Neuroretinal Dysfunction With Intact Blood-Retinal Barrier

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Diabetes Volume 63, November 2014
Aldina Reis,1,2,3 Catarina Mateus,1 Pedro Melo,1,2 João Figueira,1,2,3 José Cunha-Vaz,1,2,3 and
Miguel Castelo-Branco1
Neuroretinal Dysfunction With
Intact Blood-Retinal Barrier and
Absent Vasculopathy in Type 1
Diabetes
COMPLICATIONS
Diabetes 2014;63:3926–3937 | DOI: 10.2337/db13-1673
It is unknown whether independent neural damage may
occur in the pre-/absent vascular diabetic retinopathy
(DR). To exclude vasculopathy, it is important to measure
the integrity of the blood-retinal barrier (BRB). This crosssectional study addressed this problem in type 1 diabetic
patients with normal ocular fundus and absent breakdown of the BRB (confirmed with vitreous fluorometry).
These were compared with a group with disrupted BRB
(with normal fundus or initial DR) and normal controls.
Multifocal electroretinography and chromatic/achromatic
contrast sensitivity were measured in these 42 patients
with preserved visual acuity. Amplitudes of neurophysiological responses (multifocal electroretinogram) were
decreased in all eccentricity rings in both clinical groups,
when compared with controls, with sensitivity >78% for
a specificity level of 90%. Implicit time changes were also
found in the absence of initial DR. Impaired contrast sensitivity along chromatic axes was also observed, and achromatic thresholds were also different between controls
and both clinical groups. The pattern of changes in the
group without baseline BRB permeability alterations, as
probed by psychophysical and electrophysiological measurements, does thereby confirm independent damage
mechanisms. We conclude that retinal neuronal changes
can be diagnosed in type 1 diabetes, independently of the
breakdown of the BRB and onset of vasculopathy.
Diabetic retinopathy (DR) is a major cause of vision loss
(1). The prevalence of DR in type 1 diabetic patients is
very high (;90% after 15 years of disease duration), as
shown by long-term epidemiological studies (2). Among
the several known risk factors for DR development, disease duration is indeed one of the most relevant (3–6),
together with metabolic control (5,7). Nevertheless, a fraction of patients presenting good metabolic control (;10%)
do develop DR, and still another proportion of patients
with poor metabolic control, nevertheless, do not develop
this complication (8). The role of vasculopathic mechanisms in DR is well established. Accordingly, classification
of ocular fundus changes is relevant to the management
and treatment of complications of this disease (9–11). The
concept of a preretinopathy stage is characterized by the
absence of lesions in ocular fundus examination and of
the presence of subclinical changes in the blood-retinal
barrier (BRB) (3,12,13). Leakage of this barrier has been
proposed as the earliest manifestation in DR (3).
It is possible that independent neural damage may also
occur in diabetes. This is difficult to study in the presence
of concomitant vascular damage. However, if early neural
damage occurs, it can be studied by searching for evidence
of neuroretinal changes in the absence of retinal microvasculopathy. This is possible in the pre-DR stage (14–17).
Some functional studies have suggested visual impairment in diabetic patients without apparent DR, but these
studies did not exclude changes in integrity of the barrier
function using a quantitative method (2,18–25). The occurrence of structural changes indicating vascular damage is
also quite disputed in type 1 diabetic patients at a preclinical
1Visual Neuroscience Laboratory, Institute for Biomedical Imaging in Life Sciences,
Faculty of Medicine, University of Coimbra, Coimbra, Portugal
2Association for Innovation and Biomedical Research on Light and Image, Coimbra,
Portugal
3Coimbra University Hospital, Coimbra, Portugal
This article contains Supplementary Data online at http://diabetes
.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1673/-/DC1.
Corresponding author: Miguel Castelo-Branco, [email protected].
See accompanying article, p. 3590.
Received 29 October 2013 and accepted 6 May 2014.
© 2014 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit, and
the work is not altered.
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stage (23,26), possibly because the way patients are phenotyped may vary across studies. The criterion for determining the absence of DR lesions in type 1 diabetic
patients is indeed a common problem across studies, which
often are solely based on retinography or standard angiography. Definition of early vascular (endothelial) changes,
as defined above, concerning measures of inner barrier
function, may provide a more clear pathophysiological cutoff. Thus we aimed at establishing objective criteria for the
absence or presence of initial vasculopathy by including
the quantitative measurements of the permeability of
the BRB.
