Construction method of animal cerebral ischemia reperfusion model

Abstract

The invention relates to a construction method of an animal cerebral ischemia reperfusion model, which is characterized in that V-shaped incision plugging after ICA direct near-end and far-end ligation is realized for a mouse for the first time, a traditional ECA plugging path is abandoned, ligation is not carried out in the whole process, ECA is not cut off, and ECA blood supply is completely reserved; meanwhile, an ICA incision is closed through a 11-0 suture line in a single needle mode, and minimally invasive repair of the blood vessel is achieved. According to the scheme, the ECA does not need to be subjected to any dissociation, traction or catheterization operation, modeling is completed only through ICA local minimally invasive operation, the ECA keeps a natural physiological state, and blood supply is not interfered at all; meanwhile, foreign matter does not need to be additionally placed, ICA incision closing is achieved only through single-needle suture, and no device is left after modeling. According to the application, craniotomy is not needed, MCA near-end ischemia can be realized only through neck minimally invasive operation, and the application is closer to an ICA-MCA occlusion mechanism of clinical human ischemic stroke; mCA is accurately blocked through the suture, the ischemia range is controllable, reperfusion is minimally invasive thrombus withdrawing and blood vessel suturing, and secondary brain tissue damage caused by craniotomy is avoided.

Description

Technical Field

[0001] This invention relates to the field of animal model technology, and in particular to a method for constructing an animal brain ischemia-reperfusion model. Background Technology

[0002] Stroke is the second leading cause of death and disability worldwide, with ischemic stroke being the most common. This condition occurs due to narrowing or blockage of the arteries supplying blood to the brain, leading to insufficient cerebral blood flow (CBF) and oxygenation. Most ischemic strokes occur within the territory of the middle cerebral artery (MCA), hence many animal models of focal cerebral ischemia focus on this region. Current main methods for model construction include open craniotomy with mechanical occlusion, photochemical occlusion, the wire method, and embolization. Among these, the wire method, developed in 1981, has been widely used as one of the most accurate and classic models for simulating human ischemic stroke. It offers advantages such as no need for craniotomy, highly controllable reperfusion, and the ability to simulate the core and penumbra of the stroke.

[0003] This method involves making a small incision in the common carotid artery, inserting a suture into the MCA, or directly cutting open the external carotid artery (ECA), straightening it, and then inserting the suture into the MCA. Currently, disadvantages of this method include significant welfare effects, subarachnoid rupture, and hypothalamic problems. However, ischemic problems in the inferior branch arteries due to ECA occlusion are rarely mentioned. Most ischemic stroke cases involve occlusion of the internal carotid artery (ICA), MCA, or tandem occlusion; concurrent ECA embolism and ICA occlusion are rare, likely due to embolism collapse at the bifurcation of the common carotid artery. Traditional surgical methods obstruct ECA blood flow in experimental animals, leading to insufficient blood supply to the superior thyroid artery, lingual artery, facial artery, etc., resulting in a series of functional changes in organs such as the thyroid and larynx, ultimately reducing the survival rate of animal models.

[0004] Chinese patent CN110612941A discloses a method for creating a guinea pig middle cerebral artery (MCA) occlusion model. The method includes the following steps: preparing an anesthetic, weighing the guinea pig, and administering a certain amount of anesthetic via intraperitoneal injection based on the guinea pig's weight to anesthetize it; placing the guinea pig on a temperature-controlled blanket and securing it to an operating table, ensuring the guinea pig's body is straight and tension-free; shaving the guinea pig's neck hair with a clipper and disinfecting the area with iodine solution; making an incision along the midline of the guinea pig's neck, cutting the anterior cervical fascia to expose the right sternocleidomastoid muscle, and then separating the anterior neck muscles and the sternocleidomastoid muscle, using fine sutures to separate the muscle tissue until the complete carotid artery (CCA) is observed. This method constructs a middle cerebral artery (MCA) occlusion model by performing surgery on the guinea pig's neck and using silicone sutures, and directly restoring blood flow through the carotid artery after reperfusion, ensuring effective reperfusion. However, this protocol uses guinea pigs as experimental animals. While guinea pigs are also used as experimental animals, their applications are not as widespread as those of mice. Guinea pigs and mice are different animal models, with fundamental differences in physiological structure and surgical procedures. Furthermore, this protocol requires ligating and cutting the ECA and inserting an ICA through the ECA stump, which can still cause ischemia in the ECA-supply area, leading to thyroid and facial tissue dysfunction.

