Lower limb chronic ischemia rat model and construction method thereof

Abstract

The invention belongs to the technical field of medical animal model construction, and discloses a lower limb chronic ischemia rat model and a construction method thereof.The method comprises the steps that chloral hydrate dosage data for constructing a module SD rat anesthesia experiment is obtained; constructing a construction module SD rat lower limb ischemia modeling experiment scheme; after the lower limb ischemia modeling experiment of the constructed module SD rat is finished, respiration and activity information of the constructed module SD rat is monitored, and improved blood vessel color Doppler blood flow imaging scanning is utilized to obtain data information of blood flow peak velocity and blood flow acceleration time in blood flow perfusion of the affected side limb of the constructed module SD rat. The effect of the lower limb chronic ischemia modeling experiment is evaluated. According to the construction method and the construction system for the rat model with chronic lower limb ischemia, a reference basis is provided for subsequent research, and a proper intervention opportunity is selected during experimental design, so that the experimental effect is optimized.

Description

Technical Field

[0001] The present invention belongs to the technical field of constructing medical animal models, and particularly relates to a rat model of chronic lower limb ischemia and a method for constructing the same. Background Art

[0002] When studying lower limb ischemic diseases, selecting a suitable ischemic model is helpful for the experimental effect, and the correct model selection can help better understand the development of lower limb ischemic diseases. In the prior art, when preparing acute and chronic limb ischemia models, it is considered that for limb ischemia models, acute ischemia is still the main research direction and mainstream method, and rabbits are the earliest animals used in ischemic models. Restricting blood supply by ligating or embolizing blood vessels can be used to simulate limb ischemia. The vascular ligation and cutting method is a method of temporarily interrupting arterial blood supply and achieving acute and persistent vascular occlusion by ligating or cutting the blood supply artery or its branches, thereby inducing an ischemic state. Since the blood flow change is similar to peripheral arterial disease in clinical practice, the severe ischemia time of this method lasts up to 4 weeks. Therefore, this method has been widely used in the preparation of limb ischemia models in the prior art and has become one of the commonly used methods. Although there are other different methods, such as particulate embolization method, physical arterial constriction, pneumatic tourniquet, McGivney hemorrhoid ligator, orthodontic rubber band, etc. to prepare lower limb ischemia models, external compression methods such as pneumatic tourniquet or orthodontic rubber band can also cause vascular compression and stenosis. Cytological examination cannot prove whether plaque formation in the blood vessel is caused by external extrusion or ischemia. Currently, there is still no widely accepted rat model of chronic lower limb ischemia. The high-fat diet method can induce the formation of arterial plaques, which can then lead to blood vessel blockage. Currently, a commonly used method for preparing a lower limb ischemia model is to ligate or excise the femoral artery or iliac artery of the rat's lower limb, and simulate the ischemic compensation stage as a process of chronic ischemia. This is one of the commonly used measures for preparing a lower limb ischemia model.

[0003] By simulating an animal model, the disease can be better understood, and the best treatment plan can be explored. Through a suitable animal model, the progression process of the disease can be more comprehensively revealed, so as to study and screen reliable therapeutic drugs and strategies in animal experiments. It is very important for finding the best treatment method for peripheral vascular diseases. Establishing an ideal animal model of lower limb ischemia is a prerequisite for studying lower limb vascular lesions. Currently, there is no ideal animal model for lower limb vascular lesions, and there is a lack of effective evaluation of lower limb ischemia models. Summary of the Invention

[0004] To overcome the problems existing in the related art, the disclosed embodiments of the present invention provide a rat model of chronic lower limb ischemia and a method for constructing the same, specifically relating to a rat model of chronic lower limb ischemia constructed based on clamping the common iliac artery for 1 minute and 30 seconds and ligating the internal iliac artery.

[0005] The technical solution is as follows: A method for constructing a rat model of chronic lower limb ischemia, comprising the following steps:

[0006] S1. Obtain the dose data of chloral hydrate for anesthetizing SD rats in the construction module;

[0007] S2. Based on the obtained dose data of chloral hydrate, construct an experimental protocol for creating a lower limb ischemia model in SD rats in the construction module;

[0008] S3. After the experimental procedure for creating a lower limb ischemia model in SD rats in the construction module is completed, monitor the breathing and activity information of the SD rats in the construction module, and use improved vascular color Doppler flow imaging to obtain data on the peak blood flow velocity and blood flow acceleration time in the blood perfusion of the affected limb of the SD rats in the construction module, and evaluate the effect of the experimental procedure for creating a chronic lower limb ischemia model.

[0009] In step S1, the SD rats in the construction module are 4-week-old SD rats weighing 350 - 400 g;

[0010] The dose data of chloral hydrate is: a 10% chloral hydrate solution at a dose of 0.3 ml per 100 g, where the 10% chloral hydrate solution contains 100 mg of chloral hydrate per milliliter of normal saline.

[0011] In step S2, the experimental protocol for creating a lower limb ischemia model in SD rats in the construction module includes: using a vascular injury forceps to clamp the common iliac artery for 1 minute and 30 seconds to form a lower limb ischemia model, and experimental operation steps of dissecting and completely ligating the internal iliac artery.

[0012] In step S3, after the experimental procedure for creating a lower limb ischemia model in SD rats in the construction module is completed, on the 2nd, 4th, 10th, 16th, 22nd, and 28th days respectively, use improved vascular color Doppler flow imaging to obtain data on the peak blood flow velocity and blood flow acceleration time in the blood perfusion of the affected limb of the SD rats in the construction module.

