Sensitivity of cerebellar glutathione system to neonatal ionizing radiation exposure.

Reactive oxygen species (ROS) are relevant components of living organisms that, besides their role in the regulation of different important physiological functions, when present in excess are capable to affect cell oxidative status, leading to damage of cellular molecules and disturbance of normal cell function. ROS accumulation has been associated with a variety of conditions such as neurodegenerative diseases and ionizing radiation exposure. Cell ability to counteract ROS overproduction depends on the capacity of the endogenous antioxidant defenses--which includes the glutathione (GSH) system--to cope with. Since developing central nervous system (CNS) is especially sensitive to ROS-induced damage, the aim of the present work was to evaluate ROS, reduced GSH and oxidized glutathione (GSSG) levels in the cerebellum at different developmental ages after irradiation, in order to test if any changes were induced on these key oxidative stress-related cellular markers that might explain the high cerebellar vulnerability to radiation-induced injury. Since intracellular levels of GSH are maintained by glutathione reductase (GSHr), this enzymatic activity was also evaluated. Newborn Wistar rats were irradiated in their cephalic ends and the different parameters were measured, from 1h to 90 days post-irradiation. Results showed that an early transient increase in ROS levels followed by a decrease in cerebellar weight at 3-5 days post-irradiation were induced. An increase in cerebellar GSH levels was induced at 30 days after irradiation, together with a decrease in GSHr activity. These results support the hypothesis that ROS may represent a marker of damage prior to radiation-induced cell death. In contrast, it would be suggested that GSH system might play a role in the compensatory mechanisms triggered to counteract radiation-induced cerebellar damage.

The effect of X-radiation on cerebellar granule cells grown in culture. Ganglioside GM1 neuroprotective activity.

In this paper we describe the effects of X-radiation on the viability of cerebellar granule cells grown in culture. Cell cultures were exposed to X-rays 2 h after plating and then grown for 1-7 days. Two days after X-ray exposure with a dose-range of 0.1-2 Gy (acute effect), a significant decrease in neuronal number was observed. The magnitude of the lethal effect was directly correlated to the dose of X-ray applied. When the interval between plating and irradiation was increased, the acute lethal effect of X-rays decreased. 3H-thymidine incorporation was maximal during the first 24 h in vitro and decreased to nearly blank levels, after 72 h. In some experiments, cells present in each culture dish were counted at day 2 and at day 7. We observed that the number of cells present in sham-irradiated cultures decreased from day 2 to day 7, reflecting cell death after several days in vitro. The cell loss observed in X-irradiated cultures was significantly greater as compared with sham-irradiated cultures, confirming the deleterious effect of X-ray on cell survival. This effect was completely prevented by GM1 (6.5, 10 and 30 microM) added 48 h after X-ray exposure, but not 1 h after plating. We conclude that X-rays induce two different effects: an acute effect related to impaired DNA synthesis which is very active during the first 24 h in vitro, and a long-term effect owing to a sublethal damage in the surviving neuronal population.

Long-term changes in noradrenaline (NA) and dopamine (DA) contents of rat cerebellum following neonatal X-irradiation.

The effects of neonatal X-irradiation on cerebellar cathecholamine levels in rats were studied at different postnatal intervals. Since synaptogenesis in the cerebellar cortex is basically a postnatal phenomenon, changes in noradrenaline (NA) and dopamine (DA) levels induced by X-rays on the cerebella (CE) of adult rats (60 days old) were also studied. With 200 rad at birth there was an increase in both NA (+ 75%) and DA (+ 40%) levels at day 30, with a return to control values by day 90. CE weight did not change with this dose. Both 500 and 700 rad given at birth induced a persistent increase in NA levels, even when studied at day 390 (+ 127%) and a long-term decrease in DA levels. A marked atrophy of CE was found, even at day 390 (a 61% decrease in weight). Histologic analysis showed that the cerebellar cortex lacked its interneurons (agranular cerebellar cortex) and that Purkinje cells were randomly arranged. Rats showed dystonia, fine tremor, posterior train ataxia and microcephalia. On the other hand, X-irradiation of adult rats did not change cerebellar catecholamine levels or produced cerebellar atrophy. These animals did not show motor deficits or microcephalia. Taken together, these results suggest that the long-term changes in cerebellar catecholamine levels induced by neonatal X-irradiation may be somehow related to the loss of cerebellar interneurons which develop early in the postnatal period, although a primary change(s) in the activity of noradrenergic neurons can not be excluded.

WR-2721 (amifostine, ethyol) prevents motor and morphological changes induced by neonatal X-irradiation.

