Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Summary

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm³.

Abstract

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm³ wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

Introduction

Our understanding of the developing placenta and its pathologies is, in large measure, limited to inferred spatial relationships between adjacent villi and contained capillaries derived from histological sections. In this study, we have addressed this issue by developing the means to generate three-dimensional (3D) renderings of human placental capillary networks that are suitable for analyses of capillary network features (e.g. branching, solidity). To do this we have combined immunofluorescent staining with two commercial tissue clearing products, Visikol-1 and Visikol-2 (referred to below as Solution-1 and Solution-2).

The human placenta is a vast complex of blood vessels located at the interface between the mother's intervillous blood and the developing fetus. Extending out from its insertion into the chorionic plate, the umbilical cord branches into a network of arteries and veins that ramify to cover the chorionic surface with an elaborate vascular network. Their ends then penetrate down into the interior or depth of the placental disk where they undergo several more branching generations and end in the terminal villi and their contained capillary networks, the site of the exchange of gases, nutrients, and metabolic waste between fetal and maternal blood.

Insults to the placental capillary network during development have lasting consequences for the health of the fetus, newborn infant and the emergent adult ^1,2,3. In view of pregnancy-related pathologies such as miscarriage, intrauterine growth restriction, pre-eclampsia and maternal diabetes ^4,5,6 there is a high value placed on developing methods of measurement and characterization of placental villous capillary networks. A major roadblock is that placental vascular networks encompass a broad range of scale. The surface vascular networks can be as great as 4-5 mm in diameter. The terminal villous capillaries are on the order of 10 -20 µm in diameter; the placenta contains over 300 km of blood vessels ⁷. At present, there are few easy to use and rapid techniques that can capture these extremes of vessel scale. To date, only a small number of villi can be rendered by microscopy. For example, Jirkovska et al. focused on the placental villus at term, combining confocal microscopy with serial optical sections at 1 µm intervals obtained from 120 µm thick sections; no data on the number of samples studied nor statistics were provided ⁸. Capillary structures were identified, and contours of villi and capillaries were hand-drawn, with tracings exported for image analysis. While the authors discuss the implications of their findings for the "growing villous vascular network", such conclusions are problematic when only "term" (36+ week gestational age) tissue is studied. Similarly, Mayo et al. and Pearce et al. relied on the tissue of the same age, for their simulations of blood flow and oxygen transfer, but their analyses were limited to only a few term, terminal villi ^9,10. Stereology has also been applied to the study of the structure of villous vessels. But again, the focus has generally been on later pregnancies delivering liveborn infants with one or more pregnancy complications ^11,12.

Until recently, confocal microscopy was limited to imaging to tissue depths of 100-200 µm because of absorption of the excitation and emission of the fluorescence by the overlying tissue ¹³ . Though tissue clearing and 3D histology have been widely described in the literature and there are numerous methods for tissue clearing many are unsuitable for use with tissues in general, as they irreversibly damage cellular morphology through the hyperhydration of proteins or removal of lipids. Therefore, it is not possible to validate that these results are indicative of the tissue itself and not artifacts from processing whereas our tissue clearing process is a reversible technique that is able to validate against traditional histology. Tissue clearing generally involves one of three major approaches: 1) uniform matching of the refractive index (RI) of tissue components by submersion in RI matching solutions, which removes the cumulative light scattering caused from lensing due to continuous fluctuation of low RI (cytosol) and high RI (protein/lipids) constituents; 2) removing lipid components by way of embedding into hydrogel and utilizing electrophoresis/diffusion to remove lipid components; 3) expansion/denaturation of protein structure to allow increased penetration of solvents to encourage uniformity of RI ¹³. While these approaches can render tissues transparent and allow for 3D representations of biomarkers to be generated, these 3D representations are of questionable clinical value as it is challenging to determine if these images are indicative of tissue properties or of the tissue clearing process. On the other hand, since our tissue clearing is reversible standard histological and/or immunohistochemistry can be applied to the same tissue to evaluate whether the alterations are clinically significant.

This study presents an analysis of 47 distal villous samples obtained from a total of 23 clinically normal and electively terminated pregnancies between 9-23 completed weeks' gestation and two full-term normal deliveries. Immunofluorescence labeling of trophoblast and endothelia has enabled a quantitative and automated analysis of changes in the villous vascular networks and their complexity.

