Morphologic changes of red blood cells during hemorrhagic shock replicate changes of aging.

Blood loss leads to the reduction in vitality of red blood cells (RBCs). However, the changes in morphology at different stages of hemorrhagic shock have not been studied. Thus, the aim of this study was to identify and quantitate the sequence of morphological changes in RBCs during hemorrhage. This study was performed on 15 adult inbred dogs. Blood samples were taken before hemorrhage, when the mean arterial pressure reached 40 mm Hg (initial stage of shock), and at a mean arterial pressure level of 20 mm Hg (decompensated stage of shock). The volume of blood removed averaged 33.6+/-8.9 and 55.1+/-6.9 mL/kg, respectively. Evaluation of RBC morphology was performed by computerized light microscopic morphometry and scanning electron microscopy. At the early stage of hemorrhage the number of "young-appearing" RBCs with large visible surface areas (41-50 microm2) increased from 17.7%+/-3.1% to 26.6%+/-3.5% (P < 0.05). Concomitantly, the number of "old-appearing" RBCs with small visible surface area (20-30 microm2) significantly decreased from 5.3%+/-2.7% to 2.7%+/-2.3% (P < 0.01). At the stage of decompensated blood loss, the opposite phenomenon was observed. The number of "old-appearing" RBCs increased to 8.2%+/-1.1% (P < 0.01), whereas the number of "young-appearing" RBCs decreased to 12.3%+/-4.2% (P< 0.01). The changes in visible surface area of RBCs was accompanied by significant alterations in their shape. The percentage of abnormal shaped RBCs increased from 8.9%+/-1.1% before the hemorrhage to 36.4%+/-5.8% at the stage of decompensated hemorragic shock (P < 0.01). Thus, during the late decompensated stage of hemorrhagic shock, RBCs assume shape and surface area changes that are similar to those seen in aging. These changes in RBC size and shape may be due to the effects of shock-induced oxidative stress.

Effect of hemorrhagic shock duration on the activity of nuclear Ca2+/Mg(2+)-dependent DNA-endonucleases in canine cerebral cortex.

A direct correlation between the duration of arterial hypotension (40 mmHg) and the activity value of nuclear Ca2+/Mg(2+)-dependent DNA-endonucleases was revealed in dogs exposed to hemorrhagic shock. Thus, an increase in the activity of endonucleases was significant after 2 h of arterial hypotension. During that period there activity amounted to 4149 +/- 295 units/mg of protein instead of 2839 +/- 231 units/mg protein in the control (P < 0.01). With the prolonged hemorrhagic shock, the activity of DNA-endonucleases continued to increase. By 4 h of hemorrhagic shock it reached 5021 +/- 443 units/mg protein and by 6 h, 8661 +/- 338 units/mg protein which was three times the control values (P < 0.01). An electron-microscopic study of the structure of neuron nuclei karioplasm showed that by 2-4 h of hemorrhagic shock some neurons showed reduced chromatin content, and the nuclear matrix had clarified with the formation of electron-translucent empty zones. We think that the sharp increase in the nuclear endonuclease activity and the subsequent endonucleolysis can be regarded as a pathogenetic factor in the destructive processes taking place in the nuclei of cortex neurons during prolonged periods of hemorrhagic shock.

Ischemic brain edema as a complication of decompensated hypovolemic shock.

Four-hour arterial hypotension (decompensated phase) in dogs caused by acute blood loss, when the level of arterial pressure of 40 mmHg was maintained beginning from the 2nd and 3rd h by intra-arterial intermittent blood transfusion is an adequate model for reproducing a moderate ischemic edema of the brain. Pronounced hypoperfusion and considerable disturbances in blood fluidity testified to greater severity of hypoxic and hemorheological disturbances in the microvessles of the brain in decompensated animals than in their compensated counterparts. The formation of a brain edema is based on disturbed fluid circulation closely related to the structural pathology of the organelle membranes of the cells of the nervous and vascular tissues. The pathology of the organelle membranes of the cell elements of the vascular tissue leads to direct diffusion of the liquid through the vessel wall into the brain parenchyma thus disturbing intertissue relations. The pathology of the cell element membranes of the nervous tissue (neurocytes, oligodendrogliocytes and astrocytes) leading to intracellular disturbance of fluid circulation makes its contribution to changes in inter-tissue interactions. Brain hyperhydration was revealed only in animals that had sustained decompensated hypovolemic shock.

Metabolic and ultrastructural characteristics of biosynthetic processes in the cerebral cortex at 3 months following a prolonged hemorrhagic shock.

