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Published ahead of print on December 14, 2006, doi:10.1165/rcmb.2006-0256OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 36, pp. 541-551, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0256OC

Sensory Receptors in the Visceral Pleura

Neurochemical Coding and Live Staining in Whole Mounts

Isabel Pintelon, Inge Brouns, Ian De Proost, Frans Van Meir, Jean-Pierre Timmermans and Dirk Adriaensen

Laboratory of Cell Biology and Histology, University of Antwerp, Antwerp, Belgium

Correspondence and requests for reprints should be addressed to Dirk Adriaensen, Ph.D., Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Groenenborgerlaan 171, BE-2020 Antwerp, Belgium. E-mail: dirk.adriaensen{at}ua.ac.be


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Today, diagnosis and treatment of chest pain related to pathologic changes in the visceral pleura are often difficult. Data in the literature on the sensory innervation of the visceral pleura are sparse. The present study aimed at identifying sensory end-organs in the visceral pleura, and at obtaining more information about neurochemical coding. The immunocytochemcial data are mainly based on whole mounts of the visceral pleura of control and vagally denervated rats. It was shown that innervation of the rat visceral pleura is characterized by nerve bundles that enter in the hilus region and gradually split into slender bundles with a few nerve fibers. Separate nerve fibers regularly give rise to characteristic laminar terminals. Because of their unique association with the elastic fibers of the visceral pleura, we decided to refer to them as "visceral pleura receptors" (VPRs). Cryostat sections of rat lungs confirmed a predominant location on mediastinal and interlobar lung surfaces. VPRs can specifically be visualized by protein gene product 9.5 immunostaining, and were shown to express vesicular glutamate transporters, calbindin D28K, Na+/K+-ATPase, and P2X3 ATP-receptors. The sensory nerve fibers giving rise to VPRs appeared to be myelinated and to have a spinal origin. Because several of the investigated proteins have been reported as markers for sensory terminals in other organs, the present study revealed that VPRs display the neurochemical characteristics of mechanosensory and/or nociceptive terminals. The development of a live staining method, using AM1-43, showed that VPRs can be visualized in living tissue, offering an interesting model for future physiologic studies.

Key Words: lung • P2X3 ATP-receptors • vesicular glutamate transporters • visceral pleura • receptors



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Visceral pleura receptors (VPRs) likely mediate the sensory transduction of mechanical and/or nociceptive stimuli. VPRs are potentially involved in the hitherto unknown mechanisms of pain sensation and (reflex) dyspnea regularly described to result from pleural disease.

 
The visceral pleura is often considered to be insensitive to painful stimuli, and, as a consequence, is believed to lack a sensory innervation (1–3). In general medical literature, assumptions have been made about the innervation of the visceral pleura without referring to conclusive morphologic or physiologic data. Several authors indeed describe the visceral pleura as noninnervated (1–3), whereas others presume that nerves are present, but no sensory endings (4). Although early physiologic experiments (5) demonstrated that potential afferent fibers in the visceral pleura do not react to tactile, thermal, or pain stimuli, some more recent publications do suggest an autonomic innervation of the visceral pleura, containing afferent fibers traveling together with sympathetic nerve fibers (6, 7). Although the available data may be rather sparse and conflicting, the visceral pleura are unquestionably innervated, as, in the early 1900s, classical morphologists reported extensive nerve bundles and terminals (8–11). The latter studies, based on general nerve staining methods, such as silver impregnation methods and methylene blue staining, described different types of nerve endings in various animal species, the morphologic characteristics of which were suggestive of sensory end-organs (8–11). The applied general neurohistologic stainings, however, did not provide specific neurochemical information that would allow the differentiation of different populations of motor and sensory nerve terminals.

Commonly reported symptoms of both benign and malignant pleural tumors, the majority of which is associated with the visceral pleura, are chest pain and dyspnea (12–14). Pulmonary embolism and pleuritis often lead to "pleuritic chest pain," the diagnostic evaluation of which is problematic (15–17). Adequate relief of the often angina-like chest pain associated with pleural disease is typically difficult (14, 18). One of the main reasons for this difficulty is undoubtedly a lack of knowledge concerning the location, morphology, and, especially, the neurochemical characteristics of potential sensory receptors involved in afferent pathways from the visceral pleura. Although, recently, considerable attention has been paid to the identification of parietal pleura afferents in rabbits (19–21), very few data are available in rats.

Because the limited physiologic studies have not been able to provide conclusive information, and because the available morphologic data are insufficient to fully characterize the nerve fiber populations in the visceral pleura, there is a clear need for further investigation. The aim of the present study was to explore the possibility that the visceral pleura may harbor specific sensory receptor end-organs. We therefore performed an advanced immunocytochemical study. A method was developed to prepare whole mount preparations of the visceral pleura of rat lungs, which, in addition to cryostat sections of rat lungs, were suited to immunocytochemical staining. Multiple immunostaining methods were optimized and, when necessary, tyramid signal amplification (TSA) procedure was applied to optimize the detection limit of the antibodies for some of the neuronal markers. Antibodies to general neuronal markers were combined with known markers for the pulmonary sensory and motor innervation to characterize the neurochemical coding of the innervation of the rat visceral pleura. More specifically, we applied a recently established panel of antibodies that is known to selectively characterize sensory receptors in the lungs (22, 23) and in other organs (24–26). To verify the selective location of the studied nerve fiber populations in the visceral pleura, elastic fibers were additionally stained. Because the conduction velocity of nerve fibers is an important discriminating feature, myelin sheaths were visualized. Denervation studies were applied to identify the origin of the pleural nerve terminals. Finally, efforts were made to optimize a vital staining method for the visualization of sensory receptors in living whole mount preparations of the visceral pleura.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Animals
Studies were performed on 10-d-old (n = 10), and on 3- (n = 6), 4- (n = 14) and 5- (n = 3) wk-old Wistar rats (Charles River, Brussels, Belgium) of both sexes. The animals were kept with their mothers (10-d-old) or in separate acrylic cages (3-, 4-, and 5-wk-old) in an acclimatized room (12/12 h light/dark cycle; 22 ± 3°C), and were provided with water and food ad libitum. National and international principles of laboratory animal care were followed, and the experiments were approved by the local ethics committee of the University of Antwerp. All animals were killed by intraperitoneal injection of an overdose of sodium pentobarbital (200 mg/kg Nembutal; CEVA Santé Animale, Brussels, Belgium) containing 500 U/kg heparin (256S68F12; Rhône Poulenc Rorer, Brussels, Belgium).

