Yen Chin Liu

Microglia: a promising target for treating neuropathic and postoperative pain, and morphine tolerance.

Management of chronic pain, such as nerve-injury-induced neuropathic pain associated with diabetic neuropathy, viral infection, and cancer, is a real clinical challenge. Major surgeries, such as breast and thoracic surgery, leg amputation, and coronary artery bypass surgery, also lead to chronic pain in 10-50% of individuals after acute postoperative pain, partly due to surgery-induced nerve injury. Current treatments mainly focus on blocking neurotransmission in the pain pathway and have only resulted in limited success. Ironically, chronic opioid exposure might lead to paradoxical pain. Development of effective therapeutic strategies requires a better understanding of cellular mechanisms underlying the pathogenesis of neuropathic pain. Progress in pain research points to an important role of microglial cells in the development of chronic pain. Spinal cord microglia are strongly activated after nerve injury, surgical incision, and chronic opioid exposure. Increasing evidence suggests that, under all these conditions, the activated microglia not only exhibit increased expression of microglial markers CD 11 b and Iba 1, but also display elevated phosphorylation of p38 mitogen-activated protein kinase. Inhibition of spinal cord p38 has been shown to attenuate neuropathic and postoperative pain, as well as morphine-induced antinociceptive tolerance. Activation of p38 in spinal microglia results in increased synthesis and release of the neurotrophin brain-derived neurotrophic factor and the proinflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α. These microglia-released mediators can powerfully modulate spinal cord synaptic transmission, leading to increased excitability of dorsal horn neurons, that is, central sensitization, partly via suppressing inhibitory synaptic transmission. Here, we review studies that support the pronociceptive role of microglia in conditions of neuropathic and postoperative pain and opioid tolerance. We conclude that targeting microglial signaling might lead to more effective treatments for devastating chronic pain after diabetic neuropathy, viral infection, cancer, and major surgeries, partly via improving the analgesic efficacy of opioids.

The c-Jun N-terminal kinase 1 (JNK1) in spinal astrocytes is required for the maintenance of bilateral mechanical allodynia under a persistent inflammatory pain condition.

&NA; Peripheral inflammation induces persistent central sensitization characterized by mechanical allodynia and heat hyperalgesia that are mediated by distinct mechanisms. Compared to well‐demonstrated mechanisms of heat hyperalgesia, mechanisms underlying the development of mechanical allodynia and contralateral pain are incompletely known. In this study, we investigated the distinct role of spinal JNK in heat hyperalgesia, mechanical allodynia, and contralateral pain in an inflammatory pain model. Intraplantar injection of complete Freunds adjuvant (CFA) induced bilateral mechanical allodynia but unilateral heat hyperalgesia. CFA also induced a bilateral activation (phosphorylation) of JNK in the spinal cord, and the phospho JNK1 (pJNK1) levels were much higher than that of pJNK2. Notably, both pJNK and JNK1 were expressed in GFAP‐positive astrocytes. Intrathecal infusion of a selective peptide inhibitor of JNK, D‐JNKI‐1, starting before inflammation via an osmotic pump, reduced CFA‐induced mechanical allodynia in the maintenance phase but had no effect on CFA‐induced heat hyperalgesia. A bolus intrathecal injection of D‐JNKI‐1 or SP600126, a small molecule inhibitor of JNK also reversed mechanical allodynia bilaterally. In contrast, peripheral (intraplantar) administration of D‐JNKI‐1 reduced the induction of CFA‐induced heat hyperalgesia but did not change mechanical allodynia. Finally, CFA‐induced bilateral mechanical allodynia was attenuated in mice lacking JNK1 but not JNK2. Taken together, our data suggest that spinal JNK, in particular JNK1 plays an important role in the maintenance of persistent inflammatory pain. Our findings also reveal a unique role of JNK1 and astrocyte network in regulating tactile allodynia and contralateral pain.

