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Painful diabetic neuropathy (PDN) is a common complication of diabetes mellitus with obscure underlying mechanisms. The adaptor protein APPL1 is critical in mediating the insulin sensitizing and insulin signaling. In neurons, APPL1 reportedly affects synaptic plasticity, while its role in the pathogenesis of PDN is masked. Our western blotting revealed significantly decreased APPL1 expression in the dorsal horn in streptozocin (STZ)-induced rats versus the control rats, coupled with concomitant mechanical and thermal hyperalgesia. Afterwards, the determination of exact localization of APPL1 in spinal cord by immunofluorescent staining assay revealed highly expressed APPL1 in the lamina of spinal dorsal horn in control rats, with the overexpression in neurons, microglia and underexpression in astrocytes. The APPL1 expression in laminae I and II was significantly downregulated in PDN rats. Additionally, APPL1 deficiency or overexpression contributed to the increase or decrease of Map and Bassoon, respectively. The localization and immunoactivity of APPL1 and mammalian target of rapamycin (mTOR) were determined in spinal dorsal horn in PDN rats and control rats by immunohistochemistry, suggesting pronounced decrease in APPL1 expression in the superficial layer of the spinal cord in PDN rats, with p-mTOR expression markedly augmented. APPL1 knockdown by infection with lentiviral vector facilitated the activation of mTOR and abrogated mechanical withdrawal threshold (MWT) values in PDN rats. Genetically overexpressed APPL1 significantly eliminated the activation of mTOR and resulted in the augmented MWT values and thermal withdrawal latency (TWL) values. Further, the APPL1 levels affect phosphorylation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), and Akt, the phosphorylation of AMPK and Akt as well as the small GTPase, Rab5 expression in PDN rats. Our results uncovered a novel mechanism by which APPL1 deficiency facilitates the mTOR activation, and thus exacerbates the hyperalgesia in STZ-induced diabetic rats, presumably via the regulation of Rab5/Akt and AMPK signaling pathway.
Learn More >An essential prerequisite for the survival of an organism is the ability to detect and respond to aversive stimuli. Current belief is that noxious stimuli directly activate nociceptive sensory nerve endings in the skin. We discovered a specialized cutaneous glial cell type with extensive processes forming a mesh-like network in the subepidermal border of the skin that conveys noxious thermal and mechanical sensitivity. We demonstrate a direct excitatory functional connection to sensory neurons and provide evidence of a previously unknown organ that has an essential physiological role in sensing noxious stimuli. Thus, these glial cells, which are intimately associated with unmyelinated nociceptive nerves, are inherently mechanosensitive and transmit nociceptive information to the nerve.
Learn More >Headache disorders are highly prevalent and debilitating, with limited treatment options. Previous studies indicate that many pro-inflammatory immune cells contribute to headache pathophysiology. Given the well-recognized role of regulatory T (Treg) cells in maintaining immune homeostasis, we hypothesized that enhancing Treg function may be effective to treat multiple headache disorders. In a mouse model of chronic migraine, we observed that repeated nitroglycerin (NTG, a reliable trigger of migraine in patients) administration doubled the number of CD3 T cells in the trigeminal ganglia without altering the number of Treg cells, suggesting a deficiency in Treg-mediated immune homeostasis. We treated mice with low-dose interleukin-2 (ld-IL2) to preferentially expand and activate endogenous Treg cells. This not only prevented the development of NTG-induced persistent sensitization, but also completely reversed the established facial skin hyper-sensitivity resulting from repeated NTG administration. The effect of ld-IL2 was independent of mouse sex and/or strain. Importantly, ld-IL2 treatment did not alter basal nociceptive responses, and repeated usage did not induce tolerance. The therapeutic effect of ld-IL2 was abolished by Treg depletion and was recapitulated by Treg adoptive transfer. Furthermore, treating mice with ld-IL2 1-7 days after mild traumatic brain injury effectively prevented as well as reversed the development of behaviors related to acute and chronic post-traumatic headache. In a model of medication overuse headache, Ld-IL2 completely reversed the cutaneous hyper-sensitivity induced by repeated administration of sumatriptan. Collectively, the present study identifies ld-IL2 as a promising prophylactic for multiple headache disorders with a mechanism distinct from the existing treatment options.
Learn More >Aldosterone is believed to be synthesized exclusively in the adrenal gland through the processing enzyme aldosterone synthaseMineralocorticoid receptors are predominantly expressed in peripheral nociceptive neurons whose activation leads to increased neuronal excitability and mechanical sensitivity WHAT THIS ARTICLE TELLS US THAT IS NEW: Extra-adrenal production of aldosterone by aldosterone synthase within peripheral sensory neurons contributes to ongoing mechanical hypersensitivity via intrinsic activation of neuronal mineralocorticoid receptorsIntrathecally-applied aldosterone synthase inhibitor reduced aldosterone content in peripheral sensory neurons and subsequently attenuated enhanced mechanical hypersensitivity resulting from local inflammation BACKGROUND:: Recent emerging evidence suggests that extra-adrenal synthesis of aldosterone occurs (e.g., within the failing heart and in certain brain areas). In this study, the authors investigated evidence for a local endogenous aldosterone production through its key processing enzyme aldosterone synthase within peripheral nociceptive neurons.
