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Patients with Fabry disease suffer from chronic debilitating pain and peripheral sensory neuropathy with minimal treatment options, but the cellular drivers of this pain are unknown. Here, we propose a novel mechanism by which altered signaling between Schwann cells and sensory neurons underlies the peripheral sensory nerve dysfunction we observe in a genetic rat model of Fabry disease. Usingin vivoandin vitroelectrophysiological recordings, we demonstrate that Fabry rat sensory neurons exhibit pronounced hyperexcitability. Schwann cells likely contribute to this finding as application of mediators released from cultured Fabry Schwann cells induces spontaneous activity and hyperexcitability in naïve sensory neurons. We examined putative algogenic mediators using proteomic analysis and found that Fabry Schwann cells release elevated levels of the protein p11 (S100-A10) which induces sensory neuron hyperexcitability. Removal of p11 from Fabry Schwann cell media causes hyperpolarization of neuronal resting membrane potential, indicating that p11 contributes to the excessive neuronal excitability caused by Fabry Schwann cells. These findings demonstrate that rats with Fabry disease exhibit sensory neuron hyperexcitability caused in part by Schwann cell release of the protein p11.Graphical Abstract

Increased excitability of primary sensory neurons after peripheral nerve injury may cause hyperalgesia and allodynia. Dorsal root ganglion field stimulation (GFS) is effective in relieving clinical pain associated with nerve injury and neuropathic pain in animal models. However, its mechanism has not been determined. We examined effects of GFS on transmission of action potentials (APs) from the peripheral to central processes by in vivo single-unit recording from lumbar dorsal roots in sham injured rats and rats with tibial nerve injury (TNI) in fiber types defined by conduction velocity. Transmission of APs directly generated by GFS (20 Hz) in C-type units progressively abated over 20 seconds, whereas GFS-induced Aβ activity persisted unabated, while Aδ showed an intermediate pattern. Activity generated peripherally by electrical stimulation of the sciatic nerve and punctate mechanical stimulation of the receptive field (glabrous skin) was likewise fully blocked by GFS within 20 seconds in C-type units, whereas Aβ units were minimally affected and a subpopulation of Aδ units was blocked. After TNI, the threshold to induce AP firing by punctate mechanical stimulation (von Frey) was reduced, which was reversed to normal during GFS. These results also suggest that C-type fibers, not Aβ, mainly contribute to mechanical and thermal hypersensitivity (von Frey, brush, acetone) after injury. Ganglion field stimulation produces use-dependent blocking of afferent AP trains, consistent with enhanced filtering of APs at the sensory neuron T-junction, particularly in nociceptive units.

Many patients with chronic pain conditions suffer from depression. The mechanisms underlying pain-induced depression are still unclear. There are critical links of medial prefrontal cortex (mPFC) synaptic function to depression, with signaling through the endocannabinoid (eCB) system as an important contributor. We hypothesized that afferent noxious inputs after injury compromise activity-dependent eCB signaling in the mPFC, resulting in depression. Depression-like behaviors were tested in male and female rats with traumatic neuropathy [spared nerve injury (SNI)], and neuronal activity in the mPFC was monitored using the immediate early gene c-fos and in vivo electrophysiological recordings. mPFC eCB Concentrations were determined using mass spectrometry, and behavioral and electrophysiological experiments were used to evaluate the role of alterations in eCB signaling in depression after pain. SNI-induced pain induced the development of depression phenotypes in both male and female rats. Pyramidal neurons in mPFC showed increased excitability followed by reduced excitability in the onset and prolonged phases of pain, respectively. Concentrations of the eCBs, 2-arachidonoylglycerol (2-AG) in the mPFC, were elevated initially after SNI, and our results indicate that this resulted in a loss of CB1R function on GABAergic interneurons in the mPFC. These data suggest that excessive release of 2-AG as a result of noxious stimuli triggers use-dependent loss of function of eCB signaling leading to excessive GABA release in the mPFC, with the final result being behavioral depression. SIGNIFICANCE STATEMENT Pain has both somatosensory and affective components, so the complexity of mechanisms underlying chronic pain is best represented by a biopsychosocial model that includes widespread CNS dysfunction. Many patients with chronic pain conditions develop depression. The mechanism by which pain causes depression is unclear. Although manipulation of the eCB signaling system as an avenue for providing analgesia per se has not shown much promise in previous studies. An important limitation of past research has been inadequate consideration of the dynamic nature of the connection between pain and depression as they develop. Here, we show that activity-dependent synthesis of eCBs during the initial onset of persistent pain is the critical link leading to depression when pain is persistent.

A major hurdle preventing effective interventions for patients with mild traumatic brain injury (mTBI) is the lack of known mechanisms for the long-term cognitive impairment that follows mTBI. The closed head impact model of repeated engineered rotational acceleration (rCHIMERA), a non-surgical animal model of repeated mTBI (rmTBI), mimics key features of rmTBI in humans. Using the rCHIMERA in rats, this study was designed to characterize rmTBI-induced behavioral disruption, underlying electrophysiological changes in the medial prefrontal cortex (mPFC), and associated mitochondrial dysfunction. Rats received 6 closed-head impacts over 2 days at 2 Joules of energy. Behavioral testing included automated analysis of behavior in open field and home-cage environments, rotarod test for motor skills, novel object recognition, and fear conditioning. Following rmTBI, rats spent less time grooming and less time in the center of the open field arena. Rats in their home cage had reduced inactivity time 1 week after mTBI and increased exploration time 1 month after injury. Impaired associative fear learning and memory in fear conditioning test, and reduced short-term memory in novel object recognition test were found 4 weeks after rmTBI. Single-unit in vivo recordings showed increased neuronal activity in the mPFC after rmTBI, partially attributable to neuronal disinhibition from reduced inhibitory synaptic transmission, possibly secondary to impaired mitochondrial function. These findings help validate this rat rmTBI model as replicating clinical features, and point to impaired mitochondrial functions after injury as causing imbalanced synaptic transmission and consequent impaired long-term cognitive dysfunction.

Neonatal hypoxic-ischemic encephalopathy (HIE) causes serious neurological disability; there are, however, currently few promising therapies for it. We have recently shown that δ-opioid receptor (DOR) is neuroprotective by downregulating TNF-α. Since hypoxia-ischemia (HI) triggers a robust inflammatory response, which exacerbates HI brain damage, we investigated, in this study, whether DOR activation could regulate inflammatory cytokine expression, thereby playing a protective effect on the neonatal brain under HI. Twenty-five neonatal rats were randomly divided into five groups: (1) control (control); (2) HI; (3) HI with saline (HI + NS); (4) DOR activation with UFP-512 (a potent and specific DOR agonist) under HI conditions (HI + U); and (5) DOR inhibition using NT treatment under HI conditions (HI + NT). The rats were sacrificed by decapitation at 24 h after HI, and their brains were rapidly removed for measurements. The protein expression of TNF-α, IL-6, ICAM-1, IL-10, IL-18, NQO-1, Nrf-2, and HO-1 was measured using Western blot. In the hemispheres exposed to HI, DOR activation significantly decreased the expressions of TNF-α, IL-6, and ICAM-1 in the cortex, while it significantly increased IL-10 and had no effect on IL-18 in the same region. In contrast, DOR had no appreciable effect on inflammatory cytokine expression in non-cortical tissues including hippocampal, subcortical, and cerebellar tissues. Moreover, HI stress triggered an upregulation of Nrf-2 nuclear protein as well as some of its downstream anti-inflammatory genes such as HO-1 and NQO-1 in the cortex, while DOR activation further augmented such a protective reaction against HI injury. DOR plays an important role in protecting against HI by regulating the expression of inflammatory and anti-inflammatory cytokines in the cortex, which is likely mediated by the Nrf-2/HO-1/NQO-1 signaling.