TLR3 deficiency impairs spinal cord synaptic transmission, central sensitization, and pruritus in mice

Itch, also known as pruritus, is a common, intractable symptom of several skin diseases, such as atopic dermatitis and xerosis. TLRs mediate innate immunity and regulate neuropathic pain, but their roles in pruritus are elusive. Here, we report that scratching behaviors induced by histamine-dependent and -independent pruritogens are markedly reduced in mice lacking the Tlr3 gene. TLR3 is expressed mainly by small-sized primary sensory neurons in dorsal root ganglions (DRGs) that coexpress the itch signaling pathway components transient receptor potential subtype V1 and gastrin-releasing peptide. Notably, we found that treatment with a TLR3 agonist induces inward currents and action potentials in DRG neurons and elicited scratching in WT mice but not Tlr3(-/-) mice. Furthermore, excitatory synaptic transmission in spinal cord slices and long-term potentiation in the intact spinal cord were impaired in Tlr3(-/-) mice but not Tlr7(-/-) mice. Consequently, central sensitization-driven pain hypersensitivity, but not acute pain, was impaired in Tlr3(-/-) mice. In addition, TLR3 knockdown in DRGs also attenuated pruritus in WT mice. Finally, chronic itch in a dry skin condition was substantially reduced in Tlr3(-/-) mice. Our findings demonstrate a critical role of TLR3 in regulating sensory neuronal excitability, spinal cord synaptic transmission, and central sensitization. TLR3 may serve as a new target for developing anti-itch treatment.

Autologous Transplantation of Endothelial Progenitor Cells Attenuates Acute Lung Injury in Rabbits

Background:Acute lung injury (ALI) and end-stage acute respiratory distress syndrome (ARDS) are among the most common causes of death in intensive care units. Activation and damage of pulmonary endothelium is the hallmark of ALI/ARDS. Recent studies have demonstrated the importance of circulating endothelial progenitor cells (EPCs) in maintaining normal endothelial function as well as endothelial repairing after vascular injury. Here, the authors present the first study demonstrating the therapeutic potential of EPCs in a rabbit model of ALI/ARDS. Methods:Circulating EPCs were obtained from rabbits using Ficoll centrifugation. One week after culturing, ALI was induced in rabbits by oleic acid (75 mg/kg, intravenous), and autologous EPCs were transplanted intravenously. Vasomotor function of isolated pulmonary artery and degrees of lung injury were assessed 2 days later. Results:Endothelial dysfunction in the pulmonary artery was significantly attenuated in rabbits treated with EPCs, whereas the endothelium-independent relaxation responses were not different. Expression of inducible nitric oxide synthase was suppressed in the pulmonary artery of EPC-treated animals. Infiltration of leukocytes in the lung parenchyma was significantly reduced after EPC transplantation. EPCs also decreased water content, hyaline membrane formation, and hemorrhage in lungs. Conclusion:The authors demonstrated that autologous transplantation of EPCs preserves pulmonary endothelial function and maintains the integrity of pulmonary alveolar–capillary barrier. Transplantation of EPCs can be a novel cell-based, endothelium-targeted therapeutic strategy for prevention and treatment of ALI/ARDS.

Extracellular caspase-6 drives murine inflammatory pain via microglial TNF-α secretion

Increasing evidence indicates that the pathogenesis of neuropathic pain is mediated through spinal cord microglia activation. The intracellular protease caspase-6 (CASP6) is known to regulate neuronal apoptosis and axonal degeneration; however, the contribution of microglia and CASP6 in modulating synaptic transmission and pain is unclear. Here, we found that CASP6 is expressed specifically in C-fiber axonal terminals in the superficial spinal cord dorsal horn. Animals exposed to intraplantar formalin or bradykinin injection exhibited CASP6 activation in the dorsal horn. Casp6-null mice had normal baseline pain, but impaired inflammatory pain responses. Furthermore, formalin-induced second-phase pain was suppressed by spinal injection of CASP6 inhibitor or CASP6-neutralizing antibody, as well as perisciatic nerve injection of CASP6 siRNA. Recombinant CASP6 (rCASP6) induced marked TNF-α release in microglial cultures, and most microglia within the spinal cord expressed Tnfa. Spinal injection of rCASP6 elicited TNF-α production and microglia-dependent pain hypersensitivity. Evaluation of excitatory postsynaptic currents (EPSCs) revealed that rCASP6 rapidly increased synaptic transmission in spinal cord slices via TNF-α release. Interestingly, the microglial inhibitor minocycline suppressed rCASP6 but not TNF-α-induced synaptic potentiation. Finally, rCASP6-activated microglial culture medium increased EPSCs in spinal cord slices via TNF-α. Together, these data suggest that CASP6 released from axonal terminals regulates microglial TNF-α secretion, synaptic plasticity, and inflammatory pain.

