Immature Glial Cells Research Articles

The Amount of Immature Glial Cells in Organotypic Brain Slices Determines the Susceptibility to Murine Cytomegalovirus Infection

Cytomegalovirus (CMV) is the most common infectious cause of congenital anomalies of the brain and also causes brain damage in immunocompromised individuals. We investigated the effects of murine cytomegalovirus (MCMV) infection on the developing mouse brain in terms of susceptible cells and age-related resistance to MCMV in brain slice cultures. Brain slices from BALB/c mice at different developmental stages were infected with recombinant MCMV in which the lacZ gene was inserted into a late gene. The subventricular zone and cortical marginal region were the sites most susceptible to MCMV infection, and the susceptibility declined with the development of the brain. Immunohistochemical staining showed that the virus-susceptible cells were positive for GFAP, nestin, and Musashi-1, and that most of the infected cells were positive for the proliferative cell nuclear antigen and labeled with bromodeoxyuridine. These results suggest that the susceptible cells in the subventricular zone are immature glial cells, including neural progenitor cells. Immature glial cells proliferated when the brain slices were cultured for a prolonged time and furthermore, they showed themselves to be susceptible to virus infection even under serum-free conditions. These results suggest that the amount of immature glial cells, which include neural progenitor cells, in the developing brain or in the damaged brain with neural proliferation may be closely associated with the susceptibility of the brain to CMV infection in humans.

Chronic hypoxia up-regulates fibroblast growth factor ligands in the perinatal brain and induces fibroblast growth factor-responsive radial glial cells in the sub-ependymal zone

A number of signaling molecules have been implicated in the acute response to hypoxia/ischemia in the adult brain. In contrast, the reaction to chronic hypoxemia is largely unexplored. We used a protocol of chronic hypoxia in rat pups during the first three postnatal weeks, encompassing the period of cellular plasticity in the cerebral cortex. We find that the levels of fibroblast growth factor 1 (FGF1) and FGF2, two members of the FGF family, increase after 2 weeks of chronic hypoxia. In contrast, members of the neurotrophin family are unaffected. FGF2 is normally expressed in the nucleus of mature, glial fibrillary acidic protein (GFAP)-containing astrocytes. Under hypoxia, most FGF2-containing cells do not express detectable levels of GFAP, suggesting that chronic low O 2 induces their transformation into more immature glial phenotypes. Remarkably, hypoxia promotes the appearance of radial glia throughout the sub-ventricular and ependymal zones. Most of these cells express vimentin and brain lipid binding protein. A subset of these radial glial cells expresses FGF receptor 1, and are in close contact with FGF2-positive cells in the sub-ventricular zone. Thus, FGF receptor signaling in radial glia may foster cell genesis after chronic hypoxic damage. From the results of this study we suggest that after the chronic exposure to low levels of oxygen during development, the expression of radial glia increases in the forebrain periventricular region. We envision that astroglia, which are the direct descendants of radial glia, are reverting back to immature glial cells. Alternatively, hypoxia hinders the normal maturation of radial glia into GFAP-expressing astrocytes. Interestingly, hypoxia increases the levels of expression of FGF2, a factor that is essential for neuronal development. Furthermore, chronic hypoxia up-regulated FGF2’s major receptor in the periventricular region. Because radial glia have been suggested to play a key role in neurogenesis and cell migration, our data suggests that hypoxia-induced FGF signaling in radial glia may represent part of a conserved program capable of regenerating neurons in the brain after injury.

Migration and Proliferation of Motile Immature Glial Cells in the Developing Cerebral Cortex of Infantile Rat.

Using immunohistochemistry of thymosin β4 (Tβ4), GFAP, vimentin 9 and lectin histochemistry of Griffonia simplicifolia B4 isolectin (IB4), we investigated migration and proliferation of immature glial cells in the infantile rat cerebral cortex from P0 to P60. Although the immature glial cells in the cortex are commonly Tβ4 positive, dynamic behavior of the immature glial cells are dramatically different before and after P11; between P0 and P11, they are actively migrating, highly proliferative and differentiate other types of glial cells, while after 12 days of birth, they turn out to react positively with Thiamine pyrophosphatase (TTPase), a definitive marker of mature microglia, and stop migration and proliferation. We concluded that the Tβ4 positive immature glial cells in the rat cortex before P11, although they have been called the microglia, are glioblasts, common precursors of neuroglia, and they are differentiated into microglia during the second postnatal week.

