A Comparative Study of Brain Injury Biomarker S100β During General and Spinal Anesthesia for Caesarean Delivery: A Prospective Study

2. Materials and Methods

2.1. Study Design and Participants

The study was a single-centre prospective, controlled study. In accordance with the inclusion and exclusion criteria, the parturient women who undergo C-section in The Obstetrics and Gynaecology Hospital of The National Center of the Maternal and Child Health of Mongolia from July 2021 to December 2023 enrolled in this study. All procedures performed in the study complied with the Declaration of Helsinki and adhered to the Consolidated Standards of Reporting Trials (CONSORT) guidelines. The terms general anaesthesia (GA) and spinal anaesthesia (SA) were used to designate two distinct groups. Women giving birth by caesarean-section in hospital granted permission to take part in the study and were registered. Figure 1 shows patient recruitment and flow. The sample size of the study was calculated using Open Epi software. The sample size was calculated based on the difference in means under the hypothesis of a difference between the two groups. A minimum of 29 subjects was required per study group. The study adhered to the following specific inclusion and exclusion criteria for the participants. The inclusion criteria were as follows: (1) aged between 18-40 (2) American Society of Anaesthesiologists physical status I or II (3) term gestation at 37 weeks (4) patients whose haemoglobin >100 g/L (5) women with uncomplicated singleton pregnancies who were advised to undergo elective caesarean sections due to factors such as a previous caesarean delivery, a history of primary infertility, or other reasons. Exclusion criteria were as follows: (1) less than height 150 cm (2) body mass index (BMI) ≥ 30 kg/m2 (3) parturient women who suffered from severe internal, surgical, or obstetric comorbidities (4) preeclampsia (5) known fetal neurologic deficit, intrauterine growth retardation (6) patients who received analgesic and sedative medicine before C-section (7) patients who suffering from a severe mental illness (8) emergency C-section for delivery (9) classification as ASA status ≥ III (10) patients who were unwilling to partake in the study (11) patients who were allergic to anaesthetics (12) patients who had contraindications to general/ or spinal anaesthesia.

2.2. Methods of Anaesthesia

In preparation, Ranitidine 150 mg orally (H2-blocker), and metoclopramide 10 mg intravenously. To avoid supine hypotension syndrome, women in both groups were maintained in a 15-degree left lateral tilt position until birth. An observer gathered all the intraoperative data. Hypoxia was defined as SpO2 <95%. A 20% decrease in systolic arterial pressure below the baseline value was defined as hypotension in this study and treated with intravenous ephedrine. Additionally, oxygen was administered at a rate of approximately five L/min through a transparent face mask during the operation. The anaesthetists performing the anaesthesia had similar levels of expertise and were unaware of the study protocol.

GA procedure: In the instances in the general anaesthesia group, pre-oxygenation was carried out using 100% oxygen for five minutes before the administration of general anaesthetic. Patients were induced with propofol 2 mg/kg (manufacturer: Guangdong Jiabo Pharmaceutical Co., Ltd.; specification: 20 ml/ 200 mg) and succinylcholine 2 mg/kg intravenously. After anaesthesia was induced tracheal intubation was performed. After intubation the patients were administered 0.5 mg/kg of atracurium besylate (manufacturer: Flagship Biotech International, India; specification: 2.5 ml/25 mg), 2.0 μg/kg of fentanyl (manufacturer: IVCO, Mongolia, specification: 0.005%-2 ml) and infusions of propofol at 2.5 mg/kg per and fentanyl at 0.05 mg/kg per minute. Surgery began after verification that the endotracheal tube was appropriately placed and positioned through capnography. After delivery, the patients received intravenous injection of five units of oxytocin then continuous pump infusion 15 units (manufacturer: HBM Pharma, Slovakia, specifications: 1 ml/5 mg). Anaesthesia maintained with continuous inhalation of 1 MAC of isoflurane and propofol intravenously at a dosage of 1.5 mg/kg.

