Qinghao Xu

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.

Delta-opioid receptors (DOR) are present in the superficial dorsal horn and are believed to regulate the release of small afferent transmitters as evidenced by the effects of spinally delivered delta-opioid preferring peptides. Here we examined the effects of intrathecal SNC80 [(+)-4-[α(R)-α-[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-(methoxybenzyl)-N,N-diethylbenzamide], a selective nonpeptidic DOR agonist, in three preclinical pain models, acute thermal escape, intraplantar carrageenan-tactile allodynia, and intraplantar formalin flinches, and on the evoked release of substance P (SP) from small primary afferents. Rats with chronic intrathecal catheters received intrathecal vehicle or SNC80 (100 or 200 μg). Intrathecal SNC80 did not change acute thermal latencies or carrageenan-induced thermal hyperalgesia. However, SNC80 attenuated carrageenan-induced tactile allodynia and significantly reduced both phase 1 and phase 2 formalin-induced paw flinches, as assessed by an automatic flinch counting device. These effects were abolished by naltrindole (3 mg/kg i.p.), a selective DOR antagonist, but not CTOP (10 µg i.t.), a selective MOR antagonist. Furthermore, intrathecal SNC80 (200 μg) blocked formalin-induced substance P release otherwise evoked in the ispilateral superficial dorsal horn as measured by NK1 receptor internalization. In conclusion, intrathecal SNC80 alleviated pain hypersensitivity after peripheral inflammation in a fashion paralleling its ability to block peptide transmitter release from small peptidergic afferents, which by its pharmacology appears to represent an effect mediated by a spinal DOR.

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.

Purpose of review The causes of inflammatory pain and neuropathic pain are fundamentally different. There are, however, common mechanisms underlying the generation of each pain state. We will discuss some specific elements observed in both tissue and nerve injury pain states and consider the hypothesis that these two states actually demonstrate a convergence over time. Recent findings The increased pain sensation following tissue and nerve injury results from several mechanisms, including altered ion channel expression in dorsal root ganglion neurons, enhanced dorsal horn glutamate release from primary afferents, enhanced glutamate receptor function in second-order neurons, disinhibition in the dorsal horn and glia cell activation. The role of specific subtypes of receptors, ion channels and glutamate transporters is revealed at peripheral and central sites. Importantly over time, a number of changes, in the dorsal root ganglion and in dorsal horn observed after tissue injury resemble changes observed after nerve injury. Summary Recognition of mechanisms common to both inflammatory pain and neuropathic pain might shed light on the understanding of the transition from acute pain to persistent pain.

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.