Cell Death In Slices Research Articles

Modeling traumatic injury in organotypic spinal cord slice culture obtained from adult rat.

Nowadays there are various models of spinal cord injury (SCI) that recreate mechanisms of human SCI. The ex vivo modeling of injury is a robust approach, confronts with less experimental and ethical challenges. Currently almost all ex vivo models are obtained either from embryonic or postnatal animals, which can hardly mimic features of human SCI. This study was designed to develop SCI in slice culture of adult rats. Here, the lumbar enlargement of an adult rat was sliced and cultured. After seven days in vitro, a weight was dropped to simulate the injury. The result showed that although the rate of cell death in first days of in vitro was high, it reduced after 7 days and dropping a weight at the time caused significant rate of cell death in slices. It was shown that injury can disturb histological features and neuronal integrity in the slices. Treating the injured slices with valproic acid resulted in a significant decrease of TNF-α and increase of BDNF expression. Collected data revealed obtained slices from adult rat were able to adjust to the culture environment after 7 days and dropping a weight at the time point could simulate the injury. Besides mimicking the disturbing features of human SCI, this model can response to VPA pharmacological treatment.

In‐vitro Analysis of Catalase‐, Oxidase‐ and SOD‐mimetic Activity of Commercially Available and Custom‐synthesized Cerium Oxide Nanoparticles and Assessment of Neuroprotective Effects in a Hippocampal Brain Slice Model of Ischemia

Oxidative stress has been implicated in many neurological conditions, from stroke to Alzheimer's disease. Cerium oxide (CeO2) nanoparticles (nanoceria or CeNPs) are potent antioxidants that are being explored as a potential therapeutic for these and other pathologies involving oxidative stress. Nanoceria are small, capable of catalytically neutralizing multiple types of reactive oxygen species (ROS), and, due to cycling between the Ce3+ and Ce4+ oxidation states, they can continually regenerate their ROS‐neutralizing capacity depending on the redox conditions. However, both therapeutic and toxic effects of CeNPs have been reported and we hypothesize that this stems from differences in the types of CeNPs used because the method of synthesis as well as the choice of stabilizers coating the particles can lead to disparate biological effects. Understanding which physical/chemical properties correlate with biological activity is critical in order to gain insight into the conditions that promote the positive effects and to understand under what circumstances toxic properties emerge. In the present study we begin to address these issues in a systematic way. Specifically, we tested the enzyme‐mimetic properties of two commercially available (Treibacher Industries and Nanophase) and one custom‐synthesized (CeNRx) CeNP formulation using commercially available enzymatic assay kits. These particles differed in size and method of stabilization. We detected significant differences in the catalase‐ and SOD‐mimetic activities across the three formulations with the Treibacher formulation displaying low levels of catalase‐mimetic activity and undetectable levels of SOD‐mimetic activity. The Nanophase formulation displayed intermediate levels of catalase‐mimetic and low levels of SOD mimetic activity. The custom‐synthesized CeNRx formulation displayed the highest levels of both catalase and SOD‐mimetic activity of the three formulations tested. None of the nanoparticle formulations displayed significant oxidase mimetic activity, demonstrating that there is not a high level of non‐selective oxidation with these CeNPs. We then tested the effects of these CeNPs in a mouse hippocampal brain slice model of ischemia where cell death was assessed using the fluorescence indicator SYTOX green. Fluorescence levels in the slices treated with Treibacher CeNPs were not significantly different from that in untreated slices. In contrast, there was a ~23% increase in cell death in slices treated with Nanophase CeNPs, and a 34% decrease in cell death in slices treated with CeNRx CeNPs. These results demonstrate that three CeNP formulations display different enzyme‐mimetic activity in vitro which correlates with different neuroprotective profiles in brain slices. Future studies will assess further CeNP modifications in order maximize beneficial and minimize toxic effects.Support or Funding InformationSome funding for these studies was provided by CeNRx.

Endoplasmic reticulum stress signaling involvement in manganese-induced nerve cell damage in organotypic brain slice cultures

Overexposure to manganese (Mn) has been known to induce neuronal damage. However, the mechanisms underlying the neurotoxicity of Mn are still incompletely understood but seem to involve endoplasmic reticulum (ER) stress. The current study investigated whether ER stress signaling was involved in Mn-induced neurotoxicity in organotypic brain slices. After the brain slices were respectively exposed to 400μM Mn for 0, 6, 12, 18, 24h, there was a time-dependent increase in apoptotic cell death in slices and levels of lactate dehydrogenase (LDH) in the culture medium. Moreover, Mn was found to upregulate GRP78/94, CHOP and caspase-12 expression. Furthermore, PERK phosphorylation, PERK-mediated phosphorylation of eIF2a and ATF4 mRNA expression increased. IRE1 activation and Xbp1 mRNA splicing also increased. However, ATF6 p90 levels did not change. The findings clearly demonstrated that Mn induced the ER stress via activation of PERK and IRE1 signaling pathway, which contributed to the occurrence of apoptosis in cultured slices.

Pathological consequences of inducible nitric oxide synthase expression in hippocampal slice cultures

The generation of toxic concentrations of nitric oxide by the inducible nitric oxide synthase expressed in microglia and other brain cell types is frequently invoked as a causative factor in neurodegeneration. Experiments were carried out on slice cultures of rat hippocampus to test this hypothesis. Exposure of the slices to bacterial lipopolysaccharide plus interferon-gamma led to a time-dependent expression of functional inducible nitric oxide synthase that was found only in microglia. Microglial activation by other means, such as physical damage, was not associated with inducible nitric oxide synthase expression. Damage and cell death in slices expressing inducible nitric oxide synthase was evaluated over a period of 6 days, but none was found. Consistent with this result, cGMP measurements indicated that the average local nitric oxide concentration remained in the low nanomolar range. When the microglial population was expanded to a density three-fold above normal by applying granulocyte-macrophage colony stimulating factor, however, lipopolysaccharide plus interferon-gamma provoked neurodegeneration that could be blocked by an inducible nitric oxide synthase inhibitor. The associated nitric oxide concentration in the slices was saturating for guanylyl cyclase-coupled nitric oxide receptors, signifying at least 10nM. It is concluded that inducible nitric oxide synthase is expressed in microglia only in response to specific stimuli involving the innate immune system, and that the resulting level of nitric oxide in intact brain tissue is normally too low to inflict damage directly. Quantities of nitric oxide sufficient to contribute directly or indirectly to pathology could be produced should the density of microglia become high enough, although caution must be exercised in extrapolating this finding to the human brain in vivo.