Effects of ventricular unloading on apoptosis and atrophy of cardiac myocytes

BACKGROUND: Ventricular unloading decreases cardiac ventricular mass. This loss of ventricular mass can be due to either atrophy (a reversible process) or apoptosis (an irreversible process) of the cardiac myocytes. We investigated the effect of ventricular unloading on atrophy and apoptosis of cardiac myocytes, using working and nonworking transplant heart models in rats. MATERIALS AND METHODS: ACI rats underwent heterotopic heart transplantation with two different techniques to create working and nonworking cardiac grafts. Cardiac grafts were harvested at different time points after transplantation. TUNEL, caspase-3 assay, and electron microscopy were used to assess the degree of apoptosis while cellular atrophy was estimated by calculation of the cytoplasmic index (CI = mean sectional cytoplasmic area/nucleus). RESULTS: Ventricular mass reduction was more pronounced in nonworking than in working hearts (P < 0.05). Apoptotic index and caspase-3 activities increased in both groups, peaking at 3 days after transplantation, but were not significantly different between the two models. The cytoplasmic index was significantly lower in nonworking than in working grafts (P < 0.05). CONCLUSIONS: These data suggest that cellular atrophy is the primary mechanism that accounts for myocardial weight reduction following ventricular unloading. The inference is that ventricular unloading by ventricular assist devices may not cause permanent loss of cardiac myocytes, thus allowing for functional recovery.     

Influence of indocyanine green staining on the biomechanical strength of porcine internal limiting membrane

PURPOSE: Indocyanine green (ICG) has recently been introduced to stain selectively the internal limiting membrane (ILM) of the retina for ILM peeling. The aim of the present in vitro study was to examine the effect of ICG staining on the biomechanical properties of porcine ILM. METHODS: Two parallel 10 x 7 mm strips of central retina were prepared from each of the 40 porcine eyes. 0.005% ICG staining combined with white light illumination for 3 min was performed. Unstained, nonilluminated and 0.1% glutaraldehyde-treated specimens were used as controls. Biomechanical-force elongation measurements were performed using an automated material tester. The absorption spectrum of the ICG solution and the emission spectrum of the light source was measured. RESULTS: After ICG staining of the retina combined with 3 min of illumination, a significant increase in ultimate force by 45% and a decrease in ultimate elongation by 24% was found. Without light exposure, there was no such effect, suggesting a light-dependent process. After 30 min of 0.1% glutaraldehyde treatment, there was an increase in ultimate force by 107% and a decrease in ultimate elongation by 66.6%. The absorption spectrum of the light source was continuous in the range from 400 to 800 nm including the absorption peak of ICG at 700 nm. CONCLUSIONS: ICG staining of the retina including the ILM causes a significant increase in the biomechanical stiffness thereby facilitating ILM peeling. The effect is due to a photosensitizing effect of ICG leading to collagen cross-linking.     

Mouse strain-dependent heterogeneity of resting limbal vasculature

PURPOSE: Heterogeneity of the extent of angiogenesis induced by exogenous growth factors may be determined by genetic influences. Because angiogenesis is the formation of new vessels from preexisting ones, strain-related influences on na?ve resting limbal vessel phenotype and gene expression were determined in mice having divergently low and high angiogenic responses. METHODS: Resting limbal vessel surface area and density and extent of bFGF-induced corneal angiogenesis were determined in C57BL/6J, BALB/cJ, F1 intercross identical with C57BL/6J X 129S3/SvIM, and 129S3/SvIM mouse strains by quantitative three-dimensional reconstruction confocal microscopy. Strain-related influences on pro- and antiangiogenic gene expression in na?ve cornea were determined by quantitative real-time RT-PCR. RESULTS: The strain-dependent rank order of resting limbal vessel surface area and resting vessel density paralleled bFGF-induced neovascularization: 129S3/SvIM > BALB/cJ, F1 > C57BL/6J (P < 0.0006). Pigment epithelium-derived factor (PEDF) was increased more than 67-fold compared to Ang-2 in resting cornea of both C57BL/6J and 129S3/SvIM strains (P < 0.0001; P < 0.0001), suggesting a strongly antiangiogenic environment. The corneas of the C57BL/6J mice demonstrated 1.8-, 1.5-, and 1.7-fold increased mRNA levels for Flt-1, VEGF, and bFGF, respectively (P < 0.02; P < 0.04; P < 0.02); however, TSP-1 expression was increased 2.4-fold compared with 129S3/SvIM (P < 0.0004). CONCLUSIONS: Strain-dependent differences in the resting limbal vessel surface area and density correlated with heterogeneity in the extent of bFGF-induced angiogenesis. Differences in pro- and antiangiogenic gene expression levels in resting cornea may influence vascular limbal phenotype during quiescence and may predict susceptibility to angiogenesis-dependent diseases.     