Type 1 diabetic patients without clinical evidence for
DR may show impaired objective responses to stimuli of
distinct spatial frequencies (19), sometimes concomitantly associated with different contrast levels (18). A
link of such deficits with parvo- and magnocellular impairment is, however, missing. Besides the changes in achromatic contrast sensitivity (20,22), there are also studies
showing changed psychophysical and physiologic changes
in these patients without DR, related to color contrast
sensitivity (24,25) and multifocal electroretinogram
(mfERG) responses (21). It remains unclear whether these
changes can be present independently of vascular damage
(e.g., if they could reflect direct neural damage).
The current study aimed to circumvent the abovementioned methodological limitations by providing, as
stated above, a clear-cut pathophysiological cutoff between presence and absence of BRB damage (quantitative
analysis of BRB permeability with vitreous fluorometry
[VF]). We combined this measure with objective structural
and functional (electrophysiological) measures of visual
function, as well as psychophysical thresholds. In summary, we investigated retinal neural dysfunction in a
population of type 1 diabetic patients, from the psychophysical and neurophysiological point of view, with
characterization of BRB leakage and metabolic control
level. The goal was to identify whether neural damage can
occur independently of alterations of the BRB and normal
fundus images.
RESEARCH DESIGN AND METHODS
Patient Selection, Demographic Characteristics, and
Ophthalmological Examination
The study followed the tenets of the Declaration of
Helsinki and was approved by our ethics committee.
Prior to the inclusion in the study, informed consent was
obtained from all subjects after a full explanation.
We tested 42 eyes of 42 patients (mean age 6 SD =
26.6 6 5.3 years) with preserved visual acuity (VA) and
divided them into two groups: one group without any
clinical signs of DR and normal BRB permeability as
assessed by VF (n = 14 eyes; VA = 1.11 6 0.15) and
another group with BRB breakdown/vasculopathy with no
clinical signs of DR or mild nonproliferative DR (n = 28 eyes;
VA = 1.06 6 0.14). These data were compared with those
obtained in 25 age-matched controls (27.4 6 5.8 years).
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Sample sizes were sufficient to probe evidence for isolated
neural damage as assessed by electrophysiological measures
as the main outcome (amplitude of central retinal responses,
R1, sample size calculation for a a = 0.05 and power = 0.8;
predicted effect size of 30 nV/deg2 between controls and
patients, as expected from our previous data and the literature cited in the article’s introduction and DISCUSSION).
The criterion for choosing the study eye was the one
with larger number of lesions, when applicable. If no DR
signs were visible and BRB permeability values were
normal, the right eye was chosen if the year of the patient’s
birthday was an even number and the left eye if it was an
odd number. This last principle (to prevent bias) was also
applied to healthy controls.
Participants were submitted to a complete ophthalmological examination, including the best-corrected VA, slit
lamp examination of anterior chamber, intraocular pressure measurement (Goldmann applanation tonometer),
angle and fundus examination (noncontact lens), and
subjective visual complaints. Exclusion criteria included
media opacities, neuroophthalmological/retinal diseases
(besides DR), and high ammetropies (sphere . 64D; cylinder .6 2D).
Setting
Data were collected at the Institute for Biomedical
Imaging in Life Sciences, the Association for Innovation
and Biomedical Research on Light and Image, and the
University Hospital of Coimbra until 2011.
Laboratory Analysis—Glycated Hemoglobin
In order to evaluate the patients’ metabolic control level,
blood samples were collected for analysis of glycated hemoglobin (HbA1c ). This was assessed by high-performance
liquid chromatography (Variant II, Bio-Rad).
Characterization of Ocular Fundus and BRB Status
Color Fundus Photography
Color fundus photographs (35°) of both eyes were taken
in seven standard fields of the retina according to Early
Treatment Diabetic Retinopathy Study (ETDRS) procedures (9), with a Topcon TRC-50IA Retinal Ophthalmic
Camera with a Sony DXC-950P digital camera with a resolution of 0.5 MPixels/768 3 576. These photographs
were always obtained after psychophysical and electrophysiological assessment to prevent artifactual changes
in retinal adaptation.
Fluorescein Angiography
Besides color fundus photography, fluorescein angiography (FA) was sequentially performed (TRC-50 IA Topcon;
digital camera: B/W Megaplus 1.4i, resolution of 1
MPixel/1,340 3 1,037) to increase sensitivity in detecting
topographic changes on BRB permeability.
A red-free photograph was taken before 10% fluorescein sodium solution administration, which was injected
in the antecubital vein in a volume adjusted to the patients’
weight prior to VF.
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Pure Neuroretinal Dysfunction in Diabetes
A series of 20 early-phase pictures during the arterial
and capillary filling were taken in the eye with larger
number of lesions, when applicable. If no DR signs were
visible, the right eye was chosen if the year of the
patient’s birthday was an even number and the left eye
if it was an odd number. After this series, a maculacentered photograph was taken from the contralateral
eye, followed by acquisition of quadrantic field images
of the selected and the contralateral eyes, respectively.
