Effect Of Ceftriaxone On Bee Venom-induced Downregulation Of GLT-1 And Inflammatory Pain And Hyperalgesia In Rats

Objective: Glial glutamate transporter-1(GLT-1) plays an important role in spinal nociceptive transmission, but its function differs in various pain models. In chronic pathological pain states such as chronic nerve constriction injury (CCI) model, chronic morphine resisted hyperalgesia etc., upregulating the expression and function of GLT-1 depressed the development of hyperalgesia, while inhibiting GLT-1 exacerbated it. However, in acute pain processes such as formalin test or intrathecal injection of PGE2, PGF2αand NMDA, inhibiting expression and function of GLT-1 resulted in antinociceptic effect. Is the discrepancy caused by the diverse characteristics of different pain models? Or the discrepancy indicated different role of GLT-1 in various pain models? Finding activators more selective for GLT-1 and pain model more similar to clinical pathological pain might be contributable to clarify the questions.Bee venom (BV) test is a new pain model with multi- phenotype of pain. Subcutaneous injection of BV into plantar surface of the hindpaw of rats can induce inflammatory manifestations such as edema and redness in the injected region. Following the inflammatory manifestations, monophasic and long lasted (for 1~2 h) spontaneous flinches of the injected paw, and primary mechanical and heat hyperalgesia in the injected area and secondary heat hyperalgesia in the corresponding area of contrlateral side can be induced after the BV injection. The hyperalgesia usually lasts 48 h or more. Therefore, the BV test shows characteristics more similar to clinical pathological pain and is believed to be more suitable for study in inflammatory pathological pain. Rothstein et al. reported in Nature (2005) thatβ-lactam antibiotics, such as Ceftriaxone (Cef) can greatly and selectively promote GLT-1 expression and function. The result has opened 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 influences of Cef on BV-induced spontaneous pain and hyperalgesia and changes of GLT-1 expression in the process in order to further elucidate the role of GLT-1 in the development and maintenance of inflammatory pain and hyperalgesia. The results to be obtained will provide new clue for study in prevention and treatment of pathological pain using Cef.Methods: Seventy-seven male Sprague-Dawley rats weighing 230±30g were randomly divided into the following 5 groups(n=11):1 Control group The thermal withdrawal latency and mechanical withdrawal threshold were measured;2 BV1h group The rats were injected subcutaneously with BV solution of 50μl (4g·L-1) into the plantar surface of the right hindpaw and the number of flinches during 0~55 min after the BV injection were counted;3 BV4h group The thermal withdrawal latency and mechanical withdrawal threshold at 4 h after the BV injection were measured. The others were the same as those in the BV1h group.4 BV24h group The thermal withdrawal latency and mechanical withdrawal threshold were measured at 24 h after the BV injection. The others were the same as those in the BV1h group.5 Cef group Cef was administrated (i.p.) for 7 days, once a day. On 7th day, the rats were injected with the BV solution. The number of flinches within 0~55 min and thermal withdrawal latency and mechanical withdrawal threshold at 4 h after the BV injection were measured to reflect development of spontaneous pain and hyperalgesia, respectively. Two doses of Cef in 100 mg/kg (Cef100+BV4h) and 200 mg/kg (Cef200+BV4h) was used. Equal volume of normal saline (NS) was injected as control (NS+BV4h).After the above experimental procedures were completed, rats in each group were sacrificed and the lumber 5 (L5) segment of the spinal cord were removed for assaying GLT-1 expression using immunohistochemical staining (n=6) and Western Blot analysis (n=5).Data were expressed as mean±SD ( x±s) and analyzed by One-Way ANOVE and t-test using SPSS statistical software. The confidence level was set at P<0.05.Results1 The number of spontaneous flinchesThe injection of BV produced a long lasted and monophasic nociceptive behavior responses characterized with continuously flinching and lifting/licking the injected hindpaw for more than 1 h. The maximum number of flinches could reach 80～90 times per 5 min. The number reduced to about 25 times at 55 min after the BV injection. There was no difference in the number between BV1h, NS+BV4h and Cef100+BV4h groups. While both the total number during 55 min observed and the number per 5 min at the time points of 30, 35 and 40 min in Cef200+BV4h group was increased compared with NS+BV4h group(P<0.05).2 Thermal withdrawal latencyThe latency was significantly decreased at 4 h after the BV injection in the injected site compared with the control rats (P<0.05). Simultaneously, the decrease was also found in the corresponding area of hindpaw contralateral to the injected one. And the decreases lasted to 24 h after the BV injection. These changes indicated that heat hyperalgesia was induced and maintained in a certain period. There was no difference in the latency between NS+BV4h and BV4h groups. After administration of Cef, the primary decrease in the latency in the injected site was significantly increased compared with NS+BV4h group (P<0.05) in a dose-dependent manner. But the latency did not reach the control level even in the dose of 200 mg/kg of Cef. The secondary decrease in the latency in the corresponding area of hindpaw contralateral to the injected one was also increased dose-dependently after Cef treatment (P<0.05), and it was almost approaching the control level in the dose of 200 mg/kg of Cef.3 Mechanical withdrawal thresholdThe threshold was decreased apparently in the injected site at 4 h after the BV injection and the decreases lasted to 24 h after the BV injection compared with the control rats (P<0.05). These changes showed that mechanical hyperalgesia was induced and maintained in a certain period. Mechanical hyperalgesia did not appear in the corresponding area of the uninjected site. There was no significance in the threshold between NS+BV4h group and BV4h group. After Cef treatment, the decrease of the threshold in the injected site was increased in a dose-dependently manner but the threshold did not reach the control level even in the dose of 200 mg/kg of Cef(P<0.05). The threshold in the corresponding area of hindpaw contralateral to the injected site was not affected by Cef administration.4 GLT-1 expressionImmunohistochemistry staining results showed that in control rats, brown, fine GLT-1 immunoreactive particles distributed in laminae, especially in the laminaeⅠ~Ⅱof the dorsal horn the L5 spinal segment. Scattered distribution of the particles was also found in the white matter of the same spinal segment. There was no difference in GLT-1 expression between two sides of the dorsal horn. No changes were found in the expression at 1 h after the BV injection compared to control group. The expression of GLT-1 in the dorsal horn ipsilateral to the BV injection was significantly down-regulated at 4 h and 24 h, especially at 4 h, represented with decreases in the area and IOD of GLT-1 immunoreactive staining in the dorsal horn ipsilateral to the BV injection compared with Control group. There was no difference in the expression of GLT-1 immunoreactive staining between NS+BV4h group and BV4h group. While Cef treatment in a dose-dependent manner enhanced the GLT-1 expression in the dorsal horn, especially in the superficial laminae, which was represented with increases in the area and IOD of GLT-1 immunoreactive staining compared with NS+BV4h group (P <0.05).Western blot analysis also showed the downregulation of GLT-1 expression in the L4-L5 segments of the spinal cord at 4 h and 24 h after the BV injection. After Cef treatment, GLT-1 expression was increased in a dose-dependent manner compared with NS+BV4h group (P <0.05).Conclusion: Cef inhibited downregulation of GLT-1 expression and heat and mechanical hyperalgesia induced by the BV injection, which suggested that Cef, to some extent, has an effect of anti-hyperalgesia by upregulating expression of GLT-1. However, high dose of Cef could enhance spontaneous pain behaviour induced by the BV injection.