AUTNEU-01643; No of Pages 5 Autonomic Neuroscience: Basic and Clinical xxx (2014) xxx–xxx
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Effects of Lewis lung carcinoma and B16 melanoma on the innervation of the mouse trachea Tilman Graulich a, Suman Kumar Das b, Lars Wessels a, Wolfgang Kummer a,c, Gerald Hoefler b, Christian Mühlfeld a,d,e,⁎ a
Institute of Anatomy and Cell Biology, Justus-Liebig-University Giessen, Aulweg 123, 35385 Giessen, Germany Institute of Pathology, Medical University Graz, Auenbruggerplatz 25, 8036 Graz, Austria University of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany d Institute of Functional and Applied Anatomy, Hannover Medical School, 30625 Hannover, Germany e Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany b c
a r t i c l e
i n f o
Article history: Received 6 December 2013 Received in revised form 4 March 2014 Accepted 12 March 2014 Available online xxxx Keywords: Cancer Autonomic nervous system Airways Stereology Electron microscopy
a b s t r a c t Cancer patients often suffer from dyspnea the pathogenesis of which is incompletely understood. Both dyspnea and pulmonary diseases are closely linked to airway innervation. Recently, it was shown that Lewis lung carcinoma induces cardiac hypoinnervation in the mouse. We hypothesized that airway innervation undergoes similar changes as myocardial innervation and that this effect occurs in different mouse models of cancer. C57Bl6 mice were randomly assigned to subcutaneous injection of Lewis lung carcinoma cells (LLC, n = 6), B16 melanoma cells (B16, n = 6), or saline (control group, C, n = 10). After 16 or 21 days, respectively, the trachea was processed for light and electron microscopic design-based stereology and the volume, surface area and length of axons ramifying in the tracheal wall were estimated. Body weight was reduced both in LLC and B16 vs. C. Hypoinnervation was present in both tumor groups compared to controls as volume and surface area of axons were significantly reduced in LLC and B16. However, the total length of tracheal axons and the mean number of axons per nerve fiber were reduced only in LLC but not in B16 compared to C indicating a differentially pronounced effect of cancer on tracheal innervation. In conclusion, reduced innervation of the trachea was observed in two different murine tumor models. These findings add to the pathophysiological concepts explaining cancer-related dyspnea and open new perspectives of treating this symptom. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Dyspnea occurs frequently in cancer patients and is often difficult to control (Ben-Aharon et al., 2012). The subjective sensation of the patient is a feeling of breathlessness to which a number of cancerrelated factors contribute, such as anemia, loss of skeletal muscle mass, inflammation, pain and psychosocial factors (Cachia and Ahmedzai, 2008; McMillan, 2009). The feeling of breathlessness is related to the patient's perception of air hunger, increased effort of breathing and chest tightness (Thomas et al., 2011). As such, the concepts to explain the occurrence of dyspnea in cancer patients are strongly related to the afferent information from the peripheral nervous system (sensory innervation of the airways and chemoreceptors) and the central integration of the effort of breathing (Thomas et al., 2011).
⁎ Corresponding author at: Institute of Functional and Applied Anatomy, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. E-mail address: [email protected] (C. Mühlfeld).
Surprisingly, there is very little information about changes of the airway innervation of cancer patients or in adequate animal models. Airways receive both sensory (afferent) and autonomic motor (efferent) innervation (Kummer et al., 1992; Canning, 2006; Undem and Nassenstein, 2009). Each nerve fiber consists of a varying number of axons which can only be determined at the electron microscopic level. The sensory fibers are mainly derived from the sensory vagal ganglia (nodose and jugular) and monitor the chemical composition of the inhaled air and of the airway lining fluid, mechanical irritation of the mucosa and airway wall tension. They provide this input to the respiratory regulatory centers of the brainstem thereby regulating both respiratory pattern and initiating reflexes governing airway function (Canning, 2006; Taylor-Clark and Undem, 2006; Krasteva et al., 2011). The efferent innervation is dominated by parasympathetic fibers that regulate the airway smooth muscle cell tone (Kobayashi et al., 2004; Canning, 2006) and the secretion of mucus but are also addressing several other targets such as the local immune system (Cavalotti et al., 2004; Weigand et al., 2009). Consequently, structural and functional alteration of airway innervation is associated with both
http://dx.doi.org/10.1016/j.autneu.2014.03.005 1566-0702/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Graulich, T., et al., Effects of Lewis lung carcinoma and B16 melanoma on the innervation of the mouse trachea, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.005
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T. Graulich et al. / Autonomic Neuroscience: Basic and Clinical xxx (2014) xxx–xxx
respiratory diseases and breathing discomfort (Undem and Nassenstein, 2009). Interestingly, in the cancer cachectic mouse, a reduction of myocardial innervation to about 50% of control values was observed (Mühlfeld et al., 2011). We hypothesized that this phenomenon is not specific for the heart but affects the airways as well. Therefore, the present study was designed to test this hypothesis in two experimental models of extrapulmonary cancer by quantification of tracheal innervation using stereology.
