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Purpose: Retinal Vessel Analyser (RVA) is a validated instrument to measure retinal vessel diameter in humans. The purpose of this study was to assess the reproducibility (inter-observer reliability) and the repeatability (test–retest reliability) of RVA with a microscope-mounted fundus camera to determine retinal vessel diameter in minipigs.
Methods: Ocular fundus image from five anaesthetized minipigs was recorded in a digital videotape for approximately 5 min, under stable systemic arterial pressure and gas conditions. To evaluate the reproducibility, each one of two investigators used RVA to measure the diameter of the superior temporal retinal artery on five separate 30-second video sequences from each minipig, which were the same video sequences for both investigators. To evaluate the repeatability, one investigator performed five measurements on a single, randomly selected, 30-second video sequence from each minipig. The reproducibility was determined using the intra-class correlation coefficient (ICC), and the repeatability was assessed using the coefficient of variation (COV). Bland–Altman plots were also used to assess agreement between the two investigators.
Results: Retinal arteriolar diameter measurements with RVA in minipigs were highly reproducible. Differences between the two investigators were lower than 0.7%. The ICC was 1.00, indicating perfect reproducibility, and the mean COV was 0.18%, reflecting excellent repeatability of the measurements with RVA.
Conclusion: Retinal vessel diameter can reliably be determined not only in humans, but also in minipigs, using the commercially available RVA apparatus and a microscope-mounted fundus camera.
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The knowledge of retinal haemodynamics and blood flow regulation is of fundamental importance for the understanding of mechanisms underlying retinal diseases. In ischaemic microangiopathies of the inner retina, such as diabetic retinopathy and retinal vein occlusions, impairment of retinal circulation results in blood flow alterations that, in turn, affect the delivery of oxygen and metabolic substrates necessary for the maintenance of retinal structure and function (Pournaras et al. 2008). To cope with this, various possible therapeutic agents are under investigation using protocols in animal models and in humans. Thus, there is considerable interest in studying the parameters of retinal blood flow regulation in health and in disease.
Vessel diameter constitutes an important parameter of blood flow (Pournaras et al. 2008). In the last decades, the development of non-invasive techniques to evaluate retinal haemodynamics has led to new and important information about retinal blood flow regulation in the healthy and diseased human eye. However, most of these techniques are limited by the fact that only information about blood velocity is available but not blood flow per se. Hence, it is difficult to decide whether an increase in blood velocity is caused by an increase in blood flow or by vasoconstriction within the measured vascular bed (Schmetterer & Garhofer 2007). Therefore, to determine blood flow, the exact determination of vessels diameter is crucial.
Retinal vessels diameter has been measured in past years mainly from magnified ocular fundus photographs using a calliper or by scanning across the vessel (Pournaras et al. 2008). Nowadays, Retinal Vessel Analyser (RVA), a newly developed device, has allowed online and continuous, non-invasive measurements of retinal vessels diameter (Blum et al. 1999; Lanzl et al. 2000; Nagel et al. 2000; Kiss et al. 2002). This device consists of a fundus camera, a charge-coupled device (CCD) measuring camera for electronic online image acquisition, and a computer for system control analysis and archiving of the diameter data (Garhofer et al. 2010). In general, a vessel section of about 1.5 mm in length is scanned at a frequency of 25/second (Pournaras et al. 2008).
Retinal Vessel Analyser is a validated instrument to measure retinal vessel diameter in humans (Polak et al. 2000; Pache et al. 2002; Seifert & Vilser 2002). However, a great number of protocols studying the pathophysiology of retinal circulation and testing possible therapeutic agents require the use of animal models, in which the reproducibility of RVA has not been evaluated. Therefore, the purpose of this study was to assess the reproducibility as well as the repeatability of RVA with a microscope-mounted fundus camera to determine retinal vessel diameter in an animal model.
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Systemic blood pressure, pH, PaCO2 and PaO2 remained practically stable during each 5-min recording from any of the animals studied, as shown in Table 1.
Table 1. Systemic blood pressure (BP), pH, arterial partial pressure of carbon dioxide (PaCO2) and arterial partial pressure of oxygen (PaO2) at the beginning and at the end of each 5-min fundus recording from the five minipigs studied. Data regarding BP, PaCO2 and PaO2 are expressed in mmHg.
|Animal||0 min||5 min|
The data used to evaluate the reproducibility of the measurements with RVA are presented in Table 2. As five different retinal vessels were studied, vessel diameter values ranged from 149.1 to 214.0 AU, but those corresponding to the same video sequences were very close to each other when the two investigators were compared. The intra-class correlation coefficient (κ) of the 25 pairs of these measurements was 1.00, indicating perfect reproducibility of the method in minipigs.
Table 2. Retinal vessel diameter in AU measured on video sequences by Investigators A and B, independently, with RVA. The intra-class correlation coefficient calculated from the 25 pairs of the corresponding measurements was 1.00, indicating high reproducibility of RVA in minipigs.
|Investigator||Animal||Time periods (min)|
Bland–Altman plot analysis showed an excellent inter-observer agreement. Differences between the two investigators were lower than 0.7%. The mean paired difference between the measurements of the two investigators was −0.1 AU, that is, near zero, and all the differences of the 25 pairs of individual measurements were within the limits of agreement (Fig. 1).
