Pelvic embolization for treatment of hemorrhage related to spontaneous and induced abortion.

OBJECTIVE: Presentation of outcomes of pelvic arterial embolization for hemorrhage after spontaneous or induced abortion. STUDY DESIGN: We collected case reports of embolization after spontaneous or induced abortion from oral presentations and from members of the National Abortion Federation. RESULTS: Pelvic arterial embolization was performed for 11 women who had hemorrhage after spontaneous or induced abortion, and it was initially successful for all women. One woman ultimately required a hysterectomy after unsuccessful repeated embolization. Prophylactic embolization was done for 8 women who were at risk for hemorrhage from placenta accreta; 4 of these women had subsequent hysterectomies. CONCLUSIONS: Selective pelvic arterial embolization may be a successful treatment for hemorrhage associated with spontaneous and induced abortion. Embolization can be considered before hysterectomy is undertaken for control of hemorrhage. There may be a role for prophylactic catheterization or embolization when there is a risk of severe hemorrhage.

High-yield selection and extraction of two promoter-defined phenotypes of neural stem cells from the fetal human brain.

Neural stem and precursor cells reside in the ventricular lining of the fetal forebrain, and may provide a cellular substrate for brain repair. To selectively identify and extract these cells, we infected dissociated fetal human brain cells with adenoviruses bearing the gene for green fluorescence protein (GFP), placed under the control of enhancer/promoters for two genes (nestin and musashi1) that are expressed in uncommitted neuroepithelial cells. The cells were then sorted by fluorescence-activated cell sorting (FACS) on the basis of E/nestin- or P/musashi1-driven GFP expression. Both P/musashi1:hGFP- and E/nestin:EGFP-sorted cells were multipotent: limiting dilution with clonal expansion as neurospheres, in tandem with retroviral lineage analysis and xenograft to E17 and P0-2 rat forebrain, revealed that each phenotype was able to both self-renew and co-generate neurons and glia. Thus, fluorescent genes placed under the control of early neural promoters allow neural stem cells to be specifically targeted, isolated, and substantially enriched from the fetal human brain.

Transplantation of CD34 human cells into mice with severe combined immunodeficiency results in functional T cells 4 weeks after transplantation.

OBJECTIVE: Our purpose was to determine whether transplantation of fetal human CD34(+) cells into mice with severe combined immunodeficiency results in functional T cells. STUDY DESIGN: The cells used in this study were isolated from fetal human liver tissue obtained after elective termination of normal 18- to 24-week pregnancies. Women with medical conditions that could confound the outcome were excluded. Cells were labeled with fluorochrome-conjugated antibodies that recognized CD34 or other cell surface antigens. The cells were then sorted with the use of a fluorescein-activated cell sorter. The human sorted cells were injected intraperitoneally in mice with severe combined immunodeficiency. Four groups of mice were studied: group 1, injected with 10(5) CD34(+) cells (n = 17); group 2, injected with 10(5) CD34(-) cells (n = 14); group 3, injected with 10(6) unsorted cells (n = 19); and group 4, sham-injected with phosphate-buffered saline solution as controls (n = 14). At 1, 2, and 4 weeks after transplantation, the peripheral blood monocytes of the study mice were analyzed for functional T cells. Aliquots of cells (10(5)) were incubated for 48 hours with 0, 5, 10, and 20 micrograms of phytohemagglutinin. Thereafter the cells were treated with 1 microCi of tritiated thymidine. Subsequently the incorporation of tritiated thymidine was determined by liquid scintillation counting. RESULTS: Cells from mice transplanted with either unsorted cells, sorted CD34(+) cells, or CD34(-) cells showed a response to phytohemagglutinin that varied with time and with the mitogen concentration. Even though unsorted fetal human liver cells had a maximal response at 2 weeks, this posttransplantation response was not statistically significant. CD34(+) cell response to phytohemagglutinin was significant at 4 weeks after transplantation. CD34(-) cells also had a peripheral blood cell response at 4 weeks after transplantation; however, this response was not statistically significant. In addition, all mice transplanted with fetal human liver cells had some functional T cells at 4 weeks; however, this response was statistically significant only for CD34(+) cells. CONCLUSION: Transplantation of either sorted CD34 (positive or negative) cells or unsorted fetal human liver cell preparations into mice with severe combined immunodeficiency results in functional T cells. However, only the mice with transplanted CD34(+) cells demonstrated a statistically significant response.

