# 3D Imaging and Quantitative Characterization of Mouse Capillary Coronary Network Architecture

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## Abstract

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## Simple Summary

## Abstract

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Animal Model

#### 2.2. Arterial Pressure Measurements

^{®}High Throughput System (Kent Scientific corporation, CT, USA). This non-invasive method is based on the analysis of the caudal blood flow by a sensor cuff after the complete occlusion of the blood flow and its progressive recovery controlled by an occlusion cuff placed at the basis of the tail. Each mouse was placed in an animal holder on a warming support in order to vasodilate the caudal vasculature. The tail external temperature was monitored and the recording began when it reached 32 °C. Measurements were realized on 15 consecutive cycles over 15 min, and the mean SAP, DAP, and MAP were calculated on these 15 cycles.

#### 2.3. Mice Preparation

^{®}1 mg/mL, Vector Labs, Eurobio Scientific, Les Ulis, France) with an insulin syringe. Ten minutes after the injection, an intraperitoneal 100 µL injection of isosorbide dinitrate (Risordan

^{®}10 mg/10mL, Medisol, Lyon, France) was made with 25G needle to dilate the vessels. The mouse was then euthanized by an intraperitoneal injection of 300 µL sodium pentobarbital (Exagon

^{®}à 400 mg/mL, Axience, Pantin, France) diluted in physiological saline solution. After death, a sternotomy was carried out to catheterize the left ventricle. A perfusion of physiological solution at 80 mm Hg pressure for 3 min was done to remove the blood from the vasculature. A second perfusion of 4% formalin (10% neutral buffered formalin, DiaPath, Martinengo, Italy) was done to fix the tissues. The heart was delicately removed and placed in paraformalin overnight at 4 °C. A negative control was done in similar conditions, but without lectin injection.

#### 2.4. Optical Clearing

^{®}, VWR Chemicals, Fontenay-sous-Bois, France) at room temperature. Permeabilization and lipid removal were then performed in a 2/3 dichloromethane (Dichloromethane, anhydro ≥ 99.8%, with 40–150 ppm amylene, Sigma-Aldrich, Lyon, France) and 1/3 methanol solution for 3 h at room temperature. The remaining methanol was removed by two 15 min baths in pure dichloromethane solutions at room temperature. Last, RI matching was done by leaving the sample in a dibenzyl ether solution (Benzyl ether 98%, Sigma-Aldrich) for a few hours at room temperature, then kept at 4 °C until imaging.

#### 2.5. Shrinkage Measurement

#### 2.6. Image Acquisition

#### 2.6.1. Light Sheet Microscopy

^{3}parallelepipedic sections in the left ventricle (LV), the septum (S), and the right ventricle (RV) of the heart. The system spatial resolution was 1 µm (x, y) and 4 µm (z). Step size used was 2 µm. Voxel dimensions were 0.5 µm (x, y) and 2 µm (z).

#### 2.6.2. Confocal Microscopy

^{3}parallelepipedic sections. The system spatial resolution was 279 nm (x, y) and 1284 nm (z). The step size used was 630 nm. Voxel dimensions were 380 nm (x, y) and 630 nm (z).

#### 2.7. Image Processing

#### 2.7.1. Segmentations

#### 2.7.2. Filtering

#### 2.7.3. Skeletonization and Distance Mapping

#### 2.8. Data Analysis

#### 2.8.1. Cardiac Volumes and Vascular Density

^{3}, was calculated by multiplying the number of white voxels, corresponding to the volume object in voxels, by the volume of one voxel in mm

^{3}.

#### 2.8.2. Fractal Dimension

#### 2.8.3. Normalized Number of Segments

^{3}).

#### 2.8.4. Normalized Total Capillary Length

^{-3}, was calculated by summing up all segment lengths converted in meter, normalized to the total cardiac tissue volume in mm

^{3}.

#### 2.8.5. Normalized Number and Percentage of Nodes

^{3}). The percentage of nodes was calculated by dividing the number of nodes by the number of segments. The node/segments ratio depends on the number of segments connected at each node, and can hence be used as an index of the connectivity of the network.

#### 2.8.6. Segment Diameter

#### 2.8.7. Tortuosity

#### 2.9. Statistical Analysis

^{®}software (Prism 9.0.1 version), (San Diego, CA, USA).

#### 2.9.1. Global Parameters

#### 2.9.2. Topological Parameters

## 3. Results

#### 3.1. Cardiovascular Parameters

#### 3.2. Shrinkage

#### 3.3. Global Parameters

#### 3.3.1. Vascular Density

#### 3.3.2. Fractal Dimension

#### 3.3.3. Normalized Number of Segments

#### 3.3.4. Total Capillary Length

#### 3.3.5. Number and Percentage of Nodes

#### 3.4. Topological Parameters

#### 3.4.1. Segment Length

^{−1}). From the rate constant K the length constant λ was defined as 1/K, in µm, and used as an index of length distribution. The shorter λ is, the higher the proportion of sort segments is. Curves are presented in Figure 3a and λ values are given in Table 4. The median values are 12.34, 12.88, and 12.01 for LV, RV, and S, respectively. Statistical comparisons showed no significant differences for λ between RV, S, and LV.

