ACTA HISTOCHEMICA ET CYTOCHEMICA

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How Does the Ca²⁺-paradox Injury Induce Contracture in the Heart?—A Combined Study of the Intracellular Ca²⁺ Dynamics and Cell Structures in Perfused Rat Hearts—

Hiroki Mani, Hideo Tanaka, Tetsuya Adachi, Masaya Ikegawa, Ping Dai, N ...

Article type: Regular Article
2015 Volume 48 Issue 1 Pages 1-8
Published: February 28, 2015
Released on J-STAGE: February 28, 2015
Advance online publication: January 15, 2015

DOIhttps://doi.org/10.1267/ahc.14059

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The calcium (Ca²⁺)-paradox injury of the heart, induced by restoration of extracellular Ca²⁺ after its short-term depletion, is known to provoke cardiomyocyte contracture. However, undetermined is how the Ca²⁺-paradox provokes such a distinctive presentation of myocytes in the heart. To address this, we imaged sequential intracellular Ca²⁺ dynamics and concomitant structures of the subepicardial ventricular myocytes in fluo3-loaded, Langendorff-perfused rat hearts produced by the Ca²⁺ paradox. Under rapid-scanning confocal microscopy, repletion of Ca²⁺ following its depletion produced high-frequency Ca²⁺ waves in individual myocytes with asynchronous localized contractions, resulting in contracture within 10 min. Such alterations of myocytes were attenuated by 5-mM NiCl₂, but not by verapamil, SEA0400, or combination of ryanodine and thapsigargin, indicating a contribution of non-specific transmembrane Ca²⁺ influx in the injury. However, saponin-induced membrane permeabilization of Ca²⁺ showed no apparent contracture despite the emergence of high-frequency Ca²⁺ waves, indicating an essential role of myocyte-myocyte and myocyte-extracellular matrix (ECM) mechanical connections in the Ca²⁺ paradox. In immunohistochemistry Ca²⁺ depletion produced separation of the intercalated disc that expresses cadherin and dissipation of β-dystroglycan located along the sarcolemma. Taken together, along with the trans-sarcolemmal Ca²⁺ influx, disruption of cell-cell and cell-ECM connections is essential for contracture in the Ca²⁺-paradox injury.

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New Grocott Stain without Using Chromic Acid

Kazuya Shiogama, Kayo Kitazawa, Yasuyoshi Mizutani, Takanori Onouchi, ...

Article type: Regular Article
2015 Volume 48 Issue 1 Pages 9-14
Published: February 28, 2015
Released on J-STAGE: February 28, 2015
Advance online publication: February 03, 2015

DOIhttps://doi.org/10.1267/ahc.14045

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We established a new “ecological” Grocott stain for demonstrating fungi, based upon a 4R principle of refusal, reduction, reuse, and recycle of waste management. Conventional Grocott stain employs environmentally harsh 5% chromic acid for oxidization. Initially, we succeeded in reducing the concentration of chromic acid from 5% to 1% by incubating the solution at 60°C and using five-fold diluted chromic acid solution at which point it was reusable. Eventually, we reached the refusal level where 1% periodic acid oxidization was efficient enough, when combined with preheating of sections in the electric jar, microwave oven, or pressure pan. For convenience sake, we recommend pressure pan heating in tap water for 10 min. Stainability of fungi in candidiasis and aspergillosis was comparable with conventional Grocott stain, while Mucor hyphae showed enhanced staining. The modified sequence was further applicable to detecting a variety of mycotic pathogens in paraffin sections. Our environmentally-friendly Grocott stain also has the advantage of avoiding risk of human exposure to hexavalent chromium solution in the histopathology laboratory. The simple stain sequence is can be easily applied worldwide.

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Immunohistochemical Analysis of Collagen IV and Laminin Expression in Spontaneous Melanoma Regression in the Melanoma-Bearing Libechov Minipig

Daniela Planska, Monika Burocziova, Jan Strnadel, Vratislav Horak

Article type: Regular Article
2015 Volume 48 Issue 1 Pages 15-26
Published: February 28, 2015
Released on J-STAGE: February 28, 2015
Advance online publication: February 24, 2015

DOIhttps://doi.org/10.1267/ahc.14020

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Spontaneous regression (SR) of human melanoma is a rare, well-documented phenomenon that is not still fully understood. Its detailed study cannot be performed in patients due to ethical reasons. Using the Melanoma-bearing Libechov Minipig (MeLiM) animals of various ages (from 3 weeks to 8 months) we implemented a long-term monitoring of melanoma growth and SR. We focused on immunohistochemical detection of two important extracellular matrix proteins, collagen IV and laminin, which are associated with cancer. We showed that SR of melanoma is a highly dynamic process. The expression of collagen IV and laminin correlated with changes in population of melanoma cells. Tumours of 3-week-old animals consisted primarily of melanoma cells with a granular expression of collagen IV and laminin around them. Thereafter, melanoma cells were gradually destroyed and tumour tissue was rebuilt into the connective tissue. Collagen IV expression slightly increased in tumours of 10-week-old pigs showing extracellular fibrous appearance. In tumours of older animals, areas lacking melanoma cells demonstrated a low expression and areas still containing melanoma cells a high expression of both proteins. We considered the age of 10 weeks as a turning point in the transition between tumour growth and SR of the MeLiM melanoma.

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