Aldehyde fixation is not necessary for the formation of “dark” neurons

Abstract

Kherani and Auer [6] recently reported a study in Acta Neuropathologica relating to the mechanism of the formation of “dark” neurons. Their observations led them to suggest that (1) both in vivo and during the early postmortem period, glutamate release and transmembrane ion Xuxes cause some perturbation in a proportion of neurons, which become shrunken, hyperchromatic under the light microscope, and condensed, hyperelectron-dense at the ultrastructural level, but only while aldehyde Wxation is underway, (2) the shrinkage/condensation is executed by transmembrane ion Xuxes or by actin Wlaments, and (3) everything considered, the formation of “dark” neurons is a biotic process. This idea was logically deduced in part from the results of an experiment dealing with the formation of the artefactual “dark” neurons of Cammermeyer (i.e., neurons formed even in normal brain tissue when removed from the skull without previous transcardial aldehyde Wxation followed by a 24-h delay [1]), and in part from the earlier experience of neuropathologists that artefactual “dark” neurons are generally not encountered either in autopsy materials when the postmortem period is long or after nonaldehyde Wxation. Furthermore, they assumed that this idea also applies to the nonartefactual “dark” neurons produced in vivo by ischemia, hypoglycemia, or epilepsy. In a number of communications (reviewed by [3]), our team have postulated another mechanism, which unfortunately is not mentioned in their study. This mechanism postulates that, in each neuron, the intracellular spaces among the ultrastructural elements of conventional transmission electron microscopy are Wlled with a continuous trabecular gel consisting of proteins, ions and water; this gel stores noncovalent (mechanical) free energy, at the expense of which a volume-shrinking phase transition can spread throughout its whole soma-dendrite domain in response to a chemical or a physical noxa (for the existence and importance of such gels in cell biology, see [10]). The pivotal observations supporting this mechanism were as follows: (1) if initiated by physical (nonbiotic) noxae, the formation of “dark”-neurons took place even under conditions that were extremely unfavorable for enzyme-mediated (biotic) processes [4, 7, 9], (2) independently of the nature (physical or chemical) of the initiating noxa and of the circumstances (favorable or unfavorable for enzymatic processes) of the “dark”-neuron formation, the excess water appeared in neighboring astrocytic elements, but not in the surrounding extracellular space [2–9] (the “destination” of transmembrane ion Xuxes), and (3) in each in-vivo case we investigated [2, 5, 8], both the recovering and the dying “dark” neurons displayed ultrastructural signs proving indisputably that they had been compact and electron-dense even long before aldehyde Wxation. To obtain direct evidence against the role of aldehyde Wxation in the formation of the nonartefactual “dark” neurons, we produced “dark” granule neurons in the hippocampal dentate gyrus of four anesthetized living rats via a single condenser discharge, and in the neocortex of four other anesthetized living rats via a pin puncture head injury (for the methodological details and the animal care, see [2]). In each group, two rats were immediately Wxed by the transcardial perfusion of 500 ml of a 3:1 mixture of methanol and chloroform, while the cut-oV heads of the other rats were subjected to Wxation by microwave irradiation (continuous 750 W, controlled 80°C, 1 min). The brains were removed from the skull 1 day later [1] and processed (without aldehyde postWxation) for electron microscopy as usual. P. Bukovics · J. Pal · F. Gallyas (&) Department of Neurosurgery, University of Pecs, 7623 Ret utca 2, Pecs, Hungary e-mail: ferenc.gallyas.sen@aok.pte.hu