Bethany Fitzsimmons

Intrathecal minocycline attenuates peripheral inflammation-induced hyperalgesia by inhibiting p38 MAPK in spinal microglia.

Activation of p38 mitogen‐activated protein kinase (p38) in spinal microglia is implicated in spinal nociceptive processing. Minocycline, a tetracycline derivative, displays selective inhibition of microglial activation, a function that is distinct from its antibiotic activity. In the present study we examined antinociceptive effects of intrathecal (IT) administration of minocycline in experimental models of inflammation‐evoked hyperalgesia in addition to the effect of minocycline on stimulation‐induced activation of p38 in spinal microglia. Intrathecal minocycline produced a dose‐dependent reduction of formalin‐evoked second‐phase flinching behaviour in rats, and prevented thermal hyperalgesia induced by carrageenan injection into the paw. In contrast, systemic delivery (intraperitoneally) of minocycline inhibited the first but not the second phase of formalin‐induced flinching, and it had no effect on carrageenan‐induced hyperalgesia. Centrally mediated hyperalgesia induced by IT delivery of N‐methyl‐d‐aspartate was completely blocked by IT minocycline. An increase in phosphorylation (activation) of p38 (P‐p38) was observed in the dorsal spinal cord after carrageenan paw injection, assessed by both Western blotting and immunohistochemistry. The increased P‐p38 immunoreactivity was seen primarily in microglia but also in a small population of neurons. Minocycline, at the IT dose that blocked carrageenan‐induced hyperalgesia, also attenuated the increased P‐p38 in microglia. In addition, minocycline suppressed lipopolysaccharide‐evoked P‐p38 in cultured spinal microglial cells. Taken together, these findings show that minocycline given IT produces a potent and consistent antinociception in models of tissue injury and inflammation‐evoked pain, and they provide strong support for the idea that this effect is mediated by direct inhibition of spinal microglia and subsequent activation of p38 in these cells.

Spinal p38β isoform mediates tissue injury‐induced hyperalgesia and spinal sensitization

Antagonist studies show that spinal p38 mitogen‐activated protein kinase plays a crucial role in spinal sensitization. However, there are two p38 isoforms found in spinal cord and the relative contribution of these two to hyperalgesia is not known. Here we demonstrate that the isoforms are distinctly expressed in spinal dorsal horn: p38α in neurons and p38β in microglia. In lieu of isoform selective inhibitors, we examined the functional role of these two individual isoforms in nociception by using intrathecal isoform‐specific antisense oligonucleotides to selectively block the expression of the respective isoform. In these rats, down‐regulation of spinal p38β, but not p38α, prevented nocifensive flinching evoked by intraplantar injection of formalin and hyperalgesia induced by activation of spinal neurokinin‐1 receptors through intrathecal injection of substance P. Both intraplantar formalin and intrathecal substance P produced an increase in spinal p38 phosphorylation and this phosphorylation (activation) was prevented when spinal p38β, but not p38α, was down‐regulated. Thus, spinal p38β, probably in microglia, plays a significant role in spinal nociceptive processing and represents a potential target for pain therapy.

Spinal phosphinositide 3-kinase-Akt-mammalian target of rapamycin signaling cascades in inflammation-induced hyperalgesia.

Phosphinositide 3-kinase (PI3K), Akt, and their downstream kinase, mammalian target of rapamycin (mTOR), are implicated in neural plasticity. The functional linkages of this signaling cascade in spinal dorsal horn and their role in inflammatory hyperalgesia have not been elucidated. In the present work, we identified the following characteristics of this cascade. (1) Local inflammation led to increase in rat dorsal horn phosphorylation (activation) of Akt (pAkt) and mTOR (pmTOR), as assessed by Western blotting and immunocytochemistry. (2) Increased pAkt and pmTOR were prominent in neurons in laminae I, III, and IV, whereas pmTOR and its downstream targets (pS6, p4EBP) were also observed in glial cells. (3) Intrathecal treatment with inhibitors to PI3K or Akt attenuated Formalin-induced second-phase flinching behavior, as well as carrageenan-induced thermal hyperalgesia and tactile allodynia. (4) Intrathecal rapamycin (an mTORC1 inhibitor) displayed anti-hyperalgesic effect in both inflammatory pain models. Importantly, intrathecal wortmannin at anti-hyperalgesic doses reversed the evoked increase not only in Akt but also in mTORC1 signaling (pS6/p4EBP). (5) pAkt and pmTOR are expressed in neurokinin 1 receptor-positive neurons in laminae I–III after peripheral inflammation. Intrathecal injection of Substance P activated this cascade (increased phosphorylation) and resulted in hyperalgesia, both of which effects were blocked by intrathecal wortmannin and rapamycin. Together, these findings reveal that afferent inputs trigged by peripheral inflammation initiate spinal activation of PI3K–Akt–mTOR signaling pathway, a component of which participates in neuronal circuits of facilitated pain processing.

