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Advances in spine surgery enable technically safe interventions in older patients with disabling spine disease, yet postoperative delirium (POD) poses a serious risk for postoperative recovery. This study investigates biomarkers of pro-neuroinflammatory states that may help objectively define the pre-operative risk for POD. This study enrolled patients aged ≥60 scheduled for elective spine surgery under general anesthesia. Biomarkers for a pro-neuroinflammatory state included S100 calcium-binding protein β (S100β), brain-derived neurotrophic factor (BDNF), Gasdermin D, and the soluble ectodomain of the triggering receptor expressed on myeloid cells 2 (sTREM2). Postoperative changes of Interleukin-6 (IL-6), Interleukin-1β (IL-1β), and C-reactive protein (CRP) were assessed as markers of systemic inflammation preoperatively, intraoperatively, and early postoperatively (up to 48 h). Patients with POD (n = 19, 75.7 ± 5.8 years) had higher pre-operative levels of sTREM2 (128.2 ± 69.4 pg/mL vs. 97.2 ± 52.0 pg/mL, p = 0.049) and Gasdermin D (2.9 ± 1.6 pg/mL vs. 2.1 ± 1.4 pg/mL, p = 0.29) than those without POD (n = 25, 75.6 ± 5.1 years). STREM2 was additionally a predictor for POD (OR = 1.01/(pg/mL) [1.00–1.03], p = 0.05), moderated by IL-6 (Wald-χ2 = 4.06, p = 0.04). Patients with POD additionally showed a significant increase in IL-6, IL-1β, and S100β levels on the first postoperative day. This study identified higher levels of sTREM2 and Gasdermin D as potential markers of a pro-neuroinflammatory state that predisposes to the development of POD. Future studies should confirm these results in a larger cohort and determine their potential as an objective biomarker to inform delirium prevention strategies.

Background: Post-stroke delirium (PSD) is a modifiable predictor for worse outcome in stroke. Knowledge of its risk factors would facilitate clinical management of affected patients, but recently updated national guidelines consider available evidence insufficient. Aims: The study aimed to establish risk factors for PSD incidence and duration using high-frequency screening. Methods: We prospectively investigated patients with ischemic stroke admitted within 24 h. Patients were screened twice daily for the presence of PSD throughout the treatment period. Sociodemographic, treatment-related, and neuroimaging characteristics were evaluated as predictors of either PSD incidence (odds ratios (OR)) or duration (PSD days/unit of the predictor, b), using logistic and linear regression models, respectively. Results: PSD occurred in 55/141 patients (age = 73.8 ± 10.4 years, 61 female, National Institutes of Health Stroke Scale (NIHSS) = 6.4 ± 6.5). Age (odds ratio (OR) = 1.06 (95% confidence interval (CI): 1.02–1.10), b = 0.08 (95% CI = 0.04–0.13)), and male gender ( b = 0.99 (95% CI = 0.05–1.93)) were significant non-modifiable risk factors. In a multivariable model adjusted for age and gender, presence of pain (OR mvar = 1.75 (95% CI = 1.12–2.74)), urinary catheter (OR mvar = 3.16 (95% CI = 1.10–9.14)) and post-stroke infection (PSI; OR mvar = 4.43 (95% CI = 1.09–18.01)) were predictors of PSD incidence. PSD duration was impacted by presence of pain ( b mvar = 0.49 (95% CI = 0.19–0.81)), urinary catheter ( b mvar = 1.03 (95% CI = 0.01–2.07)), intravenous line ( b mvar = 0.36 (95% CI = 0.16–0.57)), and PSI ( b mvar = 1.60 (95% CI = 0.42–2.78)). PSD (OR = 3.53 (95% CI = 1.48–5.57)) and PSI (OR = 5.29 (95% CI = 2.92–7.66)) independently predicted inferior NIHSS at discharge. Insular and basal ganglia lesions increased the PSD risk about four- to eight-fold. Discussion/Conclusion: This study identified modifiable risk factors, the management of which might reduce the negative impact PSD has on outcome.

