BACKGROUND: Gabapentin binds at the extracellular α2δ1 subunit of voltage- sensitive calcium channels. Some voltage-sensitive calcium channels regulate substance P release from small primary afferents. We sought to determine in vivo whether spinal and systemic gabapentin at antihyperalgesic doses will attenuate substance P release.
METHODS: Rats prepared with chronic intrathecal (IT) catheters received IT vehicle or gabapentin 10 minutes before intraplantar formalin (5%, 50 μL) injection. For systemic studies, vehicle or gabapentin was delivered intraperitoneally (IP) 15 minutes before formalin injection. In separate groups of rats, to assess the effect of IT or IP gabapentin upon formalin-evoked substance P release, animals received similar treatment for assessment of flinching, but underwent transcardial perfusion with 4% paraformaldehyde 10 minutes after the formalin injection. Substance P release was determined by the incidence of neurokinin 1 receptor (NK1r) internalization in the ipsilateral and contralateral superficial dorsal horn in immunofluorescent stained tissues.
RESULTS: Unilateral intraplantar formalin evoked biphasic hindpaw flinching. IT gabapentin (100 and 200 μg) and IP gabapentin (100 and 200 mg/kg) resulted in a dose-dependent reduction in phase 2, but not phase 1, flinching in comparison with vehicle-treated rats. Intraplanatar formalin resulted in NK1r internalization in the ipsilateral, but not contralateral, superficial dorsal horn. IT gabapentin (200 μg, but not 100 μg) and IP gabapentin (200 mg/kg, but not 100 mg/kg) significantly reduced ipsilateral NK1r internalization in comparison with vehicle-treated control. Importantly, internalization evoked by IT substance P was not blocked by IT gabapentin.
CONCLUSION: Systemic and spinal gabapentin have an acute inhibitory effect on the release of substance P from small primary afferents and a concurrent effect upon the initiation of facilitated pain states.
Published ahead of print March 8, 2011
From the *Department of Anesthesiology, Dokkyo Medical University, School of Medicine, Tochigi, Japan; and †Department of Anesthesiology, University of California, San Diego, La Jolla, California.
Funding: National Institutes of Health: NIH-DA02110, Bethesda, Maryland (to TLY).
The authors declare no conflict of interest.
Preliminary data were presented as a poster at the American Society of Anesthesiology Annual meeting, San Diego, California, 2010.
Reprints will not be available from the authors.
Tony L. Yaksh is one of the Section Editors of Pain Mechanisms for the Journal. This manuscript was handled by Quinn H. Hogan, the second Section Editor of Pain Mechanisms, and Dr. Yaksh was not involved in any way with the editorial process or decision.
Address correspondence to Tony L. Yaksh, PhD, Department of Anesthesiology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92103-0818. Address e-mail to email@example.com.
