Abstract
Background:
Recent studies showed partial reversal of opioid-induced respiratory depression in the preBötzinger Complex and the Parabrachial Nucleus/Kölliker-Fuse Complex. We hypothesized that opioid antagonism in the Parabrachial Nucleus/Kölliker-Fuse Complex plus preBötzinger Complex completely reverses respiratory depression from clinically relevant opioid concentrations.
Methods:
Experiments were performed in 48 adult, artificially ventilated, decerebrate rabbits. We decreased baseline respiratory rate ~50% with intravenous, “analgesic” remifentanil infusion or produced apnea with remifentanil boluses and investigated the reversal with naloxone microinjections (1mM, 700nl) into the Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex. In another group of animals, we injected naloxone only into the preBötzinger Complex to determine whether prior Parabrachial Nucleus/Kölliker-Fuse Complex injection impacted the naloxone effect. Lastly, we injected the μ-opioid receptor agonist [D-Ala,2N-MePhe,4Gly-ol]-enkephalin (DAMGO, 100μM, 700nl) into the Parabrachial Nucleus/Kölliker-Fuse Complex. Data are presented as median (25–75%).
Results:
Remifentanil infusion reduced respiratory rate from 36 (31–40) to 16 (15–21) breaths per minute (bpm). Naloxone microinjections into the bilateral Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex increased rate to 17 (16–22, n=19, p=0.005), 23 (19–29, n=19, p<0.001), and 25 (22–28) bpm (n=11, p<0.001), respectively. Naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex prevented apnea in 12/17 animals, increasing respiratory rate to 10 (0–12) bpm (p<0.001); subsequent preBötzinger Complex injection prevented apnea in all animals (13 (10–19) bpm, n=12, p=0.002). Naloxone injection into the preBötzinger Complex alone increased respiratory rate to 21 (15–26) bpm during “analgesic” concentrations (n=10, p=0.008), but not during apnea (0 (0–0) bpm, n=9, p=0.500). DAMGO injection into the Parabrachial Nucleus/Kölliker-Fuse Complex decreased respiratory rate to 3 (2–6) bpm.
Conclusions:
Opioid reversal in the Parabrachial Nucleus/Kölliker-Fuse Complex plus preBötzinger Complex only partially reversed respiratory depression from “analgesic” and even less from “apneic” opioid doses. The lack of recovery pointed to opioid-induced depression of respiratory drive that determines the activity of these areas.
Introduction
Recent studies of opioid-induced respiratory depression have expanded the focus from the respiratory rhythm generator in the preBötzinger Complex to areas that provide inputs to those preBötzinger Complex neurons that mediate inspiratory on- and off-switch and thus determine inspiratory and expiratory phase duration (1). We have previously shown in acute in vivo rabbit studies that naloxone injection into the Parabrachial Nucleus partially reversed respiratory rate depression from “analgesic” remifentanil concentrations (2) while injections into the preBötzinger Complex did not (3). These results were supported by similar studies in dogs (4, 5). Our last study supported the idea of an additional opioid-sensitive source of inputs to the respiratory rhythm generator outside of the Parabrachial Nucleus and preBötzinger Complex (2). Since then, the importance of the Kölliker-Fuse Nucleus for the control of respiratory phase duration (6–8) as well as for opioid-induced respiratory depression (9–11) has been highlighted by multiple investigators. In particular, a novel mouse model showed that respiratory depression including from high morphine doses was substantially attenuated when μ-opioid receptors were knocked out selectively in Kölliker-Fuse Nucleus neurons (9–11).
We recently showed in our in vivo rabbit model that glutamatergic disfacilitation of the Parabrachial Nucleus and Kölliker-Fuse Nucleus was necessary to achieve maximal respiratory rate depression (8). We thus hypothesized that opioid-induced respiratory depression was due to combined depression of Parabrachial Nucleus and Kölliker-Fuse Nucleus activity. After initial experiments showed that the opioid effect could not be fully reversed in these areas, we extended our hypothesis to include the preBötzinger Complex. In contrast to our previous studies that used non-vagotomized animals (2, 3), the current experiments were performed in vagotomized animals to prevent that small changes in respiratory parameters were confounded by ventilator-induced respiratory rate entrainment.
We used local microinjections of the opioid antagonist naloxone into the bilateral Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex to antagonize the respiratory depression from intravenous remifentanil. To determine whether the magnitude of opioid reversal depended on the opioid dose (11), we used both “analgesic” concentrations, i.e., steady-state remifentanil infusions that depressed respiratory rate by ~50% (2, 3, 11–14), as well as “apneic” concentrations, i.e., a remifentanil bolus that just resulted in apnea under control conditions (10). To clarify whether the observed preBötzinger Complex effect depended on prior opioid reversal in the Parabrachial Nucleus/Kölliker-Fuse Complex, we added experiments where we injected naloxone solely into the preBötzinger Complex. Once we had determined that naloxone reversal of the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex prevented apnea from the “apneic” remifentanil bolus, we further investigated if naloxone injection also prevented apnea from “very high” remifentanil concentrations, i.e., up to 10x of the threshold “apneic” bolus.
Lastly, to gauge the degree to which Parabrachial Nucleus/Kölliker-Fuse Complex neurons could be depressed by μ-opioid receptor agonists we injected the μ-opioid receptor agonist [D-Ala,2N-MePhe,4Gly-ol]-enkephalin (DAMGO) at high, supraclinical concentrations into the bilateral Parabrachial Nucleus/Kölliker-Fuse Complex.
Methods
2.1. Surgical preparation
The research was approved by the Subcommittee on Animal Studies of the Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin, and the Institutional Animal Care and Use Committee, Medical College of Wisconsin, in accordance with provisions of the Animal Welfare Act, the Public Health Service Guide for the Care and Use of Laboratory Animals, and Veterans Affairs policy. Experiments were carried out on adult (3–4 kg), pathogen-free, New Zealand White rabbits of either sex. Anesthesia was induced with 5 vol% sevoflurane via facemask and ventilated via tracheotomy with an anesthesia machine (Ohmeda CD, GE, Datex Ohmeda, Madison, WI). Anesthesia was maintained with 1.5–3% isoflurane. Inspiratory oxygen fraction, expiratory carbon dioxide concentration and expiratory isoflurane concentration were continuously displayed with an infrared analyzer (POET II, Criticare Systems, Waukesha, WI). Skin was infiltrated with lidocaine 1% before each skin incision. Femoral arterial and venous lines were used for blood pressure monitoring, infusion of solutions, and bolus drug administration, respectively. Care was taken to increase anesthetic depth for any signs of “light anesthesia”, e.g., an increase in blood pressure or lacrimation. Lactated Ringer’s solution with 3 mcg/ml epinephrine was continuously infused at 1 ml/h. At this rate, the infusion did not result in appreciable changes in heart rate and blood pressure from baseline. Infusion rate was increased as needed to counteract or prevent hypotension in response to drug injections and/or from blood loss but maintained as constant as possible during the recording phase. The animal was maintained at 37.0 ± 0.5°C with a warming blanket. The animal was placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA), and blunt precollicular decerebration with complete removal of the forebrain was performed through a parietal craniotomy. After decerebration, isoflurane was either discontinued or continued at subanesthetic levels (0.3–0.4 vol%) for blood pressure control. This sedative concentration equals ~0.2 minimum alveolar concentration (15), which is associated with a ≤10% decrease in respiratory rate and peak phrenic activity (16, 17). Volatile anesthetics add to but do not amplify the remifentanil effect (16), i.e., slight variation in isoflurane concentration between experiments would affect the baseline respiratory rate and peak phrenic activity, but not the dose-dependency of the remifentanil effect. Isoflurane concentration was not changed during the experimental protocol. Decerebration eliminates the need for further general anesthesia but it may reduce forebrain and midbrain inputs to the respiratory center and thus cause a minor increase in apneic threshold (18, 19). The brainstem was exposed via occipital craniotomy and partial removal of the cerebellum. Animals were paralyzed with rocuronium (15 mg/kg subcutaneous bolus), followed by pancuronium 2 mg/h infusion to avoid motion artifacts during neural/neuronal recording. Bilateral vagotomy was performed to achieve peripheral deafferentation to avoid interference of the mechanical ventilation with the underlying central respiratory rhythm and respiratory neuronal activity. Respiratory rates after vagotomy were comparable to our previous studies in non-vagotomized animals (2, 3). The phrenic nerve and in some experiments the vagus nerve were recorded with fine bipolar electrodes through a posterior neck incision. The complete surgical preparation required 6–7h.
Throughout the experiment animals were ventilated with a hyperoxic gas mixture (FiO2 0.6) to achieve functional denervation of the peripheral chemoreceptors and thus rule out that the observed drug effects were due to effects on the carotid bodies. Mild hypercapnia (expiratory carbon dioxide: 45–55 mmHg) was used to emulate the hypercarbia encountered clinically during opioid use in patients. Mild hypercapnia may also have compensated for the loss of respiratory drive with decerebration since respiratory rates were similar to rabbit preparations using normocapnia in anesthetized, non-decerebrate preparations (12, 20). Blood pressure was maintained stable throughout the experiment by adjusting the intravenous infusion rate. At the end of the experiment, animals were euthanized with intravenous potassium chloride.
