Name: Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary (Video) | JoVE Journal
Uploaded: 2017-12-10T14:00:00.000+00:00
Duration: 12 min 38 s
Description: Learn passive CLARITY for three-dimensional reconstruction of vascular architecture in intact mouse ovaries. This method reveals capillary distribution in follicles and advances reproductive biology research.

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5 minutes

This study was supported by grants from the Chinese Special Fund for Postdocs (No. 2014T70392 to YF), the National Natural Science Foundation of China (No. 81673766 to YF), the New Teacher Priming Fund, the Zuoxue Foundation of Fudan University, and the Development Project of Shanghai Peak Disciplines-Integrative Medicine (20150407).

All procedures involving animal subjects followed the guidelines of the Animal Ethics Committee at Shanghai Medical College, Fudan University (Approval number 20160225-013).
1. Preparation of the Transparent Mouse Ovary
<ol>
	<li>Preparation of the solutions
	<ol>
		<li>Prepare phosphate-buffered saline (PBS) solution (1 M, pH 7.6) with 0.1% Triton X-100 (PBST). To make 1 L of 10x PBS stock solution, mix 87 g of NaCl, 3.1 g of NaH2PO4 &#183; 2H2O, and 28.7 g of Na2HPO4 &#183;12H2O. Add dH2O to 1 L and stir ~2 h. Adjust to pH 7.4 with 1 N NaOH and store at room temperature. Dilute this 10x stock solution by 1/10 using dH2O and then make 0.1% Triton X-100 in PBS.</li>
		<li>Prepare the hydrogel solution (pH 7.5) by mixings 4% (wt/vol) paraformaldehyde (PFA) solution, 4% (wt/vol) acrylamide, 0.05% (wt/vol) bisacrylamide, and 0.25% (wt/vol) VA-044 initiator in double-distilled water on ice.</li>
		<li>Prepare the clearing solution (pH 8.5) by mixing 200 mM sodium borate buffer containing 4% (wt/vol) sodium dodecyl sulfate (SDS) in double-distilled water.</li>
	</ol>
	</li>
	<li>Transcardial perfusion and ovary preparation
	<ol>
		<li>Keep all solutions on ice during all procedures.</li>
		<li>Anesthetize adult female wild type C57BL/6 mouse with an intraperitoneal injection of pentobarbital (0.35 mg/kg) solution. Once it shows no toe-pinch response, the mouse is properly anesthetized.</li>
		<li>Use sticky tape to fix the mouse with limbs stretched out on a flat foam board.</li>
		<li>Expose the heart by an incision on the xiphoid-process through the chest, skin, and bones with scissors.</li>
		<li>Make an incision into the animal&#39;s right atrium, and then perfuse the left ventricle with 20 mL of ice-cold 1x PBS at 10 mL/min.</li>
		<li>Perfuse the animal with at least 20 mL of hydrogel solution at 10 mL/min.</li>
		<li>Incise the skin over the abdomen along the medioventral line with scissors, which exposes the whole enterocoelia, and remove the intestines. Collect the ovaries as well as the uterus to maintain the integrity of ovarian morphology.</li>
		<li>Remove the fat tissues around the ovary under a microscope.</li>
	</ol>
	</li>
	<li>Degassing
	<ol>
		<li>Place the ovaries in 10 mL hydrogel solution at 4 &#176;C for fixation for 3 days.</li>
		<li>Transfer the ovary into a 5-mL tube and fill the tube with hydrogel solution. Stretch parafilm over the top of the tube and then wrap parafilm around the neck of the tube. 
		Caution: Ensure that there are no bubbles between the parafilm and the hydrogel solution.</li>
		<li>Place the tube on a shaker in the incubator at 37 &#176;C for a maximum of 3 h to allow the hydrogel to polymerize.</li>
		<li>Remove the ovary from the gel using a spatula and carefully remove the excess gel from around the ovary by tissue paper. Wash the ovary four times with 1x PBS.</li>
	</ol>
	</li>
	<li>Passive clearing
	<ol>
		<li>Submerge the ovary in 10 mL of the clearing solution. Gently shake at 80 rpm at 37 &#176;C to start the clearing process.</li>
		<li>In the first week, change the solution every 3 days. After one week, refresh the clearing solution once a week until the ovary becomes clear. Usually this process takes about 2-3 weeks. 
		NOTE: The ovary can be directly immunostained or can be stored in sodium azide (0.02%) in PBS at 4 &#176;C for several weeks before immunostaining.</li>
	</ol>
	</li>
</ol>
2. Immunostaining
<ol>
	<li>Wash the ovary twice in PBST on the day 1 at 37 &#176;C on a shaker.</li>
	<li>Incubate the ovary on a shaker in primary antibodies/PBST solution (Table 1) on day 2-3 at 37 &#176;C. In the work presented here, the primary antibodies were platelet-endothelial cell adhesion molecule 1 (CD31) and tyrosine hydroxylase diluted at 1:10 and 1:50 in PBST, respectively. 
	Caution: If two or more kinds of primary antibody are used, make sure there are different animal sources.</li>
	<li>Wash off the primary antibodies with PBST buffer at 37 &#176;C on day 4 on a shaker.</li>
	<li>Incubate the ovary with the desired secondary antibodies in PBST on day 5-6 at 37 &#176;C on a shaker. In the work presented here, the secondary antibodies were Alexa Flour 488 for tyrosine hydroxylase and Alexa Flour 594 for CD31 diluted at 1:50 in PBST.</li>
	<li>Wash off the secondary antibodies with PBST buffer at 37 &#176;C on day 7 on a shaker.</li>
	<li>Transfer the tissue into refractive index matching solution for refractive index homogenization on day 8. Put the tissue on a paper with gridlines to check its visual clarity by checking whether the gridlines can be seen. Once the tissue has been fully clarified, it can be imaged.</li>
	<li>Make a putty cylinder that could just surrounds the tissue, and then shape it into a horseshoe shape and press the outer ridge tightly onto a glass slide in order to make a sealed space. The height of the putty should be a little taller than the ovary.</li>