2. Material and methods All the animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol (GZ 66.010/0085-II/10b/2009) was approved by the Austrian committee on the ethics of animal experiments (Bundesministerium für Wissenschaft und Forschung). Female 8–9-week-old C57BL6/J mice were maintained on a 12 h light–12 h dark cycle and were fed a standard laboratory chow diet (4.5% w/w fat). Food intake and body mass were monitored daily throughout the experiments. Mice were randomly distributed to one of the following three groups: 1. control group (C, n = 10, injection of saline solution); 2. Lewis lung carcinoma group (LLC, n = 6, injection of 2 ∗ 106 Lewis lung carcinoma cells); 3. B16 melanoma group (B16, n = 6, injection of 2 ∗ 106 B16 melanoma cells). Both saline and tumor cell injections were performed subcutaneously in the neck region of the mice. Tumor cells were taken from an exponentially proliferating cell culture grown in Dulbecco's Modified Eagle Medium (DMEM) with high glucose concentration containing 10% (v/v) fetal bovine serum, 2 mM L-glutamine und 1% penicillin–streptomycin. LLC and B16 mice were sacrificed 21 or 16 days after tumor implantation, respectively. Control animals were either killed 16 (n = 4) or 21 (n = 6) days after saline injection. As the body weight gain did not differ between control animals killed after 16 or 21 days, the data were pooled and combined as one control group. After sacrifice, several organs were harvested for other studies (Das et al., 2011; Mühlfeld et al., 2011; Graulich et al., 2012). The lungs and the trachea were fixed by instillation fixation through the larynx using a fixative containing 4% paraformaldehyde in PBS at a hydrostatic pressure of 20 cm H2O. The whole trachea (from the lower end of the larynx to the bifurcation) was cut into rings of approximately 0.5 mm thickness to enable later tissue orientation. Using systematic uniform random sampling the rings were randomly assigned to epoxy resin embedding according to standard protocols or cryofixation. The samples embedded in epoxy resin were used for obtaining semithin sections (1 μm) stained with Richardson's solution to quantify the overall composition of tracheal wall components. From the cryofixed samples, 40 μm thick cryostat sections were obtained and subjected to immunolabeling for proteingene-product 9.5 (PGP9.5) as a pan neuronal marker (Gulbenkian et al., 1987). Unspecific protein binding sites were blocked before incubation with the primary antibody (rabbit polyclonal anti-PGP9.5 antibody, diluted 1:4000, Biotrend, Köln, Germany) for 18 h. After washing, sections were incubated with the secondary antibody (peroxidase-linked donkey-anti-rabbit IgG, diluted 1:100, Amersham International Biotechnology, Little Chalfont, Buckinghamshire, UK) for 1 h. Visualization of the immunoreaction was performed by the 3,3′-diaminobenzidine reaction enhanced by 1.5% nickel ammonium sulfate. The sections were then osmicated, contrasted with halfsaturated uranyl acetate, dehydrated in an ascending ethanol series, and finally flat embedded in epoxy resin. The embedded section was glued on a resin blind block and ultrathin sections were cut and poststained with lead citrate and uranyl acetate.
Stereological analyses of semithin sections were carried out with an Olympus BX51 light microscope (Olympus, Hamburg, Germany), equipped with an Olympus DP72 digital camera and a computer with the newCast software (Visiopharm, Horsholm, Denmark) and of ultrathin sections using a LEO 906 transmission electron microscope (Zeiss, Oberkochen, Germany), respectively. Two semi- and two ultrathin sections per animal were analyzed. All fields of view (FOV) for analysis were gathered by systematic uniform random sampling (SURS) (Gundersen and Jensen, 1987). Consistent with Gundersen and Jensen (1987) a minimum number of 100–200 counting events per parameter and animal was regarded as sufficient. Tracheal wall composition was evaluated by quantification of the volume fractions of epithelium, smooth muscle cells, connective tissue and cartilage by the point counting method (Weibel, 1979; Mühlfeld and Ochs, 2013) at an objective lens magnification of 10×. The total sections were used for quantification. The total axon length ramifying in the tracheal wall layers was estimated using a modification of the method recently described by Mühlfeld et al. (2010). In short, at a magnification of 21,560× the sections were sampled yielding a total number of FOV of 772 ± 359 in C, 989 ± 211 in LLC and 453 ± 359 in B16. The volume and surface density of the axons were estimated using a combined point and line grid. The line grid consisted of cycloid arches and was positioned on the FOV parallel to a virtual axis from the adventitial layer to the epithelium. As the staining intensity varied, all axons that were identifiable either by staining or morphology were used for counting (Fig. 1). Under the simplistic geometric assumption of axons being cylindrical we calculated the length of axons contained within a piece of trachea of 1 mm length from the volume and surface area of the axons.
3. Statistics Data are presented as mean (standard deviation) or as individual data for each animal in the figures. Differences between control and tumor bearing mice were analyzed using Kruskal Wallis rank sum test and subsequently, if a significant difference was observed, if applicable, the Bonferroni's post hoc test was used to compare the groups pairwise. Differences were regarded as significant if p b 0.05.
Fig. 1. Tracheal nerve fiber next to smooth muscle cells. A nerve fiber (arrows) embedded in collagen fibrils (col) from a control animal is shown next to a smooth muscle cell (SMC). The image demonstrates that the immunostaining is very variable among the axons.
