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Purpose: To undertake the first ultrastructural characterization of human retinal pigment epithelial (RPE) differentiation from fetal development to adolescence., Methods: Ten fetal eyes and three eyes aged six, nine, and 17 years were examined in the temporal retina adjacent to the optic nerve head by transmission electron microscopy. The area, number, and distribution of RPE organelles were quantified and interpreted within the context of adjacent photoreceptors, Bruch's membrane, and choriocapillaris maturation., Results: Between eight to 12 weeks' gestation (WG), pseudostratified columnar epithelia with apical tight junctions differentiate to a simple cuboidal epithelium with random distribution of melanosomes and mitochondria. Between 12 to 26 WG, cells enlarge and show long apical microvilli and apicolateral junctional complexes. Coinciding with eye opening at 26 WG, melanosomes migrate apically whereas mitochondria distribute to perinuclear regions, with the first appearance of phagosomes, complex granules, and basolateral extracellular space (BES) formation. Significantly, autophagy and heterophagy, as evidenced by organelle recycling, and the gold standard of ultrastructural evidence for autophagy of double-membrane autophagosomes and mitophagosomes were evident from 32 WG, followed by basal infoldings of RPE cell membrane at 36 WG. Lipofuscin formation and deposition into the BES evident at six years increased at 17 years., Conclusions: We provide compelling ultrastructural evidence that heterophagy and autophagy begins in the third trimester of human fetal development and that deposition of cellular byproducts into the extracellular space of RPE takes place via exocytosis. Transplanted RPE cells must also demonstrate the capacity to subserve autophagic and heterophagic functions for effective disease mitigation.

Purpose: We investigated the relationship between inflammation, neuronal loss, and expression of indoleamine 2, 3-dioxygenase (IDO) and quinolinic acid (QUIN) in the retina of subjects with type 1 diabetes (T1D) and type 2 diabetes (T2D) and in the retina of rats with T1D., Methods: Retinas from T1D (n = 7), T2D (n = 13), and 20 age-matched nondiabetic human donors and from T1D (n = 3) and control rats (n = 3) were examined using immunohistochemistry for IDO, QUIN, cluster of differentiation 39 (CD39), ionized calcium-binding adaptor molecule (Iba-1, for macrophages and microglia), Vimentin (VIM; for Müller cells), neuronal nuclei (NeuN; for neurons), and UEA1 lectin (for blood vessels)., Results: Based on morphologic criteria, CD39+/ionized calcium binding adaptor molecule 1(Iba-1+) resident microglia and CD39-/Iba-1+ bone marrow-derived macrophages were present at higher density in T1D (13% increase) and T2D (26% increase) human retinas when compared with controls. The density and brightness of IDO+ microglia were increased in both T1D and T2D human retinas. The intensity of QUIN+ expression on CD39+ microglia and VIM+ Müller cells was greatly increased in both human T1D and T2D retinas. T1D retinas showed a 63% loss of NeuN+ neurons and T2D retinas lost approximately 43% when compared with nondiabetic human retinas. Few QUIN+ microglia-like cells were seen in nondiabetic retinas, but the numbers increased 18-fold in T1D and 7-fold in T2D in the central retina. In T1D rat retinas, the density of IDO+ microglia increased 2.8-fold and brightness increased 2.1-fold when compared with controls., Conclusions: Our findings suggest that IDO and QUIN expression in the retinas of diabetic rats and humans could contribute to the neuronal degeneration that is characteristic of diabetic retinopathy.

