A. I. Krivchenko

Nitric oxide-mediated central sympathetic excitation promotes CNS and pulmonary O2 toxicity

In hyperbaric oxygen (HBO(2)) at or above 3 atmospheres absolute (ATA), autonomic pathways link central nervous system (CNS) oxygen toxicity to pulmonary damage, possibly through a paradoxical and poorly characterized relationship between central nitric oxide production and sympathetic outflow. To investigate this possibility, we assessed sympathetic discharges, catecholamine release, cardiopulmonary hemodynamics, and lung damage in rats exposed to oxygen at 5 or 6 ATA. Before HBO(2) exposure, either a selective inhibitor of neuronal nitric oxide synthase (NOS) or a nonselective NOS inhibitor was injected directly into the cerebral ventricles to minimize effects on the lung, heart, and peripheral circulation. Experiments were performed on both anesthetized and conscious rats to differentiate responses to HBO(2) from the effects of anesthesia. EEG spikes, markers of CNS toxicity in anesthetized animals, were approximately four times as likely to develop in control rats than in animals with central NOS inhibition. In inhibitor-treated animals, autonomic discharges, cardiovascular pressures, catecholamine release, and cerebral blood flow all remained below baseline throughout exposure to HBO(2). In control animals, however, initial declines in these parameters were followed by significant increases above their baselines. In awake animals, central NOS inhibition significantly decreased the incidence of clonic-tonic convulsions or delayed their onset, compared with controls. The novel findings of this study are that NO produced by nNOS in the periventricular regions of the brain plays a critical role in the events leading to both CNS toxicity in HBO(2) and to the associated sympathetic hyperactivation involved in pulmonary injury.

Baroreflex-mediated cardiovascular responses to hyperbaric oxygen.

The cardiovascular system responds to hyperbaric hyperoxia (HBO2) with vasoconstriction, hypertension, bradycardia, and reduced cardiac output (CO). We tested the hypothesis that these responses are linked by a common mechanism-activation of the arterial baroreflex. Baroreflex function in HBO2 was assessed in anesthetized and conscious rats after deafferentation of aortic or carotid baroreceptors or both. Cardiovascular and autonomic responses to HBO2 in these animals were compared with those in intact animals at 2.5 ATA for conscious rats and at 3 ATA for anesthetized rats. During O2 compression, hypertension was greater after aortic or carotid baroreceptor deafferentation and was significantly more severe if these procedures were combined. Similarly, the hyperoxic bradycardia observed in intact animals was diminished after aortic or carotid baroreceptor deafferentation and replaced by a slight tachycardia after complete baroreceptor deafferentation. We found that hypertension, bradycardia, and reduced CO--the initial cardiovascular responses to moderate levels of HBO2--are coordinated through a baroreflex-mediated mechanism initiated by HBO2-induced vasoconstriction. Furthermore, we have shown that baroreceptor activation in HBO2 inhibits sympathetic outflow and can partially reverse an O2-dependent increase in arterial pressure.

Dynamics and Control Mechanisms in Maintenance of Regional Cerebral Blood Flow

Experiments with rabbits, cats, and monkeys during recording of complex physiological processes (LEPG, ThG, Po2, Pco2, and ECoG) in functionally discrete brain regions of awake animals have shown that functional changes, expressed as desynchronization effects on ECoG, are followed by an increase of local blood flow (LCBF) in regional brain cortex up to 0.3 to 0.4 ml per minute per 1 gm brain tissue or an increase of 35% to 45% of resting levels of LCBF. Under normal physiological conditions LCBF and Po2 change periodically without any external interference at frequency ranges 0.005 to 0.2 cps. This is characteristic of all brain regions and all species of animals investigated. These variations range in amplitude as much as 28% of the mean level of LCBF. Changes of LCBF have no correlation with changes of systemic blood pressure (SAP). Local control mechanisms appear to be responsible for them. The interrelationships of changes of functional activity and CBF in local regions of awake brain are characterized by a very short time delay (less than one second), but under narcosis it increased up to ten seconds. All these facts taken together suggest that control mechanisms responsible for local brain vascular reactions have two components; the first is metabolic and the second is neurogenic in nature.

A current viewpoint on structure and evolution of collagens. I. Fibrillar collagens

This review summarizes current data of structure of the most representative group of superfamily of collagens—fibrillar collagens. The attention is focused on structural organization of individual domains and their functional role in the hierarchical stacking of collagen α-chains. There are presented characteristics of the main stages of biosynthesis and the supramolecular processing of fibrillar collagens. Also considered are some aspects of evolution of fibrillar collagens. The role of duplication of genome and genes, intergene combination, and translocation of exons in evolution of collagen genes is discussed.

