Upregulation of T-type Ca2+ channels in long-term diabetes determines increased excitability of a specific type of capsaicin-insensitive DRG neurons.

BACKGROUND: Previous studies have shown that increased excitability of capsaicin-sensitive DRG neurons and thermal hyperalgesia in rats with short-term (2-4 weeks) streptozotocin-induced diabetes is mediated by upregulation of T-type Ca(2+) current. In longer-term diabetes (after the 8th week) thermal hyperalgesia is changed to hypoalgesia that is accompanied by downregulation of T-type current in capsaicin-sensitive small-sized nociceptors. At the same time pain symptoms of diabetic neuropathy other than thermal persist in STZ-diabetic animals and patients during progression of diabetes into later stages suggesting that other types of DRG neurons may be sensitized and contribute to pain. In this study, we examined functional expression of T-type Ca(2+) channels in capsaicin-insensitive DRG neurons and excitability of these neurons in longer-term diabetic rats and in thermally hypoalgesic diabetic rats. RESULTS: Here we have demonstrated that in STZ-diabetes T-type current was upregulated in capsaicin-insensitive low-pH-sensitive small-sized nociceptive DRG neurons of longer-term diabetic rats and thermally hypoalgesic diabetic rats. This upregulation was not accompanied by significant changes in biophysical properties of T-type channels suggesting that a density of functionally active channels was increased. Sensitivity of T-type current to amiloride (1 mM) and low concentration of Ni(2+) (50 µM) implicates prevalence of Cav3.2 subtype of T-type channels in the capsaicin-insensitive low-pH-sensitive neurons of both naïve and diabetic rats. The upregulation of T-type channels resulted in the increased neuronal excitability of these nociceptive neurons revealed by a lower threshold for action potential initiation, prominent afterdepolarizing potentials and burst firing. Sodium current was not significantly changed in these neurons during long-term diabetes and could not contribute to the diabetes-induced increase of neuronal excitability. CONCLUSIONS: Capsaicin-insensitive low-pH-sensitive type of DRG neurons shows diabetes-induced upregulation of Cav3.2 subtype of T-type channels. This upregulation results in the increased excitability of these neurons and may contribute to nonthermal nociception at a later-stage diabetes.

Specific functioning of Cav3.2 T-type calcium and TRPV1 channels under different types of STZ-diabetic neuropathy.

Streptozotocin (STZ)-induced type 1 diabetes in rats leads to the development of peripheral diabetic neuropathy (PDN) manifested as thermal hyperalgesia at early stages (4th week) followed by hypoalgesia after 8weeks of diabetes development. Here we found that 6-7 week STZ-diabetic rats developed either thermal hyper- (18%), hypo- (25%) or normalgesic (57%) types of PDN. These developmentally similar diabetic rats were studied in order to analyze mechanisms potentially underlying different thermal nociception. The proportion of IB4-positive capsaicin-sensitive small DRG neurons, strongly involved in thermal nociception, was not altered under different types of PDN implying differential changes at cellular and molecular level. We further focused on properties of T-type calcium and TRPV1 channels, which are known to be involved in Ca(2+) signaling and pathological nociception. Indeed, TRPV1-mediated signaling in these neurons was downregulated under hypo- and normalgesia and upregulated under hyperalgesia. A complex interplay between diabetes-induced changes in functional expression of Cav3.2 T-type calcium channels and depolarizing shift of their steady-state inactivation resulted in upregulation of these channels under hyper- and normalgesia and their downregulation under hypoalgesia. As a result, T-type window current was increased by several times under hyperalgesia partially underlying the increased resting [Ca(2+)]i observed in the hyperalgesic rats. At the same time Cav3.2-dependent Ca(2+) signaling was upregulated in all types of PDN. These findings indicate that alterations in functioning of Cav3.2 T-type and TRPV1 channels, specific for each type of PDN, may underlie the variety of pain syndromes induced by type 1 diabetes.

