Macrophage activation associated with chronic murine cytomegalovirus infection results in more severe experimental choroidal neovascularization.

The neovascular (wet) form of age-related macular degeneration (AMD) leads to vision loss due to choroidal neovascularization (CNV). Since macrophages are important in CNV development, and cytomegalovirus (CMV)-specific IgG serum titers in patients with wet AMD are elevated, we hypothesized that chronic CMV infection contributes to wet AMD, possibly by pro-angiogenic macrophage activation. This hypothesis was tested using an established mouse model of experimental CNV. At 6 days, 6 weeks, or 12 weeks after infection with murine CMV (MCMV), laser-induced CNV was performed, and CNV severity was determined 4 weeks later by analysis of choroidal flatmounts. Although all MCMV-infected mice exhibited more severe CNV when compared with control mice, the most severe CNV developed in mice with chronic infection, a time when MCMV-specific gene sequences could not be detected within choroidal tissues. Splenic macrophages collected from mice with chronic MCMV infection, however, expressed significantly greater levels of TNF-α, COX-2, MMP-9, and, most significantly, VEGF transcripts by quantitative RT-PCR assay when compared to splenic macrophages from control mice. Direct MCMV infection of monolayers of IC-21 mouse macrophages confirmed significant stimulation of VEGF mRNA and VEGF protein as determined by quantitative RT-PCR assay, ELISA, and immunostaining. Stimulation of VEGF production in vivo and in vitro was sensitive to the antiviral ganciclovir. These studies suggest that chronic CMV infection may serve as a heretofore unrecognized risk factor in the pathogenesis of wet AMD. One mechanism by which chronic CMV infection might promote increased CNV severity is via stimulation of macrophages to make pro-angiogenic factors (VEGF), an outcome that requires active virus replication.

Neuronal NAD(P)H oxidases contribute to ROS production and mediate RGC death after ischemia.

PURPOSE: To study the role of neuronal nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase-dependent reactive oxygen species (ROS) production in retinal ganglion cell (RGC) death after ischemia. METHODS: Ischemic injury was induced by unilateral elevation of intraocular pressure via direct corneal cannulation. For in vitro experiments, RGCs isolated by immunopanning from retinas were exposed to oxygen and glucose deprivation (OGD). The expression levels of NAD(P)H oxidase subunits were evaluated by quantitative PCR, immunocytochemistry, and immunohistochemistry. The level of ROS generated was assayed by dihydroethidium. The NAD(P)H oxidase inhibitors were then tested to determine if inhibition of NAD(P)H oxidase altered the production of ROS within the RGCs and promoted cell survival. RESULTS: It was reported that RGCs express catalytic Nox1, Nox2, Nox4, Duox1, as well as regulatory Ncf1/p47phox, Ncf2/p67phox, Cyba/p22phox, Noxo1, and Noxa1 subunits of NAD(P)H oxidases under normal conditions and after ischemia. However, whereas RGCs express only low levels of catalytic Nox2, Nox4, and Duox1, and regulatory Ncf1/p47, Ncf2/p67 subunits, they exhibit significantly higher levels of catalytic subunit Nox1 and the subunits required for optimal activity of Nox1. It was observed that the nonselective NAD(P)H oxidase inhibitors VAS-2870, AEBSF, and the Nox1 NAD(P)H oxidase-specific inhibitor ML-090 decreased the ROS burst stimulated by OGD, which was associated with a decreased level of RGC death. CONCLUSIONS: The findings suggest that NAD(P)H oxidase activity in RGCs renders them vulnerable to ischemic death. Importantly, high levels of Nox1 NAD(P)H oxidase subunits in RGCs suggest that this enzyme could be a major source of ROS in RGCs produced by NAD(P)H oxidases.

Retinoblastoma treatment: utilization of the glycolytic inhibitor, 2-deoxy-2-fluoro-D-glucose (2-FG), to target the chemoresistant hypoxic regions in LH(BETA)T(AG) retinal tumors.

