Neurological outcome in school-age children after in utero exposure to coumarins.

The effect of prenatal exposure to coumarins (acenocoumarol, phenprocoumon) on neurological outcome was assessed in a cohort of 306 children aged 7-15 years. Findings were compared with those in a non-exposed cohort of 267 children, matched for sex, age, and demographic region. We used a neurological examination technique which pays special attention to minor neurological dysfunction (MND). None of the children was found to be neurologically abnormal. However, exposure to coumarins during gestation increases the risk for MND in children of school age, odds ratio (OR) 1.9 (CI(95) 1.1-3.4), predominantly after exposure in the second or third trimester, odds ratio 2.1 (CI(95) 1.2-3.8). We found a dose-response relationship with an odds ratio of 1.2 (CI(95) 1.0-1.5) per mg coumarin derivative prescribed per day. The results suggest that coumarins have an influence on the development of the brain which can lead to mild neurological dysfunctions in children of school age.

Coumarins during pregnancy: long-term effects on growth and development of school-age children.

Anticoagulation during pregnancy is complicated because of potential risks for mother and foetus. Unfractionated or low-molecular-weight heparin is used for most anticoagulant indications. Its efficacy, however, in pregnant women with prosthetic heart valves is questioned, therefore coumarins are preferred for this indication. We studied long-term effects of prenatal coumarin-exposure on growth and on neurological, behavioural and cognitive development in 274 school-age children in comparison with 231 age-matched non-exposed controls. No major abnormalities were found. The exposed children had an increased risk for minor neurological dysfunction and for a low intelligence quotient (IQ below 80). The risk for a combination of two or more (minor) abnormalities was higher for the exposed children, RR = 7.6. We conclude that prenatal exposure to coumarins is associated with an increased risk for disturbances in development in school-age children. However, for the vast majority of children there is no clinical significant effect on growth and long-term development.

In utero exposure to coumarins and cognition at 8 to 14 years old.

OBJECTIVE: To assess the cognitive abilities in school-aged children who have been exposed to coumarins in utero. BACKGROUND: Coumarin derivatives are an effective option for anticoagulant therapy in pregnant women. However, case reports describe anomalies of the fetal central nervous system after in utero exposure to coumarins. It is unclear whether prenatal exposure has an effect on cognitive functioning later in childhood. METHODS: The exposed cohort consisted of 291 children from mothers who were prospectively registered because of coumarin treatment during pregnancy. The nonexposed cohort included 253 age-matched peers. An IQ was estimated using subtests of the Weschler Intelligence Scale for Children-Revised. Educational achievement was examined with tests for reading, spelling, and arithmetic. In addition, schoolteachers were asked to judge performance on language and arithmetic. The observers were not aware of the exposure status of the child. RESULTS: No differences in mean IQ were found between the exposed and nonexposed cohort (mean difference: -1.1; 95% confidence interval [CI]: -3.2-1.1), but an IQ score below 80 was found in 11 children in the exposed compared with 3 children in the nonexposed cohort (odds ratio [OR] = 3.1; CI: 0.8-11.6). Regarding the tests for educational achievement, exposed children as a group performed as well as nonexposed controls. Exposed boys, in comparison with nonexposed boys, showed a higher frequency of poor performance on reading (OR = 2.9; CI: 1.1-7.4) and spelling (OR = 2.5; CI: 1.0-6.0). CONCLUSION: Cognitive functioning in coumarin-exposed children does not differ from nonexposed controls, but a minority of children seem to be prone to the potential negative effects of coumarins during pregnancy.

Behavioural outcome of school-age children after prenatal exposure to coumarins.

