Anti-Inflammatory and Anti-Oxidative Effects of AM404 in IL-1β-Stimulated SK-N-SH Neuroblastoma Cells

An emerging number of studies address the involvement of neuroinflammation and oxidative stress in the pathophysiology of central nervous system (CNS) disorders such as depression, schizophrenia, anxiety, and neurodegenerative diseases. Different cytokines and molecules, such as prostaglandin (PG) E2, are associated with neuroinflammatory processes. The active acetaminophen metabolite AM404 has been shown to prevent inflammation and neuroinflammation in primary microglia and organotypic hippocampal slice cultures. However, its effects on pathophysiological conditions in the CNS and especially on neurons are still poorly understood. In this study, we therefore evaluated the effects of AM404 and acetaminophen on the arachidonic acid cascade and oxidative stress induced by interleukin (IL)-1β in human SK-N-SH neuronal cells. We observed that AM404 and acetaminophen significantly and concentration-dependent inhibited IL-1β-induced release of PGE2, independent of cyclooxygenases (COX)-1 and COX-2 enzymatic activity as well as COX-2 mRNA and protein levels in SK-N-SH-cells. The reduction of IL-1β-induced PGE2-release by AM404 and acetaminophen treatment might be mediated by the 8-iso-PGF2α pathway since IL-1β-induced synthesis of this free radical marker is dose-dependently reduced by both compounds, respectively. Therefore, understanding of the potential therapeutic properties of AM404 in neuroinflammation and oxidative stress might lead to future treatment options of different neurological disorders.

Variants of Oxidized Regenerated Cellulose and Their Distinct Effects on Neuronal Tissue

Based on oxidized regenerated cellulose (ORC), several hemostyptic materials, such as Tabotamp®, Equicel® and Equitamp®, have been developed to approach challenging hemostasis in neurosurgery. The present study compares ORC that differ in terms of compositions and properties, regarding their structure, solubility, pH values and effects on neuronal tissue. Cytotoxicity was detected via DNA-binding fluorescence dye in Schwann cells, astrocytes, and neuronal cells. Additionally, organotypic hippocampal slice cultures (OHSC) were analyzed, using propidium iodide, hematoxylin-eosin, and isolectin B4 staining to investigate the cellular damage, cytoarchitecture, and microglia activation. Whereas Equicel® led to a neutral pH, Tabotamp® (pH 2.8) and Equitamp® (pH 4.8) caused a significant reduction of pH (p < 0.001). Equicel® and Tabotamp® increased cytotoxicity significantly in several cell lines (p < 0.01). On OHSC, Tabotamp® and Equicel® led to a stronger and deeper damage to the neuronal tissue than Equitamp® or gauze (p < 0.01). Equicel® increased strongly the number of microglia cells after 24 h (p < 0.001). Microglia cells were not detectable after Tabotamp® treatment, presumably due to an artifact caused by strong pH reduction. In summary, our data imply the use of Equicel®, Tabotamp® or Equitamp® for specific applications in distinct clinical settings depending on their localization or tissue properties.

Rapid astroglial stress response and elevation of endogenous BiP levels promote neuronal survival in hippocampal slice cultures

A hallmark of neurodegenerative diseases is the accumulation of protein aggregates, the formation of which is prevented by chaperone proteins. BiP is the central chaperone in the endoplasmic reticulum. In this study we investigated the pattern of BiP in tunicamycin-stressed murine organotypic hippocampal slice cultures (OHCs). In stressed OHCs highest apoptotic rates occur in neurons of the CA1 regions and the dentate gyrus, in which we found BiP levels to be lowest. Highest BiP protein levels were found in astrocytes. Cell culture experiments indicated that the stress response of glial cells is faster and stronger than in neuronal cells. We hypothesize that the rapid and pronounced BiP expression in astrocytes helps to maintain the fine-balanced micromilieu necessary for survival of neurons. SubAB is a toxin, which cleaves and inactivates BiP. Low dosages of SubAB did not elicit a specific glial response and apoptosis was not induced in a specific hippocampal subfield. Mild prestressing with SubAB promoted neuronal viability in tunicamycin-treated OHCs. We conclude that preconditioning of hippocampal tissue with stressors that elevate endogenous chaperone levels exert a protective effect thereby promoting neuronal survival. These experiments strengthen the thesis that preconditoning with mild stressors positively affects the survival of neuronal cells.

In vitro ictogenesis is stochastic at the single neuron level

Seizure initiation is the least understood and most disabling element of epilepsy. Studies of ictogenesis require high speed recordings at cellular resolution in the area of seizure onset. However, in vivo seizure onset areas can’t be determined at the level of resolution necessary to enable such studies. To circumvent these challenges, we used novel GCaMP7-based calcium imaging in the organotypic hippocampal slice culture model of post-traumatic epilepsy in mice. Organotypic hippocampal slice cultures generate spontaneous, recurrent seizures in a preparation in which it is feasible to image the activity of the entire network (with no unseen inputs existing). Chronic calcium imaging of the entire hippocampal network, with paired electrophysiology, revealed 3 patterns of seizure onset: low amplitude fast activity, sentinel spike, and spike burst + low amplitude fast activity onset. These patterns recapitulate common features of human seizure onset, including low voltage fast activity and spike discharges. Weeks-long imaging of seizure activity showed a characteristic evolution in onset type and a refinement of the seizure onset zone. Longitudinal tracking of individual neurons revealed that seizure onset is stochastic at the single neuron level, suggesting that seizure initiation activates neurons in non-stereotyped sequences seizure to seizure. This study demonstrates for the first time that transitions to seizure are not initiated by a small number of neuronal “bad actors” (such as overly connected hub cells), but rather by network changes which enable the onset of pathology among a large populations of neurons.

