Preliminary study on the use of perfluoropropane contrast agent in monitoring drug diffusion during fascial plane blocks

Abstract

Objectives

This research aims to preliminarily verify the feasibility of utilizing a novel perfluoropropane ultrasound contrast agent (UCA) for observing the spread of drugs within the fascial plane.We demonstrated the feasibility of this method by conducting transverse abdominis plane block(TAPB) using two-dimensional (2D) ultrasound.

Methods

Firstly, to explore the optimal dilution ratio of UCA mixed with local anesthetics (LA), this study conducted in vitro simulation experiments by diluting the UCA with 0.375% ropivacaine hydrochloride(ROP) at various ratios (0, 10x, 30x, 100x, 300x, 1000x).The contrast of images under 2D ultrasound was observed and measured in a six-well plate. After selecting two relatively suitable doses, TAPB was performed using rabbits to determine the best dilution ratio. Next, 0.375% ROP was mixed with UCA at the selected optimal dilution ratio (with the addition of methylene blue). TAPB was performed, and the diffusion area of the contrast agent was recorded in real-time under 2D ultrasound. After dissection, photographs were taken to record the spread range of methylene blue, and the correlation and consistency between the two methods of observing drug diffusion were compared. Finally, we conducted in vitro and in vivo experiments to evaluate the muscular and neural toxicity of the novel UCA when combined with 0.375% ROP.

Results

Using 2 ml of 0.375% ROP in combination with perfluoropropane UCA, without further dilution, produced stable and high-contrast 2D ultrasound images in an in vitro simulation experiment and TAPB in rabbits.This mixture ratio was subsequently used to observe drug diffusion and toxicity in further studies. A paired t-test analysis showed no statistically significant difference in the measured area between the spread of 0.375% ROP + perfluoropropane UCA in 2D ultrasound imaging and the spread of methylene blue (MB) after dissection.The area recorded by ultrasound images exhibited a strong correlation with the distribution range of LA as reflected by MB after dissection (R = 0.70, P = 0.02).Bland-Altman analysis showed that the mean difference in the measured area between the two methods was 0.65 cm2, and the 95% confidence interval (CI) of the difference was [-1.38 cm2, 1.16 cm2]. Only one data point was outside the 95% CI.Neither in vivo nor in vitro studies have found that perfluoropropane UCA increases the known muscular and neural toxicity of 0.375%ROP.

Conclusions

Our preliminary study demonstrates the potential feasibility and safety of real-time monitoring of LA diffusion in rabbits using the novel perfluoropropane UCA under 2D ultrasound.

Introduction

Fascial plane blocks (FPBs) are techniques used to alleviate pain by injecting local anesthetic (LA) into the space between two discrete fascial layers, thereby diffusing the LA into the peripheral nerves and surrounding tissues [1]. The efficacy of nerve conduction blockade through LA is closely linked to drug diffusion. Observing drug diffusion is helpful in understanding the analgesic mechanisms of block techniques. With its advantages of non-invasiveness, visualization, and portability, ultrasound has rapidly developed in clinical practice as a guidance technique for nerve blocks. However, both 2D and 3D ultrasound observations of LA have their own limitations, and their results are inconsistent [2, 3], Therefore, it is necessary to find a new method to judge the diffusion range of LA in ultrasonic images.

Ultrasound contrast agents (UCA) are composed of suspended microbubbles that interact strongly with ultrasound beams, making them easily detectable using ultrasound imaging systems. However, based on its safety, UCA is currently used more and more widely in clinical applications, but most studies are still limited to intravenous use.It is worth exploring whether UCA can be applied to FPB drug diffusion monitoring by local injection. Several Japanese studies [4, 5] have demonstrated that the perfluorobutane UCA solution called Sonazoid^® can provide clear, real-time, and continuous visualization of LA diffusion under contrast-enhanced ultrasonography (CEUS) mode without complications. This finding suggests the feasibility of using UCA for nerve blocks. However, there are limitations in translating these findings into clinical settings. First, the UCA is mostly observed through the CEUS mode, but this mode has a lower imaging quality. Moreover, most portable ultrasound devices do not use the CEUS mode. Second, a CEUS mode for peripheral nerve block (PNB) has not yet been developed. Thirdly, raw materials for Sonazoid^® is perfluorobutane, which is expensive and difficult to obtain. Finally, there are no detailed reports on the toxicity of UCA mixed with LA at the cellular level.

