Evaluation of a Morphometric Density Approach for Quantitation of Pneumonia Severity using a Murine Influenza Model

Floyd D. Wilson^1*, Lanny W. Pace¹ and Frederic J. Hoerr^2
*Correspondence: Floyd D. Wilson, Mississippi Veterinary Research and Diagnostic Laboratory, Mississippi State University, Pearl, MS 39208, USA, Tel: 601-707-7499, Email:

Keywords

Mice • Pneumonia • Lung density • Morphometrics

Introduction

Digital microscopy is often applied for the interpretation and quantification of pathological evaluations using experimental animals [1-9] and humans [10]. However, many studies utilize expensive commercial image analysis systems having proprietary software that preclude routine standard applications or employ labor- and time-intensive procedures. We evaluated the use of a relatively simple inexpensive morphometric method to quantify the histologic lung inflammatory response using a murine pneumonia model. For this purpose, the free, downloadable NIH ImageJ image analysis software program was utilized in conjunction with a standard microscope and attached camera. The results of this study are reported.

Materials and Methods

General methods

The lung sections used in the study were retrieved from our histology archives of previous studies. The samples represented noninfected control (NC) and infected control (IC) populations from a large therapeutic drug trial that utilized a mouse influenza pneumonia model.] A detailed description of the mouse pneumonia model was previously published by other investigators [11]. Briefly, the IC mice were infected intranasally with 5 x 104 H1N1 influenza virus suspended in 50ul media, while the NC mice were given only media. A total of 177 lung samples were included in the study. Groups of mice were sacrificed and evaluated at 3-, 5-, 7-, 10- and 15-days following virus exposure. Samples of lungs were removed and placed in neural buffered formalin for histology slide processing. Our routine histology and morphometric studies utilized standard 5 μm thick H&E-stained sections.

Microscopic evaluations were performed using an Olympus CX31 microscope (Olympus Corporation, Breinigsville, PA). Photomicrographs were taken with an AmScope MU1403 camera (AmScope, Irvine, CA) using the associated software-based program. The microscope lamp setting was 5 and the software was set at medium density (2048 x 1644 pixels).

Morphometrics

The morphometric approach used for quantifying lung inflammation represents a modification of the method we previously reported for quantification of avian bone marrow cellularity using density measurements [9]. The lung sections were first photographed at 25-X (1 image) and 400-X (3 images) magnifications. For each histology section the entire lung area present at 25-X was outlined and measured. Three microscopic fields from the most affected regions were used for the 400-X evaluations, each field measuring 15,510 μm² with a total area of 46,530 μm².

The colored photographed images were converted to binary format using the downloadable National Institutes of Health (NIH) ImageJ software program. The pixel densities of the converted binary images were then calculated by the image analysis software. The presence of “morphometric pneumonia” (MP) was arbitrarily defined as a density value that was above the upper 95% confidence interval for the NC control values and for a particular magnification. The 95% confidence intervals are given in (Table 1). The percentage of pneumonia area was also determined for some images.

The morphometric results were also compared to those obtained by semiquantitative pneumonia severity scoring. A six -level scoring system for grading pneumonia was employed in which: 0=absent, pneumonia not observed; 1=minimal severity; 2=mild; 3=moderate; 4=marked and 5=severe. When uncertainty existed between scoring groups in assigning a value, half values were given. All evaluations were made in a “blind” manner without reference to experimental treatments.

Statistical evaluations

Statistical analysis utilized the GraphPad Prism version 9 software program (GraphPad Software, Inc. San Diego, CA.). Statistical differences between the groups were determined using the Kruskal-Wallis ANOVA test and the Dunn’s test for multiple comparisons. Statistical differences in pneumonia occurrence utilized the Fisher’s exact test. Correlation coefficients were calculated using the Pearson test.

