Tandospirone Prevents Anesthetic-Induced Respiratory Depression through 5-HT1A Receptor Activation in Rats

doi:10.21203/rs.3.rs-3966570/v1

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Tandospirone Prevents Anesthetic-Induced Respiratory Depression through 5-HT1A Receptor Activation in Rats

https://doi.org/10.21203/rs.3.rs-3966570/v1

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Background: Respiratory depression is a side effect of anesthetics such as fentanyl, dexmedetomidine, and midazolam. Clinical treatment with specific antagonists or respiratory stimulants interferes with the sedative effects of anesthetics; therefore, drugs that ameliorate respiratory depression without affecting the sedative effects of anesthetics are needed. Previous studies have suggested that tandospirone may be one such candidate. Therefore, we performed pharmacological studies in rats to evaluate this issue.

Methods: The pharmacodynamic ability of tandospirone to ameliorate respiratory depression and its effects on arterial oxygen saturation (SaO₂) were evaluated in a rat model under anesthesia. The protein kinase A redistribution method was used to determine whether tandospirone activated α_2a/2cand μ receptors. The effects of tandospirone on current modulation of α₁β₂γ₂ and α₄β₂δ gamma amino-butyric acid (GABA) receptors were explored using the two-electrode voltage clamp technique.

Results: Prophylactic administration of tandospirone reduced respiratory depression caused by fentanyl, dexmedetomidine, and midazolam in rats. Tandospirone increased SaO₂ in rats treated with fentanyl or midazolam. The ability of tandospirone to prevent respiratory depression was completely inhibited by the 5-hydroxytryptamine (5-HT)_1A receptor antagonist WAY100635. Co-administration of tandospirone with dexmedetomidine or fentanyl did not affect the activation of α_2a/2c or μ receptors by dexmedetomidine or fentanyl. Tandospirone did not affect the modulation of α₁β₂γ₂ and α₄β₂δ GABA receptors by midazolam.

Conclusion: Tandospirone ameliorates respiratory depression caused by anesthetics in rats through 5-HT_1A receptor activation. Future studies should validate these findings and evaluate whether tandospirone has clinical application value for ameliorating respiratory depression in patients receiving anesthetics.

Biological sciences/Drug discovery/Pharmacology

Biological sciences/Drug discovery/Pharmacology/Pharmacodynamics

Biological sciences/Drug discovery/Pharmacology/Receptor pharmacology

5-HT1A

tandospirone

respiratory depression

fentanyl

midazolam

dexmedetomidine

Anesthetic drugs are widely used for anesthetic induction and intraoperative stabilization, and they play a crucial role in surgical practice. Widely used anesthetic agents include opioids, such as fentanyl (Zaki et al. 2023); benzodiazepines, such as midazolam ( Nordt SP, Clark RF 1997); and α₂-adrenergic receptor agonists, such as dexmedetomidine (Zaki et al. 2023). However, respiratory depression is common when using anesthetic drugs (Kienitz et al. 2022). Compared with the normal breathing pattern, the respiratory rhythm during depression is fast and shallow with transient pauses. Respiratory depression can lead to death in people with opioid overdose. When high doses of midazolam or dexmedetomidine are administered in a single session, respiratory depression can occur, especially with combined use of anesthetics, which increases the body’s tolerance to respiratory depression. This phenomenon seriously threatens patient health and safety and increases the medical burden.

Physiological antagonists can reverse life-threatening respiratory depression (France et al. 2020). Examples include naloxone (often used with fentanyl), flumazenil (often used with midazolam), and atipamezole (often used with dexmedetomidine). Although these antagonists can reverse respiratory depression, they also reverse the sedative and analgesic effects of anesthetics (Britch and Walsh 2022). Moreover, naloxone, which is an opioid antagonist, is associated with acute withdrawal symptoms (Longnecker et al. 1973). Abdelal et al. suggested that multiple naloxone administrations are needed to reverse opioid overdose (Abdelal et al. 2022), which increases the potential for violent behavior in patients after reversal of sedation with intravenous opioid overdose (Gaddis and Watson 1992). Similarly, the antagonistic effects of flumazenil are not limited to respiratory depression. Flumazenil also blocks the sedative effects of midazolam (Gross et al. 1991). When flumazenil blocks the effects of high-dose midazolam and other long-acting benzodiazepines, the danger of re-sedation should be considered, which occurs as the concentration of antagonist decreases (Kamohara et al.) demonstrated that atipamezole causes severe hypotension and bradycardia at 200 µg/kg in cats (Kamohara et al. 2021), which is not desirable. However, respiratory depression caused by dexmedetomidine has not received much research attention.

