Netrin-1 Ameliorates Postoperative Delirium-Like Behavior in Aged Mice by Suppressing Neuroinflammation and Restoring Impaired Blood-Brain Barrier Permeability

Postoperative delirium (POD) is a common and serious postoperative complication in elderly patients, and its underlying mechanism is elusive and without effective therapy at present. In recent years, the neuroinflammatory hypothesis has been developed in the pathogenesis of POD, in which the damaged blood-brain barrier (BBB) plays an important role. Netrin-1 (NTN-1), an axonal guidance molecule, has been reported to have strong inflammatory regulatory and neuroprotective effects. We applied NTN-1 (45 μg/kg) to aged mice using a POD model with a simple laparotomy to assess their systemic inflammation and neuroinflammation by detecting interleukin-6 (IL-6), interleukin-10 (IL-10), and high mobility group box chromosomal protein-1 (HMGB-1) levels. We also assessed the reactive states of microglia and the permeability of the BBB by detecting cell junction proteins and the leakage of dextran. We found that a single dose of NTN-1 prophylaxis decreased the expression of IL-6 and HMGB-1 and upregulated the expression of IL-10 in the peripheral blood, hippocampus, and prefrontal cortex. Nerin-1 reduced the activation of microglial cells in the hippocampus and prefrontal cortex and improved POD-like behavior. NTN-1 also attenuated the anesthesia/surgery-induced increase in BBB permeability by upregulating the expression of tight junction-associated proteins such as ZO-1, claudin-5, and occludin. These findings confirm the anti-inflammatory and BBB protective effects of NTN-1 in an inflammatory environment in vivo and provide better insights into the pathophysiology and potential treatment of POD.

Microglia and infiltrating T-cells adopt long-term, age-specific, transcriptional changes after traumatic brain injury

Introduction: Traumatic brain injury (TBI) afflicts over 3 million Americans every year. Patients over 65 years of age suffer increased mortality as well as greater long-term neurocognitive and neuropsychiatric morbidity compared to younger adults. Microglia, the resident macrophages of the brain, are complicit in both. Our published and preliminary data have demonstrated a significant age-effect in which aged microglia are more prone to adopt a constitutively activated state associated with worse neurocognitive and neuropsychiatric outcomes. Therefore, we hypothesized that aged microglia would fail to return to a homeostatic state after TBI but instead adopt a long-term injury-associated state within the brain of aged mice as compared to young-adult mice after TBI. Methods: Young-adult (14-weeks) and aged (80-weeks) C57BL/6 mice underwent TBI via controlled cortical impact vs. sham injury. We utilized single-cell RNA sequencing to examine age-associated cellular responses after TBI. Four months post-TBI or sham injury, brains were harvested, and CD45+ cells (N=4,000 cells) were isolated via florescence-activated cell sorting. cDNA libraries were prepared via the 10x Genomics Chromium Single Cell 3' Reagent Kit, followed by sequencing on a HiSeq 4000 instrument. The raw data were processed using the Cell Ranger pipeline mapped to the mm10 mouse reference genome and Seurat following standard workflow. Seurat and GOrilla were used for downstream clustering, differential gene expression, and pathway analysis. All cell types were annotated using canonical markers and top expressed genes. ProjecTILs was additionally used to interpret T cell states. Results: Microglia from young-adult and aged mice have distinct transcriptional profiles pre-injury and markedly different transcriptional responses post-injury compared to young-adult mice. Pre-injury, aged mice demonstrated a disproportionate immune cell infiltration, including T cells, as compared to young-adult mice (aged versus young: 45.5% vs. 14.5%). Post-injury, the disparity was amplified with a proportional decrease in homeostatic microglia and greater increased infiltrating T cells compared to young-adult mice (Microglia: 27.5% vs. 71%; T cell: 45.5% vs. 4.5%). Of note, aged mice post-injury had a subpopulation of unique, age-specific, immune-inflammatory microglia resembling gene profiles of neurodegenerative disease-associated microglia (DAM) with enriched pathways involved in leukocyte recruitment and Alzheimer’s disease pathogenesis (FDR < 0.05). Contrastingly, post-injury, aged mice demonstrate a heterogenous T-cell infiltration with gene profiles corresponding to CD8 effector memory, CD8 native-like, CD4, and double-negative T cells (75.9%, 2.5%, 12.9%, and 8.6%, respectively) and enriched pathways including tau protein binding, macromolecule synthesis, and cytokine-mediated signaling pathways (FDR < 0.05). Conclusion: We hypothesized that aged microglia would fail to return to a homeostatic state after TBI and adopt a long-term, injury-associated state within the brain of aged mice as compared to young-adult mice after TBI. In particular, our data suggest an age-dependent reduction of homeostatic microglia post-injury yet an upregulation in a unique microglial subpopulation with a distinct immuno-inflammatory profile. Furthermore, aged subjects demonstrated a markedly disproportionate inflammatory infiltrate after TBI predominated by the presence of CD8+ T cells. In addition, post-injury, brain trauma reorganized the T cell milieu, especially CD8 effector memory T cells, via upregulating genes associated with macromolecule biosynthesis process and negative regulation of neuronal death, possibly linking TBI with its long-term sequelae and complications. Taken together, our data showed that age-specific gene signature changes in the T-cell infiltrates and the microglial subpopulation contributes to increased vulnerability of the aged brain to TBI. Age should be an a priori consideration in future TBI clinical trials.

