Chelatable Fe (II) is generated in the rat kidneys exposed to ischemia and reperfusion, and a divalent metal chelator, 2, 2'-dipyridyl, attenuates the acute ischemia/reperfusion-injury of the kidneys: a histochemical study by the perfusion-Perls and -Turnbull methods.

The perfusion-Perls and -Turnbull methods supplemented by diaminobenzidine intensification demonstrated the generation and localization of chelatable Fe (II) which can catalyze the generation of cytotoxic hydroxyl radicals (OH.) during the Fenton reaction in rat kidneys exposed to 40 min ischemia or 40 min-ischemia followed by 60 min-reperfusion. The kidneys exposed to 40 min-ischemia showed Fe (II)-deposits largely localized in the deeper half of the cortex, where the deposits densely filled the tubular cell nuclei, with a small amount of them in the cytoplasm of the proximal convoluted tubules (PCT). Intraluminally protruded or exfoliated tubular cell nuclei were also filled with the deposits. The kidneys subjected to 40 min-ischemia/ 60 min-reperfusion showed a more extensive distribution of Fe (II)-deposits, including most depths of the cortex. Furthermore, there were numerous exfoliated, Fe (II)-positive nuclei surrounded by a small amount of cytoplasm in the lumen of the PCT. These cells appeared to undergo apoptotic cell death since the lumen of strongly dilated, down-stream, proximal straight tubules were obstructed with numerous apoptotic cells in the kidneys exposed to 40 min-ischemia and 24 h-reperfusion. Pretreatment with a divalent metal chelator, 2, 2'-dipyridyl, effectively inhibited Fe (II)-staining, decreased the number of exfoliated cells in the kidneys with 40 min-ischemia/ 60 m-reperfusion, and decreased the number of apoptotic cells in the kidneys with 40 min-ischemia/24 h-reperfusion. The generation of highly reactive OH. during the Fe2+-catalyzed Fenton reaction was suggested to play a crucial role in ischemia/reperfusion-induced kidney injury.

Cellular and subcellular localizations of nonheme ferric and ferrous iron in the rat brain: a light and electron microscopic study by the perfusion-Perls and -Turnbull methods.

Iron in the brain is utilized for cellular respiration, neurotransmitter synthesis/degradation, and myelin formation. Iron, especially its ferrous form, also has the potential for catalyzing the Fenton reaction to generate highly cytotoxic hydroxyl radicals. The amount of iron in the brain must therefore be strictly controlled. In this study, we focused on the cellular and subcellular localizations of nonheme ferric (Fe(III)) and ferrous (Fe(II)) iron in the adult female rat brain using light and electron microscopic histochemistry. Although Fe(II) deposition was much less dominant than Fe(III), the brain contained iron in both forms. Among the cellular elements of the brain, oligodendrocytes were numerically the most prominent and heavily iron-storing cells. Pericapillary astrocytes and sporadic microglial cells also showed dense iron accumulation. Large neurons involved in the motor system were relatively strongly iron-positive. Subcellularly, Fe(III) and Fe(II) were mainly localized in lysosomes, and occasionally in the cytosol and mitochondria. Furthermore, capillary endothelial cells had Fe(III)-positive reactions in lysosomes and the cytosol, with Fe(II)-positive reactions on the luminal membrane. With advancing age, both Fe(III) and Fe(II) became more extensively distributed and accumulated more numerously in oligodendrocytes and astrocytes. These findings suggest that age-related increases in Fe(II) accumulation may raise the risk of tissue damage in the normal brain.

Nonheme-iron histochemistry for light and electron microscopy: a historical, theoretical and technical review.

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.

Visualization of non-heme ferric and ferrous iron by highly sensitive non-heme iron histochemistry in the stress-induced acute gastric lesions in the rat.

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.

The presence of ferric and ferrous iron in the nonheme iron store of resident macrophages in different tissues and organs: histochemical demonstrations by the perfusion-Perls and -Turnbull methods in the rat.

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.

Perfusion-Perls and -Turnbull methods supplemented by DAB intensification for nonheme iron histochemistry: demonstration of the superior sensitivity of the methods in the liver, spleen, and stomach of the rat.

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.