Liposomal Bupivacaine: A Delay Rather Than Elimination of Rebound Pain in Mouse Postoperative Model

Liposomal Bupivacaine: A Delay Rather Than Elimination of Rebound Pain in Mouse Postoperative Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Liposomal Bupivacaine: A Delay Rather Than Elimination of Rebound Pain in Mouse Postoperative Model Hao Wu, Jiayu Jin, Di Zhou, Lihai Chen, Liu Han This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7259947/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Nov, 2025 Read the published version in BMC Anesthesiology → Version 1 posted 13 You are reading this latest preprint version Abstract Background: Rebound pain, a postoperative surge in pain after peripheral nerve block (PNB) resolution, leads to increased opioid use and delayed recovery. Liposomal bupivacaine (LB) resolution prolongs analgesia, but its impact on rebound pain remains unclear. We developed a mouse model of sciatic/femoral nerve block to study LB’s effects. Methods: Male C57 mice (n=24) underwent tibial fracture surgery and were divided into sham, surgery (S), or LB (1.3%) groups. LB was administered near sciatic/femoral nerves using nerve stimulator guidance. Mechanical (von Frey) and thermal (Hargreaves) nociception were assessed. Histology evaluated nerve inflammation at days 2 and 28. Nerve block success rates were confirmed via methylene blue injection (n=15). Results: LB extended thermal (24h, P <0.05) and mechanical (36h, P <0.05) analgesia. Transient thermal rebound hyperalgesia occurred at 48h ( P =0.003), but no mechanical rebound was observed. Mild neural inflammation was more frequent with LB at day 2 ( P <0.05). Nerve stimulator-guided blocks had high success rates (sciatic: 93.3%, P =0.035; femoral: 73.3%, P =0.030). Conclusions: LB delayed but did not eliminate rebound pain, possibly due to neuroinflammation. A reliable mouse model of combined sciatic/femoral nerve block was established. Rebound Pain Regional anesthesia Liposomal bupivacaine Acute postoperative pain Nerve stimulator Sciatic nerve Femoral nerve Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Rebound pain is a common postoperative phenomenon, which usually occurs after the release of peripheral nerve block (PNB) and is characterized by a sudden increase in pain intensity, mainly within 24 hours after surgery [ 1 ]. Epidemiological data suggest a relatively high incidence of rebound pain, but the incidence varies across studies. For example, in Ethiopia, the overall incidence of rebound pain after PNB remission was found to be as high as 61.7%[ 2 ]. In China, 49.6% of day surgery patients develop rebound after PNB[ 3 ]. The epidemiological data of rebound pain also reveal its correlation with patient prognosis. A study found that rebound pain increases postoperative opioid use, prolongs hospitalization, reduces recovery quality, and lowers patient satisfaction [ 4 , 5 ]. Poor postoperative pain management can extend acute pain duration, increase chronic pain risk, and diminish quality of life. Thus, preventing and treating rebound pain is crucial. The mechanisms underlying rebound pain are multifaceted. Firstly, hyperalgesia may contribute to this phenomenon. In a rat model of sciatic nerve block with ropivacaine, transient thermal hyperalgesia occurs in the hind limbs and persists for 5 to 7 hours after the sensory block subsides[ 6 ]. Secondly, the local inflammatory response also appears to be a key driver of rebound pain. Studies have shown that peripheral nerve block can exacerbate the acute inflammatory response at the surgical incision site, characterized by neutrophil and macrophage infiltration, as well as upregulation of tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) [ 7 ]. Once the nerve block effect wanes, these inflammatory mediators can activate peripheral nociceptors and trigger rebound pain[ 8 ]. Additionally, the neurotoxicity of local anesthetics may also play a role. Studies have demonstrated that bupivacaine-induced sciatic nerve block in mice can lead to early peripheral nerve injury, marked by Wallerian degeneration and axonal demyelination[ 9 ]. Local anesthetics have also exhibited neurotoxic and cytotoxic effects. Finally, the activation of C-type fibers is thought to be associated with rebound pain. While the surgical stimulus is temporarily blunted by the nerve block, it may leave a "pain memory" that is reactivated when the block fades, resulting in severe pain[ 10 ]. Since rebound pain was originally defined as severe pain occurring within 24 hours after peripheral nerve block (PNB) intervention, some clinicians have attempted to mitigate the adverse effects of rebound pain by prolonging analgesia to cover the time points when it typically occurs. For example, continuous nerve block techniques can prolong analgesia duration, thereby reducing postoperative inflammatory responses and rebound pain incidence. Salviz et al. demonstrated that in patients undergoing rotator cuff repair surgery, the incidence of severe pain on the first postoperative day was significantly lower (15%) in those receiving continuous interscalene brachial plexus block [ 11 ]. However, continuous nerve block is technically challenging, with a high failure rate and increased management costs, limiting its wider application. Secondly, the use of local anesthetic adjuvants serves as an effective alternative to continuous nerve block. In a mouse model of sciatic nerve block with bupivacaine, perineural addition of dexamethasone prevents thermal hyperalgesia and reduces neurotoxicity[ 9 ]. Additionally, multimodal analgesia strategies integrate nerve blocks with systemic analgesics (e.g., NSAIDs and opioids) and adjunctive analgesics (e.g., corticosteroids and anticonvulsants) to optimize postoperative pain management[ 12 ]. In recent years, new ultra-long-acting local anesthetics such as liposomal bupivacaine have been used clinically to prolong postoperative analgesia[ 13 ]. A clinical study shows that liposomal bupivacaine can significantly lower pain scores, prolong analgesia, and potentially reduce rebound pain[ 14 ]. Another randomized controlled trial indicates that liposomal bupivacaine reduces pain scores and opioid use in the first 24 hours post-surgery versus placebo, with no sign of rebound pain. These findings highlight the potential of liposomal bupivacaine to eliminate rebound pain and prompt further investigation into its mechanisms. However, these studies only examined rebound pain within 24 hours post-surgery. Despite the controversial analgesic duration of liposomal bupivacaine[ 15 , 16 ], extending the observation period is necessary to prevent late-onset rebound pain and its adverse consequences. Thus, this study observed pain changes in mice for five days after peripheral nerve block with 1.3% liposomal bupivacaine, covering its theoretical analgesic period, and improved the mouse nerve block model to enhance nerve block success rate and nerve localization accuracy. Materials and Methods The experimental procedure (DWSY-23062366) was approved by the Institutional Animal Care and Use Committee (IACUC) at Nanjing Medical University. All experimental methods followed the Guidelines for the Institutional Animal Care and Use Committee at Nanjing Medical University. Animals All experiments described here were conducted in strict accordance with institutional guidelines. Male C57BL/6J mice (weighing 25–30g; aged 8 weeks; Wandong Biotechnology Corporation of Nanjing; n = 67) were housed in the same room at a constant temperature (22°C ± 2°C) and humidity (55%±5%) and maintained on a 12:12 hours light-dark cycle with free access to water and food. 24 mice were randomly allocated into the following groups: (1) sham group, (2) surgery group (S), and (3) liposomal bupivacaine group (LB). These mice were used to assess postoperative pain behaviors. Most studies involve dissecting nerves before administering local anesthetics, which can cause additional trauma. Additionally, studies using anatomical landmark-guided nerve blocks often report lower success rates. To ensure the effectiveness of our experiments, we used methylene blue staining in 15 mice to validate which method could achieve effective nerve blockade. 28 mice were assigned to the surgery group and the liposomal bupivacaine group to assess early (day 2) and late (day 28) postoperative neural injury and neuroinflammation. Animal model of Tibial Fracture The Tibial Fracture model was established using the method described by Khajuria et al[ 17 ]. Mice were anesthetized with 2% isoflurane and fixed in a right lateral recumbent position. and open mid-diaphyseal tibial fractures were induced in the left hindlimb as previously described. Postoperatively, the LB group was administered 35 mg/ kg 1.3% liposomal bupivacaine solution in the vicinity of the sciatic and femoral nerves [ 18 ], guided by a nerve stimulator. Concurrently, the S group only received a tibial fracture surgery. After sealing the injection hole and closing the wound, mice were resuscitated on a heated blanket. Combined sciatic and femoral nerve block under nerve stimulator guidance A combined sciatic and femoral nerve block was executed under nerve stimulator guidance[ 18 , 19 ]. For the sciatic nerve block, the anesthetized mouse was positioned in right lateral recumbency, with the left hind limb and inguinal region prepared by shaving and cleansing, aligning the limb perpendicular to the torso. The puncture site was identified in the posterior lateral thigh muscle, and a 22G, 5cm needle (B. Braun Melsungen AG, model 4894146N) was utilized. The needle was attached to a nerve stimulator (Stimuplex HNS12, B. Braun Melsungen AG, Germany), and the tip was directed towards the posterior mid-femur. The initial nerve stimulator current is set at 0.2 mA, frequency at 2 Hz, aiming for ankle contraction or extension in mice. The needle position is adjusted until, at 0.08–0.12 mA current, ankle movement occurs. After confirming no blood return, 35 mg /kg 1.3% liposomal bupivacaine is injected, causing ankle movement loss. If, upon resetting the current to 0.2 mA, the response doesn't recover, the sciatic nerve block is successful. For the femoral nerve block, the mouse was repositioned to supine, with the puncture site chosen at the mid-to-lower inguinal region. The needle was inserted at a 30° angle to the skin, toward the midpoint of the inguinal region. The initial current was set at 0.2 mA. The target motor responses in mice were quadriceps contraction and knee extension. The needle position was adjusted until these responses occurred at 0.08–0.12 mA. After confirming no blood return, 35 mg /kg 1.3% liposomal bupivacaine was injected, causing knee movement loss. If, upon resetting the current to 0.2 mA, the response didn't recover, the nerve block was successful. Sciatic nerve and femoral nerve block under anatomical landmark location A combined sciatic and femoral nerve block was executed under anatomical landmark location. For the sciatic nerve block, the anesthetized mouse was positioned in right lateral recumbency, with the left hind limb and inguinal region prepared by shaving and cleansing, aligning the limb perpendicular to the torso. The puncture site was identified in the posterior lateral thigh muscle, and a 29G needle (B. Braun Melsungen AG, model 4894146N) was utilized. The needle was introduced posteromedially towards the greater trochanter in an anteromedial direction. After encountering the ischial tuberosity, 35 mg/ kg 1.3% liposomal bupivacaine was injected [ 20 ]. For the femoral nerve block, referring to the method of femoral nerve