Oxytocin attenuates respiratory depression and reduces mortality from fentanyl and the combination of xylazine-fentanyl in rats

Abstract

Opioid addiction and misuse are a serious national crisis that affects public health, as well as social and economic welfare. Mortality due to opioid misuse is further exasperated by the combination of opioids with non-opioid respiratory depressants such as xylazine that are resistant to mu opioid receptor antagonists such as naloxone. This study tested the hypothesis that oxytocin can mitigate the severe opioid induced respiratory depression (OIRD) and mortality induced by high doses of fentanyl or the combination of fentanyl with xylazine. Our results show OXT can improve survival and respiratory function in both male and female rats with opioid induced respiratory depression caused by fentanyl, as well as a combination of fentanyl and xylazine. The improvement in respiratory function by OXT post fentanyl-xylazine was significantly greater than the recovery using only naloxone. Chemogenetic activation of OXT receptor positive neurons in the ventral respiratory group (VRG) provided similar benefits to that of OXT administration in reversing OIRD. These results indicate OXT is a promising therapeutic target for reversing OIRD and the respiratory depression that occurs with the combination of opioids and xylazine, a situation where naloxone is only partially effective. Additional translational benefits of OXT include it can be repurposed as it is already a FDA approved drug for other uses, has a high safety profile, and is unlikely to induce the withdrawal or reversal of analgesia that occurs with naloxone.

1. Introduction

Opioid addiction and misuse are a serious national crisis that affects public health, as well as social and economic welfare. The opioid crisis has been greatly exacerbated by the increased availability of synthetic opioids, such as fentanyl, and by the increased prescribing of opioid pain relievers [1,2]. Opioid overdose kills 130 people in the United States every day according to the CDC/NCHS National Vital Statistics System. Causes of mortality with opioids include cardiac dysrhythmias, hypothermia, and opioid induced respiratory depression (OIRD) with decreases in breathing frequency and airway obstructions.

Naloxone, a competitive antagonist of mu-opioid receptors (MORs), has a rapid onset and helps reverse OIRD but is short acting and re-dosing is necessary to reverse long-acting synthetic opioids [3–5]. In addition, the toxic effects of fentanyl and its analogues also can include compromised breathing due to mechanisms not reversible by MOR antagonists [6]. In addition, in patients taking high-dose opioids for chronic pain and individuals with opioid use disorder naloxone could block the analgesic effects of opioids aggravating pain [7,8], and increase the risk of opioid withdrawal [9,10]. Another MOR antagonist, nalmefene, has longer half-life (8–11 h), but it has limitations and adverse effects similar to naloxone [5].

In addition, fentanyl is also being increasingly combined with other non-opioid respiratory depressants. Xylazine, an alpha-2 adrenergic receptor agonist is typically used as a sedative and analgesic in veterinary medicine, is not a controlled substance and is naloxone-resistant [11]. A fentanyl-xylazine mixture (i.e., “tranq-dope”) represents a rapidly emerging public health threat and was present in 6.7% of opioid related overdose deaths in 2020. In mice a dose of xylazine of 100 mg/kg produces a synergistic interaction with fentanyl and decreased the estimated LD50 dose for fentanyl by approximately 100-fold [12]. Alternative, non-opioid receptor antagonist-based approaches for opioid and other drug induced respiratory depression is needed.

Recent work has suggested that oxytocin can act as a respiratory stimulant in patients with obstructive sleep apnea [13,14]. Furthermore, in contrast to naloxone which reverses opioid induced analgesia [7,8], and increase the risk of opioid withdrawal in chronic opioid users [9, 10], a recent randomized, double-blind placebo-controlled clinical trial has shown that IN oxytocin can reduce opioid craving and withdrawal scores, as well as the elevations of the stress hormone cortisol that occur with withdrawal [15]. Other work has shown oxytocin attenuates tolerance to morphine and mitigates naloxone-induced morphine withdrawal [16].

