Thomas Bertelmann

Proteomics of Vitreous Humor of Patients with Exudative Age-Related Macular Degeneration

Background There is absence of specific biomarkers and an incomplete understanding of the pathophysiology of exudative age-related macular degeneration (AMD). Methods and Findings Eighty-eight vitreous samples (73 from patients with treatment naïve AMD and 15 control samples from patients with idiopathic floaters) were analyzed with capillary electrophoresis coupled to mass spectrometry in this retrospective case series to define potential candidate protein markers of AMD. Nineteen proteins were found to be upregulated in vitreous of AMD patients. Most of the proteins were plasma derived and involved in biological (ion) transport, acute phase inflammatory reaction, and blood coagulation. A number of proteins have not been previously associated to AMD including alpha-1-antitrypsin, fibrinogen alpha chain and prostaglandin H2-D isomerase. Alpha-1-antitrypsin was validated in vitreous of an independent set of AMD patients using Western blot analysis. Further systems biology analysis of the data indicated that the observed proteomic changes may reflect upregulation of immune response and complement activity. Conclusions Proteome analysis of vitreous samples from patients with AMD, which underwent an intravitreal combination therapy including a core vitrectomy, steroids and bevacizumab, revealed apparent AMD-specific proteomic changes. The identified AMD-associated proteins provide some insight into the pathophysiological changes associated with AMD.

Evaluation of a posterior vitreous detachment: a comparison of biomicroscopy, B-scan ultrasonography and optical coherence tomography to surgical findings with chromodissection

Purpose:  To find the most reliable and efficient noninvasive technique to clinically detect a posterior vitreous detachment.

The vitreomacular interface in retinal vein occlusion

Purpose:  To evaluate the posterior vitreous adhesion status in patients with a history of central or branch retinal vein occlusion and to compare the results with the natural time‐course of posterior vitreous detachment in healthy age‐related controls.

Head-to-head comparison of ranibizumab PRN versus single-dose dexamethasone for branch retinal vein occlusion (COMRADE-B)

To compare the efficacy and safety of ranibizumab 0.5 mg versus dexamethasone 0.7 mg according to their European labels in macular oedema secondary to branch retinal vein occlusion (BRVO) in a 6‐month, phase IIIb, randomized trial.

The impact of posterior vitreous adhesion on ischaemia in eyes with retinal vein occlusion.

To evaluate whether posterior vitreous adhesion (PVA) contributes to retinal ischaemia in eyes suffering from central (CRVO) or branch retinal vein occlusion (BRVO).

Intravitreal Functional Plasminogen Is Elevated in Central Retinal Vein Occlusion

Purpose: To detect intravitreal functional plasminogen in vitreous samples of patients with recent onset of central retinal vein occlusion (CRVO) and to demonstrate significantly higher intravitreal plasminogen in CRVO patients in comparison to controls. Methods: Prospective clinical case series of 13 consecutive patients with recent onset of CRVO scheduled for core pars plana vitrectomy and 10 consecutive patients undergoing standard pars plana vitrectomy for routine macular surgery or vitreal floater removal. In all 23 cases, vitreous taps were extracted from the central vitreous body, and plasminogen was functionally determined in a new ultrasensitive p-nitroanilide reaction after activation with streptokinase (100% of normal, %N = functional plasminogen in pooled normal citrated plasma). Results: Plasminogen was detected in all analyzed samples (n = 23), and mean plasminogen was revealed to be 1.33 ± 1.73% (mean ± SD), with a range of 0.03-7.8%N. Patients with recent onset of CRVO exhibited significantly higher intravitreal plasminogen (2.19 ± 1.89%N) in comparison to controls (0.20 ± 0.21%N; p < 0.001, Mann-Whitney U test). Conclusion: Due to significantly increased intravitreal plasminogen in patients with recent onset of CRVO, intravitreally administered tissue plasminogen activator might be an option to induce posterior vitreous detachment (enzymatic vitreolysis) in CRVO patients.

IMPACT OF VITREORETINAL INTERFACE ARCHITECTURE ON SUCCESSFUL VITREOMACULAR TRACTION RESOLUTION IN EYES SCHEDULED FOR INTRAVITREAL OCRIPLASMIN THERAPY

Purpose: To evaluate the impact of the vitreoretinal interface architecture, in specific the angle between the posterior vitreous cortex and the internal limiting membrane, on vitreomacular traction (VMT) resolution in eyes treated with intravitreally injected ocriplasmin (Jetrea). Methods: Retrospective, multicenter cohort study and exploratory data analysis. Spectral domain optical coherence tomography assessments were performed before scheduled ocriplasmin injections. General (age and sex) as well as ocular variables (lens status, presence of epiretinal membrane formations, horizontal diameter of VMT, central retinal thickness, and in particular various prespecified angles between the posterior vitreous cortex and internal limiting membrane) were analyzed to evaluate their impact on successful VMT resolution. Results: Fifty-nine eyes of 59 patients were included. Univariate analysis of age (odds ratio [OR]: 0.881; 95% CI: [0.812–0.955]; P = 0.0022) and lens status (OR: 11.03; 95% CI: [2.23–54.57]; P = 0.0033) had a significant impact on successful VMT resolution, whereas sex (OR: 0.668; 95% CI: [0.126–2.065]; P = 0.4906), epiretinal membrane formation (OR: 0.581; 95% CI: [0.168–2.006]; P = 0.3903), horizontal diameter of VMT (OR: 0.99930; 95% CI: [0.99825–1.00035]; P = 0.1886), and central retinal thickness (OR: 0.9985; 95% CI: [0.9934–1.00436]; P = 0.56) failed. The angle at 500 &mgr;m apart from the fovea centralis, irrespective if measured nasally (OR: 1.135; 95% CI: [1.013–1.272]; P = 0.0289) or temporally (OR: 1.099; 95% CI: [1.001–1.208]; P = 0.0485), showed a significant correlation with VMT resolution. Conclusion: The angle between the posterior vitreous cortex and the internal limiting membrane 500 &mgr;m apart from the fovea centralis correlates with VMT resolution and may be a clinically useful marker for selection of patients to be treated with ocriplasmin. This observation needs to be proven in a prospective confirmatory investigation.

