Quantitative Assessment of Capillary Permeability in Deep Intracardiac Capillaries Using Fluorescent Dextran

Materials and reagents

Biological materials

1. Wild-type female mouse (Japan SLC, Inc., Shizuoka, Japan, C57BL/6NSlc)

Notes:

1. Mice were housed in separate cages, with a maximum of 5 mice per cage, in a specific-pathogen-free, temperature-controlled vivarium. They were maintained under a 12/12 h light/dark cycle with ad libitum access to food and water. The ambient room temperature was maintained at 22 ± 2 °C, and humidity was controlled at 55% ± 5%.

2. In this study, 6–7-month-old female mice (body weight: 20–25 g), whose body size had nearly reached maturity, were used as young mice, while 24-month-old female mice (body weight: 30–40 g) were used as aged mice.

3. C57BL/6NCrSlc strain (derived from NIH/Charles River, often referred to as B6N) was introduced to Japan SLC, Inc. from the Institute of Medical Science at the University of Tokyo in 1975.

4. All animal experiments were approved by the Institutional Animal Care and Use Committee at Tokyo Metropolitan Institute for Geriatrics and Gerontology (No. 20010 and 23016) and strictly adhered to the guidelines of Tokyo Metropolitan Institute for Geriatrics and Gerontology for animal experiments.

Reagents

1. Dulbecco’s phosphate-buffered saline (DPBS) (Fujifilm Wako Pure Chemical Co., catalog number: 045-29795)

2. Isoflurane (VIATRIS, catalog number: 901036504)

3. Domitor (Zenoaq, catalog number: AHE1)

4. Mitazolam (Sandoz K.K., catalog number: 614243022)

5. Vetorphale (Meiji Animal Health Co. Ltd., catalog number: VETLF5)

6. Tetramethylrhodamine (TRITC)-conjugated dextran 40 (average weight 40,000) (TdB Labs AB, catalog number: TD40)

7. Tragacanth gum, powder (FUJIFILM Wako Pure Chemical Co., catalog number: 200-2245)

8. Saline (Otsuka Pharmaceutical Factory Inc, catalog number: 3311401A2018)

9. Tissue-Tek^® OCT compound (Sakura Finetek Japan Co., Ltd., catalog number: 4560133241825)

10. Isopentane (2-methylbutane) (FUJIFILM Wako Pure Chemical Co., Japan, catalog number: 166-00615)

Solutions

1. Three-drug anesthetic mixture (MMB) (see Recipes)

2. TRITC-conjugated dextran 40 solution (see Recipes)

3. Tragacanth gum solution (see Recipes)

Recipes

1. Three-drug anesthetic mixture (MMB)

Note: Add 0.75 mL of Domitor, 0.8 mL of Midazolam, and 1 mL of Vetorphale to 7.45 mL of saline to make 10 mL of MMB. The administrative volume of MMB is 100 μL per 10 g of body weight.

2. TRITC-conjugated dextran 40 solution

Note:

1. Store TRITC-conjugated dextran at -20 °C. Avoid repeated freeze–thaw cycles.

2. The administrative volume of the TRITC-conjugated dextran 40 solution is 100 µL per 10 g of body weight.

3. Tragacanth gum solution

Note: Gradually add saline solution to the gum while mixing with a medicine spoon. Once it reaches a suitable softness, pack it into the syringe. Suitable softness means it should be firm enough to be squeezed out when the syringe is pressed.

Laboratory supplies

1. Microtube 1.5 mL (e.g., BM Equipment Co. Ltd., catalog number: NT-175)

2. Microtube 0.6 mL (e.g., Corning Inc., catalog number: MCT-060-C-S)

3. 1 mL disposable syringe (e.g., Terumo Co., catalog number: SS-01T10)

4. 5 mL disposable syringe (e.g., Terumo Co., catalog number: SS-05SZ)

5. 27 G (or 26 G) disposable needle (e.g., Terumo Co., catalog number: NN-2719S)

6. 30 G disposable needle (e.g., NIPRO Co., catalog number: 01-134)

