Norman Michaud

Tumor Necrosis Factor-α Mediates Oligodendrocyte Death and Delayed Retinal Ganglion Cell Loss in a Mouse Model of Glaucoma

Glaucoma is a widespread ocular disease characterized by a progressive loss of retinal ganglion cells (RGCs). Previous studies suggest that the cytokine tumor necrosis factor-α (TNF-α) may contribute to the disease process, although its role in vivo and its mechanism of action are unclear. To investigate pathophysiological mechanisms in glaucoma, we induced ocular hypertension (OH) in mice by angle closure via laser irradiation. This treatment resulted in a rapid upregulation of TNF-α, followed sequentially by microglial activation, loss of optic nerve oligodendrocytes, and delayed loss of RGCs. Intravitreal TNF-α injections in normal mice mimicked these effects. Conversely, an anti-TNF-α-neutralizing antibody or deleting the genes encoding TNF-α or its receptor, TNFR2, blocked the deleterious effects of OH. Deleting the CD11b/CD18 gene prevented microglial activation and also blocked the pathophysiological effects of OH. Thus TNF-α provides an essential, although indirect, link between OH and RGC loss in vivo. Blocking TNF-α signaling or inflammation, therefore, may be helpful in treating glaucoma.

Photodynamic Therapy of Experimental Choroidal Melanoma Using Lipoprotein-delivered Benzoporphyrin

BACKGROUND Benzoporphyrin derivative monoacid (BPD) is a new photosensitizer currently undergoing clinical trial for cutaneous malignancies. Compared with the clinically most frequently used sensitizer, Photofrin, BPD may offer higher tumor phototoxicity, better tissue penetration, and absence of significant skin sensitization. Low-density lipoprotein (LDL) carriers heighten efficiency and selectivity of BPD because neovascular and tumor cells express an increased number of LDL receptors. Hence, in addition to the vaso-occlusive effects similar to most other photosensitizers, LDL-BPD also has been shown to cause direct tumor cell damage. METHODS Benzoporphyrin derivative monoacid was complexed with human LDL and used in photodynamic treatment of choroidal melanomas experimentally induced in eight albino rabbits. Five rabbits served as controls. Three hours after intravenous injection of 2 mg/kg body weight of LDL-BPD, eight tumors were irradiated at 692 nm and 100 J/cm2 via an argon-pumped dye laser coupled into a slit lamp. RESULTS Angiography and histologic findings showed immediate photothrombosis after disintegration of endothelial membranes. After complete necrosis of tumor cells within 24 hours, a small fibrotic scar slowly developed. No tumor regrowth was noted up to 6 weeks when animals were killed. CONCLUSION These data suggest that photodynamic treatment with LDL-BPD may be a promising modality for multiple clinical applications, including tumors and neovascularizations II.

Verteporfin Photodynamic Therapy Retreatment of Normal Retina and Choroid in the Cynomolgus Monkey

OBJECTIVE This study evaluated the effect of repeated photodynamic therapy (PDT) applications on normal primate retina and choroid using an intravenous infusion of liposomal benzoporphyrin derivative (verteporfin). DESIGN This was an experimental study in a primate model. ANIMALS/CONTROLS: Six cynomolgus monkeys were used as experimental subjects and one monkey was used as a control subject. INTERVENTION Three consecutive PDT treatments at 2-week intervals were applied over the center of the fovea or the optic nerve of each eye. Verteporfin was delivered by intravenous infusion at a dose of 6 mg/m2, 12 mg/m2, or 18 mg/m2. Laser irradiation was then applied using a diode laser (689 nm) with light doses and spot sizes kept constant. MAIN OUTCOME MEASURES Findings were documented by fundus photography, fluorescein angiography, and light and electron microscopy. RESULTS A cumulative dose response was seen angiographically and histologically with more severe damage to the retina and choroid noted at higher dye doses. Photodynamic therapy applied to the macula using the 6-mg/m2 verteporfin dose showed recovery of choriocapillaris, with mild retinal pigment epithelium and outer photoreceptor damage at 6 weeks. At this dose, the optic nerve showed few focal sites of axon atrophy and capillary loss. Treatments over the macula using the 12-mg/m2 and 18-mg/m2 doses led to chronic absence of choriocapillaris and photoreceptors at 6 weeks. One of two optic nerves became atrophic after PDT applications using dye doses of 12 mg/m2, and both optic nerves became atrophic in the 18-mg/m2 dye dose group. CONCLUSION Limited damage to the retina, choroid, and optic nerve was present in primates treated with multiple PDT sessions using 6 mg/m2 verteporfin with light doses and the timing of irradiation kept constant. However, PDT using higher dye doses of 12 mg/m2 and 18 mg/m2 led to significant chronic damage to the normal retina, choroid, and optic nerve.

