Contribution of vagal afferents to breathing pattern in rats with lung fibrosis

Respiration Physiology 108 (1997) 45 – 61

Contribution of vagal afferents to breathing pattern in rats with lung fibrosis Jim K. Mansoor, Dallas M. Hyde, Edward S. Schelegle * Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, Uni6ersity of California, Da6is CA 95 616, USA Accepted 28 October 1996

Abstract In anesthestized male Wistar rats with bleomycin-induced lung fibrosis we examined the influence of lung vagal non-myelinated and myelinated afferents in setting breathing pattern. Fourteen days after intratracheal instillation of bleomycin, lung compliance, total lung capacity (TLC) and inspiratory capacity were reduced while functional residual capacity and residual volume were increased. Baseline tidal volume (VT) was decreased and frequency (fR) increased in the bleomycin treated rats compared with controls. Selective vagal C-fiber blockade did not affect fR or VT in any group. Vagotomy resulted in an increase in VT and decrease in fR in both groups with the percent increase in VT/TLC and decrease in fR being significantly greater in the bleomycin rats compared with controls. Vagotomy also attenuated the significantly elevated PCO2 in the bleomycin treated rats suggesting that bleomycin-induced alterations in breathing pattern contribute to blood gas abnormalities. We conclude that vagal myelinated afferents contribute to the rapid shallow breathing in bleomycin treated rats. © 1997 Elsevier Science B.V. Keywords: Pulmonary fibrosis; Bleomycin; Vagal afferents; Rapid shallow breathing

1. Introduction Pulmonary fibrosis is characterized by an excessive amount of connective tissue in the interstitial spaces of the lung (Crystal et al., 1984). In both human patients (Crystal et al., 1984) and animal models (Thrall and Scalise, 1995) of this disease, * Corresponding author. Tel.: + 1 916 7521171/+ 1 916 7588376; fax: +1 916 7527690.

early alveolar and interstitial inflammation may be present. The disease is further characterized by a decrease in total lung capacity, vital capacity and compliance. Additionally, patients with pulmonary fibrosis usually exhibit a rapid shallow breathing pattern both at rest and during exercise (Crystal et al., 1984; Savoy et al., 1981). It has been hypothesized that lung vagal afferents play a role in this abnormal breathing pattern (Guz et al., 1970). This hypothesis is based upon the

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location of vagal afferent endings within the airways and parenchyma and their known reflex responses evoked by changes in lung compliance and inflammatory mediators (Coleridge and Coleridge, 1986). Guz et al. (1970) examined the contribution of pulmonary vagal afferents to changes in breathing pattern in two patients with diffuse pulmonary fibrosis. Upon application of 2% lignocaine to both vagus nerves resting tachypnea decreased and tidal volume increased with some resolution of dyspnea. Winning et al. (1988) observed a nonsignificant 9.1% increase in tidal volume at rest upon inhalation of 5% bupivicaine aerosol in six patients with interstitial lung disease. The resulting vagal block had no effect on maximal exercise hyperventilation in these patients. Savoy et al. (1981) found no change in the breathing pattern of patients with pulmonary fibrosis after inhalation of 4% lignocaine aerosol. These studies of patients with pulmonary fibrosis are limited by the small number of patients studied and the difficulty in assessing the degree and selectivity of vagal blockade used. These limitations make it difficult to identify which, if any, pulmonary vagal afferents are responsible for the observed rapid shallow breathing patterns in patients with interstitial lung disease. A few animal studies have examined the role of pulmonary vagal afferents in breathing pattern changes associated with models of prolonged pulmonary inflammation and injury. Phillipson et al. (1975) demonstrated that total vagal blockade reversed the excess increase in breathing frequency during exercise in dogs with experimentally-induced pneumonitis and pulmonary granulomatosis. Vizek et al. (1983), using graded vagal cooling, found that vagal myelinated fibers were responsible for the rapid shallow breathing in rats with paraquat-induced pneumonia. In contrast, Trenchard et al. (1972), using anodal blockade, demonstrated in rabbits that the increase in breathing frequency following intratracheal administration of carageenin was dependent upon intact vagal C-fibers. These animal studies indicate that one or more pulmonary vagal afferents are in part or wholly responsible for the breathing pattern changes observed in these models of lung injury.

In the present study, we examine the role that non-myelinated and myelinated vagal nerve fibers play in setting the breathing pattern in rats with bleomycin-induced pulmonary fibrosis. We chose this model of experimentally-induced pulmonary fibrosis because bleomycin is commonly used to study the pathogenesis of pulmonary fibrosis and because of its use as an antineoplastic agent in humans with the clinically relevant side-effect of pulmonary fibrosis (Lazo et al., 1990; Thrall and Scalise, 1995). We have recently developed and validated a selective vagal C-fiber blocking technique in the dog (Schelegle et al., 1995). We validate this technique of vagal perineural capsaicin treatment (VPCT) for use in the rat and use it, along with vagotomy, to test the hypothesis that both vagal non-myelinated C-fibers and myelinated A-fibers contribute to the rapid shallow breathing pattern observed in rats with bleomycin-induced pulmonary fibrosis.

2. Methods

2.1. Validation of VPCT 2.1.1. General Eleven male Wistar rats (3559 79.1 g-Charles River) were anesthetized with a combination of urethane and a-chloralose (25% urethane, 2.5% a-chloralose IP (1.0 g urethane/kg; 0.1 g a-chloralose/kg)). The strap muscles were excised and the trachea was cannulated with a 14-gauge catheter. The chest was opened in the midsternal line. The lungs were ventilated (tidal volume, 1.5– 2.0 ml; frequency, 60 cycles/min) by a Harvard rodent ventilator (model 683), with a resistance placed on the expiratory flow to provide positive end-expiratory pressure of 3–6 cm H2O. Twenty to twenty-five millimeters of the right vagus nerve was carefully isolated from the carotid sheath. In some rats the left vagus nerve was also isolated. The nerve was placed on a stimulating electrode caudally and recording electrode cranially. The nerve was crushed between the two poles of the recording electrode in order to obtain a monophasic compound action potential. The distance between the electrodes was approximately 15 mm. A

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thin piece of plastic was placed under the nerve in this region and a combination of petroleum jelly and mineral oil was applied to the nerve to prevent desiccation.