Context Fetal zone steroids (FZSs) are excreted in high concentrations in preterm infants. Experimental data suggest protective effects of FZSs in models of neonatal disease. Objective We aimed to characterize the postnatal FZS metabolome of well preterm and term infants. Methods Twenty-four-hour urinary FZS excretion rates were determined in early preterm (37 weeks) infants. Pregnenolone and 17-OH-pregnenolone metabolites (n = 5), and dehydroepiandrosterone sulfate and metabolites (n = 12) were measured by gas chromatography mass spectrometry. Postnatal concentrations of FZSs were compared with already published prenatal concentrations in amniotic fluid. Results Excretion rates of total FZSs and most of the single metabolites were highest in early preterm infants. In this group, excretion rates approach those of term infants at term equivalent postmenstrual age. Preterm infants of 30-36 weeks had more than half lower median excretion rates of FZSs than early preterm infants at the same time of postmenstrual age. Postnatal concentrations of FZSs were partly more than 100-fold higher in all gestational age groups than prenatal concentrations in amniotic fluid at midgestation. Conclusion The excretion rates of FZSs as a proxy of the involution of the fetal zone of the most immature preterm infants approached those of term infants at term equivalent. In contrast, the fetal zone in more mature preterm infants undergoes more rapid involution. These data in exclusively well neonates can serve as a basis to investigate the effects of illness on the FZS metabolome in future studies.

Preterm birth causes neurological deficits. Previously, we demonstrated that fetal zone steroids reduce hyperoxia-mediated cell death in vitro. In immature oligodendrocytes (OLN-93 cells), dehydroepiandrosterone + 17β-estradiol co-treatment had synergistic beneficial effects while signals were transduced through different receptors. In immature astrocytes (C6 cells), both hormones compete for the same receptor and no synergistic effects were observed. 17β-estradiol and progesterone drastically decrease while fetal zone steroids, mainly dehydroepiandrosterone, remain persistently high within preterm infants until term. Substitution of 17β-estradiol and progesterone does not improve neurological outcomes. We investigated the influence of dehydroepiandrosterone, 17β-estradiol or dehydroepiandrosterone + 17β-estradiol treatment in C6 or OLN-93 cells on steroid receptor availability and activation of intracellular signaling molecules in hyperoxic cell culture. We sought explanations of the observed synergistic effect in preliminary study. In C6 cells, the generated signaling of dehydroepiandrosterone + 17β-estradiol treatment has no synergistic effects. The combined effect on this particular pathway does not potentiate cell survival. In OLN-93 cells, we observed significant differences in the early generated signaling of 17β-estradiol + dehydroepiandrosterone treatment to either 17β-estradiol dehydroepiandrosterone alone but never to both at the same time. The latter finding needs, therefore, further investigation to explain synergistic effects. Nevertheless, we add insight into the receptor and signaling cascade alterations induced by 17β-estradiol, dehydroepiandrosterone or 17β-estradiol + dehydroepiandrosterone treatment of C6 and OLN-93 cells in hyperoxia.

The global increase in neurodegenerative disorders is one of the most crucial public health issues. Oral polyamine intake was shown to improve memory performance which is thought to be mediated at least in part via increased autophagy induced in brain cells. In Alzheimer’s Disease, T-cells were identified as important mediators of disease pathology. Since autophagy is a central regulator of cell activation and cytokine production, we investigated the influence of polyamines on T-cell activation, autophagy, and the release of Th1/Th2 cytokines from blood samples of patients (n=22) with cognitive impairment or dementia in comparison to healthy controls (n=12) ex vivo. We found that spermine downregulated all investigated cytokines in a dose-dependent manner. Spermidine led to an upregulation of some cytokines for lower dosages, while high dosages downregulated all cytokines apart from upregulated IL-17A. Autophagy and T-cell activation increased in a dose-dependent manner by incubation with either polyamine. Although effects in patients were seen in lower concentrations, alterations were similar to controls. We provide novel evidence that supplementation of polyamines alters the function of T-cells. Given their important role in dementia, these data indicate a possible mechanism by which polyamines would help to prevent structural and cognitive decline in aging.

