Method for constructing renal ischemia reperfusion mouse model

Abstract

The invention discloses a method for constructing a renal ischemia reperfusion mouse model, and relates to the technical field of animal model construction, and the method is characterized by comprising the following steps: S1, anesthetizing a mouse; s2, fixing and disinfecting the mouse; s3, cutting the skin, separating layer by layer, and exposing kidney tissues and blood vessels; the kidney is rotated by 360 degrees with the renal artery as the axis, the rotation state is maintained for 30 minutes, and then the kidney is reset to open renal blood flow; and S5, suturing the surgical incision. According to the constructed mouse model, on the basis of effectively blocking artery blood flow, the complexity of surgical operation is reduced, the total renal artery is protected to the maximum extent, renal blood flow supply after recanalization is guaranteed, and the renal artery blood flow is blocked within a certain time without damaging blood vessels. And a good and stable animal model is provided for better researching the mechanism of acute kidney injury and innovative treatment schemes in the future.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of constructing animal models, and more particularly to a method for constructing a kidney ischemia-reperfusion mouse model. BACKGROUND

[0002] Acute kidney injury (AKI) is a clinical syndrome characterized by rapid decline in renal function within a short period of time, with nitrogenous waste accumulation and water and electrolyte disorders. During the process of acute kidney injury, hypoxia and hypotension can cause insufficient perfusion of the kidney, which is one of the main mechanisms of AKI.

[0003] Although clinical studies have provided important epidemiological and pathophysiological data for AKI, there are still some limitations. For example, it is difficult to conduct interventional studies in humans to determine causality, and it is also difficult to obtain kidney tissue for in-depth mechanism research. Therefore, it is crucial to establish a reliable animal model for in-depth study of AKI. The commonly used kidney ischemia-reperfusion animal model is the artery clamp model. The existing model has a high degree of difficulty in surgical operation: the mouse is small in size, and the operation space is limited, requiring a high level of technical skill and experience from the experimental personnel. Precise clamping and unclamping of the renal artery requires skilled microsurgical techniques, and any slight mistake can lead to damage to the renal artery, bleeding, or damage to the surrounding tissue of the renal pedicle, affecting the success rate of the model and the accuracy of the experimental results. The model has poor repeatability: differences in surgical operation details, experimental condition control, and other aspects between different laboratories and different operators can lead to poor repeatability of the model. This makes it difficult to compare and verify the results between different studies. In view of this, the present inventors propose a method for constructing a kidney ischemia-reperfusion mouse model. SUMMARY

[0004] The purpose of the present application is to provide a method for constructing a kidney ischemia-reperfusion mouse model, which solves the above problems.

[0005] The above technical purpose of the present application is achieved by the following technical solution:

[0006] The present application provides a method for constructing a kidney ischemia-reperfusion mouse model in the first aspect, comprising the following steps:

[0007] S1. Anesthetizing the mouse;

[0008] S2. Fixing and disinfecting the mouse;

[0009] S3. Incising the skin, separating and exposing the kidney tissue and blood vessels layer by layer;

[0010] S4. Twisting the kidney and resetting: rotating the kidney 360° around the renal artery, maintaining the rotated state for 30 minutes, and then resetting the kidney to open the renal blood flow;

[0011] S5. Suture the surgical incision.

[0012] The application is further configured that in step S1, the anesthetized mouse is placed in an anesthesia induction box, and isoflurane and oxygen mixed gas is input.

[0013] The application is further configured that the concentration of isoflurane is 3-5%.

[0014] The application is further configured that in step S2, the fixation is right lateral recumbency fixation after the mouse loses the righting reflex, and the disinfection area is the left flank area and the left waist.

[0015] The application is further configured that the disinfection uses iodophor.

[0016] The application is further configured that in step S3, the incision of the skin is parallel to the spine, and the incision depth of the skin is about 1 cm.

[0017] The application is further configured that in step S4, the torsion and reset of the kidney are specifically: rotating the kidney 360° around the renal artery, maintaining the rotation state for 30 minutes, and then resetting the kidney to open the renal blood flow.

[0018] In summary, the application has the following beneficial effects: the application realizes ischemia-reperfusion of the kidney by temporarily blocking the blood flow of the renal artery through "kidney torsion and reset", and establishes a new mouse model that can simulate the pathological and physiological process of RIRI, i.e. a renal torsion (RT) model. The effectiveness and stability of the model are verified by hemodynamics and pathology. The model can effectively block the arterial blood flow, reduce the complexity of surgical operation, maximize the protection of the total renal artery and ensure the renal blood flow supply after recanalization, and realize the blocking of the renal artery blood flow without damaging the blood vessels within a certain time. It provides a good and stable animal model for better exploring the mechanism of acute kidney injury and future innovative treatment programs. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a method flowchart for constructing a renal ischemia-reperfusion mouse model in an embodiment of the application Figure 1 ;

[0020] Figure 2 is a laser speckle imaging display of a renal torsion model in an embodiment of the application;

[0021] Figure 3 is a small animal live imaging display of a renal torsion model in an embodiment of the application;

[0022] Figure 4 is a Pearson correlation coefficient graph and a PCA analysis graph in an embodiment of the application;

[0023] Figure 5 These are the pathological tests of the three groups in the embodiments of the present invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1:

[0026] Laboratory animals:

[0027] Eight-week-old male C57BL / 6 mice, weighing 20-25 g, were purchased from Viton Leverage Laboratory Animal Center (Beijing, China). The animals were housed in an environment with a relative humidity of 50%-60%, a temperature of 26±2℃, and good ventilation.

