两种微透析途径检测外周疼痛刺激大鼠脊髓谷氨酸释放水平的比较

Comparison of two microdialysis approaches in determining peripheral nociceptive input-evoked glutamate release in the spinal cord of rats

目的 比较两种脊髓微透析途径检测外周疼痛刺激大鼠脊髓谷氨酸(Glu)的释放水平。方法 28只成年雄性Wistar大鼠随机分为二组,A组(n=16)、B组(n=12),分别植入LM-3于脊髓后角或环形探针于脑脊液中。24 h后启动微透析,透析液为改良林格液,流速5μl·min-1。灌注1 h后开始每10 min收集一次Glu基础浓度样本,持续1 h,取其平均值作为Glu的基础值。然后注射5％福尔马林50μl或生理盐水于大鼠后爪,并在随后的90 min内每10 min采样一次。另将A组中8只大鼠随机分为两个亚组,进一步分析灌注液的流速及成分对Glu基础释放的影响。结果 A、B组Glu基础值分别是0.82±0.09、(5.96±0.22)μmol·L-1,A组低于B组(P<0.01)。A组当灌注速度由5μl.min-1降至2 μl·min-1时,Glu浓度增至基础值的223％±7％;而当透析液改为人造脑脊液时,Glu浓度降至基础值的62％±10％。注射福尔马林后引起Glu浓度短暂显著性增加,且两种微透析途径Glu的时间浓度分布曲线相似。结论 脊髓后角和脑脊液两种微透析途径均可有效检测脊髓Glu的释放,外周疼痛刺激可引起脊髓Glu释放短暂显著性增加。后角途径可提供神经递质释放的精确定位,而脑脊液途径则更具可重复性并便于同时在常规药物作用的研究中应用。

Objective To validate and compare the two spinal microdialysis techniques: a linear tissue probe (LM-3) in the spinal dorsal horn and a loop probe in the cerebral spinal fluid (CSF) in determining peripheral nociceptive stimulation-evoked glutamate (Glu) release in the spinal cord of freely moving rats. Methods Twenty-eight adult male Wistar rats weighing 300-350 g were randomly divided into two groups: in group A a LM-3 probe was implanted into the spinal dorsal horn and in group B a loop probe was placed in the CSF. Twenty-four hours after the implantation of the probe, microdialysis was initiated with perfusion of modified Ringer' s solution at a low flow rate of 5 μl·min-1 . Following an 1 h equilibration phase the baseline Glu concentrations were measured every 10 min for 1 h. Thereafter 50 μlof 5% formalin was injected into one hindpaw of the rats and samples were collected every 10 min for 90 min. Furthermore 8 rats in group A were further divided into 2 subgroups to investigate the effects of the flow rate of microdialysis and composition of perfusate on the baseline Glu release.Results The baseline levels of Glu were (0.82±0.09) μmol·L-1 with LM-3 probe and (5.96±0.22) μmol·L-1 with the loop probe. In group A (LM-3 probe) when the flow rate of the modified Ringer's solution was decreased from 5 to 2 μl·min-1 the extracellular Glu concentrations were increased to 223%±7% of the baseline (n = 4) , whereas perfusion with artificial CSF reduced Glu concentrations to 62% ±10% of the baseline (n = 4) . Injection of formalin into the hindpaw induced a short-lasting but significant increase in Glu concentration with a similar profile and time course using either of the two microdialysis approaches. Conclusion Microdialysis in the dorsal horn or in the CSF are both effective techniques to assess Glu release in the spinal cord of rats. Peripheral nociceptive input induces a short-lasting increase in Glu release with a similar profile and time course using either of the two microdialysis approaches. The microdialysis of the dorsal horn provides a useful tool to precisely locatewhere the release of the neurotransmitter occurs, whereas the loop probe in CSF is more reproducible for simultaneous investigation of drug effects.