[0005] Chinese patent CN112107387A discloses a method for establishing a mouse model of distal middle cerebral artery (dMCAO) ischemia-reperfusion. The method involves ligating the distal MCAO vessel with a suture parallel to it to achieve occlusion. The suture is untied after 50 minutes. This method not only reduces the risk of vessel damage but also effectively achieves vascular occlusion and facilitates subsequent reperfusion. It shortens the time required to establish a transient dMCAO model in mice, reduces modeling costs, and can stably and effectively achieve a dMCAO ischemia-reperfusion model with good repeatability and low mortality. However, this method, which targets the distal middle cerebral artery (MCA), requires craniotomy and drilling to expose the vessel, making it highly invasive and causing significant brain tissue damage. It achieves ischemia by ligating the distal MCA and common carotid artery without embolization, limiting the ischemic area to the distal MCA and failing to simulate the ischemic characteristics of clinical ICA-MCA tandem occlusion. Furthermore, it avoids manipulation of the neck vessels.

[0006] Chinese patent CN104983480A discloses a method for establishing a rat model of cerebral ischemia-reperfusion injury with a catheter-preserved external carotid artery. The method involves: freeing the rat's external carotid artery to align it with or parallel to the direction of the internal carotid artery; inserting a suture plug through an incision in the external carotid artery to induce cerebral ischemia in the rat's neck; removing the suture plug 1-2 hours after inducing cerebral ischemia, and then inserting a reusable drug-injecting catheter. This method of establishing a rat ischemia-reperfusion model using the external carotid artery suture plug method preserves the normal structure of the common carotid and internal carotid arteries on the operated side, while retaining a reusable drug-injecting catheter within the external carotid artery, allowing for repeated injections into the internal carotid artery at different time points. It also enables post-model neurological function assessment, ensuring that only qualified animals are included in the experimental intervention, greatly improving the scientific rigor of the experiment. The experimental animals used in this protocol are rats, and the surgical procedures and suture specifications are completely different from those in mouse models. Although the ECA is preserved in this protocol, it needs to be freed and pulled to be in a straight line with the ICA, which will still damage the ECA vessels and surrounding tissues. In addition, a catheter injection balloon needs to be inserted, making the operation complex and highly invasive. The embolization path is still the ECA.

[0007] Chinese patent CN109758258A discloses a method for creating an experimental animal model of cerebral ischemia-reperfusion with reduced complications, comprising the following steps: carotid artery exposure procedure; ligation of the proximal end of the external carotid artery with a slipknot, without cutting; arterial incision at the proximal end of the internal carotid artery; insertion of a suture to induce ischemia in the middle cerebral artery; untying the suture at the distal end of the internal carotid artery upon reaching the predetermined ischemia time; removal of the suture; implantation of an arterial tube at the incision site; fixation of the common carotid artery incision with glue; avoiding contact with surrounding tissues for 1 minute, allowing it to cure naturally; after 2 minutes, as the glue gradually dries and cures, and the exterior no longer adheres to surrounding tissues, internal reperfusion of blood flow to the internal carotid artery is maintained through the insertion of a tube; and a final procedure. This method does not require ligation or cutting of the external carotid artery, superior thyroid artery, etc., making the operation relatively simple and convenient for subsequent animal care, especially suitable for long-term drug effect observation models. Although the proposed approach involves a proximal incision of the ICA, it does not perform double ligation of the ICA at both the proximal and distal ends. The incision has no precise range limitation, which can easily lead to vascular rupture and bleeding. In addition, the approach requires the implantation of an arterial tube and fixation with glue at the incision site. The glue is prone to adhesion to surrounding tissues, and the arterial tube can cause damage to the vascular intima, increasing the risk of thrombosis.

[0008] Therefore, finding an improved new method to establish a model of middle cerebral artery occlusion (MCAO) that is superior to traditional models is crucial for scientific research. Summary of the Invention

[0009] Based on the problems of existing methods for constructing animal brain ischemia-reperfusion models described above, such as the need to process the ECA, the impact on the blood supply of the external carotid artery (ECA), and the complexity of the operation, this invention provides a method for constructing an animal brain ischemia-reperfusion model.

[0010] This invention uses the internal carotid artery one-needle method to construct an animal model of cerebral ischemia-reperfusion.