2.1.2. Application of perineural capsaicin Small square pledglets (approximately 1 mm2) of laboratory tissue (Kimwipes®) were soaked in a mixture of 99% mineral oil, 1% Tween 80 and 0.1% capsaicin. The petroleum jelly/mineral oil was wiped off the nerve and the pledglet of capsaicin was placed directly on the nerve for a period of 15 sec. The nerve was allowed to sit for 10 min and the efficacy of the block was tested (see below). If the block was not complete, a new pledglet was added to the nerve for 15 more seconds. This was continued until a nearly complete or complete block was obtained. In order to obtain successful C wave blocks with minimal reductions of the A wave we maintained the application site free of blood and kept the tension placed on the nerve to a minimum. If these conditions were met we consistently obtained successful C fiber blocks with only minimal effects on the A wave. 2.1.3. Protocol Stimulating parameters for the A and C waves were initially obtained by varying stimulus strength and duration. Stimulating parameters for the A and C waves fell within the range of 10–80 V at 50 msec and 25 – 60 V at 300 – 350 msec, respectively. These parameters gave maximal amplitude waveforms. The vagus nerve was stimulated once every 15 sec for a total of ten each control A and C waves. The ten waves were then averaged. The recording electrode was connected to a differential preamplifier (WP Instruments, model DAM-6A) and the amplified signal was digitized using a MacLab 2e (AD Instruments) interfaced with a Macintosh SE computer. The data was processed using Scope (AD Instruments, Version 3.2) software. The capsaicin block was then applied as explained above and the vagus nerve was stimulated once every 15 sec at the preblock C wave parameters in order to observe the effect of the block on the C wave. Once the C wave was greatly attenuated or appeared to have

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been abolished, another series of 10 A and C waves were recorded and averaged using the preblock (control) stimulating parameters established for that given animal. The nerve was then crushed between the stimulating and recording electrode and stimulated again to observe the stimulus artifact alone.

2.2. Bleomycin study 2.2.1. General Thirty-one male Wistar rats (Charles River) were split into three groups consisting of an absolute control group (2429 5 g, 11 rats, ABS), a saline control group (24195 g, 11 rats, SAL) and a bleomycin group (2269 4 g, 9 rats, BLEO). Each animal had its breathing pattern measured (frequency, inspiratory time and expiratory time) while conscious prior to intratracheal instillation of either nothing (ABS), saline (SAL, 0.5 ml/animal) or bleomycin sulfate (BLEO, 0.5 U/animal). Fourteen days after intratracheal instillation each rat had its breathing pattern measured while conscious. Each rat was then anesthetized and the trachea was cannulated. The rat was then placed into a whole body plethysmograph and pulmonary function measurements were performed. Catheters were placed into the femoral artery and jugular vein and the rat was attached via the tracheal cannula to a flow-by air stream in series with a pneumotachograph. Breathing patterns (tidal volume (VT), frequency (fR), inspiratory time (TI), expiratory time (TE) and minute ventilation (VE)) were recorded prior to and after VPCT and after vagotomy. The lungs were then excised and fixed for morphologic and morphometric study. 2.2.2. Instillation of bleomycin sulfate The rats were anesthetized with a combination of ketamine (50 mg/ml) and xylazine (10 mg/ml) at a dose of 0.8–1.6 ml/kg IP with atropine (0.02 mg/ml) to prevent excessive mucous build-up in the airway. A 14-gauge sterile plastic tracheal tube (modified angiocatheter—Jelco™) was inserted into the trachea. Either 0.5 ml of sterile saline or 0.5 U of bleomycin sulfate in 0.5 ml of sterile saline was instilled into the trachea and allowed to

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flow into the lung. The instillation of the solution into the lung was verified by observing an immediate rapid shallow breathing pattern. The tracheal tube was removed and the rats were allowed to recover.

2.2.3. Pulmonary function measurements Pulmonary function measurements were performed using methods similar to those of Likens and Mauderly (1982). In brief, a rat was anesthetized with a combination of urethane and achloralose (25% urethane, 2.5% a-chloralose IP (1.0 g urethane/kg; 0.1 g a-chloralose/kg)). The strap muscles were excised and the trachea was cannulated with a 14-gauge catheter. A water filled catheter was placed into the esophagus at the level of the chest. Transpulmonary pressure was estimated by measuring the difference between the pressure in the tracheal cannula and midthoracic esophagus using a differential pressure transducer (Validyne model DP 15 – 26). The rat was placed into a whole-body plethysmograph and attached to a port linked to a flow-by air stream in series with a pneumotachograph (Hans Rudolph, series 8300). The box temperature was allowed to equilibrate. While the rat breathed spontaneously, the box was closed to the atmosphere and the changes in box pressure were measured using a differential pressure transducer (Validyne MP45-14). The rat’s tidal volume was obtained by integrating the respiratory flow signal from the pneumotachograph. All pressure and volume signals were recorded and saved for later processing using a MacLab 4e (AD Instruments) and Macintosh SE computer (Apple). The box pressure signal was corrected and converted to a volume while being calibrated against the integrated pneumotachograph signal. Functional residual capacity (FRC) was calculated using Boyle’s Law from measurements obtained by occluding the rat’s airway at the end of expiration and measuring changes in mouth pressure (Validyne MP45-24-871) and box pressure (representative of volume) while the rat attempted to breath. Lung pressure volume curves were obtained by deflating the lung from +30 – − 30 cm H2O transpulmonary pressure (the difference between mouth and esophageal pressures) at a flow rate of

3–5 ml/sec while simultaneously measuring the changes in volume and transpulmonary pressures. Quasistatic (QSC) compliance was obtained by calculating the slope of the linear portion of the lung pressure volume curve above FRC. Vital capacity (VC) was obtained by calculating the change in volume from + 30– − 30 cm H2O. Inspiratory capacity (IC) was obtained by calculating the volume change from +30 cm H2O to FRC. Total lung capacity (TLC) was obtained by adding FRC and IC. Residual volume (RV) was obtained by subtracting VC from TLC. Expiratory reserve volume (ERV) was obtained by subtracting RV from FRC.

2.2.4. Protocol for studying anaesthetized breathing pattern After measuring pulmonary functions, the rat’s vagus nerves were isolated in the neck and a thin piece of plastic was placed under the nerves. A mixture of petroleum jelly and mineral oil was placed on the nerve to prevent desiccation. A catheter was placed into the left jugular vein and situated near the right atrium. A second catheter was placed into the right femoral artery and attached to a Spectramed DTX transducer for the measurement of blood pressure. A water filled catheter was placed into the esophagus at the level of the chest and attached to a Validyne (model DP 15–26) pressure transducer for the measurement of transpulmonary pressure. A rectal probe thermocouple (Physitemp) was placed in the rectum of the rat for measurement of body temperature. The animal breathed off a bias air flow and the change in bias flow indicating inspiratory and expiratory flow was measured by a pneumotachograph (Hans Rudolph, series 8300). The output from the pneumotachograph, blood pressure transducer and transpulmonary pressure transducer were fed into a Buxco Pulmonary Mechanics Analyzer (model 6) data acquisition system and displayed on an Astro-Med (model MT 95K2) chart recorder. Breathing pattern was allowed to stabilize and a control recording was obtained. Regular blood samples were taken for the measurement of arterial PO2, PCO2 and pH using a Ciba-Corning 178 pH/blood gas analyzer. Body temperature was

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monitored and maintained between 36 – 37°C. A series of Hering – Breuer reflexes (HBRs) and pulmonary chemoreflexes (PCRs) were evoked to determine near maximal reflex responses. Five to 20 cm H2O was used to evoke the HBR, while 1–10 mg/kg capsaicin was used to evoke the PCR. Saline was also injected into the right atrium as a control. After measuring the reflex responses, the rat was allowed to recover prior to VPCT. Vagal perineural capsaicin was applied in the same manner as discussed above (Section 2.1.2). The effectiveness of the VPCT block was tested after 10 min. The VPCT block was considered effective when bradycardia and apnea evoked from the PCR was greatly attenuated or abolished and the Hering–Breuer Inflation reflex was left intact. If the block was not effective, a new pledglet of capsaicin was added to the nerve for 15 more seconds and tested 10 min later. Once an effective block was obtained, breathing pattern and pulmonary reflexes were recorded. The vagi were then cut and again breathing pattern and reflex measurements were obtained.

