Mitochondrial damage pathway

Mitochondria are the main organelles for oxidative energy supply, which convert glucose into adenosine triphosphate (ATP) through oxidative phosphorylation. Studies have shown that when SH-SY5Y nerve cells were treated with 2% propofol for 48 h, the number of mitochondria decreased, the oxygen consumption of cells decreased, the respiratory rate decreased by 35%, the activity of cytochrome C decreased, and the release of ATP decreased. These phenomena indicate that propofol damages mitochondria and impairs mitochondrial function in mature neurons. ^{Reference Qin, Li and Wang18} Propofol also affects neurons in the development stage. In 7-day-old SD rats injected with 100 mg/kg propofol, it was found by electron microscopy that the mitochondria of neurons were swollen, some mitochondrial inner membranes were broken, mitochondrial cristae were loose or even broken, matrix density was reduced, and ATP production was reduced. ^{Reference Xiao, Lv and Liang19} Propofol not only damages the mitochondria of developing neurons but also induces mitochondrial deformation, vacuolation and swelling and reduces the mitochondrial membrane potential of neural stem cells, which ultimately leads to their proliferation, differentiation and apoptosis. ^{Reference Liang, Sun, Zhong, Miao and Han20} Mitochondrial damage is involved in propofol-induced developmental neurotoxicity and could be an important target for further studies of propofol-induced developmental neurotoxicity.

Oxidative stress pathway

The production and clearance of reactive oxygen species are dynamically balanced in the normal body. Once the balance is broken by endogenous or exogenous stimulation, a large amount of reactive oxygen species (ROS) will be produced, which cause oxidative stress. Oxidative stress leads to neutrophil inflammatory infiltration, increased protease secretion and the production of a large number of oxidative intermediates, which eventually damage cellular proteins, lipids and nucleic acids. Many neurological diseases and injuries are related to oxidative stress, such as Alzheimer’s disease, Huntington’s disease, Parkinson’s disease and cerebral ischemia–reperfusion injury. ^{Reference Liu and Shirley21–Reference Araceli, Patricia and Antonio24} Some studies have found that anesthetics can cause an increase in ROS, mitochondrial damage and neuronal apoptosis. ^{Reference Zhang, Dong and Wu25} Primary hippocampal neurons were isolated from neonatal SD rats and treated with 20 μM propofol for 6 h. ROS production by these cells increased, which promoted the production of the apoptotic proteins cleaved caspase3 and Bax and reduced the expression of the B-cell lymphoma-2 (Bcl-2) protein. TUNEL staining showed that the number of apoptotic neurons increased. ^{Reference Liang, Du, Cang and Xue26} Both superoxide dismutase (SOD) and malondialdehyde (MDA) can be detected as indicators of oxidative stress. When oxidative stress occurs, the release of SOD, an oxygen free radical scavenging enzyme, decreases, causing lipid peroxidation and producing the harmful substance MDA. After exposing hippocampal neurons of neonatal rats to propofol, ROS and MDA increased and SOD decreased. ^{Reference Tang, Zhao and Yu27} These results suggest that oxidative stress is involved in the process by which propofol damages developing neurons.

Calcium overload process

Calcium ions (Ca²⁺) are an important component of the human body and a significant second messenger in cells, which is critical in the central nervous system. The balance of calcium is very important to maintaining normal nervous system function. ^{Reference Wei and Xie28} An imbalance in calcium homeostasis can lead to neuronal damage. The endoplasmic reticulum is the main source of calcium ion release in neurons, maintaining the balance of Ca²⁺ within them through recovery and release. In the mitochondrial matrix, an appropriate amount of Ca²⁺ can regulate the process of oxidative phosphorylation, but excessive Ca²⁺ will hinder mitochondrial respiration and eventually lead to cell damage. Sevoflurane can cause an increase in Ca²⁺ in primary hippocampal neurons of rats and then cause apoptosis. ^{Reference Zhu, Yao, Guo, Li and Lin29} The same effect has been found for propofol. The primary neurons of E17 Wistar rats were exposed to the therapeutic dose of propofol, and the calcium concentration and cell survival rate were measured at 3, 7 and 13 days in vitro (DIV). The results showed that the concentration of Ca²⁺ and the number of apoptotic neurons treated with propofol increased on DIV 3 and 7, but not 13, under therapeutic concentrations of propofol in primary cultured neurons obtained from E17 Wistar rats. ^{Reference Satoshi, Tomotaka and Jun30} In mouse embryonic fibroblasts, it was also found that propofol caused a large outflow of Ca²⁺ in the endoplasmic reticulum and thus cell death. ^{Reference Xu, Wang and Liang31} Therefore, calcium overload is closely related to the neurotoxicity of propofol. These findings may provide a new way to alleviate the developmental neurotoxicity caused by propofol by correcting the dysregulation of calcium concentration.