Respiratory depression is a common side effect of widely used anesthetic drugs; therefore, agents that prevent respiratory depression without reducing the sedative and analgesic effects of these anesthetics are urgently needed. With this goal in mind, we performed a preliminary literature search, which revealed that tandospirone may have the potential to improve respiratory depression without affecting sedation and analgesia (Fan et al. 2022). Tandospirone has shown promising anxiolytic effects in animal models, and it is now marketed for the treatment of anxiety disorders (Pollard et al. 1992). Tandospirone exerts its anxiolytic effects by activating postsynaptic 5-hydroxytryptamine (5-HT)_1A receptors coupled with G-proteins (G_i/o) (Huang et al. 2017), which inhibit the activity of adenylate cyclase, thereby reducing cyclic adenosine monophosphate (cAMP) and inhibiting protein kinase A (PKA)-mediated protein phosphorylation (Polter and Li 2010). However, the mechanism of action by which tandospirone improves respiratory depression remains unclear. In this study, we used a rat model to evaluate whether tandospirone prevents respiratory depression caused by anesthetics without affecting sedation and analgesia.

2.1 Animals and Cells

Male Sprague–Dawley rats (weighing 180–220 g) were provided by Beijing Huafukang Biotechnology Co. Ltd. (Beijing, China). The rats were housed in a controlled environment with a 12-hour/12-hour light–dark cycle at a constant temperature of 24°C ± 2°C in transparent boxes. The lights were switched on at 07:00, and the animals had unrestricted access to food and water and could move around freely. All experiments were approved by the Institutional Animal Care and Use Committee (approval number: IACUC-DWZX-2023P562), and the study protocol conforms to the ethical guidelines of the ARRIVE guidelines 2.0 (July, 2020 Publication). All methods were carried out in accordance with relevant guidelines and regulations.

Enhanced green fluo⁃rescent protein (EGFP) marked CHO-PKAcat cells were provided by Thermo Fisher Scientific.

2.2 Drugs

Tandospirone citrate and dexmedetomidine citrate were prepared by TargetMol Biotechnology Co. (Boston, MA, USA). Midazolam hydrochloride was purchased from Yichang Renfu Pharmaceutical Co. (Hubei, China). Fentanyl hydrochloride (purity > 99%) and atipamezole (purity > 99%) were synthesized at our institution. WAY100635 was purchased from Biyuntian Biotechnology Co. (Shanghai, China). Naloxone was obtained from McLean Biochemical Technology Co. (Shanghai, China). Nikethamide was purchased from Boselle Biotechnology Co. (Beijing, China). Flumazenil was obtained from the China Institute for Food and Drug Control (Beijing, China). Fetal bovine serum was purchased from Life Technologies Co. (Shanghai, China). Phosphate-buffered saline and 0.25% trypsin solution were obtained from Sai Aomei Cell Technology Co. (Beijing, China). Forskolin was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Bis-benzimide H-33342 trihydrochloride nuclear staining solution was obtained from Abe Medical Equipment Trading Co. (Shanghai, China). Hydrochloric acid (HCl), formaldehyde, sodium chloride (NaCl), potassium chloride (KCl), HEPES buffer, magnesium chloride (MgCl₂), calcium chloride (CaCl₂), MgCl₂ hexahydrate, and sodium pyruvate were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China).

Tandospirone, dexmedetomidine, fentanyl, nikethamide, and naloxone were dissolved individually in saline (0.9% NaCl), and flumazenil was dissolved in 2.5% dimethyl sulfoxide and Tween-80 (2.5%) prior to the experiment. Midazolam was first dissolved in deionized water and adjusted to pH 3 with HCl. All drugs were injected intravenously at a volume of 0.1 mL/100 g.

