Determining the Presence of Laminin as a Growth Promoting Molecule in Goldfish Spinal Cord Regeneration

In mammalian systems, a spinal cord injury results in loss of function to the central nervous system (CNS) (Coumons et al., 2001). Goldfish, on the other hand, exhibit a remarkable capacity to regenerate neurons and the CNS environment after spinal cord injury in vivo. This could be due to the presence of the extracellular matrix glycoprotein laminin, as it has been shown that laminin is associated with the regeneration of the optic nerve (McLoon et al., 1988; Liesi, 1985). Though laminin has been demonstrated in the goldfish optic nerve, it has not yet been studied in the goldfish spinal cord. Immunocytochemical analysis revealed the distribution and intensity of laminin-positive processes expressed after spinal cord crush, which can be correlated to the amount of regeneration observed indirectly through behavioral observations. Similarly, immunocytochemical analysis with glial fibrillary acidic protein (GFAP) immunoreactivity shows the reactivity of astrocytes, the neuron support cells responsible for producing laminin, after a spinal cord crush (McLoon et al., 1988). In the following experiment, 9 goldfish were separated into three experimental groups, 5-, 15- or 30-days post-crush, and their spinal cords crushed with a forceps; 3 goldfish were placed in a control group that received surgery but no spinal cord crush. Each fish brainstem and spinal cord was sectioned, processed immunocytochemically, and visualized with a confocal microscope. A visual assessment of representative sections of the three experimental groups revealed that the processes appeared most intense at 5- and 30-days post crush, though spread out and shorter at 5-days and concentrated more in the medial brainstem and longer at 30-days. GFAP longitudinal and radial processes appeared similar in intensity through each experimental group, with varying results for the central canal due to apparent differences in sectioning or regrowth. ImageJ analysis provided numerical results for intensity, demonstrating a shift in the peak, combined laminin and GFAP intensity from lateral to medial in the spinal cord and the opposite trend in the brainstem; this discrepancy between ImageJ and visual analysis may be due to the way in which the ImageJ results were collected or differences between sections themselves (some of the sections analyzed may be more ventral than others). The laminin expression might become more localized as regeneration progresses; however, the axons could not be visualized due to time constraints, so this could not be confirmed. It is also possible that the fish required more time to recover than was given, which would change the laminin reactivity. Additional fish in each experimental group would be necessary to confirm these results. Future experiments could include larger sample sizes, neuronal antibody markers rather than behavioral observations to observe regeneration, and an investigation of other common central nervous system proteins