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Nancy J Millenbaugh,1 Jonathan B Baskin,1 Mauris N DeSilva,1 W Rowe Elliott,1 Randolph D Glickman2 1Maxillofacial Injury and Disease Department, Naval Medical Research Unit San Antonio, Joint Base San Antonio-Fort Sam Houston, TX, USA; 2Department of Ophthalmology, The University of Texas Health Science Center at San Antonio, San Antonio, TX, USAPurpose: The continued emergence of multidrug resistant bacterial infections and the decline in discovery of new antibiotics are major challenges for health care throughout the world. This situation has heightened the need for novel antimicrobial therapies as alternatives to traditional antibiotics. The combination of metallic nanoparticles and laser exposure has been proposed as a strategy to induce physical damage to bacteria, regardless of antibiotic sensitivity. The purpose of this study was to test the antibacterial effect of antibody-targeted gold nanoparticles combined with pulsed laser irradiation.Methods: Gold nanoparticles conjugated to antibodies specific to Staphylococcus aureus peptidoglycan were incubated with suspensions of methicillin-resistant and methicillin-sensitive S. aureus (MRSA and MSSA). Bacterial suspensions were then exposed to 8 ns pulsed laser irradiation at a wavelength of 532 nm and fluences ranging from 1 to 5 J/cm2. Viability of the bacteria following laser exposure was determined using colony forming unit assays. Scanning electron microscopy was used to confirm the binding of nanoparticles to bacteria and the presence of cellular damage.Results: The laser-activated nanoparticle treatment reduced the surviving population to 31% of control in the MSSA population, while the survival in the MRSA population was reduced to 58% of control. Significant decreases in bacterial viability occurred when the laser fluence exceeded 1 J/cm2, and this effect was linear from 0 to 5 J/cm2 (r2=0.97). Significantly less bactericidal effect was observed for nonfunctionalized nanoparticles or functionalized nanoparticles without laser activation.Conclusion: Laser-activated nanoparticles targeted to S. aureus surface antigens significantly reduced the percentage of viable organisms and represents a promising new treatment modality that could be used either alone or as an adjunct to existing, conventional antibiotic therapy. Keywords: MRSA, bacteria, pulsed laser, nanospheres, photoacoustic

Traumatic optic neuropathy (TON) is a common cause of irreversible blindness following head injury. TON is characterized by axon damage in the optic nerve followed by retinal ganglion cell death in the days and weeks following injury. At present, no therapeutic or surgical approach has been found to offer any benefit beyond observation alone. This is due in part to the lack of translational animal models suitable for understanding mechanisms and evaluating candidate treatments. In this study, we developed a rat model of TON in which the eye is rapidly rotated, inflicting mechanical stress on the optic nerve and leading to significant visual deficits. These functional deficits were thoroughly characterized up to one week after injury using electrophysiology and immunohistochemistry. The photopic negative response (PhNR) of the light adapted full field electroretinogram (LA ffERG) was significantly altered following injury. This correlated with increased biomarkers of retinal stress, axon disruption, and ganglion cell death. Together, this evidence suggests the utility of our model for mimicking clinically relevant TON and that the PhNR may be an early diagnostic for TON. We also found indirect evidence that ketamine, which was used for anesthesia, may ameliorate TON. Future studies will utilize this animal model for evaluation of candidate treatments., Competing Interests: Author(s’) Disclosure Statements: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Purpose: Photoacoustics (optoacoustics) is a hybrid technology utilizing light excitation of acoustic responses in targets of interest. It has found numerous applications in biomedicine, including eye research, because of its ability to report both morphological and functional data about the interrogated tissue. This presentation will give an overview of current applications. Methods: Wavelength-dependent absorption of light in a tissue chromophore causes local heating, leading to a thermoelastic expansion-contraction cycle. If nanosecond pulses of light are used to excite this process, the resulting pressure wave is an ultrasound signal propagating through the tissue and detectable at the tissue surface. This is highly advantageous because of the known properties of ultrasound propagation in tissue and the ability to use standard, medical ultrasound equipment for detection. The time of arrival and amplitude of ultrasound signals provide information about the location and nature of the absorber. Results: Due to the wavelength dependence of the photoacoustic response, functional and physiological applications are possible. For example, retinal oximetry can be determined from the different optical absorption properties of oxy- and deoxyhemoglobin. Multispectral imaging of the posterior segment can identify pigments such as melanin or lipofuscin or the nature of foreign bodies. The technique can be combined with other imaging modalities such as ultrasound and optical coherence tomography to produce high-resolution images of retinal structures along with functional information. Conclusion: Photoacoustic technology is a powerful noninvasive tool for ocular research and to study ocular morphology, fundamental physiological parameters, cellular responses, and molecular expression.

Purpose: To determine the long-term biocompatibility of HyStem ® hydrogel in the rabbit eye for use as a carrier for cell or drug delivery into the ocular space. Methods: HyStem hydrogel formulation solidifies ∼20 min after reconstitution, thus can potentially form a solid deposit after injection in situ . To study the ocular disposition of fluorescein-labeled HyStem, we delivered 50 μL/eye over 1 min into the vitreous space of the rabbit. We used 3 Dutch-Belted and 3 New Zealand-pigmented rabbits, all females, delivered the gel into the right eyes, and injected 50 μL BSS Plus into the left eyes as a control. Retinal morphology was assessed by optical coherence tomography (OCT) and white light fundus photography. Fluorescence fundus photography enabled measurement of the clearance of the labeled hydrogel from the posterior chamber. Visual function was evaluated using flash and flicker electroretinography (ERG) pre- and postinjection and at weekly intervals thereafter for 6 weeks. Retinal immunohistochemistry for microglial inflammatory markers was carried out with antiglial fibrillary acidic protein (GFAP) antibody, isolectin B4 (IB4), and 4',6-diamidino-2-phenylindole (DAPI). Results: The gel was successfully delivered into the vitreous space without the formation of a discrete retinal deposit. Fundus imaging, OCT measurements of retinal thickness, and immunohistochemical data indicated an absence of retinal inflammation, and ERG indicated no impact on retinal function. The half-time of HyStem clearance calculated from the loss of fundus fluorescence was 3.9 days. Conclusions: HyStem hydrogel appears to be biocompatible in the ocular space of a large eye and safe for long-term intraocular application.

Rationale and Objectives: In this study, we assessed the radiation dose to the lens and the impacts of various eye shields using either a fixed or modulated tube current., Materials and Methods: Patients undergoing head computed tomography (CT) examinations were recruited, and each was randomly assigned to one of five imaging groups, either without a CT eye shield or with one of two types of shielding and topogram-based tube current modulation (TCM). The radiation dose at the eye lens was estimated using Gafchromic films. All CT images were analyzed for quality in the orbit and brain areas. Two radiologists also qualitatively assessed image artifacts and their impacts on image quality using three-point Likert scales., Results: Both barium sulfate and bismuth-antimony shields significantly reduced radiation dose to the lens (by 28.60%-31.92% and 43.87%-47.00%, respectively) while significantly inducing image artifacts. The image quality of the intraocular structure, but not the intracranial structure, was significantly degraded by shielding. In addition, discriminating the periocular tissues was improved using a bismuth-antimony shield and topogram-based TCM. Compared to fixed tube current, topogram-based TCM provided better signal-to-noise and contrast-to-noise ratios in the intracranial structures when the bismuth-antimony and barium sulfate shields were applied, respectively., Conclusion: Artifacts resulting from the application of eye shields during head CT examinations can be reduced by using topogram-based TCM instead of a fixed tube current. This could be an alternative approach for maintaining image quality in CT scans that do not encompass organ-based TCM., (Copyright © 2020 The Association of University Radiologists. Published by Elsevier Inc. All rights reserved.)