Therefore, on the basis of previous studies, we selected domestically developed perfluoropropane (Beijing Feirida Medical Technology Co., Ltd., 20210801) from China. Currently, UCA is an allergen-free option suitable for a wide range of ultrasonic probes at various frequencies. At present, it is in the phase III clinical experiment stage. The previous clinical application are mostly used for organ development through intravenous injection, but there is a lack of research ideas and related experiments on the application of nerve block. Therefore in pre-experiments conducted earlier we conducted, we demonstrated that the size of the novel UCA utilized in this investigation complies with the specifications of commercially available UCAs (i.e., less than 10 μm) through meticulous observation of drug microvesicle morphology using a microscope. Furthermore, pre-experiments confirmed that mixing UCA with 0.375% ROP did not alter the pH value.

The primary objective of this study was to conduct a preliminary investigation into the use of perfluoropropane contrast agents for monitoring drug diffusion during fascial surface block. To achieve this, we first determined the optimal concentration of a novel UCA mixed with 0.375% ROP as a contrast agent. Furthermore, given the novelty of this technique, we aimed to evaluate its safety profile by investigating muscle damage and neurotoxicity following solution injection.

Materials and methods

Optimal concentration of perfluoropropane UCA in combination with 0.375% ROP was screened

Selecting optimal ratio of perfluoropropane UCA and 0.375% ROP through simulation experiments

To prepare a stock solution, 2 mL of 0.375% ROP was added to an appropriate amount of powdered. The final volume of the solution is 2 ml. UCA and oscillated. Subsequently, the stock solution was diluted(10-fold, 30-fold, 100-fold, 300-fold, 1000-fold) using 0.375% ROP. The stock solution and five dilutions of the solution were transferred to a six-well plate under 2D ultrasound (Fig. 1A). Images were captured at 6-minute intervals for a total of 30 min using a high-frequency ultrasound probe (10-23Mhz, X10-23 L, VINNO, Suzhou, China).Testing of each test condition was performed in triplicate. The two dilution ratios with the highest contrast values (measured using grayscale) and the least variation within the observation period were selected. These dilution ratios are designated as Solution A and Solution B, respectively.

Performing Rabbit Transverse Abdominis Plane Block (TAPB) to Determine the Optimal Ratio of Perfluoropropane UCA mixed with 0.375% ROP

Animal experiments were approved by the Ethics Committee of Soochow University (Ethics Protocol No.: SUDA20221128A04). All animals were purchased from Hangzhou Yuhang Kelian Rabbit Industry Professional Cooperative. Six SPF male New Zealand white rabbits weighing 1.2 ~ 1.5 kg were selected. The rabbits were housed in SPF-grade animal facilities at a temperature of 20 ~ 25 °C and 60–65% humidity, with a 12-hour light-dark cycle. The rabbits were divided into two groups, with n = 3 in each group. Group A was injected with 0.5 mL solution A and group B was injected with 0.5 mL Solution B. Anesthesia was performed by injection of pentobarbital sodium (45 mL/kg) into the auricular vein. Based on the observed imaging effects, an appropriate mixing ratio was selected, and a portable ultrasound device (VINNO6, Suzhou, China) was used by an experienced researcher for all ultrasound examinations.

For TAPB procedure, the rabbits were placed in a supine position, and a high-frequency linear probe (X10-23 L, VINNO, Suzhou, China; 10–23 MHz) was used. The transducer was placed transversely along the spine at the mid-axillary line between the iliac crest and rib margin. Fascial layers of the rabbit abdomen were identified (Fig. 1B). Under ultrasound guidance, the skin was penetrated in the caudal to cranial direction by puncturing. The needle tip was visualized and positioned in the fascial plane between the internal oblique (IO) and transversus abdominis (TA) muscles (Fig. 1C). The solution (0.5 mL) was injected while the needle was held stationary (Fig. 1D).

