Three-Dimensional Mapping of Sensory Innervation with Substance P ...

Three-Dimensional Mapping of Sensory Innervation with Substance P in Porcine Bronchial Mucosa Comparison with Human Airways Jasmine P. Lamb and Malcolm P. Sparrow Department of Physiology; and The Asthma Institute of Asthma and Allergy, Department of Medicine, University of Western Australia, Nedlands, Western Australia

In asthma, neurogenic inflammation in bronchial airways may occur though the release of neuropeptides from C fibers via an axon reflex. Structural evidence for this neural pathway was sought in the pig and in humans by three-dimensional mapping of substance P–immunoreactive (SP-IR) nerves in whole mounts of mucosa using immunofluorescent staining and confocal microscopy. To show continuity, nerves were traced with 1,1ⴕ-didodecyl-3,3,3ⴕ,3ⴕ-tetramethyl indocarbocyanine perchlorate from their epithelial endings through the mucosa. The pan-neuronal marker protein gene product 9.5 revealed an extensive apical and basal plexus of nerves in the epithelium; 94% of these were varicose SP-IR fibers. Apical SP-IR fibers had a length density of 88 mm/mm2. Varicose apical processes followed closely around the circumference of goblet cells. Calcitonin gene–related peptide was colocalized with SP-IR in varicosites. The epithelial fibers converged into bundles as they entered the lamina propria where lateral branches ran along arterioles, often contiguous with the vascular smooth muscle. 1,1ⴕ-didodecyl-3,3,3ⴕ,3ⴕ-tetramethyl indocarbocyanine perchlorate tracing showed that they projected to the epithelium. SP-IR fibers were rare near postcapillary venules. In human bronchial epithelium, protein gene product 9.5 revealed a similar apical and basal plexus of varicose fibers that weakly stained for SP-IR. Thus, a continuous sensory nerve pathway from the epithelium to arterioles provides structural support for a local axon reflex. Keywords: neurogenic inflammation; substance P; afferent pathways; bronchi/innervation; human/physiology; swine/physiology; neuropeptides.

The sensory innervation of the bronchial mucosa comprises mainly varicose, unmyelinated, nociceptive C fibers that detect chemical substances present in the airway lumen (1). Sensory nerve endings in the epithelium respond to stimulation by generating impulses that are conducted centrally, and locally with the release of the proinflammatory neuropeptides, calcitonin gene-related peptide (CGRP), substance P (SP), and neurokinin A (2, 3). In the airway mucosa, neuropeptides cause microvascular plasma leakage (4–6), arteriolar dilation (7), mucous secretion (8), and recruitment of inflammatory cells (9, 10), collectively described as neurogenic inflammation. There is functional evidence that part of the neurogenic response, for example, plasma extravasation in rats (11) and bronchial vasodilatation in pigs (12) and dogs (13), can be attributed to a local axon reflex in tracheal and bronchial mucosa. This suggests that a sensory nerve pathway

(Received in original form December 5, 2001; accepted in final form July 30, 2002) Supported by an Australian Research Council Grant. Correspondence and requests for reprints should be addressed to Jasmine P. Lamb, Department of Physiology, University of Western Australia, 35 Stirling Highway, Nedlands, Western Australia 6009. E-mail: [email protected] Am J Respir Crit Care Med Vol 166. pp 1269–1281, 2002 DOI: 10.1164/rccm.2112018 Internet address: www.atsjournals.org

extends from nerve endings in the epithelium to underlying target vessels and glands where neuropeptides are released. This mechanism has been proposed as a cause of human asthma (14, 15), a condition in which increased levels of tachykinins and albumin in bronchial lavage fluid are found, indicating that tachykinin release and vascular leakage occurs (2). SP-immunoreactivity (SP-IR) and CGRP-IR nerves are found in the mucosa of airways in humans (16, 17), pigs (18), cats (16, 19), dogs (20), guinea pigs (16, 21, 22), and rats (4, 16) by immunohistochemistry. There is an abundance of SPcontaining nerves in the bronchial epithelium in rats and guinea pigs (4, 16), but the occurrence of these nerves in larger mammals and in humans is controversial. Guinea pig and human mucosa have been reported to contain similar amounts of SP determined by radioimmunoassay (6, 16), whereas SP-IR nerves determined immunohistochemically were sparse in large mammals, including pigs and humans (23). Furthermore, although Lundberg and colleagues (16) and Ollerenshaw and colleagues (17) showed SP in the human bronchial epithelium, many later studies have found little or no SP-IR nerves (24–28). There remains strong functional evidence that in large mammals the bronchi are innervated by neuropeptide-containing sensory nerves. In response to capsaicin, a vasodilator response is mediated locally via an axon reflex and centrally via vagal cholinergic and noncholinergic parasympathetic nerves in pigs (12), dogs (13, 20), and cats (29). This suggests that the absence of SP, CGRP, or neurokinin A fibers mentioned previously here may have been methodological. A delay in fixation may lead to SP degradation by endogenous neutral endopeptidase. SP levels may decline with age (26) and exposure to irritants such as cigarette smoke (30). Conventional histology, which uses thin sections, is not suited for following nerve pathways with a complex three-dimensional arrangement. Neurogenic inflammation involving C fiber–mediated reflexes continues to be viewed as a cause of bronchial hyperresponsiveness and asthma (3, 10, 14, 31). With respect to local reflexes, it is hypothesized that there are continuous nerves pathways between the sensory nerve endings in the epithelium and mucosal blood vessels. To provide structural evidence for this mechanism, these fibers in the bronchial mucosa in humans and large animals need to be mapped. The aim of this study was threefold: first to characterize the distribution of SP- and CGRP-containing nerves in the young pig where loss of sensory nerves in the epithelium through age or environmental influences would be minimal. Pig lung tissues were therefore fixed rapidly in situ by perfusion via the trachea to avoid the breakdown of tachykinins by endopeptidases. The second aim, in the human bronchial mucosa, was to show that morphologically similar nerves were present in the epithelium by their immunoreactivity to protein gene product 9.5 (PGP-IR), a protein found in all nerve cells. The

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third aim was to demonstrate that the projections of the epithelial nerves to the blood vessels in the lamina propria are continuous using 1,1⬘-didodecyl-3,3,3⬘,3⬘-tetramethyl indocarbocyanine perchlorate (DiI), a lipophilic carbocyanine tracer (32). This study uses a new approach: tracing nerve pathways in three dimensions in whole mounts of mucosa from the epithelial surface to their target tissues in the lamina propria using the confocal microscope.

