Intraneural Application of microRNA-1 Mimetic Nucleotides Does Not Resolve Neuropathic Pain After Chronic Constriction Injury in Rats

Abstract

Background: Alterations of the expression of microRNAs (miRNAs) in chronic pain models seem to play a crucial role in the development of neuropathic pain, with microRNA-1 (miR-1) being of particular interest. Recently, we were able to show that decreased miR-1 levels were associated with increased expression of brain-derived neurotrophic factor (BDNF) and Connexin 43 (Cx43). We hypothesized that miR-1 mimetic nucleotides could alleviate neuropathic pain caused by chronic constriction injury in rats.

Methods: MiR-1 mimetic nucleotides were evaluated for effectiveness, functionality, and intracellular stability by transfecting human glioblastoma cells (U-87 MG). In vivo transfection with miR-1 mimics and corresponding scrambled miRNAs serving as control was performed by repetitive injection (days 0, 3, and 7) into the sciatic nerve following chronic constriction injury (CCI) in rats. Quantitative PCR was used to measure miR-1 content. Cx43 expression was determined by Western blot analysis. Effects on neuropathic pain were assessed by detecting paw withdrawal thresholds using an automated filament application.

Results: Transfection of miR-1 mimics was confirmed in U-87 MG cells, with miR-1 content being increased significantly after 48 h and after 96 h (p<0.05). Effective downregulation of Cx43 expression was observed 48 and 96 h after transfection (-44 ± 0.07% and -40 ± 0.11%; p<0.05). In vivo, repetitive transfection with miR-1 mimetic nucleotides led to a 17.9-fold (± 14.2) increase of miR-1 in the sciatic nerve. However, the protein expression of Cx43 in sciatic nerves as well as paw withdrawal thresholds for mechanical stimulation was not significantly increased 10 days after chronic constriction injury.

Conclusion: While transfection with miR-1 mimics effective reduces Cx43 expression in vitro and restores miR-1 after CCI, we did neither observe altered levels of Cx43 protein level in nerves nor a beneficial effect on mechanical allodynia in vivo, most likely caused by insufficient Cx43 suppression.

Keywords: chronic pain; microRNAs; neuropathic pain; peripheral nerve.

Figure 1

Expression level of microRNA-1 (miR-1) 48h and 96h in U87 cells after transfection with scrambled microRNA-mimic (Scr) or microRNA-1 mimics (miR-1). Absolute qPCR results (raw Cq values); n=3, *p < 0.05.

Figure 2

Protein expression level of Connexin 43 (Cx43) in U87 cells 48 h and 96h after transfection with scrambled microRNA-mimic (Scr) or microRNA-1 mimic (miR-1). Determined by Western blot analysis; n=3, *p < 0.05.

Figure 3

(A) Relative microRNA-1 (miR-1) expression in sciatic nerve (n=4) and dorsal root ganglia (DRG; n=5) of CCI rats that received intraneural miR-1-mimic injections on day 0, 3, and 7 vs rats that received scrambled-microRNA mimics (Scr). (B) Operation site during intraneural injections of miR-1. As depicted, injections were placed proximally to the ligatures; *p < 0.05 vs Scrambled.

Figure 4

(A) Protein levels of Connexin 43 (Cx43) in sciatic nerves after microRNA-1 (miR-1) or scrambled (Scr) miRNA-injection. No significant changes were found in CX43 protein levels in sciatic nerves treated with miR-1 mimetic nucleotides in comparison to nerves treated with scrambled miRNAs. (B) Paw withdrawal thresholds of rats after chronic constriction injury (CCI) of the sciatic nerve and intraneural injection with scrambled microRNA mimic (Scr) or microRNA-1 mimic (miR-1). Mimics were injected at day 0, 3, 7 after CCI. Thresholds were detected at day 10 after CCI (n=16–21, n.s.).