Spectral fundus autofluorescence peak emission wavelength in ageing and AMD.

PURPOSE: To investigate the spectral characteristics of fundus autofluorescence (FAF) in AMD patients and controls. METHODS: Fundus autofluorescence spectral characteristics was described by the peak emission wavelength (PEW) of the spectra. Peak emission wavelength (PEW) was derived from the ratio of FAF recordings in two spectral channels at 500-560 nm and 560-720 nm by fluorescence lifetime imaging ophthalmoscopy. The ratio of FAF intensity in both channels was related to PEW by a calibration procedure. Peak emission wavelength (PEW) measurements were done in 44 young (mean age: 24.0 ± 3.8 years) and 18 elderly (mean age: 67.5 ± 10.2 years) healthy subjects as well as 63 patients with AMD (mean age: 74.0 ± 7.3 years) in each pixel of a 30° imaging field. The values were averaged over the central area, the inner and the outer ring of the ETDRS grid. RESULTS: There was no significant difference between PEW in young and elderly controls. However, PEW was significantly shorter in AMD patients (ETDRS grid centre: 571 ± 26 nm versus 599 ± 17 nm for elderly controls, inner ring: 596 ± 17 nm versus 611 ± 11 nm, outer ring: 602 ± 16 nm versus 614 ± 11 nm). After a mean follow-up time of 50.8 ± 10.8 months, the PEW in the patients decreased significantly by 9 ± 19 nm in the inner ring of the grid. Patients, showing progression to atrophic AMD in the follow up, had significantly (p ≤ 0.018) shorter PEW at baseline than non-progressing patients. CONCLUSIONS: Peak emission wavelength (PEW) is related to AMD pathology and might be a diagnostic marker in AMD. Possibly, a short PEW can predict progression to retinal and/or pigment epithelium atrophy.

Spectral and lifetime resolution of fundus autofluorescence in advanced age-related macular degeneration revealing different signal sources.

PURPOSE: To determine the fundus autofluorescence (FAF) lifetimes and spectral characteristics of individual drusen and hyperpigmentation independent of those with retinal pigment epithelium (RPE) in geographic atrophy (GA) areas in late-stage age-related macular degeneration (AMD). METHODS: Three consecutive patients with complete RPE and outer retinal atrophy (cRORA) exhibiting drusen that were calcified or associated with hyperpigmentation were investigated with multimodal non-invasive ophthalmic imaging including colour fundus photography (CFP), optical coherence tomography (OCT), near-infrared reflectance (NIR), blue FAF and fluorescence lifetime imaging ophthalmoscopy (FLIO). Fluorescence lifetimes were measured in two spectral channels (short-wavelength spectral channel (SSC): 500-560 nm and long-wavelength spectral channel (LSC): 560-720 nm). RESULTS: Drusen lacking RPE coverage, as confirmed by CFP and OCT, had longer FAF lifetimes than surrounding cRORA by 127 ± 66 ps (SSC) and 113 ± 48 ps (LSC, both p = 0.008 in Wilcoxon test, N = 9) and by 209 ± 100 ps (SSC) and 121 ± 56 ps (LSC, p < 0.001, N = 14) in two patients. Hyperpigmentation in CFP in a third patient shows strong FAF with prolonged lifetimes. In the SSC, persistent FAF was found inside cRORA. A crescent-shaped hyperfluorescence in an area of continuous RPE but lacking outer retina was seen in one eye with a history of anti-VEGF treatment. CONCLUSIONS: Short-wavelength fluorescence in cRORA points to fluorophores beyond RPE organelles. Fluorescence properties of drusen within cRORA differ from in vivo drusen covered by RPE. These limited findings from three patients give new insight into the sources of FAF that can be further elucidated in larger cohorts.

Progressive Dysmorphia of Retinal Pigment Epithelium in Age-Related Macular Degeneration Investigated by Fluorescence Lifetime Imaging.

