Altered nitric oxide synthase and PKC activities in cerebellum of gamma-irradiated neonatal rats.

In this study, we show that one single dose of gamma-irradiation at birth induces an inhibition of the cerebellar calcium dependent nitric oxide synthase (NOS) activity, probably correlated to the motor abnormalities and the disarrangement in the cerebellar cytoarchitecture observed in adult rats. This decrease in calcium dependent NOS activity could be associated with an increased protein kinase C (PKC) activity. PKC inhibition partially restores calcium dependent NOS activity, indicating that PKC activity could be negatively modulating the catalytic activity of calcium dependent NOS. These findings suggest that a decrease in nitric oxide (NO) production and the related increase in PKC activity could be intracellular events that participate in the onset of motor and cerebellar abnormalities induced by postnatal gamma-irradiation at early stages of life.

Loss of neuronal protein expression in mouse hippocampus after irradiation.

The effects of radiation on neurons are incompletely characterized. We evaluated changes in the expression of neuronal nuclear and other proteins in the mouse hippocampus after 17-Gy whole-brain irradiation. Expression of neuronal nuclei (NeuN), neuron-specific enolas, prospero-related homeobox 1 (Prox1), calbindin D28k, and synaptophysin 1 in the CA1, CA3, and dentate gyrus of the hippocampus was determined by immunohistochemistry; neuronal numbers were estimated by design-based stereology. At 7 days after irradiation, there was a marked reduction of NeuN neurons in CA3. Stereologic estimates confirmed a significant reduction in NeuN neurons in CA3 at 7 days, in the dentate gyrus at 7 days, 3 weeks and 2 months, and in CA1 at 2 months compared with controls; neuron-specific enolase and prospero-related homeobox 1-positive neurons in the CA3 subregion were also decreased at 7 days. The numbers of granule and pyramidal cells identified by 4'6-diamidino-2-phenylindole nuclear staining, however, remained unchanged, and there were no changes in calbindin D28k or synaptophysin 1 immunoreactivity after irradiation. We conclude that irradiation may result in a temporary loss of neuronal protein expression in mouse hippocampus. These changes do not necessarily indicate loss of neurons and indicate the need for caution regarding the use of phenotypic markers such as NeuN to estimate changes in neuronal numbers after irradiation.

Long-term impact of radiation on the stem cell and oligodendrocyte precursors in the brain.

BACKGROUND: The cellular basis of long term radiation damage in the brain is not fully understood. METHODS AND FINDINGS: We administered a dose of 25Gy to adult rat brains while shielding the olfactory bulbs. Quantitative analyses were serially performed on different brain regions over 15 months. Our data reveal an immediate and permanent suppression of SVZ proliferation and neurogenesis. The olfactory bulb demonstrates a transient but remarkable SVZ-independent ability for compensation and maintenance of the calretinin interneuron population. The oligodendrocyte compartment exhibits a complex pattern of limited proliferation of NG2 progenitors but steady loss of the oligodendroglial antigen O4. As of nine months post radiation, diffuse demyelination starts in all irradiated brains. Counts of capillary segments and length demonstrate significant loss one day post radiation but swift and persistent recovery of the vasculature up to 15 months post XRT. MRI imaging confirms loss of volume of the corpus callosum and early signs of demyelination at 12 months. Ultrastructural analysis demonstrates progressive degradation of myelin sheaths with axonal preservation. Areas of focal necrosis appear beyond 15 months and are preceded by widespread demyelination. Human white matter specimens obtained post-radiation confirm early loss of oligodendrocyte progenitors and delayed onset of myelin sheath fragmentation with preserved capillaries. CONCLUSIONS: This study demonstrates that long term radiation injury is associated with irreversible damage to the neural stem cell compartment in the rodent SVZ and loss of oligodendrocyte precursor cells in both rodent and human brain. Delayed onset demyelination precedes focal necrosis and is likely due to the loss of oligodendrocyte precursors and the inability of the stem cell compartment to compensate for this loss.

Binding kinetics of calbindin-D(28k) determined by flash photolysis of caged Ca(2+)

We have used UV flash photolysis of DM-nitrophen in combination with model-based analysis of Oregon Green 488 BAPTA-5N fluorescence transients to study the kinetics of Ca(2+) binding to calbindin-D(28K). The experiments used saturated DM-nitrophen at a [Ca(2+)] of 1.5 microM. Under these conditions, UV laser flashes produced rapid steplike increases in [Ca(2+)] in the absence of calbindin-D(28K), and in its presence the decay of the flash-induced fluorescence was due solely to the Ca(2+) buffering by the protein. We developed a novel method for kinetic parameter derivation and used the synthetic Ca(2+) buffer EGTA to confirm its validity. We provide evidence that calbindin-D(28K) binds Ca(2+) in at least two distinct kinetic patterns, one arising from high-affinity sites that bind Ca(2+) with a k(on) comparable to that of EGTA (i.e., approximately 1 x 10(7) M(-1) s(-1)) and another with lower affinity and an approximately eightfold faster k(on). In view of the inability of conventional approaches to adequately resolve rapid Ca(2+) binding kinetics of Ca(2+) buffers, this method promises to be highly valuable for studying the Ca(2+) binding properties of other biologically important Ca(2+) binding proteins.