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An LC-MS/MS APPROACH FOR GANGLIOSIDES PROFILING IN BRAIN AND RETINAL TISSUE OF MICE: APPLICATION TO GLAUCOMA MICE AGE STUDIES

Abstract Details

2017, Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, College of Sciences and Health Professions.
Gangliosides are membrane lipids with a complex sugar head containing sialic acid (polar) and a ceramide moiety made up of a sphingosine and a fatty acid (non-polar). Typically, gangliosides are part of the ganglion, a group of nerve cell bodies where they act as neuroprotective agents, support, and maintenance of the mature neuronal cells. Glaucoma is an age-related neurodegenerative disorder of the eye that leads to blindness. Retinal ganglion cells (RGCs) in retinal tissue of the eye, degeneration is seen followed by the optic nerve head damage. From immunohistochemistry studies with cholera toxin-B, gangliosides which are part of the RGCs were lost during the RGCs degeneration. Non-neuronal cells, astrocytes, which do not express gangliosides in normal retinal tissue surprisingly express/uptake gangliosides in correlation to the RGCs degeneration. Astrocyte expression/uptake of gangliosides can play protective, adverse or combined effects at different times. An identification and quantitation of individual gangliosides that are expressed/uptake provide an insight into understanding the pathophysiology of glaucoma. A specific, sensitive and quantitative liquid-chromatography-mass spectrometry (LC-MS/MS) analytical techniques were developed to identify heterogeneous molecular species of gangliosides. Of all of them, new LC-MS/MS method using a phenyl-hexyl reverse-phase chromatography was successful in resolving eight major gangliosides and ten minor gangliosides. An optimized ganglioside isolation technique was developed which is a combination of liquid-liquid extraction for lipid phase separation followed by a solid-phase extraction for desalting and removal of debris. This new LC-MS/MS method was validated for linearity, repeatability, matrix effect and recovery studies. Age studies (young vs old mice) were performed for the first time to correlate the changes of gangliosides in retinal tissue level in comparison to the superior colliculus of the mid-brain using the new LC-MS/MS method in normal control mice (C57BL/6J) versus glaucoma model mice (DBA/2J). LC-MS/MS results show an increase of major gangliosides in the glaucoma retinal tissue from old to young age mice groups whereas little change in the superior colliculus. Nevertheless, these gangliosides changes were statistically insignificant as p-value is > 0.05. The optic nerve studies using electron microscopy was undertaken to establish the glaucoma disease progression. Electron microscopy study results show no change in the optic nerve count which represents no glaucoma in old age animal samples. Thus, a non-correlation was seen with the ganglioside changes compared to optic nerve counts. A greater animal sample with much older age provides a better understanding of gangliosides role in glaucoma.
Anderson David (Advisor)
Long-Sun Xue (Committee Member)
Zhou Aimin (Committee Member)
Zhou Xiang (Committee Member)
Holland Nolan (Committee Member)
215 p.

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Citations

  • Gobburi, A. L. P. (2017). An LC-MS/MS APPROACH FOR GANGLIOSIDES PROFILING IN BRAIN AND RETINAL TISSUE OF MICE: APPLICATION TO GLAUCOMA MICE AGE STUDIES [Doctoral dissertation, Cleveland State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=csu1521174979758726

    APA Style (7th edition)

  • Gobburi, Ashta Lakshmi Prasad. An LC-MS/MS APPROACH FOR GANGLIOSIDES PROFILING IN BRAIN AND RETINAL TISSUE OF MICE: APPLICATION TO GLAUCOMA MICE AGE STUDIES. 2017. Cleveland State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=csu1521174979758726.

    MLA Style (8th edition)

  • Gobburi, Ashta Lakshmi Prasad. "An LC-MS/MS APPROACH FOR GANGLIOSIDES PROFILING IN BRAIN AND RETINAL TISSUE OF MICE: APPLICATION TO GLAUCOMA MICE AGE STUDIES." Doctoral dissertation, Cleveland State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=csu1521174979758726

    Chicago Manual of Style (17th edition)