Denervation Study
Wistar rats (10-d-old [n = 2] and 3-wk-old [n = 4]) were anaesthetized by intramuscular injections of 0.83 mg/kg droperidol and 0.017 mg/kg fentanyl (Janssen Pharmaceuticals, Beerse, Belgium) 20 min after sedation with diazepam (0.5 ml/kg Valium; Roche, Brussels, Belgium). The left (n = 3) or right (n = 3) cervical vagal nerve was exposed by a ventral approach ~15 mm caudal to the larynx, and ~5 mm of the nerve was removed. At the completion of the surgical procedure, the muscle and skin were sutured. The animals were killed 7 d after the surgical procedure, and whole mount preparations of the visceral pleura (see below) were prepared from both lung lobes, ipsilateral to the vagal denervation, and from lung lobes of the contralateral control side. All tissues were further processed for immunocytochemistry as described subsequently here.

Tissue Processing
Cryostat sections of whole rat lungs. Because the investigation of sensory lung receptors is much more efficient in neonatal animals because of a higher density of airway receptors due to the smaller lung volume (27), nerve terminals of the visceral pleura were studied in cryostat sections of 10-d-old rats. The pulmonary circulation was perfused via the right ventricle with a Kreb's solution (117 mM NaCl, 4.96 mM KCl, 2.54 mM CaCl2·2H2O, 1.2 mM MgSO4·7H2O, 1.2 mM NaH2PO4·2H2O, 25 mM NaHCO3, 10 mM D-glucose; pH 7.4). Lungs were then intratracheally instilled via a tracheal cannula with Zamboni's fixative (0.2% saturated picric acid, 4% paraformaldehyde, 0.1 M phosphate buffer, pH 7.4), ligated, dissected en bloc, degassed in a vacuum chamber, and further fixed in the same solution for 30 min. After a rinse in PBS (0.01 M, pH 7.4), lungs were treated for improvement of the immunocytochemical conditions (28), stored overnight in 30% sucrose (in PBS, 4°C), and mounted in Tissue Tek (Sakura Finetek Europe, Zoeterwoude, The Netherlands). Cryostat sections (30-µm thick) were thaw-mounted on poly-L-lysine–coated microscope slides, dried at 37°C for 2 h, and further processed for immunolabeling.

Whole mount preparations of the visceral pleura of the rat. Lungs from 3-, 4-, and 5-wk-old rats were used to prepare whole mount preparations of the visceral pleura. Studying these age groups combined: (1) the advantage of making the preparation of whole mounts much easier than those from adult animals; with (2) the possibility of studying the myelinization of the nerve fibers, which does not reach the terminal areas until ~ 2 wk after birth. Whole mounts of the visceral pleura were either made from the mediastinal (n = 19), interlobar (n = 11), costal (n = 3), or diaphragmatic (n = 2) surface of the lung lobes. Because immunostaining on cryostat sections clearly revealed that the nerve endings of interest in the visceral pleura are more numerous on the mediastinal and interlobar surfaces of the lung lobes, whole mounts were preferentially prepared from these areas of the visceral pleura. Left, right cranial, and right caudal lung lobes were included in this study. After perfusion of the pulmonary circulation with Kreb's solution, the lungs were intratracheally instilled with Kreb's solution via a tracheal cannula. Filled lungs were dissected en bloc, immersed in Zamboni's fixative, degassed, and further immersion fixed for 30 min. After rinsing in PBS, whole mount preparations of lung lobes were prepared by pinning the lung lobe with the side of interest to the bottom of a Sylgard-coated Petri dish, and by removing all lung tissue until the visceral pleura was almost all that remained. The latter was accomplished by first cutting away most of the lung lobe with microdissection scissors, and further by gently pulling off the remaining alveolar tissue with watchmaker's forceps. Some larger pieces of the removed lung tissue were fixed for 30 min in Zamboni's solution and were used as positive control tissue in the immunocytochemical procedures. Whole mounts of the visceral pleura and the pieces of lung tissue were treated for improvement of the immunocytochemical conditions (28), and were incubated simultaneously for the various immunocytochemical staining procedures.

Immunocytochemical Procedures
Immunocytochemical procedures for cryostat sections of lungs, whole mount preparations of the visceral pleura or lung pieces were performed under the same incubation conditions and at room temperature. Unless otherwise indicated, all primary and secondary antisera were diluted in PBS containing 10% normal goat serum, 0.1% BSA, 0.05% thimerosal and 0.01% NaN3 (PBS*). To minimize nonspecific binding of secondary antibodies, and to enhance penetration of antisera, a preincubation was performed for 1 h with PBS* containing 1% Triton X-100. Characteristics and sources of the applied primary antisera are listed in Table E1 in the online supplement, and those of the secondary and tertiary antisera in Table E2. The combinations of primary, secondary and tertary antisera used for multiple immunocytochemical labeling are listed in Table E3.