Purpose: The purpose of this study was to observe changes of the retinal pigment epithelium (RPE) on the transition from dysmorphia to atrophy in age-related macular degeneration (AMD) by fluorescence lifetime imaging ophthalmoscopy (FLIO). Methods: Multimodal imaging including color fundus photography (CFP), optical coherence tomography (OCT), fundus autofluorescence (FAF) imaging, and FLIO was performed in 40 eyes of 37 patients with intermediate AMD and no evidence for geographic atrophy or macular neovascularization (mean age = 74.2 ± 7.0 years). Twenty-three eyes were followed for 28.3 ± 18.3 months. Seven eyes had a second follow-up after 46.6 ± 9.0 months. Thickened RPE on OCT, hyperpigmentation on CFP, hyper-reflective foci (HRF) on OCT, attributed to single or clustered intraretinal RPE, were identified. Fluorescence lifetimes in two spectral channels (short-wavelength spectral channel [SSC] = 500-560 nm, long-wavelength spectral channel [LSC] = 560-720 nm) as well as emission spectrum intensity ratio (ESIR) of the lesions were measured by FLIO. Results: As hyperpigmented areas form and RPE migrates into the retina, FAF lifetimes lengthen and ESRI of RPE cells increase. Thickened RPE showed lifetimes of 256 ± 49 ps (SSC) and 336 ± 35 ps (LSC) and an ESIR of 0.552 ± 0.079. For hyperpigmentation, these values were 317 ± 68 ps (p < 0.001), 377 ± 56 ps (P < 0.001), and 0.609 ± 0.081 (P = 0.001), respectively, and for HRF 337 ± 79 ps (P < 0.001), 414 ± 50 ps (P < 0.001), and 0.654 ± 0.075 (P < 0.001). Conclusions: In the process of RPE degeneration, comprising different steps of dysmorphia, hyperpigmentation, and migration, lengthening of FAF lifetimes and a hypsochromic shift of emission spectra can be observed by FLIO. Thus, FLIO might provide early biomarkers for AMD progression and contribute to our understanding of RPE pathology.

Enrichment and characterization of extracellular vesicles from ex vivo one-sided human placenta perfusion.

PROBLEM: Extracellular vesicles (EVs) released by the placenta are packed with biological information and play a major role in fetomaternal communication. Here, we describe a comprehensive set-up for the enrichment and characterization of EVs from human placenta perfusion and their application in further assays. METHOD OF STUDY: Human term placentas were used for 3 h ex vivo one-sided perfusions to simulate the intervillous circulation. Thereafter, populations of small (sEVs) and large EV (lEVs) were enriched from placental perfusate via serial ultracentrifugation. Following, EV populations were characterized regarding their size, protein concentration, RNA levels, expression of surface markers as well as their uptake and miRNA transfer to recipient cells. RESULTS: The sEV and lEV fractions from an entire perfusate yielded, respectively, 294 ± 32 µg and 525 ± 96 µg of protein equivalents and 2.6 ± 0.5 µg and 3.6 ± 0.9 µg of RNA. The sEV fraction had a mean diameter of 117 ± 47 nm, and the lEV fraction presented 236 ± 54 nm. CD63 was strongly detected by dot blot in sEVs, whereas only traces of this marker were found in lEVs. Both EV fractions were positive for the trophoblast marker PLAP (placental alkaline phosphatase) and annexin A1. EV internalization in immune cells was visualized by confocal microscopy, and the transfer of placental miRNAs was detected by quantitative real-time PCR (qPCR). CONCLUSIONS: Enriched EV populations showed characteristic features of sEVs and lEVs. EV uptake and transfer of miRNAs to recipient cells demonstrated their functional integrity. Therefore, we advocate the ex vivo one-sided placenta perfusion as a robust approach for the collection of placental EVs.

Fluorescence Lifetimes and Spectra of RPE and Sub-RPE Deposits in Histology of Control and AMD Eyes.

