Aims: To investigate the efficacy and safety of intravitreal Dexamethasone implant (DEX-I) therapy in the treatment of diabetic macular edema (DME) refractory to intravitreal bevacizumab (IVB). Material and methods: This retrospective and cross-sectional study included 37 eyes of 37 patients who received 3 loading doses of IVB injections for DME with no response and underwent DEX-I implant. Best-corrected visual acuity (BCVA), intraocular pressure (IOP) measurements and central foveal thickness (CFT) measured by spectral domain optical coherence tomography (SD-OCT) were recorded and compared before DEX-I, at the first week, first, second, third and sixth months. Duration of DME, glycated hemoglobin (HbA1c) levels, DME types and lens status (phakic, pseudophakic) were also recorded. Results: The mean age of the patients was 61.14 ±8.69 years (59.5% male, 40.5% female). 35.1% of the patients had cystoid macular edema, 64.9% had diffuse macular edema and 73 % were phakic and 27% were pseudophakic. BCVA, CFT and IOP values before DEX-I injection were 0.78 ±0.16 LogMAR, 493.73 ±107.6 μm and 13.05 ±2.59 mmHg, respectively. At 6 months after DEX-I, BCVA, CFT and IOP values were 0.64 ±0.11 LogMAR, 397.35 ±59.72 μm and 16.3 ±2.51 mmHg, respectively. In all follow-ups, there was a significant improvement in BCVA, a significant decrease in CFT and a significant increase in IOP compared to pre-injection. Ocular hypertension was observed in 0.8 % of patients and progression of cataract progression in 1% of patients after treatment. Conclusion: DEX-I therapy is an effective and safe treatment option for DME refractory to IVB treatment.
BACKGROUND AND PURPOSE: The primary objective was to compare diffusion tensor imaging (DTI) scalar parameters of peripheral nerves between subjects with type 2 diabetes mellitus (T2DM) and those without diabetes. Secondarily, we aimed to correlate DTI scalar parameters with nerve morphometric properties. METHODS: Median, tibial, and sural nerves were harvested from 34 male cadavers (17 T2DM, 17 nondiabetic). Each nerve was divided into three segments. The initial segment was scanned using 9.4 Tesla MRI system (three-dimensional pulsed-gradient spin-echo sequence). DTI scalars were calculated from region-average diffusion-weighted signals. Second segment was optically cleared, acquired with optical projection tomography (OPT), and analyzed for morphometrical properties. Toluidine-stained sections were prepared from last segment, and axon- and myelin-related properties were evaluated. RESULTS: DTI scalar parameters of median and tibial nerves were comparable between the groups, while sural nerves of T2DM exhibited on average 41% higher mean diffusivity (MD) (p = 0.03), 38% higher radial diffusivity (RD) (p = 0.03), and 27% lower fractional anisotropy (FA) (p = 0.005). Significant differences in toluidine-evaluated parameters of sural nerves were observed between the groups, with a positive correlation between FA with fiber density (p = 0.0001) and with myelin proportion (p < 0.0001) and an inverse correlation between RD and myelin proportion (p = 0.003). OPT-measured morphometric properties did not correlate with DTI scalar parameters. CONCLUSIONS: High-field DTI shows promise as an imaging technique for detecting axonal and myelin-related changes in small sural nerves ex vivo. The reduced fiber density and decreased myelin content, which can be observed in T2DM, likely contribute to observed FA reduction and increased MD/RD.
- MeSH
- Diabetes Mellitus, Type 2 * diagnostic imaging pathology MeSH
- Diabetic Neuropathies diagnostic imaging pathology MeSH
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Cadaver * MeSH
- Median Nerve diagnostic imaging pathology MeSH
- Sural Nerve * diagnostic imaging pathology MeSH
- Tibial Nerve diagnostic imaging pathology MeSH
- Reproducibility of Results MeSH
- Aged MeSH
- Sensitivity and Specificity MeSH
- Diffusion Tensor Imaging * methods MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Publication type
- Journal Article MeSH
The GPCR signalling cascade is a key pathway responsible for the signal transduction of a multitude of physical and chemical stimuli, including light, odorants, neurotransmitters and hormones. Understanding the structural and functional properties of the GPCR cascade requires direct observation of signalling processes in high spatial and temporal resolution, with minimal perturbation to endogenous systems. Optical microscopy and spectroscopy techniques are uniquely suited to this purpose because they excel at multiple spatial and temporal scales and can be used in living objects. Here, we review recent developments in microscopy and spectroscopy technologies which enable new insights into GPCR signalling. We focus on advanced techniques with high spatial and temporal resolution, single-molecule methods, labelling strategies and approaches suitable for endogenous systems and large living objects. This review aims to assist researchers in choosing appropriate microscopy and spectroscopy approaches for a variety of applications in the study of cellular signalling. LINKED ARTICLES: This article is part of a themed issue Complexity of GPCR Modulation and Signaling (ERNST). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v182.14/issuetoc.
