Victor K. Han

Identification of somatomedin/insulin-like growth factor immunoreactive cells in the human fetus.

ABSTRACT: Somatomedins/insulin-like growth factors (Sm/IGFs) are present in blood and in extracts from multiple tissues of the human fetus and induce the proliferation of cultured human fetal cells. To identify the cellular location of immunoreactive Sm/IGF in human fetal tissues, we have performed immunocytochemistry in tissues from prostaglandin-induced human fetal abortuses of 12 to 20 wk in gestation. Every tissue studied except the cerebral cortex contains Sm/IGF immunoreactive cells. Cells staining positively include hepatocytes, hepatic hemopoietic cells, columnar epithelia of the pulmonary airways, intestine and kidney tubules, adrenal cortical cells, dermal cells, skeletal and cardiac muscle fibers, and pancreatic islet and acinar cells. Immunostaining was specific for Sm/IGFs, but because of the cross-reactivity of the antibodies it was not possible to determine whether the immunoreactivity represented Sm-C/IGF I, IGF II, or both. Liver contained the greatest proportion of immunoreactive cells, while the thymus and spleen had only a few immunostained cells. With the possible exception of dermal and some adrenal cortical cells, the immunoreactive cells do not appear to be the primary sites of Sm/IGF synthesis, because parallel in situ hybridization histochemical studies using Sm/IGF oligodeoxyribonucleotide probes show that Sm/IGF mRNAs are localized predominantly to fibroblasts and mesenchymal cells. Therefore the immunoreactive cells identified in this study may define sites of action of Sm/IGFs.

Regional expression of transforming growth factor-α mRNA in the rat central nervous system

Abstract The expression of transforming growth factor-α (TGFα) mRNA in various regions of the adult rat central nervous system (CNS), as well as in selected peripheral tissues was examined using Northern blot analysis. The highest expression was found in the cervical spinal cord, with the levels of TGFα mRNA being 5- to 6-fold higher in this tissue than in any other examined. Significant expression was also found in the olfactory bulb, anterior olfactory nucleus, corpus striatum, hippocampus, ventral mesencephalon and caudal brainstem. Of the peripheral tissues examined, only adrenal gland expressed TGFα mRNA at similar levels. Lower, but detectable, expression was found in prefrontal cortex, cerebral cortex and cerebellum. The latter levels were similar to those observed in lung, liver, kidney and, variably, salivary gland. These data demonstrate a widespread but differential distribution of TGFα mRNA throughout the rat CNS, and indicate relatively high levels of expression of this growth factor in central versus peripheral tissues.

IGF-Binding Protein mRNAs in the Human Fetus: Tissue and Cellular Distribution of Developmental Expression

Insulin-like growth factors (IGF-I and IGF-II) are synthesized by most embryonic and fetal tissues, and regulate cellular growth and differentiation as autocrine/paracrine factors. A family of six IGF-binding proteins (IGFBPs) modulate IGF biological actions as both negative (inhibitory) and positive (potentiating) modulators. To determine the tissue distribution of IGFBP mRNA expression, we performed Northern blot analysis and in situ hybridization of human fetal tissues during gestational ages 10-16 weeks (n = 8). IGFBP-1 mRNA was expressed only in the liver, whereas other IGFBP mRNAs were expressed in variable abundance in every tissue examined. IGFBP-2 mRNA was expressed in moderate abundance in every tissue with the highest level observed in the liver; IGFBP-3 mRNA was expressed most abundantly in the skin, muscle and heart; IGFBP-4 mRNA was expressed in moderate abundance equally in all tissues; IGFBP-5 mRNA was expressed most abundantly in the skin, muscle and stomach, and IGFBP-6 mRNA was expressed in low abundance in all tissues. Notable exceptions were that liver expressed little or no IGFBP-4, -5 and -6 mRNAs, spleen and thymus expressed low levels of IGFBP-5 mRNA, and brain expressed little or no IGFBP-5 and IGFBP-6 mRNA. In situ hybridization of human fetal tissues showed IGFBP mRNAs were expressed in both epithelial and mesenchymal cells depending on the specific IGFBP and the stage of development. IGFBP-3, -4, and -5 mRNAs were localized mainly in the mesenchymal cells, and IGFBP-2 mRNA was localized predominantly in the epithelial cells. IGFBP-6 mRNA was localized in low abundance in both epithelial and mesenchymal cells. These studies indicate that IGFBPs are important paracrine modulators of IGF action on cellular growth and differentiation, by modulating IGF-dependent or IGF-independent actions.