Conventional immunocytochemical labelings. For conventional single labeling, whole mount preparations and cryostat sections were incubated overnight with rabbit polyclonal primary antisera (see Table E1). After rinsing in PBS the tissues were incubated for 4 h with either Cy3-conjugated Fab fragments of goat anti-rabbit IgG (GAR-Fab-Cy3, diluted 1:2,000) or FITC-conjugated Fab fragments of goat anti-rabbit IgG (GAR-Fab-FITC, diluted 1:100). In conventional double immunocytochemical stainings, the tissue was then incubated for a successive night with a second primary antibody, followed by incubation with the appropriate secondary antibody, as indicated in Table E3. To allow combination of two primary antisera raised in rabbit, binding sites of the first primary antibody were blocked by GAR-Fab-Cy3 or -FITC and unlabeled GAR-Fab. The second primary antibody was detected in a conventional way, using GAR-Fab-FITC or -Cy3, respectively.

Multiple staining using TSA. To obtain enhanced sensitivity and to allow uncomplicated combination of antisera raised in the same species (29), a biotin-conjugated TSA kit (NEL700; PerkinElmer LAS, Zaventem, Belgium) was applied. Before the immunocytochemical staining procedures, endogenous peroxidase activity was blocked by hydrogen peroxide (3% in 50% methanol/PBS; 10 or 30 min for cryostat sections and whole mounts, respectively). After the first primary incubation, polyclonal rabbit anti–vesicular glutamate transporter (VGLUT) 2 or guinea pig anti-VGLUT1 (overnight incubation; see Tables E1 and E3) were detected using a 1-h incubation with GAR-Fab-BIOT or DAGP-BIOT, respectively. Subsequent incubations with ExtrAvidin-horseradish peroxidase (in PBS, 1 h or 2 h for cryostat sections and whole mounts, respectively), biotin-conjugated tyramide (diluted 1:100 in "amplification solution"; 10 min), and Cy3-conjugated streptavidin (diluted 1:6,000; 10 min) or FITC-conjugated streptavidin (diluted 1:1,000; 10 min) were applied to visualize the reaction. In double immunocytochemical procedures and triple immunostaining with antibodies raised in different species, the sections and whole mount preparations were subjected to an additional conventional immunostaining with appropriate second and third primary antibodies. For triple immunocytochemical stainings with antibodies raised in the same species, procedures were applied as previously published (29). TSA-enhanced immunostaining for the ATP receptor, P2X3, was performed as described previously by Brouns and coworkers (30).

Control experiments for the immunocytochemical procedures. Negative staining controls for all immunocytochemical procedures were performed by substitution of nonimmune sera for the primary and/or secondary antisera. The general specificity of the primary antibodies for their respective antigens was tested by the providing companies, and as described previously (30–32). To check for possible cross reactivity after consecutive multiple staining when using two or three rabbit primary antisera, the results of single immunostaining for each of the antigens were evaluated and compared with those from multiple labeling. Controls for the amplification-based multiple staining were performed by omission of the primary antiserum of the second and third incubation. In addition, nonamplified stainings with primary antibodies, using the same concentrations as for the TSA-enhanced reactions, were routinely included. For the triple immunocytochemical staining using three rabbit antibodies, all control stainings were performed as previously described (29).

Immunocytochemical stainings were simultaneously performed on whole mount preparations of the visceral pleura and on the small pieces of lung tissue obtained during whole mount preparation. Because the staining patterns of the antibodies used have been described for the innervation of airways and pulmonary neuroepithelial bodies in our earlier publications, small pieces of lung tissue served as an appropriate positive control (22, 23, 30, 31). Similarly, aside from the visceral pleura, cryostat sections of whole lungs invariably harbored large amounts of lung tissue for positive control.

Live Staining of Nerve Terminals in Whole Mounts
Whole mounts of rat (n = 4) visceral pleura used for in vitro staining were prepared as described above, but without the fixation step, and under continuous perfusion with ice-cold Kreb's solution. The visceral pleura of the mediastinal surface of the left and right caudal lung lobes were used. Whole mounts were incubated for 15 min with 20 µM of the fluorescent styryl pyridinium dye, AM1-43 (Biotium/Gentaur Molecular Products, Brussels, Belgium) in Dulbecco's modified Eagle's medium (DMEM)-F12 (Gibco/Invitrogen, Merelbeke, Belgium) at 37°C. After rinsing for 15 min in DMEM-F12 with 1 mM 4-sulfonato calix[8]arene sodium salt (Biotium/Gentaur Molecular Products, Brussels, Belgium) to reduce background staining, whole mounts were transferred to fresh DMEM-F12 for 30 min before evaluation. Subsequent to imaging of the AM1-43 labeled structures in live visceral pleura whole mounts, the tissues were fixed and further processed for immunolabeling of VGLUT2, as described above.

Microscopic Imaging and Data Analysis
Immunocytochemical and vital stainings were briefly checked using an epi-fluorescence microscope (Zeiss Axiophot; Carl Zeiss, Jena, Germany) equipped with filters for the visualization of FITC and Cy3. An inverted microscope (Axiovert 200; Carl Zeiss), attached to a microlens-enhanced dual spinning disk confocal system (UltraVIEW ERS; PerkinElmer LAS; Seer Green, UK), equipped with a three-line (488, 568, and 647 nm) argon–krypton laser for excitation of the FITC, Cy3, and Cy5 labels, respectively, was used for multicolor high-resolution imaging. AM1-43 (excitation maximum: 479 nm) was excited by the 488-nm line. Images were analyzed and processed using Volocity3.5 (Improvision, Coventry, UK) and Adobe Photoshop 7.0 software (Adobe Systems Benelux BV, Amsterdam, The Netherlands).

Diameters of the nerve fibers were measured on images of nerve fibers and their terminals taken from protein gene product (PGP)9.5/myelin basic protein (MBP) double immunolabeling in the whole mount preparations of the visceral pleura. All images were maximum-intensity projections of confocal optical sections. Because diameters often differed strongly within a single nerve fiber, the smallest and largest diameters were measured for each myelinated nerve fiber (n = 10) that gave rise to a specific laminar nerve terminal. Data are presented as a set of diameters, ranging from the smallest to the largest diameter measured, and numbers should be regarded as indicative rather than absolute.