Distinct expression pattern of microtubule-associated protein-2 in human oligodendrogliomas and glial precursor cells.

Microtubule-associated protein 2 (MAP2), a protein linked to the neuronal cytoskeleton in the mature central nervous system (CNS), has recently been identified in glial precursors indicating a potential role during glial development. In the present study, we systematically analyzed the expression of MAP2 in a series of 237 human neuroepithelial tumors including paraffin-embedded specimens and tumor tissue microarrays from oligodendrogliomas, mixed gliomas, astrocytomas, glioblastomas, ependymomas, as well as dysembryoplastic neuroepithelial tumors (DNT), and central neurocytomas. In addition, MAP2-immunoreactive precursor cells were studied in the developing human brain. Three monoclonal antibodies generated against MAP2A-B or MAP2A-D isoforms were used. Variable immunoreactivity for MAP2 could be observed in all gliomas with the exception of ependymomas. Oligodendrogliomas exhibited a consistently strong and distinct pattern of expression characterized by perinuclear cytoplasmic staining without significant process labeling. Tumor cells with immunoreactive bi- or multi-polar processes were mostly encountered in astroglial neoplasms, whereas the small cell component in neurocytomas and DNT was not labeled. These features render MAP2 immunoreactivity a helpful diagnostic tool for the distinction of oligodendrogliomas and other neuroepithelial neoplasms. RT-PCR, Western blot analysis, and in situ hybridization confirmed the expression of MAP2A-C (including the novel MAP2+ 13 transcript) in both oligodendrogliomas and astrocytomas. Double fluorescent laser scanning microscopy showed that GFAP and MAP2 labeled different tumor cell populations. In embryonic human brains, MAP2-immunoreactive glial precursor cells were identified within the subventricular or intermediate zones. These precursors exhibit morphology closely resembling the immunolabeled neoplastic cells observed in glial tumors. Our findings demonstrate MAP2 expression in astrocytic and oligodendroglial neoplasms. The distinct pattern of immunoreactivity in oligodendrogliomas may be useful as a diagnostic tool. Since MAP2 expression occurs transiently in migrating immature glial cells, our findings are in line with an assumed origin of diffuse gliomas from glial precursors.

Sox8 gene expression identifies immature glial cells in developing cerebellum and cerebellar tumours

Sox8 is a member of the E subgroup of Sox genes, the other members of which are Sox9 and Sox10, both of which are implicated in specific human disorders. Recently, Sox8 homologues have been cloned in chick, mouse and human and have been shown to be strongly expressed in the embryonic and adult brain. Nevertheless, the cell types that express Sox8 have not been determined. We show here that Sox8 is expressed in immature glia in the developing cerebellum. Sox8 is also expressed in scattered cells in the cerebellar tumour, medulloblastoma. This gene therefore provides an early glial marker that may provide more detailed insight into the cellular makeup and consequent behaviour of medulloblastomas.

Immunohistochemical localization of substance P receptors in the midline glia of the developing rat medulla oblongata with special reference to the formation of raphe nuclei

Immunohistochemical localization of the substance P receptor (SPR) was examined in the developing rat medulla oblongata, with special reference to the development of substance P (SP)-immunoreactive neurons which form the medullary raphe nuclei. During development, SPR immunoreactivity was detected in cells lying lateral to the medullary midline from embryonic day 13 (E13) to postnatal day 5 (P5). The SPR-positive cell bodies were located close to the fourth ventricle, and bore long processes extending to the ventral pial surface. This SPR immunoreactivity co-localized with staining for monoclonal antibody 1D11, a specific marker of immature astrocytes. Substance P (SP)-immunoreactive neurons were first detected at E14 in the ventrolateral part of the medulla. By E16 their number had increased and they were arrayed in two rows closely parallel to the SPR-immunoreactive processes of non-neuronal cells. By P1, two separate SP-immunoreactive cell clusters could be recognized at the midline, representing dorsally the nascent raphe pallidus and ventrally the raphe obscurus. In addition, many SP-immunoreactive fibers traveled rostrocaudally in the medulla oblongata, juxtaposed to the midline sheets of SPR-immunoreactive long processes. SPR-immunoreactive processes at the midline were also immunoreactive for S-100, a glia-specific calcium-binding protein that is known to promote axonal growth of raphe neurons. These results suggest that SPR-expressing immature glial cells at the medullary midline are involved in the development of SP-immunoreactive raphe neurons, both in the formation of the medullary raphe nuclei and in axon guidance and growth.

