Globally, the number of people with diabetes is expected to reach 552 million in 2030
(1). Both the macrovascular (coronary artery disease, peripheral artery disease, and
stroke) and microvascular (retinopathy, nephropathy, and neuropathy) complications
of diabetes are major causes of morbidity and mortality (2). In theory, the long preclinical
phase should provide a window to apply interventions that preempt progression to clinical
disease, but to date no strategy achieves this aim for diabetes complications. Intensive
control of hyperglycemia and blood pressure implemented with the imperfect means available
today delays the appearance and reduces the severity of most complications, but does
not prevent their development (3). Drugs such as fenofibrate have reduced the risk
of diabetic retinopathy (DR) progression in type 2 diabetic patients, and inhibitors
of the renin-angiotensin system (e.g., enalapril, losartan) have reduced the risk
of progression in type 1 diabetic patients, but have not reduced the risk of DR incidence.
Thus, new approaches are required to derive further benefit from current interventions
at an earlier stage of diabetes and to support the development of new interventions.
One such approach is to identify biomarkers that will enable systematic patient surveillance
and the identification of high-risk patients and also surrogate end points that will
accelerate the discovery of new interventions.
The retinal vessels are early and prevalent targets of diabetic damage. They can be
seen, measured, and tested by noninvasive means (4). Hence, investigators have attempted
over the decades to identify early changes in the retinal vessels in diabetes and
determine whether they could inform about the development of retinopathy and other
complications. New measuring techniques have steadily come along, and others are in
development. It seems timely to assess which concepts have emerged and which needs
must be met toward fulfilling the expectation that retinal vessels may be able to
predict the clinical onset of complications. The National Institutes of Health (NIH)/JDRF
convened a workshop on 3–4 October 2011, in Bethesda, Maryland, titled “Seeing the
Development of Diabetes Complications: Retinal Vessels as Biomarkers.” From the presentations
and discussions at the workshop emerged that, to date, changes in retinal vascular
caliber appear to be among the earliest changes detected in the retina in diabetes.
In addition, changes in vascular caliber have implications relevant to the pathophysiology
of the vascular and end-organ damage observed in the eye and elsewhere in diabetes.
Thus they fulfill important requirements for being investigated as biomarkers of diabetes
complications (3). In this review we will focus on retinal vascular caliber as a potential
biomarker for the three major microvascular complications of diabetes: retinopathy,
nephropathy, and neuropathy.
CURRENT STATUS OF EARLY BIOMARKERS FOR DIABETES COMPLICATIONS
Biomarker definition and assessment
A biomarker is defined as “a characteristic that is objectively measured and evaluated
as an indicator of normal biological processes, pathogenic processes, or pharmacological
responses to a therapeutic intervention” (5). A biomarker useful in the prevention
of a clinical end point (e.g., retinopathy) would 1) noninvasively detect early preclinical
disease before the first clinical signs of the end point (e.g., microaneurysms) appear,
2) be causally linked or be an indicator of a causal mechanism that leads to the development
of this end point, and 3) be consistently and strongly associated with the end point
(5). Additional characteristics would expand its clinical usefulness: a marker informative
for an individual patient would facilitate clinical decision making while a marker
that reliably measures reversibility of the disease process could become a surrogate
end point in drug development and preventative treatment. Finally, the incremental
value of a novel biomarker over standard available biomarkers should be assessed not
only with standard statistical measures such as the area under the curve (AUC) of
the receiver operating characteristic curve or C-statistic, but also with more recently
developed measures such as weighted net reclassification index (wNRI) and net benefit
(NB) (5). Standard measures such as AUC give information on the overall improvement
in discrimination over the full range of potential decision thresholds, whereas decision/analytic
performance measures such as wNRI and NB indicate clinical usefulness over a smaller
range of medically relevant thresholds. Furthermore, it is important to realize the
distinction between the assessment of the quality of predictions from a model and
decisions (or classifications) from a rule (5). This distinction between a “prediction
model” and a “prediction rule” is unclear in most of the current literature. The key
element is that going from a prediction model to a prediction rule requires the definition
of a decision threshold or cutoff. Standard measures such as AUC deal with models
and not rules. In contrast, these novel measures such as wNRI and NB try to incorporate
medically relevant thresholds. A good model is, however, the first step in creating
a good rule (5).
