Vascular retinal biomarkers improves the detection of the likely cerebral amyloid status from hyperspectral retinal images.

INTRODUCTION: This study investigates the relationship between retinal image features and ß-amyloid (Aß) burden in the brain with the aim of developing a noninvasive method to predict the deposition of Aß in the brain of patients with Alzheimer's disease. METHODS: Retinal images from 20 cognitively impaired and 26 cognitively unimpaired cases were acquired (3 images per subject) using a hyperspectral retinal camera. The cerebral amyloid status was determined from binary reads by a panel of 3 expert raters on 18F-florbetaben positron-emission tomography (PET) studies. Image features from the hyperspectral retinal images were calculated, including vessels tortuosity and diameter and spatial-spectral texture measures in different retinal anatomical regions. RESULTS: Retinal venules of amyloid-positive subjects (Aß+) showed a higher mean tortuosity compared with the amyloid-negative (Aß-) subjects. Arteriolar diameter of Aß+ subjects was found to be higher than the Aß- subjects in a zone adjacent to the optical nerve head. Furthermore, a significant difference between texture measures built over retinal arterioles and their adjacent regions were observed in Aß+ subjects when compared with the Aß-. A classifier was trained to automatically discriminate subjects combining the extracted features. The classifier could discern Aß+ subjects from Aß- subjects with an accuracy of 85%. DISCUSSION: Significant differences in texture measures were observed in the spectral range 450 to 550 nm which is known as the spectral region known to be affected by scattering from amyloid aggregates in the retina. This study suggests that the inclusion of metrics related to the retinal vasculature and tissue-related textures extracted from vessels and surrounding regions could improve the discrimination performance of the cerebral amyloid status.

Non-invasive in vivo hyperspectral imaging of the retina for potential biomarker use in Alzheimer's disease.

Studies of rodent models of Alzheimer's disease (AD) and of human tissues suggest that the retinal changes that occur in AD, including the accumulation of amyloid beta (Aß), may serve as surrogate markers of brain Aß levels. As Aß has a wavelength-dependent effect on light scatter, we investigate the potential for in vivo retinal hyperspectral imaging to serve as a biomarker of brain Aß. Significant differences in the retinal reflectance spectra are found between individuals with high Aß burden on brain PET imaging and mild cognitive impairment (n = 15), and age-matched PET-negative controls (n = 20). Retinal imaging scores are correlated with brain Aß loads. The findings are validated in an independent cohort, using a second hyperspectral camera. A similar spectral difference is found between control and 5xFAD transgenic mice that accumulate Aß in the brain and retina. These findings indicate that retinal hyperspectral imaging may predict brain Aß load.

Preliminary investigation of multispectral retinal tissue oximetry mapping using a hyperspectral retinal camera.

Oximetry measurement of principal retinal vessels represents a first step towards understanding retinal metabolism, but the technique could be significantly enhanced by spectral imaging of the fundus outside of main vessels. In this study, a recently developed Hyperspectral Retinal Camera was used to measure relative oximetric (SatO2) and total hemoglobin (HbT) maps of the retina, outside of large vessels, in healthy volunteers at baseline (N = 7) and during systemic hypoxia (N = 11), as well as in patients with glaucoma (N = 2). Images of the retina, on a field of view of ∼30°, were acquired between 500 and 600 nm with 2 and 5 nm steps, in under 3 s. The reflectance spectrum from each pixel was fitted to a model having oxy- and deoxyhemoglobin as the main absorbers and scattering modeled by a power law, yielding estimates of relative SatO2 and HbT over the fundus. Average optic nerve head (ONH) saturation over 8 eyes was 68 ± 5%. During systemic hypoxia, mean ONH saturation decreased by 12.5% on average. Upon further development and validation, the relative SatO2 and HbT maps of microvasculature obtained with this imaging system could ultimately contribute to the diagnostic and management of diseases affecting the ONH and retina.

Hyperspectral multiplex single-particle tracking of different receptor subtypes labeled with quantum dots in live neurons.