Dorsal root ganglion field stimulation (GFS) relieves evoked and spontaneous neuropathic pain by use-dependent blockade of impulse trains through the sensory neuron T-junction, which becomes complete within less than 1 minute for C-type units, also with partial blockade of Aδ units. We used this tool in the spinal nerve ligation (SNL) rat model to selectively block sensory neuron spontaneous activity (SA) of axotomized neurons at the fifth lumbar (L5) level vs blockade of units at the L4 level that remain uninjured but exposed to inflammation. In vivo dorsal root single-unit recordings after SNL showed increased SA in L5 units but not L4 units. Ganglion field stimulation blocked this SA. Ganglion field stimulation delivered at the L5 dorsal root ganglion blocked mechanical hyperalgesia behavior, mechanical allodynia, and ongoing spontaneous pain indicated by conditioned place preference, whereas GFS at L4 blocked evoked pain behavior but not spontaneous pain. In vivo single-unit recordings of spinal cord dorsal horn (DH) wide-dynamic-range neurons showed elevated SA after SNL, which was reduced by GFS at the L5 level but not by GFS at the L4 level. In addition, L5 GFS, but not L4 GFS, increased mechanical threshold of DH units during cutaneous mechanical stimulation, while L5 GFS exceeded L4 GFS in reducing evoked firing rates. Our results indicate that SA in injured neurons supports increased firing of DH wide-dynamic-range neurons, contributing to hyperalgesia, allodynia, and ongoing pain. Ganglion field stimulation analgesic effects after nerve injury are at least partly attributable to blocking propagation of this SA.

Objectives Electrical stimulation of the dorsal root ganglion (DRG), referred to here as ganglionic field stimulation (GFS), is effective in reducing clinical pain, probably by interrupting transmission of afferent impulse trains on sensory neurons as they pass through the DRG. We therefore tested whether efferent impulse trains conveyed by sensory neurons, which contribute to neurogenic inflammation, may also be interrupted by GFS. Materials and Methods Collagen-induced arthritis, a model of clinical rheumatoid arthritis, was initiated in rats concurrently with the insertion of an electrode for GFS at the fourth lumbar DRG. Continuous GFS (20 Hz pulse rate, current at 80% of the motor threshold) was initiated 6 days later and continued for 14 days. Plantar pain sensitivity, ankle arthritis score, and dimensions of the foot and ankle were determined one hour after termination of GFS. Results The foot/ankle contralateral to GFS developed hypersensitivity to threshold and noxious mechanical stimulation, swelling, and high arthritis score, all of which were normalized in the foot/ankle ipsilateral with GFS. Histology showed GFS limited joint destruction. Electrophysiological recording showed GFS can block efferent impulse trains. Conclusions Our findings show that GFS can reduce neurogenic inflammation and the resulting joint damage in a model of rheumatoid arthritis, probably by blocking the transit of impulse trains through the DRG. GFS may have clinical utility in limiting joint destruction in inflammatory arthritis such as rheumatoid arthritis.

Background: Hypoxic/ischemic injury to the heart is a frequently encountered clinical problem with limited therapeutic options. Since microRNAs (miRNAs) are involved in hypoxic/ischemic events, and δ-opioid receptor (DOR) activation is known to protect against hypoxic/ischemic injury, we speculated on the involvement of DOR activation in altering miRNA expression in the heart under hypoxic conditions. The present study aimed to test our hypothesis. Methods: Male Sprague Dawley rats were exposed to hypoxia (9.5-10% O2) for 1, 5, or 10 days with or without DOR activation. The target miRNAs were selected from TaqMan low-density array (TLDA) data and were further analyzed by quantitative real-time PCR. Results: We found that: 1) hypoxia alters the miRNA expression profiles depending on the hypoxic duration; 2) DOR activation shifts miRNA expression profiles in normoxic conditions and upregulates miR-128a-3p, miR-134-5p, miR-135a, miR-193a-3p, miR-196a, miR-324-3p, and miR-338; and 3) DOR activation modifies hypoxia-induced changes in miRNA expression and increases the levels of miR-128a-3p, miR-134-5p, miR-135a, miR-193a-3p, miR-196a, miR-324-3p, miR-141, miR-200b, and miR-324-3p. For example, miR-196c-5p decreased by 50% while miR-135a-5p increased 2.9 fold after 10 days under hypoxic conditions. Moreover, DOR activation further strengthened the hypoxia-induced increase of the levels of miR-7a-5p. When DOR was activated using UFP-512, the level of miR-107-3p significantly increased 1 day after the administration of UFP-512, but gradually decreased back to normal under normoxia. Conclusion: Hypoxia significantly modifies the miRNA profile in the heart, which can be mimicked or modified by DOR activation. Defining the targeted pathways that regulate the diverse cellular and molecular functions of miRNAs may provide new insights into potential therapies for hypoxic/ischemic injury of the heart.

Parkinson's disease (PD), a neurodegenerative disorder, is traditionally classified as a movement disorder. Patients typically suffer from many motor dysfunctions. Presently, clinicians and scientists recognize that many non-motor symptoms are associated with PD. There is an increasing interest in both motor and non-motor symptoms in clinical studies on PD patients and laboratory research on animal models that imitate the pathophysiologic features and symptoms of PD patients. Therefore, appropriate behavioral assessments are extremely crucial for correctly understanding the mechanisms of PD and accurately evaluating the efficacy and safety of novel therapies. This article systematically reviews the behavioral assessments, for both motor and non-motor symptoms, in various animal models involved in current PD research. We addressed the strengths and weaknesses of these behavioral tests and their appropriate applications. Moreover, we discussed potential mechanisms behind these behavioral tests and cautioned readers against potential experimental bias. Since most of the behavioral assessments currently used for non-motor symptoms are not particularly designed for animals with PD, it is of the utmost importance to greatly improve experimental design and evaluation in PD research with animal models. Indeed, it is essential to develop specific assessments for non-motor symptoms in PD animals based on their characteristics. We concluded with a prospective view for behavioral assessments with real-time assessment with mobile internet and wearable device in future PD research.

Perinatal hypoxic-ischemic (HI) brain injury results in death or profound long-term neurologic disability in both children and adults. However, there is no effective pharmacological therapy due to a poor understanding of HI events, especially the initial triggers for hypoxic-ischemic injury such as disrupted ionic homeostasis and the lack of effective intervention strategy. In the present study, we showed that neonatal brains undergo a developmental increase in the disruption of K+ homeostasis during simulated ischemia, oxygen-glucose deprivation (OGD) and neonatal HI cortex has a triple phasic response (earlier attenuation, later enhancement, and then recovery) of disrupted K+ homeostasis to OGD. This response partially involves the activity of the δ-opioid receptor (DOR) since the earlier attenuation of ischemic disruption of K+ homeostasis could be blocked by DOR antagonism, while the later enhancement was reversed by DOR activation. Similar to DOR activation, acupuncture, a strategy to promote DOR activity, could partially reverse the later enhanced ischemic disruption of K+ homeostasis in the neonatal cortex. Since maintaining cellular K+ homeostasis and inhibiting excessive K+ fluxes in the early phase of hypoxic-ischemic insults may be of therapeutic benefit in the treatment of ischemic brain injury and related neurodegenerative conditions, and since many neurons and other cells can be rescued during the "window of opportunity" after HI insults, our first findings regarding the role of acupuncture and DOR in attenuating ischemic disruption of K+ homeostasis in the neonatal HI brain suggest a potential intervention therapy in the treatment of neonatal brain injury, especially hypoxic-ischemic encephalopathy.

