INTRODUCTION

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The innervation of mammalian airways includes a dense plexus of nerve fibers just beneath the epithelium (1). Arising from this nerve plexus, numerous fibers can be observed penetrating the basement membrane and extending into the epithelial layer, often nearly reaching the lumen (2). In humans, it has been estimated that up to 10 such fibers are present in each millimeter of airway surface area (3). This plexus of afferent nerve fibers participates in the defense of the airways by its ability to initiate defensive respiratory reflexes such as coughing, and autonomic reflexes including mucus secretion, vasodilatation, and bronchoconstriction (5).

The epithelial nerve plexus is predominantly afferent in nature. Using retrograde neuronal labeling techniques it has been found that the vast majority of afferent nerve fibers in the guinea pig trachea are derived from neurons with cell bodies located in the nodose and jugular ganglia of the vagus nerve (8, 9). Because these ganglia are derived from distinct embryological structures, it is not surprising that the physiological responsitivity and neurochemistry of their nerve endings are quite different (9). Thus, about 30% of the afferent fibers innervating the guinea pig trachea are C fibers containing substance P (SP), neurokinin A, and calcitonin gene related peptide. Virtually all of these fibers are derived from neurons with cell bodies in the jugular ganglia, with the nodose neurons contributing less than 2% of the neuropeptide-containing afferent fibers innervating the guinea pig airways (8, 9).

The heterogeneity in the chemistry and physiology of nodose and jugular neurons underlies the importance of ascertaining whether the afferent nerves fibers within the epithelial layer are derived from cell bodies located in the nodose ganglia, jugular ganglia, or both. In this study we describe experiments in which retrograde tracing studies were carried out using two neuronal tracers, one that diffuses throughout the airway wall, the other where diffusion is limited to the epithelial layer. The results support the hypothesis that the nerve fibers specifically within the epithelial layer of the guinea pig trachea are derived almost exclusively from neurons situated within the jugular ganglia.

	METHODS

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A total of 12 male guinea pigs (100 to 200 g, Harlan Sprague-Dawley Inc., Indianapolis, IN) were used for retrograde tracer studies designed to identify and characterize the nodose and jugular ganglion neurons that innervate the tracheal epithelium. The experimental design consisted of three groups: a control group of four guinea pigs that received 30 µl of saline into the proximal trachea, one experimental group of four guinea pigs instilled with 30 µl of rhodamine-labeled latex microspheres into the proximal trachea, and one experimental group of four guinea pigs instilled with 30 µl of fast blue dye into the proximal trachea. After a 7-d transport period, the experimental and control animals were killed. The nodose and jugular ganglia (Figure 1) were removed and prepared for SP immunocytochemistry and the trachea was also removed to assess the diffusion of the retrograde tracers.

View larger version (93K): [in this window] [in a new window]   Figure 1.   Photograph of the rostral portion of vagus nerve (X), associated cranial nerves (IX, XI, and XII), and the two sensory ganglia, after being dissected from the guinea pig (kindly provided by Dr. Wolfgang Kummer). Note: in this species the jugular and nodose ganglia are easily recognized as two completely separate entities.

Tracheal Instillation of Retrograde Tracers

Guinea pigs were anesthetized by intramuscular injection of ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (2.5 mg/kg). Thirty µl of rhodamine-labeled latex microspheres (Luma Fluor Inc., Naples, FL), 5% fast blue dye (Sigma Chemical Co., St. Louis, MO), or 10% saline were instilled into the proximal region of the trachea. The instillation technique involved a 50-µl Hamilton microsyringe with a curved metal feeding tube connected to the syringe. In addition, flexible wire covered with gauze was connected to a plexiglass board elevated 45°. The guinea pig was placed on the board by positioning the gauze wire behind the upper incisors to aid in the extension of the head and the opening of the mouth. Gauze-covered forceps were used to deflect the tongue outward and ventral. A light was positioned for optimal visualization of the posterior oral cavity. The feeding tube connected to the microsyringe was deflected downward past the posterior region of the tongue and into the laryngeal orifice. Once an increase in resistance was felt, the feeding tube was pushed through the laryngeal orifice and the tracer was immediately instilled into the proximal trachea. Upon entrance into the trachea, the microsyringe was rotated in a circular pattern to enable a greater chance of tracer distribution around the circumference of the trachea. In these experiments the fluorescent dyes were limited to the rostral region (approximately upper 25%) of the trachea. The animal was then removed from the plexiglass board and positioned supine during recovery from anesthesia.

