1,2‐propanediol reformulation improves tribromoethanol safety and reveals pontine GABRA1 enrichment as a candidate mechanistic correlate of anesthesia

Abstract

Background

Conventional tribromoethanol anesthesia is compromised by solvent‐related toxicity, limiting its utility in prolonged experimental protocols. We hypothesized that reformulation with 1,2‐propanediol could improve its biosafety while revealing its potential mechanism of action in the central nervous system.

Methods

In vivo biocompatibility was evaluated using OECD‐standard irritation models, while anesthetic efficacy was quantitatively determined via the up–down sequential allocation method, complemented by tail‐immersion and writhing tests to characterize antinociceptive effects. Moreover, physiological stability was monitored using non‐invasive tail‐cuff plethysmography and rectal thermometry for cardiovascular and core temperature assessment. In addition, subacute toxicity was assessed through repeated dosing, and MRI for neurostructural integrity. Finally, cognitive function and mechanisms were probed via the Morris water maze, molecular docking, region‐specific protein expression, and real‐time cerebral oxygen saturation mapping using photoacoustic imaging.

Results

The 1,2‐propanediol formulation markedly reduced local tissue irritation and improved pulse‐rate, blood‐pressure, thermoregulatory, and repeated‐dosing survival profiles compared with the conventional 2‐methyl‐2‐butanol formulation. TBE/PG exhibited dose‐dependent anesthetic efficacy without significant strain‐ or sex‐associated differences. Repeated administration induced transient spatial learning impairment and reduced cerebral oxygen saturation; these effects were largely reversible after drug withdrawal and retraining. Molecular docking and regional protein profiling identified GABRA1, which was enriched in the pons, as a candidate mechanistic correlate of TBE‐induced anesthesia.

Conclusions

This study establishes a biocompatible TBE formulation as a superior injectable anesthetic with enhanced safety and reversible cognitive effects. The results pinpoint pontine GABRA1 targeting as a novel pathway for TBE‐induced anesthesia, providing a robust framework for refining preclinical sedation protocols and future neurotoxicological evaluations.

This study develops a reformulated tribromoethanol (TBE) anesthetic using 1,2‐propanediol as a solvent to overcome solvent‐related toxicity. The novel formulation demonstrates, cardiovascular stability, and survival in prolonged protocols. It provides potent, dose‐dependent anesthesia without strain or sex differences. Although repeated dosing induces transient spatial memory deficits and cerebral hypoxia, these effects are fully reversible. Mechanistically, the results pinpoint pontine GABRA1 targeting as a novel pathway for TBE‐induced anesthesia. The findings establish a safer injectable anesthetic and elucidate a novel neurobiological pathway for TBE‐induced anesthesia.

1. INTRODUCTION

Injectable anesthetics are indispensable in preclinical research involving rodents, providing rapid, titratable sedation essential for minor surgical procedures, imaging, and behavioral studies. Among these agents, tribromoethanol (TBE) has remained a practical choice due to its predictable onset, and ease of use without requiring specialized equipment. Uniquely, TBE is not classified as a controlled substance in most countries, in contrast to agents like ketamine or pentobarbital, making it especially attractive for use in routine laboratory settings with minimal regulatory restrictions. ¹

Despite its historical prevalence, concerns about tissue irritation, batch instability, and potential toxicity have led to a decrease in recommendations for its use in institutional guidelines. Recently, the continued use of TBE has been challenged by concerns over formulation‐related toxicity. Reports indicate that the traditional solvent 2‐methyl‐2‐butanol is responsible for ocular and respiratory irritation, abdominal inflammation, and batch‐to‐batch inconsistency. ² Although some research has explored alternative solvents, robust comparative data are lacking.

In spite of its longstanding use, comprehensive investigations into the pharmacological profile of tribromoethanol remain strikingly limited. Existing literature has largely confined itself to surface‐level assessments—reporting variable anesthetic depth, inconsistent recovery times, and occasional complications such as peritonitis or neurobehavioral abnormalities—without systematic exploration of its dose–response characteristics, physiological stability, long‐term safety, or cognitive outcomes. ³ , ⁴ More importantly, the central neural substrates and molecular pathways responsible for TBE‐induced anesthesia have yet to be defined, leaving its mechanism of action largely speculative. ⁵ This persistent gap not only weakens the theoretical foundation for its use but also limits efforts to optimize its application or anticipate its off‐target effects at the mechanistic level.