[0011] The objective of this invention can be achieved through the following technical solutions: This application provides a method for constructing an animal cerebral ischemia-reperfusion model, specifically a method for constructing an animal cerebral ischemia-reperfusion model using the internal carotid artery single-needle technique, comprising the following steps: Step 1: Animal pretreatment and anesthesia, and exposure of the common carotid artery (CCA) and internal carotid artery (ICA). Step 2, ICA incision: Use sutures to tie slip knots at the proximal and distal ends of the ICA, and make a V-shaped incision between the ligation points; Step 3: Insertion of a thrombus to block the MCA: A microvascular thrombus adapted to the diameter of the ICA is inserted into the ICA through the V-shaped incision to block the middle cerebral artery (MCA); Step 4, Ischemia and Reperfusion: After maintaining the ischemic state of the MCA occlusion, first release the ligation at the distal end of the ICA and remove the microvascular embolus, then release the ligation at the proximal end of the ICA to achieve cerebral ischemia-reperfusion. Step 5: Single-needle closure of the ICA incision: Perform a single-needle suture and tie a knot at the V-shaped incision to achieve complete closure of the ICA incision; Throughout steps one through five, the external carotid artery blood supply is preserved: the external carotid artery (ECA) is not ligated, cut, or freed during the entire modeling process, maintaining the natural physiological state and smooth blood flow of the ECA.

[0012] In one embodiment of the present invention, in step one, 8-week-old mice are selected.

[0013] In one embodiment of the present invention, in step one, the mice undergo a 3-day adaptation period before modeling. The rearing environment is a temperature of 22-26°C, a relative humidity of 40-70%, and a 12-hour light / 12-hour dark cycle. Each cage contains 4-5 mice, who have free access to irradiated sterilized mouse food and filtered sterilized drinking water.

[0014] In one embodiment of the present invention, in step one, 8-week-old mice are anesthetized with isoflurane and fixed supine on the operating table. The neck is prepared and disinfected. The skin is longitudinally cut 1-1.5cm along the left side of the trachea, 2-3mm away from the neck. A retractor is placed to fully expose the operating area. The common carotid artery (CCA) and internal carotid artery (ICA) are separated and exposed.

[0015] In one embodiment of the present invention, in step two, a live knot is tied at the origin of the maxillary artery distal to the ICA.

[0016] In one embodiment of the present invention, in step two, the suture is a medical surgical suture, and the tightness of the slip knot is based on blocking ICA blood flow without damaging the vascular endothelium.

[0017] In one embodiment of the present invention, in step two, the depth of the V-shaped incision only penetrates the adventitia and media of the blood vessel, without damaging the intima.

[0018] In one embodiment of the present invention, in step two, the range of the V-shaped incision does not exceed 1 / 3 of the circumference of the blood vessel.

[0019] In one embodiment of the present invention, in step two, a V-shaped incision is made in the ICA tube wall of the ligation zone using microsurgical scissors.

[0020] In one embodiment of the present invention, in step three, the microvascular embolus is a silicone suture embolus used in mouse MCAO models. During insertion, the suture embolus is kept aligned with the direction of the ICA to avoid bending or puncturing the vascular endothelium.

[0021] In one embodiment of the present invention, in step three, a microvascular embolus adapted to the diameter of the ICA is inserted into the ICA until slight resistance is felt and then insertion is stopped.

[0022] In one embodiment of the present invention, in step four, the ischemic state of the MCA occlusion is maintained for 2 hours.

[0023] In one embodiment of the present invention, in step five, 11-0 suture thread is used for single-needle suturing and knotting to achieve complete closure of the ICA incision.

[0024] In one embodiment of the present invention, in step five, after the ICA incision is sutured, the neck muscles of the mouse are repositioned, the skin incision is sutured in layers, and the mouse is kept in a single cage and kept warm after the operation. After the mouse recovers from anesthesia, it is returned to the normal breeding environment.

