[REORGANIZATION OF THE ULTRASTRUCTURE OF NEOCORTICAL NEURONS IN RATS TREATED WITH CELL-FREE DNA].

Neuron ultrastructure was studied in layers III-V of rat brain neocortex 24 hours after intraperitoneal (n=3) or intravenous (n=3) injection of cell-free DNA (7.7x10(-5) g/kg body weight). A plastic restructuring of nuclear chromatin, nucleolar hypertrophy, deep invaginations of nuclear envelope, hyperplasia of mito- chondria and their close contact with other organelles and the nucleus, formation of cytoplasmic tubulovesicular bodies which may promote enhanced synaptic vesicle transport to presynaptic axonal terminals, activation of astrocyte glia were found. The data obtained suggest that injection of cell-free DNA lead to pronounced ultrastructural reorganization in neocortical neurons directed to protein synthesis activation, enhancement of synaptic transmission efficiency, as well as intensification of energy metabolism, that may contribute to reparative and compensatory restorative processes in cerebral ischemic pathology.

[Circulating in blood plasma cell-free DNA in the pathogenesis of ischemic stroke: the role of the transcribed region of ribosomal repeat].

It has been established that DNA, in addition to its basic functions (storage and realization of genetic information), also carries CpG-rich sequences having immunopotentiating properties. In this study we investigated the dynamics of the quantitative and qualitative characteristics of cell-free DNA (cfDNA) circulating in blood plasma of patients with acute ischemic stroke compared with the biomechanics of their blood samples flow, cerebral infarct volume and dynamics of neurological disorders. The results obtained revealed a new drag-reducing function of the circulating cfDNA and its important role in a regulation of blood flow hydrodynamic resistance in conditions of disturbed cerebral circulation. Moreover, our results showed a dependence of cerebral infarct volume and clinical picture dynamics on the plasma concentration of transcribed region of ribosomal repeat CpG-rich sequences (rDNA). It was established a new function of rDNA fragments circulating in the total pool cfDNK, i.e., generation of the intercommunication between blood and brain cells to induce neuroprotection in ischemic stroke.

Properties of extracellular DNA from the cerebrospinal fluid and blood plasma during Parkinson's disease.

The cerebrospinal fluid of patients with Parkinson's disease was shown to contain extracellular DNA. Extracellular DNA concentration in the cerebrospinal fluid was 3.3-fold lower than in blood plasma from these patients. HPLC-mass spectrometry analysis showed that the pool of extracellular DNA from the liquor is characterized by a lower content of deoxythymidine, but greater amounts of deoxycytidine and deoxyguanosine than the pool of extracellular DNA from the plasma. The level of deoxyguanosine was 2 times lower than that of deoxycytidine (as differentiated from plasma extracellular DNA with similar content of these substances). Our findings indicate that extracellular DNA from the cerebrospinal fluid contains considerable amounts of modified deoxyguanosine. These data attest to significant differences in the quantitative and qualitative characteristics of extracellular DNA from the blood and cerebrospinal fluid of patients. Specific features of extracellular DNA from the cerebrospinal fluid of patients suggest its involvement in the pathogenesis of Parkinson's disease.

Emotional stress in rats changes concentration and composition of extracellular DNA circulating in blood plasma under normal conditions and in cerebral ischemia.

We studied quantitative and qualitative characteristics of extracellular DNA circulating in the blood plasma of Wistar rats under normal conditions, in psychoemotional stress (after 18 hours of aggressive conflict situation), and in acute cerebral ischemia. It was found that animals predisposed to psychoemotional stress normally have increased levels of antibodies against low excreted fragment of transcribed region of ribosomal DNA repeat rich in cytosine-guanine (CpG). A sharp increase in the level of circulating extracellular DNA was noted. Its increase was more pronounced during ischemia against the background of psychoemotional stress than in the control. These data suggest that multiple stress exposures experienced during the life can result in accumulation of GpG-rich sequences in the plasma of individuals predisposed to psychoemotional stress.

Extracellular DNA affects NO content in human endothelial cells.