SA procedure: With the patient lying in a left lateral position, spinal anaesthesia was performed at the L2–L3 or L3–L4 interspace with 10 mg of 0.5% heavy bupivacaine (manufacturer: Troikaa, Pharmaceuticals Ltd, India specification: 4 ml/20 mg) plus 10 micrograms of fentanyl. The level of the sensory block was modified to be approximately T5–T6.

2.3. Follow-Up

Preoperative: Eligible patients were contacted and provided with written informed consent prior to surgery. Preoperative assessments were carried out by main investigators, the researchers gathered demographics data and basal health questionnaires.

Intraoperative: Radial arterial cannulation was performed for all the patients using 20G Surflo® (Terumo China holding Co., Ltd.) under local anaesthesia (2% lidocaine), and arterial blood pressure was monitored. Invasive blood pressure readings including systolic, diastolic, and mean blood pressures were obtained. Heart rate and SpO2 were measured by the fingertip photoelectric sensor (manufactured: Guangzhou Sichuang Hongyi Electronic Technology Co., Ltd.). Time from anaesthesia induction to delivery, total operative time, intraoperative blood loss, urine output, and complications during surgery were recorded in both groups. All vital signs were recorded.

Postoperative: For the two hours of the postoperative stage, patients were monitored in the recovery room. Complications after C-section within two hours were recorded in both groups including postoperative nausea, vomiting, headache, fever, and pain. The patients were assessed using the Numeric pain rating scale (NPS), which showed the severity of the postoperative level of pain from a scale of 0 to 10, with 0 = no pain and 10 being the worst pain ever. The onset time was also recorded.

Neonatal assessment: Following the delivery of the baby, pediatricians assessed the neonatal condition, assigned Apgar scores at one and five minutes, and determined the need for admission to the Neonatal intensive care unit (NICU). An Apgar score of 7 to 10 was considered normal; 4 to 6, mild neonatal asphyxia; and 3 and below, severe neonatal asphyxia. The Apgar score was assessed based on 5 criteria: activity, pulse rate, grimace (reflex irritability), skin color, and respiratory effort. Following the delivery of the baby, the cord was cut between the two clamps placed approximately 10 to 12 cm apart and away from the placenta and newborn. One to three ml of blood was collected from the umbilical artery from between the clamps immediately following placental delivery. This protocol was designed to prevent contamination of S100β levels in the cord arterial blood by placental S100β, ensuring that the measured concentrations accurately reflect those originating from the newborn.

2.4. Laboratories Outcome Measurements

Measurement of serum s100 β: The maternal serum S100β protein levels of the 30 patients from each group were analyzed before and at the end of the operation, with 5 ml of blood was withdrawn from the mother via the arterial line. The blood samples were immediately centrifuged at 2500 RPM for 10 minutes and the supernatant was gathered and stored at minus 80-degree temperature until the measurement of S100β levels. The concentration of S100β protein in the blood serum was determined by the enzyme-linked antibody reaction (ELISA) using the Human S100 calcium binding protein B (S100B) ELISA kit (Catalog Number SL2183Hu) album of Sunlong Biotech CO., Ltd (China). According to the manufacturer’s protocol, each sample was incubated with the tracer from the kit for 2 hours, following the instructions precisely to maintain intra-batch variation below 10% and inter-batch variation below 15%.

Arterial blood gas analysis: The blood gas analysis was performed from umbilical cord blood immediately drawn after the delivery. A sample of the blood was immediately inserted to a blood gas/electrolyte analyzing system. (COBAS B-221, ROSHE) for рН, рСО2, рО2, Hb, Hct, O2Hb, COHb, Ca2+, K+, Na+, CI-, SO2, BB, BE, HCO3, Osm measure and compared between the 2 groups, and the remaining blood sample was then used for the S100β study assay.

