Development of spontaneous optic neuropathy in NF-kappaBetap50-deficient mice: requirement for NF-kappaBetap50 in ganglion cell survival.

Although the transcription factor NF-kappaBeta is known to regulate cell death and survival, its precise role in cell death within the central nervous system remains unknown. The purpose of this study was to investigate the role of NF-kappaBetap50 in the age-related survival of retinal ganglion cells (RGCs). Eyes of mice with a deleted NF-kappaBetap50 gene and its wild-type mice at each of age were studied by histopathological studies. The number of RGCs was counted using retrograde labelling methods. Mice were subjected to intravitreous injection of N-methyl-D aspartate (NMDA) to induce RGC death. In p50-deficient mice, the number of RGCs significantly decreased with age in total independence of intraocular pressure measurement. Optic nerves of p50-deficient mice showed hypertrophy astrocytes and enlargement of the axons, together with a decreased number of axons. Immunohistochemistry showed a strong expression of glial fibrillary acidic protein. The histological results show obvious excavation of the optic nerve head in p50-deficient mice at 10 months of age. Intravitreal injection of NMDA in young p50-deficient mice damaged RGCs more intensively than in control animals. We further noticed that autoantibodies against RGCs were produced in p50-deficient mice. Our results show that p50 deficiency induced age-related RGC death, indicating a new insight into the role of p50 in the pathophysiology of neuropathy, and further experiments with p50-deficient mice may provide new targets for therapeutic intervention for human glaucoma.

Protein kinase C and the development of diabetic vascular complications.

Hyperglycemic control in diabetes is key to preventing the development and progression of vascular complications such as retinopathy, nephropathy and neuropathy. Increased activation of the diacylglycerol (DAG)-protein kinase C (PKC) signal transduction pathway has been identified in vascular tissues from diabetic animals, and in vascular cells exposed to elevated glucose. Vascular abnormalities associated with glucose-induced PKC activation leading to increased synthesis of DAG include altered vascular blood flow, extracellular matrix deposition, basement membrane thickening, increased permeability and neovascularization. Preferential activation of the PKCbeta isoform by elevated glucose is reported to occur in a variety of vascular tissues. This has lead to the development of LY333531, a PKCbeta isoform specific inhibitor, which has shown potential in animal models to be an orally effective and nontoxic therapy able to produce significant improvements in diabetic retinopathy, nephropathy, neuropathy and cardiac dysfunction. Additionally, the antioxidant vitamin E has been identified as an inhibitor of the DAG-PKC pathway, and shows promise in reducing vascular complications in animal models of diabetes. Given the overwhelming evidence indicating a role for PKC activation in contributing to the development of diabetic vascular complications, pharmacological therapies that can modulate this pathway, particularly with PKC isoform selectivity, show great promise for treatment of vascular complications, even in the presence of hyperglycemia.

Lipid peroxidation and peroxynitrite in retinal ischemia-reperfusion injury.

PURPOSE: To investigate whether lipid peroxides play a role in retinal cell death due to ischemia-reperfusion injury, whether recombinant human thioredoxin (rhTRX) treatment reduces production of lipid peroxides of the retina, and whether such treatment reduces the number of cells expressing c-Jun and cyclin D1. METHODS: Retinal ischemia was induced in rats by increasing the intraocular pressure to 110 mm Hg for 60 minutes. After reperfusion, immunohistochemical staining for lipid peroxide, peroxynitrite, c-Jun, and cyclin D1 and propidium iodide (PI) staining were performed on retinal sections from animals treated intravenously with and without rhTRX, a free radical scavenger. Quantitative analyses of PI-, c-Jun-, and cyclin D1-positive cells were performed after the ischemic insult. Concentration of lipid peroxides in the retina was determined by the thiobarbituric acid assay. RESULTS: Specific immunostaining for lipid peroxides was seen in the ganglion cell layer at 6 hours after reperfusion, in the inner nuclear layer at 12 hours, and in the outer nuclear layer at 48 hours. Time course studies for PI-positive cells in the three nuclear layers coincided with those of specific immunostaining for lipid peroxides. The specific immunostaining was weakened by pre- and posttreatment with 0.5 mg of rhTRX. The number of PI-, c-Jun-, and cyclin D1-positive cells and the concentration of lipid peroxides were significantly decreased by treatment with rhTRX compared with those of vehicle-treated control rats (P: < 0. 01). CONCLUSIONS: Lipid peroxides formed by free radicals may play a role in neuronal cell death in retinal ischemia-reperfusion injury.