Inflammatory factors

The main feature of neuroinflammation is the overexpression of pro-inflammatory factors caused by glial cell activation or immune cell infiltration and the suppression of anti-inflammatory factor expression. ^{Reference Peng, Li, Yu, Liu and Qi32} In the brain, glial cells are divided into microglia, astrocytes and oligodendrocytes. Microglia are the main neuroimmune cells and the first sensor of pathophysiological changes. They are very important for monitoring and defense, and trigger signaling cascades. When experiencing trauma or brain disease, microglia are stimulated and activated, polarized to become M1-activated microglia, and secrete proinflammatory factors, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6, thereby stimulating the deterioration characteristic of neurological diseases. ^{Reference Liu, Li and Xia33–Reference Wang, Fu and Duan35} Inflammatory factors are also important in the process of propofol-induced neurotoxicity. Wang et al. exposed Sprague–Dawley rats (SD rats) and PC12 cells to propofol. Propofol caused nerve cell damage in the brains of rats by increasing proinflammatory factors, including IL-1β, IL-6, IL-17, and IL-18, which was also found in vitro. ^{Reference Wang, Suo, Yang and Zhang36} After neonatal rats were exposed to propofol, TNF-α expression levels were found to be elevated in the cerebrospinal fluid, hippocampus and prefrontal cortex, resulting in neuronal damage. ^{Reference Chen, Deng, Wang and Liu37} Propofol can produce inflammatory effects in the hippocampus of the developing brain by enhancing astrogliosis activation (as measured by GFAP level) and increasing the levels of neuronal nitric oxide synthase (nNOS) and the proinflammatory cytokines IL-6 and TNF-α. ^{Reference Yang, Yi, Pan, Hu and Duan38} The Fas ligand/Fas death receptor pathway, as an inflammatory mediator in central nervous system pathology, is also involved in the neurotoxicity of propofol. This pathway leads to the activation of Caspase-8 and Caspase-9 through the mediated exogenous and Bcl-2-dependent endogenous apoptosis pathways, respectively, to result in neurodevelopmental abnormalities. ^{Reference Milanovic, Pesic and Loncarevic-Vasiljkovic39} The above results indicate that neuroinflammation may be involved in the neurotoxicity of propofol, providing meaningful insights for finding therapeutic targets in neuroinflammatory signaling cascades.

Noncoding RNA

In mammals, more than 80% of DNA is transcribed, but less than 2% of RNA is transcribed into proteins, and the rest are RNAs without coding ability. Among all human organs, the central nervous system has the highest content of noncoding RNA. ^{Reference Mehta, Chokkalla and Vemuganti40} Noncoding RNAs play an important role in brain neurodevelopment and neurological diseases. ^{Reference Ang, Trevino and Chang41} microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) have been found to be involved in the neurotoxicity process of propofol. ^{Reference Xiu, Luan, Gu, Liu and Xu42,Reference Xu and Luo43}

MicroRNAs (miRNAs) are endogenous noncoding RNA molecules with a length of approximately 22 nucleotides that regulate cellular processes by inhibiting the translation of messenger RNAs. ^{Reference He and Hannon44} MiRNAs have become a research hotspot on the neurotoxicity of anesthetics in recent years. So far, more than 40 miRNAs have been found to be involved in the neurotoxicity process of anesthetics. ^{Reference Bahmad, Darwish, Dargham, Machmouchi and Chamaa45} The expression of miR-363-3p and miR-34a in SH-SY5Y cells was dysregulated after propofol treatment, resulting in a decrease in the cell survival rate. ^{Reference Li, Li, Zhuang and Li46,Reference Yao and Zhang47} Propofol can cause neuronal injury during brain development by causing an imbalance of miRNA expression. ^{Reference Zhang, Xu, Chi and Cui10,Reference Tang, Zhao and Yu27,Reference Mao, Wang, Gong, Wu, Zhang and Wang48} Zhu et al. exposed hippocampal neurons of neonatal rats to 50 μM propofol for 6 h. PCR showed that after propofol treatment, the expression of miR-455-3p decreased and the expression of its target protein EphA4 increased, leading to a decrease in neuronal proliferation and an increase in apoptosis. ^{Reference Zhu, Li, Tian, Zhou, He and Zhou49}

Long noncoding RNAs (lncRNAs), with a length of more than 200 nucleotides, lack an open reading frame. ^{Reference Jxcab, Ypw, Xin, Gxla, Kuang and Czda50} Studies have shown that lncRNAs participate in many biological processes, including neurodevelopment and neurological diseases. ^{Reference Chen, Wang, Zhang, Li, Zheng and Duan51,Reference Aliperti, Skonieczna and Cerase52} Transcription of the lncRNA called HOX antisense RNA (HOTAIR) is involved in nervous system diseases and is related to sevoflurane-mediated brain dysfunction. ^{Reference Khani-Habibabadi, Zare, Sahraian, Javan and Behmanesh53,Reference Wang, Feng, Fu and Liu54} It was found that the miR-455-3p/NLRP1 axis regulated by HOTAIR was also involved in the neurotoxicity of propofol. ^{Reference Gong, Wan, Zhang and Liang55} The lncRNA BNDF-AS can reverse the expression of brain-derived neurotrophic factor (BDNF). Xu et al. found that the expression of BDNF-AS decreased after propofol treatment in neurons, and reversing this BDNF-AS change reduced the apoptosis induced by propofol. ^{Reference Xu and Luo43} In addition, recent studies have found that neonatal animals with high levels of lncRNA LCRF are more vulnerable to nervous system damage caused by propofol. ^{Reference Zeng, Yao and Zhong56}

