Acta Ophthalmologica 2014
Retinal vessel oxygen saturation and its correlation with structural changes in retinitis pigmentosa Cengiz T€ urksever,1,* Christophe Valmaggia,2,* Selim Org€ ul,1 Daniel F. Schorderet,3,4,5 Josef Flammer1 1 and Margarita G. Todorova 1
Department of Ophthalmology, University of Basel, Basel, Switzerland Department of Ophthalmology, Cantonal Hospital, St. Gallen, Switzerland 3 IRO – Institut for Research in Ophthalmology, Sion, Switzerland 4 Department of Ophthalmology, University of Lausanne, Lausanne, Switzerland 5 School of Life Sciences, Federal Institute of Technology, Lausanne, Switzerland 2
ABSTRACT. Purpose: To study the influence of retinal structural changes on oxygen saturation in retinitis pigmentosa (RP) patients. Methods: Oximetry measurements were performed on 21 eyes of 11 RP patients and compared to 24 eyes of 12 controls. Retinal oxygen saturation was measured in all major retinal arterioles (A-SO2) and venules (V-SO2) with an oximetry unit of the retinal vessel analyser (IMEDOS Systems UG, Jena, Germany). Oximetry data were compared with morphological changes measured by Cirrus optical coherence tomography (OCT) (Carl Zeiss Meditec, Dublin, CA, USA, macular thickness protocol). Results: In RP patients, the retinal A-SO2 and V-SO2 levels were higher at 99.3% (p = 0.001, ANOVA based on mixed-effects model) and 66.8% (p < 0.001), respectively, and the difference between the two (A-V SO2) was lower at 32.5% (p < 0.001), when compared to the control group (92.4%; 54.0%; 38.4%, respectively). With the RP group, the A-V SO2 correlated positively, not only with central macular thickness, but also with retinal thickness, in zones 2 and 3 (p = 0.006, p = 0.007, p = 0.014). Conclusion: These data indicate that oxygen metabolism was altered in RP patients. Based on our preliminary results, retinal vessel saturation correlated with structural alterations in RP. This method could be valuable in monitoring disease progression and evaluating a potential therapeutic response. Key words: inner/outer segment junction line – optical coherence tomography – oxygen – retinal vessel oximetry – retinitis pigmentosa – saturation
Acta Ophthalmol. 2014: 92: 454–460 ª 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
doi: 10.1111/aos.12379 *These authors contributed equally to this work
Introduction Retinitis pigmentosa (RP), the most commonly recognized hereditary retinal dystrophy of the outer retina, occurs worldwide with a frequency of
454
1/4000 (Ammann et al. 1965; Puech et al. 1991; Hamel 2006). The disease is well known for its genetic heterogeneity and common clinical symptoms: initial night blindness, centripetal progression of visual field constriction resulting in
tunnel vision and reduced central vision at the end stage (Hamel 2006; Berger et al. 2010). Gene mutations expressed in photoreceptors or retinal pigment epithelium (Berger et al. 2010) trigger neuronal degeneration through various metabolic pathways, including apoptosis (Marc & Jones 2003; Cottet & Schorderet 2009). Following atrophy, the inner retina responds with secondary remodelling, proximal intraretinal pigment migration as well as neuronal and glial migration (Marc & Jones 2003; Marc et al. 2003; Jones & Marc 2005). As dystrophy progresses, the optic disc becomes pale and arterial vessels attenuate (Marc & Jones 2003; Marc et al. 2003; Hamel 2006; Sahaboglu et al. 2013). Optical coherence tomography (OCT) has enabled the identification of different patterns of structural involvement in RP. In stages with high retinal pigment epithelium metabolic demand due to the loss of photoreceptor integrity, distortions of retinal microstructure and/or macular oedema have been observed (Sandberg et al. 2005; Iriyama & Tanagi 2012; Mitamura et al. 2012). Further OCT studies have confirmed a correlation between retinal microstructure and visual function in RP patients, in particular at the junction between the inner and outer segments, the so-called inner/outer segment junction (IS/OS) line (Aizawa et al. 2009; Kim et al. 2013; Mitamura et al. 2013). Retinal hemodynamics impairment in the context of reduced arterial and
Acta Ophthalmologica 2014
venous blood velocities has been confirmed, not only in the central retina (Li et al. 2009) but also in the periphery (Cellini et al. 1997; Beutelspacher et al. 2011). Quantitative analysis of retinal vessel attenuation has demonstrated a significant correlation with the severity of retinopathy (Cellini et al. 1997; Ma et al. 2012). Even in early stages, RP patients have shown reduced systolic velocities in posterior ciliary arteries, as measured by colour Doppler (Cellini et al. 1997; Ma et al. 2012) and confirmed by quantitative blood-flow MRI measurements (Zhang et al. 2013). Retinal oximetry has been established to analyse retinal vessel oxygen saturation (Schweitzer et al. 2001; Geirsdottir et al. 2012; Hardarson 2013). Until now, this method has only been studied in few vascular entities and in glaucoma (Hammer et al. 2009; Olafsdottir et al. 2011; Boeckaert et al. 2012; Hardarson & Stef ansson 2012; Hardarson 2013; Hardarson et al. 2013; Vandewalle et al. 2013b). When structural changes occur, including neurovascular remodelling as in the case of RP, modification of retinal blood oxygenation is to be expected. In this study, we aimed to investigate the oxygen saturation in retinal arterioles and venules in RP patients. Also, to investigate the oxygen saturation of the retinal vessels as an indicator of metabolic retinal dysfunction in RP patients, we correlated the oximetry data with the morphological changes measured by OCT. If the degree of retinal vessel saturation disturbances correlates with the structural alterations in RP, the method would help in understanding the disease. It would also add new tools in monitoring disease progression and evaluating potential therapeutic responses.