2.2.5. Histopathology After breathing pattern was studied, the lungs of each rat were fixed for morphologic and morphometric study. The lungs were fixed in situ using a vascular perfusion technique. In brief, the rat’s chest was opened in the midline and the rat was placed on a ventilator. Heparin was injected into the right ventricle. An incision was made into the right ventricle and a 14-gauge catheter was placed into the pulmonary artery. Tubing from a peristaltic pump was attached to the catheter. The vasculature of the lung was perfused with a solution of phosphate buffered saline at a maximum pressure of 20 cm H2O. To establish a constant volume history, the lung was inflated to 30 cm H2O three times and then held constant at 12 cm H2O on the deflation curve. The vasculature of the lung was then perfused with a 10% solution of zinc–formalin (Z-fix, Anatech) at a maximum pressure of 20 cm H2O for a minimum period of 20–40 min. The trachea was tied off and the heart and lungs were removed and placed in zinc –formalin solution until the lungs could be sectioned and embedded in paraffin blocks.

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Each rats’ right cranial lung lobe, right caudal lung lobe and left lung lobe were fractionated into 5 mm blocks, embedded in paraffin and cut into 5 mm thick sections. The sections were stained with hematoxylin and eosin and evaluated for volume of lesion using light microscopy. All fields in a given section (about 5–20) were evaluated at a magnification of × 40 with a 42 point multipurpose test system. Volume of lesion (VL in mm3) for each lobe was calculated using a fractionator, the volume of a sampling field (Vlung field) and the volume density of lesion in the lung (VVL,L) (Bolender et al., 1993): 1 1 VL = × × Vlungfield × VVL,L f1 f2 where f1 is the fraction of blocks sampled per lobe, f2 is the fraction of fields sampled per block in volume, Vlung field is the volume of a sampling field in lung tissue (area ×section thickness) and VVL,L is the volume density of lesion per lung field which is a ratio of the volume of lesion to the volume of a lung field in mm3/mm3.

2.3. Calculations and data analysis The compound action potential A and C waves were analyzed by measuring the changes in the peak amplitudes, areas under the waves and conduction velocities before and after VPCT. Statistical analysis was performed using a Students’ paired t-test (Statview, version 4.01). The HBR data were analyzed by calculating the inhibitory ratio: IRHBR = (THBRapnea − TE )/TE where THBRapnea is the duration of apnea during an increase in peak end-inspiratory pressure and TE is the mean expiratory time for the three breaths prior to the pressure increase. Likewise the strength of the pulmonary chemoreflex (PCR) was quantified by expressing the apnea evoked following right atrial injection of capsaicin as an inhibitory ratio: IRPCR = (TPCRapnea − TE)/TE where TPCRapnea is the duration of apnea following right atrial injection of capsaicin and TE is the

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mean expiratory time for the three breaths prior to right atrial injection of capsaicin. Further analysis of the PCR included comparisons of the changes in heart rate and mean arterial blood pressure after right atrial injection of capsaicin. The percent change in heart rate was calculated by the following formula: fH =[(fHafter −fHbefore/fHbefore]× 100 where fHbefore is the average heart rate for the minute prior to right atrial capsaicin injection and fHafter is the average heart rate of the five slowest heart beats after capsaicin injection. The percent change in blood pressure was calculated using the following formula: MAP =



n

(MAPafter −MAPbefore) ×100 MAPbefore

where MAPbefore is the average mean arterial blood pressure for the minute prior to right atrial capsaicin injection and MAPafter is 1/3 times the minimum systolic blood pressure plus 2/3 times the minimum diastolic blood pressure after capsaicin injection. In order to analyze the effects of bleomycin on lung structure and function, a 1-way ANOVA (Statview 4.0, Abacus Concepts, Berkeley, CA) was used. The variables analyzed included all pulmonary function variables, conscious breathing pattern variables, before VPCT HBR and PCR variables, before VPCT breathing pattern variables and before VPCT blood gases and pH variables. Tidal volume data was examined both as absolute values and relative to total lung capacity (VT/TLC). Post-hoc analysis was performed using a Scheffe’s test. After this initial analysis, it was found that there were no significant differences in any of the variables between the absolute and saline control groups, thus, these groups were combined to form a single control group (n= 22; CON). The 1-way ANOVA was then rerun as described above using control and bleomycin treated groups as the two levels of the independent variable. An univariate 2-way ANOVA with one repeated factor was used to initially examine the effect of VPCT and vagotomy on bleomycin-induced breathing pattern, with condition (CON vs.

BLEO) being the group factor and treatment (before VPCT, after VPCT, vagotomy) being the within factor. The dependent variables analyzed included all breathing pattern variables, blood gases and pH. Significant interactions were analyzed using repeated mean contrasts. Data are presented as means9 S.E. Significance was set at p5 0.05 for all statistical analysis.

3. Results

3.1. Validation of VPCT VPCT had no significant affect on the A wave of the compound action potential in the 11 rats studied, but significantly reduced the C wave. The mean peak amplitude and area under the A wave was 4469134 mV and 228960 mV sec, respectively prior to VPCT, and 3579 100 mV and 203951 mV sec, respectively after VPCT (Fig. 1(A) and (B)). Similarly, A wave conduction velocity was unaffected by VPCT (pre-treatment, 51.295.8 ms − 1; post-treatment, 45.89 5.5 ms − 1). In contrast, the C wave peak amplitude and area under the wave were significantly affected by VPCT. The mean peak amplitude and area under the C wave prior to VPCT was 729 14 mV and 5709 132 mV sec, respectively and 79 3 mV and 899 40 mV sec, respectively after VPCT (Fig. 1(C) and (D)). In six of 11 cases we were unable to distinguish the reduced C wave after VPCT from the baseline noise; in these cases, the integral was recorded as zero. In addition to examining the selectivity of VPCT by comparing the changes in the compound action potential before and after VPCT, the PCR and HBR responses evoked by the highest tolerated dose of capsaicin (1–10 mg/kg) and positive pressure applied to the lung at the peak of inspiration (5–15 cm H2O) were compared before and after VPCT in all of the rats (22 CON and 9 BLEO) in the bleomycin study. There were significant decreases in the IRPCR, fH and MAP when eliciting the PCR after VPCT compared to before VPCT (Table 1). There was no difference in the IRHBR when eliciting the HBR before and after VPCT.