Retinal organoid technology enables generation of an inexhaustible supply of three-dimensional retinal tissue from human pluripotent stem cells (hPSCs) for regenerative medicine applications. The high similarity of organoid-derived retinal tissue and transplantable human fetal retina provides an opportunity for evaluating and modeling retinal tissue replacement strategies in relevant animal models in the effort to develop a functional retinal patch to restore vision in patients with profound blindness caused by retinal degeneration. Because of the complexity of this very promising approach requiring specialized stem cell and grafting techniques, the tasks of retinal tissue derivation and transplantation are frequently split between geographically distant teams. Delivery of delicate and perishable neural tissue such as retina to the surgical sites requires a reliable shipping protocol and also controlled temperature conditions with damage-reporting mechanisms in place to prevent transplantation of tissue damaged in transit into expensive animal models. We have developed a robust overnight tissue shipping protocol providing reliable temperature control, live monitoring of the shipment conditions and physical location of the package, and damage reporting at the time of delivery. This allows for shipping of viable (transplantation-competent) hPSC-derived retinal tissue over large distances, thus enabling stem cell and surgical teams from different parts of the country to work together and maximize successful engraftment of organoid-derived retinal tissue. Although this protocol was developed for preclinical in vivo studies in animal models, it is potentially translatable for clinical transplantation in the future and will contribute to developing clinical protocols for restoring vision in patients with retinal degeneration., (© 2019 John Wiley & Sons, Ltd.)

Purpose: Our study aimed to determine whether the altered expression of biomarkers linked to corneal injuries, such as the edema-regulating proteins aquaporin-1 and aquaporin-5 (AQP1 and AQP5), occurred following primary blast exposure., Methods: Adult male Dutch Belted rabbits were anesthetized and exposed to blast waves with peak overpressures of 142.5-164.1 kPa (20.4-23.4 psi). These exposure groups experienced peak blast overpressure-specific impulses (impulse per unit surface area) of 199.6-228.5 kPa-ms. Unexposed rabbits were included as controls. The animals were euthanized at 48 h post-exposure. Corneas obtained from the euthanized blast-exposed and control rabbits were processed for quantitative PCR and western blot to quantify mRNA and the protein expression of AQP1 and AQP5. Immunohistochemical analysis was conducted to determine the cellular localization of AQP1 and AQP5., Results: Corneal thickness increased up to 18% with the peak blast overpressure-specific impulses of 199.6-228.5 kPa-ms at 48 h after blast exposure. mRNA levels of AQP1 and AQP5 increased in the whole cornea lysates of blast-exposed rabbits relative to those of the controls. Western blot analyses of whole cornea lysates revealed that the expression levels of AQP1 and AQP5 were approximately 2- and 1.5-fold higher, respectively, in blast-exposed rabbits compared to controls. The extent of AQP1 immunostaining (AQP1-IS) increased in the epithelial cell layer after blast exposure. The AQP5-IS pattern changed from a mixed membrane and cytoplasmic expression in the controls to predominantly cytoplasmic expression in the basally located cornea epithelial cells of blast-exposed rabbits., Conclusions: Primary blast exposure resulted in edema-related changes in the cornea manifested by the altered expression of the edema-regulating proteins AQP1 and AQP5 with blast overpressure-specific impulses. These findings support potential acute corneal injury mechanisms in which the altered regulation of water permeability is caused by primary blast exposure.

Adipose tissue dysregulation, a hallmark of obesity, contributes to a chronic state of low-grade inflammation and is associated with increased risk and progression of several breast cancer subtypes, including claudin-low breast tumors. Unfortunately, mechanistic targets for breaking the links between obesity-associated adipose tissue dysfunction, inflammation, and claudin-low breast cancer growth have not been elucidated. Ovariectomized female C57BL/6 mice were randomized (n = 15/group) to receive a control diet, a diet-induced obesity (DIO) diet, or a DIO + resveratrol (0.5% wt/wt) diet. Mice consumed these diets ad libitum throughout study and after 6 weeks were orthotopically injected with M-Wnt murine mammary tumor cells, a model of estrogen receptor (ER)-negative claudin-low breast cancer. Compared with controls, DIO mice displayed adipose dysregulation and metabolic perturbations including increased mammary adipocyte size, cyclooxygenase-2 (COX-2) expression, inflammatory eicosanoid levels, macrophage infiltration, and prevalence of crown-like structures (CLS). DIO mice (relative to controls) also had increased systemic inflammatory cytokines and decreased adipocyte expression of peroxisome proliferator-activated receptor gamma (PPARγ) and other adipogenesis-regulating genes. Supplementing the DIO diet with resveratrol prevented obesity-associated increases in mammary tumor growth, mammary adipocyte hypertrophy, COX-2 expression, macrophage infiltration, CLS prevalence, and serum cytokines. Resveratrol also offset the obesity-associated downregulation of adipocyte PPARγ and other adipogenesis genes in DIO mice. Our findings suggest that resveratrol may inhibit obesity-associated inflammation and claudin-low breast cancer growth by inhibiting adipocyte hypertrophy and associated adipose tissue dysregulation that typically accompanies obesity., (© 2017 Wiley Periodicals, Inc.)

Background: Exposure of cells to very short (<1 µs) electric pulses in the megavolt/meter range have been shown to cause a multitude of effects, both physical and molecular in nature. Physically, nanosecond electrical pulses (nsEP) can cause disruption of the plasma membrane, cellular swelling, shrinking and blebbing. Molecularly, nsEP have been shown to activate signaling pathways, produce oxidative stress, stimulate hormone secretion and induce both apoptotic and necrotic death. We hypothesize that studying the genetic response of primary human dermal fibroblasts exposed to nsEP, will gain insight into the molecular mechanism(s) either activated directly by nsEP, or indirectly through electrophysiology interactions., Methods: Microarray analysis in conjunction with quantitative real time polymerase chain reaction (qRT-PCR) was used to screen and validate genes selectively upregulated in response to nsEP exposure., Results: Expression profiles of 486 genes were found to be significantly changed by nsEP exposure. 50% of the top 20 responding genes coded for proteins located in two distinct cellular locations, the plasma membrane and the nucleus. Further analysis of five of the top 20 upregulated genes indicated that the HDFa cells' response to nsEP exposure included many elements of a mechanical stress response., Conclusions: We found that several genes, some of which are mechanosensitive, were selectively upregulated due to nsEP exposure. This genetic response appears to be a primary response to the stimuli and not a secondary response to cellular swelling., General Significance: This work provides strong evidence that cells exposed to nsEP interpret the insult as a mechanical stress.

The objective of this study was to determine whether clinically significant ocular trauma can be induced by a survivable isolated primary blast using a live animal model. Both eyes of 18 Dutch Belted rabbits were exposed to various survivable low-level blast overpressures in a large-scale shock tube simulating a primary blast similar to an improvised explosive device. Eyes of the blast-exposed rabbits (as well as five control rabbits) were thoroughly examined before and after blast to detect changes. Clinically significant changes in corneal thickness arose immediately after blast and were sustained through 48 h, suggesting possible disruption of endothelial function. Retinal thickness (RT) increased with increasing specific impulse immediately after exposure. Intraocular pressure (IOP) was inversely correlated with the specific impulse of the blast wave. These findings clearly indicate that survivable primary blast causes ocular injuries with likely visual functional sequelae of clinical and military relevance.

Nanosecond electrical pulse (nsEP) exposure activates signaling pathways, produces oxidative stress, stimulates hormone secretion, causes cell swelling and induces apoptotic and necrotic death. The underlying biophysical connection(s) between these diverse cellular reactions and nsEP has yet to be elucidated. Using global genetic analysis, we evaluated how two commonly studied cell types, U937 and Jurkat, respond to nsEP exposure. We hypothesized that by studying the genetic response of the cells following exposure, we would gain direct insight into the stresses experienced by the cell and in turn better understand the biophysical interaction taking place during the exposure. Using Ingenuity Systems software, we found genes associated with cell growth, movement and development to be significantly up-regulated in both cell types 4 h post exposure to nsEP. In agreement with our hypothesis, we also found that both cell lines exhibit significant biological changes consistent with mechanical stress induction. These results advance nsEP research by providing strong evidence that the interaction of nsEPs with cells involves mechanical stress.

