Microvascular and cellular responses in the optic nerve of rats with acute experimental allergic encephalomyelitis (EAE).

The optic nerve of rats with EAE was examined at various times to determine the integrity of the blood-brain barrier (BBB) and to assess monocyte-macrophage, T cell, and microglial responses. In naive control animals, leakage of horseradish peroxidase (HRP) and the presence of cells expressing major histocompatibility complex (MHC) class II antigen were evident in the meninges of the retrobulbar optic nerve. In rats with EAE, microglia in the region of the lamina cribrosa and in the regions adjacent to the meninges became activated from day 7 to 8 postinduction (pi). HRP leakage was also evident in the region of the lamina cribrosa from day 7 to 8 pi. On day 8 pi, infiltration of inflammatory cells and Monastral blue leakage were apparent in the myelinated region of the optic nerve. The intensity of these cellular and vascular changes peaked at day 12 pi, when signs of clinical disease became manifest. Monocytes-macrophages expressing MHC class II and the ED1 antigen, together with lymphocytes expressing the alphabetaT cell receptor, constituted the major proportion of cells associated with inflammatory lesions. Thus: (i) the inherent weakness of the BBB as well as the presence of both antigen (myelin) and MHC class II+ cells in the retrobulbar optic nerve are likely susceptibility factors for the frequent involvement of this region in EAE and multiple sclerosis; and (ii) activation of microglia occurs early in the pathogenesis of experimental optic neuritis.

Microvascular and cellular responses in the retina of rats with acute experimental allergic encephalomyelitis (EAE).

The microvascular and cellular responses in the retina during acute EAE were characterized using whole-mount preparations. The earliest detectable event was the accumulation of monocytes and T cells within veins on day 7 postinduction (pi). Mild breakdown of the blood-retinal barrier (BRB), activation of microglia and infiltration of monocytes and T cells into the retinal parenchyma were first evident on days 7 to 8 pi. Monocyte adhesion to the vessel wall and breakdown of the BRB were colocalized in the same vessel segments and occurred predominantly in veins. The marked difference in response observed in the retina versus the myelinated region of the optic nerve suggests that two types of inflammatory cascades are initiated. A mild response, characterised by very low numbers of T cells and monocytes and an absence of expression of MHC class II by resident microglia, is initiated when only small amounts of the encephalitogenic antigen are present in the perivascular space or associated with perivascular antigen-presenting cells. A full blown inflammatory reaction, as observed in the optic nerve, is initiated in the presence of substantial amounts of encephalitogenic antigen. This severe response is characterised by the infiltration of large numbers of CD4+, CD8+ T cells and ED1+ monocytes, and by abundant MHC class II expression by resident microglia as well as other cell types. Thus, MHC class II expression by resident microglia may be a possible effective amplifier mechanism if the encephalitogenic antigen is encountered in the tissue parenchyma.

Factors determining the migration of astrocytes into the developing retina: migration does not depend on intact axons or patent vessels.

Astrocytes migrate into the cat retina from the optic nerve, beginning from embryonic day (E) 52. Once they have entered the retina they concentrate along major axon bundles and fail to enter regions of the retina with high densities of neurones, in particular the area centralis region of the ganglion cell layer. These nonuniformities appear as the astrocytes spread over the retina during development, and in this study we have examined factors that might control their spread. First we examined astrocytes in a retina in which the axon bundles had degenerated following an optic nerve lesion at birth. The area over which astrocytes had spread was normal, suggesting that their spread does not depend on the presence of intact axons. Second, we noted that, despite the degeneration of all ganglion cells following the nerve lesion, astrocytes still did not spread over the area centralis. Their spread is apparently not inhibited by concentrations of neurones. Third, we examined astrocytes in retinas of animals raised in an atmosphere containing 70-80% oxygen, which prevents the formation of retinal vessels. Again, the area over which the astrocytes had spread was normal, suggesting that their spread does not depend on the presence of patent blood vessels. These negative findings led us to compare the distribution of spindle cells (precursors of retinal vasculature) and astrocytes in the cat during development. The close correspondence in their topographical distribution and the earlier spread of the spindle cells lead us to suggest that spindle cells provide a basal lamina component that may guide the migration of astrocytes.