Because receptor-like laminar nerve terminals in the visceral pleura seemed to have a preferential location in specific regions of the rat lung surface, their incidence at different locations was assessed in serial cryostat sections of whole lung/heart/esophagus complexes (10-d-old; n = 3) immunostained for VGLUT2. To avoid double counting, one out of five of the serial sections was evaluated for each rat. Receptor-like nerve terminals in the visceral pleura were systematically counted (n = 234) while the location of each terminal was carefully determined. Results revealed that laminar endings could be subdivided into four categories based on their specific locations in the visceral pleura (i.e., interlobar, mediastinal facing the heart, mediastinal facing the esophagus, and costal). Unfortunately, whole lung cryosections appeared to be tricky for the clear identification of diaphragmatic surfaces, which were, therefore, not included in this quantification. To estimate the occurrence of receptor end-organs in particular regions of the lung surface, the percentage of the total number of counted terminals present in each group was calculated. The numbers are relative, and only intended as a solid indication of the incidence of the laminar nerve terminals in particular regions of the visceral pleura.

Unless specifically denoted otherwise in the figure legends, all images shown were taken from rat visceral pleura whole mounts of the mediastinal and interlobar surfaces of the lung lobes.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Immunostaining for PGP9.5 on Whole Mount Preparations and Cryostat Sections
Immunocytochemical staining for the general neuronal marker, PGP9.5, on whole mount preparations revealed thick nerve bundles that enter the rat visceral pleura at the hilus of each lung lobe and cross the mediastinal surface. The bundles continuously branch into smaller nerve trunks that also reach the other lung surfaces, thereby giving rise to a wide-meshed network over the entire lung surface (but that is clearly more dense at the mediastinal side). More distally, the nerve bundles consist of just a few fibers. At all levels, the nerve trunks appeared to repeatedly split off single PGP9.5-immunoreactive (ir) nerve fibers (Figures 1A–1C), which in turn revealed dichotomous branches: one of the branches terminating as laminar end-organs with the appearance of sensory receptors (Figures 1D–1F), whereas the other traveled further in the visceral pleura, frequently forming more receptor-like terminals along its way (Figures 1A–1C). The latter was best evaluated using camera lucida drawings to follow single fibers in an electronic composite of low-magnification images of a PGP9.5-immunostained visceral pleura (Figure 2).


Figure 1
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  		Figure 1. (A) Immunocytochemical staining for PGP9.5 in a whole mount preparation of rat (4W, 4-wk-old) visceral pleura, showing a nerve bundle that gives rise to several collateral branches consisting of one, or just a few, nerve fibers (arrows). These branch further, and finally terminate as laminar nerve terminals (open arrows) that will be referred to as VPRs. Projection of 48 confocal images (0.5-µm intervals). (B) PGP9.5 immunostaining in rat (4W) visceral pleura whole mount. A PGP9.5-ir nerve fiber bundle gives rise to several branches (arrows) that form laminar VPRs (open arrows). Projection of 62 confocal images (0.5-µm intervals). (C) Whole mount preparation of rat (5W, 5-wk-old) visceral pleura immunostained for PGP9.5. Compact laminar endings (open arrows) of VPRs are formed by collateral branches of PGP9.5-ir nerve fibers that, afterward, continue to travel in the small pleural nerve bundle (arrows). Projection of 54 confocal images (0.5-µm intervals). (D) Typical laminar endings of VPRs as they are frequently seen in whole mount preparations of the rat (5W) pleura after PGP9.5 immunostaining. A PGP9.5-ir nerve fiber branches into several slender fibers that further subdivide and terminate as VPR complexes. Projection of 38 confocal images (0.5-µm intervals). (E) Confocal image clearly revealing the morphology of VPRs as networks of very thin arborizations of nerve fibers that terminate as separate laminar end-organs. Whole mount preparation of rat (4W) visceral pleura. Projection of 44 confocal images (0.5-µm intervals). (F) High-magnification image of a PGP9.5-immunostained whole mount preparation of rat (4W) visceral pleura, showing several laminar nerve terminals that arise from a thin, single-branching nerve fiber to form a VPR. Projection of 40 confocal images (0.5-µm intervals).

 

Figure 2
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  		Figure 2. Camera lucida drawing of a computer-based reconstruction of 14 neighboring maximal-intensity confocal images of nerve fibers and terminals in a whole mount preparation of rat (4W) visceral pleura immunostained for PGP9.5. The displayed nerve fibers, representing a small part of the extensive network of branching nerve fibers (arrows) that traverse the visceral pleura, regularly give rise to VPRs (open arrows).

 
Comparison of visceral pleura whole mounts of the mediastinal, diaphragmatic, interlobar, and costal lung surfaces revealed the presence of laminar end-organs on all surfaces, but with a seemingly higher density in the mediastinal and interlobar areas. Meticulous evaluation of a high number of cryostat sections of whole rat lungs showed similar receptor-like laminar nerve endings in the visceral pleura. Despite the low probability of sectioning the terminals of this wide-meshed network, assessment of the incidence of receptor end-organs in cryostat sections clearly revealed that the majority of receptor-like terminals are located at the interlobar and mediastinal surfaces of the lung lobes (Figures 3A–3D). Only a small percentage of the receptor-like terminals was present at the costal surface of the lung lobes (Figure 3A).


Figure 3
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  		Figure 3. (A) Graph showing the percentages of the total number of VPRs (n = 234) that are located in different regions of the visceral pleura. VPRs were categorized by evaluating serial cryostat sections of whole rat lungs immunostained for VGLUT2. The location of VPRs was classified as interlobar, mediastinal facing the heart, mediastinal facing the esophagus, or costal. (B–D) PGP9.5 immunostaining on cryostat sections of rat lungs (PD10, Postnatal Day 10) showing PGP9.5-ir laminar nerve terminals close to the lung surface (open arrows), reminiscent of the presence of VPRs. The three images are taken from specific regions of the lung where VPRs are most frequently found (i.e., at the mediastinal surface facing the heart ["H" in (B)] and the esophagus ["O" in (C)], or in the interlobar regions [D]). Projection of 30 (B), 28 (C), and 33 (D) confocal images (1-µm intervals). P, visceral pleura.

