The effects of intranasal implantation of mesenchymal stem cells on nitric monoxide levels in the hippocampus, control of cognitive functions and motor activity in a model of cerebral ischemia in rats

doi:10.21203/rs.3.rs-2075480/v1

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The effects of intranasal implantation of mesenchymal stem cells on nitric monoxide levels in the hippocampus, control of cognitive functions and motor activity in a model of cerebral ischemia in rats

https://doi.org/10.21203/rs.3.rs-2075480/v1

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published 22 Feb, 2023

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Hypoxia occurs in situations of disbalancebetween metabolic needs and the supply of oxygen to organs and tissues of the body. In this regard, tissue hypoxia and ischemia are essential components of the pathogenesis of many diseases. One of the promising areas of research into the mechanisms of ischemia is attempting to weaken the negative effect of hypoxia and ischemia in the brain by using a variety of techniques that activate neuroprotective mechanisms. Here, we aimed to assess the dynamics of restoration of motor activity control in an experimental model of ischemic stroke in rats (cerebral ischemia – CI) after intranasal perineural implantation of mesenchymal stem cells into the receptive field of the olfactory nerve.

It was found that the perineural administration of MSCs to rats in the acute period of cerebral ischemia was accompanied by clear signs of recovery of cognitive and motor functions within 1 and 3 days after the operation. On the seventh day after ischemia modeling, rats with the introduction of MSCs had no distinctive features in the control of motor activity compared to the period before the operation in the same rats. In the hippocampus of rats after modeling ischemia, a significant decrease in the content of NO by about 50% relative to the initial level is observed after one day. In the hippocampus of rats in which ischemia was modelled with simultaneous intranasal administration of MSC, a significant decrease in NO content by 39% relative to the initial level was also observed after one day. The content of NO increases slightly, but the difference in the level of NO relative to ischemic rats was not significant. The copper content in the hippocampus in the rats of these two groups did not change. There was a tendency to increase the efficiency of the antioxidant system 1 day after ischemia in both studied groups, and this effect was more pronounced with intranasal administration of MSC.

mesenchymal stem cells

ischemic brain stroke

nitric oxide

cooper

electron paramagnetic resonance

The issues of an effective and sustainable increase in the resistance of the human body to the development of hypoxia and ischemia have not yet been resolved. Hypoxia occurs in situations of discrepancies between the metabolic needs of the whole organism and/or various organs and tissues and their oxygen supply [1, 2, 3]. In this regard, tissue hypoxia and ischemia are key components of the pathogenesis of many diseases [4, 5]. The problem of hypoxia and ischemia is especially relevant for the neural networks of the brain, which control the somatic and visceral functions of the body and can function optimally only with a stable supply of oxygen. A significant number of studies have been devoted to seeking mechanisms and effective approaches to adapting to hypoxia in the whole organism and various organs and tissues [2, 6, 7, 8]. One of the directions in this aspect is an attempt to reduce the negative effects of hypoxia and ischemia on the brain using a variety of techniques that activate endogenous neuroprotective mechanisms [9, 10, 11]. For this purpose, it seems promising to use mesenchymal stem cells (MSCs), which have a high reparative and immunosuppressive potential, as well as a directed migratory ability to reach damaged tissues to activate proliferative processes in damaged areas [12, 13, 14]. It is believed that MSCs manifest their reparative potential due to the secretion of highly active molecules, the formation and release of exosomes containing miRNAs, as well as factors that have an immunosuppressive effect [13, 15, 16, 17]. The positive effects of MSCs are also hypothetically explained by the inhibition of lipid peroxidation, an increase in the antioxidant properties of cells and tissues, which is accompanied by an active release of pro-angiogenic and anti-apoptotic cytokines from MSCs, control of inflammation and immune responses, activation of reparative processes in cells and tissues, accompanied by an increase in resistance to hypoxia [13, 18].

