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Recived: 5 May 2018 / Accepted: 10 June 2018 / Published online: 30 June 2018 UDC 616.341-614.876-616-092.4+576.344
MORPHOMETRIC INDICATORS OF THE SMALL INTESTINE OF IRRADIATED RATS
Darkhan E. Uzbekov 1, http://orcid.org/0000-0003-4399-460X Dariya M. Shabdarbaeva % http://orcid.org/0000-0001-9463-1935 Nailya Zh. Chaizhunusova 2, http://orcid.org/0000-0002-6660-7118 Kairat A. Almissaev 1, http://orcid.org/0000-0001-6650-4971 Saltanat E. Uzbekova 3, http://orcid.org/0000-0001-9006-120X Ruslan M. Saporov 1, http://orcid.org/0000-0003-3152-8759 Madina M. Apbasova 4, http://orcid.org/0000-0003-3215-1076 Masaharu Hoshi 5, http://orcid.org/0000-0001-6978-0883
1 Department of Pathological anatomy and Forensic medicine,
2 Department of Nutrition and Hygienic disciplines,
3 Department of Anatomy and Histology,
4 Department of Anesthesiology and Reanimatology,
Semey State Medical University, Semey, The Republic of Kazakhstan;
5 Research Institute for Radiation Biology and Medicine, Hiroshima, Japan
Abstract
Introduction. It's known that persons exposed to p- and Y-rays, together with a different of damaging effects, particular importance is also attached to the digestive system. The dominant role of neutron-activated radionuclide Manganese-56 (56Mn) was noted in the treatises of scientists who studied the atomic bomb effects of Japanese cities, deserving the interest today.
The research purpose. Investigate and compare the morphometric indicators in the small intestine of rats exposed to low dose by p- and Y-rays.
Materials and methods. In experiment, male sex «Wistar» rats in amount of 90, weighting approximately 270-350 g. Three groups were identified: 1) 56Mn which obtained by neutron activation of 100 mg Mn02 powder using the «Baikal-1» atomic reactor with a neutrons fluence of 4*1014 n/cm2; 2) 60Co Y-rays; 3) control group. Necropsy of the animals were on the 3rd, 14th and 60th days after irradiation, then the small intestine removed, after which it was fixed in 10% formalin. Tissues fragments embedded in paraffin, then sections are manufactured serial transverse 4 thickness, which were subsequently stained by hematoxylin and eosin (H&E). For stereoscopic changes, a microphotometric system with Avtandilov's ocular measuring grid was used. In each micropreparation, 20 fields with a total area of 181 points were counted. Statistical processing of the results was processed using licensed packages of application programs «SPSS 2.0». All quantitative variables are described using the mean (M), median (Me) and interquartile interval (IQR). In their comparison, depending on the factors studied, the Kruskel-Wallis criterion was used. The critical level of significance p in testing the statistical hypotheses in this study was taken to be 0,05.
Results. On the basis of the histological research methods characterizing the structural state of the small intestine of irradiated rats, pronounced histomorphological changes are both reactive and destructive in later terms. Structural rearrangement of the tissue composition of the examined organ which persists several weeks after the termination of the influence of 56Mn, indicates a toxic and sensitizing effect of internal irradiation as compared to 60Co effects. The most pronounced dystrophic, inflammatory and necrotic changes are observed in the late periods after irradiation of 56Mn when studying histostructural processes occurring in the tissues of the studied animal organs after exposure to neutron-activated manganese dioxide and external irradiation identified by the totality of morphometrical indicators. The studied parameters of the small intestine have statistically significant
differences in the effect groups of the factors and the control group (p<0,001). It was found that the greatest difference in thickness of the mucosa compared to the control group after exposure to 56Mn by 6,3 mcm, whereas after 60Co effect at 6,25 mcm. The greatest decrease in the enterocytes number of the intestinal villi is observed in the I-group at 17,39%, and in the II-group at 3,99%. After exposure to 60Co, the leukocytes number compared to the control group is 4,76% higher, and after 56Mn at 3,77%. The greatest deviations in the lymphocytes number are observed after y-, and then p—irradiation.
Conclusion. Thus, 56Mn effect on the small intestine of rats showed a high level of risk p—ray exposure, which is confirmed by the morphometric indicators.
Keywords: radioactive 56Mn, gastrointestinal syndrome, intestinal cells, morphometry.