Results

Microscopic examples of a noninfected normal control lung (NC) and infected pneumonia lungs (Infected control or IC) are shown in (Figure 1A-F). The results for routine histologic and morphometric evaluations are given in (Table 1) and are presented graphically in (Figures 2-4).

Histologically the viral pneumonia was focal to multifocal, but sometimes diffuse (Figure 1). The inflammation was characterized by thickening and hypercellularity of the alveolar walls and flooding of the alveolar lumens with mainly mononuclear inflammatory cells. Cuboidal metaplasia of type II alveolar pneumocytes was sometimes present resulting in an adenomatous appearance (Figures 1C and 1D).

A suggestion of an increase in the mean density or “morphometric pneumonia” (MP) for infected group (IC) compared to the non-infected lungs (NC) group using the 25-X images occurred as early as experimental day 3. A significant difference between means of the IC and NC groups at this magnification was not apparent until day 10 and persisted on day 15 (Table 1 and Figure 2). Significant elevations in mean density in IC compared to NC when using 400-X images occurred from 7 day onward (Figure 3). Note that there was a reduction in density for NC at the 25X compared to 400-X measurements (Table 1).

The strength of correlations between the pneumonia severity scores and mean densities was generally good (Table 1). While the strength of the correlation coefficient using the 25-X density data was moderate (r=0.55), there was a strong correlation using the 400-X evaluation data (r=0.75).

Discussion

The general approach of using binary digital image density measurements to quantitate pneumonia severity in mice appears valid. It provides a simple and inexpensive method for quantification of pneumonia parameters using routine histology sections, standard microscopy and ImageJ, a free downloadable software program. The method is rapid, and results can be obtained in a few seconds. The procedure j provides an approach for evaluating archived histology specimens. The basic morphometric approach reported could be further refined by application of other procedures such as special staining or immunohistochemistry. For example, the % area of pulmonary fibrosis in mice has been measured using special staining (Masson’s trichrome) for collagen detection 7. The color-threshold was adjusted for blue to detect collagen staining and the % blue area determined by scanning for blue coloration [7].

Our approach for measuring pneumonia intensity is based on the method we previously developed for quantitation of bone marrow cellularity in chickens [12]. It is also similar to another method reported for quantitation of bone marrow cellularity in chickens with chicken anemia virus (CAV) previously described by others [1]. Those authors used a positive pixel count algorithm for quantification of bone marrow cellularity which measured area and staining intensity. However, that method also requires an image analysis optic system using proprietary software. In contrast, our density approach is performed with a standard microscope using a software program provided free for download from NIH.

The density results using 25-X images were generally different from the results with the 400-X images. The difference mainly reflects the incorporation of the entire lung section area for the 25-X density measurements which includes normal lung regions. This allows for estimation of overall pneumonia involvement that reflects both intensity and the lung area involved. In contrast, the 400-X density measurements exclusively reflect the severity or intensity of pneumonia in the involved lung regions. This was also an apparent difference in strength of correlation between density measurements made at different magnifications to severity scores for pneumonia. Thus, measurements at the two magnifications can potentially provide different types of useful quantitative information on pneumonia involvement.

Pneumonia severity scoring in this study was performed by an experienced investigator. However, differences in semiquantitative lesion scoring can vary between observers. Digital density measurements allow for verification or standardization of the severity scoring. The calibration of lesion scoring results with lung density measurements facilitates the establishment of normal ranges and the standardization of severity scoring between different observers.

Acknowledgments

We gratefully acknowledge the contribution of Nirmal Pugh, Tahir Mir and Mohammad K. Ashfaq from the Center for Natural Products Research at the University of Mississippi for allowing use of the cases described herein. The cases comprise archived histology specimens of client-submitted diagnostic pathology study from mice in a therapeutic drug trial. We are thankful for the excellent technical efforts of Tariq Muhammad for the preparation of histology slides.

Declaration of Conflicting Interests Statement

Dr Wilson has served as a paid consultant to the Center for Natural Products Research at the University of Mississippi who provided samples for this study.

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