Haksu Kyung

Quantitative Analysis of Retinal Ganglion Cell Survival with Rbpms Immunolabeling in Animal Models of Optic Neuropathies

PURPOSE To investigate whether a recently described retinal ganglion cell (RGC) marker Rbpms (RNA binding protein with multiple splicing) could be used for RGC quantification in various models of RGC degeneration. METHODS Optic nerve crush, excitotoxicity, and elevated intraocular pressure (IOP) rat models were used. Topographic analysis of Rbpms immunolabeling was performed on retinal wholemounts. Retrograde labelings with Fluorogold (FG) and III β-tubulin immunohistochemistry were compared. RESULTS In the optic nerve crush model, 37%, 87%, and 93% of Rbpms-positive cells were lost 1, 2, and 4 weeks, respectively. Significant loss of Rbpms-positive cells was noted 1 week after intravitreal injection of 12, 30, and 120 nmol N-methyl-d-aspartate (NMDA), whereas coinjection of 120 nmol of NMDA along with MK-801 increased the cell number from 10% to 59%. Over 95% of Rbpms-positive cells were FG- and III β-tubulin-positive after injury caused by optic nerve crush and NMDA injection. In rats with elevated IOP, induced by trabecular laser photocoagulation, there was a significant loss of Rbpms-positive cells compared with that of contralateral controls (P = 0.0004), and cumulative IOP elevation showed a strong linear relationship with the quantification of RGCs by Rbpms immunolabeling and retrograde labeling with FG. More than 99% of the remaining Rbpms-positive cells were double-labeled with FG. CONCLUSIONS Rbpms can reliably be used as an RGC marker for quantitative evaluation in rat models of RGC degeneration, regardless of the nature and the location of the primary site of the injury and the extent of neurodegeneration.

The Neuronal EGF-Related Gene Nell2 Interacts with Macf1 and Supports Survival of Retinal Ganglion Cells after Optic Nerve Injury

Nell2 is a neuron-specific protein containing six epidermal growth factor-like domains. We have identified Nell2 as a retinal ganglion cell (RGC)-expressed gene by comparing mRNA profiles of control and RGC-deficient rat retinas. The aim of this study was to analyze Nell2 expression in wild-type and optic nerve axotomized retinas and evaluate its potential role in RGCs. Nell2-positive in situ and immunohistochemical signals were localized to irregularly shaped cells in the ganglion cell layer (GCL) and colocalized with retrogradely-labeled RGCs. No Nell2-positive cells were detected in 2 weeks optic nerve transected (ONT) retinas characterized with approximately 90% RGC loss. RT-PCR analysis showed a dramatic decrease in the Nell2 mRNA level after ONT compared to the controls. Immunoblot analysis of the Nell2 expression in the retina revealed the presence of two proteins with approximate MW of 140 and 90 kDa representing glycosylated and non-glycosylated Nell2, respectively. Both products were almost undetectable in retinal protein extracts two weeks after ONT. Proteome analysis of Nell2-interacting proteins carried out with MALDI-TOF MS (MS) identified microtubule-actin crosslinking factor 1 (Macf1), known to be critical in CNS development. Strong Macf1 expression was observed in the inner plexiform layer and GCL where it was colocalizied with Thy-1 staining. Since Nell2 has been reported to increase neuronal survival of the hippocampus and cerebral cortex, we evaluated the effect of Nell2 overexpression on RGC survival. RGCs in the nasal retina were consistently more efficiently transfected than in other areas (49% vs. 13%; n = 5, p<0.05). In non-transfected or pEGFP-transfected ONT retinas, the loss of RGCs was approximately 90% compared to the untreated control. In the nasal region, Nell2 transfection led to the preservation of approximately 58% more cells damaged by axotomy compared to non-transfected (n = 5, p<0.01) or pEGFP-transfected controls (n = 5, p<0.01).

Celastrol supports survival of retinal ganglion cells injured by optic nerve crush.

