Photodynamic therapy with PhotoPoint photosensitiser MV6401, indium chloride methyl pyropheophorbide, achieves selective closure of rat corneal neovascularisation and rabbit choriocapillaris.

AIM: The new photosensitiser PhotoPoint MV6401, indium chloride methyl pyropheophorbide, was assessed as a possible ocular photodynamic therapy agent in a rat model of experimentally induced corneal neovascularisation and in choriocapillaris closure in the rabbit. Optimal drug and light activation parameters were determined. METHODS: MV6401 (Miravant Pharmaceuticals, Inc, Santa Barbara, CA, USA) was activated at 664 nm using a DD3-0665 (Miravant Systems Inc) 0.5 W diode laser. Corneal neovascularisation in rats was induced using an N-heptanol technique. The evaluated drug dosages, light dosages, and post-injection activation times ranged from 0.01-0.1 micromol/kg, 5-25 J/cm(2), and 10-60 minutes, respectively. The efficacy of MV6401 on normal choriocapillaris and choroidal vessels was evaluated in rabbits with indirect ophthalmoscopy, fundus photography, fluorescein angiography, and histology. In rabbits, the evaluated drug dosages, light dosages, and post-injection activation times ranged from 0.025-0.25 micromol/kg, 3.3-20 J/cm(2), and 10 minutes, respectively. RESULTS: In the rat corneal neovascularisation model, an optimal intravenous drug dosage of 0.075 micromol/kg was activated by a 20 J/cm(2) light dose at 10 minutes after drug administration, the results of which demonstrated early evidence of efficacy in ocular neovascularisation. In rabbits, closure of the normal choriocapillaris was selectively achieved at a drug dosage of 0.15 micromol/kg using light doses from 3.3 to 20 J/cm(2). CONCLUSION: PhotoPoint MV6401 is a potent photosensitiser that demonstrates both efficacy and selectivity in experimental ocular models.

Choroidal neovascular membrane inhibition in a laser treated rat model with intraocular sustained release triamcinolone acetonide microimplants.

AIM: To determine if intravitreal microimplants containing triamcinolone acetonide (TAAC) inhibit experimental fibrovascular proliferation (FVP) induced by laser trauma in a rat as a model of choroidal neovascular membranes (CNVMs). METHODS: 20 anaesthetised male Brown Norway rats received a series of eight krypton red laser lesions per eye (647 nm, 0.05 s, 50 micro m, 150 mW). Three types of sterilised TAAC microimplant designs were evaluated: implant A consisting of 8.62% TAAC/20% polyvinyl alcohol (PVA) matrix (by dry weight); implant B consisting of 3.62% TAAC/20% PVA matrix; and implant C consisting of a dual 8.62% TAAC/20% PVA matrix design combined with a central core (0.5 mm) of compressed TAAC to extend the implant release time. For each animal studied, one eye received one of the three aforementioned TAAC implant designs, while the fellow eye received a control implant consisting of PVA but without TAAC. The animals were sacrificed at day 35 and ocular tissues were processed for histological analysis. Serial histological specimens were methodically assessed in a masked fashion to analyse each laser lesion for the presence or absence of FVP; maximum FVP thickness for each lesion was measured from the choriocapillaris. RESULTS: All three types of TAAC implants inhibited FVP relative to controls in a statistically significant fashion. In the eyes that received implant A (n = 8), the mean thickness of the recovered lesions (n = 36) measured 32 (SD 22) micro m, compared to 52 (30) micro m (p <0.005) for the recovered lesions (n = 40) from the fellow control eyes. In the eyes that received implant B (n = 6), the mean thickness of the recovered lesions (n = 31) measured 28 (15) micro m, compared to 50 (29) micro m (p <0.001) for the lesions (n = 19) recovered from the fellow control eyes. In the eyes that received implant C (n = 6), the mean thickness of the recovered lesions (n = 21) measured 39 (24) micro m, compared to 65 (30) micro m (p <0.001) for the lesions (n = 39) recovered from the fellow control eyes. CONCLUSIONS: All three of the tested TAAC microimplant designs produced potent inhibition of FVP in a rat model of CNVMs. There were no differences in inhibition of FVP between the three different types of implants evaluated. This study provides evidence that: (1) corroborates previous investigations that propose TAAC as a potential treatment for CNVMs in humans, and (2) demonstrates TAAC can be effectively delivered via long acting sustained release intraocular microimplants. It should be noted, however, that the FVP observed in this rat laser trauma may not reflect the CNVM observed in human with exudative age related macular degeneration (AMD).