[0013] Furthermore, obtaining the data on the peak blood flow velocity and blood flow acceleration time in the blood perfusion of the affected limb of the SD rats in the construction module includes:

[0014] Step 1. Construct an initial marked model for the improved vascular color Doppler flow imaging ultrasonic system;

[0015] Step 2. Perform speed update and measurement update for the peak blood flow velocity recognition method;

[0016] Step 3. Calculate the vector square root distance between the observed quantity and the observed information, and calculate the corresponding threshold of the observed information;

[0017] Step 4. Based on the vector square root distance method, introduce a deviation correction factor λ k, correct the estimated abnormal frequency band wave spectrum deviation matrix R;

[0018] Step 5, perform deviation correction blood flow peak velocity identification measurement update, and use the corrected abnormal frequency band wave spectrum deviation matrix R to perform measurement update on the blood flow peak velocity identification method;

[0019] Step 6, use the deviation correction blood flow peak velocity to identify the current blood flow peak velocity state error and correct the error of the conventional vascular color Doppler blood flow imaging.

[0020] In Step 1, construct an improved initial marking model for the vascular color Doppler blood flow imaging ultrasonic system, including:

[0021] (1) Initial marking state equation. Without considering the velocity and position information of the blocked channel, select the state quantity expression as:

[0022]

[0023] In the formula, f is the state quantity, kH and kλ are the position latitude error and position longitude error respectively, and ku E , ku N are the ipsilateral common iliac velocity error and ipsilateral external iliac velocity error respectively; is the ultrasonic platform error angle of the vascular color Doppler blood flow imaging; is the constant drift of the vascular color Doppler blood flow imaging ultrasonic instrument; Δ a is the zero bias of the acceleration module of the vascular color Doppler blood flow imaging ultrasonic instrument;

[0024] Based on the combination of YPU and DVL, improve the initial marking of the vascular color Doppler blood flow imaging ultrasonic system, and the corresponding state equation is:

[0025]

[0026] In the formula, is the state value after the improvement of the system abnormal frequency band wave spectrum, G YPU is the state transition matrix, f YPU is the system abnormal frequency band wave spectrum deviation matrix, v YPU is the system abnormal frequency band wave spectrum;

[0027] The state transition matrix G established by the state equation YPU is:

[0028]

[0029] In the formula, G is a 9×9 matrix and S is a 9×8 matrix;

[0030] (2) Initial marking measurement equation; The YPU and DVL are combined, the velocity error is selected as the observable, and the velocity in the navigation system measured by the YPU and the velocity u in the vehicle body coordinate system measured by the DVL a in the projection difference are used as the observables. Then, the measurement equation corresponding to the initial marking of the improved vascular color Doppler blood flow imaging ultrasonic system based on the YPU / DVL combination is:

[0031]

[0032] In the formula, Ψ u is the measurement value corresponding to the initial marking of the improved vascular color Doppler blood flow imaging ultrasonic system, is the observation matrix of the velocity projection in the vehicle body coordinate system, B u is the measurement matrix for measuring the abnormal frequency band wave spectrum; A u is the abnormal frequency band wave spectrum matrix;

[0033] Under the condition of a small misalignment angle, where J is the misalignment angle of the vascular color Doppler blood flow imaging ultrasonic platform; If the total iliac velocity error ku E on the operative side and the external iliac velocity error ku N on the operative side are selected as the observables, then there is:

[0034]

[0035] In the formula, is the observation matrix of the velocity in the vehicle body coordinate system, is the velocity value under the DVL, k is the velocity coefficient, u n is the external iliac velocity value on the operative side, is the velocity of the qth order under the DVL;

[0036] Using to replace to obtain the measurement matrix B u is:

[0037]

[0038] In the formula, represents the first two rows of the matrix .

[0039] In step two, the velocity update and measurement update of the blood flow peak velocity identification method are carried out, including the following steps:

[0040] (1) Velocity update equation:

[0041]

[0042] In the formula, is the prior estimate of the state quantity, is the prior estimate under the self-update of the k-1 state, is the reciprocal of the DVL velocity in the k-1 state, I is the blood flow image, IP k-1 is the blood flow peak image velocity value in the k-1 state, is the state transition matrix of the Lth order at time k-1, G k-1 is the state transition matrix at time k-1, P k|k-1 is the prior estimate of the current blood flow peak velocity state error deviation matrix;

[0043] (2) Measurement update equation, the expression is:

[0044]

[0045] In the formula, IP k|k is the information matrix, L is the Lth order, is the updated measurement abnormal frequency band wave spectrum matrix, K k is the frequency band wave spectrum state value at time k, is the prior estimate of the self-state quantity update at time k, ψ k is the measurement value corresponding to the initial mark at time k, B k is the measurement abnormal frequency band wave spectrum matrix at time k.

[0046] In step three, calculate the vector square root distance between the observed quantity and the observed information, and calculate the corresponding threshold of the observed information, including:

[0047] Select the observed quantity at time k and the prior estimate of the observed quantity The vector square root distance between them is used as the evaluation index, then the evaluation index at time k is defined as:

[0048]

[0049] In the formula, M k is the vector square root distance, P ee,k|k-1 is the prior estimate of the blood flow peak velocity state error deviation matrix from time k to time k-1 in the evaluation, v k is the observed information vector, P zz,k|k-1 is the prior estimate of the measurement error deviation matrix.

[0050] In step four, based on the vector square root distance method, introduce the deviation correction factor λ k , and correct the estimated abnormal frequency band wave spectrum deviation matrix R matrix, the expression is:

[0051]

[0052] In the formula, is the correction value of the abnormal frequency band wave spectrum matrix;

[0053] Convert the expression of the new evaluation index at time k into a non - linear problem of solving γ k , and the expression is:

[0054]

[0055] In the formula, d(γ k ) is the evaluation value under non - linearity converted from the new time k for solving γ k , γ k is the non - linear frequency band wave spectrum matrix coefficient, A k is the value of the abnormal frequency band wave spectrum matrix at time k, is the evaluation index under the second order;

[0056] γ k is solved by the Newton iteration method, then the relationship between γ k (i + 1) and γ k (i) is:

[0057]

[0058] In the formula, γ k (i + 1) is the non - linear frequency band wave spectrum matrix value at the (i + 1) - th node, γ k (i) is the non - linear frequency band wave spectrum matrix value at the i - th node, v k (i) is the abnormal frequency band wave spectrum at the i - th node at time k, is the state error deviation of the blood flow peak velocity solved at the i - th node under the prior estimate of the update of its own state quantity at time k.