2.2. Neuronal Recording, microinjection procedures and measured variables
All neuronal recording and microinjection techniques have been well established by our research group and have been previously described in detail (21, 22). In short, extracellular neuronal recordings were obtained using multibarrel micropipettes (20–40 μm tip diameter) consisting of three drug barrels and a recording barrel containing a 7 μm thick carbon filament. Barrels were filled with the glutamatergic agonist α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 50 μM, 70 nl/ injection) and the opioid receptor antagonist naloxone (1mM, 700nl/injection), which were dissolved in artificial cerebrospinal fluid. The microinjected volume was determined via height changes in the meniscus in the respective pipette barrel with a 100x monocular microscope and calibrated reticule (resolution ~3.5 nl). Respiratory neuronal discharge was recorded extracellularly from neuronal aggregates and individual neurons and classified by the temporal relationships relative to the phrenic neurogram. The neuronal and neural activity and pressure microejection marker signals were recorded using a digital acquisition system. These variables were also continuously displayed and recorded along with the phrenic neurogram, vagal neurogram, discharge rate-meter, respiratory rate, arterial blood pressure, and airway carbon dioxide concentration on a computerized chart recorder (Powerlab/16SP; ADInstruments, Castle Hill, Australia). Before and after drug injection, steady-state conditions were obtained for respiratory parameters. Post experiment LabChart data was exported to SigmaPlot 11 (Systat Software, San Jose, CA) for data reduction, data plotting and statistical analysis. Between 10 and 50 consecutive respiratory cycles were averaged over 1–2 min with the number of cycles dependent on the respiratory rate. Using the phrenic neurogram we determined respiratory rate, inspiratory and expiratory duration, and peak phrenic activity. Using the vagal neurogram we determined peak vagus activity. In rabbits, inspiratory phase timing of the vagal neurogram closely matches the phrenic neurogram without the post-inspiratory activity typically observed in rats (10) but with minor activity during mid-expiration. Peak vagus activity was calculated as the amplitude between minimal vagus nerve activity before start of inspiration and peak vagus nerve activity during inspiratory phase. Since changes in peak phrenic activity closely reflect changes in respiratory tidal volume but the absolute value does not correspond with the absolute tidal volume (23), peak phrenic activity and peak vagus activity were normalized to the respective control values for all calculations.
2.3. Identification of the Parabrachial Nucleus, Kölliker-Fuse Nucleus and preBötzinger Complex.
We previously characterized the locations of the Kölliker-Fuse Nucleus, Parabrachial Nucleus (8) and preBötzinger Complex (22) in our model through stereotaxic coordinates, neuronal recordings and typical respiratory rate response to AMPA injection. For protocols investigating only the Parabrachial Nucleus and Kölliker-Fuse Nucleus, we inserted the micropipette in a grid-wise fashion starting at the caudal end of the inferior collicle at 1.5mm lateral from midline and moved lateral and caudal with step size 0.5mm (0.47mm rostro-caudal, corrected for the 20-degree angle of the stereotaxic frame). In areas where neuronal activity was encountered, we microinjected AMPA (50 μM, 70nl) starting at the ventral limit of the tonic neuronal activity and then in 1 mm steps more dorsally. The area of maximal AMPA-induced tachypnea was labeled as “Parabrachial Nucleus” and the area of maximal bradypnea was labeled as “Kölliker-Fuse Nucleus” (fig. 1). The Parabrachial Nucleus was located 1.0±0.9mm caudal from the inferior collicle, 2.6±0.7mm lateral to midline and 7.7±1.9mm ventral to the dorsal surface, and the Kölliker-Fuse Nucleus was located 1.1±0.2mm caudal, 0.7±0.3mm lateral and 1.8±0.7mm ventral to the Parabrachial Nucleus (n=12). Since the Parabrachial Nucleus - Kölliker-Fuse Nucleus distance was consistent and matched our previous study (8), in later experiments we functionally identified the Parabrachial Nucleus and used the location 1mm caudal, 0.5mm lateral and 2mm ventral to the Parabrachial Nucleus for Kölliker-Fuse Nucleus injections.
Fig. 1. Brainstem locations of naloxone microinjection.
A: Phrenic neurogram tracings illustrate the functional identification of the Parabrachial Nucleus, Kölliker-Fuse Nucleus, and preBötzinger Complex through typical responses to injection of the glutamate receptor agonist α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, vertical arrows). B: Dorsal view of the brainstem. Superimposed are the approximate distribution areas of the naloxone injections into the Parabrachial Nucleus (blue), Kölliker-Fuse Nucleus (green) and preBötzinger Complex (red). We estimate an effective spherical diffusion radius for our injection volume of 1–1.2mm (22). There is little overlap between the Parabrachial Nucleus and Kölliker-Fuse Nucleus injections in the brainstem as the Kölliker-Fuse Nucleus is located 2mm ventral to the Parabrachial Nucleus. C: Stereotaxic coordinates of the naloxone injection sites for Parabrachial Nucleus injections (blue squares) and Kölliker-Fuse Nucleus injections (green squares), projected over coronal slices of the rostral pons. Coordinates for the bilateral injections were averaged for each animal. For clarity, injection sites <1.5mm caudal to the inferior collicle are summarized in the slice “−1.0mm”, coordinates between 1.5 and 2.5mm caudal to the inferior collicle are summarized in the slice “−2.0mm”, and injection sites ≥2.5mm caudal to the inferior collicle are summarized in the slice “−3.0mm”. To account for residual cerebellar tissue covering the dorsal brainstem in our preparation, we subtracted 5mm from the measured stereotaxic depth coordinate in all animals, i.e., the depicted depth of injection is an approximation. D: Stereotaxic coordinates of the naloxone injections into the preBötzinger Complex after injection into the Parabrachial Nucleus and Kölliker-Fuse Nucleus (yellow squares, Cohort A) or solely into the preBötzinger Complex (red squares, Cohort B), projected over coronal slices of the caudal medulla oblongata. Coordinates for the bilateral injections were averaged for each animal. Injection sites <1.5mm rostral to obex are summarized in the slice “+1.0mm”. Injection sites ≥1.5mm rostral to obex are summarized in the slice “+2.0mm”. Please see section 2.3. for average stereotaxic coordinates. The outlines of the maps are redrawn from histological sections obtained for our previous studies in adult rabbits (8, 22). The atlas of Meessen and Olszewski (46) was used for comparison. Please note different scale for D. SC: superior collicle, IC: inferior collicle, CP: cerebellar peduncle, LPBN: lateral parabrachial nucleus, MPBN: medial parabrachial nucleus, LC: locus coeruleus, CS: Calamus Scriptorius, nAmb: nucleus Ambiguus, preBotC: preBötzinger Complex, nTS: nucleus Tractus Solitarii, XII: hypoglossal motor nucleus, X: vagal dorsal motor nucleus, py: pyramidal tract.
For protocols including the preBötzinger Complex, we identified the preBötzinger Complex as the area with inspiratory and expiratory neuronal activity where AMPA injection caused maximal tachypnea (fig. 1). The preBötzinger Complex was located on average 2.1±0.7mm rostral to obex, 2.7±0.4mm lateral from midline and 5.0±0.5mm ventral to the dorsal surface (n=32). The Parabrachial Nucleus was located 9.6±0.7mm rostral to the preBötzinger Complex at the same distance lateral from midline and, dependent on the thickness of the residual cerebellar peduncle, 3.2±1.2mm ventral to the preBötzinger Complex (n=23).
Complete, bilateral functional identification of all areas including the time required for respiratory rate to return to baseline after each AMPA injection required 4–5h.
2.4. Opioid effect sites at “analgesic” IV remifentanil concentrations
Experimenters were not blinded to the experimental conditions, and we did not perform formal randomization to experimental protocols. Since the effect of naloxone microinjection persists >2h, only one complete protocol was performed per animal.
To determine how much opioid effects in the Kölliker-Fuse Nucleus and Parabrachial Nucleus contributed to systemic opioid-induced respiratory depression at “analgesic” opioid doses, we infused remifentanil intravenously at 0.15±0.08 mcg/kg/min until respiratory rate was depressed by approximately 50% (fig. 2). In rabbits, a ~50-% respiratory rate depression was associated with loss of response to ear-pinch and pedal withdrawal reflex (14, 24). Its fast onset and short half-life that is independent of the duration of infusion (12, 25) make remifentanil the ideal drug to investigate opioid effects at steady-state concentrations. After reaching steady-state effect for 10–15 min, naloxone (1mM, 700nl) was microinjected bilaterally into the Kölliker-Fuse Nucleus, and 5 min were allowed to obtain maximal effect. Subsequently, naloxone was injected into the bilateral Parabrachial Nucleus. After interim analysis revealed that naloxone injections into the Kölliker-Fuse Nucleus and Parabrachial Nucleus did not lead to complete reversal of respiratory rate depression, for all subsequent protocols, we additionally injected naloxone into the bilateral preBötzinger Complex. Volume and timing for Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex microinjections had been established in previous studies (8, 22). Animals where naloxone was injected into the Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex are included in Cohort A.
Fig. 2. Injection sequence for intravenous remifentanil and local naloxone microinjections.
The initial intravenous remifentanil bolus (red arrow) was chosen to cause apnea >30 seconds. Once respiratory rate returned, the remifentanil infusion was started (red line). In many animals, one or more adjustments of the infusion rate were necessary to achieve the targeted respiratory rate depression of 50%. Steady-state respiratory rate depression was confirmed for 15 minutes before start of the brainstem injections. The remifentanil dose-rate was continued unchanged throughout the entire injection sequence. Cohort A: Naloxone (blue arrows) was microinjected into the bilateral Kölliker-Fuse Nucleus and Parabrachial Nucleus in 19 animals and subsequently into the preBötzinger Complex in 12 of these animals. “Apneic” IV remifentanil boluses (red arrows) were given after naloxone injections into the pons and after injection into the preBötzinger Complex. After each IV remifentanil bolus, we awaited recovery of respiratory rate to the pre-bolus level before subsequent injections. In six animals, we also tested a “very high” IV remifentanil dose. Cohort B: Naloxone was microinjected only into the bilateral preBötzinger Complex. Since the subsequent “apneic” IV remifentanil bolus continued to cause apnea in the majority of animals, no “very high” IV remifentanil bolus was given in this cohort. IV REMI: intravenous remifentanil, KF: Kölliker-Fuse Complex, PBN: Parabrachial Nucleus, preBötC: preBötzinger Complex.
To determine whether the effect of preBötzinger Complex injections depended on prior naloxone reversal in the Parabrachial Nucleus and Kölliker-Fuse Nucleus, in a separate set of animals (Cohort B) we infused remifentanil until steady-state and injected naloxone solely into the preBötzinger Complex. At the end of the experiments, naloxone 15–40 mcg/kg was injected intravenously to document that respiratory rate returned to pre-remifentanil control. We have previously described that aCSF and naloxone injections into the Parabrachial Nucleus or preBötzinger Complex did not have any independent effects and did not repeat those control injections in the present study (2, 3, 26).
