Overdose Effects of Medetomidine and Fentanyl in Rats: Reflex, Vital and Analgesic Parameters as Predictive Markers

Introduction

Medetomidine (MT) is a selective α2-adrenergic receptor agonist widely used in veterinary medicine for its sedative, analgesic, and anxiolytic properties. By acting on the central nervous system, MT inhibits norepinephrine release, leading to decreased neuronal activity and profound sedation.¹ However, its use is not without risks. In cases of overdose, severe respiratory depression, loss of reflexes, and even death may occur.² Thus, the management of MT overdose (OD) remains a critical concern.

The increasing use of opioids, particularly in combination with α2-adrenergic agonists for recreational purposes, has been associated with a rising number of human fatalities due to their high potentiating effects.³ Fentanyl (FT), a highly potent opioid, is frequently combined with MT to enhance analgesia and achieve deeper anesthesia. While clinically effective, this combination substantially increases the risk of OD because of their synergistic respiratory and cardiovascular depressant effects.⁴

Recent reports highlight a concerning shift in recreational drug use trends. Xylazine, an α2-adrenergic agonist for veterinary use, previously found in illicit opioid mixtures, is increasingly replaced by MT in combination with FT.⁵ This has resulted in a growing number of cases of recreational substance abuse involving MT.^6–8 Compared with xylazine, MT produces stronger sedation, bradycardia, hypotension, and central nervous system depression, with effects that may persist longer than those of either FT or xylazine.⁹ Medetomidine, an α₂-adrenergic agonist, despite being used only in veterinary medicine, is increasingly implicated in FT related overdose cases. During May 2024, Philadelphia public health officials began testing for this drug in overdose cases of FT, and MT was found in 29% of FT samples. Six months later this prevalence increased to 87% of FT samples.¹⁰ In Michigan, between March 1, 2024 and May 16, 2025, 4290 post-mortem blood samples were tested, MT being detected in 17 specimen, 16 of 17 samples contained FT in combination with MT.¹¹ Despite its clinical and toxicological importance, there is limited information in veterinary medicine regarding the dose ranges of MT, alone or in combination with FT that may induce OD.^6–8

Most published studies involving MT evaluate its effects as part of balanced anesthetic protocols, in which drug interactions, inhalant anesthetics, and clinical titration obscure the independent contribution of α2-agonist exposure to cardiorespiratory depression.^2,12,13 In contrast, experimentally defined overdose thresholds for MT alone, or in simple combination with FT, remain poorly characterized.^4,14,15 The combination of MT with FT is mentioned in several articles in which the administration was carried out intraperitoneally in rats,^4,13 the rest of the studies being carried out on other animal species.^16–20

The importance of this study extends beyond veterinary medicine, providing additional information relevant to understanding the clinical use and increasing number of recreational abuse cases involving MT and opioids.¹⁶ This knowledge gap is increasingly relevant in the context of the current opioid crisis in the United States and the increasing reports of recreational use of α2-agonists and fentanyl, the results obtained contribute to the foundation of better-informed measures regarding the safety, monitoring, and dosing of these pharmacological combinations.^6–8

Materials and Methods

The study was carried out with the approval of the USAMV Cluj-Napoca Bioethics Commission, no. 438 of 17.04.2024 and the project authorization no. 407 of 21.05.2024 from the Romanian National Sanitary Veterinary and Food Safety Authority (NSVFSA). All experimental procedures were performed in accordance with EU Directive 2010/63/EU on the protection of animals used for scientific purposes and Romanian Law No. 43/2014. The study was carried out in an authorized facility for laboratory animal research at the University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Romania (Authorization No. 926, issued on October 6, 2021).

The animals were not euthanized and allowed to recover. None of the rats died during or after the experiment. This decision was made in accordance with the 3Rs (Replacement, Reduction and Refinement) principle and the requirements of Directive 2010/63/EU on the protection of animals used for scientific purposes. The procedures performed in this study did not cause moderate or severe suffering, and the handling did not cause stress or suffering. Intramuscular administration of the substances was performed using a 1 mL syringe and a 27G needle, a mild procedure (Article 15, Directive 2010/63/EU). All animals were monitored for at least 60 minutes according to the experimental protocol, or until the complete recovery of reflexes and physiological parameters. This approach reflects the ethical commitment to refine experimental protocols by minimizing unnecessary harm and avoiding the euthanasia of healthy animals when scientifically and ethically justified.

