthalmol Proc Ser 1977; 14: 75-83. Gloor B, Gloor E. Die Erfal3barkeit glaukomat6ser Gesichtsfeldausffille mit dem automatischen Perimeter Octopus. Klin Mbl Augenheilk 1986; 188: 33-8. Schweitzer D, Klein S, Guenther S. Early diagnosis of glaucoma by means of fundus spectrometry. Proceedings of the International Glaucoma Symposium, Jerusalem, Israel August 18th-22nd 1991. Lemberg R, Legge JW. Hematin compounds and bile pigments. In: Richterich R, editor. Klinische Chemie 3. Auflage, Basel: S. Karger, 1971.
257
13. Gabel VP, Hillenkamp F. Visible and near infrared light absorption in pigment epithelium and choroid. In: Shimizu K, editor. XXIII. Concilium Ophthalmologicum, Excerpta Medica, Amsterdam, 1978: 658-62. 14. Wyszecki G, Stiles WS. Color science. New York: Wiley, 1967. 15. Brown PK, Wald G. Visual pigments in single rods and cones of the human retina. Science 1964; 144: 45-52. 16. Schweitzer D, Klein S, Deufrains A, Koenigsdoerffer E. Limits of fundus reflectometry. In: Nasemann J, Burk R, editors. Scanning laser ophthalmoscopy and tomography. Muenchen: Quintessenz Verlag GmbH, 1990. 17. Flamer J. Neigung zu vasospastischen Reaktionen bei Patienten mit Glaukom und glaukom~ihnlichen Erkrankungen. In: Stodtmeister R, Pillunat LE, editors. Mikrozirkulation in Gehirn und Sinnesorganen. Stuttgart: F. Enke Verlag, 1991. 18. Jonas JB, Gusek GC, Naumann GOH. Die parapapillfire Region in Normal- und Glaukomaugen. I. Planimetrische Werte von 312 Glaukom- und 125 Normalaugen. Kiln Mbl Augenheilk 1988; 193: 52-61. 19. Snodderly MD, Auran F, Delori F. The macular pigment. II. Spatial distribution in primate retinas. Invest Ophthalmol Vis Sci 1984; 25: 674-85. 20. Shahidi M, Zeimer RC, Mori M. Topography of the retinal thickness in normal subjects. Ophthalmology 1990; 97: 1120-4. 21. Glovinsky Y, Quigley HA, Brown AE, Pease ME. Macular ganglion cell loss in size dependent in experimental glaucoma. Proceedings of the International Glaucoma Symposium, Jerusalem, Israel, August 18th-22nd, 1991.
Spectrometric investigations in ocular hypertension and early stages of primary open angle glaucoma and of low tension glaucoma multisubstance analysis Dietrich Schweitzer, Sabine Guenther, Mateusz Scibor & Martin Hammer
Department of Ophthalmology, University of Jena, Bachstrasse 18, 0-6900 Jena, Germany
Key words: glaucoma, microcirculation, reflectometry, scattering, spectra deconvolution, xanthophyll Abstract
The approximation of logarithmic difference spectra between the reflectance of the normal fundus and the fundus reflectance in different stages of glaucoma is demonstrated by a model. The influences of fundus pigments like oxihemoglobin, melanin, xanthophyll and rhodopsin as well as the intensity and the exponent of the scattered light are optimized. Glaucomatous alterations in the extinction of these pigments and of the scattering parameters are different in the macula, in the papillo-macular bundle and in the parapapillary region temporal to the optic disc. A lack of oxihemoglobin only in the papillo-macular bundle in first relative losses in the visual field function points to a damaged microcirculation in early POAG. In progressive glaucoma the extinction spectrum of xanthophyll is detectable in the papillo-macular bundle. A decreased intensity of the scattered light and an altered scattering exponent are suggestive of a damage in the nerve fiber layer at early stages of glaucoma.
