X-Ray Microprobe Analysis of Subcellular Elemental Distribution in Normal and Injured Peripheral Nerve Axons

Abstract

Studies involving X-ray microanalysis of liver, heart, and other nonneuronal cells have contributed significantly to our understanding of submembrane distribution of elements (e.g., Na, K, Ca) and their role in cellular injury (Trumpet al., 1979). However, with respect to elemental distribution in nerve cells, there is little direct information available from either X-ray microprobe analysis or biochemical studies. Relevant data from the few published microprobe studies are difficult to interpret due to certain methodological problems. In some cases the results were not quantitated (Ricket al., 1976; Schloteet al., 1981), whereas in other studies the tissue was chemically fixed and/or cytochemical markers such as pyroantimonate were used (Hillman and Llinas, 1974; Oschmanet al., 1974; Duce and Keen, 1978; Ellismanet al., 1979; Chanet al., 1984). These chemical procedures can produce artefactual translocation of elements (Morgan, 1979; Somlyo, 1985). To provide information concerning the subcellular distribution of elements in nerve cells, electron probe X-ray microanalysis was used in this study. The microprobe data are generated by visually identifying a cellular compartment, followed by manual placement of the microprobe electron beam over the compartment and subsequent analysis. However, subtle patterns of elemental distribution which might exist within a compartment cannot be determined conveniently because the beam is placed manually and the large amount of time required to produce the necessary data. To circumvent this problem, digital X-ray imaging with computer-controlled beam placement was used. This method can analyze a cellular region point by contiguous point and thereby provide a chemical map of elemental distribution. In the present study, selected compartments of normal rat sciatic nerve axons were examined by both X-ray microprobe analysis and digital imaging. In addition, we determined how elemental distribution was altered in axons injured by hypoxia or transection. To prevent artefactual translocation of elements, frozen, unfixed sections of nerves were analyzed. The results from this study indicate that each axonal compartment examined displayed a characteristic distribution of elemental content and that following injury this distribution was altered.