Sepsis-associated encephalopathy is one of the most common complications of sepsis and is associated with increased mortality and poor prognosis in patients with sepsis; early diagnosis and treatment are of clinical relevance. The pathogenesis is multifactorial and involves neuroinflammation, destruction of the blood-brain barrier, and changes in the neurotransmitter levels [16]. NAC is an anti-inflammatory and antioxidant substance that plays an essential role in neurological diseases. This study aimed to investigate the effects of NAC on sepsis-associated encephalopathy-related pathological features in a rat model and detect early pathological changes in the brain using various MRI modalities. NAC treatment resulted in significant protective effects against sepsis-associated encephalopathy pathologies in the rats, while the brain-related changes were correlatively detected using DKI and Glu CEST.
Patients with sepsis-associated encephalopathy are known to have anxiety and depression, which are related to cognitive impairment due to damages in the hippocampus region of the brain [17]. In the OFT, we observed that rats injected with LPS were more active at the periphery of the field and had more obvious anxiety and depression; NAC treatment alleviated these symptoms. In such cases, NAC may normalise the release of neurotransmitters from the Glu nerve endings, restoring metabolism to achieve the antioxidant and antidepressant effects [18]. Previous studies have also suggested that NAC may act as an antidepressant by regulating the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor, metabolic Glu receptor, and Glu exchanger of the astrocytes [19, 20]. Furthermore, the antidepressant effect of chronic NAC treatment may be related to the loss of average volume in the hippocampal CA1, dentate gyrus, and hippocampal phylum subregion. NAC has also been shown to restore the decline of hippocampal monoamine levels and enhance metabolism in the hippocampus [21, 22].
During sepsis-associated encephalopathy, Glu metabolism-related changes occurred early in the disease (within 24 hours); Glu-related excitatory toxicity occurs when Glu is over-released with insufficient synaptic clearance and may aggravate injury [23]. In the early stages of sepsis- associated encephalopathy, detecting early abnormalities is challenging using conventional imaging modalities such as MRI. The combined use of emerging modalities (DKI and Glu CEST) may help in the successful detection and monitoring of sepsis-associated encephalopathy-related features in the early stage. The results of this study demonstrated the utility of Glu CEST in detecting the pathologies before and after NAC treatment. In addition, our results also showed that after NAC treatment, glutathione levels were increased. Glutathione possibly combines with free radicals and heavy metals to transform harmful toxins into harmless substances and excretes them out of the body [24]. Glutathione is regulated by excitatory amino acid carrier 1 on the cell membrane, which encourages extracellular Glu and cysteine to enter the cell for glutathione synthesis, thereby reducing the excitatory toxicity of Glu and improving the antioxidant level in cells [25, 26]. The primary physiological function of glutathione is to scavenge free radicals in the human body. As an essential antioxidant, glutathione protects the sulfhydryl groups in many proteins and enzymes [27]. NAC might be involved in the molecular synthesis and regulation of glutathione during inflammation, thus improving the antioxidant levels in cells, alleviating cytotoxic damage, and reducing brain injury in sepsis-associated encephalopathy [28, 29].
Glu is converted into glutamine in the astrocytes and recycled into neurons to synthesise Glu and maintain synaptic transmission. Hence, Glu levels may be closely related to the astrocyte function [30]. Glu levels are associated with dysfunction in the astrocytes, which release transmitters such as ATP and adenosine that regulate synaptic transmission and plasticity. Continuous synaptic transmission is regulated by the astrocytes’ neurotransmitter uptake transporters that remove Glu from the synaptic cleft [31]. Astrocytes are also part of the blood-brain barrier and maintain the homeostasis of the CNS. It has been reported that the LPS stimulation causes extensive structural changes in the astrocytes, such as the structural remodelling and loss of foot processes, resulting in blood-brain barrier destruction [32]. Higher glutathione levels in the astrocytes can improve the blood-brain barrier function by maintaining tight junction protein in the brain tissue and inhibiting injury-induced tight junction phosphorylation[33]. Our GFAP immunohistochemistry and Nissl staining results showed that NAC reduced the sepsis-associated encephalopathy-related nervous tissue damages, which may be related to alleviating the structural damage of the astrocytes. The changes in blood-brain barrier permeability and the regulation of inflammatory factors by sepsis-associated encephalopathy may be related to the foot process swelling and astrocyte dysfunction, which may also affect the release of neurotransmitters (Glu). Our EB results indicate that the sepsis- associated encephalopathy group had the most serious impairment of the blood-brain barrier permeability, while NAC alleviated this damage. We speculate that the salvage effects of NAC on the astrocytes may be the possible mechanism behind this observation as astrocytes are important components of the blood-brain barrier.
