House dust mite-treated PAR2 over-expressor mouse: A novel model of atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a complex skin disease involving causative effects from both intrinsic and extrinsic sources. Murine models of the disease often fall short in one of these components and, as a result, do not fully encapsulate these disease mechanisms. OBJECTIVE: We aimed to determine whether the protease-activated receptor 2 over-expressor mouse (PAR2OE) with topical house dust mite (HDM) application is a more comprehensive and clinically representative AD model. METHODS: Following HDM extract application to PAR2OE mice and controls, AD clinical scoring, itching behaviour, skin morphology and structure, barrier function, immune cell infiltration and inflammatory markers were assessed. Skin morphology was analysed using haematoxylin and eosin staining, and barrier function was assessed by transepidermal water loss measurements. Immune infiltrate was characterised by histological and immunofluorescence staining. Finally, an assessment of AD-related gene expression was performed using quantitative RT-PCR. RESULTS: PAR2OE mice treated with HDM displays all the characteristic clinical symptoms including erythema, dryness and oedema, skin morphology, itch and inflammation typically seen in patients with AD. There is a significant influx of mast cells (P < .01) and eosinophils (P < .0001) into the dermis of these mice. Furthermore, the PAR2OE + HDM mice exhibit similar expression patterns of key differentially expressed genes as seen in human AD. CONCLUSION: The PAR2OE + HDM mouse presents with a classic AD pathophysiology and is a valuable model in terms of reproducibility and overall disease representation to study the condition and potential therapeutic approaches.

Pharmacologic inhibition of hypoxia-inducible factor (HIF)-hydroxylases ameliorates allergic contact dermatitis.

BACKGROUND: When an immune cell migrates from the bloodstream to a site of chronic inflammation, it experiences a profound decrease in microenvironmental oxygen levels leading to a state of cellular hypoxia. The hypoxia-inducible factor-1α (HIF-1α) promotes an adaptive transcriptional response to hypoxia and as such is a major regulator of immune cell survival and function. HIF hydroxylases are the family of oxygen-sensing enzymes primarily responsible for conferring oxygen dependence upon the HIF pathway. METHODS: Using a mouse model of allergic contact dermatitis (ACD), we tested the effects of treatment with the pharmacologic hydroxylase inhibitor DMOG, which mimics hypoxia, on disease development. RESULTS: Re-exposure of sensitized mice to 2,4-dinitrofluorobenzene (DNFB) elicited inflammation, edema, chemokine synthesis (including CXCL1 and CCL5) and the recruitment of neutrophils and eosinophils. Intraperitoneal or topical application of the pharmacologic hydroxylase inhibitors dymethyloxalylglycine (DMOG) or JNJ1935 attenuated this inflammatory response. Reduced inflammation was associated with diminished recruitment of neutrophils and eosinophils but not lymphocytes. Finally, hydroxylase inhibition reduced cytokine-induced chemokine production in cultured primary keratinocytes through attenuation of the JNK pathway. CONCLUSION: These data demonstrate that hydroxylase inhibition attenuates the recruitment of neutrophils to inflamed skin through reduction of chemokine production and increased neutrophilic apoptosis. Thus, pharmacologic inhibition of HIF hydroxylases may be an effective new therapeutic approach in allergic skin inflammation.

A novel therapeutic with two SNAP-25 inactivating proteases shows long-lasting anti-hyperalgesic activity in a rat model of neuropathic pain.

A pressing need exists for long-acting, non-addictive medicines to treat chronic pain, a major societal burden. Botulinum neurotoxin type A (BoNT/A) complex - a potent, specific and prolonged inhibitor of neuro-exocytosis - gives some relief in several pain disorders, but not for all patients. Our study objective was to modify BoNT/A to overcome its inability to block transmitter release elicited by high [Ca2+]i and increase its limited analgesic effects. This was achieved by fusing a BoNT/A gene to that for the light chain (LC) of type/E. The resultant purified protein, LC/E-BoNT/A, entered cultured sensory neurons and, unlike BoNT/A, inhibited release of calcitonin gene-related peptide evoked by capsaicin. Western blotting revealed that this improvement could be due to a more extensive truncation by LC/E of synaptosomal-associated protein of Mr = 25 k, essential for neuro-exocytosis. When tested in a rat spared nerve injury (SNI) model, a single intra-plantar (IPL) injection of LC/E-BoNT/A alleviated for ∼2 weeks mechanical and cold hyper-sensitivities, in a dose-dependent manner. The highest non-paralytic dose (75 U/Kg, IPL) proved significantly more efficacious than BoNT/A (15 U/Kg, IPL) or repeated systemic pregabalin (10 mg/Kg, intraperitoneal), a clinically-used pain modulator. Effects of repeated or delayed injections of this fusion protein highlighted its analgesic potential. Attenuation of mechanical hyperalgesia was extended by a second administration when the effect of the first had diminished. When injected 5 weeks after injury, LC/E-BoNT/A also reversed fully-established mechanical and cold hyper-sensitivity. Thus, combining advantageous features of BoNT/E and/A yields an efficacious, locally-applied and long-acting anti-hyperalgesic.

Changes of voltage-gated sodium channels in sensory nerve regeneration and neuropathic pain models.

