Increased levels of neurosteroids in response to injury: analysis by gas chromatography/mass spectrometry.

SCHUMACHER M; LIERE P; LABOMBARDA F; MEFFRE, D; STEIN D; DE NICOLA A; GUENNOUN, R

Toronto Canada

Congreso; The Endocrine Society ' s 89th Annual Meeting; 2007

The Endocrine Society

Sensitive methods for the accurate analysis and quantification of steroids in plasma and tissues are an important issue. Indeed, the reliability of methods which continue to be used for the analysis of steroids remains a serious problem. This concerns both the methods for quantitative analysis, such as immunoassays, and the methods for pretreatment of the biological samples, including steroid extraction, purification and fractionation. The danger of indirect measurements of conjugated steroids is well illustrated by the finding that sulfate esters of 3 -hydroxy- 5 steroids have been artefactually detected in rodent plasma and brain during decades. We have used a sample workup method coupled to gas chromatography/mass spectrometry (GC/MS) analysis for the precise and sensitive measure of unconjugated and sulfated steroids. Results confirm the absence of steroid sulfates in rat brain and plasma. The measure of unconjugated steroids by GC/MS has allowed to provide reference values for steroid levels in different parts of the nervous system, and to show that an increase in the synthesis of progesterone and its 5 -reduced metabolites is part of the response of neural cells to injury. This was shown by using two lesion models in the adult male rat : transection of the spinal cord and contusion injury of the prefrontal cortex. Levels of pregnenolone and progesteronewere increased in the spinal cord of castrated and adrenalectomized male rats in response to transection. As the animals were deprived of their steroidogenic endocrine glands, these findings strongly suggest an increase in the local synthesis of these steroids. Moreover, increased levels of 5 -dihydroprogesterone and allopregnanolone were measured in the spinal cord of lesioned animals, whithout changes in their plasma levels. Within only six hours after contusion injury, levels of pregnenolone, progesterone and 5 -dihydroprogesterone were significantly increased and those of testosterone decreased in the brain of gonadally intact male rats. In contrast, plasma levels of 5 -dihydroprogesterone did not change in reponse to injury, suggesting the local activation of the 5 -reduction pathway of progesterone in the brain. These findings strongly suggest that increased neurosteroid synthesis is a mechanism by which neural cells cope with neurodegeneration. This is consistent with the neuroprotective and promyelinating effects of progesterone and allopregnanolone.