IMEs face limitations associated with chronic failure, predominantly attributed to the prolonged neuroinflammatory response 2. The characterization of the neuroinflammatory response has evolved over decades, embracing advanced techniques such as genomics and spatially resolved transcriptomics 11,12,14,27-33. While gene expression studies enhance our broad comprehension of the interplay between IMEs and cortical tissue, there is a notable absence of reported advanced proteomic methods to explain the intricate changes in protein expression specific to neuroinflammation, neurodegeneration, or the viability of tissue and cells. This shortfall has likely hampered the development of targeted intervention strategies to enhance IME functionality.
Fig. 6 shows a review of how the biological response to neural implants is typically quantified. The search terms used in PubMed were: "microelectrode" AND ("biological response" OR "inflammation" OR "tissue response" OR "inflammatory response" OR "foreign body response" OR "failure") AND ("brain" OR "cortical" OR "intracortical"). This search output a reasonably sized representation of the literature, but this is not an exhaustive list of all IME papers in the field. All papers from this search that were published in 2000 or later were included in the review (n = 256). The search terms were intended to target experimental papers that are developing or characterizing microelectrodes and implanting them into either the brain or live neural cells. Any papers that did not fit these requirements (n=72) were removed.
Of the remaining 184 papers, 40% (73 papers) did not mention the biological response to the implant. Of the 60% of papers (111 papers) that did mention the biological response, only 73% (81 papers) of that subgroup used any quantitative metric to determine the effect of the implant on the tissue. Of the 81 papers that used quantitative methods to characterize the biological response to the implant, 67% (54 papers) used protein expression assays to quantify the response. The 54 proteomic papers represent a relatively high number of papers using protein expression as an indication of the state of the brain tissue. However, 98% of papers (53 in total) that looked into protein expression used a method that measured intensity, such as immunohistochemistry. Analyzing intensity measurements means that the papers were not able to look at large numbers of proteins at once due to a limited number of microscope channels, and it means that the protein counts were not counting the actual protein concentration, but were instead estimating based on intensity. The average number of proteins quantified in the subgroup that used intensity measurements was 4.3 proteins. In this literature review we only found one paper that measured protein counts, and this paper quantified the expression of 3 proteins through cytometric bead arrays 34. Our study expands on current methods by using the actual protein counts rather than intensity readings and by quantifying 83 proteins at once.
Figure 6: Results from literature review show of 184 papers that characterized implanted microelectrodes. Only 1 paper quantified protein expression with counts, rather than typical intensity readings. This paper measured the expression of 3 proteins. The search terms used in PubMed were: "microelectrode" AND ("biological response" OR "inflammation" OR "tissue response" OR "inflammatory response" OR "foreign body response" OR "failure") AND ("brain" OR "cortical" OR "intracortical").
In this investigation, we meticulously profiled the expression of approximately 80 proteins within a 180 μm radius of the IME implantation site at 4, 8, and 16 weeks post-implantation to better understand the sub-chronic and chronic neuroinflammatory response to IME implantation. Overall, of the three time points investigated in this study (4WK, 8WK, and 16WK), 4WK demonstrates the strongest changes in innate immune marker expression, while 8WK and 16WK exhibit deficits in local neurons and oligodendrocytes. (Fig. 1-5, Table 2). All ten proteins that are only differentially expressed in the 4WK vs naïve control comparison are associated with microglia, macrophages, or peripheral immune cells. The 8WK time point has the largest downregulation of neuronal health and autophagy proteins (Fig. 1C, 2, 3). By 16 weeks post-implantation (16WK), most of the 8WK effects still linger, but several proteins (ATG12, NeuN, SYP) are no longer downregulated. This could mean that the tissue is healing, but time points past 16 weeks post-implantation would be needed to confirm to what extent the tissue is able to heal.
