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On my research projects ...

Following an insult to the spinal cord, astrocytes at the site of the injury undergo a phenomenon known as astrogliosis, which creates a large mass known as the glial scar (Silver and Miller 2004). The glial scar is inhibitory to nerve regeneration, not simply acting as a physical barrier that neurons cannot cross, but by expressing extracellular matrix proteins that trigger an inhibitory growth response in neurons. One such extracellular matrix molecule family is the chondroitin sulfate proteoglycans (CSPGs), which are found in relatively high concentrations in the glial scar following injury.

Previous assays in vitro and in vivo have demon-strated the inhibitory potential of isolated and purified CSPGs (Asher, Morgenstern et al. 2001). However, in order to transfer this knowledge base to clinical applications, CSPGs as they are produced and released from astrocytes in vivo must be understood, i.e., we need to develop and analyze a physiologically-relevant model.

The hypothesis for the study is: CNS injury induces the upregulation of astrocyte produced CSPGs that subsequently inhibits neural regeneration. The goals of this study were to 1) develop a physiologically relevant in vitro model using adenovirus technology; 2) determine the precise outgrowth behaviors in vitro of sensory neurons contacting astrocytes that over-express inhibitory CSPGs, such as brevican, neurocan, NG2, and phosphocan; and 3) determine the factors that regulate astrocyte upregulation of CSPGs in vitro.

Using an adenovirus vector that encoded for the desired CSPG (i.e., brevican) astrocytes were trans-fected in alternating lanes of brevican + cells and brevican (-) cells. This novel model closely mimics previous stripe-assay paradigms, while creating a more physiologically relevant model in which to examine neurite behavior. Chick (E9-10) dorsal root ganglion (DRG) neurons were added to this cell paradigm and inhibition was quantified using a simple crossing vs. turning standard. A "crossing" neurite breached the surface of a brevican + cell and a "turning" neurite exhibited some type of avoidance behavior in the presence of a brevican + cell. There was a significant (p < 0.0001) increase in inhibition in the brevican + cells compared to controls.

This technique provides a useful and simple way to express a molecule of interest from a localized area

within a dish of confluent cells. Applications of these methods include the ability to create an astrocytic scar in which high levels of CSPGs are expressed, such as that expressed by glial scars in vivo. This model represents a useful tool for assaying neuronal behavior j in response to CSPGs in their innate cell surface con- i formation. Furthermore, localized expression of molecules of interest in culture is useful for axon guidance assays, but may also be extended to the study of synapses or any interaction between two cell types in which one is induced to express a modulatory factor. Presently, studies are being conducted to examine the morphological behavior of growth cones coming into contact with CSPG + astrocytes using microinjection/ micromanipulation techniques and time-lapse micros-copy. These studies should help elucidate some of the underlying behavioral mechanisms behind the inhibition that has been seen up until this point.

Subsequent studies will add more complex pieces to the story. In vivo, it is known that certain growth factors, present in the glial scar, can up-regulate a variety of molecules. We will use our model to characterize the up-regulation of astrocytic PCs by transforming growth factor (TGF-fil) and epidermal growth factor (EGF). Further, once we identify the mechanisms using our model in vitro, we will be able to test these mechanisms in vivo. Thus, a future series of experiments will determine if CSPG over-expressing astrocytes in vivo have the same inhibitory properties as they do in vitro and what factors, in vivo, regulate inhibition.

Ultimately, the long-term goal of this project is to better understand the physiological role that CSPGs play in response to injury. Such an understanding will allow researchers and clinicians to manipulate the molecules that regulate the expression of CSPGs (e.g., antagonist or antibodies), to restore connectivity following spinal cord injury or traumatic brain injury, to improve motor and sensory function.

References

Asher, R. A., D. A. Morgenstern, et al. (2001). "Chondroitin sulfate proteoglycans: inhibitory components of the glial scar." Prog Brain Res 132: 611-619.

Silver, J. and J. H. Miller (2004). "Regeneration Beyond the Glial Scar." Nature 5: 146-156.