PURPOSE: To analyze the effect of the glycolytic inhibitor 2-deoxy-2-fluoro-d-glucose (2-FG) on tumor burden, hypoxia, and blood vessels in LH(BETA)T(AG) retinal tumors. METHODS: Seventeen-week-old LH(BETA)T(AG) retinal tumor eyes (n = 36) were treated with 2-FG and analyzed at 1 day and 1 week post a single treatment, and 1 day post a biweekly treatment for 3 weeks. Tumor sections were analyzed for hypoxia, tumor burden, and vasculature. To assess tumor burden, sections were processed for standard hematoxylin-eosin (H&E) staining. Immunofluorescent techniques were used to stain for new and mature blood vessels. RESULTS: Hypoxia and tumor burden reduction are significantly different between the treatment schedules used (P < 0.001). Eyes treated with 2-FG for 3 weeks showed a significant decrease in hypoxia (P = 0.001) and tumor burden (P = 0.009); whereas those treated with one injection and evaluated at 1 day and 1 week postinjection did not show a decrease in either hypoxia (P = 0.373 and P = 0.782, respectively) or tumor burden (P = 0.203 and P = 0.836, respectively). When evaluating the spatial distribution of hypoxic regions in the different areas of the tumor, 2-FG showed a differential effect on hypoxia depending on the area. Hypoxia was most decreased in the base of the treated eyes with a 95% reduction (P < 0.001). CONCLUSIONS: This is the first study to elucidate that 2-FG treatment in retinoblastoma produces an impact on hypoxia and a concomitant decrease on tumor burden. In this study, the authors validate their previous studies by revealing that glycolytic inhibitors effectively target hypoxia in retinoblastoma tumors. The future application of 2-FG as an adjuvant treatment to standard chemotherapy may enhance the treatment of retinoblastoma.

The high-mobility group box-1 nuclear factor mediates retinal injury after ischemia reperfusion.

PURPOSE: High-mobility group protein B1 (Hmgb1) is released from necrotic cells and induces an inflammatory response. Although Hmgb1 has been implicated in ischemia/reperfusion (IR) injury of the brain, its role in IR injury of the retina remains unclear. Here, the authors provide evidence that Hmgb1 contributes to retinal damage after IR. METHODS: Retinal IR injury was induced by unilateral elevation of intraocular pressure and the level of Hmgb1 in vitreous humor was analyzed 24 hours after reperfusion. To test the functional significance of Hmgb1 release, ischemic or normal retinas were treated with the neutralizing anti-Hmgb1 antibody or recombinant Hmgb1 protein respectively. To elucidate in which cell type Hmgb1 exerts its effect, primary retinal ganglion cell (RGC) cultures and glia RGC cocultures were treated with Hmgb1. To clarify the downstream signaling pathways involved in Hmgb1-induced effects in the ischemic retina, receptor for advanced glycation end products (Rage)-deficient mice (RageKO) were used. RESULTS: Hmgb1 is accumulated in the vitreous humor 24 hours after IR. Inhibition of Hmgb1 activity with neutralizing antibody significantly decreased retinal damage after IR, whereas treatment of retinas or retinal cells with Hmgb1 induced a loss of RGCs. The analysis of RageKO versus wild-type mice showed significantly reduced expression of proinflammatory genes 24 hours after reperfusion and significantly increased survival of ganglion cell layer neurons 7 days after IR injury. CONCLUSIONS: These results suggest that an increased level of Hmgb1 and signaling via the Rage contribute to neurotoxicity after retinal IR injury.

Toll-like receptor 4 contributes to retinal ischemia/reperfusion injury.

PURPOSE: We investigated whether retinal ischemia and inflammation produced by raising the intraocular pressure above normal systolic levels differs in mice that lack a functional toll-like receptor 4 (Tlr4) signaling pathway. METHODS: In this work we used the murine strain B6.B10ScN-Tlr4(lps-del)/JthJ, which does not express functional Tlr4. C57BL/6J was considered as the control. We induced retinal ischemia by unilateral elevation of intraocular pressure for 1 h by direct corneal cannulation. The changes in expression of proinflammatory genes 24 h postreperfusion were assessed by quantitative PCR. Corresponding changes in protein abundances were analyzed by western blot and immunohistochemistry. Cell death was evaluated by direct counting of neurons in the ganglion cell layer of flat-mounted retinas seven days postreperfusion. RESULTS: We showed that Tlr4-deficient mice display significantly reduced expression of proinflammatory genes, including RelA, tumor necrosis factor (Thf), interleukin 6 (Il6), chemokine (C-C motif) ligand 2 (Ccl2), chemokine (C-C motif) ligand 5 (Ccl5), chemokine (C-X-C motif) ligand 10 (Cxcl10), Cybb, nitric oxide synthase 2 (Nos2), and intercellular adhesion molecule 1 (Icam1) 24 h after reperfusion. The mice that lacked Tlr4 showed significantly increased survival of neurons in the ganglion cell layer following ischemic injury, as compared to wild-type controls. CONCLUSIONS: Our results indicate that Tlr4 signaling is involved in retinal damage and inflammation triggered by ischemic injury.

