‘Functional histology’ in brain ischemia studies

Classical histology and biochemistry constitute two very different ways to study biology. Classical histology allows direct visualization of tissue, but is limited by the non-specificity of organic dyes and the need to stain fixed tissue in postmortem samples. Classical biochemistry allows direct observation of the activities of tissue constituents, however, at the expense of complete cellular disruption, and is, in a sense, ‘blind' in that kinetic or equilibrium expressions are required intermediaries between data and interpretation. But advances in microscope technology, protein engineering, and transgenic animal technology have blurred this classical distinction. One can today speak of a ‘functional histology' that goes considerably beyond that discussed by Salthouse (1976) many years ago. It is well known that brain ischemia induces morphological alterations in postischemic neurons. Studies using classical histology cataloged end-stage morphologies such as ischemic cell change (Rosenblum, 1997). However, the past decade has seen increasing application of ‘functional histology' methods that literally illustrate ischemia-induced morphological changes at earlier time periods of the injury course. Two notable examples are the Hu et al (2000) discovery of the accumulation in postischemic neurons of ubiquitin–protein clusters (Ge et al, 2007) and our own work on mRNA-containing structures (DeGracia et al, 2008), both of which occur within minutes of reperfusion. In this issue of the Journal of Cerebral Blood Flow and Metabolism, the article by Krzysztof et al (2011) advances the application of ‘functional histology' to a completely new level in the field. The authors utilized fluorescent photobleaching technology and transgenic animals bred to contain a version of green fluorescent protein fused with the calreticulin endoplasmic reticulum-targeting sequence. Using this system, the authors visualized from the cortical surface of intact whole animals the real-time dynamics of the endoplasmic reticulum of cortical neurons during the ischemic period. On the basis of the time of recovery of fluorescent signal after photobleaching, the authors elegantly and convincingly demonstrated that fragmentation of the endoplasmic reticulum occurs within minutes of the onset of global ischemia. The results are illustrated with striking three-dimensional images of fragmented endoplasmic reticulum in cortical neurons. The techniques and analysis are straightforward and unambiguous, leaving little room for controversy. To my knowledge, this study now marks the earliest observation of a clear-cut ‘functional morphological' alteration in postischemic neurons. It is also notable the authors attribute this change to activation of ‘stress responses' in the postischemic neurons. This interpretation reflects the increasing realization that postischemic neuronal death may not be because of only ischemia-induced damage but also because of a competition between ischemia-induced damage and the cell's innate ability to respond to damage (Endres et al, 2008; Lo, 2008; DeGracia, 2010). In sum, the paper by Krzysztof et al (2011) literally illustrates the power of a ‘functional histological' approach by demonstrating the very rapid response of the neuron to the ischemic perturbation. We expect the general approach used here to become increasingly prevalent, and the current paper represents an important milestone on this path.