Contribution of the caudal medullary raphe to opioid induced respiratory depression

Abstract

Background: Opioid-induced respiratory depression can be partially antagonized in the preBötzinger Complex and Parabrachial Nucleus/Kölliker-Fuse Complex. We hypothesized that additional opioid antagonism in the caudal medullary raphe completely reverses the opioid effect.

Methods: In adult ventilated, vagotomized, decerebrate rabbits, we administrated remifentanil intravenously at "analgesic", "apneic", and "very high" doses and determined the reversal with sequential naloxone microinjections into the bilateral Parabrachial Nucleus/Kölliker-Fuse Complex, preBötzinger Complex, and caudal medullary raphe. In separate animals, we injected opioid antagonists into the raphe without intravenous remifentanil.

Results: Sequential naloxone microinjections completely reversed respiratory rate depression from "analgesic" and "apneic" remifentanil, but not "very high" remifentanil concentrations. Antagonist injection into the caudal medullary raphe without remifentanil independently increased respiratory rate.

Conclusions: Opioid-induced respiratory depression results from a combined effect on the respiratory rhythm generator and respiratory drive. The effect in the caudal medullary raphe is complex as we also observed local antagonism of endogenous opioid receptor activation, which has not been described before.

Keywords: Caudal medullary raphe; Opioid-induced respiratory depression; Parabrachial Nucleus/ kölliker-fuse complex; Respiratory phase timing; Respiratory rhythm generator; preBötzinger complex.

Figure 1,

A+B: Examples of the Phrenic Neurogram (a.u., arbitrary units) with AMPA injection into (A) the Caudal Medullary Raphe, the area comprised of the raphe pallidus and obscurus, and (B) the Rostral Medullary Raphe, which is located towards the caudal end of the facial nucleus. AMPA injection (black arrow) into the Caudal Medullary Raphe increased respiratory rate (A1, breaths/min, bpm) and peak phrenic activity (A2, %, normalized to control). Inspiratory duration was not changed (A3), but expiratory duration was decreased (A4). AMPA injection into the Rostral Medullary Raphe severely decreased respiratory rate (B1) and peak phrenic activity (B2), consistent with activation of the GABAergic neurons of the Raphe Magnus. Inspiratory duration was often variable (B3) while expiratory duration was increased (B4). C: Contour plots for the changes in respiratory parameters with AMPA injection, based on six animals. AMPA (70nl) was injected in the midline with stepsize 0.47mm rostro-caudal (0.5mm, corrected for an angle of 20° between brainstem and head holder) and 0.5mm ventro-dorsal, starting at the ventral limit of neuronal discharge. Values for percent change from baseline were aligned relative to the functionally identified preBötzinger Complex (preBötC) and the average plotted for an assumed preBötC location at 2.5mm rostral to obex (obex=0). An increase in respiratory rate was observed between 2mm caudal to and 1.5mm rostral to the preBötC. This area was considered the caudal medullary raphe (CMR, arrow) for study purposes. The area was rostral to the nucleus of the solitary tract (NTS) where AMPA injection caused a prompt increase in respiratory rate and peak phrenic activity and ~1.5mm caudal to the caudal end of the facial nucleus (7N). Scale: ≥40% (red) to ≤−40% (purple) from baseline, green: no change.

Figure 2:

Locations of the caudal end of the Caudal Medullary Raphe (lower), the preBötzinger Complex (preBötC, middle), and the caudal end of the facial nucleus (7N, upper) as identified by injection of fluorescent latex microspheres in four rabbits. The left panel shows diagrammatic hemisections of the brainstem at each rostro-caudal level with the individual injection sites superimposed in red squares. The right panels show the corresponding hemisections from one animal with immunoreactivity for 5-hydroxytryptophan (5HT), representing serotonergic neurons and choline acetyltransferase (ChAT), representing cholinergic (motor) neurons, superimposed over nuclear staining with DAPI. Red microspheres mark the injection sites. Injection protocols did not differentiate between raphe pallidus (RPa) and raphe obscurus (ROb). IO: inferior olive; py: pyramidal tract; 12N: nucleus hypoglossus; 10N: nucleus nervi vagi; NA: nucleus ambiguus; RMg: raphe magnus. Bar = 1mm. The inserts show zoomed images of the injection sites. Bar = 0.25mm.

