Stain and Evaluation Method for Microvascular Perfusion Staining

Abstract

The present invention discloses a staining agent, a method for preparing a heart section, and a method for evaluating microvascular perfusion staining. The mixed staining agent is used for microvascular perfusion staining, and the mixed staining agent includes DiIC 12 and DiIC 18 . In the mixed staining agent, the concentration ratio of DiIC 12 to DiIC 18 is 90-60:10-40. Experiments show that a certain proportion of the mixed staining agent can effectively solve the problems of poor solubility and uneven staining of DiIC 18 in coronary microcirculation perfusion staining, and does not affect the biological morphology and structure of myocardial cells and microvessels.

Description

Technical Field

[0001] The present invention relates to the technical field of microvascular observation, and particularly to a staining agent and a method for evaluating microvascular perfusion staining. Background Art

[0002] For a long time, myocardial ischemia caused by stenosis of the main coronary artery has been considered as the main cause of coronary heart disease, while the clinical importance of coronary microvascular (also known as microcirculation) dysfunction (CMD) has been ignored. The latest clinical research has found that CMD is a potential pathological factor of ischemic heart disease and found that about 20%-30% of patients with chest pain have no obvious stenosis or occlusion of the main coronary artery and its main branches, but are caused by CMD. Therefore, the pathological changes and treatment of coronary microvessels have also attracted the attention of life science workers. However, since the heart is a dense solid tissue, and the microvascular system is perpendicular to the heart surface and parallel to the myocardial fibers, it is difficult to observe the morphological structure of coronary microvessels. Therefore, there is an urgent need for a better method for staining and observing the morphological structure of coronary microvessels, which is helpful for further research on coronary microvascular dysfunction.

[0003] So far, there have been various methods for labeling or staining coronary microvessels, such as endothelial cell-specific markers and ink perfusion methods, etc., but they all have certain defects, mainly including complex operation steps, weak signal intensity, inability to completely visualize coronary microvessels, and problems such as damage to the microvascular morphological structure and too fast fluorescence signal attenuation, and the morphological structure of microvessels cannot be accurately observed. Summary of the Invention

[0004] In view of the above situation, it is necessary to provide a method for microvascular perfusion staining and evaluation aiming at the inability to accurately observe the morphological structure of microvessels in the existing microvascular staining technology.

[0005] A mixed staining agent for microvascular perfusion staining, the mixed staining agent comprising DiIC 12 and DiIC 18 .

[0006] Further, in the above mixed staining agent, the concentration ratio of DiIC 12 to DiIC 18 is 90-60:10-40.

[0007] Further, in the above mixed staining agent, the concentration ratio of DiIC 12 to DiIC 18 is 80:20.

[0008] The present invention also discloses a method for preparing a heart section, including:

[0009] Inject a PBS solution into the heart ventricle of a living body, clamp the aorta, perfuse a staining agent into the heart ventricle, and then inject a formaldehyde solution. The staining agent uses the above-mentioned mixed staining agent;

[0010] Take out the heart tissue and place it in a formaldehyde solution for fixation;

[0011] Dehydrate the fixed heart tissue;

[0012] Embed the dehydrated heart tissue and then perform cryosectioning.

[0013] Furthermore, in the above method for preparing a heart section, the heart tissue is fixed in a formaldehyde solution with a formaldehyde content of 10% for 24 h, and the heart tissue is dehydrated in a sucrose solution with a sucrose content of 30% for 12 h.

[0014] The present invention also discloses a method for evaluating microvascular perfusion staining, including:

[0015] Prepare mixed staining agents with different concentration gradients of DiIC 12 and DiIC 18 ;

[0016] Inject the prepared mixed staining agent into the heart ventricle of a living body and prepare heart sections to obtain multiple sample groups. Among them, each sample group uses a mixed staining agent with the same concentration gradient, and each sample group includes multiple heart sections;

[0017] Obtain the Z-stack images of each heart section and perform three-dimensional image construction to obtain a three-dimensional image;

[0018] Detect the three-dimensional images of each heart section respectively to obtain the measured values of each evaluation index in each heart section and perform standardization processing;

[0019] Calculate the proportion of each sample group under each evaluation index according to the standardized measured values, and calculate the weight coefficients of each evaluation index;

[0020] Calculate the comprehensive score of each sample group according to the proportion of each heart section under each evaluation index and the weight coefficients of each evaluation index.

