Molecular characterization and functional analysis of phagocytosis by human embryonic stem cell-derived RPE cells using a novel human retinal assay.

PURPOSE: To examine the ability of retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (HESC) to phagocytose photoreceptor outer segments, and to determine whether exposure to human retina induces any morphological changes in these cells. METHODS: HESC-RPE cells were derived from a super-confluent preparation of the Shef1 HESC line. Pigmented colonies were isolated and expanded into pigmented monolayers on Matrigel matrix-coated dishes or filters. Cells were exposed to fluorescently labeled outer segments isolated from the porcine eye and assessed for phagocytic activity at regular intervals. Expression of molecules associated with RPE phagocytosis was analyzed by RT-PCR, immunocytochemistry, and western blot. The role of Mer Tyrosine Kinase (MERTK) in the phagocytosis of outer segments was investigated using antibodies directed against MERTK to block function. In a novel approach, cells were also exposed to fresh human neural retina tissue then examined by electron microscopy for evidence of phagocytosis and changes in cell morphology. RESULTS: HESC-derived RPE cells are capable of phagocytosing isolated porcine outer segments and express molecules associated with RPE-specific phagocytosis, including MERTK. Pre-incubation with antibodies against MERTK blocked phagocytosis of photoreceptor outer segments, but not polystyrene beads. HESC-RPE cells also phagocytosed outer segments in a novel human retinal explant system. Furthermore co-culture adjacent to human retina tissue in this preparation resulted in the appearance of features in HESC-derived RPE cells normally observed only as the RPE matures. CONCLUSIONS: The ingestion of photoreceptor outer segments from an isolated population and an artificial ex vivo human retina system demonstrates HESC-derived RPE cells are functional. HESC-derived RPE possess the relevant molecules required for phagocytosis, including MERTK, which is essential for the phagocytosis of outer segments but not latex beads. Furthermore, some changes observed in cell morphology after co-culture with human retina may have implications for understanding the full development and differentiation of RPE cells.

Severe cough: a cause of late bleb leak.

PURPOSE: To highlight coughing as a potential cause for late postoperative trabeculectomy bleb leaks. METHODS: A retrospective study of 2 patients in whom late bleb leaks developed after a bout of severe coughing. Perioperative 5-fluorouracil was used at the time of the initial filtration procedure for one of the patients, while the second patient had had an ordinary trabeculectomy. Both patients required surgical repair of their bleb leak to manage the resultant hypotony. RESULTS: Postoperatively, both patients had good intraocular pressure control and maintained their visual fields without topical medications. Both patients demonstrated bleb avascularity after leak repair. CONCLUSIONS: Trabeculectomy bleb leaks are well recognized. Bleb leaks in the late postoperative period are more common after the perioperative use of antimetabolites, and spontaneous leaks after minimal trauma have been described. The authors describe 2 cases of late spontaneous bleb leaks after severe coughing, both of which responded well to surgical correction. The importance of inquiring about a history of coughing with late bleb leaks in the absence of other causative factors is stressed.

Degeneration of cortical function in the Royal College of Surgeons rat.

The purpose of the current study was to determine the progress of cortical functional degeneration in the Royal College of Surgeons (RCS) rat. Cortical responses were measured with optical imaging of intrinsic signals using gratings of various spatial frequencies. Subsequently, electrophysiological recordings were also taken across cortical layers in response to a pulse of broad-spectrum light. We found significant degeneration in the cortical processing of visual information as early as 4 weeks of age. These results show that degeneration in the cortical response of the RCS rat starts before development has been properly completed.