Takeo Fukuchi

Experimental glaucoma model in the rat induced by laser trabecular photocoagulation after an intracameral injection of India ink.

A simple and reproducible rodent glaucoma model is required to elucidate the pathophysiology of damage to the optic nerve. We developed chronically elevated intraocular pressure (IOP) unilaterally in rats by injecting india ink into the anterior chamber of one eye using a 30-gauge needle. One week later, trapped carbon particles in the chamber angle formed a black band along the corneal limbus in the injected eyes. We performed direct laser photocoagulation without a gonio lens, and selectively burned the trabecular meshwork. Intraocular pressure was measured every week and laser photocoagulation was repeated until mean IOP in the experimental eyes rose above 25 mmHg. Unilateral IOP elevation was attained in all rats within 4 weeks. Twelve weeks after ink injection, we sacrificed the rats and excised the eyes for histologic analysis. The anterior chamber angle showed peripheral anterior synechia caused by laser photocoagulation, and carbon particles were engulfed by macrophages that infiltrated the ciliary cleft. In the optic nerve head, a remarkable decrease in the nerve fiber layer and cavernous degeneration were observed, suggesting glaucomatous optic nerve damage. This experimental rodent model should facilitate the study of the complex mechanisms involved in glaucoma.

Tumor Necrosis Factor-α Concentrations in the Aqueous Humor of Patients with Glaucoma

PURPOSE To investigate the concentration of tumor necrosis factor (TNF)-alpha in the aqueous humor of patients with glaucoma, including correlations with glaucoma subtypes and intraocular pressure. METHODS The study population comprised 84 patients with open-angle glaucoma who were scheduled for filtration or cataract surgery. Glaucoma subgroups included 29 cases of primary open-angle glaucoma (POAG), 28 cases of normal-tension glaucoma (NTG), and 27 cases of exfoliation glaucoma (ExG). Seventy-nine patients with senile cataract were recruited as control subjects. The concentrations of TNF-alpha in the aqueous humor were measured with an enzyme-linked immunosorbent assay. The percentages of samples positive for TNF-alpha and the measured concentrations in the glaucoma and cataract groups were compared. In addition, the relationships with the glaucoma subtypes, intraocular pressure, and glaucoma severity were analyzed. RESULTS A significantly higher percentage of subjects in the glaucoma group were positive for TNF-alpha compared with the cataract group (P = 0.011). The mean TNF-alpha concentrations among the positive cases were not different between the groups (P = 0.689). TNF-alpha-positive samples were higher in the POAG (13.7%) and NTG (10.7%) subgroups than in the cataract group without significance, but higher in ExG subgroup (29.6%) with significance (P = 0.001). Relationships between the TNF-alpha concentration and the intraocular pressure or the clinical stage of glaucoma were not observed. CONCLUSIONS TNF-alpha levels were significantly higher in the glaucoma group than in the cataract group, with a particularly large difference observed in those with ExG. The results suggest that TNF-alpha plays a key role in the progression of glaucoma.

Ultrastructural Localization of Myocilin in Human Trabecular Meshwork Cells and Tissues

We examined ultrastructurally the localization of myocilin (formerly called trabecular meshwork inducible glucocorticoid response, or TIGR) protein in cultured human trabecular meshwork (TM) cells and in normal human TM tissues. The TM, a specialized tissue located at the chamber angle of the eye, is believed to be responsible for the development of glaucoma. The myocilin gene has been directly linked to both juvenile and primary open-angle glaucomas, and multiple mutations have been identified. Human TM cells were treated with 0.1 mM of dexamethasone (DEX) to induce myocilin expression. This protein was immunolocalized by colloidal gold electron microscopy using an anti-human myocilin polyclonal antibody. Double labeling with different sizes of gold particles was also performed with additional monoclonal antibodies specific for cell organelles and structures. In both DEX-treated and untreated cultured cells, myocilin was associated with mitochondria, cytoplasmic filaments, and vesicles. In TM tissues, myocilin was localized to mitochondria and cytoplasmic filaments of TM cells, elastic-like fibers in trabecular beams, and extracellular matrices in the juxtacanalicular region. These results indicate that myocilin is localized both intracellularly and extracellularly at multiple sites. This protein may exert diverse biological functions at different sites.

Müller Cells as a source of brain-derived neurotrophic factor in the retina: noradrenaline upregulates brain-derived neurotrophic factor levels in cultured rat Müller cells.

Müller cells, the predominant glial cells in the retina, are thought to play important roles in the survival of retinal neurons. Previous studies have demonstrated that Müller cells express brain-derived neurotrophic factor (BDNF), which has a pronounced neurotrophic effect on retinal ganglion cells. In this study, we investigated whether Müller cells express and release BDNF in culture. Reverse transcriptase-PCR, immunocytochemistry and Western blotting revealed that Müller cells produce BDNF mRNA and protein. Using the enzyme-linked immunosorbant assay, BDNF protein levels in Müller cells and their conditioned medium were quantified, demonstrating that Müller cells produce and release high levels of BDNF. Noradrenaline administration caused an upregulation of BDNF mRNA and protein expression by cultured Müller cells. These results suggest that Müller cells may act as an endogenous source of BDNF in the retina. Furthermore, induction of BDNF expression by adrenergic agonists may provide a therapeutic approach to retinal neurodegenerative disorders.