PURPOSE: The present study was conducted to determine changes in the expression of voltage-gated sodium channels (VGSCs) α-subunits after nerve injury and their relation with development of neuropathic pain. METHODS: We used the crush injury model of regeneration of the sciatic nerve (Crush) and the spared nerve injury (SNI) model of neuropathic pain in the rat. Measurements of thermal and mechanical pain thresholds were performed until 3 months after injury. Real-time PCR and immunohistochemistry of VGSC α-subunits were used to evaluate the mRNA and protein expression in the DRG. RESULTS: Both nerve injuries induced similar alterations in the VGSCs expression at 7 dpi, with upregulation of Nav1.3, and downregulation of Nav1.7, Nav1.8 and Nav1.9. These changes persisted until 28 days, when hyperalgesia was still present in SNI but not in Crush rats. At 90 days, mRNA expression of all analyzed α-subunits returned to basal levels in the Crush group. However, SNI rats still showed altered expression of VGSCs, and neuropathic pain responses. Immunohistochemical staining revealed that Nav1.8 and Nav1.9 were widely expressed in IB4-positive neurons of the DRG, relevant in pain processing. The population of neurons coexpressing each α-subunit and IB4 was also affected by the injury, more markedly after the Crush. CONCLUSION: Shifts in VGSCs expression occur in parallel to neuropathic pain behavior in rats early after injury, while at later times they appear to be more related to sensory nerve degeneration and regeneration processes.

Differential effects of activity dependent treatments on axonal regeneration and neuropathic pain after peripheral nerve injury.

Activity treatments are useful strategies to increase axonal regeneration and functional recovery after nerve lesions. They are thought to benefit neuropathy by enhancing neurotrophic factor expression. Nevertheless the effects on sensory function are still unclear. Since neurotrophic factors also play a fundamental role in peripheral and central sensitization, we studied the effects of acute electrical stimulation and early treadmill exercise on nerve regeneration and on neuropathic pain, and the relation with the expression of neurotrophins. After sciatic nerve section and suture repair, rats were subjected to electrical stimulation (ES) for 4h after injury, forced treadmill running (TR) for 5 days, or both treatments combined. Sciatic nerve section induced hyperalgesia in the medial area of the plantar skin in the injured paw. TR and ES differently but positively reduced adjacent neuropathic pain before and after sciatic reinnervation. ES enhanced motor and sensory reinnervation, and combination with TR induced strong agonistic effects in relieving pain. The differential effects of these activity treatments were related to changes in neurotrophic factor mRNA levels in sensory and motor neurons. ES speeded up expression of BDNF and GDNF in DRG, and of BDNF and NT3 in the ventral horn. TR reduced the levels of pro-nociceptive factors such as BDNF, NGF and GDNF in DRG. Combination of ES and TR induced intermediate levels suggesting an optimal balancing of treatment effects.

FGF-2 low molecular weight selectively promotes neuritogenesis of motor neurons in vitro.

In this study, we screened in vitro the different capabilities of trophic factors with promising effect for enhancing selective regeneration and thus promoting specific reinnervation of target organs after peripheral nerve regeneration. We found that FGF-2 (18 kDa) was the trophic factor that exerted the most selective effect in promoting neurite outgrowth of spinal motoneurons both in terms of elongation and arborization. The mechanism underlying this effect on neuritogenesis seems related to FGF-2 enhancing the interaction between FGFR-1 and PSA-NCAM. The interaction of these two receptors is important during the early stages of neuritogenesis and pathfinding, while integrin alpha7B subunit seems to play a role during neurite stabilization.

Nociceptive responses and spinal plastic changes of afferent C-fibers in three neuropathic pain models induced by sciatic nerve injury in the rat.

Peripheral nerve injuries induce plastic changes on primary afferent fibers and on the spinal circuitry, which are related to the emergence of neuropathic pain. In this study we compared three models of sciatic nerve injury in the rat with different degrees of damage and impact on regeneration capability: crush nerve injury, chronic constriction injury (CCI) and spared nerve injury (SNI). All three models were characterized by means of nerve histology, in order to describe the degenerative and regenerative process of injured axons. Nociceptive responses were evaluated by mechanical and thermal algesimetry tests. Crush animals displayed higher withdrawal thresholds on the ipsilateral paw compared to the contralateral during the time of denervation, while CCI and SNI animals showed mechanical and thermal hyperalgesia. Central plasticity was evaluated by immunohistochemical labeling of non-peptidergic (IB4-positive) and peptidergic (substance P-positive) nociceptive C-fibers on L4-L6 spinal cord sections. After crush nerve injury and SNI, we observed progressive and sustained reduction of IB4 and SP immunolabeling at the sciatic projection territory in the superficial laminae of the dorsal horn, which affected only the tibial and peroneal nerves projection areas in the case of SNI. After CCI, changes on SP-immunoreactivity were not observed, and IB4-immunoreactive area decreased initially but recovered to normal levels on the second week post-injury. Thus, nociceptive responses depend on the type of injury, and the immunoreactivity pattern of afferent fibers at the spinal cord display changes less pronounced after partial than complete sciatic nerve injury. Although signs of neuropathic pain appear in all three lesion models, nociceptive responses and central plasticity patterns differ between them.