Резюме
МОРФОМЕТРИЧЕСКИЕ ПОКАЗАТЕЛИ ТОНКОЙ КИШКИ ОБЛУЧЕННЫХ КРЫС
Дархан Е. Узбеков 1, http://orcid.org/0000-0003-4399-460X Дария М. Шабдарбаева 1, http://orcid.org/0000-0001-9463-1935 Найля Ж. Чайжунусова 2, http://orcid.org/0000-0002-6660-7118 Кайрат A. Альмисаев % http://orcid.org/0000-0001-6650-4971 Салтанат Е. Узбекова 3, http://orcid.org/0000-0001-9006-120X Руслан M. Сапоров \ http://orcid.org/0000-0003-3152-8759 Мадина M. Апбасова 4, http://orcid.org/0000-0003-3215-1076 Масахару Хоши 5, http://orcid.org/0000-0001-6978-0883
1 Кафедpа na^om4ec^ анатомии и судебнoй медицины, 2 Кафедpа питания и гигиенических дисциплин, 3 Кафeдpа анатомии и rnc^orn^ 4 Кафeдpа анeстeзиoлoгии и peаниматoлoгии, Гoсудаpствeнный медицинский унивepситeт гopoда Семей, г. Семей, Республика Казахстан;
5 Научнo-исслeдoватeльский институт pадиациoннoй биoлoгии и медицины, г. Хиpoсима, Япoния
Введение. Извeстнo, 4to у лиц, noдвepгавшихся вoздeйствию в— и Y—^y4eH^ наряду с различными пoвpeждающими эффeктами oco6oe MecTo oтвoдится и пищeваpитeльнoй систeмe. Дoминиpyющая poль нeйтpoннo—aктивиpoвaннoгo paдиoнyклида — Марганца—56 (56Mn) oтмeчалаcь в трудах yчeных, изучавших пocлeдcтвия атoмнoй бoмбаpдиpoвки в япoнcких гopoдах и вызываeт oгpoмный интepec no ceгoдняшний дeнь.
Цель исслeдoвания. Изучить и сравнить мopфoмeтpичecкиe noказатeли тонгай кишки крыс, noдвepгавшихcя вoздeйcтвию малых дoз в— и Y—излyчeния.
Матepиалы и методы. В экcnepимeнтe иcnoльзoваны 90 крыс oбoих noлoв линии «Вистар», маccoй 270—350 гр. Были выдeлeны 3 группы: 1) 56Mn, noлyчeнный путём нeйтpoннoй активации 100 мг nopoшка Mn02 на атoмнoм peактope «Байкал—1» при флюeнce нeйтpoнoв 4*1014 н/cм2; 2) 60Co Y—излyчeниe; 3) кoнтpoльная группа. Живoтных noдвepгали нeкponcии 4epe3 3, 14 и 60 днeй nocлe oблyчeния, затeм извлeкали тoнкий кишeчник, nocлe 4ero фикcиpoвали eгo в 10% фopмалинe. Фpагмeнты тканeй заливали в парафин, затeм изгoтoвливали nonepeчныe cepийныe cpeзы тoлщинoй 4 мкм, кoтopыe в дальнeйшeм oкpашивали гeматoкcилинoм и эoзинoм (H&E). Для cтepeocкonичecких измeнeний иcnoльзoвали микpoфoтoмeтpичecкyю cиcтeмy с oкyляpнoй измepитeльнoй ceткoй Автандилoва. В каждoм микponpenаpатe npocчитывали 20 noлeй cyммаpнoй nлoщадью 181 тoчка. Статиcтичecкyю oбpабoткy peзyльтатoв npoвoдили с иcnoльзoваниeм лицeнзиpoванных пактов прикладных npoгpамм «SPSS 2.0». Bœ изyчаeмыe кoличecтвeнныe nepeмeнныe noказатeли onиcаны при noмoщи cpeднeй (М), мeдианы (Me) и мeжкваpтильнoгo интepвала (IQR). При их cpавнeнии в завиcимocти oт изyчаeмых фактopoв был
использован критерий Краскела-Уоллиса. Критический уровень значимости р при проверке статистических гипотез в данном исследовании принимался равным 0,05.
Результаты. По совокупности гистологических методов исследования, характеризующих структурное состояние тканей тонкой кишки экспериментальных животных, выраженные гистоморфологические изменения в поздние сроки носят как реактивный, так и деструктивный характер. Структурная перестройка тканевого состава исследованного органа крыс, развивающаяся в ранние и поздние сроки после прекращения действия 56Мп, указывает на токсическое и сенсибилизирующее действие внутреннего облучения по сравнению с эффектами 60Со. При сравнении гистоструктурных процессов, возникающих в тканях тонкой кишки крыс после воздействия нейтронно-активированного диоксида марганца и внешнего облучения, наиболее выраженные дистрофические, воспалительные и некротические изменения отмечаются в поздние сроки после облучения 56Мп, выявляемые по совокупности морфометрических показателей. Изучаемые параметры тонкой кишки имеют статистически значимые различия в группах воздействия факторов и контрольной группе (р<0,001). Установлено, что наибольшая разница по толщине слизистой оболочки по сравнению с контрольной группой после воздействия 56Мп отмечается на 6,3 мкм, тогда как после экспозиции 60Со на 6,25 мкм. Наибольшее уменьшение количества энтероцитов в ворсинках кишечника отмечается в 1-ой группе на 17,39%, а во 11-ой на 3,99%. После воздействия 60Со количество лейкоцитов по сравнению с контрольной группой превышает на 4,76%, а после 56Мп на 3,77%. Наибольшие отклонения по количеству лимфоцитов отмечается после Y-, а затем в-облучения.
Выводы. Таким образом, воздействие 56Мп на тонкую кишку крыс выявил высокий уровень риска облучения, что подтверждено морфометрическими показателями.
Ключевые слова: радиоактивный 56Мп, желудочно-кишечный синдром, кишечные клетки, морфометрия.