2.5. Opioid effect sites at “apneic” IV remifentanil concentrations
To determine whether the contributions of the Kölliker-Fuse Nucleus, Parabrachial Nucleus and preBötzinger Complex to opioid-induced respiratory depression depended on the opioid concentration, we injected a remifentanil bolus that was sufficient to cause apnea >30sec (fig. 2). Since the exact dose to achieve apnea varied between animals, we chose 10mcg (~3mcg/kg) as a standard dose. However, boluses were repeated with larger doses when the initial bolus did not result in apnea. Since repeat doses required return of respiratory rate to baseline, this added 30–60 min to the experiment. In Cohort A, the “apneic” bolus was repeated after naloxone injection into the PBN/KF and again after naloxone injection into the preBötzinger Complex. In Cohort B, the “apneic” bolus was repeated after naloxone injection solely into the preBötzinger Complex.
To compare the effect sites of “analgesic” and “apneic” remifentanil concentrations in the same animals, the entire experimental sequence consisted of the “apneic” intravenous remifentanil bolus after which we started the remifentanil infusion and waited until respiratory rate reached steady-state depression (~50% control) for 10–15 min. If respiratory rate was substantially higher or lower than 50%, we adjusted the remifentanil infusion rate and waited until a new steady-state was obtained for >10min, i.e., for an additional 20–30 min. Once a satisfactory infusion rate was established, the rate was not changed throughout the entire naloxone injection protocol. In Cohort A, at steady-state respiratory depression, we performed bilateral naloxone microinjections into the Kölliker-Fuse Nucleus and, after a 5-min wait, into the Parabrachial Nucleus. When respiratory parameters had reached steady-state after the Parabrachial Nucleus injection (5–10 min), we repeated the “apneic” bolus. The concurrent remifentanil infusion meant that the plasma concentrations after the repeat “apneic” bolus were somewhat higher than after the initial bolus, however, the time requirement to discontinue the remifentanil infusion, inject and recover from the “apneic” bolus, restart the infusion and achieve the same steady-state conditions as before would have been prohibitive. To obtain paired data for “analgesic” and “apneic” concentrations, we chose to continue the remifentanil infusion. After the “apneic” bolus we waited for respiratory parameters to recover to pre-bolus values and performed bilateral naloxone microinjections into the preBötzinger Complex. After the effects of the naloxone injection had reached steady-state (5 min), we repeated the “apneic” remifentanil bolus. After recovery of respiratory rate to pre-bolus rate, we injected naloxone intravenously to document that the control respiratory rate had not changed. Only then was the remifentanil infusion discontinued.
In Cohort B, the initial remifentanil bolus and infusion rate were determined in the same fashion, but naloxone was injected only into the preBötzinger Complex. The entire experiment including surgical preparation, functional identification of the injection sites and the remifentanil/ naloxone protocol required 16–18h. Depending on whether the experiments required functional identification of the Parabrachial Nucleus and Kölliker-Fuse Nucleus in addition to the preBötzinger Complex, the first remifentanil injection occurred between 18:00 and 20:00, and experiments often lasted past midnight. Hemodynamics, end-tidal CO2 and nerve recordings were remarkably stable throughout the entire experiment.
2.6. Effects of “very high” remifentanil concentrations on areas outside the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex
Initial experiments showed that naloxone injection into the PBN/KF and preBötzinger Complex reliably prevented apnea from the “apneic” remifentanil bolus (10 mcg). We sought to determine, whether naloxone reversal of these areas would be able to prevent apnea including from “very high” remifentanil doses, or whether “very high” remifentanil doses would also depress other respiratory-related areas, e.g., respiratory drive to the PBN/KF and preBötzinger Complex or respiratory motor output. In a subgroup of Cohort A, we completed the naloxone injection sequence into the Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex to test the reversal of “analgesic” and “apneic” remifentanil concentrations as described in 2.4. and 2.5.. After the final “apneic” remifentanil bolus, we allowed respiratory rate to recover to pre-bolus values with remifentanil infusion running. We then injected 10–50 mcg bolus doses of remifentanil intravenously in short sequence until we observed apnea in the phrenic neurogram or to a maximal dose of 100 mcg remifentanil. For this analysis, we also determined peak vagus activity.
2.7. Effects of supraclinical opioid concentrations, compared to glutamate receptor blockade in the Parabrachial Nucleus and Kölliker-Fuse Nucleus
We investigated whether maximal opioid receptor activation in the Parabrachial Nucleus and Kölliker-Fuse Nucleus resulted in similar effects as disfacilitating neuronal activity with glutamate antagonist injection (8). In a separate set of animals, we injected the μ-opioid receptor agonist DAMGO at supraclinical concentrations (100μM, 700nl) bilaterally into the functionally identified Parabrachial Nucleus and Kölliker-Fuse Nucleus. We statistically compared the opioid effects with results from our previous study where Parabrachial Nucleus and Kölliker-Fuse Nucleus activity was eliminated using microinjections of the non- N-methyl-D-aspartate (NMDA) receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX; 1 mM, 700nl/ injection) and NMDA receptor antagonist d(–)-2-amino-5-phosphonopentanoic acid (AP5; 5 mM, 700 nl/ injection) (8).
Initial comparison of the two datasets showed a difference in baseline respiratory rate. Since both, DAMGO and AP5/NBQX injections reduced respiratory rate to the single digits, a difference in the change in respiratory parameters between groups could have been due to the difference in baseline rate. In response to peer review, we matched baseline respiratory rates in both groups: We performed two additional experiments using DAMGO injections and excluded one animal with a respiratory rate of <20 breaths per minute (bpm), and we excluded animals with respiratory rates >37 bpm from the original AP5/NBQX dataset (6–8, 27). Of note, the selection of data subsets may have biased the results towards the properties of these animals. In our adult outbred rabbit model, we observe a natural variation in baseline respiratory rate between ~20–40 bpm that remains remarkably constant over the course of many hours. Individual baseline rate does not clearly correlate with sex, age, or weight, and varies even between animals of the same litter when experiments are performed in the same week. However, we cannot rule out that despite our efforts at consistency in surgical preparation and experimental conditions, respiratory control and rate were influenced by unrecognized factors that may have changed since our previous study (6–8, 27). It is also possible that pontine function is different in animals with very high or low baseline respiratory rates, and that the interpretation of our data is limited to animals with baseline respiratory rates between 20–37 bpm.
2.8. Control Studies: Effects of naloxone or artificial cerebrospinal fluid injections into the Parabrachial Nucleus, Kölliker-Fuse Nucleus, and preBötzinger Complex
To ensure that the effect of local naloxone microinjections we observed during remifentanil infusion represented a reversal of the extrinsic opioid effect rather than antagonism of endogenous opioid receptor activation or γ-aminobutyric acid (GABA)A receptor antagonism (28), we injected naloxone (1mM, 700nl) sequentially into the bilateral Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex. We also injected artificial cerebrospinal fluid (700nl), which was used as solvent for all injected drugs, into the bilateral Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex to rule out an independent effect of the solvent.
2.9. Statistical analysis
Statistical analysis was performed using SigmaPlot 11 (Systat Software, USA). We did not perform a formal power analysis, and no adjustments were made for interim analyses. Comparable studies have used 4 to 9 rats (10) ((29), 4 to 11 mice (11, 13), 8 to 16 rabbits (2, 3), and 10 to 21 dogs (4, 5) per protocol.
Since experiments were technically very difficult and labor intensive, we included data from a few animals where one single data point (injection of the “apneic” remifentanil dose into either the Parabrachial Nucleus/Kölliker-Fuse Complex or preBötzinger Complex) was missing. The total number of animals is indicated for each comparison. To eliminate the problem of “missing values”, we calculated the difference (“delta”) for each variable between naloxone injection into the Kölliker-Fuse Nucleus and IV remifentanil, between naloxone injection into the Parabrachial Nucleus and Kölliker-Fuse Nucleus, between naloxone injection into the preBötzinger Complex and Parabrachial Nucleus, or between naloxone injection into the preBötzinger Complex and IV remifentanil. Testing revealed that not all “deltas” were normally distributed (Shapiro-Wilk test). For paired data, we uniformly used the Wilcoxon Signed Rank test to test each “delta” against no change (Null-Hypothesis). Within the same protocol, we used the Mann-Whitney-U test to compare the effects of naloxone injection into the preBötzinger Complex between animals where naloxone had been previously injected into the Parabrachial Nucleus/Kölliker-Fuse Complex and those where naloxone was solely injected into the preBötzinger Complex (unpaired data). Hypothesis testing was two-tailed. The critical value for significant differences was adjusted according to the number of comparisons for each protocol according to Bonferroni, i.e., p<0.01 for “analgesic” remifentanil concentrations (5 comparisons) and p<0.0125 for “apneic” remifentanil concentrations (4 comparisons). Results for the different remifentanil concentrations were analyzed separately without additional correction for multiple comparisons. Inputs to inspiratory on- and off-switch were calculated from the values for inspiratory and expiratory duration as described in the Appendix. For 3.4., values for inspiratory duration and peak phrenic activity were log transformed, and the adjusted correlation coefficients (R2, squares of Pearson’s correlation) were compared using bootstrap analysis (R 3.5.0, R Foundation for Statistical Computing, Vienna, Austria). In accordance with the exploratory nature of the study, for additional comparisons, e.g., between different remifentanil concentrations, between respiratory parameters, or between peak phrenic and peak vagus activity we used Cohen’s d. This is defined as the difference between two means (μ1-μ2), divided by the pooled standard deviation (s), i.e., d= (μ1-μ2)/s. The pooled standard deviation is weighted for the number of samples (n) in each group, i.e., s = (s12 *(n1-1) + s22 *(n2-1))/(n1+n2-2))1/2. A Cohen’s d ≥0.5 is considered a “medium”, ≥0.8 a “large”, and ≥2 a “huge” difference (30). In addition, groups were compared using the Mann-Whitney-U test without corrections for multiple comparisons. Parameters are presented as median (25%−75% range).
