Ceftriaxone Plays An Anti-nociceptive Role Through Up-regulating Spinal GLT-1 Expression And Function In Chronic Neuropathic Pain Model In Rats

Glutamate is a primary type of excitatory amino acid neurotransmitters in the central nervous system and participates in the transmission of nociceptive information and the development of hyperalgesia in the spinal cord by activating various pre- or post- synaptic glutamate receptors. Because there is no enzyme to decompose the glutamate in extracellular fluid, it is a high-affinity, sodium-dependent, efficient high-capacity glutamate transporter system within the central nervous system that regulated the extracellular glutamate homeostasis. To date, five types of cell membrane glutamate transporters have been cloned, which include EAAT1 (GLAST), EAAT2 (glial glutamate transpor-1, GLT-1), EAAT3 (EAAC1), EAAT4 and EAAT5. Although each of them exhibits a distinct distribution and function, GLT-1, distributing primarily in astrocytes, may play a predominant role in the clearance of glutamate in the synaptic cleft and the termination of synaptic transmission. It has been shown that GLT-1 implicated in the development and maintenance of pathological pain. For example, the development of the hyperalgesia was accompanied by the down-regulation of GLT-1 expression and/or the decrease of its glutamate uptake in spinal dorsal horn in chronic constriction injury of sciatic nerve and morphine tolerance models. Administration of glutamate transporters activator riluzole could alleviate the hyperalgesia induced by CCI, while administration of glutamate transporters inhibitors TBOA (DL-threo-beta-benzyloxyaspartate) could induce hyperalgesia in normal rats. These results indicated that the inhibition of the expression and the uptake function of GLT-1 might exacerbate the hyperalgesia, while the activation of GLT-1 might have anti-nociceptive effect. Thus, regulating the expression and glutamate uptake of GLT-1 protein might be a new target for the management of neuropathic pain in clinic. However, more specific, but less-side-effect GLT-1 activator that promotes the expression and/or glutamate uptake of GLT-1 is primarily necessary for the regulation.Rothstein et al. reported in Nature (2005) thatβ-lactam antibiotics, such as Ceftriaxone (Cef) can greatly and selectively promote GLT-1 expression and its uptake function. The property of Cef showed some benefits to some pathological processes induced by excitotoxic effect of glutamate such as amyotrophic lateral sclerosis, stroke, brain tumors and epilepsy, etc. The findings have opened up a new vista for using Cef as a specific activator of GLT-1 in the study of pain.Therefore, the present study was undertaken to observe anti-nociceptive effects of Cef on chronic neuropathic pain model induced by chronic constrictive nerve injury (CCI) of sciatic nerve, and to identify the role of changes in the spinal GLT-1 expression and its uptake of glutamate in the process in rats. The results obtained would shed new light on the prevention and therapy of neuropathic pain using Cef in clinic and provide evidence for the elucidation of the role that GLT-1 plays in the neuropathic pain.1 Intraperitoneal injection of Cef antagonizes the neuropathic pain and up-regulates GLT-1 expression in the spinal dorsal horn of CCI ratsThe effects of intraperitoneal administration of Cef at different time point on the neuropathic pain behaviors and GLT-1 expression in spinal dorsal horn of CCI rats were observed in order to provide evidence to elucidate the anti-nociceptive effect of Cef may result from regulating spinal GLT-1.Method: One hundred and thirty male Sprague-Dawley rats were randomly divided into 5 groups:①Sham group (n=25): First, 5 rats were subjected to sham operation of CCI of right sciatic nerve, in which all procedures of CCI were performed except for the ligation of the right sciatic nerve. The thermal withdrawal latency and mechanical withdrawal threshold of the rats were continuously measured at time points of 1d before, and 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d, 19d and 21d after the sham operation. After the last time of the test, the spinal GLT-1 expression of the rats was observed with Western blotting analysis. Furthermore, in order to provide control for dynamic change of GLT-1 expression during the process in CCI or other groups, another 20 rats subjected to the sham treatments were used to observe spinal GLT-1 expression on another 2 time points of postoperative day 7 and 15 (10 rats were included in each time point, and 5 of them were used for Western blotting analysis and 5 for immunohistochemisty) besides the postoperative day 21.