Nociceptive neurons differentially express fast and slow T-type Ca²âº currents in different types of diabetic neuropathy.

T-type Ca²âº channels are known as important participants of nociception and their remodeling contributes to diabetes-induced alterations of pain sensation. In this work we have established that about 30% of rat nonpeptidergic thermal C-type nociceptive (NTCN) neurons of segments L4-L6 express a slow T-type Ca²âº current (T-current) while a fast T-current is expressed in the other 70% of these neurons. Streptozotocin-induced diabetes in young rats resulted in thermal hyperalgesia, hypoalgesia, or normalgesia 5-6 weeks after the induction. Our results show that NTCN neurons obtained from hyperalgesic animals do not express the slow T-current. Meanwhile, the fraction of neurons expressing the slow T-current did not significantly change in the hypo- and normalgesic diabetic groups. Moreover, the peak current density of fast T-current was significantly increased only in the neurons of hyperalgesic group. In contrast, the peak current density of slow T-current was significantly decreased in the hypo- and normalgesic groups. Experimental diabetes also resulted in a depolarizing shift of steady-state inactivation of fast T-current in the hyperalgesic group and slow T-current in the hypo- and normalgesic groups. We suggest that the observed changes may contribute to expression of different types of peripheral diabetic neuropathy occurring during the development of diabetes mellitus.

Novel peptide from spider venom inhibits P2X3 receptors and inflammatory pain.

P2X3 purinoreceptors expressed in mammalian sensory neurons play a key role in several processes, including pain perception. From the venom of the Central Asian spider Geolycosa sp., we have isolated a novel peptide, named purotoxin-1 (PT1), which is to our knowledge the first natural molecule exerting powerful and selective inhibitory action on P2X3 receptors. PT1 dramatically slows down the removal of desensitization of these receptors. The peptide demonstrates potent antinociceptive properties in animal models of inflammatory pain.

Peripheral Inflammation Results in Increased Excitability of Capsaicin-Insensitive Nociceptive DRG Neurons Mediated by Upregulation of ASICs and Voltage-Gated Ion Channels.

Previously, we have characterized the capsaicin-insensitive low pH-sensitive (caps-lpH+) subtype of small-sized nociceptive dorsal root ganglion (DRG) neurons that express acid-sensing ion channels, T-type Ca2+ channels, and have isolectin B4-negative phenotype. These neurons demonstrated increased excitability in a model of long-term diabetes, contributing to chronic pain sensation. Here we studied changes in the excitability of the caps-lpH+ neurons and underlying changes in the functional expression and gating properties of ion channels under complete Freund's adjuvant (CFA)-induced peripheral inflammation. We have found that, under these pathological conditions, the functional expression of the acid-sensing ion channels (ASICs) and voltage-gated Na+ channels, was increased. In addition, T-type Ca2+ current was significantly increased in the neurons at the membrane potentials close to its resting value. Altogether, the observed changes in the channel functioning shifted a pH level evoking an action potential (AP) toward its physiological value and led to an increase of evoked and spontaneous excitability of the caps-lpH+ neurons that may contribute to hyperalgesia and chronic inflammatory pain.

The efficacy of immediate implantation of macroporous poly(N-[2-hydroxypropyl]-methacrylamide) hydrogel after laceration spinal cord injury in young rats / Eficacia de la implantación inmediata de hidrogel macroporoso de poli (N- [2-hidroxipropil] - metacrilamida) después de una lesión de la médula espinal en ratas jóvenes