Primary outcomes were the changes in respiratory parameters with local microinjection of naloxone at one remifentanil concentrations, or microinjection of DAMGO, resp.. Secondary outcomes were comparisons between respiratory parameters (e.g., inspiratory and expiratory duration) at the same remifentanil concentration, between the effects on different areas (i.e., Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex) at the same remifentanil concentration, between different opioid concentrations, and between phrenic and vagal nerve activity.
Results
In total, 48 animals were used in our studies. Three animals died during the surgical preparation, and four animals developed significant respiratory slowing during AMPA mapping and were removed from further study. No animal died during the remifentanil/ naloxone injection sequence.
3.1.: Opioid effect sites at “analgesic” IV remifentanil concentrations
To determine the effect of “analgesic” remifentanil concentrations on the Parabrachial Nucleus/ Kölliker-Fuse Complex and preBötzinger Complex, we microinjected naloxone into the Parabrachial Nucleus/ Kölliker-Fuse Complex and preBötzinger Complex (Cohort A), or preBötzinger Complex alone (Cohort B) during remifentanil infusion, dosed to depress baseline respiratory rate by 50%. Cohort A consisted of eight female (3.5±0.9kg) and 11 male (2.8±0.4kg) animals, and cohort B consisted of four female (3.7±0.6kg) and six male (3.3±0.4kg) animals. In Cohort A, the continuous remifentanil infusion depressed respiratory rate from 36 bpm to 16 bpm (fig. 3B, left). Sequential, bilateral microinjections of naloxone into the bilateral Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex (n=12) increased respiratory rate to 17 (p=0.005), 23 (p<0.001), and 25 bpm (p<0.001), respectively. For 25–75% range, please see Table 1. In Cohort B, remifentanil infusion decreased respiratory rate from 33.5 to 17 bpm (fig. 3B, right). Naloxone microinjection solely into the preBötzinger Complex increased respiratory rate to 20.5 bpm (p=0.005). The effect of naloxone injection into the preBötzinger Complex was similar with or without prior naloxone injection into the PBN/KF (blue bracket, p=0.242, fig. 3B).
Fig. 3. “Analgesic” remifentanil concentrations.
Bilateral naloxone injections into the Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex significantly reversed the respiratory rate depression from intravenous remifentanil. The “analgesic” remifentanil dose-rate was chosen to achieve ~50% respiratory rate depression. A: Phrenic neurogram (PNG; a.u. [arbitrary units]) tracings during control conditions and sequential drugs injections in an individual rabbit. B-E: Pooled data for changes in respiratory rate and other respiratory parameters. F-G: Values for inputs to inspiratory on- and off-switch were derived from the values for inspiratory and expiratory duration and are presented in % of apneic threshold with apneic threshold=100% (Appendix 1). Data for Cohort A are presented on the left side of each panel: Sequential naloxone injections into the Kölliker-Fuse Nucleus and Parabrachial Nucleus were performed in 19 animals. In 12 of these animals, naloxone was subsequently injected into the preBötzinger Complex. Data for Cohort B are presented on the right side of each panel (shaded): In a separate group of 10 animals, naloxone was injected only into the preBötzinger Complex. Black brackets: The difference (“delta”) between values from two sequential injections was tested against no change (Wilcoxon signed rank test). Blue brackets: Comparison of the “deltas” from preBötzinger Complex injection with and without prior naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex (Mann-Whitney rank sum test). Levels of significance below the critical p=0.01 are highlighted in red. Phrenic neurogram traces and pooled data are color coded for the same condition to facilitate reader orientation. NAL: naloxone, KF: Kölliker-Fuse Nucleus, PBN: Parabrachial Nucleus, preBötC: preBötzinger Complex, IV REMI: intravenous remifentanil.
Table 1:
Summary data for “analgesic” and “apneic” remifentanil concentrations in Cohorts A and B. Mean (25–75%). “Analgesic” remifentanil concentrations are given as IV infusion. “Apneic” concentrations are given as IV bolus. Inputs are normalized to the apneic threshold. Peak phrenic activity is normalized to control.
| Cohort A: “analgesic” remifentanil concentration, naloxone injection into the Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex | ||||||
|---|---|---|---|---|---|---|
| Control | IV remifentanil | Naloxone Kölliker-Fuse Nucleus | Naloxone Parabrachial Nucleus | Naloxone preBötzinger Complex | IV Naloxone | |
| Number of animals | 19 | 19 | 19 | 19 | 12 | 19 |
| Breaths per minute | 36 (31 – 40) | 16 (15 – 21) | 17 (16 – 22) | 23 (19 – 29) | 25 (22 – 28) | 33 (28.5 – 40) |
| Inspiratory duration (sec) | 0.78 (0.69 – 0.89) | 1.5 (1.23 – 1.72) | 1.43 (1.21 – 1.60) | 1.20 (1.01 – 1.38) | 1.16 (1.02 – 1.26) | 0.88 (0.70 – 0.95) |
| Input to inspiratory off-switch (%) | 185 (169 – 201) | 129 (122 – 141) | 131 (125 – 143) | 143 (134 – 157) | 146 (140 – 157) | 174 (164 – 199) |
| Expiratory duration (sec) | 0.85 (0.74 – 1.06) | 2.22 (1.80 – 2.48) | 2.01 (1.61 – 2.24) | 1.50 (1.15 – 1.79) | 1.28 (1.15 – 1.52) | 0.96 (0.82 – 1.11) |
| Input to inspiratory on-switch (%) | 175 (153 – 192) | 112 (109 – 120) | 116 (112 – 125) | 129 (120 – 146) | 138 (128 – 146) | 163 (149 – 179) |
| Peak phrenic activity (%) | 100 | 84 (77 – 96) | 87 (80 – 95) | 88 (81 – 99) | 91 (86 – 104) | 109 (91 – 119) |
| Cohort B: “analgesic” remifentanil concentration, naloxone injection into the preBötzinger Complex | ||||||
| Control | IV remifentanil | Naloxone Kölliker-Fuse Nucleus | Naloxone Parabrachial Nucleus | Naloxone preBötzinger Complex | IV Naloxone | |
| Number of animals | 10 | 10 | -- | -- | 10 | 10 |
| Breaths per minute | 34 (22 – 35) | 17 (11 – 18) | -- | -- | 21 (15 – 26) | 33 (28.5 – 40) |
| Inspiratory duration (sec) | 0.85 (0.79 – 1.09) | 1.32 (1.18 – 1.94) | -- | -- | 1.35 (1.00 – 1.71) | 0.80 (0.78 – 0.94) |
| Input to inspiratory off-switch (%) | 161 (135 – 177) | 136 (117 – 144) | -- | -- | 134 (122 – 158) | 183 (164 – 188) |
| Expiratory duration (sec) | 1.08 (0.84 – 1.61) | 2.26 (2.02 – 3.57) | -- | -- | 1.68 (1.39 – 2.40) | 0.96 (0.85 – 1.37) |
| Input to inspiratory on-switch (%) | 152 (125 – 176) | 112 (103 – 115) | -- | -- | 122 (104 – 133) | 162 (134 – 175) |
| Peak phrenic activity (%) | 100 | 89 (84 – 96) | -- | -- | 96 (84 – 103) | 108 (94 – 127) |
| Cohort A: “apneic” remifentanil concentration, naloxone injection into the Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex | ||||
|---|---|---|---|---|
| Control | IV remifentanil | Naloxone Kölliker-Fuse+Parabrachial Nucleus | Naloxone preBötzinger Complex | |
| Number of animals | 17 | 17 | 17 | 12 |
| Breaths per minute | 36 (31 – 40) | 0 (0 – 0) | 10 (0 – 12) | 13 (10 – 19) |
| Inspiratory duration (sec) | 0.78 (0.69 – 0.89) | 2.96 (1.96 – 4.24) | 1.87 (1.35 – 2.19) | 1.92 (1.41 – 2.57) |
| Input to inspiratory off-switch (%) | 185 (169 – 201) | 105 (102 – 117) | 118 (113 – 135) | 117 (109 – 133) |
| Expiratory duration (sec) | 0.85 (0.74 – 1.06) | 17.5 (14.2–19.9) | 3.73 (2.97 – 14.5) | 2.73 (1.58 – 4.58) |
| Input to inspiratory on-switch (%) | 175 (153 – 192) | 100 (100 – 100) | 102 (100 – 105) | 107 (102 – 126) |
| Peak phrenic activity (%) | 100 | 47 (42 – 65) | 56 (52 – 77) | 63 (56 – 78) |
| Cohort B: “apneic” remifentanil concentration, naloxone injection into the preBötzinger Complex | ||||
| Control | IV remifentanil | Naloxone Kölliker-Fuse+Parabrachial Nucleus | Naloxone preBötzinger Complex | |
| Number of animals | 9 | 9 | -- | 9 |
| Breaths per minute | 33 (20 – 35) | 0 (0 – 0) | -- | 0 (0 – 0) |
| Inspiratory duration (sec) | 0.84 (0.77 – 1.13) | 2.2 (1.7 – 2.9) | -- | 4.65 (2.89 – 7.88) |
| Input to inspiratory off-switch (%) | 176 (147 – 186) | 112 (106 – 122) | -- | 101 (100 – 106) |
| Expiratory duration (sec) | 1.14 (0.85 – 1.71) | 21.2 (13.1 – 24.8) | -- | 20.6 (6.90 – 22.0) |
| Input to inspiratory on-switch (%) | 147 (122 – 175) | 100 (100 – 100) | -- | 100 (100 – 100) |
| Peak phrenic activity (%) | 100 | 61 (32 – 71) | -- | 69 (65 – 72) |
In Cohort A, remifentanil infusion decreased peak phrenic activity from 100 to 84% of control (fig. 3C, left). Naloxone injections into the Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex increased peak phrenic activity to 87 (p=0.022), 88 (p<0.001), and 91% (p=0.898), respectively. In Cohort B, remifentanil infusion decreased peak phrenic activity from 100 to 89%, and naloxone injection into the preBötzinger Complex did not reverse the depression (96%, p=0.039, fig. 3C, right). The effect of naloxone injection into the preBötzinger Complex was similar with or without prior naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex (blue bracket, p=0.193, fig. 3C).