Transpupillary thermotherapy for subfoveal occult choroidal neovascularization: effect on ocular perfusion.

PURPOSE: To perform a descriptive analysis of the effects on ocular blood flow of transpupillary thermotherapy (TTT) for occult subfoveal choroidal neovascular membranes (CNVMs) in age-related macular degeneration (AMD). METHODS: Eleven subjects with occult subfoveal CNVM due to AMD were assessed in a masked fashion by color Doppler imaging (CDI) within 24 hours before, 24 hours after, and 1 month after undergoing TTT. RESULTS: In the posterior ciliary arteries (PCAs), there were no statistically significant changes observed in the peak systolic velocity (PSV), end diastolic velocity (EDV), or resistive index (RI) at 24 hours. At 1 month, the mean EDV decreased 36% (P = 0.0105) and the mean RI increased 3.8% (P = 0.0305) in the nasal PCA. Although there was a similar trend in the temporal PCA, the differences did not reach statistical significance. In the central retinal artery (CRA), the mean PSV decreased 16% (P = 0.0137), and the mean EDV decreased 21% (P = 0.0222) at 24 hours after treatment. There were no statistically significant differences in the CRA blood flow indices at 1 month after treatment. In the ophthalmic artery, there were no statistically significant differences observed in the mean PSV, EDV, or RI at 24 hours or 1 month after treatment. CONCLUSIONS: TTT is associated with transiently decreased volumetric blood flow in the retinal circulation 24 hours after treatment. In the posterior ciliary arteries that supply the choroid, there were no changes observed at 24 hours, but at 1 month, there was a decrease in the mean EDV and an increase in the RI in the nasal and temporal PCAs, reaching statistical significance in the nasal PCA only. This study suggests that TTT could lead to alterations in choroidal blood flow, as assessed by CDI. Further study is warranted.

Hyperthermic, supersaturated humidification in the treatment of xerostomia.

OBJECTIVES: To investigate the role of hyperthermic, supersaturated humidification in the treatment of radiation-induced xerostomia. STUDY DESIGN: A randomized, controlled, crossover pilot study of patients with symptomatic xerostomia following radiotherapy for head and neck cancer. Patients compared a standard bedside humidifier with a new device delivering hyperthermic, supersaturated humidification through a nasal cannula. METHODS: The patients were randomized to a 2-week course of standard, cool air bedside humidification or to hyperthermic, supersaturated humidification through a nasal cannula (Vapotherm Inc., Annapolis, MD). After a 1-week washout period, patients were crossed over to the opposite device for another 2 weeks. The patients underwent physical examination initially and after each trial period using an objective xerostomia scale, and then completed a questionnaire quantifying their symptoms. Patients additionally rated their symptoms at home, twice daily, using a visual analogue scale. RESULTS: Using the Oral Assessment Guide, lip scores went from 1.67 initially to 1.67 after control and 1.67 after Vapotherm. Tongue scores were 1.67, 1.5, and 1.83, respectively. Saliva scores were 1.67, 1.5, and 1.5, respectively. Mucous membranes scores were 1.5, 1.67, and 1.5, respectively. The physical examination scores at these four sites were not significantly different between control and Vapotherm (P =.78,.78,.72, and.37, respectively). The patient symptom questionnaire and visual analogue scores also revealed no significant difference between the two devices. CONCLUSION: The Vapotherm MT-3000 device appears to provide minimal or no additional relief from radiation xerostomia over standard bedside humidifiers. Further investigation may be warranted with newer models of the device and with disorders of mucociliary clearance.

