Challenges in the Development of Intravenous Neurokinin-1 Receptor Antagonists: Results of a Safety and Pharmacokinetics Dose-Finding, Phase 1 Study of Intravenous Fosnetupitant.

Oral NEPA is the fixed-combination antiemetic comprising netupitant (neurokinin-1 receptor antagonist [NK1 RA]) and palonosetron (5-hydroxytryptamine-3 receptor antagonist [5-HT3 RA]). Intravenous (IV) NEPA, containing fosnetupitant, a water-soluble N-phosphoryloxymethyl prodrug of netupitant, has been developed. Fosnetupitant does not require excipients or solubility enhancers often used to increase IV NK1 RA water solubility, preventing the occurrence of hypersensitivity and infusion-site reactions associated with these products. In this phase 1 study, subjects received a 30-minute placebo or fosnetupitant (17.6-353 mg) infusion and an oral NEPA or placebo capsule, with 2-sequence crossover treatment for fosnetupitant 118- to 353-mg dose cohorts. IV fosnetupitant safety and pharmacokinetics were evaluated, and its equivalence to an oral netupitant 300-mg dose was defined. Overall, 158 healthy volunteers were enrolled. All adverse events (AEs) were mild or moderate in intensity. Doppler-identified infusion-site asymptomatic thrombosis occurred in 5.4% (fosnetupitant) and 1.2% (oral NEPA) of subjects. The frequency or number of treatment-related AEs did not increase with ascending fosnetupitant doses. The most common treatment-related AEs were headache (fosnetupitant, 8.1%; oral NEPA, 12.7%) and constipation (fosnetupitant, 1.4%; oral NEPA, 7.5%). A fosnetupitant 235-mg dose was equivalent, in terms of netupitant exposure, to 300-mg oral netupitant. The safety profile of a single fosnetupitant 235-mg infusion was similar to that of single-dose oral NEPA.

Phase 3 Study of Palonosetron IV Infusion Vs. IV Bolus for Chemotherapy-Induced Nausea and Vomiting Prophylaxis After Highly Emetogenic Chemotherapy.

CONTEXT: Palonosetron (PALO) is one of the two active components of NEPA, the fixed-combination antiemetic comprising netupitant (oral)/fosnetupitant (IV) and PALO. To increase the convenience of NEPA administration, especially for patients with swallowing difficulties, an IV NEPA formulation has been developed, where PALO is administered as a 30-minute infusion instead of the approved 30-second bolus. OBJECTIVES: To determine the efficacy and safety of the PALO component used in IV NEPA. METHODS: Noninferiority, double-blind, and randomized Phase 3 trial in chemotherapy-naive adult patients with cancer requiring highly emetogenic chemotherapy. Patients were randomized to receive a single dose of PALO 0.25 mg administered IV either as a 30-minute infusion or as a 30-second bolus before highly emetogenic chemotherapy. The primary objective was to demonstrate noninferiority of the 30-minute infusion vs. 30-second bolus in terms of complete response (CR; no emesis and no rescue medication) in the acute phase. Secondary efficacy endpoints were CR in the delayed and overall phases and no emesis and no rescue medication in all phases. Safety was a secondary endpoint. RESULTS: Overall, 440 patients received study treatment. In the infusion group, 186 (82.7%) patients reported CR in the acute phase vs. 186 (86.5%) patients in the bolus group, demonstrating the noninferiority of PALO infusion vs. bolus (P < 0.001). Secondary endpoints showed similar results between the two treatment groups. CONCLUSION: PALO 0.25-mg 30-minute IV infusion was noninferior to 30-second IV bolus in terms of CR rate in the acute phase. These results support the use of PALO 0.25 mg as a component of IV NEPA.

Phase IIIb Safety and Efficacy of Intravenous NEPA for Prevention of Chemotherapy-Induced Nausea and Vomiting (CINV) in Patients with Breast Cancer Receiving Initial and Repeat Cycles of Anthracycline and Cyclophosphamide (AC) Chemotherapy.

