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Liver Freezing Response of the Freeze-Tolerant Wood Frog, Rana sylvatica, in the Presence and Absence of Glucose. I. Experimental Measurements
Cryobiology 38, 310 –326 (1999) Article ID cryo.1999.2175, available online at http://www.idealibrary.com on
Liver Freezing Response of the Freeze-Tolerant Wood Frog, Rana sylvatica, in the Presence and Absence of Glucose I. Experimental Measurements Ramachandra V. Devireddy,* Paul R. Barratt,* Kenneth B. Storey,† and John C. Bischof* ,1 *Bioheat and Mass Transfer Laboratory, Department of Mechanical Engineering, University of Minnesota, Minneapolis, Minnesota 55455, U.S.A.; and †Department of Biology, Carleton University, Ottawa, Ontario K1S 5B6, Canada In this study, two methods are used to assess the equilibrium and dynamic cell volumes in Rana sylvatica liver tissue during freezing in the presence and absence of a cryoprotectant (glucose). The first is a “two-step” low-temperature microscopy (equilibrium and dynamic) freezing method and the second is a differential scanning calorimeter (DSC) technique. These two techniques were used to study (i) the in vitro architecture of R. sylvatica frog liver tissue and to measure its characteristic Krogh cylinder dimensions; (ii) the “equilibrium” (infinitely slow) cooling behavior and the osmotically inactive cell volume (V b) of R. sylvatica liver cells; and (iii) the dynamic water transport response of R. sylvatica liver cells in the presence and absence of the CPA (glucose) at a cooling rate of 5°C/min. Stereological analysis of the slam frozen (.1000°C/min) micrographs led to the determination that 74% of the liver tissue in control frogs was cellular versus 26% that was extracellular (vascular or interstitial). Mapping the stereological measurements onto a standard Krogh cylinder geometry (Model 1) yielded distance between adjacent sinusoid centers, DX 5 64 m m; original sinusoid (vascular) radius, r vo 5 18.4 mm; and length of the Krogh cylinder, L 5 0.71 m m (based on an isolated frog hepatocyte cell diameter of 16 mm). A significant observation was that ;24% of the frog hepatocyte cells are not in direct contact with the vasculature. To account for the cell– cell contact in the frog liver architecture a modified Krogh cylinder geometry (Model 2) was constructed. In this model (Model 2) a second radius, r 2 5 28.7 m m, was defined (in addition to the original sinusoid radius, r vo 5 18.4 mm, defined above) as the radius of the membrane between the adjacent cells (directly adjacent to vascular spaces) and embedded cells (removed from vascular spaces). By plotting the two-step equilibrium cooling results on a Boyle–van’t Hoff plot, the osmotically inactive cell volume, V b was obtained as 0.4 z V o (where V o is the isotonic cell volume). The two-step dynamic micrographs and the heat release measurements from the DSC were used to obtain water transport data during freezing. The DSC technique confirmed that R. sylvatica cells in control liver tissue do not dehydrate completely when cooled at 5°C/min but do so when cooled at 2°C/min. © 1999 Academic Press Key Words: cryopreservation; R. sylvatica; liver tissue; directional solidification stage; low temperature microscopy; differential scanning calorimetry; Krogh cylinder.
Wood frogs overwinter in the leaf litter on the forest floor. Here they may experience multiple freezing episodes over the winter months and can endure freezing temperatures as low as 26 to 28°C and freezing times of 2 weeks or more. Freezing is typically initiated near the equilibrium freezing point (FP) of body fluids (approx. 20.5°C in the absence of cryoprotective agents (CPAs)) as a result of contact with environmental ice that seeds nucleation of body fluids
Received January 28, 1999; accepted April 22, 1999. 1 To whom correspondence should be addressed.
0011-2240/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
across the frog’s skin. However, frog skin and gut also exhibit ice-nucleating bacteria (7) and plasma also contains ice-nucleating proteins that could help guide ice formation through the vasculature (25, 26, 27). Hence, multiple mechanisms ensure that freezing occurs close to the FP, thereby minimizing the osmotic shock to cells. As ice forms in the vasculature, the osmolality of the unfrozen solution in the vascular space rises above that of the intracellular fluid, causing an increased osmotic pressure which results in water outflow from the cells. To help counteract these volume changes, freezing also triggers the synthesis of a CPA. In wood frogs
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this involves the rapid breakdown of liver glycogen reserves to produce large amounts of glucose which is then circulated to all organs of the body (5, 27). Several studies in cells and tissues of the freeze-tolerant wood frog have focused on the freezing response and the ensuing biochemical alterations; however, studies of the cellular water transport involved have been few. Storey et al. (26) confirmed a relationship between maintaining a critical water volume within the cells and surviving the freeze. Studies in the Storey lab also showed that the hyperglycemic response to freezing could also be triggered by dehydration of the animal in the absence of freezing (27). Further work using magnetic resonance imaging (MRI) allowed imaging of ice formation in the wood frog, providing a noninvasive and real-time analysis of the mode of ice propagation through the body and visual confirmation that organs shrink during freezing due to water loss (23), a finding also supported by measurements of organ masses in control versus frozen frogs (6). More recent studies have focused on the biochemical reactions occurring within the frog at the onset of freezing as well as during the entire freeze–thaw cycle (3, 4, 16). However, little research has been done with tissues of a freeze-tolerant animal to investigate the manipulation and control of water transport which ultimately defines water volume. In the present study, two new methods are used to assess the equilibrium and dynamic cell volumes in R. sylvatica liver tissue during freezing. The first is a low-temperature microscopy technique which will simply be called “twostep” dynamic freezing method (20) and the second is a differential scanning calorimeter (DSC) technique (8, 9). These techniques were used to study the in vitro architecture of Rana sylvatica frog liver tissue and to measure its characteristic dimensions (Krogh cylinder dimensions). In addition, the “equilibrium” (infinitely slow) cooling behavior and the osmotically inactive cell volume (V b) of R. sylvatica liver cells were also quantified by a complimentary low-temperature microscopy method (i.e., two-step equilibrium freezing). And finally, the
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dynamic water transport response of R. sylvatica liver cells in the presence and absence of the CPA (glucose) was obtained at a cooling rate of 5°C/min, using either the two-step dynamic freezing method or the DSC technique or both. In Part II of this study (11), two models of water transport are compared to the dynamic water transport data to obtain the water permeability parameters of R. sylvatica liver cells, both in the presence and in the absence of CPA (glucose). MATERIALS AND METHODS
Animals Experiments were performed on freshly isolated livers from 23 wood frogs collected near Ottawa, Ontario, Canada, during the spring of 1997. For control tissue experiments, the freshly excised livers were placed in chilled (on ice at 4°C) RPMI culture medium (Celox, Inc., Hopkins, MN, U.S.A.) and then cut into small sections (#1 mm 3) using a razor blade. For the glucose experiments, livers were placed in chilled RPMI culture medium with 0.4 M glucose and the whole liver was then sectioned into #1-mm 3 pieces which were then equilibrated with the 0.4 M glucose solution for ;30 min. The sectioned samples were then frozen as described below for the two-step freezing experiments. Ischemic time of the liver on ice was always ,2.5 h. Two-Step Low-Temperature Microscopy Freezing Methods Sectioned liver samples were frozen by one of the three following methods, described previously (20). Slam freezing. Five to six tissue samples were placed on a microscope microslide and then slam-frozen (.1000°C/min) by bringing the microslide into intimate contact with a highly polished copper block which, until use, was in thermal equilibrium with liquid nitrogen at 2196°C. The microslide with the frozen samples was then quickly transferred to a petri dish containing liquid nitrogen. While immersed in liquid nitrogen the samples were carefully de-
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FIG. 1. Schematic of the two-step freezing “directional cooling stage” and the projected thermal history of the sample: Step 1 shows the freezing of the tissue sample from a base temperature above freezing (T H) to an intermediate temperature below freezing (T L). Step 2 shows the freezing of the tissue sample at a very high cooling rate (slam cooling or B . 10008C/min).
tached from the microslide and stored in liquid nitrogen prior to processing via the freeze substitution process. The slam freezing process preserves the morphological structure of the tissue since the submicroscopic trapped intracellular ice crystals and the tissue space cannot be separated and are thus measured as one giving the fully hydrated dimensions of the cellular space. Two-step equilibrium freezing. Constant and controlled rates of freezing were obtained by cooling samples on a directional solidification stage (Fig. 1) built in our laboratory, based on the design of Rubinsky and Ikeda (22) and discussed in detail elsewhere (20). Briefly, the stage consists of two copper platforms fitted with type ‘T’ thermocouples (Omega Tech.