Background and purpose Stroke therapy still lacks successful measures to improve post stroke recovery. Neurotrophin-3 (NT-3) is one promising candidate which has proven therapeutic benefit in motor recovery in acute experimental stroke. Post stroke, the immune system has opposing pathophysiological roles: pro-inflammatory cascades and immune cell infiltration into the brain exacerbate cell death while the peripheral immune response has only limited capabilities to fight infections during the acute and subacute phase. With time, anti-inflammatory mechanisms are supposed to support recovery of the ischemic damage within the brain parenchyma. However, interestingly, NT-3 can improve recovery in chronic neurological injury when combined with the pro-inflammatory stimulus lipopolysaccharide (LPS). Aim We elucidated the impact of NT-3 on human monocyte and T cell activation as well as cytokine production ex vivo after stroke. In addition, we investigated the age-dependent availability of the high affinity NT-3 receptor TrkC upon LPS stimulation. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from acute stroke patients and controls and incubated with different dosages of NT-3 (10 and 100 ng/mL) and with or without LPS or anti-CD3/CD28 for 48 h. Total TrkC expression and cell activation (CD25, CD69 and HLA-DR) were assessed by FACS staining. IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21 and IL-22 were quantified by cytometric bead array. Results Most monocytes and only a small proportion of T cells expressed TrkC in blood from humans without stroke. Activation of cells from young humans (without strokes) using anti-CD3/CD28 or LPS partially reduced the proportion of monocytes expressing TrkC whilst they increased the proportion of T cells expressing TrkC. In contrast, activation of cells from elderly humans (without strokes) did not affect the proportion of monocytes expressing TrkC and only anti-CD3/CD28 led to an increase in the proportion of CD4+ T cells expressing TrkC. In blood from stroke patients or controls, NT-3 treatment reduced the percentage of monocytes and CD4+ and CD8+ T cells that were activated and reduced all cytokines investigated besides IL-21. Conclusions NT-3 attenuated immune responses in cells from stroke patients and controls. The mechanism whereby human immune cells respond to NT-3 may be via TrkC receptors whose levels are regulated by stimulation. Further work is required to determine whether the induction of sensorimotor recovery in rodents by NT-3 after CNS injury is caused by this attenuation of the immune response.

Background and Purpose— The contribution of neuroinflammation and, in particular, the infiltration of the brain by lymphocytes is increasingly recognized as a substantial pathophysiological mechanism after stroke. The interaction of lymphocytes with endothelial cells and platelets, termed thromboinflammation, fosters microvascular dysfunction and secondary infarct growth. Siponimod is an S1PR (sphingosine-1-phosphate receptor) modulator, which blocks the egress of lymphocytes from lymphoid organs and has demonstrated beneficial effects in multiple sclerosis treatment. We investigated the effect of treatment with siponimod on stroke outcome in a mouse model of cerebral ischemia. Methods— Transient middle cerebral artery occlusion was induced in middle-aged wild-type mice. Animals were either treated with siponimod (3 mg/kg; intraperitoneal) or vehicle for 6 days. Stroke outcome was assessed by magnetic resonance imaging (spleen volume: prestroke, day 3, and day 7; infarct volume: days 1, 3, and 7) and behavioral tests (prestroke, day 2, and day 6). Immune cells of the peripheral blood and brain-infiltrating cells ipsilateral and contralateral were analyzed by VETScan and by flow cytometry. Results— Siponimod significantly induced lymphopenia on day 7 after transient middle cerebral artery occlusion and reduced T-lymphocyte accumulation in the central nervous system. No effect was detected for lesion size. Conclusions— For siponimod administered at 3 mg/kg in transient middle cerebral artery occlusion mouse model, our findings do not provide preclinical evidence for the use of S1PR1/5 modulators as neuroprotectant in stroke therapy.

