Cerebral infarction severity scoring method based on brain slice TTC staining of mouse cerebral infarction model

Abstract

The invention belongs to the technical field of mouse brain slice image processing, and particularly discloses a mouse cerebral infarction model-based brain slice TTC staining cerebral infarction severity scoring method, which comprises the following steps of: constructing a mouse cerebral infarction model; the method comprises the following steps: anatomically dissecting an euthanasia mouse to obtain a mouse brain, slicing, and carrying out TTC staining; using a scanner to collect brain slice images; then, the brain slice image is subjected to image algorithm processing, and the cerebral infarction degree can be rapidly and accurately evaluated; meanwhile, the ischemic penumbra damage degree (namely, TTC staining saturation) is taken as a quantitative index, so that the consistency with mouse neurobehavioral performance is improved.

Description

Technical Field

[0001] The invention relates to the technical field of mouse brain slice image processing, in particular to a cerebral infarction severity scoring method based on TTC staining of brain slices of a mouse cerebral infarction model. Background Art

[0002] Stroke is one of the cerebrovascular diseases that seriously endangers human health. According to the type of stroke, it can be divided into ischemic stroke, hemorrhagic stroke and transient ischemic attack; among them, ischemic stroke is the most common type. When ischemic stroke occurs, the ischemic area of ​​the brain can be divided into the ischemic core area and the ischemic penumbra. The former is irreversible neuronal damage caused by ischemia; the latter is active due to collateral circulation, but is in a low metabolic and electrical inactivity state. As the ischemic time prolongs, the ischemic penumbra will gradually transform into the ischemic core area, causing the symptoms of stroke to gradually worsen. When thrombolysis or thrombectomy is performed on stroke patients in a timely manner, the ischemic penumbra can gradually restore some or all of its functions. Therefore, the clinical efficacy evaluation of stroke patients focuses on the dynamic monitoring of the ischemic penumbra rather than the range changes of the ischemic core area by using computed tomography (CT) and magnetic resonance imaging (MRI).

[0003] In order to study the injury mechanism and prevention and treatment measures of stroke, the mouse cerebral infarction model is widely used, that is, by artificially blocking the mouse cerebral blood supply artery, the model effect of cerebral infarction is achieved. TTC staining is a commonly used detection method to evaluate the extent of cerebral infarction in mice. The principle is that TTC reacts with succinate dehydrogenase in the mitochondria of living cells to generate red formazan products, thereby reflecting whether the cells are alive or not. In inactive tissues (such as the ischemic core area), due to the inactivation of succinate dehydrogenase, it does not change and appears white; in the ischemic penumbra, the nerve cells still have low activity, so they synthesize less formazan products, making the tissue appear pink. Therefore, when the cerebral infarction time is short and the ischemic penumbra still exists, the mouse brain at this time is sliced ​​and stained with TTC, such as Figure 1 As shown in the figure, the brain slice can show the distribution of three areas: red (normal brain tissue), pink (ischemic penumbra), and white (ischemic core area). The existing analysis method is to obtain the area of ​​pink and white areas in the slice by manual or computer recognition, and then calculate the cerebral infarction area or volume of the whole brain to assess the severity of cerebral infarction.

[0004] However, different researchers use different techniques for TTC staining, and the quality of the TTC reagents they use is inconsistent, which results in uneven staining, poor contrast, and unclear boundaries between regions, which increases the workload of subsequent slice analysis and affects the accuracy of the analysis. In addition, the existing TTC staining analysis method only analyzes a single factor, the infarct area or volume, while ignoring the differences in the degree of damage to the ischemic penumbra, making it difficult to better reflect the actual severity of cerebral infarction.

[0005] Therefore, it is necessary to design a cerebral infarction severity scoring method based on TTC staining of brain sections in a mouse cerebral infarction model, so as to quickly and accurately evaluate the extent of cerebral infarction and further promote basic research on stroke. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for scoring the severity of cerebral infarction based on TTC staining of brain slices of a mouse cerebral infarction model.