Baroreceptor afferents modulate brain excitation and influence susceptibility to toxic effects of hyperbaric oxygen

Unexplained adjustments in baroreflex sensitivity occur in conjunction with exposures to potentially toxic levels of hyperbaric oxygen. To investigate this, we monitored central nervous system, autonomic and cardiovascular responses in conscious and anesthetized rats exposed to hyperbaric oxygen at 5 and 6 atmospheres absolute, respectively. We observed two contrasting phases associated with time-dependent alterations in the functional state of the arterial baroreflex. The first phase, which conferred protection against potentially neurotoxic doses of oxygen, was concurrent with an increase in baroreflex sensitivity and included decreases in cerebral blood flow, heart rate, cardiac output, and sympathetic drive. The second phase was characterized by baroreflex impairment, cerebral hyperemia, spiking on the electroencephalogram, increased sympathetic drive, parasympatholysis, and pulmonary injury. Complete arterial baroreceptor deafferentation abolished the initial protective response, whereas electrical stimulation of intact arterial baroreceptor afferents prolonged it. We concluded that increased afferent traffic attributable to arterial baroreflex activation delays the development of excessive central excitation and seizures. Baroreflex inactivation or impairment removes this protection, and seizures may follow. Finally, electrical stimulation of intact baroreceptor afferents extends the normal delay in seizure development. These findings reveal that the autonomic nervous system is a powerful determinant of susceptibility to sympathetic hyperactivation and seizures in hyperbaric oxygen and the ensuing neurogenic pulmonary injury.

Antiepileptic Drugs Prevent Seizures in Hyperbaric Oxygen: A Novel Model of Epileptiform Activity.

Breathing oxygen at sufficiently elevated pressures can trigger epileptiform seizures. Therefore, we tested the hypothesis that pre-treatment with FDA-approved antiepileptic drugs could prevent seizure onset in hyperoxia at 5 atmospheres absolute. We selected drugs from two putative functional categories, Na+-channel antagonists and GABA enhancers, each administered intraperitoneally at four doses in separate groups of C57BL/6 mice. The drugs varied in efficacy at the doses used. Of the five tested Na+-channel antagonists, carbamazepine and lamotrigine more than tripled seizure latency compared to values seen in vehicle controls. Primidone, zonisamide and oxcarbazepine were less effective. Of the four GABA reuptake inhibitors, tiagabine and vigabatrin also increased seizure latency by more than three times control values; valproic acid was less effective, and the GABA synthesis promoter gabapentin was intermediate in effectiveness. We infer that Na+-channel function and GABA neurotransmission may be critical targets in the pathophysiology of CNS O2 toxicity. Because these essential components of neuronal excitation and inhibition are also implicated in the pathogenesis of other seizure disorders, including generalized epilepsy, we propose that, at some level, common pathways are involved in these pathologies, although the initiating insults differ. Furthermore, hyperoxic exposures are not known to cause the spontaneously-recurring seizures that characterize true clinical epilepsy. Nonetheless, experimental studies of hyperbaric oxygen toxicity could provide new insights into molecular mechanisms of seizure disorders of various etiologies. In addition, the neuropathology of hyperbaric oxygen is particularly relevant to the hypothesis held by some investigators that oxidative stress is an etiological factor in clinical epilepsies.

Limit of Change in Unsaturation Index of Fatty Acid Composition of Phospholipids at Adaptation of Molluscs to Biogenic and Abiogenic Environmental Factors

Comparative study of fatty acid composition of total phospholipids, as well as of phosphatidylcholine and phosphatidylethanolamine from hepatopancreas and leg muscle was carried out on several representatives of gastropod molluscs (Gastropoda) and on the bivalve mussel Mytilus edulus (Bivalvia). The objects of our study were marine littorins Littorina saxsatilis adapted to different temperature conditions of White Sea and Barents Sea, the freshwater lymnaea Lymnaea stagnalis infested by Trematoda, and mussels from the White and Black Seas. It was shown that depending on the existence conditions of the studied tissue or lipid, the maximal changes occurred in the percentage of saturated acids (4–83%); the percentage of unsaturated acids was less expressed (1–14%) and the changes in unsaturation index (UI), on average, did not exceed 20%. It was suggested that the revealed quantitative restriction of the UI change under the action of various external factors is a limit for maintenance of the membrane fluidity necessary for normal cell viability, specifically in the studied ectothermic molluscs.

