Local delivery of minocycline-loaded PLGA nanoparticles from gelatin-coated neural implants attenuates acute brain tissue responses in mice.

BACKGROUND: Neural interfaces often elicit inflammatory responses and neuronal loss in the surrounding tissue which adversely affect the function and longevity of the implanted device. Minocycline, an anti-inflammatory pharmaceutics with neuroprotective properties, may be used for reducing the acute brain tissue responses after implantation. However, conventional administration routes require high doses which can cause adverse systemic side effects. Therefore, the aim of this study was to develop and evaluate a new drug-delivery-system for local and sustained administration of minocycline in the brain. METHODS: Stainless steel needles insulated with Parylene-C were dip-coated with non-crosslinked gelatin and minocycline-loaded PLGA nanoparticles (MC-NPs) were incorporated into the gelatin-coatings by an absorption method and subsequently trapped by drying the gelatin. Parylene-C insulated needles coated only with gelatin were used as controls. The expression of markers for activated microglia (CD68), all microglia (CX3CR1-GFP), reactive astrocytes (GFAP), neurons (NeuN) and all cell nuclei (DAPI) surrounding the implantation sites were quantified at 3 and 7 days after implantation in mice. RESULTS: MC-NPs were successfully incorporated into gelatin-coatings of neural implants by an absorption method suitable for thermosensitive drug-loads. Immunohistochemical analysis of the in vivo brain tissue responses, showed that MC-NPs significantly attenuate the activation of microglial cells without effecting the overall population of microglial cells around the implantation sites. A delayed but significant reduction of the astrocytic response was also found in comparison to control implants. No effect on neurons or total cell count was found which may suggest that the MC-NPs are non-toxic to the central nervous system. CONCLUSIONS: A novel drug-nanoparticle-delivery-system was developed for neural interfaces and thermosensitive drug-loads. The local delivery of MC-NPs was shown to attenuate the acute brain tissue responses nearby an implant and therefore may be useful for improving biocompatibility of implanted neuro-electronic interfaces. The developed drug-delivery-system may potentially also be used for other pharmaceutics to provide highly localized and therefore more specific effects as compared to systemic administration.

Impact of degradable nanowires on long-term brain tissue responses.

BACKGROUND: A promising approach to improve the performance of neural implants consists of adding nanomaterials, such as nanowires, to the surface of the implant. Nanostructured interfaces could improve the integration and communication stability, partly through the reduction of the cell-to-electrode distance. However, the safety issues of implanted nanowires in the brain need to be evaluated and understood before nanowires can be used on the surface of implants for long periods of time. To this end we here investigate whether implanted degradable nanowires offer any advantage over non-degradable nanowires in a long-term in vivo study (1 year) with respect to brain tissue responses. RESULTS: The tissue response after injection of degradable silicon oxide (SiOx)-coated gallium phosphide nanowires and biostable hafnium oxide-coated GaP nanowires into the rat striatum was compared. One year after nanowire injection, no significant difference in microglial or astrocytic response, as measured by staining for ED1 and glial fibrillary acidic protein, respectively, or in neuronal density, as measured by staining for NeuN, was found between degradable and biostable nanowires. Of the cells investigated, only microglia cells had engulfed the nanowires. The SiOx-coated nanowire residues were primarily seen in aggregated hypertrophic ED1-positive cells, possibly microglial cells that have fused to create multinucleated giant cells. Occasionally, degradable nanowires with an apparently intact shape were found inside single, small ED1-positive cells. The biostable nanowires were found intact in microglia cells of both phenotypes described. CONCLUSION: The present study shows that the degradable nanowires remain at least partly in the brain over long time periods, i.e. 1 year; however, no obvious bio-safety issues for this degradable nanomaterial could be detected.

The influence of porous silicon on axonal outgrowth in vitro.

xonal outgrowth on smooth and porous silicon surfaces was studied in organ culture. The pore size of the silicon substrata varied between 100 and 1500 nm. We found that axons preferred to grow and elongate on porous silicon surfaces only when pores of (150-500 nm) are available.

Neurite guidance on protein micropatterns generated by a piezoelectric microdispenser.

In this study, we developed a microdispenser technique in order to create protein patterns for guidance of neurites from cultured adult mouse dorsal root ganglia (DRG). The microdispenser is a micromachined silicon device that ejects 100 picolitre droplets and has the ability to position the droplets with a precision of 6-8 microm. Laminin and bovine serum albumin (BSA) was used to create adhesive and non-adhesive protein lines on polystyrene surfaces (cell culture dishes). Whole-mounted DRGs were then positioned close to the patterns and neurite outgrowth was monitored. The neurites preferred to grow on laminin lines as compared to the unpatterned plastic. When patterns were made from BSA the neurites preferred to grow in between the lines on the unpatterned plastic surface. We conclude that microdispensing can be used for guidance of sensory neurites. The advantages of microdispensing is that it is fast, flexible, allows deposition of different protein concentrations and enables patterning on delicate surfaces due to its non-contact mode of operation. It is conceivable that microdispensing can be utilized for the creation of protein patterns for guiding neurites to obtain in vitro neural networks, in tissue engineering or rapid screening for guiding proteins.

Galectin-3 inhibits Schwann cell proliferation in cultured sciatic nerve.

The production of galectin-3, a carbohydrate-binding mammalian lectin, is upregulated in Schwann cells after peripheral nerve injury in areas where Schwann cells proliferate. Here we tested if galectin-3 affected proliferation of Schwann cells in cultured sciatic nerve segments. Galectin-3 significantly decreased the number of bromodeoxyuridine-labelled Schwann cell nuclei. Neither lactose nor a synthetic inhibitor directed against the carbohydrate-binding region abolished the effects of galectin-3. In addition, a mutant galectin-3 unable to bind endogenous carbohydrates had similar effects as normal galectin-3. We conclude that galectin-3 reduces proliferation of Schwann cells in cultured sciatic nerve segments by a mechanism which is independent of its carbohydrate-binding moiety.