Focal, periocular delivery of 2-deoxy-D-glucose as adjuvant to chemotherapy for treatment of advanced retinoblastoma.

PURPOSE: The aim of this study was to evaluate the changes in tumor burden and hypoxia in the LH(BETA)T(AG) retinal tumors after treatment with a focal, single-modality, and combination therapy using periocular carboplatin and 2-deoxy-d-glucose (2-DG). METHODS: Seventeen-week-old LH(BETA)T(AG) transgenic mice (n = 25) were treated with periocular injections of varying doses of 2-DG (62.5, 125, 250, 500 mg/kg) to obtain a dose response. Same-aged mice (n = 30) received periocular injections of saline, carboplatin, and 2-DG. Mice were divided into six groups: saline; carboplatin (31.25 µg/20 µL, subtherapeutic dose); 2-DG (250 mg/kg); 2-DG (500 mg/kg); carboplatin (31.25 µg/20 µL) and 2-DG (250 mg/kg); and carboplatin (31.25 µg/20 µL) and 2-DG (500 mg/kg). Injections were administered twice weekly for three consecutive weeks. Eyes were enucleated at 20 weeks of age, snap frozen, and analyzed for hypoxic regions and tumor volume. RESULTS: The difference in percentage of hypoxia after treatment with 500 mg/kg 2-DG was statistically significant from the other dose groups (P < 0.015). The difference in tumor burden was statistically significant from the 250 mg/kg dose (P < 0.015) and 500 mg/kg dose (P < 0.001). Highly significant differences were found between the treatment types for tumor burden, percentage of hypoxia, and pimonidazole intensity (P < 0.001). Tumor burden decreased significantly after all forms of treatment (P < 0.001); however, tumor burden became significantly more reduced after treatment with combination therapy of carboplatin and 2-DG than with either treatment alone (P < 0.001). The percentage of hypoxia and pimonidazole intensity decreased after treatment with 2-DG alone and in combination with carboplatin (P < 0.001) in all treatment groups using 2-DG regardless of the 2-DG dose used. There was no percentage reduction of hypoxia after treatment with carboplatin alone (P = 0.25). CONCLUSIONS: This study demonstrates the efficacy of focal, periocular 2-DG as an adjunct to carboplatin chemotherapy to decrease both intratumoral hypoxia and tumor burden. Hypoxia is increasingly present in advanced disease of LH(BETA)T(AG) retinal tumors. The use of glycolytic inhibitors as a therapeutic strategy has the potential to enhance current retinoblastoma treatments.

Spectral domain optical coherence tomography in a murine retinal detachment model.

Spectral domain optical coherence tomography (SD-OCT) was used to image retinal detachments in vivo, in a murine model of retinal detachment (RD). Subretinal injections of hyaluronic acid (Healon) were delivered to the right eye of seventeen 10-20 week-old C57Bl6 mice. Evaluation of the fundus with an operating microscope and fundus photography were performed. In vivo, non-contact, ultra high resolution SD-OCT imaging was performed on day 0, day 1-2, day 5-6 and day 15-16. The retinal morphology at the edge and in the area of maximal RD was evaluated. Eyes were enucleated for histologic analysis. The retinal detachment was confirmed by microscopy in all mice. The extent of the retinal detachment was evaluated by measuring the height of the retinal detachment. The retinal layers, including the photoreceptor layer, were evaluated. Retinal layers appeared indistinct soon after RD (day 1, 5), particularly over areas of maximal detachment. By day 5 and 15 the external limiting membrane was no longer visible and there was increased reflectivity of the photoreceptor layer and undulation of the outer retina in areas of RD on both SD-OCT and histology. The thickness of the outer nuclear layer and photoreceptor outer segments decreased on day 5 and 15. SD-OCT is a promising technology to follow retinal detachment and outer retinal abnormalities in a murine model.

Estrogen receptor beta protects against in vivo injury in RPE cells.