Biochemical indicators (the content of ATP, total RNA and DNA, acid and alkaline phosphatase activity) and ultrastructural changes in cells of the nerve tissue were studied in the cerebral cortex of anesthetized dogs 3 months after they had sustained a 4-h hemorrhagic shock (arterial pressure = 40 mmHg). A new variant of reconstruction of cell membranes and organelles that develops in neurons and gliocytes of the brain in the process of adaptation 3 months after resuscitation is identified. This reconstruction variant is described by (a) the presence of a monolayer of a substance with a medium electron density with the incorporated dense granules instead of the common three-layer organisation; (b) the ability displayed by the organellar structure to detect "errors" in its organisation; (c) changes in the character of intercellular relations. The development of this variant of cell structure in the postresuscitation period is likely to be based on the information disintegration of a cell as a system, resulting in distortion of biosynthesis of supracellular ensembles and biological membranes of nerve cells in particular.

[Evaluation of blood flow heterogeneity in the limbs of dogs by hydrogen clearance in venous blood]. / Ob otsenke neodnorodnosti krovotoka v konechnosti u sobak po klirensu vodoroda v venoznoi krovi.

On the basis of Fick's principle, concept of the tissue blood flow heterogeneity and H2 concentration changes in arterial blood after short-term hydrogen inhalation, a method of calculating some blood flow indices in the limbs by H2 content desaturation changes in venous blood was developed. The suggested method allows to calculate the mean regional flow rate, its compartmentation, and blood flow/tissue volume ratio for the measured components of the regional blood flow. In the limb tissue of anesthetized dogs, two components of the blood flow with the rates 0.79 +/- 0.07 and 0.12 +/- 0.01 were measured, their mean rate being 0.16 +/- 0.01 ml/min. g. The share of the "slow" component in total blood flow and tissue volume was 70 +/- 3 and 93 +/- 1 per cent respectively.

[Detection of a steady increase in endonuclease activity in dog liver cell nuclei after long term arterial hypotension and in post-resuscitation period]. / Obnaruzhenie ustoichivogo povysheniia éndonukleaznoi aktivnosti v kletochnykh iadrakh pechini sobak pri dlitel'noi arterial'noi gipotenzii i v postreanimatsionnom periode.

Presence of Ca2+, Mg(2+)-dependent endonuclease activity was found in dog liver cell nuclei. Specific characteristics of chromatin autolysis were also studied in liver cell nuclei after 2-4 hrs long arterial hypotension as compared with that within the later restoration period 1-3 months. The rate of DNA acid-soluble fraction accumulation correlated directly with the arterial hypotension duration. Quantitative evaluation of the liver tissue Ca2+, Mg(2+)-dependent endonuclease activity was undertaken under conditions of hemorrhagic shock. The enzymatic activity was not normalized both after death and in the postresuscitation period. Analysis of the chromatin autolysis and of alterations in the enzymatic activity during postresuscitation period enabled to suggest that the isozyme spectrum of Ca2+, Mg(2+)-dependent endonuclease was altered.

[Acute and massive hemorrhage: mechanisms of compensation and damage]. / Ostraia massivnaia krovopoteria: mekhanizmy kompensatsii i povrezhdeniia.

To evaluate the body's compensatory reserves, experiments were made on anesthetized (with heparine) mongrel dogs of both sexes weighing 8-25 kg. The experiments have indicated that early monitoring of physiological parameters (external respiration, cardiovascular performance, hemostasis, red blood cell morphometry) in the first 5-10 minutes of acute massive hemorrhage is of high informative value. The generalization of vasoconstriction resulting in higher overall blood volume, the maintenance of venous return, tachypnea, active spontaneous hemodilution, and increased heart rate are the leading mechanisms of perfusion pressure maintenance in early acute hemorrhage. The severity of acute damage to the membranes of red blood cells and endotheliocytes and hence changes occurring in the suspension structure of blood, which drastically impairs its rheological parameters and fluidity, can play the key role in the pathogenesis of decompensatory cardiovascular and visceral functions in prolonged arterial hypotension. The findings open up new possibilities for early evaluation and prediction of the ensurina course of delayed massive hemorrhage.

[Effect of verapamil on the activity of Ca2+-, Mg2+-dependent endonucleases in hypothalamic cell nuclei in long-term hypovolemic shock]. / Vliianie verapamila na aktivnost' Ca2+-, Mg+-zavisimykh éndonukleas v kletochnykh iadrakh gipotalamusa pri dlitel'nom gipovolemicheskom shoke.