The parameters of DTI are related to the demyelination changes in the brain under physiological or pathological conditions, while the parameters of DKI are more complex. DKI technology is an extension of DTI, and DKI is suitable for grasping functional tissue microstructural changes under non-normal distribution [34, 35]. Recent studies using electron microscopy have detected less myelination in the subcortical white matter and axons of the hippocampus region during sepsis, explaining the reduced myelin formation in sepsis-associated encephalopathy as detected by DTI [36]. Moreover, the myelin sheath in the prefrontal cortex and hippocampal axons is reduced during sepsis, possibly as the differentiation/maturation of the microglia is affected and the expression of transcription factors Olig1 and Olig2 is inhibited [37]. Our results suggest damages in the brain microstructures of animals in the sepsis-associated encephalopathy group, which may be related to the pathophysiological mechanism. Furthermore, these results also suggest that NAC treatment had a protective effect on the animals from the deleterious effects of sepsis-associated encephalopathy. The most critical parameter in DKI is MK, which is independent of the spatial orientation of the organisation structure. A larger MK value indicates a more complex structure with a more pronounced diffusion limit of abnormal distribution of water molecules. Therefore, we employed DKI to investigate early signals related to sepsis-associated encephalopathy and found that MK in the cortical region was significantly higher in the sepsis-associated encephalopathy group than that in the NAC and control groups. Previous studies have also confirmed that the pathophysiological mechanism of MK may be related to the proliferation of reactive astrocytes after brain injury [38]. In addition, previous studies have also shown that TNF-α blockers can inhibit demyelinating changes in the CNS. In this study, we also found a significant correlation between DKI and TNF-α levels that not only indicates that NAC may improve neuroinflammatory progression by inhibiting TNF-α levels but also that DKI has excellent potential to detect sepsis-associated encephalopathy-related early inflammatory markers.
Life-threatening organ dysfunction caused by the abnormal immune response is often a fundamental cause of the onset and development of sepsis. NAC can inhibit sepsis-associated encephalopathy-related inflammation and enhance the antioxidant levels. In the beginning of sepsis-associated encephalopathy, associated inflammatory signals can reach different brain areas by stimulating peripheral nerves and blood circulation. Astrocytes play a crucial role in driving inflammatory brain injury by monitoring and integrating inflammatory signals and coordinating the role of immune cells in the CNS [39–42]. Activation of astrocytes in the brain tissues can be detected in the early stage of sepsis. The activated astrocytes can release various inflammatory mediators (e.g., TNF-α, IL-1β, IL-6, IL-18) and enhance the expression of NFκB, promoting the inflammatory process [43]. Excessive production of inflammatory cytokines such as TNF-α leads to further worsening of neuroinflammation and massive loss of the brain cells. TNF-α appears to be a key mediator of sepsis-associated encephalopathy, directly associated with blood-brain barrier disruption, brain oedema, neutrophil infiltration, astrocyte proliferation, and brain cell apoptosis. TNF-α is a mononuclear factor mainly produced by the monocytes and macrophages [44]. The level of TNF-α is positively correlated with the severity of inflammation, and its gene expression is regulated by NFκB [45]. NFκB is a protein complex that controls transcribed DNA, cytokine production, and cell survival. It plays a crucial role in regulating the immune response to infection and is identified as a vital mediator of blood-brain barrier in sepsis-associated encephalopathy. The NF-κB family consists of five members: RelA (p65), RelB, c-Rel, NF-κB1 (p50/p105), and NF-κB2 (p52/p100). These members form isomers or heterodimers that play different roles in regulating the expression of inflammatory genes, cell survival, and neuron differentiation in the CNS [46]. Bacterial LPS activates NF-kB by binding to the toll-like receptor 4 receptor on the surface of the cell membrane and by degrading NF-kB inhibitor family [47]. The classical pathway starts with the activation of NFκB1(p50/p105), which then enters the nucleus, binds to the DNA, and promotes transcription of NFκB dependent genes such as NLRP3, Pro-IL-1β, and Pro-IL-18, subsequently triggering the inflammasome and causing cellular damage such as the mitochondrial damage and oxidative stress [48, 49]. However, this phenomenon was not observed in mice lacking TNF-receptor 1 gene [50].
This study is limited by the fact that clinical conditions are often complex, and many patients have other underlying diseases. Therefore, it is difficult for ordinary animal models to simulate the symptoms seen in different patients accurately. However, it can provide a basic background for further clinical studies.