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Modulation Of Nociception-induced Plasticity By NO-mediated Signal Pathways In The Spinal Cord

Posted on:2006-08-11Degree:DoctorType:Dissertation
Country:ChinaCandidate:X C ZhangFull Text:PDF
GTID:1100360155960729Subject:Neurobiology
Abstract/Summary:PDF Full Text Request
In addition to tetanic stimulation of the sciatic nerve, natural noxious stimulation of skin or nerve injury is also able to induce LTP of C-fiber-evoked response in the spinal cord. Given that hippocampal LTP is considered as a fundamental mechanism of learning and memory, it is possible that spinal LTP of C-fiber-evoked response is a form of pain memory. Several lines of evidence have suggested that the hypersensitivity of spinal pain-related neurons, termed central sensitivity, plays a critical role in production of hyperalgesia and allodynia induced by inflammation or nerve injury. Hence, it is believed that spinal LTP may be a substrate of central sensitivity. Some studies have suggested that the formation of NO, as well as activation of NO-mediated signaling pathways, involve in the spinal mechanisms underlying hyperalgesia and allodynia. However, the potential role of NO in spinal LTP associated with nociception has not been explored. The present study was designed to investigate whether contribution of NO, as well as NO-activated down-stream targets, to the spinal LTP of C-fiber-evoked field potentials. NO involves in the plasticity of nociceptive responses in the spinal dorsal hornThe effects of the NO synthase (NOS) inhibitor L-NAME and hemoglobin, a membrane impermeant scavenger of NO, on LTP of C-fiber-evoked field potentials were observed. In six rats, L-NAME (1mM, 20μ1 in volume) was applied directly onto the dorsal surface of recording segments 30 min before tetanic stimulation. L-NAME completely blocked the induction of LTP, but had no affect on the baseline responses. Similarly, hemoglobin also prevented the induction of spinal LTP when applied onto the spinal dorsal surface at 30 min before tetanic stimulation. However, D-NAME (lmM, 20ul), an inactive form of L-NAME, had little effect on the induction of spinal LTP in six other rats, arguing against a nonspecific pharmacological effect of L-NAME being responsible for the prevention of spinal LTP. When L-arginine (2mM, 20μl), a substrate for NOS, was applied to the recording segments prior to the introduction of L-NAME, the inhibition by L-NAME on spinal LTP was completely reversed in five rats tested. Thus, these results demonstrate that NO is implicated in induction of spinal LTP of C-fiber-evoked field potentials.Although the tetanic sciatic stimulation could induce LTP of C-fiber-evoked field potentials, the effects of tetanic sciatic stimulation on pain-related behavior were seldom reported. In the present study paw withdrawal latency (PWL) to radiant heat stimulation was assessed followed the tetanic sciatic stimulation. Before operation, there was no obvious difference in PWL to radiant heat stimuli between the left and right paw (9.83±0.62 vs. 9.77±0.66s, p>0.05). After the tetanic electrical stimulation (10 trains of 2 s duration, 100 Hz 0.5 ms rectangular pulses, 10 s intervals between trains, 35 V), the ipsilateral PWL decreased markedly on day 1 post-stimulation (6.51±0.88s) and the decrease was maintained for 6 days. In contrast, the contralateral PWL was not significantly changed. In the sham group, there was not any significant change in bilateral PWLs. To clarify the involvement of NO in thermal hyperalgesia by sciatic tetanic stimulation, the NOS inhibitor L-NAME (2mM, 10ul) was intrathecally injected at 30 min prior to tetanic sciatic stimulation. L-NAME completely reversed tetanic sciatic stimulation-induced thermal hyperalgesia, although L-NAME had no effects on the basic PWL to radiant heat stimulation in the control group.To further determine the role of NO in spinal LTP of C-fiber-evoked field potentials, the NADPH diaphorase activity of lumbar spinal cord was observed in different time after tetanic sciatic stimulation. At 5 min after stimulation, a robust dense staining of granules and NADPH diaphorase-reactive neurons was detected in the ipsilateral spinal cord with predominant intensity in the superficial layer , suggesting that the NOS-contained neurons was largely activated. At 30 min after tetanic sciatic stimulation, the increased staining density of granules and NADPH diaphorase-reactive neurons showed a moderate attenuation, and then a clear decrease approaching to baseline values at 2 h after stimulation, implicating that NOS-activated neurons recruit to basic level. There was no detectable change in staining dense of granules and NADPH diaphorase-reactive neurons in the contralateral spinal cord.These results suggest that the involvement of NO in the spinal LTP of C-fiber-evoked field potentials and related behavior changes. Furthermore, endogenous NO production is required for the induction of spinal LTP of C-fiber-evoked field potentials and the onset of thermal hyperalgesia induced by tetanic sciatic stimulation. This also provides further evidence that spinal LTP is a substrate of central sensitization of nociception.Different roles of NO-activated two pathways in the plasticity of