Imaging of placental transport mechanisms: A review.

Functional analysis of material transfers requires precise statement of residence times in each tissue compartment. For the placenta, neither extractive biochemistry, isotope partitioning, nor mass-based quantitative assays provide adequate spatial resolution to allow the necessary precision. Dual-perfusion assays of material transfer in isolated placental cotyledons provide time-series data for two compartments, the maternal and fetal blood, but fail to distinguish the two cellular compartments (syncytiotrophoblast, fetal endothelium) which actively regulate rates of transfer in each direction for essentially every important molecule type. At present, no definitive technology exists for functional analysis of placental transfer functions. The challenge in developing such a technology lies in the exquisitely small and delicate structures involved, which are scaled at cellular and subcellular sizes (between 50 nm and 50 microm). The only available technologies attaining this high spatial resolution are imaging technologies, primarily light and electron microscopy. To achieve the high-quality images necessary, confocal laser scanning microscopy (CLSM) is required, to provide a uniform optical sectioning plane. In turn, this requires relatively high fluorescence intensities. Design of an adequate technology therefore bases on CLSM imaging fluorochrome-tagged tracers. The temporal resolution necessary to analyse placental material transfers is expected to be of the order of a few seconds, so that conventional wet-fixation protocols are too slow. For adequately rapid fixation, snap-freezing is required. As part of this review we report results obtained from an appropriately designed experimental protocol, analysed by CLSM and transmission electron microscopy (TEM). The images acquired were tested for uniformity of illumination and fluorescence emission strength. Relevant data was encoded in the green channel of the trichrome images obtained, and this was thresholded by application of strict quantitative criteria. The thresholding procedure is suitable for automation and produces reproducible, objectifiable results. Thresholded images were subjected to image calculation procedures designed to highlight image elements (pixels) containing (green) fluorescence associated with the tracer protein; all other sources of fluorescence were visualised in the final images only if no green fluorescence was detectable in that pixel. The resulting images were maps, showing the distribution of tracer molecules at a predefined time interval after perfusion of the tracer into the vital (term) cotyledon. Spatial resolution was routinely better than 1 microm and temporal resolution was approximately 5s. At timepoints up to 10 min after intravital application into the fetal vascular circulation, tracer was associated with capillaries in the villous structures, and no tracer was observed in the syncytiotrophoblast. Clear distinction was achieved between the four tissue compartments relevant to placental transfers, thus providing a novel technology capable of generating high-quality data concerning the regulation of transfers of any molecule that can bear a fluorescent tag. The potential applications of this methodology lie in analyses of factors influencing the rates of fetomaternal and maternofetal exchanges (for example, drugs), and of functional responses of the placental regulation to pathophysiological conditions such as hypoxia

Molecular mapping deep within a living human organ: Analysis of microvessel function on the timescale of seconds and with sub-micrometre spatial resolution.

Visualising vascular endothelial cell function in individual blood microvessels allows elucidation of molecular interactions at the vascular wall, the first barrier between blood-borne therapeutic agent and its target. Functional analysis in situ requires sub-micrometer spatial resolution and tagged molecules generating contrast in living blood vessels. Light microscopy fulfills these requirements, particularly if fluorescent tags deliver the contrast. However, vascular arborisations in living organs defy morpho-functional analysis, filling tissues with closely meshed three-dimensional networks which are inaccessible to optical imaging. We protocol here successful morpho-functional analysis of microvascular processing in a living organ, the human placental cotyledon. Fluorescence-tagged tracer was positionally fixed by snap-freezing, frozen sections were cut, freeze-dried and heat-fixed. A brief histochemical procedure then labelled all vascular elements in the sections, providing fluorescence contrast in two colour channels. Mosaic monochromatic images acquired in both channels delivered high-resolution maps of centimeter-wide tissue areas. Quantitative analysis of the images’ greyscale histograms defined objectifiable, reproducible thresholds, used to reduce the images to colour-coded wide-area functional maps tracking placental vascular processing of the tagged molecules. Rapid positional fixing of tracer with reduction of images to maps was combined with ultrastructural tracking to elucidate vascular processing at scales of nanometres and seconds

Fine localization of the Nijmegen breakage syndrome gene to 8q21: evidence for a common founder haplotype.

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, a birdlike face, growth retardation, immunodeficiency, lack of secondary sex characteristics in females, and increased incidence of lymphoid cancers. NBS cells display a phenotype similar to that of cells from ataxia-telangiectasia patients, including chromosomal instability, radiation sensitivity, and aberrant cell-cycle-checkpoint control following exposure to ionizing radiation. A recent study reported genetic linkage of NBS to human chromosome 8q21, with strong linkage disequilibrium detected at marker D8S1811 in eastern European NBS families. We collected a geographically diverse group of NBS families and tested them for linkage, using an expanded panel of markers at 8q21. In this article, we report linkage of NBS to 8q21 in 6/7 of these families, with a maximum LOD score of 3.58. Significant linkage disequilibrium was detected for 8/13 markers tested in the 8q21 region, including D8S1811. In order to further localize the gene for NBS, we generated a radiation-hybrid map of markers at 8q21 and constructed haplotypes based on this map. Examination of disease haplotypes segregating in 11 NBS pedigrees revealed recombination events that place the NBS gene between D8S1757 and D8S270. A common founder haplotype was present on 15/18 disease chromosomes from 9/11 NBS families. Inferred (ancestral) recombination events involving this common haplotype suggest that NBS can be localized further, to an interval flanked by markers D8S273 and D8S88