Proteins Associated with Astrocytes or Microglia:
The formation of the tight glial scar that encapsulates the implant is achieved through the migration and expansion of astrocytes upon activation. Two proteins involved in the cytoskeletal expansion of astrocytes are VIM and GFAP 35. Here, we found GFAP expression to be heavily upregulated at all time points (4WK, 8WK, 16WK), with a ~350-450% increase in implanted tissue compared to naïve control (Fig. 1). We expected a similar trend with vimentin (VIM), an intermediate filament that plays a similar role in astrocytic activation 36. However, we found the exact opposite to be true, as vimentin was downregulated in all three measured time points (4WK, 8WK, 16WK) (Fig. 1, Table 2). Vimentin has another role in the motor cortex: it helps to form tight junctions between endothelial cells in blood vessels of the BBB, maintaining the structure that separates the brain parenchyma from circulating blood 37,38. Implantation of the IME breaks these blood vessels, and the healing process is incredibly slow due to the persistence of the device in the tissue. The BBB is reported to reestablish its integrity at approximately 8 weeks post-implantation, with minor leakage continuing into 16 weeks post-implantation 7. With vimentin being downregulated at 4WK, 8WK, and 16WK time points, the loss around the implant site likely contributes to the leaky vasculature. It is possible that vimentin’s overall downregulation in the blood vessels is outweighing the upregulation in activated astrocytes. Aldehyde dehydrogenase 1 family member L1 (ALDH1L1) is an enzyme that regulates astrocyte metabolism, cell division, and cell growth. It was only differentially expressed (downregulated) in the 16WK mice (Fig. 1D, Table 2). The function of ALDH1L1in the CNS is not entirely known, but its downregulation in the 16WK mice may be an indication of injured or diseased state astrocytes 39.
In the current study, many proteins associated with microglia, specifically CD11b, CD11c, CD45, IBA1, Ki-67, MHC II, SPP1, and TMEM119, were found to be upregulated in the 4WK time point (Fig. 1B, 2, Table 2). Microglia are the main phagocytic cell type in neural tissue 40, and the upregulation of these proteins validates the presence of microglia at the implant site. CD11b has a known role in cell adhesion during inflammation 40,41. It is likely upregulated to allow for the adhesion of activated microglia and macrophages to the implanted electrodes and to one another, as they aggregate and form a thin layer on the surface of the electrode 42. CD11c is expressed in a subset of microglia that are believed to have neuroprotective qualities 43. Moreover, both CD11c and CD11b are expressed by peripheral immune cells such as dendritic cells, while peripheral macrophages also express CD11b. CD45 is considered to have low expression in microglia compared to macrophages and T-cells, and plays a role in adhesion in myeloid cells 44. Therefore, upregulation of CD45 could be indicative of T-cells and other peripheral immune cells at the microelectrode interface. IBA1 is involved in the membrane ruffling process and phagocytosis in activated microglia 45, and has been a common marker for total microglial and macrophage population in cortical tissue in immunohistochemical evaluation of the tissue-electrode interfaces 46,47. MHC II is a protein involved in antigen presentation that is expressed by microglia, astrocytes, and other immune cells 48,49. Secreted phosphoprotein 1 (SPP1), also known as osteopontin, is secreted by microglia, macrophages, and T-cells, and is involved in the toll-like receptor signaling pathway. It is pro-inflammatory and activates and recruits more microglia to the implant site 50. Together, MHC II and SPP1 may implicate the roles of innate and adaptive immune system in response to IME implantations. TMEM119 is a protein expressed on microglia that is mainly abundant in resting cells. Upon activation, TMEM119 concentrations are reduced in microglia 51. TMEM119 is upregulated at only the 4WK time point, which could indicate that by the 8WK time point the microglia have fully activated and lost the surface TMEM119 abundance. Taken together, the listed microglial and astrocytic proteins could be further investigated as a target to mitigate the inflammatory cascade. Ki-67 is a ubiquoous marker for cell proliferation, and suggest the active cell proliferation, likely by immune cells, at the site of implantation 41,52.