Objectives Correct identification of inflammatory etiologies of stroke is of outmost importance as they require treatment of the underlying disease. Aim of this study was to determine the prevalence of inflammatory changes in cerebrospinal fluid (CSF) observed in young cryptogenic stroke patients. Materials and methods Of 6476 records of patients diagnosed with ischemic stroke, 278 had confirmed ischemia in brain imaging and received lumbar puncture. A total of 122 were classified as young stroke (≤55 years), and 156 were classified as older stroke patients; lumbar puncture in this cohort was indicated due to atypical clinical presentation. Results An infectious etiology was detected in 2.5% of young stroke patients (n = 3: vasculitis due to opportunistic infection, vasculitis due to neuroborreliosis, secondary vasospasm after viral meningitis) and in 1.9% (n = 3) in the older stroke cohort (vasculitis due to neurotuberculosis, septic embolic ischemia, vasculitis post-haemophilus influenza meningoencephalitis). Isolated vasculitis was evident in one patient of the older stroke cohort (0.6%). Non-specific alterations in CSF included increased cell count in 10% in young and in 9.3% in the older stroke cohort. Intrathecal Ig synthesis was present in 3.4% of the younger and in 4% of the older stroke cohort. Conclusions The prevalence of an infectious etiology in young stroke is modest but slightly higher in comparison with older stroke patients. As brain imaging is not always sufficient for suspecting vasculitis, we recommend implementation of lumbar puncture in young cryptogenic stroke patients. If an infectious disease is present in ischemic stroke, it is of high therapeutic relevance.

Preterm birth causes neurological deficits. Previously, we demonstrated that fetal zone steroids reduce hyperoxia-mediated cell death

Human donor milk (HDM) provides appropriate nutrition and offers protective functions in preterm infants. The aim of the study is to examine the impact of different storage conditions on the stability of the human breast milk peptidome. HDM was directly frozen at &minus, 80 &deg, C or stored at &minus, 20 &deg, C (120 h), 4 &deg, C (6 h), or room temperature (RT for 6 or 24 h). The milk peptidome was profiled by mass spectrometry after peptide collection by ultrafiltration. Profiling of the peptidome covered 3587 peptides corresponding to 212 proteins. The variance of the peptidome increased with storage temperature and time and varied for different peptides. The highest impact was observed when samples were stored at RT. Smaller but significant effects were still observed in samples stored at 4 &deg, C, while samples showed highest similarity to those immediately frozen at &minus, C when stored at &minus, C. Peptide structures after storage at RT for 24 h point to the increased activity of thrombin and other proteases cleaving proteins at lysine/arginine. The results point to an ongoing protein degradation/peptide production by milk-derived proteases. They underline the need for immediate freezing of HDM at &minus, C or &minus, C to prevent degradation of peptides and enable reproducible investigation of prospectively collected samples.

Background Stroke induces immune alterations such as impaired oxidative burst and reduced release of neutrophil extracellular traps (NETs). We hypothesised that key enzymes of these defence mechanisms may be altered in ischaemic stroke. Therefore, we analysed the intra- and extracellular amounts of myeloperoxidase (MPO) and neutrophil elastase (NE) in patient sera and granulocytes and monocytes. Because the autonomous nervous system is thought to mediate stroke-induced immune alterations, we also studied the influence of stress hormones and acetylcholine on MPO and NE. Rapid recanalization by recombinant tissue plasminogen activator (r-tPA) is the only available treatment for ischaemic stroke besides thrombectomy, and its influence on antibacterial defence mechanisms of granulocytes and monocytes were addressed here. Methods Ex vivo: Intracellular and serum MPO and NE were measured on days 0, 1, 3 and 5 post-stroke by either flow cytometry or enzyme-linked immunosorbent assay (ELISA) and compared to controls. In vitro: Blood from healthy donors was incubated with catecholamines, dexamethasone and acetylcholine, and the percentage of NET-producing cells and the area covered by NETs were quantified immunohistochemically. Intra- and extracellular MPO and NE were quantified by flow cytometry or ELISA. Blood samples from healthy donors were incubated with r-tPA, and oxidative burst, phagocytosis, NETosis, cytokine release, MPO and NE were quantified by flow cytometry, ELISA and microscopy. Results MPO was reduced in granulocytes but increased in sera obtained from stroke patients compared to controls. NE was not altered intracellularly but was elevated in patient sera. The percentage of NET-producing neutrophils was decreased by stress hormones and increased by acetylcholine. Neither intracellular MPO nor NE was altered by hormone treatment; however, adrenaline and acetylcholine induced NE release. r-tPA led to reduced phagocytosis and oxidative burst in granulocytes and monocytes in vitro. NETosis, MPO release and cytokines were not altered, whereas NE release was enhanced by r-tPA. Conclusions Intracellular reduction of MPO might be responsible for reduced NETosis in stroke patients. The impact of enhanced MPO and NE serum levels in stroke patients should be addressed in future studies. r-tPA impaired antibacterial defence function in vitro. Therefore, patients who undergo unsuccessful recanalization therapy might be at higher risk for infection, which should be analysed in future investigations. Immune alterations due to r-tPA effects in stroke patients should also be investigated. Electronic supplementary material The online version of this article (doi:10.1186/s12974-017-0914-6) contains supplementary material, which is available to authorized users.

