Understanding the Role of Innate Immunity in the Response to Intracortical Microelectrodes

Abstract

Intracortical microelectrodes exhibit enormous potential for researching the nervous system, steering assistive devices and functional electrode stimulation systems for severely paralyzed individuals, and augmenting the brain with computing power. Unfortunately, intracortical microelectrodes often fail to consistently record signals over clinically useful periods. Biological mechanisms, such as the foreign body response to intracortical microelectrodes and self-perpetuating neuroinflammatory cascades, contribute to the inconsistencies and decline in recording performance. Unfortunately, few studies have directly correlated microelectrode performance with the neuroinflammatory response to the implanted devices. However, of those select studies that have, the role of the innate immune system remains among the most likely links capable of corroborating the results of different studies, across laboratories. Therefore, the overall goal of this review is to highlight the role of innate immunity signaling in the foreign body response to intracortical microelectrodes and hypothesize as to appropriate strategies that may become the most relevant in enabling brain-dwelling electrodes of any geometry, or location, for a range of clinical applications.

I. INTRODUCTION

A. Failure of Intracortical Microelectrodes

In order to become a clinical solution for many brain interfacing applications, intracortical microelectrodes should ideally operate consistently over time ranges of years to decades to prevent the repetition of highly invasive brain surgeries. Unfortunately, intracortical microelectrodes fail to consistently record neural signals over extended periods. Many labs studying various chronically implanted intracortical microelectrodes have demonstrated trends of the decreasing number of units detected,^1–6 the number of channels detecting units,^1,2,7–11 signal amplitudes,^4,10,12 and decoder performance,⁴ as well as generally inconsistent recording performance^1,7,13 and recording longevity,^8,10,13 regardless of the animal model. Together, such characteristics are not ideal for the long-term performance of brain-computer and brain-machine interfaces.

Declining and inconsistent recording performance has been ascribed to many failure mechanisms, typically grouped into mechanical, material, or biological mechanisms.¹⁰ In their review, Barrese et al. defined mechanical failures as physical relocation or damage of the electrode array and hardware, material failures as breakdown of electrode array materials, and biological failures as a consequence of the foreign body response or implantation trauma following device insertion.¹⁰ Mechanical and materials related failure modes have recently been reviewed and will not be discussed here (see Prasad et al., Barrese et al., Jorfi et al., and Kozai et al. for examples^7,9,10,14,15). Biological failures are typically associated with the interruption of electrical signals between cortical neurons and recording contacts. They will be further described and discussed in the sections that follow.

B. Foreign Body Response to Intracortical Microelectrodes

Intracortical microelectrodes fail, in part, due to biological mechanisms, many of which result from the foreign body response to the implant and subsequent chronic inflammatory cascades (for review, see Jorfi et al.¹⁴). As a microelectrode array is inserted in the brain, blood vessels are ruptured,¹⁶ releasing blood proteins into the brain parenchyma. Several components of blood, including proteins, can be neurotoxic.¹⁷ Furthermore, tissue is displaced and neurons and glia cells along the insertion path are damaged or killed.¹⁸ Blood proteins will adsorb to the surface of the electrode array and denature, upon which they are recognized by the brain’s major inflammatory cells, the microglia.^19,20 Subsequently, microglia transition from a dormant ramified state to a pro-inflammatory amoeboid phenotype by retracting processes and upregulating lytic enzymes.¹⁸ In the activated state, microglia are able to release a variety of soluble pro-inflammatory and cytotoxic factors, such as nitric oxide, reactive nitrogen species, and reactive oxygen species.^21–24 Evidence of oxidative gene expression and cellular oxidative damage have been detected at the electrode tissue interface.²⁵ Factors released by pro-inflammatory microglia likely contribute to neuronal death and neurodegeneration (Fig. 1).^21–24 Additionally, soluble factors released by microglia may damage the blood-brain barrier by disrupting tight adherens junctions connecting endothelial cells of the vasculature.²⁶ Furthermore, oxidative factors may also contribute to material degradation (Fig. 1).²⁷ Thus, consequences of electrode implantation and early inflammatory activation may contribute to the decline or unreliability of electrode performance.

FIG. 1:

Oxidative stress following neural probe implantation. The implantation of neural probes leads to the over-production of reactive oxygen species (ROS), which can consequently (1) perpetuate the foreign body response, (2) facilitate neuronal death, and (3) facilitate corrosion and delamination of the microelectrode surface. (Reprinted from Ereifej et al.²⁵ under a Creative Commons Attribution International License, Copyright 2018: https://creativecom-mons.org/licenses/by/4.0/.)

Attracted by the release of soluble factors by nearby microglia, more microglia migrate from the parenchyma. Likewise, additional monocytes and other myeloid cells are recruited from circulation across the leaky blood-brain barrier.^28,29 Blood proteins and necrotic cells from insertion damage, as well as the early inflammatory response and leaky blood-brain barrier promote pro-inflammatory activation in the newly recruited inflammatory cells.^20,28,30 One pathway by which microglia and macrophages recognize such damage and enact pro-inflammatory responses is through Toll-like receptors (TLRs).³¹ Later sections of this paper will discuss TLRs in depth. Soluble factors from the total collection of inflammatory cells around the implant may cause further damage to neurons, recording materials (Fig. 1), and the blood-brain barrier.^21–24 Repeated damage to cells and blood-vessels resulting in the recruitment and activation of more inflammatory cells is hypothesized to propagate self-perpetuated chronic inflammatory cascades indefinitely.^20,28 The neuronal and material damage resulting from self-perpetuating inflammatory cascades may contribute to long-term biological failure mechanisms.^20,28

As the microglial and macrophage response matures, within days to weeks, the fibroblast of the brain, astrocytes, begin their response to the implantation of the microelectrode. Astrocytes proliferate, become hypertrophic, and migrate to the implantation site, ultimately resulting in a dense encapsulation layer around the electrode.¹⁸ Astrocytic responses typically start wider and more diffuse, and then become denser and more compact over the course of several weeks.¹⁸ Astrocytic encapsulation isolates the inflammatory or damaged region around the electrode from the surrounding parenchyma. In addition to astrocytes, meningeal fibroblasts may migrate to the electrode-tissue interface and contribute to electrode encapsulation.^6,10 Together, encapsulation of the electrode may interfere with diffusion of neuroactive substances,³² increase the electrical impedance between the electrode and neurons,^13,33 increase the distance between neurons and electrodes,¹³ or in extreme cases promote extrusion of electrode arrays.¹⁰

Overall, chronic inflammation, neuronal loss, blood-brain barrier permeability, and encapsulation of microelectrode arrays may stem from the foreign body response to implanted intracortical microelectrodes and can be implicated in the recording performance of the implanted device.

C. Studies Linking Neuroinflammation to Electrode Failure

Despite all the efforts to overcome biological intracortical failure mechanisms (see Jorfi et al. or Hermann^14,34), only a handful of studies have directly compared neuroinflammation to electrode performance. Interestingly, each study can be linked to the innate immunity, the body’s fast-acting response to pathogenic threats. Here we briefly discuss the studies directly comparing neuroinflammation to electrode performance and their links to innate immunity.

