Fetal Rat Central Nervous System Research Articles

The Role of Fetal Neurology in the Progress of Perinatal Medicine

The prevalence of cerebral palsy has not decreased despite major improvements in clinical care in antenatal/ neonatal period as well as intrapartum period. In about 70% of cases, cerebral palsy results from events occurring before birth that can disrupt normal development of the brain. The antepartum risk factors should include fetal brain mal development and intrauterine brain injuries, which are unclassifiable into congenital brain anomalies and may exist inconspicuously during pregnancy and even after birth. Especially, neuronal migration disorder and acquired brain damage in utero should be responsible for postnatal neurological impairment. Imaging technologies including three dimensional ultrasound have been remarkably improved and contributed to prenatal evaluation of fetal Central Nervous System (CNS) development and assessment of CNS abnormalities in utero. In this article, objective and precise imaging diagnoses of fetal CNS including migration disorders and acquired brain damages. Furthermore, 3D bidirectional power Doppler angiography has depicted fine cerebral vessels of medullary veins which may relate with timing of insult as well as with postnatal neurological prognosis. It is promising to clarify the developmental mechanism of CNS damages with advanced ultrasound diagnostic techniques in the near future. Postnatal unexplained neurological deficits may strongly relate with intrauterine brain development therefore fetal neurology has great responsibility and an important role in perinatal medicine.

Ultrasound versus MRI in the diagnosis of fetal head and trunk anomalies

Objective. To compare antenatal sonography and magnetic resonance (MR) imaging (MRI) in the diagnosis of fetal head and trunk anomalies.Methods. Forty pregnant women with fetal anomalies on ultrasound (US) examination underwent MRI. The MR examination was done by a radiologist who was provided with the US data. The MR images were then read by one of the two radiologists who were blinded to the US data. They were however told that the region of interest was (1) head–neck or (2) trunk or (3) both. Antenatal US and MRI findings were compared with postnatal diagnosis. Postnatal evaluation included plain radiograph, US, computed tomography, MRI, surgery, physical evaluation and autopsy.Results. Sixty anomalies were detected in the 40 women studied. This included 36 central nervous system (CNS), 7 thoracic, 7 gastrointestinal, 8 genitourinary and 2 face–neck anomalies. In the evaluation of CNS and thoracic anomalies, more number of confident diagnoses could be obtained by MRI when compared with that by US. In the detection of gastrointestinal and genitourinary anomalies, there was no significant difference between the two modalities.Conclusion. More number of confident diagnoses could be obtained by MRI when compared with that by US, in the evaluation of fetal CNS and thoracic anomalies. MRI can be used in complex fetal anomalies as a supplementary tool following US.

Generation of inner ear hair cell immunophenotypes from neurospheres obtained from fetal rat central nervous system in vitro.

Neural stem cells are suggested to possess a highly plastic ability to differentiate into several specific cell types, not only neuronal lineages but also other germ layer tissue-specific cell lineages. To examine whether hair cell immunophenotypes could be derived from the central nervous system (CNS), we established cell cultures from embryonic day 16.5 fetal rat brain tissues, and analyzed changes in immunohistochemical features of the CNS cell cultures by induction of differentiation. The results of this study showed that neural progenitors obtained from fetal rat CNS generated hair cell immunophenotypes with expression of both epitopes of hair cell marker proteins Brn-3c and myosin VIIa in vitro. These findings indicate that immature neural progenitors possess the potential to differentiate into hair cell phenotypes. Immature neural progenitors may be useful as materials for cell transplantation therapy for replacement of damaged inner ear hair cells.

Ontogeny of calcitonin receptor mRNA and protein in the developing central nervous system of the rat.

In this study, the expression of receptors for calcitonin (CTR), the CTR C1a and C1b isoforms, was investigated during development of the fetal rat central nervous system (CNS) by using in situ hybridization and immunohistochemistry. Coincident expression with both techniques was evident. Immunohistochemical evidence for the expression of the C1a isoform alone was found. Expression was first observed at embryonic day 12/13 (E12/E13) within and adjacent to the ventricular zones known to include primary matrices of proliferation, in regions of the preoptic area, anterior and posterior hypothalamus, anterior and posterior pons, medulla, and spinal cord. At later times, with the decline in the density of immunoreactivity at these loci (E15), expression in primary matrices was found later at distinct loci within the ventricular zones of cerebellum (E17), and at E19, the tectum, lateral ventricle, and cortical subplate. By E19, the density of staining had increased and was widespread throughout the expanding CNS. In the rostral domains, moderate to high density was found in the external plexiform layer; the medial preoptic area and nucleus; the ventromedial, dorsomedial, and arcuate hypothalamic nuclei; and the lateral and posterior hypothalamic areas. In the midbrain, similar levels of expression were noted in the central nucleus of raphe; the deep mesencephalic, dorsal raphe, and laterodorsal tegmental nuclei; and the ventral periaqueductal gray. In the pons, positive loci included the locus coeruleus and the gigantocellular and pontine reticular nuclei. In the medulla, high expression was evident in the gigantocellular, intermediate, magnocellular, and medullary reticular, spinal trigeminal and cuneate nuclei; and the nucleus tractus solitarius. In the spinal cord, moderate to high density of staining was found in the ventral, dorsal, and lateral horns, and in the ventral, dorsal, and cuneate funiculi. On the other hand, transitory expression was found in the diagonal band, bed nucleus of the stria terminalis, amygdala, and the lateral mamillary and anterobasal nuclei of the hypothalamus. These studies indicate a role for CTR in the activation of some premigratory neuroblasts in the CNS as well as a possible role later in an undefined function associated with mature neurons of particular nuclei.