Accepted January 7, 2011
Published ahead of print March 8, 2011
Gabapentin has a prominent antihyperalgesic action after either systemic or spinal (intrathecal: IT) delivery.1–5 Previous work emphasized that the molecule binds with high affinity to the α2δ1 extracellular auxiliary subunit of voltage-sensitive calcium channels (VSCCs), particularly the N- and L-, but not T-, types.6,7 Point mutation studies of the α2δ1 protein showed that such changes had no effect upon calcium channel function, but completely prevented the binding and actions of gabapentin.8 Although the analgesic efficacy of gabapentin in various facilitated pain states is well known, its mechanism of action remains uncertain. Given the structural link between α2δ1 and the pore-forming elements of the VSCCs and the presence of α2δ1 in dorsal root ganglion (DRG),9 a prevalent hypothesis is that gabapentin regulates afferent terminal transmitter release through regulation of calcium channel function. However, this hypothesis is complex. Thus, although an acute regulation by gabapentin of DRG calcium currents has been reported,10–12 gabapentin had only modest effects upon the frequency and amplitude of excitatory postsynaptic currents in superficial dorsal horn neurons.13 That work suggests that ongoing presynaptic neurotransmitter release is not regulated by this drug. Direct measurement of release in spinal slices has shown that gabapentin can diminish release of glutamate and several peptides,14,15 though with the caveat that the effects appear limited to conditions associated with prior sensitization or lengthy drug exposures.16,17 Other work has emphasized that gabapentin's actions may be dependent upon the up-regulation of α2δ1 as occurs after nerve injury9 or may alter calcium channel trafficking.18 Such hypotheses imply the likelihood that the antihyperalgesic actions of gabapentin are limited to events that may require exposures of hours to days. Yet it is evident from the acute antihyperalgesic effects of gabapentin in vivo that other acute mechanisms must be in play. The present experiments aimed to determine the effects of gabapentin in vivo on the spinal release of substance P evoked by intraplantar formalin injection. Because dorsal horn substance P is primarily contained in small TRPV1 (+) (transient receptor potential vanilloid 1 receptor) primary afferents, changes in its release reflect an effect upon the releasing function of that small afferent terminal, in contrast to a transmitter such as glutamate, which may arise from many neurons and even glia. In the present work, substance P release was examined in vivo by assessing the internalization of the neurokinin/receptor (NK1r) in the superficial dorsal horn produced by unilateral intraplantar formalin injection. NK1rs are densely located on lamina I projection neurons, but are largely absent from lamina II neurons.19,20 Finally, we sought to compare the effects of gabapentin given IT and intraperitoneally (IP) at equianalgesic doses. Previous work with morphine interestingly demonstrated that internalization was blocked at analgesic doses after IT delivery, but supra-analgesic doses after systemic delivery.21 As intraplantar formalin injection evokes a well-defined biphasic flinching response, the in vivo activity of gabapentin on release and pain behavior can be compared.
Male Holtzman Sprague–Dawley rats (250 to 300 g; Harlan, Indianapolis, IN) were individually housed and maintained on a 12-hour light/dark cycle with testing occurring only during the light cycle. Food and water were available ad libitum. Animal care was in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 85 to 23, Bethesda, MD) and as approved by the institutional Animal Care and Use Committee of the University of California, San Diego.
Intrathecal Catheter Implantation
Rats were implanted with a single IT catheter for drug delivery.22,23 In brief, rats anesthetized with isoflurane in an air/oxygen mixture (1:1) were implanted with a polyethylene (outside diameter 0.36 mm) catheter passed through the cisternal membrane into the IT space to the level of the L2 to 3 spinal segments (approximately 8.5 cm) and externalized on top of the head. Animals were given 5 mL of lactated Ringer's solution subcutaneously, and they recovered under a heat lamp. Rats without motor deficit were allowed to recover for 5 to 7 days before study.
Formalin-Induced Paw-Flinching Behavior
To assess formalin-evoked flinching, we placed a metal band around the left hindpaw of the animal, and we allowed the rat to acclimate 30 minutes before experimental manipulation. At time 0, formalin (5%, 50 μL) was delivered into the left hindpaw. Immediately after the formalin injection, rats were placed into the test chamber and nociceptive behavior (flinching and shaking of the injected paw) was quantified by an automatic flinch counting device (UARDG, Department of Anesthesiology, University of California, San Diego).24 Flinches were counted in 1-minute intervals for 60 minutes. The data are expressed as total number of flinches observed during phase 1 (0 to 10 minutes) and phase 2 (11 to 60 minutes).
Drugs and Delivery
Gabapentin (Sigma Chemical, St. Louis, MO) was dissolved in 0.9% saline to be delivered in a volume of 10 μL for IT delivery or to be delivered IP in a volume of 1.5 mL/kg. Drugs were administered IT 10 minutes or IP 15 minutes before the intraplantar delivery of formalin (5%, 50 μL).
Effects of Intrathecal Gabapentin on NK1r Internalization Induced by Exogenous Substance P
To exclude the possibility that gabapentin directly blocked the NK1r internalization mechanism, we administered IT saline or gabapentin to rats before IT substance P (30 nmol). Thirty minutes later, rats were killed and fixed. Spinal NK1r immunoreactive neurons, with or without NK1r internalization, were counted.