[0007] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0008] A method for scoring the severity of cerebral infarction based on TTC staining of brain sections of a mouse cerebral infarction model comprises the following steps:

[0009] S1. Establishment of mouse cerebral infarction model;

[0010] S2. After the model was established, the mice were euthanized at the time point to be studied, and the mouse brains were dissected and sliced ​​for TTC staining;

[0011] S3, placing the TTC-stained brain slice on a cover glass to ensure that the brain slice fits the cover glass, and using a scanner to collect images of the brain slice;

[0012] S4, filling the background of the brain slice image with black, and dividing it into the infarct side image and the non-infarct side image from the brain midline, and converting the infarct side image and the non-infarct side image into the infarct side saturation image and the non-infarct side saturation image;

[0013] S5. According to the statistical distribution of the pixel values ​​of the saturation image on the infarct side, the pixel values ​​are divided into three equal intervals. The areas with the highest, middle and lowest pixel values ​​correspond to the normal brain tissue, ischemic penumbra and ischemic core area, respectively. The average saturation values ​​of the whole image on the infarct side and the ischemic core area are obtained, respectively. ipsi 、Core ipsi At the same time, the saturation image of the non-infarct side is also divided using the same pixel value interval to obtain the average saturation of the non-infarct side Slide contra ;

[0014] S6. Use the following formula to correct the saturation values ​​of the infarct side saturation image and the non-infarct side saturation image:

[0015] Sat Corrected =Slide ipsi or contra -Core ipsi ;

[0016] Calculate the saturation value ratio after correction:

[0017] R=Sat Corrected-ipsi / Sat Corrected-contra ;

[0018] Finally, the severity score of cerebral infarction in mice was calculated according to the following formula:

[0019] Severity = (1-R) ​​* 100.

[0020] As a preferred solution of the present invention, step S1 comprises:

[0021] S11, mice were anesthetized with 2.5% isoflurane to ensure that the righting reflex was absent;

[0022] S12. After disinfecting the neck with 75% alcohol, cut the skin longitudinally and separate the tissue to expose the right carotid sheath.

[0023] S13. After separating and ligating the external carotid artery, internal carotid artery and common carotid artery, use microscissors to make a small cut in the external carotid artery and insert the suture. Send the suture from the internal carotid artery to the beginning of the middle cerebral artery through the bifurcation of the common carotid artery, and use the feeling of resistance as the standard;

[0024] S14. After fixing the suture, close the neck incision, wait for 1 hour before anesthetizing again and removing the suture to complete the construction of the mouse cerebral infarction model.

[0025] As a preferred solution of the present invention, step S2 comprises:

[0026] S21. Prepare 2% TTC solution with PBS at a dosage of 5 mL / piece. Use it immediately after preparation and keep it away from light.

[0027] S22, place the mouse brain in the brain trough of the brain slice, and freeze it in a -20°C refrigerator for 10 minutes; during this time, add 5 mL / well of 2% TTC solution to the 6-well plate and keep it away from light;

[0028] S23. Use a blade to cut the mouse brain into 6 slices with a thickness of 1 mm, and immediately transfer the slices to a 6-well plate containing TTC solution. Stain for 12 minutes in the dark. During this period, turn all brain slices over at the 6th minute to ensure uniform staining.

[0029] S24. After staining, discard the TTC solution, wash with PBS for 3 minutes, and discard the PBS;

[0030] S25. Soak the brain slices in 4% paraformaldehyde for 12-24 hours for fixation; do not destroy the integrity of the brain slices during the entire staining process to reduce slice deformation.

[0031] As a preferred solution of the present invention, in step S3, the resolution of the brain slice image is above 600 PPI.

[0032] Compared with the prior art, the present invention can objectively and efficiently perform TTC staining and then perform image algorithm processing, which can quickly and accurately evaluate the degree of cerebral infarction; at the same time, the degree of damage to the ischemic penumbra (i.e., TTC staining saturation) is incorporated as a quantitative indicator to improve the consistency with the neurobehavioral performance of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of different regions of brain slices after TTC staining in the prior art.

[0034] Figure 2 It is a technical flow chart of the present invention.

[0035] Figure 3 Brain slice images were manually segmented for the present invention.

[0036] Figure 4 The brain slice image of the present invention is converted into a brain slice saturation image.

[0037] Figure 5 The ischemic penumbra and ischemic core area are marked for the image algorithm system.

[0038] Figure 6 This is the cerebral infarction severity score obtained by analyzing different TTC staining results and image algorithm systems in the present invention.

