Experimental Studies On Anti-hyperalgesia Mechanism Of Ketamine

BackgroundTissue damage and inflammation usually produce hyperalgesia and allodynia. It has known that central sensitization is related to hyperalgesia and allodynia. At present, hyperalgesia has been thought to being created by a cascade of neuronal events within the spinal cord dorsal horns. Although this neuronal model provided an excellent framework, however, there recently are mounting evidences that spinal cord glia (microglia and astrocytes) and proinflammatory cytokines as key players in the creation and maintenance of hyperalgesia states of diverse etiologies. Dorsal horn astrocytes and mocroglia are now known to be activated in response to many matters, such as subcutaneous inflammation, intraperitoneal bacteria, peripheral nerve injury, lumbar spinal root constriction, spinal nerve transection, immune activation within the spinal cord, and so forth. Similar to other immune cells, release the proinflammatory cytokines, activated glia, besides releasing substances that excite spinal-responsive neurons, such as reactive oxgen species (ROS), NO, PGs, EAAs, and growth factors. Blocker of proimflammatory cytokines can alleviate hyperalgesia states induced by diverse etiologies. Furthermore, the drugs that blocked astrocytes activation can block hyperalgesia induced by above-mentioned factors.The mechanism underlying hyperalgesia companied by morphine tolerance is believed to involve complex changes in neuronal plasticity. Recent many observations suggested that celluar mechanisms between thermal hyperalgesia and allodynia induced by neuropathic pain and morphine tolerance were similar in spinal dorsal horn. Since thermal hyperalgesia and allodynia induced by neuropathic pain and morphine tolerance share similar cellular mechanisms in spinal dorsal horn, we speculate that action of glia may involve in development of morphine tolerance. Infusion of LPS (i.c.v.) and LPS (i.t.) can induce hyperalgesia, however, mechanism to produce hyperalgesia is not very clear at present. Significant evidence now exists supporting an important role for N-methyl-D-aspartate (NMDA) receptor activation in spinal cord sensitivity. NO is thought to be one of the mediator of at least some of the effects of NMDA receptor activation. Activated glia increases synthesis of NO. Therefore, proimflammatory cytokines and NO maybe also play an important role in hyperalgesia induced by central inflammation. It was reported that NMDA receptor antagonist MK801 and ketamine prevented morphine tolerance and hyperalgesia. MK-801 also blocked astrocytes activation in spinal cord horn and hyperalgesia and allodynia induced by neuropathy.Ketamine is a non-competitive N-methy-D-aspartate receptor antagonist widely used in clinical anesthesia. There were some researchful results that ketamine inhibited hyperalgesia companied with morphine tolerance and decreased neuropathic hyperalgesia. It is necessary for deeply investigating the anti-hyperalgesia mechanisms of ketamine. In this thesis, we used three different animal models, i.e. chronic morphine tolerance of spinal cord, L5 spinal nerve transection, and central inflammation induced by intrathecal injection (i.t.) of LPS, and studied the possible anti-hyperalgesia mechanisms of ketamine through observing effects of this agent on painful behavior and astrocytes, proinflammatory cytokines, NO, and NOS.Part oneEffects of intrathecal co-adminstration of ketamine with morphine on astrocytes and proinflammatory cytokines of spinalcord in morphine tolerance ratsObjectiveTo investigate the effects of intrathecal co-administration of ketamine, a non-competitive N-methy-D-aspartate receptor antagonist, with morphine on pain behaviours, activation of astrocytes, and releases of IL-1β and IL-6 from spinal cord in the rats ofmorphine tolerance. MethodsExperimental animal Twenty-seven male Sprague-Dawley male rats weighing 170-250 g were randomly divided averagely into three groups averagely: control group (group C), morphine tolerance group (group M), and ketamine + morphine group (group MK). By incision at the lower back, polyethylene catheter was threaded into subarachnoid space. Paw-withdrawal latency (PWL) of the left hind was measured for determining thermal pain sensitivity. By