In Cohort A, remifentanil infusion increased inspiratory duration from 0.78 sec to 1.5 sec (fig. 3D, left). Naloxone injections into the Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex decreased inspiratory duration to 1.43 (p=0.012), 1.2 (p<0.001), and 1.16 sec (p=0.007), respectively. In Cohort B, remifentanil increased inspiratory duration from 0.84 to 1.32 sec, which naloxone injection into the preBötzinger Complex did not change (1.35 sec, p=0.375, fig. 3D, right). The effect of naloxone injection into the preBötzinger Complex was similar with and without prior naloxone injection into the PBN/KF (blue bracket, p=0.149, fig. 3D).
In Cohort A, remifentanil infusion increased expiratory duration from 0.84 to 2.22 sec (fig. 3E, left). Naloxone injections into the Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex decreased expiratory duration to 2.01 (p=0.008), 1.50 (p<0.001), and 1.28 sec (p<0.001), respectively. In Cohort B, remifentanil infusion increased expiratory duration from 1.08 to 2.26 sec, which was decreased by naloxone injection into the preBötzinger Complex to 1.68 sec (p=0.002, fig. 3E, right). The decrease in expiratory duration with naloxone injection into the preBötzinger Complex alone was greater than the decrease after prior naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex (blue bracket, p<0.001, fig. 3E).
In Cohort A, remifentanil infusion decreased inputs to inspiratory off-switch from 185% to 129% of apneic threshold (fig. 3F, left). Naloxone injections into the Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex increased these inputs to 131% (p=0.002), 143% (p<0.001), and 146% (p=0.024), respectively. In Cohort B, remifentanil decreased inputs to inspiratory off-switch from 161 to 136%, which naloxone injection into the preBötzinger Complex did not change (134%, p=0.064, fig. 3F, right). The effect of naloxone injection into the preBötzinger Complex was similar with and without prior naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex (blue bracket, p=0.460, fig. 3F).
In Cohort A, remifentanil decreased inspiratory on-switch from 175 to 112% of apneic threshold (fig. 3G, left). Naloxone injections into the Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex increased these inputs to 116 (p=0.007), 129 (p<0.001), and 138% of apneic threshold (p<0.001), respectively. In Cohort B, inputs to inspiratory on-switch decreased with remifentanil infusion from 152 to 112 % of apneic threshold and increased with naloxone injection into the preBötzinger Complex to 122% (p=0.002, fig. 3G, right). The effect of naloxone injection into the preBötzinger Complex was similar with and without prior naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex (blue bracket, p=0.699, fig. 3G) suggesting that the observed difference in expiratory duration may have been due to the slower respiratory rate and longer expiratory duration before naloxone injection (see Appendix 1).
Additional analysis showed that naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex (fig. 3B, left) increased respiratory rate more than injection into the preBötzinger Complex (data pooled for Cohort A+B, fig. 3B, left+right, Cohen’s d 0.8, p=0.033). Naloxone microinjection into the PBN/KF decreased expiratory duration (fig. 3E, left) more than inspiratory duration (fig. 3D, left, Cohen’s d 0.9, p=0.008) as did the subsequent injection into the preBötzinger Complex (fig. 3E+D, left, Cohen’s d 1.6, p=0.004). Naloxone injection into the preBötzinger Complex alone decreased expiratory duration (fig. 3E, right) more than inspiratory duration (fig. 3D, right, Cohen’s d 1.6, p<0.001).
3.2.: Opioid effect sites at “apneic” IV remifentanil concentrations
To determine the effect of “apneic” remifentanil concentrations on the Parabrachial Nucleus/ Kölliker-Fuse Complex and preBötzinger Complex, we administered an intravenous remifentanil bolus that caused apnea under control conditions before and after microinjection of naloxone into the Parabrachial Nucleus/ Kölliker-Fuse Complex and after additional naloxone injection into the preBötzinger Complex (Cohort A, 17 animals), or after naloxone injection into the preBötzinger Complex alone (Cohort B, nine animals). In Cohort A, in 12/17 animals naloxone microinjection into the PBN/KF prevented apnea from the intravenous remifentanil bolus that had caused apnea under control conditions. Respiratory rate increased from 0 to 10 bpm (p<0.001, fig. 4B, left). For 25–75% range, please see Table 1. After additional naloxone microinjection into the preBötzinger Complex (n=12), the repeat remifentanil bolus did not cause apnea in any animal, and respiratory rate was increased to 13 bpm (p=0.002). In Cohort B, naloxone microinjection into the preBötzinger Complex alone prevented apnea from the IV remifentanil bolus only in 2/9 animals (fig. 4B, right), and median respiratory rate was not increased (0 bpm, p=0.500). Compared to the effect of naloxone microinjection into the preBötzinger Complex alone, preBötzinger Complex injection after naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex caused a larger increase in respiratory rate (blue bracket, p=0.006, fig. 4B).
Fig. 4, “Apneic” remifentanil concentrations.
Bilateral naloxone injections into the Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex prevented respiratory rate depression and apnea from an intravenous remifentanil bolus (~10mcg, IV REMI). A: Phrenic neurogram (PNG; a.u. [arbitrary units]) tracings from the same rabbit shown in fig. 3 show that the same remifentanil bolus (remi arrow) that elicited apnea during control conditions (left) only moderately depressed respiratory rhythm after naloxone injection into the Kölliker-Fuse Nucleus and Parabrachial Nucleus (middle) and even less after subsequent naloxone injection into the preBötzinger Complex (right). B-E: Pooled data for changes in respiratory rate and other respiratory parameters. Please note the different time scale for inspiratory (D) and expiratory (E) duration. F-G: Values for inputs to inspiratory on- and off-switch were derived from the values for inspiratory and expiratory duration and are presented in % of apneic threshold with apneic threshold=100% (Appendix 1). Data for Cohort A are presented on the left side of each panel: Data were available for apneic bolus after naloxone injection into the Kölliker-Fuse Nucleus and Parabrachial Nucleus in 17 animals and for 12 animals after additional injection into the preBötzinger Complex. Data for Cohort B are presented on the right side of each panel (shaded): In a separate group of nine animals, naloxone was injected only into the preBötzinger Complex. Black brackets: The difference (“delta”) between values from two sequential injections was tested against no change (Wilcoxon signed rank test). Blue brackets: Comparison of the “deltas” from preBötzinger Complex injection with and without prior naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex (Mann-Whitney rank sum test). Levels of significance below the critical p=0.0125 are highlighted in red. NAL: naloxone, KF: Kölliker-Fuse Nucleus, PBN: Parabrachial Nucleus, preBötC: preBötzinger Complex, IV REMI: intravenous remifentanil.
Since inspiratory duration, expiratory duration, and peak phrenic activity cannot be measured during apnea, we extrapolated these variables from the first breath after apnea (see fig. 4A, “control”). If no apnea was observed, we averaged parameters for approximately six to eight breaths at the lowest respiratory rate following the bolus (fig. 4A, “NAL KF+PBN” and “NAL KF+PBN+preBötC”). In Cohort A, the “apneic” remifentanil bolus reduced peak phrenic activity from 100 to 47% (fig. 4C, left). Naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex (56%, p=0.030) and preBötzinger Complex (63%, p=0.064) did not significantly increase peak phrenic activity. In Cohort B, the remifentanil bolus depressed peak phrenic activity from 100 to 61% (fig. 4C, right), and naloxone injection into the preBötzinger Complex did not change peak phrenic activity (69%, p=0.250). The naloxone effect was similar with and without prior naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex (blue bracket, p=0.596, fig. 4C).
The “apneic” remifentanil bolus increased inspiratory duration from 0.78 to 2.96 sec (fig. 4D, left). Naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex reduced the increase to 1.87 sec (p=0.009), while naloxone injection into the preBötzinger Complex caused no further change (1.92 sec, p=0.375). In Cohort B, the “apneic” remifentanil bolus increased inspiratory duration from 0.84 to 2.20 sec (fig. 4D, right). Naloxone injection into the preBötzinger Complex further increased inspiratory duration to 4.65 sec (p=0.008). The naloxone effect on inspiratory duration was not significantly different with prior Parabrachial Nucleus/Kölliker-Fuse Complex reversal (blue bracket, p=0.016, fig. 4D).
In Cohort A, the “apneic” remifentanil bolus increased expiratory duration from 0.85 to 17.48 sec (fig. 4E, left). Naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex reduced the increase to 3.73 sec (p=0.007), and injection into the preBötzinger Complex further reduced the increase to 2.73 sec (p=0.002). In Cohort B, the “apneic” remifentanil bolus increased expiratory duration from 1.14 to 21.2 sec (fig. 4E, right), and naloxone injection into the preBötzinger Complex did not change it (20.58 sec, p=0.426). The naloxone effect on expiratory duration was not different after prior Parabrachial Nucleus/Kölliker-Fuse Complex reversal (blue bracket, p=0.079, fig. 4E).
In Cohort A, the “apneic” remifentanil bolus decreased inspiratory off-switch from 185 to 105% of apneic threshold (fig. 4F, left). Naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex (118%, p=0.268) and preBötzinger Complex (117%, p=0.625) did not significantly change these inputs. In Cohort B, remifentanil decreased inputs to inspiratory off-switch from 176 to 112%, and naloxone injection into the preBötzinger Complex further decreased inputs to inspiratory off-switch to 101% (p=0.039, fig. 4F, right). The naloxone effect on inputs to inspiratory off-switch was not significantly different with prior Parabrachial Nucleus/Kölliker-Fuse Complex reversal (blue bracket, p=0.066, fig. 4F).