Corp., Stamford, CT, U.S.A.) which are differentially cooled by an internal liquid nitrogen flow and heated by an external foil heater. The copper platform temperatures are regulated by control of the heater voltages by Fuji digital controllers (Total Temperature Instrumentation, Williston, VT, U.S.A.). The sectioned tissue sample rested on a normal microslide which in turn rested on the two constant temperature bases. When freezing began, the microslide with the sample was propelled at a constant velocity V, across the gap D gap, from the base maintained at a temperature above freezing (T H) to the base maintained at an intermediate temperature below freezing (T L). The cooling rate within the sample was maintained by control-
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ling the temperature of the two bases of the stage, the velocity of the microslide, and the distance of the gap, i.e., B (8C/min) 5 (T H 2 T L/D gap) z V. For equilibrium freezing, the specimens were cooled at 2°C/min from 10°C to preset end temperatures of 24, 26, 28, 210, and 220°C, respectively. Upon reaching the low-temperature base, the samples were allowed to equilibrate for 10 min. Tissue samples were then slam-frozen and stored in liquid nitrogen prior to processing via the freeze substitution process. Two-step dynamic freezing. For dynamic freezing, liver tissue samples were cooled using the directional solidification stage at a cooling rate of 5°C/min from 10°C to end temperatures of 24, 26, 28, 210, and 220°C for control tissue (absence of glucose) and to 26, 28, 210, and 220°C for tissue equilibrated with 0.4 M glucose. The presence of 0.4 M glucose in the medium did not allow for ice to be consistently nucleated on the directional cooling stage at temperatures above 26°C; thus experimental data could not be obtained at 24°C for tissue equilibrated with 0.4 M glucose. Upon reaching the end temperature, the samples were immediately slam-frozen (with no time for equilibration at the end temperature) and stored in liquid nitrogen prior to processing via the freeze substitution process. Since the samples were immediately slam-frozen upon reaching the end temperature, the morphological structure of the tissue is preserved at the instant of freezing. In contrast, the two-step equilibrium freezing protocol allows the tissue cells to dehydrate at the chosen end temperature. Thus the two-step equilibrium freezing micrographs show an enlarged vascular/extracellular ice crystals when compared to the two-step dynamic freezing micrographs at the same end temperatures. Freeze Substitution The freeze substitution protocol involved suspending the samples in 1 ml of freeze substitution medium (2g OsO 4 in 100 ml acetone) in a freeze substitution device (Bal-Tec, Middlebury, CT, U.S.A.). The protocol required holding the device at 290°C for 24 h, 260°C for 2 h, 230°C for 2 h, and 0°C for 30 min,
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before equilibrating to room temperature (20). During this process the acetone replaced the ice in the sample, and the osmium fixed the lipid or membranous structure (12, 21). Embedding and sectioning. After freeze substitution, the samples were dehydrated at room temperature in 10 –20 ml of 100% acetone before being infiltrated with Quetol resin (Ted Pella Inc., Redding, CA, U.S.A.). The resulting hard blocks of resin-embedded tissue were then sectioned on a Reichert ultracut microtome (Leica, Vienna, Austria) to a thickness of about 0.5 mm. The tissue sections were mounted on microslides, heated on a hot plate at 50°C, and then stained with toluidine blue in preparation for image analysis (20). Photomicrography and image analysis. Photomicrographs of the tissue sections were obtained by imaging through a Newvicon camera (Hamamatsu-Photonics, Bridgewater, NJ, U.S.A.) attached to a Nikon Labophot light microscope (Nikon Inc., Melville, NY, U.S.A.) and digitized on a workstation computer with a frame grabber (SGI, Mountainview, CA, U.S.A.). Image analysis of the photomicrographs was then performed to obtain the cellular and vascular/ extracellular volumes using NIH Image software (NIH, Bethesda, MD, U.S.A.), as described previously (20). In the image analysis, ice regions are seen as white spaces and are generally assumed to be vascular/extracellular in nature. In viewing micrographs of dynamically cooled (5°C/min) tissue, white spaces that were of equal or smaller diameter than a typical frog hepatocyte cell diameter ;16 mm were considered to be intracellular ice. In an attempt to account for intracellular ice crystals, these particular white spaces were darkened and counted with the intracellular volume. These intracellular ice spaces were found to be significant in the analysis of 5°C/min dynamic freezing micrographs (both in the presence and in the absence of glucose). Model 1: Standard Krogh Cylinder Geometry Dimensions The standard Krogh cylinder dimensions (Fig. 2A), DX, the average (shortest) distance
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tissue (20). The cellular space with volume V is represented in the Krogh model as the box sur2 rounding the cylinder (LDX 2 2 p r vo L) and A c is the effective membrane surface area available for water transport (5 2 p r voL), where r vo is the original vascular radius (assumed to be a constant during the water transport process). The length of the Krogh cylinder, L, was selected by imposing the constraint that the tissue cells must have the same overall cellular volume as that of the isolated frog hepatocyte cell (;2145 mm 3, based on a cell diameter of 16 mm). Differential Scanning Calorimetry Experiments
FIG. 2. (A) Schematic of the standard Krogh cylinder geometry (Model 1). The dimensions are DX, the distance between adjacent sinusoid centers (64 6 17.9 mm); r vo, the original sinusoid (vascular) radius (18.4 6 5.2 mm); and L, the axial length of the vascular cylinder (0.71 mm). (B) Schematic of the modified Krogh cylinder geometry (Model 2): The Krogh model has been modified to account for multiple cells between vascular cylinders. The dimensions are DX, the distance between adjacent sinusoid centers (64 6 17.9 mm); r 1 , the original sinusoid (vascular) radius (18.4 6 5.2 mm); r 2 , inner cell border radius (28.7 6 8.0 mm); and L, the axial length of the vascular cylinder (0.71 mm).
between vascular sinusoids (center– center) and r vo, the vascular (sinusoid) radius, were obtained by averaging at least 25 measurements made per slam-frozen tissue micrograph (n 5 4 images) and correlating these with the in vitro vascular dimensions of the slam-frozen control
Sample preparation. Experiments were performed using freshly excised liver tissue slices as previously described for the two-step freezing experiments. The liver tissue was cut into small pieces using a razor blade and patted externally using a blotting paper. The liver tissue slices (;1–1.5 mm 3 or ;1–1.5 mg) were then placed in standard aluminum DSC sample pans (Perkin–Elmer Corporation, Norwalk, CT, U.S.A.) and a small drop (,8 ml) of isotonic phosphate-buffered saline solution (PBS) (Celox, Inc.) was added to keep the tissue in an isotonic environment. A natural ice-nucleating agent, Pseudomonas syringae (ATCC, Rockville, MD, U.S.A.), was added (0.5 to 1 mg) before the pans were sealed and reweighed to measure the total sample weight (#10 mg). The ice-nucleating agent always nucleated the vascular/extracellular space at temperatures $22.5°C in order to avoid damaging intracellular ice formation, which occurs predominantly at temperatures below 25°C in most cell systems (28). DSC dynamic cooling protocol. The DSC measures heat released during phase changes as a function of time and temperature. Our lab has developed a DSC cooling protocol to measure water transport during freezing of a cell suspension (Epstein–Barr virus transformed, EBVT human lymphocyte) and a normal mammalian tissue (rat liver) system (8, 9). To ensure the accuracy and repeatability of the experimental data, the limitations of the DSC machine were
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FIG. 3. Heat flow vs temperature (DSC) curve: The superimposed heat flow thermograms obtained at a cooling rate of 5°C/min are shown. The bottom and top curves correspond to the heat flow thermograms measured in Step 3 and Step 7 of the DSC cooling protocol, respectively. A secondary heat release is also evident during the initial cooling (Step 3) run between 214 and 216°C. The heat flow (mW/mg) is plotted along the y axis and the subzero temperatures (°C) are plotted along the top x axis. The negative axis for the heat flow on the x axis implies an exothermic heat release in the DSC sample.
studied and a set of calibration and control experiments was performed, as previously described (8). In this study DSC experiments were conducted at two cooling rates (2 and 5°C/min) on R. sylvatica liver tissue slices (control tissue without glucose). A brief outline of the DSC dynamic cooling protocol is presented here: Step 1: The sample (liver slices 1 PBS 1 0.5 to 1 mg of P. syringae) initially at 4°C was cooled at 5°C/min until the vascular/extracellular ice nucleated (usually around 22.5°C). This nucleation temperature was approximately 1 to 1.5°C higher than that found in EBVT lymphocyte cell suspensions (8), normal mammalian (rat) liver slices in PBS (9), and tumor (rat prostate) tissue slices in PBS (10). The higher nucleation temperature might be due to the presence of ice nucleating bacteria (7) or proteins (25–27) in the frog liver tissue slice. Step 2: At
the time of nucleation the sample was manually triggered to thaw at a warming rate (10°C/min) such that T ph (20.53°C) was reached (but not overshot) and ice remained in the extracellular solution. Step 3: The sample was then cooled to 250°C at the specified cooling rate (2 or 5°C/ min) and the initial heat release due to the medium mixed with osmotically active cells was measured (q initial) (see Fig. 3, for 5°C/min). A secondary heat release shown between 216 and 214°C in Fig. 3 was found at a cooling rate of 5°C/min, but not at the slower cooling rate of 2°C/min. This secondary heat release was interpreted as trapped intracellular water changing phase inside the cells as will be described later. Step 4: The sample was reequilibrated at 20.53°C by thawing at 100°C/min. Step 5: To help differentiate between the heat released by the medium and the intracellular fluid in Step 3,
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the liver cells were rendered osmotically inactive by cooling the sample at a high cooling rate (200°C/min) down to 250°C. Step 6: Step 4 was repeated. Since Step 5 resulted in all the cells having compromised membranes, the intracellular water, proteins, and salts now are continuous with the extracellular solution (no membrane barriers exist), as previously suggested by Ko¨rber et al. (17). Step 7: The sample was then cooled to 250°C at the specified cooling rate (2 or 5°C/min) to measure the final heat release due to lysed or osmotically inactive cells mixed with medium (q final) (see Fig. 3, for 5°C/ min). Translation of heat release to cell volume data. The total difference in the heat release between the two cooling runs (Step 3 and Step 7) is denoted as Dq dsc (5 q initial 2 q final) and is shown in Fig. 3 for a cooling rate of 5°C/min. The fractional difference up to a subzero temperature, T, is denoted by Dq(T) dsc (5 q(T) initial 2 q(T) final ). The measurements of interest, Dq dsc and Dq(T) dsc, were obtained using the DSC-7 (Perkin–Elmer) software after selection of an appropriate baseline. These differences in heat release can be related to the changes in the cell volume as previously shown (8 –10), V o 2 V~T! Dq~T! dsc 5 Vo 2 Vb Dq dsc
[1]
We can write a simplified equation to measure water transport data from the DSC measured heat releases as V~T! 5 V o 2
Dq~T! dsc z ~V o 2 V b!. Dq dsc
[2]
Thus, Eq. [2] can be used to generate the water transport data from DSC heat release readings. The unknowns needed in Eq. [2] are the Krogh cylinder (Model 1) dimensions (L, DX, r vo), the isotonic cell volume, V o, and the osmotically inactive cell volume, V b, which were all obtained using two-step freezing results (either from slam-frozen images or two-step equilibrium cooled micrographs).
RESULTS AND DISCUSSION
Model 1: Standard Krogh Cylinder Geometry Dimensions The dimensions of the Krogh cylinder (Model 1, Fig. 2A), DX, the average (shortest) distance between vascular sinusoids (center– center) and r vo, the vascular (sinusoid) radius were chosen to correlate with the in vitro vascular dimensions of the slam-frozen control tissue (20). Using NIH Image software it was first determined that 74% of the frog liver tissue was cellular space, while the remaining 26% was vascular/extracellular space and mean DX was 64 6 17.9 mm based on an average of at least 25 measurements per slam-frozen micrographs from three different animals. The vascular (sinusoid) radius, r vo, was found using two different methods. The first was to measure the vascular radius using NIH Image software and a value of 12.2 6 5.3 mm was obtained. Slam tissue images showed vascular spaces to be oblong in shape. This was because the section cut was not always exactly perpendicular to the vascular sinusoids; thus the direct measurement of vascular radius was not considered accurate. The second method was by direct sterological area measurements of cellular and extracellular spaces in the slam tissue images. The vascular area of the Krogh cylinder (Model 1) can be 2 and since 26% of the total expressed as p r vo area is vascular/extracellular, one could express 2 p r vo 5 0.26 z DX 2 and solve for r vo. Using this method, r vo was found to be 18.4 6 5.2 mm. The radius calculated using the second method was deemed to be more accurate and hence was used in this analysis. The length of the Krogh cylinder, L, was selected by imposing the constraint that the cells in intact liver tissue must have the same overall cell volume as that of isolated hepatocytes which is ;2145 mm 3 (based on a frog hepatocyte cell diameter of 16 mm obtained from the projected area of in vivo hepatocyte cells). Hence the dimensions of the Krogh cylinder (Model 1) were found to be: distance between adjacent vascular sinusoids, DX 5 64 6 17.9 m m, original vascular radius,
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initial cell volumes of the cells adjacent to vasculature and embedded cells were equal; i.e., L z 2 (DX 2 2 p r 22 ) 5 L z ( p r 22 2 p r vo ). Using the dimensions obtained earlier (DX 5 64 6 17.9 m m and r 1 5 r vo 5 18.4 6 5.2 mm), the outer radius r 2 was determined to be 28.7 6 8.0 mm. The modified Krogh cylinder geometry (Fig. 2B) is described further, in Part II of this study (11). Two-Step Equilibrium Freezing Response: Boyle–van’t Hoff Plot FIG. 4. R. sylvatica liver anatomy: A magnified section of the slam-frozen image shows that more than two hepatocyte cells can be found between adjacent vascular sinusoids. Approximately 24% of the frog hepatocyte cells were found to be at least one cell removed from vascular sinusoids.
