As mentioned above, all the adhesions examined were well vascularized and contained both blood and lymphatic vessels (Fig 3, top, A, and center, B). Angiogenesis and lymphangiogenesis were early events, occurring in the first week after talc instillation. By means of immunolocalization of the endothelial cell marker PECAM-1, sprouting angiogene-sis was detected and blood vessels were observed stemming from both bases of the adhesions (Fig 3, bottom, C). Moreover, nonsprouting angiogenesis by intussusception was also observed in the newly formed capillary blood vessels of the adhesions. Lymphangiogenesis occurred contemporaneously with angiogenesis, and lymphatic vessels were also present at 1 week (Fig 3, center, B). Here
Newly developed vessels such as arterioles, capillaries, and venules were uniformly distributed throughout all the adhesions, including both the visceral and parietal segments. Larger blood and lymphatic vessels were commonly found at the edges of the adhesion. Newly formed vessels exhibited a normal morphologic and histologic appearance and, for example, all arterioles examined already displayed well-developed intimal, medial, and adventitial layers at 1 week after instillation (Fig 3, top, A).
Examination of semithin sections of resin-embedded adhesion samples clearly revealed the presence of myelinated nerve fibers in adhesions from 20% (2 of 10 rabbits) [Fig 4]. Nerves were already observed at 1 week after instillation, and no differences in the percentage of adhesions containing nerve fibers were observed among experimental times. Thus, in both rabbits, the percentage of adhesions containing nerves was 67%. Moreover, the site of adhesion formation did not appear to influence their presence, given that nerves were observed in adhesions joining visceral pleura with either costal, diaphragmatic, or mediastinal parietal pleura.
In all adhesions examined, nerves consisted of a single fascicle containing 5 to 20 thin myelinated axons of various diameters (1 to 6 ^m) uniformly distributed throughout the nerve section (Fig 4, top, A, and center, B). Nerves were always observed in association with blood vessels, mainly arterioles (Fig 4, top, A, and center, B), and followed a sinuous course along the longitudinal axis of the adhesion. Moreover, mast cells were frequently found in close proximity to both nerves and vascular structures (Fig 4, center, B). Morphometric evaluation of nerves revealed that both the nerve profile diameter and number of axons varied along the length of the nerve. In all cases, the magnitude of these two parameters, nerve diameter and axon number, was always greater in the parietal than in the visceral segment of the adhesion. On ultrastructural examination, nerves in adhesions showed well-developed myelinated axons with adjacent Schwann cells and surrounding epineurium (Fig 4, bottom, C).
Figure 3. Neovascularization. Top, A, and center, B: Maturation of pleural adhesion parallelled rapid growth and high development of a vascular network, including arterioles (top, A) [hema-toxylin-eosin; bar = 25 |j,m] and lymphatic vessels (center, B) [hematoxylin-eosin; bar = 50 |j,m]. Bottom, C: Immunolocaliza-tion of the endothelial cell marker PECAM-1 by indirect immunofluorescence reveals the parenchymal origin of newly formed vessels (arrows) that vascularize the pleural adhesion (ba = basis of the adhesion; lp = lung parenchyma; bar = 50 |j,m).
Figure 4. Innervation. Top, A, and center, B: Pleural adhesion innervation resulted from the growth of nerves containing a variable number of thin myelinated axons (methylene blue; bars = 10 |j,m). Note the association of these nerves with both arterioles (top, A, and center, B) and mast cells (mc) [center, B]. Bottom, C: Ultrastructural observation of the proximal section of a newly formed nerve exhibiting a near-mature structure with a well-organized epineurium and endoneuria (bar = 0.5 |j,m).