Ewelina Rojewska

Importance of glial activation in neuropathic pain

Glia plays a crucial role in the maintenance of neuronal homeostasis in the central nervous system. The microglial production of immune factors is believed to play an important role in nociceptive transmission. Pain may now be considered a neuro-immune disorder, since it is known that the activation of immune and immune-like glial cells in the dorsal root ganglia and spinal cord results in the release of both pro- and anti-inflammatory cytokines, as well as algesic and analgesic mediators. In this review we presented an important role of cytokines (IL-1alfa, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-15, IL-18, TNFalpha, IFNgamma, TGF-beta 1, fractalkine and CCL2); complement components (C1q, C3, C5); metaloproteinases (MMP-2,-9) and many other factors, which become activated on spinal cord and DRG level under neuropathic pain. We discussed the role of the immune system in modulating chronic pain. At present, unsatisfactory treatment of neuropathic pain will seek alternative targets for new drugs and it is possible that anti-inflammatory factors like IL-10, IL-4, IL-1alpha, TGF-beta 1 would fulfill this role. Another novel approach for controlling neuropathic pain can be pharmacological attenuation of glial and immune cell activation. It has been found that propentofylline, pentoxifylline, minocycline and fluorocitrate suppress the development of neuropathic pain. The other way of pain control can be the decrease of pro-nociceptive agents like transcription factor synthesis (NF-kappaB, AP-1); kinase synthesis (MEK, p38MAPK, JNK) and protease activation (cathepsin S, MMP9, MMP2). Additionally, since it is known that the opioid-induced glial activation opposes opioid analgesia, some glial inhibitors, which are safe and clinically well tolerated, are proposed as potential useful ko-analgesic agents for opioid treatment of neuropathic pain. This review pointed to some important mechanisms underlying the development of neuropathic pain, which led to identify some possible new approaches to the treatment of neuropathic pain, based on the more comprehensive knowledge of the interaction between the nervous system and glial and immune cells.

Differential activation of spinal microglial and astroglial cells in a mouse model of peripheral neuropathic pain.

The pharmacological attenuation of glial activation represents a novel approach for controlling neuropathic pain, but the role of microglial and astroglial cells is not well established. To better understand the potential role of two types of glial cells, microglia and astrocytes, in the pathogenesis of neuropathic pain, we examined markers associated with them by quantitative RT-PCR, western blot and immunohistochemical analyses in the dorsal horn of the lumbar spinal cord 7days after chronic constriction injury (CCI) to the sciatic nerve in mice. The mRNA and protein of microglial cells were labeled with C1q and OX42(CD11b/c), respectively. The mRNA and protein of astrocytes were labeled with GFAP. The RT-PCR results indicated an increase in C1q mRNA that was more pronounced than the increased expression of GFAP mRNA ipsilateral to the injury in the dorsal spinal cord. Similarly, western blot and immunohistochemical analyses demonstrated an ipsilateral upregulation of OX42-positive cells (72 and 20%, respectively) and no or little (8% upregulation) change in GFAP-positive cells in the ipsilateral dorsal lumbar spinal cord. We also found that chronic intraperitoneal injection of the minocycline (microglial inhibitor) and pentoxifylline (cytokine inhibitor) attenuated CCI-induced activation of microglia, and both, but not fluorocitrate (astroglial inhibitor), diminished neuropathic pain symptoms and tactile and cold sensitivity. Our findings indicate that spinal microglia are more activated than astrocytes in peripheral injury-induced neuropathic pain. These findings implicate a glial regulation of the pain response and suggest that pharmacologically targeting microglia could effectively prevent clinical pain syndromes in programmed and/or anticipated injury.

