Previous studies on the neuroprotective effects of ISO focused mainly on cerebral ischemia-reperfusion models. Whether ISO function in neuroinflammation caused by systemic inflammation that may be induced by clinical surgery remains unexplored. Considering the critical role of neuroinflammation in the pathogenesis of ischemic brain injury [36], we investigated the effects of ISO on neuroinflammation motivated by systemic inflammation in LPS-modelled mice. As a common inhalation anesthetic used in clinical anesthesia, ISO has both neurotoxic and neuroprotective properties based on exposure concentration, time, and brain age[31]. Studies have shown that exposure to ISO in adult mice differs from that in young or old mice [4]. ISO has been shown to diminish cell injury and death at clinical concentrations in the cerebral ischemia-reperfusion model [58]. Multiple Studies confirmed that ISO treatment has a neuroprotective effect [25, 30, 59, 60]. Our study shows that ISO remarkably alleviates the neuroinflammation incurred by LPS-induced systemic inflammation, BBB damage, microglia activation, and peripheral immune cell infiltration. In addition, ISO regulates microglial polarization through JAK2/STAT3/IRF8 signaling pathway to a certain extent.
As an essential feature of various brain diseases, neuroinflammation is involved in multiple CNS diseases [55]. Systemic inflammation produced by surgical trauma disrupts the BBB and triggers neuroinflammation [5]. LPS brings about systemic inflammation through various pathways, and the choice of LPS intraperitoneal injection is a more clinical approach [12, 61]. Therefore, in this paper, we chose the method of intraperitoneal injection of LPS to simulate neuroinflammation. Inflammatory processes act on synapses in the hippocampus, giving rise to the loss of synapses and impairment of learning and cognitive memory[50, 52]. Our results demonstrated that LPS-induced spatial memory impairment in mice was effectively improved by ISO posttreatment, and ISO alone had no disadvantageous effect. Excessive microglial cell overactivation lead to neuronal damage, triggering detrimental effects such as exacerbating neurodegenerative diseases[47, 62, 63]. The data verify that LPS-induced neuroinflammation activates severe neuronal death, markedly reversed by ISO treatment. NO produced when the CNS is affected by inflammation may exacerbate neuronal apoptosis, reflecting the degree of inflammation to some extent [49]. We found that ISO treatment retard LPS-induced NO production in the brain. This suggested that ISO post-treatment had neuroprotective effects, and we subsequently explored whether ISO played a protective role by attenuating neuroinflammation. At the same time, LPS-induced BV2 microglia cells were used to construct a cell model of neuroinflammation in vitro [64]. Pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, play an essential role in neuroinflammation as regulatory factors mediating inflammatory diseases[65, 66]. Both in vivo and in vitro experiments indicate that LPS substantially up-regulated the expression of pro-inflammatory factors in the system. Concurrently, ISO treatment inversed the up-regulated tendency, suggesting that ISO could decrease the degree of neuroinflammation.
Neuroinflammation is characterized by the activation of microglial cells and the expression of proinflammatory mediators in the CNS, in which microglial cells play a crucial role [61, 64]. Microglia are an inactivated phenotype in the healthy brain and are involved in maintaining homeostasis [67]. Peripheral LPS stimulates the innate immune system, releases cytokines and chemokines through various pathways, and ultimately mediates the activation of microglia cells. Multiple phenotypes exist simultaneously depending on the type of stimulus in the environment [53, 68]. In vitro experiments demonstrated that ISO dwindled the number of BV2 microglia activated by LPS. Activated microglia are divided into M1 pro-inflammatory type and M2 anti-inflammatory type by specific surface markers, in which the M1 type releases pro-inflammatory factors, aggravating inflammation and impairment. M2 releases neuroprotective factors that promote injury repair and functional improvement [13, 22, 69]. Past research showed that extensive inhibition of microglia is harmful activity, and activating the transformation of the M1 type into the M2 type is favorable for neurological diseases [15, 70]. That is, the protective effect of microglia cells is realized by altering their phenotype [16, 17]. Previous studies have shown that ISO treatment changes the polarization state of microglia cells in cerebral ischemia-reperfusion models [27, 32, 33, 71]. This is similar to our results. LPS treatment trigger microglia activation in both in vivo and in vitro. Compared with the control group, M1-type surface marker CD86, iNOS vastly increased, and pro-inflammatory factors and chemokines IL-1β, TNF-α, and CCL2 were released. While ISO post-treatment significantly lessens M1 markers and grows M2 surface markers CD206 and Arg-1, producing neuroprotective factors such as IL-10 and TGF-β. In other words, ISO decreased M1-type microglia and augmented M2-type microglia under LPS modeling. This suggests that ISO may mitigate neuroinflammation by regulating microglia polarization.