r vo 5 18.4 6 5.2 mm, and length of the Krogh cylinder, L 5 0.71 m m. Model 2: Modified Krogh Cylinder Geometry Dimensions In analyzing the slam tissue images (Fig. 4), counts were also made of the number of cells spanning the distance between adjacent vascular sinusoids. Of a total of 87 individual hepatocyte cells analyzed in the slam tissue images (n 5 4 images from four different frogs) 21 were found to be at least one cell removed from the vascular spaces or were completely bordered by other cells. Thus our measurements suggest that ;24% (21 of 87) of frog hepatocyte cells were not in direct contact with vasculature and although area measurements can be deceiving and some vascular sinusoids may exist over or under the cells analyzed, this measurement is taken as strong evidence that some percentage of the frog hepatocyte cells are not in direct contact with vascular spaces. To account for these embedded cells the Krogh cylinder geometry, Model 1, (Fig. 2A) was modified to Model 2 (Fig. 2B) which accounts for cells bordering a vascular conduit (adjacent) and cells which do not (embedded). The embedded cell radius r 2 was determined such that the
The equilibrium (infinitely slow) cooling behavior and the osmotically inactive cell volume (V b) of R. sylvatica liver cells were obtained using the two-step equilibrium freezing method. Figure 5 shows the micrographs from liver tissue cooled slowly on the directional stage and then equilibrated for 10 min at an end temperature of 0, 24, 26, 28, 210, and 220°C before slam freezing to 2196°C. Tissue cooled to 0°C before slam freezing is used as the reference (Fig. 5, slam) for comparison of equilibrium freezing effects at the other five temperatures. It can be seen that cell volume progressively decreases whereas extracellular ice content increases with decreasing freezing temperature. A Boyle–van’t Hoff (BVH) plot (Fig. 6) was constructed by examining the cellular volumes depicted in the freeze-dehydrated tissue micrographs (Fig. 5), as previously described (20). The inverse of the osmolality experienced by the tissue cells (Osm 5 DT/1.858, where DT 5 273.15 2 T, K) is plotted on the x-axis while the normalized equilibrium cell volume is plotted on the y-axis. By extrapolating to the volume at infinite osmolality, the osmotically inactive cell volume, V b 5 0.4 z V o, was obtained. Two-Step Dynamic Freezing Response Figure 7 shows the control liver tissue (absence of glucose) response to the two-step dynamic freezing method. The first image, on the top left-hand corner, is of the tissue slam-frozen (.1000°C/min) from 0°C, before freeze dehydration of the cells began. The next five images are freeze-substituted micrographs of tissue fro-
FIG. 5. Two-step equilibrium-cooled light micrographs of R. sylvatica liver tissue: Tissue slices were cooled on the directional stage at 2°C/min and equilibrated for 10 min at various end temperature (0, 24, 26, 28, 210, and 220°C) followed by slam-freezing to 2196°C. In the micrograph of control tissue cooled to 0°C, the Rs indicate the trapped red blood cells in vascular spaces, surrounded by cells (C). In the other micrographs, the optically dense spaces (stars) and the light areas (arrows) correspond to tissue components and extracellular ice crystals, respectively. Bar in the slam-frozen image, 50 mm.
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FIG. 6. Boyle–van’t Hoff plot constructed at constant subzero temperatures in the presence of external ice for R. sylvatica liver tissue: The filled circles represent the average “equilibrium” volumetric data at various end temperatures (0, 24, 26, 28, 210, and 220°C) obtained using the micrographs shown in Fig. 5. The linear curve extrapolates to infinite osmolality, giving the osmotically inactive volume of the R. sylvatica liver cells as 0.4V o. The error bars represent standard deviations in the data (n 5 4).
zen to various end temperatures, 24, 26, 28, 210, and 220°C (as denoted in the top lefthand corner of each image) before immediate slam freezing. In the 0°C tissue (slam-frozen image, Fig. 7A), individual hepatocyte cells are discernible (indicated by Cs) surrounding the trapped red blood cells in vascular spaces (indicated by Rs). As freezing begins the cells begin to dehydrate and water diffuses out of the cells and into the vasculature. As this happens, the cells begin to coalesce and flatten out (Fig. 7B–F, stars), while the vasculature expands (Fig. 7B–F, arrows). In comparison to the equilibrium-cooled micrographs (Fig. 5), the size of the vascular ice crystals is in general smaller at comparable end temperatures in the dynamically cooled micrographs (Fig. 7). The presence of smaller ice crystals suggests that a higher fraction of intracellular water remains within the cells and formed intracellular ice, as will be described below. Cellular dehydration (and therefore water transport from the cells) appears to cease at 210°C, as no further reduction in
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projected cell area is discernible by image analysis in the micrograph cooled to 220°C. Figure 8 shows the response of R. sylvatica liver tissue slices equilibrated with 0.4 M glucose using the two-step dynamic freezing method. The first image, on the top left-hand corner, is of the tissue slam-frozen (.1000°C/ min) from 0°C, before freeze dehydration began. The next five images are freeze-substituted micrographs of tissue frozen to various end temperatures, 26, 28, 210, and 220°C. As before, in the tissue cooled only to 0°C and slam-frozen, the Rs in Fig. 8A indicate the trapped red blood cells in vascular spaces, surrounded by cells (indicated by Cs) and as freezing progresses the cells begin to dehydrate and coalesce together (Fig. 8B–E, dark spaces, denoted by stars) and the vasculature expands (Fig. 8B–E, arrows). As mentioned earlier, no experimental data could be obtained at 24°C, as the presence of 0.4 M glucose in the medium did not allow for ice to be consistently nucleated on the directional cooling stage at temperatures above 26°C. Note that ice crystals are in general smaller in Fig. 8 (presence of 0.4 M glucose) than in Fig. 7 (absence of glucose) for comparable end temperatures (especially at temperatures #210°C). The presence of smaller ice crystals likely indicates that increased incidence of intracellular ice is occurring in the presence of 0.4 M glucose than in its absence. As in the case of control tissue, cellular dehydration appears to cease at 210°C in the presence of 0.4 M glucose, as no further reduction in the cell volumes is discernible by image analysis in the micrograph cooled to 220°C. Estimation of Intracellular Ice during TwoStep Dynamic Cooling In viewing the micrographs of the dynamically cooled tissue at end temperatures, #28°C (in control tissue, Fig. 7) and #26°C (in tissue equilibrated with 0.4 M glucose, Fig. 8), white voids (ice crystals) were found that were of smaller diameter than the typical frog hepatocyte cell (;16 mm). These ice crystals likely formed and grew during the dynamic cooling process and not during the slam-frozen step
FIG. 7. Two-step dynamically cooled light micrographs of R. sylvatica liver tissue: Tissue slices were cooled on the directional stage at 5°C/min to various end temperature (0, 24, 26, 28, 210, and 220°C) immediately followed by a second slam-freezing step to 2196°C. As before, in the control micrograph (0°C) the Rs indicate the trapped red blood cells in vascular spaces, surrounded by cells (C) while in the remaining micrographs the optically dense spaces (stars) and the light areas (arrows) correspond to tissue components and extracellular ice crystals, respectively. Bar in the slam-frozen image, 50 mm.
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FIG. 8. Two-step dynamically cooled light micrographs of R. sylvatica liver tissue: Tissue slices were equilibrated with 0.4 M glucose and cooled at 5°C/min to various end temperatures (0, 26, 28, 210, and 220°C) immediately followed by a second slam-freezing step to 2196°C. As before, in the control micrograph (0°C) the Rs indicate the trapped red blood cells in vascular spaces, surrounded by cells (C) while in the remaining micrographs the optically dense spaces (stars) and the light areas (arrows) correspond to tissue components and extracellular ice crystals, respectively. Bar in the slam-frozen image, 50 mm.
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FIG. 9. Micrographs of R. sylvatica control liver tissue cooled at 5°C/min to 220°C showing intracellular ice crystals: Intracellular ice formation in dynamic freezing (5°C/min) was accounted for by measuring white (assumed vascular) spaces in the light micrograph (A). Those spaces with characteristic size that measured to be less than the diameter of a typical hepatocyte cell (;16 mm) were assumed to be intracellular ice, and these spaces were darkened (B) and included as contributing to the cellular volume. (A) Scale bar, 25 mm.
which leaves only submicroscopic ice crystals that cannot be distinguished from the tissue spaces. Since the vascular sinusoids had a typical radius twice that of the typical hepatocyte cell (r vo 5 18.4 mm vs cell radius 5 8.0 mm), these white voids (#16 mm in the largest dimension) were assumed to represent intracellular ice formed during dynamic cooling. These areas were then filled in black (using NIH Image software) to be counted as cellular space. Figure 9A shows a micrograph of control tissue cooled dynamically at 5°C/min to 220°C and Fig. 9B shows the same micrograph with intracellular crystals (white voids, #16 mm) filled in as gray, to be counted as cellular spaces. This modification led to an increase in the nondimensional cell volume of ;5% at 28°C and ;10% at 220°C. This suggests that the anatomy of the frog liver prevents a total dehydration in all of the cells (at 5°C/min) and a fraction of the intracellular water is trapped inside the cell once water transport ceases (at approx. 210°C as seen in Figs. 7 and 8) and changes into intracellular ice on sufficient supercooling. In support of the above interpretation, the DSC measured a secondary heat release while cooling control tissue at 5°C/min at 214 to 216°C (as shown in Fig. 3). This secondary peak was measured and translates to ;20% of the heat release due to the total water volume, a larger fraction than the ;10% increase seen in
the dynamically cooled micrographs (as discussed above). Note that the secondary heat release occurs after water transport has ceased (T , 2108C) and hence the biophysical events (water transport and intracellular ice formation) are decoupled. Therefore, while translating the 5°C/min DSC heat release readings into volumetric shrinkage data (between 0 and 210°C) using Eq. [2], an end volume of 0.6 z V o (instead of V b or 0.4 z V o) was used (see Fig. 10B). Since the DSC did not measure a secondary heat release when cooled dynamically at 2°C/min, the osmotically inactive cell volume (V b or 0.4 z V o) was used in Eq. [2] to translate the DSC heat release readings at 2°C/min to volumetric shrinkage data (see Fig. 10B). Volumetric Shrinkage Response with and without Glucose The dynamic water transport data for R. sylvatica control liver tissue cells cooled dynamically at 2°C/min (DSC data) and at 5°C/min (DSC and two-step dynamic data) are shown in Figs. 10A– 10C. In Fig. 10A, the filled and open circles represent the two-step dynamic water transport data, with and without accounting for the effect of intracellular ice crystals (i.e., with and without “blackening” out the white voids less than the typical frog hepatocyte cell diameter, ;16 mm). In Fig. 10B, the open triangles and open squares represent the 5 and 2°C/min dynamic DSC water
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FIG. 10. Temperature versus volumetric responses of the cells in R. sylvatica liver tissue: The Krogh cylinder (Model 1) simulated equilibrium volumetric response (---) is shown for reference, in all the figures. The error bars represent standard deviations in the data (n 5 3 for two-step and n 5 6 for DSC data). (A) The dynamic two-step water transport data in R. sylvatica control liver tissue is shown, with (F) and without (E) accounting for the effect of intracellular ice. (B) The open triangles and open squares represent the 5 and 2°C/min dynamic DSC water transport data, respectively. (C) A comparison of the data shown in (A) and (B). The symbols and the data are the same as in (A) and (B). (D) The two-step dynamic water transport data in R. sylvatica liver tissue equilibrated with 0.4 M of glucose is shown, with (■) and without (h) accounting for the effect of intracellular ice.
