archives@tulane.edu / Experimental stroke in endothelial nitric oxide synthase (eNOS) and neuronal
nitric oxide synthase (nNOS) knockout mice showed diverse effects on brain
injury. nNOS and eNOS have been shown to uncouple in pathological conditions
to produce superoxide. Oxidative stress is believed to be the underlying cause of
several cardiovascular diseases including ischemic stroke. However, the role of
eNOS and nNOS uncoupling in ischemic stroke is not well studied. Our objective
of the study was to determine the effect of eNOS and nNOS inhibition on reactive
oxygen species (ROS), NO, viability and mitochondrial bioenergetics in rat brain
microvascular endothelial cells (BMECs) and rat cortical neurons following
oxygen-glucose deprivation-reoxygenation (OGD/R). We found that non-specific
inhibition of NOS in endothelial cells reduced ROS levels in BMECs but
increased ROS levels in neurons under normoxia. This suggests that a pool of
uncoupled NOS exists in the BMECs whereas the dominant functional NOS in
neurons produces NO. We observed increased levels of ROS following OGD/R
that is sensitive to NOS inhibition in both BMECs and neurons indicating eNOS
and nNOS uncoupling during OGD/R. Furthermore, NOS inhibition reduced
mitochondrial respiration while it improved cell survival rate in both BMECs and
neurons following OGD/R. Thus, it is possible that decreased mitochondrial
respiration in the immediate aftermath (4 hours) of OGD/R could be protective
against reoxygenation injury.
Moreover, we identified the expression of nNOS in BMECs from rat, human, and
mouse. We observed that the nNOS in the BMECs constitutively produces
superoxide under physiological conditions instead of NO. In contrast, nNOS in
the neurons produces NO and doesn’t contribute to ROS. We also confirmed the
nNOS expression and its function in freshly isolated rat brain microvessels. In
addition, we developed a novel method to measure mitochondrial respiration in
freshly isolated mouse brain microvessels using Seahorse XFe24 Analyzer. We
validated the method by demonstrating impaired mitochondrial respiration in
cerebral microvessels isolated from old mice compared to young mice. In
summary, the present doctoral research investigated the distinct role of NOS
isoforms in BMECs and Neurons leading to the identification of novel functional
variant of nNOS in BMECs and brain microvessels. / 1 / RAMARAO SVNL
| Identifer | oai:union.ndltd.org:TULANE/oai:http://digitallibrary.tulane.edu/:tulane_88064 |
| Date | January 2018 |
| Contributors | Sure, Venkata Naga Lakshmi Ramarao (author), (author), (author), Katakam, Prasad VG (Thesis advisor), (Thesis advisor), School of Medicine Biomedical Sciences Graduate Program (Degree granting institution), NULL (Degree granting institution) |
| Publisher | Tulane University |
| Source Sets | Tulane University |
| Language | English |
| Detected Language | English |
| Type | Text |
| Format | electronic, pages: 179 |
| Rights | No embargo, Copyright is in accordance with U.S. Copyright law. |
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