In utero exposure to coumarin derivatives may affect the development of the central nervous system of the child, irrespective of the period of exposure in pregnancy. Little is known about effects on development in the long term. The aim of the present study was to determine whether prenatal exposure to coumarins affects behavioural outcome in children at school age. Behavioural outcome was assessed in a cohort of 305 exposed children, aged 7-15 years. Findings were compared with those in a cohort of 263 non-exposed controls, matched for sex, age, and demographic region. Behaviour was rated by parents and teachers using standardized questionnaires: the Groningen Behaviour Checklist Family situation (GBF) and the Groningen Behaviour Checklist School situation (GBS), respectively. The findings of the GBF were supported by the results of the GBS, filled in by teachers who were blind for the exposure status of the child. In comparison to the non-exposed children, the coumarin-exposed children scored lower on the cluster 'positive task orientation' (GBF P<0.05, GBS P<0.01), they scored higher on 'emotional instability' (GBF P<0.001, GBS P<0.05), and they had more problems on the social clusters (P<0.01). Based on the results of both questionnaires, we conclude that behavioural development may be negatively influenced in school-age children after in utero exposure to coumarins, leading to less favourable task-oriented and social-emotional behaviour. However, the frequency of clinically relevant 'problem behaviour' (GBF) was not increased in relation to coumarin exposure, the odds ratio was 1.2 (CI(95) 0.7-1.8).

The human hyaloid system: cell death and vascular regression.

The present study had investigated the roles of apoptosis and necrosis in the regression of the human fetal hyaloid vasculature. Normal human fetal hyaloid specimens (n = 67) ranging from 10 to 20 weeks' gestation were studied. Specimens were either immunolabeled with anti-von Willebrand factor and major histocompatibility complex class I antibodies or investigated using the terminal-deoxyribonucleotidyl transferase-mediated dUTP-biotin DNA nick-end labeling technique. A fluorescent DNA-binding dye acridine orange/ethidium bromide mixture was also applied to unfixed flat mounts of hyaloid vasculature and some specimens were processed for transmission electron microscopy. Vascular regression including cell loss in the connecting vessels, stretching and thinning of the vasa hyaloidea propria, tunica vasculosa lentis and the pupillary membrane was clearly evident after 13 weeks' gestation. Cresyl violet staining revealed condensed cells and pyknotic bodies throughout the hyaloid system; cell death occurred either in single cells or along small capillary segments associated with vascular regression. Acridine orange/ethidium bromide staining showed DNA condensation at early and late stages of cell death. Similarly, DNA nick-end labeling was positive in endothelial cells, pericytes and vessel and non-vessel associated hyalocytes. The observation of hyalocytes juxtaposed to cytolysed endothelial cells may indicate a role for these cells in vascular regression. Features of apoptosis were more evident during early vascular regression whilst necrosis was increasingly evident at later stages.

Growth until puberty after in utero exposure to coumarins.

Anticoagulation with coumarins is an effective therapy during pregnancy. Fetal exposure to coumarin derivatives during the first trimester, however, is associated with skeletal anomalies (warfarin or coumarin embryopathy). Information about long-term effects of prenatal coumarin exposure on the skeletal development is not available. We investigated growth and body proportions at school age of children exposed to coumarins in utero. A blind population-based cohort study was conducted on 307 exposed children and 267 non-exposed controls ages 8-15 years. The exposed cohort was based on a prospective registry of coumarin-treated pregnant women. Anthropometric data included height, weight, head circumference, and measurements to evaluate body proportions. The mean height of exposed children did not differ from that of the non-exposed children (mean difference 0.01 SD). In addition, no differences were found for the proportional measures. As a group, children exposed in the first trimester showed no evidence of growth impairment. Two children in this group, however, were born with signs of coumarin embryopathy and one of these displayed a deficit in height at school age. Long-term growth was not affected by a high cumulative dosage or exposure after the first trimester. We conclude that, when exposure during the first trimester is avoided, coumarin therapy during pregnancy has no demonstrable risk for the child's skeletal development.

Human retinal microglia: expression of immune markers and relationship to the glia limitans.