SUMMARY: Spinal cord regeneration after mechanical injury is one of the most difficult biomedical problems. This article evaluates the effect of poly(N-[2-hydroxypropyl]-methacrylamide) hydrogel (PHPMA-hydrogel) on spinal cord regeneration in young rats after lateral spinal cord hemi-excision (laceration) at the level of segments T12-T13 (TrGel group). The locomotor function score (FS) and the paretic hindlimb spasticity score (SS) were assessed according to Basso-Beattie-Bresnahan (BBB) and Ashworth scales, respectively, and compared to a group of animals with no matrix implanted (Tr group). Regeneration of nerve fibers at the level of injury was evaluated at ~5 months after spinal cord injury (SCI). One week after the SCI induction, the FS on the BBB scale was 0.9±0.5 points in the Tr group and 3.6±1.2 points in the TrGel group. In the Tr group, the FS in 5 months was significantly lower than in 2 weeks after SCI, while no significant changes in FS were detected in the TrGel group over the entire observation period. The final FS was 0.8±0.3 points in the Tr group and 4.5±1.8 points in the TrGel group. No significant changes in SS have been observed in the TrGel group throughout the experiment, while the Tr group showed significant increases in SS at 2nd week, 6th week, 3th month and 5th month. The SS in 5 months was 3.6±0.3 points on the Ashworth scale in the Tr group and 1.8±0.7 points in the TrGel group. Throughout the observation period, significant differences in FS between groups were observed only in 5 weeks after SCI, whereas significant differences in SS were observed in 2, 3 and 6-8 weeks post-injury. Glial fibrous tissue containing newly formed nerve fibers, isolated or grouped in small clusters, that originated from the surrounding spinal cord matter have been found between the implanted hydrogel fragments. In conclusion, PHPMA-hydrogel improves recovery of the hindlimb locomotor function and promotes regenerative growth of nerve fibers. Further research is needed to clarify the mechanism of this PHPMA-hydrogel effect.

RESUMEN: La regeneración de la médula espinal después de una lesión mecánica es uno de los problemas biomédicos más difíciles. Este artículo evalúa el efecto del hidrogel de poli (N- [2-hidroxipropil] -metacrilamida) (PHPMA-hidrogel) sobre la regeneración de la médula espinal en ratas jóvenes después de la hemiescisión lateral de la médula espinal (lesión) a nivel de los segmentos T12 - T13 (Grupo TrGel). La puntuación de la función locomotora (FS) y la puntuación de espasticidad parética de las patas traseras (SS) se evaluaron de acuerdo con las escalas de Basso- Beattie-Bresnahan (BBB) y Ashworth, respectivamente, y se compararon con un grupo de animales sin matriz implantada (grupo Tr). Se evaluó la regeneración de las fibras nerviosas al nivel de la lesión ~ 5 meses después de la lesión de la médula espinal (LME). Una semana después de la inducción de SCI, el FS en la escala BBB fue 0,9 ± 0,5 puntos en el grupo Tr y 3,6 ± 1,2 puntos en el grupo TrGel. En el grupo Tr, el FS en 5 meses fue significativamente menor que en 2 semanas después de SCI, mientras que no se detectaron cambios significativos en FS en el grupo TrGel durante el período de observación. El FS final fue de 0,8 ± 0,3 puntos en el grupo Tr y de 4,5 ± 1,8 puntos en el grupo TrGel. No se han obser- vado cambios significativos en SS en el grupo TrGel durante el experimento, mientras que el grupo Tr mostró aumentos significativos en SS en la 2ª semana, 6ª semana, 3º mes y 5º mes. La SS en 5 meses fue de 3,6 ± 0,3 puntos en la escala de Ashworth en el grupo Tr y de 1,8 ± 0,7 puntos en el grupo TrGel. A lo largo del período de observación, se observaron diferencias significativas en FS entre los grupos solo en 5 semanas después de la LME, mientras que se observaron diferencias significativas en SS en 2, 3 y 6-8 semanas después de la lesión. Entre los fragmentos de hidrogel implantados se observó tejido fibroso glial que contenía fibras nerviosas recién formadas, aisladas o agrupadas en pequeños grupos, que se originaban a partir de la materia de la médula espinal circundante. En conclusión, PHPMA-hydrogel mejora la recuperación de la función locomotora de las patas traseras y promueve el crecimiento regenerativo de las fibras nerviosas. Se requieren más estudios para aclarar el mecanismo del efecto de hidrogel PHPMA.