Aim of review: δ-opioid receptor (DOR) is a member of opioid receptor superfamily, and is widely distributed throughout the central nervous system and plays diverse functions. Accumulated evidence has demonstrated that activation of DOR is neuroprotective against hypoxic/ischemic insults and DOR signaling may be one of the key factors determining neuronal survival during hypoxic/ischemic stress.Method: In this review, we summarized the research progress of DOR-mediated neuroprotection against hypoxic/ischemic injury, and then discussed the underlying mechanisms.Recent findings: We have made a special focus on DOR action against hypoxic/ischemic disruption of ionic homeostasis since it is an initial and key step in hypoxic/ischemic neuronal injury and death. DOR stabilization of ion homeostasis against hypoxic/ischemic stress involves multiple strategies such as ionic interplays, protein kinase mechanism, and decrease in excitability and excitatory transmission-associated ionic derangement.Summary: The findings of DOR stabilization of hypoxic/ischemic disruption of ionic homeostasis provide us a new concept of neuroprotection against neurological disorders and have potentially significant impacts on the prevention and treatment of some serious neurological conditions, such as stroke. Citation: Dongman Chao, Ying Xia. δ-opioid receptor induced neuroprotection against hypoxic/ischemic injury: regulation of ionic homeostasis. J Anesth Perioper Med 2015; 2: 325-35. doi: 10.24015/JAPM.2015.0043This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Neonatal hypoxic-ischemic encephalopathy (HIE) causes serious neurological disability; there are, however, currently few promising therapies for it. We have recently shown that δ-opioid receptor (DOR) is neuroprotective by downregulating TNF-α. Since hypoxia-ischemia (HI) triggers a robust inflammatory response, which exacerbates HI brain damage, we investigated, in this study, whether DOR activation could regulate inflammatory cytokine expression, thereby playing a protective effect on the neonatal brain under HI. Twenty-five neonatal rats were randomly divided into five groups: (1) control (control); (2) HI; (3) HI with saline (HI + NS); (4) DOR activation with UFP-512 (a potent and specific DOR agonist) under HI conditions (HI + U); and (5) DOR inhibition using NT treatment under HI conditions (HI + NT). The rats were sacrificed by decapitation at 24 h after HI, and their brains were rapidly removed for measurements. The protein expression of TNF-α, IL-6, ICAM-1, IL-10, IL-18, NQO-1, Nrf-2, and HO-1 was measured using Western blot. In the hemispheres exposed to HI, DOR activation significantly decreased the expressions of TNF-α, IL-6, and ICAM-1 in the cortex, while it significantly increased IL-10 and had no effect on IL-18 in the same region. In contrast, DOR had no appreciable effect on inflammatory cytokine expression in non-cortical tissues including hippocampal, subcortical, and cerebellar tissues. Moreover, HI stress triggered an upregulation of Nrf-2 nuclear protein as well as some of its downstream anti-inflammatory genes such as HO-1 and NQO-1 in the cortex, while DOR activation further augmented such a protective reaction against HI injury. DOR plays an important role in protecting against HI by regulating the expression of inflammatory and anti-inflammatory cytokines in the cortex, which is likely mediated by the Nrf-2/HO-1/NQO-1 signaling.

We have recently demonstrated that δ-opioid receptor (DOR) activation attenuates α-synuclein expression/aggregation induced by MPP(+) and/or severe hypoxia. Since α-synuclein plays a critical role in the pathogenesis of Parkinson's disease, DOR activation may trigger an antiparkinson pathway(s) against α-synuclein-induced injury. However, the underlying mechanism is unknown yet. In HEK293T and PC12 cells, we investigated the effects of DOR activation on the oligomer formation induced by α-synuclein overexpression and mutation in normoxic and hypoxic conditions and explored the potential signaling pathways for DOR protection. We found that (1) increased expression of both wild-type and A53T-mutant α-synuclein led to the formation of α-synuclein oligomers and cytotoxic injury; (2) DOR activation largely attenuated the formation of toxic α-synuclein oligomers induced by α-synuclein overexpression/mutation and/or hypoxia; (3) DOR activation attenuated α-synuclein-induced cytotoxicity through TORC1/SIK1/CREB, but not the phospho-CREB pathway, while DOR activation reduced hypoxic cell injury through the phospho-CREB mechanism; and (4) the interaction of α-synuclein and the DJ-1 was involved in the mechanisms for DOR-mediated protection against α-synuclein oligomer formation. Our findings suggest that DOR attenuates the formation of toxic α-synuclein oligomers through the phos-CREB pathway under hypoxic conditions, and through TORC1/SIK1/CREB pathways in the conditions of α-synuclein overexpression and mutation. The DJ-1 gene was involved in the DOR protection against parkinsonian injury.

Background and Purpose Hypoxia/reoxygenation induces synthesis of reactive oxygen species (ROS) which can attack macromolecules and cause brain injury. The transcription factor, nuclear factor (erythroid-derived 2)-like 2, (Nrf2), ia potent activator of genes with an antioxidant responsive element and Nrf2 can counteract oxidative injury by increasing expression of several antioxidative genes in response to ROS stress. Here, we show that activation of the δ-opioid receptor (DOR) increasedNrf2 protein expression and translocation, thereby leading to cytoprotection. Experimental Approach We used HEK293t cells exposed to 0.5% O2 for 16 h and then reoxygenated for 4 h as a model of hypoxia-reperfusion (H/R) injury. Real time PCR, Western blotting, siRNA and immunohistochemical techniques were used to follow Nrf2 expression and activity. Cell viability and damage (as LDH leakage) were also measured. Key Results H/R injury triggered Nrf2 translocation into the nucleus and up-regulated expression of several downstream genes, relevant to antioxidation, such as NAD(P)H:quinone oxidoreductase (NQO1). Incubation with the DOR agonist UFP-512 enhanced Nrf2 protein expression and translocation and up-regulated its downstream genes in normoxia and further increased Nrf2 expression and translocation after H/R, protecting the cells against loss of viability and damage. The effect of UFP-512 on Nrf2 nuclear translocation was blocked by the DOR antagonist, naltrindole. Also, DOR–mediated cytoprotection was strongly inhibited after transfection of HEK293t cells with Nrf2 siRNA. Conclusions and Implications The DOR agonist UFP-512 was cytoprotective against H/R injury and this effect was partly dependent on DOR-mediated increase in Nrf2 function.

Background and Purpose Astrocytic excitatory amino acid transporters (EAATs) regulate extracellular glutamate concentrations and play a role in preventing neuroexcitotoxicity. As the δ-opioid receptor (DOP receptor) is neuroprotective against excitotoxic injury, we determined whether DOP receptor activation up-regulates EAAT expression and function. Experimental Approach We measured mRNA and protein expression of EAAT1, EAAT2 and EAAT3 in cultured mouse astrocytes exposed to a specific DOP receptor agonist (UFP-512) with or without a DOP receptor antagonist, DOP receptor siRNA or inhibitors of PKC, PKA, PI3K, p38, MAPK, MEK and ERK, and evaluated the function of EAATs by measuring glutamate uptake. Key Results Astrocytic DOP receptor mRNA and protein were suppressed by DOP receptor siRNA knockdown. DOP receptor activation increased mRNA and protein expression of EAAT1 and EAAT2, but not EAAT3, thereby enhancing glutamate uptake of astrocytes. DOP receptor-induced EAAT1 and EAAT2 expression was largely reversed by DOP receptor antagonist naltrindole or by DOP receptor siRNA knockdown, and suppressed by inhibitors of MEK, ERK and p38. DOP receptor-accelerated glutamate uptake was inhibited by EAAT blockers, DOP receptor siRNA knockdown or inhibitors of MEK, ERK or p38. In contrast, inhibitors of PKA, PKC or PI3K had no significant effect on DOP receptor-induced EAAT expression. Conclusions and Implications DOP receptor activation up-regulates astrocytic EAATs via MEK-ERK-p38 signalling, suggesting a critical role for DOP receptors in the regulation of astrocytic EAATs and protection against neuroexcitotoxicity. As decreased EAAT expression contributes to pathophysiology in many neurological diseases, including amyotrophic lateral sclerosis, our findings present a new platform for potential treatments of these diseases.

Emerging evidence suggests that the δ-opioid receptor (DOR) is neuroprotective against hypoxic stress. Since inflammatory factors play an important role in hypoxic injury, we sought to determine if DOR modulates cellular inflammatory process in hypoxia. Since astrocytes produce inflammatory cytokines and play a major role in inflammatory response to hypoxia, we exposed cultured rat astrocytes to severe hypoxia (0.1 % O2) for 3–6 h to examine if DOR activation altered hypoxia-induced expression of proinflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) along with cell viability. To examine a differential DOR-mediated modulation between glial and neuron-like cells, we also conducted the same experiments in differentiated PC-12 cells. We found that hypoxia reduced cell viability in both astrocytes and PC-12 cells, and DOR activation with UFP-512 (5 μM) effectively protected both the cells against hypoxic insult. Hypoxia markedly increased TNF-α levels in the astrocytes (p < 0.05); however, the same hypoxic stress reduced TNF-α levels in the PC-12 cells. Exogenous addition of TNF-α to the culture media further worsened the hypoxia-induced damage in PC-12 cells. Hypoxia increased the DOR-expression in astrocytes but not in PC-12 cells. DOR-activation reversed the hypoxia-induced changes in TNF-α levels in both the cells. In contrast, the IL-1β levels did not significantly alter in astrocytes and PC-12 cells when exposed to the same hypoxic conditions or following DOR activation. Our novel data suggest that DOR activation inhibits TNF-α mediated inflammatory processes following exposure to severe hypoxia with a differential regulation between glial vs. neuron-like cells.