Immunophenotypic characterization of human fetal liver hematopoietic stem cells during the midtrimester of gestation.

OBJECTIVE: Our purpose was to define the extent to which gestational age influences the number of fetal liver cells that coexpress phenotypic markers associated with hematopoietic stem cells and major histocompatibility antigens. STUDY DESIGN: Fetal liver cells from abortuses of 9 to 24 weeks of gestation were studied (n = 61). Low-density nucleated liver cells were isolated on a discontinuous density gradient and subsequently incubated with antibodies that recognize markers of hematopoietic stem cells (i.e., CD33, CD34, CDw90, CD117, and CD123). Human leukocyte antigen class I (A, B, C) and class II (DR) antigens were also determined on these cells. Each sample was analyzed by immunocytochemistry and flow cytometry. Analysis of variance was used for statistical analysis. RESULTS: Of the markers measured, only the percentage of CD123-positive cells increased significantly with gestational age (p < 0.01). The percentage of triple-positive cells (CD34+, CD117+, and CD123+) increased with age but did not reach significance (p = 0.05). Human leukocyte A, B, and C antigens were expressed on all nucleated cells from 9 to 24 weeks of gestation. Human leukocyte DR antigen, however, was expressed only on 50% of these cells. The percentage of cells that expressed both hematopoietic stem cell markers and DR antigen did not vary with gestational age. CONCLUSIONS: From 9 to 24 weeks of gestation the number of human fetal liver hematopoietic stem cells that coexpress major histocompatibility antigens increases with advancing gestational age, largely because the percentage of these cells remains constant while the liver mass increases.

HIV infection of human fetal neural cells is mediated by gp120 binding to a cell membrane-associated molecule that is not CD4 nor galactocerebroside.

HIV infection of central nervous system (CNS) tissue is a common finding in both adult and pediatric AIDS. Because most children are believed to be infected perinatally, we have developed a model of HIV CNS infection that utilizes explant organotypic cultures of human fetal CNS tissue. Using this model we previously reported that both lymphocytotropic and monocytotropic HIV isolates infect microglia and astrocytes. However, the mechanism by which HIV infects these cells remains to be elucidated. We have observed that neural cell infection in these cultures may be the result of receptor-mediated endocytosis. In order to confirm this observation and to determine the ligand responsible for this process, organotypic cultures were exposed to untreated HIV, HIV pretreated with soluble CD4 (sCD4) or, as a control, heat-inactivated HIV. To address the question of a putative receptor for HIV infection, CNS cultures were either untreated or pretreated with gp120 or with the deglycosylated form of this protein. Other cultures were treated with antibodies to CD4 (anti-T4A) or to galactocerebroside (GC). Results demonstrate that pretreatment of either HIV with sCD4 or CNS cultures with gp120 significantly inhibits HIV infection. The inhibition of infection was demonstrated by a reduction in the number of cells positive for HIV proteins and by decreases in HIV proviral DNA and p24 production. Pretreatment of CNS cultures with deglycosylated gp120, anti-T4A or anti-GC antibodies did not inhibit HIV infection. These data suggest that HIV gp120 is needed for binding to a surface molecule on CNS cells that is not CD4 nor GC and that this molecule may function as a receptor and lead to infection of neural cells.

Oligodendrocyte gene expression in the human fetal spinal cord during the second trimester of gestation.