2.3 Rat model of respiratory depression

The MouseOX Murine Plus Oximeter System was used to measure real-time arterial oxygen saturation (SaO₂) in rats. Before the experiment, the neck was shaved to expose the skin, and the rats were placed in a clear Plexiglas tank with bedding. The rats wore a clear neck collar to simulate the study condition and were allowed to move freely. After at least 30 minutes of acclimatization, the basal SaO₂ was recorded using an infrared probe. Tandospirone was intravenously injected at 0.1, 0.5, 1, 2, 4 or 8 mg/kg 10 minutes before starting the experiment. Intravenous fentanyl (80 µg/kg) was administered 5 minutes after injection of tandospirone, and continuous recording of SaO₂ began 5 minutes after tandospirone injection. The vital signs were transmitted to the computer in real-time, including SaO₂, respiratory rate, and heart rate. The parameters were measured for 15 seconds every 5 minutes for a total of 45–60 minutes, and the average of each 15-second recording was obtained.

2.4 PKA redistribution

CHO-µ-PKAcatEGFP and CHO-α_2a/2c-PKAcatEGFP cells were cultured in disposable 200 µL F12 medium with 10% fetal bovine serum under standard conditions (37℃, 5% carbon dioxide) to regulate cell fusion up to approximately 80%. After preparation of the cell suspension, a small portion of the mixture (one-third to one-quarter) was mixed with a small volume of medium in a pipette sink. The CHO cells were inoculated into black opaque 96-well plates and cultured overnight. The next day, drug administration began once the cell attachment rate had reached 80%.

In the experimental group, 40 µM tandospirone was added to each well. In the positive control group, 40 µM dexmedetomidine or fentanyl was added to each well. The co-administration group received 40 µM tandospirone and 40 µM dexmedetomidine or fentanyl. Additionally, 40 µM forskolin was added to each well and left to react for 10 minutes. The control group were untreated, and 200 µL F12 medium was added to complete the reaction system. Once the reaction was complete, 12% formaldehyde solution was applied to each well for 20 minutes for fixation. The samples were stained with nucleic acid dye and kept in the dark for 3 hours. Fluorescence detection was performed using high-content screening equipment, and the particle size and area were calculated.

2.5 Two-electrode voltage clamp

Sexually mature female African clawed frogs (Xenopus laevis) were purchased from the Kunming Institute of Zoology, Chinese Academy of Sciences. The frogs were reared according to standard experimental procedures, changing water three times per week and maintaining a temperature of 18–23℃. The frogs had a 12-hour/12-hour light–dark cycle and were fed three times per week. To obtain Xenopus oocytes, the frogs were anesthetized by covering the entire body with crushed ice for at least 30 minutes. Then, the skin and muscle layers of the right leg were cut through at 1-cm intervals on the ventral side to fully expose the ovary. The oocytes were removed and placed in OR-2 solution (82 mM NaCl, 2.5 mM KCl, 5 mM HEPES buffer, and 1 mM MgCl₂ at pH 7.6). The clumps of oocytes were treated with 1 mg·mL^− 1 collagenase (Sigma, Boston, US) in OR-2 for 1 hour. Stage V mature cells were observed and carefully selected under the microscope with a 10-fold volume of OR-2 solution. The selected oocytes were cultured in ND-96 (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES buffer at pH 7.6) overnight at 16°C. The next day, microelectrode glass capillary tubes (#5206, VitalSense, Wuhan, China) were pulled using a two-stage glass micropipette puller (Narishige, Tokyo, Japan) for microinjection. The complementary RNA mixture was collected in sterile, enzyme-free centrifuge tubes at a ratio of α₁:β₂:γ₂ = 1:1:1 or α₄:β₂:δ = 1:1:1, and 40 ng of the mixture was injected into each oocyte using the microinjection pump. The cells were then cultured in OR-2 solution for 2–4 days, and the cell culture solution was changed daily. Current recordings were made after 2 days of synaptic α₁β₂γ₂ gamma amino-butyric acid (GABA) receptor expression. Current recordings were made after 4 days of extrasynaptic expression of α₄β₂δ GABA receptors.

2.6 Statistical analysis

The MouseOx Plus software was used to disaggregate, filter, average, and export Microsoft Excel spreadsheets from the .txt files generated by MouseOx. The statistical analyses and figure preparation were performed using GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). All data are presented as the average of 15-second recordings obtained at 5-minute intervals. The groups were compared by two-way analysis of variance (dose × time) followed by Bonferroni’s post hoc test. The data are expressed as the mean ± standard error of the mean.^*P < 0.05 was considered statistically significant.