Figure 3:

Injection sequence for intravenous remifentanil and local naloxone microinjections. The initial intravenous remifentanil bolus (red arrow) was chosen to cause apnea for more than 60s. Once respiratory activity returned, the remifentanil infusion was started (red line) until steady-state respiratory rate depression of 50%. This dose-rate was continued unchanged throughout the entire injection sequence. For Cohort A (n=11), naloxone (dark blue arrows) was microinjected into the bilateral Kölliker-Fuse Nucleus, Parabrachial Nucleus, preBötzinger Complex (including premotor neurons), and Caudal Medullary Raphe. Apneic remifentanil boluses (red arrows) were given after naloxone injections into the pons and preBötzinger Complex, and after subsequent naloxone injection into the raphe. After each “apneic” remifentanil bolus, we awaited complete recovery of the respiratory rate to prebolus levels before continuing the protocol. The “very high” remifentanil bolus was administered shortly after the last “apneic” bolus to maximize effect (light blue arrow). In Cohort B (n=10), naloxone was microinjected in the reverse order. In Cohort C, naloxone was microinjected only into the preBötzinger Complex including the premotor neurons and caudal medullary raphe. This protocol was truncated after five animals.

Figure 4:

Analgesic remifentanil concentrations. Bilateral naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex (PBN/KF), the preBötzinger Complex including the premotor neurons (preBötC), and the Caudal Medullary Raphe (raphe) completely reversed the respiratory rate depression from intravenous remifentanil at analgesic concentrations (50% respiratory rate depression). A: Phrenic neurogram tracings during control conditions and sequential drug injections in one rabbit. (B-E) Pooled data for measured respiratory parameters and (F,G) values for the inputs to inspiratory off-switch and on-switch, derived from inspiratory and expiratory duration. Data are presented separately for Cohort A (left panels in white, n=11), Cohort B (center panels in grey, n=10), and Cohort C (right panels in blue, n=5). Bars indicate that the difference between values from two subsequent injections or control was tested against no change (Wilcoxon signed rank test). The levels of significance below the critical P=0.0125 are highlighted in red. The dotted line indicates the threshold value, set at 1 in our model, which the sum of inputs must exceed to result in phase switch (see Appendix 1 in (1)).

Figure 4:

Analgesic remifentanil concentrations. Bilateral naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex (PBN/KF), the preBötzinger Complex including the premotor neurons (preBötC), and the Caudal Medullary Raphe (raphe) completely reversed the respiratory rate depression from intravenous remifentanil at analgesic concentrations (50% respiratory rate depression). A: Phrenic neurogram tracings during control conditions and sequential drug injections in one rabbit. (B-E) Pooled data for measured respiratory parameters and (F,G) values for the inputs to inspiratory off-switch and on-switch, derived from inspiratory and expiratory duration. Data are presented separately for Cohort A (left panels in white, n=11), Cohort B (center panels in grey, n=10), and Cohort C (right panels in blue, n=5). Bars indicate that the difference between values from two subsequent injections or control was tested against no change (Wilcoxon signed rank test). The levels of significance below the critical P=0.0125 are highlighted in red. The dotted line indicates the threshold value, set at 1 in our model, which the sum of inputs must exceed to result in phase switch (see Appendix 1 in (1)).

Figure 5:

“Apneic” remifentanil concentrations. Bilateral naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex (PBN/KF), the preBötzinger Complex including the premotor neurons (preBötC), and the Caudal Medullary Raphe (raphe) completely prevented the respiratory rate depression from an intravenous remifentanil bolus that caused apnea >60sec under control conditions. A: Phrenic neurogram tracings from the same rabbit shown in figure 4 shows that sequential naloxone injections increasingly reduced respiratory rate depression from the “apneic” bolus. (B-E) Pooled data for measured respiratory parameters and (F,G) values for the inputs to inspiratory off-switch and on-switch, derived from inspiratory and expiratory duration. Data are presented separately for Cohort A (left panels in white, n=10), Cohort B (center panels in grey, n=10), and Cohort C (right panels in blue, n=4). Bars indicate that the difference between values from two subsequent injections or control was tested against no change (Wilcoxon signed rank test). The levels of significance below the critical P=0.0167 are highlighted in red. The dotted line indicates the threshold value, set at 1 in our model, which the sum of inputs must exceed to result in phase switch (see Appendix 1 in (1)).

