| Opioids are the most effective analgesic drugs and have been used in clinic commonly, but accompanying many side effects. Intrathecal or epidural injection of opioids have varieties advantages when used in the postoperative analgesia and malignant and non-malignant chronic pain treatment, such as the smaller dosage, the long-lasting analgesic effect and the low incidence of adverse reactions, but opioid tolerance would be induced more likely. How to enhance analgesic effect of opioids, reduce adverse reactions and drugs consumption has become a research focus. Agmatine (agmatine, AG), the endogenous ligand of the imidazoline receptor(imidazoline receptor, IR), is widely distributed in the the central nervous system. AG has identified as a new neurotransmitter and/or neuromodulator for it can act with imidazoline receptors,α2 adrenergic receptors and NMDA receptors. Studies have shown that AG can not only improve the analgesic effect of opioids significantly but also prevent and reverse the morphine tolerance and dependence effectively. AG are involved in certain neurotransmitters(e.g. catecholamines)and hormone release by acting on imidazoline receptors, and resulting in many biological effects, such as regulation of hypertension, high blood sugar and diuresis. However, the effects of intraspinal administration of AG were not studied systematicaliy. The study is to investigate the feasibility of agmatine as a assisted drug for intraspinal opioids.Part I TO observe the effects of agmatine(AG)on primarily cultured spinal cord neuron growth and its influence on neurons injury induced with glutamate (Glu)and to further explore possible underlying mechanisms, primarily cultured spinal cord neurons from embryonic rat were prepared. After 3 days in vitro, AG was added into neurons culture at various concentrations ranging from 0.5 to 80μg/ml and maintained for another 12, 24 and 36hrs. Subsequently, the effect of AG on mitochondrial activity, reflecting cell survival or toxicity was assessed by 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and neutral red assay respectively. Parallelingly, following addition of AG was carried out. Glu at final concentration of 2mmol/L was then administrated into cultures. 12, 24 and 36hrs in vitro latter, the morphological changes of the cells as well asβ-tubulinⅢimmunopositive cells were then observed under microscope. On the other hand, neuronal viability was determined by MTT assay.Moreover, Hoechst33342 and PI staining were undertaken to examine cell apoptosis and death,respectively, and malondialdehyde(MDA)of production in cultured neurons was detected. Eventually, these changes were analyzed and compared among different groups. We found that in normal culture condition, AG has no influence on cellular survival and growth, even no any neurotoxicity occured at concentration lower than 40μg/ml. Whereas neuronal survival and growth were significantly attenuated at the presence of 80μg/ml AG., When they are exposed to Glu, cells showed poor growth, even degeneration and further celluar viability decreased markedly. Additionally, cell apoptosis and death rate significantly increased (P<0.05 or 0.01). In the presence of AG, AG can significantly impede Glu-induced injury to neurons in morphology, elevate neuronal survival and viability. In addition, production of MDA in cultured neurons treated with AG can also be attenuated compared with control (P<0.01). These results suggested that AG, a novel neural transmitter or modulator has no toxical influence on neuron growth, and can protect neurons against peroxidative damage caused by exposure to Glu. The underlying mechanisms of neuroprotective effects of AG may be relate to their antagonistic action on NMDA receptors, and further repression of cascade damage induced by excitatory amino acids.Part II To study the action of intrathecal agmatine on the analgesia effects of morphine in BV rats. Thirty SD rats were divided into three groups randomly as follows:①C1 group in which the rats were only i.t. saline(10μl);②M1 group in which the rats were i.t. morphine(15μg/10μl);③AM1 group in which the rats were i.t. morphine 15μg and AG 12.5μg(10μl).All rats were subcutaneous injection(s.c.)of bee venom(0.2mg/50μl)in the left plantar after 5 min after intrathecal administration and the number of spontaneous flinches were observed and record within 1 hour. Compared with that of C1 group, the number of flinches of M1 group were significantly reduced(P<0.05), and these of AM1 group were further down(P<0.05), indicated that intrathecal morphine could inhibit the spontaneous pain induced by s.c.BV and intrathecal AG could enhanced the inhibitory effects of morphine. The synergistic effect was found between AG and morphine in BV test.Part III To observe the reverse effects of intrathecal agmatine on morphine tolerance. Twenty four SD rats were divided into three groups randomly as follows:①C2 Group in which the rats were intrathecal saline only(15μl);②M2 Group in which the rats were i.t. morphine(15μg/15μl)twice a day for 4 days consecutively;③AM2 group in which the rats were i.t.15μg/15μl morphine twice a day for 3 days, i.t. morphine15μg and AG 12.5μg(15μl)on the fourth day. All rats were measured thermal paw withdrawal latency(TWL)and mechanical withdrawal threshold(MWT)10min after i.t. treatment. Another 24 rats were recepted same grouped and administrated. But all rats of group C3, M3 and AM3 were s.c. BV 50μl(0.2mg)5 min after intrathecal injection of drug, and the number of spontaneous paw withdrawal reflex within 1 hour were observed and recorded. After 3 days administration consecutively, the thermal and mechanical stimulation threshold inhanced by morphine of rats of M2 group were get down, and there was no significant difference when compared with that of C2 group(P>0.05), suggested that the morphine toleranc of pain threshold has been established. AG could reverse the morphine toleranc significantly (P<0.05). There was no different between the number of flinches of M3 group(morphine group)and those of C3 group(P>0.05), suggested that the morphine toleranc of BV-induced pain has been established. Compared with C3 group and morphine tolerance group, i.t. AG and morphine could decrease the number of flinches induced by BV significantly(P<0.05), indicated that AG could reverse the morphine toleranc of inflammatory pain too.In summary, the results of this study show that AG has no influence on cellular survival and growth, even no any neurotoxicity occured at concentration lower than 40μg/ml, and could protect neurons against peroxidative damage caused by exposure to Glu.Intrathecal AG could enhanced the inhibitory effects of morphine on BV-induced spontaneous pain, and reverse the morphine toleranc of pain threshold and inflammatory pain significantly, may be able to become a new assisted drug for intraspinal opioids. |
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