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ТЕОРЕТИЧЕ ( КАЯ И ЭКСПЕРИМЕНТАЛЬНАЯ МЕДИЦИНА
© КОЛЛЕКТИВ АВТОРОВ, 2017 UDC: 616.681-007.21:614.876
D. Ye. Uzbekov1, M. Hoshi2, K. Shichijo3, N. Zh. Chaizhunusova4, D. M. Shabdarbayeva1, N. B. Sayakenovi, Y. O. Kairkhanova5, A. Zh. Saimova5, S. A. Apbasova1
COMPARATIVE CHARACTERISTICS OF HISTOMORPHOLOGIC CHANGES IN THE LUNG OF RATS EXPOSED TO GAMMA- AND NEUTRON RADIATION
department of pathological anatomy and forensic medicine of Semey state medical university (Semey, Kazakhstan),
2Research Institute for Radiation Biology and Medicine (Hiroshima, Japan), 3Atomic Bomb Disease Institute (Nagasaki, Japan),
4Department of nutrition and hygienic disciplines of Semey state medical university (Semey, Kazakhstan), 5Semey state medical university (Semey, Kazakhstan)
It is known that persons exposed to ionizing radiation, together with a different of damaging effects, particular importance is also attached to the respiratory system. The dominant role of neutron-activated radionuclide Manganese-56 (56Mn) was noted in the treatises of Japanese scientists who studied the A-bomb effects of Hiroshima and Nagasaki, deserving the interest today. In this regard, the research purpose was to study the microscopic changes in the lung of rats exposed to y- and neutron radiation.
In experiment, both sexes «Wistar» rats in amount of 36, weighting approximately 220-330 g. Necropsy of the animals were on the 3rd, 14th and 60th days after irradiation, then the lung removed, after which it was fixed in 10% formalin. Tissues fragments embedded in paraffin, then sections are manufactured serial transverse 4 mm thickness, which were subsequently stained by hematoxylin and eosin (H&Е). Our experimental studies have shown that the majority of animals exposed to 56Mn and 60Co starting from the 2nd week observed signs of fibrosis and chronic inflammation, foci of emphysematous dilated alveoli, whereas after inhalation by MnО2 in rats prevailed thickening of interalveolar septa due to cellular elements. Foci of hemosiderosis, signs of vasculitis, lymphadenitis and hyalinosis were typically for neutron-irradiated rats.
Thus, 56Mn effect on the rat lung revealed a high risk of neutron radiation, which confirmed the presence of inflammatory processes. The findings support a role of ionizing radiation in the formation of morphologic signs depending on both dose and type of radiation.
Key words: ionizing radiation, pulmonary tissue, morphologic changes, pneumonitis, fibrosis, rats
It is known that among the neutron-induced radioisotopes 56Mn is a dominant element found after an A-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 radiation. People who returned early to Hiroshima and Nagasaki after A-bombing were reported to suffer from the symptoms of acute radiation effects [16]. Currently, particular interest represents a comparative assessment of pulmonary morphologic changes in persons exposed to 60Co and 56Mn [10]. Most diseases attributable to radiation in A-bomb survivors and nuclear reactor workers are lung pathology [16]. Pulmonary damage was identified as an important sphere of interest in radiation research after a significant number of sacrifice died early from radiation induced lung injury (RILI) [3]. Scientists are continuing to develop the mitigators for radiation injuries to the lung caused by a single dose of ionizing radiation at doses pertinent to a radiologic attack or nuclear accident. Therefore, animal models that reproduce radiation injuries in humans are necessary [5]. Average or se-
vere RILI significantly exerts on patients life quality, and may even lead to death. In spite of decennary of studying the morphogenesis for lethal RILI at the organ level remain insufficiently defined [3].
Objective of the study - to identify and compare the microscopic changes in the rat lung after exposure by single dose of y-radiation and neutron-activated 56Mn powder.
MATERIALS AND METHODS
For this study, it was purchased and raised in a specific-pathogen-free facility six-month-old both sexes «Wistar» rats in an amount of 36 with mean whole body weight 220-330 g. All experimental animals were acclimatized for 2 weeks before initiation of experiments and kept under normal conditions and fed pellets concentrated diet and vitamin mixtures. They were maintained at constant temperature (22±1°C) on 8 hour light -dark cycle.