Bilateral late-phase pictures (5 and 10 min) were finally
taken in order to first classify in a qualitative manner the
presence or absence of leakage to help substantiate the
classification of the DR level (10).
VF
VF is a valuable tool for studying the intraocular fluid
dynamics and the permeability of the blood-ocular barriers, namely, the BRB. VF brought an important clinical
contribution for quantifying BRB permeability in DR and
is of particular interest in the preclinical stage since
diabetic eyes show increased permeability values even in
apparently normal ocular fundi (27,28).
For this purpose, we used a commercial fluorophotometer, the FM-2 Fluorotron Master (Coherent, Palo
Alto, CA) and followed the standard protocol for BRB
permeability measurement (29). A dose of 14 mg/kg was
injected, and measurements of fluorescein concentration
in the posterior vitreous were obtained 1 h after injection,
after discounting for the natural fluorescence of the ocular tissues (preinjection scan). Blood samples were collected
at 10, 15, and 50 min after injection for posterior measurements of the plasmatic fluorescein concentration (a
cuvette that was adapted to the optical head of the Fluorotron was used for measuring the concentration of fluorescein in plasmatic solutions). With all these parameters,
it was possible to obtain the penetration ratio (PR) of the
fluorescein across the BRB (30).
Functional Assessment
We have applied several psychophysical and electrophysiological methods to characterize visual function and estimate
neurophysiological parameters that could be used to study
structure-function correlations in our groups.
Psychophysics
Chromatic Contrast Sensitivity. Chromatic contrast sen-
sitivity was tested along three parallel, randomly interleaved staircases (31) (Cambridge Colour Test, CRS,
Rochester, U.K.). We simultaneously assessed the three
cone confusion axes, modulated in the CIE 1976 u’v’ color
space. The chromatic stimulus (a Landolt-like C-shaped
ring) appears over the noisy pattern of gray circles of
different sizes/luminance levels, and participants chose
the gap position in a forced four-choice task. Stimuli were
presented monocularly on a 21-inch g-corrected monitor
(GDM-F520; Sony, Tokyo, Japan; refresh rate 100 Hz) at
a viewing distance of 1.8 m in a darkened room. Far
vision refractive correction was worn by participants,
Diabetes Volume 63, November 2014
and tinted contact/spectacle lenses were replaced by
lenses in a trial frame when necessary. The test ended
after 11 reversals of each adaptive staircase, and the
mean of the last 7 reversals was obtained as the threshold expressed in confusion vector length for each protan,
deutan, and tritan axes in CIE 1976 u’v’ color space units
(32,33).
Achromatic Contrast Sensitivity. Achromatic contrast sen-
sitivity within the magnocellular pathway was probed using
a perimetric test based on frequency-doubling technology
(FDT) (33–35). Vertically oriented sinusoidal grating stimuli
(0.25 cpd, 25 Hz) were presented monocularly using a Humphrey Matrix Perimeter (Carl Zeiss Meditec Inc.).
An N-30-F (nasal 30°) threshold testing strategy was
chosen. This procedure consisted of a modified binary
search using a four-reversal rule to determine the threshold level at each of the 19 tested locations. The range of
possible raw data are between 0 dB (maximum contrast/
lowest patient sensitivity) and 56 dB. The formula to
calculate sensitivity in dB units was log10*(2,048/c)*10*H,
where c ranges from 1 to 2,048 (scaled minimum and
maximum contrast, respectively) and H (Humphrey scaling
factor) is ;2.
Statistical analysis was performed considering both
global parameters (mean deviation [MD] and pattern SD
[PSD]) and contrast sensitivity pooled from five regions:
the 5° central area (C) and the four visual field quadrants
(superior temporal [ST], superior nasal [SN], inferior nasal [IN], and inferior temporal [IT]).
Neurophysiology
The electrophysiological procedures mentioned below
were performed with pupil dilation.
mfERG
mfERG was recorded using RETIscan (Roland Consult,
Germany) with DTL-Plus electrodes to measure local
photopic activity within photoreceptor/bipolar cell circuitry (36). The stimulus consisted in a 30° central visual
field hexagonal pattern with 61 elements adjusted for the
magnification factor, pseudo-randomly presented binocularly according to the standard m-sequence on a 20-inch
cathode ray tube monitor (rate 60 Hz; viewing distance
33 cm). Participants’ far vision refractive correction was
compensated, when applicable, according to the adjustment scale for this purpose, and near refractive correction
was added if necessary.