Contribution of ginsenosides Rg1, Rb1 to the neuroprotective effect of Panax notoginseng in mouse organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation

We previously demonstrated that Panax notoginseng (pNG) root extract treatments exertedneuroprotective effects on brain injuries using middle cerebral artery occlusion in mice. The present study aims to investigate the neuroprotective effects of PNG extract and its ginsenosides Rg1 and Rb1 on ischemic neuronal damage caused by oxygen and glucose deprivation (OGD) in mouse organotypic hippocampal slice cultures (OHSCs). Before the experiments, hippocampal slices collected from 7-day-old Swiss mice were cultured for 7 days. OGD was triggered in OHSCs for 30, 60, or 90 min with the aim of finding the optimal period of OGD for drug testing. PNG extract (10, 30 μg/ml), ginsenosides Rg1 and Rb1 (5, 25 μM), or MK-801 25 μM, a reference drug, was added to the culture medium 24 h before OGD and these treatments were continued for 24 h after the optimum 60-min period of OGD. After 24 h of OGD exposure, the measurement of propidium iodide uptake was analysed in OHSCs to evaluate neuronal cell damage. The results showed that OGD time-dependently increased PI uptake of the OHSCs. PNG 30 μg/ml treatment reduced the OGD-induced neuronal cell damage in OHSCs. Ginsenosides Rg1 25 μM, Rb1 (5, 25 μM), as well as MK-801 (25 μM) significantly inhibited PI uptake 24 h after OGD exposure. However, ginsenoside Rg1 5 μM did not show any significant effects on the OGD-induced neuronal cell damage. These findings indicated that ginsenosides Rg1 and Rb1 contributed to the neuroprotective effects of PNG against ischemic damage in OHSCs and the neuroprotective effect of ginsenoside Rb1 was stronger than that of ginsenoside Rg1.

Development of P301S tau seeded organotypic hippocampal slice cultures to study potential therapeutics

Intracellular tau inclusions are a pathological hallmark of Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration and other sporadic neurodegenerative tauopathies. Recent in vitro and in vivo studies have shown that tau aggregates may spread to neighbouring cells and functionally connected brain regions, where they can seed further tau aggregation. This process is referred to as tau propagation. Here we describe an ex vivo system using organotypic hippocampal slice cultures (OHCs) which recapitulates aspects of this phenomenon. OHCs are explants of hippocampal tissue which may be maintained in culture for months. They maintain their synaptic connections and multicellular 3D architecture whilst also permitting direct control of the environment and direct access for various analysis types. We inoculated OHCs prepared from P301S mouse pups with brain homogenate from terminally ill P301S mice and then examined the slices for viability and the production and localization of insoluble phosphorylated tau. We show that following seeding, phosphorylated insoluble tau accumulate in a time and concentration dependent manner within OHCs. Furthermore, we show the ability of the conformation dependent anti-tau antibody, MC1, to compromise tau accrual in OHCs, thus showcasing the potential of this therapeutic approach and the utility of OHCs as an ex vivo model system for assessing such therapeutics.

LAG3 is not expressed in human and murine neurons and does not modulate α-synucleinopathies

While the initial pathology of Parkinson's disease and other α-synucleinopathies is often confined to circumscribed brain regions, it can spread and progressively affect adjacent and distant brain locales. This process may be controlled by cellular receptors of α-synuclein fibrils, one of which was proposed to be the LAG3 immune checkpoint molecule. Here, we analyzed the expression pattern of LAG3 in human and mouse brains. Using a variety of methods and model systems, we found no evidence for LAG3 expression by neurons. While we confirmed that LAG3 interacts with α-synuclein fibrils, the specificity of this interaction appears limited. Moreover, overexpression of LAG3 in cultured human neural cells did not cause any worsening of α-synuclein pathology ex vivo. The overall survival of A53T α-synuclein transgenic mice was unaffected by LAG3 depletion and the seeded induction of α-synuclein lesions in hippocampal slice cultures was unaffected by LAG3 knockout. These data suggest that the proposed role of LAG3 in the spreading of α-synucleinopathies might not be universally valid.

Innovative approach for the application of MTT, LDH and bis-ANS

The acute hippocampus slice-based models can serve as a feasible tool to gain deeper understanding on how pathological events in AD, in particular Aβ oligomerization, influence hippocampal functionality and therefore paving the way to develop and test related hypothesis but also to screen potential protective agents or validate results seen in vivo or predicted in silico. To overcome the shortcomings of the in vitro and in vivo methods, we developed a cost effective and simple apparatus for maintaining the viability of acute tissue slices, named: ExViS (Ex Vivo System; Universal Mini-Chamber Tube System for Acute Tissue Slices). The system allowed us to further develop methods that use acute (ex vivo) hippocampal slices to model AD-related patomechanisms. Over the course of my PhD studies we have developed the following techniques and ex vivo models based on the features of acute hippocampal slices: 1. Rapid, reliable and quantitative determination of Aβ1-42 toxicity in ageing (OGD) acute hippocampal slice model using MTT and LDH assays. 2. Quantitative determination of zinc-induced Aβ1-42 oligomerization toxicity in acute hippocampal slice model using MTT assay. 3. Fluorescent imaging of neurite cross-sections and detailed imaging of neurite structures in acute hippocampal slices via a novel application of bis-ANS and its co-staining variations. 4. Modelling the effect of Zn2+ released in glutamergic synaptic cleft in the hippocampus on Aβ1-42 aggregation and its consequences on cell viability and synaptic functionality, learning and memory (LTP).