Diabetic macular edema (DME) is a common complication of diabetic retinopathy (DR) and a leading cause of visual loss in this population [
VEGF causes conformational changes in the tight junctions of the retinal vascular endothelial cells and plays a major role in the increased vascular permeability and BRB breakdown in diabetic eyes [
In our earlier study, we found that also the intravitreal uric acid (UA) concentrations correlated significantly with degree of DR [
Optical coherence tomography (OCT) has enabled clinicians to noninvasively evaluate the effect of DR on retinal thickness in a standard clinical setting [
The aim of our study was to analyse the vitreous and serum of diabetic patients with DME and severe NPDR and compare them to nondiabetic controls. The analysis focused on VEGF and UA as two possible pathogenetic factors in the development of DME. We compared blood and vitreous levels of VEGF, UA, and protein between the two study groups and describe their correlation with the changes seen in OCT.
This consecutive, prospective study involved 29 patients divided into two groups. First group involved 16 subjects with type 2 diabetes mellitus (DM) with NPDR and cystoid DME. In this group, the mean duration of DM was
Clinical and laboratory characteristic of diabetic subjects and nondiabetic controls.
Parameter | DME ( |
Control ( |
|
---|---|---|---|
Number of patients (men/women) | 4/12 | 1/12 | ns |
Age (years) | 71 (61–77) | 71 (66–74) | ns |
LogMAR BCVA | 1.0 (0.6–1.0) | 0.5 (0.5–0.6) |
|
Chronic kidney disease | 1 (6.2%) | 0 (0.0%) | ns |
Dyslipidemia | 8 (50.0%) | 4 (30.8%) | ns |
Hypertension | 14 (87.5%) | 10 (76.9%) | ns |
HbA |
51.5 (43.0–63.3) | NA | NA |
Serum albumin (g/L) | 42.7 (41.1–44.6) | 44.4 (39.6–45.2) | ns |
CRP (mg/L) | 1.6 (1.0–3.7) | 2.0 (0.8–3.6) | ns |
Data are expressed as median ± interquartile range or total number and %.
ns: not significant;
The diagnosis of DM was based on the WHO criteria [
At the time of the study, all patients were in a stable clinical condition without clinical or laboratory signs of acute inflammation. The research was approved by the Local Institutional Ethics Committee, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic. Data and sample collection was independent of all treatment decisions. It did not affect a patient’s access to treatment and fully complied with all ethical and legal requirements for noninterventional data collection in the Czech Republic. All patients gave written informed consent to the treatment, as well as data collection. The reported investigations were in accordance with the principles of the current version of the Declaration of Helsinki.
OCT examinations were performed one day before vitrectomy with spectral domain OCT (Cirrus HD-OCT, Carl Zeiss Meditec AG, Jena, Germany) using macular cube acquisition according to the manufacturer’s protocol. The macular cube 512 × 128 scan consists of 128 raster scans with 512 A-scans, within a 6 × 6 mm macular area. The mean central retinal thickness (CRT, i.e., central subfield thickness) from the internal limiting membrane to the retinal pigment epithelium at the fovea was defined as the mean retinal thickness in a 1 mm diameter circular zone concentred on the fovea. Also cube volume (CV) and cube average thickness (CAT) of the scanned area were calculated by Cirrus HD-OCT software and checked for accuracy. The CV is calculated from the 1 mm diameter zone and CAT from the central 6 mm diameter zone concentred on the fovea.
Based on previous studies that evaluated morphological changes in DME [
Vitrectomy was performed to improve visual acuity and to decrease retinal thickness in the macula. Each patient underwent standard three-port therapeutic pars plana vitrectomy using current surgical techniques (the Alcon CONSTELLATION Vision System). Before opening the infusion port at the start of the vitrectomy, undiluted vitreous samples were obtained and collected in sterile tubes (cca. 0.3 mL). Overnight fasting blood samples were drawn from the antecubital vein at the time of vitrectomy and used for biochemical assay. Samples of vitreous and serum were rapidly frozen after collection at −80°C.
Routine biochemical parameters of serum were determined by standard clinical-chemistry methods. The concentration of UA was estimated using enzymatic methods (uricase-peroxidase) with photometric detection (Modular, Roche, Germany). The low detection limit of the method was 30
All statistical analyses were performed using the SPSS version 16 (SPSS Inc., Chicago, IL, USA). We calculated the median with 1st and 3rd quartile (IQR, interquartile range). In 16 subjects, the intravitreal VEGF and in 3 subjects the intravitreal UA concentration were under the detection limit; these subjects were included in the statistical analysis to avoid selection bias. Hence, we used the nonparametric analysis for ordinal variables, and the concentrations under the detection limit were assigned “minor than other.” The comparison between DME group and control group was done by Mann-Whitney
Biochemical analysis of the vitreous showed significant differences between DM and control group in the concentration of VEGF, UA, and total protein but not albumin as shown in Table
Laboratory analysis of vitreous of diabetic subjects and nondiabetic controls.
Parameter | DME ( |
Control ( |
|
---|---|---|---|
VEGF (pg/mL) | 192.7 (140.9–523.5) | <LOD |
|
UA ( |
156.0 (86.0–209.0) | 70.0 (48.5–138.0) |
|
Albumin (mg/L) | 1050 (618–1780) | 550 (295–1495) | ns |
Total protein (g/L) | 6.3 (4.9–9.1) | 3.6 (3.1–4.2) |
|
Data are expressed as median with interquartile range.
ns: not significant,
LOD: limit of detection (31.2 pg/mL).