Tissue Preparation and Immunocytochemistry
For tissue harvest (10 minutes after intraplantar formalin), rats were deeply anesthetized with isoflurane and transcardially perfused with 0.9% NaCl, followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PBS), pH 7.4. The lumbar cord was removed and postfixed overnight. After cryoprotection in 30% sucrose, coronal sections were taken using a sliding microtome (30 μm). To assess NK1r expression in the spinal cord dorsal horn, we incubated sections with a rabbit anti-NK1r polyclonal antibody (1:3000 in 0.01 M PBS containing 10% normal goat serum and 10% Triton X-100 (Advanced Targeting Systems, San Diego, CA) overnight at room temperature. After a rinse in PBS, sections were incubated for 120 minutes at room temperature in a goat antirabbit secondary antibody conjugated with Alexa Fluor 488 (1:1000 in same buffer; Invitrogen, Eugene, OR). All sections were rinsed, mounted on glass slides, and coverslipped with ProLong mounting medium (Fisher Scientific, Pittsburgh, PA).
Quantification of NK1r Internalization
NK1r internalization was counted using an Olympus BX-51 fluorescence microscope (Olympus Optical, Tokyo, Japan), and a 60× oil-immersion objective lens and internalization was assessed according to the standard of previous reports.19,25 The filter set was Omega Optical XF100 to 2 Green Bandpass Filter. Neuronal profiles that had 10 or more endosomes were considered to have internalized NK1rs. The total number of NK1r immunoreactive neurons in lamina I/II, with and without NK1r internalization, was counted and taken to calculate the fraction of cells showing internalization. Counting was done without knowledge of treatments. Mean counts from 2 to 5 sections per segment were used as representative counts for a given animal.26 Images were taken using Magna FIRE SP and processed by Adobe Photoshop CS4. Nomenclature for drugs and receptors conforms with the guide to receptors and channels of the British Journal of Pharmacology.27
Changes in paw-flinching behavior were analyzed using 1-way analysis of variance (ANOVA) for phases 1 and 2 with Tukey post hoc tests performed for pairwise comparisons. Analysis of NK1r internalization data consisted of 1-way or 2-way ANOVAs with Tukey post hoc analysis. Significance was defined as P < 0.05.
Effects of Intrathecal and Intraperitoneal Gabapentin on Formalin Evoked NK1r Internalization
NK1r immunoreactivity was typically observed outlining the cytosolic membrane in many superficial dorsal horn neurons (Fig. 1B). Unilateral intraplantar injection of formalin (5%, 50 μL) produced a robust NK1r internalization in ipsilateral superficial dorsal horn (Figs. 1A, 2C) but not contrallateral dorsal horn (ipsilateral L4: 26 ± 5%, L5: 62 ± 5%, L6: 62 ± 8%; contralateral L4: 12 ± 4%, L5: 5 ± 2%, L6: 14 ± 6%, P < 0.05). IT gabapentin (200 μg but not 100 μg) showed a prominent reduction in the ipsilateral L5 and L6 increase in formalin-evoked internalization (P < 0.05) (Fig. 2C). IP gabapentin (200 mg/kg, but not 100 mg/kg) significantly reduced the formalin-induced NK1r internalization at L5 and L6 levels of the superficial lumbar spinal cord (P < 0.05) (Fig. 3C).
Effects of Intrathecal Gabapentin on NK1r Internalization Induced by Exogenous Substance P
IT substance P (30 nmol) produced widespread NK1r internalization at L4 to 6 levels of spinal cord lamina I in comparison with IT saline (P < 0.05) (Fig. 4, A–C). IT gabapentin (200 μg), a dose that completely blocked formalin-induced NK1r internalization, did not affect exogenous substance P–induced NK1r internalization (Fig. 4, B and D).