Optoacoustic (OA) microscopy using an all-optical system based on the probe beam deflection technique (PBDT) for detection of laser-induced acoustic signals was investigated as an alternative to conventional piezoelectric transducers. PBDT provides a number of advantages for OA microscopy including (i) efficient coupling of laser excitation energy to the samples being imaged through the probing laser beam, (ii) undistorted coupling of acoustic waves to the detector without the need for separation of the optical and acoustic paths, (iii) high sensitivity and (iv) ultrawide bandwidth. Because of the unimpeded optical path in PBDT, diffraction-limited lateral resolution can be readily achieved. The sensitivity of the current PBDT sensor of 22 μV/Pa and its noise equivalent pressure (NEP) of 11.4 Pa are comparable with these parameters of the optical micro-ring resonator and commercial piezoelectric ultrasonic transducers. Benefits of the present prototype OA microscope were demonstrated by successfully resolving micron-size details in histological sections of cardiac muscle.

The mechanism(s) responsible for the breakdown (nanoporation) of cell plasma membranes after nanosecond pulse (nsEP) exposure remains poorly understood. Current theories focus exclusively on the electrical field, citing electrostriction, water dipole alignment and/or electrodeformation as the primary mechanisms for pore formation. However, the delivery of a high-voltage nsEP to cells by tungsten electrodes creates a multitude of biophysical phenomena, including electrohydraulic cavitation, electrochemical interactions, thermoelastic expansion, and others. To date, very limited research has investigated non-electric phenomena occurring during nsEP exposures and their potential effect on cell nanoporation. Of primary interest is the production of acoustic shock waves during nsEP exposure, as it is known that acoustic shock waves can cause membrane poration (sonoporation). Based on these observations, our group characterized the acoustic pressure transients generated by nsEP and determined if such transients played any role in nanoporation. In this paper, we show that nsEP exposures, equivalent to those used in cellular studies, are capable of generating high-frequency (2.5 MHz), high-intensity (>13 kPa) pressure transients. Using confocal microscopy to measure cell uptake of YO-PRO®-1 (indicator of nanoporation of the plasma membrane) and changing the electrode geometry, we determined that acoustic waves alone are not responsible for poration of the membrane.

Purpose: A computational model of the porcine eye was developed to simulate primary blast exposure. This model facilitates understanding of blast-induced injury mechanisms., Methods: A computational model of the porcine eye was used to simulate the effects of primary blast loading for comparison with experimental findings from shock tube experiments. The eye model was exposed to overpressure-time histories measured during physical experiments. Deformations and mechanical stresses within various ocular tissues were then examined for correlation with pathological findings in the experiments., Results: Stresses and strains experienced in the eye during a primary blast event increase as the severity of the blast exposure increases. Peak stresses in the model occurred in locations in which damage was most often observed in the physical experiments., Conclusions: Blast injuries to the anterior chamber may be due to inertial displacement of the lens and ciliary body while posterior damage may arise due to contrecoup interactions of the vitreous and retina. Correlation of modeling predictions with physical experiments lends confidence that the model accurately represents the conditions found in the physical experiments., Translational Relevance: This computational model offers insights into the mechanisms of ocular injuries arising due to primary blast and may be used to simulate the effects of new protective eyewear designs.

Accumulating evidence suggests that ubiquitin E3 ligases are involved in cancer development as their mutations correlate with genomic instability and genetic susceptibility to cancer. Despite significant findings of cancer-driving mutations in the BRCA1 gene, estrogen receptor (ER)-positive breast cancers progress upon treatment with DNA damaging-cytotoxic therapies. In order to understand the underlying mechanism by which ER-positive breast cancer cells develop resistance to DNA damaging agents, we employed an estrogen receptor agonist, Erb-041, to increase the activity of ERβ and negatively regulate the expression and function of the estrogen receptor α (ERα) in MCF-7 breast cancer cells. Upon Erb-041-mediated ERα down-regulation, the transcription of an ERα downstream effector, BCA2 (Breast Cancer Associated gene 2), correspondingly decreased. The ubiquitination of chromatin-bound BCA2 was induced by ultraviolet C (UVC) irradiation but suppressed by Erb-041 pretreatment, resulting in a blunted DNA damage response. Upon BCA2 silencing, DNA double-stranded breaks increased with Rad51 up-regulation and ataxia telangiectasia mutated (ATM) activation. Mechanistically, UV-induced BCA2 ubiquitination and chromatin binding were found to promote DNA damage response and repair via the interaction of BCA2 with ATM, γH2AX and Rad51. Taken together, this study suggests that Erb-041 potentiates BCA2 dissociation from chromatin and co-localization with Rad51, resulting in inhibition of homologous recombination repair.

Ultraviolet (UV) light is a leading cause of diseases, such as skin cancers and cataracts. A main process mediating UV-induced pathogenesis is the production of reactive oxygen species (ROS). Excessive ROS levels induce the formation of DNA adducts (e.g., pyrimidine dimers) and result in stalled DNA replication forks. In addition, ROS promotes phosphorylation of tyrosine kinase-coupled hormone receptors and alters downstream energy metabolism. With respect to the risk of UV-induced photocarcinogenesis and photodamage, the antitumoral and antioxidant functions of natural compounds become important for reducing UV-induced adverse effects. One important question in the field is what determines the differential sensitivity of various types of cells to UV light and how exogenous molecules, such as phytochemicals, protect normal cells from UV-inflicted damage while potentiating tumor cell death, presumably via interaction with intracellular target molecules and signaling pathways. Several endogenous molecules have emerged as possible players mediating UV-triggered DNA damage responses. Specifically, UV activates the PIKK (phosphatidylinositol 3-kinase-related kinase) family members, which include DNA-PKcs, ATM (ataxia telangiectasia mutated) and mTOR (mammalian target of rapamycin), whose signaling can be affected by energy metabolism; however, it remains unclear to what extent the activation of hormone receptors regulates PIKKs and whether this crosstalk occurs in all types of cells in response to UV. This review focuses on proteomic descriptions of the relationships between cellular photosensitivity and the phenotypic expression of the insulin/insulin-like growth receptor. It covers the cAMP-dependent pathways, which have recently been shown to regulate the DNA repair machinery through interactions with the PIKK family members. Finally, this review provides a strategic illustration of how UV-induced mitogenic activity is modulated by the insulin sensitizer, ursolic acid (UA), which results in the metabolic adaptation of normal cells against UV-induced ROS, and the metabolic switch of tumor cells subject to UV-induced damage. The multifaceted natural compound, UA, specifically inhibits photo-oxidative DNA damage in retinal pigment epithelial cells while enhancing that in skin melanoma. Considering the UA-mediated differential effects on cell bioenergetics, this article reviews the disparities in glucose metabolism between tumor and normal cells, along with (peroxisome proliferator-activated receptor-γ coactivator 1α)-dependent mitochondrial metabolism and redox (reduction-oxidation) control to demonstrate UA-induced synthetic lethality in tumor cells.

The signaling pathways via mTOR (mammalian target of rapamycin) and AMPK (AMP-activated protein kinase) play key roles in transcription, translation and carcinogenesis, and may be activated by light exposure. These pathways can be modulated by naturally occurring compounds, such as the triterpenoid, ursolic acid (UA). Previously, the transcription factors p53 and NF-κB, which transactivate mitochondrial apoptosis-related genes, were shown to be differentially modulated by UA. UA-modulated apoptosis, following exposure to UV-VIS radiation (ultraviolet to visible light broadband radiation, hereafter abbreviated to UVR), is observed to correspond to differential levels of oxidative stress in retinal pigment epithelial (RPE) and skin melanoma (SM) cells. The cellular response to this phytochemical was characterized using western blot, flow cytometry, microscopy with reactive oxidative species probes MitoTracker and dihydroethidium, and membrane permeability assay. UA pretreatment potentiated cell cycle arrest and UVR-induced apoptosis selectively in SM cells while reducing photo-oxidative stress in the DNA of RPE cells presumably by antioxidant activity of UA. Mechanistically, the nuclear transportation of p65 and p53 was reduced by UA administration prior to UVR exposure while the levels of p65 and p53 nuclear transportation in SM cells were sustained at a substantially higher level. Finally, the mitochondrial functional assay showed that UVR induced the collapse of the mitochondrial membrane potential, and this effect was exacerbated by rapamycin or UA pretreatment in SM preferentially. These results were consistent with reduced proliferation observed in the clonogenic assay, indicating that UA treatment enhanced the phototoxicity of UVR, by modulating the activation of p53 and NF-κB and initiating a mitogenic response to optical radiation that triggered mitochondria-dependent apoptosis, particularly in skin melanoma cells. The study indicates that this compound has multiple actions with the potential for protecting normal cells while sensitizing skin melanoma cells to UV irradiation.

