Embryo Brain Research Articles

Enhanced tissue differentiation in the developing mouse brain using magnetic resonance micro‐histology

Worldwide efforts to understand developmental processes demand new high-resolution 3D imaging methods to detect the consequences of gene function in embryo development and diseases. Encouragingly, recent studies have shown that MRI contrast agents can highlight specific tissue structures in ex vivo adult mouse brains. MR imaging of mouse embryos is currently limited by a lack of tissue staining capabilities that would provide the flexibility and specificity offered by histological stains conventionally used for mouse embryo phenotyping. The MRI staining properties of two readily available contrast agents, Mn-DPDP and Gd-DTPA, were investigated in mid-gestation mouse embryos. Brain tissue substructures not normally visible using MRI were detected. Mn-DPDP and Gd-DTPA provided spatially distinct tissue staining patterns. An initial assessment indicated that these agents utilized independent contrast enhancement mechanisms. Mn-DPDP was identified as a potential MRI contrast agent for enhancement of mouse embryonic cellular density and enabled identification of regions containing populations of neural stem and progenitor cells within the intact embryo brain. Different contrast agents may be used to provide tissue-specific contrast enhancement, suggesting that a host of specialized MRI stains may be available for probing the developing mouse brain and investigating developmental and disease mechanisms.

Spatial expression and functional flexibility of monocarboxylate transporter isoforms in the zebrafish brain

The present study provides in vivo evidence to prove the functional plasticity of monocarboxylate transporters (MCTs) in brains of vertebrates using zebrafish (Danio rerio) as a model. In the mammalian central nervous system (CNS), energy demands are largely met by oxidation of glucose. In recent studies, in addition to glucose, lactate is also considered an energy substrate for the CNS. Astrocytes were demonstrated to play an important role in transporting lactate as metabolic substrate from capillaries to neurons through monocarboxylate transporters (MCTs). The present study was to use zebrafish as an in vivo model to test the hypothesis of whether the various MCT homologues play differential roles in the development and functioning of the CNS. Using RT-PCR and double in situ hybridization coupling with immunocytochemical staining experiments, zebrafish MCTs1~4 were all found to be expressed in brains of embryos, and were further elucidated to be localized in both neurons and astrocytes. Loss-of-functions by morpholino knockdown further provided in vivo evidences to infer that zMCTs1, -2, and -4 may be involved in metabolite transport and functioning in the developing brain. Subsequent rescue experiments with capped mRNAs of specific isoforms further indicated that zMCT2 is an indispensable monocarboxylate-transporting route for CNS development and function in zebrafish. This information is essential for identifying proper candidates of MCT isoforms that are involved in the development and functioning of the CNS.

Human melanoma cells in the rhombencephalon of the chick embryo: a novel model for brain metastasis

Malignant melanoma has the highest propensity to metastasize to the brain of all primary neoplasms in adults. Here, we describe invasive growth and the development of melanoma metastases from suspensions of human melanoma cells in the brain of the chick embryo. Patient-derived melanoma cells and established melanoma cell lines were injected into the rhombencephalic brain vesicle of the two-day-chick embryo. After 48 and 96 h, tumor formation was studied in serial paraffin sections with melanoma-specific HMB45 and human-specific MIB1 proliferation markers. The majority of the cells injected into the embryonic liquor cavity perished. Only melanoma cells in newly formed aggregates or when attached to the dorsal roof plate escaped apoptosis. Local invasion occurred not in the ventral differentiating neural epithelium but only in the roof plate. Although after 48 h melanoma cells invaded the rhombencephalic roof plate profusely at both sides, after 96 h typically one large tumor developed in the midline between roof plate and the dorsal surface epithelium. From the tumor, single cells invaded the mesenchyme and blood vessels. Cell lines with different invasive properties retained their graded invasive behaviour. Maximally invasive cells formed continuous tracks via vessels and along nerve fibres. The central tumor in the roof plate of the chick embryo rhombencephalon resembles a metastatic nodule in the patients with melanoma. Penetration of the roof plate epithelium, tumor formation and invasion of surrounding tissues by single cells can reliably be reproduced. The chick embryo model can be used for molecular studies of early phases of melanoma brain metastasis.

Use of RNA interference by in utero electroporation to study cortical development: the example of the doublecortin superfamily.

