The Effect Of Sulbactam On Chronic Neuropathic Pain And Hyperalgesia Of CCI Rats

Objective: chronic neuropathic pain, induced by the damage and dysfunction of nervous system, is a form of chronic pain which seriously affects the patients’ physical and mental health and quality of life. However, the pathogenesis of neuropathic pain is not very clear and is still lack of effective clinical treatments. Therefore, exploring the mechanism of neuropathic pain has always been a hot topic of pain research.Glutamate is the primary excitatory amino acid neurotransmitters in central nervous system. The excessive accumulation of glutamate in spinal cord participates in the transmission of nociceptive information and the development of hyperalgesia. Glial glutamate transporter 1(GLT-1), mainly distributing on the astrocytes, plays a major role in maintaining the dynamic balance of extracellular glutamate concentration via uptaking glutamate from extracellular fluid. Some studies have shown that the down-regulation of GLT-1 function or expression participated in the development and maintenance of pain and hyperalgesia in a variety of pain models, such as chronic constriction injury(CCI) of sciatic nerve model, spinal nerve ligation model and taxol induced pain model etc. Moreover, application of gene transfection method to increase the expression of GLT-1, could effectively relieve the visceral pain. Therefore, exploring the ideal GLT-1 agonists to upregulate the expression and function of GLT-1, might be a potential target for the treatment of chronic neuropathic pain.Previous studies in our lab suggest that beta-lactam antibiotics such as ceftriaxone play an anti-nociceptive role during chronic neuropathic pain via upregulating the expression and glutamate uptake of GLT-1. This finding provides a new clue for the treatment of chronic neuropathic pain. But direct application of ceftriaxone for clinical prevention and treatment of neuropathic pain still faces many problems, such as dysbacteriosis or bacteria resistant etc. It is important for the clinical application of the above research results to look for substitute, which could not only play an anti-nociceptive role like ceftriaxone but also avoid the side effects induced by ceftriaxone.Sulbactam is a kind of atypical beta-lactam antibiotics and possesses a beta-lactam ring which is similar to ceftriaxone. So it might be able to play an anti-nociceptive role like ceftriaxone. Furthermore, sulbactam alone has no antimicrobial capability, which can avoid side effects produced by long-term use of antibiotics. Therefore, the present study was undertaken to observe the effect of sulbactam on chronic neuropathic pain and hyperalgesia of CCI rats and the GLT-1 expression in spinal dorsal horn of CCI rats, in order to provide new clue and evidence for the research of prevention and treatment of neuropathic pain in clinical.Methods: One hundred and seventy healthy male Sprague-Dawley rats, weighing 280±20 g, were randomly divided into the following four groups:①CCI group(n=31): the CCI of the right sciatic nerve were performed. The thermal withdrawal latency and mechanical withdrawal threshold of the rats were continuously measured at the time points of 1 d before, and 1 d, 5 d, 9 d, 13 d, 17 d and 21 d after the CCI operation.②Sham group(n=23): rats were subjected to sham operation of right sciatic nerve, in which all procedures of CCI were performed except for the ligation of the right sciatic nerve. Other procedures were the same as those in the CCI group.③CCI+Sul prevention group(n=33): the rats were intrathecally injected with sulbactam once daily for 5 consecutive days beginning immediately after the CCI operation. Five subgroups were gained according to the doses of sulbactam, which were 150 nmol, 225 nmol, 300 nmol, 400 nmol and 800 nmol subgroups. Other procedures were the same as those in the CCI group. Meanwhile, rats with intrathecal injection of normal saline(NS) were designed as vehicle control.