7. Cork sheet, 3 mm (e.g., Green Stuff World SL, catalog number: 8436574509571ES)

8. White micro slide glass (Matsunami Glass Ind. Ltd., catalog number: FF-001)

9. Dry ice block (2 kg)

Equipment

1. Laboratory Animal Anesthesia System (Shinano Manufacturing Co., LTD., model: SN-487-OT Air)

2. Scissor (e.g., Kenis, Ltd., catalog number: 3-154-0262)

3. Tweezer (e.g., Kenis, Ltd., catalog number: 3-319-0281)

4. Ring tweezer (e.g., Kenis, Ltd., catalog number: 3-345-0555)

5. -80 °C freezer (e.g., Panasonic, catalog number: MDF-U33V-PJ)

6. Cryostat (e.g., Leica Microsystems, model: CM3050 S)

7. Upright fluorescence microscope (e.g., Leica Microsystems, model: DM6 B)

8. Microscope camera (e.g., Leica Microsystems, model: DFC9000 GT sCMOS camera)

Software and datasets

1. Leica Application Suite X (LAS X) (Leica, version 3.7.4.23463)

2. ImageJ (NIH, Version 1.54 g)

Note: To perform quantitative processing on multiple image files simultaneously, we create and execute the ImageJ macro shown in Figure S1.

3. GraphPad Prism (GraphPad Software, version 9.5.1)

Procedure

A. Animal experiments (Figure 1)

1. Anesthesia (Figure 1A)

a. Supply 4%–5% isoflurane to the anesthesia chamber.

b. Place the mouse in the anesthesia chamber (approximately 3 min).

c. Confirm that the mouse has lost consciousness and that its breathing is stable (approximately 5 min).

d. Remove the mouse from the anesthesia chamber.

e. Administer MMB intraperitoneally to the mouse with a 26 G or 27 G needle syringe at 0.1 mL per 10 g body weight.

f. After confirming that the mice are sufficiently anesthetized (approximately 5 min after MMB administration), proceed to the next step.

2. Dextran administration and isolation of the heart (Figure 1B)

a. Lay the mouse on its side (with the caudal vein facing upward).

b. Using a 30 G needle syringe, administer fluorescent dextran at 0.1 mL per 10 g of body weight (injection rate: 30 μL/s) intravenously into the mouse via the tail vein. Inject negative-control mice with an equivalent volume of DPBS (or saline).

c. Place the mouse in a prone position.

d. Nine minutes later, fix the mouse in a supine position.

e. Spray ethanol onto the mouse’s abdomen.

f. Lift the abdomen with tweezers, cut the skin with scissors, and make an incision from the abdomen to the throat.

g. Further incise the peritoneum and diaphragm to expose the internal organs.

h. Cut the central ribs to expose the heart.

i. Ten minutes after administration, remove the heart by inserting scissors between the great artery and vein, the pulmonary artery and vein, and the heart tissue. At this time, perfusion is not performed.

Note: When tissue is perfused, fluorescent substances are washed out, making it difficult to detect fluorescence during subsequent observations.

j. Remove the heart with ring forceps.

k. Quickly freeze the removed heart.

Note: Steps A2i–j must be completed within 30 s.

Figure 1. Animal experiments. (A) Anesthesia. Place the mouse in the anesthesia box and anesthetize it with isoflurane. Once anesthesia takes effect and the mouse stops moving, administer MMB intraperitoneally (0.1 mL per 10 g of body weight). (B) Dextran administration and isolation of the heart. Mice are placed on their side, and TRITC-conjugated dextran (TD 40) is administered intravenously with a 30 G syringe (0.1 mL per 10 g of body weight). Negative-control mice are injected with an equivalent volume of DPBS (or saline). After administration, the mice are kept on their stomachs. After 9 min, they are placed in a supine position. 10 min after administration, the abdomen is opened, and the heart is quickly removed (within 30 s).

3. Preparation of cardiac tissue slices (Figure 2)

Figure 2. Preparation of cardiac tissue slices for permeability assessment. (A) Using a 5 mL syringe, apply the Tragacanth gum solution to a 1 cm square piece of cork board. The extracted heart is quickly fixed to tragacanth gum with the apex facing upward and frozen in isopentane pre-cooled with dry ice (freezing should be completed within 1 min of heart extraction). Sections should be prepared from the frozen tissue using a cryostat within 6 months. (B) Place a small amount of fresh OCT compound on the chilled sample stage in the cryostat, then place the frozen block on top to freeze it in place. Trim the frozen tissue block until the right and left ventricles are visible within the block, then section at 10 μm and collect the sections on a slide. Up to three sections can be included on each slide. The slides are dried thoroughly at room temperature (at least 2 h). Three sections are selected. The slides are observed for fluorescence under the upright microscope within 24 h (no later than 3 days) of preparation.