Active voltage range of the signal was 6200 mV using
a band-pass filter of 5–100 Hz and amplification at a gain
of 100,000. Eight 47-s cycles were obtained for averaging
at an artifact rejection level of 10%.
Analyses were performed using the first-order kernels.
For each hexagon and concentric ring (eccentricities, in
diameter of visual angle: ring 1, 4.4°; ring 2, 4.4–13.6°;
ring 3, 13.6–25.8°; ring 4, 25.8–40.8°; ring 5, 40.8–58.7°),
the amplitude (nV/deg2) and implicit times (ms) of the N1
trough and the P1 peak were calculated.
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Reis and Associates
Table 1—Demographic and clinical characterization of the 42 included diabetic patients
HbA1c
Age
(years)
Diabetes
duration (years)
%
mmol/ mol
Vascular lesions
(no/yes)
ETDRS grading
(study eye)
1
29
12
6.6
49
No
10
2
28
17
8.4
68
Yes
20
3
25
15
6.2
44
No
10
4
23
15
8.7
72
Yes
20
5
25
12
7.2
55
No
10
6
14
7
7.8
62
No
10
7
15
13
9.6
81
Yes*
10
8
25
17
9.3
78
Yes*
10
9
30
24
7.0
53
No
10
10
23
12
11.6
103
Yes
35D
11
26
15
8.7
72
No
10
12
27
15
13.0
119
Yes
35D
13
28
17
11.1
98
Yes
20
14
29
14
6.7
50
No
10
15
28
18
10.0
86
Yes
20
16
25
17
7.6
60
Yes
20
17
16
12
9.9
85
No
10
18
23
12
6.8
51
No
10
19
25
17
6.5
48
Yes
20
20
31
27
8.1
65
Yes
35D
21
29
24
9.1
76
No
10
22
29
22
9.1
76
Yes
35D
23
23
10
8.9
74
No
10
24
33
22
7.3
56
Yes
35C
25
27
19
7.3
56
No
10
26
29
21
8.1
65
Yes
20
27
19
13
7.6
60
No
10
28
24
14
6.4
46
Yes*
10
29
31
15
8.2
66
Yes
20
30
23
12
8.4
68
Yes
20
31
18
16
7.9
63
Yes
20
32
31
13
8.0
64
Yes
35D
33
29
25
7.1
54
Yes
20
34
35
19
6.9
52
Yes
20
35
37
29
8.9
74
Yes
35D
36
34
26
10.1
87
Yes
20
37
35
25
6.5
48
Yes
35C
38
32
29
7.4
57
Yes
20
39
24
17
8.6
70
Yes
35C
40
29
13
8.5
69
No
10
41
22
20
11.1
98
Yes
35C
42
31
20
9.6
81
Yes
20
Patient ID
*Criterion for vascular lesions based on VF results.
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Statistical Analysis
Given the observed relative violation of ANOVA assumptions, nonparametric analyses (Mann-Whitney U testing/
Spearman rank correlation) at a significance level of P ,
0.05 were performed using StatView (SAS, Cary, NC), with
correction for the number of group comparisons.
Given the conservative approach of only including one
eye per participant (see RESEARCH DESIGN AND METHODS), we
also replicated this analysis with an approach that uses all
eyes, with correction for dependence using generalized
estimating equations (see Supplementary Data with this
replication analysis).
Receiver operating characteristic (ROC) analyses (MedCalc, version 12.2.1.0, Mariakerke, Belgium) were performed to determine sensitivities at defined levels of
specificity (;90%) and to compare differences between
controls and patients without signs of DR and preserved
BRB. Diagnostic accuracies of psychophysical and electrophysiological tests were assessed by comparing areas under the ROC curves (AUCs). Statistic significance was set
at P , 0.05.
RESULTS
Table 1 provides a global summary of the demographic
and clinical data of the included diabetic patients (age,
diabetes duration, HbA1c, and presence/degree of vascular ocular fundus lesions). Group analyses are presented
below.
Demographic Characteristics and Ophthalmological
Characterization
The study group included 42 eyes of 42 type 1 diabetic
patients (14–37 years; mean age 6 SD = 26.6 6 5.3 years)
with preserved VA (best-corrected values ranging from
0.9 to 1.3) divided into two subgroups according to the
values of BRB permeability assessed by VF and the ETDRS
classification system (based on color fundus photography
and FA analysis): one group with normal BRB permeability (#3.1 3 1026 min21) and no fundus signs of DR (n =
14 eyes; VA = 1.11 6 0.15) and another group that included patients with mild nonproliferative DR, or no DR
but increased BRB permeability values (n = 28 eyes; VA =
1.06 6 0.14).