that included refraction, best-corrected visual acuity (Snellen charts), intraocular pressure examination by applanation (Goldmann tonometer), slit-lamp examination, biomicroscopy and fundoscopy. Retinitis pigmentosa patients from our diagnostic unit were enrolled in the study after informed consent. Their clinical phenotype was characterized following clinical and electrophysiological examination. Inclusion criteria for RP patients were as follows: Caucasian origin, characteristic fundoscopic findings of RP, reduced or non-detectable a- and b-wave amplitudes of the scotopic full-field ERG. Inclusion criteria for controls were as follows: Caucasian origin, best-corrected Snellen visual acuity at distance ≥0.8. Exclusion criteria for patients and controls were as follows: above inclusion criteria not fulfilled; presence of ocular and/or systemic pathology that could influence the OCT and retinal vessels oximetry data; unstable fixation; fundus oximetry images with inadequate quality; and refusal to participate in the study. To evaluate oxygen saturation of the retinal vessels, we used the spectrophotometric oximetry tool of the retinal vessel analyser, coupled to a fundus camera FF450 (IMEDOS Systems UG, Jena, Germany; Carl Zeiss Meditec AG, Jena, Germany) and to a digital camera system (KY-F75; JVC Inc., Yokohama, Japan). This digital spectrophotometric imaging system allows for valuable assessment of retinal vessel oxygenation (Hammer et al. 2002; Hardarson 2013). Fundus images were
taken with the help of a dual band-pass filter imaging system with two different transmission bands, the first a nonsensitive to oxygen 548 nm wavelength and the second oxygen-sensitive with 610-nm wavelength filters. In this way, two retinal images were taken simultaneously at two different wavelengths. The software compared the two photographs, according to the optical density ratio (ODR): the colour-coded oxygen saturation of the retinal vessels and the photometric vessel diameter alterations along the selected vessel segment. Specialized software (VISUALIS; IMEDOS Systems UG, Jena, Germany) incorporated in the retinal vessel analyzer allows then for automatic evaluation of the acquired digital retinal images. Only images fulfilling the following criteria were further analysed: optic disc located at the image centre (Man et al. 2013); clearness of the images; appropriate brightness. VESSELMAP software incorporated in Imedos system enables to correct the effect of vessel diameter on retinal vessels saturation measurements. Imaging
Patients and controls were adapted for 10 min to mesopic conditions as described elsewhere (Hammer et al. 2002; Hardarson 2013). Images of both eyes were obtained, starting with the right eye. At least four images were taken and more images added if needed. The oxygen saturation was measured in all major retinal arterioles (A-SO2) and venules (V-SO2) within a distance of 0.5–1.0 optic disc diameter from the optic disc edge. In addition,
Material and Methods This pilot study adhered to the Tenets of the Declaration of Helsinki. Informed consent was obtained from all participants before performing oximetry imaging after explanation of the nature and possible consequences of the study. Subjects
All controls as well as RP patients had a complete ophthalmic examination
(A)
(B)
Fig. 1. Fundus oximetry images (map) of a control (A, left eye) and of a patient with retinitis pigmentosa (RP) (B, right eye). Oxygen saturation is measured along the main retinal vessels, within 0.5–1.0 optic disc diameter distance from the optic disc and up to the first vessel branching. The colours in the oximetry images indicate the relative oxygen saturation (%) in retinal vessels. Note the increased A-SO2 and V-SO2 in RP patients.
