Nerve regulation method for improving memory and cognitive ability

Abstract

The invention relates to the field of nerve regulation and control, in particular to a nerve regulation and control method for improving memory and cognitive competence, which comprises the following steps: injecting adenovirus into a PVT brain region of a mammal, and activating the adenovirus through chemical stimulation to inhibit neuronal activity of the PVT brain region, so that the cognitive competence and memory of the mammal can be improved.

Description

Technical Field

[0001] The present invention relates to the field of neural regulation technology, and in particular to a neural regulation method for improving memory and cognitive ability. Background Art

[0002] Cognition refers to the process by which people acquire or apply knowledge, or the process of information processing, which includes sensation, perception, memory, thinking, execution, imagination and language. The brain receives information input from the outside world and controls behavior after processing. This process is the process of information processing, that is, the cognitive process. Cognitive ability is plastic and will be affected and regulated by many factors, including learning, training, living environment, age and health status. Memory is an important part of cognitive function and is regulated by multiple brain regions, including related neural networks such as the hippocampus, prefrontal cortex and thalamus. Appropriate and accurate brain neural stimulation will regulate the excitability of neurons and the way of signal transmission, thereby improving physiological function. Therefore, many researchers have conducted research on neural regulation to explore its potential non-drug treatment intervention for cognitive ability. Protective pathways and mechanisms.

[0003] Neuromodulation technology refers to the technology that uses electrical, magnetic, chemical or other physical means to regulate and control the activities of the nervous system. This technology has the advantages of being non-invasive or minimally invasive, highly accurate and highly adjustable, and is particularly suitable for groups with drug or food intolerance. However, if the target brain area of ​​neuromodulation training is not accurately positioned or the conditions are not properly controlled, it may cause neurological damage. There are currently no reports on the use of neuromodulation technology to specifically regulate the PVT brain area. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a neuromodulation method for improving memory and cognitive ability. The neuromodulation method provided by the present invention can alleviate cognitive function damage in chronic cerebral ischemia model mice and improve memory and cognitive ability by inhibiting PVT and its neural function circuit.

[0005] In a first aspect, the present invention provides a neuromodulatory method for improving memory and cognitive ability, the method comprising the following steps: injecting adenovirus into the PVT brain region of a mammal, and activating the adenovirus through chemical stimulation to inhibit neurons in the PVT brain region.

[0006] The present invention uses the CNO-hM4Di / hM3Dq system in chemical genetics technology to regulate neural activity. hM4Di / hM3Dq is an artificially designed protein receptor, which is transformed from a human muscarinic receptor. Muscarinic receptors are a type of G protein-coupled receptor. When acetylcholine binds to muscarinic receptors, the receptor is activated, the conformation changes, and the coupled G protein is further activated. The G protein then triggers downstream reactions, ultimately activating or inhibiting neurons. In the CNO-hM4Di / hM3Dq system, hM4Di / hM3Dq only binds exclusively to the artificially designed drug clozapine N-oxide (CNO), and can activate or inhibit neuronal discharge after binding thereto. Whether neurons are activated or inhibited depends on the specific subtype of muscarinic receptors. If it is an M4 type muscarinic receptor, it is coupled to the Gi protein, and the subsequent reaction will eventually inhibit neurons. The artificial receptor transformed from this receptor is hM4Di. If it is an M3 muscarinic receptor, Gq protein is recruited, and the downstream reaction will eventually activate neurons. The artificial receptor modified from this receptor is hM3Dq. CNO can be administered by intraperitoneal injection or drinking water. After metabolism, the function of neurons will be restored.

[0007] The neuroregulatory method provided by the present invention injects a protein receptor loaded by adenovirus into the PVT brain region of mice with decreased cerebral blood flow, and injects hM4Di and hM3Dq respectively. Two weeks after viral expression, CNO is injected intraperitoneally to activate hM4Di and hM3Dq, thereby causing inhibition and activation of PVT neurons, respectively. By controlling the frequency of chemical stimulation, it is found that in mice expressing hM4Di, once a day injection of CNO can improve the cognitive function and memory of mammals, and reduce pathological damage to the prefrontal cortex and hippocampus. Therefore, appropriate inhibition of PVT neuron activity can improve memory ability.

