B. H. Choi

Glial cell line-derived neurotrophic factor protects against ischemia/hypoxia-induced brain injury in neonatal rat.

Abstract Ischemic/hypoxic brain damage induced in 7-day-old rats was significantly attenuated in a dose-dependent manner by intracerebral injection of glial cell line-derived neurotrophic factor (GDNF; 2 or 4 μg) within 30 min after the insult. Whereas the great majority of the vehicle-treated animals showed massive infarction involving more than 75% of the affected cerebral hemisphere, GDNF injection resulted in a remarkable reduction in both the incidence and severity of the brain damage (incidence ranging from 76% to 93% in controls to 34% to 64% in the 2.0-μg group and 7% to 29% in 4.0-μg group). The induction of immunoreactive 70-kDa heat shock protein (HSP70) in cerebral cortical neurons was also significantly reduced in GDNF-treated animals as compared to controls. The mechanisms responsible for the neuroprotective effects of GDNF remain unknown, although it has been speculated that these may be endogeneous. The higher expression of GDNF and its mRNA in developing brains may be one of the factors responsible for the relative resistance to ischemia of fetal and neonatal as opposed to adult brains. GDNF may possibly act by protecting against oxidative stress or by scavenging free radicals generated during ischemia. The results of our study strongly suggest that GDNF may prove to be an effective and potent protective agent against perinatal ischemic/hypoxic encephalopathy.

Repair and reconstruction of the cortical plate following closed cryogenic injury to the neonatal rat cerebrum

SummaryA cryogenic lesion was induced in the parietal cortex of neonatal rats at postnatal day 2, and the chronological sequence of cellular events during repair and reconstruction of the cortical plate examined. Serial sections of cerebra obtained at varying intervals ranging from 1 to 60 days postinjury were studied by light and electron microscopy and by immunocytochemistry for fibronectin, laminin, type IV collagen, vimentin and glial fibrillary acidic protein. In addition, localization of heavily labeled neurons (generated on embryonic day 20) in the cerebral cortical plate was examined by [3H]thymidine radioautography. Repair of a welldefined coagulative lesion was accomplished with little or no mesenchymal cell proliferation in either the necrotic zone or the leptomeninges. Eventually, fusion of the adjacent cortical plates took place with the formation of a microsulcus. Migration of neurons continued to take place along the outer margins of the lesion, and postmigratory neurons accumulated within the upper cortical layers. Around the microsulcus, heavily labeled neurons aligned themselves with layers II–III of the adjacent normal cortical plate. Irregular clusters of neurons closely abutting the leptomeningeal surface were frequently noted when repair took place without an intervening molecular layer and/or a welldefined pial-glial barrier. Supplementing intrinsic information inherent in migrating neurons, local environmental signals provided by the radial glia, glia limitans, basal lamina and pial-glial barrier appear to influence the polarity and final positioning of postmigratory neurons within the cortical plate. The necrotic zone within the deeper layers of the cortex eventually healed with a cell-sparse gliotic layer. The end result was a histological pattern that, in many respects, resembled that of human micropolygyria. In contrast, identical cryogenic lesions induced in the brains of rats at postnatal day 10 healed with fibrous scar formation and/or caviation, and a micropolygyric pattern did not develop.

Cortical dysplasia associated with massive ectopia of neurons and glial cells within the subarachnoid space

SummaryA detailed neuropathological study of the brain of a 31-day-old premature newborn infant revealed the presence of massive ectopia of neurons and glial cells within the subarachnoid space. The extrusion of neural tissue into the subarachnoid space appeared to have taken place through multiple pialglial bridges. The laminar cortical pattern was also severely disturbed at these sites. Narrow strips of normal and dysplastic cortex alternated in direct relationship to the presence or absence of the pial-glial gaps. Migration of postmitotic neurons and the final positioning of postmigratory neurons appear to take place within highly specified and restricted pathways entrained in a radial direction. Our findings suggest that the pial-glial barrier plays an important role in the control of neuronal migration, and that its disruption may lead to the development of neuronal and glial cell ectopias in the subarachnoid space. The crucial role played by radial glia, the glia limitans and the basal lamina during cortical neurogenesis is emphasized.