Please cite this article as: Graulich, T., et al., Effects of Lewis lung carcinoma and B16 melanoma on the innervation of the mouse trachea, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.005
T. Graulich et al. / Autonomic Neuroscience: Basic and Clinical xxx (2014) xxx–xxx
4. Results
Table 2 Tracheal wall composition.
The initial body mass at the time-point of tumor cell or saline injection was not statistically different between the groups. The final body mass showed a similar loss of body mass in both tumor groups and a gain of body mass in the same period in the control group. The tumor cell injection induced a tumor mass which was similar in both tumor groups (Table 1). The composition of the tracheal wall with respect to the volume of the epithelium, connective tissue, cartilage and smooth muscle cells did not exhibit major differences among the group. The only statistically significant differences were related to a higher volume of smooth muscle cells in the LLC group (Table 2). In all groups, the majority of nerve fibers stained by PGP 9.5 were found in the smooth muscle layer and around blood vessels (Fig. 1). Minor amounts of nerve fibers were observed in the lamina propria, the epithelium and the connective tissue around the cartilage rings. In both tumor groups, the volume and surface area of axons ramifying in the tracheal wall were statistically significantly reduced indicating hypoinnervation. The model-based, calculated parameter “length of axons” showed a significant reduction only in the LLC group but not in the B16 group. The higher scattering of data and the stronger decrease in axon surface area and volume in the LLC group may provide an explanation for the non-significance of the B16 group. The mean number of axons per nerve fiber was not significantly reduced in the tumor groups although a trend towards a reduction was observed in LLC compared with C (p = 0.094) (Fig. 2).
5. Discussion The present study for the first time shows that an extrapulmonary tumor induced by injection of tumor cells in mice quantitatively affects the innervation of the trachea. Moreover, the reduction was present in two tumor models based on tumor cell lines with completely different origin. This suggests that the observed effect is rather based on the systemic effect of the tumor than on a cell-type specific characteristic. A similar effect on the innervation of the mouse left ventricle was recently shown in one of these tumor models (LLC), indicating that this phenomenon is not specific to one organ but may represent a reaction of the autonomic and/or sensory nervous system in general (Mühlfeld et al., 2011). Here, we used two independent, well-established murine tumor models, namely Lewis lung carcinoma (Budzynski, 1982) and B16 melanoma (Graves et al., 2006). Both models caused a tumor burden of 10–15% of body mass and a significant reduction of body mass consistent with the development of cachexia. Mice of the control group had a significant increase of body mass in the same period of time. The duration of the experiments was 21 days for LLC and only 16 days for B16. This discrepancy was caused by the greater suffering of the B16 group compared with LLC which made a further duration of the experiments until 21 days both practically and ethically impossible. The shorter duration of the experiments in the B16 group may be a reason for the less pronounced effect on the tracheal innervation in this group.
Table 1 Body and tumor mass.
Initial body mass Final body mass Mass difference Tumor mass
3
C (n = 10)
LLC (n = 6)
B16 (n = 6)
19.3 (1.4) 21.4 (1.7) 2.14 (0.72) 0
20.0 (0.5) 17.4 (0.5) ⁎⁎⁎ −2.47 (0.56) ⁎⁎⁎ 2.47 (0.56) ⁎⁎⁎
18.2 (1.7) 16.0 (0.5) ⁎⁎⁎ −2.20 (2.03) ⁎⁎⁎ 1.92 (0.46) ⁎⁎⁎
Data are given as mean (SD). C, control group; LLC, Lewis lung carcinoma group; B16, B16 melanoma group. ⁎⁎⁎ p b 0.001.
VV(cart/trachea) VV(con/trachea) VV(SMC/trachea) VV(epi/trachea) V(cart) [μm3/μm] V(con) [μm3/μm] V(SMC) [μm3/μm] V(epi) [μm3/μm]
C (n = 10)
LLC (n = 6)
B16 (n = 6)
0.36 (0.07) 0.32 (0.05) 0.11 (0.05) 0.21 (0.08) 93,290 (36,310) 81,700 (33,140) 29,260 (16,999) 48,640 (10,755)
0.33 (0.06) 0.30 (0.04) 0.18 (0.03)⁎, ⁎⁎⁎ 0.19 (0.03) 88,983 (32,387) 79,167 (27,734) 46,233 (16,937)⁎⁎
0.32 (0.06) 0.36 (0.07) 0.08 (0.02)⁎⁎⁎ 0.24 (0.04) 75,367 (15,467) 83,917 (17,472) 19,317 (6406)⁎⁎
49,717 (15,408)
55,733 (10,384)
Data are given as mean (SD). All data were referred to 1 μm length of trachea. C, control group; LLC, Lewis lung carcinoma group; B16, B16 melanoma group; VV, volume density related to tracheal volume; cart, cartilage; con, connective tissue; SMC, smooth muscle cells; epi, epithelium; ⁎ p b 0.05 vs. control group. ⁎⁎ p b 0.05 B16 vs. LLC. ⁎⁎⁎ p b 0.01 B16 vs. LLC.