block guided by nerve stimulator, the mice were placed in supine position, and the puncture needle was inserted into the middle third of the groin at 30° from the skin. The depth of the puncture needle was 5 mm, and 35 mg /kg 1.3% liposomal bupivacaine was injected. Staining Study To assess the success rate of nerve blocks, 15 mice were injected around both the femoral and sciatic nerves on each side. One side was randomly selected for injection using the anatomical landmark guidance method(Group A)[ 18 ], while the other side used the nerve-stimulator guidance method (Group N). A volume of 0.085 mL of 0.1% methylene blue solution was injected around each nerve[ 21 ] . The anatomical landmark-guided approach for sciatic and femoral nerve block is as follows: Sciatic Nerve Block: Referring to Liljana's research [ 18 ], the mouse is fixed in a lateral decubitus position, and the middle and posterior-lateral muscle area of the thigh is chosen as the puncture site. A 29-gauge needle is directed posterior to the midpoint of the femur. After the puncture needle encounters the femur and no blood is aspirated, 0.085 mL of 0.1% methylene blue is injected. Femoral Nerve Block procedure: Referring to the anatomical structure of the femoral nerve and the muscle contraction response under neurostimulation guidance, targeted improvements are made. Except for not using a neurostimulator, the rest of the operation is the same as above, with the puncture needle depth set to 5mm, and 0.085 mL of 0.1% methylene blue is injected. This procedure led to a total of 30 methylene blue stainings for the sciatic nerves and 30 methylene blue stainings for the femoral nerves across the left and right hind limbs. One hour after injection, the mice were euthanized under isoflurane anesthesia and dissected to assess whether the nerve staining was successful and to determine the success rates of sciatic and femoral nerve blocks. This assessment was utilized to determine the efficacy of nerve stimulation-guided blocks of the sciatic and femoral nerves in mice. The presence of staining on the nerves indicated a successful block, while the absence of staining suggested a failed block[ 21 ]. Nociceptive Behavioral Tests Two tests were used to assess pain behavior: (i) Mechanical nociception assessed by the withdrawal response to von Frey filament application, (ii) Thermal nociception assessed by the withdrawal response to thermal stimulus. Mechanical nociception Unrestricted mice were positioned under a clear plastic chamber that was elevated on a mesh floor, allowing them to acclimate to the environment. Mechanical withdrawal responses were assessed using a series of calibrated von Frey filaments (ranging from 0.04 to 4 g)[ 22 , 23 ]. These filaments were applied from beneath the cage, through the mesh floor openings, to the plantar surface of the hind paw, specifically targeting the middle area of the left leg. The filament was pressed until it produced a slight bend and was then held in this position for 6 seconds. Beginning with a filament force of 0.6 g, we determine the 50% paw withdrawal threshold utilizing Dixon’s up-down method (with cutoff thresholds of 4g [maximum] and 0.04g [minimum]). Thermal nociception Thermal withdrawal latencies were evaluated utilizing the Hargreaves method. Animals were placed in a plastic chamber on an elevated glass platform. A radiant heat source (SH-1000 Plantar Hot Plate Tester, Shanghai Vax Biotech Co., Ltd, Shanghai, China) was positioned beneath the glass to direct heat towards the plantar surface of the paw. The intensity of the radiant heat was calibrated to achieve an average withdrawal latency of approximately 10 seconds for the paws of naive animals. Each paw was subjected to heat stimulation three times with a minimum interval of 3 minutes between applications, and the average withdrawal latency was calculated by averaging these values (with a cutoff time of 15 seconds) [ 9 , 24 ]. Histological Evaluation(HE) Twenty-eight mice were divided into the liposomal bupivacaine and surgical groups, and 7 mice in each group were selected for detection on day 2 and day 28, respectively. These mice were anesthetized as described above, followed by gentle exposure and removal of approximately 8–10 millimeters of the sciatic and femoral nerves. This time interval was chosen correspond to short-term and long-term nerve tissue damage. The specimens were fixed in 4 formalin for 48 hours, washed with ethanol, and embedded in paraffin. A pathologist, blinded to the experimental treatment, analyzed the slides using standardized scales for perineural inflammation. As shown in Fig. 1 , edema and lymphocyte infiltration are characteristic features of inflammation. Inflammation was classified as follows: no inflammation (absence of inflammatory changes), mild inflammation (areas with less than 50% edema or cellular infiltrate), and severe inflammation (diffuse areas with greater than 50% edema or cellular infiltrate)[ 25 ]. Wallerian degeneration, reflecting abnormal myelin and axonal degeneration due to nerve injury, was used to assess nerve damage. The classic histological appearance of Wallerian degeneration includes axonal swelling and myelin breakdown (Fig. 4 ). Wallerian degeneration was classified as follows: no Wallerian degeneration (absence of lesions), mild Wallerian degeneration (0–50% of fibers showing Wallerian degeneration), and severe Wallerian degeneration (greater than 50% of fibers showing Wallerian degeneration) [ 26 ]. Longitudinal sections of 5mm thickness were prepared and stained with hematoxylin and eosin to assess inflammation[ 26 ]. Each specimen was scored by a blinded pathologist in the treatment group for the degree of lymphocytic infiltration (Fig. 4 ): no inflammation, indicating no signs of inflammation. mild inflammation, with swelling and lymphocytic infiltration covering up to 50% of the surface. severe inflammation, with swelling and lymphocytic infiltration covering more than 50% of the surface. Statistical Analysis Statistical analysis was performed using SPSS 22.0 via a repeated-measures one-way analysis of variance (ANOVA) followed by Bonferroni’s post-tests for evaluating the effects of liposomal bupivacaine (LB) on PWL and 50% paw withdrawal threshold. The dependent variable for these analyses was time in hours. Chi-square tests and Fisher's exact probability method were used to examine the success rates of sciatic nerve and femoral nerve blocks between nerve stimulator guidance and anatomical landmark localization methods. The Mann-Whitney test was applied to compare the histopathology scores between time points. The statistical significance was determined as P < 0.05. All data were presented as means ± standard error of mean (SEM). Results Nerve stimulator-guided nerve block enhances the accuracy of nerve localization To choose a more appropriate nerve block method for mice, we compared the femoral and sciatic nerve block guided by a nerve stimulator with that located by anatomical landmarks, to construct a suitable mouse nerve block model. We used fifteen C57 mice (8 weeks old). On each mouse's left hind leg, nerve stimulator - guided block (Group N) was applied, while anatomical - landmark - guided block (Group A) was on the right. We used 0.1% bilateral methylene blue instead of local anesthetic for nerve block. After blocking, the mice were euthanized and dissected to observe the methylene blue location and drug distribution of the sciatic and femoral nerves. The range of nerve staining was used as the criterion to determine the success of sciatic and femoral nerve blocks (Fig. 1 A-D). The results showed that in Group N, compared with Group A, both the sciatic and femoral nerves exhibited more precise nerve localization and a wider range of drug distribution. The nerve block success rate in Group N was significantly higher than in Group A (Table 1 ), with no vascular rupture during the block. Our study shows that in mice, compared with traditional anatomical landmark - located nerve blocks, the femoral and sciatic nerve block guided by nerve stimulator has a higher success rate and improves the accuracy of nerve localization. This method is precise and highly reproducible. In follow - up studies, we used nerve stimulator guidance to construct a mouse model of femoral and sciatic nerve block. Table 1 Comparison of Sciatic and Femoral Nerve Block Success Rates between Nerve Stimulator-Guided and Anatomical Landmark Techniques Nerve Group Staining success YES/NOT success rate (%) YES NOT sciatic nerve Group A 8 7 53.3 Group N χ2 P 14 1 93.3 6.136 0.035 femoral nerve Group A 3 12 20.0 Group N χ2 P 11 4 73.3 8.571 0.030 Peripheral nerve block with 1.3% liposomal bupivacaine delays the onset of rebound pain To investigate whether liposomal bupivacaine can eliminate rebound pain, 24 mice (8 weeks old) were randomly divided into three groups: blank control group (sham), operation group (S), and operation + nerve block group (LB). LB mice underwent femoral and sciatic nerve block with 1.3% liposomal bupivacaine one hour before surgery. Samples without analgesia within one hour were excluded. Preoperative baseline measurements were taken, and mechanical/thermal pain thresholds were tested at 1 h, 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 96 h, and 120 h post - surgery. After tibial plateau fracture surgery, the LB group exhibited higher thermal and mechanical pain thresholds within 24 hours (Fig. 2 – 3 ), indicating that 1.3% liposomal bupivacaine provides effective postoperative analgesia during this period. However, at 48 hours post - modeling, the LB group's thermal pain threshold dropped significantly compared to the S group, indicating hyperalgesia and rebound pain onset. After 48 hours, there were no significant differences in pain thresholds between the two groups. These results show that LB, though having a longer analgesic effect than traditional local anesthetics, delays but doesn't eliminate rebound pain. rebound pain may be related to nerve injury and neuroinflammation To preliminarily explore the mechanism of rebound pain, we euthanized LB group mice with rebound pain at 48 h and corresponding S group mice. Their femoral and sciatic nerves on the surgical side were removed and stained with HE. Results showed that both nerves had inflammatory cell infiltration and Wallerian degeneration, indicating nerve injury and neuroinflammation (Fig. 4 A-B). To determine the permanence of the injury and inflammation, HE staining of femoral and sciatic nerves in LB mice was done on postoperative day 28. Nerve injury was classified based on the proportion of Wallerian degeneration: no injury (0%), mild injury (0–50%), and severe injury (> 50%). On postoperative day 2, LB mice had mild to severe nerve injury, while no injury was observed in either group on day 28 (Table 2 ). Similarly, neuroinflammation was assessed based on inflammatory cell infiltration: no inflammation (0%), mild inflammation (0–50%), and severe inflammation (> 50%). On postoperative day 2, LB mice had mild to severe neuroinflammation, which disappeared by day 28 (Table 3 ). These results suggest that rebound pain may be associated with nerve injury and neuroinflammation, but the damage is reversible. Table 2 Neural Toxicity After Single-Shot Regional Anesthesia at day 2 and at day 28. Nerve Group Wallerian degeneration at day 2 Wallerian degeneration at day 28 None Mild Severe None Mild Severe sciatic nerve LB 0 7 0 7 0 0 S 5 2 0 7 0 0 femoral nerve LB 0 3 4 7 0 0 S 5 2 0 7 0 0 Table 3 Perineural Inflammation After Single-Shot Regional Anesthesia at day 2 and at day 28. Nerve