Name: a high-throughput in situ method for estimation of hepatocyte nuclear ploidy in mice
Uploaded: 2020-04-19T13:00:00
Description: Watch this Scientific Journal Video about A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice at JoVE.com

This work was funded by the Spanish MINECO Government grants BFU2014-58686-P (LAN) and SAF-2017-84708-R (DJB). LAN was supported by a national MINECO Ram&#243;n y Cajal Fellowship RYC-2012-11700 and Plan GenT award (Comunitat Valenciana, CDEI-05/20-C), and FMN by a regional ValI+D studentship of the Valencian Generalitat ACIF/2016/020. RP would like to acknowledge Prof. Ewa K. Paluch for funding. We thank Dr. Alicia Mart&#237;nez-Romero (CIPF Cytometry service) for help with the IN Cell Analyzer platform.

All animal experiments were previously approved by the CIPF ethics&#160;committee. Mice were housed in a pathogen-free facility at the Centro de Investigaci&#243;n Pr&#237;ncipe Felipe (Valencia, Spain), registered as an experimental animal breeder, user, and supply centre (reg. no. ES 46 250 0001 002) under current applicable European and Spanish animal welfare regulations (RD 53/2013).
1. Tissue harvesting and sample preparation
NOTE: This protocol describes how to ...

<ol>
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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+hepatocyte+ploidy+levels+with+body+size+and+growth+rate+in+mammals.">Relationship of hepatocyte ploidy levels with body size and growth rate in mammals.</a> Genome. 44 (3), 350-360 (2001).</li><li>Tanami, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+zonation+of+liver+polyploidy.">Dynamic zonation of liver polyploidy.</a> Cell and Tissue Research. 368 (2), 405-410 (2017).</li><li>Kudryavtsev, B. N., Kudryavtseva, M. V., Sakuta, G. A., Stein, G. 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A high-content, high-throughput approach for the analysis of tissue remodeling and estimation of hepatocyte nuclear ploidy in the murine liver is described. Once familiar with the procedure, a user can process, image and analyze multiple samples in a 3&#8722;5 day period, generating large testable datasets that provide a detailed signature of liver health. Given the simplicity of the sample preparation method, together with the large numbers of cells and tissue area analyzed (on average 14 mm2/sample), results...

