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EFFECTS OF RADIATION ON THE DEVELOPMENT OF THE CENTRAL NERVOUS SYSTEM

Objective

The deleterious effects of low doses (10 to 1000 mGy) of ionizing radiation on the development of the central nervous system represent a much debated issue. In contrast with recent work from us and others, some authors claim that doses as high as 500 mGy are largely innocuous to the developing organisms. The aims of the present project are to compare and analyze the effects of protracted versus short low dose radiation exposures (less than 500 mGy) using a number of approaches. These range from ultrastructural observations to nerve cell cultures. The experiments and analyses will be performed in Mol, Barcelona and Bruyeres-le-Chatel. Frequent contacts between contractors and other workers in the field will occur within the frame of EULEP and during the congresses of the European Society of Radiobiology. In addition, one-day working sessions including the 3 contractors and EEC representatives have been planned.
Research was carried out into the evaluation of the deleterious effects of exposure to a low dose of ionizing radiation during fetal development. The study involved monitoring of the acute effects of an exposure to 600 KeV neutrons on day 15 postconception (PC) in the Wistar rat and evaluation of the protracted effects of a low dose rate of gamma exposure during whole or part of the pregnancy.

With respect to exposure to 600 KeV neutrons on day 15 postconception, brain weights of prenatally irradiated rats decreased linearly according to dose in 3, 15 and 24 month old rats. The slight microcephaly (a 2% brain weight loss) found after an exposure to only 1 cGy was found to be significant.

Evaluation of the protracted effects of a gamma exposure (at a low dose rate) during whole or part of the pregnancy involved a large amount of superposition between the numerous dose effect curves but some interesting conclusions can be drawn. If, in general, protracted exposures produce less effects than acute ones, it also appears that a careful selection of the period of irradiation during the pregnancy (eg by irradiating between day 12 and day 16 PC) produces a brain atrophy at least similar to the one produced by an acute exposure to the same dose given in 20 s during day 15 PC. Brain atrophy may not always represent the best available estimator of the effects of a prenatal irradiation of the brain. The decrease of the volume of the cingulum (a purely white matter area located above the corpus callosum) in relation to the dose can be much more important than the loss in brain weight. Here also, an exposure of a few seconds to X-rays does not cause more effect than a 5h gamma irradiation. The reasons for the very high radiosensitivity of the cingulum are unclear. A reduction of a neuronal population (calbindin positive) was found to selectively occur in the cingular cortex, an area of the cerebral cortex located just above the cingulum.

A study has been made of naturally occurring cell death during postnatal development in the cerebral cortex of the normal rat and in micrencephalic rats induced by prenatal X-irradiation. Dendritic spines have been developed on cortical pyramidal cells in rats after X-ray exposure during the fetal period. A technical approach has been made to the immunocytochemical identification of local circuit neurons in the cerebral cortex using antibodies to parvalbumin, calbindin, gamma aminobutyric acid (GABA) anddifferent neuropeptides. Preliminary studies has also been carried out in rats irradiated with 5 cGy and 100 cGy at embryonic ages 15 days, 17 days, 19 days.

An in vitro model was previously developed to study the radiosensitivity of the freshly isolated mesencephalic and striatal rat cells. This radiosensitivity was assessed using 3 parameters:
nerve cells lethality;
neurites growth;
dopamine (DA) and gamma amino butyric acid (GABA) uptake in 3 day old cell culture irradiated on day 0.

Preliminary results on the identification of neurons and glial cells by immunohistochemistry of their particular cytoskeleton proteins have been obtained.
The relative number of living cells decreased significantly after 0.50 Gy or higher doses. The longest neurite was reduced with as little as 0.25 Gy. The exposure dose of 0.25 Gy with gamma rays seems to be near the threshold value for a deleterious effect on nerve cells. The neurotransmitter uptake and neurite growth seem to be parameters more sensitive than the cell lethality.
Two protocols of irradiation exposure are used by the 3 different contractors: chronical and acute. In chronic experiments, pregnant rats are exposed with from 0 to 1000 mGy during the whole or only the last week of pregnancy. Acute irradiations are attempts not only to detect the most sensitive phases of brain development but mainly to find the reasons for such radiosensitivity. Collection of material (autopsies) will take place from a few days before birth to 3 months after.

Morphology (CEN/SCK)
Six different endpoints will be considered:
an ultrastructural analysis of the development of synapses in the cingular cerebral cortex of the young adult rat;
electron microscopic analysis of the development of the lamellar bodies and Nissl blocks in the neurons from cortical layers II to V;
semiautomatic image analysis of myelination in the white matter (cingulum bundle and corpus callosum);
assessment of the density of the glial progenitors (residual embryonic cells) after irradiation at day 15 post conception in 2 different rat strains, Wistar and SpragueADawley;
measurements of the cytokines interleukin 1 (ILA1) and ILA6 in normal versus irradiated rats; treatment with indomethacin to prevent IL formation. (in collaboration with R. Hooghe); .SP 0 analysis of the development of radial glia in prenatal brains.

Physiological cell death in early postnatal neurons (Barcelona Univ.)
Four different parameters will be considered (part of the material to be studied was irradiated and perfusion fixed in Mol in January 1990):
comparison of the physiological neuronal cell death in normal versus irradiated early postnatal brains;
changes in the adult GABAergic neuronal cell populations after prenatal irradiation;
changes in dendritic arborization and spines in 1 month old pyramidal nerve cells;
immunocytochemical study of transitory populations of parvalbumin containing neurons in the future corpus callosum (cortical embryonic subplate).

The following questions will be addressed: is physiological cell death restricted to certain classes of nerve cells or general ?; is there a differential radiosensitivity for this class of cells ?.

Behaviour of irradiated neuroblasts in tissue culture (CEA-Bruyeres-le-Chatel)
influence of prenatal irradiation on the in vitro lethality of the nerve cells;
in vitro assessment of neurotransmitter uptake;
a quantitative appraisal of the dendritic arborization of the dopaminergic and gabaergic neurons (identified by autoradiography);
in situ immunocytochemical analysis of the distribution of the dopamine and GABA neurotransmitters (in collaboration with Y. Vernois).
a study of 2.5 MeV (nonmonochromatic) neutrons using brain weight as an endpoint (after prenatal irradiation at day 15).

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

BELGIAN NUCLEAR RESEARCH CENTRE
Address
200,Herrmann Debrouxlaan 40-42
1160 Bruxelles
Belgium

Participants (2)

COMMISSARIAT A L'ENERGIE ATOMIQUE
France
Address
Rue De La Federation 31-33
75015 Paris
Hospital Principes de Espana
Spain
Address
7,Calle Feixa Llarga
08907 Barcelona