Joanne J. Steinauer

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.

Role of spinal microglia in visceral hyperalgesia and NK1R up-regulation in a rat model of chronic stress.

BACKGROUND & AIMS Chronic psychological stress is associated with visceral hyperalgesia and increased expression of spinal NK1 receptors (NK1Rs). We aimed to identify the role of spinal microglia in this process. METHODS Male Wistar rats were exposed to water avoidance (WA) or sham stress 1 hour each day for 10 days and given daily injections of minocycline, the p38 inhibitor SB203580, or saline. Phosphorylation levels of the kinase p38 (P-p38), the microglia marker OX42, NK1R, and IkappaBalpha were assessed by immunoblotting and/or immunostaining of spinal samples collected at day 11. The visceromotor response to colorectal distention at baseline and following WA were also assayed in rats given injections of minocycline, SB203580, or vehicle. The effects of fractalkine were assessed on the visceromotor response in rats exposed to minocycline or vehicle. RESULTS P-p38 protein levels and immunoreactivity were increased in stressed rats and colocalized with OX42-positive cells and neurons in the dorsal horn. This increase was reversed by minocycline or SB203580 exposure. Stress-induced increased NK1R expression was blocked by minocycline but not SB203580. WA-induced decreased IkappaBalpha expression was blocked by minocycline and SB203580. WA-induced hyperalgesia was blocked by minocycline and SB203580 intrathecally. Fractalkine-induced hyperalgesia was blocked by minocycline. CONCLUSIONS This is the first demonstration that stress-induced activation of spinal microglia has a key role in visceral hyperalgesia and associated spinal NK1R up-regulation.

Botulinum toxin B in the sensory afferent: transmitter release, spinal activation, and pain behavior.

Summary Peripheral botulinum toxin‐B attenuates peripheral and central terminal neurotransmitter release and nociceptive processing via a presynaptic effect with a possible postsynaptic component. ABSTRACT We addressed the hypothesis that intraplantar botulinum toxin B (rimabotulinumtoxin B: BoNT‐B) has an early local effect upon peripheral afferent terminal releasing function and, over time, will be transported to the central terminals of the primary afferent. Once in the terminals it will cleave synaptic protein, block spinal afferent transmitter release, and thereby prevent spinal nociceptive excitation and behavior. In mice, C57Bl/6 males, intraplantar BoNT‐B (1 U) given unilaterally into the hind paw had no effect upon survival or motor function, but ipsilaterally decreased: 1) intraplantar formalin‐evoked flinching; 2) intraplantar capsaicin‐evoked plasma extravasation in the hind paw measured by Evans blue in the paw; 3) intraplantar formalin‐evoked dorsal horn substance P (SP) release (neurokinin 1 [NK1] receptor internalization); 4) intraplantar formalin‐evoked dorsal horn neuronal activation (c‐fos); 5) ipsilateral dorsal root ganglion (DRG) vesicle‐associated membrane protein (VAMP); 6) ipsilateral SP release otherwise evoked bilaterally by intrathecal capsaicin; 7) ipsilateral activation of c‐fos otherwise evoked bilaterally by intrathecal SP. These results indicate that BoNT‐B, after unilateral intraplantar delivery, is taken up by the peripheral terminal, is locally active (blocking plasma extravasation), is transported to the ipsilateral DRG to cleave VAMP, and is acting presynaptically to block release from the spinal peptidergic terminal. The observations following intrathecal SP offer evidence for a possible transsynaptic effect of intraplantar BoNT. These results provide robust evidence that peripheral BoNT‐B can alter peripheral and central terminal release from a nociceptor and attenuate downstream nociceptive processing via a presynaptic effect, with further evidence suggesting a possible postsynaptic effect.

Safety assessment and pharmacokinetics of intrathecal methylprednisolone acetate in dogs.