With this protocol, we isolated and analyzed terminal villi and their capillary networks on a scale not possible previously. This approach, when applied to villous and capillary network development across gestation will identify those properties that are the foundation for the birth of a healthy child. When applied to studies of complicated pregnancies, it will also clarify when and how the placental pathologies modify the villous trees and the capillary networks they sheath, and how these impinge on fetal well-being.

Protocol

This protocol follows the guidelines of New York State Institute for Basic Research in Developmental Disabilities human research ethics committee.

1. Villous Tree Dissection

1. Rinse formalin fixed placenta tissue with phosphate buffed saline (PBS)¹⁴ to remove the formalin and place in a Petri dish on the stage of a dissection microscope. Use scalpel and fine forceps to tease the placenta tissue apart to locate villous trees (white threadlike branching structures). Dissect out pieces of villus tree (several mm long) and transfer the tissue to a micro-tube containing ~1 mL PBS.

2. Immunofluorescent Staining

1. With a pipette, remove the PBS and replace with fresh PBS. Let stand for 10 min with occasional swirling. Repeat once more.
   ​NOTE: This step and all further steps are done at room temperature except the primary antibody incubation.
2. Using a pipette, remove and replace the PBS with PBS +0.1% Triton-X100 + 2% goat serum (PBS-T-GS) to permeabilize the tissue and block non-specific binding of secondary antibodies. Incubate for 30 min.
3. Replace the PBS-T-GS with PBS containing 2% goat serum, both mouse monoclonal anti-CK7 and rabbit anti-CK7.
   1. Incubate tissue overnight at 4 °C.
   2. Remove the remaining antibodies by washing with PBS-GS for 10 minutes with occasional swirling.
   3. Remove the PBS-GS and replace with PBS-GS containing both goat anti-mouse IgG coupled with a green emitting (520 nm) fluorophore and goat anti-rabbit IgG coupled an infrared emitting (652 nm) fluorophore.
   4. Remove the antibodies by repeating step 2.1.
   5. Store the immunolabeled tissue at room temperature in the dark.

3. Clarification of the Tissue

1. Wash the samples 3 times with PBS at 10 minute intervals.
2. Dehydrate the tissue by removing the PBS with a pipette and replacing it with 50% ethanol (methanol may be used as well). Incubate for 10 min. Replace with fresh 50% ethanol and incubate for 10 min. Repeat this once more for a total of 3 incubations. Repeat incubations with 70%. Then replace with 100% ethanol and incubate for 15 minutes (alternatively methanol may be used).
3. With a fine-tipped forceps, transfer tissue to a fresh micro-tube containing ~ 1 mL Solution-1 for 4 h.
4. With fine-tipped forceps, transfer tissue to a fresh micro-tube containing Solution-2 for ~ 4 h.
   NOTE: When stored in the dark, the immunofluorescence is stable at room temperature for at least 6 months.
5. Do not mix with any aqueous solution, (e.g. mounting medium) as the clearing will lost.

4. Mounting for Microscopy

1. Do not mount the tissue between a standard glass slide and a coverslip as the tissue is soft and easily deformed.
2. Refer to Figure 5B and mount the tissue in a Sykes-Moore chamber as follows;
   1. With fine-tipped forceps place a 25-mm round coverslip in the chamber base.
   2. With a fine-tipped forceps place the rubber gasket in the base on the coverslip.
   3. With a pipette place a drop (~40 µL) of Solution-2 in the center of the coverslip.
   4. With the fine-tipped forceps transfer a piece tissue from Solution-2 to the drop in the center of the coverslip.
   5. Place a second coverslip on the top of the gasket.
   6. Thread the locking ring into the top of the base compressing the coverslips and gasket until firm.
      NOTE: Be careful not to over-tighten or the coverslip will shatter.
   7. Insert a 22-gauge needle into a port on the base and through the gasket.
   8. Fill a 3-mL syringe with ~ 1.5 mL of Solution-2.
   9. Mount a 25-gauge needle on the syringe.
   10. Insert the syringe needle into the opposite port and through the gasket.
   11. Ensure that the air in the chamber is venting through the other 25-gauge needle, gently inject solution-2and fill the chamber.
   12. Remove the needle and syringe and place the chamber on a large glass slide on the confocal microscope stage.
3. Alternatively, cut custom wells out of PDMS silicone sheets.
   1. With scissors cut a 25.4 × 25.4 mm² piece from the PDMS sheet.
   2. With a scalpel cut ~ 12.7 × 12.7 mm² well in the piece.
   3. Press the piece firmly onto a microscope slide.
   4. Fill the well to the top with Solution-2.
   5. Transfer the tissue piece from the micro-tube to the center of the well.
   6. With fine-tipped forceps place a coverslip on the top of the well such that the bottom of the coverslip is wetted with Solution-2.
   7. Mount the Sykes Moore Chamber assembly with the tissue on the confocal microscope stage.

