[40] In vivo fluorescent imaging of NADH redox state in brain

2002 ◽  
pp. 482-494 ◽  
Author(s):  
Robert E. Anderson ◽  
Fredric B. Meyer
Keyword(s):  
Redox State ◽  
Fluorescent Imaging ◽  
Nadh Redox State

In vivo monitoring of cellular energy metabolism using SoNar, a highly responsive sensor for NAD+/NADH redox state

2016 ◽  
Vol 11 (8) ◽  
pp. 1345-1359 ◽  
Author(s):  
Yuzheng Zhao ◽  
Aoxue Wang ◽  
Yejun Zou ◽  
Ni Su ◽  
Joseph Loscalzo ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Redox State ◽  
Cellular Energy ◽  
In Vivo Monitoring ◽  
Cellular Energy Metabolism ◽  
Nadh Redox State

Multiparametric monitoring of the awake brain exposed to carbon monoxide

1995 ◽  
Vol 78 (3) ◽  
pp. 1188-1196 ◽  
Author(s):  
A. Mayevsky ◽  
S. Meilin ◽  
G. G. Rogatsky ◽  
N. Zarchin ◽  
S. R. Thom
Keyword(s):  
Carbon Monoxide ◽  
Blood Flow ◽  
Redox State ◽  
Ionic Homeostasis ◽  
Awake Rat ◽  
Brain Oxidative Stress ◽  
The Brain ◽  
Measurable Change ◽  
Nadh Redox State

We have applied in vivo real-time techniques to monitor the physiological changes associated with exposure to a pattern of carbon monoxide (CO) known to cause brain oxidative stress. Using a multiparametric monitoring device connected to the brain, we exposed unanesthetized rats to two levels of CO, 0.1 and 0.3% in air. Energy metabolism was evaluated by the optical monitoring of relative cerebral blood flow (CBF) and intramitochondrial redox state. Ionic homeostasis was assessed by measurements of K+,Ca2+, and H+ or Na+ levels in the extracellular space. The electrical parameters monitored were the electrocorticogram and direct current steady potential. Under 1,000 ppm of CO, the CBF was increased significantly without any measurable change in the NADH redox state, suggesting that the cause for the increased CBF was not hypoxia. Exposing the awake rat to 1,000 ppm of CO (40 min) followed by 3,000 ppm of CO (20 min) led to an increase in CBF followed by episodes of spontaneous brain depolarizations characterized by changes in ionic homeostasis and blood flow. These changes were similar to those recorded under cortical spreading depression. In most animals exposed to 3,000 ppm of CO, spontaneous oscillations in CBF and NADH redox state that were negatively correlated were recorded. The results indicate that an inspired CO level of 0.1% had effects largely restricted to blood flow, whereas at a higher CO level an additional impairment in energy supply resulted in a complex pattern of effects similar to that caused by brain ischemia.

scholarly journals Cytosolic Free Calcium, NAD/NADH Redox State and Hemodynamic Changes in the Cat Cortex during Severe Hypoglycemia

1989 ◽  
Vol 9 (2) ◽  
pp. 149-155 ◽  
Author(s):  
Daisuke Uematsu ◽  
Joel H. Greenberg ◽  
Martin Reivich ◽  
Andrea Karp
Keyword(s):  
Redox State ◽  
Mean Transit Time ◽  
Recovery Period ◽  
Control Level ◽  
Severe Hypoglycemia ◽  
Free Calcium ◽  
Cytosolic Free Calcium ◽  
High Level ◽  
Nadh Redox State

Summary Using indo-1, a fluorescent Ca2+ indicator, in vivo fluorometric measurements were made of changes in cytosolic free Ca2+, NAD/NADH redox state, and hemodynamics directly from the cat cortex during and after severe insulin-induced hypoglycemia. Cytosolic free Ca2+ started to increase when the EEG became isoelectric, remained at a significantly high level (p < 0.05) during the period of isoelectric EEG (IEEG), and recovered to the control level 6 min following an intravenous infusion of glucose. The NAD/NADH redox state oxidized significantly during IEEG and then recovered rapidly to the control level after the glucose infusion. Local cortical blood volume (LCBV) increased gradually during the progression of hypoglycemia, reaching the maximal level (146 ± 7%) at the end of IEEG, and then started to recover. The mean transit time (MTT) through the cortical microcirculation was shortened during the IEEG (control: 3.84 ± 0.41 s versus IEEG: 2.73 ± 0.17 s, p < 0.05), whereas it was prolonged during the 30-min recovery period (5.68 ± 0.58 s, p < 0.05). Local cortical blood flow calculated from the LCBV and MTT showed a twofold increase 5 min into IEEG (201 ± 27% of control, p < 0.05), recovered 15 min into the recovery period, and then decreased to 77% of control (p < 0.05) by 30 min. The data support the hypothesis that hypoglycemic brain damage might be mediated by an elevation of cytosolic free calcium.

scholarly journals In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences

2015 ◽  
Vol 112 (9) ◽  
pp. 2876-2881 ◽  
Author(s):  
Xiao-Hong Zhu ◽  
Ming Lu ◽  
Byeong-Yeul Lee ◽  
Kamil Ugurbil ◽  
Wei Chen
Keyword(s):  
Human Brain ◽  
Redox State ◽  
Ex Vivo ◽  
Healthy Human ◽  
Nad Metabolism ◽  
Mitochondrial Functions ◽  
Age Dependent ◽  
Nadh Redox State

NAD is an essential metabolite that exists in NAD+or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD+/NADH redox state and modulating cellular signaling processes through the activity of the NAD+-dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD+and NADH contents and the NAD+/NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD+, total NAD contents, and NAD+/NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ.

