[Development of the iridocorneal angle and congenital glaucoma]. / Entwicklung des Kammerwinkels und kongenitales Glaukom.

The trabecular meshwork originates from cells of the neural crest which migrate to the iridocorneal angle during embryonic and fetal development of the eye. Correct morphogenesis of trabecular outflow pathways requires the differentiation of the cells to a porous and lamellate meshwork as well as the ingrowth of Schlemm's canal and posterior movement of the iris root. A failure in these processes is responsible for primary congenital or infantile glaucoma which presents with increased resistance to aqueous humor outflow resulting in increased intraocular pressure. Most cases appear to be of a sporadic nature but hereditary cases are often caused by mutations in the CYP1B1 gene which encodes for the enzyme cytochrome P450 1B1. Mutations cause a reduction in enzymatic activity which probably leads to diminished turnover of an as yet unidentified metabolite taking part in the signaling processes essential for formation of the trabecular meshwork and Schlemm's canal. More rarely, mutations in latent transforming growth factor beta binding protein 2 (LTBP2) or in the transcription factor FOXC1 have been described as causative for primary congenital glaucoma.

Human anterior chamber angle development without cell death or macrophage involvement.

PURPOSE: The iridocorneal angle in the mammalian eye including the trabecular meshwork (TM) develops from undifferentiated mesenchyme/neural crest between the iris root and cornea. The precise mechanisms underlying anterior angle development are unclear, and the contribution of cell death and phagocytic resorption by macrophages in angle development is controversial. In this study, we examined the human anterior chamber angle during various stages of development for evidence of cell death and phagocytic resorption. METHODS: Eyes from the human fetus (F) of 7, 8, 9, 10, 11, 13, 15, 18, 19, 21, 22, 23, and 27 weeks as well as eyes from 5- and 11-month-old children and donors 24, 48, and 67 years of age were obtained. Formalin-fixed and paraffin-embedded sections were examined by hematoxylin and eosin (H&E) staining. Immunohistochemistry was performed using polyclonal antibodies against CD68. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) labeling was also performed to evaluate cell death. RESULTS: By light microscopy, the development of human angle structures appeared to progress as previously described. Histological evidence of cellular death or resorption by macrophages was not observed. Furthermore, the chamber angle tissues did not stain with CD68 at any stage of development. Few CD68 positive cells were observed in the iris stroma and the anterior ciliary body between fetal weeks 10 and 18 (F10w and F18w). TUNEL labeled nuclei were not detected in the anterior chamber angle in any fetal or infant eyes. By contrast, TUNEL positive nuclei in TM cells were observed in the examined adult donor specimens. CONCLUSIONS: The results suggest that at the time points examined, neither cell death nor phagocytic resorption with macrophages appear to play a role in the development of the human anterior chamber angle.

Perinatal hypoxia induces anterior chamber changes in the eyes of offspring fish.

Hypoxia is a consistent challenge for aquatic animals. It is a pressing environmental problem; hypoxia can cause cranial edema and ovarium dysfunction in fish. Although several studies have reported the effect of hypoxic insult to the visual system, the hypoxic effect on perinatal animals and in particular their offspring has yet to be elucidated. In this study, activated caspase-3 activity was investigated using immunohistochemistry in order to examine the perinatal hypoxic damage in offspring fish. Offspring were divided into groups based on different time points of sacrifice. This allowed assessment of ocular development for different age groups. The results indicated that perinatal hypoxia induced ocular developmental defects in the offspring. The defects took the form of trabecular cell death and fibre degeneration, corneal thinning and lens fibre derangement. A concomitant change in intraocular pressure was recorded by tonometer in the experimental animals compared with the controls. Further investigation should be initiated to develop strategies to prevent developmental disability due to perinatal hypoxia and to increase survivability of the offspring.

[Glaucoma with aniridia and isolated congenital glaucoma in siblings: contribution and limits of genetics]. / Glaucome secondaire à une aniridie et glaucome congénital isolé dans une même fratrie: apports et limites de la génétique.

