Respiratory Physiology & Neurobiology 152 (2006) 143–151
Supranodose vagotomy eliminates anandamide-evoked cardiorespiratory depression in anaesthetized rats Beata Kopczy´nska ∗ , Małgorzata Szereda-Przestaszewska Laboratory of Respiratory Reflexes, PAS Medical Research Centre, 5 Pawi´nskiego Street, 02-106 Warsaw, Poland Accepted 29 July 2005
Abstract Respiratory effects of an intravenous injection of anandamide were investigated in 19 urethane-chloralose anaesthetised and spontaneously breathing rats. In 10 neurally intact rats the effects of anandamide were checked to establish appropriate dose of the drug. In the second group, nine rats were challenged with anandamide while intact, following bilateral midcervical vagotomy and after subsequent supranodose vagotomy. Bolus injection of 1 mg kg−1 of anandamide into the right femoral vein pre- and post-midcervical vagotomy induced in all nine rats prompt apnoea of similar duration: 2.97 ± 0.5 and 3.2 ± 0.4 s, respectively. In post-apnoeic breaths tidal volume decreased below the control level by 25% (P < 0.01) prior to and by 43.4% (P < 0.001) after midcervical vagotomy. Supranodose vagotomy precluded the respiratory response to anandamide. Anandamide-induced decrease in mean arterial blood pressure in nerve-intact and vagotomised rats was abolished by supranodose vagotomy. Results indicate that the cardio-respiratory depression evoked by anandamide administered via the peripheral circulation requires intact supranodose vagi. © 2005 Elsevier B.V. All rights reserved. Keywords: Control of breathing; Anandamide; Apnoea; Vagotomy; Nodose ganglion; Rat
1. Introduction Anandamide is a natural, endogenous agonist of cannabinoid CB1 receptors present on the neurones of the central and peripheral nervous system (Freund et al., 2003). They were found in the sympathetic and ∗ Corresponding author. Tel.: +48 22 6086522; fax: +48 22 6685532. E-mail address:
[email protected] (B. Kopczy´nska).
1569-9048/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2005.07.007
parasympathetic ganglia and on axon terminals of airway nerves (Buckley et al., 1998; Pertwee, 1999; Calignano et al., 2000). Unfortunately, few physiological studies address the contribution of the peripheral CB1 receptors to the reflex control of breathing. It is generally held that activation of CB1 cannabinoid receptors with the natural 9 -tetrahydrocannabinol (-9-THC) or synthetic agonists induces cardiovascular depression and markedly impairs ventilation, both in conscious and anaesthetised animals (Doherty et al.,
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1983; Vivian et al., 1998; Schmid et al., 2003). Difficulty with anandamide inheres the fact that it is also the ligand of the vanilloid receptors VR1 (Zygmunt et al., 1999; Smart et al., 2000). In rats activation of VR1 receptors by their classical agonist, capsaicin, induces the pulmonary chemoreflex, consisting of apnoea, hypotension, bradycardia and post-apnoeic stimulation of breathing, based mainly on an increased tidal volume (Kaczy´nska and Szereda-Przestaszewska, 2000). Recent study by Lin and Lee (2002) on the effects of anandamide in rats has shown that its administration into the jugular vein evoked hypotension and apnoea of the chemoreflex. In the breaths that followed the apnoea, the tidal volume and the frequency of breathing remained near the baseline. The competitive antagonist of the VR1 receptors eliminated coincident intensive activation of unmyelinated vagal C-fibres by anandamide reported in this paper. Different pattern of the respiratory response evoked by local intraarterial challenge of anandamide into the hindlimb vasculature showed an enhanced ventilation and hypotension, which appeared to be mediated by VR1 receptors (Smith and McQueen, 2001). On the other side, it was shown that the third phase of post-anandamide hypotension was the only phenomenon clearly ascribed to the activation of CB1 receptors (Varga et al., 1995; Malinowska et al., 2001). Analysis of the published data provides inconsistent evidence as to the role of the feedback from vagally innervated pulmonary receptors. Respiratory depression due to -9-THC challenge occurred in nerveintact and vagotomised cats (Doherty et al., 1983). On the other side, perivagal capsaicin treatment blocked the response to anandamide in rats (Lin and Lee, 2002). Considering the presence of CB1 receptors on the vagal pathway and in the nodose ganglia (Buckley et al., 1998), we hypothesised that the nodose ganglia mediate the reflex changes evoked by anandamide. Present experiments were designed to: (1) characterize the post-anandamide pattern of breathing following an intravenous injection, which has not been addressed; (2) to examine the effectiveness of midcervical and subsequent supranodose vagotomy in precluding anandamide-induced cardiorespiratory effects. The objective of the present study was to evaluate the pattern of cardiorespiratory effects produced by anandamide and its reflex pathway by measuring the ventilatory and blood pressure responses in the intact
rats subsequently treated by an infra- and supranodose vagotomy.
