Ilene K. Sugino

Decreased choriocapillaris perfusion following surgical excision of choroidal neovascular membranes in age-related macular degeneration

AIMS/BACKGROUND To evaluate macular changes following surgical excision of subfoveal choroidal neovascular membranes (CNVs) in age-related macular degeneration (AMD). METHODS The clinical records, fluorescein angiograms, and CNV histopathology of 12 patients with AMD who underwent surgical excision of subfoveal CNV were reviewed. RESULTS New areas of decreased choriocapillaris perfusion were noted by fluorescein angiography in the previous location of the CNV in 8/12 (75%) cases. Surgically excised tissue contained retinal pigment epithelium (RPE) in 11/11 specimens and choriocapillaris in 1/11 specimen studied. CONCLUSIONS Choriocapillaris atrophy may partly underlie the limited visual outcome following subfoveal surgery for AMD. Abnormal choriocapillaris perfusion following CNV excision may be due to pre-existing choriocapillaris atrophy, to choriocapillaris damage or removal at the time of surgery, or to RPE removal at surgery with abnormal RPE repopulation of the dissected area and subsequent choriocapillaris degeneration.

Comparison of FRPE and Human Embryonic Stem Cell–Derived RPE Behavior on Aged Human Bruch's Membrane

PURPOSE To compare RPE derived from human embryonic stem cells (hES-RPE) and fetal RPE (fRPE) behavior on human Bruchs membrane (BM) from aged and AMD donors. METHODS hES-RPE of 3 degrees of pigmentation and fRPE were cultured on BM explants. Explants were assessed by light, confocal, and scanning electron microscopy. Integrin mRNA levels were determined by real-time polymerase chain reaction studies. Secreted proteins in media were analyzed by multiplex protein analysis after 48-hour exposure at culture day 21. RESULTS hES-RPE showed impaired initial attachment compared to fRPE; pigmented hES-RPE showed nuclear densities similar to fRPE at day 21. At days 3 and 7, hES-RPE resurfaced BM to a limited degree, showed little proliferation (Ki-67), and partial retention of RPE markers (MITF, cytokeratin, and CRALBP). TUNEL-positive nuclei were abundant at day 3. fRPE exhibited substantial BM resurfacing at day 3 with decreased resurfacing at later times. Most fRPE retained RPE markers. Ki-67-positive nuclei decreased with time in culture. TUNEL staining was variable. Increased integrin mRNA expression did not appear to affect cell survival at day 21. hES-RPE and fRPE protein secretion was similar on equatorial BM except for higher levels of nerve growth factor and thrombospondin-2 (TSP2) by hES-RPE. On submacular BM, fRPE secreted more vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor, and platelet-derived growth factor; hES-RPE secreted more TSP2. CONCLUSIONS Although pigmented hES-RPE and fRPE resurfaced aged and AMD BM to a similar, limited degree at day 21, cell behavior at earlier times was markedly dissimilar. Differences in protein secretion may indicate that hES-RPE may not function identically to native RPE after seeding on aged or AMD BM.

Preparation and transplantation of photoreceptor sheets

PURPOSE Photoreceptor (PR) transplantation may be a treatment for blindness secondary to PR degeneration. We studied different technical aspects of PR-sheet preparation. METHODS Geographic variation in the thickness of the cat PR layer (from the outer segments to the outer plexiform layer) and inner retina (width of the remainder of the retina) was studied. PR sheets (cat and human) were prepared through gelatin embedding and subsequent vibratoming or excimer laser ablation. Cat PR sheets were evaluated after transplantation. RESULTS The thickness of the cat PR layer and inner retina varied in different regions. The superior central retina, including the area centralis, was thickest (PR layer: 115-123 microm, entire retina: 225-230 microm, in fixed tissue). The peripheral retina was approximately 40% thinner than the center. Fresh retina was approximately 7.9% thicker than the fixed retina. Both vibratomy and excimer laser ablation removed the inner retina, leaving a PR-layer sheet with good morphology. To produce good quality PR sheets with vibratomy, use of different gelatin concentrations (2% to 35%) at various stages of sheet preparation was crucial. To produce PR sheets of uniform thickness with excimer laser ablation, control of fluid on the retinal surface was critical. Twenty-four hours after PR transplantation surgery, donor PR cells were well oriented and in close contact with host retinal pigment epithelial cells. Gelatin supporting the transplant dissolved as early as 100 min after surgery. CONCLUSIONS We confirmed and expanded the work of previous investigators and showed that cat and human PR sheets can be manufactured using vibratomy or excimer laser ablation. This preparation provides a well oriented and organized PR cell layer after transplantation.