Effects of Intrathecal and Intraperitoneal Gabapentin on Formalin Evoked Flinching Behavior
Intraplantar formalin resulted in a robust biphasic incidence of flinching over a 60-minute period. Pretreatment with IT gabapentin (100 and 200 μg) resulted in a dose-dependent suppression of phase 2 flinching with no effect on phase 1, in comparison with vehicle-treated control (Fig. 2, A and B). Similarly, after IP delivery, gabapentin (100 and 200 mg/kg) diminished phase 2, but not phase 1, in comparison with vehicle control (Fig. 3, A and B). Importantly, IT gabapentin at the highest dose did not produce adverse effects on behavior or motor function, in comparison with vehicle control (Table 1). IP 100 and 200 mg/kg gabapentin were observed to be associated with reduced spontaneous activity in some rats. No other motor effects were noted.
As reviewed, binding to the α2δ1subunit associated with VSCCs is likely essential for the spinal antihyperalgesic actions of gabapentin and its analogues.28 The association of α2δ1subunits with VSCCs in DRGs, along with the likelihood that one or more of these VSCCs may be instrumental in mediating depolarization-evoked transmitter release, raises the possibility that gabapentin may regulate release from primary afferents.29
Afferent Substance P Release
The present results indeed indicate that gabapentin given either IT or IP exerts a potent, short latencied effect in vivo upon small afferent-evoked substance P release. Previous work showed that IT gabapentin reduced formalin-induced release of glutamate in spinal dorsal horn30 and systemic gabapentin reduced spinal glutamate release after a visceral irritant.31 However, unlike the present work with substance P, the effects of gabapentin on spinal glutamate release cannot necessarily be ascribed to primary afferents, given the ubiquitous nature of spinal glutamate distribution. The present effects of gabapentin on substance P release are in accordance with recent work in which we showed that substance P release in spinal dorsal horn can be attenuated in vivo by IT N-type, but not T- or L-type, calcium channel blockers.32
The use of NK1r internalization as an index of primary afferent substance P release has been validated by several criteria and reviewed previously.21,33 An important caveat is the concern that the agent has a direct effect upon the postsynaptic internalization process. We showed that internalization produced by exogenous IT substance P was, however, not altered by IT gabapentin, indicating that the suppressant effects on evoked release was not due to a direct effect on the internalization process.
Facilitated Pain States
The present work confirmed a potent antinociceptive effect of gabapentin given IT or IP upon phase 2, but not phase 1, of the formalin-evoked flinching, a finding consistent with those previously reported.1–5 Consistent with a spinal action of gabapentin, the effective total dose was 200 μg IT versus 60 mg IP (e.g., total dose for 200 mg/kg in a 300-g rat).
Correlation Between Effects on Substance P Release and Antinociception
The functional relevance of the IT/IP gabapentin effects on release is suggested by the parallel between behaviorally relevant doses and those required to reduce release. We note, however, that after both IT and IP delivery, phase 2 flinching was reduced at doses lower than those required to produce reduction in NK1r internalization. Whether this represents a component of gabapentin's action on the flinching model, which is not mediated by effects on peptidergic C-fibers, an effect upon other spinal systems, or even nonspinal systems34 is not known.
Effect of Gabapentin on Acute Facilitation
One feature of gabapentin's actions is its effects upon facilitated pain states, which may be initiated within minutes of stimulation (phase 2 flinching and tissue injury-evoked hyperalgesia) but not upon acute nociception (phase 1 flinching, thermal thresholds).1–5,35 An example of the acuity of this effect is the observation that although IT or IP gabapentin or pregabalin (S(+)-3-isobutyl-γ aminobutyric acid) has no effect upon acute thermal escape latencies, they reverse the thermal hyperalgesia evoked in seconds to minutes by IT substance P, an effect reflecting a postsynaptic effect mediated by substance P on the NK1r. The specificity of this effect is emphasized by the observation that an isomer of pregabalin (R(-)-3-isobutyl-γ aminobutyric acid) with lower α2δ1 binding affinity was less active in vivo.36 This differential effect upon acute versus facilitated states distinguishes the actions of gabapentin from those of other drugs that block small afferent release, such as opiates.37 The observation that gabapentin did not block NK1r internalization evoked by IT substance P, and yet blocked the hyperalgesia evoked by IT substance P, raises the interesting likelihood that gabapentin has several sites of spinal action, one of which is presynaptic on the afferent (to block release of substance P and likely other afferent transmitters such as glutamate) and one of which appears not to be on the primary afferent.13 Where the nonafferent action is we do not know, but the lack of effect upon internalization evoked by IT substance P emphasizes that the gabapentin effect was not due to an artifact of an action on the internalization process.