[0039] Figure 7 The results of correlation analysis between different analysis results and mouse behavioral performance: (a) is the method of the present invention; (b) is the result of manual identification of cerebral infarction volume; (c) is the result of automatic identification of cerebral infarction volume by ImageJ software. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0041] like Figure 1As shown, this embodiment exemplarily demonstrates a method for scoring the severity of cerebral infarction based on TTC staining of brain sections of a mouse cerebral infarction model, comprising the following steps:

[0042] S1. Establishment of mouse cerebral infarction model;

[0043] In this embodiment, the suture embolism method was used as an example to construct a mouse cerebral infarction model, and the specific construction process was as follows:

[0044] S11, mice were anesthetized with 2.5% isoflurane to ensure that the righting reflex was absent;

[0045] S12. After disinfecting the neck with 75% alcohol, cut the skin longitudinally and separate the tissue to expose the right carotid sheath.

[0046] S13. After separating and ligating the external carotid artery, internal carotid artery and common carotid artery, use microscissors to make a small cut in the external carotid artery and insert the suture. Send the suture from the internal carotid artery to the beginning of the middle cerebral artery through the bifurcation of the common carotid artery, and use the feeling of resistance as the standard;

[0047] S14. After fixing the suture, close the neck incision, wait for 1 hour before anesthetizing again and removing the suture to complete the construction of the mouse cerebral infarction model.

[0048] S2. After the model is constructed, the mice are euthanized at the time point to be studied, and the mouse brains are obtained by dissection, and the slices are subjected to TTC staining; the exemplary process of TTC staining in this embodiment is as follows:

[0049] S21. Prepare 2% TTC solution with PBS at a dosage of 5 mL / piece. Use it immediately after preparation and keep it away from light.

[0050] S22, place the mouse brain in the brain trough of the brain slice, and freeze it in a -20°C refrigerator for 10 minutes; during this time, add 5 mL / well of 2% TTC solution to the 6-well plate and keep it away from light;

[0051] S23. Use a blade to cut the mouse brain into 6 slices with a thickness of 1 mm, and immediately transfer the slices to a 6-well plate containing TTC solution. Stain for 12 minutes in the dark. During this period, turn all brain slices over at the 6th minute to ensure uniform staining.

[0052] S24. After staining, discard the TTC solution, wash with PBS for 3 minutes, and discard the PBS;

[0053] S25. Soak the brain slices in 4% paraformaldehyde for 12-24 hours for fixation; do not destroy the integrity of the brain slices during the entire staining process to reduce slice deformation.

[0054] S3. Place the TTC-stained brain slice on a coverslip to ensure that the brain slice fits the coverslip well. Use a scanner to capture the brain slice image. To ensure image clarity, the resolution of the brain slice image needs to be above 600 PPI.

[0055] S4. Use image software (such as Photoshop software) to fill the background of the brain slice image with black, and divide it into the infarct side image and the non-infarct side image from the midline of the brain. The specific process is as follows: Figure 3 As shown, the infarct side image and the non-infarct side image are converted into the infarct side saturation image and the non-infarct side saturation image. The results are shown in Figure 4 As shown;

[0056] S5. According to the statistical distribution of pixel values ​​in the saturation image on the infarct side, the pixel values ​​are divided into three equal intervals. The areas with the highest, middle and lowest pixel values ​​correspond to normal brain tissue, ischemic penumbra and ischemic core area, respectively. For details, see Figure 5 , and the average saturation values ​​of the whole image on the infarct side and the ischemic core area were obtained, respectively. ipsi 、Core ipsi At the same time, the saturation image of the non-infarct side is also divided using the same pixel value interval to obtain the average saturation of the non-infarct side Slide contra ;

[0057] S6. Use the following formula to correct the saturation values ​​of the infarct side saturation image and the non-infarct side saturation image:

[0058] Sat Corrected =Slide ipsi or contra -Core ipsi ;

[0059] Calculate the saturation value ratio after correction:

[0060] R=Sat Corrected-ipsi / Sat Corrected-contra ;

[0061] Finally, the severity score of cerebral infarction in mice was calculated according to the following formula:

[0062] Severity = (1-R) ​​* 100;

[0063] Several examples of mouse cerebral infarction severity scores are shown in Figure 6 As shown by Figure 6 It can be seen that with the increase of the cerebral infarction area of ​​mice and the decrease of the saturation value of the ischemic penumbra, the severity score of cerebral infarction gradually increased.

