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Свидко Е.Н., Демин Ю.А. Морфологическое подтверждение экспериментальной модели лимбальной клеточной недостаточности
27.05.2014, 14:03

Резюме
Свидко К.М.1, Дьомін Ю.А.2 Морфологічне підтвердження експериментальної моделі лимбальної клітинної недостатності.
У даній роботі доведена практичність використання експериментальної моделі лімбальної недостатності з використанням мітоміцина С, встановлений час його виведення з рогівки кроликів – 26,2 години. Морфологічно підтверджено відсутність повноцінної регенерації рогівки при створеній недостатності клітин лімба.
Ключові слова: експериментальна модель, лімбальна недостатність, мітоміцин С, передній епітелій рогівки.

Резюме
Свидко Е.Н., Демин Ю.А. Морфологическое подтверждение  экспериментальной модели лимбальной клеточной недостаточности.
В данной работе доказана практичность использования экспериментальной модели лимбальной недостаточности с использованием митомицина С, установленное время его выведения из роговицы кроликов - 26,2 часы. Морфологически подтверждено отсутствие полноценной регенерации роговицы при созданной недостаточности клеток лимба.
Ключевые слова: экспериментальная модель, лимбальная недостаточность, митомицин С, передний эпителий роговицы.

Summary
Svidko K.M., Dyomin U.A. Morphological confirmation of experimental model of limbal stem cells deficiency.
In this research practical using of experimental model of limbal cells deficiency with mitomycin C was proved. Period of elimination of mitomycin C from rabbit's cornea was 26.2 hours. Absence of a complete regeneration of the cornea after creation of limbal stem cells deficiency was morphologically confirmed.
Key words: experimental model, limbal stem cells deficiency, mitomycin C, anterior epithelium of the cornea.

Рецензент: д.біол.н., проф. І.О. Іванюра

УДК 617.713-092.4.068

1Институт проблем криобиологии и криомедицины НАН Украины (Харьков)

61015, Харьков, ул. Переяславская 23

Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine

23, Pereyaslavskaya str., Kharkov, Ukraine 61015

2Харьковская медицинская академия последипломного образования

61176, г. Харьков, ул.Корчагинцев, 58

Kharkov medical academy of Postgraduate Education

61176, Kharkov, 58 str.Korchahyntsev

v.teryshin_lsmu@mail.ru

Currently, the role of limbal eye area in corneal regeneration and self-renewal   during life is proved. It was found that in the basal layer of the limbal epithelium exist cells with properties of the stem cells. During studies it was confirmed that the localization of stem cells is in the limbal zone and these cells have high proliferative capacity and longer period of existence in comparison with the central corneal cells [6, 8, 9]. Diseases of the eye, especially chronic, are characterized by the involvement of limb zone cells in the pathogenesis [3, 7, 10]. We can see  decline in number of the limbal stem cells, deterioration of the limbal cells' microenvironment due to the lack of specific growth regulators such as epithelial growth factor, transforming growth factor, a variety of neurotrophic growth factors.
It is required an experimental model for the study of alternative treatment of the limbal stem cells deficiency. The most using model of the limbal stem cells deficiency is the model with 0.04% mitomycin C [2]. However, it is known that mitomycin C has cytotoxic activity [12], so it is necessary to know the period of drug's illumination from the animal’s cornea for obtaining right and truthful results.

The aim of our study was to determine the mitomycin C priod of illuminatin in animal's cornea, and research changes, that were caused during establishing of the experimental model.

Materials and methods. In experiment took part 13 rabbits (26eyes), breed "Chinchilla", weight 4-5 kg.  During working with animals "General ethical principles of experiments on animals" were taken into account [4]. To investigate the mitomycin C period of elimination were taken 3 rabbits (6 eyes) with the same diameter of the cornea (10 mm). Ultrasound pachymetry with Pachymeter Ocuscan (company Alcon) was performed for each animal. At first filter paper discs (diameter 10 mm) with 10% ethanol solution were put on the cornea for 20 seconds. Then necrotic cells of the corneal epithelium were removed with microtupfer. For control was used 1% solution of fluorescein.

