Thesis
Sensory Neurons Respond to Neurocan Knock-down in Reactive Astrocytes
(2015)
Abstract
Spinal Cord Injury (SCI), in many cases due to trauma related events, is a potentially incapacitating affliction that can lead to immense physical, psychosocial and financial burdens for both individuals and society. Understanding the cellular and molecular changes that occur following SCI may lead to therapies for improved treatment. At the cellular level, contusive SCI often results in the formation of a glial (astrocyte) scar that blocks successful axonal regeneration, partly due to the up-regulation of inhibitory extracellular matrix (ECM) molecules like chondroitin sulfate proteoglycans (CSPGs). The long-term goal is to encourage neuroregeneration in vivo by targeting specific inhibitory CSPGs thereby promoting neuronal reconnection and restoration of function. To accomplish this goal, we are investigating the mechanisms by which CSPGs inhibit axonal outgrowth. Since previous studies have shown that the most highly regulated CSPG following SCI is neurocan, the hypothesis for this study was: Effective silencing of the NCAN gene, which encodes the CSPG neurocan, will increase neurite outgrowth on injured (reactive) astrocytes. In this study, knock-down of neurocan was accomplished using shRNA technology. Postnatal rat primary astrocytes were transfected with neurocan shRNA, which reduces neurocan expression. Transforming Growth Factor Beta (TGF-β), a reagent known to mimic nervous system injury, was used to stimulate CSPG production. Embryonic day 8-12 chicken dorsal root ganglion (DRG) sensory neurons were then co-cultured with neurocan shRNA transfected (neurocan-deficient) or GFP control transfected (neurocan-expressing) astrocytes at 15-20% confluency on glass coverslips coated with a growth-permissive substrate, poly-L-ornithine (PLO). Using epifluorescence microscopy, initial qualitative assessments were completed to document the behavior of neuronal growth cones (leading edge of elongating axon) upon contact with neurocan shRNA transfected and GFP control transfected astrocytes. DRG neurons were identified using an antibody to a neurospecific isoform of tubulin (anti-beta-III-tubulin) followed by a rhodamine-conjugated secondary antibody. Astrocytes were identified using the GFP tag present on both the neurocan shRNA construct and the control construct. Hoechst staining was employed to identify the nuclei of each cell in the cultures. Quantitative assessment involved a rubric including criteria for measuring the number of transfected astrocytes with neurites growing through the ECM, the number of transfected astrocytes without neurites, the total number of neurites, and the ratio of neurites to transfected astrocytes. Preliminary data showed that DRG neurons attached well to PLO and elongated among both populations of astrocytes. Results from this study did not support the hypothesis. Differences between treatment and control groups were statistically insignificant when a two-tailed t test was computed with p-values all above 0.05. The cause of this phenomenon may be the compensatory mechanisms elicited by the up-regulation of other CSPGs in response to the neurocan knock-down. Future studies will investigate the effects of other CSPGs on neurite outgrowth post-injury. Collectively, these data and future experiments will reveal mechanisms of CSPG inhibition after SCI and may lead to the identification of specific CSPG targets to augment neuronal regeneration in the central nervous system after tissue damage.
Keywords
- spinal cord injury,
- astrocyte,
- neurocan,
- chondroitin sulfate proteoglycan
Disciplines
Publication Date
Spring March 27, 2015
Degree
Bachelor of Science
Field of study
Neuroscience
Department
Spinal Cord and Brain Injury Research Center. Anatomy & Neurobiology.
Comments
Recommended Citation
Khandpur, Umang. “Sensory Neurons Respond to Neurocan Knock-down in Reactive Astrocytes.” University of Kentucky, Berkeley Electronic Press, 27 Mar. 2015, works.bepress.com/UmangKhandpur/2/.
Citation Information
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Creative Commons license
This work is licensed under a Creative Commons CC_BY-NC-ND International License.