Purpose: To investigate fluorescence lifetimes as well as spectral characteristics of drusen and RPE autofluorescence in AMD. Methods: Fluorescence lifetimes and spectra of five eyes with AMD and nine control eyes were analyzed in cryosections by means of two-photon excited fluorescence at 960 nm. Spectra were detected at 490 to 647 nm. Lifetimes were measured using time-correlated single photon counting in two spectral channels: 500 to 550 nm and 550 to 700 nm. Fluorescence decays over time were approximated by a series of three exponential functions. The amplitude-weighted mean fluorescence lifetime was determined. Results: We identified 196 sub-RPE deposits (AMD, n = 76; control, n = 120) and recorded 241 RPE sites. The peak emission wavelength of sub-RPE deposits was significantly green shifted compared with RPE (peak at 570 nm vs. 610 nm), but did not differ between AMD and control donors. Sub-RPE deposits showed considerably longer mean fluorescence lifetimes than RPE (ch1, 581 ± 163 ps vs. 177 ± 25 ps; ch2, 541 ± 125 ps vs. 285 ± 31 ps; P < 0.001). Sub-RPE deposits found in AMD eyes had longer lifetimes than deposits of controls (ch1, 650 ± 167 ps vs. 537 ± 145 ps; ch2, 600 ± 125 ps vs. 504 ± 111 ps; P < 0.001). In AMD eyes, sub-RPE deposits showed a more homogenous autofluorescence distribution and more deposits were larger than 63 µm than in control eyes. Conclusions: Ex vivo fluorescence imaging of sub-RPE deposits in cross-sections enables the separation of their autofluorescence from that of over- or underlying structures. Our analysis showed considerable variability of sub-RPE deposit lifetimes but not spectra. This indicates that sub-RPE deposits either consist of a variety of different fluorophores or expose the same fluorophores to different microenvironments.

Fundus Autofluorescence Lifetimes and Spectral Features of Soft Drusen and Hyperpigmentation in Age-Related Macular Degeneration.

Purpose: To investigate the autofluorescence lifetimes as well as spectral characteristics of soft drusen and retinal hyperpigmentation in age-related macular degeneration (AMD). Methods: Forty-three eyes with nonexudative AMD were included in this study. Fluorescence lifetime imaging ophthalmoscopy (FLIO), which detects autofluorescence decay over time in the short (SSC) and long (LSC) wavelength channel, was performed. The mean autofluorescence lifetime (τm) and the spectral ratio (sr) of autofluorescence emission in the SSC and LSC were recorded and analyzed. In total, 2760 soft drusen and 265 hyperpigmented areas were identified from color fundus photographs and spectral domain optical coherence tomography (SD-OCT) images and superimposed onto their respective AF images. τm and sr of these lesions were compared with fundus areas without drusen. For clearly hyperfluorescent drusen, the local differences compared to fundus areas without drusen were determined for lifetimes and sr. Results: Hyperpigmentation showed significantly longer τm (SSC: 341 ± 81 vs. 289 ± 70 ps, P < 0.001; LSC: 406 ± 42 vs. 343 ± 42 ps, P < 0.001) and higher sr (0.621 ± 0.077 vs. 0.539 ± 0.083, P < 0.001) compared to fundus areas without hyperpigmentation or drusen. No significant difference in τm was found between soft drusen and fundus areas without drusen. However, the sr was significantly higher in soft drusen (0.555 ± 0.077 vs. 0.539 ± 0.081, P < 0.0005). Hyperfluorescent drusen showed longer τm than surrounding fundus areas without drusen (SSC: 18 ± 42 ps, P = 0.074; LSC: 16 ± 29 ps, P = 0.020). Conclusions: FLIO can quantitatively characterize the autofluorescence of the fundus, drusen, and hyperpigmentation in AMD. Translational Relevance: The experimental FLIO technique was applied in a clinical investigation. As FLIO yields information on molecular changes in AMD, it might support future diagnostics.

Influence of Lens Fluorescence on Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO) Fundus Imaging and Strategies for Its Compensation.