Chemicals

Fentanyl (Fentanyl Kalceks, 50 µg/mL, 10 mL ampoule, total 500 µg, Kalceks, Riga, Latvia) and medetomidine (Domitor, 1 mg/mL, 10 mL vial, total 10 mg, Orion Pharma, Espoo, Finland) were purchased from commercial sources.

Animals

A total of 40 Wistar rats (Rattus norvegicus) were purchased from the authorized laboratory animal facility, the Centre for Experimental Medicine and Practical Skills, “Iuliu Hațieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania. The cohort included both males and non-pregnant females, aged 8–12 weeks and weighting 185–418g.

The rats were housed in a controlled environment at 22 °C and approximately 60% humidity. Based on cage size, 3–5 rats were assigned per cage, separated by sex. Animals were acclimatized for six days prior to the start of the experiment. A 12-hour light–dark cycle was maintained, and food and water were provided ad libitum.

Experimental Design

A total of eight groups of rats were formed. Four groups, each comprising 2 males and 2 females, were used to determine the OD of MT, while the remaining four groups, with the same sex composition, were used to assess the OD of the MT and FT combination. All groups were homogeneous with respect to body weight and sex ratio.

MT was administered intramuscularly in doses of 0.01, 0.03, 0.05 and 0.1 mg/kg. The standard dose of MT (0.1 mg/kg) determined in phase one of the study as most effective, was combined and administered intramuscularly with the following doses of FT: 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, and 0.1 mg/kg (Figure 1). No rats required the administration of antagonistic substances.

Figure 1 Study design.

In the groups receiving only MT, body weight (g) standard deviation ranged from ±22.68 to 36.57, whereas in the MT + FT groups, it ranged from ±29.54 to 48.95. In Phase 1, multiple doses were screened (n=4 per group; 2 males/2 females) to identify the most efficient MT dose. In Phase 2, different doses of FT were tested in combination with the most efficient dose of MT from phase 1. The groups producing the most consistent and clinically relevant effects (MT 0.1 mg/kg and MT 0.1 mg/kg + FT 0.1 mg/kg) were expanded by adding four additional animals (balanced by sex) to improve the estimate precision and strengthen internal replication of the primary values while limiting animal use. A single expanded group improves robustness of the key regimen’s effect estimate, but it should be interpreted as supportive internal confirmation rather than definitive confirmatory validation.

Clinical and Neurophysiological Monitoring

Overdose (OD) state was confirmed when all five standard reflexes were completely abolished, and the HR and RR values showed significant percentage decreases, ranging between 15.7–31% for HR and 15.4–28% for RR compared to the values obtained at the initial time point (At0). The establishment of the recovery time was based on the complete reappearance of the five standard reflexes, the presence of spontaneous mobility, reactivity to painful, tactile or acoustic stimuli and the return of physiological parameters to values close to those recorded at the At0 time.

The evaluation of vital signs was through monitorization of respiratory rate (RR), heart rate (HR), rectal temperature (RT) and tissular oxygen saturation (SpO2). For these four physiological parameters the percentage reduction was calculated at the time of OD and at the end of the monitoring period (Pt60) compared to the pre-therapeutic (At0) values, using the formula: .²¹

Neurophysiological state of rats was assessed by the monitoring of five standard reflexes: alertness (AN), astasia (AT), palpebral reflex (PR), righting reflex from lateral decubitus position (Rref), and sternal recumbency (SR) - the rat has no tendency to rise from the sternal recumbency position, in a support position on the 4 limbs.²²

The antinociceptive evaluation (ANT) was performed by the cold ethanol (at −20°C) tail flick test (TFT),²³ which is a method frequently used in pharmacological research to evaluate antinociceptive effects in rats.^23,24

Assessment Schedule

Before intramuscular administration of the test substances, each rat was placed in an individual cage and allowed a 20-minute acclimatization period (Figure 2). Clinical monitoring and evaluation of physiological parameters (RR, HR, RT and SpO₂) as well as neurological reflexes and nociceptive response were carried out over a 60-minute observation period. In the first 10 minutes post-injection, assessments were performed every 2 minutes in order to capture rapid changes in vital functions and reflex responses. Subsequently, evaluations were performed every 10 minutes, until the 60-minute mark (Figure 2). Parameters were also assessed at baseline (T0), prior to drug administration, to allow comparison with post-administration values.

