Adverse long-term impact of anesthesia and sedative agents on the developing brain in young children, such as anesthesia-induced developmental neurotoxicity, is a widely discussed topic since the 2016 Food and Drug Administration (FDA) drug safety communication (FDA 2016). The results of a multi-center randomized controlled clinical study, comparing GA and awake-regional anesthesia (GAS trial) in infants aged 2 years, showed absence of neurotoxicity following GA of less than one h at 2- and 5-years follow-up (Davidson et al. 2016; McCann et al. 2019). The above study was in-line with the Paediatric Anaesthesia Neurodevelopment Assessment (PANDA) study, which assessed the developmental outcomes in matched-siblings (aged 8–15 years) with or without GA exposure, before the age of 3 years (Sun et al. 2016). Furthermore, the results of the Mayo Anaesthesia Safety in Kids (MASK) assessing neuropsychological of children aged either 8 to 12 or 15 to 20 years old with a history of no exposure, single exposure, or multiple GA exposure prior to the age 3 years reported no difference in intelligence quotient (IQ) scores between all groups. They further concluded that multiple, but not single, GA exposure could be associated with behavioral and learning difficulties (Warner et al. 2018). Therefore, neurotoxicity and cognitive changes associated with anesthetic exposure remains debatable (Vutskits et al. 2012).

The utilization of biomarkers as an outcome measure of anesthetic agents’ effect on neural tissues has been reviewed to assist in the development in safe and effective anesthetic strategies for young children (Levy et al. 2016). Overall, serum biomarkers have been shown to be a reliable indicator of a biological processes but not necessarily a clinical endpoint (Strimbu and Tavel 2010). Serum biomarkers validity as a predictive tool for neurological damage in mild traumatic brain injury cases has been proven (Shahim et al. 2016; Lee et al. 2021). At a cellular level, they are regarded as surrogate markers for physiological processes, such as apoptosis, glial/astrocyte damage, neuronal damage, neural inflammation, and synaptogenesis inhibition. The baseline values of these biomarkers vary with genetic and environmental factors. Despite variable baseline biomarker levels, an increase or decrease in these surrogate markers with anesthesia and surgery denotes protein activity changes within the neurons. Each biomarker must be correlated in conjugation with a range of other biomarkers, hence a panel of biomarkers are recommended.

According to the literature, changes in serum Caspase-3 and NfL levels have been reported following GA exposure (Balasubramanian et al. 2021). Additionally, specific biomarkers have received considerable interest, such as NSE and S100B protein, for predicting post-operative cognitive dysfunction (Silva et al. 2016; Stojanovic Stipic et al. 2017). Therefore, changes in serum levels of four plasma biomarkers: Caspase-3, NSE, NfL and S100B protein were assessed in this study as surrogate markers of neurotoxicity in children. Overall results of this study report a reduction in Caspase-3 levels in otherwise healthy children aged between 3 and 6 years old following short duration DGA exposure, thus suggesting an absence of subclinical neurotoxicity.

Serum Caspase-3 protein, also known as CPP32 or apopain, is a member of the endoproteases (cysteine-aspartic proteases) family (Asadi et al. 2021). Caspase-3 has been identified as a key biomarker of apoptosis, activated in apoptotic cells via both extrinsic and intrinsic pathways. Furthermore, non-apoptotic functions, such as neural development, tissue differentiation, and regeneration, have been correlated to Caspase-3 levels (Shalini et al. 2015; Asadi et al. 2021). Caspase-3 levels denote the rate of apoptosis with cell death caused by proteolysis of many cellular proteins (Yang et al. 2020). Of clinical importance is the reported rapid increase in Caspase-3 activity post insult, which has been linked to stroke and traumatic brain injury in the adult population (Lorente et al. 2015; Glushakova et al. 2017). Raised serum Caspase-3 levels have also been seen in degenerative neurological conditions. Therefore, levels of Caspase-3 are measured as a marker of prognosis in intensive care units (Balasubramanian et al. 2021).