BACKGROUND: NEPA, a combination antiemetic of a neurokinin-1 (NK1 ) receptor antagonist (RA) (netupitant [oral]/fosnetupitant [intravenous; IV]) and 5-HT3 RA, palonosetron] offers 5-day CINV prevention with a single dose. Fosnetupitant solution contains no allergenic excipients, surfactant, emulsifier, or solubility enhancer. A phase III study of patients receiving cisplatin found no infusion-site or anaphylactic reactions related to IV NEPA. However, hypersensitivity reactions and anaphylaxis have been reported with other IV NK1 RAs, particularly fosaprepitant in patients receiving anthracycline-cyclophosphamide (AC)-based chemotherapy. This study evaluated the safety and efficacy of IV NEPA in the AC setting. MATERIALS AND METHODS: This phase IIIb, multinational, randomized, double-blind study enrolled females with breast cancer naive to highly or moderately emetogenic chemotherapy. Patients were randomized to receive a single 30-minute infusion of IV NEPA or single oral NEPA capsule on day 1 prior to AC, in repeated (up to 4) cycles. Oral dexamethasone was given to all patients on day 1 only. RESULTS: A total of 402 patients were included. The adverse event (AE) profiles were similar for IV and oral NEPA and consistent with those expected. Most AEs were mild or moderate with a similarly low incidence of treatment-related AEs in both groups. There were no treatment-related injection-site AEs and no reports of hypersensitivity or anaphylaxis. The efficacy of IV and oral NEPA were similar, with high complete response (no emesis/no rescue) rates observed in cycle 1 (overall [0-120 hours] 73.0% IV NEPA, 77.3% oral NEPA) and maintained over subsequent cycles. CONCLUSION: IV NEPA was highly effective and safe with no associated hypersensitivity and injection-site reactions in patients receiving AC. IMPLICATIONS FOR PRACTICE: As a combination of a neurokinin-1 (NK1 ) receptor antagonist (RA) and 5-HT3 RA, NEPA offers 5-day chemotherapy-induced nausea and vomiting prevention with a single dose and an opportunity to improve adherence to antiemetic guidelines. In this randomized multinational phase IIIb study, intravenous (IV) NEPA (fosnetupitant/palonosetron) was safe and highly effective in patients receiving multiple cycles of anthracycline-cyclophosphamide (AC)-based chemotherapy. Unlike other IV NK1 RAs, the IV NEPA combination solution does not require any surfactant, emulsifier, or solubility enhancer and contains no allergenic excipients. Hypersensitivity reactions and anaphylaxis have been reported with other IV NK1 RAs, most commonly with fosaprepitant in the AC setting. Importantly, there were no injection-site or hypersensitivity reactions associated with IV NEPA.

PKCγ interneurons, a gateway to pathological pain in the dorsal horn.

Chronic pain is a frequent and disabling condition that is significantly maintained by central sensitization, which results in pathological amplification of responses to noxious and innocuous stimuli. As such, mechanical allodynia, or pain in response to a tactile stimulus that does not normally provoke pain, is a cardinal feature of chronic pain. Recent evidence suggests that the dorsal horn excitatory interneurons that express the γ isoform of protein kinase C (PKCγ) play a critical role in the mechanism of mechanical allodynia during chronic pain. Here, we review this evidence as well as the main aspects of the development, anatomy, electrophysiology, inputs, outputs, and pathophysiology of dorsal horn PKCγ neurons. Primary afferent high-threshold neurons transmit the nociceptive message to the dorsal horn of the spinal cord and trigeminal system where it activates second-order nociceptive neurons relaying the information to the brain. In physiological conditions, low-threshold mechanoreceptor inputs activate inhibitory interneurons in the dorsal horn, which may control activation of second-order nociceptive neurons. During chronic pain, low-threshold mechanoreceptor inputs now activate PKCγ neurons that forward the message to second-order nociceptive neurons, turning thus tactile inputs into pain. Several mechanisms may contribute to opening this gate, including disinhibition, activation of local astrocytes, release of diffusible factors such as reactive oxygen species, and alteration of the descending serotoninergic control on PKCγ neurons through 5-HT2A serotonin receptors. Dorsal horn PKCγ neurons, therefore, appear as a relevant therapeutic target to alleviate mechanical allodynia during chronic pain.