Rennaker et al. were the first to demonstrate a correlation between microelectrode recording performance and the inflammatory response.³⁵ Specifically, in their study, Rennaker et al. investigated the effects of minocycline, chosen for its neuroprotective and neurorestorative effects,³⁶ on intracortical microelectrode recording performance.³⁵ Rats that were administered minocycline via water for two days before surgery through five days after surgery exhibited improved recording performance over controls after the first week of implantation, upon which the signal-to-noise ratio of controls steadily dropped.³⁵ End-point histological analyses revealed that astrocytic encapsulation was decreased at one- and four-week time points after implantation in animals treated with minocycline.³⁵ Improved recording performance was hypothesized to be caused by decreased inflammation, neuronal dieback, and microglia activation in addition to the observed decrease in astrocyte encapsulation. Minocycline was later shown to inhibit the pro-inflammatory phenotype of macrophages.³⁷ Alternative explanations could factor in the antibiotic activity of minocycline. Perhaps a lower bacterial load activated less inflammation via innate immunity pathways. Regardless of the mechanism of action, minocycline is not suitable for long-term administration due to detrimental side effects including bone discoloration.^38,39 The long-term clinical significance of minocycline induced bone discoloration remains unknown, yet is reported to be worrisome to orthopedic surgeons while further characterization is completed.^40,41

In contrast to the previous study administering an anti-inflammatory compound, Harris⁴² examined the effects of a pro-inflammatory compound on intracortical microelectrode performance in an unpublished thesis study. At the end of a four-week study, rats administered a one-time surgical dose of the pro-inflammatory agent lipopolysaccharide (LPS) exhibited significantly lower neuronal density within the first 50 μm away from the implant.⁴² Additionally, administration of LPS significantly reduced firing rates in evoked neural recordings.⁴² Thus, inflammation is associated with poor neuronal survival and recording performance. Interestingly, the pro-inflammatory agent LPS is derived from the bacterial cell walls of gram-negative bacteria and predominantly recognized by the innate immunity receptors cluster of differentiation 14 (CD14) and Toll-like receptor 4 (TLR4) to induce robust inflammatory responses.⁴³ The results of Harris⁴² indicate that activation of innate immunity can result in detrimental effects in recording performance and tissue integration.

Furthermore, Saxena et al. examined the effects of blood-brain barrier permeability on recording performance.²⁸ In this study, extravasation of blood proteins and myeloid cells into the brain parenchyma around intracortical microelectrodes implanted in rats coincided with poor recording performance.²⁸ The team hypothesized that chronic blood-brain barrier permeability following intracortical microelectrode implantation was part of a positive feedback loop with chronic inflammation, resulting in neurodegeneration,²⁸ similar to the self-perpetuating neurodegenerative response described by Potter et al.²⁰ One mechanism by which microglia and macrophages may recognize blood proteins and promote inflammation is through the innate immunity receptor Toll-like receptors.^31,44 Thus, innate immunity may play a role in the positive feedback loop of blood-brain barrier permeability and chronic inflammation that leads to intracortical microelectrode failure.

Finally, Kozai et al. investigated the role of caspase-1 in the recording performance of intracortical microelectrodes.¹¹ Caspase-1 was studied for its role in the activation of the pro-inflammatory cytokine IL-1β, as well as its role in neuronal death related to ischemia and chronic neurodegeneration.¹¹ Knockout mice lacking caspase-1 implanted with intracortical microelectrodes exhibited significantly improved single unit yields over wild type controls out to ~ 150 days after implantation with similar yields extending to the end of the experiment around 180 days after implantation.⁴⁵ Coincidentally, caspase-1 is involved in several innate immunity mechanisms, including inflammasome, RIG-like receptor, and TLR signaling.⁴⁵ In this instance, attenuation of innate immunity improved chronic intracortical microelectrode performance. Thus, innate immunity appears to play a role in the failure of intracortical microelectrodes.

Overall, studies linking inflammation to intracortical microelectrode performance reveal several connections by which innate immunity may be involved in intracortical microelectrode performance.

II. DISCUSSION

A. Innate Immunity

As indicated above, innate immunity has ties to the foreign body response to intracortical microelectrodes. Thus, further understanding of the innate immunity may inform strategies to mitigate intracortical microelectrode failures.

Innate immunity is often described as the body’s first line of defense against pathogenic threats.⁴⁶ Innate immunity should be distinguished from the body’s other defense system, adaptive immunity, by several characteristics. Innate immunity activates much more quickly, responding within minutes or hours, as compared to several days.^46,47 The quickness in responses is, in part, due to the widely distributed germline encoded effectors.⁴⁷ In contrast, the adaptive immunity requires genetic recombination and clonal expansion of specific effectors.^46,47 The disadvantage of germline encoded effectors is that innate immunity has less diversity and specificity of responses.⁴⁷ However, the recognition of general patterns conserved among classes of pathogens allows the innate immunity to be versatile and have capable effector cells distributed throughout the body.^46,47 In further contrast, the adaptive immunity features memory, meaning that adaptive responses to a previously encountered threat may be activated more quickly and with greater intensity.⁴⁷ Innate immunity is not traditionally thought to feature memory; thus, innate immune responses enact with a relatively consistent activation time and intensity. However, there is growing evidence that innate immunity may feature some degree of memory.⁴⁸ Innate and adaptive immunity are not completely independent, as the innate immunity often aids in the activation and regulation of adaptive immunity effectors.⁴⁷

Innate immunity is comprised of physical barriers, chemical barriers, and cellular responses.⁴⁷ Physical barriers include epithelial layers, mucosal tissues, and glandular tissues.⁴⁷ Chemical barriers include acidic fluids, anti-microbial proteins, and anti-microbial peptides that reside near the physical barriers.⁴⁷ Cellular innate immune responses are typically mediated by phagocytic cells, such as macrophages, neutrophils, dendritic cells, monocytes, and microglia, but can also involve natural killer cells, leukocytes, epithelial cells, and endothelial cells.⁴⁷ Additionally, complement glycoproteins found in serum are included in innate immunity.⁴⁷

In the event that pathogens bypass the physical and chemical barriers of the body, cellular effectors may address the threat. Cellular effectors of the innate immunity are generally activated via pattern-recognition receptors (PRRs), which recognize molecular patterns common to categories of pathogens referred to as pathogen associated molecular patterns (PAMPs). Some PRRs may also recognize molecular patterns on endogenous molecules released by the body called damage (or danger) associated molecular patterns (DAMPs).⁴⁷ The family of PRRs employed by the innate immunity are TLRs,⁴⁹ C-type lectin receptors,⁵⁰ Retinoic acid-inducible gene-I-like receptors (RLRs),^51,52 and Nod-like receptors (NLRs).^53,54 The TLRs and CLRs are transmembrane proteins expressed across plasma membranes; however, TLRs may also be expressed on endosomes and lysosomes.⁴⁷ In contrast, RLRs and NLRs are expressed in the cytosol.⁴⁷ Both TLRs and NLRs have demonstrated recognition of DAMPs in addition to PAMPs.⁴⁷ Activation of the various PRRs may result in pro-inflammatory activation, apoptosis, phagocytosis, coagulation cascades, opsonization, or complement activation.⁴⁶

In the context of foreign body responses to implanted intracortical microelectrodes, TLRs may be the most relevant. Some TLRs are expressed on plasma membranes,^55,56 so they may be able to respond to external threats directly. The TLRs also have the capability to recognize DAMPs and PAMPs,^31,57 enabling the potential detection of tissue, cellular, and vascular damage associated with intracortical microelectrode implantation, in addition to pathogens introduced with the microelectrode. Thus, we will also discuss TLRs and their adaptor molecule, CD14.