This study tested the hypothesis that oxytocin can mitigate severe OIRD and mortality in unrestrained conscious animals induced by high doses of fentanyl or the combination of fentanyl with xylazine. This work also examined if there are any sex differences in OIRD caused by fentanyl and treatment with OXT. To identify possible sites of action of oxytocin we also tested if chemogenetic activation of oxytocin receptor positive neurons (OXTR+) in the ventral respiratory group (VRG) could reverse OIRD.

2. Methods

All animal experiments were performed in accordance with National Institutes of Health guidelines and were approved by the George Washington University Institutional Animal Care and Use Committee (IACUC; protocols #2022–028, #2022–035, and #2025–141). Adult male and female Sprague-Dawley rats (250–320 g) and adult male and female mice (20–28 g) were used. Sprague Dawley rats were obtained from Hilltop Laboratory Animals, Inc. (Scottdale, PA), and mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Animals were housed under standard environmental conditions (24–26 °C; 12-h light/dark cycle, lights on from 7:00 a.m.–7:00 p.m.) with food and water available ad libitum.

Respiratory function was assessed in conscious, unrestrained animals using whole-body plethysmography (Scireq, Montreal, Canada). Following a 30-min acclimation period, animals received an intraperitoneal injection of fentanyl (0.5 or 1.3 mg/kg) or fentanyl combined with xylazine (0.5 and 0.1 mg/kg, respectively). Ten minutes later, animals were injected intraperitoneally with saline, oxytocin (200 nmol/kg), naloxone (0.1 mg/kg), the combination of oxytocin and naloxone, or the DREADDs agonist clozapine-N-oxide (CNO 1 mg/kg), as indicated.

For chemogenetic activation of OXTR+ neurons in the VRG the transgenic OXT receptor (OXTR)-Cre mouse line (B6.Cg-Oxtrtm1.1(cre) Hze/J, Common Name: Oxtr-T2A-Cre-D, Jackson Labs stock # 031303) was used in combination with DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) that are engineered G protein–coupled receptors (GPCRs) that do not respond to natural neurotransmitters and only respond to synthetic ligands, such as CNO. We expressed hM3DGq excitatory DREADDs in OXTR+ neurons using an AAV floxed excitatory DREADDs vector (AAV8-hSyn-DIO-hM3D(Gq)-mCherry, UNC Core) that was injected into the VRG to selectively express excitatory DREADDs in OXTR+ VRG neurons in male and female mice.

For this injection mice were anesthetized using a mixture of keta-mine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.), then secured in a stereotaxic frame with the neck bent at 45 ° to expose the calamus scriptorius (David Kopf Instruments, CA, USA). Using stereotaxic guidance of a pulled glass capillary (40 μm tip diameter) (G1, Narishige, UK) attached to a pneumatic microinjector (IM-11–2, Narishige, UK), 200nL of AAV floxed DREADDs was bilaterally injected into the VRG (AP 0.4 mm from the Obex, ML 1.5 mm, DV 4.5 mm). The capillary remained in place for 5 min to allow diffusion and was then removed slowly to avoid dispersion to neighboring brainstem regions. DREADDs-mCherry expression was observed in the VRG in all mice used for these experiments after the mice were sacrificed.

