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Quantitative Measurement of Bleomycin-induced Lung Fibrosis in Rabbits Using Sequential in vivo Regional Analysis and High-Resolution Computed Tomography

Posted May 31, 2011 Updated Apr 10, 2023
By Yukihiro Nagatani MD , Norihisa Nitta MD PhD , Hideji Otani MD , Kenichi Mukaisho MD PhD , Akinaga Sonoda MD PhD , Ayumi Nitta-Seko MD PhD , Masashi Takahashi MD , Kiyoshi Murata MD PhD
13 min read

Rationale and Objectives

To examine the feasibility of quantitative high-resolution computed tomography (HRCT) findings to monitor the stage of bleomycin-induced pulmonary fibrosis in rabbits by correlating HRCT and pathologic scores and analyzing sequential changes on HRCT images using regional volume histograms.

Materials and Methods

Lung fibrosis was induced by injecting bleomycin intratracheally into 23 Japanese white rabbits. Rabbits were randomly separated into seven groups depending on follow-up period (12-hour, 24-hour, 3-day, 7-day, 14-day, 21-day, and 28-day). Four-row HRCT examinations were performed at any of the seven time points in each follow-up period and just after bleomycin administration in addition to pre-bleomycin administration as the baseline scan. Scores of consolidation, homogenous ground-glass opacity (GGO), inhomogeneous GGO, reticulolinear shadow, and honey-comb formation were recorded as ratio of affected area to total cross-section in four transaxial planes. Inflammatory and fibrous changes were scored histopathologically. Correlations between HRCT and pathologic findings were assessed. Sequential changes on HRCT images in areas with pathologically confirmed fibrosis were assessed on volume histograms of cubic regions of interest (ROI) using quantitative parameters.

Results

Consolidation and inhomogeneous GGO exhibited fair correlations with inflammation scores ( r = 0.273, P = .009, and r = 0.393, P < .001); reticulolinear shadow and inhomogeneous GGO had a fair and good correlation, respectively, with fibrous scores ( r = 0.327, P = .001, and r = 0.579, P < .001). Inhomogeneous GGO was hardly detected on regional images at 21 and 28 days after bleomycin administration. As to mean computed tomography attenuation, skewness, and kurtosis, inhomogeneous GGO differed from reticulolinear shadow and consolidation.

Conclusion

Using appropriate ROI settings, quantitative assessment of inhomogeneous GGO with regional volume histograms enables us to monitor the progression of lung fibrosis by sequential observations.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive and fatal lung disease; average survival from the time of diagnosis ranges from 3 to 5 years . It is the most common form of idiopathic interstitial pneumonia and is associated with a pathologic pattern known as usual interstitial pneumonia (UIP). Typical high-resolution computed tomography (HRCT) findings of IPF are reticular abnormality, subpleural honeycombing, and traction bronchiectasis of spatially patchy distribution with minimal ground-glass opacity (GGO) . The underlying etiology of IPF and its optimal treatment remain to be identified .

Although the pathogenesis of IPF is not completely understood, proposed factors are injury to the alveolar epithelium followed by not only persistent inflammatory stimulation but also lymphocyte-monocyte interactions that sustain the production of growth factors such as tumor growth factor-β1 , proteolytic enzymes such as arginase-1 and tissue inhibitor of matrix metalloproteinase , and pro-fibrotic cytokines such as interleukin-4 and 13 under the influence of a T H 2 to T H 1 response . The deposition of connective tissue elements that progressively remodel and destroy the normal tissue architecture plays an important role.

