CC BY
17
ISSN2079-8334. Ceim Meduuuuu ma 6w№ii. 2018. № 2(64)
DOI 10.26724 / 2079-8334-2018-2-64-153-156 UDC 616.24-06:616.45-001.1/3]-091.8:616-076
THE USE OF SEMI-THIN SECTION METHOD IN THE STUDY OF STRESS-INDUCED
STRUCTURAL CHANGES IN LUNGS
E-mail: mn_koptev@ukr.net
The perfect knowledge of the microscopic structure of the appropriate organ is crucial in the solution of many urgent problems of clinical medicine. Better preservation of tissue structures is achieved when the tissues are embedded into epoxy resins followed up with the semi-thin section making. In contrast to the thick sections that are used for light microscopy, due to the greater awareness and relative cost effectiveness without the need of complicated expensive equipment, the method of semithin sections today is currently used in morphological studies. The paper was aimed at the study of the prospects of using the semi-thin section method to evaluate the immobilization stress-induced structural changes in the lungs. The resulting study showed the appropriateness of the use of semi-thin sections to study structural changes in lungs, induced by the experimental stress. The study of the semi-thin sections is the beneficial technique of the morphological analysis that permits to evaluate the structure of the studied material at the tissue and cellular level.
Keywords: semi-thin sections, lungs, stress.
The paper has been written within the research scientific study "Determination of mechanisms of morphogenesis of organs, tissues and vascular-nerve structures of the body in normal condition, during experiment and under the influence of external factors. Morphoexperimental grounding of the efficacy of new surgical suture material used in clinical practice", state registration number: 0113U00124.
The perfect knowledge of the microscopic structure of the appropriate organ is crucial in the solution of many urgent problems of clinical medicine. In this respect the special techniques of morphological studies that require sectioning of biological tissues with subsequent staining and studying using the light microscope is of great importance. Currently, the method of embedment of tissues into paraffin, celloidin or gelatin to obtain histological specimens that can be stained in various ways for subsequent histological studies is widely used. However, the disadvantage of such techniques is the middling quality of preservation of tissue structures and the relatively small area of the histological sections [1, 5]. Better preservation of tissue structures is achieved when the tissues are embedded into epoxy resins followed up with the semi-thin section making [6]. The obtained epoxy fragments can be sectioned into the 2-3^m slices using the ultramicrotome and glass knives, which are quite easily stained in various ways, designed for semi-thin sections [1-3, 5-7]. In contrast to the thick sections that are used for light microscopy, due to the greater awareness and relative cost effectiveness without the need of complicated expensive equipment, the method of semi-thin sections is currently used in morphological studies, carried out by the researchers from the Higher State Educational Establishment of Ukraine "Ukrainian Medical Stomatological Academy" [1, 6-9].
Purpose of the paper was the study of prospects for using the semi-thin section method to evaluate the immobilization stress-induced structural changes in the lungs.
Materials and methods. 60 Wistar white male rats of 240-260 g body weight, aged 8-10 months were involved into study. Animals from Group I were used to represent the experimental model of the acute immobilization stress. Rats from Group II were subjected to the chronic immobilization stress. Intact animals were assigned into Group III (control), who were housed in the common conditions at vivarium and were not involved into any other experiments or studies. The experimental part of the research has been carried out in compliance with the requirements of the international principals of the "European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes" (Strasbourg, 1985) and corresponding Law of Ukraine "For the Protection of Pet Animals" (No.3446-IV, as of 21.02.2006, Kyiv), as well as ethic code of the Ukrainian physician and ethic code of the scientist [4, 10]. Experimental model of the acute immobilization stress was simulated by immobilization of non-anaesthetized rats, lying supine, for 6 hours. Chronic immobilization stress was simulated by immobilization of the rats in a similar way for 40 minutes daily during 21 days. The animals were killed by decapitation under intraperitoneal thiopentone anesthesia within two hours after immobilization period.