- MeSH
- Humans MeSH
- Microscopy * methods MeSH
- Receptors, G-Protein-Coupled * chemistry metabolism MeSH
- Signal Transduction MeSH
- Spectrum Analysis * methods MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
This study aimed to compare the fascicular anatomy of upper limb nerves visualized using in situ high-resolution ultrasound (HRUS) with ex vivo imaging modalities, namely, magnetic resonance microscopy (MRM), histological cross-sections (HCS), and optical projection tomography (OPT). The median, ulnar, and superficial branch of radial nerve (n = 41) were visualized in 14 cadaveric upper limbs using 22-MHz HRUS. Subsequently, the nerves were excised, imaged with different microscopic techniques, and their morphometric properties were compared. HRUS accurately differentiated 51-74% of fascicles, while MRM detected 87-92% of fascicles when compared to the referential HCS. Among the compared modalities, HRUS demonstrated the smallest fascicular ratios and fascicular cross-sectional areas, but the largest nerve cross-sectional areas. The probability of a fascicle depicted on HRUS representing a cluster of multiple fascicles on the referential HCS increased with the fascicular size, with some differences observed between the larger median and ulnar nerves and the smaller radial nerves. Accordingly, HRUS fascicle differentiation necessitates cautious interpretation, as larger fascicles are more likely to represent clusters. Although HCS is considered the reference modality, alterations in nerve cross-sectional areas or roundness during sample processing should be acknowledged.
- MeSH
- Upper Extremity * innervation diagnostic imaging MeSH
- Middle Aged MeSH
- Humans MeSH
- Magnetic Resonance Imaging methods MeSH
- Microscopy methods MeSH
- Cadaver MeSH
- Median Nerve diagnostic imaging MeSH
- Radial Nerve * diagnostic imaging anatomy & histology MeSH
- Ulnar Nerve * diagnostic imaging anatomy & histology MeSH
- Aged MeSH
- Ultrasonography * methods MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Remodeling of a microvascular network is common part of pathological changes associated with wide spectrum of diseases. Quantitative analysis of these alterations relies often on analysis of a point-pattern on the histological slide, i.e. on sections through the microvascular network only. Common techniques are based on the estimation of the average density of points representing section through microvessels on the histological image. This approach inherently omits the information about the regularity of the pattern. Thus, we used approach based on the Voronoi segmentation and chose the best statistical model of areas of Voronoi cells surrounding microvessels on 20 samples of human myocardium. The best model is based on the log-normal distribution. Parameters of the model for given data can be estimated as a mean and a standard deviation of logarithms of areas of Voronoi cells. Moreover, these parameters can be transformed to the widely used measure called the microvascular density.
- MeSH
- Histological Techniques MeSH
- Cardiovascular Diseases MeSH
- Humans MeSH
- Microvessels anatomy & histology MeSH
- Microscopy methods MeSH
- Myocardium * ultrastructure MeSH
- Image Processing, Computer-Assisted methods MeSH
- Vascular Remodeling MeSH
- Pattern Recognition, Automated MeSH
- Check Tag
- Humans MeSH
OBJECTIVE: The highly infiltrative growth of glioblastoma (GBM) makes distinction between the tumor and normal brain tissue challenging. Therefore, fluorescence-guided surgery is often used to improve visual identification of radiological tumor margins. The aim of this study was to evaluate the ability of recently developed molecularly targeted near-infrared (NIR) protease-activated probes to visualize GBM tissue and to compare the most promising candidate with the gold standard, 5-aminolevulinic acid (5-ALA). METHODS: Single-substrate probes 6QC-ICG and 6QC-Cy5 (cysteine cathepsin cleavable), double-substrate probes AG2-FNIR and AG2-Cy5 (cysteine cathepsin and caspase 3 cleavable), and 5-ALA were administered intravenously to mice with orthotopic tumors. Activation of the probes was also evaluated in cell cultures in vitro and in biopsy material from patients with GBM ex vivo. The tumor to normal brain tissue fluorescence ratio (TNR) was quantified in brain sections using preclinical and clinical visualization platforms, and in tissue homogenates and cell suspensions using spectrofluorimetry. Subcellular localization of the fluorophores was visualized by confocal microscopy. RESULTS: In vitro, the single-substrate probe 6QC-ICG was cleaved in glioma cells and macrophages, and the resulting fluorophore accumulated intracellularly. In experimental GBMs, both single- and double-substrate probes visualized tumor tissue, while in healthy brain tissue the signal was minimal. TNR was highest for 6QC-ICG and AG2-FNIR, but the signal intensity was higher for 6QC-ICG. Using xenograft and syngeneic mouse models, as well as human GBM biopsy material ex vivo, the authors confirmed the ability of 6QC-ICG to specifically visualize the glioma tissue using preclinical and clinical visualization platforms. Finally, a comparison with 5-ALA in animals coadministered with both compounds revealed a higher TNR for 6QC-ICG in experimental GBMs. CONCLUSIONS: The cysteine cathepsin-cleavable probe 6QC-ICG is activated by glioma cells and tumor-associated macrophages, leading to a high contrast between tumor and nontumorous brain tissue that is superior to that of the current standard, 5-ALA. In addition to a well-defined mechanism of action, protease-activated probes that use NIR fluorophores (e.g., indocyanine green) have the advantage of low absorption and scattering of the NIR light and lower tissue autofluorescence. These results suggest that 6QC-ICG has the potential to become the targeted agent in intraoperative detection of GBM tissue using fluorescence imaging.