	<li>Place the clarified ovary carefully into the center of the putty and add refractive index matching solution. Put a glass-bottom dish over the putty tightly and use putty to fix the dish.</li>
</ol>
3. Confocal Microscopy
<ol>
	<li>For imaging with a confocal microscope, in the &#34;Ni-E&#34; panel choose the lens (in this case water immersion 25X objective lens, 1.1-NA, 2 mm-WD) with the proper working distance (in this case 2.0 mm).</li>
	<li>In the &#34;Acquisition&#34; panel, select the number of channels. Here, use three channels.
	<ol>
		<li>First, use the &#34;eye port&#34; tab and the XY controller joystick to move the tissue to the center of field by checking through the microscope eyepieces.</li>
		<li>After turning off the shutter light, click the &#34;live&#34; tab to adjust the &#34;laser power&#34;, &#34;HV (Gain)&#34;, and &#34;offset&#39; for each channel separately. Higher laser power and HV can lead to higher signals, and higher offset can reduce the background noise.</li>
		<li>Select the proper &#34;Scan size&#34; and &#34;Count&#34;. Use a scan size of 1024 x 1024 &#181;m2 and a count of 4 to 8.</li>
	</ol>
	</li>
	<li>After setting the image quality for defining the depth of the tissue in the panel &#34;Z intensity correction&#34;, and after locating the lens on the top-most layer of the ovary (in the center of the tissue) and adjusting the acquisition for the top layer, click the &#34;plus&#34; button to define the top layer.
	<ol>
		<li>Move the lens down and set the appropriate HV and laser power for the bottom of the tissue and add the lowest layer information into the Z-intensity correction table. Click on &#34;To ND&#34; to synchronize the setting with the &#34;ND acquisition&#34; panel.</li>
	</ol>
	</li>
	<li>In the &#34;ND acquisition&#34; panel, first set the number of scanning steps. Then, tick the tabs &#34;Z&#34; and &#34;Large Image&#34;.
	<ol>
		<li>Go to the &#34;Scan large image&#34; window to define the size of the field according to the border of the tissue with the help of tissue visualization through the eyepieces. Go back to the &#34;ND acquisition&#34; panel to input the size of the scanning area (&#34;fields&#34;) and choose the overlap between 10% and 15%.</li>
		<li>Click &#34;Run Z correction&#34;, not &#34;Run now&#34;, to automatically set the proper HV, laser power, and offset for each layer, and wait for the image to process.</li>
	</ol>
	</li>
</ol>
4. 3D Reconstruction of Individual Follicles and Their Vasculatures
<ol>
	<li>First, use ImageJ5 to open the original NIS file. Click the &#34;Image&#34; button and choose the &#34;Color&#34; and the &#34;Split Channels&#34;. Then, click &#34;File&#34; and separately save the channels as tiff file series by using the &#34;Image Sequences&#34; option in &#34;Save as&#34;.</li>
	<li>Use Imaris to open one of the tiff file series. Click the &#34;Edit&#34; button and choose &#34;Add channels&#34; to add the rest of the channels. The name and color of the channel can be changed in the &#34;Display Adjustment&#34; tabs. The thickness of the tissue might need to be corrected in the &#34;Image Properties&#34; panel in the drop-down list under &#34;Edit&#34;.</li>
	<li>For the 3D cropping of the final images and removal of excess parts, click the &#34;Edit&#34; button and choose &#34;Crop 3D&#34;. The size of the field can be adjusted by dragging the border.</li>
	<li>To reconstruct the vessel signals with the Filaments algorithm in the Surpass panel, click the &#34;Filaments&#34; button to create a new Filament and click next.
	<ol>
		<li>In order to define the largest and the thinnest diameter, go to the &#34;Slice&#34; panel and find the tracing vessel. The distance will be automatically shown on the &#34;Measure&#34; panel by selecting two points at the maximum width of the vessel.</li>
		<li>Go back to the &#34;Surpass&#34; panel and input the measured width and click next.</li>
	</ol>
	</li>
	<li>Adjust the starting and seed point threshold. In the &#34;Camera&#34; panel, choose &#34;Select&#34;. If some of the automatically produced spheres need to be removed, press shift and click on the point. By choosing &#34;Navigate&#34; and moving the mouse, the structure can be rotated to ensure that all the correct points have been retained, and then click next.</li>
	<li>Choose the highest threshold for the local contrast and click next. Do not select &#34;Detect Spines&#34;, and continue to finalize the reconstruction of the blood vessel cylinder. The color can be edited by clicking the &#34;Color&#34; tab. The statistical data can be extracted in the &#34;Statistic&#34; panel of the reconstructed Filament. Excess parts can be removed in the &#34;Edit&#34; panel. 
	NOTE: Other algorithms can be used for measuring the volume of total follicles or follicular walls. In the work presented here, the oocytes were marked by the autofluorescence of both secondary antibodies. Because of the large amount of data, a workstation server was used for the data analysis.</li>
</ol>
5. Statistical Analysis
NOTE: Image analysis data are presented as means &#177; standard errors.
<ol>
	<li>Perform comparisons between the follicles using independent sample t-test, and use Pearson&#39;s correlation test to compare correlation coefficients of follicular wall and capillary length. Set a value of P &#60;0.05 as the limit for statistical significance.</li>
</ol>