3.1 Tandospirone reduced fentanyl-induced respiratory depression in rats

Fentanyl-induced respiratory depression was significantly reduced in the tandospirone group compared with the vehicle control group (^ΦP < 0.0001, ^ΩP < 0.001, ^$P < 0.01, ^*P < 0.05 versus vehicle control group). Tandospirone at 0.1, 0.5, 1, 2, 4, or 8 mg/kg significantly reduced respiratory depression in a dose-dependent manner over 60 minutes (Fig. 1A). Within 5 minutes of fentanyl injection, SaO₂ decreased significantly in the vehicle control group from 95.78% ± 0.52–37.62% ± 1.44% (39.28% below baseline). However, the SaO₂ in the 2 mg/kg tandospirone group decreased from 95.78% ± 0.26–63.60% ± 2.38% (66.40% below baseline) (^ΦP < 0.0001 versus vehicle control group).

The 5-HT_1A receptor antagonist WAY100635 (Fig. 1B) completely blocked the ameliorative effects of tandospirone on respiratory depression. Figure 1B shows that WAY100635 alone did not change the baseline SaO₂ in rats, and the drug did not improve respiratory depression when used alone. In the treatment group receiving intravenous injection of tandospirone (Fig. 1C), the marked decrease in SaO₂ (from 94.94% ± 0.51–43.53% ± 3.30% [45.85% below baseline]) 5 minutes after fentanyl injection (80 µg/kg intravenous) indicated that the respiratory depression model had been successfully established, and there was no significant difference between the groups. However, the SaO₂ increased from 50.19% ± 3.07–79.66% ± 1.67% (increase of 33.05%) when injected 10 minutes after 2 mg/kg tandospirone (^ΦP < 0.0001). Respiratory depression caused by fentanyl was reversed by naloxone, resulting in the rats regaining consciousness.

3.2 Tandospirone improved dexmedetomidine-induced respiratory depression in rats

Three minutes after dexmedetomidine administration (Fig. 2A), SaO₂ decreased from 95.75% ± 0.46–62.89% ± 2.83%, representing a decrease of 65.68% from baseline. SaO₂ recovered to 77.19% ± 3.91% at 33 minutes (80.62% of baseline). Dexmedetomidine decreased SaO₂ from 95.78% ± 0.30–84.51% ± 3.10% at 3 minutes (^ΦP < 0.0001) (88.23% of baseline). SaO₂ recovered to 81.89% ± 3.46% at 33 minutes (85.50% of baseline). There was no significant change in SaO₂ after administration of 8 mg/kg tandospirone. Compared with the vehicle control group, pre-injection of WAY100635 completely blocked the ability of tandospirone to improve respiratory depression (Fig. 2B). A 6-minute baseline measurement was made prior to the experiment. A considerable reduction in SaO₂ was recorded 6 minutes after atipamezole injection in all groups (Fig. 2C), implying that the model of respiratory depression was proficiently established. The experimental results demonstrate that atipamezole blocked respiratory depression caused by dexmedetomidine.

3.3 Tandospirone improved midazolam-induced respiratory depression in rats

No significant differences in SaO₂ were observed between the groups of rats prophylactically injected with tandospirone (Fig. 3A). In the vehicle control group, 3 minutes after midazolam administration, SaO₂ decreased from 94.91% ± 0.86–69.82% ± 3.01% (73.56% of baseline). In the group that received 2 mg/kg tandospirone, the SaO₂ decreased from 96.18% ± 0.44–95.67% ± 0.28% after 3 minutes (99.47% of baseline) (^ΦP < 0.0001). Tandospirone at 4 or 8 mg/kg was also effective at reducing the respiratory depression induced by midazolam. SaO₂ decreased more in rats administered WAY100635 before tandospirone and then injected with midazolam than in the vehicle control group, suggesting that WAY100635 completely blocked the ability of tandospirone to reduce respiratory depression(Fig. 3B). A 6-minute baseline measurement was made prior to the experiment, and a considerable reduction in SaO₂ was recorded 6 minutes after flumazenil injection in all groups (Fig. 3C), implying that the model of respiratory depression was proficiently established. After 9 minutes, the drugs were administered. The graph illustrates that tandospirone (2 mg/kg) was as effective as the specific antagonist flumazenil (1.8 mg/kg) at reducing respiratory depression and surpassed the effect of nikethamide (80 mg/kg).

