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. Author manuscript; available in PMC: 2016 Jun 1.
Published in final edited form as: Acta Ophthalmol. 2014 Nov 27;93(4):e275–e280. doi: 10.1111/aos.12593

Conjunctival Microvascular Hemodynamics in Sickle Cell Retinopathy

Ali Kord Valeshabad 1, Justin Wanek 1, Ruth Zelkha 1, Jennifer I Lim 1, Nicole Camardo 1, Bruce Gaynes 2, Mahnaz Shahidi 1
PMCID: PMC4437847  NIHMSID: NIHMS635099  PMID: 25429907

Abstract

Purpose

To determine alterations in bulbar conjunctival microvascular hemodynamics in sickle cell retinopathy (SCR) subjects with focal macular thinning (FMT).

Methods

Conjunctival microcirculation imaging and spectral domain optical coherence tomography (SD-OCT) were performed in 22 subjects (eyes) diagnosed with SCR. Based on evaluation of SD-OCT retinal thickness maps, eyes were assigned to one of two groups: with or without FMT. Conjunctival venule diameter and axial blood velocity were measured in multiple venules in each eye by customized image analysis algorithms. Measurements were then categorized into two vessel size groups (vessel size 1 and 2) and compared between FMT groups. A Pearson correlation coefficient was computed to assess the relationship between retinal thickness and axial blood velocity.

Results

Mean age, hematocrit, sickle cell hemoglobin type, and median retinopathy score were not significantly different between the two groups (p ≥ 0.1). Retinal thickness in parafoveal and perifoveal temporal subfields was significantly lower in eyes with FMT as compared to eyes without FMT (p ≤ 0.04). There was a significant effect of FMT on axial blood velocity (P = 0.04), while the effect of vessel size was not significant (P = 0.4). In vessel size 1, axial blood velocity was lower in eyes with FMT than in eyes without FMT (P = 0.03), while in vessel size 2, there was no statistically significant difference between FMT groups (P = 0.1). In vessel size 1, there was a significant positive correlation between axial blood velocity and retinal thickness in the perifoveal (r = 0.48, P = 0.02) and parafoveal (r = 0.43, P = 0.04) temporal subfields.

Conclusion

Conjunctival axial blood velocity in small venules is reduced in SCR subjects with focal macular thinning.

Keywords: Bulbar Conjunctiva, Hemodynamics, Sickle cell disease, Retina

Introduction

Sickle cell disease (SCD) is a hereditary hemoglobinopathy characterized by the formation of rigid, sickle shaped erythrocytes (Lei & Karniadakis 2012). The pathophysiology of SCD is based on the polymerization of deoxyhemoglobin S, which leads to hemodynamic abnormalities such as sludging, vascular occlusion and ischemia in the microvasculature, including that of the retina (Emerson et al. 2006). Sickle cell retinopathy (SCR) is typified by a number of ischemic events, including central and branch retinal artery occlusion, chorioretinal infarctions, and proliferative retinopathy (Acacio & Goldberg 1973; Nagpal et al. 1977). Ongoing, intermittent microvascular occlusions occurring in subjects with SCR create chronic inflammation via adhesion of leukocytes to the endothelium. The resultant chemokine and oxidants production can lead to macular infarctions and retinal thinning, as demonstrated by fluorescein angiography (Knapp 1972; Ryan 1974; Goldberg 1976; Asdourian et al. 1982; Merritt et al. 1982; Westrich & Feman 1986; Murthy et al. 2011), and spectral domain optical coherence tomography (SD-OCT) imaging, respectively (Witkin et al. 2006; Chow et al. 2011; Hoang et al. 2011; Murthy et al. 2011; Lim 2012). Furthermore, peripheral retinal ischemia has been associated with temporal macular thinning in subjects with SCR (Murthy et al. 2011).