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Fig. 1. Effect of VPCT on the compound action potential of the vagus nerve in a rat. Note the absence of the C wave after VPCT (C, D) whereas the A wave is not affected (A, B). Arrow represents the onset of the stimulus.

3.2. Bleomycin study We examined the effects of (1) bleomycin on lung structure and function; and (2) VPCT and vagotomy on breathing pattern in control and bleomycin-induced fibrotic rats. There were no significant differences between the absolute and saline control groups for any of the variables, therefore these two groups were combined to form a single control group (n = 22; CON) that was used in comparisons with the bleomycin treated group. Fourteen days after instillation of bleomycin sulfate, the bleomycin treated rats showed significantly less weight gain than the

control rats (CON: before, 2419 3 g; after, 3549 6 g; BLEO: before, 2269 4 g; after, 2899 13 g).

3.2.1. Histopathology and morphometry Rats that received either no treatment or intratracheal saline (CON) had normal pulmonary parenchymal tissue with little or no fibrosis (Fig. 2(A)). The rare lesions in control rats were comprised of aggregations (four or more) of alveolar macrophages in the airspaces. In contrast, rats that received intratracheal bleomycin sulfate showed multifocal interstitial and peribronchiolar fibrosis (Fig. 2(B)). The volume of lesion (VL) of the three lung lobes measured in the bleomycin

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Table 1 Pulmonary chemoreflex and HBR responses before and after VPCT Reflex

Variable

Before VPCT

After VPCT

PCR

IRPCR Change in fH (%) Change in MAP (%)

5.09 0.6 (BL TE, 0.55 90.02 sec) −68.99 2.6 (BL, 3849 9 b/min) −53.19 2.4 (BL, 90.89 3.6 mmHg)

2.1 90.4* (BL TE, 0.53 90.02 sec) −11.7 92.1* (BL, 42899 b/min) −23.7 9 2.6* (BL, 97.893.2 mmHg)

HBR

IRHBR

55.39 5.9 (BL TE, 0.54 90.03 sec)

51.2 9 5.3 (BL TE, 0.49 90.03 sec)

BL, baseline; IRHBR, Hering–Breuer reflex inhibitory ratio. Note the significant reduction in the pulmonary chemoreflex response but little change in the Hering–Breuer inflation reflex response after VPCT. Values represent mean 9SEM. Significance level set at pB0.05. * Significant difference between before and after VPCT.

treated rats was significantly greater than those of the control rats (CON, 19 911 mm3; BLEO, 14699222 mm3). The mean volume density of lesion (VVL) of the three lung lobes was also significantly greater in the bleomycin treated rats as compared with control rats (CON, 0.0019 0.0007; BLEO, 0.13290.0270).

3.2.2. Pulmonary function There were significant differences between the two groups of rats in all of the mean pulmonary function values (Table 2). Quasistatic compliance, TLC, VC and IC were significantly less in the bleomycin treated rats than in the control rats. Functional residual capacity and RV were significantly greater in the bleomycin treated rats than in control rats. 3.2.3. Reflexes Prior to any vagal nerve treatment we compared the PCR at capsaicin doses of 1 and 5 mg/kg and the HBR at inflation pressures of 5, 10 and 15 cm H2O between the control and bleomycin treated rats. We found that both the PCR and the HBR were blunted in the bleomycin treated rats Table 3. Following the injection of 1 mg/kg of capsaicin into the right atrium, the IRPCR and the fH were significantly reduced in the bleomycin treated group as compared to the control group. In addition, there was a near significant (p = 0.069) reduction in the MAP in the bleomycin treated group compared with the controls. When 5 mg/kg of capsaicin was injected into 12 of 22 control rats

that would tolerate this dose and in all of the bleomycin treated rats the IRPCR, fH and the MAP were significantly reduced in the bleomycin treated group. None of the control rats tolerated 10 mg/kg capsaicin and therefore no comparison was made at this dose. The IRHBR was significantly reduced in the bleomycin treated rats for inflation pressures of 5 and 10, but not for 15 cm H2O.

3.2.4. Breathing pattern in conscious rats Prior to instillation of bleomycin there were no significant differences in fR (CON, 97.69 3.2 br/min; BLEO, 102.59 4.6 br/min), TI (CON, 0.2490.010 sec; BLEO, 0.23 90.010 sec) or TE (CON, 0.399 0.015 sec; BLEO, 0.359 0.021 sec) between the conscious control and subsequently bleomycin treated rats. Fourteen days after instillation of saline or bleomycin the conscious bleomycin treated rats had a significantly greater fR (CON, 109.49 4.0 br/min; BLEO, 303.29 25.7 br/min) and significantly reduced TI (CON, 0.2290.006 sec; BLEO, 0.129 0.016 sec) and TE (CON, 0.3590.018 sec; BLEO, 0.099 0.006 sec) compared with conscious control rats. 3.2.5. Effects of VPCT and 6agotomy on bleomycin-induced breathing pattern Breathing patterns of representative rats from the control group and bleomycin group for the different treatment conditions are shown in Fig. 3. Note in both rats little change in breathing pattern after VPCT (Fig. 3(A), (B), (D) and (E)). Note also that vagotomy almost completely

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Fig. 2. A representative micrograph of a rat lung intratracheally instilled with saline (A) or bleomycin sulfate (B). Rats were sacrificed 14 – 17 days after intratracheal instillation. Note the peribronchiolar fibrosis in the bleomycin treated lung (B). AD, alveolar duct; F, fibrosis; TB, terminal bronchiole. Line represents 100 mm.

reversed the rapid shallow breathing pattern in the bleomycin treated rat (Fig. 3(F)). Prior to any vagal treatment but after anesthesia administration, bleomycin treatment signifi-

cantly reduced VT, TI and TE and significantly increased fR (Fig. 4(A), (E), (F), (C)). Bleomycin treatment had no effect on VT/TLC or VE (Fig. 4(B) and (D)).

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Table 2 Mean pulmonary function values for control and bleomycin treated rats

Control Bleomycin

QSC (ml/cm H2O)

TLC (ml)

VC (ml)

FRC (ml)

IC (ml)

RV (ml)

1.059 0.06 0.449 0.10*

14.0490.28 11.089 0.81*

11.02 90.25 6.49 90.77*

4.31 9 0.10 6.06 9 0.25*

9.74 9 0.24 5.02 90.67

3.02 9 0.14 4.59 9 0.25*

QSC, quasi-static compliance; TLC, total lung capacity; VC, vital capacity; FRC, functional residual capacity; IC, inspiratory capacity; RV, residual volume. Values represent mean 9SE. Significance level set at p50.05. Note the significant differences between the two groups for all variables. * Significant difference between control and bleomycin treated rats.