④CCI+Sul therapy group(n=25): the rats were intrathecally injected with sulbactam once daily for 5 consecutive days beginning from the CCI postoperative day 9. Three subgroups were gained according to the doses of sulbactam, which were 300 nmol, 400 nmol and 800 nmol subgroups. Other procedures were the same as those in the CCI group. Meanwhile, rats with intrathecal injection of NS were designed as vehicle control.The rats were sacrificed based on the time points: the rats in sham group and CCI group were sacrificed on postoperative day 5, 9, 13, 17 and 21. The rats in sulbactam prevention group(800 nmol subgroup) were sacrificed on postoperative day 5, 9 and 13. The rats in sulbactam therapy group(800 nmol subgroup) were sacrificed on postoperative day 13, 17 and 21. The lumbar 4 to lumbar 6(L4-L6) segments of right spinal dorsal horn were separated to observe the changes of GLT-1 expression using western blot.Results:1 The effect of sulbactam on the chronic neuropathic pain and hyperalgesia of CCI ratsThe thermal withdrawal latency and mechanical withdrawal threshold of sham group have no obvious change during the observed period of 21 d after the sham operation. Compare with sham group, no obvious change in the thermal withdrawal latency and mechanical withdrawal threshold was observed 1 d after CCI operation. The significant decrease of thermal withdrawal latency and mechanical withdrawal threshold was observed on postoperative day 5(P<0.05), peaked on postoperative day 9 and lasted to the observed postoperative day 21(P<0.05), which indicated that CCI induced the thermal hyperalgesia and mechanical allodynia of rats. Compare with CCI group, preventive administration of sulbactam 150 nmol, 225 nmol, 300 nmol, 400 nmol and 800 nmol did not significantly improve the thermal withdrawal latency and mechanical withdrawal threshold of CCI rats on postoperative day 5, 9, 13, 17, and 21(P>0.05). And therapeutic administration of sulbactam 300 nmol, 400 nmol and 800 nmol also did not significantly improve the thermal withdrawal latency and mechanical withdrawal threshold of CCI rats on postoperative day 13, 17, and 21 compared with CCI group(P>0.05). Intrathecal injection of NS had no significant effect on the development of thermal hyperalgesia and mechanical allodynia in CCI rats(P>0.05).2 The effect of sulbactam on the expression of GLT-1 in spinal dorsal horn of CCI rats.Western blot analysis showed that there was some basic expression of GLT-1 in L4-L6 segments of spinal dorsal horn in sham rats. Compared with sham group, CCI induced significant down-regulation in GLT-1 expression of spinal dorsal horn on postoperative day 5, 9, 13, 17 and 21, which was represented by the decrease in the ratio of the IOD of band for GLT-1 immunoblot to that of β-actin(P<0.05). Compared with CCI group, preventive administration of sulbactam 800 nmol significantly prevented the down-regulation of GLT-1 expression on postoperative days 5, which was represented by the increase in the ratio of the IOD of band for GLT-1 immunoblot to that of β-actin(P<0.05). However, on the postoperative day 9 and 13, GLT-1 expression decreased again and showed no significant difference compared with CCI group(P>0.05). The therapeutic administration of sulbactam 800 nmol didn’t reverse the down-regulation of GLT-1 expression induced by CCI on postoperative day 13, 17 and 21(P>0.05). Intrathecal injection of NS had no significant effect on the GLT-1 expression of CCI rats.Conclusions:1 Preventive administration of sulbactam increased the expression of GLT-1 on CCI postoperative day 5. But the GLT-1 expression decreased again to the CCI level on CCI postoperative day 9 and 13. Simultaneously, preventive administration of sulbactam did not significantly relieve the thermal hyperalgesia and mechanical allodynia induced by CCI.2 Therapeutic administration of sulbactam did not reverse the down-regulation of GLT-1 expression induced by CCI. Simultaneously, therapeutic administration of sulbactam did not significantly relieve the thermal hyperalgesia and mechanical allodynia induced by CCI.3 The results suggested that sulbactam had no significant anti-nociceptive effect during chronic neuropathic pain, which might be related to its failing in effective up-regulation of GLT-1 expression in spinal dorsal horn during CCI.