Туйшдеме
СЭУЛЕЛЕНГЕН ЕГЕУК¥ЙРЫКТАР Ж1Н1ШКЕ 1ШЕПН1Н МОРФОМЕТРИЯЛЫК К6РСЕТК1ШТЕР1
Дархан Е. Узбеков 1, http://orcid.org/0000-0003-4399-460X Дария М. Шабдарбаева 1, http://orcid.org/0000-0001-9463-1935 Найля Ж. Чайжунусова 2, http://orcid.org/0000-0002-6660-7118 Кайрат А. Альмисаев 1, http://orcid.org/0000-0001-6650-4971 Салтанат Е. Узбекова 3, http://orcid.org/0000-0001-9006-120X Руслан М. Сапоров 1, http://orcid.org/0000-0003-3152-8759 Мадина М. Апбасова 4, http://orcid.org/0000-0003-3215-1076 Масахару Хоши 5, http://orcid.org/0000-0001-6978-0883
1 Патологиялык анатомия жэне сот медицина кафедрасы, 2 Тагамтану жэне гигиеналык пэндер кафедрасы, 3 Анатомия жэне гистология кафедрасы,
4 Анестезиология жэне реаниматология кафедрасы, Семей каласынын мемлекетлк медицина университет^ Семей к-, Казахстан Республикасы
5 Радиациялык биология жэне медицина гылыми-зерттеу институты, Хиросима к., Жапония
^спе. в- мен Y-сэулелеу эсерше душар болгандардьщ кептеген бYлiндiргiш салдарымен коса аскорыту жYЙесiне да ерекше мэн белшедк Жапон калаларындагы атомдык; бомбалаудыц салдарын зерттеген галымдардыц ецбектершдеп нейтронды-белсендi Марганец-56 (56Мп) радионуклидшщ басым релi заманауи жагдайда зор кызыгушылык; арттырады.
Зеpттеу максаты. Шагын дозалы в- мен Y-сэулелеу эсерше ушыраган егеукуйрыктардыц жНшке iшегiндегi морфометриялык; керсеткiштердi аныктау жэне салыстыру.
Материалдар мен эд^тер. Тэжiрибе жYзiнде «Вистар» ту;ымды 270-350 гр салмагы бар аталы; жэне аналы; жынысты 90 егеу^уйры^ пайдаланылган. 3 топ;а ipiKTey жYргiзiлдi: 1) 56Mn, ягни 100 мг Mn02 унтагын «Байкал-1» атом реакторы ар;ылы 4*1014 н/см2 нейтрон флюенсшде нейтронды; белсендiру жYзiнде алынган элемент; 2) 60Со Y-сэулелеу; 3) ба;ылау тобы. Жануарларга сэулелеуден кейш 3-шi, 14-шi жэне 60-шы тэулттерде некропсия жYргiзу барысында жНшке шегш алып, 10%-ш формалинде фиксациялады;. Тш фрагменттерiн парафинге ;уйып, калыццыгы 4 мкм келденец сериялы; кесiндiлер дайындап, эрi карай гематоксилин мен эозинмен (Н&Е) бояды;. Стереоскопиялы; езгерiстер жYзiнде Автандиловтыц окулярлы; елшегiш тор сызыгы бар микрофотометриялы; жYЙесi ;олданылган. Эрбiр микропрепаратта 181 нYктелi жиынты; келемi жYзiнде 20 алацы есептелген. Зерттеу нэтижелершщ статистикалы; ец^ «SPSS 2.0» ;олданбалы багдарламаныц лицензияланган пакеттерi кемепмен жYзеге асырылган. БYкiл зерттелген санды; керсетюштердН статистикалы; ецдеуi кезiнде олар орташа керсеткiш (М) жэне медиана (Ме), сондай-а; квартиль аралы; интервал (IQR) жYзiнде сипатталган. Зерттеуге алынган факторлардыц эсерiн салыстырмалы тYPде багалау барысында Краскел-УоллистН Н-елшемi ;олданылган. Нелдт статистикалы; гипотеза на;тылыгыныц критикалы; децгей 0,05-ке тец деп саналган.