VPCT had no effect on VT, VT/TLC, fR, TI or TE in the control and bleomycin treated rats (Fig. 4(A), (B), (C), (E) and (F)). In contrast, VPCT resulted in a significant increase in VE (Fig. 4(D)) in the bleomycin treated rats. This increase in VE was the result of non-significant increases in VT (p = 0.454) and fR (p = 0.140) after VPCT. Vagotomy resulted in a significant increase in VT, VT/TLC, TI and TE (Fig. 4(A), (B), (E), (F)) and a significant decrease in fR (Fig. 4(C)) in the control and bleomycin treated rats. Vagotomy had no effect on VE in the control rats while vagotomy reduced VE in the bleomycin treated rats to before VPCT values (Fig. 4(D)). The VT/TLC and fR responses to the vagal treatments was different in the bleomycin treated rats as compared to the control rats. Further analysis revealed a significantly greater percent increase in VT/TLC and percent decrease in fR in the bleomycin treated rats as compared to controls after vagotomy (Fig. 5(A) and (B)).

3.2.6. Blood gases and pH before and after 6agal treatments Prior to any vagal treatment but after anesthesia administration, bleomycin treatment resulted in a significant decrease in arterial PO2 and a significant increase in arterial PCO2; pH was not affected (Table 4). VPCT had no effect on PO2, PCO2 or pH in either of the groups. Vagotomy, however, resulted in a significant decrease in PCO2 and increase in pH in both control and bleomycin treated rats compared to the after VPCT condition. The PCO2 response to the vagal treatments was different in the bleomycin treated rats as compared to the control rats. Post-hoc analysis revealed a significantly greater percent decrease in

arterial PCO2 in the bleomycin treated rats as compared to the control rats after vagotomy. This interaction mirrored the interaction between bleomycin treatment and vagotomy observed for VT/TLC and fR.

4. Discussion This is the first study that examines the role of lung vagal afferents in the control of respiration in any animal model of bleomycin-induced lung fibrosis. We examined the effects of (1) bleomycin on lung structure, pulmonary function and conscious breathing pattern and (2) VPCT and vagotomy on breathing pattern in control and bleomycin treated rats. We found bleomycin treatment resulted in a rapid shallow breathing pattern that was associated with significant alterations in lung structure and pulmonary function. We also found that selectively blocking the conduction in non-myelinated vagal afferents had little or no effect on breathing pattern in bleomycin treated rats, but that eliminating both non-myelinated and myelinated vagal afferent conduction virtually reversed the bleomycin-induced rapid shallow breathing. These observations support the hypothesis that myelinated vagal fibers contribute significantly to the rapid shallow breathing pattern observed in this rat model of pulmonary fibrosis.

4.1. Validation of VPCT In order to justify the use of perineural capsaicin as a method of blocking C-fiber conduction in the vagus nerve we felt we had to clearly show the

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Table 3 The effect of bleomycin treatment on the responses to the pulmonary chemoreflex and Hering – Breuer inflation reflex Reflex

Variable

Dose/inflation pressure

Control

Bleomycin treated

PCR

IRPCR

1 mg/kg 5 mg/kg

2.259 0.53 (BL TE, 0.59 90.03 sec) 6.639 0.97 (BL TE, 0.64 90.03 sec)

0.23 90.14* (BL TE, 0.43 90.03 sec) 2.09 9 0.81* (BL TE, 0.43 90.03 sec)

Change in fH (%)

1 mg/kg 5 mg/kg

−39.39 6.5 (BL, 3699 10 b/min) −76.8 91.9 (BL, 363910 b/min)

−4.70 92.80* (BL, 389914 b/min) −50.09 8.6* (BL, 4209 16 b/min)

Change in MAP (%)

1 mg/kg

−35.39 4.3 (BL, 83.29 4.6 mmHg)

−21.7 93.60 (BL, 90.79 5.4 mmHg)

5 mg/kg

−57.993.9 (BL, 103.396.1 mmHg)

−39.9 96.4* (BL, 88.99 4.8 mmHg)

5 cm H2O 10 cm H2O 15 cm H2O

5.59 1.0 (BL TE, 0.60 90.3 sec) 29.79 3.5 (BL TE, 0.60 90.03 sec) 51.09 5.2 (BL TE, 0.64 90.03 sec)

1.490.2* (BL TE, 0.41 90.04 sec) 11.19 2.1* (BL TE, 0.41 90.04 sec) 52.4 93.5 (BL TE, 0.36 90.03 sec)

HBR

IRHBR

IRPCR, pulmonary chemoreflex inhibitory ratio; BL, baseline; IRHBR, Hering – Breuer inhibitory ratio. Note that both reflexes were significantly affected by bleomycin treatment. Values represent mean9SEM. Significance level is set at p50.05. * Significant difference between control and bleomycin groups.

effects of this treatment on the compound action potential of the vagus nerve. We saw an 89.7% decrease in the mean peak amplitude of the C wave and an 84.3% decrease in the mean area under the curve of the C wave which was associated with a 20.0% decrease in the mean peak amplitude of the A wave and an 11.0% decrease in the mean area under the curve of the A wave. In six out of 11 cases we were unable to discern the C wave from the baseline noise after the block was applied and the decrease in the peak amplitude and the area under the curve of these C waves was calculated as 100%. The changes that

Fig. 3. The effect of vagal perineural capsaicin treatment (VPCT) and vagotomy on breathing pattern in an anesthetized saline and bleomycin treated rat. (A, D) Breathing pattern before VPCT; (B, E), breathing pattern after VPCT; (C, F), breathing pattern after vagotomy. Note that VPCT had little effect on breathing pattern in these rats, but that vagotomy reversed most of the rapid shallow breathing seen in the bleomycin treated rat.

we observed in the compound action potential with VPCT agree with the results of others (Baranowski et al., 1986; Waddell and Lawson, 1989; Petsche et al., 1983) who observed similar decreases in the amplitude of the C waves with only small non-significant decreases of the A waves after topical application of capsaicin on the vagus and peripheral nerves. Further validation of the selectivity of VPCT used in this study can be obtained from examining pulmonary reflexes known to be elicited by specific nerve fiber populations travelling in the vagus nerves. The PCR, consisting of bradycardia, hypotension and apnea, followed by rapid shallow breathing, is mediated by stimulation of pulmonary and bronchial C-fibers (Coleridge and Coleridge, 1986). The HBR, on the other hand, consists of prolonged apnea due to the stimulation of vagal A-fibers (slowly adapting receptors) by hyperinflation of the lung (Coleridge and Coleridge, 1986). In this study, VPCT resulted in a significant attenuation of the PCR with little or no effect on the HBR. These findings are similar to those obtained in previous studies (Jansco and Such, 1983; Schelegle et al., 1995) that demonstrated a selective abolition of the lung C-fiber evoked PCR.

