Nanomedicine-driven neuropathic pain relief in a rat model is associated with macrophage polarity and mast cell activation

Abstract

We explored the immune neuropathology underlying multi-day relief from neuropathic pain in a rat model initiated at the sciatic nerve, by using a nanoemulsion-based nanomedicine as a biological probe. The nanomedicine is theranostic: both therapeutic (containing celecoxib drug) and diagnostic (containing near-infrared fluorescent (NIRF) dye) and is small enough to be phagocytosed by circulating monocytes. We show that pain-like behavior reaches a plateau of maximum hypersensitivity 8 days post-surgery, and is the rationale for intravenous delivery at this time-point. Pain relief is evident within 24 h, lasting approximately 6 days. The ipsilateral sciatic nerve and associated L4 and L5 dorsal root ganglia (DRG) tissue of both nanomedicine and control (nanoemulsion without drug) treated animals was investigated by immunofluorescence and confocal microscopy at the peak of pain relief (day-12 post-surgery), and when pain-like hypersensitivity returns (day-18 post-surgery). At day-12, a significant reduction of infiltrating macrophages, mast cells and mast cell degranulation was observed at the sciatic nerve following treatment. In the DRG, there was no effect of treatment at both day-12 and day-18. Conversely, at the DRG, there is a significant increase in macrophage infiltration and mast cell degranulation at day-18. The treatment effect on immune pathology in the sciatic nerve was investigated further by assessing the expression of macrophage cyclooxygenase-2 (COX-2)—the drug target—and extracellular prostaglandin E2 (PGE2), as well as the proportion of M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages. At day-12, there is a significant reduction of COX-2 positive macrophages, extracellular PGE2, and a striking reversal of macrophage polarity. At day-18, these measures revert to levels observed in control-treated animals. Here we present a new paradigm of immune neuropathology research, by employing a nanomedicine to target a mechanism of neuropathic pain—resulting in long-lasting pain relief--whilst revealing novel immune pathology at the injured nerve and associated DRG. Electronic supplementary material The online version of this article (10.1186/s40478-019-0762-y) contains supplementary material, which is available to authorized users.

Targeted cyclooxygenase-2 inhibiting nanomedicine results in pain-relief and differential expression of the RNA transcriptome in the dorsal root ganglia of injured male rats

“…Peripheral nerve injury was induced (this was designated “day 0”) using CCI as first described by Bennett and Xie 9 and adapted previously. 4 , 5 , 14 , 15 Briefly, CCI causes inflammation and subsequent pain when chromic gut sutures are loosely tied around the common sciatic nerve in the animal’s hindleg. Animals are anesthetized with inhaled isoflurane and oxygen.…”

“…Mechanical allodynia testing of both hind-paws was conducted using von Frey monofilaments of increasing diameters using the up-down method as described previously. 5 , 14 , 15 , 26 Behavioral testing was conducted twice before surgery (day 0), and days 2 through 12 after surgery, with a rest day on the day of and after surgery. The 50% paw withdrawal threshold was calculated and treatment groups were analyzed by two-way analysis of variance (ANOVA) and post hoc Tukey testing using GraphPad Prism 8.0.…”

“…The 50% paw withdrawal threshold was calculated and treatment groups were analyzed by two-way analysis of variance (ANOVA) and post hoc Tukey testing using GraphPad Prism 8.0. 5 , 15 …”

“…We have previously shown that localized delivery of a single micro-dose (0.24 mg/kg) of CXB in nanoemulsion, which is incorporated in circulating macrophages/monocytes that then naturally accumulate at the site of injury provides up to six days of pain relief. 5 , 14 , 15 , 25 An important feature is that the nanomedicine delivers drug to the affected injured nerve. 5 , 14 , 15 This creates a unique opportunity to assess drug effects on cellular expression of RNAs when the drug is present at the distal injury and virtually no other parts of the peripheral nervous system.…”

“… 5 , 14 , 15 , 25 An important feature is that the nanomedicine delivers drug to the affected injured nerve. 5 , 14 , 15 This creates a unique opportunity to assess drug effects on cellular expression of RNAs when the drug is present at the distal injury and virtually no other parts of the peripheral nervous system. With this approach, we have been able to reveal transcriptome changes in the DRG associated with CXB-NE.…”