Due to the systemic nature of SCD, microvascular alterations likely occur in all microcirculatory systems of the human body, including the conjunctiva and retina (Nagpal et al. 1977; Noguchi & Schechter 1981). The conjunctiva and retina may have comparable hemodynamic properties since they are supplied by different branches of the ophthalmic artery (Gray 1918). In SCR, the primary site of microvascular occlusions and altered hemodynamics is retinal microvessels and capillaries. However, due to limited resolution and contrast of retinal images, hemodynamic properties have been predominantly evaluated in large retinal blood vessels by research instruments (Riva et al. 1981; Cuypers et al. 2000; Yazdanfar et al. 2003; Yoshida et al. 2003; Landa et al. 2012), and have only been reported in retinal microvessels by prototype adaptive optics systems (Martin & Roorda 2005; Zhong et al. 2008; Tam & Roorda 2011; Zhong et al. 2011). On the other hand, the conjunctival microcirculation is assessable for high resolution imaging by standard slitlamp biomicroscopy, offers high contrast, and does not require the use of local mydriatic agents or intravenous injections. In fact, pathognomonic vaso-occlusions in isolated vascular segments (comma signs) (Paton 1961; Roy et al. 1985; Cheung et al. 2002) and hemodynamic abnormalities of the conjunctiva have been documented in SCD subjects (Cheung et al. 2002; Lima et al. 2006; Cheung et al. 2010; Wanek et al. 2013).

SCR subjects with higher incidences of retinal microvascular occlusions likely have more systemic hematological abnormalities (Roy et al. 1995). In addition, the severity of microvascular occlusions in the conjunctiva has been shown to be correlated with hematological alterations in SCD subjects (Serjeant et al. 1972). Therefore, we hypothesize that increased systemic hematological abnormalities that cause microvascular occlusive ischemic events and subsequent retinal thinning may also manifest in alterations in conjunctival microvascular hemodynamics. The purpose of the current study is to assess conjunctival microvascular hemodynamics in SCR subjects with and without macular thinning with the use of our previously established prototype optical imaging device (Shahidi et al. 2010; Gaynes et al. 2012; Wanek et al. 2013).

Patients and Methods

Subjects

This prospective research study was approved by an Institutional Review Board of the University of Illinois at Chicago. Prior to subject enrollment, the research study was explained to the subjects, and informed consents were obtained according to the tenets of the Declaration of Helsinki. Twenty two subjects with a clinical diagnosis of sickle cell anemia (SS, N = 12) or sickle cell-hemoglobin C disease (SC, N = 10) participated in the study. Subjects did not have uncontrolled systemic hypertension, diabetes mellitus, clinical evidence of other maculopathies, or history of retinal treatments, except peripheral scatter laser photocoagulation which was performed in 3 subjects. The subjects did not use local sympathomimetic or para-sympatholytic medications prior to conjunctival imaging. The severity of SCR was evaluated by a retina specialist (JIL)) based on the Goldberg classification (Goldberg 1971). The range of SCR stages was from 0 to IV, and the median SCR stage was II. The majority of SCR subjects who participated in the study were those in our previous published studies (Chow et al. 2011; Chow et al. 2012; Wanek et al. 2013).

Clinical data including the subjects' ages, and hematocrit (HCT) were obtained from the clinical charts. Blood pressure was measured either at the time of imaging (16 of 22 subjects) or within a few hours of imaging during clinic visits (6 of 22 subjects). The mean systolic (SBP) and diastolic blood (DBP) pressures were then converted to mean arterial blood pressure (MAP) using a standard formula: MAP = 1/3(SBP) + 2/3(DBP).

Retinal Thickness Mapping

SD-OCT imaging was performed using a commercially available instrument (Spectralis, Heidelberg Engineering, Inc, Carlsbad, California, USA). Nineteen horizontal raster B scans were obtained over a 30° × 25° retinal area centered on the fovea, and retinal thickness maps were generated using the instrument's software. From the retinal thickness map, the presence of focal macular thinning (FMT) was identified as an abrupt decrease in retinal thickness in one or more of the 9 Early Treatment Diabetic Retinopathy Study (ETDRS) subfields, as previously described (Chow et al. 2011; Chow et al. 2012). SCR eyes were categorized into two groups (with or without FMT) based on the presence or absence of FMT. Examples of retinal thickness maps in two SCR eyes with and without FMT are shown in Figure 1. In the eye with FMT, macular thinning was evident in the perifoveal temporal subfield, while the eye without FMT displayed a uniform normal thickness map. Mean retinal thickness in each ETDRS subfield was compared between groups to confirm retinal thinning in subjects with FMT.

Figure 1.

Figure 1

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Examples of retinal thickness maps obtained in two sickle cell retinopathy subjects without (Left) and with (Right) focal macular thinning (FMT). The Early Treatment Diabetic Retinopathy Study (ETDRS) macular subfields are displayed on each thickness map. Macular thinning is observed in perifoveal temporal and inferior subfields in the subject with FMT.

