Chromatische Pupillometrie bei ehemaligen Extremfrühgeborenen mit und ohne Frühgeborenenretinopathie in der Anamnese

Die Entwicklung der Netzhaut beginnt im 1. Monat der Schwangerschaft und persistiert bis zu einigen Jahren nach der Geburt. Eine verfrühte Entbindung stört diesen Reifungsprozess und kann zu postpartalen retinalen Schäden und einem erhöhten Risiko für Ametropien und Amblyopien im Kindesalter führen. Besonders Frühgeborene vor der 32. Gestationswoche sind von einer Frühgeborenenretinopathie (ROP) bedroht und bedürfen regelmäßiger ophthalmologischer Kontrollen und einer Therapie bei Progredienz der Erkrankung. Die Auswirkungen von Frühgeburt und ROP auf die Netzhaut sind bisher noch nicht vollständig geklärt und werden bisweilen in zahlreichen Studien erforscht. Ziel der vorliegenden Arbeit war es die kindliche Netzhaut durch Applikation rezeptorspezifischer Lichtstimuli und Messung ihrer Aktivität über den Pupillenlichtreflex näher zu beschreiben (Pupillometrie). Die in dieser Studie durchgeführte Pupillometrie ist Teil einer prospektiven Langzeitnachverfolgungsstudie ehemals frühgeborener Kinder, die nach ihrer Geburt im Rahmen einer multizentrischen Studie in den Jahren 2001-2007 mittels digitaler Weitwinkelkamera (RetCam) untersucht wurden. Der Status der Netzhaut bezüglich akuter ROP sowie Geburtsgewicht, Gestationsalter und Komorbiditäten der Kinder wurde in einer digitalen Datenbank festgehalten. Diese diente als Grundlage der aktuellen Studie, welche 2011 initiiert wurde. Hierfür wurden 191 Kinder im Alter von 6 bis 13 Jahre rekrutiert und in vier Gruppen eingeteilt: Gesunde Frühgeborene ohne ROP, Frühgeborene mit spontan zurückgebildeter ROP, Frühgeborene mit behandlungsbedürftiger ROP und termingeborene Kinder. Durch Reizung der Netzhaut mittels kurzwelliger Strahlung geringer Leuchtdichte bei dunkler Umgebung wurde vornehmlich das Stäbchensystem angesprochen, durch Applikation langwelliger Strahlung hoher Leuchtdichte bei Raumhelligkeit vornehmlich das Zapfensystem. Die Auswertung der Daten ergab eine signifikant geminderte Aktivität der Zapfen bei Frühgeborenen mit spontan zurückgebildeter ROP in der Anamnese. Eine makuläre Reifungsstörung im Rahmen der Frühgeburt scheint ebenfalls die Photorezeptoren längerfristig negativ zu beeinflussen. Zahlreiche Studien unterstützen unsere Ergebnisse. Die chromatische Pupillometrie ermöglicht als schnelles und nicht invasives Verfahren ohne Weittropfen des Patientenauges die isolierte Messung der Aktivität von Stäbchen, und Zapfen über den Pupillenlichtreflex. Hierbei könnten in Zukunft nach Etablierung von Standards das Ausmaß retinaler Erkrankungen und deren Therapieverfahren objektiviert werden.Retinal development begins 4 weeks post conception and lasts until early childhood. Preterm birth disrupts ocular development and may cause postpartal retinal dysfunction, ametropia and amblyopia. Especially preterm infants born before the 32nd week of gestation are at risk of developing retinopathy of prematurity (ROP) and thus need frequent ophthalmological eye examinations and, in cases where ROP persists, further treatment. The effect of ROP on the human retina is still not completely understood and is being evaluated in several clinical trials. We aimed to assess retinal function in young children who were born premature to blue and red light stimuli in order to measure rod and cone mediated pupillary responses (pupillometry). Our pupillometric examinations are part of a long-term follow up study of former preterm children that had previously been screened for ROP employing digital wide- field retinal imaging (RetCam) between 2001 and 2007 and were part of a mulicenter study. Status of acute ROP, birth weight, gestational age and comorbidities were compiled in a database used for our study launched in 2011. We examined 191 children between the age of 6 to 13 years and categorised them into four groups: healthy preterms without ROP, preterms with spontaneously regressed ROP, preterms with severe laser treated ROP and term born children. We applied short wavelength low intensity flash light in the dark to assess rod function and long wavelength high intensity flash light at day light to assess cone function. Our data suggest a significantly attenuated cone driven light response in former preterms with spontaneously regressed ROP. In addition, macular developmental arrest in children with and without ROP was associated with decreased cone function in comparison to term borns. Our findings are supported by several other studies. Chromatic pupillometry provides an easy and non-invasive way to examine the eyes without dilating patients’ pupils. Different light conditions and stimuli enable the examiner to assess rod and cone mediated retinal activity through measurement of the pupillary light reflex. Further research is needed to standardize chromatic pupillometry for clinical use and could contribute to assess the extent of retinal diseases and the effect of treatments