Group Neural inflammation at day 2 Neural inflammation at day 28 None Mild Severe None Mild Severe sciatic nerve LB 1 6 0 7 0 0 S 6 1 0 7 0 0 femoral nerve LB 1 5 1 7 0 0 S 6 1 0 7 0 0 Discussion Liposomal bupivacaine (LB) is a novel ultra-long-acting local anesthetic approved by the US Food and Drug Administration. Its theoretical analgesic duration can reach 72 hours [ 27 ], and it has the potential to reduce rebound pain. However, current basic research shows controversial duration of analgesia. For example, Liljana et al. [ 18 ] reported that in a mouse model of sciatic nerve block using 1.3% LB, the average duration of sensory block was 118 minutes. Wenling Zhao et al. [ 27 ] observed a median duration of sensory block of 4.83 hours in a rat model of postoperative sciatic nerve block. Most current basic studies use peripheral nerve block guided by traditional anatomical landmarks. The shortened efficacy of LB may be due to low nerve block success rates or incomplete block. Whether this is related to the method of peripheral nerve block has not been reported. According to prior research, some researchers used nerve blocks under anatomical vision, causing additional damage to animal models. Others used nerve blocks guided by anatomical landmarks, which had low success rates and effectiveness. In our study, we performed sciatic and femoral nerve blocks in mice using nerve stimulator guidance. The duration of sensory block with LB was prolonged to 24 h. Compared with anatomical - landmark - guided peripheral nerve block, bilateral methylene blue nerve staining showed that nerve stimulator - guided peripheral nerve block allowed better drug encapsulation of nerves and had higher and more stable success rates. This was mainly because nerve stimulator guidance improved needle placement accuracy[ 28 ]. This method relied on evoked muscle contraction to accurately position the needle tip near the target nerve. The results highlight that nerve stimulator - guided peripheral nerve block is superior to traditional anatomical - landmark - guided nerve block. Notably, LB showed significant analgesic effects within 24 h post - surgery, with no rebound pain during this period. However, rebound pain was observed at 48 h, indicating it was delayed rather than eliminated. Past studies did not report this phenomenon, possibly due to insufficient observation time or different models from our study. Our research strongly suggests extending the observation window when using new long - acting local anesthetics. The original definition of rebound pain was limited to severe pain occurring within 24 hours after peripheral nerve block (PNB) [ 29 ], typically observed with traditional local anesthetics such as bupivacaine or ropivacaine. Our study extends this definition and emphasizes the importance of aligning the duration of surgical nociceptive stimulation with that of PNB action. With the clinical introduction of new ultra-long-acting local anesthetics and adjuvants like dexamethasone to prolong PNB duration, the monitoring time window for rebound pain should be correspondingly extended. Failure to do so may result in severe hyperalgesia post-rebound pain, leading to adverse outcomes such as increased chronic pain incidence. The mechanism of rebound pain is still unclear. Relevant studies have shown that liposomal bupivacaine exerts an anesthetic effect by blocking sodium channels in nerve cell membranes. However, long-term or high-concentration exposure can lead to neurotoxicity and trigger neuroinflammation[ 30 ]. This neurotoxicity may involve multiple pathways, including disruption of cell membrane integrity, increased intracellular calcium levels, activation of inflammatory signaling pathways (such as PI3K/Akt and oxidative stress), and induction of apoptosis[ 31 ]. Neuroinflammation enhances the sensitivity of nerve fibers, especially C fibers, which become overexcited in an inflammatory state, leading to thermal hyperalgesia [ 10 ]. Due to the diminished analgesic effect of liposomal bupivacaines, persistent local inflammation may further lower the pain threshold, resulting in rebound pain. Notably, adding anti-inflammatory agents like dexamethasone to bupivacaine preparations has been proven to reduce the incidence of rebound pain. Dexamethasone may alleviate neuroinflammation and C-fiber overexcitation by inhibiting inflammatory pathways (such as PI3K/Akt) and reducing oxidative stress[ 32 , 33 ]. In this study, we preliminarily explored the mechanism of LB-induced rebound pain. HE staining revealed nerve swelling and lymphocyte infiltration in both the sciatic and femoral nerves during rebound pain, indicating a close relationship between rebound pain and neuroinflammation. This study has certain limitations. Firstly, it has only preliminarily explored the mechanism of rebound pain. Future research will need to delve deeper into its molecular mechanisms to provide more reliable evidence for targeted interventions to mitigate the adverse effects of rebound pain. Secondly, although nerve staining confirmed the success of femoral nerve block, the sensory block areas detected by the von Frey and thermal pain tests are innervated by the sciatic nerve. This means these tests only demonstrate the effectiveness of sciatic nerve block over time, not that of femoral nerve block. Thus, more suitable methods to verify femoral nerve block effectiveness in mice need to be developed. Finally, as this study only examined basic level results and given the differences between animal models and humans, further clinical research is essential to validate our findings. Conclusion Our findings suggested that rebound pain was involved in the development of postoperative hyperalgesia. The delay of rebound pain by liposomal bupivacaine was associated with the inhibition of neuroinflammation-mediated Wallerian degeneration in mice. Abbreviations LB Liposomal bupivacaine PNB peripheral nerve block ELISA Enzyme-linked immunosorbent assay HE Histological Evaluation Declarations Availability of data and materials All data presented in this study are available from corresponding author on reasonable request. Ethical Approval Statement "This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University (Approval No. DWSY-23062366). All experimental procedures were performed in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the institutional ethical guidelines." Funding This work was supported by the 2021 Nanjing Municipal Health Science and Technology Development Special Fund Project Plan (YKK21128). Conflicts of Interest No potential conflict of interest relevant to this article was reported. Author Contributions Hao Wu (Conceptualization; Investigation; Data acquisition and analysis; Methodology; Formal analysis; Writing – original draft) Jiayu Jin (Conceptualization; Investigation; Methodology; Writing – review & editing) Di Zhou (Data curation; Investigation; Validation; Visualization) Lihai Chen (Conceptualization; Supervision; Writing – review & editing) Liu Han(Conceptualization; Supervision; Formal analysis; Validation; Writing – review & editing) ORCID Hao Wu, https://orcid.org/0009-0003-1624-3795 Jiayu Jin, https://orcid.org/0009-0005-9353-454X Di Zhou, https://orcid.org/0009-0005-2490-8974 Lihai Chen, https://orcid.org/0000-0001-8535-9239 Liu Han, https://orcid.org/0000-0003-2993-0394 Additional information Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations References Muñoz-Leyva F, Cubillos J, Chin KJ. Managing rebound pain after regional anesthesia. Korean J Anesthesiol. 2020;73(5):372–83. Admassie BM, Debas SA, Admass BA. Prevention and management of rebound pain after resolution of regional block: a systematic review. Ann Med Surg (Lond). 2024;86(8):4732–7. Barry GS, Bailey JG, Sardinha J, Brousseau P, Uppal V. Factors associated with rebound pain after peripheral nerve block for ambulatory surgery. Br J Anaesth. 2021;126(4):862–71. Luebbert E, Rosenblatt MA. Postoperative Rebound Pain: Our Current Understanding About the Role of Regional Anesthesia and Multimodal Approaches in Prevention and Treatment. Curr Pain Headache Rep. 2023;27(9):449–54. Dada O, Gonzalez Zacarias A, Ongaigui C, Echeverria-Villalobos M, Kushelev M, Bergese SD, et al. Does Rebound Pain after Peripheral Nerve Block for Orthopedic Surgery Impact Postoperative Analgesia and Opioid Consumption? A Narrative Review. Int J Environ Res Public Health. 2019;16(18):3257. Kolarczyk LM, Williams BA. Transient heat hyperalgesia during resolution of ropivacaine sciatic nerve block in the rat. Reg Anesth Pain Med. 2011 May-Jun;36(3):220–4. Yamada T, Hasegawa-Moriyama M, Kurimoto T, Saito T, Kuwaki T, Kanmura Y. Peripheral Nerve Block Facilitates Acute Inflammatory Responses Induced by Surgical Incision in Mice. Reg Anesth Pain Med. 2016 Sep-Oct;41(5):593–600. Pogatzki-Zahn EM, Segelcke D, Schug SA. Postoperative pain-from mechanisms to treatment. Pain Rep. 2017;2(2):e588. An K, Elkassabany NM, Liu J. Dexamethasone as adjuvant to bupivacaine prolongs the duration of thermal antinociception and prevents bupivacaine-induced rebound hyperalgesia via regional mechanism in a mouse sciatic nerve block model. PLoS ONE. 2015;10(4):e0123459. Kleggetveit IP, Namer B, Schmidt R, Helås T, Rückel M, Ørstavik K, et al. High spontaneous activity of C-nociceptors in painful polyneuropathy. Pain. 2012;153(10):2040–7. Salviz EA, Xu D, Frulla A, Kwofie K, Shastri U, Chen J, et al. Continuous interscalene block in patients having outpatient rotator cuff repair surgery: a prospective randomized trial. Anesth Analg. 2013;117(6):1485–92. Joshi GP. Rational Multimodal Analgesia for Perioperative Pain Management. Curr Pain Headache Rep. 2023;27(8):227–37. Peng H, Wen J, Chen M, Xia Z, Jiang Y, Xie K, et al. Preoperative Analgesia Efficacy of Liposomal Bupivacaine Following Pericapsular Nerve Group (PENG) Block in Patients with Hip Fracture: A Randomized Controlled Observer-Blinded Study. Pain Ther. 2025;14(1):283–96. Gong R, Tan G, Huang Y. The Efficacy of Liposomal Bupivacaine in Thoracic Surgery: A Systematic Review and Meta-Analysis. J Pain Res. 2024;17:4039–51. Hussain N, Brull R, Sheehy B, Essandoh MK, Stahl DL, Weaver TE, et al. Perineural Liposomal Bupivacaine Is Not Superior to Nonliposomal Bupivacaine for Peripheral Nerve Block Analgesia. Anesthesiology. 2021;134(2):147–64. Dinges HC, Wiesmann T, Otremba B, Wulf H, Eberhart LH, Schubert AK. The analgesic efficacy of liposomal bupivacaine compared with bupivacaine hydrochloride for the prevention of postoperative pain: a systematic review and meta-analysis with trial sequential analysis. Reg Anesth Pain Med. 2021;46(6):490–8. Khajuria DK, Karuppagounder V, Nowak I, Sepulveda DE, Lewis GS, Norbury CC, et al. Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice. J Bone Min Res. 2023;38(11):1560–76. Markova L, Cvetko E, Ugwoke CK, Horvat S, Umek N, Stopar Pintarič T. The Influence of Diabetic Peripheral Neuropathy on the Duration of Sciatic Nerve Block with 1.3% Liposomal Bupivacaine and 0.25% Bupivacaine Hydrochloride in a Mouse Model. Pharmaceutics. 2022;14(9):1824. Shikanov A, Domb AJ, Weiniger CF. Long acting local anesthetic-polymer formulation to prolong the effect of analgesia. J Control Release. 