Роль мозгового нейротрофического фактора BDNF и его рецептора TrkB в устойчивости нейронов гиппокампа к ишемии-реперфузии (экспериментальное исследование)

The purpose of the study: to assess the content of BDNF and its TrkB receptor in the populations of hippocampal pyramidal neurons in the post-resuscitation period and to identify the contribution of these factors to the neuron resistance to ischemia. Material and methods. The condition of populations of pyramidal neurons of the CA1 and CA4 hippocampus fields was investigated in white mature male rats that underwent a 10-minute cardiac arrest at different periods of the post-resuscitation period (1st, 4th, 7th, 14th day). Animals after a sham surgery served as a reference group. Immunocytochemical methods were used to determine immunoreactivity to BDNF and TrkB proteins. Based on the visual inspection and analysis of the optical density, the following types of neurons with different color intensity were distinguished: weak (BDNF–, TrkB–), moderate (BDNF+, TrkB+) and strong (BDNF++, TrkB++). The total density of neurons and the number of cells with different immunoreactivity to the studied proteins per 1 mm of length were determined. We used the Olympus BX-41 microscope and Image Scope M, ImageJ 1.48 v, MS Excel software. Statistical data processing was performed using Statistica 7.0 software. Results. There was a decrease in the overall density of the population of pyramidal neurons in both studied fields of the hippocampus of the resuscitated animals as compared to the reference group: in the CA1field, on the 4th day after cardiac arrest (26%); in the CA4 field, on the 7th day (38.5%). It was found that the number of BDNF+ neurons doubled in the CA4 field on the 4th day, and the number of BDNF++ neurons decreased. On the 7th day, the number of BDNF– cells decreased sharply, the number of BDNF+ cells decreased to the reference level, and the number of BDNF++ neurons remained reduced vs. the reference group. There was a decrease in the number of BDNF– and BDNF+ cells in the CA1 field on the 4th day, while the number of BDNF++ neurons remained the same. The observed changes remained on Day 14. The analysis TrkB protein expression in the CA4 field on the 7th day of the post-resuscitation period as revealed by reactivity with anti-TrkB antibody demonstrated a decrease in the number of TrkB– cells as compared to the reference group. By the 14th day, the number of not only TrkB– neurons, but also TrkB+ cells was reduced, while the number of TrkB++ neurons remained at the level of the reference group. There was a decrease in the number of TrkB+ neurons was observed in the CA1 field on the 4th day after resuscitation. On day 7, there was a decreased numbers of both TrkB+ and TrkB–-neurones. The number of TrkB–-neurones remained decreased up to day 14. At that, the number of TrkB++ neurons persisted at the reference level throughout the observation period. Conclusion. The obtained results demonstrate that the resistance of neurons to ischemia-reperfusion is associated with the intracellular expression of BDNF and TrkB proteins. The reduction of the overall density of neurons in the post-resuscitation period was obsereved both in hippocampal fields CA1 and СА4; only cells with minimal and moderate content of the studied proteins died. Neurons with the highest BDNF and TrkB protein content survived.


Introduction
The interest in growth factors as potential therapeutic agents to combat post-ischemic brain disorders has increased significantly over recent years [1,2]. The efficacy of a therapy that stimulates local production of these factors and their receptors in the damaged brain tissue is actively studied [3,4].
Neurotrophic factors are polypeptides that play an important role in maintaining the function of neurons that are involved in the survival processes, growth, differentiation of neurons, as well as in the mechanisms of neural plasticity, learning and memory. The brain-derived neurotrophic factor (BDNF) is the most studied member of this group, which has neuroprotective properties and plays an important role in restoring brain function in various neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's chorea [3,5].
However, the literature lacks data on the level and nature of TrkB protein expression in areas of the brain that are most sensitive to hypoxia, in particular, in the hippocampus, under normal conditions and in ischemia caused by temporary cardiac arrest. Since the neuroprotective effect of BDNF and its mimetics is mediated by TrkB and there is a close relationship with the functional state of the central nervous system, it is recommended to study the dynamics of the BDNF and TrkB content in neuronal populations of the brain in the post-resuscitation period. This will provide an opportunity to assess how neuroprotective properties of BDNF are implemented in neurons with high sensitivity to hypoxia. It is known that the pyramidal neurons of the hippocampus, the part of the brain associated with learning and memory, are most susceptible to ischemia [9].
The purpose of this study is to assess the content of BDNF and its TrkB receptor in the populations of hippocampal pyramidal neurons in the post-resuscitation period and to identify the contribution of these factors to the neuron resistance to ischemia.