79.89 1.7 71.49 5.3**

82.3 9 1.3 70.1 94.7**

80.0 9 2.1 67.6 94.3**

45.6 91.0 49.8 91.5**

45.0 9 1.0 48.8 91.7**

After VPCT

42.991.0** 44.4 91.2*,**

Vagotomy

7.3890.01 7.399 0.01

Before VPCT

pH

7.3990.02 7.409 0.01

After VPCT

7.41 90.01** 7.429 0.01**

Vagotomy

Note that arterial PO2 was significantly less and PCO2 was significantly greater in bleomycin treated rats for all conditions. Note also that PCO2 and pH were significantly different from the other treatment conditions after vagotomy. Values represent mean9 SE. Significance level is set at p50.05. * Significant difference between vagotomy and both before and after VPCT. ** Significant difference between control and bleomycin treated groups.

Control Bleomycin

Before VPCT

Vagotomy

Before VPCT

After VPCT

PCO2

PO2 (mmHg)

Table 4 Mean blood gas and pH values for control and bleomycin treated rats before and after VPCT and after vagotomy

56 J.K. Mansoor et al. / Respiration Physiology 108 (1997) 45–61

J.K. Mansoor et al. / Respiration Physiology 108 (1997) 45–61

57

Fig. 4. Mean values for tidal volume (A), VT/TLC (B), respiratory frequency (C), minute ventilation (D), inspiratory time (E) and expiratory time (F) in control and bleomycin treated rats before and after vagal perineural capsaicin treatment (VPCT) and vagotomy. Symbols indicate significant difference (p5 0.05) between: , control and bleomycin treated rats before VPCT; D, vagotomy and both before VPCT and after VPCT for control and bleomycin treated rats; †, after VPCT and before VPCT for bleomycin treated rats; ‡, vagotomy and after VPCT for bleomycin treated rats.

Our compound action potential and reflex data in combination with the observations of others (Baranowski et al., 1986; Waddell and Lawson, 1989; Petsche et al., 1983) supports the efficacy of using perineural capsaicin treatment as a selective C-fiber block. However, this data also points to limitations of this technique. First, like other researchers, we were unable to totally abolish the C wave of the compound action potential with capsaicin treatment. Second, though small, there was a consistent drop in the peak amplitude and area under the wave of the A wave even in the most successful blocks. Third, the time required to obtain a satisfactory selective C fiber block without significantly affecting the A wave appears to be somewhat variable. In the present study there was a fine line between blocking C waves and blocking both C and A waves and in many cases we had a marked decrement in the compound action potential of the A wave which reversed after about 10 min. Indeed, the VPCT protocol used in this study was based on preliminary studies in which we completely vagotomized rats by leaving the capsaicin soaked pledglet on the nerve longer than 1 min. In contrast, other investigators have applied similar concentrations of capsaicin for 30

min with only small reversible decrements in the A wave (Baranowski et al., 1986 and Petsche et al., 1983. We are unable to explain this discrepancy between our observations and those of others. Fourth, the C-fiber block does not appear to be easily reversed having a duration of 100 min or greater (Petsche et al., 1983). These limitations clearly indicate the need to evaluate the effectiveness of this block with either reflexes or compound action potentials before and after perineural capsaicin treatment.

4.2. Effects of bleomycin on lung structure, pulmonary function and conscious breathing pattern Fibrotic changes due to bleomycin treatment is well documented in both experimental animals (Lazo et al., 1990; Thrall and Scalise, 1995) and humans undergoing bleomycin therapy for cancer (Lazo et al., 1990). Our bleomycin treated rats had large fibrotic lesions compared to our control rats receiving only saline. Bleomycin is thought to exert a cytotoxic effect on cells by cleaving DNA in the presence of molecular oxygen and a metal ion as well as causing lipid peroxidation of cell

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membranes. This cellular damage ultimately results in lung inflammation that culminates in pulmonary fibrosis (Lazo et al., 1990). Human subjects with idiopathic pulmonary fibrosis show decreases in lung compliance and VC and small increases in RV that are highly variable (Myre et al., 1988). Similar changes in lung capacities and compliance have been observed in rabbits (Berend et al., 1985) and hamsters (Goldstein et al., 1979). We observed in our bleomycin treated rats that quasistatic lung compliance, TLC, VC and IC were significantly reduced, while FRC and RV were increased. This would indicate that the bleomycin treated rats in this study had reduced respiratory reserves and that the work of breathing for this group of rats was considerable. Patients with idiopathic pulmonary fibrosis also show increases in the work of

breathing (Thrall and Scalise, 1995; Renzi et al., 1982). The full effects of bleomycin treatment on breathing pattern in animal models has not been previously documented. Rapid breathing was observed in the conscious bleomycin treated rats as compared to the control rats. Tidal volume was not measured in the conscious rats, but in anesthetized rats the bleomycin treated group had a significantly smaller tidal volume and greater frequency than controls. The rapid breathing pattern in our conscious bleomycin treated rats mimics the breathing pattern observed in humans with idiopathic lung fibrosis (Lazo et al., 1990; Renzi et al., 1982; Savoy et al., 1981) and parallels the observations in human cancer patients receiving bleomycin intravenously as chemotherapy (Lazo et al., 1990). In order to examine the relationship between increases in breathing frequency, volume density of lesion and pulmonary functions in the conscious bleomycin treated rats we further analyzed our data using correlation analysis. In the nine rats treated with bleomycin the increase in conscious breathing frequency was significantly correlated with the volume density of lesion (r= 0.82; p 50.01), VC (r= −0.76; p 5 0.02), IC (r= − 0.74; p5 0.02) and TLC (r= − 0.76; p5 0.02). These significant correlations are consistent with the notion that alterations in lung mechanics in this model of pulmonary fibrosis contribute to the rapid shallow breathing pattern observed.

4.3. Role of lung 6agal afferents in the rapid shallow breathing pattern in bleomycin-induced pulmonary fibrosis

Fig. 5. Percent change in VT/TLC (A) and breathing frequency (B) after vagal perineural capsaicin treatment and vagotomy in control and bleomycin treated rats. , indicates a significant (p 50.05) difference between control and bleomycin treated rats.