2. Methods Nineteen adult male Wistar rats (195–250 g body weight) were anaesthetised with an intraperitoneal injection of 600 mg kg−1 of urethane (Sigma) and 120 mg kg−1 of ␣-chloralose (Fluka AG). Additional doses of urethane and ␣-chloralose were administered intravenously (i.v.), if necessary—dependent on response to applying pressure to a limb joint and the basal heart rate and blood pressure (BP). Rats were placed supine and breathed spontaneously room air. An incision was made in the trachea below the larynx, and the cannula inserted into the caudal end was connected to a pneumotachograph. Femoral vein and artery were catheterized for administration of supplemental anaesthesia and drugs and to monitor blood pressure, respectively. The vagus nerves in the midcervical region were cleared from the adjacent tissue, removed from the sheath and prepared for section later in the experiment. Rostral vagal trunks were carefully separated from the superior cervical ganglia. The nodose ganglia were dissected free from the surrounding tissue; attention was paid to preserve their blood supply intact. At the last stage of the experiment the supranodose vagi were transected as far from the ganglion as possible, i.e. at least 5 mm distal from its rostral pole. Ethical approval for the experimental procedures used in this study was obtained from the local animal care committee. All animal procedures were in accordance with NIH Guide for the Care and Use of Laboratory Animals. Arterial pressure was measured with BP-2 pressure monitor (Columbus Instruments) and mean arterial pressure (MAP) was calculated. Tidal volume signals were recorded from a pneumotachograph (RSS 100HR Research Pneumotach System). End-tidal CO2 concentration was measured with a capnograph (Engstr¨om Eliza plus, Gambro). Electromyogram of the costal diaphragm was recorded with bipolar electrodes, amplified with NL 104 amplifier (Digitimer) filtered and measured with a model AS 101 (Asbit) leaky integrator (time constant = 100 ms). All recordings were registered on an Omnilight 8M 36 apparatus (Honey-
B. Kopczy´nska, M. Szereda-Przestaszewska / Respiratory Physiology & Neurobiology 152 (2006) 143–151
Fig. 1. (A) Relationship between the dose of anandamide and mean prolongation of the expiratory time in intact rats. Values 1 – presence of apnoea. *** P < 0.001, * P < 0.05 vs. the dose of 0.3 mg kg−1 of anandamide (ANOVA); n = 10. (B) Dose related depressive effect on tidal volume (VT ) evoked by anandamide (0.3–1 mg kg−1 ) in intact rats. Note the most apparent response to the dose of 1 mg kg−1 . *** P < 0.001 vs. control (preanandamide values), Duncan’s test; n = 10.
