Date on Master's Thesis/Doctoral Dissertation

5-2020

Document Type

Master's Thesis

Degree Name

M. Eng.

Department

Bioengineering

Degree Program

JB Speed School of Engineering

Committee Chair

Magnuson, David

Committee Co-Chair (if applicable)

Giridharan, Guruprasad

Committee Member

Kopechek, Jonathan

Author's Keywords

matlab; tracing; rat; spinal cord; propriospinal neurons

Abstract

The objective of this research project is to evaluate the efficiency of traditional chemical tracing compared to dual-viral tracing for labeling long ascending propriospinal neurons (LAPNs) in the uninjured rat spinal cord, and to develop a MATLAB program which will accurately quantify this labeling.

To evaluate chemical tracing, Fluoro-Ruby (FR) was injected at the level of the axon terminals, and the number of ipsilateral labeled cell bodies at lumbar level was quantified. Similarly, two dual-viral systems were evaluated, by either injecting retro-AAV-Cre or HiRet-Lenti-Cre unilaterally in combination with a Cre-dependent adeno-associated virus. The HiRet-Lenti and FR groups labeled significantly greater numbers of LAPNs than the retro-AAV group, showing that dual-viral systems utilizing retro-AAVs do not provide robust labeling when tracing LAPNs. Additionally, dual-viral tracing utilizing the HiRet-lentivirus provides more specificity than traditional chemical tracing with FR. Thus, dual-viral labeling utilizing HiRet-lentiviruses is preferred due to greater specificity and more prominent labeling.

To automate the cell counting process, a MATLAB program was developed to accurately determine the number of ipsilateral cell bodies labeled by each of the tracing techniques. To validate the accuracy of the MATLAB program, the number of labeled cells counted for each tracing technique by manual counting was compared to the number generated by the MATLAB program. The number of LAPNs counted manually did not significantly differ from the number of LAPNs counted by the MATLAB application between labeling groups, and there was a highly significant correlation between the two methods.

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