Oxidative stress is among the major factors that cause nerve cell death under severe hypoxia. It involves intense formation of free radicals and reduced activity of the antioxidant defense [1–3]. The induction of reactive oxygen species (ROS) and further activation of peroxidation result in damage of biopolymers: nucleic acids, proteins, and polysaccharides. They also disturb intercellular and intracellular signaling [4–9]. Even short-term ischemia or severe hypoxia may injure parts of brain and thereby cause neurologic impairment and behavioral dysfunction. It has been found that oxidative stress contributes greatly to neurodegenerative processes noted in strokes, brain infarctions, craniocerebral injuries, and various neurodegenerative diseases [10–16]. In other words, hypoxia disrupts a variety of processes by launching a cascade of irreversible metabolic changes in parts of the brain. This disruption manifests itself, in particular, as functional aberrations in the central nervous system (CNS) [17, 18].

Neuron injury and death is a complex process, which includes many interdependent factors: oxygen shortage (hypoxia), energy shortage, membrane depolarization, ionic homeostasis disruption, hyperactivation of exciting amino acid receptors, calcium intake into neurons, excessive ROS formation, and tissue acidosis [19, 20]. The possibility of controlling the effects of hypoxia with pharmaceuticals may rest upon their ability to affect metabolic processes and CNS functioning.

Therefore, the development of new drugs and study of their ability to prevent excessive radical accumulation and support the antioxidant defense of the body are topical. Substances with antioxidant properties are of special interest, as they add to neuron protection from the action of universal insults, which underlie the majority of neurodegenerative diseases [21, 22].

In this regard, use of water with a modified isotope composition, namely, deuterium-depleted water (DDW), is considered as a possible approach to the correction of metabolic and functional CNS disorders.

It has been found that addition of DDW to the animal diet reduces the deuterium content in blood plasma and tissues of various organs, including the brain [23–27]. The change in the content of deuterium owing to its replacement by protium may affect many biologic processes [28]. In experiments, decreased deuterium content in the body increased the function of antioxidant and detoxifying systems [29, 30] and affected the functional state of the CNS. Specifically, DDW is beneficial for stress tolerability and anxiety level in laboratory animals long exposed to a stress factor, reduces anxiety in strange environment [31], improves long-term memory, and stimulates exploratory behavior [32, 33].

These facts prompted us to investigate the DDW effect on oxidative processes in the brain and CNS functioning in hypoxia.

In this work, we studied the DDW influence on the anxiety level and learning ability of rats in normoxia and acute hypercapnic hypoxia.

MATERIALS AND METHODS

Deuterium-depleted water (50 ppm) for the experiments was produced in a device designed at Kuban State University [34, 35]. Physiologically adequate drinking water was obtained by supplementing the DDW with mineral salts (mg/L): total salts 314–382, bicarbonates 144–180, sulfates < 1, chlorides 60–76, calcium 6, magnesium 3, sodium 50–58, and potassium 50–58. Distilled water with the natural deuterium concentration of 150 ppm and the same mineral salt supplements was prepared as a control. Animals were kept in a vivarium under natural illumination with free access to food and water.

Experiments were conducted with 28 male rats of the Wistar breed at ages of 2.5 months weighing 240 to 270 g. The effect of DDW on the CNS function was assessed under normoxic conditions without exposure to hypoxia and 1 day after experimental hypoxia.

Acute hypercapnic hypoxia was modeled by placing rats into 1-L air-tight vessels. The animals were kept there until the first agonal breath. The rats were then taken from the vessels and placed in standard cages [36].

Experiments were done in the first half of the daytime. The animals were divided into four groups, seven in each:

Group 1 (control). Intact rats that received water with the natural deuterium concentration (150 ppm) for 6 weeks and that did not that experience hypoxia.

Group 2. Rats that received DDW (50 ppm) for 6 weeks and that did not experience hypoxia.

Group 3. Rats that received water with the natural deuterium concentration for 6 weeks and that experienced acute hypoxia on day 43 of the experiment.

Group 4. Rats that received DDW for 6 weeks and that experienced experimental acute hypoxia on day 43 of the experiment.