Purpose: To examine the effect of free insulin-like growth factor (IGF) binding protein-3 (IGFBP-3), independent of the effect of insulin-like growth factors, in modulating retinal vascular permeability., Methods: We assessed the ability of a form of IGFBP-3 that does not bind IGF-1 (IGFBP-3NB), to regulate the blood retinal barrier (BRB) using two distinct experimental mouse models, laser-induced retinal vessel injury and vascular endothelial growth factor (VEGF)-induced retinal vascular permeability. Additionally, in vitro studies were conducted. In the animal models, BRB permeability was quantified by intravenous injection of fluorescein labeled serum albumin followed by digital confocal image analysis of retinal flat-mounts. Claudin-5 and vascular endothelial-cadherin (VE-cadherin) localization at interendothelial junctions was studied by immunofluorescence. In vitro changes in transendothelial electrical resistance (TEER) and flux of fluorescent dextran in bovine retinal endothelial monolayers (BREC) were measured after IGFBP-3NB treatment. Acid (ASMase) and neutral (NSMase) sphingomyelinase mRNA levels and activity were measured in mouse retinas., Results: Four days postinjury, laser-injured mouse retinas injected with IGFBP-3NB plasmid demonstrated reduced vascular permeability compared with retinas of laser-injured mouse retinas injected with control plasmid. IGFBP-3NB administration resulted in a significant decrease in laser injury-associated increases in ASMase and NSMase mRNA and activity when compared with laser alone treated mice. In vivo, intravitreal injection of IGFBP-3NB reduced vascular leakage associated with intravitreal VEGF injection. IGFBP-3NB partially restored VEGF-induced in vivo permeability and dissociation of claudin-5 and VE-cadherin at junctional complexes. When IGFBP-3NB was applied basally to bovine retinal endothelial cells (BREC) in vitro, TEER increased and macromolecular flux decreased., Conclusions: Intravitreal administration of IGFBP-3NB preserves junctional integrity in the presence of VEGF or laser injury by reducing BRB permeability in part by modulating sphingomyelinase levels.

Purpose: To examine mural cell differentiation and pericyte ensheathment during human choroidal vascular formation and into adulthood., Methods: Triple- and double-labeled immunohistochemistry (alpha-smooth muscle actin [αSMA], desmin, NG2, calponin, caldesmon, CD44, CD34, and CD39) were applied to human fetal (8-32 weeks' gestation) and adult choroidal and retinal wholemounts and histologic cross-sections. Transmission electron microscopy (TEM) was also undertaken., Results: Early in development CD44+ stem cells also stained with αSMA and CD39, suggesting a common precursor. At 12 weeks' gestation, αSMA+ mural precursor cells, confirmed by TEM, were found scattered and isolated over the primordial vascular tree. During development, αSMA+ cells formed a continuous sheath around large arterioles; in veins there were gaps in αSMA expression. The choriocapillaris had an extensive vascular bed but limited coverage by αSMA+ and NG2+ mural cells. Calponin was expressed only on large vessels, and no caldesmon was detected. Pericyte ensheathment of adult capillaries was 11% for choroid versus 94% for retina. Remarkably, choroidal pericytes had no visible intermediate filaments (IFs) on TEM, though IFs were present in retinal pericytes. Neither retinal nor choroidal pericytes stained with desmin., Conclusions: CD44+ stem cells are involved in the formation of mural cells in the human choroidal vasculature. A marked reduction in pericyte ensheathment of human choroidal vessels suggests a permanently open "plasticity window" and a predisposition to vascular instability and poor autoregulatory ability.

Purpose: Dilated and tortuous vessels (plus disease) in ROP is a grim prognostic indicator of visual outcome. The purpose of this study was to determine whether alterations in pericytes and smooth muscle cells (SMCs), are associated with the pathogenesis of ROP, including plus disease., Methods: Kittens were exposed to either 4 (standard obliterative model) or 2 (modified model) days of hyperoxia, resulting in vaso-obliteration or localized vessel regression, respectively, and returned to room air. The modified model more closely resembles human ROP. Desmin and alpha-smooth muscle actin (SMA) immunohistochemistry and lectin labeling were used to label mural cells and vessels. The desmin ensheathment ratio (DER), a quantitative measure of vessel stability, was determined., Results: In the neovasculature of the standard model and surviving vasculature of the modified model, radial arterioles and venules were dilated and SMCs attenuated. SMA expression on venules was decreased, and the difference in desmin expression normally observed between arterioles and venules was lost, indicating altered SMC differentiation. The DER was reduced in both ROP models, consistent with highly unstable vascular plexuses, receptive to angiogenic and vascular regression signals., Conclusions: The results provide compelling evidence of significant changes in arteriolar and venular SMCs in both experimental models of ROP. The delayed differentiation and apparent dedifferentiation of SMCs during the hypoxic phases would result in an impaired ability to regulate blood flow, contributing to the vasodilation and tortuosity, hallmarks of plus disease. Vessel tortuosity was seen only in the nonobliterative model, suggesting that tortuosity may be due to increased capillary resistance resulting from capillary closure.