Contact-spacing among astrocytes is independent of neighbouring structures: in vivo and in vitro evidence.

We have examined the morphology of astrocytes and the arrays they form in two situations, in retinas from which ganglion cells and blood vessels have been caused to degenerate, and in vitro. These observations were made to test whether the regularity of the spacing of astrocytes within normal central nervous tissue results from interaction among astrocytes, or from interaction between astrocytes and other elements of that tissue. Both in the partially degenerated cat retina, and in cultures of astrocytes from neonatal rat cortex, astrocytes make and maintain contact with neighbouring astrocytes, yet space their somas apart, giving regularity to the arrays. These results support the hypothesis that the regularity observed in arrays of astrocytes in intact tissue results from an interaction among astrocytes, independent of neighbouring structures, and lead us to suggest that the cell-cell interactions involved in contact spacing serve to distribute astrocytes through the central nervous system, and may, in other tissues, underlie the formation of epithelia.

Structure of the macroglia of the retina: sharing and division of labour between astrocytes and Müller cells.

A detailed comparison is made between astrocytes and Müller cells of the cat's retina, with emphasis on their structural specialisations. Evidence is presented that astrocytes and Müller cells both contribute to the formation of the inner glia limitans of the retina, the glia limitans of vessels, and the glial sheaths of neurones. In particular, it was noted that both astrocytes and Müller cells wrap bundles of ganglion cells axons, that both contribute processes to the glial convergence on the initial segments and node-like structures of axons, and that both wrap the somas of neurones in the ganglion cell layer. Further, it was noted that adherent junctions form between astrocytes, between Müller cells, and between astrocytes and Müller cells, but not between these cells and neurones, or among neurones. These similarities suggest that astrocytes and Müller cells function interchangeably in many respects, and we suggest that they be regarded as variants of macroglia. Quantitative differences between astrocytes and Müller cells were noted in their ensheathment of neurones. In particular, the glial sheaths around the somas of ganglion cells are formed predominantly by Müller cells, and the glial processes attached to node-like specialisations of their axons are formed mainly by astrocytes. One qualitative difference was noted between the two cell classes. The gap junctions which form between astrocytes do not form between Müller cells or between cells of the two classes. From these differences, and previously established features of their shape, orientation, distribution and origin, a hypothesis is developed of the specialisation of macroglia represented by Müller cells.

Differentiation and morphogenesis in pellet cultures of developing rat retinal cells.

We previously developed a reaggregate cell culture system (pellet cultures) in which retinal neuroepithelial cells proliferate and give rise to rod photoreceptor cells (rods) in vitro (Watanabe and Raff, 1990, Neuron 4:461-467). In the present study, we analyzed cell differentiation and morphogenesis in pellet cultures by using both cell-type-specific markers with immunofluorescence and electron microscopy. We demonstrated that, in addition to rods, the other major retinal cell types, including amacrine cells, bipolar cells, Müller cells, and ganglion cells were all present in the pellets, where most were able to develop from dividing precursor cells in vitro. The different cell types in the pellets became organized into two distinct structures: dark rosettes and pale rosettes. The cellular composition of these structures indicated that the dark rosettes correspond to the outer nuclear layer and the pale rosettes to the inner nuclear layer of the normal retina. Ultrastructural studies have indicated that the thin layer of neuronal processes surrounding the dark rosettes correspond to the outer plexiform layer, and the central region of the pale rosettes correspond to the inner plexiform layer of the normal retina. Other features of normal retinal development also occurred in the pellets, including programmed cell death and the formation of inner and outer rod cell segments and synapses. Thus, pellet cultures provide a convenient way to study different aspects of retinal development where one can control the size and the cellular composition of the initial reaggregate.

The role of Müller cells in the formation of the blood-retinal barrier.