DR
DR does not currently have risk biomarkers that satisfy the above criteria, but there
is research in several areas. Measurements of retinal hemodynamics at steady state
show variable directions of the abnormalities in diabetic patients (6), in part attributable
to the influence of blood glucose levels at the time of testing (7). Measurements
of retinal hemodynamics in response to stimuli have shown abnormal autoregulation
of retinal blood flow in DR patients, with flow increasing in parallel with the severity
of retinopathy (8). However, further development of abnormal autoregulation as a biomarker
will need to address the scant data available in patients without retinopathy, the
inconsistency of abnormalities detected, and the longitudinal validation with the
development of clinical retinopathy.
Measurements of retinal oxygen metabolism—which is thought to be affected in DR—using
a noninvasive retinal oxymeter have shown elevated oxygen saturation levels in both
retinal arterioles and venules in DR patients, suggesting that maldistribution and
shunting and thus decreased delivery of oxygen from blood to tissue may be a feature
of clinically overt DR. However, not all studies have been able to demonstrate these
elevated oxygen saturation levels. Furthermore, it is not known whether retinal oxymetry
is useful in the preclinical phase of DR as an indicator of disease risk. Controversial
is whether markers of inflammation and endothelial dysfunction increase in the systemic
circulation before the appearance of clinical retinopathy, especially in type 1 diabetic
patients (9,10), and it is unknown whether one or more of those abnormalities can
predict clinical DR.
Fluorescein angiography has aided in the assessment of DR for several decades by enhancing
the sensitivity of detection of the classical DR lesions: microaneurysms, increased
capillary permeability, focal areas of capillary nonperfusion, and retinal neovascularization.
However, the invasive nature of angiography discourages the use of this technique
in the search for preclinical abnormalities with biomarker potential.
Diabetic nephropathy
Similar efforts are underway to discover biomarkers for diabetic nephropathy (11).
Biomarkers such as albuminuria, which can predict rate of progression of overt nephropathy,
have limited predictive precision at earlier stages of disease. Microalbuminuria,
even after persisting for up to two years or more, can spontaneously regress to normoalbuminuria,
and patients may remain microalbuminuric for many years without progression to overt
diabetic nephropathy. Therefore, microalbuminuria, although a strong indicator of
increased diabetic nephropathy risk, is not a sufficiently reliable predictor of diabetic
nephropathy, and other biomarkers are needed to supplement the measure of urinary
albumin in diabetic patients (12). Kidney biopsy samples can show early disease but
the predictive power is limited and the biopsy procedure is too invasive for routine
use (13,14). Some promising diabetic nephropathy biomarkers currently being studied
include the decline of glomerular filtration rate and serum uric acid (11).
Diabetic neuropathy
Diabetic neuropathy encompasses a wide array of clinical conditions including autonomic
neuropathy and distal sensorimotor polyneuropathy. For the latter, two biomarkers
show potential in the detection of damage to small, unmyelinated fibers, an early
sign of diabetic neuropathy (15). The intraepidermal nerve fiber density (IENFD) from
3-mm skin biopsies is a sensitive but invasive method for detecting early changes
in neuropathy (16). Corneal confocal microscopy is a noninvasive ophthalmic method
that detects corneal nerve fiber abnormalities in early and more advanced neuropathy
and that relates to IENFD and measures of neuropathy (17). More recently corneal confocal
microscopy has shown an improvement in corneal nerve abnormalities but with no change
in IENFD or electrophysiology 12 months after pancreas transplantation in patients
with type 1 diabetes (18).
