Proteomic analysis of diabetic retinopathy identifies potential plasma-protein biomarkers for diagnosis and prognosis.

OBJECTIVES: To identify molecular pathways and prognostic- and diagnostic plasma-protein biomarkers for diabetic retinopathy at various stages. METHODS: This exploratory, cross-sectional proteomics study involved plasma from 68 adults, including 15 healthy controls and 53 diabetes patients for various stages of diabetic retinopathy: non-diabetic retinopathy, non-proliferative diabetic retinopathy, proliferative diabetic retinopathy and diabetic macular edema. Plasma was incubated with peptide library beads and eluted proteins were tryptic digested, analyzed by liquid chromatography-tandem mass-spectrometry followed by bioinformatics. RESULTS: In the 68 samples, 248 of the 731 identified plasma-proteins were present in all samples. Analysis of variance showed differential expression of 58 proteins across the five disease subgroups. Protein-Protein Interaction network (STRING) showed enrichment of various pathways during the diabetic stages. In addition, stage-specific driver proteins were detected for early and advanced diabetic retinopathy. Hierarchical clustering showed distinct protein profiles according to disease severity and disease type. CONCLUSIONS: Molecular pathways in the cholesterol metabolism, complement system, and coagulation cascade were enriched in patients at various stages of diabetic retinopathy. The peroxisome proliferator-activated receptor signaling pathway and systemic lupus erythematosus pathways were enriched in early diabetic retinopathy. Stage-specific proteins for early - and advanced diabetic retinopathy as determined herein could be 'key' players in driving disease development and potential 'target' proteins for future therapies. For type 1 and 2 diabetes mellitus, the proteomic profiles were especially distinct during the early disease stage. Validation studies should aim to clarify the role of the detected molecular pathways, potential biomarkers, and potential 'target' proteins for future therapies in diabetic retinopathy.

Blindness is decreasing among children born preterm during the last four decades in Denmark.

PURPOSE: Children born preterm are believed to be at increased risk of visual impairment (VI). The increased survival rate of extremely preterm children may have changed the spectrum of diseases occurring postnatally. The aim of this study was to analyse the prevalence and causes of VI in an ex-preterm Danish population during the last 4 decades. METHODS: The study was based on the National Register of Blind and Visually Impaired Children (NRVIC). Ex-preterms born at gestational age (GA) <32 weeks and enrolled in NRVIC at any time between 1988 and 2020 were included. The main cause of VI, the severity of VI and systemic comorbidities were analysed for temporal changes. RESULTS: A total of 335 patients were included. The prevalence of VI decreased from 26/1000 preterm children in the 1980s to 15/1000 in 2000s. Blindness due to preterm birth is almost eliminated affecting 1/1000 today. Sequelae to retinopathy of prematurity (ROP) was the most common cause of VI (51% of cases) in the 1980s but decreased to 34% in 2010s, whereas non-ocular causes of VI increased from 1% to 36%, respectively. More than half of the children (64%) had combined comorbidities and 36% had isolated VI. CONCLUSION: The improved monitoring in neonatal intensive care units and management of ROP has reduced the prevalence of severe VI due to ROP and almost eliminated blindness over the last decades in Denmark. However, preterm children are still at risk of non-ocular causes of VI and comorbidities following the premature delivery.

Blood-Biomarkers for Glucose Metabolism in Preterm Infants.

This was an exploratory, prospective, longitudinal, cohort study that aimed to establish "healthy" reference levels related to growth parameters and glucose metabolites in preterm infants. This was conducted to further investigate growth and metabolic disturbances potentially related to neonatal illness. The study sample consisted of 108 preterm infants born before 32 weeks in 2018-2019 in the Capital Region of Denmark. Repetitive blood samples were acquired at the neonatal wards, while clinical data were obtained from the regional hospital medical record system. Thirty-four "healthy" preterm infants (31%) were identified. The "ill" infants were divided into four subgroups dependent on gestational age and small for gestational age. Reference levels for the growth parameters and metabolic biomarkers glucose, albumin, and adiponectin, and two glucose control indicators, glycated albumin and fructosamine, were determined for the "healthy" and "ill" subgroups. The "ill" extremely preterm infants had increased glucose levels (mean difference 0.71 mmol/L, 95% CI 0.23; 1.18 mmol/L) and glycated albumin (corrected; %) (mean difference 0.92 mmol/L, 95% CI 0.38 mmol/L;1.47 mmol/L) compared to the "healthy" infants. In "ill" extremely preterm infants and "ill" very preterm infants born small for gestational age, levels of biomarkers containing proteins were decreased. In the "Ill" extremely preterm infants and infants born small for gestational age, postnatal growth was continuously decreased throughout the postconceptional period. The short-term glucose-control indicator, glycated albumin (corrected; %), reflected well the high glucose levels due to its correction for the depleted plasma-protein pool.