Figure 2 Design of the experiment: Monitored clinical parameters: respiratory rate (RR), heart rate (HR), rectal temperature (RT) and tissue oxygen saturation (SpO2); and standard reflex alertness (AN), ataxia (AT), palpebral reflex (PR), righting reflex from lateral decubitus position (Rref), and sternal recumbency (SR). TFT was performed 20 minutes prior At0 and 40 minutes post-administration of MT or MT combined with FT. Parameters were also assessed at baseline prior to drug administration (At0), to allow comparison with post-administration (Pt60) values.

For ANT, rats (male and female) were acclimatized prior to testing to reduce stress-related variables. The initial measurement was performed 20 minutes before (At −20) the administration of the therapeutic protocol, and the next measurement, 40 minutes’ post-administration (Pt +40) (Figure 2). To prevent tail and skin injuries, a time limit of 40 seconds (cutoff) was established. Analgesic efficacy was expressed by the percentage of maximum possible analgesia (%MPA), calculated according to the formula:.²² The values obtained reflect the withdrawal latency and the degree of analgesia induced by the tested substances. A significant increase in the response time indicates the antinociceptive potential of the administered drugs.

The time points corresponding to OD induction and full recovery were noted individually for each animal based on the presence or absence of standard reflexes and the return of vital parameters to baseline levels. All measurements were carried out by the same investigator who was blinded to the treatment protocol.

Instrumentation and Monitoring Procedures

For HR monitoring, the EDAM - IM8 VET system (manufacturer Cardiac direct, 4204 Jutland Drive, Suite A, San Diego, USA) was used. The conscious rat was placed in a cage that limited its movements. HR and ECG sensors were placed on the two forelimbs and the left hind limb. In situations where the rat tended to move during monitoring, which could have affected the recorded values, these were compared and correlated with the values obtained by measuring the pulse (applying the sensor to the base of the tail). For monitoring RR, sensors were applied to the extremity of the two front limbs and on the left hind limb. To avoid errors, the method of direct observation of chest movements was also used - as animals breathe, their rib cage moves to inflate and deflate their lungs. The movements of the rib cage, visible to the eye, can be monitored as a measure of respiration.²⁵

M3T Vet Monitor (producer Tootoo Meditech co, LTD, ShenZhen, China) was used to monitor SpO2 via a sensor applied to the distal end of the right hind limb RT temperature was measured using a rectal probe.

For ANT, the Neslab RTE 7 apparatus (ThermoElectron, Newington, CT, USA) with an adjustable temperature of between −25°C and +150°C (±0.01°C) and 70% ethanol solution were used for (TFT).

Statistical Analysis

All data were statistically analyzed using GraphPad InStat 3.10 software. The unpaired t-test with Welch’s correction was applied to compare physiological parameters between treatment protocols or genders at specific time points (eg, OD or Pt60). Effect sizes were also calculated using eta squared (η²), which complements p-values by quantifying how much of the observed variance is attributable to the effect of MT 0.1 mg/kg or MT 0.1 mg/kg combined with FT 0.1 mg/kg.

To assess the evolution of physiological parameters over time in response to treatment, a two-way repeated measures ANOVA was conducted. When significant differences were detected through two-way ANOVA, Bonferroni post-hoc corrections were applied to adjust for multiple comparisons. In addition to p-values, 95% confidence intervals (CIs) were reported.

Results

Medetomidine Standard Overdose Determination

Testing the four doses of MT (0.01, 0.03, 0.05 and 0.1 mg/kg) revealed that only the dose of 0.1 mg/kg caused the complete abolition of the five standard reflexes in all individual, from the OD moment until the end of the 60-minute monitoring period.

