Jennifer E. Slemmer

Causal Role of Apoptosis-Inducing Factor for Neuronal Cell Death Following Traumatic Brain Injury

Traumatic brain injury (TBI) consists of two phases: an immediate phase in which damage is caused as a direct result of the mechanical impact; and a late phase of altered biochemical events that results in delayed tissue damage and is therefore amenable to therapeutic treatment. Because the molecular mechanisms of delayed post-traumatic neuronal cell death are still poorly understood, we investigated whether apoptosis-inducing factor (AIF), a pro-apoptotic mitochondrial molecule and the key factor in the caspase-independent, cell death signaling pathway, plays a causal role in neuronal death following TBI. Using an in vitro model of neuronal stretch injury, we demonstrated that AIF translocated from mitochondria to the nucleus of neurons displaying axonal disruption, chromatin condensation, and nuclear pyknosis in a caspase-independent manner, whereas astrocytes remained unaffected. Similar findings were observed following experimental TBI in mice, where AIF translocation to the nucleus coincided with delayed neuronal cell death in both cortical and hippocampal neurons. Down-regulation of AIF in vitro by siRNA significantly reduced stretch-induced neuronal cell death by 67%, a finding corroborated in vivo using AIF-deficient harlequin mutant mice, where secondary contusion expansion was significantly reduced by 44%. Hence, our current findings demonstrate that caspase-independent, AIF-mediated signaling pathways significantly contribute to post-traumatic neuronal cell death and may therefore represent novel therapeutic targets for the treatment of TBI.

Combined effects of mechanical and ischemic injury to cortical cells: secondary ischemia increases damage and decreases effects of neuroprotective agents.

Traumatic brain injury (TBI) involves direct mechanical damage, which may be aggravated by secondary insults such as ischemia. We utilized an in vitro model of stretch-induced injury to investigate the effects of mechanical and combined mechanical/ischemic insults to cultured mouse cortical cells. Stretch injury alone caused significant neuronal loss and increased uptake of the dye, propidium iodide, suggesting cellular membrane damage to both glia and neurons. Exposure of cultures to ischemic conditions for 24h, or a combination of stretch and 24h of ischemia, caused greater neuronal loss compared to stretch injury alone. Next, we tested the neuroprotective effects of superoxide dismutase (SOD), and the nitric oxide (NO) synthase inhibitors 7-nitroindazole (7-NINA) and lubeluzole. In general, these agents decreased neuronal loss following stretch injury alone, but were relatively ineffective against the combined injury paradigm. A combination of SOD with 7-NINA or lubeluzole offered no additional protection than single drug treatment against stretch alone or combined injury. These results suggest that the effects of primary mechanical damage and secondary ischemia to cortical neurons are cumulative, and drugs that scavenge superoxide or reduce NO production may not be effective for treating the secondary ischemia that often accompanies TBI.

Cell death, glial protein alterations and elevated S-100β release in cerebellar cell cultures following mechanically induced trauma

Recent studies in vivo have shown that cells of the cerebellum, and particularly Purkinje neurons (PNs), are susceptible to damage following traumatic brain injury (TBI). To investigate more closely the effects of TBI at the cellular level, we subjected cerebellar cell cultures to injury using an in vitro model of stretch-induced mechanical trauma and found increased cell damage and neuronal loss with increasing levels of injury and time post-injury. The release of neuron-specific enolase and S-100 beta were also elevated after injury. Compared to our previous findings in hippocampal cells, S-100 beta levels were much higher in cerebellar cultures after injury, suggesting that cells from different brain regions show variable responses to mechanical trauma. Lastly, the addition of exogenous S-100 beta to uninjured cerebellar cells caused no overt change in cell viability or overall neuronal number; there were, however, fewer calbindin-positive PNs, similar to findings after stretch injury.