3.4 CHO-α_2a/2c-PKAcat-enhanced green fluorescent protein (EGFP) cell redistribution experiments

In inactive cells, fluorescent particles exist in a highly aggregated state. After activation by forskolin, intracellular adenylate cyclase is activated, increasing intracellular cAMP and activating PKA, and the fluorescent fusion protein changes from aggregated to dispersed. The results show that the number of fluorescent particles in the cells treated with forskolin alone was markedly decreased in CHO-α_2a-PKAcat-EGFP cells compared with cells from the vehicle control group (Fig. 4A&B). In the tandospirone treatment group (1 × 10^− 5 to 1 × 10^− 10 M), the number of fluorescent particles significantly decreased at a concentration of 10^− 9 mol·L^− 1, indicating that forskolin fully activated the cells. PKA shifted from aggregated to dispersed, indicating that tandospirone did not interact with α_2a/2c receptors. Dexmedetomidine (1 × 10^− 5 to 1 × 10^− 9 mol·L^− 1 M) counteracted the activation by forskolin, indicating that dexmedetomidine binds to α_2a/2c receptors and inhibits PKA redistribution (Fig. 4C&D). In the tandospirone group (1 × 10^− 5 to 1 × 10^− 11 mol·L^− 1), there was no significant difference in the number of fluorescent particles after the addition of dexmedetomidine (1 × 10^− 7 mol·L^− 1) compared with the forskolin alone group. Moreover, tandospirone did not influence the α_2a/2c receptor binding effect of dexmedetomidine, indicating that tandospirone does not act directly on α_2a/2c receptors.

3.5 CHO-µ-PKAcat-EGFP cell redistribution experiments

Fentanyl activated µ receptors on CHO-PKA-cat-EGFP cells in a concentration-dependent manner(Fig. 4E&F). Significant redistribution of cellular fluorescent particles compared with the forskolin group was observed in the fentanyl group (1 × 10^− 6 to 1 × 10^− 10 mol·L^− 1). In the tandospirone group (1 × 10^− 5 to 1 × 10^− 9 mol·L^− 1), there was no significant difference in the number of fluorescent particles compared with the forskolin group. In both the tandospirone group (1 × 10^− 5 to 1 × 10^− 11 mol·L^− 1) and the fentanyl group (1 × 10^− 7 mol·L^− 1), there was no significant difference in the number of fluorescent particles when compared with the forskolin group. Moreover, tandospirone prophylaxis did not impact the µ-receptor binding effect of fentanyl, suggesting that tandospirone does not act directly on µ receptors.

3.6 Effect of tandospirone on α₁β₂γ₂ and α₄β₂δ GABA_A receptor currents

In Xenopus oocytes, which express synaptic and extrasynaptic receptors (Fig. 5A and 5B, respectively), the inward currents induced by GABA (1 µM) through the α₁β₂γ₂ GABA receptor subtype were − 89.44 ± 23.73 nA, and the inward currents through the α₄β₂δ GABA receptor subtype were − 208.49 ± 40.17 nA, as measured by dual-motor voltage clamp with the cells clamped at − 70 V. Midazolam (100 µM) increased the 1 µM GABA-evoked current amplitude, and both GABA receptor subtypes showed the same enhancement effect at this dose (Fig. 5D–5E). Tandospirone (10 µM) decreased the 1 µM GABA-mediated inward chloride current, and for α₁β₂γ₂ and α₄β₂δ GABA receptor modulation, the inhibition rate was − 5.59% ± 6.28% and − 38.07% ± 7.80%, respectively. Tandospirone reduced the modulation of GABA receptor currents by endogenous GABA, with a significant decrease in extrasynaptic receptor currents. The combination of tandospirone (10 µM) and midazolam (100 µM) reduced the 1 µM GABA-induced current amplitude, and for α₁β₂γ₂ receptor and α₄β₂δ receptor modulation, the inhibition rate was 85.54% ± 18.74% and 62.68% ± 24.16%, respectively. However, for α₁β₂γ₂ receptors, the difference in current amplitude induced by 1 µM GABA compared with 10 µM tandospirone was not statistically significant. The results suggest that tandospirone may regulate GABA receptor current, but not enough to affect the efficacy of midazolam.

Respiratory depression is an unavoidable side effect of currently used clinical anesthetics, and treatment of respiratory depression is still unsatisfactory in the clinical setting. We formulated an effective non-invasive technique for identifying respiratory depression in rats. We used this model of respiratory depression to investigate the pharmacodynamics of tandospirone, and we used CHO cells and Xenopus oocytes as vectors. Additionally, we explored the mechanism of action of tandospirone based on PKA redistribution in conjunction with two-electrode voltage clamp experiments.