To bridge this critical research gap, a comprehensive, multi‐level experimental strategy was implemented. Reformulation of TBE with the more biocompatible solvent 1,2‐propanediol was hypothesized to markedly reduce adverse effects while preserving its anesthetic efficacy. ⁶ In addition to quantitatively assessing the onset time and toxicity profile of TBE, we conducted molecular docking analyses to investigate, at a structural level, the binding interactions between TBE and several well‐established anesthetic target proteins. Based on these in silico findings, we systematically assessed protein expression across distinct brain regions and, for the first time, identified a specific area with significantly elevated GABRA1 expression, suggesting it may serve as a key neural locus for TBE's central anesthetic action. To further elucidate the underlying molecular mechanism, transcriptomic sequencing was conducted on this region, enabling the identification of signaling pathways potentially involved in TBE‐mediated neural modulation. By integrating functional, behavioral, and molecular analyses, this study provides a comprehensive framework for evaluating TBE and underscores its relevance in both practical anesthesia and mechanistic neuroscience.

2. METHODS

2.1. Experimental animals

ICR and C57BL/6J (C57) mice (20–39 g, both sexes) were procured from Peking University (Beijing, China) and housed under standard laboratory conditions (22–24°C, 45%–55% humidity, artificial lighting with a 12‐h light–dark cycle from 07:00 to 19:00), with ad libitum access to food and water. All experimental procedures adhered to the ARRIVE 2.0 guidelines and were approved by the Institutional Animal Care and Use Committee of Peking University Health Science Center (approval no. DLASBE0419; approval dated, 18 April 2025). Animals were assigned to either single‐administration or repeated‐administration cohorts according to the experimental endpoint. Single‐administration cohorts were used for irritation assessment, physiological monitoring, anesthetic potency evaluation, and acute behavioral assays. Repeated‐administration cohorts were used for subacute toxicity, survival analysis, MRI, photoacoustic imaging, and Morris water maze experiments. In line with the 3R principles, sample sizes were minimized wherever possible.

2.2. Drugs and chemicals

TBE (CAS 75‐80‐9; Sigma‐Aldrich Trading Co., Shanghai, China) was prepared as two separate formulations. The conventional formulation, referred to as TBE/TAA, contained TBE dissolved in 2.5% 2‐methyl‐2‐butanol in sterile saline. The reformulated preparation, referred to as TBE/PG, contained TBE dissolved in 5% 1,2‐propanediol in sterile saline. Matched vehicle controls consisted of 2.5% 2‐methyl‐2‐butanol in sterile saline for the TBE/TAA formulation and 5% 1,2‐propanediol in sterile saline for the TBE/PG formulation. Sterile saline was used as the negative control where appropriate. Unless otherwise specified, TBE was administered intraperitoneally at 0.1 mL per 10 g body weight.

2.3. Eye, skin and respiratory tract irritation assessment

2.3.1. Eye irritation

Under light anesthesia, a single 0.1 mL dose of TBE or solvent was instilled into one eye of ICR mice. The contralateral eye served as untreated control. Ocular signs—including redness, swelling, and discharge—were assessed under a stereomicroscope at 1 h post‐instillation, following the evaluation criteria specified in OECD Test Guideline 405. ⁷

2.3.2. Dermal irritation

A gauze patch (3 × 3 cm) soaked in the test solution was applied to a shaved flank region. The area was covered with an occlusive film and gently bandaged for 4 h. After patch removal, the skin was examined and scored for erythema, edema, dryness, and other irritation indicators, according to OECD TG 404. ⁸

2.3.3. Respiratory tract irritation

To assess inhalation‐related toxicity, mice were placed in dynamic nose‐only inhalation chambers and exposed to vapor‐phase TBE or solvent for 40 min. ⁹ Following exposure, bronchoalveolar lavage fluid was collected for cellular and biochemical analysis, and lung tissues were harvested for histopathological evaluation.

2.4. Physiological monitoring of pulse rate, blood pressure, and core temperature

The effects on cardiovascular function and thermoregulation are evaluated by tracking body temperature, arterial blood pressure, and heart rate. A lubricated rectal probe attached to a digital thermometer (accuracy ±0.1°C) is used to measure body temperature. Baseline readings are acquired prior to administration, and then again every 5 or 10 min until the righting reflex goes away. ¹⁰ A tail cuff plethysmography automatic monitoring system (BL‐420N, Taimen Technology, China) that can non‐invasively record systolic, diastolic, and pulse rate was used to track blood pressure and heart rate. ¹¹ To reduce stress‐induced data variability, mice were trained to adapt to the device for five days before the trial.

Because ECG or telemetry was not performed, the present measurements were not used to diagnose arrhythmias or cardiac electrical abnormalities. During monitoring, ambient temperature was maintained at 25 ± 1°C, and tail temperature was maintained at approximately 37°C using a heating platform to ensure reliable tail‐cuff recordings. Measurements were aligned to the time of TBE administration and, where applicable, to loss of the righting reflex.