[0025] In one embodiment of the present invention, after the animal brain ischemia-reperfusion model is constructed, a model effectiveness verification step is further included, the verification step comprising: (1) Cerebral blood flow detection: Before reperfusion, laser speckle imaging technology was used to detect the blood flow status of the head, face and neck of mice to confirm that the blood flow in the MCA occlusion area was significantly reduced and the blood flow in the ECA supply area was normal. (2) Neurological function scoring: The Longa five-point scoring system was used to assess the degree of neurological deficit in mice after surgery; (3) Detection of cerebral infarction volume: After the operation, the mice were sacrificed and the brain tissue was taken for 2.0% TTC staining. The percentage of cerebral infarction volume to total brain volume was calculated by image analysis software. (4) Thyroid function test: Plasma was collected from mice 24 hours after surgery, and the concentrations of plasma T3 and T4 were determined by enzyme-linked immunosorbent assay. (5) Long-term prognosis detection: record the changes in body weight and survival rate of mice within 30 days after surgery.

[0026] In one embodiment of the present invention, the experimental data for validating the model were statistically analyzed using Prism10 software. Quantitative data were expressed as x±s. One-way ANOVA was used to compare the means of multiple groups, and the Turkey method was used for pairwise comparisons between groups. P<0.05 was considered statistically significant.

[0027] This invention provides a method for constructing a mouse internal carotid artery single-needle cerebral ischemia-reperfusion (MCAO) model. The core innovation is to directly insert embolization through a V-shaped incision in the internal carotid artery (ICA), preserve the blood supply of the external carotid artery (ECA) throughout the process, and close the ICA incision with a single needle suture.

[0028] This application method is the first to achieve direct proximal and distal ligation of the ICA followed by V-shaped incision embolization in mice, abandoning the traditional ECA embolization path, and without ligating or cutting the ECA throughout the process, completely preserving the ECA blood supply; at the same time, the ICA incision is closed with a single needle using 11-0 sutures to achieve minimally invasive vascular repair.

[0029] This approach requires no detachment, traction, or catheterization of the ECA. Modeling is achieved solely through minimally invasive procedures on the ICA, maintaining the ECA in its natural physiological state with uninterrupted blood supply. Furthermore, no additional foreign objects are required; the ICA incision is closed with a single-needle suture, leaving no residual devices after modeling.

[0030] This proposed approach eliminates the need for craniotomy, achieving proximal MCA ischemia through minimally invasive neck surgery, closely mimicking the ICA-MCA occlusion mechanism in clinical human ischemic stroke. By precisely occluding the MCA with suture embolization, the ischemic range is controllable, and reperfusion is achieved through minimally invasive thrombectomy followed by vascular suturing, avoiding secondary brain tissue damage caused by craniotomy.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The proposed method eliminates invasive procedures such as craniotomy, vascular catheterization, and glue fixation. The ICA incision is ≤1 / 3 of the circumference and a single-needle suture is used, which significantly reduces the vascular injury rate, leaves no foreign body residue, and the risk of postoperative vascular adhesion and thrombosis is almost zero.

[0032] 2. The proposed method preserves the natural blood supply of the ECA throughout the process, avoiding insufficient blood supply to the superior thyroid artery, facial artery, etc., with no significant abnormalities in thyroid hormone levels. Mice recover their weight quickly after surgery, and the 30-day survival rate is much higher than that of traditional ECA ligation / cutting models, significantly improving the survival rate of model animals.

[0033] 3. The animal model provided in this application directly simulates the ischemic mechanism of ICA-MCA tandem occlusion in human clinical practice, and has no non-clinical interfering factors of ECA ischemia. The infarct volume is stable (33.8%), the neurological function score is more accurate, the model has a higher clinical fit, and is more suitable for the preclinical evaluation of stroke drugs.

[0034] 4. The proposed method does not require complicated procedures such as catheter placement, craniotomy, and vascular traction. It is completed in just five minimally invasive steps in the neck: ligation, incision, embolization, suturing, and reperfusion. The procedure is short, simple, and reproducible, making it suitable for large-scale laboratory modeling. Attached Figure Description

[0035] Figure 1 A schematic diagram of the process of constructing an animal model of cerebral ischemia-reperfusion using the one-needle method of the internal carotid artery; Figure 2 Blood flow speckle images of the brain, neck, and face before and after ICA surgery in the ICA group; Figure 3 Blood flow speckle images of the brain, neck, and face before and after ECA surgery; Figure 4 Representative TTC-stained brain sections and percentages of infarct area relative to total brain area for the ECA and ICA groups; Figure 5 Neurological scores for the ECA and ICA groups; Figure 6 Quantitative thyroid hormone levels in the ECA, ICA, and control group before and after surgery; Figure 7 Changes in body weight of mice in the ECA and ICA groups 30 days after surgery; Figure 8 The changes in survival rate of mice in the ECA and ICA groups 30 days after surgery. Detailed Implementation