INTRODUCTION: Congenital glaucoma associated with aniridia and primary congenital glaucoma are regarded as different entities. Indeed, the abnormalities of the angle's structures as well as the genes involved are different. We report the observation of two sisters presenting these two types of glaucoma with particular attention paid to the importance and the difficulty of genetic counseling. OBSERVATIONS: Child L, with no particular family history, had presented bilateral aniridia complicated by bilateral glaucoma since birth. In addition to medical and surgical treatment, general and genetic investigations were undertaken that revealed no abnormalities. No microdeletion of the gene PAX6 responsible for the aniridia was found. Consequently, the genetic advice was in favor of a second pregnancy for this couple. At birth, L's sister also presented bilateral congenital glaucoma, which was isolated, without aniridia. New genetic investigations were carried out but no abnormalities in PAX6, nor in FOXC1 or PITX2 involved in the development of the anterior chamber, were found. Moreover, the haplotypes for aniridia locus AN2 inherited by the two sisters were different, proof that this gene could not be responsible for the glaucoma. DISCUSSION: At L's birth, the hypothesis retained was that she was a sporadic case whose gene mutation could not be identified (which happens in 50% of sporadic cases). The risk for the second pregnancy was negligible, although not null. The primary congenital glaucoma presented by L's sister remains unexplained in the context of aniridia and the role of the PAX6 gene was eliminated. The study of PITX2 and FOXC1 genes involved in anterior segment dysgenesis proved that they were also not involved. Thus, this observation evokes the responsibility of a gene other than PAX6 in aniridia, which could also have a role in isolated congenital glaucoma. CONCLUSION: Analysis of congenital pathologies from a more genetic than clinical point of view seems to progressively break down the barriers established between the various phenotypes of hereditary congenital anomalies. Even if the association of aniridia and primary congenital glaucoma in siblings is reported here for the first time, it does not appear so extraordinary if one considers the complexity of the anterior chamber's development, which involves many genes, most of them still unidentified to date.

The mouse anterior chamber angle and trabecular meshwork develop without cell death.

BACKGROUND: The iridocorneal angle forms in the mammalian eye from undifferentiated mesenchyme between the root of the iris and cornea. A major component is the trabecular meshwork, consisting of extracellular matrix organized into a network of beams, covered in trabecular endothelial cells. Between the beams, channels lead to Schlemm's canal for the drainage of aqueous humor from the eye into the blood stream. Abnormal development of the iridocorneal angle that interferes with ocular fluid drainage can lead to glaucoma in humans. Little is known about the precise mechanisms underlying angle development. There are two main hypotheses. The first proposes that morphogenesis involves mainly cell differentiation, matrix deposition and assembly of the originally continuous mesenchymal mass into beams, channels and Schlemm's canal. The second, based primarily on rat studies, proposes that cell death and macrophages play an important role in forming channels and beams. Mice provide a potentially useful model to understand the origin and development of angle structures and how defective development leads to glaucoma. Few studies have assessed the normal structure and development of the mouse angle. We used light and electron microscopy and a cell death assay to define the sequence of events underlying formation of the angle structures in mice. RESULTS: The mouse angle structures and developmental sequence are similar to those in humans. Cell death was not detectable during the period of trabecular channel and beam formation. CONCLUSIONS: These results support morphogenic mechanisms involving organization of cellular and extracellular matrix components without cell death or atrophy.

Axial growth of the fetal eye and evaluation of the hyaloid artery: in utero ultrasonographic study.

The aims of this prospective, cross-sectional study were to report axial ocular growth during human gestation, to determine the presence of the hyaloid artery (HA) and its blood flow, and to provide a timetable for HA regression. The study group comprised 231 low-risk singleton pregnancies between 14 and 38 weeks' gestation. Ocular axial length (OAL), anterior chamber depth (ACD) and posterior chamber depth (PCD) were measured using high-resolution ultrasound. The growth of these eye segments in correlation with gestational age (GA) was established. The presence of the HA and its regression were determined. By using power Doppler, ultrasound blood flow within the HA was estimated. HA regression is a gradual process that is not evident before 18 weeks' gestation. In all fetuses beyond 29 weeks' gestation, no HA could be detected (P<0.001). Blood flow within the HA was documented only until the 16th week of gestation. The correlation coefficients, r=0.924, 0.784 and 0.929, for OAL, ACD and PCD, respectively, were found to be highly statistically significant (P<0.0001). The present data offer normative measurements of the fetal axial eye lengths, timetable for HA regression and flow cessation.

