Indian Journal of Experimental Biology Vol. 39, December 2001, pp. 1299-1301 Antinociceptive activity of a neurosteroid tetrahydrodeoxycorticosterone (5cx-pregnan-3cx-21-diol-20-one) and its possible mechanism(s) of action P K Mediratta, M Gambhir, K K Sharma & M Ray Department of Pharmacology, University College of Medical Sciences and GTB Hospital, Delhi II 0095, India Fax: 91-11-2299495; E-mail: [email protected]/[email protected] Received 9 Apri/2001; revised 2 July 2001 The present study investigates the effects of a neurosteroid tetrahydrodeoxycorticosterone (5a-pregnan-3a-21-diol-20- one) in two experimental models of pain sensitivity in mice. Tetrahydrodeoxycorticosterone (2.5, 5 mg!kg, ip) dose dependently decreased the licking response in formalin test and increased the tail flick latency (TFL) in tail flick test. Bicuculline (2 mg/kg, ip), a GABAA receptor antagonist blocked the antinociceptive effect of tetrahydrodeoxy­ corticosterone in TFL test but failed to modulate licking response in formalin test. Naloxone (I mglkg, ip), an opioid antagonist effectively attenuated the analgesic effect of tetrahydrodeoxycorticosterone in both the models. Tetrahydrodeoxycorticosterone pretreatment potentiated the anti nociceptive response of morphine, an opioid compound and nimodipine, a calcium channel blocker in formalin as well as TFL test. Thus, tetrahydrodeoxycorticosterone exerts an analgesic effect, which may be mediated by modulating GABA-ergic and/or opioid-ergic mechanisms and voltage-gated calcium channels. It is now well known that some of the steroids like Allopregnanolone has been shown to exhibit progesterone can act on central nervous system (CNS) antinociceptive effect against an aversive thermal to produce a number of endocrine and behavioral stimulus 10 and in a rat mechanical visceral pain 11 effects. Further, enzymes involved in the biosynthesis model • However, the role of tetrahydrodeoxy­ of some of these steroidal hormones are found in cell corticosterone in mediation of pain sensitivity has not specific areas in the brain. These steroids synthesized been well defined. The present study investigates the 1 de novo in the brain are known as neurosteroids • effect of tetrahydrodeoxycorticosterone on tail flick Some of the important steroids characterized in the latency test, a model of acute pain and formalin CNS include progesterone, allopregnanolone (5a­ induced pain response, a model of tonic (continuous) pregnan-3a-ol-20-one), tetrahydrodeoxycorticoster­ pain. one (5a-pregnan-3a-21-diol-20-one), dehydroepian­ Animals-Swiss albino male mice weighing drosterone and pregn-anolone sulfate. It has been 25-30 g were obtained from the Central Animal suggested that neurosteroids may be playing a House of the Institution and housed in 12hr light-12 neuromodulatory role especially the allosteric hr dark cycle at 22°±2°C with food and water bimodal regulation of GABAA receptor chloride available freely except l hr before and during the 2 3 channel , inhibition of glycine chloride channel and experiment. The animals were maintained as per the voltage gated calcium channels (VGCCs)4 and 'Guidelines for the Care and Use of Animals in potentiatiOn of N-methyl-0-aspartate (NMDA) Scientific Research' prepared by Indian National 5 12 receptor responses . Science Academy, Delhi and the approval from the Increased levels of progesterone are reportedly Institutional Ethical Committee was taken. 7 associated with decrease in pain sensitivitl· . Drugs and Chemicals-Tetrahydrodeoxycortico­ However, the degree and mechanism by which sterone, naloxone, bicuculline methiodide (Sigma progesterone exerts its antinociceptive effects are not Chemical Co., USA), nimodipine (Cipla Ltd.) and clearly understood. Progesterone per se is devoid of morphine sulfate were used in the study. binding capacity to GABAA receptors, but in the Tetrahydrodeoxycorticosterone was dispersed in 1% neuronal glia of the brain it is metabolized to Tween 80 and diluted with saline, nimodipine was allopregnanolone and tetrahydrodeoxycortico­ dissolved in 100% ethanol and morphine sulfate, sterone8·9 which are among the most potent of the naloxone and bicuculline were dissolved in distilled 2 known neurosteroids active at GABAA receptors . water. Formalin (0.5%; v/v) solution was prepared by 1300 INDIAN J EXP BIOL , DECEMBER 2001 Table !