Retinal microvascular complexity as a putative biomarker of biological age: a pilot study.

Physiological changes associated with aging increase the risk for the development of age-related diseases. This increase is non-specific to the type of age-related disease, although each disease develops through a unique pathophysiologic mechanism. People who age at a faster rate develop age-related diseases earlier in their life. They have an older "biological age" compared to their "chronological age". Early detection of individuals with accelerated aging would allow timely intervention to postpone the onset of age-related diseases. This would increase their life expectancy and their length of good quality life. The goal of this study was to investigate whether retinal microvascular complexity could be used as a biomarker of biological age. Retinal images of 68 participants ages ranging from 19 to 82 years were collected in an observational cross-sectional study. Twenty of the old participants had age-related diseases such as hypertension, type 2 diabetes, and/or Alzheimer's dementia. The rest of the participants were healthy. Retinal images were captured by a hand-held, non-mydriatic fundus camera and quantification of the microvascular complexity was performed by using Sholl's, box-counting fractal, and lacunarity analysis. In the healthy subjects, increasing chronological age was associated with lower retinal microvascular complexity measured by Sholl's analysis. Decreased box-counting fractal dimension was present in old patients, and this decrease was 2.1 times faster in participants who had age-related diseases (p = 0.047). Retinal microvascular complexity could be a promising new biomarker of biological age. The data from this study is the first of this kind collected in Montenegro. It is freely available for use.

Timing Is Important-Management of Metabolic Syndrome According to the Circadian Rhythm.

Physiological processes occur in accordance with a rhythm regulated by the endogenous biological clock. This clock is programmed at the molecular level and synchronized with the daily light-dark cycle, as well as activities such as feeding, exercise, and social interactions. It consists of the core clock genes, Circadian Locomotor Output Cycles Protein Kaput (CLOCK) and Brain and Muscle Arnt-Like protein 1 (BMAL1), and their products, the period (PER) and cryptochrome (CRY) proteins, as well as an interlocked feedback loop which includes reverse-strand avian erythroblastic leukemia (ERBA) oncogene receptors (REV-ERBs) and retinoic acid-related orphan receptors (RORs). These genes are involved in the regulation of metabolic pathways and hormone release. Therefore, circadian rhythm disruption leads to development of metabolic syndrome (MetS). MetS refers to a cluster of risk factors (RFs), which are not only associated with the development of cardiovascular (CV) disease (CVD), but also with increased all-cause mortality. In this review, we consider the importance of the circadian rhythm in the regulation of metabolic processes, the significance of circadian misalignment in the pathogenesis of MetS, and the management of MetS in relation to the cellular molecular clock.

The role of miRNA in colorectal cancer diagnosis: A pilot study.

Despite recent advances in diagnosis and treatment, colorectal cancer (CRC) remains the third most common cancer worldwide, and has both a poor prognosis and a high recurrence rate, thus indicating the need for new, sensitive and specific biomarkers. MicroRNAs (miRNAs/miRs) are important regulators of gene expression, which are involved in numerous biological processes implicated in tumorigenesis. The objective of the present study was to investigate the expression of miRNAs in plasma and tissue samples from patients with CRC, and to examine their potential as CRC biomarkers. Using reverse transcription-quantitative PCR, it was revealed that miR-29a, miR-101, miR-125b, miR-146a and miR-155 were dysregulated in the formalin-fixed paraffin-embedded tissues of patients with CRC, compared with the surrounding healthy tissue, and these miRNAs were associated with several pathological features of the tumor. Bioinformatics analysis of overlapping target genes identified AGE-RAGE signaling as a putative joint regulatory pathway. miR-146a was also upregulated in the plasma of patients with CRC, compared with the healthy control group, and had a fair discriminatory power (area under the curve, 0.7006), with 66.7% sensitivity and 77.8% specificity. To the best of our knowledge, this distinct five-miRNA deregulation pattern in tumor tissue, and upregulation of plasma miR-146a, were shown for the first time in patients with CRC; however, studies on larger patient cohorts are warranted to confirm their potential to be used as CRC diagnostic biomarkers.

Quality of Life of Elementary School Students with Type 1 Diabetes in a Developing Country during the COVID Pandemic.