Epilepsy is one of the most common neurological disorders affecting about 1% of population. Although the precise mechanism of its pathophysiological changes in the brain is unknown, epilepsy has been recognized as a disorder of brain excitability characterized by recurrent unprovoked seizures that result from the abnormal, excessive, and synchronous activity of clusters of nerve cells in the brain. Currently available therapies, including medical, surgical, and other strategies, such as ketogenic diet and vagus nerve stimulation, are symptomatic with their own limitations and complications. Seeking new strategies to cure this serious disorder still poses a big challenge to the field of medicine. Our recent studies suggest that acupuncture may exert its antiepileptic effects by normalizing the disrupted neuronal and network excitability through several mechanisms, including lowering the overexcited neuronal activity, enhancing the inhibitory system, and attenuating the excitatory system in the brain via regulation of the interaction betweenδ-opioid receptors (DOR) and Na+channels. This paper reviews the progress in this field and summarizes new knowledge based on our work and those of others.

Hypoxia-ischemia (HI) insult induces inflammatory/oxidative injury and causes neurological disability and mortality. However, there are limited therapeutic options at present. Our recent studies have shown that the δ-opioid receptor (DOR) is neuroprotective against hypoxic-ischemic stress. In this work, we asked if DOR activation protects the brain against hypoxic-ischemic encephalopathy (HIE) by regulating inflammatory cytokines and Nrf-2, a basic leucine zipper protein that regulates the expression of antioxidant proteins. Our results showed that DOR activation with UFP-512 treatment significantly decreased TNF-α, IL-6, and ICAM-1 expression (P0.05). In the control group, no significant difference was detected after the vehicle treatment. Furthermore, we investigated the role of Nrf-2 in the DOR neuroprotection since our recent data suggest that DOR signaling promotes the expression and relocation of Nrf2 and thus induces cytoprotection. HI significantly increased nuclear Nrf-2 protein, while DOR activation by pretreatment with UFP-512 before HI further elevated the level of nuclear Nrf-2 protein by 70% (P This work was supported by NIH (AT-004422), Memorial Hermann’s Foundation and Vivian L Smith Neurologic Foundation. GD was supported by 973 Program (2012CB518502) and NSFC (81574053 & 81590953) of China.

Activation of delta-opioid receptors (DOR) is neuroprotective against hypoxic/ischemic injury in the cortex, which is at least partially related to its action against hypoxic/ischemic disruption of ionic homeostasis that triggers neuronal injury. Na(+) influx through TTX-sensitive voltage-gated Na(+) channels may be a main mechanism for hypoxia-induced disruption of K(+) homeostasis, with DOR activation attenuating the disruption of ionic homeostasis by targeting voltage-gated Na(+) channels. In the present study we examined the role of DOR in the regulation of Na(+) influx in anoxia and simulated ischemia (oxygen-glucose deprivation) as well as the effect of DOR activation on the Na(+) influx induced by a Na(+) channel opener without anoxic/ischemic stress and explored a potential PKC mechanism underlying the DOR action. We directly measured extracellular Na(+) activity in mouse cortical slices with Na(+) selective electrodes and found that (1) anoxia-induced Na(+) influx occurred mainly through TTX-sensitive Na(+) channels; (2) DOR activation inhibited the anoxia/ischemia-induced Na(+) influx; (3) veratridine, a Na(+) channel opener, enhanced the anoxia-induced Na(+) influx; this could be attenuated by DOR activation; (4) DOR activation did not reduce the anoxia-induced Na(+) influx in the presence of chelerythrine, a broad-spectrum PKC blocker; and (5) DOR effects were blocked by PKCβII peptide inhibitor, and PKCθ pseudosubstrate inhibitor, respectively. We conclude that DOR activation inhibits anoxia-induced Na(+) influx through Na(+) channels via PKC (especially PKCβII and PKCθ isoforms) dependent mechanisms in the cortex.

Maintenance of ionic balance is essential for neuronal functioning. Hydrogen sulfide (H(2)S), a known toxic environmental gaseous pollutant, has been recently recognized as a gasotransmitter involved in numerous biological processes and is believed to play an important role in the neural activities under both physiological and pathological conditions. However, it is unclear if it plays any role in maintenance of ionic homeostasis in the brain under physiological/pathophysiological conditions. Here, we report by directly measuring Na(+) activity using Na(+) selective electrodes in mouse cortical slices that H(2)S donor sodium hydrosulfide (NaHS) increased Na(+) influx in a concentration-dependent manner. This effect could be partially blocked by either Na(+) channel blocker or N-methyl-D-aspartate receptor (NMDAR) blocker alone or almost completely abolished by coapplication of both blockers but not by non-NMDAR blocker. These data suggest that increased H(2)S in pathophysiological conditions, e.g., hypoxia/ischemia, potentially causes a disruption of ionic homeostasis by massive Na(+) influx through Na(+) channels and NMDARs, thus injuring neural functions. Activation of delta-opioid receptors (DOR), which reduces Na(+) currents/influx in normoxia, had no effect on H(2)S-induced Na(+) influx, suggesting that H(2)S-induced disruption of Na(+) homeostasis is resistant to DOR regulation and may play a major role in neuronal injury in pathophysiological conditions, e.g., hypoxia/ischemia.

Neurons in the mammalian central nervous system are extremely vulnerable to oxygen deprivation and blood supply insufficiency. Indeed, hypoxic/ischemic stress triggers multiple pathophysiological changes in the brain, forming the basis of hypoxic/ischemic encephalopathy. One of the initial and crucial events induced by hypoxia/ischemia is the disruption of ionic homeostasis characterized by enhanced K(+) efflux and Na(+)-, Ca(2+)- and Cl(-)-influx, which causes neuronal injury or even death. Recent data from our laboratory and those of others have shown that activation of opioid receptors, particularly delta-opioid receptors (DOR), is neuroprotective against hypoxic/ischemic insult. This protective mechanism may be one of the key factors that determine neuronal survival under hypoxic/ischemic condition. An important aspect of the DOR-mediated neuroprotection is its action against hypoxic/ischemic disruption of ionic homeostasis. Specially, DOR signal inhibits Na(+) influx through the membrane and reduces the increase in intracellular Ca(2+), thus decreasing the excessive leakage of intracellular K(+). Such protection is dependent on a PKC-dependent and PKA-independent signaling pathway. Furthermore, our novel exploration shows that DOR attenuates hypoxic/ischemic disruption of ionic homeostasis through the inhibitory regulation of Na(+) channels. In this review, we will first update current information regarding the process and features of hypoxic/ischemic disruption of ionic homeostasis and then discuss the opioid-mediated regulation of ionic homeostasis, especially in hypoxic/ischemic condition, and the underlying mechanisms.

Hypoxic/ischemic disruption of ionic homeostasis is a critical trigger of neuronal injury/death in the brain. There is, however, no promising strategy against such pathophysiologic change to protect the brain from hypoxic/ischemic injury. Here, we present a novel finding that activation of delta-opioid receptors (DOR) reduced anoxic Na+ influx in the mouse cortex, which was completely blocked by DOR antagonism with naltrindole. Furthermore, we co-expressed DOR and Na+ channels in Xenopus oocytes and showed that DOR expression and activation indeed play an inhibitory role in Na+ channel regulation by decreasing the amplitude of sodium currents and increasing activation threshold of Na+ channels. Our results suggest that DOR protects from anoxic disruption of Na+ homeostasis via Na+ channel regulation. These data may potentially have significant impacts on understanding the intrinsic mechanism of neuronal responses to stress and provide clues for better solutions of hypoxic/ischemic encephalopathy, and for the exploration of acupuncture mechanism since acupuncture activates opioid system.

