Melatonin Decreases Acute Inflammatory Response to Neural Probe Insertion

Abstract

Neural electrode insertion trauma impedes the recording and stimulation capabilities of numerous diagnostic and treatment avenues. Implantation leads to the activation of inflammatory markers and cell types, which is detrimental to neural tissue health and recording capabilities. Oxidative stress and inflammation at the implant site have been shown to decrease with chronic administration of antioxidant melatonin at week 16, but its effects on the acute landscape have not been studied. To assess the effect of melatonin administration in the acute phase, specifically the first week post-implantation, we utilized histological and q-PCR methods to quantify cellular and molecular indicators of inflammation and oxidative stress in the tissue surrounding implanted probes in C57BL/6 mice as well as two-photon microscopy to track the microglial responses to the probes in real-time in transgenic mice expressing GFP with CX3CR1 promotor. Histological results indicate that melatonin effectively maintained neuron density surrounding the electrode, inhibited accumulation and activation of microglia and astrocytes, and reduced oxidative tissue damage. The expression of the pro-inflammatory cytokines, TNF-α and IL-6, were significantly reduced in melatonin-treated animals. Additionally, microglial encapsulation of the implant surface was inhibited by melatonin as compared to control animals following implantation. Our results combined with previous research suggest that melatonin is a particularly suitable drug for modulating inflammatory activity around neural electrode implants both acutely and chronically, translating to more stable and reliable interfaces.

Keywords: anti-inflammatory; antioxidant; glial scar; insertion trauma; melatonin.

Figure 1

Melatonin (MT) administration preserves neuron density near the electrode site. (a) Representative images for NeuN (neurons) and NF200 (axons) staining in control and MT groups. (b) Plot of neuron body density (counted neurons per square μm) versus distance from implanted electrode center. (c) Bar graph depicting neuron density at 20 μm from electrode site. (d) NF200 intensity normalized to image corners versus distance from electrode. (e) Bar graph depicting normalized NF200 intensity at 20 μm from electrode site. (**: p < 0.01, ***: p < 0.001, two-way ANOVA).

Figure 2

Melatonin (MT) decreases Caspase-3 expression in acute electrode implantation. (a) Representative images for Caspase-3 (apoptosis marker) staining in control and MT groups. White arrows indicate areas with Caspase-3 expression. Overlay with NeuN (grey) and Caspase-3 (red) to indicate neuron death. (b) Caspase-3 counts for both groups at different distances from electrode center (n.s., Two-way ANOVA).

Figure 3

Melatonin (MT) administration prevents astrocytic activation near the electrode site. (a) Representative images for GFAP (astrocytes) and tomato lectin (vasculature and microglia) staining in control and MT groups. (b) Plot of GFAP intensity normalized to image corners versus distance from electrode center. (c) Bar graph depicting normalized GFAP intensity at 50 μm from electrode site. (d) Tomato lectin intensity normalized to image corners versus distance from electrode. (e) Bar graph depicting normalized tomato lectin intensity at 50 μm from electrode site. (*: p < 0.05, **: p < 0.01, ****: p < 0.0001, Two way ANOVA).

Figure 4

Melatonin (MT) administration prevents microglial activation and lipid peroxidation near the electrode site. (a) Representative images for Iba-1 (microglia) and 4HNE (lipid peroxidation) staining in control and MT groups. (b) Plot of Iba-1 intensity normalized to image corners versus distance from electrode center. (c) Bar graph depicting normalized Iba-1 intensity at 50 μm from electrode site. (d) 4HNE intensity normalized to image corners versus distance from electrode. (e) Bar graph depicting normalized 4HNE intensity at 50 μm from electrode site. (*: p < 0.05, ***: p < 0.001, ****: p < 0.0001, two-way ANOVA).

Figure 5

PCR relative quantification of TNF-α and IL-6-fold change indicative of inflammatory response under various conditions. Fold change is relative to assay standards. (a) Comparison of TNF-α fold change near electrode implant between groups. (b) Comparison of IL-6-fold change near electrode implant between groups. (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. One way ANOVA). Groups: Control (untouched neural tissue), MT(+P) (MT-treated neural tissue with probe implanted), MT(−P) (MT-treated neural tissue without probe implanted), Saline(+P) (saline-treated neural tissue with probe implanted), and Saline(−P) (saline-treated neural tissue without probe implanted).

Figure 6

Chronic TPM imaging of dummy electrodes over time. (a) Representative TPM images showing the surface of electrodes over 7 days post insertion. Green, CX3CR1-GFP; red, blood vessel; blue, second-harmonic signal from collagen. Yellow dotted boxes indicate the location of electrodes. (b) Quantification of microglial coverage on the electrode surface over time. Mean ± SD are plotted. Two-way ANOVA followed by multiple comparisons at each time point (*: p < 0.05, ***: p < 0.001). (c) The movement speed of microglial end-feet after electrode implantation. Mean ± SD are plotted.