The way we study cortical development has undergone a revolution in the last few years following the ability to use shRNA in the developing brain of the rodent embryo. The first gene to be knocked-down in the developing brain was doublecortin (Dcx). Here we will review knockdown experiments in the developing brain and compare them with knockout experiments, thus highlighting the advantages and disadvantages using the different systems. Our review will focus on experiments relating to the doublecortin superfamily of proteins.

HuC–eGFP mosaic labelling of neurons in zebrafish enables in vivo live cell imaging of growth cones

The field of axon guidance is taking advantage of the powerful genetic and imaging tools that are now available to visualise growth behaviour in living cells, both in vivo and in real time. We have developed a method to visualise individual neurons within the living zebrafish embryo which provides exceptional cellular resolution of growth cones and their filopodia. We generated a DNA construct in which the HuC promoter drives expression of eGFP. Injection of the plasmid into single cell fertilised zebrafish egg resulted in mosaic expression of eGFP in neurons throughout the developing embryo. By manipulating the concentration of injected plasmid, it was possible to optimise the numbers of neurons that expressed the construct so that individual growth cones could be easily visualised. We then used time-lapse high magnification widefield epifluorescence microscopy to visualise the growth cones as they were exploring their environment. Growth cones both near the surface of the embryo as well as deep within the developing brain of embryos at 20 h post fertilisation were clearly imaged. With time-lapse sequence imaging with intervals between frames as frequent as 1 s there was minimal loss of fluorescence intensity and the dynamic nature of the growth cones became evident. This method therefore provides high magnification, high resolution time-lapse imaging of living neurons in vivo and by use of widefield epifluorescence rather than confocal it is a relatively inexpensive microscopy method.

AT motif binding factor 1 (ATBF1) is highly phosphorylated in embryonic brain and protected from cleavage by calpain-1

ATBF1 is a transcription factor that regulates genes responsible for repairing tissues and the protection of cells from oxidative stress. Therefore reduction of ATBF1 promotes susceptibility to varieties of human diseases including neurodegenerative diseases and malignant tumors. The instability of the protein was found to be an important background of diseases. Because ATBF1 is composed of a large 404-kDa protein, it can be easily targeted by proteinases. The protein instability should be a serious problem for the function in the cells and practically for our biochemical study of ATBF1. We have found that calpain-1 is a protease responsible for the degeneration of ATBF1. We observed distinct difference between embryo and adult brain derived ATBF1 regarding the sensitivity to calpain-1. The comparative study showed that eight phosphorylated serine residues (Ser1600, Ser2634, Ser2795, Ser2804, Ser2900, Ser3431, Ser3613, Ser3697) in embryonic brain, but only one site (Ser2634) in adult brain. As long as these amino acids were phosphorylated, ATBF1 derived from embryonic mouse brain showed resistance to cleavage; however, treatment with calf intestine alkaline phosphatase sensitized ATBF1 to be digested by calpain-1. An inhibitor (FK506) against calcineurin, which is a serine/threonine specific phosphatase enhanced the resistance of ATBF1 against the digestion by calpain-1. Taken together, these results demonstrate that these phosphorylation sites on ATBF1 function as a defensive shield to calpain-1.

Acetoacetyl-CoA synthetase is essential for normal neuronal development

Cholesterol and fatty acids are essential, abundant components of neuronal tissue. Acetoacetyl-CoA synthetase (AACS) is a ketone body-utilizing enzyme for the synthesis of cholesterol and fatty acids and is highly expressed in the brain. In this study, we investigated the regulation of AACS during neurite outgrowth to clarify the physiological role of AACS in neurogenesis. Messenger RNA levels and the expression of AACS were increased during neurite outgrowth in Neuro-2a cells. The expression of HMG-CoA reductase, a key enzyme of cholesterol biosynthesis, was also increased. ChIP assays showed that the amount of SREBP-2, a key transcription factor of cholesterol synthesis, interacted with the AACS promoter was increased during neurite outgrowth, and knockdown of SREBP-2 down-regulated the mRNA levels of AACS in Neuro-2a cells. The expression of AACS in the brains of mouse embryos was dramatically increased between E16.5 and E18.5. Moreover, knockdown of AACS in primary neurons caused decreases in the expression of MAP-2 and NeuN, which are markers of neuronal differentiation, as well as synaptopodin, a marker of spine apparatus. These results suggest that AACS is regulated by SREBP-2 and involves in the normal development of neurons.