The morphometric quantification of the innervation in this study was based on the principles of stereology (for a review see Ochs and Mühlfeld (2013)), however, there is one limitation that should be mentioned here. Surface area estimations require the use of isotropic uniform random sections or a combination of vertical sections and cycloid arcs (Baddeley et al., 1986). Our method of using tracheal rings and cycloid arcs does not completely fulfill the criteria for vertical sections which may impose a minimal bias to our data. Due to Baddeley et al. (1986) it would have been necessary to cut the trachea longitudinally and randomize the orientation in a flat position, however, this would have caused morphological deterioration of the tracheal wall due to the cartilage rings, so we had to accept a potential bias here. However, as the volume estimation does not require randomization of tissue orientation and our data on volume and surface area are well correlated we think that the potential bias has not influenced our results substantially. As mentioned above, hypoinnervation due to cancer was also observed in a recent study on the mouse left ventricle (Mühlfeld et al., 2011). The left ventricle is mainly supplied by sympathetic, noradrenergic nerve fibers with only limited amounts of parasympathetic, cholinergic or sensory nerve fibers (Peters et al., 1980; Momose et al., 2001). The tracheal innervation is more complex with afferent, cholinergic and nitrergic efferent nerve fibers contributing a large proportion to tracheal innervation (Canning, 2006; Undem and Nassenstein, 2009). Thus, the tracheal hypoinnervation observed in this study reveals that the effect of the tumor is not restricted to the sympathetic nervous system. The functional significance of these findings, particularly for the development of dyspnea, however, is unclear and cannot be answered from the present study. Additional information on the type of nerve fibers or the tracheal wall layer where nerve fibers are reduced could provide helpful insights in future studies. As such, given the information on the function of airway innervation (Undem and Nassenstein, 2009), effects of reduced/altered innervation may be related to changes in smooth muscle tone, reduced chemosensory information from the epithelium, changes in sputum amount and composition, among others. In addition to the subjective feeling of breathlessness, however, some studies have also revealed a decrease in pulmonary function in patients with extrapulmonary tumors (Bachmann et al., 2009). In a recent study, it was shown that the alveolar surface area was not reduced in mice subjected to LLC despite the similarities between cancer cachexia and caloric restriction (Graulich et al., 2012). In the latter condition, also termed nutritional emphysema, a reduction of alveolar surface area has been described (Harkema et al., 1984; Karlinsky et al., 1986). Changes of the airway innervation in caloric restriction have not been investigated so far. It is likely, however, that the reduction of airway innervation observed in the present cancer
Please cite this article as: Graulich, T., et al., Effects of Lewis lung carcinoma and B16 melanoma on the innervation of the mouse trachea, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.005
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T. Graulich et al. / Autonomic Neuroscience: Basic and Clinical xxx (2014) xxx–xxx
Fig. 2. Stereological data on tracheal innervation. A: volume of axons. B: surface area of axons. C: length of axons. D: mean number of axons per nerve fiber. Volume, surface area and length were related to 1 μm length of trachea wall. Each data point represents one animal, horizontal bars represent group mean.
mouse models is not due to body weight loss as the left ventricular innervation of the mouse heart was not altered by caloric restriction (Gruber et al., 2012) in contrast to the findings in the cancer cachectic mouse (Mühlfeld et al., 2011). To see whether cancer affects the overall composition of the tracheal wall, the volume fractions of epithelium, cartilage, smooth muscle cells and connective tissue were determined. In both tumor groups, none of the compartments showed signs of atrophy. Surprisingly, a significantly higher smooth muscle cell volume fraction and total volume per unit length of trachea were observed in the LLC group compared with C and B16. Qualitatively, the volume of individual smooth muscle cells did not appear to be different between the groups. Thus, this observation may rather be associated with factors influencing the quantification of smooth muscle cells (e.g. a different contraction status) in the different groups than with the experimental model and, overall, the composition of the tracheal wall appeared similar in all groups. In contrast, the myocardium of mice subjected to LLC was characterized by a strong atrophic phenotype mainly because of myofibrils (Mühlfeld et al., 2011). The investigation of the lung in the LLC model had also shown a reduction of the intracellular surfactant pool despite normal intraalveolar surfactant composition (Graulich et al., 2012). Taken together, our study shows that tracheal innervation is significantly reduced in two murine tumor models. These data may provide a potential additional explanation for the occurrence of dyspnea in cancer patients.
Competing interests statement The authors declare that they have no competing interests. Author contributions TG, SKD, LW have contributed to data acquisition, analysis and interpretation of the data, WK, GH and CM have contributed to the conception and design of the study, and the analysis and interpretation of the data. All authors were involved in drafting and revising the manuscript critically for important intellectual content and have read and approved the final manuscript. Funding The study was funded by the Verein zur Förderung der Krebsforschung in Gießen e.V. and the DFG via the excellence cluster Rebirth (CM) and by the PhD program Molecular Medicine of the Medical University of Graz (SD). Acknowledgments The authors wish to thank Gerd Magdowski, Gerhard Kripp, Tamara Papadakis (Gießen) and Silvia Schauer (Graz) for the excellent technical support.