Introduction
Methods for the measurement of glaucomatous alterations in the thickness of the nerve fiber layer were recently demonstrated by several authors [1-4]. The results of such geometric measurements are important in the diagnosis of glaucoma. But they are limited on changes in the number of axons in the nerve fiber bundles. More evidence for the early diagnosis of glaucoma can be obtained if functional parameters for characterizing the metabolism in the fundus are available. The glaucomatous alteration of the optical density in the nerve fiber layer was studied in the redfree light by Eikelboom et al. [5]. The statistical discrimination between normals and glaucoma suspects, based on calculated values for mean deviation of density and pattern standard deviation of density, results in sensitivity and specificity rates of 80% and 100%. The reflec-
tance from the retina is lower than normal in diffuse nerve fiber layer losses. A high value in the pattern of the density indicates localized nerve fiber layer defects. The most important region for this measurement was situated temporal to the optic disc. Spectral measurements of the nerve fiber layer in monkey eyes with artificially induced arcuate defects were presented by Knighton et al. [6]. The absolute reflectance of a damaged nerve fiber layer decreases as its thickness decreases. The separated reflectance of the intact nerve fiber layer shows a scattering influence with a scattering exponent of 2.56 to 3.05. The aim of our study is to find out alterations in the extinction of fundus pigments or of blood and of scattering properties which occur with increasing severity of glaucoma. This investigation might be a first step in the perception of pathological aspects of metabolism in glaucoma.
252
D. Schweitzer et al.
Methods and materials
Spectral measurements were made with the Jena ophthalmospectrometer whose arrangement has been described in [7]. The spectral dispersed light of a 100 W Xenon lamp illuminated a spot in the fundus. The spot diameter was 0.13 mm. Spectral measurements were done serially in steps of 10 nm within the wavelength range from 430nm to 700 nm. The measured light was related to the intensity, measured in a white standard fundus of a model eye. Such measurements result in reflectance spectra which are different at diverse fundus sites. These reflectance spectra are sum spectra. They are influenced by the absorption of substances in the fundus and the reflexion or scattering of intermediate layers. According to a previous study [8], the locations macula (M), papillo-macular bundle (PMB) and parapapillary region (PP) temporal to the optic disc were investigated. As shown in Table 1, the reflectance spectra at these locations were measured in normals, in ocular hypertension (OH), primary open angle glaucoma (POAG) and low tension glaucoma (LTG), The inclusion criteria for the selection of subjects were determined by a corrected visual acuity better than 0.6 and a refractive error lower than + - 6Dsph and + - 3Dcy I. No clinically relevant pathological alterations could be detected at the anterior segment (e.g. cataract) or in the fundus (e.g. diabetic retinopathy). All subjects had no history of relevant ocular or systemic
diseases. One randomly selected eye was measured in each proband. Only when the eyes of the same proband had different stages of glaucoma, both eyes were included in the study. The ocular hypertension was characterized by a maximal intraocular pressure higher than 21.5 mmHg at several times. The optic disc and the visual field must be normal. For the classification of POAG and of LTG, the increasing severity of the visual field losses was used. The distinctive features mainly correspond to the classification of Aulhorn and Karmeyer [9]. Stage 4 and 5 of that classification are drawn together. A relative defect was considered to be present when at least two adjacent or three non-adjacent test points had a reduction of the sensitivity of 5-9 db or a single test point had a deviation of 10 db in comparison with the normal age-corrected sensitivity [10]. The investigation of the visual field was carried out with the 'OCTOPUS 500 EZ' perimeter in program G1. As shown in [8] and in [11], the statistical comparison of reflectance spectra leads especially in the papillo-macular bundle to a good discrimination between normals and POAG patients suffering from first relative losses in the visual field (sensitivity 77.3%, specificity 79.2%). In case of first absolute defects the separation between normals and POAG patients leads to a sensitivity and a specificity of 88,9% and 86,4%. Based on the Lambert-Beer's law, an estimation of alterations in the tissue structure and in the extinction of pigments in the glaucomatous fundus
Table 1. Survey of the subjects.
Subjects
No.
Normals OH POAG 1 POAG 2 POAG 3 POAG 4 LTG1 LTG2
Papillo-macular bundle PMB
Macula MAC
33 14 35 27 7 5 7 6
No.
Age (years) Mean
Range
41.0 44.2 51.2 57.2 51.4 60.2 47.1 47.0
(23-64) (14-64) (19-72) (26-75) (26-62) (55-65) (16-66) (16-69)
22 11 24 18 5 6 7 5
Age (years)
Parapapillary (halo)-region PP No.