Vertical Transmission of Avian Bornavirus in Psittaciformes: Avian Bornavirus RNA and Anti-Avian Bornavirus Antibodies in Eggs, Embryos, and Hatchlings Obtained from Infected Sun Conures (Aratinga solstitialis)

Fertilized eggs were obtained from four pairs of sun conures (Aratinga solstitialis) infected with avian bornavirus (ABV) genotype 2, as determined by the sequence of the P24 gene. ABV RNA could be detected in early embryos of all four pairs. ABV RNA also was detected in brain, liver, and eyes of late-stage embryos of one of the pairs (Pair 4) and in blood of a 2-wk-old hatchling of this pair, demonstrating that vertical transmission can occur. ABV RNA could be detected in the liver but not in the brain or eyes of the late-stage embryos of another pair (Pair 3). Although it could be detected in the undeveloped eggs of the female parent and 8-day-old embryos, bornaviral RNA could not be found in the brain and liver of the late-stage embryos or in feathers and blood of young (5-9-wk-old) hatchlings of a third pair (Pair 2). At 11 wk, ABV RNA could be detected again in feathers and blood of these hatchlings and in the brain of one of the hatchlings of Pair 2 that suddenly died. ABV RNA could however be detected in throat swabs of the 5- and 9-wk-old hatchlings and their parents (Pair 2). Although the continued presence of ABV RNA in feathers and blood below the detection level of the reverse transcription-PCR used cannot be excluded, this result also may be attributable to feeding by the infected parents. Analysis by enzyme-linked immunosorbent assay showed that egg yolks and serum of late-stage embryos contain variable amounts of non-neutralizing anti-ABV-P40, -P10, -P24, and -P16 antibodies, the ratio of which reflected the antibody ratio in the serum of the female parent. Antibodies against the viral glycoprotein, which are considered neutralizing in mammals, and against ABV RNA polymerase were not detected. Whereas 5-wk-old hatchlings of the pair (Pair 2) that produced ABV RNA-free late-stage embryos were free of anti-ABV antibodies, such antibodies could be detected again in the serum of these hatchlings at 9 wk of age, before the age that bornaviral RNA could again be detected in feathers and blood. At 16 wk, these antibodies became abundant. The finding that late-stage embryos, presumably free of ABV RNA, can be obtained from eggs from infected parents suggests that hand- or foster-raising of such birds may be a method to obtain birnavirus-free offspring from some infected birds.

Comparison of Spheroids Formed by Rat Glioma Stem Cells and Neural Stem Cells Reveals Differences in Glucose Metabolism and Promising Therapeutic Applications

Cancer stem cells (CSCs) are thought to be partially responsible for cancer resistance to current therapies and tumor recurrence. Dichloroacetate (DCA), a compound capable of shifting metabolism from glycolysis to glucose oxidation, via an inhibition of pyruvate dehydrogenase kinase was used. We show that DCA is able to shift the pyruvate metabolism in rat glioma CSCs but has no effect in rat neural stem cells. DCA forces CSCs into oxidative phosphorylation but does not trigger the production of reactive oxygen species and consecutive anti-cancer apoptosis. However, DCA, associated with etoposide or irradiation, induced a Bax-dependent apoptosis in CSCs in vitro and decreased their proliferation in vivo. The former phenomenon is related to DCA-induced Foxo3 and p53 expression, resulting in the overexpression of BH3-only proteins (Bad, Noxa, and Puma), which in turn facilitates Bax-dependent apoptosis. Our results demonstrate that a small drug available for clinical studies potentiates the induction of apoptosis in glioma CSCs.

Postmortem stability of brain GABAergic and glutamatergic receptors and enzymes under ecological conditions