To rationally use MSCs, methods for their targeted delivery to damaged areas of the brain are being actively developed [17, 19]. For example, systemic administration of stem cells into venous or arterial vessels of the bloodstream of patients has been used in cases of developing destructive processes in the CNS [20, 21]. The disadvantage of this technique is the diffuse distribution of MSCs throughout the circulatory system of the whole organism, so a relatively small number of cells reach the blood-brain barrier, and an even smaller number passes it (about 1–3%) [22]. The introduction of MSCs into the cerebrospinal fluid pathways during brain injuries was tested, most often by lumbar puncture [23], without taking into account the need for MSCs to overcome the intense cerebrospinal fluid flow from the brain to the spinal cord. Another method of MSCs implantation is based on the local injection of cells during neurosurgical operations into the damaged area of the brain [24].

The indicated shortcomings of the abovementioned methods of treating brain injuries and stroke using MSCs are pushing the search for more effective ways to implant MSCs in the brain. In previous studies, it was found that within no more than 1 hour after implanting MSCs into the submucosal space of the nasal cavity of rats in the region of the terminals of the olfactory nerve, MSCs begin to accumulate in damaged areas of the brain and continue to accumulate throughout the day [25, 26]. It has been shown that MSCs migration is not carried out randomly throughout the brain, but rather in somatotopic dependence from the endings of the olfactory nerve - in the nasal cavity to the olfactory bulb into the anterior cranial fossa, and from the terminals of the trigeminal nerve - to its nuclei in the brainstem (in the posterior cranial fossa) due to their perineural migration [19, 25, 26, 27].

Thus, here we have studied and established the conditions for experimental substantiation of the selective methods of perineurally implanting stem cells for their subsequent migration into the anterior or posterior cranial fossa to activate reparative and metabolic processes in the brain aiming at restoring damaged neural networks and controlling impaired brain functions after a stroke [26, 27].

Studies of stroke models reveal that restoration of brain function involves many mechanisms, including cerebral angiogenesis, axonal and dendritic growth, oligodendrogenesis, neurogenesis, and restoration of the balance of neurotransmitters and signalling molecules. At present, a group of diseases has been experimentally and clinically identified, in the development of which pronounced changes in the exchange of the gaseous mediator nitric oxide (NO) are observed. Among them, most notably, diseases of the cardiovascular system including hypertension, myocardial infarction, pulmonary hypertension, atherosclerosis, as well as stroke [28, 29, 30, 31]. NO is capable of exerting both activating and inhibitory effects on various metabolic processes in the human body [32, 33]. NO plays an essential role in the functioning of neurons in the brain and the potentiation of long-term memory [29, 34].

Elucidation of the role of NO in the physiology and pathology of the cell makes it possible to modulate the NO generation systems to enhance the protective and physiological action of NO and simultaneously limit its damaging effects. It should noted that, according to scientific literature, the effects of NO depend not only on its concentration, but also on the site of its production by isoforms of NO synthase (NOS), diffusion in cells and tissues, interaction with reactive oxygen species, and many other factors [32, 33]. Therefore, both the concentration and the site of NO generation in body tissues from substrates or pharmacological donors of NO should be taken into account when predicting the results of its biological action.

Recently, electron paramagnetic resonance (EPR) has been considered one of the most effective methods for the detection and quantitative determination of NO in biological tissues [35, 36]. This is due to the development of the technique by Vanin et al. [35] using the spin capture method, which is based on the reaction of a radical (in this case, NO) with a spin trap. As a result of this reaction, an adduct with a characteristic EPR spectrum is formed. The authors used the Fe^{2 +} complex with diethyldithiocarbamate (DETC) to capture NO and form a stable ternary complex (DETC)₂-Fe^{2 +}-NO in various animal tissues. This complex is characterized by an easily recognizable EPR spectrum with a g-factor g = 2.035–2.040 and a triplet hyperfine structure. This method has a sensitivity of 0.04–0.4 nM allowing direct measurements and is highly sensitive due to the use of spin traps.