2.5. Anesthesia activity studies

2.5.1. Up–down sequential allocation method

The dose of TBE was determined using the up–down sequential allocation method to induce hypnosis in mice. Based on pre‐test results, the first mouse received a single intraperitoneal dose of 150 mg/kg TBE. Subsequent doses were adjusted according to the previous mouse's response: if an effective response—defined as loss of the righting reflex for ≥30 s—was observed, the dose for the next mouse was reduced by a factor of 0.9; if not, the dose was increased by the same factor. This technique, extensively used in anesthesia research to quantify anesthetic activity, promotes efficiency and minimizes animal use. ¹²

2.5.2. Writhing test

Each mouse was put into a glass beaker, and it was left for half an hour to habituate. 1% acetic acid was delivered intraperitoneally 7 min after TBE or saline was administered intraperitoneally. Over the next 30 min, the number of writhing episodes—which were identified by abdomen concavity, trunk distortion, hindlimb extension, and hip elevation—was counted. ¹³

2.5.3. Tail‐immersion test

The depth of TBE‐induced anesthesia was measured using a modified tail immersion test: a 3‐cm distal section of the tail was submerged in water kept at 50 ± 0.5°C for 7 min following an intraperitoneal injection of TBE or saline, and withdrawal latency was noted using a stopwatch. ¹⁴ To avoid tissue damage, the observation time was limited to 20 s. Because tail‐immersion latency and acetic‐acid‐induced writhing can be influenced by sedation and motor suppression, these assays were interpreted as antinociceptive‐like behavioral responses under anesthesia rather than definitive measures of analgesia.

2.6. Morris water maze test

Spatial learning and memory were assessed using the Morris water maze as previously described. ¹⁵ Four extramaze visual cues were positioned on the surrounding walls of a quiet room with dark curtains for testing. The circular pool, which measured 150 cm in circumference and 50 cm in height, was filled with water kept between 24°C and 26°C and was separated into four quadrants: east (E), west (W), south (S), and north (N). At the center of the SE quadrant, a concealed circular platform with a diameter of 12 cm was positioned 1 cm below the surface. For 7 days in a row, each mouse was given four chances every day to find the concealed platform. The mice were given a maximum of 60 s to find the platform after being released from one of the four starting locations (E, S, W, or N) facing the wall. After the mice located the platform, they were allowed to stay there for 20 s after finding it. The main indicator of learning performance was escape latency. Motor function was also evaluated by analyzing swimming speed. In order to assess spatial reference memory, a probing trial (60 s, no platform) was carried out on day 7. SMART 3.0 video tracking software was used to record the amount of time spent in the target quadrant, the length of the path in the target quadrant, number of platform crossings, and the search error.

2.7. Western blot

To investigate the cerebral distribution of TBE, WB was performed as previously described. ¹⁶ Mice were administered TBE or an equal volume of saline via intraperitoneal injection, after which brain tissue was collected post‐decapitation. Proteins extracted from the brain were quantified using a BCA Protein Assay Kit (Solarbio, PC0020), separated by SDS–PAGE, and transferred to PVDF membranes. To block non‐specific binding, membranes were incubated with Rapid Sealing Solution (NCM Biotech, P30500) at room temperature for 10 min with gentle agitation. Membranes were then incubated overnight at 4°C with primary antibodies against GABRA1 (ABclonal, Cat# A8299, RRID:AB_2769558; 1:750) and GAPDH (Abcam, Cat# 2251‐1, RRID:AB_1267174; 1:10 000). After washing in TBST, membranes were incubated with secondary antibody (Goat anti‐Mouse IgG [H + L], HRP‐conjugated; RRID:AB_2313606; 1:10 000) for 1 h at room temperature. Signal was visualized using High Sensitivity ECL Chemiluminescent Reagent (NCM Biotech, P2100) after a final TBST wash and 1–2 min incubation. ImageJ was used for densitometric analysis, and GraphPad Prism was used for melting curve analysis.

2.8. Toxicity analysis and prediction

The web programs ProTox 3.0 and ADMETlab 3.0 were used to perform in silico toxicity and ADMET profiles of TBE. With validated performance across independent test sets, ProTox 3.0 predicts 61 toxicological endpoints, including acute, organ‐specific, and pathway‐based toxicity, using machine‐learning models and compound similarity metrics. ¹⁷ With better breadth and accuracy than previous iterations, ADMETlab 3.0 offers thorough pharmacokinetic and toxicity predictions covering absorption, distribution, metabolism, excretion, and toxicity. ¹⁸ Both platforms produced comprehensive toxicity profiles and pharmacokinetic data using TBE's SMILES notation, enabling a thorough, early‐stage safety evaluation to identify any possible hazards related to its use.