Tracing of Neural Pathways

METHODS

Imunohistochemistry

Porcine Bronchial Mucosa Landrace pigs (n ⫽ 6, 4 weeks old and 9–11 kg in body weight) were obtained from Medina Agricultural Farm (Medina, WA, Australia). Pigs were sedated with tiletamine/zolazepam (0.4 mg/kg intramuscularly; Virbac, Peakhurst, NSW, Australia) and xylazine (0.2 mg/kg; Troy Laboratories, Smithfield, NSW, Australia) and euthanized with sodium pentabarbitone (163 mg/kg intravenously). Within 5 minutes, an endotracheal tube (6.0 mm inside diameter) was installed into the distal trachea, and the lungs were deflated by carefully puncturing the chest wall and reinflated in situ to 15–20 cm H2O with fixative 4% paraformaldehyde in phosphate-buffered saline (pH 7.2) via the tracheal tube. Constant pressure was maintained for 1 hour. The trachea was ligated, and the lung was excised and immersed in fixative. The main stem bronchi were dissected out between second and fifth laterals (33). The internal diameter of each bronchus ranged from approximately 2–8 mm. The bronchus was opened out and pinned epithelial side up onto a petri dish lined with Sylgard (Dow Corning, Midland, MI). The mucosa was separated at the lamina propria/smooth muscle junction by peeling it away from the wall. Procedures were approved by the Animal Ethics Committee of the University of Western Australia.

Human Bronchial Mucosa Samples of bronchi were donated with the consent of patients (n ⫽ 5) undergoing lobectomy for carcinoma. One female patient was 39 years old and was a nonsmoker. All others had a history of smoking and were elderly, with an average age of 65 years. Following lobectomy, small segments of bronchi (3–7 mm in internal diameter) were obtained and fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7.2) as soon as practical. There was an unavoidable delay between excision and fixation of 1.5–4.0 hours. The bronchus was opened, and the mucosa was separated at the lamina propria/smooth muscle junction. In the 39-year-old nonsmoker, the mucosa could be readily peeled away from the wall, but in the older patients, separation required a scalpel. Sections of fixed bronchi were also routinely frozen in isopentane, cooled in liquid nitrogen, and cryosectioned in 30-␮m thick sections. The use of human lung tissue was approved by the Human Ethical Committee of Western Australia.

The fluorescent dye DiI (Molecular Probes, Eugene, OR) was evaporated from a solution in methanol onto 100-␮m glass beads (Sigma, St. Louis, MO). These beads were gently placed with a glass spreader onto the luminal surface of freshly fixed porcine bronchi segments pinned out in a petri dish (as described previously here). The preparation was immersed in fixative and incubated at room temperature for 18 days to allow for diffusion of the dye along the nerves before the mucosa was processed for immunohistochemistry.

Antibodies to the pan-neuronal marker, PGP; sensory neuropeptides, CGRP and SP; smooth muscle and myoepithelial cells, ␣-actin; and epithelial cell, cytokeratin 18, were used (Table 1). Phalloidin Oregon green 488 conjugate (Molecular Probes), a toxin that stains F-actin in epithelial cells, was also used. The staining protocol was adapted from Weichselbaum and Sparrow (34). Specimens were permeabilized in dimethyl sulfoxide with the exception of tissues already labeled with DiI where 70% glycerol buffered with 0.5 M bicarbonate, pH 8 (32), was substituted because the lipophilic tracer is insoluble in this solution. The tissue was immersed in the respective permeabilizing agent for 5 ⫻ 10 minutes, agitated in washing buffer (phosphate-buffered saline, pH 7.2) for 5 ⫻ 10 minutes, and incubated for 30 minutes in 1% skim milk powder in buffer to block nonspecific binding. Specimens were then incubated overnight in a humidified chamber at 4⬚C with primary antibodies at dilutions of 1:200 except CGRP, 1:400, and then washed 5 ⫻ 30 minutes. This step was repeated for secondary antibodies (1:200) and phalloidin (100 U/ml, incubated at room temperature). The specimens, suspended in 90% glycerol containing p-phenylethylenediamine to reduce bleaching of the fluorochromes, were mounted in custommade chambers to reduce compression of the whole mount and to enable the specimen to be viewed from both sides.

Confocal Laser Scanning Microscopy The confocal microscope used was MRC1024 with Laser Sharp acquisition version 3.2 software (BIORAD; Hemel Hempsted, Hertfordshire, UK) and a Diaphot inverted microscope (Nikon, Japan) focusing a Krypron/Argon laser (American Laser Corp., Salt Lake City, UT). The excitation wavelengths of the laser were 488, 568, and 647 nm. Fluorophores Oregon Green 488, CY3, DiI, CY5, and Alexa Fluor 633 (Table 1) had absorption (excitation) maxima at 496, 554, 551, 649, and 632 nm, and their emission maxima were 524, 566, 585, 666, and 647 nm, respectively. In double- and triple-labeling experiments, the fluorophores were selected for maximal separation, and no bleed through occurred. Each whole mount was scanned at increasing depths of focus (0.5-␮m steps) producing a z series of xy images. From this z series the brightest pixel at each xy location was projected to give a twodimensional image using Confocal Assistant 4.2. The z series was also viewed in cross-section with National Institutes of Health image software. Cross-sections (x series of zy images and y series of zx images)

TABLE 1. ANTIBODIES Primary Antibodies PGP 9.5: polyclonal, raised in rabbit (UltraClone, Isle of White, UK) CGRP: polyclonal, raised in rabbit (Amersham, Buckinghamshire, UK)

Fluorophore-labeled Secondary Antibodies Anti–rabbit IgG raised in goat conjugated to Oregon Green 488 or Alexa Fluor 633 (Molecular Probes, Eugene, OR) or CY5 (Zymed, San Francisco, CA)

SP: monoclonal, raised in rat (Chemicon, Temecula, CA)

Anti–rat IgG raised in the goat conjugated to CY3 (Amersham) or Alexa Fluor 633 (Molecular Probes)

␣-Actin: monoclonal, raised in mouse (Sigma, St. Louis, MO).