Нэтижелер. Эксперимента жануарлар жНшке шегшН ;урылымды; жагдайын аны^тайтын гистологиялы; зерттеу эдiстерi кешеуiлдеу мерзiмi аясында туындаган ай;ын гистоморфологиялы; езгерютердН реактивтi жэне деструкциялы; сипатын керсеткен. 56Mn эсерiне ушыраган егеукyйрыктардыц зерттеуге алынган агзасында ;алыптас;ан ерте жэне кешеуiлдеу мерзiмi аясындагы тiндiк ;урамыныц езгеруi, негiзiнен 60Со эсерiне ;араганда, iшкi иондаушы сэулелеудщ анагурлым улы жэне сенсибилизациялаушы ы;палын бiлдiредi. Нейтронды-белсендiрiлген марганец диоксидi мен сырт;ы иондаушы сэулелеудщ егеукуйрыктардыц ш агзаларына эсерiн салыстырмалы тYPде багалау барысында, морфометриялы; эдiстi ;олдану ар;ылы сэйкес санды; керсеткiштер кемегiмен кешеутдеу мерзiмi аясында бас;а тэжiрибелiк топтардагы жануарлар агзаларында ацгарылган патологиялы; езгерютермен салыстырганда, ай;ын дистрофиялы;, ;абынулы; пен некрозды; Yдерiстердiц 56Mn эсерiнен кейш жYзеге асатыны дэлелденген. ЖНшке шектН зерттелген параметрлерi эсер ету факторлар топтары мен ба;ылау топтары арасында статистика жYзiнде мэндi екенi ацгарылган (р<0,001). Сiлемейлi ;абы;шаныц ;алыцдыгы бойынша анагурлым ай;ын айырмашылы; белгiлерi ба;ылау топтарына ;араганда, 56Mn эсерiнен кейш 6,3 мкм-ге, ал 60Со экспозициясынан кейiн 6,25 мкм-ге ;алыцдаганы аны^талган. 1шек бYPлерiндегi энтероциттер саны I^i топта - 17,39%-га, ал ll^i топта - 3,99%-га азайганы ;упталган. 60Со эсерiнен кейiн лейкоциттер саны ба;ылау тобымен салыстырганда 4,76%-га, ал 56Mn эсерiн алгандарда 3,77%-га жогарылаганы дэлелденген. Лимфоциттер саны бойынша анагурлым ай;ын ауыт;улар Y-, содан соц в-сэулелеуден кейiн бай;алган.
Корытынды. Сонымен, егеу;уйры;тардыц жiцiшке iшiгiне 56Mn эсерi морфометриялы; керсеткiштермен расталатын в-сэулелену ^утнИ жогары децгейiн керсеттi.
Негiзгi сездер: paduo^ceHdi 56Mn, асцазанчшек синдромы, шек жасушалары, морфометрия.
Библиографическая ссылка:
Узбеков Д.Е., Шабдарбаева Д.М., Чайжунусова Н. Ж., Альмисаев К.А., Узбекова С.Е., Сапоров P.M., Апбасова M.M., Хоши M. Морфометрические показатели тонкой кишки облученных крыс / / Наука и Здравоохранение. 2018. 3 (Т.20). С. 5-19.
D.E., Shаbdаrbаеvа D.M., Chaizhunusоva N.Zh., Almissaev K.A., ^Ье^а S.E., Saporov R.M., Apbasova M.M., H^shi M. МогрЬютеИс indicators of №е small intеstinе of irradiatеd rats. Nauka i Zdravookhranenie [Science & Healthcare]. 2018, (Vol.20) 3, pp. 5-19.
Узбеков Д.Е., Шабдарбаева Д.М., Чайжунусова Н.Ж., Альмисаев К.А., Узбекова С.Е., Сапоров P.M., Апбасова M.M., Хоши M. Сэулеленген егеукуйрыктар жНшке шегУц морфометриялы; керсеткiштерi / / Гылым жэне Денсаулы; са;тау. 2018. 3 (Т.20). Б. 5-19.
Introduction
It's generally known that neutron-induced radioisotope such as 56Mn was dominant element found after an atomic bomb explosion. Since 56Mn and the other neutron-activated radioisotopes were present in dust after bombings, people have inhaled these radioactive materials and been internally exposed to ionizing radiation [23]. People who returned early to Hiroshima and Nagasaki after atomic bombing were reported to suffer from the symptoms of acute radiation effects [28]. Consequently, atomic bomb effects on health of survivors have been correlated with delayed p-and Y-rays [11]. The accidental high-dose radiation exposure induces a series of injury levels in multiple organs. Most of studies regarding the fast neutron effect have focused at intestinal changes [9, 31]. Thereby, increasing attention has been given to the radiation effect on the gastrointestinal (GI) tract due to concerns about exposure to radiation after an accident [24]. It's known that nuclear factor is pronounced in GI tract those that are exposed to the external environment, therefore one of outcomes of radiation effects is GI-syndrome [12], which characterized clinically by haemorrhage, endotoxemia, bacterial infection, anorexia, nausea, vomiting, diarrhoea, loss of electrolytes and fluid, dehydration, systemic infection, septic shock and even death. In spite of the significant advances that have occurred in research on underlying mechanisms over the last two decades, the overall morphogenesis of the gastrointestinal syndrome still remains unclear. According to morphologists, these symptoms are probably due to a rapid modification of the intestinal motility and to the structural alteration of the intestinal mucosa [16, 21, 37]. Presently, particular interest is a peculiarity of histomorphological changes in the small intestine of persons exposed to 56Mn and 60Co, allowing in the future to work out the diagnostic criteria for assessing of internal and external radiation effect on the digestive system [6].
The objective of study
To identify and compare the morphometric indicators in the rats small intestine after exposure by single small dose by 56Mn and 60Co.
