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Keywords:

  • hypoxia;
  • ischaemia;
  • oximetry;
  • oxygen saturation;
  • retina;
  • retinal vein occlusion

Abstract.

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Source of Funding
  9. Financial Disclosure
  10. References

Purpose:  The aim of this study was to test whether oxygen saturation in retinal blood vessels is affected by branch retinal vein occlusion (BRVO).

Methods:  The spectrophotometric retinal oximeter is based on a fundus camera. It simultaneously captures images of the retina at 586 and 605 nm and calculates optical density (absorbance) of retinal vessels at both wavelengths. The ratio of the two optical densities is approximately linearly related to haemoglobin oxygen saturation. Relative oxygen saturation was measured in retinal blood vessels in 24 patients with BRVO. Friedman’s test and Dunn’s post test were used for statistical analyses.

Results:  Oxygen saturation in occluded venules ranged from 12% to 93%. The median oxygen saturation was 59% (range 12–93%, n = 22) in affected retinal venules, 63% (23–80%) in unaffected venules in the BRVO eye and 55% (39–80%) in venules in the fellow eye (p = 0.66). Corresponding values for arterioles were 101% (89–115%, n = 18), 95% (85–104%) (p < 0.05) and 98% (84–109%).

Conclusions:  Venular saturation in BRVO is highly variable between patients. Hypoxia is seen in some eyes but not in others. This may reflect variable severity of disease, degree of occlusion, recanalization, collateral circulation, tissue atrophy, arteriovenous diffusion or vitreal transport of oxygen.


Introduction

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Source of Funding
  9. Financial Disclosure
  10. References

Branch retinal vein occlusion (BRVO) reduces blood flow to a part of the retina (Fujio et al. 1994; Kang & Lee 1995; Avila et al. 1998; Yoshida et al. 2003; Horio & Horiguchi 2004; Noma et al. 2009a,b). The retinal circulation supplies the inner retina with oxygen (see for example Linsenmeier 1986) and animal studies have shown that following experimental BRVO, the (inner) retina does indeed become hypoxic. Low partial pressure of oxygen (pO2) is reported in the preretinal vitreous over retinal areas affected by BRVO in miniature pigs (Pournaras et al. 1990a), pigs (Noergaard et al. 2008), cats (Stefansson et al. 1990) and monkeys (Pournaras et al. 1997), although no difference was found in one study on monkeys (Ernest & Archer 1979). Pournaras et al. (1990b) made a direct measurement of pO2 in the inner retina with intraretinal electrodes and found it to be lower in experimental BRVO.

Retinal hypoxia may contribute to production of vascular endothelial growth factor and development of macular oedema and neovascularization. Measurement of retinal oxygenation in patients with BRVO may help to assess the severity of the occlusion and to monitor treatment results. In this study, we use a retinal oximeter to measure retinal vessel oxygen saturation in patients with BRVO.

Materials and Methods

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Source of Funding
  9. Financial Disclosure
  10. References

Patients

The study was approved by the National Bioethics Committee of Iceland and The Icelandic Data Protection Authority. Patients signed informed consent for participation in the study and the study adhered to the tenets of the Declaration of Helsinki.

Oxygen saturation measurements were performed on 24 consecutive patients with BRVO. Retinal vessels were divided into three categories: (1) Vessels affected by the occlusion, (2) vessels in the BRVO eye, which are not affected by the occlusion and (3) vessels in the fellow eye. The measured vessels not affected by the occlusion (categories 2 and 3) were chosen so that they were comparable in location to the affected vessels in the same patient. For example, if a major superotemporal venule was occluded, a major inferotemporal venule was chosen for comparison in the same eye and a major temporal venule (preferably superotemporal) in the fellow eye. The affected arteriole (category 1) was chosen as the arteriole that supplied the affected area to the greatest degree. All measurements were made before treatment of the affected eye.

Measurements could be made in all categories of arterioles in 18 patients and in all categories of venules in 22 patients. The mean age of the patients at the time of measurement was 70 years for arteriolar measurements and 67 years for venular measurements (range 44–86 years for both). The mean duration of the occlusion at the time of measurement was 3 months, range 0–9 months.