2.1. Brain slice preparation, Immunohistochemistry, and confocal images

After completion of Whole-Body Plethysmography recordings, animals were anesthetized by isoflurane and transcardially perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA). Brains were dissected, post-fixed in 4% PFA overnight at room temperature, then washed 3 × 10 min in PBS. A series of 50 μm coronal section medullary slices containing the VRG were obtained using a Leica dissection vibratome (Leica VT 1000S). 10–20 VRG medullary slices were obtained from each animal. All slice sections were processed using immunohistochemistry for mCherry and Phox2b to identify Cre-dependent VRG and phox2b positive-expressing neurons, respectively. After blocking nonspecific proteins in 10% normal goat serum (NGS) in PBS with 0.3% Triton X-100 (PBST) for 4 h at room temperature, slices were incubated in primary antibody at 4^◦ C for 24 h (Chicken anti-RFP antibody (1:1000, Millipore Sigma, Cat. # AB3528), and Mouse monoclonal anti Phox2b (1:200, sc-376997; Santa Cruz Biotechnology, Inc., Santa Cruz, CA)). Secondary antibodies were applied for 4 h at room temperature. Secondary antibodies were Goat anti Chicken Alexa Fluor^™ Plus 555, (Invitrogen, Cat. # A32932) and goat anti mouse Alexa Fluor 647 (Invitrogen, Cat. # A-21241), both 1:200 dilution. Slides were mounted with Fluorogel (Electron Microscopy Sciences, SKU: 17985–10) and imaged with a Leica TCS SP8 multi-photon confocal microscope equipped with supercontinuum white laser source and single molecule detection hybrid detectors (SMD HyD, Leica, Wetzlar, Germany). Leica TCS SP8 MP and Zeiss LSM 980 confocal microscopy were used to assess colocalization of mCherry and Phox2b labeled neurons in the VRG. Brain tissue slices containing VRG were examined with 555 and 647 nm wavelengths to visualize mCherry-Alexa Fluor 555 and the Phox2b-Alexa Fluor 647, respectively. Images were captured with a DFC365FX camera at 2048 by 2048-pixel resolution. Full field images of the entire slice were taken at 10x to localize the VRG. Z-stacks were then taken with 20x/0.75 oil- immersion objective, at z-step size of 0.9 μm to produce image volumes allowing for the identification of colocalization of mCherry and Phox2b positive neurons. Images were processed and analyzed using Imaris 10 software (Oxford Instruments Oxford, UK).

2.2. Data analysis

All data are presented as individual points, and the mean ± standard deviation (SD). For ex vivo experiments, ‘n’ is reported as number of identified neurons in the VRG. For in vivo experiments, ‘n’ is the number of animals. These values are stated throughout the Results and Figure Legends. Statistical comparisons were made using repeated-measurements (RM) one-way and two-way ANOVA with Dunnett’s and Tukey’s multiple comparisons, paired or unpaired Student’s tests, as appropriate. Specific statistical tests are noted in the results. Differences were considered statistically significant if p-value < 0.05. Plethysmography data was obtained using the Emka IOX software (version 2.10.5.28, Emka Technologies, Sterling VA). Graph creation and statistical analyses were conducted using GraphPad Prism 9 (GraphPad Software, San Diego, CA).

3. Results

Injection of fentanyl (0.5 mg/kg) in male and female rats induced rapid respiratory depression with decreases in respiratory rate (Fig. 1, top) as well as an increased incidence of apneas (Fig. 1, bottom). Injection of oxytocin (OXT, 200 mmol/kg, ip) significantly improved respiratory rate and decreased the incidence of apneas (Fig. 1). There were no significant differences between males (Fig. 1 left) and females (Fig. 1, right) in either the responses to fentanyl or beneficial actions of OXT.

Fig. 1.

Effects of intraperitoneal oxytocin on fentanyl-induced respiratory depression in male and female rats. Respiratory rate (RR, breaths per minute; top panels) and apnea frequency (events per hour; bottom panels) were measured in conscious, unrestrained male (left panels) and female (right panels) rats at baseline, 5–10 min following intraperitoneal fentanyl administration, and 1 h after treatment with saline or oxytocin. Oxytocin was administered intraperitoneally at a dose of 200 nmol/kg. Individual symbols represent single animals; horizontal lines denote group means and error bars indicate ± standard deviation (SD). Statistical comparisons between saline- and oxytocin-treated groups at the 1-hour post-treatment time point are indicated by brackets. p < 0.05, p < 0.01, p < 0.001, p < 0.0001. n = 8 animals per group.

Using a high dose of fentanyl (1.3 mg/kg) respiratory depression was more severe and without treatment the survival at 5 h post fentanyl administration in male rats was 55%, whereas with OXT treatment there was 100% survival (Fig. 2, left). Female rats had a significantly higher rate of survival than males without treatment at this high dose (1.3 mg/kg), and, similar to males, with OXT treatment there was 100% survival in female rats (Fig. 2, right).

