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.