Type 1 diabetes (T1D) is a condition that affects all aspects of life, and thus is closely related to the quality of life itself. Dealing with it during the COVID-19 pandemic is a big challenge. A case-control study conducted in Montenegro at the end of 2021 included 87 elementary school students with T1D and 248 of their peers as controls matched by gender. Standardized questionnaires were distributed to participants (Peds-QL Generic core 4.0 questionnaire for all participants and Peds-QL Diabetes Module 3.2 only for cases). Based on them, the results of obtained scores were measured and compared using non-parametric statistical methods in relation to gender, region and type of household. Children with T1D reported lower quality of life comparing to matching controls with lower scores in almost all domains. Differences in the same domains among patients and their classmates were also observed in the different gender subgroups, environment type subgroups and in the central region. Results of the study provide insights to prioritizing actions for children with diabetes care as well as for public healthcare planning.

Inflammation-Related microRNAs-146a and -155 Are Upregulated in Mild Cognitive Impairment Subjects Among Older Age Population in Montenegro.

BACKGROUND: Pathological and clinical features of Alzheimer's disease (AD) are in temporal discrepancy and currently accepted clinical tests provide the diagnosis decades after the initial pathophysiological events. In order to enable a more timely detection of AD, research efforts are directed to identification of biomarkers of the early symptomatic stage. Neuroinflammatory signaling pathways and inflammation-related microRNAs (miRNAs) could possibly have a crucial role in AD, making them promising potential biomarkers. OBJECTIVE: We examined the expression of circulatory miRNAs with a documented role in AD pathophysiology: miR-29a/b, miR-101, miR-125b, miR-146a, and miR-155 in the plasma of AD patients (AD, n = 12), people with mild cognitive impairment (MCI, n = 9), and normocognitive group (CTRL, n = 18). We hypothesized that these miRNA expression levels could correlate with the level of participants' cognitive decline. METHODS: The study participants completed the standardized interview, neurological examination, neuropsychological assessment, and biochemical analyses. miRNA expression levels were assessed by RT-PCR. RESULTS: Neurological and laboratory findings could not account for MCI, but miR-146a and -155 were upregulated in the MCI group compared to the control. miR-146a, known to mediate early neuroinflammatory AD events, was also upregulated in the MCI compared to AD group. ROC curve analysis for miRNA-146a showed 77.8% sensitivity and 94.4% specificity and 66.7% sensitivity and 88.9% specificity for miR-155. CONCLUSION: Determination of circulatory inflamma-miRs-146a and -155 expression, together with neuropsychological screening, could become a non-invasive tool for detecting individuals with an increased risk for AD, but research on a larger cohort is warranted.

Trends in nationwide incidence of pediatric type 1 diabetes in Montenegro during the last 30 years.

Significant and unexplained variations in type 1 diabetes (T1D) incidence through the years were observed all around the world. The update on this disorder's incidence is crucial for adequate healthcare resource planning and monitoring of the disease. The aim of this study was to give an update on the current incidence of pediatric T1D in Montenegro and to analyze incidence changes over time and how the exposure to different factors might have affected it. This retrospective cohort study included a total of 582 patients younger than 15 years who were newly diagnosed with T1D during the past 30 years. The average age at diagnosis was 8.4 ± 3.91 years. The mean annual incidence of T1D in the Montenegro population during the whole study period of 30 years was 15.2/100,000 person-years. Slightly higher incidence rates were observed in male compared to female individuals, and the incidence increased with age, with the highest incidence in the 10-14 age group. If the model is observed as one without jointpoints, the annual percentage change (APC) for the total population is 3.1 (1.8-4.4); for male individuals, 3.8 (2.1-5.5); and for female individuals, 2.1 (0.6-3.5). In 2020, the first year of the coronavirus disease of 2019 (COVID-19) pandemic, in comparison to 2019, the incidence rate increased from 19.7/100,000 to 21.5/100,000, with the highest increase in the age group of 5-9 years. This is the first nationwide report on a 30-year period of T1D incidence trend in Montenegro. It suggests that T1D incidence among Montenegrin children is rising again and that there is a short-term influence of COVID-19 on new-onset T1D.

Antioxidant defense system in the prefrontal cortex of chronically stressed rats treated with lithium.