[0008] As a preferred technical solution of the present invention, the mammals include mice, horses, cows, sheep, dogs, rabbits, pigs, monkeys or humans.

[0009] As a preferred technical solution of the present invention, the adenovirus is AAV2 / 9-syn-hM4Di-mcherry.

[0010] As a preferred technical solution of the present invention, the chemical stimulation is intraperitoneal injection of clozapine N-oxide. The combination of clozapine-N-oxid (CNO) and hM4Di can effectively inhibit neuronal activity in the PVT brain region.

[0011] As a preferred technical solution of the present invention, the frequency of the chemical stimulation is once a day, and the injection amount of the clozapine N-oxide each time is 2 mg / kg.

[0012] As a preferred technical solution of the present invention, the chemical stimulation is performed continuously for 13-15 days (eg, 13, 14, 15 days, etc.).

[0013] In the present invention, if the number of stimulations (ie, the number of stimulation days) is insufficient, the endogenous protective mechanism of the body may not be activated, or the endogenous protective mechanism may not be sufficient to produce a lasting protective effect.

[0014] In the present invention, the effect time of a single chemical stimulation on the cell state is 4-6 hours.

[0015] In the present invention, if the effect time of a single chemical stimulation on the cell state (i.e., the PVT inhibition activity time) is too long, it will cause damage to the nerve function, and if the PVT inhibition activity time is too short, the cell activity is relatively light and insufficient to bring about significant physiological changes. The technical solution provided by the embodiment of the present invention has the following advantages over the prior art:

[0016] The neuromodulation method provided by the present invention acts on PVT and can reduce pathological damage to the prefrontal cortex and hippocampus and improve memory and cognitive ability by appropriately inhibiting the activity of PVT neurons. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0019] Figure 1 This is a graph of laser speckle results of mice before and after BCAS surgery described in Example 1 of the present invention;

[0020] Figure 2 This is a graph showing the behavioral results of mice one month after BCAS surgery described in Example 1 of the present invention;

[0021] Figure 3 This is a diagram showing the staining result of the cell activation marker cFOS described in Example 1 of the present invention;

[0022] Figure 4 This is an experimental flow chart of PVT regulation of the present invention;

[0023] Figure 5 This is a graph showing the results of a novel object recognition test in which the cognitively impaired mice were subjected to different PVT regulation methods as described in Example 2 of the present invention;

[0024] Figure 6 This is a diagram showing the results of the Y-maze test of cognitively impaired mice subjected to different PVT regulation methods as described in Example 2 of the present invention;

[0025] Figure 7 This is a graph showing the results of an open field test in which the cognitively impaired mice described in Example 2 of the present invention were subjected to different PVT regulation methods;

[0026] Figure 8 This is a diagram of pathological changes in the prefrontal cortex of cognitively impaired mice subjected to different PVT regulation methods as described in Example 3 of the present invention;

[0027] Fig. 9 This is a statistical diagram of pathological damage to the prefrontal cortex of cognitively impaired mice subjected to different PVT regulation methods as described in Example 3 of the present invention;

[0028] Fig.10 This is a diagram of the pathological changes in the motor cortex of the cognitively impaired mice subjected to different PVT regulation methods described in Example 3 of the present invention;

[0029] Fig.11 This is a statistical diagram of motor cortex pathological damage in cognitively impaired mice subjected to different PVT regulation methods as described in Example 3 of the present invention;

[0030] Fig.12 This is a diagram of the pathological changes in the sensory cortex of the cognitively impaired mice subjected to different PVT regulation methods described in Example 3 of the present invention;

[0031] Fig.13 This is a statistical diagram of sensory cortex pathological damage in cognitively impaired mice subjected to different PVT regulation methods as described in Example 3 of the present invention;