To obtain the semi-thin sections, fragments of lung tissue were poured into vials with cold phosphate buffered 4% glutaraldehyde solution (pH 7,4) and stored in the refrigerator. Within one hour after the prior fixation the slices were minced in a drop of the fresh fixative on the plastic plate. The obtained 1 mm3 tissue blocks were removed into the fresh 4% glutaraldehyde solution, supplemented with 1% calcium chloride solution. Thereafter the blocks were washed during 12 hours in the several
© M.M.Koptev, N.I.Vynnyk, 2018
ISSN2079-8334. Ceim медицини ma ôwnoziï. 2018. № 2(64)
fresh portions of 0,1 M phosphate buffer (pH 7.4), supplemented with calcium chloride. Within the next 2 hours the obtained specimens were fixed in osmic fixative. Thereafter the specimens were washed in the buffer during 1 hour, dehydrated in spirits of the ascending densities and embedded into the EPON-812. The semi-thin sections were obtained using the UMTP-7ultramicrotome. Before staining the slide plates with sections were stored in the thermostat at (45-50)°C. The obtained specimens were stained by the phosphate buffered 0,1% toluidine blue (pH 7.8). Thereafter the stained sections were put into polystyrene under the cover glasses and after polymerization were studied under the light microscope.
Results of the study and their discussion. The resulting study showed the appropriateness of the use of semi-thin sections to study structural changes in lungs, induced by the experimental stress. Particularly, the study of the lung interstitial tissue of the rats from control group and the subsequent analysis of the semi-thin sections permitted to determine its cellular composition. The resident cells -fibroblasts, and migrant cells - macrophages, lymphocytes, plasmacyte and mastocytes have been found. Fibroblasts were large and their shape varied from the oblong to regular orbicular. The cytoplasm of the fibroblasts was basophilic. Nuclei were clear, prolate with decondensed chromatin and 1-2 big nucleoli. Irregular-shaped macrophages were detected perivascularly, containing numerous digitate processes. Their cytoplasm was weakly basophilic and contained numerous optically clear vacuoles. The nuclei were small, orbicular, fabaceous or irregular-shaped, containing large lumps of chromatin. Plasmacytes were orbicular or oblong with sharply basophilic cytoplasm, except for small clear zone near the nucleus. The nucleus of a plasmacyte was relatively small, orbicular or prolate, located eccentrically (Fig. 1).
The analysis of the semi-thin sections of the lungs of rats that were subjected to the acute immobilization stress has shown the increase in number of migrant cells in the interstitium, namely, macrophages, plasmacytes, mastocytes (Fig. 2). The enlarged cisterns of the endoplasmic reticulum granules were detected in the basophilic cytoplasm of plasmacytes at large magnifications of the light microscope.
Fig. 1. Lungs of the rat fom control group. Semi-thin section. Toluidine blue stain: Lens: Fig. 2. Rat lungs after simulation of the model of the acute immobilization stress. Semi-100: Ocular lens:15: 1 - interstitium; 2 - plasmacyte; 3 - venule; 4 - lumen of the thin section. Toluidine blue stain: Lens: 100: Ocular lens: 15: 1 - lumen of the alveolus; 2 alveolus. - interstitium; 3 - plasmacyte; 4 - venule.
The increase in number of macrophages, plasmacytes and lymphocytes, which formed the perivascular aggregations in the connective tissue of the interalveolar septa, was also detected in the interstitium of rats who were subjected to the chronic stress (Fig. 3).
Hin
m
a
Fig. 3. Rat lungs after simulation of the model of the chronic immobilization stress. Semi-thin section. Toluidine blue stain: Lens: 100: Ocular lens:15: 1 - plasmacyte; 2 -medium-sized lymphocytes; 3 - venule; 4 - macrophage; 5 - glycogen-containing cells; 6 - capillary.
Fig. 4. Rat lungs after simulation of the experimental model of the chronic immobilization stress. Semi-thin section. Toluidine blue stain: Lens: 100: Ocular lens:15: 1 - lumen of the alveolus; 2 - lumen of the venule; 3 - basal membrane; 4 -macrophage; 5 - postcapillary.
