The Expression Of Glutamate Transporter In Spinal Dorsal Horn And Mechanism Of Botulinum Toxin A On Neuropathic Pain In Rats

Part oneThe expression of GLAST and GLT-1 of spinal dorsal horn and the effect of botulinum toxin A on neuropathic pain in ratsObjective To observe the expression of GLAST and GLT-1 in spinal dorsal horn on neuropathic pain (NP). To detect the location of GLAST and GLT-1 on astroglia and confirm the effect and safe concentration of botulinum toxin A against NP.Methods One hundred and twenty male SD rats (weigh:150 g-180 g) were randomly divided into seven groups. Two groups, which included sham (n=30) and PSNL (n=60) groups, were involved in step one. The rats in the PSNL group underwent partial sciatic nerve ligation. The surgical procedure of the rats in the sham group was identical except partial sciatic nerve ligation. For step two, there were five groups (n=6) afer PSNL established, that is BTXA5, BTXA10, BTXA20, BTXA40 and vehicle groups, what accepted single subcutaneous injection with botulinum toxin A 5U/kg,10U/kg,20U/kg and 40U/k, respectively and group vehicle just accepted saline injection. The expression of GLAST and GLT-1 in the ipsilateral dorsal horn was examined by immunofluorescence. Double immunofluorescence were applied to detect he location of GLAST and GLT-1 on astroglia. To quantify the GLAST and GLT-1 in the ipsilateral dorsal horn via western blot. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured at 1 d before PSNL (baseline) and at 1,3,5,7,14 d after PSNL.Results Comparing to the sham group, the PWT and PWL significantly decreased at 3 d after PSNL and lasted to 14 d (p?0.05). On the contrary, the PWT and PWL of sham group hadn't changed between per-operation and post-operation (P>0.05).The expression of GLAST increased on 3 d after PSNL and a small amount of expression presented until 14 d (P?0.05). The expression of GLT-1 increased on 5 d and decreased sharply on 10 d (P?0.05). There were not change of the GLAST and GLT-1 on sham group.Double immunofluorescence showed that GLT-1 but no GLAST were colocalized with astroglia in the ipsilateral spinal dorsal horn after PSNL. The PWT and PWL significantly increased and lasted to 14 d after BTXA subcutaneous injection expect BTXA40 group. There were significantly difference comparing to vehicle group (P<0.05).However, all rats were sacrificed on 2 d after administration of BTXA in BTXA40 group.Conclusion The GLT-1 locate in astroglia after PSNL. Astroglia may contribute to NP due to the GLT-1 dropped. BTXA have an great effect on NP between 5 and 20 U/kg administration. Meanwhile, BTXA10 show more stability of pain threshold compared to the other two groups, but administration of 40 U/kg BTXA is fatality.Part two Botulinum toxin A attenuated neuropathic pain by the expression up-regulation of GLT-1Objective To investigated whether BTXA regulated the expression of GLT-1 with neuropathic pain in rats.Methods Ninety male SD rats (weigh:150 g-180 g) were randomly divided into three groups (n=30) which all them underwent PSNL. Group A accepted vehicle administration. Group B applied single subcutaneous injection on ipsilateral paw with BTXA (10 U/kg), Group C accepted intraperitoneal injection with GLT-1 inhibitor base on group B, especially it would cease administration of GLT-1 inhibitor on 6d. The PWT and PWL were assessed on 1d before PSNL and 1,3,5,7,14 d after PSNL. Six rats were sacrificed in 1,3,5,7,14d after PSNL, to detect the expression of GLT-1 in the ipsilateral L5 spinal dorsal horn by immunofluorescence and qualify it with Western blot.Results Comparing to the group A, the PWT and PWL increased at 3d after PSNL (P?0.05). The PWT and PWL of group C significantly decreased at 3d after PSNL and until to 6d (P?0.05), but them gradually increased in 7d after PSNL (P>0.05) what comparing to the group B. The expression of GLT-1 ascended on 3d and reached the peak on 7d until to 14d in group B which compared to group A (P?0.05) Group B. Comparing with group B, the expression of GLT-1 in group C descended on 3d until to 6d (P?0.05) however, it increased gradually on 7d (P>0.05).Conclusion Single subcutaneous injection of BTXA can attenuate neuropathic pain and last to 14d. On the contrary, GLT-1 inhibitor reverse the effect of BTXA via intraperitoneal injection, however, the effect of BTXA present again when cease administration of GLT-1 inhibitor. BTXA raise the expression of GLT-1 against neuropathic pain.