Anti–mouse IgG raised in the goat conjugated to Oregon Green 488 (Molecular Probes)

Cytokeratin 18: monoclonal, raised in mouse (Dako, Denmark) Definition of abbreviations: CGRP ⫽ calcitonin gene-related peptide; IgG ⫽ immunoglobulin G; PGP ⫽ protein gene product; SP ⫽ substance P.

Lamb and Sparrow: Sensory Nerves in Bronchial Mucosa

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were reconstructed and projected. Adjacent fields were montaged using Adobe Photoshop 5.0 software. In multiple-labeling experiments, single fields were scanned for each marker, and the images were colored and superimposed with Adobe Photoshop 5.0 software.

Nerves in the Epithelium Colocalization of SP-IR and PGP-IR nerves in the porcine bronchial epithelium was compared in double-staining experiments. In seven projected fields from three pigs, a count of the number of times nerve fibers crossed the lines of a grid (total line length 1.4 mm) was performed for each nerve marker. The difference between the counts of the SPIR and PGP-IR nerves was compared using a one-tailed, paired t test. In the epithelium, the length of the SP-IR nerves in the apical and basal layers was compared for each of six fields (three pigs). This was done with reference to three-dimensional images created from their z series. In the apical layer, the fibers were traced from their nerve endings to the point at which the axon began to descend through the epithelium (denoted “X”). The basal layer was traced from X to the point at which nerves descended into the lamina propria. These were measurements of horizontal length; it was not feasible to measure the vertical components. The nerve tracings were measured with National Institutes of Health image software to calculate the total length per unit area and were expressed as mean ⫾ SEM. The number of nerve processes in the apical layer and the number of fibers rising up from the basal layer were also counted. The size of each field was 31,605 ␮m2.

RESULTS Mapping of Sensory Nerves in Porcine Bronchial Mucosa

An extensive innervation of the bronchial mucosa was demonstrated by immunofluorescence labeling of the nerves for PGP 9.5, a pan-neuronal marker, and confocal imaging to depths of 100 ␮m through whole mounts of mucosa. All images shown are orientated with the mucosal folds running from left to right. Figure 1A is a representative field of PGP-IR nerves imaged from the luminal surface. Tissue autofluorescence resulting from 488-nm excitation faintly reveals the epithelial cells enabling the depth of the epithelium to be determined during scanning (approximately 20 ␮m), and this allowed the distribution of nerves to be partitioned above and below the basal lamina. Figure 1B shows an abundance of varicose fibers in the epithelium, with fewer nerves in the trough of the mucosal fold. The lamina propria contained many nerve bundles that predominantly ran along the length of the airways (Figure 1C). Specific staining for SP-IR demonstrated that the mucosa was innervated by fine fibers and a few nerve bundles (Figure 2A). In the epithelium, these nerves had characteristically fine, varicose axons (approximately 1 ␮m in diameter), and their endings often had one to five large terminal varicosities (approximately 3 ␮m) (Figure 2B). For each of 20 fields (four pigs), the three-dimensional information contained in the z series was used to show the arrangement of SP-IR nerves in cross-section by digital reconstruction. Figure 3 shows a lumen view (large panel ) together with projected cross-sections (right and bottom panels). These cross-sections showed SP-IR fibers lying in two plexi in the epithelium. An apical layer of SP-IR nerves lays just beneath the luminal border (0–6 ␮m) comprised of many fibers and nerve endings and is shown as a line diagram (Figure 3B) where X marks the point at which fibers leave this layer. These axons descended to the base of the epithelium, where they ran laterally forming a second plexus, also shown as a tracing (Figure 3C). In the apical layer, there were 1,904 ⫾ 303 terminal processes per mm2, and the apical network was supplied by 709 ⫾ 155 ascending nerves per mm2, which arose from a near continuous basal nerve network, that is, approximately three apical processes per (ascending) axon. The length of the processes in the apical layer ranged from 12 to 105 ␮m in Figure 3, and the maximum length was 126 ␮m for six fields from three pigs. The length–density of fibers was approximately fourfold greater in

Figure 1. Confocal projection of the mucosa from a main bronchus of a pig showing nerves immunofluorescently labeled for the pan-neuronal marker PGP 9.5 (PGP-IR). (A ) The mucosa has been scanned to a depth of 50 ␮m from the lumen. Thin varicose fibers and thicker nerve bundles are present. Mucosal fold is shown (double-headed arrow, bar ⫽ 100 ␮m). (B ) Inset field in (A). The depth is 0–20 ␮m from luminal surface (corresponds to the epithelium). Fine varicose nerves are seen (bar ⫽ 100 ␮m). (C ) Inset field in (A). The depth is 20–50 ␮m. The underlying lamina propria has thick nerve bundles that run longitudinally along of the airway (bar ⫽ 100 ␮m).

the apical layer compared with the basal layer, 87.5 ⫾ 5.15 and 21 ⫾ 6 mm/mm2, respectively. We did not attempt to measure the vertical component of fibers passing through the epithelium to the basal layer.

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Figure 2. (A ) Confocal projection of SP-IR nerves in the bronchial mucosa of a pig. Varicose fibers and nerve endings are abundant with fewer nerves in the mucosal fold (doubleheaded arrow). Some nerve bundles are present (arrowheads). The image depth is 75 ␮m from the lumen (bar ⫽ 50 ␮m). (B ) A projection showing fine varicose SP-IR nerves in the bronchial epithelium of a pig. Nerve endings have several enlarged terminal varicosities (arrow). Some nerve endings have curved profiles where they encircle goblet cells (arrowhead) (see Figure 5). The image depth is 21 ␮m (bar ⫽ 50 ␮m).

