Qi deficiency and blood stasis model and application thereof

Abstract

The invention belongs to the technical field of medicines, and discloses a qi deficiency and blood stasis model and application thereof. A mouse qi deficiency and blood stasis model is prepared by combining temporary occlusion of bilateral common carotid artery with systemic hypotension surgery and sleep deprivation, and the model is detected through gripping power, lingual surface image analysis, open field behavioristics, new object recognition experiments, electron microscope ultrastructure analysis and functional nuclear magnetism. The results of ultrastructure and functional nuclear magnetism show that the model conforms to the clinical common qi deficiency and blood stasis characterization such as weakness, weakness, white tongue, memory deterioration and the like, and the model conforms to the clinical actual pathological conditions of qi deficiency and blood stasis, and is a qi deficiency and blood stasis syndrome. And a reliable animal model is provided for screening medicines and health-care products for preventing and recovering ischemic stroke.

Description

Technical Field

[0001] The present invention relates to a method for constructing a Qi deficiency and blood stasis model. The method mainly comprises a method of temporarily clamping bilateral common carotid arteries combined with systemic hypotension surgery (BCCAO) and sleep deprivation to prepare a mouse Qi deficiency and blood stasis model (CCI), which can be used for the development of Qi deficiency and blood stasis related drugs or health products, and belongs to the field of medical technology. Background Art

[0002] Qi deficiency and blood stasis syndrome is a disease caused by weak qi and stagnant blood circulation. Traditional Chinese medicine believes that qi is the leader of blood, and the power of blood transportation throughout the body comes from the promotion of qi. Wang Qingren, a great Chinese medicine expert in the Qing Dynasty, described it in his book "Correcting Errors in Medicine" as follows: "Since the original qi is weak, it cannot reach the blood vessels. If the blood vessels have no qi, they will stagnate and stagnate." He believed that people are based on yang qi, and diseases are based on qi deficiency. It can be seen that qi deficiency is the pathogenesis of blood stasis, and blood stasis is a manifestation of long-term qi deficiency. The clinical manifestations of qi deficiency and blood stasis syndrome mainly include less qi and laziness to speak, fatigue and weakness, dark tongue with white fur, pain like thorns, pain that does not move, dark and pale complexion, etc., showing obvious signs of blood stasis.

[0003] Qi deficiency and blood stasis syndrome is common in people who have suffered an acute ischemic stroke. It is a kind of stroke sequelae. Middle-aged and elderly people are prone to Qi deficiency and blood stasis syndrome due to aging, long-term overwork, irregular diet, etc., and long-term Qi deficiency and blood stasis are prone to induce acute ischemic stroke. With the increasing aging of my country's population, chronic middle-aged and elderly diseases such as Qi deficiency and blood stasis syndrome have become a problem that needs to be urgently solved in clinical prevention and treatment.

[0004] At present, the mouse models simulating qi deficiency and blood stasis mainly include drug modeling, surgical intervention and biological stress method, among which the drug modeling animals are prone to drug tolerance, which is not conducive to long-term research; the surgical intervention method is committed to the preparation of blood stasis characterization, which does not conform to the actual situation of clinical qi deficiency and blood stasis; the biological stress method can only make animals produce partial signs of qi deficiency and blood stasis, which is different from the intrinsic mechanism of clinical qi deficiency and blood stasis patients. The present invention provides a more accurate model preparation method for simulating clinical qi deficiency and blood stasis patients, and constructs a qi deficiency and blood stasis mouse model by using bilateral common carotid artery temporary clamping combined with systemic hypotension surgery (BCCAO) combined with sleep deprivation (SD), and compares the two models of BCCAO and sleep deprivation alone, and finds that it is more in line with the clinical qi deficiency and blood stasis symptoms. Summary of the invention

[0005] In order to overcome the shortcomings of existing mouse Qi deficiency and blood stasis syndrome, the present invention provides a new modeling method for mouse Qi deficiency and blood stasis model. The new modeling method is to prepare a mouse Qi deficiency and blood stasis model (CCI) by combining chronic cerebral ischemia (BCCAO) with sleep deprivation.