2.5. Statistical Analysis

The primary outcome of the study involved collecting blood samples for the analysis of brain injury biomarker S100β. These samples were obtained from the arterial line of maternal blood, both preoperative and postoperative, as well as from the umbilical artery of C-section deliveries performed under either spinal or general anaesthesia. The secondary outcomes were invasive hemodynamic monitoring, surgery and anaesthesia outcomes, umbilical cord blood gas values, Apgar scores, neonatal asphyxia rate, and maternal postoperative numeric pain rating scale (NPRS) compared in two groups. Categorical variables were expressed as frequency and percentages. Continuous variables were assessed for distribution using the Shapiro-Wilk test, and dependent variables were normally distributed. Differences between the two groups were calculated using a parametric test. Differences between the means of two groups were evaluated by the T test. Proportional differences between groups were calculated using the Chi-square test. In case of abnormal distribution, differences between three groups were determined by Friedman’s test, and Wilson Signed Rank test was used for differences between two groups

3. Results

Sixty patients were recruited for the study. This study included a total of 60 pregnant women undergoing caesarean section, equally divided into two anaesthesia groups: Spinal Anaesthesia (SA) and General Anaesthesia (GA) (n=30 per group). To ensure comparability before assessing anesthetic effects on fetal outcomes, maternal demographic and obstetric characteristics were analyzed between the two groups. They were comparable among the study groups, with no statistically significant differences (p > 0.05), as shown in Table 1.

3.1. S100β Marker and Methods of Anaesthesia

The mean serum S100β levels was 194.1 ± 45.8 ng/L in all women pre-operatively with no significant difference observed between the two groups (p=0.231). Furthermore, there were no significant difference between SA and GA group S100β protein levels post-surgery (p=0.375) and in newborn blood serum (p=0.143) (Table 2).

The changes in S100β protein levels before and after surgery were different for each group. The maternal blood S100β post-surgery and neonatal umbilical cord blood protein levels were different in the GA group (p=0.047), there were lacked a difference in the SA group. In the SA group, preoperative S100β concentration in maternal blood was 195.1±36.2 ng/L, then increased to 200.9±42.9ng/L at the end of operation. Also, in the GA group, preoperative S100β concentration in maternal blood was 193.0±54.3 ng/L, then increased to 197.0±42.7 at the end of operation (Figure 2).

3.2. Perioperative Characteristics by Anaesthesia Groups

The perioperative outcomes, intraoperative time, drug dosage and postoperative complications between the 2 groups, are shown in Table 3 and Table 4. There were zero noteworthy differences in time from anaesthesia to incision, the operation duration, infused crystalloid volume, blood loss, and urine output (Table 3).

However the frequency of headache and nausea were more common in the SA group (p<0.0001, p<0.0001) while hypertension and tachycardia were more frequent in the GA group (p=0.079, p<0.0001) during surgery. Postoperative complications such as nausea, headache and weakness were significantly difference in the SA and GA groups (p=0.044). We detected that the time of onset of pain was significantly shorter in GA group was 13 (43.3%) patients felt pain after surgery within ≤ 60 minutes. The NPRScore and length of stay in hospital between the two groups were also alike. (p=0.105, p=0.232) (Table 4)

3.3. Neonatal Clinical and Laboratory Outcomes Following Spinal Versus General Anaesthesia

No significant differences were found in Apgar scores and neonatal asphyxia rates between the two groups (p=0.476) and blood gas outcomes of the umbilical artery (UA) in Table 5. It shows pH and Ca²⁺ levels of UA were lower in the GA group (p=0.009, p=0.0001).

4. Discussion

The stress response to surgery triggers various physiological and biochemical changes, including sympathetic nervous system activation, the release of stress hormones such as cortisol and catecholamines, and modulation of immune function [25]. Exposure to anaesthetic agents has been shown to induce programmed cell death (apoptosis) in glial and neuronal cells of the central nervous system, potentially leading to neurotoxicity and brain injury. Numerous animal and in vitro studies have reported that anaesthetic agents can exert harmful effects on the developing brain [26], particularly barbiturates, ketamine, propofol, and inhaled anaesthetics [27].