[0059] Another object of the present invention is to provide a rat model of chronic lower limb ischemia, which is constructed by using the construction method of the rat model of chronic lower limb ischemia.

[0060] Combining all the above - mentioned technical solutions, the beneficial effects of the present invention are as follows: The construction method and construction system of the rat model of chronic lower limb ischemia provided by the present invention provide a reference basis for subsequent research, help to select an appropriate intervention time during experimental design to optimize the experimental effect. At the same time, it also provides new ideas and directions for the construction of clinical treatment models of chronic limb ischemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The drawings here are incorporated into the specification and form a part of this specification, showing embodiments in line with the present disclosure, and are used together with the specification to explain the principles of the present disclosure;

[0062] Figure 1It is a flowchart of the method for constructing a rat model of chronic lower limb ischemia provided by an embodiment of the present invention;

[0063] Figure 2 It is a schematic diagram of the skin temperature of the rat limb after surgery provided by the present invention;

[0064] Figure 3 It is a schematic diagram of the leg circumference of the rat limb after surgery provided by the present invention;

[0065] Figure 4 It is a schematic diagram of the Tarlov function score of the rat after surgery provided by the present invention;

[0066] Figure 5 It is a schematic diagram of the appearance score of the rat limb after surgery provided by the present invention;

[0067] Figure 6 It is a schematic diagram of the vascular anatomy of male rats provided by the present invention;

[0068] Figure 7 It is a schematic diagram of weighing the rat after grasping provided by the present invention;

[0069] Figure 8 It is a schematic diagram of disinfection and anesthesia according to body weight provided by the present invention;

[0070] Figure 9 It is a schematic diagram of fixing and draping the operating table provided by the present invention;

[0071] Figure 10 It is a schematic diagram of free abdominal cavity blood vessels provided by the present invention;

[0072] Figure 11 It is a schematic diagram of clamping the common iliac artery provided by the present invention;

[0073] Figure 12 It is a schematic diagram of ligating the internal iliac artery provided by the present invention;

[0074] In the figure: 1. Ureter; 2. Internal spermatic artery; 3. Posterior mesenteric artery; 4. Rectum; 5. Superior artery; 6. Superior vesical artery; 7. Seminal vesicle; 8. Renal artery; 9. Iliolumbar artery; 10. Genitofemoral nerve; 11. Common iliac artery; 12. Median sacral artery; 13. Internal iliac artery; 14. External iliac artery; 15. Deep circumflex iliac artery. Detailed implementation manners

[0075] To make the above objects, features, and advantages of the present invention more apparent and understandable, the following provides a detailed description of the specific embodiments of the present invention with reference to the accompanying drawings. Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0076] Example 1, as shown in the figure, the method for constructing a rat model of chronic lower limb ischemia provided by the embodiment of the present invention includes:

[0077] S1. Obtain the data of the chloral hydrate dosage for anesthetizing SD rats in the construction module;

[0078] S2. Based on the obtained chloral hydrate dosage data, construct an experimental protocol for creating a lower limb ischemia model in SD rats in the construction module;

[0079] S3. After the experimental procedure for creating a lower limb ischemia model in SD rats in the construction module is completed, monitor the breathing and activity information of the SD rats in the construction module, and use improved vascular color Doppler flow imaging scanning to obtain the data of the peak blood flow velocity and blood flow acceleration time in the blood perfusion of the affected limb of the SD rats in the construction module, and evaluate the effect of the experimental procedure for creating a chronic lower limb ischemia model.

[0080] Exemplarily, in step S1, the SD rats in the construction module are 4-week-old SD rats weighing 350 - 400 g;

[0081] The data of the chloral hydrate dosage is: 10% chloral hydrate at a dosage of 0.3 ml per 100 g, where the 10% chloral hydrate solution contains 100 mg of chloral hydrate per milliliter of normal saline.

[0082] In step S2, the experimental protocol for simulating the creation of a lower limb ischemia model in SD rats in the construction module includes: using a vascular injury forceps to clamp the common iliac artery for 1 minute and 30 seconds to form a lower limb ischemia model, and the experimental operation steps of dissecting and completely ligating the internal iliac artery.

[0083] Exemplarily, in step S3, after the experimental procedure for creating a lower limb ischemia model in SD rats in the construction module is completed, on the 2nd, 4th, 10th, 16th, 22nd, and 28th days respectively, use improved vascular color Doppler flow imaging scanning to obtain the data of the peak blood flow velocity and blood flow acceleration time in the blood perfusion of the affected limb of the SD rats in the construction module.

[0084] Using improved vascular color Doppler flow imaging scanning to obtain the data of the peak blood flow velocity in the blood perfusion of the affected limb of the SD rats in the construction module includes:

[0085] Step 1: Construct the initial marking model of the improved vascular color Doppler flow imaging ultrasonic system;

[0086] Step 2: Perform velocity update and measurement update of the blood flow peak velocity recognition method;

[0087] Step 3: Calculate the vector square root distance between the observed quantity and the observed information, and calculate the corresponding threshold of the observed information;

[0088] Step 4: Based on the vector square root distance method, introduce the deviation correction factor λ k , and correct the estimated abnormal frequency band wave spectrum deviation matrix R;

[0089] Step 5: Perform deviation correction blood flow peak velocity recognition measurement update, and use the corrected abnormal frequency band wave spectrum deviation matrix R to perform blood flow peak velocity recognition method measurement update;

[0090] Step 6: Use the deviation correction blood flow peak velocity to identify the current blood flow peak velocity state error, and correct the error of the conventional vascular color Doppler flow imaging.

