Neuroimmune interactions in neuropathic pain. Involvement of tnf-alpha and voltage-gated sodium channels in sensory neurons after peripheral nerve injury
- Autores: Laura Casals Díaz
- Directores de la Tesis: Xavier Navarro Acebes (dir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2011
- Idioma: inglés
- Tribunal Calificador de la Tesis: Rafael Maldonado López (presid.), M. M. Puig Riera de Conías (secret.), José Miguel Vela Hernández (voc.)
- Materias:
- Enlaces
- Tesis en acceso abierto en: TESEO
- Resumen
Pain is a protective sensation needed for the survival and well-being of the individual. However, damage to the nervous system can produce a neuropathic pain syndrome, where pain is no longer protective, arises spontaneously and is characterized by a complete lack of correlation between the intensity of peripheral noxious stimuli and of pain sensation. Multiple mechanisms, occurring at both the peripheral and central nervous system, contribute to the neuropathic pain syndrome. We focused our research on the mechanisms triggered by sciatic nerve injury on primary afferent neurons. On the one hand, the literature provides evidence for a role of the altered expression in the dorsal root ganglion (DRG) of voltage-gated sodium channels (VGSCs), determinants of neuronal excitability and propagation of the action potential, in inflammatory and neuropathic pain conditions. Their altered expression may be a contributing factor to the hyperexcitability and subsequent pain and dysaesthesia that can develop after nerve injury. On the other hand, it has been demonstrated that Wallerian degeneration following axonal injury is tightly correlated with the development of neuropathic pain. The proinflammatory cytokine tumor necrosis factor-¿ (TNF-¿) is upregulated and released immediately at the site of nerve injury, and triggers the early events of Wallerian degeneration. TNF-¿ is able to induce ectopic activity in peripheral nerve fibers and sensitisation of C-fibers. Moreover, pain-related behavior can be effectively reduced in rodent models of painful neuropathy using TNF-¿ neutralizing agents. TNF-¿ activates signalling pathways which end up in the activation of transcription of NF-kappa B-responsive genes. However, the modulation of the transcription of elements directly related to the excitability of sensory neurons, such as VGSCs has not been assessed. Our aim was to characterize the changes in ion channels in experimental models of neuropathic pain, as well as in relation to the nerve injury-induced release of TNF-¿.
We first characterized three models of sciatic nerve injury in the rat with different degrees of damage and impact on regeneration capability: crush nerve injury, chronic constriction injury (CCI) and spared nerve injury (SNI). All three models of sciatic nerve injury induced a neuropathic pain syndrome characterized by the decrease of mechanical and thermal thresholds, with a different temporal pattern depending on the injury. Immunohistochemical labeling of the two populations of nociceptive C-fibers on the superficial dorsal horn of L4-L6 spinal cord sections was performed, and revealed that only the reduced staining of the non-peptidergic C-fiber population (isolectin B4-positive) occurred in parallel to the process of axonal degeneration and alterations of normal nociceptive behavior, while it was not the case of the peptidergic population (substance P-positive).
As nociceptive behaviour and labeling of primary afferents in the spinal cord were more markedly affected by the Crush and SNI, we selected both injury models for the subsequent experiment. In parallel to the assessment of nociceptive behaviour, we then compared the temporal profile of transcriptional changes of VGSCs developed after a nerve injury in which regeneration and reinervation of distal targets was allowed (Crush), with those changes occurring after a chronic nerve injury (SNI). We focused on the mRNA expression of TTX-sensitive (TTX-S) Nav1.3 and Nav1.7, and the TTX-resistant (TTX-R) Nav1.8 and Nav1.9, because of their presence in nociceptive peripheral neurons. SNI rats showed sustained hypersensitivity along 3 months of follow-up, while nociception in Crush rats started to normalize by 28 days post-injury (dpi). Results obtained by real-time PCR showed that both injuries induced similar early alterations in the VGSCs expression, with upregulation of Nav1.3, and downregulation of Nav1.7, Nav1.8 and Nav1.9. These changes persisted until 28 dpi, when hyperalgesia was still present in SNI, but not in Crush rats. mRNA expression of all analyzed ¿-subunits returned to basal levels after reinnervation of peripheral distal targets (Crush), while remained altered in SNI rats. Therefore, transcriptional changes of VGSCs correlated with hyperalgesia only at early stages of the neuropathy, while they fit with the progression of regeneration after the severance of sciatic nerve axons.
Subsequently, we investigated the influence of TNF-¿ blockade on neuropathic pain behavior in the rat SNI model, and then we analyzed its potential consequences on the mRNA expression of Nav1.3, Nav1.7, Nav1.8 and Nav1.9, in sensory neurons of the DRG. Thalidomide was used as a blocker of nerve injury-induced TNF-¿ production, which resulted in the attenuation of mechanical and hot pain hyperalgesia, which was more marked until 14 dpi. Real-time PCR analysis showed that thalidomide treatment significantly enhanced the upregulation of Nav1.3 and downregulation of Nav1.8 and Nav1.9 induced by the SNI in pooled ipsilateral L4/L5 DRGs. However, at 28 dpi VGSC expression was similar between treated and untreated injured rats.
As we demonstrated that both pain behavior and the early changes in VGSCs expression were modulated by TNF-¿ inhibition, we investigated the involvement of TNF signaling via TNF receptor 1, and its downstream mediators nuclear factor kappa B (NF-kB) and the cellular Fas-associated death domain-like interleukin-1-ß-converting enzyme-inhibitory protein (c-FLIP) on these alterations. We performed the SNI on wild-type (WT) C57BL6/J mice, mice deficient of TNF receptor 1 (KO mice), on transgenic mice where NF-kB pathway was inactivated by overexpression of a dominant negative (dn) form of the inhibitor of kappa B (I¿B¿) in neurons (dnIkB), and on mice overexpressing neuronal c-FLIP (FLIP). We observed a long-lasting absence of post-injury thermal hyperalgesia and a reduction of mechanical hypersensitivity in KO mice. In dnIkB mice hypersensitivity was attenuated at 7, 21 and 28 dpi; however they were present at 3 and 14 dpi. In contrast, FLIP mice showed hyperalgesia similarly to that observed in WT mice. On the other hand, mRNA analysis in KO mice suggests that TNF-alpha through the TNFR1-NF-kB pathway mediated the downregulation of Nav1.7 and Nav1.9 subunits at 7 dpi in lumbar DRG neurons, and also the downregulation of Nav1.7 at 28 dpi. Interestingly, all these events coincided when hyperalgesia was reverted and allodynia attenuated. Neuronal FLIP did not seem to be involved in the regulation of VGSCs alpha subunits. Therefore, NF-kB activated by TNFR1 can control the transcription of genes whose protein products are key determinant of sensory neurons excitability.