2007;117(1):97–103. Markova L, Umek N, Horvat S, Hadžić A, Kuroda M, Pintarič TS, et al. Neurotoxicity of bupivacaine and liposome bupivacaine after sciatic nerve block in healthy and streptozotocin-induced diabetic mice. BMC Vet Res. 2020;16(1):247. Zhang Y, Cui B, Gong C, Tang Y, Zhou J, He Y, et al. A rat model of nerve stimulator-guided brachial plexus blockade. Lab Anim. 2019;53(2):160–8. Zhang L, Wang Z, Song C, Liu H, Li Y, Li J, et al. Spinal NR2B phosphorylation at Tyr1472 regulates IRE(-)DMT1-mediated iron accumulation and spine morphogenesis via kalirin-7 in tibial fracture-associated postoperative pain after orthopedic surgery in female mice. Reg Anesth Pain Med. 2021;46(4):363–73. Gonzalez-Cano R, Boivin B, Bullock D, Cornelissen L, Andrews N, Costigan M. Up-Down Reader: An Open Source Program for Efficiently Processing 50% von Frey Thresholds. Front Pharmacol. 2018;9:433. Mitsui K, Hishiyama S, Jain A, Kotoda Y, Abe M, Matsukawa T, et al. Role of macrophage autophagy in postoperative pain and inflammation in mice. J Neuroinflammation. 2023;20(1):102. Papadopoulos G, Duckwitz V, Doherr MG. Femoral and sciatic nerve blockade of the pelvic limb with and without obturator nerve block for tibial plateau levelling osteotomy surgery in dogs. Vet Anaesth Analg. 2022;49(4):407–16. Marty P, Bennis M, Legaillard B, Cavaignac E, Ferre F, Lebon J, et al. A New Step Toward Evidence of In Vivo Perineural Dexamethasone Safety: An Animal Study. Reg Anesth Pain Med. 2018;43(2):180–5. Quaye A, McAllister B, Garcia JR, Nohr O, Laduzenski SJ, Mack L, et al. A prospective, randomized trial of liposomal bupivacaine compared to conventional bupivacaine on pain control and postoperative opioid use in adults receiving adductor canal blocks for total knee arthroplasty. Arthroplasty. 2024;6(1):6. Zhao W, Yang J, Zhang Y, Liu J, Zhang W. QX-OH/Levobupivacaine: Fixed-dose combination to provide a long-acting postoperative pain of knee surgery in rodents. Eur J Pharm Sci. 2018;111:418–24. Lim JA, Sung SY, Lee JH, Lee SY, Kwak SG, Ryu T, et al. Comparison of ultrasound-guided and nerve stimulator-guided interscalene blocks as a sole anesthesia in shoulder arthroscopic rotator cuff repair: A retrospective study. Med (Baltim). 2020;99(35):e21684. Admassie BM, Tegegne BA, Alemu WM, Getahun AB. Magnitude and severity of rebound pain after resolution of peripheral nerve block and associated factors among patients undergoes surgery at university of gondar comprehensive specialized hospital northwest, Ethiopia, 2022. Longitudinal cross-sectional study. Ann Med Surg (Lond). 2022;84:104915. Ma R, Wang X, Lu C, Li C, Cheng Y, Ding G, et al. Dexamethasone attenuated bupivacaine-induced neuron injury in vitro through a threonine-serine protein kinase B-dependent mechanism. Neuroscience. 2010;167(2):329–42. Fang J, Shi Y, Du F, Xue Z, Cang J, Miao C, et al. The effect of perineural dexamethasone on rebound pain after ropivacaine single-injection nerve block: a randomized controlled trial. BMC Anesthesiol. 2021;21(1):47. Morita S, Oizumi N, Suenaga N, Yoshioka C, Yamane S, Tanaka Y. Dexamethasone added to levobupivacaine prolongs the duration of interscalene brachial plexus block and decreases rebound pain after arthroscopic rotator cuff repair. J Shoulder Elb Surg. 2020;29(9):1751–7. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 26 Nov, 2025 Read the published version in BMC Anesthesiology → Version 1 posted Editorial decision: Revision requested 28 Aug, 2025 Reviews received at journal 26 Aug, 2025 Reviews received at journal 26 Aug, 2025 Reviews received at journal 18 Aug, 2025 Reviewers agreed at journal 16 Aug, 2025 Reviewers agreed at journal 14 Aug, 2025 Reviewers agreed at journal 14 Aug, 2025 Reviewers agreed at journal 14 Aug, 2025 Reviewers invited by journal 13 Aug, 2025 Editor invited by journal 12 Aug, 2025 Editor assigned by journal 07 Aug, 2025 Submission checks completed at journal 07 Aug, 2025 First submitted to journal 31 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7259947","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":501940035,"identity":"90a38369-10bd-44a9-8e66-3622416ee53a","order_by":0,"name":"Hao Wu","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Wu","suffix":""},{"id":501940036,"identity":"eb72786b-62da-46be-806b-9c24737702b8","order_by":1,"name":"Jiayu Jin","email":"","orcid":"","institution":"the Affiliated Nanjing Hospital of Nanjing Medical University (Nanjing First Hospital)","correspondingAuthor":false,"prefix":"","firstName":"Jiayu","middleName":"","lastName":"Jin","suffix":""},{"id":501940037,"identity":"7d945ba8-ce1f-499d-9ab4-77a28c9d6fa4","order_by":2,"name":"Di Zhou","email":"","orcid":"","institution":"the Affiliated Nanjing Hospital of Nanjing Medical University (Nanjing First Hospital)","correspondingAuthor":false,"prefix":"","firstName":"Di","middleName":"","lastName":"Zhou","suffix":""},{"id":501940038,"identity":"943539b8-ed09-4883-8278-372b801f8327","order_by":3,"name":"Lihai Chen","email":"","orcid":"","institution":"the Affiliated Nanjing Hospital of Nanjing Medical University (Nanjing First Hospital)","correspondingAuthor":false,"prefix":"","firstName":"Lihai","middleName":"","lastName":"Chen","suffix":""},{"id":501940039,"identity":"290837be-69dd-4fcb-9d30-3e8ed0002a2d","order_by":4,"name":"Liu Han","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYBACAwhlwdgAoniAmJ8BwiakRQKojBmsRUKygWQtBgcIOMyc/ewBhh8VErIN/OePSbypuFNnfP5w24MfDHZyujgss+zJS2DsOSNh3CCRzCY558wzCbMbie2GPQzJxmY4rDM4kGPAzNgmkdggwcwmzdt2GKgFyOVhOJC4DZeW82+gWvgPA7X8Oyxh3H+wTfIPPi03YLYwJAO1NByWMGBIbJPGa8uNNwZgv7RJJBtbzjl2WHLGDaAWGQM8fjmfYwAMMRvZfv6DD2+8qTnMz99//Jnkmwo7OVxagID9B4hkQzMKp/JRMApGwSgYBUQAAOtwVtuM88cMAAAAAElFTkSuQmCC","orcid":"","institution":"the Affiliated Nanjing Hospital of Nanjing Medical University (Nanjing First Hospital)","correspondingAuthor":true,"prefix":"","firstName":"Liu","middleName":"","lastName":"Han","suffix":""}],"badges":[],"createdAt":"2025-07-31 08:38:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7259947/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7259947/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12871-025-03430-2","type":"published","date":"2025-11-26T15:57:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89641750,"identity":"af2a8788-e825-4206-a8d8-803c461c70d7","added_by":"auto","created_at":"2025-08-22 08:11:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":351481,"visible":true,"origin":"","legend":"\u003cp\u003eStaining results of the sciatic nerve and femoral nerve in mice. （A）Failure in sciatic nerve staining；（B）Success in sciatic nerve staining；（C）Failure in femoral nerve staining；（D）Success in femoral nerve staining.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7259947/v1/4a6e92657f09763ec81a7199.png"},{"id":89640068,"identity":"28ac6b78-8eff-4959-9182-08bb15f712f0","added_by":"auto","created_at":"2025-08-22 08:03:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":111335,"visible":true,"origin":"","legend":"\u003cp\u003ePaw Withdrawal Latency (PWL) induced by heat stimuli after combined sciatic and femoral nerve block with liposomal bupivacaine.This is an illustration of nociceptive responses of the treated (left) hindlimb to heat stimuli as a function of treatment and time (\u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05). The LB group showed anesthesia from 1 hour to 12 hour postoperatively (P\u0026lt;0.001),and at 24 hour（P=0.020）, compared to the S group, postoperatively. The LB group showed rebound pain at 48 hours postoperatively (\u003csup\u003e++\u003c/sup\u003eP=0.003), compared to the S group .The LB group showed hyperalgesia to mechanical stimuli （\u003csup\u003e# \u003c/sup\u003eP\u0026lt;0.05，\u003csup\u003e##\u003c/sup\u003e P\u0026lt;0.01）,compared to the Sham group. the S group\u0026nbsp; showed hyperalgesia to \u0026nbsp;heat stimuli（\u003csup\u003e$$\u003c/sup\u003e P\u0026lt;0.01）,compared to the Sham group .\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7259947/v1/896bee2684f192e24c677a42.png"},{"id":89641751,"identity":"6e457285-b556-422e-ae4a-e57f25808697","added_by":"auto","created_at":"2025-08-22 08:11:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":67425,"visible":true,"origin":"","legend":"\u003cp\u003eThe 50% paw withdrawal threshold (g)induced by mechanical stimuli after combined sciatic and femoral nerve block with liposomal bupivacaine. This is an illustration of nociceptive responses of the treated (left) hindlimb to mechanical stimuli as a function of treatment and time (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). The LB group showed anesthesia from 1 hour to 24 hour postoperatively (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001),and at 36 hour（\u003cem\u003eP\u003c/em\u003e=0.003）, compared to the S group, postoperatively. The LB group showed hyperalgesia to mechanical stimuli （\u003csup\u003e#\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05，\u003csup\u003e## \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01）,compared to the Sham group. the surgery group (S) showed hyperalgesia to mechanical stimuli（\u003csup\u003e$$\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01）,compared to the Sham group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7259947/v1/a1f5bca8ebfe66fccea98d1f.png"},{"id":89641754,"identity":"4da9aaf6-b9af-4a7e-a89e-bc57a85d828f","added_by":"auto","created_at":"2025-08-22 08:11:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":286094,"visible":true,"origin":"","legend":"\u003cp\u003eHistological examination of a sciatic nerve（A） and a femoral nerve （B）showing mild inflammatory response and Wallerian degeneration. Neural inflammation is characterised by nerve swelling and lymphocytic infiltration，and the typical histological appearance of Wallerian degeneration is a balloon-like feature(arrow).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7259947/v1/25f53f62692c5d0205fe3ca5.png"},{"id":97179439,"identity":"0da291d8-42dd-491b-b28b-ad2030291956","added_by":"auto","created_at":"2025-12-01 16:15:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1689115,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7259947/v1/af4945a2-28a5-473b-8b2d-d7e6495116af.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Liposomal Bupivacaine: A Delay Rather Than Elimination of Rebound Pain in Mouse Postoperative Model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRebound pain is a common postoperative phenomenon, which usually occurs after the release of peripheral nerve block (PNB) and is characterized by a sudden increase in pain intensity, mainly within 24 hours after surgery [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Epidemiological data suggest a relatively high incidence of rebound pain, but the incidence varies across studies. For example, in Ethiopia, the overall incidence of rebound pain after PNB remission was found to be as high as 61.7%[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In China, 49.6% of day surgery patients develop rebound after PNB[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The epidemiological data of rebound pain also reveal its correlation with patient prognosis. A study found that rebound pain increases postoperative opioid use, prolongs hospitalization, reduces recovery quality, and lowers patient satisfaction [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Poor postoperative pain management can extend acute pain duration, increase chronic pain risk, and diminish quality of life. Thus, preventing and treating rebound pain is crucial.