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Acta Ophthalmologica 2014
we computed the arterio-venous difference (A-V SO2), known to be proportional to the oxygen saturation. Examples of oximetry readings of a control individual and of an RP patient are shown in Fig. 1A,B, respectively. For evaluation of in vivo retinal structures (Sakata et al. 2009), we performed a frequency domain OCT on Cirrus OCT (Carl Zeiss Meditec. Dublin, CA, USA). The OCT images were taken using a macular thickness (Macular Cube 512 9 128) and high definition image protocol (HD 5 Line Raster). The software of the Cirrus OCT provided a macular thickness map divided into nine subfields. To analyse the data as an indicator for metabolic retinal dysfunction, in particular in RP patients where a significant constriction of visual fields occurs, we computed the mean macular thickness in three areas: zone 1 at 3°; zone 2 between 3° and 8°; and zone 3 between 8° and 15° (Fig. 2A,B, centre). The highly reflective line above the retinal pigment epithelium (RPE) layer indicates the photoreceptor IS/OS. The presence of an intact IS/OS line in OCT images is an indicator of proper photoreceptor integrity and function (Gloesmann et al. 2003; van Velthoven
et al. 2007). In RP patients, the integrity of the IS/OS line has been well associated with better visual function, brighter fundus autofluorescence parafoveolar density and higher retinal sensitivity (Iriyama & Tanagi 2012; Mitamura et al. 2012, 2013). For this reason, we measured the length of the intact IS/OS line for every eye on HD 5 Line Raster OCT images using a measurement tool from the Cirrus OCT (Fig. 2B, right). In addition, we assessed the relationship between retinal vessel oxygen saturation values and the length of intact IS/OS line, as well as the OCT data averaged in three zones, as described above. Statistical analysis
For statistical analysis of Oximetry data, ANOVA based on mixed-effects model, which allows taking the dependency of the left and right eye in the same subject into account, was applied. p-values of less than or equal to 0.05 were considered statistically significant. To correlate the oximetry data (the mean A-SO2; V-SO2; A-V SO2 %) to the OCT measurements (the mean macula thickness and retinal thickness
(lm) averaged in circles: zone 1: at 3°; zone 2: 3°–8°; zone 3: 8°–15°), a linear mixed-effects model, suitable for the repeated measurements data, was used. To predict the effect of the structural alterations on oximetry measurements, the eye and the group effects were taken into account, where the eye and the group were treated as a fixed factors and the subject as a random factor.
Results A total of 11 patients (5 ♂: 6 ♀; 21 eyes) with clinical and electrophysiological evidence of RP were enrolled in the study. One patient was evaluated only monocularly due to post-traumatic corneal haze with irregular astigmatism and aphakia in the other eye. Twelve age-matched controls (3 ♂: 9 ♀; 24 eyes) were recruited from our data bank. Demographic data of our patients and controls are given in Table 1. Oximetry data
The reproducibility of the oximetry measurements was excellent and comparable to previous studies on control
(A)
(B) Fig. 2. Represents an example of optical coherence tomography (OCT) measurements of a control (A) and of a retinitis pigmentosa (RP) patient (B). We averaged the mean macular thickness in three circles: zone 1 at 3°; zone 2 between 3° and 8°; and zone 3 between 8° and 15°, as exemplified in A, in the middle. The IS/OS line is only partially visible in the presented RP patient (B, right). The intact area, visible only at the foveal centre, is labelled with red line. Outside the measured area, the inner/outer segment junction (IS/OS) line is completely absent and the retinal thickness is reduced.
456
Acta Ophthalmologica 2014
Table 1. Represents the demographic data for the studied groups.
Demographic characteristics
Controls
Retinitis pigmentosa patients
Number of patients (eyes examined) Age, mean SD, years Sex (♂:♀) Snellen BCVA, mean SD
12 (24) 36.08 (10.69) 3:9 1.0 (0.000)
11 (21) 35.33 (10.81) 5:6 0.49 (0.30)
eyes (Hammer et al. 2008; Geirsdottir et al. 2012; Lasta et al. 2012). In the control group, we calculated a test– retest mean standard deviation for A-SO2 (1.52%) and for V-SO2 (2.50%). In the RP group, the measurements also showed a reliable reproducibility: the mean test–retest SD values were as follows: for the A-SO2 1.87% and for the V-SO2 2.27%. Comparison of retinal vessel oxygen measurements in groups
We then compared the retinal vessel oxygen saturation measurements in controls against those of RP patients. In controls, the SO2 values were as follows: the mean A-SO2 and V-SO2 of the retina were measured at 92.4% and 54.0%, respectively. The mean A-V SO2 in controls was 38.4%. In RP patients, the retinal A-SO2 and V-SO2 levels increased significantly to 99.3% (p = 0.001) and 66.8% (p < 0.001). In consequence, the A-V SO2 values decreased to 32.5% (p < 0.001; Fig. 3A–C; Table 2). There was no statistically significant difference between the right and left eye analysed in each group separately (p > 0.507, ANOVA, based on mixed-effects model; Table 2). Retinal vessel diameter and its influence on oximetry measurements
To rule out the effect of retinal vessel diameter on oxygen saturation measurements (Beach et al. 1999; Vandewalle et al. 2013a), even having a VESSELMAP software incorporated in the Imedos system, we evaluated the relationship between the A-SO2 and the diameter of arterioles, as well as between the V-SO2 and the diameter of venules. Results of our study showed no influence of retinal vessel diameters on vessel SO2 measurements (linear mixed-effects model). This was the case for the venous measurements in the
p-values (one-way ANOVA)
0.816 0.000
combined group (p = 0.527), as well as separately in controls (p = 0.298) and in RP group (p = 0.159). A-SO2 neither in the combined group, nor in controls or RP patients correlated with the diameter of arterioles (p > 0.137).
