Anti-nociceptive Effect Of Ceftriaxone, A Selective GLT-1 Activator, In Chronic Neuropathic Pain In Rats

Objective Glutamate is a primary excitatory amino acid neurotransmitter in the central nervous system and participates in the transmission of nociceptive information and the development of hyperalgesia in the spinal cord. Because there is not enzyme for decomposing glutamate in extracellular fluid, extracellular glutamate homeostasis is regulated by an efficient high-capacity glutamate transporter (GT) system within the the central nervous system. To date, at least five cell membrane GT proteins have been cloned: GLAST(EAAT1), GLT-1 (EAAT2), AAC1(EAAT3), EAAT4 and EAAT.Recent studies demonstrated that GLT-1 participates in the development and maintenance of pathological pain and hyperalgesia. For example, in the model of CCI(chronic constriction injury)of sciatic nerve and morphine tolerance, the GLT-1 downregulation induced the spontaneous pain and hyperalgesia. The riluzole, a positive GT activity regulator, attenuated pain and hyperalgesia, while the l-trans- pyrrolidine-2,4-dicarboxylate(PDC), a GT inhibitor, exacerbated spontaneous nociceptive behaviors and hyperalgesia. The results suggested that upregulating GLT-1 expression and activity might produce antinociceptive effects, while blocking the GLT-1 expression and activity might exacerbate spontaneous pain behaviors and hyperalgesia. Thus, regulating of GLT-1 may be a new site to therapy chronic neuropathic pain.Now, the selectivity of the well-known and used activators of GLT-1is not satisfactory, and their roles are very multiplex. New drug which can upregulate the expression and function of GLT-1 is needed for demonstrating the above presumption. Rothstein et al reports in the journal of Nature in 2005 that beta-lactam antibiotics such as ceftriaxone (Cef) could increase the expression of GLT-1 and its glutamate uptake. Based on the above, we hypothesized that the beta-lactam antibiotics may have anti-nociceptive effect in CCI pain model. Therefore, the present study was undertaken to observe effects of Cef on thermal hyperalgesia and mechanical allodynia and the expression of GLT-1 within the spinal cord after CCI of the sciatic nerve. The results to be obtained will provide new evidence for the role of GLT-1 in neuropathic pain and hyper- algesia, and new clues for the study about new pharmacologic effect of the drugs in the treatment of pathological pain.Methods Ninety male Spague-Dawley rats were divided randomly into four groups:Sham group: The right sciatic nerve was exposured, but without CCI. Thermal withdrawal latency and mechanical withdrawal threshold were measured on preoperative day 1, postoperative days 1, 3, 5, 7, 9, 11 and 14;CCI 14 d group: The CCI of the right right sciatic nerve was performed. Other procedures were identical to sham group. In addition, CCI 1 d, CCI 4 d and CCI 7 d group were designed to observe dynamic change in GLT-1 expression after the CCI;Cef prevention group: Intraperitoneal injection with Cef (200 mg/kg) once a day for 7 days began immediately after the CCI. The change of thermal withdrawal latency and mechanical withdrawal threshold were measured on preoperative day 1, postoperative day 1, 3, 5 and 7. At the same time, a group with intraperitoneal injection with NS was designed as control;Cef therapy group: Intraperitoneal injection with Cef (200 mg/kg) once a day for 7 days began from 7th day after the CCI. Other procedures were the same to Sham group. At the same time, a group with intraperitoneal injection with NS was designed as control;After completing the above-mentioned experimental procedures, the dorsal horn of spinal cord was dissected out to observe the change of the expression of GLT-1 with immunohistochemistry and Western blot.Results There was no change in the thermal withdrawal latency and mechanical withdrawal threshold in the sham rats during the observed period for 14 d after the sham operation. The CCI induced the development of thermal hyperalgesia and mechanical allodynia represented with decreases in the thermal withdrawal latency and mechanical withdrawal threshold in the hindpaw ipsilateral to the CCI. The decreases initiated on the third day(P<0.05), reached to the lowest level of 55% and 20% of normal values on postoperative days 5 and 7, and continued to the postoperative days 14 observed. Intraperitoneal injection with NS had no effect on the development of thermal hyperalgesia and mechanical allodynia. In Cef prevention group, the thermal withdrawal latency and mechanical withdrawal threshold in the hindpaw ipsilateral to the CCI were increased on postoperative days 5~7 and on day 7, respectively, compared with NS control group(P<0.05). The results suggested that the administration of Cef prevented the development of the thermal hyperalgesia and mechanical allodynia induced by the CCI. In Cef therapy group, the thermal withdrawal latency and mechanical withdrawal threshold in the hindpaw ipsilateral to the CCI were increased on postoperative days 11~14 and on day 14, respectively, compared with NS control group(P<0.05). The results suggested that the administration of Cef reversed the developed thermal hyperalgesia and mechanical allodynia induced by the CCI.Immunohistochemical staining showed that brown, fine GLT-1 immunoreactive (IR) particles distributed in all laminae of the dorsal horn in the L5 spinal segmentin rats of sham group. There was no difference between both sides of the dorsal horn. CCI induced a biphasic change in the expression of GLT-1 in the dorsal horn of the spinal cord. There was an initial increase in GLT-1 expression on postoperative days 1 and 4, which was represented with increase in the area,integral optical density (IOD) and average optical density (AOD) of GLT-1 immunoreactive staining (P<0.05). The initial upregulation was then followed by a downregulation on postoperative days 7 and 14, which was represented with decrease in the IOD, AOD and area. Intraperitoneal injection with NS had no effect on the changes of expression of GLT-1 induced by the CCI. The preventive and therapeutic administration of Cef in the same protocols as mentioned above significantly prevented and reversed the downregulation of GLT-1 expression normally induced on 7th and 14th day after the CCI, respectively, which was represented with increase in the IOD,AOD and area of GLT-1 immunoreactive staining compared with the NS control group (P<0.05).Western blotting analysis showed the expression of GLT-1 in the dorsal horn of the L4~L5 spinal segment in sham rats. Compared with sham group, CCI induced an initial upregulation in GLT-1 expression on postoperative days 1 and 4, and a followed downregulation on the postoperative days 7 and 14 in the spinal dorsal horn. Intraperitoneal injection with NS had no effect on the expression of GLT-1 induced by the CCI. The preventive and therapeutic administration of Cef in the same protocols as mentioned above significantly prevented and reversed the downregulation of GLT-1 expression normally induced on 7th and 14th day after the CCI, respectively, which was represented with the increase in the ratio of the IOD of band for GLT-1 immunoblot to that ofβ-actin(P<0.05).Conclusion CCI induced thermal hyperalgesia and mecha- nical allodynia and downregulation in GLT-1 expression. Cef, the selective promoter for GLT-1 expression and function, reduced the development of the hyperpathia and allodynia, prevented and reversed the downregulation of GLT-1. The results suggested that Cef could prevent and therapy the chronic neuropathic pain through upregulating the expression and function of GLT-1.