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Published on: October 23, 2014
Tissue plasminogen activator protects hippocampal neurons from oxygen-glucose deprivation injury
1Department of Pediatrics, Queen's University, Kingston, Ontario, Canada. mf6@post.queensu.ca
Abstract:
We have previously shown that tissue plasminogen activator (tPA) participates in the neurotoxicity of microglial conditioned medium (MgCM). Killing of hippocampal neurons by MgCM was prevented by both plasminogen activator inhibitor-1 (PAI-1) and anti-tPA antibody. An N-methyl-D-aspartate (NMDA) receptor blocker protected neurons from MgCM, suggesting that this subtype of glutamate receptor is involved. Whereas glutamate receptor-mediated events are important in cerebral ischemia and tPA has previously been shown to enhance excitotoxicity in hippocampus, we hypothesized that tPA would exaggerate oxygen glucose deprivation (OGD) injury in cultures of hippocampal neurons. Dissociated rat hippocampal cells were grown under conditions designed to optimize neuronal growth while minimizing glial replication. At 7--10 days, cultures were subjected to OGD for 2.5 hr. Recombinant human tPA (1,000 IU) was added immediately after OGD. Viability was assessed 24 hr later. Viable, apoptotic, and necrotic cells were classified and quantified based on staining patterns of acridine orange and ethidium bromide under fluorescence microscopy. tPA alone did not alter neuronal integrity. OGD produced significant neuronal death (viability reduced by 45%, P < 0.001). tPA completely protected OGD-exposed cultures. Potential mechanisms of tPA protection were explored. Whereas tPA antibody abolished the protective effect of tPA, its proteolytic inhibitor PAI-1 did not alter the effect. The effect of tPA was tested in separate free radical and excitatory amino acid insults. It did not protect neurons from hydrogen peroxide (1 microM), S-nitro-acetylpenicillamine (10 microM), glutamate (50 microM), or NMDA (10 microM) damage but significantly attenuated injury caused by 250 microM kainate. We conclude that tPA is capable of protecting hippocampal neurons from OGD by a nonproteolytic action. The mechanism of protection was not defined, although attenuation of AMPA/kainate glutamate receptors may play a role.
Insights
Tissue plasminogen activator (tPA) surprisingly protected hippocampal neurons from oxygen-glucose deprivation (OGD) injury. This neuroprotective effect of tPA was not dependent on its enzymatic activity.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Tissue plasminogen activator (tPA) has been implicated in neurotoxicity, particularly in microglial conditioned medium (MgCM) effects on hippocampal neurons.
- Glutamate receptor-mediated excitotoxicity is a key factor in cerebral ischemia and hippocampal injury.
- Previous research suggested tPA might enhance excitotoxicity, prompting investigation into its role during oxygen-glucose deprivation (OGD).
Purpose of the Study:
- To investigate the hypothesis that tPA exacerbates injury in hippocampal neurons subjected to OGD.
- To determine the effect of recombinant human tPA on neuronal viability following OGD.
- To explore the mechanisms underlying tPA's action, differentiating between proteolytic and non-proteolytic effects.
Main Methods:
- Dissociated rat hippocampal cultures were subjected to 2.5 hours of OGD.
- Recombinant human tPA was administered immediately post-OGD, with viability assessed 24 hours later.
- Cell death was quantified using acridine orange and ethidium bromide staining, and effects of tPA antibody and PAI-1 were evaluated.
Main Results:
- OGD significantly reduced neuronal viability by 45%.
- Contrary to the hypothesis, tPA completely protected OGD-exposed hippocampal neurons.
- tPA's protective effect was abolished by tPA antibody but not by the proteolytic inhibitor PAI-1, indicating a non-proteolytic mechanism.
Conclusions:
- Tissue plasminogen activator (tPA) demonstrates a potent neuroprotective effect against OGD-induced injury in hippocampal neurons.
- The protective action of tPA is independent of its proteolytic activity.
- While the exact mechanism remains undefined, tPA may attenuate injury through modulation of AMPA/kainate glutamate receptors.