[0091] Exemplarily, in Step 1, the construction of the initial marking model of the improved vascular color Doppler flow imaging ultrasonic system includes:

[0092] (1) The initial marking state equation, without considering the velocity and position information of the blocked channel, selects the state quantity expression as:

[0093]

[0094] In the formula, f is the state quantity, kH and kλ are the position latitude error and position longitude error respectively, ku E , ku N are the velocity error of the ipsilateral common iliac artery and the velocity error of the ipsilateral external iliac artery respectively; is the error angle of the vascular color Doppler flow imaging ultrasound platform; is the constant drift of the vascular color Doppler flow imaging ultrasonic instrument; Δ a is the zero bias of the acceleration module of the vascular color Doppler flow imaging ultrasonic instrument;

[0095] The state equation corresponding to the initial marking of the improved vascular color Doppler flow imaging ultrasonic system based on the YPU / DVL combination is:

[0096]

[0097] In the formula, is the state value after the improvement of the system abnormal frequency band wave spectrum, G YPU is the state transition matrix, f YPUis the spectral deviation matrix of the system abnormal frequency band wave, v YPU is the spectral of the system abnormal frequency band wave;

[0098] The state transition matrix G established by the state equation YPU is:

[0099]

[0100] In the formula, G is a 9×9 matrix, and S is a 9×8 matrix; the non-zero elements in G are as follows:

[0101] G 1,4 = 1 / A e , G 2,1 = (u E / A e )tanH secH, G 2,3 = secH / A e , G 3,3 = (u N / A e )tanH; G 3,4 = 2ω ie sinH + (u E / A e )tanH, Q 3,6 = -d U , G 3,7 = d N ;

[0102] G 4,3 = -[2ω ie sinH + (u E / A e )tanH], G 4,5 = d U ; G 4,7 = -d E , G 5,4 = -1 / A e , G 5,6 = ω ie sinH + (u E / A e )tanH, G 5,7 = -[ω ie cosH + (u E / A e )]; G 6,1 = -ω ie sinH, G 6,3 = 1 / A e , G 6,5 = -[ω ie sinH + (u E / A e)tanH], G 6,7 = -u N / A e ; G 7,1 = ω ie cosH + (u E / A e )(secH) 2 ), G 7,3 = tanH / A e ), G 7,5 = ω ie cosH + u E / A e , …;

[0103] G 9,8 = u N / A e ;

[0104] In the formula, S is a 9×8 matrix, and its expression form is as follows:

[0105]

[0106] In the formula, represents the first two rows of the attitude matrix Y a n ;

[0107] (2) Initial marked measurement equation; When YPU and DVL are combined, the velocity error is selected as the observable, and the velocity measured by YPU in the navigation system and the velocity u a measured by DVL in the body-fixed system are selected as the observable, then the measurement equation corresponding to the initial mark of the improved vascular color Doppler blood flow imaging ultrasonic system based on the YPU / DVL combination is:

[0108]

[0109] In the formula, Ψ u is the measured value corresponding to the initial mark of the improved vascular color Doppler blood flow imaging ultrasonic system, is the observation matrix of the velocity projection in the body-fixed system, B u is the measurement matrix used to measure the abnormal frequency band wave spectrum; A u is the abnormal frequency band wave spectrum matrix;

[0110] Under the condition of linear small misalignment angle, where J is the misalignment angle of the vascular color Doppler blood flow imaging ultrasonic platform; If the velocity errors ku E of the common iliac artery on the operative side and ku N of the external iliac artery on the operative side are selected as the observables, then there is:

[0111]

[0112] Wherein, is the velocity observation matrix under the carrier system, is the velocity value under DVL, k is the velocity coefficient, and u n is the velocity value of the ipsilateral external iliac artery, is the velocity of the q-th order under DVL;

[0113] Use to replace to obtain the measurement matrix B u which is:

[0114]

[0115] Wherein, represents the first two rows of the matrix .

[0116] In step two, the velocity update and measurement update of the blood flow peak velocity identification method include:

[0117] (1) Velocity update equation:

[0118]

[0119] Wherein, is the prior estimate of the state quantity, is the prior estimate under the self-update of the k-1 state, is the reciprocal of the velocity of DVL in the k-1 state, I is the blood flow image, and IP k-1 is the blood flow peak image velocity value in the k-1 state, is the state transition matrix at the k-1 moment in the L-th order, and G k-1 is the state transition matrix at the k-1 moment, and P k|k-1 is the prior estimate of the current blood flow peak velocity state error deviation matrix;

[0120] (2) Measurement update equation, the expression is:

[0121]

[0122] Wherein, IP k|k is the information matrix, L is the L-th order, is the updated measurement abnormal frequency band wave spectrum matrix, and K k is the frequency band wave spectrum state value at the k moment, is the prior estimate of the self-state quantity update at the k moment, ψ k is the measurement value corresponding to the initial mark at the k moment, and B k is the measurement abnormal frequency band wave spectrum matrix at the k moment.

[0123] In step 3, calculate the vector root-mean-square distance between the observed quantity and the observation information. Calculating the corresponding threshold of the observation information includes:

[0124] Select the observed quantity at time k and the prior estimate of the observed quantity The vector root-mean-square distance between them is used as the evaluation index. Then the evaluation index at time k is defined as:

[0125]

[0126] In the formula, M k is the vector root-mean-square distance, P ee,k|k-1 is the prior estimate of the state error deviation matrix of the blood flow peak velocity from time k to time k - 1 in the evaluation, v k is the observation information vector, and P zz,k|k-1 is the prior estimate of the measurement error deviation matrix.