1. Toll-Like Receptors and CD14

One major group of effectors in innate immunity is a class of PRRs called TLRs.⁵⁸ The TLRs are transmembrane proteins that recognize molecular patterns associated with either pathogens or tissue damage and enact downstream inflammatory activation.^58,59 The TLRs are expressed on peripheral immune cells, such as macrophages and dendritic cells, as well as several cells of the central nervous system, including microglia, astrocytes, and neurons.^58,60,61

Toll-like receptors are named for their similarity to Toll,^62,63 a protein responsible for directing dorsal-ventral patterning in Drosophila embryos⁶⁴ and involved in innate immunity in adult Drosophila.⁶⁵ The TLRs were later identified in humans based on their shared molecular structure and downstream signaling pathways.⁶⁶

Toll-like receptors are type 1 transmembrane proteins that feature leucine-rich repeat (LRR) domains on the extracellular side and a Toll/IL-1 receptor (TIR) domain.^58,62,66,67 The LRRs are sequence motifs with frequent interspersed instances of the amino acid leucine and are found on many proteins involved in protein-protein interactions such as signal transduction.^58,68 The other major component of Toll-like receptors, the TIR domain, is a protein-protein interaction module found involved in the host responses of both plants and animals.^58,69

Building off the common structural elements of LRR and TIR domains, the TLR family gives rise to at least 12 different functional members in mice and 10 different functional members in humans.^49,51,58 The TLRs 1–9 are functional in both mice and humans, TLR10 is functional in humans but not mice,⁷⁰ and TLRs 11–13 have been identified in mice but not humans.⁴⁹ The members of the TLR family vary in membrane location and ligand specificity.⁵⁸ Some TLRs, such as TLR 1, 2, 4–6, 8, and 11, are located on the cell membrane, with the LRR domain facing the extracellular environment and the TIR domain facing the cytosol.^49,71,72 With an outward-facing ligand-binding domain, TLRs on the cell membrane monitor for external threats like bacteria. Other TLRs, such as TLR 3, 7, 8, and 9, are located on intracellular vesicles, with the LRR domains facing the interior of the vesicle and the TIR domain facing the cytosol.^49,71,72 With a vesicle-facing ligand-binding domain, TLRs on vesicle membranes monitor for internal threats, such as viruses. Each TLR recognizes its own set of ligands, which may be pathogen associated molecular patterns (PAMPs) or damage/danger associated molecular patterns (DAMPs).

The PAMPs recognized by TLRs and other PRRs are molecular patterns shared among broad categories of pathogens, allowing rapid and versatile innate immune responses with a handful of widely available receptor types.⁵⁸ The broad, fast-acting nature of TLRs contrasts against the highly specialized receptors and antibodies of the adaptive immunity, which require time-consuming clonal expansion to distribute effector cells to combat pathogens.⁵⁸ The PAMPs are effective targets for immune recognition because they occur in pathogens but not host cells, allowing inflammatory effector cells to discriminate between self and non-self.⁵⁸ Furthermore, PAMPs are typically molecular patterns involved in critical pathogenic survival mechanisms.⁵⁸ Mutations in pathogens removing PAMPs are typically deadly, allowing TLR signaling to remain effective across many generations of evolution.⁵⁸

Of note, PAMPs can be utilized in the recognition of bacterial, fungal, parasitic, and viral pathogens (for a detailed summary, see Akira et al.⁷³). As opposed to PAMPs, DAMPs are patterns found in endogenous molecules released in the event of noninfectious tissue damage (Table 1) from Pineau and Lacroix,³¹ Tsan and Gao,⁵⁷ and Beg.⁵⁹ The DAMPs recognized by TLRs include heat shock proteins (HSP), extracellular matrix (ECM) components, components of necrotic cells, surfactant protein A, RNA, DNA, and fibrinogen.^31,59 Heat shock proteins are molecules released when cells are exposed to various stresses.^31,57,74,75 TLRs can recognize HSP22,⁷⁶ HSP60,^77–79 HSP70,^80,81 and Gp96.⁸² Molecules may be cleaved from the ECM by proteolytic enzymes during the inflammatory response to traumatic injury,³¹ and TLRs can recognize heparan sulfate,^83–85 hyaluronan-derived oligosaccharide,^86,87 biglycan,⁸⁸ and fibronectin extra domain A.⁸⁹ Necrotic cells promote inflammation via TLR recognition of the released chromatin binding protein high mobility group box 1 (HMGB1)^30,90–93 or unidentified ligands.^94,95 Surfactant protein A is a component of pulmonary surfactant and may activate TLRs.⁹⁶ Host RNA^97–99 and DNA^97,100 can activate TLRs, but the method of endosome localization is unclear.⁹⁷ Fibrinogen escapes vasculature during inflammation and can bind TLRs to further propagate inflammatory mechanisms.⁴⁴

TABLE 1:

Summary of major damage associated molecular patterns (DAMPs) recognized by Toll-like receptors (TLRs), based on data from Pineau and Lacroix,³¹ Tsan and Gao,⁵⁷ and Beg⁵⁹

DAMP	TLR	Factors Produced	Refs.
Heat shock proteins (HSPs)
HSP60	TLR2, TLR4	TNF, NO	77–79
HSP70	TLR2, TLR4	TNF, IL-1b, IL-6, IL-12	80, 81
Gp96	TLR2, TLR4	TNF, IL-12	82
HSP22	TLR4	TNF, IL-6, IL-12	76
Extracellular matrix components
Heparan sulfate	TLR4	TNF	83–85
Hyaluronan-derived oligosaccharide	TLR2, TLR4	TNF, MIP-1a, MIP-2, KC	86, 87
Biglycan	TLR2, TLR4	TNF, MIP-2	88
Fibronectin extra domain A	TLR4	MMP-9	89
Decorin	TLR2, TLR4	PDCD4, IL-10	101
Versican	TLR2, TLR6	TNF-α, IL-6	102
Necrotic cells	TLR2, TLR3	TNF, IL-8, MIP-2, KC, MMP-3, iNOS	94, 95
RNA	TLR3, TLR7	IL-12, IFN-α	97, 98
DNA (in the form of immune complexes)	TLR9	IFN-α	97, 100
High mobility group box 1 protein (HMGB1)	TLR2, TLR4	TNF, IL-1a,IL-1b, IL-6, IL-8, MIP-1a, MIP-1b, COX-2, iNOS	30, 90–93
Lung surfactant protein A	TLR4	TNF-α, IL-10	96
Fibrinogen	TLR4	MIP-1α, MIP-1β, MIP-2, MCP-1	44
S100A8	TLR4	TNF-α	103
S100A9	TLR4	IL-1β, TNF-α, IL-6, IL-8 and IL-10	104
Fibrillar β-amyloid	TLR2, TLR4	Superoxide radical	105
α-synuclein	TLR4	TNF-α, IL-6, CXCL1, ROS	106

Upon binding of a PAMP or DAMP to the sequence of LRR domains on a TLR, cytoplasmic signaling events occur (for a more detailed review, see Akira and Takeda¹⁰⁷). Genes induced by TLR activation have been implicated in the production of pro-inflammatory cytokines, chemokines, major histocompatibility complex (MHC), co-stimulatory molecules, and antimicrobial peptides.⁵⁸ Although cytokines and chemokines promote innate immunity and inflammatory responses, MHC and co-stimulatory molecules facilitate adaptive immune responses. Cytokines released in response to TLR activation include the interleukins (IL) IL-1α,⁹⁰ IL-1β,⁹⁰ IL-6,^76,108 and IL-12,^81,97 tumor necrosis factor (TNF, also listed as TNF-α),^{78,86–88,90,92,94,97,108} interferon-α (IFN- α),97 and the chemokines macrophage inflammatory protein-1α (MIP-1α),^86,90,95 MIP-1β,⁹⁰ MIP-2,^86,88,95 MCP-1,¹⁰⁹ and KC.^86,95 Cytokines can promote further inflammatory activation and edema, and chemokines can promote cellular extravasation and trafficking.¹¹⁰

Other factors released after TLR activation include the free radical signaling molecule nitric oxide (NO)^77,78 and an enzyme that produces NO, inducible nitric oxide synthetase (iNOS).^92,94 Additionally, TLR activation may result in release of the matrix metalloprotease (MMP) 3 or MMP9,⁹⁵ which are involved in tissue repair.⁸⁹ Finally, TLR activation may release the pro-inflammatory enzyme cyclooxygenase 2 (COX-2),⁹² which activates arachidonic acid/prostaglandin inflammatory mechanisms.¹¹¹ Overall, TLR signaling pathways utilize a broad range of downstream effectors to respond to pathogenic and endogenous threats, which can be a likely source of the self-perpetuating inflammatory response to microelectrodes described above.