Materials and methods
For this study, it was purchased and raised in a specific-pathogen-free facility six-month-old sexes «Wistar» rats from Animal Laboratory of Karaganda State Medical University in an amount
of 90 with mean whole body weight 270-350 g. All animals were acclimatized for two weeks before initiation of experiments and kept under normal conditions and fed pellets concentrated diet. The rats were maintained at constant temperature (22±1°C) on 8 hour light-dark cycle. Then, animals were allocated into 3 groups. The first group of rats (n=30) were subjected to 0,2 Gy 56Mn which was obtained by neutron activation of 100 mg of Mn02 (Rare Metallic Co., Ltd., Japan) powder using the «Baikal-1» nuclear reactor with neutron flux 4*1014 n/cm2. Activated powder with total activity of 56Mn 2,75*108 Bq was sprayed pneumatically over animals placed in the special box. The moment of exposition beginning of experimental animals by 56Mn powder is 6 minute after finishing of neutron activation. Duration of exposition of rats to 56Mn radioactive powder was 4,0 hour [2].
The second group of rats (n=30) were irradiated with a total dose of 2 Gy was performed at a dose rate of 2.6 Gy/min using 60Co Y-ray by radiotherapy device «Teragam K-2 unit» (Czech Republic). After irradiation, rats were taken back to the animal facility and routinely cared. The experiment was followed our institution's guide for the care and use of laboratory animals. During the exposure, rats were placed in a specially engineered cage made of organic glass with individual compartments for each animal.
The third group consisted of control rats (n=30) which were placed on shelves in the same facility and shielded from the radiation. All animal procedures were approved by Ethical Committee of Semey Medical University, Kazakhstan (Protocol №5 dated 16.04.2014). The rats were housed in a moderate security barrier.
The rats were sacrificed on the 3rd, 14th and 60th day after irradiation and the small intestine was immediately surgically extracted for further histological and morphometric study. The small intestine sections were deparaffinized and dehydrated in graded 10% formalin solutions. Paraffin sections performed with 4 ^im thickness. For routine pathology, sections were hydrated and stained by hematoxylin-eosin (H&E). The specimens were examined under a Leica DM 1000 microscope (Germany) and images were captured with a charge-coupled device camera. Qualitative histological assessment of intestinal injury was carried out to obtain an overall damage severity result.
For stereoscopic changes, a microphotometric system was used with Avtandilov's ocular measuring grid [1]. In the histological sections of the tissue of the small intestine, the thickness of the mucosa, the number and volume of enterocytes, the number of leukocytes and lymphocytes were analyzed. In each slide glasses, 20 fields with a total area of 181 points were counted. The studies were performed with *10 and *40 magnifications.
Statistical processing of the results was processed using licensed packages of application programs «SPSS 2.0». All quantitative variables are described using the mean (M), median (Me) and interquartile interval (IQR). In their comparison, depending on the factors studied, the Kruskel-Wallis criterion was used. The critical level of significance p in testing the statistical hypotheses in this study was taken to be 0,05 [18].
Results
In this study, we have performed experiment with 56Mn-exposed Wistar rats. Although the radioactivity level received from 56Mn was rather low, the observed biological effects were consistent in our experiment. That was previously reported the internal dose estimates in the gastrointestinal organs of rats exposed to 56Mn [30]. According to dosimetry study the highest doses were fixed in the small intestine [3, 29]. To assess the health of rats after radiation, we
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evaluated activity, posture, dehydration and pelage of the rats. Radiation induced a decrease in health score in all groups of irradiated animals.
Algorithm description of slide glasses of the small intestine included: presence of a layers in the intestinal wall; degree of vascular hyperemia; state of mucosa, submucosa, muscle membrane and serosa. Light microscopic examination of the intestinal mucosa of control rats showed a normal architecture of villi, crypts and enterocytes. Intestinal tissues were collected on the 3rd, 14th and 60th day post-radiation, time associated with complete crypt ablation in small intestine after radiation exposure. H&E sections of intestine revealed not damage in control groups. However, in segment radiated rats, partial loss of crypts were observed exclusively within the targeted segment, while the rest of the intestine was unaffected.
The study of slide glasses of the small intestine in rats on the 3rd day after 56Mn effect shown presence the severe degenerative changes of glands, mild accumulations of leukocytes in the stroma and uneven hyperemia of the stromal capillaries (Fig. 1-B). In rats that exposed to 60Co was found focal accumulations in the glandular lumen the cellular elements, preferably desquamated epithelial cells and reactive nature cells (Fig. 1 -C) in compared with
control rats (Fig ---
, • ij til
1-A).
• * .
■is' : --S - *
cm W/nrl
Fig. 1. Photomicrograph of rat small intestine on the 3rd day after exposure. Control (A), 56Mn (B) and 60Co (C); H&E staining, original magnification xiQ.
The results of changes in the small intestine compared with the control group, it's the highest
under the influence of the studied factors on the when 56Mn irradiation and more than the
3rd day are presented in Table 1. As can be seen, parameters of the control group by 3,31%. When
the thickness of the mucosa is not statistically exposed to 60Co, an increase of 1,48% is noted in
different when exposed to ionizing radiation. The comparison with the control group (p<0,001). number of enterocytes in the villus of the intestine
Table 1.
