Guidance of dorsal root ganglion neurites and Schwann cells by isolated Schwann cell topography on poly(dimethyl siloxane) conduits and films.

Biomimetic replicas of cellular topography have been utilized to direct neurite outgrowth. Here, we cultured postnatal rat dorsal root ganglion (DRG) explants in the presence of Schwann cell (SC) topography to determine the influence of SC topography on neurite outgrowth. Four distinct poly(dimethyl siloxane) conduits were fabricated within which DRG explants were cultured. To determine the contribution of SC topographical features to neurite guidance, the extent of neurite outgrowth into unpatterned conduits, conduits with randomly oriented SC replicas, and conduits with SC replicas parallel or perpendicular to the conduit long axis was measured. Neurite directionality and outgrowth from DRG were also quantified on two-dimensional SC replicas with orientations corresponding to the four conduit conditions. Additionally, live SC migration and neurite extension from DRG on SC replicas were examined as a first step toward quantification of the interactions between live SC and navigating neurites on SC replicas. DRG neurite outgrowth and morphology within conduits and on two-dimensional SC replicas were directed by the underlying SC topographical features. Maximal neurite outgrowth and alignment to the underlying features were observed into parallel conduits and on parallel two-dimensional substrates, whereas the least extent of outgrowth was observed into perpendicular conduits and on perpendicular two-dimensional replica conditions. Additionally, neurites on perpendicular conditions turned to extend along the direction of underlying SC topography. Neurite outgrowth exceeded SC migration in the direction of the underlying anisotropic SC replica after two days in culture. This finding confirms the critical role that SC have in guiding neurite outgrowth and suggests that the mechanism of neurite alignment to SC replicas depends on direct contact with cellular topography. These results suggest that SC topographical replicas may be used to direct and optimize neurite alignment, and emphasize the importance of SC features in neurite guidance.

Micropatterns of positive guidance cues anchored to polypyrrole doped with polyglutamic acid: a new platform for characterizing neurite extension in complex environments.

This paper describes a method for preparing substrates with micropatterns of positive guidance cues for the purpose of stimulating the growth of neurons. This method uses an oxidizing potential, applied to a micropattern of indium tin oxide in the presence of pyrrole and polyglutamic acid, to electrodeposit a matrix consisting of polypyrrole doped with polyglutamic acid. The resulting matrix subsequently can be modified with positive guidance cues via standard amide coupling reactions. Cells adhered to the micropatterned substrates can be stimulated electrically by the underlying electrodeposited matrix while they are in contact with positive guidance cues. This method can be extended to include both positive and negative guidance cues in a variety of combinations. To demonstrate the suitability of this method in the context of nerve guidance, dorsal root ganglia were grown in the presence of a micropatterned substrate whose surface was modified with molecules such as polylysine, laminin, or both. Cell adhesion and neurite extension were found to occur almost exclusively in areas where positive guidance cues were attached. This method is easy to execute and is of general utility for fundamental studies on the behavior of neurons in the presence of complex combinations of guidance cues as well as advanced bioelectronic devices such as neuronal networks.

Reproducible echocardiography in juvenile sheep and its application in the evaluation of a pulmonary valve homograft implant.

Increased use of the ovine animal model in cardiovascular surgical research has created a salient need for standardized echocardiography techniques. To demonstrate a reproducible image in this species and confirm the validity of echocardiography as a diagnostic tool, we implanted 10 sheep with a pulmonary valve homograft and monitored them through weekly echocardiographic examinations until 20 weeks after implantation. We obtained good images from the left cranial and the left caudal transducer windows without needing to sedate the animals. Sedated sheep yielded adequate views from the right apical window. Echocardiographic data on the implanted homografts (including functional capacity, presence of calcification, and hemodynamic information and measurements), completely agreed with the results of the post-explantation examinations.

Patterns of chondroitin sulfate immunoreactivity in the developing tectum reflect regional differences in glycosaminoglycan biosynthesis.

The glycosaminoglycan chondroitin sulfate (CS) is expressed in many parts of the developing brain, both in regions where axons preferentially grow and in areas that axons distinctly avoid. Some in vitro studies suggest that CS and proteoglycans (PGs) that carry CS enhance axon growth, whereas others suggest that CS and CSPGs inhibit it. In the developing hamster, there is evidence that midbrain raphe cells act as a barrier to prevent growth of optic axons across the tectal midline. Here we show that in the newborn hamster, CS immunoreactivity is substantially higher in midline than in lateral tectum, raising the possibility that CSPGs play a role in the unilateral containment of optic axons. However, analysis of tectal PGs by anion exchange chromatography and denaturing gel electrophoresis failed to detect substantial differences between midline and lateral tectum in either the types or relative amounts of CSPG and heparan sulfate PG protein cores. In contrast, metabolic labeling of tectal slices in vitro documented that incorporation of 35S-sulfate into macromolecules is significantly increased at the tectal midline, in a pattern resembling chondroitin sulfate immunoreactivity. This difference was evident whether slices were labeled for 1 hr or overnight and was not paralleled by a difference in overall protein synthesis, suggesting that the rate of synthesis of sulfated macromolecules is specifically elevated in midline tectum. We propose that the concentration of CS at the midline of the developing tectum is a reflection of a higher rate of synthesis or sulfation of glycosaminoglycans by midline cells, rather than a higher level of production of any particular CSPG. These results suggest that the distribution of some axon guidance signals in development may be controlled by differential regulation of glycosaminoglycan biosynthetic enzymes.

Unilateral containment of retinal axons by tectal glia: a possible role for sulfated proteoglycans.

(1) A distinct group of radial glia resides along the roofplate of the mesencephalon. Results of experiments, in which the neonatal tectum is manipulated surgically, point to the involvement of these glia in compartmentalizing retinotectal axons to one side of the midbrain. (2) Immunohistochemical studies document that the GAGs CS and KS are expressed along these midline glia during development: their expression occurs after the intertectal axons grow across the midline, but is coincident with the time of ingrowth of retinotectal axons, which fail to cross the midline. Together with results of in vitro experiments from other laboratories, these observations suggest that CS and KS are involved in the barrier function of the midline cells. (3) Preliminary data on biochemical characterization of PGs in developing tectum indicate that similar PG core proteins are found in the midline region as well as in the lateral tectum, whereas metabolic labeling shows a significantly higher uptake of radioactive sulfates along the midline. Thus differential glycosylation of proteins along the midline is likely, along with the possibility that it is the sugar chains which contribute to the barrier function of the raphe glia. Taken in the context of what we currently know about the biochemical heterogeneity of PGs, their developmental expression, and their functions in relation to the growth of axons from a variety of different neuronal cell types, it is clear that the analyses of interactions between PGs and growing axons must occur at several different levels, not the least of which involves a detailed understanding of the milieu in vivo within which these interactions take place.