Medication overuse reinstates conditioned pain modulation in women with migraine.

Background This study investigated the effects of medication overuse and withdrawal on modulation of pain processing in women with migraine. Temporal summation of laser-evoked thermal pain was used to measure the effects of conditioned pain modulation. Methods 36 female participants (12 healthy volunteers, 12 with episodic migraine and 12 with medication overuse headache) were included in a two session protocol. Medication overuse headache subjects were also tested three weeks after medication overuse headache withdrawal. Mechanical and laser-evoked thermal pain thresholds were measured on the back of the non-dominant hand where, later, temporal summation of laser-evoked thermal pain to repetitive thermal stimuli was elicited for 30 min, at an intensity producing moderate pain. Between the 10th and 20th minutes, the contralateral foot was immersed into a water bath at a not painful (30℃) or painfully cold (8℃; conditioned pain modulation) temperature. Results Episodic migraine, medication overuse headache and medication overuse headache withdrawal were associated with an increase in extracephalic temporal summation of laser-evoked thermal pain as compared to healthy volunteer subjects, while there was no alteration of laser-evoked thermal and mechanical extracephalic pain thresholds in these subjects. Conditioned pain modulation was highly efficient in temporal summation of laser-evoked thermal pain in healthy volunteer subjects, with a solid post-effect (reduction of pain). Conditioned pain modulation was still present, but reduced, in episodic migraine. By contrast, conditioned pain modulation was normal in medication overuse headache and strongly reduced in medication overuse headache withdrawal. Furthermore, in medication overuse headache withdrawal, the post-effect was no longer a decrease, but a facilitation of pain. Conclusions These data show that a decrease in conditioned pain modulation does not underlie medication overuse headache in women. On the contrary, medication overuse reinstated conditioned pain modulation in female migraine patients. They also identify different phenotypes of pain modulation in migraine patients. Registration number N° 2008-A00471-54.

Effects of glia metabolism inhibition on nociceptive behavioral testing in rats.

Fluoroacetate has been widely used to inhibit glia metabolism in vivo. It has yet to be shown what the effects of chronic intrathecal infusion of fluoroacetate on nociceptive behavioral testing are. The effects of chronic infusion of fluoroacetate (5 nmoles/h) for 2 weeks were examined in normal rats. Chronic intrathecal fluoroacetate did not alter mechanical threshold (von Frey filaments), responses to supra-threshold mechanical stimuli (von Frey filaments), responses to hot (hot plate) or cool (acetone test) stimuli and did not affect motor performance of the animals, which was tested with rotarod. This suggests that fluoroacetate at appropriate dose did not suppress neuronal activity in the spinal cord.

ΔN-TRPV1: A Molecular Co-detector of Body Temperature and Osmotic Stress.

Thirst and antidiuretic hormone secretion occur during hyperthermia or hypertonicity to preserve body hydration. These vital responses are triggered when hypothalamic osmoregulatory neurons become depolarized by ion channels encoded by an unknown product of the transient receptor potential vanilloid-1 gene (Trpv1). Here, we show that rodent osmoregulatory neurons express a transcript of Trpv1 that mediates the selective translation of a TRPV1 variant that lacks a significant portion of the channel's amino terminus (ΔN-TRPV1). The mRNA transcript encoding this variant (Trpv1dn) is widely expressed in the brains of osmoregulating vertebrates, including the human hypothalamus. Transfection of Trpv1dn into heterologous cells induced the expression of ion channels that could be activated by either hypertonicity or by heating in the physiological range. Moreover, expression of Trpv1dn rescued the osmosensory and thermosensory responses of single hypothalamic neurons obtained from Trpv1 knockout mice. ΔN-TRPV1 is therefore a co-detector of core body temperature and fluid tonicity.

Neuropathic pain depends upon D-serine co-activation of spinal NMDA receptors in rats.

