Position sensitive measurement of lithium traces in brain tissue with neutrons.

PURPOSE: The application of lithium is well known to have an antimanic-depressive effect, however, the influence it has on the human brain is still insufficiently known. The aim of our work is to develop a method to investigate the lithium concentration in the human brain with a very high sensitivity and a submillimeter resolution. Present methods either do not provide spatial resolution or are not sensitive enough to measure the naturally occurring lithium content in the human brain. Our method provides the opportunity to perform postmortem series measurements and obtain a detailed map of the lithium distribution in the human brain. This way possible correlations of the lithium distribution in the human brain and biological reasons for affective disorder can be clarified. METHODS: To study the lithium distribution in different regions of the human brain the authors developed a method to measure lithium traces postmortem with a submillimeter spatial resolution using the neutron capture reaction (6)Li(n, α)(3)H. The lithium is measured by coincident detection of the alpha particles and tritons, emitted in opposite directions. The general concept, the preparation of the brain samples, the experimental setup at the measurement station of the Forschungs-Neutronenquelle Heinz Maier-Leibnitz, and a first measurement on human brain tissue are presented. RESULTS: A first measurement on a brain tissue sample nicely showed a spatial distribution of lithium down to a few hundreds of pg∕cm(3) with a maximal resolution of about σ(x) = σ(y) ≈ 200 µm. Also a direct correlation of lithium and optical tissue structure is observable. Typical measurement times of a few minutes allow for series measurements of up to 20 × 20 mm(2) large samples with a thickness of w = 10-20 µm in medical studies. CONCLUSIONS: The combination of a very high lithium sensitivity with position resolving measurement makes this method well suited for postmortem studies of the microscopic lithium distribution in the human brain and thus to form a microscopic picture of the impact of lithium in different areas of the human brain.

Spatial dynamics of DNA damage response protein foci along the ion trajectory of high-LET particles.

High-linear energy transfer (LET) ion irradiation of cell nuclei induces complex and severe DNA lesions, and foci of repair proteins are formed densely along the ion trajectory. To efficiently discriminate the densely distributed/overlapping foci along the ion trajectory, a focus recognition algorithm called FociPicker3D based on a local fraction thresholding technique was developed. We analyzed high-resolution 3D immunofluorescence microscopic focus images and obtained the kinetics and spatial development of γ-H2AX, 53BP1 and phospho-NBS1 foci in BJ1-hTERT cells irradiated with 55 MeV carbon ions and compared the results with the dynamics of double-strand break (DSB) distributions simulated using the PARTRAC model. Clusters consisting of several foci were observed along the ion trajectory after irradiation. The spatial dynamics of the protein foci supports that the foci clusters are not formed by neighboring foci but instead originate from the DSB cluster damage induced by high-LET radiations.

Competition effect in DNA damage response.

We have built an ion-microbeam for studies of the nuclear topography and kinetics of double-strand break repair at the single cell level. Here, we show that a first and a second, delayed single ion exposure at different nuclear sites led to comparable accumulations of phospho-ATM, gamma-H2AX and Mdc1 at both earlier (e) and later (l) microirradiated sites. In contrast, accumulations of 53BP1 and the recombination protein Rad51 were strongly reduced at l-sites. This apparent competition effect is accompanied by a reduced amount of 53BP1 in undamaged areas of the irradiated nuclei. We suggest that a critically limited pool size combined with strong binding at irradiated sites leads to the exhaustion of unbound factors freely roaming the nuclear space. The undersupply of these factors at l-sites requires in addition a long-lasting binding at e-sites or a weaker binding at l-sites. The observed effects suggest that DNA damage response at individual nuclear sites depends on the time course of damage load. This may have implications for therapeutic radiation treatments.

Quantitative analysis of DNA-damage response factors after sequential ion microirradiation.

Several proteins are known to form foci at DNA sites damaged by ionizing radiation. We study DNA damage response by immunofluorescence microscopy after microirradiation of cells with energetic ions. By using microirradiation, it is possible to irradiate different regions on a single dish at different time-points and to differentiate between cells irradiated earlier and later. This allows to directly compare immunofluorescence intensities in both subsets of cells with little systematic error because both subsets are cultivated and stained under identical conditions. In addition, by using irradiation patterns such as crossing lines, it is possible to irradiate individual cells twice and to differentiate between immunofluorescence signals resulting from the cellular response to the earlier and to the later irradiation event. Here, we describe the quantitative evaluation of immunofluorescence intensities after sequential irradiation.