Figure 1. Bland–Altman plots of the inter-observer agreement in retinal vessel diameter measurements with the Retinal Vessel Analyser. The difference ‘retinal vessel diameter by Investigator A minus retinal vessel diameter by Investigator B’ was plotted against the mean retinal vessel diameter of both investigators. The mean paired difference is shown by the solid line, the limits of agreement (±1.96 SD) by the dashed lines. An excellent inter-observer agreement is shown: the mean paired difference is near zero (i.e. −0.1 arbitrary units), and all the differences of the 25 pairs of individual measurements are within the limits of agreement.
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The data used to evaluate the repeatability of the measurements with RVA are presented in Table 3. As five different retinal vessels were studied, vessel diameter values ranged from 190.0 to 230.6 AU, but those corresponding to the same animal were very close to each other. Individual coefficients of variation ranged from 0.12% to 0.24% (mean coefficient of variation = 0.18%), reflecting excellent repeatability of the measurements with RVA in minipigs.
Table 3. Retinal vessel diameter in AU, repeatedly measured on video sequences by Investigator B with RVA. The mean coefficient of variation of the measurements was 0.18%, reflecting excellent repeatability of RVA in minipigs.
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The current study evaluated the reproducibility and repeatability of RVA to measure retinal vessel diameter in minipigs. The results showed perfect reproducibility and excellent repeatability of RVA when used in minipigs, indicating for the first time that retinal vessel diameter can reliably be determined in this animal model using the commercially available RVA apparatus and a microscope-mounted fundus camera. This device may thus be considered validated to be used in protocols investigating retinal vessel diameter changes under various conditions in minipigs.
The reproducibility of RVA to measure retinal vessel diameter in humans has previously been defined as high. Polak et al. (2000) performed measurements in nine healthy young volunteers and reported intra-class correlation coefficients of 0.98 and 0.96 for retinal veins and retinal arteries, respectively, concluding that short-term measurements of retinal vessel diameter with the Zeiss RVA are highly reproducible. They also calculated short-term coefficients of variation to be 1.3% and 2.6% for retinal veins and retinal arteries, respectively (Polak et al. 2000). In another study, Seifert and Vilser used RVA to measure retinal vein diameters in 12 healthy volunteers continuously for 5 min, and the measurements were repeated at the same vessel locations after 2 hr. This resulted in a short-term coefficient of variation of 1.5% (Seifert & Vilser 2002). Finally, to evaluate the short-term reproducibility of RVA, Pache et al. (2002) measured retinal vessel diameter in 20 healthy subjects at baseline and after 2 hr. Short-term variability of vessel diameter was only 1%, and the intra-class correlation coefficient was 0.97 and 0.96 for retinal veins and retinal arteries, respectively.
Thanks to the above favourable results, RVA is considered a reliable device for both analysis and follow-up of retinal vessel diameter changes in humans. The current study demonstrates that RVA can also be considered reliable for the study of retinal vessel diameter changes in minipigs. The intra-class correlation coefficient for retinal arteries was 1.00, quite comparable with that calculated in humans. Our study provides also information about repeatability, calculating a 0.18% mean coefficient of variation of repeated measurements by the same person, a number which adds to the excellent reliability of RVA in minipigs.
Some limitations about the present study need to be mentioned. Both investigators performed measurements on the same video sequences, so what was evaluated was the reproducibility (or inter-observer reliability) of RVA measurements on a specific video sequence and not that of the whole recording process (e.g. manual positioning of the animal, set-up of the microscope, etc.). In addition, retinal vessel diameter measurements were limited to retinal arterioles. This was done because, from the physiopathological point of view, changes in retinal blood flow are mainly related to changes of the retinal arteriolar diameter (Pournaras et al. 2008).
Clinical interest of the results of the current study is high. Retinal vessel response to drugs already used in ophthalmology has recently begun to draw interest (Papadopoulou et al. 2009; Lanzl et al. 2011). However, several protocols investigating physiopathological pathways of the retina, or new drugs, cannot be directly applied to humans, because of their interventional nature and safety concerns. For instance, RVA has been used in minipigs to evaluate the vasomotor effect of intra-vitreal juxta-arteriolar injection of endothelin-A receptor inhibitor (Stangos et al. 2010), l-arginine (Mendrinos et al. 2010) and l-lactate (Mendrinos et al. 2011), on retinal arteriolar diameter after acute branch retinal vein occlusion. A scientific basis for validation of RVA measurements in animals is provided by the current study.
In conclusion, retinal vessel diameter can reliably be determined not only in humans, but also in minipigs, using the commercially available RVA apparatus and a microscope-mounted fundus camera. An excellent reproducibility and repeatability of this method make it appropriate to evaluate retinal vessel diameter changes in short-term experimental protocols.