Neurochemical biomarkers have emerged as useful tools for assessing the subclinical neurological impacts of environmental toxicants in birds and other wildlife. Careful consideration of biomarker stability is necessary before implementing their use on tissues from ecological studies, as receptors and enzymes in the brain may be affected by postmortem conditions. The goal of this study was to evaluate the postmortem stability of key GABAergic and glutamatergic receptors (N-methyl-d-aspartate (NMDA), gamma-aminobutyric acid (GABAA-benzodiazepine)) and enzymes (glutamine synthetase (GS), glutamic acid decarboxylase (GAD)) under environmentally relevant field and storage conditions to determine their suitability as biomarkers. We exposed chicken embryo brains to postmortem environmental and storage conditions typical for ecological studies (12, 24, and 48h at 7°C or 25°C; 1, 4, and 8 weeks at −80°C or −20°C; 1 or 2 freeze thaw cycles), and measured [3H] MK-801 binding to the NMDA receptor, [3H] flunitrazipam binding to the GABAA-benzodiazepine receptor, GS activity, and GAD activity.We found that [3H] MK-801 binding is stable under all conditions studied. GAD activity was fairly stable under each storage and environmental temperatures for all durations, but was significantly less stable when stored at −20°C than at −80°C. [3H] flunitrazipam binding and GS activity were both impacted by environmental and storage temperature and duration, and might best be utilized in studies of samples with similar histories. Our findings here demonstrate that caution is warranted when comparing samples with different collection and storage histories, but that some biomarkers are fairly stable under various conditions.

Seboxplays an important role during the early mouse oogenesisin vitro

Oogenesis is a highly complex process that requires the exquisite temporal and spatial regulation of gene expression at multiple levels. Skin-embryo-brain-oocyte homeobox (Sebox) gene encodes a transcription factor that is highly expressed in germinal vesicle stage oocytes and that plays an essential role in early embryogenesis at the 2-cell stage in the mouse. As Sebox is also expressed in mouse fetal ovaries, the aim of the present study was to study its role during the early oogenesis in vitro. Expression of Sebox was low in 15.5 to 17.5 days post coitum (dpc) ovaries, showed a peak at 18.5 dpc and then its expression decreased dramatically in newborn ovaries. Sebox expression was efficiently knocked down (>80%) in fetal mouse ovary explants in culture using RNAi technology. When fetal ovary explants were transfected with Sebox-specific RNAi, the number of oocytes at germinal vesicle stage and showing a diameter of 40-70 μm was decreased significantly to 75% after 7 days of culture relative to the negative control, and to 22.4% after 10 days of culture, thus indicating that Sebox plays an important role in the early oogenesis in mice.

α-Tocopherol mitigates ethanol induced malformations and cell damage in the eye and brain of the chick embryo

The goal of this study is to investigate the ability of vitamin E in the active form α-tocopherol to mitigate the ethanol induced damaging effects and malformations in the developing chick embryo. Fertilized eggs were divided into five groups; the control group, and treated groups. The experimental groups were injected, in the air sac, before incubation with a single dose of 100μl saline solution, 100μl of 10% ethanol in saline solution, a mixture of 100μl of 10% ethanol and 200ppm vitamin E or 10% ethanol and 400ppm vitamin E. Treatment of developing chick embryos with 10% ethanol resulted in growth retardation and malformations in the eye, brain, limbs and other body parts. Ethanol exerted its degenerative effects probably via increasing membrane fluidity leading to membrane damage and significantly increased levels of lipid peroxidation. Ethanol also induced significant reduction in nitric oxide levels resulting in reduced body weight of the treated embryo probably due to restricted bold flow. Ethanol significantly increased glutathione level as a defense response. Vitamin C levels were significantly decreased after ethanol treatment due to oxidation or utilization. Vitamin E in the active form α-tocopherol partially mitigated the ethanol damaging effects either by exerting its antioxidant properties leading to a significant reduction of lipid peroxidation levels, retaining normal levels of nitric oxide or maintaining normal levels of endogenous antioxidants, glutathione and vitamin C.

Waking-like Brain Function in Embryos

Experience-dependent plastic changes in the brain underlying complex forms of learning are generally initiated when organisms are awake, and this may limit the earliest developmental time at which learning about external events can take place. It is not known whether waking-like brain function is present prenatally in higher vertebrate (bird or mammal) embryos, or whether embryos have brain circuitry that can selectively turn on a waking-like state in response to salient external sensory stimulation. Combining submillimeter-resolution brain positron emission tomography (PET), structural X-ray computed tomography (CT) of the skeleton for fine-scale embryo aging, and noninvasive behavioral recording of chicken embryos in the egg revealed unexpectedly wide variation in prenatal brain activity, inversely related to behavioral activity, which developed into different sleep-like fetal brain states. Brief prenatal exposure to a salient chicken vocalization (eliciting strong postnatal behavioral responses) increased higher-brain activity significantly more than a spectrally and temporally matching "nonvocal" noise analog. Patterns of correlated activity between the brainstem and higher-brain areas resembling awake, posthatching animals were seen exclusively in chicken-stimulated embryos. Waking-like brain function is present in a latent but inducible state during the final 20% of embryonic life, selectively modulated by context-dependent monitoring circuitry. These data also reveal the developmental emergence of sleep-like behavior and its linkage to metabolic brain states and highlight problems with assigning embryo brain states based on behavioral observations.