In Cohort A, the “apneic” remifentanil bolus decreased inputs to inspiratory on-switch from 175 to 100% of apneic threshold (fig. 4G, left). Naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex increased inputs to 102% (p=0.020), and subsequent injection into the preBötzinger Complex increased inputs to 107% (p=0.002). In Cohort B, inputs to inspiratory on-switch decreased with the remifentanil bolus from 147 to 100% of apneic threshold (fig. 4G, right) and did not change with naloxone injection (100%, p=0.82). Inputs to inspiratory on-switch increased more after prior Parabrachial Nucleus/Kölliker-Fuse Complex reversal (blue bracket, p=0.002, fig. 4G).
Additional analysis showed that naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex decreased expiratory duration (fig. 4E, left) more than inspiratory duration (fig. 4D, left, Cohen’s d 0.9, p=0.057) as did the subsequent injection into the preBötzinger Complex (fig. 4E+D, left, Cohen’s d 1.6, p<0.001). Naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex increased inputs to inspiratory on-switch more at “analgesic” (fig. 3G, left) than “apneic” remifentanil concentrations (fig. 4G, left, Cohen’s d 1.2, p<0.001) as did the subsequent injection into the preBötzinger Complex (fig. 3 and 4G, left, Cohen’s d 2.4, p<0.001). Inputs to inspiratory off-switch also increased more with naloxone injection into the preBötzinger at analgesic than at apneic concentrations (fig. 3 and 4F, left, Cohen’s d 1.3, p=0.003). After naloxone injection solely into the preBötzinger Complex, there was a substantial difference between the lack of effect on expiratory duration (fig. 4E, right) and the increase in inspiratory duration (fig. 4D, right, Cohen’s d 1.1, p=0.093) and also between the corresponding decrease in inputs to inspiratory off-switch (fig. 4F, right) and the lack of effect on inspiratory on-switch (fig. 4G, right, Cohen’s d 1.0, p=0.005). The percentage of control inputs to inspiratory on- and off-switch above the apneic threshold that could be recovered with naloxone injections into the respective brainstem areas at “analgesic” and “apneic” concentrations is presented in Table 2.
Table 2:
Percentage of inputs to respiratory phase duration that was restored with naloxone injections into the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex at “analgesic” and “apneic” remifentanil concentrations. Values are relative to the total inputs above the apneic threshold during control conditions. For example, “analgesic” remifentanil concentrations depressed inputs to expiratory duration by ~88% (equaling a decrease in inputs from 2 to 1.12). 22% of the input was reversed with naloxone injection into the preBötzinger Complex and 25% with naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex, suggesting that 41% was due to depression of respiratory drive. Original data and statistics are provided in Table 1, 3.1., 3.2., and fig. 3 and 4. Cohort B did not receive naloxone reversal of the Parabrachial Nucleus/Kölliker-Fuse Complex.
| Remifentanil | Respiratory phase | Respiratory drive | Parabrachial Nucleus/ Kölliker-Fuse Complex | preBötzinger Complex |
|---|---|---|---|---|
| Cohort A “analgesic” | inspiratory duration | 29 (9–49)% | 24 (13–30)% | 4 (2–11)% |
| expiratory duration | 41 (25–55)% | 25 (16–33)% | 22 (8–24)% | |
| Cohort A “apneic” | inspiratory duration | 77 (62–92)% | 14 (3–25)% | −3 ((−7)-10)%* |
| expiratory duration | 90 (82–98)% | 0.002 (0–4.7)% | 7 (0.1–16)% | |
| Cohort B “analgesic” | inspiratory duration | 44 (26–52)% | 12 (6–17)% | |
| expiratory duration | 54 (52–62)% | 26 (19–30)% | ||
| Cohort B “apneic” | inspiratory duration | 98 (95–100)% | −12 ((−69)-(−4))%* | |
| expiratory duration | 100 (100–100)% | 0 (0–0)% | ||
negative values indicate a decrease in inspiratory duration by “apneic” remifentanil concentrations in the preBötzinger Complex. Median (25–75% range).
3.3.: The quotient of peak phrenic activity / inspiratory duration and respiratory drive
To determine whether the quotient of peak phrenic activity and inspiratory duration (PPA/TI) is an adequate surrogate for “respiratory drive”, we analyzed how intravenous remifentanil bolus injections affected peak phrenic activity and inspiratory duration. As illustrated for a single animal in fig. 5A–C, inspiratory duration and peak phrenic activity changed concomitantly after an “apneic” remifentanil bolus, however, the remifentanil-induced increase in inspiratory duration was substantially reduced with naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex while the maximal depression of peak phrenic activity did not change much. Correlation analysis using data from all animals, log-transformed to allow linear regression analysis, and analyzed separately for opioid dose and naloxone injection (fig. 5D–F) showed that the predictive properties of ln(TI) for ln(PPA/TI) were consistently higher than ln(PPA) for all but one dataset. We conclude that systemic opioids affect excitatory inputs to phase-switching mechanisms differently from peak phrenic activity. Consequently, the quotient of peak phrenic activity and inspiratory duration is not a reliable reflection of drive to all components of the respiratory rhythm and pattern generator. In the following analysis and discussion, we will use the term “respiratory drive” more broadly as inputs to the mechanisms of phase switch and motor activity, and not limited to the concept of drive as the quotient of peak phrenic activity and inspiratory duration.
Fig. 5. Opioids depress inspiratory phase timing differently from peak phrenic activity.
A-C: Data from the rabbit shown in fig. 3A and 4A. A: The increase in inspiratory duration from the “apneic” remifentanil bolus (remi arrow), plotted for each breath vs. time, was smaller after naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex (blue, lowest respiratory rate 12 breaths per minute, bpm) and preBötzinger Complex (red, lowest respiratory rate 19 bpm). B: The decrease in peak phrenic activity, normalized to control, was attenuated less. Please note that inspiratory duration was increased, and peak phrenic activity was decreased before remifentanil injection after naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex (“Naloxone PBN/KF”) and preBötzinger Complex (“Naloxone PBN/KF+preBötC”) because of the continuous remifentanil infusion (see 2.5.). C: Naloxone reversal greatly decreased the prolongation of inspiratory duration from the “apneic” remifentanil bolus from 4 sec to ~1.5 sec while peak phrenic activity was always depressed 30–40%. D-F: Pooled data from all remifentanil protocols (n=171) illustrate the correlation between inspiratory duration, peak phrenic activity, and respiratory drive, here defined as quotient of peak phrenic activity and inspiratory duration (PPA/TI). The predictive properties of ln(TI) for ln(PPA/TI) were higher than ln(PPA) in all but one dataset. F: Correlation coefficients (R2, squares of Pearson’s correlation) for each dataset, and bootstrap analysis for adjusted correlation coefficients. TI: inspiratory duration, PPA: peak phrenic activity.
3.4.: Effects of “very high” remifentanil concentrations on areas outside the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex
To determine whether “very high” remifentanil concentrations affect respiratory rate and tidal volume outside the respiratory rhythm generator, we injected up to 100 mcg remifentanil IV after naloxone reversal of the Parabrachial Nucleus/ Kölliker-Fuse Complex and preBötzinger Complex. In six animals, the “very high” remifentanil bolus substantially decreased respiratory rate from 23.5 (20.5–28) bpm to 11 (7.2–12.5) bpm (p=0.031). Peak phrenic activity was depressed to 5 (0–38)% of the pre-bolus amplitude (p=0.031). In 3/6 animals, peak phrenic activity was completely abolished by the remifentanil bolus (phrenic apnea, fig. 6A). The “very high” remifentanil bolus depressed peak vagal activity to 50 (41–55)% of pre-bolus amplitude (p=0.031), but rhythmic vagal respiratory activity continued during phrenic apnea (fig. 6B). In comparison, the initial “apneic” remifentanil bolus before naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex (see 2.5.) generated apnea in the phrenic and vagal neurogram (fig. 6C). At baseline, vagal inspiratory activity closely matched phrenic activity, which confirms that the central respiratory pattern generator controls the phase timing of respiratory pump muscles as well as airway motor activity (31). The concomitant phrenic and vagal apnea observed after the initial “apneic” remifentanil bolus indicated that remifentanil completely depressed the respiratory rhythm generator, i.e., Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex function. The observation that after naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex, the “very high” remifentanil bolus still completely depressed phrenic activity (fig. 6A) but that that the respiratory rhythm continued as reflected in the continued phasic vagal activity (fig. 6B) suggests a) that naloxone successfully prevented complete depression of the respiratory rhythm generator, and b) that the “very high” remifentanil concentration directly depressed inspiratory premotor and/ or phrenic motoneurons. Vagal premotor and motoneurons appeared to be less sensitive to direct opioid depression. Before naloxone microinjections into the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex, the initial “apneic” remifentanil bolus depressed peak phrenic activity more than peak vagus activity (both calculated from the first breath after apnea, relative to the respective peak activity before the bolus) (Cohen’s d: 2.1, p=0.015, fig. 6C). This difference was also observed at the maximal remifentanil effect after the “very high” remifentanil bolus (Cohen’s d: 1.1, p=0.065, fig. 6D).
Fig. 6. Effect of “very high” remifentanil concentrations on areas outside the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex.
