Combined Ultrasound and Nerve Stimulator-Guided deep nerve block may decrease the rate of local anesthetics systemic toxicity especially in HBV carriers: A Randomized clinical trial

Background: Ultrasound-guidance might decrease the incidence of local anesthetics systemic toxicity(LAST) for many peripheral nerve blocks compared with nerve stimulator-guidance. However, it remained uncertain whether ultrasound-guidance would be superior to the nerve stimulator-guidance for deep nerve block in the lower extremity. This study was designed to investigate that whether ultrasound-guided deep nerve block would decrease the incidence of LAST comparing with those with nerve stimulator-guidance, and to find out associated risk factors for LAST. Methods: Three hundred patients for elective lower limb surgery and desiring lumbar plexus blocks(LPBs) and sciatic nerve blocks(SNBs) were enrolled in this study. Patients were randomly assigned to receive LPBs and SNBs with ultrasound-guidance (Group U), nerve stimulator-guidance (Group N) or dual-guidance (Group M). The primary outcome was the incidence of the LAST. The secondary outcomes were the number of needle redirection, motor and sensory block onset and restoration time in the nerve distributions, and associated risk factors. Results: There were 18 patients occurring with LAST, including 12 in group U, 4 in group N and 2 in group M. For multiple comparisons among the three groups, we found that the incidence of LAST in group U(12%) was significantly higher than that in group N(4%)(P=0.037) and group M(2%)(P=0.006). The OR of LAST with hepatitis B HBV infection and female gender were 3.352(95% CI,1.233-9.108, P=0.013 ) and 9.488(95% CI,2.142-42.093, P=0.0004), respectively. Conclusions: Ultrasound-guidance, HBV infection and female gender were risk factors for LAST in LPBs and SNBs. For patients with HBV infection or female gender undergoing LPBs and SNBs, we recommended that combined ultrasound and nerve stimulator-guidance should be used to improve the safety. Trial registration: This study was approved by the Ethical Committee from the first affiliated hospital of Army Medical University. The protocol was registered prospectively with Chinese Clinical Trial Registry

(ChiCTR-IOR-16008099) on March 15th 2016. Key words: ultrasound; nerve stimulation; nerve block; female; HBV; LAST Background Although peripheral nerve blocks have been a safe and effective way to provide analgesia for procedures in a variety of settings, using this type of anesthesia did have risks that should not be overlooked. The incidence of the Local anesthetics systemic toxicity (LAST) was reported to be 0.04/1000 to 1.8/1000 in a latest summary [1]. LAST, life-threatening and sometimes fatal condition, was related to patient characteristics(such as aging, low muscle mass, liver disease, cardiac disease, renal disease or diabetes), local anesthetic characteristics and practice settings [1].
The lumbar plexus blocks (LPBs) combined with sciatic nerve blocks(SNBs) for lower extremity surgery are more and more popular nowadays. Because of the depth of lumbar plexus and sciatic nerve, LPBs and SNBs were advanced regional anesthesia techniques and may be more susceptible to the LAST [2]. LPBs and SNBs were traditionally performed by surface anatomical landmarks and nerve stimulator-guidance. Ultrasound could offer a direct visualization of nerve structures, needle pathway and local anesthetics(LAs) spread in real time so that it was widely used in peripheral nerve blocks. Accumulating data suggested higher efficacy and safety with ultrasound-guidance(US) for nerve blocks [3,4], specifically for interscalene [5], supraclavicular [6], infraclavicular [7], axillary [8] blocks.
Michael et al [9] reported that use of ultrasound reduced the risk of LAST throughout its continuum by 60%to 65% as compared with that not use. However, most of the studies were focused on upper extremity . Due to the deep location of lumbar plexus and sciatic nerve, whether use of ultrasound in LPBs and SNBs would be beneficial for efficacy and safety was argued. Most of the published studies suggested that ultrasound-guidance would shorten the performance time and onset time [10][11][12]. However, studies comparing the incidence of LAST between ultrasound-guidance and nerve stimulator-guidance specific to LPBs and SNBs were limited.
We designed this study to determine whether ultrasound-guided LPBs and SNBs would decrease the incidence of LAST comparing with those with nerve stimulator-guidance(NS), and to find out associated risk factors for LAST.