[0021] Furthermore, in the above method for evaluating microvascular perfusion staining, the evaluation indexes include:

[0022] The percentage of highly fluorescent spots, the percentage of weakly fluorescent area, the total length of fluorescent microvessels, the fluorescence attenuation rate of microvessels, and the porosity of fluorescent microvessels.

[0023] Further, in the above microvascular perfusion staining evaluation method, the step of calculating the proportion of each sample group under each evaluation index according to the measured values after standardization includes:

[0024] Calculating the proportion of each slice under each evaluation index according to the measured values of the evaluation indexes of each heart slice, and obtaining the proportion value corresponding to each heart slice, wherein the calculation formula of the proportion value corresponding to the heart slice is:

[0025] Z ij is the value after standardization of the measured value of the j-th evaluation index of the i-th heart slice;

[0026] Calculating the mean value of the proportion values corresponding to each slice in the same slice group under each evaluation index, and obtaining the proportion of the sample group under each evaluation index.

[0027] Further, in the above microvascular perfusion staining evaluation method, the step of calculating the weight coefficients of each evaluation index includes:

[0028] Calculating the entropy value of each index according to the proportion of each sample group under each evaluation index, and the calculation formula of the entropy value ej is

[0029] where ej is the entropy value, k is a constant, P ij is the proportion of the i-th sample group under the j-th evaluation index, M is the number of sample groups, and n is the number of evaluation indexes;

[0030] Calculating the effective information value of each evaluation index according to the entropy value of each evaluation index, and the calculation formula of the effective information value gi is

[0031] where

[0032] Calculating the weight coefficient of each evaluation index according to the effective information value of each evaluation index, and the calculation formula of the weight coefficient Wj is

[0033]

[0034] Further, in the above microvascular perfusion staining evaluation method, the formula for calculating the comprehensive score Si of each sample group is:

[0035] where, wj is the weight coefficient of the j-th evaluation index, p ij is the proportion of the i-th sample group under the j-th evaluation index, M is the number of sample groups, and n is the number of evaluation indexes.

[0036] Further, in the above-mentioned microvascular perfusion staining evaluation method, after obtaining the measured values of each evaluation index in each of the cardiac sections, it further includes:

[0037] Performing null value and outlier rejection processing on the obtained measured values.

[0038] The beneficial effects of the present invention are mainly reflected in that the present invention mainly determines the proportion range of the dyes DiIC 12 and DiIC 18 Experiments show that a certain proportion of the mixed dye can effectively solve the problems of poor solubility and uneven staining of DiIC 18 in coronary microcirculation perfusion staining, does not affect the biological morphology and structure of myocardial cells and microvessels, can visualize the morphology and structure of the coronary microcirculation completely, and can accurately measure related indexes such as the number and density of microvessels. This method is helpful for the further study of coronary microvascular dysfunction. Description of the Drawings

[0039] Figure 1 is the effect of the ratio of DiIC 12 / DiIC 18 on the staining effect of rat coronary microcirculation (laser confocal microscope, 400x);

[0040] Figure 2a is the effect of different ratios of DiIC 12 / DiIC 18 on the percentage of highly fluorescent spots in the indexes of rat coronary microvascular ring staining;

[0041] Figure 2b is the effect of different ratios of DiIC 12 / DiIC 18 on the percentage of weakly fluorescent area in the indexes of rat coronary microvascular ring staining;

[0042] Figure 2c is the effect of different ratios of DiIC 12 / DiIC 18 on the total length of fluorescent microvessels in rat coronary microvascular ring staining;