ABNORMALITIES OF RETINAL VASCULAR CALIBER AS A BIOMARKER OF DIABETES MICROVASCULAR
COMPLICATIONS
Since the invention of the ophthalmoscope nearly 150 years ago and the first reports
of retinal vascular changes (e.g., retinal arteriolar narrowing, venular widening,
hemorrhages, microaneurysms) being linked with systemic diseases, numerous studies
have advanced the concept that retinal measurements may predict a range of systemic
diseases. Within the last decade, these retinal measurements have become increasingly
sophisticated and computer based, leading to the development of software that measures
quantitatively retinal arteriolar and venular calibers. Typically, these softwares
(e.g., Interactive Vessel Analysis [IVAN]) measure the width of the erythrocyte column,
which approximates the internal lumen diameter. These are usually summarized and expressed
as the central retinal artery (CRAE) and the central retinal vein (CRVE) equivalents
(Fig. 1) (4). These software methods have been applied in multiple population-based
studies, comprising more than 50,000 people from different racial/ethnic groups in
the general population and diabetic cohorts. These data have provided a clearer understanding
of epidemiology, risk factor associations, and prognostic significance of “static
measurements of retinal vascular caliber.” The broad understanding is that changes
in retinal vascular caliber reflect a range of subclinical pathophysiologic responses
to hyperglycemia, hypertension, inflammation, hypoxia, and endothelial dysfunction,
and can predict not only different diabetes microvascular complications, but also
stroke and coronary heart disease (19–22). This review will not cover the latter relationship
of retinal vascular caliber with stroke and coronary heart disease.
Figure 1
Retinal fundus photograph assessed quantitatively by the IVAN software (University
of Wisconsin). The measured area of retinal vascular parameters was standardized as
the region from 0.5 to 1.0 disc diameters away from the disc margin. Retinal arteriolar
and venular calibers were summarized as CRAE and CRVE, respectively, from retinal
fundus photograph. CRAE and CRVE were defined based on the revised Knudtson-Parr-Hubbard
formula.
Retinal vascular caliber and DR
In terms of diabetes microvascular complications, studies have primarily focused on
the association between retinal vascular calibers and the risk of DR both in type
1 and type 2 diabetes. Table 1 presents an overview of these studies (23–27). For
type 1 diabetes, the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR)
was the first to investigate the relationship of arteriolar and venular caliber to
the incidence and progression of DR (23). In this study, while controlling for glycosylated
hemoglobin A1c, duration of diabetes, blood pressure level, and other risk factors,
larger retinal arteriolar and venular calibers measured at baseline were associated
with an increased risk of progression of DR but were not associated with the 4-year
incidence of DR. Similarly, in the New Jersey 725 study, which included 468 African
Americans, neither arteriolar nor venular calibers were related to incident DR (24).
In contrast, a study of 645 Australian children and adolescents (aged 12–20 years)
with type 1 diabetes who were initially free of retinopathy showed that larger retinal
arteriolar caliber at baseline was associated with a more than threefold higher risk
of DR after a median follow-up of 2.5 years (hazard rate ratio [HR] 3.44 [95% CI 2.08–5.66])
(25). The increased risk was recorded after adjusting for a variety of vascular risk
factors, diabetes duration, glycemic control, and retinal arteriolar caliber (25).
In this study no association was found for retinal venular caliber with incident retinopathy.
In summary, there are indications that retinal vascular calibers may be associated
with the risk of both incidence and progression of DR-related outcomes in type 1 diabetes,
though the data are not entirely consistent.