②CCI group (n=25): The CCI of the right sciatic nerve was performed. Other procedures and animal assignment were the same as those in the sham group.③Cef group(n=5): The naive rats were intraperitoneally injected with Cef (200 mg/kg) once daily for 7 days, and the thermal withdrawal latency and mechanical withdrawal threshold were continuously measured at time points of 1d before, and 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d, 19d and 21d after the Cef administration. Meanwhile, a group, consisted of another 5 rats with intraperitoneal injection of normal saline (NS) by the same means was designed as vehicle control.④CCI+i.p.Cef prevention group (n=20): The rats subjected to CCI were intraperitoneally injected with Cef (200 mg/kg) once daily for 7 postoperative days. The first time of the injection was performed immediately after the CCI operation. Other procedures of treatments were the same as those in the sham group, except for the spinal GLT-1 expression on CCI postoperative day 15 (n=5) was assayed with Western blotting analysis only. Meanwhile, a group, consisted of another 20 rats assigned in the same way as those in above, with intraperitoneal injection of NS by the same means was designed as vehicle control.⑤CCI+i.p.Cef therapy group (n=15): The rats subjected to CCI were intraperitoneally injected with Cef (200 mg/kg) once daily for 7 postoperative days. The first time of the injection was performed on the postoperative day 9. Other procedures of treatments were the same as those in the sham group, except for the observation of spinal GLT-1 expression, which was only performed on CCI postoperative day 15 and 21. Meanwhile, a group, consisted of another 15 rats assigned in the same way as those in above, with intraperitoneal injection of NS was designed as vehicle control.Results: Cef alone did not change the baseline responses of rats to thermal and mechanical stimulation. No change in the thermal withdrawal latency and mechanical withdrawal threshold was observed in the sham rats during the observed period of 21 d after the sham operation. Thermal hyperalgesia and mechanical allodynia were induced after CCI which were represented by the decrease of thermal withdrawal latency and mechanical withdrawal threshold. The decrease of the latency and threshold was initiated on CCI postoperative day 3 (P<0.05), peaked on postoperative day 7 and continued to postoperative day 21 observed in the hind paw ipsilateral to CCI. Compared with CCI group, intraperitoneal preventive administration of Cef significantly increased the thermal withdrawal latency (from postoperative day 3 to day 21) and the mechanical withdrawal threshold (from postoperative day 5 to day 21) (P<0.05). Alternatively, intraperitoneal therapeutic administration of Cef significantly reversed the CCI-induced decreases in thermal withdrawal latency (from postoperative day 11 to day 21) and the mechanical withdrawal threshold (from postoperative day 15 to day 21) (P<0.05). Neither preventive nor therapeutic administration of NS (vehicle of Cef) had significant effect on the thermal withdrawal latency and mechanical withdrawal threshold in CCI rats. The results indicated that intraperitoneal administration of Cef had anti-nociceptive effect on CCI rats. However, the effectiveness of the preventive and therapeutic effects of Cef on mechanical allodynia was less than those on thermal hyperalgesia.Immunohistochemical staining showed that brown, fine GLT-1 immunoreactive particles distributed in all laminae of the dorsal horn in the L5 spinal segment of rats in sham group. CCI induced a significant decrease of GLT-1 immunoparticles in the spinal dorsal horn ipsilateral to CCI on postoperative day 7 and 15. Western