We have compared the ability of Müller cells and astrocytes to induce the formation of barrier properties in blood vessels. Müller cells cultured from the rabbit retina, and astrocytes and meningeal cells cultured from the rat cerebral cortex, were injected into the anterior chamber of the rat eye, where they formed aggregates on the iris. We have examined the barrier properties of the vessels in those aggregates and, for comparison, the barrier properties of vessels in the retina, ciliary processes and iris. Two tracers were perfused intravascularly to test barrier properties. The movement of Evans Blue was assessed by light microscopy, and the movement of horseradish peroxidase by light and electron microscopy. Our results indicate that Müller cells share the ability of astrocytes to induce the formation of barrier properties by vascular endothelial cells, and we suggest that Müller cells play a major role in the formation of barrier properties in retinal vessels.

Sensitivity and neural organization of the cat cornea.

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.

Degeneration of astrocytes in feline retinopathy of prematurity causes failure of the blood-retinal barrier.

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.

The effect of oxygen on vasoformative cell division. Evidence that 'physiological hypoxia' is the stimulus for normal retinal vasculogenesis.

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.

Vascularization of the human fetal retina: roles of vasculogenesis and angiogenesis.

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.

Diurnal variation of corneal thickness in the cat.

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.

Vascular changes and their mechanisms in the feline model of retinopathy of prematurity.

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.

Long-term neural regeneration in the rabbit following 180 degrees limbal incision.

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.

Roles of vascular endothelial growth factor and astrocyte degeneration in the genesis of retinopathy of prematurity.

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.

Glial, vascular, and neuronal cytogenesis in whole-mounted cat retina.

A method was developed for detecting cytogenesis in retinal whole-mount preparations by bromodeoxyuridine (BrdU) immunohistochemistry. Because BrdU is a nonspecific marker that labels all cells in the S phase of the cell cycle, it is ideally combined with other cell-specific markers to study the cytogenesis of specific cell types. Double-label protocols to visualize mitotically active astrocytes and cells associated with the forming vasculature have been developed and applied to the retina. This approach revealed that, during normal development of the kitten retina, vascular mitogenesis occurs predominantly in the ganglion cell and nerve fiber layers, where the inner retinal plexus is formed by a process involving transformation of mesenchymal precursor cells and division of vascular endothelial cells. The peak density of vascular mitogenesis moved in a central-to-peripheral manner and was associated with the leading edge of the forming capillary plexus. A small number of dividing vascular endothelial cells was also associated with angiogenesis, the process responsible for the formation of the outer retinal plexus, vessels at the area centralis, and the radial peripapillary capillaries. Cytogenesis associated with astrocytes occurred in the ganglion cell and nerve fiber layers but was apparent predominantly at or close to the optic nerve head. Confirming earlier studies, neuronal mitogenesis was shown to occur predominantly at the ventricular zone, first at the area centralis and spreading peripherally with increasing maturity. A second region of neuronal cytogenesis, at the subventricular zone, was also apparent. Tissue hyperoxia decreased the rate of vasculogenic cell division but had no apparent effect on neurogenic or astrocytic cell division. Four distinct zones of cell generation were therefore identified within the retina, each associated with either glial, vascular, or neuronal cytogenesis. Thus, BrdU immunohistochemistry in whole-mounted retinal preparations offers a fast and reliable alternative to [3H]thymidine autoradiography for the study of the topography of cytogenesis during development.

Changes in corneal endothelial morphology in cats as a function of age.

A cross-sectional study of changes in cat corneal endothelial cell morphology with age was conducted. The central corneal endothelium of 12 kittens and 70 adult cats was photographed using specular microscopy. Endothelial cell density (ECD), coefficient of variation of cell size (used as an index of polymegethism), and cellular shape factor (perimeter 2/area) were determined for each animal and analysed as a function of age. We found a rapid non-linear decrease in ECD and polymegethism in the first nine months of post-natal life. Subsequently there was a slight central cell loss of 11 cells/mm2 or 0.37% per year during adult life which was not statistically significant. However, polymegethism increased significantly with age during adult life. The shape factor for endothelial cells was 13.61 throughout adult life, indicating that the cat corneal endothelium consists predominantly of six-sided cells. Possible explanations for the finding of no significant decrease in cell density with age could include the higher peripheral ECD in the cat which may compensate for central loss, the short life expectancy of the cat and the large individual variation in corneal diameter.