The efficacy of existing therapies and the discovery of innovative treatments for central nervous system (CNS) diseases have been limited by the lack of appropriate methods to investigate complex molecular processes at the synaptic level. To improve our capability to investigate complex mechanisms of synaptic signaling and remodeling, we designed a fluorescence hyperspectral imaging platform to simultaneously track different subtypes of individual neurotransmitter receptors trafficking in and out of synapses. This imaging platform allows simultaneous image acquisition of at least five fluorescent markers in living neurons with a high-spatial resolution. We used quantum dots emitting at different wavelengths and functionalized to specifically bind to single receptors on the membrane of living neurons. The hyperspectral imaging platform enabled the simultaneous optical tracking of five different synaptic proteins, including subtypes of glutamate receptors (mGluR and AMPAR) and postsynaptic signaling proteins. It also permitted the quantification of their mobility after treatments with various pharmacological agents. This technique provides an efficient method to monitor several synaptic proteins at the same time, which could accelerate the screening of effective compounds for treatment of CNS disorders.

A prototype hyperspectral system with a tunable laser source for retinal vessel imaging.

PURPOSE: To describe the technology and determine the within-session repeatability of manual retinal reflectance measurements of arterioles and venules using a prototype hyperspectral retinal camera. METHODS: Six healthy young volunteers (three males, average age 26 ± 4 years) had five repeated sets of retinal images captured between 500 and 600 nm at 5-nm intervals using a newly developed hyperspectral retinal camera. Optical densities were manually extracted for first-degree arterioles and venules and the repeatability of retinal reflectance was compared sequentially. The SDs of the differences between sequential mean values were used as an indication of the variance, while the coefficient of repeatability (COR) and intraclass correlation coefficient (ICC) were used to assess repeatability. RESULTS: The mean difference between each sequential measure was calculated using 21 images from each of the five spectral cubes. The SDs of these values ranged from 0.01 to 0.06 OD units and from 0.01 to 0.07 OD units for first-degree arterioles and venules, respectively. The COR ranged from 0.02 to 0.11 OD units (relative to a mean OD of 0.15 [0.06-0.23] OD units) for arterioles and 0.03 to 0.14 OD units (relative to a mean OD of 0.25 [0.17-0.31] OD units) for venules. Good reliability (P < 0.001) was found for arterioles (ICC: 78.8%-94.4% with a Cronbach's α of 89.6%-97.6%) and for venules (ICC: 63.7%-92.1% with a Cronbach's α of 86.2%-98.1%). CONCLUSIONS: Manual optical density determination with this novel hyperspectral camera showed very good intrasession (and intraobserver) repeatability with a small degree of variance that should form the basis of reliable retinal oxygen saturation values in future imaging research studies. Future automation of retinal vessel reflectance image analyses will likely further improve this repeatability.

Fabrication of paclitaxel nanocrystals by femtosecond laser ablation and fragmentation.

Nanonization, which involves formulating the drug powder as nanometer-sized particles, is a known method to improve drug absorption and allow the intravenous administration of insoluble drugs. This study investigated a novel femtosecond (fs) laser technique for the fabrication of nanocrystals in aqueous solution of the insoluble model drug paclitaxel. Two distinct methods of this technology, ablation and fragmentation, were investigated and the influence of laser power, focusing position and treatment time on the particle size, drug concentration, and degradation was studied. The colloidal suspensions were characterized with respect to size, chemical composition, morphology, and polymorphic state. Optimal laser fragmentation conditions generated uniformly sized paclitaxel nanoparticles (<500 nm) with quantifiable degradation, while ablation followed by fragmentation was associated with a larger polydispersity. Laser treatment at higher powers produced smaller particles with larger amount of degradation. The crystalline morphology of the drug was retained upon nanonization, but the anhydrous crystals were converted to a hydrated form, a phenomenon also observed during bead milling. These findings suggest that drug nanocrystals can be produced with fs laser technology using very little drug quantities, which may be an asset for preclinical evaluation of new drug candidates.

Nanonization of megestrol acetate by laser fragmentation in aqueous milieu.