Background: Intrathecal methylprednisolone acetate (MPA) has been used in patients with chronic pain syndromes. Its safety has been debated after reports of adverse events. No systematic preclinical evaluation of MPA has been reported. In the current study, the acute and long-term effects of intrathecal MPA on dog spinal tissue was studied with the injectate reformulated to include minimal adjuvants. Methods: Seventeen dogs were implanted with intrathecal catheters and randomized to three groups: vehicle (lidocaine; 4 dogs), MPA 20 mg/ml (human dose; 7 dogs), and MPA 80 mg/ml (maximum deliverable dose; 6 dogs). In parallel with the human protocols, dogs received four injections at 7-day intervals. Clinical observations and plasma methylprednisolone measurements were done before and at intervals after intrathecal delivery. One week (acute) or 6 weeks (long-term) after the last injection, animals were sacrificed and spinal tissues harvested for histopathology. Results: Other than a brief motor block, no adverse clinical event occurred in any animal. Group A (vehicle) showed minimal histologic changes (median histology-score; acute: 1.3, long-term: 1.0). Group B (MPA 20 mg/ml) had a diffuse inflammatory reaction (acute: 2.0, long-term: 3.0), group C (MPA 80 mg/ml) a severe inflammatory response, with large inflammatory masses (acute: 4.0, long-term: 7.0) The severity of the inflammatory reaction increased significantly with increasing dose at long-term sacrifice (acute P = 0.167, long-term P = 0.014). No neuronal injury, demyelination, or gliosis was seen in any animal. Conclusion: These results, showing dose-dependent intrathecal inflammatory reactions at MPA doses and injectate concentrations comparable to those used in humans, indicate that the continued use of this modality in humans is not recommended.

Systematic analysis of rat 12/15-lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia

Previously, we observed significant increases in spinal 12‐lipoxygenase (LOX) metabolites, in particular, hepoxilins, which contribute to peripheral inflammation‐induced tactile allodynia. However, the enzymatic sources of hepoxilin synthase (HXS) activity in rats remain elusive. Therefore, we overexpressed each of the 6 rat 12/15‐LOX enzymes in HEK‐293T cells and measured by LC‐MS/MS the formation of HXB3, 12‐HETE, 8‐HETE, and 15‐HETE from arachidonic acid (AA) at baseline and in the presence of LOX inhibitors (NDGA, AA‐861, CDC, baicalein, and PD146176) vs. vehicle‐treated and mock‐transfected controls. We detected the following primary intrinsic activities: 12‐LOX (Alox12, Alox15), 15‐LOX (Alox15b), and HXS (Alox12, Alox15). Similar to human and mouse orthologs, proteins encoded by rat Alox12b and Alox12e possessed minimal 12‐LOX activity with AA as substrate, while eLOX3 (encoded by Aloxe3) exhibited HXS without 12‐LOX activity when coexpressed with Alox12b or supplemented with 12‐HpETE. CDC potently inhibited HXS and 12‐LOX activity in vitro (relative IC50s: CDC, ~0.5 and 0.8 μM, respectively) and carrageenan‐evoked tactile allodynia in vivo. Notably, peripheral inflammation significantly increased spinal eLOX3; intrathecal pretreatment with either siRNA targeting Aloxe3 or an eLOX3‐selective antibody attenuated the associated allodynia. These findings implicate spinal eLOX3‐mediated hepoxilin synthesis in inflammatory hyperesthesia and underscore the importance of developing more selective 12‐LOX/HXS inhibitors.—Gregus, A. M., Dumlao, D. S., Wei, S. C., Norris, P. C., Catella, L. C., Meyerstein, F. G., Buczynski, M. W., Steinauer, J. J., Fitzsimmons, B. L., Yaksh, T. L., Dennis, E. A. Systematic analysis of rat 12/15‐lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia. FASEB J. 27, 1939–1949 (2013). www.fasebj.org

Role of spinal p38α and β MAPK in inflammatory hyperalgesia and spinal COX-2 expression

Pharmacological studies indicate that spinal p38 mitogen-activated protein kinase plays a role in the development of hyperalgesia. We investigated whether either the spinal isoform p38α or p38β is involved in peripheral inflammation evoked pain state and increased expression of spinal COX-2. Using intrathecal antisense oligonucleotides, we show that hyperalgesia is prevented by downregulation of p38β but not p38α, whereas increases in spinal COX-2 protein expression at 8 hours are mediated by both p38α and β isoforms. These data suggest that early activation of spinal p38β isoform may affect acute facilitatory processing, and both p38β and α isoforms mediate temporally delayed upregulation of spinal COX-2.

Intrathecal substance P-saporin in the dog: distribution, safety, and spinal neurokinin-1 receptor ablation.