The doses of 0.05 and 0.1 mg/kg caused complete suppression of standard reflexes and consistent physiological changes compatible with OD (Figure 3A). At doses of 0.01 and 0.03 mg/kg, OD criteria were not reached, and reflexes were partially or completely maintained throughout the entire monitoring period. At the dose of 0.05 mg/kg, all rats (n = 4) became overdosed, but showed partial recovery of reflexes and vital functions by minute 60. The dose of 0.1 mg/kg induced complete OD, with no recovery during the monitoring period, but no antidote was required, as the animals did not show any clinical signs of life-threatening conditions (eg critical oxygen saturation or severe bradycardia). For this dose, an additional batch (n = 4) was added, for robust confirmation of the observed effect.

Figure 3 Results of MT dose testing: (A) OD induction time results. (B) Comparative results of HR, at time At0, OD and Pt60, depending on the dose of MT used. (C) Comparative results of RR, at time At0, OD and Pt60, depending on the dose of MT used.

At the 0.05 mg/kg dose, the mean time to OD was 1357.5 ± 527.1 seconds, with considerable interindividual variability. In contrast, at the 0.1 mg/kg dose, the mean time for OD was 468.75 ± 140.4 seconds, indicating a faster and more uniform response (Figure 3A). The difference in the dispersion of values highlights that the 0.1 mg/kg dose has a more predictable pharmacodynamic profile, justifying its selection as the standard OD dose for subsequent testing.

In the case of HR at the time of OD, mean values of 231 ± 6.32 bpm were obtained for the dose of 0.05 mg/kg, the percentage decrease was 33.8%, reaching an average of 210 ± 29.16 bpm by the end of the 60 minutes, being 40.2% lower than the At0 value. HR decreased significantly at the 0.1 mg/kg MT dose, from a mean value of 331.9 ± 35.04 bpm recorded at the At0, to 238.1 ± 32.51 bpm in the OD time decreasing by approximately 28.2%. At Pt60, HR continued to decrease, reaching a mean of 109.62 ± 24.59 bpm, reflecting a 66.1% decrease compared to the pre-therapeutic value, suggesting a sustained inhibition of cardiac activity (Figure 3B).

At the dose of 0.1 mg/kg MT, RR was affected, decreasing by 20.3% at the time of OD, from a mean value of 90.8 ± 9.73 brpm at At0, to 72.4 ± 12.25 brpm during OD. Subsequently at the time of Pt60 (49.5 ± 10.01brpm), the RR showed a decrease of 45.5% compared to the At0 (Figure 3C). These data highlight a progressive respiratory depressant effect, without reaching a critical threshold that would have required emergency therapeutic intervention.

Tissue oxygen saturation (SpO₂ - Figure 4A) and rectal temperature (RT - Figure 4B) were maintained throughout the experiment within physiological limits, without significant variations between evaluation times or doses of MT administered.

Figure 4 Results of MT and MT + FT dose testing: (A) SpO2 values. (B) Rectal temperature values.

Following the analysis of the results obtained by testing the four doses of MT, the dose of 0.1 mg/kg MT was selected as the standard overdose, due to the shortest induction time, the complete abolition of standard reflexes even after Pt60, as well as the marked decrease in RR and HR.

Fentanyl Standard Overdose Determination Combined with 0.1 mg/kg MT

The administration of the combination of 0.1 mg/kg MT with four different doses of FT (0.025, 0.05, 0.075 and 0.1 mg/kg) revealed a marked synergistic effect, especially with the 0.1 mg/kg FT dose. All doses of FT used in combination with MT resulted in complete abolition of standard reflexes and induced OD. The shortest time required for complete abolition of reflexes (243.75 ± 119.39 seconds) was obtained at the dose of 0.1 mg/kg of FT combined with the standard dose of MT (Figure 5A).

Figure 5 Results of testing the dose of 0.1mg/kg MT combined with 0.025, 0.05, 0.075, 0.1 mg/kg of FT: (A) OD induction time results (** p=0.0058). (B) Comparative results of HR, at time At0, OD and Pt60, depending on the FT dose. (C) Comparative results of RR, at time At0, OD and Pt60, depending on FT dose.