For preparation of the test drug solution 2 mg of mitomycin C was used. The contents of the vial were dissolved in 5 ml of purified water (concentration of mitomycin C - 0.04%). Then filter paper was cut in 10 mm diameter circles. On the circle with dimensional auto-sampler was applied 7mkl of previously prepared mitomycin C solution. Paper circles were carried out on the cornea during different time intervals: 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes. After exposure paper circle was placed in 20 ml bottle and sealed with a rubber stopper. To each bottle with the same pipette class 1 was added 2 mL of methanol. Then bottle was placed for 5 minutes in the ultrasound bath. Quantitative determination of mitomycin C in the samples was performed by using liquid chromatography with diode matrix detection. As analytical wavelength was used maximum of mitomycin C absorption at 360 nm. In this way sufficiently high selectivity of the signal in conjunction with a low detection threshold of material in the samples was provided.

To study morphological features of the limbal stem cells deficiency 10 rabbits were selected.  Model of the limbal stem cells deficiency was created. 

Animals were taken from the experiment under anesthesia by air embolism at 3rd and 7th days. Fixing of the cornea was performed in 10% neutral formalin aqueous solution, then it was degreased in alcohols with increasing concentration and put in paraffin [1]. Paraffin blocks were sectioned in 3-4microns layers, which were stained with hematoxylin-eosin. Microscopic studies were carried out using a microscope BIOREX 3 "KONUS". Photomicrography - with a microscope company BIOREX 3 "KONUS" with adapted programs for this study.

Results and discussion: During this study we obtained the results of quantitative elimination of mitamicin C in samples (Table 1).

Table 1

N

Concentration, %

Pick area

Exposure time, mins

  1.  

0,03971519

703648

0,5

  1.  

0,03715364

658264

1

  1.  

0,03551022

629147

2

  1.  

0,03221181

570708

3

  1.  

0,03001634

531810

4

  1.  

0,03001182

531730

5

  1.  

0,04

708694

0

 

The diffusion rate (v) was 1,99764 E-05 g per minute, 19.97635 mg per

minute.

Volume can be calculated by the standard formula: V=2pR

And then elimination time of mitamycin C from the cornea t=V/v

t= 2pR/v=2*3,14*0,021/1,99764E-05=26,2 hours

From this study we can make conclusion that after this period it is possible to use the cryopreserved cord blood cells.

During histological studies on the third day we observed  a defect of anterior epithelium (80% of histology). The epithelium is thin, it’s surface is uneven, epithelial cells are arranged in 1-2 rows (Pic. 1). On the periphery of the cornea, close to the limb, more number of cell layers were observed. Epithelial cells had irregular hyperchromatic nuclei and edematous, optically clear cytoplasm. Different layers of cells were not different morphologically, so it was impossible to distinguish the layers of anterior epithelium.

Рic. 1. Animal’s cornea on the 3rd day after creating of the experimental limbal stem cells deficiency. A thin layer of the anterior epithelium. Hematoxylin and eosin. x10

During the preparation of histology epithelial layer was easily peeled from the basement membrane (Pic. 2).

In the stroma of the cornea there was a significant edema, which led to separation of collagen fibers and appearance of the gaps between them. Keratocytes were elongated, with hyperchromatic nuclei. Migration of the segmented leukocytes more on the periphery were observed, indicating the intensity of inflammation.

Entocornea was presented by dense layer of arranged collagen fibers.

In the back epithelium was visible a single layer of cells with irregular polygonal shape. They had an oval nucleus, clear cytoplasm. On histology was a significant increase in cell size due to swelling of the cytoplasm. The contour of the endothelium was uneven, wavy.

Pic. 2. Cornea animals on the third day after creating of the model of limbal stem cells deficiency. Front epithelium partially flaked off from the basal membrane. Hematoxylin and eosin. x10.