Fig. 2.

Mortality following IP fentanyl injection and subsequent treatment with oxytocin in male and female rats. Survival of conscious, unrestrained male (left panel) and female (right panel) rats following intraperitoneal fentanyl administration (1.3 mg/kg) and subsequent treatment with saline or oxytocin (200 nmol/kg, intraperitoneal). Survival was monitored for up to 6 h following fentanyl injection. Survival curves were compared using the Mantel–Cox (log-rank) test. p < 0.05 was considered statistically significant. n = 9 animals per group.

Administration of a combination of fentanyl and xylazine (0.5 mg/kg and 0.1 mg/kg, respectively) induced a more severe respiratory depression of respiratory rate and incidence of apneas compared to fentanyl by itself (Fig. 3).

Fig. 3.

Respiratory depression in male rats induced by intraperitoneal fentanyl alone or in combination with xylazine. Respiratory rate (RR, breaths per minute; left panel) and apnea frequency (events per hour; right panel) were measured in conscious, unrestrained male rats at baseline, following intraperitoneal administration of fentanyl (0.5 mg/kg) alone, or fentanyl combined with xylazine (0.5 and 0.1 mg/kg, respectively). Individual symbols represent single animals; horizontal lines denote group means and error bars indicate ± standard deviation (SD). Statistical comparisons between fentanyl alone and fentanyl + xylazine groups are indicated by brackets. p < 0.05, p < 0.0001. n = 8 animals per group.

To compare the treatment of OXT with naloxone animals were given the combination of fentanyl and xylazine ((0.5 mg/kg and 0.1 mg/kg, respectively) followed by either naloxone (0.1 mg/kg, ip), OXT (200 mmol/kg, ip) or the combination of naloxone with OXT (0.1 mg/kg and 200 mmol/kg, ip, respectively). As expected, both naloxone and OXT improved respiratory rate and reduced the incidence of apneas (Fig. 4). The treatment of OIRD caused by fentanyl and xylazine with the combination of naloxone and OXT was not significantly greater than OXT by itself (Fig. 4).

Fig. 4.

Effects of intraperitoneal naloxone, oxytocin, and their combination on respiratory depression induced by fentanyl and xylazine in male rats. Effects of intraperitoneal naloxone (0.1 mg/kg), oxytocin (200 nmol/kg), or their combination on respiratory depression in male rats induced by intraperitoneal fentanyl + xylazine (0.5 and 0.1 mg/kg, respectively). Respiratory rate (RR, breaths per minute; left panel) and apnea frequency (events per hour; right panel) were measured in conscious, unrestrained male rats at baseline, 5–10 min following fentanyl + xylazine administration, and 1 h after treatment with saline, naloxone, oxytocin, or naloxone + oxytocin. Individual symbols represent single animals; horizontal lines denote group means and error bars indicate ± standard deviation (SD). Statistical comparisons among treatment groups at the 1-hour post-treatment time point are indicated by brackets. p < 0.05, p < 0.01, p < 0.001, p < 0.0001. n = 8 animals per group.

To better compare the benefits on OXT and naloxone we examined respiratory rate in 15 min periods following fentanyl-xylazine injection. Surprisingly the improvement in respiratory rate seen with OXT was either not significantly different from that with naloxone, or greater than that with naloxone at one hour post fentanyl-xylazine injection (Fig. 5). The mortality caused by the combination of fentanyl and xylazine (1.3 mg/kg and 10 mg/kg, respectively) was prevented by either naloxone or OXT (Fig. 6).

Fig. 5.

