Accepted

Advanced glycation end-products (AGEs) are proteins/lipids that are glycated upon sugar exposure and are often increased during inflammatory diseases such as osteoarthritis and neurodegenerative disorders. Here, we developed an extracellular matrix (ECM) using glycated type I collagen (ECM-GC), which produced similar levels of AGEs to those detected in the sera of arthritic mice. In order to determine whether AGEs were sufficient to stimulate sensory neurons, dorsal root ganglia (DRGs) cells were cultured on ECM-GC or ECM-NC-coated plates. ECM-GC or ECM-NC were favorable for DRG cells expansion. However, ECM-GC cultivated neurons displayed thinner F-actin filaments, rounded morphology, and reduced neuron interconnection compared to ECM-NC. In addition, ECM-GC did not affect RAGE expression levels in the neurons, although induced rapid p38, MAPK and ERK activation. Finally, ECM-GC stimulated the secretion of nitrite and TNF-α by DRG cells. Taken together, our in vitro glycated ECM model suitably mimics the in vivo microenvironment of inflammatory disorders and provides new insights into the role of ECM impairment as a nociceptive stimulus.

The great majority of neurons in the superficial dorsal horn of the spinal cord are excitatory interneurons, and these are required for the normal perception of pain and itch. We have previously identified 5 largely non-overlapping populations among these cells, based on the expression of four different neuropeptides (cholecystokinin, neurotensin, neurokinin B and substance P) and of green fluorescent protein driven by the promoter for gastrin-releasing peptide (GRP) in a transgenic mouse line. Another peptide (neuropeptide FF, NPFF) has been identified among the excitatory neurons, and here we have used an antibody against the NPFF precursor (pro-NPFF) and a probe that recognises Npff mRNA to identify and characterise these cells. We show that they are all excitatory interneurons, and are separate from the five populations listed above, accounting for ~6% of the excitatory neurons in laminae I-II. By examining phosphorylation of extracellular signal-regulated kinases, we show that the NPFF cells can respond to different types of noxious and pruritic stimulus. Ablation of somatostatin-expressing dorsal horn neurons has been shown to result in a dramatic reduction in mechanical pain sensitivity, while somatostatin released from these neurons is thought to contribute to itch. Since the great majority of the NPFF cells co-expressed somatostatin, these cells may play a role in the perception of pain and itch.

p53 and parkin are involved in mitochondrial quality control. The present study aimed to characterize the functional significance of parkin/p53 in the development of mitochondrial dysfunction and the pathophysiology of neuropathic pain in type I diabetes. Type I diabetes was induced in mice (N = 170) using streptozotocin (STZ). Pifithrin-α, a selective p53 inhibitor, was administered to assess its effects on diabetic pain hypersensitivity, parkin expression and mitochondrial function. Expressions of parkin and p53, mitochondrial number and level of reactive oxygen species (ROS) in the dorsal root ganglion (DRG) were analyzed by immunohistochemistry, western blotting and real time PCR. Separately, mice were treated using intravenous methylglyoxal, then pain hypersensitivity and p53/parkin expression in the DRG were assessed. Mitochondrial membrane potential was also analyzed in cultured DRG neurons treated with methylglyoxal. Mice developed pain hypersensitivity for 3 weeks after STZ treatment. p53 expression was significantly increased (control, 0.68 ± 0.122; STZ, 1.88 ± 0.21) whereas parkin expression was significantly reduced (control, 1.02 ± 0.17; STZ, 0.59 ± 0.14), in the DRG after STZ treatment. Inhibition of p53 by pifithrin-α prevented STZ-induced pain hypersensitivity and parkin downregulation. Pifithrin-α also inhibited STZ-induced reductions in mitochondrial number and accumulation of mitochondrial ROS. Methylglyoxal elicited pain hypersensitivity and alteration of p53/parkin expression, similar to STZ. Methylglyoxal also decreased mitochondrial membrane potential in cultured DRG neurons. Alteration of p53/parkin expression produces mitochondrial dysfunction and ROS accumulation, leading to pain hypersensitivity in diabetic or methylglyoxal treated mice. Methylglyoxal produces neurological derangements similar to diabetes, via direct mechanisms on DRG neurons.

Complex regional pain syndrome I (CRPS-I) is a chronic painful pathology still undertreated. CTK 01512-2 is a recombinant version of the spider peptide Phα1β, and it functions as a voltage-gated calcium channel blocker and a transient receptor potential ankyrin 1 (TRPA1) antagonist with antinociceptive effect in different pain models. Here, we investigate the mechanisms involved in the acute and chronic nociceptive phases of a model of CPRS-I in mice and assess the antinociceptive effect of CTK 01512-2 using this model. Adult male and female mice C57BL/6 (20-30 g) were used to determine mechanical (von Frey test) or cold (acetone test) allodynia induction. Inflammatory parameters (serum and tibial nerve lactate levels, hind paw temperature and edema, or tissue cell infiltration) were evaluated after chronic post-ischemia pain (CPIP, a model of CPRS-I) induction. Anti-inflammatory and anti-neuropathic drugs or CTK 01512-2 were tested. First, we detected that CPIP-induced mechanical and cold allodynia in male and female mice in a similar way. In the acute phase (1 day after CPIP), an increase in inflammatory parameters were observed, as well as the anti-allodynic effect of anti-inflammatory compounds. In the chronic phase (17 days after CPIP), mice exhibited mechanical and cold allodynia, and anti-neuropathic drugs induced antinociception, while no inflammatory alterations were found. CTK 01512-2 reversed the CPIP allodynic effect in both nociceptive phases. Thus, this CPRS-I model can be used to understand the mechanisms involved in CPRS-I induced pain and inflammation. Besides, we observed that CTK 01512-2 has a valuable antinociceptive effect in this pain model.