We reviewed the methods of nonheme-iron histochemistry with special focus on the underlying chemical principles. The term nonheme-iron includes heterogeneous species of iron complexes where iron is more loosely bound to low-molecular weight organic bases and proteins than that of heme (iron-protoporphyrin complex). Nonheme-iron is liberated in dilute acid solutions and available for conventional histochemistry by the Perls and Turnbull and other methods using iron chelators, which depend on the production of insoluble iron compounds. Treatment with strong oxidative agents is required for the liberation of heme-iron, which therefore is not stained by conventional histochemistry. The Perls method most commonly used in laboratory investigations largely stains ferric iron, but stains some ferrous iron as well, while the Turnbull method is specific for the latter. Although the Turnbull method performed on sections fails in staining ferrous iron or stains only such parts of the tissue where iron is heavily accumulated, an in vivo perfusion-Turnbull method demonstrated the ubiquitous distribution of ferrous iron, particularly in lysosomes. The Perls or Turnbull reaction is enhanced by DAB/silver/gold methods for electron microscopy. The iron sulfide method and the staining of redox-active iron with H(2)O(2) and DAB are also applicable for electron microscopy. Although the above histochemical methods have advantages for visualizing iron by conventional light and electron microscopy, the quantitative estimation of iron is not easy. Recent methods depending on the quenching of fluorescent divalent metal indicators by Fe(2+) and dequenching by divalent metal chelators have enabled the quantitative estimation of chelatable Fe(2+) in isolated viable cells.

Perfusion-Perls and -Turnbull methods supplemented by the intensification with 3,3'-diaminobenzidine (+ DAB) enabled stronger and more extensive staining of nonheme iron than the Perls + and Turnbull + DAB methods carried out on tissue sections fixed with 10% formalin in 0.9% saline or PBS. The section- and perfusion-Perls + DAB methods are not specific for the demonstration of nonheme ferric iron but also stain nonheme ferrous iron. However, owing to its high sensitivity, the perfusion-Perls + DAB method would provide useful information about nonheme iron deposition regardless of oxidation states in normal and pathological conditions. The perfusion-Turnbull + DAB method is specifically demonstrable of nonheme ferrous iron and the results from this method showed significant stores of nonheme ferrous iron in the hepatocytes, Kupffer cells, splenic macrophages, and gastric parietal cells of the rat. Since nonheme ferrous iron is considered to be critically involved in free radical generation, the perfusion-Turnbull + DAB method would visualize such populations of cells that are at risk from free radical damage.

We previously developed the highly sensitive perfusion-Perls and -Turnbull methods to visualize nonheme ferric (Fe (III)) and ferrous (Fe (II)) iron, respectively. The present study used these methods to investigate the possible presence of nonheme iron in the redox (ferric/ferrous) state in the noneheme iron store (phagolysosomes and siderosomes) of resident macrophages in the rat. The perfusion-Perls and -Turnbull methods at pH 0.6 supplemented by DAB intensification intensely stained resident macrophages of different tissues and organs of normal and iron-overloaded rats. The perfusion-Turnbull method, which is specific for nonheme Fe (II), partly stained hemosiderin at pH 5.3, but hardly stained it at the physiological pH, suggesting the presence of some iron in the reduced form, free Fe2+ and/or loosely bound Fe (II), at the intravacuolar pH (5.4+/-0.2) of the phagolysosomes of macrophages. Electron microscopy of the splenic and hepatic macrophages treated by the perfusion-Perls or -Turnbull method showed that Fe (II) deposits were largely distributed along the margin of hemosiderin masses while Fe (III) deposits could also be found within hemosiderin masses.

[Purpose] This study aimed to determine the differences in the excitability of spinal motor neurons during motor imagery of a muscle contraction at different contraction strengths. [Methods] We recorded the F-wave in 15 healthy subjects. First, in a trial at rest, the muscle was relaxed during F-wave recording. Next, during motor imagery, subjects were instructed to imagine maximum voluntary contractions of 10%, 30%, and 50% while holding the sensor of a pinch meter, and F-waves were recorded for each contraction. F-waves were recorded immediately and at 5, 10, and 15 min after motor imagery. [Results] Both persistence and F/M amplitude ratios during motor imagery under maximum voluntary contractions of 10%, 30%, and 50% were significantly higher than that at rest. In addition, persistence, F/M amplitude ratio, and latency were similar during motor imagery under the three muscle contraction strengths. [Conclusion] Motor imagery under maximum voluntary contractions of 10%, 30%, and 50% can increase the excitability of spinal motor neurons. The results indicated that differences in muscle contraction strengths during motor imagery are not involved in changes in the excitability of spinal motor neurons.

Redox-active non-heme iron catalyzes hydroxyl radical [Formula: see text] generation through Haber-Weiss reaction. Oxidative tissue damage by OH* has been suggested in the development of stress-induced gastric lesion. Using highly sensitive non-heme iron histochemistry, the perfusion-Perls and -Turnbull methods plus DAB intensification, we studied the distribution of non-heme ferric and ferrous iron (NHF[III] and NHF[II]) in the normal stomach and its changes in the acute gastric lesions induced by restraint water immersion (RWI) stress in the rat. Both NHF[III] and NHF[II] staining increased in the oncotic parietal cells located at the erosive lesion which developed on the gastric mucosal folds after 3 h RWI. It was considered that increase in non-heme iron in these cells catalyzed OH* generation under the presence of O(2)(*-) released from abundant injured mitochondria. This was supported by the increase in H(2)O(2) staining in the erosive region and the obvious reduction of the gastric lesion following administration of deferoxamine before RWI. NHF[II] was stained in the arterial endothelium in the tela submucosa of the normal gastric wall and increase in the entire gastric mucosa after 3 h RWI suggests that the changes in the vascular non-heme iron metabolism were also involved in the response of the stomach to stressful conditions.