The mean duration of disease ranged from 7 to 29
years (group with no DR, mean 6 SD = 14.43 6 4.86
years; group with initial DR, mean 6 SD = 18.93 6
5.04 years).
All participants completed the protocol, so no cases of
missing data were present.
Diabetes Volume 63, November 2014
to 9.9% (85 mmol/mol; mean 6 SD values = 7.74 6 1.11%
[61 6 12.1 mmol/mol]). In the group with disruption
of BRB, HbA1c varied from 6.2% (44 mmol/mol) to
13.0% (119 mmol/mol; mean 6 SD values = 8.70% 6
1.64% [72 6 17.9 mmol/mol]). This difference did
not reach statistical significance (P = 0.07).
Characterization of BRB Status
Color Fundus Photography and FA
These two methods were jointly used to classify the DR
level: 10 (17 eyes, 40.5%), 20 (15 eyes, 36%), 35C (4 eyes,
9.5%), and 35D (6 eyes, 14%). See Table 1 for more details
(right column).
Measures of BRB Permeability Using VF
In the group with disrupted BRB, PR values (1026 min21)
ranged from 1.78 to 12.39 (mean 6 SD = 4.78 6 2.79),
while in the other group, these values ranged from 1.08 to
2.81 (mean 6 SD = 2.12 6 0.49). These are below the
limit for normal values (#3.1 3 1026 min21) identified in
the literature, thereby enabling us to define the participants in this group as having normal BRB permeability
(37) according to the inclusion criteria.
Table 2 provides a detailed characterization of the diabetic group characterized by no changes in BRB and,
simultaneously, by the absence of signs of DR.
Functional Assessment
Figure 1 shows different examples of psychophysical and
neurophysiological data in three patients with different
patterns of damage (patients 5 and 29 present no DR,
patient 5 with minimal functional changes and patient
29 with clear impairment on visual function testing; patient 4 had initial DR).
Table 2—Characterization of the diabetic patient group with
no changes in BRB (£3.1 3 1026 min21) and DR ETDRS
grading of 10
VA
Disease
BRB PR
(study
duration
(study
Patient
Sex
eye)
(years)
eye)
1
Male
1.3
12
1.88
3
Male
1.3
15
1.72
5
Male
1.0
12
1.53
6
Female
1.0
7
2.81
9
Male
1.3
24
1.82
11
Male
1.3
15
2.20
14
Male
1.3
14
1.08
17
Male
1.0
12
2.34
Analysis of Metabolic Control—HbA1c
18
Male
1.0
12
2.64
These patients had thorough and systematic local surveillance of metabolic control levels since childhood, at the
time of their diagnosis of diabetes: one visit to the
endocrinology department, every 6 months in the initial
follow-up, and one visit yearly thereafter. In the group
with normal BRB, HbA1c varied from 6.2% (44 mmol/mol)
21
Female
1.0
24
1.91
23
Male
1.0
10
2.63
25
Male
1.0
19
2.05
27
Female
1.0
13
2.50
40
Female
1.0
13
2.51
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Figure 1—Representative examples of some results of two patients without DR (patient 5 without DR and minimal changes on functional
testing; patient 29 without DR with more changes than patient 5) and a patient with initial DR (patient 4), with three microaneurisms
identified. A: FA. B: Achromatic contrast sensitivity (FDT). C: Standard mfERG (C1: plots; C2: analysis by concentric rings).
Psychophysics
Chromatic Contrast Sensitivity—Evidence for Damage
of Both Parvocellular and Koniocellular Pathways Even
in Patients with Absent Leakage and DR. As illustrated
in Fig. 2, chromatic contrast sensitivity measures were
significantly different between controls and patients.
Considering the group with disrupted BRB, the comparison with controls showed higher thresholds for all chromatic axes (protan, P = 0.007; deutan, P , 0.0001;
tritan, P , 0.0001). Importantly, significantly higher
thresholds were also found, but only for the tritan axis
in the group with normal BRB permeability and no DR
(P = 0.009). When comparing both patient groups, significant differences are only found for the deutan axis
(P = 0.01).
Achromatic Contrast Sensitivity—Evidence for Early
Magnocellular Impairment. MD, a global parameter of
magnocellular performance (FDT testing; see RESEARCH
yielded significant differences between controls and each group of patients (with normal
BRB permeability and no DR, P = 0.002; with leakage
and/or DR, P = 0.0002), with no significant effect
detected between patient groups. Concerning PSD (field
heterogeneity measure), a significant difference was
found between controls and patients with diabetes without leakage and no DR (P = 0.02) and no difference
between patients groups. For details, see Fig. 3A.