Activation of N-methyl-d-aspartate (NMDA) receptors is critical for hypersensitivity in chronic neuropathic pain. Since astroglia can regulate NMDA receptor activation by releasing the NMDA receptor co-agonist d-serine, we investigated the role of NMDA receptor and d-serine in neuropathic chronic pain. Male Wistar rats underwent right L5-L6 spinal nerve ligation or sham surgery and were tested for mechanical allodynia and hyperalgesia after 14 days. Acute intrathecal administration of the NMDA receptor antagonist d-AP5 as well as chronic administration of the glia metabolism inhibitor fluoroacetate significantly reduced mechanical allodynia in neuropathic rats. The effect of fluoroacetate was reversed by acutely administered intrathecal d-serine. Degrading d-serine using acute intrathecal administration of d-aminoacid oxidase also reduced pain symptoms. Immunocytochemistry showed that about 70% of serine racemase, the synthesizing enzyme of d-serine, was expressed in astrocyte processes in the superficial laminae of L5 dorsal horn. Serine racemase expression was upregulated in astrocyte processes in neuropathic rats compared to sham rats. These results show that neuropathic pain depends upon glial d-serine that co-activates spinal NMDA receptors.

High salt intake increases blood pressure via BDNF-mediated downregulation of KCC2 and impaired baroreflex inhibition of vasopressin neurons.

The mechanisms by which dietary salt promotes hypertension are unknown. Previous work established that plasma [Na(+)] and osmolality rise in proportion with salt intake and thus promote release of vasopressin (VP) from the neurohypophysis. Although high levels of circulating VP can increase blood pressure, this effect is normally prevented by a potent GABAergic inhibition of VP neurons by aortic baroreceptors. Here we show that chronic high salt intake impairs baroreceptor inhibition of rat VP neurons through a brain-derived neurotrophic factor (BDNF)-dependent activation of TrkB receptors and downregulation of KCC2 expression, which prevents inhibitory GABAergic signaling. We show that high salt intake increases the spontaneous firing rate of VP neurons in vivo and that circulating VP contributes significantly to the elevation of arterial pressure under these conditions. These results provide the first demonstration that dietary salt can affect blood pressure through neurotrophin-induced plasticity in a central homeostatic circuit.

Extracellular signal-regulated kinase phosphorylation in forebrain neurones contributes to osmoregulatory mechanisms.

Vasopressin secretion from the magnocellular neurosecretory cells (MNCs) is crucial for body fluid homeostasis. Osmotic regulation of MNC activity involves the concerted modulation of intrinsic mechanosensitive ion channels, taurine release from local astrocytes as well as excitatory inputs derived from osmosensitive forebrain regions. Extracellular signal-regulated protein kinases (ERK) are mitogen-activated protein kinases that transduce extracellular stimuli into intracellular post-translational and transcriptional responses, leading to changes in intrinsic neuronal properties and synaptic function. Here, we investigated whether ERK activation (i.e. phosphorylation) plays a role in the functioning of forebrain osmoregulatory networks. We found that within 10 min after intraperitoneal injections of hypertonic saline (3 m, 6 m) in rats, many phosphoERK-immunopositive neurones were observed in osmosensitive forebrain regions, including the MNC containing supraoptic nuclei. The intensity of ERK labelling was dose-dependent. Reciprocally, slow intragastric infusions of water that lower osmolality reduced basal ERK phosphorylation. In the supraoptic nucleus, ERK phosphorylation predominated in vasopressin neurones vs. oxytocin neurones and was absent from astrocytes. Western blot experiments confirmed that phosphoERK expression in the supraoptic nucleus was dose dependent. Intracerebroventricular administration of the ERK phosphorylation inhibitor U 0126 before a hyperosmotic challenge reduced the number of both phosphoERK-immunopositive neurones and Fos expressing neurones in osmosensitive forebrain regions. Blockade of ERK phosphorylation also reduced hypertonically induced depolarization and an increase in firing of the supraoptic MNCs recorded in vitro. It finally reduced hypertonically induced vasopressin release in the bloodstream. Altogether, these findings identify ERK phosphorylation as a new element contributing to the osmoregulatory mechanisms of vasopressin release.

Cancer pain is not necessarily correlated with spinal overexpression of reactive glia markers.