(A)
(B)
Correlation between the OCT data (averaged in circles) and the oximetry data
To correlate the oximetry measurements in RP patients with structural measurements, the oximetry data were further compared to the OCTs. Asymmetric and unilateral forms of RP have been reported in the literature (Farrell 2009; Mukhopadhyay et al. 2011). Therefore, to test the fixed effect (eye and group) and the random effect (subject) on oximetry and OCT data, a linear mixed-effects model was applied. Categorical predictors are presented as differences of means with corresponding 95% C.I. and p-values. For continuous predictors, results are expressed as differences of means (slope, %/lm) increasing the predictor one unit (percentage points/micrometre). Within RP group, the A-V SO2 correlated significantly with the central macular thickness [p = 0.006; with the slope of 0.089%/lm (CI 0.029–0.149, %/lm)], but also with the retinal thickness in zone 3 [p = 0.014; with the slope of 0.090%/lm (CI 0.021– 0.160, %/lm)]. In zone 2, the A-V SO2 showed a significant trend to decrease as the retina thickness reduced [p = 0.074; with the slope of 0.039%/ lm (CI 0.004–0.082, %/lm)]. The correlation slopes for V-SO2 versus OCT parameters (macula thickness; zone 1; zone 2; zone 3) in the RP group were negative ( 0.043; 0.013; 0.037; 0.035), however, not statistically significant (Fig. 4). For A-SO2 of the RP patients, none of the tested OCT parameters showed statistically significant correlations: the slope was positive but also not statistically significant.
(C)
Fig 3. Depicts from A to C the box plots of the retinal vessels oxygen saturation for arterioles (A-SO2), venules (V-SO2), as well as their difference (A-V SO2). The box length is the interquartile range, the line in bold depicts the median. In each graph, the control group is plotted to the left and the retinitis pigmentosa group is plotted to the right. The box plots of right eyes are white-coloured, and the box plots of the left eyes are grey-coloured. The respective p-values are given in Table 2 (ANOVA based on mixed-effects model).
In our study, the length of intact IS/ OS line, an indirect measure for integrity of the inner and outer photoreceptors’ segments (Gloesmann et al. 2003; van Velthoven et al. 2007), was significantly shorter in RP patients (mean 2645 lm; 95% CI 2123–3168 lm; p < 0.001), when compared to controls (mean 5979 lm; 95% CI 5494–6465 lm, ANOVA based on linear-effects model). With progression of the RP disease, the length of the IS/OS line showed a trend for negative correlation with the A-SO2 and V-SO2 and a trend for positive correlation with the A-V SO2 values. Nerveless, within RP group the correlation between the oximetry parameters and the length of the IS/OS, line did not reach the significant level (p > 0.05). In controls, no significant correlations were found between the SO2 values and the OCT data.