Formation of corneal endothelium is essential for anterior segment development - a transgenic mouse model of anterior segment dysgenesis.

The anterior segment of the vertebrate eye is constructed by proper spatial development of cells derived from the surface ectoderm, which become corneal epithelium and lens, neuroectoderm (posterior iris and ciliary body) and cranial neural crest (corneal stroma, corneal endothelium and anterior iris). Although coordinated interactions between these different cell types are presumed to be essential for proper spatial positioning and differentiation, the requisite intercellular signals remain undefined. We have generated transgenic mice that express either transforming growth factor (alpha) (TGF(alpha)) or epidermal growth factor (EGF) in the ocular lens using the mouse (alpha)A-crystallin promoter. Expression of either growth factor alters the normal developmental fate of the innermost corneal mesenchymal cells so that these cells often fail to differentiate into corneal endothelial cells. Both sets of transgenic mice subsequently manifest multiple anterior segment defects, including attachment of the iris and lens to the cornea, a reduction in the thickness of the corneal epithelium, corneal opacity, and modest disorganization in the corneal stroma. Our data suggest that formation of a corneal endothelium during early ocular morphogenesis is required to prevent attachment of the lens and iris to the corneal stroma, therefore permitting the normal formation of the anterior segment.

Ocular abnormalities in transgenic mice harboring mutations in the type II collagen gene.

PURPOSE: To characterize the morphological changes in the eyes of transgenic mice harboring different mutations in type II collagen gene to elucidate the function of this collagen in the eye, and to find out whether these animals could function as models for the human arthro-ophthalmopathies of the Kniest, Stickler and Wagner types. METHODS: Three genetically engineered mouse lines representing two types of mutations in the triple-helical domain of type II collagen and their nontransgenic littermates used as controls were analyzed on day 18.5 embryonic development. After genotyping by polymerase chain reaction (PCR) and Southern hybridization the embryos were prepared for routine histology. Polarization microscopy was done on hyaluronidase-treated sections. RESULTS: Histological analysis revealed several genotype-dependent abnormalities in the eyes of the transgenic mice. Most striking changes were observed in the vitreous architecture; in one line of mice the vitreous was tightly packed in the posterior region of the vitreous space with thick fibrils, empty cavities and dense membrane-like material. The other mutation resulted in reduced filament density of the vitreous. In the most severely affected phenotype the internal limiting membrane was detached from the retinal layers and was markedly thickened, and the posterior lens capsule was thickened. The anterior chamber was shallow or absent in all transgenic lines but was well formed in the normal animals. Changes were also observed in the lens, corneal and scleral structures. CONCLUSIONS: The ocular changes observed in transgenic mice harboring mutations in type II collagen gene show similarities to the human ocular findings in Kniest dysplasia, and in Stickler and Wagner syndromes. We therefore propose that these animals could serve as models for systematic analysis of vitreoretinal degeneration and other abnormalities, as seen in these syndromes.

Glial cell line-derived neurotrophic factor enhances survival and growth of prenatal and postnatal spinal cord transplants.