The purpose of the study was to evaluate the dynamics of restoring the control of cognitive and motor functions as well as NO production in the hippocampus of rats in an experimental model of ischemic stroke in the hippocampus after intranasal implantation of MSCs into the receptive field of the olfactory nerve for subsequent perineural migration of MSCs to damaged areas of the brain located in the anterior and middle cranial fossae.

The experiments were carried out on male rats (n = 90), weighing 200–250 grams. The animals were kept under standard vivarium conditions with access to food and water ad libitum. All surgical procedures were performed on anaesthetized animals (55.6 mg/kg ketamine, 5.5 mg/kg xylazine, 1.1 mg/kg acepromazine, intraperitoneally).

Cerebral ischemia (CI) was modelled by ligation of both common carotid arteries at the bifurcation level (Brain Center, Institute of Physiology of the National Academy of Sciences of Belarus, Minsk). Three groups of animals were formed: 1) group 1 – CI (in this group, rats were anaesthetized and surgical access to the area of the carotid arteries was established, with further bilateral ligation of the common carotid arteries; n = 30). 2) group 2 – CI + MSC (included animals after ligation of the common carotid arteries with simultaneous intranasal perineural administration of MSCs; n = 30). 3) Control group (anesthesia and surgical access to the carotid arteries area is used for this, but without ligation of the common carotid arteries) (control, n = 30). In each group, 10 animals underwent behavioral tests, and the hippocampus was extracted from 10 animals after decapitation for subsequent measurements of NO and Cu by EPR spectroscopy. Another 10 animals in each group did not participate in the experiments, they served as a survival control during the entire time of the experiments.

For implantation of stem cells, primary culture of MSCs obtained from the adipose tissue of female rats (n = 13), weighing 250–300 grams, was used according to the method approved by the Scientific Council of the Institute of Physiology of the National Academy of Sciences of Belarus (protocol No. 8, 26.08.2010). MSCs were cultured for seven days. The concentration of living cells in the suspension ready for implantation was 50,000 cells in 50 µl of phosphate buffer solution (counting was carried out in a Goryaev chamber). To initiate the movement of implanted cells along the olfactory nerve, a suspension of MSCs was injected using an insulin syringe under the mucous membrane of the upper nasal cavity of rats [25, 37, 38]. Unilateral injection of a suspension of MSCs (50 µl) into the receptive area of the olfactory nerve was performed 10 min after stroke modelling.

Using the computerized certified technique elevated plus maze (a neurophysiological method for assessing the effectiveness of cognitive and motor function control of rodents), we studied the features of the regulation of motor activity and maintaining habitual body positions in space for rodents at rest and during movement within several days after modelling CI and intranasal administration of MSCs. This test was chosen due to the features of the setup, which combines both open (“dangerous”) and closed “mink” areas, as well as a large number of various parameters (more than 200) that the ANY-Maze program allows to calculate (distance travelled, average speed, number of visits, time spent in sectors, etc.). Each animal was tested for 5 minutes. At the same time, using a Logitech Webcam 905 video camera (Logitech, China), located at a height of 2.4 m above the installation, the behaviour of rats in the maze and the following parameters were constantly recorded: the distance travelled, the average speed of movement of animals, the number of visits and the time spent in open, closed and central sectors, the time of verticalization (racks) in closed sectors, the nature of grooming and several other indicators. The obtained data were converted from analogue to digital form using the ANY-Maze licensed software package for animal behaviour visualization (Stoelting Inc., USA) (version 4.82, serial number: DBUT-VNJJ-JD8S-SFGU; license number: PK8W-NBWV -HTHT-BYHB dated February 28, 2013). The ANY-Maze software package allows automatic statistical processing of the received data.