Morphometric indicators of the smal intestine of experimental animals on the 3rd day
Morphometric 56Mn 60Co Control Kruskal-Wallis test р value
indicators М Ме IQR М Ме IQR М Ме IQR
Thickness of the mucosa, mcm 36,16 35,92 2,96 35,56 35,32 3,02 35,02 35,36 2,52 H=2,802 0,422
Enterocytes number, % 76,42 76,54 1,98 74,58 74,54 1,72 73,10 73,22 0,88 H=31,106 <0,001
Enterocytes volume, mcm2 138,28 138,22 1,56 137,02 136,56 2,52 134,86 134,88 2,62 H=25,244 <0,001
Leukocytes number, % 20,46 20,28 1,46 19,96 20,06 1,72 11,28 11,26 2,04 H=47,258 <0,001
Lymphocytes number, % 24,06 24,22 1,4 24,14 24,28 1,58 25,02 24,88 1,16 H=27,688 <0,001
There were similar situation for leukocytes: irradiation by 56Mn the number of leukocytes exceeds the control group by 9,14%; when exposed to 60Co an increase of 8,67% that observed compared with the control group (p<0,001).
The number of enterocytes were the highest with irradiation of 56Mn and 60Co by 3,42% and 2,17%, respectively in comparison with the control group (p<0,001).
Changes of the number of lymphocytes depended on the studied factor. When exposure by 56Mn and 60Co the number of lymphocytes have decreased by 0,95% and 0,88%, respectively, relative to the control group (p<0,001).
Microscopic picture of animals on the 14th day after 56Mn exposure provided on figure 2, where we drew attention to the presence of gaps semi-collapsing glands with degeneration, as well as optically empty vacuoles in the glands. Mild cluster of cellular elements, predominantly leukocytes. Considerable importance should be given to presence in the lumen of the individual glands accumulation of lymphocytes, modified leukocytes, mainly neutrophils. Histological studies of 60Co-exposed rats revealed radiation-induced prominent degeneration. Moreover, we are registered the inflammatory response manifested by moderate of macrophages and lymphocytes infiltration in the stroma of the mucous and in lumen of some glands.
Fig. 2. Photomicrograph of the small intestine in 56Mn-exposed rat on the 14th day. H&E staining, original magnification x40.
The parameters of the small intestine on the 14th day after irradiation are presented in Table 2, from which it's evident that the numbers of enterocytes in the villus of the intestine became statistically insignificant on the 14th day between the groups. The thickness of the mucous membrane is different when exposed to various factors. It's the largest in comparison with the
control group after irradiation by 56Mn by 5,97 mcm, whereas after exposure to 60Co more than the control group at 5,36 mcm. The trend in the number of enterocytes and on the 14th day coincide with the indicators of the 3rd day. Also, the greatest differences were noted after exposure to 56Mn.
Table 2.
Morphometric indicators of the small intestine of experimental animals on the 14th day.
Morphometric indicators 56Mn 60Co Control Kruskal-Wallis test p value
M Me IQR M Me IQR M Me IQR
Thickness of the mucosa, mcm 40,06 40,44 2,34 39,44 39,56 2,24 34,08 34,02 1,22 H=45,018 <0,001
Enterocytes number, % 73,08 73,82 3,98 73,58 73,58 1,96 73,30 73,42 2,24 H=0,4958 0,920
Enterocytes volume, mcm2 141,24 140,84 2,42 139,94 140,48 1,62 134,11 134,38 2,18 H=36,146 <0,001
Leukocytes number, % 20,84 20,48 1,54 20,02 20,28 1,36 13,10 13,34 2,52 H=42,862 <0,001
Lymphocytes number, % 27,48 28,46 5,42 27,54 28,44 5,73 25,31 24,36 3,74 H=20,322 <0,001
The same situation was specially for the leukocytes. Their number in absolute values is lower than on the 3rd day, but the trend persists: the highest number after exposure to 56Mn and 60Co was 7,74% and 6,93%, respectively (p<0,001). The trend of the number of lymphocytes persists and on the 14th day after irradiation. However, in comparison with the 3rd and 14th day, the amount increases after all irradiation methods and exceeds the control group values when exposed to 56Mn by 2,17% and after 60Co by 2,24% (p<0.001).
Figure 3 shows that p-radiation effect induces on the 60th day moderate severe degenerative changes of the mucosa and submucosa. The sections are represented mainly by edematous
stroma. Epithelial glands exposed to collapse, in the lumen observed mild accumulations of cellular elements with a predominance the neutrophils. The glands are shrouded by optical empty vacuoles. Mostly celebrated the formation of necrotic foci. In contrast to the internal p- and external Y-radiation, inhalation of nonactivated manganese in rats intestine after 2 month contributes to the appearance narrowing and swollen glands, epithelial desquamation of some glands. Regarding experimental animals exposed to Y-radiation it should be noted the presence of marked degenerative and necrobiotic changes of surface mucous layer and uneven hyperemia of vessels. Here and there observed the foci of necrosis and reactive changes in the mucosa.
Fig. 3. Light microscopy of 56Mn-induced rat small intestine on the 60th day after exposure;
H&E staining, original magnification x40.
The above data are consistent with our study results the small intestine in both 56Mn- and 60Co-exposed rats showed a similar changes. Nevertheless, according to results of histologic examination most pronounced changes were observed in the small intestine of rats from 56Mn group, indicating that neutron radiation has a significant biologic effect on examined organ [32].
