The thesis is devoted to the problem of improving diagnosis efficiency in case of primary open-angle glaucoma progression by studying apoptosis and mitochondrial dysfunction of blood mononuclear cells (ВМСs) and developing new criteria for the disease prognosis on the basis of the obtained results. Blood mononuclear cells (BMCs) were studied to evaluate mitochondrial dysfunction and cells'inclination to undergo apoptosis. Studying spontaneous apoptosis and dexamethasone - induced apoptosis of BMCs in vitro and apoptosis induction index (AII) makes it possible to detect such internal disturbances of the organism which cannot be revealed by conventional methods. It also may contribute to determine the degree of body exhaustion or body сell functional reserve. The level of apoptosis, an apoptosis induction index (viability) of BMCs, changes in mitochondrial activity show common pathological tendencies in the body for POAG occurrence and progression. Blood mononuclear cells (BMCs) were isolated by using the Ficoll-Urografin density gradient method (d = 1,077). The isolated cells were incubated by using the method of "Cultivating cells to determine an apoptosis induction index". Apoptosis was induced with apoptosis-inducing concentration of dexamethasone. The apoptosis induction index was evaluated according to the Annexin V/ PI protocol by using flow cytometry procedure. The PAS flow cytometer (Partec, Germany) and AnnexinV-FITC Apoptosis detection Kit I (BD Bioscience Pharmingen, USA) were used during the study. The spontaneous apoptosis, induced apoptosis were studied and apoptosis induction index (AII) was determined by the formula: AІІ=АІsp/АІind The common method based on using Rhodamine 123 and propidium iodide (Fluka) was applied to evaluate the mitochondrial membrane potential (MMP).All samples were analyzed by using the PAS flow cytometer (Partec, Germany). The number of cells with mitochondrial hyperpolarization was detected while determining changes in the mitochondrial membrane potential on the cell division graphs by the amount of accumulated Rhodamine 123. Mitochondrial hyperpolarization was detected as more intense fluorescence of Rhodamine123 - stained cells. The percentage of such cells in the population was calculated. The peak analysis was made automatically with the program Flo Max. The decrease in cell functional reserve in patients with primary open-angle glaucoma has been detected. The induced apoptosis level of mononuclear cells (МС) was found to be lower (25,80±5,01%) (р<0,05) if compared to values in the reference group (28,34±4,55%) as well as an apoptosis induction index (AII) was higher (0,88±0,19 versus 0,79±0,12) (р<0,05). Studying spontaneous apoptosis, induced apoptosis and apoptosis induction index according to the stages has revealed the peculiarities of the POAG course that is an improvement of some values in patients with stage II. They are as following: a decrease in the spontaneous apoptosis level (p<0,05) in patients with stage II (19,57±2,92%) compared to the reference group (22,39±4,81%), a lower apoptosis induction index (AII) in patients with stage II of POAG (p> 0.05) and a higher AII in patients with stages I, III and IV(stage I - 0.92±0.16, stage III - 0.92±0.15, stage IV - 0.99±0.26) compared to those in the reference group (0.79±0.12) (p<0.05). The studies have shown a decrease in the mitochondrial function in patients with POAG. There has been detected an increase in the number of leukocytes (WBCs) with a reduced mitochondrial membrane potential (MMP) in patients with stage IV of POAG (1,84·109/l), compared to the values from the reference group (1,09·109/l) (p<0.05). The decrease in the number of cells with a high mitochondrial hyperpolarization in patients with POAG compared to that in the reference group (0.94±0.43% vs 1.59±0.43%) (p<0.05) has been detected. The progressive decrease in the number of cells with high level of hyperpolarized mitochondria has been determined: stage I - 1,24±0,71%; stage II- 1,12±0,40%, stage III - 0,79±0,40%, and stage IV - 0,76±0,40%. The number of cells with a high level of hyperpolarized mitochondria in patients with stages II, III and IV was lower than that in the reference group (1.59±0.43%) (p<0.05). A strong negative correlation was determined between the POAG stage and number of cells with high level of hyperpolarized mitochondria (the Spearman correlation coefficient R = -0.84 at p<0.05). The reduction in the number of cells with hyperpolarized mitochondria indicates the low adaptive capacity of patient cells. The number of equations have been developed to determine the POAG severity (stage) dependence on: 1) the number of mononuclear cells (ВМСs) with signs of necrosis - y1=-0,64+5,579x-3,37 x2+0,54x3; 2) the number of ВМС with signs of mitochondrial membrane hyperpolarization - y2=1,43-1,60log(x); 3) the amount of ВМСs with signs of apoptosis - y3=1,19-0,001x2+5,36*105x3; 4) the apoptosis induction index - y4=-1,19+9,21x-14,02x2+7,46x3. The multivariate mathematic model for the prognosis of POAG progression has been developed, where y1, y2, y3, and y4 are prognostic coefficients. According to the suggested equations, the prognosis is negative if the sum of all prognostic coefficients (y1 + y2 + y3 + y4) obtained after calculating exceeds the value of 5. The method for the POAG prognosis based on values of apoptosis induction index for ВМСs has been developed. The apoptosis induction index(x) was determined in the isolated mononuclear cell culture. The obtained values were introduced into the formula y=-1,19+9,21x-14,02x2+7,46x3 and disease deterioration was prognosticated at "y"> 1,5, a risk of the disease deterioration was elevated at "y">2, a significant risk was at "y">3 and dramatically high risk of the disease deterioration was prognosticated at "y">4.