Please cite this article as: Graulich, T., et al., Effects of Lewis lung carcinoma and B16 melanoma on the innervation of the mouse trachea, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.005
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Please cite this article as: Graulich, T., et al., Effects of Lewis lung carcinoma and B16 melanoma on the innervation of the mouse trachea, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.005
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Autonomic Neuroscience: Basic and Clinical journal homepage: www.elsevier.com/locate/autneu
Effects of Lewis lung carcinoma and B16 melanoma on the innervation of the mouse trachea Tilman Graulich a, Suman Kumar Das b, Lars Wessels a, Wolfgang Kummer a,c, Gerald Hoefler b, Christian Mühlfeld a,d,e,⁎ a
Institute of Anatomy and Cell Biology, Justus-Liebig-University Giessen, Aulweg 123, 35385 Giessen, Germany Institute of Pathology, Medical University Graz, Auenbruggerplatz 25, 8036 Graz, Austria University of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany d Institute of Functional and Applied Anatomy, Hannover Medical School, 30625 Hannover, Germany e Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany b c
a r t i c l e
i n f o
Article history: Received 6 December 2013 Received in revised form 4 March 2014 Accepted 12 March 2014 Available online xxxx Keywords: Cancer Autonomic nervous system Airways Stereology Electron microscopy
a b s t r a c t Cancer patients often suffer from dyspnea the pathogenesis of which is incompletely understood. Both dyspnea and pulmonary diseases are closely linked to airway innervation. Recently, it was shown that Lewis lung carcinoma induces cardiac hypoinnervation in the mouse. We hypothesized that airway innervation undergoes similar changes as myocardial innervation and that this effect occurs in different mouse models of cancer. C57Bl6 mice were randomly assigned to subcutaneous injection of Lewis lung carcinoma cells (LLC, n = 6), B16 melanoma cells (B16, n = 6), or saline (control group, C, n = 10). After 16 or 21 days, respectively, the trachea was processed for light and electron microscopic design-based stereology and the volume, surface area and length of axons ramifying in the tracheal wall were estimated. Body weight was reduced both in LLC and B16 vs. C. Hypoinnervation was present in both tumor groups compared to controls as volume and surface area of axons were significantly reduced in LLC and B16. However, the total length of tracheal axons and the mean number of axons per nerve fiber were reduced only in LLC but not in B16 compared to C indicating a differentially pronounced effect of cancer on tracheal innervation. In conclusion, reduced innervation of the trachea was observed in two different murine tumor models. These findings add to the pathophysiological concepts explaining cancer-related dyspnea and open new perspectives of treating this symptom. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Dyspnea occurs frequently in cancer patients and is often difficult to control (Ben-Aharon et al., 2012). The subjective sensation of the patient is a feeling of breathlessness to which a number of cancerrelated factors contribute, such as anemia, loss of skeletal muscle mass, inflammation, pain and psychosocial factors (Cachia and Ahmedzai, 2008; McMillan, 2009). The feeling of breathlessness is related to the patient's perception of air hunger, increased effort of breathing and chest tightness (Thomas et al., 2011). As such, the concepts to explain the occurrence of dyspnea in cancer patients are strongly related to the afferent information from the peripheral nervous system (sensory innervation of the airways and chemoreceptors) and the central integration of the effort of breathing (Thomas et al., 2011).
⁎ Corresponding author at: Institute of Functional and Applied Anatomy, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. E-mail address: [email protected] (C. Mühlfeld).
Surprisingly, there is very little information about changes of the airway innervation of cancer patients or in adequate animal models. Airways receive both sensory (afferent) and autonomic motor (efferent) innervation (Kummer et al., 1992; Canning, 2006; Undem and Nassenstein, 2009). Each nerve fiber consists of a varying number of axons which can only be determined at the electron microscopic level. The sensory fibers are mainly derived from the sensory vagal ganglia (nodose and jugular) and monitor the chemical composition of the inhaled air and of the airway lining fluid, mechanical irritation of the mucosa and airway wall tension. They provide this input to the respiratory regulatory centers of the brainstem thereby regulating both respiratory pattern and initiating reflexes governing airway function (Canning, 2006; Taylor-Clark and Undem, 2006; Krasteva et al., 2011). The efferent innervation is dominated by parasympathetic fibers that regulate the airway smooth muscle cell tone (Kobayashi et al., 2004; Canning, 2006) and the secretion of mucus but are also addressing several other targets such as the local immune system (Cavalotti et al., 2004; Weigand et al., 2009). Consequently, structural and functional alteration of airway innervation is associated with both
http://dx.doi.org/10.1016/j.autneu.2014.03.005 1566-0702/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Graulich, T., et al., Effects of Lewis lung carcinoma and B16 melanoma on the innervation of the mouse trachea, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.005
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T. Graulich et al. / Autonomic Neuroscience: Basic and Clinical xxx (2014) xxx–xxx
respiratory diseases and breathing discomfort (Undem and Nassenstein, 2009). Interestingly, in the cancer cachectic mouse, a reduction of myocardial innervation to about 50% of control values was observed (Mühlfeld et al., 2011). We hypothesized that this phenomenon is not specific for the heart but affects the airways as well. Therefore, the present study was designed to test this hypothesis in two experimental models of extrapulmonary cancer by quantification of tracheal innervation using stereology.
