Intrathecal neurosteroids and a neurosteroid antagonist: Effects on inflammation-evoked thermal hyperalgesia and tactile allodynia

doi:10.1016/j.neulet.2013.05.027

Neuroscience Letters

Volume 548, 26 August 2013, Pages 27-32

https://doi.org/10.1016/j.neulet.2013.05.027 Get rights and content

Highlights

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Spinal allopregnanolone and alphaxalone increase thermal threshold in normal and inflamed paw.
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Effects of allopregnanolone were prevented by neurosteroid antagonist 17PA.
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Reversal of an equi-analgesic dose of alphaxalone occurred only at higher antagonist dosing.
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Spinal neurosteroid binding site(s) acted upon by 17PA regulates spinal pain processing

Abstract

Neurosteroids regulate neuronal excitability though binding sites associated with the ionotropic γ-aminobutyric acid (GABA_A) receptor. We sought to characterize the spinal analgesic actions in rats of two 5α-reduced neurosteroids, allopregnanolone and alphaxalone, on nociceptive processing and to determine whether a putative neurosteroid antagonist attenuates this effect: (3α,5α)-17-phenylandrost-16-en-3-ol (17PA). Intrathecal (IT) injection of allopregnanolone (1–30 μg/10 μL in 20% cyclodextrin) delivered through lumbar catheters produced a dose-dependent analgesia in rats as measured by thermal thresholds in the ipsilateral (inflamed by intraplantar carrageenan) and in the contralateral (un-inflamed paws). Similar observations were made with alphaxalone (30–60 μg in 20% cyclodextrin). Effective doses were not associated with suppressive effects on pinnae, blink or placing and stepping reflex. Effects of allopregnanolone (30 μg) on the normal and hyperalgesic paw were completely prevented by IT 17PA (30 μg). Reversal by IT 17PA of an equi-analgesic dose of alphaxalone occurred only at higher antagonist dosing. These results suggest that a spinal neurosteroid-binding site with which 17PA interacts may regulate spinal nociceptive processing in normal and inflamed tissue.

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 GABA_A 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 GABA_A 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.