Tissue Removal and Preparation

Seven days after retrograde tracer or saline instillation, the experimental and control animals were killed by 100% CO₂ asphyxiation and exsanguinated. The nodose ganglia, jugular ganglia, and trachea were removed. In the guinea pig, the jugular and nodose ganglia are easily recognized as two completely separate entities (Figure 1). However in other species (for example, the rat), the ganglia have considerable overlap in their boundaries. All tissues were fixed in picric acid- formaldehyde fixative for 3 h at 4° C. The fixative consisted of 2% paraformaldehyde, 15% saturated picric acid, and 0.15 M phosphate buffer. The tissues were rinsed 3 times with a 0.1 M phosphate-buffered saline containing 0.3% Triton-X-100 (PBS-Tx). During the final rinse, the tissues remained in the PBS-Tx overnight at 4° C.

The nodose and jugular ganglia were mounted with a specified orientation on corks so that the first section collected was of the ventral surface. The ganglia and trachea were frozen in isopentane, cooled with liquid nitrogen, and stored in airtight plastic bags at 60° C.

Continuous serial cryostat sections (12-µm thickness) of the nodose and jugular ganglia were collected sequentially on five different subbed coverslips. The first coverslip had sections 1, 6, 11 . . . , the second coverslip had sections 2, 7, 12 . . . and so on. The entire ganglia and trachea were sectioned; however, not every section was evaluated. The first coverslip was used to determine the localization of rhodamine-labeled latex microspheres or fast blue in the ganglia and trachea. Coverslips 2 and 3 were used for SP immunocytochemistry. The fourth and fifth coverslips were used for unrelated studies.

Localization of Rhodamine-labeled Latex Microspheres in the Nodose Ganglia, Jugular Ganglia, and Trachea

The coverslips containing alternate sections of the nodose or jugular ganglia were mounted using fluoromount. Without further processing, the sections were analyzed for the distribution of microsphere-labeled cell bodies. Twenty-four sections were observed and used to count the total number of nerve cell bodies in each vagal ganglia. The sections were counted by direct observation using a laboratory cell counter. The nerve cell bodies were counted by starting in the right hand corner of each section and moving in a serpentine manner to ensure counting all cell bodies in the section. The percentage of rhodamine-labeled neurons was calculated for each vagal ganglia by dividing the number of rhodamine-labeled cell bodies by the total number of cell bodies. The main criterion used to identify a nerve cell body was the observation of a defined nucleus in each cell body. Recounting the same cell body was avoided by collecting every fifth section and only counting nucleated cell bodies.

Sections of the trachea were mounted and observed in a similar manner. The localization of rhodamine-labeled latex microspheres in the tracheal epithelium and/or lower submucosa was also determined by direct observation.

Localization of Fast Blue in the Nodose Ganglia, Jugular Ganglia, and Trachea

The localization of fast blue in nerve cell bodies in the nodose ganglia and jugular ganglia were observed, counted, and calculated in the same manner as described in the previous section.

In addition, the average number of tracheal-specific neurons derived from the nodose and the jugular ganglia was calculated by dividing the number of labeled neurons containing either red beads or fast blue by the total number of tracheal neurons labeled by the retrograde tracers.

Sections of the trachea were mounted and observed in a similar manner. The localization of fast blue in the tracheal epithelium and/or lower submucosa was also determined by direct observation.

Immunocytochemical Procedures

Immunocytochemical procedures for localizing a single antigen are identical to those described previously (4). Briefly, cryostat sections on gelatin-coated coverslips were covered with a diluted primary antiserum, rabbit anti-SP (Peninsula, Belmont, CA; 1:200 working dilution). The coverslips were incubated in a humidified chamber at 37° C for 30 min. The coverslips were rinsed with a 1% bovine serum albumin-phosphate saline buffer containing triton-X solution (PBS-Tx + BSA) three times, allowing 5 min for each rinse. The sections on the coverslips were covered with a diluted secondary antiserum, fluorescein isothiocyanate-labeled goat anti-rabbit (ICN Immunobiologicals, Costa Mesa, CA; 1:200 working dilution) and incubated at 37° C for 30 min. The coverslips were rinsed again in PBS-Tx + BSA, three times for 5 min. Controls for specificity of primary antisera consisted of absorption of 1 µg/ml antisera with SP. Nonspecific background labeling was determined by omission of primary antiserum. Even after these controls were employed, however, cross-reactivity of antisera with other known or unknown peptides present in the tissue cannot be excluded by immunocytochemical procedures. Therefore, the term peptide immunoreactivity (i.e., SP-IR) is used here when referring to labeled structures.