Activation of delta-opioid receptors (DOR) attenuates anoxic K(+) leakage and protects cortical neurons from anoxic insults by inhibiting Na(+) influx. It is unknown, however, which pathway(s) that mediates the Na(+) influx is the target of DOR signal. In the present work, we found that, in the cortex, (1) DOR protection was largely dependent on the inhibition of anoxic Na(+) influxes mediated by voltage-gated Na(+) channels; (2) DOR activation inhibited Na(+) influx mediated by ionotropic glutamate N-methyl-D-aspartate (NMDA) receptors, but not that by non-NMDA receptors, although both played a role in anoxic K(+) derangement; and (3) DOR activation had little effect on Na(+)/Ca(2+) exchanger-based response to anoxia. We conclude that DOR activation attenuates anoxic K(+) derangement by restricting Na(+) influx mediated by Na(+) channels and NMDA receptors, and that non-NMDA receptors and Na(+)/Ca(2+) exchangers, although involved in anoxic K(+) derangement in certain degrees, are less likely the targets of DOR signal.

Perinatal hypoxic-ischemic (HI) brain injury results in death or profound long-term neurologic disability in both children and adults. However, there is no effective pharmacological therapy due to a poor understanding of HI events, especially the initial triggers for hypoxic-ischemic injury such as disrupted ionic homeostasis and the lack of effective intervention strategy. In the present study, we showed that neonatal brains undergo a developmental increase in the disruption of K

Parkinson’s disease (PD) is the second most common age-related neurodegenerative disease resulting from the death of dopaminergic neurons in the substantia nigra pars compacta (SNc) which innervate all of the neuclei of the basal ganglia. Cell loss in the SNc causes a consequent striatal dopamine (DA) deficiency, leading to a cascade of functional changes in basal ganglia circuitry and ultimately the development of the cardinal features of PD, including resting tremor, bradykinesia, postural instability and rigidity, along with non-motor symptoms including cognitive, affective, psychiatric, and autonomic problems. DA replacement with the Levodopa (L-DOPA) is still a gold standard for symptomatic therapy of PD. However, the side effects of long-term use of L-DOPA are obvious. The emerging non-dopaminergic treatments for PD are becoming a scheduling program for the pipe-lines of drug development and clinical trials. The abundant expression of opioid receptors and their endogenous ligands, especially those for the δ-opioid system within the basal ganglia, has attracted much attention for its contribution to neurodegenerative diseases such as PD. In this article, we first discussed the relevant functional neuroanatomy and circuitry of the basal ganglia, and the evolving PD models of the basal ganglia. We then retrospected the studies on the location and expression of the δ-opioid system within the basal ganglia in health and PD, opioid-induced regulation of neurotransmitter release in basal ganglia, effects of pharmacological δ-opioid receptor (DOR) manipulation on PD and levodopa-induced dyskinesia (LID), and the effects of DOR activation on dopaminergic neuron injury/cellular model of PD. Based on these analyses, we believe that δ-opioid signaling is generally a beneficial, not detrimental factor for motor complications in PD. However, more in-depth investigations are expected to provide more solid and direct evidence for this benefit and to develop therapeutic strategies against PD by targeting δ-opioid system.

Neurons in the mammalian central nervous system are extremely vulnerable to oxygen deprivation and blood supply insufficiency. Hypoxia/ischemia can produce considerably longer or sustained changes in ionic concentrations that are characterized by enhanced K+ efflux and Na+-, Ca2+- and Cl− influx. The hypoxic/ischemic disruption of ionic homeostasis is an initial and key step for hypoxic/ischemic neuronal injury and death. Limiting hypoxic/ischemic ion dysregulation and stabilization of ionic homeostasis in the initial stage of hypoxia/ischemia is an efficient strategy in the treatment of stroke and ischemia-related neurodegenerative conditions. Substantial studies from our and many independent laboratories have demonstrated that activation of the δ-opioid receptor (DOR) is neuroprotective against hypoxic/ischemic insults. In this chapter, we first updated the recent progress in the studies of DOR neuroprotection against hypoxic/ischemic insults and the features and potential mechanisms of hypoxia/ischemia-induced disruption of major cation homeostasis. On the basis of this discussion, we addressed the important role of DOR in the homeostatic maintenance of these ions and the underlying mechanisms. Based on our recent work and current literature, we have drawn an initial picture of how DOR protects neurons against hypoxia/ischemia through the regulation of ionic homeostasis. In brief, DOR signals inhibit Na+ influx and reduce the increase in intracellular Ca2+, thus decreasing the excessive K+ leakage. This DOR-mediated action involves a PKC-dependent and PKA-independent signaling pathway.

Epilepsy is one of the most common neurological disorders, affecting about 1 % of the population. Although the precise mechanism of its pathophysiological changes in the brain is unknown, epilepsy has been recognized as a disorder of brain excitability characterized by recurrent unprovoked seizures that result from the abnormal, excessive, and synchronous activity of clusters of nerve cells in the brain. Currently available therapies, including medical, surgical, and other strategies such as ketogenic diet and vagus nerve stimulation, are all used for the control of epileptic symptoms with their own limitations and complications. Seeking new strategies to cure this serious disorder still poses a big challenge in the field of medicine. Recent studies suggest that acupuncture may exert its antiepileptic effects by normalizing the disrupted neuronal and network excitability through several mechanisms, including lowering the overexcited neuronal activity, enhancing the inhibitory system, and attenuating the excitatory system in the brain via regulation of the interaction between δ-opioid receptors (DOR) and Na+ channels. This chapter reviews the progress in this field and summarizes new knowledge based on our work and the work of others.

Opioids and their analogues have been used as clinical analgesics for several centuries, with reports dating back to as early as 4000 B.C. Their anti-pain action is primarily accomplished through three major opioid receptor subtypes: μ-opioid receptor (MOR), δ-opioid receptor (DOR), and κ-opioid receptor (KOR). Although these opioid receptors have been cloned in many species, their complex functions, other than pain modulation, are still largely unknown in both physiological and pathophysiological conditions. However, recent studies have uncovered fresh insights into opioid receptor-mediated functions and their underlying mechanisms. Studies suggest that these opioid receptors participate in various physiological activities. Therefore, their dysfunction may play a role in numerous diseases, including hypoxic/ischemic injury in brain, epileptic seizures, Parkinson’s disease, Multiple Sclerosis, cardiovascular disease, respiratory depression, pulmonary artery hypertension, malignancies, diabetic cutaneous wounds, immune disease, chronic kidney disease, uremic pruritus, renal ischemia-reperfusion injury, and pre-eclampsia. There is also strong evidence showing that opioid receptors are involved in the regulation of stress, feeding and obesity, and play an essential role in hibernation in some species. One of the most exciting findings in the past decade is DOR-mediated neuroprotection and cardioprotection. The upregulation of DOR expression and activity increases the neuronal and myocardial tolerance to hypoxic and/or ischemic stress. The DOR signal triggers different mechanisms at multiple levels, depending on stress duration and severity, to preserve neuronal and myocardial survival, including the stabilization of homeostasis and increase in pro-survival signaling. DOR-mediated neuroprotection and cardioprotection may potentially influence clinical pharmacology in terms of prevention and treatment of life-threatening conditions like stroke and myocardial infarction. The main purpose of this chapter is to update recent research on opioids and their various functions, particularly in pathophysiological conditions. This chapter aims to deliver an informative reference for better understanding the opioid system from a/the pathophysiological point of view.