The BBB is fundamental support for maintaining the function and stability of the CNS. Its integrity ensures that the CNS is protected from invasion by toxins, pathogens, and immune cells [72]. Peripheral administration of LPS rarely crosses the BBB directly but binds to corresponding receptors on the outside [4]. Coupled with LPS-induced systemic inflammation, endothelial cell inflammation and consequent BBB inflammation occur. Besides, pro-inflammatory cytokines in peripheral blood directly disturb the BBB and alter its function, allowing immune cells and pro-inflammatory molecules to enter the brain [50, 61]. This is one of the hallmarks of neuroinflammation -- recruitment of immune cells [10]. Following, the above stimulation caused microglia activation, and the synthesis of NO and other factors further destroyed the integrity of the BBB [19]. In addition, the BBB increases permeability due to pro-inflammatory factors, resulting in cerebral edema [37]. Similar to our results, LPS treatment aggrandized cerebral water content, and ISO post-treatment alleviated this symptom. Flow cytometry was used to determine the proportion of immune cells in the brains of mice given various treatments. The data indicated that the number of peripheral immune cells and microglia in the brain tissue treated with LPS significantly differed from that treated with ISO and minocycline. This suggests that LPS-induced systemic inflammation greatly disrupts the BBB, leading to the entry of peripheral immune cells into the CNS. ISO treatment protects the BBB and dwindles its injury, thereby slowing the entry of peripheral immune cells into the brain and preventing microglia activation. Moreover, we examined indicators affecting BBB integrity, such as tight junction proteins and adhesion factors, which showed that ISO decreased the expression of BBB-destroying factors such as VCAM and MMP9. In this research, we used microglia inhibitor and the neuroinflammatory treatment drug minocycline as the anti-inflammatory standard; the trend of ISO was similar to minocycline. These results confirmed that ISO plays a protective role in LPS-induced systemic inflammation by protecting BBB and lowering the degree of CNS stimulation and neuroinflammation.
Afterward, we attempted to explore the signal transduction mechanism of ISO regulating microglia polarization. Previous papers have found that the JAK2/STAT3 pathway is vital in baffling microglia polarization [10, 17]. Therefore, we preliminarily proposed the hypothesis that the JAK2/STAT3 pathway may be involved in the ISO regulation of microglia polarization. Both in vivo and in vitro experiments demonstrated that LPS modeling activated up-regulation of JAK2/STAT3 expression in microglia cells; Consistent with the JAK/STAT inhibitor AG490, ISO reverses the up-regulation of JAK2/STAT3. Studies have shown that the JAK2/STAT3 pathway is a positive regulator of neuroinflammation and plays an essential role in regulating the phenotypic polarization of microglia cells [73, 74]. Activation of STAT3 promoted M1-type polarization of microglia [75]. This is similar to our outcomes. Besides, studies demonstrated that the transcription factor IRF8 controls the reaction process of microglia, which is upregulated after nerve injury and activates an expression program that determines the signaling of reactive microglia [76]. Our study verifies that IRF8 acts on the JAK2/STAT3 pathway and administers the polarization state of microglia cells. This is consistent with previous evidence that IRF8 is essential in the morphology and typing of microglial cells [77]. Based on the above results, we speculated that ISO might directly regulate microglia polarization through the JAK2/STAT3/IRF8 signaling pathway and retard neuroinflammation.
Our study suggests that ISO plays a neuroprotective role by controlling BBB damage and regulating microglia polarization to alleviate the degree of neuroinflammation. The limitation of this study is that the purpose of the paper was to investigate the role of ISO in neuroinflammation and the BBB caused by systemic inflammation that may occur during clinical surgery. The long-term effects of isoflurane require further investigation. Meanwhile, we looked into some potential signaling pathways. We only investigated the JAK2/STAT3/IRF8 axis in which ISO attenuates M1 polarization expression, while the M2 polarization up-regulation pathway didn’t explore and needed further study.