The study of the semi-thin sections enabled better visualization of structural changes in the alveoli of rats of this group, too. Type I alveolocytes were located on the basal membrane, which in
ISSN2079-8334. Ceim медицини та бюлоги. 2018. № 2(64)
toluidine blue stain was presented in the form of basophilic stripe and was enlarged as compared with the controls. The difference between nucleus-containing and respiratory portions of the type I alveolocytes was leveled due to the enlargement of the latter. Pinocytic vesicles were detected in the cytoplasm. After simulation of the experimental model of the chronic immobilization stress the blood stasis was detected in all sections of the microcirculatory blood flow: the lumens were filled with red blood cells. The wall of the capacitance vessels was thinned, the basal membrane was visualized in the form of thin basophilic stripe with preserved continuity. Blood plasma was of increased optical density (Fig.4).
The foci of the red blood cells diapedesis from the dilated blood microvessels into the interstitial connective tissue and lumens of the alveoli was detected in the peripheral parts of the lungs. Additionally, numerous interstitial and alveolar macrophages were found in the described areas.
Thus, the findings show that the use of the semi-thin sections for evaluation of the lung structure in normal condition and after exposure to stress permits better study of their histological structure, the state of microcirculatory blood flow and gives the characteristic to the cellular elements of the interstitium and alveoli. The use of semi-thin section method provides with prior macro-microscopical analysis of the histological structures for the subsequent study of the selected objects using the maximal magnifications of the light microscope with the application of wide range of histological stains or the follow up electron-microscopic study that was also reported by our colleagues [1].
The study of the semi-thin sections of the normal rat lungs and after exposure to immobilization stress has established that, microscopically, the immobilization stress induces the development of the marked morphofunctional changes in rat lungs, namely, a significant dilatation of the alveoli, destruction and thinning of the interalveolar septa, microcirculation disorder with the development of the events of blood stasis and occurrence of hemorrhages into interstitial connective tissue and lumens of the alveoli, hyperhydration and infiltration of interstitium by the white blood cells.
Currently, the study of the semi-thin sections is the beneficial technique of the morphological analysis that permits to evaluate the structure of the studied material at the tissue and cellular level that was demonstrated by the example of the analysis of the stress-induced structural changes of lungs.
Prospects of further research will encompass the study of the potentialities of the semi-thin section method to be used in morphological studies of various organs and systems.
1. Belokon SA, Vitko YuN, Tkachenko PI, Starchenko II, Gryn VG. Optimizatsiya issledovaniya strukturnykh elementov biologicheskikh tkaney na gistograficheskikh shlifakh. Molodoy uchenyy. 2014; (15): 134-7. [in Russian]
2. Bilash SM, Shepitko VI, Yeroshenko HA, Kostylenko YuP, vynakhidnyky; Vyshchyi derzhavnyi navchalnyi zaklad Ukrayiny "Ukrayinska medychna stomatolohichna akademiya", patentovlasnyk. Sposib identyfikatsiyi endokrynotsytiv shlunkovo-kyshkovoho traktu na epoksydnykh shlifakh. Patent Ukrayiny № 81382. 2013 Cherven' 25. [in Ukrainian]
3. Bilash SM, Shepitko VI, Ivanochko VM, Yeroshenko HA, Bilash VP, vynakhidnyky; Vyshchyi derzhavnyi navchalnyi zaklad Ukrayiny "Ukrayinska medychna stomatolohichna akademiya", patentovlasnyk. Sposib doslidzhennya morfolohiyi mozochka na epoksydnykh shlifakh. Patent Ukrayiny № 98541. 2015 Sichen 15. [in Ukrainian]
4. Zakon Ukrayiny "Pro zakhyst tvaryn vid zhorstokoho povodzhennya" № 3447 - IV vid 21.02.2006. Kyyiv; 2006. 18 s. [in Ukrainian]
5. Korzhevskiy DE, Kirik OV, Karpenko MN. Teoreticheskiye osnovy i prakticheskoye primeneniye metodov immunogistokhimii. Sankt Peterburg: SpetsLit; 2012. 112 s. [in Russian]
6. Kostylenko YuP, Kovalev YeV. Metody raboty s polutonkimi epoksidnymi srezami v gistologicheskoy praktike. Arkhiv anatomii, gistologii, embriologii. 1978; 75 (12): 68-72. [in Russian]
7. Kostylenko YuP, Boyko IV, Starchenko II, Prilutskiy AK. Metod izgotovleniya gistologicheskiy preparatov, ravnotsennykh polutonkim srezam s bolshoy obzornoy poverkhnosti, dlya mnogotselevykh morfologicheskikh issledovaniy. Morfologiya. 2007; (5): 94-6. [in Russian]
8. Starchenko II, Vitko YUN. Osobennosti topografii troynichnogo uzla cheloveka vo vnutriutrobnom periode razvitiya. Visnik problem biologii' i meditsini. 2013; 98 (1-1): 208-10. [in Russian]
9. TkachenkoPI, Starchenko II, Bilokon SO. Pro pokhodzhennya ta morfolohichni vidminnosti shchyto-yazychnoyi i zobno-hlotkovoyi protok yak anatomichnykh utvoren, shcho mozhut staty dzherelom rozvytku kist shyyi. Visnyk problem biolohiyi i medytsyny. 2014; 108 (2-2): 179-82. [in Ukrainian]
10. European convention for the protection of vertebrate animals used for experimental and other scientific purposes. Council of Europe, Strasbourg; 1986. 53 p.