\u003c/p\u003e\u003cp\u003eThe mechanisms underlying rebound pain are multifaceted. Firstly, hyperalgesia may contribute to this phenomenon. In a rat model of sciatic nerve block with ropivacaine, transient thermal hyperalgesia occurs in the hind limbs and persists for 5 to 7 hours after the sensory block subsides[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Secondly, the local inflammatory response also appears to be a key driver of rebound pain. Studies have shown that peripheral nerve block can exacerbate the acute inflammatory response at the surgical incision site, characterized by neutrophil and macrophage infiltration, as well as upregulation of tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Once the nerve block effect wanes, these inflammatory mediators can activate peripheral nociceptors and trigger rebound pain[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, the neurotoxicity of local anesthetics may also play a role. Studies have demonstrated that bupivacaine-induced sciatic nerve block in mice can lead to early peripheral nerve injury, marked by Wallerian degeneration and axonal demyelination[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Local anesthetics have also exhibited neurotoxic and cytotoxic effects. Finally, the activation of C-type fibers is thought to be associated with rebound pain. While the surgical stimulus is temporarily blunted by the nerve block, it may leave a \"pain memory\" that is reactivated when the block fades, resulting in severe pain[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSince rebound pain was originally defined as severe pain occurring within 24 hours after peripheral nerve block (PNB) intervention, some clinicians have attempted to mitigate the adverse effects of rebound pain by prolonging analgesia to cover the time points when it typically occurs. For example, continuous nerve block techniques can prolong analgesia duration, thereby reducing postoperative inflammatory responses and rebound pain incidence. Salviz et al. demonstrated that in patients undergoing rotator cuff repair surgery, the incidence of severe pain on the first postoperative day was significantly lower (15%) in those receiving continuous interscalene brachial plexus block [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, continuous nerve block is technically challenging, with a high failure rate and increased management costs, limiting its wider application. Secondly, the use of local anesthetic adjuvants serves as an effective alternative to continuous nerve block. In a mouse model of sciatic nerve block with bupivacaine, perineural addition of dexamethasone prevents thermal hyperalgesia and reduces neurotoxicity[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Additionally, multimodal analgesia strategies integrate nerve blocks with systemic analgesics (e.g., NSAIDs and opioids) and adjunctive analgesics (e.g., corticosteroids and anticonvulsants) to optimize postoperative pain management[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn recent years, new ultra-long-acting local anesthetics such as liposomal bupivacaine have been used clinically to prolong postoperative analgesia[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. A clinical study shows that liposomal bupivacaine can significantly lower pain scores, prolong analgesia, and potentially reduce rebound pain[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Another randomized controlled trial indicates that liposomal bupivacaine reduces pain scores and opioid use in the first 24 hours post-surgery versus placebo, with no sign of rebound pain. These findings highlight the potential of liposomal bupivacaine to eliminate rebound pain and prompt further investigation into its mechanisms. However, these studies only examined rebound pain within 24 hours post-surgery. Despite the controversial analgesic duration of liposomal bupivacaine[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], extending the observation period is necessary to prevent late-onset rebound pain and its adverse consequences. Thus, this study observed pain changes in mice for five days after peripheral nerve block with 1.3% liposomal bupivacaine, covering its theoretical analgesic period, and improved the mouse nerve block model to enhance nerve block success rate and nerve localization accuracy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e The experimental procedure (DWSY-23062366) was approved by the Institutional Animal Care and Use Committee (IACUC) at Nanjing Medical University. All experimental methods followed the Guidelines for the Institutional Animal Care and Use Committee at Nanjing Medical University.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnimals\u003c/b\u003e\u003c/p\u003e\u003cp\u003e All experiments described here were conducted in strict accordance with institutional guidelines. Male C57BL/6J mice (weighing 25\u0026ndash;30g; aged 8 weeks; Wandong Biotechnology Corporation of Nanjing; n\u0026thinsp;=\u0026thinsp;67) were housed in the same room at a constant temperature (22\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and humidity (55%\u0026plusmn;5%) and maintained on a 12:12 hours light-dark cycle with free access to water and food. 24 mice were randomly allocated into the following groups: (1) sham group, (2) surgery group (S), and (3) liposomal bupivacaine group (LB). These mice were used to assess postoperative pain behaviors. Most studies involve dissecting nerves before administering local anesthetics, which can cause additional trauma. Additionally, studies using anatomical landmark-guided nerve blocks often report lower success rates. To ensure the effectiveness of our experiments, we used methylene blue staining in 15 mice to validate which method could achieve effective nerve blockade. 28 mice were assigned to the surgery group and the liposomal bupivacaine group to assess early (day 2) and late (day 28) postoperative neural injury and neuroinflammation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnimal model of Tibial Fracture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe Tibial Fracture model was established using the method described by Khajuria et al[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Mice were anesthetized with 2% isoflurane and fixed in a right lateral recumbent position. and open mid-diaphyseal tibial fractures were induced in the left hindlimb as previously described. Postoperatively, the LB group was administered 35 mg/ kg 1.3% liposomal bupivacaine solution in the vicinity of the sciatic and femoral nerves [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], guided by a nerve stimulator. Concurrently, the S group only received a tibial fracture surgery. After sealing the injection hole and closing the wound, mice were resuscitated on a heated blanket.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCombined sciatic and femoral nerve block under nerve stimulator guidance\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA combined sciatic and femoral nerve block was executed under nerve stimulator guidance[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For the sciatic nerve block, the anesthetized mouse was positioned in right lateral recumbency, with the left hind limb and inguinal region prepared by shaving and cleansing, aligning the limb perpendicular to the torso. The puncture site was identified in the posterior lateral thigh muscle, and a 22G, 5cm needle (B. Braun Melsungen AG, model 4894146N) was utilized. The needle was attached to a nerve stimulator (Stimuplex HNS12, B. Braun Melsungen AG, Germany), and the tip was directed towards the posterior mid-femur. The initial nerve stimulator current is set at 0.2 mA, frequency at 2 Hz, aiming for ankle contraction or extension in mice. The needle position is adjusted until, at 0.08\u0026ndash;0.12 mA current, ankle movement occurs. After confirming no blood return, 35 mg /kg 1.3% liposomal bupivacaine is injected, causing ankle movement loss. If, upon resetting the current to 0.2 mA, the response doesn't recover, the sciatic nerve block is successful. For the femoral nerve block, the mouse was repositioned to supine, with the puncture site chosen at the mid-to-lower inguinal region. The needle was inserted at a 30\u0026deg; angle to the skin, toward the midpoint of the inguinal region. The initial current was set at 0.2 mA. The target motor responses in mice were quadriceps contraction and knee extension. The needle position was adjusted until these responses occurred at 0.08\u0026ndash;0.12 mA. After confirming no blood return, 35 mg /kg 1.3% liposomal bupivacaine was injected, causing knee movement loss. If, upon resetting the current to 0.2 mA, the response didn't recover, the nerve block was successful.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSciatic nerve and femoral nerve block under anatomical landmark location\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA combined sciatic and femoral nerve block was executed under anatomical landmark location. For the sciatic nerve block, the anesthetized mouse was positioned in right lateral recumbency, with the left hind limb and inguinal region prepared by shaving and cleansing, aligning the limb perpendicular to the torso. The puncture site was identified in the posterior lateral thigh muscle, and a 29G needle (B. Braun Melsungen AG, model 4894146N) was utilized. The needle was introduced posteromedially towards the greater trochanter in an anteromedial direction. After encountering the ischial tuberosity, 35 mg/ kg 1.3% liposomal bupivacaine was injected [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. For the femoral nerve block, referring to the method of femoral nerve block guided by nerve stimulator, the mice were placed in supine position, and the puncture needle was inserted into the middle third of the groin at 30\u0026deg; from the skin. The depth of the puncture needle was 5 mm, and 35 mg /kg 1.3% liposomal bupivacaine was injected.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStaining Study\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the success rate of nerve blocks, 15 mice were injected around both the femoral and sciatic nerves on each side. One side was randomly selected for injection using the anatomical landmark guidance method(Group A)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], while the other side used the nerve-stimulator guidance method (Group N). A volume of 0.085 mL of 0.1% methylene blue solution was injected around each nerve[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] .\u003c/p\u003e\u003cp\u003eThe anatomical landmark-guided approach for sciatic and femoral nerve block is as follows: Sciatic Nerve Block: Referring to Liljana's research [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], the mouse is fixed in a lateral decubitus position, and the middle and posterior-lateral muscle area of the thigh is chosen as the puncture site. A 29-gauge needle is directed posterior to the midpoint of the femur. After the puncture needle encounters the femur and no blood is aspirated, 0.085 mL of 0.1% methylene blue is injected. Femoral Nerve Block procedure: Referring to the anatomical structure of the femoral nerve and the muscle contraction response under neurostimulation guidance, targeted improvements are made. Except for not using a neurostimulator, the rest of the operation is the same as above, with the puncture needle depth set to 5mm, and 0.085 mL of 0.1% methylene blue is injected.\u003c/p\u003e\u003cp\u003eThis procedure led to a total of 30 methylene blue stainings for the sciatic nerves and 30 methylene blue stainings for the femoral nerves across the left and right hind limbs. One hour after injection, the mice were euthanized under isoflurane anesthesia and dissected to assess whether the nerve staining was successful and to determine the success rates of sciatic and femoral nerve blocks. This assessment was utilized to determine the efficacy of nerve stimulation-guided blocks of the sciatic and femoral nerves in mice. The presence of staining on the nerves indicated a successful block, while the absence of staining suggested a failed block[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eNociceptive Behavioral Tests\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTwo tests were used to assess pain behavior: (i) Mechanical nociception assessed by the withdrawal response to von Frey filament application, (ii) Thermal nociception assessed by the withdrawal response to thermal stimulus.