Chih-Ju Lin

Visualizing and quantifying difference in cytoplasmic and nuclear metabolism in the hepatobiliary system in vivo

Abstract. 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.

Direct visualization of functional heterogeneity in hepatobiliary metabolism using 6-CFDA as model compound

Hepatobiliary metabolism is one of the major functions of the liver. However, little is known of the relationship between the physiological location of the hepatocytes and their metabolic potential. By the combination of time-lapse multiphoton microscopy and first order kinetic constant image analysis, the hepatocellular metabolic rate of the model compound 6-carboxyfluorescein diacetate (6-CFDA) is quantified at the single cell level. We found that the mouse liver can be divided into three zones, each with distinct metabolic rate constants. The sinusoidal uptake coefficients k1 of Zones 1, 2, and 3 are respectively 0.239 ± 0.077, 0.295 ± 0.087, and 0.338 ± 0.133 min-1, the apical excreting coefficients k2 of Zones 1, 2, and 3 are 0.0117 ± 0.0052, 0.0175 ± 0.0052, and 0.0332 ± 0.0195 min-1, respectively. Our results show not only the existence of heterogeneities in hepatobiliary metabolism, but they also show that Zone 3 is the main area of metabolism.

Human Hepatocellular Carcinoma Diagnosis by Multiphoton Autofluorescence Microscopy

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.

Multiphoton dynamic imaging of the effect of chronic hepatic diseases on hepatobiliary metabolism in vivo

In this study, intravital multiphoton microscopy was used to quantitatively investigate hepatobiliary metabolism in chronic pathologies of the liver. Specifically, through the use of the probe molecule 6-carboxyfluorescein diacetate, 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 that 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. The approach demonstrated in this work allows the study of the response of hepatic functions to different pathologies in real time and is useful for studying processes such as pharmacokinetics through direct optical imaging.

Dynamic visualization of the recovery of mouse hepatobiliary metabolism to acetaminophen-overdose damage

Acetaminophen (APAP) overdose is one of the worlds leading causes of drug-induced hepatotoxicity. Although traditional methods such as histological imaging and biochemical assays have been successfully applied to evaluate the extent of APAP-induced liver damage, detailed effect of how APAP overdose affect the recovery of hepatobiliary metabolism and is not completely understood. In this work, we used intravital multiphoton microscopy to image and quantify hepatobiliary metabolism of the probe 6-carboxyfluorescein diacetate in APAP-overdose mice. We analyzed hepatobiliary metabolism for up to 7 days following the overdose and found that the excretion of the probe molecule was the most rapid on Day 1 following APAP overdose and slowed down on Days 2 and 3. On Day 7, probe excretion capability has exceeded that of the normal mice, suggesting that newly regenerated hepatocytes have higher metabolic capabilities. Our approach may be further developed applied to studying drug-induced hepatotoxicity in vivo.

In vivo multiphoton imaging and quantification of cytoplasmic and nuclear metabolism in the hepatobiliary system

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.

Direct visualization of functional heterogeneity in hepatobiliary metabolism

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.

In vivo N-acetyl cysteine reduce hepatocyte death by induced acetaminophen

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, thats 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%.