Table 1
Relationship of baseline retinal vascular calibers with incidence and progression
of DR in type 1 and type 2 diabetes
For type 2 diabetes, data from WESDR and Australian Diabetes, Obesity and Lifestyle
(AusDiab) studies show that neither retinal arteriolar nor venular calibers measured
at baseline were associated with the incidence or progression of DR (26,27). Several
factors may underlie the discrepancy between the findings in patients with type 1
and 2 diabetes. Although four out of these five studies used the same procedures and
definitions to obtain DR cases, differences across these studies in the distribution
of age and other cardiovascular risk factors, sample sizes, and follow-up periods
may partially explain this discrepancy. In these studies, patients with type 1 diabetes
were below the age of 30 years, whereas those with type 2 diabetes had a mean age
of >60 years. Accompanying this wide age distribution, the most important difference
between patients with type 1 and 2 diabetes was the prevalence of hypertension. In
WESDR, for example, 74% of the patients with type 2 diabetes were diagnosed with hypertension,
whereas the corresponding figure in type 1 diabetes patients was about 17% (23,27).
Because the effect of hypertension on retinal vascular calibers may be in the opposite
direction than found in diabetes, this confounding effect of hypertension may have
led to the inconclusive results found in type 2 diabetes.
In addition to measuring the predictive capabilities of baseline retinal vascular
calibers, two studies have examined whether longitudinal changes in these calibers
are related to the risk of DR. Widening of the venules over the first 4 years of WESDR
was independently associated with increased risk of subsequent 6-year incidence and
progression of DR as well as incidence of proliferative DR and diabetic macular edema
in both type 1 and 2 diabetes, which is similar to the results from a Swedish study
(28,29). In view of the wide range of calibers normally exhibited by retinal vessels,
changes occurring over time in the same individual may be more robust and sensitive
informers of ongoing vascular processes than the mere caliber at baseline. Additional
prospective studies should be undertaken to confirm and investigate a role for longitudinal
changes in retinal vascular calibers in predicting the risk of DR.
Retinal vascular caliber and diabetic nephropathy
The Renin Angiotensin System Study (RASS), a randomized controlled clinical trial
of angiotensin-converting enzyme inhibition or angiotensin receptor blockade versus
placebo in normoalbuminuric, normotensive people with type 1 diabetes, showed that
both retinal arteriolar caliber at baseline and changes in retinal arteriolar and
venular calibers over a 5-year period are independently related to changes in renal
structural parameters (such as glomerular basement membrane width, mesangial matrix
fractional volume, and glomerulopathy index) measured in sequential biopsies over
the same time period (30). These renal structural parameters reflect important pathological
processes which, if progressive, ultimately lead to renal dysfunction in diabetic
nephropathy. Among 927 persons with type 2 diabetes from WESDR, retinal venular caliber
was independently associated with the 10-year incidence of diabetic nephropathy, which
was defined as the development of gross proteinuria, initiation of renal dialysis,
or renal transplantation during follow-up (26). Compared with persons in the first
quartile of retinal venular caliber, those in the fourth were nearly twice as likely
to develop diabetic nephropathy (HR 2.08 [95% CI 1.47–2.94]). In contrast, retinal
arteriolar caliber was not related to the risk of diabetic nephropathy (26). In the
same WESDR study, these associations were also confirmed in 557 patients with type
1 diabetes and 16-year incidence of gross proteinuria and renal insufficiency (31).
Retinal vascular caliber and diabetic neuropathy
Thus far, only a few studies have examined the link between retinal vascular caliber
and diabetic neuropathy. A study of a multiethnic Asian population with diabetes showed
that wider arteriolar caliber, but not venular caliber, was independently and cross-sectionally
associated with peripheral neuropathy as defined from neurothesiometer or monofilament
sensory testing (multivariable adjusted odds ratio [OR] 2.81 [95% CI 1.38–5.73]) (32).
In contrast, a population-based Danish study among 208 long-surviving type 1 diabetic
patients showed that persons with smaller retinal arteriolar caliber were more likely
to have nephropathy (OR per SD decrease 2.17 [95% CI 1.29–3.68]), but not neuropathy
(1.10 [0.70–1.71]) (31). Retinal venular caliber was not associated with nephropathy
or neuropathy (33). Prospective studies are needed to define the link between retinal
vascular caliber and diabetic neuropathy.