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMechanical nociception\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUnrestricted mice were positioned under a clear plastic chamber that was elevated on a mesh floor, allowing them to acclimate to the environment. Mechanical withdrawal responses were assessed using a series of calibrated von Frey filaments (ranging from 0.04 to 4 g)[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These filaments were applied from beneath the cage, through the mesh floor openings, to the plantar surface of the hind paw, specifically targeting the middle area of the left leg. The filament was pressed until it produced a slight bend and was then held in this position for 6 seconds. Beginning with a filament force of 0.6 g, we determine the 50% paw withdrawal threshold utilizing Dixon\u0026rsquo;s up-down method (with cutoff thresholds of 4g [maximum] and 0.04g [minimum]).\u003c/p\u003e\u003cp\u003e\u003cb\u003eThermal nociception\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThermal withdrawal latencies were evaluated utilizing the Hargreaves method. Animals were placed in a plastic chamber on an elevated glass platform. A radiant heat source (SH-1000 Plantar Hot Plate Tester, Shanghai Vax Biotech Co., Ltd, Shanghai, China) was positioned beneath the glass to direct heat towards the plantar surface of the paw. The intensity of the radiant heat was calibrated to achieve an average withdrawal latency of approximately 10 seconds for the paws of naive animals. Each paw was subjected to heat stimulation three times with a minimum interval of 3 minutes between applications, and the average withdrawal latency was calculated by averaging these values (with a cutoff time of 15 seconds) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eHistological Evaluation(HE)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTwenty-eight mice were divided into the liposomal bupivacaine and surgical groups, and 7 mice in each group were selected for detection on day 2 and day 28, respectively. These mice were anesthetized as described above, followed by gentle exposure and removal of approximately 8\u0026ndash;10 millimeters of the sciatic and femoral nerves. This time interval was chosen correspond to short-term and long-term nerve tissue damage. The specimens were fixed in 4 formalin for 48 hours, washed with ethanol, and embedded in paraffin.\u003c/p\u003e\u003cp\u003eA pathologist, blinded to the experimental treatment, analyzed the slides using standardized scales for perineural inflammation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, edema and lymphocyte infiltration are characteristic features of inflammation. Inflammation was classified as follows: no inflammation (absence of inflammatory changes), mild inflammation (areas with less than 50% edema or cellular infiltrate), and severe inflammation (diffuse areas with greater than 50% edema or cellular infiltrate)[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWallerian degeneration, reflecting abnormal myelin and axonal degeneration due to nerve injury, was used to assess nerve damage. The classic histological appearance of Wallerian degeneration includes axonal swelling and myelin breakdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Wallerian degeneration was classified as follows: no Wallerian degeneration (absence of lesions), mild Wallerian degeneration (0\u0026ndash;50% of fibers showing Wallerian degeneration), and severe Wallerian degeneration (greater than 50% of fibers showing Wallerian degeneration) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLongitudinal sections of 5mm thickness were prepared and stained with hematoxylin and eosin to assess inflammation[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Each specimen was scored by a blinded pathologist in the treatment group for the degree of lymphocytic infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e):\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eno inflammation, indicating no signs of inflammation.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003emild inflammation, with swelling and lymphocytic infiltration covering up to 50% of the surface.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003esevere inflammation, with swelling and lymphocytic infiltration covering more than 50% of the surface.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed using SPSS 22.0 via a repeated-measures one-way analysis of variance (ANOVA) followed by Bonferroni\u0026rsquo;s post-tests for evaluating the effects of liposomal bupivacaine (LB) on PWL and 50% paw withdrawal threshold. The dependent variable for these analyses was time in hours. Chi-square tests and Fisher's exact probability method were used to examine the success rates of sciatic nerve and femoral nerve blocks between nerve stimulator guidance and anatomical landmark localization methods. The Mann-Whitney test was applied to compare the histopathology scores between time points. The statistical significance was determined as P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All data were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean (SEM).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eNerve stimulator-guided nerve block enhances the accuracy of nerve localization\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo choose a more appropriate nerve block method for mice, we compared the femoral and sciatic nerve block guided by a nerve stimulator with that located by anatomical landmarks, to construct a suitable mouse nerve block model. We used fifteen C57 mice (8 weeks old). On each mouse's left hind leg, nerve stimulator - guided block (Group N) was applied, while anatomical - landmark - guided block (Group A) was on the right. We used 0.1% bilateral methylene blue instead of local anesthetic for nerve block. After blocking, the mice were euthanized and dissected to observe the methylene blue location and drug distribution of the sciatic and femoral nerves. The range of nerve staining was used as the criterion to determine the success of sciatic and femoral nerve blocks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D). The results showed that in Group N, compared with Group A, both the sciatic and femoral nerves exhibited more precise nerve localization and a wider range of drug distribution. The nerve block success rate in Group N was significantly higher than in Group A (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), with no vascular rupture during the block. Our study shows that in mice, compared with traditional anatomical landmark - located nerve blocks, the femoral and sciatic nerve block guided by nerve stimulator has a higher success rate and improves the accuracy of nerve localization. This method is precise and highly reproducible. In follow - up studies, we used nerve stimulator guidance to construct a mouse model of femoral and sciatic nerve block.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of Sciatic and Femoral Nerve Block Success Rates between Nerve Stimulator-Guided and Anatomical Landmark Techniques\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNerve\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eStaining success YES/NOT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003esuccess rate (%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYES\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNOT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003esciatic nerve\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGroup A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e53.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGroup N\u003c/p\u003e\u003cp\u003eχ2\u003c/p\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e93.3\u003c/p\u003e\u003cp\u003e6.136\u003c/p\u003e\u003cp\u003e0.035\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003efemoral nerve\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGroup A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGroup N\u003c/p\u003e\u003cp\u003eχ2\u003c/p\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e73.3\u003c/p\u003e\u003cp\u003e8.571\u003c/p\u003e\u003cp\u003e0.030\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePeripheral nerve block with 1.3% liposomal bupivacaine delays the onset of rebound pain\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate whether liposomal bupivacaine can eliminate rebound pain, 24 mice (8 weeks old) were randomly divided into three groups: blank control group (sham), operation group (S), and operation\u0026thinsp;+\u0026thinsp;nerve block group (LB). LB mice underwent femoral and sciatic nerve block with 1.3% liposomal bupivacaine one hour before surgery. Samples without analgesia within one hour were excluded. Preoperative baseline measurements were taken, and mechanical/thermal pain thresholds were tested at 1 h, 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 96 h, and 120 h post - surgery. After tibial plateau fracture surgery, the LB group exhibited higher thermal and mechanical pain thresholds within 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), indicating that 1.3% liposomal bupivacaine provides effective postoperative analgesia during this period. However, at 48 hours post - modeling, the LB group's thermal pain threshold dropped significantly compared to the S group, indicating hyperalgesia and rebound pain onset. After 48 hours, there were no significant differences in pain thresholds between the two groups. These results show that LB, though having a longer analgesic effect than traditional local anesthetics, delays but doesn't eliminate rebound pain.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003erebound pain may be related to nerve injury and neuroinflammation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo preliminarily explore the mechanism of rebound pain, we euthanized LB group mice with rebound pain at 48 h and corresponding S group mice. Their femoral and sciatic nerves on the surgical side were removed and stained with HE. Results showed that both nerves had inflammatory cell infiltration and Wallerian degeneration, indicating nerve injury and neuroinflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). To determine the permanence of the injury and inflammation, HE staining of femoral and sciatic nerves in LB mice was done on postoperative day 28. Nerve injury was classified based on the proportion of Wallerian degeneration: no injury (0%), mild injury (0\u0026ndash;50%), and severe injury (\u0026gt;\u0026thinsp;50%). On postoperative day 2, LB mice had mild to severe nerve injury, while no injury was observed in either group on day 28 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similarly, neuroinflammation was assessed based on inflammatory cell infiltration: no inflammation (0%), mild inflammation (0\u0026ndash;50%), and severe inflammation (\u0026gt;\u0026thinsp;50%). On postoperative day 2, LB mice had mild to severe neuroinflammation, which disappeared by day 28 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results suggest that rebound pain may be associated with nerve injury and neuroinflammation, but the damage is reversible.