Glial cell line-derived neurotrophic factor was first described as a trophic factor for developing dopamine neurons. However, it has been shown that glial cell line-derived neurotrophic factor messenger RNA is also expressed in several areas of the developing brain and spinal cord, suggesting that it may have additional roles in the nervous system. Intraocular transplantation of neural tissue provides a unique method to examine in vivo effects of trophic factors. We have therefore studied the effects of glial cell line-derived neurotrophic factor on spinal cord survival and development following grafting to the anterior chamber of the eye of adult rats. We used spinal cord tissue from fetal stages (embryonic days 14 and 18) and postnatal days 1 and 14 as donors. The spinal cord tissue was allotransplanted to the anterior eye chamber of Sprague-Dawley host rats after incubation in buffered saline containing 100 micrograms glial cell line-derived neurotrophic factor/ml or 100 micrograms cytochrome C/ml. One group of postnatal day 1 spinal cord grafts was also treated with concentrations of 20 and 10 micrograms glial cell line-derived neurotrophic factor/ml. In all cases, 5 microliters of the same solution was injected into the anterior eye chamber on postgrafting days 5, 10, 15 and 20 (total amounts 0.5, 0.1 and 0.05 microgram/eye/injection, respectively). We found that all glial cell line-derived neurotrophic factor-treated spinal cord grafts grew more than controls. The effect of glial cell line-derived neurotrophic factor was most prominent in grafts from newborn rats. In these grafts we found a dose-dependent effect of glial cell line-derived neurotrophic factor on growth. Moreover, grafts treated with the highest dose (0.5 microgram) grew to sizes exceeding the initial size at transplantation. In these transplants we also found greater numbers of large neurons compared to controls. Glial fibrillary acidic protein immunoreactivity, in contrast, showed increased gliosis in controls. Similar results were found with syngeneic spinal cord postnatal day 1 grafts in Fisher hosts. Spinal cord tissue grafts from two-week-old rats treated with the highest glial cell line-derived neurotrophic factor dose every fifth day, through day 35 postgrafting, responded with increased growth and less necrotic tissue compared with controls; however, we could not detect neurofilament immunoreactivity in these transplants. Taken together, these results suggest that glial cell line-derived neurotrophic factor may be a potent trophic factor for neurons in the spinal cord and in spinal cord transplants. Of particular importance is that glial cell line-derived neurotrophic factor treatment can be used to obtain survival of postnatal spinal cord tissue, that would otherwise show minimal or no survival. Thus, glial cell line-derived neurotrophic factor allows successful transplantation of more mature spinal cord tissue, which may have important implications for both basic and clinical neuroscience.

Apoptosis during macrophage-dependent ocular tissue remodelling.

We have characterized the nature and pattern of cell death during regression of the pupillary membrane, a developmentally transient capillary network found in the anterior chamber of the eye. This analysis has revealed that the cellular components of the pupillary membrane include vascular endothelial cells in an intricate network of fine capillaries as well as attendant macrophages. The capillaries are situated on the anterior surface of the lens and held in relative position by a cobweb-like meshwork of extracellular matrix fibres that regress along with the cellular components of this structure. Cell death during regression of the pupillary membrane is characteristic of apoptosis. Specifically, apoptotic bodies containing condensed chromatin can be observed in vascular endothelial cells and genomic DNA isolated from the pupillary membrane shows the nucleosomal fragmentation pattern typical of apoptotic cells. Using a method for labelling fragmented DNA in tissue preparations (TUNEL), we have assessed the overall pattern of apoptotic cell death during pupillary membrane regression. We find that apoptosis occurs either in single cells in healthy vessels or synchronously along the entire length of a capillary segment. Both morphological and TUNEL analysis indicate that capillary regression occurs from junction to junction one segment at a time. We propose a model to explain the pattern of capillary regression observed and conclude from these and previous experiments (Lang and Bishop (1993) Cell 74, 453-462), that during regression of the pupillary membrane, the macrophage elicits target cell death by inducing apoptosis.

[Ocular anomalies in Alagille's syndrome]. / Anomalies oculaires dans le syndrome d'Alagille.

The authors examine a family, in which two brothers were affected by a severe expression of arteriohepatic dysplasia (ADH, Alagille syndrome), an autosomal dominant disorder associated with intrahepatic cholestasis, characteristic facial appearance, congenital embryotoxon. One of these two cases presented a keratoconus and both had retinal pigmentary degeneration with pigment clumping. The father showed a benign expression of ADH, including the characteristic facial appearance and posterior embryotoxon. Another brother presented only retinal pigmentary abnormalities and a bilateral arcus senilis-like corneal opacity, without any other clinical sign of ADH. The presence of posterior embryotoxon in all the cases of Alagille syndrome confirms that this sign is a hallmark of ADH, also in its benign expression.