Minocycline reduces the injury-induced expression of prodynorphin and pronociceptin in the dorsal root ganglion in a rat model of neuropathic pain

A role of neuropeptides in neuropathic pain development has been implicated; however, the neuroimmune interactions that are involved in the underlying mechanisms may be more important than previously thought. To examine a potential role of relations between glia cells and neuropeptides in neuropathic pain, we performed competitive reverse-transcription polymerase chain reaction (RT-PCR) from the dorsal lumbar spinal cord and the dorsal root ganglion (DRG) after chronic constriction injury (CCI) in the rat sciatic nerve. The RT-PCR results indicated that complement component 1, q subcomponent (C1q) mRNA expression was higher than glial fibrillary acidic protein (GFAP) in the spinal cord 3 and 7 days post-CCI, suggesting that spinal microglia and perivascular macrophages are more activated than astrocytes. In parallel, we observed a strong upregulation of prodynorphin mRNA in the spinal cord after CCI, with no changes in the expression of proenkephalin or pronociceptin. Conversely, the expression of GFAP mRNA in the DRG was higher than C1q, which suggests that the satellite cells are activated shortly after injury, followed by the macrophages and polymorphonuclear leukocytes infiltrating the DRG. In the DRG, we also observed a very strong upregulation of prodynorphin (1387%) as well as pronociceptin (122%) and a downregulation of proenkephalin (47%) mRNAs. Interestingly, preemptive and repeated i.p. injection of minocycline reversed the activation of microglia/macrophages in the spinal cord and the trafficking of peripheral immune cells into the DRG, and markedly diminished the upregulation of prodynorphin and pronociceptin in the DRG. We thus provide novel findings that inhibition of C1q-positive cells by minocycline can diminish injury-induced neuropeptide changes in the DRG. This suggests that immune cells-derived pronociceptive factors may influence opioid peptide expression. Therefore, the injury-induced activation of microglia and leukocytes and the subsequent activation of neuropeptides involved in nociception processes are potential targets for the attenuation of neuropathic pain.

Effects of selective and non-selective inhibitors of nitric oxide synthase on morphine- and endomorphin-1-induced analgesia in acute and neuropathic pain in rats

Nitric oxide (NO) has been reported to be involved in the mechanisms of pain generation throughout the nervous system. We examined the effects of intrathecally (i.t.) administered nitric oxide synthase (NOS) inhibitors on the antinociceptive effects of morphine and endomorphin-1 during acute pain and in chronic constriction injury (CCI)-exposed rats. We used N(G)-nitro-l-arginine methyl ester (l-NAME), a non-selective NOS inhibitor; 7-nitroindazole (7-NI) or 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM), selective inhibitors of neuronal NOS (NOS1); and 1400W dihydrochloride, a selective inhibitor of inducible NOS (NOS2). Morphine (0.5-2.5 μg) and endomorphin-1 (2.5-20 μg) in acute pain and morphine (10-40 μg) and endomorphin-1 (5-20 μg) after CCI-injury were combined with NOS inhibitors. For acute pain, the ED50 for endomorphin-1 (7.1 μg) was higher than that of morphine (1.3 μg) in the tail-flick test. For neuropathic pain, the ED50 value for morphine was much higher (43.2 μg) than that of endomorphin-1 (9.2 μg) in von Frey test. NOS inhibitors slightly influenced pain thresholds in both pain models. Moreover, in neuropathic pain, the effects of morphine were more potentiated by L-NAME, TRIM, 7-NI and 1400W (12×, 8.6×, 4.1× and 5.3×, respectively) than were the effects of endomorphin-1 (2.7×, 4.3×, 3.4× and 2.1×, respectively) in the von Frey test. Minocycline which is known to enhance the efficiency of morphine in neuropathic pain, decreased the mRNA expression of NOS1 in the DRG and NOS2 and C1q in the spinal cord after CCI. Both NOS2 and IBA-1 protein levels in the spinal cord and NOS1, NOS2 and IBA1 protein levels in DRG decreased after minocycline administration. In conclusion, our results provide evidence that both neuronal and non-neuronal NOS/NO pathways contribute to the behavioural pain responses evoked by nerve injury. The NOS inhibitors regardless of the type of pain enhanced morphine antinociception and, to a lesser extent, altered the effects of endomorphin-1, an opioid ligand with a peptidergic structure.