Nanonization is a simple and effective method to improve dissolution rate and oral bioavailability of drugs with poor water solubility. There is growing interest to downscale the nanocrystal production to enable early preclinical evaluation of new drug candidates when compound availability is scarce. The purpose of the present study was to investigate laser fragmentation to form nanosuspensions in aqueous solution of the insoluble model drug megestrol acetate (MA) using very little quantities of the drug. Laser fragmentation was obtained by focusing a femtosecond (fs) or nanosecond (ns) laser radiation on a magnetically stirred MA suspension in water or aqueous solution of a stabilizing agent. The size distribution and physicochemical properties of the drug nanoparticles were characterized, and the in vitro dissolution and in vivo oral pharmacokinetics of a laser fragmented formulation were evaluated. A MA nanosuspension was also prepared by media milling for comparison purpose. For both laser radiations, smaller particles were obtained as the laser power was increased, but at a cost of higher degradation. Significant nanonization was achieved after a 30-minfs laser treatment at 250mW and a 1-hns laser treatment at 2500mW. The degradation induced by the laser process of the drug was primarily oxidative in nature. The crystal phase of the drug was maintained, although partial loss of crystallinity was observed. The in vitro dissolution rate and in vivo bioavailability of the laser fragmented formulation were similar to those obtained with the nanosuspension prepared by media milling, and significantly improved compared to the coarse drug powder. It follows that this laser nanonization method has potential to be used for the preclinical evaluation of new drug candidates.

Reverse iontophoresis of amino acids: identification and separation of stratum corneum and subdermal sources in vitro.

PURPOSE: To differentiate the stratum corneum (SC) and subdermal sources of amino acids (AAs) extracted by reverse iontophoresis. METHODS: 13 zwitterionic AAs were quantified in this in vitro study. Repetitive tape-stripping permitted the distribution of the analytes to be determined in the SC. Iontophoresis experiments were performed in which the subdermal chamber contained either phosphate-buffered saline (PBS) only, or a mixture of the 13 AAs in PBS. RESULTS: AAs were homogeneously distributed across the SC and broadly divided into three groups (high, medium, low) in terms of total amount present. As expected, extraction to the cathode for the essentially neutral analytes involved was more efficient. Initial samples obtained during the first hour of iontophoresis primarily extracted AAs from the SC. The fluxes observed in the latter half of the 6-h experiment, on the other hand, correlated well with the corresponding subdermal concentrations. CONCLUSION: A relatively short extraction period (approximately 1 h) by reverse iontophoresis can be used to evaluate the content of AAs in the SC. Once this 'reservoir' has been depleted, reverse iontophoresis can then monitor the subdermal concentrations of the AAs. The latter appears most useful for compounds which are present at lower levels in the SC.

In vitro optimization of dexamethasone phosphate delivery by iontophoresis.

BACKGROUND AND PURPOSE: This study was designed to evaluate the effects of competing ions and electroosmosis on the transdermal iontophoresis of dexamethasone phosphate (Dex-Phos) and to identify the optimal conditions for its delivery. METHODS: The experiments were performed using pig skin, in side-by-side diffusion cells (0.78 cm(2)), passing a constant current of 0.3 mA via Ag-AgCl electrodes. Dex-Phos transport was quantified for donor solutions (anodal and cathodal) containing different drug concentrations, with and without background electrolyte. Electrotransport of co-ion, citrate, and counterions Na(+) and K(+) also was quantified. The contribution of electroosmosis was evaluated by measuring the transport of the neutral marker (mannitol). RESULTS: Electromigration was the dominant mechanism of drug iontophoresis, and reduction in electroosmotic flow directed against the cathodic delivery of Dex-Phos did not improve drug delivery. The Dex-Phos flux from the cathode was found to be optimal (transport number of approximately 0.012) when background electrolyte was excluded from the formulation. In this case, transport of the drug is limited principally by the competition with counterions (mainly Na(+) with a transport number of approximately 0.8) and the mobility of the drug in the membrane. DISCUSSION AND CONCLUSION: Dex-Phos must be delivered from the cathode and formulated rationally, excluding mobile co-anions, to achieve optimal iontophoretic delivery.

Stabilization and size control of gold nanoparticles during laser ablation in aqueous cyclodextrins.

Femtosecond laser radiation has been used to ablate a gold target in aqueous beta-cyclodextrin (CD) solutions to produce stable gold nanoparticle colloids with extremely small size (2 to 2.4 nm) and size dispersion (1 to 1.5 nm). On the basis of XPS and zeta-potential measurements, we propose a model involving chemical interactions between the gold and the CDs. The model includes both the hydrophobic interaction of the Au0 with the interior cavity of the CD and the hydrogen bonding of O- groups on the partially oxidized gold surface with -OH groups of the CDs.