(K-417) Spatial Proteomic Analysis of Intracortical Microelectrode Implant Site

Intracortical microelectrodes (IMEs) are devices implanted in the brain’s cortex that can record neuronal activity. One application of these recorded signals can be used to help individuals with motor deficits operate prosthetic devices or wheelchairs [1]. Currently, recording quality for IMEs decays over chronic time points, with significant evidence attributing this failure to the neuroinflammatory response following implantation [2].  

Upon insertion, microelectrodes rupture blood vessels, breaking the blood-brain barrier (BBB). The implanted electrode activates microglia, resident immune cells in the brain, causing them to accumulate around the electrode. Implantation also activates astrocytes, causing them to mobilize towards the implant and envelope the electrode in a glial scar [3]. Inflammatory cells that respond to electrode implantation, such as microglia and astrocytes, secrete proinflammatory and cytotoxic factors that prolong inflammation and kill local neurons [4]. Thus, neuronal death combined with glial scar impedance results in chronic failure of recording microelectrodes [5]. 

Currently, there is no treatment to fully prevent inflammation and electrode failure, potentially because the underlying biological pathways are yet to be fully understood. Our proteomic study aims to bridge this gap by analyzing the differential expression of 42 proteins around the implant site. The chosen proteins are associated with microglial activation, glial scar formation, and neuronal death, making this the first large-scale proteomic study of IME implanted tissue. Our results provide new information about pathways activated during neuroinflammation, which could reveal potential therapeutic targets to improve IME performance.

C57BL/6J wild-type mice were implanted with four non-recording Michigan-style probes in the motor cortex and sacrificed 8 weeks following implantation. Non-surgical animals were used as controls. Following perfusion with PBS and 30% sucrose, brain extraction, and slicing, brain sections stained with custom antibodies from NanosString Technologies conjugated with UV-cleavable fluorescent barcodes unique to each protein of interest.

After staining, the tissue was imaged in Nanostring’s GeoMx Digital Spatial Profiler, which produced fluorescent images used to locate the implant site. Using a custom Fiji code, the tissue around the implant was segmented into three concentric rings called areas of interest (AOIs), each 90 µm wide (Figure 1). After AOI selection, the GeoMx used a UV light to cleave and collect barcodes from each of the three AOIs. The fluorescent barcodes were counted to capture information about the expression of each of the 42 proteins within each AOI. This process was repeated for control animals, but rather than an implant site, a region of healthy tissue was chosen and segmented into AOIs. 

Protein counts from the inner, middle, and outer AOIs of implanted animals were compared to the respective rings in control animals and to each other. Differential expression was quantified through fold change. Using an unpaired, two-tailed t-test for equal variance, statistically significant differentially expressed proteins were identified. To account for random significance, a Benjamini-Hochberg correction was used with a false discovery rate of 0.05. 

Protein expression analysis indicated that markers for microglial activation (CD11b and CD45) were significantly upregulated in the implanted inner ring compared to control (Figure 2). This panel of proteins also measured the expression of astrocytic markers such as GFAP and vimentin. Our results indicate significantly more GFAP expression in the implanted inner ring compared to the middle and outer (Figure 3), which is consistent with previous findings that show upregulation of GFAP and vimentin to be a hallmark of astrocyte activation and glial scar formation [6]. However, all implanted AOIs show downregulation of vimentin, indicating that another pathway besides astrocytic activation is dominating vimentin expression (Figure 2). Vimentin is abundantly expressed in vascular endothelium [7], which is ruptured during implantation. Thus, downregulation of vimentin could indicate BBB damage, outweighing the increased expression during astrocyte activation. Alternatively, markers for neuron viability, such as myelin basic protein and neurofilament, were downregulated in all AOIs of implanted animals, indicating a loss after device implantation.

Another significant group of proteins measured was related to autophagy, a survival mechanism important for clearing intracellular protein aggregates. Autophagy proteins were downregulated across all implanted AOIs (ULK1, ATG5) (Figure 2), except for CTSD, which was upregulated in the inner ring (Figure 3). Previous studies have reported that CTSD is essential for lysosomal formation during autophagy [8,9], while another study has linked CTSD expression to apoptosis [10].  Though the role of CTSD expression has not been studied in the brain, the expression of CTSD without other autophagy proteins suggests that apoptotic pathways may be overriding autophagy, inducing cell death rather than prolonging survival.

Our results provide insight into potential regulators of microglial activation, glial scar formation, and neuronal death, all hallmarks of the neuroinflammatory response to intracortical microelectrodes. In the future, we aim to further segment implanted tissue to understand cell-specific changes in protein expression following microelectrode implantation. Targeting any or a combination of the proteins identified here, on a spatial or cell-specific level, could become a viable strategy to improve the chronic recording performance of intracortical microelectrodes and effectively improve outcomes for clinical applications.

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