Homeostasis is one of the most fundamental concepts in understanding the physiological functions of bodies as well as the pathophysiology of diseases, and in a global view of system biology, is also a vital guiding principle for medicine. Because of the particular importance of ionic environment in neuronal functions, ion homeostasis is the most fundamental mechanism among all the brain homeostasis regulations, and most of the functional activities of the brain are highly dependent on the dynamic and relatively steady state of ionic environment. The concentrations of ions are in dynamic balance under physiological conditions, which depend on the complicated, but elaborate, regulatory mechanisms. The δ-opioid receptor (DOR) regulates a diverse array of physiological functions. DOR and its cognate endogenous opioids are widely expressed throughout the central nervous system and have an extensive interaction with ion channels, receptors, and transporters. DOR can regulate the release of many neurotransmitters, modify neuronal electrical activities and synaptic plasticity, and thereby tightly regulates ion homeostasis in neural activities. In this chapter, we will focus on the regulation of homeostasis of the key cations such as Na+, K+, Ca2+ in the brain under physiological conditions and the role of DOR in such homeostatic regulation. Almost all the previous studies regarding DOR-mediated regulation of ionic homeostasis under normoxic condition focused on intracellular Ca2+ activity. Few studies highlighted the regulation of K+ and Na+ homeostasis despite the functional coupling of DOR with K+ channels and Na+ channels. Overall, the predominant effect of DOR activation on Ca2+ entry is inhibitory. However, some studies show either mobilization of intracellular Ca2+ or stimulation of Ca2+ entry with opioid activation. In general, DOR signaling is inhibitory to Na+ influx and K+ efflux. In regard to this issue, we have recently made the first finding that DOR activation and expression reduces Na+ currents by targeting voltage-sensitive Na+ channels. The DOR effects have been demonstrated to occur through activation of different kind of G proteins, including Gi, Go, and even Gs classes.

Central neurons are extremely vulnerable to hypoxic/ischemic insult, which is a major cause of neurologic morbidity and mortality as a consequence of neuronal dysfunction and death. Our recent work has shown that δ-opioid receptor (DOR) is neuroprotective against hypoxic and excitotoxic stress, although the underlying mechanisms remain unclear. Because hypoxia/ischemia disrupts ionic homeostasis with an increase in extracellular K+, which plays a role in neuronal death, we asked whether DOR activation preserves K+ homeostasis during hypoxic/ischemic stress. To test this hypothesis, extracellular recordings with K+-sensitive microelectrodes were performed in mouse cortical slices under anoxia or oxygen–glucose deprivation (OGD). The main findings in this study are that (1) DOR activation with [D-Ala2, D-Leu5]-enkephalinamide attenuated the anoxia- and OGD-induced increase in extracellular K+ and decrease in DC potential in cortical slices; (2) DOR inhibition with naltrindole, a DOR antagonist, completely abolished the DOR-mediated prevention of increase in extracellular K+ and decrease in DC potential; (3) inhibition of protein kinase A (PKA) with N-(2-[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide dihydrochloride had no effect on the DOR protection; and (4) inhibition of protein kinase C (PKC) with chelerythrine chloride reduced the DOR protection, whereas the PKC activator (phorbol 12-myristate 13-acetate) mimicked the effect of DOR activation on K+ homeostasis. These data suggest that activation of DOR protects the cortex against anoxia- or ODG-induced derangement of potassium homeostasis, and this protection occurs via a PKC-dependent and PKA-independent pathway. We conclude that an important aspect of DOR-mediated neuroprotection is its early action against derangement of K+ homeostasis during anoxia or ischemia.

This study explored the association between cerebral metabolic rates of glucose (CMRGlc) and the severity of Vascular Parkinsonism (VP) and Parkinson's disease (PD). A cross-sectional study was performed to compare CMRGlc in normal subjects vs. VP and PD patients. Twelve normal subjects, 22 VP, and 11 PD patients were evaluated with the H&Y and MMSE, and underwent 18F-FDG measurements. Pearson's correlations were used to identify potential associations between the severity of VP/PD and CMRGlc. A pronounced reduction of CMRGlc in the frontal lobe and caudate putamen was detected in patients with VP and PD when compared with normal subjects. The VP patients displayed a slight CMRGlc decrease in the caudate putamen and frontal lobe in comparison with PD patients. These decreases in CMRGlc in the frontal lobe and caudate putamen were significantly correlated with the VP patients' H&Y, UPDRS II, UPDRS III, MMSE, cardiovascular, and attention/memory scores. Similarly, significant correlations were observed in patients with PD. This is the first clinical study finding strong evidence for an association between low cerebral glucose metabolism and the severity of VP and PD. Our findings suggest that these changes in glucose metabolism in the frontal lobe and caudate putamen may underlie the pathophysiological mechanisms of VP and PD. As the scramble to find imaging biomarkers or predictors of the disease intensifies, a better understanding of the roles of cerebral glucose metabolism may give us insight into the pathogenesis of VP and PD.

Conditions that limit O2 supply for a prolonged period lead to cardiac injury. However, current strategies against hypoxic cardiac injury are limited in clinical settings. It is of utmost importance to find novel clues for protecting cells from hypoxic injury. Our work has recently shown that δ-opioid receptor (DOR) activation strongly regulates microRNA expression in the heart exposed to prolonged hypoxia, suggesting a role of DOR in cardiac adaptation to hypoxic stress. To further determine DOR's action on cell survival under hypoxia and the underlying mechanisms, we exposed HEK293t cells to hypoxia for a prolonged period and then investigated cellular viability and changes in mitochondrial membrane potential and the nuclear factor erythroid 2-related factor 2 (Nrf2), which are thought to be involved in the regulation of cell survival under hypoxia. Our data show that prolonged hypoxia (0.5% of O2 for 16-48 hours) caused serious cellular injury. DOR activation with UFP-512, a potent and specific DOR agonist, prevented the collapse of mitochondrial membrane potential and markedly attenuated cell injury in the hypoxic condition. The DOR-mediated protection was largely reversed by DOR antagonism with naltrindole. Furthermore, we observed that DOR activation increased Nrf2 translocation from cytoplasm to nucleus. The DOR-mediated cytoprotection was largely abolished by the "knock-down" of Nrf2. Our results suggest that DOR activation increases Nrf2 nuclear translocation and stabilizes mitochondrial membrane potential during the process of cellular protection against hypoxic stress. SC and TC are equal contributors. Supported by HD-034852, AT-004422, NSFC 81060096 and 81260204.

Astrocytic excitatory amino acid transporters (EAATs) regulate extracellular glutamate concentrations and play a role in preventing neuroexcitotoxicity. As the δ-opioid receptor (DOP receptor) is neuroprotective against excitotoxic injury, we determined whether DOP receptor activation up-regulates EAAT expression and function.We measured mRNA and protein expression of EAAT1, EAAT2 and EAAT3 in cultured mouse astrocytes exposed to a specific DOP receptor agonist (UFP-512) with or without a DOP receptor antagonist, DOP receptor siRNA or inhibitors of PKC, PKA, PI3K, p38, MAPK, MEK and ERK, and evaluated the function of EAATs by measuring glutamate uptake.Astrocytic DOP receptor mRNA and protein were suppressed by DOP receptor siRNA knockdown. DOP receptor activation increased mRNA and protein expression of EAAT1 and EAAT2, but not EAAT3, thereby enhancing glutamate uptake of astrocytes. DOP receptor-induced EAAT1 and EAAT2 expression was largely reversed by DOP receptor antagonist naltrindole or by DOP receptor siRNA knockdown, and suppressed by inhibitors of MEK, ERK and p38. DOP receptor-accelerated glutamate uptake was inhibited by EAAT blockers, DOP receptor siRNA knockdown or inhibitors of MEK, ERK or p38. In contrast, inhibitors of PKA, PKC or PI3K had no significant effect on DOP receptor-induced EAAT expression.DOP receptor activation up-regulates astrocytic EAATs via MEK-ERK-p38 signalling, suggesting a critical role for DOP receptors in the regulation of astrocytic EAATs and protection against neuroexcitotoxicity. As decreased EAAT expression contributes to pathophysiology in many neurological diseases, including amyotrophic lateral sclerosis, our findings present a new platform for potential treatments of these diseases.