Methods
This study was approved by Committee from the hospital of Medical University the Ethical Patients scheduled for elective lower limbs surgery in the Southwest Hospital and desiring LPBs and SNBs were offered enrollment. Written informed consent was obtained from the participants for publication of this article and any accompanying tables. A copy of the written consent was available for review by the Editor of this journal. Inclusion criteria were as following: willingness to participate in the study (written informed consent), ASA classification of I to III, elder than 18 years old. Exclusion criteria were as following: refusal to participate, history of neurological diseases, coagulopathy and infection at the site of block, allergy to local anesthetics(LAs), and any contraindication to peripheral nerve blockade noted by the attending anesthesiologist. All patients were randomly allocated to Group U (ultrasound-guidance), Group N (nerve stimulator-guidance) or group M(combinedguidance) by random number table, respectively.

Blinding
The anesthesiologist performed LPBs and SNBs was strictly blinded to patients' group assignment before the procedure. Only when the anesthesiologist commenced with the block, was a prepared sealed opaque envelope containing the patient's group assignment opened. Then the anesthesiologist completed the block with the indicated technique. There were two investigators in the study. One investigator blinded to the technique used during the procedure was present in the block area to assess procedure-related outcomes. To ensure the blindness of the patient to the anesthesia method preferred, all procedures performed behind an opaque screen and investigators were required not to say anything about the technique in use. Another investigator assessing block quality was blinded to the group allocation and remained outside the block area until completion of the procedure.
Finally, a statistician blinded to the whole process did statistic analysis, with group data labeled only as numbers until all analyses were completed.

Block preparation
LPBs and SNBs were performed preoperatively by 1 of 3 attending anesthesiologists who were skilled in peripheral nerve blockade with both US and NS guidance. Meanwhile all of them had been in clinical practice with an effort on regional anesthesia for at least 5 years.
After arriving the operating room, patients were placed in the lateral decubitus position with the surgical limb uppermost and monitored continuously via electrocardiography, SpO2 measurement, and noninvasive blood pressure monitoring during nerve blockade and surgery. Both the ultrasound and nerve stimulation systems were prepared and positioned conventionally in each group. The ultrasound machine and nerve stimulator were turned on, and a grounding lead was placed on the lateral aspect of the leg being blocked for each group. Patient's group allocation was given to the anesthesiologist only after preparation of both systems and just before the block procedure. Patients were pretreated with 0.05mg/kg midazolam and 1.5μg/kg fentanyl. The injection site was prepared with chlorhexidine gluconate. 5 ml of 0.5% lidocaine was injected subcutaneously at the site of needle insertion. The LA mixture was made up of 200mg ropivacaine,200mg lidocaine and 20ml of 0.9% sodium chloride solution. The concentration of ropivacaine and lidocaine was 0.4%.
The total amount of LAs used was determined by the dosage of ropivacaine needed, namely 3mg/kg. Patients and the investigator assessing the block quality were kept away from seeing both the block procedure itself and the sonographic displayed by an opaque screen.
According to group allocation, patients received their nerve blocks under one of the following three techniques.

