Evaluating the in vivo glial response to miniaturized parylene cortical probes coated with an ultra-fast degrading polymer to aid insertion

Abstract

Objective: Despite the feasibility of short-term neural recordings using implantable microelectrodes, attaining reliable, chronic recordings remains a challenge. Most neural recording devices suffer from a long-term tissue response, including gliosis, at the device-tissue interface. It was hypothesized that smaller, more flexible intracortical probes would limit gliosis by providing a better mechanical match with surrounding tissue.

Approach: This paper describes the in vivo evaluation of flexible parylene microprobes designed to improve the interface with the adjacent neural tissue to limit gliosis and thereby allow for improved recording longevity. The probes were coated with an ultrafast degrading tyrosine-derived polycarbonate (E5005(2K)) polymer that provides temporary mechanical support for device implantation, yet degrades within 2 h post-implantation. A parametric study of probes of varying dimensions and polymer coating thicknesses were implanted in rat brains. The glial tissue response and neuronal loss were assessed from 72 h to 24 weeks post-implantation via immunohistochemistry.

Main results: Experimental results suggest that both probe and polymer coating sizes affect the extent of gliosis. When an appropriate sized coating dimension (100 µm × 100 µm) and small probe (30 µm × 5 µm) was implanted, a minimal post-implantation glial response was observed. No discernible gliosis was detected when compared to tissue where a sham control consisting of a solid degradable polymer shuttle of the same dimensions was inserted. A larger polymer coating (200 µm × 200 µm) device induced a more severe glial response at later time points, suggesting that the initial insertion trauma can affect gliosis even when the polymer shuttle degrades rapidly. A larger degree of gliosis was also observed when comparing a larger sized probe (80 µm × 5 µm) to a smaller probe (30 µm × 5 µm) using the same polymer coating size (100 µm × 100 µm). There was no significant neuronal loss around the implantation sites for most device candidates except the group with largest polymer coating and probe sizes.

Significance: These results suggest that: (1) the degree of mechanical trauma at device implantation and mechanical mismatches at the probe-tissue interface affect long term gliosis; (2) smaller, more flexible probes may minimize the glial response to provide improved tissue biocompatibility when used for chronic neural signal recording; and (3) some degree of glial scarring did not significantly affect neuronal distribution around the probe.

Figure 1.

Parylene probe fabrication protocol, polymer coating procedure. First, a thin layer of parylene was deposited. A thin aluminum mask layer that defined the probe geometry was deposited onto the top of the parylene layer with a lift-off method. An oxygen plasma etch was performed to pattern the probe. The mask layer was dissolved with a chemical etchant to complete probe fabrication. The probe was coated with the polymer through MIMIC, and the whole device was dried and released from the substrate [71].

Figure 2.

Surgical procedure for the animal study. (A) Two craniotomies were performed. (B), (C) Two probes from each of the five groups were implanted simultaneously via a custom designed surgical holder. (D) Dental cement was used to fix the surgical holder, and the wound was sutured. The animal was placed on a heating pad until recovery from anesthesia. (E) Location and dimension of the two craniotomies and the ten probes (table 1 for number reference) with different dimensions. Black circle display the location of the four skull screws. A = 4 mm; b = 4 mm; c = 6 mm; d = 3 mm.

Figure 3.

Representative image of (1) a montage of images with a grid for implantation site identification. Scale bar: 500 μm. (2) Inset: magnified images of several implantation sites for downstream image analysis process. Sample ID: 72 h control animal sectioned 2 mm deep from the surface of the brain stained with GFAP (astrocytes). R and L indicate right hemisphere and left hemisphere for the animal. Scale bar: 200 μm.

Figure 4.

(A) Schematic of the rotational intensity sweep profile anaysis. (B) Represensative data showing normalized intensity versus distance from the center of the implantation site. The intensity values were normalized by the undamaged area, which was denoted with a value of 1. Area under the curve and above 1 was obtained to indicate the relative image area where the cell density was greater compared to the undamaged area within the region of interest.

Figure 5.

Representative images of GFAP immunostaining for different devices at different time points. Scale bar: 200 μm.

Figure 6.

(A) Normalized GFAP intensity at different time points for the five probe groups. *Significance between different groups (p < 0.05). **Significance between different groups (p < 0.01). ***Significance between different groups (p < 0.001). GFAP intensity over the distance from the center of the implantation site for different groups. (B) 72 h post device implantation. (C) 24 weeks post device implantation. The shaded area represents the standard error for each data point.

Figure 7.

Representative images of Iba-1 immunostaining for different groups at different time points. Scale bar: 200 μm.

Figure 8.

(A) Normalized intensity of Iba-1 staining at different time points for the five probe groups. **Significance between different groups (p < 0.01), Iba-1 intensity over the distance from the center of the implantation site for different groups. (B) 72 h post device implantation. (C) 24 weeks post device implantation. The shaded area represents the standard error for each data point.

Figure 9.

NeuN intensity over the distance from the center of the implantation site for different groups. (A) 72 h post device implantation. (B) 24 weeks post device implantation. The shaded area represents the standard error for each data point.