Original Paper Jot~alof
Biomedical Science
Received: April 28, 2003 Accepted: July 25, 2003
J Biomed Sci 2003;10:718-724 DOI: 10.1159/000073958
Supranodose Vagotomy Precludes Reflex Respiratory Responses to Serotonin in Cats Beata Kopczyr~ska
Ma{gorzata Szereda-Przestaszewska
Laboratory of Respiration Physiology, PAS Medical Research Centre, Warsaw, Poland
Key Words Apnoea • Feline breathing control • Nodose ganglion • Serotonin • Vagotomy
Abstract Mediation of the respiratory reflex effects of an exogenous serotonin challenge goes beyond the lung vagi and is suggested to involve the nodose ganglia. In the present experiments the effects of an intravenous serotonin challenge on breathing pattern were studied in 8 pentobarbitone-chloralose anaesthetised cats. Bolus injection of serotonin oxalate (50 gg/kg) into the right femoral vein evoked prompt apnoea of 19.2 (_+ 2.4)-second duration in all 8 cats while intact; the apnoea was much shorter after midcervical vagal section (8.1 + 0.9 s, p < 0.001), and was abolished by supranodose vagotomy. In post-apnoeic breaths, the tidal volume was reduced from a baseline of 34.1 + 4.0to 13.5 _+ 1.1 ml (p< 0.001) prior to, and from a baseline of 43.9 +_ 5.4 to 33.8 + 6.6 ml (p < 0.01) after midcervical vagotomy; the serotonin challenge did not affect tidal volume following supranodose vagal section (p = 0.4). The increase in respiratory rate found in intact (p < 0.001) and midcervically vagotomised cats (p < 0.01) was eliminated by the neurotomy above the nodose ganglia. Supranodose va-
K A rkG E rk Fax+4l 61 306 1234 E-Mall karger@karger,ch www.karger.com
© 2003 National Science Council, ROC S, Karger AG, Basel 1021-7770/03/0106-0718519.50/0 Accessible online at: www.karger.com/jhs
gotomy altered cardiovascular response to serotonin by replacing the fall in blood pressure with an increase. These data suggest that the sequelae of serotonininduced pulmonary chemoreflex, i.e. respiratory arrest, cardiovascular changes and post-apnoeic pattern of breathing require intact nodose ganglia. Copyright © 2003 National Science Council, ROCand S, Karger AG, Basel
Introduction Serotonin (5HT) is known to evoke a variety of respiratory responses in animal species. Injection of 5HT into the pulmonary circulation of spontaneously breathing cats evokes prompt apnoea, associated with hypotension and bradycardia and followed by rapid shallow breathing. This constellation of cardiorespiratory responses has been termed pulmonary chemoreflex [6]. It has been shown that the respiratory effects of 5HT are due to stimulation of pulmonary C fibre endings, endowed with 5 HT3 receptors [3, 17] in cats and rabbits; however, 5HT is acting at reduced level following midcervical section of the vagi [8]. Moreover, apnoea induced by injecting 5HT into the common carotid artery is abolished by supranodose vagotomy in cats [4, 16].
Beata Kopczyfiska Laboratoi7 of Respiration Physiology, PAS Medical Research Centre 5 Pawifiskiego St, PL-02-106 Warsaw (Poland) Tel. +48 22 6086522, t;'ax +48 22 6685532, E-Mail
[email protected]
Sensory receptors that are responsible for the emergence of the ventilatory effects of intravascular serotonin after section of the cervical portion of the vagi may be present in areas supplied by both pulmonary vessels and branches of ascending aorta. Nodose ganglia forming a continuity with the vagi are the relay station between sensory neurones in the respiratory organs and central axons transmitting to the nucleus of the solitary tract (NTS) of the medulla. Feline nodose ganglion includes serotonergic neurones endowed with 5HT3 receptors [14, 33]. Earlier investigations on respiratory effects of 5HT in cats have not focused on the ventilatory changes; however, the respective records indicated a decline in tidal volume during post-apnoeic breathing [8]. Records in the paper by Jacobs and Comroe [ 16] also showed moderate decreases in tidal volume following intracarotid 5HT administration, both in intact and midcervically vagotomised cats. Yet, there are no data comparing ventilatory changes mediated via infranodose and supranodose inputs during post-apnoeic breathing in 5HT-challenged animals. It has been evidenced that inputs from neither laryngeal afferents [28] nor carotid sinus nerves [4, 16, 27, 30] contribute to post-serotonin apnoea and ventitatory effects in cats. The present study was designed to evaluate the contribution of the other pathways, namely the pulmonary vagal afferents and nodose ganglion to 5HT-induced ventilatory effects.
