Carfentanil exposure could cause severe ventilatory depression associated with muscle stiffness, hypoxemia/hypercapnia (due to hypoventilation) and hypothermia; however, its effect on inspiratory motor drive alone (without these associated effects) has not been investigated. This, along with the inhibitory impact of opioids on hypoxic and hypercapnic ventilation, allows us to determine the impacts of carfentanil on the phrenic nerve (PN) activities before and during hypoxia and hypercapnia in this study.

Two groups of anesthetized and spontaneously breathing rats were initially exposed to aerosolized vehicle (Ctrl) and carfentanil (1.7 mg/m³) for 10 min. After vagotomy, paralysis and artificial ventilation, the peak and frequency of integrative PN activity (∫PN and f_R), minute PN (MPN = ∫PN × f_R), arterial blood pressure (ABP) and heart rate (HR) were recorded before and during hypoxia (10% O₂ for 1 min) and hypercapnia (10% CO₂ for 3 min).

Our results showed that:

1. Carfentanil significantly reduced MPN and ∫PN by ∼50% and led to hypertension without change in f_R and HR.
2. Hypoxia enhanced MPN, ∫PN and f_R and decreased ABP, and hypercapnia brought about similar PN responses with HR decreased in the Ctrl rats.
3. Carfentanil strikingly attenuated the MPN, f_R and ∫PN during both chemical challenges and exacerbated the hypotension during hypoxia and the bradycardia during hypercapnia.

Our results suggest that carfentanil greatly suppresses the inspiratory motor drive, at least partially, via attenuating the chemoreflexes to substantially contribute to carfentanil-induced ventilatory depression and failure.

• Carfentanil

• Depression

• Hypothermia

• Motor Drive

Zhuang J, Gao X, Shi S, Xu F (2026) Carfentanil Depresses Inspiratory Motor Drive. J Subst Abuse Alcohol 13(2): 1111.

Abbreviations:

ABP: Arterial Blood Pressure; f_R: Frequency of ∫PN; HR: Heart Rate; MABP: Mean Arterial Blood Pressure; MOR: Mu-Opioid Receptor; MPN: Minute Phrenic Activity; P_ETCO₂: Partial Pressure of End-Tidal Carbon Dioxide; PN: Phrenic Nerve; ∫PN: Integrated Phrenic Nerve Activity; SpO₂: Peripheral Capillary Oxygen Saturation.

Opioid overdose induces 44,685 deaths from August 2024 to August 2025 in the United States (https://www. cdc.gov/nchs/nvss/vsrr/drug-overdose-data.htm), in which overdose fentanyl is the deadest [1-3]. Carfentanil is the second most frequently used opioid after fentanyl [4], with the potency 100-fold higher compared to that of fentanyl [5], and a highly absorbable ability [6]. Acute exposure to aerosolized carfentanil in the Moscow theater to incapacitate local Chechen rebels led to 125 deaths in 2002 [7,8]. However, the mechanisms underlying the carfentanil exposure-induced ventilatory depression and death are still not fully understood.

Our recent studies have established a model of sudden death in the spontaneously breathing rats exposed to overdose carfentanil. Acute exposure to aerosolized carfentanil for 10-15 min gives rise to severe ventilatory depression, leading to death via activation of mu-opioid receptors (MORs) [9,10]. In these cases, carfentanil could also induce several associated effects that secondarily alter ventilation, such as chest wall rigidity [11,12], hypoxemia and hypercapnia as the result of the ventilatory depression [13-15], and hypothermia [9-16]. These associated effects have been demonstrated to limit the ventilation and promote cardiorespiratory failure and arrest [17-22]. However, the effect of carfentanil on inspiratory motor drive alone (i.e., the intensity of the signal generated by the brainstem’s respiratory center to the diaphragm) without the interferences of those associated effects is still unknown. In addition, an adequate hypoxic (HVR) and hypercapnic ventilatory response (HCVR) is essential for maintaining blood gas homeostasis. It is generally accepted that opioids are able to attenuate these chemoreflexes via acting on MORs in humans [23-25], and mammalian species, including dogs [26], cats [27], rabbits [28], rats [29-32] and mice [33,34]. Importantly, the blunted chemoreflexes are known to significantly contribute to opioid-induced ventilatory depression [29-35]. To date, the investigation is lacking to define if carfentanil affects the response of inspiratory motor drive to hypoxia and hypercapnia.