Epidemiological data suggest that estrogen deficiency in postmenopausal women may contribute to the severity of AMD. We discovered that 17beta-estradiol (E2) was a crucial regulator of the severity of extracellular matrix turnover (ECM) dysregulation both in vivo and in vitro. We also found in vitro that the presence of estrogen receptor (ER)beta regulates MMP-2 activity. Therefore in an attempt to delineate the role of the ER subtypes, female estrogen receptor knockout (ERKO) mice were fed a high-fat diet, and the eyes were exposed to seven 5-second doses of nonphototoxic levels of blue-green light over 2 weeks. Three months after cessation of blue light treatment, transmission electron microscopy was performed to assess severity of deposits, Bruchs membrane changes, and choriocapillaris endothelial morphology. We found that changes in the trimolecular complex of pro-MMP-2, MMP-14 and TIMP-2 correlated with increased Bruch's membrane thickening or sub-retinal deposit formation (basal laminar deposits) in ERKObeta mice. In addition RPE isolated from ERKObeta mice had an increase in expression of total collagen and a decrease in MMP-2 activity. Finally we found that ERK an intermediate signaling molecule in the MMP pathway was activated in RPE isolated from ERKObeta mice. These data suggest that mice which lack ERbeta are more susceptible to in vivo injury associated with environmental light and high fat diet.

Liposome-delivered ATP effectively protects the retina against ischemia-reperfusion injury.

PURPOSE: We investigated the effect of ATP (ATP) encapsulated in liposomes (ATP-liposomes) on the level of inflammation and neuronal death in the retina induced by ischemia reperfusion (IR). METHODS: Primary retinal ganglion cells treated with ATP-liposomes, empty liposomes, and phosphate buffer solution (PBS) were deprived of oxygen and glucose (OGD) for 6 h in vitro, in an anaerobic chamber. Plates were assessed for the proportion of necrotic versus apoptotic cells and for cell survival 12 h after OGD. For in vivo experiments, we induced retinal ischemia by unilateral elevation of intraocular pressure for 1 h by direct corneal canulation. Mice were injected with liposomes or PBS 24 h before IR, at the time of surgery, and every 24 h until sacrifice. Transmission electron microscopic analysis was used to identify necrotic and apoptotic cells in ischemic retinas. The changes in expression of pro-inflammatory genes 24 h post reperfusion were assessed by quantitative reverse transcription polymerase chain reaction (RT-PCR). Corresponding changes in protein abundances were analyzed by immunohistochemistry. Cell death was evaluated by direct counting of neurons in the ganglion cell layer (GCL) of flatmounted retinas 7 days post reperfusion. RESULTS: Treatment with ATP-liposomes increases retinal ganglion cell (RGC) survival and decreases necrotic cell death following OGD. Injection of ATP-liposomes markedly decreased necrotic cell death in the GCL following retinal ischemia. The ATP-liposome treatment reduced the expression of pro-inflammatory genes, including that of interleukin 1ß (Il1ß), interleukin 6 (Il6), tumor necrosis factor (Thf), chemokine (C-C motif) ligand 2 (Ccl2), chemokine (C-C motif) ligand 5 (Ccl5), chemokine (C-X-C motif) ligand 10 (Cxcl10), intercellular adhesion molecule 1 (Icam1), and nitric oxide synthase 2 (Nos2), in the retina 24 h after IR and significantly reduced the GCL neuron death rate 7 days after reperfusion. CONCLUSIONS: ATP-liposome treatment of IR-challenged neural tissues suppressed necrosis and correlated with a significantly reduced level of inflammation and retinal damage.

Phosphatidylserine-containing liposomes promote maximal survival of retinal neurons after ischemic injury.

We investigated the systemic effect of liposomes bearing apoptotic signals on the level of inflammation and neuronal death induced by ischemia-reperfusion (IR). Using a model of retinal ischemia, we showed that treatment with phosphatidylserine (PS) and phosphatidylcholine (PC) liposomes significantly reduced the expression of proinflammatory genes, including that of Il1b, Il6, Ccl2, Ccl5, Cxcl10, and Icam1, 24 h after reperfusion. Phosphatidylserine liposome treatment was the most efficient and correlated with significantly reduced neuronal death in the retina 7 days after reperfusion. The results of our study indicate that therapeutic strategy based on mimicking a systemic increase in apoptotic signaling can significantly reduce central nervous system damage induced by IR and improve neurologic outcome.

Inactivation of astroglial NF-kappa B promotes survival of retinal neurons following ischemic injury.