Conjunctival Microcirculation Imaging

Conjunctival microcirculation imaging was performed with the use of our optical imaging system (EyeFlow™) as previously described (Shahidi et al. 2010). In brief, a slit lamp biomicroscope equipped with a digital charged coupled device camera (Photometrics, Tuscon, AZ) was utilized to capture image sequences of red blood cell movement within the conjunctiva microcirculation. During imaging, an external fixation target was presented to minimize eye movement. Several image sequences, each 2 seconds in duration, were acquired. From each conjunctival microcirculation image sequence, 10 or more consecutive frames were manually selected based on image focus and the absence of large eye movements. In order to minimize the potential effect of variable light exposure and heating of the conjunctival surface on hemodynamics measurements, image acquisition was standardized in all subjects by keeping the light illumination level constant, placing a heat absorbing filter (ThorLabs Inc, Newton, New Jersey, USA) in the light illumination path, and restricting the light exposure time to approximately 15 minutes while multiple locations were imaged.

Our image analysis method for deriving conjunctival hemodynamic properties has been previously validated in a healthy human subject (Shahidi et al. 2010) and in acute hypertension in rabbit (Gaynes et al. 2012). Image analysis was performed on image sequences to first register them semi-automatically for eye motion correction and then determine diameter and axial blood velocity of several conjunctival venules, as previously described (Shahidi et al. 2010). Conjunctival venules were selected for hemodynamic measurements since they are more numerous, have minimal pulsatility (Koutsiaris et al. 2007; Koutsiaris et al. 2010), and are visualized with higher contrast as compared to arterioles. Venules were distinguished from arterioles by observing the direction of blood flow in conjunctival microcirculation image sequences and verifying that blood in the venule drained into a larger vessel. Briefly, a vessel of interest was manually identified and the centerline of the selected venule was automatically extracted. Venule diameter was derived as the full width at half maximum (FWHM) of intensity profiles generated perpendicular to the vessel centerline, averaged along the vessel length. Axial blood velocity was measured by tracking the motion of red blood cells along the vessel centerline in consecutive frames. Tracking of red blood cell motion was performed by generating a spatial-temporal image, which displayed intensity variation along the centerline of the vessel (rows) as a function of time (columns), thus revealing diagonal bands corresponding to the motion of aggregated red bloods cells (or plasma gaps). Axial blood velocity was derived by determining the slope of the prominent diagonal bands of the spatial temporal image (Koutsiaris et al. 2007; Shahidi et al. 2010; Gaynes et al. 2012).

In each SCR subject, one eye was randomly selected for data analysis. Diameter and axial blood velocity measurements were obtained in multiple venules, ranging in number between 5 and 17 venules in each eye. Conjunctival venule diameter and axial blood velocity measurements were obtained in 119 and 105 conjunctival venules of 12 and 10 eyes with and without FMT, respectively. The observers who performed conjunctival imaging and analyzed the images were masked to the retinal thickness status of the subjects.

Repeatability of Measurements

To determine the repeatability of conjunctival venular diameter and axial blood velocity measurements, imaging was performed in one eye of 5 healthy African-American subjects (age; 41 ± 6 years). Five repeated diameter and axial blood velocity measurements were obtained in 2 - 5 venules per subject. The mean and standard deviation (SD) of the repeated measurements were calculated in each venule and averaged in each subject. The repeatability was determined as the SD averaged over all healthy subjects. The SD (mean) of diameter and axial blood velocity measurements was 1.5 μm (19.8 μm) and 0.09 mm/s (0.50 mm/s), respectively.

Statistical Analysis

Kolmogorov-Smirnov test confirmed normal distributions of continuous variables including age, HCT, MAP, retinal thickness, venule diameter and axial blood velocity. These continuous variables were compared between groups using unpaired Student's t-test. SCD genotype and percent of subject who received transfusion were compared using a Chi square test. To account for differences in blood velocity according to vessel size, data were categorized into two groups comprised of vessel size 1 (≤ 19 μm) or vessel size 2 (>19 μm) based on the median of diameter measurements in all subjects (19 μm). Diameter and velocity measurements were averaged in each vessel size group, yielding 2 data points per subject. Diameter and velocity measurements were compared between vessel sizes in each FMT group using a paired t-test. A repeated measures two-way analysis of variance (ANOVA) was performed to determine the effects of FMT (with and without) and vessel size (size 1 and size 2) on axial blood velocity. A Pearson correlation coefficient was computed to assess the relationship between retinal thickness and axial blood velocity. Statistical analysis was performed using SPSS version 21 (SPSS Inc, Chicago, IL, USA). Statistical significance was accepted at P ≤ 0.05.