BackgroundNeonate immune cell functions lack full protection against pathogens. This could be either defect or protective mechanism against overshooting proinflammatory immune responses.We here analysed the function of classical, pro- and anti-inflammatory monocytes and granulocytes from neonates in comparison with adults to investigate if suppressed functions of subpopulations are causative for the unique neonatal immune status. Therefore, reactive oxygen species (ROS) and surface activation markers were quantified in subpopulations.MethodsIn a prospective, longitudinal study granulocyte and monocyte subpopulations were analysed in healthy term infants (> 37 week;n = 13) in comparison with healthy young adults (n = 11). Percentage (%) of cells expressing surface marker (HLA-DR, CD11b, CD62L, CD32, Toll-Like-Receptor-2) and expression per cell, determined by mean fluorescence intensity (MFI), were measured by flow cytometry. ROS production was induced by fMLP, PMA andE. coliin term neonates (> 37 week;n = 13).ResultsClassical granulocytes were down- and proinflammatory granulocytes upregulated in neonates compared with adults. Percentage of TLR-2 expressing granulocytes was increased in neonates. Granulocytic ROS production depended on stimulation. The percentage of anti-inflammatory monocytes was increased, while classical monocytes were reduced in neonates. HLA-DR (%, MFI) showed reduction for all monocyte subpopulations, while CD32, CD11b, CD62L and TLR-2 were differently regulated in comparison with adults.ConclusionsDifferentially regulated granulocyte and monocyte subpopulations indicate a unique state of neonatal immunity to fight infections and prevent dysregulation. Further studies are needed to investigate the role of reduced granulocytic ROS formation and reduced monocytic HLA-DR in active disease.

Background and Purpose. Regulatory T cells (Tregs) have been suggested to modulate stroke-induced immune responses. However, analyses of Tregs in patients and in experimental stroke have yielded contradictory findings. We performed the current study to assess the regulation and function of Tregs in peripheral blood of stroke patients. Age dependent expression of CD39 on Tregs was quantified in mice and men. Methods. Total FoxP3+ Tregs and CD39+FoxP3+ Tregs were quantified by flow cytometry in controls and stroke patients on admission and on days 1, 3, 5, and 7 thereafter. Treg function was assessed by quantifying the inhibition of activation-induced expression of CD69 and CD154 on T effector cells (Teffs). Results. Total Tregs accounted for 5.0% of CD4+ T cells in controls and + Tregs were most strongly reduced in stroke patients. On day 3 the Treg-mediated inhibition of CD154 upregulation on CD4+ Teff was impaired in stroke patients. CD39 expression on Treg increased with age in peripheral blood of mice and men. Conclusion. We demonstrate a loss of active FoxP3+CD39+ Tregs from stroke patient’s peripheral blood. The suppressive Treg function of remaining Tregs is impaired after stroke.

