HISTOLOGY AND HISTOPATHOLOGY OF BLOOD VESSELS: BACKSCATTERED SCANNING ELECTRON MICROSCOPY APPROACH
Aim. To study the technical possibility of em-ploying the original technique of long-term osmium tetroxide postfixation and uranyl acetate/lead citrate counterstaining with the following embedding into epoxy resin for the investigation of histological and histopathological features of the blood vess...
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Kemerovo State Medical University
2019-03-01
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| Series: | Фундаментальная и клиническая медицина |
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| Online Access: | https://fcm.kemsmu.ru/jour/article/view/125 |
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| author | RINAT A. Mukhamadiyarov ANTON G. Kutikhin |
| author_facet | RINAT A. Mukhamadiyarov ANTON G. Kutikhin |
| author_sort | RINAT A. Mukhamadiyarov |
| collection | DOAJ |
| description | Aim. To study the technical possibility of em-ploying the original technique of long-term osmium tetroxide postfixation and uranyl acetate/lead citrate counterstaining with the following embedding into epoxy resin for the investigation of histological and histopathological features of the blood vessels. Materials and Methods. The study was carried out on rat abdominal aorta, carotid atherosclerot-ic plaque excised during endarterectomy, and stent excised during total surgical correction of Fallot’s tetralogy. After the 24-hour fixation in 10% neutral phosphate buffered formalin, samples were stained with 2% osmium tetroxide for 60 hours, dehydrated in ascending ethanol series (50, 60, 70, 80, and 95% ethanol, 99,7% isopropanol, acetone), impregnated into acetone : epoxy resin (Epon-Araldite, 1:1), and embedded into fresh epoxy res-in with the following grinding and polishing of polymerised epoxy resin blocks. Counterstain-ing with 2% uranyl acetate was performed during the incubation in 95% ethanol while counterstain-ing with Reynolds’ lead citrate was conducted af-ter the grinding. Samples were then sputter coated with carbon and were finally visualised by back-scattered scanning electron microscopy.Results. Our technique retained integrity of the blood vessels, preserving all vascular layers (intima, media, adventitia, and perivascular adipose tissue) and allowed visualisation of all vessel structures (endothelial cells, elastic fibers, smooth muscle cell layers, fibroblasts, perivascular adipose tissue cells, vasa vasorum, and lymphoid follicles) at x5000 magnification. In addition, it was possible to differentiate even small caliber vasa vasorum from lymphoid follicles. The developed technique considerably ex-pands quality of visualization as compared to the conventional approach (paraffin embedding, routine histological staining, and light microscopy) with a limited magnification not permit-ting quantitative analysis of vasa vasorum and lymphoid follicles. Further, it makes possible to analyse tissues containing metal implants or large amounts of calcified tissue without their disintegration.Conclusions. Out technique may be suggested for the evaluation of physiological and pathological neovascularisation and assessment of adventitial and perivascular inflammation. |
| format | Article |
| id | doaj-art-6d2c7551e4c044f683e3f1487c4590e0 |
| institution | DOAJ |
| issn | 2500-0764 2542-0941 |
| language | Russian |
| publishDate | 2019-03-01 |
| publisher | Kemerovo State Medical University |
| record_format | Article |
| series | Фундаментальная и клиническая медицина |
