Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2026 Mar 12.
Published in final edited form as: J Pharmacol Exp Ther. 2025 May 27;392(7):103616. doi: 10.1016/j.jpet.2025.103616

Xylazine exacerbates fentanyl-induced respiratory depression and bradycardia

Catherine Demery 1, Sierra C Moore 1, Erica S Levitt 1,2, Jessica P Anand 1,*, John R Traynor 1,3,*
PMCID: PMC12977009  NIHMSID: NIHMS2142779  PMID: 40543302

Abstract

Fatal opioid overdoses in the United States have nearly tripled during the past decade, with greater than 92% involving a synthetic opioid like fentanyl. Fentanyl potently activates the μ-opioid receptor to induce both analgesia and respiratory depression. The danger of illicit fentanyl has recently been exacerbated by adulteration with xylazine, an α2-adrenergic receptor agonist typically used as a veterinary anesthetic. In 2023, over a 1000% increase in xylazine-positive overdoses was reported in some regions of the United States. Xylazine has been shown to potentiate the lethality of fentanyl in mice, yet a mechanistic underpinning for this effect has not been defined. Herein, we evaluate fentanyl, xylazine, and their combination in whole-body plethysmography (to measure respiration) and pulse oximetry (to measure blood oxygen saturation and heart rate) in male and female CD-1 mice. We show that xylazine decreases breathing rate more than fentanyl by increasing the expiration time. In contrast, fentanyl primarily reduces breathing by inhibiting inspiration, and xylazine exacerbates these effects. Fentanyl but not xylazine decreased blood oxygen saturation, and when combined, xylazine did not change the maximum level of fentanyl-induced hypoxia. Xylazine also reduced heart rate more than fentanyl at higher doses. Finally, loss in blood oxygen saturation correlated with the frequency of fentanyl-induced severe apneas, but not breathing rate. Together, these findings provide insight into how the addition of xylazine to illicit fentanyl may increase the risk of overdose.

Keywords: Opioid overdose, Plethysmography, Fentanyl, Xylazine, Respiratory depression

1. Introduction

Fatal opioid overdoses in the United States have drastically increased over the past decade, exceeding 82,000 in 2023. Over 92% of these overdoses involve a synthetic opioid such as fentanyl (https://www.cdc.gov/nchs/nvss/vsrr/drug-overdose-data.htm). Fentanyl, along with other illicit and clinically important opioids, binds to the μ-opioid receptor to induce analgesia as well as the life-threatening effect of respiratory depression. Fentanyl is more dangerous than classical opioids like morphine for reasons including greater potency at the μ-opioid receptor, increased lipophilicity that facilitates rapid blood–brain barrier penetration, and induction of respiratory muscle rigidity (Hill et al, 2020; Haouzi and Tubbs, 2022; Kelly et al, 2023; Marchette et al, 2023). The danger of illicit fentanyl has recently been exacerbated by the replacement of less harmful adulterants (ie, mannitol) with xylazine, an α2-adrenergic receptor (α2AR) agonist used as a veterinary anesthetic (Leconte and Sethi, 2023; Spencer et al, 2023; Papudesi et al, 2024). Activation of α2AR autoreceptors leads to central nervous system depression that can increase the likelihood of overdose when combined with opioids. Furthermore, xylazine is not used in humans due to its induction of severe bradycardia (Ruiz-Colón et al, 2014; Papudesi et al, 2024). The codetection of fentanyl with xylazine has risen starkly over the past few years, both in Drug Enforcement Agency seizures and postmortem toxicology reports (https://www.nflis.deadiversion.usdoj.gov/home.xhtml), leading the White House to declare fentanyl-xylazine combinations an emerging threat to the United States (Gupta, 2023).

Xylazine is rarely abused independently of opioids, with over 99% of xylazine detection occurring in the presence of fentanyl (Spencer et al, 2023). Yet, 89% of illicit opioid users who are aware of potential xylazine adulteration do not want xylazine in their fentanyl samples (Hochheimer et al, 2024), likely due to the risk of xylazine-induced skin ulcers, which are hypothesized to result from peripheral α2AR activation (Papudesi et al, 2024). Information regarding the ratio of fentanyl-to-xylazine in illicit samples is lacking, but postmortem toxicology studies have reported an approximate 1:1 median ratio (Hays et al, 2024) or a 1:4 mean ratio (Truver et al, 2024) of fentanyl to xylazine. In mice, xylazine has previously been shown to potentiate the lethality of fentanyl at ratios of 1:5 and 1:10 (fentanyl-to-xylazine) (Acosta-Mares et al, 2023). Furthermore, isobolographic analysis of fentanyl- and xylazine-induced lethality in mice showed that the two drugs synergize when combined and that naloxone (an opioid receptor antagonist) but not yohimbine (an α2AR antagonist) was able to reverse the lethal effect of a fentanyl-xylazine combination (Smith et al, 2023). Xylazine was also shown to prolong fentanyl-induced brain hypoxia (Choi et al, 2023), an effect that was fully rescued by coadministration of naloxone together with the α2AR antagonist, atipamezole (Choi et al, 2024). Finally, other preclinical studies have found that xylazine does not increase the rewarding effects of fentanyl but can alter symptoms of withdrawal (Acosta-Mares et al, 2023; Khatri et al, 2024; Sadek et al, 2024).

In this study, we investigated whether xylazine alters respiratory depression, the lethal effect of fentanyl. We considered which components of the respiratory process were affected using whole-body plethysmography and measured vital signs using pulse oximetry in male and female CD-1 mice. Fentanyl was held constant at a dose (3.2 mg/kg i.p.) that produces significant but not maximal respiratory depression. Fentanyl was combined with xylazine at ratios of 1:1 up to 1:10, to best reflect the relative quantities of both drugs identified in human overdose postmortem samples. Changes in breathing rate, inspiration, expiration, apneas, blood oxygen saturation, and heart rate were evaluated. We found that high doses of xylazine reduced breathing rate and heart rate more than fentanyl, and in combination, the effect was not different from xylazine alone. As expected, fentanyl decreased blood oxygen levels, whereas xylazine had no effect despite reducing breathing rate to a greater extent than fentanyl. Reductions in blood oxygen levels correlated with the frequency of fentanyl-induced severe apneas. These findings shed light on the mechanisms by which the addition of xylazine to illicit fentanyl may increase the risk of overdose in humans.