2. Material and methods All the animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol (GZ 66.010/0085-II/10b/2009) was approved by the Austrian committee on the ethics of animal experiments (Bundesministerium für Wissenschaft und Forschung). Female 8–9-week-old C57BL6/J mice were maintained on a 12 h light–12 h dark cycle and were fed a standard laboratory chow diet (4.5% w/w fat). Food intake and body mass were monitored daily throughout the experiments. Mice were randomly distributed to one of the following three groups: 1. control group (C, n = 10, injection of saline solution); 2. Lewis lung carcinoma group (LLC, n = 6, injection of 2 ∗ 106 Lewis lung carcinoma cells); 3. B16 melanoma group (B16, n = 6, injection of 2 ∗ 106 B16 melanoma cells). Both saline and tumor cell injections were performed subcutaneously in the neck region of the mice. Tumor cells were taken from an exponentially proliferating cell culture grown in Dulbecco's Modified Eagle Medium (DMEM) with high glucose concentration containing 10% (v/v) fetal bovine serum, 2 mM L-glutamine und 1% penicillin–streptomycin. LLC and B16 mice were sacrificed 21 or 16 days after tumor implantation, respectively. Control animals were either killed 16 (n = 4) or 21 (n = 6) days after saline injection. As the body weight gain did not differ between control animals killed after 16 or 21 days, the data were pooled and combined as one control group. After sacrifice, several organs were harvested for other studies (Das et al., 2011; Mühlfeld et al., 2011; Graulich et al., 2012). The lungs and the trachea were fixed by instillation fixation through the larynx using a fixative containing 4% paraformaldehyde in PBS at a hydrostatic pressure of 20 cm H2O. The whole trachea (from the lower end of the larynx to the bifurcation) was cut into rings of approximately 0.5 mm thickness to enable later tissue orientation. Using systematic uniform random sampling the rings were randomly assigned to epoxy resin embedding according to standard protocols or cryofixation. The samples embedded in epoxy resin were used for obtaining semithin sections (1 μm) stained with Richardson's solution to quantify the overall composition of tracheal wall components. From the cryofixed samples, 40 μm thick cryostat sections were obtained and subjected to immunolabeling for proteingene-product 9.5 (PGP9.5) as a pan neuronal marker (Gulbenkian et al., 1987). Unspecific protein binding sites were blocked before incubation with the primary antibody (rabbit polyclonal anti-PGP9.5 antibody, diluted 1:4000, Biotrend, Köln, Germany) for 18 h. After washing, sections were incubated with the secondary antibody (peroxidase-linked donkey-anti-rabbit IgG, diluted 1:100, Amersham International Biotechnology, Little Chalfont, Buckinghamshire, UK) for 1 h. Visualization of the immunoreaction was performed by the 3,3′-diaminobenzidine reaction enhanced by 1.5% nickel ammonium sulfate. The sections were then osmicated, contrasted with halfsaturated uranyl acetate, dehydrated in an ascending ethanol series, and finally flat embedded in epoxy resin. The embedded section was glued on a resin blind block and ultrathin sections were cut and poststained with lead citrate and uranyl acetate.
Stereological analyses of semithin sections were carried out with an Olympus BX51 light microscope (Olympus, Hamburg, Germany), equipped with an Olympus DP72 digital camera and a computer with the newCast software (Visiopharm, Horsholm, Denmark) and of ultrathin sections using a LEO 906 transmission electron microscope (Zeiss, Oberkochen, Germany), respectively. Two semi- and two ultrathin sections per animal were analyzed. All fields of view (FOV) for analysis were gathered by systematic uniform random sampling (SURS) (Gundersen and Jensen, 1987). Consistent with Gundersen and Jensen (1987) a minimum number of 100–200 counting events per parameter and animal was regarded as sufficient. Tracheal wall composition was evaluated by quantification of the volume fractions of epithelium, smooth muscle cells, connective tissue and cartilage by the point counting method (Weibel, 1979; Mühlfeld and Ochs, 2013) at an objective lens magnification of 10×. The total sections were used for quantification. The total axon length ramifying in the tracheal wall layers was estimated using a modification of the method recently described by Mühlfeld et al. (2010). In short, at a magnification of 21,560× the sections were sampled yielding a total number of FOV of 772 ± 359 in C, 989 ± 211 in LLC and 453 ± 359 in B16. The volume and surface density of the axons were estimated using a combined point and line grid. The line grid consisted of cycloid arches and was positioned on the FOV parallel to a virtual axis from the adventitial layer to the epithelium. As the staining intensity varied, all axons that were identifiable either by staining or morphology were used for counting (Fig. 1). Under the simplistic geometric assumption of axons being cylindrical we calculated the length of axons contained within a piece of trachea of 1 mm length from the volume and surface area of the axons.
3. Statistics Data are presented as mean (standard deviation) or as individual data for each animal in the figures. Differences between control and tumor bearing mice were analyzed using Kruskal Wallis rank sum test and subsequently, if a significant difference was observed, if applicable, the Bonferroni's post hoc test was used to compare the groups pairwise. Differences were regarded as significant if p b 0.05.
Fig. 1. Tracheal nerve fiber next to smooth muscle cells. A nerve fiber (arrows) embedded in collagen fibrils (col) from a control animal is shown next to a smooth muscle cell (SMC). The image demonstrates that the immunostaining is very variable among the axons.