Nerve stimulation technique
In the operating room, LPB was performed using the chayen's approach [ 13,14]. The puncture site was located 4-5 cm lateral from posterior midline along the intercristal line.
A 110-mm, 22-G stimulating needle connected to a nerve stimulator (Stimuplex HNS 11, B. Braun) was advanced perpendicular to skin. The nerve stimulator was set to a pulse duration of 0.1 ms, current intensity of 1.0 mA, and frequency of 2 Hz. When the stimulating intensity was progressively reduced to 0.4 mA or less while maintaining the twitch in the quadriceps distribution. The total volume of LAs was determined by the amount of LA mixture according to patient's weight as we mentioned above. Each point was given half of the calculated total volume of LA mixture. When the correct needle position was achieved based on evoking the desired motor response, the amount of LAs described above was injected. SNB was performed with the classic Labat's approach [15].
The needle was inserted 5 cm below the midpoint of a line connecting the posterior superior iliac spine and the greater trochanter. After an appropriate stimulus was localized in the sciatic distribution, LAs described above was injected.

Ultrasound-guided technique
We chose "Shamrock Method" for LPBs [ 16]. A sterile cover was put on a 3MHz low- the same volume occurred as described previously until circumferential spread around the nerve was obtained. The needle was redirected, when required, to achieve this goal. As for group M, initially, needle-to-nerve guidance was applied according to group U. Maintaining the needle nerve position, the nerve stimulator was set as described for group N. When the correct needle position was achieved based on evoking the desired motor response, LAs described above was injected.

Block evaluation
Evaluation of nerve block was performed by an investigator blinded to those who administered LPBs and SNBs. The motor and sensory response in the nerve distribution area were assessed every 5 minutes until complete motor and sensory effects were achieved. If it took more than 30 minutes to achieve sensory loss in both distributions after the end of injection with LAs, we identified that it was failed block. The attending anesthesiologist had the right to perform general anesthesia, rescue blockade, or supplementation with a local field block when came across with a failed block. The motor block was assessed with a modified Bromage Scale; 2, full motor strength, 1, decreased strength; and 0, no strength. Similarly, the sensory block was evaluated with ice: 2, full sensation (no change): 1, decreased sensation; and 0, no sensation.
Postoperative follow-ups were performed in the post-anesthesia care unit and by telephone within 72 hours after the procedure by clinical personnel in addition to studyrelated procedures.

Outcomes
The primary outcome was the incidence of LAST. LAST can present with both central nervous system (CNS) and cardiovascular system (CVS) clinical manifestations. The CNS symptomsincluded tongue numbness, tinnitus, light-headedness, metallic taste, nystagmus, confusion, tremors, agitation, seizures, coma, and respiratory arrest [18]. The CVS symptoms include tachycardia, arrhythmias, hypertension, and later toxic symptoms such as bradycardia, cardiac depression, cardiovascular collapse, and asystole [18]. The Demographic and perioperative data were expressed as the means and standard deviations.
Parametric and non-parametric Kolmogorov-Smirnov tests were applied to assess normality. The primary outcome (incidence of LAST) and potential risk factors were compared by 2 test or Fisher exact test when appropriate (n<5 in any field). In the 2 test, we tested whether there were differences in the incidence of the LAST and the odds ratios of potential risk factors among different groups. The demographic and secondary outcomes were compared among the three groups by one-way ANOVA, following by multiple comparisons using LSD test or Welch and Dunett's T3 test for unequal variances. In the oneway ANOVA, we tested whether there were differences in the patients' characteristics and block quality among the three groups. This analysis was followed by 95% CIs with Bonferroni correction to adjust for multiple comparisons (three different methods for nerve block for motor and sensory onset and restoration time data and demographic data) to minimize the chance of a type error I (0.05). For all comparisons, 2-tailed P <0.05 was considered statistically significant.
The incidence of LAST was low according to published data. So we did a test for the power of test regarding the incidence of the LAST in three groups after the experiment. In the current study, there were 319 patients randomly allocated into the three groups. Finally, data from 100 patients for each group were analyzed. We did a test for the power of test regarding the primary outcome after experiment. We calculated the effect size (0.182) in the software PASS. Then we set the significant level as 0.05. Referring to the result, we found that when the total sample size was 300, the power(1-β) of the test was 0.81.