Abstract 2689: Inhibition of metastasis of circulating tumor cells by extracellular matrix secreted by human embryonic cells

Abstract We have previously demonstrated the increased metastatic potential of human prostate cancer PC-3 CTCs compared to their parental counterparts in both chick embryo and mouse models. In the present study, we tested the effects of human embryonic extracellular matrix (ECM), secreted by human embryonic cells including conditioned medium (CM) and a semi-solid form (hECM), on metastasis of PC-3 CTC in the chick embryo model. The chorioallentoic membrane (CAM) of 18 chicken embryos were inoculated with either 106 PC-3 human prostate cancer cells or PC-3 CTCs, both stably expressing GFP. Twelve hours later, embryos were divided into 6 groups each containing three embryos: PC-3 parental control; PC-3 + CM; PC-3 + hECM; CTC control; CTC + CM; and CTC+ hECM. Twelve hours following inoculation of the cells, a single dose of 100 μL of either treatment was given to the appropriate group. Embryo brains were removed on day 8 post-inoculations, and processed for cryo-sectioning, generating 3 slides of brain tissue per embryo taken at various depths. Imaging was performed using the IV-100 scanning laser microscope (Olympus Corp, Tokyo, Japan) in order to count metastatic foci. PC-3 controls had an average of 11.1 metastatic foci compared to 2.55 in the PC-3 + hECM group (p< 0.0001) and 2.76 in the PC-3 + CM group (p< 0.0001) The treatment showed even greater response on the CTC cells which an average of 30.9 metastatic foci in the CTC controls compared to 4.38 in the CTC + hECM group (p< 0.0001) and 4.18 in the CTC + CM group (p< 0.0001). Thus, human embryonic secreted ECM compound drastically decreased the metastatic potential of human prostate cancer CTCs in the chick embryo model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2689. doi:1538-7445.AM2012-2689

Abstract 5552: Macrophages increase brain metastasis in a chick embryo model

Abstract Macrophages are known to promote tumor growth by various mechanisms. In the present study, we determined if macrophages could promote metastasis in the chick embryo models. The chorioallentoic membrane (CAM) of six chicken embryos was inoculated with 106 PC-3 human prostate cancer cells stably expressing GFP. Twelve hours later, 106 macrophages isolated from the peritoneal washings of three nude mice were added to the CAMs of three embryos. Embryo brains were removed on day 8 post-inoculation, and processed for cryosectioning. Imaging was performed using the IV-100 scanning laser microscope (Olympus Corp, Tokyo, Japan). Images were processed using Image J software in order to calculate the number, size, and total area of brain metastases in the control and macrophage groups. The macrophage group resulted in an average of 43.3 brain metastases compared to 5.5 in the control group (p=0.000001). There was also a significantly greater total metastatic area in the macrophage group of 504.8 mm2 compared to 100.5 mm2 in the control group (p= 0.000001). However, the control group showed a significantly larger size of individual metastases of 28.9 mm2 compared to 19.3 mm2 in the macrophage group (p= 0.0007). Macrophages, therefore, promote brain metastasis in the chick embryo model of human prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5552. doi:1538-7445.AM2012-5552