In this study, Caspase-3 persistently reduced after anesthetic exposure suggesting no neurological damage within the anesthetic dosages used in this study, in this age group following short anesthetic exposures (106 ± 28 min). These findings correlate with the primate study, in which exposure to isoflurane induced a significant increase in neurodegenerative apoptotic biomarker S100B and Caspase-3 levels as opposed to the equipotent exposure of sevoflurane (Liang et al. 2010). The immediate cause for dynamic changes in serum Caspase-3 noted in our study is hard to pinpoint without associated nucleotide imaging of the brain. However, findings of this study reiterate the usefulness of this biomarker as a neuromonitoring tool for the various anesthetic agents and various durations of anesthesia in this age group.

Serum NSE biomarker, a critical glycolytic protein, is considered as a well-known biomarker of neuronal damage (Shaik et al. 2019). The glycolytic protein pathway of NSE is known to increase following brain injury, trauma, and epilepsy (Shaik et al. 2019; Lee et al. 2021). Normal serum levels of NSE are within the range of 2–20 ng/ml, with a pathological level recorded at more than 30 ng/ml (Cata et al. 2011). Results of this study revealed that NSE levels were within normal serum levels, with a lower detection limit of 6.49 to 8.37 ng/ml. Therefore, no clinical significance can be attributed to NSE changes since the overall levels were within the normal physiological range reported previously.

S100B protein, a calcium-binding protein, is known to be present in astrocytes and glial cells, hence a potential role as a biomarker in cases of degenerative and inflammatory neurological diseases (Cata et al. 2011). Additionally, Serum NfL, a neuron-specific marker of neurological injury is reported to be the most abundant and soluble proteins of the neurofilament family (Thebault et al. 2020). This feasibility study reported no statistically significant changes in S100B and NfL levels among the three measured time points. Such results could be attributed to the short post-operative time at which the blood samples were collected. Although longer post-operative sample collection might show different results, obtaining blood samples post-DGA might be difficult in such young group of patients.

Of clinical interest, child recovery following DGA could be associated with variable interchangeable behavioral disturbances, such as post-anesthetic agitation, delirium, and excitement (Vlajkovic and Sindjelic 2007; Reduque and Verghese 2012). As this study included children aged between 3 and 6 years old, only fine motor sub-domains of significance in the ‘schedule of growing skills-2’ were used to assess neurological changes effecting fine motor function. All included children were scored twice; just before and then two weeks after DGA. The maximum score possible was 28 (21–23 expected for 3-year-old, 24–26 expected for 4-year-old and 27–28 expected for 5 years old). All patients, except one, scored within the expected ranges indicating no effect on children’s motor function two weeks post-DGA.

Limitations of this study include the lack of a control group, short assessment period and low sample size in which the effect of confounding factors, such as type of dental treatment, pre-existing pain, stress, and post-operative time, were not assessed and could have affected the outcomes of this study. Although patients with previous experience of GA were not excluded from this feasibility study, only one patient had such experience. Future controlled studies should be conducted to further understand the effect of multiple anesthetic exposure. Additionally, lack of long-term sampling of the biomarkers (i.e., sample collection at regular intervals, such as 3, 6, 12, and 24 h) might provide better assessment to the changes in serum level of biomarkers. Despite the above, this study adds to the scarce human studies on the safety neurotoxicity of DGA agents, especially in the dental pediatric field where the majority of procedures do not exceed 1–2 h. This study as such can be considered as a stepping stone to a larger study looking at biomarkers for neurotoxicity to anesthesia exposure of three h or more in duration. Therefore, future prospective well-designed studies of sufficient sample size are required to ascertain which serum biomarkers can be used to compare toxic effects of GA agents in comparison to patients receiving dental treatment under local analgesia. Furthermore, studies assessing the effect of GA on children below the age of 3 years are of interest as neurotoxicity could be higher due to immaturity of neural tissue. These future studies assessing serum biomarkers should consider including NSE and Caspase-3 as both have been shown to be sensitive to neurological insult in the immediate pre-operative period in our this study. Additionally, the primary end point to look for is also important and more objective end points, such as biomarkers and neuroimaging, is recommended in future studies.