Reactive astrocytes have been implicated in neuronal loss following ischemic stroke. However, the molecular mechanisms associated with this process are yet to be fully elucidated. In this work, we tested the hypothesis that astroglial NF-kappaB, a key regulator of inflammatory responses, is a contributor to neuronal death following ischemic injury. We compared neuronal survival in the ganglion cell layer (GCL) after retinal ischemia-reperfusion in wild-type (WT) and in GFAP-IkappaBalpha-dn transgenic mice, where the NF-kappaB classical pathway is suppressed specifically in astrocytes. The GFAP-IkappaBalpha-dn mice showed significantly increased survival of neurons in the GCL following ischemic injury as compared with WT littermates. Neuroprotection was associated with significantly reduced expression of pro-inflammatory genes, encoding Tnf-alpha, Ccl2 (Mcp1), Cxcl10 (IP10), Icam1, Vcam1, several subunits of NADPH oxidase and NO-synthase in the retinas of GFAP-IkappaBalpha-dn mice. These data suggest that certain NF-kappaB-regulated pro-inflammatory and redox-active pathways are central to glial neurotoxicity induced by ischemic injury. The inhibition of these pathways in astrocytes may represent a feasible neuroprotective strategy for retinal ischemia and stroke.

Laser trabeculoplasty induces changes in the trabecular meshwork glycoproteome: a pilot study.

Laser trabeculoplasty (LT) is a commonly used modality of treatment for glaucoma. The mechanism by which LT lowers the intraocular pressure (IOP) is unknown. With the use of cat eyes, selective laser trabeculoplasty (SLT) with a Q-switched frequency doubled Nd:YAG laser was used to treat the trabecular meshwork (TM). Laser treated TM was then subjected to proteomic analysis for detection of molecular changes and histological analysis for the detection of structural and protein expression patterns. In addition, the protein glycosylation patterns of laser treated and nontreated TM was assessed and differentially glycosylated proteins were proteomically identified. SLT laser treatment to the TM resulted in elevated glycosylation levels compared to nonlasered TM. TM laser treatment also resulted in protein expression levels changes of several proteins. Elevated levels of biglycan, keratocan and prolargin were detected in laser treated TM compared to nonlasered controls. Further investigation is anticipated to provide insight into how glycosylation changes affect TM proteins and TM regulation of aqueous outflow in response to laser trabeculoplasty.

Retinal tumor imaging and volume quantification in mouse model using spectral-domain optical coherence tomography.

We have successfully imaged the retinal tumor in a mouse model using an ultra-high resolution spectral-domain optical coherence tomography (SD-OCT) designed for small animal retinal imaging. For segmentation of the tumor boundaries and calculation of the tumor volume, we developed a novel segmentation algorithm. The algorithm is based on parametric deformable models (active contours) and is driven by machine learning-based region classification, namely a Conditional Random Field. With this algorithm we are able to obtain the tumor boundaries automatically, while the user can specify additional constraints (points on the boundary) to correct the segmentation result, if needed. The system and algorithm were successfully applied to studies on retinal tumor progression and monitoring treatment effects quantitatively in a mouse model of retinoblastoma.

Mouse retinal pigmented epithelial cell lines retain their phenotypic characteristics after transfection with human papilloma virus: a new tool to further the study of RPE biology.

Development of immortalized mouse retinal pigmented epithelial cell (RPE) lines that retain many of their in vivo phenotypic characteristics, would aid in studies of ocular diseases including age related macular degeneration (AMD). RPE cells were isolated from 18-month-old (estrogen receptor knockout) ERKOalpha and ERKObeta mice and their C57Bl/6 wildtype littermates. RPE65 and cellular retinaldehyde binding protein (CRALBP) expression, in vivo markers of RPE cells, were detected by real-time RT-PCR and western analysis. We confirmed the presence of epithelial cell markers, ZO1, cytokeratin 8 and 18 by immunofluorescence staining. In addition, we confirmed the distribution of actin filaments and the expression of ezrin. To develop cell lines, RPE cells were isolated, propagated and immortalized using human papilloma virus (HPV) 16 (E6/E7). RPE-specific markers and morphology were assessed before and after immortalization. In wildtype littermate controls, there was no evidence of any alterations in the parameters that we examined including MMP-2, TIMP-2, collagen type IV, and estrogen receptor (ER)alpha and ERbeta protein expression and ER copy number ratio. Therefore, immortalized mouse RPE cell lines that retain their in vivo phenotype can be isolated from either pharmacologically or genetically manipulated mice, and may be used to study RPE cell biology.

