Effects of X-irradiation on nerve growth factor in the developing mouse brain.

The involvement of neurotrophins after radiation injury during brain development were studied in pregnant mice (C 57/B1) exposed on gestation day 15 to X-ray doses of 0.02-2 Gy. Nerve growth factor protein (NGF) and different cholinergic markers were investigated on postnatal day 1 (P1) and day 21 (P21); in situ hybridization with brain-derived neurotrophic factor (BDNF) and trkC (receptor serving to bind neurotrophin-3) probes was investigated on P21 in cortex, hippocampus, septum and cerebellum. The level of NGF protein was increased in irradiated forebrain on P1 in a dose-related manner. However, on P21 the NGF protein dropped down below the control levels in irradiated hippocampus and cerebellum. The response of acetylcholine esterase (AChE) activity in cerebellum at P21 was correlated with the changes in the amount of NGF. The intensity of cell labelling with trkC probe decreased after irradiation in the region of the hippocampus at P21, especially in dentate gyrus. The expression of BDNF mRNA was increased at P21 by low doses of irradiation (0.02-1 Gy) but was decreased by a high dose (2 Gy) in the same area. Thus, the radiation induced an alteration of neurotrophins, and the changes varied depending on the dose or time after irradiation. Such alterations in the pattern of growth factor production may modulate the response of cells to radiation. Furthermore, NGF protein levels and the expression of BDNF and trkC mRNA were affected by radiation doses as low as 0.02 Gy, indicating that during development the neurotrophins and their receptors are very sensitive to radiation.

Effects of single dose and fractionated irradiation on foetal mouse brain aggregates.

Foetal mouse brain cells were grown in vitro. 1 Gy X-irradiation was given as a single dose on culture day 2 (corresponding to embryonic day 17 in vivo) or in daily fractions (4 x 0.25 Gy), starting on day 2 in culture. The growth (size) and biochemical differentiation of the aggregates were studied up to day 40 in culture. Aggregates given a single dose (1 Gy) were smaller with lower protein and DNA amount than controls up to day 30-40 in culture. When the dose was fractionated, a partial recovery occurred by day 20, and values returned to control levels by day 30-40. The specific activity of acetylcholinesterase (AChE) was significantly higher after 10-20 days, in the irradiated (1 Gy) aggregates. In fractionated irradiated aggregates, the increase of specific activity of AChE was evident only after 10 days in culture. Compared with fractionated X-irradiation, where recovery was observed after 20-40 days in culture, a single dose of irradiation produced more permanent changes in the amounts of DNA and protein and in the size and neurochemical activities of aggregates.

Neurochemical effects of a 20 kHz magnetic field on the central nervous system in prenatally exposed mice.

C57/B1 mice were exposed during pregnancy (gestation days 0-19) to a 20 kHz magnetic field (MF). The asymmetric sawtooth-waveform magnetic field in the exposed racks had a flux density of 15 microT (peak to peak). After 19 days, the exposure was terminated, and the mice were housed individually under normal laboratory conditions. On postnatal day (PD) 1, PD21, and PD308, various neurochemical markers in the brains of the offspring were investigated and the brains weighed. No significant difference was found in the whole brain weight at PD1 or PD21 between exposed offspring and control animals. However, on PD308, a significant decrease in weight of the whole brain was detected in exposed animals. No significant differences were found in the weight of cortex, hippocampus, septum, or cerebellum on any of the sampling occasions, nor were any significant differences detected in protein-, DNA-level, nerve growth factor (NGF), acetylcholine esterase- (AChE), or 2',3'-cyclic nucleotide 3'-phosphodiesterase- (CNP; marker for oligodendrocytes) activities on PD21 in cerebellum. Cortex showed a more complex pattern of response to MF: MF treatment resulted in a decrease in DNA level and increases in the activities of CNP, AChE, and NGF protein. On PD308, the amount of DNA was significantly reduced in MF-treated cerebellum and CNP activity was still enhanced in MF-treated cortex compared to controls. Most of the effects of MF treatment during the embryonic period were similar to those induced by ionizing radiation but much weaker. However, the duration of the exposure required to elucidate the response of different markers to MF seems to be greater and effects appear later during development compared to responses to ionizing radiation.

Effects of irradiation on cholinergic neurons and nerve growth factor mRNA in mouse foetal brain aggregation cultures.

Neurochemical and morphological markers were used to study the dose-response relationship and effects of different doses (0.5, 1.0 and 2.0 Gy) of X-irradiation on foetal brain cells in reaggregating cell cultures. Cells, prepared from mouse forebrain were irradiated on culture day 2, corresponding to embryonic day 16 in vivo. The cell reaggregates were monitored continually up to day 40 in vitro. The level of a neuronal marker for cholinergic neurons, namely acetylcholine esterase (AChE), increased linearly with the dose of irradiation after 10 days in vitro. Furthermore, by day 30 the activity of AChE had decreased to a level below that found in the control aggregates. In the 2-Gy treatment the level of choline acetyltransferase (ChAT), another cholinergic-neuron marker, increased during the first 20-30 days in culture but had declined to control levels by day 40. Using Northern blot analysis of total RNA prepared from these cell reaggregates, we determined relative changes in the level of expression of mouse nerve growth factor (NGF) mRNA subsequent to irradiation after 10 and 30 days in culture. The ratio between the level of expression of NGF and that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was calculated and it was found that by day 10 in vitro the level of NGF had increased compared with control levels, in a dose-related manner. After 30 days in culture, the level of NGF mRNA was still high after the low-dose (0.5 Gy) treatment, whereas it had decreased below control levels after the higher-dose (1.0 and 2.0 Gy) treatment. The aggregates showed a tendency to deform and fuse together after irradiation: furthermore, the number of aggregates occurring as two or more grown together, increased with the X-ray dose. Aggregate size decreased with time in vitro and with irradiation dose. Thus, we showed that cholinergic and morphological markers are affected by irradiation and the alteration in AChE activity induced by irradiation correlated well with the changes in NGF mRNA.