Neuronal and immunological basis of action of antidepressants in chronic pain – clinical and experimental studies

The current knowledge of the pharmacological actions of the tricyclic antidepressants (TCAs) has slowly evolved through their over 40-year history. Chronic pain represents one of the most important public health problems, and antidepressants are an essential part of the therapeutic strategy in addition to classical analgesics. This article reviews the available evidence on the efficacy and safety of antidepressants in chronic pain conditions; namely, headaches, low back pain, fibromyalgia, cancer pain and especially neuropathic pain. TCAs are traditionally the main type of depression medication used to treat chronic pain. Recently, new antidepressants were introduced into clinical use, with a significant reduction in side effects and equivalent efficacy on mood disorders. These new drugs that are effective for chronic pain belong to the tetracyclic antidepressants (TeCAs) group (amoxapine, maprotiline), the serotonin and noradrenaline reuptake inhibitors (SNRIs) group (duloxetine, venlafaxine, milnacipran) and the atypical antidepressants group (bupropion, trazodone, mirtazapine, nefazodone). In this review, we present the available publications on TCAs (amitriptyline, doxepin, imipramine, desipramine, nortriptyline), TeCAs (amoxapine, maprotiline), selective serotonin reuptake inhibitors (SSRIs) (citalopram, fluoxetine, paroxetine), SNRIs (duloxetine, venlafaxine, milnacipran) and atypical antidepressants (bupropion) for the treatment of neuropathic pain. We also review analgesics acting as both opioid receptor agonists and also acting as aminergic reuptake inhibitors. Existing data are insufficient to conclude which of these new classes of antidepressants has the best clinical profile and will be the most effective in the treatment of neuropathic pain; in addition, a lower incidence of side effects should be considered. Increased experimental and translational research is a key for further improvement of the treatment of chronic pain with antidepressants. However, evidence from basic science is needed to improve our understanding of the mechanisms of action and their possible pharmacodynamic interactions.

Mechanisms and pharmacology of diabetic neuropathy – experimental and clinical studies

Neuropathic pain is the most common chronic complication of diabetes mellitus. The mechanisms involved in the development of diabetic neuropathy include changes in the blood vessels that supply the peripheral nerves; metabolic disorders, such as the enhanced activation of the polyol pathway; myo-inositol depletion; and increased non-enzymatic glycation. Currently, much attention is focused on the changes in the interactions between the nervous system and the immune system that occur in parallel with glial cell activation; these interactions may also be responsible for the development of neuropathic pain accompanying diabetes. Animal models of diabetic peripheral neuropathy have been utilized to better understand the phenomenon of neuropathic pain in individuals with diabetes and to define therapeutic goals. The studies on the effects of antidepressants on diabetic neuropathic pain in streptozotocin (STZ)-induced type 1 diabetes have been conducted. In experimental models of diabetic neuropathy, the most effective antidepressants are tricyclic antidepressants, selective serotonin reuptake inhibitors, and serotonin norepinephrine reuptake inhibitors. Clinical studies of diabetic neuropathy indicate that the first line treatment should be tricyclic antidepressants, which are followed by anticonvulsants and then opioids. In this review, we will discuss the mechanisms of the development of diabetic neuropathy and the most common drugs used in experimental and clinical studies.

Involvement of pro- and antinociceptive factors in minocycline analgesia in rat neuropathic pain model

In neuropathic pain the repeated minocycline treatment inhibited the mRNA and protein expression of the microglial markers and metalloproteinase-9 (MMP-9). The minocycline diminished the pronociceptive (IL-6, IL-18), but not antinociceptive (IL-1alpha, IL-4, IL-10) cytokines at the spinal cord level. In vitro primary cell culture studies have shown that MMP-9, TIMP-1, IL-1beta, IL-1alpha, IL-6, IL-10, and IL-18 are of microglial origin. Minocycline reduces the production of pronociceptive factors, resulting in a more potent antinociceptive effect. This change in the ratio between pro- and antinociceptive factors, in favour of the latter may be the mechanism of minocycline analgesia in neuropathy.