Purpose: We qualitatively describe the anatomic features of primary blast ocular injury observed using a postmortem porcine eye model. Porcine eyes were exposed to various levels of blast energy to determine the optimal conditions for future testing., Methods: We studied 53 enucleated porcine eyes: 13 controls and 40 exposed to a range of primary blast energy levels. Eyes were preassessed with B-scan and ultrasound biomicroscopy (UBM) ultrasonography, photographed, mounted in gelatin within acrylic orbits, and monitored with high-speed videography during blast-tube impulse exposure. Postimpact photography, ultrasonography, and histopathology were performed, and ocular damage was assessed., Results: Evidence for primary blast injury was obtained. While some of the same damage was observed in the control eyes, the incidence and severity of this damage in exposed eyes increased with impulse and peak pressure, suggesting that primary blast exacerbated these injuries. Common findings included angle recession, internal scleral delamination, cyclodialysis, peripheral chorioretinal detachments, and radial peripapillary retinal detachments. No full-thickness openings of the eyewall were observed in any of the eyes tested. Scleral damage demonstrated the strongest associative tendency for increasing likelihood of injury with increased overpressure., Conclusions: These data provide evidence that primary blast alone (in the absence of particle impact) can produce clinically relevant ocular damage in a postmortem model. The blast parameters derived from this study are being used currently in an in vivo model. We also propose a new Cumulative Injury Score indicating the clinical relevance of observed injuries.

Purpose: To determine the penetration of orally administered trimethoprim (TMP)-sulfamethoxazole (SMX) into the aqueous and vitreous cavity of noninflamed human eyes., Methods: Nine adult patients undergoing cataract surgery and 10 adult patients undergoing pars plana vitrectomy were given 3 doses of oral TMP-SMX every 12 hours before the surgery. Aqueous and blood samples were collected from patients undergoing cataract surgery; vitreous and blood samples were collected from patients undergoing vitrectomy. The levels of TMP and SMX were analyzed using high-performance liquid chromatography and were compared with the mean minimum inhibitory concentrations (MIC) of potential ocular pathogens., Results: TMP-SMX was present in all samples. Among eyes undergoing cataract surgery, the mean concentrations of TMP in aqueous and blood were 0.341 ± 0.141 μg/mL (mean ± SD) and 1.501 ± 0.433 μg/mL and of SMX were 5.259 ± 0.929 μg/mL and 11.835 ± 2.100 μg/mL, respectively. Among eyes undergoing vitrectomy, the mean concentrations of TMP in vitreous and blood were 1.864 ± 0.807 μg/mL and 4.591 ± 2.979 μg/mL and of SMX were 5.910 ± 2.705 μg/mL and 39.289 ± 15.469 μg/mL, respectively. MIC levels were achieved against many bacterial pathogens, including methicillin-resistant Staphylococcus aureus., Conclusions: TMP-SMX penetrates both the aqueous and vitreous cavities when given orally. The components reach therapeutic inhibitory concentrations in the ocular cavity against many potential pathogens.

Optoacoustic imaging is an emerging medical technology that uniquely combines the absorption contrast of optical imaging and the penetration depth of ultrasound. While it is not currently employed as a clinical imaging modality, the results of current research strongly support the use of optoacoustic-based methods in medical imaging. One such application is the diagnosis of the presence of soft tissue foreign bodies. Because many radiolucent foreign bodies have sufficient contrast for imaging in the optical domain, laser-induced optoacoustic imaging could be advantageous for the detection of such objects. Common foreign bodies have been scanned over a range of visible and near infrared wavelengths by using an optoacoustic method to obtain the spectroscopic properties of the materials commonly associated with these foreign bodies. The derived optical absorption spectra compared quite closely to the absorption spectra generated when using a conventional spectrophotometer. By using the probe-beam deflection technique, a novel, pressure-wave detection method, we successfully generated optoacoustic spectroscopic plots of a wooden foreign body embedded in a tissue phantom, which closely resembled the spectrum of the same object obtained in isolation. A practical application of such spectra is to assemble a library of spectroscopic data for radiolucent materials, from which specific characteristic wavelengths can be selected for use in optimizing imaging instrumentation and provide a basis for the identification of the material properties of particular foreign bodies.

Certain phytochemicals, such as the stilbene, resveratrol (RES, found in red grapes and berries), and the triterpenoid, ursolic acid (UA, found in waxy berries and herbs such as rosemary and oregano), have antioxidant, anti-inflammatory and antiproliferative effects. Two human-derived cell lines, hTERT-RPE with a nonmalignant phenotype derived from retinal pigment epithelium, and ATCC CRL-11147 derived from a malignant skin melanoma, were used as in vitro models of photooxidative stress produced by exposure to the broadband output of a 150 W Hg vapor arc lamp at an irradiance of 19-26 mW cm(-2). In untreated cells, UV-VIS broadband light exposure produced a loss of proliferative ability, an activation of NF-κB and an increase in protein carbonyl adducts at 24 h postexposure. Pretreatment of the cells with RES or UA at 1-2 μmsignificantly reduced the amount of phosphorylated NF-κB at 24 h postexposure. RES pretreatment reduced the burden of light-induced protein carbonyl adducts by up to 25% in exposed cells. UA treatment markedly increased the sensitivity of melanoma cells to UV radiation, while conferring some photoprotection to RPE cells. These observations indicate that phytochemicals modulate the cellular response to photochemical stress by interacting with specific cell-signaling pathways., (© 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.)

Fluorescent nanodiamonds (FNDs) have drawn much attention in recent years for biomedical imaging applications due to their desired physical properties including excellent photostability, high biocompatibility, extended far-red fluorescence emission, and ease of surface functionalization. Here we explore a new feature of FNDs, i.e. their photoacoustic emission capability, which may lead to potential applications of using FNDs as a dual imaging contrast agent for combined fluorescence and photoacoustic imaging modalities. We observed significant enhancement of photoacoustic emission from FNDs when they were conjugated with gold nanoparticles (GNPs).

Objective: This overview of ultraviolet (UV) phototoxicity considers the interaction of UVA and short-wavelength VIS light with the retina and retinal pigment epithelium., Methods: The damage mechanisms underlying UV retinal phototoxicity are illustrated with a literature survey and presentation of experimental results., Results: Depending on the wavelength and exposure duration, light interacts with tissue by three general mechanisms: thermal, mechanical, or photochemical. Although the anterior structures of the eye absorb much of the UV component of the optical radiation spectrum, a portion of the UVA band (315-400 nm) penetrates into the retina. Natural sources, such as the sun, emit energetic UV photons in relatively long durations, which typically do not result in energy confinement in the retina, and thus do not produce thermal or mechanical damage but are capable of inducing photochemical damage. Photochemical damage in the retina proceeds through Type 1 (direct reactions involving proton or electron transfers) and Type 2 (reactions involving reactive oxygen species) mechanisms. Commonly used drugs, such as certain antibiotics, nonsteroidal anti-inflammatory drugs, psychotherapeutic agents, and even herbal medicines, may act as photosensitizers that promote retinal UV damage, if they are excited by UVA or visible light and have sufficient retinal penetration., Conclusions: Although the anterior portion of the eye is the most susceptible to UV damage, the retina is at risk to the longer UV wavelengths that propagate through the ocular media. Some phototoxicity may be counteracted or reduced by dietary intake of antioxidants and protective phytonutrients.