a. Using a 5 mL syringe, apply the Tragacanth gum solution to a 1 cm square piece of cork board.

b. Place a small stainless steel bowl in the bottom of a Styrofoam box and fill the space around the bowl with dry ice pellets. Place some pellets in the bowl and slowly add isopentane/2-methylbutane.

c. Steps A3a–b (Figure 2A) are performed between dextran administration and cardiac tissue extraction (9 minutes; Figure 1B).

d. Quickly secure the extracted heart tissue to a corkboard with the apex facing upward (within 10 s of excision).

e. Place the heart tissue on the corkboard in the cooled isopentane for 1 min using a forceps. Transfer the heart to a cryovial cooled on dry ice.

Note: To prevent further diffusion of the tracer, even ex vivo, steps 3Ac and d must be completed within 30 s.

f. The heart cryoblocks should be stored in a plastic container at -80 °C. It is advisable to protect them from light using aluminum foil or a similar material.

Note: Store for up to 6 months. It is preferable to prepare and observe sections within one month.

g. Place a small amount of fresh OCT compound on the chilled sample stage (set temperature: -25 °C) in the cryostat (set temperature: -25 °C); then, place the frozen block on top to freeze it in place (Figure 2B).

h. Trim the frozen tissue block until the right and left ventricles are visible within the block; then, section at 10 μm and collect the sections on a slide. Up to three sections can be included on each slide.

i. Dry the slides thoroughly in the dark at room temperature (for at least 2 h).

j. Select three non-consecutive sections.

k. Observe the slide as is under a fluorescence microscope.

Notes:

1. Do not place any mounting medium or cover glass on the slide. This is to prevent the fluorescent substance that has leaked from the capillaries from spreading and flowing out across the entire section.

2. The entire process, from section preparation and air-drying to observation, should be completed within 24 h (no later than 3 days).

B. Data analysis for the permeability of cardiac microvascular vessels

B1. Data acquisition using fluorescence microscopy (Figure S2)

1. Launch the fluorescence microscope (DM6B).

2. Launch the image analysis software, LasX.

3. Settings (DM6B): Objective lens: 5×; excitation filter and absorption filter: select a filter equivalent to TRITC fluorescence.

4. Observation: Obtain an image of the entire section (the entire heart tissue).

5. Outline the section image (heart tissue) to be scanned with a line.

6. Switch the objective lens to 40×.

7. Select 3–4 areas in the tissue image to optimize focus.

8. Image acquisition (Microscope camera: DFC9000 GT sCMOS camera): Exposure: 100 ms; intensity: 17%.

9. Scan an image.

10. Save it with a file name.

11. Load the saved file.

12. Extract 20 regions of interest (150 μm × 150 μm) per section from the left ventricular wall using the cropping function.

13. Save images as TIFF (922 × 922 pixels).

B2. Calculation of background (Figure 3A, Figure S3)

1. Here, the images of the negative control [DPBS (or saline) injection group] are used.

2. Launch the image analysis software, ImageJ.

3. Load the files in the saved folder.

4. Open the image(s) and convert the color image (RGB) to 16-bit grayscale mode (tonal range: 0-65535).

5. Perform image processing using the ImageJ macro in Figure S1.

6. After running this macro, the following values are displayed in the Excel file for each section.

7. To calculate the background, the “MAX intensity” value in Table 1 is used.

8. Set the median of the 60 N “MAX intensity” values as the background.

Figure 3. Data analysis of permeability. (A) Calculation of background. Here, the images of the negative control [DPBS (or saline) injection group] are used. Twenty 150 μm × 150 μm regions of interest per section were extracted from the left ventricular wall, where capillaries were observed parallel to the myocardial layer, using LAS X software and saved as TIFF. Then, open the image(s) in 16-bit grayscale mode in ImageJ. Three sections per mouse, across N mice, yielded a total of 60N images. The median maximum fluorescence intensity value obtained at 60N locations is used as the background for the entire left ventricular region in each age group of mice. (B) Calculation of leakage area from the microvascular vessel in the left ventricular wall. Here, the images of the TRITC-conjugated dextran-injection group are used. Twenty 150 μm × 150 μm regions of interest per section were extracted from the left ventricular wall, where capillaries were observed parallel to the myocardial layer, using LAS X software and saved as TIFF. Next, open the image(s) in grayscale mode in ImageJ. The leakage fluorescence area was calculated as the sum of pixels with fluorescence intensity values between the background fluorescence intensity (BG) and BG × a (2.2 for the mouse left ventricular wall; see Validation of protocol section), derived from the fluorescence intensity image. The ratio relative to the total area of this region was calculated.