Early postnatal hyperglycaemia is a risk factor for treatment-demanding retinopathy of prematurity.

BACKGROUND: To investigate whether neonatal hyperglycaemia in the first postnatal week is associated with treatment-demanding retinopathy of prematurity (ROP). METHODS: This is a Danish national, retrospective, case-control study of premature infants (birth period 2003-2006). Three national registers were searched, and data were linked through a unique civil registration number. The study sample consisted of 106 cases each matched with two comparison infants. Matching criteria were gestational age (GA) at birth, ROP not registered and born at the same neonatal intensive care unit. Potential 'new' risk factors were analysed in a multivariate logistic regression model, while adjusted for previously recognised risk factors (ie, GA at birth, small for gestational age, multiple birth and male sex). RESULTS: Hospital records of 310 preterm infants (106 treated; 204 comparison infants) were available. Nutrition in terms of energy (kcal/kg/week) and protein (g/kg/week) given to the preterm infants during the first postnatal week were statistically insignificant between the study groups (Mann-Whitney U test; p=0.165/p=0.163). Early postnatal weight gain between the two study groups was borderline significant (t-test; p=0.047). Hyperglycaemic events (indexed value) were statistically significantly different between the two study groups (Mann-Whitney U test; p<0.001). Hyperglycaemia was a statistically independent risk factor (OR: 1.022; 95% CI 1.002 to 1.042; p=0.031). CONCLUSION: An independent association was found between the occurrence of hyperglycaemic events during the first postnatal week and later development of treatment-demanding ROP, when adjusted for known risk factors.

Neonatal Risk Factors for Treatment-Demanding Retinopathy of Prematurity: A Danish National Study.

PURPOSE: One goal of the study was to identify "new" statistically independent risk factors for treatment-demanding retinopathy of prematurity (ROP). Another goal was to evaluate whether any new risk factors could explain the increase in the incidence of treatment-demanding ROP over time in Denmark. DESIGN: A retrospective, register-based cohort study. PARTICIPANTS: The study included premature infants (n = 6490) born in Denmark from 1997 to 2008. METHODS: The study sample and the 31 candidate risk factors were identified in 3 national registers. Data were linked through a unique civil registration number. Each of the 31 candidate risk factors were evaluated in univariate analyses, while adjusted for known risk factors (i.e., gestational age [GA] at delivery, small for gestational age [SGA], multiple births, and male sex). Significant outcomes were analyzed thereafter in a backward selection multiple logistic regression model. MAIN OUTCOME MEASURES: Treatment-demanding ROP and its associations to candidate risk factors. RESULTS: Mechanical ventilation (odds ratio [OR], 2.84; 95% confidence interval [CI], 1.99-4.08; P < 0.01) and blood transfusion (OR, 1.97; 95% CI, 1.20-3.14; P = 0.01) were the only new statistically independent risk factors, in addition to GA at delivery, SGA, multiple births, and male sex. Modification in these prognostic factors for ROP did not cause an increase in treatment-demanding ROP. CONCLUSIONS: In a large study population, blood transfusion and mechanical ventilation were the only new statistically independent risk factors to predict the development of treatment-demanding ROP. Modification in the neonatal treatment with mechanical ventilation or blood transfusion did not cause the observed increase in the incidence of preterm infants with treatment-demanding ROP during a recent birth period (2003-2008).

Refraction and visual acuity in a national Danish cohort of 4-year-old children of extremely preterm delivery.

PURPOSE: A recent threefold increase in laser treatment for advanced retinopathy of prematurity (ROP) triggered a nationwide preschool ophthalmic and developmental status among extremely preterm survivors. Here, we discuss refraction and visual acuity. METHODS: Survivors (n = 178) from a national birth cohort (February 2004 to March 2006) of gestational age <28 weeks (PT) and 56 full-term (FT) controls attended for evaluation at age 4 years. Cycloplegic refraction and keratometry were achieved by Retinomax autokeratorefractor and visual acuities by symbol recognition (HOTV, logMAR). RESULTS: The refractive distribution presented a myopic tail (4.5%) and a hyperopic tail (11.9% ≥+2.5 D) as special preterm features, and corneas were more curved. Astigmatism and anisometropia were only marginally increased, and visual acuities were generally good. Best-corrected binocular median logMAR visual acuity was 0.1 in FT and 0.2 in PT, in Snellen equivalents 0.8 and 0.63. Snellen acuity ≤0.5 occurred across the ROP subgroups, but mainly in those with at least ROP stage 3. Two children had low vision. CONCLUSIONS: The overall fair outcome for refraction and function is in accordance with other recent northern Europe experience. The results differ in particular from the poorer ophthalmic outcomes reported in the pioneer US treatment studies (cryotherapy for ROP and ETROP). The diode laser ablations (n = 32) appeared effective in our series; except one child, all treated subjects had good or fair social vision at the age of 4 years.