Purpose: To identify and apply a range of suitable mural cell markers and undertake an in vivo characterization of pericyte and smooth muscle cell (SMC) differentiation in the developing rat retina., Methods: Pericyte and SMC differentiation was characterized by immunohistochemistry with antibodies to NG2, desmin, alpha-smooth muscle actin (SMA), calponin, and caldesmon., Results: Immature mural precursor cells (MPCs) were scattered throughout the primitive capillary plexus in the rat retina at embryonic day (E)20. The postnatal differentiation of pericytes and arteriolar and venous SMCs followed with distinct intermediate phenotypes. SMC differentiation coincided with selection of major vessels from the primordial capillary bed. Maturation of radial arteriolar SMCs was indicated by the expression of calponin and caldesmon, proteins that play a role in the regulation of SMC contraction. The mere presence of immature mural cells did not confer vessel stability; rather vessel stability in the developing rat retina coincided with caldesmon and calponin expression in arteriolar SMCs., Conclusions: This normative data and the identification of suitable in vivo markers of pericytes and SMCs will allow meaningful interpretation of the changes in these cell types. When examining the role of mural cells in developmental and pathologic vascularization, the results show that there is a need to use multiple-marker immunohistochemistry because of significant mural cell heterogeneity. The observation that the expression of caldesmon and calponin in arteriolar SMCs coincides with resistance to hyperoxia in the developing rat retina, lead us to suggest that maturation of SMCs and their consequent ability to regulate blood flow may play a key role in vessel stabilization.

Purpose: To evaluate evidence for the presence of vascular precursor cells (angioblasts) and astrocyte precursor cells (APCs) in the developing human retina and determine their relationship., Methods: Pax-2/GFAP/CD-34 triple-label immunohistochemistry was applied to four retinas aged 12, 14, 16, and 20 weeks of gestation (WG) to label APCs, astrocytes, and patent blood vessels. APCs are Pax-2(+)/GFAP(-), whereas astrocytes are Pax-2(+)/GFAP(+). Adenosine diphosphatase (ADPase) enzyme histochemistry, which identifies endothelial cells and vascular precursors, was applied to human retinas aged 12, 16, 17, and 19 WG. Nissl stain, a nonspecific cell soma marker, was applied to 14.5-, 18-, and 21-WG retinas. Established blood vessels were visualized with CD34 and ADPase., Results: Topographical analysis of the distribution of Nissl-stained spindle cells and ADPase(+) vascular cells showed that these two populations have similar distributions at corresponding ages. ADPase(+) vascular precursor cells preceded the leading edge of patent vessels by more than 1 millimeter. In contrast, Pax-2(+)/GFAP(-) APCs preceded the leading edge of CD34(+) blood vessels by a very small margin, and committed astrocytes (Pax-2(+)/GFAP(+)) were associated with formed vessels and nerve fiber bundles. Two populations of ADPase(+) cells were evident, a spindle-shaped population located superficially and a deeper spherical population. The outer limits of these populations remain static with maturation., Conclusions: A combination of Pax-2/GFAP/CD34 immunohistochemistry, Nissl staining, and ADPase histochemistry showed that the vascular precursor cells (angioblasts), identified using ADPase and Nissl, represent a population distinct from Pax-2(+)/GFAP(-) APCs in the human retina. These results lead to the conclusion that formation of the initial human retinal vasculature takes place through vasculogenesis from the prior invasion of vascular precursor cells.

Purpose: To characterize the topography of and the cellular processes that underlie vascularization of the human retina., Methods: The vasculature of human eyes obtained from fetuses ranging in age from 14 to 38 weeks of gestation (WG) was examined in Nissl-stained, whole-mount preparations and by anti-CD34 immunohistochemistry., Results: The first event in retinal vascularization, apparent before 15 WG, was the migration of large numbers of spindle-shaped mesenchymal precursor cells from the optic disc. These cells proliferated and differentiated to produce cords of endothelial cells. By 15 WG, some cords were already patent and formed an immature vascular tree in the inner retinal layers that was centered on the optic disc. These processes are consistent with vessel formation by vasculogenesis. Angiogenesis then increased the vascular density of this immature plexus and extended it peripherally and temporally. Maturation of the plexus was characterized by substantial remodeling, which involved the withdrawal of endothelial cells into neighboring vascular segments. The outer plexus was formed as a result of the extension of capillary-sized buds from the existing inner vessels, a process that began around the incipient fovea between 25 and 26 WG., Conclusions: These observations suggest that the formation of primordial vessels in the central retina is mediated by vasculogenesis, whereas angiogenesis is responsible for increasing vascular density and peripheral vascularization in the inner retina. In contrast, the outer plexus and the radial peripapillary capillaries are formed by angiogenesis only. These mechanisms of retinal vascularization appear similar to those of vascularization of the central nervous system during development.