Paclitaxel in the treatment of retinal tumors of LH beta-Tag murine transgenic model of retinoblastoma.

PURPOSE: The purpose of this study was to investigate tumor control efficacy of paclitaxel in the treatment of retinal tumors harbored by the LH beta-Tag murine transgenic model of retinoblastoma. METHODS: LH beta-Tag-positive mice (n = 5) 10 weeks of age received two 20-microL subconjunctival injections delivered with a 72-hour interval to right eyes only. Paclitaxel was dissolved in 100% dimethyl sulfoxide (DMSO) and delivered at doses of 0.5 mg, 0.25 mg, 0.125 mg, 0.0625 mg, 0.0313 mg, and 0.0152 mg in a 20-microL volume. Control mice received two subconjunctival injections of DMSO or of saline solution to right eyes only or they remained untreated. Eyes were analyzed at 16 weeks of age for residual tumor burden, which was measured by gauging the cross-sectional area of largest tumor focus. RESULTS: Linear regression analysis of tumor burden demonstrates a statistically significant (P < 0.001) dose-response relationship between paclitaxel concentration and tumor size. Transient ocular toxicities were observed after treatment, but most had subsided at the time of analysis, 6 weeks after injection. After histologic assessment, the 0.25-mg paclitaxel dose had effectively reduced tumor burden and was associated with minimal toxicity, including mild conjunctival chemosis and fibrosis, corneal epitheliopathy, and corneal edema. CONCLUSIONS: Subconjunctival delivery of paclitaxel effectively inhibits intraocular tumor burden in the LH beta-Tag model of retinoblastoma. This treatment modality may represent a novel strategy for the clinical management of retinoblastoma.

Simultaneous fundus imaging and optical coherence tomography of the mouse retina.

PURPOSE: To develop a retinal imaging system suitable for routine examination or screening of mouse models and to demonstrate the feasibility of simultaneously acquiring fundus and optical coherence tomography (OCT) images. METHODS: The imaging system is composed of a photographic slit lamp for biomicroscopic examination of the fundus, an OCT interferometer, an OCT beam delivery system designed for the mouse eye, and a mouse positioning stage. Image acquisition was controlled with software that displays the fundus and OCT images in real time, and allows the user to control the position of the OCT beam spot on the fundus image display. The anesthetized mouse was placed in a cylindrical holder on the positioning stage, and a single operator adjusted the position of mouse. RESULTS: Fundus images and OCT scans were successfully acquired in both eyes of 8 C57BL/6 mice. Once the animal is anesthetized and placed in the holder, a typical imaging experiment takes less than 2 minutes. The retinal vasculature, pigmentation, nerve fiber arrangement, and optic nerve head were clearly visible on the fundus images. The quality of the OCT images was sufficient to allow measurement of the total, inner, and outer retinal thicknesses and to visualize the optic nerve head excavation. CONCLUSIONS: The study demonstrates the feasibility of acquiring simultaneous fundus and OCT images of the mouse retina, by a single operator, in a manner suitable for routine evaluation of mouse models of retinal disease.

Evaluation of an integrated orbital tissue expander in an anophthalmic feline model.

PURPOSE: To evaluate the anatomical effects and tissue biocompatibility in a feline model of an integrated orbital tissue expander (OTE) designed to stimulate bone growth in an anophthalmic socket. DESIGN: An animal study was performed in cats to assess orbital bone growth with and without an OTE. METHODS: The OTE is an inflatable (0.5 to >6.0 cm(3)) polymeric globe sliding on a titanium T plate secured to the lateral orbital rim with screws. Eight cats had left eye enucleation at age two weeks, with five orbits receiving an OTE and the remaining three serving as nonimplanted controls. Serial transconjunctival implant inflation was performed by injecting normal saline solution into the OTE to a final volume of 3.5 ml. Serial computed tomographic scans were obtained to assess socket growth. All eight cats were euthanized at 18 weeks and dry skulls prepared. The effective orbital volume was measured by inflating an OTE in the orbit of a dry skull until it filled the cavity completely. RESULTS: Three cats periodically scratched open the tarsorrhaphy and conjunctiva to rupture the OTE, which resulted in implant exchanges. At 18 weeks, the OTE expanded orbital volume was approximately 18% smaller than the normal contralateral side. In the control animals, the anophthalmic orbital volume was approximately 66% smaller than the contralateral orbit. Histopathology of orbital tissues showed no evidence of foreign body reaction. CONCLUSIONS: This proof-of-concept pilot study demonstrated implant efficacy in cats, and no implant-related adverse effects were observed. OTE has the potential to stimulate bone growth in human anophthalmic orbits.