Prolonged use of high-dose morphine impairs angiogenesis and mobilization of endothelial progenitor cells in mice.

BACKGROUND: Morphine is one of the most commonly prescribed analgesics for treating wound pain. Using a mouse model of excisional wound injury, we determined the effects of high-dose morphine on angiogenesis and mobilization of endothelial progenitor cells. METHODS: An excisional wound was created on mice treated with placebo or morphine (20 mg/kg, i.p. injection for 14 days). Wound healing was compared by measuring the final-to-initial wound area ratio. Generation of superoxide anions in the wound was determined by luminol-enhanced chemiluminescence. Circulating mononuclear cells were isolated and measured for endothelial progenitor cell (defined as CD34+/CD133+ cell) counts. In vivo and in vitro measurements of angiogenesis after morphine treatment were performed using the Matrigel assay. RESULTS: Mice treated with morphine had reduced wound closure and higher wound superoxide ions concentrations than control mice. Morphine reduced the number of postwound circulating endothelial progenitor cells. Matrigel assay showed impaired angiogenesis in animals and reduced capillary tube formation in cultured endothelial cells treated with morphine. CONCLUSION: High-dose morphine impaired angiogenesis, increased systemic oxidative stress, and impaired mobilization of endothelial progenitor cells. This study emphasizes the potential detrimental effect of high-dose morphine on angiogenesis after systemic administration.

Transplantation of endothelial progenitor cells improves pulmonary endothelial function and gas exchange in rabbits with endotoxin-induced acute lung injury.

BACKGROUND:Circulating endothelial progenitor cells (EPCs) have been therapeutically applied to aid vascular repair and myocardial regeneration. The number of circulating EPCs also provides invaluable outcome prediction for fatal diseases such as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). However, evidence for the therapeutic potential of EPCs in subjects with ALI/ADRS is limited. METHODS:Circulating EPCs were obtained from rabbits using Ficoll centrifugation. One week after culturing EPCs in endothelial growth medium-2, ALI was induced in rabbits by intratracheal instillation of lipopolysaccharide (500 &mgr;g/kg). Autologous EPCs or saline were administered IV after induction of ALI and animals were killed 2 days later. Pulmonary artery endothelial function and gas exchange were determined. Degrees of lung injury were assessed by alveolocapillary permeability, lung hemoglobin content, and myeloperoxidase activity. RESULTS:In comparison with controls, PO2 in arterial blood was significantly elevated and pulmonary artery endothelium-dependent relaxation response was restored in rabbits receiving EPC transplantation. Lung water, Evans blue, and bronchoalveolar lavage protein contents were significantly reduced in the EPC transplanted group, indicating a better preservation of the alveolocapillary membrane. Transplantation of EPCs decreased the lung hemoglobin level. Furthermore, expressions of CD11b and myeloperoxidase activity were also suppressed after administration of EPCs. CONCLUSIONS:Transplantation of EPCs restored pulmonary endothelial function, preserved integrity of the alveolocapillary barrier and suppressed the lung inflammatory response, thereby improving pulmonary gas exchange in rabbits with intratracheal lipopolysaccharide-induced ALI. Transplantation of EPCs can be a novel cell-based, endothelium-targeted therapeutic strategy for prevention and treatment of ALI/ARDS.