The effect of botulinum neurotoxin A on sciatic nerve injury-induced neuroimmunological changes in rat dorsal root ganglia and spinal cord

Botulinum neurotoxin serotype A (BoNT/A) acts by cleaving synaptosome-associated-protein-25 (SNAP-25) in nerve terminals to inhibit neuronal release and shows long-lasting antinociceptive action in neuropathic pain. However, its precise mechanism of action remains unclear. Our study aimed to characterize BoNT/A-induced neuroimmunological changes after chronic constriction injury (CCI) of the sciatic nerve. In the ipsilateral lumbar spinal cords of CCI-exposed rats, the mRNA of microglial marker (complement component 1q, C1q), astroglial marker (glial fibrillary acidic protein, GFAP), and prodynorphin were upregulated, as measured by reverse transcription-polymerase chain reaction (RT-PCR). No changes appeared in mRNA for proenkephalin, pronociceptin, or neuronal and inducible nitric oxide synthase (NOS1 and NOS2, respectively). In the dorsal root ganglia (DRG), an ipsilateral upregulation of prodynorphin, pronociceptin, C1q, GFAP, NOS1 and NOS2 mRNA and a downregulation of proenkephalin mRNA were observed. A single intraplantar BoNT/A (75 pg/paw) injection induced long-lasting antinociception in this model. BoNT/A diminished the injury-induced ipsilateral spinal upregulation of C1q mRNA. In the ipsilateral DRG a significant decrease of C1q-positive cell activation and of the upregulation of prodynorphin, pronociceptin and NOS1 mRNA was also observed following BoNT/A admistration. BoNT/A also diminished the injury-induced upregulation of SNAP-25 expression in both structures. We provide evidence that BoNT/A impedes injury-activated neuronal function in structures distant from the injection site, which is demonstrated by its influence on NOS1, prodynorphin and pronociceptin mRNA levels in the DRG. Moreover, the silence of microglia/macrophages after BoNT/A administration could be secondary to the inhibition of neuronal activity, but this decrease in neuroimmune interactions could be the key to the long-lasting BoNT/A effect on neuropathic pain.

Delta-Opioid Receptor Analgesia Is Independent of Microglial Activation in a Rat Model of Neuropathic Pain

The analgesic effect of delta-opioid receptor (DOR) ligands in neuropathic pain is not diminished in contrast to other opioid receptor ligands, which lose their effectiveness as analgesics. In this study, we examine whether this effect is related to nerve injury-induced microglial activation. We therefore investigated the influence of minocycline-induced inhibition of microglial activation on the analgesic effects of opioid receptor agonists: morphine, DAMGO, U50,488H, DPDPE, Deltorphin II and SNC80 after chronic constriction injury (CCI) to the sciatic nerve in rats. Pre-emptive and repeated administration of minocycline (30 mg/kg, i.p.) over 7 days significantly reduced allodynia and hyperalgesia as measured on day 7 after CCI. The antiallodynic and antihyperalgesic effects of intrathecally (i.t.) administered morphine (10–20 µg), DAMGO (1–2 µg) and U50,488H (25–50 µg) were significantly potentiated in rats after minocycline, but no such changes were observed after DPDPE (10–20 µg), deltorphin II (1.5–15 µg) and SNC80 (10–20 µg) administration. Additionally, nerve injury-induced down-regulation of all types of opioid receptors in the spinal cord and dorsal root ganglia was not influenced by minocycline, which indicates that the effects of opioid ligands are dependent on other changes, presumably neuroimmune interactions. Our study of rat primary microglial cell culture using qRT-PCR, Western blotting and immunocytochemistry confirmed the presence of mu-opioid receptors (MOR) and kappa-opioid receptors (KOR), further we provide the first evidence for the lack of DOR on microglial cells. In summary, DOR analgesia is different from analgesia induced by MOR and KOR receptors because it does not dependent on injury-induced microglial activation. DOR agonists appear to be the best candidates for new drugs to treat neuropathic pain.

Minocycline influences the anti-inflammatory interleukins and enhances the effectiveness of morphine under mice diabetic neuropathy

A single streptozotocin (STZ) injection in mice can induce significant neuropathic pain along with an increase in plasma glucose levels and a decrease in body weight. Seven days after the administration of STZ, an upregulation of C1q-positive cells was observed. Additionally, interleukins (IL-1beta, IL-3, IL-4, IL-6, IL-9, IL12p70, IL-17); proteins of the tumor necrosis factor (TNF) family, e.g., IFNgamma and sTNF RII, were upregulated. Chronic administration of minocycline increases antinociceptive factors (IL-1alpha, IL-2, IL-10, sTNFRII) in diabetic mice. Minocycline also reduces the occurrence of neuropathic pain and significantly potentiates the antiallodynic and antihyperalgesic effects of morphine.