The natural polyphenol resveratrol (RSV) displays a wide spectrum of health beneficial activities, yet the precise mechanisms remain to be fully elucidated. Here we show that RSV promotes the multimerization and cellular levels of adiponectin in 3T3-L1 adipocytes. The stimulatory effect of RSV was not affected by knocking out Sirt1, but was diminished by suppressing the expression levels of DsbA-L, a recently identified adiponectin-interactive protein that promotes adiponectin multimerization. Suppression of the Akt signaling pathway resulted in an increase in the expression levels of DsbA-L and adiponectin. On the other hand, knocking out FOXO1 or suppressing the activity or expression levels of the AMP-activated protein kinase (AMPK) down-regulated DsbA-L and adiponectin. The stimulatory effect of RSV on adiponectin and DsbA-L expression was completely diminished in FOXO1-suppressed and AMPK-inactivated 3T3-L1 adipocytes. Taken together, our results demonstrate that RSV promotes adiponectin multimerization in 3T3-L1 adipocytes via a Sirt1-independent mechanism. In addition, we show that the stimulatory effect of RSV is regulated by both the Akt/FOXO1 and the AMPK signaling pathways. Last, we show that DsbA-L plays a critical role in the promoting effect of RSV on adiponectin multimerization and cellular levels.

Four different antibiotics, delivered individually to rabbit eyes via hydrophilic intraocular lenses soaked in the drug solution prior to implantation, were measured in aqueous and vitreous humor samples from the eyes. To meet this analytical need, we developed a sensitive, high performance liquid chromatographic (HPLC) method for measuring the concentrations of moxifloxacin, gatifloxacin, linezolid, and cefuroxime in the ocular tissue. Separations were carried out on a LichroSpher RP-18 column, maintained at room temperature. The fluoroquinolones were eluted with a mobile phase consisting of 20% acetonitrile, in 0.1% trifluoroacetic acid (pH 3.0) with 30 mM tetrabutylammonium chloride. Linezolid and cefuroxime were eluted with 25% acetonitrile in 25 mM Na acetate buffer, pH 5.0. All elutions were isocratic. With ultraviolet detection, the lower limit of quantitation (LLOQ) for these compounds approached 1 ng (on-column injection). By using fluorescence detection, the LLOQ for the fluoroquinolones improved to 200 pg. The overall accuracy of the method was >or=90%. With minor modifications, the method was optimized for each of the agents, and the resulting analytical sensitivity made the method suitable for clinical investigations of the ocular penetration of these drugs., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

PURPOSE. In Old World primates, the retina receives input from histaminergic neurons in the posterior hypothalamus. They are a subset of the neurons that project throughout the central nervous system and fire maximally during the day. The contribution of these neurons to vision, was examined by applying histamine to a dark-adapted, superfused baboon eye cup preparation while making extracellular recordings from peripheral retinal ganglion cells. METHODS. The stimuli were 5-ms, 560-nm, weak, full-field flashes in the low scotopic range. Ganglion cells with sustained and transient ON responses and two cell types with OFF responses were distinguished; their responses were recorded with a 16-channel microelectrode array. RESULTS. Low micromolar doses of histamine decreased the rate of maintained firing and the light sensitivity of ON ganglion cells. Both sustained and transient ON cells responded similarly to histamine. There were no statistically significant effects of histamine in a more limited study of OFF ganglion cells. The response latencies of ON cells were approximately 5 ms slower, on average, when histamine was present. Histamine also reduced the signal-to-noise ratio of ON cells, particularly in those cells with a histamine-induced increase in maintained activity. CONCLUSIONS. A major action of histamine released from retinopetal axons under dark-adapted conditions, when rod signals dominate the response, is to reduce the sensitivity of ON ganglion cells to light flashes. These findings may relate to reports that humans are less sensitive to light stimuli in the scotopic range during the day, when histamine release in the retina is expected to be at its maximum.

Light emitted from a femtosecond laser is capable of plasma-induced ablation of various materials. We tested the feasibility of utilizing femtosecond-pulsed laser radiation (lambda=800 nm, 140 fs, 0.9 mJ/pulse) for ablation of urinary calculi. Ablation craters were observed in human calculi of greater than 90% calcium oxalate monohydrate (COM), cystine (CYST), or magnesium ammonium phosphate hexahydrate (MAPH). Largest crater volumes were achieved on CYST stones, among the most difficult stones to fragment using Holmium:YAG (Ho:YAG) lithotripsy. Diameter of debris was characterized using optical microscopy and found to be less than 20 microm, substantially smaller than that produced by long-pulsed Ho:YAG ablation. Stone retropulsion, monitored by a high-speed camera system with a spatial resolution of 15 microm, was negligible for stones with mass as small as 0.06 g. Peak shock wave pressures were less than 2 bars, measured by a polyvinylidene fluoride (PVDF) needle hydrophone. Ablation dynamics were visualized and characterized with pump-probe imaging and fast flash photography and correlated to shock wave pressures. Because femtosecond-pulsed laser ablates urinary calculi of soft and hard compositions, with micron-sized debris, negligible stone retropulsion, and small shock wave pressures, we conclude that the approach is a promising candidate technique for lithotripsy.

Over the past two decades, bioactive natural compounds have been shown to be a plausible adjunct to the treatment of breast cancer, the second leading cause of cancer death among American women. This study was designed to investigate the effects of ursolic acid (UA), a pentacyclic triterpene found in many foods and herbs, in a model of postmenopausal breast cancer. Ovariectomized C57BL/6 mice (n = 40) were randomized to receive control diet (AIN-93G) or diet supplemented with UA at 1 of 3 doses (wt/wt): 0.05%, 0.10%, or 0.25% (≈54, 106, or 266 mg/kg body weight/day, respectively). After 3 wk, syngeneic MMTV-Wnt-1 mammary tumor cells were injected in the mammary fat pad, and mice continued on their respective diets for 5 more wk. All UA doses decreased tumor cell proliferation, as assessed by Ki67 immunostaining; nevertheless, UA at 0.10% was most effective in inhibiting tumor take and decreasing tumor final tumor size. Modulation of Akt/mTOR signaling and induction of apoptosis appeared to mediate these effects on tumor growth. UA potently disrupted cell cycle progression and induced necrosis in a clonal MMTV-Wnt-1 mammary tumor cell line in vitro. This study supports the potential of UA as an antitumorigenic agent.

NF-kappaB signaling pathway has been known to play a major role in the pathological process of atherogenesis. Unlike high shear stress, in which the NF-kappaB activity is transient, our earlier studies have demonstrated a persistent activation of NF-kappaB in response to low shear stress in human aortic endothelial cells. These findings partially explained why low shear regions that exist at bifurcations of arteries are prone to atherosclerosis, unlike the relatively atheroprotective high shear regions. In the present study, we further investigated 1) the role of NF-kappaB signaling kinases (IKKalpha and beta) that may be responsible for the sustained activation of NF-kappaB in low shear stress and 2) the regulation of these kinases by reactive oxygen species (ROS). Our results demonstrate that not only is a significant proportion of low shear-induced-kinase activity is contributed by IKKbeta, but it is also persistently induced for a prolonged time frame. The IKK activity (both alpha and beta) is blocked by apocynin (400 microM), a specific NADPH oxidase inhibitor, and diphenyleneiodonium chloride (DPI; 10 microM), an inhibitor of flavin-containing oxidases like NADPH oxidases. Determination of ROS also demonstrated an increased generation in low shear stress that could be blocked by DPI. These results suggest that the source of ROS generation in endothelial cells in response to low shear stress is NADPH oxidase. The DPI-inhibitable component of ROS is the primary regulator of specific upstream kinases that determine the persistent NF-kappaB activation selectively in low shear-induced endothelial cells.