Background: This study aimed to investigate the effects of lithium treatment on gene expression and activity of the prefrontal antioxidant enzymes: copper, zinc superoxide dismutase (SOD1), manganes superoxide dismutase (SOD2), catalase (CAT), and glutathione peroxidase (GPx) in animals exposed to chronic restraint stress (CRS). Methods: The investigated parameters were quantified using real-time RT-PCR, Western blot analyses, and assays of enzyme activities. Results: We found that lithium treatment decreased gene expression of SOD2, as well as the activities of SOD1 and SOD2 in chronically stressed rats to the levels found in unstressed animals. However, lithium treatment in animals exposed to CRS increased prefrontal GPx activity to the levels found in unstressed animals. Conclusions: These findings confirm that treatment with lithium induced the modulation of prefrontal antioxidant status in chronically stressed rats. Our results may be very important in biomedical research for understanding the role of lithium in maintaining the stability of prefrontal antioxidant defense system in neuropsychiatric disorders caused by chronic stress.

Effects of mood stabilizer lithium on noradrenergic turnover in the prefrontal cortex of chronically stressed rats.

OBJECTIVE: Data about the dynamics of noradrenaline (NA) transmission, storage and degradation may be very important for understanding the reduction of functional deficiency of NA and maintaining the stability of NA levels in animals with depressive-like behavior treated with lithium. This study aimed to investigate the effects of mood stabilizer lithium on concentrations of NA in the prefrontal cortex (PFC), as well as behavior rats exposed to chronic restraint stress (CRS). In addition, this study examined the effects of lithium on protein levels of noradrenaline transporter (NET), vesicular monoamine transporter 2 (VMAT2) and catechol-O-methyltransferase (COMT), as well as the enzyme activity of monoamine oxidase A (MOA) in the PFC of chronically stressed rats. METHODS: The investigated parameters were quantified by Western blot analysis, CAT Research ELISA kits, and an assay of enzyme activity. Also, the forced swim test (FST) was used to assess the behavior of animals. RESULTS: We found that lithium treatment decreased high protein levels of NET and VMAT2, as well as the enzyme activity of MOA in chronically stressed rats to the levels found in unstressed animals. In addition, lithium treatment decreased the concentration of NA (24%) and immobility in animals exposed to CRS. CONCLUSION: Our data confirm that lithium-induced modulation of prefrontal noradrenergic turnover and stabilized the behavior of chronically stressed rats.

TREND database: Retinal images of healthy young subjects visualized by a portable digital non-mydriatic fundus camera.

Topological characterization of the Retinal microvascular nEtwork visualized by portable fuNDus camera (TREND) is a database comprising of 72 color digital retinal images collected from the students of the Faculty of Medicine at the University of Montenegro, in the period from February 18th to March 11th 2020. The database also includes binarized images of manually segmented microvascular networks associated with each raw image. The participant demographic characteristics, health status, and social habits information such as age, sex, body mass index, smoking history, alcohol use, as well as previous medical history was collected. As proof of the concept, a smaller set of 10 color digital fundus images from healthy older participants is also included. Comparison of the microvascular parameters of these two sets of images demonstrate that digital fundus images recorded with a hand-held portable camera are able to capture the changes in patterns of microvascular network associated with aging. The raw images from the TREND database provide a standard that defines normal retinal anatomy and microvascular network geometry in young healthy people in Montenegro as it is seen with the digital hand-held portable non-mydriatic MiiS HORUS Scope DEC 200.This knowledge could facilitate the application of this technology at the primary level of health care for large scale telematic screening for complications of chronic diseases, such as hypertensive and diabetic retinopathy. In addition, it could aid in the development of new methods for early detection of age-related changes in the retina, systemic chronic diseases, as well as eye-specific diseases. The associated manually segmented images of the microvascular networks provide the standard that can be used for development of automatic software for image quality assessment, segmentation of microvascular network, and for computer-aided detection of pathological changes in retina. The TREND database is freely available at https://doi.org/10.5281/zenodo.4521043.

Simulium reptans (Linnaeus, 1758) and Simulium reptantoides Carlsson, 1962 from the Balkan Peninsula.

