Expression patterns of chloride transporters in the auditory brainstem of developing chicken.

GABAergic transmission changes from depolarization to hyperpolarization in most vertebrate brain regions during development. By contrast, in the auditory brainstem of chicken a depolarizing effect of GABA persists after hatching. Since auditory brainstem neurons that receive GABAergic input have a Cl- reversal potential above resting membrane potential, a specifically tuned activity of Cl- transporters is likely. We here present a developmental study of the expression patterns of several members of the SLC12 family (NKCC1, NKCC2, KCC1, KCC2, KCC4, CCC6, CCC9) and of AE3 at developmental ages E7, E10, E12, E15, E17, and P1 with quantitative RT-PCR. NKCC2 and CCC9 were not detected in auditory brainstem (positive control: kidney). KCC1, CCC6 and AE3 were expressed, but not regulated, while NKCC1, KCC2 and KCC4 were regulated. The expression of the latter transporters increased, with KCC2 exhibiting the strongest expression at all time points. Biochemical analysis of the protein expression of NKCC1, KCC2 and KCC4 corroborated the findings on the mRNA level. All three transporters showed a localization at the outer rim of the cells, with NKCC1 and KCC2 expressed in neurons, and KCC4 predominantly in glia. The comparison of the published chloride reversal potential and expression of transporter proteins suggest strong differences in the efficiency of the three transporters. Further, the strong KCC2 expression could reflect a role in the structural development of auditory brainstem synapses that might lead to changes in the physiological properties.

Cell-free artificial implants of electrospun fibres in a three-dimensional gelatin matrix support sciatic nerve regeneration in vivo.

Surgical repair of larger peripheral nerve lesions requires the use of autologous nerve grafts. At present, clinical alternatives to avoid nerve transplantation consist of empty tubes, which are only suitable for the repair over short distances and have limited success. We developed a cell-free, three-dimensional scaffold for axonal guidance in long-distance nerve repair. Sub-micron scale fibres of biodegradable poly-ε-caprolactone (PCL) and collagen/PCL (c/PCL) blends were incorporated in a gelatin matrix and inserted in collagen tubes. The conduits were tested by replacing 15-mm-long segments of rat sciatic nerves in vivo. Biocompatibility of the implants and nerve regeneration were assessed histologically, with electromyography and with behavioural tests for motor functions. Functional repair was achieved in all animals with autologous transplants, in 12 of 13 rats that received artificial implants with an internal structure and in half of the animals with empty nerve conduits. In rats with implants containing c/PCL fibres, the extent of recovery (compound muscle action potentials, motor functions of the hind limbs) was superior to animals that had received empty implants, but not as good as with autologous nerve transplantation. Schwann cell migration and axonal regeneration were observed in all artificial implants, and muscular atrophy was reduced in comparison with animals that had received no implants. The present design represents a significant step towards cell-free, artificial nerve bridges that can replace autologous nerve transplants in the clinic. Copyright © 2017 John Wiley & Sons, Ltd.

Characterisation of cell-substrate interactions between Schwann cells and three-dimensional fibrin hydrogels containing orientated nanofibre topographical cues.

The generation of complex three-dimensional bioengineered scaffolds that are capable of mimicking the molecular and topographical cues of the extracellular matrix found in native tissues is a field of expanding research. The systematic development of such scaffolds requires the characterisation of cell behaviour in response to the individual components of the scaffold. In the present investigation, we studied cell-substrate interactions between purified populations of Schwann cells and three-dimensional fibrin hydrogel scaffolds, in the presence or absence of multiple layers of highly orientated electrospun polycaprolactone nanofibres. Embedded Schwann cells remained viable within the fibrin hydrogel for up to 7 days (the longest time studied); however, cell behaviour in the hydrogel was somewhat different to that observed on the two-dimensional fibrin substrate: Schwann cells failed to proliferate in the fibrin hydrogel, whereas cell numbers increased steadily on the two-dimensional fibrin substrate. Schwann cells within the fibrin hydrogel developed complex process branching patterns, but, when presented with orientated nanofibres, showed a strong tendency to redistribute themselves onto the nanofibres, where they extended long processes that followed the longitudinal orientation of the nanofibres. The process length along nanofibre-containing fibrin hydrogel reached near-maximal levels (for the present experimental conditions) as early as 1 day after culturing. The ability of this three-dimensional, extracellular matrix-mimicking scaffold to support Schwann cell survival and provide topographical cues for rapid process extension suggest that it may be an appropriate device design for the bridging of experimental lesions of the peripheral nervous system.

Three-dimensional configuration of orientated fibers as guidance structures for cell migration and axonal growth.

Peripheral nerve injuries can be surgically repaired by suturing the transected nerve stumps or, in case of larger lesions, by the transplantation of an autologous nerve graft. To avoid donor site morbidity, the development of artificial implants is desired. Clinically, hollow conduits have been used for this purpose but are inferior to the autograft because they lack internal guidance cues for Schwann cells and regenerating axons. In this article, we describe the design of a three-dimensional (3D) scaffold consisting of parallel fibers embedded in a collagen matrix. For this purpose, an electrospinning device was developed to produce and manipulate a 3D array of aligned poly(ɛ-caprolactone) (PCL) microfibers. This fiber array was then incorporated into biodegradable PCL tubes to serve as artificial nerve bridges. Using primary cultures of embryonic chicken dorsal root ganglia, we show that PCL microfibers in the 3D matrix of our composite scaffold guide the direction of Schwann cell migration and axonal growth.