transport data, respectively. A good agreement is found between the two-step dynamic water transport data accounting for the presence of intracellular ice crystals (filled circles) and the DSC data (open triangles) at a cooling rate of 5°C/min, as shown in Fig. 10C. The data were found to be statistically similar with a confidence level of $92.5% (by Student’s t test). The dynamic water
transport data for frog liver tissue cells cooled at 5°C/min in the presence of 0.4 M glucose are shown in Fig. 10D. The filled and open squares represent the two-step dynamic water transport data, with and without accounting for the effect of intracellular ice crystals. The Krogh cylinder (Model 1)-predicted equilibrium cooling response (cells cooled infinitely slowly) in the absence
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(Figs. 10A–C) and presence of 0.4 M glucose (Fig. 10D) is also shown for reference as a dashed line. R. sylvatica Liver Freeze Response It should be pointed out that the cooling rates (2 and 5°C/min) and the end temperatures (0 to 220°C) investigated in this study are not physiologically relevant. Frogs in nature would experience much slower cooling rates (;1–2°C/h) and also much milder end temperatures (.28°C). The wood frogs in nature will, therefore, dehydrate fully to the equilibrium cooled end volume at any temperature, as shown by the two-step equilibrium cooled micrographs (Fig. 5) and the normalized equilibrium volumes in the Boyle–van’t Hoff plot (Fig. 6), obtained in this study. In addition to equilibrium behavior, this study determined the volumetric shrinkage data (water transport) for frog liver tissue (hepatocyte) cells under “nonequilibrium” (or dynamic) cooling conditions at a cooling rate of 5°C/min. In Part II of this study (11), the experimentally determined dynamic water transport data (shown in Fig. 10) is fit to a mathematical model of water transport, to predict the water permeability parameters of R. sylvatica liver tissue cells in the presence and absence of CPA (glucose). R. sylvatica Liver Architecture: Comparison to Mammalian (Rat) Liver One of the aims of this study was to study the in vitro architecture of R. sylvatica frog liver tissue and to measure its characteristic dimensions (Krogh cylinder dimensions). These can then be compared to those of the mammalian (rat) liver (20) to provide insight into how the liver structure of a freeze-tolerant amphibian differs from that of a freezeintolerant mammal (rat). This study found that the structural makeup of the two livers was remarkably different: (i) ;26% of the frog liver was vascular/extracellular space vs ;15% in rat liver; (ii) the radius of the vascular sinusoids in the frog were approximately five times larger than in the rat (18.4 vs 3.8 mm); (iii) the average distance between
the sinusoids in the frog was approximately three times larger than in the rat (64 vs 22 mm); (iv) the diameter of the frog hepatocytes was slightly smaller than the rat hepatocytes (16 vs 21 mm); and (v) ;24% of the cells in the frog liver were not directly adjacent to vascular spaces as opposed to 0% of rat liver cells. An extensive literature search was performed to check if any previous studies have reported the morphology/architecture of the R. sylvatica liver tissue observed in this study. Several excellent review articles and studies have been presented in the literature on the function, development, and morphology of the liver of amphibians and vertebrates, including Bennett and Glenn (1), Beresford and Henninger (2), Elias (13), Godula (14), Gumucio (15), Jones (18), Moore (19), and Spornitz (24). However, we were unable to find any previous reference to the observed morphology/architecture of the R. sylvatica liver tissue, obtained in this study. Preliminary results on Rana pipiens suggest that the architecture of this non-freeze-tolerant frog liver is very similar to R. sylvatica (unpublished data). CONCLUSIONS
This study investigates the water transport characteristics during freezing in the liver tissue of the freeze-tolerant wood frog R. sylvatica in the presence and absence of glucose. Experiments were performed using two-step equilibrium and dynamic freezing methods as well as a differential scanning calorimetry technique. Following the two-step (equilibrium and dynamic) freezing protocol, the frozen tissue slices were freeze substituted and embedded in Quetol resin for sectioning on a microtome. Stereological analysis of the slam-frozen (.1000°C/min) results led to the measurements that 74% of the control tissue is cellular (26% extracellular–interstitial and vascular); osmotically inactive cell volume, V b , is 0.4 z V o ; and the Krogh cylinder (Model 1) dimensions are DX 5 64 m m, r vo 5 18.4 mm, and L 5 0.71 m m (assuming the typical diameter of the frog hepatocyte cells is 16
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mm). In addition, stereological measurements suggest that ;24% of the frog hepatocyte cells are not in direct contact with the vasculature (as opposed to 0% in mammalian rat liver tissue). A parallel study was also done using the DSC in which control liver tissue was cooled dynamically at 2 and 5°C/min. The two-step dynamic freezing micrographs and the measured heat releases from the DSC were used to obtain the volumetric shrinkage data during freezing. The DSC technique confirmed that R. sylvatica cells in control liver tissue do not dehydrate completely when cooled at 5°C/min but do so when cooled at 2°C/min. It was previously shown that the mammalian (rat) liver tissue cells undergo complete cellular dehydration when cooled at 5°C/min (9, 20). A modified Krogh cylinder (Model 2) was constructed to account for the architecture with more than one cell between vascular spaces. In this model a second radius, r 2 5 28.7 m m, was defined (in addition to the original sinusoid radius, r vo 5 18.4 mm, defined above) as the radius of the membrane between the adjacent cells (directly adjacent to vascular spaces) and embedded cells (removed from vascular spaces). In Part II of this study (11), a mathematical model of water transport is presented and fit to the experimentally determined water transport data to obtain the biophysical permeability parameters of R. sylvatica liver tissue cells in the presence and absence of CPA (glucose). ACKNOWLEDGMENTS This work was supported by a grant from the National Science Foundation (NSF-BES 9703326) and a grant from the Whitaker Foundation to J.C.B. REFERENCES 1. Bennett, T. P., and Glenn, J. S. Fine structural changes in liver cells of Rana catesbeiana during natural metamorphosis. Dev. Biol. 22, 535–560 (1970). 2. Beresford, W. A., and Henninger, J. M. A tabular comparative histology of the liver. Arch. Histol. Jpn. 49, 267–281 (1986). 3. Cai, Q., Greenway, S. C., and Storey, K. B. Differential regulation of the mitochondrial ADP/ATP translocase gene in wood frogs under freezing stress. Biochim. Biophys. Acta 1353, 69 –78 (1997).
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4. Cai, Q., and Storey, K. B. Freezing-induced genes in wood frog (Rana sylvatica): Fibrinogen upregulation by freezing and dehydration. Am. J. Physiol. 272, R1408 –R1492 (1997). 5. Costanzo, J. P., Lee, R. E., Jr., and Lortz, P. H. Physiological responses of freeze-tolerant and -intolerant frogs: clues to evolution of anuran freeze tolerance. Am. J. Physiol. 265, R721–R725 (1993). 6. Costanzo, J. P., Lee, R. E., and Wright, M. F. Cooling rate influences cryoprotectant distribution and organ dehydration in freezing wood frogs. J. Exp. Zool. 261, 373–378 (1992). 7. Costanzo, J. P., and Lee, R. E. Mini review: Ice nucleation in freeze-tolerant vertebrates. Cryo-Lett. 17, 111–118 (1996). 8. Devireddy, R. V., Raha, D., and Bischof, J. C. Measurement of water transport during freezing in cell suspensions using a differential scanning calorimeter. Cryobiology 36(2), 124 –155 (1998). 9. Devireddy, R. V., and Bischof, J. C. Measurement of water transport during freezing in mammalian liver tissue. II. The use of differential scanning calorimetry. ASME J. Biomech. Eng. 120(5), 559 –569 (1998). 10. Devireddy, R. V., Smith, D. J., and Bischof, J. C. Mass transfer during freezing in rat prostate tumor tissue. AIChE J. 45(3), 639 – 654 (1999). 11. Devireddy, R. V., Barratt, P. R., Storey, K. B., and Bischof, J. C. Liver freezing response of the freezetolerant wood frog Rana sylvatica, in the presence and absence of glucose. II. Mathematical modeling. Cryobiology 38, 327–338 (1999). 12. Echlin, P. “Low Temperature Microscopy and Analysis,” Chapters 3 and 7, Plenum, New York/London, 1992. 13. Elias, H. Liver morphology. Biol. Rev. 30, 263–310 (1955). 14. Godula, J. Quantitative investigations of cellular organelles from the liver of amphibia: Xenopus laevis (Daudin) and Ambystoma mexicanum (Cope). Acta Biol. Cracov., Ser. Zool. 13, 225–236 (1970). 15. Gumucio, J. J. Hepatocyte heterogeneity: The coming of age from the description of a biological curiosity to a partial understanding of its physiological meaning and regulation. Hepatology 9, 154 –160 (1989). 16. Holden, C. P., and Storey, K. B. Signal transduction, second messenger, and protein kinase responses during freezing exposures in wood frogs. Am. J. Physiol. 271, R1205–R1211 (1996). 17. Ko¨rber, C. H., Englich, S., and Rau, G. Intracellular ice formation: Cryomicroscopical observation and calorimetric measurement. J. Microsc. 161, 313–325 (1991). 18. Jones, A. L. Anatomy of the normal liver. In “Hepatology: A Textbook of Liver Disease” (D. Zakim
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and T. D. Boyer, Eds.), Vol. 1, pp. 3–30, Saunders, Philadelphia, 1990. Moore, J. A. (Ed.). “Physiology of Amphibia,” Academic Press, New York/London, 1964. Pazhayannur, P. V., and Bischof, J. C. Measurement and simulation of water transport during freezing in mammalian liver tissue. ASME J. Biomech. Eng. 119, 269 –277 (1997). Robards, A. W., and Sleytr, U. B. “Practical Methods in Electron Microscopy,” Vol. 10, Chapter 7, Elsevier, Amsterdam, 1985. Rubinsky, B., and Ikeda, M. A cryomicroscope using directional solidification for controlled freezing of biological material. Cryobiology 22, 55– 68 (1985). Rubinsky, B., Wong, S. T. S., Hong, J. S., Gilbert, J., Roos, M., and Storey, K. B. 1H magnetic resonance imaging of freezing and thawing in freeze-tolerant frogs. Am. J. Physiol. 266, R1771–R1777 (1994).