[0036] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0037] Example This embodiment provides a method for constructing an animal cerebral ischemia-reperfusion model using a single-needle technique along the internal carotid artery, specifically a method for constructing a mouse middle cerebral artery ischemia (MCAO) model. A schematic diagram of the process is provided below. Figure 1 The specific steps are as follows: S1, Pretreated mice: S1-1. Animal Reception, Health Assessment, and Adaptation Period Upon arrival, animal staff transferred the animals from their transport packages to cages and examined each animal. The examination included checking their appearance, limbs, and orifices, as well as observing for any abnormal behavior while at rest or moving. The acclimatization period was 3 days. Eight-week-old mice were used in this example.

[0038] S1-2, Environmental Protection: Mice will be housed in transparent resin plastic cages (420mm × 240mm × 240mm), 4-5 mice per cage. The cage bedding will be autoclaved corn cob bedding (Jiangsu Medison Biomedical Co., Ltd., China), changed twice weekly. The room number for each animal will be recorded in the experimental log throughout the experiment. The animal rooms will be equipped with HEPA filters, with ventilation at 10-20 times per hour. The temperature will be maintained between 22-26°C (66-79°F), and the relative humidity between 40-70%. Temperature and humidity will be continuously monitored and recorded. Lighting conditions will consist of 12 hours of fluorescent lighting (08:00-20:00) and 12 hours of no lighting per day.

[0039] S1-3, Food and Drinking Water: Laboratory mice had unlimited access to specialized rodent feed (irradiated and sterilized, Jiangsu Medison Biomedical Co., Ltd., China). Each batch of animal feed received was accompanied by a corresponding batch analysis certificate from the supplier, which was archived. Throughout the experiment, the laboratory mice had unlimited access to internally treated drinking water, which consisted of local municipal water filtered and sterilized using a Mol ultrapure water system. Drinking water was tested twice a year, including tests for heavy metals, nitrates, dissolved minerals, total bacterial count, and E. coli. Test reports were archived.

[0040] Within the foreseeable future, the levels of known contaminants in animal food and drinking water will not affect the purpose or procedures of this experiment.

[0041] S1-4, Cages and Animal Identification Each laboratory mouse is assigned a unique number. Before grouping the animals, the cages should be labeled with the project number, species / strain, sex, cage number, and animal number. After grouping, the cages should be labeled with color-coded group information and the above information. Grouping details should be recorded in the randomization file. Cage racks should be used to stratify the cages to mitigate the influence of environmental factors on the experiment.

[0042] S2. Constructing the MCAO model S2-1, Anesthetized mice Mice were anesthetized with isoflurane, their necks were prepared, and they were fixed in a supine position on the operating table. The surgical area was then disinfected.

[0043] S2-2. Make a longitudinal incision of the skin, approximately 1-1.5 cm long, 2-3 mm to the left of the trachea. Place the retractor to fully expose the operating area.

[0044] S2-3. Dissect and expose the internal carotid artery, and tie a slipknot at the origin of the internal carotid artery. Tie a slipknot at the distal end of the internal carotid artery before the origin of the pterygopalatine artery.

[0045] S2-4. Make a V-shaped cut between the two knots (no more than one-third of the vessel circumference), insert the corresponding specification suture through the cut, and stop when you encounter slight resistance.

[0046] S2-5. After ischemia, open the distal dead knot, pull out the suture, sew a stitch at the cut with 11-0 suture thread and tie a knot, and the cut will be completely closed.

[0047] S2-6. After two hours of ischemia, the dead knot at the origin of the internal carotid artery is loosened to achieve reperfusion.

[0048] S3, Cerebral blood flow measurement S3-1. Before reperfusion, anesthetize the mice and shave their heads, faces and necks.

[0049] S3-2. Subsequently, the skull was exposed, and blood flow in the face and brain of both groups of mice was assessed using laser speckle imaging (Sansbio, Jiangsu, China). Figure 2 and Figure 3 ) S3-3. The original images are processed using computer algorithms to create blood flow comparison charts. Figure 4 ) S4, Neurological Deficit Score The two groups of MCAO models were assessed for neurological deficits using the Longa five-point scoring system, with scores ranging from 0 to 4, where 0 indicates no damage and 4 indicates severe damage.