Table 1. Raw intensity data per region for calculating background

Three sections per mouse, for N mice results in a total of 60 N images.

Note: For initial screening, we recommend conducting experiments with N ≥ 4 per group.

B3. Calculation of leakage area (Figure 3B, Figure S3)

1. Here, the images of the TRITC-conjugated dextran-injection group are used.

2. Launch the image analysis software, ImageJ.

Note: Converting from LASX to ImageJ results in a 150 × 150 (=22,500) μm² frame area of 22,461.544 μm².

3. Load the files in the saved folder.

4. Open the image(s) and convert the color image (RGB) to 16-bit grayscale mode (tonal range: 0-65535).

5. Perform image processing using the ImageJ macro in Figure S1.

6. After running this macro, the following values are displayed in the Excel file for each section.

7. To calculate the leakage area, the “Area” value in Table 2 is used.

Note: For initial screening, we recommend a minimum of N = 4 per group, with power calculation shown in Validation.

Table 2. Raw data of the leakage area per region

Three sections per mouse, for N mice, results in a total of 60 N images.

Note: For initial screening to determine the a-value, we recommend conducting experiments with N ≥ 4 per group.

8. Analyze the values of the 60 N “area” values.

C. Quantification and statistical analysis

1. All statistical analyses were performed using GraphPad Prism software.

2. All data are represented as mean values ± standard error of the mean (SEM).

3. For comparison between two groups, a two-tailed Student’s t-test for unpaired data was used. For more than two groups, data were evaluated by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. Multiple and independent experiments are performed to validate the reproducibility of findings. For initial screening, we recommend a minimum of N = 4 per group, with power calculation shown in Validation.

Validation of protocol

This protocol has been used and validated in the following research article:

1. Nakamura M et al. [23] Assessment of permeability in deep tissue capillaries using a new method reflects the nutrient supply status in a healthy heart. Regen Ther.

In the aforementioned paper [23], in addition to dextran, GSL-IB4 was administered intravenously to precisely localize blood vessels (Figure 4A), after which capillary permeability was assessed. This method requires two intravenous administrations and uses two fluorescent dyes. Consequently, it poses challenges in experiments such as immunofluorescence staining with multiple antibodies to investigate permeability mechanisms, as it requires selecting secondary antibodies with non-overlapping emission wavelengths, thereby limiting analysis. Therefore, we modified the protocol to evaluate permeability without administering GSL-IB4 (Figure 4B). We focused on the a-value (Figure 3B).

In the case of administration in combination with dextran and GSL-IB4 (Figure 4A), the a-values used to calculate the upper threshold for the extravasation area were 1.8, 2.0, or 2.2 [23]. Here, we optimized the a-value to accurately extract the intravascular dextran area in the absence of GSL-IB4.

Figure 4. Modification of the protocol to evaluate permeability without administering GSL-IB4. Previously reported (A) [23] and modified (B) animal experimental protocols for permeability assessment. (C) The influence of the a-value on the brightness of fluorescent molecules remaining in blood vessels, due to differences in protocols. Fluorescence intensity values of fluorescent dextran remaining within blood vessels in each section area were calculated, and the most frequent intensity value was used as the representative intensity for that area. After background correction, all data (N = 20 images per section; 3–7 sections per mouse; 3 mice per group; Table S1) were presented as a beeswarm plot and as mean values ± standard error of the mean (SEM). P values were determined using a two-tailed Student’s t-test for unpaired data. P < 0.05 was considered significant. At 2.2, no effect of intensity or GSL-IB4 administration was observed across generations. ns indicates not significant; *P < 0.05; ****P < 0.0001.