Bone cancer pain is a common and disruptive symptom in cancer patients. In cancer pain animal models, massive reactive astrogliosis in the dorsal horn of the spinal cord has been reported. Because astrocytes may behave as driving partners for pathological pain, we investigated the temporal development of pain behavior and reactive astrogliosis in a rat bone cancer pain model induced by injecting MRMT-1 rat mammary gland carcinoma cells into the tibia. Along with the development of bone lesions, a gradual mechanical and thermal allodynia and hyperalgesia as well as a reduced use of the affected limb developed in bone cancer-bearing animals, but not in sham-treated animals. Dorsal horn Fos expression after nonpainful palpation of the injected limb was also increased in bone cancer-bearing animals. However, at any time during the evolution of tumor, there was no increase in glial fibrillary acidic protein (GFAP) immunoreactivity in the dorsal horn. Further analysis at 21days after injection of the tumor showed no increase in GFAP and interleukin (IL) 1ß transcripts, number of superficial dorsal horn S100ß protein immunoreactive astrocytes, or immunoreactivity for microglial markers (OX-42 and Iba-1). In contrast, all these parameters were increased in the dorsal horn of rats 2weeks after sciatic nerve ligation. This suggests that in some cases, bone cancer pain may not be correlated with spinal overexpression of reactive glia markers, whereas neuropathic pain is. Glia may thus play different roles in the development and maintenance of chronic pain in these 2 situations.

Multicenter phase IV study of palonosetron in the prevention of chemotherapy-induced nausea and vomiting (CINV) in patients with non-Hodgkin lymphomas undergoing repeated cycles of moderately emetogenic chemotherapy.

Antiemetic therapy for chemotherapy-induced nausea and vomiting in patients with non-Hodgkin lymphoma (NHL) receiving moderately emetogenic chemotherapy (MEC) generally includes a serotonin-type 3 (5-HT3) receptor antagonist (RA). The efficacy and safety of the second-generation 5-HT3 RA, palonosetron, in patients with NHL receiving MEC was assessed. Patients received a single iv bolus injection of 0.25 mg palonosetron and chemotherapy on day 1 of the first chemotherapy cycle, and up to three further consecutive cycles. Eighty-eight patients were evaluable for efficacy and safety. The primary endpoint, the percentage of patients with a complete response in the overall phase (0-120 h after chemotherapy in each cycle), increased from 68.2% (cycle 1) to 80.5% (cycle 2), remaining high for the following cycles, and > 90% patients were emesis-free without using aprepitant during therapy. Across all cycles, 78.4% of patients experienced treatment-emergent adverse events, but only 8% related to study drug, confirming palonosetron's good safety profile (EudraCT Number: 2008-007827-14).

Efficacy of oral palonosetron compared to intravenous palonosetron for the prevention of chemotherapy-induced nausea and vomiting associated with moderately emetogenic chemotherapy: a phase 3 trial.

BACKGROUND: Palonosetron (Aloxi(®), Onicit(®)) is a pharmacologically unique 5-HT3 receptor antagonist (RA) approved as a single IV injection for the prevention of nausea and vomiting induced by chemotherapy (CINV) of either moderate or highly emetogenic potential (MEC and HEC, respectively). An oral palonosetron formulation has been developed and compared to the IV formulation. METHODS: In this multinational, multicenter, double-blind, double-dummy, dose-ranging trial, 651 patients were randomly assigned to receive one of the following as a single dose prior to moderately emetogenic chemotherapy: oral palonosetron 0.25, 0.50, and 0.75 mg or IV palonosetron 0.25 mg. Patients were also randomized (1:1) to receive dexamethasone 8 mg IV or matched placebo on day 1. The primary endpoint was complete response (CR; no emesis, no rescue therapy) during the acute phase (0-24 h). RESULTS: Acute CR rates were 73.5, 76.3, 74.1, and 70.4 % for all patients receiving the palonosetron 0.25, 0.50, and 0.75 mg oral doses, and for IV palonosetron 0.25 mg, respectively; delayed CR (24-120 h) rates were 59.4, 62.5, 60.1, and 65.4 %, and overall CR (0-120 h) rates were 53.5, 58.8, 53.2, and 59.3 %, respectively. The addition of dexamethasone improved emetic control (acute CR rate) by at least 15 % for all groups except oral palonosetron 0.25 mg, where the acute CR improvement was approximately 7 %. Adverse events were similar in nature, incidence, and intensity for all oral and IV palonosetron groups, and were the expected adverse events for 5-HT3 RAs (primarily headache and constipation). CONCLUSION: Oral palonosetron has a similar efficacy and safety profile as IV palonosetron 0.25 mg and may be the preferred formulation in certain clinical situations. Among the tested oral treatments, a palonosetron 0.50-mg oral dose has been favored for the prevention of CINV in patients receiving moderately emetogenic chemotherapy due to a numerical gain in efficacy without a side effect disadvantage.