blotting analysis showed that there was basal expression of GLT-1 in sham group. Compared with sham group, the GLT-1 protein was significantly down-regulated on CCI postoperative day 7, 15 and 21 (P<0.05). Compared with CCI group, intraperitoneal administration of NS had no significant effect on GLT-1 expression in CCI rats, but the Cef significantly increased the GLT-1 immunoparticles in the superficial laminae, and up-regulated GLT-1 protein expression (western blot) of the spinal dorsal horn on postoperative day 7, 15 and 21 (P<0.05). Alternatively, intraperitoneal therapeutic administration of Cef showed similar up-regulating effects on GLT-1 immunoparticles and protein expression in the superficial laminae of the spinal dorsal horn ipsilateral to CCI on postoperative day 15 and 21 (P<0.05). The up-regulating effect of Cef on GLT-1 expression in CCI rats was coincident in time course with its anti-nociceptive effect.The above results suggested that Cef had anti-nociceptive effect on CCI rats and the anti-nociceptive effect may result from its up-regulating effect on spinal GLT-1 expression.2 Intrathecal administration of Cef shows similar anti-neuropathic pain effect on CCI rats to that of intraperitoneal injection of CefThe effect of intrathecal administration of Cef at different time point on the neuropathic pain induced by CCI was observed in order to certify that Cef plays an anti-nociceptive role in CCI induced neuropathic pain through modulating spinal GLT-1.Method: Sixty male Sprague-Dawley rats were randomly divided into 7 groups (n=5 in each group):①Sham group: Rats were subjected to sham operation of CCI in which all procedures of CCI were performed except for the ligation of the right sciatic nerve. The thermal withdrawal latency and mechanical withdrawal threshold were continuously measured at time points of 1d before, and 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d, 19d and 21d after the sham operation.②CCI group: The CCI of the right sciatic nerve was performed. Other procedures were the same as those in the sham group. ③Cef group: The naive rats were intrathecally injected with Cef (502 nmol/kg) once daily for 5 days, and the thermal withdrawal latency and mechanical withdrawal threshold were continuously measured at time points of 1d before, and 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d, 19d and 21d after the Cef administration. Meanwhile, a group, consisted of another 5 rats with intrathecal injection of NS by the same means was designed as vehicle control.④CCI+i.t.Cef prevention group: The rats subjected to CCI were intrathecally injected with Cef (502 nmol/kg) once daily for 5 postoperative days beginning immediately after the CCI operation, when thermal and mechanical hyperalgesia had not been induced. Other procedures were the same as those in the sham group. Meanwhile, a group, consisted of another 5 rats with intrathecal injection of NS by the same means was designed as vehicle control.⑤CCI+i.t.Cef therapy group: The rats were treated identically with those in CCI+i.t.Cef prevention group, except for the beginning of administration of Cef was on the postoperative day 9, when thermal and mechanical hyperalgesia had already been induced. Other procedures were the same as those in the sham group. Meanwhile, a group, consisted of another 5 rats with intrathecal injection of NS by the same means was designed as vehicle control.⑥As-ODNs+CCI+i.t.Cef prevention group: Besides CCI and intrathecal administration of Cef in the same way as that in CCI+ i.t.Cef prevention group, the rats were intrathecally injected with GLT-1 antisense oligodeoxy- nucleotides (As-ODNs) in a dose of 18 nmol once daily on the CCI postoperative day 1.5 and 3.5. Other procedures were the same as those in sham group. Meanwhile, a group consisted of another 5 rats with intrathecal injection of distilled water (DW, vehicle of GLT-1 As-ODNs) was designed as vehicle control.