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Резюме Резюме Summary 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. 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
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 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. | |||||||||||||||||||||||||||||||||
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