Because of established roles of particular PRR in neurodegenerative disorders, we have taken particular interest in the potential role of TLR2, TLR4, and the co-receptor cluster of differentiation 14 (CD14) in the foreign body response to intracortical microelectrodes.

The cell-surface receptor TLR2 is expressed on endothelial cells and antigen-presenting cells, including macrophages and microglia.^58,112,113 Rather than forming homodimers to activate downstream signaling pathways, TLR2 typically forms heterodimers with TLR1 or TLR6.⁵⁸ The main function of TLR2 is the recognition of Gram-positive bacteria via peptidoglycan (disputed by Travassos et al.¹¹⁴) and lipoteichoic acid,^115–119 but it also binds the PAMPs lipoproteins/lipopeptides,¹²⁰ lipoarabinomannan,¹²¹ phenol-soluble modulin,¹²² glycoinositolphospholipids, ¹²³ porins,¹²⁴ atypical LPS,^125,126 and zymosan.^107,113 In addition to the aforementioned PAMPs, TLR2 recognizes the DAMPs HSP60,⁷⁹ HSP70,^80,81 GP96,⁸² hyaluronan-derived oligosaccharide,⁸⁶ biglycan,⁸⁸ necrotic cells,^94,95 and HMGB1.^91,93 The broad range of recognized ligands makes TLR2 an effective innate immunity activator throughout the body, but the important role of TLR2 in CNS inflammation and neurodegeneration will be discussed below.

In contrast to TLR2, TLR4 communicates via homodimerization and recognizes Gram-negative bacteria.^58,127–129 The cell-surface receptor TLR4 is expressed on macrophages, microglia, and several immune cells.^58,66,130 In addition to bacterial recognition via LPS, TLR4 also recognizes several other PAMPs,^73,107 including mannan,¹³¹ glucuronoxylomannan,^73,132 viral fusion protein,¹³³ and viral envelope proteins.¹³⁴ Also, TLR4 is also involved in the recognition of several DAMPs,^31,59 such as HSP60,^77,78 HSP70,^80,81 Gp96,⁸² HSP22,⁷⁶ heparan sulfate,^83–85 hyaluronan-derived oligosaccharide,^86,87 biglycan,⁸⁸ fibronectin extra domain A,⁸⁹ HMGB1,^91,93 and fibrinogen.⁴⁴ Involvement of TLR4 signaling in CNS inflammation and neurodegeneration will also be discussed in below.

The co-receptor CD14, a 55 kD glycoprotein closely associated with TLR2 and TLR4, is predominantly known for its role in the recognition of LPS.¹³⁵ Briefly, CD14 binds LPS monomers extracted from LPS aggregates by LPS binding protein (LBP)^136,137 and the TLR4-MD2 complex subsequently binds LPS.^135,138 Although the exact mechanism of LPS transfer from CD14 to TLR4-MD2 has not been elucidated, the presence of CD14 greatly improves the sensitivity of LPS recognition by effector cells.¹³⁹ Macrophages and to a lesser extent microglia express CD14,^140–142 either linked to a membrane by a glycophosphoinositol or released in a soluble form.^58,135 In addition to LPS recognition, CD14 acts as a co-receptor to TLR2 in the recognition of the various PAMPs, such as virions¹⁴³ and the cell wall components peptidoglycan (disputed by Travassos et al.¹¹⁴) and LTA.^119,144 The co-receptor CD14 is thought to be an adaptor molecule to the TLRs for the binding of both PAMPs and DAMPs.¹⁴⁴ For example, CD14 acts as a co-receptor to TLR2 and/or TLR4 in the recognition of HMGB1,¹⁴⁵ β amyloid plaques,¹⁰⁵ and HSP70.^80,146 Furthermore, CD14 signals independently of TLR2 and TLR4, such as in the endosomal recognition of viral nucleic acids by TLR7 and TLR9,¹⁴⁷ and the non-TLR mediated recognition of necrotic cells¹⁴⁴ and apoptotic cells.^148–150 Finally, CD14 facilitates the internalization of several molecules, such as phosphatidylinositol.¹⁵¹ The role of CD14 in CNS inflammation and neurodegeneration will also be discussed further in below.

2. TLR2, TLR4, and CD14 in Neurodegenerative Disorders

The innate immunity receptors TLR2, TLR4, and CD14 play a prominent role in several neurodegenerative disorders due to their expression in CNS tissue and ability to promote potent inflammatory mechanisms.¹⁵² The receptors TLR2 and TLR4 are expressed on microglia and astrocytes in both human and mice and on neurons in mice only.^60,153 Additionally, TLR2 is expressed on oligodendrocytes.¹⁵³ Although constitutive expression of CD14 in the brain is limited to perivascular, leptomeningeal, and choroid plexus macrophages, it can be upregulated in parenchymal microglia following injury.^154,155 Subsequently, activation of TLR2, TLR4, and/or CD14 on microglia can induce neurodegeneration.^156–158 In addition to parenchymal cells of the brain, TLR2 and TLR4 are expressed on cerebral endothelial cells¹⁵⁹ and soluble CD14 may participate in the ligand recognition of endothelial cells lacking membrane-bound CD14.¹⁶⁰ Injuries of the CNS may also recruit CD14 positive macrophages to enact and propagate inflammatory responses.¹⁵⁴ The receptors TLR2 and TLR4 are commonly expressed in macrophages as well.

Neuroinflammatory mechanisms involving TLR2, TLR4, and CD14 activation have been demonstrated to contribute to neurodegeneration.^156–158 Resident microglia or infiltrating macrophages may respond to TLR2, TLR4, and CD14 ligands linked to CNS disorders and injuries, such as α-synuclein,^106,161 fibrillar β-amyloid,¹⁰⁵ heparan sulfate proteoglycans,¹⁶² heat shock proteins,^77,163,164 necrotic neurons,^94,165 and whole and partial bacteria.^156,166 Activation of TLRs on microglia can lead to microglial activation and the subsequent release of cytokines, chemokines, reactive oxygen species,¹⁰⁶ and nitric oxide,¹⁵⁶ as well as induce phagocytosis of both damaged and viable neurons.¹⁶⁷ The release of reactive oxygen species by activated microglia can cause damage to neuronal membranes and dendrites.¹⁶⁸ Additionally, activation of TLRs has been shown to interfere with remyelination.¹⁶⁹ In summary, overreaction of microglia to TLR2, TLR4, and CD14 can cause neuronal damage in many ways.

Contrary to the major role of TLR2, TLR4, and CD14 signaling in neurodegeneration, TLR2, TLR4, and CD14 have also been linked to neuroprotection.¹⁷⁰ Activation of TLR2, TLR4, and CD14 may protect neurons by reverting microglia to a pro-healing activation state,¹⁷¹ inducing cytokine release in a beneficial context,¹⁷² or to facilitate clearing of dangerous molecules from the CNS.¹⁷³ Thus, well-regulated innate immune signaling is useful for maintaining neuronal health. These results contradict many reports of the current understanding of TLR2, TLR4, and CD14, but should be further explored for a more complete understanding of the role of innate immunity in microelectrode performance.