Using EPR spectroscopy NO production was assessed in intact animals along with rats on the first day after stroke[39, 40]. To form the complex (DETC)₂-Fe²⁺-NO in the tissues of rats, DETC-Na was injected intraperitoneally at a dose of 500 mg/kg in 0.5 ml of water (per animal); a mixture of solutions: ferrous sulphate (FeSO₄x7 H₂O, Sigma, USA) at a dose of 37.5 mg/kg and sodium citrate at a dose of 187.5 mg/kg (in a volume of 1 ml of water per 300 g of animal weight), prepared immediately before introduction, were injected intramuscularly at three points - the right and left thighs and at the withers. DETC-Na and iron citrate are distributed throughout the body and upon interaction form a water-insoluble DETC-Fe²⁺ complex, which can interact with NO to form a stable radical (DETC)₂-Fe²⁺-NO [35, 41]. Typical EPR spectra of frozen rat organ samples containing (DETC)₂-Fe²⁺-NO represent an EPR signal with g_⊥ = 2.038, g_|| = 2.01 and a triplet hyperfine structure at g_⊥. The results obtained make it possible to quantify the NO content in biological tissues. Tissues were harvested 40 min after the introduction of the spin trap components. Due to their small volume, the left and right parts of the hippocampus were combined into one sample (100 mg).

The spectra of the (DETC)₂-Fe²⁺-NO complex were measured using an EMX/plus X-band (9.50 GHz) spectrometer with the temperature controller Bruker ER 4112HV at a temperature of 77° K with a magnetic field modulation of 100 kHz, modulation amplitude 2G, microwave power 30 mW, the time constant 200 ms. The modulation amplitude, amplification, and microwave power in all experiments were selected so that there was no overmodulation and saturation of the EPR signal and they remained the same throughout all measurements. The ready samples, truncated in the shape of the measurement cuvette, were immediately weighed before the measurement. The sample weights were about 100 mg. The amplitude of the EPR spectra was normalized to the sample weight and the amplitude of the EPR signal of a reference sample with a known concentration [30].

The results are presented as the mean and error of the mean (M ± m). To assess the significance of differences between the samples, the Mann-Whitney U-test and the Wilcoxon T-test were used. Differences were considered significant at a significance level of p < 0.05.

The results of testing the studied groups of animals on the 1st day after CI are shown in Fig. 1. In the CI group without MSCs implantation, on the first day after modelling cerebral ischemia, a significant decrease was observed in the total time of mobility of rats to reach on average up to 39% of the total time of mobility before ligation of common carotid arteries (control). On the other hand, in the CI + MSC group, the animals in which were perineurally implanted with MSCs, the decrease of the total mobility time on the first day after CI was less pronounced and averaged 67% of the control level (Fig. 1). When assessing the average distance travelled for 5 minutes on the first day in rats of the CI group, a statistically significant decrease was observed (on average, up to 28% of the distance travelled by intact animals). In the CI + MSC group, which were injected with MSCs after CI, the total distance travelled significantly differed from that of animals without therapy and averaged 65% of the control level (Fig. 1). Similar changes were observed for the total number of intersections of maze lines by rats: in the CI group, on day 1 after CI, a decrease by more than 6 times - on average in this parameter was recorded, to reach a level of 16% of the initial one, while in the CI + MSC group with the use of MSCs the decrease in this parameter was insignificant - by an average of 7%. The average speed of movement of animals before surgery and 24 hours after modelling CI is a kind of resulting general motor activity of animals. A day after CI, a progressive recovery of the average speed of movement through the maze was observed precisely in those rats that were injected with MSCs. In particular, the average speed of movement of animals in the maze on the first day after CI simulation decreased by 73% in rats that were not injected with MSCs and by 37% in rats from the CI + MSC group, which were injected with MSCs (Fig. 1). The total number of “freezes” of animals on the first day after CI modelling increased significantly in both groups: the increase was on average 83 ± 10% in rats that did not receive MSCs, and in the group of rats with MSCs, the increase was less pronounced (average by 63 ± 8%, p < 0.05).