Cerebral damage may be the primary risk factor for visual impairment in preschool children born extremely premature.

OBJECTIVES To investigate the importance of cerebral damage and retinopathy of prematurity (ROP) for visual impairment in preschool children born extremely premature and to determine the primary risk factor of the two. METHODS A clinical follow-up study of a Danish national cohort of children born extremely premature (gestational age, <28 weeks). The study sample consisted of 262 extremely preterm children born between February 13, 2004, and March 23, 2006, of whom 178 children (67.9%) participated. A matched control group consisted of 56 term-born children (gestational age, 37 to <42 weeks). All participants were identified through the National Birth Register and invited to participate in a clinical examination. The children were evaluated with regard to visual acuity, foveal sequelae, and maximum ROP stage and the presence of global developmental deficits (an indicator for cerebral damage) that was measured by the Ages and Stages Questionnaire. RESULTS Global developmental deficits and foveal sequelae occurred more often in extremely preterm children than in term-born control children and increased with ROP severity (χ2 test; P = .11 and P < .001, respectively). Global developmental deficits, moderate to severe foveal abnormality, and ROP treatment were independently associated with visual impairment (P < .05, for better and worse eyes). A stepwise multiple logistic regression for better-eye logarithmic visual acuities of 0.3 or greater (Snellen scale, ≤0.5) yielded an odds ratio of 8.7 (95% CI, 3.0-25.2; P < .001) for global developmental deficit and 6.3 (95% CI, 2.2-18.5; P < .001) for moderate to severe foveal sequelae. CONCLUSION Cerebral damage and ROP are independent risk factors for visual impairment in children born extremely premature, and cerebral damage may be the primary risk factor.

Experts do not agree when to treat retinopathy of prematurity based on plus disease.

OBJECTIVES: To investigate inter-reader agreement on five severity levels of central vascular changes (none, mild, moderate, severe pre-plus disease, plus disease) and aggressive posterior retinopathy of prematurity (ROP), and to see whether an unintended shift in indication for treatment occurred. METHODS: Four international ROP readers participated. Before the grading of the photographs, the readers were informed that a high proportion of advanced ROP cases were included. In total, 243 photographs/948 quadrants were available from 136 infants. As a standard series of photographs was available, grading was performed under optimised conditions. RESULTS: The four readers agreed on the quadrant scores of only 70 (7.38%) of the 948 quadrants--that is, on 1, 5, 15, 4 and 45 quadrants for scores 0, 1, 2, 3 and 4, respectively. The mean scores differed systematically between the readers (permutation test, p<0.0001). Agreement on presence of aggressive posterior ROP from all four readers was not obtained for any of the photographs. Readers scored plus disease in at least two quadrants in 95.5% of the eyes for which treatment was indicated. All four readers agreed on the scoring of indication for treatment for 195 eyes (80.2%); however, treatment was only recommended in 18 (7.4%) eyes. One reader was found to differ systematically from the others in indicating treatment (Rasch analysis; p=0.0001). Finally, a significant shift in indication for treatment occurred between birth period 2000-2002 and 2003-2006 (Mann-Whitney rank sum test, p<0.001). CONCLUSIONS: Inter-reader agreement on central vascular changes is poor, especially when based on more than two rating categories. The subjective nature of diagnosing such vascular changes possibly resulted in earlier treatment of preterm infants in Denmark over the entire study period (1997-2006). The recent increased incidence of treated infants in Denmark is, at least in part, explained by a significant shift in indication for treatment.

A new risk-based screening criterion for treatment-demanding retinopathy of prematurity in Denmark.