⑦As-ODNs+CCI+i.t.Cef therapy group: The rats were treated identically with those in As-ODNs+CCI+i.t.Cef prevention group, except for GLT-1 As-ODNs was administered on the CCI postoperative day 10.5 and 12.5. Other procedures were the same as those in sham group. Meanwhile, a group consisted of another 5 rats with intrathecal injection of DW (vehicle of GLT-1 As-ODNs) was designed as vehicle control.Results: Cef alone did not change the baseline responses to thermal and mechanical stimulation. Compared with CCI group, intrathecal preventive administration of Cef (CCI+i.t.Cef prevention group) significantly increased the thermal withdrawal latency from postoperative day 3 to day 21 observed in the hind paw ipsilateral to CCI (P<0.05). Alternatively, intrathecal therapeutic administration of Cef significantly reversed the CCI-induced decreases in the latency from postoperative day 11 to day 21 (P<0.05). The mechanical withdrawal threshold was also significantly increased by intrathecal preventive or therapeutic administration of Cef (P<0.05), though the effectiveness of the preventive and therapeutic effects of Cef on mechanical allodynia was less than those of Cef on thermal hyperalgesia. Neither intrathecal preventive nor therapeutic administration of NS had significant effect on the development of the thermal hyperalgesia and mechanical allodynia. Intrathecal administration of GLT-1 As-ODNs significantly inhibited the preventive and therapeutic effects of Cef on neuropathic pain induced by CCI, which was represented by the decreases in thermal withdrawal latency and mechanical withdrawal threshold appeared again (P<0.05). Intrathecal administration of DW had no significant effect on the pain behaviors. Meanwhile, Western blotting analysis showed that the intrathecal administered GLT-1 As-ODNs really significantly inhibited the up-regulation of GLT-1 expression in spinal dorsal horn induced by Cef in CCI rats (see the results in the third part).The above results indicated that intrathecal administration of Cef had anti-nociceptive effect on CCI rats.3 Intrathecal administration of Cef significantly up-regulates the GLT-1 expression in spinal dorsal horn of CCI ratsThe effect of intrathecal administration of Cef at different time point on the GLT-1 expression in spinal dorsal horn of CCI rats was observed in order to explore the possible mechanism of the anti-nociceptive effect of Cef on CCI rats.Method: One hundred and ninety-five male Sprague-Dawley rats were randomly divided into 6 groups:①Sham group (n=35): Rats were subjected to sham operation of CCI and were sacrificed on postoperative day 5, 7, 13, 15 and 21 to observe the GLT-1 expression in L4-L6 segments of spinal dorsal horn. Ten rats were included in each group of day 7 and 15, and 5 of them were used for Western blotting analysis and 5 for immunohistochemisty. Five rats were included in each group of day 5, 13 and 21 and used for Western blotting analysis only.②CCI group (n=30): The CCI of the right sciatic nerve was performed. Other procedures were the same as those in the sham group, except for the observation of the spinal GLT-1 expression, which was only performed on CCI postoperative day 7, 15 and 21. In addition, GLT-1 expression in the cupular part of postcentral cortex in the brain of the rats was observed on postoperative day 5 (another 5 rats) and 7 (used the same 5 rats that had been used for spinal GLT-1 expression analysis) with Western blotting analysis as a control for the effect of intrathecal administration of Cef on spinal GLT-1 expression.③CCI+i.t.Cef prevention group (n=25): The rats subjected to CCI were intrathecally injected with Cef (502 nmol/kg) once daily for 5 postoperative days beginning immediately after the CCI operation when thermal and mechanical hyperalgesia had not been induced. Other procedures were the same as those in the sham group, except for the observation of the spinal GLT-1 expression, which was only performed on CCI postoperative day 5, 7, 15 and 21, and 5 rats were included in group of day 15 for Western blotting analysis only. In addition, GLT-1 expression in the cupular part of postcentral cortex in the brain of the rats was also observed on postoperative day 5 and 7 with Western blotting analysis. Meanwhile, a group, consisted of another 25 rats assigned in the same way as those in above, with intrathecal injection of NS was designed as vehicle control.