The rapid shallow breathing pattern present in our conscious bleomycin treated rats was still present following a-chloralose anesthesia. Rapid shallow breathing patterns have been observed in anesthetized rabbits intratracheally instilled with carrageenin (Trenchard et al., 1972) and in rats instilled with paraquat (Vizek et al., 1983). The consistently greater breathing frequency and reduced tidal volume across all vagal treatments observed in the bleomycin treated rats suggest the presence of a strong nonvagal component con-

J.K. Mansoor et al. / Respiration Physiology 108 (1997) 45–61

tributing to the bleomycin-induced rapid shallow breathing. We believe that three nonvagal components contributed to the observed rapid shallow breathing in bleomycin treated rats. First, the bleomycin treated rats were significantly smaller than the control rats. Second, the decrements in total lung capacity and lung compliance were considerable suggesting a mechanical limitation to inspiration in the bleomycin group. These first two possibilities are strongly supported by our observation that the bleomycin-induced effect on tidal volume was eliminated when tidal volume was normalized to total lung capacity. Third, we observed a greater arterial PCO2 and a lower arterial PO2 in the bleomycin treated rats in all conditions. This hypercapnia combined with the mild hypoxemia in the bleomycin treated rats would be expected to provide a tachypneic drive that would survive the vagal treatments used in the present study. However, these chemical drives for ventilation would not be expected to reduce tidal volume. The fact that the percent decrease in breathing frequency and the increase in normalized tidal volume was greater following vagotomy in the bleomycin treated rats suggests an active role for vagal myelinated fibers in driving the bleomycininduced rapid shallow breathing. The significant decrease in the HBR inhibitory ratio at 5 and 10 cm H2O in the bleomycin treated rats suggests that there is a decrease in the discharge of slowly adapting receptors at lower lung inflation pressures. This finding taken together with the results of Yu et al. (1991) that decreases in lung compliance cause a decrease in the deflation discharge of slowly adapting receptors would seem to lend evidence to the argument that a decrease in slowly adapting receptor activity during expiration contributed to the observed rapid shallow breathing pattern in the bleomycin treated rats. Rapidly adapting receptors, on the other hand, are known to be stimulated by decreases in lung compliance (Pisarri et al., 1990) as well as inflammatory mediators (Coleridge and Coleridge, 1986). When stimulated rapidly adapting receptors have been shown to evoke reflex rapid shallow breathing (Green and Kaufman, 1990) and augmented breaths (Coleridge and Coleridge, 1986). The

59

bleomycin treated rats had marked reductions in lung compliance which lends support to the hypothesis that rapidly adapting receptors as well as slowly adapting receptors contribute to the bleomycin-induced rapid shallow breathing. We reasoned that the inflammation associated with bleomycin treatment (Goto et al., 1984; Thrall et al., 1979; Thrall and Scalise, 1995) would stimulate lung C-fibers (Coleridge and Coleridge, 1986) and contribute to the rapid shallow breathing in the rats treated with this drug. To our surprise there was little or no change in breathing pattern following VPCT in the bleomycin treated rats. This suggests that lung C-fibers play little or no role in the bleomycin-induced rapid shallow breathing in this rat model of lung fibrosis. One possibility that would explain this apparent lack of a significant C-fiber influence is that in our rats at 14–17 days post bleomycin treatment the intense early inflammatory response had begun to resolve and C-fiber activity was no longer significantly elevated. This possibility is supported by animal studies that demonstrate initial intense inflammation (1–3 days post bleomycin) that gradually subsides and becomes minimal 21 days post-treatment (Thrall and Scalise, 1995). If this were indeed the case, the rapid shallow breathing induced by bleomycin treatment would be solely due to nonvagal components and pulmonary vagal myelinated afferents. A second possibility is that C-fiber stimuli were present in our bleomycin treated rats but that bleomycin treatment in someway destroys C-fiber endings and/or interferes with their receptor function. This possibility is supported by the blunted PCRs we observed at both 1 and 5 mg/kg of capsaicin injection. We believe that the blunted PCR was due, in part, to the dilution of the bolus injection of capsaicin in the engorged great veins of the bleomycin treated rats and the destruction of the pulmonary capillary bed (Crystal et al., 1984) reducing the number of C-fibers accessible from the pulmonary vasculature. Consistent with this was our observation upon necropsy of engorgement of the right and left external jugular veins, superior vena cava and right atrium and ventricle. These observations agree with those of

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Sato et al. (1993) who found right heart hypertrophy and elevated pulmonary arterial pressures in bleomycin treated rats. Finally, it is possible that the discharge of lung vagal C-fibers is increased but that the discharge of lung myelinated afferents is increased to such an extent that their discharge alone is sufficient to produce the rapid shallow breathing observed in our bleomycin treated rats. If this were the case, both sets of fibers would have to be blocked in order to reverse the bleomycin-induced rapid shallow breathing, and thus the methodology employed in this study would not be sufficient to see any changes in breathing pattern after C-fiber block. The possibility that lung C-fiber discharge is increased in our bleomycin-treated rats is supported by the observed increase in minute ventilation following VPCT that was reversed by vagotomy. One possible interaction that would account for this change in minute ventilation is an increase in respiratory drive mediated through an increase in rapidly adapting receptor activity (Coleridge and Coleridge, 1986) and an increase in lung C-fiber activity causing an inhibition of a and g motorneurons that innervate inspiratory muscles (Koepchen et al., 1977; Schmidt and Wellhoner, 1970). In all conditions (before VPCT, after VPCT, vagotomy) PO2 was significantly less in the bleomycin treated rats than in the control rats. Sato et al. (Sato et al., 1993) also observed reduced PO2 in bleomycin treated rats. Arterial CO2 tension, on the other hand, was greater in all conditions in the bleomycin treated rats. These observations are consistent with a severe diffusion limitation or physiological shunt being present in the bleomycin treated rats. With vagotomy there was a significantly greater attenuation of the elevated PCO2 levels in the bleomycin rats as compared to the control rats suggesting that the elevated PCO2 in the bleomycin treated rats is in part being determined by the vagally mediated rapid shallow breathing. The reduced tidal volume in the vagally intact bleomycin treated rats in combination with increases in RV and FRC suggests that there is an increase in dead space ventilation without a compensatory increase in

alveolar ventilation which would lead to an elevated PCO2. 5. Conclusion The morphometric and physiologic data presented in this study indicates that the rat is an appropriate model for studying the breathing pattern abnormalities seen in human lung fibrosis. We have shown that the rapid shallow breathing pattern in bleomycin treated rats relies on intact vagal myelinated nerve fibers and that a non-vagal component also plays a role in bleomycin-induced breathing pattern changes in the rat. Non-myelinated C-fibers, on the other hand, do not seem to contribute to the rapid shallow breathing pattern in bleomycin treated rats, but may play a modulatory role.

Acknowledgements The authors would like to acknowledge the assistance of Tan Pham, Jennifer Ward, Neil Rabara and Chin Yee Loh. This work was supported by NIH R29 HL49406.