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Acta Ophthalmologica 2014
Table 2. depicts the oxygen saturation (%) in retinal arterioles and venules evaluated separately for both eyes, the respective p-values between eyes and between groups (ANOVA, based on mixed-effects model). All retinitis pigmentosa (RP) patients had higher arteriolar and venular saturation than the healthy group, resulting in lower arterio-venous difference (p < 0.05). 95% Confidence interval (lower limit-upper limit)
Mean,% Variables
Group
Right eye
Left eye
Right eye
Left eye
p-values (between eyes)
A-SO2
Controls RP Controls RP Controls RP
92.4 100.1 53.8 66.8 38.5 33.3
92.4 98.4 54.2 66.9 38.3 31.5
90.1–94.7 95.2–105.1 51.9–55.9 63.8–69.9 36.6–40.5 28.9–37.7
90.0–94.8 94.8–102.0 52.4–55.9 62.9–70.9 36.4–40.5 27.4–35.6
0.991 0.528 0.806 0.990 0.854 0.507
V-SO2 A-V SO2
p-values between groups 0.001
Retinal vessel oxygen saturation and its correlation with structural changes in retinitis pigmentosa Cengiz T€ urksever,1,* Christophe Valmaggia,2,* Selim Org€ ul,1 Daniel F. Schorderet,3,4,5 Josef Flammer1 1 and Margarita G. Todorova 1
Department of Ophthalmology, University of Basel, Basel, Switzerland Department of Ophthalmology, Cantonal Hospital, St. Gallen, Switzerland 3 IRO – Institut for Research in Ophthalmology, Sion, Switzerland 4 Department of Ophthalmology, University of Lausanne, Lausanne, Switzerland 5 School of Life Sciences, Federal Institute of Technology, Lausanne, Switzerland 2
ABSTRACT. Purpose: To study the influence of retinal structural changes on oxygen saturation in retinitis pigmentosa (RP) patients. Methods: Oximetry measurements were performed on 21 eyes of 11 RP patients and compared to 24 eyes of 12 controls. Retinal oxygen saturation was measured in all major retinal arterioles (A-SO2) and venules (V-SO2) with an oximetry unit of the retinal vessel analyser (IMEDOS Systems UG, Jena, Germany). Oximetry data were compared with morphological changes measured by Cirrus optical coherence tomography (OCT) (Carl Zeiss Meditec, Dublin, CA, USA, macular thickness protocol). Results: In RP patients, the retinal A-SO2 and V-SO2 levels were higher at 99.3% (p = 0.001, ANOVA based on mixed-effects model) and 66.8% (p < 0.001), respectively, and the difference between the two (A-V SO2) was lower at 32.5% (p < 0.001), when compared to the control group (92.4%; 54.0%; 38.4%, respectively). With the RP group, the A-V SO2 correlated positively, not only with central macular thickness, but also with retinal thickness, in zones 2 and 3 (p = 0.006, p = 0.007, p = 0.014). Conclusion: These data indicate that oxygen metabolism was altered in RP patients. Based on our preliminary results, retinal vessel saturation correlated with structural alterations in RP. This method could be valuable in monitoring disease progression and evaluating a potential therapeutic response. Key words: inner/outer segment junction line – optical coherence tomography – oxygen – retinal vessel oximetry – retinitis pigmentosa – saturation
Acta Ophthalmol. 2014: 92: 454–460 ª 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
doi: 10.1111/aos.12379 *These authors contributed equally to this work
Introduction Retinitis pigmentosa (RP), the most commonly recognized hereditary retinal dystrophy of the outer retina, occurs worldwide with a frequency of
454
1/4000 (Ammann et al. 1965; Puech et al. 1991; Hamel 2006). The disease is well known for its genetic heterogeneity and common clinical symptoms: initial night blindness, centripetal progression of visual field constriction resulting in
tunnel vision and reduced central vision at the end stage (Hamel 2006; Berger et al. 2010). Gene mutations expressed in photoreceptors or retinal pigment epithelium (Berger et al. 2010) trigger neuronal degeneration through various metabolic pathways, including apoptosis (Marc & Jones 2003; Cottet & Schorderet 2009). Following atrophy, the inner retina responds with secondary remodelling, proximal intraretinal pigment migration as well as neuronal and glial migration (Marc & Jones 2003; Marc et al. 2003; Jones & Marc 2005). As dystrophy progresses, the optic disc becomes pale and arterial vessels attenuate (Marc & Jones 2003; Marc et al. 2003; Hamel 2006; Sahaboglu et al. 2013). Optical coherence tomography (OCT) has enabled the identification of different patterns of structural involvement in RP. In stages with high retinal pigment epithelium metabolic demand due to the loss of photoreceptor integrity, distortions of retinal microstructure and/or macular oedema have been observed (Sandberg et al. 2005; Iriyama & Tanagi 2012; Mitamura et al. 2012). Further OCT studies have confirmed a correlation between retinal microstructure and visual function in RP patients, in particular at the junction between the inner and outer segments, the so-called inner/outer segment junction (IS/OS) line (Aizawa et al. 2009; Kim et al. 2013; Mitamura et al. 2013). Retinal hemodynamics impairment in the context of reduced arterial and
Acta Ophthalmologica 2014
venous blood velocities has been confirmed, not only in the central retina (Li et al. 2009) but also in the periphery (Cellini et al. 1997; Beutelspacher et al. 2011). Quantitative analysis of retinal vessel attenuation has demonstrated a significant correlation with the severity of retinopathy (Cellini et al. 1997; Ma et al. 2012). Even in early stages, RP patients have shown reduced systolic velocities in posterior ciliary arteries, as measured by colour Doppler (Cellini et al. 1997; Ma et al. 2012) and confirmed by quantitative blood-flow MRI measurements (Zhang et al. 2013). Retinal oximetry has been established to analyse retinal vessel oxygen saturation (Schweitzer et al. 2001; Geirsdottir et al. 2012; Hardarson 2013). Until now, this method has only been studied in few vascular entities and in glaucoma (Hammer et al. 2009; Olafsdottir et al. 2011; Boeckaert et al. 2012; Hardarson & Stef ansson 2012; Hardarson 2013; Hardarson et al. 2013; Vandewalle et al. 2013b). When structural changes occur, including neurovascular remodelling as in the case of RP, modification of retinal blood oxygenation is to be expected. In this study, we aimed to investigate the oxygen saturation in retinal arterioles and venules in RP patients. Also, to investigate the oxygen saturation of the retinal vessels as an indicator of metabolic retinal dysfunction in RP patients, we correlated the oximetry data with the morphological changes measured by OCT. If the degree of retinal vessel saturation disturbances correlates with the structural alterations in RP, the method would help in understanding the disease. It would also add new tools in monitoring disease progression and evaluating potential therapeutic responses.