Fig. 1

Investigating the suitable mixing ratio of 0.375% ROP and perfluoropropane UCA: Operation diagram. A: The ultra high frequency ultrasonic probe is shown to collect images at different dilution multiples. B: Figure shows the stratification diagram of the abdominal fascia of rabbits, EO, the external oblique muscle of the abdomen; IO, internal obliquus abdominal muscle; TA, transverse abdominalis; C: Schematic diagram of block puncture path at the transverse abdominis plane is shown. The red arrow indicates that the needle tip enters the transverse abdominis plane. D: The area highlighted by the orange arrow in the figure is the perfluoropropane UCA development area

Local anesthetic diffusion was assessed by injection of perfluoropropane UCA

Eleven male New Zealand white rabbits, weighing 1.2 ~ 1.5 kg, were selected for the experiment. Housing conditions, anesthesia method, and nerve block protocol were the same as those described above. A total of 0.6 mL of medication which included 0.5 mL of the mixture of 0.375% ROP + perfluoropropane UCA (prepared as described previously) and 0.1 mL of MB, was injected into the transversus abdominis plane. After the injection, a high-frequency probe (6.5-18Mhz, X6-16 L, VINNO, Suzhou, China) was gently placed along the cranio-caudal direction on the body surface, with the puncture site as the center point of probe placement. The probe was moved towards the cranial end up to the border where imaging ends, recorded as point “a”(Fig. 2A). Then, the probe was moved towards the caudal side up to the border where imaging ends, recorded as point “b” (Fig. 2B). The area between points “a” and “b” was scanned using the ultrasound imaging mode. The UCA diffusion area under ultrasound was simulated using Photoshop (Fig. 2B). Subsequently, the rabbits were dissected to observe the clear outlines of the contrast agent (Fig. 2C).

Fig. 2

Area measurement diagram. A: illustrates the furthest point of diffusion of UCA along the caudal side of the skull. B: shows a simulated diagram of UCA diffusion area using Photoshop software. C: The diagram illustrates the diffusion range of MB after dissection. Point a represents the furthest point of diffusion towards the head side, while point b represents the furthest point of diffusion towards the tail side

In Vivo and In Vitro studies were conducted to evaluate neurotoxicity and muscular toxicity of perfluoropropane UCA

In vivo study

Twenty four rabbits were randomly divided into two groups of 12 rabbits each. One group underwent unilateral sciatic nerve(SNB) and the other group underwent unilateral TAPB. Each group was further divided into four subgroups with three rabbits per subgroup: 0.9% NaCl (N group), 0.9% NaCl + perfluoropropane UCA (N + U group), 0.375% ROP (R group), and 0.375% ROP + perfluoropropane UCA (R + U group).The solution configuration method is the same as described above. After completing all experimental procedures, euthanasia must be humanely administered to rabbits retaining vital signs. The specific procedure is to inject pentobarbital anesthesia into the abdomen of the rabbit to induce death (100-150 mg/kg). The concentration of its solution is 20%.

SNB Group: After rabbits were anesthetized, they were placed in the right lateral position. The nerve puncture site was selected approximately 1 cm below the line connecting ischial tubercle and greater trochanter of femur. An ultrahigh-frequency (UHF) ultrasound probe was used to identify the location of the sciatic nerve (Fig. 3A). When the needle tip reached the sciatic nerve trunk (Fig. 3B), it was quickly inserted and withdrawn. The sciatic nerve appeared as a bright, hyperechoic band (Fig. 3C). A dark liquid area surrounding the sciatic nerve in the rabbits was observed immediately after drug infusion without the use of a contrast agent (Fig. 3D). Movement disorders were monitored after the rabbits regained consciousness. The tissue samples were collected 72 h later. Nerve injury classification: [6] Grade 0 indicates no pathological changes, Grade 1 represents 0–2% of nerve fibers with demyelination, Grade 2 represents 2–5% of nerve fibers with demyelination, and Grade 3 indicates demyelination of ≥ 5% of nerve fibers.

TAPB Group: The TAPB procedure was conducted as described above, and tissue samples were collected 72 h post-procedure from the region of drug diffusion, excluding the puncture site. The excised fascial samples were examined under an optical microscope using the scoring system established by Benoit [5]. Grade 0, no damage; grade 1, localized or scattered fiber disruptions; grade 2, extensive necrosis of the primary connective tissue involving numerous muscle bundles; and grade 3, near-total destruction of the muscle mass.

luxol fast blue(LFB) staining: ① Approximately 0.5 cm of nerve tissue was carefully dissected from the vicinity of the sciatic nerve block, avoiding the puncture site. Subsequently, the dissected nerve tissue was immersed in a 4% paraformaldehyde solution ( volume ratio of 1:10) for 72 h for fixation. ② Dehydration treatment was conducted. ③Liquid paraffin was poured into an embedding mold, and the tissue specimen was placed in the mold prior to solidification. Subsequently, the mold was allowed to cool. The paraffin-embedded tissue block was positioned on a glass slide submerged in distilled water, and the slide was subsequently transferred to an oven for drying. ④ Deparaffinization and clearing. ⑤ The sample was then rehydrated. ⑥LFB staining with a 0.1% solution. ⑦ Differentiation. ⑧ Counterstaining.⑨ Observing.