Experiments on anesthetized non-inbred 8-17 kg b.w.dogs have demonstrated that cerebral ischemia consequent to 4-hour hypovolemic shock activates Ca(2+)-, Mg(2+)-dependent endonucleases in cell nuclei of the hypothalamus. Intravenous injection of calcium channels blocker verapamil (Orion) in a dose 0.1 mg/kg 30 min before blood loss prevents activation of the above endonucleases and makes it possible to avoid internucleosome fragmentation of genome DNA.

[DNA-endonucleolysis in cell nuclei of brain and liver in patients dying from hemorrhage and after resuscitation: general patterns and differences]. / DNK-endonukleoliz v kletochnykh iadrakh mozga i pecheni pri urmiranii ot korovopoteri i posle reanimatsii: obshchie zakonomernosti i razlichiia.

Specific features of Ca2+, Mg2+ dependent DNA endonucleolysis in the nuclei of the cerebral cortex, hypothalamus and liver were investigated in mongrel anesthetized male and female dogs. The endonucleolysis was studied in different periods of long-term arterial hypotension and in postresuscitation period, with strain pUC 19 plasmids as substrate for determination of nuclear endonuclease activity. It was established that nuclear DNA-endonucleases coupled with chromatin activated earlier in brain cortical and hepatic neurons than in the hypothalamus. Changes in activity of the enzymes directly correlated with duration of CNS ischemia. Active endonucleolysis occurred in cerebral and hepatic nuclei even 3 months after the blood loss and resuscitation. Postresuscitation changes in Ca2+ and Mg2+ dependent endonucleases in cortical nuclei are phasic while in the liver their activity for three months did not differ much from that in the end of hypotension. The activity of nuclear endonucleases in the hypothalamus returned to normal after beginning of resuscitation and did not change later. The data obtained evidence for active involvement of apoptosis mechanisms in brain and liver cell degeneration in massive blood loss and in postresuscitation period including a late one.

[Effect of laser exposure on hemostatic parameters in the preagonal and post-resuscitation period]. / Deistvie lazernogo izlucheniia na parmetry gemostaza v preagonal'nom i postreanimatsionnom periode.

Effects of intravascular low-intensive laser exposure of the blood on the hemostasis during acute blood loss and the early postresuscitation period after 4-min clinical death were studied on narcotized dogs (8-17 kg) of both sexes with different initial levels of heparin. During the preagonal period laser exposure caused hypercoagulation in animals with initial heparin content below 60 micrograms/ml. This acceleration of blood clotting prevented a drop in the activity of antithrombin III and hypercoagulation by the third hour of postresuscitation period. In animals with initial heparin content more than 60 micrograms/ml laser exposure caused hypocoagulation in the presence of reduced activity of antithrombin III by the third hour of the resuscitation period.

[Early prognostic signs of disorders of the regional pulmonary circulation in the dynamics of hemorrhagic shock and in the post-resuscitation period]. / Rannie prognosticheskie priznaki narushenii regionarnogo krovoobrashcheniia legkikh v dinamike gemorragicheskogo shoka i v postreanimatsionnom periode.

The earliest significant unfavourable signs of shock are as follows: an increase in total pulmonary resistance, reduced pulmonary blood flow and tachypnea. Ventilation-perfusion disturbances in the lungs precede the onset of circulation decompensation, which in the pulmonary flow manifests as enhanced pulmonary blood content and elevated pressure in the pulmonary artery. Pulmonary flow damage in the early postresuscitation period is to a great extent predetermined by the degree of circulation disturbances in the lungs, developing during hypotension period, the leading among them being congestive processes in the lungs (increased blood content, hypertension) which progressed in case of unfavourable outcome of resuscitation and reduction of the microvascular bed.

[Significance of DNA endonucleases in the pathogenesis of ischemic lesion of the brain during massive hemorrhage]. / Znachenie DNK-éndonukleoliza v patogeneze ishemicheskogo porazheniia mozga pri massivnoi krovopotere.

Experiments on dogs exposed to hemorrhagic shock demonstrated a direct relationship between the duration of arterial hypertension and activities of Ca(2+)-Mg(2+)-dependent DNA-endonucleases in the cellular nuclei of the cerebral cortex. Appreciable differences in the kinetics of accumulation of acid-soluble products of DNA chromatin in the cellular nuclei of the cerebral cortex were detected: chromatin DNA degradation was the most rapid in the nervous tissue of dogs exposed to 6 h hemorrhagic shock. Verapamil, a Ca blocker, injected to animals 30 min before massive hemorrhage in a dose of 0.1 mg/kg, reliably reduced the activities of Ca(2+)--Mg(2+)-dependent endonucleases of the cellular nuclei of the cerebral cortex by the 4th h of hemorrhagic shock. The detected physicochemical changes in the chromatin DNA of the cerebral cortex cellular nuclei and a progressive increase of activities of Ca(2+)--Mg(2+)-dependent endonucleases during a prolonged hemorrhagic shock are by their nature close to the phenomenon of genomic programmed cellular death.

