INTRODUCTION

In previous studies [1, 2], a hypothesis was put forward about the relationship between the composition of blood cells and the charge of their membranes in patients with glioblastomas. This phenomenon is of considerable interest in connection with the transition of reparative processes in body tissues into regenerative processes, which is observed in malignant tumors of any genesis, characterized by necrotic cell death [3, 4]. A large number of works study the interaction between pools of blood cells without taking into account their charge characteristics [5-8].

The role of the chronic inflammatory process in the development and progression of malignant tumors is beyond doubt [9-12], while the significance of the transition of   repair mechanisms to regeneration involving stem cells of mesenchymal origin from the bone marrow has not been extensively studied in case of   brain gliomas.

In this work we utilized the effect of low concentrations of verapamil hydrochloride and electromagnetic oscillations of   low-level laser radiation (LLLR) with a frequency of 1.2 Hz on the change in the level of aggregation of   blood cells taken from patients with gliomas and meningiomas before surgical treatment and on day 7 after surgery under in vitro conditions. Laser radiation with an oscillation frequency of 1.2 Hz is used in medical institutions with a positive effect on the rehabilitation of patients [13].

Then we studied the correlation between changes in the level of aggregation of blood cells without exposure and under the influence of a 0.25% solution of verapamil hydrochloride and LLLR, and the cellular composition of blood in the same samples. The blood composition in each sample remained unchanged, and the charge of blood cell membranes and their correlation with the quantitative composition of blood cells were analyzed.

The work aimed to study the change in the correlation coefficient between the blood cells quantities and their level of aggregation, affected by the influence of verapamil hydrochloride diluted with water by a factor of 1:10.000 and LLLR with a frequency of 1.2 Hz in patients with grade III of gliomas and meningiomas with I grade of malignancy before and after surgery.

MATERIALS AND METHODS

The object of the study was the peripheral blood cells taken from patients during the neurosurgical operations for the removal of tumors (in compliance with the norms of the Committee on Bioethics). A survey of 11 patients undergoing a course of complex treatment at the A. P. Romodanov Neurosurgery Institute of National Academy was performed to obtain the indicators of the degree of aggregation of blood cells before the start of therapeutic and surgical interventions, as well as on the 7th day after the operation. As a control, a similar blood sampling was performed in healthy donors (12 people) who underwent a clinical examination at the blood transfusion point. The study groups were formed subject to the condition of the absence of concomitant inflammatory diseases. For instance, the common clinical blood tests (ESR, the number of erythrocytes and other cellular elements of the blood) showed the absence of a pronounced inflammatory process in patients with gliomas of   III grade of malignancy and meningiomas of I grade of malignancy. Patients accompanied by changes in the processes of blood cells aggregation (hypoproteinemia, autoimmune diseases, and diseases requiring the use of salicylates) were not included in the study groups.

Heparinized venous blood samples taken from patients with gliomas and meningiomas before the start of treatment and on the 7th day after the operation, were separated by centrifugation (1500 rpm for 10 minutes) into blood cells and plasma. The cell fraction was used to determine the surface plasmon resonance (SPR) angle shift in degrees, which reflects the degree of aggregation of blood cells. The study of the initial processes of blood cell aggregation has become possible with the opening of a new direction in biosensorics, using the SPR effect [14, 15], which does not require a radioactive or fluorescent label. The SPR phenomenon consists of the resonant excitation of surface plasmons at the dielectric-metal (gold or silver) interface at a certain angle of incidence of a TM-polarized electromagnetic (laser) wave on this interface. The value of the resonant angle of incidence is sensitive to the refractive index of the external medium, which is employed in this sensor. The unit of measurement is considered to be the SPR shift in degrees, which depends not only on the number of cells deposited on a glass plate coated with a thin layer of gold, but also on the total area of the membranes of these cells directly interacting with the plasmon. A plasmon is a cloud of free electrons generated by the action of a laser beam passing through a glass prism on a thin layer of gold. As is known, during agglutination or aggregation of blood cells, the ratio of the surface of cell particles to their volume decreases, while the total number of cells, compared to the control, may be greater, and the area of contact with the gold plate may be smaller, which leads to a decrease in the shift in the SPR readings, measured in degrees. The study of SPR parameters with blood cells in healthy individuals and patients with neurosurgical pathology was carried out using a PLASMON SPR spectrometer (Biosuplar) with an optical excitation source (GaAs laser, λ=650 nm), developed at the Institute of Semiconductor Physics of National Academy of Sciences of Ukraine. Glass plates (n=1.612) with a layer of gold (45-50 nm) deposited onto an intermediate adhesive layer of chromium (~ 5 nm) were fixed on a retroreflecting glass prism (n = 1.612) of the spectrometer using an immersion liquid (polyphenyl ether, n = 1.612). In the course of work with blood cells, plates with a deposited layer of gold were reused after repeated washing with ion-free water. The interaction of the gold layer with the charge of blood cell membranes leads to a shift in the plasmon resonance curve (SPR shift in degrees), which was measured and processed by the device with the BSS55 software package, with the results displayed in graphical form on a computer monitor [14].