Results

Twelve and 10 subjects (eyes) were included in groups with and without FMT, respectively. Demographic and clinical data of SCR subjects are listed in Table 1. HCT levels were significantly lower in SS (25 ± 2%) than in SC (34 ± 6%) subjects (P = 0.008), but HCT levels of subjects with and without FMT were not significantly different (P = 0.1, unpaired t-test). Likewise, mean age and SCD genotype did not differ significantly between the two groups (p ≥ 0.1). The difference in MAP between subjects with and without FMT was marginally significant (p = 0.05). Central foveal subfield thickness was not significantly different between the two FMT groups (p = 0.1), while retina was significantly thinner in parafoveal and perifoveal temporal subfields of the subjects with FMT as compared to the subjects without FMT (p ≤ 0.04).

Table 1.

Demographic and clinical data of sickle cell subjects with and without focal macular thinning (FMT). Data are presented as mean and standard deviation or number and percentage.

Variables With FMT (N = 12) Without FMT (N = 10) P-Value
Age (years) 41 ± 13 35 ± 14 0.3
Genotype SS 6 (50%) 6 (60%) 0.6
Hematocrit (%) 32 ± 7 27 ± 4 0.1
Mean Arterial Blood Pressure (mmHg) 96 ± 12 87 ± 8 0.05
Central Foveal Subfield Thickness (μm) 255 ± 27 303 ± 84 0.1
Parafoveal Temporal Subfield Thickness (μm) 295 ± 25 334 ± 56 0.04
Perifoveal Temporal Subfield Thickness (μm) 258 ± 21 290± 42 0.03
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Figure 2 shows examples of conjunctival microcirculation images in two eyes with and without FMT. In two venules with the same diameter of 19 μm, axial blood velocity measurements were 0.35 mm/s and 0.75 mm/s in the eye with and without FMT, respectively. In eyes with FMT, venule diameter ranged between 12 and 25 μm and axial blood velocity ranged between 0.30 and 0.84 mm/s. In eyes without FMT, venule diameter ranged between 12 and 26 μm, and axial blood velocity ranged between 0.25 and 1.08 mm/s.

Figure 2.

Figure 2

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Examples of conjunctival microcirculation images obtained in two sickle cell retinopathy subjects without (Left) and with (Right) focal macular thinning (FMT). The outlined edges of two selected conjunctival venules (red lines) were automatically identified for diameter measurements (19 μm in both subjects). Space time images (inserts) were derived based on the movement of red blood cells or plasma gaps along the vessel lengths, depicting prominent diagonal bands. Axial blood velocity was measured as the slope of the overlaid red line which represents the prominent bands. Axial blood velocity was lower in the subject with FMT.

Mean values of diameter and axial blood velocity, categorized by vessel size and FMT groups are listed in Table 2. As expected, vessel diameter was significantly larger in vessel size 2 as compared with vessel size 1 in both FMT groups (P < 0.001, paired t-test). Vessel diameter measurements were not significantly different between the FMT groups for both vessel sizes (P > 0.1). There was a significant effect of FMT on axial blood velocity (P = 0.04, ANOVA), while the effect of vessel size on axial blood velocity was not significant (P = 0.4). There was not a significant interaction between FMT and vessel size (P = 0.6). In vessel size 1, axial blood velocity was lower in eyes with FMT than in eyes without FMT (P = 0.03), while in vessel size 2, axial blood velocity did not differ significantly between FMT groups (P = 0.1). In vessel size 1, there was a significant positive correlation between axial blood velocity and retinal thickness in the perifoveal (r = 0.48, P = 0.02) and parafoveal (r = 0.43, P = 0.04) temporal subfields. In vessel size 2, axial blood velocity was not significantly correlated with retinal thickness in perifoveal or parafoveal temporal subfields (P ≥ 0.8).

Table 2.

Diameter and axial blood velocity, categorized by vessel size, in with and without FMT groups.