Masson trichrome staining: The tissue was fixed and embedded as described previously. ① Deparaffinization and hydration.② Hematoxylin staining. ③ Eosin staining. ④ The sections were soaked in phosphomolybdic acid solution. ⑤ The sections were treated with 1% acetic acid for 1 min, stained with aniline blue for 5 min, and then subjected to another 1-minute treatment with 1% acetic acid. Dehydration and clearing. ⑦ The sections were mounted on glass slides. ⑧ Observing.

Hematoxylin and eosin (HE) staining: The tissues were fixed and embedded as described above. Deparaffinization and clearing. ② Hydration of the sections. ③ Staining with hematoxylin ④ Staining with eosin. ⑤ The sections were dehydrated and mounted on glass slides. ⑥ Observing.

Enzyme-linked immunosorbent assay (ELISA) protocol: ① PBS (9 µL) was added to every 1 mg of tissue sample. ② Sample was homogenized and the homogenate was placed in a centrifuge at 4℃. Sample was centrifuged at 3000 rpm for 10 min. The supernatant was removed and kept on ice, and the precipitate was discarded. ③ ELISA kit reagents and plate strips were allowed to equilibrate at room temperature for 20 min. ④ The standard solution (50 µL) was added to each well. ⑤ Horseradish peroxidase (HRP) antibody (10 µL) was then added to each well. ⑥ Solution A (50 µL) was added to each well. ⑦ Then, stop solution (50 µL) was added to each well. ⑧ A standard curve was created using ELISA curve fitting software.

Fig. 3

Before and after SNB procedure. A: Illustration of the sciatic nerve in a high-frequency ultrasound image, indicated by the red arrow. B: Illustration of the needle insertion in the plane, indicated by the red arrow pointing to the needle tip. C: Illustration of the ultrasound image after injecting perfluoropropane UCA, with the red arrow pointing to the hyperechoic band indicating the area of drug diffusion. D: Illustration of the ultrasound image after injecting saline, with the red arrow pointing to the hypoechoic area indicating the formation of a fluid-filled space after drug injection

In vitro study

Four cell lines used in this experiment were purchased from Procell(Shanghai, China) and included NCTC Clone 929 mouse fibroblasts, SH-SY5Y human neuroblastoma, RSC96 Schwann, and L6 rat myoblast cell lines. The experimental groups consisted of the following: the 0.9% NaCl group (N group), 0.9% NaCl + perfluoropropane UCA group (N + U group), 0.375% ROP group (R group), and 0.375% ROP + perfluoropropane UCA group (R + U group).

CCK-8 assay: The cells were counted using a blood cell count plate. Each experiment included three blank and negative control wells. Five time points were set for drug treatment: 0, 30, 60, 90, and 120 min. When the desired level of cell adhesion was achieved, each drug combination was added to the aforementioned groups as applicable with six replicate wells per group. The processing time was as previously described. After drug treatment, the medium was discarded, and each well was supplemented with 100 µL of fresh medium and 10 µL of CCK-8 reagent. The cells were then incubated for 1 h and the absorbance was measured at 450 nm to calculate the cell survival rate.

Annexin V-FITC/PI apoptosis detection: Cells were inoculated into a six-well plate at a density of 1 × 10^5 cells/mL. Once the cells had adhered, they were treated with the drugs corresponding to the pre-group and incubated for 1 h. The cells were digested using pancreatic enzymes (without EDTA) and subsequently centrifuged at 1000 rpm (300×g) for 5 min. The cells were washed twice with pre-cooled PBS and centrifuged at 1000 rpm (300×g) for 5 min after each wash. The 1× Binding Buffer (100 µL) was added to each centrifuge tube and gently mixed using a pipette. Finally, 4 µL of Annexin V-FITC and 4 µL of PI dye were added to each tube and gently mixed. Samples were incubated at room temperature protected from light for 10 min. The 1× Binding Buffer (400 µL) was added, mixed gently, and transferred into a flow tube. The samples were analyzed within 1 h using flow cytometry.

