A cerebral infarction severity scoring method based on TTC staining of brain sections in a mouse cerebral infarction model

Abstract

The present invention belongs to the technical field of mouse brain slice image processing, and specifically discloses a method for scoring the severity of cerebral infarction based on TTC staining of brain slices in a mouse cerebral infarction model. The method comprises: establishing a mouse cerebral infarction model; euthanizing the mouse, dissecting the mouse brain, and then performing TTC staining on the slices; acquiring brain slice images using a scanner; and then performing image algorithm processing on the brain slice images to quickly and accurately assess 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 consistency with the neurobehavioral performance of the mouse.

Description

Technical Field

[0001] The present 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 most serious cerebrovascular diseases that threatens human health. Stroke can be categorized by type: ischemic stroke, hemorrhagic stroke, and transient ischemic attack (TIA); ischemic stroke is the most common type. During an ischemic stroke, the ischemic area can be divided into an ischemic core and an ischemic penumbra. The former represents irreversible neuronal damage caused by ischemia; the latter, while maintained by collateral circulation, is in a state of hypometabolic, electrical inactivity. With prolonged ischemia, the ischemic penumbra gradually transforms into an ischemic core, exacerbating stroke symptoms. With timely thrombolysis or thrombectomy, the ischemic penumbra can gradually recover partial or full function. Therefore, clinical evaluation of treatment efficacy in stroke patients, using computed tomography (CT) and magnetic resonance imaging (MRI), focuses on the dynamic monitoring of the ischemic penumbra rather than changes in the extent of the ischemic core.

[0003] In order to study the damage mechanism and prevention and treatment measures of stroke, the mouse cerebral infarction model is widely used, that is, the cerebral blood supply artery of the mouse is artificially blocked to achieve the model effect of cerebral infarction. TTC staining is a commonly used detection method to evaluate the degree 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, no changes occur and it appears white; in the ischemic penumbra, nerve cells still have low activity, and therefore synthesize fewer formazan products, which makes 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 TTC stained, such as Figure 1 As shown, brain slices can generally display three areas: red (normal brain tissue), pink (ischemic penumbra), and white (ischemic core). Existing analysis methods use manual or computer identification to determine the areas of the pink and white areas in the slices, and then calculate the infarct area or volume of the entire brain to assess the severity of cerebral infarction.

[0004] However, TTC staining techniques vary among researchers, and the quality of the TTC reagents used can be inconsistent. This can lead to uneven staining, poor contrast, and unclear demarcations between different regions, increasing the workload for subsequent slice analysis and compromising analytical accuracy. Furthermore, existing TTC staining analysis methods focus solely on infarct size or volume, ignoring differences in the extent of ischemic penumbra damage. Consequently, they struggle to accurately 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 assess 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 sections 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 in a mouse cerebral infarction model comprises the following steps:

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

[0010] S2. After the model is established, the mice are euthanized at the time point to be studied, and the brains of the mice are dissected and sliced ​​for TTC staining.

[0011] S3. Place the TTC-stained brain slice on a coverslip to ensure that the brain slice fits the coverslip well, and use a scanner to capture images of the brain slice.

[0012] S4. Fill the background of the brain slice image with black, and divide it into the infarct side image and the non-infarct side image along the brain midline, and convert 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. Divide the pixel values ​​into three equal intervals according to the statistical distribution of the pixel values ​​of the saturation image on the infarct side. 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 image Slide contra ;

[0014] S6. Correct the saturation values ​​of the infarct-side saturation image and the non-infarct-side saturation image using the following formula:

[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 includes:

[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 isolating and ligating the external carotid artery, internal carotid artery, and common carotid artery, use microscissors to make a small incision in the external carotid artery and insert the suture plug. The suture plug is passed from the internal carotid artery through the common carotid bifurcation to the origin of the middle cerebral artery, with resistance being the standard.

[0024] S14. After fixing the suture bolt, close the neck incision. After 1 hour, anesthetize again and remove the suture bolt to complete the construction of the mouse cerebral infarction model.

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

[0026] S21. Prepare 2% TTC solution in PBS at a volume of 5 mL / piece. Use immediately after preparation and store in a dark place.

[0027] S22. Place the mouse brain in the brain slice trough and quickly freeze in a -20°C refrigerator for 10 minutes. During this time, add 5 mL / well of 2% TTC solution to a 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. Immediately transfer the slices to a 6-well plate containing TTC solution and stain in the dark for 12 minutes. During this period, all brain slices were turned 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. Fix the brain slices by soaking them in 4% paraformaldehyde for 12-24 hours. Do not damage the integrity of the brain slices during the staining process to reduce slice deformation.