Simulium reptans (Linnaeus, 1758) and Simulium reptantoides Carlsson, 1962 are two species of the Simulium reptans group whose distribution is unclear because of their confusing taxonomy and systematics. Their genetic variability is well known for populations in northern and central Europe and shows that both species have two forms; however, the genetic variability of these species in southern and eastern Europe is unknown. To identify the status of these two species in southeast Europe, mtDNA was extracted from 19 individuals from 12 localities across the Balkan Peninsula. Phylogenetic analysis confirmed the existence of two species with 7.38-7.94% divergence. Each species was comprised of two clades, with 2.31% and 1.43% interclade divergence for S. reptans and S. reptantoides, respectively. This study revealed the presence of both species across the Balkans and that S. reptans occurs in this area in only one form (S. reptans B), while S. reptantoides is found in two genetic forms (A and B).

The effect of antioxidant status on overall survival in renal cell carcinoma.

INTRODUCTION: The oxidative stress contributes to all three phases of carcinogenesis and represents a concomitant condition in renal cell carcinoma (RCC). RCC is the most common type of neoplasm of the kidney, and despite numerous studies the set of predictive and prognostic markers of survival are still unknown. The aim of our study was to examine the relation between antioxidant (AO) status and overall survival (OS) in RCC patients. MATERIAL AND METHODS: Our study included 95 patients with RCC, who underwent radical nephrectomy. We analysed the prognostic role of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, glutathione reductase, glutathione, and malondialdehyde) and other clinicopathological factors (size, grade, stage, and histological subtype) on the OS of RCC patients. RESULTS: The 5-year OS was 54.6%. The survival analysis related to AO parameters showed no significant difference in survival of RCC patients. The concentration of malondialdehyde, an indicator of lipid peroxidation, also had no significant effect on the survival rate of RCC patients. Univariate and multivariate analysis confirmed the significance of clinicopathological parameters (size, p < 0.001; Fuhrman grade, p = 0.001, and stage, p < 0.001) for patients' survival. CONCLUSIONS: In our cohort of patients, different antioxidant parameters were not found to be predictors for OS of patients with RCC, who underwent radical nephrectomy.

Regional Patterns in Retinal Microvascular Network Geometry in Health and Disease.

The study explores the regional differences in microvascular geometry between the optic disc (O) and the macular area (M) in health and disease. Skeletonized manually segmented vascular networks from 15 healthy, 15 retinas with diabetic retinopathy (DR), and 15 retinas with glaucoma from publicly available High-Resolution Fundus (HRF) image database were used. When visualized by a digital fundus camera, O has a substantial proportion of small arteries and larger arterioles, while M contains smaller arterioles at the periphery and avascular zone in the center. We hypothesized that in pathological conditions the vascular network remodelling patterns in these two regions may be different. The analysis of box-counting fractal dimension (Db), lacunarity (Λ), and microvascular density showed that in healthy retinas, Λ and vessel density were lower in the M compared to the O, while the Db did not change. In retinas with DR, the Db was the lowest in the M, which was different from all other groups. The vessel density followed this trend. Lacunarity was the highest in the O of DR group compared to all other groups. The results show that in DR various regions of retinal microvascular network remodel in a different manner and to different extent.

Phylogeography of Simulium Subgenus Wilhelmia (Diptera: Simuliidae)-Insights From Balkan Populations.

Many morphologically similar species of the simuliid (Diptera: Simuliidae) subgenus Wilhelmia, Enderlein are difficult to distinguish. Thus, the revision of the subgenus using various morphological, cytogenetic, and genetic analyses has been attempted. Neglected until now, the Balkan Peninsula, a crossroad between Europe and Anatolia, provides insight which could resolve problematic interrelationships of the taxa within this subgenus. To uncover the status and relations within the subgenus Wilhelmia, mtDNA was extracted from 47 individuals of six morphospecies: Simulium balcanicum (Enderlein, 1924), Simulium turgaicum Rubtsov, 1940, Simulium lineatum (Meigen, 1804), Simulium pseudequinum Séguy, 1921, Simulium equinum (Linnaeus, 1758), and Simulium paraequinum Puri, 1933 from 21 sites throughout the Balkan Peninsula. Phylogenetic analysis of the Wilhelmia species using mitochondrial DNA barcoding (COI) gene showed two major branches, the lineatum branch, which includes the lineages sergenti, paraequinum, and lineatum, and the equinum branch. In the equinum branch, the mtDNA sequences formed six clades, with high genetic distances, suggesting the existence of different species. Historically, the clades of the equinum branch appeared at numerous islands, perhaps as a result of allopatric speciation. The paraequinum lineage (lineatum branch) is composed of two species. However, six clades of the lineatum lineage overlapped with intra- and interspecific genetic distances. Our results revealed that the species S. balcanicum, S. pseudequinum B, and S. equinum were omnipresent in the Balkans. The results point to not only the fair diversity of Wilhelmia species in the Balkans, but also indicate that most Wilhelmia species live in sympatry.