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eNeural Toxicity After Single-Shot Regional Anesthesia at day 2 and at day 28.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNerve\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003eWallerian degeneration at day 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e\u003cp\u003eWallerian degeneration at day 28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMild\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSevere\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMild\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eSevere\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003esciatic nerve\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003efemoral nerve\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003ePerineural Inflammation After Single-Shot Regional Anesthesia at day 2 and at day 28.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNerve\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003eNeural inflammation at day 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e\u003cp\u003eNeural inflammation at day 28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMild\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSevere\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMild\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eSevere\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003esciatic nerve\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003efemoral nerve\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eLiposomal bupivacaine (LB) is a novel ultra-long-acting local anesthetic approved by the US Food and Drug Administration. Its theoretical analgesic duration can reach 72 hours [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and it has the potential to reduce rebound pain. However, current basic research shows controversial duration of analgesia. For example, Liljana et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] reported that in a mouse model of sciatic nerve block using 1.3% LB, the average duration of sensory block was 118 minutes. Wenling Zhao et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] observed a median duration of sensory block of 4.83 hours in a rat model of postoperative sciatic nerve block. Most current basic studies use peripheral nerve block guided by traditional anatomical landmarks. The shortened efficacy of LB may be due to low nerve block success rates or incomplete block. Whether this is related to the method of peripheral nerve block has not been reported.\u003c/p\u003e\u003cp\u003eAccording to prior research, some researchers used nerve blocks under anatomical vision, causing additional damage to animal models. Others used nerve blocks guided by anatomical landmarks, which had low success rates and effectiveness. In our study, we performed sciatic and femoral nerve blocks in mice using nerve stimulator guidance. The duration of sensory block with LB was prolonged to 24 h. Compared with anatomical - landmark - guided peripheral nerve block, bilateral methylene blue nerve staining showed that nerve stimulator - guided peripheral nerve block allowed better drug encapsulation of nerves and had higher and more stable success rates. This was mainly because nerve stimulator guidance improved needle placement accuracy[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This method relied on evoked muscle contraction to accurately position the needle tip near the target nerve. The results highlight that nerve stimulator - guided peripheral nerve block is superior to traditional anatomical - landmark - guided nerve block. Notably, LB showed significant analgesic effects within 24 h post - surgery, with no rebound pain during this period. However, rebound pain was observed at 48 h, indicating it was delayed rather than eliminated. Past studies did not report this phenomenon, possibly due to insufficient observation time or different models from our study. Our research strongly suggests extending the observation window when using new long - acting local anesthetics.\u003c/p\u003e\u003cp\u003eThe original definition of rebound pain was limited to severe pain occurring within 24 hours after peripheral nerve block (PNB) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], typically observed with traditional local anesthetics such as bupivacaine or ropivacaine. Our study extends this definition and emphasizes the importance of aligning the duration of surgical nociceptive stimulation with that of PNB action. With the clinical introduction of new ultra-long-acting local anesthetics and adjuvants like dexamethasone to prolong PNB duration, the monitoring time window for rebound pain should be correspondingly extended. Failure to do so may result in severe hyperalgesia post-rebound pain, leading to adverse outcomes such as increased chronic pain incidence.\u003c/p\u003e\u003cp\u003eThe mechanism of rebound pain is still unclear. Relevant studies have shown that liposomal bupivacaine exerts an anesthetic effect by blocking sodium channels in nerve cell membranes. However, long-term or high-concentration exposure can lead to neurotoxicity and trigger neuroinflammation[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This neurotoxicity may involve multiple pathways, including disruption of cell membrane integrity, increased intracellular calcium levels, activation of inflammatory signaling pathways (such as PI3K/Akt and oxidative stress), and induction of apoptosis[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Neuroinflammation enhances the sensitivity of nerve fibers, especially C fibers, which become overexcited in an inflammatory state, leading to thermal hyperalgesia [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Due to the diminished analgesic effect of liposomal bupivacaines, persistent local inflammation may further lower the pain threshold, resulting in rebound pain. Notably, adding anti-inflammatory agents like dexamethasone to bupivacaine preparations has been proven to reduce the incidence of rebound pain. Dexamethasone may alleviate neuroinflammation and C-fiber overexcitation by inhibiting inflammatory pathways (such as PI3K/Akt) and reducing oxidative stress[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this study, we preliminarily explored the mechanism of LB-induced rebound pain. HE staining revealed nerve swelling and lymphocyte infiltration in both the sciatic and femoral nerves during rebound pain, indicating a close relationship between rebound pain and neuroinflammation.\u003c/p\u003e\u003cp\u003eThis study has certain limitations. Firstly, it has only preliminarily explored the mechanism of rebound pain. Future research will need to delve deeper into its molecular mechanisms to provide more reliable evidence for targeted interventions to mitigate the adverse effects of rebound pain. Secondly, although nerve staining confirmed the success of femoral nerve block, the sensory block areas detected by the von Frey and thermal pain tests are innervated by the sciatic nerve. This means these tests only demonstrate the effectiveness of sciatic nerve block over time, not that of femoral nerve block. Thus, more suitable methods to verify femoral nerve block effectiveness in mice need to be developed. Finally, as this study only examined basic level results and given the differences between animal models and humans, further clinical research is essential to validate our findings.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur findings suggested that rebound pain was involved in the development of postoperative hyperalgesia. The delay of rebound pain by liposomal bupivacaine was associated with the inhibition of neuroinflammation-mediated Wallerian degeneration in mice.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLiposomal bupivacaine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePNB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eperipheral nerve block\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEnzyme-linked immunosorbent assay\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHistological Evaluation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data presented in this study are available from corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026quot;This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University (Approval No. DWSY-23062366). All experimental procedures were performed in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the institutional ethical guidelines.\u0026quot;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the 2021 Nanjing Municipal Health Science and Technology Development Special Fund Project Plan (YKK21128).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo potential conflict of interest relevant to this article was reported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHao Wu (Conceptualization; Investigation; Data acquisition and analysis; Methodology; Formal analysis; Writing \u0026ndash; original draft) Jiayu Jin (Conceptualization; Investigation; Methodology; Writing \u0026ndash; review \u0026amp; editing) Di Zhou (Data curation; Investigation; Validation; Visualization) Lihai Chen (Conceptualization; Supervision; Writing \u0026ndash; review \u0026amp; editing) Liu Han(Conceptualization; Supervision; Formal analysis; Validation; Writing \u0026ndash; review \u0026amp; editing)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHao Wu, https://orcid.org/0009-0003-1624-3795\u003c/p\u003e\n\u003cp\u003eJiayu Jin, https://orcid.org/0009-0005-9353-454X\u003c/p\u003e\n\u003cp\u003eDi Zhou, https://orcid.org/0009-0005-2490-8974\u003c/p\u003e\n\u003cp\u003eLihai Chen, https://orcid.org/0000-0001-8535-9239\u003c/p\u003e\n\u003cp\u003eLiu Han, https://orcid.org/0000-0003-2993-0394\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher\u0026rsquo;s Note\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMu\u0026ntilde;oz-Leyva F, Cubillos J, Chin KJ. Managing rebound pain after regional anesthesia. Korean J Anesthesiol. 2020;73(5):372\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAdmassie BM, Debas SA, Admass BA. Prevention and management of rebound pain after resolution of regional block: a systematic review. Ann Med Surg (Lond). 2024;86(8):4732\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBarry GS, Bailey JG, Sardinha J, Brousseau P, Uppal V. Factors associated with rebound pain after peripheral nerve block for ambulatory surgery. Br J Anaesth. 2021;126(4):862\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLuebbert E, Rosenblatt MA. Postoperative Rebound Pain: Our Current Understanding About the Role of Regional Anesthesia and Multimodal Approaches in Prevention and Treatment. Curr Pain Headache Rep. 2023;27(9):449\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDada O, Gonzalez Zacarias A, Ongaigui C, Echeverria-Villalobos M, Kushelev M, Bergese SD, et al. Does Rebound Pain after Peripheral Nerve Block for Orthopedic Surgery Impact Postoperative Analgesia and Opioid Consumption? A Narrative Review. Int J Environ Res Public Health. 2019;16(18):3257.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKolarczyk LM, Williams BA. Transient heat hyperalgesia during resolution of ropivacaine sciatic nerve block in the rat. Reg Anesth Pain Med. 2011 May-Jun;36(3):220\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYamada T, Hasegawa-Moriyama M, Kurimoto T, Saito T, Kuwaki T, Kanmura Y. Peripheral Nerve Block Facilitates Acute Inflammatory Responses Induced by Surgical Incision in Mice. Reg Anesth Pain Med. 2016 Sep-Oct;41(5):593\u0026ndash;600.