24. Spornitz, U. M. Studies of the liver of Xenopus laevis. III. The ultrastructure and the glycogen content of the developing liver. Anat. Embryol. 154, 1–25 (1978). 25. Storey, K. B., and Storey, J. M. Freeze tolerance in animals. Physiol. Rev. 68(1), 27– 84 (1988). 26. Storey, K. B., Bischof, J. B., and Rubinsky, B. Cryomicroscopic analysis of freezing in liver of the freeze-tolerant wood frog. Am. J. Physiol. 263, R185–R194 (1992). 27. Storey, K. B., and Storey, J. M. Cellular adaptations for freezing survival by amphibians and reptiles. In “Advances in Low-Temperature Biology” (P. L. Steponkus, Ed.), Vol. 2, pp. 101–129, JAI Press, London, 1993. 28. Toner, M. Nucleation of ice crystals in biological cells. In “Advances in Low Temperature Biology” (P. L. Steponkus, Ed.), Vol. 2, pp. 1–52, JAI Press, London, 1993.
Liver Freezing Response of the Freeze-Tolerant Wood Frog, Rana sylvatica, in the Presence and Absence of Glucose I. Experimental Measurements Ramachandra V. Devireddy,* Paul R. Barratt,* Kenneth B. Storey,† and John C. Bischof* ,1 *Bioheat and Mass Transfer Laboratory, Department of Mechanical Engineering, University of Minnesota, Minneapolis, Minnesota 55455, U.S.A.; and †Department of Biology, Carleton University, Ottawa, Ontario K1S 5B6, Canada In this study, two methods are used to assess the equilibrium and dynamic cell volumes in Rana sylvatica liver tissue during freezing in the presence and absence of a cryoprotectant (glucose). The first is a “two-step” low-temperature microscopy (equilibrium and dynamic) freezing method and the second is a differential scanning calorimeter (DSC) technique. These two techniques were used to study (i) the in vitro architecture of R. sylvatica frog liver tissue and to measure its characteristic Krogh cylinder dimensions; (ii) the “equilibrium” (infinitely slow) cooling behavior and the osmotically inactive cell volume (V b) of R. sylvatica liver cells; and (iii) the dynamic water transport response of R. sylvatica liver cells in the presence and absence of the CPA (glucose) at a cooling rate of 5°C/min. Stereological analysis of the slam frozen (.1000°C/min) micrographs led to the determination that 74% of the liver tissue in control frogs was cellular versus 26% that was extracellular (vascular or interstitial). Mapping the stereological measurements onto a standard Krogh cylinder geometry (Model 1) yielded distance between adjacent sinusoid centers, DX 5 64 m m; original sinusoid (vascular) radius, r vo 5 18.4 mm; and length of the Krogh cylinder, L 5 0.71 m m (based on an isolated frog hepatocyte cell diameter of 16 mm). A significant observation was that ;24% of the frog hepatocyte cells are not in direct contact with the vasculature. To account for the cell– cell contact in the frog liver architecture a modified Krogh cylinder geometry (Model 2) was constructed. In this model (Model 2) a second radius, r 2 5 28.7 m m, was defined (in addition to the original sinusoid radius, r vo 5 18.4 mm, defined above) as the radius of the membrane between the adjacent cells (directly adjacent to vascular spaces) and embedded cells (removed from vascular spaces). By plotting the two-step equilibrium cooling results on a Boyle–van’t Hoff plot, the osmotically inactive cell volume, V b was obtained as 0.4 z V o (where V o is the isotonic cell volume). The two-step dynamic micrographs and the heat release measurements from the DSC were used to obtain water transport data during freezing. The DSC technique confirmed that R. sylvatica cells in control liver tissue do not dehydrate completely when cooled at 5°C/min but do so when cooled at 2°C/min. © 1999 Academic Press Key Words: cryopreservation; R. sylvatica; liver tissue; directional solidification stage; low temperature microscopy; differential scanning calorimetry; Krogh cylinder.
Wood frogs overwinter in the leaf litter on the forest floor. Here they may experience multiple freezing episodes over the winter months and can endure freezing temperatures as low as 26 to 28°C and freezing times of 2 weeks or more. Freezing is typically initiated near the equilibrium freezing point (FP) of body fluids (approx. 20.5°C in the absence of cryoprotective agents (CPAs)) as a result of contact with environmental ice that seeds nucleation of body fluids
Received January 28, 1999; accepted April 22, 1999. 1 To whom correspondence should be addressed.
0011-2240/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
across the frog’s skin. However, frog skin and gut also exhibit ice-nucleating bacteria (7) and plasma also contains ice-nucleating proteins that could help guide ice formation through the vasculature (25, 26, 27). Hence, multiple mechanisms ensure that freezing occurs close to the FP, thereby minimizing the osmotic shock to cells. As ice forms in the vasculature, the osmolality of the unfrozen solution in the vascular space rises above that of the intracellular fluid, causing an increased osmotic pressure which results in water outflow from the cells. To help counteract these volume changes, freezing also triggers the synthesis of a CPA. In wood frogs
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this involves the rapid breakdown of liver glycogen reserves to produce large amounts of glucose which is then circulated to all organs of the body (5, 27). Several studies in cells and tissues of the freeze-tolerant wood frog have focused on the freezing response and the ensuing biochemical alterations; however, studies of the cellular water transport involved have been few. Storey et al. (26) confirmed a relationship between maintaining a critical water volume within the cells and surviving the freeze. Studies in the Storey lab also showed that the hyperglycemic response to freezing could also be triggered by dehydration of the animal in the absence of freezing (27). Further work using magnetic resonance imaging (MRI) allowed imaging of ice formation in the wood frog, providing a noninvasive and real-time analysis of the mode of ice propagation through the body and visual confirmation that organs shrink during freezing due to water loss (23), a finding also supported by measurements of organ masses in control versus frozen frogs (6). More recent studies have focused on the biochemical reactions occurring within the frog at the onset of freezing as well as during the entire freeze–thaw cycle (3, 4, 16). However, little research has been done with tissues of a freeze-tolerant animal to investigate the manipulation and control of water transport which ultimately defines water volume. In the present study, two new methods are used to assess the equilibrium and dynamic cell volumes in R. sylvatica liver tissue during freezing. The first is a low-temperature microscopy technique which will simply be called “twostep” dynamic freezing method (20) and the second is a differential scanning calorimeter (DSC) technique (8, 9). These techniques were used to study the in vitro architecture of Rana sylvatica frog liver tissue and to measure its characteristic dimensions (Krogh cylinder dimensions). In addition, the “equilibrium” (infinitely slow) cooling behavior and the osmotically inactive cell volume (V b) of R. sylvatica liver cells were also quantified by a complimentary low-temperature microscopy method (i.e., two-step equilibrium freezing). And finally, the
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dynamic water transport response of R. sylvatica liver cells in the presence and absence of the CPA (glucose) was obtained at a cooling rate of 5°C/min, using either the two-step dynamic freezing method or the DSC technique or both. In Part II of this study (11), two models of water transport are compared to the dynamic water transport data to obtain the water permeability parameters of R. sylvatica liver cells, both in the presence and in the absence of CPA (glucose). MATERIALS AND METHODS
Animals Experiments were performed on freshly isolated livers from 23 wood frogs collected near Ottawa, Ontario, Canada, during the spring of 1997. For control tissue experiments, the freshly excised livers were placed in chilled (on ice at 4°C) RPMI culture medium (Celox, Inc., Hopkins, MN, U.S.A.) and then cut into small sections (#1 mm 3) using a razor blade. For the glucose experiments, livers were placed in chilled RPMI culture medium with 0.4 M glucose and the whole liver was then sectioned into #1-mm 3 pieces which were then equilibrated with the 0.4 M glucose solution for ;30 min. The sectioned samples were then frozen as described below for the two-step freezing experiments. Ischemic time of the liver on ice was always ,2.5 h. Two-Step Low-Temperature Microscopy Freezing Methods Sectioned liver samples were frozen by one of the three following methods, described previously (20). Slam freezing. Five to six tissue samples were placed on a microscope microslide and then slam-frozen (.1000°C/min) by bringing the microslide into intimate contact with a highly polished copper block which, until use, was in thermal equilibrium with liquid nitrogen at 2196°C. The microslide with the frozen samples was then quickly transferred to a petri dish containing liquid nitrogen. While immersed in liquid nitrogen the samples were carefully de-
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FIG. 1. Schematic of the two-step freezing “directional cooling stage” and the projected thermal history of the sample: Step 1 shows the freezing of the tissue sample from a base temperature above freezing (T H) to an intermediate temperature below freezing (T L). Step 2 shows the freezing of the tissue sample at a very high cooling rate (slam cooling or B . 10008C/min).
tached from the microslide and stored in liquid nitrogen prior to processing via the freeze substitution process. The slam freezing process preserves the morphological structure of the tissue since the submicroscopic trapped intracellular ice crystals and the tissue space cannot be separated and are thus measured as one giving the fully hydrated dimensions of the cellular space. Two-step equilibrium freezing. Constant and controlled rates of freezing were obtained by cooling samples on a directional solidification stage (Fig. 1) built in our laboratory, based on the design of Rubinsky and Ikeda (22) and discussed in detail elsewhere (20). Briefly, the stage consists of two copper platforms fitted with type ‘T’ thermocouples (Omega Tech.