[0028] A method for constructing a mouse model of renal ischemia-reperfusion, such as Figure 1 , Figure 2 As shown, it includes the following steps:

[0029] S1. Anesthetize mice: Place mice in an anesthesia induction chamber and introduce a mixture of 4% isoflurane and oxygen (flow rate 1L / min).

[0030] S2. Fix and disinfect the mouse: After the mouse loses its righting reflex, fix it in the right lateral decubitus position, and disinfect the left hypochondrium and left lumbar region with iodine solution, with a disinfection area of ​​2-3 cm in diameter.

[0031] S3. Make an incision in the skin to expose the kidney tissue: Make an incision about 1 cm in the skin parallel to the spine, and separate and expose the left kidney tissue and blood vessels layer by layer;

[0032] S4. Kidney Twist and Repositioning: Rotate the kidney 360° around the renal artery (clockwise or counterclockwise) and maintain the rotation for 30 minutes. Then reposition the kidney to restore renal blood flow.

[0033] S5. After the surgery, suture the surgical incision closed. Figure 1 ).

[0034] Comparative Example 1:

[0035] Sham group: The animals were placed in the anesthesia induction box, and 4% isoflurane and oxygen mixed gas (flow rate 1 L / min) was introduced. After the animals lost the righting reflex, they were fixed in the right lateral position. The left flank and left waist were disinfected with iodophor, and the disinfection range was 2-3 cm in diameter. A surgical incision about 1 cm in length was made in the skin parallel to the spine. The left kidney tissue and blood vessels were exposed by layer separation. After the operation, the surgical incision was sutured and closed.

[0036] Comparative example 2:

[0037] Arterial clamp group: The animals were placed in the anesthesia induction box, and 4% isoflurane and oxygen mixed gas (flow rate 1 L / min) was introduced. After the animals lost the righting reflex, they were fixed in the right lateral position. The left flank and left waist were disinfected with iodophor, and the disinfection range was 2-3 cm in diameter. A surgical incision about 1 cm in length was made in the skin parallel to the spine. The left kidney tissue and blood vessels were exposed by layer separation. The renal artery was blocked by using an arterial clamp to block blood flow. After blocking the renal blood flow for 30 minutes, the arterial clamp was loosened to open the renal blood flow. After the operation, the surgical incision was sutured and closed.

[0038] Hemodynamic analysis

[0039] Small animal live imaging showed that before torsion, the kidney was visualized after injecting fluorescent dye into the heart; after torsion, the kidney was not visualized after injecting fluorescent dye into the heart; after the reduction of torsion, the kidney was visualized after injecting fluorescent dye into the heart. Laser speckle imaging showed that before kidney torsion, the kidney blood flow was unobstructed; after kidney torsion, the kidney blood flow stopped; after the reduction of kidney torsion, the kidney blood flow recovered again. The results of laser speckle imaging and small animal live imaging experiments showed that the kidney torsion model could effectively block the renal blood flow, and the blood flow recovered after reperfusion. Figure 2 , Figure 3 )。

[0040] Histopathological changes

[0041] HE staining showed that the glomerular structure was complete, the tubular brush border was clear, and there was no obvious inflammatory cell infiltration in the sham group, while obvious renal tubular injury, including lumen dilation, loss of brush border, and cast formation, occurred in the kidney torsion group and the arterial clamp group. Figure 5 )。

[0042] Transcriptome analysis

[0043] Correlation analysis

[0044] According to the Pearson correlation coefficient diagram and the PCA analysis diagram, on the one hand, the correlation within the groups of the sham group, the AC group and the RT group is large, on the other hand, the correlation between the RT group and the AC group is large, and the correlation with the sham group is small. Figure 4 A、 Figure 4 B).

[0045] It should be pointed out finally that the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for constructing a mouse model of renal ischemia-reperfusion, characterized in that: Includes the following steps: S1. Anesthetize mice; S2. Fix and disinfect the mice; S3. Cut open the skin, separate and expose the kidney tissue and blood vessels layer by layer; S4. Kidney rotation and repositioning: Rotate the kidney 360° around the renal artery as the axis, maintain the rotation for 30 minutes, then reposition the kidney to open the renal blood flow; S5. Suture the surgical incision.

2. The method for constructing a mouse model of renal ischemia-reperfusion according to claim 1, characterized in that: in In step S1, the anesthetizing of the mouse involves placing the mouse in an anesthesia induction chamber and introducing a mixture of isoflurane and oxygen.

3. The method for constructing a mouse model of renal ischemia-reperfusion according to claim 2, characterized in that: The concentration of the isoflurane is 3-5%.

4. The method for constructing a mouse model of renal ischemia-reperfusion according to claim 1, characterized in that: in In step S2, the fixation is performed by fixing the mouse in a right lateral decubitus position after it loses its righting reflex, and the disinfection area is the left hypochondrium and left lumbar region.

5. The method for constructing a mouse model of renal ischemia-reperfusion according to claim 4, characterized in that: The disinfection process uses iodine solution.

6. The method for constructing a mouse model of renal ischemia-reperfusion according to claim 1, characterized in that: In step S3, the skin incision is made parallel to the spine, and the incision depth is approximately 1 cm.

7. The method for constructing a mouse model of renal ischemia-reperfusion according to claim 1, characterized in that: in In step S4, the kidney is twisted and repositioned as follows: the kidney is rotated 360° around the renal artery and maintained in the rotated state for 30 minutes, and then the kidney is repositioned to open the renal blood flow.

8. The method for constructing a mouse model of renal ischemia-reperfusion according to claim 7, characterized in that: The rotation of the kidney 360° around the renal artery as an axis can be either clockwise or counterclockwise.

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