The CNS function was tested in the animals in an elevated plus maze (EPM) and a T maze [37, 38]. The EPM test assessed the anxiety of the experimental animals. The EPM had two open and two closed 90-cm long arms with 15-cm high walls. The numbers of entries to the closed and open arms, rearings, head dips, groomings, and times spent in the closed arms, open arms, and center of the maze were recorded for 5 min. Learning was assessed in the T maze test with a positive reward. Food and water consumption are the best-studied behavior patterns; therefore, they serve as the conventional model of motivated behavior [37].

The effect of long-term DDW uptake on the conditioning with positive reward was assessed after hypoxia in comparison with rats that were not exposed to hypoxia. The control group consisted of intact rats that received standard water with the normal deuterium content. After habituation to the maze, rats learned to alternate right and left runs with a positive reward. The food reward (bread balls) was placed alternately in feeders of the right and left arm. To increase motivation, the learning occurred after food deprivation. Rats that fasted for 48 h were placed into the start arm of the T maze, and a feeder with food was placed into one of the goal arms. The door of the start arm was opened 30 s after the animal was placed. The click of the opening door served as a conditioned stimulus. The following parameters were recorded: the percentage of correct runs and the time of the conditioned response (the time from the leaving of the starting chamber to taking food or the time of a wrong run). Each animal made 20 runs within 4 days of the learning to alternate right and left runs. The preservation of the memory trace was tested in 2 days. Daily run results were averaged.

The results were assessed with STATISTICA 10 software. The Kruskal–Wallis H test was used to test the hypothesis of equal medians in the multiple comparison of several independent samples. The significance of differences in quantitative parameters between two independent small samples was assessed by the nonparametric Mann–Whitney U test. Differences between two small samples with pairwise measurements were tested by the Wilcoxon W test. Differences between samples were considered significant at p < 0.05.

RESULTS

In experiments on the influence of water with an altered isotope composition (D : H = 50 ppm) on rat anxiety in the EPM test under normal conditions and after experimental hypercapnic hypoxia, rats of group 3, which received water with the natural isotope ratio, showed a higher level of anxiety than the control group 1 (Fig. 1). They less frequently (by 37.5%) entered the closed arms of the EPM, reared less frequently(by 46.7%), and never dipped their heads from the open arms of the maze. They spent less time (by 66.7%) in the center and more time in the closed arms. They did not enter the open arms, whereas the control animals of group 1 spent less than 2% of the test time there (4 s).

Fig. 1.
figure 1

The influence of deuterium-depleted water on rat anxiety in the EPM test in the norm and 1 day after hypoxia. (a) Time spent in the center, closed arms, and open arms. (b) Number of entries into closed and open arms, rearings, head dips, and groomings. The data are presented as M ± m. Differences are significant: * at p < 0.05 compared to group 1 (150 ppm, control), # at p < 0.05 compared to group 3 (150 ppm, hypoxia).

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Rats of group 4 (50 ppm, hypoxia) were much less anxious than group 3 (150 ppm, hypoxia). In particular, rats of group 4 spent more time in the center and less in closed arms (p < 0.05). They showed a higher level of exploratory activity (The number of rearings was 41.2% higher) and a lower level of grooming frequency (by 73.3%). As well, head dips were noted. Like group 3, they did not enter the open arms.

In animals of group 2 (50 ppm, no hypoxia), nearly all anxiety indices were significantly lower than in the control.

Thus, the analysis of behavior patterns in the EPM test 1 day after experimental hypercapnic hypoxia revealed high anxiety in rats that received water with the natural isotope ratio that experienced hypoxia. Long (42 days) DDW consumption significantly decreased anxiety in animals with or without hypoxia.

The study of the effect of DDW (50 ppm) on conditioning with positive reward in the T maze in rats that did and did not experience hypercapnic hypoxia (Fig. 2) shows that normally DDW did not affect animal learning. The number of correct runs in group 2 (50 ppm) was practically the same as in the control on all days of the learning phase. The number of correct runs in animals of group 3 (150 ppm, hypoxia) was significantly (by 23%) lower than in the control. On day 2 of the learning, the percentage of correct runs increased to 57% but it was still less than in the control (68%). The trend towards learning improvement in group 3 rats (150 ppm, hypoxia) was observed in the days that followed as well. On day 4 of the learning phase, the percentages of correct runs in groups 3 and 1 were practically equal, whereas the number of correct runs in group 4 (50 ppm, hypoxia) was at the control level in all days of the learning phase, and on day 1 it significantly exceeded that in group 3.