The percentage of rhodamine-labeled or fast blue-labeled nerve cell bodies containing SP was calculated by dividing the number of rhodamine-labeled or fast blue-labeled nerve cell bodies containing SP-IR by the total number of rhodamine-labeled or fast blue-labeled nerve cell bodies, respectively.

Statistical Analysis

The mean values of rhodamine-labeled neurons and rhodamine- labeled neurons containing SP were compared by one-way ANOVA using the Macintosh based software Statview II. The same procedure was used for the mean values of the fast blue-labeled neurons and fast blue-labeled neurons containing SP. Significance was set at p < 0.05. Upon obtaining significant F values, the individual means were compared using the least significant difference test.

Photomicrographic Illustrations

After the coverslips of the nodose ganglia, jugular ganglia, and trachea were mounted with fluoromount, the sections were observed using an Olympus DX60 fluorescence microscope equipped with fluorescein (excitation 465 nm and emission 515 nm), rhodamine (excitation 535 nm and emission 590 nm), and ultraviolet (excitation 350 nm and emission 420 nm) filters. The sections were photographed with an Olympus PM-C35PX camera. The slides were scanned into Adobe PhotoDeluxe 1.0 program for the Power Macintosh using the Polaroid Sprint Scan 35 System. Digital images were printed on an Epson 700 Stylus Printer without additional image processing.

	RESULTS

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Retrograde Tracing of Fast Blue from the Tracheal Mucosa

Fast blue dye, after intratracheal instillation, labeled the epithelium and also diffused through the basement membrane and into the lamina propria of the trachea. Therefore, the fast blue was used to label neurons innervating the entire tracheal wall (Figures 2A and 2B). In the histological sections evaluated, the average number of neurons retrogradely labeled by tracheal instillation of fast blue dye was 71 ± 6 in the jugular ganglia and 106 ± 8 in the nodose ganglia (Figure 3). Thus, 40 ± 1% of the tracheal neurons taking up the dye were derived from jugular neurons, and 60 ± 1% were derived from the nodose neurons. This represented 3.2 ± 0.2% and 4.8 ± 0.1% of the total number of neurons in the jugular and nodose ganglia, respectively (n = 4).

View larger version (108K): [in this window] [in a new window]   Figure 2.   (A) Photomicrograph demonstrating the deposition of fast blue dye throughout the entire tracheal wall. The fast blue transported from the tracheal wall was localized in the epithelial layer and passed through the basement membrane into the submucosa. Arrowheads point to the fast blue that penetrated the basement membrane and entered the submucosa. (B) Photomicrograph of jugular ganglion cell bodies labeled by retrograde tracing of fast blue from the tracheal wall. Arrowheads point to a jugular cell body labeled with fast blue.

View larger version (44K): [in this window] [in a new window]   Figure 3.   The number of cell bodies in the jugular and nodose ganglia that were labeled by tracheal instillation of fast blue (which diffuses throughout the tracheal wall). The number of cell bodies in the jugular and nodose ganglia were counted 7 d after instillation and every fifth section of each ganglion was used for counting. Bars are mean ± SEM, n = 4 ganglia.

We evaluated the fast blue-labeled neurons for SP immunoreactivity. Consistent with previous studies (8, 9), approximately half the fast blue-labeled neurons in the jugular ganglia were SP-positive. In sections obtained from four jugular ganglia the fraction of fast blue-labeled neurons that were SP-positive was 163/329; this corresponded to an average of 49 ± 4%. By contrast, virtually none (4/399; 1 ± 0.05%) of the labeled neurons in the nodose ganglia were positive for SP immunoreactivity (Figure 4).