| spelling | doaj-art-6d2c7551e4c044f683e3f1487c4590e02025-08-20T03:19:11ZrusKemerovo State Medical UniversityФундаментальная и клиническая медицина2500-07642542-09412019-03-0141614124HISTOLOGY AND HISTOPATHOLOGY OF BLOOD VESSELS: BACKSCATTERED SCANNING ELECTRON MICROSCOPY APPROACHRINAT A. Mukhamadiyarov0ANTON G. Kutikhin1Research Institute for Complex Issues of Cardiovascular DiseasesResearch Institute for Complex Issues of Cardiovascular DiseasesAim. To study the technical possibility of em-ploying the original technique of long-term osmium tetroxide postfixation and uranyl acetate/lead citrate counterstaining with the following embedding into epoxy resin for the investigation of histological and histopathological features of the blood vessels. Materials and Methods. The study was carried out on rat abdominal aorta, carotid atherosclerot-ic plaque excised during endarterectomy, and stent excised during total surgical correction of Fallot’s tetralogy. After the 24-hour fixation in 10% neutral phosphate buffered formalin, samples were stained with 2% osmium tetroxide for 60 hours, dehydrated in ascending ethanol series (50, 60, 70, 80, and 95% ethanol, 99,7% isopropanol, acetone), impregnated into acetone : epoxy resin (Epon-Araldite, 1:1), and embedded into fresh epoxy res-in with the following grinding and polishing of polymerised epoxy resin blocks. Counterstain-ing with 2% uranyl acetate was performed during the incubation in 95% ethanol while counterstain-ing with Reynolds’ lead citrate was conducted af-ter the grinding. Samples were then sputter coated with carbon and were finally visualised by back-scattered scanning electron microscopy.Results. Our technique retained integrity of the blood vessels, preserving all vascular layers (intima, media, adventitia, and perivascular adipose tissue) and allowed visualisation of all vessel structures (endothelial cells, elastic fibers, smooth muscle cell layers, fibroblasts, perivascular adipose tissue cells, vasa vasorum, and lymphoid follicles) at x5000 magnification. In addition, it was possible to differentiate even small caliber vasa vasorum from lymphoid follicles. The developed technique considerably ex-pands quality of visualization as compared to the conventional approach (paraffin embedding, routine histological staining, and light microscopy) with a limited magnification not permit-ting quantitative analysis of vasa vasorum and lymphoid follicles. Further, it makes possible to analyse tissues containing metal implants or large amounts of calcified tissue without their disintegration.Conclusions. Out technique may be suggested for the evaluation of physiological and pathological neovascularisation and assessment of adventitial and perivascular inflammation.https://fcm.kemsmu.ru/jour/article/view/125аортастентыэпоксидная смолаvasa vasorumнеоваскуляризацияaortastentsepoxy resinvasa vasorumneovascularization |
| spellingShingle | RINAT A. Mukhamadiyarov ANTON G. Kutikhin HISTOLOGY AND HISTOPATHOLOGY OF BLOOD VESSELS: BACKSCATTERED SCANNING ELECTRON MICROSCOPY APPROACH Фундаментальная и клиническая медицина аорта стенты эпоксидная смола vasa vasorum неоваскуляризация aorta stents epoxy resin vasa vasorum neovascularization |
| title | HISTOLOGY AND HISTOPATHOLOGY OF BLOOD VESSELS: BACKSCATTERED SCANNING ELECTRON MICROSCOPY APPROACH |
| title_full | HISTOLOGY AND HISTOPATHOLOGY OF BLOOD VESSELS: BACKSCATTERED SCANNING ELECTRON MICROSCOPY APPROACH |
| title_fullStr | HISTOLOGY AND HISTOPATHOLOGY OF BLOOD VESSELS: BACKSCATTERED SCANNING ELECTRON MICROSCOPY APPROACH |
| title_full_unstemmed | HISTOLOGY AND HISTOPATHOLOGY OF BLOOD VESSELS: BACKSCATTERED SCANNING ELECTRON MICROSCOPY APPROACH |
| title_short | HISTOLOGY AND HISTOPATHOLOGY OF BLOOD VESSELS: BACKSCATTERED SCANNING ELECTRON MICROSCOPY APPROACH |
| title_sort | histology and histopathology of blood vessels backscattered scanning electron microscopy approach |
| topic | аорта стенты эпоксидная смола vasa vasorum неоваскуляризация aorta stents epoxy resin vasa vasorum neovascularization |
| url | https://fcm.kemsmu.ru/jour/article/view/125 |
| work_keys_str_mv | AT rinatamukhamadiyarov histologyandhistopathologyofbloodvesselsbackscatteredscanningelectronmicroscopyapproach AT antongkutikhin histologyandhistopathologyofbloodvesselsbackscatteredscanningelectronmicroscopyapproach |