Purpose: To explore the contribution of crystalline lens fluorescence to fluorescence lifetimes measured with fluorescence lifetime imaging ophthalmoscopy (FLIO) and to propose a computational model to reduce the lens influence. Methods: FLIO, which detects autofluorescence decay over time in a short-wavelength spectral channel (SSC, 498-560 nm) and a long-wavelength spectral channel (LSC, 560-720 nm), was performed on 32 patients before and after cataract extraction. The mean autofluorescence lifetime (τ m ) of the fundus was determined from a three-exponential fit of the postoperative fluorescence decays. The preoperative measurements were fit with series of exponential functions in which one fluorescence component was time-shifted in order to represent lens fluorescence. Results: Postoperatively, τ m was 185 ± 22 ps in the SSC and 209 ± 34 ps in the LSC at the posterior pole. These values were best reproduced by fitting the postoperative measurements with a three-exponential model with a time-shifted third fluorescence component (SSC, 203 ± 45 ps; LSC, 215 ± 29 ps), whereas disregarding time-shifted lens fluorescence resulted in significantly (P < 0.001) longer τ m values (SSC, 474 ± 206 ps; LSC, 215 ± 29 ps). The fluorescence of the cataract lens contributed to the total fluorescence by 54.2 ± 10.6% (SSC) and 29.5 ± 9.9% (LSC). Conclusions: Cataract lens fluorescence greatly alters fluorescence lifetimes measured at the fundus by FLIO, resulting in an overestimation of the lifetimes; however, this may be compensated for considerably by taking lens influence into account in the fitting model. Translational Relevance: This study investigates cataract fluorescence in FLIO and a mathematical model for compensation of this influence.

Simplified approach to least-square fitting of fluorescence lifetime ophthalmoscopy (FLIO) data by fixating lifetimes.

Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new imaging modality in ophthalmology. For clinical investigations, the amplitude-weighted mean of two or three lifetime components is usually analyzed. In this study, we investigated the effects of fixation of lifetime components. This resulted in slightly higher fit errors but mean lifetimes were highly correlated to those from fits with variable individual lifetimes. Furthermore, this approach resulted in a similarly good discrimination of diabetic retinopathy patients from controls, a reduction of the computational workload, a de-noising of the mean lifetime images and allows higher local resolution. Thus, fixation of lifetimes in the fit of FLIO data could be superior for clinical routine analysis of FLIO data.

Fundus autofluorescence beyond lipofuscin: lesson learned from ex vivo fluorescence lifetime imaging in porcine eyes.

Fundus autofluorescence (FAF) imaging is a well-established method in ophthalmology; however, the fluorophores involved need more clarification. The FAF lifetimes of 20 post mortem porcine eyes were measured in two spectral channels using fluorescence lifetime imaging ophthalmoscopy (FLIO) and compared with clinical data from 44 healthy young subjects. The FAF intensity ratio of the short and the long wavelength emission (spectral ratio) was determined. Ex vivo porcine fundus fluorescence emission is generally less intense than that seen in human eyes. The porcine retina showed significantly (p<0.05) longer lifetimes than the retinal pigment epithelium (RPE): 584 ± 128 ps vs. 121 ± 55 ps 498-560 nm, 240 ± 42 ps vs. 125 ± 20 ps at 560-720 nm. Furthermore, the lifetimes of the porcine RPE were significantly shorter (121 ± 55 ps and 125 ± 20 ps) than those measured from human fundus in vivo (162 ± 14 ps and 179 ± 13 ps, respectively). The fluorescence emission of porcine retina was shifted towards a shorter wavelength compared to that of RPE and human FAF. This data shows the considerable contribution of fluorophores in the neural retina to total FAF intensity in porcine eyes.

Frataxin overexpression in Müller cells protects retinal ganglion cells in a mouse model of ischemia/reperfusion injury in vivo.

Müller cells are critical for retinal function and neuronal survival but can become detrimental in response to retinal ischemia and increased oxidative stress. Elevated oxidative stress increases expression of the mitochondrial enzyme frataxin in the retina, and its overexpression is neuroprotective after ischemia. Whether frataxin expression in Müller cells might improve their function and protect neurons after ischemia is unknown. The aim of this study was to evaluate the effect of frataxin overexpression in Müller cells on neuronal survival after retinal ischemia/reperfusion in the mouse in vivo. Retinal ischemia/reperfusion was induced in mice overexpressing frataxin in Müller cells by transient elevation of intraocular pressure. Retinal ganglion cells survival was determined 14 days after lesion. Expression of frataxin, antioxidant enzymes, growth factors and inflammation markers was determined with qRT-PCR, Western blotting and immunohistochemistry 24 hours after lesion. Following lesion, there was a 65% increase in the number of surviving RGCs in frataxin overexpressing mice. Improved survival was associated with increased expression of the antioxidant enzymes Gpx1 and Sod1 as well as the growth factors Cntf and Lif. Additionally, microglial activation was decreased in these mice. Therefore, support of Müller cell function constitutes a feasible approach to reduce neuronal degeneration after ischemia.