well). Rectal temperature was maintained between 37 and 39 ◦ C throughout the experiment. The stock solution of anandamide (Arachidonylethanolamide, C22 H37 NO2 , Sigma) was in 96% ethanol. Prior to the use, dilutions were made with saline in order to minimalize the effect of the solvent and to obtain the final 9.6% concentration of ethanol, containing the proper dose of the drug. The dose-response curve of the respiratory variables to intravenously injected anandamide was determined with three different doses of anandamide (0.3, 0.6 and 1 mg kg−1 ) in 10 intact rats in preliminary experiments (Fig. 1). The effective dose of 1 mg kg−1 was chosen and applied in the proper experiments in nine remaining rats. This dose used in our study was identical with previously applied (Malinowska et al., 2001; Lin and
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Lee, 2002). Drug was administered at intervals at least 15 min to avoid any effect of accumulation. Injections were completed within 2 s. All injections had a volume of 0.2 ml and were followed by a flush of 0.1 ml saline. A 9.6% ethanol in saline was used as a control for anandamide. Test i.v. injections of 0.1–0.3 ml aliquots of such prepared solvent showed no cardiorespiratory effects. The respiratory effects of anandamide challenge were recorded in nine rats while (i) initially neurally intact, (ii) following bilateral section of the cervical vagi, and (iii) subsequently treated by supranodose vagotomy. Each individual value of tidal volume (VT ), ventilation (VE ), respiratory rate (f) was taken as an average over five consecutive breaths. The ventilatory parameters were assessed prior to anandamide injection, during early post-apnoeic phase, and at 30 and 60 s after the challenge. The expiratory time (TE ) was determined from the record of integrated diaphragm activity. Prolongation of the expiratory time (TE ) was measured as the ratio of maximal TE during post-anandamide apnoea (TE test ) to control expiration (TE control ), TE test /TE control . The duration of apnoeic period in diaphragm activity was taken as the time of apnoea (respiratory inhibition). Ventilatory responses were assessed by comparing the mean of five consecutive breaths during the period of breathing ensuing anandamide injection to the mean of five preceding breaths (control = pre-challenge values), and were expressed as absolute changes. All experimental data were analysed by repeated measures two-way ANOVA with time (pre-challenge, early post-apnoeic phase, 30 and 60 s after the challenge) and innervation status (intact, cervical vagi cut, supranodose vagi cut) as repeated measures factors. In rats vagotomised at the supranodose level (absence of apnoea) instead of “early post-apnoeic phase” the parameters registered 2 s after drug administration were taken into account. Differences between individual time points and experimental states were evaluated by Duncan or Scheff´e’s test. Prolongation of the expiratory time (TE test /TE control ) and the duration of apnoea were analysed by one-way ANOVA with innervation status as repeated measures factor. In all cases, a P < 0.05 was considered significant. All results shown are mean ± S.E.M.
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B. Kopczy´nska, M. Szereda-Przestaszewska / Respiratory Physiology & Neurobiology 152 (2006) 143–151
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Table 1 Initial mean values for parameters before injection of anandamide Parameter VT (ml) VE (ml min−1 ) f (min−1 ) MAP (mmHg)
Mean baseline value 2.8 ± 0.3 171.1 ± 20.2 62.5 ± 3.6 81.4 ± 4.5
All values are mean ± 1 S.E.M.; n = 19.
3. Results The dose-response curves of Fig. 1 illustrate changes in the respiratory variables produced by a series of i.v. injections of anandamide, ranging from 0.3 to 1 mg kg−1 , in intact animals. The data clearly indicate a vivid impact on expiratory inhibition and post-apnoeic decline in VT resulting from a dose of 1 mg kg−1 . This dose was selected in the following experiments. Values for each individual parameter was taken as an average over five consecutive breaths to set baseline values before administration of the drug (Table 1). Mean preliminary values for VT and f, VE and MAP were similar to those previously prescribed (Lin and Lee, 2002; McQueen et al., 2004; Schmid et al., 2003). Intravenous anandamide challenge produced uniform cardiorespiratory effects, comprising an apnoea followed by slower breathing of decreased VT and a fall in arterial blood pressure in intact and midcervically vagotomised rats. There was no respiratory effect when similar volumes of the solvent were administered. As illustrated in Fig. 2 anandamide injected at a dose of 1 mg kg−1 evoked immediate expiratory apnoea of mean duration of 2.97 ± 0.5 s (A, intact) and of 3.2 ± 0.4 s (B, vagotomised rats), showing a similar levels of expiratory inhibition (P = 0.9). Vagotomy performed at the supranodose level precluded the occurrence of apnoea (C). In the apnoeic pause, the expiratory time was significantly elongated: the mean prolongation of TE being 4.3 ± 0.9- and 4.2 ± 0.5-fold prior to and after midcervical vagotomy, respectively (Fig. 3). Supranodose vagotomy effectively lessened the pro-
Fig. 3. Mean prolongation of the expiratory time (TE test /TE control ) induced by anandamide challenge in the intact rats and subsequently vagotomised at infra- and supranodose levels: (a) P < 0.05 compared with the intact state; (b) P