References Baranowski, R., B. Lynn and A. Pini (1986). The effects of locally applied capsaicin on conduction in cutaneous nerves in four mammalian species. Br. J. Pharmacol. 89: 267 – 276. Berend, N., D. Feldsien, D. Cederbaums and R.M. Cherniack (1985). Structure-function correlation of early stages of lung injury induced by intratracheal bleomycin in the rabbit. Am. Rev. Respir. Dis. 132: 582 – 589. Bolender, R.P., D.M. Hyde and R.T. Dehoff (1993). Lung morphometry: a new generation of tools and experiments for organ, tissue, cell, and molecular biology. Am. J. Physiol. 265: L521 – L548. Coleridge, H.M. and J.C.G. Coleridge (1986). Reflexes evoked from tracheobronchial tree and lungs. In: Handbook of Physiology, Section 3: The Respiratory System, Vol. II: Control of Breathing, part 1, edited by N.S. Cherniack and J.G. Widdicombe. Bethseda, MD: American Physiological Society, pp. 395 – 429. Crystal, R.G., P.B. Bitterman, S.I. Rennard, A.J. Hance and B.A. Keogh (1984). Interstitial lung diseases of unknown cause (Part 1). N. Engl. J. Med. 310: 154 – 166.

J.K. Mansoor et al. / Respiration Physiology 108 (1997) 45–61 Goldstein, R.H., E.C. Lucey, C. Franzblau and G.L. Snider (1979). Failure of mechanical properties to parallel changes in lung connective tissue composition in bleomycin-induced pulmonary fibrosis in hamsters. Am. Rev. Respir. Dis. 120: 67 – 73. Goto, T., D. Befus, R. Low and J. Bienenstock (1984). Mast cell heterogeneity and hyperplasia in bleomycin-induced pulmonary fibrosis of rats. Am. Rev. Respir. Dis. 130: 797 – 802. Green, J.F. and M.P Kaufman (1990). Pulmonary afferent control of breathing as end-expiratory lung volume decreases. J. Appl. Physiol. 68(5): 2186–2194. Guz, A., M.I.M. Noble, J.H. Eisele and D.W. Trenchard (1970). Experimental results of vagal block in cardiopulmonary disease. In: Breathing: Hering–Breuer Centenary Symposium, edited by R. Porter. London: Churchill, pp. 315 – 328. Jansco, G. and G. Such (1983). Effects of capsaicin applied perineurally to the vagus cerve on cardiovascular and respiratory functions in the cat. J. Physiol. 341: 359– 370. Koepchen, H.P.. M. Kalia, D. Sommmer and D. KlussenDorf (1977). Action of type J afferents on the discharge of medullary respiratory neurons. In: Krogh Centenary Symposium on Respiratory Adaptations, Capillary Exchange and Reflex Mechanism, edited by A.S. Paintal and P. Gill-Kumar. Delhi: Vallabhbhai Patel Chest Institute, pp. 407 – 425. Lazo, J.S., D.G. Hoyt, S.M. Sebti and B.R. Pitt (1990). Bleomycin: a pharmacologic tool in the study of the pathogenesis of interstitial pulmonary fibrosis. Pharmacol. Ther. 47: 347 – 358. Likens, S.A. and J.L. Mauderly (1982). Effect of elastase or histamine on single-breath N2 washouts in the rat. J. Appl. Physiol. 52: 141–146. Myre, M., S. Allard, C. Bernard and R.R. Martin (1988). Clinical, functional and pathological correspondence in early stage idiopathic pulmonary fibrosis: evidence for small airway obstruction 1–2. Respiration 53: 174–186. Petsche, U, E. Fleischer, F. Lembeck and H.O. Handwerker (1983). The effect of capsaicin application to a peripheral nerve on impulse conduction in functionally identified afferent nerve fibres. Brain Res. 265: 233–240. Phillipson, E.A., E. Murphy, L.F. Kozar and R.K. Schultze (1975). Role of vagal stimuli in exercise ventilation in dogs with experimental pneumonitis. J. Appl. Physiol. 39: 76 – 85. Pisarri, T.E., A. Jonzon, J.C.G. Coleridge and H.M. Coleridge

.

61

(1990). Rapidly adapting receptors monitor lung compliance in spontaneously breathing dogs. J. Appl. Physiol. 68: 1997 – 2005. Renzi, G., J. Milic-Emili and A.E. Grassino (1982). The pattern of breathing in diffuse lung fibrosis. Bull. Eur. Physiopathol. Respir. 18: 461 – 472. Sato, S., S. Kato, Y. Arisaka, H. Takahashi, K. Takahashi and H. Tomoike (1993). Changes in pulmonary hemodynamics during normoxia and hypoxia in awake rats treated with intratracheal bleomycin. Tohoku J. Exp. Med. 169: 233 – 244. Savoy, J., S. Dhingra and N.R. Anthonisen (1981). Role of vagal airway reflexes in control of ventilation in pulmonary fibrosis. Clin. Sci. 61: 781 – 784. Schelegle, E.S., J.K. Mansoor and J.F. Green (1995). Influence of background vagal C-fiber activity on eupneic breathing pattern in anesthetized dogs. J. Appl. Physiol. 79: 600 – 606. Schmidt, T. and H.H. Wellhoner (1970). The reflex influence of a group of slowly conducting vagal afferents on a and g discharges in cat intercostal nerves. Pfluegers Arch. 318: 335 – 345. Thrall, R.S., J.R. McCormick, R.M. Jack, R.A. McReynolds and P.A. Ward (1979). Bleomycin-induced pulmonary fibrosis in the rat. Am. J. Pathol. 95: 117 – 130. Thrall, R.S. and P.J. Scalise. (1995). Bleomycin. In: Pulmonary Fibrosis, edited by S.H. Phan and R.S. Thrall. New York: Marcel Dekker, pp. 231 – 293. Trenchard, D.W., D. Gardner and A. Guz (1972). Role of pulmonary vagal afferent nerve fibres in the development of rapid shallow breathing in lung inflammation. Clin. Sci. 42: 251 – 263. Vizek, M., S. Frydrychova, S. Houstek and F. Palecek (1983). Effect of vagal cooling on lung function residual capacity in rats with pneumonia. Bull. Eur. Physiopathol. Respir. 19: 23 – 26. Waddell, P.J. and S.N. Lawson (1989). The C-fibre conduction block caused by capsaicin on rat vagus nerve in vitro. Pain 39: 237 – 242. Winning, A.J., R.D. Hamilton and A. Guz (1988). Ventilation and breathlessness on maximal exercise in patients with interstitial lung disease after local anesthetic aerosol inhalation. Clin. Sci. 74: 275 – 281. Yu, J., T.E. Pisarri, J.C.G. Coleridge, and H.M. Coleridge (1991). Response of slowly adapting pulmonary stretch receptors to reduced lung compliance. J. Appl. Physiol. 71: 425 – 431.