[0006] Furthermore, the sleep deprivation is between 16 and 20 hours per day, and the total period of long-term deprivation is not less than 30 days.

[0007] Furthermore, the chronic cerebral ischemia is hypotension induced by changes in isoflurane concentration, combined with bilateral common carotid artery ligation.

[0008] The above method can be used to screen or evaluate the preparation of active substances and drugs for preventing or treating diseases related to qi deficiency and blood stasis.

[0009] Furthermore, the Qi deficiency and blood stasis include symptoms such as fatigue, shortness of breath, worsening with movement, slightly puffy face, or hemiplegia, loose stools or constipation, clear urine, and dark tongue and pulse.

[0010] Furthermore, the qi deficiency and blood stasis may be caused by long-term insomnia induced by various reasons, or by blood circulation disorders caused by cerebral ischemia, myocardial ischemia, etc.

[0011] Furthermore, the active substances may include substances obtained from animals, plants, minerals, microorganisms, biosynthesis, chemical synthesis, etc.

[0012] Furthermore, the drugs may include drugs derived from animals, plants, minerals, microorganisms, biosynthesis, chemical synthesis, etc., and combinations thereof.

[0013] Furthermore, the active substance or drug can be taken alone or in combination with other therapeutic drugs or symptomatic drugs.

[0014] The beneficial effects of the present invention are as follows: the present invention proves that the model causes physical weakness and fatigue through gripping experiments; tongue surface image analysis determines that the model can cause dark tongue quality; open field behavioral tests show that the model's autonomous behavior ability and exploration ability are significantly reduced; new object recognition experiments show that the model's memory is significantly reduced. Further electron microscopic ultrastructural analysis found that the myelin lesions in the corpus callosum and striatum of the model animals were obvious, leading to neuronal information conduction disorders, which is the main reason for the memory loss in mice. Furthermore, the functional nuclear magnetic resonance results of the model also showed that the functional connectivity between memory-related brain areas and somatic movement and sensory brain areas was significantly weakened. Compared with the control group mice, the mouse model of Qi deficiency and blood stasis constructed in this study showed significant physical weakness, dark tongue and white fur, memory loss and other manifestations of Qi deficiency and blood stasis and pathological damage mechanisms, which fits the characteristics of the actual clinical situation and provides a reliable animal model for simulating the screening of drugs and health products before and during the recovery period of acute ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Effects of bilateral common carotid artery temporary occlusion combined with systemic hypotension surgery (BCCAO) on global cerebral blood flow in mice Baseline: cerebral blood flow before BCCAO surgery; BCCAO: cerebral blood flow at the completion of BCCAO surgery; 1Day: cerebral blood flow 1 day after BCCAO surgery. (A) Representative images of cerebral blood flow detected by laser speckle detection at baseline before surgery, at the completion of BCCAO surgery, and 1 day after surgery; (B) Comparison of cerebral blood flow in mice at baseline before surgery, at the completion of BCCAO surgery, and 1 day after surgery; **P<0.01 compared with Baseline; ***P<0.001 compared with Baseline.

[0016] Figure 2 Effect of CCI model on forelimb grip strength of mice Experimental groups: The mice were randomly divided into four groups. The mice in the Sham group were fed normally for 35 days; the mice in the BCCAO group were fed normally for 7 days and then underwent 5% isoflurane-induced systemic hypotension combined with bilateral common carotid artery temporary clamping surgery, and then continued to be fed normally for 28 days; the mice in the SD group were sleep deprived for 18 hours every day from 12:00 to 8:00 the next day for 35 days; the mice in the CCI group were treated with sleep deprivation in the same way as the SD group for 7 days, and then underwent 5% isoflurane-induced systemic hypotension combined with bilateral common carotid artery temporary clamping surgery, and then continued to be sleep deprived in the same way as the SD group for 28 days. Grip: Mouse forelimb grip strength; ***P<0.001 compared with Sham.