2. Materials and methods

2.1. Animals

Male and female CD-1 mice aged 6–12 weeks were used for all experiments (n = 84 total). Mice were either bred in-house at the University of Michigan or purchased from Envigo. All experiments were performed during the light phase of the 12-hour light/dark cycle on which mice were maintained. Mice were housed by sex in groups of 2–5 in clear polypropylene home cages with corncob bedding and free access to food, water, and enrichment. All studies followed the Guide for the Care and Use of Laboratory Animals (National Research Council Committee, 2011) and were approved by the University of Michigan Institutional Animal Care and Use Committee. Sample sizes of n = 6 animals per group were determined prior to data collection. Numbers of animals of each sex (male or female; referring to a set of biological attributes) are listed for each experiment.

2.2. Drugs

Fentanyl powder (Cayman Chemical Company) was dissolved in saline. Xylazine (Cayman Chemical Company) powder was dissolved in DMSO, which was then diluted in castor oil followed by saline to achieve a 1:1:8 ratio of DMSO, castor oil, and saline, respectively. For the combined doses of fentanyl plus xylazine, fentanyl was prepared in the saline component of the diluent and added last to the preparation of xylazine in DMSO and castor oil to achieve the desired drug concentrations. The vehicle contained 1:1:8 proportions of DMSO, castor oil, and saline, respectively. Each animal received only one injection at 10 mL/kg of body weight.

2.3. Whole-body plethysmography

Respiratory measures were recorded using vivoFlow whole-body plethysmography chambers by SCIREQ. Chambers were supplied with 0.5 L/min of medical-grade air USP (Cryogenic Gases). All mice were given 30 minutes to acclimate to the chambers the day prior to test day. On test day, weights and temperatures (Braun digital infrared thermometer) for all mice were recorded. Body temperature (35–37 °C), chamber temperature (20–22 °C), and chamber humidity (10%) were used in volume calculations. Awake, free-moving mice were then placed in chambers for 10 minutes of habituation (data not saved) and 20 minutes of baseline recording prior to removal from chambers to administer an intraperitoneal injection of xylazine, fentanyl plus xylazine, or vehicle. Mice were returned to the chambers for 60 minutes of post-treatment recording. Emka Technologies iox 2.10.8.6 software was used to collect data in 30-second intervals, which were then collapsed into 5-minute bins for analysis. Apneas and expiratory flow 50 (EF50) values were calculated after exporting data as individual breaths. The same vehicle group is shown in xylazine and fentanyl plus xylazine time course graphs. Equal numbers of male (n = 3) and female (n = 3) mice were used for all groups, with the following exceptions: fentanyl + 10 mg/kg xylazine, n = 4 females and n = 2 males; fentanyl + 32 mg/kg xylazine, n = 4 females and n = 2 males.

2.4. Pulse oximetry

Blood oxygen saturation and heart rate were measured using the MouseOx Plus Small Animal Pulse Oximeter from Starr Life Sciences. Awake, free-moving animals were habituated to enclosures and practice collars for 60 minutes prior to experiment start. Practice collars were exchanged for MouseOx collars, and baseline recordings were taken for 60 minutes before mice were removed from enclosures to administer an intraperitoneal injection of xylazine, fentanyl plus xylazine, or vehicle. Data were collected for 60 minutes after injection and collapsed into 5-minute bins for analysis. The same vehicle group is shown in xylazine and fentanyl plus xylazine time course graphs. Equal numbers of male (n = 3) and female (n = 3) mice were used for all groups except the xylazine 32 mg/kg group, which contained n = 5 females and n = 1 male.

2.5. Data analysis

Data from male and female mice in a given treatment group were combined for analysis, as no obvious differences were observed, although we were not statistically powered to detect differences.

For breathing rate, inspiration time (Ti) and expiration time (Te), peak inspiratory flow (PIF) and peak expiratory flow (PEF), blood oxygen saturation, and heart rate, data were averaged into 5-minute bins for analysis. Data are presented as % baseline of each mouse during the 20-minute period prior to injection (t= −20 to 0 minutes). Raw baseline data by treatment group are shown in Supplemental Table 1; group averages were compared via one-way ANOVA. All groups were compared from t = 0–60 minutes after injection via two-way ANOVA (or a mixed effect model if any values were missing). If the two-way ANOVA main effect of treatment was P < .05, average group values from t = 10–15 minutes were then compared via one-way ANOVA with Tukey’s post hoc test. GraphPad Prism v.10 (GraphPad) was used for all statistical analyses.

For apneas and EF50, data were exported as individual breaths. Frequency of apneas was calculated in Microsoft Excel using the following criteria: Apnea=Te>2[Avg:Tepost-injection];SevereApnea=Te>2[Avg:Ti+Tepost-injection]. The average lengths of Ti and Te from t = 0–60 minutes were used to define an apnea and calculated for each mouse individually. Outliers were identified via nonlinear regression and outlier removal method (Q = 1%) and removed (Motulsky and Brown, 2006). The average number of apneas per treatment group from t = 0–15 minutes after injection was compared via one-way ANOVA with Tukey’s post hoc test, if the main effect was significant at P < .05. Pearson correlation between blood oxygen saturation (t = 10–15 minutes) and total or severe apneas (t = 0–15 minutes) was computed. EF50 values were calculated as % baseline of each mouse during the baseline period (t = −20 to 0 minute). Average EF50 values among groups were compared from t = 0–15 minutes or t = 0–60 minutes after injection using one-way ANOVA with Tukey’s post hoc test if the main effect was significant with P < .05. GraphPad Prism v.10 was used for all statistical analyses except linear trend lines and SEM for correlation analyses, which were calculated in Excel.