The goal of this study is to determine the impacts of acute exposure to aerosolized carfentanil on the phrenic nerve (PN) activity and its responses to hypoxia and hypercapnia in the anesthetized, vagotomized, paralyzed and artificially ventilated rats. This preparation was used to avoid the other carfentanil-induced effects that can secondarily alter ventilation as mentioned above.

Animal Use

Twenty-five pathogen-free Sprague-Dawley adult male rats were purchased from Charles River Laboratories, Inc. (Wilmington, MA). All rats were housed in filter top cages in the animal facility of Lovelace Biomedical Research Institute with a 12:12 h light/dark cycle and provided with water and food ad libitum. The rooms were constantly ventilated and the temperature was kept at 24-25°C. The animals were quarantined for one week before experiments. Experiments were performed during 9:00 and 17:00 hours to avoid influence of the circadian rhythm. The experimental protocols (FY23-010) were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (IACUC), which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, USA.

General Animal Preparation

The animals were anesthetized with urethane (1.2 g/ kg, ip) and supplemental doses (0.4 g/kg, ip) administered as needed to completely suppress eye-blink and limb withdrawal reflexes. The rats were placed in a supine position for tracheotomy and tracheal cannulation [30]. The latter was loosely jacked by a tube (diameter 2.0 cm) that was connected to a gas mixing flow-meter (GF-3MP, Cameron Instrument Co., Port Aransas, TX) providing a gas mixture of 30% O₂ in nitrogen (1.0 L/min). A CO₂-sensor (Nonin LifeSense® Vet) was used for sampling the air from a branch of the cannula to measure partial pressure of end-tidal carbon dioxide (P_ETCO2) and a paw wrap sensor (Nonin Pulse Oximeter 2500V) was employed to analyze peripheral capillary oxygen saturation (SpO₂).The right femoral artery was cannulated (PE-50) for recording arterial blood pressure (ABP) and heart rate (HR) via a reusable BP transducer (ADInstruments Inc. item # MLT0380/D). Subsequently, the left cervical PN isolated and marked by placing a piece of thread loosely around the isolated nerve [36]. The animal’s core temperature (T_B) was monitored with a rectal probe and maintained at 36.5–37.5°C by a heating pad and radiant heat lamp throughout the experiment.

Experiment Protocols

After isolation of the left PN and bilateral vagus nerves and cannulation of the right femoral artery, the anesthetized and spontaneously breathing rats were placed in a standard exhaust fume hood and randomly exposed to aerosolized vehicle (Ctrl, n = 12) or carfentanil (1.7 mg/m³, n = 11) for 10 min. This concentration was selected since carfentanil concentration at 4 mg/m³ for 10 min induced 100% mortality during exposure in anesthetized and spontaneously breathing rats [10]. Immediately after the exposure, the rats were moved out from the fume hood and the cardiorespiratory activities (PN, HR and ABP),SpO₂,P_ETCO₂ and TB were recorded following vagotomy, paralysis and artificial ventilation.

It is well known that the initial HVR (within 1 min of hypoxia) is mediated primarily by the carotid body [37,38], while 3-5 min hypercapnia-induced HCVR is mainly mediated by central CO₂-chemoreceptors [39,40]. Moreover, we found that after completion of carfentanil exposure, the depressed PN activity was stable for at least 15 min in our Pilot study (n = 2). Therefore, once the baseline activity became stable, hypoxic challenge (10% O₂ for 1 min) and then hypercapnic challenge (10% CO₂ for 3 min) with a-5 min interval were applied to the Ctrl and carfentanil-exposed rats. We previously found that the PN response to the same hypoxic or hypercapnic challenge in the anesthetized, vagotomized, paralyzed and ventilated rats was fully recovered within 5 min [41]. It is worthy to note that bilateral vagotomy per se has little effect on the ventilatory responses to hypoxia and hypercapnia in animals as indicated in the previous studies [42,43].

Aerosol exposure

The vehicle solutions (saline with 0.5% methanol) without or with carfentanil (5 µg/ml) were aerosolized by a vibrating mesh nebulizer (Ireland Ltd., Galway Ireland, AG-AL1000) [10], that was connected to the end of gas mixture tubing (containing 30% O2 at the baseline) via a T-type tube. The latter loosely jacketed the tracheal cannula opening. The aerosol exposure duration was 10 minutes, with an effective nebulizer output of 0.33 ml/min for both vehicle and carfentanil solutions. The aerosol had a volume median diameter of 2.5-4.0 µm, as indicated by the manufacturer. A funnel positioned over the tracheal cannula was connected to the vacuum with inline filters (Pall BB50T Breathing Circuit Filters, Pall Corp, NY) to filter the exhausted aerosol for decontamination. The aerosol exposure was completed within the standard exhaust fume hood (size: 3 x 6 ft).

