Studies on pain-related messengers and receptors in dorsal root ganglia and spinal cord

Abstract

Pain is the most common reason why people seek medical advice. A large group of patients suffering from so-called neuropathic pain, due to injury to the nervous system itself, is often not helped by the currently available therapies. Classical analgesic drugs, such as opioids and non-steroidal anti-inflammatory drugs, may have severe side effects. There is a need and interest to study basic mechanisms involved in pain and to evaluate new putative targets for therapies. We have studied the expression of some messenger molecules following axotomy, a neuropathic pain model, in the guinea pig. Substance P was, in analogy to other species studied, down- regulated in dorsal root ganglia (DRGs). The immunoreactivity of galanin (GAL), a neuropeptide with inhibitory properties, was significantly increased but not to the same extent as seen in the rat and monkey. NPY-like immunoreactivity (-LI ) revealed a highly significant increase in small nociceptive neurons. No significant changes were seen regarding the nitric oxide synthase, vasoactive intestinal polypeptide, and calcitonin gene- related peptide (CGRP)-Lls.

All in a all, substantial species differences are seen in neuropeptide expression following nerve injury. NPY may constitute an important autoregulatory inhibitory factor in neurotransmission in the guinea pig. The excitatory messenger SP is generally down-regulated in DRGs after axotomy. DRG cultures were used to study the release of putative neurotransmitters such as CGRP and glutamate (GLU). CGRP was released in a Ca2+-dependent manner following stimulation by potassium, capsaicin (CAPS), and nicotine. Ruthenium red attenuated both CAPS and nicotine-induced release of CGRP. NPY reduced the nicotine-stimulated outflow of CGRP, but did not affect CAPS-induced release. The excitatory amino acids GLU and aspartate did not elicit release a of CGRP. GAL was rapidly up-regulated in DRG cultures, which suggests that DRG cultures constitute a model of axotomized neurons, instead of being a model of 'normal' sensory neurons.

We have examined the effect of different growth factors on GAL expression in DRG cultures as well as on GLU release. Basic fibroblast growth factor (bFGF) and brain-derived neurotrophic factor (BDNF) suppressed GAL upregulation in DRG cultures in line with previous findings made in vivo. GLU release was significantly increased by bradykinin (BK), which release was not seen following stimulation with CAPS and potassium. The BK-induced release was attenuated by adding growth factors to the culture medium. bFGF was the most potent, reducing GLU release to basal levels. bFGF also decreased BK-induced Cal+ transients in DRG neurons but did not affect potassium-induced Ca2+ transients.

DRG cultures constitute a suitable model for studying release of neurotransmitters, such as neuropeptides and GLU. The culture system may, however, be regarded as a model of axotomized neurons and not a normal phenotype. Growth factors, and in particular bFGF, suppress GAL up-regulation, BK- induced GLU release, and Ca2- transients.

The N-methyl-D-aspartate-(NMDA) receptor is an ion channel associated with pain transmission in the spinal cord. However, NMDA receptors have been postulated to play a role already at the peripheral site. Five subunits denoted NMDAR1, 2A-D are cloned. NMDAR1 transcripts were found in approximately 50% of the human DRG neuron profiles (NPs), while 100% of the rat and mouse DRG neurons express this subunit. Several pain conditions are associated with tissue acidification, such as ischemia and inflammation. A family of acid-sensing ion channel (ASIC )receptors was cloned. We have analyzed the expression of one of these channels, ASIC 3, also called DRASIC, which is known to be exclusively expressed in DRG neurons in the rat. However, expression is more widespread in human tissues. Approximately 20% of human DRG NPs expressed this subunit.

DRASIC can in parallel to NMDAR1 be regarded as a potential peripheral target for pain therapy. Antisense (AS) oligodeoxynucleotides (ODNs) may serve as a potential tool to knock down the expression of a specific protein by interfering with the translation of mRNA into protein. We have evaluated the spinal use of phosphodiester ODNs and two stable ODN modifications, 2'-O-allyl and C-5-propyne-phophorothioate ODNs, in the mice formalin test. ODNs failed to significantly decrease pain behavior and receptor binding. C-5-propyne decreased receptor binding and pain behavior only in doses where severe side effects were seen. A rapid uptake was detected in all layers of the spinal cord with the aid of fluorescein (FITC)-conjugated ODNs. Phosphodiester ODNs produced a significant decrease in receptor binding and pain behavior, although the cellular uptake and tissue distribution seemed poor. A minor contribution of non-AS-specific effects was revealed in mismatch ODNs.

2'-O-allyl and C-5-propyne-ODNs did not prove to be more advantageous than PHD ODNS for spinal use. AS may provide a valuable tool for studying the physiological role of a protein for which no specific antagonist has been developed. The need for well controlled AS studies is emphasized in order to be able to claim true AS effects.