Expression of glial cell line-derived neurotrophic factor in the brain and cerebrospinal fluid of the developing rat

Expression of GDNF in developing rat brain from PND 1 to 14 and on PND 21 was examined immunocytochemically. At PND 1, intense diffuse immunoreactivity was noted within the cytoplasm of a diverse group of neuronal and nonneuronal cells, including choroid plexus epithelial cells, ependymal cells, tanycytes of the third ventricle, and cellular elements in the subarachnoid compartment. GDNF expression became more localized among these cells from PND 7–14 and was almost undetectable by PND 21. Although GDNF‐positive small glial cells were scattered within the cerebral cortical plate and the striatum already at PND 1, GDNF expression among astroglial cells within the corpus callosum and in the white matter adjacent to the lateral ventricles was more prominent between PND 5 and 8. GDNF expression among the pyramidal neurons of the cerebral cortex was evident relatively early in the postnatal period, but the neurons of the hippocampus and thalamus showed more intense immunoreactivity at later periods between PND 8–14. ELISA of the CSF revealed a rapid rise in GDNF levels from 71.4±10.9 pg/ml (mean±S.E.M.) at PND 1 to peak levels of 138.4±18.5, 135.1±5.4 and 132.9±8.0 pg/ml, at PND 5, 7 and 9, respectively. Peak CSF levels of GDNF occurred when GDNF expression was intense within astroglial cells in the corpus callosum and cerebral white matter. Thereafter, the levels gradually decreased to 76.5±9.7 pg/ml at PND 21. The widespread expression of GDNF among different cellular elements in the developing brain suggest that GDNF probably plays diverse functional roles in many different neuronal systems in addition to its known effects on the dopaminergic system. Developmental shifts in GDNF expression further suggest that GDNF may be of critical importance at different stages of brain growth and differentiation.

Developmental events during the early stages of cerebral cortical neurogenesis in man

SummaryThe telencephalic vesicles of early human embryos and fetuses ranging from 6–18 weeks of ovulation age were subjected to correlative light and electron microscopic, immunocytochemical and Golgi analysis. The cellular events that take place during the early stages of cortical neurogenesis are characterized by the early maturation of layer I. The deployment of radial glia, the formation of the glia limitans, the establishment of the pial-glial barrier, and the migration and maturation of Cajal-Retzius cells within the marginal zone (MZ) all take place by 6–7 weeks. Neurons destined to populate the cortical plate (CP) arrive within the MZ by 7–8 weeks of age and appear to divide the MZ into two layers. Progressive compaction of CP neurons and widening of the intermediate zone characterize the next stage. It is suggested that the early establishment of the pial-glial barrier and the early maturation of the layer I are critical to the subsequent growth and development of the CP.

The behavior of the extracellular matrix and the basal lamina during the repair of cryogenic injury in the adult rat cerebral cortex.

SummaryA well-defined coagulative lesion was produced in the fronto-parietal cortex of adult rats by application of a cold probe, and the chronological sequence of events during the healing process, particularly the relationship between astroglial processes and the newly forming basal lamina (BL) and the behavior of the extracellular matrix (ECM) was examined immunocytochemically and ultrastructurally. By electron microscopy, new BL synthesis was first noted 7 days following injury, and a continuous and well-defined BL was present from 14 days onward. These findings correlated well with the pattern of immunoreactivity for laminin and for type IV collagen. Both laminin and type IV collagen appeared both to be produced primarily by mesenchymal cells within and around the wound as well as those of the blood vessels, and to become an integral part of the new BL. Although there was no immunocytochemical evidence to indicate secretion of laminin, type IV collagen or fibronectin by astrocytes, a well-defined BL appeared to form only in tight apposition with astroglial processes over the pial surface. This suggests that the BL is formed by subpial astrocytes in close interaction with ECM components at the pial surface. Fibronectin appears to contribute significantly to the formation of the BL by providing a suitable substratum for the coordinated cellular interaction necessary for successful regeneration of the BL.

Bimodal upregulation of glial cell line-derived neurotrophic factor (GDNF) in the neonatal rat brain following ischemic/hypoxic injury.

