Short and long term biocompatibility of NeuroProbes silicon probes

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Abstract

Brain implants provide exceptional tools to understand and restore cerebral functions. The utility of these devices depends crucially on their biocompatibility and long term viability. We addressed these points by implanting non-functional, NeuroProbes silicon probes, without or with hyaluronic acid (Hya), dextran (Dex), dexamethasone (DexM), Hya + DexM coating, into rat neocortex. Light and transmission electron microscopy were used to investigate neuronal survival and glial response. The surface of explanted probes was examined in the scanning electron microscope. We show that blood vessel disruption during implantation could induce considerable tissue damage. If, however, probes could be inserted without major bleeding, light microscopical evidence of damage to surrounding neocortical tissue was much reduced. At distances less than 100 μm from the probe track a considerable neuron loss (∼40%) occurred at short survival times, while the neuronal numbers recovered close to control levels at longer survival. Slight gliosis was observed at both short and long term survivals. Electron microscopy showed neuronal cell bodies and synapses close (<10 μm) to the probe track when bleeding could be avoided. The explanted probes were usually partly covered by tissue residue containing cells with different morphology. Our data suggest that NeuroProbes silicon probes are highly biocompatible. If major blood vessel disruption can be avoided, the low neuronal cell loss and gliosis should provide good recording and stimulating results with future functional probes. We found that different bioactive molecule coatings had small differential effects on neural cell numbers and gliosis, with optimal results achieved using the DexM coated probes.

Introduction

Recent neocortical prostheses begin to offer exceptional possibilities for restoring cerebral function (Schwartz, 2004). Furthermore, multiple microprobes for cerebral recording and stimulation are vital tools to advance understanding of cellular and systems level function in the central nervous system. While significant success has been achieved with these approaches, the interface between brain tissue and implanted prostheses or probes can still be greatly improved. Important aspects of this interface include: (1) reaching neurons at the desired location in the brain; (2) ensuring consistent electrical recording and stimulation conditions in the long term; and (3) rendering the devices as inert as possible in terms of biocompatibility, biostability and biofouling.

The European Project NeuroProbes (http://www.neuroprobes.eu) aims to achieve an optimal neural tissue-probe interface. Single and multiple shank silicon-based NeuroProbes microprobes are developed and combined with chemical sensors and microfluidic systems (Frey et al., 2007, Neves et al., 2007, Ruther et al., 2008, Spieth et al., 2008). The specific microsystem integration of NeuroProbes is designed so that these features can be integrated into a common platform in a modular fashion (Aarts et al., 2008). This will permit a consistent and combined use of biosensors, drug delivery paths, electrical recording and stimulation to understand brain function.

Long term viability and biocompatibility is a key attribute of implanted microprobes. Even materials considered to be biocompatible can induce adverse brain reactions. Two major factors determine the degree of tissue response. First, when a probe is inserted, it causes mechanical damage, damaging brain cells, disrupting blood vessels and thus compromising the blood–brain barrier. Second, an inflammatory neural tissue reaction is induced by both the implant and the injury it causes (for review see Cheung, 2007). This usually results in the development of a glial scar around the probe, which tends to reduce the efficacy of neuronal recording and stimulation (Edell et al., 1992, Fawcett and Asher, 1999, Polikov et al., 2005, Schwartz, 2004, Shain et al., 2003, Szarowski et al., 2003, Turner et al., 1999).

Probes are usually implanted for long term recordings lasting weeks or months. Therefore they should not be susceptible to attack by biological fluids, proteases, macrophages, brain metabolic factors. Silicon seems to be a good candidate as resistant material for long term recording probes. Surface damage is usually reported if stimulation has been performed through the electrodes of the probe (for review see Merrill et al., 2005). At the same time, scanning electron microscopy shows adherent cells and tissue residue on the surface of the explanted probes (Biran et al., 2005, Hoogerwerf and Wise, 1994, Turner et al., 1999).