Proliferation and Differentiation of Progenitor Cells Throughout the Intact Adult Rat Spinal Cord

The existence of multipotent progenitor populations in the adult forebrain has been widely studied. To extend this knowledge to the adult spinal cord we have examined the proliferation, distribution, and phenotypic fate of dividing cells in the adult rat spinal cord. Bromodeoxyuridine (BrdU) was used to label dividing cells in 13- to 14-week-old, intact Fischer rats. Single daily injections of BrdU were administered over a 12 d period. Animals were killed either 1 d or 4 weeks after the last injection of BrdU. We observed frequent cell division throughout the adult rodent spinal cord, particularly in white matter tracts (5-7% of all nuclei). The majority of BrdU-labeled cells colocalized with markers of immature glial cells. At 4 weeks, 10% of dividing cells expressed mature astrocyte and oligodendroglial markers. These data predict that 0.75% of all astrocytes and 0.82% of all oligodendrocytes are derived from a dividing population over a 4 week period. To determine the migratory nature of dividing cells, a single BrdU injection was given to animals that were killed 1 hr after the injection. In these tissues, the distribution and incidence of BrdU labeling matched those of the 4 week post injection (pi) groups, suggesting that proliferating cells divide in situ rather than migrate from the ependymal zone. These data suggest a higher level of cellular plasticity for the intact spinal cord than has previously been observed and that glial progenitors exist in the outer circumference of the spinal cord that can give rise to both astrocytes and oligodendrocytes.

Surgical Resection Alone Is Effective Treatment for Ovarian Immature Teratoma in Children and Adolescents: A Report of the Pediatric Oncology Group and the Children’s Cancer Group

This study was conducted to evaluate the routine use of chemotherapy, with its attendant long-term effects, in children and adolescents after the surgical removal of ovarian immature teratoma. Forty-four girls who underwent laparotomy for complete removal of an ovarian tumor containing pure immature teratoma between 1995 and 1996 served as subjects. Patients were classified as having stage I cancer when disease was found in the ovary only with negative peritoneal washings. Among the patients who had adequate staging, no stage II disease was observed. Follow-up for the first 2 years included frequent measurements of serum levels of α-fetoprotein (AFP) and diagnostic imaging studies of the chest and pelvis. Thereafter, patients returned annually for regular physical examinations. At diagnosis, the age range of the subjects was 1.5 to 20 years. Four girls were lost to follow-up, two shortly after surgery and two within 2 years. Nineteen patients were followed for more than 5 years, and four had undergone surgery less than 2 years earlier. Histologic examination found no embryonal carcinoma, choriocarcinoma, or germinoma. Fifteen patients were classified as having grade 1 tumors, 12 had grade 2 tumors, and 2 had grade 3 tumors. On central pathology review, 13 patients were found to have tumors containing microscopic foci of a yolk sac tumor that was previously undetected. Unilateral salpingo-oophorectomy was the treatment selected in 28 patients (64 percent). Only one hysterectomy was necessary. Lymph node samples, obtained from 16 patients, showed mature gliomatosis in three patients. Three subjects had positive peritoneal washings, all of which contained only mature teratomatous or immature glial cells. The 13 patients with ovarian immature teratoma plus yolk sac tumor were slightly older (median, 12 years) than those with pure teratoma (median, 10 years) and were more likely to have larger tumors. Fifty-five percent of the girls with immature teratoma had grade 1 disease, compared with only 8 percent of those whose teratoma contained a yolk sac tumor. Conversely, 46 percent of the children with immature teratoma plus yolk sac tumor were classified as having grade 3 disease, compared with 6 percent of those without yolk sac tumor. Ten of the 13 girls (80 percent) with yolk sac components had measurable levels of AFP, compared with only 10 of 31 of the girls (34 percent) with no yolk sac tumor elements. Furthermore, the median level of AFP was 252 among children with yolk sac tumor, compared with 25 among those without yolk sac tumor. Ovarian immature teratoma containing yolk sac tumor was less likely to rupture during removal than immature teratoma (8 vs. 32 percent). No patients died. There was one malignant recurrence in a 12-year-old girl who had a large ovarian immature teratoma with yolk sac tumor, an initial serum AFP level of 1045 ng/ml, and peritoneal washings and lymph nodes positive for gliomatosis. A sharp rise in her serum AFP level at 18 weeks signaled a recurrence. Two nodules containing grade 2 immature teratoma with yolk sac tumor were found in her liver. She was disease free 57 months after four courses of cisplatin, etoposide, and bleomycin therapy. Two other patients required additional surgical procedures. Both girls underwent exploratory laparotomy for a pelvic mass. One contained ovarian mature cystic teratoma and mature glial implants. The other was composed of multiple mature glial implants on the peritoneal surfaces and on the surface of the remaining ovary, which was removed. Both patients had normal serum AFP levels that did rise, and both continue to be well without additional symptoms. Normal adolescent follicular cysts were frequently observed among the entire study group. Am J Obstet Gynecol 1999;181:353–358