Development of retinal vasculature in the cat: processes and mechanisms.

Two principal processes can be distinguished in the development of the retinal circulation in the cat. One process, which forms most of the inner layer of vasculature, involves three stages. First, beginning prior to E (embryonic day) 26, spindle cells of mesenchymal origin spread over the inner surface of the retina. Second, beginning at approximately E48, a network of coarse capillaries forms, apparently derived from spindle cells. Third, major vessels differentiate from the capillary plexus, and the capillaries become thinner and more widely spaced. All three stages begin at the optic disc and spread towards the margin of the retina. The other process involves budding of capillary sized vessels from existing vasculature. This process forms the inner layer of vasculature at the area centralis, the outer layer of vasculature, and the radial peripapillary capillaries. It begins between P (postnatal day) 7 and P10 at the area centralis and spreads to the margins of the retina. The radial peripapillary capillaries form at a later stage (P20). The different topographies of the two processes suggest that they are controlled by distinct mechanisms. In the first process, the formation of vessels follows a pattern set by the early migration of spindle cells. In the second process, the vessels form in a pattern determined by the metabolic needs of the developing retina.

Endometriosis of the inguinal region: magnetic resonance imaging (MRI) findings.

A case of endometrioma of the right inguinal canal region, diagnosed preoperatively, is presented. The diagnosis was made on the basis of cyclical symptoms relating to menstrual periods, in combination with demonstration of blood products within an enhancing focal lesion in the inguinal region with magnetic resonance imaging. The case presented here is unique, as it is the first case, to our knowledge, of an endometriotic lesion in the inguinal canal to demonstrate the characteristic 'shading sign' at magnetic resonance imaging.

Concentration of astrocytic filaments at the retinal optic nerve junction is coincident with the absence of intra-retinal myelination: comparative and developmental evidence.

The structure of the lamina cribrosa (LC) and astrocytic density were examined in various species with and without intra-retinal myelination. Sections of optic nerve from various species were stained with Milligan's trichrome or antibodies to glial fibrillary acidic protein, myelin basic protein (MBP) and antibody O4. Marmoset, flying fox, cat, and sheep, which lack intraretinal myelination, were shown to possess a well-developed LC as well as a marked concentration of astrocytic filaments distal to the LC. Rat and mouse, which lack intraretinal myelination, lacked a well-developed LC but exhibited a marked concentration of astrocytic filaments in this region. Rabbit and chicken, which exhibit intraretinal myelination, lacked both a well-developed LC and a concentration of astrocytes at the retinal optic nerve junction (ROJ). A marked concentration of astrocytes at the ROJ of human fetuses was also apparent at 13 weeks of gestation, prior to myelination of the optic nerve; in contrast, the LC was not fully developed even at birth. This concentration of astrocytes was located distal to O4 and MBP immunoreactivity in human optic nerve, and coincided with the site of initial myelination of ganglion cell axons in marmoset and rat. Myelination proceeded from the chiasm towards the retinal end of the human optic nerve. Moreover, the outer limit of oligodendrocyte precursor cells (OPC) migration into the rabbit retina was restricted by the outer limit of astrocyte spread. These observations indicate that a concentration of astrocytic filaments at the ROJ is coincident with the absence of intraretinal myelination. Differential expression of tenascin-C by astrocytes at the ROJ appears to contribute to the molecular barrier to OPC migration (see Bartsch et al., 1994), while expression of the homedomain protein Vax 1 by glial cells at the optic nerve head appears to inhibit migration of retinal pigment epithelial cells into the optic nerve (see Bertuzzi et al., 1999). These observations combined with our present comparative and developmental data lead us to suggest that the astrocytes at the ROJ serve to regulate cellular traffic into and out of the retina.