3. Results

3.1. Fentanyl combined with xylazine reduces breathing rate to a greater degree than 3.2 mg/kg fentanyl alone

We first considered how fentanyl, xylazine, and their combination affected breathing rate using whole-body plethysmography (Fig. 1A). The dose of 3.2 mg/kg fentanyl was chosen because it produces significant but nonmaximal effects on respiration. Xylazine was combined at a 1:1 up to 10:1 ratio with fentanyl. Given the rapid onset of fentanyl-induced respiratory depression, we compared the average respiration rate of the different treatment groups from 10 to 15 minutes after injection (Fig. 1B, bar graph). All doses of xylazine (3.2, 10, and 32 mg/kg) drastically reduced breathing rate relative to vehicle (P < .0001). Fentanyl (3.2 mg/kg) also reduced breathing rate relative to vehicle (P < .0001) but to a lesser extent than the 32 mg/kg dose of xylazine (P < .05). The combination of fentanyl and 3.2, 10, or 32 mg/kg xylazine further reduced breathing rate relative to fentanyl alone (P < .01, P < .0001, and P < .0001, respectively) but did not significantly differ from the corresponding dose of xylazine alone (Fig. 1B). However, as shown in Fig. 1C, fentanyl, xylazine, and their combination induce different breathing patterns that are not captured by the breathing rate metric.

Fig. 1.

Fig. 1.

Open in a new tab

Fentanyl combined with xylazine reduces breathing rate more than 3.2 mg/kg fentanyl alone. (A) Graphical representation of study design. Breathing rate of male and female CD-1 mice (n = 6 per group) was recorded using vivoFlow whole-body plethysmography chambers supplied with air according to the timeline. (B) Effect of different doses of xylazine or xylazine-fentanyl combinations on breathing rate. Arrows and dotted lines indicate intraperitoneal injection (t = 0). Breathing rate was compared among all groups via two-way ANOVA (t = 0–60 minutes; main effect of treatment, F(7, 40) = 31.93, P < .0001; main effect of time, F(4.192, 167.7) = 75.42, P < .0001; main effect of subject, F(40, 440) = 6.868, P < .0001; time × treatment interaction, F(77, 440) = 2.391, P < .0001). Average values by treatment group from t = 10–15 minutes after injection were compared via one-way ANOVA (F(7, 40) = 37.26, P < .0001) with Tukey’s post hoc test: ****P < .0001 versus vehicle; ⊹P < .05, ⊹⊹P < .01, ⊹⊹⊹⊹P < .0001 versus fentanyl alone. Females are shown as circles and males as squares. (C) Representative breathing traces 10–15 minutes after injection of xylazine, fentanyl, or their combination compared with saline.

3.2. Xylazine exacerbates fentanyl-induced changes in inspiration

As opioids primarily inhibit brainstem regions that control the process of inspiration (Sun et al, 2019; Bachmutsky et al, 2020; Bateman and Levitt, 2023; Bateman et al, 2023), we next evaluated how xylazine alters fentanyl-induced changes in Ti and PIF, as depicted in Fig. 2A. Relative to the vehicle, xylazine did not affect Ti at any dose tested (Fig. 2B). As expected, fentanyl alone and in combination with xylazine (3.2, 10, or 32 mg/kg) increased Ti compared to vehicle (P < .0001, t = 10–15 minutes after injection; Fig. 2B, bar graph). Only fentanyl combined with 10 mg/kg xylazine produced a significant increase in Ti relative to fentanyl alone (P < .05). Similarly, xylazine did not affect PIF (Fig. 2C), whereas fentanyl alone and in combination with xylazine decreased PIF relative to vehicle (P < .05 and P < .0001, t = 10–15 minutes after injection; Fig. 2C, bar graph). The combination of fentanyl and xylazine (3.2, 10, or 32 mg/kg) reduced PIF below that of fentanyl (P < .01, P < .01, and P < .05, respectively), potentially suggesting a synergistic effect on PIF (Fig. 2C). Taken together, these results show that xylazine lacks an independent effect on inspiration but enhances fentanyl-induced changes in inspiration.

Fig. 2.

Fig. 2.

Open in a new tab

Xylazine exacerbates fentanyl-induced changes in inspiration. (A) Graphical representation of study design. Inspiratory parameters of male and female CD-1 mice (n = 6 per group) were recorded using vivoFlow whole-body plethysmography chambers supplied with air according to the timeline. (B) Effects of different doses of xylazine or fentanyl-xylazine combinations on inspiration time. Arrows and dotted lines indicate intraperitoneal injection point. Time course of inspiration time (Ti) from 0 to 60 minutes by the treatment group. Bar graph displays the group values from t = 10–15 minutes. Females are shown as circles and males as squares. Inspiratory rates were compared via two-way ANOVA (main effect of treatment, F(7, 40) = 23.82, P < .0001; main effect of time, F(4.868, 194.7) = 24.41, P < .0001; main effect of subject, F(40, 440) = 26.01, P < .0001; time × treatment interaction, F(77, 440) = 2.841, P < .0001). Average values from t = 10–15 minutes after injection were compared via one-way ANOVA (F(7, 40) = 31.99, P < .0001) with Tukey’s post hoc test. (C) Effects of different doses of xylazine or fentanyl–xylazine combinations on PIF over time were compared by two-way ANOVA main effect of treatment, F(7, 40) = 22.59, P < .0001; main effect of time, F(4.079, 163.2) = 59.96, P < .0001; main effect of subject, F(40, 440) = 31.23, P < .0001; time × treatment interaction, F(77, 440) = 3.156, P < .0001. Bar graphs display the group values from t = 10–15 minutes (females as circles, males as squares). Average values from t = 10–15 minutes after injection were compared via one-way ANOVA (F(7, 40) = 25.36, P < .0001) with Tukey’s post hoc test. *P < .05, ****P < .0001 versus vehicle; ⊹P < .05, ⊹⊹P < .01 versus fentanyl alone.