On the 3rd day after CI, in the CI + MSC group, there was a tendency for a more rapid recovery of control of orienting motor activity, which is presented in Table 1. In the CI group of rats (without the introduction of MSCs) decrease in the distance traveled over 5 minutes of observation was found at this time from 7.63 ± 0.85 to 3.27 ± 0.53 meters (by 57%) compared to control values. The animals also decreased the distance traveled in the closed zone, the number of episodes of mobility, the total distance traveled in the closed zone, the average speed of movement and the total time of mobility in the closed zone, and there was no visit to the open areas of the maze. A similar picture was observed in animals of this group (group CI) on the seventh day after the operation. This pattern reflects a decrease in orienting-motor activity in laboratory animals without the introduction of MSCs after ligation of the common carotid arteries; moreover, these changes persisted both in the initial period (third day) and in a longer period (seventh day) after modeling ischemic stroke.

Table 1

Evaluation of the effectiveness of cognitive and motor function control in rodents on the 3rd day after modelling cerebral ischemia with simultaneous intranasal perineural administration of MSCs. Shown are the values before and after cerebral ischemia (CI), as well as before and after cerebral ischemia with intranasal perineural administration of MSCs (CI + MSC). mean±SEM values of the distance travelled (Distance), the total time of mobility of rats (Time mobile), the number (Number of verticalization) and time (Time of verticalization) of verticalization in closed sections, as well as the time of stays in the central sector, are given.
	CI		CI + MSC
	before	3 days	before	3 days
Distance (m)	7.63±0,85	3.27±0,53	7.20±0,66	4.94±0,63
Time mobile (s)	143.9±8,8	58.2±9,8	138.9±9,8	88.3±16,4
Number of verticalizations	13.6±1,7	5.2±1,9	10.7±1,7	7.5±2,5
Time of verticalization (s)	19.9±4,1	6.2±2,6	17.1±1,7	9.9±3,2
Time in the center zone (s)	65.9±17,1	27.6±11,6	70.6±12,0	42.4±6,9

In comparison, in the group of rats that were perineurally implanted with MSCs during the acute period of stroke, it was found that this therapy is accompanied by a faster recovery of the time of motor activity in animals. On the third day of observation, the distance covered in 5 minutes decreased only by 32%, while in the group without therapy - by 57%. In animals without treatment, on the third day of observation, a decrease in the time of motor activity by 59% was noted, and in the group of rats that were injected with MSCs, the decrease in this indicator reached only 36%. A similar pattern was noted when comparing the number and time of verticalization in rodents in the pre- and postoperative periods. Thus, on the third day of observation, the number of upright stances of rats for 5 minutes in the CI group decreased in comparison with the preoperative period by an average of 62%, and in the CI + MSC group - only by 30%. The comparison of the verticalization time in rats of the two groups with CI modeling revealed a more rapid recovery of this indicator in those animals that were intranasally implanted with MSCs after occlusion of the common carotid arteries (Table 1). In the CI group, on the third day of observation, a decrease in the time spent by animals in the central zone of the plus maze by an average of 59% compared with the same indicator in the same rats in the preoperative period (control) was found. For comparison, for the same periods in patients with intranasal implantation of MSCs after CI, a less pronounced decrease in the time spent by animals in the central zone of the plus maze was found - by an average of 42% compared with control indicators. It should also be noted that on the seventh day after the simulation of ischemia in rats of the CI + MSC group, there were actually no obvious distinguishing signs in the control of orienting-motor activity in comparison with the preoperative period in the same rats.

Thus, the introduction of MSCs in the acute phase of stroke was accompanied by a significant protective effect on the preservation and/or restoration of control of cognitive functions and motor activity of animals. Considering that hippocampal neural networks play a significant role in the processes of memorization and in the process of controlling the approximate motor activity of animals [34], it was decided to emphasize the detailing of metabolic events in CI at the NO system level in the hippocampus.

