Effect of neuroprotective agents against
mechanical/ischemic injury in vitro.
D. C. Engel1,2, J. E. Slemmer1, C. I. De Zeeuw1, A. I.R. Maas2 and J. T. Weber1
Departments of Neuroscience1 and Neurosurgery2, Erasmus Medical Center, Rotterdam, The Netherlands
Traumatic brain injury (TBI) leads to cell damage by direct mechanical disruption of the brain and by secondary insults such as ischemia.
We utilized an in vitro model of stretch-induced injury to investigate the effects of mechanical, ischemic and combined mechanical/ischemic insults to cultured mouse cortical cells. Stretch injury alone increased uptake of the dye, propidium iodide (PrI), 15 min after injury, suggesting cellular membrane damage. Uptake of PrI was dependent on the level of stretch and decreased with time post-injury up to 48 hr. Stretch injury also caused a significant reduction in the amount of MAP2-positive neurons in cultures. Exposure of cultures to ischemic conditions for 24 hr, or a combination of stretch followed by 24 hr of ischemia, caused no change in PrI uptake when compared to stretch injury alone. However, both ischemia and the combined insult paradigm caused a greater reduction in neurons compared to stretch alone.
Next, we tested the effects of the potential neuroprotective agents, 7-nitroindazole (7-NINA), lubeluzole, and superoxide dismutase (SOD) on injured cells. Post-treatment (addition of agents 15 min after stretch-induced injury) by these agents provided no protection against combined insults, as measured by the amount of MAP2-positive neurons. Pre-treatment of cultures by lubeluzole (100 nM) and SOD provided modest protection against stretch injury alone; pre-treatment by SOD provided somewhat protection against ischemia alone; pre-treatment by 7-NINA (1μM and 10μM) and SOD provided modest protection in the combined insult paradigm, though less than in the stretch injury alone.
These results suggest that degenerative mechanisms caused by secondary ischemic insults may be too devastating to the cortical neurons in vitro, which are already compromised from primary mechanical damage following TBI.
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