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</ol>

The authors have nothing to disclose.

In the current study, we present 3D imaging to evaluate the relationships between capillaries and individual growing follicles. In our previous work using the same protocol 9, we studied the roles of large vasculature, interactions between follicles, and the location of follicles in intact mouse ovaries. The passive CLARITY approach allowed us to study micro- and macro-vasculatures, folliculogenesis, and the interrelationships between the corpora lutea and follicles as well as to reconstruct the o...

The follicle is the fundamental structural and functional unit of the ovary, and its development is highly related to the vasculature within the ovary. Blood vessels supply nutrition and hormones to the follicles and thus play important roles in the growth and maturation of follicles1.
A combination of technologies, including selective blood vessel markers, transgenic mouse models, and pharmaceutical development, have increased our knowledge about ovarian vascular networks, angiogenesis, and the function of blood vessels in folliculogenesis. The ovary is known as an active organ because it remodels various tissues and vascular networks during folliculogenesis and ovulation. Such active remodeling in the size and structure of vessels is required for the biological function of developing and recruiting follicles.
Traditional histological and histomorphometric methods using ovarian sections and immunolabeling of blood vessels are limited to two-dimensional (2D) images2. With the development of three-dimensional (3D) reconstruction technologies, 2D images of tissue slices can be overlapped to make a 3D structure, but this method still has some limitations &#8212; sectioning of the tissue can destroy the microstructures, some parts of the tissue are often missing, and significant labor is involved in making 3D reconstructions from images obtained from slices. Whole-tissue 3D imaging with confocal microscopy can overcome many of these limitations, but these methods are limited to the evaluation of angiogenesis in the embryonic ovary3. Using whole tissue clearing methods such as CLARITY4 can increase the visualized volume so as to solve these problems in postnatal and adult ovaries, and such methods provide optical clearance of the ovary without any structural deformations. Imaging of the 3D architecture of the intact ovary provides an accurate image database for image analysis software, such as the Imaris software package used in this work.
Remodeling of the ovary throughout adulthood is part of a dynamic physiological system, and this makes the ovary an excellent model for investigations into the regulation of angiogenesis. Furthermore, evaluating the role of ovarian blood vessels in pathologic conditions of the female reproductive system such as polycystic ovary syndrome or ovarian cancers can be studied through whole ovarian tissue imaging. The development of the passive CLARITY method and the use of advanced image analysis software have provided detailed spatial information on the relationships between blood vessels and ovarian structures such as follicles.