[0032] Fig.14 This is a diagram of hippocampal pathological changes in cognitively impaired mice subjected to different PVT regulation methods as described in Example 3 of the present invention;

[0033] Fig.15 This is a statistical diagram of hippocampal pathological damage in cognitively impaired mice subjected to different PVT regulation methods as described in Example 3 of the present invention;

[0034] Fig.16 This is a diagram showing the effect of inhibiting PVT regulation on brain white matter in cognitively impaired mice described in Example 3 of the present invention;

[0035] Fig.17 This is a statistical diagram of the effects of inhibiting PVT regulation on brain white matter in cognitively impaired mice described in Example 3 of the present invention. DETAILED DESCRIPTION

[0036] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the scheme of the present invention will be further described below. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0037] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all of the embodiments.

[0038] Example 1 Determining the brain regions that respond to fluctuations in cerebral blood flow

[0039] Cerebral blood flow is the source of oxygen and nutrition for neurons. When cerebral blood flow decreases, cognitive function will be impaired. Therefore, identifying the brain areas that are specifically active when cerebral blood flow decreases will help identify the target brain areas for neuromodulation.

[0040] (1) Prepare an animal model to simulate decreased cerebral blood flow.

[0041] First, mice were used as experimental animals to simulate the decrease in cerebral blood flow by preparing a typical mouse bilateral common carotid artery stenosis (BCAS) model. Eight-week-old male mice were divided into two groups: a control group (Sham group) and a BCAS group. The mice in the BCAS group were anesthetized and the skin was prepared for disinfection. The ventral skin of the neck was incised in the middle of the neck, with a length of about 2-3 cm. The muscles on both sides of the neck were carefully bluntly separated, and the common carotid artery was freed without injuring the vagus nerve. The left common carotid artery was narrowed with a 0.18mm titanium alloy coil, and the right common carotid artery was narrowed with the same method after waiting for 30 minutes. The common carotid artery of the control group mice was not narrowed, and the rest of the operations were the same as those of the BCAS group. The cerebral blood flow was detected with a laser speckle Doppler instrument before and after modeling to visualize and clarify the decrease in cerebral blood flow.

[0042] (2) Behavioral testing

[0043] Next, the cognitive function of mice with decreased blood flow was evaluated using an automatic behavioral detection system (including a display screen, a control interface, and a detector. Open the control interface to select different behaviors and set conditions). Neurological function was tested 4 weeks after modeling to confirm that the modeling was successful. Neurological function mainly tested motor function and cognitive function, which were tested using behavioral tests, including open field, new object recognition, and Y maze.

[0044] Open field test (measurement of motor function and anxiety-like behavior): A 40×40 cm box was used in a soundproof room with a light intensity of 100-500 lux, a temperature of 22-25°C, and a humidity of 40-60%. The recording started immediately after the mouse was placed in the center of the box and lasted for 10 min. The total distance moved and the time spent in the center area were measured and recorded using EthoVision XT 17 software (Noldus IT, Netherlands).

[0045] New object recognition experiment (to detect the exploration desire and memory ability of mice): During the adaptation stage, mice were placed in a box without any objects for 15 minutes every day for 3 consecutive days. The test was carried out on the fourth day. Two identical objects were placed in the box and the mice were allowed to explore freely for 10 minutes. After 4 hours, a familiar object was replaced with a new object and the mice were allowed to explore for another 5 minutes. 70% alcohol was used to remove odor traces after each experiment. Climbing up objects and sitting on objects are not counted as exploration, but sniffing with the nose or touching with the front paws are counted as exploration. Recognition index = time to explore new objects / total time to explore two objects. The recognition index is proportional to the exploration desire and memory of the mice. The higher the recognition index, the better the cognitive ability of the mice.

[0046] Y-maze test (to test spatial recognition ability): The Y-maze consists of three arms (marked as A, B, and C). During the adaptation stage, mice were adapted for 3 consecutive days. The mice were placed alone at the junction of the three arms, the C arm was blocked, and they were allowed to move freely for 10 minutes. The test was conducted on the fourth day, the C arm was opened, and they were allowed to explore freely for 5 minutes. The time and moving distance of the mice in the three arms were recorded. Exploration index = distance explored in the C arm / total time explored in the three arms. The exploration index is proportional to the spatial recognition ability of the mice. The higher the exploration index, the better the spatial cognition ability of the mice.