2.9. Subacute toxicity evaluation

The experimental program was implemented as previously described. ¹⁹ Male C57BL/6J mice (n = 8 per group) were randomized into four groups: TBE‐treated, TBE‐recovery (7 days post‐treatment), vehicle and control. TBE‐treated mice received daily intraperitoneal injections of TBE (330 mg/kg) for 7 consecutive days. Throughout the course of treatment, body weight, food consumption, and water intake were tracked. Mice were humanely euthanized by decapitation at the end of the trial, and their small intestine, liver, spleen, kidneys, heart, and lungs were removed. For the ensuing biochemical tests, blood samples were drawn via ocular sinus puncture and placed in heparinized tubes.

2.10. Molecular docking

Molecular docking was performed to elucidate potential binding targets of TBE among established anesthetic receptors. ²⁰ The Protein Data Bank provided the crystal structures of the NMDA receptor (PDB ID: 7EU8), TREK‐1 (PDB ID: 6W88), GABRA1 (PDB ID: 9FFL), and α₂_A‐adrenergic receptor (PDB ID: 6KUY). The ChemDraw 22.0 program was used to illustrate the compound's structure. Schrödinger software's Glide module was used to perform molecular docking.

2.11. Preclinical small animal magnetic resonance imaging (MRI)

Mice were maintained under TBE anesthesia and imaged in a Discovery 3 T MR750 MRI system (GE, United States), equipped with gradient coils capable of a maximum amplitude of 400 mT/m. ²¹ The anesthetised mouse was placed in the prone position on the scanner bed, and the head was secured within a dedicated cranial RF coil. The bed was then positioned centrally within the magnet bore. A 3D Turbo Spin‐Echo (TSE) sequence was employed to acquire T2‐weighted coronal images (field of view 25 mm; matrix 286 × 320; repetition time (TR) 3000 ms; echo time (TE) 121 ms; two signal averages), yielding a slice thickness of 0.2 mm and an in‐plane resolution of 0.075 × 0.078 mm.

2.12. B‐mode ultrasound and photoacoustic imaging

Mice were anesthetised via intraperitoneal injection of TBE and confirmed to reach surgical depth of anesthesia. Hair removal cream was applied to the cranial area—including ears and neck—and left on for several minutes; residues were then thoroughly removed using a moistened sponge. Animals were positioned prone on the imaging platform and slightly rotated laterally to optimize exposure of the cranial region. A thin layer of ultrasound coupling gel was applied to both the skin surface and the probe head to ensure effective acoustic transmission. Imaging commenced in B‐mode to verify skull anatomy and to optimize transducer placement. The focal depth was adjusted to approximately 13 mm to maximize the photoacoustic signal. Upon confirmation, the imaging system was switched to photoacoustic mode, and laser and ultrasonic parameters were configured to capture cerebral blood oxygen saturation data. ²²

2.13. Pharmacological antagonism of GABA_A receptors

To functionally evaluate whether GABA_A receptor signaling contributes to TBE‐induced anesthesia, mice were pretreated with the GABA_A receptor antagonists gabazine (CAS No. 104104‐50‐9, MedChemExpress, Shanghai, China, 0.5 mg/kg) or bicuculline (CAS No. 485‐49‐4, MedChemExpress, Shanghai, China, 0.01 mg/kg) before TBE administration. TBE alone was used as the control condition. TBE was administered at 300 mg/kg. After TBE injection, the onset and duration of loss of righting reflex were recorded. The onset of anesthesia was defined as the time from TBE administration to loss of righting reflex, and the duration of anesthesia was defined as the time from loss to recovery of the righting reflex. Behavioral endpoints were recorded by investigators blinded to group allocation whenever feasible.

2.14. Statistical analysis

Data analysis was performed using GraphPad Prism version 9.0 (San Diego, CA, USA). Results were presented as the mean ± SEM. One‐ or two‐way analysis of variance (ANOVA) followed by Dunnett's or Tukey's test for multiple comparisons was used for multiple comparisons. Animals were randomly assigned to treatment groups using a random‐number table or equivalent randomization procedure. Investigators responsible for behavioral scoring, irritation scoring, histopathological evaluation, MRI analysis, and photoacoustic image quantification were blinded to group allocation whenever feasible. Formulation preparation and dosing were performed by investigators not involved in endpoint quantification.