To determine the population of nerves in the epithelium that contain SP, the mucosa was stained for PGP and SP concurrently (Figure 4). In seven fields from three pigs, 94% of the PGP-IR nerves were SP-IR (significant, p ⬍ 0.05). Fluorescently labeled phalloidin toxin binding to F-actin outlined the cells in the apical epithelium, enabling unstained goblet cells to be distinguished among the epithelial cells (Figure 5A). At high power, tufts of cilia at their luminal border were observed (Figure 5Ai). In this layer, epithelial nerves, both SP-IR and PGP-IR positive, frequently passed around the apex of goblet cells (Figures 5B and 5C). Epithelial cells were visualized by their autofluorescence when excited by the 488-nm laser used to reveal PGP-IR (Figures 5C and 5D), whereas goblet cells appeared as black circles. Fibers passed between epithelial cells (Figure 5D). SP-IR fibers in the basal epithelium converged into larger bundles as they penetrated the basement membrane and entered the lamina propria (Figure 6). At 20–30 ␮m into the lamina propria, these bundles ran longitudinally along the airway, whereas other bundles continued to penetrate deeper into the lamina propria. In Figure 6, nerves deep in the lamina propria were faint because the laser penetration was at its limit. In subsequent preparations, this was overcome by separating the mucosa just below these nerve trunks and scanning from the lamina propria side of the whole mount. Figure 7 shows large SP-IR nerve bundles in the lamina propria from which smaller bundles branched upward to give rise to the epithelial nerves. These nerves ran longitudinally along the airways together with other large bundles revealed by PGP-IR (Figures 8A and 1C). These SP-IR nerves retained their distinctive varicose appearance. At intervals along the airway some bundles passed through the muscularis to the adventitia (data not shown). In the lamina propria, SP-IR fibers occurred in the proximity of the mucosal circulation and mucous glands (Figures 8B–8D). Antibodies against smooth muscle ␣-actin stained the muscularis and pericytes of blood vessels and the myoepithelial cells of glands. SP-IR nerves followed small arterioles, located approxi-

mately 30 ␮m below the basement membrane, that had closely packed smooth muscle bundles encircling the vessel. These small bundles followed the contours of the outer surface of the vascular smooth muscle at the same optical depth, indicating that they lay beside the muscularis (Figure 8B). Occasionally SP-IR fibers were seen to cross (less than 5 ␮m) precapillary arterioles or the capillary bed underneath the epithelium. SP-IR fibers were seldom seen within 10 ␮m of the postcapillary venules identified by ␣-actin staining of the pericytes that surround the vessel, incompletely leaving claw–like spaces (Figure 8B). Larger bundles of SP-IR nerves ran along arterioles at approximately 10 ␮m (Figure 8C), and these were on occasions followed to mucous glands. SP-IR fibers were observed within mucous glands that had cells with unstained, cytoplasmic regions (Figure 8D). Whole mounts of mucosa stained for CGRP-IR (Figure 9A) had nerves with the same appearance and distribution as SPIR (Figure 6). Concurrent staining for SP-IR and CGRP-IR demonstrated colocalization (Figures 9B and 9C). Mapping of Sensory Nerves in Human Bronchial Mucosa

There was a marked difference in the amount of PGP-IR nerves in the 39-year-old patient compared with the elderly patients. The former patient had an abundance of PGP-IR varicose fibers in the epithelium with larger terminal varicosities (Figure 10A). A view of the whole mount in cross-section was obtained by digital reconstruction of the confocal z series (Figure 10A) and also by directly imaging the whole mount from the side (Figure 10B). Together, these views demonstrated the threedimensional arrangement of the nerves lying in apical and basal plexi in the epithelium with nerves traversing between these layers. The apical layer of fibers with nerve endings lies just beneath the luminal border with nerves following around the apex of the goblet cells (Figures 10C–10E). The basal nerves converged into bundles that entered the lamina propria. Many nerve bundles were seen deep in the lamina propria by imaging from the muscularis side of the lamina propria (data not shown). In elderly patients, few PGP-IR epithelial nerves were seen.


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Figure 3. (A, large panel) Projected image of SP-IR fibers in the bronchial epithelium of a pig viewed from lumen. Fibers branch laterally and most nerve endings have an enlarged terminal varicosity. (Right and bottom panels) Projected crosssections show nerves in the epithelium arranged in two plexi. An apical plexus lies immediately below the luminal surface (lumen ⫽ *L) with fibers descending to the base of the epithelium to form a second lateral layer. Cross-sections were reconstructed from a confocal z series with optical sections 0.5 ␮m apart. Consecutive images could not always be perfectly superimposed in the zx and zy projections because the mucosa is not completely flat because of mucosal folds (bar ⫽ 50 ␮m). (B ) Tracing of the apical fibers in large panel. Processes (12–105 ␮m in length) radiate from the top of the ascending fibers labeled X. (C ) Tracing of basal fibers in large panel to X includes the horizontal component of the ascending axons.