The administration of MT 0.1 mg/kg in combination with four progressive doses of FT caused a dose-dependent decrease in HR in all protocols investigated (Table 1; Figure 5B). All doses of FT combined with MT induced progressive bradycardia (Table 1) but the values were maintained within limits compatible with physiological functioning in all rats.

Table 1 The Mean Values of HR (Bpm) Measured at At0, OD, Pt60, and the Percentage Decrease (%) of the Parameter Compared to the At0 Value

RR followed a downward trend in all groups of rats administered the combination of MT with FT, indicating a cumulative effect of OD. At baseline (At0), mean values were comparable between groups, but at Pt60 there were significant differences. Doses of 0.075 and 0.1 mg/kg FT produced the most pronounced decreases in RR (Table 2; Figure 5C), without inducing apparent respiratory distress, suggesting superior sedative efficacy in the absence of immediate life-threatening risk.

Table 2 The Mean Values of RR (Brpm) Measured at At0, OD, Pt60, and the Percentage Decrease (%) of the Parameter Compared to the At0 Value

Similar to MT protocol, tissue oxygen saturation (SpO₂ - Figure 4A) and rectal temperature (RT - Figure 4B) were maintained throughout the experiment within physiological limits, without significant variations.

Based on the results, of the four doses of FT, 0.1 mg/kg was selected as the standard overdose in combination with 0.1 mg/kg MT.

Comparative Analysis

Comparative statistical analysis of the OD time, HR, RR and differences between genders were performed between the rats groups treated with the dose of 0.1 mg/kg MT versus the group treated with 0.1 mg/kg MT in combination with 0.1 mg/kg FT.

A shorter time was highlighted for OD induction time in the case of the combination of MT with FT (243.75 seconds), compared to the administration of MT alone (468.75 seconds), being statistically significant (unpaired Welch’s t-test: t (13.23) = 3.28, p = 0.0058) (Figure 6A). The mean difference between groups was −225.0 ± 68.6 seconds, with a 95% CI ranging from −372.9 to −77.1, confirming that the effect is robust and unlikely due to chance. The effect size was moderate to large (η² = 0.45), indicating that approximately 45% of the variability in induction time could be explained by administered medication. These results demonstrate that the addition of FT to MT significantly accelerates the onset of overdose, reducing induction time by 48%.

Figure 6 Comparative results between MT versus MT combined with FT. (A) OD induction time (**p=0.0058). (B) Comparative results of HR, at time At0, OD and Pt 60 (** p=0.0021; *** p<0.0001). (C) Comparative results of RR, at time At0, OD and Pt60.

Comparison of HR values revealed statistically significant differences at OD and Pt60. Unpaired t-test with Welch correction indicated a significant HR difference at OD between the MT and MT+FT groups (p = 0.0021; 95% CI: 23.99–87.26), with a moderate to strong effect (η² = 0.5039) (Figure 6B). The impact of FT is extremely significant at Pt60 (p <0.0001; 95% CI: 111–168.4) with a strong effect (η² = 0.8866), where the values obtained at this time are closer to the At0 values, unlike the group with MT alone (Figure 6B). Bidirectional ANOVA analysis with repeated measurements confirmed a significant effect of time (p < 0.0001), with HR varying significantly between At0, OD and Pt60. The effect of administrated medication (p < 0.0001) is significant, indicating clear differences between MT versus MT combined with FT. Furthermore, there is a significant interaction between time and medication (p < 0.0001), suggesting that the evolution of HR over time is influenced by the administered medication and the time of measurement. Bonferroni post-hoc tests showed significant decreases in HR over time for both protocols. In the MT group, all differences between At0, OD and Pt60 were significant (p < 0.0001), indicating a potent and progressive effect of MT on HR. In the MT+FT group, although the differences between At0 versus OD and versus Pt60 were significant (p < 0.001). The decrease between OD and Pt60 did not reach statistical significance (p = 0.0551), suggesting a possible stabilization of the effect after association with FT.