Fragments of extracellular DNA are permanently released into the blood flow due to cell apoptosis and possible de novo DNA synthesis. To find out whether extracellular DNA can affect the synthesis of nitric oxide (NO), one of key vascular tone regulators, we studied in vitro effects of three artificial DNA probes with different sequences and 10 samples of extracellular DNA (obtained from healthy people and patients with hypertension and atherosclerosis) on NO synthesis in endothelial cell culture (HUVEC). For detection of NO in live cells and culture medium, we used a NO-specific agent CuFL penetrating into the cells and forming a fluorescent product FL-NO upon interaction with NO. Human genome DNA fragments affected the content of NO in endothelial cells; this effect depended on both the base sequence and concentration of DNA fragments. Addition of artificial DNA and extracellular DNA from healthy people into the cell culture in a low concentration (5 ng/ml) increased the detected NO concentration by 4-fold at most. Cytosine-guanine (CG)-rich fragment of the transcribed sequence of ribosomal repeat was the most powerful NO-inductor. The effect of DNA fragments on NO synthesis was comparable with that of low doses of oxidizing agents, H(2)O(2) and 17ß-estradiol. Extracellular DNA samples obtained from patients with hypertension and atherosclerosis decreased NO content in cells and medium by 1.3-28 times compared to the control; the effect correlated with the content of CG-rich sequences.

[Delayed appearance of hypertension in spontaneously hypertensive rat (SHR) injected with CpG-rich DNA early in ontogenesis].

In this study we have investigated properties of blood serum extracellular DNA (cell-free DNA) from patients with essential arterial hypertension (AH). Cell-free DNA concentration was not changed in the control AH group compared to norma (healthy donors) but fragments of CpG-rich cell-free DNA marker content were increased at transcribed area of ribosomal repeat (TArDNA, CpG-DNA). To evaluate effect of CpG-DNA on AH development in 2-day SHR line and in control normotensive line (WKY), 700 ng of human TArDNA single subcutaneous injection were inoculated to obtain anti-CpG-DNA polyclonal antibodies. These antibodies could change CpG-DNA contents in total cell-free DNA. Blood pressure (BP) in 9-week SHR line rats immunized with CpG-DNA was equal to BP of WKY rats. Then BP of immunized SHR steadily increased with age and reached high value 8 weeks later compared to control SHR rats. Cell-free DNA analysis in 17-week SHR line rats showed significantly reduced concentrations of cell-free DNA and also showed decrease in small DNA fragments content, but increased content of CpG-DNA (rat TArDNA). These changes were accompanied with 3.5-fold blood endonuclease activity increase and decrease of free (unbound to cell-free DNA) anti-CpG-DNA antibodies quantity. Total anti-CpG-DNA antibodies quantity in immunized rats wasn't changed compared to control animals. Thus, observed effect of increase in stable BP elevation age in immunized SHR line rats doesn't relate to increase of anti-CpG-DNA antibody production. Possible reason of this effect is further discussed.

[Hemodynamic mechanisms of negative impact of emotional stress on the development of cerebral ischemia in stressor load-sensitive rats].

Emotional stress aggravates the course of brain ischemia that has developed in the presence of the former. The investigation studied the functional state of parietooccipital leptomeningeal anastomoses by vital biomicroscopy, as well as the linear blood flow velocity in the a. cerebri media and a. basilaris by high-frequency (38.5 MHz) Doppler ultrasonography before and after occlusion of the common carotid artery in normal (control) Wistar rats and ones that had experienced 18-hour aggressively conflict emotional stress and that were sensitive to the latter. Hemodynamic differences were found in the rats having a varying sensitivity to the used model of cerebral ischemia. Inadequate collateral blood supply along the circle of Willis was shown when this model of brain ischemia was used in the control animals. It was ascertained that in emotional stress, blood flow considerably reduced in the a. basilaris; the contiguous blood supply area displayed evolving brain edema that compressed leptomeningeal anastomoses and prevented the formation of collateral circulation, followed by occlusion of the common carotid arteries. With this, unilateral occlusion was followed by a short-term reduction in systemic blood pressure (BP) that was not seen in the controls and bilateral occlusion was by the development of collapse. Cerebral blood flow became dependent of systemic BP. The obtained experimental data suggest that it is expedient to include antistressor agents into therapy for chronic cerebrovascular diseases. This is particularly relevant to patients with occlusive carotid artery lesion and Willis circle anomalies since emotional stress may be fatal to them.

[Haemodynamic role of blood-plasma circulating cell-free DNA and contained therein high-molecular-weight CpG-rich fraction in pathogenesis of arterial hypertension and atherosclerosis obliterans of carotid arteries].