Please cite this article as: Graulich, T., et al., Effects of Lewis lung carcinoma and B16 melanoma on the innervation of the mouse trachea, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.005
T. Graulich et al. / Autonomic Neuroscience: Basic and Clinical xxx (2014) xxx–xxx
4. Results
Table 2 Tracheal wall composition.
The initial body mass at the time-point of tumor cell or saline injection was not statistically different between the groups. The final body mass showed a similar loss of body mass in both tumor groups and a gain of body mass in the same period in the control group. The tumor cell injection induced a tumor mass which was similar in both tumor groups (Table 1). The composition of the tracheal wall with respect to the volume of the epithelium, connective tissue, cartilage and smooth muscle cells did not exhibit major differences among the group. The only statistically significant differences were related to a higher volume of smooth muscle cells in the LLC group (Table 2). In all groups, the majority of nerve fibers stained by PGP 9.5 were found in the smooth muscle layer and around blood vessels (Fig. 1). Minor amounts of nerve fibers were observed in the lamina propria, the epithelium and the connective tissue around the cartilage rings. In both tumor groups, the volume and surface area of axons ramifying in the tracheal wall were statistically significantly reduced indicating hypoinnervation. The model-based, calculated parameter “length of axons” showed a significant reduction only in the LLC group but not in the B16 group. The higher scattering of data and the stronger decrease in axon surface area and volume in the LLC group may provide an explanation for the non-significance of the B16 group. The mean number of axons per nerve fiber was not significantly reduced in the tumor groups although a trend towards a reduction was observed in LLC compared with C (p = 0.094) (Fig. 2).
5. Discussion The present study for the first time shows that an extrapulmonary tumor induced by injection of tumor cells in mice quantitatively affects the innervation of the trachea. Moreover, the reduction was present in two tumor models based on tumor cell lines with completely different origin. This suggests that the observed effect is rather based on the systemic effect of the tumor than on a cell-type specific characteristic. A similar effect on the innervation of the mouse left ventricle was recently shown in one of these tumor models (LLC), indicating that this phenomenon is not specific to one organ but may represent a reaction of the autonomic and/or sensory nervous system in general (Mühlfeld et al., 2011). Here, we used two independent, well-established murine tumor models, namely Lewis lung carcinoma (Budzynski, 1982) and B16 melanoma (Graves et al., 2006). Both models caused a tumor burden of 10–15% of body mass and a significant reduction of body mass consistent with the development of cachexia. Mice of the control group had a significant increase of body mass in the same period of time. The duration of the experiments was 21 days for LLC and only 16 days for B16. This discrepancy was caused by the greater suffering of the B16 group compared with LLC which made a further duration of the experiments until 21 days both practically and ethically impossible. The shorter duration of the experiments in the B16 group may be a reason for the less pronounced effect on the tracheal innervation in this group.
Table 1 Body and tumor mass.
Initial body mass Final body mass Mass difference Tumor mass
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C (n = 10)
LLC (n = 6)
B16 (n = 6)
19.3 (1.4) 21.4 (1.7) 2.14 (0.72) 0
20.0 (0.5) 17.4 (0.5) ⁎⁎⁎ −2.47 (0.56) ⁎⁎⁎ 2.47 (0.56) ⁎⁎⁎
18.2 (1.7) 16.0 (0.5) ⁎⁎⁎ −2.20 (2.03) ⁎⁎⁎ 1.92 (0.46) ⁎⁎⁎
Data are given as mean (SD). C, control group; LLC, Lewis lung carcinoma group; B16, B16 melanoma group. ⁎⁎⁎ p b 0.001.
VV(cart/trachea) VV(con/trachea) VV(SMC/trachea) VV(epi/trachea) V(cart) [μm3/μm] V(con) [μm3/μm] V(SMC) [μm3/μm] V(epi) [μm3/μm]
C (n = 10)
LLC (n = 6)
B16 (n = 6)
0.36 (0.07) 0.32 (0.05) 0.11 (0.05) 0.21 (0.08) 93,290 (36,310) 81,700 (33,140) 29,260 (16,999) 48,640 (10,755)
0.33 (0.06) 0.30 (0.04) 0.18 (0.03)⁎, ⁎⁎⁎ 0.19 (0.03) 88,983 (32,387) 79,167 (27,734) 46,233 (16,937)⁎⁎
0.32 (0.06) 0.36 (0.07) 0.08 (0.02)⁎⁎⁎ 0.24 (0.04) 75,367 (15,467) 83,917 (17,472) 19,317 (6406)⁎⁎
49,717 (15,408)
55,733 (10,384)
Data are given as mean (SD). All data were referred to 1 μm length of trachea. C, control group; LLC, Lewis lung carcinoma group; B16, B16 melanoma group; VV, volume density related to tracheal volume; cart, cartilage; con, connective tissue; SMC, smooth muscle cells; epi, epithelium; ⁎ p b 0.05 vs. control group. ⁎⁎ p b 0.05 B16 vs. LLC. ⁎⁎⁎ p b 0.01 B16 vs. LLC.
