Intrathecal neurosteroids and a neurosteroid antagonist: Effects on inflammation-evoked thermal hyperalgesia and tactile allodynia☆
Introduction
Neurosteroids have analgesic, anesthetic and anticonvulsant effects [14], [26]. Spinal delivery of 5α-reduced neurosteroids such as allopregnanolone (3α,5α-tetrahydroprogesterone or 3α,5α-THP) reduces the facilitated nociceptive response produced by repetitive nerve stimulation [4], [20], [23] or visceral inflammation [31]. Antagonism of these effects by IT bicuculline [9] suggests that these agents facilitate activation by γ-amino butyric acid (GABA) of the GABAA chloride ionophore and at higher concentrations by directly activating the ionophore [1], promoting the channel open state [2]. Molecular studies have shown two neurosteroid binding sites that mediate the potentiating and direct channel activating effect of neurosteroids [12]. Ample evidence supports the ability of neurosteroids to amplify dorsal horn GABAA receptor activity regulating components relevant to dorsal horn pain processing [6], [24], [28], [33].
Evidence for the action of neurosteroids at a specific site is further provided by the observation that neurosteroid activity is antagonized by agents such as (3α,5α)-17-phenylandrost-16-en-3-ol (17PA). 17PA has little effect on baseline GABA-mediated Cl flux but antagonizes gating and conductance augmentation produced by 5α-reduced steroids, such as allopregnanolone [15], [18]. We note that two 5α-reduced steroids, allopregnanolone and alphaxalone ((3α,5α)-3-hydroxypregnane-11,20-dione), both produce sedation but only the effects of allopregnanolone are reversed by 17PA when administered systemically in vivo. This differential effect has been taken to support different neurosteroid binding sites [15]. Whether this differential effect is observed viz. the analgesic effects of these two neurosteroids is not known. Thus, we examined: (i) spinal actions of allopregnanolone and alphaxalone in regulating inflammation evoked hyperalgesia; (ii) dose dependency of this spinal action; and (iii) whether IT 17PA would antagonize spinal actions of IT allopregnanolone and/or alphaxalone, supporting an interaction with a spinal neurosteroid binding site.
Section snippets
Animals
Adult male rats (Holtzman, 200–250 g) (12/12 day–night cycle, ad libitum access to food/water) were used in accordance with protocols approved by the Institutional Animal Care and Use Committee at the University of California, San Diego. To allow IT delivery, rats were prepared with chronic lumbar IT catheters placed 8.5 cm from the cisterna magna and externalized on the top of the head [17], [35].
Drugs
The 5α-reduced neurosteroid agonists, allopregnanolone and alphaxalone, and the neurosteroid
Agonist effects upon thermal hyperalgesia
Baseline thermal escape latencies prior to drug treatment was similar for all treatment groups (approximately 9–11 s) in vehicle treated animals, intraplantar carrageenan reduced the thermal escape latency of the inflamed (ipsilateral) paw, which persisted undiminished through the 4 h time point (approximately 3–5 s, see Fig. 1), with no change in the normal (contralateral) paw withdrawal latency. IT cyclodextrin (20%/10 μL) had no effect upon the thermal withdrawal latency of the ipsilateral vs.
Discussion
Intrathecal delivery of 5α-reduced neurosteroid agonists allopregnanolone and alphaxalone in a cyclodextrin-water-based vehicle reverse, in a dose-dependent fashion, thermal and mechanical hyperalgesia. These effects occurred at doses that are not systemically active and which have no effect upon general function or motor function. These results are in accord with previous work showing that IT delivery of several neurosteroid-like molecules produces a significant effect upon facilitated pain
Conclusions
We demonstrated that two structurally similar 5α-reduced neurosteroids produce a potent antinociception and antihyperalgesic action. A putative neurosteroid receptor antagonist had distinguishable potency in reversing the effects of the two agents, consistent with the hypothesis of possible multiple targets for spinal neurosteroids.
Acknowledgments
Funding was provided by NIDA 02110. We would like to thank Damon McCumber for performing some of the studies shown here.
References (36)
- et al.
Mechanisms of neurosteroid interactions with GABAA receptors
Pharmacol. Ther.
(2007) - et al.
Quantitative assessment of tactile allodynia in the rat paw
J. Neurosci. Methods
(1994) - et al.
Characterization of variables defining hindpaw withdrawal latency evoked by radiant thermal stimuli
J. Neurosci. Methods
(1997) - et al.
Caveolin-3 expression and caveolae are required for isoflurane-induced cardiac protection from hypoxia and ischemia/reperfusion injury
J. Mol. Cell. Cardiol.
(2008) - et al.
Conserved site for neurosteroid modulation of GABAA receptors
Neuropharmacology
(2009) - et al.
Antagonism of neurosteroid modulation of native gamma-aminobutyric acid receptors by (3alpha,5alpha)-17-phenylandrost-16-en-3-ol
Eur. J. Pharmacol.
(2007) - et al.
New evidence that both T-type calcium channels and GABAA channels are responsible for the potent peripheral analgesic effects of 5alpha-reduced neuroactive steroids
Pain
(2005) - et al.
Neuroactive steroids inhibit spinal reflex potentiation by selectively enhancing specific spinal GABA(A) receptor subtypes
Pain
(2009) Neurosteroids: endogenous role in the human brain and therapeutic potentials
Prog. Brain Res.
(2010)- et al.
The interaction of anaesthetic steroids with recombinant glycine and GABAA receptors
Br. J. Anaesth.
(2004)
Analgesic effects of intrathecally administered 3 alpha-hydroxy-5 alpha-pregnan-20-one in a rat mechanical visceral pain model
Life Sci.
The utility of 2-hydroxypropyl-beta-cyclodextrin as a vehicle for the intracerebral and intrathecal administration of drugs
Life Sci.
Chronic catheterization of the spinal subarachnoid space
Physiol. Behav.
Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence misconceptions and control strategies
Biochim. Biophys. Acta
Neurosteroids: endogenous regulators of the GABAA receptor
Nat. Rev.
Laminar compartmentalizationof GABAA-receptor subtypes in the spinal cord: an immunohistochemical study
J. Neurosci.
Acute neurosteroids inhibit the spinal reflex potentiation via GABAergic neurotransmission
Am. J. Physiol. Renal Physiol.
Junctional versus extrajunctional glycine and GABAA receptor-mediated IPSCs in identified lamina I neurons of the adult rat spinal cord
J. Neurosci.
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- ☆
Work was undertaken in partial fulfillment of the requirement for the Master thesis in pharmaceutical science by ES and JP at the University of Uppsala School of Pharmacy.
- 1
These authors contributed equally to this work.
- 2
Present address: Svartbäcksgatan 83B, 75333 Uppsala, Sweden.
- 3
Present address: St. Johannesgatan 32B, 75233 Uppsala, Sweden.