3.3. Xylazine produces robust effects on expiration that differ from those induced by fentanyl

The effects of fentanyl and xylazine on Te and PEF, illustrated in Fig. 3A, were also considered. We found that xylazine drastically increased Te, while fentanyl had no effect (Fig. 3B). From 10 to 15 minutes after injection, all doses of xylazine produced significant increases in Te relative to vehicle (3.2 mg/kg, P < .01; 10 mg/kg, P < .001; 32 mg/kg, P < .01), as did fentanyl in combination with 10 or 32 mg/kg xylazine (P < .01 and P < .0001, respectively). Fentanyl did not increase the effect of any dose of xylazine on Te. The large effect of xylazine on Te accounts for the xylazine-driven reduction in breathing rate observed in Fig. 1B. Xylazine decreased PEF relative to control (Fig. 3C) at all doses tested (P < .01). Fentanyl significantly increased PEF relative to control (P < .0001). When fentanyl was combined with xylazine, PEF was significantly decreased relative to fentanyl alone (P < .0001, t = 10–15 minutes after injection) to levels comparable to xylazine alone. We observed similar findings for EF50, the flow rate when half of the volume of air in the lungs has been expired (Fig. 3D). Fentanyl increased EF50 (P < .0001), whereas xylazine at 3. 2 mg/kg (P < .05 for t = 0–15 minutes; P < .001 for t = 0–60 minutes), 10 mg/kg (P < .01 for t = 0–15 minutes; P < .001 for t = 0–60 minutes), and 32 mg/kg (P < .0001 for t = 0–15 minutes and t = 0–60 minutes) decreased this parameter relative to control. As with PEF, the combination of fentanyl and either 3.2, 10, or 32 mg/kg xylazine yielded a significant reduction in EF50 relative to fentanyl alone (P < .0001 for all at t = 0–15 minutes and t = 0–60 minutes) to levels comparable to control from 0 to 15 minutes after injection. Only the 32 mg/kg dose of xylazine produced a significantly lower EF50 alone versus in com- bination with fentanyl from t = 0–15 minutes (P < .01) and t = 0–60 minutes (P < .05). In summary, xylazine greatly affects the process of expiration and reverses fentanyl-induced increases in expiratory flow.

Fig. 3.

Fig. 3.

Open in a new tab

Xylazine produces robust effects on expiration that differ from those induced by fentanyl. (A) Graphical representation of study design. Expiratory parameters in male and female CD-1 mice (n = 6 per group) were recorded using vivoFlow whole-body plethysmography chambers supplied with air according to the timeline. Expiratory parameters were compared independently among groups via two-way ANOVA or mixed effect model (t = 0–60 minutes). The average values by treatment group from t = 10–15 minutes after injection were compared via one-way ANOVA (P < .0001 for both parameters) with Tukey’s post hoc test (P-values shown in bar graphs). (B) Time course of Te from 0 to 60 minutes by treatment group (mixed-effects model: main effect of treatment, F(7, 40) = 7.490, P < .0001; main effect of time, F(3.993, 159.3) = 9.751, P < .0001; time × treatment interaction, F(77, 439) = 1.953, P < .0001). Arrows and dotted lines indicate intraperitoneal injection point. The bar graph displays the group values from t = 10–15 minutes (average ± SEM). Females are shown as circles and males as squares. (C) Time course of PEF from 0 to 60 minutes by treatment group (two-way ANOVA: main effect of treatment, F(7, 40) = 20.12, P < .0001; main effect of time, F(6.303, 252.1) = 164.6, P < .0001; main effect of subject, F(40,600) = 11.05, P < .0001; time × treatment interaction, F(105, 600) = 6.692). Arrows and dotted lines indicate intraperitoneal injection point. The bar graph displays the group values from t = 10–15 minutes. Females are shown as circles and males as squares. (D) EF50 (females shown as circles and males as squares) values by treatment group from t = 0–15 minutes (filled bar graph, one-way ANOVA: F(7,37) = 25.91, P < .0001) or t = 0–60 minutes after injection (open bar graph, one-way ANOVA: F(7,37) = 36.26, P < .0001) with Tukey’s post hoc test. *P < .05, **P < .01, ***P < .001, ****P < .0001 versus vehicle; ⊹⊹P < .01, ⊹⊹⊹P < .001, ⊹⊹⊹⊹P < .0001 versus fentanyl alone.

3.4. Fentanyl but not xylazine decreases blood oxygen saturation and both drugs lower heart rate

We also examined how fentanyl and xylazine affect blood oxy- gen levels using pulse oximetry (Fig. 4A) because depleted blood oxygen levels can be fatal. Percent blood oxygen saturation is shown for xylazine, fentanyl, and fentanyl combined with xylazine (Fig. 4B). Xylazine had no impact on blood oxygen saturation alone, whereas fentanyl or fentanyl plus xylazine reduced blood oxygen levels compared with vehicle at t = 10–15 minutes (P < .0001, Fig. 4B, bar graph). There were no differences in the maximal reduction in blood oxygen saturation (t = 10–15 minutes) between fentanyl and either combination of fentanyl plus xylazine. We also compared the area under the curve from t = 0–60 minutes, which again showed no effect of xylazine alone, whereas fentanyl alone or in combination with 3.2 or 32 mg/kg xylazine comparably reduced oxygen saturation relative to vehicle (P < .0001, Fig. 4D). These results show that doses of 3.2–32 mg/kg xylazine do not contribute to blood oxygen saturation loss as measured by pulse oximetry.

Fig. 4.

Fig. 4.

Open in a new tab

Fentanyl but not xylazine decreases blood oxygen saturation and both drugs lower heart rate. (A) Graphical representation of study design. Blood oxygen saturation and heart rate of male and female CD-1 mice (n = 6 per group) were measured using the MouseOx pulse oximetry system according to the timeline shown. (B–C) Arrows and dotted lines indicate intraperitoneal injection point. Parameters were independently compared among all groups via two-way ANOVA (t = 0–60 minutes). Average values by treatment group from t = 10–15 minutes after injection were then compared via one-way ANOVA with Tukey’s post hoc test (females shown as circles, males as squares). (B) Time course of blood oxygen saturation (%) by treatment group (mixed-effects model: main effect of treatment, F(5, 30) = 29.48, P < 0.0001; main effect of time, F(3.362, 98.10) = 11.41, P < .0001; time × treatment interaction, F(55, 321) = 2.764, P < .0001) with a bar graph displaying group values from t = 10–15 minutes (one-way ANOVA: F(5, 30) = 30.22, P < .0001). (C) Time course of heart rate by treatment group (two-way ANOVA: main effect of treatment, F(5, 30) = 26.98, P < .0001; main effect of time, F(4.476,132.7) = 25.33, P < .0001; time × treatment interaction, F(55, 326) = 4.663, P < .0001) with a bar graph displaying group values from t = 10–15 minutes (one-way ANOVA: F(5, 30) = 16.81, P < .0001). (D–E) Area under the curve values by treatment group from t = 0–60 minutes after injection were compared via one-way ANOVA with Tukey’s post hoc test. Females are shown as circles and males as squares. For (D) blood oxygen saturation (%), one-way ANOVA: F(5, 30) = 27.76, P < .0001 and (E) heart rate (% baseline), one-way ANOVA: F(5, 30) = 60.42, P < .0001. ****P < .0001 versus vehicle; ⊹P < .05, ⊹⊹P < .01, ⊹⊹⊹⊹P < .0001 versus fentanyl alone.