Ki-67 is upregulated in only the 4WK time point compared to naïve control (Fig. 1, Table 2). In the adult motor cortex, neurons no longer proliferate, meaning that the Ki-67 is likely expressed in astrocytes, microglia, and infiltrating peripheral immune cells, not neurons. Ki-67 -/- mice have been shown to reduce tumor growth while also inhibiting major histocompatibility complex expression (see MHC II, Table 2). However, Ki-67 is not essential for proliferation to occur 53. It is not known how Ki-67 targeting would affect traumatic brain injury or IME implantation, but reducing the efficiency of proliferation in immune cells as well as inhibiting major histocompatibility complexes may be beneficial to neural injuries by reducing the amplification of the inflammatory response in the first 4 weeks following the injury. Other proteins in the proliferation pathway, such as NOX4 or CSF1R, may reduce proliferation more effectively compared to Ki-67 knockout.
Proteins Associated with the Peripheral Immune System
Peripheral immune protein expression can be used to quantify the extent of immune activity at the implant site. These proteins are mainly expressed by T-cells, macrophages, dendritic cells, and neutrophils that circulate through the blood and are recruited into the IME implant site. The BBB is reported to reestablish its integrity at approximately 8 weeks post-implantation, with minor leakage continuing into 16 weeks post-implantation 7. Peripheral immune cells can be both passively and actively recruited to the site of injury following microelectrode implantation. IBA1, Ki-67, MHC II, and SPP1 are upregulated at only the 4WK time point (Fig. 1, Table 2). All four of these proteins are found on microglia and macrophages, and were discussed in the Astrocyte or Microglia Proteins section. Four proteins are downregulated at the 4WK time point: CD3E, CD86, CTLA4, and Ly6G/Ly6C. CD86 expressed on innate cells binds and activates CTLA4, and it is believed that this binding dampens T-cell activation by keeping CD86 from binding with CD28 54. The levels of CTLA4 are low in resting T-cells, and the protein is upregulated by T cells as a self-regulating mechanism of the immune system to prevent run-away inflammation 55. CTLA4 is downregulated, but CD40L, another protein involved in T-cell activation, is upregulated at the 8WK time point (KEGG:04660) (Fig. 1, Table 2) 56-58. The combined low CTLA4 and high CD40L demonstrates that at 8WK time point there might be higher infiltration of activated T-cells at the implant site.
The mice implanted with IMEs for 4WKs or 8WKs have seven proteins associated with peripheral immune cells upregulated compared to naïve control mice (Fig. 1B, C, 3, Table 2). However, by 16WK, only three of these proteins remains upregulated (Fig. 1D, 3, Table 2). The three peripheral immunity associated proteins upregulated at all three time points are CD11b, CD11c, and CD45 (Fig. 1, 3). All three of these proteins are expressed in microglia and macrophages (Table 2). Ly6G/Ly6C is expressed in both myeloid-derived suppressor cells and neutrophils 59,60. This data indicates the persistence of potential innate immune cells such as macrophages, neutrophils, dendritic cells until 16 weeks of this study.
The upregulation of peripheral pathways indicates the presence of specific cell types, mainly macrophages and T-cells. On the other hand, downregulation of peripheral pathways indicated that these cells do not migrate into the implant site or are selectively cleared by the 4WK time point. T-cells and other peripheral cells such as dendritic cells and neutrophils are relatively uncharacterized in the context of IME implantation, and represent a potential emerging area for immunomodulation of the neuroinflammatory response to intracortical microelectrodes. In fact, immunomodulation of many diseases and injury states though T-cell programing is becoming an emerging area for immunoengineering 61-63. Perhaps similar strategies can be adopted for the neural interface.