Activation of TLR2, TLR4, and CD14 has been implicated in the neurodegeneration caused by a variety of CNS diseases and injuries, including Parkinson’s disease (PD),¹⁷⁴ dementia with Lewy bodies (DLB),¹⁷⁴ multiple system atrophy (MSA),¹⁷⁴ multiple sclerosis (MS),¹⁷⁵ amyotrophic lateral sclerosis (ALS),¹⁷⁶ Alzheimer’s disease,^105,177 neuropathic pain,¹⁷⁸ subarachnoid hemorrhage,^179,180 traumatic brain injury,¹⁸¹ focal cerebral ischemia,¹⁸² spinal cord injury,¹⁸³ and brain injury.¹⁸⁴ Synucleinopathies (PD, DLB, and MSA), Alzheimer’s disease, and ALS will be discussed in further detail due to the involvement of multiple receptors of interest and heavy innate immunity involvement.

Alzheimer’s disease is a memory disorder involving plaques made of β amyloid in the parenchyma of the brain.^185,186 Bystander damage caused by the chronic inflammatory mechanisms directed against β amyloid plaques is hypothesized to be a major source of neurodegeneration.¹⁸⁷ The receptors TLR2, TLR4, and CD14 on microglia recognize fibrillar β amyloid and induce a pro-inflammatory state involving phagocytosis and ROS release.¹⁰⁵ Inhibiting TLR2 and TLR4 by various means has resulted in decreased generation of pro-inflammatory factors and subsequent neurotoxicity.^{158,185,188–190} On the other hand, activation of TLR2, TLR4, and CD14 signaling has been shown to facilitate the clearance of β amyloid,¹⁹¹ and the removal of neurons damaged by Aβ.¹⁹² Thus, some degree of TLR activation while limiting neuronal damage is likely the optimal scenario for treating Alzheimer’s disease.

Recent studies have identified participation of TLR2 and TLR4 in synucleinopathies, such as Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy.^{106,161,173,174} Synucleinopathies involve misfolding and buildup of the protein α-synuclein, resulting in TLR-mediated neuroinflammation and neurodegeneration.^106,193 Activation of TLR2 disrupts the clearance of α-synuclein via autophagy.¹⁶¹ Contrarily, activation of TLR4 promotes the clearance of α-synuclein via phagocytosis, while simultaneously promoting neurodegenerative inflammatory mechanisms.^106,173 Thus, a proper balance of clearing harmful materials and limiting self-inflicted damage must be maintained to combat synucleinopathies. Alternatively, a TLR4 ligand similar to LPS, mono-phosphoryl lipid A, has been identified to selectively promote clearance and not destructive inflammatory cascades.¹⁷⁴ Therefore, approaches more nuanced than simply shutting down the signaling of entire TLRs must be considered in the mitigation of TLR-mediated neuroinflammation and neurodegeneration.

Amyotrophic lateral sclerosis is a disorder involving death of motoneurons in the motor cortex and spinal cord resulting in muscle atrophy and paralysis.^185,194,195 Although the initial cause of motoneuron damage is unclear, activated microglia with mutations in SOD1 have been shown to exacerbate neurodegeneration via the release of superoxide, nitrate, and nitrite.^185,196 TLR2, TLR4, and CD14 have been shown to activate microglia by binding extracellular mutated SOD1.¹⁹⁷ Furthermore, knocking out TLR4 resulted in increased survival and improved functional outcomes in a mouse model of ALS.¹⁹⁸ Recently, natural and synthetic TLR4 antagonists have been shown to mitigate neurodegeneration and nitric oxide release (natural TLR4 antagonist only) by microglia, in vitro.¹⁷⁶ Overall, persistent activation of microglia via TLR2, TLR4, and CD14 in ALS leads to further neurodegeneration, and inhibiting these pathways mitigates harmful outcomes.

In summary, TLR2, TLR4, and CD14 are expressed on tissues of the CNS, recognize PAMPS and DAMPS in the CNS, and promote inflammatory mechanisms involved in neurodegenerative disorders. Temporal and regulated inhibition of TLR2, TLR4, and CD14 holds the potential to mitigate neurodegenerative disorders, because clearance of the ligands that generate inflammation must be considered for proper healing to occur.

B. TLR2, TLR4, and CD14 in Foreign Body Response to Intracortical Microelectrodes

Although there is currently little direct evidence for TLR2, TLR4, and CD14 signaling in the foreign body response to intracortical microelectrodes, several connections exist that suggest their role in the recognition of damage to promote and perpetuate inflammation. Particularly, TLR2, TLR4, and CD14 are expressed in the cells at the electrode tissue interface;^60,153–155 ligands recognized by TLR2, TLR4, and CD14 have been reported at the electrode tissue interface;^89,199 and outcomes associated with the activation of TLR2, TLR4, and CD14 pathways have been reported by several groups.^{61,66,157,200–205} Together, the concurrent presence of receptors, ligands, and downstream effectors strengthens the likelihood for participation of TLR2, TLR4, and CD14 in the foreign body response to intracortical microelectrodes.

As discussed above, resident parenchymal microglia express TLR2 and TLR4 constitutively,^60,153 and may express CD14 following injury.^154,155 Additionally, infiltrating macrophages constitutively express TLR2, TLR4, and CD14. Following intracortical microelectrode implantation, both resident microglia and infiltrating macrophages accumulate at the electrode-tissue interface and revert to a pro-inflammatory activated state.²⁹ Therefore, the receptors TLR2, TLR4, and CD14 may play a role in the pro-inflammatory activation of microglia and macrophages through the recognition of ligands.

Several of the ligands that activate TLR2, TLR4, and CD14 may be present at the electrode tissue interface. Starting with PAMPs, residual endotoxin (LPS) has been detected at levels above FDA guidelines for CNS devices on intracortical microelectrode probes even following standard sterilization protocols with ethylene oxide.¹⁹⁹ Ravikumar et al. compared sterilization methods and found that probes with higher endotoxin levels exhibited higher microglial activation, astrocytic encapsulation, blood-brain barrier permeability, and neuronal dieback at acute time points.¹⁹⁹ Since TLR4 and CD14 are involved in the main pathway of endotoxin recognition,⁵⁸ their findings indicate that activation of TLR4 and CD14 may contribute to unfavorable inflammatory mechanisms at the electrode tissue interface. Immunohistochemical differences between sterilization methods were not observed 16 weeks after implantation, suggesting that endotoxin or LPS may not be a significant activating ligand for chronic inflammatory responses once the LPS introduced by the electrode has been cleared. Additionally, as mentioned above, Harris administered LPS systemically to rats at the time of intracortical microelectrode implantation and observed decreased neuronal survival paired with lower firing rates in evoked recordings compared to control rats,⁴² indicating that activation of TLR4 and CD14 may contribute to declining recording performance as well as the foreign body response to the electrode. Of note, Gram-positive bacterial cell wall components, recognized by TLR2, were not quantified in the sterilization study but may be present following imperfect sterilization methods.

