Defense Date

6-12-2020

Graduation Date

Summer 8-8-2020

Availability

One-year Embargo

Submission Type

dissertation

Degree Name

PhD

Department

Biological Sciences

Committee Chair

John Archie Pollock

Committee Member

Jelena Janjic

Committee Member

Benedict Kolber

Committee Member

Sarah Woodley

Keywords

chronic pain, neuroinflammation, nanomedicine, neuropathic pain, RNA sequencing, celecoxib

Abstract

The neuroinflammatory response to peripheral nerve injury is associated with chronic pain and significant changes in the expression profiles of RNAs in neurons, glia and infiltrating immune cells: a neuro-immune triad. Chronic constriction injury (CCI) of the rat sciatic nerve provides an opportunity to mimic neuropathic injury and quantitatively assess behavior and differential gene expression in individual animals. Macrophages that phagocytose intravenously injected nanoemulsion carrying the non-steroidal anti-inflammatory, NSAID, Celecoxib, naturally accumulate at the site of injury resulting in relief of CCI behavioral hyper-sensitivity. It is not known beyond the inhibition of cyclooxygenase-2 (COX-2) activity and the reduction in prostaglandin E2 (PGE2), what gene expression may be altered by this treatment. Previously, we showed that a single intravenous injection of nanoemulsion containing celecoxib (0.24 mg/kg) reduced inflammation of the sciatic nerve and relieved pain-like behavior for up to 6 days. To elucidate aspects of the molecular mechanisms underlying CCI pain as well as pain relief, I assessed an NSAID-loaded nanoemulsion macrophage-targeted therapy to explore changes in RNA expression in both pain and pain-relieved states. Sciatic nerve and dorsal root ganglia (DRG) tissue from CCI animals was used to evaluate the expression profiles utilizing quantitative polymerase chain reactions (qPCR) and RNA sequencing, respectively. In the injured sciatic nerve treated with drug-loaded nanoemulsion, I observed mRNA changes consistent with the reduced recruitment of macrophages evident by a reduction in chemokine and cytokine mRNA expression. Furthermore, genes associated with selective adhesion of macrophages, as well as changes in the neuronal and glial associated mRNAs were observed. Moreover, several neuroactive genes were found to respond to the celecoxib loaded nanoemulsion in animals receiving pain relief as compared to animals that received drug-free vehicle including: Monoamine oxidase B (MAOB), NMDA-Receptor 2b (Grin2b), Calcium channel TRPV3, Interleukin IL-6, Voltage-dependent Calcium channel subunit alpha 1B (Cacna1b/C2v2.2), Integrin alpha M (Itgam/Cd11b), Sodium channel Nav1.7 (Scn9a), and Preprotachykinin-1 (Tac1), which produces neurokinin A and substance P.

The transcriptome of the ipsilateral DRGs was assessed using high throughput RNA sequencing to provide insight into the molecular mechanisms involved in the underlying neuroimmune response. Comparing the DRG transcriptome of CCI animals treated with celecoxib-loaded nanoemulsion (CXB-NE) as well as drug-free (vehicle) nanoemulsion (DF-NE) to naïve, unoperated rats revealed significant differential RNA expression of 115 genes. Differential expression of particular transcripts was validated by quantitative PCR in the DRG and the bioinformatics tool PANTHER (protein annotation through evolutionary relationship) revealed that the differentially expressed genes involved in the neuroinflammatory response are associated with several key signaling mechanisms. This study showed that by directly inhibiting COX-2 activity in infiltrating macrophages at the site of injury on the sciatic nerve, there was a subsequent change in RNA expression in cell bodies of the DRG. While it is not surprising that peripheral nerve injury is associated with extensive changes in the expression profile of RNAs in the associated DRG, it is clear that attenuating the activity of COX-2 and the corresponding reduction in PGE2 at the site of injury results in changes in the transcriptome of the associated DRG reflecting pain relief as well as the partial reversal of the gene expression profiles in the injured sciatic nerve associated with chronic pain. Overall, it can be concluded that by solely targeting COX-2 production in macrophages in the CCI model, there is a direct effect of gene expression changes related to cells associated with the neuroimmune triad, the axon regeneration process, and the chronic pain response.

Language

English

Additional Citations

A. Stevens, L. Liu, D. Bertovich, J.M. Janjic, J.A. Pollock. Differential expression of neuroinflammatory mRNAs in the rat sciatic nerve following chronic constriction injury and pain-relieving nanoemulsion NSAID delivery to infiltrating macrophages. International Journal of Molecular Sciences 2019; 20(21): 5269. Doi.org/10.3390/ijms20215269.

A. Stevens, M. Saleem, B. Deal, J.M. Janjic, J.A. Pollock. Targeted COX-2 inhibiting nanomedicine results in pain-relief and differential expression of RNA in the dorsal root ganglia of injured male rats. Molecular Pain In Press 2020.

M. Saleem*, A. Stevens*, B. Deal, L. Liu, J. M. Janjic, J. A. Pollock. A new best practice for validating tail vein injections in rat with near infrared labeled agents. Journal of Visualized Experiments 2019; e59295. *Co-authors contributed equally.

J. M. Janjic, K. Vasudeva, M. Saleem, A. Stevens, L. Liu, S. Patel, J. A. Pollock. Low-dose NSAIDs reduce pain via macrophage targeted nanoemulsion delivery to neuroinflammation of the sciatic nerve in rat. Journal of Neuroimmunology 2018; 318: 72-79.

Download

Share

COinS