To study the dynamics of NO content in the hippocampus by EPR spectroscopy using the spin trap technique, spectra were recorded before and after modelling of CI and simultaneous intranasal administration of MSCs. Figure 2 shows examples of EPR spectra of the hippocampus of a control rat compared to rats after 1 day of ischemia caused by ligation of the carotid arteries both with simultaneous intranasal administration of MSCs and without administration of MSCs. The spectra show a characteristic triplet signal from the (DETC)₂-Fe²⁺-NO complex. Additionally, in the same region, a signal from the (DETC)₂-Cu complex can also be detected by EPR spectroscopy [42]. Figure 3 shows statistical data on the integral signal intensities of (DETC)₂-Fe²⁺-NO in the spectra of the studied samples (hippocampus). The results show a significant reduction in NO content one day after modelling CI caused by ligation of the carotid arteries. In the hippocampus of rats in which ischemia was modelled with simultaneous intranasal administration of MSCs, no significant difference was found in the NO content relative to ischemic rats.

As already mentioned, in the EPR spectra of hippocampal tissues, in addition to the characteristic triplet signal from the (DETC)₂-Fe²⁺-NO complex, signals from the (DETC)₂-Cu complex were analyzed. The copper content in the rat hippocampus 1 day after modelling CI caused by ligation of the carotid arteries tended to decrease (Fig. 4). In the group with the MSCs implantation, there was a tendency to restore copper concentrations to levels that are characteristic of intact animals. Interesting results were obtained by calculating the Cu/NO ratio in the hippocampus of rats, which indicates the characteristics of correlation between the states of the NO system and antioxidant system (Fig. 5): a tendency to increase in the efficiency of the antioxidant system 1 day after ischemia in both studied groups was observed. This effect was more pronounced with intranasal administration of MSCs.

The problem of restoring lost brain functions after circulatory disorders is of great relevance in the clinical aspect [23]. And since technologies for effectively restoring control of impaired somatic and autonomic brain functions after circulatory disorders have not yet been developed, experimental developments in this direction are being carried out intensively in many countries across the globe [15, 22].

Our study showed that the central control of the orienting-motor activity of rats is recovered faster in the group of animals in which MSCs migrate to the ischemic areas of the brain after implanting them intranasally in the region of the olfactory nerve terminals. The data obtained in the plus maze showed that perineural implantation of MSCs attenuates the negative consequences of ischemia on the brain systems that control the motor activity of animals, which manifests as a faster recovery of animal activity, an increase in the average speed of movement and the distance travelled during the implementation of orienting behavior in the maze and a series of other indicators of tentative-motor activity of rats. The fact that after perineural implantation MSCs migrate in the direction of damaged areas of the nervous tissue is well established [26, 27, 38]. Previously, it was shown that when modeling local neurodestruction in the sensorimotor zone of the brain and subsequent injection of MSCs into the submucosal region of the nasal cavity of rats, MSCs move along the fibres of n. olfactorius into the central structures of the olfactory system and then get distributed in areas of brain destruction in the anterior and middle cranial fossae [25, 26, 27]. The data obtained form a basis for analysing the mechanisms of the revealed MSC reparative phenomenon in the nervous tissue of the brain.

Under hypoxic conditions, the impaired control of metabolic processes is accompanied by neurodestruction in various parts of the brain. With occlusion of the external carotid arteries, the structures of the neocortex and hippocampus are primarily affected due to the weakening of cerebral blood flow and deterioration of conditions for adequate oxygenation [43, 44, 45]. It is self-evident that neurons are more sensitive to a drop in tissue oxygen saturation compared to glial components [43]. In the process of evolution, protective processes have evolved in the nervous tissue of the brain to ensure plasticity under different conditions, including disruption of the redox potential and energy processes, which in brain cells are controlled by signalling molecules such as the gaseous mediators NO [6, 8, 11, 46]. One of the mechanisms contributing to the positive effects of MSCs in the brain tissue is an adaptive change in the NO system, which promotes the activation of reparative processes in the brain tissue during hypoxia induced by occlusion of the common carotid arteries.