As xylazine is known to induce severe bradycardia in humans, we also evaluated heart rate over time following administration of xylazine, fentanyl, or a combination (Fig. 4C). Xylazine, fentanyl, and both combinations of fentanyl plus xylazine (3.2 or 32 mg/kg) significantly decreased heart rate relative to vehicle at t = 10–15 minutes (P < .0001; Fig. 4C, bar graph). We also calculated the area under the curve for each treatment and found that all significantly reduced heart rate relative to vehicle (P < .0001, Fig 4E). There was no difference in heart rate reduction between xylazine alone or in combination with fentanyl at either dose, which could indicate a subadditive effect. The dose of 32 mg/kg xylazine significantly decreased heart rate, alone or in combination with fentanyl, relative to fentanyl alone (P < .01 and P < .05). Thus, over time, fentanyl in combination with high doses of xylazine reduces breathing rate more than fentanyl alone.

3.5. Fentanyl induces severe apneas that correlate with loss in oxygen saturation

Given that fentanyl in combination with xylazine reduced breathing rate (Fig. 1B), PIF (Fig. 2C), and PEF (Fig. 3C) compared with fentanyl alone, we were surprised that xylazine did not worsen fentanyl-induced changes in oxygen saturation (Fig. 4B). It is known that sleep apneas in humans are associated with reduced blood oxygen saturation (Yadollahi et al, 2010; Wali et al, 2020); thus, we next evaluated whether apneas could explain this discrepancy. As depicted in Fig. 5A, an apnea was defined as a breath with Te longer than twice the average postinjection Te calculated for each individual mouse: Apnea=Te>2[Avg:Tepost-injection]. A severe apnea was classified as a breath in which the Te exceeded twice the average breath length (Te and Ti) after injection for each mouse: SevereApnea=Te>2[Avg:Ti+Tepost-injection]. The total number of apneas (Fig. 5B) and severe apneas (Fig. 5C) are shown for the first t = 0–15 minutes after injection, during which oxygen saturation is maximally decreased. As shown in Fig. 5B, fentanyl alone or in combination with 3.2 mg/kg xylazine induced a significant number of total apneas compared with control (P < .0001 and P < .01, respectively). A significant correlation between the number of total apneas and blood oxygen saturation in the first 15 minutes after injection was found (P < .05, R2 = 0.7088; Fig. 5D). By restricting the analysis to severe apneas only, we found that fentanyl alone (P < .01) or in combination with xylazine (P < .05) induced significantly more apneas than vehicle (Fig. 5C). There was a strong correlation between the number of severe apneas and oxygen saturation loss (P < .0001, R2 = 0.9996; Fig. 5E). Taken together, the fentanyl-specific decrease in blood oxygen saturation may be explained by the greater frequency of severe apneas induced by fentanyl during the first 15 minutes after injection, during which time oxygen saturation was maximally reduced.

Fig. 5.

Fig. 5.

Open in a new tab

Fentanyl induces severe apneas that correlate with loss in oxygen saturation. (A) Graphical representation of study design. The frequency of apneas in male and female CD-1 mice (n = 6 per group) was determined using vivoFlow whole-body plethysmography chambers supplied with air. (B) The number of total apneas from t = 0–15 minutes after injection by treatment group (females shown as circles, males as squares) was compared via one-way ANOVA (F(5, 29) = 13.54, P < .0001) with Tukey’s post hoc test. Apnea=Te>2[Avg:Tepost-injection]. (C) The number of severe apneas from t = 0–15 minutes after injection by the treatment group (females shown as circles, males as squares) was compared via one-way ANOVA (F(5, 28) = 7.583, P < .001) with Tukey’s post hoc test. SevereApnea=Te>2[Avg:Ti+Tepost-injection]. (D) Pearson correlation (r=−0.84, R2 = 0.71, P < .05) between the percentage of oxygen saturation (t = 10–15 minutes after injection) and the number of total apneas (t = 0–15 minutes after injection). Each data point is the average ± SEM per group. Linear equation: y = −0:44x + 93:97. (E) Pearson correlation (r=−0.99, R2 = 0.99, P < .0001) between the percentage of oxygen saturation (t = 10–15 minutes after injection) and the number of severe apneas (t = 0–15 minutes after injection). Linear equation: y = −2:54x + 95:94. (F) Representative apneas from t = 0–15 minutes after injection. *P < .05, **P < .01, ****P < .0001 versus vehicle; ⊹P < .05, ⊹⊹P < .01, ⊹⊹⊹⊹P < .0001 versus fentanyl alone.

4. Discussion

In mammals, breathing rate is governed by active inspiration and passive expiration. Herein, we show that fentanyl and xylazine depress breathing rates via different mechanisms. In contrast to fentanyl, which increases Ti, xylazine reduces breathing rate by extending Te. Although xylazine did not produce any changes in inspiration alone, it did exacerbate the effects of fentanyl. Alone, fentanyl but not xylazine reduced blood oxygen saturation at the doses tested, and xylazine did not further reduce blood oxygen saturation when combined with fentanyl. The reduction in blood oxygen caused by fentanyl strongly correlated with the frequency of severe apneas; xylazine did not cause apneas or exacerbate the effects of fentanyl. Finally, both fentanyl and xylazine induced bradycardia, although when fentanyl was combined with a high dose of xylazine, the effect was not different from xylazine alone. Altogether, these results show that xylazine exacerbates fentanyl-induced depression of respiration and heart rate.