[Molecular and cellular mechanisms of ischemic damage of the brain in hemorrhagic shock]. / Molekuliarnye i kletochnye mekhanizmy ishemicheskogo povrezhdeniia mozga pri gemorragicheskom shoke.

General lightening of caryoplasma, the appearance of destructive areas in which electron-optic density was undetectable, ruptures in chromatin chains, changes in the nature of structural organization of euchromatic areas at the expense of density redistribution have been found in the nuclei of brain cortex oligodendrogliocytes and astrocytes by electron microscopy of caryoplasma of the nuclei from light neurons during experiments on anesthetized adult mongrel dogs weighing 9 to 26 kg recovered after a 4-hour hemorrhagic shock (BP 40 mm Hg). The nuclei of dark neurons and oligodendrogliocytes had matrix structure similar to control. The nuclei of astrocytes had marked matrix lightening with the formation of large zones in which electron-optic density was undetectable. It is evident that the degree of chromatin destruction in different neurons was not identical in hemorrhagic shock and depended on the functional neuronal activity. It has been established that there is a significant increase in the activity of nuclear Ca2+, Mg(2+)-depended endonucleases associated with chromatin and irreversibly cleaving ds- and ss-chromatin DNA chains. We believe that an increase in Ca2+, Mg(2+)-depended DNA-endonuclease activity should be considered an important pathogenetic factor in destructive processes that take place in the nuclei of brain cortex neurons in severe hemorrhagic shock.

[Changes in the functional status of the left-ventricular myocardium during dying and in the early postresuscitation period]. / Izmeneniia funktsional'nogo sostoianiia levogo zheludochka pri umiranii i v rannii postreanimatsionnyi period.

Experiments on dogs have shown that left-ventricular contractility reduces and the processes of contraction and relaxation are discoordinated during the first minutes of dying from acute blood loss with the mean arterial pressure of 40 mm Hg. During the postreanimation period following a ten-min clinical death spontaneous respiration is resumed late (after 15.1 +/- 3.2 min), as are the corneal reflexes (after 46.1 +/- 8.5 min). If there are no changes in left-ventricular contractility in the course of dying, vital functions of the organism and neurological reflexes recover sooner (respiration after 6.1 +/- 2.0 min and corneal reflexes after 32.6 +/- 4.1 min). A relationship has been revealed between the severity of the postreanimation period and the degree of reduction of left-ventricular myocardial contractility during hypoperfusion (1 to 6 hrs after reanimation): the contractility index in animals with late neurologic recovery was lower than in those with a relatively early neurologic recovery.

[The neurochemical, molecular and ultrastructural mechanisms of the formation of latent postresuscitation encephalopathy]. / Neirokhimicheskie, molekuliarnye i ul'trastrukturnye mekhanizmy formirovaniia skrytoi postreanimatsionnoi éntsefalopatii.

The content of total RNA and DNA, activity of Ca2+, Mg(2+)-dependent DNA endonuclease, and ultrastructural changes in nerve tissue cells were examined in the brain cortex of narcotized dogs 1 to 3 months after a 4-hour hemorrhagic shock (arterial pressure 40 mm Hg). A new variant of reconstruction of cell membranes and organelles formed by them was revealed, developing in the brain neurons in the course of adaptation during the first-third months of the postshock period. Evidently, the molecular base of development of an atypical variant of cell structure rearrangement in the remote period after shock is the internucleosomal fragmentation of a part of the DNA of nerve cells resultant from DNA endonucleolysis and subsequent information disintegration of a cell as a system. This distorts the process of biosynthesis of supramolecular ensembles, specifically, of nerve cell biomembranes.

[Effect of perftoran on cerebral blood flow in the reperfusion period after global transient brain ischemia]. / Vliianie perftorana na tserebral'nyi krovotok v reperfuzionnyi period posle global'noi prekhodiashchei ishemii golovnogo mozga.