Descending serotonergic facilitation of spinal ERK activation and pain behavior

Serotonin (5‐HT) derived from bulbo‐spinal projection is released by nociceptive input into the spinal dorsal horn. Here we report that formalin injection in the paw produced pain behavior (flinching) and phosphorylation of spinal ERK1/2 (P‐ERK1/2, indicating activation) in rats. Depletion of spinal 5‐HT by intrathecal (IT) 5,7‐DHT, a serotonergic neurotoxin, profoundly reduced formalin evoked flinching and the increase in P‐ERK1/2. Ondansetron (a 5‐HT3 receptor antagonist) at IT doses that inhibited flinching also attenuated spinal ERK activation. These findings reveal that primary afferent‐evoked activation of spinal ERK requires the input from an excitatory 5‐HT descending pathway.

Spinal 12-lipoxygenase-derived hepoxilin A3 contributes to inflammatory hyperalgesia via activation of TRPV1 and TRPA1 receptors

Peripheral inflammation initiates changes in spinal nociceptive processing leading to hyperalgesia. Previously, we demonstrated that among 102 lipid species detected by LC-MS/MS analysis in rat spinal cord, the most notable increases that occur after intraplantar carrageenan are metabolites of 12-lipoxygenases (12-LOX), particularly hepoxilins (HXA3 and HXB3). Thus, we examined involvement of spinal LOX enzymes in inflammatory hyperalgesia. In the current work, we found that intrathecal (IT) delivery of the LOX inhibitor nordihydroguaiaretic acid prevented the carrageenan-evoked increase in spinal HXB3 at doses that attenuated the associated hyperalgesia. Furthermore, IT delivery of inhibitors targeting 12-LOX (CDC, Baicalein), but not 5-LOX (Zileuton) dose-dependently attenuated tactile allodynia. Similarly, IT delivery of 12-LOX metabolites of arachidonic acid 12(S)-HpETE, 12(S)-HETE, HXA3, or HXB3 evoked profound, persistent tactile allodynia, but 12(S)-HpETE and HXA3 produced relatively modest, transient heat hyperalgesia. The pronociceptive effect of HXA3 correlated with enhanced release of Substance P from primary sensory afferents. Importantly, HXA3 triggered sustained mobilization of calcium in cells stably overexpressing TRPV1 or TRPA1 receptors and in acutely dissociated rodent sensory neurons. Constitutive deletion or antagonists of TRPV1 (AMG9810) or TRPA1 (HC030031) attenuated this action. Furthermore, pretreatment with antihyperalgesic doses of AMG9810 or HC030031 reduced spinal HXA3-evoked allodynia. These data indicate that spinal HXA3 is increased by peripheral inflammation and promotes initiation of facilitated nociceptive processing through direct activation of TRPV1 and TRPA1 at central terminals.

Systemic and Intrathecal Effects of a Novel Series of Phospholipase A2 Inhibitors on Hyperalgesia and Spinal Pge2 Release

Phospholipase A2 (PLA2) forms are expressed in spinal cord, and inhibiting spinal PLA2 induces a potent antihyperalgesia. Here, we examined the antihyperalgesic effects after systemic and i.t. delivery of four compounds constructed with a common motif consisting of a 2-oxoamide with a hydrocarbon tail and a four-carbon tether. These molecules were characterized for their ability to block group IVA calcium-dependent PLA2 (cPLA2) and group VIA calcium-independent PLA2 (iPLA2) in inhibition assays using human recombinant enzyme. The rank ordering of potency in blocking group IVA cPLA2 was AX048 (ethyl 4-[(2-oxohexadecanoyl)amino]butanoate), AX006 (4-[(2-oxohexadecanoyl)amino]butanoic acid), and AX057 (tert-butyl 4-[(2-oxohexadecanoyl)amino]butanoate) > AX010 (methyl 4-[(2-oxohexadecanoyl)amino]butanoate) and for inhibiting group VIA iPLA2 was AX048, AX057 > AX006, and AX010. No agent altered recombinant cyclooxygenase activity. In vivo, i.t. (30 μg) and systemic (0.2-3 mg/kg i.p.) AX048 blocked carrageenan hyperalgesia and after systemic delivery in a model of spinally mediated hyperalgesia induced by i.t. substance P (SP). The other agents were without activity. In rats prepared with lumbar i.t. loop dialysis catheters, SP evoked spinal prostaglandin E2 (PGE2) release. AX048 alone inhibited PGE2 release. Intrathecal SR141617, a cannabinoid CB1 inhibitor at doses that blocked the effects of i.t. anandamide had no effect upon i.t. AX048. These results suggest that AX048 is the first systemically bioavailable compound with a significant affinity for group IVA cPLA2, which produces a potent antihyperalgesia. The other agents, although demonstrating enzymatic activity in cell-free assays, appear unable to gain access to the intracellular PLA2 toward which their action is targeted.

Inflammatory hyperalgesia induces essential bioactive lipid production in the spinal cord.