The rats were allocated into 4 groups. The first group of animals (n=9) were subjected to 56Mn which was obtained by neutron activation of 100 mg of manganese dioxide (MnО2) powder using nuclear reactor («Baikal-1») with neutron
Figure 1 - Photomicrograph of uniform airiness in control rat lung (a).
flux 4x1014 n/cm2. Activated powder with total activity of 56Mn 2.75x108 Bq was sprayed pneumatically over rats placed in the special box. The moment of exposition beginning of experimental animals by 56Mn powder is 6 minute after finishing of neutron activation. The second group of rats (n=9) were exposed to not irradiated Mn02. The spray powder was carried out in a chemical box, which contained boxes of 9 rats. Each portion of Mn02 was sprayed in box with lots of biologic objects. Then unirradiated powder and incubated biologic objects in a container for hour. The third group of rats (n=9) were irradiated with a single dose of 2 Gy was performed at a dose rate of 2.6 Gy/min using 60Co Y-ray by czech radio-therapeutic device «Teragam K-2 unit». During the exposure, animals were placed in a specially engineered cage made of organic glass with individual compartments for each rat. After irradiation, rats were taken back to the animal facility and routinely cared. The fourth group consisted of control rats (n=9) which were placed on shelves in the same facility and shielded from radiation. All animal procedures were approved by Ethical Committee of Semey State Medical University, Kazakhstan (Protocol №5 dated 16.04.2014) in accordance with Directive of the European Parliament and the Council on the Office in animals protection.
The rats were sacrificed on the 3rd, 14th, 60th day after irradiation and the lung was immediately surgically extracted for further histologic study. The lung sections were deparaffinized and dehydrated in graded 10% formalin solutions. Paraffin sections performed with 4 mm thickness. Stained by hematoxylin-eosin (H&E) slide glasses were examined under a Leica DM 1000 microscope (Germany) and images were captured with
a charge-coupled device camera (Visitron Systems, Puchheim, Germany) at x10 and x40 magnifications. Qualitative histological assessment of lung injury was carried out to obtain an overall damage severity result.
RESULTS AND DISCUSSION
In the present study, we performed experiment with neutron-activated 56Mn powder exposed rats. It was previously reported the internal dose estimates in organs of 56Mn-exposed rats. The highest doses were recorded in the lung [13].
The study of lung components in control rats revealed an uniform airiness of pulmonary tissue, shown on figure 1 a. In the lung parenchyma of animals inhaled Mn02, starting from the 2nd week reveals changes in the vascular walls with signs of productive vasculitis. Status of the pulmonary parenchyma is characterized by focal emphysema moderate severity. Intra-alveolar septa are thickened due to lymphocytes, alveolar macrophages and less number of neutro-phils. Changes in the structure of the interstitial tissue are manifested by presence of inflammatory foci (fig. 1 b).
Microscopic changes in the lung tissue of Mn02-inhaled rat (b). H&E staining, original magnification xl0
Research of different pathologists established that radiation causes tissue damage via sensitization of autoreactive lymphocytes which react with lung tissue [18]. On the 3rd day after y -radiation exposure, authors registers the inflammatory response manifested by moderate of cellular infiltration of interstitial lung tissue [7]. The above data are consistent with our study results the lung tissue in 60Co-exposed rats showed a similar changes.
TeopeTHqecicag h экспернмента^bнаg меaнцнна
Figure 2 - Light microscopy of 56Mn-induced (a, b, c) magnification xio and x40
According to histologic examination most pronounced changes were observed in the lung of 56Mn-exposed rats, indicating that neutron radiation has a significant biologic effect on examined organ. In experimental animals exposed to 56Mn and 60Co were observed thickening of intraalveolar septa in virtue of leucocytes, erythrocytes, lymphocytes, histiocytes, alveolocytes, and rupture of the intraalveolar septa [10].
Figure 2 a shows an uneven blood filling with a predominance of capillary-venous plethora. It is necessary to pay attention to changes that occur in the bloodstream characterized by presence of erythrocytes in vascular lumen. Distruc-tion of pulmonary parenchyma manifested by formation of fibrosis. It has been observed pronounced bronchitis with coverage of the lung parenchyma and lymph node, bronchial lumen contains stratum of desquamated ciliated epithelium,
Y-ray-induced rat lung (d). H&E staining, original
a small amount of mucus strands. It is also noted transition the inflammation to peribronchial tissue. By magnificating x40, can be traced diape-detic microhemorrhage, several small focal intra-alveolar hemorrhage of intensive red color with a small amount of leukocytes. Foci of acute alveolar emphysema varying degree of severity and prevalence with a thinning and defects of intraalveolar septa. In some fields of weak-moderate congestion hemosiderophages (fig. 2 b, c), observed on the 14th day in rats exposed 56Mn. Comparative characteristics of changes in the lung after influence of Y- and neutron radiation showed uniformity of manifestation. Microscopic picture of animals on the 2nd week after 60Co exposure provided on figure 2 d, where we are noted signs of thickening of the vascular walls, as confirmed by research which describes pulmonary vasculopathy after a single dose of Y-irradiation rats [8]. Considerable importance should be given
Figure 3 - Histologic sections of the pulmonary magnification x10
to presence of the thickened walls of the interstitial tissue, alternating with emphysematous changed portions of the lung, presented keenly dilated alveoli. Mostly intra-alveolar septa are thickened. They determined cell infiltrates. Peribronchial infiltration is expressed. The sections are represented mainly by medium caliber bronchi which in a state of mild dystonia, a subtotal desquamation of ciliated epithelium and signs of mild mucus hypersecretion.
