| ObjectiveGlial cells express little or only a small amount of COX-2 in the resting state, but the expression of microglia COX-2 m RNA and protein increased rapidly while microglia are activated to morphological changes, releasing tumor necrosis factor(TNF-α), interleukin-1, interleukin-6 and NO when stimulated by cytokines, LPS and other proinflammatory substances. The expression of COX-1 essentially remains stable in astrocytes after nerve injury, while the expression of COX-2 increases. These suggest that COX-2 and glial cells may play important roles in the nervous inflammatory response system. But there is no clear relationship between the activation of spinal glial cells and the development of Neuropathic pain. So, this study used high spinal nerve ligation rat model to investigate the relationship between the expression of microglia and astrocytes with a COX-2 inhibitor tepoxalin. We used behavioral methods and immunofluorescence staining technique for the experiment.MethodsExperiment 1: Manufacture SNL neuropathic pain models and measures the mechanical paw withdrawal threshold of rats after spinal nerve ligation20 male SD rats were randomly divided into two groups, sham group and the surgery group. SNL neuropathic pain model were made according the following procedure, first, anesthetized the rats with 10% chloral hydrate(300mg/Kg, ip). Second, slited the waist iliac crest and exposed L5 spinal processus transverses then ligated the L5 spinal nerve with the 6th silk tightly. Finally, prinkled penicillin to prevent infection followed by stitching muscle and skin. Mechanical paw withdrawal threshold were determined with von Frey Filament everyday.Experiment 2: Changes of mechanical withdrawal threshold after a single oral administration of tepoxalin in rats40 male rats were randomly divided into five groups after L5 spinal nerve ligation. The groups were as follow:(1) CMC group: 0.5% sodium carboxymethylcellulose(20ml/kg), as the negative control group;(2) diclofenac sodium Group: 2.58mg/kg of diclofenac sodium, as the positive control group;(3) tepoxalin low, medium and high dose groups(0.78mg/kg, 1.56mg/kg and 3.12mg/kg), as drug groups. According to the most sensitive time point of mechanical pain(day 4) in the experiment 1, 1h after oral administration on day 4, we measured mechanical withdrawal threshold with von Frey Filament fiber, the results were expressed as 50% mechanical paw withdrawal threshold.Experiment 3: The effect of tepoxalin on the mechanical withdrawal threshold and the activation of the glial cells of early neuropathic rats18 male rats were randomly divided into three groups after L5 spinal nerve ligation. The groups were as follows:(1) CMC Group: 0.5% sodium carboxymethylcellulose(20ml/kg), as the negative control group;(2) diclofenac group: 2.58mg/kg of diclofenac, as the positive control group;(3) tepoxalin group: 3.12mg/kg tepoxalin. All groups were administered orally daily from 1h before SNL to day 7 postoperation, then measured the mechanical withdrawal threshold 1h after oral administration from day 3 to day 7 and detected the activation of spinal microglia and astrocyte by immunofluorescence.Experiment 4: The effect of tepoxalin on mechanical withdrawal threshold and the activation of the glial cells of late neuropathic rats18 male rats were randomly divided into three groups after L5 spinal nerve ligation. The groups were the same as the experiment 3. All groups were administered orally daily from day 15 to day 20 postoperation. Mechanical allodynia threshold were measured on day 15, 17, 20. The activation of microglia and astrocyte on day 20 was detected by immunofluorescence.Results1. Mechanical withdrawal threshold after spinal nerve ligation over timeThe mechanical paw withdrawal threshold(PWT) reduced significantly after spinal nerve ligation in rats, the PWT of ipsilateral side remaining below 5g from day 3 to day 15. Mechanical paw withdrawal threshold was significantly decreased(P<0.01), compared with sham-operated and contralateral side. The PWT of ipsilateral side from day 16 to day 21 was also significantly decreased(P <0.05).2. Mechanical paw withdrawal threshold after a single oral adminisration of tepoxalinTepoxalin significantly improved mechanical paw withdrawal threshold in rats. The mechanical paw withdrawal threshold of CMC group, diclofenac group and tepoxalin 0.78,1.56,3.12mg/kg groups were 4.07 ± 1.69 g, 4.01 ± 0.26 g, 3.91 ± 0.77 g, 3.97±1.2g, 4.15±1.06 g respectively before the administration,and 4.25±1.05 g, 10.85 ±1.86 g, 6.2±0.77 g, 8.08±1.47 g, 11.15±1.02 g respectively 1 hour after the administration.The PWT of tepoxalin high-dose group and diclofenac group increase significantly(P <0.01), compared with the CMC control group; the PWT of tepoxalin medium group increase significantly(P <0.05), compared with the CMC control group; the PWT of tepoxalin high-dose group and the medium group increase(respectively for P <0.05 and P <0.01), compared with the tepoxalin low-dose group.3. The effects of pre-administration and continuous oral administration of tepoxalin for 7 days on mechanical withdrawal threshold and the activation of OX-42 and GFAPContinuous oral administration of tepoxalin inhibited the development of mechanical withdrawal threshold in SNL rats. The mechanical PWT of the CMC control group, diclofenac group and tepoxalin(3.12mg/kg) group were 3.12±1.76 g, 10.85 ± 1.52 g, 10.42 ± 2.47 g on day 3 after administration; 3.66 ± 0.26 g,11.63 ± 0.66 g,12.87 ± 0.51 g on day 5 after administration; and 3.33 ± 0.73 g,12.67 ± 0.29 g,12.74 ± 0.65 g on day 7 after administration. From the statistics, we found oral administration of tepoxalin and diclofenac can effectively suppress pain threshold from day 3 to day 7(P<0.01).Immunofluorescence staining showed that SNL induced the expression of spinal microglia and astrocyte, and continuous oral administration of tepoxalin could inhibit the activation of microglia and astrocyte. The immunofluorescence staining intensity of the optical density(IOD) of OX-42 was significantly lower on day 3 and day 7(P<0.01 and P<0.05 respectively), compared with the CMC control group; the IODof GFAP was lower(P<0.05) on day 3 and day 7, compared with the CMC control group.4. The effects of continuous oral administration of tepoxalin from day 15 to day 20 after SNL on mechanical withdrawal threshold and the activation of microglia and astrocyteContinuous oral administration of tepoxalin from day 15 after spinal nerve ligation could inhibit the mechanical withdrawal threshold latency, but the activation of glial cells in the spinal cord was not inhibited. The mechanical PWT of the CMC control group, diclofenac group and tepoxalin(3.12mg/kg) group were 5.57 ± 1.65 g,11.4 ± 1.77 g,10.06 ± 1.5g on day 15 after administration, 6.91 ± 1.82 g,10.9 ± 1.9g,11.38 ± 0.96 g on day 17 after administration; and 7.9 ± 0.7g,11.66 ± 0.61 g,11.84 ± 0.84 g on day 20 after administration. The immunofluorescence staining on day 20 after SNL showed that the expression of OX-42 and GFAP did not changed compared with the CMC control group.Conclusions1. Selective COX-2 inhibitors tepoxalin dose-dependently inhibits mechanical pain induced by SNL in rats;2. Spinal COX-2 and glial activation are involved in early stage of neuropathic pain. |
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