Ryohei Koide

Cytotoxicity of ophthalmic solutions with and without preservatives to human corneal endothelial cells, epithelial cells and conjunctival epithelial cells

Purpose:  The cytotoxicity of a range of commercial ophthalmic solutions in the presence and absence of preservatives was assessed in human corneal endothelial cells (HCECs), corneal epithelia and conjunctival epithelia using in vitro techniques.

In vitro Testing of Antioxidants and Biochemical End-Points in Bovine Retinal Tissue

Lipid peroxidation in aliquots of bovine retina (without rod outer segments, ROS), purified ROS and retinal pigment epithelium (RPE) was initiated with 5 mM ferric iron and 80 mM ADP. After 30 min of oxidation at 37°C, the concentration of thiobarbituric-acid-reacting substances (TBARS) which approximates lipid hydroperoxide (LHP), increased in the ROS from 2.0 ± 3.6 to 90.2 ± 34.5 nmol malondialdehyde (MDA)/mg protein and in the RPE from 0.54 ± 0.2 to 51.5 ± 15.8 nmol MDA/mg protein. Sixteen lipid and aqueous antioxidants (AOX) from natural or synthetic sources, including five flavonoids, were evaluated for their ability to inhibit the oxidative reaction. Palm-oil-derived vitamin E showed significant protection in retina, ROS and RPE (64, 68 and 74%), respectively. Of the flavonoids tested, good protection in the retina was found at 10–5M for epigallocatechin gallate (50%) and at 50 ng/ml for pycnogenol (61%) and catechin (52%). When catechin and palm oil vitamin E, catechin and coenzyme Q10 or coenzyme Q10 and pycnogenol were combined, the individual effect was enhanced. TBARS as an indirect measure of LHP level and hemoglobin-methylene blue determination for direct LHP were used as alternative end-point determinations of lipid peroxidation. These measure different aspects of AOX reactions. The results demonstrate the usefulness of an in vitro model system that can rapidly and accurately determine the capacity of a single AOX against lipid peroxidation or be used to show synergistic efficacy.

Distribution and ultrastructural localization of a receptor for pituitary adenylate cyclase activating polypeptide and its mRNA in the rat retina.

Localization and gene expression of pituitary adenylate cyclase activating polypeptide receptor (PACAPR) in the rat retina were studied by immunocytochemistry and in situ hybridization, respectively. Antisera were raised against a synthetic peptide that corresponds to the carboxy-terminal cytoplasmic domain which is found in all subtypes of PACAPR. Strong PACAPR mRNA expression and PACAPR-like immunoreactivity (PACAPR-LI) were detected in ganglion cells, amacrine cells, and in the inner plexiform layer. PACAPR-LI appeared to be concentrated predominantly in the neuronal perikarya and processes. At the ultrastructural level, strong immunostaining for PACAPR was visible in the plasma membranes, rough endoplasmic reticulum and cytoplasmic matrix in neurons. This study provides the basis for a better understanding of the functions of PACAP in the rat retina.

Electron microscopic observation of pituitary adenylate cyclase-activating polypeptide (PACAP)-containing neurons in the rat retina☆☆

The distribution and localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the rat retina were studied by immunocytochemistry with both light and electron microscopy. PACAP-like immunoreactivity (PACAP-LI) was detected in the amacrine and horizontal cells as well as in the inner plexiform layer, the ganglion cell layer and the nerve fiber layer. PACAP-LI seemed to be concentrated predominantly in the neuronal perikarya and their processes, but not in other cells in the retina. At the ultrastructural level, PACAP-LI was visible in the plasma membranes, rough endoplasmic reticulum, and cytoplasmic matrix in the PACAP-positive neurons in the inner nuclear layer. In the inner plexiform layer, PACAP-positive amacrine cell processes made synaptic contact with immunonegative amacrine cell processes, bipolar cell processes, and ganglion cell terminals. These findings suggest that PACAP may function as a neurotransmitter and/or neuromodulator.

Gene Expression for PACAP Receptor mRNA in the Rat Retina by in Situ Hybridization and in Situ RT‐PCR

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor (PAC 1 -R) are localized in the rat retina by in situ hybridization and in situ RT-PCR. PAC 1 -R is distributed in the rat brain and eye. 1,2 PAC 1 -R exists in at least eight splice variants that are differentially coupled to signal transduction pathways, 3 but the distribution and localization of these splice variants in the retina remain to be determined. There are at least eight isoforms of the PAC 1 -R, and the signal transduction system is different for each subtype. We studied gene expression of PAC 1 -R mRNA in the rat retina by in situ hybridization. In addition, we tried to identify PAC 1 -R subtypes in the rat retina by in situ RT-PCR.