Glycine inhibitory dysfunction turns touch into pain through astrocyte-derived D-serine.

Glycine inhibitory dysfunction provides a useful experimental model for studying the mechanism of dynamic mechanical allodynia, a widespread and intractable symptom of neuropathic pain. In this model, allodynia expression relies on N-methyl-d-aspartate receptors (NMDARs), and it has been shown that astrocytes can regulate their activation through the release of the NMDAR coagonist d-serine. Recent studies also suggest that astrocytes potentially contribute to neuropathic pain. However, the involvement of astrocytes in dynamic mechanical allodynia remains unknown. Here, we show that after blockade of glycine inhibition, orofacial tactile stimuli activated medullary dorsal horn (MDH) astrocytes, but not microglia. Accordingly, the glia inhibitor fluorocitrate, but not the microglia inhibitor minocycline, prevented allodynia. Fluorocitrate also impeded activation of astrocytes and blocked activation of the superficial MDH neural circuit underlying allodynia, as revealed by study of Fos expression. MDH astrocytes are thus required for allodynia. They may also produce d-serine because astrocytic processes were selectively immunolabeled for serine racemase, the d-serine synthesizing enzyme. Accordingly, selective degradation of d-serine with d-amino acid oxidase applied in vivo prevented allodynia and activation of the underlying neural circuit. Conversely, allodynia blockade by fluorocitrate was reversed by exogenous d-serine. These results suggest the following scenario: removal of glycine inhibition makes tactile stimuli able to activate astrocytes; activated astrocytes may provide d-serine to enable NMDAR activation and thus allodynia. Such a contribution of astrocytes to pathological pain fuels the emerging concept that astrocytes are critical players in pain signaling. Glycine disinhibition makes tactile stimuli able to activate astrocytes, which may provide d-serine to enable NMDA receptor activation and thus allodynia.

Glycine inhibitory dysfunction induces a selectively dynamic, morphine-resistant, and neurokinin 1 receptor- independent mechanical allodynia.

Dynamic mechanical allodynia is a widespread and intractable symptom of neuropathic pain for which there is a lack of effective therapy. We recently provided a novel perspective on the mechanisms of this symptom by showing that a simple switch in trigeminal glycine synaptic inhibition can turn touch into pain by unmasking innocuous input to superficial dorsal horn nociceptive specific neurons through a local excitatory, NMDA-dependent neural circuit involving neurons expressing the gamma isoform of protein kinase C. Here, we further investigated the clinical relevance and processing of glycine disinhibition. First, we showed that glycine disinhibition with strychnine selectively induced dynamic but not static mechanical allodynia. The induced allodynia was resistant to morphine. Second, morphine did not prevent the activation of the neural circuit underlying allodynia as shown by study of Fos expression and extracellular-signal regulated kinase phosphorylation in dorsal horn neurons. Third, in contrast to intradermal capsaicin injections, light, dynamic mechanical stimuli applied under disinhibition did not produce neurokinin 1 (NK1) receptor internalization in dorsal horn neurons. Finally, light, dynamic mechanical stimuli applied under disinhibition induced Fos expression only in neurons that did not express NK1 receptor. To summarize, the selectivity and morphine resistance of the glycine-disinhibition paradigm adequately reflect the clinical characteristics of dynamic mechanical allodynia. The present findings thus reveal the involvement of a selective dorsal horn circuit in dynamic mechanical allodynia, which operates through superficial lamina nociceptive-specific neurons that do not bear NK1 receptor and provide an explanation for the differences in the pharmacological sensitivity of neuropathic pain symptoms.

