Beschreibung
Pain or the treatment of pain plays a serious role in both human and veterinary medicine. In particular, chronic neuropathic pain poses great challenges to treating physicians. Studies suggest that micro RNAs (miRs), and especially micro RNA-1 (miR-1), play a key role in the onset and development of chronic neuropathic pain. Our group has previously demonstrated a link between the reduction of miR-1 expression in neural tissue and the induction of brain derived neurotrophic factor (BDNF) and connexin 43 (Cx43). Both BDNF and Cx43 are closely related to the development and maintenance of chronic pain conditions. This led to the hypothesis that miR-1-mimetic nucleotides (miR-1-mimic) alleviate chronic constriction injury (CCI)-induced neuropathic pain in rats.
Firstly, the miR-1-mimic was tested for efficacy, functionality and intracellular stability using a transfection experiment on human glioblastoma cells (U-87 MG). The transfected cells were analyzed 48 h and 96 h after transfection by qRT-PCR and Western blot to determine the expression level of miR-1 and the relative expression of Cx43 (n = 3). U-87 MG cells transfected in the same manner with a noncoding version of the molecule with the same chemical properties (scrambled miR-1 mimics) served as control group (n = 3).
After obtaining permission from the responsible authority to perform animal experiments, the miR-1 mimic was used in vivo in male Wistar rats. To induce chronic peripheral mononeuropathy of the left sciatic nerve, animals underwent CCI surgery. Subsequently, the animals were divided into two groups. Animals in the first group received an intraneural injection with miR-1 mimics three times (day 0, 3, and 7) into the left sciatic nerve (n=6). The control group was treated accordingly with scrambled miR-1 mimics (n=6). Measurement of allodynia as a sign of the development and severity of neuropathy was performed using Dynamic plantar Aesthesiometer® in both groups on the left hind paw on the tenth post-operative day. Subsequently, all animals were euthanized so that a section of the sciatic nerve of the left side, and the associated dorsal ganglia (L4 to L6), could be removed from them. The obtained neural tissue was analyzed by qRT-PCR or Western blot to measure the expression level of miR-1 and the relative expression of Cx43.
Differences between animal groups were compared using Student's t-test and Bonferroni-Holms-correction. In vitro experiments were performed in triplicates. A p value of < 0.05 was considered statistically significant.
In the transfection experiment, there was a significantly higher expression level of miR-1 in U-87-MG cells treated with miR-1 mimics after 48 h and 96 h compared to the control group. In parallel, the relative expression of Cx43 in U-87-MG cells treated with miR-1 mimics was significantly lower than the control group at both time points.
In vivo, qRT-PCR revealed significantly higher miR-1 expression in the neural tissue (sciatic nerve) of animals treated with miR-1 mimics compared to control animals. In contrast, there were no significant differences in miR-1 expression between the groups in the dorsal ganglia. Examination of neural tissue (sciatic nerve) by Western blot analysis revealed no statistically significant differences between groups in relative expression of Cx43. Furthermore, the paw withdrawal threshold determined as a measure of mechanical allodynia showed no significant difference between the two groups.
In the transfection experiment, miR-1 mimics were shown to be effective, functional, and intracellularly stable, as they were able to reduce Cx43 expression in a prolonged and effective manner. However, a similar result could not be achieved in the living organism with the experimental set up outlined here. Nor was relief of neuropathic pain achieved.
Nonetheless, miR-1 remains a promising candidate for the therapy of neuropathic pain and an interesting subject for future research.