In this study, we established the quantitative and temporal ability of tandospirone to improve respiratory depression caused by anesthetics in rats. Prophylactic administration of tandospirone significantly reduced respiratory depression caused by fentanyl/midazolam and dexmedetomidine, increased SaO₂, and accelerated recovery in rats. In the PKA redistribution experiment, dexmedetomidine activated CHO cells in a concentration-dependent manner, and intracellular fluorescent particles remained aggregated. We established that tandospirone did not act directly on the α_2a and α_2c receptors, resulting in a decrease in the area of fluorescent particles and causing PKA redistribution Meanwhile, fentanyl led to significant PKA redistribution. As a result, the intracellular fluorescent particles changed from an aggregated to a diffuse appearance. These results suggest that dexmedetomidine activates both α_2a and α_2c receptors, and fentanyl activates the µ receptor. However, tandospirone failed to block the activating effect of forskolin, indicating minimal binding to α_2a/2c and µ receptors. When we fixed the dexmedetomidine concentration in combination with tandospirone, it did not affect the activation of α_2a/2c receptors by dexmedetomidine. Similarly, our results confirm that the combination of tandospirone and fentanyl has no impact on the activation of µ receptors by fentanyl. Thus, tandospirone does not appear to regulate respiration by directly acting on µ receptors. Midazolam is a benzodiazepine that positively modulates the GABA receptor, which is expressed as a pentameric protein. A previous study suggested that midazolam binds to the GABA receptor only in the presence of the γ2 subunit (Hong et al. 2011). To validate the modulatory effects of tandospirone and midazolam on both intrinsic and extrinsic subunits at the synapse, we implanted the α₁β₂γ₂ subunit and the α₄β₂δ subunit in Xenopus oocytes. To investigate how tandospirone and midazolam affected GABA receptors, we conducted two-electrode voltage clamp experiments. We found that 1 µM GABA had a significant modulatory effect on GABA_A receptors, resulting in inward chloride current and hyperpolarization. The current amplitude was reduced by tandospirone, while midazolam increased the amplitude of GABA-induced currents. The overall current amplitude was additive when tandospirone was applied with midazolam, and tandospirone produced the same effect on current modulation for both α₁β₂γ₂ and α₄β₂δ GABA receptors. The original current-modulating effect of midazolam on α₁β₂γ₂ and α₄β₂δ GABA receptors was not altered by co-administration of tandospirone.

Tandospirone is primarily a 5-HT_1A receptor agonist. Therefore, we aimed to explore ways to enhance respiratory depression by inhibiting 5-HT_1A receptors (Huang et al. 2017). The results showed that the 5-HT_1A receptor antagonist WAY100635 greatly impeded the ability of tandospirone to ameliorate fentanyl- and midazolam-induced respiratory depression and partially blocked dexmedetomidine-induced respiratory depression. Neither tandospirone nor nikethamide had any effect. Other researchers have demonstrated 5-HT receptor-mediated activation of inwardly rectifying potassium channels in rat dorsal raphe neurons (Penington et al. 1991). Tandospirone is a G-protein-coupled receptor agonist (Lefkowitz 2000). Activation of 5-HT receptors involves G-proteins in the transduction pathway, and G-proteins may interact directly with potassium channels (Katayama et al. 1997).

Tandospirone is widely prescribed for the clinical management of anxiety disorders because of its efficacy and minimal side effects. It has been available in Japan since 1996 and in China since 2004. When metabolized in vivo, tandospirone produces 1-[2‐pyrimidyl]‐piperazine(1-PP) (Hu et al. 2019). The pharmacodynamic effects of 1-PP remain unclear; however, on the basis of the current literature, it is hypothesized that 1-PP acts as an α₂-adrenergic receptor antagonist (Myers et al. 2004). After 5 minutes of administration of 1-PP, there was no significant increase in SaO₂ and no difference in the block of dexmedetomidine compared with the lysophospholipid control group. These results suggest that tandospirone may not exert its effects through 1-PP. We also previously investigated the protective effects of 1-PP against acute death in mice caused by dexmedetomidine. No positive results were obtained; thus, the results have not been published.