BDNF diminishes caspase-2 but not c-Jun immunoreactivity of neurons in retinal ganglion cell layer after transient ischemia.

PURPOSE: Retinal ischemia-reperfusion injury induces apoptosis of retinal neurons. The purpose of this study was to examine the association of c-Jun, caspase-1, -2, and -3 immunoreactivities and neuronal apoptosis in the retinal ganglion cell layer (GCL) and to study the effects of intravitreal brain-derived neurotrophic factor (BDNF) on the expression of these gene products in a rat model of retinal ischemia-reperfusion injury. METHODS: After 60 minutes of ischemia, eyes were enucleated after 3, 6, 12, 24, and 168 hours of reperfusion. The numbers of c-Jun-, caspase-1-, caspase-2-, caspase-3, and TdT-dUTP terminal nick-end labeling (TUNEL)-positive cells in the GCL were counted. Recombinant human BDNF (5 microg) or vehicle was injected intravitreally immediately after reperfusion. At 6, 24, and 168 hours, the numbers of immunoreactive cells in BDNF- and vehicle-treated groups were compared. RESULTS: Expression of c-Jun and caspase-2 was found in dying cells in flat-mounted retinas. The numbers of caspase-1- and caspase-3-positive cells were fewer than c-Jun- or caspase-2-positive cells. Cell death in the retinal GCL was suppressed by an intravitreal injection of BDNF. The numbers of TUNEL- and caspase-2-positive cells were lower in the BDNF-treated group at 6 hours after reperfusion (P<0.01). The number of c-Jun-positive cells in the treated retinas was not altered by the treatment. CONCLUSIONS: Expression of c-Jun and caspase-2 is associated with neuronal cell apoptosis in the GCL. Suppression of caspase-2 expression may explain the neuroprotective effects of BDNF.

Apoptotic retinal neuronal death by ischemia-reperfusion is executed by two distinct caspase family proteases.

PURPOSE: To evaluate possible roles of caspase-1 and caspase-3 in retinal ischemia-reperfusion injury. METHODS: Retinal ischemia was induced in rats by increasing the intraocular pressure to 110 mm Hg for 60 minutes. Expression of caspase-1 and caspase-3 was studied at the mRNA and protein levels using immunohistochemical staining, western blot analysis, semiquantitative reverse transcription-polymerase chain reaction, and assay of the enzymatic activities. Apoptotic retinal neurons were detected by the TdT-dUTP terminal nick-end labeling (TUNEL) method. To study the roles of the caspases in retinal ischemia-reperfusion injury, an inhibitor of caspase-1, acetyl-tyrosyl-valyl-alanyl-aspart-1-al (Ac-YVAD-CHO; total dose, 10(-7) moles) and that of caspase-3, acetyl-aspartyl-glutamylvalyl-aspart-1-al (Ac-DEVD-CHO; total dose, 10(-7) moles) was injected intravitreally and the number of TUNEL-positive cells was compared with the number in sections not treated with the inhibitors. RESULTS: In the inner nuclear layer (INL), caspase-3-like immunoreactivity was predominantly detected, whereas caspase-1-like immunoreactivity was more predominant in the outer nuclear layer (ONL). Expression of caspase-1 and -3 was upregulated at the protein and gene levels 24 hours after reperfusion. Intravitreal injection of Ac-DEVD-CHO decreased the number of TUNEL-positive cells more significantly in the INL than in the ONL (P < 0.01) at 24 hours, whereas, intravitreal injection of Ac-YVAD-CHO was more effective in decreasing the number in the ONL (P < 0.05). CONCLUSIONS: These findings suggest a possibility that cell-type-specific activation of caspases takes place in retinal ischemia-reperfusion injury, and such caspase may induce retinal neuronal cell death.

Caspaselike proteases activated in apoptotic photoreceptors of Royal College of Surgeons rats.