Proteins Associated with Neurons and Oligodendrocytes
All six measured proteins associated with maintaining the structure of neurons and oligodendrocytes: MAP2, NfL, SYP, NeuN, MBP, and OLIG2, are downregulated in at least one timepoint (Fig. 2, Table 2). The broad downregulation of markers for neuronal health indicates significant deficits in the health and functionality of both neurons and oligodendrocytes. Two neuronal health proteins: synaptophysin (SYP) and neuronal nuclear protein (NeuN), are downregulated in implanted animals at the 8WK time point and are not significantly differentially expressed at the 4WK or 16WK time points. SYP is a protein that lines the synaptic vesicles 25. Synaptic vesicles are used to transport neurotransmitters to the synaptic terminals. The release of the neurotransmitters from the synaptic vesicles allows for signal transmission from neuron to neuron 64. Decreased synapse could indicated a decrease in neurons or a decrease in synaptic connections between neurons. NeuN is a protein found in neuronal nuclei involved in mRNA splicing 65 and is the most common marker in the IME histology literature for neuronal health and survival 65. The fact that SYP and NeuN are both downregulated at 8WK and are not significantly differentially expressed at the 16WK time point could indicate neuronal healing of some capacity between 8 weeks and 16 weeks post-implantation. Similar trend of fluctuations in NeuN density in histological evaluation of the IME-tissue interface were reported by Potter et al. 66.
MAP2, NfL, MBP, and OLIG2 are all downregulated at both the 8WK and 16WK time points (Fig. 1, 2, Table 2). Neurofilament light (NfL) and microtubule-associated protein 2 (MAP2) are both proteins that make up the neuronal cytoskeleton. Degradation of the neuronal cytoskeleton has been linked to the transition between reversible and irreversible damage to brain tissue 67,68. NfL is downregulated by ~50% (FC= 2-1) in implanted animals by 8WK and ~62% (FC= 2-1.4) by 16WK (Fig. 1C, D). The increasing decline in NfL expression could be directly linked to decreases in neuron recording performance with time. However, NfL does not represent a target for immunomodulation approaches to mitigate IME performance. The integrity of the oligodendrocytes, which make up the myelin that allows for efficient transmission of action potentials, are also compromised following IME implantation. Myelin basic protein (MBP) is the second most abundant protein in the myelin cells of the central nervous system 69, and is considered to be essential for the formation of tight myelin sheaths around an axon 70. MBP is approximately four times more abundant in naïve control animals compared to implanted mice at 4WK, 8WK, and 16WK time points (Fig. 1B, C, D). This means that ~75% (FC = 2-2) of MBP is lost following IME implantation, which would significantly impact the functionality of the cortical neurons and by extension, the ability of IMES to detect single-unit activity. MBP is the only neuronal health protein that begins downregulation as early as the 4WK time point. Oligodendrocyte transcription factor 2 (OLIG2), is a protein that regulates the transcription for myelin-associated proteins. In traumatic brain injury, OLIG2 is upregulated immediately after injury and remains upregulated for up to 3 months, aiding in the remyelination of the tissue 71,72. In the case of IME implantation, we see the opposite effect with OLIG2 downregulation at both 8 and 16 weeks post-implantation (Fig. 1, 2, Table 2). The permanent presence of the electrode may be preventing successful remyelination.
Overall, the neuronal health proteomic data indicates that the health and functionality of neurons and oligodendrocytes is likely the lowest at approximately the 8WK time point. Degradation of the neuronal cytoskeleton (NfL, MAP2) as well as the oligodendrocytes (MBP, OLIG2) begins at the 8WK time point and continues into the 16WK mice. Some components, including SYP and NeuN, are at least partially regenerated by 16 weeks post implantation, but other components of the cytoskeleton have endured what may be irreversible damage. These structural components (NfL, MAP2, MBP) are degrading and are not being regenerated by 16 weeks post-implantation. Our more complete data set further questions the validity of using NeuN as the sole marker of neuronal health which has been common practice in the IME literature for some time, as NeuN is not downregulated at the 16WK time point, yet we still see deficits in other neuronal proteins, and 16WKs is associated with chronic recording failure.