The present study evaluates the effect of celastrol on the survival of retinal ganglion cells (RGCs) injured by optic nerve crush (ONC). Celastrol, a quinine methide triterpene extracted from the perennial vine Tripterygium wilfordii (Celastraceae), has been identified as a potential neuroprotective candidate in a comprehensive drug screen against various neurodegenerative diseases. Two weeks after ONC, the average density of remaining RGCs in retinas of animals treated with daily intraperitoneal (i.p.) injections of celastrol (1mg/kg) was approximately 1332 cells/mm(2), or 40.8% of the Celastrol/Control group. In retinas of the Vehicle/ONC group about 381 RGCs/mm(2) were counted, which is 9.6% of the total number of RGCs in the DMSO/Control group. This corresponds to approximately a 250% increase in RGC survival mediated by celastrol treatment compared to Vehicle/ONC group. Furthermore, the average RGC number in retinas of ONC animals treated with a single intravitreal injection of 1mg/kg or 5mg/kg of celastrol was increased by approximately 80% (760 RGCs/mm(2)) and 78% (753 RGCs/mm(2)), respectively, compared to Vehicle/ONC controls (422 cells/mm(2)). Injection of 0.2mg/kg of celastrol had no significant effect on cell survival, with the average number of RGCs being 514 cells/mm(2) in celastrol-treated animals versus 422 cells/mm(2) in controls. The expression levels of Hsp70, Hsf1, Hsf2, HO-1 and TNF-alpha in the retina were analyzed to evaluate the roles of these proteins in the celastrol-mediated protection of injured RGCs. No statistically significant change in HO-1, Hsf1 and Hsp70 levels was seen in animals with ONC. An approximately 2 fold increase in Hsf2 level was observed in celastrol-treated animals with or without injury. Hsf2 level was also increased 1.8 fold in DMSO-treated animals with ONC injury compared to DMSO-treated animals with no injury suggesting that Hsf2 induction has an injury-induced component. Expression of TNF-alpha in retinas of celastrol-treated uninjured and ONC animals was reduced by approximately 2 and 1.5 fold compared to vehicle treated animals, respectively. The observed results suggest that mechanisms underlying celastrol׳s RGC protective effect are associated with inhibition of TNF-alpha-mediated cell death.

Disc haemorrhage is associated with the fast component, but not the slow component, of visual field decay rate in glaucoma

Background/aims To investigate the association of disc haemorrhage (DH) with regional visual field (VF) decay in glaucoma. Methods Retrospective longitudinal study was performed. Patients from the University of California, Los Angeles, glaucoma database were assigned to two groups based on the presence or absence of a DH. Pointwise exponential regression was used to identify the fast and slow rate components of VF decay. Associations between patient demographics, ocular and systemic factors, and visual field rates were analysed. Results A total of 185 patients were included, 54 of whom were documented to have a DH at some point during their course. DH group had a higher female preponderance (p=0.017, OR 2.23), a higher incidence of peripapillary atrophy (p=0.002, OR 4.46), more advanced disease (p=0.016) and a higher fast rate component of VF decay (p<0.001) than non-DH group. With multivariate logistic regression analysis, only the fast rate component showed a statistically significant relationship with DH. Conclusions The presence of DH is associated with a greater fast component rate of VF decay. The identification and monitoring of the fast component of VF decay may prove useful in the identification and management of glaucoma patients at high risk of progression.

Location of Initial Visual Field Defects in Glaucoma and Their Modes of Deterioration.

PURPOSE To describe the location of initial visual field defects (VFD) in glaucoma, their modes of deterioration, and those factors associated with different modes of deterioration. METHODS Patients with POAG were categorized into four groups based on three consecutive initial VFD: (1) superior paracentral defects (PD), (2) inferior PD, (3) superior nasal defects (ND), and (4) inferior ND. According to the worsening of the VF, four further subgroups were identified: (1) superior central worsening (CW), (2) inferior CW, (3) superior peripheral or nasal worsening (NW), and (4) inferior NW. Systemic and ocular factors were analyzed for each of the subgroups to identify possible associations. RESULTS One hundred sixty-two eyes of 162 subjects were analyzed. Superior PD (n = 40) were more frequent in females and associated with disc hemorrhage (DH), and were less frequent in patients with systemic hypertension (HT). Inferior PD (n = 35) showed a significant association with cup shape measure and axial length. Superior ND (n = 37) were more highly associated with HT and diabetes. Inferior ND (n = 50) showed a lower incidence of DH. With binary logistic regression analysis, superior PD showed a significant association with both HT and DH. With respect to VF worsening, superior CW showed a significant association with HT and diabetes, whereas superior NW was associated with a high minimum IOP during follow-up, and inferior NW was associated with a high maximum IOP during follow-up. CONCLUSIONS The initial location and subsequent direction of worsening of VFD were associated with different systemic and ocular factors.