ROS detection: The cells were inoculated into a six-well plate, and the experiment commenced once the cell density reached 80%. DCFH-DA was diluted by a factor of 1000 using the corresponding serum-free medium required for each cell culture. The final concentration of DCFH-DA is 10 µmol/L. The old cell culture medium was discarded and 1 mL of diluted DCFH-DA was added to each well. The six-well plates containing DCFH-DA were incubated at 37℃ in a cell incubator for 20 min. Each well was thoroughly washed with a serum-free culture solution to ensure the removal of noncellular DCFH-DA. Based on the pre-grouping, the corresponding drug treatment was added and incubated for 1 h. Then, the drug was removed by washing the cells twice with serum-free medium and the cells were collected. Reactive oxygen species (ROS) levels were quantified using flow cytometry with excitation at 488 nm and emission at 525 nm.

Statistical nalysis

Changes in UCA levels at different time points under various dilution factors were analyzed using a two-way ANOVA., and the Paired T-test was used to compare the development area of perfluoropropane UCA and MB diffusion area. Spearman’s correlation analysis was used to assess the correlation between the two methods. A Bland-Altman diagram was generated to examine the consistency of the two measurement methods. Multiple groups of samples in the cell experiment were compared using One-way ANOVA. P < 0.05 was considered statistically significant.

Results

Suitable mixing ratios of 0.375% ROP and perfluoropropane UCA

UCA exhibited a high echo at 0 min for different dilution ratios (Fig. 4A). The undiluted UCA with 0.375% ROP, which represents the assumed stock solution, immediately exhibited imaging in a six-well plate due to the high ultrasonic concentration of microbubbles hindering the imaging process. Over time, the imaging effect gradually improved and reached a stable state within 30 min. As the UCA dilution ratio increased, the images of each group transitioned quickly from a high echo to a low echo within 6 min and eventually reached a state of no echo after 30 min. Statistical analysis revealed that the gray value was the highest for 0.375% ROP and UCA diluted 10 fold at 0 min, indicating the optimal imaging effect at the initial time point (Fig. 4B). Considering the requirements for imaging stability and contrast, we selected undiluted perfluoropropane UCA and UCA diluted 10-fold with 0.375% ROP to evaluate imaging effects in animals.

Fig. 4

isualization of different dilutions of UCA in a six-well plate. A: Illustration of images of UCA at different dilution factors at various time points. B: Illustration of a trend chart showing the variation of grayscale values of UCA at different dilution factors over time. Each group has n = 3. The data is presented as mean ± standard deviation

Optimal mixing ratio of perfluoropropane UCA and 0.375% ROP by TAPB in rabbits

Based on the results of six-well plate imaging, the imaging effect of undiluted perfluoropropane UCA mixed with 2 mL of 0.375% ROP and subsequent oscillation was the most stable over time. This was followed by 10-fold dilution of UCA with 0.375% ROP. Solution A was prepared using undiluted perfluoropropane UCA, whereas solution B was prepared by diluting UCA 10-fold with 0.375% ROP, both used for rabbit TAPB. Following the administration of solution A, the drug diffusion area formed a continuous, distinct, and high-echo zone, effectively blocking deep tissue imaging below it (Fig. 5A). No noticeable stratification was observed within a 30-minute period. When solution B was administered, the drug injection plane exhibited stratification (Fig. 5B). Therefore, perfluoropropane UCA without further dilution was selected for follow-up studies.

Fig. 5

The imaging effects of two different dilution factors of UCA in rabbit TAPB. A: Illustration of the injection effect of undiluted mixture of perfluoropropane UCA and 0.375% ROP. B: Illustration of the injection effect of perfluoropropane UCA diluted 10 times with 0.375% ROP. The red arrow indicates the imaging area of UCA, and the yellow arrow indicates the dark area caused by the drug solution

Comparison of drug diffusion range measured by 2D ultrasound and assessed by MB diffusion

This study included a total of 11 samples. The simulated drug diffusion area determined by ultrasound imaging was (Mean ± SD) 2.74 ± 0.74 cm [2]. The visually observed MB diffusion area after dissection was (Mean ± SD) 2.85 ± 0.90 cm [2]. A paired t-test analysis indicated no statistically significant differences between the two measurement methods (Fig. 6A). There was a significant correlation between the distribution range of LA indicated by UCA and MB methods, with a Spearman rank correlation coefficient of R = 0.70 and P = 0.02 (Fig. 6B).