The immunoreactivity, morphology and relationship to the glia limitans of microglia were investigated in flatmounts and sections of normal human retina, using immunogold histochemistry, electron microscopy (EM), and antibodies directed against CD45, major histocompatability complex class I (MHC-I), MHC-II, and human macrophage antigens. Immunoreactivity was evident for all antibodies tested, including MHC-I, which labeled both microglia and retinal vascular endothelium. Most consistent labeling was obtained using antibodies to CD45, MHC-II, and anti-human macrophage (S22) antigen. Immunoreactive cells were seen in the perivascular space (perivascular cells), where they were closely adherent to the vessel profile, and in the retinal parenchyma (microglia). Some parenchymal microglia were also vessel associated and by EM were seen to be closely related to the glia limitans (paravascular microglia). Paravascular microglia were shown by optical densitometry, to express higher levels of MHC antigens than neighboring, non-vessel associated, parenchymal microglia. In addition, paravascular microglia were macrophage (S22) antigen positive, while other parenchymal microglia did not express macrophage antigens. Quantitative data indicate that similar populations of microglia are immunoreactive to CD45, MHC-I, and MHC-II, while relatively few microglia (approximately 10%) are immunoreactive for human macrophage (S22) antigens, supporting previous suggestions that microglia are a heterogeneous population.

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.

Angiogenesis in normal human retinal development: the involvement of astrocytes and macrophages.

Recent studies have suggested a role for mononuclear phagocytes series (MPS) cells in neovascularisation associated with retinal pathology and experimentally induced subretinal neovascularisation. The present study is concerned with the normal development of the human retinal vasculature. Morphological details are provided of developing vascular structures including the formation of tight junctions and canalisation of angioblast cords. The relationships of astrocytes and pericytes to developing structures and the presence of a perivascular collagenous matrix are described. Ultrastructural and histochemical analyses reveal an association between MPS cells and developing vascular structures. It is suggested that MPS cells may influence angiogenesis in normal retinal development, as well as in retinal pathology.

Early differentiation of ganglion, amacrine, bipolar, and Muller cells in the developing fovea of human retina.

We examined the differentiation and maturation of neurons and glia of the inner nuclear layer (INL) and ganglion cell layer (GCL) in the retina of a human fetus of 15 weeks gestation. Serial, ultrathin sections were cut from a resin-embedded specimen from the posterior pole of the retina. The region of the putative fovea was defined by the absence of rod photoreceptors from the outer nuclear layer; only sections through the putative fovea were studied. Cell somata were classified on the basis of morphological criteria and, through the analysis of serial sections, morphological characteristics of the cell processes were established. In the inner plexiform layer (IPL), the types of synapses were analysed. The majority of cells in the INL and GCL were differentiated and could be identified. Ganglion cell somata were observed in the GCL and INL. Of 186 somata analysed in the INL, 66 were Muller cells, 21 amacrine cells, and 2 ganglion cells; a further 7 cells were classified as either amacrine or ganglion. Bipolar cells were thought to comprise the majority of the remaining 90 somata, but these could not be positively identified, as it was not possible to trace bipolar cell axons to their cell bodies deep in the INL. A detailed description of the morphological characteristics of the identified cells and their processes, and of the axonal processes of bipolar cells, is provided. Puncta adherentia and other simple intercellular junctions were commonly seen in the IPL and involved all cell types. Amacrine cell synapses and immature, monad bipolar cell synapses were common within the IPL. Dyad bipolar synapses were uncommon at this stage of development. A possible sequence of synaptogenesis in the IPL is discussed.

Morphology of intraretinal new vessels in the PETH rat.

The mature stages of retinal dystrophy in PETH rats are characterised by loss of the photoreceptor layer and invasion of the retinal pigment epithelium by new capillaries derived from the retinal vessels. The new capillaries are fenestrated where they are adjacent to the basement membrane of the retinal pigment epithelium and are surrounded by cells of the disrupted pigment epithelium, which follow the course of the capillaries into the inner retina. Abnormal basement membrane deposits are common within the retinal pigment epithelium.

Human fetal optic nerve: overproduction and elimination of retinal axons during development.