[0127] In step 4, based on the vector root-mean-square distance method, introduce the deviation correction factor λ k , and correct the estimated abnormal frequency band wave spectrum deviation matrix R matrix. The expression is:

[0128]

[0129] In the formula, is the corrected value of the abnormal frequency band wave spectrum matrix;

[0130] Convert the expression of the new evaluation index at time k into a non-linear problem of solving γ k . The expression is:

[0131]

[0132] In the formula, d(γ k ) is the non-linear evaluation value converted from the new time k to solve γ k , γ k is the non-linear frequency band wave spectrum matrix coefficient, A k is the value of the abnormal frequency band wave spectrum matrix at time k, is the evaluation index under the second order;

[0133] γ k is solved by the Newton iteration method. Then the relationship between γ k (i + 1) and γ k (i) is:

[0134]

[0135] In the formula, γk (i + 1) is the spectral array value of the non-linear frequency band wave at the (i + 1)-th node, γ k (i) is the spectral array value of the non-linear frequency band wave at the i-th node, v k (i) is the spectral of the abnormal frequency band wave at the i-th node at time k, is the state error deviation of the blood flow peak velocity solved at the i-th node under the prior estimate of the update of its own state quantity at time k.

[0136] Exemplarily, using the improved vascular color Doppler blood flow imaging scan, obtaining the data information of the blood flow acceleration time in the blood perfusion of the affected limb of the SD rat in the construction module includes: obtaining the blood flow acceleration data information through the method steps of the same principle, and then using the conventional formula for conversion to obtain the data information of the blood flow acceleration time.

[0137] The present invention uses more advanced improved vascular color Doppler blood flow imaging technology and molecular biological means and methods, improves the reliability and repeatability of the model, and finally realizes the in-depth understanding and effective intervention of the mechanism, pathophysiology, diagnosis and treatment of chronic limb ischemia diseases.

[0138] Example 2, combined with the construction method of the chronic lower limb ischemia rat model provided by the embodiments of the present invention, specific experimental operations and implementations are carried out, including:

[0139] Step 1, Feed 4-week-old SD rats until they reach 350 - 400 g, weigh them, and anesthetize them by intraperitoneal injection of 10% chloral hydrate at a dose of 100 g / 0.3 ml. Among them, the chloral hydrate ratio: the 10% chloral hydrate solution contains 100 mg of chloral hydrate per milliliter of normal saline.

[0140] Experiments show that performing surgical anesthesia with an anesthetic appropriately proportioned according to the rat body weight shows that generally, for rats with a larger body weight, according to the anesthetic dose of 100 g / 0.3 ml, the anesthetic effect is good.

[0141] Step 2, After the rats are fully anesthetized, take the supine position, fix the four limbs on the animal operating table, depilate and prepare the skin of both lower limbs and the abdomen, disinfect the abdominal skin with 75% alcohol, and then cover it with a sterile surgical drape.

[0142] Step 3, Make an incision with a length of 2 - 3 cm beside the midline of the rat abdomen, and use surgical instruments such as a scalpel, vascular separation forceps, and tweezers to bluntly separate the tissues, avoiding damaging the intestinal tract and other abdominal organs.

[0143] Step 4, Separate the common iliac artery to the external iliac artery and the accompanying veins and their branches.

[0144] Step 5: For the model group of SD rats, use a vascular injury forceps to clamp the common iliac artery for 1 minute and 30 seconds (this is the modeling method. By clamping the common iliac artery, blood flow is temporarily blocked and vascular injury is caused. By controlling the clamping time and degree of injury, the purpose of lower limb ischemia in rats is achieved, forming a lower limb ischemia model). Free and completely ligate the internal iliac artery (the internal iliac artery mainly supplies blood to the pelvis, but its branches such as the superior gluteal artery and the inferior gluteal artery also supply blood to part of the lower limb. After ligation, it may cause local ischemia in the lower limb, especially in the buttocks and proximal lower limb areas) to reduce collateral circulation compensation; for the normal control group of rats, only free the common iliac artery and surrounding blood vessels, and perform sham surgery without clamping and ligation (perform sham surgery for comparison with the model group).

[0145] Experiments have shown that clamping the blood vessels for 1 minute and 30 seconds in the constructed rat model is the best way to achieve the effect.

[0146] Step 7: Press the wound with a sterile cotton ball to stop bleeding for 5 minutes, then suture the skin with 5-0 thread, disinfect it once with 75% alcohol, and suture it simply continuously with silk thread to close the surgical incision, and then disinfect it. For 3 consecutive days after the operation, inject penicillin (2.0×10 6 U / kg) through the marginal vein of the tail (to prevent abnormal conditions in rats caused by incision and intra-abdominal infection); after the operation, place the rats in a warm and quiet environment until they fully recover. Monitor the breathing and activity of the rats to ensure their safety.

[0147] Step 8: Immediately perform improved vascular color Doppler flow imaging scans on the 2nd, 4th, 10th, 16th, 22nd, and 28th days after the operation to detect the blood flow perfusion of the affected limb of the rats (peak blood flow velocity, blood flow acceleration time) and evaluate the surgical effect.

[0148] Example 3: The present invention provides a rat model of chronic lower limb ischemia. The gene similarity between rats and other mammals is higher than that of animals such as rabbits, and it can better simulate the disease states and physiological responses of other mammals. The present invention takes rats as an example to evaluate the lower limb ischemia model as follows in the experiment.

[0149] Experiment 1.

[0150] (1) General indicators: During the process of establishing the rat ischemia model, the Tarlov function of the rat's lower limbs, the degree of limb appearance ischemia, skin temperature changes, leg circumference changes should be observed regularly, and whether there are situations such as lameness and necrosis. Tarlov function score: No movement is 0 points, hardly perceptible movement, no weight-bearing is 1 point, frequent and intense movement, no weight-bearing is 2 points, supporting weight, can walk 1 or 2 steps is 3 points, only slightly defective walking is 4 points, normal but slow walking is 5 points, complete and fast walking is 6 points. Limb appearance ischemia score: Leg auto-amputation is 0 points, leg necrosis is 1 point, foot necrosis is 2 points, discoloration of two or more toes is 3 points, discoloration of one toe is 4 points, discoloration of two or more nails is 5 points, discoloration of one nail is 6 points, no necrosis is 7 points.