Purpose: To assess the role of vascular endothelial growth factor (VEGF) in the feline model of retinopathy of prematurity (ROP)., Methods: Retinopathy of prematurity was induced in neonatal cats by raising them in an oxygen-enriched (70% to 80%) atmosphere for 4 days to suppress vessel formation and then returning them to room air for 3 to 27 days. In situ hybridization was used to detect the expression of VEGF and its high-affinity receptor, flk-1, in the retina of neonatal cats, and glial fibrillary acidic protein immunocytochemistry was used to assess astrocyte status., Results: The expression of VEGF in the innermost layers of retina fell in hyperoxia and increased on return to room air. Vascular endothelial growth factor expression was transient; it was maximal where vessels were about to form, and it rapidly downregulated after vessels had formed. During the proliferative vasculopathy of ROP, VEGF expression was stronger than in the normally developing retina, and the astrocytes that normally express VEGF degenerated. After the degeneration of astrocytes, VEGF was expressed by neurones of the ganglion cell layer. flk-1 was expressed by intraretinal and preretinal vessels. Supplemental oxygen therapy reduced or eliminated the overexpression of VEGF expression, astrocyte degeneration, and formation of preretinal vessels., Conclusions: Regulation of VEGF by tissue oxygen mediates the inhibition of vessel growth during hyperoxia and the subsequent proliferative vasculopathy. Degeneration of retinal astrocytes creates conditions for the growth of preretinal vessels.

Purpose: To assess the role of oxygen in normal retinal vasculogenesis., Methods: A new preparation for studying cytogenesis in retinal wholemounts was developed. Nuclei of dividing cells were labeled with a monoclonal antibody to bromodeoxyuridine (BrdU), and vascular cells were covisualized with Griffonia simplicifolia lectin. The topography and time course of vasculogenic cell division and vessel formation were determined in the kitten retina during normal development and under experimental hyperoxia and hypoxia., Results: During normal development, vasculogenic cell division was maximal at the leading edge of the forming vessels. Normal vessel formation was initially proliferative, and cell division was high. However, after vessel formation occurred, which presumably relieved tissue hypoxia, the mitogenic process was markedly reduced, and many excess capillary segments underwent retraction. The rate of vasculogenic cell division and vessel formation increased when the inner layers of the retina were made avascular after exposure to hyperoxia, and it decreased when there was an increase in inspired oxygen., Conclusions: The authors have shown that between 17% and 45% oxygen, the extent of vasculogenic cell division is inversely proportional to the level of oxygen in the inspired gas mixture. They have further shown that dividing vascular cells have a peak density in a region proximal to the edge of the forming vasculature. The density is maximal between P7 and P8, a time when formation of photoreceptor outer segment begins, only a few days before the onset of retinal function. These results led the authors to conclude that the stimulus for normal vasculogenesis is a transient but physiological level of hypoxia induced by the increasing activity of retinal neurons.

This study addresses the role of astrocytes in the genesis of retinopathy of prematurity, examined in the feline model of this condition. Evidence is presented that the hypoxia of retinopathy of prematurity, in addition to inducing vasoproliferation, damages the retina directly. Retinal neurons survive the hypoxia, but the astrocytes, which are involved in the formation of the glia limitans of the retinal vessels, degenerate. Astrocytes subsequently recolonize the retina after a delay that matches the period of leakiness of the proliferative vasculature (described in the companion article). Given the evidence from other studies that the barrier properties of vessels are induced by their glia limitans, the authors suggest that the initial lack of barrier properties in the new vasculature is caused by the degeneration of astrocytes and that the subsequent formation of those properties is induced by the astrocytes that recolonize the retina some days later. The observation that astrocytes are more sensitive to hypoxia than neurons, at least in developing tissue, was unexpected. The literature reporting on the damage caused to central nervous tissue by hypoxia is consistent in assessing neurons as more sensitive and glial changes as a reaction to neuronal damage. The sensitivity of astrocytes found in this study and earlier in vitro research suggests that degenerated astrocytes can be replaced and their structural and functional relationships reestablished.