NOVEL STATIC RETINAL VESSEL CHARACTERISTICS AS BIOMARKERS FOR DIABETES COMPLICATIONS
While most current studies have focused on the measurement of retinal vascular caliber,
there are other measurable parameters in the retinal vascular architectural network
(34). Changes in the retinal vascular architecture may result in impaired space-filling
and microcirculatory transport, nonuniform shear distribution across bifurcations,
and reduced energy efficiency in blood flow and may therefore be an indicator of early
disease (35,36).
A number of retinal vascular “architectural” parameters, including fractal dimension,
tortuosity, branching angle and length-to-diameter ratio, and measures of bifurcation
optimality have been studied using computer-assisted software (37,38). Figures 1–4
show a currently available semiautomated computer-based program, the Singapore I Vessel
Assessment (SIVA), that measures a spectrum of retinal vascular parameters in addition
to vascular caliber.
At present, the clinical utility of changes in retinal vascular architecture is limited,
as it remains unknown what constitutes an abnormal or pathological value of these
retinal vascular parameters. Recent studies indicate that some of the newer quantitative
retinal vascular parameters are associated with diabetes (39) and associated complications,
such as hypertension (40), stroke (41), and cardiovascular mortality (42). However,
at present there are few studies on the relationship of these measures with DR and
other microvascular complications. Furthermore, the small number of studies, lack
of overall consistency, the relatively small increase in predictive value, and the
costs of measuring these additional vascular characteristics, limit extending their
use more broadly and point to the need for more research in this area.
For example, fractal geometry can be used to quantify the branching pattern that exhibits
the property of self-similarity (Fig. 2). Fractal parameters may be a useful way of
determining microvascular density by assessing its space-filling properties. To date,
although fractal analysis studies of the retinal vasculature have demonstrated excellent
reproducibility (43), the ability of this method to predict clinical outcomes is less
clear. Cross-sectional studies have identified fairly weak relationships (relative
risks ranging from 1.37–1.45 per SD decrease in fractal dimension) with clinical disease
(chronic kidney disease, diabetic macroalbuminuria, and diabetic neuropathy) (39,44),
while a prospective study found no association between retinal vascular fractal dimension
and incident retinopathy (45). The use of fractals in assessing retinal vasculature
may be limited by several factors 1) vascular beds are multifractal rather than single
fractal and 2) differences in the various methods used to image and compute fractal
dimension (43,45
46).
Figure 2
a: Fractal dimension is a measure of a fractal structure that exhibits the property
of self-similarity, characterizing the distribution of the branching vascular system
in two-dimensional space. In SIVA, fractal dimension is calculated from a skeletonized
line tracing that uses the box-counting method and divides each digital photograph
into a series of squares for various side lengths, and the number of boxes is counted.
b: Low fractal dimension. c: High fractal dimension.
The tortuosity of vessels is difficult to quantify (Fig. 3) and multiple measures
have been described (e.g., simple, curvature) (42). Nonetheless, some cross-sectional
studies show modest associations between tortuosity and DR, and between tortuosity
and diabetic kidney disease (47,48). Other geometric parameters (Fig. 4), such as
the length-to-diameter ratio, optimality deviation, and branching angle, also demonstrate
modest associations with retinopathy in cross-sectional and prospective studies (49),
as well as with cerebral white matter hyperintensities and coronary heart disease
(50). Thus, although advances in image analysis provide an opportunity to quantify
additional aspects of retinal vasculature, the value of these measurements as biomarkers
or surrogate end points is not yet known.
Figure 3
a: Simple tortuosity and curvature tortuosity are defined to quantify retinal vascular
tortuosity in SIVA. Simple tortuosity is estimated as the actual path length of the
vessel segment divided by the straight line length. Curvature tortuosity is derived
from the integral of the curvature square along the path of the vessel, normalized
by the total path length, which takes into account the bowing and points of inflection.
b: Low tortuosity. c: High tortuosity.