Within the NeuroProbes project, we emphasize long term chronic use of the silicon microprobes and seek actively to diminish inflammatory and glial responses to implantation. The silicon surface of the probes was coated by different molecules, in order to produce more biocompatible surfaces for use in brain implants. We followed three main directions. First, we attempted to create a surface resembling the habitual environment of the neural tissue using hyaluronic acid (Hya) coating. Hya is a naturally occurring polysaccharide, which forms an important component of the extracellular matrix (Fraser et al., 1997). In addition, Hya has an important role in biological processes such as cell motility, cell differentiation and wound healing (Pasqui et al., 2007, Pouyani and Prestwich, 1994). Injecting hyaluronic acid into an injured area was shown to reduce glial scar formation in the rat neocortex (Lin et al., 2009). Second, we aimed to decrease bleeding due to mechanical damage by coating probes with dextran (Dex). Dex is a complex polysaccharide, used medicinally as an antithrombotic agent to reduce blood viscosity and vascular thrombosis (Gallus and Hirsh, 1976). It binds erythrocytes, platelets, and vascular endothelium, so reducing erythrocyte aggregation and platelet adhesiveness (for reviews see Abir et al., 2004, Johnson and Barker, 1992). Clots formed in the presence of Dex are more easily lysed due to an enhanced fibrinolysis (Jones et al., 2008). Furthermore, surface immobilized Dex limits cell adhesion and spreading (Massia et al., 2000). Thirdly we attempted to reduce tissue reaction by coating probes with the anti-inflammatory drug dexamethasone (DexM). DexM is a synthetic and potent steroid hormone of the glucocorticoid class. Systemic DexM administration attenuates glial responses in rat neocortex (Shain et al., 2003, Spataro et al., 2005), but may also have serious side effects (Kaal and Vecht, 2004, Koehler, 1995, Twycross, 1994). Previous work suggests that implanted silicon probes coated with DexM reduces the tissue reaction and lowers neuronal loss (Zhong and Bellamkonda, 2007, Zhong et al., 2005).

Here we used several microscopical methods to quantify short and long term neuron loss and glial reaction in response to the implantation into rat neocortex of NeuroProbes silicon probes. Light and transmission electron microscopy, as well as stereological methods were used to examine cellular responses and scanning electron microscopy was used to reveal the modifications of the probe surface caused by the brain tissue. The effect on the tissue reaction of different coatings, including Hya, Dex, DexM and Hya/DexM, was investigated.

Section snippets

Probe implantation and explantation

The biocompatibility of NeuroProbes silicon probes was tested in vivo, in the neocortex of Wistar rats (n = 13). All implanted multiple microprobe had four, 2-mm-long shanks, and were non-functional, i.e. were not equipped with output cables. Three different types of probes were implanted (Fig. 1, Table 1). (1) E100P probes had shanks with a cross section of 140 μm × 100 μm at the connector part gradually decreasing to 100 μm × 100 μm close to the tip, with an opening angle of 17°, five Pt electrode

Results

The biocompatibility and the effect of different coatings on NeuroProbes silicon probes were investigated in the rat neocortex in vivo, at short and long terms. Three different types of probes were implanted into the dorsal neocortical areas of rats (see Section 2, Table 1, Fig. 1; Herwik et al., 2009). Glial reaction was described with qualitative analysis around the probe track, whereas neuronal numbers were quantified with manual stereological methods. Qualitative light microscopic analysis

Discussion

Implanted recording and stimulation microprobes are indispensable tools for research on the function of the intact brain, but their utility depends on biocompatibility and long term viability. Silicon-based surfaces are known to be highly biocompatible (Rutten, 2002). However, acute and chronic inflammatory processes seem likely to affect the surrounding neural tissue (for review see Polikov et al., 2005).

In this study we explored the biocompatibility of NeuroProbes silicon probes, and also

Conclusions

Biocompatibility and long term viability are crucial questions for chronic applications of implanted devices. Our data shows that the silicon-based microprobes developed in the framework of the NeuroProbes project are highly biocompatible. Neuronal loss around the probes was evident at 1 week after implantation within distances of 100 μm from the probe track. However, it was considerably reduced with time, with neuronal densities returning to 90% of control levels at 2–4 weeks after

Acknowledgements

We wish to thank Ms K. Iványi, K. Lengyel, E. Simon, K. Faddi, Mr P. Kottra and Gy. Goda for excellent technical assistance. This study was supported by the Hungarian Government OTKA K81357 grant and Bolyai János Research Fellowship grant and European Union NeuroProbes EU IP IST-027017 grant.

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