Tissue Interactions that Regulate Facial Morphogenesis

Molecular signaling among the forebrain, neural crest mesenchyme, and facial ectoderm regulate morphogenesis of the upper jaw. The forebrain and neural crest mesenchyme regulate expression of Sonic Hedgehog in the Frontonasal Ectodermal Zone (FEZ), which controls proximodistal extension and dorsoventral polarity of the upper jaw. The FEZ is highly conserved among vertebrates, but the spatial pattern of Shh expression is varied and highly correlated with the shape of the facial primordia in the developing upper jaw. In birds Shh expression spans the entire Frontonasal Process, while in mammals (human and mouse) Shh is restricted to lateral domains associated with the Median Nasal Processes. These expression patterns appear to result from molecular signals, including SHH itself, from the forebrain. From these observations we predicted that the signaling axis among the brain, mesenchyme, and ectoderm could be used to generate variation during evolutionary and disease processes. We have tested this by modulating SHH signaling in the brain of chick embryos to varying degrees and assessing facial shape using 3‐dimensional morphometrics and multivariate statistical analyses. This perturbation produced embryos with continuous phenotypic variation. The shape outcomes were highly correlated with SHH signaling, cell proliferation rates, and the organization of the FEZ in treated embryos. Further, these results revealed that phenotypic variation may result from non‐linear mechanisms by which signaling pathways operate. For instance, when the SHH pathway was blocked maximally, a highly penetrant phenotype resulted, while at lower levels of blockade the phenotypes rapidly returned to more normal shapes. Thus, large phenotypic variation was generated by small changes in receptor activation over certain ligand concentrations.

Systemic virus distribution and host responses in brain and intestine of chickens infected with low pathogenic or high pathogenic avian influenza virus

BackgroundAvian influenza virus (AIV) is classified into two pathotypes, low pathogenic (LP) and high pathogenic (HP), based on virulence in chickens.Differences in pathogenicity between HPAIV and LPAIV might eventually be related to specific characteristics of strains, tissue tropism and host responses.MethodsTo study differences in disease development between HPAIV and LPAIV, we examined the first appearance and eventual load of viral RNA in multiple organs as well as host responses in brain and intestine of chickens infected with two closely related H7N1 HPAIV or LPAIV strains.ResultsBoth H7N1 HPAIV and LPAIV spread systemically in chickens after a combined intranasal/intratracheal inoculation. In brain, large differences in viral RNA load and host gene expression were found between H7N1 HPAIV and LPAIV infected chickens. Chicken embryo brain cell culture studies revealed that both HPAIV and LPAIV could infect cultivated embryonic brain cells, but in accordance with the absence of the necessary proteases, replication of LPAIV was limited. Furthermore, TUNEL assay indicated apoptosis in brain of HPAIV infected chickens only. In intestine, where endoproteases that cleave HA of LPAIV are available, we found minimal differences in the amount of viral RNA and a large overlap in the transcriptional responses between HPAIV and LPAIV infected chickens. Interestingly, brain and ileum differed clearly in the cellular pathways that were regulated upon an AI infection.ConclusionsAlthough both H7N1 HPAIV and LPAIV RNA was detected in a broad range of tissues beyond the respiratory and gastrointestinal tract, our observations indicate that differences in pathogenicity and mortality between HPAIV and LPAIV could originate from differences in virus replication and the resulting host responses in vital organs like the brain.