Pseudohypopyon after management of posterior capsule rupture using intracameral triamcinolone injection in cataract surgery.

Dear Editor, Rupture of the posterior capsule and prolapse of the vitreous body are relatively common complications of cataract surgery. When posterior capsule rupture (PCR) occurs, the vitreous body prolapsed into anterior chamber should be removed completely. However, the surgical procedure to remove the vitreous body is difficult and troublesome because the vitreous structure is transparent under an operating microscope and has sticky and amorphous characteristics. Triamcinolone acetonide (TA) has been used to visualize the vitreous body prolapsed into the anterior chamber after PCR during cataract surgery [1]. Occasionally, following an intracameral or intravitreal injection, TA particles may migrate into the anterior chamber, lay in the inferior angle, and form a pseudohypopyon [2]. We present a patient who developed a pseudohypopyon following intracameral TA injection for the verification of prolapsed vitreous body during management of PCR in cataract surgery. A 57-year-old, healthy, woman presented with decreased vision in her left eye. She was diagnosed with age-related left cataract and scheduled for cataract operation. Under topical anesthesia, standard phacoemulsification was performed in the operating room. When the posterior capsule was ruptured and the vitreous body was prolapsed into the anterior chamber, anterior vitrectomy was done to remove the prolapsed vitreous. TA solution (4 mg/0.1 mL) was injected at the location where the vitreous body was likely to have been placed using a syringe with a dull-edged needle. Immediately, the prolapsed vitreous appeared as a white-colored gel. Then, vitreous with TA granules was easily removed with a vitreous cutter. After removing the prolapsed vitreous, an intraocular lens was inserted by using sulcus fixation. At the end of operation, TA was clearly eliminated from the anterior chamber and anterior vitreous cavity. On the 1st day postoperatively, she was noted to have 3 × 4-mm sized yellow-whitish deposits in the inferior portion of the anterior chamber (Fig. 1). The visual acuity of her left eye was finger counting at 30 cm. She did not report any pain, redness or lid edema. Only topical antibiotic was applied, and the patient was followed-up on a weekly basis. The yellow-whitish deposits in the anterior chamber cleared spontaneously on the 5th week postoperatively. The visual acuity of her left eye was improved to 20 / 20 without any specific complications or treatments. Fig. 1 Yellow-whitish deposits seen in the anterior chamber 1 day following cataract surgery with triamcinolone acetonide for visualizing prolapsed vitreous body due to posterior capsule rupture. Some triamcinolone acetonide particles were seen on the intraocular ... Although intracameral and intravitreal TA is widely used in anterior or posterior vitrectomy to help visualize the vitreous body, it is associated with the potential risk of developing postoperative complications including endophthalmitis and intraocular pressure elevation. However, in this case, endophthalmitis was not observed; instead we observed a TA-related pseudohypopyon. The pseudohypopyon can be explained due to the passage of remnant TA from the vitreous cavity to the anterior chamber as a result of eye movements. Another theory which explains this condition is an aseptic inflammatory reaction against the excipient of the TA [3]. The development of endophthalmitis following intracameral or intravitreal TA injection must be ruled out when a pseudohypopyon is present. Usually a pseudohypopyon is distinguishable from an infective hypopyon by its ground glass appearance, the presence of refractile particles, and its shifting position, which is dependent upon the patients head position. The presence of other associated clinical symptoms and signs may help to establish the correct diagnosis. In conclusion, we report a case of pseudohypopyon after intracameral TA injection for the verification of remnant vitreous during the management of PCR in phacoemulsification and intraocular lens insertion, from which the patient completely recovered without any specific treatment. However, care should be taken that pseudohypopyon are distinguished from endophthalmitis and any other pathologic conditions after anterior vitrectomies with TA in cataract surgery.