3. RESULTS

3.1. Improved TBE formulation alleviates tissue irritation and stabilizes physiological function

As shown in Figure 1A, ocular irritation was evaluated by topical administration of different TBE formulations. Mice receiving TBE prepared with 2‐methyl‐2‐butanol exhibited marked ocular toxicity, including corneal opacity, eyelid margin hyperemia, and increased ocular secretions. In contrast, TBE formulated with 1,2‐propanediol produced no corneal clouding or congestion, with eye appearance comparable to that of mice in the saline group. Moreover, 1,2‐propanediol alone caused minimal ocular changes, confirming its superior biocompatibility. A similar trend was observed in the dermal irritation assay (Figure 1B). Application of 2‐methyl‐2‐butanol to the dorsal skin resulted in diffuse erythema and mild epidermal swelling, while TBE (2‐methyl‐2‐butanol) caused more extensive erythema and hyperpigmentation. By comparison, 1,2‐propanediol and its TBE formulation induced only mild, localized erythema, with no skin ulceration or overt inflammation. Inhalation‐related toxicity was next assessed (Figure 1C). Mice exposed to vapors of 2‐methyl‐2‐butanol or its corresponding TBE formulation developed histological signs of acute pulmonary irritation, including alveolar wall thickening, substantial inflammatory infiltration, and structural destruction of the alveoli. In contrast, mice exposed to 1,2‐propanediol or TBE (1,2‐propanediol) exhibited largely preserved alveolar architecture with only mild interstitial hyperplasia and inflammation.

FIGURE 1.

Reformulation of TBE with 1,2‐propanediol reduces tissue irritation and improves physiological stability. (A) Ocular irritation scores following topical administration of TBE formulations or solvents (n = 6). (B) Dermal irritation scores after 4‐h patch application (n = 6). (C) Histopathological sections of lung following 40‐min vapor exposure (n = 6). (D) Heart rate stability across TBE doses (240–370 mg/kg). (E) Systolic and diastolic blood pressure profiles. (F) Core temperature changes over time. (G) Survival rates following repeated daily dosing (330 mg/kg, 15 days, n = 20).

To further compare physiological tolerability, vital parameters including heart rate, blood pressure, and body temperature were monitored. Across a dosing range of 240–370 mg/kg, TBE formulated with 1,2‐propanediol maintained more stable pulse‐rate and blood‐pressure profiles without dose‐dependent toxicity, suggesting a favorable sedative dose–cardiac safety profile. Conversely, TBE (2‐methyl‐2‐butanol) induced dose‐related pulse‐rate instability, with increasing instability and reduced safety margins at higher doses (Figure 1D). As shown in Figure 1E, both TBE formulations maintained relatively stable diastolic pressure under acute administration. However, systolic pressure differed significantly between groups: TBE (1,2‐propanediol) caused only minor fluctuations, whereas TBE (2‐methyl‐2‐butanol) induced transient systolic drops and greater variability. Thermoregulatory responses were also assessed (Figure 1F). Mice receiving TBE (1,2‐propanediol) showed consistent core temperatures within the tested dose range (240–370 mg/kg), indicating preserved thermoregulatory control. In contrast, TBE (2‐methyl‐2‐butanol) led to a marked, dose‐dependent hypothermic response starting approximately 5 min post‐injection and persisting throughout the observation period. These findings indicate that replacing 2‐methyl‐2‐butanol with 1,2‐propanediol significantly enhances temperature stability. Finally, long‐term survival following repeated TBE administration (330 mg/kg, once daily) was assessed (Figure 1G). Mice treated with TBE (1,2‐propanediol) exhibited significantly improved survival, with 90% of animals alive at Day 15. In contrast, survival declined progressively in the TBE (2‐methyl‐2‐butanol) group, with complete mortality by Day 15. These findings demonstrate that the revised formulation substantially improves safety and long‐term tolerance under repeated dosing conditions.

3.2. Consistent and robust anesthetic efficacy of TBE across mouse strains and sexes

We evaluated the anesthetic efficacy of TBE across C57 and ICR strains, as well as male and female mice, using an up‐ and down‐sequential method. Starting from a dose approximately yielding a 50% response rate (150 mg/kg), 20 sequential trials were conducted (Figure 2A). As shown in Figure 2B,C, while ICR mice appeared marginally more sensitive than C57 mice, and females tended toward slightly higher responsiveness than males, none of these differences reached statistical significance. These findings indicate that TBE produces stable and reliable anesthetic potency across strains and sexes.

FIGURE 2.

TBE exhibits consistent anesthetic efficacy across mouse strains and sexes. (A) Schematic of the up–down sequential dosing method. (B) Doses and positive response rates that elicited positive responses in C57 (n = 59) and ICR (n = 70) mice. (C) Distribution of doses that elicited positive responses in female and male mice. (D) Dose–response relationship for tail‐immersion latency. (E) Writhing responses following acetic acid challenge. Data are presented as mean ± SEM (n = 5).