The parameters of the rat's small intestine after 60 days are presented in Table 3. As can be seen, all the studied parameters of the small
intestine have statistically significant differences in the effect groups of the factors and the control group (p<0,001). The thickness of the mucosa continues to increase in comparison with 3 and 14 days. As before, the largest thickness difference with the control group after exposure to 56Mn was 6,3 mcm, then after exposure to 60Co at 6,25 mcm. The number of enterocytes in the intestinal villi continues to decrease in comparison with the control group. The greatest decrease is noted in the 56Mn exposure group at 17,39% and after exposure to 60Co by 3,99%.
Table 3.
Morphometric indicators of the small intestine of experimental animals on the 60th day.
Morphometric indicators 56Mn 60Co Control Kruskal-Wallis test р value
М Ме IQR М Ме IQR М Ме IQR
Thickness of the mucosa, mcm 40,56 40,22 1,38 40,52 40,22 1,64 34,26 34,48 1,6 H=36,348 <0,001
Enterocytes number, % 56,86 55,28 8,2 70,26 70,56 1,28 74,24 73,98 1,04 H=48,328 <0,001
Enterocytes volume, mcm2 138,34 138,64 4,86 138,02 137,78 3,34 133,78 133,34 1,4 H=29,392 <0,001
Leukocytes number, % 16,52 17,48 5,24 17,54 17,94 1,26 12,74 12,12 3,82 H=27,894 <0,001
Lymphocytes number, % 30,14 29,98 1,06 29,68 29,82 2,06 24,32 24,22 1,4 H=50,022 <0,001
The number of leukocytes gradually decreases and reaches the lowest values on the 60th day in comparison with acute and subacutes effects. After exposure to 60Co, its excess of the control group remains 4,76%, after 56Mn was 3,77%. The number of lymphocytes continues to increase by 60 days. As well as on the 3rd and 14th days, the greatest deviations in comparison with the control group were observed after exposure to 56Mn and 60Co.
The summary table of the 56Mn effect on the parameters of the small intestine is presented in Table 4. As can be seen from this table, the thickness of the mucosa in the dynamics increases, the number of enterocytes in the villi of the intestine decreases, the enterocyte klemmi practically does not change, the number of leukocytes increases up to 14 days, then are decreasing. The number of lymphocytes in the dynamics is studied increases.
Table 4.
Morphometric indicators on different days in the 56Mn-induced rat small intestine.
Morphometric 3 ird day 14th day 30th day Kruskal-Wallis test р value
indicators М Me IQR M Me IQR M Me IQR
Thickness of the mucosa, mcm 36,16 35,94 2,96 40,06 40,44 2,34 40,56 40,22 1,38 H=28,462 <0,001
Enterocytes number, % 76,42 76,54 1,98 73,08 73,82 3,98 56,86 55,28 8,2 H=35,854 <0,001
Enterocytes volume, mcm2 138,28 138,22 1,56 141,24 140,82 2,4 138,34 138,64 4,86 H=16,732 <0,001
Leukocytes number, % 20,44 20,28 1,46 20,84 20,48 1,54 16,52 17,48 5,24 H=24,186 <0,001
Lymphocytes number, % 24,06 24,22 1,22 27,48 28,47 5,42 30,14 29,98 1,06 H=25,352 <0,001
The conducted research confirms the assumption that the controlled effect factor has a high degree of influence on all the resulting signs of morphometry. This suggests that a single exposure to small dose 56Mn and 60Co has a direct damaging effect to the small intestine of Wistar rats at a later date. A damaging mechanism acting of the small intestinal tissue can be the hyperactivation of lipoperoxidation under the influence of neutron-activated manganese dioxide capable of damaging the gastrointestinal tract organs. Another controlled exposure factor is external irradiation of rats which had the least pronounced effect on the parameters of the small intestine in comparison with internal irradiation. The revealed indicators of the degree of influence of the controlled factor demonstrates that the effect of this factor doesn't affect the most significant indicators of morphometry but affect the most dynamic indicators of morphometry.
Discussion
In this study, we have shown the sequence of histomorphologic changes in the rat small intestine from early to late stage after a single influence of 56Mn and 60Co at small dose, which were the initiators of radiation-induced intestinal injury (RIII). Results of morphologic studies have shown that structural changes in the small intestine observed in irradiated rats little differed from the previously published results using different radiation models.
In segment-radiated rats, the radiated intestinal segment was still identifiable by H&E staining at 14th day post-radiation and didn't exhibit complete normalization of architecture of the mucosa and bowel wall as compared with non-irradiated tissue adjacent to the radiated segment. Histological analysis showed that radiation could induce epithelial degeneration, that's characterized by the loss of intestinal structural integrity, on the 14th day post-radiation. A complete understanding of the mechanisms driving epithelial regeneration and repair, as well as the complications due to exposure of the small intestine to 56Mn would benefit from the ability to study later phases of regeneration involving intestinal epithelial hyperplasia and hyper-proliferation and ideally, times associated with complete normalization of the intestinal epithelial architecture.