A comprehensive evaluation of myelination during normal human development is essential to understand the pathology of congenital diseases of white matter. The present study establishes quantitative values for normal oligodendrocyte-specific gene expression during the early stages of myelination in the human fetal spinal cord. Complementary techniques of Northern and immunoblotting were used to determine relative amounts of oligodendrocyte-specific mRNAs and proteins between 12 and 24 gestational weeks. Values were determined for myelin basic protein, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and proteolipid protein. The relative amount of myelin-associated glycoprotein mRNA was also estimated. To compare gene expression between glial cell types, the relative amounts of mRNA and protein were determined for glial fibrillary acidic protein (GFAP), a cell-type specific marker for astrocytes. All oligodendrocyte-specific genes expressed similar developmental kinetics. Between 12 and 15 gestational weeks, less than a five-fold increase was detected in the expression of these genes and their protein products. Between 15 and 22 gestational weeks, the relative amounts of mRNA and protein for the myelin genes increased more than 80-fold. The kinetics of GFAP expression were similar to those of the myelin-associated genes. Absolute values for the increase in mass of the human fetal spinal cord were also obtained. These results provide data that may aid in the neuropathologic assessment and characterization of myelin disorders in the preterm, neonatal, and pediatric spinal cord.

Quantification of myelin basic protein in the human fetal spinal cord during the midtrimester of gestation.

The amount of myelin basic protein (MBP) was quantified in human fetal spinal cords from 12 to 24 gestational weeks (GW). MBP expression was determined by Northern blot, quantitative immunoblot, and immunocytochemistry. The development of compact myelin was analyzed by electron microscopy. Thirty-eight human fetal spinal cords were obtained after elective termination of intrauterine pregnancies from healthy women. Northern blot analysis showed a 15.8-fold increase in MBP mRNA between 12 and 18 GW. From 18 to 24 GW, MBP mRNA increased by 2.2-fold. The mRNA data paralleled immunoblot results that showed a 90.5-fold increase in MBP (0.147 ng/mg to 13.3 ng/mg tissue) between 12 and 18 GW and an approximately 11.5-fold increase between 18 and 24 GW (13.3 ng/mg to 154 ng/mg tissue). Immunocytochemical analysis also showed increased staining for MBP with advancing gestational age. At 12 GW, MBP immunoreactivity was observed in all three spinal cord funiculi. By 18 GW, MBP was expressed throughout the spinal cord white matter with the exception of the lateral corticospinal tracts and in the rostral levels of the fasciculus gracilis. With respect to myelin, at 12 GW, rare, noncompacted myelin lamellae were observed by electron microscopy. By 18 GW, discrete areas of compact myelin were observed in areas that showed MBP immunoreactivity, and at 24 GW, compact myelin was prominent throughout the white matter of the spinal cord. This study demonstrates a quantitative increase in MBP expression that is associated with myelin formation during the second trimester of human gestation. This information may provide normative data that can aid in the diagnosis of myelin disorders of the preterm, neonatal, and pediatric spinal cord.

Temporal and spatial expression of major myelin proteins in the human fetal spinal cord during the second trimester.

Immunohistochemical identification of myelin basic protein (MBP) is a sensitive method for assessing myelination in the human fetal central nervous system (CNS). However, the temporospatial relationship of expression of two other major myelin proteins, proteolipid protein (PLP) and myelin-associated glycoprotein (MAG) to that of MBP during fetal development has not been assessed in human tissues. Vibratome sections of cervical, thoracic and lumbosacral levels from 37 normal spinal cords of < or = 10 to 24 gestational week (GW) fetuses were analyzed using immunohistochemical methods. Using light microscopy, MBP was the first oligodendrocyte marker detected, present by 10 GW at more rostral levels. PLP and MAG were detected rostrally between 12 to 14 GW. All myelin proteins were expressed in anterior to posterior and rostral to caudal gradients. By the late second trimester, expression of MBP, PLP and MAG was noted in all locations in the spinal white matter except for the corticospinal tract. Expression of MAG was particularly marked in the posterior root entry zone and propriospinal tracts. The results suggest that PLP and MAG are expressed later than MBP but follow similar spatial gradients.

Muscarinic receptor-dependent activation of phospholipase C in human fetal central nervous system organotypic tissue culture.