PURPOSE: To study the role of caspase-like proteases, especially roles of more extensively characterized caspase-1 and caspase-2, in apoptotic photoreceptor cell degeneration in Royal College of Surgeons (RCS) rats. METHODS: Both RCS and Sprague-Dawley rats were used. Cryosections of the retinas at various postnatal times were immunostained with antibodies against caspase-1 (interleukin-1beta-converting enzyme, ICE) and caspase-2 (Nedd2/Ich-1). Double staining with TdT-dUTP terminal nick-end labeling (TUNEL), propidium iodide, and the antibodies was also performed. To evaluate the time course of protein expression, western blot analysis was carried out. The temporal profile of caspase-like protease activity was studied using a fluorogenic tetrapeptide substrate, acetyl-tyrosyl-valyl-alanyl-aspartic acid alpha (4-methyl-coumaryl-7-amide) (Ac-YVAD-MCA). Intravitreal injection of a caspase-1 inhibitor, acetyl-tyrosyl-valyl-alanyl-aspartic-aldehyde (Ac-YVAD-CHO), at postnatal days 21 (P21) and P26 was performed to see if this caused a decrease in apoptotic cell number at P28. RESULTS: TUNEL-positive photoreceptors of RCS rats stained strongly with antibodies against caspase-1 and caspase-2. Double staining studies revealed that caspase-1 and caspase-2 were coexpressed in apoptotic cells. Western blot analysis showed that active forms of caspase-1-like and caspase-2-like proteases were upregulated at P28, concurrent with the peak in TUNEL-positive cells. The enzymatic activity of caspase-1-like protease was elevated in RCS rat retinas at P28, and the inhibitor of caspase-1 transiently reduced the number of the apoptotic photoreceptors. CONCLUSIONS: Activation of caspase-like proteases plays an important role in photoreceptor apoptosis of RCS rats.

Progressive brown discoloration of silicone intraocular lenses after vitrectomy in a patient on amiodarone.

A patient who was treated with amiodarone for 3 years developed brown discoloration of the intraocular lenses in both eyes. Contrast sensitivity and blue perception were reduced in the right eye. After vitrectomy for a vitreoretinal traction syndrome in the left eye, the discoloration appeared to increase. The apparent progression may have been related to breakdown of the blood-aqueous barrier after vitrectomy. However, because the discoloration developed before surgery and was bilateral, long-term administration of amiodarone may also have played a role.

A possible role for p16INK4 in neuronal cell death after retinal ischemia-reperfusion injury.

PURPOSE: To study whether cell type-specific death occurs in retinal ischemia-reperfusion injury and the possible roles of p16INK4 in the determination of cell death. METHODS: Retinal ischemia-reperfusion injury was induced in rats by a ligation method. After 1 hour of ischemia and a time of reperfusion that varied, rat eyes were enucleated. Cell death in the retina was studied by the TdT-dUTP terminal nick-end labeling method and propidium iodide (PI) staining. Electron microscopic observation of the retina was also performed. Immunohistochemical studies using antibodies against syntaxin and calbindin were performed to detect amacrine cells and horizontal cells, respectively, and immunohistochemical studies using an antibody against p16INK4 were performed to study whether this cell cycle-related protein was expressed in dying cells. RESULTS: Most of the calbindin-positive horizontal cells in the outer aspect of the inner nuclear layer (INL) showed morphologic features of necrosis. In contrast, syntaxin-positive amacrine cells in the inner aspect of the INL showed features of apoptosis. Of 320 calbindin-positive horizontal cells, only 11 (3.4%) showed positive PI staining. Those calbindin-positive, horizontal cells were p16INK4 positive. In contrast, 746 of 910 (82.0%) syntaxin-positive amacrine cells showed condensed PI staining, and none were p16INK4 positive. CONCLUSIONS: Expression of p16INK4 may regulate the fate of retinal neurons in ischemia-reperfusion injury, and cell type-specific death thus occurs in the retina after such injury.

Protective effect of adult T-cell leukemia-derived factor on retinal ischemia-reperfusion injury in the rat.

PURPOSE: To evaluate the protective effects of recombinant adult T-cell leukemia- derived factor (ADF)-human thioredoxin against ischemia-reperfusion injury in the rat retina. METHODS: Retinal ischemia was induced in rats by increasing the intraocular pressure to 110 mm Hg for 60 minutes. Various doses of recombinant human ADF (rhADF) or vehicle were administered intravenously before ischemia induction and immediately after reperfusion. The degree of retinal damage was assessed by electroretinogram (ERG) recording, by measuring the inner retinal thickness, and by counting the number of TdT-dUTP terminal nick-end labeling (TUNEL)-positive cells in the inner nuclear layer. RESULTS: The amplitudes of the ERG b-wave and oscillatory potentials were increased significantly by treatment before ischemia and after reperfusion with 0.5 mg or 5 mg rhADF and by treatment after reperfusion with 1 mg rhADF, compared with those of vehicle-treated control rats (P < 0.01). On day 28 after reperfusion, the thickness of the inner retina of control rats and of rats treated before ischemia and after reperfusion with 0.5 mg rhADF were 46.1+/-6.4 microm and 78.5+/-8.9 microm, respectively (P < 0.01). The number of TUNEL-positive cells on days 1 and 2 after reperfusion was decreased significantly by treatments with 0.5 mg rhADF compared with the number of TUNEL-positive cells in control rats (P < 0.01). CONCLUSIONS: Electrophysiologic and histologic studies showed that ischemia for 60 minutes produces severe damage in vehicle-treated control rat retina, particularly in the inner retinal layer. Intravenous injection of rhADF protects the rat retina from ischemia-reperfusion injury.