Neurologic, Gastric, and Opthalmologic Pathologies in a Murine Model of Mucolipidosis Type IV

Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder caused by mutations in the MCOLN1 gene, which encodes the 65-kDa protein mucolipin-1. The most common clinical features of patients with MLIV include severe mental retardation, delayed motor milestones, ophthalmologic abnormalities, constitutive achlorhydria, and elevated plasma gastrin levels. Here, we describe the first murine model for MLIV, which accurately replicates the phenotype of patients with MLIV. The Mcoln1(-/-) mice present with numerous dense inclusion bodies in all cell types in brain and particularly in neurons, elevated plasma gastrin, vacuolization in parietal cells, and retinal degeneration. Neurobehavioral assessments, including analysis of gait and clasping, confirm the presence of a neurological defect. Gait deficits progress to complete hind-limb paralysis and death at age ~8 mo. The Mcoln1(-/-) mice are born in Mendelian ratios, and both male and female Mcoln1(-/-) mice are fertile and can breed to produce progeny. The creation of the first murine model for human MLIV provides an excellent system for elucidating disease pathogenesis. In addition, this model provides an invaluable resource for testing treatment strategies and potential therapies aimed at preventing or ameliorating the abnormal lysosomal storage in this devastating neurological disorder.

Photodynamic therapy inhibition of experimental intimal hyperplasia: Acute and chronic effects

PURPOSE Intimal hyperplasia (IH) is a focal arterial problem that still eludes successful therapy. We have previously demonstrated the feasibility of use of photodynamic therapy (PDT) for the acute treatment of experimental IH with light to activate an otherwise biologically inert photosensitizer. The purpose of this study was to determine the acute and long-term effects of PDT inhibition of IH on the artery wall. METHODS Segmental IH was induced by balloon injury localized to the cervical common carotid artery of 33 rats. The photosensitizer chloroaluminum sulfonated phthalocyanine (5 mg/kg) for the experimental group or saline solution for the control group was administered intravenously. Twenty-four hours later, all instrumented portions of arteries were irradiated at 675 nm to induce cytotoxic injury in the PDT-treated arteries as compared with laser only-treated arteries for controls. Animals were killed at 1, 2, 4, and 16 weeks. RESULTS There were no untoward side effects in either group. All PDT-treated arteries were devoid of smooth muscle or inflammatory cells in the treated media. There was no evidence of arterial degeneration of PDT-treated arteries. Only three arteries in the PDT group developed IH, whereas it was universal in all controls. In control arteries, immunocytochemistry with bromodeoxyuridine revealed maximal intimal and medial cell proliferation at 1 week, and morphometric analysis demonstrated a maximal IH at 2 weeks. Immunocytochemistry staining for smooth muscle cell actin was positive for the IH in control and when present in PDT-treated arteries, whereas the adventitia of PDT-treated arteries were positive after 2 weeks. Electron microscopy demonstrated early myofibroblast migration to the adventitia, and at 16 weeks occasional myofibroblasts were noted in the media of PDT-treated arteries. There was complete reendothelial cell covering of the intima by 4 weeks. CONCLUSIONS These in vivo data demonstrate that PDT is an effective local method for the treatment of experimental IH. There is no evidence of significant recurrence of IH or arterial degeneration. Further studies with PDT may provide novel approaches to the understanding and treatment of arterial IH.

Transducin γ-subunit sets expression levels of α- and β-subunits and is crucial for rod viability

Transducin is a prototypic heterotrimeric G-protein mediating visual signaling in vertebrate photoreceptor cells. Despite its central role in phototransduction, little is known about the mechanisms that regulate its expression and maintain approximately stoichiometric levels of the α- and βγ-subunits. Here we demonstrate that the knock-out of transducin γ-subunit leads to a major downregulation of both α- and β-subunit proteins, despite nearly normal levels of the corresponding transcripts, and fairly rapid photoreceptor degeneration. Significant fractions of the remaining α- and β-subunits were mislocalized from the light-sensitive outer segment compartment of the rod. Yet, the tiny amount of the α-subunit present in the outer segments of knock-out rods was sufficient to support light signaling, although with a markedly reduced sensitivity. These data indicate that the γ-subunit controls the expression level of the entire transducin heterotrimer and that heterotrimer formation is essential for normal transducin localization. They further suggest that the production of transducin β-subunit without its constitutive γ-subunit partner sufficiently stresses the cellular biosynthetic and/or chaperone machinery to induce cell death.

HIV protease inhibitors provide neuroprotection through inhibition of mitochondrial apoptosis in mice.