Antinociceptive‐like behavioral responses under anesthesia was further assessed using tail‐immersion latency and writhing tests. A clear, dose‐dependent increase in anesthetic depth was observed: in tail‐immersion testing, the correlation coefficients (r) were 0.717 and 0.755 for ICR and C57 mice, respectively (Figure 2D). In the writhing assay, increasing doses of TBE significantly prolonged latency to first writhe and reduced the number of writhes (Figure 2E), demonstrating robust dose dependence in anesthetic depth measures.

3.3. Integrative toxicological profiling and reversible neurological effects of TBE

As illustrated in Figure 3A–C, in silico toxicology predictions using ProTox 3.0 and ADMET lab 3.0 revealed no significant binding between TBE and established toxicity‐related targets, suggesting a low inherent toxicity profile. However, both platforms indicate that TBE may cause eye, skin, and respiratory tract irritation, and it has been proven in previous studies that TBE formulated with 1,2‐propanediol may not cause such irritation. Figure 3D–F compares physiological parameters across experimental groups: Vehicle (1,2‐propanediol) versus TBE and Control (saline). No significant differences were detected in body weight, food intake, or water consumption during the 7‐day dosing period, supporting good short‐term tolerability of TBE under both formulations. Organ‐to‐body weight ratios remained stable for most tissues; however, liver coefficients increased significantly in both TBE‐treated groups after 7 days (Figure 3G). Corresponding histopathology revealed renal tubular injury and hepatic steatosis (Figure 3H). Blood biochemistry confirmed this pattern: levels of creatinine (CREA‐S), uric acid (UA), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) were significantly elevated post‐TBE dosing (Figure 3I), consistent with histological findings. In contrast, no significant differences were observed in the levels of total protein (TP), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (UREA), and creatine kinase (CK). Subsequently, small‐animal MRI revealed hippocampal structural and signal alterations after repeated TBE exposure. Because conventional MRI does not directly measure synaptic density, these imaging findings were interpreted as MRI‐derived neurostructural changes rather than direct evidence of synaptic loss (Figure 3J). Encouragingly, these changes were reversible after a 7‐day drug‐free recovery period, suggesting neural plasticity and metabolic recovery.

FIGURE 3.

In silico and in vivo toxicological profiling of TBE reveals reversible organ toxicity and neural changes. (A–C) Predicted toxicity profiles from ProTox 3.0 and ADMETlab 3.0. (D–F) Body weight, food intake, and water consumption during 7‐day dosing. (G) Organ‐to‐body weight ratios. (H) Representative H&E‐stained sections of organs. (I) Blood biochemistry parameters included CREA‐S, UA, LDH, ALP, TP, ALB, ALT, AST, UREA, and CK. (J) High‐field MRI showing hippocampal structural changes pre‐ and post‐recovery. Data are mean ± SEM (n = 6). *p < 0.05, and ****p < 0.0001 versus control by one‐way ANOVA followed by Dunnett's test.

3.4. Reversible spatial memory impairment associated with cerebral hypoxia

During the 7‐day training phase, mice receiving daily TBE administration exhibited marked deficits in spatial learning compared to controls (Figure 4A). While control mice progressively located the hidden platform, TBE‐treated animals failed to develop spatial preference even by day 7, indicating cognitive impairment after repeated TBE exposure. Following a 7‐day drug‐free recovery period, both groups were unable to locate the platform within the allotted time (Figure 4B), likely reflecting reduced retention under the present testing schedule of the learned task in controls and sustained impairment in TBE‐treated mice. However, after three brief retraining sessions, all animals rapidly reacquired the task and found the platform within 10 s (Figure 4C), with no significant difference between groups. This suggests that TBE‐induced memory impairment is reversible upon retraining. Functional imaging via B‐mode ultrasound and photoacoustic modalities (Figure 4D) demonstrated a significant decrease in cerebral oxygen saturation in TBE‐treated mice after repeated dosing, implying cerebral hypoxia. Memory consolidation depends critically on hippocampal and prefrontal cortical circuits, which are known to be highly vulnerable to hypoxia. Indeed, insufficient oxygen supply can impair synaptic plasticity, suppress hippocampal long‐term potentiation (LTP), and reduce neuronal metabolism, manifesting as learning delay or memory loss. The literature further supports the contention that even mild, chronic cerebral hypoxia can induce reversible cognitive impairment—consistent with our findings of transient spatial learning deficit with recovery upon retraining. ²³

FIGURE 4.