PupilEXT: Flexible Open-Source Platform for High-Resolution Pupillometry in Vision Research

The human pupil behavior has gained increased attention due to the discovery of the intrinsically photosensitive retinal ganglion cells and the afferent pupil control path’s role as a biomarker for cognitive processes. Diameter changes in the range of 10–2 mm are of interest, requiring reliable and characterized measurement equipment to accurately detect neurocognitive effects on the pupil. Mostly commercial solutions are used as measurement devices in pupillometry which is associated with high investments. Moreover, commercial systems rely on closed software, restricting conclusions about the used pupil-tracking algorithms. Here, we developed an open-source pupillometry platform consisting of hardware and software competitive with high-end commercial stereo eye-tracking systems. Our goal was to make a professional remote pupil measurement pipeline for laboratory conditions accessible for everyone. This work’s core outcome is an integrated cross-platform (macOS, Windows and Linux) pupillometry software called PupilEXT, featuring a user-friendly graphical interface covering the relevant requirements of professional pupil response research. We offer a selection of six state-of-the-art open-source pupil detection algorithms (Starburst, Swirski, ExCuSe, ElSe, PuRe and PuReST) to perform the pupil measurement. A developed 120-fps pupillometry demo system was able to achieve a calibration accuracy of 0.003 mm and an averaged temporal pupil measurement detection accuracy of 0.0059 mm in stereo mode. The PupilEXT software has extended features in pupil detection, measurement validation, image acquisition, data acquisition, offline pupil measurement, camera calibration, stereo vision, data visualization and system independence, all combined in a single open-source interface, available at https://github.com/openPupil/Open-PupilEXT

Mechanotransduction of substrate stiffness in endothelial cell collective migration

Endothelial damage during life-saving percutaneous angioplasty contributes to re-stenosis rates of nearly 20% within 5 years. Re-endothelialization, the collective endothelial cell migration over exposed extracellular matrix (ECM) and stent struts, can restore a continuous, functional endothelium. During atherosclerotic disease, vascular ECM becomes stiffer. ECM stiffness affects epithelial cell collective migration in other pathogenic contexts. However, substrate stiffness effects on endothelial cell collective migration have yet to be explored. We developed quantitative computational image processing algorithms for assessing collective migration. We then used these image analysis techniques to measure the effect of substrate stiffness on critical aspects of porcine aortic endothelial cell (PAEC) two-dimensional collective migration: (1) migration distance, (2) directedness, and (3) togetherness. PAEC were seeded on collagen-coated polyacrylamide hydrogels (4-50 kPa) in a 5 mm cloning ring and then allowed to migrate outwards. We found that migration distance increased with substrate stiffness and that there was a concomitant increase in PAEC alignment. We found that decreased togetherness on stiffer substrates led to enhanced proliferation at the migratory interface. We used the specific Rho kinase (ROCK) inhibitor Y27632 to show that ROCK-mediated contractility limited endothelial cell collective migration on soft substrates. We observed that PAEC secrete and remodel fibronectin on collagen-coated substrates. Interestingly, α5 integrin, but not fibronectin, was important for directed collective migration on stiff substrates. These findings provide insight into how substrate stiffness affects endothelial cell collective migration. This work will inform how the mechanical properties of tissue and tissue engineered construct could be designed to promote a functional endothelium.Ph.D., Biomedical Engineering -- Drexel University, 201