In addition to PAMPs, several DAMPs may be present at the electrode tissue interface due to the damage caused by insertion trauma, micromotion, and chronic inflammatory mechanisms.¹⁹⁹ Enhanced expression of fibronectin, a ligand to TLR4,⁸⁹ has been detected in the reactive tissue around implanted intracortical microelectrodes,²⁰⁶ as well as cortical impact injures.²⁰⁷ HMGB1, recognized by TLR2,^91,93 TLR4,^91,93 and CD14,¹⁴⁵ was observed to be upregulated around implanted intracortical microelectrodes²⁰⁸ as well as in the brain following ischemia.^92,209,210 Furthermore, necrotic cells, recognized by TLR2^94,95 and CD14,¹⁴⁴ have been observed around implanted intracortical microelectrodes.²¹¹ Other DAMPs have been observed in various brain injuries and illnesses. A ligand to TLR4, HSP70, has been observed in the brain following focal cerebral ischemia.^212,213 In vitro studies of CNS cells also indicate evidence of DAMP release following nervous system injuries. The ligands HSP60 and HSP70, recognized by TLR2,^79–81 TLR4,^77,78,80,81 and CD14 (HSP60 only),^80,146 were observed on the axons of cultured DRG neurons that were explanted from rats following sciatic nerve injury.^163,164 Furthermore, HSP60 was observed being released from apoptotic and necrotic cells in mixed CNS culture (embryonic forebrain-derived cells of unspecified composition).⁷⁷ Additionally, fibrinogen, a blood protein that activates TLR4,⁴⁴ has been speculated to be present around implanted intracortical microelectrodes.^14,21,214 The disruption of vasculature upon intracortical microelectrode implantation¹⁶ and chronic permeability of the blood brain barrier likely results in the presence of fibrinogen in the brain at both acute and chronic time points.^28,215 Overall, a variety of DAMPs may be released in the brain following intracortical microelectrode implantation.

Finally, the downstream consequences of TLR2, TLR4, and CD14 activation may indicate a role in the foreign body response to implanted intracortical microelectrode. Activation of TLR2, TLR4, and CD14 on microglia and macrophages leads to the activation of NF-κB, which mediates pro-inflammatory genes responsible for the release of cytokines, chemokines, cyclooxygenase-2, and matrix metalloproteinase-9 (MMP-9).^66,216,217 The factors generated following NF-κB activation may perpetuate chronic inflammatory mechanisms around the implanted device. Activated microglia and macrophages are present at the electrode tissue interface long after device implantation.^29,215 Activation of TLRs has the capability to induce neuronal damage,^61,157 and neuronal dieback is commonly observed along with the foreign body response to intracortical microelectrodes.^21,215,218 Activation of TLRs by DAMP recognition may contribute to the loss of neurons around the implant. Additionally, the neurons killed around implanted intracortical microelectrodes may provide further stimulus for TLR activation via necrosis or the release of HSP60, HSP70, or HMGB1, thus self-perpetuating chronic inflammation. Furthermore, activation of TLRs may contribute to chronic blood-brain barrier permeability via the release of nitric oxide pro-inflammatory cytokines, NO, and reactive oxygen species (ROS), and MMP-9.^{26,66,200–205} Blood-brain barrier permeability is a persistent problem of the foreign body response to intracortical microelectrodes and has been associated with poor recording performance.^28,215

Overall, the evidence presented here suggests that TLR2, TLR4, and CD14 play a role in the foreign body response to intracortical microelectrodes via the recognition of PAMPs and DAMPs by microglia and macrophages and subsequent pro-inflammatory, neurotoxic, and blood-brain barrier damaging actions (Fig. 2).

FIG. 2:

Potential role of TLR2, TLR4, and CD14 in the foreign body response to intracortical microelectrodes. Microglia and macrophages at the electrode tissue interface may recognize PAMPs and DAMPs at the electrode tissue interface via TLR2, TLR4, and CD14, resulting in the release of soluble factors such as cytokines, chemokines, reactive oxygen species, and reactive nitrogen species. The released soluble factors may damage neurons, blood vessels, and electrode materials, leading to poor intracortical microelectrode performance. Cells damaged and killed by the soluble inflammatory factors may release DAMPs that activate microglia and macrophages. Blood proteins released following damage to the blood brain barrier may also act as DAMPs. Inflammatory cells infiltrating across the blood-brain barrier permeability may become activated by DAMPs and contribute to the foreign body response. Thus, TLR2, TLR4, and CD14 signaling may contribute to the activation and perpetuation of chronic inflammatory mechanisms in the foreign body response to intracortical microelectrodes.

1. Current Strategies to Inhibit TLR2, TLR4, and CD14

Several strategies exist for inhibiting TLR2-, TLR4-, and CD14-mediated signaling, varying in target and molecular composition. The most direct strategies target the receptors themselves, however, inhibition strategies may target other components of the TLR2/TLR4/CD14 signal transduction pathways. A variety of peptides, antibodies, and RNA sequences may disrupt signaling at the various targets. In this section, several TLR2/TLR4/CD14 inhibition strategies under different stages of investigation will be reviewed (see Table 2).

TABLE 2:

Strategies for the inhibition of TLR2, TLR4, and CD14

Inhibition Target Mechanism	Type of Therapeutic	Receptor Targeted	Therapeutic	Application(s)
Ligand binding	Small molecule	CD14/TLR4	IAXO-101	Sepsis,233 neuropathic pain,178 malaria234
Ligand binding	Small molecule	CD14/TLR4	IAXO-102	Aneurysm235
Ligand binding/ receptor dimerization	Small molecule	TLR2	SSL3236	N/A
Ligand binding	Antibody	CD14	IC14	LPS responsiveness,241 sepsis242,243
Co-receptors (non-CD14)	Small molecule	TLR4 (via MD-2)	Eritoran	Sepsis250
Co-receptors (non-CD14)	Small moleculee	TLR4 (via MD-2)	TAP2	Inflammation251
Co-receptors (non-CD14)	Small molecule	TLR4 (via MD-2) (natural)	Curcumin	Integration of intracortical microelectrodes208
TLR4-MyD88 Complex formation	Small molecule	TLR4	Wogonin	Inflammation in DRG neurons,255 traumatic brain injury216
Intracellular TLR domain interactions	Small molecule	TLR4	TAK-242/resatorvid	Sepsis,260 endotoxic shock,259 traumatic brain injury,261 cerebral ischemia,257 intracerebral hemorrhage,257 optic nerve crush262
Receptor expression at cell surface	siRNA	TLR4	N/A	Neuropathic pain220
Receptor expression at cell surface	Small molecule	TLR4	Oxymatrine	Traumatic brain injury181
Receptor expression at cell surface	Small molecule	TLR4	PACAP	Traumatic brain injury223
Receptor expression at cell surface	Small molecule	TLR2	Glycyrrhizin	Autoimmune thyroiditis221
Receptor expression at cell surface	Small molecule	TLR4	Melatonin	Subarachnoid hemorrhage,224 integration of intracortical microelectrode228
Receptor expression at cell surface	Small molecule	TLR4	Apigenin	Subarachnoid hemorrhage225
Receptor expression at cell surface	Small molecule	TLR4	Ursolic acid	Subarachnoid hemorrhage226