ВИКОРИСТАННЯ МЕТОДУ НАП1ВТОНКИХ ЗР1З1В ДЛЯ ДОСЛ1ДЖЕННЯ СТРУКТУРНИХ ЗМ1Н ЛЕГЕНЬ НА ТЛ1 СТРЕСОВОГО ВПЛИВУ М.М. Коптев, Н.1. Винник, О.В. Коковська, Б.М. Фшеню С.М. Бшаш
Виршення багатьох актуальних проблем клшчно!
ИСПОЛЬЗОВАНИЕ МЕТОДА ПОЛУТОНКИХ СРЕЗОВ ДЛЯ ИССЛЕДОВАНИЯ СТРУКТУРНЫХ ИЗМЕНЕНИЙ ЛЕГКИХ НА ФОНЕ СТРЕССОРНОГО ВОЗДЕЙСТВИЯ М.Н. Коптев, Н.И. Винник, О.В. Коковская, Б.Н. Филенко, С.М.
Билаш
Решение многих актуальных проблем клинической
ISSN2079-8334. Ceim медицини та бюлогп. 2018. № 2(64)
медицини можливе лише за умови досконалих знань про мкроскошчну будову вщповщних оргашв. Краще збереження тканинних структур досягаеться при заливцi тканин в епоксидш смоли з подальшим виготовленням натвтонких зрiзiв. Завдяки бiльшiй тформативносп, порiвняно iз товстими зрiзами, вiдноснiй економiчностi без потреби у складному вартюному обладнанш, метод напiвтонких зрiзiв на сьогодш широко використовуеться у морфолопчних дослiдженнях. Метою роботи стало вивчення можливостей використання методу напiвтонких зрiзiв для оцiнки структурних змш, яю виникають у легенях на та iмобiлiзацiйного стресу. Виконана робота показала доцшьшсть використання напiвтонких зрiзiв для вивчення структурних змш у легенях, яю виникають пiсля впливу експериментального стресу. Вивчення напiвтонких зрiзiв е корисним прийомом морфологтчного аналiзу, який дозволяе ощнити структуру дослiджуваного матерiалу на тканинному i клiтинному рiвнi.
Ключовi слова: напiвтонкi зрiзи, легеш, стрес.
Стаття надiйшла 26.01.18р.
медицины возможно только при условии совершенных знаний микроскопического строения соответствующих органов. Лучшее сохранения тканевых структур достигается при заливке тканей в эпоксидные смолы с последующим изготовлением полутонких срезов. Благодаря большей информативности, по сравнению с толстыми срезами, относительной экономичности без необходимости использования сложного оборудовании, метод полутонких срезов сегодня широко используется в морфологических исследованиях. Целью работы стало изучение возможностей использования метода полутонких срезов для оценки структурных изменений, которые возникают в легких на фоне иммобилизационного стресса. Проделанная работа показала целесообразность использования полутонких срезов для изучения структурных изменений в легких, которые возникают после воздействия экспериментального стресса. Изучение полутонких срезов является ценным приемом морфологического анализа, который позволяет оценить структуру исследуемого материала на тканевом и клеточном уровне.