[0043] Figure 2d is the effect of different ratios of DiIC 12 / DiIC 18 on the fluorescence attenuation rate of microvessels in rat coronary microvascular ring staining;

[0044] Figure 2e For different DiIC 12 / DiIC 18 ratio on the fluorescence microvascular porosity in the coronary microvascular ring staining of rats;

[0045] Figure 3a For the Pearson correlation and linear regression analysis of the percentages of DiIC 12 and DiIC 18 under the high - light fluorescence spot percentage index;

[0046] Figure 3b For the Pearson correlation and linear regression analysis of the percentages of DiIC 12 and DiIC 18 under the weak fluorescence area percentage index;

[0047] Figure 3c For the Pearson correlation and linear regression analysis of the percentages of DiIC 12 and DiIC 18 under the total length of fluorescent microvessels index;

[0048] Figure 3d For the Pearson correlation and linear regression analysis of the percentages of DiIC 12 and DiIC 18 under the microvascular fluorescence attenuation rate index;

[0049] Figure 3e For the Pearson correlation and linear regression analysis of the percentages of DiIC 12 and DiIC 18 under the fluorescence microvascular porosity index;

[0050] It should be noted that for the sake of simplicity in writing, C 12 and C 18 in the attached drawings are respectively represented as DiIC 12 and DiIC 18 . Specific implementation manners

[0051] To make the objectives, features and advantages of the present invention more obvious and understandable, the following describes in detail the specific implementation manners of the present invention with reference to the embodiments. Several embodiments of the present invention are given in the embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0052] An embodiment of the present invention provides a mixed staining agent for microvascular perfusion staining. The staining agent used for microvascular perfusion staining is DiIC 18 and DiIC 12 mixed staining agent. The heart perfusion method is used for microvascular staining, and after 3D image reconstruction, the microvascular structure is observed.

[0053] DiIC 18 Full name is 1,1'-di-octadecyl-3,3,3',3'-tetra-methylammonium carbocyanine perchlorate, which is a lipophilic dye and has been widely used to label cell membranes and trace neuronal connections in living and fixed tissues. In the cell membrane, the two 18-alkyl chains of the DiI molecule can be buried in the lipid bilayer parallel to the phosphatidyl chains, while the chromophore group is located on the surface of the bilayer. Through cardiac perfusion, DiI quickly enters the cell membrane, that is, the endothelial cell membrane lining the blood vessels, and can quickly label microvessels, and can be excited to produce a strong fluorescence signal. However, it is found in experiments that due to the long C 18 alkyl chain of DiI and strong hydrophobicity, aggregation is likely to occur during the perfusion process, and the aggregation rate is closely related to the environmental temperature. The lower the temperature, the faster the aggregation, resulting in microvascular blockage. After the blood vessels are blocked, the blood vessels rupture due to pressure, causing cardiomyocytes to be stained and affecting the observation of microvessels (see the picture corresponding to the ratio of DiIC Figure 1 and DiIC 18 and DiIC 12 being 100:0 in

[0054] DiIC 12 Full name is 1,1′-di-dodecyl-3,3,3′,3′-tetra-methylammonium carbocyanine perchlorate, which is one of the members of the DiI carbocyanine dye family. Its two alkyl chains are shorter, so its hydrophobicity is smaller than that of DiIC 18 and it can maintain a long enough dissolution time to be effectively incorporated into the cell membrane, making the vascular perfusion more uniform, stable, easy to operate, and with good observation effect (see the picture corresponding to the ratio of DiIC Figure 1 and DiIC 18 and DiIC 12 being 0:100 in 12 Since DiI C 12 is directly delivered to the endothelial cell membrane, a high concentration is achieved on the cell membrane, increasing the signal-to-noise ratio. However, the DiI C

[0055] Example 1

[0056] A method for preparing a heart section, comprising the following steps:

[0057] In vivo preparation: Rats, male, weighing 300 - 350 g, were anesthetized with 0.7 g / 100 g of 20% urethane;

