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

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.