PACAP Attenuates NMDA-Induced Retinal Damage in Association with Modulation of the Microglia/Macrophage Status into an Acquired Deactivation Subtype

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been known as a neuroprotectant agent in several retinal injury models. However, a detailed mechanism of this effect is still not well understood. In this study, we examined the retinoprotective effects and associated underlying mechanisms of action of PACAP in the mouse N-methyl-d-aspartic acid (NMDA)-induced retinal injury model, focusing on the relationship between PACAP and retinal microglia/macrophage (MG/MΦ) status. Adult male C57BL/6 mice received an intravitreal injection of NMDA to induce retinal injury. Three days after NMDA injection, the number of MG/MΦ increased significantly in the retinas. The concomitant intravitreal injection of PACAP suppressed NMDA-induced cell loss in the ganglion cell layer (GCL) and significantly increased the number of MG/MΦ. These outcomes associated with PACAP were attenuated by cotreatment with PACAP6-38, while the beneficial effects of PACAP were not seen in interleukin-10 (IL-10) knockout mice. PACAP significantly elevated the messenger RNA levels of anti-inflammatory cytokines such as transforming growth factor beta 1 and IL-10 in the injured retina, with the immunoreactivities seen to overlap with markers of MG/MΦ. These results suggest that PACAP enhances the proliferation and/or infiltration of retinal MG/MΦ and modulates their status into an acquired deactivation subtype to favor conditions for neuroprotection.

A case of retinal light damage by green laser pointer (Class 3b)

In previously reported cases of retinal injury from red (He–Ne; 632 nm) laser pointers, the maximum output was 5 mW or lower (Class 3a), and gazing time was 10 s or longer [1–3]. Higher-energy, green (532 nm) laser pointers are increasingly displacing red lasers and, here also, injuries have been reported [4]. We report a case of retinal light damage caused, after a moment’s gaze, by a high-output (Class 3b) green laser pointer unavailable to the general public in Japan that was brought from overseas.

Glutamate-stimulated proliferation of rat retinal pigment epithelial cells

We investigated the effects of glutamate on cell proliferation and the expression of basic fibroblast growth factor (bFGF) and its receptor (FGF-R1) mRNA in cultured rat retinal pigment epithelial (RPE) cells. The number of primary RPE cells was significantly higher after treatment with 0.2 to 1.0 mM glutamate (maximum at 1.0 mM) for 7 days than in controls. Glutamate-stimulated cell proliferation was abolished by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), but not by 6,7-dinitroquinoxaline-2,3-dione or L(+)-2-amino-3-phosphonopropionic acid. Proliferation was increased to a similar extent by N-methyl-D-aspartate (NMDA), but not by kainate, alpha-amino-3-hydroxy-3-methyl-4-isoxazolepropionic acid or trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid. NMDA-receptor-like immunoreactivity was detected in most cells cultured. Treatment of cells with glutamate increased the level of bFGF mRNA and, to a lesser extent, that of FGF-R1 mRNA, which peaked 2 and 4 days, respectively, after glutamate was added. The increase in bFGF mRNA induced by glutamate was inhibited by MK-801. These findings suggest that glutamate might stimulate proliferation of RPE cells through activation of NMDA receptors and expression of bFGF and further suggest that glutamate may be involved in the proliferative changes of RPE cells in retinal wound healing.

Diplopia after cataract surgery

PURPOSE To evaluate the cause of diplopia after cataract surgery. SETTING Cataract surgery at 7 hospitals and examination of diplopia at a central eye hospital. METHODS This study comprised 18 eyes of 17 patients with diplopia that developed after cataract surgery in which retrobulbar anesthesia was used. The Hess screen test was done to diagnose oculomotor dysfunction. RESULTS Several cases showed superior or inferior deviation of the globe, but most patients had nonuniform disturbances of eye movement. Examination of 3 patients by the Hess chart within 1 week after surgery showed paralysis of eye muscles but an overaction at a later stage, evident by reversal of eye position 1 month later. Surgery for strabismus was performed in 6 cases. One case with diplopia improved spontaneously 3 months after cataract surgery and achieved good alignment. CONCLUSIONS The Hess screen test was useful for comparing changes in oculomotor function before and after surgery. Oculomotor dysfunction after cataract surgery may be caused directly by traumatic injury during administration of anesthesia or surgery using bridle sutures or indirectly from sensitivity to anesthetic agents.

Blue LED light exposure develops intracellular reactive oxygen species, lipid peroxidation, and subsequent cellular injuries in cultured bovine retinal pigment epithelial cells

Abstract The effects of blue light emitter diode (LED) light exposure on retinal pigment epithelial cells (RPE cells) were examined to detect cellular damage or change and to clarify its mechanisms. The RPE cells were cultured and exposed by blue (470 nm) LED at 4.8 mW/cm2. The cellular viability was determined by XTT assay and cellular injury was determined by the lactate dehydrogenase activity in medium. Intracellular reactive oxygen species (ROS) generation was determined by confocal laser microscope image analysis using dihydrorhodamine 123 and lipid peroxidation was determined by 4-hydroxy-2-nonenal protein-adducts immunofluorescent staining (HNE). At 24 h after 50 J/cm2 exposures, cellular viability was significantly decreased to 74% and cellular injury was significantly increased to 365% of control. Immediately after the light exposure, ROS generation was significantly increased to 154%, 177%, and 395% of control and HNE intensity was increased to 211%, 359%, and 746% of control by 1, 10, and 50 J/cm2, respectively. These results suggest, at least in part, that oxidative stress is an early step leading to cellular damage by blue LED exposure and cellular oxidative damage would be caused by the blue light exposure at even lower dose (1, 10 J/cm2).