Figure 4
A series of parameters are defined to quantify bifurcation of retinal vessels in SIVA
(not bounded by the measured zone). At each bifurcation, the widths of the trunk vessel
(d0) and its two branching vessels (d1, d2) are measured, as well as the branching
angle. Branching angle (θ1 + θ2) is defined as the first angle subtended between two
daughter vessels at each bifurcation. Angular asymmetry is defined as the difference
between two daughter angles (θ1 − θ2). Branching coefficient is defined as the ratio
of the branching vessel widths to trunk vessel width (d1 + d2)2/d0
2. Asymmetry ratio is defined as the ratio of the two branching vessel widths d2
2/d1
2. Junctional exponent deviation is defined using the value of three in healthy vascular
networks [(|d0
3 − d1
3 − d2
3|1/3)]/d0. Length-to-diameter ratio is defined as the ratio of the length between
two branching points to the trunk vessel width (L/d0).
ABNORMALITIES OF RETINAL VASCULAR CALIBER AS BIOMARKERS OF DIABETES COMPLICATIONS–DYNAMIC
MEASUREMENTS
Recent functional studies have tested the behavior of retinal vessels in response
to stimuli and measured the changes in vascular diameter with the laser Doppler flowmeter
or the dynamic retinal vessel analyzer (IMEDOS, Germany). Their results complement
the static measurements in identifying retinal vascular caliber changes as early consequences
of diabetes with the potential to become biomarkers of risk for diabetes complications.
The functional abnormalities detected in patients with relatively well-controlled
type 1 diabetes and no retinopathy consist of reduced or absent dilation of retinal
vessels in response to light stimulation and reduced or absent constriction of retinal
arteries in response to increased intraluminal pressure (51–54).
Flickering light stimulates activity of the neural retina and the increased metabolic
demand activates autoregulatory mechanisms that dilate retinal blood vessels and enhance
blood flow. Whereas in healthy individuals retinal arterioles and venules increase
their diameter by 2–4% in response to diffuse luminance flicker, diabetic patients
show a minimal or absent response to this stimulus (51,52). The relative reduction
in retinal vessel dilation seen in diabetic patients occurs in vessels with resting
diameters not different from those of normal control subjects and may therefore precede
morphological widening of retinal vessels. The dilation of retinal vessels to flickering
light reflects both retinal neural activity and the vascular response, hence the reduced
or absent dilation seen in diabetes could be due to defects in either or both of these
components.
Responses to increased intraluminal pressure are easier to attribute to vascular mechanisms.
Normal arteries respond to elevations in intraluminal pressure with constriction and
to pressure reduction with dilation via mechanisms that are inherent to vascular smooth
muscle and independent of neural, metabolic, and hormonal influences. This behavior
is termed the myogenic response (53). Figure 5 illustrates the role of the normal
myogenic response in maintaining constant retinal blood flow when ocular perfusion
pressure is increased by a change in posture from sitting to reclining. A clinical
study showed that when such postural change was tested in well-controlled patients
with type 1 diabetes without retinopathy only about half of the 17 patients examined
manifested the normal vasoconstrictor response to increased pressure. The other half
reproducibly failed to constrict the retinal arteries, and some showed paradoxical
vasodilation (54). Of note, in the same study, only the patients who failed to vasoconstrict
in response to pressure showed an increase in resting arterial diameter over a 12-month
period (54), suggesting that the loss of myogenic responsiveness may be an initial
step in the process that widens the caliber of retinal vessels. According to Poiseuille
law, flow is related to the fourth power of the vessel radius. Thus, the loss of myogenic
response and subsequent increased arterial diameter would permit unphysiologically
high blood flow to reach the capillaries and affect the regulation of hydrostatic
pressure and eventually the integrity of the capillary wall. Further studies are required
to test if the defective myogenic response is in fact causally linked to the development
of clinical retinopathy and to validate its usefulness as a biomarker of risk. Also
to be investigated is whether an abnormal myogenic response of retinal vessels to
pressure could be a biomarker for the development of diabetic nephropathy because
the kidney, akin to the retina, has autoregulatory mechanisms to maintain stable blood
flow despite variations in systemic blood pressure (55).