applying 12 g von Frey filments on the plantar of left hind paw, the total number of paw withdrawals in three sets of 10 stimulations for each set separated by 5 min expressed mechanical sensitivity (allodynia). At 10 a.m. on day 4 post- catheterization, 10 μ l of normal water (i.t.) was administrated in group C, morphine (20 μ g /10 μ l, i.t.) in group M, 20μ g of morphine in combination with ketamine (250μ g.10μ l^-1, i.t.) in group MK once daily for 5 consecutive days.Observation parameters Basic or pre-drug values of PWL and total number of paw withdrawals to were measured on day 3 post-catheterization. PWL and a percentage of maximal possible effect (%MPE) were determined 30 minutes after delivery of the scheduled drugs (i.t.) on day 1, 3, and 5. 24 hours after 5th intrathecal injection of the drug, chronic morphine withdrawal-induced PWL and the total number of paw withdrawals were recorded before morphine challenge test. Then, all the group received 10 μ l of morphine (5 μ g, i.t.) for morphine challenge test. At 30 min after the morphine challenge test, PWL was observed once more. Subsequently, for each group, lumbar spinal cord were obtained for measurement of the average optical density (AOD), the integral optical density (IOD) of glial fibrillary acidic protein (GFAP) immuno-reactive cells in the dorsal horn (n=4) by means of immunohistochemistry SABC methods and for determining IL-1 β and IL-6 withELISA method. ResultsBasic values of PWL in three groups were almost similar, they were 9.31 ± 0.90 seconds, 9.23±0.89 seconds and 9.19±0.28 seconds respectively. Before the morphine challenge on day 6, PWL of group C was almost the same as basic values, but PWL values of group M and group MK decreased significantly as compared with basic values. Relative to basic values of PWL, however, reduced value was larger in group M (2.71 ± 0.20 seconds) than in group MK (1.37 ±0.33 seconds ) (P<0.01) and reduced percent was larger in group M (29.44 ± 2.12%) than in group MK (14.89 ± 2.85% ) (P<0.01). After the morphine challenge on day 6, %MPE was significantly higher in group C (59.20±4.0%) than in group M (8.86±1.42%) (P<0.01) and group MK (21.32±3.92%) (P<0.01), and furthermore, %MPE was also significantly high in group MK as compared with in group M (P<0.01). Basic total withdrawal numbers of three groups were about 0.11±0.33. Before morphine challenge, total withdrawal numbers was significantly higher in group M (10.66±1.41)than in group MK(5.22 ±1.22, P<0.01) and in group C (0.22±0.44, P<0.01).Astrocytic responses in lumbar spinal cord Astrocytes in spinal dorsal horn were activated by morphine and became hypertrophy. Ketamine could decrease the degree of activation of astrocytes by morphine. The average area (AA), the average optical density (AOD) and the integral optical density (IOD) of GFAP immuno-reactive cells in the dorsal horn were significantly lower in group MK than the responding values in group M respectively.IL-1β and IL-6 in lumbar spinal cord Taking pg. mg protein^-1 as measuring unit, IL-1 β (25.68±4.03) and IL-6 (31.37±2.06) were higher in group M in than IL-1β (9.18 ± 2.65, P<0.01) and IL-6 (12.97±1.84, P<0.01) in group C. Although IL-1β (16.54 ±4.08) and IL-6 (20.95 ±1.85) were significantly higher in group MK than in group C respectively, both were significantly lower than the responding values in group M. ConclusionIntrathecal repeated morphine may result in antinociceptive decrease of morphine (morphine tolerance) and abnormal pain sensitivity (thermal hyperalagesia and allodynia ). Activation of astrocytes and release of proinflammatory may involve in intrathecal morphine tolerance.Intrathecal coadministration of morphine with ketamine may prevent morphine tolerance and abnormal pain sensitivity including thermal hyperalagesia and allodynia induced by intrathecal repeated morphine. These results suggested that NMDA receptor antagonist prevent morphine tolerance and abnormal pain sensitivity.Intrathecal coadministration of morphine with ketamine may inhibit activation of astrocytes in spinal dorsal horn induced by chronic morphine tolerance.Ketamine may inhibit release of IL-1β and IL-6 of lumbar spinal cord induced by chronic intrathecal morphine. Part twoEffects of ketamine on proinflammatory cytokines of the spinal cord in rats with neuropathic painObjectiveTo investigate the effects of ketamine on the pain behavior and proinflammatory cytokines of the spinal cord in rats with neuropathic pain. MethodsExperimental animal 27 male Sprague-Dawley rats weighing 170g-250g were randomly divided into sham-operated, L5 spinal nerve transection, and L5 spinal nerve transection plus ketamine (i.p.) (treatment group). 