-Effect oftetrahydrodeoxycorticosterone (THDOC) and its interaction with GABAergic, opioidergic and a calcium channel blocker, nimodipine in tail flick latency and formalin tests (Values are mean± SE from 8- 10 animals in each group) Treatment Tail flick latency Licking response (sec) (mg/kg) (sec) Earl y phase (0-5 min) Late phase (25-30 min) Vehi cle 2.8 ± 0.3 132.2 ± 10.4 92.5 ± 8.6 THDOC (2.5) 4.4 ± 0.8 90.6 ± 8.4'• 54.4 ± m 8'• THDOC (5) 6.4 ± 0.8'"• 62 .4 ± 7.4" 'a 26.6 ± 11 .2"'• THDOC (5) + Bicuculline (2) 3.0 ± o.5" b 65.5 ± 9.4'"• 30.4 ± 8.2"'• THDOC (5) + Naloxone ( I) 3.2 ± 0.6" b 112.4 ± 7.2'"b 90.4 ± 8.5'"b Morphine (2.5) 4.8 ± o.8·· 88.6 ± 8.4'• 52 .6 ± 11.2'• Morphine (5) 7.8 ± J.8' "a 50.4 ± 9.6"'a 24.6 ± 7.2"'a THDOC (2.5) +Morphine (2.5) 6.8 ± 0.2'c 58.8 ± 6.8'c 22.4 ± 8.4'c Nimodipine (20) 2.9 ± 0.6 85.4 ± 8.8·· 42.6 ± 11.2"• Nimodipine (40) 3.2 ± 0.4 58.6 ± 6.7'"• 34.6 ± 9.:("• THDOC (2.5) + Nimodipine (20) 7.2 ± 1.6" 'c 52.4 ± 12.4'c 32.8 ± 6.8'c P va lues: *< 0.05; **< 0.01; ***< 0.001 • Compared to vehicle-treated group h Compared 10 THDOC-treated group c Compared to respective THDOC/Morphine/Nimodipine-treated group adding normal saline to the stock solution of 4% Student's t test and a P value of less than 0.05 was formaldehyde in water. considered as significant. Animals were randomly divided into different The results are summarized 111 Table 1. groups of I 0 mtce 111 each group. Tetrahydrodeoxycorticosterone (2.5, 5 mglkg, ip) Tetrahydrodeoxycorticosterone was injected 1 hr, produced a dose dependent antinociceptive effect in morphine, bicuculline and nimodipine 30 min, both tail flick latency (TFL) test, a model for acute whereas naloxone 15 min, before performing the pain, and formalin induced pain response, a model for analgesic test. The drugs/vehicle were administered tonic continuous pain. There was a significant intraperitoneall y (ip). increase in the TFL and a decrease in time an animal Tail flick latency test- The tail of the animal was spent in licking the formalin injected paw duri ng early placed in the slot of water circulated jacket of as well as late phase in formalin test. The increase in analgesiorneter (TECHNO) which had a tungsten wire TFL produced by tetrahydrodeoxycorticosterone was l mm below the tail surface. The heat was adjusted effectively blocked by both bicuculline (2 mg/kg, ip), dai ly to provide normal reaction time of 2-4 sec. For a GABAA receptor antagonist and naloxone ( l mg/kg, drug studies a cut off time of I 0 sec was used and if ip) an opioid antagoni st. Thus, the analgesic effect of th e mouse did not respond in I 0 sec, it was removed tetrahydrodeoxycorticosterone on TFL seems to be and given a score of 10. mediated via modulation of GABAA chloride channel Fo rmalin test- The test was performed by inj ecting complex as well as opioid-ergic pathways. Unlike its 0.2 ml of 0.5 % formalin under the plantar surface of effect on TFL, the decrease in paw li cking response ri gh t hi nd paw of mouse. The left paw was injected by tetrahydrodeoxycorticosterone was antagonized with 0.2 ml of 0.9% saline and acted as control. The significantly only by naloxone and not by bicuculline animal was th en kept in an open mouse perspex cage indicating thereby th at the opioid-ergic mechanisms and the time spent by the animal licking the injected may be playing a more important role in the paw was recorded. Two di stinct periods of intensive antinociceptive effect of tetrahydrodeoxycorti co­ licking, i.e. an early phase (0-5 min) and late phase sterone. Morphine (2.5, 5 mg/kg, ip) produced a dose (25-30 min) were observed and scored separately for dependent analgesic response in both TFL and assessing drug effect. formalin test. This antinociceptive effect of morphine Sta1istical analysis-All results are expressed as was potentiated by pretreating the animals with n1ean ± SE. The data were analyzed using unpaired tetrahydrodeoxycorticosterone (Table I). This NOTES 1301 observation lends further credence to the hypothesis 3 Wu FS, Gibbs IT & Farb DH. Inverse modulation of y­ that opioid-ergic pathways are playing a significant aminobutyric acid and glycine-induced currents by progesterone. Mol Pharmacal, 37 (1990) 597. role in mediating the analgesic effect of 4 Ffrench-Mullen JMH, Danks P & Spencer KT. Neurosteroids tetrahydrodeoxycorticosterone. modulate calcium currents in hippocampal CAl neurons via a 4 Neurosteroids have been shown to inhibit VGCCs . pertussis toxin-sensitive G protein-coupled mechanism. 1 Further, various calcium channel blockers are Neurosci, 14 (1994) 1963. 15 5 Wu FS, Gibbs IT & Farb DH. Pregnenolone sulfate: a reported to exhibit an antinociceptive effect • Hence, posi tive allosteric modulator at the N-methyl-D-aspartate tetrahydrodeoxycorticosterone may be exerting an receptor. Mol Pharmacal, 40 ( 1991 ) 333. analgesic effect by inhibiting VGCCs also. In the 6 Gintzler JH & Bohan M. Pain threshold are elevated during present study nimodipine, a CCB per se decreased the pseudopregnancy. Brain Res, 507 ( 1990) 312. paw licking response in formalin test but failed to 7 Frye CA, Cuevas CA. Crystal S & Kanarek RB. Diet and estrus cycle influence pain sensiti vity in rats. Pharmacal modulate TFL. This corroborates earlier observations, Biochem Behav, 45 ( 1993) 255.