[0017] Figure 3 .CCI model for RGB values ​​of mouse tongue Experimental groups: The mice were randomly divided into four groups. The mice in the Sham group were fed normally for 35 days; the mice in the BCCAO group were fed normally for 7 days and then underwent 5% isoflurane-induced systemic hypotension combined with bilateral common carotid artery temporary clamping surgery, and then continued to be fed normally for 28 days; the mice in the SD group were sleep deprived for 18 hours every day from 12:00 to 8:00 the next day for 35 days; the mice in the CCI group were treated with sleep deprivation in the same way as the SD group for 7 days, and then underwent 5% isoflurane-induced systemic hypotension combined with bilateral common carotid artery temporary clamping surgery, and then continued to be sleep deprived in the same way as the SD group for 28 days. R: R value of mouse tongue surface image; G: G value of mouse tongue surface image; B: B value of mouse tongue surface image; *P<0.05 compared with Sham; **P<0.01 compared with Sham; ***P<0.001 compared with Sham.

[0018] Figure 4 Effects of CCI modeling on the autonomous activity, cognitive ability and memory of mice Experimental grouping is shown in Example 2; Total Distance: the total distance of the mouse in the open field box; Time in center: the time the mouse stayed in the center area of ​​the open field box; Entries in center: the number of times the mouse entered the center area of ​​the open field box; RI: recognition index; Escape time: escape time; T zone exploration time: T zone exploration time. (A) Open field test, novel object recognition test and Barnes maze test; (B) Movement trajectory of mice in the open field test; (C) Comparison of total movement distance, center area residence time and center area entry times in the open field test; **P<0.01 compared with Sham; ***P<0.001 compared with Sham; (D) Comparison of recognition index in the novel object recognition test; *P<0.05 compared with Sham; ***P<0.001 compared with Sham; (E) Comparison of escape time and exploration time in the target box area in the Barnes maze test; ***P<0.001 compared with Sham.

[0019] Figure 5 Fast blue staining was used to detect the effect of CCI modeling on myelin sheath in the cc, cg, ec brain regions of the corpus callosum and the CPu brain region of the striatum of mice. The experimental groups were the same as those in Example 2. cc: cc region of the corpus callosum; cg: cg region of the corpus callosum; ec: ec region of the corpus callosum; CPu: CPu region of the striatum.

[0020] Figure 6 Effects of CCI model on the ultrastructure of myelin sheath in mice Experimental groups: Mice were randomly divided into two groups. The mice in the Sham group were fed normally for 35 days; the mice in the CCI group were subjected to CCI modeling. g-ratio: the ratio of the inner radius of the myelin sheath to the outer radius of the myelin sheath; (A) Representative images of myelin sheaths in each group taken with a transmission electron microscope; (B) Comparison of g-ratio values ​​of myelin sheaths in each group; **P<0.01 compared with the Sham group.

[0021] Figure 7 .Effects of CCI modeling on the functional connectivity strength between brain regions in mice The experimental grouping is the same as in Example 6. CA1: CA1 area of ​​hippocampus; CA2: CA2 area of ​​hippocampus; CA3: CA3 area of ​​hippocampus; ccg: Genu of corpus callosum; DG: Dentate gyrus; ec: External capsule; ee: Corpuscallosum, extreme capsule; GU: Gustatory areas; LS: Lateral septal nucleus; MOp: Primary motor area; ORB: Orbitalarea, medial part; SSs: Supplemental somatosensory area, supplement the somatosensory area; 12 brain areas on the left and right sides of the mouse brain were selected to form a 24×24 matrix diagram; (A) Functional connection matrix diagram of each brain area; (B) Visualization results of functional connection strength; (C) The different sites of functional connection brain areas between Sham group and CCI group mice (P<0.05). DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings, but the present invention is not limited to the illustrated implementation scope.