Time-course of respiratory depression post fentanyl + xylazine administration and recovery with intraperitoneal naloxone or oxytocin treatment in male rats. Effects of intraperitoneal naloxone (0.1 mg/kg) or oxytocin (200 nmol/kg) on respiratory rate in male rats induced by intraperitoneal fentanyl + xylazine. Respiratory rate (RR, breaths per minute) was measured in conscious, unrestrained male rats following fentanyl + xylazine administration and at 15-min intervals for up to 120 min after treatment with naloxone or oxytocin. Individual symbols represent single animals; horizontal lines denote group means and error bars indicate ± standard deviation (SD). Data were analyzed using repeated-measures two-way ANOVA with Tukey’s post hoc multiple-comparisons testing to assess treatment effects over time. Asterisks indicate significant differences between treatment groups at the indicated time points. p < 0.05, p < 0.01, p < 0.001, p < 0.0001. n = 8 animals per group.

Fig. 6.

Effects of naloxone and oxytocin on mortality following fentanyl and xylazine administration in male rats. Survival of conscious, unrestrained male rats following intraperitoneal administration of fentanyl + xylazine (1.3 and 0.1 mg/kg, respectively) and subsequent treatment with saline, naloxone (0.1 mg/kg, intraperitoneal), or oxytocin (200 nmol/kg, intraperitoneal). Survival was monitored for up to 6 h after fentanyl + xylazine injection. Survival curves were compared using the Mantel–Cox (log-rank) test. p < 0.05 was considered statistically significant. n = 8 animals per group.

To identify possible sites of action of oxytocin we tested if chemogenetic activation of oxytocin receptor positive (OXTR+) neurons in the VRG could reduce OIRD. Floxed excitatory DREADDs was injected into the VRG in OXTR+cre mice (Fig. 7A). After at least 4 weeks of recovery selective chemogenetic excitation of OXTR+ VRG neurons was accomplished by activating the excitatory DREADDs with the DREADDs ligand CNO (1 mg/kg), Chemogentic activation of OXTR+ VRG neurons significantly reversed the respiratory depression induced by fentanyl (10 mg/kg), see Fig. 7B. We next explored if OXTR+ VRG neurons were Phox2B positive, as Phox2B is a commonly used marker of chemosensitive VRG neurons, and prior work has shown optogenetic stimulation of Phox2b+ VRG neurons improved breathing activity after fentanyl induced OIRD [17]. Surprisingly confocal image analyses show very few, only 4.3 % of OXTR+ mCherry positive neurons in the VRG overlap with Phox2b positive cells (Fig. 7C, representative OXTR+/mCherry+ expression seen in a total of 771 VRG neurons from 27 slices (5–7 slices/animal from 5 animals in total)). Phox2b positive neurons were more densely located medial to the OXTR+ neurons (Fig. 7C).

Fig. 7.

Chemogenetic activation of oxytocin receptor–expressing neurons in the ventral respiratory group attenuates fentanyl-induced respiratory depression. (A) Schematic illustrating bilateral injection of AAV8-hSyn-DIO-hM3D(Gq)-mCherry into the ventral respiratory group (VRG) of OXTR-Cre mice. (B) Respiratory rate (RR, breaths per minute) measured at baseline, 5–10 min following intraperitoneal fentanyl administration, and 1 h after activation of excitatory DREADDs with clozapine-N-oxide (CNO, 1 mg/kg, intraperitoneal) or saline. Individual symbols represent single animals; horizontal lines denote group means and error bars indicate ± standard deviation (SD). Statistical comparisons between treatment groups at the 1-hour post-treatment time point are indicated by brackets. (C) Representative coronal brainstem section showing the VRG and higher-magnification images demonstrating mCherry-labeled DREADDs expression (red) and Phox2b immunoreactivity (green). Limited overlap between mCherry-positive and Phox2b-positive neurons was observed. Scale bars as indicated. p < 0.05, p < 0.01, p < 0.001, p < 0.0001. n = 7–8 animals per group.