Hepatocytes in the mammalian liver can undergo stalled cytokinesis to produce binuclear cells, and DNA endoreplication to produce polyploid nuclei containing up to 16N DNA content. Overall cellular and nuclear ploidy increase during postnatal development, ageing and in response to diverse cellular stresses1. The process of polyploidization is dynamic and reversible2, although its precise biological function remains unclear3. Increased ploidy is associated with reduced proliferative capacity4, genetic diversity2, adaptation to chronic injury5 and cancer protection6. Hepatocyte ploidy alterations occur as a result of altered circadian rhythm7, and weaning8. Most notably, the ploidy profile of the liver is altered by injury and disease9, and compelling evidence suggests that specific ploidy changes, such as increased &#8805;8N nuclei or loss of 2N hepatocytes, provide useful signatures for tracking non-alcoholic fatty liver disease (NAFLD) progression3,10, or the differential impact of viral infections11.
In general terms, liver injury and regeneration are associated with increased hepatocyte cell size and nuclear area12, together with reduced overall numbers of hepatocytes, particularly those with 2N DNA content10,11. Parenchymal injury in the liver is also frequently accompanied by expansion of non-parenchymal cells (NPCs), including stromal myofibroblasts, inflammatory cells and bipotent liver progenitor cells. High-throughput methods that provide a quantitative cytological profile of parenchymal cell number and nuclear ploidy, whilst also accounting for changes in NPCs, therefore have considerable potential as research and clinical tools to track the response of the liver during injury and disease. Compelling recent in situ analysis of ploidy spectra in human samples of hepatocellular carcinoma also demonstrate that nuclear ploidy is dramatically increased within tumors and is specifically amplified in more aggressive tumor subtypes with reduced differentiation and loss of TP5313. Hence, there is a strong possibility that methodological advances in quantitative assessment of nuclear ploidy will assist in future prognostic profiling of liver cancer.
In this protocol, a flexible high-throughput methodology for the comparative analysis of mouse liver tissue sections is described, which provides detailed cytometric profiling of hepatocyte numbers, the NPC response and an internally calibrated method for estimating nuclear ploidy (Figure 1). Hepatocytes are distinguished from NPCs by hepatocyte nuclear factor 4 alpha (HNF4&#945;) immunolabelling, prior to characterization of nuclear size and nuclear morphometry. &#34;Minimal DNA content&#34; is estimated for all circular nuclear masks by integrating mean Hoechst 33342 intensity (a proxy for DNA density) with interpolated three-dimensional (3D) nuclear volume. Hepatocyte minimal DNA content is then calibrated using NPCs to generate a nuclear ploidy profile.
Image acquisition, nuclear segmentation and image analysis are performed using high-content imaging, enabling large areas of two-dimensional (2D) liver sections containing tens of thousands of cells to be screened. A custom-written program is provided for automated post-processing of high-content image analysis data to produce a sample-wide ploidy profile for all circular hepatocyte nuclei. This is performed using free to download software to calculate nuclear ploidy based on stereological image analysis (SIA)10,11,14,15. The SIA methodology has been previously validated by flow cytometry as an accurate, albeit laborious, method for estimating hepatocyte nuclear ploidy in the liver14, assuming circular nuclear morphology and a monotonic relationship between nuclear size and DNA content. In this protocol, both nuclear parameters are measured by assessment of nuclear morphometry and Hoechst 33342 labelling. Calculation of &#34;minimal DNA content&#34; for each nuclear mask is followed by calibration of hepatocyte nuclear ploidy using NPCs, which have a known 2&#8722;4N&#160;DNA content and therefore serve as a useful internal control.
Compared to conventional flow cytometry methods16 the approach described enables hepatocyte nuclear ploidy to be assessed in situ and does not require access to fresh tissue or disaggregation methods that can bias outcomes and be difficult to standardize. As with all SIA-based approaches, nuclear ploidy subclasses &#62;2N are underrepresented by 2D sampling due to the sectioning of larger nuclei outside of the equatorial plane. The tissue-wide ploidy profile also describes minimum DNA content for all circular hepatocyte nuclear masks, and does not directly discriminate between mononuclear hepatocytes and binuclear cells that have two discrete (&#34;non-touching&#34;) nuclei of the same ploidy. However, the simplicity of this protocol allows considerable scope for it to be adapted to account for additional parameters such as internuclear spacing or cell perimeter analysis, that would facilitate identification of binuclear cells providing a more detailed assessment of cellular ploidy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3,5-diethoxycarboxynl-1,4-dihydrocollidine diet (DDC)</td>
			<td>TestDiet</td>
			<td>1810704</td>
			<td>Modified LabDiet mouse diet 5015 with 0.1% DDC</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-goat IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A11055</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat Leica CM1850 UV</td>
			<td>Leica biosystems</td>
			<td>CM1850 UV</td>
			<td>Tissue sectioning</td>
		</tr>
		<tr>
			<td>Fluorescent Mounting medium</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad Software</td>
			<td>Prism 8</td>
			<td>Statistical software for graphing data</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma-Aldrich</td>
			<td>B2261</td>
			<td>Final concentration 5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>IN Cell Analyzer 1000</td>
			<td>GE Healthcare Bio-Sciences Corp</td>
			<td></td>
			<td>High-Content Cellular Imaging and Analysis System</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>R2019a</td>
			<td>Data analytics software for automated analysis of nuclear ploidy</td>
		</tr>
		<tr>
			<td>Microscope coverslides</td>
			<td>VWR International</td>
			<td>630-2864</td>
			<td>Size of 24 x 60 mm</td>
		</tr>
		<tr>
			<td>Microsoft Office Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>Speadsheet software</td>
		</tr>
		<tr>
			<td>OCT Tissue Tek</td>
			<td>Pascual y Furi&#243;</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Panreac AppliChem</td>
			<td>141451.121</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen for immunostaining</td>
			<td>Sigma-Aldrich</td>
			<td>Z377821-1EA</td>
			<td>5mm tip width</td>
		</tr>
		<tr>
			<td>Polysine Microscope Slides</td>
			<td>VWR International</td>
			<td>631-0107</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polyclonal Anti-HNF4&#945;</td>
			<td>Thermo Fisher Scientific</td>
			<td>PA5-79380</td>
			<td>Dilution 1:250 (alternative)</td>
		</tr>
		<tr>
			<td>Rabit polyclonal Anti-HNF4&#945;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-6556</td>
			<td>Dilution 1:200 (antibody used in the study)</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P5927</td>
			<td></td>
		</tr>
	</tbody>
</table>

a high-throughput in situ method for estimation of hepatocyte nuclear ploidy in mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

This method has been used to measure the impact of cholestatic injury on the adult mouse liver by feeding animals for 0&#8722;21 days with a hepatotoxic diet containing 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)17. Chronic DDC feeding results in hepatocellular injury increased ploidy and periportal expansion of NPCs. The user should be aware that mouse strain and age-dependent differences may exist in nuclear ploidy and that all analyses have been perform...

Watch this Scientific Journal Video about A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice at JoVE.com

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...