The first major option for inhibiting TLR2/TLR4/CD14 signaling we will discuss utilizes methods to alter the cell-surface expression of TLR2/TLR4/CD14, either by downregulation or internalization. Administration of rosmarinic acid,²¹⁹ siRNA,²²⁰ oxymatrine,¹⁸¹ glycyrrhizin,²²¹ argon,²²² pituitary adenylate cyclase-activating polypeptide (PACAP),²²³ melatonin,²²⁴ apigenin,²²⁵ and ursolic acid,²²⁶ resulted in reduced TLR2, TLR4, and/ or CD14 expression; whereas, walnut extract was shown to promote receptor internalization.²²⁷ Of note, intrathecal siRNA that reduces TLR4 expression attenuated neuropathic pain in a rat model.²²⁰ Oxymatrine and PACAP conferred neuroprotection in a rat models of traumatic brain injury by inhibiting the expression and upregulation of TLR4.^181,223 Glycyrrhizin inhibited TLR2-HMGB1 signaling, resulting in reduced lymphocyte trafficking in experimental autoimmune thyroiditis in mice.²²¹ Melatonin, apigenin, and ursolic acid attenuated the effects of subarachnoid hemorrhage, including neurobehavioral deficits, blood-brain barrier permeability, and neuronal apoptosis.^224–226 Even more promising, administration of melatonin has resulted in improved intracortical microelectrode recording and neuronal viability.²²⁸ Although melatonin was chosen for its ability to inhibit caspase activation, cytochrome c release, and oxidative stress, inhibition of TLR4 may have contributed to improved microelectrode integration and performance.^224,228 Reducing the expression of TLR2, TLR4, and/or CD14 effectively attenuates the negative effects of overactive TLR signaling. Again, the success of cell-surface expression in CNS injuries and disorder demonstrate a potential application for integrating intracortical microelectrodes. Therefore, we utilized transgenic TLR2, TLR4, or CD14 knockout mice to investigate the effects of removal of each receptor in the integration and performance of intracortical microelectrodes. Studies with TLR2/TLR4 utilized dummy implants and investigated only the histological response.²²⁹ Endpoint histology at 2 and 16 weeks after implantation demonstrated that Tlr4^−/− mice exhibited significantly lower BBB permeability at acute and chronic time points but also demonstrated significantly lower neuronal survival at the chronic time point. Additionally, inhibition of the TLR2 pathway had no significant histological effect compared to control animals. Together, our results indicate that complete genetic removal of TLR4 was detrimental to the integration of intracortical neural probes, while inhibition of TLR2 had no impact within the tests performed in this study. Implanting functional recording intracortical microelectrodes into Cd14^−/− mice exhibited acute but not chronic improvements in intracortical microelectrode performance without significant differences in end point histology.²³⁰ However, when we used a mouse bone marrow chimera model to selectively knockout CD14 from either brain resident microglia or blood-derived macrophages, we were able to understand the most effective targets for future therapeutic options—brain-derived microglia or infiltrating blood-derived cells.²³¹ Using single-unit recordings, we demonstrate that inhibiting CD14 from the blood-derived macrophages improves recording quality over the 16-week long study. We conclude that targeting CD14 in blood-derived cells should be part of the strategy to improve the performance of intracortical microelectrodes and that the daunting task of delivering therapeutics across the blood-brain barrier may not be needed to increase intracortical microelectrode performance. Knowing that both TLR4 and CD14 are important mediators of intracortical microelectrode integration and performance, other strategies to inhibit their activation must be explored.

Our next step was to consider inhibiting TLR2/TLR4/CD14 signaling via antagonism of the binding domain with a small molecule. The glycolipid IAXO-101 has demonstrated success as a CD14-TLR4 antagonist by competitively occupying CD14,²³² resulting in mitigation of sepsis, neuropathic pain, and experimental malaria in mice.^178,233,234 Therefore, we explored the use of IAXO-101 in an intracortical microelectrode model. Interestingly, mice receiving IAXO-101 exhibited significant improvements in recording performance over the entire 16-week duration without significant differences in endpoint histology.²³⁰ Combined with our chimera studies, we hypothesized that some degree of CD14 signaling may be necessary over chronic time ranges to facilitate wound healing mechanisms. We also remained unsure if IAXO-101 was simply not the most optimal method for small molecule inhibition and are exploring other options. For example, a similar compound IAXO-102 attenuated the development of an experimental form of aneurysm.²³⁵ The naturally derived antagonist to TLR2 staphylococcal superantigen-like protein 3 (SSL3) may inhibit TLR2 signaling by both blocking binding sites and disrupting heterodimerization.²³⁶ Because of the diversity of molecular structures recognized by the same TLR, the effectiveness of small molecule antagonists to interfere with ligand-receptor interactions may vary between ligand.²³⁷ Small molecule antagonists to the TLR2/TLR4/CD14 pathways have demonstrated varying degrees of success. The capability of small molecule antagonists to mitigate TLR2/TLR4/CD14 activity and attenuate detrimental inflammatory outcomes is promising for their utilization in intracortical microelectrode integration. When contemplating the effectiveness of a small molecule antagonist approach, it is important to consider delivery to the brain. Depending on the approach, delivery through the blood-brain barrier is complicated, despite the known “leakiness” associated with the neuroinflammatory response to intracortical microelectrodes.

Another mechanism of inhibiting TLR2/TLR4/CD14 signaling by blocking the ligand-receptor interactions utilizes blocking antibodies. Antibodies that bind a cellular receptor and prevent ligand binding have demonstrated success in various inflammatory disorders.²³⁸ Anti-CD14 antibodies may disrupt CD14 signaling by blocking the binding of LPS to CD14^239,240 or through mechanisms independent of LPS binding.²³⁹ The commercially available monoclonal antibody IC14 recognizes human CD14 and saturates CD14 on monocytes and granulocytes.²⁴¹ Intravenous administration of IC14 in humans reduced responsiveness to LPS, specifically attenuating the release of pro-inflammatory cytokines (TNF, IL-6, and IL-10), the activation of leukocytes and endothelial cells, the acute phase protein response, and various symptoms.²⁴¹ The administration of IC14 has produced less promising results in severe cases of sepsis.^242,243 One potential setback for the use of blocking antibodies is activation of the complement pathway by the Fc region of the antibody.²⁴⁴ Recent studies employing hybrid antibodies with inert Fc regions have been successful in attenuating CD14-mediated inflammatory responses without activating complement-related side effects in both in vivo porcine models and ex vivo human whole blood models of sepsis.²⁴⁴ Furthermore, pairing complement inhibition with hybrid anti-CD14 antibodies has demonstrated enhanced survival in a porcine model of polymicrobial sepsis.²⁴⁵ Finally, hybrid anti-CD14 antibodies with and without concurrent complement inhibition has outperformed the synthetic small molecule antagonist eritoran in mitigating monocyte activation in response to sepsis.²⁴⁶ Therapeutic antibodies have demonstrated success in mitigating TLR2/TLR4/CD14 mediated disorders despite potential complications. Thus, therapeutic antibodies may be applicable for integrating intracortical microelectrodes within the brain.

Alternative inhibition strategies to inhibit TLR2 and TLR4 focus on the non-CD14 co-receptors. The protein MD-2 (myeloid differentiation 2) is the other major cell-surface co-receptor involved in the recognition of LPS. Where CD14 is thought to facilitate recognition of both PAMPs and DAMPs, MD-2 is thought to facilitate response to PAMPs only.¹⁴⁴ Limiting the types of ligands inhibited will likely result in different outcomes. Small molecule antagonists to TLR4-MD-2 may be derived from synthetic and natural sources, such as olive oil and curcumin.²³⁷ Synthetic antagonists to TLR4-MD-2 are typically similar in structure to the LPS component lipid A that is recognized by the TLR4 receptor complex.²³⁷ Eritoran is a synthetic small molecule antagonist derived from lipid A that binds MD-2 without promoting the dimerization of TLR4.^237,247 Despite promising in vitro²⁴⁸ and animal model results,²⁴⁹ eritoran was unable to reduce mortality in clinical trials of severe sepsis.²⁵⁰ More recently, the TLR4/MD-2 antagonist peptide TAP2, hypothesized to bind the LPS-binding pocket of MD-2, was shown to attenuate LPS-induced inflammatory activation in vitro and in mice.²⁵¹ However, clinical trials with TAP2 have not been reported. Becaust of the predominant interaction of MD-2 with PAMPS,¹⁴⁴ the authors did not initially expect TLR4-MD-2 antagonists to be effective in mitigating chronic inflammation associated with the damage caused by intracortical microelectrodes. However, Potter et al. previously demonstrated that the natural TLR4-MD-2 antagonist curcumin improves neuronal survival and blood-brain barrier stability around intracortical microelectrodes implanted in rats.²⁰⁸ Their results may indicate a larger role of pathogens at the electrode tissue interface, involvement of MD-2 in DAMP recognition, or promiscuous activity of curcumin as an antioxidant, which was the author’s intended application. Regardless of the mechanism, this finding indicates the potential for TLR4-MD-2 inhibitors in the integration of intracortical microelectrodes.