Imaging and quantification of calcified corneal lesions with optical coherence tomography

PURPOSE: The purpose of this study was to image and quantify calcified corneal lesions with optical coherence tomography (OCT) and to evaluate this diagnostic method to assess this corneal disease. METHODS: In a clinical study 15 eyes of 14 patients with calcified corneal lesions in the anterior portions of the cornea were assessed with slit-lamp biomicroscopy and slit-lamp-adapted OCT. The qualitative and quantitative optical changes were compared with the clinical findings. RESULTS: In corneal OCT all calcified lesions revealed marked hyperreflective changes that correlated well with macroscopic findings. Dense calcified lesions resulted in partial or complete shadowing of the posterior corneal structures. The thickness values for the hyperreflective calcified lesions ranged from 27 to 344 microm. CONCLUSION: Noncontact slit-lamp-adapted OCT may be of value in assessing calcified corneal lesions. Corneal OCT could also be used to precisely measure the depth of these corneal processes and evaluate treatment modalities.     

Continuous monitoring of corneal thickness changes during LASIK with online optical coherence pachymetry

PURPOSE: To assess the continuous intraoperative monitoring of central corneal thickness (CCT) changes during laser in situ keratomileusis (LASIK) using online optical coherence pachymetry (OCP). SETTING: Department of Ophthalmology, Vivantes Klinikum Neukolln, Berlin, Germany. METHODS: In this prospective nonrandomized comparative clinical case series of consecutive patients, 32 eyes having LASIK for myopia, myopic astigmatism, or hyperopia were continuously monitored intraoperatively in real time with online OCP integrated into a clinical excimer laser. The intraoperative values were compared to the postoperative flap and residual stromal thicknesses measured with corneal optical coherence tomography (OCT) as well as the calculated myopic ablation depth. RESULTS: Continuous monitoring with online OCP enabled intraoperative visualization of the CCT changes during LASIK. The CCT, flap thickness after the microkeratome pass, time-resolved ablation, and residual stromal thickness were assessed. Intraoperatively, the mean flap thickness was 135 microm +/- 38 (SD) and the mean residual stromal thickness, 286 +/- 59 microm. The mean intraoperative flap and residual stromal thickness values were 43.7 microm and 15.4 microm lower, respectively, than the postoperative values assessed with corneal OCT (P<.001 and P=.005, respectively). The optically determined myopic ablation depth was 118 +/- 37 microm, which was 28 microm higher than the nominal ablation depth. There was a significant correlation (P<.001) between the postoperative flap (r=0.79) and residual (r=0.88) thickness measured with corneal OCT as well as the calculated myopic ablation depth (r=0.95). CONCLUSIONS: Intraoperative online OCP could be an important safety feature to monitor the flap and residual stromal thicknesses during LASIK. The individual ablation depth and possible dehydration effects were also monitored continuously.     