Alterations in the synthesis of insulin-like growth factor binding proteins and insulin-like growth factors in rat C6 glioma cells transfected with a gap junction connexin43 cDNA

When C6 glioma cells are stably transfected with a connexin43 cDNA and gap junctions are increased, the rate of cellular proliferation is decreased. To determine if this phenomenon is related to alterations in IGFBP and IGF synthesis, we have compared IGFBPs and IGFs in the conditioned media from primary rat astroglia, C6, and transfected C6 clones Cx43-13 (high expresser), and Cx43-12 and Cx43-14 (intermediate expressers). Primary astroglia produced IGFBP-2 (34 kDa) and IGFBP-3 (40-45 kDa). C6 cells synthesized high levels of IGFBP-3 and low levels of IGFBP-2, and a 24 kDa IGFBP (IGFBP-4). Cx43-13 cells did not synthesize IGFBP-3, but produced low levels of IGFBP-2 and high levels of IGFBP-4. Cx43-12 and Cx43-14 secreted IGFBP profiles similar to the parent C6 line, but with reduced levels of IGFBP-2. The lack of IGFBP-3 in Cx43-13 cells was not due to the presence of proteases. Northern analysis showed IGFBP-2 mRNA to be readily detectable only in the primary astroglia. IGFBP-3 mRNA was detected in the primary astroglia, C6, Cx43-12 and Cx43-14, but not in Cx43-13. In contrast, IGFBP-4 mRNA was readily detected only in the Cx43-13. IGF-II concentrations in the media were low to undetectable for both C6 and transfected cells. IGF-I concentrations were significantly lower in the media from transfected cells compared to the C6 cells. Stable mRNA levels for IGF-I were lower in transfected cells, with the lowest levels observed in the Cx43-13 cells. Although C6 cells did not respond mitogenically to exogenous IGF-I or IGF-II, Cx43-13 cells responded to IGF-I or IGF-II in a dose dependent manner. Conditioned media from Cx43-13 cells decreased the DNA synthesis of C6 cells, and this effect could be reversed by the addition of IGF-II. The decreased synthesis of the autocrine/paracrine growth factor IGF-I together with decreased levels of a positive modulator IGFBP-3, and the increased levels of a negative modulator IGFBP-4 in the extracellular milieu, may be responsible for the reduced proliferative capacity in cells expressing abundant connexin43.

Control of growth and development of the feto-placental unit

Classical gene targeting has identified many genes important for fetal and placental development. Null mutation of these genes may lead to fetal growth restriction, malformation or embryonic death. Growth restriction of epigenetic basis can predispose to adult-onset diseases. The mechanisms underlying this process, termed fetal programming, are beginning to be understood.

Cerebral resuscitation therapy in pediatric near-drowning.

N E A RO R O W N I N G is a major cause of neurologic morbidity and mortality in children. ~ ~ Some investigators believe this morbidity and mortality is related to uncontrolled rises in intracranial pressure and concomitant derangement in cerebral blood flow and metabolism? Control of ICP and reduction of cerebral metabolic demands has therefore been the goal of therapeutic efforts to reduce the morbidity and mortality seen in near-drowning. However, because the therapies necessary to achieve these goals remain unproved and carry significant morbidity, many authors have thought that they should be reserved for severely comatose children. 4 Our group investigated all severely comatose near-drowned children for ICP as defined by Orlowski score and, more recently, Glasgow coma Scale, and administered treatment according to a protocol including hyperventilation and dexamethasone in an effort to control ICP and maintain cerebral perfusion. Our results with this regimen are presented.

Perinatal assessment of cerebral flow velocity wave forms in the human fetus and neonate.

