RUSHian Roulette: The Impact of Alkyl Nitrites on the Blood-Brain Barrier Restricted; Files Only

Abstract

Human immunodeficiency virus (HIV) is a sexually transmitted infection that weakens the body’s immune system, eventually progressing to the life-threatening acquired immunodeficiency syndrome (AIDS). Antiretroviral therapy (ART) is a groundbreaking therapeutic that greatly improves morbidity and mortality by reducing plasma viral loads to undetectable levels. However, dissemination of these compounds to the brain, a major viral reservoir, is poor due to the blood-brain barrier (BBB), a specialized semi-permeable membrane that serves as a unique point of regulation for ions, molecules, and possible pathogens within the body. Alkyl nitrites, colloquially known as “poppers” or “rush,” are liquid drugs utilized recreationally as inhalants, predominantly by men who have sex with men due to their ability to relax smooth muscle tissue throughout the body. Such vasodilative properties spark great interest, and whether they could modulate BBB integrity or permeability to affect distribution of therapeutic compounds, like ART, to the brain has yet to be determined. Thus, we assessed the impact of tert-butyl, isopentyl, and isobutyl nitrites on BBB integrity and permeability. First, identity and purity of each nitrite were confirmed by infrared spectroscopy, and testing ionization and solubility determined methanol as the optimal solvent for cell culture experiments. Second, human brain microvascular endothelial cells (BMVECs), a key component of the BBB, were treated with tert-butyl or isopentyl nitrite and probed by western blot for claudin-5 (CLDN5) or occludin (OCLN), tight junction (TJ) proteins crucial for maintenance of BBB integrity. tert-Butyl nitrite significantly downregulated expression of CLDN5 at the lowest (p=0.0249) and highest (p=0.0016) doses, and there were no significant impacts on TJ protein expression for any additional treatments. Lastly, a tricellular BBB model using transwell culture of BMVECs, astrocytes, and pericytes to integrate multiple key cellular components of the BBB was treated with tert-butyl, isopentyl, or isobutyl nitrite and assessed for changes in permeability. tert-Butyl nitrite significantly decreased permeability at the lowest dose (p=0.0001), whereas permeability remained unchanged with isopentyl or isobutyl nitrite treatment. Collectively, these data elucidate the impact of alkyl nitrites on BBB integrity and permeability, and informs their influence on access of ART and other therapeutic compounds to the brain. 

Table of Contents

Introduction – 1

Methods – 6

Results – 17

Discussion – 31

Limitations – 36

Future Directions – 38

Conflicts of Interest Statement – 40

References – 41

 

Figures:

-      Figure 1. Chemical structures of tert-butyl nitrite, isopentyl nitrite, and isobutyl nitrite – 3

-      Figure 2. Proposed metabolic pathways of isobutyl (alkyl) nitrite to produce free radical nitric oxide – 4

-      Figure 3. Chemical properties of Bradford assay to determine total protein concentration – 11

-      Figure 4. BMVEC confluency and morphology remains unchanged after treatment with tert-butyl or isopentyl nitrite – 21 

-      Figure 5. Astrocyte confluency and morphology remains unchanged after treatment with tert-butyl, isopentyl, and isobutyl nitrite – 22

-      Figure 6. Pericyte confluency and morphology remains unchanged after treatment with tert-butyl, isopentyl, and isobutyl nitrite – 23

-      Figure 7. Expression of CLDN5 in BMVECs 24 hours post-treatment with tert-butyl nitrite – 25 

-      Figure 8. Expression of CLDN5 in BMVECs 24 hours post-treatment with isopentyl nitrite – 26

-      Figure 9. Expression of OCLN in BMVECs 24 hours post-treatment with tert-butyl nitrite – 27

-      Figure 10. Expression of OCLN in BMVECs 24 hours post-treatment with isopentyl nitrite – 28

-      Figure 11. in vitro model of the blood-brain barrier – 29 

-      Figure 12. Permeability of tricellular blood-brain barrier transwell model – 30

 

Tables:

-      Table 1. Confirmation of tert-butyl, isopentyl, and isobutyl nitrite identity and purity by infrared spectroscopy – 18 

-      Table 2. Methanol is the optimal solvent for alkyl nitrite treatments – 19

-      Table 3. Biological concentrations of tert-butyl, isopentyl, and isobutyl nitrite for BMVEC treatments – 20

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry Biology, Neuroscience
Keyword	BMVECs Astrocytes Pericytes MSM Poppers Alkyl Nitrites Permeability Blood-Brain Barrier ART Rush HIV Men who have sex with men Integrity
Committee Chair / Thesis Advisor	Dr. Dionna Williams, Emory University
Committee Members	Dr. Matthew Weinschenk, Emory University Dr. Richard Himes, Emory University

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