Mean
Range
38.5 43.8 50.5 54.1 54.6 61.8 47.1 42.6
(23-54) (14-64) (19-68) (26-72) (49~52) (55-68) (16-66) (16-53)
17 13 27 20 7 6 6 5
Age (years) Mean
Range
43.8 41.0 49.4 55.1 51.4 61.3 44.0 42.6
(23-64) (14-54) (19-72) (26-72) (26-62) (55--68) (16-58) (16-53)
Spectrometric investigations in ocular hypertension can be obtained. At an appointed location of the healthy fundus the reflectance Ro(1) can be described by a simple model as Ro(1 ) = Rg * 10 2~'E(l)*c*d * Ko,[lo/liln0
Results
In the case of glaucoma the reflectance RI(1) at the same location is (2)
In these equations are: Ro.l(1) - sum reflectance (normal, pathologic), Rg - underground reflectance (sclera), e(1) - spectral extinction coefficient, c - concentration of a pigment, d - thickness of a layer, Do,1 - optical double density (normal, pathologic), K*[lo/l~] - scattering, K - scattering intensity, 1- wavelength, n - scattering exponent. If one forms the logarithms of equations (1) and (2), and, after that, the difference of both, only glaucomatous alterations in the extinctions and in the scattering are documented in equation (3): log(Ro/R0 = + (D1-Do) + log[Ko/K0 + (no-nl)*log[lo/l~]
(3)
The parts of substance-specific extinction alterations and of a changed scattering are estimable quantitatively by means of the multisubstance analysis [7]. The multisubstance analysis is the approximation of a measured spectrum or a calculated logarithmic difference spectrum by a model function. Equation (4) shows a model function which leads to good fits of the logarithmic difference spectra between normal and glaucomatously altered reflectance spectra of the same location: log(RflR 0 = PA[0] + PA[1]*log[lo/li] - PAl2]* EHBO2 + PA[3]*t~Mel + PA[4]*Exar, t - PA[5]*ep, n
ing intensities, PA[1] is the alteration of the scattering exponent and PA[2-5] are the alterations of the substance-specific extinctions.
(1)
= Rg * 10 -D~ * Ko*[lo/li] n~
Rl(1) = Rg * 10 - m * K,*[lo/li] nl
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(4)
In this equation, EHBO2is the extinction coefficient of oxihemoglobin [12], ~Me~is the extinction coefficient of melanin [13], eXantis the extinction coefficient of xanthophyll [14], eRh is the extinction coefficient of rhodopsin [15], PA[0] is the logarithm of the wavelength independent relation of the scatter-
The reproducibility of spectra measurements at very low light levels depends on subject specific influences, on the adjustment between subject and measuring device and on the photon noise of the measured light. The photon noise is proportional to the square root of the intensity of the measured light. This error acts as a natural limitation in measurements of the optical density in the fundus [16]. In the shortwave range (450 rim) the reproducibility is in the order of 10-15% and has values of 5-10% at 700 nm. So the reproducibility of spectral measurements in the same subject depends on the wavelength and on the fundus pigmentation. In the investigated groups of normals and patients the variation coefficient is in the order of 40% at 450nm and 25% at 700nm. The multisubstance analysis in this study is applied to the logarithmic difference spectra which were obtained from the mean reflectance spectra of all normals and of all patients suffering from the considered stage of glaucoma. These compared spectra are measured at the same fundus site. As an example, Fig. 1 shows the fit of the logarithmic difference spectrum between normals and patients in the POAG stage 1 measured in the papillomacular bundle. The model function is formed by equation 4. It is assumed that a parameter has an essential influence in fitting if its uncertainty is at least lower than 50% after optimization. The parameters PA[2-5] correspond to the pathologically altered extinction of the substances used in the model function, related to their normal values. The results of the multisubstance analysis are shown in Table 2. In this table, the first value of a parameter (extinction or scattering) is followed by the uncertainty of the corresponding parameter. The correlation coefficient is a measure of the quality which is reached by the approximation.
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Table 2. Alterations in extinctions and in scattering.
