Investigating cell turnover in the healthy and diseased adult human brain

For decades it was thought that cells that lost in the human central nervous system because of ageing or disease – different from other cell tissues – cannot be replaced and that in humans all neurons are generated during prenatal development. However, over the last 20 years, it became obvious that there is a certain level of adult neurogenesis in most mammals that mainly occurs in the dentate gyrus and the subventricular zone. Whether or not findings from animal studies also hold true in humans was difficult to study as direct evidence – as obtained in animals from genomic labeling using for instance nucleosides like BrdU – was not feasible in humans because of ethical considerations. The establishment of the so-called radiocarbon technique, a method taking advantage of the above-ground nuclear bomb tests during the Cold War to retrospectively birth date cells by determination of the 12C/14C ratio in genomic DNA – allowed to investigate the age and the turnover dynamics of cells in various human tissues. Applying this technique we here (i) studied whether there is adult neurogenesis in the healthy human brain, specifically within the hippocampus, (ii) studied whether there is adult neurogenesis in the diseased human brain, specifically in response to cortical stroke, and (iii) investigated the age and growth dynamics of brain tumors, specifically benign meningiomas. In essence we demonstrate (i) that there is a lifelong adult neurogenesis within the human hippocampus and provide an integrated model of hippocampal cell turnover dynamics, (ii) that there is no significant induction of cortical neurogenesis following ischemic cortical stroke in humans, and (iii) that the age of benign meningiomas is significantly older than that of more malignant brain tumors. The clinical implications of these findings are discussed and research projects for future studies identified

Preconditioning concepts for the therapeutic use of extracellular vesicles against stroke

Various preclinical stroke models have demonstrated the neuroprotective effects of extracellular vesicles (EVs) obtained from several types of cells, including neurons, astrocytes, microglia, neuronal progenitor cells, bone marrow stem cells, and mesenchymal stem cells. EVs interfere with key mechanisms in stroke pathophysiology such as cell death, neuroinflammation, autophagy, and angiogenesis. The mode of action and efficacy depend on the specific EV content, including miRNAs, proteins, and lipids, which can be modified through (I) bioengineering methods, (II) choice of source cells, and (III) modification of the source cell environment. Indeed, modifying the environment by preconditioning the EV-secreting cells with oxygen-glucose deprivation or medium modification revealed superior neuroprotective effects in stroke models. Although the concept of preconditioned EVs is relatively novel, it holds promise for the future treatment of ischemic stroke. Here, we give a brief overview about the main mechanisms of EV-induced neuroprotection and discuss the current status of preconditioning concepts for EV-treatment of ischemic stroke

Reversal of oral anticoagulation in patients with acute intracerebral hemorrhage

In light of an aging population with increased cardiovascular comorbidity, the use of oral anticoagulation (OAC) is steadily expanding. A variety of pharmacological alternatives to vitamin K antagonists (VKA) have emerged over recent years (direct oral anticoagulants, DOAC, i.e., dabigatran, rivaroxaban, apixaban, and edoxaban) which show a reduced risk for the occurrence of intracerebral hemorrhage (ICH). Yet, in the event of ICH under OAC (OAC-ICH), hematoma characteristics are similarly severe and clinical outcomes likewise substantially limited in both patients with VKA- and DOAC-ICH, which is why optimal acute hemostatic treatment in all OAC-ICH needs to be guaranteed. Currently, International Guidelines for the hemostatic management of patients with OAC-ICH are updated as several relevant large-sized observational studies and recent trials have established treatment approaches for both VKA- and DOAC-ICH. While the management of VKA-ICH is mainly based on the immediate reversal of elevated levels of international normalized ratio using prothrombin complex concentrates, hemostatic management of DOAC-associated ICH is challenging requiring specific antidotes, notably idarucizumab and andexanet alfa. This review will provide an overview of the latest studies and trials on hemostatic reversal agents and timing and summarizes the effects on hemorrhage progression and clinical outcomes in patients with OAC-ICH

Peak Troponin I Levels Are Associated with Functional Outcome in Intracerebral Hemorrhage