We have estimated the number of axons in the optic nerves of human fetuses ranging in gestational age from approximately 10 to 33 weeks. At 10-12 weeks of gestation there were an estimated 1.9 million axons in the optic nerve. A peak count of 3.7 million axons was obtained from a specimen of 16-17 weeks gestation. The estimated number of axons then declined, stabilizing at an estimated 1.1 million axons by about week 29 of gestation. This figure is in close agreement with an estimate of 1.1-1.3 million optic axons in the human adult optic nerve. The results indicate that at least 70% of optic axons generated during development of the primary visual pathway are lost during fetal life. Part of this loss probably occurs as a result of the refinement of the terminal distribution of ganglion cell projections within their target nuclei. The significance of the relatively prolonged period of axonal loss is discussed.

Retinal development in humans: the roles of differential growth rates, cell migration and naturally occurring cell death.

The distribution of ganglion cells throughout the retinal ganglion cell layer is non-uniform in adult mammals. This paper reviews some of our data describing the development of retinal ganglion cell topography in the human fetus. Results indicated that early in the fetal period the distribution of cells in the ganglion cell layer is almost uniform, but by the end of gestation there is a gradient in cell density of about 10:1 (central:peripheral). Peripheral retina grows more rapidly than the central retina prior to about 23 weeks gestation, but this differential growth rate apparently has little effect on the development of a centro-peripheral density gradient. The gradient appears between about 18 and 30 weeks gestation, and during this period there appears to be a greater rate of cell death in the ganglion cell layer of the peripheral retina. Cell density at the developing fovea is less than the perifoveal cell density at all ages, suggesting that ganglion cells migrate from foveal into perifoveal regions throughout the fetal period.

Development of the human retina: patterns of cell distribution and redistribution in the ganglion cell layer.

Neurogenesis in the ventricular layer and the development of cell topography in the ganglion cell layer have been studied in whole-mounts of human fetal retinae. At the end of the embryonic period mitotic figures were seen over the entire outer surface of the retina. By about 14 weeks gestation mitosis had ceased in central retina and differentiation of photoreceptor nuclei was evident within a well-defined area which constituted about 2% of total retina area. This area was approximately centered on the site of the putative fovea, identified by the exclusive development of cone nuclei at that location. The area of retina in which mitosis had ceased increased as gestation progressed. By mid-gestation mitosis in the ventricular layer occupied about 77% of the outer surface of the retina and by about 30 weeks gestation mitosis in the ventricular layer had ceased. Cell density distributions in the ganglion cell layer were nonuniform at all stages studied (14-40 weeks). Densities were highest at about 17 weeks gestation, and by mid-gestation the adult pattern of cell topography was present with maps showing elevated cell densities in posterior retina and along the horizontal meridian. Cell densities generally declined throughout the remainder of the gestation period, except in the posterior retina, where densities in the perifoveal ganglion cell layer remained high during the second half of gestation. There is a rapid decline in cell density in the foveal ganglion cell layer toward the end of gestation, and it is suggested that the persistence of high densities in the perifoveal region may be related to migration of cells away from the developing fovea. The total population of cells in the ganglion cell layer was highest (2.2-2.5 million cells) between about weeks 18 and 30 of gestation. After this the cell population declined rapidly to 1.5-1.7 million cells. It is suggested that naturally occurring neuronal death is largely responsible for this decline.

Softening of drusen and subretinal neovascularization.

On the basis of clinico-pathological examination, drusen were divided into hard and soft. A few small, hard drusen with a hyaline composition were found in 50 per cent of the normal aged eyes examined. The appearance of drusen of softer consistency often heralded the onset of senile macular degeneration. Soft drusen resulted either from the breakdown of the hyaline content of hard drusen or from the focal aggregation into mounds of membranous debris which accumulated as a widespread shallow layer between the basement membrane of the pigment epithelium and Bruch's membrane. Softening of drusen was most evident when the retinal pigment epithelium demonstrated the greatest proliferative activity, with migration of cells into the retina and the formation of the basal laminar deposit. It was at this stage that subretinal neovascularization was first detected. A patient in whom the gradual development of small soft drusen and an associated pigmentary disturbance was followed by subretinal new vessel formation is reported. The eye was subsequently found to have five separate breaks in Bruch's membrane and the ultrastructural findings are described.