Learn More >Cortical spreading depression (CSD) is a wave of transient network hyperexcitability leading to long lasting depolarization and block of firing, which initiates focally and slowly propagates in the cerebral cortex. It causes migraine aura and it has been implicated in the generation of migraine headache. Cortical excitability can be modulated by cholinergic actions, leading in neocortical slices to the generation of rhythmic synchronous activities (UP/DOWN states). We investigated the effect of cholinergic activation with the cholinomimetic agonist carbachol on CSD triggered with 130 mM KCl pulse injections in acute mouse neocortical brain slices, hypothesizing that the cholinergic-induced increase of cortical network excitability during UP states could facilitate CSD. We observed instead an inhibitory effect of cholinergic activation on both initiation and propagation of CSD, through the action of muscarinic receptors. In fact, carbachol-induced CSD inhibition was blocked by atropine or by the preferential M1 muscarinic antagonist telenzepine; the preferential M1 muscarinic agonist McN-A-343 inhibited CSD similarly to carbachol, and its effect was blocked by telenzepine. Recordings of spontaneous excitatory and inhibitory post-synaptic currents in pyramidal neurons showed that McN-A-343 induced overall a decrease of the excitatory/inhibitory ratio. This inhibitory action may be targeted for novel pharmacological approaches in the treatment of migraine with muscarinic agonists.
Learn More >Growing evidence suggests a critical role of astrocytes for pain regulation. The water channel protein aquaporin 4 (AQP4), a functional regulator of astrocytes, is involved in various neurological disorders. However, the pathophysiological roles of AQP4 in pain conditions remain unclear. In the present study, we investigated the effect of AQP4 gene knockout in central sensitization induced by complete Freund's adjuvant (CFA). The behavioral analysis revealed that mechanical allodynia and thermal hyperalgesia were more severe in AQP4 null mice than those of wild-type controls over the course of 11 days following CFA intraplantar injection. CFA caused activation of astrocytes with upregulated expression levels of AQP4 and glutamate transporter 1 (GLT1) in the dorsal horn of the spinal cord. AQP4 deficiency reduced GLT1 up-regulation, causing persistent expression of the neuronal activation marker Fos within superficial dorsal horn neurons, including glutamatergic neurons. However, AQP4 deletion did not affect CFA-evoked proinflammatory cytokine expression in the spinal cord. Together, these results have shown that AQP4 absence intensifies CFA-induced spinal central sensitization, which is associated with reduced compensatory up-regulation of GLT1, subsequently increasing glutamatergic overexcitation. Therefore, targeting spinal cord AQP4 may serve as a potential strategy for treatment of peripheral inflammation-evoked hyperalgesia.
Learn More >Neuropathy is major source of chronic pain that can be caused by mechanically or chemically induced nerve injury. Intraplantar formalin injection produces local necrosis over a two-week period and has been used to model neuropathy in rats. To determine whether neuropathy alters dopamine (DA) receptor responsiveness in mesolimbic brain regions, we examined dopamine D-like and D-like receptor (DR) signaling and expression in male rats 14 days after bilateral intraplantar formalin injections into both rear paws. DR-mediated G-protein activation and expression of the DR long, but not short, isoform were reduced in nucleus accumbens (NAc) core, but not in NAc shell, caudate-putamen or ventral tegmental area of formalin-compared to saline-treated rats. In addition, DR-stimulated adenylyl cyclase activity was also reduced in NAc core, but not in NAc shell or prefrontal cortex, of formalin-treated rats, whereas DR expression was unaffected. Other proteins involved in dopamine neurotransmission, including dopamine uptake transporter and tyrosine hydroxylase, were unaffected by formalin treatment. In behavioral tests, the potency of a DR agonist to suppress intracranial self-stimulation (ICSS) was decreased in formalin-treated rats, whereas DR agonist effects were not altered. The combination of reduced DR expression and signaling in NAc core with reduced suppression of ICSS responding by a DR agonist suggest a reduction in D autoreceptor function. Altogether, these results indicate that intraplantar formalin produces attenuation of highly specific DA receptor signaling processes in NAc core of male rats and suggest the development of a neuropathy-induced allostatic state in both pre- and post-synaptic DA receptor function.
Learn More >Non-steroidal anti-inflammatory drugs (NSAIDs), commonly known as COX-1/COX-2 inhibitors, can be effective in treating mild to moderate migraine headache. However, the mechanism by which these drugs act in migraine is not known, nor is the specific contribution of COX-1 versus COX-2 known. We sought to investigate these unknowns using celecoxib, which selectively inhibits the enzymatic activity of COX-2, by determining its effects on several migraine-associated vascular and inflammatory events. Using in vivo two-photon microscopy, we determined intraperitoneal celecoxib effects on CSD-induced blood vessel responses, plasma protein extravasation, and immune cell activation in the dura and pia of mice and rats. Compared to vehicle (control group), celecoxib reduced significantly CSD-induced dilatation of dural arteries and activation of dural and pial macrophages but not dilatation or constriction of pial arteries and veins, or the occurrence of plasma protein extravasation. Collectively, these findings suggest that a mechanism by which celecoxib-mediated COX-2 inhibition might ease the intensity of migraine headache and potentially terminate an attack is by attenuating dural macrophages activation and arterial dilatation outside the blood brain barrier (BBB), and pial macrophages activation inside the BBB.
Learn More >Neuronal activity can be modulated by mechanical stimuli. To study this phenomenon quantitatively, we mechanically stimulated rat cortical neurons by shear stress and local indentation. Neurons show 2 distinct responses, classified as transient and sustained. Transient responses display fast kinetics, similar to spontaneous neuronal activity, whereas sustained responses last several minutes before returning to baseline. Local soma stimulations with micrometer-sized beads evoke transient responses at low forces of ∼220 nN and pressures of ∼5.6 kPa and sustained responses at higher forces of ∼360 nN and pressures of ∼9.2 kPa. Among the neuronal compartments, axons are highly susceptible to mechanical stimulation and predominantly show sustained responses, whereas the less susceptible dendrites predominantly respond transiently. Chemical perturbation experiments suggest that mechanically evoked responses require the influx of extracellular calcium through ion channels. We propose that subtraumatic forces/pressures applied to neurons evoke neuronal responses via nonspecific gating of ion channels.
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