Background: Troponin I is a widely used and reliable marker of myocardial damage and its levels are routinely measured in acute stroke care. So far, the influence of troponin I elevations during hospital stay on functional outcome in patients with atraumatic intracerebral hemorrhage (ICH) is unknown. Methods: Observational single-center study including conservatively treated ICH patients over a 9-year period. Patients were categorized according to peak troponin I level during hospital stay (≤0.040, 0.041–0.500, > 0.500 ng/mL) and compared regarding baseline and hematoma characteristics. Multivariable analyses were performed to investigate independent associations of troponin levels during hospital stay with functional outcome – assessed using the modified Rankin Scale (mRS; favorable 0–3/unfavorable 4–6) – and mortality after 3 and 12 months. To account for possible confounding propensity score (PS)-matching (1: 1; caliper 0.1) was performed accounting for imbalances in baseline characteristics to investigate the impact of troponin I values on outcome. Results: Troponin elevations (> 0.040 ng/mL) during hospital stay were observed in 308 out of 745 (41.3%) patients and associated with poorer status on admission (Glasgow Coma Scale/National Institute of Health Stroke Scale). Multivariable analysis revealed troponin I levels during hospital stay to be independently associated with unfavorable outcome after 12 months (risk ratio [95% CI]: 1.030 [1.009–1.051] per increment of 1.0 ng/mL; p = 0.005), but not with mortality. After PS-matching, patients with troponin I elevation (≥0.040 ng/mL) versus those without had a significant higher rate of ­unfavorable outcome after 3 and 12 months (mRS 4–6 at 3 months: < 0.04 ng/mL: 159/265 [60.0%] versus ≥0.04 ng/mL: 199/266 [74.8%]; p < 0.001; at 12 months: < 0.04 ng/mL: 141/248 [56.9%] versus ≥0.04 ng/mL: 179/251 [71.3%]; p = 0.001). Conclusions: Troponin I elevations during hospital stay occur frequently in ICH patients and are independently associated with functional outcome after 3 and 12 months but not with mortality

Preconditioned extracellular vesicles from hypoxic microglia reduce poststroke AQP4 depolarization, disturbed cerebrospinal fluid flow, astrogliosis, and neuroinflammation

Background: Stroke stimulates reactive astrogliosis, aquaporin 4 (AQP4) depolarization and neuroinflammation. Preconditioned extracellular vesicles (EVs) from microglia exposed to hypoxia, in turn, reduce poststroke brain injury. Nevertheless, the underlying mechanisms of such effects are elusive, especially with regards to inflammation, AQP4 polarization, and cerebrospinal fluid (CSF) flow. Methods: Primary microglia and astrocytes were exposed to oxygen-glucose deprivation (OGD) injury. For analyzing the role of AQP4 expression patterns under hypoxic conditions, a co-culture model of astrocytes and microglia was established. Further studies applied a stroke model, where some mice also received an intracisternal tracer infusion of rhodamine B. As such, these in vivo studies involved the analysis of AQP4 polarization, CSF flow, astrogliosis, and neuroinflammation as well as ischemia-induced brain injury. Results: Preconditioned EVs decreased periinfarct AQP4 depolarization, brain edema, astrogliosis, and inflammation in stroke mice. Likewise, EVs promoted postischemic CSF flow and cerebral blood perfusion, and neurological recovery. Under in vitro conditions, hypoxia stimulated M2 microglia polarization, whereas EVs augmented M2 microglia polarization and repressed M1 microglia polarization even further. In line with this, astrocytes displayed upregulated AQP4 clustering and proinflammatory cytokine levels when exposed to OGD, which was reversed by preconditioned EVs. Reduced AQP4 depolarization due to EVs, however, was not a consequence of unspecific inflammatory regulation, since LPS-induced inflammation in co-culture models of astrocytes and microglia did not result in altered AQP4 expression patterns in astrocytes. Conclusions: These findings show that hypoxic microglia may participate in protecting against stroke-induced brain damage by regulating poststroke inflammation, astrogliosis, AQP4 depolarization, and CSF flow due to EV release

A Specific Reduction in A beta(1-42) vs. a Universal Loss of A beta Peptides in CSF Differentiates Alzheimer's Disease From Meningitis and Multiple Sclerosis

