Alfred K. Yu

The Comparative Histologic Effects of Subthreshold 532- and 810-nm Diode Micropulse Laser on the Retina

PURPOSE Therapeutic retinal laser photocoagulation can damage the neurosensory retina and cause iatrogenic visual impairment. Subthreshold micropulse photocoagulation may decrease this risk by selective tissue treatment. The aim of this study was to compare subthreshold 810-nm diode micropulse laser and subthreshold 532-nm micropulse laser on the retina by histologic examination and differential protein expression. METHODS Fourteen Dutch-belted rabbits received subthreshold 810-nm diode micropulse laser photocoagulation in their right eye and subthreshold 532-nm micropulse laser photocoagulation in their left eye. Histology and immunohistochemical detection of stromal cell-derived factor-1 (SDF-1), β-actin, vascular endothelial growth factor (VEGF), glial fibrillary acidic protein (GFAP), and insulin-like growth factor 1 (IGF-1) were analyzed 12 hours, 3 days, 14 days, and 28 days post-laser photocoagulation. RESULTS Histologically, all time points produced a similar degree of retinal disruption in both wavelengths. Immunohistochemically, SDF-1 expression was greatest at the 12-hour time point and decreased thereafter. SDF-1, VEGF, and β-actin up-regulation was detected at early time points in both the 810- and 532-nm micropulse laser-treated animals. CONCLUSIONS Subthreshold micropulse retinal laser photocoagulation caused equivalent histologic changes from both 532- and 810-nm diode lasers. Differential protein expression was not evident between the different laser conditions.

Ocular Safety of Infliximab

Dear Editor: We read with interest the recent paper by Giansanti et al. on the intraocular safety of infliximab (Remicade ) in animal and cell culture models. Infliximab safety is an important factor as intraocular use of anti-Tumor Necrosis Factor (TNF)-a agents have been proposed as a potential therapeutic approach for patients with ocular inflammation. However, we feel that the intraocular safety data presented in this paper using infliximab in rabbits is misleading, as the manufacturer has published that infliximab does not bind rabbit TNF-a. The authors stated that the aim of the study ‘‘was to confirm the retinal safety of intravitreal infliximab at 2 mg in a rabbit model and to undertake safety testing of different doses of infliximab using a cell culture model [retinal ganglion (RGC-5) and retinal pigment epithelial (RPE) cell lines].’’ The rabbits were given intraocular injections of either infliximab or sterile saline solution. The authors found ‘‘slit-lamp biomicroscopy, indirect funduscopy, and electroretinogram (ERG) evidenced no significant difference between control and infliximab-injected eyes.’’ Histological results also showed no difference between the eyes injected with infliximab and those injected with sterile saline solution. The authors concluded that this result ‘‘suggests that infliximab has no direct retinal toxicity using rabbit [model].’’ However, to demonstrate safety of the antibody they assumed that infliximab (anti-human TNF-a) cross reacts with rabbit TNF-a, which they did not establish or discuss. In support of their work we also found that intravitreal infliximab showed no apparent toxicity in 6 New Zealand albino rabbits when analyzed by clinical examination, ERG, and histologic evaluation (unpublished data). However, we also discovered that according to the Remicade package insert, infliximab does not cross react with TNF-a in species other than humans and chimpanzees. This was confirmed by personal communication with the manufacturer (Centocor) stating that they tested ‘‘neutralization of rabbit TNF-a by infliximab using a bioassay, and it was negative.’’ Therefore, this issue of binding the host TNF-a is critical when assessing the safety of a therapeutic agent in another species, which the authors do not bring up in their discussion. They do state in their discussion that infliximab has been reported in an Association for Research in Vision and Ophthalmology (ARVO) abstract to reduce uveitis. This report has not been substantiated in the peer review literature and further classification of mechanism of action would be necessary. The data do demonstrate the absence of nonspecific ocular toxicity of the carrier vehicle in infliximab, but do not demonstrate in vivo safety for the anti-TNF-a antibody. The in vitro results of Giansanti et al. showed no effect of infliximab on two cell lines: ARPE-19 (human RPE cell line) and RGC-5 (Sprague-Dawley rat ganglion cell line). The data using the ARPE-19 cell line do suggest in vivo safety as this cell line is human and can express TNF-a; however, infliximab does not cross-react with the rat TNF-a. Without binding of the target tissue TNF-a, the safety of the infliximab drug carrier can be concluded only from the RGC-5 data. When testing biologics such as anti-TNF-a in animals or cell culture, it is essential to establish that the agent binds the target tissue before relevant safety data can be inferred. This study (and our unpublished work) does demonstrate safety of the drug carrier in rabbits and cell culture. However, additional testing would be needed to infer intraocular safety. For example, either testing of another anti-TNF-a antibody that binds the animal used for the toxicity study, or using a different species that does have cross-reactivity with the agent. In this case, chimpanzees could be used for toxicity studies of infliximab. Cross-reactivity has been a common assumption in many toxicity studies, but it is essential to establish especially when future patient care may be affected.