Vitreous concentrations of VEGF in diabetic versus control group. DM group
Vitreous concentrations of uric acid in diabetic versus control group. DM group
Vitreous concentrations of total protein in diabetic versus control group. DM group
In the diabetic group, UA concentration in vitreous correlated significantly with vitreous VEGF concentration (
Relationship between vitreous VEGF and vitreous UA concentrations in diabetic versus control group. Dashed lines represent limits of detection (VEGF = 31.2 pg/mL, UA = 30
Median of serum concentration of UA in diabetic patients was significantly elevated compared with the control group (337.0
There was a significant correlation between UA concentrations in serum and vitreous (
The median CRT, CAT, and CV did not differ significantly between both groups and are listed in Table
OCT parameters of diabetic subjects and nondiabetic controls.
Parameter | DME ( |
Control ( |
|
---|---|---|---|
CRT ( |
479.0 (421.5–661.3) | 498.0 (374.5–540.5) | ns |
CAT ( |
392.0 (329.8–414.3) | 332.0 (316.0–346.5) | ns |
CV (mm3) | 14.2 (11.9–15.0) | 12.0 (11.4–12.5) | ns |
SRD | 6 (37.5%) | 0 (0%) |
|
Data are expressed as median with interquartile range or total number and %.
ns: not significant,
CAT: cube average thickness, CRT: central retinal thickness, CV: cube volume, and SRD: serous retinal detachment.
In the diabetic group, there was a significant correlation between CRT and CAT (
Further, among all OCT parameters, only CV correlated significantly with the concentration of vitreous VEGF in the DM group (
The correlation of logMAR BCVA with changes in OCT parameters and vitreous content was also evaluated and we found it to be nonsignificant in both groups. There was also no correlation between OCT parameters and serous concentrations of UA or VEGF.
The results demonstrate that biochemical analysis of the vitreous showed significant higher concentrations of VEGF, UA, and total protein in DM and control group. Moreover, in patients with DME intravitreal levels of UA correlate significantly with intravitreal levels of VEGF. Furthermore, we found that the CV measured with Cirrus HD-OCT correlate significantly with the concentration of VEGF in the vitreous of patients with NPDR and DME.
In our earlier study, we showed that the levels of intravitreal UA correlated significantly with the degree of DR [
UA is a degradation product of metabolism and under normal conditions UA acts as an antioxidant. In diabetics, hyperglycaemia induces redox stress, which leads to consumption of the naturally occurring local antioxidants protecting capillary endothelium [
On the other side, high UA concentration in the vitreous of diabetic patients may also be a compensatory protective factor. Under experimental conditions, the VEGF-induced production of reactive oxygen species was attenuated by urate; however, it did not modify the VEGF-induced changes in permeability of monolayers [
It has to be elucidated whether UA is originating from leakage of retinal vessels, which is increased in DR, or from local production. Although total vitreous protein was significantly higher in the diabetic group compared to controls, its level did not correlate with both UA and VEGF. Furthermore, in the diabetic subjects we found no correlation between serum and vitreous level of UA. These findings support the local production of UA in DR. However, to be able to distinguish the origin of increased UA in the vitreous, further analyses, for example, with tagged UA, should be done.
Since there was a correlation between vitreous UA and VEGF but no correlation between vitreous UA and OCT parameters, we conclude that UA has a probable relation with diabetic microangiopathy and accordingly DR but not directly with the development of DME.
Recent studies have shown that VEGF causes conformational changes in the tight junctions of retinal vascular endothelial cells and plays a major role in the elevated vascular permeability in diabetic eyes with DME. It is well known that the vitreous VEGF levels correlate significantly with the severity of DR [
In the present study, there was no significant correlation between vitreous VEGF levels and CRT of diabetic patients. However, the results show that in DME increase in CV correlated with increased concentration of VEGF in the vitreous. Nevertheless, one caveat is that previous studies defined central retinal thickness differently. The retinal thickness at the central fovea in Funatsu et al. [
The mean CRT in DME is widely accepted as the new surrogate marker for evaluating treatment efficacy [
Like us, Sonoda et al. showed that there was no significant correlation between intravitreal VEGF levels and the amount of subretinal fluid in DME [
The strength of the study described here is that it determined the relationship between the levels of intravitreal biochemical parameters and retinal morphology at the same time. The limitation was the small sample size (29 eyes). This was caused by decreasing use of vitrectomy for DME and this curtailed collection of vitreous samples. Although our vitrectomy for DME might be considered overtreatment, it was a comparatively effective method as it stabilized the intraocular condition of DME and the efficacy was maintained for a long period [
Vitreous concentrations of uric acid and VEGF were significantly higher in DM subjects than in controls. Moreover, vitreous UA concentration correlated significantly with the vitreous VEGF concentrations in patients with NPDR and cystoid DME. Increased VEGF concentrations are known to be involved in the pathogenesis of DME. Our results suggest that, apart from VEGF, the role of UA in the pathogenesis and progression of DR should also be considered.
Comparing OCT parameters to the vitreous levels of UA and VEGF, we found that increased concentration of intravitreal VEGF in patients with NPDR and cystoid DME correlated with increase of cube volume calculated by Cirrus HD-OCT. Since DME usually affects the macular area and not only the foveal region, the assessment of the VEGF concentration in clinical practice using the cube volume is comprehensible. This OCT parameter could be used to assess the efficacy of anti-VEGF therapy.
No financial relationship with the organisation that sponsored the research exists.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was supported by research Projects DRO VFN64165, Prvouk P25/LF1/2, and SVV 260032-2015. The authors thank the laboratory staff for technical assistance.