[0058] The chest cavity of the in vivo animal was opened to expose the heart. The descending aorta was clamped with a hemostatic forceps, and 5 ml of PBS solution was slowly injected into the left ventricle, the aorta was clamped, and 2 ml of a mixed staining agent of DiIC with a ratio gradient of 100:0 was perfused. Finally, 5 ml of 10% formaldehyde solution was injected; 12 and DiIC 18 The mixed staining agent was injected, and finally 5 ml of 10% formaldehyde solution was injected;

[0059] The heart tissue was removed and fixed in 10% formaldehyde solution for 24 h (protected from light), and then the fixed heart was taken out;

[0060] The tissue was cut into 4 - mm - thick slices, dehydrated with 30% sucrose solution, embedded in OCT compound, and frozen;

[0061] The frozen sections were cut into 60 - μm - thick slices to obtain heart sections, and the prepared heart sections were mounted with glycerol containing DAPI for standby.

[0062] In this example, DiIC12 and DiIC18 were prepared into a 5 - μg / ml solution. Before the start of heart perfusion, 2 three - way stopcocks and 3 10 - ml syringes were connected. PBS, DiI working solution, and 10% formaldehyde solution were respectively filled in them, and the air bubbles in the syringes were emptied. The rats were anesthetized, the chest cavity was opened to expose the heart, the descending aorta was clamped with a hemostatic forceps, PBS solution was slowly injected into the left ventricle, the aorta was clamped, and DiI C 12 and DiIC 18 The mixed staining agent with a ratio gradient of 100:0 was perfused. The working dilution of this mixed staining agent was prepared by mixing PBS and 5% sucrose solution in a ratio of 1:4. Then, 10% formaldehyde solution was injected into the left ventricle of the heart, and the heart was taken out and fixed in 10% formaldehyde solution. The fixed heart was taken out and cut into 4 - mm - thick tissue. After dehydration with 30% sucrose solution, it was embedded in OCT and frozen for standby.

[0063] It should be noted that the mixed staining agent of DiIC 12 and DiIC 18 with a ratio gradient of 100:0 means taking 100 parts by mass of DiIC 12 and 0 parts of DiIC 18 .

[0064] Example 2

[0065] This example is basically the same as Example 1, except that:

[0066] In the ventricle, a mixed staining agent of DiIC 12 and DiIC 18mixed staining agent.

[0067] Example 3

[0068] This example is basically the same as Example 1, except that:

[0069] DiIC with a perfusion ratio gradient of 80:20 in the ventricle 12 and DiIC 18 mixed staining agent.

[0070] Example 4

[0071] This example is basically the same as Example 1, except that:

[0072] DiIC with a perfusion ratio gradient of 60:40 in the ventricle 12 and DiIC 18 mixed staining agent.

[0073] Example 5

[0074] This example is basically the same as Example 1, except that:

[0075] DiIC with a perfusion ratio gradient of 40:60 in the ventricle 12 and DiIC 18 mixed staining agent.

[0076] Example 6

[0077] This example is basically the same as Example 1, except that:

[0078] DiIC with a perfusion ratio gradient of 20:80 in the ventricle 12 and DiIC 18 mixed staining agent.

[0079] Example 7

[0080] This example is basically the same as Example 1, except that:

[0081] DiIC with a perfusion ratio gradient of 0:100 in the ventricle 12 and DiIC 18 mixed staining agent.

[0082] The heart sections prepared in Examples 1 to 7 were observed under a laser confocal microscope. The laser confocal microscope was set to the Z-stack mode with a 40x eyepiece. The sample was excited with light at 570 nm, and signals in the range of 570 - 590 nm were collected to generate images with a pixel resolution of 1024*1024. Then, a layer scan of 0.63 μm was performed once, and 3D construction of the Z-stack images was carried out using the LAS function of the laser confocal microscope, that is, 3D image construction of the Z-stack images was performed using the LAS function of the laser confocal microscope to obtain three-dimensional images.