Anti-angiogenic therapy of proliferative diabetic retinopathy.

Proliferative diabetic retinopathy (PDR) remains one of the major causes of acquired blindness in developed nations. This is true despite the development of laser treatment, which can prevent blindness in the majority of those who develop this complication. The hallmark of PDR is neovascularisation (NV), abnormal angiogenesis that may ultimately cause severe vitreous cavity bleeding and/or retinal detachment. Pharmacologic therapy aimed at preventing NV, as an adjunct to laser treatment, or as an alternative to laser treatment, would be a welcome addition to the armamentarium. PDR could be prevented by improved metabolic control or by pharmacologically blunting the biochemical consequences of hyperglycaemia (e.g., with aldose reductase inhibitors, inhibitors of non-enzymatic glycation or by protein kinase C (PKC) inhibition). The angiogenesis in PDR could be treated via growth factor (e.g., vascular endothelial growth factor (VEGF), insulin like growth factor-1) blockade, integrin (e.g., alpha-v beta-3) blockade or extracellular matrix alteration (e.g., with steroid compounds), or interference with intracellular signal transduction pathways (e.g., PKC and mitogen activated protein kinase pathway proteins). Numerous potentially useful anti-angiogenic compounds are in development, but two drugs are presently in clinical trials for the treatment of the preproliferative stage of PDR.

Intravitreal triamcinolone acetonide inhibits choroidal neovascularization in a laser-treated rat model.

OBJECTIVE: To determine if intravitreal triamcinolone acetonide (TAAC) inhibits experimental choroidal neovascular membranes induced by laser trauma in a rat model. METHODS: Nineteen anesthetized male Brown Norway rats received a series of 8 krypton red laser lesions per eye (647 nm, 0.05 seconds, 50 microm, and 150 mW in 17 rats, and 200 mW in 2 rats). One eye received an intravitreal injection of triamcinolone acetonide (20 microL, 0.8 mg) and the other eye received an injection of isotonic sodium chloride solution. Fundus and fluorescein angiography examinations occurred just before euthanasia and tissue processing for histopathology on day(s) 0, 1, 3, 7, 14, 21, 28, and 35. RESULTS: From the control eyes that underwent photocoagulation at 150 mW, 57 discrete lesions with definitive fibrovascular proliferations were observed at 21, 28, and 35 days, arising from a total of 72 spots placed (79% yield). From the control eyes that underwent photocoagulation at 200 mW, 11 discrete lesions with definitive fibrovascular proliferations were observed at 28 days, arising from a total of 16 spots placed (69% yield). In the TAAC-treated group, no fibrovascular proliferations were observed in the 72 lesions and in the 16 lesions created with 150 mW and 200 mW, respectively. CONCLUSION: Intravitreal TAAC is a potent inhibitor of fibrovascular proliferations in a rat model of choroidal neovascular membranes induced by laser trauma. CLINICAL RELEVANCE: This study corroborates previous investigations that propose TAAC as a potential treatment for choroidal neovascular membranes in humans.

Changing therapeutic paradigms for exudative age-related macular degeneration: antiangiogenic agents and photodynamic therapy.

