Dose-dependent Respiratory Depression by Remifentanil in the Rabbit Parabrachial Nucleus/Kölliker-Fuse Complex and Pre-Bötzinger Complex

Abstract

Background: Recent studies showed partial reversal of opioid-induced respiratory depression in the pre-Bötzinger complex and the parabrachial nucleus/Kölliker-Fuse complex. The hypothesis for this study was that opioid antagonism in the parabrachial nucleus/Kölliker-Fuse complex plus pre-Bötzinger complex completely reverses respiratory depression from clinically relevant opioid concentrations.

Methods: Experiments were performed in 48 adult, artificially ventilated, decerebrate rabbits. The authors decreased baseline respiratory rate ~50% with intravenous, "analgesic" remifentanil infusion or produced apnea with remifentanil boluses and investigated the reversal with naloxone microinjections (1 mM, 700 nl) into the Kölliker-Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex. In another group of animals, naloxone was injected only into the pre-Bötzinger complex to determine whether prior parabrachial nucleus/Kölliker-Fuse complex injection impacted the naloxone effect. Last, the µ-opioid receptor agonist [d-Ala,2N-MePhe,4Gly-ol]-enkephalin (100 μM, 700 nl) was injected into the parabrachial nucleus/Kölliker-Fuse complex. The data are presented as medians (25 to 75%).

Results: Remifentanil infusion reduced the respiratory rate from 36 (31 to 40) to 16 (15 to 21) breaths/min. Naloxone microinjections into the bilateral Kölliker-Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex increased the rate to 17 (16 to 22, n = 19, P = 0.005), 23 (19 to 29, n = 19, P < 0.001), and 25 (22 to 28) breaths/min (n = 11, P < 0.001), respectively. Naloxone injection into the parabrachial nucleus/Kölliker-Fuse complex prevented apnea in 12 of 17 animals, increasing the respiratory rate to 10 (0 to 12) breaths/min (P < 0.001); subsequent pre-Bötzinger complex injection prevented apnea in all animals (13 [10 to 19] breaths/min, n = 12, P = 0.002). Naloxone injection into the pre-Bötzinger complex alone increased the respiratory rate to 21 (15 to 26) breaths/min during analgesic concentrations (n = 10, P = 0.008) but not during apnea (0 [0 to 0] breaths/min, n = 9, P = 0.500). [d-Ala,2N-MePhe,4Gly-ol]-enkephalin injection into the parabrachial nucleus/Kölliker-Fuse complex decreased respiratory rate to 3 (2 to 6) breaths/min.

Conclusions: Opioid reversal in the parabrachial nucleus/Kölliker-Fuse complex plus pre-Bötzinger 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.

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.

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.

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.

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.

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.

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 (–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.

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.

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.