Fig. 6

Statistical analysis of the diffusion area of two types of drug solutions. A: Illustration of paired t-test statistical graph for two measurement methods. B: Illustration of Spearman correlation analysis for two measurement area methods. C: Illustration of Bland-Altman plot analyzing the agreement between the two methods. n = 11. Data are presented as mean ± standard deviation

The Bland-Altman plot (Fig. 6C) showed that the mean difference between the two methods was 0.65 cm [2], with a 95% confidence interval for the difference ranging from − 1.38 cm [2] to 1.16 cm [2].

Evaluation of nerve and muscular toxicities of perfluoropropane UCA using animal studies

The animals were divided into four groups and injected with 0.9% NaCl (N group), 0.9% NaCl + perfluoropropane UCA (N + U group), 0.375% ROP (R group), or 0.375% ROP + perfluoropropane UCA (R + U). No significant activity disorder or death was observed in all four groups after a 72-hour period.

Results from Masson staining showed densely arranged and undamaged neural fibers with intact nerve sheaths in both the N and N + U groups. Normal neural fibers appeared red and were surrounded by thick myelin sheaths that were stained red (Fig. 7A and C). In both the R and R + U groups, partial or complete demyelination of the nerve sheaths was observed, resulting in a neuropathological score of 1 (Fig. 7E and G). LFB staining also revealed densely arranged neural fibers with intact nerve sheaths in both the N and N + U groups. Normal neural fibers appeared blue and were surrounded by thick myelin sheaths stained in blue (Fig. 7B and D). Both the R and R + U groups exhibited partial or complete demyelination of nerve sheaths, resulting in a neuropathological score of 1 (Fig. 7F and H).

Muscle HE staining demonstrated a neat arrangement of muscle fibers with minimal fibrous tissue in the N and N + U groups. A few small blood vessels within, without significant infiltration of inflammatory cells were observed (Fig. 8A and B). Partial muscle fiber loosening and edema were observed in the R and R + U groups, with occasional infiltration of inflammatory cells but without muscle fiber necrosis. Both the N and N + U groups had six samples each, all with a pathological score of zero, indicating no damage. The R and R + U groups also had six samples each with pathological scores of 1, indicating mild muscle fiber damage (Fig. 8C and D).

The ELISA results showed that both in the neural and muscle tissues, the levels of inflammatory factors TNF-α and IL-1β were increased in the R and R + U groups compared to the N group. However, there were no significant differences between the N and N + U groups and the R and R + U groups (Fig. 9). This indicated that the use of perfluoropropane UCA did not further increase the release of inflammatory factors induced by 0.375% ROP in neural and muscle tissues.

Fig. 7

Neuro-pathological changes after treatment with different drugs under two staining methods. A, C, E, and G images show neuro Masson staining. B, D, F, and H images show neuro LFB staining. A and B show the 0.9% NaCl treatment group. C and D show the 0.9% NaCl + perfluoropropane UCA treatment group. E and F show the 0.375% ROP treatment group. G and H show the 0.375% ROP + perfluoropropane UCA treatment group. The red arrow indicates the nerve outer membrane. The green arrow indicates demyelination. The black arrow indicates normal nerve fibers. Each group n = 3

Fig. 8

The effects of different drugs on inflammatory factors in neural tissue. A: The levels of TNF-α in different treatment groups after 72 h of administration. B: The levels of IL-1β in different treatment groups after 72 h of administration. Each group n = 3. Data are presented as mean ± standard deviation. *P < 0.05, **P < 0.01

Fig. 9

Pathological changes in the muscular fascia layer after treatment with different drugs as observed through HE staining. A: The 0.9% NaCl treatment group. B: The 0.9% NaCl + perfluoropropane UCA treatment group. C: The 0.375% ROP treatment group. D: The 0.375% ROP + perfluoropropane UCA treatment group. The red arrow indicates muscle fibers. The green arrow indicates the nucleus of muscle fiber cells. The black arrow indicates blood vessels. The orange arrow indicates mildly damaged vacuolar fibers. The yellow arrow indicates inflammatory cells. Each group n = 3

Effect of perfluoropropane UCA on different cells

The CCK-8 assay results showed no statistically significant differences in the viability of the four cell types at 0, 30, 60, 90, and 120 min between the R and R + U groups. Furthermore, there was no significant decrease in the cell viability of any of the four cell types following treatment with 0.9% NaCl + perfluoropropane UCA in comparison with the N group (Fig. 10). To assess cell apoptosis 1 h after drug treatment, Annexin V-FITC/PI double staining combined with flow cytometry was performed. In this examination, Q1 quadrant denoted necrosis and partial late-stage apoptosis, Q2 quadrant denoted early-stage apoptosis, and Q3 quadrant denoted late-stage apoptosis. Statistical analyses were conducted on the combined apoptotic percentages in quadrants Q2 and Q3 for each cell type. No significant differences in apoptosis were observed between the R and R + U groups or between the N and N + U groups for any of the four cell types (Fig. 11).