Retinal oximetry

The retinal oximeter (Oxymap ehf., Reykjavik, Iceland) has been described previously (Hardarson et al. 2006, 2009a,b; Traustason et al. 2009; Hardarson & Stefánsson 2010). It is based on a fundus camera (Canon CR6-45NM; Canon Inc., Tokyo, Japan), which is coupled with a beam splitter (MultiSpec Patho-Imager; Optical Insights, Tucson, AZ, USA) and a digital camera (SBIG ST-7E; Santa Barbara Instrument Group, Santa Barbara, CA, USA). It yields fundus images with four wavelengths of light simultaneously. Specialized software automatically selects measurement points on the oximetry images and calculates the optical density (absorbance) of retinal vessels at two wavelengths, 605 and 586 nm. Optical density is sensitive to oxygen saturation at 605 nm but not at the reference wavelength, 586 nm. The ratio of these optical densities is approximately linearly related to haemoglobin oxygen saturation (Beach et al. 1999; Harris et al. 2003). The oximeter is calibrated to yield relative oxygen saturation values (Hardarson et al. 2006). However, the calibration is not perfect and in some cases the measurements exceed 100%. The oximeter has been shown to be sensitive to changes in oxygen saturation and to yield repeatable results (Hardarson et al. 2006). The oximetry values obtained may therefore be used for comparison, even if they may differ from the absolute saturation values.

Infrared light was used to align the fundus camera (oximeter) and the images were taken in a dark room. The time between images (flashes) of the same eye was on average about 1 min. Pupils were dilated with 1% tropicamide (Mydriacyl®; S.A. Alcon-Couvreur N.V., Puurs, Belgium), which was in some cases supplemented with 10% phenylephrine hydrochloride (AK-DilateTM; Akorn Inc., Lake Forest, IL, USA). The fellow eye was not dilated in two cases (adequate dilation of pupil in darkness).

The oximeter estimates light absorbance by measuring light intensity outside and inside retinal vessels (Hardarson et al. 2006). Extravascular haemorrhages may therefore interfere with measurements. Care was taken to avoid measuring vessel segments with adjacent haemorrhages to reduce possible artefacts.

Statistical analysis was performed with Prism, version 5 (GraphPad Software Inc., LaJolla, CA, USA). Friedman’s test and Dunn’s post test were used.

Results

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Source of Funding
  9. Financial Disclosure
  10. References

Table 1 shows the results of saturation measurements. Some of the affected venular segments in Table 1 received blood also from non-occluded branches and in some cases, the image quality was poor. A separate analysis was performed on a subgroup where occluded venules could be reliably measured. The saturation (median and range) in occluded venules was 44% (12–93%), 63% (40–75%) in unaffected venules in the same eye and 55% (48–72%) in the fellow eye (n = 7, p = 0.96, Friedman’s test). Figure 1 shows individual values from this subgroup. Figure 2 shows an example of oxygen measurements in an eye affected by BRVO.

Table 1.   Retinal vessel oxygen saturation (%) in patients with branch retinal vein occlusion (median and range).
 Affected eyeFellow eyeFriedman’s test for differences in saturation (p)
Affected vesselUnaffected vessel
  1. * Significant difference between affected arterioles and unaffected arterioles in affected eye according to Dunn’s post test (p < 0.05).

Arterioles (n = 18)101% 89–115%95% 85–104%98% 84–109%0.024*
Venules (n = 22)59% 12–93%63% 23–80%55% 39–80%0.66
image

Figure 1.  Oxygen saturation (%) in retinal venules in patients with branch retinal vein occlusion. The lines connect measurements on the same patients. Measurements, which may be affected by several confounding factors, have been excluded.

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image

Figure 2.  Oxygen saturation map of a patient with branch retinal vein occlusion. The frame on the colour fundus photograph indicates the retinal area on the oxygen saturation maps.

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Discussion

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Source of Funding
  9. Financial Disclosure
  10. References

This study shows that oxygen saturation in occluded branch retinal venules is very variable between patients. Some occluded vessels are hypoxic, whereas others are not. Oxygen saturation in occluded retinal venules, ranged from 12% to 93%. Low venular oxygen saturation is the result of decreased supply of oxygen. Low venular saturation conforms with studies on animal models, which indicate low partial pressure of oxygen (pO2) in the retinal tissue in BRVO (Stefansson et al. 1982, 1990; Pournaras et al. 1990a,b, 1997; Noergaard et al. 2008). pO2 in the vitreous above an area affected by BRVO has for example been found to be 7.5 mmHg in the pig (Noergaard et al. 2008), 15.1 mmHg (Pournaras et al. 1990a) and 15.5 mmHg (Pournaras et al. 1990b) in the minipig and 6 mmHg in the cat (Stefansson et al. 1990). The saturation in occluded venules, found in this study (12–93%) equals approximately13–65 mmHg according to a formula by Kelman (1966) and assuming venous pH and carbon dioxide tension within normal range. Our lower pO2 values are comparable with the animal experiments. The animal experiments measure vitreal (pre-retinal) pO2, which may be slightly lower than pO2 in retinal venules. Moreover, the experimental vein occlusions may be more complete than the human disease.