<table><tbody><tr><td>Acrylamide</td><td>Vetec</td><td>v900845</td><td>http://www.sigmaaldrich.com/catalog/product/vetec/v900845</td></tr><tr><td>Alexa Flour 488 (Dilution 1:50)&amp;nbsp;</td><td>Life Technologies</td><td>A11039</td><td>https://www.thermofisher.com/antibody/product/Goat-anti-Chicken-IgY-H-L-Secondary-Antibody-Polyclonal/A-11039</td></tr><tr><td>Alexa Flour 594 (Dilution 1:50)</td><td>Life Technologies</td><td>A11012</td><td>https://www.thermofisher.com/antibody/product/Goat-anti-Rabbit-IgG-H-L-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A-11012</td></tr><tr><td>Bisacrylamide</td><td>Amresco</td><td>172</td><td>http://www.amresco-inc.com/BIS-ACRYLAMIDE-0172.cmsx</td></tr><tr><td>Black wall glass bottom dish (Willco-Dish)</td><td>Ted Pella</td><td>14032</td><td>http://www.tedpella.com/section_html/706dish.htm#black_wall</td></tr><tr><td>Boric acid</td><td>Sinopharm Chemical Reagent</td><td>10004818</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=10004818</td></tr><tr><td>Disodium hydrogen phosphate dodecahydrate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; 12H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sinopharm Chemical Reagent</td><td>10020318</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=10020318</td></tr><tr><td>FocusClear</td><td>Celexplorer</td><td>FC-102</td><td>http://www.celexplorer.com/product_list.asp?MainType=107&amp;amp;BRDarea=1</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM996</td><td>http://www.parafilm.com/products</td></tr><tr><td>Paraformaldehyde</td><td>Sinopharm Chemical Reagent</td><td>80096618</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=80096618</td></tr><tr><td>PECAM1/CD31, platelet-endothelial cell adhesion molecule 1 (Dilution 1:10)</td><td>Abcam</td><td>ab28364</td><td>http://www.abcam.com/cd31-antibody-ab28364.html</td></tr><tr><td>Photoinitiator VA044</td><td>Wako</td><td>va-044/225-02111</td><td>http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm</td></tr><tr><td>Sodium azide</td><td>Sigma</td><td>S2002</td><td>http://www.sigmaaldrich.com/catalog/product/sial/s2002?lang=en&amp;amp;region=US</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sinopharm Chemical Reagent</td><td>10019318</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=10019318</td></tr><tr><td>Sodium dihydrogen phosphate dihydrate (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; 2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sinopharm Chemical Reagent</td><td>20040718</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=20040718</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sinopharm Chemical Reagent</td><td>30166428</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=30166428</td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sinopharm Chemical Reagent</td><td>10019718</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=10019718</td></tr><tr><td>Triton X-100</td><td>Sinopharm Chemical Reagent</td><td>30188928</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=30188928</td></tr><tr><td>Tyrosine hydroxylase (TH, Dilution 1:50)</td><td>Abcam</td><td>ab76442</td><td>http://www.abcam.com/tyrosine-hydroxylase-phospho-s40-antibody-ab51206.html</td></tr></tbody></table>

three-dimensional reconstruction of the vascular architecture of the passive clarity-cleared mouse ovary

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

We adapted the passive CLARITY method into a quick and simple method for passive ovary clearing while preserving the follicular and vascular architecture and obtaining the highest fluorescent signal from labeled markers of vessels and follicles. The 3D architecture of the follicular vasculature was determined by immunostaining for CD31, a marker for endothelial cells6. CD31 staining in the ovaries of adult mice was traced using the Filament algorithm and showed int...