Additional inhibition strategies to inhibit TLR2/TLR/CD14 signaling target downstream intracellular signal transduction pathways. Antagonists have been developed or identified to target the adaptor molecules MyD88 adaptor-like (Mal) and TRIF-related adaptor molecule (TRAM),²⁵² the TLR2 TIR domain,²⁵³ JNK,¹⁶⁶ ubiquitin chains,²⁵⁴ TLR4-MyD88 interactions,²⁵⁵ TLR1-TLR2 heterodimerization,²⁵⁶ and the intracellular domain of TLR4.^257,258 Of note, administration of the flavonoid wogonin attenuated LPS-induced inflammation in dorsal root ganglion neurons²⁵⁵ and improved outcomes in a model of traumatic brain injury²¹⁶ by interfering with TLR4MyD88 complex formation.²⁵⁵ Furthermore, cyclohexene-derived TAK-242/resatorvid inhibits TLR4 signaling by binding the intracellular domain of TLR4 and preventing interactions with downstream adapter molecules.^257,259 In vivo administration of TAK-242 for the treatment of sepsis,²⁶⁰ endotoxic shock,²⁵⁹ traumatic brain injury,²⁶¹ cerebral ischemia,²⁵⁷ intracerebral hemorrhage,²⁵⁷ and optic nerve crush²⁶² resulted in positive outcomes, such as attenuated inflammation and neuroprotection. A phase III clinical trial (NCT00633477) investigated the utilization of TAK-242 to treat sepsis, but the trial was discontinued due to business concerns unrelated to safety.²⁶³ Strategies to inhibit TLR2/TLR4/CD14 signaling have demonstrated preliminary efficacy but have not demonstrated clinical success to this date. Nonetheless, many of the in vivo experiments have demonstrated efficacy in conditions related to the foreign body response to intracortical microelectrodes. Traumatic brain injury involves similar phenomena exhibited in the foreign body response to intracortical microelectrodes, such as blood-brain barrier permeability, neuroinflammation, and neuronal death.²⁶⁴ Additionally, ischemic conditions have been observed around intracortical microelectrodes.²⁶⁵ The efficacy of inhibiting intracellular signal transduction pathways downstream of TLR2/TLR4/CD14 in CNS applications is promising for the utilization in the integration of intracortical microelectrodes.

Overall, TLR2, TLR4, and CD14 may be attenuated at different points along their respective signaling pathways (Table 2). Many promising findings have been observed in vitro and in vivo, but strong clinical performance in humans has not been demonstrated. Human clinical trials mainly failed in severe cases of sepsis, which is likely not the best predictor of efficacy mitigating the foreign body response to intracortical microelectrodes. However, promising in vivo results in CNS injuries and diseases, coupled with our preliminary work in the role of TLR4/CD14 in microelectrode performance clearly indicate the potential success of additional strategies for integrating intracortical microelectrodes.

III. CONCLUSION: FUTURE PERSPECTIVES AND DIRECTIONS

Electrical signals recorded from the neurons of human patients by intracortical microelectrodes have been used to communicate with computers, control robotic limbs, and to control the patient’s own arm. The signal quality and the length of time that useful signals can be recorded are inconsistent. The widely held view of the community is that the inflammatory response of neural tissue that surrounds the microelectrodes, at least in part, compromises electrode reliability. Several studies have demonstrated the connection between neuroinflammation and microelectrode performance.

Inflammation is initiated when inflammatory cells recognize foreign biologics (i.e., damaged/infiltrating proteins and cells). Serum proteins and blood-derived cells invade the central nervous system following microelectrode implantation and aggravate the neuroinflammatory response. Cells and tissue are damaged from the trauma of microelectrode implantation. At the microelectrode surface, accumulation of pro-inflammatory molecules causes neuronal degeneration and increases the permeability of the blood-brain barrier, self-perpetuating the process. In rodents, the neuroinflammatory response to implanted microelectrodes in the motor cortex has been linked to decreased fine motor skills.²⁶⁶

Many labs, including ours, have worked to develop new microelectrode designs to reduce to neuroinflammatory response to intracortical microelectrodes. The extent to which neuroinflammation has been altered by electrode design varies between labs and designs. In the near term, the field must remember the effort that was taken to obtain FDA and CE approvals for clinical trials. The most direct route to translation is working with the devices further along in development. Ultimately, one electrode design will be unable to reach all the targets of the brain that may require interfacing. Therefore, simply making the intracortical microelectrode smaller, thinner, more flexible, mechanically dynamic, or inert may not be enough. We believe that the most likely route to enabling intracortical microelectrodes to interface with any region of the brain is to intimately understand the neuroinflammatory process, to allow for the most directed strategies for mitigation without interruption of the normal healthy response to disease or injury.

Here, we have outlined the rationale behind our hypothesis for the importance of the innate immune system in intracortical microelectrode performance. We have introduced our preliminary efforts and attempted to inspire others to also implement approaches that target the controlled immunosuppression of the localized and specific parts of the innate immune response to intracortical microelectrodes, as opposed to broader approaches that may lead to greater side effects. Much of the work reported here in understanding the role of specific innate immunity pathways was performed in mice. Although most basic neuroscience studies of the brain are also performed in mice, the majority of brain computer interface studies in rodents chose the rat over the mouse.²⁶⁷ We have previously reported that there is no difference in the inflammatory response to microelectrode in the mouse and rat models²⁶⁸ and thus chose to focus on the mouse as more transgenic models are available in mice than rats. The number and type of TLRs in mice varies to that found in humans; so as with all animal work, precaution should be taken in overanalyzing results from transgenic studies in mice when deciding pathways to clinical translation. Additionally, as chemists and biomaterialists, we appreciate the role of new materials and electrode designs in the overall picture. In the end, no one approach will work alone, indefinitely. Only after we understand the body’s temporally driven process for rejection of implanted devices can we better engineer the neural interface and our complete strategy for long-term integration.

ABBREVIATIONS:

ALS

amyotrophic lateral sclerosis

CE

Conformité Européene

COX-2

cyclooxygenase 2

CD14

cluster of differentiation 14

DAMPs

damage (or danger) associated molecular patterns

DLB

dementia with Lewy bodies

ECM

extracellular matrix

FDA

Food and Drug Administration

HSP

heat shock proteins

HNMGB1

High Mobility Group Box 1

iNOS

inducible nitric oxide synthetase

IFN-α

interferon-α

IL

interleukin

LPS

lipopolysaccharide

LRR

leucine-rich repeat

MIP-1α

macrophage inflammatory protein-1α

MHC

major histocompatibility complex

MMP

matrix metalloprotease

MS

multiple sclerosis

MSA

multiple system atrophy

NLRs

Nod-like receptors

NO

nitrous oxide

PD

Parkinson’s disease

PAMPs

pathogen associated molecular patterns

PRRs

pattern-recognition receptors

PACAP

pituitary adenylate cyclase-activating polypeptide

ROS

reactive oxygen species

RLRs

retinoic acid-inducible gene-I-like receptors

SSL3

staphylococcal superantigen-like protein 3

TLRs

Toll-like receptors

TNF or TNF-α

tumor necrosis factor