CULTURE OF HUMAN RETINAL PIGMENT EPITHELIAL CELLS FROM PERIPHERAL SCLERAL FLAP BIOPSIES

PURPOSE We studied various methods for harvesting retinal pigment epithelium (RPE) biopsies from cadaver human eyes of donors over age 60 years. Our goal was to harvest cells for possible autologous RPE cell transplantation in patients with age-related macular degeneration and to test the viability of the RPE after isolation by evaluating explant growth in culture. METHODS Choroid-RPE biopsies were excised from enucleated human eyes. The RPE was separated from the choroid by treatment with type IV collagenase. RPE patches were cultured. After 100-500 cells had grown out from the explant, the primary cultures were passaged. RESULTS There was no clear effect of donor age on the ability to establish primary RPE cultures with good morphology from biopsies 2 x 2-10 x 10 mm2 in size. Biopsies 6 x 6 mm2 or larger produced satisfactory primary cultures more than 70% of the time. The number of viable RPE cells (defined as the number of cells adherent to the culture dish 24 h after plating) obtained after enzymatic separation of the RPE and choroid was an important determinant of our ability to establish primary cultures and passage the cells. Primary cultures with good cellular morphology were obtained 100% of the time when RPE explants > 4 mm2 in size were obtained from the biopsy specimen. Seventy-three percent of the biopsies yielding explants > 4 mm2 in size were successfully passaged. CONCLUSIONS These results suggest that peripheral scleral flap biopsies in aging donors can be used to establish RPE explant primary cultures. These cultures may be suitable as a source for autologous RPE transplantation in patients.

Progressive presumed choriocapillaris atrophy after surgery for age-related macular degeneration.

PURPOSE The authors sought to evaluate the progression of presumed choriocapillaris atrophy after surgical excision of a subfoveal choroidal neovascular membrane (CNV) in an 80-year-old man with age-related macular degeneration. METHODS The CNV was excised using a conventional three-port vitrectomy with subretinal dissection. The excised tissue was studied with light and electron microscopy. Preoperative and serial postoperative fluorescein angiograms (FA) and fundus photographs were obtained to study the dissection bed. RESULTS Seven days after surgery, the FA showed hyperfluorescence in the area previously occupied by the CNV. Six weeks after surgery, this area showed retinal pigment epithelium (RPE) depigmentation, atrophy, or both on clinical examination, and the FA showed presumed choriocapillaris nonperfusion. Seven months after surgery, the area of the RPE depigmentation or atrophy and the corresponding area of presumed choriocapillaris nonperfusion had enlarged. The area of depigmentation or atrophy continued to enlarge for 1 year after surgery. Histologically, the excised CNV specimen disclosed RPE cells but no choriocapillaris. CONCLUSIONS Presumed choriocapillaris nonperfusion after CNV excision may be due to RPE removal at surgery and may progress postoperatively.

Cell-Deposited Matrix Improves Retinal Pigment Epithelium Survival on Aged Submacular Human Bruch's Membrane

PURPOSE To determine whether resurfacing submacular human Bruchs membrane with a cell-deposited extracellular matrix (ECM) improves retinal pigment epithelial (RPE) survival. METHODS Bovine corneal endothelial (BCE) cells were seeded onto the inner collagenous layer of submacular Bruchs membrane explants of human donor eyes to allow ECM deposition. Control explants from fellow eyes were cultured in medium only. The deposited ECM was exposed by removing BCE. Fetal RPE cells were then cultured on these explants for 1, 14, or 21 days. The explants were analyzed quantitatively by light microscopy and scanning electron microscopy. Surviving RPE cells from explants cultured for 21 days were harvested to compare bestrophin and RPE65 mRNA expression. Mass spectroscopy was performed on BCE-ECM to examine the protein composition. RESULTS The BCE-treated explants showed significantly higher RPE nuclear density than did the control explants at all time points. RPE expressed more differentiated features on BCE-treated explants than on untreated explants, but expressed very little mRNA for bestrophin or RPE65. The untreated young (<50 years) and African American submacular Bruchs membrane explants supported significantly higher RPE nuclear densities (NDs) than did the Caucasian explants. These differences were reduced or nonexistent in the BCE-ECM-treated explants. Proteins identified in the BCE-ECM included ECM proteins, ECM-associated proteins, cell membrane proteins, and intracellular proteins. CONCLUSIONS Increased RPE survival can be achieved on aged submacular human Bruchs membrane by resurfacing the latter with a cell-deposited ECM. Caucasian eyes seem to benefit the most, as cell survival is the worst on submacular Bruchs membrane in these eyes.