Genomic structure and promoter analysis of the ecto-phosphodiesterase I gene ( PDNP3) expressed in glial cells

PDNP (phosphodiesterase I/nucleotide pyrophosphatase) is one of a series of ectoenzymes that are involved in hydrolysis of extracellular nucleotides. PDNP possesses ATPase (EC 3.6.1.3) and ATP pyrophosphatase (EC 3.6.1.8) activities. Mammalian PDNP consists of three closely related family proteins (PDNP1, -2, and -3), and they are expressed in different cell types and at different developmental stages. Rat PDNP3 is expressed in a subset of immature glial cells and in the alimentary tract. Human PDNP3 is expressed in glioma cells, prostate, and uterus, but not in the alimentary tract. We have cloned genomic DNA containing the whole coding region of the human PDNP3 gene and determined its exon–intron structure. The human PDNP3 gene spans over 60 kb and is organized into 25 exons and 24 introns. We determined the nucleotide sequence of the 5′-flanking region of human and rat PDNP3 genes. The upstream region of both species lacks a canonical TATA box and contains a putative binding site for CCAAT enhancer-binding proteins near the transcription start site. Promoter activity analysis of the 5′-flanking region revealed that the sequence around the CCAAT box is required for its transcriptional activity in 9L rat glioma cells. A gel shift assay demonstrated that 9L nuclear extract contains proteins that bind to this region.

Valproic acid for the treatment of pediatric malignant glioma.

Despite surgery and adjuvant cytotoxic therapy anaplastic astrocytoma, glioblastoma and diffuse intrinsic brain stem glioma continue to have dismal prognosis. Differentiation induction is a new approach taking into account that malignant glioma cells share many features with immature glial progenitor cells that are capable of terminal differentiation. The concept of differentiation therapy is currently evaluated for several pediatric malignancies with or without multimodal standard therapy. Valproic acid (VPA) is a branched chain fatty acid that is able to inhibit proliferation of neuroectodermal cells and to induce these cells along neuronal or glial lineage. Preclinical studies have shown that VPA inhibits growth of human and rodent glial tumor cells in vitro and induces a distinct mature glial phenotype. In addition, growth of human neuroblastoma cells is inhibited in vitro and in vivo and exhibits marked evidence of differentiation. Treatment of neuroblastoma and glioma cells with VPA was accompanied by changes of surface molecule expression that enhance immunogenicity and reduce their capability to metastasize. The antitumoral effects observed in preclinical studies were reached at concentrations that are readily achieved in patients treated with VPA for epilepsy. Epilepsy patients receiving VPA have significantly enhanced hemoglobin F levels, supporting the hypothesis that nontoxic levels of VPA can induce cellular differentiation. Broad clinical experience with VPA and its low toxicity encourage the evaluation of VPA in patients that have been submitted to postoperative combined chemo- and radiotherapy for pediatric malignant glioma.

Localization of urokinase-type plasminogen activator, its receptor, and inhibitors in mouse forebrain during postnatal development

Proteolytic enzymes are postulated to play a role in cell migration and synapse organization during brain development. Among these, urokinase-type plasminogen activator (uPA) has been studied in neoplastic and cultured brain cells extensively. We hypothesized that uPA, its receptor, and its inhibitors would be expressed in immature glial and neuronal cells in postnatal mouse forebrain. Immature cortical neurons were immunoreactive for uPA, its receptor, and its substrate plasminogen peaking at the end of postnatal week two, consistent with the postulated role in synaptogenesis. Immunoreactivity for uPA receptor was also observed on astroglial cells in vitro. Neither it nor uPA were convincingly detected in subventricular zone precursor cells, immature white matter or pre-labeled immature cells that had been transplanted into brain. Plasminogen activator inhibitor type 1 immunoreactivity was observed on endothelia up to 12days age, and type 2 was observed to surround immature cells.We conclude, based on the spatial and temporal distribution of immunoreactivity, that uPA and its receptor may be relatively more important for synaptogenesis, remodeling, and reactive processes than for cell migration in developing mouse brain.

