Inhibition of Na+/H+exchanger modulates microglial activation and scar formation following microelectrode implantation

Abstract

Objective.Intracortical microelectrodes are an important tool for neuroscience research and have great potential for clinical use. However, the use of microelectrode arrays to treat neurological disorders and control prosthetics is limited by biological challenges such as glial scarring, which can impair chronic recording performance. Microglia activation is an early and prominent contributor to glial scarring. After insertion of an intracortical microelectrode, nearby microglia transition into a state of activation, migrate, and encapsulate the device. Na⁺/H⁺exchanger isoform-1 (NHE-1) is involved in various microglial functions, including their polarity and motility, and has been implicated in pro-inflammatory responses to tissue injury. HOE-642 (cariporide) is an inhibitor of NHE-1 and has been shown to depress microglial activation and inflammatory response in brain injury models.Approach.In this study, the effects of HOE-642 treatment on microglial interactions to intracortical microelectrodes was evaluated using two-photon microscopyin vivo.Main results.The rate at which microglia processes and soma migrate in response to electrode implantation was unaffected by HOE-642 administration. However, HOE-642 administration effectively reduced the radius of microglia activation at 72 h post-implantation from 222.2µm to 177.9µm. Furthermore, treatment with HOE-642 significantly reduced microglial encapsulation of implanted devices at 5 h post-insertion from 50.7 ± 6.0% to 8.9 ± 6.1%, which suggests an NHE-1-specific mechanism mediating microglia reactivity and gliosis during implantation injury.Significance.This study implicates NHE-1 as a potential target of interest in microglial reactivity and HOE-642 as a potential treatment to attenuate the glial response and scar formation around implanted intracortical microelectrodes.

Keywords: brain-computer interface; foreign body response; gliosis; inflammation; intravital imaging.

Figure 1.

Experimental setup for two-photon imaging. (a) Timeline of experimental procedures. HOE-642 was administered immediately prior to surgery for acutely observed mice. (b) Schematic of headcap and imaging window installation suitable for sub-chronic in vivo two-photon imaging of a microelectrode implanted in the mouse cortex. The imaging window was sealed with a transparent elastomer and glass coverslip to protect the brain surface while allowing for longitudinal imaging. (c) 2D representative image visualizing microglia (green) and cortical vasculature (red) around an implanted microelectrode array (shaded blue). (d) A microglial cell of ramified morphology. (e) A microglial cell of transitional (activated) morphology.

Figure 2.

Microglia extend processes in the direction of the microelectrode at velocities unaffected by HOE-642 treatment. (a) Microglial process extension around an implanted probe (outlined blue) over time in a HOE-treated mouse. Consecutive timepoints are labeled in red (preceding) and green (subsequent). Yellow (overlap of red and green) indicates no spatial movement of cells or processes between consecutive time points. Immediately following insertion, microglia processes move toward the electrode as indicated by layers of green end-feet followed by layers of red end-feet (white triangles). Multiple processes extend toward the probe surface until they initiate contact and stop moving (yellow overlap). (b) Velocity of actively migrating microglia processes in the direction of the electrode over time demonstrate active migration until 40–50 min after insertion, at which point most processes make contact with probe surface and stop moving. The velocities of microglia processes in HOE-treated (blue solid) and control (black dashed) animals follow similar trajectories, suggesting that HOE-642 does not influence rate at which processes migrate toward the electrode. Data is plotted as mean ± SEM.

Figure 3.

HOE-642 administration does not influence microglia migration velocities following microelectrode implantation. (a), (b) Merged 2D images of microglia at 12 h (red) and 24 h (green) migrating toward an implanted microelectrode (shaded blue). Processes and cell soma of activated microglia migrate toward the surface of the probe in both control (a) and HOE (b) mice. Note the less activated morphology of microglia in HOE-treated mice compared to the control. (c), (d) Microglia migration begins around 12–24 h post-implantation. Microglia cell body velocity between HOE-treated and control mice remains fairly constant with respect to time (c) and distance (d) from the probe suggesting that, similar to microglia process extension, HOE-642 does not influence rate of cellular migration following microelectrode implantation. Data is plotted as mean ± SEM.

Figure 4.

HOE-642 treatment reduces morphological activation of microglia in a spatiotemporal manner. (a), (b) Representative two-photon images from control (a) and HOE-treated (b) mice depict more ramified (R) and less transitional (T) microglia closer to the probe at 12 h post-implantation following HOE-642 treatment. Red dotted lines show the axis separating processes extending off the hemispheres toward and away from the probe surface (shaded blue). (c), (d) Average ramification indices between HOE-treated (circles and solid lines) and control (asterisks and dashed lines) mice at different time points post-insertion is shown with fitted logistic regression curves across distance away from the probe surface. Acutely implanted mice (c) were measured from 2 h to 6 h post-insertion (3, 4, and 5 h data omitted from plot for clarity). Sub-chronically implanted mice (d) and (e) were evaluated from 6 to 72 h. Plots were separated into 6–24 and 48–72 h to ease readability. For a given spatial bin, ramification generally decreased with time, indicating progressive microglia activation. Activation radii were defined as the distance at which there was a 50% probability for ramification (0.5 ramification index). HOE-642 administration visibly shifted the ramification curve leftwards (less activation) compared to controls indicating a reduction in microglia reactivity after implantation. (f) Difference in activation radii, defined as the distance at which ramification is 50% (0.5) at a given timepoint, between HOE-treated and control groups in sub-chronic mice over time. Error bars are not shown because values are not averages, but rather results from logistic regression analyses. Positive differences in activation radii suggest a greater radius of activation in control mice compared to HOE-treated mice.

Figure 5.

HOE-642 induces changes in number and length of processes directed towards and away from implants. Morphological T- and D-indices between HOE-treated (circles and solid lines) and control (asterisks and dashed lines) mice. (a)–(c) T-index calculates degree of activation depending on length of most prominent leading versus lagging microglia process with respect to the electrode. (d)–(f) D-index calculates degree of activation depending on number of leading versus lagging microglia processes with respect to the electrode. Values near one indicate ramification (equal in length or number of leading vs. lagging processes) and values near zero suggest activation with preferred orientation toward the electrode. Acutely implanted mice were observed from 2 to 6 h post-insertion (3, 4, and 5 h data omitted from plot for clarity). Values are fitted with custom dual sigmoidal functions and error bars indicate standard error. For a given spatial bin, T- and D-indices generally decreased with time, indicating a greater degree of microglia polarization. HOE-642 administration shifted T- and D-index curves upwards (more ramified) compared to controls suggesting attenuation microglia activation in response to electrode implantation.

Figure 6.

HOE-642 administration attenuates severe microglial encapsulation of intracortical electrodes. (a), (b) Thresholded microglial (GFP-positive) signal up to 20 μm above the surface of the implant (blue outline) in control (a) and HOE-treated (b) mice demonstrate an extensive reduction of microglia encapsulation in the HOE-treated mouse 5 h post-implantation. (c), (d) Percent of microglial surface coverage between HOE-treated and control mice at acute (c) and chronic (d) time points. HOE-642 treatment significantly lowered surface coverage for both acute (c) and chronic (d) mice (two-way ANOVAs; p < 0.001). Note that surface coverage in HOE-treated mice increased relative to control mice during the sub-chronic phase resulting in no significant difference in surface coverage between the two groups, suggesting a drop in efficacy of chronic HOE-642 administration. Statistical differences in surface coverage between HOE and control mice at individual timepoints are shown (Bonferroni corrected Welch’s t-tests; * p < 0.05). Data is plotted as mean ± SEM.