These occurred in small infrequent patches. Some profiles were seen ascending toward the apical surface in cryosections, but there were lengths of epithelium in which no fibers were observed. Compared with PGP-IR staining of epithelial nerves, SP-IR was very faint and required greater laser excitation resulting in a high background. Figure 11A shows strings of varicosities detected in the epithelium of the 39-year-old patient. Some colocalization of PGP-IR and SP-IR was seen in epithelial nerves where SP-IR was detected (Figures 11B and 11C). Tracing Neural Pathways in Porcine Bronchial Mucosa

To demonstrate the continuity of the pathways of sensory nerves in the bronchial mucosa of the pig, DiI tracing and CGRP immunostaining were used to follow and identify these fibers, respectively. DiI was deposited on to the luminal surface of segments of pig bronchus. There it entered the endings of varicose nerve fibers in the apical layer of the epithelium and revealed a network of fine fibers (0.5–1.8 ␮m) that had enlarged terminal varicosities (2–2.5 ␮m) (Figure 12A). Receptive fields, comprising apical epithelial fibers stemming from a common nerve, were distin-

guished with DiI. Figure 12B shows a typical receptive field (maximum span 89 ␮m) with five or more processes, and Figure 12C is a corresponding side view (a projection of y–z cross-sections) showing these fibers all originated from a bundle that had ascended from the lamina propria. DiI diffusion into nerve bundles and trunks in the lamina propria demonstrated continuity with fine epithelial fibers (Figure 12D). These trunks in the lamina propria also gave rise to nerve bundles running longitudinally along the airway (approximately 45 ␮m below the lumen). DiI-labeled trunks were comparable in diameter to those seen with PGP-IR (Figure 8A) and were much larger than SPIR bundles observed (Figures 7 and 8A). This indicates that DiI diffused across the membranes of individual fibers into adjacent nerves. The dye also diffused into unidentified cells that must be in close contact with the nerve fibers. Two principle shaped cells were noted: round (Figures 12A) and star-shaped with long processes (data not shown). Varicose epithelial nerves traced with DiI also stained positively for CGRP-IR (Figure 13A). Colocalization of these labels confirmed that the DiI was mainly tracing sensory nerves in the epithelium. Immunostaining was weaker in glycerol-permeablized tissues compared with dimethyl sulfoxide treatment (Fig-

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Figure 5. (A ) Confocal projection of the apical bronchial epithelium of a pig stained with phalloidin reveals the margins of epithelial cells. Goblet cells are unstained (scan depth 1 ␮m, bar ⫽ 10 ␮m). (Ai) Tufts of cilia on the epithelial cells are seen as white speckles (bar ⫽ 10 ␮m). (B ) SP-IR fibers in the apical bronchial epithelium of a pig (bar ⫽ 10 ␮m). (C and D) The same field as (B) concurrently stained for PGP-IR nerves. Background autofluorescence (488-nm excitation) reveals the epithelial cells but not goblet cells. Varicose fibers with nerve endings pass around the apices of goblet cells (arrowhead) and between epithelial cells (arrows). The depth of B and C is 6 ␮m. D is a projection of two optical sections at 3 ␮m of depth (bar ⫽ 10 ␮m).

Figure 4. Projected image of nerves in the bronchial epithelium of a pig concurrently immunostained for PGP and SP. (A ) The total innervation is revealed by PGP-IR. (B ) In the same field, SP-IR labels almost all of the same nerves. There are few PGP-IR nerves that are not SP-IR (arrow). The image depth is 19 ␮m (bar ⫽ 50 ␮m).

ure 9); however, this compromise was unavoidable as dimethyl sulfoxide dissolves DiI. The intensity of the CGRP-IR was strongest in the varicosities and was variable where DiI had previously diffused along the nerve. As a result, continuity of these strings of varicosities was often masked in projections. These nerve pathways were more readily followed by examining the optical sections of the z series. Continuity between epithelial nerves and axons that run along blood vessels in the lamina propria was demonstrated by merging DiI tracings of nerves through the mucosa and the corresponding ␣-actin-IR that visualized blood vessels. Figure 13B shows a DiIlabeled nerve in the lamina propria that had been traced from the epithelium (data not shown). It branches into two nerves that run longitudinally along the airway on either side of an arteriole. Concurrent CGRP immunostaining identified sensory fibers within DiI-labeled bundles with continuous nerve pathways from the epithelium to blood vessels. Figure 13C is a view of the lamina propria that shows two DiI/CGRP-IR–labeled bundles that branch from a nerve trunk and follow an arteriole. Another example of these pathways is shown through the whole mucosa in Figure 13D, where the field corresponds to Figure 12D. As a consequence of the thickness of this projection (82 optical sections), the appearance of the finer CGRP-IR nerves was poor for the reasons given previously here (Figure 13Di).

In this large montage, CGRP-IR/DiI–labeled epithelial fibers join bundles that enter the lamina propria and pass through large nerve trunks into smaller bundles that follow blood vessels running longitudinally along the airway (Figure 13Dii). In most tissue samples, sensory nerves appeared predominantly to follow arterioles (Figures 13B and 13C) and rarely venules and post capillary venules (Figures 13Dii and 13Diii). These bundles often touched the vessels or followed them closely (less than 1–10 ␮m apart). Bundles labeled by DiI were often wider than the CGRPIR stain, particularly in large nerve trunks consistent with Figure 8A where PGP-IR demonstrates a substantial number of the fibers are not SP-IR.

DISCUSSION In this study, we have mapped the distribution of SP-IR and CGRP-IR nerves in whole mounts of porcine bronchial mucosa by imaging with confocal microscopy through depths of 100 ␮m. A comprehensive view of the three-dimensional organization in the mucosa and their anatomical relationship to the bronchial circulation and mucous-secreting cells has been obtained in a way that has not been previously possible using thin sections. It can be reasonably concluded that these varicose fibers are sensory nerves based on their distribution and neuropeptide immunoreactivity. In the lamina propria, varicose SP-IR and CGRP-IR nerves lie in nerve trunks adjacent to the airway smooth muscle and give rise to bundles that travel toward the epithelium. These nerves have lateral branches to within approximately 20 ␮m of the epithelium that run along the arterioles and go to mucous glands; smaller bundles are contiguous with the vascular smooth muscle of smaller arterioles. The nerves that reach the epithelium pass through the basement membrane and spread laterally to form a network at the base of the epithelium. From this, plexus fibers ascend between epithelial cells to the apex where they arborize to form a layer of varicose processes within 3 ␮m of the luminal surface. Many processes follow very closely around the circumference of goblet cells.