J. Neurochem. (2010) 114, 981–993.

Inhibition of spinal constitutive NOS-2 by 1400W attenuates tissue injury and inflammation-induced hyperalgesia and spinal p38 activation

Nitric oxide (NO) and its synthesizing enzymes, including NO synthase‐2 (NOS‐2, also called inducible NOS, iNOS), have been implicated in spinal nociception. 1400W is a highly selective NOS‐2 inhibitor, as compared with either NOS‐1 (neuronal NOS, nNOS) or NOS‐3 (endothelial NOS). Here we examined the anti‐nociceptive effects of intrathecal (IT) administration of 1400W in two experimental models of hyperalgesia (formalin and carrageenan models), in addition to the effect of 1400W on stimulation‐induced activation of spinal p38 mitogen‐activated protein kinase (p38). IT treatment of rats with 1400W produced a dose‐dependent inhibition of paw formalin‐induced phase II flinches, and attenuated carrageenan‐induced thermal hyperalgesia. In contrast, IT injection of a selective inhibitor of NOS‐1, nNOS inhibitor‐I, had no effect in either model. Furthermore, 1400W at a dose that suppressed formalin‐induced flinching behavior also blocked formalin‐evoked p38 phosphorylation (activation) in the spinal cord, while nNOS inhibitor‐I displayed no activity. The prompt effects of IT 1400W suggest involvement of constitutively expressed NOS‐2 in spinal nociception. The NOS‐2 protein in rat spinal cords was undetectable by Western blotting. However, when the protein was immunoprecipitated prior to Western blotting, NOS‐2‐immunoreactive bands were detected in the tissues, including naïve spinal cords. The presence of constitutive spinal NOS‐2 was further confirmed by reverse transcriptase‐polymerase chain reaction. Taken together, the present studies suggest that constitutively expressed spinal NOS‐2 mediates tissue injury and inflammation‐induced hyperalgesia, and that activation of p38 is one of the downstream factors in NO‐mediated signaling in the initial processing of spinal nociception.

Mechanisms of antinociception of spinal galanin: how does galanin inhibit spinal sensitization?

Galanin by a spinal action has been shown to have an antihyperalgesic action. Thus, in rats with lumbar intrathecal (IT) catheters, the thermal hyperalgesia evoked by carrageenan paw injection was blocked by IT delivery of galanin(1-29) (Gal(1-29)) and galanin(2-11) (Gal(2-11)) with the rank order of activity being Gal(1-29)>Gal(2-11). We sought to determine whether this spinal action reflects an effect upon afferent transmitter release, e.g., substance P (SP), and/or on secondary neurons, e.g., signaling postsynaptic to neurokinin 1 (NK1) receptor activation. To address the question on afferent release, we investigated the effect of IT administration of galanin on tissue injury-induced spinal NK1 internalization (an indicator of SP release). Noxious stimulation (paw compression) produced an increase in NK1 internalization in dorsal horn lamina I. IT pretreatment of rats with Gal(1-29) and Gal(2-11) significantly attenuated the evoked NK1 internalization, with the rank order of activity being Gal(1-29)>Gal(2-11)>saline. To address the question of postsynaptic action, we examined the effects of IT galanin upon IT SP-induced thermal hyperalgesia and spinal PGE2 release. Application of SP (30 nmol) directly to spinal cord led to a decrease in thermal thresholds and a profound increase in PGE(2) concentration in spinal dialysates. Both phenomena were reversed by Gal(1-29) and Gal(2-11) (10nmol, IT). These findings suggest that the antihyperalgesic effect of spinal galanin is due to its action on sites both presynaptic (inhibition of SP release) and postsynaptic (blockade of SP-evoked hyperalgesia and PGE2 production) to the primary afferents.

In Vivo Gene Knockdown in Rat Dorsal Root Ganglia Mediated by Self-Complementary Adeno-Associated Virus Serotype 5 Following Intrathecal Delivery

We report here in adult rat viral vector mediate-gene knockdown in the primary sensory neurons and the associated cellular and behavior consequences. Self-complementary adeno-associated virus serotype 5 (AAV5) was constructed to express green fluorescent protein (GFP) and a small interfering RNA (siRNA) targeting mammalian target of rapamycin (mTOR). The AAV vectors were injected via an intrathecal catheter. We observed profound GFP expression in lumbar DRG neurons beginning at 2-week post-injection. Of those neurons, over 85% were large to medium-diameter and co-labeled with NF200, a marker for myelinated fibers. Western blotting of mTOR revealed an 80% reduction in the lumbar DRGs (L4–L6) of rats treated with the active siRNA vectors compared to the control siRNA vector. Gene knockdown became apparent as early as 7-day post-injection and lasted for at least 5 weeks. Importantly, mTOR knockdown occurred in large (NF200) and small-diameter neurons (nociceptors). The viral administration induced an increase of Iba1 immunoreactivity in the DRGs, which was likely attributed to the expression of GFP but not siRNA. Rats with mTOR knockdown in DRG neurons showed normal general behavior and unaltered responses to noxious stimuli. In conclusion, intrathecal AAV5 is a highly efficient vehicle to deliver siRNA and generate gene knockdown in DRG neurons. This will be valuable for both basic research and clinic intervention of diseases involving primary sensory neurons.