Figure 5
Normal response of retinal arterial diameter, blood speed, and blood flow to a postural
change that increases ocular perfusion pressure. The hemodynamic parameters were measured
using a laser Doppler flowmeter in the superior temporal retinal artery of healthy
subjects while sitting (baseline), after lying down for 30 min, and again after sitting
for 20 min. The change in posture from sitting to reclining increased ocular perfusion
pressure and elicited the myogenic response, measured as a decrease in the arterial
diameter. The arterial diameter decreased in each subject, the decrease ranging from
−2.8 to −9.9% of baseline values. Blood speed increased in each subject, and blood
flow—calculated as the product of the cross-sectional area of the artery at the measurement
site and the average blood speed—did not change. Values are the means ± SD of measurements
in 11 subjects. (A high-quality color representation of this figure is available in
the online issue.)
NEWER METHODS FOR DYNAMIC STUDIES OF RETINAL VESSELS
Further, the development of retinal vascular caliber changes as a robust biomarker
of risk for retinopathy and other microvascular complications of diabetes depends,
in part, on the capability to detect in vivo subclinical changes caused by diabetes.
Instruments and strategies that can probe the vessels with enhanced resolution and
functional yield will be able to reconstruct mechanisms and consequences of the early
widening of retinal vascular caliber and to identify additional early abnormalities
with biomarker potential. The newer approaches briefly described below are poised
to widen the accessibility of hemodynamic measurements, permit observations of discrete
vascular structures at very high resolution, and test molecular events occurring on
the vascular endothelium.
Doppler Fourier domain optical coherence tomography to measure retinal blood flow
Doppler Fourier domain optical coherence tomography (OCT) imaging is now widely used
for clinical purposes, and the continuous enhancement of the speed of image acquisition
and resolution are making OCT an important tool for dynamic measurements. As a coherent
detection technique, OCT can detect the Doppler frequency shift of backscattered light
and thus measure the speed of moving objects such as erythrocytes that scatter light
and thereby provide information on blood flow velocity. The high-speed and ultrahigh-speed
Fourier domain OCT enables acquisition of blood velocity and vessel volumes that permit
measurements of pulsatile blood flow. Comparisons in normal and diseased human retinas
have been performed (56). The Doppler OCT, which is still in development at this time,
may eventually offer significant advantages over the methods used to date to measure
retinal blood flow. By measuring flow over the central retinal artery or at vessels
at the optic nerve head, Doppler OCT can provide total retinal blood flow as opposed
to flow in individual vessels. The measurements are depth-resolved, increasing the
accuracy of the data. The blood flow measuring capability is compatible with existing
standard OCT patient interfaces, and new strategies and software will likely render
the blood flow calculations completely automatic and facilitate the testing of its
use as a risk biomarker for diabetes complications (57). This will make it possible
to test and compare retinal autoregulatory responses and their longitudinal changes
in larger cohorts of patients using standardized instruments and methods as opposed
to instruments with different characteristics developed in different research laboratories.
Adaptive optics for detailed live imaging of human retinal vessels
The adaptive optics approach is based upon the optical properties of the eye and is
applied to small fields at any particular instant of measurement. Derived from astronomical
research tools, the technique offers the opportunity to visually “correct” for the
imperfect optics of the eye that result in a blurred point spread function. Real-time,
confocal scanning laser ophthalmoscope with adaptive optics currently achieves with
research-grade instruments resolution on the order of 2 microns in the human retina.
Structural imaging of the retina using adaptive optics enables the visualization of
vascular walls, vascular crossings, vascular cells, nerve fiber crossings, cells in
the vicinity of arcades, and capillaries (Fig. 6) (58). Dynamic blood flow can also
be measured with this approach, and flow maps can be obtained (59,60). Observations
are restricted to small fields at any given time, but this potential limitation is
counterbalanced by the high resolution and dynamic nature of the imaging. Adaptive
optics could be used to gain new insights into diabetes microvascular complications
through correlations between vascular cells or vessel function and topography within
the vascular network and within the retina. Currently only a few adaptive optics systems
exist worldwide, but with the recent appearance of commercial instruments there will
be more widespread testing of this technology in the pursuit of identifying the subclinical
changes that may become biomarkers of diabetes complications.