Materials and Methods
The brains of white nonlinear mature male rats weighing 190-250 g who underwent a 10-minute cardiac arrest (n=21) (intra-thoracic clamping of the vascular bundle) were studied [10]. 1, 4, 7, 14 days after resuscitation (indirect cardiac massage in combination with mechanical ventilation in the hyperventilation mode using «Animal Respirator» device manufactured by «SMT Geratehandel», with intra-tracheal injection of epinephrine solution at a dose of 0.1 mg/kg), the animals were withdrawn from the experiment by decapitation under anesthesia (5-7 animals for each term of the post-resuscitation period). Rats after a sham surgery served as a reference group (n=5). Experiments were carried out in accordance with the recommendations of the Ethics Committee of the V. A. Negovsky Research Institute of General Reanimatology, in accordance with the «Guidelines for works involving experimental animals» (Order No. 755 of the Ministry of Health of the USSR dated 12.08.1977).
Immediately after decapitation, the brain was isolated and fixed in 4% paraformaldehyde solution in 0.1 M phosphate buffer pH 7.4 (PBS) for 1 day at 4°C. After standard treatment, brain samples were embedded into paraffin. 5-6 μm sections were made from paraffin blocks. Post-resuscitation changes in populations of pyramidal neurons of the CA1 and CA4 hippocampal fields were investigated.
Images were obtained using the Olympus BX-41 microscope and ImageScopeМ software. The intensity of the immunohistochemical reaction in the neuronal cytoplasm was evaluated using the ImageJ 1.48 v imaging software. The «Mean Gray Value» was determined and the optical density мационного периода общая плотность обеих нейрональных популяций оставалась сниженной.
Statistical data processing was performed in the Statistica 7.0 software using the ANOVA method for multiple comparisons (post-hoc comparisons of means).

Results and Discussion
It was found that a 10-minute cardiac arrest resulted in a decrease in the total density of neurons in the CA4 field (on 7 th day after resuscitation, 38.5% vs. the reference group) and in the CA1 field (on 4th day post-resuscitation, 26% vs. the reference group). indicating the death of pyramidal cells ( fig. 2). Therefore, neuronal loss in the CA1 field occurs earlier than in the CA4 field, which is consistent with the data of other researchers on the high vulnerability of CA1 neurons in the post-ischemic period [9,11]. On the 14 th day of the post-resuscitation period, the total density of both neuronal populations remained reduced.
The immunohistochemical assay showed that neurons with moderate and strong immunoreactivity to the BDNF protein prevail in the CA4 field ( fig. 3, a), and neurons with moderate immunoreactivity to the BDNF protein prevail in the CA1 field under normal conditions (in the reference group animals) ( fig. 3, b).
It was found that the number of BDNF+ neurons increased in the CA4 field on the 4 th day after resuscitation; the number of BDNF ++ cells decreased, and the number of BDNFneurons did not change compared to the reference group ( fig. 3, a). The data indicate a decreased BDNF protein expression in this neuronal population.
Analysis of the TrkB protein content showed that neurons with mild and moderate immunoreactivity prevail in the reference group animals in both studied hippocampal fields ( fig. 3 c, d).
There was a decrease in the number of TrkBcells in the CA4 field on the 7 th day of the post-resuscitation period. At that, the number of TrkB + and TrkB ++ neurons did not change in comparison with the reference group. By the 14 th day, the number of not only TrkBneurons, but also TrkB + cells was reduced, while the number of TrkB ++ neurons remained unchanged ( fig. 3, c). It is obvious that cells with mild and moderate immunoreactivity to TrkB die.
In the CA1 field, the number of TrkB + cells decreased on the 4 th day after resuscitation in the resuscitated rats compared to the reference group animals. There were no significant differences from the reference group in the number of TrkBand TrkB ++ neurons. The obtained data indicate the death of neurons