 
Double Immunostaining for PGP9.5 and Elastin-{alpha}
Double immunostaining for PGP9.5 and elastin-{alpha} on whole mount preparations of the visceral pleura of rat lungs showed that the laminar end-organs, arising from branching PGP9.5-ir nerve fibers, appeared to invariably protrude between the abundant elastic fibers of the visceral pleura (Figure 4A). The receptor-like terminals did not reveal obvious contacts with blood or lymphatic vessels in the connective tissue layer of the visceral pleura, or with any other specialized cells or structures. Cryostat sections, double labeled for PGP9.5 and elastin-{alpha}, confirmed that the laminar endings were always intermingled with elastic fibers, and provided more clear images of the elastic networks in the visceral pleura (Figure 4B). Because of the sensory receptor–like morphology of the laminar endings, and their unique relationship with the visceral pleura, they will subsequently be referred to here as "visceral pleura receptors" (VPRs).


Figure 4
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  		Figure 4. (A) Double immunostaining for PGP9.5 (red, Cy3 fluorescence) and elastin-{alpha} (green, FITC fluorescence) in a whole mount preparation of rat (4W) pleura. A VPR (open arrows), composed of diffuse laminar nerve terminals, is seen to arise from a branching PGP9.5-ir nerve fiber (arrows). Note that the VPR is located between the elastic fibers that are typically abundant in the visceral pleura. Projection of 16 confocal images (0.5-µm intervals). (B) Cryostat section of a rat lung (PD10) double-immunostained for PGP9.5 (red, Cy3 fluorescence) and elastin-{alpha} (green, FITC fluorescence). Confocal image, taken from an interlobar lung surface, showing PGP9.5-ir laminar nerve terminals (open arrows) protruding between the abundant elastic fibers that form a characteristic network in the visceral pleura. Projection of 22 confocal images (0.5-µm intervals). A, lung alveole; P, visceral pleura. (C and D) Whole mount preparation of rat (4W) visceral pleura double stained for PGP9.5 (green, FITC fluorescence) and MBP (red, Cy3 fluorescence). Some of the PGP9.5-ir nerve fibers in the nerve bundle show an MBP-ir myelin sheath (arrowheads) that ends just before the dichotomous branching point (asterisks), where one of the branches gives rise to a VPR (open arrows), and the other branch (arrows), now unmyelinated, continues its trajectory together with the nerve bundle. Projection of 38 (C) and 42 (D) confocal images (0.5-µm intervals).

 
Double Immunostaining for PGP9.5 and MBP
Combined immunostaining for PGP9.5 and MBP revealed MBP-ir myelin sheaths surrounding a substantial population of nerve fibers present in the branching nerve bundles in the visceral pleura (Figures 4C and 4D). Myelinated nerve fibers were seen to lose their myelin sheaths in the immediate neighborhood of dichotomous branching points, where collateral PGP9.5-ir fibers arose and terminated as VPRs (Figures 4C and 4D). Subsequently, the other unmyelinated nerve branch often further traversed the visceral pleura for considerable distances, thereby regularly giving rise to more collateral branches that also showed receptor-like endings (Figures 4C and 4D). Myelinated nerve fibers giving rise to VPRs had diameters ranging between 1.4 and 3.5 µm.

Neurochemical Characterization of VPRs
Multiple immunocytochemical staining showed that Na+/K+-ATPase-{alpha}3 is present both in the laminar terminals of VPRs and in the nerve fibers from which they arise (Figures 5A and 5B).


Figure 5
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  		Figure 5. (A–C) Immunocytochemical triple staining for PGP9.5 (red, Cy3 fluorescence), Na+/K+-ATPase-{alpha}3 (artificial blue color of Cy5 fluorescence in far red) and VGLUT1 (green, FITC fluorescence) in rat visceral pleura (4W; whole mount preparation). (A) Combined red, blue, and green channels revealing that the VPRs (open arrows), formed by collaterals of a PGP9.5-ir nerve fiber (arrows), express IR for both Na+/K+-ATPase-{alpha}3 and VGLUT1. (B) The blue channel illustrates that Na+/K+-ATPase {alpha}3 is present in the laminar terminals (open arrows) and in the nerve fiber (arrows) from which they arise. (C) VGLUT1 is present in the receptor-like endings (open arrows) as seen in the green channel. Although IR for VGLUT1 is clear in the branching nerve fiber (arrows), the staining is rather weak in the receptor terminals (open arrows). Projection of 16 confocal images (1-µm intervals). (D and E) Double immunostaining for PGP9.5 (green, FITC fluorescence) and VGLUT2 (red, Cy3 fluorescence) of a VPR, observed at the level of the visceral pleura in a cryostat section of rat (PD10) lungs. (D) Combination of the red and green channel shows a PGP9.5-ir nerve fiber (arrows) that forms a VPR (open arrow). The laminar terminals also express VGLUT2 IR. (E) The red channel shows that VGLUT2 IR can be found both in the nerve fibers (arrows) that give rise to the VPR, and in the arborizations of the end-organ (open arrow). Note a more intense staining, located at the level of the receptor terminals. Projection of 56 confocal images (0.5-µm intervals). (F) Cryostat section of a rat (PD10) lung double labeled for VGLUT2 (red, Cy 3 fluorescence) and elastin-{alpha} (green, FITC fluorescence) showing that VGLUT2 IR is clearly displayed both in the nerve fiber (arrow) and in the numerous arborizations that form a VPR (open arrow). This confocal image illustrates that the receptor end-organs are typically formed in the area near the surface of the lung lobe where abundant elastic fibers characterize the presence of the visceral pleura. Projection of 70 confocal images (0.5-µm intervals). A, lung alveole; P, visceral pleura. (G–I) Confocal image of several VPRs that arise from a network of branching nerve fibers in a whole mount preparation of rat (4W) visceral pleura. Triple immunostaining for PGP9.5 (red, Cy3 fluorescence), VGLUT2 (artificial blue color of Cy5 fluorescence in far red) and P2X3 receptor (green, FITC fluorescence). (G) Combined image showing that VGLUT2 and P2X3 receptor IR are displayed with variable intensity in the same PGP9.5-ir laminar endings (open arrows), but that their IR is very weak in the branching PGP9.5-ir nerve fibers (arrows). (H) The blue channel reveals that VGLUT2 IR is only weak in the nerve fibers (arrows) that give rise to VPRs, while much stronger in the end-organs (open arrows). (I) Green channel demonstrating that P2X3 receptors are mainly expressed on the laminar terminals of most of the VPRs (open arrows). Note that not all receptor end-organs appear to exhibit detectable IR for the P2X3 receptor (open arrowhead in [I]). Projection of 52 confocal images (1-µm intervals).