A: Phrenic neurogram tracings from an individual rabbit show that high bolus doses of intravenous remifentanil (total of 100mcg, red arrows) after naloxone microinjection into the bilateral Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex decreased peak phrenic activity to zero. B: However, during phrenic apnea the respiratory rhythm continued in the vagus neurogram. The continued rhythm confirmed that opioid antagonism in the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex successfully prevented inhibition of the respiratory rhythm generator even by “very high” remifentanil concentrations. The depression of peak phrenic activity (A) was likely due to direct inhibition of inspiratory premotor and/or motoneurons. In all animals, respiratory rate decreased by 15 (14–16) breaths per minute (n=6). The slowing in respiratory rate after the initial remifentanil bolus suggests that respiratory drive to the respiratory rhythm generator was decreased by “very high” remifentanil concentrations. C-D: We performed additional analysis to determine whether opioids depressed peak phrenic activity more than peak vagus activity. Peak phrenic and peak vagus activity was calculated relative to peak activity before the intravenous remifentanil bolus and pooled for six animals (mean±SD). C: Peak phrenic and peak vagus activity for each breath after recovery from apnea from the 10-mcg remifentanil bolus before naloxone injection into the brainstem showed that phrenic activity was more depressed by remifentanil. D: Peak phrenic and peak vagus activity for each breath starting at maximal depression from the 100-mcg remifentanil bolus after naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex again showed that phrenic activity was more depressed. Complete loss of phrenic motor output (apnea) was observed in 3/6 animals. Statistical difference between pooled peak phrenic and peak vagus activity for the first breath after apnea/ at maximal depression: #: Cohen’s d: 2.1, p=0.015; *: Cohen’s d: 1.1, p=0.065. Mann-Whitney-U test.
We also observed that, although naloxone reversal in the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex prevented cessation of the respiratory rhythm, the “very high” remifentanil bolus still caused significant depression of respiratory rate. Assuming that the naloxone injections were sufficient to locally antagonize all opioid effects, this suggests that remifentanil depressed the activity of the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex by decreasing the respiratory drive to these areas.
3.5.: Effects of supraclinical opioid concentrations, compared to glutamate receptor blockade in the Parabrachial Nucleus and Kölliker-Fuse Nucleus
To determine the effect of maximal opioid receptor activation, in six animals, the mu-opioid agonist DAMGO was injected into the bilateral Parabrachial Nucleus and Kölliker-Fuse Nucleus (fig. 7). These data were compared to glutamate antagonist injections in a subset of 13 animals from our previous study (8), which were selected to match the average respiratory rate at control. The levels of significance for comparisons between the effects of DAMGO and glutamate antagonists in the two studies are indicated by “blue brackets” in fig. 7. DAMGO injection depressed respiratory rate from 29 (28–31) to 6 (4–7) bpm (p=0.031, fig. 7B, left). This was similar to the effect of the glutamate receptor antagonists NBQX+AP5, which depressed respiratory rate from 29 (26–33) to 2 (1–2) bpm (blue bracket, p=0.160, fig. 7B). DAMGO injection did not depress peak phrenic activity (100% to 96 (81–105)%, p=0.688, fig. 7C, left), which was similar to NBQX+AP5 (100% to 97 (84–99)%; blue bracket, p=0.511, fig. 7C). DAMGO increased inspiratory duration from 0.98 (0.90–1.04) to 5.03 (4.45–6.66) sec (p=0.031, fig. 7D, left), which was less than the increase from the glutamate antagonists (0.81 (0.77–0.90) to 17.5 (11.4–22.6) sec; blue bracket, p=0.020, fig. 7D). DAMGO increased expiratory duration from 1.32 (1.08–1.64) to 5.41 (4.53–9.83) sec (p=0.031, fig. 7E, left), which was less than the increase from the glutamate antagonists (1.16 (0.98–1.59) to 22.1 (17.1–29.3) sec; blue bracket, p=0.007, fig. 7E). Similar to inspiratory duration, the inputs to inspiratory off-switch were decreased by DAMGO from 161 (155–169) to 101 (100–101)% of apneic threshold (p=0.031, fig. 7F, left), which was less than the decrease from the glutamate antagonists (180 (170–188) to 100 (100–100)% of apneic threshold; blue bracket, p=0.010, fig. 7F). DAMGO decreased inputs to inspiratory on-switch from 151 (130–154) to 101 (100–101)% of apneic threshold (p=0.031, fig. 7G, left), which was similar to the decrease from glutamate antagonists (146 (126–162) to 100 (100–100)%; blue bracket, p=0.965, fig. 7G). The discrepancy between the significant difference in effect on expiratory duration and no difference in effect on inputs to inspiratory on-switch may have been due to the fact that with very long respiratory phases, large differences in phase duration can be caused by only very small differences in inputs to phase duration (Appendix 1).
Fig. 7.
Microinjection of supraclinical concentrations of the mu-opioid agonist [D-Ala,2N-MePhe,4Gly-ol]-enkephalin (DAMGO, 100μM, 700nl) into the bilateral Parabrachial Nucleus/Kölliker-Fuse Complex severely depressed respiratory rate. We compared the effect size with data from 13 animals from our previous study using microinjections of the non-NMDA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX; 1 mM, 700nl) and NMDA receptor antagonist d(–)-2-amino-5-phosphonopentanoic acid (D-AP5; 5 mM, 700 nl)(9). Animals were selected to match control respiratory rate. A: Phrenic neurogram (PNG; a.u. [arbitrary units]) tracings from one individual animal. B-E: Pooled data for changes in respiratory rate and other respiratory parameters. Respiratory rate depression was similar between DAMGO (n=6) and NBQX/AP5 injection (n=13), as was the effect on peak phrenic activity. F-G: Values for inputs to inspiratory on- and off-switch were derived from the values for inspiratory and expiratory duration and are presented in % of apneic threshold with apneic threshold=100% (Appendix 1). Black brackets: The difference (“delta”) between DAMGO injection and control was tested against no change (Wilcoxon signed rank test). Blue brackets: Comparison of the “deltas” from DAMGO injection vs. control with NBQX/AP5 injection vs. control (Mann-Whitney rank sum test). Levels of significance below the critical p=0.025 are highlighted in red.
The prominent respiratory rate depression by DAMGO injection into the Parabrachial Nucleus/Kölliker-Fuse Complex that is similar to the effects of glutamate antagonists in scale and pattern (i.e., significant prolongation of inspiratory and expiratory duration) suggests that many of the Parabrachial Nucleus/Kölliker-Fuse Complex neurons that contribute to respiratory phase timing are opioid-sensitive. As described above (2.7.) this may only apply for animals with baseline respiratory rates between 20–37 bpm.
3.6. Control Studies: Effects of naloxone or artificial cerebrospinal fluid injections; potential desensitization to remifentanil
To rule out endogenous opioid receptor activation, in four animals, naloxone was microinjected into the bilateral Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex without remifentanil infusion. Naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex or preBötzinger Complex, resp., did not have any effect on respiratory rate (vs. control, p>0.999, or p>0.999, resp.; data not shown), inspiratory duration (p=0.625 or p=0.625, resp.), expiratory duration (p=0.250 and p=0.250, resp.), or peak phrenic activity (p=0.375 and p=0.250, resp.). At high concentrations (>100μM), naloxone can act as a γ-aminobutyric acid type A (GABAA) receptor antagonist (28). However, we did not observe any changes in respiratory rate with any naloxone injections into the Parabrachial Nucleus/Kölliker-Fuse Complex or preBötzinger Complex, i.e., respiratory nuclei, which are under GABAAergic control. Therefore, naloxone mediated GABAA receptor antagonism did not appear to be a confounder in our experiments.
Injection of artificial cerebrospinal fluid, which was used as solvent, into the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex did not affect respiratory rate (Parabrachial Nucleus/Kölliker-Fuse Complex vs. control, p=0.500; preBötzinger Complex vs. control, p>0.999; n=3, data not shown), inspiratory duration (p=0.250 and p=0.750, resp.), expiratory duration (p=0.250 and 0.500, resp.), or peak phrenic activity (p=0.500 and p=0.500, resp.).
Post hoc, we reviewed our entire experimental data for signs of desensitization to the remifentanil effect due to prolonged remifentanil infusion and repeated bolus injections. We found that even after long remifentanil infusions when multiple adjustments, i.e., increases or decreases, in infusion rate were required to achieve 50% respiratory rate depression, respiratory rate did not change during the 15 minutes of steady-state before the first naloxone injection. After “apneic” bolus injections of remifentanil that followed naloxone injections into the Parabrachial Nucleus/Kölliker-Fuse Complex or preBötzinger Complex, respiratory rate reliably returned to pre-bolus values (difference pre- to post-bolus rate 0 (−1, 1) bpm, n=26) including after the last remifentanil bolus injection in Cohort A, which was the third remifentanil bolus per animal (difference 0 (−1,1) bpm, n=8). Other authors have shown a decrease in respiratory rate depression during continuous remifentanil infusion starting after 90 minutes and resulting in only ~60% of the maximal depression after 300 minutes in freely behaving rabbits with variable PCO2 and decreasing sedation level (24). However, in our decerebrate rabbit model with controlled PCO2 and PO2, there was no obvious attenuation of the respiratory rate depression after prolonged remifentanil infusion and repeated bolus injections. We conclude that desensitization to the respiratory depressant effects of remifentanil was not a relevant confounding factor in our experiments. The complete recovery of respiratory rate to control values with intravenous naloxone injection at the end of the experiment confirmed that there was also no systematic decrease in respiratory rate due to a deterioration of the preparation.
Discussion
This study explored the contributions of the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex to opioid-induced respiratory depression in an acute, in vivo rabbit model. Remifentanil decreased respiratory rate by depressing inspiratory on-switch through effects in the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex. Remifentanil also depressed inspiratory off-switch through effects on the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex. However, “apneic” concentrations decreased inspiratory duration through an effect in the preBötzinger Complex. Sequential naloxone injection into the Kölliker-Fuse Nucleus, Parabrachial Nucleus, and preBötzinger Complex could not completely reverse the respiratory depression from “analgesic” remifentanil concentrations and even less so from “apneic” doses (Table 2). This suggests that opioids significantly depressed respiratory drive to the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex, and that the depression of drive reduced the activity of these areas especially at high opioid concentrations (fig. 8).