Adenosine 5′ triphosphate evoked mobilization of intracellular calcium in central nervous system white matter of adult mouse optic nerve

Although it has been established that immature glial cells express functional purinergic receptors, the responsiveness of mature glial cells in vivo had not been elucidated. This question was addressed using fura-2 ratiometric measurements of [Ca2+]i in the adult mouse optic nerve, a central nervous system (CNS) white matter tract, taking advantage of the facts that (i), the optic nerve contains glial cells but not neurons and (ii), that fura-2 loads primarily astrocytes in isolated intact optic nerves. We show that adenosine 5′ triphosphate (ATP) evoked an increase in [Ca2+]i in a concentration-dependent manner with a half-maximal effect at 3 μm ATP, and with a rank order of agonist potency of ATP>ADP>α,β-methyline-ATP>UDP>adenosine. The results indicate mainly P2Y and P2X components, consistent with the in vitro astroglial purinergic receptor profile. The in vivo response of mature glia to ATP may be important in their response to CNS damage.

Expression of DSD-1-PG in primary neural and glial-derived cell line cultures, upregulation by TGF-?, and implications for cell-substrate interactions of the glial cell line Oli-neu

DSD-1-PG is a chondroitin sulfate proteoglycan with neurite-outgrowth promoting properties expressed during development and upon lesion of neural tissues which has been defined with the specific monoclonal antibody 473HD. Double immunofluorescence studies performed on primary cerebellar cultures document that the proteoglycan is expressed on the surface of immature glial cells and the neural cell line Oli-neu, a model of mouse oligodendrocyte progenitors. Biochemical and immunoprecipitation studies performed with biosynthetically labelled Oli-neu and primary neural cells demonstrated that DSD-1-PG is expressed in vitro as a proteoglycan of 1000 kD apparent Mr with two core glycoproteins of 250 kD and 400 kD. In order to study the regulation of DSD-1-PG expression, an in vitro enzyme-linked immunosorbent assay based on Oli-neu and mAb 473HD was established. TGF-beta1-3 induced up-regulation of the proteoglycan, while various growth factors and cytokines did not significantly affect DSD-1-PG expression in both the supernatant and the extract of the culture monolayer. FACSCAN analysis suggested that the proteoglycan is upregulated on the surface of Oli-neu. Cell substrate adhesion assays revealed that this enhanced expression correlates with a selective reduction of adhesion to laminin, but not fibronectin or merosin, which could specifically be neutralized by antibodies to DSD-1-PG. We conclude that the proteoglycan contributes to the regulation of glial precursor interactions with the extracellular matrix.

A role of adhesion molecules in neuroglial plasticity

Glial cells are exquisitely sensitive to changes in neuronal activity, and their capacity for structural plasticity including migration is critical for remodeling and repair of nervous tissue. Our in vitro studies suggest that isoforms of the neural cell adhesion molecule (NCAM) carrying an unconventional carbohydrate polymer, polysialic acid (PSA), are involved in these events. We have demonstrated that neurohypophyseal explants from newborn rats generate cellular outgrowth of immature astrocytes displaying the characteristics of oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells previously identified in the optic nerve. Treatment of O-2A cells with the enzyme Endo N, which specifically removes PSA from the cells surface, produced a complete blockade of the dispersion of the O-2A cell population from the explant. Identical effects of Endo N were observed in migration assays using cortical O-2A cells. Neurohypophyseal O-2A cells express functional NMDA class of glutamate receptors and the pharmacological blockade of these receptors inhibit PSA-NCAM biosynthesis and dramatically diminish O-2A cell migration from neurohypophyseal explants. This suggests a potential mechanism through which neuronal activity via glutamate release may regulate PSA-NCAM expression on immature glial cells, which in turn is critical for their migration.