The role of gaseous neurotransmitters during conditions of normoxia, hypoxia, and cerebral ischemia is traditionally regarded as a topic of highest priority in experimental research and clinical observations [10, 32, 33]. Taking into account the importance of the neural networks of the brain and specifically the hippocampus in the processes of memorization [34] and the properties of the control of locomotor behaviour patterns in the process of orienting-motor activity of animals, it was decided to focus on detailing the metabolic events during CI in terms of the NO levels in the hippocampus.

The results obtained by us earlier using EPR spectroscopy [30, 35, 36, 40, 47] in a model of ischemia caused by ligation of the external carotid arteries demonstrate that brain hypoxia is accompanied by a significant decrease in NO production by 36% in the hippocampus within the first 3 days after ischemic stroke modelling. We also showed a decrease in copper content by an average of 24%. In the same model of ischemia, a significant decrease in the content of NO in the olfactory bulb of the brain of rats was found two times 1 and 2 days after modelling the cerebral ischemia [47]. The level of NO production in rats in which ischemia was modelled with a simultaneous intranasal administration of MSCs was also reduced 1 and 2 days after brain ischemia. No significant difference was found in the NO content in rats that underwent modelling of ischemia with simultaneous intranasal administration of MSCs relative to ischemic rats. The copper content, which corresponds to the level of superoxide dismutases 1 and 3, in the olfactory bulb of rats tended to increase after modelling ischemia and persisted for two days of observation [48].

Previously, we also showed that after the onset of signs of stroke induced by 5-minutes in hypobaric hypoxic conditions (conditional rise to a height of 4500 m above sea level), already after 5 hours of ischemia, the formation of NO in the hippocampus decreases by 2–3 times and this decrease persists within 24 and 72 hours [49]. It is believed that during transient ischemic attacks and ischemic stroke, not only neuronal and glial elements are damaged, but the structure of the endothelium of blood vessels is also disturbed. Disruption of the endothelial structure is accompanied by a decrease in the production of endothelial NO, which leads to vasoconstriction and, consequently, impairs brain oxygen saturation [31, 50].

Earlier, we have shown that changes in the NO system in the brain during hypoxia have a complex nature – on the one hand, the NO level decreases due to a decrease in the activity and content of endothelial NO synthase, on the other hand, there is a compensatory NO increase in the structures of the nervous tissue, mainly due to the expression of neuronal and inducible NO synthase. This last aspect in cerebral ischemia is one of the key incentives to seek methods to control the level of NO in the brain tissue by correcting the activity of NO synthase since, under different metabolic conditions, pathological processes in tissues are enhanced both with a lack of NO and with an excess of its formation [28, 29, 49, 51].

Therapeutic strategies that reduce energy consumption and reduce the severity of oxidative stress are currently one of the most developed methods of primary and secondary neuroprotection of postischemic cerebral disorders [51]. These strategies are primarily based on the activation of the body's own antioxidant resources under conditions of hypoxia and ischemia, where Zn-Cu-superoxide dismutase is of the greatest importance. The effectiveness of the neuroprotective action of superoxide dismutases increases during hypoxia and glutamate cytotoxicity; therefore, in the culture of cortical neurons and transgenic mice genetically predisposed to hypersecretion of superoxide dismutases, there is practically an absolute resistance of cells to NMDA toxicity and focal ischemia [5, 52]. It has been established that the number of dead neurons is higher in mice with a genetically determined deficiency of superoxide dismutases during ligation of the middle cerebral artery since it has been demonstrated that they are less resistant to elevated concentrations of glutamate, hydrogen peroxide, and NO donors in experiments in vitro [53]. It is important to note that the degree of activity and protective properties of superoxide dismutase are predetermined depending on the level of NO production, the formation of superoxides, and the concentration of Ca²⁺ ions. So, in animals with the receiving MSCs, the activity of the antioxidant system after modelling cerebral ischemia was more pronounced. The results obtained indicate that even a single perineural administration of MSCs into the receptive fields of the cranial nerves is sufficient for achieving selective accumulation of MSCs in areas of brain damage and, as a result, activating the restoration of functions impaired due to brain damage. The basis for the activation of reparative processes in the nervous tissue of the brain after CI modelling is the secretion of numerous neurotrophic factors by MSCs in the areas of neurodestruction after disrupting the blood supply to the brain [54].