This method enables the user to generate a profile of hepatocyte nuclear DNA content from 2D tissue sections for the study of liver development, regeneration, and chronic disease. This automated high throughput approach can be applied to all 2D liver tissue sections, providing an internally calibrated readout of nuclear ploidy for all simple circular hepatocyte nuclei. Changes in hepatocyte nuclear ploidy are associated with aging, chronic liver disease, and cancer.This technique provides a simple means to profile nuclear ploidy in fixed or cryopreserved liver specimens. In situ ploidy profiling methods have considerable potential as research and clinical tools for tracking liver disease progression as they do not require tissue desegregation or access to fresh material. After harvesting the murine liver tissue sample, embed the liver lobule into an optimal cutting temperature medium filled cryomold and immediately place the mold on dry ice to ensure rapid freezing.To obtain slices of the frozen liver lobule sample, section the sample at a six micrometer thickness, placing a labeled polyamide coated slide over each sample for five seconds to let each sample stick to the slide. When all of the slices have been collected, allow the samples to equilibrate for three to five minutes at room temperature before immunolabeling. At the end of the equilibration, fix the tissue sections in a fume hood with one milliliter of 4%paraformaldehyde in PBS for 10 minutes at room temperature.At the end of the fixation, rinse the slides with three three minute washes in PBS with gentle agitation. After the last wash, dry the area around each tissue section and use a hydrophobic pen to draw a circle around each sample. To permeabilize the samples, treat the sections with a 0.5%non-ionic surfactant in PBS for 15 minutes at room temperature.At the end of the incubation, wash the samples for three minutes in PBS two times with gentle agitation as demonstrated before blocking the non-specific binding with a filtered solution of 1%bovine serum albumen, 5%horse serum, and 0.2%non-ionic surfactant in PBS for at least one hour at room temperature. Next, incubate the slides with the HHF4-alpha antibody diluted in blocking buffer overnight at four degrees Celsius in a humid staining chamber protected from light. The next morning, wash the slides four times in PBS with gentle agitation before incubation with an appropriate fluorescence conjugated secondary antibody and Hoechst diluted in filtered 1%bovine serum albumen and 0.2%non-ionic surfactant in PBS for two hours at room temperature in a humid staining chamber protected from light.At the end of the incubation, wash the slides four times as demonstrated, followed by two three minute washes in double distilled water with gentle agitation. After the last wash, mount the samples with two drops of fluorescence mounting medium on a coverslip and apply gentle pressure as necessary to eliminate any bubbles. Then, check the slides using a conventional fluorescence microscope to ensure good fixation and immunolabeling.For fluorescence image acquisition, set the parameters on a high content imaging platform to acquire fluorescence images using the appropriate excitation for the fluorophores used. Then scan samples to acquire sufficient images to obtain complete coverage of the tissue section. At the end of the scan, adjust the nuclear segmentation parameters of the software to ensure that the nuclei are optimally segregated and modify the threshold intensity to ensure optimal gating of the HNF4-alpha positive hepatocytes and HNF4-alpha negative non-parenchymal cells.For nuclear quantification, set the nuclear area in square micrometers based on the Hoechst staining, the main nuclear Hoechst intensity, the nuclear elongation factor, the mean nuclear roundness index, the HNF4-alpha status, and the nuclear X, Y coordinates based on the center of gravity. To perform a hepatocyte nuclear ploidy analysis, first download and install the ploidy quantification analysis program. In MATLAB, navigate to the App tab of the tool strip and click Install App.Open the Ploidy Application ML App Install program. A message will appear to confirm the successful installation. In each data analysis file, include a sheet termed, Cell Measures, containing all of the data required for the ploidy analysis set out in columns as indicated.For each experimental condition, provide a control dataset that will be used to calculate the internal control for two to four N nuclear ploidy calibration. For biological replicates, store each spreadsheet in its own folder, naming the folder prefixes incrementally. Next, launch the Ploidy application.The Ploidy application graphical user interface will appear. Click the Path to Control Data button to navigate to the folder in which the control data replicates reside. This data path will then appear in the interface.In Folder Prefix, type the name to be given to the output files. Click the Path to Other Data button and navigate to the folder in which the comparative data replicates reside. This data path will then appear in the interface.Then click Run. When the analysis is complete, the Status bar will read Analysis Complete. After immunolabeling, it is important to check all of the slides by conventional fluorescence microscopy to confirm that a good quality fixation and staining has been obtained.Smearing or blurring of the Hoechst dye can indicate inadequate fixation or sample degradation prior to fixation. Nuclear segregation and HNF4-alpha threshold parameters should be carefully optimized prior to automatic image analysis to broadly reflect the visual pattern of the immunostaining observed by fluorescence microscopy. Following image analysis, the data should reflect the increasing numbers of non-parenchymal cells and the small, but significant, reduction in HNF4-alpha positive nuclei numbers within the liver with DDC injury.In healthy control livers, approximately 63%of HNF4-alpha positive nuclei exhibit a simple circular morphometry. Comparison of the relative nuclear ploidy between control and DDC treated groups should reflect a significant loss of 2C and 4C hepatocyte nuclei with injury together with increased numbers of greater than 8C cells. The relative positional information for each ploidy sub-group can be interrogated by retrieving the 2D location of particular hepatocyte subsets within the high content image analysis software.Immunolabeling with additional antibodies, such as Ki-67, can be performed to assess cell replication and hepatocyte nuclear morphometry data can be further interrogated to compliment the ploidy analysis. The tissue-wide ploidy profile generated by this method describes the minimum DNA content for all circular hepatocyte nuclei, but does not discriminate between mononuclear cells and binuclear hepatocytes. The method can potentially be adapted to account for parameters such as cell perimeter, to facilitate identification of binuclear cells and provide an additional readout of cellular ploidy.

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Medicine

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

A Simple Method of Mouse Lung Intubation

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Creation of Abdominal Adhesions in Mice

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and ...