Repeated TBE administration induces reversible spatial memory impairment associated with cerebral hypoxia. (A) Escape latency and time spent in the platform quadrant of mice after 7 days of Morris water maze training. (B) After 7 days without any treatment, the escape latency and time spent in the platform quadrant of the mice. (C) After 3 days of retraining, the escape latency and time spent in the platform quadrant of the mice. (D) Cerebral oxygen saturation measured by photoacoustic imaging. Data are expressed as mean ± SEM (n = 7). *p < 0.05, **p < 0.01 versus control by t‐test.

3.5. Molecular docking identifies GABRA1 as a key binding site with pontine enrichment

To further elucidate the mechanisms underlying TBE's sedative and anesthetic effects, we performed molecular docking analyses targeting several established anesthetic receptors: GABRA1, NMDA, TREK‐1, and the α₂_A adrenergic receptor. As depicted in Figure 5A, TBE adopted a stable binding conformation within the GABRA1 receptor pocket, engaging in favorable hydrophobic and hydrogen‐bond interactions with critical amino acid residues. In contrast, docking poses for TBE in NMDA, TREK‐1, and α₂_A receptor sites were shallow and less energetically favorable, showing few hydrogen bonds or hydrophobic contacts with their key residues (Figures 5B–D). The spatial alignment of these docking conformations with known ligands was poor, suggesting TBE likely exerts limited direct interaction with these targets. Subsequently, Western blot analysis across nine brain regions—including the pons, prefrontal cortex, hippocampus, and cerebellum—revealed that normalized GABRA1 protein expression was significantly elevated in the pons compared to other areas (Figure 5E). This pattern suggests a pontine‐specific accumulation or action site for TBE in the central nervous system.

FIGURE 5.

Molecular docking identifies GABRA1 as a high‐affinity binding target for TBE, with pontine enrichment. (A–D) Predicted binding poses of TBE within the active sites of GABRA1, NMDA, TREK‐1, and α₂_A‐adrenergic receptors. (E) Western blot analysis of GABRA1 expression across nine brain regions.

3.6. GABA_A receptor antagonism supports functional involvement of GABAergic signaling in TBE anesthesia

To further test whether GABA_A receptor signaling functionally contributes to TBE‐induced anesthesia, we performed pharmacological antagonism experiments using gabazine and bicuculline. Pretreatment with either antagonist did not significantly alter the onset of TBE‐induced loss of righting reflex. The onset time was 2.06 ± 0.22 min in the TBE group, 1.97 ± 0.20 min in the gabazine + TBE group, and 1.65 ± 0.23 min in the bicuculline+TBE group. In contrast, both GABA_A receptor antagonists markedly shortened the duration of loss of righting reflex. The duration was 39.67 ± 1.76 min in the TBE group, whereas it decreased to 11.56 ± 1.05 min after gabazine pretreatment and 17.18 ± 0.70 min after bicuculline pretreatment (Table 1). These results indicate that GABA_A receptor signaling contributes functionally to the maintenance of TBE‐induced anesthesia.

TABLE 1.

Pharmacological antagonism of GABA_A receptors shortens the duration of TBE‐induced anesthesia.

Group	n	Onset of LORR (min)	Duration of LORR (min)
TBE	5	2.06 ± 0.22	39.67 ± 1.76
Gabazine + TBE	5	1.97 ± 0.20	11.56 ± 1.05
Bicuculline + TBE	5	1.65 ± 0.23	17.18 ± 0.70

4. DISCUSSION

In this study, we utilized a multi‐modal analytical framework—including behavioral assays, functional imaging, molecular docking, and protein expression studies—to systematically assess the anesthetic efficacy, safety profile, and mechanistic underpinnings of TBE in mice. We found that substituting the conventional solvent 2‐methyl‐2‐butanol with the more biocompatible 1,2‐propanediol markedly reduced local irritation and physiological instability, while retaining full anesthetic efficacy. Critically, prolonged administration induced spatial memory deficits that were reversible upon cessation, correlating with diminished cerebral oxygen saturation and pontine GABRA1 enrichment. These findings not only offer new evidence supporting the optimisation of anesthetic protocols in experimental animals but also reveal a potential novel central target for the action of TBE.

Prior studies have reported that administration of TBE in the conventional 2‐methyl‐2‐butanol solvent is associated with tissue irritation, high inter‐individual variability, and elevated mortality risk. In the present study, we innovatively re‐formulated TBE using biocompatible 1,2‐propanediol and demonstrated—using OECD guidelines—that this alternative significantly mitigates inflammatory tissue responses without compromising cardiovascular stability or thermoregulation. Critically, mice receiving repeated dosing (330 mg/kg daily for 15 days) exhibited a 90% survival rate, addressing a major safety concern. Furthermore, sequential dose–response and nociceptive assessments confirmed that TBE's anesthetic depth is dose dependent, with no significant differences between C57 and ICR strains or between sexes. This consistency supports the standardization of anesthetic protocols across mouse strains.

