Intracellular free-calcium concentration ([Ca2+]i) was measured in lamprey spinal axons using the fluorescent calcium indicator fura 2. We used both a photomultiplier tube and a video-image processing system to measure the temporal and spatial distributions of [Ca2+]i in the proximal segments of transected axons. Within 3 min following transection, a spatially graded increase in the [Ca2+]i was apparent in the last few millimeters of the axons. Superimposed on the initial gradient was a moving front of calcium that progressed up the axon, reaching 1.6 mm from the cut end in 3 hr. The [Ca2+]i behind the moving front exceeded 10 microM. This movement of Ca2+ was greatly reduced by an externally applied electrical field with the cathode distal to the lesion and was increased by an applied field of the opposite polarity. When axons were transected in Ca2(+)-free medium, no increases in [Ca2+]i occurred. One d after transection, [Ca2+]i was at or below the precut levels, except in the distal 250 microns, where it remained slightly elevated. Therefore, axons can survive the high levels of [Ca2+]i that occur after transection and can reestablish normal [Ca2+]i levels within 24 hr. Measurements of both the diffusion coefficient and the fluorescence polarization of fura 2 indicate that the dye is not significantly bound to axoplasmic components.

Many reports have demonstrated inflammation after the placement of dental restorations. To explain this side-effect, we studied a biomarker in the inflammatory response. The intercellular adhesion molecule-1 (ICAM-1) is a key mediator for recruitment of leukocytes to the site of inflammation. Therefore, we investigated whether methacrylates (a BISGMA-based dental resin, BISGMA, and MAA) and Cyracure UVR 6105, an epoxy monomer, could alter ICAM-1 expression in unstimulated and TNF-alpha-stimulated endothelial cells. Six-well plates with monolayers of human umbilical vein cells, ECV 304 (ATCC CRL 1998), were exposed to TNF-alpha (1 ng/mL) in the presence and absence of subtoxic and TC50 doses of chemicals for 24 hrs at 37 degrees C/5% CO2. Several doses of TNF-alpha (0.5-2 ng/mL) were coincubated with 100 microL of undiluted aqueous dental resin extracts. Cells were harvested and stained with mAB FITC-conjugated anti-human ICAM-1 (CD54). ICAM-1 expression was measured by flow cytometry. Cells expressed basal levels of ICAM-1, which was up-regulated by TNF-alpha but was not changed by all samples studied. Except for UVR 6105, the methacrylates significantly decreased ICAM-1 expression in TNF-alpha-stimulated cells. These findings suggest that methacrylates may decrease the recruitment of leukocytes to sites of inflammation.

We have used the three major techniques for measuring cytoplasmic calcium concentration to quantify calcium changes in a variety of cells during a number of developmental processes. In this paper, we describe our experiences with aequorin, calcium electrodes, and fluorescent calcium indicators. The Xenopus oocyte is used as the prime example throughout this discussion, as we have done all three types of measurements in oocytes. Although each of these methods gives roughly the same value for the cytoplasmic calcium concentration in oocytes, each method has its own advantages and disadvantages when it comes to practical experiments. The particular problems with each of the methods are discussed in detail. We conclude that the choice of method depends on the type of information that is required. Calcium electrodes are probably the most precise method but are also the most technically difficult and only provide very localized measurements. Aequorin is quite suitable when an overall average of calcium levels in a cell is required but it is difficult to get good information on the spatial distribution of calcium. Fluorescent indicators are the most straightforward method to use and provide much information about the spatial distribution of calcium in cells as well as average levels in cell suspensions, however they are the most difficult to calibrate.