ABSTRACTS: The temporal relationship between changes in cerebral Doppler flow velocity wave forms, ductal patency, blood gases, and blood pressure during the transition from intrauterine to newborn life was assessed longitudinally in 16 healthy term fetuses and newborns. Doppler flow velocity wave forms were obtained from fetal cerebral arteries (anterior cerebral, internal carotid, and basilar) before birth, within 8 h after birth, and again at 24 and 48 h after birth. The resistance index was used as a measure of vascular resistance. The resistance index of the cerebral arteries studied increased significantly between the antenatal and 8-h study periods. This was followed by a significant decrease below fetal levels by the 24-h study period, with little change thereafter. We conclude that in the newborn human, as in the newborn lamb, the transition from fetal to immediate newborn life is associated with an increase in cerebral vascular resistance and thus a decrease in cerebral blood flow in response to the increase in arterial oxygenation. The subsequent decrease in the cerebral resistance index between 8 and 24 h of life cannot be explained by a loss of ductal shunting nor by associated changes in newborn blood gases or blood pressure, but may rather reflect a remodeling of the circulation due to impedance matching.

Females Follow a More “Compact” Early Human Brain Development Model Than Males. A Case-Control Study of Preterm Neonates

The pattern of sexual differentiation of the human brain is not well understood, particularly at the early stages of development when intense growth and multiple maturational phenomena overlap and interrelate. A case-control study of 20 preterm males and females matched for age was conducted. Three-dimensional images were acquired with 3 T MRI. The cerebral volume and the cortical folding area (FA), defined as the surface area of the interface between cortical gray and white matter, were compared between males and females. Females had smaller cerebra than males even after removing the influence of overall size differences between the subjects. The cortical FA increased in relation to volume by a power of 4/3 in both groups. Females had larger cortical FA compared with males with similar cerebral volumes. The study provides in vivo evidence of sexually dimorphic early human brain development. The relatively more “compact” female model may well relate to sex differences in neural circuitry and cognitive domains.

Effects of hyperventilation, hypothermia, and altered blood viscosity on cerebral blood flow, cross-brain oxygen extraction, and cerebral metabolic rate for oxygen in cats.

Therapies including hyperventilation (HV) and hypothermia (HT) are currently simultaneously used in brain-injured children at risk for cerebral swelling to reduce cerebral blood flow (CBF) and alter cerebral metabolic rate for oxygen (CMRO2). Since HV and HT may contribute to significant patient morbidity, we evaluated the effects of these treatments in combination on CBF, CMRO2, and cross-brain oxygen extraction (CBO2) using the Kety-Schmidt technique before controlled bleeding to alter blood viscosity in 20 lightly anesthetized, paralyzed cats, and after bleeding in another 17 cats. The degree of HV (PaCO2 24 to 26 torr) and HT (32 degrees and 30 degrees C) used were representative of that employed in pediatric neurointensive care. HV at normothermia resulted in a significant decline in CBF (P less than .05) and an unchanged CMRO2. HV and HT together to 32 degrees C resulted in a further significant fall in CBF and CMRO2 (p less than .05), but an unchanged CBO2. Further cooling of the animal to 30 degrees C during HV, both before and after controlled bleeding, resulted in no further significant fall in CBF, CBO2, or CMRO2. This relationship was found despite a significant fall in Hgb (p less than .001), suggesting that blood viscosity did not significantly influence CBF at this temperature. Our data suggest that HT to 32 degrees C during HV may have therapeutic benefit by decreasing CBF and CMRO2, but further cooling to 30 degrees C may not result in further cerebral protective effects.

Optimizing stimulation and analysis protocols for neonatal fMRI.

The development of brain function in young infants is poorly understood. The core challenge is that infants have a limited behavioral repertoire through which brain function can be expressed. Neuroimaging with fMRI has great potential as a way of characterizing typical development, and detecting abnormal development early. But, a number of methodological challenges must first be tackled to improve the robustness and sensitivity of neonatal fMRI. A critical one of these, addressed here, is that the hemodynamic response function (HRF) in pre-term and term neonates differs from that in adults, which has a number of implications for fMRI. We created a realistic model of noise in fMRI data, using resting-state fMRI data from infants and adults, and then conducted simulations to assess the effect of HRF of the power of different stimulation protocols and analysis assumptions (HRF modeling). We found that neonatal fMRI is most powerful if block-durations are kept at the lower range of those typically used in adults (full on/off cycle duration 25-30s). Furthermore, we show that it is important to use the age-appropriate HRF during analysis, as mismatches can lead to reduced power or even inverted signal. Where the appropriate HRF is not known (for example due to potential developmental delay), a flexible basis set performs well, and allows accurate post-hoc estimation of the HRF.