Recording of cardiorespiratory activities

Animals were placed on a surgery board in supine position, paralyzed with pancuronium (0.1-0.3 mg/kg for induction and 0.1 mg/kg/h for maintenance) and artificially ventilated (7025 Rodent Ventilator, UGO Basile SRL, Italy). The artificial ventilation (V_T, ~10 ml/kg at a rate of 60 -70 breaths/min) was adjusted to maintain P_ETCO₂ at approximately 40 torr. A positive end-expiratory pressure of 1.0 cmH₂ O was maintained to prevent atelectasis. The inlet of the ventilator was controlled by a 3-way valve so that the animal was allowed to inhale 30% O₂ (baseline), hypoxic or hypercapnic gas mixtures. Subsequently, transection of bilateral vagus nerves was conducted to prevent entrainment of phrenic motor output with the ventilator. During paralysis, the absence of nociception was evaluated and adjusted by ensuring a lack of the ABP and HR response to firmly pinching the hind toes.

The PN was cut distally, desheathed and its central end was mounted on a bipolar recording electrode and then covered with petroleum jelly to prevent drying. Raw signals of PN were band-pass filtered (100 - 3k Hz), amplified and recorded. Integrated PN (∫PN) was simultaneously calculated from the raw signal using a 0.1 s time decay. All of the recording settings were kept the same throughout the experiment in the individual rats. ∫PN (i.e., the height), frequency of ∫PN (f_R) and minute PN activity (MPN = ∫PN × f_R), equivalent to tidal volume (V_T), respiratory frequency (f_R) and minute ventilation (V_E ), were measured and calculated [44]. The ∫PN and MPN were expressed in arbitrary units (a.u. and a.u./min) in the results. These signals, coupled with airflow, ABP, HR, SpO₂,P_ETCO₂ and T_B were digitized and recorded using a PowerLab/8sp unit (model ML 785, ADInstruments Inc., Colorado Springs, CO) and a computer with LabChart Pro 7 software. An apnea is defined as the absence of PN discharges for a period equivalent to or greater than three complete respiratory cycles [16].

Chemicals

Urethane (item # U2500) and methanol (item # 34860-100ML-R) were purchased from Sigma-Aldrich (St. Louis, MO). Carfentanil was purchased from Cayman Chemical (item # 31340 in the form of 1 mg/mL solution in methanol). All of these chemicals were subsequently diluted with normal saline to desired concentrations.

Data Acquisition and Statistical Analysis

The data (MPN, PN, f_R, ABP, HR, SpO₂ ,P_ETCO₂ and T_B) obtained before hypoxia and hypercapnia were collected and expressed as the absolute values. The responses to hypoxia were determined by the largest response during the earlier phase of hypoxia and those to hypercapnia defined by the greatest response during hypercapnia. These data were presented as Δchange from the corresponding baseline data. Group data were reported as means ± SE. Student-t test was employed to analyze the difference of the data in the rats exposed to vehicle and carfentanil. Two-way ANOVA with repeated measures was used to analyze the differences of the baseline data between the rats exposed to vehicle and carfentanil, and the hypoxia- or hypercapnia-induced the change (Δchange) vs. “0” (no change) in each group and between the two groups. The Benjamini-Hochberg method to control the false discovery rate, with P-values < 0.05, was considered significant.

Inhalation of aerosolized carfentanil inhibits inspiratory motor drive

The representative recordings of cardiorespiratory activities in the Ctrl and carfentanil-exposed rats are compared in Figure 1.

Figure 1: Typical recordings to show the impact of carfentanil exposure on cardiorespiratory activities in two anesthetized, vagotomized, paralyzed and ventilated rats exposed to vehicle (Ctrl) and carfentanil (CRF), respectively. The cardiorespiratory activities include phrenic nerve (PN) activity, ʃPN, f_R, MPN, partial pressure of end-tidal carbon dioxide (P_ETCO₂), arterial blood pressure (ABP), heart rate (HR), peripheral capillary oxygen saturation (SpO₂), and body temperature (T_B), which are the same for the following figures.

Compared to the Ctrl, carfentanil exposure induced robust changes in cardiorespiratory activities. These changes were characterized by significantly reducing MPN, ʃPN and raising MABP without remarkable changes in f_R and HR. Moreover, T_B, SpO₂ and P_ETCO₂ were the same in both groups. The corresponding group data displayed the same results, and vehicle exposure failed to affect cardiorespiratory activities (Figure 2).