The hydrodynamic resistance (HR) of blood is one of the components of the total peripheral resistance. High-molecular-weight DNA appears to decrease the HR in accordance with the Toms's effect. The present study was undertaken to investigate the HR and properties of cell-free DNA circulating in the blood plasma (hereinafter referred to as pDNA) of the control donors, patients suffering from either arterial hypertension (AH) alone or that combined with atherosclerotic lesions of the carotid arteries (CAs). Within the normal concentrations of pDNA, we revealed an inverse dependence of the HR thereupon and upon the content in pDNA of the high-molecular-weight CpG-rich fraction (CpG-DNA), i. e., a transcribed region of the ribosomal repeat (rDNA). A decrease or an increase in the pDNA concentration in all the patients examined was accompanied by an elevation of the rDNA concentration in the blood plasma. Exceeding a certain level thereof appeared to give rise to an increase in both the HR and arterial pressure (AP). Patients presenting with degree I essential AH were found to have a decreased endonuclease activity of the blood plasma, with the pDNA concentration being more than two-fold higher with no change in the rDNA content. Their HR appeared to be increased (p<0.01). Patients diagnosed as having degree II AH were characterized by a normal or decreased level of pDNA and an elevated content of pDNA, with the HR being slightly lowered. In patients presenting with atherosclerosis obliterans of the ACs, the initial manifestations of the lesions of the carotid arteries were typically revealed on the background of a lowered HR (p<0.05). All patients suffering from atherosclerotic lesions of the ACs could be subdivided into two groups, which in our opinion is probably associated with different various mechanisms of destructive damage to the arterial intima. In some of them, the pDNA concentration does not differ from the normal values, but in its composition, there is an increased content of rDNA, elevating as obliteration of the vessels' lumen increases, with the HR being decreased. The majority of them have degree II AH. In others, the pDNA concentration is by an order of magnitude higher than the normal values, while the rDNA content in pDNA is decreased, with the HR being elevated. Most of them have degree III AH. Pronounced and rough stenoses take an asymptomatic course in patients with decreased values of the HR and a slightly elevated level of pDNA and/or rDNA in the blood plasma. A higher level thereof leads to a rise in the HR and to the appearance of neurological symptomatology. Hence, CpG-DNA circulating in the composition of pDNA is a constantly acting endogenous blood factor decreasing the HR (the Toms's effect) and normalizing AP under physiological conditions, being however a cause of their increase and impairment of blood circulation in the pathogenesis of AH and atherosclerosis obliterans of the CAs.

Transcranial evaluation of blood flow velocity in the basilar artery in rats by high frequency ultrasonic Doppler technique.

The possibility of recording the blood flow velocity in the basilar artery (a. basilaris) in rats through the occipital membrane by a two-channel ultrasonic Doppler system with single-element transducers was demonstrated. A. basilaris was located with a contact transducer working at a frequency of 33 MHz in two modes: pulsed and permanent. The mean blood flow velocity in narcotized male Wistar rats was 4.93+/-0.78 cm/sec. Functional loading tests (acute elevation of blood pressure and occlusion of the common carotid arteries) showed that this method allows recording dynamic characteristics of the blood flow reactions in a. basilaris with evaluation of its pulse wave pattern and analyzing their correlations with blood flow parameters in the carotid arteries using simultaneously the second (bandage) transducer.

Delta sleep-inducing peptide and Deltaran: potential approaches to antistress protection.

The aims of the present work were to perform a comparative study of the effects of delta sleep-inducing peptide and Deltaran on neurons in emotiogenic brain structures and to address the question of whether it is possible to prevent or decrease the negative influences of stress loads on the severity of subsequent cerebral ischemia in rats, using glycine with delta sleep-inducing peptide combined in the neuroprotective formulation Deltaran. The results showed that Deltaran and delta sleep-inducing peptide had largely the same actions on the nature of spike activity of neurons in the dorsal hippocampus, paraventricular nucleus of the hypothalamus, and ventral anterior nuclei of the thalamus, evoking activation of some of the neurons in these brain structures. The dorsal hippocampus was dominated by activation of spike activity in response to administration of delta sleep-inducing peptide; Deltaran produced activation mainly in the paraventricular nuclei of the hypothalamus. In all animals given Deltaran, the index of brain blood supply was significantly greater than in animals not given Deltaran. The survival rate of cerebral ischemia was 100% in animals given Deltaran. Death occurred in 38% of animals not given Deltaran.