Fig. 10

The effects of different drugs on inflammatory factors in muscle fascia tissue. A: graph shows the levels of TNF-α in different treatment groups after 72 h of administration. B: graph shows the levels of IL-1β in different treatment groups after 72 h of administration. Each group n = 3. Data are presented as mean ± standard deviation. * indicates P < 0.05, ** indicates P < 0.01

Fig. 11

The effects of different drug treatments on cell viability. A: graph compares the cell viability of NCTC Clone 929 cells at different time points. B: graph compares the cell viability of L6 cells at different time points. C: graph compares the cell viability of SH-SY5Y cells at different time points. D: graph compares the cell viability of RSC96 cells at different time points.Each group is composed of 6 replicate wells. Data are presented as mean ± standard deviation

Fig. 12

The effects of different drug treatments on cell viability. A graph, B graph, C graph, and D graph respectively represent the apoptotic rate of four types of cells after 1 h of drug treatment, as determined by Annexin-V/PI double staining. E graph, F graph, G graph, and H graph show the statistical analysis of the apoptotic rate of the four types of cells after 1 h of drug treatment. The experiment was repeated 3 times. Data are presented as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001

Discussion

In recent years, there have been several reports investigating the feasibility of using ultrasound contrast agents (UCA) for nerve blockade and monitoring the diffusion of LA [4, 5, 7]. However, as previously mentioned, these studies have certain limitations. Therefore, our study aims to build on previous research to improve the technique. The most commonly used LA for FPB are bupivacaine and ropivacaine, with concentrations ranging from 0.0625 to 0.75%. However, because of its lower cardiac toxicity compared to bupivacaine, ropivacaine has been increasingly used as a substitute for bupivacaine in clinical practice to achieve a longer duration of nerve blockade [8]. Considering the concentrations commonly used in clinical practice, we selected 0.375% ropivacaine and MB to simulate the diffusion of the drug for our study.

Our pre-experiments results have already demonstrated that the particle size of perfluoropropane UCA conforms to clinical requirements. Mixing it with 0.375% ropivacaine did not alter the stability or particle size of the microbubbles, nor did it affect the pH of 0.375% ropivacaine. Therefore, there is no risk of vascular occlusion even in the event of absorption into the bloodstream.

Simulated in vitro imaging was performed using the UCA dilution factor described previously [4]. We found that undiluted perfluoropropane UCA, oscillated with 2 mL of 0.375% ropivacaine, caused ultrasound shadowing, consistent with the observations of a previous study [4]. Although this phenomenon was observed only for 90 s in the previous study, we extended the observation time to 30 min. This extension is relevant in clinical practice; 90 s may not provide sufficient time to observe LA diffusion. With prolonged observation time, we further discovered that while the imaging effect of the undiluted UCA remained stable up to 30 min, the imaging quality from other UCA dilutions decreased rapidly within 6 min. Taking into account the disparities between in vivo and in vitro imaging, a combination of undiluted UCA and 10-fold diluted UCA employed in a previous study was chosen for the TAPB in rabbits. We chose the TAPB technique based on the results of our pre-experiments, which indicated that the fascial layer of the transversus abdominis muscle, located specifically at the level of the rectus abdominis sheath, was more clearly distinguishable and accessible to operators than other anatomical locations in rabbits. In the transversus abdominis plane of rabbits, a 10-fold dilution of UCA led to drug stratification. However, undiluted UCA did not exhibit any layering within 30 min. Furthermore, it should be noted that the distribution of UCA within the fascial layer remained observable throughout the entire 30-minute observation period.

Additionally, Onishi et al. [5] diluted the UCA by a factor of 100 and utilized CEUS to observe the distribution of the UCA following a rectus sheath block. However, owing to the limited contrast between the injected fluid area and surrounding tissues in 2D ultrasound, it was difficult to determine the diffusion location of LA.