It was established, while modeling the global transient cerebral ischemia in experiments with 51 anesthetized nonlinear white rats (weight 300-330 g), that a preliminary administration of perftoran in the discussed pathology does not only improve the reperfusion process in the brain but also levels the deficits of crystalloid solutions used as blood substitutes (by the example of physiological solution). Therefore, the drug has a cytoprotective action on the vascular-system endothelium. A direct admixture of perftoran to the autoblood introduced after global transient cerebral ischemia improves essentially the reperfusion-period course in the brain versus the combination of physiological solution and autoblood.

[Low-energy laser irradiation of blood as a method of prevention of blood circulation decompensation in hemorrhagic shock]. / Nizkoénergeticheskoe lazernoe obluchenie krovi kak sposob profilaktiki dekompensatsii krovoobrashcheniia pri gemorragicheskom shoke.

Effect of low-intensity laser exposure of the blood on the central hemodynamics, oxygen transporting function of the blood, oxygen balance of the organism, and surface configuration of erythrocyte membranes was studied in dogs exposed to 2-h arterial hypotension (arterial pressure 40 mm Hg). Blood exposure was started from the tenth min of hypotension and went on for 45 min (group 2) or 120 min (group 3); group 1 was control. The best results were attained after 45-min laser exposure. A longer exposure creates prerequisites for complications, such as dysadaptation of the vascular tone and delayed decrease of hemoglobin concentration.

[The effect of intravascular helium-neon laser blood irradiation on the state of the compensatory processes in the acute period of hemorrhagic shock and after resuscitation]. / Vliianie vnutrisosudistogo geliineonovogo lazernogo oblucheniia krovi na sostoianie kompensatornykh protsessov v ostrom periode gemorragicheskogo shoka i posle reanimatsii.

The effect of low-intensity intravascular laser blood irradiation on morphofunctional characteristics of erythrocytes and circulation parameters has been studied experimentally on 22 adult inbred anesthetized dogs of both sexes 9 to 23 kg of weight during a 2-hour hemorrhagic shock and in the first hours after resuscitation. It has been established that the use of intravascular laser blood irradiation during 45 min of hemorrhagic shock stabilizes erythrocyte membranes and improves myocardial function. Hyperdynamic and hyperperfusion syndromes were significantly more pronounced in the postresuscitation period in animals treated by intravascular laser blood irradiation than in the control. Thus, the efficacy of intravascular laser blood irradiation was associated both with the recovery of erythrocyte cytoarchitectonics and the enhancement of myocardial contractility.

[Changes in the oxygen balance of the body in the acute period of hemorrhagic shock and after resuscitation under the effect of intravascular helium-neon laser irradiation of the blood]. / Izmeneniia kislorodnogo balansa organizma v ostrom periode gemorragicheskogo shoka i posle reanimatsii pod vliianiem vnutrisosudistogo gelii-neonovogo lazernogo oblucheniia krovi.

The effect of intravascular laser irradiation of the blood (ILIB) on the oxygen budget of the body and blood acid-base balance in the acute period of hemorrhagic shock and after resuscitation was studied in experiments on dogs. Duration of hypotension was 2 h. Laser therapy was started from the 5th-8th min of hypotension and lasted for 45 min; it was performed with a He-Ne laser (ALOK-1, 1 mWt power at the tip of the light guide, wavelength 633 nm) introduced in the right jugular vein. Exposure to ILIB in the acute period of hemorrhagic shock was conducive to creation of favorable conditions for realization of the compensatory mechanisms aimed at improvement of circulation and gas exchange in the lungs, at provision of delivery of oxygen to tissues and its utilization, and, hence, promoted the survival of animals by triggering the regulatory mechanisms of redistribution of the regional bloodflow, improvement of tissue perfusion, and possibly, of oxyhemoglobin dissociation processes in red cells.

[The effect of low-intensity laser radiation on the parameters of the blood anticoagulation system in the early postresuscitation period]. / Vliianie nizkointensivnogo lazernogo izlucheniia na parametry protivosvertyvaiushchei sistemy krovi v rannem postreanimatsionnom periode.

The effect of low-intensive laser exposure at a wavelength of 634 nm and power 1 mWt at the tip of the light-guide on the blood anticoagulative system was studied in mongrel narcotized dogs of both sexes weighing 8 to 16 kg during the postresuscitation period after 4-min clinical death from massive blood loss. The anticoagulative system in the blood plasma was depleted during the reanimation period (the activities of plasmin and fibrinogen-heparin complexes and the level of heparin dropped). Intravascular laser exposure of the blood (for 30 min during blood loss after drop of the mean arterial pressure to 40 mm Hg and at the beginning of the second hour of the postreanimation period) boosted the activities of plasmin and fibrinogen-heparin complexes but failed to increase the level of heparin in the postreanimation period.