Background and Purpose: Lower-pole ureteronephroscopy requires transmission of holmium:YAG energy along a deflected fiber. Current ureteroscopes are capable of high degrees of deflection, which may stress laser fibers beyond safe limits during lower-pole use. We hypothesized that optical fiber and safety measures differ among manufacturers., Materials and Methods: Small (200-273-microm) and medium-diameter (300-400-microm) Ho:YAG fibers were tested in a straight and 180 degrees bent configuration. Energy transmission was measured by an energy detector. Fiber durability was assessed by firing the laser in sequentially tighter bending diameters. The fibers were bent to 180 degrees with a diameter of 6 cm and run at 200- to 4000-mJ pulse energy to determine the minimum energy required to fracture the fiber. The bending diameter was decreased by 1-cm increments and testing repeated until a bending diameter of 1 cm was reached. The maximum deflection of the ACMI DUR-8E ureteroscope with each fiber in the working channel was recorded. The flow rate through the working channel of the DUR-8E was measured for each fiber., Results: The mean energy transmission differed among fibers (P < 0.001). The Lumenis SL 200 and the InnovaQuartz 400 were the best small and medium-diameter fibers, respectively, in resisting thermal breakdown (P < 0.01). The Dornier Lightguide Super 200 fractured repeatedly at a bend diameter of 2 cm and with the lowest energy (200 mJ). The other small fibers fractured only at a bend diameter of 1 cm. The Sharplan 200 and InnovaQuartz Sureflex 273T were the most flexible fibers, the Lumenis SL 365 the least. The flow rate was inversely proportional to four times the power of the diameter of the fiber., Conclusions: Optical performance and safety differ among fibers. Fibers transmit various amounts of energy to their cladding when bent. During lower-pole nephroscopy with the fiber deflected, there is a risk of fiber fracture from thermal breakdown and laser-energy transmission to the endoscope. Some available laser fibers carry a risk of ureteroscope damage.

This study was conducted to evaluate the effectiveness of a new antifungal drug, micafungin, and standard antifungal drugs against endophthalmitis induced in a rabbit by intravitreal injection of Aspergillus fumigatus, an important fungal pathogen. Effectiveness was evaluated by the preservation of b-wave amplitude at 72 h after injection of the fungus relative to the b-wave amplitude at baseline before any intravitreal injections. A 0.06 ml inoculum of 10(6) conidia of A. fumigatus was injected into the vitreous of the right eye of all rabbits; and, 12 h later, a 0.06 ml solution containing one of 3 antifungal drugs or saline was injected into the vitreous of both eyes. All three antifungal drugs produced significant b-wave preservation at 72 h in infected eyes compared to that in infected eyes receiving saline injections. There was no statistically significant difference between the effects of micafungin and amphotericin B in the right eyes with fungal endophthalmitis, and both produced significantly more preservation of b-wave amplitude than voriconazole. Amphotericin B, but neither micafungin nor voriconazole produced significant reduction of the b-wave amplitude in the left eyes.

Mammalian retinas receive input from the posterior hypothalamus, and the neurotransmitter in this pathway is histamine. To determine whether histamine influences ganglion cells, we analyzed the effects of histamine on their maintained and light-evoked activity in vitro. In monkeys, histamine increased the maintained firing rate in 42% of ganglion cells, decreased it in 38%, and had no effect in 20%. When histamine and the HR3 agonist, methylhistamine, were applied to the same cells in succession, their effects were sometimes different, a finding suggesting that at least one other histamine receptor is present. In addition, the responses of some ganglion cells to full-field light stimuli were decreased by histamine and methylhistamine. In rats, the effects of histamine were somewhat different. Histamine increased the maintained firing rate of 82% of ganglion cells. Methylhistamine and the HR2 agonist, dimaprit, had the same effects as histamine. In some cells, histamine increased the light responses, but in others it decreased them. Histamine had no effect on ganglion cells in either species when synaptic transmission was blocked by low Ca2(+)/high Mg2+ Ames medium. Thus, the major effects of histamine were on the maintained activity of retinal ganglion cells. In both rats and monkeys, 80% or more of the ganglion cells were affected by histamine, and these responses were mediated by at least two of the histamine receptor subtypes.

Holmium:YAG lithotripsy requires transmission of optical energy using laser fibers. Optical fibers may be subjected to severe angular deflection and bending during flexible nephroscopy or flexible ureteronephroscopy. Irradiation of a deflected fiber in a tight bending radius may produce fiber failure and consequent irradiation of the ureteroscope, with risk of instrument damage and patient injury. We review the physics and engineering aspects of optical fibers to explain why fibers fail.

Laser-induced heating in an ocular phantom is measured with magnetic resonance thermography (MRT) using temperature-dependent phase changes in proton resonance frequency. The ocular phantom contains a layer of melanosomes isolated from bovine retinal pigment epithelium. The phantom is heated by the 806-nm output of a continuous wave diode laser with an irradiance of 2.4 to 21.6 W/cm2 in a beam radius of 0.8 or 2.4 mm, depending on the experiment. MRT is performed with a 2 T magnet, and a two-turn, 6-cm-diam, circular radio frequency coil. Two-dimensional temperature gradients are measured within the plane of the melanin layer, as well as normal to it, with a temperature resolution of 1 degrees C or better. The temperature gradients extending within the melanin layer are broader than those orthogonal to the layer, consistent with the higher optical absorption and consequent heating in the melanin. The temperature gradients in the phantom measured by MRT closely approximate the predictions of a classical heat diffusion model. Three-dimensional temperature maps with a spatial resolution of 0.25 mm in all directions are also made. Although the temporal resolution is limited in the prototype system (22.9 s for a single image "slice"), improvements in future implementations are likely. These results indicate that MRT has sufficient spatial and temperature resolution to monitor target tissue temperature during transpupillary thermotherapy in the human eye.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in people over 60 years of age in the US and many other developed countries. Increasingly sophisticated methods for the diagnosis and treatment of macular degeneration are not effective for the majority of patients in whom late stage disease is present at the time of diagnosis. Research to elucidate the changes in RPE cell biology during aging has stimulated interest in preventive and prophylactic therapies for earlier intervention in the degenerative process. In the normal retina, the RPE performs the functions of barrier, macrophage, and neuroprotective cell layer. During aging and in the presence of disease the robustness of each of these functions diminishes. The utility of telomerase-mediated cell and gene therapy to prevent the decline in function of RPE cells during aging is evaluated.

Purpose: To assess the feasibility of conjugation of verteporfin (Visudyne, Parkedale Pharmaceuticals, Rochester, Minnesota, USA) to antibody against vascular endothelial growth factor., Design: Experimental study., Methods: Rabbit antimouse vascular endothelial growth factor polyclonal antibody was conjugated to verteporfin. Fluorescence excitation-emission spectra of verteporfin and conjugate were examined. Vascular endothelial growth factor-expressing murine endothelial cells were incubated with saline, verteporfin, or conjugate, followed by laser exposure or no laser exposure. Cell viability at 1 and 24 hours was assessed via trypan blue exclusion. Results were analyzed by two-way analysis of variance with replication and the Bonferroni multiple comparison test., Results: The fluorescence excitation-emission spectrum of the conjugate was similar to that of verteporfin. After laser exposure, cell viability in conjugate-treated cells was reduced to 6% at 1 hour (P <.0001) and to 4% at 24 hours (P <.0001), compared with approximately 40% in nonlaser-exposed, conjugate-treated cells. The cytotoxicity in the conjugate-treated cells was higher than in verteporfin-treated cells exposed to laser, although the difference did not reach statistical significance., Conclusions: The conjugation of verteporfin to polyclonal antibody is possible without the loss of its photosensitizing properties. Conjugated verteporfin destroys cellular targets at least as effectively as verteporfin alone.

