Investigations into central mechanisms of pain transmission
The pain transmission system is inherently plastic in nature; plasticity of nociceptive processing in the dorsal hom of the spinal cord is believed to contribute to clinical states of post-injury pain hypersensitivity. Both enhancement and tachyphylaxis of nociceptive processing have been reported previously following repeated carrageenan-induced inflammation. The present study aimed to investigate central mechanisms involved in the transformation of pain transmission from 'physiological' to 'pathophysiological' in adult rats, using a model of mild intraplantar inflammation induced by intraplantar administration of carrageenan at doses markedly lower than those standardly used in research into central mechanisms of inflammatory pain transmission. Changes in plantar inflammation, thermal and mechanical sensitivity were assessed following intraplantar injection of repeated doses of carrageenan (0.5%, corresponding to a dose of 0.25 mg and 0.1 %, corresponding to a dose of 0.05 mg), administered at weekly (0.5% and 0.1 %) and daily (0.1%) intervals. Expression of mRNA of key genes implicated in plasticity of central spinal pain transmission in laminae I, II and V of the dorsal hom of the lumbar spinal cord (laminae involved in central nociceptive transmission) was investigated using in-situ hybridisation techniques. The genes investigated were calcium calmodulin kinase IIa (CaMKIla), a key intracellular molecule instantaneously activated by neuronal stimulation; alterations in CaMKIla expression can rapidly induce nociceptive plasticity through modulation of many excitatory and inhibitory nociceptive mediators; the cyclooxygenase enzymes COX-1 and COX-2, which catalyse prostaglandin synthesis and are implicated in the modulation of the central nociceptive response to inflammatory injury; the immediate early genes zif11268, junD and tissue plasminogen activator (tPA), which have been implicated in the induction and maintenance of neuronal plasticity in higher centres, and the precursors for the inhibitory neurotransmitter molecules y-amino butyric acid (GABA), enkephalin and dynorphin. A method for organotypic culture of neonatal spinal cord was developed and characterised with the aim of providing a useful technique for more detailed study of the molecular basis of nociceptive plasticity. Mild inflammatory injury induced by 0.5% and 0.1% carrageenan treatment induced consistent hyperalgesic behaviour, which did not change following weekly repeated injection. Temporary attenuation of hyperalgesia developed following daily repetitive administration of 0.1 % carrageenan, but hyperalgesia returned when this repetitive inflammatory stimulation was maintained. Preliminary studies on the role of NMDA receptors, opioid receptors and a 2A adrenoreceptors in the mediation of this tachyphylaxis suggest that these receptor systems did not playa major role in the observed tachyphylaxis In-situ hybridization studies did not identify changes in gene expression induced by repetitive carrageenan treatment in lamina V. In laminae I1II, changes were observed in expression of certain genes (notably CaMKIla, COX-2 and proenkephalin), but not of immediate early genes, GAD 67 or prodynorphin. Hyperalgesia associated with weekly carrageenan treatment correlated closely with significantly enhanced transcription of CaMKIla mRNA in laminae IIII; moreover, tachyphylaxis of hyperalgesic behaviour correlated with attenuation of CaMKIla upregulation. Since increased expression of CaMKIla, leading to regulation of expression of a range of kinase-dependent receptors and intracellular mediators, is a hallmark of neuronal plasticity in higher centers, this suggests that central plasticity of nociceptive transmission in the dorsal hom could have contributed to the development of hyperalgesia following carrageenan treatment. Weekly administration of carrageenan also consistently induced significant upregulation of COX-2 and proenkephalin mRNA expression in laminae I1II, suggesting that ultimate modulation of pain sensation following inflammatory injury is determined by the interaction of excitatory and inhibitory transmitter pathways. COX-I, prodynorphin and GAD 67 mRNA expression were not significantly changed in relation to the intensity of inflammatory injury or in relation to changes in nociceptive responses. This would suggest that these mediators did not play a key role in the modulation of spinal nociceptive transmission associated with mild inflammatory injury. With the possible exception of CaMKIla, changes in gene expression did not correlate closely with plasticity of nociceptive behaviour induced by daily repeated carrageenan treatment. 200 !lm transverse slices of postnatal spinal cord were cultured successfully for up to 5 days using a simple interface culture system. Histochemical and immunocytochemical assays indicated that the architecture of organotypically cultured spinal cord closely resembled that observed in-vivo. This study presents a new approach to the investigation of neuronal plasticity associated with tissue injury and inflammation. Different mechanisms underlying plasticity of nociceptive responses may be induced by induced by high intensity as opposed to lowintensity injury. The observation of tachyphylaxis of hyperalgesia following daily repeated carrageenan treatment may represent engagement of endogenous 'anti-hyperalgesic' mechanisms. Further investigation of the molecular basis of endogenous 'antihyperalgesia', facilitated by organotypic slice culture techniques, may identify new targets for the treatment and prevention of persistent pathological nociceptive transmission following inflammatory injury.