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author:("Berta, temin")
1.  Neuropathic Pain Is Constitutively Suppressed in Early Life by Anti-Inflammatory Neuroimmune Regulation 
The Journal of Neuroscience  2015;35(2):457-466.
Peripheral nerve injury can trigger neuropathic pain in adults but not in infants; indeed, for unknown reasons, neuropathic pain is rare before adolescence. We show here that the absence of neuropathic pain response in infant male rats and mice following nerve injury is due to an active, constitutive immune suppression of dorsal horn pain activity. In contrast to adult nerve injury, which triggers a proinflammatory immune response in the spinal dorsal horn, infant nerve injury triggers an anti-inflammatory immune response, characterized by significant increases in IL-4 and IL-10. This immediate anti-inflammatory response can also be evoked by direct C-fiber nerve stimulation in infant, but not adult, mice. Blockade of the anti-inflammatory activity with intrathecal anti-IL10 unmasks neuropathic pain behavior in infant nerve injured mice, showing that pain hypersensitivity in young mice is actively suppressed by a dominant anti-inflammatory neuroimmune response. As infant nerve injured mice reach adolescence (postnatal day 25–30), the dorsal horn immune profile switches from an anti-inflammatory to a proinflammatory response characterized by significant increases in TNF and BDNF, and this is accompanied by a late onset neuropathic pain behavior and increased dorsal horn cell sensitivity to cutaneous mechanical and cold stimuli. These findings show that neuropathic pain following early life nerve injury is not absent but suppressed by neuroimmune activity and that “latent” pain can still emerge at adolescence, when the neuroimmune profile changes. The data may explain why neuropathic pain is rare in young children and also why it can emerge, for no observable reason, in adolescent patients.
PMCID: PMC4293402  PMID: 25589741
anti-inflammatory; development; dorsal horn; infant; neuropathic; pain
2.  Glia and pain: Is chronic pain a gliopathy? 
Pain  2013;154(0 1):S10-S28.
Activation of glial cells and neuro-glial interactions are emerging as key mechanisms underlying chronic pain. Accumulating evidence has implicated 3 types of glial cells in the development and maintenance of chronic pain: microglia and astrocytes of the central nervous system (CNS), and satellite glial cells of the dorsal root and trigeminal ganglia. Painful syndromes are associated with different glial activation states: (1) glial reaction (ie, upregulation of glial markers such as IBA1 and glial fibrillary acidic protein (GFAP) and/or morphological changes, including hypertrophy, proliferation, and modifications of glial networks); (2) phosphorylation of mitogen-activated protein kinase signaling pathways; (3) upregulation of adenosine triphosphate and chemokine receptors and hemichannels and downregulation of glutamate transporters; and (4) synthesis and release of glial mediators (eg, cytokines, chemokines, growth factors, and proteases) to the extracellular space. Although widely detected in chronic pain resulting from nerve trauma, inflammation, cancer, and chemotherapy in rodents, and more recently, human immunodeficiency virus-associated neuropathy in human beings, glial reaction (activation state 1) is not thought to mediate pain sensitivity directly. Instead, activation states 2 to 4 have been demonstrated to enhance pain sensitivity via a number of synergistic neuro-glial interactions. Glial mediators have been shown to powerfully modulate excitatory and inhibitory synaptic transmission at presynaptic, postsynaptic, and extrasynaptic sites. Glial activation also occurs in acute pain conditions, and acute opioid treatment activates peripheral glia to mask opioid analgesia. Thus, chronic pain could be a result of “gliopathy,” that is, dysregulation of glial functions in the central and peripheral nervous system. In this review, we provide an update on recent advances and discuss remaining questions.
PMCID: PMC3858488  PMID: 23792284
Astrocytes; ATP receptors; Chemokines; Cytokines; Human; Microglia; Rodents; Satellite glial cells; Spinal cord
3.  Tissue plasminogen activator contributes to morphine tolerance and induces mechanical allodynia via astrocytic IL-1β and ERK signaling in the spinal cord of mice 
Neuroscience  2013;247:376-385.
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 (10 mg/kg/day, s.c.), is abrogated in tPA−/− mice. Chronic morphine induces tPA expression in 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 IBA-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.
PMCID: PMC3722295  PMID: 23707980
acute opioid analgesia; chronic morphine exposure; extracellular signal-regulated kinase (ERK); interleukin-1 beta (IL-1β); protease; tPA knockout mice
4.  Neuroprotectin/Protectin D1 protects neuropathic pain in mice after nerve trauma 
Annals of neurology  2013;74(3):490-495.