nociceptive response in the spinal dorsal hornTwo potential targets of NO in the nervous system are soluble guanylyl cyclase (sGC) and ADP-ribosyltransferases (ADPRTs). Through binding to the heme region of sGC, NO triggers the production of cGMP. In the present study we observed the roles of the NO-activated two signal pathways in spinal LTP of C-fiber-evoked field potentials.In view of the previous finding that NO mediates the induction of hippocampus LTP in part by activating sGC, we examined the possible involvement of cGMP in soinal LTP using two different sGC inhibitors, MB (a nonspecific inhibitor) and ODQ (a specific inhibitor). In agreement with the results from hippocampus studies, both MB (4mM, 20ul) and ODQ (lOuM, 20ul) completely blocked the induction of spinal LTP of C-fiber-evoked field potentials (n=4, n=5, respectively), but failed to affect the baseline responses. When 8-Br-cGMP (lOOuM, 20^1), a phosphodiesterase-resistant membrane-permeant analog of cGMP, was applied to the spinal cord before introduction of MB or ODQ, MB- or ODQ-induced inhibition of spinal LTP was completely prevented (n=4, n=5, respectively), confirming the specific effect of MB or ODQ on spinal LTP.As the NO-activated sGC participates in the induction of spinal LTP, whether NO or NO-related signaling modulate the ongoing LTP of C-fiber-evoked field potentials? We observed the role of NO in the maintenance of spinal LTP. In five rats, L-NAME (lmM, 20ul) was applied directly onto the dorsal surface of recording segments at 120 min after tetanic stimulation. No detectable changes of potentiation in C-fiber-evoked field potentials were found during the recording periods after L-NAME treatment (p>0.05, n=5). Similarly, spinal application of hemoglobin (2mg/ml, 20ul) at 120 min after the induction of LTP, didn't alter spinal LTP (p>0.05, n=6). These findings suggest that no effect of L-NAME and hemoglobin on the maintenance of LTP of C-fiber-evoked field potentials.Although L-NAME or hemoglobin fails to alter the maintenance of spinal LTP, it can not exclude that the events of down-stream of activation of NO are involved in spinal LTP. Early studies have demonstrated that NO-donating compounds stimulate the ADP-ribosylation of proteins or auto-ADP-ribosylation of the poly-ADPRT, suggesting ADPRTs may be another target of NO action. To test whether the activity of ADPRTs participates in spinal LTP formation, two different ADPRT inhibitors,nicotinamide, an inhibitor of the mono-ADPRT, and benzamide, a more potent inhibitor of the poly-ADPRT, were used. Spinal administration of nicotinamide affected neither baseline responses nor LTP of C-fiber-evoked field potentials (n=5). Differently, the poly-ADPRT inhibitor benzamide significantly suppressed the maintenance but not the induction of spinal LTP and its early phase, as well as the baseline responses of C-fiber-evoked field potentials (n=7). No statistically difference was found between the mean potentiation of C-fibre-evoked field potentials in benzamide- (209.39 ±13.22 %) and saline-treated rats (237.77 ± 14.82 %) at 60 min after tetanic stimulation (P>0.05, n=7). However, from 90 min after tetanic stimulation, spinal LTP was gradually reduced in benzamine-treated group. At 120 min after tetanic stimulation, the potentiation of C-fiber-evoked field potentials was significantly lower in benzamine-treated group than that in saline-treated group (P<0.05). At 240 min after tetanic stimulation, the mean C-fiber-evoked field potentials approached to baseline. This result indicates that poly-ADPRT primarily links to the maintenance of spinal LTP.In summary, the data demonstrate that activation of NO-induced sGC contributes to the induction of spinal LTP, supporting an involvement of NO-induced sGC in hypersensitization of pain. A new finding that NO-related poly-ADPRT activity is primarily responsible for the maintenance of spinal LTP provides a novel mechanism underlying spinal synaptic plasticity.NO-mediated the plasticity of nociceptive response in the spinal dorsal horn by enhancement of excitability of primary afferent terminalsAs a possible fashion, NO modulates the function of primary afferent terminals in the spinal dorsal horn. The present study showed that SNP (5mM) obviously increased the excitability of DRG neurons. The number of evoked action potentials (APs) increased from 9±2 to 17±4, following 3min washout, it returned to 8±2, suggesting that NO can increase the excitability of DRG neurons. In contrast, the excitability of DRG neurons has no significant change throughout the whole recording period when no drug was applied.There is considerable evidence implicating that K+ Channel is a key factor in modulating the excitability of neuron. Hence, the effects of SNP on outward K+ currents in DRG neurons were observed. The results showed that SNP (5mM) significantly inhibited the outward K+ currents in DRG neurons (PO.05), after a 3 min or more long time washout, the K+ currents was little recovered. Moreover, the...
Keywords/Search Tags:long-term potentiation, nitric oxide, soluble guanylyl cyclase, ADP-ribosyltransferase, dorsal root ganglion, field potential, action potential, K~+ channel, patch clamp, spinal cord, rat
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