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Svensson et al. studied the comparative effect of allopregnanolone and alphaxalone linked with cyclodextrin for the spinal analgesic actions in rats using the IT route. The results suggested that the IT administration of both actives in combination with cyclodextrin regulated the spinal nociceptive processing in inflamed tissue, whereas the complex formation enhanced the penetration across the thick epithelial tissue and spinal neurosteroid-binding site [81]. Ken et al. demonstrated the prolongation action of local anesthetic of levobupivacaine-complexed with maltosyl-β-cyclodextrin (G2-β-CD) administered at spinal and sciatic nerve blocks in rats.
The modulation of delivery systems using subarachnoid space is considered a breakthrough for direct administration of active to the targeted receptor for treating central nervous system ailments such as epilepsy, Alzheimer's disease, dementia, migraine, stroke, Parkinsonism, cancer, etc. This review article focuses on various insights to tweak the modulations for subarachnoid space delivery, nanocarrier systems for subarachnoid delivery and clinical trials to enlighten the applications of nanomedicine for the treatment of brain conditions. The strategic modulation in the subarachnoid delivery, such as changing the selective parameters of drug possessing the biophysical property, is considered a compatible way for distribution of drug through subarachnoid space to overcome the issue of a longer period for cerebrospinal fluid clearance, thus reducing the frequency of administration. Another parameter, such as conjugation of active with nanocarriers such as liposomes, dendrimers, cyclodextrins, polymers, stem cells, etc., will help in increasing the solubility of the hydrophobic drug and promoting the entry of such nano-molecules into lipophilic cell membranes to enhance the bioavailability. Direct delivery to subarachnoid space offers an immediate action in case of receptor-oriented delivery at a desirable site with a lower dose and lesser associated side effects. However, limitations of drug-like improper pharmacokinetics and inadequate tissue distribution, development of diseases like fibrosis due to repeated frequency of administration and high risk of infection remain serious concerns. In near future, nanocarriers will highlight the burgeoning of delivery at subarachnoid space for immediate and long-term therapy with higher safety in treatment of various central nervous system conditions such as subarachnoid hemorrhage, cancer, Alzheimer's disease, Parkinsonism, etc.
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As discussed (Section 2), electrophysiological and immunohistochemistry studies reveal mouse VB neurons to express neurosteroid-sensitive synaptic (α1β2γ2) and extrasynaptic (α4β2δ) GABAARs (Peden et al., 2008; Herd et al., 2013; Brown et al., 2015 – Fig. 1). Allopregnanolone is active in numerous models of acute and pathological pain states, with evidence of a neuroprotective effect (Kavaliers and Wiebe, 1987; Svensson et al., 2013; Borowicz et al., 2011; Rey and Coirini, 2015). The antinociceptive effects of allopregnanolone and ganaxolone are impaired in the δ−/- mice (Mihalek et al., 1999).
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In addition to the sigma-1 receptors, we also evaluated the potential effects of DHEAS and PREGS on peripheral GABAA and NMDA receptors, which are critical modulatory receptors in neuronal excitability (Chisari et al., 2010; Harrison and Simmonds, 1984; Monnet et al., 1995; Zhu and Paoletti, 2015). In the spinal cord, GABAA or NMDA receptors are involved in neurosteroid-induced pain hypersensitivity (Svensson et al., 2013; Yoon et al., 2009, 2010). However, the potential action of neurosteroids on peripheral GABAA or NMDA receptors has not been reported.
The role of peripheral neurosteroids and their related mechanisms on nociception have not been thoroughly investigated. Based on emerging evidence in the literature indicating that neurosteroids and their main target receptors, i.e., sigma-1, GABA_A and NMDA, affect P2X-induced changes in neuronal activity, this study was designed to investigate the effect of peripherally injected dehydroepiandrosterone sulphate (DHEAS) and pregnenolone sulfate (PREGS) on P2X receptor-mediated mechanical allodynia in rats. Intraplantar injection of either neurosteroids alone did not produced any detectable changes in paw withdrawal frequency to the innocuous mechanical stimulation in naïve rats. However, When DHEAS or PREGS were co-injected with a sub-effective dose of αβmeATP, mechanical allodynia was developed and this was dose dependently blocked by pre-injection of the P2X antagonist, TNP-ATP. These results demonstrates that DHEAS and PREGS potentiate the activity of P2X receptors which results in the enhancement of αβmeATP-induced mechanical allodynia. In order to investigate the potential role of peripheral sigma-1, GABA_A and NMDA receptors in this facilitatory action, we pretreated animals with BD-1047 (a sigma-1 antagonist), muscimol (a GABA_A agonist) or MK-801 (a NMDA antagonist) prior to DHEAS or PREGS + αβmeATP injection. Only BD-1047 effectively prevented the facilitatory effects induced by neurosteroids on αβmeATP-induced mechanical allodynia. Collectively, we have shown that peripheral neurosteroids potentiate P2X-induced mechanical allodynia and that this action is mediated by sigma-1, but not by GABA_A nor NMDA, receptors.

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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.

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Published by Elsevier Ireland Ltd.

Intrathecal neurosteroids and a neurosteroid antagonist: Effects on inflammation-evoked thermal hyperalgesia and tactile allodynia☆

Highlights

Abstract

Introduction

Section snippets

Animals

Drugs

Agonist effects upon thermal hyperalgesia

Discussion

Conclusions

Acknowledgments

Pharmacol. Ther.

J. Neurosci. Methods

J. Neurosci. Methods

J. Mol. Cell. Cardiol.

Neuropharmacology

Eur. J. Pharmacol.

Pain

Pain

Prog. Brain Res.

Br. J. Anaesth.

Life Sci.

Life Sci.

Physiol. Behav.

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.

Neurosteroids: endogenous regulators of the GABA_A receptor

Laminar compartmentalizationof GABA_A-receptor subtypes in the spinal cord: an immunohistochemical study

Junctional versus extrajunctional glycine and GABA_A receptor-mediated IPSCs in identified lamina I neurons of the adult rat spinal cord