Extinction
Scattering
HBO2
Melanin
Xanthophyll
0
-0,111 0,01 - 0,475 0,116 0,351 0,077 - 0,37 0,059 0,23 0,03 0,679 0,07 - 0,69 0,19 0
0
- 0,17 0,054 - 0,09 0,042 0 - 0,21 0,02 0 - 0,09 0,08 - 0,21 0,02 0 0,18 0,05 - 0,17 0,019 - 0,36 0,06 0 -
- 0,23 0,058 - 0,36 0,06 0 0 - 0,36 0,02 0 0 0
0,32 0,089 0 0 1,05 0,1 - 0,459 0,09 0,32 0,08 0,79 0,1 0 - 0,446 0,01 0 - 0,52 0,016 0 - 0,08 0,02
0,105 0,074 0 0,21 0,046 0,12 0,03 0 0,21 0,11 0,25 0,03 0
Rhodopsin
Intensity 1,106 0,01 1,156 0,026 1,122 0,026 - 1,096 0,021 -
- 0,859 0,141 0
- 0,3 0,04 0,93 0,16
-
-
-
0 0,93 0,19
0,098 0,07 0,174 0,027 0
-
0 0,26 0,087 0 0 0,24 0,06 0
0 0
- 0,42 0,16 0
0 0,26 0,046 0
1 , 0 9 6
0,007 1,069 0,027 0
- 0,37 0,11 0
-
1,101 0,015 0 1,148 0,026 1,38 0,032 0
0 - 1,089 0,027 0,186 0,02 - 0,349 0,174 - 0,256 0,029 - 0,219 0,01
Correl. Coeff.
Fundus site
Glaucoma stage
0
0,903
MAC
OH
2,087 0,319 1,34 0,27 0,546 0,02 0
0,914
PMB
0,715
PP
0,87
MAC
0,98
PMB
2,69 0,32 2,6 0,36 1,15 0,07 0,81 0,35 0,4 0,17 1,07 0,09 1,9 0,3 2,16 0,37 0
0,92
PP
0,89
MAC
0,98
PMB
0,89
PP
0,67
MAC
0,98
PMB
0,93
PP
0,77
MAC
0,972
PMB
0,43 0,3 0 - 1,44 0,39 0,44 0,11 0
0,95
PP
0 0,67
MAC PMB
0,977
PP
0,987
MAC
0,818
PMB
0,98
PP
Exponent
0,62 0,15 0,36 0,086
POAG1
POAG2
POAG3
POAG4
LTG1
LTG2
Glaucomatous alterations in the extinction of oxihemoglobin (HBO2), melanin, xanthophyU and rhodopsin, as well as in the scattering intensity and in the scattering exponent in the fundus. The values of the extinction and of the scattering intensity are relative alterations to the healthy status. In each field the value of the optimized parameter is followed by its uncertainty. The scattering exponent in this table is the difference between the scattering exponents under normal and under pathologic conditions. In the multisubstance analysis were used the logarithmic difference spectra of the mean reflectance spectra of normals and of single pathologic stages at the locations macula, papillo-macular bundle and parapapillary region temporal to the optic disc.
Spectrometric investigations in ocular hypertension (I,~
D0-D)"
g.2tl
0.15
0 .IO
0,0"5 -
0,00 450
500
550
600
650
700 nm
Fig. 1. Approximation of the logarithmic difference spectrum of normals and P O A G 1 patients of the papillo-macular bundle (PMB) by the model according equation 4 .. + . . + . logarithmic difference spectrum, ... model function.