Prevalence of neuropathic pain is high after major surgeries. However, effective treatment for preventing neuropathic pain is lacking. Here we report that peri-surgical treatment of Neuroprotectin D1/protectin D1 (NPD1/PD1), derived from docosahexaenoic acid, prevents nerve injury-induced mechanical allodynia and ongoing pain in mice. Intrathecal post-treatment of NPD1/PD1 also effectively reduces established neuropathic pain and produces no apparent signs of analgesic tolerance. Mechanistically, NPD1/PD1 treatment blocks nerve injury-induced long-term potentiation, glial reaction, inflammatory responses, and reverses synaptic plasticity in the spinal cord. Thus, NPD1/PD1 and related mimetics might serve as a new class of analgesics for preventing and treating neuropathic pain.
PMCID: PMC3791159  PMID: 23686636
5.  Resolvin E1 inhibits neuropathic pain and spinal cord microglial activation following peripheral nerve injury 
Accumulating evidence indicates that activation of spinal cord microglia plays an important role in the genesis of neuropathic pain. Resolvin E1 (E1) is derived from omega-3 polyunsaturated fatty acid and exhibits potent anti-inflammatory, pro-resolution, and anti-nociceptive effects. We further examined whether RvE1 could reduce neuropathic pain and modulate spinal cord microglial activation. Intrathecal pre-treatment of RvE1 (100 ng) daily for 3 days partially prevented the development of nerve injury-induced mechanical allodynia and up-regulation of IBA-1 (microglial marker) and TNF-α in the spinal cord dorsal horn. Furthermore, intrathecal post-treatment of RvE1 (100 ng), 3 weeks after nerve injury, transiently reduced mechanical allodynia and heat hyperalgesia. Finally, RvE1 blocked lipopolisaccharide-induced microgliosis and TNF-α release in primary micoglial cultures. Our data suggest that RvE1 may attenuate neuropathic pain via inhibiting microglial signaling. Targeting the anti-inflammatory and pro-resolution lipid mediators may offer new options for preventing and treating neuropathic pain.
PMCID: PMC3671904  PMID: 22878925
Eicosapentaenoic acid (EPA); Tumor necrosis factor-alpha TNF-α; lipopolisacride (LPS); nerve injury; omega-3 poly unsaturated fatty acids; spinal cord
6.  Extracellular caspase-6 drives murine inflammatory pain via microglial TNF-α secretion 
The Journal of Clinical Investigation  2014;124(3):1173-1186.
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.
PMCID: PMC3934175  PMID: 24531553
7.  Microglia and Spinal Cord Synaptic Plasticity in Persistent Pain 
Neural Plasticity  2013;2013:753656.
Microglia are regarded as macrophages in the central nervous system (CNS) and play an important role in neuroinflammation in the CNS. Microglial activation has been strongly implicated in neurodegeneration in the brain. Increasing evidence also suggests an important role of spinal cord microglia in the genesis of persistent pain, by releasing the proinflammatory cytokines tumor necrosis factor-alpha (TNFα), Interleukine-1beta (IL-1β), and brain derived neurotrophic factor (BDNF). In this review, we discuss the recent findings illustrating the importance of microglial mediators in regulating synaptic plasticity of the excitatory and inhibitory pain circuits in the spinal cord, leading to enhanced pain states. Insights into microglial-neuronal interactions in the spinal cord dorsal horn will not only further our understanding of neural plasticity but may also lead to novel therapeutics for chronic pain management.
PMCID: PMC3759269  PMID: 24024042
8.  TLR3 deficiency impairs spinal cord synaptic transmission, central sensitization, and pruritus in mice 
The Journal of Clinical Investigation  2012;122(6):2195-2207.
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.
PMCID: PMC3366391  PMID: 22565312
9.  Acute morphine induces matrix metalloproteinase-9 up-regulation in primary sensory neurons to mask opioid-induced analgesia in mice 
Molecular Pain  2012;8:19.
Despite decades of intense research efforts, actions of acute opioids are not fully understood. Increasing evidence suggests that in addition to well-documented antinociceptive effects opioids also produce paradoxical hyperalgesic and excitatory effects on neurons. However, most studies focus on the pronociceptive actions of chronic opioid exposure. Matrix metalloproteinase 9 (MMP-9) plays an important role in neuroinflammation and neuropathic pain development. We examined MMP-9 expression and localization in dorsal root ganglia (DRGs) after acute morphine treatment and, furthermore, the role of MMP-9 in modulating acute morphine-induced analgesia and hyperalgesia in mice.