[Anterior chamber cleavage syndromes]. / Les syndromes de clivage du segment antérieur.

Authors analyse the anatomic findings in the anterior chamber cleavage syndrome and emphasize necessity of genetics (heredity being well known) and supervision for detecting glaucoma which occurs in about 50% of these patients and usually appears between 5 and 30 years of age. (Juvenile glaucoma).

A morphological study of the inner surface of the anterior chamber angle in pre and postnatal human eyes.

The morphological changes in the inner surface of the human anterior chamber angle during pre and postnatal development were studied by light microscopy, scanning and transmission electron microscopy. Seventeen human foetal eyes (12-22 weeks) and ten infant and juvenile eyes (2.5 months--8 years) were investigated with the aim of establishing whether the trabecular meshwork is covered by an uninterrupted membrane at any stage in development. In 12-14 week old foetal eyes cuboidal corneal endothelial cells cover the inner surface of the anterior half to one third of the trabecular anlage. In this region there is a transition to more flattened uveal trabecular endothelial cells. Only rarely did corneal endothelial cells extend completely to the angle apex. The transition zone between corneal and uveal trabecular endothelial cells becomes located more anteriorly as development progresses. Intercellular gaps, which occur between uveal trabecular endothelial cells as early as 12-14 weeks in development, enlarge and become more frequent during development thus providing a route of communication between the anterior chamber and the developing intertrabecular spaces or channels. In the first months of life only a few cord-like uveal trabeculae orientated predominantly in a meridional direction overlie the more lamellate corneoscleral trabeculae. By the second year typical uveal trabeculae are more prominent and form a web-like arrangement. This is accompanied by a gradual decrease in the frequency of the cytoplasmic extensions of uveal trabecular endothelial cells which circumscribe the intratrabecular gaps. The timing of this remodelling and maturation appears to be remarkably variable between individuals. The implications of these findings on the theories of the pathogenesis of congenital glaucoma are discussed.

A re-appraisal of the development of the anterior chamber.

The development of the anterior chamber passes through distinct well orchestrated phases. It is a dynamic transition from an embryonic cleft to the adult anterior chamber. The cornea, lens, tunica vasculosa lentis, the iris, the ciliary epithelium, and the trabecular meshwork all play important roles in the development of the anterior chamber. Apart from these morphological changes that occur, the hydrodynamics of the transudate (from the tunica vasculosa lentis) and the aqueous humor from the ciliary epithelium appear to be essential components in the maintenance of the integrity of the anterior and posterior chambers.

A morphologic and morphometric analysis of the aqueous outflow system of the developing cat eye.

The cellular and tissue changes accompanying the development and growth of the aqueous outflow system of the cat were investigated by quantitative light microscopy and by scanning and transmission electron microscopy. As in primates, the trabecular beams and sheets of the cat aqueous outflow system developed by reorganization of cells and extracellular matrix within the tissue filling the anterior chamber angle recess. Enlargement and coalescence of intercellular spaces gave rise to intertrabecular channels. From 3 to 9 days after birth, communications were established between the anterior chamber and intertrabecular spaces by perforation and resorption of tissue which initially covered the angle apex and appeared to be a peripheral extension of Descement's membrane and the corneal endothelium. Macrophage-like cells could be involved in this process. A rapid increase in the volume of the intertrabecular spaces and in the number of trabecular cells coincided with the opening of the trabecular meshwork to the anterior chamber. The trabecular meshwork grew 150-fold in volume from birth to adulthood, mainly as a result of a similar-fold expansion of its connective tissue components. The volume of the intertrabecular spaces increased 24-fold and trabecular cell number increased 14-fold during this same period. The disproportionate increase in volume of the various components of the trabecular meshwork was responsible for the decreased cell density and rarefaction displayed by this tissue as development progressed. Development of the aqueous outflow system of the cat is thus a complex, but highly co-ordinated, process, that depends on continued proliferation of cells and extracellular matrix, a progressive ordering of these components, and selective atrophy and removal of specific tissue components.