At the present, the two clinical biomarkers used to monitor diabetic progression are blood glucose and HbA1c. However, advanced glycation end products (AGEs) have been shown to contribute to diabetic pathogenesis, and there is interest in the use of AGEs in tissues as long-term glycemic markers. In this study, we investigate the in vitro rate of fluorescent AGEs (fAGEs) formation with multiphoton microscopy in different porcine tissues (aorta, cornea, kidney, dermis, and tendon) from glucose, galactose, and fructose, three primary monosaccharides found in human diets. These results may be of value in developing long-term glycemic biomarkers for diabetes.
Advanced glycation end products (AGEs) contribute to diabetic complications. Proteins undergo a series of complex and chain of biochemical actions in producing AGEs, some of which are fluorescent. In our study, we investigated the effects of glucose, fructose, and galactose in forming AGEs. Porcine tissues such as corneas, tendons, skin, aortas, and kidney were treated in the D-glucose, D-galactose, and D-fructose solutions, and multiphoton microscopy was performed on the glycated tissue specimens. We found that the rate of fAGEs formation from fructose was the highest.
Exposure of tissues to sugar lead to the formation of advanced glycation end products (AGEs), contributing to diabetic complications. In human physiology, the vasculature is in direct contact with blood, thus the effect of diabetes is expected to be most severe with the vasculature. In this study, we incubated excised porcine aorta in D-glucose, Dgalactose, and D-fructose solutions. Multiphoton microscopy shows that for Days 4 to 48 incubation, autofluorescence is constant along the aorta sections, suggesting that monosaccharide diffusion is rapid when compared to the rate of fluorescent AGE formation.
In this study, we explore the use of red blood cell and hemoglobin autofluorescence ad potential long-term biomarkers for diabetes. We found that under 370 nm excitation, both red blood cells (RBC) and Hb fluorescence in the 420-600 nm region. At early time points following diabetic induction in rats, autofluorescence increases in lysed Hb is more dramatic compared to that of RBC. Moreover, we found significance variance of Hb autofluorescence despite relatively constant HbA1c levels. The results of our study suggest that with additional development, RBC and hemoglobin autofluorescence from may be used as long-term glycemic markers for monitoring diabetic complications in the clinical setting.
Cardiovascular complication is a major health concern for diabetic patients. Elevated blood sugar levels lead to the formation of advanced glycation end products (AGEs), which are implicated in diabetic pathogenesis. In this study, we investigate the effect of prolonged exposure to elevated sugar level by studying the combined effect of diffusion and glycation rate in arteries from different commonly-consumed simple sugars. Since some AGEs are autofluorescent, we will perform multiphoton autofluorescence imaging to quantify the rates of fluorescent AGEs formation in elastic fibers and collagen fibers. Cross-section imaging of arteries and spatial and temporal profiles of autofluorescence of the blood vessel will be investigated.
The formation and accumulation of advanced glycation end products (AGEs) contribute to diabetic complications such as retinopathy, neuropathy, nephropathy, and cardiovascular diseases. It is clear that the development of effective technique in AGEs detection and the establishing the correlation of measured AGE parameters to diabetic pathogenesis are invaluable in the monitoring of disease progression and drug discovery of anti-AGE compounds. Since some AGE are fluorescent, we propose to investigate the degree of tissue glycation in forming fluorescent AGEs (fAGEs). In this preliminary study, we will investigate the effects of glucose, fructose, and galactose, three of the most abundant dietary simple sugars, in fAGEs production. Excised tissues will be treated in solutions containing the three sugar types; multiphoton autofluorescence imaging will then be performed on the treated tissues to determine their autofluroescence levels.
We used intravital multiphoton microscopy to study the recovery of hepatobiliary metabolism following carbon tetrachloride (CCl4) induced hepatotoxicity in mouse. Our images were processed by a first order kinetic model to generate rate constant resolved images of the mouse liver. At Day 14 following induction, a restoration of the mouse hepatobiliary function was found. Our approach allows the study of the response of hepatic functions to chemical agents in real time and is useful for studying pharmacokinetics of drug molecules through optical microscopic imaging.
Intravital multiphoton microscopy was used to study hepatobiliary metabolism in chronic pathologies of the liver. Through the use of the probe molecule 6-carboxyfluorescein diacetate (6-CFDA), the effects of liver fibrosis, fatty liver, and hepatocellular carcinoma on the metabolic capabilities of mouse liver were investigated. After the acquisition of time lapse images, a first order kinetic model was used to calculate rate constant resolved images of various pathologies. It was found the ability of the liver to metabolically process the probe molecules varies among different pathologies, with liver fibrosis and fatty liver disease negatively impacted the uptake, processing, and excretion of molecules.