Neonatal X-irradiation induces permanent abnormalities in cerebellar cortex cytoarchitecture and neurochemistry, as well as impairment in motor gait. The aim of the present work was to examine the potential protective properties of WR-2721 (Amifostine, Ethyol), a free radical scavenger, against the above mentioned alterations by using a previously described neuroprotection assessment protocol. Pre-irradiation treatment with amifostine was effective in partially preventing the cerebellar morphological damage and the motor gait impairment induced by ionizing radiation. No changes in cerebellar noradrenaline (NA) levels were detected in amifostine-treated irradiated animals. These results suggest that it is possible to counteract radiation-induced damage in the cerebella and motor gait of neonatal rats through oxygen free radical scavenger administration prior to irradiation. The presence of the agent before the injury occurs, favors the efficacy of amifostine neuroprotective activity. Clinical implications of this model are related to the daily exposure of many people to different sources of radiation (accidental, diagnostical or therapeutical).

Activities of monoamine oxidase-A and -B in adult rat cerebellum following neonatal X-irradiation.

The activities of monoamine oxidases, MAO-A and MAO-B, were separately determined in the cerebellum (CE) from adult rats neonatally exposed to 5 Gy X-irradiation. They were found to be markedly reduced: 58% and 66% of values from nonirradiated, littermate controls. Since the specific activities of both isoenzymes (per mg tissue weight) were not significantly different from controls, the reduction of activity per CE is basically explained by the irradiation-induced cerebellar atrophy. The unmodified MAO-A specific activity makes it highly improbable that the increase in the cerebellar noradrenaline content, characteristic of neonatally X-irradiated rats, could be due to a decreased neuronal metabolism of noradrenaline by this enzyme.

6-hydroxydopa during development of central adrenergic neurons produces different long-term changes in rat brain noradrenaline.

6-hydroxydopa (6-OH-DOPA) administered to rats during their early development produces long-term modifications in the content of brain noradrenaline (NA) which have regional differences. An increase in brain stem NA is observed when the rats are exposed to the drug between the day 14 of gestation and the 9th postnatal day. When 6-OH-DOPA is injected subcutaneously on the 13th postnatal day or later, there is a decrease in brain stem NA. On the other hand, the content of NA in the telediencephalon is depleted for the first time in rats exposed to the drug during the day 16 of gestation, the decrease is more evident when the injection is done on days 17 or 18 and the effect is also marked when the drug is administered in the period between the day of birth and the 20th day of age. These results indicate that 6-OH-DOPA exerts different effects during the process of development and that the increase in brain stem NA is not solely dependent on the depletion produced in the forebrain because both phenomena are temporally dissociated. The adrenergic neurons injured by the drug, most probably respond in such a way that leads to an increase in brain stem NA only during the period in which they are under the influence of the factors controlling their physiologic development.

DSP-4: a novel compound with neurotoxic effects on noradrenergic neurons of adult and developing rats.

The pharmacological actions of the compound N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4) are compatible with a specific neurotoxic effect on both peripheral and central noradrenergic neurons. The systemic injection of DSP-4 to adult rats transiently alters sympathetic neurons in the periphery but in the central nervous system the compound determines a marked and prolonged reduction of noradrenaline (NA) levels in all brain regions studied. When DSP-4 was injected systemically to rats at birth in doses ranging from 6.25 to 100 micrograms/g, no changes were found in peripheral sympathetic neurons 40 days later. On the contrary, in the same conditions and in relation to the dose injected, there were marked and persistent changes in the levels of NA in different regions of the brain. In the cerebral cortex and the spinal cord, the neonatal injection of SDP-4 produced a marked and long-lasting depletion of NA levels, similar to that observed after injection of the compound to adult rats. These changes were accompanied by a moderate increase in brain stem NA and a marked elevation of the amine in the cerebellum. These changes, different from the depletion observed in both regions when the compound was given to adult rats, are however similar to those observed after the neonatal injection of the neurotoxic compounds 6-hydroxydopamine or its precursor amino acid, 6-hydroxydopa. This indicates that probably central noradrenergic neurons respond in the same manner after different chemical injuries. DSP-4 crosses the placental barrier because when it was given to pregnant rats at the end of gestation, long-term changes were found in brain NA levels in their offspring, similar to those produced by the neonatal administration of the compound. This new neurotoxic compound provides a very useful tool for the study of noradrenergic neurons both in adult animals and during ontogenesis.

GM1 ganglioside treatment protects against long-term neurotoxic effects of neonatal X-irradiation on cerebellar cortex cytoarchitecture and motor function.