Fig. 2.
figure 2

The influence of deuterium-depleted water (50 ppm) on the percentage of correct runs in the T maze test in the norm and after exposure to hypoxia. The data are presented as M ± m. Differences are significant: * at p < 0.05 compared to group 1 (150 ppm, control), at # p < 0.05 compared to group 3 (150 ppm, hypoxia).

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As well, group 3 (150 ppm, hypoxia) showed a pronounced difference in the time of the conditioned response (Fig. 3). Although the time of the conditioned response was the longest on the first day of the learning phase in all animals, this index in group 3 significantly exceeded that in the control group, by 66.7%, and exceeded it by 52.7% in group 4 (50 ppm, hypoxia). Although the conditioned response time progressively shortened each day in all groups, on days 1 to 3 it was longer in group 3 than in the control and significantly longer than in group 4. The times of conditioned response in all groups became the same as in the control group 1 only on day 4 of the learning phase.

Fig. 3.
figure 3

The influence of deuterium-depleted water on the time of conditioned reaction in the T maze test. The data are presented as M ± m. Differences are significant: * at p < 0.05 compared to group 1 (150 ppm, control), at # p < 0.05 compared to group 3 (150 ppm, hypoxia).

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Two days after the end of the learning phase, all animals preserved the habit of the conditioned alternation of right and left runs. No significant variation among groups was noted. The times of the conditioned response did not vary either.

Thus, we found that long-term DDW consumption did not affect animal learning under normal conditions, whereas its consumption prior to hypoxic treatment favored the preservation of the ability to learn and memory at the control level; that is, it exerted a notable protective antiamnesic action.

DISCUSSION

As mentioned above, the induction of ROS and subsequent activation of peroxidation in ischemia–hypoxia damages biopolymers, that is, nucleic acids, proteins and polysaccharides, and induces changes in integrating inter- and intracellular signaling [4, 7]. Even short-time ischemia or severe hypoxia may cause injuries in parts of the brain accompanied by neurologic impairment and behavioral dysfunction. Thus, hypoxia disrupts a variety of processes, including those related to behavior, by triggering a cascade of irreversible metabolic changes in parts of the brain [18]. Antioxidant agents are considered promising in the pharmacological treatment of brain ischemia [4]. It has been shown that oxidative processes are intensified in the brain of stressed rats. The presence of water with a modified D : H ratio in the rat diet prior to the hypoxic event inhibits oxidative processes in the brain. All parameters recorded (peroxidation level, malonic dialdehyde content, and catalase activity) remain at the control level.

Hypoxia significantly impairs animal behavioral responses, causes amnesia, increases emotional anxiety, and weakens the orientational and explorational activity [36, 39]. Studies of behavioral responses in the EPM and T maze tests show that long-term DDW consumption prior to the hypoxia event reduces emotional anxiety in rats, is beneficial to orientational and exploratory activity, and favors learning and memory preservation at the control level; that is, it exerts pronounced anxiolytic and protective antiamnesic effects.

Earlier, it was found that long-term DDW consumption reduced the D : H ratio in the rat brain and increased its antioxidative potential [40]. It is also known that oxidative stress disrupts cognitive functions of the CNS [41]. We presume that the anxiolytic and antiamnesic effects of DDW consumption detected in our study are due mainly to the substitution of protium for deuterium in brain tissues and to the effect of this substitution on the antioxidant systems of the brain, thereby improving the resistance to oxidative stress factors.

We propose that the effect of DDW consumption on metabolic processes is mediated by isotopic substitution (H for D) in active and allosteric centers of enzymes and by replacement of HDO molecules with H2O in the hydration shells of proteins and nucleic acids [42]. The selective changes in active and allosteric centers of enzymes associated with hydrogen isotope exchange in easily dissociating groups (hydroxy –OH, thiol –SH, and primary –NH2 and secondary =NH amino groups), which increase the protium content, may alter the rates of catalytic processes by lowering the activation energies of the transition states of molecules in biocatalysis [42].

In addition, the change of the isotopic D : H ratio in tissues and biologic fluids may alter the functional state of the body associated with general nonspecific adaptive processes responding to any endogenous or exogenous factor [43]. This effect may also be among the mechanisms that mediate the anxiolytic and antiamnesic action of long-term DDW consumption.

Our data indicate that DDW is promising as an antiamnesic and anxiolytic pharmaceutical and that further studies of its action are required.