Discussion
In Fig. 2 the change in the extinction of the oxigensaturated blood is demonstrated depending on progressive glaucoma. A reduced extinction of blood in early stage P O A G 1 is most distinctive in the PMB. From stage P O A G 3 a strong reduction of 3 6 + - 6 % in the extinction of blood is also detectable in the PP. That means, in P O A G the retinal microcirculation seems to be earlier damaged than the ciliary system. In LTG 1 the largest reduction in the extinction of blood is detectable in the PP. As the parapapillary region is mainly supplied by the short posterior ciliary vessels, this behaviour points to a damage in the ciliary vessel system in LTG. Ciliary vasospasms could be the reason for this pathologic stage, as supposed by Flamer [17[. From the spectrometric measurements it can be concluded that already in O H the microcirculation is reduced in both the retina and the choroid. As demonstrated in Fig. 3, the pigmentation in PP shows an increasing tendency in POAG, whereas the relative extinction of melanin is decreased in the macula. The pigmentation in PP is lower in P O A G 2 than in P O A G 1. This alteration in the pigmentation is in accordance with the observations of Jonas [18]. In the PMB the pigmentation is faintly altered with progressive POAG. It is an interesting fact, that xanthophyll appears in the papillo-macular bundle in both P O A G and LTG. As shown in Fig. 4, the extinction of xanthophyll in the macula is
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particularly enlarged in POAG 1 and P O A G 2. According to Snodderly et al. [19], who investigated the isolated retina by microspectrophotometry, xanthophyll is detectable also outside the fovea. This pigment is located mostly in the receptor axon layer and in the inner plexiform layer. In a healthy eye the nerve fiber layer acts as a scattering screen and hinders the penetration of the measuring light in these layers which contain xanthophyll. Probably in glaucoma the nerve fiber layer is thin and the reflected light is diminished also by the absorption of xanthophyll. As demonstrated in Fig. 5, the glaucomatous alteration of the scattering intensity shows different courses at the investigated fundus sites. In OH the scattering intensity is decreased at all fundus sites. In P O A G 1, the scattering intensity is reduced in both the papillo-macular bundle and the macula, but it is increased in the parapapillary region. With increasing severity of glaucoma the scattering intensity shows an increased course in the macula, whereas it has a decreased tendency in the PMB. A reduction in the wavelength-independent scattering intensity arises from a lower concentration of scattering particles in the measured volume. If the scattering is mainly caused by particles in the nerve fiber layer, a reduced wavelength-independent scattering corresponds to a thinning of this layer. This thinning is easily detectable in the papillo-macular bundle, because the retina thickness has a maximum in the middle of the distance between optic disc and fovea in normals [20]. In this area also the concentration of the ganglion cell axons has a maximum [21]. Alterations in the size of the scattering particles correspond to the difference of the scattering exponents in normal subjects and glaucoma patients. As demonstrated in Fig. 6, the scattering exponent is reduced in both the PMB and the macula. In the parapapillary region the scattering exponent is enlarged. The weakly decreased scattering exponent as measured at the PMB suggests to a loss of large scattering particles in POAG 2 and P O A G 3. The strongly reduced scattering exponent in the macula indicates a loss of small particles in the stages POAG 2 and POAG 4. The increased scattering exponent in PP is caused by the appearance of scattering particles. In POAG 1 a large number of
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Fig. 2. Fig. 3. Fig. 4. Fig. 5.
Change Change Change Change
of the of the of the of the
extinction of oxihemoglobin at different fundus sites in progressive glaucoma. extinction of melanin at different fundus sites in progressive glaucoma. extinction of xanthophyll at different fundus sites in progressive glaucoma. scattering intensity at different fundus sites in progressive glaucoma.
small particles leads to a highly increased scattering exponent in the parapapillary region. The results in this study are in agreement with the investigations of Knighton [6]. It was found by this group that the scattering in artificially induced nerve fiber losses is reduced and the scattering coefficient is in the order of 2.5 to 3.1. In scattering measurements the
influence of a turbid lense is negligible in first order. In aperture diaphragm separation between illuminating and reflection light the common measuring volume is in the fundus. The influence of age-related alterations must be taken into account. The demonstrated glaucomatous alterations in the extinction of fundus pigments and in the light scattering can be considered as a first step in the in vivo investigation of the pathologic metabolism.
References
Fig. 6. Change of the scattering exponent at different fundus sites in progressive glaucoma.