Clinicopathological correlation of primary and recurrent choroidal neovascularisation following surgical excision in age related macular degeneration

AIMS/BACKGROUND Fluorescein angiography and histopathological findings were correlated in two patients with recurrent choroidal neovascular membranes (CNVs) in an attempt to gain insight into the possible causes of recurrent CNVs and into the healing response after CNV excision. METHODS Two patients with recurrent CNVs underwent repeat excision, and the excised tissue was studied with light and electron microscopy. RESULTS Incomplete CNV excision probably led to the recurrences. The portion initially excised appears to have been anterior to the RPE in case 1. In both cases, recurrent CNVs contained RPE-like cells suggesting that native RPE can repopulate the dissection bed. The tissue excised at the second operation contained areas with hyperplastic RPE and fragments of Bruch’s membrane (external to the RPE basement membrane) in a matrix of fibrillar collagen and fibrocytes, suggesting that initial removal of the CNV can be followed by an abnormal anatomical arrangement of RPE and scarring of Bruch’s membrane. CONCLUSIONS Abnormal resurfacing of the dissection bed by RPE and fibroblasts may underlie, in part, the limited visual outcome often seen after surgical excision of CNVs in age related macular degeneration.

A method to enhance cell survival on Bruch's membrane in eyes affected by age and age-related macular degeneration.

PURPOSE To determine whether conditioned medium (CM) derived from bovine corneal endothelial cells (BCECs) can support transplanted cells on aged and age-related macular degeneration (AMD) Bruchs membrane (BM). METHODS Retinal pigment epithelium (RPE) cells derived from human embryonic stem cells (hES-RPE) and cultured fetal and aged adult RPE were seeded onto the inner collagenous layer of submacular BM-choroid-sclera explants generated from aged and AMD human donor eyes. Paired explants were cultured in BCEC-CM or CM vehicle. To assess cell behavior after attachment to BM was established, explants were harvested after 21 days in culture. To assess whether sustained exposure to BCEC-CM was necessary for improved cell survival on BM, short exposure to BCEC-CM (3, 7, 14 days) was compared with 21-day exposure. Explants were harvested and evaluated by scanning electron and light microscopy. Extracellular matrix (ECM) deposition after exposure to BCEC-CM was evaluated following RPE cell removal after day 21 on tissue culture dishes or on BM. RESULTS BCEC-CM significantly enhanced hES-RPE, fetal RPE, and aged adult RPE survival on BM, regardless of submacular pathology. Although shorter BCEC-CM exposure times showed significant improvement in cell survival compared with culture in CM vehicle, longer BCEC-CM exposure times were more effective. BCEC-CM increased RPE ECM deposition on tissue culture plastic and on BM. CONCLUSIONS The results of this study indicate that RPE survival is possible on AMD BM and offer a method that could be developed for enhancing transplanted cell survival on AMD BM. Increased ECM deposition may account for improved cell survival after culture in BCEC-CM.

Abnormal collagen fibrils in nanophthalmos: a clinical and histologic study

PURPOSE To report the successful treatment of choroidal detachment in a patient with nanophthalmos and to report histopathologic findings in this patients sclera. METHODS Choroidal detachment, secondary angle closure, and nanophthalmos were diagnosed using biomicroscopy, indirect ophthalmoscopy, and echography. Full-thickness sclerectomies in four quadrants were made on the right eye. Sclerae from these sclerectomies were studied ultrastructurally. RESULTS Best-corrected visual acuity improved to RE, 20/60 from 20/100 preoperatively; the anterior chamber deepened, and the choroidal detachment resolved. Histopathologic studies of each of the three scleral layers disclosed abnormal collagen fibrils that were frayed, split, and contained lightly stained cores. CONCLUSION New findings include the identification of collagen with lightly stained centers and identification of differences in collagen morphology in different areas of the sclera in a nanophthalmic eye.

Clinicopathological correlation of an excised choroidal neovascular membrane in pseudotumour cerebri

AIMS/BACKGROUND To correlate the histopathology of an excised choroidal neovascular membrane (CNV) with the clinical and angiographic findings in a 32-year-old woman with pseudotumour cerebri and a peripapillary CNV with subfoveal extension. METHODS The patient’s visual acuity was assessed by individuals experienced in low vision refraction and who were not members of the surgical team. The CNV was excised via a conventional three port vitrectomy with subretinal dissection. The excised tissue was studied with light and electron microscopy. Preoperative and serial postoperative fluorescein angiograms (FAs) and fundus photographs were obtained to study the dissection bed. RESULTS One week after surgery, the FA showed mottled subfoveal choriocapillaris perfusion. Three weeks after surgery, this area showed retinal pigment epithelium (RPE) atrophy clinically, and the FA showed choriocapillaris non-perfusion. Six months after surgery, the area of RPE atrophy and the corresponding area of choriocapillaris non-perfusion had expanded. Histologically, the excised CNV disclosed hyperplastic RPE, fibrovascular tissue, and no choriocapillaris. Fragments of RPE basement were present along the external edge of the specimen. The patient’s visual acuity did not improve significantly after surgery. CONCLUSIONS Choriocapillaris non-perfusion can develop even in young patients following CNV excision. In this particular case, it is believed that choriocapillaris atrophy was caused by incomplete ingrowth of RPE into the dissection bed following RPE removal with CNV excision. As far as is known, this is the first report describing the results of surgery for CNV secondary to papilloedema associated with pseudotumour cerebri.