The morphometric quantification of the innervation in this study was based on the principles of stereology (for a review see Ochs and Mühlfeld (2013)), however, there is one limitation that should be mentioned here. Surface area estimations require the use of isotropic uniform random sections or a combination of vertical sections and cycloid arcs (Baddeley et al., 1986). Our method of using tracheal rings and cycloid arcs does not completely fulfill the criteria for vertical sections which may impose a minimal bias to our data. Due to Baddeley et al. (1986) it would have been necessary to cut the trachea longitudinally and randomize the orientation in a flat position, however, this would have caused morphological deterioration of the tracheal wall due to the cartilage rings, so we had to accept a potential bias here. However, as the volume estimation does not require randomization of tissue orientation and our data on volume and surface area are well correlated we think that the potential bias has not influenced our results substantially. As mentioned above, hypoinnervation due to cancer was also observed in a recent study on the mouse left ventricle (Mühlfeld et al., 2011). The left ventricle is mainly supplied by sympathetic, noradrenergic nerve fibers with only limited amounts of parasympathetic, cholinergic or sensory nerve fibers (Peters et al., 1980; Momose et al., 2001). The tracheal innervation is more complex with afferent, cholinergic and nitrergic efferent nerve fibers contributing a large proportion to tracheal innervation (Canning, 2006; Undem and Nassenstein, 2009). Thus, the tracheal hypoinnervation observed in this study reveals that the effect of the tumor is not restricted to the sympathetic nervous system. The functional significance of these findings, particularly for the development of dyspnea, however, is unclear and cannot be answered from the present study. Additional information on the type of nerve fibers or the tracheal wall layer where nerve fibers are reduced could provide helpful insights in future studies. As such, given the information on the function of airway innervation (Undem and Nassenstein, 2009), effects of reduced/altered innervation may be related to changes in smooth muscle tone, reduced chemosensory information from the epithelium, changes in sputum amount and composition, among others. In addition to the subjective feeling of breathlessness, however, some studies have also revealed a decrease in pulmonary function in patients with extrapulmonary tumors (Bachmann et al., 2009). In a recent study, it was shown that the alveolar surface area was not reduced in mice subjected to LLC despite the similarities between cancer cachexia and caloric restriction (Graulich et al., 2012). In the latter condition, also termed nutritional emphysema, a reduction of alveolar surface area has been described (Harkema et al., 1984; Karlinsky et al., 1986). Changes of the airway innervation in caloric restriction have not been investigated so far. It is likely, however, that the reduction of airway innervation observed in the present cancer
Please cite this article as: Graulich, T., et al., Effects of Lewis lung carcinoma and B16 melanoma on the innervation of the mouse trachea, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.005
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T. Graulich et al. / Autonomic Neuroscience: Basic and Clinical xxx (2014) xxx–xxx
Fig. 2. Stereological data on tracheal innervation. A: volume of axons. B: surface area of axons. C: length of axons. D: mean number of axons per nerve fiber. Volume, surface area and length were related to 1 μm length of trachea wall. Each data point represents one animal, horizontal bars represent group mean.
mouse models is not due to body weight loss as the left ventricular innervation of the mouse heart was not altered by caloric restriction (Gruber et al., 2012) in contrast to the findings in the cancer cachectic mouse (Mühlfeld et al., 2011). To see whether cancer affects the overall composition of the tracheal wall, the volume fractions of epithelium, cartilage, smooth muscle cells and connective tissue were determined. In both tumor groups, none of the compartments showed signs of atrophy. Surprisingly, a significantly higher smooth muscle cell volume fraction and total volume per unit length of trachea were observed in the LLC group compared with C and B16. Qualitatively, the volume of individual smooth muscle cells did not appear to be different between the groups. Thus, this observation may rather be associated with factors influencing the quantification of smooth muscle cells (e.g. a different contraction status) in the different groups than with the experimental model and, overall, the composition of the tracheal wall appeared similar in all groups. In contrast, the myocardium of mice subjected to LLC was characterized by a strong atrophic phenotype mainly because of myofibrils (Mühlfeld et al., 2011). The investigation of the lung in the LLC model had also shown a reduction of the intracellular surfactant pool despite normal intraalveolar surfactant composition (Graulich et al., 2012). Taken together, our study shows that tracheal innervation is significantly reduced in two murine tumor models. These data may provide a potential additional explanation for the occurrence of dyspnea in cancer patients.
Competing interests statement The authors declare that they have no competing interests. Author contributions TG, SKD, LW have contributed to data acquisition, analysis and interpretation of the data, WK, GH and CM have contributed to the conception and design of the study, and the analysis and interpretation of the data. All authors were involved in drafting and revising the manuscript critically for important intellectual content and have read and approved the final manuscript. Funding The study was funded by the Verein zur Förderung der Krebsforschung in Gießen e.V. and the DFG via the excellence cluster Rebirth (CM) and by the PhD program Molecular Medicine of the Medical University of Graz (SD). Acknowledgments The authors wish to thank Gerd Magdowski, Gerhard Kripp, Tamara Papadakis (Gießen) and Silvia Schauer (Graz) for the excellent technical support.
Please cite this article as: Graulich, T., et al., Effects of Lewis lung carcinoma and B16 melanoma on the innervation of the mouse trachea, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.005
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Please cite this article as: Graulich, T., et al., Effects of Lewis lung carcinoma and B16 melanoma on the innervation of the mouse trachea, Auton. Neurosci. (2014), http://dx.doi.org/10.1016/j.autneu.2014.03.005