Our results showing that fentanyl primarily affects the process of inspiration are consistent with a body of prior literature and reflect opioid inhibition of neurons in the pre-Bötzinger complex and Kölliker-Fuse nucleus of the brainstem (Levitt et al, 2015; Bachmutsky et al, 2020; Hill et al 2020; Varga et al, 2020; Baertsch et al, 2021; Bateman and Levitt, 2023; Marchette et al, 2023). Fentanyl increases Ti, thereby lowering breathing rate, and reduces PIF. When combined with fentanyl, all doses of xylazine administered further reduced PIF in a possible synergistic manner. This may reflect a greater reliance on brainstem respiratory rhythm generation, which is inhibited by opioids when sleeping (McKay et al, 2005) or while sedated by drugs like xylazine.

During passive expiration, neurons in the ventral rostral group of the brainstem fire intrinsically as part of the central pattern generator (Segers et al, 2015; West and Luks, 2021). Xylazine could inhibit expiration through α2AR autoreceptor activation in these neurons (Zanella et al, 2007) or in chemosensory neurons within the locus coeruleus (Magalhães et al, 2018) or nucleus tractus solitarius (Favero et al, 2011). Xylazine also reduced PEF and EF50, whereas fentanyl increased these parameters at a dose that did not increase locomotion. In rodents, changes in EF50 have been observed in response to respiratory challenges such as SARS-CoV-2 infection (Menachery et al, 2015) and allergic asthma (Glaab et al, 2002). In line with our results, fentanyl was shown to induce a prolonged increase in EF50 in rats (Seckler et al, 2022), yet this finding has not been consistent across all studies (Baby et al, 2024). It is possible that the opposing effects of fentanyl and xylazine on EF50 reflect the respiratory muscle rigidity caused by fentanyl (Haouzi and Tubbs, 2022) versus the muscle relaxant effects of xylazine (Ko et al, 1998), but more studies are needed to better understand this in the context of opioid use.

The correlation between the fentanyl-induced reduction in blood oxygen saturation and number of severe apneas is consistent with human data that show a greater decrease in blood oxygen levels associated with more severe obstructive sleep apnea (Yadollahi et al, 2010; Wali et al, 2020). Consistent with our findings using pulse oximetry, anesthetic doses of xylazine were observed to decrease breathing rate with little effect on peripheral blood oxygen saturation in mice (Tsukamoto et al, 2015). Moreover, it has been demonstrated that xylazine does not worsen the maximum level of hypoxia induced by fentanyl in the nucleus accumbens of rats as measured by an oxygen-sensing electrode (Choi et al, 2023; Kiyatkin and Choi, 2024). However, these studies also found that xylazine on its own decreased oxygenation of the nucleus accumbens (Choi et al, 2023; Kiyatkin and Choi, 2024), whereas the current experiments show no effect on blood oxygen saturation, potentially reflecting tissue-specific effects, species differences, or different sensitivities of the techniques employed. It is also worth noting that mice have a much higher specific metabolic rate than humans (Demetrius, 2005; Perlman, 2016), which allows them to better adapt to changes in blood oxygen saturation.

As a sedative, xylazine is known to decrease metabolic rate (Xiao et al, 2013; Mohammed et al, 2018), which decreases breathing rate. It is therefore possible that xylazine does not directly inhibit breathing circuits but instead reduces breathing rate by decreasing metabolic rate. This may account for the lack of effect on blood oxygen saturation: the lower breathing rate induced by xylazine may be sufficient under conditions of decreased metabolic demand. Separately, high CO2 conditions (eg, 5%) mimic increased metabolic demand (Robba et al, 2020; Rawat et al, 2024) and are utilized in many opioid-induced respiratory depression studies to increase ventilatory drive (Bachmutsky et al, 2020; Hill et al, 2020). This is a confounding variable because hypercapnia-driven breathing is mechanistically distinct, and opioids inhibit the ventilatory response to hypercapnia, which can magnify a drug’s perceived effect. It is worth noting that we used air in these studies to better reflect the context of human overdose and avoid these confounds.

Finally, our data, showing that xylazine alone or in combination with fentanyl-induced bradycardia to a greater degree than fentanyl alone, are consistent with previous literature reporting decreased heart rate and hypotension as clinical symptoms of fentanyl-xylazine overdose (Ayub et al, 2023). We have established that fentanyl lowers blood oxygen levels and xylazine decreases heart rate, reducing oxygen supply to critical tissues. In a fatal overdose, inadequate oxygenation of the brain and heart causes brain damage and eventual cardiac arrest (Kiyatkin, 2019; Bateman et al, 2023). Thus, the xylazine-driven changes in the cardiovascular system coupled with the low blood oxygen levels induced by fentanyl could greatly increase the risk of overdose in humans.

Altogether, we show that xylazine worsens fentanyl-induced respiratory depression via direct effects on expiration, as well as exacerbating fentanyl’s effects on inspiration. Despite the combination of fentanyl and xylazine reducing breathing rate more than fentanyl alone, this did not translate to a further reduction in blood oxygen saturation. Instead, blood oxygen depletion correlated with the number of fentanyl-induced severe apneas. Additionally, at high doses, xylazine reduced heart rate more than fentanyl. The extent of heart rate reduction when fentanyl was combined with xylazine did not differ from the effect of xylazine alone. Thus, xylazine produces independent effects on respiration and heart rate while also exacerbating some effects produced by fentanyl. These results provide preclinical insight into the mechanisms underlying xylazine’s contribution to overdoses involving opioids in humans. Future work should delineate how these effects manifest in the human population with regard to risk of fatal overdose.

Supplementary Material

Supplemental

This article has supplemental material available at jpet.aspetjournals.org.

Significance Statement:

Xylazine, found in illicit fentanyl samples, exacerbates fentanyl-induced respiratory depression in mice. Fentanyl-induced reduction in blood oxygen levels correlates with the number of severe apneas. The findings show how the addition of xylazine to fentanyl may increase the risk of overdose.

Acknowledgments

Financial support

This work was supported by the National Institutes of Health [Grants UG3 DA056884, R21DA051723, R01 DA061320, R01 HL174547, and T32 GM132046].

Abbreviations

α2AR

α2-adrenergic receptor

EF50

expiratory flow 50

PEF

peak expiratory flow

PIF

peak inspiratory flow

Te

expiration time

Ti

inspiration time

Footnotes

Laboratory of Origin: Edward F. Domino Research Center, Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48104.

Conflict of interest

The authors declare no conflicts of interest.

