The distribution of small and intermediate conductance calcium-activated potassium channels in the rat sensory nervous system.

Small (SK) and intermediate (IK) conductance calcium-activated potassium channels are candidate ion channels for the regulation of excitability in nociceptive neurones. We have used unique peptide-directed antisera to describe the immunocytochemical distribution of the known isoforms of these ion channels in dorsal root ganglia (DRG) and spinal cord of the rat. These investigations sought to characterize further the phenotype and hence possible functions of nociceptive neurone subpopulations in the rat. In addition, using Western blotting, we sought to determine the level of protein expression of SK and IK channels in sensory nervous tissues following induction of inflammation (Freund's Complete Adjuvant (FCA) arthritis model) or nerve injury (chronic constriction injury model). We show that SK1, SK2, SK3 and IK1 are all expressed in DRG and spinal cord. Morphometric analysis revealed that SK1, SK2 and IK1 were preferentially localized to neurones having cell bodies <1000 microm2 (putative nociceptors) in DRG. Dual labeling immunocytochemistry showed that these ion channels co-localize with both CGRP and IB4, known markers of nociceptor sub-populations. SK2 was localized almost exclusively in the superficial laminae of the spinal cord dorsal horn, the region in which many sensory afferents terminate; the distribution of SK1 and IK1 was more widespread in spinal cord, although some preferential labeling within the dorsal horn was observed in the case of IK1. Here we show evidence for a distinctive pattern of expression for certain members of the calcium-activated potassium channel family in the rat DRG.

Techniques of advanced light microscopy and their applications to morphological analysis of human extra-embryonic membranes.

The science of light microscopy has advanced dramatically in recent years through the introduction of new technology. A brief description of scanning light microscopes, laser illumination, the confocal principle, digital imaging, and image processing reveals a number of theoretical advantages which are particularly useful in improving epifluorescence microscope images. Examples of results from several studies of human extra-embryonic membranes conducted in our laboratory show how the application of these techniques has been used to describe structures such as microtrabeculae and rivets for the first time, to map the microscopic distribution of a wide range of proteins, and to observe the activity of placental villi at the microscopic level in an environmentally controlled microscope stage. High-sensitivity detectors have permitted the "super-resolution" detection of structures smaller than the theoretically calculated limits of light microscope resolution. Rendering images in false colour is demonstrably useful in detecting subtle variations in fluorescence intensity at different intracellular sites and at different sites within tissues of fetal membranes. Processing stacks of digital images using appropriate software allows the 3-D reconstruction of suitably sized extra-embryonic membrane components. These digital images created from optical sections through the tissue are obtained non-destructively, and the relationships in space of the components are well preserved.

Behaviour of two IgG subclasses in transport of immunoglobulin across the human placenta.

The human IgG subclasses are a family of highly related yet distinct molecules. Each of these four subclasses performs a discrete function within the human immune system. Previous studies have shown that one of these molecules, hIgG2, may be discriminated against in transport across the human placenta. We have aimed to elucidate the mechanism of this discrimination in order to gain a more comprehensive understanding of the process of transport of immunoglobulin across the human placenta. We have used a combination of immunocyctochemical localisation and biochemical analysis to detail the behaviour of hIgG2. Confocal laser scanning microscopy was used to compare the localisation of hIgG1 (chosen as representative of the efficiently transported subclasses) and hIgG2 in term and first trimester chorionic villi. Complementary evidence was provided from immunoblot analysis of isolated placental coated vesicles. The data presented here suggest that the hIgG2 is transported into the syncytiotrophoblast and appears to accumulate in the stroma of the villi. This leads us to the hypothesis that the fetal capillary endothelium is the cellular impediment to the transport of hIgG2 into the fetal circulation.

Cytokine and free radical responses of alveolar macrophages in vitro to asbestos fibres.

A method for culturing primary rat alveolar macrophages (AMs) for 14 days was used to compare their responses to crocidolite and chrysotile asbestos fibres. Exposure to crocidolite increased production of tumour necrosis factor-alpha (TNF-alpha) and interleukin 1beta (IL-1beta), whereas exposure to chrysotile did not; neither fibre altered the production of interleukin 6 (IL-6). IL-1beta production increased progressively, while TNF-alpha was fully elevated from day 1. Conversely, chrysotile, but not crocidolite, increased production of superoxide anion and nitric oxide (NO) radicals. These differential responses were only observed by extending the culture beyond the usual 1-3 days.

A novel synthetic erionite fibre radiolabelled with (57)Co.

The aim was to produce a radiolabelled fibre suitable for long-term carcinogenesis studies. To this end, we have successfully synthesised erionite fibres by a method adapted to incorporate (57)Co into the crystal framework. Morphologically the fibres are straight, of median length 2.5 microm, with 11% of fibres > 8 microm long, and median width 0.32 microm. These values are comparable to natural Oregon erionite. Autoradiography confirmed that the (57)Co was associated with the fibres themselves. The stability of the radiolabel in vivo was examined by instilling 1 mg of synthetic erionite into the lungs of F-344 rats. About half of the thoracic content of (57)Co was cleared in the first week, and over the following 5 weeks the remainder was cleared slowly with a half-time of 120 days. After 6 weeks the urinary excretion of (57)Co was only 0.054% of the initial lung content per day. This represented fibre dissolution plus any leaching of (57)Co from the fibres. It can be concluded that the (57)Co is bound into the erionite fibres with sufficient stability in vivo for studying their effects in relation to translocation to the pleura.