[0083] The three-dimensional images of each heart section were respectively detected to obtain the measured values of the evaluation indexes. Specifically, the measurement of DiIC 12 and DiIC 18 The ratio gradient dye solution was used to evaluate the microvascular perfusion effect: the percentage of bright fluorescent spots, the percentage of weak fluorescence area, the total length of fluorescent microvessels, the fluorescence attenuation rate of microvessels, and the porosity of fluorescent microvessels.

[0084] The measurement of each evaluation index of the three-dimensional image can be carried out using conventional image processing software, such as Image J and Angio Tool.

[0085] Among them, the measurement steps of the percentage of bright fluorescent spots: Open the software Image J, then open the three-dimensional picture, use the magic wand tool to automatically select "Analyze" for analysis, and measure to obtain the Area. Select the Vessels area within the fluorescence threshold of 10 - 255 in Index A. The index value is equal to Area / Vessels area. In this calculation step, the fluorescence intensity of the bright fluorescent spots should be greater than or equal to 20, and the diameter should be greater than the ratio of the area of the microvessel to the total fluorescence area, and then multiply by 100%.

[0086] The measurement steps of the percentage of weak fluorescence area: Open the software Angio Tool, then open the three-dimensional picture. Select the fluorescence threshold of 0 - 10, turn on the noise reduction and hole filling functions, and click Run analysis. Then open the picture again, select the fluorescence threshold of 10 - 255, turn on the noise reduction and hole filling functions, and click Run analysis. Take the ratio of the Vessels area selected twice to obtain the percentage of weak fluorescence area. Among them, the fluorescence intensity of the weak fluorescent spots should be less than 200, and the diameter should be less than the ratio of the area of the microvessel to the total fluorescence area, and then multiply by 100%.

[0087] Measurement steps for the total length of fluorescent microvessels: Open the software Angio Tool, then open the 3D image, select the fluorescence threshold of 10 - 255, turn on noise reduction and hole filling, Run Analysis, and select Total Vessels Length.

[0088] The fluorescence decay rate of microvessels is calculated by the software Image J. Measure the intensity of the fluorescence quenching of microvessels within 30 minutes, that is, the index = (MeanT0 - MeanT30) / 30 min, where MeanT0 and MeanT30 are the fluorescence intensities at time T0 and T0 plus 30 min respectively.

[0089] Measurement steps for the porosity of fluorescent microvessels; Use the software Angio Tool - open the 3D image, select the fluorescence threshold of 10 - 255, turn on the noise reduction and hole filling functions, Run Analysis, and select Mean E Lacunarity (porosity).

[0090] Experimental results:

[0091] DiIC 12 / DiIC 18 For the effect of the ratio gradient dye solution of DiIC Figure 1 .DiIC 12 and DiIC 18 on the staining effect of rat coronary microvessel perfusion, see 12 For the effect of the ratio gradient dye solution of DiIC 18 and DiIC