Centenarian-Sourced Lactobacillus casei Combined with Dietary Fiber Complex Ameliorates Brain and Gut Function in Aged Mice

Dietary intervention could modulate age-related neurological disorders via the gut–brain axis. The potential roles of a probiotic and the dietary fiber complex (DFC) on brain and gut function in aged mice were investigated in this study. Lactobacillus casei LTL1361 and DFC were orally administrated for 12 weeks, and the learning and memory ability, as well as the oxidative parameters, inflammatory markers, gut barrier function and microbial metabolite short-chain fatty acids (SCFAs), were investigated. LTL1361 and DFC supplementation ameliorated cognitive ability, attenuated oxidative stress in brain and inflammation in serum and colon, ameliorated gut barrier function, and increased the SCFA concentrations and gene expression of SCFA receptors. The protective effect was more significantly enhanced in aged mice treated with the combination of LTL1361 and DFC than treated with LTL1361 or DFC alone. These results could be associated with the protected morphology of pyramidal nerve cells in hippocampus of mice brain and the downregulation of apoptosis marker caspase-3 in brain and upregulation of tight junction proteins in small intestine and colon. The results indicated that Lactobacillus casei LTL1361 and DFC alleviated age-related cognitive impairment, as well as protected brain and gut function. Lactobacillus casei LTL1361 and DFC might be used as novel and promising antiaging agents in human.

G-quadruplexes Stabilization Upregulates CCN1 and Accelerates Aging in Cultured Cerebral Endothelial Cells

Senescence in the cerebral endothelium has been proposed as a mechanism that can drive dysfunction of the cerebral vasculature, which precedes vascular dementia. Cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) is a matricellular protein secreted by cerebral endothelial cells (CEC). CCN1 induces senescence in fibroblasts. However, whether CCN1 contributes to senescence in CEC and how this is regulated requires further study. Aging has been associated with the formation of four-stranded Guanine-quadruplexes (G4s) in G-rich motifs of DNA and RNA. Stabilization of the G4 structures regulates transcription and translation either by upregulation or downregulation depending on the gene target. Previously, we showed that aged mice treated with a G4-stabilizing compound had enhanced senescence-associated (SA) phenotypes in their brains, and these mice exhibited enhanced cognitive deficits. A sequence in the 3′-UTR of the human CCN1 mRNA has the ability to fold into G4s in vitro. We hypothesize that G4 stabilization regulates CCN1 in cultured primary CEC and induces endothelial senescence. We used cerebral microvessel fractions and cultured primary CEC from young (4-months old, m/o) and aged (18-m/o) mice to determine CCN1 levels. SA phenotypes were determined by high-resolution fluorescence microscopy in cultured primary CEC, and we used Thioflavin T to recognize RNA-G4s for fluorescence spectra. We found that cultured CEC from aged mice exhibited enhanced levels of SA phenotypes, and higher levels of CCN1 and G4 stabilization. In cultured CEC, CCN1 induced SA phenotypes, such as SA β-galactosidase activity, and double-strand DNA damage. Furthermore, CCN1 levels were upregulated by a G4 ligand, and a G-rich motif in the 3′-UTR of the Ccn1 mRNA was folded into a G4. In conclusion, we demonstrate that CCN1 can induce senescence in cultured primary CEC, and we provide evidence that G4 stabilization is a novel mechanism regulating the SASP component CCN1.