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

The overall goal of this protocol is to provide a complete three-dimensional view of the capillary distribution in mouse ovarian follicles and show their relationships. The ovary is an essential organ in reproduction and in endocrinology. Count each different developmental stage of follicle and the somatic cells.In addition, unlike other organs, ovary does not show the repeated branching of the functional unit. The local vascular system may be together with the nervous system are also capable of undergoing major changes in each menstrual cycle in adult females. Applying the passive clarity in the ovary allows us to see the overall distribution and the connection among those cells and follicles in the ovary.The most of the one key of this measure is that it has the least effect on the ovary, and accounts for the clearing of various markers. Through the reconstruction of the microscopic image we provided a map of the ovary. Some details of the vessels, neurons, and follicles can be observed clearly.For image analysis, some options of Imaris were used to evaluate the relationships between the ovary and the structures. Prepare profusion solutions according to the article instruction and keep them on ice. Anesthetize the mice and make sure the mouse has no toe pinch response.Spray ethanol, make an incision on the right atrium, then instantly profuse the left ventricle with 20ml of ice cold PBS at the speed of 10ml per minute to flush out the blood. Then profuse the animal with cold Hydrogel Solution with the same speed. Collect the ovaries as well as the uterus to maintain the integrity of ovarian morphology.Transfer the separated ovaries to the cold Hydrogel Solution. Under a stereo microscope remove the fatty tissues around the ovary. For better result of tissue fixation, we recommend removing of ovarian bursa.Keep the ovaries in Hydrogel Solution for three days. After three days, transfer the ovaries into a glass tube and fill the tube with fresh Hydrogel Solution. Stretch the parafilm over the top of the tube and then wrap another parafilm around the neck of the tube.Ensure there are no bubbles between the parafilm and the Hydrogel Solution. Place the glass tube on a shaker incubator at 37 degrees Centigrade for two hours until gel forms. Carefully remove the excess gel around the ovaries with a paper filter.Put the ovaries in a 15ml tube and fill the tube with 10ml of clearing solution. Place the tube on a shaker incubator at 37 degrees Centigrade for at least two months. Change the clearing solution every day in the first three days and then weekly until the ovary becomes cleared.All immunostaining procedures are done at 37 degrees Centigrade on a shaker. First, wash the ovary in PBST for 24 hours. Second incubate ovary in primary antibody diluted by PBST for two days.Third, wash off the primary antibodies with PBST buffer. Fourth, incubate the ovary with the desired secondary antibody in PBST for two days. Fifth, wash off the secondary antibodies with PBST.Sixth, transfer the ovary into a refractive index matching solution for RI homogenization. Check the visual transparency, when the ovary has been fully clarified it can be imaged. First, prepare a clean glass bottom dish.Make a putty cylinder and shape it into a horseshoe shape. Press the outer edge tightly onto glass slide to make a sealed space. The height of the putty should be a little higher than the ovary.Move the ovary carefully to the center and add mounting solution. Push a dish over the putty and use two putty pieces for fix it. When the ovary is ready to image, Nikon confocal microscope with 16X lens can be selected to image it.For imaging with confocal microscope, first choose the lens in the Ni-E panel. In the panel acquisition, select the number of channels. First, use Eye Port tab and XY controller joystick to move the tissue to the center of field through checking by microscope eyepieces.Second, after turning off the shutter light, click live tab to adjust the laser power, HV, and offset for each channel. In the panel Z Intensity Correction adjust acquisition for top layer, click the plus button to define the top, move the lens down lens and set appropriate HV and laser power to the bottom of tissue and add the lowest layer information click To ND to synchronize the setting with the panel and the acquisition. And in acquisition, first set the number of scanning steps.Click the tabs of Z and large image. Second, go to the scan large image window to define the size of field according to the border of the tissue. Third, back to the ND acquisition to input the size of the scanning fields and choose the overlap within 10 and 50%Then click run Z correction and wait for the imaging process.First use imagic to open the original NIS file. Click image button, choose color and display channels, then click file, choose image sequences and separately save them as tiff files. Second, use Imaris to open one of the tiff files.Click edit button and choose the add channels to add the rest. The name and color can be changed in the display adjustment. The thickness of the tissue should also be corrected in image properties.Third, click edit button and choose Crop 3D to cut the extra part. First, click the filament button and click next. Go to the slice panel, find a tracing vessel and distance, then back to the Surpass, input the static and click next.Second, adjust the starting and set point threshold. In the camera panel, choose the select, press shift and click on the point to remove the extra part. Third, choose the highest threshold and click next.Don't select detects points and go next. Some excess part can be removed in edit panel. The color can be edited by clicking the color.Ovarian vasculature 3D architecture was investigated following immunostaining CD31. The 3D structure can be either observed by slices through other slices or by entirety through volume rendering. CD 31 is staining of adult mouse follicular vasculature was done.Through the reconstruction of capillaries by the filaments, we could show relationship between the individual follicles and their capillaries. The ovary, like a woman, is always active and interchangeable. However, due to the limitation of traditional histological analysis, it's very hard to describe the relationships among such complex constituents.The newly developed method, passive clarity may help up us to overcome the optical obstacle, keep the integrity of the whole tissues, and provide the functional clues.

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Research

JoVE Journal

Biology

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Learn passive CLARITY for three-dimensional reconstruction of vascular architecture in intact mouse ovaries. This method reveals capillary distribution in follicles and advances reproductive biology research.

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

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