[0092] on various indicators of rat coronary microvessel perfusion staining, see Figure 2. For the Pearson correlation and linear regression analysis of various indicators with the ratio of DiIC 12 / DiIC 18 see Figure 3. 12 / DiIC 18 The results show that when the ratio of DiIC 12 / DiIC 18 is 80:20, the microvessel staining is uniform, the fluorescence intensity is moderate, there are few high - brightness red spots, and the fluorescence quenching rate is slow. Secondly, the staining effects from good to bad are the DiIC 12 ratio gradient dye solutions of 90:10, 60:40, 100:0, 40:60, 20:80, and 0:100. The percentage of high - brightness fluorescence spots in the coronary microvessel staining of SD rats is negatively correlated with the ratio of DiIC 18 in DiIC 12 / DiIC18 The ratio of DiIC in 12 showed a negative correlation (DiIC 18 On the contrary), and the Pearson coefficient was -0.59795; the total length of fluorescent microvessels was positively correlated with the ratio of DiIC in 12 / DiIC 18 The ratio of DiIC in 12 showed a positive correlation (DiIC 18 On the contrary), and the Pearson coefficient was -0.74265; the fluorescence attenuation rate of microvessels was negatively correlated with the ratio of DiIC in 12 / DiIC 18 The ratio of DiIC in 12 showed a negative correlation (DiIC 18 On the contrary), and the Pearson coefficient was 0.63833; the porosity of fluorescent microvessels was positively correlated with the ratio of DiIC in 12 / DiIC 18 The ratio of DiIC in 12 showed a positive correlation (DiIC 18 On the contrary), and the Pearson coefficient was 0.67964. Usually, when the absolute value of the Pearson coefficient is between 0.8 - 1.0, it indicates a very strong correlation; between 0.6 - 0.8, it indicates a strong correlation; between 0.4 - 0.6, it indicates a medium correlation; between 0.2 - 0.4, it indicates a weak correlation; and between 0.0 - 0.2, it indicates a very weak correlation or no correlation. Therefore, in the DiI staining of coronary microvessels in SD rats, the ratio of DiIC12 in DiIC12 / DiIC18 was strongly correlated with the percentage of bright fluorescent spots, the total length of fluorescent microvessels, the fluorescence attenuation rate of microvessels, and the porosity of fluorescent microvessels, and was moderately correlated with the percentage of weak fluorescence area.

[0093] Another embodiment of the present invention also provides a method for evaluating microvascular perfusion staining, comprising the following steps:

[0094] S1, preparing a mixed staining agent of DiIC 12 and DiIC 18 with different concentration gradients;

[0095] S2, injecting the prepared mixed staining agent into the ventricle of the living heart and preparing heart sections to obtain multiple sample groups, wherein each sample group uses a mixed staining agent with the same concentration gradient, and each sample group includes multiple heart sections;

[0096] S3, obtaining the Z-stack images of each heart section and performing three-dimensional image construction to obtain a three-dimensional image;

[0097] S4, respectively detecting the three-dimensional images of each heart section to obtain the measured values of each evaluation index in each heart section and performing standardized processing;

[0098] S5. Calculate the proportion of each of the sample groups under each of the evaluation metrics based on the measured values after standardization processing, and calculate the weight coefficients of each of the evaluation metrics.

[0099] S6. Calculate the comprehensive score of each of the sample groups based on the proportion of each of the cardiac sections under each of the evaluation metrics and the weight coefficients of each of the evaluation metrics.

[0100] In this embodiment, DiIC with gradient ratios of 100:0, 60:40, 90:10, 80:20, 40:60, 20:80, and 0:100 12 and DiIC 18 gradient ratio of the mixed stain. Inject the gradient ratio of DiIC12 and DiIC18 stains into the left ventricle of the heart, fix in 10% formaldehyde solution for 24 h, dehydrate in 30% sucrose solution for 12 h, embed in OCT, observe after 3D reconstruction after cryosectioning, measure the evaluation metrics, perform Pearson correlation analysis, and comprehensively evaluate the coronary microvascular perfusion staining effect by the entropy weight method.

[0101] Specific implementation: Prepare one sample group for each gradient ratio of the mixed stain, that is, there are 7 sample groups. Each sample group requires three living hearts, and 5 cardiac sections are prepared from each living heart, that is, one sample group has 15 cardiac sections. Perform 3D reconstruction for each cardiac section to generate a three-dimensional image, and collect the values of five evaluation metrics for the three-dimensional image. The five evaluation metrics are: percentage of bright fluorescent spots, percentage of weak fluorescence area, total length of fluorescent microvessels, fluorescence microvessel attenuation rate, and fluorescence microvessel porosity.

[0102] Perform standardization processing on the values of the five collected evaluation metrics. Data standardization refers to homogenizing heterogeneous metrics. For positive metrics: the total length of fluorescent microvessels is processed by the following formula (1) for standardization. For reverse metrics: the percentage of bright fluorescent spots, percentage of weak fluorescence area, fluorescence microvessel attenuation rate, and fluorescence microvessel porosity are processed by the following formula (2) for standardization.