[0047] (3) cFOS staining, a cell activation marker, identifies brain regions that respond to decreased cerebral blood flow

[0048] Finally, the control group and BCAS group mice were perfused by apical perfusion, and the brain tissue was fixed with PFA at 4°C for 24h. The brain tissue was cut into 70μm slices using a vibrating slicer and immersed in a 24-well plate with PBS buffer in sequence. The brain slices were perforated with 0.2% Triton X-100, incubated at room temperature for 20min, and washed with PBST for 5min×3 times. Blocked with 5% BSA and incubated at room temperature for 60min. The primary antibody cFOS (CST, 2250T, 1:1000) was diluted with blocking solution, incubated at 4°C overnight, rewarmed at room temperature for 20min the next day, and washed with PBST for 5min×3 times. The secondary antibody (Thermo, 1:1000) and DAPI (Abcom, 1:1000) were diluted with blocking solution, incubated at room temperature in the dark for 2h, and washed with PBST for 5min×3 times. The patch was dried and then dripped with anti-quenching sealing agent to avoid bubbles. The images were collected using a confocal microscope.

[0049] The results are as follows Figure 1-Figure 3 As shown: Figure 1 This is the laser speckle result of mice before and after BCAS surgery. Figure 1 It can be seen that compared with the control group, the cerebral blood flow in the BCAS group of mice was significantly reduced after surgery. Figure 2 This is a graph showing the behavioral results of mice one month after BCAS surgery. Figure 2 It can be seen that compared with the control group, the mice in the BCAS group showed decreased new object recognition ability and spatial recognition ability 40 days after surgery, indicating that the chronic hypoperfusion model was successfully prepared. Figure 3 This is the result of staining the cell activation marker cFOS. Figure 3 It can be seen that the PVT, BNST, LSV and other brain areas of the mice in the BCAS group were activated at 40 days.

[0050] Example 2: Clarifying the effect of PVT neuron activity on cognitive function

[0051] The PVT is located in the midline of the thalamus and receives information input from multiple brain regions. It integrates interoceptive and exteroceptive signals to guide adaptive homeostatic regulation. This example uses a neural regulation method to control PVT activity to clarify the activity pattern of neurons in the brain region that has a cognitive protective effect. The experimental process of PVT regulation is as follows: Figure 4 shown.

[0052] In this example, mice were used as experimental animals and divided into a control group, a model group, a PVT neuron activity activation group, and a PVT neuron activity inhibition group, which were named as follows: Sham group, BCAS 40d group, BCAS+hM3Dq group, and BCAS+hM4Di group.

[0053] To clarify the intervention effect of PVT activity, the BCAS+hM3Dq group and the BCAS+hM4Di group were injected with viruses in situ in the PVT brain area two weeks before modeling, with excitatory AAV2 / 9-syn-hM3Dq-mcherry virus and inhibitory AAV2 / 9-syn-hM4Di-mcherry virus injected respectively.

[0054] (1) First, inject adenovirus and mark the brain area. ① Anesthesia and fixation. Use isoflurane gas to anesthetize the mouse and fix its head on the stereotaxic fixture so that the tail cannot fall off when lifted. ② Level the skull surface. Place the needle tip of the injection pump on the skull surface. Set it as 0 point just above Bregma. The skull surface is considered flat if the z-axis difference between 1.5 on the left and right and ≤0.03 between the anterior and posterior bregma. ③ Inject the virus at the PVT point. Use a skull drill to open a bone window of appropriate size. Perform virus injection at the following stereotaxic coordinate points: PVT, with Bregma as the reference point, move backward (AP) -0.5mm, move left and right (ML) ±0.65mm, and the needle insertion depth (DV) -3.65mm. Wait 2 weeks for virus expression.

