Measurement of Water Transport During Freezing in Mammalian Liver Tissue: Part II—The Use of Differential Scanning Calorimetry

1998 ◽  
Vol 120 (5) ◽  
pp. 559-569 ◽  
Author(s):  
R. V. Devireddy ◽  
J. C. Bischof
Keyword(s):  
Water Transport ◽  
Rat Liver ◽  
Liver Tissue ◽  
Freeze Substitution ◽  
Sprague Dawley ◽  
Transport Parameters ◽  
Biophysical Parameters ◽  
Scanning Calorimetry ◽  
Tissue Slices ◽  
Low Temperature Microscopy

There is currently a need for experimental techniques to assay the biophysical response (water transport or intracellular ice formation, IIF) during freezing in the cells of whole tissue slices. These data are important in understanding and optimizing biomedical applications of freezing, particularly in cryosurgery. This study presents a new technique using a Differential Scanning Calorimeter (DSC) to obtain dynamic and quantitative water transport data in whole tissue slices during freezing. Sprague-Dawley rat liver tissue was chosen as our model system. The DSC was used to monitor quantitatively the heat released by water transported from the unfrozen cell cytoplasm to the partially frozen vascular/extracellular space at 5°C/min. This technique was previously described for use in a single cell suspension system (Devireddy, et al. 1998). A model of water transport was fit to the DSC data using a nonlinear regression curve-fitting technique, which assumes that the rat liver tissue behaves as a two-compartment Krogh cylinder model. The biophysical parameters of water transport for rat liver tissue at 5°C/min were obtained as Lpg = 3.16 x 10−13 m3/Ns (1.9 μm/min-atm), ELp = 265 kJ/mole (63.4 kcal/mole), respectively. These results compare favorably to water transport parameters in whole liver tissue reported in the first part of this study obtained using a freeze substitution (FS) microscopy technique (Pazhayannur and Bischof, 1997). The DSC technique is shown to be a fast, quantitative, and reproducible technique to measure dynamic water transport in tissue systems. However, there are several limitations to the DSC technique: (a) a priori knowledge that the biophysical response is in fact water transport, (b) the technique cannot be used due to machine limitations at cooling rates greater than 40°C/min, and (c) the tissue geometric dimensions (the Krogh model dimensions) and the osmotically inactive cell volumes Vb, must be determined by low-temperature microscopy techniques.

Measurement and Simulation of Water Transport During Freezing in Mammalian Liver Tissue

1997 ◽  
Vol 119 (3) ◽  
pp. 269-277 ◽  
Author(s):  
P. V. Pazhayannur ◽  
J. C. Bischof
Keyword(s):  
Water Transport ◽  
Rat Liver ◽  
Liver Tissue ◽  
Subzero Temperature ◽  
Biophysical Parameters ◽  
Cylinder Model ◽  
Chi Squared ◽  
Controlled Freezing ◽  
Cellular Water ◽  
Krogh Cylinder

Optimization of cryosurgical procedures on deep tissues such as liver requires an increased understanding of the fundamental mechanisms of ice formation and water transport in tissues during freezing. In order to further investigate and quantify the amount of water transport that occurs during freezing in tissue, this study reports quantitative and dynamic experimental data and theoretical modeling of rat liver freezing under controlled conditions. The rat liver was frozen by one of four methods of cooling: Method 1—ultrarapid “slam cooling” (≥ 1000° C/min) for control samples; Method 2—equilibrium freezing achieved by equilibrating tissue at different subzero temperatures (−4, −6, −8, −10°C); Method 3°-two-step freezing, which involves cooling at 5°C/min. to −4, −6, −8, −10 or −20°C followed immediately by slam cooling; or Method 4—constant and controlled freezing at rates from 5–400°C/min. on a directional cooling stage. After freezing, the tissue was freeze substituted, embedded in resin, sectioned, stained, and imaged under a light microscope fitted with a digitizing system. Image analysis techniques were then used to determine the relative cellular to extracellular volumes of the tissue. The osmotically inactive cell volume was determined to be 0.35 by constructing a Boyle van’t Hoff plot using cellular volumes from Method 2. The dynamic volume of the rat liver cells during cooling was obtained using cellular volumes from Method 3 (two-step freezing at 5°C/min). A nonlinear regression fit of a Krogh cylinder model to the volumetric shrinkage data in Method 3 yielded the biophysical parameters of water transport in rat liver tissue of: Lpg = 3.1 X 10−13 m3/Ns (1.86 μ/min-atm) and ELP = 290 kJ/mole (69.3 kcal/mole), with chi-squared variance of 0.00124. These parameters were then incorporated into the Krogh cylinder model and used to simulate water transport in rat liver tissue during constant cooling at rates between 5–100°C/min. Reasonable agreement between these simulations and the constant cooling rate freezing experiments in Method 4 were obtained. The model predicts that the water transport ceases at a relatively high subzero temperature (−10°C), such that the amount of intracellular ice forming in the tissue cells rises from almost none (=extensive dehydration and vascular expansion) at ≤5°C/min to over 88 percent of the original cellular water at ≥50°C/min. The theoretical simulations based on these experimental methods may be of use in visualizing and predicting freezing response, and thus can assist in the planning and implementing of cryosurgical protocols.