Electrophysiological Changes That Accompany Reactive GliosisIn Vitro

An in vitro injury model was used to examine the electrophysiological changes that accompany reactive gliosis. Mechanical scarring of confluent spinal cord astrocytes led to a threefold increase in the proliferation of scar-associated astrocytes, as judged by bromodeoxyuridine (BrdU) labeling. Whole-cell patch-clamp recordings demonstrated that current profiles differed absolutely between nonproliferating (BrdU-) and proliferating (BrdU+) astrocytes. The predominant current type expressed in BrdU- cells was an inwardly rectifying K+ current (KIR; 1.3 pS/pF). BrdU- cells also expressed transient outward K+ currents, accounting for less than one-third of total K+ conductance (G). In contrast, proliferating BrdU+ astrocytes exhibited a dramatic, approximately threefold reduction in KIR (0.45 pS/pF) but showed a twofold increase in the conductance of both transient (KA) (0.67-1. 32 pS/pF) and sustained (KD) (0.42-1.10 pS/pF) outwardly rectifying K+ currents, with a GKIR:GKD ratio of 0.4. Relative expression of GKIR:GKD led to more negative resting potentials in nonproliferating (-60 mV) versus proliferating astrocytes (-53 mV; p = 0.015). Although 45% of the nonproliferating astrocytes expressed Na+ currents (0.47 pS/pF), the majority of proliferating cells expressed prominent Na+ currents (0.94 pS/pF). Injury-induced electrophysiological changes are rapid and transient, appearing within 4 hr postinjury and, with the exception of KIR, returning to control conductances within 24 hr. These differences between proliferating and nonproliferating astrocytes are reminiscent of electrophysiological changes observed during gliogenesis, suggesting that astrocytes undergoing secondary, injury-induced proliferation recapitulate the properties of immature glial cells. The switch in predominance from KIR to KD appears to be essential for proliferation and scar repair, because both processes were inhibited by blockade of KD.

Remyelination in the rat dorsal funiculus following demyelination by laser irradiation

Excimer laser (KrF excimer laser, 248 nm wavelength) was used to damage cellular components in the dorsal funiculus at the lumbar level (L2) of the rat spinal cord. An open lesion was not found at the irradiation site on the spinal cord. However, the cytological examination revealed that cellular components were damaged to the depth of 200–500 μm from the pial surface. The characteristic feature was that at the border of the lesion, many axons remained naked but intact after their myelin sheaths had been completely disintegrated. Such naked axons were subsequently remyelinated by mature or immature glial cells. Mature oligodendrocytes, while retaining their cytoplasmic processes connected with the myelin sheaths of unaffected axons, extended new cytoplasmic processes on nearby naked axons and made new myelin sheaths around them. In contrast, 7 days after the irradiation, numerous immature glial cells appeared in association with naked axons, and some of them were differentiated into oligodendrocytes forming thin myelin sheaths on naked axons. These findings suggest that demyelinated axons can cause the proliferation and probably dedifferentiation of the oligodendrocyte lineage. The use of lasers provides a unique experimental model of demyelination and remyelination in the central nervous system of adult mammals.

Early effects of iodine deficiency on radial glial cells of the hippocampus of the rat fetus. A model of neurological cretinism.

The most severe brain damage associated with thyroid dysfunction during development is observed in neurological cretins from areas with marked iodine deficiency. The damage is irreversible by birth and related to maternal hypothyroxinemia before mid gestation. However, direct evidence of this etiopathogenic mechanism is lacking. Rats were fed diets with a very low iodine content (LID), or LID supplemented with KI. Other rats were fed the breeding diet with a normal iodine content plus a goitrogen, methimazole (MMI). The concentrations of -thyroxine (T4) and 3,5,3'triiodo--thyronine (T3) were determined in the brain of 21-d-old fetuses. The proportion of radial glial cell fibers expressing nestin and glial fibrillary acidic protein was determined in the CA1 region of the hippocampus. T4 and T3 were decreased in the brain of the LID and MMI fetuses, as compared to their respective controls. The number of immature glial cell fibers, expressing nestin, was not affected, but the proportion of mature glial cell fibers, expressing glial fibrillary acidic protein, was significantly decreased by both LID and MMI treatment of the dams. These results show impaired maturation of cells involved in neuronal migration in the hippocampus, a region known to be affected in cretinism, at a stage of development equivalent to mid gestation in humans. The impairment is related to fetal cerebral thyroid hormone deficiency during a period of development when maternal thyroxinemia is believed to play an important role.