[0103]

[0104] where Z ij is the value after standardization processing of the measured value of the j-th evaluation metric of the i-th cardiac section, xij is the measured value of the j-th evaluation metric of the i-th cardiac section, max(xj) is the maximum value of the measured values of each cardiac section under the j-th evaluation metric, and min(xj) is the minimum value of the measured values of each cardiac section under the j-th evaluation metric.

[0105] Calculate the proportion pij of each sample under each index to the index through the following formula (3).

[0106]

[0107] Among them, m is the total number of heart sections. There are 15 heart sections in each sample group. Therefore, m is 90 in this embodiment, and n is the total number of evaluation indexes, which is 5 in this embodiment. Calculate the mean value of the proportion values corresponding to the 15 heart sections in the same sample group under each evaluation index to obtain the proportion of the sample group under each evaluation index. The calculation results are shown in Table 1 for example.

[0108] Table 1 Proportion Pij of the i-th sample group in the j-th index to the index ( The number of heart sections in the sample group is 15)

[0109]

[0110] Calculate the entropy value ej of each index through the following formula (4). The results are shown in Table 2. Among them, M is the number of sample groups, n is the number of evaluation indexes, k>0, ln is the natural logarithm, and ej>0. The constant k in the formula is related to the number of sample groups M. Generally, k = 1 / ln(M), then 0≦e≦1;

[0111]

[0112] Calculate the information utility value gj of each index through the following formula (5). The results are shown in Table 2.

[0113]

[0114] Calculate the weight coefficient Wj of each index through the following formula (6). The results are shown in Table 2.

[0115]

[0116] Table 2 Evaluation of DiIC 12 / DiIC 18 Ratio of the information entropy value ej, information utility value gj, and weight coefficient wj of the microvascular DiI perfusion effect index ( The number of heart sections in the sample group is 15)

[0117]

[0118] Calculate DiIC through the following formula (7) 12 and DiIC 18 The comprehensive scores Si of the gradient ratios 100:0, 90:10, 80:20, 60:40, 40:60, 20:80, and 0:100 are shown in Table 3;

[0119]

[0120] Table 3 DiIC 12 / DiIC 18 Ratio's comprehensive score Si and ranking results of microvascular perfusion staining effect ( The number of heart slices in the sample group is 15)

[0121]

[0122] In this embodiment, the entropy weight method is used to evaluate DiIC 12 / DiIC 18 Ratio's results of information entropy value ej, information utility value gj and weight coefficient wj of microvascular DiI perfusion effect indicators show that the weight coefficients of each indicator from large to small are: fluorescence microvascular porosity 0.2878 ± 0.0548, percentage of bright fluorescence spots (%) 0.2758 ± 0.0704, percentage of weak fluorescence area (%) 0.2540 ± 0.0700, microvascular fluorescence decay rate (ΔIntDen / Time) 0.2410 ± 0.0657 and total length of fluorescence microvessels × 104 (μm) 0.0377 ± 0.0156; DiIC 12 / DiIC 18 The group with the best comprehensive score Si of coronary microvascular DiI perfusion effect for the DiIC 12 / DiIC 18 mixed stain is the DiIC with a concentration ratio of 80:20, Si = 0.7602 ± 0.1081, followed by those with concentration ratios of 60:40, 90:10, 100:0, 40:60, 20:80 and 0:100 from good to poor; DiIC 12 / DiIC 18 mixed stains.

[0123] Furthermore, before standardizing the measured values of each evaluation indicator in each of the obtained heart slices, it further includes:

[0124] Removing null values and outliers from the obtained measured values.

[0125] Among them, the null value processing is: if the indicator value contains a null value, the entire data is removed;

[0126] The outlier processing is: calculate the mean and standard deviation of the data under each indicator respectively. If the data is greater than the mean + 3 * standard deviation or less than the mean - 3 * standard deviation, the entire data is removed.