Results
Study flow diagram was presented in Fig 1. A total of 319 patients were evaluated for eligibility and offered enrollment in this study. 18 were excluded, including 3 not meeting inclusion criteria, 13 declined to participate and 2 other reasons. There were no failed or aborted blocks in either group. One patient in group U was lost of follow-up.
Patient's characteristics were presented in Tab 1. There was no statistically significant difference in the age, gender, weight and height among the three groups. Only one patient in group M was with ASA Ⅲ status. Most of the operations were performed in knee and ankle.
There was statistically significant difference insurgical duration time in group U (41.0±24.21minutes) comparing with that in group M (51.5±30.8 minutes).
The primary and secondary outcomes were shown in Tab 2. The incidence of LAST in all the three groups was 6%. What's more, there was statistically significant in the incidence of LAST among the three groups. For multiple comparisons among the three groups, we found that the incidence of LAST in group U(12%) was significantly higher than that in group N(4%) We analyzed risk factors such as age, gender, liver disease, diabetes according to The Third

American Society of Regional Anesthesia and Pain Medicine Practice Advisory on Local
Anesthetic Systemic Toxicity [1]. In the current study, 52 patientsinfected with HBV and 7 of them occurred with LAST. As shown in Tab

Discussion
There were three main findings in the current study. Firstly, use of ultrasound did not improve the quality of deep nerve block. Secondly, use of ultrasound did not improve the quality of deep nerve block. Thirdly, use of ultrasound, HBV infection and female gender may be risk factors for LAST.
In the present study, we found that LPBs and SNBs with US were not superior to those with NS in the onset and restoration time. Spencer S. Liu et al [19] summarized that 8 of 10 RCTs reported use of ultrasound would shorter the onset time of the lower extremity block, 2 of 10 found no difference, and no RCT reported slower onset with ultrasound.
However, most of the RCTs were about the femoral and peroneal nerve. Recently, Arnuntasupakul et al [12] reported that ultrasound with nerve stimulator for LPBs resulted a shorter total anaesthesia time comparing with US alone. Due to the size and location of the lumbar plexus and sciatic nerve, LPBs and SNBs were advanced regional anesthesia techniques. Different admission passage for the nerve block may result in different outcome. Furthermore, due to the comorbidities, some of the enrolled patients may already have minor pathological changes in the targeted nerve which may affect the onset and restoration time.
The incidence of LAST was 6% which was much higher than previously reported [ 1]. LAST can occur as a result of the patient's risk factors and current medications, inadvertent injection of LAs directly into the vascular system, an exceeded maximum dose of the LAs, or immediate absorption of the LAs when it was injected into an extremely vascular area [18]. It had been widely reported that US was safer comparing with NS because US could provide direct visualization of the target nerve, surrounding tissues, and injectate spread [9,20]. However, in our study, about two-thirds of the patients occurring with LAST were from the group U. There were two main reasons. Firstly, a fair amount of patients in our study were with HBV infection, renal diseases who may be more susceptible to LAST [21]. Secondly, the lumbar plexus and sciatic nerves were difficult to be visualized due to its depth. In order to get a better vision of tissues near the nerve, block needle and the deposition of LAs, ultrasonic probe was required a pressure near the injection site. The pressure would make the blood flow of the deeply located small vessels slower. It was difficult for the Doppler ultrasonography to exam the deeply placed small vessels especially with slow flow [22]. The continuous pressure caused by the ultrasonic probe made small deeply located vessels "invisible" thus it harder to avoid injecting into Patient's weight, comorbidities, use of other medications, genetics, allergies, and other physiologic limitations also affected the incidence of LAST [ 23]. Factors affected the systemic absorption of LAs, its peak plasma concentration and the time to reach that peak were all related to LAST. Bupivacaine and ropivacaine were degraded in the liver by α1acid glycoprotein(AAG) [21]. Patients with liver diseases would have a decreased clearance rate of LAs due to the low concentration of AAG, which may