View larger version (21K): [in this window] [in a new window]   Figure 4.   The percentage of jugular and nodose cell bodies labeled from the entire tracheal wall (fast blue-labeled) that contained SP immunoreactivity. Bars are mean ± SEM, n = 4.

Retrograde Tracing of Rhodamine-labeled Latex Microspheres from the Tracheal Epithelium

The rhodamine-labeled latex microspheres were localized only in the tracheal epithelial layer. They did not pass through the basement membrane and thus were not observed in the tracheal submucosa. Therefore, the rhodamine-labeled latex microspheres were used to label only those neurons innervating the epithelial compartment of the trachea (Figures 5A and 5B).

View larger version (82K): [in this window] [in a new window]   Figure 5.   (A) Photomicrograph demonstrating the localization of rhodamine-labeled latex microspheres in the epithelial trachea. The rhodamine-labeled latex microspheres transported from the tracheal wall were localized in the epithelial layer and did not diffuse into the lamina propria. Arrowheads point to the rhodamine beads that penetrated the epithelium, but do not enter the submucosa. (B) Photomicrograph of jugular ganglion cell bodies labeled by retrograde tracing of rhodamine-labeled latex microspheres from the tracheal epithelium. Arrowheads point to a jugular cell body containing rhodamine beads.

The average number of rhodamine-labeled tracheal neurons was 56 ± 4 in the jugular ganglia and 1 ± 0.2 in the nodose ganglia (Figure 6). Thus, 97 ± 1% of the tracheal neurons taking up the dye were derived from jugular neurons, and 2 ± 1% were derived form the nodose neurons. This represented 1.9 ± 0.3% and 0.06 ± 0.02% of the total neurons in the jugular and nodose ganglia, respectively (n = 4).

View larger version (18K): [in this window] [in a new window]   Figure 6.   The number of cell bodies in the jugular and nodose ganglia that were labeled by tracheal instillation of rhodamine- labeled latex microspheres (which are limited to the tracheal epithelium). The number of cell bodies in the jugular and nodose ganglia were counted 7 d after tracer instillation and every fifth section of each ganglion was used for counting. Bars are mean ± SEM, n = 4.

As with fast blue labeling, both SP-positive and SP-negative neurons were labeled with the rhodamine-labeled microspheres. The percentage of jugular neurons in four ganglia labeled from the tracheal epithelial compartment (rhodamine-microsphere-labeled) that contained SP immunoreactivity was 60 ± 6% (133/221). The few nodose neurons labeled with rhodamine-labeled latex microspheres were SP-negative (Figure 7).

View larger version (20K): [in this window] [in a new window]   Figure 7.   The percentage of jugular and nodose cell bodies labeled from the tracheal epithelium (rhodamine bead-labeled) that contained SP immunoreactivity. Bars are mean ± SEM, n = 4.

	DISCUSSION

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Sensory nerve fibers in the tracheal epithelium are thought to play an important role in the defense of the airways (7, 13). These fibers come into direct contact with inhaled particles and chemicals, and when activated can evoke powerful defensive reflexes that include bronchoconstriction and cough. To better understand the mechanisms that regulate the activity of these nerve fibers, it is important to first identify the sensory neurons from which they arise.

Retrograde neuronal tracing techniques have been used to identify the sensory neurons that project fibers to the guinea pig airways (8). This involves applying a dye to the airway that is taken up by the fibers and transported back to their soma in various ganglia. Using this strategy, it has been demonstrated that the vast majority of sensory innervation to the guinea pig airways arises from cell bodies located in either the nodose or jugular vagal ganglia (8, 9). In these studies, the dyes used to label the neurons diffused throughout the airway wall, and thus were used to indiscriminately label neurons throughout the mucosa.

Recently, rhodamine-labeled latex microspheres have been successfully employed to label neurons projecting specifically to the rat nasal epithelium (14). This strategy is based on two observations. First, nerve endings avidly take up rhodamine-labeled latex microspheres and retrogradely transport them to their cell body (15, 16). Second, the size of the microspheres (~ 0.04 mm) renders them unable to penetrate the airway basement membrane (14). The results of the present studies confirm both of these observations. The rhodamine-labeled microspheres, following intratracheal instillation, were localized above the basement membrane within the epithelial compartment, and a week later the microspheres were readily observed within the cell bodies located in the vagal sensory ganglia. The major conclusion derived from this work is that, whereas cell bodies in both nodose and jugular ganglia project fibers to the guinea pig trachea (as determined using diffusible fast blue dye instillation), only the jugular ganglion neurons projected fibers to the epithelial compartment of the airway wall.