[Cell-free plasmic DNA as a blood factor determining hemodynamics in health and in vascular pathology].

The article reviews investigations performed in the Research Neurology Institute on blood flow biomechanics which resulted in discovery of a universal permanent theological blood factor--circulating cell-free plasmic DNA reducing hydrodynamic blood resistance in accordance with Toms' effect. In vitro and in vivo experiments were made with use of drag-reducing polymers for correction of circulation. Basic mechanisms were discovered by which cell-free DNA regulates normal and affected circulation. Clinico-experimental investigations demonstrated the role of imbalance between DNA concentration in blood plasma and length of its fragments caused by their change in cerebrovascular diseases, in disorder of circulation pattern. It was shown in animal experiments that it is possible to correct abnormal circulation characteristics in ischemic disorders of brain circulation by intravenous injections of high-molecular DNA solutions.

[Delta-sleep inducing peptide and the drug deltaran: possible approaches to antistress protection].

An aim of the present study was a comparative investigation of a delta-sleep inducing peptide and the drug deltaran on the neural activity of the brain structures involved in emotional processing. Another goal was to analyze the possibility to prevent negative effects of emotional stress on brain ischemia using, along with deltaran, glycine and a delta-sleep inducing peptide. Deltaran and the delta-sleep inducing peptide exert in general similar effect on the burst activity of neurons in the dorsal hippocampus, hypothalamic paraventricular nucleus and ventral anterior thalamic nucleus, inducing amplification of the majority of recorded units. The activation of neuronal activity was seen mostly after the delta-sleep inducing peptide microiontophoresis in the dorsal hippocampus and after the deltaran application in the hypothalamic paraventricular nuclei. The index characterizing blood supply was significantly higher in all rats receiving deltaran as compared to the controls. Animals receiving deltaran survived experimental brain ischemia in 100% cases versus 38% in those not exposed to this drug.

Effect of emotional stress on hemoglobin oxygen affinity in low resistant animals under normal conditions and during cerebral ischemia.

Hemoglobin oxygen affinity within the estimated physiological range plays an adaptive antioxidant role during acute cerebral ischemia. This range depends on individual emotional resistance. Brain ischemia induced by common carotid artery occlusion in low resistant Wistar rats increased hemoglobin oxygen affinity by 12% during the acute period. Emotional stress also increased hemoglobin oxygen affinity and determined shifts in this parameter during the development of cerebral ischemia: moderate increase in hemoglobin oxygen affinity (<25%) was followed by further increase in this parameter, while more pronounced shift (>25%) resulted in a significant drop in this parameter due to hemoglobin deoxygenation. Adaptation to stress shifted the upper physiological limit for self-regulation of this process.

Deltaran prevents an adverse effect of emotional stress on the course of cerebral ischemia in low-resistant animals.

Local cerebral blood flow in the left hemisphere decreased most significantly in low-resistant Wistar rats preexposed to emotional stress. Deltaran selectively increased blood flow in the left hemisphere and improved blood supply to neuronal activity unit of the brain in these animals. This drug prevented progressive decrease in local cerebral blood flow in both hemispheres during the acute stage of ischemia. The effect of Deltaran was related to modulation of collateral blood flow and adequate blood supply to neuronal activity unit in the brain tissue. Deltaran decreased the mortality rate (by 62%) and alleviated the symptoms of cerebral ischemia. The positive effect of Deltaran was more pronounced in the left hemisphere.

Correction of cerebral ischemia in low-resistant animals with an antistress drug Deltaran.

Deltaran decreased the amplitude of EEG slow waves and restored neuronal reactivity after carotid artery occlusion in Wistar rats sensitive to cerebral ischemia. Deltaran had no effect on local cerebral blood flow. This drug increased blood supply to a unit of neuronal activity in the brain of intact animals during the acute stage of cerebral ischemia, provided 100% survival rate of rats with cerebral ischemia, and prevented the development of neurological symptoms in survivors. Animal experiments proved the possibility of correcting cerebral ischemia with antistress drug Deltaran.

[Long-fragment DNA of blood plasma as one of the criteria of individual sensitivity to emotional stress and to cerebral ischemia].