Effects of nerve growth factor on X-irradiated reaggregation cultures of rat brain cells.

The effects of exogenously added nerve growth factor (NGF) on reaggregation cultures of foetal rat brain cells after X-irradiation with 2 Gy were studied. Irradiation caused decreased protein and DNA levels, which was not prevented by NGF. The activities of the cholinergic marker enzymes choline acetyl transferase and acetylcholine esterase were increased in irradiated cultures. However, no difference in the activities of these enzymes was found between irradiated and unirradiated NGF-treated cultures. Irradiation did not affect the activity of the marker enzyme for oligodendrocytes (2',3'-cyclic nucleotide 3'-phosphodiesterase), but caused an increase in the astrocyte marker (glutamine synthetase) activity. This effect on astrocytes was prevented by NGF.

Effects of low-dose X-irradiation on mouse-brain aggregation cultures.

Biochemical and morphological differentiation in reaggregating mouse-brain cell cultures after low-dose radiation (0.5 Gy) in vitro was studied. Cells were irradiated on culture day 2, corresponding to embryonic day 15-16, and different glial and neuronal markers were followed through development to postnatal day 40. The shape and size of irradiated aggregates were more irregular and smaller compared with controls. Total amounts of DNA and protein were significantly lower in irradiated aggregates than in controls between days 8 and 20. After 30 days in culture activities of the glial markers glutamine synthetase (GS) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) were lower in X-irradiated aggregates than in controls. However, after 40 days the CNP activity in irradiated aggregates increased to levels above those of the controls. Irradiated and control aggregates did not differ significantly in neuronal marker enzyme activities, i.e. choline acetyltransferase (ChAT), acetylcholine esterase (AChE) and glutamic acid decarboxylase (GAD) measured on a per mg protein basis. On days 20 and 30 the amount of nerve growth factor (NGF) was two-fold higher in irradiated aggregates compared with non-irradiated ones, suggesting that, after irradiation, surviving cells in culture were induced to produce more NGF. After 40 days the amount of NGF in irradiated aggregates had decreased to the level found in the control aggregates.

Effects of injecting antibodies to mouse nerve growth factor into the chick embryo.

The function of NGF in chick embryos was studied by injecting antibodies to mouse nerve growth factor (NGF). The uptake of mammalian antibodies into the 8- to 15-day-old chick embryo was followed by an enzyme-linked immunoassay. Normal rabbit antibodies (250 micrograms) were administered to the yolk, of which less than 5% was found in the embryo (300 ng of IgG per g wet wt of embryo). The concentration was proportionally lower when 100 micrograms anti-NGF antibodies were injected (40 ng/g). The concentration of anti-NGF antibodies was 1.5 times higher following injection directly into the body of the embryos. The effects of injecting antibodies at days 3-7 were studied in 10-day-old embryos by measuring the diameter frequencies of neurons in sympathetic and sensory ganglia. In comparison with controls, significantly smaller neurons were found in the sympathetic ganglia in embryos directly injected with anti-NGF. In the spinal ganglia, distribution of neuron diameters did not differ between anti-NGF-treated embryos and controls. Finally, the ability of different antibodies to mouse NGF to inhibit the in vitro activity of recombinant chick NGF was investigated. Total block was found at 1000-2000 ng of IgG per ml for most of the antibodies tested, levels not reached when injecting the embryo with antibodies to NGF. We conclude that the main reason for the limited effects in chick embryos by injection of NGF antibodies is due to the low levels of penetration of the anti-NGF IgG into the embryo.

Effects of nerve growth factor on autonomic neurons in the chick embryo: a stereological study.

Quantitative effects of nerve growth factor (NGF) on the sympathetic, Remak and ciliary ganglia in chicken embryos were investigated. Purified mouse beta NGF was injected (80 micrograms per day for three or four consecutive days) into the yolk sac at different stages (starting on days 6, 8, 10 and 13) of embryonic development. Ganglia were taken for fixation and embedding one day after the last NGF injection. The number of neurons belonging to the different size classes was determined by a computer aided stereological method based on unfolding of cell diameter frequencies. The volume of sympathetic ganglia was increased at all stages with a maximum of 8-fold occurring on day 10. The ganglion of Remak showed a 3-fold volume increase up to embryonic days 10 and 12. Ciliary ganglia did not exhibit any differences in volume or neuron size between the controls and the embryos injected with NGF. The number of neurons was increased in younger sympathetic and Remak ganglia in response to NGF, as was the recruitment of neurons to the larger size classes.

Effects of nerve growth factor on sensory neurons in the chick embryo: a stereological study.

Chicken embryos on days 6-13 of incubation received injections of purified beta NGF (80 micrograms/day) for 3 or 4 days and were then killed. Sensory ganglia were fixed and taken for embedding and sectioning. A stereological method based on unfolding of cell-diameter frequencies was used to determine the number of neurons of different size in the spinal, trigeminal and nodose ganglia. The total volume of the ganglia was also determined. NGF induced increases in diameter of the neural crest-derived dorsomedial (DM) neurons in spinal and trigeminal ganglia. Injected NGF did not influence ventrolateral (VL) neurons of neural crest origin in the spinal ganglia nor the ventrolateral neurons of placodal origin in the trigeminal ganglion. The volumes of spinal and trigeminal ganglia increased by 50 and 100%, respectively. The volume of the nodose ganglion and the total number and size of the placodal nodose neurons were unaffected by NGF. The results demonstrate a clear difference in the response to NGF in vivo between smaller and larger sensory neurons.