Please note that MD and PSD provide essentially
different information, and MD is more useful as a lesion
indicator since it defines absolute/global damage. This
value is higher in the group of initial DR, as expected.
DESIGN AND METHODS)
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Figure 2—Chromatic contrast thresholds in patients and in controls, where impairment can be already observed in the group with no DR
and normal BRB permeability. Percentile data depicted. mNPDR, mild nonproliferative DR.
To probe whether regional differences were underlying
these effects, we then analyzed magnocellular achromatic
contrast sensitivity in distinct regions. Significant differences were found in all five locations, between controls
and both patient groups, except in the C region and IT
quadrant of the visual field in patients with no lesions
(group with preserved BRB and no DR: ST, P = 0.04; SN,
P = 0.01; IN, P = 0.058—group with disrupted BRB: C, P =
0.02; ST, P = 0.01; SN, P = 0.003; IN, P , 0.0001).
Comparisons between both patient groups showed no significant differences (see Fig. 3B for details).
Neurophysiology
mfERG—Direct Evidence for Neural Dysfunction in the
Absence of Leakage and DR. The mfERG showed a signif-
icant decrease in amplitude in all concentric rings in both
patient groups when compared with control subjects (P1
wave, all eccentricity rings, P , 0.0001 in the group of
Figure 3—Magnocellular contrast sensitivity expressed in whole-field deviation (MD) and in regional homogeneity (PSD) measures in
patients and in controls (A), as well as in quadrantic thresholds (B). Percentile data depicted. mNPDR, mild nonproliferative DR; Temp.
Sup., ST; Nasal Sup., SN; Nasal Inf., IN; Temp. Inf., IT.
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disrupted BRB—group with preserved BRB, R1, P , 0.0001;
R2, P = 0.0003; R3–R5, P , 0.0001) (Fig. 4A), suggesting
a neural basis for damage at the outer retinal level (photoreceptor and bipolar cell circuit level) since dysfunction
occurs irrespective of evidence for early vascular damage.
Analysis of implicit times showed a significant decrease
in patients with preserved BRB and no DR, as compared
with controls, but only in ring 2 (P = 0.006). This difference is also evident in ring 2 (P = 0.02) in the group with
disrupted BRB (see Fig. 4B for details).
A similar pattern was found concerning the N1 component:
decrease in amplitude values in both patient groups (with
disrupted BRB, R2, P = 0.001; R3, P = 0.0003; R4, P = 0.0006;
R5, P , 0.0001—with preserved BRB, rings 2–5, P , 0.0001),
with no changes in implicit times (Fig. 5A and B).
It is important to point out that no significant differences were obtained for amplitude and implicit time
values of P1 and N1 waves between both clinical groups.
Structure and Function Correlations
Correlations with BRB Permeability
Significant correlations could be observed between PR
and functional measures in the group with leakage, namely,
in chromatic thresholds (deutan, r = 0.39, P = 0.04; tritan,
r = 0.37, P = 0.05) and in mfERG P1-wave implicit time
(most peripheral rings, R5, r = 0.46, P = 0.002).
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Correlations with Duration of Diabetes
Duration of disease correlated with implicit time of
mfERG, namely, P1 wave (most peripheral rings, R4,
r = 0.43, P = 0.03; R5, r = 0.44, P = 0.02; and marginally
lost in ring 1, r = 0.25, P = 0.06) and N1 wave (ring 1, r =
0.44, P = 0.02), in the group with BRB changes. In the
other clinical group, duration of diabetes only correlates
with a chromatic protan thresholds axis: r = 0.72, P =
0.002. These findings suggest distinct pathophysiological
mechanisms in the presence or absence of BRB breakdown.
ROC Curve—Sensitivity/Specificity Analysis
ROC curves were generated for all included tests, allowing us to compare sensitivities (probability of positive
testing results when the clinical condition is present) for
detecting impairment at fixed specificity levels. In our
analysis, the chosen cutoff for this parameter was set
near 90%. The highest sensitivity values were found in
mfERG amplitude values (P1 wave, sensitivities of 78%
across the five concentric rings). N1 wave amplitude
values also showed high sensitivities (up to 82%) in
paracentral rings, except in the central ring. Chromatic
contrast tests showed the lowest sensitivity, in particular, concerning protan and deutan axes (Table 3 and
Fig. 6).
Figure 4—P1 wave amplitude (A) and implicit time (B) of mfERG across concentric rings. Decreased amplitude values are found in both
patient groups, showing a different pattern in implicit time (decreased values in the group with no DR). Percentile data depicted. mNPDR,
mild nonproliferative DR.
