Astrocyte Reactivity in Female Rats with Persistent Low Back Pain Following Spinal Mobilization

Abstract

Low back pain (LBP) is a physiologically complex and highly disabling condition with poorly understood pathophysiology. Glial reactivity has been reported in patients with chronic LBP and astrocyte reactivity has been suggested as a potential contributor to LBP chronicity.1 Spinal mobilization (SM) is a non‐pharmacological approach with mild to moderate efficacy in treating LBP but physiological mechanisms responsible are unknown.PurposeTo investigate changes in astrocyte reactivity in the upper lumbar spinal cord following SM treatment in a Nerve‐Growth Factor (NGF)‐induced LBP pre‐clinical model. We hypothesize that the administration of trunk NGF will increase astrocyte reactivity, while SM treatment will decrease this reactivity in the upper lumbar spinal cord of female rats.MethodsFemale Sprague Dawley rats (212‐252g) were unilaterally injected with either NGF (50μl;2.0μM) or phosphate buffer saline ‐ PBS (50μl; control) on Days 0 and 5 in the left multifidus muscle under isoflurane anesthesia (1‐2%/2L O2). Ten days lapsed allowing for muscular LBP to develop in NGF animals. Treatment groups (NGF + SM [N=5]; PBS + SM [N=4]) received daily passive SM (10 min) via a feedback motor (1.2Hz, 0.9N) from Days 10 to 16, while the NGF only (N=5) group received no SM treatment. On Day 17, lumbar spinal cord (L1–L3) segments were harvested, fixated, and sectioned at 14μm. Sections were blocked and permeabilized (0.1% Triton X‐100 in 5% donkey serum) for 20 min at room temperature (RT), rinsed in PBS (6 x 5 min), and incubated with primary (Polyclonal Rabbit Anti‐Glial Fibrillary Acidic Protein ‐ 3h at RT) and secondary antibodies (IgG Highly Cross‐Adsorbed Donkey anti‐Rabbit, Alexa Fluor® 488 ‐ 1h at RT). Sections were then mounted on Fluorogel II containing 4′,6‐diamidino‐2‐phenylindole. Images of the superficial dorsal‐horn (laminas I‐III) and deep dorsal‐horn (lamina V) were captured at 20x magnification (Nikon Eclipse). Astrocyte mean fluorescent intensity (MFI) was quantified using FIJI/ImageJ® by blinded lab personnel. Analysis of variance was used to assess the differences in MFI between groups on the left and right sides of the spinal cord dorsal‐horn.ResultsNo significant differences between groups at the superficial horn level was found (p>0.05). Astrocyte MFI was statistically significantly higher in the NGF‐only group when compared to the PBS +SM group at the L3 spinal cord deep‐horn level (p=0.03). SM did not appear to decrease astrocyte reactivity at superficial or deep‐horn spinal cord levels.ConclusionOverall, these preliminary findings suggest that superficial‐horn astrocyte reactivity does not play a large mechanistic role in this female NGF‐induced LBP model despite astrocytic hyperreactivity being demonstrated among male rats with NGF‐induced LBP. Biological sex differences in glial signaling have been reported in other pre‐clinical pain models which may provide an explanation for the non‐significant NGF findings among females. Whether SM acts to decrease astrocyte reactivity in male NGF‐induced LBP rats remains to be investigated.