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

[0032] Compared with the existing technology, the present invention can objectively and efficiently perform TTC staining followed by image algorithm processing, which can quickly and accurately assess the extent of cerebral infarction; at the same time, it incorporates the degree of damage to the ischemic penumbra (i.e., TTC staining saturation) as a quantitative indicator to improve 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 solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0041] like Figure 1As shown, this embodiment exemplifies 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 example, the suture embolization method was used as an example to construct a mouse cerebral infarction model. The specific construction process is 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 isolating and ligating the external carotid artery, internal carotid artery, and common carotid artery, use microscissors to make a small incision in the external carotid artery and insert the suture plug. The suture plug is passed from the internal carotid artery through the common carotid bifurcation to the origin of the middle cerebral artery, with resistance being the standard.

[0047] S14. After fixing the suture bolt, close the neck incision. After 1 hour, anesthetize again and remove the suture bolt to complete the construction of the mouse cerebral infarction model.

[0048] S2. After the model is established, the mice are euthanized at the time point to be studied, and the mouse brains are dissected and sliced ​​for TTC staining. The exemplary process of TTC staining in this example is as follows:

[0049] S21. Prepare 2% TTC solution in PBS at a volume of 5 mL / piece. Use immediately after preparation and store in a dark place.

[0050] S22. Place the mouse brain in the brain slice trough and quickly freeze in a -20°C refrigerator for 10 minutes. During this time, add 5 mL / well of 2% TTC solution to a 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. Immediately transfer the slices to a 6-well plate containing TTC solution and stain in the dark for 12 minutes. During this period, all brain slices were turned 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. Fix the brain slices by soaking them in 4% paraformaldehyde for 12-24 hours. Do not damage the integrity of the brain slices during the 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 must be above 600 PPI.

[0055] S4. Use image software (e.g., Photoshop) 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 along the brain midline. 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, and the results are shown in Figure 4 As shown;

[0056] S5. Divide the pixel values ​​into three equal intervals based on the statistical distribution of the pixel values ​​in the saturation image on the infarct side. The areas with the highest, middle, and lowest pixel values ​​correspond to normal brain tissue, ischemic penumbra, and ischemic core, respectively. For details, see Figure 5 , and the average saturation values ​​of the whole image of 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 image Slide contra ;

[0057] S6. Correct the saturation values ​​of the infarct-side saturation image and the non-infarct-side saturation image using the following formula:

[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 scoring results are shown below: Figure 6 As shown by Figure 6 It can be seen that as the cerebral infarction area of ​​mice increases and the saturation value of the ischemic penumbra decreases, the severity score of cerebral infarction gradually increases.

[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 that of the currently commonly used ImageJ software automatic recognition and analysis method (0.8525 vs 0.7366), and is similar to the results 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 assess the extent of cerebral infarction.

[0066] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any 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 in a mouse cerebral infarction model, characterized in that: The following steps are involved: S1. Establishment of mouse cerebral infarction model; S2. After the model is established, the mice are euthanized at the time point to be studied, and the brains of the mice are dissected and sliced ​​for TTC staining. S3. Place the TTC-stained brain slice on a coverslip to ensure that the brain slice fits the coverslip well, and use a scanner to capture images of the brain slice. S4. Fill the background of the brain slice image with black, and divide it into the infarct side image and the non-infarct side image along the brain midline, and convert the infarct side image and the non-infarct side image into the infarct side saturation image and the non-infarct side saturation image; S5. Divide the pixel values ​​into three equal intervals according to the statistical distribution of the pixel values ​​of the saturation image on the infarct side. 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 image Slide contra ; S6. Correct the saturation values ​​of the infarcted and non-infarcted saturation images using the following formula: 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, wherein: 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 isolating and ligating the external carotid artery, internal carotid artery, and common carotid artery, a small incision is made in the external carotid artery using microscissors and a suture plug is inserted. The suture plug is then passed from the internal carotid artery through the bifurcation of the common carotid artery to the origin of the middle cerebral artery, with the sensation of resistance being the standard. S14. After fixing the suture bolt, close the neck incision. After 1 hour, anesthetize again and remove the suture bolt 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, wherein: The step S2 comprises: S21. Prepare 2% TTC solution in PBS at a volume of 5 mL / piece. Use immediately after preparation and store in a dark place. S22. Place the mouse brain in the brain slice trough and quickly freeze in a -20°C refrigerator for 10 minutes. During this time, add 5 mL / well of 2% TTC solution to a 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. Immediately transfer the slices to a 6-well plate containing TTC solution and stain in the dark for 12 minutes. During this period, all brain slices were turned 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. Fix the brain slices by soaking them in 4% paraformaldehyde for 12-24 hours. Do not damage the integrity of the brain slices during the staining process to reduce slice deformation.

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

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