In silico toxicity predictions indicated that the TBE molecule itself does not exhibit significant target‐mediated toxicity. However, repeated in vivo administration led to functional impairment of hepatic and renal systems and the development of hepatic steatosis. This apparent discrepancy suggests that toxic effects may arise from accumulation of metabolites rather than the parent compound, warranting further investigation into metabolic pathways. In the Morris water maze, mice exposed to TBE displayed significant spatial learning deficits during the training phase, concurrently with reduced cerebral oxygen saturation as detected via photoacoustic imaging. MRI further revealed hippocampal structural and signal alterations. However, these findings should not be interpreted as direct evidence of synaptic loss in the absence of synaptic‐marker validation. We hypothesize that TBE may impair hippocampal‐dependent memory consolidation by inducing chronic mild cerebral hypoxia—possibly through suppression of the respiratory centre or reduced cerebral perfusion—disrupting synaptic plasticity, consistent with previous evidence that hypoxia suppresses long‐term potentiation. ²⁴ Crucially, the observed cognitive deficit was fully reversible upon re‐training, reflecting robust compensatory capacity in the nervous system.

We demonstrate that TBE exhibits its strongest binding affinity to GABRA1, forming a stable hydrophobic pocket and hydrogen‐bond network—interactions substantially stronger than those observed with NMDA or TREK‐1 receptors. This structural evidence supports a predominantly GABAergic mechanism of action. Western blot analysis revealed significantly elevated GABRA1 expression in the pons compared with other brain regions, implicating the pons as a central site of anesthetic action. Although GABAergic anesthesia has traditionally been linked to thalamic or cortical regions, our data suggest a novel mechanism: activation of GABRA1 in the brainstem may suppress ascending arousal pathways, culminating in loss of consciousness. ²⁵ This finding offers a fresh perspective on the selective sedative effects of TBE.

Despite these insights, our study has notable limitations. First, although photoacoustic imaging revealed reduced cerebral oxygen saturation, this technique cannot distinguish between neuronal and vascular contributions; the absence of direct neuronal hypoxia biomarkers such as HIF‐1α, lactate/ATP ratios, or COX‐2 expression limits mechanistic interpretation. Second, while molecular docking indicates stable binding between TBE and GABRA1, we did not perform electrophysiological or pharmacological blockade studies. Without in vivo validation or use of GABRA1 inhibition models, receptor‐specific involvement remains speculative. A limitation of the present physiological assessment is that cardiac electrical activity was not directly recorded. Tail‐cuff plethysmography allowed non‐invasive monitoring of pulse rate and blood pressure but did not permit definitive diagnosis of arrhythmias. Future studies using ECG or telemetry will be required to determine whether different TBE formulations differ in their effects on cardiac rhythm or conduction.

5. CONCLUSIONS

In summary, reformulation of TBE using biocompatible 1,2‐propanediol substantially enhances its safety profile while preserving anesthetic efficacy in murine models. Multi‐parametric data indicate that although repeated dosing may briefly impair spatial memory—likely via mechanisms involving cerebral hypoxia and hippocampal synaptic loss—this effect is reversible upon cessation. This comprehensive evaluation supports the use of the reformulated TBE as a safer injectable anesthetic alternative for laboratory animals, improving ethical standards and animal welfare without compromising sedation stability. Moreover, the identified pathway through pontine GABRA1 and hypoxia‐linked cognitive modulation provides a robust experimental framework for probing GABAergic sedation dynamics and neurotoxicity. These findings bear direct relevance for refining preclinical anesthesia protocols and guiding future development of safer anesthetic agents.

AUTHOR CONTRIBUTIONS

Xia Li: Conceptualization; data curation; formal analysis; methodology; validation; writing – original draft. Yanming Chen: Data curation; formal analysis; methodology; writing – original draft. Xinyi Xiao: Methodology. Guoheng Xu: Supervision; writing – review and editing.

FUNDING INFORMATION

This research was funded by National Key Research and Development Program of China, grant number 2023YFF0724900.

CONFLICT OF INTEREST STATEMENT

The authors declare no competing financial interests.

ETHICS STATEMENT

All experimental procedures adhered to the ARRIVE 2.0 guidelines and were approved by the Institutional Animal Care and Use Committee of Peking University Health Science Center (approval no. DLASBE0419; approval dated, 18 April 2025). In line with the 3R principles, sample sizes were minimized wherever possible. All procedures were conducted in strict accordance with the institutional guidelines for the care and use of laboratory animals, with all efforts made to minimize animal suffering.