Stem cell mediated retinal repair: models and mechanisms

Aim: I aimed to investigate methods of delivering neuroprotection to the retina: a) using magnetofection (a non-viral transfection technique) in ocular tissue, b) using stem cells as a vector of neuroprotection delivery and c) harnessing galvanotaxis to direct migration of stem cells into the retina. Methods: For the recipient test bed of the experiments in my thesis, I used retinal explants from mice. The viability of retinal explants was determined using retinal ganglion cell health as a read-out in the form of Sholl plots. I also explored magnetofection with oscillation as a non-viral technique for neuroprotection delivery in ocular tissue using cornea and retinal explants. Using the galvanotaxis technique, I explored its use in directing neural stem cells in-vitro with an electrotactic chamber and ex-vivo on a retinal explant in a modified Boyden chamber. Finally, I magnetofected neural stem cells to over-express BDNF and directed their migration into the retina using galvanotaxis. Results: The retinal explants had a viability of up to 3 days based on the Sholl plots of retinal ganglion cells. Magnetofection with oscillation transfected cornea endothelium and the retinal ganglion cell layer with GFP in the explant models. It also transfected neural stem cells with BDNF-myc. Directed migration with neural stem cells occurred in the electrotactic chamber as well as in the retinal explant model. In the absence of electric field, no migration into the retina occurred. Neurospheres transfected with BDNF-myc also migrated into the retina when exposed to an electric field. Conclusion: Within the period of up to 3 days, retinal explants can be used to investigate neuroprotective therapeutic agents using Sholl plots of retinal ganglion cells. Magnetofection with oscillation is a novel non-viral technique for potentially transfecting the eye in the anterior and posterior segments. Neurospheres can be directed to migrate into the retina using an electric field in the ex-vivo model

Sholl analysis: A quantitative comparison of semi-automated methods

Background: Sholl analysis remains one of the most commonly used methods to quantify neuronal dendritic complexity and is therefore a key analysis tool in neurobiology. While initially proposed when the quantification of neuronal structure was undertaken manually, the advent of software packages allowing automated analysis has resulted in the introduction of several semi and fully automated methods to quantify dendritic complexity. Unfortunately results from these methods have not in all cases been consistent. We therefore compared the results of five commonly used methods (Simple Neurite Tracer, manual, Fast Sholl, Bitmap, and Ghosh lab) using manual analysis as a ground truth. New method: Comparison of four semi-automated methods to the manual method using diolistically labelled mouse retinal ganglion cells. Results: We report consistency across a range of published techniques. While the majority perform well (Simple Neurite Tracer and Fast Sholl profiles have areas under the curve within 4.5% of the profile derived using the manual method), we highlight two areas in two of the methods (Bitmap and Ghosh lab methods) where errors may occur, namely undercounting (>20% relative to the manual profile) and a second peak. Comparison with existing methods: Our results support published validation of the Fast Sholl method. Conclusions: Our study highlights the importance of manual calibration of automated analysis software

Automated registration of multimodal optic disc images: clinical assessment of alignment accuracy

Purpose: To determine the accuracy of automated alignment algorithms for the registration of optic disc images obtained by 2 different modalities: fundus photography and scanning laser tomography. Materials and Methods: Images obtained with the Heidelberg Retina Tomograph II and paired photographic optic disc images of 135 eyes were analyzed. Three state-of-the-art automated registration techniques Regional Mutual Information, rigid Feature Neighbourhood Mutual Information (FNMI), and nonrigid FNMI (NRFNMI) were used to align these image pairs. Alignment of each composite picture was assessed on a 5-point grading scale: “Fail” (no alignment of vessels with no vessel contact), “Weak” (vessels have slight contact), “Good” (vessels with 50% contact), and “Excellent” (complete alignment). Custom software generated an image mosaic in which the modalities were interleaved as a series of alternate 5×5-pixel blocks. These were graded independently by 3 clinically experienced observers. Results: A total of 810 image pairs were assessed. All 3 registration techniques achieved a score of “Good” or better in >95% of the image sets. NRFNMI had the highest percentage of “Excellent” (mean: 99.6%; range, 95.2% to 99.6%), followed by Regional Mutual Information (mean: 81.6%; range, 86.3% to 78.5%) and FNMI (mean: 73.1%; range, 85.2% to 54.4%). Conclusions: Automated registration of optic disc images by different modalities is a feasible option for clinical application. All 3 methods provided useful levels of alignment, but the NRFNMI technique consistently outperformed the others and is recommended as a practical approach to the automated registration of multimodal disc images