This study used a reliable, consistent, and safe model of respiratory depression in rats. However, this study had several limitations. First, we did not evaluate the correlations of respiratory rate and tidal volume with SaO₂. Second, the mechanism by which tandospirone reduced respiratory depression without compromising the effects of sedation and analgesia was not evaluated in this study. Finally, the male Sprague–Dawley rats used in this experiment were not differentiated based on their degree of sensitivity to respiratory depression compared with female rats. In other experiments in male and female rats treated with the opioids heroin and fentanyl, it was found that heroin elicited more extensive and prolonged (lasting for 45–60 minutes) respiratory depression in female than in male rats (Marchette et al. 2023). These findings will be evaluated in future experiments, where additional opioids will be tested to investigate the differences in SaO₂ between male and female rats.

The effectiveness of tandospirone at ameliorating respiratory depression caused by anesthetics was examined in this study. Tandospirone displayed low toxicity, stable efficacy, and ameliorated the respiratory depression induced by a variety of anesthetics. Therefore, tandospirone may have application value as a broad-spectrum drug to protect against respiratory depression caused by anesthetics. Further studies should be performed to validate our findings. We used a well-established experimental model, and thus the procedures can be reproduced by other researchers to test the impact of drugs on arterial blood gases in animal models. The findings provide a foundation for more effective drug evaluation experiments in the future.

Tandospirone ameliorates respiratory depression caused by anesthetics in rats through activation of the 5-HT_1A receptor. Future studies should validate these findings and evaluate whether tandospirone has clinical application value for ameliorating respiratory depression in patients receiving anesthetics.

Declaration of interest

None.

Acknowledgments

We recognise the outstanding academic assistance provided by Shu-zhuo Zhang and Yu-lei Li in the experiments.

Data Availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request. Please contact us at [email protected] if you wish to retrieve the data.

Author Contribution

MRS: Investigation, Methodology, Validation, Formal analysis, Data Curation, Writing - Original Draft. MZH: Validation, Formal analysis. WJT: Formal analysis, Writing - Original Draft. ZY: Methodology, Writing - Review & Editing, Supervision, Funding acquisition. RBS: Conceptualization, Resources, Supervision, Funding acquisition.

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No competing interests reported.

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You are reading this latest preprint version

Tandospirone Prevents Anesthetic-Induced Respiratory Depression through 5-HT1A Receptor Activation in Rats

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Animals and Cells

2.2 Drugs

2.3 Rat model of respiratory depression

2.4 PKA redistribution

2.5 Two-electrode voltage clamp

2.6 Statistical analysis

3. Results

3.1 Tandospirone reduced fentanyl-induced respiratory depression in rats

3.2 Tandospirone improved dexmedetomidine-induced respiratory depression in rats

3.3 Tandospirone improved midazolam-induced respiratory depression in rats

3.4 CHO-α_2a/2c-PKAcat-enhanced green fluorescent protein (EGFP) cell redistribution experiments

3.5 CHO-µ-PKAcat-EGFP cell redistribution experiments

3.6 Effect of tandospirone on α₁β₂γ₂ and α₄β₂δ GABA_A receptor currents

4. Discussion

5. Conclusion

Declarations

Author Contribution

References

Additional Declarations

Supplementary Files

Status:

Version 1

Tandospirone Prevents Anesthetic-Induced Respiratory Depression through 5-HT1A Receptor Activation in Rats

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Animals and Cells

2.2 Drugs

2.3 Rat model of respiratory depression

2.4 PKA redistribution

2.5 Two-electrode voltage clamp

2.6 Statistical analysis

3. Results

3.1 Tandospirone reduced fentanyl-induced respiratory depression in rats

3.2 Tandospirone improved dexmedetomidine-induced respiratory depression in rats

3.3 Tandospirone improved midazolam-induced respiratory depression in rats

3.4 CHO-α2a/2c-PKAcat-enhanced green fluorescent protein (EGFP) cell redistribution experiments

3.5 CHO-µ-PKAcat-EGFP cell redistribution experiments

3.6 Effect of tandospirone on α1β2γ2 and α4β2δ GABAA receptor currents

4. Discussion

5. Conclusion

Declarations

Author Contribution

References

Additional Declarations

Supplementary Files

Status:

Version 1

3.4 CHO-α_2a/2c-PKAcat-enhanced green fluorescent protein (EGFP) cell redistribution experiments

3.6 Effect of tandospirone on α₁β₂γ₂ and α₄β₂δ GABA_A receptor currents