The coupling of muscarinic-cholinergic receptors (mAchR) with the phospholipase C (PLC) second messenger system has been demonstrated in central nervous system (CNS) tissue of many animal species. However, little information exists regarding this association in the developing human CNS. Due to the suggested role of acetylcholine in the regulation of development and differentiation of neural cells, the knowledge of these relationships during human fetal development acquires singular importance. Because of this, we examined the cholinergic stimulation of PLC in human fetal CNS organotypic tissue cultures. Agonist treatment of cultures, in the presence of lithium, resulted in a 4-6-fold increase in inositol phosphates formation. This increase was caused principally by the formation of inositol phosphate (IP). However, kinetic studies demonstrated that the levels of IP2, IP3 and IP4 also increased rapidly after stimulation reaching maximum levels before IP. These results support the hypothesis that muscarinic receptor activation results in an increase in the hydrolysis of PIP2. The inositol phosphate formation was dependent on agonist concentration. The obtained EC50 values were approximately 57 +/- 15 microM for carbachol, 8 +/- 2 microM for acetylcholine and 49 +/- 15 microM for oxotremorine. The agonist-dependent formation of inositol phosphates was inhibited by the muscarinic antagonists atropine and pirenzepine. Pirenzepine inhibited carbachol stimulation with high affinity (Ki = 2.90 +/- 1.15 nM), indicating that PLC activation is the result of activation of the m1 subtype of muscarinic receptors. Treatment of cultures with pertussis toxin did not result in inhibition of agonist-dependent activation of PLC. This result suggests that the m1 muscarinic receptor is coupled to PLC through Gq.

Placenta accreta encountered during dilation and evacuation in the second trimester.

OBJECTIVE: To assess the frequency of placenta accreta encountered during dilation and evacuation (D&E) in the second trimester. METHODS: Among 16,827 second-trimester D&E procedures performed at our hospitals and clinics, seven cases of placenta accreta, either suspected clinically or proven histologically, were encountered. These cases were analyzed for history of prior cesarean delivery, placenta localization, and histology of hysterectomy specimens. RESULTS: Six of the seven cases suspected clinically were confirmed histologically. All placenta accreta patients had at least one cesarean delivery (mean 1.7), and five had a preoperative sonogram demonstrating some form of placenta previa. The prevalence of clinical placenta accreta encountered during D&Es in the second trimester was 0.04%, the same as that reported for placenta accreta diagnosed clinically in the third trimester. CONCLUSION: Placenta accreta can be a potential complicating factor in the patient undergoing D&E in the second trimester.

Neural cell targets of human immunodeficiency virus type 1 in human fetal organotypic cultures.

Some children infected by HIV-1 demonstrate nervous system disease. Because a significant percentage of these children are believed to be infected during gestation and it is thought that HIV-1 may infect distinct glial populations, this work tested the hypothesis that different HIV-1 isolates can infect cells of the developing human fetal central nervous system (CNS). Central nervous system organotypic tissue cultures derived from human fetal brain enable the study of complex interactions between CNS cell types. Central nervous system organotypic cultures were exposed to lymphocytotropic (L-tropic) or monocytotropic (M-tropic) HIV-1 isolates and monitored for viral infection. HIV-1 gp41 and p24 antigens were detected by immunocytochemistry (ICC), HIV-1 RNA was localized in the cytoplasm of CNS cells by in situ hybridization (ISH), and viral DNA was detected by polymerase chain reaction (PCR) in HIV-1-exposed cultures. Double-label ICC identified HIV-1 antigens in both microglia and astrocytes. These results demonstrate that both L- and M-tropic isolates infect microglia and astrocytes in human fetal organotypic cultures. In addition, HIV-1 infection was detected in culture supernatants up to day 57 postinfection and at 90 days by coculture with susceptible CEM cells. HIV-1 infection of neural cells appears to be productive. This model may permit further examination of the interaction of HIV-1 with the developing human CNS and the mechanisms of AIDS-associated neuropathology.

Muscarinic receptor-dependent activation of phospholipase C in the developing human fetal central nervous system.