4. Discussion

This study shows OXT can improve survival and respiratory function in animals with opioid induced respiratory depression caused by fentanyl, as well as a combination of fentanyl and xylazine. The improvement in respiratory function by OXT post fentanyl-xylazine was either equal to or significantly greater than the recovery using naloxone. Chemogenetic activation of OXT receptor positive neurons in the VRG provided similar benefits to that of OXT administration in reversing OIRD. While the benefits of OXT was similar in both males and females, we found female rats had a higher survival after high doses of fentanyl compared to males. However unlike the results in this study, in which fentanyl was given via intraperitoneal injection, rats given a high intravenous dose (25 microg/kg) of fentanyl produced a respiratory depression that was slightly more severe in females, suggesting the route of administration might also play a role in sex differences [18].

The findings in this study extends prior work that has shown OXT can stimulate respiration. OXT microinjected into the pre-Bötzinger complex within the VRG, a brainstem site postulated to be essential for generating inspiratory rhythm, increased respiratory frequency and diaphragm EMG activity [19]. In addition, OXT may serve as a respiratory stimulant by activating neurons in the rostral ventrolateral medulla (RVLM) as microinjection of OXT into the RVLM also increased respiratory frequency and diaphragm muscle activity [20]. Stimulating Phox2b+ retrotrapezoid nucleus (RTN) neurons, important in chemosensitivity, enhances breathing after fentanyl administration, whereas their inhibition exacerbates fentanyl induced hypoventilation [17]. However as very few, only 4.3 %, of OXTR+ mCherry positive neurons in the VRG overlap with Phox2b positive cells (Phox2b+ neurons were more densely located medial to the OXTR+ neurons), this work suggests OXTR+ neurons are a distinct separate population from Phox2b+ neurons. Consistent with the results from this study, application of an oxytocin receptor agonist and optogenetic activation of oxytocinergic receptors/varicosities in the RTN stimulated respiration by increasing breathing amplitude [21].

In addition to prior work that showed IV administration of oxytocin dose-dependently rescued fentanyl induced OIRD [22] additional work has demonstrated intranasal (IN) OXT significantly increased the amplitude of inspiratory-related tongue muscle activity, and increased the firing of protruder hypoglossal motorneurons that would provide tongue protrusion and upper airway opening [23]. In clinical studies intranasal (IN) oxytocin reduces duration of apneas and hypopneas in patients with Obstructive Sleep Apnea (OSA) [13]. The arterial oxygen desaturation that occurred during obstructive events was significantly (p < 0.001) less severe and the risk of bradycardia associated with obstructive events was also significantly (p < 0.002) reduced with IN OXT. In addition, IN OXT acted as a respiratory stimulant, increasing respiratory rate during non-obstructive periods [13].

It is worth noting there are limitations for using OXT to reverse OIRD. Naloxone is FDA approved and often readily available for reversing acute OIRD overdose. However OXT may be as effective, or even more effective, as naloxone in reversing respiratory depression caused by the combination of opioids with other drugs of abuse, such as xylazine. OXT may also be highly beneficial for OIRD prevention in patients taking high-dose opioids for chronic pain or chronic opioid users in which naloxone would likely cause opioid withdrawal and/or aggravate pain. Numerous studies have reported oxytocin exerts analgesic effects in mice and rats [24–26]. In clinical studies, oxytocin induced analgesia in low back pain [27], increased pain threshold in patients with irritable bowel syndrome [28] and relieved headache [29]. In addition, IN oxytocin can reduce opioid craving and withdrawal scores, as well as the elevations of the stress hormone cortisol that occur with withdrawal [15]. Other work has shown oxytocin attenuates tolerance to morphine and mitigates naloxone-induced morphine withdrawal [16]. This suggest OXT may be better tolerated and particularly beneficial in preventing OIRD in chronic opioid users with chronic pain rather than in naïve-opioid users.

In summary, these results indicate OXT is a promising therapeutic target for reversing opioid induced respiratory depression, as well as reversing the more severe respiratory depression that occurs with the combination of opioids and xylazine. Additional translational benefits of OXT include it can be repurposed as it is already a FDA approved drug for other uses, has a high safety profile, and is unlikely to induce the withdrawal or the reversal of analgesia that can occur with using MOR antagonists in chronic opioid users.

Data availability

Data will be made available on request.