Corp., Stamford, CT, U.S.A.) which are differentially cooled by an internal liquid nitrogen flow and heated by an external foil heater. The copper platform temperatures are regulated by control of the heater voltages by Fuji digital controllers (Total Temperature Instrumentation, Williston, VT, U.S.A.). The sectioned tissue sample rested on a normal microslide which in turn rested on the two constant temperature bases. When freezing began, the microslide with the sample was propelled at a constant velocity V, across the gap D gap, from the base maintained at a temperature above freezing (T H) to the base maintained at an intermediate temperature below freezing (T L). The cooling rate within the sample was maintained by control-
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ling the temperature of the two bases of the stage, the velocity of the microslide, and the distance of the gap, i.e., B (8C/min) 5 (T H 2 T L/D gap) z V. For equilibrium freezing, the specimens were cooled at 2°C/min from 10°C to preset end temperatures of 24, 26, 28, 210, and 220°C, respectively. Upon reaching the low-temperature base, the samples were allowed to equilibrate for 10 min. Tissue samples were then slam-frozen and stored in liquid nitrogen prior to processing via the freeze substitution process. Two-step dynamic freezing. For dynamic freezing, liver tissue samples were cooled using the directional solidification stage at a cooling rate of 5°C/min from 10°C to end temperatures of 24, 26, 28, 210, and 220°C for control tissue (absence of glucose) and to 26, 28, 210, and 220°C for tissue equilibrated with 0.4 M glucose. The presence of 0.4 M glucose in the medium did not allow for ice to be consistently nucleated on the directional cooling stage at temperatures above 26°C; thus experimental data could not be obtained at 24°C for tissue equilibrated with 0.4 M glucose. Upon reaching the end temperature, the samples were immediately slam-frozen (with no time for equilibration at the end temperature) and stored in liquid nitrogen prior to processing via the freeze substitution process. Since the samples were immediately slam-frozen upon reaching the end temperature, the morphological structure of the tissue is preserved at the instant of freezing. In contrast, the two-step equilibrium freezing protocol allows the tissue cells to dehydrate at the chosen end temperature. Thus the two-step equilibrium freezing micrographs show an enlarged vascular/extracellular ice crystals when compared to the two-step dynamic freezing micrographs at the same end temperatures. Freeze Substitution The freeze substitution protocol involved suspending the samples in 1 ml of freeze substitution medium (2g OsO 4 in 100 ml acetone) in a freeze substitution device (Bal-Tec, Middlebury, CT, U.S.A.). The protocol required holding the device at 290°C for 24 h, 260°C for 2 h, 230°C for 2 h, and 0°C for 30 min,
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before equilibrating to room temperature (20). During this process the acetone replaced the ice in the sample, and the osmium fixed the lipid or membranous structure (12, 21). Embedding and sectioning. After freeze substitution, the samples were dehydrated at room temperature in 10 –20 ml of 100% acetone before being infiltrated with Quetol resin (Ted Pella Inc., Redding, CA, U.S.A.). The resulting hard blocks of resin-embedded tissue were then sectioned on a Reichert ultracut microtome (Leica, Vienna, Austria) to a thickness of about 0.5 mm. The tissue sections were mounted on microslides, heated on a hot plate at 50°C, and then stained with toluidine blue in preparation for image analysis (20). Photomicrography and image analysis. Photomicrographs of the tissue sections were obtained by imaging through a Newvicon camera (Hamamatsu-Photonics, Bridgewater, NJ, U.S.A.) attached to a Nikon Labophot light microscope (Nikon Inc., Melville, NY, U.S.A.) and digitized on a workstation computer with a frame grabber (SGI, Mountainview, CA, U.S.A.). Image analysis of the photomicrographs was then performed to obtain the cellular and vascular/ extracellular volumes using NIH Image software (NIH, Bethesda, MD, U.S.A.), as described previously (20). In the image analysis, ice regions are seen as white spaces and are generally assumed to be vascular/extracellular in nature. In viewing micrographs of dynamically cooled (5°C/min) tissue, white spaces that were of equal or smaller diameter than a typical frog hepatocyte cell diameter ;16 mm were considered to be intracellular ice. In an attempt to account for intracellular ice crystals, these particular white spaces were darkened and counted with the intracellular volume. These intracellular ice spaces were found to be significant in the analysis of 5°C/min dynamic freezing micrographs (both in the presence and in the absence of glucose). Model 1: Standard Krogh Cylinder Geometry Dimensions The standard Krogh cylinder dimensions (Fig. 2A), DX, the average (shortest) distance
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tissue (20). The cellular space with volume V is represented in the Krogh model as the box sur2 rounding the cylinder (LDX 2 2 p r vo L) and A c is the effective membrane surface area available for water transport (5 2 p r voL), where r vo is the original vascular radius (assumed to be a constant during the water transport process). The length of the Krogh cylinder, L, was selected by imposing the constraint that the tissue cells must have the same overall cellular volume as that of the isolated frog hepatocyte cell (;2145 mm 3, based on a cell diameter of 16 mm). Differential Scanning Calorimetry Experiments
FIG. 2. (A) Schematic of the standard Krogh cylinder geometry (Model 1). The dimensions are DX, the distance between adjacent sinusoid centers (64 6 17.9 mm); r vo, the original sinusoid (vascular) radius (18.4 6 5.2 mm); and L, the axial length of the vascular cylinder (0.71 mm). (B) Schematic of the modified Krogh cylinder geometry (Model 2): The Krogh model has been modified to account for multiple cells between vascular cylinders. The dimensions are DX, the distance between adjacent sinusoid centers (64 6 17.9 mm); r 1 , the original sinusoid (vascular) radius (18.4 6 5.2 mm); r 2 , inner cell border radius (28.7 6 8.0 mm); and L, the axial length of the vascular cylinder (0.71 mm).
between vascular sinusoids (center– center) and r vo, the vascular (sinusoid) radius, were obtained by averaging at least 25 measurements made per slam-frozen tissue micrograph (n 5 4 images) and correlating these with the in vitro vascular dimensions of the slam-frozen control
Sample preparation. Experiments were performed using freshly excised liver tissue slices as previously described for the two-step freezing experiments. The liver tissue was cut into small pieces using a razor blade and patted externally using a blotting paper. The liver tissue slices (;1–1.5 mm 3 or ;1–1.5 mg) were then placed in standard aluminum DSC sample pans (Perkin–Elmer Corporation, Norwalk, CT, U.S.A.) and a small drop (,8 ml) of isotonic phosphate-buffered saline solution (PBS) (Celox, Inc.) was added to keep the tissue in an isotonic environment. A natural ice-nucleating agent, Pseudomonas syringae (ATCC, Rockville, MD, U.S.A.), was added (0.5 to 1 mg) before the pans were sealed and reweighed to measure the total sample weight (#10 mg). The ice-nucleating agent always nucleated the vascular/extracellular space at temperatures $22.5°C in order to avoid damaging intracellular ice formation, which occurs predominantly at temperatures below 25°C in most cell systems (28). DSC dynamic cooling protocol. The DSC measures heat released during phase changes as a function of time and temperature. Our lab has developed a DSC cooling protocol to measure water transport during freezing of a cell suspension (Epstein–Barr virus transformed, EBVT human lymphocyte) and a normal mammalian tissue (rat liver) system (8, 9). To ensure the accuracy and repeatability of the experimental data, the limitations of the DSC machine were
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FIG. 3. Heat flow vs temperature (DSC) curve: The superimposed heat flow thermograms obtained at a cooling rate of 5°C/min are shown. The bottom and top curves correspond to the heat flow thermograms measured in Step 3 and Step 7 of the DSC cooling protocol, respectively. A secondary heat release is also evident during the initial cooling (Step 3) run between 214 and 216°C. The heat flow (mW/mg) is plotted along the y axis and the subzero temperatures (°C) are plotted along the top x axis. The negative axis for the heat flow on the x axis implies an exothermic heat release in the DSC sample.
studied and a set of calibration and control experiments was performed, as previously described (8). In this study DSC experiments were conducted at two cooling rates (2 and 5°C/min) on R. sylvatica liver tissue slices (control tissue without glucose). A brief outline of the DSC dynamic cooling protocol is presented here: Step 1: The sample (liver slices 1 PBS 1 0.5 to 1 mg of P. syringae) initially at 4°C was cooled at 5°C/min until the vascular/extracellular ice nucleated (usually around 22.5°C). This nucleation temperature was approximately 1 to 1.5°C higher than that found in EBVT lymphocyte cell suspensions (8), normal mammalian (rat) liver slices in PBS (9), and tumor (rat prostate) tissue slices in PBS (10). The higher nucleation temperature might be due to the presence of ice nucleating bacteria (7) or proteins (25–27) in the frog liver tissue slice. Step 2: At
the time of nucleation the sample was manually triggered to thaw at a warming rate (10°C/min) such that T ph (20.53°C) was reached (but not overshot) and ice remained in the extracellular solution. Step 3: The sample was then cooled to 250°C at the specified cooling rate (2 or 5°C/ min) and the initial heat release due to the medium mixed with osmotically active cells was measured (q initial) (see Fig. 3, for 5°C/min). A secondary heat release shown between 216 and 214°C in Fig. 3 was found at a cooling rate of 5°C/min, but not at the slower cooling rate of 2°C/min. This secondary heat release was interpreted as trapped intracellular water changing phase inside the cells as will be described later. Step 4: The sample was reequilibrated at 20.53°C by thawing at 100°C/min. Step 5: To help differentiate between the heat released by the medium and the intracellular fluid in Step 3,
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the liver cells were rendered osmotically inactive by cooling the sample at a high cooling rate (200°C/min) down to 250°C. Step 6: Step 4 was repeated. Since Step 5 resulted in all the cells having compromised membranes, the intracellular water, proteins, and salts now are continuous with the extracellular solution (no membrane barriers exist), as previously suggested by Ko¨rber et al. (17). Step 7: The sample was then cooled to 250°C at the specified cooling rate (2 or 5°C/min) to measure the final heat release due to lysed or osmotically inactive cells mixed with medium (q final) (see Fig. 3, for 5°C/ min). Translation of heat release to cell volume data. The total difference in the heat release between the two cooling runs (Step 3 and Step 7) is denoted as Dq dsc (5 q initial 2 q final) and is shown in Fig. 3 for a cooling rate of 5°C/min. The fractional difference up to a subzero temperature, T, is denoted by Dq(T) dsc (5 q(T) initial 2 q(T) final ). The measurements of interest, Dq dsc and Dq(T) dsc, were obtained using the DSC-7 (Perkin–Elmer) software after selection of an appropriate baseline. These differences in heat release can be related to the changes in the cell volume as previously shown (8 –10), V o 2 V~T! Dq~T! dsc 5 Vo 2 Vb Dq dsc
[1]
We can write a simplified equation to measure water transport data from the DSC measured heat releases as V~T! 5 V o 2
Dq~T! dsc z ~V o 2 V b!. Dq dsc
[2]
Thus, Eq. [2] can be used to generate the water transport data from DSC heat release readings. The unknowns needed in Eq. [2] are the Krogh cylinder (Model 1) dimensions (L, DX, r vo), the isotonic cell volume, V o, and the osmotically inactive cell volume, V b, which were all obtained using two-step freezing results (either from slam-frozen images or two-step equilibrium cooled micrographs).
RESULTS AND DISCUSSION
Model 1: Standard Krogh Cylinder Geometry Dimensions The dimensions of the Krogh cylinder (Model 1, Fig. 2A), DX, the average (shortest) distance between vascular sinusoids (center– center) and r vo, the vascular (sinusoid) radius were chosen to correlate with the in vitro vascular dimensions of the slam-frozen control tissue (20). Using NIH Image software it was first determined that 74% of the frog liver tissue was cellular space, while the remaining 26% was vascular/extracellular space and mean DX was 64 6 17.9 mm based on an average of at least 25 measurements per slam-frozen micrographs from three different animals. The vascular (sinusoid) radius, r vo, was found using two different methods. The first was to measure the vascular radius using NIH Image software and a value of 12.2 6 5.3 mm was obtained. Slam tissue images showed vascular spaces to be oblong in shape. This was because the section cut was not always exactly perpendicular to the vascular sinusoids; thus the direct measurement of vascular radius was not considered accurate. The second method was by direct sterological area measurements of cellular and extracellular spaces in the slam tissue images. The vascular area of the Krogh cylinder (Model 1) can be 2 and since 26% of the total expressed as p r vo area is vascular/extracellular, one could express 2 p r vo 5 0.26 z DX 2 and solve for r vo. Using this method, r vo was found to be 18.4 6 5.2 mm. The radius calculated using the second method was deemed to be more accurate and hence was used in this analysis. The length of the Krogh cylinder, L, was selected by imposing the constraint that the cells in intact liver tissue must have the same overall cell volume as that of isolated hepatocytes which is ;2145 mm 3 (based on a frog hepatocyte cell diameter of 16 mm obtained from the projected area of in vivo hepatocyte cells). Hence the dimensions of the Krogh cylinder (Model 1) were found to be: distance between adjacent vascular sinusoids, DX 5 64 6 17.9 m m, original vascular radius,
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initial cell volumes of the cells adjacent to vasculature and embedded cells were equal; i.e., L z 2 (DX 2 2 p r 22 ) 5 L z ( p r 22 2 p r vo ). Using the dimensions obtained earlier (DX 5 64 6 17.9 m m and r 1 5 r vo 5 18.4 6 5.2 mm), the outer radius r 2 was determined to be 28.7 6 8.0 mm. The modified Krogh cylinder geometry (Fig. 2B) is described further, in Part II of this study (11). Two-Step Equilibrium Freezing Response: Boyle–van’t Hoff Plot FIG. 4. R. sylvatica liver anatomy: A magnified section of the slam-frozen image shows that more than two hepatocyte cells can be found between adjacent vascular sinusoids. Approximately 24% of the frog hepatocyte cells were found to be at least one cell removed from vascular sinusoids.