Variable Vessel Size With FMT (N = 12) Without FMT (N =10) P Value
Diameter (μm) 1 16 ± 2 16 ± 2 0.4
2 23 ± 3 26 ± 5 0.1
P Value < 0.001 < 0.001
Velocity (mm/s) 1 0.45 ± 0.11 0.64 ± 0.24 0.03
2 0.49 ± 0.23 0.78 ± 0.59 0.1
P Value 0.5 0.5
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Discussion

Prevention of vision loss in subjects with SCR is of clinical importance. The underlying causes of retinal ischemia in SCR are increased viscosity, venostasis, and microvascular occlusion due to disease-related abnormalities in hematological and hemodynamic properties (Emerson et al. 2006). Previous studies have demonstrated the presence of vaso-occlusions and hemodynamic alterations in the conjunctiva of SCD subjects (Paton 1961; Roy et al. 1985; Cheung et al. 2002; Lima et al. 2006; Cheung et al. 2010; Wanek et al. 2013). However, to our knowledge, the conjunctival microcirculation has not been assessed in relation to the presence and absence of macular thinning. In the present study, we demonstrated reduced conjunctival venule blood velocity in small caliber vessels in SCR subjects with focal macular thinning.

The finding of reduced conjunctival venular blood velocity in SCR subjects with FMT supports our hypothesis that systemic hematological abnormalities that cause more severe or frequent retinal microvascular occlusions and consequent retinal thinning also may manifest in alterations of conjunctival microvascular hemodynamics. In fact, SCD subjects with severe conjunctival vaso-occlusive signs have been reported to have greater range of red blood cell density and slower macular capillary blood flow (Roy et al. 1985; Roy et al. 1995). In the current study, reduced blood velocity was observed in smaller venules, which are more prone to occlusions than large vessels. In addition, the finding of a significant positive correlation between retinal thickness and axial blood velocity in smaller venules suggests that factors which contribute to retinal ischemia and thinning may also play a role in conjunctival microvascular hemodynamics. Furthermore, diameter measurements were similar between subjects with and without macular thinning in both vessel size categories. This finding may suggest that the reduced blood flow was sufficient to meet the presumably lower metabolic requirements of the conjunctiva, therefore not requiring vasodilation. Further studies are needed to establish a direct association between alterations in retinal microvascular hemodynamics and retinal thinning.

Alterations in blood velocity were observed in conjunctival venules, even though microvascular occlusive events occur mostly in the arteriole side of the capillary network. Hematological abnormalities due to SCD result in arteriole stenosis and erythrocyte aggregation in venules, thereby increasing vascular resistance (Bishop et al. 2001), and reducing the overall perfusion pressure. These factors which are likely more prevalent in subjects with SCR and retinal thinning may account for the reduced blood velocity observed in venules of the conjunctiva.

Axial blood velocity in the microvasculature has been previously reported to be different between SCD genotype and also dependent on systemic factors (Pries et al. 1996; Koutsiaris et al. 2007; Wanek et al. 2013). It is unlikely that these factors significantly affected our finding of reduced blood velocity in eyes with macular thinning because age, HCT, vessel diameter, and the percentage of SCD genotypes were similar between the two groups. Additional studies in a larger population will be useful to assess differences in conjunctival hemodynamics in groups stratified by both retinal thinning and SCD genotype. In the current study, there was a significant difference in blood pressure between subjects with and without macular thinning. The higher mean blood pressure in SCR subjects with macular thinning may be indicative of higher peripheral resistance due to widely disseminated occlusions in the microvasculature throughout the body. Future studies are needed to substantiate the presence of increased resistance due to vaso-occlusions by quantifying conjunctival occlusions (Roy et al 1985) and calculating microvascular areas in the conjunctiva and the retina from fluorescein angiograms.

One limitation of the current study is that fluorescein angiography was not performed to document peripheral retinal ischemia, since it is not a standard of care for stage 2 SCR. However, a previous study has shown a relationship between retinal ischemia and thinning, which were assessed by fluorescein angiography and OCT, respectively (Murthy et al. 2011). Moreover, the effects of conjunctival comma signs, extreme tortuosity or kinking in adjacent vascular segments on conjunctival blood velocity were not investigated in the current study. Future prospective studies are needed to evaluate their effects on conjunctival hemodynamics in SCR subjects. Overall, assessment of conjunctival microvascular hemodynamic properties may be of potential value for gauging microcirculatory abnormalities due to SCR.

Acknowledgments

Supported by NIH research grants EY017918 (MS), NIH core grant EY001792, senior scientific investigator award (MS) and an unrestricted departmental grant from Research to Prevent Blindness, and Gerhard Cless Retina Research Fund (JIL). The authors would like to thank Dr. Norman P. Blair for fruitful discussions.

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