STUDY OF THE INFLUENCE OF HUMORAL FACTORS OF FETAL NERVOUS STEM CELLS ON THE CYTOTOXIC ACTIVITY OF RATH SPLENOCYTES WITH TRAUMATIC BRAIN INJURY

Abstract

In the pathogenesis of trauma, there are, traditionally, 2 phases. The first phase is caused by damage to the brain under the direct action of a traumatic factor, which leads to mechanical damage to the integrity of neurons, glia, blood vessels of the brain. The second phase, where the so-called secondary disorders occur, begins a few hours after TBI and lasts from several days to several weeks, characterized by calcium in neurons, free radical formation, oxidative stress, synthesis of proinflammatory cytokines and the development of immunopathological reactions leading to necrosis. and neuronal apoptosis. One of the approaches to the treatment of the consequences of trauma is cell therapy with various stem cells (SC), and their humoral factors, which have proven to be a promising method of treatment.

The aim – to study the effect on cytotoxin activity of rat splenocytes in the first and 5th day after TBI humoral factors produced in cell culture in vitro by embryonic neuronal cells.

Material and Methods. The work used mature rats (n=36), bred in the vivarium of the State Institution "INH NAMS". All work with experimental animals was carried out in compliance with legal norms and requirements of the Law of Ukraine №3447 IV "On protection of animals from cruel treatment", "European Convention for the protection of vertebrate animals used for research and other scientific purposes" (Strasbourg, 1986), with taking into account the principles of bioethics and biosafety standards. The animals were kept in standard conditions of an accredited vivarium. Traumatic brain injury in rats was modeled according to the recommendations of Romanov GA, and others (2015) for modeling TBI in animals by dropping a load (weighing 100 g) from a height of 120 cm on the head of rats that were anesthetized intraperitoneally 0.5 ml mixtures of ketamine (70.0 mg / kg) and sedazine (15 mg / kg) [22].

Determination of cytotoxic activity of spleen cells in rats. The cytotoxic activity of splenocytes against sheep erythrocytes was determined by the spectrophotometric method described [23].

Obtaining fractions of conditioned medium from cultures of embryonic cells of the brain of rats was performed according to the developed method [24]. The brain was removed under sterile conditions from the fruits of ether-anesthetized females (E16-18 days of pregnancy), freed from blood vessels and membranes, and suspended in the medium by repeated picketing [24]. The resulting cell suspensions were seeded on the bottom of plastic Petri dishes and cultured for 48 hours in nutrient medium without embryonic calf serum. Supernatants of brain cell cultures, so-called conditioned media (CS), were used to treat animals with trauma.

Results. In traumatic brain injury in rats there is an increase in direct and antibody-dependent cytotoxicity of spleen cells, which is registered 24 hours after injury with a gradual further increase to 5 days. A single injection of trophic factors, which are produced in cell culture by embryonic nerve cells in a conditioned environment, rats immediately after injury causes a probable decrease in both types of cytotoxicity. Administration of conditioned media of embryonic nerve cells to rats with trauma for 1, 2, 3 days after injury causes, for 5 days, probable inhibition of increased direct and antibody-dependent cytotoxicity, which indicates the immunosuppressive effect of these trophic factors.

Conclusions. In traumatic brain injury in rats, there is an increase in the cytotoxic activity of splenocytes as early as 24 hours after injury, which may be inhibited by the introduction of trophic factors produced by embryonic cells of the rat brain.