Cytosolic free calcium and NAD/NADH redox state in the cat cortex during in vivo activation of NMDA receptors

1989 ◽  
Vol 482 (1) ◽  
pp. 129-135 ◽  
Author(s):  
Daisuke Uematsu ◽  
Joel H. Greenberg ◽  
Martin Reivich ◽  
Andrea Karp
Keyword(s):  
Nmda Receptors ◽  
Redox State ◽  
Free Calcium ◽  
Cytosolic Free Calcium ◽  
Nadh Redox State

Nitric oxide synthase inhibition by L-NAME during repetitive focal cerebral ischemia in rabbits

1996 ◽  
Vol 271 (2) ◽  
pp. H588-H594 ◽  
Author(s):  
R. E. Anderson ◽  
F. B. Meyer
Keyword(s):  
Nitric Oxide ◽  
Cerebral Ischemia ◽  
Nitric Oxide Synthase ◽  
Redox State ◽  
Focal Cerebral Ischemia ◽  
Control Group ◽  
Reperfusion Period ◽  
Oxide Synthase ◽  
Nadh Redox State

The effects of nitric oxide synthase inhibition on brain acidosis, regional cortical blood flow (rCBF), and NADH redox state were examined using in vivo fluorescence imaging during four 15-min periods of moderate focal cerebral ischemia, each separated by three 5-min reperfusion periods followed by a final 3-h reperfusion period. Fasted rabbits under 1.5% halothane were divided into six groups of seven animals each: nonischemic controls, ischemic controls, and the following drug groups receiving NG-nitro-L-arginine methyl ester (L-NAME) intravenously 20 min before repetitive ischemia (as follows: 0.1 mg/kg, 1 mg/kg, 10 mg/kg, and 1 mg/kg + 5 mg/kg L-arginine). L-NAME at 0.1 and 1 mg/kg prevented the development of significant brain acidosis throughout the four ischemic insults. L-NAME at 10 mg/kg reduced preischemic rCBF by 21% (P < 0.05) and did not mitigate brain acidosis after the third and fourth ischemic insults. Brain intracellular pH returned toward baseline after the 3-h final reperfusion in all groups. NADH redox state was significantly (P < 0.05) elevated from baseline controls in all groups during the last three ischemic insults. During the final reperfusion period, NADH redox state returned toward baseline values only in the 0.1 mg/kg L-NAME and ischemic control group. In conclusion, low-dose L-NAME attenuated brain acidosis independent from rCBF changes during intermittent, moderate focal cerebral ischemia.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light &gt;600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

scholarly journals O23: CHARACTERISING PATIENT-DERIVED COLORECTAL CANCER TISSUE-ORIGINATED ORGANOIDAL SPHEROIDS FOR HIGH-THROUGHPUT MICROFLUIDIC APPLICATIONS

2021 ◽  
Vol 108 (Supplement_1) ◽  
Author(s):  
MI Khot ◽  
M Levenstein ◽  
R Coppo ◽  
J Kondo ◽  
M Inoue ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Fluid Flow ◽  
High Throughput ◽  
Microfluidic Devices ◽  
In Vitro Models ◽  
Fluorescent Imaging ◽  
Cancer Tissue ◽  
Cell Models

Abstract Introduction Three-dimensional (3D) cell models have gained reputation as better representations of in vivo cancers as compared to monolayered cultures. Recently, patient tumour tissue-derived organoids have advanced the scope of complex in vitro models, by allowing patient-specific tumour cultures to be generated for developing new medicines and patient-tailored treatments. Integrating 3D cell and organoid culturing into microfluidics, can streamline traditional protocols and allow complex and precise high-throughput experiments to be performed with ease. Method Patient-derived colorectal cancer tissue-originated organoidal spheroids (CTOS) cultures were acquired from Kyoto University, Japan. CTOS were cultured in Matrigel and stem-cell media. CTOS were treated with 5-fluorouracil and cytotoxicity evaluated via fluorescent imaging and ATP assay. CTOS were embedded, sectioned and subjected to H&E staining and immunofluorescence for ABCG2 and Ki67 proteins. HT29 colorectal cancer spheroids were produced on microfluidic devices using cell suspensions and subjected to 5-fluorouracil treatment via fluid flow. Cytotoxicity was evaluated through fluorescent imaging and LDH assay. Result 5-fluorouracil dose-dependent reduction in cell viability was observed in CTOS cultures (p&lt;0.01). Colorectal CTOS cultures retained the histology, tissue architecture and protein expression of the colonic epithelial structure. Uniform 3D HT29 spheroids were generated in the microfluidic devices. 5-fluorouracil treatment of spheroids and cytotoxic analysis was achieved conveniently through fluid flow. Conclusion Patient-derived CTOS are better complex models of in vivo cancers than 3D cell models and can improve the clinical translation of novel treatments. Microfluidics can streamline high-throughput screening and reduce the practical difficulties of conventional organoid and 3D cell culturing. Take-home message Organoids are the most advanced in vitro models of clinical cancers. Microfluidics can streamline and improve traditional laboratory experiments.

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