[0151] (2) Imaging examination: After the model establishment, all SD rats underwent lower limb color Doppler flow imaging examination, and the detection sites were the affected / healthy iliac and femoral arteries. Conventional ultrasound was used to observe the course of the femoral artery (between the inguinal region and the knee joint), whether there was vascular distortion, observe the echogenicity in the lumen of the femoral artery and whether there was thrombus formation, and at the same time measure the lumen diameter of the femoral artery. Improved color Doppler flow imaging (CDFI) was used to observe the blood flow situation of the entire femoral artery, whether there was a colorful mosaic-like disordered blood flow signal and filling defect. At the same time, improved color Doppler flow imaging (CDFI) was used to collect the blood flow spectrum, observe its morphology, and detect the peak velocity and acceleration time of blood flow at the starting segments of the common iliac artery and external iliac artery.

[0152] (3) Pathological examination: After anesthetizing the SD rats in the model group with 1% sodium pentobarbital solution (80 mg / kg), the bilateral triceps surae, quadriceps femoris, and blood vessels from the iliac artery to the femoral artery were quickly taken, and the rats were sacrificed, and HE staining and immunohistochemical SP staining were performed respectively. Take the triceps surae specimens fixed with 10% formaldehyde, embed them in paraffin and make continuous sections with a thickness of 5 μm for the longitudinal and transverse sections of the muscle, perform hematoxylin-eosin staining, and observe the changes in vascular structure, inflammatory cells, connective tissue, etc. Observe the pathological changes of muscle fibers under the microscope (100 times) and take pictures.

[0153] (4) Immunohistochemical SP staining: Take the quadriceps femoris specimens fixed with 10% formaldehyde, make continuous cross-sectional sections with a thickness of 3 μm, and perform SP staining. Observe under a high-power microscope (400 times), and the cells stained brown-yellow are vascular endothelial cells. Select 1 section for each rat, select 5 fields of view for each section, calculate the capillary density (number of vascular endothelial cells / HPF), and at the same time detect the expression of CD34 in the tissue.

[0154] Such as Figure 2 The skin temperature of the rat's limb after surgery, Figure 3 The leg circumference of the rat's limb after surgery, Figure 4 The Tarlov function score of the rat after surgery, Figure 5As shown by the limb appearance score of rats after surgery. In the results of iliac artery ultrasound examination of rats, the peak velocity of arterial blood flow in rats is shown in Table 1, and the acceleration time of arterial blood flow in rats is shown in Table 2.

[0155] Table 1

[0156] Peak blood flow velocity (cm / s) Statistical value Iliac common on the operative side 27.10 ± 10.7 P＝0.046 Iliac common on the healthy side 37.67+4.10 P＝0.046 Iliac external on the operative side 17.62 ± 2.98 P＝0.003 Iliac external on the healthy side 28.76 ± 6.66 P＝0.003

[0157] Table 2

[0158]

[0159]

[0160] Pathological results:

[0161] Two days after surgery, blood vessels: Focal exfoliation of intimal endothelial cells, no obvious abnormality in the media, and a small amount of inflammatory cell infiltration in the adventitia. Muscle: No obvious abnormality was seen;

[0162] Four days after surgery, blood vessels: The intimal endothelial cells of the blood vessels were swollen and peeled off from the vessel wall. Connective tissue hyperplasia occurred in the media, and fibrous connective tissue hyperplasia and focal infarction occurred in the adventitia. Muscle: Muscle atrophy, and individual muscle cell nuclei disappeared;

[0163] Ten days after surgery, blood vessels: The intimal endothelial cells were swollen and peeled off from the vessel wall. No obvious abnormality was seen in the media, and fibrous connective tissue hyperplasia with a small amount of inflammatory cell infiltration occurred in the adventitia. Muscle: Muscle cell atrophy, and interstitial connective tissue hyperplasia;

[0164] Sixteen days after surgery, blood vessels: No obvious abnormality was seen in the intima. Connective tissue hyperplasia with calcium salt deposition occurred in the media, and connective tissue hyperplasia with interstitial small vessel hyperplasia and edema occurred in the adventitia. Muscle: Muscle atrophy, and fibrous connective tissue hyperplasia;

[0165] Twenty-two days after surgery, blood vessels: Interstitial cell hyperplasia with small vessel hyperplasia was seen under the intima of the blood vessels, and calcium salt deposition occurred in the media. Muscle: No widening of the muscle gap was seen, no muscle atrophy and necrosis were seen, and no inflammatory infiltration and fibrous necrosis were seen;

[0166] Twenty-eight days after surgery, blood vessels: Focal hyperplasia of interstitial cells under the intima. Muscle: No widening of the muscle gap was seen, no muscle atrophy and necrosis were seen, and no inflammatory infiltration and fibrous necrosis were seen.

[0167] Result analysis:

[0168] (1) The skin temperature of SD rats after construction module surgery (such as Figure 2) It decreased compared with the control group on the day after surgery, and on the 16th, 22nd, and 28th days after surgery, and the differences were statistically significant (P < 0.05). However, it increased compared with the control group on the 4th day after surgery, which was considered to be due to the inflammatory stimulation caused by the relatively short modeling time after surgery, resulting in an increase in skin temperature. Overall, the skin temperature of the modeling group decreased compared with the control group, and the reason was considered to be caused by lower limb vascular ischemia after modeling.

[0169] (2) The leg circumference of the SD rats in the modeling group (such as Figure 3 ) decreased compared with the control group on the 10th, 16th, 22nd, and 28th days after surgery, and the differences were all statistically significant (P < 0.05). Combining with pathological HE staining, the reason was considered to be muscle atrophy caused by lower limb ischemia, resulting in a certain degree of ischemic tissue damage.