This study documents changes to retinal vasculature during the feline form of retinopathy of prematurity (ROP). The authors describe the closure and obliteration of retinal vessels during exposure to high oxygen, the pattern and tempo of growth of proliferative vasculature, which, after the return of the animal to room air, extends from the optic disc in a spectacular "rosette" pattern, the formation of preretinal vascular growths, and an initial lack of barrier properties in the new vessels. Finally, the response of the vasculature to the relief of hypoxia is reported, including the gradual establishment of barrier properties in the intraretinal vessels, the partial normalization of the proliferative vessels, and the abnormalities that persist. It is suggested that the vascular changes occur in successive stages: closure and obliteration during hyperoxia, vasoproliferation induced by hypoxia, and normalization after the relief of hypoxia with distinct cellular mechanisms and stimuli. It is argued that the same stages can be seen in the human form of ROP; two possible stimuli for the fibroplasia that damages the retina in human ROP are discussed.

Penetrating 180 degrees superior limbal incisions were made on the right eye of four adult New Zealand albino rabbits. The contralateral eye served as control. Corneal touch thresholds (CTT) for the central, superior and inferior cornea (2-3 mm from limbus) were determined 3, 9, 15, 24 and 30 months after surgery. In all animals, the CTT was significantly elevated in the superior region of the cornea throughout the measurement period. CTT was elevated in the central and inferior cornea 3 months following surgery and was not affected in the inferior cornea on all other occasions. The animals were then sacrificed and the corneas subjected to histochemical demonstration of acetylcholinesterase corneal nerves. All rabbits showed a reduction in the number of histochemically detectable stromal nerve trunks in the operated region. These stromal nerve trunks showed regenerative changes including abnormally curved course and a subnormal number of axons within a nerve trunk. Epithelial nerve fiber defects included absence or distorted architecture of the basal epithelial plexus and intra-epithelial terminals. These results indicate that although extensive stromal reinnervation had occurred, the extent and quality of stromal nerves was inadequate to restore a normal epithelial plexus and corneal sensitivity.

The innervation of the adult cat cornea was investigated both psychophysically and histologically. Mean corneal touch threshold (CTT) for 25 adult domestic cats was 43 +/- 9 mg in the center of the cornea and 100 +/- 32 and 94 +/- 33 mg in the superior and inferior cornea, respectively. Gold chloride impregnation showed that the cat cornea is innervated by 16-20 radial nerve trunks that enter the mid-posterior stroma at various sites around the corneal circumference. As these trunks travel anteriorly toward the center of the cornea they give off collaterals that form the anterior stromal and subepithelial plexus. Fibers from the subepithelial plexus penetrate the epithelial basement membrane and give off numerous long fibers that ramify in the basal epithelial layer. Intraepithelial terminals arise from these, penetrating between the epithelial cells, ending with a terminal enlargement at the wing cell level. A distinct pattern of neural organization was found in the periphery of the cat cornea. This consisted of finer nerve fibers that entered the cornea at the subepithelial and basal epithelial levels at numerous sites around the corneal circumference. These fibers branched after a short distance in the cornea and appeared to innervate the anterior stroma and epithelium in the periphery of the cornea. This study thus provides direct evidence of two types of neural organization in the cornea of the domestic cat. Stromal nerves appear to be the main source of innervation to the epithelium in the center of the cornea while conjunctival nerves supply the peripheral epithelium.

Central corneal thickness of both eyes of seven cats was measured hourly for 72 hr using ultrasonic pachometry. The mean corneal thickness was 569 +/- 36 micron (mean +/- SD), and the diurnal variation was 49 +/- 14 micron (8.6% of corneal thickness). In a separate experiment, the corneal thickness of one eye of each of five cats was measured following 2 hr of natural sleep; 2 1/4 hr after eye opening, the corneas had thinned an average of 43 +/- 22 micron. The authors conclude that corneal swelling induced by eye closure during periods of sleep is the prime determinant of the diurnal variation in cat corneal thickness. In studies where corneal thickness is to be monitored over a period of time, it is possible to control against this large diurnal variation by ensuring that the cat is active for a period of two hours prior to pachometry measurements.