Neuroprotection can be achieved by preventing apoptotic death of postmitotic cells. Apoptotic death can occur by either a caspase-dependent mechanism, involving cytochrome c, apoptosis protease-activating factor-1 (Apaf-1), and caspase-9, or a caspase-independent mechanism, involving apoptosis-inducing factor (AIF). HIV protease inhibitors (PIs) avert apoptosis in part by preventing mitochondrial outer membrane permeabilization (MOMP), but the precise mechanism by which they work is not known. Here, we evaluated the impact of the PIs in a mouse model of retinal detachment (RD) in vivo and in murine primary retinal cell cultures in vitro. Oral administration of the PIs nelfinavir and ritonavir significantly inhibited photoreceptor apoptosis, while preventing the translocation of AIF from mitochondria to the nucleus as well as the activation of caspase-9. RD-induced photoreceptor apoptosis was similarly inhibited in mice carrying hypomorphic mutations of the genes encoding AIF or Apaf-1. Nelfinavir attenuated apoptosis as well as mitochondrial release of AIF and cytochrome c, and subsequent activation of caspase-9 in vitro, in photoreceptor cultures exposed to starvation or monocyte chemoattractant protein-1-stimulated (MCP-1-stimulated) macrophages. Our results suggest that the MOMP inhibition by PIs involved interruption of both caspase-dependent and caspase-independent apoptosis pathways and that PIs may be clinically useful for the treatment of diseases caused by excessive apoptosis.

Overexpression of Rhodopsin Alters the Structure and Photoresponse of Rod Photoreceptors

Rhodopsins are densely packed in rod outer-segment membranes to maximize photon absorption, but this arrangement interferes with transducin activation by restricting the mobility of both proteins. We attempted to explore this phenomenon in transgenic mice that overexpressed rhodopsin in their rods. Photon capture was improved, and, for a given number of photoisomerizations, bright-flash responses rose more gradually with a reduction in amplification--but not because rhodopsins were more tightly packed in the membrane. Instead, rods increased their outer-segment diameters, accommodating the extra rhodopsins without changing the rhodopsin packing density. Because the expression of other phototransduction proteins did not increase, transducin and its effector phosphodiesterase were distributed over a larger surface area. That feature, as well as an increase in cytosolic volume, was responsible for delaying the onset of the photoresponse and for attenuating its amplification.

Characterization of Retrokeratoprosthetic Membranes in the Boston Type 1 Keratoprosthesis

OBJECTIVE To evaluate retroprosthetic membranes that can occur in 25% to 65% of patients with the Boston type 1 keratoprosthesis (KPro). METHODS Two patients with Peter anomaly and 2 with neurotrophic scarred corneas underwent revisions of their type 1 KPros because of visually compromising retroprosthetic membranes. The excised membranes were studied by light microscopy with hematoxylin-eosin, periodic acid-Schiff, and toluidine blue stains. Immunohistochemical and transmission electron microscopic examination were also used. RESULTS Light microscopic examination revealed that the retro-KPro fibrous membranes originated from the hosts corneal stroma. These mildly to moderately vascularized membranes grew through gaps in the Descemet membrane to reach behind the KPro back plate and adhere to the anterior iris surface, which had undergone partial lysis. In 2 cases, the fibrous membranes merged at the pupil with matrical portions of metaplastic lens epithelium, forming a bilayered structure that crossed the optical axis. Retro-KPro membranes stained positively for α-smooth muscle actin but negatively for pancytokeratin. Electron microscopy confirmed the presence of actin filaments within myofibroblasts and small surviving clusters of metaplastic lens epithelial cells. CONCLUSIONS Stromal downgrowth, rather than epithelial downgrowth, was the major element of the retro-KPro membranes in this series. Metaplastic lens epithelium also contributed to opacification of the visual axis. Florid membranous inflammation was not a prominent finding and thus probably not a requisite stimulus for membrane development. Further advances in prosthetic design and newer antifibroproliferative agents may reduce membrane formation.

Liposomal Benzoporphyrin Derivative Verteporfin Photodynamic Therapy

PURPOSE The authors have previously shown that photodynamic therapy (PDT) using lipoprotein-delivered benzoporphyrin derivative mono-acid (BPD) effectively closed experimental choroidal neovascularization (CNV). In the current study, the authors used a clinical preparation, liposomal BPD verteporfin in the same model, with experiments designed to establish optimal dye and light doses, and the timing of laser light irradiation after dye injection, for effective and selective closure of CNV. METHODS Experimental CNV was induced in the maculae of cynomolgus monkeys. Liposomal BPD verteporfin was injected intravenously at doses of 1.0, 0.5, 0.375, and 0.25 mg/kg. Laser light at 692 nm then was applied to CNV, with an irradiance of 600 mW/cm2 and fluence of 150 J/cm2, at various times after dye injection, ranging from 5 to 120 minutes. Treatment effect was assessed by fundus photography and fluorescein angiography and confirmed by light and electron microscopy. The PDT of experimental CNV was studied to assess efficacy; PDT performance on normal eyes was studied to investigate selectivity. RESULTS The CNV closure was demonstrated by fluorescein angiography and histopathologic findings at all tested dye doses. A dye dose of 0.375 mg/kg, with laser light irradiation applied 20 to 50 minutes after dye injection, optimized CNV closure with minimal retinal and choroidal damage. No major local adverse effects were noted, and the drug was well tolerated systematically. CONCLUSIONS Liposomal BPD verteporfin is a potent photosensitizer, and PDT using this dye is a potentially effective and selective treatment for CNV.