Expression of Antioxidant Enzymes in Patients with Uterine Polyp, Myoma, Hyperplasia, and Adenocarcinoma.

We previously found that compared to patients with benign uterine diseases (polyps, myomas), patients with premalignant (hyperplasia simplex and complex) and malignant (adenocarcinoma) lesions had enhanced lipid peroxidation and altered uterine antioxidant enzyme (AOE) activities. To further elucidate the mechanism of the observed changes, we examined protein and mRNA levels of copper-zinc superoxide dismutase (CuZnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and transcription factor Nrf2. We also examined correlations of AOE expression with AOE activity, lipid hydroperoxides (LOOH) level, and level of Nrf2. Our results showed decreased CuZnSOD, CAT, and Nrf2 levels, and increased GPx and GR levels in hyperplasias, while in patients with adenocarcinoma, the level of CAT was decreased and GR was increased, compared to benign groups. Similar changes in mRNA levels were also detected, indicating predominantly translational control of the AOE expression. The positive correlation of enzyme expression/activity was recorded for CuZnSOD, GPx, and GR, but only among groups with benign diseases. Only GR and GPx expressions were positively correlated with LOOH. Nrf2 protein was positively correlated with mRNA levels of CuZnSOD and GR. Observed results indicate involvement of diverse redox mechanisms in etiopathogenesis of different gynecological diseases, and may improve redox-based approaches in current clinical practice.

Modulation of Hippocampal Antioxidant Defense System in Chronically Stressed Rats by Lithium.

This study examined the effects of lithium on gene expression and activity of the antioxidant enzymes copper zinc superoxide dismutase (SOD1), manganese superoxide dismutase (SOD2), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) in the hippocampus of chronically stressed rats. In addition, we examined the effects of lithium on anxiety behaviors, hippocampal concentrations of dopamine (DA) and malondialdehyde (MDA), protein levels of brain-derived neurotrophic factor (BDNF), tyrosine hydroxylase (TH), dopamine transporter (DAT), and catechol-O-methyltransferase (COMT), as well as activity of monoamine oxidase (MAO) in chronically stressed rats. The investigated parameters were quantified by real-time RT-PCR, Western blot analyses, and assays of enzyme activities. We found that lithium did not change gene expression of SOD1, CAT, GPx, and GR but decreased gene expression of SOD2 in chronically stressed rats. A very important result in this study was that lithium treatment decreased the enzyme activities of SOD1 and SOD2 but increased the enzyme activities of GPx and GR in stress condition, which indicates the control of redox balance. The reduced concentration of MDA confirms this. In addition, we found that lithium treatment decreased high protein levels of BDNF and DAT in chronically stressed rats to the level found in unstressed animals. Also, lithium treatment increased the expression of TH but decreased the enzyme activity of MAO B, which contributed to the increase of hippocampal concentration of DA in chronically stressed rats to the level of unstressed animals. Finally, lithium treatment in animals exposed to chronic stress increased the time spent in open arms. Lithium-induced modulation of hippocampal antioxidant status and attenuation of oxidative stress stabilized behavior in animals with high anxiety index. In addition, reduced oxidative stress was followed by the changes of both turnover of DA and levels of BDNF protein in chronically stressed rats treated with lithium. These findings may be important in preclinical research of the effects of lithium on oxidative stress level in pathological conditions.

Fractal characterization of retinal microvascular network morphology during diabetic retinopathy progression.

OBJECTIVE: The study aimed to characterize morphological changes of the retinal microvascular network during the progression of diabetic retinopathy. METHODS: Publicly available retinal images captured by a digital fundus camera from DIARETDB1 and STARE databases were used. The retinal microvessels were segmented using the automatic method, and vascular network morphology was analyzed by fractal parametrization such as box-counting dimension, lacunarity, and multifractals. RESULTS: The results of the analysis were affected by the ability of the segmentation method to include smaller vessels with more branching generations. In cases where the segmentation was more detailed and included a higher number of vessel branching generations, increased severity of diabetic retinopathy was associated with increased complexity of microvascular network as measured by box-counting and multifractal dimensions, and decreased gappiness of retinal microvascular network as measured by lacunarity parameter. This association was not observed if the segmentation method included only 3-4 vessel branching generations. CONCLUSIONS: Severe stages of diabetic retinopathy could be detected noninvasively by using high resolution fundus photography and automatic microvascular segmentation to the high number of branching generations, followed by fractal analysis parametrization. This approach could improve risk stratification for the development of microvascular complications, cardiovascular disease, and dementia in diabetes.