Editorial: Intraventricular hemorrhage and posthemorrhagic hydrocephalus

In this issue, Dr. Robinson provides a comprehensive overview of intraventricular hemorrhage (IVH) of prematurity and posthemorrhagic hydrocephalus (PHH).1 The article covers epidemiology, pathophysiology, management, and current research in these fields. Relevant background material is obtained from a comprehensive MEDLINE database search and from additional references found in the identified articles. There is little to add to this excellent overview, but I would like to highlight and expand on several points raised by the author. The article describes stability in the number of preterm infants in the neonatal intensive care unit of the author’s institution, but a 3-fold drop in surgical intervention for PHH. As suggested, there are probably many factors contributing to this decrease, but changing attitudes among surgeons is probably one of them. In the Hydrocephalus Clinical Research Network (HCRN), we have documented significant variation in the management of these patients from one center to another across 4 centers (Riva-Cambrin et al., unpublished data). Among 110 neonates surgically treated for IVH, the strongest factor predicting whether they needed treatment was the center at which they were treated. As a result, we have developed standardized protocols for making decisions about treatment of these children and are now applying these prospectively. The pathophysiology of both IVH and PHH are described by Dr. Robinson. In addition to the biomarkers discussed in the article, some new data are relevant. In a mouse model of intracranial hemorrhage, the brains of mouse embryos were exposed to lysophosphatidic acid (LPA). This exposure resulted in the development of fetal hydrocephalus, which was dependent on the expression of the LPA1 receptor. Administration of an LPA1 antagonist blocked the development of fetal hydrocephalus.8 The LPA signaling pathway therefore appears to have a role in causing fetal hydrocephalus associated with hemorrhage. It is interesting to note that Dr. Robinson’s group has seen a reduced incidence of CSF shunting with serial lumbar punctures. This topic was the subject of a Cochrane database review4 in 2002 that identified 4 controlled trials, 3 of which were randomized. Two of these studies evaluated lumbar punctures and 2 evaluated reservoir tapping. The relative risks for shunt placement, death, and disability in the pooled data were very close to 1.0, with no significant effect. These measures have not been formally reconsidered in the last 10 years, so it is interesting to see that Dr. Robinson’s group has had success with serial lumbar punctures. The recent work by Whitelaw et al.6 on drainage, irrigation, and fibrinolytic therapy (DRIFT) is described by Dr. Robinson. As noted in the section on nonsurgical treatment, a Phase I trial7 suggested a benefit compared with historical controls, but a randomized trial5 was stopped early because of an increased hemorrhage rate. It should be emphasized, as mentioned later in the section on neuropsychological outcome, that the delayed followup of those patients demonstrated a clear benefit from the therapy.6,7 This trial6 was conducted at 3 centers, but most of the patients were from a single center, and further experience with this technique is required before it is more universally adopted. The temporary treatment option of subgaleal shunt versus subgaleal reservoir is discussed. This has been studied by Wellons et al.,3 whose results appeared to favor the use of a reservoir, although the variability in baseline factors did not allow strong conclusions. Within the HCRN, further prospective work is leading to a randomized trial to compare these 2 treatment options. Dr. Robinson describes an increased shunt infection rate in patients in the PHH group compared with those with other causes of hydrocephalus. Our data have demonstrated an increased risk of shunt failure in addition to the risk of infection. Among 554 children with various causes of hydrocephalus, IVH was an independent risk factor for shunt revision.2 Intraventricular hemorrhage and PHH continue to provide significant challenges for children, their families, and their surgeons. The field is broad, and there are many areas of active investigation. We are indebted to Dr. Robinson for this comprehensive review of the current status of this condition. (http://thejns.org/doi/abs/10.3171/2011.11.PEDS11412) 239 241

The Expression of Neuronal Nitric Oxide Synthase in the Brain of the Mouse During Embryogenesis

The distribution of neuronal nitric oxide synthase (nNOS) in the process of normal mouse brain growth from embryonic (E) Day 11 to postnatal (P) Day 1 was investigated by means of immunohistochemical and immunofluorescent methods. Our results demonstrated that nNOS positive neurons appeared early in superficial cortex at E11. At E13, nNOS positive neurons were located in lateral hypothalamus and amygdala, and temporarily in medullar and ventral hypothalamic neuroepithelia. From E15 to P0, nNOS positive neurons were distributed in superior and inferior colliculi, positive staining could also be seen in superior and inferior tectal neuroepithelium at E15. From E17 to birth, the medial geniculate nucleus had a high density of nNOS labeling. The distribution of nNOS gradually increased and extended laterally in embryo brain, which in turn implies that NO might be involved in the development of mouse brain.

Human Embryo Neuronal Culture <i>in Vitro</i>: A Model to Study Cellular Physiology, Receptors, Power and Toxicity of Cytostatic Drugs for Human Use

Neural cells cultures from human embryo brain of 9° - 11°W gestational age have been used to study ERα (Estrogens Receptor α) and to perform toxicity test for Mitomycin C and Methotrexate. Histochemical confirmation of cellular neuronal phenotype was based on histochemical evidence of NSE (Neuron Specific Enolase).The detection of ERα in neuronal cells was performed with a rabbit Monoclonal Antibody. ERα was absent both on neurons grown in vitro and on tissue brain specimens. This finding is apparently in contrast with the positive immunoreactivity of ERα and ERβ reported by other Authors on foetal and adult CNS (Central Nervous System). The absence of nuclear ERα on neurons in culture and in brain tissue specimens in our experiment is not in contrast with the relevant physiologic role of estrogens on nervous central system, but it could be correlated to the embryonic period of life and could represent a protection of male brain from an undue estrogens imprinting. The mitomycin C, alkylation agent, has shown in our experiment a major neurotoxic and cytostatic power in comparison with methotrexate. Our conclusion is that human embryo neuronal culture in vitro is a powerful instrument for physiology and human therapy for cancer and neurodegenerative diseases.