Histopathologic and immunohistochemical Analysis
Lung tissues which were immersed in formalin for at least 24 hr were analyzed for histopathology. Hematoxylin-Eosin (H-E) staining was used for analysis of the pulmonary arterioles in terms of medial wall thickness under a microscope at a magnification of 400X. The methods were described in our previous published study.21,22and the medial wall thickness of each arteriole was expressed as follows:
Percent wall thickness = (medial thickness × 2 /external diameter) × 100.
Verhoeff-van Gieson staining was performed for evaluating the severity of neointimal formation using a modified scoring system published before.23-25 The degree of vascular remodeling was determined by measuring 30 transversally cut vessels (outer diameter: 50-100 µm) from each rat and expressing the thickness of the combined intimal and medial layers as a percentage of total vessel diameter. The percentage of vascular occlusion of these 30 vessels was categorized as Grade 0: no evidence of luminal obstruction; Grade 1: the presence of partial (<50%) luminal occlusion; and Grade 2: the present of luminal occlusion greater than 50%. An average Grade score for 30 vessels was calculated for each animal. Afterwards, a quantitative analysis of luminal obstruction in 30 consecutive small pulmonary arteries from all rats in each group was performed.
In addition, double immunohistochemical staining was performed to present the smooth muscle actin (SMA) by using formalin-fixed, paraffin-embedded tissue. Experiments were performed by double staining polymer detection systems (TADS03, BioTnA, Taiwan) using rabbit polyclonal anti-SMA (1:100, Cell signaling, MA, USA) and anti-PCNA (Proliferating Cell Nuclear Antigen)(1:100, Cell signaling, MA, USA) antibodies.