Subcutaneous morphine induced a marked up-regulation of MMP-9 protein in DRGs but not spinal cords. Morphine also increased MMP-9 activity and mRNA expression in DRGs. MMP-9 up-regulation peaked at 2 h but returned to the baseline after 24 h. In DRG tissue sections, MMP-9 is expressed in small and medium-sized neurons that co-express mu opioid receptors (MOR). In DRG cultures, MOR agonists morphine, DAMGO, and remifentanil each increased MMP-9 expression in neurons, whereas the opioid receptor antagonist naloxone and the MOR-selective antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) suppressed morphine-induced MMP-9 expression. Notably, subcutaneous morphine-induced analgesia was enhanced and prolonged in Mmp9 knockout mice and also potentiated in wild-type mice receiving intrathecal injection of MMP-9 inhibitors. Consistently, intrathecal injection of specific siRNA targeting MMP-9 reduced MMP-9 expression in DRGs and enhanced and prolonged morphine analgesia. Subcutaneous morphine also produced heat hyperalgesia at 24 h, but this opioid-induced hyperalgesia was not enhanced after MMP-9 deletion or inhibition.
Transient MMP-9 up-regulation in DRG neurons can mask opioid analgesia, without modulating opioid-induced hyperalgesia. Distinct molecular mechanisms (MMP-9 dependent and independent) control acute opioid-induced pronociceptive actions (anti-analgesia in the first several hours and hyperalgesia after 24 h). Targeting MMP-9 may improve acute opioid analgesia.
PMCID: PMC3353172  PMID: 22444868
Dorsal root ganglion; Metalloprotease; MMP-9; mu opioid receptor (MOR); Opioid-induced analgesia; Opioid-induced hyperalgesia (OIH); Spinal cord
10.  Acute morphine activates satellite glial cells and up-regulates IL-1β in dorsal root ganglia in mice via matrix metalloprotease-9 
Molecular Pain  2012;8:18.
Activation 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.
Subcutaneous 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.
Acute 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.
PMCID: PMC3352126  PMID: 22439811
11.  TNF-α contributes to spinal cord synaptic plasticity and inflammatory pain: Distinct role of TNF receptor subtype 1 and 2 
Pain  2010;152(2):419-427.
Tumor necrosis factor-alpha (TNF-α) is a key proinflammatory cytokine. It is generally believed that TNF-α exerts its effects primarily via TNF receptor subtype-1 (TNFR1). We investigated distinct role of TNFR1 and TNFR2 in spinal cord synaptic transmission and inflammatory pain. Compared to wild-type (WT) mice, TNFR1 and TNFR2 knockout (KO) mice exhibited normal heat sensitivity and unaltered excitatory synaptic transmission in the spinal cord, as revealed by spontaneous excitatory postsynaptic currents (sEPSCs) in lamina II neurons of spinal cord slices. However, heat hyperalgesia after intrathecal TNF-α and the second-phase spontaneous pain in the formalin test were reduced in both TNFR1- and TNFR2-KO mice. In particular, heat hyperalgesia after intraplantar injection of complete Freund's adjuvant (CFA) was decreased in the early phase in TNFR2-KO mice but reduced in both early and later phase in TNFR1-KO mice. Consistently, CFA elicited a transient increase of TNFR2 mRNA levels in the spinal cord on day 1. Notably, TNF-α evoked a drastic increase in sEPSC frequency in lamina II neurons, which was abolished in TNFR1-KO mice and reduced in TNFR2-KO mice. TNF-α also increased NMDA currents in lamina II neurons, and this increase was abolished in TNFR1-KO mice but retained in TNFR2-KO mice. Finally, intrathecal injection of the NMDA receptor antagonist MK-801 prevented heat hyperalgesia elicited by intrathecal TNF-α. Our findings support a central role of TNF-α in regulating synaptic plasticity (central sensitization) and inflammatory pain via both TNFR1 and TNFR2. Our data also uncover a unique role of TNFR2 in mediating early-phase inflammatory pain.
PMCID: PMC3022092  PMID: 21159431
proinflammatory cytokine; central sensitization; TNFR1; TNFR2; formalin; complete Freund's adjuvant
12.  Toll-like Receptor-7 Mediates Pruritus 
Nature neuroscience  2010;13(12):1460-1462.
Toll-like receptors (TLRs) are typically expressed in immune cells to regulate innate immunity. Here we report that functional TLR7 is expressed in C-fiber primary sensory neurons and important for inducing itch (pruritis) but not necessary for eliciting mechanical, thermal, inflammatory and neuropathic pain in mice. Thus, we have uncovered TLR7 as a novel itch mediator and a potential therapeutic target for anti-itch treatment in skin disease conditions.
PMCID: PMC2991508  PMID: 21037581
13.  Resolvins RvE1 and RvD1Attenuate Inflammatory Pain via Central and Peripheral Actions 
Nature medicine  2010;16(5):592-597.