 
Using antibodies against VGLUTs to stain glutamatergic nerves, VGLUT1 immunoreactivity (IR), which was detectable in only a subpopulation of VPRs, was strong in nerve fibers but rather weak in the laminar endings (Figures 5A and 5C). In cryostat sections of 10-d-old rats, double labeling for PGP9.5 and VGLUT2 demonstrated that VGLUT2 IR can be detected both in nerve fibers that give rise to complex receptor-like terminals and in the end-organs of VPRs (Figures 5D–5F). In whole mounts of 3- to 5-wk-old rats, combination of PGP9.5 and VGLUT2 revealed that the PGP9.5-ir nerve fibers, from which the VPRs branch, exhibited only a faint VGLUT2 IR (Figures 5G and 5H). Intense staining for VGLUT2, however, was invariably located at the level of the laminar endings of all VPRs (Figures 5G and 5H).

A subpopulation of the PGP9.5-ir VPRs appeared to express the P2X3 ATP-receptor. P2X3 receptor IR was predominantly displayed in the laminar terminals and nearly undetectable in the branching nerve fibers (Figures 5G and 5I).

IR for the calcium-binding protein, calbindin D28k (CB), though often weak, appeared to be perfectly colocalized with the PGP9.5 IR, and could be demonstrated both in the nerve fibers and in the VPRs (Figures 6A and 6B).


Figure 6
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  		Figure 6. (A and B) VPR complex in a whole mount preparation of rat (4W) pleura, double-immunostained for PGP9.5 (red, Cy3 fluorescence) and CB (green, FITC fluorescence). (A) Red channel showing a branching nerve fiber (arrows) forming VPRs (open arrows), stained with PGP9.5. (B) Green channel illustrating the rather weak expression of CB in VPRs. Projection of 30 confocal images (0.5-µm intervals). (C–E) Double immunocytochemical staining for PGP9.5 (red, Cy3 fluorescence) and CGRP (green, FITC fluorescence) in rat visceral pleura (4W; whole mount preparation). A PGP9.5-ir nerve bundle in the visceral pleura gives rise to many branching nerve fibers, which are seen to terminate in VPRs (open arrows). CGRP-ir nerve fibers (arrowheads) are present in the nerve bundle, but do not appear to form receptor-like endings in the imaged area. (C) Combined two-channel overview. Projection of 58 confocal images (0.5-µm intervals). (D and E) Single-channel images of the framed area in (C). (D) VPR stained with PGP9.5 (open arrow), as seen in the red channel. (E) Green channel illustrating that CGRP-ir nerve fibers (arrowheads) in the visceral pleura are rather rare, varicose, and not associated with the VPR (open arrow). Projection of 32 confocal images (0.5-µm intervals). (F) Confocal image of TH immunostaining on a whole mount preparation of rat (4W) visceral pleura, showing that VPRs (open arrows) may express IR for TH, although very rarely. The nerve fiber (arrow) that gives rise to this VPR also shows IR for TH. Projection of 44 confocal images (0.5-µm intervals). (G) Whole mount preparation of rat (3W, 3-wk-old) visceral pleura double labeled for PGP9.5 (red, Cy3 fluorescence) and TH (green, FITC fluorescence). Combination of the red and green channel reveals TH IR in a nerve fiber (arrows) in a pleural nerve bundle and in the terminals of a visceral pleura receptor–like end-organ (open arrow) that is formed by one of its branches. Note that the orange to yellow color results from the colocalization of TH and PGP9.5 IR. Projection of 24 confocal images (0.5-µm intervals).

 
Thin varicose calcitonin gene-related peptide (CGRP)-ir nerve fibers were observed in the nerve bundles traversing the visceral pleura (Figures 6C–6E). These CGRP-ir nerve fibers often appeared to give rise to collateral branches that, however, seemed to be unrelated to the VPRs (Figures 6C and 6E). In addition, none of the observed VPRs was seen to express CGRP (Figures 6C–6E).

To label potential motor fibers in the branching pleural nerve bundles, immunostaining for the vesicular acetylcholine transporter (VAChT), as a marker for parasympathetic cholinergic fibers, and tyrosine hydroxylase (TH), as a marker for sympathetic adrenergic nerves, were used. No VAChT IR was found in nerve fibers traversing the visceral pleura, or in any of the receptor-like laminar endings. TH IR was found to be present in a limited number of the PGP9.5-ir nerve fibers in nerve bundles crossing the visceral pleura (Figures 6F and 6G). Moreover, TH IR could be observed in just a few receptor-like terminals that appear to originate from TH-ir nerve fibers (Figures 6F and 6G).

Unilateral Cervical Vagal Denervation
At 7 d after unilateral cervical vagal denervation, no notable differences could be observed between denervated and control animals in the number, distribution, or morphology of the laminar receptor endings in whole mounts of the visceral pleura of rat lung lobes ipsilateral to the denervation side. In addition, as expected, the visceral pleura of lung lobes contralateral to the denervated side did not show any changes in the presence of VPRs. Denervation data appeared to be identical in 10-d-old and 4-wk-old rats.