Age related macular degeneration (AMD) is the leading cause of irreversible visual loss in the United States. Overall, approximately 10 - 20% of patients with AMD exhibit the exudative form, which is responsible for most of the estimated 1.2 m cases of severe visual loss from AMD. Visual loss develops in the exudative form of AMD due to abnormal choroidal neovascular membranes (CNVM) that develop under the retina, leak serous fluid and blood, and ultimately cause a blinding disciform scar in, and under, the retina. Currently, the only well-studied and widely accepted method of treatment is laser photocoagulation of the CNVM. However, only a minority of patients with exudative AMD show well-demarcated 'classic' CNVM amenable to laser treatment, and at least half of these patients suffer persistent or recurrent CNVM formation within two years. In addition, since the treatment itself causes a blinding central scotoma when the CNVM is located subfoveally, many clinicians do not treat subfoveal CNVM. With these treatment limitations, there has been a great deal of interest in alternative therapies for AMD, including anti-angiogenic agents and photodynamic therapy. Angiogenesis involves a complex interplay of cellular events involving a cascade of factors that are both inhibitory and stimulatory. Soluble growth factors have been the best-known cell modulating agents in ophthalmology, but there are a multitude of potential sites for inhibition of angiogenesis by pharmacological agents. With regard to photodynamic therapy, a photosensitising dye is injected intravascularly and low power laser light is used to activate the dye within the CNVM to cause vascular occlusion by a photochemical reaction. Closure of the CNVM is achieved without severe collateral damage to the non-vascular tissues as occurs with laser photocoagulation.

Displaced starburst amacrine cells of the rabbit retina contain the 67-kDa isoform, but not the 65-kDa isoform, of glutamate decarboxylase.

The cholinergic identity of retinal starburst amacrine neurons is well established, but recent evidence suggests that these cells are GABAergic as well. Confirmation of this dual transmitter function requires the demonstration of glutamate decarboxylase (GAD), the biosynthetic enzyme for GABA, within starburst cells. The current work was undertaken to determine whether rabbit retinal starburst amacrine neurons contain either of the two known isoforms of GAD. To do this, we have examined the localization of the following: (1) the 65-kDa isoform of GAD; (2) the 67-kDa isoform of GAD; (3) choline acetyltransferase; and (4) the fluorescent dye DAPI, a marker for cholinergic amacrine cells. In addition, we labeled displaced starburst neurons directly, by injecting them with Lucifer Yellow in vitro. Four strata within the inner plexiform layer contained immunoreactive GAD65. A non-GAD65-immunoreactive zone separated the two innermost strata (G3 and G4); this zone contained (1) the dendrites of individual Lucifer Yellow-injected, displaced starburst amacrine cells; (2) dendrites immunoreactive for choline acetyltransferase; and (3) processes of DAPI-labeled amacrine cells. Immunoreactive GAD67 appeared in the same strata that contained GAD65, and in at least two additional strata, one of which lay at precisely the same depth as the proximal cholinergic stratum. In addition, the somas of displaced starburst cells were strongly immunoreactive for GAD67, but not for GAD65. These results demonstrate (1) that displaced starburst amacrine cells contain the 67-kDa isoform of GAD, but not the 65-kDa isoform; and (2) that the dendrites of starburst (67-kDa GAD) amacrines, and the dendrites of 65-kDa-GAD-containing amacrines, occupy different strata within the inner plexiform layer. Thus, displaced starburst cells do contain GAD, and can, presumably, manufacture GABA. The reasons for their preferential use of the 67-kDa GAD isoform remain to be elucidated.

Acetylcholinesterase and choline acetyltransferase localization patterns do correspond in cat and rat retinas.

Is acetylcholinesterase (AChE) a reliable marker for cholinergic activity in the cat and rat retinas? To evaluate this question, radial sections, labeled for AChE, have been compared to sections labeled for choline acetyltransferase (ChAT). Within the inner plexiform layer (IPL) of each species, two lightly-stained AChE bands are revealed which correspond to the depths of ChAT immunoreactivity. Although retinal AChE is not limited exclusively to sites where ChAT is present, AChE and ChAT activity do occur in the same IPL sublaminae. Used with proper caution, AChE is a reliable secondary indicator of cholinergic activity.

Cholinergic and GABAergic neurons occur in both the distal and proximal turtle retina.