#### 3.4.2. Diameter

#### 3.4.3. Tortuosity

^{1}). From the rate constant K the tortuosity constant τ was defined as 1/K, and used as an index of tortuosity distribution. Curves are presented in Figure 3c and τ values are given in Table 4. τ was below 1, indicating that the capillaries were almost straight. The median values are 1.22, 1.33, and 1.24 for LV, RV, and S, respectively. Statistical comparisons showed non significant differences between RV, S, and LV.

#### 3.5. Confocal Microscopy

## 4. Discussion

#### 4.1. 3D Imaging and Image Processing

#### 4.2. Global and Archhitectural Parameters

^{3}of cardiac tissue, with an average length around 15 µm, representing nearly 30% of the cardiac volume, and a total capillary length around 10m/mm

^{3}of cardiac tissue. To our knowledge, there are no previously published 3D data on the coronary capillary volume and number density. The coronary capillary number density has been calculated in 2D with histological images, and was estimated at 2500 capillaries/mm² [5,19]. Considering an average capillary length of 15 µm, this corresponds to 170,000 capillaries/mm

^{3}, a value lower but in the same range of our own values. The total capillary length/mm

^{3}of cardiac tissue has already been calculated on rats by 2D measurements and mathematical 3D reconstruction at 3.5 m/mm

^{3}[20]. It has also been calculated on mouse cleared brain with values near to 1m/mm

^{3}of health cerebral tissue [7]. More recently, total vessel length has been calculated on mouse heart by 3D imaging and found to be 3m/mm

^{3}[18]. This value is consistent with our own, though a bit lower. The difference may be explained by differences in image resolution. Indeed, in these studies, voxel size was higher than in our study, and hence may have failed to identify small segments.

#### 4.3. Light Sheet Microscopy Versus Confocal Microscopy

## 5. Conclusions

## Supplementary Materials

## Author Contributions

## Funding

## Institutional Review Board Statement

## Informed Consent Statement

## Data Availability Statement

## Acknowledgments

## Conflicts of Interest

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**Figure 1.**Image processing. (

**a**,

**b**) Representative frequency distribution of pixel population of non-labelled (negative control) (

**a**) and lectin-labelled (

**b**) left ventricle after 3D imaging by light sheet microscopy. 1: background. 2: non-labelled cardiac tissue. 3: lectin-labelled capillary network. (

**c**), Gaussian fit of the subset of pixels corresponding to the cardiac tissue from curve (

**a**). (

**d**), Gaussian fit of the subset of pixels corresponding to the cardiac tissue from curve (

**b**). (

**e**), Representative segmented image of left ventricle capillary network. The image was obtained by overlaying the original capillaries image and the binarized image of the capillaries obtain after segmentation. (

**f**), Skeletonization and image mapping of the capillary network of (

**e**). Average radii of each segment are encoded in false colors from blue to red. The image was obtained by overlaying the skeleton and the distance map images of the capillary network.

**Figure 2.**Global parameters. Data obtained from the left ventricle (blue), septum (red) and right ventricle (green) of six hearts. Columns are means and error bars are standard deviations. Black dots are individual values. Data were compared by Kruskal–Wallis test with post-hoc Dunn’s test. ns = non significant. * p < 0.05. (

**a**). Volume capillary density, in %. (

**b**). Fractal dimension. (

**c**). Number of capillary segments per mm

^{3}of cardiac tissue. (

**d**). Total length of capillary segments (m) per mm

^{3}of cardiac tissue. (

**e**). Number of nodes per mm

^{3}of cardiac tissue. (

**f**). Percentage of nodes on the number of segments.

**Figure 3.**Architectural parameters. Data obtained from the left ventricle (blue), septum (red), and right ventricle (green) of six hearts. (

**a**). Relative frequency distribution of segment length. Data were fitted by one-phase exponential decay. (

**b**). Relative frequency distribution of segment diameter. Data were fitted by Gaussian equation. (

**c**). Relative frequency distribution of segment tortuosity. Data were fitted by one-phase exponential decay. Curves between LV, S, and RV were compared by F tests.

**Figure 4.**Confocal microscopy. (

**a**). Segmented image of left ventricle capillary network. (

**b**). Relative frequency distribution of segment length. Data were fitted by one-phase exponential decay. (

**c**). Relative frequency distribution of segment diameter. Data were fitted by Gaussian equation. (

**d**). Relative frequency distribution of segment tortuosity. Data were fitted by one-phase exponential decay.

**Table 1.**Cardiovascular parameters. Systolic (ASP), diastolic (DAP), and mean (MAP) vascular pressure in mmHg and cardiac rhythm in beat per minute (BPM) were measured on six mice.