[0170] (3) The Tarlov function score of the modeling group (such as Figure 4 ) Data analysis showed that it decreased compared with the normal control group on the day after surgery, and on the 4th, 10th, and 16th days after surgery, and the differences were statistically significant (P < 0.05). The degree of decrease was the most severe on the day after surgery, which was considered to be due to the relief of the effects of anesthesia and analgesic drugs and pain. The recovery was rapid from the 16th to the 22nd day after surgery, and it was basically the same as the control group on the 28th day after surgery. Analysis of limb appearance data (such as Figure 5 ) showed that the modeling group decreased compared with the control group on the 4th, 10th, and 16th days after surgery, and the differences were statistically significant (P < 0.05). It began to recover from the 16th to the 22nd day after surgery, but severe ischemic changes such as amputation and leg necrosis were not observed in the degree of limb appearance change in the early postoperative period, so it was considered that the lower limb ischemic state caused by acute ischemia could be excluded.

[0171] (4) The results of rat ultrasound (Table 1, Table 2) showed that: compared with the control group, the arterial diameter of the modeling group was smaller, the intracavitary echo was poorer, and no obvious thrombus formation was observed; the blood of the rats in the modeling group was viscous, the blood flow was less, and the intima was not smooth; the arterial blood flow of the rats in the normal group was rich and the filling was good. There were statistically significant differences in the peak blood flow velocity and blood flow acceleration time of the arteries between the modeling group and the normal control group. The peak blood flow velocity of the modeling group was lower than that of the control group, and the acceleration time was slower than that of the control group.

[0172] (5) By analyzing the pathology at different time points and combining general situation indicators, color Doppler flow imaging, etc., the conclusion was drawn: the pathological changes at different time points. According to the research results, on the 10th and 16th days after surgery, the lower limb ischemia symptoms of the rats were the most severe. On the 22nd day after surgery, vascular endothelial cell hyperplasia and collateral circulation establishment were observed in the vascular intima, indicating that the lower limb ischemia of the rats began to gradually recover from the 16th to the 22nd day. On the 28th day after surgery, no ischemic changes in blood vessels and muscles were found through pathological examination, which indicated that the ischemic period mainly concentrated between 16 - 22 days.

[0173] It has been reported in the existing literature that the chronic limb ischemia model should have an ischemic duration of at least 2 weeks, and the observation period requires at least 14 days, preferably 4 weeks. This is because to enable the model to more accurately simulate the pathophysiological process of chronic limb ischemia, it is necessary to ensure that the ischemic duration and the observation period are long enough to fully observe the processes such as tissue damage, repair, and collateral circulation formation caused by ischemia.

[0174] Although many different models have been developed and improved, there are still technical difficulties in the prior art to obtain a chronic ischemic limb model similar to pathological occlusion. It can be further explored in experiments. By optimizing surgical methods, adjusting feeding conditions, using more advanced imaging techniques and molecular biology means, etc., the reliability and repeatability of the model are improved, and finally an in-depth understanding and effective intervention of the disease mechanism, pathophysiology, diagnosis, and treatment of chronic limb ischemia are achieved.

[0175] Experiments show that the present invention improves the reliability and repeatability of the model, and finally achieves an in-depth understanding and effective intervention of the disease mechanism, pathophysiology, diagnosis, and treatment of chronic limb ischemia.

[0176] In the above embodiments, the descriptions of each embodiment have their own emphases. For the parts not detailed or recorded in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0177] The above is only a relatively preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for constructing a rat model of chronic lower limb ischemia, characterized in that: The construction method includes the following steps: S1, obtain the chloral hydrate dosage data for the anesthesia experiment of SD rats in the construction module; S2, based on the obtained chloral hydrate dosage data, construct the experimental scheme of SD rat lower limb ischemia model; S3. After the lower limb ischemia modeling experiment of the SD rats with the construction module is completed, the breathing and activity information of the SD rats with the construction module are monitored, and the data information of the peak blood flow velocity and blood flow acceleration time in the affected limb blood perfusion of the SD rats with the construction module are obtained by using the improved vascular color Doppler blood flow imaging scan to evaluate the effect of the lower limb chronic ischemia modeling experiment.

2. The method for constructing a rat model of chronic ischemia of lower limbs according to claim 1, characterized in that: In step S1, the module SD rats were constructed using 4-week-old SD rats weighing 350-400 g; The dosage data of chloral hydrate is: 100g / 0.3ml dosage of 10% chloral hydrate solution, wherein the 10% chloral hydrate solution contains 100mg of chloral hydrate per ml of normal saline.

3. The method for constructing a rat model of chronic ischemia of lower limbs according to claim 1, characterized in that: In step S2, a construction module SD rat lower limb ischemia modeling experimental scheme is constructed, including: using a vascular injury clamp to clamp the common iliac artery for 1 minute and 30 seconds to form a lower limb ischemia model, and freeing and completely ligating the internal iliac artery experimental operation steps.

4. The method for constructing a rat model of chronic ischemia of lower limbs according to claim 1, characterized in that: In step S3, after the lower limb ischemia modeling experiment of the construction module SD rats is completed, the improved vascular color Doppler blood flow imaging scan is used to obtain data information on the peak blood flow velocity and blood flow acceleration time in the affected limb blood perfusion of the construction module SD rats on the 2nd, 4th, 10th, 16th, 22nd and 28th days respectively.

5. The method for constructing a rat model of chronic ischemia of lower limbs according to claim 4, characterized in that: Obtain data information on blood flow peak velocity and blood flow acceleration time in the affected limb blood perfusion of the construction module SD rat, including: Step 1, constructing an initial marking model of an improved vascular color Doppler blood flow imaging ultrasound system; Step 2, performing speed update and measurement update of the blood flow peak velocity identification method; Step 3: Calculate the vector square root distance between the observed value and the observed information, and calculate the threshold corresponding to the observed information; Step 4: Based on the vector square root distance method, introduce the deviation correction factor λ k , correct the estimated abnormal frequency band wave spectrum deviation matrix R; Step 5: Perform deviation correction and blood flow peak velocity identification measurement update, and use the corrected abnormal frequency band wave spectrum deviation matrix R to perform blood flow peak velocity identification method measurement update; Step six, using the deviation to correct the blood flow peak velocity, identifying the current blood flow peak velocity state error, and correcting the conventional vascular color Doppler blood flow imaging error.