Fig. 3. Changes in the number of neurons with different levels of of BDNF (a, b) and TrkВ (c, d) protein expression in the population of pyramidal neurons of the CA4 and CA1 hippocampal fields in the post-resuscitation period, M±m.
Note. a, b -the data for the СА4 field; c, d -the data for the CA1 field. Примечание. a, b -данные для поля СА4; c, d -данные для поля СА1.
Therefore, the decrease in the total density of hippocampal pyramidal neurons in the post-resuscitation period (within 14 days) was due to cells with low and moderate content of both TrkB and BDNF protein.
According to the obtained results, there was a decrease in expression of both BDNF and TrkB proteins in the neuronal populations of the hippocampus 4-7 days after the cardiac arrest, which was probably related to the decrease in the their expression level in the post-ischemic period. This assumption is confirmed by a number of studies. For instance, a decrease in the expression of BDNF and its TrkB receptor was found in the CA1 field of the hippocampus 4 hours to 1 day after isolated cerebral ischemia in gerbils [12], as well as in the cerebral cortex and hippocampus 1 day after occlusion of the middle cerebral artery in mice [13]. In vitro experiments demonstrated that hypoxia-hypoglycemia caused a decrease in the BDNF and TrkB expression in hippocampal neurons [14]. By means of PCR-analysis and ELISA techniques it was shown that there was a decrease in the content of BDNF mRNA and protein in the CA1 hippocampal field after temporary occlusion of the middle cerebral artery in rats, which was accompanied by neuronal degeneration [9]. Similar changes in the hippocampus were observed on the model of global cerebral ischemia [15]. Slightly different data were obtained in a study by Ferrer et al., 1998 [16]. For instance, a 5-minute brain ischemia in gerbils led to a decrease in the number of BDNF-immunoreactive neurons in the CA1 field, but TrkB-immunoreactive cells survived. At that, the authors noted that 95% of surviving neurons expressing BDNF also produced TrkB [16].

Заключение
Полученные результаты свидетельствуют о том, что устойчивость нейронов к ишемии-реперфузии ассоциирована с содержанием в них белков BDNF и TrkB. Наименьшей устойчивостью к гибели в течение 14 дней после остановки сердца обладали нейроны с минимальным и умеренным содержанием исследуемых белков. При этом лучшую выживаемость продемонстрировали нейроны с наибольшим содержанием BDNF и TrkB. Выявленные факты раскрывают перспективность исследо-We found that normally only a small number of the pyramidal neurons in rat's hippocampus highly express the TrkB protein. This observation was typical for both CA1 and CA4 fields. Some differences between these areas were found in the BDNF content. For instance, while the number of neurons with a high BDNF content (BDNF ++ ) was relatively small in the CA1 field of the reference group animals, the number of such cells in the CA4 field was much higher than that of neurons with a low and moderate BDNF content (BDNFand BDNF + ). In gerbil studies, a small content of TrkB immunoreactive neurons in the CA1 field was also found under normal conditions, but most cells expressed BDNF [16]. Data obtained in this study show that the process the post-resuscitation neuronal death in field CA1 develops a little bit earlier than in field СА4. It may be due to the difference in the baseline level of BDNF expression.
Many studies have shown that BDNF is a potential therapeutic agent for managing the ischemic brain damage [1,3,4]. Activation of endogenous neurotrophic factors and their receptors is one of the most promising areas in the development of brain protection techniques [2]. At present, a variety of ways to stimulate endogenous BDNF production are proposed [4,9,12]. For example, Mokhtary (2017) showed that intraventricular injection of triiodothyronine 24 hours after brain ischemia led to an increase in the BDNF content in the hippocampus [9]. Positive results were obtained in experimental studies of neuroprotective action of synthetic and herbal BDNF mimetics and TrkB receptor agonists [7,[17][18][19]. Physical exercises [4,20], as well as methods of non-invasive stimulation of the brain by alternating currents are also considered as ways to increase the BDNF level [21].

Conclusion
The obtained results indicate that the resistance of neurons to ischemia-reperfusion is associated with the expression of BDNF and TrkB proteins. Neurons with minimal and moderate content of the studied proteins had the least resistance to death within 14 days after cardiac arrest. At the same time, neurons with the highest expression of BDNF and TrkB demonstrated the best survival rate. These facts demonstrate that studies of various ways to increase the BDNF expression and the activation of its TrkB receptor in neurons for its protection against ischemic damage are promising.