[0023] Example 1: Effects of BCCAO surgery on whole brain blood flow in mice

[0024] BCCAO surgery: BCCAO surgery uses 5% isoflurane to induce systemic hypotension in mice combined with temporary clamping of bilateral common carotid arteries to prepare a mouse global cerebral ischemia model. Mice were deprived of water and food for one night before surgery, 2% isoflurane was used to induce anesthesia in mice, and 1.5% isoflurane was used to maintain anesthesia. After anesthesia, the mice were fixed in a supine position, the neck was prepared and disinfected, a midline incision was made, and the bilateral common carotid arteries of the mice were exposed and separated. 5% isoflurane was used to induce systemic hypotension in mice and maintained for 12 minutes. At 2 minutes, the bilateral common carotid arteries were clamped with a mouse artery clamp, and the clamping was maintained for 10 minutes. After completion, the isoflurane concentration was adjusted to 0%, the artery clamp was released, penicillin powder was applied to the wound, and the skin was aligned and sutured.

[0025] Laser speckle real-time blood flow detection: The principle of laser speckle blood flow imaging technology is that when the target is irradiated with laser, a random interference image is formed after reflection. When the blood flows, the interference image also moves, thereby achieving real-time, high-resolution imaging. Laser speckle is used to detect changes in cerebral blood flow in mice before surgery, at the completion of surgery, and 1 day after surgery. During the experiment, 2% isoflurane was used to induce anesthesia in mice, and 1.5% isoflurane was used to maintain anesthesia. After anesthesia, the mice were fixed in a supine position, the top of the head was prepared and disinfected, a midline incision was made, and the skull was exposed. During the test, the animal was placed on the stage, and the cerebral blood flow was detected after the hair adhering to the skull was wiped off with a cotton swab.

[0026] Statistics and analysis: All data are The t-test was used to perform statistical analysis on the preoperative baseline blood flow and the blood flow at the completion of the operation, and the preoperative baseline blood flow and the blood flow 1 day after the operation. One-way analysis of variance (Prism8.0) was used, and P<0.05 was used as the statistical difference standard.

[0027] Results: Figure 1 The results of the effect of BCCAO surgery on the whole brain blood flow of mice showed that the cerebral blood flow of the same mouse was significantly reduced immediately after BCCAO surgery (P<0.001), which was about 25% of the baseline value. When tested one day after surgery, the cerebral blood flow had recovered, but was still significantly lower than the baseline value detected before surgery (P<0.001), which was about 80% of the baseline value, indicating that this method has the conditions for preparing a blood stasis model.

[0028] Table 1 Changes of cerebral blood flow detected by laser speckle during BCCAO surgery

[0029]

[0030] **P<0.01, ***P<0.001 vs Baseline, n=4.

[0031] Example 2: Effect of Qi Deficiency and Blood Stasis Model on Physical Fatigue Indications in Mice

[0032] Preparation of Qi deficiency and blood stasis model: C57BL / 6J male mice were placed in a sleep deprivation apparatus for sleep deprivation (SD). The sleep deprivation apparatus was used for sleep intervention from 12 noon to 8 am the next day, and sleep deprivation was performed for 20 hours a day for 7 consecutive days. On the 8th day, a chronic cerebral ischemia model was created by using a surgical modeling method of temporary clamping of bilateral common carotid arteries combined with systemic hypotension. The BCCAO model operation method is as follows: 2% isoflurane induced anesthesia in mice, 1.5% isoflurane maintained anesthesia, mice were fixed in a supine position after anesthesia, the neck was prepared and disinfected, a midline incision was made, the bilateral common carotid arteries of mice were exposed and separated, 5% isoflurane induced systemic hypotension in mice for 12 minutes, and the bilateral common carotid arteries were clamped with a mouse artery clamp at 2 minutes, and the clamping was maintained for 10 minutes. After completion, the isoflurane concentration was adjusted to 0%, the artery clamp was released, penicillin powder was applied to the wound, and the skin was aligned and sutured. One day after the animal surgical modeling was completed, sleep deprivation was performed using a sleep deprivation apparatus from 12 noon to 8 am the next day, for 20 hours a day for 28 days.

