Background & Aims

The need for novel, non-opioid analgesics is glaringly urgent. Chronic pain is characterized by neuroplastic pathologies that produce maladaptive refinement of pain circuits, which are dependent on activation of the N-methyl-D-aspartate (NMDA) receptor (1). Additionally, microglia, the resident immune cells of the nervous system, act as essential neuroplastic regulators (2) and likely contribute to the sex differences seen in chronic pain (3-5). Agmatine, an endogenous decarboxylated form of L-arginine, is an antagonist of the NMDA receptor and has demonstrated significant analgesic efficacy in preclinical models (6-8). We have observed that agmatine appears to be associated with elevation in microglial residents, specifically in female subjects, however, it is still unclear whether microglial recruitment contributes to inhibitory effects of agmatine in pain. The aim of this study was to characterize the effect of agmatine on microglial function in inflammatory pain.

Methods

Male and female ICR CD-1 mice (21-30 g) were intrathecally administered (5 ul) with either saline or 30 nmol agmatine (9). Half of the mice from each group were administered complete Freund’s adjuvant (CFA) into the left hind paw to induce an inflammatory state. Hypersensitivity was tested by using an electronic von Frey device. Mice were sacrificed and perfused with 4% paraformaldehyde. Spinal cords were extracted and prepared for immunohistochemical analysis with the microglia marker, Iba1. An Olympus BX34 microscope was used to take 10x images of the lumbar dorsal horn. ImageJ2 version 2.14.0/1.54 was used to quantify Iba1 immunoreactivity. ImageJ2 plug in Microglia Morphometry (10) was used to generate image intensities and label masks to score various morphological parameters of microglia. R studio was used to generate solidity, somaness, and branchiness scores. Code was adapted from Martinez et al., 2023. GraphPad Prism version 10.0.2 (171) was used to statistically analyze data

Results

One-way ANOVA determined a significant reduction in hypersensitivity of female mice treated with agmatine and CFA compared to female mice treated with saline and CFA (P= 0.023). Males treated with agmatine and CFA showed a trend towards attenuated hypersensitivity. Female, but not male, mice that received both agmatine alone and agmatine + CFA showed significantly increased Iba1 immunoreactivity when compared to all other groups, as determined by one-way ANOVA and Tukey’s post-hoc test. One-way ANOVA determined a significant difference between agmatine and saline groups treated with CFA for the morphological solidity score (P= 0.0060), which is the area of the cell divided by the convex area. No other statistically significant morphological differences were found; however, female subjects treated with agmatine alone showed a trend towards increased branchiness, determined by dividing the number of branch points of a cell by its geodesic diameter.

Conclusions

These data show that agmatine is analgesic in murine inflammatory pain in male and female mice. Agmatine alone is sufficient to induce a significant increase in Iba1 immunoreactivity in female, but not male, spinal cord. Moreover, agmatine induces distinct microglial morphological differences in female spinal cord that indicate agmatine may be preventing the proinflammatory actions of microglia that contribute to inflammatory pain. Future studies will focus on the origin (central vs. peripheral) of the increase in Iba1+ cells in female subjects administered with agmatine and their specific roles (phagocytic vs not).

References

References

1.Qu XX, Cai J, Li MJ, Chi YN, Liao FF, Liu FY, et al. Role of the spinal cord NR2B-containing NMDA receptors in the development of neuropathic pain. Exp Neurol. 2009 Feb;215(2):298–307.

2.Zhou LJ, Peng J, Xu YN, Zeng WJ, Zhang J, Wei X, et al. Microglia Are Indispensable for Synaptic Plasticity in the Spinal Dorsal Horn and Chronic Pain. Cell Rep. 2019 Jun 25;27(13):3844–59.e6.

3.Silveira Prudente A, Hoon Lee S, Roh J, Luckemeyer DD, Cohen CF, Pertin M, et al. Microglial STING activation alleviates nerve injury-induced neuropathic pain in male but not female mice. Brain Behav Immun. 2024 Mar 1;117:51–65.

4.Kuhn JA, Vainchtein ID, Braz J, Hamel K, Bernstein M, Craik V, et al. Regulatory T-cells inhibit microglia-induced pain hypersensitivity in female mice. Elife [Internet]. 2021 Oct 15;10. Available from: https://dx.doi.org/10.7554/eLife.69056

5.5. Huck NA, Donovan LJ, Shen H, Jordan CE, Muwanga GPB, Bridges CM, et al. Sex-distinct microglial activation and myeloid cell infiltration in the spinal cord after painful peripheral injury. Neurobiol Pain. 2022 Oct 12;12:100106.

6.Fairbanks CA, Schreiber KL, Brewer KL, Yu CG, Stone LS, Kitto KF, et al. Agmatine reverses pain induced by inflammation, neuropathy, and spinal cord injury. Proc Natl Acad Sci U S A. 2000 Sep 12;97(19):10584–9.

7.Peterson CD, Waataja JJ, Kitto KF, Erb SJ, Verma H, Schuster DJ, et al. Long-term reversal of chronic pain behavior in rodents through elevation of spinal agmatine. Mol Ther. 2023 Apr 5;31(4):1123–35.

8.Peterson CD, Kitto KF, Verma H, Pflepsen K, Delpire E, Wilcox GL, et al. Agmatine requires GluN2B-containing NMDA receptors to inhibit the development of neuropathic pain. Mol Pain. 2021 Jan;17:17448069211029171.

9.Hylden JL, Wilcox GL. Intrathecal morphine in mice: a new technique. Eur J Pharmacol. 1980 Oct 17;67(2-3):313–6.

10.Martinez A, Hériché JK, Calvo M, Tischer C, Otxoa-de-Amezaga A, Pedragosa J, et al. Characterization of microglia behaviour in healthy and pathological conditions with image analysis tools. Open Biol. 2023 Jan;13(1):220200.

Presenting Author

Cecilia Barajas

Poster Authors

Cecilia Barajas

OTHR

University of Minnesota

Lead Author

George Wilcox

University of Minnesota

Lead Author

Cristina Peterson

University of Minnesota

Lead Author

Kelley F. Kitto

University of Minnesota, Dept. of Pharmaceutics

Lead Author

Carolyn A. Fairbanks

PhD

University of Minnesota, Dept. of Pharmaceutics

Lead Author

Topics

  • Treatment/Management: Interventional Therapies – Intrathecal Drug Delivery