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Figure 6. SP-IR nerve fibers in the bronchial mucosa of a pig imaged from the lumen. (Large panel) Projection showing fine varicose SP-IR fibers in the mucosa. Nerve terminals have swollen varicosities (image depth of 104 ␮m). (Right and bottom panels) Projected crosssections show SP-IR nerves in the epithelium converging into bundles as they enter the lamina propria. Some bundles run longitudinally along the airway 20–30 ␮m below the epithelium (arrow). Others penetrate deeper (arrowhead). Cross-sections were reconstructed from a confocal z series with optical sections 0.5 ␮m apart (bar ⫽ 50 ␮m). Note that the apical and basal plexuses are not resolved here because the mucosal strip did not lie completely flat.

The apical layer of epithelial nerves was the most densely innervated region of the mucosa and comprised single, varicose, SP-IR, and CGRP-IR processes spanning up to 126 ␮m that terminated in one or more enlarged varicosities. These appeared to be arborizations originating from a central axon having one to five terminal processes, confirmed by DiI tracing, which spread out laterally, forming irregularly shaped receptive fields that were elongated in the direction of the mucosal folds. The nerves ramifying the apical epithelium were most dense on the ridges of the mucosal folds. In this layer, the mean density of sensory nerves was 1,900 terminal processes per mm2, and they arose from 709 central fibers per mm2 that had ascended through the epithelium from the basal plexus. The average SP-IR length– density of axons in the apical epithelium was 88 and 22 mm/ mm2 in the basal layer. The pig bronchus is more densely innervated than the rat trachea, which has 44 mm/mm2 SP-IR axons in the epithelium, but an apical layer was not distinguished (4). In human bronchi, PGP-IR epithelial nerves were revealed, most strikingly in the 39-year-old nonsmoker, where they were distributed along the length of the bronchus. The arrangement of nerves was very similar to that of the pig. An apical layer of varicose processes terminated in enlarged varicosities, and these fibers encircled goblet cells. These processes arose from fibers that had crossed the epithelium from a basal plexus that was supplied by nerve bundles in the lamina propria. By demonstrating these layers of nerves in the epithelium that are common

to the human and pig and by identifying sensory neuropeptides in these porcine nerves, this study has strengthened the case for this arrangement of epithelial sensory nerves in humans. Ultrastructural studies of human bronchial epithelium have also revealed axon profiles situated close to the basement membrane and to the airway lumen, with some fibers laying between the epithelial cells that were characteristic of sensory nerves (35). In our much older patients with a history of smoking, there were fewer PGP-positive epithelial nerves, and they tended to be unevenly spread across the epithelium. SP-IR epithelial nerves were faintly stained in human bronchi. The weak signal suggests low concentrations of SP-IR in the varicosities. This could be due to break down of the tachykinins by neuroendopeptidase, SP being particularly sensitive, during the unavoidable delay before fixation. Some studies have been able to demonstrate a significant number of SP-IR nerves in human bronchial epithelium in thin sections (16, 17, 26). However, in the majority of studies, SP-IR nerves are described as “negative” (25, 27, 28, 36) or “rare” (24, 26) in human bronchial epithelium. It appears that the difference lies mainly in the age and medical history of the subjects (26, 28) and sensitivity of the method. There are obvious limitations in obtaining young airways that have been rapidly fixed. This shows that some improvement in demonstrating SP nerves can be obtained using whole mounts in conjunction with confocal microscopy where high-quantum yield, long-wavelength fluorophores that emit

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Figure 7. A montage of projected cross-sections showing SP-IR nerves deep in the bronchial lamina propria of a pig. The mucosa was scanned from the muscularis side of the lamina propria through a depth of 85 ␮m to the luminal surface. Thick SP-IR nerve bundles run along the airway deep in the lamina propria and give rise to the fibers in the epithelium (bar ⫽ 25 ␮m).

Figure 8. (A ) Montage of a nerve trunk of a pig imaged from muscularis side of the lamina propria in a bronchial airway of a pig. PGP-IR has been colorized blue and SP-IR green, and additive color mixing of these primary colors of light produces cyan thus demonstrating colocalization. Thick PGP-IR nerve bundles run along the airway and some of these bundles contain varicose SP-IR nerves (bar ⫽ 50 ␮m). (B ) A montage showing vessels of the mucosal circulation imaged from the muscularis side of the lamina propria in a bronchial airway of a pig. The vascular smooth muscle (red) is stained by ␣-actin. An arteriole (*A) located approximately 30 ␮m below the basement membrane is identified by closely packed smooth muscle bundles encircling the vessel. An SPIR bundle (cyan) follows the contours of the arteriole wall and lies on the vascular smooth muscle (between arrowheads). Fibers also came within less than 5 ␮m of the vascular smooth muscle of a precapillary arteriole (*PCA) (arrow). Fibers were more than 10 ␮m from collecting venules (*CV) and postcapillary venules (*PCV, bar ⫽ 50 ␮m). (C ) A bundle of SP-IR nerves follows an arteriole approximately 10 ␮m from the vascular muscularis in the bronchial lamina propria of a pig (bar ⫽ 50 ␮m). (D ) Confocal image shows varicose SP-IR fibers (cyan) within a mucous gland in the bronchial lamina propria of a pig. Faint cytokeratin-18 staining (red) reveals a mucous gland containing cells with unstained cytoplasmic regions (bar ⫽ 50 ␮m).