6. The method for constructing a rat model of chronic ischemia of lower limbs according to claim 5, characterized in that: In step 1, an initial marking model of an improved vascular color Doppler blood flow imaging ultrasound system is constructed, including: (1) Initial marking state equation, ignoring the speed and position information of the blocked channel, the state quantity expression is selected as: In the formula, f is the state quantity, kH and kλ are the position latitude error and position longitude error respectively, and ku E ,ku N They are the common iliac velocity error on the surgical side and the external iliac velocity error on the surgical side; is the error angle of the ultrasound platform for vascular color Doppler blood flow imaging; is the constant value drift of vascular color Doppler blood flow imaging ultrasound instrument; Δ a Zero bias for the acceleration module of the vascular color Doppler blood flow imaging ultrasound instrument; Based on the combination of YPU and DVL, the initial marking of the vascular color Doppler blood flow imaging ultrasound system is improved, and the corresponding state equation is: In the formula, is the improved state value of the abnormal frequency band spectrum of the system, G YPU is the state transfer matrix, f YPU is the spectrum deviation matrix of the system abnormal frequency band wave, v YPU It is the spectrum of abnormal frequency band of the system; The state transfer matrix G established by the state equation YPU for: Where G is a 9×9 matrix and S is a 9×8 matrix; (2) Initial marking measurement equation; YPU and DVL are combined, the velocity error is selected as the observation quantity, and the navigation system velocity measured by YPU is selected The speed u of the load system measured by DVL a Taking the difference in projection as the observed quantity, the measurement equation corresponding to the initial mark of the improved vascular color Doppler blood flow imaging ultrasound system based on the YPU / DVL combination is: In the formula, Ψ u The measurement values ​​corresponding to the initial markings of the improved vascular color Doppler blood flow imaging ultrasound system are is the observation matrix of velocity projection under the carrier system, B u A is the measurement matrix, used to measure the spectrum of abnormal frequency band waves; u To measure the spectrum array of abnormal frequency band; Under the condition of linear small misalignment angle, Where J is the misalignment angle of the vascular color Doppler blood flow imaging ultrasound platform; if the iliac velocity error ku on the surgical side is selected E and the iliac velocity error ku N As an observation, we have: In the formula, is the velocity observation matrix under the load system, is the speed value under DVL, k is the speed coefficient, u n is the external iliac velocity value on the surgical side, is the speed of the qth order under DVL; use replace The measurement matrix B is obtained u for: In the formula, Representative Matrix The first two lines of .

7. The method for constructing a rat model of chronic lower limb ischemia according to claim 6, characterized in that: In step 2, the speed update and measurement update of the blood flow peak velocity identification method are performed, including the following steps: (1) Velocity update equation: In the formula, is the prior estimate of the state quantity, is the prior estimate under the k-1 state self-update, is the inverse of the speed of DVL in the k-1 state, I is the blood flow image, IP k-1 is the peak image velocity value of blood flow in the k-1 state, is the L-order state transfer matrix at time k-1, G k-1 is the state transfer matrix at time k-1, P k|k-1 is the prior estimate of the current blood flow peak velocity state error deviation matrix; (2) Measurement update equation, expressed as: Where, IP k|k is the information matrix, L is the Lth order, is the updated spectrum array of the measured abnormal frequency band, K k is the spectrum state value of the frequency band wave at time k, is the prior estimate of the state update at time k, ψ k is the measurement value corresponding to the initial mark at time k, B k is the spectrum array of abnormal frequency band measured at time k.

8. The method for constructing a rat model of chronic lower limb ischemia according to claim 7, characterized in that: In step 3, the vector square root distance between the observed quantity and the observed information is calculated, and the threshold corresponding to the observed information is calculated, including: Select the observed quantity at time k A priori estimate of the observed quantity The vector square root distance between them is used as the evaluation index, then the evaluation index at time k is is defined as: Where M k is the vector square root distance, P ee,k|K-1 is the prior estimate of the blood flow peak velocity state error matrix from time k to time k-1 in the judgment, v k is the observation information vector, P zz,k|k-1 is the prior estimate of the measurement error bias matrix.

9. The method for constructing a rat model of chronic lower limb ischemia according to claim 8, characterized in that: In step 4, based on the vector square root distance method, the deviation correction factor λ is introduced k , the estimated abnormal frequency band wave spectrum deviation array R array is corrected, and the expression is: In the formula, It is the correction value of the spectrum array of abnormal frequency band; The new k-time evaluation index The expression is transformed into solving γ k The nonlinear problem is expressed as: In the formula, d(γ k ) is the new k moment and is transformed into solving γ k The nonlinear judgment value, γ k is the nonlinear frequency band spectrum array coefficient, A k is the spectrum array value of the abnormal frequency band at time k, It is the evaluation index under the second order; γ k Solved by Newton's iteration method, γ k (i+1) and γ k The relationship (i) is: In the formula, γ k (i+1) is the spectrum array value of the nonlinear frequency band of the i+1th node, γ k (i) is the spectrum array value of the nonlinear frequency band of the i-th node, v k (i) is the spectrum of abnormal frequency band wave at the i-th node at time k, It is the blood flow peak velocity state error deviation solved by the ith node under the prior estimate of its own state quantity update at time k.

10. A rat model of chronic lower limb ischemia, characterized in that: The model is constructed by using the method for constructing a rat model of chronic lower limb ischemia as described in any one of claims 1 to 9.

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