increase the incidence of the LAST. However, even patients with advanced liver dysfunction, synthesis of AAG was still maintained [21,24]. In patients with hepatic dysfunction, single-dose blocks can usually be performed safely with normal dose of the LAs [25]. It indicated that decreased clearance of LAs caused by isolated hepatic dysfunction was not the main reason here. However, as shown in Tab 4, HBV infectious patients had a higher risk of LAST in this study. Patients who had been infected with HBV may result in chronic liver disease. Patients with chronic liver disease usually had vascular dysfunction, especially angiogenesis, microvascular derangements and microcirculatory dysfunction [26,27]. Cirrhosis caused numerous microscopic vessel aberrations, may become entangled with each other, such as sharp bends, anomalous branching patterns, abnormal branching angles and tortuosity [28].
McAvoy et al [29] demonstrated that cirrhosis patients had selective regional increases in blood flow in the splanchnic and hepatic circulations, yet diminished flow in the peripheral limbs. Neovascularization and slower blood flow made it easier to inject with LAs into extremely vascular area especially for the use of ultrasound, resulting in an increasing incidence of LAST. The vascular endothelial growth factor(VEGF) and bone morphogenetic protein 9 (BMP-9) had been widely reported to promote angiogenesis [30]. A higher BMP-9 levels in human serum accompanied with advanced stages of liver fibrosis, meanwhile overexpression BMP-9 accelerating the liver fibrosis and BMP-9 knockdown attenuated the liver fibrosis in a mouse model [31]. Plasma VEGF was elevated in cirrhosis patients, especially in those with spider angiomas [27]. A higher level of serum BMP-9 and VEGF in patients with HBV indicated a more advanced stage of liver dysfunction and more formation of new blood vessels. But it need our further efforts to determine the relationship of the VEGF and BMP9 between the HBV infection. VEGF and BMP9 may be promising prognostic indicators for the incidence of LAST for patients with HBV in deep nerve block.
Herein women were more likely to occur with the LAST, which was in consistence with the latest regional anesthesia and pain medicine practice advisory on LAST [1]. Some enrolled patients with LAST were HBV carries. The increase risk of LAST in female may be related to HBV infection. In physiological conditions estrogen/estrogen receptor α (ER/ER α) axis had a protective effect on HBV-associated liver damage and taking postmenopausal hormone replacement therapy resulted in a lower risk of hepatocellular carcinoma development for HBV positive women carriers [32].In a female cirrhosis rat model the mRNA expression of ER α was lower in that of a sham rats and the ability of 17β-estrodiol to alleviate of relevant complications was diminished [33]. It indicated that HBV women carriers might have a lower level of ER/ER α that made them more susceptible to LAST. It need further efforts to investigate the underlying mechanism.
There are a number of limitations to this study. Firstly, it was not possible to blind the anesthesiologist performing the nerve block, we couldn't exclude the potential influence of a performance bias in this study. Secondly, although we made our efforts to maintain blinding among the investigators, patients, and statistician, it may be partial blinding due to muscle contractions elicited by nerve stimulation, collecting the needle redirections and so on. We attempted to minimize this bias by only involving staff anesthesiologists experienced in peripheral nerve blockades using both guidance modalities. Thirdly, part of the limitation was related to techniques and equipment used in this single-center study, so that it could not generalized to other techniques or peripheral nerve block locations.
The degree of advantages and disadvantages provided by ultrasound-guided deep nerve block especially in HBV carriers was likely to vary by nerve block site as well. We only demonstrated some interesting phenomenon found in a single-center study. The underlying mechanisms were not illuminated in the present study. A multicenter and more detailed experiments were needed to certify our discovery and illuminate the mechanisms.

Conclusions
Our results suggested that ultrasound-guidance, HBV infection and female gender were

Availability of data and materials
The raw data and materials are available from the corresponding author upon reasonable request.

Authors' contributions
Xu-hao Zhang helped design and conduct the study; data collection and manuscript writing.