1. Caprioli J, Ortiz-Colberg R, Miller JM, Tressler Ch. Measurements of peripapillary nerve fiber layer contour in glaucoma. Am J Ophthalmol 1989; 108: 404-13. 2. Zeimer RC, Shahidi M, Mori MT, Benhamon E. In vivo evaluation of a noninvasive method to measure the retinal thickness in primates. Arch Ophthalmo11989; 107: 1006-9. 3. Cristini G, Cennamo G, Daponte P. Choroidal thickness in primary glaucoma. Ophthalmologica 1991; 202: 81-5. 4. Weinreb RN, Dreher AW, Coleman A, Quigley H, Shaw
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B, Reiter K. Histopathologic validation of fourier-ellipsometry measurements of retinal nerve fiber layer thickness. Arch Ophthalmol 1990; 108: 557-60. Eikelboom RH, Cooper RL, Barry ChJ. A study of variance in densitometry of retinal nerve fiber layer photographs in normals and glaucoma suspects. Invest Ophthalmol Vis Sci 1990; 31: 2373-83. Knighton RW, Jacobson SG, Kemp CM. The spectral reflectance of the nerve fiber layer of the macaque retina. Invest Ophthalmol Vis Sci 1989; 30: 2393~t02. Schweitzer D, Tr6ger G, Koenigsdoerffer E, Klein S. Multisubstanzanalyse - Nachweis yon Substanzen in einzelnen Schichten des Augenhintergrundes. Fortschr Ophthalmol 1991; 88: 554-61. Schweitzer D, Klein S, Stein A, Truckenbrodt C. Glaukomdiagnostik mittels Fundusspektrometrie in Bereichen auBerhalb der Papille. Klin Mbl Augenheilk 1991; 198: 544-9. Aulhorn E, Karmeyer H. Frequency distribution in early glaucomatous visual field defects. 2nd Internat Visual Field Symposium, Tfibingen (1976). In: Doe Ophthalmol Proc Ser 1977; 14: 75-83. Gloor B, Gloor E. Die Erfal3barkeit glaukomat6ser Gesichtsfeldausffille mit dem automatischen Perimeter Octopus. Klin Mbl Augenheilk 1986; 188: 33-8. Schweitzer D, Klein S, Guenther S. Early diagnosis of glaucoma by means of fundus spectrometry. Proceedings of the International Glaucoma Symposium, Jerusalem, Israel August 18th-22nd 1991. Lemberg R, Legge JW. Hematin compounds and bile pigments. In: Richterich R, editor. Klinische Chemie 3. Auflage, Basel: S. Karger, 1971.
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13. Gabel VP, Hillenkamp F. Visible and near infrared light absorption in pigment epithelium and choroid. In: Shimizu K, editor. XXIII. Concilium Ophthalmologicum, Excerpta Medica, Amsterdam, 1978: 658-62. 14. Wyszecki G, Stiles WS. Color science. New York: Wiley, 1967. 15. Brown PK, Wald G. Visual pigments in single rods and cones of the human retina. Science 1964; 144: 45-52. 16. Schweitzer D, Klein S, Deufrains A, Koenigsdoerffer E. Limits of fundus reflectometry. In: Nasemann J, Burk R, editors. Scanning laser ophthalmoscopy and tomography. Muenchen: Quintessenz Verlag GmbH, 1990. 17. Flamer J. Neigung zu vasospastischen Reaktionen bei Patienten mit Glaukom und glaukom~ihnlichen Erkrankungen. In: Stodtmeister R, Pillunat LE, editors. Mikrozirkulation in Gehirn und Sinnesorganen. Stuttgart: F. Enke Verlag, 1991. 18. Jonas JB, Gusek GC, Naumann GOH. Die parapapillfire Region in Normal- und Glaukomaugen. I. Planimetrische Werte von 312 Glaukom- und 125 Normalaugen. Kiln Mbl Augenheilk 1988; 193: 52-61. 19. Snodderly MD, Auran F, Delori F. The macular pigment. II. Spatial distribution in primate retinas. Invest Ophthalmol Vis Sci 1984; 25: 674-85. 20. Shahidi M, Zeimer RC, Mori M. Topography of the retinal thickness in normal subjects. Ophthalmology 1990; 97: 1120-4. 21. Glovinsky Y, Quigley HA, Brown AE, Pease ME. Macular ganglion cell loss in size dependent in experimental glaucoma. Proceedings of the International Glaucoma Symposium, Jerusalem, Israel, August 18th-22nd, 1991.