Neurons located in the jugular ganglia project both neuropeptide-containing C fibers and neuropeptide-negative A fibers to the guinea pig airways (9). It is possible that one or the other of these subpopulations of jugular fibers preferentially innervates the epithelium. The observation that both SP-positive and SP-negative neurons were labeled with rhodamine-microspheres indicates, however, that both fiber populations are present in the epithelial layer.

The results do not allow for an accurate determination of what percentage of jugular fibers that go to the trachea end up in the epithelium. However, by comparing the number of jugular neurons that were labeled with the diffusible fast blue dye with the number labeled with rhodamine-labeled microspheres it would appear that over 80% of the jugular fibers in the trachea are in the epithelial layer. The relative efficacy of neuronal labeling between fast blue- and rhodamine-labeled microspheres is not known, therefore such interpretation must be cautiously regarded. It should also be pointed out that in the present study the dye was applied only to the rostral aspect of the trachea; the extent to which the findings pertain to the epithelium of the bronchial tree is not known.

An assumption in this study is that each nerve ending that comes in contact with fast blue- or rhodamine-labeled microspheres will take up the dye and transport it to the cell body. It is possible that nodose nerve endings are in the epithelial layer, but for some unknown reason fail to take up the rhodamine-labeled microspheres. This is unlikely, however, because in other experiments we have injected the rhodamine-labeled microspheres directly into the airway wall (beneath the basement membrane) and 1 wk later found them within numerous neurons in the nodose ganglia (data not shown).

The explanation for why jugular, but not nodose nerve fibers are found in the guinea pig tracheal epithelium is not known. It is reasonable to speculate, however, that the epithelial cells produce a neurotrophic molecule that interacts with jugular, but not nodose nerve endings. This possibility is supported by the observations that the neurotrophin biology is distinct between neurons in these two vagal ganglia (17).

Not only is the neurotrophin biology distinct between nodose and jugular ganglia, but their embryological origin, and their physiology is also different. Although it has not been systematically studied, it is likely that the central connections will also be different between nodose and jugular ganglion neurons (18). With respect to embryology, it appears that nodose ganglion neurons are derived from the ectodermal placodes, whereas the jugular ganglia, like the dorsal root ganglia, are derived from the neural crest (19, 20). Physiologically, the nodose nerve fibers in the guinea pig trachea conduct action potential primarily in the A range. They are rapidly adapting fibers that have a mean somal diameter of about 40 ± 3.2 µm and are exquisitely sensitive to mechanical stimulation (9, 21). They are relatively unresponsive to changes in osmolarity, or to chemicals such as capsaicin (12, 21). Jugular nerve endings in the guinea pig trachea, on the other hand, conduct action potentials in the C and A range. The C and A fibers have a mean somal diameter of approximately 23 ± 1.3 µm and 33 ± 0.06 µm, respectively (9). Regardless of conduction velocity or somal diameters, they are less sensitive than nodose fibers to mechanical stimulation, are less adapting to suprathreshold stimulation, and are responsive to hypertonic saline and capsaicin (12, 21). Thus, with respect to their physiological response, it seems to matter less if the fiber is a C fiber or an A fiber than whether its cell body is located in the nodose or jugular ganglion.

The difference in the physiology of the neurons found within the two vagal sensory ganglia underscores the potential relevance of the observation that the tracheal epithelium is innervated specifically with jugular nerve fibers. Thus, by understanding the jugular neuron phenotype, more insights may be gained into the mechanisms by which epithelial nerve fiber density and physiology are regulated. In this context it is useful to note that the rhodamine-labeled microspheres do not interfere with the electrophysiology of the neurons (16). Therefore, by combining elecrophysiological techniques with the tracing techniques described here, new information about the electrophysiological regulation of epithelial fibers may be ascertained. An epithelial-derived neurotrophin selective for jugular neurons could conceivably underlie the increase in neuropeptide-containing nerve density present in patients with persistent cough syndromes (22).