Remy Wiertz

Bifurcating microchannels as a scaffold to induce separation of regenerating neurites

Many neural interfacing strategies, such as the sieve electrode and the cultured probe, rely on neurite growth to establish neural contact. But this growth is subject to natural fasciculation, compromising the effectiveness of these interfacing strategies by reducing potential selectivity. This in vitro study shows that the fasciculation mechanism can be manipulated by providing an appropriate microchannel scaffold to guide and influence growth, thereby achieving a high degree of selectivity. The microchannels employed have a bifurcation from a primary channel into two secondary channels. This bifurcating microstructure was able to support and promote fasciculated growth over 70% of the time for microchannels widths of 2.5, 5, 10 and 20 microm. Fasciculation is shown to be a strong force during ingrowth, with the initiation of neurite separation related to random spatial exploration. Narrower microchannels initiate separated growth better. Once separated growth starts fasciculation results in an even distribution of neurite growth across the bifurcation. The reduction from 20 microm to 10 microm wide channels also resulted in a 3-fold decrease in ingrowing neurites performing 180 degrees turns to exit the microchannel via the entrance. No neurite turning was observed for both the 5 and 2.5 microm wide channels.

In vitro Verification of a 3-D Regenerative Neural Interface Design: Examination of Neurite Growth and Electrical Properties Within a Bifurcating Microchannel Structure

Toward the development of neuroprosthesis, we propose a 3-D regenerative neural interface design for connecting with the peripheral nervous system. This approach relies on bifurcating microstructures to achieve defasciculated ingrowth patterns and, consequently, high selectivity. In vitro studies were performed to validate this design by showing that fasciculation during nerve regeneration can be influenced by providing a scaffold to guide growth appropriately. With this approach, neurites can be separated from one another and guided toward specific electrode sites to create a highly selective interface. The neurite separation characteristics were examined for smaller microchannel structures (2.5 and 5 ¿m wide) and larger microchannels (10 and 20 ¿m wide), with smaller microchannels shown to be statistically more effective at initiating separation. Electrodes incorporated at different locations within the microchannels allowed for the recording and tracking of action potential propagation. Microchannel size was also found to play an important role in this regard, with smaller microchannels amplifying the recordable extracellular signal; a twofold increase in the signal to noise ratio was found for 5 ¿m wide microchannels.

Impedance Sensing for Monitoring Neuronal Coverage and Comparison With Microscopy

We investigated the applicability of electric impedance sensing (IS) to monitor the coverage of adhered dissociated neuronal cells on glass substrates with embedded electrodes. IS is a sensitive method for the quantification of changes in cell morphology and cell mobility, making it suitable to study aggregation kinetics. Various sizes of electrodes were compared for the real-time recording of the impedance of adhering cells, at eight frequencies (range: 5 Hz-20 kHz). The real part of the impedance showed to be most sensitive at frequencies of 10 and 20 kHz for the two largest electrodes (7850 and 125 600 μm2). Compared to simultaneous microscopic evaluation of cell coverage and cell spreading, IS shows more detail.

Inhibition of neuronal cell–cell adhesion measured by the microscopic aggregation assay and impedance sensing

Microscopic aggregation assay and impedance sensing (IS) were used to monitor a change in in vitro neuron-neuron adhesion in response to blocking of cell adhesion molecules. By blocking neuron-neuron adhesion, migration and aggregation of neuronal cells can be inhibited. This leads to better control of spatial arrangement of cells in culture. In the literature N-CAM, L1 and N-cadherin proteins are pointed out as main regulators of neuronal adhesion. In this study, these three main cell adhesion molecules were used to inhibit neuron-to-neuron adhesion and aggregation. Both soluble extracellular domains and antigen antibodies were added to these adhesion molecules. They were investigated for their blocking ability in neuronal cultures. First, in a 96 h aggregation assay on a low-adhesive substrate, the effect of inhibition of the three proteins on aggregation of cortical neurons was investigated optically. Both L1 antibody and L1 protein had no effect on the degree of aggregation. An N-cadherin antibody however was shown to be effective in aggregation inhibition at concentrations of 1 and 3 µg ml(-1). Up to 96 h no aggregation occurred. A similar effect was achieved by the N-cadherin protein, although less distinct. N-CAM blocking revealed no inhibition of aggregation. Second, results from IS corresponded to those of the aggregation assays. In these experiments neuron-neuron adhesion was also inhibited by blocking N-CAM L1 and N-cadherin. Cortical neurons were cultured in small wells containing circular 100 µm diameter gold electrodes, so small changes in cell-cell interactions in monolayers of neurons could be monitored by IS. Impedances of neuron-covered electrodes were significantly lower in the presence of the N-cadherin antibody and protein at concentrations of 1, 3 and 10 µg ml(-1), indicating a less profound binding between adjacent neurons. Results from the aggregation assays and impedance measurements demonstrate the applicability of blocking cell adhesion molecules for inhibition of cell-cell adhesion and aggregation.

