Kim, Sung-Min
(2018)
Zebrafish Extracellular Matrix as a Therapeutic Agent for Adult Mammalian Central Nervous System Regeneration.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Mammalian central nervous system (CNS) has limited capacity for regeneration. Despite numerous efforts in the last few decades, CNS-related injuries remain as detrimental as they were 50 years ago. While some functional recovery can occur, most regenerations are limited by an extracellular matrix (ECM) that actively inhibits axonal repair and promotes glial scarring. In most tissues, the ECM is an architectural foundation that plays an active role in supporting cellular development and regenerative response after injury. In mammalian CNS, however, this is not the case - its composition is not conducive for regeneration, with various molecules restricting plasticity and neuronal growth. In fact, the CNS-ECM alters its composition dramatically following an injury to restrict regeneration and to prioritize containment of injury as well as preservation of intact neural circuitry. Therefore, an ideal solution to limited CNS regeneration would be to modify and supplement the inhibitory extracellular environment so that it becomes more regeneration-permissive. Mammalian nervous tissue cannot provide such ECM, and synthesizing it in a laboratory is beyond current technology. Remarkably however, evolutionarily primitive species possess robust regenerative neural tissue. For example, small tropical freshwater dwelling zebrafish (Danio rerio) can completely regenerate severed spinal cord, re-gaining full motor function in a week. We believe their ECM contributes to its regenerative capability and that it can be harnessed to induce regeneration even in mammalian CNS. The objective of this dissertation was to evaluate the tissue-specific properties of zebrafish CNS-ECM for CNS injuries in terms of (1) influence on neuron viability and network formation, (2) potential for evoking regenerative CNS traits, and (3) capacity to restore axon connections that translate to meaningful behavioral recovery.
Scaffolds enriched with ECM derived from zebrafish brains (zf-brECM) were used to grow a population of primary cortical neurons. The scaffolds were compared to others that were enriched with ECM derived from mammalian tissue, such as pig brain (p-brECM), pig urinary bladder (p-UBM), and rat brain (r-brECM). The scaffolds themselves were designed to help control the distribution of neuronal bodies and axonal sprouting. Ultimately, zf-brECM promoted significantly more neuron survival and growth than mammalian ECMs. Additionally, zf-brECM significantly increased the overall formation of new axon networks. More importantly, axon networks formed in the presence of zf-brECM resulted in functional propagation of action potential signals. Building upon this, therapeutic efficacy of zf-brECM was explored in vivo using a rodent model of optic nerve crush (ONC). ONC was chosen as a representative model of CNS injury model for two reasons: 1) ONC surgery is reliably replicable, lending itself to minimal surgical variability and intra-animal data noise; 2) optic nerves are small and their neural circuitries are simple, making the resident retinal ganglion cells (RGCs) and their axons easy to analyze. We compared zf-brECM against commercially available mammalian ECMs by looking at the biological response of the optic nerves and observing traits that are considered hallmarks of CNS regeneration: glial scar deposition and expression level of axon inhibitors, namely chondroitin sulfate proteoglycans (CSPG). While ECM technology for CNS injuries has been limited and virtually unstudied in the visual system, it is known that mammalian optic nerves cannot regenerate under normal physiological circumstances. Therefore, we also observed long-distance growth of repairing axons traversing the lesion sites and compared the resulting behavioral recovery. The final behavioral assays revealed that only zf-brECM was able to restore pupil response as well as depth perception to damaged rodent eyes.
This body of work demonstrates the regenerative potential of zf-brECM, combined with its affordability, easy handling, and fast reproduction, positions zebrafish as an excellent candidate for a novel ECM source in the future. Specifically, it shows that zebrafish ECM holds promising regenerative potential for application in adult mammalian CNS injuries. Future research is necessary to determine the specific factors in zebrafish CNS-ECM responsible for the regenerative events.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
20 June 2018 |
| Date Type: |
Publication |
| Defense Date: |
24 January 2018 |
| Approval Date: |
20 June 2018 |
| Submission Date: |
9 April 2018 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
135 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Swanson School of Engineering > Bioengineering |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Zebrafish, CNS regeneration, Optic nerve, ECM, Biomaterials, Axon repair |
| Date Deposited: |
20 Jun 2018 16:08 |
| Last Modified: |
20 Jun 2018 16:08 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/34194 |
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