Fig. 8:
Schematic of opioid effects on respiratory rate and tidal volume. Tonic respiratory drive determines the activity of the Parabrachial Nucleus/ Kölliker-Fuse Complex, the preBötzinger Complex, and inspiratory premotor and motoneurons. Part of the drive that determines respiratory phase duration (blue) is relayed by the Parabrachial Nucleus/Kölliker-Fuse Complex, while other drive projects directly to the respiratory rhythm generator in the preBötzinger Complex. Drive that determines the magnitude of the tidal volume (green) is partially relayed through the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex but also directly projects to respiratory premotor neurons and phrenic motoneurons. Expiratory motoneurons were not recorded in this study. “Analgesic” opioid doses depress mostly Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex activity (bold red frames) and thus respiratory rate. The magnitude of the opioid effect that can be reversed in each area is presented in Table 2. Higher opioid doses directly affect respiratory drive and premotor and motoneurons, resulting in additional decrease in tidal volume.
Opioids depress drive to the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex
We used changes in inspiratory and expiratory duration with naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex or preBötzinger Complex to calculate the relative inputs to inspiratory on- and off-switch from these areas (Table 2). Two observations stood out: Naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex or preBötzinger Complex restored inputs more at “analgesic” than “apneic” remifentanil concentrations (Table 2), suggesting that the level of Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex activity that can be recovered with naloxone injection depends on respiratory drive to these areas, and that this drive is opioid-sensitive (fig. 8).
Secondly, naloxone reversal of the Parabrachial Nucleus/Kölliker-Fuse Complex did not always prevent apnea from the “apneic” remifentanil bolus, and reversal of the preBötzinger Complex rarely did. However, reversal of both areas together reliably prevented apnea, and respiratory rhythm persisted even after “very high” remifentanil bolus doses, albeit with decreased rate. This suggests that the inputs from both areas to inspiratory on-switch are additive and that respiratory drive to these areas is opioid-sensitive, but still sufficient to sustain respiratory rhythm even at “very high” remifentanil concentrations (3.4., fig. 6).
In a freely behaving mouse model, morphine dose-dependently depressed respiratory rate. Mu-opioid receptor deletion in the Kölliker-Fuse nucleus prevented respiratory rate depression by ~20% of baseline rate at every dose, and μ-opioid receptor deletion in the preBötzinger Complex did not reduce respiratory depression at doses ≥30mg/kg ((11), their fig. 3), suggesting that the increasing depression was due to a reduction in respiratory drive. Apneic doses were not tested. A similar study found no additional reduction of respiratory depression after μ-opioid receptor deletion in the preBötzinger Complex and Parabrachial Nucleus/Kölliker-Fuse Complex in the same animals, however, the study was likely underpowered ((13), their fig. 3). Species differences may exist between mammals, e.g., in dogs naloxone injection into the Parabrachial Nucleus fully reversed respiratory rate depression (5) while no reversal was observed in the preBötzinger Complex (4).
Potential sources of opioid-sensitive respiratory drive
In our decerebrate, hyperoxic and moderately hypercapnic preparation, both supratentorial “awake” drive (32) as well as carotid body inputs were eliminated. This left the prevailing hypercapnia (tissue CO2 tension) and the medullary and pontine arousal centers of the raphe as the main source of respiratory drive (for review, see (1)). Chemosensitive neurons in the retrotrapezoid nucleus, considered the main source and integrator of respiratory chemodrive (33, 34), were unaffected even by “apneic” morphine doses (35). However, in rats, injection of the opioid antagonist CTAP into the caudal medullary raphe reduced respiratory rate depression by intravenous DAMGO from 30% to 15% (36), making this area a promising candidate for future research.
Opioid effects on post-inspiratory activity
Vagal nerve recordings in rats display prominent post-inspiratory activity, which controls motor output to airway muscles (31, 37). It also allows gauging the contribution of post-inspiratory activity to respiratory phase timing. Volumetric mapping of the brainstem respiratory network showed highly synchronized activity during respiratory phase transitions in the areas relevant for phase timing (31). Prominent activity during inspiratory on- and off-switch was observed in the preBötzinger Complex and the main post-inspiratory – expiratory phase transition was in the dorsal respiratory group (31). In the in-situ rat model, vagal post-inspiratory activity was completely abolished by systemic opioids suggesting a mechanism for the observed increase in inspiratory duration (10). In rabbits, vagal activity is mostly inspiratory, and post-inspiratory activity – though present in the preBötzinger Complex – cannot be determined from vagal recordings. We thus limit our discussion of opioid effects to inspiratory-expiratory phase timing.
Potential neuronal targets for respiratory opioid effects
Opioids prolong expiratory duration, and apnea always results from a failure of inspiratory on-switch (1). In vitro, μ-opioid receptor agonists depressed >60% of Kölliker-Fuse Complex neurons (9, 11). In our preparation, DAMGO injection into the Parabrachial Nucleus/Kölliker-Fuse Complex resulted in severe depression of inspiratory on-switch (fig. 7), suggesting that opioids depressed neurons that promote inspiratory on-switch in the preBötzinger Complex (7). DAMGO also directly inhibited Dbx+ preBötzinger Complex neurons in medullary slices (38), i.e., pre-inspiratory and inspiratory neurons whose stimulation generates inspiratory bursts in vivo (39). Both mechanisms, depression of pontine inputs to the preBötzinger Complex as well as direct inhibition of preBötzinger Complex neurons results in prolonged expiratory duration.
DAMGO injection into the Parabrachial Nucleus/Kölliker-Fuse Complex increased inspiratory duration (fig. 7D), and naloxone in the Parabrachial Nucleus/Kölliker-Fuse Complex reversed the increase in inspiratory duration from systemic remifentanil, suggesting that remifentanil depressed inputs to preBötzinger Complex neurons that promote inspiratory off-switch (7, 8), e.g., SST+ post-inspiratory neurons (39). Naloxone injection into the preBötzinger Complex decreased inspiratory duration (fig. 3) as did μ-opioid receptor deletion in the preBötzinger Complex in mice (11), pointing to additional, direct inhibition of post-inspiratory neurons.
In contrast, at “apneic” remifentanil concentrations, naloxone injection solely into the preBötzinger Complex increased inspiratory duration (fig. 4). Opioids directly depressed inspiratory preBötzinger Complex neurons in vitro (29), and DAMGO injection into the preBötzinger Complex shortened inspiratory duration in rabbits in vivo (26, 40). We hypothesize that the moderate increase in inspiratory duration at “apneic” remifentanil doses (fig. 4) was the net effect of depressed pontine inputs to inspiratory off-switch and direct inhibition of preBötzinger Complex inspiratory neurons. All neuron types have been described in the preBötzinger Complex in multiple species in vivo (7, 39, 41–43), but opioid effects on individual neuron types must yet be systematically investigated.
Remifentanil effects on phrenic nerve motor output
We found that depression of peak phrenic activity did not closely correlate with changes in respiratory phase timing (fig. 5). After opioid reversal in the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex, “very high” remifentanil doses depressed phrenic motor output completely while rhythmic respiratory activity continued (fig. 6). Single neuron recordings in vivo showed no direct depression of respiratory premotor neurons at “analgesic” systemic opioid concentrations (26, 44) but at near-apneic fentanyl doses (44) doses. Direct depression of spinal motoneurons was observed in vitro at “clinical” DAMGO concentrations (100nM) (45). Direct depression of motor output from very high opioid doses may reduce the effectiveness of drugs designed specifically to stimulate respiratory rhythm.
Methodological considerations
Naloxone injections:
Modeling suggests that the spread of our injection volume (700nl) resulted in an effective naloxone concentration of 50μM (5% of 1mM barrel concentration) within a spherical radius of 1–1.2mm at 5 min after injection (22). We believe that this concentration was sufficient to fully antagonize the remifentanil effect in the entire Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex area at “analgesic” (~50nM (25)) and “apneic” plasma concentrations. We have discussed elsewhere that the neuronal population of the Parabrachial Nucleus/Kölliker-Fuse Complex that contributes to phase timing may be located between our injection sites, i.e., in the medial Parabrachial Nucleus (8, 46), which matches other studies (13). Projections from this area to the preBötzinger Complex were shown functionally and histologically (7, 47, 48). Pilot data showed an unchanged naloxone effect 2h after microinjection suggesting that sequential injections completely blocked the remifentanil effect in all areas. The lack of change in respiratory rate starting 3–5 minutes after naloxone injection indicated that the area of effective naloxone concentration did not increase after that point. In a subset of nine animals in the current study, naloxone injection 0.5mm caudal to the preBötzinger Complex caused minor increases in respiratory rate (1 (0–3) bpm) while more caudal injections did not, suggesting that our preBötzinger Complex injections did not reach the rostral ventral respiratory group.
Opioid dosing:
We chose a 50% depression of respiratory rate as surrogate for an “analgesic” remifentanil dose since veterinary (14) and respiratory studies (13, 24) showed a ~50% rate depression at opioid doses that suppressed pain responses in spontaneously breathing animals. These doses are likely higher than “analgesic” doses in humans where analgesics can be dosed to “acceptable” pain levels while animal studies generally measure complete lack of movement to pain stimulus. However, a 40–60% depression of minute ventilation was observed in human volunteers after 0.15 mg/kg (49) or 0.2 mg/kg (50) morphine, i.e., a dose sufficient to provide effective analgesia for patients after major surgery.
Conclusion
Opioid reversal in the Parabrachial Nucleus/Kölliker-Fuse Complex and preBötzinger Complex does not completely reverse opioid-induced respiratory depression, suggesting that depression of respiratory drive limits the activity that can be recovered in these areas. This mechanism must be taken into account during the development of drugs designed to stimulate the respiratory rhythm generator.
Supplementary Material
Summary statement:
Opioids dose–dependently depress respiratory rate through effects on the Parabrachial Nucleus/ Kölliker-Fuse Complex, the preBötzinger Complex, and excitatory drive to these areas. Motor output is also depressed through direct effects on inspiratory premotor and/or motoneurons.
Acknowledgements:
The authors thank Andrew Williams (Engineering Technician) for his outstanding support with the experimental setup.
Funding statement: This work was supported by the NIH (R01-GM112960, Dr. Stucke). The statistical analysis was aided by the CTSI Biostatistics Program with funding from the National Center for Advancing Translational Sciences, NIH, award number UL1TR001436. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Footnotes
Conflicts of interest: The authors declare no competing interests.
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