Intravenous injection ofpolyethylenoxide WSR-301 reducing hydrodynamic blood resistance (Toms effect) improves gas exchange in the lungs and halved lethality of the animals with cerebral ischemia. The aim of the study was to establish whether free plasma DNA influences blood gases and lethality of the animals with brain ischemia. Common carotid arteries were ligated for 15 min in intact stressed and tested in the open field Wistar male rats, then some of the rats received intravenous solution of homologous long-fragment DNA (20x10(-6) g/ml of blood). Cerebral circulation, acid-base equilibrium, paO2, paCO2, asymptotic blood viscosity, plasmic concentration and length of DNA fragments in plasma, lethality and neurological status of the survivors were studied. It was found that long fragments of rat DNA show hydrodynamic Toms effect. In normal passive rats sensitive to cerebral ischemia part of plasm DNA is fragmented, gas composition and blood viscosity of blood is worse (p < 0.05) than in active animals. There is a direct correlation between the level of long-fragment DNA in plasm and paO2 (r = 0.55) and inverse--with paCO2 (r = -0.84). Intravenous injection of long-fragment DNA improved the course and reduced lethality of brain ischemia 2-3-fold. Thus, qualitative and quantitative characteristics of plasma circulating DNA are responsible for differences in blood gases in rats differently tolerable to cerebral ischemia and can serve as one of the criteria of individual sensitivity to it being essential in pathogenesis of ischemic stroke.

Individual resistance to cerebral ischemia and negative effect of emotional stress on the course of this disorder.

The ratio of low-activity and high-activity rats differed in autumn, winter, and spring litters. Initially more intensive cerebral blood flow in low-active rats and its more pronounced decrease after common carotid artery occlusion determined their higher sensitivity to cerebral ischemia (compared to high-activity animals). After 18-h immobilization stress cerebral blood flow decreased by 10-15%, which abolished the difference in the individual resistance to cerebral ischemia. Independently on emotional resistance, cerebral ischemia was not accompanied by the development of collateral blood flow in the acute period and caused death of 90% rats.

[Disregulation of collateral blood flow and lipid peroxidation abnormalities as a cause of negative influence of emotional stress on the course of cerebral ischemia]. / Dizreguliatsiia kollateral'nogo krovoobrashcheniia i narushenie perekisnogo okisleniia lipidov -- osnova negativnogo vliianiia émotsional'nogo stressa na techenie tserebral'noi ishemii.

Under normal conditions, local brain blood flow in young rats determined their individual resistance to ischemia and emotional stress. In low-activity rats predisposed to emotional stress, a level of blood flow was significantly (p<0.05) higher, comparing to high-active animals resistant to emotional stress. An exposure of rats to aggressive- and conflict situation for 18 h was followed by pronounced emotional stress accompanied by the reduction of local cerebral blood flow by 10-15% and abolished individual differences in resistance to cerebral ischemia. Collateral blood flow did not develop in pre-stressed rats during the acute period of cerebral ischemia. The mortality rate reached 90% independently of the animal emotional resistance. In ischemia after 2 h exposure to stress, differences were revealed in the intensity of lipid peroxidation in the brain of animals with divergent emotional resistance. Comparing to stress-resistant rats, in stress-predisposed animals, a level of malonic dialdehyde in the amygdale basal nuclei was 2-fold lower. The results suggest the differences in stress response in animals with various resistance to the effect of adverse factors.

[New findings concerning pathogenesis of disorders of cerebral circulation]. / Novoe v patogeneze narushenii mozgovogo krovoobrashcheniia.

The data about the role of blood flow in atherosclerosis development were examined. The aim of the study was determination of hydrodynamic blood resistance and Toms effect in patients with ischemic disorders of cerebral circulation in different periods of the disease. 45 patients with atherosclerotic damages of major arteries of head were observed. Such damages were combined with arterial hypertension in 32 individuals. The value of hematocrit, asymptotic viscosity, hydrodynamic resistance and Toms' effect were determined in blood as well as levels of cholesterol, beta-lipoproteins and platelets. The presence of Toms' effect was found in blood samples of both patients and controls. There was a significant increase of hydrodynamic blood resistance in all the patients as compared to control group. The patients with ischemic disorders of cerebral circulation were compensated by their own blood factor in less degree in acutest and acute periods than later. Low values of Toms' effect in separate patients were prognostic sings for good restoration of neurologic functions; higher values were observed in patients with arterial hypertension. The conclusion was made that hemodynamic properties of blood flow may be considered as predictors of the severity of the course of cerebral circulation disorders.