Experimental evaluation of online optical coherence pachymetry for corneal refractive surgery

Purpose Online optical coherence pachymetry (OCP) allows to monitor central changes of the corneal cross section intraoperatively. In this experimental evaluation the validity of the optical measurements for corneal refractive surgery was assessed.Methods Online OCP based on low-coherence interferometry with a wavelength of 1310 nm and a measurement frequency of 74 Hz was directly integrated in a clinical excimer laser. In 16 patients the central corneal thickness was measured with online OCP and ultrasound pachymetry (US). Furthermore, the ablation characteristics were assessed in corneoscleral discs unsuitable for transplantation (n=12) and PMMA samples (n=18).Results Online OCP was possible in all patients and materials studied. The mean central corneal thickness was 537±31 m (OCP) and 546±33 m (US). The corneal reproducibility was ±4.3 m (coefficient of variation [CV] 0.8%) with online OCP and ±3.7 m (CV 0.68%) with US. The reproducibility in PMMA samples was ±1.0 m (CV 0.16%). There was a significant correlation between online OCP and US measurements (r=0.93, P<0.001). The mean difference was 9.1 m or 1.69% (P=0.01), and the limits of agreement (95% CI) ranged from –15 m to 33 m. There was a significant linear relationship (r=0,95; P<0.001) between the calculated and the optically determined ablation depth with online OCP. Also ablation depth measurements in PMMA correlated positively with spectrophotometric values (r=0.98; P<0.001).Conclusion In this experimental evaluation, online OCP revealed to be a precise and reproducible method to assess the central corneal thickness and its changes intraoperatively. This could be important to monitor incisional and excimer laser-based corneal refractive procedures, such as PRK or LASIK.The authors have no commercial, proprietary or financial interest in any research or devices described in the presented study     

Biomechanical changes in the anterior lens capsule after trypan blue staining

PURPOSE: To examine the effect of trypan blue staining on the biomechanical behavior of the porcine anterior lens capsule. SETTING: Department of Ophthalmology, Technical University of Dresden, Dresden, Germany. METHODS: Fifty-five anterior lens capsules from porcine cadaver eyes were used. Two parallel 8.0 mm x 4.0 mm large capsule strips were prepared from each capsule. After trypan blue staining for various time intervals combined with exposure to white light (6000 lux) or with no light exposure, biomechanical stress-strain measurements were performed using an automated material tester. Untreated specimens and specimens treated with glutaraldehyde 0.1% were used as controls. The absorption spectrum of trypan blue 0.1% solution and the emission spectrum of the light source were measured. RESULTS: After treatment with light and trypan blue, at 25% strain, there was a statistically significant increase in stress of up to 70.1% and in elastic stiffness of 47% and a decrease in the ultimate mechanical strain of up to 13%. There were no biomechanical changes in capsules with trypan blue staining in the absence of light or after a short illumination time of 30 seconds, indicating a light-dependent process. After 30 minutes of glutaraldehyde 0.1% treatment, there was an increase in stress of 321.6% at 25% strain and a decrease in the ultimate strain of 47.6%. The emission spectrum of the light source included the absorption peak for trypan blue at 580 nm. CONCLUSIONS: Trypan blue staining of the lens capsule combined with light irradiation for at least 1 minute led to an increase in elastic stiffness at 25% strain and a reduction in the ultimate extensibility. This effect is probably due to the photosensitizing action of trypan blue, leading to light-induced collagen crosslinking of the capsule collagen similar to age-related crosslinking. Nucleus expression might be impeded by the increased capsule stiffness. Continuous curvilinear capsulorhexis is facilitated.     

Reproduzierbarkeit der Goniometrie mittels spaltlampen-adaptierter optischer Kohärenztomographie

