| Background:Morphine is one of the most commonly used drugs in the treatment of severe and chronic pain such as the pain inducted by cancer. Its use for chronic pain conditions is limited by the development of tolerance, a loss of analgesic effectiveness of the drug during repeated use. In addition, prolonged use of morphine could also lead to physical dependence, a need for continuing use of the drug to prevent the symptoms of withdrawal. People usually have to use increasing doses to achieve the desired effect. because of the development of tolerance and physical dependence, which increased the side effects such as respiratory depression, nausea, vomiting, cough suppression, and sedation. As such, the possible mechanisms for these phenomena have been intensively studied in an attempt to understand and prevent them. Despite the existence of a large body of information on the subject, the molecular and cellular mechanisms underlying morphine tolerance and dependence are not yet completely understood. Over the last decade, compelling evidence has accumulated indicating that glial cells activation plays an important role in morphine-induced tolerance and dependence. The activation of glial cells and enhanced proinflammatory cytokine expression at the spinal cord has been implicated in the development of morphine tolerance, and morphine withdrawal-induced hyperalgesia. Not only microglia, but also astrocytes, are activated in the spinal cord after morphine tolerance, and these activated glial cells participate in the maintenance of morphine-induced tolerance and dependence. Morphine tolerance and pathological pain are predicted to involve similar cellular and molecular mechanisms. Increasing evidence shows that inhibition of astrocytic activation attenuated the development of morphine tolerance, and withdrawal-induced hyperalgesia in rats. Propentofylline, a glial modulator which depressed the activation of microglia and astrocytes, attenuated the development and maintenance of morphine tolerance. We therefore predicted that microglia activation is crucial for the development of the morphine tolerance, and astrocyte activation is crucial for the maintenance of the morphine tolerance.Miyoshi et al. reported that IL-18-mediated microglia/astrocyte interactions in the spinal cord have a substantial role in the generation of tactile allodynia. They show that both the IL-18and IL-18receptor (IL-18R), which are induced in spinal dorsal horn, are crucial for tactile allodynia after nerve injury. Nerve injury induced a striking increase in IL-18and IL-18R expression in the dorsal horn, and IL-18and IL-18R were upregulated in hyperactive microglia and astrocytes, respectively. The functional inhibition of IL-18signaling pathways suppressed injury-induced tactile allodynia and decreased the phosphorylation of nuclear factor κB in spinal astrocytes and the induction of astroglial markers. Conversely, intrathecal injection of IL-18induced behavioral, morphological, and biochemical changes similar to those observed after nerve injury. First, TLR4triggers microglial activation through the p38MAPK pathway. IL-18produced by the TLR4/p38MAPK signaling cascade in activated microglia stimulates IL-18R on astrocytes in a paracrine manner. IL-18binding to IL-18R increases NF-kB phosphorylation in astrocytes and causes GFAP upregulation. IL-18R expression is upregulated by IL-18R stimulation, thus accelerating this signaling pathway in astrocytes. IL-18or other cytokines might stimulate microglia and astrocytes in an autocrine or paracrine manner and induce production of pro-inflammatory molecules, such as IL-1β, IL-6and TNF-a, as well as COX-2and inducible nitric oxide synthase. These pro-inflammatory molecules induced in glial cells sensitize dorsal horn neurons in the spinal cord.Miyoshi et al. showed that IL-18and IL-18Rs are induced in the spinal cord and participate in neuropathic pain after the L5spinal nerve ligation (SNL). They showed that nerve injury induced by the L5spinal nerve ligation induces an increase in IL-18and IL-18R expression in microglia and astrocytes, respectively, in the dorsal horn and that the IL-18mediated microglia/astrocyte interaction is crucial for the development and maintenance of tactile allodynia through glial cell-specific signal transduction cascades. IL-18upregulation in spinal microglia occurs through the TLR4/p38MAPK pathway. They suggested that upregulation of IL-18in spinal microglia have an important role in mechanical hypersensitivity after nerve injury. And they reported that suppressing IL-18in the spinal cord could prevent nerve injury-induced mechanical hypersensitivity. They found that anti-IL-18antibody significantly inhibited the injury-induced tactile allodynia at days1,3,5, and7after surgery. And blocking the IL-18R in the spinal cord by anti-IL-18R antibodies inhibited nerve injury-induced mechanical hypersensitivity. Increasing evidence shows that NF_B activation in spinal glial cells contributes to the generation of neuropathic pain. We found that both anti-IL-18and anti-IL-18R antibod ies inhibited the injury-induced increase in NF_B phosphorylation at day7after SNL. In addition, the immunofluorescence intensity of p-NFkB and GFAP in individual cells in the dorsal horn of anti-IL-18-and anti-IL-18R-treated rats was significantly lower than in those of vehicle-treated rats. In conclusion, they demonstrated that IL-18-mediated microglia/astrocyte interaction in the dorsal horn enhances neuropathic pain processing after nerve injury. Blocking IL-18signaling cascade in the spinal cord may represent a new approach to effectively treat clinical neuropathic pain.IL-18is a member of the IL-1family; IL-1β and IL-18are related closely, and both require intracellular cysteine protease caspase-1for biological activity. Shavit et al. Reported chronic morphine treatment could induce production of pro-inflammatory molecules, such as IL-1β. Morphine tolerance and pathological pain are predicted to involve similar cellular and molecular mechanisms. Miyoshi et al. demonstrated that IL-18-mediated microglia/astrocyte interaction in the dorsal horn