Turtle retinas were processed immunocytochemically and histochemically to detect the presence of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and glutamate decarboxylase (GAD). We observed cholinergic and gamma-aminobutyric acid (GABA)ergic neurons in the proximal retina, as expected, and in the distal retina as well. ChAT immunoreactivity in the distal retina was observed within the axons and pedicles of numerous cone photoreceptors, suggesting that a population of turtle cone photoreceptors uses ACh as a neurotransmitter. Type L2 horizontal cells were immunoreactive for GAD, and their dendrites invaginated into cone pedicles. AChE histochemistry revealed processes within the outer plexiform layer which formed a loosely organized lattice. In the proximal retina, labeling for ChAT and GAD was similar to that reported by previous investigators. Processes from ChAT-labeled amacrine cells in the inner nuclear layer formed a stratum within the distal inner plexiform layer (IPL) (at 16-21% relative IPL depth), and processes from ChAT-labeled amacrines in the ganglion cell layer formed a proximal ChAT stratum (at 55-58% relative IPL depth). In addition, six AChE-labeled bands and five GAD-labeled bands were observed within the IPL of stained retinas. Therefore, we determined that the two broadest AChE-labeled bands and the two broadest GAD-labeled bands overlapped the two labeled ChAT strata. The evidence for cholinergic and GABAergic processes in both the inner plexiform layer and the outer plexiform layer, combined with electrophysiological evidence from other investigators, raises the possibility that distal retinal neurons may be involved in the encoding of directional information.

Antiserum to lucifer yellow: preparation, characterization, and use for immunocytochemical localization of dye-filled retinal neurons.

We present a new method for the preparation of antisera to Lucifer Yellow, and these antisera are here shown to be particularly suitable for immunocytochemical localization of multiple dye-injected cells in large pieces of vertebrate retina. The method involves the preparation of covalent conjugates of the VS isomer of Lucifer Yellow with keyhole limpet hemocyanin (KLH) or rabbit serum albumin (RSA), and their use as immunogens in rabbits. Both carrier protein conjugates yielded robust antibody responses. Antiserum to the KLH-LY conjugate contained precipitating antibodies against LY and KLH, although activity to the latter did not interfere with immunocytochemical staining. Rabbit antiserum to the RSA-LY conjugate contained precipitating antibody only against LY. When used for immunocytochemical staining of large retinal pieces containing many LY-filled cells, both antisera yielded well-stained, darkly filled cells similar to those seen with the Golgi technique; even very fine dendritic processes of retinal ganglion cells could be followed for long distances. LY immunocytochemistry provides a useful alternative to photooxidation for the analysis of multiple dye injected cells, especially in whole mounts. This approach may also be useful for immunocytochemical identification of cells filled with LY after tissue fixation.

Myopia development in nonhuman primates--a literature review.

This paper is a review of and commentary on the available literature dealing with myopia production in various types of monkeys and in tree shrews by experimental manipulations of their physical, visual, and/or bodily environments. The results from these studies are compared with hypotheses and models which purport to explain myopia development. Possible relations are also noted between conditions, created experimentally in myopic eyes of primates, and similar pathological states, clinically observed in certain human myopes.

Laminar separation of light-evoked K+ flux and field potentials in frog retina.

Light-evoked changes in [K+] 0 and field potentials were recorded from the retinas of grass frogs. In the proximal retina, light induced an increase in [K+]0. This increase had components at light onset and offset, was maximal with small spot stimulation, and reached greatest amplitude at the same depth as the proximal negative response (PNR). Extracellular dye marking revealed that this depth was within the inner plexiform layer. The off-components of both the K+ increase and PNR occurred distal to the on-components, thus supporting recent proposal that "off" synapases lie distal to "on" synapses. Since a well-developed M-wave, having a time course nearly identical to the K+ increase, was also seen in the proximal retina, this field potential appears to be a normal component of the intraretinal electroretinogram.