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Diabetes Volume 63, November 2014
Figure 5—N1 wave amplitude (A) and implicit time (B) of mfERG across concentric rings. Percentile data depicted. mNPDR, mild nonproliferative DR.
Diagnostic Accuracy by AUC
The AUC summarizes the diagnostic accuracy of each
parameter (AUC = 1 would represent a perfect discrimination between controls and diabetic patients with no
signs of DR/preserved BRB; AUC = 0.5 means discrimination at the chance level). Our results showed AUCs
between 0.946 and 0.532 (Table 3). The higher AUC values (P , 0.0001) correspond to the mfERG test (P1 and
N1 wave amplitudes, except ring 1 of the N1 wave).
DISCUSSION
In this study, we have found evidence for an independent
neural phenotype in pre- or absent vascular DR in type 1
diabetes. This was possible to establish by using a clear
pathophysiological cutoff defining the onset of endothelial lesions. This cutoff is based on in vivo objective
measures of BRB permeability.
Retinal neuronal changes occurring in type 1 diabetic
patients with no breakdown of the BRB or onset of
vasculopathy were probed by psychophysical (achromatic/
chromatic contrast sensitivity) tests of ganglion cell
magno-, parvo- and koniocellular pathways and electrophysiological recordings (mfERG). The latter suggest
a neural correlate of dysfunction at the outer retina.
Interestingly, we found that neurophysiological changes
correlate with increased BRB permeability in the group
of patients with leakage, which confirms the existence of
an independent mechanism in the absence of BRB disruption. This corroborates the idea that isolated neural
damage can occur even when the (usually dominant)
vascular damage is not present. The fact that these
measures were more tightly linked with disease time
duration in the group with vascular lesions suggests that
vascular damage superimposed on primary and secondary
neuronal changes can dominate during the subsequent
period in the natural history of disease. These issues can
best be clarified in a longitudinal study.
Concerning the functional pathways that were affected
even in the prevascular stage, we identified damage of
both parvocellular and koniocellular pathways (as demonstrated by chromatic contrast sensitivity testing). Some
previous studies also report significant color vision
defects in type 1 diabetes (24,25). However, these studies
did not provide a clear cutoff between prevascular and
vascular stages.
Impairment of magnocellular function was also observed (as revealed by decreased thresholds in FDT in C
and peripheral regions), even in the group with normal
BRB measures and without clinical signs of DR. Previous
studies reported a nonselective decrease in contrast sensitivity (18,19,22,38). Our study used a specific perimetric
test biased to the activation of the magnocellular performance, probing central and peripheral vision, and our
results are consistent with a previous study documenting
diabetes.diabetesjournals.org
Reis and Associates
3935
Table 3—AUC and associated 95% CI for the studied testing variables comparing controls and diabetic patients with ETDRS
grading of 10 and preserved BRB
Parameters
AUC
95% CI
P value
Sensitivity/
specificity (%)
Criterion value for
90% specificity
Cambridge Colour Test
Protan
Deutan
Tritan
0.646
0.586
0.794
0.498–0.776
0.438–0.724
0.656–0.895
0.1164*
0.3893*
0.0001
21/89
14/89
50/89
51
49
73
FDT
MD
PSD
C
ST
SN
IN
IT
0.805
0.738
0.691
0.721
0.760
0.711
0.670
0.676–0.899
0.602–0.847
0.552–0.808
0.584–0.834
0.625–0.865
0.573–0.825
0.530–0.791
,0.0001
0.0072
0.03189
0.0098
0.0020
0.0118
0.0540
43/90
57/90
36/88
50/90
50/90
36/90
36/93
21.51
4.26
29.50
28.38
29.00
29.60
29.50
ERG—P1 amplitude
Ring 1
Ring 2
Ring 3
Ring 4
Ring 5
0.938
0.867
0.900
0.912
0.921
0.822–0.988
0.730–0.950
0.772–0.970
0.787–0.976
0.799–0.981
,0.0001
,0.0001
,0.0001
,0.0001
,0.0001
79/90
79/90
79/90
71/90
79/90
63.1
33.87
22.76
13.74
10.40
ERG—P1 IT
Ring 1
Ring 2
Ring 3
Ring 4
Ring 5
0.500
0.790
0.721
0.693
0.702
0.346–0.654
0.641–0.898
0.566–0.846
0.536–0.823
0.546–0.831
1.0000*
0.0011
0.0169
0.0478
0.0272
14/93
71/90
50/90
50/90
43/90
42.4
34.38
33.69
32.91
33.23
ERG—N1 amplitude
Ring 1
Ring 2
Ring 3
Ring 4
Ring 5
0.721
0.881
0.912
0.926
0.891
0.562–0.848
0.743–0.960
0.784–0.977
0.801–0.984
0.757–0.966
0.0201
,0.0001
,0.0001
,0.0001
,0.0001
46/90
85/90
85/90
85/90
85/90
11.20
9.59
7.12
4.18
3.54
ERG—N1 IT
Ring 1
Ring 2
Ring 3
Ring 4
Ring 5
0.580
0.715
0.586
0.554
0.603
0.418–0.730
0.555–0.843
0.424–0.736
0.393–0.708
0.441–0.751
0.5013*
0.0302
0.3982*
0.5869*
0.3576*
39/93
46/90
31/93
15/90
31/86
18.75
13.80
12.80
12.80
13.30
Sensitivities were obtained for each parameter at ;90% specificity. Criterion values related with this specificity are also presented.