Figure 6
Example of adaptive optics imaging results from diabetic patients. a: Example of the
variability of capillary diameter in a diabetic patient. A single capillary can range
from a normal caliber (black-filled arrow) to distended (white-filled arrow) within
a few hundred microns. Scale bar = 50 microns. b: Examples of capillary tortuosity
and remodeling. Here we see capillaries that are tortuous and reverse directions.
Also visible are the variations in capillary diameter as seen in panel a. Scale bar
= 50 microns.
Fluorescent nanoparticle imaging to probe retinal vascular endothelium in vivo
The transition from physiology to pathology is likely to involve first a sequence
of molecular changes, eventually followed by cellular changes and structural damage.
Therefore, capturing the initial or very early molecular changes occurring in the
endothelium of retinal vessels in diabetes is of keen interest to the field. Recently,
new tools have been introduced that measure in vivo monomolecular interactions using
highly sensitive probes (61). The probes mimic the interaction of immune cells with
the retinal endothelium, a common event in the pathogenesis of several eye diseases,
including early DR. The transient interaction of these probes with the endothelial
ligands is detected by light-based fundus imaging, such as in scanning laser ophthalmoscopy.
This technology has permitted the quantitative evaluation of discrete molecular interactions
on the endothelium of rodent retinal vessels with a sensitivity that is comparable
to immunohistochemistry or PCR (62). A current challenge is to bring the technique
to testing in humans. The strategy is to custom design molecular probes using biodegradable
polymers in combination with fluorophores that are approved for human use. The advantages
of sensitivity and specificity inherent in the molecular probes, the quantitative
nature of the attained signal, and the automated image processing could make this
technique a unique tool for screening and monitoring early cellular events in diabetic
retinal microangiopathy, as well as measuring early responses to therapeutic interventions.
These, in turn, could expand the number of potential biomarkers of retinopathy risk
and early interventions to be tested.
CONCLUSIONS
Retinal blood vessels can be noninvasively observed and monitored and show changes
reflecting early pathophysiology of diabetes, supporting the concept that these changes
could translate into biomarkers of risk and surrogate targets for preemptive interventions.
In experimental diabetic animals, the investigation has yielded a large number of
biochemical, molecular, and cellular changes, and some of the changes were proven
to be of pathogenic importance in the development of experimental DR. But translation
to human diabetes has been slow and difficult, with the result that to date clinical
medicine does not have one validated and usable biomarker of retinopathy risk nor
drugs to help prevent the development of retinopathy.
Over the last decade, advances in retinal imaging techniques have allowed the measurement
and quantitative analysis of static retinal vessel characteristics. Testing of these
characteristics in large population-based studies shows that the caliber of the retinal
vessels provides information on the risk for DR and other microvascular complications
beyond traditional risk factors. Dynamic studies contribute complementary and mechanistic
data showing that defective vasoconstrictor and vasodilator responses to stimuli are
often the first detectable changes in the retinal vessels of diabetic patients without
retinopathy. One direction that builds on this information is to continue to develop
better and newer approaches to retinal vascular imaging and to enhance the power of
detection and the accuracy of quantification of retinal microvascular changes. A second
direction is to seek validation for the promising candidate biomarkers. For example,
longitudinal changes in retinal vascular caliber and dynamic responses to stimuli
should be tested against the development of clinical retinopathy as well as other
microvascular complications of diabetes. A third strategy is to quantify early molecular
changes using highly sensitive and specific molecular probes. The results of these
studies will tell whether retinal vessels can be used to “see” the early development
of diabetes complications. Positive results will generate the much sought-after opportunity
to implement cost-effective preventative and interventional strategies.