Insular cortex representation of dynamic mechanical allodynia in trigeminal neuropathic rats.

Dynamic mechanical allodynia is a widespread symptom of neuropathic pain for which mechanisms are still poorly understood. The present study investigated the organization of dynamic mechanical allodynia processing in the rat insular cortex after chronic constriction injury to the infraorbital nerve (IoN-CCI). Two weeks after unilateral IoN-CCI, rats showed a dramatic bilateral trigeminal dynamic mechanical allodynia. Light, moving stroking of the infraorbital skin resulted in strong, bilateral upregulation of extracellular-signal regulated kinase phosphorylation (pERK-1/2) in the insular cortex of IoN-CCI animals but not sham rats, in whose levels were similar to those of unstimulated IoN-CCI rats. pERK-1/2 was located in neuronal cells only. Stimulus-evoked pERK-1/2 immunopositive cell bodies displayed rostrocaudal gradient and layer selective distribution in the insula, being predominant in the rostral insula and in layers II-III of the dysgranular and to a lesser extent, of the agranular insular cortex. In layers II-III of the rostral dysgranular insular cortex, intense pERK also extended into distal dendrites, up to layer I. These results demonstrate that trigeminal nerve injury induces a significant alteration in the insular cortex processing of tactile stimuli and suggest that ERK phosphorylation contributes to the mechanisms underlying abnormal pain perception under this condition.

Dorsal horn NK1-expressing neurons control windup of downstream trigeminal nociceptive neurons.

Windup is a progressive, frequency-dependent increase in the excitability of trigeminal and spinal dorsal horn wide dynamic range (WDR) nociceptive neurons to repetitive stimulation of primary afferent nociceptive C-fibers. Superficial dorsal horn neurokinin 1 receptor (NK1R)-expressing neurons were recently shown to regulate sensitization of WDR nociceptive neurons through activation of a defined spino-bulbo-spinal loop. However, the windup of WDR nociceptive neurons was not regulated through this loop. In the present study, we sought to identify the alternative circuit activated by dorsal horn NK1Rs that mediates WDR neuron windup. As a model we used the rat spinal trigeminal nucleus, in which the subnucleus oralis (Sp5O) contains a pool of WDR neurons that receive their nociceptive C-input indirectly via interneurons located in the medullary dorsal horn (MDH). First, we found that intravenous injection of NK1R antagonists (SR140333 and RP67580) produced a reversible inhibition of Sp5O WDR neuron windup. Second, we anatomically identified in the MDH lamina III a subpopulation of NK1R-expressing local interneurons that relay nociceptive information from the MDH to downstream Sp5O neurons. Third, using microinjections of NK1R antagonists during in vivo electrophysiological recordings from Sp5O WDR neurons, we showed that WDR neuron windup depends on activation of NK1Rs located in the MDH laminae I-III. We conclude that, in contrast to central sensitization that is controlled by a spino-bulbo-spinal loop, Sp5O WDR neuron windup is regulated through a local circuit activated by MDH lamina III NK1Rs.

Glycine inhibitory dysfunction turns touch into pain through PKCgamma interneurons.

Dynamic mechanical allodynia is a widespread and intractable symptom of neuropathic pain for which there is a lack of effective therapy. During tactile allodynia, activation of the sensory fibers which normally detect touch elicits pain. Here we provide a new behavioral investigation into the dynamic component of tactile allodynia that developed in rats after segmental removal of glycine inhibition. Using in vivo electrophysiological recordings, we show that in this condition innocuous mechanical stimuli could activate superficial dorsal horn nociceptive specific neurons. These neurons do not normally respond to touch. We anatomically show that the activation was mediated through a local circuit involving neurons expressing the gamma isoform of protein kinase C (PKCgamma). Selective inhibition of PKCgamma as well as selective blockade of glutamate NMDA receptors in the superficial dorsal horn prevented both activation of the circuit and allodynia. Thus, our data demonstrates that a normally inactive circuit in the dorsal horn can be recruited to convert touch into pain. It also provides evidence that glycine inhibitory dysfunction gates tactile input to nociceptive specific neurons through PKCgamma-dependent activation of a local, excitatory, NMDA receptor-dependent, circuit. As a consequence of these findings, we suggest that pharmacological inhibition of PKCgamma might provide a new tool for alleviating allodynia in the clinical setting.