r vo 5 18.4 6 5.2 mm, and length of the Krogh cylinder, L 5 0.71 m m. Model 2: Modified Krogh Cylinder Geometry Dimensions In analyzing the slam tissue images (Fig. 4), counts were also made of the number of cells spanning the distance between adjacent vascular sinusoids. Of a total of 87 individual hepatocyte cells analyzed in the slam tissue images (n 5 4 images from four different frogs) 21 were found to be at least one cell removed from the vascular spaces or were completely bordered by other cells. Thus our measurements suggest that ;24% (21 of 87) of frog hepatocyte cells were not in direct contact with vasculature and although area measurements can be deceiving and some vascular sinusoids may exist over or under the cells analyzed, this measurement is taken as strong evidence that some percentage of the frog hepatocyte cells are not in direct contact with vascular spaces. To account for these embedded cells the Krogh cylinder geometry, Model 1, (Fig. 2A) was modified to Model 2 (Fig. 2B) which accounts for cells bordering a vascular conduit (adjacent) and cells which do not (embedded). The embedded cell radius r 2 was determined such that the
The equilibrium (infinitely slow) cooling behavior and the osmotically inactive cell volume (V b) of R. sylvatica liver cells were obtained using the two-step equilibrium freezing method. Figure 5 shows the micrographs from liver tissue cooled slowly on the directional stage and then equilibrated for 10 min at an end temperature of 0, 24, 26, 28, 210, and 220°C before slam freezing to 2196°C. Tissue cooled to 0°C before slam freezing is used as the reference (Fig. 5, slam) for comparison of equilibrium freezing effects at the other five temperatures. It can be seen that cell volume progressively decreases whereas extracellular ice content increases with decreasing freezing temperature. A Boyle–van’t Hoff (BVH) plot (Fig. 6) was constructed by examining the cellular volumes depicted in the freeze-dehydrated tissue micrographs (Fig. 5), as previously described (20). The inverse of the osmolality experienced by the tissue cells (Osm 5 DT/1.858, where DT 5 273.15 2 T, K) is plotted on the x-axis while the normalized equilibrium cell volume is plotted on the y-axis. By extrapolating to the volume at infinite osmolality, the osmotically inactive cell volume, V b 5 0.4 z V o, was obtained. Two-Step Dynamic Freezing Response Figure 7 shows the control liver tissue (absence of glucose) response to the two-step dynamic freezing method. The first image, on the top left-hand corner, is of the tissue slam-frozen (.1000°C/min) from 0°C, before freeze dehydration of the cells began. The next five images are freeze-substituted micrographs of tissue fro-
FIG. 5. Two-step equilibrium-cooled light micrographs of R. sylvatica liver tissue: Tissue slices were cooled on the directional stage at 2°C/min and equilibrated for 10 min at various end temperature (0, 24, 26, 28, 210, and 220°C) followed by slam-freezing to 2196°C. In the micrograph of control tissue cooled to 0°C, the Rs indicate the trapped red blood cells in vascular spaces, surrounded by cells (C). In the other micrographs, the optically dense spaces (stars) and the light areas (arrows) correspond to tissue components and extracellular ice crystals, respectively. Bar in the slam-frozen image, 50 mm.
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FIG. 6. Boyle–van’t Hoff plot constructed at constant subzero temperatures in the presence of external ice for R. sylvatica liver tissue: The filled circles represent the average “equilibrium” volumetric data at various end temperatures (0, 24, 26, 28, 210, and 220°C) obtained using the micrographs shown in Fig. 5. The linear curve extrapolates to infinite osmolality, giving the osmotically inactive volume of the R. sylvatica liver cells as 0.4V o. The error bars represent standard deviations in the data (n 5 4).
zen to various end temperatures, 24, 26, 28, 210, and 220°C (as denoted in the top lefthand corner of each image) before immediate slam freezing. In the 0°C tissue (slam-frozen image, Fig. 7A), individual hepatocyte cells are discernible (indicated by Cs) surrounding the trapped red blood cells in vascular spaces (indicated by Rs). As freezing begins the cells begin to dehydrate and water diffuses out of the cells and into the vasculature. As this happens, the cells begin to coalesce and flatten out (Fig. 7B–F, stars), while the vasculature expands (Fig. 7B–F, arrows). In comparison to the equilibrium-cooled micrographs (Fig. 5), the size of the vascular ice crystals is in general smaller at comparable end temperatures in the dynamically cooled micrographs (Fig. 7). The presence of smaller ice crystals suggests that a higher fraction of intracellular water remains within the cells and formed intracellular ice, as will be described below. Cellular dehydration (and therefore water transport from the cells) appears to cease at 210°C, as no further reduction in
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projected cell area is discernible by image analysis in the micrograph cooled to 220°C. Figure 8 shows the response of R. sylvatica liver tissue slices equilibrated with 0.4 M glucose using the two-step dynamic freezing method. The first image, on the top left-hand corner, is of the tissue slam-frozen (.1000°C/ min) from 0°C, before freeze dehydration began. The next five images are freeze-substituted micrographs of tissue frozen to various end temperatures, 26, 28, 210, and 220°C. As before, in the tissue cooled only to 0°C and slam-frozen, the Rs in Fig. 8A indicate the trapped red blood cells in vascular spaces, surrounded by cells (indicated by Cs) and as freezing progresses the cells begin to dehydrate and coalesce together (Fig. 8B–E, dark spaces, denoted by stars) and the vasculature expands (Fig. 8B–E, arrows). As mentioned earlier, no experimental data could be obtained at 24°C, as the presence of 0.4 M glucose in the medium did not allow for ice to be consistently nucleated on the directional cooling stage at temperatures above 26°C. Note that ice crystals are in general smaller in Fig. 8 (presence of 0.4 M glucose) than in Fig. 7 (absence of glucose) for comparable end temperatures (especially at temperatures #210°C). The presence of smaller ice crystals likely indicates that increased incidence of intracellular ice is occurring in the presence of 0.4 M glucose than in its absence. As in the case of control tissue, cellular dehydration appears to cease at 210°C in the presence of 0.4 M glucose, as no further reduction in the cell volumes is discernible by image analysis in the micrograph cooled to 220°C. Estimation of Intracellular Ice during TwoStep Dynamic Cooling In viewing the micrographs of the dynamically cooled tissue at end temperatures, #28°C (in control tissue, Fig. 7) and #26°C (in tissue equilibrated with 0.4 M glucose, Fig. 8), white voids (ice crystals) were found that were of smaller diameter than the typical frog hepatocyte cell (;16 mm). These ice crystals likely formed and grew during the dynamic cooling process and not during the slam-frozen step
FIG. 7. Two-step dynamically cooled light micrographs of R. sylvatica liver tissue: Tissue slices were cooled on the directional stage at 5°C/min to various end temperature (0, 24, 26, 28, 210, and 220°C) immediately followed by a second slam-freezing step to 2196°C. As before, in the control micrograph (0°C) the Rs indicate the trapped red blood cells in vascular spaces, surrounded by cells (C) while in the remaining micrographs the optically dense spaces (stars) and the light areas (arrows) correspond to tissue components and extracellular ice crystals, respectively. Bar in the slam-frozen image, 50 mm.
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FIG. 8. Two-step dynamically cooled light micrographs of R. sylvatica liver tissue: Tissue slices were equilibrated with 0.4 M glucose and cooled at 5°C/min to various end temperatures (0, 26, 28, 210, and 220°C) immediately followed by a second slam-freezing step to 2196°C. As before, in the control micrograph (0°C) the Rs indicate the trapped red blood cells in vascular spaces, surrounded by cells (C) while in the remaining micrographs the optically dense spaces (stars) and the light areas (arrows) correspond to tissue components and extracellular ice crystals, respectively. Bar in the slam-frozen image, 50 mm.
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FIG. 9. Micrographs of R. sylvatica control liver tissue cooled at 5°C/min to 220°C showing intracellular ice crystals: Intracellular ice formation in dynamic freezing (5°C/min) was accounted for by measuring white (assumed vascular) spaces in the light micrograph (A). Those spaces with characteristic size that measured to be less than the diameter of a typical hepatocyte cell (;16 mm) were assumed to be intracellular ice, and these spaces were darkened (B) and included as contributing to the cellular volume. (A) Scale bar, 25 mm.
which leaves only submicroscopic ice crystals that cannot be distinguished from the tissue spaces. Since the vascular sinusoids had a typical radius twice that of the typical hepatocyte cell (r vo 5 18.4 mm vs cell radius 5 8.0 mm), these white voids (#16 mm in the largest dimension) were assumed to represent intracellular ice formed during dynamic cooling. These areas were then filled in black (using NIH Image software) to be counted as cellular space. Figure 9A shows a micrograph of control tissue cooled dynamically at 5°C/min to 220°C and Fig. 9B shows the same micrograph with intracellular crystals (white voids, #16 mm) filled in as gray, to be counted as cellular spaces. This modification led to an increase in the nondimensional cell volume of ;5% at 28°C and ;10% at 220°C. This suggests that the anatomy of the frog liver prevents a total dehydration in all of the cells (at 5°C/min) and a fraction of the intracellular water is trapped inside the cell once water transport ceases (at approx. 210°C as seen in Figs. 7 and 8) and changes into intracellular ice on sufficient supercooling. In support of the above interpretation, the DSC measured a secondary heat release while cooling control tissue at 5°C/min at 214 to 216°C (as shown in Fig. 3). This secondary peak was measured and translates to ;20% of the heat release due to the total water volume, a larger fraction than the ;10% increase seen in
the dynamically cooled micrographs (as discussed above). Note that the secondary heat release occurs after water transport has ceased (T , 2108C) and hence the biophysical events (water transport and intracellular ice formation) are decoupled. Therefore, while translating the 5°C/min DSC heat release readings into volumetric shrinkage data (between 0 and 210°C) using Eq. [2], an end volume of 0.6 z V o (instead of V b or 0.4 z V o) was used (see Fig. 10B). Since the DSC did not measure a secondary heat release when cooled dynamically at 2°C/min, the osmotically inactive cell volume (V b or 0.4 z V o) was used in Eq. [2] to translate the DSC heat release readings at 2°C/min to volumetric shrinkage data (see Fig. 10B). Volumetric Shrinkage Response with and without Glucose The dynamic water transport data for R. sylvatica control liver tissue cells cooled dynamically at 2°C/min (DSC data) and at 5°C/min (DSC and two-step dynamic data) are shown in Figs. 10A– 10C. In Fig. 10A, the filled and open circles represent the two-step dynamic water transport data, with and without accounting for the effect of intracellular ice crystals (i.e., with and without “blackening” out the white voids less than the typical frog hepatocyte cell diameter, ;16 mm). In Fig. 10B, the open triangles and open squares represent the 5 and 2°C/min dynamic DSC water
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FIG. 10. Temperature versus volumetric responses of the cells in R. sylvatica liver tissue: The Krogh cylinder (Model 1) simulated equilibrium volumetric response (---) is shown for reference, in all the figures. The error bars represent standard deviations in the data (n 5 3 for two-step and n 5 6 for DSC data). (A) The dynamic two-step water transport data in R. sylvatica control liver tissue is shown, with (F) and without (E) accounting for the effect of intracellular ice. (B) The open triangles and open squares represent the 5 and 2°C/min dynamic DSC water transport data, respectively. (C) A comparison of the data shown in (A) and (B). The symbols and the data are the same as in (A) and (B). (D) The two-step dynamic water transport data in R. sylvatica liver tissue equilibrated with 0.4 M of glucose is shown, with (■) and without (h) accounting for the effect of intracellular ice.
