Immune cells display a high degree of phenotypic plasticity, which may facilitate their participation in both the progression and resolution of injury-induced inflammation. The purpose of this study was to investigate the temporal expression of genes associated with classical and alternative polarization phenotypes described for macrophages and to identify related cell populations in the brain following neonatal hypoxia-ischemia (HI). HI was induced in 9-day old mice and brain tissue was collected up to 7 days post-insult to investigate expression of genes associated with macrophage activation. Using cell-markers, CD86 (classic activation) and CD206 (alternative activation), we assessed temporal changes of CD11b+ cell populations in the brain and studied the protein expression of the immunomodulatory factor galectin-3 in these cells. HI induced a rapid regulation (6 h) of genes associated with both classical and alternative polarization phenotypes in the injured hemisphere. FACS analysis showed a marked increase in the number of CD11b+CD86+ cells at 24 h after HI (+3667%), which was coupled with a relative suppression of CD11b+CD206+ cells and cells that did not express neither CD86 nor CD206. The CD11b+CD206+ population was mixed with some cells also expressing CD86. Confocal microscopy confirmed that a subset of cells expressed both CD86 and CD206, particularly in injured gray and white matter. Protein concentration of galectin-3 was markedly increased mainly in the cell population lacking CD86 or CD206 in the injured hemisphere. These cells were predominantly resident microglia as very few galectin-3 positive cells co-localized with infiltrating myeloid cells in Lys-EGFP-ki mice after HI. In summary, HI was characterized by an early mixed gene response, but with a large expansion of mainly the CD86 positive population during the first day. However, the injured hemisphere also contained a subset of cells expressing both CD86 and CD206 and a large population that expressed neither activation marker CD86 nor CD206. Interestingly, these cells expressed the highest levels of galectin-3 and were found to be predominantly resident microglia. Galectin-3 is a protein involved in chemotaxis and macrophage polarization suggesting a novel role in cell infiltration and immunomodulation for this cell population after neonatal injury.

We investigated the effects of ionizing radiation on microvessel structure and complexity in the hippocampus. We also assessed neurogenesis and the neurovascular niche. Postnatal day 14 male C57BL/6 mice received a single dose of 8 Gy to the whole brain and were killed 6 hours, 1 week, 7 weeks, or 1 year later. Irradiation decreased the total number of microvessels and branching points from 1 week onwards and decreased the total microvessel area 1 and 7 weeks after irradiation. After an initial increase in vascular parameter densities, concomitant with reduced growth of the hippocampus, the densities normalized with time, presumably adapting to the needs of the surrounding nonvascular tissue. Irradiation decreased the number of neural stem and progenitor cells in the hippocampus. The relative loss increased with time, resulting in almost completely ablated neurogenesis (DCX þ cells) 1 year after irradiation (77% decreased 1 week, 86% decreased 7 weeks, and 98% decreased 1 year after irradiation compared with controls). After irradiation, the distance between undifferentiated stem cells and microvessels was unaffected, and very few dying endothelial cells were detected. Taken together, these results indicate that the vasculature adjusts to the surrounding neural and glial tissue after irradiation, not vice-versa.

A blood biomarker to monitor individual susceptibility to neuronal injury from cranial radiotherapy could potentially help to individualize radiation treatment and thereby reduce the incidence and severity of late effects. An important feature of such a blood biomarker is that its concentration is not confounded by varying degrees of release from the brain into the blood across the blood-brain barrier (BBB). In this study, we investigated serum neurofilament light protein (NFL) concentrations in 21-day old mice following a single dose of cranial irradiation (8Gy). Cranial irradiation resulted in acute cell injury measured as a 12.9-fold increase in caspase activity 6h after irradiation; activation of inflammation measured by levels of CCL2 and increased BBB permeability measured by C-sucrose concentration ratios in brain and cerebrospinal fluid (CSF). Serum levels of NFL peaked at 6h after both anesthesia and cranial irradiation, but no timely correlation of serum NFL concentration with BBB permeability was found. Further, three groups of patients with different degrees of BBB impairment (measured as the CSF/serum albumin ratio) were investigated. There was no correlation between serum NFL concentration and CSF/serum albumin ratio (r=0.139, p=0.3513), however a strong correlation was found for NFL concentration in serum and NFL concentration in CSF (r=0.6303, p<0.0001). In conclusion, serum NFL appears to be a reliable blood biomarker for neuronal injury, and its concentration is not confounded by BBB permeability.

Posterior fossa tumors are the most common childhood intracranial tumors, and radiotherapy is one of the most effective treatments. However, irradiation induces long-term adverse effects that can have significant negative impacts on the patient’s quality of life. The purpose of this study was to characterize irradiation-induced cellular and molecular changes in the cerebellum. We found that irradiation-induced cell death occurred mainly in the external germinal layer (EGL) of the juvenile rat cerebellum. The number of proliferating cells in the EGL decreased, and 82.9% of them died within 24 h after irradiation. Furthermore, irradiation induced oxidative stress, microglia accumulation, and inflammation in the cerebellum. Interestingly, blood-brain barrier damage and blood flow reduction was considerably more pronounced in the cerebellum compared to other brain regions. The cerebellar volume decreased by 39% and the migration of proliferating cells to the internal granule layer decreased by 87.5% at 16 weeks after irradiation. In the light of recent studies demonstrating that the cerebellum is important not only for motor functions, but also for cognition, and since treatment of posterior fossa tumors in children typically results in debilitating cognitive deficits, this differential susceptibility of the cerebellum to irradiation should be taken into consideration for future protective strategies.

BackgroundNeurogranin (Ng) is a small 7.6 kDa postsynaptic protein that has been detected at elevated concentrations in cerebrospinal fluid (CSF) of patients with Alzheimer’s disease (AD), both as a full-length molecule and as fragments from its C-terminal half. Ng is involved in postsynaptic calcium (Ca) signal transduction and memory formation via binding to calmodulin in a Ca-dependent manner. The mechanism of Ng secretion from neurons to CSF is currently unknown, but enzymatic cleavage of Ng may be of relevance. Therefore, the aim of the study was to identify the enzymes responsible for the cleavage of Ng, yielding the Ng fragment pattern of C-terminal fragments detectable and increased in CSF of AD patients.MethodsFluorigenic quenched FRET probes containing sequences of Ng were utilized to identify Ng cleaving activities among enzymes known to have increased activity in AD and in chromatographically fractionated mouse brain extracts.ResultsHuman Calpain-1 and prolyl endopeptidase were identified as the candidate enzymes involved in the formation of endogenous Ng peptides present in CSF, cleaving mainly in the central region of Ng, and between amino acids 75_76 in the Ng sequence, respectively. The cleavage by Calpain-1 affects the IQ domain of Ng, which may deactivate or change the function of Ng in Ca2+/calmodulin -dependent signaling for synaptic plasticity. While shorter Ng fragments were readily cleaved in vitro by prolyl endopeptidase, the efficiency of cleavage on larger Ng fragments was much lower.ConclusionsCalpain-1 and prolyl endopeptidase cleave Ng in the IQ domain and near the C-terminus, respectively, yielding specific fragments of Ng in CSF. These fragments may give clues to the roles of increased activities of these enzymes in the pathophysiology of AD, and provide possible targets for pharmacologic intervention.Electronic supplementary materialThe online version of this article (10.1186/s13024-018-0279-z) contains supplementary material, which is available to authorized users.