[0055] (2) After viral expression, CNO (injection standard 3.3 mg / kg) was injected intraperitoneally to activate the virus and regulate the neuronal activity in the PVT brain area. Stimulate once a day, maintain activation for 5 hours, and then stimulate again after an interval of 24 hours for 14 consecutive days. During the neuroregulation period, place the mice in the treatment chamber to ensure sufficient food and activity space. Observe the environmental conditions in the treatment chamber, including temperature, humidity, light, oxygen content, etc., and observe the respiratory rate and activity frequency of the mice.

[0056] (3) The BCAS group underwent behavioral testing 40 days after surgery (the content and method of the behavioral testing were the same as those in Example 1).

[0057] The results are as follows Figure 5-7 As shown, Figure 5 This is the result of the novel object recognition test of cognitively impaired mice receiving different PVT regulation methods (n=4-8, **p<0.01). Figure 6 The results of the Y-maze test of cognitively impaired mice receiving different PVT regulation methods (n=4-8, ****p<0.0001, **p<0.01) are shown in Figure 1. Figure 5 and Figure 6 It can be seen that compared with the Sham group, the mice in the BCAS 40d group showed obvious declines in exploration desire, memory, novel object recognition ability, and spatial recognition ability, while the BCAS+hM4Dq group inhibited the activity of PVT neurons and alleviated the neurological damage caused by BCAS. The BCAS+hM3Dq group activated PVT excitatoryly, and the cognitive function of mice was not improved. Figure 7This is the result of the open field test in which cognitively impaired mice were subjected to different PVT regulation methods. There was no significant difference between the groups, indicating that no difference in motor ability and anxiety state of the mice was observed. Figure 5-7 The results show that appropriate inhibition of PVT activity can protect cognitive function during cerebral hypoperfusion, while activating PVT activity has no protective effect.

[0058] Example 3 Effect of PVT activity on neuropathological changes in mice

[0059] The brain regions responsible for cognitive functions are mainly the prefrontal cortex and hippocampus. In this example, HE staining and analysis were performed on each group of cognitively impaired mice in Example 2 to determine whether PVT activity would cause nerve damage.

[0060] First, the fixed and dehydrated tissue samples are embedded in paraffin and sliced ​​to a thickness of 4-5 μm. The paraffin sections are spread on glass slides and baked to make the tissue firmly adhere. Then, the tissue is dewaxed with xylene, followed by dehydration through a series of increasing concentrations of alcohol (70%, 80%, 95%, 100%), and then fully hydrated with distilled water. Next, hematoxylin is used to stain the cell nucleus to make it bluish purple, followed by differentiation and blueing steps to improve the staining effect. After that, eosin is used to stain the cytoplasm and extracellular matrix to make it pink. Finally, it is dehydrated again through gradient alcohol, transparentized with xylene, covered with a coverslip and sealed, and observed under a microscope.

[0061] The results are as follows Figure 8-17 As shown:

[0062] Figure 8 This is a picture of pathological changes in the prefrontal cortex of cognitively impaired mice that received different PVT regulation methods (the red arrows point to cells with irregular shapes and degeneration and necrosis). Fig. 9 The following is a statistical diagram of pathological damage to the prefrontal cortex of cognitively impaired mice subjected to different PVT regulation methods (n=4, *p<0.05, **p<0.01, ***p<0.001). Figure 8 and Fig. 9 The staining results of the prefrontal cortex showed that the neurons in the Cg, PL, and IL regions of the Sham group were relatively complete and clear. In the BCAS 40d group, irregular cell shapes, degeneration, necrosis and other pathological changes were generally observed. Compared with the BCAS 40d group, the neurons in the Cg, PL, and IL regions of both the BCAS+hM4Di group and the BCAS+hM3Dq group were improved.