BACKGROUND: Visualization of the anterior chamber angle (ACA) is an important diagnostic part of evaluating patients with glaucoma. The purpose of this study was to evaluate the intra- and interobserver variability and reliability of the ACA and angle opening distance (AOD) measurements using optical coherence tomography (OCT). METHODS: To evaluate the intra- and interobserver variability, ACA and AOD were both measured five times and in three consecutive images in 22 patients (24 eyes) by two experienced observers. The intraclass correlation coefficient (ICC) as a measure of reliability was determined to estimate the intra- and interobserver variability. The main outcome measures were accuracy, reproducibility assessed with the coefficient of variation (CV), and the limits of agreement of ACA and AOD. RESULTS: The intraobserver variability of five replicate measurements was +/-1.4 degrees for ACA (CV 6.2%) and +/-11 micro m for AOD (CV 4%). The ICC for the intraobserver reliability was 0.99 for both ACA and AOD. The interobserver variability of three intersessional measurements was +/-2.5 degrees for ACA (CV 10.9%) and +/-24 micro m (CV 8.3%) for AOD. The ICC was 0.95 for ACA and 0.98 for AOD. There was no difference ( p>0.05) between the two observers measuring ACA and AOD. CONCLUSIONS: Two-dimensional visualization of the ACA and its assessment with slitlamp-adapted OCT yielded reliable ACA and AOD measurements in a clinical setting. Thus, OCT goniometry could provide an objective method to assess the anterior chamber angle.     

Molecular cloning and characterization of a glutathione S-transferase from largemouth bass (Micropterus salmoides) liver that is involved in the detoxification of 4-hydroxynonenal

We are currently investigating the role of detoxification pathways in protecting against the sublethal effects of chemicals in largemouth bass (Micropterus salmoides). To this end, previous work in our laboratory indicated a remarkable ability of bass liver glutathione S-transferases (GSTs) to detoxify 4-hydroxynonenal (4HNE), a common mutagenic and cytotoxic alpha,beta-unsaturated aldehyde produced during the peroxidation of lipids. In the current study, we observed that GST-mediated 4HNE conjugation in bass liver follows high efficiency single-enzyme Michaelis-Menten kinetics, suggesting that an individual GST isoform is involved in 4HNE detoxification. Using 5' and 3' rapid amplification of cDNA ends (RACE), a full-length GST cDNA of 957 base pairs (bp) in length, containing an open reading frame of 678 bp and encoding a polypeptide of 225 amino acids, has been cloned. Interestingly, a search of the BLAST protein database revealed the presence of homologous GST proteins in the plaice (Pleuronectes platessa), European flounder (Platichthys flesus) and fathead minnow (Pimephales promelas), but not in other fish species. Furthermore, the bass GST protein exhibited little homology with the mammalian GSTA4 subclass of proteins which rapidly metabolize 4HNE. The recombinant 6 x His-tagged expressed GST protein showed high catalytic activity towards 4HNE, while showing moderate or low activity toward other class specific GST substrates. HPLC-GST subunit analysis, followed by sequencing, demonstrated that the isolated bass liver GST subunit constitutes the major GST protein in bass liver, with a molecular mass of 26.4 kDa. In summary, the presence of a highly expressed GST isozyme in bass and several evolutionarily divergent fish species indicates the conservation of an important and distinct detoxification protein that protects against oxidative damage in certain aquatic organisms.     

Antioxidant effect of hydroxytyrosol, a polyphenol from olive oil: scavenging of hydrogen peroxide but not superoxide anion produced by human neutrophils

Hydroxytyrosol (HT) (also known as dihydroxyphenylethanol (DPE)) is a polyphenol extracted from virgin olive oil. HT is known to exert an antioxidant effect but the mechanism of action and the identity of the reactive oxygen molecule(s) targeted are not known. In this study, we show that HT inhibits luminol-amplified chemiluminescence of human neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA) and opsonized zymosan. This effect was dose-dependent and occurred immediately after the addition of HT. However, HT had no effect on lucigenin-amplified chemiluminescence, suggesting that it does not inhibit NADPH oxidase activation or scavenge superoxide anions. Furthermore, HT inhibited H(2)O(2)-dependent-dichlorofuoroscein (DCFH) fluorescence of activated neutrophils, as measured by flow cytometry. Finally, HT inhibited luminol-amplified chemiluminescence in a cell-free system consisting of H(2)O(2) and HRPO. These results suggest that HT, a polyphenol derived from olive oil, could exert its antioxidant effect by scavenging hydrogen peroxide but not superoxide anion released during the respiratory burst.