Figure 5:

“Apneic” remifentanil concentrations. Bilateral naloxone injection into the Parabrachial Nucleus/Kölliker-Fuse Complex (PBN/KF), the preBötzinger Complex including the premotor neurons (preBötC), and the Caudal Medullary Raphe (raphe) completely prevented the respiratory rate depression from an intravenous remifentanil bolus that caused apnea >60sec under control conditions. A: Phrenic neurogram tracings from the same rabbit shown in figure 4 shows that sequential naloxone injections increasingly reduced respiratory rate depression from the “apneic” bolus. (B-E) Pooled data for measured respiratory parameters and (F,G) values for the inputs to inspiratory off-switch and on-switch, derived from inspiratory and expiratory duration. Data are presented separately for Cohort A (left panels in white, n=10), Cohort B (center panels in grey, n=10), and Cohort C (right panels in blue, n=4). Bars indicate that the difference between values from two subsequent injections or control was tested against no change (Wilcoxon signed rank test). The levels of significance below the critical P=0.0167 are highlighted in red. The dotted line indicates the threshold value, set at 1 in our model, which the sum of inputs must exceed to result in phase switch (see Appendix 1 in (1)).

Figure 6:

“Very high” remifentanil concentrations. A: After naloxone injection into the bilateral Parabrachial Nucleus/Kölliker-Fuse Complex, preBötzinger Complex including premotor neurons, and the Caudal Medullary Raphe, remifentanil was injected intravenously until apnea or to a maximal dose of 100 mcg. In 19 animals, the bolus caused a significant change in all parameters (Wilcoxon signed rank test, critical P<0.05). B: To determine whether the effect of “very high” remifentanil was due to insufficient antagonism of above brainstem regions, we plotted the difference between respiratory rate after the “very high” remifentanil bolus and baseline respiratory rate versus the difference from baseline for “analgesic” (black) and “apneic” (red) remifentanil concentrations at the end of the naloxone injection sequence. Values <0 indicate a decreased respiratory rate compared to baseline, i.e., −100% indicates apnea. Values >0 indicate an increased respiratory rate compared to baseline, which was not infrequently seen after naloxone injection into the raphe. Linear regression analysis showed little correlation between the “very high” remifentanil effect and level of reversal of “analgesic” and “apneic” remifentanil concentrations, suggesting that the naloxone injections correctly antagonized the study areas and that “very high” remifentanil concentrations depressed other areas of respiratory drive.

Figure 7:

Opioid antagonist injections into the Caudal Medullary Raphe without systemic remifentanil. Naloxone injection (n=7, left) and, in a separate set of animals, sequential injections of CTAP (n=7, middle, light grey) and subsequently naltrindole (right, dark grey) significantly increased respiratory rate. To illustrate that this effect occurred over the entire extent of the Caudal Medullary Raphe, we display the respiratory parameters at baseline (black), after injections into the area caudal to the preBötC level (green), at the preBötC level (brown), and rostral to the preBötC level (red). Statistical comparisons were performed solely between baseline and the values at the end of the injection sequence (red) for naloxone and CTAP, and between the last CTAP injection and end of the naltrindole injection sequence for naltrindole. Wilcoxon-signed-rank test, critical P=0.017.

Figure 8:

Pooled data for injections of the mu-opioid receptor agonist DAMGO into the caudal medullary raphe in seven animals. DAMGO depressed respiratory rate through an increase in inspiratory and expiratory phase duration. The effect was reversed by intravenous (IV) naloxone. This shows that endogenous opioids cause only submaximal receptor activation in the raphe. Wilcoxon-signed-rank test, critical P<0.05.

Figure 9:

Pooled data for the changes (delta) in inputs to (A) the inspiratory off-switch (derived from the inspiratory duration) and (B) inspiratory on-switch (derived from the expiratory duration) with naloxone injections into the Caudal Medullary Raphe (raphe) or the respiratory rhythm generator, i.e., the Parabrachial Nucleus/Kölliker Fuse Complex (PBN/KF) plus preBötzinger Complex (preBötC). Deltas are compared between naloxone injections without remifentanil infusion (data from section 3.4., white boxes) or at “analgesic” (section 3.1.) or “apneic” systemic remifentanil concentrations (section 3.2.). Deltas from different cohorts (naloxone injection orders) were pooled for matching injections (same location and remifentanil concentration) when they were not statistically different. Comparison of inputs to inspiratory off- and on-switch showed no difference between naloxone injections at different remifentanil concentrations (black bracket). C: Statistical comparisons between deltas from different cohorts for matching injections (see above) mostly showed no significant differences, and data were thus pooled and used for the analyses in A and B. Inputs to inspiratory on-switch during “apneic” remifentanil concentrations increased more with naloxone injection into the PBN/KF+preBötC after prior naloxone injection into the raphe and are displayed separately (B, blue bracket, p<0.001). *: Mann-Whitney-U test for two comparisons, ^: Kruskal-Wallis test for three comparisons.