Delta-opioid receptors (DOR) are involved in cardioprotection under hypoxia/ ischemia. However, the underlying mechanism is not well understood yet. We have recently shown that DOR activation significantly modify the expression of hypoxia-sensitive microRNAs (miRNAs) in the brain and kidney. In extension, we further investigated if DOR activation can regulate cardiac miRNAs in hypoxia. We compared global changes in miRNA gene expression in the rat heart following exposure to hypoxia for 10 days with those in normoxia using a low density miRNA microarray to delineate hypoxia-induced changes in the miRNA expression. The up-regulated miRNAs from the microarray analysis were selected for further investigating their response to hypoxia and DOR activation with a DOR agonist, UFP-512 after 1, 5, and 10 days. We found that 1) miR-376a increased after 1-day hypoxia and progressively increased with hypoxic duration; 2) miR-7b, miR-107, miR-134, miR-135a, miR-193a-3p, miR-196a and miR-324-3p progressively increased after 1-day hypoxia and reached a peak after 5-days; 3) miR-7a, miR-141, miR-196c, miR-200a, miR-200b, miR-203a and miR-324 increased only after 1-day hypoxia and significantly decreased after 5-10 days of hypoxia; 4) miR-128a did not significantly change after 1-10 days of hypoxia. After DOR activation with UFP-512, miR-128a, miR-135a, miR-193a-3p, miR-196a, miR-200a, miR-324-3p and miR-338 continued to increase after 1-day hypoxia, and then decreased after 5-10 days of hypoxia; 5) miR-7b and miR-134, in the condition of DOR activation, increased after 1-day hypoxia and further increased after 5-day hypoxia, while significantly decreased after 10-day exposure. In contrast, some miRNAs like miR-350 decreased in response to hypoxia, while DOR activation almost completely reversed such hypoxic reduction. These results suggest that cardiac miRNAs differentially respond to hypoxic stress with a variable degree directly related to the duration of hypoxic exposure, and DOR activation can regulate such hypoxic responses. Supported by HD034852 , AT004422 , NSFC31071046, CS20102010, ZD201007 , CY 20119004,CY20120003, STCSM10DZ1975800,NBRP09CB522901 & 12CB518502.

We investigated whether δ-opioid receptor (DOR)-induced neuroprotection involves the brain-derived neurotrophic factor (BDNF) pathway. We studied the effect of DOR activation on the expression of BDNF and other proteins in the cortex of C57BL/6 mice exposed to hypoxia (10% of oxygen) for 1–10 days. The results showed that: (1) 1-day hypoxia had no appreciable effect on BDNF expression, while 3- and 10-day hypoxia progressively decreased BDNF expression, resulting in 37.3% reduction (p < 0.05) after 10-day exposure; (2) DOR activation with UFP-512 (1 mg/kg, i.p., daily) partially reversed the hypoxia-induced reduction of BDNF expression in the 3- or 10-day exposed cortex; (3) DOR activation partially reversed the hypoxia-induced reduction in functional TrkB (140-kDa) and attenuated hypoxia-induced increase in truncated TrkB (90-kDa) in the 3- or 10-day hypoxic cortex; and (4) prolonged hypoxia (10 days) significantly increased TNF-α level and decreased CD11b expression in the cortex, which was completely reversed following DOR activation; and (5) there was no significant change in pCREB and pATF-1 levels in the hypoxic cortex. We conclude that prolonged hypoxia down-regulates BDNF-TrkB signaling leading to an increase in TNF-α in the cortex, while DOR activation up-regulates BDNF-TrkB signaling thereby decreasing TNF-α levels in the hypoxic cortex.

Hypoxic/ischemic injury to kidney is a frequently encountered clinical problem with limited therapeutic options. Since microRNAs are differentially involved in hypoxic/ischemic events and δ-opioid receptor (DOR) activation is known to protect against hypoxic/ischemic injury, we speculated on the involvement of DOR activation in altering the microRNA (miRNA) expression in kidney under hypoxic condition. We selected 31 miRNAs based on microarray data for quantitative PCR analysis. Among them, 14 miRNAs were significantly altered after prolonged hypoxia, DOR activation or a combination of both. We found that 1) DOR activation alters miRNA expression profiles in normoxic conditions; 2) hypoxia differentially alters miRNA expression depending on the duration of hypoxia; and 3) DOR activation can modify hypoxia-induced changes in miRNA expression. For example, 10-day hypoxia reduced the level of miR-212 by over 70%, while DOR activation could mimic such reduction even in normoxic kidney. In contrast, the same stress increased miR-29a by >100%, which was reversed following DOR activation. These first data suggest that hypoxia comprehensively modifies the miRNA profile within the kidney, which can be mimicked or modified by DOR activation. Ascertaining the targeted pathways that regulate the diverse cellular and molecular functions of miRNA may provide new insights into potential therapies for hypoxic/ischemic injury of the kidney.

Cardiovascular dysfunction often causes blood/oxygen insufficiency in the kidney that is very sensitive to changes in oxygen/blood delivery. Indeed, hypoxic/ischemic kidney stress is a frequent problem in clinical settings. There is, however, no promising strategy for prevention and treatment of such injury. Since recent studies suggest that microRNAs are differentially involved in hypoxic/ischemic events and delta-opioid receptor (DOR) activation increases antioxidant capacity and protects against hypoxic/ischemic injury, we asked if DOR activation regulates microRNA expression in the kidney under hypoxic condition. We isolated microRNAs from normoxic and hypoxic rat kidneys and used the paired microRNAs for microarray analysis. Thirty-one microRNAs were selected for quantitative PCR analysis based on the microarray data. Among them, 14 microRNAs were significantly altered in response to prolonged hypoxia for 1, 5 and 10 days, DOR activation with UFP-512 (1 mg/kg/day, ip, at days 0, 4 and 8) or a combination of both. Our novel data show that 1) DOR activation shifts miRNA expression profiles in normoxic conditions; 2) hypoxia differentially alters the miRNA expression profiles depending on hypoxic durations; and 3) DOR activation modifies hypoxia-induced changes in miRNA expression. For example, 10-day hypoxia reduced the level of miR-212 by >70% (p100% (p

Cardiovascular disorders may cause hypoxic/ischemic injury to the cortex, leading to dysfunction of cortical cognition with a worse outcome in the older population. Although damage to cognitive cortex has been implicated in cognitive dysfunctions, the exact pathophysiology of a hypoxic/ischemic insult to this region is not well understood yet at the cellular and molecular levels. We used the whole-cell patch clamp in mouse slice preparations to characterize the basic electrophysiological properties of pyramidal cells of prefrontal cortex (PFC) and determine the response of these neurons to hypoxic stress in young adult (24±4 days old, n=7) and relatively older (42±12 days old, n=17) mice. In relatively older adult neurons (42±12 days old, n=17), the currents evoked by stepping from -140 mV to 20 mV (IV, holding at -60 mV) were shifted upward by hypoxic stress, indicating an increase in membrane conductance. Whole cell currents (holding at -60 mV) were found to decrease with a 3-min hypoxia, while increased with 5-min and 7-min hypoxia, suggesting that prolonged hypoxia increased open probability of ion channels. Resting membrane potential (MP) shifted upward in all three hypoxic conditions with a larger change seen with the longer duration of hypoxia. Action potentials (APs) showed the same tendency with 7-min hypoxia, showing a significant reduction in the number of hypoxia-stimulated pulses. However, all hypoxic conditions blocked the APs generation, suggesting hypoxic inhibition of neuronal excitability. In contrast, hypoxia-induced IV was apparently reversed in the younger neurons as compared to the older neurons, especially after 5 minutes of hypoxia (99.26 pA vs. -14.91 pA at -100 mV, P