The orofacial formalin test in the mouse: a behavioral model for studying physiology and modulation of trigeminal nociception.

UNLABELLED: The aim of the current study was to adapt the orofacial formalin pain model previously developed in rats for use in mice and to characterize as fully as possible the behavioral changes in this species. The effects of subcutaneous injection of different formalin concentrations (.5%, 1%, 2%, 4%, and 8%) were examined on the face-rubbing response. In mice, formalin injection into the upper lip induced sustained face-rubbing episodes with vigorous face-wash strokes directed to the perinasal area. A positive linear relationship between formalin concentration and amplitude of the rubbing activity was observed during the first and second phase of the test with concentration up to 4%. With the highest concentration used (8%), the amplitude of both phases had plateaued. Systemic administration of morphine and paracetamol induced a dose-dependent inhibition of the rubbing behavior during the second phase. Although both paracetamol and morphine inhibited the first phase, a dose-dependent inhibition was found only for morphine. The ED50 value (95% confidence interval) for suppressing the rubbing response during the first phase was 2.45 mg/kg (1.90-3.08 mg/kg) for morphine. The ED50 values for suppressing the rubbing response during the second phase were 3.52 mg/kg (2.85-4.63 mg/kg) for morphine and 100.66 mg/kg (77.98-139.05 mg/kg) for paracetamol. Heterosegmental nociceptive stimulation evoked by subcutaneous injection of capsaicin into the back of the animal 10 min before the formalin test produced a dose-dependent inhibition of the second phase of the rubbing response. The ED50 values for suppressing the rubbing response during the first and second phases were 9.04 microg (1.36-65.13 microg) and 0.92 microg (0.28-2.99 microg), respectively. In conclusion, the mouse orofacial formalin test appears to be a reliable model for studying the behavioral encoding of the intensity of nociceptive orofacial stimulation and the counter-irritation phenomenon and for testing analgesic drugs. PERSPECTIVE: To further exploit the new opportunities of investigating nociceptive processing at the molecular level with the transgenic "knockout" approach, we require suitable behavioral models in mice. The presented mouse orofacial formalin test appears to be a reliable model for studying the behavioral encoding of the intensity of nociceptive stimulation and the counter-irritation phenomenon and for testing analgesic drugs.

Both oral and caudal parts of the spinal trigeminal nucleus project to the somatosensory thalamus in the rat.

Recent evidence has been accumulated that not only spinal trigeminal nucleus caudalis (Sp5C) neurons but also spinal trigeminal nucleus oralis (Sp5O) neurons respond to noxious stimuli. It is unknown, however, whether Sp5O neurons project to supratrigeminal structures implicated in the sensory processing of orofacial nociceptive information. This study used retrograde tracing with Fluorogold in rats to investigate and compare the projections from the Sp5O and Sp5C to two major thalamic nuclei that relay ascending somatosensory information to the primary somatic sensory cortex: the ventroposteromedial thalamic nucleus (VPM) and the posterior thalamic nuclear group (Po). Results not only confirmed the existence of contralateral projections from the Sp5C to the VPM and Po, with retrogradely labelled neurons displaying a specific distribution in laminae I, III and V, they also showed consistent and similar numbers of retrogradely labelled cell bodies in the contralateral Sp5O. In addition, a topographic distribution of VPM projections from Sp5C and Sp5O was found: neurons in the dorsomedial parts of Sp5O and Sp5C projected to the medial VPM, neurons in the ventrolateral Sp5O and Sp5C projected to the lateral VPM, and neurons in intermediate parts of Sp5O and Sp5C projected to the intermediate VPM. All together, these data suggest that not only the Sp5C, but also the Sp5O relay somatosensory orofacial information from the brainstem to the thalamus. Furthermore, trigemino-VPM pathways conserve the somatotopic distribution of primary afferents found in each subnucleus. These results thus improve our understanding of trigeminal somatosensory processing and help to direct future electrophysiological investigations.