5. Confocal Microscopy

1. Bring the tissue in the chamber into focus with a 10x objective.
2. Move the microscope stage up and down through the focal plane to measure the distance between the top and bottom of the tissue.
3. Move the stage in 2.5 µm steps to collect a confocal image file containing a set of images through the tissue from top to bottom.

6. Reversing the Clearing

1. Reverse the clearing by transferring the tissue to a micro-tube containing > 20x the volume of 100% ethanol. At 1 h intervals replace with 70% ethanol, then 50% ethanol and finally PBS. Store at 4 °C in the dark.
   NOTE: Now process the tissue for standard paraffin embedding, sectioning and histological or immuno-histochemical staining.

7. Deconvolution

NOTE: For the following steps, software Commands, Tabs and drop-down menus are italicized.

1. Start the deconvolution software and open the image file.
2. Enter parameters listed in the 'Summary' window.
   1. For 'Spacings' enter the voxel x, y, and z dimensions in µm.
   2. For each color channel, select the fluorescent dye used for the immunostaining from the drop-down menu.
   3. For the 'Modality', select Laser Scanning Confocal from the drop-down menu.
   4. For the 'Objective Lens', select 10x from the drop-down menu.
   5. For the 'Immersion Medium', select Air from the drop-down menu.
   6. Click on the 'Apply' button.
3. Click on the 'Deconvolution' tab.
4. Click on '3D Deconvolution' in the drop-down menu.
5. In the '3D -Deconvolution' window insure that the settings are: 1) Adaptive PSF Blind, 2) Theoretical PSF, 3) Use (Default Adaptive PSF, 10 iterations, Medium noise), 4) Base Name.
6. Click on the 'green Check button' to start the program.
7. When finished, repeat steps 7.3 -7.6 to restart the program. When finished restart it for third and last time.
8. In the 'Data Manager' window, click on the file prefixed with "10-10-10-".
9. Click on the 'File' tab, then click on 'Save As'.
10. In the 'Save As' window select '8bit unsigned integer' from the 'Data Type' drop-down menu and click on Save.

8. Image Processing

1. Start Fiji software.
   1. Click on 'File', click on 'Open' and select the Deconvolved image file and click on the 'Open' button in the 'Open' window.
   2. Click on 'Image/Color/Split channels' and convert the three-color image file into three new files containing only one color. Save the red image file that contains the immunolabeled villous capillary image set. Delete the green and blue image files.
   3. Subtract the background fluorescence from the red image file with: 'Process/Subtract background'.
   4. In the drop-down menu set Rolling ball radius to 10 pixels.
   5. Leave the 4 menu options unchecked and click 'OK'.
   6. Click 'Yes' on the Process Stack menu.
   7. Remove inherent tissue fluorescence (autofluorescence) by opening the 'Image/Adjust/Brightness-Contrast' menu and adjusting the minimum, maximum, brightness and contrast settings. Be careful not to remove any of the capillary immunofluorescence.
   8. Minimize remaining irrelevant fluorescence by opening the 'Image/Adjust/Threshold' menu and adjusting the sliders such that the capillary immunofluorescence is highlighted. Check the 'Dark background' box and click on 'Apply'. On the 'convert to binary drop-down' menu check the 'Black background' box and click on 'OK'. Save the black and white (binary) image file for analyses below.