Phospholipids composition of cell nuclei of rat’s brain in dynamics of normal ontogenesis and after hypoxia

The phospholipid composition of cell nuclei was examined in different animals and at various states of the body differing in the organizational level [1, 2]. However, the composition of lipids of higher vertebrates during ontogeny (prenatal and postnatal), as well as the phospholipid composition of brain cell nuclei at different pathological states, has not been studied sufficiently. For example, hypoxia, which is frequent during different periods of ontogeny and is a stress factor, affects fatty acid composition of lipids [3] and, hence, membrane fluidity [4]. Taking into account the important role of phospholipids (located predominantly in nuclear membranes) in the structural and functional organization of cell nuclei, we studied phospholipids and the unsaturation index of their fatty acids in the cell nuclei of the rat brain during different periods of ontogeny and after hypoxia that occurred during these periods. The study was carried out on 19-day-old rat embryos and young (5and 45-day-old), adult (3.5to 4-month-old) and old (1.4to 1.5-year-old) rats. At these stages of development, part of animals was exposed to hypoxia, and the remaining animals served as a control. The brains of several embryos were pooled into a single sample; there were at least five such samples. The brains of five-day-old rats were subjected to the same procedure. In other age groups, every brain was analyzed separately. There were at least five rats in each group. One-hour hypoxia was induced by passing a nitrogen–oxygen mixture containing 8% of oxygen through a chamber into which pregnant females or animals of the corresponding age groups had been put. During the exposure, room temperature was maintained in the chamber, and the concentrations of oxygen and carbon dioxide were controlled by means of an SF-101 gas analyzer (France). After brain homogenization, cell nuclei were isolated by centrifugation and phospholipids were extracted from them by the standard method [5]. Phospholipids were divided into classes by means of two-dimensional thin-layer chromatography on silica gel [6]. Methyl esters of fatty acids of all phospholipids were analyzed by means of gas–liquid chromatography and identified with the use of a mass-spectrometer (LKB, Sweden). Then, the unsaturation index (the number of double bonds in 100 fatty acid molecules) was calculated.

The effect of a collagen tripeptide fragment (GER) on fibroblast adhesion and spreading depends on properties of an adhesive surface

The effect of collagen tripeptide fragment GER on the adhesion and spreading of mouse embryonic fibroblasts STO to different substrates (polystyrene plastic, poly-L-lysine, fibronectin, gelatin) has been studied. It was found that tripeptide GER was involved in fibroblast adhesion and spreading. The cell response depended both on the mode of tripeptide addition to culture medium and the substrate type. Coincubation of fibroblasts with tripeptide stimulated the cell attachment and spreading to untreated plastic and plastic coated with fibronectin or gelatin but did not change cell adhesion to immobilized poly-L-lysine. Preincubation of cells with tripeptide resulted in partial inhibition of fibroblast adhesion and spreading on fibronectin- and gelatin-coated substrata. It was shown that activation and inhibition of adhesive processes after tripeptide treating was higher on fibronectin than gelatin. The data obtained support the assumption about concerted action of tripeptide GER (activity was dependent both on the used concentration of the tripeptide and the mode of tripeptide addition to culture medium) and chemical characteristics of substrate (polymers of styrene and L-lysine, ECM proteins in native (fibronectin) or partly denatured (gelatin) form) on the cell adhesion and spreading. The main targets that GER peptide may affect during the formation of cell-substrate interactions are discussed.

Effect of Total Hypothermia on the Fatty Acid Composition of Blood Phospholipids of Rats and Ground Squirrels and of the Light Irradiation on Chemical Processes in Lipid Extract

Effect of hypothermia on the fatty acid composition of rat and ground squirrel blood phospholipids is studied. Different reaction of these animals to cooling is revealed; in rats no changes were observed in the fatty acid composition of blood phospholipids, whereas in the winterhibernating ground squirrels there were significant changes in the content of individual fatty acids (FA). The content of monoenic acids in ground squirrels decreased almost by 50%, while the content of saturated acid (C18) and of polyenic acids C18: 2ω6 and C20: 4ω6 rose significantly. Such changes seem to be the mechanism that promotes maintenance of the organism viability under conditions of a decreased level of metabolism, heart rhythm, and body temperature and is evolutionary acquired. At the same time, the observed changes in the content of individual FA do not lead to sharp changes in such integrative parameters as the total non-saturation of phospholipids, which determines liquid properties of chylomicrons and other lipolipoprotein transport particles of the ground squirrel blood. There are studied absorption spectra of blood lipid extracts of rats and ground squirrels under effect of light as well as effect of light upon the FA composition of lipid extracts of these animals. The FA composition of lipid extracts has been established to remain practically constant, whereas the character of changes of spectra under action of light indicates the presence in the extracts of oxidation-reduction reactions. The obtained data allow suggesting that in the lipid extract there occurs cooperation of both the phospholipids molecules themselves and of them with other organic molecules, which makes it possible for fatty acids to participate in processes of transport both of electrons and of protons. This novel role of FA as a participant of the electron transfer might probably be extrapolated to chemical reactions (processes) occurring inside the membrane.