The coupling of muscarinic-cholinergic receptors (mAChR) to adenylate cyclase and phospholipase C (PLC) second messenger systems has been demonstrated in many animal species. However, little is known about this association in the developing human central nervous system. Because of the proposed role of acetylcholine in the regulation of development and differentiation of neural cells, an understanding of these relationships during human fetal development gains importance. We report, in this communication, the coupling of mAChR with PLC in the human fetal brain. This coupling was determined using two independent approaches that relied upon estimating the accumulation of inositol phosphates (IPs) and cytidine diphosphate diacylglycerol (CDP-DAG). Carbachol treatment of brain slices, in the presence of lithium, resulted in the accumulation of IPs. Analysis of the kinetics of this accumulation showed that IP3 and IP2 increased rapidly, reaching a peak or plateau before IP. The results also showed that agonist-stimulated PLC produced two second messengers, IP3 and DAG. The production of DAG was strongly supported by the carbachol-dependent increase of CDP-DAG. The accumulation of IP and CDP-DAG was dependent on agonist concentration. The obtained EC50 values were approximately: carbachol 47 microM; acetylcholine 6 microM; and oxotremorine 25 microM. Unexpectedly, all three agonists demonstrated a similar efficacy. The cholinergic stimulation of inositide hydrolysis appears to be the result of activation of the m1 muscarinic receptor.

Patterns of glial development in the human foetal spinal cord during the late first and second trimester.

Although the presence of radial glia, astrocytes, oligodendrocytes and microglia has been reported in the human foetal spinal cord by ten gestational weeks, neuroanatomic studies employing molecular probes that describe the interrelated development of these cells from the late first trimester through the late second trimester are few. In this study, immunocytochemical methods using antibodies to vimentin and glial fibrillary acidic protein were used to identify radial glial and/or astrocytes. An antibody to myelin basic protein was used for oligodendrocytes and myelin; and, an antibody to phosphorylated high and medium molecular weight neurofilaments identified axons. Lectin histochemistry using Ricinus communis agglutinin-I was employed to identify microglia. Vibratome sections from 35 human foetal spinal cord ranging in age from 9-20 gestation weeks were studied. By 12 gestational weeks, vimentin-positive radial glia were present at all three levels of the spinal cord. Their processes were easily identified in the dorsal two-thirds of cord sections, and reaction product for vimentin was more intense at cervical and thoracic levels than lumbosacral sections. By 15 gestational weeks, vimentin-positive processes were radially arranged in the white matter. At this time, glial fibrillary acidic protein-positive astrocytes were more obvious in both the anterior and anterolateral funiculi than in the dorsal funiculus, and the same rostral to caudal gradient was seen for glial fibrillary acidic protein as it was for vimentin. Myelin basic protein expression followed similar temporal and spatial patterns. Ricinus communis agglutinin-I labelling revealed more microglia in the white matter than in grey matter throughout the spinal cord from 10-20 gestational weeks. By 20 gestational weeks, the gradients of glial fibrillary acidic protein and vimentin expression were more difficult to discern. White matter contained more microglia than grey matter. These results suggest that astrocytes as well as oligodendrocytes follow anterior-to-posterior and rostral-to-caudal developmental patterns in the human foetus during middle trimester development.

Immunohistochemical detection of myelin basic protein is a sensitive marker of myelination in second trimester human fetal spinal cord.

The Luxol fast blue (LFB) technique is widely used for the assessment of myelination. Lectin histochemistry using peanut agglutinin (PNA) has also been employed for this purpose. Recently, immunohistochemical methods using antibodies to several myelin-related proteins have been adopted to study myelination in humans. However, the relative sensitivities of these different methods for the detection of early myelination in the human fetal central nervous system have not been determined. Vibratome sections of cervical spinal cord from 15 human abortuses ranging in age from 15 to 24 gestational weeks (GW) were probed with immunohistochemical methods using antibodies to myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), and myelin-associated glycoprotein (MAG). In addition, LFB and PNA histochemistry was employed. The degree of myelination observed in immunohistochemically stained sections was compared to that found in corresponding LFB- and PNA-stained paraffin-embedded tissues. The intensity of myelination was graded by two observers on a scale of 0 (none), +1 (mild), +2 (moderate), and +3 (marked). At all ages examined, the MBP immunohistochemical method revealed more myelin than LFB or MAG staining. CNPase could not be reliably detected until after 18 GW. Peanut agglutinin stained myelin, but subpial astrocytes and the intervening neuropil were also stained. These results suggest that MBP is a more sensitive marker for early human fetal myelination than CNPase, MAG, PNA or LFB.