Exposure of neonatal rats to a 5 Gy dose of X-irradiation induces permanent abnormalities in cerebellar cortex cytoarchitecture (disarrangement of Purkinje cells, reduction of thickness of granular cortex) and neurochemistry (late increase in noradrenaline levels), and motor function (ataxic gait). The neuroprotective effects of gangliosides have been demonstrated using a variety of CNS injuries, including mechanical, electrolytic, neurotoxic, ischemic, and surgical lesions. Here, we evaluated whether systemically administered GM1 ganglioside protects against the long-term CNS abnormalities induced by a single exposure to ionizing radiation in the early post-natal period. Thus, neonatal rats were exposed to 5 Gy X-irradiation, and subcutaneously injected with one dose (30 mg/kg weight) of GM1 on h after exposure followed by three daily doses. Both at post-natal days 30 and 90, gait and cerebellar cytoarchitecture in X-irradiated rats were significantly impaired when compared to age-matched controls. By contrast, both at post-natal days 30 and 90, gait in X-irradiated rats that were treated with GM1 was not significantly different from that in non-irradiated animals. Furthermore, at post-natal day 90, cerebellar cytoarchitecture was still well preserved in GM1-treated, X-irradiated animals. GM1 failed to modify the radiation-induced increase in cerebellar noradrenaline levels. Present data indicate that exogenous GM1, repeatedly administered after neonatal X-irradiation, produces a long-term radioprotection, demonstrated at both cytoarchitectural and motor levels.

Antiserum to nerve growth factor does not prevent the increase of brain stem noradrenaline after neonatal 6-hydroxydopa.

Antiserum to nerve growth factor (anti-NGF) given intraventricularly to newborn rats systemically injected with 6-hydroxydopa (6-OH-DOPA), did not prevent the long-term increase of brain stem noradrenaline produced by 6-OH-DOPA when given alone. Since the anti-NGF was biologically active and penetrated into the brain parenchyma, the role played by NGF in the outgrowth of central noradrenergic neurons, responsible for the elevation of brain stem noradrenaline, does not seem to be important.

Different effects of neonatal vinblastine on peripheral and central noradrenaline neurons.

The systemic injection of newborn rats of the mitotic inhibitor vinblastine sulfate (0.25 microgram/s.c. 48 h after birth), produces marked and persistent changes in peripheral sympathetic neurons. Approximately half the neuronal population of the superior cervical ganglia was destroyed already at 16 days of age and this was accompanied by a partial but persistent depletion of noradrenaline (NA) from peripheral organs receiving a rich sympathetic nerve supply such as the heart, salivary glands and spleen. After the systemic injection of vinblastine to newborn rats, the content of NA in several brain regions remained unaltered at 45-60 days of age. To overcome the obstacle that the blood-brain barrier could represent to vinblastine penetration into the brain, the compound was injected directly into the brain of rat pups at 2 days of age (0.25-1.0 microgram). When these animals were killed 45-60 days later, no changes were found in the concentration of NA in the cerebral cortex, the spinal cord or the cerebellum but NA levels were increased in the brain stem. Besides producing a partial but persistent peripheral sympathectomy, vinblastine injected either systemically or intracerebrally to newborn rats, provides a useful tool for the analysis of similarities and differences between the ontogenesis of central and peripheral NA neurons.

Differential effect of various 6-hydroxydopa treatments on the development of central and peripheral noradrenergic neurons.

6-Hydroxydopamine or 6-hydroxydopa injected systemically into newborn rats produced marked changes in the development of central and peripheral noradrenergic neurons. Noradrenaline concentration was elevated in the brain stem, particularly in the pons, and decreased in the cerebral cortex and the spinal cord while in the cerebellum, the effects were dependent on the mode of administration. The changes produced by 6-hydroxydopa in brain regional noradrenaline were related to the dose injected at birth. Similar modifications in the development of central noradrenergic neurons were found in the offspring of rats which had received 6-hydroxydopa at 16 days of gestation. The involvement of peripheral sympathetic neurons varied with the compound used and the form of its administration. Thus, 6-hydroxydopamine produced a permanent although partial peripheral sympathectomy, an effect which was less evident following multiple injections of 6-hydroxydopa after birth and almost minimal after a single injection. The prenatal administration of 6-hydroxydopa did not alter peripheral sympathetic neurons. It is concluded that with the appropriate treatment schedule, it is possible to lesion selectively the noradrenergic neurons in the central nervous system.

Stimulation of beta-adrenergic receptors in the pineal gland increases the noradrenaline stores of its sympathetic nerves.

