MOLECULAR, GENETIC, AND IN VITRO TOOLS

Techniques have been developed that allow researchers to isolate and grow populations of neurons to investigate the effects of specific proteins and molecules on neuronal injury and repair. Neurons can be grown in isolation or with glial cells such as oligodendrocytes or Schwann cells to study the processes of axonal outgrowth and myelination. Investigators use molecular biology-based techniques, such as DNA or protein analysis, that can be used to easily visualize or analyze outcomes.

Demonstrating the power of a cell culture experiment, the simple growth-cone turning assay led to the discovery that altering various molecules inside the growing axon regulates protein and cyclic nucleotide activities, which, in turn, can convert an axon’s response to a growth-inhibiting molecule from one of repulsion to one of attraction (Song et al., 1998). When this application is applied to regenerating axons in the rat spinal cord, investigators showed that the regrowth of transected neurons has the potential to be enhanced considerably (Neumann et al., 2002; Qiu et al., 2002). Furthermore, the recent elucidation of the signaling pathways responsible for this switch in response may lead to the discovery of a strategy for enhancing axon regeneration (Wen et al., 2004).

Often, in vitro assays can be used in experiments with animal models, thus allowing researchers to verify and examine the effects detected in vitro to be evaluated in a more complex system. For example, chondroitin sulfate proteoglycans were found to inhibit neurite outgrowth in in vitro experiments (Snow et al., 1990). Analysis with animal models demonstrated that the levels of these proteoglycans are enhanced, or up-regulated, during central nervous system (CNS) injury (Snow et al., 1990) and led to the development of a strategy to break down these substances and promote the regrowth of axons in the intact rat spinal cord after an injury (Bradbury et al., 2002).

Animal Models for Molecular and Genetic Studies

Models consisting of multiple-transgenic animals have been developed to investigate molecular mechanisms and to identify the molecules critical for specific processes (Table 3-1). These models provide a better understanding of the genetic and molecular basis by which spinal cord circuits, specific neuronal subtypes, and synapses are formed (Shirasaki and Pfaff, 2002; Lanuza et al., 2004). For example, by studying the development of the nervous system of the fruit fly (Drosophila melanogaster), researchers have identified numerous molecules that can regulate the growth of the axon and the formation of neuronal connections (Vaessin et al., 1991; Kidd et al., 1998; Kraut et al., 2001; Jin, 2002). This information should provide



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