Expression of cell cycle-related genes in dying cells in retinal ischemic injury.

PURPOSE: To investigate whether cell cycle-related genes play a role in neuronal cell death in retinal ischemia-reperfusion injury. METHODS: Retinal ischemia-reperfusion injury was induced in rats by a ligation method and also by increasing the intraocular pressure. After 1 hour-of ischemia, cell death in the retina was studied using the TdT-dUTP terminal nick-end labeling (TUNEL) method, propidium iodide (PI) staining, DNA ladder formation, and ultrastructural studies. Immunohistochemical studies using antibodies against cell cycle-related genes were conducted. Changes in expression of cyclin D1 mRNA were quantitated using competitive quantitative polymerase chain reaction. RESULTS: At 3 hours after reperfusion, cells in the ganglion cell layer were the first to die, followed by those in the inner nuclear layer (at 6 hours) and outer nuclear layer (at 9 hours). Ultrastructural studies revealed condensed nuclei and relatively preserved mitochondria; DNA ladder formation was also detected. Immunostaining was positive for the cell cycle-related gene products c-Jun, cyclin B1, and cyclin D1. The time course of TUNEL-positive cells and that of cells positive for c-Jun or cyclin D1 in the inner nuclear layer was similar. A double-labeling study, using PI or TUNEL, and immunohistochemical analysis revealed that dying cells expressed c-Jun and cyclin D1, whereas cyclin B1 expression was observed in Müller cells. Quantitation of cyclin D1 mRNA revealed an approximate 4-fold increase at 24 hours after reperfusion. CONCLUSIONS: Aberrant expression of cell cycle-related genes may play an important role in the cell death that accompanies retinal ischemia-reperfusion injury.

Diagnosis of intraocular lymphoma by polymerase chain reaction.

BACKGROUND: Cytopathological examinations have been used in the diagnosis of intraocular lymphoma. However, sometimes it is not easy to detect malignant cells in the biopsy specimens. We applied a method that identified monoclonal proliferation of B lymphocytes by using polymerase chain reaction (PCR) in the diagnosis of patients suspected to have intraocular B-cell lymphoma. METHODS: Three specimens of the diagnostic vitrectomy were studied by cytological examination and by PCR to amplify the complementary determining region (CDR3) of immunoglobulin heavy chain (IgH) gene. As a positive control, a biopsy specimen of an orbital lymphoma was examined; four vitrectomy specimens from patients with diabetic retinopathy, proliferative vitreo-retinopathy, acute retinal necrosis (ARN) and macular hole were negative controls. RESULTS: On cytologic examination, no malignant cells were found in three specimens: suspected intraocular lymphoma and one ARN. In contrast, a discrete band that reflected monoclonal proliferation of B lymphocytes was detected by PCR in specimens from two patients and the positive control. Vitrectomy specimens from the negative controls, including ARN, did not show a discrete band on PCR. CONCLUSIONS: Two cases of ocular malignant lymphoma were diagnosed by PCR identification of monoclonal proliferation of B lymphocytes. This method may be an additional diagnostic tool in the diagnosis of intraocular B-cell lymphoma.

CHARGE association with congenital glaucoma due to maldevelopment of the anterior chamber angle.

PURPOSE: We examined the clinicopathologic features of a male infant with the CHARGE association and bilateral congenital glaucoma. METHODS: Trabeculectomy specimens were obtained from the anterior chamber angle and examined by light and electron microscopy. RESULTS: Histopathologic examination of the trabeculectomy specimens showed immature development of the trabecular meshwork that was covered by the ciliary muscles. There were few intertrabecular spaces because the meshwork was almost completely filled with cells and extracellular substances. Deposition of a granular and/or homogeneous substance was observed in the subendothelial area of Schlemm's canal. CONCLUSIONS: Our patient exhibited features typical of the CHARGE association, but also had congenital glaucoma. We hypothesize that these clinical findings are all mediated by a neurocristopathic mechanism. Our findings suggest that the CHARGE association may predispose to anterior chamber angle maldevelopment, which can lead to congenital glaucoma.