[0127] Since there are no null values in each index of this method, null value processing is not required; and the data under each index are all within the range of plus or minus 3 times the mean, so outlier processing is not required either.

[0128] It can be known from the experiments that the comprehensive score of the staining effect reaches above 0.7, the microvascular staining is uniform, the fluorescence intensity is moderate, there are few high-brightness red spots, and the fluorescence quenching rate is slow, and the structure of the microvessels can be observed better. Therefore, using DiIC 12 and DiIC 18 in a concentration ratio of 90 - 60:10 - 40 for the mixed staining agent can obtain good staining effects.

[0129] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0130] The above-described embodiments merely represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the appended claims.

Claims

1. A mixed staining agent for microvascular perfusion staining, characterized in that, The mixed staining agent includes DiIC 12 and DiIC 18 , and the concentration ratio of DiIC 12 to DiIC 18 is 90 to 60:10 to 40.

2. The mixed staining agent according to claim 1, characterized in that, DiIC 12 The concentration ratio with DiIC 18 is 80:

20.

3. A method for evaluating microvascular perfusion staining, characterized in that, comprising: Prepare DiIC with different concentration gradients 12 and DiIC 18 mixed staining agent; Obtaining a plurality of sample groups, wherein each of the sample groups includes a plurality of cardiac sections, and the cardiac sections of each sample group are stained with the same concentration gradient of the mixed staining agent; Obtaining Z-stack images of each of the cardiac sections and performing three-dimensional image construction to obtain a three-dimensional image; Detecting the three-dimensional images of each of the cardiac sections respectively to obtain the measured values of each evaluation index in each of the cardiac sections and performing standardization processing; Calculating the proportion of each sample group under each evaluation index according to the measured values after standardization processing, and calculating the weight coefficients of each evaluation index; Calculating the comprehensive score of each sample group according to the proportion of each cardiac section under each evaluation index and the weight coefficients of each evaluation index.

4. The method for evaluating microvascular perfusion staining according to claim 3, characterized in that, The evaluation indexes include: Percentage of bright fluorescent spots, percentage of weak fluorescence area, total length of fluorescent microvessels, fluorescence attenuation rate of microvessels, and fluorescence microvascular porosity.

5. The method for evaluating microvascular perfusion staining according to claim 3, characterized in that, The step of calculating the proportion of each sample group under each evaluation index according to the measured values after standardization processing includes: Calculating the proportion of each section under each evaluation index according to the measured values of the evaluation indexes of each cardiac section to obtain the proportion value corresponding to each cardiac section, wherein the calculation formula of the proportion value corresponding to the cardiac section is: Z ij is the value after standardization of the measured value of the j-th evaluation index of the i-th heart slice; Calculating the mean value of the proportion values corresponding to each section in the same section group under each evaluation index to obtain the proportion of the sample group under each evaluation index.

6. The method for evaluating microvascular perfusion staining according to claim 3, characterized in that, The step of calculating the weight coefficients of each evaluation index includes: Calculating the entropy value of each index according to the proportion of each sample group under each evaluation index, and the calculation formula of the entropy value ej is Among them, ej is the entropy value, k is a constant, Pij is the proportion of the i-th sample group under the j-th evaluation index, M is the number of sample groups, and n is the number of evaluation indexes; Calculating the effective information value of each evaluation index according to the entropy value of each evaluation index, and the calculation formula of the effective information value gj is Among them, Calculating the weight coefficient of each evaluation index according to the effective information value of each evaluation index, and the calculation formula of the weight coefficient Wj is 7. The method for evaluating microvascular perfusion staining according to claim 3, characterized in that, The formula for calculating the comprehensive score si of each sample group is: Among them, \(W_j\) is the weight coefficient of the \(j\)-th evaluation index, \(p_{ij}\) is the proportion of the \(i\)-th sample group under the \(j\)-th evaluation index, \(M\) is the number of sample groups, and \(n\) is the number of evaluation indexes.

Images