Extracellular matrix changes of the optic nerve lamina cribrosa in monkey eyes with experimentally chronic glaucoma

Using light microscopic immunohistochemistry, we studied the immunolocalization and immunoreactivity of the extracellular matrix, including collagen types III, IV, VI, laminin, and alpha elastin in the lamina cribrosa of monkey eyes with normal and experimentally chronic glaucoma. Our results showed: (1) abnormal linearlike immunodeposits of both collagen type IV and laminin in the margin of the lamina cribrosa with significant density in the glaucomatous eyes; (2) the immunoreactivity of collagen type III resembled that of the normal eye, but was slightly stronger at the laminar surface; (3) findings with collagen type VI resembled those of type III with an enhanced linearlike staining surrounding the nerve-fiber bundles. Furthermore, staining of alpha elastin demonstrated dramatic changes in both reactivity and localization.The lamina cribrosa of glaucomatous eyes showed a markedly reduced immunoreactivity as well as an irregular, interrupted pattern. These observations suggest that the changes might be a secondary to the long-standing elevation of intraocular pressure. The alteration of these macromolecules may modify the course of glaucomatous optic damage.

Immunolocalization of heat shock proteins in the retina of normal monkey eyes and monkey eyes with laser-induced glaucoma.

PURPOSE To examine the expression and localization of heat shock proteins (HSPs) in the retinas of normal and experimentally induced primate glaucoma eyes. These proteins are known to be produced in response to a variety of stresses. METHODS Experimental glaucoma was induced in the right eyes of three adult monkeys by repeated applications of argon laser to the chamber angle. Immunostaining with a panel of antibodies against HSP 90, 70, 60, 47, and 27 was performed on retinal sections prepared from the normal and glaucomatous monkey eyes. RESULTS The intensity of immunostaining for HSP 90, 60, and 27 was greatly enhanced in the retinas of glaucomatous eyes. Prominent reactivity was observed in the inner retinal layers, especially in the ganglion cell and nerve fiber layers. The staining intensity for HSP 70 was also moderately increased, while immunoreactivity against HSP 47 remained almost unchanged in glaucomatous retinas. Immunostaining against glial fibrillary acidic protein was increased and the immunolabeling pattern appeared to be identical with that of HSP 90 in glaucoma retinas. CONCLUSIONS The level of HSP 90, 70, 60, and 27 in primate retinas was increased in experimentally induced ocular hypertension. The differences in expression pattern suggest that each HSP may have its unique role in responding to damage or injury related to intraocular pressure elevation.

Establishment of a novel enzyme-linked immunosorbant assay for Thy-1 : Quantitative assessment of neuronal degeneration

In the central nervous system (CNS), Thy-1 is expressed predominantly on neurons and serves as a specific marker for neurons. In the present study, we established a two-site enzyme-linked immunosorbent assay (ELISA) that detects trace amounts of Thy-1 protein. Recombinant Thy-1 protein expressed in Escherichia coli was purified and used as a standard. Of the regions of the nervous system examined, the highest Thy-1 concentration was found in the striatum followed by the hippocampus, neocortex, cerebellum, spinal cord, retina and optic nerve. We found that injection of a neurotoxin, N-methyl-D-aspartate, into the vitreous cavity reduced the Thy-1 level in the retina. Thy-1 ELISA will be useful for quantitative assessment of neurodegeneration in the CNS.

Corneal endothelial damage after trabeculectomy with mitomycin C in two patients with glaucoma with cornea guttata

Purpose. To report two patients with glaucoma who exhibited severe damage to the corneal endothelium after a trabeculectomy with mitomycin C (MMC). Methods. This study includes clinical histories and specular microscopic pictures of the cases. Results. Both patients were middle-aged women, underwent trabeculectomy with MMC, had moderate to severe cornea guttata preoperatively, and developed a shallow to flat anterior chamber, classified as grade 2 according to Spaeth early in the postoperative period. Stromal opacity caused by corneal edema associated with severe Descemets membrane folds appeared within 2 to 5 days in both cases. The density of the corneal endothelium was decreased on specular microscopic examination. The severe corneal endothelial damage seen after the trabeculectomy with MMC was likely owing to a combination of the preexisting cornea guttata, the flat anterior chamber, and possibly the administration of MMC. Conclusion. Severe endothelial damage after trabeculectomy with MMC may occur in patients with glaucoma and associated cornea guttata. The use of tight sutures on the scleral flap or a modified operative method, nonpenetrating trabeculectomy, may be effective in preventing a shallow to flat anterior chamber postoperatively.

Topically administered timolol and dorzolamide reduce intraocular pressure and protect retinal ganglion cells in a rat experimental glaucoma model

Aims: This study sought to elucidate the effects of timolol and dorzolamide on intraocular pressure (IOP) and retinal ganglion cell (RGC) death in an experimental model of glaucoma in rat. Methods: Mild elevation of IOP was induced in rats by intracameral injection of India ink and subsequent laser trabecular photocoagulation. IOP was measured before the surgical procedures and weekly thereafter. Timolol (0.5%), timolol XE (0.5%), dorzolamide (1%), and artificial tears (vehicle) were topically applied daily. Retinal sections were prepared for histology to determine RGC number. Results: Timolol, timolol XE, and dorzolamide induced a significant reduction in IOP (p<0.05) and counteracted the reduction in RGC number that occurred in vehicle treated glaucomatous eyes (p<0.05). The coefficient of correlation between RGC number and IOP was significant in the dorzolamide treated group (r = −0.908, p<0.005), but not in other groups (p>0.05). Conclusions: Both timolol formulation and dorzolamide reduced IOP and protected RGCs in a rat model of experimental glaucoma. It cannot be ruled out that timolol might protect RGCs by additional mechanisms other than simply lowering of IOP.

Clinicopathological features in anterior visual pathway in neuromyelitis optica.

Neuromyelitis optica spectrum disorder (NMOsd) is an autoimmune disorder of the central nervous system characterized by aquaporin‐4 (AQP4) autoantibodies. The aim of this study was to elucidate the characteristics of involvement of the anterior visual pathway (AVP) and neurodegeneration via glia–neuron interaction in NMOsd.