1 ml of ketamine (50mg.kg^-1, i.p.) was given for treatment group and 1 ml of normal water (i.p.) for sham-operated and L5 spinal nerve transection group on the day of cutting L5 spinal nerve. 2h later, 2h later, rats were anesthetizd with thiopental (40 mg·kg^-1, i.p.) and left L5 spinal nerve transection was carried out in L5 spinal nerve transection and in treatment group. From on day 1 to day 7 after L5 spinal nerve transection, each rat was given 1 ml of ketamine (50mg.kg^-1, i.p.) at 10:00 a.m. once every day. 1 ml of normal water (i.p.) was given in sham-operated group and in treatment group.Observation parameters At 3:00 p.m., paw withdrawal latency (PWL) to radiant heat and total numbers of withdrwal to 30 stimuli with 12g von Frey filment was recorded on day 1, 3, 5, and 7 after L5 spinal nerve transection. At 10 a.m. on the day 8, lumbar spinal cord(n=5) was harvested for measurement of IL-1 β and IL-6 contents with ELISA. ResultsBehavioral outcome Thermal hypergalgesia and mechanical allodynia occurred onday 1 post-transection of L5 spinal nerve and became more significant along with time lengthening. In spite that PWL shortened progressively in treatment group and the corresponding PWL was significantly shorter than in sham-operated group on day 3, 5, 7, PWL was significantly longer in treatment group than in L5 spinal nerve transection group at each time point post-transection of L5 spinal nerve. Although numbers of withdrwal to 30 stimuli with 12g von Frey filments were significantly more in treatment group than in sham-operated group, the positive withdrwal numbers were significantly fewer in treatment group than in L5 spinal nerve transection group at each time point.IL-1β and IL-6 in lumbar spinal cord Taking pg. mg protein^-1 as measuring unit, both IL-1β (18.05 ± 1.99) and IL-6 (23.65±2.76) increased significantly high in L5 spinal nerve transection group as compared with IL-1β (12.54 ± 1.67, P<0.01) and IL-6 (16.01±0.93, i><0.01) in treatment and IL-1β (9.25±0.80, P<0.01) and IL-6 (11.94±1.95, P<0.01) in sham-operated group. Both IL-1β and IL-6 were significantly higher in treatment group than in sham-operated group, but the two values were significantly lower in treatment group than in L5 spinal nerve transection group respectively.ConclusionL5 spinal nerve transection produced neuropathic pain manifested characteristically asthermal hyperalgesia and allodynia in behavior and increased synthesis of IL-1β and IL-6 in spinal cord.Ketamine prevented hyperalgesia and allodynia induced by L5 spinal nerve transection, lowered levels of IL-1β and IL-6 in spinal cord. These effects may be contributed to ketamine blocking NMDA receptor and inhibiting activation of glia in spinal cord in rats.Part threeEffect of ketamine on hyperalgesia and NO/NOS and IL-1β mRNA in the lumbar spinal cord after intrathecal injection ofLPS in ratsObjectiveTo study the effect of ketamine(i.p) on hyperalgesia and content of nitric oxide(NO), activity of NOS, inducible nitric oxide synthase(iNOS), and interleukin-1β mRNA (IL-1β mRNA) in the lumbar spinal cord after intrathecal injection of LPS in rats. MethodsExperimental animal Thirty male and female Sprague-Dawley rats, weighing 220-250g, were randomly divided equally into control group, LPS group and ketamine group. 