[0050] The scoring criteria are as follows: 0 points: no neurological deficit; 1 point: inability to fully extend the paralyzed side, indicating mild neurological deficit; 2 points: the subject turns in circles towards the paralyzed side while walking, indicating moderate neurological deficit; 3 points: the subject leans towards the paralyzed side while walking, indicating severe neurological deficit; 4 points: inability to walk spontaneously and loss of consciousness. Figure 6 ) S5, TTC staining of brain slices S5-1. After euthanizing the mouse, quickly remove the brain tissue and place it in a -20°C freezer for 20 minutes.

[0051] S5-2. Cut the brain into six 2 mm thick slices.

[0052] S5-3. Immerse these slices in a 2.0% solution of 2,3,5-triphenyltetrazol chloride (TTC, Phygene, Fuzhou, China) and incubate at 37°C in the dark for 30 minutes.

[0053] S5-4. Subsequently, slides were collected, photographed, and image analysis software was used to determine the infarct volume and edema. Figure 5 ) S5, Thyroid hormone measurement Plasma was collected 24 hours after the operation, and plasma L-thyroxine (T4) and 3,5,3'-triiodothyronine (T3) (ng / ml) were quantitatively measured by enzyme-linked immunosorbent assay (Molecular Devices, Shanghai, China).

[0054] S6, survival rate and weight change The mice were weighed 30 days after surgery, and the time of death was recorded.

[0055] S7. Statistical Processing Statistical analysis was performed using Prism 10 software. Quantitative data are expressed as mean ± standard deviation (x±s). One-way ANOVA was used for comparisons of means across multiple groups, and Turkey's method was used for pairwise comparisons between groups. A p-value < 0.05 was considered statistically significant.

[0056] The evaluation results of the MCAO model constructed in this embodiment are as follows: like Figure 2-3 As shown, blood flow in the facial, neck, and cerebral regions of both groups of mice was within the normal range before surgery. Two hours after ischemia, the mean cerebral blood flow (CBF) on the surgical side was significantly reduced in both the ECA and ICA groups compared to the contralateral side. Notably, blood flow in the facial and neck regions was significantly reduced in the ECA group, while the ICA group retained most of the facial and cervical blood flow.

[0057] like Figure 4 As shown, TTC staining of brain tissue sections revealed neuropathological damage caused by cerebral ischemia, clearly distinguishing the infarcted area from normal brain tissue. In the model group, the infarcted area appeared white. Image analysis showed that the infarct volume in the ECA and ICA groups was 34.6% and 33.8%, respectively. These results confirm the successful establishment of the MCAO model using a single injection method.

[0058] like Figure 5As shown, consistent with previous reports, the Longa score in the ECA group (2.79±0.347) was higher than that in the ICA group (2.55±0.227). This indicates that the neurological condition of the mice in the ECA group was significantly worse than that in the ICA group.

[0059] like Figure 6 As shown, the mean T3 concentrations in the ICA, ECA, and SHAM groups were 0.83±0.36 ng / ml, 3.32±0.68 ng / ml, and 2.34±0.72 ng / ml, respectively. The mean T4 concentrations were 15.24±5.36 ng / ml, 45.44±4.371 ng / ml, and 31.44±6.930 ng / ml, respectively.

[0060] like Figure 7 and Figure 8 As shown, both groups of mice exhibited similar initial body weights before surgery (p>0.05). Both groups experienced weight loss at 24 hours and 2 days post-surgery, which could be partly attributed to previously reported complications associated with MCAO surgery. However, the ICA group showed a trend towards weight recovery from 1 week to 1 month post-surgery, and by day 30, the weight loss observed in the ICA group was significantly less than that in the ECA group. These findings suggest that the ICA group mice had a better prognosis than the ECA group mice.

[0061] In summary, this method preserves blood flow through the ECA, more closely resembling the actual mechanism of ischemic stroke in humans. Preliminary comparative results indicate that this method has potential advantages over traditional methods in certain classic experimental indicators. This novel approach provides an innovative solution to the challenges faced by traditional models. Future research should further investigate the modeling effectiveness of this technique and its potential new findings, ultimately providing more effective model support for the diagnosis and treatment of stroke.