However, in our study, clear images of LA diffusion were still obtained by injecting the UCA under 2D ultrasound. Possible reasons for this discrepancy may include the following: (1) The use of a higher concentration of UCA results in greater echogenicity compared to the surrounding tissues. (2) Differences in the injection sites. The anatomical structure of the rectus sheath and the plane structure of the transversus abdominis muscle are different, resulting in inconsistent echo differences in the tissue and inconsistent imaging effects.

Based on the above observations, we selected undiluted UCA to monitor LA diffusion in our experiments. Previous studies have indicated that differences in the distribution area of injected substances visible on MRI after TAPB range from 2.5 to 6-fold. This is related to technical and physiological factors [9]. Therefore, in this study, the TAPB blockade was performed by the same researcher to ensure consistency in operator factors such as injection force and direction. However, significant intragroup variability was still observed despite these efforts, which might be due to specific technical aspects that made it difficult to inject the solution into the transversus abdominis plane with complete accuracy on every occasion. Our findings suggest a strong correlation between the range of LA diffusion under 2D ultrasound and the spread observed in postmortem MB diffusion. This is consistent with the findings of a previous study [5] that compared the diffusion of LA observed under CEUS with the longitudinal diffusion length of iohexol observed under X-ray.radiography.

Studies have shown that LA is cytotoxic to fibroblasts, muscle cells, neurons, and Schwann cells both in vitro and in vivo. This includes the activation of cell apoptosis and generation of ROS [10, 11]. Although our toxicity study showed that different cells responded differently to various drug treatments, we did not observe an increase in the known cytotoxicity of LA upon the addition of perfluoropropane UCA. The inconsistent trends in cytotoxicity among the different cell types observed in this study may be attributed to either the transient nature of the treatment or differences in tolerance to drug stimulation.

This study demonstrates several innovative aspects: (1) It is the first to report the use of perfluoropropane UCA for monitoring LA diffusion in FPB. (2) This study utilized a portable ultrasound device for the first time to observe the diffusion of LA in a fascial plane blockade using 2D ultrasound. (3) This study examined the toxicity of perfluoropropane UCA mixed with ROP at a microscopic cellular level for the first time.

However, there are several limitations in this study: (1) Due to limited resources, the sample size was small, and animal experiments were only conducted using TAPB as an example. The FPB encompasses a wide area, and the imaging effect of the blockade at different anatomical positions may vary under 2D ultrasonography. Animal anatomical characteristics are not entirely consistent with those of humans and LA diffusion patterns cannot be fully generalized to humans. (2) This study only explored the neurotoxicity and myotoxicity of perfluoropropane UCA mixed with the commonly used LA, ROP. The characteristics and potential changes in toxicity when other amide- or ester-type local anesthetics are mixed with perfluoropropane UCA require further exploration. (3) In clinical practice, most FPB involve the injection of large volumes of LA into the fascial plane. As muscles typically have an abundant blood supply, there is a risk of systemic toxicity due to the absorption of LA into the bloodstream [12]. This study only investigated the local neurotoxicity and myotoxicity of perfluoropropane UCA. Further exploration is necessary to determine whether the systemic absorption of perfluoropropane UCA after mixing with LA affects LA metabolism and systemic toxicity. In conclusion, future research should focus on conducting experiments with large sample sizes, including animal and cadaver studies, to further explore the feasibility of using perfluoropropane UCA to monitor drug diffusion using 2D ultrasound. This will provide a more comprehensive and detailed understanding of the feasibility of this method and promote its application in clinical setting. Additionally, we anticipate the development of a dedicated ultrasound imaging mode that utilizes UCA to monitor drug diffusion in FPB under ultrasound guidance.

Conclusion

Stable and high-contrast images on 2D ultrasound were achieved by oscillating 2 mL of undiluted 0.375% ROP and perfluoropropane UCA. When 0.375% ROP mixed with perfluoropropane UCA and MB was used, the simulated drug diffusion area obtained through 2D ultrasound showed no statistically significant difference compared to the MB diffusion area observed after dissection, demonstrating a strong correlation. Toxicity studies in vivo and in vitro have shown that the use of perfluoropropane UCA does not increase neurotoxicity or myotoxicity caused by 0.375% ROP. This preliminary study demonstrates the potential feasibility and safety of using perfluoropropane UCA to monitor drug diffusion with 2D ultrasound. However, further studies are necessary to confirm these findings and to assess their applicability in clinical practice.