Overexpression of nestin and vimentin in the ependyma of spinal cords from hydrocephalic infants

The ependyma of the spinal central canal in cases of hydrocephalus shows abnormalities which vary with the aetiology of ventricular dilatation. To determine whether these ependymal changes are developmental or reactive in nature, immunohistochemical findings were compared between nine normal controls and 12 cases of hydrocephalus (three each of congenital aqueductal stenosis. Dandy-Walker malformation, Chiari type II malformation, and post-haemorrhagic hydrocephalus) using antisera to nestin, vimentin and glial fibrillary acidic protein. The main pathological findings were disruption of ependymal layer, apparent pseudostratification of ependyma, expansion, cleft or syrinx formation in relation to the central canal, and ependymal rosette formation. In normal developing fetal spinal cord, nestin and vimentin were expressed mainly in pseudostratified ependymal cells and radial fibres in the median septum. In cases with congenital hydrocephalus (congenital aqueductal stenosis. Dandy-Walker malformation, and Chiari type II malformation), nestin was overexpressed in immature ependymal cells, and strong vimentin immunoreactivity was detected in the long tract of radial fibres in the median septum. Nestin and vimentin were also expressed in small cells and their fibres which covered areas denuded of ependymal cells in cases of Chiari type II malformation and post-haemorrhagic hydrocephalus. Two conclusions are suggested by this report. First, the ependyma of the spinal central canal in congenital hydrocephalus shows a delay in maturation of radial glial cells into mature astrocytes and ependymal cells. Second, areas of ependymal denudation may be repaired by the immature glial cells derived from subependymal cells.

Overexpression of nestin and vimentin in ependymal cells in hydrocephalus.

In order to elucidate the immunohistochemical features of hydrocephalic ependyma, immunohistochemical examination was undertaken in 11 normal, postmortem brains (age range, 11 weeks' postconception to 6 months after birth) and 12 hydrocephalic brains (three cases each of congenital aqueductal stenosis, Dandy-Walker malformation, Arnold-Chiari type II malformation and posthemorrhagic hydrocephalus) by using antisera to nestin, vimentin and glial fibrillary acidic protein (GFAP). In normal brains, nestin was predominantly expressed in neuroepithelial cells and radial glial fibers during the period of neuronal migration. Vimentin immunoreactivity was principally detected in immature ependymal cells and their basal fibers after the period of neuronal migration, then partly replaced by GFAP reactivity during late gestation. In hydrocephalus, the areas of ependymal disruption were covered with nestin- or vimentin-positive cells. Nestin and vimentin were also expressed in immature ependymal cells or their basal processes in anatomical regions such as the roof or floor plate of the fourth ventricle or the cerebral aqueduct, and the ventral part of the third ventricle. These results suggest that the overexpression of nestin and vimentin in hydrocephalus follows two patterns: a reactive pattern of proliferating immature glial cells associated with ependymal cell loss and an abnormal developmental pattern of immunopositivity associated with anatomical regions in the midline mesencephalon.

HIV-1 Gene Expression and Replication in Neuronal and Glial Cell Lines with Immature Phenotype: Effects of Nerve Growth Factor

Encephalopathy and neurological disorders are a major manifestation of pediatric AIDS. Although HIV-1 can replicate in cells of neuronal and glial origin, it is yet unclear whether immature neural cells, which are present during nervous system development, can support HIV-1 replication and whether neurotrophic factors can modulate HIV-1 gene expression. In this study we show that a glial cell line with a phenotype closely resembling immature glial cells is more permissive to HIV-1 infection and replication than a neuroblastic cell line. After HIV-1 infection or after transfection of these cells with the HIV-1 LTR-CAT reporter gene alone or in the presence of Tat, both HIV-1 replication and viral gene expression progressively decrease in the neuronal cell line, while they increase in the glial cell line. In both cell types viral gene expression and replication are augmented by the addition to the cells of nerve growth factor (NGF) at concentrations which induce neuronal differentiation. However, these effects are again more evident with the glial cell type, suggesting that immature glial cells may represent one of the major targets and reservoirs of HIV-1 in the developing nervous system. As NGF and Tat act synergistically in inducing HIV-1 gene expression, these data also suggest that during development the presence of high levels of neural trophic factors may activate viral replication and render the CNS more susceptible to the deleterious effects of HIV-1 infection.