Figure 2: The group data to reflect the effects of carfentanil (CRF) exposure on the cardiorespiratory activities. Ctrl = 12 and CRF = 11; mean ± SE; *, P < 0.05 compared to Ctrl.

Carfentanil exposure reduces the response of inspiratory motor drive to hypoxia

The effects of carfentanil on the cardiorespiratory response to hypoxia (dropping SpO₂ by approximately 30%) are presented in Figure 3.

Figure 3: Cardiorespiratory responses to hypoxia (5% O₂ for 2 min) in anesthetized, vagotomized, paralyzed and ventilated rats exposed to vehicle (Ctrl) and carfentanil (CRF). Ctrl = 12 and CRF = 11; mean ± SE; *, P < 0.05 compared to Ctrl; †, P < 0.05 compared to before hypoxia “0”.

Hypoxic challenge (10% O₂ for 1 min) strikingly increased MPN, f_R and ʃPN and lowered MABP without effect on HR in the Ctrl rats. These responses recovered within 3–4 min after the end of the hypoxic challenge. Compared to the Ctrl, in the carfentanil-pretreated rats, the responses of MPN, f_R and ʃPN to hypoxia were substantially attenuated by 66%, 43% and 54%, respectively, and the hypotension response was aggravated. In addition, there was no difference in P_ETCO₂ and T_B between the two groups.

Carfentanil exposure diminishes the response of inspiratory motor drive to hypercapnia

As illustrated in Figure 4, hypercapnic challenge (10% CO₂ for 3 min) enhanced P_ETCO₂ from 40 torr to 70 torr. In the Ctrl rats, hypercapnia elevated MPN, f_R, ʃPN and MABP and reduced HR. The hypercapnic response fully recovered within 5 min after the exposure. In the carfentanil-pretreated rats, the responses of MPN, f_R and ʃPN to hypercapnia were profoundly blunted by 54%, 68% and 39%, respectively, and the bradycardic response was strikingly worsened. Moreover, the values of SpO₂ and T_B did not differ between the two groups.

Figure 4: Cardiorespiratory responses to hypercapnia (10% CO₂ for 3 min) in anesthetized, vagotomized, paralyzed and ventilated rats exposed to vehicle (Ctrl) and carfentanil (CRF). Ctrl = 12 and CRF = 11; mean ± SE; *, P < 0.05 compared to Ctrl; †, P < 0.05 compared to before hypercapnia “0”.

Our previous study has shown that exposure to carfentanil at 4 mg/m³ for 10 min induces severe ventilatory depression followed by sudden death in anesthetized and spontaneously breathing rats with vagal intact [45]. The cardiorespiratory depression is featured by reducing V_E, V_T and f_R by 55%, 30% and 35%, respectively, and increasing MABP without change in HR just before cardiorespiratory failure. In the present study, carfentanil exposure (1.7 mg/m³ for 10 min) inhibits inspiratory motor drive (MPN and ʃPN) by ∼50% and produces hypertension without change in f_R and HR in the anesthetized, vagotomized, paralyzed and ventilated rats. Different from the spontaneously breathing rats, there is no obvious change in f_R after carfentanil in this study (Figures 1 and 2). The lack of hypoxia/hypercapnia caused by carfentanil-induced hypoventilation may be accountable for this discrepancy. Additionally, bilateral vagotomy has been reported to significantly diminish the f_R response to opioids [46]. Consistent with our findings, carfentanil exposure (0.4–10 mg/m³ for 15–20 min) has been reported to induce dyspnea/labored breathing and ∼35–55% respiratory depression in awake mice, rats and ferrets [9–49]. Furthermore, systemic injection of carfentanil brings about ventilatory depression in rats and mice [50–53]. The advantage of our animal preparation is to avoid the carfentanil-induced associated effects, including muscle rigidity [11,12], hypoxemia and hypercapnia [13–15], and hypothermia [9,10]. More importantly, these effects can promote ventilatory depression and accelerate cardiorespiratory failure and arrest [17–54]. Thus, our results indicate a unique impact of carfentanil on inspiratory motor drive without these interferences.