On the 7th day epithelialization of the cornea was showed. During detailed study it was observed violation of differentiation of the corneal epithelium. The thickness of anterior epithelium was uneven: more on the periphery than in the center. Epithelium was stratified, and here were changes in arrangement of cells, that are usual for anterior epithelium of the cornea; goblet cells were found, that are usual  for epithelium of the conjunctiva. On the periphery conjunctival epithelium grew on the corneal surface like a shaft. Surface of the cornea was uneven.

Corneal stromal edema was decreased, collagen fibers, combined into bundles, were oriented parallel to each other. Keratocytes with basophilic nuclei were observed between them, the long axis of which was located parallel to the surface of the cornea. Ammount of the segmented leukocytes in the surface layers of the corneal stroma significantly decreased/ Attenuation of the inflammatory process was observed. In the corneal stroma there was isolated vessels. Endothelial cells had flattened basophilic nuclei and slightly swollen eosinophilic cytoplasm. Around blood vessels single lymphocytes were visible. Entocornea was not changed. Cells of the posterior epithelium had oval basophilic nuclei and light oxiphylic cytoplasm. There was a significant reduction of cytoplasm swelling.

Сonclusions
1. It was found that the most simple and reliable experimental model of limbal stem cells deficiancy is a model with 0.04% mitomycin C.

2. Experimentally it was proved that elimination period of mitomycin C from rabbit's cornea is 26.2 hours. Due to this we can use this model in researches with   cryopreserved cord blood cells.

3. Histology on the 7th day showed changes in arrangement of anterior epithelium cells, germination of the vessels, goblet cells were found that are usual  for epithelium of the conjunctiva. According to this features we can say that it was no  full regeneration of the cornea and limbal stem cells deficiency were appeared.

 

Литература

1. Микроскопическая техника: руководство / Под. ред. Д.С. Саркисова, Ю.Л. Перова. – М : Медицина, 1996. – 544 с.
2. Милюдин Е.С. Экспериментальная модель недостаточности регио-нальных стволовых клеток роговичного эпителия / Е.С. Милюдин // Вестник СамГУ. Естественнонаучная серия. – 2006. – № 9 (49). – С. 219–226.
3. Получение трехмерного трансплантанта лимбальных клеток роговицы / Н.В. Пасечникова, В.К. Гринь, Г.И. Дрожжина [и др.] // Вестник неотложной и восстановительной медицины. – 2012. – Т. 13, № 1. – С. 99–102.
4. Рєзников О.Г. Загальні етичні принципи експериментів на тваринах / О.Г. Рєзников // Ендокринологія. – 2003. – Т. 8, № 1. – С. 142–145.
5. Сухинин М.В. Реактивные изменения переднего эпителия роговицы при повреждениях ростковой зоны лимба глаза / М.В. Сухинин, В.Г. Гололобов, И.В. Гайворонский // Фундаментальные проблемы гистологии, гистогенез и регенерация тканей. – СПб.: ВМедА, 2004. – С. 136-137.
6. Трансплантация стволовых клеток лимба при патологии роговицы / Г.К. Мухамеджанова, Б.О. Сулеева, И.Е. Ольшевский [и др.] // Новые технологии в лечении заболеваний роговицы : Всероссийская научно-практич. конф. «Федоровские чтения-2004»: тезисы докл. – М., 2004. – С. 490–495.
7. Ченцова Е.В. Лимбальная клеточная недостаточность / Е.В. Ченцова, Л.Р. Николаева // Вестник офтальмологии. – 2006. – № 3. – С. 43–47.
8. Characterisation of extracellular matrix components in the limbal epithelial stem cell compartment / U. Schlotzer-Schrehardt, T. Dietrich, K. Saito [et. al.] // Exp Eye Res. – 2007. – Vol. 85 (6). – P. 845–860.
8. Grueterich M. Human limbal progenitor cells expanded on intact amniotic membrane ex vivo / M. Grueterich, S.C. Tseng // Arch. Ophthalmol. – 2002. – Vol. 120 (6). – P.783–790.
9. Histopathology of human corneas after amniotic membrane and limbal stem cell transplantation for severe chemical burn / J. Stoiber, W.H. Muss, G. Pohla-Gubo [et. al.] // Cornea. – 2002. – Vol. 21 (5). – P.482–489.
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