[0062] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for constructing an animal model of cerebral ischemia-reperfusion, characterized in that, The steps for establishing an animal model of cerebral ischemia-reperfusion using a single-needle technique along the internal carotid artery include: Step 1: Animal pretreatment and anesthesia, and exposure of the common carotid artery (CCA) and internal carotid artery (ICA); Step 2, ICA incision: Use sutures to tie slip knots at the proximal and distal ends of the ICA, and make a V-shaped incision between the ligation points; Step 3: Insertion of a thrombus to block the MCA: A microvascular thrombus adapted to the diameter of the ICA is inserted into the ICA through the V-shaped incision to block the middle cerebral artery MCA; Step 4, Ischemia and Reperfusion: After maintaining the ischemic state of the MCA occlusion, first release the ligation at the distal end of the ICA and remove the microvascular embolus, then release the ligation at the proximal end of the ICA to achieve cerebral ischemia-reperfusion. Step 5: Single-needle closure of the ICA incision: Perform a single-needle suture and tie a knot at the V-shaped incision to achieve complete closure of the ICA incision; Throughout steps one through five, the external carotid artery blood supply is preserved: the external carotid artery (ECA) is not ligated, cut, or dislodged during the entire modeling process, maintaining the natural physiological state and smooth blood flow of the ECA.

2. The method for constructing an animal cerebral ischemia-reperfusion model according to claim 1, characterized in that, In step one, 8-week-old mice were selected.

3. The method for constructing an animal cerebral ischemia-reperfusion model according to claim 1, characterized in that, In step one, the mice undergo a 3-day acclimatization period before modeling. The rearing environment is 22-26℃, relative humidity 40-70%, 12-hour light / 12-hour dark cycle, with 4-5 mice per cage. They have free access to irradiated sterilized mouse food and filtered sterilized drinking water.

4. The method for constructing an animal cerebral ischemia-reperfusion model according to claim 1, characterized in that, In step one, 8-week-old mice were anesthetized with isoflurane and fixed supine on the operating table. The neck was prepared and disinfected. The skin was longitudinally cut 1-1.5cm along the left side of the trachea, 2-3mm away from the neck. A retractor was placed to fully expose the operating area. The common carotid artery (CCA) and internal carotid artery (ICA) were separated and exposed.

5. The method for constructing an animal cerebral ischemia-reperfusion model according to claim 1, characterized in that, In step two, the depth of the V-shaped incision only penetrates the adventitia and media of the blood vessel, without damaging the intima.

6. The method for constructing an animal cerebral ischemia-reperfusion model according to claim 5, characterized in that, In step two, the V-shaped incision shall not exceed one-third of the circumference of the blood vessel.

7. The method for constructing an animal cerebral ischemia-reperfusion model according to claim 1, characterized in that, In step three, the microvascular embolus is a silicone suture embolus used in mouse MCAO models. During insertion, the suture embolus is kept aligned with the direction of the ICA to avoid bending or puncturing the vascular endothelium.

8. The method for constructing an animal cerebral ischemia-reperfusion model according to claim 1, characterized in that, In step four, the ischemic state of the MCA occlusion is maintained for 2 hours.

9. The method for constructing an animal cerebral ischemia-reperfusion model according to claim 1, characterized in that, In step five, 11-0 sutures are used for single-needle suturing and knotting to achieve complete closure of the ICA incision; In step five, after completing the ICA incision suturing, the mouse's neck muscles are repositioned, the skin incision is sutured in layers, and the mouse is kept in a single cage and kept warm after surgery. After the mouse recovers from anesthesia, it is returned to its normal breeding environment.

10. The method for constructing an animal cerebral ischemia-reperfusion model according to claim 1, characterized in that, After the animal brain ischemia-reperfusion model is established, the model effectiveness verification step is also included, which includes: (1) Cerebral blood flow detection: Before reperfusion, laser speckle imaging technology was used to detect the blood flow status of the head, face and neck of mice to confirm that the blood flow in the MCA occlusion area was significantly reduced and the blood flow in the ECA supply area was normal. (2) Neurological function scoring: The Longa five-point scoring system was used to assess the degree of neurological deficit in mice after surgery; (3) Detection of cerebral infarction volume: After the operation, the mice were sacrificed and the brain tissue was taken for 2.0% TTC staining. The percentage of cerebral infarction volume to total brain volume was calculated by image analysis software. (4) Thyroid function test: Plasma was collected from mice 24 hours after surgery, and the concentrations of plasma T3 and T4 were determined by enzyme-linked immunosorbent assay. (5) Long-term prognosis detection: record the changes in body weight and survival rate of mice within 30 days after surgery.

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