CRediT authorship contribution statement

Conceptualization, Investigation, Formal Analysis, Writing - Original Draft, Writing - Review & Editing, Visualization: Demery.

Investigation: Moore.

Conceptualization, Writing - review & editing, Supervision, Funding Acquisition: Levitt.

Conceptualization, Writing - Review & Editing, Supervision, Project Administration, Funding Acquisition: Anand.

Conceptualization, Writing - Review & Editing, Supervision, Project Administration, Funding Acquisition: Traynor.

Data availability

The authors declare that all the data supporting the findings of this study are available within the manuscript and supplementary data.

References

  1. Acosta-Mares P, Violante-Soria V, Browne T Jr, and Cruz SL (2023) Xylazine potentiates the lethal but not the rewarding effects of fentanyl in mice. Drug Alcohol Depend 253:110993. [DOI] [PubMed] [Google Scholar]
  2. Ayub S, Parnia S, Poddar K, Bachu AK, Sullivan A, Khan AM, Ahmed S, and Jain L (2023) Xylazine in the opioid epidemic: A systematic review of case reports and clinical implications. Cureus 15:e36864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baby SM, May WJ, Getsy PM, Coffee GA, Nakashe T, Bates JN, Levine A, and Lewis SJ (2024) Fentanyl activates opposing opioid and non-opioid receptor systems that control breathing. Front Pharmacol 15:1381073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bachmutsky I, Wei XP, Kish E, and Yackle K (2020) Opioids depress breathing through two small brainstem sites. Elife 9:e52694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baertsch NA, Bush NE, Burgraff NJ, and Ramirez JM (2021) Dual mechanisms of opioid-induced respiratory depression in the inspiratory rhythm-generating network. Elife 10:e67523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bateman JT and Levitt ES (2023) Opioid suppression of an excitatory pontomedullary respiratory circuit by convergent mechanisms. Elife 12:e81119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bateman JT, Saunders SE, and Levitt ES (2023) Understanding and countering opioid-induced respiratory depression. Br J Pharmacol 180:813–828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Choi S, Irwin MR, and Kiyatkin EA (2023) Xylazine effects on opioid-induced brain hypoxia. Psychopharmacology (Berl) 240:1561–1571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Choi S, Irwin MR, Noya MR, Shaham Y, and Kiyatkin EA (2024) Combined treatment with naloxone and the alpha2 adrenoceptor antagonist atipamezole reversed brain hypoxia induced by a fentanyl-xylazine mixture in a rat model. Neuropsychopharmacology 49:1104–1112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Demetrius L (2005) Of mice and men. When it comes to studying ageing and the means to slow it down, mice are not just small humans. EMBO Rep 6 Spec No(Suppl 1):S39–S44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Favero MT, Takakura AC, de Paula PM, Colombari E, Menani JV, and Moreira TS (2011) Chemosensory control by commissural nucleus of the solitary tract in rats. Respir Physiol Neurobiol 179:227–234. [DOI] [PubMed] [Google Scholar]
  12. Glaab T, Hoymann HG, Hohlfeld JM, Korolewitz R, Hecht M, Alarie Y, Tschernig T, Braun A, Krug N, and Fabel H (2002) Noninvasive measurement of mid-expiratory flow indicates bronchoconstriction in allergic rats. J Appl Physiol (1985) 93:1208–1214. [DOI] [PubMed] [Google Scholar]
  13. Haouzi P and Tubbs N (2022) Effects of fentanyl overdose-induced muscle rigidity and dexmedetomidine on respiratory mechanics and pulmonary gas exchange in sedated rats. J Appl Physiol (1985) 132:1407–1422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hays HL, Spiller HA, DeRienz RT, Rine NI, Guo HT, Seidenfeld M, Michaels NL, and Smith GA (2024) Evaluation of the relationship of xylazine and fentanyl blood concentrations among fentanyl-associated fatalities. Clin Toxicol (Phila) 62:26–31. [DOI] [PubMed] [Google Scholar]
  15. Hill R, Santhakumar R, Dewey W, Kelly E, and Henderson G (2020) Fentanyl depression of respiration:Comparison with heroin and morphine. Br J Pharmacol 177:254–266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hochheimer M, Strickland JC, Rabinowitz JA, Ellis JD, Dunn KE, and Huhn AS (2024) Knowledge, preference, and adverse effects of xylazine among adults in substance use treatment. JAMA Netw Open 7:e240572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kelly E, Sutcliffe K, Cavallo D, Ramos-Gonzalez N, Alhosan N, and Henderson G (2023) The anomalous pharmacology of fentanyl. Br J Pharmacol 180: 797–812. [DOI] [PubMed] [Google Scholar]
  18. Khatri SN, Sadek S, Kendrick PT, Bondy EO, Hong M, Pauss S, Luo D, Prisinzano TE, Dunn KE, Marusich JA, et al. (2024) Xylazine suppresses fentanyl consumption during self-administration and induces a unique sex-specific withdrawal syndrome that is not altered by naloxone in rats. Exp Clin Psychopharmacol 32:150–157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kiyatkin EA (2019) Respiratory depression and brain hypoxia induced by opioid drugs: Morphine, oxycodone, heroin, and fentanyl. Neuropharmacology 151:219–226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kiyatkin EA and Choi S (2024) Brain oxygen responses induced by opioids: focus on heroin, fentanyl, and their adulterants. Front Psychiatry 15:1354722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ko JC, Nicklin CF, Heaton-Jones TG, and Kuo WC (1998) Comparison of sedative and cardiorespiratory effects of diazepam, acepromazine, and xylazine in ferrets. J Am Anim Hosp Assoc 34:234–241. [DOI] [PubMed] [Google Scholar]
  22. Leconte CE and Sethi R (2023) The appearance of xylazine in the United States as a fentanyl adulterant. Prim Care Companion CNS Disord 25:22nr03473. [DOI] [PubMed] [Google Scholar]