Light damage to the retina occurs through three general mechanisms involving thermal, mechanical, or photochemical effects. The particular mechanism activated depends on the wavelength and exposure duration of the injuring light. The transitions between the various light damage mechanism may overlap to some extent. Energy confinement is a key concept in understanding or predicting the type of damage mechanism produced by a given light exposure. As light energy (either from a laser or an incoherent source) is deposited in the retina, its penetration through, and its absorption in, various tissue compartments is determined by its wavelength. Strongly absorbing tissue components will tend to "concentrate" the light energy. The effect of absorbed light energy largely depends on the rate of energy deposition, which is correlated with the exposure duration. If the rate of energy deposition is too low to produce an appreciable temperature increase in the tissue, then any resulting tissue damage necessarily occurs because of chemical (oxidative) reactions induced by absorption of energetic photons (photochemical damage). If the rate of energy deposition is faster than the rate of thermal diffusion (thermal confinement), then the temperature of the exposed tissue rises. If a critical temperature is reached (typically about 10 degrees C above basal), then thermal damage occurs. If the light energy is deposited faster than mechanical relaxation can occur (stress confinement), then a thermoelastic pressure wave is produced, and tissue is disrupted by shear forces or by cavitation-nonlinear effects. Very recent evidence suggests that ultrashort laser pulses can produce tissue damage through nonlinear and photochemical mechanisms; the latter because of two-photon excitation of cellular chromophores. In addition to tissue damage caused directly by light absorption, light toxicity can be produced by the presence of photosensitizing agents. Drugs excited to reactive states by ultraviolet (UV) or visible light produce damage by type I (free radical) and type II (oxygen dependent) mechanisms. Some commonly used drugs, such as certain antibiotics, nonsteroidal anti-inflammatory drugs (NSAIDs), and psychotherapeutic agents, as well as some popular herbal medicines, can produce ocular phototoxicity. Specific cellular effects and damage end points characteristic of light damage mechanisms are described.

A novel method based on liquid chromatography-mass spectrometry with electrospray ionization (LC-MS) has been developed for analysis of voriconazole in aqueous humor. The separation was achieved on a reversed-phase C(18) column eluted by 70% acetonitrile-30% water-0.01% TFA. The correlation between the concentration of voriconazole to peak area was linear (r(2)=0.9990) between 0.04 and 60 ng, with a coefficient of variance of less than 3%. Limit of quantitation (LOQ) was estimated to be 5 ng/ml voriconazole with an injection volume of 2 microl of aqueous humor. Both intra-day and inter-day imprecision were less than 3% over the whole analytical range. Parallel analyses of voriconazole samples by LC-MS and by high-performance liquid chromatography (HPLC)-UV showed that the two methods were highly correlated (r(2)=0.9985). LC-MS was used to the determine voriconazole levels achieved in the aqueous humor of the rabbit eye, following topical application of 5 or 10 microg voriconazole in the form of eyedrops for 11 days b.i.d. The lower dosage produced an aqueous humor concentration of 7.29+/-5.84 microg/ml, while the higher dosage produced a concentration of 14.56+/-12.90 microg/ml., (Copyright 2002 Elsevier Science B.V.)

Purpose: To investigate conditions promoting the differentiation of cultured human retinal pigment epithelial (RPE) cells and assess the differentiation potential of telomerase-immortalized RPE cells., Methods: Serially passaged RPE 340 (parental) cells have limited replicative ability and senesce after 50 to 60 population doublings (PD)s. RPE 340 cells transfected with the catalytic component of human telomerase (hTERT) have an extended lifespan. RPE 340 and hTERT-transfected RPE (hTERT-RPE) cells were maintained at confluence without serum for up to 12 weeks. Morphologic, immunocytochemical, flow cytometric, and spectrophotometric analyses were performed to examine the extent of RPE differentiation., Results: Parental RPE 340 and hTERT-RPE cells underwent growth arrest and differentiated in the absence of serum. In early-passage parental (PD11) and hTERT-RPE (PD115 and PD300) cells, serum deprivation for 4 weeks or more induced terminal differentiation as characterized by mature, growth-arrested, confluent sheets of polygonal and melanized cells that demonstrated diminished 5'-bromo-2'-deoxyuridine (BrdU) uptake and positive reactivity with antibodies to cellular retinaldehyde-binding protein (CRALBP), cytokeratin, and vimentin. Reintroduction of serum at 4 or 8 weeks allowed the cells to reenter the cell cycle and demelanize. Midpassage (PD 25) parental cells, however, were irreversibly arrested at G(1) after 8 weeks of serum deprivation., Conclusions: Cultured parental and hTERT-RPE cells express RPE-associated proteins and become stably melanized when density is arrested in the absence of serum. Moreover, hTERT-immortalized RPE cells retain the capacity to undergo terminal differentiation in vitro, even after long-term culture.

The optical properties of melanin have been characterized for a number of laser wavelengths in the visible region. The index of refraction of melanin is measured by the conventional method of minimum deviation using a hollow quartz prism at these wavelengths. The inverse adding doubling method based on the diffusion approximation and radiative transport theory have been employed to determine the absorption, scattering, and scattering anisotropy coefficients of melanin from the measurements of diffuse transmission, diffuse reflection and collimated transmission using double integrating spheres. The results obtained by the use of inverse adding doubling method have been compared to the Monte Carlo simulation technique.

Purpose: We test the hypothesis that erbium:YAG (Er:YAG) lithotripsy is more efficient than holmium:YAG (Ho:YAG) lithotripsy., Materials and Methods: Human calculi composed of greater than 97% calcium oxalate monohydrate and cystine were studied. Calculi were irradiated in water using Er:YAG or Ho:YAG lasers. Er:YAG lithotripsy was done with a 425 microm sapphire optical fiber at a pulse energy of 50 mJ at 10 Hz. Ho:YAG lithotripsy was performed with a 365 microm low hydroxy optical fiber at a pulse energy of 500 mJ at 10 Hz or a 425 microm sapphire optical fiber at a pulse energy of 50 mJ at 10 Hz. Fragmentation was defined as the initial stone mass minus the final dominant fragment mass and normalized for incident laser fluence (energy per unit area of fiber tip)., Results: Mean fragmentation plus or minus standard deviation for calcium oxalate monohydrate was 38 +/- 27 mg for Er:YAG and 22 +/- 6 for Ho:YAG (low hydroxy silica fiber) versus 5 +/- 1 for Ho:YAG (sapphire fiber, p = 0.001). When fragmentation was normalized for incident laser fluence given different optical fiber sizes, mean fragmentation efficiency was 53.6 +/- 38.7 g-microm2/J for Er:YAG lithotripsy compared with 22.6 +/- 6.4 for Ho:YAG (low hydroxy silica fiber) lithotripsy (p = 0.04). Mean cystine fragmentation was 15 +/- 3 mg for Er:YAG versus 9 +/- 1 for Ho:YAG (sapphire fiber, p = 0.0005)., Conclusions: Er:YAG lithotripsy is more efficient than Ho:YAG lithotripsy.

Background and Purpose: Holmium:YAG lithotripsy of uric acid calculi produces cyanide. The laser and stone parameters required to produce cyanide are poorly defined. In this study, we tested the hypotheses that cyanide production: (1) varies with holmium:YAG power settings; (2) varies among holmium:YAG, pulsed-dye, and alexandrite lasers; and (3) occurs during holmium:YAG lithotripsy of all purine calculi., Materials and Methods: Holmium:YAG lithotripsy of uric acid calculi was done using various optical fiber diameters (272-940 microm) and pulse energies (0.5-1.5 J) for constant irradiation (0.25 kJ). Fragmentation and cyanide were quantified. Cyanide values were divided by fragmentation values, and fragment sizes were characterized. To test the second hypothesis, uric acid calculi were irradiated with Ho:YAG, pulsed-dye, and alexandrite lasers. Fragmentation and cyanide were measured, and cyanide per fragmentation was calculated. Fragment sizes were characterized. Finally, Ho:YAG lithotripsy (0.25 kJ) of purine and nonpurine calculi was done, and cyanide production was measured., Results: Fragmentation increased as pulse energy increased for the 550- and 940-microm optical fibers (P < 0.05). Cyanide increased as pulse energy increased for all optical fibers (P < 0.002). Cyanide per fragmentation increased as pulse energy increased for the 272-microm optical fiber (P = 0.03). Fragment size increased as pulse energy increased for the 272-microm, 550-microm, and 940-microm optical fibers (P < 0.001). The mean cyanide production from 0.25 kJ of optical energy was Ho:YAG laser 106 microg, pulsed-dye 55 microm, and alexandrite 1 microg (P < 0.001). The mean cyanide normalized for fragmentation (microg/mg) was 1.18, 0.85, and 0.02, respectively (P < 0.001). The mean fragment size was 0.6, 1.1, and 1.9 mm, respectively (P < 0.001). After 0.25 kJ, the mean amount of cyanide produced was monosodium urate stones 85 microg, uric acid 78 microg, xanthine 17 microg, ammonium acid urate 16 microg, calcium phosphate 8 microg, cystine 7 microg, and struvite 4 microg (P < 0.001)., Conclusions: Cyanide production varies with Ho:YAG pulse energy. To minimize cyanide and fragment size, Ho:YAG lasertripsy is best done at a pulse energy < or = 1.0 J. Cyanide production from laser lithotripsy of uric acid calculi varies among Ho:YAG, pulsed-dye, and alexandrite lasers and is related to pulse duration. Cyanide is produced by Ho:YAG lasertripsy of all purine calculi.

