Neuroprotection by steroids after neurotrauma in organotypic spinal cord cultures: A key role for progesterone receptors and steroidal modulators of GABAA receptors

Abstract

Progesterone is neuroprotective after spinal cord injury, however its mechanism of action remains unexplored. Here we used organotypic spinal cord slice cultures from 3 weeks-old mice to evaluate the mechanisms of neuroprotection by progesterone and its 5α-reduced metabolites. In vitro spinal cord injury, using a weight drop model, induced a decrease in the number of motoneurons. This was correlated with an increase in the number of dying cells (PI+ cells) and in LDH release. Addition of 10 μM of progesterone, 5α-dihydroprogesterone (5α-DHP) or allopregnanolone (3α, 5α-tetrahydroprogesterone) to the medium at the time of injury rescued the spinal cord slices from the effects of damage. Progesterone prevented membrane cell damage, motoneuron loss and cell death. These effects were not due to its bioconversion to 5α-DHP nor to allopregnanolone, as supported by the finasteride, an inhibitor of 5α-reductase enzymes, and by the absence of 5α-reduced progesterone metabolites in the slices analyzed by gas chromatography–mass spectrometry. The neuroprotective effects of progesterone required PR as they could not be observed in slices from homozygous knockout PR−/− mice. Allopregnanolone treatment was also neuroprotective. Its effects were not due to its bioconversion back to 5α-DHP, which can activate gene transcription via PR, because they were still observed in slices from knockout PR−/− mice. Allopregnanolone effects involved GABAA receptors, as they were inhibited by the selective GABAA receptor antagonist Gabazine, in both PR+/+ and PR−/− mice. Altogether, these findings identify both PR and GABAA receptors as important targets for neuroprotection by progestagens after spinal cord injury,