After radiation exposure in rat lungs on the 2nd month were found small focal mild hemorrhage, cellular response in the form of leukocyte infiltration and fibroblast proliferation (fig. 3 a).
Microscopic changes also characterized by presence of vasculitis, lymphadenitis, hyalinosis and peribronchial cell infiltration. According to our research all of above changes were typically for rats irradiated 56Mn (fig. 3 b).
In this study, we have shown the sequence of morphologic changes in the rat lungs from early to late stage after a single influence of 56Mn and 60Co at 2 Gy dose, which were the initiators of radiation pneumonitis related to fibrogenesis. Results of morphologic studies have shown that structural changes in the lung observed in irradiated rats little differed from the previously published results using different radiation models. It should be noted that the most prominent microscopic changes were detected in rats exposed to 56Mn after 2 weeks.
Currently, great importance is attached to the role of Mn, inducing apoptosis or necrosis. Thus, in the literature we have examined papers revealed regarding ability of Mn to cause histo-
in rats exposed to 56Mn. H&E staining, original
morphologic changes in the lung tissue of animals [17]. In one of the papers, authors showed that the intoxication by Mn steams leads to distruction of the pulmonary parenchyma, the development of manganese pneumonia and lung edema [9], which corresponds to the data obtained in our experiment.
Identified us the signs of pneumonitis and fibrosis have been well described in models of irradiated rats [14]. According to the literature, the early phase after acute lung injury characterized by exudative inflammation complicated by interstitial pneumonia after few weeks postradiation. The later phase is characterized by chronic inflammation and tissue restructuring [2]. There are studies indicating that accumulation and activation of monocytes, macrophages and lymphocytes are a key component of RILI [11]. Radiation damage contributes to the development of chronic inflammatory processes that may predispose irradiated individuals to the formation of lung fibrosis. Although the pathogenesis of radiation-induced lung fibrosis at the molecular and cellular levels is not completely understood, initial immune and inflammatory responses to repeated irritants lead to tissue damage and progressive fibrosis [14]. Histologic analysis of irradiated lungs showed the fibrosis development by progressive collagen deposition after several month, which characterized by formation of typical fibroblast foci and accompanied by a severe second outbreak of leukocyte infiltration. The later fibrogenesis phase characterized the fibrotic foci evolved and grow together into widespread fibrosis with the pulmonary architectonics remodelling [12]. During granula-
tissue
TeopeTuneciag h экспернмента^bнаg MejHUHHa
tion growth and tissue repair, pulmonary interstitial fibrosis occurs in the late phase of RILI after fibroblast cell proliferation and collagen synthesis, inducing the development of respiratory failure, which is one of the major causes of death [15]. Our findings of pulmonary tissue changes in irradiated rats consistent with conclusion of some authors that marked thickening of alveolar walls and dilatation of intralobular septa, and also sealed foci of fibrosis, mainly in intralobular septa [6].
In contrast to the external y-radiation, internal 56Mn radiation contributes to the development of pathologic changes manifested by formation of foci of hemorrhage and emphysema [10]. The emphysema is characterized by dilatation of air space and destruction of the alveolar walls. Scientists have shown that the combination of lung fibrosis and emphysema have bad prognosis similar to idiopathic pulmonary fibrosis [4].
Our results showing about multifocal alveolar reactive changes, such as edema and moderate increase the number of cells in the alveolar walls, and also interstitial inflammation in the pulmonary tissue confirmed already known fact regarding Y-radiation effect to organism. These changes are most often found in the form of a thickening of the alveolar and vascular walls, sclerosis on the 3rd month after exposure [1].
