Selective regulation of 3 alpha-hydroxysteroid oxido-reductase expression in dorsal root ganglion neurons: a possible mechanism to cope with peripheral nerve injury-induced chronic pain.

The enzyme 3alpha-hydroxysteroid oxido-reductase (3alpha-HSOR) catalyzes the synthesis and bioavailability of 3alpha,5alpha-neurosteroids as allopregnanolone (3alpha,5alpha-THP) which activates GABA(A) receptors and blocks T-type calcium channels involved in pain mechanisms. Here, we used a multidisciplinary approach to demonstrate that 3alpha-HSOR is a cellular target the modulation of which in dorsal root ganglia (DRG) may contribute to suppress pain resulting from peripheral nerve injury. Immunohistochemistry and confocal microscope analyses showed 3alpha-HSOR-immunostaining in naive rat DRG sensory neurons and glial cells. Pulse-chase, high performance liquid chromatography and Flo/One characterization of neurosteroids demonstrated 3alpha,5alpha-THP production in DRG. Behavioral methods allowed identification of pain symptoms (thermal and mechanical hyperalgesia and/or allodynia) in rats subjected to sciatic nerve chronic constriction injury (CCI). Reverse transcription and real-time polymerase chain reaction revealed that 3alpha-HSOR mRNA concentration in CCI-rat ipsilateral DRG, 5-fold higher than in contralateral DRG, was also 4- to 6-fold elevated than that in sham-operated or naive rat DRG. Consistently, Western blotting confirmed increased 3alpha-HSOR protein levels in CCI-rat ipsilateral DRG and double immunolabeling showed that 3alpha-HSOR overexpression occurred in DRG neurons but not in glia. Functional plasticity of 3alpha-HSOR leading to increased 3alpha,5alpha-THP production was evidenced in CCI-rat DRG. Interestingly, behavioral and molecular time-course investigations revealed that 3alpha-HSOR gene upregulation was correlated to pain symptom development. Most importantly, in vivo knockdown of 3alpha-HSOR expression in healthy rat DRG using 6-carboxyfluorescein-3alpha-HSOR-siRNA exacerbated thermal and mechanical pain perceptions. This paper is the first to show that siRNA-induced knockdown of a key neurosteroid-synthesizing enzyme directly affects an important function as nociception. Hopefully, these results may be useful for the development of novel analgesics.