Aldolase C-positive cerebellar Purkinje cells are resistant to delayed death after cerebral trauma and AMPA-mediated excitotoxicity

The cerebellum has been shown to be vulnerable to global ischemic damage in tightly controlled zones of Purkinje cells (PCs) that lack aldolase C, an enzyme critical for glycolysis. Here, we investigated whether aldolase C‐negative PCs were more likely to die after cerebral trauma in vivo, and whether this death was mediated by excitotoxic [α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA)‐mediated] means in vitro. Mice were subjected to controlled cortical impact, or remained uninjured, and were killed at 6 h, 24 h or 7 days after injury. Cerebellar sections (both ipsilateral and contralateral to the site of cerebral injury) were stained against aldolase C and calbindin (a marker of PCs). The number of viable, calbindin‐positive PCs decreased significantly at 24 h and 7 days after injury, and the percentage of surviving, aldolase C‐positive PCs significantly increased at those time‐points. In addition, we subjected murine cerebellar cultures to AMPA (30 µm, 20 min), which killed a significant number of PCs at 24 h post‐treatment. A similar number of PCs was lost after transfection with aldolase C siRNA, and this effect was exacerbated in transfected cultures treated with AMPA. The results from the present study indicate that aldolase C provides marked neuroprotection to PCs after trauma and excitotoxicity.

Glutamate-induced elevations in intracellular chloride concentration in hippocampal cell cultures derived from EYFP-expressing mice

The homeostasis of intracellular Cl− concentration ([Cl−]i) is critical for neuronal function, including γ‐aminobutyric acid (GABA)ergic synaptic transmission. Here, we investigated activity‐dependent changes in [Cl−]i using a transgenetically expressed Cl−‐sensitive enhanced yellow‐fluorescent protein (EYFP) in cultures of mouse hippocampal neurons. Application of glutamate (100 µm for 3 min) in a bath perfusion to cell cultures of various days in vitro (DIV) revealed a decrease in EYFP fluorescence. The EYFP signal increased in amplitude with increasing DIV, reaching a maximal response after 7 DIV. Glutamate application resulted in a slight neuronal acidification. Although EYFP fluorescence is sensitive to pH, EYFP signals were virtually abolished in Cl−‐free solution, demonstrating that the EYFP signal represented an increase in [Cl−]i. Similar to glutamate, a rise in [Cl−]i was also induced by specific ionotropic glutamate receptor agonists and by increasing extracellular [K+], indicating that an increase in driving force for Cl− suffices to increase [Cl−]i. To elucidate the membrane mechanisms mediating the Cl− influx, a series of blockers of ion channels and transporters were tested. The glutamate‐induced increase in [Cl−]i was resistant to furosemide, bumetanide and 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulphonic acid (DIDS), was reduced by bicuculline to about 80% of control responses, and was antagonized by niflumic acid (NFA) and 5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid (NPPB). We conclude that membrane depolarization increases [Cl−]i via several pathways involving NFA‐ and NPPB‐sensitive anion channels and GABAA receptors, but not through furosemide‐, bumetanide‐ or DIDS‐sensitive Cl− transporters. The present study highlights the vulnerability of [Cl−]i homeostasis after membrane depolarization in neurons.

Assessing Antioxidant Capacity in Brain Tissue: Methodologies and Limitations in Neuroprotective Strategies

The number of putative neuroprotective compounds with antioxidant activity described in the literature continues to grow. Although these compounds are validated using a variety of in vivo and in vitro techniques, they are often evaluated initially using in vitro cell culture techniques in order to establish toxicity and effective concentrations. Both in vivo and in vitro methodologies have their respective advantages and disadvantages, including, but not limited to, cost, time, use of resources and technical limitations. This review expands on the inherent benefits and drawbacks of in vitro and in vivo methods for assessing neuroprotection, especially in light of proper evaluation of compound efficacy and neural bioavailability. For example, in vivo studies can better evaluate the effects of protective compounds and/or its metabolites on various tissues, including the brain, in the whole animal, whereas in vitro studies can better discern the cellular and/or mechanistic effects of compounds. In particular, we aim to address the question of appropriate and accurate extrapolation of findings from in vitro experiment-where compounds are often directly applied to cellular extracts, potentially at higher concentrations than would ever cross the blood-brain barrier—to the more complex scenario of neuroprotection due to pharmacodynamics in vivo.