Summing up, irradiation of normal lung tissue has two generally recognized adverse effects, including inflammatory and fibrotic processes [12]. These histomorphologic changes in the lung of rats exposed to Y- and neutron radiation make it possible to develop diagnostic criteria for assessing of radiation effect on the lung, depending on cumulative dose.
CONCLUSION
Our research results and their comparison with literature data led to the conclusion that majority of experimental animals exposed to Y- and neutron radiation more pronounced changes were observed from the 2nd week after exposure, consisting the appearance of fibrosis and chronic inflammation, foci of emphysematous dilated alveoli, whereas after inhalation by Mn02 in rats dominated thickening of intra-alveolar septa on account of leukocytes, erythrocytes, lymphocytes, histiocytes and alveolocytes. Consequently, like y-rays, 56Mn also promotes activation of inflammatory processes and stimulation of immune responses manifested by cellular infiltration. It should be noted that the most prominent histo-logic picture characterized by presence of hemo-siderosis foci, signs of vasculitis, lymphadenitis and hyalinosis were found, in particular, in rats exposed to 56Mn.
Thus, experimental studies confirm the role of neutron radiation in the formation of morphologic features which typically for radiation pneumonitis, that is a form of acute or subacute lung injury, depends on both dose and type of radiation.
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Received 26.04.2017
Д. Е Узбеков1, М. Хоши2, К Шичиджо3, Н. Ж. Чайжунусова4, Д. М. Шабдарбаева1, Н. Б. Саякенов1, Ы. О. Кайрханова5, А. Ж. Саимова5, С. А. Апбасова1
СРАВНИТЕЛЬНАЯ ХАРАКТЕРИСТИКА ГИСТОМОРФОЛОГИЧЕСКИХ ИЗМЕНЕНИЙ В ЛЕГКИХ КРЫС, ПОДВЕРГАВШИХСЯ ГАММА- И НЕЙТРОННОМУ ОБЛУЧЕНИЮ
1Кафедра патологической анатомии и судебной медицины Государственного медицинского университета г. Семей (Семей, Казахстан),
2Научно-исследовательский институт радиационной биологии и медицины/ (Хиросима, Япония), зИнститут по изучению заболеваний последствий атомной бомбардировки (Нагасаки, Япония), 4Кафедра питания и гигиенических дисциплин Государственного медицинского университета г. Семей (Семей, Казахстан),
5Государственный медицинский университет г. Семей (Семей, Казахстан)
Известно, что у лиц, подвергавшихся воздействию ионизирующего излучения, наряду с различными повреждающими эффектами особое место отводится дыхательной системе. Доминирующая роль нейтронно-активированного радионуклида (Марганца-56 (56Мп) отмечалась в трудах японских ученых, изучавших последствия атомной бомбардировки в Хиросиме и Нагасаки, и представляет научный интерес по сей день. В связи с этим целью исследования явилось изучение микроскопических изменений в легких крыс, подвергавшихся воздействию Y- и нейтронного излучения.
В эксперименте использованы крысы обоих полов линии «Вистар» в количестве 36 особей, массой 220330 гр. Животных подвергали некропсии через 3, 14 и 60 сут после облучения, затем извлекали легкие, после чего фиксировали в 10% формалине. Фрагменты тканей заливали в парафин, затем изготовливали поперечные серийные срезы толщиной 4 мкм, которые в дальнейшем окрашивали гематоксилином и эозином. Результаты экспериментального исследования показали, что у большинства животных, подвергнутых воздействию 56Мп и 60Со, начиная со 2 недели отмечались признаки фиброза и хронического воспаления, очаги эмфизематозно расширенных альвеол, тогда как после ингаляции МпО2 в легких крыс наблюдалось утолщение межальвеолярных перегородок за счет клеточных элементов. Очаги гемосидероза, признаки васкулита, лимфаденита и гиалиноза преобладали у крыс, подвергавшихся воздействию нейтронного излучения.
Таким образом, изучение воздействия 56Мп на легкие крыс выявило высокий уровень риска облучения, что подтверждено наличием воспалительных процессов. Полученные данные подтверждают роль ионизирующего излучения в формировании морфологических признаков, зависящих как от дозы, так и от типа излучения.