Impaired neurodevelopment in preterm infants is caused by prematurity itself; however, hypoxia/ischemia, inflammation, and hyperoxia contribute to the extent of impairment. Because preterm birth is accompanied by a dramatic decrease in 17β-estradiol (E2) and progesterone, preliminary clinical studies have been carried out to substitute these steroids in preterm infants; however, they failed to confirm significantly improved neurologic outcomes. We therefore hypothesized that the persistently high postnatal production of fetal zone steroids [mainly dehydroepiandrosterone (DHEA)] until term could interfere with E2-mediated protection. We investigated whether E2 could reduce hyperoxia-mediated apoptosis in three immature glial cell types and detected the involved receptors. Thereafter, we investigated protection by the fetal zone steroids DHEA, 16α-hydroxy-DHEA, and androstenediol. For DHEA, the involved receptors were evaluated. We examined aromatases, which convert fetal zone steroids into more estrogenic compounds. Finally, cotreatment was compared against single hormone treatment to investigate synergism. In all cell types, E2 and fetal zone steroids resulted in significant dose-dependent protection, whereas the mediating receptors differed. The neuroprotection by fetal zone steroids highly depended on the cell type-specific expression of aromatases, the receptor repertoire, and the potency of the fetal zone steroids toward these receptors. No synergism in fetal zone steroid and E2 cotreatment was detected in two of three cell types. Therefore, E2 supplementation may not be beneficial with respect to neuroprotection because fetal zone steroids circulate in persistently high concentrations until term in preterm infants. Hence, a refined experimental model for preterm infants is required to investigate potential treatments.

Background: Newborns are prone to infections, which are independent predictors of neonatal mortality and morbidity. Neutrophil extracellular traps (NETs) are structures composed of chromatin and antimicrobial molecules that capture and kill pathogens. NETs may play an important role in the innate immune system and, thus, might be associated with impaired neonatal immune function. Objectives: This study aimed to compare NET formation between term neonates and healthy adults. We additionally investigated the effects of gestational age, birth weight, mode of delivery, gender, and perinatal infections. Methods: We collected cord blood from 57 term infants (mean gestational age, 39.1 weeks) and 9 late preterm infants (35 weeks), and peripheral blood from 18 healthy adult donors. Neutrophils were isolated, and then NET formation was induced using three different stimulants: N-formylmethionine-leucyl-phenylalanine, phorbol 12-myristate 13-acetate (PMA), or lipopolysaccharide. NETs were immunohistochemically stained and analyzed with regard to NET percentage and NET area. Results: With all three stimuli, healthy term infants showed a lower NET percentage than the adult control group (p < 0.0001 each). The groups also differed in NET area, but the significance level was lower. Following PMA stimulation, we observed greater reductions in NET percentage and NET area in preterm than term infants. Conclusions: The lower NET formation observed in term infants compared to adults likely contributes to the reduced neonatal immune response. NET formation appeared to be even further decreased in late preterm neonates. There remains a need for further investigations of NET formation in more immature preterm infants.

Background and Purpose— Stroke-induced immune alterations predispose patients to infections. Although the relationship between stroke and the adaptive immune system has been investigated in detail, to date it is unknown whether the innate immune system, which forms the first line of antibacterial defense, is also impaired in patients with stroke. Therefore, we investigated whether chemotaxis, phagocytosis, oxidative burst, degranulation of defensins, and NETosis in monocytes and in neutrophil granulocytes are altered in patients with stroke compared with controls. Methods— Sixty-three patients having acute ischemic stroke were recruited within 12 hours of symptom onset; blood was sampled on admission and on days 1, 3, 5, and 7. Thirty-seven age-matched controls were also recruited. Cell migration, phagocytosis, and oxidative burst of phagocytes were determined in vitro. Human neutrophil peptides 1 to 3 and serum metanephrine levels were measured by enzyme-linked immunosorbent assay, and NETosis was quantified by immunohistochemistry. Results— The key mechanisms required for bacterial killing, oxidative burst, and NETosis were significantly reduced in samples taken from patients with stroke compared with controls, whereas migration, phagocytic function, and defensin production remained unimpaired in monocytes and granulocytes from patients with stroke. Conclusions— Stroke-induced immune alterations include impairment of the first-line defense performed by specialized phagocytes against bacteria. The hypothesis that these changes enhance susceptibility to acquired infections is supported by our observation that on admission oxidative burst in monocytes was more impaired in patients with stroke with subsequent stroke-associated infections.