Human fetal astrocytes induce the expression of blood-brain barrier specific proteins by autologous endothelial cells.

The blood-brain barrier (BBB) is involved in many normal regulatory mechanisms as well as in pathologic conditions of the central nervous system. Previous studies examining the development and function of the BBB in vitro have primarily utilized cell lines or cultured tissues from non-human sources. In contrast, this study used a coculture system of human fetal astrocytes and autologous endothelial cells. Astrocytes and endothelial cells (EC) were isolated and cultured on the opposite sides of a synthetic permeable membrane. The cocultures were characterized by electron and light microscopy for morphology and by immunocytochemistry for cell-type specific markers. Using these coculture conditions, astrocytes displayed characteristic morphology and expressed glial fibrillary acidic protein. When cocultured with astrocytes, endothelial cells retained factor VIII expression and expressed the BBB-specific proteins, brain-type glucose transporter (GLUT-1) and gamma-glutamyl transpeptidase. This expression was dependent on EC being in close apposition to or in direct contact with astrocytes. The model presented in this study may permit further examination of the role of the BBB in both normal human neurodevelopment and neuropathologic conditions.

Neuroanatomical localization of myelin basic protein in the late first and early second trimester human foetal spinal cord and brainstem.

The temporal and spatial expression of myelin basic protein in the first and second trimester human foetal spinal cord and brainstem from 9 to 20 gestational weeks was determined by immunocytochemistry in sections of cervical, thoracic and lumbosacral levels from 41 human foetal spinal cords and ten brainstems. Myelin basic protein-positive oligodendrocytes were observed peripheral to the ependyma at 9-10 gestational weeks. Oligodendrocytes expressing myelin basic protein were seen at 10-12 gestational weeks in the anterior and lateral funiculi. Myelin basic protein was detected later in the posterior funiculi than in the anterolateral white matter and most spinal cord tracts could not be identified by means of variation in myelin basic protein expression. Myelin basic protein was found in the midline of the brainstem at ten gestational weeks and spread laterally during the second trimester. We conclude that in the human foetal spinal cord, myelin basic protein is present by 10 gestational weeks in the anterolateral cervical spinal cord and midline of the brainstem. It is expressed in a rostral-to-caudal and anterolateral-to-posterior manner in most tracts of the spinal cord. However, an exception to these findings is that the fasciculus gracilis, upon developing into a defined region, had more myelin basic protein-positive cells at the lumbar level than in more rostral regions. Definition of the kinetics of myelin basic protein expression in the normal human foetal spinal cord provides a baseline for study of aberrant myelination and demyelination.

Human fetal myelinated organotypic cultures.

We have previously reported the establishment of organotypic cultures derived from human fetal brain tissue. Although these cultures permit the testing of multiple hypotheses about normal human neurodevelopment and neuropathologic conditions, they have the limitation of not being myelinated and therefore preclude the study of questions related to myelinogenesis and diseases of myelin. In the current communication, we describe recent developments that allow us to overcome this limitation and permit the establishment of a myelinated organotypic culture model. Sections of dorsal column dissected from the lumbar spinal cord of human fetuses ranging in age 21-23 weeks of gestation were placed in culture. The explants were maintained for up to 12 weeks during which time they were characterized and shown to express a number of CNS cell-type-specific markers including glial fibrillary acidic protein (astrocytes), nerve growth factor receptor and neurofilament protein (neurons), CD68 (microglia), and myelin basic protein, HNK-1 and galactocerebroside (oligodendrocytes). In addition, lectin histochemistry using Ricinus communis agglutinin-1 detected microglia and endothelial cells. Upon explantation, abundant myelin was seen by electron microscopy in the cultures. Although during the culture period there was degradation of myelin, there was also evidence of maintenance of intact myelin sheaths around small caliber axons and de novo myelin synthesis. This model system may permit the further use of human organotypic cultures to investigate issues related to neurodevelopment and to pathologic conditions including those relevant to dysmyelination and demyelination.