that included refraction, best-corrected visual acuity (Snellen charts), intraocular pressure examination by applanation (Goldmann tonometer), slit-lamp examination, biomicroscopy and fundoscopy. Retinitis pigmentosa patients from our diagnostic unit were enrolled in the study after informed consent. Their clinical phenotype was characterized following clinical and electrophysiological examination. Inclusion criteria for RP patients were as follows: Caucasian origin, characteristic fundoscopic findings of RP, reduced or non-detectable a- and b-wave amplitudes of the scotopic full-field ERG. Inclusion criteria for controls were as follows: Caucasian origin, best-corrected Snellen visual acuity at distance ≥0.8. Exclusion criteria for patients and controls were as follows: above inclusion criteria not fulfilled; presence of ocular and/or systemic pathology that could influence the OCT and retinal vessels oximetry data; unstable fixation; fundus oximetry images with inadequate quality; and refusal to participate in the study. To evaluate oxygen saturation of the retinal vessels, we used the spectrophotometric oximetry tool of the retinal vessel analyser, coupled to a fundus camera FF450 (IMEDOS Systems UG, Jena, Germany; Carl Zeiss Meditec AG, Jena, Germany) and to a digital camera system (KY-F75; JVC Inc., Yokohama, Japan). This digital spectrophotometric imaging system allows for valuable assessment of retinal vessel oxygenation (Hammer et al. 2002; Hardarson 2013). Fundus images were
taken with the help of a dual band-pass filter imaging system with two different transmission bands, the first a nonsensitive to oxygen 548 nm wavelength and the second oxygen-sensitive with 610-nm wavelength filters. In this way, two retinal images were taken simultaneously at two different wavelengths. The software compared the two photographs, according to the optical density ratio (ODR): the colour-coded oxygen saturation of the retinal vessels and the photometric vessel diameter alterations along the selected vessel segment. Specialized software (VISUALIS; IMEDOS Systems UG, Jena, Germany) incorporated in the retinal vessel analyzer allows then for automatic evaluation of the acquired digital retinal images. Only images fulfilling the following criteria were further analysed: optic disc located at the image centre (Man et al. 2013); clearness of the images; appropriate brightness. VESSELMAP software incorporated in Imedos system enables to correct the effect of vessel diameter on retinal vessels saturation measurements. Imaging
Patients and controls were adapted for 10 min to mesopic conditions as described elsewhere (Hammer et al. 2002; Hardarson 2013). Images of both eyes were obtained, starting with the right eye. At least four images were taken and more images added if needed. The oxygen saturation was measured in all major retinal arterioles (A-SO2) and venules (V-SO2) within a distance of 0.5–1.0 optic disc diameter from the optic disc edge. In addition,
Material and Methods This pilot study adhered to the Tenets of the Declaration of Helsinki. Informed consent was obtained from all participants before performing oximetry imaging after explanation of the nature and possible consequences of the study. Subjects
All controls as well as RP patients had a complete ophthalmic examination
(A)
(B)
Fig. 1. Fundus oximetry images (map) of a control (A, left eye) and of a patient with retinitis pigmentosa (RP) (B, right eye). Oxygen saturation is measured along the main retinal vessels, within 0.5–1.0 optic disc diameter distance from the optic disc and up to the first vessel branching. The colours in the oximetry images indicate the relative oxygen saturation (%) in retinal vessels. Note the increased A-SO2 and V-SO2 in RP patients.
