Morphometry tool for histological
assessment in COPD model in mice

Use of a digital morphometry tool for histological assessment in an Elastase-induced COPD model in mice

Loeb E¹, Zuckerman A¹, Margalit R², Alagem N ³, Sagiv Y ³. ¹Patho-Logica, Golda Meir Street 7, Scientific Park, Ness-Ziona, Israel. ²Science In Action, Sapir Street 5, Scientific park, Ness-Ziona, Israel. ³ Kamada Ltd, Holzman 2, Scientific Park, Rehovot, Israel

Introduction:
The Elastase-induced COPD (chronic obstructive pulmonary disease) mouse model is well-known for emphysema studies. Following administration of test drug, Elastase is instilled intratracheally once a week for four weeks to induce COPD. Lungs are harvested, fixed, and stained for histological evaluation. The H&E-stained slides are analyzed using a digital morphometry tool as applied to the analysis of representative pictures of the affected lung parts.

Aim:
The objective of this study was to develop a reliable quantitative tool for assessing the degree of alveolar loss and rupture damage in treated vs. control lungs of elastase-induced COPD in mice as parameters for the assessment of a treatment effect.

Materials and Methods:

• Anesthetized mice received weekly intratracheal instillations of 20 µL Test Item.
• 24 hours after each dose, 0.2U PPE in 20 µL saline was instilled.
• At the end of weeks 2, 3 & 4, five mice were sacrificed for histology.
• Lungs were fixed in 4% Formaldehyde using intra- and extra-bronchial fixation (gravitation method).
• 5-µm-thick paraffin sections were cut and H&E stained.
• Representative pictures were taken in affected areas (4 fields per lung) using 4X objective magnification

Digital morphometry analysis:
Image analysis was performed using “Image Pro Plus” Version 6.3 (Media Cybernetics, US). From each histological slide, four pictures were taken and sent for digital analysis.

Protocol:

1. 1. Spatial calibration was applied (marked calibration).
2. 2. Area of Interest (AOI) selection was chosen.
3. 3. Threshold selection was based on HSI color system (Hue, Saturation and Intensity).
4. 4. Intensity of 96-255 grey levels was depicted to show the void spaces.
5. The calibrated parameters of Area and Perimeter were measured and used for creating statistical data.

Table 1: Results of morphometry, digital evaluation of lung emphysema.

Figure 1: Illustration of a threshold image measurement of alveolar spaces in the Lung, shown in red X4 H&E.

Results:

• Morphometric quantification shows clear differences in area counts, mean and sum when comparing healthy and affected lungs through accurate comparison between affected lungs (Table 1).
• SD shows greater variation in the measurement of the affected lungs compared to the healthy lungs.
• Areas of air were quantified using a rigid threshold (Fig. 1)
• We extract the numbers from the histological image and show them in a graph for comparative means (Fig. 2)
• Area / Perimeter provide a good index for emphysema quantification (Fig. 3)

Figure 2.  Representative pictures of two normal and two affected lungs with different graph profiles

Figure 3. Summary of x4-fold measurements per animal area / perimeter.

Summary and Conclusions:
We have developed a very accurate and useful tool for the quantification of alveolar loss and rupture damage in a mouse emphysema model. This morphometry technology enables a quantification of the comparison of different treatments effects in this model, and should be applicable to other models where key morphological features can be quantified.

References:

1. Cruz F. F., Antunes M. A., Abreu S. C., Fujisaki L. C., Silva J. D., Xisto D. G., et al. . (2012). Protective effects of bone marrow mononuclear cell therapy on lung and heart in an elastase-induced emphysema model. Respir. Physiol. Neurobiol. 182, 26–36.
2. Weibel E. R. (1990). Morphometry: stereological theory and practical methods, in Models of Lung Disease-Microscopy and Structural Methods, eds Gil J., editor. (New York, NY: Marcel Dekker; ), 199–247