Inflammatory pain, such as arthritis pain, is a growing health problem1. Inflammatory pain is generally treated with opioids and cyclooxygenase (COX) inhibitors, but both are limited by side effects. Recently, resolvins, a novel family of lipid mediators including RvE1 and RvD1 derived from omega-3 polyunsaturated fatty acid, show remarkable potency in treating disease conditions associated with inflammation2, 3. Here we report that peripheral (intraplantar) or spinal (intrathecal) administration of RvE1 or RvD1 (0.3–20 ng) potently reduces inflammatory pain behaviors in mice induced by intraplantar injection of formalin, carrageenan or complete Freund’s adjuvant, without affecting basal pain perception. Intrathecal RvE1 also inhibits spontaneous pain and heat and mechanical hypersensitivity evoked by intrathecal capsaicin and TNF-α. RvE1 plays anti-inflammatory roles via reducing neutrophil infiltration, paw edema, and proinflammatory cytokine expression. RvE1 also abolishes TRPV1- and TNF-α-induced excitatory postsynaptic current increase and TNF-α-evoked NMDA receptor hyperactivity in spinal dorsal horn neurons, via inhibition of ERK signaling pathway. Thus, we demonstrate a novel role of resolvins in normalizing spinal synaptic plasticity that has been implicated in generating pain hypersensitivity. Given the remarkable potency of resolvins and well known side effects of opioids and COX inhibitors, resolvins may represent novel analgesics for treating inflammatory pain.
PMCID: PMC2866054  PMID: 20383154
14.  Large A-fiber activity is required for microglial proliferation and p38 MAPK activation in the spinal cord: different effects of resiniferatoxin and bupivacaine on spinal microglial changes after spared nerve injury 
Molecular Pain  2009;5:53.
After peripheral nerve injury, spontaneous ectopic activity arising from the peripheral axons plays an important role in inducing central sensitization and neuropathic pain. Recent evidence indicates that activation of spinal cord microglia also contributes to the development of neuropathic pain. In particular, activation of p38 mitogen-activated protein kinase (MAPK) in spinal microglia is required for the development of mechanical allodynia. However, activity-dependent activation of microglia after nerve injury has not been fully addressed. To determine whether spontaneous activity from C- or A-fibers is required for microglial activation, we used resiniferatoxin (RTX) to block the conduction of transient receptor potential vanilloid subtype 1 (TRPV1) positive fibers (mostly C- and Aδ-fibers) and bupivacaine microspheres to block all fibers of the sciatic nerve in rats before spared nerve injury (SNI), and observed spinal microglial changes 2 days later.
SNI induced robust mechanical allodynia and p38 activation in spinal microglia. SNI also induced marked cell proliferation in the spinal cord, and all the proliferating cells (BrdU+) were microglia (Iba1+). Bupivacaine induced a complete sensory and motor blockade and also significantly inhibited p38 activation and microglial proliferation in the spinal cord. In contrast, and although it produced an efficient nociceptive block, RTX failed to inhibit p38 activation and microglial proliferation in the spinal cord.
(1) Blocking peripheral input in TRPV1-positive fibers (presumably C-fibers) is not enough to prevent nerve injury-induced spinal microglial activation. (2) Peripheral input from large myelinated fibers is important for microglial activation. (3) Microglial activation is associated with mechanical allodynia.
PMCID: PMC2759920  PMID: 19772627
15.  Calcium phosphate transfection optimization for serum-free suspension culture 
Cytotechnology  2001;35(3):175-180.
Aim of this study was to identify optimal conditions for suspension transfection in the absence of serum. Transfection parameters for suspension culture can be very different to ones in adherent cells. Most transfection protocols have been developed and optimizedfor adherent culture. Using green fluorescent protein (GFP) as reporter, FCS was eliminated from the transfection process by altering critical parameters and by substituting serum with albumin. Using standard phosphate and calcium concentrations for transfection in the absence of serum resulted in titers of only 1% of those observed in the presence of serum. A reduction of the calcium concentration from 250 mM to 100 mM, yielded a 25-fold increase in the expression of the recombinant protein compared to the serum-free standard conditions. Altering the phosphate concentration, 1.4 mM in the transfection buffer, did not improve the protein expression. Interestingly, reduction of DNA quantity by half to a concentration of 0.5 μg per milliliter of culture volume resulted in a two-fold increase of protein production. Addition of albumin to serum-free medium protected the cells against the toxicity of the calcium phosphate transfection particles (CaPi) yielding higher protein expression. All the experiments were executed in a shaken multi-well system, allowing high multiplicity parameter screening to speed up optimizations. The culture system is inexpensive, simple and efficient, minimizing costs for labor and consumables.
PMCID: PMC3449700  PMID: 22358856
calcium; coprecipitation; DNA; HEK293; phosphate; small-scale culture; suspensionculture; transfection; transient geneexpression

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