④CCI+i.t.Cef therapy group (n=20): The rats were treated identically with those in CCI+i.t.Cef prevention group, except for the beginning of administration of Cef was on the postoperative day 9, when thermal and mechanical hyperalgesia had already been induced. Other procedures were the same as those in the sham group, except for the observation of the spinal GLT-1 expression, which was only performed on CCI postoperative day 13, 15 and 21. Meanwhile, a group, consisted of another 20 rats assigned in the same way as those in above, with intrathecal injection of NS was designed as vehicle control.⑤As-ODNs+CCI+i.t.Cef prevention group (n=10): Besides CCI and intrathecal administration of Cef in the same way as that in CCI+ i.t.Cef prevention group, the rats were intrathecally injected with GLT-1 As-ODNs in a dose of 18 nmol once daily on the CCI postoperative day 1.5 and 3.5. Other procedures were the same as those in the sham group, except for the observation of the spinal GLT-1 expression was only performed on CCI postoperative day 5 and 7 (n=5 in each time points) with Western blotting analysis. Meanwhile, a group consisted of another 10 rats with intrathecal injection of DW (vehicle of GLT-1 As-ODNs) was designed as vehicle control.⑥As-ODNs+CCI+i.t.Cef therapy group (n=10): The rats were treated identically with those in As-ODNs+CCI+i.t.Cef prevention group, except for the administration of AS-ODNs was performed on the CCI postoperative day 10.5 and 12.5, and the observation of GLT-1 expression was performed on CCI postoperative day 13 and 15.Results: Intrathecal preventive administration of Cef significantly increased GLT-1 immunoparticles on postoperative day 7 and up-regulated expression of GLT-1 protein (western blot) on postoperative day 7, 15 and 21 in the superficial laminae of the spinal dorsal horn ipsilateral to CCI (P<0.05). Alternatively, intrathecal therapeutic administration of Cef significantly increased GLT-1 immunoparticles on postoperative day 15 and up-regulated GLT-1 expression (western blot) on postoperative day 15 and 21 in the superficial laminae of the spinal dorsal horn ipsilateral to CCI (P<0.05), while intrathecal administration of NS had no significant effect on spinal GLT-1 expression in CCI rats. In addition, Western blotting analysis showed that there was no significant difference in the expression of GLT-1 in cupular part of postcentral cortex of the rats brain (an area outside the spinal cord) among CCI, CCI +i.t. NS, or CCI + i.t. Cef groups. The results indicated that intrathecal administration of Cef specifically up-regulated the expression of GLT-1 in the spinal cord of CCI rats.Compared with CCI+i.t.Cef group, either preventive (As-ODNs+CCI+ i.t.Cef prevention) or therapeutic (As-ODNs+CCI+i.t.Cef therapy group) administration of GLT-1 As-ODNs in a dose of 18 nmol significantly inhibited the up-regulation of GLT-1 expression in spinal dorsal horn induced by Cef in CCI rats (P<0.05). While intrathecal administration of DW had no significant effect on the spinal GLT-1 expression of Cef treated CCI rats. The results indicated that the GLT-1 As-ODNs was indeed inhibited the GLT-1 expression. Furthermore, accompanied with the inhibition of the spinal GLT-1 up-regulation in the Cef treated CCI rats, intrathecal administration of GLT-1 As-ODNs significantly inhibited the preventive and therapeutic effects of Cef on neuropathic pain induced by CCI (see the results in the second part). The inhibiting effect of GLT-1 As-ODNs on the pain behaviors was consistent temporally with its inhibiting effect on GLT-1 expression.The above results suggested that it was the up-regulating effect of Cef on spinal GLT-1 that contributed to the anti-nociceptive effect of the drug on CCI rats.4 Intrathecal administration of Cef increases the glutamate uptake of GLT-1 in spinal dorsal horn of CCI ratsThe effect of intrathecal administration of Cef at different time point on the [3H]-glutamate uptake of GLT-1 in spinal dorsal horn of CCI rats was observed in order to explore the effect of Cef on the function of GLT-1 in spinal dorsal horn and to provide further evidence to certify that it was the regulating effect of Cef on spinal GLT-1 that contributed to the anti-nociceptive effect of the drug on CCI rats.Method: Sixty male Sprague-Dawley rats were randomly divided into 6 groups:①Sham group (n=10): Rats were subjected to sham operation of CCI and were sacrificed on postoperative day 5 (n=5) and day 14 (n=5).