A new microscopy method to assess mass transfer during freezing in rat liver tissue slices

1995 ◽  
Vol 53 ◽  
pp. 1064-1065
Author(s):  
D. Raha ◽  
P. Pazhayannur ◽  
J. C. Bischof
Keyword(s):  
Water Transport ◽  
Liver Tissue ◽  
Ice Crystals ◽  
Tissue Slices ◽  
Free Zone ◽  
Vascular Space ◽  
Cellular Dehydration ◽  
Intracellular Ice ◽  
Quantitative Understanding ◽  
Microscopy Method

In cryofixation of biological samples it is typically desirable to minimize or avoid ice crystals in the sample by one of several rapid cooling techniques. Using these techniques freezing rates of 1,000 -10,000 °C/s are commonly achieved at a cooling boundary. At these rates, cellular dehydration is typically absent, and intracellular ice crystals are so small as to be considered negligible within an “ice-free” zone typically ranging 5-25 μm into the sample from the cooling boundary. However, during medical applications of freezing including cryopreservation as well as cryosurgery, the rate of freezing is orders of magnitude less than the idealized freezing procedures used in cryofixation. During slower freezing at rates between 1 - 100 °C/min, significant water transport between the cells and the extracellular/vascular space of a tissue can occur. This dehydration affects how and where the ice will form in the tissue which in turn can influence the post-freeze viability and therefore the overall success of a medical freezing treatment. The purpose of this work is to obtain a more quantitative understanding and prediction of the biophysics of water transport in liver tissue during freezing.

On the Possible Application of a Microscale Thermocouple to Measure Intercellular Ice Formation in Cells Embedded in an Extracellular Matrix

2004 ◽  
Author(s):  
Deepak Kandra ◽  
Ram V. Devireddy
Keyword(s):  
Extracellular Matrix ◽  
Water Transport ◽  
Ice Formation ◽  
Scanning Calorimetry ◽  
Microscopy Technique ◽  
Intracellular Ice ◽  
Tissue Systems ◽  
Low Temperature Microscopy ◽  
Microfabrication Techniques ◽  
In Cells

To optimize a freezing protocol for tissue systems, knowledge of intercellular ice formation and water transport is essential. Water transport during freezing can be measured using low temperature microscopy technique [1] and/or by differential scanning calorimetry method [2]. To study the formation of intracellular ice in cells embedded in an extracellular matrix we propose to design and develop an array of microscale thermocouples using microfabrication techniques [3]. The microfabricated thermocouples will be required to accurately measure the small temperature fluctuations in an embedded cell due to the formation of intracellular ice.

Acute alcohol produces hypoxia directly in rat liver tissue in vivo: role of Kupffer cells

1996 ◽  
Vol 271 (3) ◽  
pp. G494-G500 ◽  
Author(s):  
G. E. Arteel ◽  
J. A. Raleigh ◽  
B. U. Bradford ◽  
R. G. Thurman
Keyword(s):  
Rat Liver ◽  
Kupffer Cells ◽  
Large Dose ◽  
Liver Tissue ◽  
Ethanol Metabolism ◽  
Isolated Perfused Rat Liver ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Hypoxia Marker

Previous studies using liver slices and isolated perfused rat liver have suggested that ethanol causes hypoxia by increasing oxygen consumption. However, ethanol also increases blood flow to the liver, a phenomenon that may counteract the effects of hypermetabolism by increasing oxygen delivery. Thus whether ethanol causes hypoxia in vivo remains unclear. To clarify this important point, female Sprague-Dawley rats (100-125 g) simultaneously received pimonidazole (120 mg/kg ip), a 2-nitroimidazole hypoxia marker, and one large dose of ethanol (5 g/kg ig), which increase hepatic oxygen uptake dramatically and elevate ethanol metabolism (swift increase in alcohol metabolism) in 2-3 h. After 2 h, ethanol significantly increased the accumulation of bound pimonidazole in pericentral regions of the liver lobule. Treatment of animals with the Kupffer cell-specific toxicant, GdCl3 (10 mg/kg iv, 24 h before experiment), blocked ethanol-induced increases in pimonidazole binding. It is concluded that one large dose of ethanol causes pericentral hypoxia in rat liver tissue in vivo and that Kupffer cells are involved.

Metabolic Fate of 1,3-Butanediol in the Rat: Liver Tissue Slices Metabolism

1971 ◽  
Vol 101 (12) ◽  
pp. 1711-1718 ◽  
Author(s):  
M. A. Mehlman ◽  
R. B. Tobin ◽  
H. K. J. Hahn ◽  
L. Kleager ◽  
R. L. Tate
Keyword(s):  
Rat Liver ◽  
Liver Tissue ◽  
Metabolic Fate ◽  
Tissue Slices

scholarly journals Peptide-fluorophore/AuNP conjugate-based two-photon excited fluorescent nanosensor for caspase-3 activity imaging assay in living cells and tissue

MedChemComm ◽  
2017 ◽  
Vol 8 (7) ◽  
pp. 1435-1439 ◽  
Author(s):  
Qingshan Ge ◽  
Ningning Wang ◽  
Jishan Li ◽  
Ronghua Yang
Keyword(s):  
Rat Liver ◽  
Liver Tissue ◽  
Caspase 3 ◽  
Living Cells ◽  
Live Cells ◽  
Tissue Slices ◽  
Two Photon

Via the assembly of two-photon dye (TPdye)-labeled peptides on the gold nanoparticle's surface, a novel two-photon excited (TPE) fluorescent nanosensor has been developed for the measurement of caspase-3 activity in live cells and rat liver tissue slices.