In order to delineate the spatial and temporal patterns of glial cell line‐derived neurotrophic factor (GDNF) expression following ischemic/hypoxic injury in immature and neonatal brain, GDNF protein levels and immunocytochemistry were studied in rats subjected to a modified Levine procedure. Significant upregulation of GDNF protein occurred in a bimodal fashion in the damaged left cerebral cortex and hippocampus, while the levels in the right cerebral hemisphere of both control and ischemic groups remained relatively unchanged. Immunocytochemical studies indicated that the early rise in GDNF levels was most likely to be related to enhanced neuronal release of GDNF. The second rise was probably related to progressive astrogliosis that occurred in response to injury. In contrast to the lack of GDNF expression among astrocytes in normal mature brains, reactive astrocytes in the neonate appear to possess a ready capacity to express GDNF. Spatial and temporal changes in the pattern of GDNF expression following injury, as determined in this study may provide insight into the functions of GDNF in vivo and into possible therapeutic approaches toward prevention of damage or rescue of neurons following brain injury.

Abnormal neuronal distribution within the cerebral cortex after prenatal methylmercury intoxication.

SummaryC57BL/6J mice were poisoned with methylmercury during pregnancy, and the location of heavily labeled neurons generated at embryonic day 16 was determined by tritiated thymidine autoradiography of the cerebral cortex of offspring at postnatal day 10. Camera lucida plotting of the distribution of radioactively labeled cortical neurons revealed statistically significant differences between control and methylmercury-treated groups. While control animals showed regular and tight packing of labeled neurons within the upper part of the cortical layer II, in methylmercury-treated animals such neurons were irregularly distributed throughout cortical layers II and III. Short-term intermittent and long-term lowdosage regimens of intoxication produced similar results. These findings support the hypothesis that prenatal methylmercury poisoning results in abnormal neuronal migration and anomalous cortical cytoarchitectonic patterning within the developing brain and provide a possible morphological basis for some of the neurobehavioral abnormalities that may be observed in association with sublethal prenatal intoxication in humans.

Expression of glial cell line-derived neurotrophic factor (GDNF) in the developing human fetal brain.

GDNF expression was examined immunocytochemically in developing human fetal brains obtained from aborted fetuses ranging from 7 to 39 weeks in gestational age. At 7–8 weeks, strong immunoreactivity was noted within radial glial processes, glia limitans and choroid plexus of the telencephalic vesicle. By 10 weeks, ependymal cells, primitive matrix cells and early developing cortical plate neurons showed positive staining. By 15–16 weeks, migrating neurons in the subventricular and intermediate zones and in the cortical plate were strongly positive for GDNF. The glia limitans of the cerebral cortex and subependymal astrocytes remained positive at this time. As fetal age increased, GDNF expression shifted to neurons and glial cells in the deeper structures of the brain. The most prominent GDNF staining was observed in the cytoplasm and dendrites of Purkinje cells of the cerebellum by 25 weeks and thereafter. Pyramidal neurons of the CA1 region and granule cells of the dentate fascia of the hippocampus, neurons of the entorhinal cortex, and scattered neurons within the brain stem, medulla and spinal cord all showed strong GDNF staining by 25–35 weeks. Widespread GDNF expression in neuronal and non‐neuronal cells with distinct developmental shifts suggests that GDNF may play a critical role in the survival, differentiation and maintenance of neurons at different stages of development in the developing human fetal brain.

Effects of methylmercury on neuroepithelial germinal cells in the developing telencephalic vesicles of mice.

SummaryMethylmercury (MeHg) poisoning (20 mg/kg body weight) in embryonic mice resulted in significant reductions of mitotic indices in the neuroepithelial germinal cells of the telencephalon at the ventricular surface 4 to 12 h following intoxication. After 24 h, no significant difference in the mitotic indices was observed as compared to controls. However, after 48 h there was an increase in mitotic indices of MeHg group as compared to controls. Analysis of the mitotic figures revealed features suggestive of early-phase mitotic arrest in MeHg-exposed animals. Radioautographic studies suggest a disturbance in the interkinetic nuclear migration of proliferating ventricular cells in the MeHg group. Acute degenerative changes in scattered ventricular cells characterized by edema and spongy changes of cytoplasm associated with dissolution of ribosomes, clearing of the cytoplasmic matrix and loss of organelles including microtubules were observed by electron microscopy. Loss of microtubules was also evident within mitotic figures in MeHg-poisoned animals. It is suggested that reduction and arrest of mitotic activity and disturbances in the interkinetic nuclear migration of neuroepithelial germinal cells are related to cytotoxic effects of MeHg on ventricular cells, including effects on microtubules. These findings suggest in that MeHg severely affects proliferating neuroepithelial germinal cells during the acute phases of MeHg poisoning, and that these changes may eventually affect the architectonic makeup of the cortical plate as the brain matures.