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Figure 9. (A, large panel) Confocal projection showing CGRP-IR varicose fibers, many with prominent nerve endings (enlarged varicosities) and nerve bundles in the bronchial mucosa of a pig. Scanned from lumen (bar ⫽ 25 ␮m). (Right and bottom panels) Projected cross-sections show an apical plexus in the epithelium with fibers descending to a basal plexus before converging into bundles that enter the lamina propria. Some nerves descend approximately 20 ␮m before running longitudinally, whereas others descend more steeply into the lamina propria. Cross-sections were digitally reconstructed from the z series of optical sections through a depth of 72 ␮m. (B and C) Colocalization of SP-IR (B) and CGRP-IR (C) in apical epithelial nerves viewed from the lumen of the porcine bronchial airway (bar ⫽ 25 ␮m).

strongly at a wavelength well separated from tissue autofluorescence were employed. In the pig, SP-IR fibers comprised 94% of all nerves in the epithelium since almost all nerves stained by pan-neuronal marker PGP colocalized with SP. This compares favorably with rat trachea in which SP-IR varicose axons comprised 90% of those staining for PGP (4), and thus, the great majority of nerves were likely to be C-fiber afferents. The residual population of nerves (6–10%) was significant in both studies and may be a subgroup of mechanosensory, thin myelinated A␦ fibers (rapidly adapting receptors) that are thought to lie in the epithelium (37). In guinea pig trachea, Hunter and Undem (38) retrogradely traced epithelial nerves to their somata in the jugular ganglia where both SP-IR and SP-negative neurones were labeled, showing that two populations of nerves innervate the epithelium. An electrophysiologic study has shown SP-IR and SP-negative neurones in the jugular ganglia project C fibers and A␦ fibers to the trachea, respectively (39). Airway disease may profoundly influence SP expression. In airway inflammation caused by allergic challenge (40, 41) or viral infection (42) in rodents, there is a qualitative switch in nodose ganglia neurones whereby SP expression is induced. This occurs in A␦, non-nocioceptive neurones that projected to the tracheal epithelium (41, 42). Neurotrophic factors present in inflammation have also been demonstrated to cause plasticity in afferent nerves (43, 44). Recently,

endobronchial biopsies from patients with moderate asthma revealed an increase in SP staining in the bronchial epithelium, but the staining was diffuse and thus did not resemble the nerve fibers that we have described (45). In the pig and human, many of the fibers in the apical epithelium pass between epithelial cells and closely follow around the apex of goblet cells, with varicosities and terminal endings lying on their circumference. This arrangement of apical nerves is likely to facilitate the detection of chemical substances, with these nerves releasing neuropeptides that trigger local protective effects such as increased frequency of cilia beat on epithelial cells and mucous secretion from goblet cells. There is functional evidence that increased beat and secretion is mediated by SP activation of tachykinin receptors (neurokinin 1) present on these cells (46, 47). These local functions are thought to be characteristic properties of bronchial C fibers in addition to evoking centrally mediated reflexes (31, 48). SP-IR and CGRP-IR fibers in the lamina propria were seen passing through capillary beds and to follow arterioles (less than 1 to 10 ␮m separation); some fibers followed closely the vascular smooth muscle of small arterioles (less than 1 ␮m). In rat trachea and dog bronchus SP-IR nerve profiles also closely follow arterioles in the lamina propria (4). Thus, neuropeptides released from these fibers can readily diffuse over the vascular smooth muscle to cause vasodilation. These neural structures provide

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Figure 10. Nerves in a whole mount of human bronchial mucosa stained for PGP-IR in a 39-year-old nonsmoker. (A, large panel) Confocal projection reveals varicose PGP-IR fibers in the epithelium viewed from the lumen. Nerve endings have enlarged terminal varicosities (arrowhead). (Right and bottom panels) Projected crosssections show nerves in an apical plexus immediately below the luminal surface. These fibers descend to the base of the epithelium. (B ) Human bronchial mucosa scanned from the side of the whole mount through a thickness of 37 ␮m showing PGP-IR fibers in crosssection. Apical nerves descend to the base of the epithelium where they converge into bundles that enter the lamina propria. Pulmonary neuroendocrine cells lie in the epithelium (arrowhead, bar ⫽ 50 ␮m). (C and D ) The apical epithelium showing PGP-IR stained varicose fibers that pass around the apex of goblet cells (arrowhead, scan depth 14 ␮m). (E ) Autofluorescence arising from 488-nm excitation has been overlaid to show the goblet cells more clearly (black).

the basis for the functional studies that have shown stimulation of vagal C fibers releases tachykinin neuropeptides in pigs (12) and dogs (13) with an increase in tracheobronchial blood flow; a component of this vasodilation has been attributed to an axon reflex. We have shown that there is continuity of putative C fibers (CGRP-IR), necessary for an axon reflex, from the epithelium to deep in the lamina propria by tracing DiI-labeled nerves that stained for CGRP-IR. These fibers were followed from their varicose nerve endings in the apical epithelium through the lamina propria directly, or indirectly via large nerve bundles, to vascular beds where they ran along arterioles (less than 1 ␮m). Identification of some ␣-actin–stained vessels was difficult as a consequence of glycerol permeabilization and imaging them deep within the whole mount. Markers specific for the endothelium of vessels, for example, Lycopersicon esculentum tomato lectin, may be an alternate stain used to classify vessels (49, 50). DiI diffused along nerve fibers, tracing their pathways, and also diffused across their membranes into adjacent fibers. Thus,

Figure 11. SP staining of the epithelium in a whole mount of human bronchial mucosa (39-year-old nonsmoker). (A ) A projection of SP-IR staining viewed from the lumen. The mucosal fold runs obliquely (scan depth 42 ␮m, bar ⫽ 50 ␮m). (B ) PGP-IR varicose nerves in the epithelium. Viewed from lumen (scan depth, 17 ␮m, bar ⫽ 25 ␮m). (C ) Same field: some colocalization of SP-IR with PGP-IR in these varicose nerves can just be detected (bar ⫽ 25 ␮m).