Methods Eight adult cats of either sex (2.5-4.5 kg body weight) were anaesthetized with intraperitoneal (i.p.) injection of 30 mg. kg-I of sodium pentobarbitone (Sagatal, May and Baker Ltd). Additional doses (16 rag. kg- 1) of c~-chloralose (Fluka AG) were administered intravenously (i.v.) to maintain possibly constant levels of surgical anaesthesia. Cats were placed supine on a heated operating table, breathing spontaneously with room air. Femoral vein and femoral artery were catheterized for further injections and to monitor blood pressure, respectively. An incision was made in the trachea below the larynx, and the cannula inserted into the caudal end was connected to a pneumotachograph. The two recurrent laryngeal nerves were spared. The superior laryngeal nerves were prepared for sectioning prior to midcervical vagotomy. The C4-C5 root of the right phrenic nerve was cleared, cut, desheathed and prepared for recording. The vagus nerves in the mid-cervical region were cleared from the adjacent tissue, removed from the sheath, dissected from the cervical sympathetic trunks, 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 s u r -
Nodose Ganglia Mediate Serotonin Action
rounding 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 50 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 the NIH Guide for the Care and Use of Laboratory Animals. Arterial pressure was measured with a pressure manometer (CK 01 Mera-Tronik) and a blood pressure monitor (MCK 411S). Volume signals were :recorded from a pneumotachograph (Electrospirometer CS6, Mercury). End-tidal C02 was measured with a capnograph (Engstr6m Eliza plus). Action potentials of the phrenic nerve were amplified with NL 104 amplifier (Digitimer) and measured with a model AS 101 (Asbit) leaky integrator (time constant = t00 ms). All recordings were registered with Omnilight 8 M 36 apparatus (Honeywell). Rectal temperature was maintained between 37 and 39 °C throughout the experiment. Serotonin oxalate (Fluka AG, Buchs SG) in a dose of 0.05 mgkg- t (0.188 gmol/kg) dissolved in 0.9% saline (solution freshly made before the experiment) was injected as a bolus through the catheter placed in the right femoral vein. 5HT (at a concentration of 3.75 raM) was administered noncumulatively at intervals longer than 20 min to avoid tachyphylaxis [32]. Injections were completed within 1-2 s. The doses of 5HT used were derived from the doseresponse relationship established in preliminary experiments (not shown). All drug administrations were followed by a flush of 0.3 ml saline. Test i.v. injections of 0.3 aliquots of physiological saline showed no volmne effects. The respiratory effects of the 5HT challenge were recorded in all 8 cats while (i) neurally intact, (ii) following bilateral section of the superior laryngeal nerves (SLNs) and the cervical vagi, and (iii) after section of the supranodose vagi. SLNs were cut to exclude the inhibitory influence from the larynx on the ventilatory activity. Each individual value of tidal volume (VT), ventilation (VE) and respiratory rate (f) was taken as an average over 5 consecutive breaths. The ventilatory parameters were assessed prior to the 5HT 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 the integrated phrenic neurogram. Prolongation of the expiratory time (TE) was measured as the ratio of maximal Ts during post-serotonin apnoea or expiration (TE t~st) to control expiration (TE control), TE test/Tlzcontrol. The duration of the apnoeic period in phrenic activity was taken as the time of apnoea (respiratory inhibition). The ventilatory responses were assessed by comparing the mean of 5 consecutive breaths during the period of rapid, shallow breathing following the 5HT injection to the mean of 5 preceding breaths (control = pre-challenge values), and were expressed as absolute changes. VT, VE, f, TE and MAP data were analysed by two-way ANOVA with time pre, challenge, early post-challenge, 30 and 60 s after the challenge and denervation status (intact, SLNs + cervical vagi cut, supranodose vagi cut) as repeated measures' factors. Prolongation of the expiratory time (TE test/TEcontrol)and the duration of apnoea were analysed by one-way ANOVA with denervation status as repeated measures' factor. Differences between individual time points and experimental states were evaluated by Duncan test. In all cases p < 0.05 was considered significant. All results shown are means + 1 standard error.
J BiomedSci 2003;10:7t 8-724
719
Fig. 1. Respiratory response to intravenous injection of serotonin in cats treated with midcervical (a) and supranodose (b) vagotomy. 5HT injection marked by a dash above the upper record. Note the expiratory apnoea coupled with electrical silence in the phrenic integral. Breathing following apnoea is associated with decreased tidal volume. Following supranodose vagotomy (lower trace) post-serotonin apnoea is abolished. Vr: tidal volume; %CO2: end-tidal CO2; phrenic nerve: integrated neurogram of the phrenic nerve.
Results 1210-
T
8-
6-
4-
2-
a,b
Intact
Midcervical vagi cut
Supranodose vagi cut
Fig. 2. Mean prolongation of expiratory time (rE tes]T~ control)on an intravenous 5HT challenge in intact, cervical and supranodose vagicut animals, a p < 0.001 compared with intact, b p < 0.05 compared with midccrvically vagotomized cats; n = 8.
720
J Biomed Sci 2003;10:718-724
One-way ANOVA showed significant effect of denervation on apnoea duration and prolongation of TE (p < 0.001). Intravenous injection of 5HT provoked the expiratory apnoea of a mean duration of 19.2 -+ 2.4 s in all 8 cats while intact. Following midcervical vagotomy the mean duration of the 5HT-induced apnoea was decreased to 8.1 + 0.9 s (p < 0.001). When vagi were cut again at the supranodose level, no apnoeic response to 5HT challenge was seen. Figure 1 illustrates the effect of 5HT on respiratory variables in a cat vagotomized at infranodose (fig. 1a) and subsequently also supranodose level (fig. lb). Figure 2 shows the mean prolongation of T~ after 5HT administration. It was significantly diminished (p < 0.001) after midcervical vagotomy (rE test/rE control ratio = 2.72 + 0.34 exceeds I; apnoea still present) and after supranodose section of the vagi (rE test/rEcontrol ratio = 0.90 + 0.01