9. Analysis

1. Object Counting and Skeletonization.
   1. Select the binary image file created in Step 8.1.8.
   2. Click on 'Analyze/3D OC Options' and check only 'Volume' and 'Store results …' boxes in the drop-down menu. Insure that the 'Show numbers' box is unchecked. Click on the 'OK' button.
   3. Click on 'Analyze/3D Objects Counter'. In the drop-down menu, set 'Size Filter: Min.' to 5000. Insure that the 'Exclude objects on edges' box is unchecked. Check that the 'Objects, Statistics' and 'Summary' boxes are checked. Click on the 'OK' button.
      NOTE: This may take several minutes depending on file size and computer configuration.
   4. Save the Statistics table and the Objects map file.
   5. Select the Objects map file. Click on 'Plugins/Skeleton/Skeletonize 2D-3D' to skeletonize the capillary network in the binary file. Click on 'File/Save as'. Select 'Tiff…' in the drop-down menu. In the Save as TIFF window add "Skel-" to object map file name and click on the 'Save' button.
   6. Click on 'Analyze/Skeleton/Analyze Skeleton 2D-3D'. In the drop-down menu accept the default settings and click on the 'OK' button. Save the Branch 'information' table, the 'Results' table, and two output files (Skel-Objects-labeled -skeletons and Tagged skeleton).

Results

The generated 3D renderings of the capillaries in clusters of terminal villi in human placenta from 8 weeks gestational age to term delivery were counted as individual clusters and skeletonized for network analysis. The functional units of the placenta (Figure 1A) are the villus trees that are an extension of surface vessels where they have penetrated the placenta parenchyma (Figure 1B, and enlar...

Discussion

The Institutional Review Board approved the collection of placental villous tissues for formalin fixation from electively terminated pregnancies. Before the procedures were performed, a brief review of the medical record noted maternal age, parity, and confirmed the absence of any underlying medical (e.g., hypertension, diabetes, and lupus) or fetal (structural or chromosomal anomalies, abnormal growth) was performed. The data sheet and any collected specimens were labeled only with a Study ID. Thus, all the spe...

Materials

Name	Company	Catalog Number	Comments
10% Formaldehyde solution (w/v) in aqueous phosphate buffer	Macron Fine Chemicals	H-121-08	General fixation agent, ready to use formula, use caution as vapors are toxic
Scalpel blades	ThermoFisher Scientific	08-916-5B No. 11
Scalpel	ThermoFisher Scientific	08-913-5
Fine forceps	Electron Microscopy Services	78354-119
Micro Tube (1.7 mL)	PGC Scientifics	505-201
Phosphate buffered saline	Sigma	D8537	PBS
Pipette	VWR	52947-948	disposable, 3ml transfer pipette
Triton X-100	Boehriner Mannheim	789 704	Dilute to 0.1% from stock
Goat Serum	Gibco	16210-064	Dilute to 2% in PBS solution
Mouse monoclonial anti-ck7 Keratin 7 Ab-2 (Clone OV-TL 12/30)	ThermoFisher Scientific	MS-1352-RQ
Rabbit Anti-CD31 antibody	Abcam	ab28364
green emitting (520 nm) fluorochrome	Invitrogen	A11017	Alexa-Fluor 488
infrared emitting (652 nm) fluorochrome	Invitrogen	A21072	Alexa Fluor 633
Ethanol alcohol 200 proof	Pharmco-Aaper	111000200	Dilute down to lower concentrations using PBS as needed
Solution-1	Visikol Inc.		Visikol Histo-1
Solution-2	Visikol Inc.		Visikol Histo-2
Skyes-Moore chambers	BellCo Glass Inc.	P/N 1943-11111
25 gauge needle	ThermoFisher Scientific	14-826AA	BD Precision Glide Needes
3 mL syringe	ThermoFisher Scientific	14-823-40	BD disposable syringe
PDMS silicon sheets	McMaster-Carr	P/N 578T31
confocal microscope	Nikon Inc.		Nikon C1 Confocal Microscope
Deconvolution software	Media Cybernetics		AutoQuant X22
Fiji image processing software			free, Open source  software available at https://fiji.sc
Hematoxylin	Leica Biosystems	3801570	Component 1 of SelecTech H&E staining system
Alcoholic Eosin	Leica Biosystems	3801615	Component 2 of SelecTech H&E staining system
Blue Buffer	Leica Biosystems	3802918	Component 3 of SelecTech H&E staining system
Aqua Define MCX	Leica Biosystems	3803598	Component 4 of SelecTech H&E staining system
Immunohistochemistry detection system	ThermoFisher Scientific	TL-125-QHD	UltraVision Quanto Detection System HRP DAB