Acute morphine activates satellite glial cells and up-regulates IL-1β in dorsal root ganglia in mice via matrix metalloprotease-9

BackgroundActivation of spinal cord glial cells such as microglia and astrocytes has been shown to regulate chronic opioid-induced antinociceptive tolerance and hyperalgesia, due to spinal up-regulation of the proinflammatory cytokines such as interleukin-1 beta (IL-1β). Matrix metalloprotease-9 (MMP-9) has been implicated in IL-1β activation in neuropathic pain. However, it is unclear whether acute opioid treatment can activate glial cells in the peripheral nervous system. We examined acute morphine-induced activation of satellite glial cells (SGCs) and up-regulation of IL-1β in dorsal root ganglia (DRGs), and further investigated the involvement of MMP-9 in these opioid-induced peripheral changes.ResultsSubcutaneous morphine injection (10 mg/kg) induced robust peripheral glial responses, as evidenced by increased GFAP expression in DRGs but not in spinal cords. The acute morphine-induced GFAP expression is transient, peaking at 2 h and declining after 3 h. Acute morphine treatment also increased IL-1β immunoreactivity in SGCs and IL-1β activation in DRGs. MMP-9 and GFAP are expressed in DRG neurons and SGCs, respectively. Confocal analysis revealed a close proximity of MMP-9 and GFAP immunostaining. Importantly, morphine-induced DRG up-regulation of GFAP expression and IL-1β activation was abolished after Mmp9 deletion or naloxone pre-treatment. Finally, intrathecal injections of IL-1β-selective siRNA not only reduced DRG IL-1β expression but also prolonged acute morphine-induced analgesia.ConclusionsAcute morphine induces opioid receptors- and MMP-9-dependent up-regulation of GFAP expression and IL-1β activation in SGCs of DRGs. MMP-9 could mask and shorten morphine analgesia via peripheral neuron-glial interactions. Targeting peripheral glial activation might prolong acute opioid analgesia.

Inhibitory effect of lamotrigine on A-type potassium current in hippocampal neuron-derived H19-7 cells

Summary:  Purpose: We investigated the effects of lamotrigine (LTG) on the rapidly inactivating A‐type K+ current (IA) in embryonal hippocampal neurons.

Tissue plasminogen activator contributes to morphine tolerance and induces mechanical allodynia via astrocytic IL-1β and ERK signaling in the spinal cord of mice

Accumulating evidence indicates that activation of spinal cord astrocytes contributes importantly to nerve injury and inflammation-induced persistent pain and chronic opioid-induced antinociceptive tolerance. Phosphorylation of extracellular signal-regulated kinase (pERK) and induction of interleukin-1 beta (IL-1β) in spinal astrocytes have been implicated in astrocytes-mediated pain. Tissue plasminogen activator (tPA) is a serine protease that has been extensively used to treat stroke. We examined the potential involvement of tPA in chronic opioid-induced antinociceptive tolerance and activation of spinal astrocytes using tPA knockout (tPA(-/-)) mice and astrocyte cultures. tPA(-/-) mice exhibited unaltered nociceptive pain and morphine-induced acute analgesia. However, the antinociceptive tolerance, induced by chronic morphine (10mg/kg/day, s.c.), is abrogated in tPA(-/-) mice. Chronic morphine induces tPA expression in glial fibrillary acidic protein (GFAP)-expressing spinal cord astrocytes. Chronic morphine also increases IL-1β expression in GFAP-expressing astrocytes, which is abolished in tPA-deficient mice. In cultured astrocytes, morphine treatment increases tPA, IL-1β, and pERK expression, and the increased IL-1β and pERK expression is abolished in tPA-deficient astrocytes. tPA is also sufficient to induce IL-1β and pERK expression in astrocyte cultures. Intrathecal injection of tPA results in up-regulation of GFAP and pERK in spinal astrocytes but not up-regulation of ionized calcium binding adapter molecule 1 in spinal microglia. Finally, intrathecal tPA elicits persistent mechanical allodynia, which is inhibited by the astroglial toxin alpha-amino adipate and the MEK (ERK kinase) inhibitor U0126. Collectively, these data suggest an important role of tPA in regulating astrocytic signaling, pain hypersensitivity, and morphine tolerance.