Iontophoretic delivery of carboplatin in a murine model of retinoblastoma.

PURPOSE: To evaluate the efficacy and dose-response of transcorneoscleral Coulomb controlled iontophoresis (CCI) of carboplatin in the treatment of retinal tumors of a murine model of retinoblastoma. METHODS: Thirty 6-week-old LHBETATAG mice underwent a total of six, serial iontophoretic treatments administered two times per week using a current density of 2.57 mA/cm2 for 5 minutes. Fourteen animals received carboplatin treatments at concentrations of 1.4, 7.0, 10.0, or 14.0 mg/mL without current. Ten control mice underwent treatment with balanced saline solution. RESULTS: A dose-dependent inhibition of intraocular tumor was observed after repetitive iontophoretic treatment. At carboplatin concentrations of 7 mg/mL, 50% of the treated eyes (4/8) exhibited tumor control. No corneal toxicity was observed in eyes treated at carboplatin concentrations under 10 mg/mL. CONCLUSIONS: CCI delivery of carboplatin safely and effectively controls intraocular tumors in a dose-dependent manner in this murine model of retinoblastoma. CCI is a noninvasive, painless option for the focal delivery of carboplatin. However, further clinical and laboratory research is needed before this method of drug delivery is available for children with retinoblastoma.

Experimental model for proliferative vitreoretinopathy by intravitreal dispase: limited by zonulolysis and cataract.

BACKGROUND: The intravitreal injection of dispase has been shown to be a valuable method for induction of experimental PVR. The goal of the present study was to gain additional information about potential side effects associated with this method. METHODS: Twenty-one pigmented rabbits received a single injection of dispase under topical anesthesia to one eye only, contralateral eyes served as untreated control. The animals were injected with doses from 0.045 to 0.065 units of dispase: 8 animals received 0.045 units, 9 animals 0.055 units and 4 animals 0.065 units. RESULTS: Proliferative vitreoretinopathy occurred in 81% of the treated eyes. In 90% cataract formation was observed. Lens luxation was present in 47.3% of the cataract eyes. CONCLUSION: Intravitreal injection of dispase resulted in the reproducible induction of PVR in addition to cataract formation and lens luxation. Whether these effects may all be associated with a toxic reaction or whether the proliferative changes are solely triggered by endogenous reactions similar to the pathomechanism of human PVR and whether the cataract formation and the lens luxation may be avoided by changing the method of injection require further investigation.

Anecortave acetate as single and adjuvant therapy in the treatment of retinal tumors of LH(BETA)T(AG) mice.

PURPOSE: To evaluate the tumor control efficacy of the antiangiogenic agent anecortave acetate as single and combined therapy, in retinal tumor reduction using the LH(BETA)T(AG) mouse model of retinoblastoma. METHODS: Group A: Ten-week-old, LH(BETA)T(AG) mice received a single subconjunctival injection of anecortave acetate (1200, 600, 300, and 150 microg) delivered to right eyes only. Group B: Ten-week-old, LH(BETA)T(AG) mice received a single subconjunctival injection of anecortave acetate (600, 300, and 150 microg) delivered to right eyes only, either during a cycle of carboplatin (six subconjunctival deliveries) or after the completed cycle. Carboplatin was delivered at the subtherapeutic concentration of 62.5 microg. All animals were euthanatized at 16 weeks of age, and the eyes were examined histopathologically. RESULTS: A statistically significant reduction in tumor burden was detected after a single periocular injection of anecortave acetate. The reduction of tumor burden followed a U-shaped dose-response curve. Tumor burden was significantly decreased when anecortave acetate and carboplatin were combined. However, varying doses and delivery schedule of these agents had significant impact on the effectiveness of the combined treatment. The most effective scheme was delivering a low dose (150-300 microg) of anecortave acetate after a complete cycle of carboplatin. Histopathological evaluation showed no signs of retinal toxicity to anecortave acetate delivery alone or in combination with carboplatin. CONCLUSIONS: Anecortave acetate, as monotherapy or as adjuvant therapy, significantly controlled tumor burden in a murine model of retinoblastoma. Moreover, adjuvant therapy enabled the use of typically subtherapeutic carboplatin doses without decreasing efficacy of the therapy.