Laser peripheral iridoplasty for chronic angle closure

Background In at least a third of primary angle closure cases, appositional angle closure persists after laser peripheral iridotomy, and further intervention may be considered. Laser peripheral iridoplasty (LPIp) can be used in treating chronic angle closure when angle closure persists after laser peripheral iridotomy. Previous reviews have found insufficient data to determine its clinical effectiveness, compared to other interventions. This is an update of a Cochrane Review first published in 2008 and updated in 2012. It examines all studies to date to establish whether LPIp shows any effectiveness over other available treatment options. Objectives To assess the effectiveness of laser peripheral iridoplasty in the treatment of people with chronic angle closure, when compared to laser peripheral iridotomy, medical therapy or no further treatment. Search methods We searched various electronic databases. The date of the search was 20 December 2020. Selection criteria We included only randomised controlled trials (RCTs) assessing the use of LPIp in cases of suspected primary angle closure (PACS), confirmed primary angle closure (PAC), or primary chronic angle‐closure glaucoma (PACG). We applied no restrictions with respect to gender, age or ethnicity of participants. Trials evaluating LPIp for acute attacks of angle closure were not eligible. Data collection and analysis We used standard methodological procedures expected by Cochrane. Two authors independently assessed studies for risk of bias using Cochrane’s 'risk of bias' tool. We collected adverse effects information from the trials. Main results We included four RCTs involving 252 participants (276 eyes). In total, three different methods of intervention were used and 15 outcomes reported, with different time points. We used narrative synthesis to describe the majority of the findings, as meta‐analysis was only possible for a limited number of outcomes due to the variation in study design and outcomes assessed. Authors' conclusions After reviewing the outcomes of four RCTs, argon LPIp as an intervention may be no more clinically effective than comparators in the management of people with chronic angle closure. Despite a potential positive impact on anterior chamber morphology, its use in clinical practice in treating people with chronic angle closure is not supported by the results of trials published to date. Given these results, further research into LPIp is unlikely to be worthwhile

Development and validation of a standardized tool for reporting retinal findings in abusive Head trauma

PURPOSE: To develop and validate a robust standardized reporting tool for describing retinal findings in children examined for suspected abusive head trauma. DESIGN: A prospective interobserver and intraobserver agreement study. METHOD: An evidence-based assessment pro forma was developed, recording hemorrhages (location, layer, severity) and additional features. Eight consultant pediatric ophthalmologists and 7 ophthalmology residents assessed a series of 105 high-quality RetCam images of 21 eyes from abusive head trauma cases with varying degrees of retinal hemorrhage and associated findings. The pediatric ophthalmologists performed a repeat assessment of the randomized images. The images were observed simultaneously with standardized display settings. Interobserver and intraobserver agreement was assessed using free-marginal multirater kappa, intraclass correlation coefficients, and concordance coefficients. RESULTS: Almost-perfect interobserver agreement was observed for residents and pediatric ophthalmologists recording the presence and number of fundus hemorrhages (intraclass correlation coefficients 0.91 and 0.87, respectively) and the location of hemorrhages (concordance coefficients 0.86 and 0.85, respectively). Substantial agreement was observed by both groups regarding size of hemorrhage (concordance coefficients 0.73 and 0.76), moderate agreement for hemorrhage morphology (concordance coefficients 0.53 and 0.52), and other findings (concordance coefficients 0.48 and 0.59). Intraobserver agreement for pediatric ophthalmologists varied by question, ranging from substantial to perfect for the presence, number, location, size, and morphology of fundus hemorrhage. CONCLUSION: We have developed and validated a standardized clinical reporting tool for ophthalmic findings in suspected abusive head trauma, which has excellent interobserver and intraobserver agreement among consultant specialists and residents. We suggest that its use will improve standardized clinical reporting of such cases