[0033] Mouse forelimb grip test: Forelimb grip test is a common behavioral experiment to detect animal muscle strength. During the experiment, the mouse forelimbs are placed on the horizontal bar of the grip tester. After the mouse forelimbs grasp tightly, the mouse tail is gently lifted and pulled back horizontally at a constant speed. After the grip tester reads the reading, the test is considered completed. Each mouse is tested 3 times, with an interval of 15 minutes between each test to give the mouse sufficient rest time.

[0034] Statistics and analysis: All data are Dunnett's test was used to analyze the forelimb grip strength of each group (Prism 8.0), and P < 0.05 was set as the statistical difference standard.

[0035] Results: As shown in Table 2 and Figure 2 As shown in the CCI model, there was a significant statistical difference between SD and CCI in the forelimb grasping strength test of mice (P<0.001), indicating that mice in the SD group and the CCI group showed signs of weakness and fatigue of Qi deficiency and blood stasis.

[0036] Table 2 Grip test

[0037]

[0038] ***P<0.001 vs Sham, n=4.

[0039] Example 3 Effect of CCAO surgery on tongue quality in mice

[0040] Tongue surface image analysis: Tongue surface image is a common indicator of TCM examination. The tongue surface image analysis experiment is used to analyze whether mice have signs of qi deficiency and blood stasis. In the experiment, mice were anesthetized with 2% isoflurane, and tongue surface images of mice were collected using a high-definition camera. The images were collected once at 14 days and 28 days of sleep deprivation, for a total of two times for each mouse.

[0041] Statistics and analysis: All data are Dunnett's test was used to analyze the RGB values ​​of the tongue surface of each group (Prism 8.0), and P < 0.05 was set as the statistical difference standard.

[0042] Results: Figure 3 The results of the effect of the CCI model on the RGB values ​​of the tongue surface of mice showed that only the R (P<0.001), G (P<0.001), and B (P<0.01) values ​​of the CCI group showed statistical differences at the same time, indicating that the CCI group showed signs of blood stasis of qi deficiency and blood stasis, while the R, G, and B values ​​of the BCCAO group and the SD group did not show statistical differences. The above results suggest that the CCI model can induce the signs of qi deficiency and blood stasis in mice, such as weakness and dull tongue surface.

[0043] Example 4: Effects of CCI modeling on the autonomous activity, cognitive ability and memory of mice

[0044] Open field experiment: The open field experiment uses the rodents' natural instinct to explore novel places to detect the animals' autonomous behavior and exploratory behavior. The open field experiment is used to detect the damage of CCI modeling to the mice's autonomous activity ability. The open field box uses a 50cm×50cm×50cm opaque plate, and the bottom is divided into 4×4 squares. Keep the environment quiet during the experiment to reduce interference. The day before the modeling is completed, the animals are placed in the open field box alone for 5 minutes to adapt. After each animal is adapted, use 10% alcohol to wipe the open field box to remove odor. After the modeling is completed, the animals are placed in the center square, and the Smart 3.0 behavioral data acquisition software is used to record the movement trajectory and total movement distance of each group of mice within 5 minutes, the time spent in the center area, and the number of times the center area is entered. After each mouse is tested, the box needs to be cleaned before subsequent experiments.

