Alteration of pulmonary neuroendocrine cells during epithelial repair of naphthalene-induced airway injury

Abstract

Whole-mount airway preparations isolated from the lungs of mice treated by intraperitoneal injection of naphthalene and allowed to recover for 5 days were examined for the distribution and abundance of solitary pulmonary neuroendocrine cells (PNECs) and neuroepithelial bodies (NEBs) along the main axial pathway of the right middle lobe. Sham mice treated with corn oil vehicle were examined in a similar manner. An antibody to calcitonin gene-related peptide, a neuroendocrine cell marker, was used to identify the location, size, and number of PNECs and NEBs in the airways. After naphthalene treatment and epithelial repair, NEBs were significantly increased along the walls of the airways as well as on branch point ridges. The surface area covered by NEBs composed of 20 or fewer PNECs was significantly enlarged after naphthalene treatment compared with control NEBs of an equivalent cell number. The PNEC number per square millimeter was also increased more than threefold above control values after naphthalene treatment. These findings provide further support for a key role of neuroendocrine cells in the reparative process of airway epithelial cell renewal after injury.

Figure 1.

Whole-mount airway preparation of the main axial pathway of the right middle lobe of the mouse. The numerical designation of airway bifurcation ridges begins with the first intralobar bifurcation. The numerical designation of airway generations of the axial pathway also begins immediately after the first intralobar airway branch point (arrowhead). Airway generations 1 to 3, 4 to 6, 7 to 9, and 10 to 12 are identified by the vertical lines superimposed over the image. NEBs (arrows) can be visualized sporadically dotting the surfaces of the airway. Scale bar, 1 mm.

Figure 2.

NEB distribution and abundance in the main axial airway path of the right middle lobe in mice treated with corn oil (control) or naphthalene. A: The number of NEBs on airway bifurcations; B: The number of NEBs on airway generations; C: The number of NEBs per square millimeter for different airway generations, demonstrating that NEB frequency was similar along the entire axial airway path.

Figure 3.

Total NEB numbers found on the first nine bifurcations (A) or the first nine airway generations (B) of the main axial airway path of the right middle lobe in mice treated with corn oil (control) or naphthalene. The density of solitary neuroendocrine cells (PNECs) is shown (C) for control mice and for mice treated with naphthalene. An asterisk designates a significant difference from the control value (P < 0.05).

Figure 4.

Distribution of NEBs grouped by cell number per NEB for cell clusters of 60 cells or less, expressed as the percentage of the total number of NEBs. The naphthalene mice showed an increase in the smallest NEBs (2–5 cells) compared with control.

Figure 5.

NEB area for cell clusters of different numbers. Treatment of mice with naphthalene was associated with a significant increase in area for NEBs of 20 cells or less in size. An asterisk designates a significant difference from the corresponding control value (P < 0.05).

Figure 6.

Whole-mount airway images of NEBs in control (A and B) and naphthalene-treated (C and D) mice. Compact ovoid clusters of cells were present (A and C), as well as irregularly arranged clusters of cells (B and D) in both groups of mice. Scale bar, 20 μm.

Figure 7.

The percentage of irregularly arranged clustered neuroendocrine cells more than doubled in the airways of mice treated with naphthalene compared with control mice. An asterisk designates a significant difference from control value (P < 0.05).

Figure 8.

NEB with small CGRP-IR cells intermixed with CGRP-negative bronchiolar epithelial cells of the airway from a naphthalene-treated mouse whole-mount airway preparation. These unstained cells may be PNECs, not expressing this marker, or they could be PNEC precursors. A: The NEB appears to be surrounded by larger epithelial cells (arrow). In the 1.5-μm-thick plastic section from a whole-mount airway preparation that had been immunostained before embedding and sectioning (B), the nuclear density of the NEB is greater than that of the surrounding CGRP-negative epithelial cells. Scale bar, 10 μm.

Figure 9.

NEBs immunostained as whole-mount airway preparations and subsequently embedded in plastic and sectioned from a control mouse (A) and a naphthalene-treated mouse (B). The control mouse has an epithelial lining of uniform height and cellularity. In contrast, the mouse treated with naphthalene has an irregular epithelial lining of various heights and cellularity, particularly for those cells in close proximity to the NEB. Scale bar, 10 μm.