In the present study, we demonstrate that the exogenous NTN-1, an axonal guidance molecule, improve the postoperative of POD-like behavior in aged mice by its anti-inflammatory and BBB-protecting effect. Our results indicate that pretreatment with NTN-1 given through the caudal vein alleviates systemic inflammatory response and protects BBB integrity after surgery/anesthesia. In addition, the exogenous NTN-1limits neuroinflammation both in the hippocampus and prefrontal cortex, according to the expression of inflammatory cytokines and reactive states of microglia in these brain regions. As far as we know,this is the first report about the neuroprotective effect of NTN-1 in mice model of POD.
A large amount of evidence indicated that neuroinflammation plays an important role in POD. Peripheral aseptic inflammation activates innate immune system, which starts the inflammatory process and eventually lead to POD.[7, 15, 42]. In the aseptic surgery setting, cell trauma releases damage associated molecular patterns (DAMPs) that bind to Toll-like receptors (TLRs) via high mobility group box-1 (HMGB1) to activate BMDMs, which then upregulating the expression of pro-inflammatory cytokines such as TNF-α, IL-1 and IL-6[43, 44]. These cytokines can cause further activation of DAMPs in positive feedback [45, 46]and be released into the circulation and disrupt the integrity of the blood brain barrier (BBB)[42, 47]. Our results show that NTN-1 attenuates the systemic release of proinflammatory factor IL-6 and increases of anti-inflammation cytokine IL-10 after surgery, which are the vital cytokines after trauma. At the same time, NTN-1 reduced the release of HMGB-1, which is passively released from cells damaged by aseptic trauma and targets circulating BM-DMs. These findings are consistent with the potent anti-inflammatory activity of NTN-1 in many other disease models that associated with inflammation such as renal ischemia reperfusion injury, acute peritonitis, acute pancreatitis. The migration and aggregation of white blood cells to the inflammatory site is the central link of the whole inflammatory response. Early studies found that NTN-1 interacts with the UNC-5B receptor expressed on the surface of white blood cells and inhibits the migration of white blood cells. In Alzheimer's disease (AD) rats, it has been demonstrated that NTN-1 concentrations in the serum were positively correlated with the systemic expression of IL-10, one of the most important mediators in the anti-inflammatory activity. What is more, in acute peritonitis and acute colitis models, NTN-1 inhibits the migration of inflammatory cells and induces the M2 polarization phenotype of macrophages[53, 54]. This further indicates that the changes of peripheral inflammatory factors may be related to powerful anti-inflammatory effect of NTN-1.
An intact functioning blood–brain barrier (BBB) is fundamental to proper homoeostatic maintenance and perfusion of the CNS. Inflammatory damage to the unique microvascular endothelial cell monolayer that constitutes the luminal BBB surface, leading to elevated capillary permeability, has been linked to various neurological disorders ranging from ischaemic stroke and traumatic brain injury, to neurodegenerative disease and CNS infections. Moreover, the neuroinflammatory cascade that typically accompanies BBB failure in these circumstances has been strongly linked to elevated levels of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). In models of subarachnoid hemorrhage, multiple sclerosis  and stroke, NTN-1 has been shown to have a protective effect on BBB and improve neurocognitive function, which was also noted in our model. There is compelling evidence that exogenous NTN-1 significantly diminished the diffusion of dextran across mouse brain-derived endothelial cells in vitro. The barrier tightening induced by NTN-1, at least in part, is the consequence of netrin-induced tight junction molecule upregulation. It has been reported that levels of both transmembrane and intracellular components of the junctional complex increased in response to NTN-1. In addition, treatment of human brain-derived endothelial cells with NTN-1 enriched junctional proteins in lipid raft membrane microdomains, where proteins effectively interact to form functional clusters that support barrier integrity. Thus, NTN-1 reduces the incidence of POD by reducing the entry of peripheral inflammatory cytokines through impaired flow barriers.
In addition to mitigate peripheral inflammatory response, NTN-1 reduces the activation of glia cells and the expression of inflammatory cytokines in the hippocampus and prefrontal cortex. Microglia are crucially important during development involved in the phagocytosis of neural precursor cells. Under non-injurious conditions, microglia subserve important functions involved in surveillance of brain parenchyma in order to maintain homeostasis. Following release of pro-inflammatory cytokines by the innate immune response, microglia are activated by one or more pathways. Activated microglia rapidly switch to a proinflammatory phenotype with stout morphology, and enhance the production of proinflammatory molecules . These pro-inflammatory cytokines and the debris released by activated microglia can convert astrocytes into a neurotoxic A1 reactive subtype[60, 61], which cause astrocytes to lose their normal synaptic maintenance and phagocytosis along with induce rapid death of neurons and oligodendrocytes[60, 61]. In our model of POD, NTN-1 reverts the morphological changes of microglia both in the hippocampus and prefrontal cortex to their original forms, representing the transformation of the inflammatory phenotype to the resting state, thereby changing the pro-inflammatory environment by regulating the secretion of inflammatory cytokines. Herein, it is reasonable that pre-treatment with NTN-1 facilitates the improve of POD-like behavior in aged mice because hippocampus and prefrontal cortex which are in charge of shaping emotion, learning and organizing memory [62, 63].`
In addition, the regulation of lipid mediators by neuronal circuits might be an important part in the control of inflammation to sterile injury. The vagus nerve regulates the expression of the axonal guidance molecule NTN-1 can increases SPM production in vivo during acute-self limited inflammation, were this protein upregulates exudate RvD5 and PD1 concentrations[17, 64]. Our previous research has verified the anti - inflammatory and proresolving activities of PD1 in the inflammatory milieu both in vivo and in vitro and identified the role of PD1 in regulating postoperative inflammation and ensuing POD-like behavior of mice. In our study, compared with the control group, the concentration of the endogenous NTN-1 in hippocampus and prefrontal cortex at 6 hours postoperatively significantly reduced. This is most likely the result of the endogenous NTN-1 being consumed after participating in pro-resolution of inflammation by regulating SPM. So, the neuroprotective effect of Netrin-1 may be related to this mechanism. What is more, NTN-1 is involved in regulating inflammatory signaling pathways and inhibiting the production of pro-inflammatory cytokines. In previous studies, it has been observed that NTN-1 can promote the production of cAMP in immune cells and activate the cAMP/ protein kinase A (PKA) signaling pathway to inhibit the production of pro-inflammatory cytokines[65, 66]. Ranganathan found that inhibits the ischemia-reperfusion(I/R) induced acute kidney injury (AKI) NTN-1 model of renal tubular epithelial cells, polymorphonuclear neutrophils(PMN) and mononuclear cells in an enzyme called cyclooxygenase 2 (cox-2) expression. NTN-1 May inhibit the NF-κB activation lowered cox-2 expression, thus reduce the inflammatory response. Does NTN-1 also inhibit the production of pro-inflammatory mediators by other means? It is thus essential to explore the underlying mechanism of NTN-1 on inflammation in further investigation.
There are several limitations to our research. First of all, there are a number of signaling pathways that have been shown to be involved in anti-inflammatory and vascular endothelial cell protection. An in-depth study of the mechanism of NTN-1that we need to search will open up a new way for the prevention and treatment of inflammation-related lesions. Secondly, we have only demonstrated that exogenous prophylactic NTN-1 can improve POD by providing positive anti-inflammatory responses and protective BBB functions after surgery in elderly mice. However, how endogenous NTN-1 changes during this process has not been studied, NTN-1 small interfering RNA (siRNA) can be used in later study.