  23. Levitt ES, Abdala AP, Paton JFR, Bissonnette JM, and Williams JT (2015) μ opioid receptor activation hyperpolarizes respiratory-controlling Kolliker-Fuse neurons and suppresses post-inspiratory drive. J Physiol 593:4453–4469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Magalhães KS, Spiller PF, da Silva MP, Kuntze LB, Paton JFR, Machado BH, and Moraes DJA (2018) Locus coeruleus as a vigilance centre for active inspiration and expiration in rats. Sci Rep 8:15654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marchette RCN, Carlson ER, Frye EV, Hastings LE, Vendruscolo JCM, Mejias-Torres G, Lewis SJ, Hampson A, Volkow ND, Vendruscolo LF, et al. (2023) Heroin- and fentanyl-induced respiratory depression in a rat plethysmography model: potency, tolerance, and sex differences. J Pharmacol Exp Ther 385:117–134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McKay LC, Janczewski WA, and Feldman JL (2005) Sleep-disordered breathing after targeted ablation of preBotzinger complex neurons. Nat Neurosci 8:1142–1144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Menachery VD, Gralinski LE, Baric RS, and Ferris MT (2015) New metrics for evaluating viral respiratory pathogenesis. PLoS One 10:e0131451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mohammed A, Al-Hozab A, and Alshaheen T (2018) Effects of diazepam and xylazine on changes of blood oxygen and glucose levels in mice. Adv Anim Vet Sci 6:121–127. [Google Scholar]
  29. Motulsky HJ and Brown RE (2006) Detecting outliers when fitting data with nonlinear regressionda new method based on robust nonlinear regression and the false discovery rate. BMC Bioinformatics 7:123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. National Research Council Committee (2011), Guide for the Care and Use of Laboratory Animals, National Academies Press, Washington (DC). [Google Scholar]
  31. Papudesi BN, Malayala SV, and Regina AC (2024) Xylazine toxicity, in StatPearls [Internet]. StatPearls Publishing, Treasure Island. [Google Scholar]
  32. Perlman RL (2016) Mouse models of human disease: An evolutionary perspective. Evol Med Public Health 2016:170–176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rawat D, Modi P, and Sharma S (2024) Hypercapnea, in StatPearls [Internet], Stat-Pearls Publishing, Treasure Island. [Google Scholar]
  34. Robba C, Siwicka-Gieroba D, Sikter A, Battaglini D Dąbrowski W, Schultz MJ, de Jonge E, Grim C, Rocco PR, and Pelosi P (2020) Pathophysiology and clinical consequences of arterial blood gases and pH after cardiac arrest. Intensive Care Med Exp 8(Suppl 1):19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ruiz-Colón K, Chavez-Arias C, Díaz-Alcalá JE, and Martínez MA (2014) Xylazine intoxication in humans and its importance as an emerging adulterant in abused drugs: a comprehensive review of the literature. Forensic Sci Int 240:1–8. [DOI] [PubMed] [Google Scholar]
  36. Sadek SM, Khatri SN, Kipp Z, Dunn KE, Beckmann JS, Stoops WW, Hinds TD Jr, and Gipson CD (2024) Impacts of xylazine on fentanyl demand, body weight, and acute withdrawal in rats: a comparison to lofexidine. Neuropharmacology 245:109816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Seckler JM, Grossfield A, May WJ, Getsy PM, and Lewis SJ (2022) Nitrosyl factors play a vital role in the ventilatory depressant effects of fentanyl in unanesthetized rats. Biomed Pharmacother 146:112571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Segers LS, Nuding SC, Ott MM, Dean JB, Bolser DC, O’Connor R, Morris KF, and Lindsey BG (2015) Peripheral chemoreceptors tune inspiratory drive via tonic expiratory neuron hubs in the medullary ventral respiratory column network. J Neurophysiol 113:352–368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Smith MA, Biancorosso SL, Camp JD, Hailu SH, Johansen AN, Morris MH, and Carlson HN (2023) “Tranq-dope” overdose and mortality: lethality induced by fentanyl and xylazine. Front Pharmacol 14:1280289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sun X, Thörn Pérez C, Halemani DN, Shao XM, Greenwood M, Heath S, Feldman JL, and Kam K (2019) Opioids modulate an emergent rhythmogenic process to depress breathing. Elife 8:e50613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Truver MT, Brogan SC, Jaeschke EA, Kinsey AM, Hoyer JL, Chronister CW, Crosby MM, and Goldberger BA (2024) A quantitative LC-MS/MS analysis of xylazine, p-fluorofentanyl, fentanyl and fentanyl-related compounds in postmortem blood. J Chromatogr B Analyt Technol Biomed Life Sci 1237:124059. [DOI] [PubMed] [Google Scholar]
  42. Tsukamoto A, Serizawa K, Sato R, Yamazaki J, and Inomata T (2015) Vital signs monitoring during injectable and inhalant anesthesia in mice. Exp Anim 64:57–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Varga AG, Reid BT, Kieffer BL, and Levitt ES (2020) Differential impact of two critical respiratory centres in opioid-induced respiratory depression in awake mice. J Physiol 598:189–205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wali SO, Abaalkhail B, AlQassas I, Alhejaili F, Spence DW, and Pandi-Perumal SR (2020) The correlation between oxygen saturation indices and the standard obstructive sleep apnea severity. Ann Thorac Med 15:70–75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. West JB and Luks AM (2021) West’s Respiratory Physiology: The Essentials, 11th ed., Lippincott Williams & Wilkins, Wolters Kluwer, Maryland. [Google Scholar]
  46. Xiao YF, Wang B, Wang X, Du F, Benzinou M, and Wang YXJ (2013) Xylazine-induced reduction of tissue sensitivity to insulin leads to acute hyperglycemia in diabetic and normoglycemic monkeys. BMC Anesthesiol 13:33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yadollahi A, Giannouli E, and Moussavi Z (2010) Sleep apnea monitoring and diagnosis based on pulse oximetry and tracheal sound signals. Med Biol Eng Comput 48:1087–1097. [DOI] [PubMed] [Google Scholar]
  48. Zanella S, Viemari JC, and Hilaire G (2007) Muscarinic receptors and alpha2-adrenoceptors interact to modulate the respiratory rhythm in mouse neonates. Respir Physiol Neurobiol 157:215–225. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental
NIHMS2142779-supplement-Supplemental.pdf (252.8KB, pdf)

Data Availability Statement

The authors declare that all the data supporting the findings of this study are available within the manuscript and supplementary data.

RESOURCES