- MeSH
- Fluorescent Dyes MeSH
- Glioblastoma * diagnostic imaging pathology MeSH
- Aminolevulinic Acid * MeSH
- Humans MeSH
- Molecular Probes MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Brain Neoplasms * diagnostic imaging pathology MeSH
- Optical Imaging methods MeSH
- Peptide Hydrolases metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
BACKGROUND: Age-related macular degeneration (AMD) is one of the major causes of vision loss in individuals aged ≥ 65 years in developed countries. This study aimed to determine the associations between modifiable risk factors and AMD. This is the first study describing the relationship between lifestyle factors and AMD in the Czech Republic. METHODS: In this cross-sectional case-control study, 93 AMD cases and 58 controls without AMD and cataract were included. All participants were examined by Optical coherence tomography at the Clinic of Eye Treatment at the University Hospital Brno. Data were collected using a pre-tested self-report questionnaire in a face-to-face interview. RESULTS: We found significant associations between those who were living in the city (OR 95% CI: 2.19 (1.0-4.6); p = 0,039), with a positive family history of AMD (OR 95% CI: 12.75 (1.6-98.6); p = 0,015), exposure to cigarette smoke (OR 95% CI: 2.72 (1.4-5.4); p = 0,004), and daily exposure to passive smoking (OR 95% CI: 2.29 (1.0-5.1); p = 0,045) and AMD. In men, we found significant associations between daily sunlight exposure (OR 95% CI: 2.98 (1.0-8.5); p = 0,041), short or long sleep duration (OR 95% CI: 3.98 (1.2-13.2); p = 0,024) and AMD. Men daily exposed to sunlight were at a 2.98 times higher risk of AMD than men with less than daily sunlight exposure. Men with short or long sleep duration (< 6 and > 8 h) were at a 3.98 times higher risk of AMD than men with recommended sleep duration of 6-8 h. CONCLUSIONS: An increased risk of AMD was observed for living in the city, family history of AMD, exposure to cigarette smoke, and daily exposure to passive smoking. Increased risk of AMD was observed for daily sunlight exposure and short or long sleep duration; however, only in men.
- MeSH
- Smoking adverse effects epidemiology MeSH
- Middle Aged MeSH
- Humans MeSH
- Macular Degeneration * epidemiology etiology MeSH
- Tomography, Optical Coherence * MeSH
- Cross-Sectional Studies MeSH
- Surveys and Questionnaires MeSH
- Risk Factors MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Case-Control Studies MeSH
- Life Style * MeSH
- Tobacco Smoke Pollution adverse effects MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
Current methods for assessing cell proliferation in 3D scaffolds rely on changes in metabolic activity or total DNA, however, direct quantification of cell number in 3D scaffolds remains a challenge. To address this issue, we developed an unbiased stereology approach that uses systematic-random sampling and thin focal-plane optical sectioning of the scaffolds followed by estimation of total cell number (StereoCount). This approach was validated against an indirect method for measuring the total DNA (DNA content); and the Bürker counting chamber, the current reference method for quantifying cell number. We assessed the total cell number for cell seeding density (cells per unit volume) across four values and compared the methods in terms of accuracy, ease-of-use and time demands. The accuracy of StereoCount markedly outperformed the DNA content for cases with ~ 10,000 and ~ 125,000 cells/scaffold. For cases with ~ 250,000 and ~ 375,000 cells/scaffold both StereoCount and DNA content showed lower accuracy than the Bürker but did not differ from each other. In terms of ease-of-use, there was a strong advantage for the StereoCount due to output in terms of absolute cell numbers along with the possibility for an overview of cell distribution and future use of automation for high throughput analysis. Taking together, the StereoCount method is an efficient approach for direct cell quantification in 3D collagen scaffolds. Its major benefit is that automated StereoCount could accelerate research using 3D scaffolds focused on drug discovery for a wide variety of human diseases.