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Figure 12. Nerves in the bronchial mucosa of a pig traced with DiI (bar ⫽ 50 ␮m). (A ) Confocal projection of DiI-labeled epithelial fibers with enlarged terminal varicosities (arrowhead). Deposits of DiI (*D) remain on the epithelium where the bead lay with some light staining of surrounding epithelial cell membranes. Some axons also trace to an unidentified cell body (arrow) that has taken up the dye. Scanned from the lumen. (B ) DiI tracing reveals a receptive field of varicose processes in the epithelium viewed from the luminal surface. Background fluorescence reveals goblet cells (black circles). (C ) Side view of B (y projection of reconstructed x–z cross-sections) showing the pathway of the nerve fibers through the mucosa. Epithelium (*E) and lamina propria (*LP). DiI has diffused into a nerve (right) that passes down into the lamina propria to a large nerve bundle (arrowhead). The DiI traces the nerve toward the lumen and at the apical epithelium varicose fibers are seen to radiate out. (D ) Montage reveals continuity of fibers from the epithelium to nerve trunks in the lamina propria traced with DiI. (Large panel) Confocal projections viewed from lumen. DiI has diffused along a nerve trunk (top left) and continues into smaller bundles and varicose fibers. A long epithelial fiber is shown with a large terminal varicosity (arrow). (Right and bottom panels) Each sideview (montage of projected cross-sections of the large panel ) shows the varicose fibers lie in the epithelium (*E) and nerve trunks below in the lamina propria. The DiI has also diffused axons and into unidentified cell bodies in the vicinity of the basement membrane (arrowhead). Scanned from the lumen to a depth of 81 ␮m.

the entire width of nerve bundles and trunks was revealed; these were of similar dimensions as those obtained using PGP-IR. CGRP-IR staining was a minor component of large nerve trunks. DiI also labeled a few cells that were in close contact with the nerves. These cells need to be identified, but antibodies to mast cell tryptase and human leukocyte antigen-DR raised against human antigens were not cross-reactive in pigs. Although the extensive staining of nerves by DiI is useful, an alternative nerve tracer that remains confined to the fiber, which it labels initially in the epithelium, should also be sought for subsequent tracing experiments. SP-IR and DiI/CGRP-IR co-stained fibers were rarely seen close to postcapillary venules. This is consistent with studies in rat trachea and dog bronchus in which SP-IR nerves were not seen near postcapillary venules (4). The paucity of SP nerves near the postcapillary venules could be construed as consistent

with the view that leakage from them in large mammals is not a prominent feature of neurogenic inflammation as it is in rodents (51, 52). This is controversial as plasma extravasation can be induced in patients with asthma by SP inhalation (53). Plasma extravasation occurs in the rat via NK-1 receptor-mediated gap formation between endothelial cells (49, 54); the source of SP may be the extensive plexus of C fibers in the basal epithelium (4). In the pig, the postcapillary venules lay 15–20 ␮m below a plexus of SP-rich nerves in the basal epithelium. We have also shown that human bronchi have a similar neural plexus, but we could demonstrate only traces of SP-IR in these fibers. In summary, the bronchial epithelium of the pig is abundantly supplied by varicose SP-IR and CGRP-IR nerve fibers. These fibers lie in a plexus above the basal membrane and project upward between the epithelial cells to the apical epithelium, where they arborize into processes that terminate in enlarged

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Figure 13. The pathways of epithelial nerves (Ai–Aiii) traced to blood vessels in the lamina propria (B–D) with DiI (blue) in porcine bronchial mucosa. After 18 days of exposure to DiI, the mucosa was stained for sensory nerves with CGRP-IR (green/cyan) and for blood vessels with ␣-actin-IR (red). All images are confocal projections and were scanned from the lumen (bar ⫽ 50 ␮m). (A ) Part of the field from Figure 12A. (Ai) Varicose epithelial nerves have been traced with DiI and (Aii) stained for CGRP-IR. Only parts of the axons are strongly stained including the terminal varicosities. (Aiii) These images, when superimposed, show the strongest CGRP-IR staining where DiI is weak (arrow). (B ) In the lamina propria DiI has labeled nerve bundles (arrowheads) that run above and below an arteriole (*A) less than 1–10 ␮m apart. (Ci) In the lamina propria DiI has diffused into a large nerve trunk that divides into smaller nerve bundles (arrowheads) that lie less than 1–5 ␮m from an arteriole (*A). These arterioles are outlined in (Cii). Within the bundles a few varicose fibers stain for CGRP-IR. (D ) The images in (Di) and (Dii) are montages that include the area in Figure 12D (large panel ) where DiI nerve pathways are shown. The image depth is 81 ␮m, comprising the epithelium and the lamina propria. (Di) CGRP-IR is more prominent in nerve bundles compared with the smaller single nerve fibers where only the varicosities are seen. A terminal process (arrowhead) that corresponds to the varicose epithelial fiber is shown in Figure 12D (large panel, arrow). (Dii) The DiI tracing from Figure 12D (large panel), showing a continuous pathway of nerves from the epithelium into the lamina propria, has been overlaid on the CGRP-IR (Figure 13 Di) and ␣-actin-IR of this montage. The CGRPIR labeling corresponds with some of the DiI nerves. In the lamina propria CGRP-IR bundles comprise a minor component of much larger DiI-labeled bundles. ␣-Actin-IR staining reveals blood vessels in the lamina propria; the upper and lower vessels resemble a post capillary venule (*PCV) and an arteriole, respectively. A DiI- and CGRP-IR–labeled nerve is revealed running between two blood vessels and is shown at higher magnification in (Diii). It runs close to the larger vessel (less than 1–4 ␮m) and also passes by (1–3 ␮m) the smaller vessel at the far left.

varicosities adjacent to the airway lumen. The basal layer is supplied by nerve bundles from the lamina propria that arise from nerve trunks that run deep in the lamina propria adjacent to the airway smooth muscle. As these bundles pass through the microvasculature beneath the epithelium, some run along vessels, primarily arterioles, and lie within a micron of their vascular smooth muscle. The continuous nature of these pathways has been demonstrated by tracing individual nerve fibers from the lumen with DiI and provides a structural basis for the existence of a local axon reflex that could be involved in the vasodilation observed in neurogenic inflammation. In human bronchus, a similar distribution of epithelial nerves was shown

using the pan-neuronal marker PGP 9.5, but staining for SP-IR was weak or not detected. Acknowledgment : The authors thank Dr. Markus Weichselbaum for consultation on confocal imaging and image analysis and to Dr. Alan James, Dr. Neil Carroll, and Dr. Darryl Knight for human lung samples. Jasmine Lamb was the recipient of an Annie Phillip’s scholarship.

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