In Vitro AM1-43 Staining of the Rat Visceral Pleura
Incubation with the fluorescent marker AM1-43 resulted in the specific and reproducible visualization of fibers and receptor end-organs in live, whole mount preparations of the visceral pleura, suggestive of a selective accumulation of AM1-43 in VPRs and the nerve fibers that give rise to VPRs (Figures 7A and 7B). Because AM1-43 labeling was still detectable after fixation of the whole mounts (Figure 7C), subsequent immunostaining for VGLUT2 allowed us to unambiguously demonstrate that the AM1-43-labeled structures correspond to VGLUT2-ir VPRs (Figures 7D and 7E).


Figure 7
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  		Figure 7. (A and B) Live staining of the visceral pleura of a 4-wk-old rat using the fluorescent probe, AM1-43 (green fluorescence), a fixable form of the fluorescent FM dyes. (A) Computer-based reconstruction of a nerve fiber (arrows) and associated receptor end-organ (open arrows) in the visceral pleura. Projection of 18 confocal images (0.5-µm intervals). (B) Laminar terminals suggestive of the staining of VPRs. Projection of 24 confocal images (0.5-µm intervals). (C) Same pleural receptor-like endings as in (B) after fixation of the tissue. Note some slight changes in the morphology of the terminals, but a well preserved AM1-43 staining. (D) Subsequent immunostaining for VGLUT2 (artificial blue color of Cy5 fluorescence in far red) confirms the selective AM1-43 labeling of live VPRs in (B). (E) Combination of the green and blue channel. (C–E) Projection of 56 confocal images (0.5-µm intervals).

 
Control Experiments
No qualitative difference was observed in the chemical coding of the VPRs between cryostat sections and whole mount preparations, between 10-d-old and 3-, 4-, or 5-wk-old rats, or between male and female rats.

No obvious differences were observed between the various lung lobes used in this study, and, although the visceral pleura of the different lung surfaces (mediastinal, interlobar, costal, diaphragmatic) revealed considerable variation in the number of visceral pleura receptor end-organs, their morphology and chemical coding did not noticeably differ.

Results of the immunocytochemical protocols used in nerve fibers and neuroepithelial bodies of the positive control lung tissues confirmed selectivity of the staining in all cases.

Substitution of primary or secondary antisera with nonimmune sera consistently resulted in negative controls in all immunocytochemical stainings. For all studied antigens, single labeling did not show obvious differences with the results of multiple labeling. Nonamplified indirect immunostaining with primary antibodies, using the same concentrations as for TSA-enhanced reactions, gave negative staining results. No differences in localization of VGLUT2 were observed between TSA-enhanced and conventional immunodetection.


   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The present study was designed to provide extensive morphologic and neurochemical data on the innervation of the rat visceral pleura. Most conspicuous were sensory receptor–like nerve endings that appeared to be invariably intermingled with the elastic fibers of the visceral pleura, and were therefore referred to as VPRs. It was shown that VPRs can be visualized by PGP9.5 immunostaining, and specifically express VGLUTs, CB, Na+/K+-ATPase-{alpha}3, and occasionally purinergic P2X3-receptors, but do not show CGRP IR. The most likely spinal sensory nerve fibers that give rise to VPRs were found to be myelinated.

The whole mount preparation model of rat visceral pleura that was developed for this study resulted in intact preparations of one or more lung surfaces, and turned out to be an excellent tool for studying the complex organization of the innervation in the visceral pleura.

According to physiologic studies, an extensive sensory innervation was not to be expected in the visceral pleura (5). Based on their morphology, the nerve endings seen in the rat visceral pleura bear close resemblance to nonencapsulated nerve terminals described previously in the pleura of rabbits, dogs, and lambs (9, 10), which were suggested to be mainly restricted to the mediastinal and interlobar surfaces of the lung lobes (9). The discrepancy between physiologic and morphologic data may, therefore, at least partly be explained by the fact that, in most of the reported physiologic experiments (9, 33), only the costal part of the pleural surface was stimulated, and in which, in our experience, there is also a very low density of receptors.

The present study clearly illustrated, for the first time, that the laminar nerve endings invariably protrude between abundant elastic fibers in the rat visceral pleura. No selective contacts of the nerve terminals with blood or lymphatic vessels could be visualized. Also, VPRs did not reveal selective interactions with other cell types in the receptor end-organs, unlike the reported complex interactions with other cell types in, for instance, aortic baroreceptors (34).

Neurochemical characterization of VPRs in 10-d-old rats did not differ from that in 3-, 4- or 5-wk-old rats. Similar to the vagal sensory innervation of pulmonary neuroepithelial bodies (31), however, the preferential localization of VGLUT2 in VPR terminals, and not in the fibers, in older animals might be explained by the local accumulation of glutamatergic secretory vesicles and limited axonal transport. The age-unrelated predominant expression of P2X3 receptors on the surface of VPR endings is likely due to the myelinated nature of the approaching nerve fiber, as has been reported for the vagal sensory innervation of pulmonary neuroepithelial bodies (30).

Studying the presence of potential motor components in the innervation pattern revealed the absence of cholinergic motor fibers in nerve bundles traversing the visceral pleura, and that VPRs do not exhibit VAChT IR. TH IR, on the other hand, was seen in a few adrenergic nerve fibers in the pleural nerve bundles and also, exceptionally, in the terminals of rat VPRs. The presence of a typical marker for postganglionic sympathetic motor neurons in sensory endings may seem contradictory at first sight. However, catecholamines and catecholamine-synthesizing enzymes have been reported in subpopulations of mammalian (including rats) cranial and spinal sensory neurons (35–37).