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Materials and methods

Rabbit Model of Bleomycin-induced Pulmonary Fibrosis

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Experimental Protocol

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CT Data Acquisition

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HRCT Scoring

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Figure 1, (a) HRCT scan obtained 7 days after bleomycin injection. The left anterior lobe appears normal ( arrows ). In the histogram, the peak value for the cubic ROI is −850 HU. Histogram has a high sharp peak and short tail. The values for mean CT attenuation, variance, skewness, and kurtosis are −841.2, 9189, 2.463, and 6.979, respectively. (b) HRCT image obtained just after bleomycin injection. Note the homogeneous GGO in the left anterior lobe ( arrows ). In the histogram, the peak value for the cubic ROI is −708 HU. Histogram has a relatively low peak. The values for mean CT attenuation, variance, skewness, and kurtosis are −726.3, 26015, 1.172, and 2.686, respectively. (c) HRCT image obtained 7 days after bleomycin injection. Note the inhomogeneous GGO in the right posterior lobe. In the histogram, the peak value for the cubic ROI is −202 HU. Histogram has a low, blunt peak. The values for mean CT attenuation, variance, skewness, and kurtosis are −170.8, 34709, −0.259, and 0.227, respectively. (d) HRCT image obtained 28 days after bleomycin injection. Note the reticulolinear shadow in the left posterior lobe. In the histogram the peak value for the cubic ROI is -509 HU. Histogram has a low, blunt peak and bilateral long tail. The values for mean CT attenuation, variance, skewness, and kurtosis are −459.1, 60471, 0.149, and −0.446, respectively. (e) HRCT image obtained 7 days after bleomycin injection. There is consolidation in the left posterior lobe. In the histogram the peak value for the cubic ROI is 18.9 HU. Histogram has a high sharp peak. The values for mean CT attenuation, variance, skewness, and kurtosis are −63.9, 39651, −1.731, and 3.462, respectively. CT, computed tomography; GGO, ground-glass opacity; HRCT, high-resolution CT; HU, Hounsfield units; ROI, region of interest.

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Pathologic Scoring

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Serial Change of the HRCT Images in Rabbits with the Pathologically Proven Fibrosis

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Statistical Analysis

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Results

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Comparison of Values Obtained at Different Postadministration Time Points

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Table 1

HRCT and Pathologic Scores in Different Intervals

Consolidation Homogeneous Inhomogeneous Reticulolinear Honey-comb Inflammation Fibrosis GGO GGO Shadow Formation Score Score 12-hour 0.007 ± 0.034 0.096 ± 0.157 0.001 ± 0.003 0.003 ± 0.012 0.000 ± 0.000 6.17 ± 2.51 0.00 ± 0.00 24-hour 0.013 ± 0.055 0.077 ± 0.115 0.012 ± 0.051 0.004 ± 0.011 0.000 ± 0.000 7.33 ± 3.40 0.00 ± 0.00 3-day 0.044 ± 0.103 0.055 ± 0.082 0.027 ± 0.042 0.005 ± 0.012 0.000 ± 0.000 5.67 ± 3.54 0.00 ± 0.00 7-day 0.057 ± 0.093 0.083 ± 0.081 0.116 ± 0.183 0.013 ± 0.035 0.000 ± 0.000 11.08 ± 2.98 4.80 ± 2.27 14-day 0.079 ± 0.156 0.117 ± 0.112 0.124 ± 0.106 0.014 ± 0.023 0.000 ± 0.000 10.33 ± 3.24 4.67 ± 2.32 21-day 0.023 ± 0.051 0.112 ± 0.093 0.099 ± 0.104 0.116 ± 0.096 0.000 ± 0.000 10.25 ± 3.74 2.33 ± 1.80 28-day 0.008 ± 0.015 0.202 ± 0.144 0.082 ± 0.061 0.182 ± 0.109 0.000 ± 0.000 6.83 ± 3.51 2.16 ± 2.11

GGO, ground-glass opacity; HRCT, high-resolution computed tomography.

Data are mean ± standard deviation.

Figure 2, Photomicrographs by hematoxylin-eosin staining (original magnification, ×200, 3 days after (a, b) and 21 days after (c, d, e) intratracheal bleomycin administration) show inflammatory cell infiltration in the alveolar septa ( arrow in a ), intraalveolar amorphous materials (arrow in b), alveolar wall metaplastic cells lining air space ( arrow in c ), smooth muscle in stroma ( arrow in d ), and interstitial fibrosis ( arrow in e ).