All in all, taking into account the literary information [6, 8, 11, 43, 44, 45, 46] and our results published earlier [28, 29, 49, 51], after modelling CI, a decrease in NO production was observed, which was accompanied by a disruption of the control of motor activity in rats. However, in the group that received MSCs, the decrease in NO was less pronounced, this group was also characterized by better parameters of orienting-motor reactions.

Conflict of Interest

The authors declare that they have no conflicts of interest.

Ethics approval and consent to participate

The experimental procedures (using anesthesia methods) are in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985), the UK Animals Scientific Procedures Act 1986 or the European Communities Council Directive of 24 November 1986 (86/609/EEC).

Methods approved by the Scientific Council of the Institute of Physiology of the National Academy of Sciences of Belarus (protocol No. 8, 26.08.2010).

Consent for publication

All authors agree to publish the results and approved the final manuscript.

Availability of data and material

Not applicable

Competing interests

Not applicable

Funding

Ischemia modelling was carried out at the Brain Center, Institute of Physiology of the National Academy of Sciences of Belarus (Minsk, Belarus), the work was supported by the State Committee on Science and Technology of the Republic of Belarus (No. M19LITG-002). Measuring the EPR spectra of the samples was carried out at Zavoisky Physical-Technical Institute, KazSC RAS (Kazan) within the framework of a state assignment. The storage of samples and processing of the results was funded by Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030).

Authors' contributions

Guzel Yafarova carried out the interpretation of results measuring of behavioral changes and comparison them with the results of NO level, prepared Figure 1 and helped to draft the manuscript;

Yulia Tokalchik participated in simulation of ischemia and in measuring behavioral changes after this operation, participated in its interpretation and in extraction of brain samples for further measurements by EPR spectroscopy;

Tatiana Filipovich participated in simulation of ischemia and in measuring behavioral changes after this operation, participated in its interpretation and in extraction of brain samples for further measurements by EPR spectroscopy;

Viacheslav Andrianov carried out calculations and analysis of nitric oxide (NO) and cooper signals in EPR spectra of samples intensity, performed the statistical analysis, prepared Figures 2-5, participated in interpretation of results and helped to draft the manuscript;

Lyeo Bazan carried out measurements of EPR spectra of samples and participated in their interpretation;

Tatiana Bogodvid did the literature search, participated in the analysis of results and interpretation the role of NO in brain ischemia and protective actions at the NO level and helped to draft the manuscript;

Abdulla Chihab helped to draft the manuscript and responsible for its editing;

Aleksandra Zamaro participated in simulation of ischemia and in measuring behavioral changes after this operation, participated in its interpretation and in extraction of brain samples for further measurements by EPR spectroscopy;

Vladimir Kulchitsky conceived of the study, participated in its design and coordination, helped to draft the manuscript and responsible for final approval;

Khalil Gainutdinov conceived of the study, responsible for conception its design and coordination, drafting the manuscript, did the literature search and final approval.

All authors read and approved the final manuscript.

Authors thanks to colleagues S.G. Pashkevich and M.O. Dosina from Institute of Physiology of the National Academy of Sciences of Belarus for technical assistance.

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The effects of intranasal implantation of mesenchymal stem cells on nitric monoxide levels in the hippocampus, control of cognitive functions and motor activity in a model of cerebral ischemia in rats

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