Changes in RR between the MT and MT+FT rat groups did not reach statistical significance at any of the analyzed time points (OD and Pt60) (Figure 6C). Unpaired t-tests indicated at OD moment: p = 0.1657 (95% CI: −3.673–19.42), η² = 0.1326; and Pt60 moment: p = 0.1980 (95% CI: −23.28–5.285), η² = 0.1154. Two-way ANOVA confirmed a strong effect of time (p < 0.0001), but not of administrated medication (p = 0.7540). The time and medication interaction was significant (p = 0.0395), indicating that changes in RR over time are influenced by the administered medication. Subjects also contributed significantly to the variability (p = 0.0117), highlighting the diversity of individual physiological responses. Bonferroni tests showed significant decreases in RR between all-time points (At0 > OD >Pt60) in both groups (all p < 0.05). However, in the MT combine with FT group, the differences were less pronounced, suggesting a possible moderating effect of FT on the reduction in RR (Figure 6C).

Gender Statistical Analyses

Comparative analysis according to gender of rats in the group treated with MT alone and in the groups treated with MT combined with FT revealed trends suggesting gender-specific differences (Figure 7A).

Figure 7 Comparative results between MT versus MT combined with FT, gender analysis. (A) OD induction time (* p= 0.0614; **p=0.0064). (B) Comparative results of HR, at time At0, OD and Pt60 (*** male p=0.0006; female p=0.0003). (C) Comparative results of RR, at time At0, OD and Pt60.

The males from MT combined with FT group showed a shorter induction time of OD compared to MT alone (132.5 seconds vs 377.5 seconds), but the difference did not reach statistical significance (p = 0.0614). The large effect size (η² = 0.74) and unequal variances (p = 0.0006) suggest high inter-individual variability, indicating a less consistent response in males.

In females from the MT combined with FT group, the effect was statistically robust, showing a significantly shorter induction time than in the MT alone group (355.0 seconds vs 560.0 seconds; p = 0.0064). The difference of −205 s was supported by a very strong effect size (η² = 0.92), explaining over 90% of the variability. Analysis of variance (p = 0.0262) showed greater variability under FT, but the effect remained consistent and reliable.

These findings demonstrate that FT markedly potentiates the sedative effects of MT, with a gender-dependent profile: females showed a more consistent and statistically significant reduction in overdose induction time, while males exhibited a more variable response.

HR gender stratified analysis showed that the MT combined with FT protocol was consistently associated with significantly higher heart rates compared with MT alone. At OD, females treated with MT combined with FT had higher mean values than those in the MT group (282.5 vs 253.8 bpm; p = 0.0197, η² = 0.62). In males, the difference was even greater (305.0 vs 222.5 bpm; p = 0.0198, η² = 0.62). At Pt60, these differences became more pronounced. In males, heart rate was more than doubled in the MT combined with FT group compared to MT alone (251.0 vs 118.0 bpm; p = 0.0006, η² = 0.88). In females, the effect was stronger, with MT-FT animals showing over twice the heart rate of the MT group (247.8 vs 101.3 bpm; p = 0.0003, η² = 0.91). Overall, MT combined with FT significantly attenuated the bradycardia induced by MT alone, with the effect most evident at 60 minutes and more pronounced in females (Figure 7B).

Analyzed separately, RR by gender showed no robust statistical significance; however, certain patterns emerged. At OD, males showed nearly identical values across groups (78.8 vs 76.5 rpm; p = 0.639, η² = 0.04). In females, respiratory rate tended to be higher in the MT-FT group (84.0 vs 66.0 rpm), but the difference did not reach statistical significance (p = 0.088, η² = 0.41). At Pt60, males in the MT combined with FT group exhibited lower mean values than those in the MT group (39.0 vs 52.5 rpm), while females showed a similar trend (42.0 vs 46.5 rpm). However, neither difference was significant (p > 0.2 for males; p = 0.675 for females). Overall, RR reductions followed a dose- and time-dependent trend, but the addition of fentanyl did not significantly alter the magnitude of respiratory depression compared with MT alone (Figure 7C).