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePogatzki-Zahn EM, Segelcke D, Schug SA. Postoperative pain-from mechanisms to treatment. Pain Rep. 2017;2(2):e588.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAn K, Elkassabany NM, Liu J. Dexamethasone as adjuvant to bupivacaine prolongs the duration of thermal antinociception and prevents bupivacaine-induced rebound hyperalgesia via regional mechanism in a mouse sciatic nerve block model. PLoS ONE. 2015;10(4):e0123459.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKleggetveit IP, Namer B, Schmidt R, Hel\u0026aring;s T, R\u0026uuml;ckel M, \u0026Oslash;rstavik K, et al. High spontaneous activity of C-nociceptors in painful polyneuropathy. Pain. 2012;153(10):2040\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSalviz EA, Xu D, Frulla A, Kwofie K, Shastri U, Chen J, et al. Continuous interscalene block in patients having outpatient rotator cuff repair surgery: a prospective randomized trial. Anesth Analg. 2013;117(6):1485\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJoshi GP. Rational Multimodal Analgesia for Perioperative Pain Management. Curr Pain Headache Rep. 2023;27(8):227\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePeng H, Wen J, Chen M, Xia Z, Jiang Y, Xie K, et al. Preoperative Analgesia Efficacy of Liposomal Bupivacaine Following Pericapsular Nerve Group (PENG) Block in Patients with Hip Fracture: A Randomized Controlled Observer-Blinded Study. Pain Ther. 2025;14(1):283\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGong R, Tan G, Huang Y. The Efficacy of Liposomal Bupivacaine in Thoracic Surgery: A Systematic Review and Meta-Analysis. J Pain Res. 2024;17:4039\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHussain N, Brull R, Sheehy B, Essandoh MK, Stahl DL, Weaver TE, et al. Perineural Liposomal Bupivacaine Is Not Superior to Nonliposomal Bupivacaine for Peripheral Nerve Block Analgesia. Anesthesiology. 2021;134(2):147\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDinges HC, Wiesmann T, Otremba B, Wulf H, Eberhart LH, Schubert AK. The analgesic efficacy of liposomal bupivacaine compared with bupivacaine hydrochloride for the prevention of postoperative pain: a systematic review and meta-analysis with trial sequential analysis. Reg Anesth Pain Med. 2021;46(6):490\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhajuria DK, Karuppagounder V, Nowak I, Sepulveda DE, Lewis GS, Norbury CC, et al. Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice. J Bone Min Res. 2023;38(11):1560\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarkova L, Cvetko E, Ugwoke CK, Horvat S, Umek N, Stopar Pintarič T. The Influence of Diabetic Peripheral Neuropathy on the Duration of Sciatic Nerve Block with 1.3% Liposomal Bupivacaine and 0.25% Bupivacaine Hydrochloride in a Mouse Model. Pharmaceutics. 2022;14(9):1824.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShikanov A, Domb AJ, Weiniger CF. Long acting local anesthetic-polymer formulation to prolong the effect of analgesia. J Control Release. 2007;117(1):97\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarkova L, Umek N, Horvat S, Hadžić A, Kuroda M, Pintarič TS, et al. Neurotoxicity of bupivacaine and liposome bupivacaine after sciatic nerve block in healthy and streptozotocin-induced diabetic mice. BMC Vet Res. 2020;16(1):247.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang Y, Cui B, Gong C, Tang Y, Zhou J, He Y, et al. A rat model of nerve stimulator-guided brachial plexus blockade. Lab Anim. 2019;53(2):160\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang L, Wang Z, Song C, Liu H, Li Y, Li J, et al. Spinal NR2B phosphorylation at Tyr1472 regulates IRE(-)DMT1-mediated iron accumulation and spine morphogenesis via kalirin-7 in tibial fracture-associated postoperative pain after orthopedic surgery in female mice. Reg Anesth Pain Med. 2021;46(4):363\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGonzalez-Cano R, Boivin B, Bullock D, Cornelissen L, Andrews N, Costigan M. Up-Down Reader: An Open Source Program for Efficiently Processing 50% von Frey Thresholds. Front Pharmacol. 2018;9:433.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMitsui K, Hishiyama S, Jain A, Kotoda Y, Abe M, Matsukawa T, et al. Role of macrophage autophagy in postoperative pain and inflammation in mice. J Neuroinflammation. 2023;20(1):102.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePapadopoulos G, Duckwitz V, Doherr MG. Femoral and sciatic nerve blockade of the pelvic limb with and without obturator nerve block for tibial plateau levelling osteotomy surgery in dogs. Vet Anaesth Analg. 2022;49(4):407\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarty P, Bennis M, Legaillard B, Cavaignac E, Ferre F, Lebon J, et al. A New Step Toward Evidence of In Vivo Perineural Dexamethasone Safety: An Animal Study. Reg Anesth Pain Med. 2018;43(2):180\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQuaye A, McAllister B, Garcia JR, Nohr O, Laduzenski SJ, Mack L, et al. A prospective, randomized trial of liposomal bupivacaine compared to conventional bupivacaine on pain control and postoperative opioid use in adults receiving adductor canal blocks for total knee arthroplasty. Arthroplasty. 2024;6(1):6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao W, Yang J, Zhang Y, Liu J, Zhang W. QX-OH/Levobupivacaine: Fixed-dose combination to provide a long-acting postoperative pain of knee surgery in rodents. Eur J Pharm Sci. 2018;111:418\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLim JA, Sung SY, Lee JH, Lee SY, Kwak SG, Ryu T, et al. Comparison of ultrasound-guided and nerve stimulator-guided interscalene blocks as a sole anesthesia in shoulder arthroscopic rotator cuff repair: A retrospective study. Med (Baltim). 2020;99(35):e21684.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAdmassie BM, Tegegne BA, Alemu WM, Getahun AB. Magnitude and severity of rebound pain after resolution of peripheral nerve block and associated factors among patients undergoes surgery at university of gondar comprehensive specialized hospital northwest, Ethiopia, 2022. Longitudinal cross-sectional study. Ann Med Surg (Lond). 2022;84:104915.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa R, Wang X, Lu C, Li C, Cheng Y, Ding G, et al. Dexamethasone attenuated bupivacaine-induced neuron injury in vitro through a threonine-serine protein kinase B-dependent mechanism. Neuroscience. 2010;167(2):329\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFang J, Shi Y, Du F, Xue Z, Cang J, Miao C, et al. The effect of perineural dexamethasone on rebound pain after ropivacaine single-injection nerve block: a randomized controlled trial. BMC Anesthesiol. 2021;21(1):47.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMorita S, Oizumi N, Suenaga N, Yoshioka C, Yamane S, Tanaka Y. Dexamethasone added to levobupivacaine prolongs the duration of interscalene brachial plexus block and decreases rebound pain after arthroscopic rotator cuff repair. J Shoulder Elb Surg. 2020;29(9):1751\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-anesthesiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bane","sideBox":"Learn more about [BMC Anesthesiology](http://bmcanesthesiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bane","title":"BMC Anesthesiology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Rebound Pain, Regional anesthesia, Liposomal bupivacaine, Acute postoperative pain, Nerve stimulator, Sciatic nerve, Femoral nerve","lastPublishedDoi":"10.21203/rs.3.rs-7259947/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7259947/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Rebound pain, a postoperative surge in pain after peripheral nerve block (PNB) resolution, leads to increased opioid use and delayed recovery. Liposomal bupivacaine (LB) resolution prolongs analgesia, but its impact on rebound pain remains unclear. We developed a mouse model of sciatic/femoral nerve block to study LB’s effects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eMale C57 mice (n=24) underwent tibial fracture surgery and were divided into sham, surgery (S), or LB (1.3%) groups. LB was administered near sciatic/femoral nerves using nerve stimulator guidance. Mechanical (von Frey) and thermal (Hargreaves) nociception were assessed. Histology evaluated nerve inflammation at days 2 and 28. Nerve block success rates were confirmed via methylene blue injection (n=15).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e LB extended thermal (24h, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) and mechanical (36h, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) analgesia. Transient thermal rebound hyperalgesia occurred at 48h (\u003cem\u003eP\u003c/em\u003e=0.003), but no mechanical rebound was observed. Mild neural inflammation was more frequent with LB at day 2 (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). Nerve stimulator-guided blocks had high success rates (sciatic: 93.3%, \u003cem\u003eP\u003c/em\u003e=0.035; femoral: 73.3%, \u003cem\u003eP\u003c/em\u003e=0.030).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e LB delayed but did not eliminate rebound pain, possibly due to neuroinflammation. A reliable mouse model of combined sciatic/femoral nerve block was established.\u003c/p\u003e","manuscriptTitle":"Liposomal Bupivacaine: A Delay Rather Than Elimination of Rebound Pain in Mouse Postoperative Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 08:03:29","doi":"10.21203/rs.3.rs-7259947/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-28T07:41:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-27T01:35:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-26T07:25:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-18T11:57:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"56655671161796931886327375094272371957","date":"2025-08-16T15:03:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28324239863064077430464446060972079345","date":"2025-08-15T03:12:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210347260737814738650980843443050100814","date":"2025-08-14T20:33:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"288934421503420317650131431674578481348","date":"2025-08-14T14:08:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-13T22:00:58+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-12T10:59:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-07T13:17:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-07T13:16:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Anesthesiology","date":"2025-07-31T08:35:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-anesthesiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bane","sideBox":"Learn more about [BMC Anesthesiology](http://bmcanesthesiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bane","title":"BMC Anesthesiology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5baa0186-1ad0-4496-8041-6bd6b6f54011","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:10:48+00:00","versionOfRecord":{"articleIdentity":"rs-7259947","link":"https://doi.org/10.1186/s12871-025-03430-2","journal":{"identity":"bmc-anesthesiology","isVorOnly":false,"title":"BMC Anesthesiology"},"publishedOn":"2025-11-26 15:57:16","publishedOnDateReadable":"November 26th, 2025"},"versionCreatedAt":"2025-08-22 08:03:29","video":"","vorDoi":"10.1186/s12871-025-03430-2","vorDoiUrl":"https://doi.org/10.1186/s12871-025-03430-2","workflowStages":[]},"version":"v1","identity":"rs-7259947","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7259947","identity":"rs-7259947","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}