The pterygopalatine fossa: morphometric CT study with clinical implications.

PURPOSE: The pterygopalatine fossa is a deep viscerocranial space containing the maxillary artery and nerve, the pterygopalatine ganglion, and the nerve of the pterygoid canal (vidian nerve). The endoscopic approach to this area relies on adequate preoperative imaging, such as computed tomography (CT). The aim was to determine the morphometric characteristics of the pterygopalatine fossa and its communications, including several previously unpublished measurements. METHODS: 100 CT scans (56 male and 44 female patients) were analyzed. The axial, coronal, and sagittal slices, together with the three-dimensional reconstructions, were used in the study. RESULTS: The central diameter and the length of the foramen rotundum, the vertical diameter and the length of the pterygoid (vidian) canal, and the diameter of the sphenopalatine foramen were significantly larger in men. The central diameters of the foramen rotundum and the vidian canal were significantly smaller than their anterior and posterior transverse diameters. The vidian canal length of 12.1 mm indicates the presence of the type 3 VC with a sensitivity of 83% and a specificity of 85%. CONCLUSION: Several new descriptions of the pterygopalatine fossa are presented here (such as the angle between the sphenopalatine foramen and the vidian canal, a new aspect in the understanding of the FR, and the distance between the posterior wall of the maxillary sinus to the vidian canal and the foramen rotundum), which might prove useful in the comprehension of the anatomy of the pterygopalatine fossa.

Manually segmented vascular networks from images of retina with proliferative diabetic and hypertensive retinopathy.

In this article we present a data set that contains 37 image files obtained by manual vessel segmentation of raw retinal images from Structured Analysis of the Retina (STARE) database ("The STARE Project", 2018) [1]. Our expert segmented 8 images that are associated with the single diagnosis of hypertensive retinopathy and 9 images with the single diagnosis of proliferative diabetic retinopathy (Popovic et al., 2018) [2]. To validate the manual segmentation, the same expert additionally segmented a gold standard set of 20 raw images from the STARE database. Raw images of retinas associated with either diabetic proliferative retinopathy or hypertensive retinopathy display the intricate and very different morphologies of retinal microvascular networks. Very frequently, they also have pathological changes such as exudates and hemorrhages. The presence of these changes, as well as neovascularization in proliferative diabetic retinopathy, poses a significant challenge for researchers who are developing automatic methods for retinal vessel segmentation. Therefore, this data set can be useful for the development of methods for automatic segmentation. In addition, the data can be used for development of methods for quantitation of microvascular morphology of the retina in various pathological conditions.

Fractal dimension and lacunarity analysis of retinal microvascular morphology in hypertension and diabetes.

Hypertension and diabetes mellitus represent modifiable risk factors for vascular disease. They cause microvascular remodeling, and ultimately result in end-organ damage. Therefore, development of methods for noninvasive quantification of the effects of hypertension and diabetes mellitus on microvasculature is of paramount importance. The two goals of the study were: 1) to characterize the geometric complexity and inhomogeneity of retinal vasculature in hypertensive retinopathy (HR) and in proliferative diabetic retinopathy (PDR) by using box counting fractal dimension and lacunarity analysis, and 2) to determine if the combination of these two parameters can be used to describe differences in the vascular tree geometry between HR and PDR. The extended set of retinal images from the publicly available STARE database was manually segmented by our expert, validated, and made available for other researchers to use. The healthy retinal vascular network has a higher complexity (fractal dimension) compared to that in HR and in PDR. However, there is no difference in microvascular complexity between HR and PDR. The inhomogeneity of the retinal microvascular tree (lacunarity) was higher in PDR compared to HR. Lacunarity and fractal dimension together quantitatively characterize microvascular geometry in the retina with higher specificity than fractal analysis alone.