SAP (Mean ± SD) | DAP (Mean ± SD) | MAP (Mean ± SD) | BPM (Mean ± SD) |
---|---|---|---|

123.1 ± 15 mm Hg | 89.7 ± 6.9 mm Hg | 100.5 ± 9.5 mm Hg | 303.6 ± 31 |

**Table 2.**Shrinkage. Length (L), width (W), thickness (T), and ellipsoid volume (V) reduction (in %) after optical clearing were measured on seven mouse hearts.

L (Mean ± SD) | W (Mean ± SD) | T (Mean ± SD) | Volume (Mean ± SD) |
---|---|---|---|

6.70 ± 4.2% | 10.7 ± 4.2% | 2.91 ± 8.2% | 19.18 ± 7.7% |

**Table 3.**Global parameters. Values were calculated from left ventricle (LV), septum (S), and right ventricle (RV) of six mouse hearts.

Parameter | LV | S | RV |
---|---|---|---|

Vascular density | 34.4 ± 11% | 18.9 ± 4.7% | 27.8 ± 11% |

Fractal dimension | 2.46 ± 0.05 | 2.32 ± 0.11 | 2.33 ± 0.07 |

Number of segments/mm^{3} of cardiac tissue | 615,784 ± 220,000 | 399,922 ± 230,000 | 387,457 ± 200,000 |

Total length/mm^{3} of cardiac tissue | 13.01 ± 6.1 m | 11.5 ± 9.2 m | 7.11 ± 2.7 m |

Number of nodes/mm^{3} of cardiac tissue | 289,878 ± 96,000 | 169,886 ± 92,000 | 190,392 ± 100,000 |

Number of nodes/number of segments | 47.56 ± 3.0% | 42.9 ± 2.9% | 48.72 ± 2.9% |

**Table 4.**Architectural parameters. Values were obtained by non-linear regression of frequency distribution of data from left ventricle (LV), septum (S), and right ventricle (RV) of six mouse hearts (see Figure 3). λ: length constant of one-phase exponential decay. μ and σ: mean and standard deviation of Gaussian distribution. τ: tortuosity constant of one-phase exponential decay.

Parameter | LV | S | RV |
---|---|---|---|

Length | |||

λ (mean ± SEM) | 16.9 ± 0.6 µm | 15.6 ± 0.6 µm | 17.9 ± 4.4 µm |

Diameter | |||

μ (mean ± SEM) | 4.81 ± 0.24 µm | 3.78 ± 0.51 µm | 5.12 ± 0.25 µm |

σ (mean ± SEM) | 2.52 ± 0.27 µm | 2.75 ± 0.44 µm | 2.17 ± 0.26 µm |

Tortuosity | |||

τ (mean ± SEM) | 0.32 ± 0.02 | 0.35 ± 0.02 | 0.35 ± 0.02 |

**Table 5.**Light sheet microscopy versus confocal microscopy. Global and architectural parameters obtained on light sheet microscopy (LSM, voxel size 0.5 × 0.5 × 2µm) and confocal microscopy (CM, voxel size 380 × 380 × 630 nm). The difference between LSM and CM (LSM-CM) values was calculated.

Parameters | LSM | CM | (LSM-CM) |
---|---|---|---|

Vascular density | 43.3% | 31.2% | 12.1% |

Fractal dimension | 2.49 | 2.53 | 0.04 |

Number of segments/mm^{3} of cardiac tissue | 794,032 | 4,297,330 | 3,503,298 |

Number of nodes/mm^{3} of cardiac tissue | 394,296 | 1,992,201 | 1,597,905 |

Number of nodes/number of segments | 49.7% | 46.4% | 3.3% |

Total length/mm^{3} of cardiac tissue | 17.7 m | 27.1 m | 9.4 m |

Length | |||

λ (mean ± SEM) | 28.9 ± 9.8 µm | 6.89 ± 1.0 µm | 22.01 µm |

Diameter | |||

μ (mean ± SEM) | 4.61 ± 0.68 µm | 3.05 ± 0.29 µm | 1.56 µm |

σ (mean ± SEM) | 2.25 ± 0.61 µm | 2.07 ± 0.26 µm | 0.18 µm |

Tortuosity | |||

τ (mean ± SEM) | 0.31 ± 0.07 | 0.27 ± 0.02 | 0.04 |

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Nicolas, N.; Roux, E.
3D Imaging and Quantitative Characterization of Mouse Capillary Coronary Network Architecture. *Biology* **2021**, *10*, 306.
https://doi.org/10.3390/biology10040306

**AMA Style**

Nicolas N, Roux E.
3D Imaging and Quantitative Characterization of Mouse Capillary Coronary Network Architecture. *Biology*. 2021; 10(4):306.
https://doi.org/10.3390/biology10040306

**Chicago/Turabian Style**

Nicolas, Nabil, and Etienne Roux.
2021. "3D Imaging and Quantitative Characterization of Mouse Capillary Coronary Network Architecture" *Biology* 10, no. 4: 306.
https://doi.org/10.3390/biology10040306