enhances neuropathic pain processing after nerve injury, so we predicted that IL-18should participate in the development and maintenance of morphine tolerance. IL-18produced by the activated microglia stimulates IL-18R on astrocytes. IL-18binding to IL-18R increases astrocytes activation,then develop and maintain the morphine tolerance. In conclusion, in the work we set out to export the effect and the molecular and cellular mechanisms of blocking IL-18signaling cascade by intrathecal injection of anti-rat IL-18antibody in the spinal cord on the development of morphine tolerance in rats. Blocking IL-18signaling cascade in the spinal cord may represent a new approach to effectively treat clinical morphine tolerance. Aim To investigate the effect and the molecular and cellular mechanisms of blocking IL-18signaling cascade by intrathecal injection of anti-rat IL-18antibody in the spinal cord on the development of morphine tolerance in rats. In the work we set out to export a new approach to effectively treat clinical morphine tolerance.Methods Firstly all rats were implanted with intrathecal (i.t.) catheters. For intrathecal (i.t.) delivery of drugs, rats were implanted with i.t. catheters according to the improved method of Yaksh described in previous study. Fifty male adult Sprague-Dawley (SD) rats weighing150g-180g were prepared. Briefly, a sterile polyethylene (PE-10) tube filled with saline was inserted through L5/L6intervertebrae space, and the tip of the tube was placed at the spinal lumbar enlargement level. The wound was closed in two layers with4-0polyester suture. All the animals were allowed to recover for at least5days before experiments. Any rats with hind limb paralysis or paresis after surgery were excluded and euthanized with overdose pentobarbital. Drugs or vehicle were administered in volumes of20μl followed by a flush of10μl of gas to ensure drugs delivered into the subarachnoid space. Then they were tested to ensure the position of catheters five days after surgery (recorded as the first day). Because of the percentage of the success model is80%,40rats from the success models were randomly divided into5groups (n=8):saline control group (group Ⅰ), morphine tolerance group (group Ⅱ), IgG control group (group Ⅲ),0.4μg anti-rat IL-18antibody treatment group (group Ⅳ), and4μg anti-rat IL-18antibody treatment group (group Ⅴ).Tolerance to morphine antinociceptive effect was induced and drugs were injected from day3to day9. Rats were treated with subcutaneous injections of either saline or morphine (10mg/kg). The injections were given twice daily at8:00-9:00A.M. and4:00-5:00P.M. for7day, beginning on day3and ending on day9to induce opioid tolerance. Drugs were injected30min before the first morphine subcutaneous injection at8:00-9:00A.M from day3to day9. PBS was intrathecal (i.t.) injected in saline control group (group Ⅰ) and morphine tolerance group (group Ⅱ),4μg IgG,0.4μg anti-rat IL-18antibody and4μg anti-rat IL-18antibody was intrathecal (i.t.) injected in group Ⅲ, group Ⅳ and group Ⅴ. Drugs or vehicle were administered in volumes of20μl (IgG and anti-rat IL-18antibody were dissolved and diluted by PBS).Development of analgesic and antiallodynic tolerance to chronic morphine was recorded on days2and10of the morphine treatment. Chronic morphine withdrawal-induced hyperalgesia or allodynia in these animals was assessed on day10. On day2and10, paw withdrawal thermal latency(PWTL)of the basic and30min after morphine injection (4mg/kg)via tail vein were evaluated in all rats and the percentage of maximal possible antinociceptive effect (%MPE) of30min was calculated. Behavior recorded on day2and day10before the beginning of morphine treatment served as the basal latency. Lastly, the spinal cord lumbar enlargement was obtained immediately after the behavioral tests on day10to determine the expression of ERK and p-ERK by western bolt and the immunoreactivity of GFAP by immunofluorescence.Results①On day2there was no significant difference of the%MPE among five groups (P>0.05). On day10, compared with (99.70±0.85)%in saline control group(group Ⅰ), the%MPE was declined significantly in group Ⅱ-Ⅴ (P<0.05),showed that morphine tolerance was induced. Compared with (16.33±7.99)%in morphine tolerance group (group II),(49.38±17.53)%was increased significantly in4μg anti-rat IL-18antibody treatment group (group V)(P<0.05).And%MPE revealed no marked difference in IgG control group (group III) and0.4μg anti-rat IL-18antibody treatment group (group IV) compared with morphine tolerance group (group II)(P>0.05)②Immunofluorescence showed that compared with saline control group(group Ⅰ), the immunoreactivity of GFAP was markedly increased in morphine tolerance group (group II), IgG control group (group III) and0.4μg anti-rat IL-18antibody treatment group (group Ⅳ). Compared with morphine tolerance group (group II), the immunoreactivity was decreased significantly in4μg anti-rat IL-18antibody treatment group (group V). The Immunofluorescence results showed that blocking IL-18signaling cascade by intrathecal injection of anti-rat IL-18antibody in the spinal cord could induce the inhibition of astrocytic activation in the spinal cord.③The western blot analysis revealed that compared with saline control group(group I), the expression of p-ERK was increased significantly in group II-V. Compared with morphine tolerance group (group II), the expression of p-ERK was decreased significantly in4μg anti-rat IL-18antibody treatment group (group V) while showed no marked difference in IgG control group (group III) and0.4μg anti-rat IL-18antibody treatment group (group Ⅳ). The western blot results showed that blocking IL-18signaling cascade by intrathecal injection of anti-rat IL-18antibody in the spinal cord could inhibiting the phosphorylation of ERK in the spinal cord.Conclusion Blocking IL-18signaling cascade by intrathecal injection of anti-rat IL-18antibody in the spinal cord could attenuate the development of morphine tolerance in naive rats through inhibiting the phosphorylation of ERK and activation of astrocytes in the spinal cord. |
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