②CCI group (n=10): The CCI of the right sciatic nerve was performed. Other procedures were the same as those in the sham group.③CCI+i.t.Cef prevention group (n=5): The rats subjected to CCI were intrathecally injected with Cef (502 nmol/kg) once daily for 5 postoperative days beginning immediately after the CCI operation and the rats were sacrificed on postoperative day 5. Meanwhile, a group, consisted of another 5 rats with intrathecal injection of NS was designed as vehicle control.④CCI+i.t.Cef therapy group (n=5): The rats were treated identically with those in CCI+i.t.Cef prevention group, except for the beginning of Cef administration was on the postoperative day 9 and the rats were sacrificed on postoperative day 14. Meanwhile, a group, consisted of another 5 rats with intrathecal injection of NS was designed as vehicle control.⑤As-ODNs+CCI+i.t.Cef prevention group (n=5): Besides CCI and intrathecal administration of Cef in the same way as CCI+ i.t.Cef prevention group, the rats were intrathecally injected with GLT-1 As-ODNs in a dose of 18 nmol once daily on the CCI postoperative day 1.5 and 3.5. Other procedures were the same as those in CCI+i.t.Cef prevention group. Meanwhile, a group consisted of another 5 rats with intrathecal injection of DW rather than GLT-1 As-ODNs was designed as vehicle control.⑥As-ODNs+CCI+i.t.Cef therapy group (n=5): The rats were treated identically with those in As-ODNs+CCI+i.t.Cef prevention group, except for the administration of GLT-1 As-ODNs was performed on the CCI postoperative day 10.5 and 12.5, and the rats were sacrificed on postoperative day 14. Meanwhile, a group consisted of another 5 rats with intrathecal injection of DW rather than GLT-1 As-ODNs was designed as vehicle control. Rats were sacrificed at each corresponding time point and L4-L6 segments of spinal dorsal horn were separated. [3H]-glutamate was used to observe the glutamate uptake of GLT-1 in spinal dorsal horn.Results: Compared with sham group, glutamate uptake of spinal dorsal horn was significantly decreased in the CCI group (P<0.05). Compared with CCI group, either intrathecal preventive (CCI+i.t.Cef prevention group) or therapeutic (CCI+i.t.Cef therapy group) administration of Cef significantly increased the glutamate uptake of spinal dorsal horn (P<0.05), while intrathecal administration of NS had no significant effect on spinal glutamate uptake. Compared with CCI+i.t.Cef prevention group and CCI+i.t.Cef therapy group, the glutamate uptake of spinal dorsal horn was significantly decreased in As-ODNs+CCI+i.t.Cef prevention group and As-ODNs+CCI+i.t.Cef therapy group, respectively (P<0.05), while intrathecal administration of DW had no significant effect on spinal glutamate uptake.The above results indicated that intrathecal administration of Cef could increase the glutamate uptake of GLT-1 in spinal dorsal horn of CCI rats.Conclusions(1) Intraperitoneal administration of Cef alleviates the neuropathic pain induced by CCI and up-regulates the GLT-1 expression in spinal dorsal horn of CCI rats, which suggests that the anti-nociceptive effect of Cef on CCI rats may result from its up-regulating effect on spinal GLT-1.(2) Intrathecal administration of Cef shows similar effects of antagonizing nociception and up-regulating the spinal GLT-1 expression to those of intraperitoneal administration of Cef on CCI rats, and can also increase the glutamate uptake of GLT-1 in spinal dorsal horn of CCI rats. Furthermore, the above effects of intrathecal administration of Cef could be inhibited with intrathecal administration of GLT-1 As-ODNs.(3) It could be concluded from the above results that Cef plays an anti-nociceptive role through up-regulating spinal GLT-1 expression and function in chronic neuropathic pain model in rats. The results obtained would provide potent evidence for using Cef as a potential pharmacological tool for pain management in clinic, as the drug has been extensively used in clinic as antibiotic in the therapy of infectious diseases.