[0045] Novel object recognition experiment: The novel object recognition experiment is a delicate and sensitive behavioral method that uses the rodents' natural instinct to approach and explore novel objects to detect the animal's recognition memory. The novel object recognition experiment is used to detect the damage to the recognition ability of mice caused by CCI modeling. The test box for the novel object recognition experiment is made of 50cm×50cm×50cm opaque board material. Before the experiment begins, the mice are placed in the test box to get used to the test environment. After the mice freely explore the test box for 10 minutes, they are returned to the cage to rest for 1 hour before the experiment begins. The test procedure includes two stages. In the first stage, two objects A of the same shape and color are placed in the test box. The mice are allowed to explore freely for 10 minutes and then returned to the cage to rest for one hour. In the second stage, one object A is replaced with an object B of a different color and shape, and the mice are allowed to explore freely for 5 minutes. During the entire experiment, the Smart 3.0 system is used to record the time the mice spend exploring the objects. The time to explore the new objects is recorded as T new , the time to explore new objects is recorded as T old , identification index (RI) = (T new / (T new +T old )) is used to evaluate the exploration of the target object.

[0046] Barnes maze test: The Barnes maze test is an accurate behavioral method to detect animal memory by using the characteristics of rodents to avoid light and love to explore in the dark. The Barnes maze test is used to detect the damage of CCI modeling to mouse memory. The experiment lasted for 4 days. The Barnes maze was a white metal disc with a diameter of 120 cm and a height of 50 cm. There were 20 holes arranged evenly on the edge, one of which was a target box. Every day, the animal was placed in the target box for 4 minutes to adapt. After the adaptation, the animal was placed in the center of the maze. The experiment started after 3 minutes of restraining the animal in a dark box. The time taken for the animal to enter the target box was recorded. The time limit was 4 minutes. The animals that did not enter were recorded for 4 minutes. After each animal completed the experiment, it was placed in the target box for 4 minutes to adapt, and the target box was cleaned to prevent the smell from interfering with the next animal. Escape time: the total time from the animal leaving the dark box to entering the target box; T area exploration time ratio: T area is the area where the target box is located, and the ratio of the animal's activity time in T area to the escape time.

[0047] Statistics and analysis: All data are Dunnett's test was used to analyze the total distance of open field movement, the time spent in the center area, the number of entries into the center area, the cognitive index of novel object recognition, the escape time of the Barnes maze, and the proportion of time spent exploring the T area of ​​each group (Prism 8.0), with P < 0.05 as the standard of statistical difference.

[0048] Results: The effects of CCI modeling on the autonomous activity, cognitive ability and memory of mice showed that the total movement distance (P<0.001), the time spent in the central area (P<0.001) and the number of times entering the central area (P<0.01) of mice in the CCI group in the open field were significantly reduced (Table 3, Figure 4 ), CCI modeling affected the autonomous exploration ability of mice, causing a significant decline; the recognition index of mice in the CCI group decreased significantly in the new object recognition experiment (P<0.001) (Table 4, Figure 4 ), indicating that CCI modeling caused damage to the recognition ability of mice; the escape time of mice in the Barnes maze test in the CCI group was significantly prolonged (P<0.001), and the proportion of exploration time in the target box area was significantly reduced (P<0.001) (Table 5, Figure 4 ), indicating that CCI modeling affects the memory of mice, causing them to show a significant decline in memory compared to normal mice. However, there was no significant difference in the time spent in the central area and the number of times the BCCAO group entered the central area in the open field experiment, and there was no significant difference in the number of times the SD group entered the central area, the recognition index, and the escape time. The above results indicate that chronic cerebral ischemia surgery and sleep deprivation alone cannot completely induce clinical signs of qi deficiency and blood stasis in mice, while the CCI model combined with chronic cerebral ischemia and sleep deprivation can induce signs of qi deficiency and blood stasis in mice.

[0049] Table 3 Open field test

[0050]

[0051] ** P<0.01, *** P<0.001 vs Sham, n=5.

[0052] Table 4 New object recognition experiment

[0053]

[0054] *** P<0.001 vs Sham, n=5.