AIM OF THE STUDY: Comparative cross-sectional study of retinal parameters in Huntington's disease and their evaluation as marker of disease progression. CLINICAL RATIONALE FOR THE STUDY: Huntington's disease (HD) is a neurodegenerative disorder with dominant motor and neuropsychiatric symptoms. Involvement of sensory functions in HD has been investigated, however studies of retinal pathology are incongruent. Effect sizes of previous findings were not published. OCT data of the subjects in previous studies have not been published. Additional examination of structural and functional parameters of retina in larger sample of patients with HD is warranted. MATERIALS AND METHODS: This is a prospective cross-sectional study that included: peripapillary retinal nerve fiber layer thickness (RNFL) and total macular volume (TMV) measured by spectral domain optical coherence tomography (OCT) of retina, Pelli-Robson Contrast Sensitivity test, Farnsworth 15 Hue Color discrimination test, ophthalmology examination and Unified Huntington's disease Rating Scale (UHDRS). Ninety-four eyes of 41 HD patients examined in total 47 visits and 82 eyes of 41 healthy controls (HC) examined in total 41 visits were included. Analyses were performed by repeated measures linear mixed effects model with age and gender as covariates. False discovery rate was corrected by Benjamini-Hochberg procedure. RESULTS: HD group included 21 males and 20 females (age 50.6±12.0 years [mean ± standard deviation], disease duration 7.1±3.6 years, CAG triplet repeats 44.1±2.4). UHDRS Total Motor Score (TMS) was 30.0±12.3 and Total Functional Capacity 8.2±3.2. Control group (HC) included 19 males and 22 females with age 48.2±10.3 years. There was no statistically significant difference between HD and HC in age. The effect of the disease was not significant in temporal segment RNFL thickness. It was significant in the mean RNFL thickness and TMV, however not passing false discovery rate adjustment and with small effect size. In the HD group, the effect of disease duration and TMS was not significant. The Contrast Sensitivity test in HD was within normal limits and the 15-hue-test in HD did not reveal any specific pathology. CONCLUSIONS: The results of our study support possible diffuse retinal changes in global RNFL layer and in macula in Huntington's disease, however, these changes are small and not suitable as a biomarker for disease progression. We found no other structural or functional changes in retina of Huntington's disease patients using RNFL layer and macular volume spectral domain OCT and Contrast Sensitivity Test and 15-hue-test. CLINICAL IMPLICATIONS: Current retinal parameters are not appropriate for monitoring HD disease progression.
- MeSH
- Biomarkers MeSH
- Adult MeSH
- Huntington Disease * pathology MeSH
- Middle Aged MeSH
- Humans MeSH
- Nerve Fibers pathology MeSH
- Tomography, Optical Coherence * methods MeSH
- Disease Progression MeSH
- Prospective Studies MeSH
- Cross-Sectional Studies MeSH
- Retina pathology MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
To evaluate the retinal nerve fibre layer (RNFL) thickness and choroidal thickness (CT) in Parkinson disease (PD) patients. A comparative cross-sectional, hospital-based study. 39 PD and 39 controls were recruited, who were gender and age matched. Subjects that fulfilled the inclusion criteria underwent optical coherence tomography for evaluation of RNFL thickness and choroidal thickness (CT). There was significant reduction of RNFL thickness in average (adjusted mean 88.87 μm vs. 94.82 μm, P=0.001), superior (adjusted mean 110.08 μm vs. 119.10 μm, P=0.002) and temporal (adjusted mean 63.77 μm vs. 70.36 μm, P=0.004) in PD compared to controls. The central subfoveal CT was significantly thinner in PD compared to controls (adjusted mean 271.13 μm vs. 285.10 μm, P=0.003). In PD group, there was significant weak negative correlation between the duration of PD with average RNFL thickness (r=-0.354, P=0.027), moderate negative correlation between the duration of PD with central subfoveal CT (r=-0.493, P=0.001), and weak negative correlation between the stage of PD with central subfoveal CT (r=-0.380, P=0.017). PD group had significant thinner average, superior and temporal RNFL thickness and CT compared to controls.
- MeSH
- Choroid MeSH
- Humans MeSH
- Nerve Fibers MeSH
- Parkinson Disease * complications MeSH
- Cross-Sectional Studies MeSH
- Retina MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