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Correlation Between HRCT and Pathologic Scores

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Table 2

Correlation of HRCT Score with Pathologic Score

Inflammation Score Fibrosis Score r Value_P_ Value r Value_P_ Value Consolidation 0.273 ∗ .009 0.160 .128 Homogeneous GGO 0.026 .807 0.058 .580 Inhomogeneous GGO 0.393 ∗ <.001 0.579 ∗ <.001 Reticulolinear shadow 0.137 .193 0.327 ∗ .001

GGO, ground-glass opacity; HRCT, high-resolution computed tomography.

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Serial Changes on HRCT Images of Lungs with Pathologically Confirmed Fibrosis

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Table 3

Serial Change of HRCT Images in Regions with Pathologically Proven Pulmonary Fibrosis

Post 3-day 7-day 14-day 21-day 28-day FS Lobe Post 3-day 7-day 14-day 21-day 28-day FS Lobe N N N HG N RL 1 RM N N N HG HG RL 5 LP HG N HG RL RL RL 1 RM N N HG RL 5 LP N N N HG HG 2 LA HG IG C 5 LP HG HG C RL RL RL 2 LP HG N HG RL 5 LP HG HG HG IG RL RL 2 LP HG HG IG IG RL RL 5 RP N N N 2 LP HG C C C IG 5 RP HG N HG IG RL 3 LA HG IG IG IG 5 RP N HG HG N 3 LA HG HG IG 5 RP HG HG IG 3 LA N N N HG 6 LA HG N HG IG RL 3 LP HG HG IG 6 LP HG C C C HG 3 RM HG N HG IG RL RL 6 RM HG IG IG IG 3 RM HG N RL 6 RM HG N N RL IG RL 3 RP HG IG IG RL 6 RM HG N RL IG RL RL 4 LA HG HG N C RL RL 6 RP HG IG C 4 LA HG C IG IG 6 RP HG N RL IG RL RL 4 LP HG C IG 6 RP N N N HG HG 4 LP HG HG IG IG 6 RP HG N HG IG RL 4 RP N N N HG 7 LA HG IG IG IG RL 4 RM HG IG RL 7 RP HG N IG IG RL RL 5 LA HG HG RL 9 RM HG IG IG 5 LA HG RL IG RL 9 RM

C, consolidation; FS, fibrotic score; GGO, ground-glass opacity; HG, homogeneous GGO; HRCT, high-resolution computed tomography; IG, inhomogeneous GGO; LA, left anterior lobe; LP, left posterior lobe; N, normal lung; post, just after bleomycin administration; RL, reticulolinear shadow; RM, right middle lobe; RP, right posterior lobe.

Figure 3, Right middle lobe of a rabbit intratracheally injected with bleomycin. Serial changes on HRCT images and in the volume histogram parameters in an area that was most severely involved in fibrotic changes. The homogeneous GGO observed just after bleomycin administration disappeared at 7 days and the area appears normal. Note the inhomogeneous GGO observed at 14 days and the reticulolinear shadow on scans obtained 21 and 28 days after bleomycin injection. Photomicrographs of hematoxylin-eosin‒stained tissue sections obtained 28 days after bleomycin injection show honeycomb formation, interstitial fibrosis ( green arrows (1) ), and alveolar wall metaplastic cells lining the air space ( blue arrows (2) ). Original magnification ×40 ( left bottom ) and ×100 ( right bottom ). GGO, ground-glass opacity; HRCT, high-resolution computed tomography.

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Volume Histograms of 5 HRCT Findings Using Cubic ROI

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Table 4

Parameters for Volume Histogram in Different HRCT Findings

Mean Variance Skewness Kurtosis Normal lung −811.2 ± 69.3 10883 ± 5241 1.51 ± 0.64 4.34 ± 3.65 Homogeneous GGO −634.3 ± 112.5 27330 ± 19366 0.76 ± 0.54 1.39 ± 1.32 Reticulolinear Shadow −472.5 ± 90.5 48551 ± 29398 0.51 ± 0.48 0.11 ± 0.74 Inhomogeneous GGO −224.0 ± 108.9 55555 ± 28767 −0.53 ± 0.45 0.38 ± 1.08 Consolidation −64.8 ± 80.9 58304 ± 40895 −1.74 ± 0.46 4.44 ± 2.87

Data are mean ± standard deviation.

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Discussion

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