Low venous saturation is also consistent with recent results in central retinal vein occlusion, obtained with the same technology as applied here (Hardarson & Stefánsson 2010), where the oxygen saturation in occluded venules was 49%, which corresponds pO2 of approximately 26 mmHg. The venous saturation in the fellow eye was 65%.

Normal and high saturation in occluded venules were also found in the current study. High oxygen saturation in the occluded venule may be attributed to retinal atrophy (Hockley et al. 1979; Frangieh et al. 1982) and decrease in oxygen consumption coupled with a recovery of blood flow following the initial insult. The blood supply may be increased by recanalization of the thrombotic occlusion (Frangieh et al. 1982) and/or by maturation of collateral circulation (Hamilton et al. 1974, 1979; Frangieh et al. 1982; Pieris & Hill 1982; Christoffersen & Larsen 1999; Genevois et al. 2004). In some cases, diffusion of oxygen from arterioles to nearby occluded venules may raise the saturation in the occluded venule. Evidence of arteriovenous diffusion of oxygen has, for example, been found in the retina of healthy cats (Buerk et al. 1993) and humans (Schweitzer et al. 1999, Karlsson RA et al. IOVS 2007;48:ARVO E-Abstract 2290). Previous reports have shown that oxygen transport is increased through the vitreous cavity in eyes where the vitreous gel is absent and this may include eyes with posterior vitreous detachment and such transport may alleviate retinal hypoxia in BRVO (Stefansson et al. 1990; Holekamp et al. 2005; Quiram et al. 2007; Shui et al. 2009).

The high oxygen saturation in affected arterioles is difficult to explain. In general, high oxygen saturation in retinal arterioles in part of the retina could be explained by increased blood flow. It is difficult to see how this explanation could apply to the present results. It should be noted that selection of the arteriole that serves the affected area to the greatest degree was not always straightforward and the arteriolar flow will be affected to a variable extent.

Measurements on retinal vessel oxygen saturation in patients with BRVO are challenging. To isolate the effect of the occlusion, the measurement area should be free of haemorrhage and the measured vessel segment should not receive blood from non-occluded venules. Furthermore, the image quality should be good and the retinal areas, used for comparison should, ideally, be completely healthy. Strict inclusion criteria, however, entails the risk of bias. For example, the more challenging measurements may be of those, who have more severe disease. To guard against possible bias, the data was analysed in two steps, first including all possible measurements and then only the more reliable ones. Both analyses support the same main conclusion, i.e. that the saturation is variable in venules, affected by BRVO.

The retinal oximeter uses two wavelengths for calculation of relative oxygen saturation. The measurements may for example be affected by vessel diameter (Beach et al. 1999) and the velocity of blood (Schweitzer et al. 2001). The effect of vessel diameter appears to be rather small (Beach et al. 1999) and is unlikely to have changed the results of the study so that the main conclusions would be changed. The magnitude of the effect of velocity is difficult to estimate as measurements of velocity were not performed. Earlier testing of the oximeter revealed that it is sensitive to changes in oxygen saturation and gives repeatable results (Hardarson et al. 2006). Technical errors are not likely to have changed the conclusions of this study.

The oxygen saturation in occluded venules reflects the balance between supply and demand for oxygen in the affected area. The variable saturation, found in the present study, may indicate different severity of the initial occlusion and/or that the patients are at a different stage in their recovery after the initial insult. Further studies will elucidate if and how different saturation values are associated with different clinical signs and prognosis.

Acknowledgements

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Source of Funding
  9. Financial Disclosure
  10. References

The authors wish to thank Haraldur Sigurdsson, María Soffía Gottfredsdóttir, Samy Basit and Sigridur Þórisdóttir, all at Landspítali University Hospital, Reykjavik, Iceland, for their help with recruiting participants for the study. A part of the results in this article was presented at the ARVO annual meeting in May 2010.

Source of Funding

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Source of Funding
  9. Financial Disclosure
  10. References

Icelandic Center for Research (Rannís), Eimskip University Fund, University of Iceland Research Fund, Landspítali-University Hospital Research Fund and Helga Jónsdóttir and Sigurliði Kristjánsson Memorial Fund.

Financial Disclosure

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Source of Funding
  9. Financial Disclosure
  10. References

The authors have financial interest in the oximeter used in the study.

References

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Source of Funding
  9. Financial Disclosure
  10. References
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