Ключевые слова: полутонкие срезы, легкие, стресс.
Рецензент Срошенко Г.А.
DOI 10.26724 / 2079-8334-2018-2-64-156-160 УДК 615.324.615.036.8:547.857.4
ВИЗНАЧЕННЯ АНТИЦИКЛООКСИГЕНАЗНОГО КОМПОНЕНТУ В МЕХАН1ЗМ1 АНТИЕКСУДАТИВНО1 Д11 РЕЧОВИНИ КСАНТИНОВОГО РЯДУ
e-mail: kornienko-valentina1966@ukr.net
Проведет дослщження тдтвердили факт, що бенфурам пригшчуе активтсть iзоферменту ЦОГ-2 i забезпечуе протизапальну дш. При одночасному mri6ipyBaHHi бенфурамом iзоферменту ЦОГ-1 пригнiчуються простагландин-залежн механiзми захисту органiзму i збереження слизово! оболонки шлунка, дванадцятипало! кишки. Бенфурам викликав значне iнгiбування iзоферменту ЦОГ-2, що забезпечувало протизапальну дш, не впливав на активнiсть ЦОГ-1 i не перешкоджав реалiзацii' механiзмiв компенсаторного захисту слизово! оболонки шлунка та дванадцятипало! кишки. Бенфурам зменшував експресiю iзоферменту ЦОГ-2, що свщчило про його вираженi протизапальнi властивостг
Ключов! слова: бенфурам, антиексудативна активтсть, ¡зоферменти ЦОГ-1 i ЦОГ-2
Стаття е фрагментом НДР «Фармакологiчне дождження бiологiчно активних речовин i лжарських 3aco6ie синтетичного та природного походження, ix застосування в медичнш практищ» (№ державноi реестрацн 0114U003709).
Запальний процес е розповсюдженим симптомом бшьшосп захворювань у людини. В розвитку запалення важлива роль належить м1грацп лейкоципв у мшроциркуляторному русл1 тканин. В реал1зацп запального процесу приймають участь мед1атори запалення: шнтерлейкш-1, фактор некрозу пухлин, фактор активацп тромбоципв, цитокши, лейкотриен В4, а також простагландини [1]. Встановлено юнування двох ¡зоферменпв циклооксигенази (ЦОГ) - ЦОГ-1 i ЦОГ-2. 1зофермент ЦОГ-1 в норм1 контролюе утворення простагландишв (ПГ) 1з захисною функщею, яка забезпечуе гастропротекщю, посилення перфузп нирок, регулящю утворення тромбоципв. А ¡зофермент ЦОГ-2 утворюеться переважно в зон запалення, коли шдукуеться утворення ЦОГ-2 з ЦОГ-1. 1зофермент ЦОГ-2, стимулюе утворення ПГ безпосередньо в осередку запалення, яю провокують бшь, запальний процес, шдвищення температури. Нестеро'дш протизапальн засоби (НПЗЗ) за принципом впливу на активтсть ЦОГ шдрозд1ляються на неселективн шпб1тори, здатш блокувати ЦОГ-1 i ЦОГ-2, i селективш, пригшчуюч1 активтсть переважно ЦОГ-2. Саме за рахунок блокади ЦОГ-2 НПЗЗ проявляють протизапальну, болезаспокшливу i жарознижуючу д1ю, тод1 як блокада активност ЦОГ-1 призводить до пригшчення захисно! дп ПГ i розвитку небажаних поб1чних ефекпв [3]. Переважна бшьшють НПЗЗ виступае як конкуренты шпб1тори ЦОГ; тривалють дп цих сполук визначаеться швидюстю виведення самого лшарського засобу з оргашзму. Залежно вщ тривалосп д1! НПЗЗ можна роздшити на препарати з короткою i тривалою д1ею. Важливою особливютю препарапв групи НПЗЗ е те, що вони по х1м1чнш природ1 е оргашчними кислотами, а тому здатн накопичуватися в осередку запалення i сприяти прояву протизапально! дп [4, 6].
Протизапальний ефект НПЗЗ пов'язаний з пригшченням активност в осередку запалення
© В.1. Коршенко, 2018
156