Purpose: We tested the hypotheses that Proteus mirabilis viability of struvite calculi differs after exposure to different lithotripsy modalities and that the photothermal mechanism of holmium:YAG lithotripsy is antibacterial., Materials and Methods: Human calculi of known struvite composition (greater than 90% magnesium ammonium phosphate hexohydrate) were incubated with P. mirabilis. Calculi were randomly distributed and fragmented with no lithotripsy (controls), or shock wave, intracorporeal ultrasonic, electrohydraulic, pneumatic, holmium:YAG or pulsed dye laser lithotripsy. After lithotripsy fragments were sonicated and specimens were serially plated for 48 hours at 38C. Bacterial counts and the rate of bacterial sterilization were compared., Results: Median bacterial counts (colony-forming units per ml.) were 8 x 10(6) in controls and 3 x 10(6) in shock wave, 3 x 10(7) in ultrasonic, 4 x 10(5) in electrohydraulic, 8 x 10(6) in pneumatic, 5 x 10(4) in holmium:YAG and 1 x 10(6) in pulsed dye laser lithotripsy cases (p <0.001). The rate of bacterial sterilization was 50% for holmium:YAG lithotripsy treated stones versus 0% for each of the other cohorts (p <0.01)., Conclusions: P. mirabilis viability varies among lithotrites. The photothermal mechanism of holmium:YAG lithotripsy is antibacterial.

Purpose: We tested the hypothesis that holmium:YAG laser lithotripsy speed is best maximized by using low pulse energy at high pulse frequency., Materials and Methods: To demonstrate that optical fiber damage increases with pulse energy and irradiation, the 365-microm optical fiber irradiated calcium hydrogen phosphate dihydrate (CHPD), calcium oxalate monohydrate (COM), cystine, magnesium ammonium phosphate hexahydrate (MAPH), and uric acid calculi at pulse energies of 0.5 to 2.0 J. Optical energy output was measured with an energy detector after 10 J to 200 J of total energy. To demonstrate that lithotripsy efficiency varies with power, fragmentation was measured at constant power settings at total energies of 200 J and 1 kJ with the 365-microm optical fiber. Fragmentation was measured for the 272-microm optical fiber at pulse energies of 0.5 J to 1.5 J at 10 Hz. To demonstrate that low pulse energy produces smaller fragments than high pulse energy, fragment size was characterized for COM and uric acid calculi after 0.25 kJ of irradiation using the 272-microm to 940-microm optical fibers at 0.5 J to 1.5 J., Results: Damage to the 365-microm optical fiber was greatest for irradiation of CHPD, followed by MAPH, and COM (P<0.001). There was no significant optical fiber damage after cystine and uric acid lithotripsy. For the 365-microm optical fiber and CHPD, fragmentation after 200 J was greatest for pulse energies < or =1.0 J (P< 0.001). For other compositions, fragmentation was not statistically different among the power settings for constant irradiation. No significant difference was noted in fragmentation for any composition at different pulse energies (1.0 v. 2.0 J) for 1-kJ irradiation. However, for all compositions, the calculated lithotripsy speed was greatest at high power settings (P<0.001). For the 272-microm optical fiber, CHPD fragmentation was greatest for the 1.0-J pulse energy. The mean fragment size and relative quantity of fragments > or =2 mm both increased as pulse energy increased., Conclusions: Optical fiber degradation varies with stone composition, irradiation, and pulse energy. Holmium:YAG lithotripsy speed is maximized with higher power (either increased pulse energy or higher pulse frequency). Because low pulse energy may be safer and yields smaller fragments than high pulse energy, holmium:YAG lithotripsy speed is best increased by using pulse energies < or =1.0 J at a high repetition rate.

Laser induced thermal damage to the retina is investigated. The one step Arrhenius type thermal damage integral of Henriques is analyzed for its strengths and weaknesses. The zero-order activated rate process is shown to well represent the data for pulse durations greater than 10 μs. A zero-order biochemical damage mechanism involving free radical formation and thermal disruption of the melanosome's protein coat is proposed as the initial molecular process that leads to cellular damage which appears after a delay. Data are presented that show the photoactivation of melanin granule oxidative reactivity. This in vitro data is evidence for an important step in our hypothesized damage pathway. © 1999 Society of Photo-Optical Instrumentation Engineers.

The cellular pigments of the retinal pigment epithelium (RPE) have been shown to catalyze free radical activity, especially when illuminated with visible or ultraviolet light. This activity is sufficient to cause photooxidation of several major cellular components. The present investigation determined the relative ability of melanin, lipofuscin, and melanolipofuscin granules isolated from human and bovine eyes to oxidize polyunsaturated fatty acids, specifically linoleic and docosahexaenoic acids. The dark reactivity as well as the light-stimulated reactions were determined. The production of hydroperoxide derivatives of the linoleic and docosahexaenoic acids were determined by NADPH oxidation coupled to the activity of glutathione peroxidase, and also by production of thiobarbituric acid reactive substances. All RPE pigment granules stimulated fatty acid oxidation when irradiated with short wavelength (< 550 nm) visible light, with the melanosomes exhibiting the greatest light-induced activity. Only lipofuscin granules, however, caused peroxidation of fatty acids in the dark. These findings provide additional support for the role of RPE pigments in "blue light toxicity" as well as indicating that accumulation of lipofuscin may contribute to increased photooxidation in the aging RPE.

Objective: A series of experiments were conducted to test the hypothesis that the mechanism of holmium:YAG lithotripsy is photothermal., Methods and Results: To show that holmium:YAG lithotripsy requires direct absorption of optical energy, stone loss was compared for 150 J Ho:YAG lithotripsy of calcium oxalate monohydrate (COM) stones for hydrated stones irradiated in water (17+/-3 mg) and hydrated stones irradiated in air (25+/-9 mg) v dehydrated stones irradiated in air (40+/-12 mg) (P < 0.001). To show that Ho:YAG lithotripsy occurs prior to vapor bubble collapse, the dynamics of lithotripsy in water and vapor bubble formation were documented with video flash photography. Holmium:YAG lithotripsy began at 60 microsec, prior to vapor bubble collapse. To show that Ho:YAG lithotripsy is fundamentally related to stone temperature, cystine, and COM mass loss was compared for stones initially at room temperature (approximately 23 degrees C) v frozen stones ablated within 2 minutes after removal from the freezer. Cystine and COM mass losses were greater for stones starting at room temperature than cold (P < or = 0.05). To show that Ho:YAG lithotripsy involves a thermochemical reaction, composition analysis was done before and after lithotripsy. Postlithotripsy, COM yielded calcium carbonate; cystine yielded cysteine and free sulfur; calcium hydrogen phosphate dihydrate yielded calcium pyrophosphate; magnesium ammonium phosphate yielded ammonium carbonate and magnesium carbonate; and uric acid yielded cyanide. To show that Ho:YAG lithotripsy does not create significant shockwaves, pressure transients were measured during lithotripsy using needle hydrophones. Peak pressures were <2 bars., Conclusion: The primary mechanism of Ho:YAG lithotripsy is photothermal. There are no significant photoacoustic effects.