Dynamic regulation of spinal pro-inflammatory cytokine release in the rat in vivo following peripheral nerve injury.

Spinal release of cytokines may play a critical role in the maladapted nociceptive signaling underlying chronic pain states. In order to investigate this biology, we have developed a novel 'high flux' intrathecal microdialysis approach in combination with multiplex bead-based immunoassay technology to concurrently monitor the spinal release of interleukin (IL)-1beta, IL-6 and tumour necrosis factor (TNF)alpha in rats with unilateral sciatic nerve chronic constriction injury (CCI). Intrathecal microdialysis was performed under isoflurane/N(2)O anaesthesia in rats with confirmed mechanical hypersensitivity. In a first study, C-fiber strength electrical stimulation of the operated nerve in neuropathic rats was found to evoke a dramatic increase in IL-1beta efflux ( approximately 15-fold) that was significantly greater than that observed in the sham-operated group. Spinal IL-6 efflux was also responsive to primary afferent stimulation, whereas TNFalpha was not. In a second study, treatment with the glial inhibitor propentofylline for 7days normalized CCI-induced mechanical hypersensitivity. In the same animals, this treatment also significantly reduced intrathecal IL-1beta, IL-6 and TNFalpha and prevented afferent stimulation-evoked cytokine release of both IL-1beta and IL-6. These results provide support for glia as the source of the majority of intrathecal IL-1beta, IL-6 and TNFalpha that accompanies mechanical hypersensitivity in the CCI rat. Moreover, our studies demonstrate the ability of a neurone-glia signaling mechanism to dynamically modulate this release and support a role of spinal IL-1beta in the phasic transmission of abnormal pain signals.

GABA transporter type 1 (GAT-1) uptake inhibition reduces stimulated aspartate and glutamate release in the dorsal spinal cord in vivo via different GABAergic mechanisms.

Mechanisms through which the reported antinociceptive activity of GABA re-uptake inhibitors is mediated (and where on the sensory neuraxis) have not been defined. Here, microdialysis in the anaesthetised rat was used to examine the effect of selective GABA transporter type 1 (GAT-1) inhibition on basal and evoked amino acid release in the dorsal spinal cord. Reverse dialysis of the selective GAT-1 inhibitor NO-711 (10-300microM) induced a concentration-related increase in extracellular GABA (maximal approximately threefold of basal levels) without affecting other amino acids. Employing an S2/S1 paradigm, release evoked by brief high (45mM) K(+)-induced depolarisation of aspartate and glutamate, but not GABA or glycine, was found to be significantly reduced by reverse dialysis of NO-711 (300microM). Co-administration of selective antagonists for GABA(A) or GABA(B) receptors ((+)-bicuculline (100microM) or SCH 50911 (100microM), respectively) prevented the GAT-1 inhibition-induced reduction of evoked aspartate. In contrast, while (+)-bicuculline also antagonised the reduction of evoked glutamate, SCH 50911 (up to 1mM) was without effect. Inhibition of GAT-1 re-uptake was further found to play a permissive role in autoinhibitory effects on GABA release mediated through GABA(A) and GABA(B) receptors. These data demonstrate that augmentation of GABAergic neurotransmission by re-uptake inhibition activates pharmacologically distinguishable inhibitory influences on aspartate and glutamate release in the dorsal spinal cord. Thus, inhibition of spinal pro-nociceptive neurotransmitter release may contribute to the analgesic action of this drug class.

Monolithic column-based reversed-phase liquid chromatography separation for amino acid assay in microdialysates and cerebral spinal fluid.

The development of a HPLC method using a monolithic C18 column is described using fluorescence detection for the assay of 21 amino acids and related substances with derivatisation using ortho-phthaldialdehyde (OPA) in the presence of 3-mercaptopropionic acid (3-MPA). The method employs a tertiary gradient and has a run time of 24 min. Linearity (r2) for each amino acid was found to be greater than 0.99 up to a 10 microM concentration; reproducibility across all analyses (relative standard deviation (R.S.D.)) was between 0.97 and 6.7% and limit of detection (LOD) between 30 and 300 fmol on column. This method has been applied to the analysis of amino acids in both spinal microdialysis and cerebral spinal fluid samples.

Selective serotonin re-uptake inhibition attenuates evoked glutamate release in the dorsal horn of the anaesthetised rat in vivo.

Augmentation of serotonergic neurotransmission at the level of the dorsal spinal cord is proposed to contribute to the analgesic activity of selective serotonin (5-HT) reuptake inhibitors (SSRIs). In this study we have utilised microdialysis perfusion to determine the effect of two structurally unrelated SSRIs on depolarisation-induced aspartate and glutamate release in the dorsal spinal cord of the anaesthetised rat. Perfusion with artificial extracellular fluid containing 45 mM potassium produced a significant increase in aspartate and glutamate efflux. Sensitivity, at least in part, to antagonism of calcium entry by high extracellular Mg2+ indicated a neuronal origin for a proportion of stimulated release. Reverse dialysis of paroxetine (1-30 microM) reduced the increase in glutamate in a concentration dependent manner, with a significant reduction evident following inclusion in the perfusate of 30 microM. Administration of an equi-potent dose of citalopram (300 micoM) also reduced depolarisation induced glutamate release. Aspartate levels tended to decrease in the presence of paroxetine and citalopram, but this trend did not reach significance. Co-perfusion of paroxetine (30 microM) with the selective 5-HT(1A) receptor antagonist WAY 100635 (100 microM) did not prevent the reduction in depolarisation induced glutamate efflux. These results demonstrate that local administration of SSRIs has an inhibitory influence on evoked release of glutamate in the dorsal horn. This could indicate regulation of excitatory neurotransmission mediated through augmented serotonergic neurotransmission and activation of a serotonergic receptor other than the 1A subtype. Alternatively, direct inhibition with voltage dependent calcium channels, potentially a property intrinsic to molecules with high selectivity for the 5-HT transporter, may underlie this effect.