2h before intrathecal injection (i.t), 1ml of normal saline (i.p) was given in control group and LPS group, 1ml of ketamine (30mg.kg^-1) in ketamine group. Following successful puncture of lumbar, 20 μ l of LPS (0.1mg, i.t.) was administrated for each rat in LPS group and ketamine group, 20 μ l of normal saline (i.t.) was given in control group.Observation parameters At 1h, 3h, 6h, 12 h and 24h after LPS (i.t.), hot plate latency was recorded. After the hot plate latency was recorded at 24h after LPS (i.t.), lumbar spinal cord were harvested for measuring content of NO, activities of NOS and iNOS withspectrophotometry, and expression of IL—1β mRNAwith RT-PCR. ResultsBehavioral outcome Before LPS (i.t.), hot plate latency was coparative among controlgroup, LPS group and ketamine group. LPS (i.t.) was significantly shorter in LPS group than in control group respectively at each observing time point after LPS (i.t.). Hot plate latency was significantly longer in in ketamine group than the corresponding value in LPS group and in control group after LPS (i.t.). Furthermore, hot plate latency was significantlyprolonged in ketamine group after LPS (i.t.), compared with that before LPS (i.t.).Contents of NO and activity of NOS and iNOS The contents of NO and activity of NOS and iNOS were significantly higher in LPS group than in control group respectively (P<0.01). The contents of NO and activity of NOS and iNOS were significantly lower in ketamine group than in LPS group respectively (P<0.01). There was no difference in activity of iNOS between ketamine and control group (P>0.05).Expression of IL- β mRNA Expression of IL- β mRNA in ketamine group (0.4377± 0.0882) was significantly higher than that in control group (0.2667 ± 0.0732 ) (P<0.01 ) , but significantly lower than in LPS group (0.5744 ±0.0563) (P<0.05).ConclusionIntrathecal injection of LPS induced thermal hyperalgesia, enhanced activities of NOSand iNOS, increased production of NO, and upregulated expression of IL- β mRNA. The results suggested that mechanism of thermal hyperalgesia resulting from intrathecal LPS may related to increase of NO and proinflammatory cytokines.Ketamine may exert anti-hyperalgesia for the rats received intrathecal injection of LPS through preventing activation of microglial cells, inhibiting expression of IL-1β mRNA production of IL-1β , and reducing production of NO.SummaryNMDA receptor is involved in development of central sensitization. Three kinds of the animal model with hyperalgesia, i.e. intrathecal morphine tolerance, L5 spinal nerve transection, and intrathecal injection of LPS resulting in central inflammation, were used for exploring effects of ketamine, a non-competitive N-methy-D-aspartate receptor antagonist, on hyperalgesia and possible mechanism in this thesis.It was proved that intrathecally repeated administration of morphine induced antinociceptive decrease of morphine (morphine tolerance) and abnormal pain sensitivity (thermal hyperalagesia and allodynia) and the mechanism of intrathecal morphine tolerance may involve in activation of astrocytes and release of proinflammatory cytokines. The results showed that ketamine (i.t.) prevented morphine tolerance and abnormal pain sensitivity, which may be related to inhibiting activation of astrocytes in spinal dorsal horn, and decreasing release of IL-1 P and IL-6 of lumbar spinal cord induced by chronic intrathecal morphine. It is suggested that ketamine exert its effects on preventing morphine tolerance and hyperalgesia through blocking NMDA receptor. It was found that ketamine prevented hyperalgesia and allodynia induced by L5 spinal nerve transection, lowered levels of IL-1 β and IL-6 in spinal cord. These effects may be contributed to ketamine blocking NMDA receptor and inhibiting activation of glia in spinal cord in rats. Thermal hyperalgesia also occurred in the rats with central inflammation induced by LPS (i.t.). Ketamine obviously inhibited this kind of hyperalgesia, which mechanism may be through lowering activity of NOS and iNOS, decreasing the production of NO, preventing synthesis of IL-1β and IL-6 originated from microglial cels and other inflammatory cells.In summary, many factors, including activation of glia, production of NO, release of proinflammatory cytokines, were involvement in development of hyperglgesia caused by different causes. Non-competitive N-methy-D-aspartate receptor antagonist ketamine inhibited many kinds of hyperalgesia. Anti-hyperalgesia of ketamine may be related to blocking NMDA receptors, decreasing intracellular calcium, preventing activation of glia resulting from activated dorsal horn neuron and active mediators, and alleviating release of proinflammatory. The results provided new experimental evidences for clinically rational use of ketamine prevent hyperalgesia companied with chronic morphine tolerance, neuropathic pain, and central inflammation.