This study examined the brain injury biomarker S100β during cesarean section under general anaesthesia (GA; propofol, fentanyl, isoflurane) and spinal anaesthesia (SA; bupivacaine, fentanyl). Perioperative measurement of S100β levels, which reflects glial cell activity and possible neuroinjury, was performed before and after surgery [28]. Our primary finding was a slight, non-significant increase in maternal arterial S100β concentrations following C-section in both GA and SA groups. This finding aligns with those of Zhendong Xu et al., who reported no significant change in maternal venous S100β levels following epidural or general anaesthesia during C-section [29].

Additionally, our results indicated that cesarean delivery itself does not appear to significantly influence maternal S100β concentrations. However, some studies have reported higher S100β levels in vaginal deliveries compared to elective C-sections, suggesting delivery-mode dependency [30,31]. One previous study observed a significant decrease in the umbilical artery/umbilical vein (UA/UV) S100β ratio after GA compared to epidural anaesthesia, while maternal venous S100β levels remained largely unchanged [32]. In our dataset, UA S100β concentrations were consistently higher than maternal levels in both GA and SA groups, with a slightly lower level observed in the GA group.

We also established a reference range for serum S100β in third-trimester pregnant Mongolian women, identifying a baseline of 194.1 ± 45.8 ng/L. Notably, S100β levels measured beyond 37 weeks of gestation exceeded this normal range [33]. We propose two primary interpretations for our findings. Neither general nor spinal anaesthesia caused detectable neuronal injury during cesarean delivery. This may be attributed to the short duration of surgery and, consequently, limited exposure to anaesthetic agents. In fact, brief GA exposure might confer some neuroprotection under stress conditions [34], possibly explaining the slightly lower post-operative S100β levels in the GA group. The absence of maternal (e.g., CNS disorders), obstetric (e.g., diabetes, hypertension, placental insufficiency), or fetal (e.g., acute/chronic hypoxia) complications—conditions known to elevate S100β—may have influenced our results. Moreover, previous studies support the concept that fetal-origin S100β can pass into maternal circulation via a physiological gradient [35], consistent with our finding that UA S100β levels exceeded maternal concentrations. Although we did not find a significant association between S100β concentrations and perioperative variables, we observed moderate correlations with some umbilical artery blood gas parameters. In conclusion, our findings suggest that cesarean delivery under either anaesthetic technique does not significantly alter maternal S100β levels, and thus may not be associated with acute brain injury. However, the anaesthetic agents used could influence biomarker profiles.

Our study’s strengths include the use of a newly biomarker that allows for a comprehensive evaluation of the effects of two anesthesia methods on both maternal and fetal brain damage during C section. Firstly, this is the first known investigation to assess S100β levels in the maternal and umbilical circulation among pregnant Mongolian women undergoing cesarean section, thus contributing unique regional data to the global literature. Secondly, the study provides a direct comparison between general and spinal anaesthesia, offering valuable insights into their respective impacts on neonatal biochemical outcomes and maternal neurobiological markers. Thirdly, by including both maternal and fetal (umbilical arterial) blood samples, we were able to explore the potential fetal origin of S100β more comprehensively. Additionally, the use of standardized anaesthetic protocols and perioperative care ensured internal consistency, while the integration of neonatal Apgar scores and arterial blood gas analysis allowed for a broader evaluation of neonatal well-being. These elements collectively strengthen the scientific value and clinical relevance of our findings. But this study has several limitations. First, the relatively small sample size limits the statistical power and generalizability of our findings. Second, the study population consisted exclusively of pregnant women from a single tertiary hospital in Mongolia’s capital city, further restricting external applicability. Additionally, the lack of long-term follow-up prevents conclusions regarding the potential delayed neurological effects of anaesthetic exposure.

5. Conclusions

In conclusion, S100β concentrations slightly increased after C-section in both the SA and GA group. General anaesthesia has a faint impact on the umbilical cord blood’s S100β levels during C-section. Spinal and general anesthesia are considered safe for the maternal and fetal brain during cesarean sections when administered appropriately, with no evidence suggesting harmful effects. Further research needed to study associations between anaesthetic drugs, perioperative release of brain injury biomarker, and perioperative clinical outcomes are warranted.