Neurobiochemical changes in the vicinity of a nanostructured neural implant

Abstract

Neural interface technologies including recording and stimulation electrodes are currently in the early phase of clinical trials aiming to help patients with spinal cord injuries, degenerative disorders, strokes interrupting descending motor pathways, or limb amputations. Their lifetime is of key importance; however, it is limited by the foreign body response of the tissue causing the loss of neurons and a reactive astrogliosis around the implant surface. Improving the biocompatibility of implant surfaces, especially promoting neuronal attachment and regeneration is therefore essential. In our work, bioactive properties of implanted black polySi nanostructured surfaces (520-800 nm long nanopillars with a diameter of 150-200 nm) were investigated and compared to microstructured Si surfaces in eight-week-long in vivo experiments. Glial encapsulation and local neuronal cell loss were characterised using GFAP and NeuN immunostaining respectively, followed by systematic image analysis. Regarding the severity of gliosis, no significant difference was observed in the vicinity of the different implant surfaces, however, the number of surviving neurons close to the nanostructured surface was higher than that of the microstructured ones. Our results imply that the functionality of implanted microelectrodes covered by Si nanopillars may lead to improved long-term recordings.

Figure 1. Light micrographs of the electrode tracks were segmented using a custom made ImageJ macro.

From the manually defined track outlines 50 μm wide regions were segmented up to 500 μm along the selected shape of the track. Yellow-edge stripes represent the 50 μm wide manually selected ROIs. Different sides of the electrode track were also selected manually. In the case of GFAP staining (a), average pixel intensities were calculated in each ROI. On the NeuN stained images (b), cell numbers were determined manually in each ROI.

Figure 2. The two types of fabricated devices are one with nanostructured shank (right side) and a reference with polycrystalline Si front-side (left side).

The two sidewalls of both devices are microstructured with a fluorocarbon polymer layer on the Si wafer as a result of the Bosch etching step (micro-polymer). The backside of the devices are the non-polished Si wafer (microSi). Front-side of the reference device is a polycrystalline Si layer with 100–150 nm grain size (flatSi), and the front-side of the nanostructured device has 520–800 nm high pillars in a 18–70 pillars/μm² density (nanoSi).

Figure 3. Representative images of GFAP staining.

Considerable difference can be seen in the intensity of GFAP staining as a function of the distance (a). A massive glial scar is present in the vicinity of the injury. On image (b) a part of the stained tissue is presented in a distance over 1 mm from the injury which is considered to be far enough to be intact.

Figure 4. Quantification of the GFAP staining.

With increasing distance from the implantation site, the intensity of GFAP staining is decreased. In the vicinity (0–50 μm) of all sides of the electrode track, a massive glial scar was formed regardless of surface properties. From a distance of 50 μm up to 300 μm, the GFAP intensity was consistently lower by 4–5% in case of the nanoSi surfaces than all other types however, differences were not statistically significant. (N_{Micro-polymer} = 132, N_MicroSi = 66, N_FlatSi = 31, N_NanoSi = 35). Sample means and standard deviations are presented. Exact P-values are presented in the Supplementary Material.

Figure 5. Representative images of NeuN staining.

Neuron loss is observed in the vicinity of the injury (a). (b) Reference cell density at a distance of 1 mm.

Figure 6. Results of the neural cell count quantification.

Increasing neural density can be measured with the distance from the electrode track. After the first 200 μm there was no considerable change in neural cell number in function of the distance. In the first 50 μm, which is considered as the recording distance of a microelectrode, a significant difference was found in average neural cell density in case of surface properties. The highest neural cell loss was found at the microstructured fluorocarbon polymer covered surface, while the highest neural cell density appeared at the proximity of nanostructured implant surfaces. Significant difference was found between the flat and nanostructured Si surfaces implying that nanotopography is favoured by neurons within the distance relevant in neural recording. (N_{Micro-polymer} = 50, N_MicroSi = 25, N_FlatSi = 23, N_NanoSi = 23) Sample means and standard deviations are presented. Exact P-values are presented in the Supplementary Material.

Figure 7. Correlation between the GFAP-positive scar tissue and the neuronal cell loss in the first 100 microns from the implant site.

(a,b) are a pair of adjacent (60 μm distance) tissue slices stained with GFAP (a) and NeuN (b). Where strong GFAP-positivity is present, indicating the development of the glial scar, neurons are sparse or missing.