Background: Neurokinin-1 receptors (NK1-rs) located on superficial dorsal horn neurons are essential for integration of nociceptive input. Intrathecal injection of substance P-saporin (SP-SAP) leads to local loss of spinal NK1-r (+) neurons suggesting its potential as a therapeutic agent for chronic pain. The authors determined, in a canine model, effects of lumbar intrathecal SP-SAP. Methods: Distribution of SP-SAP and Saporin was determined in plasma, lumbar cerebrospinal fluid, and tissue. Safety of intrathecal SP-SAP was determined in four groups (six dogs each) administered 0 (0.9% saline), 1.5, 15, or 150 µg SP-SAP through lumbar intrathecal catheters. Behavioral, physiologic, and biochemical variables were assessed. Spinal tissues were collected at 7 and approximately 90 days, or earlier if significant morbidity developed, and analyzed for NK1-r (+) neuron loss and histopathology. Results: SP-SAP and Saporin were detectable in lumbar cerebrospinal fluid for up to 4 and 24 h, respectively. Animals receiving intrathecal saline, 1.5, or 15 µg of SP-SAP showed no persistent neurologic deficits. Three animals receiving 150 µg of SP-SAP developed pelvic limb paraparesis and were euthanized prematurely. Immunohistochemistry and in situ hybridization cell counts confirmed a significant reduction in NK1-r (+) in superficial dorsal horn neurons from lumbar spinal cord after intrathecal administration of 15 and 150 µg of SP-SAP. A significant loss of NK1-r neurons in the lumbar ventral horn occurred only with 150-µg SP-SAP. Conclusion: Intrathecal 15-µg SP-SAP reduced dorsal, but not ventral, NK1-r (+) neurons at the spinal level of delivery with minimal side effects, whereas 150-µg SP-SAP resulted in motor neuron toxicity.

Role of meningeal mast cells in intrathecal morphine-evoked granuloma formation.

Background:Intrathecal morphine forms granulomas that arise from the adjacent arachnoid membrane. The authors propose that these inflammatory cells exit the meningeal vasculature secondary to meningeal mast cell degranulation. Methods:Three sets of experiments were accomplished in dogs: (1) ex vivo meningeal mast cell degranulation (histamine release was measured ex vivo from canine dura incubated with opiates); (2) in vivo cutaneous mast cell degranulation (flare areas on the dog abdomen were measured after subcutaneous opiates); and (3) in vivo granuloma pharmacology. Dogs with lumbar intrathecal catheters received infusion of intrathecal saline or intrathecal morphine. Intrathecal morphine dogs received (1) no other treatment (control); (2) twice-daily subcutaneous naltrexone; (3) intrathecal co-infusion of cromolyn; or (4) twice-daily subcutaneous cromolyn for the 24- to 28-day study course. Results:Morphine but not fentanyl evoked dural histamine release, which was blocked by cromolyn but not naloxone. Wheal/flare was produced by subcutaneous morphine, methadone, hydromorphone, but not fentanyl, and was unaffected by naltrexone but prevented by cromolyn. Granulomas occurred in all dogs receiving intrathecal morphine (15 of 15); subcutaneous naltrexone had no effect on granulomas (six of six) but was reduced by concurrent intrathecal cromolyn (zero of five) or twice-daily subcutaneous cromolyn (one of five). Conclusions:The pharmacology of cutaneous/dural mast cell degranulation and intrathecal granulomas are comparable, not mediated by opioid receptors, and reduced by agents preventing mast cell degranulation. If an agent produces cutaneous mast cell degranulation at concentrations produced by intrathecal delivery, the agent may initiate granulomas.

Spinal release of tumour necrosis factor activates c-Jun N-terminal kinase and mediates inflammation-induced hypersensitivity

Mounting evidence points to individual contributions of tumour necrosis factor‐alpha (TNF) and the c‐Jun N‐terminal kinase (JNK) pathway to the induction and maintenance of various pain states. Here we explore the role of spinal TNF and JNK in carrageenan‐induced hypersensitivity. As links between TNF and JNK have been demonstrated in vitro, we investigated if TNF regulates spinal JNK activity in vivo.

Alfentanil: correlations between absence of effect upon subcutaneous mast cells and absence of granuloma formation after intrathecal infusion in the dog.

We hypothesize that intrathecal (IT) granulomas arising from the IT infusion of several opiates may result from the degranulation of meningeal mast cells (MC). Given functional covariance between cutaneous and meningeal MC, we propose that opioids that do not degranulate cutaneous MC will not produce a granuloma. An opioid meeting this criteria is the phenylpiperadine alfentanil HCl.