The administration of isoproterenol decreases the level of serotonin in the rat pineal gland and at the same time it increases pineal noradrenaline. These effects depend on the stimulation of a beta-adrenergic receptor because they are blocked by pretreatment of the animals with propranolol; this drug by itself does not modify either serotonin or noradrenaline levels in the pineal. The elevation of noradrenaline produced by isoproterenol is selective for the pineal because it is not observed in the salivary gland innervated by postganglionic adrenergic fibers from the same origin as pineal nerves. Pineal serotonin is stored in equilibrium in two compartments, i.e., the parenchymal cells and the adrenergic nerves and thus is most probably reduced in both sites. Since noradrenaline and serotonin are detected in pineal nerve vesicles and may coexist in them, the diminution of intravesicular serotonin, by making more storage sites available, probably determines the selective increase of pineal noradrenaline. A similar modification in the ratio of intravesicular amines as a result of the physiological stimulation of pineal beta-adrenergic receptors by the adrenergic neurotransmitter may explain some of the changes observed in the content of pineal amines.

Motor, cytoarchitectural and biochemical assessment of pharmacological neuroprotection against CNS damage induced by neonatal exposure to ionizing radiation.

Exposure of neonatal rats to a 5 Gy single dose of X-irradiation induces permanent abnormalities in cerebellar cortex cytoarchitecture and neurochemistry and motor function. This rodent model constitutes an useful tool to evaluate morphological, neurochemical and motor changes induced by ionizing radiation and the possible restorative effects of potential or clearly established neuroprotective drugs. After selection and administration of a neuroprotective agent to neonatally irradiated rats, quantitative evaluations of motor behavior (gait), cerebellar cortex cytoarchitecture and cerebellar monoamine levels are performed. Data are compared to those of both saline-injected, X-irradiated, and saline-injected, sham-irradiated controls. Evaluation of data from the different experimental groups is performed at postnatal days 30 and 90. After this postnatal interval, radiation-induced damage of cerebellar function in nonprotected rodents is considered to be permanent. The longitudinal evaluation of various parameters in the different experimental groups through a multidisciplinary approach, allows determination of the variables that are more sensitive to X-irradiation-induced damage and/or neuroprotective agent-induced restoration. Given the well-known correspondence in cerebellar developmental stages between rodents and humans, this model and related studies bring health-related implications, considering the accidental or therapeutic exposure of developing human beings to ionizing radiation.

Chronic corticosterone impairs inhibitory avoidance in rats: possible link with atrophy of hippocampal CA3 neurons.

The aim of our work was to evaluate the effect of a chronic (22 days) administration of corticosterone, which induces supraphysiological serum levels of the hormone, on an inhibitory avoidance learning in rats (one-trial step-through learning task, footshock: 0.5 mA, 2 s). We also studied hippocampal markers of neuroanatomical CA3 pyramidal neuron atrophy by using the Golgi staining method. Chronic exposure to high CORT serum levels induced a significant impairment of inhibitory avoidance learning. The CORT group also showed hippocampal glucocorticoid receptor (GR) downregulation and the decrease of hippocampal CA3 branch points and total dendritic length in the apical tree that would be causally related with the learning impairment.

Interactions of phospholipids and free fatty acids with antidepressant recognition binding sites in rat brain.

The lipid microenvironment of cell membranes has been shown to regulate both neurotransmitter and hormone receptors. Preincubation of cortical synaptosomal membranes of rat brain with phospholipase A2 (PLA2) increases the number of [3H]imipramine ([3H]IMI) high affinity binding sites without altering Kd (Bmax control: 2.53 +/- 0.28 pmol/mg protein vs Bmax PLA2: 3.66 +/- 0.26 pmol/mg protein). The displacement curves of [3H]IMI binding in synaptosomal membranes with other tricyclic antidepressants are not affected by the presence of PLA2. The effect of PLA2 was prevented by incubation with EGTA (2 x 10(-3)) or bovine serum albumin (BSA; 1:1). In addition, end products of catalytic activity of PLA2 such as unsaturated fatty acids (arachidonic or oleic acids) mimicked the effect of PLA2. These effects were entirely prevented by preincubation with BSA. The in vitro addition of the acidic phospholipid phosphatidylserine isolated from bovine brain (BC-PS) produced a similar increase in Bmax. This action was also blocked by addition of BSA. On the other hand, palmitic acid, a saturated fatty acid, and lysophosphatidylserine (lysoPS) or lysophosphatidylethanolamine (lysoPE) failed to modify [3H]IMI binding sites. The chronic administration of tricyclic antidepressant (AD) resulted in a 25% decrease in [3H]IMI binding sites in synaptosomal membranes. Preincubation of these AD-treated membranes with PLA2 did not alter [3H]IMI binding, whereas the addition of unsaturated free fatty acids (FFA) produced a greater increase in the density of [3H]IMI binding sites in comparison with control membranes. Taken together, these findings suggest that unsaturated free fatty acids could play an important role in the regulation of the number of [3H]IMI high affinity binding sites in the mammalian brain.