[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.

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.

[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.

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.

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.

[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.

[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.

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.

[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.

Blood flow hydrodynamic resistance in patients with ischaemic stroke or vascular encephalopathy and possibilities of its correction in vitro.

In order to determine the patient blood flow hydrodynamic properties in terms of the Toms-effect, the time blood flow was measured in special apparatus twice: (1) before and (2) after addition of poly(ethylene oxide) WSR-301 (Union Carbide, USA) in concentration of 2 x 10(6) g/ml of blood sample (50 ml with 500 units of heparin) taken from 26 patients with acute ischaemic stroke, 8 patients with vascular encephalopathy after stroke, 5 patients with vascular encephalopathy following atherosclerosis with or without arterial hypertension. The blood samples taken from 8 healthy persons formed the control group. Haematocrit (HCT) and asymptotic blood viscosity were studied also. It was established that hydrodynamic blood flow resistance (HBFR) did not depend on HCT and viscosity, but was significantly higher in all patient groups than in normal persons (p less than 0.05). Addition of polymer solution decreased HBFR of patient blood more intensively that in the control group. We believe that it indicates an insufficiency of an unidentified factor in native human blood.

[The enhanced efficacy of the pharmacological regulation of the processes of blood cell activation and interaction in the flow by polymer additives decreasing the hydrodynamic resistance of the blood]. / Povyshenie éffektivnosti farmakologicheskoi reguliatsii protsessov aktivatsii i vzaimodeistviia kletok krovi v potoke polimernymi dobavkami, snizhaiushchimi gidrodinamicheskoe soprotivlenie krovi.

The increase of blood cell auto- and interactivation and the decrease of aspirin antiaggregant effect were demonstrated under hemodynamic conditions of a stenotic zone in a model maximally approached to the real blood circulation. The lowering of platelet activation, of the release of active agents from platelets, and of platelet-leukocyte interactivation was attained under the conditions of the flow modified by the drag-reducing polymer solution. The recovery of aspirin antiaggregant activity and aspirin-induced reduction of the total platelet release of platelet- and leukocyte-activating agents were demonstrated under the influence of the polymer.

[Ultrastructural changes in the hemato-encephalic barrier of adult animals after acute hypoxic hypoxia]. / Izmenenie ul'trastruktury gematoéntsefalicheskogo bar'era u vzroslykh zhivotnykh posle ostroi gipoksicheskoi gipoksii.

The ultrastructure of the blood-brain barrier (BBB) was studied in different periods after the effect of acute hypoxic hypoxia. Local changes in the BBB ultrastructure indicating increased transport of blood plasma through certain areas of the vascular wall were seen already 24 hours after the hypoxia session. Four days later these changes were more marked and more diffuse. In 7 days the ultrastructure of the vessels and the surrounding brain tissue showed more significant changes. In later periods a tendency towards restoration of the BBB ultrastructure was revealed, which was also irregular in its course. The greater or lesser vulnerability of the different regions of the brain vessels may be determined both by the uneven decrease of their tonus and by the possible differences in the metabolic changes in the different areas of the brain vascular system under such conditions.

[The prevention of the development of the collagen-induced microembolic syndrome by decreasing the hydrodynamic resistance of the blood with special polymers under flow]. / Predotvrashchenie razvitiia kollagenindutsirovannogo mikroémbolicheskogo sindroma posredstvom snizheniia gidrodinamicheskogo soprotivleniia krovi spetsial'nymi polimerami v usloviiakh potoka.

The search for antiaggregatory compounds is undertaken, as a rule, under in vitro conditions which do not reflect the dynamics of the real process. The present work deals with study of the peculiarities of the development of the collagen induced microembolic syndrome and the effect produced on it by hydrodynamic resistance reducing polymers under conditions modelling real circulation. The authors show the formation of a developed microembolic syndrome under the effect of collagen with accumulation of vasoconstrictors in the environment and increase of red cell hemolysis. Reduction of blood flow turbulence by the addition of solutions of neutral high-molecular linear polymers revealed the inhibiting effect of these polymers on the development of intracellular aggregate formation and concomitant red cell microhemolysis, which was attended by sharp reduction of thromboxane A2 production in the experiment. The authors show the significance of hemodynamic conditions in the development of the microembolic syndrome and the possibility of preventing it under conditions of modification of the blood flow with polymers, which may be of high prophylactic importance.

[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.

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.

[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.

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.