Search

Pulsed radiofrequency (PRF) therapy is one of the most common treatment options for neuropathic pain, albeit the underlying mechanism has not been hitherto elucidated. In this study, we investigated the efficacy and mechanism of PRF therapy on resiniferatoxin (RTX)-induced mechanical allodynia, which has been used as a model of postherpetic neuralgia (PHN). Adult male rats were intraperitoneally injected with a vehicle or RTX. Furthermore, PRF current was applied on a unilateral sciatic nerve in all RTX-treated rats. On both ipsilateral and contralateral sides, the paw mechanical withdrawal thresholds were examined and L4-6 dorsal root ganglia (DRG) were harvested. In the DRG of rats with RTX-induced mechanical allodynia, Na

In small and large spinal dorsal root ganglion neurons, subtypes of voltage-gated sodium channels, such as NaV1.7, NaV1.8, and NaV1.9 are expressed with characteristically localized and may play different roles in pain transmission and intractable pain development. Selective stimulation of each specific subtype in vivo may elucidate its role of each subtype in pain. So far, this has been difficult with current technology. However, Optogenetics, a recently developed technique, has enabled selective activation or inhibition of specific neural circulation in vivo. Moreover, optogenetics had even been used to selectively excite NaV1.8-expressing dorsal root ganglion neurons to induce nocifensive behavior. In recent years, genetic modification technologies such as CRISPR/Cas9 have advanced, and various knock-in mice can be easily generated using such technology. We aimed to investigate the effects of selective optogenetic activation of NaV1.7-expressing afferents on mouse behavior. We used CRISPR/Cas9-mediated homologous recombination to generate bicistronic NaV1.7–iCre knock-in mice, which express iCre recombinase under the endogenous NaV1.7 gene promoter without disrupting NaV1.7. The Cre-driver mice were crossed with channelrhodopsin-2 (ChR2) Cre-reporter Ai32 mice to obtain NaV1.7iCre/+;Ai32/+, NaV1.7iCre/iCre;Ai32/+, NaV1.7iCre/+;Ai32/Ai32, and NaV1.7iCre/iCre;Ai32/Ai32 mice. Compared with wild–type mice behavior, no differences were observed in the behaviors associated with mechanical and thermal stimuli exhibited by mice of the aforementioned genotypes, indicating that the endogenous NaV1.7 gene was not affected by the targeted insertion of iCre. Blue light irradiation to the hind paw induced paw withdrawal by mice of all genotypes in a light power-dependent manner. The threshold and incidence of paw withdrawal and aversive behavior in a blue-lit room were dependent on ChR2 expression level; the strongest response was observed in NaV1.7iCre/iCre;Ai32/Ai32 mice. Thus, we developed a non-invasive pain model in which peripheral nociceptors were optically activated in free-moving transgenic NaV1.7–ChR2 mice.

Methodology to enhance intestinal absorption of macromolecular drugs is an important challenge for developing next-generation biomedicines. So far, various cationic cell-penetrating peptides have been reported to facilitate uptake of certain bioactive proteins. However, cyclic peptides might be better candidates, as they are more metabolically stable than linear peptides. Accordingly, we hypothesized that suitable cyclic peptides would promote the absorption of macromolecules across intestinal epithelium. To test this idea, we adopted Caco-2 cell permeability assay as an in vitro human intestinal absorption model, and M13 phage as a model of macromolecules. Successive screenings of a phage library displaying cyclic heptapeptides via a short GGGS linker yielded 3 hits. Among them, phage displaying cyclic heptapeptide DNPGNET (DNP-phage) showed the greatest permeability across a Caco-2 cell monolayer and mouse intestinal epithelium. Interestingly, DNPGNET (DNP) does not contain any basic amino acids. Its isoelectric point (pI) was estimated to be 2.72. It did not reduce the viability or tight-junction integrity of Caco-2 cells at concentrations up to 100μM for 24h. Uptake of either DNP-phage or a fluorescence-labeled DNP derivative (AC-DNPGNET-CGGGS modified with 5/6-FAM at the C-terminal; the cysteines serve to generate the cyclic peptide via disulfide bond formation, and GGGS is the phage linker) by Caco-2 cells was inhibited by low temperature, unlabeled AC-DNPGNET-CGGGS and EIPA, a macropinocytosis inhibitor. Thus, DNP appears to facilitate transcellular permeability of phages via macropinocytosis, but not paracellular diffusion. These findings indicate that DNP is a promising candidate as a carrier to promote intestinal absorption of macromolecular drugs.