mfERG results (in particular, the P1 wave amplitude) show the highest sensitivity values for a fixed specificity (;90%). *Nonsignificant
values.
FDT changes in diabetic patients with no clinically detectable DR (39,40). mfERG findings showed generalized decrease amplitudes that are consistent with changes at
photoreceptor/bipolar cell circuits. Recent studies (41–43)
reported local neurophysiological changes in type 1 diabetes, as assessed by mfERG. Although these local neurophysiological changes show the expected interindividual
variability (in terms of range of amplitude values), ROC and
AUC analyses showed that relatively high sensitivity is observed for electrophysiological measures at high specificities,
confirming the presence of sensitive early measures of
specific neural damage that may potentially be used as
biomarkers for detecting neural changes independently
of vascular lesions.
Accordingly, studies by the Adams group have shown
that local mfERGs are highly predictive of the subsequent
development of DR in adult patients, suggesting the
location of future lesions, based on previous local changes
in implicit time (44–46).
In our study, we also found early mfERG changes.
However, these are not directly comparable to the abovementioned results because of group definition criteria.
Changes in wave morphology and amplitude might lead to
different forms of change and even decreases in the local
implicit time values, especially in cases of amplitude
reduction. However, in patients with minimal DR, implicit
time tended to increase. As mentioned above, small
differences across studies might be due to the criterion
for defining the absence of retinal lesions. In previous
studies, it was based on fundus photography and not on
an increase in BRB permeability, which is the first sign of
vascular damage (3,12,13). In other words, BRB permeability
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Diabetes Volume 63, November 2014
Figure 6—ROC curves generated for mfERG (P1 wave amplitude of the five concentric rings) depicting that relatively high sensitivity values
correspond to a specificity of 71–79%, with AUC ;0.9.
measurements may be critical to precisely define the presence or absence of a vascular phenotype.
In summary, our study uniquely combined objective
measures of the BRB permeability and DR staging to define
diabetic patient groups with and without vascular damage.
A neural phenotype could then be investigated with
functional information gathered from psychophysical and
neurophysiological measures. We found that these could
discriminate between groups with high sensitivity and
specificity, as confirmed by ROC analyses, which support
the generalizability of our results. These findings show that
neural changes may probably occur in the retina in addition
to and independently of vascular lesions, with potential
implications for early neuroprotective treatment (47).
The main limitation of this study is that it is crosssectional and cannot infer on the relative time courses of
neural and vascular processes. Moreover, the BRB permeability assay may be differently sensitive to vascular
pathology in different regions of the retina when compared with retinal function testing across broad regions.
Future longitudinal studies should elucidate the time
course of isolated diabetes-related neural impairment in
relation to the likely dominant neural phenotype secondary to vascular damage that is also present in the natural
history of DR.
We conclude that retinal neuronal changes can occur in
type 1 diabetic patients independently of the breakdown
of the BRB or vasculopathy.
Funding. This research was supported by Fundação para a Tecnologia: FCT/
PTDC/SAU/NEU/68483/2006, COMPETE/PEst-C/SAU/UI3282/2013; the National
Brain Imaging Network of Portugal, Comissão de Coordenação Região Centro,
CENTRO-07-ST24-FEDER-00205; and Agência de Inovação, QREN-COMPETE/
DoIT/Diamarker.
Duality of Interest. No potential conflicts of interest relevant to this article
were reported.
Authors Contributions. A.R. designed the experiments, acquired and
analyzed data, and wrote and edited the manuscript. C.M. acquired and analyzed
data. P.M. and J.F. acquired data. J.C.-V. contributed to the discussion. M.C.-B.
designed the experiments, analyzed data, contributed to the discussion, and
wrote the manuscript. A.R. and M.C.-B. are the guarantors of this work and,
as such, had full access to all the data in the study and take responsibility for the
integrity of the data and the accuracy of the data analysis.
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