[0055] Table 5 Barnes maze

[0056]

[0057] ** P<0.01, *** P<0.001 vs Sham, n=5.

[0058] Example 5: Fast blue staining to detect the effect of CCI modeling on the myelin sheath in the cc, cg, ec brain regions of the corpus callosum and the CPu brain region of the striatum of mice

[0059] Fast blue staining: Myelin refers to the tubular structure wrapped around the nerve fiber, which is mainly composed of myelin. It can isolate the electrical signals of the axon from the surrounding tissues and avoid interference with the signal transmission of neurons. It has a close relationship with biological memory. Luxol Fast Blue (LFB) dye belongs to copper-phthalocyanine dye, which has the staining property of binding to myelin in alcohol solution. After the model is completed, the mice are anesthetized, and after perfusion of PBS and 4% paraformaldehyde through the heart, the brain is taken for paraffin embedding and sectioning, and the section thickness is 6μm. Dewax the paraffin sections to water: place them in xylene for 20min×2 times, anhydrous ethanol for 15min×2 times, 75% ethanol for 5min in sequence, and then rinse under running water. The sections were added with Lauk's fast blue staining solution and stained at room temperature. Ethanol was added to wash away the excess stain. After rinsing with distilled water, Luxol differentiation solution was added for color separation. Ethanol was added to differentiate until the gray and white matter were clear, and then distilled water was used for rinsing. The sections were dehydrated at room temperature, made transparent with xylene, sealed with neutral gum, and observed and photographed for preservation.

[0060] Results: Figure 5 Fast blue staining was used to detect the effect of CCI modeling on the myelin sheath in the cc, cg, ec brain regions and CPu brain regions of the corpus callosum of mice. The results showed that the myelin sheaths in the cc, cg, ec brain regions and CPu brain regions of the corpus callosum of the Sham group mice were dense, arranged neatly, darker and uniform, while those in the CCI mice were loosely arranged, lighter in color, and showed obvious myelin disintegration and vacuoles. The above results indicate that the myelin sheaths in the corpus callosum and striatum of the CCI mice were significantly damaged, which is consistent with the decline in memory in the mice.

[0061] Example 6: Effect of CCI modeling on the ultrastructure of myelin sheath in mice

[0062] Transmission electron microscopy: Transmission electron microscopy is an electron microscope that uses electron beams as light sources. It can observe fine structures smaller than 0.2 μm that cannot be observed under an optical microscope. These structures are called submicrostructures or ultrastructures. Transmission electron microscopy is used to detect the effects of CCI modeling on the ultrastructure of myelin sheaths in mice. After modeling, the mice were anesthetized and perfused with PBS and 4% paraformaldehyde through the heart. The striatum and dorsal hippocampus were separated. 1 mm2 of each brain region was taken from each animal. 3 The tissue blocks were transferred to 2% PFA-2.5% glutaraldehyde for fixation. Next, the sections were dehydrated using a gradient method and embedded in Epon resin. Finally, the tissue blocks were cut into ultrathin sections (50-60 nm) and double-stained with uranyl acetate and lead citrate. Transmission electron microscopy was used to observe and collect images. The distance from the center of the myelin sheath to the inner wall of the myelin sheath was marked as r 内 , the distance from the center of the myelin sheath to the outer wall of the myelin sheath is marked as r 外 , g-ratio = r 内 / r 外, used to evaluate myelin damage. When myelin loss occurs, the g-ratio value will become smaller.

[0063] Statistics and analysis: All data are The g-ratio of the Sham group and the CCI group was statistically analyzed using t-test (Prism8.0), with P<0.05 as the standard of statistical difference.

[0064] Results: Figure 6 The results of the effect of CCI modeling on the ultrastructure of myelin sheath in mice showed that the g-ratio of myelin sheath in CCI mice was significantly increased compared with that in Sham mice (P<0.01), indicating that CCI mice showed obvious demyelination and obvious damage to the ultrastructure of myelin sheath, verifying the previous speculation that the memory loss in mice was related to myelin damage.