Intravitreal Functional Plasminogen in Eyes with Branch Retinal Vein Occlusion

Purpose: To evaluate whether intravitreal functional plasminogen is elevated in eyes with branch retinal vein occlusion (BRVO) and to discover whether intravitreal plasminogen activities are correlated with the extent of blood-retina barrier (BRB) breakdown. Methods: Our study is a prospective case series of 20 consecutive patients with BRVO and 10 consecutive patients serving as controls. Vitreous taps were extracted from the central vitreous body and plasminogen was functionally determined in an innovative, ultrasensitive p-nitroanilide reaction after activation with streptokinase (100% of normal, %N = functional plasminogen in pooled normal citrated plasma). Intravitreal VEGF levels were assayed to estimate BRB breakdown. Results: Intravitreal functional plasminogen was detected in all analyzed samples (n = 30) and mean (±SD) plasminogen activities were found to be 0.97 ± 1.06%N (range: 0.03-3.9%N). Patients suffering from BRVO exhibited significantly higher intravitreal plasminogen (1.35 ± 1.11%N) in comparison with controls (0.20 ± 0.21%N, p < 0.001). Intravitreal VEGF concentrations in the BRVO group (576 ± 547 pg/ml) were significantly higher than these in controls (111 ± 120 pg/ml, p = 0.003). There was a significant correlation between intravitreal functional plasminogen and intravitreal VEGF levels (r = 0.519, p = 0.003). Conclusions: Intravitreal functional plasminogen is significantly elevated in eyes suffering from BRVO and correlates with the extent of BRB breakdown. The induction of posterior vitreous detachment by using intravitreally administered recombinant tissue plasminogen activator (enzymatic vitreolysis) should be explored in further investigations.

Thrombin activity in normal vitreous liquid.

An intact blood–retina barrier (BRB) ensures the homeostatic regulation of the retinal environment by preventing proteins and enzymes to pass from the blood stream into the retinal tissue as well as the vitreous cavity (’nonleaky eyes’). Nevertheless, thrombin is needed within the eye to avoid bleeding events. It might, furthermore, play an essential role in preventing BRB breakdown (’leaky eyes’). Until today, the intraocular thrombin activity as well as the source of the latter has not been investigated. The present work was conducted to evaluate whether intravitreal thrombin activity is present in eyes without BRB breakdown. Therefore, 16 vitreal taps were harvested at the beginning of a standard 23-gauge three-port pars plana vitrectomy. These 200 &mgr;l undiluted vitreous samples were instantly stabilized by 1 + 1 mixture with 5% human albumin, followed by 1 + 1 mixture of such an aliquot with 2.5 mol/l arginine, pH 8.6, and frozen at −20°C. After thawing at 23°C, thrombin activity was analyzed chromogenically in the presence of arginine protection against unspecific cleavage of the chromogenic substrate. Intravitreal thrombin was detected in all 16 analyzed samples and thrombin activity exhibited to be 1.5 ± 1.0 mIU/ml (mean value ± SD; range: 0.2–3.25 mIU/ml). Thus, our investigation is the first successful quantification of the physiologic intraocular activity of thrombin. Further studies will evaluate intravitreal thrombin activities in eyes with BRB breakdown and compare those results with the physiologic activities demonstrated herein. Standardized intraocular thrombin activity might be a new diagnostic parameter in ophthalmology.

Sampling Aqueous Humor: Anterior Segment Anatomy, Anesthetic and Surgical Technique, and Rates of Yield

Purpose: To evaluate anterior segment anatomy and anesthetic and surgical techniques with respect to the amount of aqueous humor (AH) that can be sampled out of the anterior chamber (AC) at the beginning of standard cataract removal procedures (phacoemulsification). Methods: In a prospective survey, volumes of sampled AH from 123 eyes (110 patients) were analyzed in regard to AC anatomy (anterior chamber depth, ACD) and different anesthetic techniques (local and general anesthesia). Results: 107 eyes (87%) were included into our analysis, and 16 eyes (13%) had to be excluded due to failure of AH collection. We found a significant positive association between ACD and obtained AH volume (p = 0.007). In general anesthesia, a strong trend to acquire more AH in comparison to local anesthesia was apparent, but statistical significance failed (p = 0.167). Different anesthetic techniques seem to have no significant influence on ACD (p = 0.169). No training curve for the individual surgeon was obtained. No complications were observed. Conclusion: When AH sampling is performed in eyes with a deep AC and when the procedure is performed under general anesthesia, more AH can be aspirated.