455
Acta Ophthalmologica 2014
we computed the arterio-venous difference (A-V SO2), known to be proportional to the oxygen saturation. Examples of oximetry readings of a control individual and of an RP patient are shown in Fig. 1A,B, respectively. For evaluation of in vivo retinal structures (Sakata et al. 2009), we performed a frequency domain OCT on Cirrus OCT (Carl Zeiss Meditec. Dublin, CA, USA). The OCT images were taken using a macular thickness (Macular Cube 512 9 128) and high definition image protocol (HD 5 Line Raster). The software of the Cirrus OCT provided a macular thickness map divided into nine subfields. To analyse the data as an indicator for metabolic retinal dysfunction, in particular in RP patients where a significant constriction of visual fields occurs, we computed the mean macular thickness in three areas: zone 1 at 3°; zone 2 between 3° and 8°; and zone 3 between 8° and 15° (Fig. 2A,B, centre). The highly reflective line above the retinal pigment epithelium (RPE) layer indicates the photoreceptor IS/OS. The presence of an intact IS/OS line in OCT images is an indicator of proper photoreceptor integrity and function (Gloesmann et al. 2003; van Velthoven
et al. 2007). In RP patients, the integrity of the IS/OS line has been well associated with better visual function, brighter fundus autofluorescence parafoveolar density and higher retinal sensitivity (Iriyama & Tanagi 2012; Mitamura et al. 2012, 2013). For this reason, we measured the length of the intact IS/OS line for every eye on HD 5 Line Raster OCT images using a measurement tool from the Cirrus OCT (Fig. 2B, right). In addition, we assessed the relationship between retinal vessel oxygen saturation values and the length of intact IS/OS line, as well as the OCT data averaged in three zones, as described above. Statistical analysis
For statistical analysis of Oximetry data, ANOVA based on mixed-effects model, which allows taking the dependency of the left and right eye in the same subject into account, was applied. p-values of less than or equal to 0.05 were considered statistically significant. To correlate the oximetry data (the mean A-SO2; V-SO2; A-V SO2 %) to the OCT measurements (the mean macula thickness and retinal thickness
(lm) averaged in circles: zone 1: at 3°; zone 2: 3°–8°; zone 3: 8°–15°), a linear mixed-effects model, suitable for the repeated measurements data, was used. To predict the effect of the structural alterations on oximetry measurements, the eye and the group effects were taken into account, where the eye and the group were treated as a fixed factors and the subject as a random factor.
Results A total of 11 patients (5 ♂: 6 ♀; 21 eyes) with clinical and electrophysiological evidence of RP were enrolled in the study. One patient was evaluated only monocularly due to post-traumatic corneal haze with irregular astigmatism and aphakia in the other eye. Twelve age-matched controls (3 ♂: 9 ♀; 24 eyes) were recruited from our data bank. Demographic data of our patients and controls are given in Table 1. Oximetry data
The reproducibility of the oximetry measurements was excellent and comparable to previous studies on control
(A)
(B) Fig. 2. Represents an example of optical coherence tomography (OCT) measurements of a control (A) and of a retinitis pigmentosa (RP) patient (B). We averaged the mean macular thickness in three circles: zone 1 at 3°; zone 2 between 3° and 8°; and zone 3 between 8° and 15°, as exemplified in A, in the middle. The IS/OS line is only partially visible in the presented RP patient (B, right). The intact area, visible only at the foveal centre, is labelled with red line. Outside the measured area, the inner/outer segment junction (IS/OS) line is completely absent and the retinal thickness is reduced.
456
Acta Ophthalmologica 2014
Table 1. Represents the demographic data for the studied groups.
Demographic characteristics
Controls
Retinitis pigmentosa patients
Number of patients (eyes examined) Age, mean SD, years Sex (♂:♀) Snellen BCVA, mean SD
12 (24) 36.08 (10.69) 3:9 1.0 (0.000)
11 (21) 35.33 (10.81) 5:6 0.49 (0.30)
eyes (Hammer et al. 2008; Geirsdottir et al. 2012; Lasta et al. 2012). In the control group, we calculated a test– retest mean standard deviation for A-SO2 (1.52%) and for V-SO2 (2.50%). In the RP group, the measurements also showed a reliable reproducibility: the mean test–retest SD values were as follows: for the A-SO2 1.87% and for the V-SO2 2.27%. Comparison of retinal vessel oxygen measurements in groups
We then compared the retinal vessel oxygen saturation measurements in controls against those of RP patients. In controls, the SO2 values were as follows: the mean A-SO2 and V-SO2 of the retina were measured at 92.4% and 54.0%, respectively. The mean A-V SO2 in controls was 38.4%. In RP patients, the retinal A-SO2 and V-SO2 levels increased significantly to 99.3% (p = 0.001) and 66.8% (p < 0.001). In consequence, the A-V SO2 values decreased to 32.5% (p < 0.001; Fig. 3A–C; Table 2). There was no statistically significant difference between the right and left eye analysed in each group separately (p > 0.507, ANOVA, based on mixed-effects model; Table 2). Retinal vessel diameter and its influence on oximetry measurements
To rule out the effect of retinal vessel diameter on oxygen saturation measurements (Beach et al. 1999; Vandewalle et al. 2013a), even having a VESSELMAP software incorporated in the Imedos system, we evaluated the relationship between the A-SO2 and the diameter of arterioles, as well as between the V-SO2 and the diameter of venules. Results of our study showed no influence of retinal vessel diameters on vessel SO2 measurements (linear mixed-effects model). This was the case for the venous measurements in the
p-values (one-way ANOVA)
0.816 0.000
combined group (p = 0.527), as well as separately in controls (p = 0.298) and in RP group (p = 0.159). A-SO2 neither in the combined group, nor in controls or RP patients correlated with the diameter of arterioles (p > 0.137).