In the present study, acute carfentanil exposure strikingly attenuates inspiratory motor drive during both hypoxia and hypercapnia. Particularly, carfentanil greatly suppresses the MPN, f_R and ʃPN responses to hypoxia by 66%, 43% and 47%, and hypercapnia by 54%, 68% and 39%, respectively. It also exacerbates the hypoxia-induced hypotension and hypercapnia-induced bradycardia. Our results are in line with the majority of the previous studies showing depressed HVR and HCVR [23–55]. We are aware of the lack of opioid effect on these chemoreflexes reported by some investigators [25–28]. This inconsistency may be due to the different opioid doses and agonists with different potency used. In one report, a high, but not a low, dose of fentanyl (iv) was shown to significantly depress HVR in A/J mice [33]. Owing to the preclusion of the interferences of respiratory muscle rigidity, hypothermia and hypoxemia/hypercapnia after carfentanil in this study, our data demonstrate for the first time the inhibitory impact of carfentanil on the inspiratory motor drive response to these chemoreflexes. In other words, carfentanil greatly suppresses the inspiratory motor drive, at least partially, via attenuating hypoxic and hypercapnic chemoreflexes to substantially contribute to carfentanil-induced ventilatory depression and failure.

The mechanisms responsible for the carfentanil exposure-induced respiratory depression have been investigated. Systematic antagonism of opioid receptors or MORs abrogates the respiratory depression during carfentanil exposure, while peripheral antagonism shows slight effect [9], pointing to a unique central MOR action of carfentanil. The locations of opioid central action have been extensively investigated. Opioids inhibit the respiratory-related neurons in the multiple pontomedullary regions, such as the pre-Botzinger Complex, post-inspiratory complex, nucleus tractus solitarius, medullary raphe and Kolliker–Fuse/Parabrachial nuclei [56–59]. Interestingly, a recent study implicates direct fentanyl actions on phrenic motoneurons in mice [60]. The precise mechanisms responsible for the suppressed chemoreflexes observed in our study are still unclear. HVR is triggered by stimulation of the carotid body [61], and achieved by its synaptic projection to the nucleus tractus solitarius and further projections to the respiratory network [62–64]. HCVR is primarily mediated by central CO₂/H⁺ chemosensitive neurons located in the retrotrapezoid nucleus, nucleus tractus solitarius and medullary raphe [65–67]. Studies have shown that activation of MORs in the nucleus tractus solitarius or medullary raphe blocks or diminishes HVR [32–68], and in the medullary raphe region attenuates HCVR in rats [30]. The blunted chemoreflexes are critical in the genesis of opioid-induced ventilatory depression [29–35]. Thus, it is possible that carfentanil depresses the response of inspiratory motor drive to hypoxia and hypercapnia via inhibiting these peripheral and central chemoreception and their neural circuitry in the respiratory network, which needs to be verified in future.

Significance

Opioid overdose-induced respiratory depression, especially death, in therapeutic and illicit opioid users has been a critical medical challenge [69,70]. The novelty of our findings is to reveal the direct effect of carfentanil on depression of the inspiratory motor drive and its response to hypoxia and hypercapnia without the interferences of muscle rigidity, hypoxemia/hypercapnia and hypothermia induced by carfentanil. The general similarity of depression of V_E in the spontaneously breathing rats and MPN in this study induced by carfentanil exposure suggests that the depressed inspiratory motor drive is the major contributor to carfentanil-induced ventilatory depression. Therefore, our results gain insight into the key role of depressed inspiratory motor drive in the genesis of opioid-induced ventilatory depression and thereby broaden our current knowledge about the pathophysiology of opioid-induced respiratory depression. In addition, our results may provide the foundation for development of targeted interventions to rescue the inspiratory motor drive, particularly O₂- and CO₂-chemoreflexes, and alleviate opioid-induced respiratory depression.

Prior Presentations

Part of the results were submitted to Physiology Summit Meeting, 2026, Minneapolis, under the title “Impacts of Carfentanil on the Phrenic Nerve Response to Hypoxia and Hypercapnia”.

Summary Statement

Inhalation of aerosolized carfentanil causes severe ventilatory depression, leading to death if overdosed, but the relevant mechanisms are not fully understood. Results in this study suggest that carfentanil greatly suppresses the inspiratory motor drive, at least partially, via blunting hypoxic and hypercapnic chemoreflexes to substantially contribute to carfentanil-induced ventilatory depression and failure.

Funding Statement

This study is supported by the National Institutes of Health (NIH), Bethesda. Grant numbers: HL163512 and DA059063.

Author Contribution

F. Xu, J. Zhuang and X. Gao conceived and designed the research; J. Zhuang, S. Shi and X. Gao performed the research and acquired the data; J. Zhuang, X. Gao and F. Xu analyzed and interpreted the data, and were involved in drafting and revising the manuscript.

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