Several mechanisms for this acute action on facilitated states present themselves. (i) The effects upon presynaptic release initiated by persistent small afferent input, as generated during the first phase,38 may represent distinct processes in the presynaptic terminal leading to release. Protein kinase C and adenylyl cyclase activation exert a potent facilitatory effect upon glutamate release in trigeminal slices, and this facilitation (but not acute release) is blocked by gabapentin, perhaps through reduced phosphorylation of associated VSCCs.14 (ii) α2δ1subunits are distributed in a variety of DRG cells.18 Accordingly, there are likely effects upon terminals that are not peptidergic. (iii) In dorsal horn, α2δ1 may be associated with nonafferent terminals. As has been noted, gabapentin blocks hyperalgesia initiated by spinal NK1 activation.36 In this vein, gabapentin blocks substance P–evoked NF-κB activation in cell systems by blockade of IkB-α degradation, thereby preventing translocation of p65 to the nucleus.39
Mechanisms of Actions of Gabapentins
As is discussed above, the membrane level, the role of the binding α2δ1 site in directly regulating calcium channel function, has been controversial. The present studies, however, do emphasize the presynaptic effect upon small afferent terminal-releasing properties. Other mechanisms that may be associated with the actions of gabapentin include the trafficking of intracellular VSCC, prevented by gabapentin,18 and spouting after nerve injury. Thus, α2δ1binds the thrombospondin family of extracellular matrix proteins, which play a role in intracellular trafficking and synaptogenesis.40,41 Importantly, these proteins are secreted by glia.40 Previous work has indicated that even acute tissue injury or IT substance P can initiate glial activation as measured by, for example, phosphorylation of glial P38 mitogen-activated protein kinase.42
As is outlined above, however, an important element in the present study is that it draws attention to the acuity (seconds to minutes) of the biological effects of gabapentin, including suppression of the release of at least 1 small afferent transmitter and the acute changes in facilitated spinal states. Thus, there is little doubt that up-regulation of the α2δ1 subunits occurs after nerve injury43 and that chronic gabapentin exposure may lead to a down-regulation of the associated VSCCs16 or other intracellular changes17 that alter VSCCs insertion into the cell membrane. The acute manifestations observed in vivo clearly represent an effect upon a constitutively expressed and functional spinal system that does not require additional changes in protein expression or channel mobilization or extended drug exposure.9 This profile of action thus forces us to consider the likelihood that additional mechanisms may be called into play that define the regulation exerted at the spinal level in vivo by the α2δ1 binding exhibited by gabapentin and its homologues.
In conclusion, the present work emphasizes that IT and systemic administration of gabapentin acutely regulate spinal release of substance P from small primary afferents at doses that are associated with its antihyperalgesic action. These results, along with previous electrophysiological13 and behavioral36 work consistent with a postsynaptic action, suggest that a number of mechanisms may reflect the acute actions of gabapentin and indirectly the role of the spinal α2δ1 subunit to which it binds.
Name: Toshifumi Takasusuki, MD, PhD.
Contribution: Conduct of study and data analysis.
Name: Tony L. Yaksh, PhD.
Contribution: Study design and manuscript preparation.
We would like to thank Arbi Nazarian, PhD, for his assistance in setting up the internalization protocol.
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