[0064] In some embodiments, in order to verify the feasibility of the present invention, the results of manual identification of cerebral infarction volume and the results of automatic identification of cerebral infarction volume by ImageJ software were compared with the present invention, and the results are as follows: Figure 7 As shown by Figure 7 It can be seen that the correlation coefficient with mouse neurobehavior is higher than the currently commonly used ImageJ software automatic recognition and analysis method (0.8525 vs 0.7366), and is similar to the result of manual recognition and analysis (0.8525 vs 0.8532).

[0065] In summary, the present invention can objectively and efficiently perform TTC staining followed by image algorithm processing, and can quickly and accurately evaluate the degree of cerebral infarction.

[0066] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for scoring the severity of cerebral infarction based on TTC staining of brain sections of a mouse cerebral infarction model, characterized in that: The following steps are involved: S1. Establishment of mouse cerebral infarction model; S2. After the model was established, the mice were euthanized at the time point to be studied, and the mouse brains were dissected and sliced ​​for TTC staining; S3, placing the TTC-stained brain slice on a cover glass to ensure that the brain slice fits the cover glass, and using a scanner to collect images of the brain slice; S4, filling the background of the brain slice image with black, dividing it into the infarct side image and the non-infarct side image from the brain midline, and converting the infarct side image and the non-infarct side image into the infarct side saturation image and the non-infarct side saturation image; S5. According to the statistical distribution of the pixel values ​​of the saturation image on the infarct side, the pixel values ​​are divided into three equal intervals. The areas with the highest, middle and lowest pixel values ​​correspond to the normal brain tissue, ischemic penumbra and ischemic core area, respectively. The average saturation values ​​of the whole image on the infarct side and the ischemic core area are obtained, respectively. ipsi 、Core ipsi At the same time, the saturation image of the non-infarct side is also divided using the same pixel value interval to obtain the average saturation of the non-infarct side Slide contra ; S6. Use the following formula to correct the saturation values ​​of the infarct side saturation image and the non-infarct side saturation image: Sat corrected =Slide ipsi or contra -Core ipsi ; Calculate the saturation value ratio after correction: R=Sat Corrected-ipsi / Sat Corrected-contra ; Finally, the severity score of cerebral infarction in mice was calculated according to the following formula: Severity = (1-R) ​​* 100.

2. The method for scoring the severity of cerebral infarction based on TTC staining of brain sections of a mouse cerebral infarction model according to claim 1, characterized in that: The step S1 comprises: S11, mice were anesthetized with 2.5% isoflurane to ensure that the righting reflex was absent; S12. After disinfecting the neck with 75% alcohol, cut the skin longitudinally and separate the tissue to expose the right carotid sheath. S13. After separating and ligating the external carotid artery, internal carotid artery and common carotid artery, use microscissors to make a small cut in the external carotid artery and insert the suture. Send the suture from the internal carotid artery to the beginning of the middle cerebral artery through the bifurcation of the common carotid artery, and use the feeling of resistance as the standard; S14. After fixing the suture, close the neck incision, wait for 1 hour before anesthetizing again and removing the suture to complete the construction of the mouse cerebral infarction model.

3. The method for scoring the severity of cerebral infarction based on TTC staining of brain sections of a mouse cerebral infarction model according to claim 1, characterized in that: The step S2 comprises: S21. Prepare 2% TTC solution with PBS at a dosage of 5 mL / piece. Use it immediately after preparation and keep it away from light. S22, place the mouse brain in the brain trough of the brain slice, and freeze it in a -20°C refrigerator for 10 minutes; during this time, add 5 mL / well of 2% TTC solution to the 6-well plate and keep it away from light; S23. Use a blade to cut the mouse brain into 6 slices with a thickness of 1 mm, and immediately transfer the slices to a 6-well plate containing TTC solution. Stain for 12 minutes in the dark. During this period, turn all brain slices over at the 6th minute to ensure uniform staining. S24. After staining, discard the TTC solution, wash with PBS for 3 minutes, and discard the PBS; S25. Soak the brain slices in 4% paraformaldehyde for 12-24 hours for fixation; do not destroy the integrity of the brain slices during the entire staining process to reduce slice deformation.

4. The method for scoring the severity of cerebral infarction based on TTC staining of brain sections of a mouse cerebral infarction model according to claim 1, characterized in that: In step S3, the resolution of the brain slice image is above 600 PPI.

Images