Size-dependent long-term tissue response to biostable nanowires in the brain.

Nanostructured neural interfaces, comprising nanotubes or nanowires, have the potential to overcome the present hurdles of achieving stable communication with neuronal networks for long periods of time. This would have a strong impact on brain research. However, little information is available on the brain response to implanted high-aspect-ratio nanoparticles, which share morphological similarities with asbestos fibres. Here, we investigated the glial response and neuronal loss in the rat brain after implantation of biostable and structurally controlled nanowires of different lengths for a period up to one year post-surgery. Our results show that, as for lung and abdominal tissue, the brain is subject to a sustained, local inflammation when biostable and high-aspect-ratio nanoparticles of 5 µm or longer are present in the brain tissue. In addition, a significant loss of neurons was observed adjacent to the 10 µm nanowires after one year. Notably, the inflammatory response was restricted to a narrow zone around the nanowires and did not escalate between 12 weeks and one year. Furthermore, 2 µm nanowires did not cause significant inflammatory response nor significant loss of neurons nearby. The present results provide key information for the design of future neural implants based on nanomaterials.

The density difference between tissue and neural probes is a key factor for glial scarring.

A key to successful chronic neural interfacing is to achieve minimal glial scarring surrounding the implants, as the astrocytes and microglia may functionally insulate the interface. A possible explanation for the development of these reactions is mechanical forces arising between the implants and the brain. Here, we show that the difference between the density of neural probes and that of the tissue, and the resulting inertial forces, are key factors for the development of the glial scar. Two probes of similar size, shape, surface structure and elastic modulus but differing greatly in density were implanted into the rat brain. After six weeks, significantly lower astrocytic and microglial reactions were found surrounding the low-density probes, approaching no reaction at all. This provides a major key to design fully biocompatible neural interfaces and a new platform for in vivo assays of tissue reactions to probes with differing materials, surface structures, and shapes.

Can histology solve the riddle of the nonfunctioning electrode? Factors influencing the biocompatibility of brain machine interfaces.

Neural interfaces hold great promise to become invaluable clinical and diagnostic tools in the near future. However, the biocompatibility and the long-term stability of the implanted interfaces are far from optimized. There are several factors that need to be addressed and standardized when improving the long-term success of an implanted electrode. We have chosen to focus on three key factors when evaluating the evoked tissue responses after electrode implantation into the brain: implant size, fixation mode, and evaluation period. Further, we show results from an ultrathin multichannel wire electrode that has been implanted in the rat cerebral cortex for 1 year. To improve biocompatibility of implanted electrodes, we would like to suggest that free-floating, very small, flexible, and, in time, wireless electrodes would elicit a diminished cell encapsulation. We would also like to suggest standardized methods for the electrode design, the electrode implantation method, and the analyses of cell reactions after implantation into the CNS in order to improve the long-term success of implanted neural interfaces.

Implant size and fixation mode strongly influence tissue reactions in the CNS.

The function of chronic brain machine interfaces depends on stable electrical contact between neurons and electrodes. A key step in the development of interfaces is therefore to identify implant configurations that minimize adverse long-term tissue reactions. To this end, we here characterized the separate and combined effects of implant size and fixation mode at 6 and 12 weeks post implantation in rat (n = 24) cerebral cortex. Neurons and activated microglia and astrocytes were visualized using NeuN, ED1 and GFAP immunofluorescence microscopy, respectively. The contributions of individual experimental variables to the tissue response were quantified. Implants tethered to the skull caused larger tissue reactions than un-tethered implants. Small diameter (50 µm) implants elicited smaller tissue reactions and resulted in the survival of larger numbers of neurons than did large diameter (200 µm) implants. In addition, tethering resulted in an oval-shaped cavity, with a cross-section area larger than that of the implant itself, and in marked changes in morphology and organization of neurons in the region closest to the tissue interface. Most importantly, for implants that were both large diameter and tethered, glia activation was still ongoing 12 weeks after implantation, as indicated by an increase in GFAP staining between week 6 and 12, while this pattern was not observed for un-tethered, small diameter implants. Our findings therefore clearly indicate that the combined small diameter, un-tethered implants cause the smallest tissue reactions.

Gelatine-embedded electrodes--a novel biocompatible vehicle allowing implantation of highly flexible microelectrodes.

Chronic neural interfaces that are both structurally and functionally stable inside the brain over years or decades hold great promise to become an invaluable clinical tool in the near future. A key flaw in the current electrode interfaces is that their recording capabilities deteriorate over time, possibly due to the lack of flexibility, which causes movements in relation to the neural tissue that result in small inflammations and loss of electrode function. We have developed a new neural probe using the stabilizing property of gelatine that allows the implantation of ultra-thin and flexible electrodes into the central nervous system. The microglial and astrocytic reactions evoked by implanted gelatine needles, as well as the wire bundles in combination with gelatine, were investigated using immunohistochemistry and fluorescence microscopy up to 12 weeks after implantation. The results indicate that pure gelatine needles were stiff enough to penetrate the brain tissue on their own, and evoked a significantly smaller chronic scar than stab wounds. Moreover, gelatine embedding appeared to reduce the acute reactions caused by the implants and we found no adverse effects of gelatine or gelatine-embedded electrodes. Successful electrophysiological recordings were made from very thin electrodes implanted in this fashion.