A reduced concentration of A beta(1-42) in CSF is one of the established biomarkers of Alzheimer's disease Reduced CSF concentrations of A beta(1-42) have also been shown in multiple sclerosis, viral encephalitis and bacterial meningitis As neuroinflammation is one of the neuropathological hallmarks of Alzheimer's disease, an infectious origin of the disease has been proposed According to this hypothesis, amyloid pathology is a consequence of a microbial infection and the resulting immune defense Accordingly, changes in CSF levels of amyloid-beta peptides should be similar in AD and inflammatory brain diseases A beta(1-42) and A beta(1-40) levels were measured in cerebrospinal fluid by ELISA and Western blotting in 34 patients with bacterial meningitis (n = 9), multiple sclerosis (n = 5) or Alzheimer's disease (n = 9) and in suitable controls (n = 11) Reduced concentrations of A beta(1-42) were detected in patients with bacterial meningitis, multiple sclerosis and Alzheimer's disease However, due to a concurrent reduction in A beta(1-40) in multiple sclerosis and meningitis patients, the ratio of A beta(1-42)/A beta(1-40) was reduced only in the CSF of Alzheimer's disease patients Urea-SDS-PAGE followed by Western blotting revealed that all A beta peptide variants are reduced in bacterial meningitis, whereas in Alzheimer's disease, only A beta(1-42) is reduced These results have two implications First, they confirm the discriminatory diagnostic power of the A beta(1-42)/A beta(1-40) ratio Second, the differential pattern of A beta peptide reductions suggests that the amyloid pathology in meningitis and multiple sclerosis differs from that in AD and does not support the notion of AD as an infection-triggered immunopathology

Tracking cell turnover in human brain using 15N-thymidine imaging mass spectrometry

Microcephaly is often caused by an impairment of the generation of neurons in the brain, a process referred to as neurogenesis. While most neurogenesis in mammals occurs during brain development, it thought to continue to take place through adulthood in selected regions of the mammalian brain, notably the hippocampus. However, the generality of neurogenesis in the adult brain has been controversial. While studies in mice and rats have provided compelling evidence for neurogenesis occurring in the adult rodent hippocampus, the lack of applicability in humans of key methods to demonstrate neurogenesis has led to an intense debate about the existence and, in particular, the magnitude of neurogenesis in the adult human brain. Here, we demonstrate the applicability of a powerful method to address this debate, that is, the in vivo labeling of adult human patients with 15N-thymidine, a non-hazardous form of thymidine, an approach without any clinical harm or ethical concerns. 15N-thymidine incorporation into newly synthesized DNA of specific cells was quantified at the single-cell level with subcellular resolution by Multiple-isotype imaging mass spectrometry (MIMS) of brain tissue resected for medical reasons. Two adult human patients, a glioblastoma patient and a patient with drug-refractory right temporal lobe epilepsy, were infused for 24 h with 15N-thymidine. Detection of 15N-positive leukocyte nuclei in blood samples from these patients confirmed previous findings by others and demonstrated the appropriateness of this approach to search for the generation of new cells in the adult human brain. 15N-positive neural cells were easily identified in the glioblastoma tissue sample, and the range of the 15N signal suggested that cells that underwent S-phase fully or partially during the 24 h in vivo labeling period, as well as cells generated therefrom, were detected. In contrast, within the hippocampus tissue resected from the epilepsy patient, none of the 2,000 dentate gyrus neurons analyzed was positive for 15N-thymidine uptake, consistent with the notion that the rate of neurogenesis in the adult human hippocampus is rather low. Of note, the likelihood of detecting neurogenesis was reduced because of (i) the low number of cells analyzed, (ii) the fact that hippocampal tissue was explored that may have had reduced neurogenesis due to epilepsy, and (iii) the labeling period of 24 h which may have been too short to capture quiescent neural stem cells. Yet, overall, our approach to enrich NeuN-labeled neuronal nuclei by FACS prior to MIMS analysis provides a promising strategy to quantify even low rates of neurogenesis in the adult human hippocampus after in vivo15N-thymidine infusion. From a general point of view and regarding future perspectives, the in vivo labeling of humans with 15N-thymidine followed by MIMS analysis of brain tissue constitutes a novel approach to study mitotically active cells and their progeny in the brain, and thus allows a broad spectrum of studies of brain physiology and pathology, including microcephaly