Intervention of NF-Κb Signaling Pathway and Preventing Post-Operative Cognitive Dysfunction as Well as Neuronal Apoptosis

Background: The Postoperative cognitive dysfunction (POCD) model was constructed by resection of the left hepatic lobe in aged mice to determine the behavioral effects of the POCD model in aged mice and the relationship between NF-κB and POCD in apoptosis and autophagy. Provide a theoretical basis for POCD prevention and treatment. Methods: This study was carried out in Ningbo No. 6 Hospital, Zhejiang, China, from Jun 2019 to Dec 2020. The POCD model was constructed after resection of the left extrahepatic lobe in aged mice and randomly divided into 6 groups: sham operation group, operation group (normal saline control group, solvent group, YC-1 group, PDTC group and 3-MA group). Related indicators of behavioral changes, neuronal inflammatory responses, apoptosis, and autophagy were examined. Results: The escape latency of the aged mice in the surgical group was significantly prolonged at three time points compared with the control group, and the number of insertions decreased significantly. Microglia are activated and the inflammatory response is increased, whereas PDTC has an inhibitory effect. It was demonstrated that apoptosis and necrosis of neurons can be induced by the NF-κb pathway, and autophagy can be promoted, whereas autophagy occurs before apoptosis. Conclusion: Activation of NF-κb pathway in neurons after POCD causes neuronal apoptosis and autophagy, and cognitive impairment occurs. PDTC, a NF-κb pathway inhibitor, can effectively reduce neuronal apoptosis induced by secondary brain injury after POCD. Necrosis, to protect the brain tissue.

Age-Related Impairment of Innate and Adaptive Immune Responses Following Intranasal Herpes Simplex Infection

Immune function declines with age, leading to an increased vulnerability to respiratory viral infections. The mechanisms by which aging negatively impacts the innate and adaptive immune system leading to enhanced susceptibility to infections remain to be fully elucidated. In the present study, we used a mouse model of intranasal infection with herpes simplex virus type 1 (HSV-1), a virus that can enter the lungs through the nasal route causing pneumonia, a serious health concern in the elderly. Following intranasal inoculation of young (6 weeks), adult (36 weeks), and aged (68 weeks) with HSV-1 (KOS strain) we: (i) compared the local and systemic innate and adaptive immune response to infection; and (ii) correlated the level and type of immune response to protection against HSV-1 infection. Compared to young and adult mice, aged mice displayed: (i) increased basal level activation of epithelial cells with a decreased expression of TLR3; (ii) increased activation of dendritic cells with increased expression of MHC-1, MHC-II and CD80/86; and (iii) decreased production of type-I interferons upon stimulation; (iv) a delay in cytokines and chemokines production in the lungs; and (v) an impairment in function (CD107 and IFN-g production) of HSV-specific CD8+ T cells. These impairment in innate and adaptive immune responses in aged mice following intranasal HSV-1 inoculation was associated with symptomatic herpes infection. The findings suggest an age-related impairment of both innate and adaptive immune responses which may exacerbate herpes infection and disease in the elderly.