An experimental model for the transplantation of fetal central nervous system cells to the injured spinal cord in rats.

Traumatic spinal cord injury is one of the most disabling conditions occurring in man and thus stimulates a strong interest in its histopathological, biochemical, and functional changes, primarily as we search for preventive and therapeutic methods. To develop an experimental model for transplantation of cells from the fetal rat central nervous system to the site of an injured spinal cord of an adult rat in which the transplanted cells survive and become integrated. This experimental model will facilitate investigations of factors that promote regeneration and functional recovery after spinal cord trauma. Fifteen adult Wistar rats underwent laminectomy, and an spinal cord lesion was made with microdissection. Fetal spinal cord tissue was then transplanted to the site of the injury. The rats were monitored over a 48-hour period, and then their vertebral column was completely removed for histological analysis. In 60% of transplanted rats, the fetal tissue at the injured site remained viable in the site of the lesion.

Opioid receptor and peptide mRNA expression in proliferative zones of fetal rat central nervous system

There is increasing evidence to suggest that opioid peptides may have widespread effects as regulators of growth. To evaluate the hypothesis that endogenous opioids control cellular proliferation during neural development, we have used in situ hybridization to examine opioid peptide and receptor mRNA expression in neuroepithelial zones of fetal rat brain and spinal cord. Our data show that proenkephalin mRNA is widely expressed in forebrain germinal zones and choroid plexus during the second half of gestation. In contrast, prodynorphin mRNA expression is restricted to the periventricular region of the ventral spinal cord. Little mu or delta receptor mRNA expression was detected in any regions of neuronal proliferation prior to birth. However, kappa receptor mRNA is widely expressed in hindbrain germinal zones during the 3rd week of gestation. Our present findings support the hypothesis that endogenous opioids may regulate proliferation of both neuronal and non-neuronal cells during central nervous system development. Given the segregated expression of proenkephalin mRNA in forebrain neuroepithelium and kappa receptor mRNA within hindbrain, different opioid mechanisms may regulate cell division in rostral and caudal brain regions.

Opioid receptor and peptide mRNA expression in proliferative zones of fetal rat central nervous system

Opioid receptor and peptide mRNA expression in proliferative zones of fetal rat central nervous system

Etoposide induces programmed death in neurons cultured from the fetal rat central nervous system

The effects of etoposide on the death of neurons cultured from the central nervous system (CNS) of fetal rats were examined. The cultured neurons died in the presence of 1–40 μg/ml of etoposide, which is known to induce programmed death in some kinds of cells, and this cytotoxic effect was prevented by inhibition of protein synthesis and/or RNA synthesis. Furthermore, DNA degradation, including a ladder-like pattern, became evident in these neurons 3 h after incubation with etoposide (10 μg/ml), whereas cell death commenced after about 6 h. These results indicate that etoposide-treated CNS neurons require new protein and RNA synthesis to undergo an active death programme, and that internucleosomal fragmentation of DNA mediates the etoposide-induced programmed cell death. This culture system of etoposide-treated CNS neurons is thought to be a useful model for the study of programmed neuronal cell death.

Regional development of muscarinic cholinergic binding sites in the prenatal rat brain

The ontogeny of muscarinic cholinergic binding sites was studied in rat fetal central nervous system by in vitro autoradiographic techniques using [ 3H] N-methyl scopolamine as ligand (1 nM). Nonspecific binding was determined after the addition of 1 μM atropine. The main findings of this study are the early appearance of muscarinic cholinergic binding sites in fetal rat central nervous system before gestational day 14, their subsequent spread in a caudofrontal direction and the rapid change of patterns within individual brain regions. Muscarinic cholinergic sites are present shortly after cell birth, though the time-lag between cell generation and expression of muscarinic sites differs between neuronal cell populations. High receptor densities are noted in certain brainstem nuclei that are important for early fetal and neonatal behaviors.