Ключевые слова: ионизирующее излучение, легочная ткань, морфологические изменения, пневмонит, фиброз, крысы
Теоретическая и экспгримгнтaльнaя мгдицинa
Д. Е. Узбeкoвl, M. Xoши2, К. Шичиджoз, Н. Ж. Чaйжyнycoвa4, Д. M. Шaбдapбaeвal, Н. Б. Caякeнoвl, Ы. O. Kaйpхaнoвa5, A. Ж. Caимoвa5, C. A. An6acoBai
ГДMMД-MEH НЕЙТРОНДЫ CeУЛE 6CEPIHE ¥ШЫРДРДН ЕГЕУК¥ЙРЫКТДР еКПЕШДЕП ГИаШОРФОЛОГИЯЛЫК еЗГЕР10ТЕРД1Ц CДЛЫCTЫРMДЛЫ CИПДTTДMДCЫ
^вмвй мвмлвквттк медицин yниввpcиrвriнiн ^плаг^лы;; aнaтoмия жэнв cor мeдицинa a^eipao^/ (Овмвй, К^к^н),
2Рaдиaциялы;к биалагия жэнв мвдицинa fыlлыlми-звpттвy инoтитyтыl (Xиpаcимa, Жaпания), зДюм бамбaыl эpвквriнвн тyыlндaFaн ыlpкaттapдыl звpтrвy ингитуты/ (^aca/ai, Жапaния), 4 Овмвй мвмлвквттк мвдицин yниввpcиreriнiн raFaмraнy жэнв гигивнлык пэндвp ^(p^pact^/ (Овмвй, ^aca^,
50вмвй мвмлвквттк мвдицин yниввpcиrвri (Овмвй, Kaзaкcraн)
ИoндaFыш cэyлe acepte дyшap бoлFaндapдын кeптeгeн бYлiндipгiш caлдapымeн ;oca rb^bic^y xY^ec^e дв epeкшe мэн бeлiнeдi. Xиpocимa мвн HaгacaкидaFы aтoмдык бoмбaлayдын caлдapын зepттeгeн жaпoн Faлымдapынын eнбeктepiндeгi нeйтpoнды-бeлceндi Mapгaнeц-56 (56Mn) paдиoнyaлидiнiн бacым peлi зaмaнayи жaFдaйдa !a кызыFyшылык apттыpaды. ОcыFaн CYЙeнe oтыpып, y- мвн нeйтpoнды cэyлe acepte vшыpaFaн eгeyкvйpыктapдын eкпeciндeгi микpocкoпиялык eзгepicтepдi зepттey мaкcaтымыз тyындaды.
Tэжipибe жYЗiндe <^crap» тукымды 220-ЗЗ0 гp caлмaFЫ бap aтaлык жэнв aнaлык жыныcты З6 eгeyкvйpык пaйдaлaнылFaн. ЖaнyapлapFa cэyлeлeyдeн кeйiн 3-шi, 14-шi жэнв 60-шы тэyлiктepдe нeкpoпcия жYpгiзy бapыcындa ernec^ aлып, 10%-™ фopмaлиндe фикcaциялaдык. Тм фpaгмeнттepiн пapaфингe куйып, кaлындыFЫ 4 мкм галдвнвн cepиялык кeciндiлep дaйындaп, 9pi кapaй гeмaтoкcилин мвн эoзинмeн (H&E) бoядык. Tэжipибeлiк зepттey нэтижeлepi 56Mn мвн 60Co эcepiнe vшыpaFaн жaнyapлapдын кeпшiлiгiндe 2-шi aптaдaн кв^н фибpoз бвн coзылмaлы кaбынy бeлгiлepiн, aльвeoлaлapдын эмфизeмaтoзды квнвю oшaктapы бacым бoлFaнын, aл MnО2 ингaляцияcынaн кв^н eгeyкvйpыктap eкпeciндe жacyшa элeмeнттepiнiн эcepiнeн aльвeoлa apaлык пep-дeлepдiн к^ын^ы^ кepceттi. Гeмocидepoз oшaктapы, вacкyлит, лимфaдeнит пвн гиaлинoз бeлгiлepi нeйтpoн-ды cэyлeлeнy эcepiнe шaлдыккaн eгeyкvйpыктapFa тэн бoлды.
Coнымeн, eгeyкvйpыктapдын eкпeciнe 56Mn scepi кaбынy YPДicтepiмeн pacтaлaтын cэyлeлeнy кayпiнiн жoFapы дeнгeйiн кepceттi. 3ep"iTey нэтижeлepiнe caй иoндaFыш cэyлe эcepiнeн тyындaйтын мopфoлoгиялык eзгepicтepдiн cипaты cэyлeлeнyдiн дoзacы мвн тYpiнe бaйлaныcты дaмиды.
Клт ceздвp: иoндaFыш cэyлe, erne тiнi, мopфoлoгиялык eзгepicтep, пнeвмoнит, фибpoз, eгeyкvйpыктap