Increased Frataxin Levels Protect Retinal Ganglion Cells After Acute Ischemia/Reperfusion in the Mouse Retina In Vivo.

PURPOSE: The mitochondrial protein frataxin (FXN) is highly expressed in metabolically active tissues and has been shown to improve cell survival in response to oxidative stress after ischemia. Retinal ischemia/hypoxia is a complication of ocular diseases such as diabetic retinopathy and glaucoma. There are no effective therapeutic approaches currently available. This study was performed to evaluate the neuroprotective effects of FXN after acute retinal ischemia/reperfusion in vivo. METHODS: Retinal ischemia/reperfusion was induced in adult wild-type and FXN-overexpressing mice by transient elevation of intraocular pressure (IOP) for 45 minutes. Expression of FXN was evaluated by quantitative (q)RT-PCR and Western blot analysis between 6 and 48 hours after ischemia. Retinal ganglion cell (RGC) survival was determined with immunofluorescent staining and fluorescence microscopy 14 days after lesion. Expression of hypoxia-inducible factors Hif-1α and Hif-2α and of oxidative stress markers heme oxygenase-1 (Hmox1), glutathione peroxidase 1 (Gpx1), superoxidase dismutase 1 and 2 (Sod1, Sod2), and catalase was evaluated by qRT-PCR. RESULTS: Endogenous FXN levels were upregulated for up to 24 hours after retinal ischemia in vivo. Retinal ganglion cell survival was significantly improved in FXN-overexpressing mice 14 days after ischemia. Expression of antioxidative enzymes Gpx1, Sod2, and catalase was significantly increased in FXN-overexpressing mice after lesion. CONCLUSIONS: Retinal FXN levels are increased in response to ischemia. Furthermore, elevated FXN levels had a clear neuroprotective effect as shown by increased ganglion cell survival after acute retinal ischemia/reperfusion. Frataxin's neuroprotective effect was associated with an upregulation of antioxidative enzymes. The data suggest that FXN induces neuroprotection by decreasing oxidative stress.

Spatial variation of epoxyscillirosidine concentrations in Moraea pallida (yellow tulp) in South Africa.

Moraea pallida (yellow tulp) poisoning is economically the most important intoxication of livestock in South Africa. Poisoning varies according to locality, climatic conditions and growth stage of the plant. The primary objective of this study was to determine the concentration of the toxic principle, epoxyscillirosidine, in yellow tulp leaves and to ascertain the variability of epoxyscillirosidine concentrations within and between different locations. A secondary objective was to utilise Geographic Information Systems in an attempt to explain the variability in toxicity. Flowering yellow tulp plants were collected at 26 sampling points across 20 districts of South Africa. The leaves of five plants per sampling point were extracted and submitted for liquid chromatography/mass spectrometry analysis. A large variation in mean epoxyscillirosidine concentrations, ranging from 3.32 µg/g - 238.27 µg/g, occurred between different geographical regions. The epoxyscillirosidine concentrations also varied tremendously between individual plants (n = 5) collected at the same sampling point, with up to a 24 times difference between the lowest and highest concentration detected. No generalised correlation between epoxyscillirosidine concentrations and soil elemental concentrations could be established. However, samples obtained from the north-eastern part of the sampling region tended to have higher epoxyscillirosidine concentrations compared to samples obtained from the south-western part of the sampling region. Higher toxin concentrations in the north-east were associated with statistically significant higher soil concentrations of iron, bismuth, bromide, cadmium, chromium, rubidium, tellurium, thallium, titanium and zinc, whilst soil concentrations of strontium and soil pH, were significantly lower. This study corroborated the contention that epoxyscillirosidine concentration in yellow tulp fluctuates and may explain the variability in toxicity.