Epilepsy is one of the commonest brain disorders characterized by recurrent unprovoked seizures that result from the abnormal, excessive, and synchronous activity of clusters of nerve cells in the brain. Epilepsy is very prevalent and has a high incidence. Intensive efforts have been made in the treatment for epilepsy, including medical, surgical, and other treatment strategies such as ketogenic diet and vagus nerve stimulation. However, these treatment options have their own limitations and complications. About one-third of patients show no response to pharmacological treatment. Concern regarding the adverse effects of these treatments has drawn people toward other therapies for treatment of epilepsy. Acupuncture, one of the popular therapeutic techniques in traditional Chinese medicine (TCM), has proven to be safe, efficient, and convenient with minimal side effects. Thus, it has gained popularity in treatment of various diseases, including epilepsy, in the West. However, due to the limited availability of high-quality research papers documented in English and a difference in the understanding of antiepileptic effects of acupuncture between the scientists of East and West, the application of acupuncture in the treatment of epilepsy is limited in Western community. In this chapter, we attempt to summarize the clinical observations on acupuncture in the treatment of epilepsy. Based on the underlying mechanisms of epilepsy and epileptogenesis, we have outlined the probable modes of action of acupuncture antiepilepsy in this chapter. Epileptic seizures and epileptogenesis are complex processes that result from changes in the excitability of neurons and involve synchronous discharges of a population of hyperexcitable neurons propagated through normal or pathological pathways. The increased excitability is ascribed to the changes that occur in both neurons and glial cells. In neurons, it is mainly due to (1) the innate ability of intrinsically bursting neurons in the cortex and hippocampus, (2) alterations in the membrane properties, (3) imbalance between excitatory and inhibitory transmission, and (4) alterations in existing synaptic contacts/circuits and/or establishment of new synaptic contacts/circuits. In glial cells, the most striking change is their impaired ability to maintain glutamate and K+ homeostasis. The most probable mechanism that underlies the antiepileptic effect of acupuncture is normalization of this disrupted neuronal excitability through multiple effects at different levels (from cells to molecules) with different time course (short term vs. long term) and during different stages of epilepsy (early/acute vs. later/chronic). These strategies include the modification of neuronal electrical activity and the activity of therapeutic network, the regulation of the activity of biomediators (e.g., amino acids, neuropeptides, monoamines, NO, Ca2+ and Ca2+-dependent activity, hormones, and humoral factors), the prevention from seizure-induced neuronal injury/loss and the subsequent aberrant neurogenesis and synaptic reorganization/dendritic remodeling in seizure-susceptible areas, and the regulation of ion channel activity.

Prolonged hypoxic/ischemic stress may cause cortical injury and clinically manifest as a neurological disability. Activation of the δ-opioid receptor (DOR) may induce cortical protection against hypoxic/ischemic insults. However, the mechanisms underlying DOR protection are not clearly understood. We have recently found that DOR activation modulates the expression of microRNAs (miRNAs) in the kidney exposed to hypoxia, suggesting that DOR protection may involve a miRNA mechanism. To determine if the miRNAs expressed in the cortex mediated DOR neuroprotection, we examined 19 miRNAs that were previously identified as hypoxia- and DOR-regulated miRNAs in the kidney, in the rat cortex treated with UFP-512, a potent and specific DOR agonist under hypoxic condition. Of the 19 miRNAs tested, 17 were significantly altered by hypoxia and/or DOR activation with the direction and amplitude varying depending on hypoxic duration and times of DOR treatment. Expression of several miRNAs such as miR-29b, -101b, -298, 324-3p, -347 and 466b was significantly depressed after 24 hours of hypoxia. Similar changes were seen in normoxic condition 24 hours after DOR activation with one-time treatment of UFP-512. In contrast, some miRNAs were more tolerant to hypoxic stress and showed significant reduction only with 5-day (e.g., miR-31 and -186) or 10-day (e.g., miR-29a, let-7f and -511) exposures. In addition, these miRNAs had differential responses to DOR activation. Other miRNAs like miRs-363* and -370 responded only to the combined exposure to hypoxia and DOR treatment, with a notable reduction of >70% in the 5-day group. These data suggest that cortical miRNAs are highly yet differentially sensitive to hypoxia. DOR activation can modify, enhance or resolve the changes in miRNAs that target HIF, ion transport, axonal guidance, free radical signaling, apoptosis and many other functions.

The use of opioid analgesics has a long history in clinical settings, although the comprehensive action of opioid receptors is still less understood. Nonetheless, recent studies have generated fresh insights into opioid receptor-mediated functions and their underlying mechanisms. Three major opioid receptors (μ-opioid receptor, MOR; δ-opioid receptor, DOR; and κ-opioid receptor, KOR) have been cloned in many species. Each opioid receptor is functionally sub-classified into several pharmacological subtypes, although, specific gene corresponding each of these receptor subtypes is still unidentified as only a single gene has been isolated for each opioid receptor. In addition to pain modulation and addiction, opioid receptors are widely involved in various physiological and pathophysiological activities, including the regulation of membrane ionic homeostasis, cell proliferation, emotional response, epileptic seizures, immune function, feeding, obesity, respiratory and cardiovascular control as well as some neurodegenerative disorders. In some species, they play an essential role in hibernation. One of the most exciting findings of the past decade is the opioid-receptor, especially DOR, mediated neuroprotection and cardioprotection. The upregulation of DOR expression and DOR activation increase the neuronal tolerance to hypoxic/ischemic stress. The DOR signal triggers (depending on stress duration and severity) different mechanisms at multiple levels to preserve neuronal survival, including the stabilization of homeostasis and increased pro-survival signaling (e.g., PKC-ERK-Bcl 2) and antioxidative capacity. In the heart, PKC and KATP channels are involved in the opioid receptor-mediated cardioprotection. The DOR-mediated neuroprotection and cardioprotection have the potential to significantly alter the clinical pharmacology in terms of prevention and treatment of life-threatening conditions like stroke and myocardial infarction. The main purpose of this article is to review the recent work done on opioids and their receptor functions. It shall provide an informative reference for better understanding the opioid system and further elucidation of the opioid receptor function from a physiological and pharmacological point of view.

The use of opioid analgesics has a long history in clinical settings, although the functions of opioid receptors, especially their role in the brain, are not well understood yet. Recent studies have generated abundant new data on opioid receptor-mediated functions and the underlying mechanisms. The most exciting finding in the past decade is probably the neuroprotection against hypoxic/ischemic stress mediated by delta-opioid receptors (DOR). An up-regulation of DOR expression and the release of endogenous opioids may increase neuronal tolerance to hypoxic/ischemic stress. The DOR signal triggers, depending on stress duration and severity, different mechanisms at multiple levels to preserve neuronal survival, including the stabilization of ionic homeostasis, an increase in pro-survival signaling (e.g., PKC-ERK-Bcl 2) and the enhanced anti-oxidative capacity. Recent data on DOR-mediated neuroprotection provide us a new concept of neuroprotection against neurological disorders and have a potentially significant impact on the prevention and treatment of some serious neurological conditions, such as stroke.

Past work has shown that delta-opioid receptor (DOR) activation by [D-Ala(2),D-Leu(5)]-enkephalin (DADLE) attenuated the disruption of K(+) homeostasis induced by hypoxia or oxygen-glucose deprivation (OGD) in the cortex, while naltrindole, a DOR antagonist blocked this effect, suggesting that DOR activity stabilizes K(+) homeostasis in the cortex during hypoxic/ischemic stress. However, several important issues remain unclear regarding this new observation, especially the difference between DOR and other opioid receptors in the stabilization of K(+) homeostasis and the underlying mechanism. In this study, we asked whether DOR is different from micro-opioid receptors (MOR) in stabilizing K(+) homeostasis and which membrane channel(s) is critically involved in the DOR effect. The main findings are that (1) similar to DADLE (10 microM), H-Dmt-Tic-NH-CH (CH(2)--COOH)-Bid (1-10 microM), a more specific and potent DOR agonist significantly attenuated anoxic K(+) derangement in cortical slice; (2) [D-Ala(2), N-Me-Phe(4), glycinol(5)]-enkephalin (DAGO; 10 microM), a MOR agonist, did not produce any appreciable change in anoxic disruption of K(+) homeostasis; (3) absence of Ca(2+) greatly attenuated anoxic K(+) derangement; (4) inhibition of Ca(2+)-activated K(+) (BK) channels with paxilline (10 microM) reduced anoxic K(+) derangement; (5) DADLE (10 microM) could not further reduce anoxic K(+) derangement in the Ca(2+)-free perfused slices or in the presence of paxilline; and (6) glybenclamide (20 microM), a K(ATP) channel blocker, decreased anoxia-induced K(+) derangement, but DADLE (10 microM) could further attenuate anoxic K(+) derangement in the glybenclamide-perfused slices. These data suggest that DOR, but not MOR, activation is protective against anoxic K(+) derangement in the cortex, at least partially via an inhibition of hypoxia-induced increase in Ca(2+) entry-BK channel activity.