It's known that mechanisms of injury in normal tissues after irradiation include progenitor cell depletion, microvascular injury, inflammation and cell death [8]. The major pathological change caused by RIII is architectural disorganization including inflammatory cell infiltration, villitis, desquamation and necrosis [22]. Several evidences suggest that radiation-induced dysfunctions and either changes in subcellular, cellular, histological structure of the small intestine are mediated by concerted and interrelated changes of a plethora of various extracellular mediators and their intracellular messengers [37]. Data morphologic findings were consistent with radiation enteritis. Morphological damages of radiation-induced enteritis were known as architectural changes of intestinal mucosa such as villus shortening by cell death. The acute microscopic changes of intestine by irradiation were consisted of structural changes in the villus-crypt architecture and epithelial transformations [12, 19].
Whole-body radiation can cause severe damage to the digestive system causing inflammatory processes and immediate cell death. Radiation causes inflammation and dysregulation of immune homeostasis. These histomorphologic changes in examined organ of rats exposed to p- and Y-radiation make it possible to develop diagnostic criteria for assessing of radiation effect on the small intestine depending on cumulative dose [33].
Presently, great importance is attached to the role of Mn inducing cell death [26]. Experimentally confirmed that a certain percentage of Mn enters to organism through absorption in the gastrointestinal tract. If Mn not absorbed in the stomach, it's rapidly absorbed in the small intestine. Thus, in the literature we have examined the papers revealed regarding ability of Mn to cause histomorphologic changes in the small intestine of animals [20]. Evidence obtained using genetic modification technology has convincingly shown that intestinal stem cells are columnar cells at the crypt base intermingling with Paneth cells. The molecular determinants of intestinal radiosensitivity and gastrointestinal syndrome are not well understood. Some believe that damage to stem cells plays a critical role in this process [25]. Ionizing radiation leads to the exhaustion of the stem cells pool, increases the load on the differentiated cells [22].
Few studies have focused on a biopolymer whose manipulation significantly regulates gastrointestinal syndrome via securing stem cell zones and the integrity of intestinal epithelium [27].
Cell death after radiation should be noted that damaged cells are eliminated by the adjacent epithelial cells, endothelial, fibroblasts, macrophages [13, 17, 35]. The acute morphological changes of intestine by irradiation were consisted of structural changes in the villus-crypt architecture and epithelial transformations associated with radiation-induced degeneration [12, 36]. Most authors believe that cell death resulting from toxicity of Mn is combination with cessation of ATP synthesis due to mitochondrial damage [29]. Dysfunction or death of intestinal epithelial cells caused by degeneration after radiation influence is considered as dangerous component in the pathogenesis of gastrointestinal syndrome [14]. The initiation and progression of radiation-induced intestine injury can be caused by disorder of metabolic processes and molecular mechanisms which form an compounded response [4-7, 10, 15, 34].
Summing up, the most pronounced typical pathological processes are observed in the late periods after irradiation of 56Mn then studying histostructural processes occuring in the tissues of the studied animal organs after exposure to neutron-activated manganese dioxide and external irradiation identified by the totality of morphometrical indicators.
Conclusion
The most prominent histologic picture characterized by presence the signs of inflammation and degeneration on the 3rd and 14th day, in particular, in rats exposed to 56Mn compared to rats from 60Co groups. Our research results and their comparison with literature data leds to the conclusion that majority of experimental animals exposed to p- and y-radiation more pronounced changes were observed on the 60th day after exposure consisting the appearance of chronic inflammation, signs of degeneration and necrotic foci. Consequently, like 60Co, 56Mn also promotes activation of inflammatory processes and stimulation of immune responses manifested by cellular infiltration.
Although whole-body radiation doses from 56Mn was relatively low internal doses were noted
in the small intestine, in addition to significant pathological changes that more severe and prolonged than 60Co Y-irradiation effects. These data may indicate the potential for a high risk of internal exposure to 56Mn which would have existed in airborne dust after atomic bomb explosions in Hiroshima and Nagasaki.
Thus, our data obtained from in vivo experiments provide strong evidence that в-radiation causes formation of morphologic features which typically for radiation enteritis that's a form of small intestinal injury depending on radiation type.
Interest conflict:
All authors declare that partial results of this experiment were described in the paper «Comparative characteristics of histomorphologic changes in the small intestine of rats exposed to gamma- and neutron radiation» from «European Journal of Natural History». 2017. №4. P. 38-42.
Authors contributions:
Uzbekov D. - the practical implementation of all phases of the experiment;
Shabdarbaeva D. - histological analysis and interpretation of morphometric data;
Chaizhunusova N. - administrative, technical and material support;
Almisaev K. - the practical implementation of histological staining;
Uzbekova S. - statistical analysis;
Saporov R. - the practical implementation of rats necropsy;
Apbasova M. - collection of literature review;
Hoshi M. - development of methodology.
The study was conducted according to the scientific project: «Long-term effects of internal exposure at different levels of the body: a multicenter experimental study using a nuclear reactor».
Funding for the project was carried out by Semey State Medical University.
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Correspondence:
Uzbekov Darkhan - PhD, assistant of Department of Pathological anatomy and Forensic medicine of Semey Medical University, Semey, Kazakhstan.
Address: East Kazakhstan region, 071400, Semey city, Shakarim street, 13 A - 72. Phone: 87222420532, +77055301026 E-mail: [email protected]