[0065] Example 7: Effects of CCI modeling on the strength of functional connections between brain regions in mice

[0066] Blood oxygen-dependent brain function MRI: Blood oxygen-dependent brain function MRI uses the fact that when neurons in a certain area of ​​the brain are active, the metabolic demand of the area increases accordingly. The activated neurons lead to increased oxygen consumption, which in turn changes the ratio of oxyhemoglobin and deoxyhemoglobin in the brain area. Brain function is detected through the different magnetic susceptibility of oxyhemoglobin and deoxyhemoglobin. Blood oxygen-dependent brain function MRI is used to detect the effect of CCI modeling on the strength of functional connections between brain regions in mice.

[0067] Statistics and analysis: All data are The t-test was used to analyze the brain functional connectivity between the Sham group and the CCI group (Prism8.0), and P<0.05 was set as the statistical difference standard.

[0068] Results: Figure 7 The results of the effect of CCI modeling on the strength of functional connectivity between brain regions of mice showed that the functional connectivity of memory-related brain regions (CA1, CA2, CA3, ccg, DG, ec) and somatic movement (MOp) and sensory brain regions (LS, ORB, SSs) of CCI mice was significantly weakened, which is consistent with the clinical indications of long-term chronic cerebral ischemia.

[0069] In summary, the present invention prepares a mouse Qi deficiency and blood stasis model by a method of temporary bilateral common carotid artery clamping combined with systemic hypotension surgery (BCCAO) and sleep deprivation, and detects the model through grip strength, tongue surface image analysis, open field behavior, new object recognition experiment, electron microscope ultrastructure analysis and functional nuclear magnetic resonance. The results show that the model is consistent with the common clinical manifestations of Qi deficiency and blood stasis and pathological injury mechanisms such as physical weakness, dark tongue with white fur, and memory loss, and is in line with the actual clinical situation. It provides a reliable animal model for the screening of drugs and health products for Qi deficiency and blood stasis syndrome, and before and during the recovery period of acute ischemic stroke.

[0070] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the scope of protection of the invention.

Claims

1. A Qi deficiency and blood stasis model, It is characterized in that The method for constructing the Qi deficiency and blood stasis model is prepared by combining long-term sleep deprivation with chronic cerebral ischemia.

2. The Qi deficiency and blood stasis model according to claim 1, It is characterized in that The sleep deprivation is between 16 and 20 hours per day, and the total period of long-term deprivation is not less than 30 days.

3. The Qi deficiency and blood stasis model according to claim 1, It is characterized in that The chronic cerebral ischemia is hypotension induced by changes in isoflurane concentration, combined with bilateral common carotid artery ligation.

4. Application of a Qi deficiency and blood stasis model in screening or evaluating the preparation of active substances and drugs for preventing or treating Qi deficiency and blood stasis related diseases.

5. The use according to claim 4, It is characterized in that The Qi deficiency and blood stasis include symptoms such as fatigue, shortness of breath, worsening with movement, slightly puffy face, or hemiplegia, loose stools or constipation, clear urine, and dark tongue and pulse.

6. The use according to claim 4, It is characterized in that The qi deficiency and blood stasis are caused by long-term insomnia induced by various reasons, or by blood circulation disorders caused by cerebral ischemia, myocardial ischemia, etc.

7. The use according to claim 4, It is characterized in that The active substances include substances obtained from animals, plants, minerals, microorganisms, biosynthesis, and chemical synthesis.

8. The use according to claim 4, It is characterized in that The drugs include drugs derived from animals, plants, minerals, microorganisms, biosynthesis, and chemical synthesis, and their combinations.

9. The use according to claim 4, It is characterized in that The active substance and drug can be taken alone or in combination with other therapeutic drugs or symptomatic drugs.

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