Rescue of cell death and inflammation of a mouse model of complex 1-mediated vision loss by repurposed drug molecules

Inherited mitochondrial optic neuropathies, such as Lebers hereditary optic neuropathy (LHON) and Autosomal dominant optic atrophy (ADOA) are caused by mutant mitochondrial proteins that lead to defects in mitochondrial complex 1-driven ATP synthesis, and cause specific retinal ganglion cell (RGC) loss. Complex 1 defects also occur in patients with primary open angle glaucoma (POAG), in which there is specific RGC loss. The treatment of mitochondrial optic neuropathy in the US is only supportive. The Ndufs4 knockout (Ndufs4 KO) mouse is a mitochondrial complex 1-deficient model that leads to RGC loss and rapid vision loss and allows for streamlined testing of potential therapeutics. Preceding RGC loss in the Ndufs4 KO is the loss of starburst amacrine cells, which may be an important target in the mechanism of complex 1-deficient vision loss. Papaverine and zolpidem were recently shown to be protective of bioenergetic loss in cell models of optic neuropathy. Treatment of Ndufs4 KO mice with papaverine, zolpidem, and rapamycin-suppressed inflammation, prevented cell death, and protected from vision loss. Thus, in the Ndufs4 KO mouse model of mitochondrial optic neuropathy, papaverine and zolpidem provided significant protection from multiple pathophysiological features, and as approved drugs in wide human use could be considered for the novel indication of human optic neuropathy.

Epithelial membrane protein-2 in human proliferative vitreoretinopathy and epiretinal membranes

Purpose To determine the level of epithelial membrane protein-2 (EMP2) expression in preretinal membranes from surgical patients with proliferative vitreoretinopathy (PVR) or epiretinal membranes (ERMs). EMP2, an integrin regulator, is expressed in the retinal pigment epithelium and understanding EMP2 expression in human retinal disease may help determine whether EMP2 is a potential therapeutic target. Methods Preretinal membranes were collected during surgical vitrectomies after obtaining consents. The membranes were fixed, processed, sectioned, and protein expression of EMP2 was evaluated by immunohistochemistry. The staining intensity (SI) and percentage of positive cells (PP) in membranes were compared by masked observers. Membranes were categorized by their cause and type including inflammatory and traumatic. Results All of the membranes stained positive for EMP2. Proliferative vitreoretinopathy–induced membranes (all causes) showed greater expression of EMP2 than ERMs with higher SI (1.81 vs. 1.38; P = 0.07) and PP (2.08 vs. 1.54; P = 0.09). However all the PVR subgroups had similar levels of EMP2 expression without statistically significant differences by Kruskal-Wallis test. Inflammatory PVR had higher expression of EMP2 than ERMs (SI of 2.58 vs. 1.38); however, this was not statistically significant. No correlation was found between duration of PVR membrane and EMP2 expression. EMP2 was detected by RT-PCR in all samples (n = 6) tested. Conclusions All studied ERMs and PVR membranes express EMP2. Levels of EMP2 trended higher in all PVR subgroups than in ERMs, especially in inflammatory and traumatic PVR. Future studies are needed to determine the role of EMP2 in the pathogenesis and treatment of various retinal conditions including PVR.