The effect of propofol on the fine structure and functional integrity of nerve cells

Most studies on the neurotoxicity of propofol focus on the apoptosis of nerve cells, but some experiments show that the amount of apoptosis caused by propofol accounts for only 1% - 2% of the neural cortex, while 50% of the synapses of hippocampal neurons are lost immediately after anesthesia exposure, and the number of synapses continue to decrease by 10% in the following three months. ^{Reference Briner, Nikonenko, De Roo, Dayer, Muller and Vutskits57} The occurrence of cognition and consciousness depends on the neuronal circuitries formed between the synapses of nerve cells, and synapse creation coincides with the peak time of brain development. At this time, exposure to general anesthetics can cause serious and persistent ultrastructural abnormalities of the neuropil. ^{Reference Jevtovic-Todorovic58} Xu et al. confirmed that propofol affects the formation of synapses during development through the mTOR pathway and affects cognitive function. ^{Reference Xu, Mathena and Xu59} In the process of neural development, the growth of neurons is determined by the morphology of their growth cones and axonal transport. Propofol collapses the growth cones of neurons in new-born mice by activating RhoA and affects the axonal transport of the neurotrophic factor brain-derived neurotrophic factor (BDNF), resulting in the impairment of cognitive function in mice. ^{Reference Pearn, Schilling and Jian60} Milanovic et al. found that exposure to propofol can cause damage to neural synaptic function and structure in the developing brain and can cause long-term cognitive function changes by interfering with brain plasticity changes. ^{Reference Milanovic, Pesic and Loncarevic-Vasiljkovic61} The fine structure and functional integrity of nerve cells as affected by propofol are worth exploring in depth.

The effect of propofol on neural stem cells

Neural stem cells mainly exist in the subventricular area of the lateral ventricle of the brain and the subgranular area of the dentate gyrus of the hippocampus. Neural stem cells are key to neural development because they are numerous in the developing brain and have the ability to proliferate, differentiate and migrate. ^{Reference Dennis, Suh, Rodriguez, Kril and Sutherland62,Reference Piermartiri, Dos Santos, Barros-Aragão, Prediger and Tasca63} Human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells were exposed to 20 μM, 50 μM, 100 μM and 300 μM propofol for 6 or 24 h, and 20 μM or 50 μM propofol treatment for 6 h had no significant effect on neural progenitor cells, but when the concentration of propofol was 100 μM and 300 μM, the survival rate of neural progenitor cells decreased significantly. When treated with 50 μM propofol for 24 h, the neural progenitor cells also had a significantly decreased survival rate, which indicates that long-term or high concentration propofol exposure will induce apoptosis of neural progenitor cells. ^{Reference Long, Li, Xue, Sun, Kim and Ying64} Jiang et al. isolated neural stem cells from the rat embryonic hippocampus and exposed them to 112 μM propofol for 6 h, which weakened their proliferation and migration. In addition, neuronal marker β-Tubulin III protein expression decreased, indicating that propofol inhibits the neurogenesis of neural stem cells. ^{Reference Jiang, Wang and Shi65} Propofol not only reduces the survival rate of neural stem cells and causes their apoptosis but also inhibits the proliferation of neural stem cells. A mechanistic study confirmed that propofol inhibits the proliferation of neural stem cells through the Ca2+-PKCα-ERK1/2 signal pathway. ^{Reference Hu, Huang and Zhao66}

Neural stem cells can differentiate into a variety of neural cells, including neurons, astrocytes and oligodendrocytes. Studies have clarified that the excessive production of glial cells is the pathophysiological basis of many neurological diseases. The excessive production of astrocytes leads to synaptic dysfunction and abnormal cerebrovascular function during neural development, resulting in impaired learning and memory and behavioral disorders. ^{Reference Bailey, Hou and Song67,Reference Allen, Oberdorster and Morris-Schaffer68} The use of propofol during brain development can make neural stem cells more inclined to differentiate into astrocytes, reduce the differentiation of neurons and hinder the neurogenesis of neural stem cells. ^{Reference Liang, Du, Wang, Cang and Xue69,Reference Liu, Liu and Patterson70} Cao et al. confirmed that when rat neural stem cells were cultured with 50 μM propofol for 6 h, the cell cycle stagnated, and astrocyte differentiation increased. The mechanism was that propofol interfered with neural stem cell differentiation through the microRNA-124-3p–specificity protein 1–cyclin-dependent kinase inhibitor 1B signaling pathway. ^{Reference Cao, Li, Zeng, Liu and Qin71} The above studies show that propofol induces apoptosis and impairs proliferation, differentiation and migration of neural stem cells to cause neurotoxicity, which may explain much of the propofol-induced learning and memory impairment in children. These results suggest that propofol should be used more cautiously in pregnant women and early new-borns.