Spleen Morphological Features Under Experimental Model of Multiple Organ Failure and Stem Cell Application

Надійшла 26.01.2018 Прийнята до друку 19.02.2018 Received January, 26, 2018 Accepted February, 19, 2018 Multiple organ failure syndrome (MOFS) is a complex of pathological changes, resulting in terminal stage of functioning in many organs, being characterized by such a degree of organ damage, when they are unable to support the body life. To date there are no clear clinical guidelines and efficient therapeutic products to treat MOFS consequences have been designed. In the experiment we developed the model of systemic damage of a body with tetrachloromethane [4], based on reproducting of oxidative stress, which caused MOFS development in human [6], being accompanied by pathological changes in liver, lungs, kidneys and spleen [1, 5]. At current state of medicine one considers mesenchymal fibroblast-like stem cells to be promising in correction of various pathologies because of their multipotency, capability of differentiating into cells of mesodermal organs and a simple procurement from different sources [3]. There are the reports about a successful use of mesenchymal stem cells to treat liver cirrhosis, cicatricial changes in myocardium [3]. Now, there is only scarce information about morphological changes in spleen under MOFS and stem cell application for splenic function restoration. In this context the research aim was to study the morphological features of spleen under experimental simulation of MOFS and stem cell administration.

Multiple organ failure syndrome (MOFS) is a complex of pathological changes, resulting in terminal stage of functioning in many organs, being characterized by such a degree of organ damage, when they are unable to support the body life. To date there are no clear clinical guidelines and efficient therapeutic products to treat MOFS consequences have been designed.
In the experiment we developed the model of systemic damage of a body with tetrachloromethane [4], based on reproducting of oxidative stress, which caused MOFS development in human [6], being accompanied by pathological changes in liver, lungs, kidneys and spleen [1,5].
At current state of medicine one considers mesenchymal fibroblast-like stem cells to be promising in correction of various pathologies because of their multipotency, capability of differentiating into cells of mesodermal organs and a simple procurement from different sources [3]. There are the reports about a successful use of mesenchymal stem cells to treat liver cirrhosis, cicatricial changes in myocardium [3]. Now, there is only scarce information about morphological changes in spleen under MOFS and stem cell application for splenic function restoration.
In this context the research aim was to study the morphological features of spleen under experimental simulation of MOFS and stem cell administration.
Отримані цифрові дані обробляли за допомогою статистичного пакету «GNU PSPP» («Free software foundation», США) із перевіркою на нормальність розподілу за критерієм Колмогорова-Смирнова, а також критерієм Крускала-Уолліса, для порівнянням ознак groups. The control group (n = 5) comprised the animals with intravenous injection of 0.3 ml of 0.9% NaCl, with no pathology simulation. In animals of experimental group 1 (n = 20) we simulated a multiple organ failure via intraperitoneal administration of 0.3 ml of a 30% tetrachloromethane oil solution twice a week for 12 weeks. Immediately after completing MOFS simulation (intraperitoneal administration of 0.3 ml of a 30% tetrachloromethane oil solution twice a week for 12 weeks) the mice of experimental group 2 (n = 20) were intravenously injected with 1 × 10 4 embryonic mesenchymal fibroblast-like stem cells of ICR mice, GFP gene carriers.We used here just the mentioned dose of mesenchymal fibroblast-like stem cells of ICR mice, since their higher concentration was proven to be inexpedient in previous findings [2].
The primary cell culture was derived from soft tissues of 15-17-day murine embryos. The tissue fragments were kept in Penicillin and Streptomycin solution, treated with trypsin, resuspended and inoculated in DMEM. The cells from cultures not older than passage 2 were used in the experiment.
In 3 and 9 weeks after stem cell administration, all the animals were sacrificed. Spleen fragments were taken for histological analysis. After their standard treatment, the paraffin sections were prepared, further stained with hematoxylin and eosin. The micropreparations were analyzed with microscope Olympus BX51 (Olympus, Japan). Microphotographs were processed by the Image J (version 1.50) software (National Institutes of Health, USA). An average area of lymphoid follicles and a specific one of white pulp were determined morphometrically.
Animal housing, labeling and necessary manipulations were carried out in compliance with bioethical principles and provisions of the Directive 2010/63/ EU of the European Parliament and of the Council 'On the Protection of Animals Used for Scientific Purposes'.
The obtained digital data was processed using the software for statistical analysis GNU PSPP (Free Software Foundation, USA). The normal distribution was verified with the Kolmogorov-Smirnov test and Kruskal-Wallis one as well. The Mann-Whitney U test was used to compare the characteristics between the groups.
In sections of the animals spleen of experimental group 1 the parenchyma was revealed to consist of red and white pulp. The morphological structure of spleen of animals in this group had signs of atrophy, which were more pronounced in 3 weeks after last injection of tetrachloromethane and were kept to week 9 (Fig. 1). To week 3 the area of white pulp was 42.0% and decreased down to 37.2% to week 9. The size of follicles was reduced, and there were insignificant areas of fibrosis in them. An average area of follicle to week 3 was (48,591.74 ± 2,229.70) µm 2 , being significantly lower than in the animals of control group (p < 0.05). To week 9 after completing the MOFS simulation, the average area of follicles was (25,413.46 ± 4,808.64) µm 2 , which was significantly lower than in previous observation period (p < 0.05). The red pulp had signs of fibrosis.
after stem cell injection was (93,635.66 ± 12,765.69) µm 2 , which was significantly higher than in animals group 1 (p < 0.05). To week 9 the average size of follicles was (77,467.09 ± 7,535.83) µm 2 , which was significantly lower vs. the previous observation period, and significantly higher than in animals of previous group (p < 0.05), and did not differ from the control (p = 0.23). Herewith in biological material of animals, studied in 9 weeks after injection, the size of follicles approached the norm, and the cell density of white pulp increased (Fig. 3). The red pulp was densely filled with cell elements.
The white pulp atrophy and a decreased number of lymphocyte series in spleen of animals with MOFS model may be a sign of the immune system depletion and indicate the migration of cell elements to affected organs (liver, lung, kidney). Restoring the follicle structure and increasing a number of white pulp lymphocytes in spleen of animals with simulated MOFS and administered stem cells testified to the activation of immune response.
Thus, the established morphological changes in animals spleen of control and both experimental groups indirectly testified to immunomodulatory effect of mesenchymal stem cells on a body after simulating multiple organ failure via tetrachloromethane introduction. Further determination of the presence of administered stem cells in different organs using fluorescence microscopy is promising.