We used intravital multiphoton microscopy to study the recovery of hepatobiliary metabolism following carbon tetrachloride (CCl4) induced hepatotoxicity in mice. The acquired images were processed by a first order kinetic model to generate rate constant resolved images of the mouse liver. We found that with progression of hepatotoxicity, the spatial gradient of hepatic function disappeared. A CCl4-induced damage mechanism involves the compromise of membrane functions, resulting in accumulation of processed 6-carboxyfluorescein molecules. At day 14 following induction, a restoration of the mouse hepatobiliary function was found. Our approach allows the study of the response of hepatic functions to chemical agents in real time and is useful for studying pharmacokinetics of drug molecules through optical microscopic imaging.
Liver performs xenobiotic excretion out of hepatocytes with metabolic function. However, hepatocellular metabolism was non-uniform in hepatocyte. Hepatocellular metabolism could be different in nucleus and cytoplasm. In this study, we use the molecular probe 6-carboxyfluorescein diacetate (6-CFDA) to simulate xenobiotic metabolism in hepatocytes with multi-photon fluorescence microscopy in vivo. 6-CFDA was processed by intracellular esterase to 6- carboxyfluorescein (6-CF) with green fluorescence. And this probe was used to study differences in cytoplasmic and nuclear metabolism of hepatocytes.
Hepatycotes in the liver may appear similar in morphology, however, heterogeneities may exist in cellular metabolism.
In this study, in vivo imaging of 6-carboxfluorescein diacetate (6-CFDA) metabolism in the liver was studied. We used
two-photon fluorescence microscopy and hepatic window to provide quantification in studying hepatocellular
metabolism. This model not only provides a potential platform for future study in hepatic responses and regulations, but
also contributes to the fine-tuning of organ-specific functions so as to open up a new era of exciting discoveries.
The liver is a major organ responsible for performing xenobiotic metabolism. In this process, xenobiotic is uptaken and processed in hepatocytes and subsequently excreted into the bile canaliculi. However, the intracellular heterogeneity in such metabolic processes is not known. We use the molecular probe 6-carboxyfluorescein diacetate (6-CFDA) to investigate xenobiotic metabolism in hepatocytes with intravital multiphoton fluorescence microscopy. 6-CFDA is processed by intracellular esterase to fluorescent 6-CF, which can be imaged and quantified. We found that compared to the nucleus, cytoplasmic 6-CF fluorescence intensity reached a maximum earlier (cytoplasm: 11.3±4.4 min; nucleus: 14.7±4.9 min) following 6-CFDA injection. We also found a slight difference in the rate of 6-CFDA metabolism as the rates of 6-CF decay at rates of 1.43±0.75 and 1.27±0.72 photons/min for the cytoplasm and nucleus, respectively. These results indicate that molecular transport to the nucleus is additionally hindered and can affect drug transport there.
Acetaminophen (APAP) is the famous drug in global, and taking overdose
Acetaminophen will intake hepatic cell injure. Desptie substantial progress in our
understanding of the mechanism of hepatocellular injury during the last 40 years,
many aspects of the pathophysiology are still unknown or controversial.1 In this study,
mice are injected APAP overdose to damage hepatocyte. APAP deplete glutathione
and ATP of cell, N-Acetyl Cysteine (NAC) plays an important role to protect
hepatocytes be injury. N-Acetyl Cysteine provides mitochondrial to produce
glutathione to release drug effect hepatocyte. By 6-carboxyfluorescein diacetate (6-CFDA) metabolism in vivo, glutathione keep depleting to observe the hepatocyte
morphology in time. Without NAC, cell necrosis increase to plasma membrane
damage to release 6-CFDA, that's rupture. After 6-CFDA injection, fluorescence will
be retained in hepatocyte. For cell retain with NAC and without NAC are almost the
same. With NAC, the number of cell rupture decreases about 75%.
Conventionally, the diagnosis of hepatocellular carcinoma (HCC) is performed by qualitative
examination of histopathological specimens, which takes times for sample preparation in fixation,
section and stain. Our objective is to demonstrate an effective and efficient approach to apply
multiphoton microscopy imaging the HCC specimens, with the advantages of being optical section,
label-free, subcellular resolution, minimal invasiveness, and the acquisition of quantitative information
at the same time. The imaging modality of multiphoton autofluorescence (MAF) was used for the
qualitative imaging and quantitative analysis of HCC of different grades under ex-vivo, label-free
conditions. We found that while MAF is effective in identifying cellular architecture in the liver
specimens, and obtained quantitative parameters in characterizing the disease. Our results demonstrates
the capability of using tissue quantitative parameters of multiphoton autofluorescence (MAF), the
nuclear number density (NND), and nuclear-cytoplasmic ratio (NCR) for tumor discrimination and that
this technology has the potential in clinical diagnosis of HCC and the in-vivo investigation of liver tumor development in animal models.
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