Influence of Slice Thickness and Culture Conditions on the Metabolism of 7-Ethoxycoumarin in Precision-cut Rat Liver Slices

1998 ◽  
Vol 26 (4) ◽  
pp. 541-548
Author(s):  
Roger J. Price ◽  
Anthony B. Renwick ◽  
Paula T. Barton ◽  
J. Brian Houston ◽  
Brian G. Lake
Keyword(s):  
Gas Phase ◽  
Rat Liver ◽  
Xenobiotic Metabolism ◽  
Culture Conditions ◽  
Slice Thickness ◽  
Dependent Manner ◽  
Sprague Dawley ◽  
Liver Slices ◽  
Sprague Dawley Rats ◽  
Rat Livers

This study investigated the effects of some experimental variables on the rate of xenobiotic metabolism in precision-cut rat liver slices. Liver slices of 123 ± 8μm (mean ± SEM of six slices), 165 ± 3μm, 238 ± 6μm and 515 ± 14μm thickness were prepared from male Sprague-Dawley rats, and incubated in RPMI 1640 medium in an atmosphere of 95% O2/5% CO2 by using a dynamic organ culture system. Liver slices of all thicknesses metabolised 10μM 7-ethoxycoumarin to total (free and conjugated) 7-hydroxycoumarin in a time-dependent manner. The rate of 7-ethoxycoumarin metabolism was greatest in 165μm thick slices and slowest in 515μm thick slices, being 2.74 ± 0.19pmol/minute/mg slice protein and 0.69 ± 0.07pmol/minute/mg slice protein, respectively. No marked effects on the rate of 7-ethoxycoumarin metabolism in liver slices were observed either by changing the medium to Earle's balanced salt solution (EBSS) or by changing the gas phase to 95% air/5% CO2. Moreover, the perfusion of rat livers with EBSS at 2–4°C, prior to preparation of tissue cores, did not enhance 7-ethoxycoumarin metabolism in rat liver slices. In this study, the optimal slice thickness was 175μm, with higher rates of 7-ethoxycoumarin metabolism being observed than with 250μm thick slices, which are often used for studies of xenobiotic metabolism. Variable results were obtained with slices of around 100–120μm thickness, which may be attributable to the ratio between intact hepatocytes and cells damaged by the slicing procedure in these very thin slices.

scholarly journals Postprandial Glycogen Content Is Increased in the Hepatocytes of Human and Rat Cirrhotic Liver

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 976
Author(s):  
Natalia N. Bezborodkina ◽  
Sergey V. Okovityi ◽  
Boris N. Kudryavtsev
Keyword(s):  
Rat Liver ◽  
Liver Tissue ◽  
Glycogen Content ◽  
Glycogen Synthase ◽  
Fibrous Tissue ◽  
Glycogen Metabolism ◽  
Liver Parenchyma ◽  
Dry Mass ◽  
Cirrhotic Liver ◽  
Liver Biopsies

Chronic hepatitises of various etiologies are widespread liver diseases in humans. Their final stage, liver cirrhosis (LC), is considered to be one of the main causes of hepatocellular carcinoma (HCC). About 80–90% of all HCC cases develop in LC patients, which suggests that cirrhotic conditions play a crucial role in the process of hepatocarcinogenesis. Carbohydrate metabolism in LC undergoes profound disturbances characterized by altered glycogen metabolism. Unfortunately, data on the glycogen content in LC are few and contradictory. In this study, the material was obtained from liver biopsies of patients with LC of viral and alcohol etiology and from the liver tissue of rats with CCl4-induced LC. The activity of glycogen phosphorylase (GP), glycogen synthase (GS), and glucose-6-phosphatase (G6Pase) was investigated in human and rat liver tissue by biochemical methods. Total glycogen and its labile and stable fractions were measured in isolated individual hepatocytes, using the cytofluorometry technique of PAS reaction in situ. The development of LC in human and rat liver was accompanied by an increase in fibrous tissue (20- and 8.8-fold), an increase in the dry mass of hepatocytes (by 25.6% and 23.7%), and a decrease in the number of hepatocytes (by 50% and 28%), respectively. The rearrangement of the liver parenchyma was combined with changes in glycogen metabolism. The present study showed a significant increase in the glycogen content in the hepatocytes of the human and the rat cirrhotic liver, by 255% and 210%, respectively. An increased glycogen content in cells of the cirrhotic liver can be explained by a decrease in glycogenolysis due to a decreased activity of G6Pase and GP.

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