Nerve fibers that give rise to VPRs turned out to be myelinated, with diameters ranging between 1.4 and 3.5 µm. In the old anatomic literature, myelinated nerve fibers were described in the visceral pleura of rabbits and dogs based on osmium tetroxide staining (9). Comparable to that work, in our study, myelin sheaths in the rat visceral pleura generally appeared to end just before a nerve branching point, where one of the branches gives rise to the first VPR. The other branch may, however, continue over a long distance as an unmyelinated fiber that regularly splits off additional receptor end-organs, implying that many of the VPRs are located at a considerable distance from the myelinization point.

Left or right unilateral infranodosal vagotomy revealed no reduction in the number of VPRs in the rat and, as such, confirms the nonvagal origin of VPRs. Our findings are in accordance with earlier assumptions that the receptor-like terminals in the visceral pleura may derive mainly from dorsal root ganglia of the upper thoracic spinal nerves, and reach the lungs via the sympathetic trunks (5, 9).

In a few of the earlier anatomic studies, speculations were made about a possible sensory function of the branching nerve terminals in the visceral pleura based on their morphologic resemblance to sensory receptors in other organs, such as tendons, blood vessels, and the heart (10, 11). Another argument used was the lack of identifiable "effector" structures in the visceral pleura (9, 11). A function as "stretch-receptor" of the nerve endings in the visceral pleura was suggested by Larsell and Coffey (5). The neurochemical characterization performed in the present study provides strong evidence for a sensory function of VPRs, revealing the expression of different sensory neuron–specific substances that have been used to selectively identify mechanoreceptor-like terminals in other rat organs (i.e., Na+/K+-ATPase-{alpha}3 [24], VGLUT2 [25], calcium-binding proteins [38, 39], and P2X3 receptors [26]). All VPRs in the present study appeared to originate from myelinated nerve fibers, another typical feature of peripheral low-threshold mechanosensors, but also A-fiber nociceptors (40). The preferential location of VPRs at the mediastinal and nearby interlobar lung surface suggests a potential role in sensing mechanical stress at the interface with other important organs located in the thorax.

Recently, pulmonary applied bradykinin, which is known to also stimulate the majority of electrophysiologically characterized (both mechanosensitive and nociceptive) afferents (40), was shown to induce cardiorespiratory responses that at least partly persist after vagotomy in several species (41, 42). This suggests that sympathetic pulmonary afferents, such as the population characterized in the present study, may also be involved in transmitting chemical and/or mechanical information from the lungs to the central nervous system. Multimodal mechano- and chemosensitive afferent units have been reported in the mediastinal parietal pleura (19) and, very recently, also in the costal parietal pleura (20).

A role as sensory end-organs for VPRs is further supported by their capacity to store and release glutamate. Glutamate is well known as one of the major motor transmitters in the central nervous system, and the presence of VGLUTs in sensory endings may, therefore, seem surprising. It has, however, been shown for some time that glutamate is also a neurotransmitter in the central projections of visceral sensory neurons in the brainstem (43, 44), and, recently, there is increasing evidence that glutamate is also a neurotransmitter in the peripheral projections of sensory neurons (25, 31, 45). The assumption has been made that glutamate, released from peripheral sensory nerve endings, may play an important role in modulating the excitability of sensory nerve endings (46, 47).

Very recently, a neurochemical coding, which is nearly identical to that revealed by VPRs in the present study, was described for two other sensory receptors in the rat lung (i.e., smooth muscle–associated airway receptors and the vagal sensory innervation of pulmonary neuroepithelial bodies) (22, 23). The latter studies revealed that both types of receptors also arise from 1- to 3.5-µm-thick myelinated fibers, which, in contrast to VPRs, have a vagal sensory origin. Although these three types of sensory nerve terminals in rat lungs reveal a somewhat different morphology, location, and origin, it may be hypothesized that they share complementary mechanosensory-like roles in supporting normal lung function.

Lipophylic styryl pyridinium fluorescent marker (FM) dyes typically become more fluorescent after insertion in the lipid bilayer of cell membranes, and have been widely used to observe synaptic vesicle recycling in a variety of cell types (48, 49). Recently, these FM or related dyes have also been used for vital labeling of sensory receptor cells and neurons (50–52), including pulmonary neuroepithelial bodies (53). Using FM2-10, so-called "cough receptors" could be visualized in the guinea pig trachea (40, 54). The presently demonstrated ability to visualize VPRs and to track the nerve fibers from which they arise over considerable distances using a fixable form of FM1-43 (AM1-43), in living whole mounts of the visceral pleura, opens up new perspectives for further physiologic studies of VPRs.

In conclusion, the present study provides an unambiguous morphologic and neurochemical identification of well defined sensory receptors that are uniquely associated with the visceral pleura. The use of a whole mount preparation model, and the possibility for selective live staining, create a solid basis for further physiologic studies of visceral pleural receptors. The reported characteristics suggest that VPRs may be involved in the sensory transduction of mechanical and/or chemical (nociceptive) stimuli, related to normal lung function or as a consequence of pleural disease. With respect to the potential clinical relevance, VPRs may be regarded as candidates for mediating at least certain aspects of the hitherto unknown mechanisms involved in the pain sensation and/or (reflex) dyspnea regularly described to result from visceral pleura tumors (12, 14), pulmonary embolism, and pleuritis (16, 17). Finally, it should be taken into account that the information carried by sensory terminals in the visceral pleura may give rise to sensations that are not necessarily conscious.


   Acknowledgments
 
The authors thank G. Svensson for the demanding and time-consuming preparation of the visceral pleura whole mounts, J. Van Daele and D. De Rijck for help with microscopy, imaging, and illustrations, and D. Vindevogel for aid with the manuscript.


   Footnotes
 
This work was supported by Fund for Scientific Research Flanders research grants G.0155.01 and G.0085.04 (D.A.) and by University of Antwerp grants NOI-BOF 2003 (D.A.) and KP-BOF 2006 (I.B.).

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1165/rcmb.2006-0256OC on December 14, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form July 14, 2006

Accepted in final form November 24, 2006


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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