Before the studies, the instrument micro cuvettes were washed with ion-free water. The supply of   the cell fraction, with a volume of about 400 μl, was carried out using an electric pump at a rate of 100 μl per minute. At the end of the study, blood cells were washed off the substrate with a large volume of water, while monitoring the return of the original curve on the display screen to its original position.

To reduce the level of aggregation of blood cells, a technique was developed under in vitro conditions using a blocker of NMDA-dependent Ca²⁺ ion channels, e. g. verapamil, which can change the indicators of transmembrane potential on the membranes of blood cells, mediated in our studies by their level of aggregation.

It is known that under physiological conditions, NMDA receptors are activated by millimolar concentrations of glutamate, which is present in the synaptic cleft for several milliseconds. During pathological impulses, receptors are activated by micromolar concentrations, but for a much longer time [16]. We took the same pattern as a basis concerning the effects of milli- and micromolar concentrations of verapamil hydrochloride to select its optimal concentrations to change the level of blood cell aggregation.

In the control blood samples, an equal volume of ion-free water was used for dilution instead of verapamil hydrochloride.

Blood samples were irradiated with a laser generating electromagnetic radiation with a pulse frequency of 1.2 Hz in Petri dishes for 5 minutes each. Then, in these samples, the level of aggregation of blood cells was determined individually, measured in SPR angular units.

The number of blood cells was determined in the same blood samples using an automatic hematological analyzer Mindray-3000.

Monocytes were detected in the pool of Mid cells of the peripheral bloodstream.

Statistical studies were performed using the "Statistics-10v" package.

Method for Determining the Correlation Coefficient

Spearman’s rank correlation coefficient is a nonparametric method that is used to statistically study the relationship between phenomena. In this case, the actual degree of parallelism between the two quantitative series of the studied features is determined and an assessment of the tightness of the established relationship is given using a quantitatively expressed coefficient.

To assess the tightness of interrelation, the Chaddock scale (Table 1) can be used [17]:

The statistical significance of the obtained coefficient is assessed using the Student’s t-test. If the calculated value of the t-test is less than the tabular value for a given number of degrees of freedom, the statistical significance of the observed relationship is absent. Otherwise, the correlation is considered statistically significant.

RESULTS AND DISCUSSION

We studied the change in correlation coefficient between cellular charge under the influence of low concentrations of verapamil hydrochloride and LLLR with an oscillation frequency of 1.2 Hz and cellular content in 12 blood samples from 6 patients (47±2.9 years old) with gliomas of III grade of malignancy and 5 patients with meningiomas of I grade of malignancy before and after treatment. The control group for patients with gliomas consisted of 12 practically healthy individuals (48±5.5 years).

In the group of healthy individuals, no significant correlation was found between SPR readings and blood cells. On the 7th day after the operation, the number of cells, except for granulocytes, increases: 2.5 times the number of monocytes, 1.3 - lymphocytes, 1.1 times - leukocytes, and the number of granulocytes decreases by 1.1 times (Table 2).

With grade III glioma before surgery, a significant negative correlation was observed between leukocytes and SPR readings in blood samples with the addition of verapamil hydrochloride diluted by a factor of 10.000 (Table 3).

With grade III glioma on the 7th day after the operation, a significant positive correlation was found between leukocytes in control blood samples and after exposure to LLLR and verapamil hydrochloride in combination with LLLR. A positive correlation is observed between monocytes (Mid) and SPR readings in the blood samples exposed to low concentrations of verapamil hydrochloride (1:10,000) and in combination with LLLR (Table 4).