[0063] Fig.10This is a diagram of the pathological changes in the motor cortex of cognitively impaired mice that received different PVT regulation methods (the red arrows point to cells with irregular shapes and degeneration and necrosis). Fig.11 The statistical diagram of motor cortex pathological damage in cognitively impaired mice subjected to different PVT regulation methods (n=4, *p<0.05, ***p<0.001, ****p<0.0001). Fig.10 and Fig.11 The results of motor cortex staining showed that compared with the Sham group, the BCAS 40d group showed irregular cell shapes and pathological changes such as degeneration and necrosis (arrows). Compared with the BCAS 40d group, the pathological damage of L5 neurons in the BCAS+hM4Di group was significantly improved, while the effect in the BCAS+hM3Dq group was not obvious.

[0064] Fig.12 This is a picture of the pathological changes in the sensory cortex of cognitively impaired mice that received different PVT regulation methods (the red arrows point to cells with irregular shapes and degeneration and necrosis). Fig.13 Figure 2 is a statistical diagram of sensory cortex pathological damage in cognitively impaired mice subjected to different PVT modulation methods (n=4, *p<0.05, ***p<0.001, ****p<0.0001). Fig.12 and Fig.13 The results of sensory cortex staining showed that compared with the Sham group, the BCAS 40d group showed irregular cell shapes, degeneration and necrosis and other pathological changes. Compared with the BCAS 40d group, the pathological damage of L2 / 3 and L5 neurons in the BCAS+hM4Di group was significantly improved, while the effect was not obvious in the BCAS+hM3Dq group.

[0065] Fig.14 This is a diagram of the pathological changes in the hippocampus of cognitively impaired mice that received different PVT regulation methods (the red arrows point to cells with irregular shapes and degeneration and necrosis). Fig.15 is a statistical diagram of hippocampal pathological damage in cognitively impaired mice subjected to different PVT regulation methods (n=4, **p<0.01, ***p<0.001, ****p<0.0001). Fig.14 and Fig.15 The results of hippocampal staining showed that the neurons in the CA1, CA3 and DG regions of the hippocampus in the sham group were relatively complete and clear. In the BCAS 40d group, it was observed that the cells were generally reduced in size, irregular in shape, and had pathological changes such as degeneration and necrosis. The neurons in the CA1, CA3 and DG regions of the BCAS+hM4Di group showed significant improvement, while the improvement effect of the BCAS+hM3Dq group was not significant.

[0066] White matter damage is a common pathological change in cognitive impairment. Antibody MBP is used to label white matter. Fig.16This is a graph showing the effects of inhibiting PVT regulation on the white matter of cognitively impaired mice. Fig.17 This is a statistical diagram of the effect of PVT inhibition on the white matter of cognitively impaired mice (n=3, ttest, mean±SEM, ****p<0.01, scale bar=30μm). Fig.16 and 17 It can be seen that compared with the Sham group, the white matter damage of mice in the BCAS 40d group was obvious, and after inhibiting PVT activity (BCAS+hM4di group), the white matter damage of mice could be alleviated.

[0067] In summary, the neuroregulatory method provided by the present invention can improve cognitive function and memory, and reduce pathological damage to the prefrontal cortex and hippocampus by appropriately inhibiting the activity of PVT neurons.

[0068] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0069] The above description is only a specific embodiment of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments described herein, but should conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for improving memory and cognitive ability through neural regulation, characterized in that: The method comprises the following steps: injecting adenovirus into the PVT brain region of a mammal, and activating the adenovirus through chemical stimulation to inhibit neuronal activity in the PVT brain region.

2. The method according to claim 1, characterized in that The mammals include mice, horses, cows, sheep, dogs, rabbits, pigs, monkeys or humans.

3. The method according to claim 1, characterized in that The adenovirus is AAV2 / 9-syn-hM4Di-mcherry.

4. The method according to claim 1, characterized in that: The chemical stimulation was intraperitoneal injection of clozapine N-oxide.

5. The method according to claim 4, characterized in that The frequency of the chemical stimulation was once a day, and the injection amount of clozapine N-oxide each time was 2 mg / kg.

6. The method according to claim 1, characterized in that The chemical stimulation was continued for 13-15 days.

Images