Time-dependent changes in ghrelin-immunoreactivity in dissociated neuronal cultures of the newborn rat neocortex

Ghrelin is a hormone, initially described as a gastric peptide stimulating appetite and growth hormone secretion, which also has an important role in the regulation of many other processes, including higher brain functions. Ghrelin has been described in situ in different parts of the brain, but so far there has been no data about its expression in cell cultures. Therefore, we aimed in this study to investigate the levels of ghrelin in dissociated cortical neurons at various times in culture. We applied the ABC immunocytochemical method for the detection of ghrelin in one-day-, one-week-, and two-week-old cultures. Our results clearly show that at the early stages after plating the cultures 86.2% (+/-8.93) of the neurons are ghrelin-positive and their number decreases during the culturing period. As ghrelin is present in the majority of cultured newborn neurons, when the neuronal differentiation and network formation take place, it may also influence the early synaptic formation and cell-to-cell interactions, which are both very important for network functions like learning and memory.

Neural cell–cell and cell–substrate adhesion through N-cadherin, N-CAM and L1

In this study neural (N)-cadherin, neural cell adhesion molecule (N-CAM) and L1 proteins and their antibody equivalents were covalently immobilized on a polyethylene-imine (PEI)-coated glass surface to form neuron-adhesive coatings. Impedance sensing and (supplementary) image analysis were used to monitor the effects of these CAMs. Immobilization of high concentrations of both N-cadherin protein and antibody led to good adhesion of neurons to the modified surface, better than surfaces treated with 30.0 and 100.0 µg ml(-1) N-CAM protein and antibody. L1 antibody and protein coating revealed no significant effect on neuronal cell-substrate adhesion. In a second series of combinatorial experiments, we used the same antibodies and proteins as medium-additives to inhibit cell-cell adhesion between neurons. Adhesion of neurons cultured on N-cadherin protein or antibody-modified surfaces was lowered by the addition of a soluble N-cadherin protein and antibody to the culturing medium, accelerating neuronal aggregation. The presence of a soluble N-CAM antibody or protein had no effect on the adhesion of neuronal cells on a N-cadherin protein-modified surface. On a N-cadherin antibody-coated surface, the addition of a soluble N-CAM protein led to cell death of neurons after 48 h, while a N-CAM antibody had no effect. In the presence of a soluble N-cadherin protein and antibody the aggregation of neurons was inhibited, both on N-CAM protein and N-CAM antibody-modified surfaces. Neurons cultured on immobilized antibodies were less affected by the addition of soluble CAM blockers than neurons cultured on immobilized proteins, indicating that antibody-protein bonds are more stable compared to protein-protein bonds.

Neural growth into a microchannel network: Towards A regenerative neural interface

We propose and validated a design for a highly selective “endcap” regenerative neural interface towards a neuroprosthesis. In vitro studies using rat cortical neurons determine if a branching microchannel structure can counter fasciculated growth and cause neurites to separate from one another, allowing for greater selective contact. Initial studies find that narrower branching microchannels achieve improved neurite separation. Electrical stimulation of neurites within microchannels is possible, as is recording of neurite action potentials with the microchannels acting as electrical signal amplifiers.

Ghrelin expression in dissociated cultures of the rat neocortex

Ghrelin is a hormone, initially described as a gastric peptide stimulating appetite and growth hormone secretion, which also has an important role in the regulation of many other processes including higher brain functions. Ghrelin has been described in situ in different part of the brain, but so far there are no data about it in cell cultures. Therefore, we aimed this study to investigate the developmental pattern of ghrelin in dissociated cortical neuronal cultures. We applied the ABC immunocytochemical method for detection of ghrelin in dissociated cortical neurons from newborn rats, incubated for one day-, one week-, or two week old cultures. Our results clearly show that at the early stages of development of the network, majority of the neurons are ghrelin-positive, and their number decreases during the culturing period. As ghrelin is omnipresent in the early stages, when the neuronal differentiation and network formation take place, it may also influence the early development of synaptic formation and cell-to-cell interactions, both very important for the network functions like learning and memory.

Electric impedance sensing during the inhibition of cell-cell adhesion

Electric cell impedance sensing (ECIS) was used to monitor the change of in vitro neuron-neuron adhesion in response to the blocking of N-Cam, N-Cadherin and L1. ECIS is a method in which cell morphology and cell mobility can be indirectly measured by changes in intercellular resistance.

The use of an oriented, collagen-based matrix for nerve regeneration in the central nervous system

Since surface properties of hydroxyapatite (HA) play an important role in its performance, surface modification of HA has gained much attention from researchers. Silane coupling agents have been the focus of the research. In this study, an effective surface modification method was developed using hexamethylene diisocyanate as a coupling agent. Polyethylene glycol (Mw=1500) was successfully coupled to the surface of nano-size apatite particles (nano-apatite). Various methods were used to characterize the surface-modified nano-apatite. Infra-red spectra confirmed the existence of a layer of polymer with both urethane and ether linkage on the surface of nano-apatite. The amount of grafted polymer as determined by total organic carbon analysis (TOC) and thermal gravimetric analysis (TGA) was about 20% in weight. Solid 1H MAS NMR spectra indicated that the amount of hydroxyl groups of nano-apatite was decreased by 7.7% after surface grafting reaction. It is concluded that the surface hydroxyl groups of nano-apatite have the reactivity towards isocyanate groups.