We examined whether the thresholds determined during simultaneous administration of dextran and GSL-IB4 (“BG × 1.8, 2.0, or 2.2”) could also be applied when dextran was administered alone. Here, we investigated whether vessels could be appropriately recognized by measuring the intensity of dextran within vessels (BG × “a” or higher in Figure 3B) in the dextran/GSL-IB4 co-administration group and the dextran monotherapy group. The mean intensity per pixel of the extracted images was calculated, and the most frequent value (the modal gray value) was used as the intravascular dextran intensity for each region (Figure S1 and S4). In young and aged mice, intravascular dextran brightness was compared between the dextran/GSL-IB4 co-administration group and the dextran monotherapy group (Figure 4C, Table S1). Statistical analysis using Prism9 revealed no significant differences between the young mice group when the thresholds were set to “BG × 1.8” and “BG × 2.2.” Even in aged mice, no significant differences were observed when the thresholds were set to “BG × 2.0” and “BG × 2.2.” Furthermore, the intensity of the brightness (vertical axis in Figure 4C) within blood vessels was nearly the same for both young and aged mice when a = 2.2 was set. These results demonstrate that when GSL-IB4 is not administered, the intravascular dextran brightness is equivalent in the dextran/GSL-IB4 co-administration group and the dextran-only administration group when the threshold is set to “BG × 2.2.”

Based on these results, we decided to use “BG × 2.2” as the threshold for distinguishing between intravascular and leaked dextran regions when measuring vascular permeability under conditions where dextran alone is administered.

General notes and troubleshooting

General notes

1. In the mouse strain we used, fluorescence intensity in the blood decreased over time after administration of the fluorescent substance, remaining nearly constant from 20 min onward (up to 40 min). Considering these blood dynamics and tissue detection sensitivity, we removed the heart 10 min after administration and detected fluorescence. The appropriate processing time may vary by mouse strain and the organ under observation. We suggest that users perform time-course sampling to determine the optimal time for collection.

2. When selecting tissue areas for analysis, it is best to choose regions with a consistent histological appearance across sections and with a sufficient number of areas available for analysis.

3. When evaluating capillary permeability, it is necessary to determine the a-value to distinguish between residual fluorescence and leakage areas. This value may vary depending on the microscope used for observation and the imaging conditions. In this study, for the heart, the a-value ranges from 1.8 to 2.2, but there may be differences depending on the organ, so it is necessary to determine it for each individual organ. The amount of interstitial fluid in healthy individuals is not considered to be large; so, in experiments, it is necessary to perform negative controls under the same conditions at all stages leading up to observation and to carefully observe differences in fluorescence intensity.

Troubleshooting

Supplementary information

The following supporting information can be downloaded here:

1. Figure S1. ImageJ macro for performing multi-image analysis.

2. Figure S2. Fluorescence microscope imaging conditions and data acquisition.

3. Figure S3. Acquired images.

4. Figure S4. Effect of GSL administration on the brightness of intravascular fluorescent dextran.

5. Table S1. Fluorescence intensity in the capillary of the left ventricular wall.

Acknowledgments

We would like to thank Takano R and Daisaku A for their helpful suggestions. We are grateful for the support provided by the TMIG Animal Facility. M.N., Y. Y-K., and K.S. performed the experiments, analyzed the data, and prepared the manuscript. Y.I. provided discussion and suggestions for data analysis and edited the manuscript. M.T. designed the study and wrote the manuscript. This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (grant numbers 21K19754 and 26K02492 to M.T.) and a research grant from Bristol-Myers Squibb KK to M.T. (grant number 75393673). The protocol was used in Nakamura et al. (2025) (DOI: https://doi.org/10.1016/j.reth.2025.10.005) [23].

Competing interests

The authors declare no conflicts of interest.

Ethical considerations

All animal experiments were approved by the Institutional Animal Care and Use Committee at Tokyo Metropolitan Institute for Geriatrics and Gerontology (No. 20010 and 23016) and strictly adhered to the guidelines of Tokyo Metropolitan Institute for Geriatrics and Gerontology for animal experiments. Mice were housed in separate cages at a maximum of 5 mice per cage in a specific-pathogen-free, temperature-controlled vivarium under a 12/12 h light/dark cycle with ad libitum access to food and water. Ambient room temperature was regulated at 22 ± 2 °C, and humidity was controlled at 55% ± 5%. In this study, young (6–7 months old) and aged (24–26 months old) mice were used.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used DeepL and Grammarly to write English. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.

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