(A)
(B)
Correlation between the OCT data (averaged in circles) and the oximetry data
To correlate the oximetry measurements in RP patients with structural measurements, the oximetry data were further compared to the OCTs. Asymmetric and unilateral forms of RP have been reported in the literature (Farrell 2009; Mukhopadhyay et al. 2011). Therefore, to test the fixed effect (eye and group) and the random effect (subject) on oximetry and OCT data, a linear mixed-effects model was applied. Categorical predictors are presented as differences of means with corresponding 95% C.I. and p-values. For continuous predictors, results are expressed as differences of means (slope, %/lm) increasing the predictor one unit (percentage points/micrometre). Within RP group, the A-V SO2 correlated significantly with the central macular thickness [p = 0.006; with the slope of 0.089%/lm (CI 0.029–0.149, %/lm)], but also with the retinal thickness in zone 3 [p = 0.014; with the slope of 0.090%/lm (CI 0.021– 0.160, %/lm)]. In zone 2, the A-V SO2 showed a significant trend to decrease as the retina thickness reduced [p = 0.074; with the slope of 0.039%/ lm (CI 0.004–0.082, %/lm)]. The correlation slopes for V-SO2 versus OCT parameters (macula thickness; zone 1; zone 2; zone 3) in the RP group were negative ( 0.043; 0.013; 0.037; 0.035), however, not statistically significant (Fig. 4). For A-SO2 of the RP patients, none of the tested OCT parameters showed statistically significant correlations: the slope was positive but also not statistically significant.
(C)
Fig 3. Depicts from A to C the box plots of the retinal vessels oxygen saturation for arterioles (A-SO2), venules (V-SO2), as well as their difference (A-V SO2). The box length is the interquartile range, the line in bold depicts the median. In each graph, the control group is plotted to the left and the retinitis pigmentosa group is plotted to the right. The box plots of right eyes are white-coloured, and the box plots of the left eyes are grey-coloured. The respective p-values are given in Table 2 (ANOVA based on mixed-effects model).
In our study, the length of intact IS/ OS line, an indirect measure for integrity of the inner and outer photoreceptors’ segments (Gloesmann et al. 2003; van Velthoven et al. 2007), was significantly shorter in RP patients (mean 2645 lm; 95% CI 2123–3168 lm; p < 0.001), when compared to controls (mean 5979 lm; 95% CI 5494–6465 lm, ANOVA based on linear-effects model). With progression of the RP disease, the length of the IS/OS line showed a trend for negative correlation with the A-SO2 and V-SO2 and a trend for positive correlation with the A-V SO2 values. Nerveless, within RP group the correlation between the oximetry parameters and the length of the IS/OS, line did not reach the significant level (p > 0.05). In controls, no significant correlations were found between the SO2 values and the OCT data.
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Table 2. depicts the oxygen saturation (%) in retinal arterioles and venules evaluated separately for both eyes, the respective p-values between eyes and between groups (ANOVA, based on mixed-effects model). All retinitis pigmentosa (RP) patients had higher arteriolar and venular saturation than the healthy group, resulting in lower arterio-venous difference (p < 0.05). 95% Confidence interval (lower limit-upper limit)
Mean,% Variables
Group
Right eye
Left eye
Right eye
Left eye
p-values (between eyes)
A-SO2
Controls RP Controls RP Controls RP
92.4 100.1 53.8 66.8 38.5 33.3
92.4 98.4 54.2 66.9 38.3 31.5
90.1–94.7 95.2–105.1 51.9–55.9 63.8–69.9 36.6–40.5 28.9–37.7
90.0–94.8 94.8–102.0 52.4–55.9 62.9–70.9 36.4–40.5 27.4–35.6
0.991 0.528 0.806 0.990 0.854 0.507
V-SO2 A-V SO2
p-values between groups 0.001