Autophagy Proteins:
Eight autophagy proteins, including ATG5, BAG3, ULK1, and VPS35, are downregulated in the 8WK and 16WK timepoints compared to naïve control mice (Fig. 1C, D, 5, Table 2). The downregulation of the 8 autophagy proteins indicates that autophagy is not occurring at a healthy rate in implanted mice by 8 weeks post-surgery. Autophagy removes harmful substances from the cytoplasm, allowing for the recovery of injured cells 26. Autophagy in neurons is especially important because neurons in the adult motor cortex do not divide or regenerate, so they need to survive the entire lifetime of the organism 73. The downregulation of autophagy proteins, along with the fact that neurons around the implant site are still dying up to 16 weeks post-implantation 56, suggest that by the 8WK time point the autophagy attempts to save the neurons have failed and neurons are likely resorting to apoptosis or necrosis. Though there are no apoptotic proteins quantified in this experiment, MAP2 is known to undergo proteolysis during apoptosis 74. We found MAP2 to be downregulated in both 8WK and 16WK mice compared to naïve control mice (Fig. 1C, D, 4, Table 2). MAP2 downregulation could indicate that apoptosis is occurring in local neurons. Neuronal dieback is a major concern for IME researchers as well as patients, and autophagy could be a target for the prevention of neuronal death. One study found that overexpression of ATG5, an autophagosomal protein found to be downregulated in our experiment, leads to nearly 20% longer lifespans in mice 75. A method that promotes autophagy in implanted animals before or during the 8WK and 16WK time points may prevent neuronal dieback over chronic time points.
Implications for future studies:
Proteins within the astrocytic, microglial and peripheral immune sections represent pathways that ideally would not be activated following IME implantation. The knockouts of several key inflammatory genes, including CD14 and C3, have been investigated with some success in IME applications. Ki-67, or a different protein involved in proliferation, may be worth exploring at early time points prior to 4 weeks post-implantation. Our previous understanding of immune cell proteomic activity following IME implantation comes largely from histology and western blot studies. Immunohistochemistry has mapped the timelines for microglia, macrophage, and astrocyte aggregation around the implant site. Ravikumar et al. found that microglia and macrophage populations peak at acute timepoints (2 weeks post-implantation) and slowly decrease over chronic timepoints (up to 16 weeks post-implantation). Astrocytic aggregation was shown to be highest at acute timepoints (2 weeks post-implantation), then fluctuate slightly around similar values between 4 and 16 weeks post-implantation 9. Our analysis confirms microglia and macrophage-related proteins (SPP1, MHC II, IBA1) are upregulated at the 4WK timepoint. By the 8WK time point, SPP1, MHC II, and IBA1 all lose significant differential expression, and F4/80 is a microglial/macrophage protein that becomes significantly upregulated. Astrocytic activation, quantified through GFAP, is consistently upregulated at 4-, 8-, and 16-weeks post-implantation. Other microglia, macrophage, and astrocyte-related proteins such as CD11b-c and CD45 are upregulated at all measured time points of 4-, 8-, and 16-weeks post-implantation. Our analysis confirms that microglia, macrophages, and astrocytes are present at all time points, but brings context into some molecular changes that are occuring in the protein expression of these cells over time.
Our investigation of neuronal health proteins, specifically downregulation of neuronal cytoskeletal and myelin proteins, may be an indication that irreversible damage is being done to neurons surrounding the implant. NeuN, which is typically used as a marker for neuronal health, is restored to healthy levels 50+ mm from the implant site by 16 weeks post implantation 7. Our protein expression analysis shows that approximately 62% of NfL is lost in 16WK implanted animals. The 62% of NfL lost within 180 mm is unlikely to be contained within the 50 mm radius where neuronal nuclei are depleted. This calls into question how reliable NeuN is as a marker for neuronal health. It could be that neurons are present around the implant but are not functional. Promoting autophagy, specifically ATG5 or ATG12, could improve neuronal survival or functionality.
Using functional recording studies, we can determine if the timeline of proteomic changes align with the functionality of these devices in vivo. Overall we see in the literature that units recorded and active electrode yield decline over time The observed decline in performance at the 8WK time point does not seem to have a definitive effect on recording at that time point, but may be a turning point for neuronal health that, if prevented, could improve chronic performance beyond 8 weeks post-implantation. Our recommendation for any drug with the intent to prevent neuronal defecits would be to either target continuously up to 8 weeks post-implantation or to targer between the 4WK and 8WK time points, which is when the most damage seems to occur (Fig. 4).