To evaluate if gender has an impact on the treatment response, the primary values obtained during monitorization in the group MT 0.1 mg/kg and MT 0.1 mg/kg + FT 0.1 mg/kg, were analyzed using a two-way ANOVA where gender and group treatment were fixed factors. OD time showed significant main effects of both treatment (F (1,12) = 24.88, p = 0.0003) and gender (F (1,12) = 20.16, p = 0.0007), males reaching OD earlier than females. The interaction between treatment and gender was not significant (F (1,12) = 0.1966, p = 0.6654), indicating that the treatment effect did not differ between genders. HR at OD demonstrated a strong main effect of treatment (F (1,12) = 86.92, p < 0.0001), with MT+FT resulting in markedly lower HR compared with MT alone. Neither gender (p = 0.6085) nor the interaction between treatment and gender (p = 0.1072) reached significance. Heart rate at 60 minutes (Pt60) showed a similarly robust effect of treatment (F (1,12) = 99.67, p < 0.0001), with MT+FT substantially attenuating MT-induced bradycardia. Again, neither gender (p = 0.4887) nor the treatment and gender interaction were significant (p = 0.6383), demonstrating that the cardiovascular effects of the combined protocol were consistent across genders. Respiratory rate at OD and Pt60 was not significantly affected by treatment (p = 0.1391 at OD; p = 0.2267 at Pt60) or gender (p = 0.6070 at OD; p = 0.8354 at Pt60), and no significant treatment and gender interactions were observed (p = 0.0643 and p = 0.5360, respectively).

Although these findings require validation in larger cohorts, they raise the possibility of gender-related differences in pharmacodynamics sensitivity to combined α₂-adrenergic agonist and opioid administration. Such differences, if confirmed, could have translational relevance for both veterinary and human medicine, where individualized dosing strategies may optimize safety and efficacy.

Antinociceptive Evaluation

The antinociceptive evaluation was performed for all groups of rats. The maximum value (100%) of MPA percentage was reached for the 0.1 mg/kg dose of MT and for all four different doses of FT in combination with 0.1 mg/kg MT. These results reveal the superior analgesic potential of the combination between MT and FT.

Discussion

Our findings confirm and extend previous reports in the literature by demonstrating the OD potential of medetomidine (MT) at a dose of 0.1 mg/kg in Wistar rats, as well as the modulatory effects of fentanyl (FT) when combined with MT. The cardiovascular depression induced by MT alone, characterized by a progressive and dose-dependent bradycardia, is consistent with earlier reports in humans, dogs, rabbits, and calves.^26–29 As described in experimental models, MT induces an initial hypertensive phase followed by sustained bradycardia.³⁰ The drug reduces central sympathetic outflow, increases vagal tone, and, together with an initial increase in systemic vascular resistance, produces a reflex and centrally mediated bradycardia.^31,32 In our study, the reduction in heart rate with MT alone was more pronounced at Pt60, reaching a 66% decrease compared to baseline, thereby confirming its strong cardiac inhibitory profile. Importantly, during overdose, animals metabolic demand is reduced, so a lower cardiac output can still maintain adequate tissue perfusion. Our findings showed that the animals remained hemodynamically stable and recovered uneventfully, indicating that the bradycardia represented a pharmacologically mediated, reversible adaptation rather than imminent decompensation.^19,33

Interestingly, the addition of FT appeared to modulate this bradycardic effect. While FT itself is known to induce bradycardia through vagal stimulation, particularly in multimodal anesthesia protocols,^34,35 in our study, the combination MT with FT resulted in a less steep heart rate decline compared to MT alone. This dose-dependent modulation is in line with reports that low-dose FT may not significantly impact cardiovascular dynamics during anesthesia and may counterbalance the sympathetic blockade induced by MT.^36–38

Regarding respiratory function, both MT and FT have been consistently associated with respiratory depression.^27,39 MT alone produced an evident reduction in respiratory rate during OD, while the addition of FT initially appeared to attenuate this effect at the point of OD, but by 60 minutes post-administration, respiratory depression was more pronounced in the combined protocol. This dynamic interaction parallels previous findings in dogs,^20,40 where opioids and α₂-agonists displayed both synergistic and modulatory effects on respiration depending on timing and dosage. Notably, none of the rats in our study displayed the “wooden chest” phenomenon typically associated with high-dose opioid use,⁴¹ possibly due to the muscle-relaxant properties of MT that counteract opioid-induced rigidity.⁴²