OBJECTIVE: The aim of this study was to uncover the most effective and safe criterion to implement for retinopathy of prematurity screening in Denmark. METHODS: This retrospective national cohort study is based on data from 3 national registers. These registers provided on infants treated for retinopathy of prematurity, infants in need of treatment but missed by the present screening program, and the candidate neonates for advanced retinopathy of prematurity development A nonlinear logistic regression model was fitted to the data, and various screening criteria were evaluated. RESULTS: During the study period (2002-2006), 116 infants were treated for retinopathy of prematurity, no treatment-demanding retinopathy of prematurity infants were missed by the screening program, and 182 premature infants were candidates for developing treatment-demanding retinopathy of prematurity. Screening criteria combining gestational age at delivery and birth weight limits and new risk-based criteria were compared with regards to their effectiveness. The risk-based criteria were the most effective. Use of the 0.13% risk-based criterion to define the population to be screened resulted in the detection of all treated infants in the study period and 17.4% fewer infants to screen. The model predicted this criterion to result in 1 missed case of treatment-demanding retinopathy of prematurity every 11 years and 1 case of blindness every 18 years in Denmark. CONCLUSIONS: Screening criteria based on risk estimates of developing treatment-demanding retinopathy of prematurity are the most effective for retinopathy-of-prematurity screening. The risk-based criterion of 0.13% can safely be implemented for future retinopathy-of-prematurity screening in Denmark.

Treatment for retinopathy of prematurity in Denmark in a ten-year period (1996 2005): is the incidence increasing?

OBJECTIVE: The objective of this study was to analyze the population incidence of retinopathy of prematurity treatment in Denmark in the 10-year period from 1996 to 2005. METHODS: Patient charts of infants treated for retinopathy of prematurity and the national birth registry provide information about neonatal parameters. These parameters, along with birth in the latter half of the period (2001-2005), were analyzed as risk factors for retinopathy of prematurity. The national registry for blind and visually impaired children was accessed to obtain information about visual impairment attributable to retinopathy of prematurity in both treated and untreated infants. RESULTS: The study population consisted of 5467 Danish preterm infants born in 1996 to 2005, with a gestational age of < 32 weeks, who survived for > or = 5 postnatal weeks; 2616 were born in 1996 to 2000, and 2851 were born in 2001 to 2005. The incidence of treated retinopathy of prematurity cases increased significantly from 1.3% in 1996 to 2000 to 3.5% in 2001 to 2005. Significant risk factors for retinopathy of prematurity treatment were low gestational age, small for gestational age, male gender, and multiple birth. Other, yet unknown factors contributed to the increased incidence in the latter half of the period. Of the study population, 0.6% were registered as visually impaired because of retinopathy of prematurity within 2 years after birth (early-detected visual impairment). The incidences were not significantly different between 1996 to 2000 and 2001 to 2005. Of all of the early-detected, visually impaired children, 16% had not been treated for retinopathy of prematurity and were considered screening failures. CONCLUSIONS: The incidence of retinopathy of prematurity treatment in Denmark has more than doubled during the past half-decade. This increase could not be fully explained by increased survival rates for the infants or by changes in the investigated neonatal risk factors.

Up-regulation of PK11195 binding in areas of axonal degeneration coincides with early microglial activation in mouse brain.

Increased binding of the peripheral benzodiazepine binding site (PBBS) ligand [(3)H]PK11195 in the central nervous system of patients suffering from acute and chronic neuropathology has been associated with reactive microgliosis. However, it remains uncertain which stages of microglial activation occur in conjunction with the increased [(3)H]PK11195 binding. We used quantitative autoradiography for [(3)H]PK11195 and quantitative polymerase chain reaction for PBBS mRNA and markers of early and late microglial activation to investigate the time-course of cellular responses in the hippocampus of mice with degeneration of the entorhinal-hippocampal perforant path. The axonal lesion evoked an increase in the B(max) for [(3)H]PK11195 in hippocampus which peaked at 2 days post-lesion, remained elevated at day 5 and began to decline at 10 days post-lesion. These changes occurred in the absence of significant changes in affinity in vitro. Quantitative polymerase chain reaction analysis of isolated hippocampi using exon-specific primers indicated the presence of several splice variants of PBBS mRNA, which appeared to be affected differentially by the lesion. The changes in PBBS mRNA and CD11b mRNA levels correlated with the B(max) for [(3)H]PK11195 during 10 days post-lesion, suggesting that microglial activation couples with increases in mRNA levels for these markers. In addition, the onset of changes in PBBS mRNA levels coincided with the significantly elevated tumor necrosis factor mRNA levels present during early microglial activation at 2 days post-lesion. We conclude that up-regulation of [(3)H]PK11195 binding and PBBS mRNA levels coincided with early microglial activation, characterized by concomitantly increased microglial tumor necrosis factor mRNA levels, and persisted throughout the period with reactive microgliosis.