Differential effects of nerve-growth factor and glioma-conditioned medium on neurons cultured from various regions of fetal rat central nervous system

When nerve-growth factor (NGF) was added to 17-day fetal rat central nervous system (CNS) septal neurons in culture using a defined medium, choline acetyltransferase (ChAT) activity was greatly induced. However, glioma-conditioned medium (GCM), which is expected to contain cholinergic neurotrophic factor(s) different from NGF, did not affect the ChAT activity of the cultured septal neurons. On the contrary, ChAT activity of 19-day fetal rat hippocampal neurons in culture was increased by the addition of GCM but not by NGF. These phenomena were confirmed in septal and hippocampal neuronal cultures obtained from the same embryonic day, i.e., 18-day fetal rat. The NGF-mediated increase in ChAT activity of cultured septal neurons was culture-time dependent (2.3-fold increase after 3 and 3.5-fold increase after 6 days in culture) and NGF-dose dependent (the ED 50 value was 0.8 ng/ml). The effect of NGF was completely abolished by the addition of specific anti-NGF antibodies. The differential effects of NGF and GCM on several other cultured cholinergic neurons from 17-day fetal rat spinal cord, striatum, brainstem and amygdala were measured. NGF tended to increase ChAT activities of cultured striatal and amygdala neurons but not cultured spinal cord and brainstem neurons. GCM increased ChAT activities in the latter two cultures, while having no effect on the former cultures. Although the extent of increase of NGF-mediated ChAT activities of cultured striatal and amygdala neurons were low, NGF-mediated increase of the striatal ChAT activity showed the pattern resembling that of cultured septal neurons as to time-course, dose-dependency and anti-NGF antibody sensitivity. NGF did not affect the tyrosine hydroxylase activity in the cultured brainstem catecholaminergic neurons.

Cryopreservation of primary neurons for tissue culture

We have developed new cryopreservation methods which allow storage of fetal rat central nervous system tissues for more than 1 week at 3–8°C or for several months at −70 or −90°C prior to tissue culture. For refrigeration, small brain regions (< 2 mm thick) were placed intact into 35 mm petri dishes of ‘hibernation medium’ inside a humidified chamber. Optimal preservation was obtained with hibernation media of pH 6.8–7.4, containing 30–70 mM K +, 10–30 mM Na +, 5–50 mM PO 4 2−, 20 mM lactic acid, 5 mM glucose, and less than 0.1 mM Ca 2+. The media were made approximately isotonic by addition of sorbitol. For freezing, brain tissues were dissociated by gentle trituration (without enzymes) in the above medium supplemented with 5–10% dimethylsulfoxide. After refrigeration or freezing, neurons were very sensitive to damage from mechanical stress (e.g. centrifugation, harsh rinsing or trituration). Rapid changes in osmotic pressure or excessive polylysine on the tissue culture substratum also reduced neuronal survival after cryopreservation. Pretreatment of tissue culture substrata with media from lung cell cultures, or plating of neurons at higher density (2000 cells/mm 2) improved neuronal survival after cryopreservation.

Histological effects of 6-mercaptopurine on the fetal rat central nervous system: a light-microscopic study.

Wistar rats were administered single doses of 16 or 50 mg/kg 6-mercaptopurine (6-MP) on day 12 of pregnancy. Necrosis in the fetal forebrain and spinal cord was studied by light microscope 6, 12, 14, 48, 72, and 81 h and 8 days afterward. The extent of necrosis was dose dependent. The first necroses were seen after 24 h, regardless of location (brain, spinal cord) or dose; but the extent was greatest after 48 h. All necrotic cells had a typical appearance; they were ballooned and often fragmented, their nuclei were darkly colored and frequently pyknotic, and they were often karyorhexic. Necroses appeared almost exclusively at sites of beginning cellular differentiation, i.e., in the intermediate zone. In the spinal cord the ventricular zone was also necrotic and the alar plate (dorsal horn) always affected. Phagocytizing cells (macrophages) appeared in the spinal cord after 48 h and in the brain after 72 h. After 81 h all the necrotic material had been phagocytized, at which time there was a massive congestion of the extra- and intracerebral vessels. Hemorrhages appeared in defined localizations. Eight days after exposure to 16 mg/kg 6-MP fetuses no longer showed any visible deviations. Fetuses exposed to 50 mg/kg showed deviations in the cytoarchitecture of the neopallium: an extremely broadened ventricular zone, few cells in the intermediate zone, and extensive rarefaction cells in the cortical plate with no clear layer structure. In the spinal cord, cleft formations were especially noticeable in the dorsal-horn region. All fetuses showed a hydrocephalus externus after 50 mg/kg. The mechanism leading to necrosis is discussed.

Selective necrosis in the fetal rat central nervous system produced by 5-fluoro-2'-deoxyuridine. A morphologic study.

Selective necrosis in the fetal rat central nervous system produced by 5-fluoro-2'-deoxyuridine. A morphologic study.