Body temperature and SpO₂ remained within physiological limits across all protocols, corroborating the thermoregulatory stabilizing effects of α₂-agonists,⁴³ and supporting the idea that low-dose FT in combination with MT does not exacerbate hypoxemia. The evaluation of standard reflexes confirmed that both MT and MT+FT protocols achieved profound central nervous system depression, as also observed in previous studies combining these agents.⁴ Analgesic efficacy, as demonstrated by the TFT, reached 100% for the 0.1 mg/kg dose of MT alone and for all four FT doses administered in combination with 0.1 mg/kg MT. This outcome is consistent with the documented potency of FT, which at 0.1 mg produces an analgesic effect equivalent to approximately 10 mg of morphine.^44,45

The analgesic effect of MT alone, consistent with its potent α₂-adrenergic agonist activity, which modulates nociceptive transmission at both spinal and supraspinal levels.^2,16,18

These results highlight that while MT alone produces a steep, dose-dependent bradycardia and respiratory depression, the addition of FT modulates these effects in a dose-related manner, without exacerbating hypoxemia or thermoregulatory imbalance. The translational relevance of these findings is notable: both MT and FT are widely used in clinical and experimental practice, and their interaction carries important implications not only for veterinary anesthesia but also for human medicine, particularly in the context of the ongoing opioid crisis. Understanding the complex cardiorespiratory interactions between α₂-agonists and opioids may inform safer anesthetic protocols and contribute to better strategies for minimizing the risks associated with opioid misuse and OD.

Although MT is approved for veterinary use, emerging public health and clinical toxicology reports indicate that α₂-adrenergic agonists, including MT and structurally related compounds such as xylazine, are increasingly encountered in the illicit human drug supply, often in combination with fentanyl.^6–9,11,41

The present rodent model does not aim to establish clinical overdose thresholds for humans, but rather to define reproducible, physiology-based patterns of cardiorespiratory depression and reflex impairment under controlled conditions. Given the high conservation of α₂-adrenergic and μ-opioid receptor systems across mammals, such experimental data provide mechanistic insight into drugs interactions that are increasingly relevant in human overdose settings.⁴⁶

Conclusion

The combination of 0.1 mg/kg MT with 0.1 mg/kg FT produced a more rapid and profound OD compared with MT alone, with marked effects on heart rate but a less pronounced reduction in respiratory rate. The addition of FT appeared to mitigate the progressive bradycardia induced by MT, with heart rate values at OD and Pt60 remaining closer to baseline (At0) compared with MT monotherapy. This suggests that FT potentiates the sedative and cardiorespiratory effects of MT, but may also contribute to a partial stabilization of physiological responses in the later phases of OD. The findings from the present study underscore the necessity of precise calibration when employing pharmacological combinations in both experimental and clinical contexts.

This study has several limitations that should be considered when interpreting the findings. First, overdose thresholds were defined using behavioral and physiological criteria (loss of reflexes, HR, RR). Still, invasive measurements which may have provided additional resolution on the degree of cardiorespiratory compromise, such as arterial blood pressure, blood gases, or direct oxygen saturation, were not obtained. Second, the protocol experiment was exploratory and limited to a restricted number of MT and FT doses; therefore, the thresholds identified here should be viewed as estimates within the tested range rather than definitive toxic doses. Third, all experiments were performed in healthy adult rats, and the tolerance to MT and MT+FT induced depression may differ in animals with comorbidities, altered metabolic status, or different ages. Fourth, intramuscular administration of both agents used in this model may not fully reflect clinical or illicit-use exposure patterns, which can involve variable absorption kinetics. Finally, although gender was included as a biological factor, the statistic analyses was not powered to detect subtle gender-dependent interactions beyond the main treatment effects. Collectively, these limitations indicate that the present findings define controlled experimental overdose parameters but should not be extrapolated directly to clinical or forensic contexts without further confirmatory studies.