transport data, respectively. A good agreement is found between the two-step dynamic water transport data accounting for the presence of intracellular ice crystals (filled circles) and the DSC data (open triangles) at a cooling rate of 5°C/min, as shown in Fig. 10C. The data were found to be statistically similar with a confidence level of $92.5% (by Student’s t test). The dynamic water
transport data for frog liver tissue cells cooled at 5°C/min in the presence of 0.4 M glucose are shown in Fig. 10D. The filled and open squares represent the two-step dynamic water transport data, with and without accounting for the effect of intracellular ice crystals. The Krogh cylinder (Model 1)-predicted equilibrium cooling response (cells cooled infinitely slowly) in the absence
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(Figs. 10A–C) and presence of 0.4 M glucose (Fig. 10D) is also shown for reference as a dashed line. R. sylvatica Liver Freeze Response It should be pointed out that the cooling rates (2 and 5°C/min) and the end temperatures (0 to 220°C) investigated in this study are not physiologically relevant. Frogs in nature would experience much slower cooling rates (;1–2°C/h) and also much milder end temperatures (.28°C). The wood frogs in nature will, therefore, dehydrate fully to the equilibrium cooled end volume at any temperature, as shown by the two-step equilibrium cooled micrographs (Fig. 5) and the normalized equilibrium volumes in the Boyle–van’t Hoff plot (Fig. 6), obtained in this study. In addition to equilibrium behavior, this study determined the volumetric shrinkage data (water transport) for frog liver tissue (hepatocyte) cells under “nonequilibrium” (or dynamic) cooling conditions at a cooling rate of 5°C/min. In Part II of this study (11), the experimentally determined dynamic water transport data (shown in Fig. 10) is fit to a mathematical model of water transport, to predict the water permeability parameters of R. sylvatica liver tissue cells in the presence and absence of CPA (glucose). R. sylvatica Liver Architecture: Comparison to Mammalian (Rat) Liver One of the aims of this study was to study the in vitro architecture of R. sylvatica frog liver tissue and to measure its characteristic dimensions (Krogh cylinder dimensions). These can then be compared to those of the mammalian (rat) liver (20) to provide insight into how the liver structure of a freeze-tolerant amphibian differs from that of a freezeintolerant mammal (rat). This study found that the structural makeup of the two livers was remarkably different: (i) ;26% of the frog liver was vascular/extracellular space vs ;15% in rat liver; (ii) the radius of the vascular sinusoids in the frog were approximately five times larger than in the rat (18.4 vs 3.8 mm); (iii) the average distance between
the sinusoids in the frog was approximately three times larger than in the rat (64 vs 22 mm); (iv) the diameter of the frog hepatocytes was slightly smaller than the rat hepatocytes (16 vs 21 mm); and (v) ;24% of the cells in the frog liver were not directly adjacent to vascular spaces as opposed to 0% of rat liver cells. An extensive literature search was performed to check if any previous studies have reported the morphology/architecture of the R. sylvatica liver tissue observed in this study. Several excellent review articles and studies have been presented in the literature on the function, development, and morphology of the liver of amphibians and vertebrates, including Bennett and Glenn (1), Beresford and Henninger (2), Elias (13), Godula (14), Gumucio (15), Jones (18), Moore (19), and Spornitz (24). However, we were unable to find any previous reference to the observed morphology/architecture of the R. sylvatica liver tissue, obtained in this study. Preliminary results on Rana pipiens suggest that the architecture of this non-freeze-tolerant frog liver is very similar to R. sylvatica (unpublished data). CONCLUSIONS
This study investigates the water transport characteristics during freezing in the liver tissue of the freeze-tolerant wood frog R. sylvatica in the presence and absence of glucose. Experiments were performed using two-step equilibrium and dynamic freezing methods as well as a differential scanning calorimetry technique. Following the two-step (equilibrium and dynamic) freezing protocol, the frozen tissue slices were freeze substituted and embedded in Quetol resin for sectioning on a microtome. Stereological analysis of the slam-frozen (.1000°C/min) results led to the measurements that 74% of the control tissue is cellular (26% extracellular–interstitial and vascular); osmotically inactive cell volume, V b , is 0.4 z V o ; and the Krogh cylinder (Model 1) dimensions are DX 5 64 m m, r vo 5 18.4 mm, and L 5 0.71 m m (assuming the typical diameter of the frog hepatocyte cells is 16
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mm). In addition, stereological measurements suggest that ;24% of the frog hepatocyte cells are not in direct contact with the vasculature (as opposed to 0% in mammalian rat liver tissue). A parallel study was also done using the DSC in which control liver tissue was cooled dynamically at 2 and 5°C/min. The two-step dynamic freezing micrographs and the measured heat releases from the DSC were used to obtain the volumetric shrinkage data during freezing. The DSC technique confirmed that R. sylvatica cells in control liver tissue do not dehydrate completely when cooled at 5°C/min but do so when cooled at 2°C/min. It was previously shown that the mammalian (rat) liver tissue cells undergo complete cellular dehydration when cooled at 5°C/min (9, 20). A modified Krogh cylinder (Model 2) was constructed to account for the architecture with more than one cell between vascular spaces. In this model a second radius, r 2 5 28.7 m m, was defined (in addition to the original sinusoid radius, r vo 5 18.4 mm, defined above) as the radius of the membrane between the adjacent cells (directly adjacent to vascular spaces) and embedded cells (removed from vascular spaces). In Part II of this study (11), a mathematical model of water transport is presented and fit to the experimentally determined water transport data to obtain the biophysical permeability parameters of R. sylvatica liver tissue cells in the presence and absence of CPA (glucose). ACKNOWLEDGMENTS This work was supported by a grant from the National Science Foundation (NSF-BES 9703326) and a grant from the Whitaker Foundation to J.C.B. REFERENCES 1. Bennett, T. P., and Glenn, J. S. Fine structural changes in liver cells of Rana catesbeiana during natural metamorphosis. Dev. Biol. 22, 535–560 (1970). 2. Beresford, W. A., and Henninger, J. M. A tabular comparative histology of the liver. Arch. Histol. Jpn. 49, 267–281 (1986). 3. Cai, Q., Greenway, S. C., and Storey, K. B. Differential regulation of the mitochondrial ADP/ATP translocase gene in wood frogs under freezing stress. Biochim. Biophys. Acta 1353, 69 –78 (1997).
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4. Cai, Q., and Storey, K. B. Freezing-induced genes in wood frog (Rana sylvatica): Fibrinogen upregulation by freezing and dehydration. Am. J. Physiol. 272, R1408 –R1492 (1997). 5. Costanzo, J. P., Lee, R. E., Jr., and Lortz, P. H. Physiological responses of freeze-tolerant and -intolerant frogs: clues to evolution of anuran freeze tolerance. Am. J. Physiol. 265, R721–R725 (1993). 6. Costanzo, J. P., Lee, R. E., and Wright, M. F. Cooling rate influences cryoprotectant distribution and organ dehydration in freezing wood frogs. J. Exp. Zool. 261, 373–378 (1992). 7. Costanzo, J. P., and Lee, R. E. Mini review: Ice nucleation in freeze-tolerant vertebrates. Cryo-Lett. 17, 111–118 (1996). 8. Devireddy, R. V., Raha, D., and Bischof, J. C. Measurement of water transport during freezing in cell suspensions using a differential scanning calorimeter. Cryobiology 36(2), 124 –155 (1998). 9. Devireddy, R. V., and Bischof, J. C. Measurement of water transport during freezing in mammalian liver tissue. II. The use of differential scanning calorimetry. ASME J. Biomech. Eng. 120(5), 559 –569 (1998). 10. Devireddy, R. V., Smith, D. J., and Bischof, J. C. Mass transfer during freezing in rat prostate tumor tissue. AIChE J. 45(3), 639 – 654 (1999). 11. Devireddy, R. V., Barratt, P. R., Storey, K. B., and Bischof, J. C. Liver freezing response of the freezetolerant wood frog Rana sylvatica, in the presence and absence of glucose. II. Mathematical modeling. Cryobiology 38, 327–338 (1999). 12. Echlin, P. “Low Temperature Microscopy and Analysis,” Chapters 3 and 7, Plenum, New York/London, 1992. 13. Elias, H. Liver morphology. Biol. Rev. 30, 263–310 (1955). 14. Godula, J. Quantitative investigations of cellular organelles from the liver of amphibia: Xenopus laevis (Daudin) and Ambystoma mexicanum (Cope). Acta Biol. Cracov., Ser. Zool. 13, 225–236 (1970). 15. Gumucio, J. J. Hepatocyte heterogeneity: The coming of age from the description of a biological curiosity to a partial understanding of its physiological meaning and regulation. Hepatology 9, 154 –160 (1989). 16. Holden, C. P., and Storey, K. B. Signal transduction, second messenger, and protein kinase responses during freezing exposures in wood frogs. Am. J. Physiol. 271, R1205–R1211 (1996). 17. Ko¨rber, C. H., Englich, S., and Rau, G. Intracellular ice formation: Cryomicroscopical observation and calorimetric measurement. J. Microsc. 161, 313–325 (1991). 18. Jones, A. L. Anatomy of the normal liver. In “Hepatology: A Textbook of Liver Disease” (D. Zakim
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24. Spornitz, U. M. Studies of the liver of Xenopus laevis. III. The ultrastructure and the glycogen content of the developing liver. Anat. Embryol. 154, 1–25 (1978). 25. Storey, K. B., and Storey, J. M. Freeze tolerance in animals. Physiol. Rev. 68(1), 27– 84 (1988). 26. Storey, K. B., Bischof, J. B., and Rubinsky, B. Cryomicroscopic analysis of freezing in liver of the freeze-tolerant wood frog. Am. J. Physiol. 263, R185–R194 (1992). 27. Storey, K. B., and Storey, J. M. Cellular adaptations for freezing survival by amphibians and reptiles. In “Advances in Low-Temperature Biology” (P. L. Steponkus, Ed.), Vol. 2, pp. 101–129, JAI Press, London, 1993. 28. Toner, M. Nucleation of ice crystals in biological cells. In “Advances in Low Temperature Biology” (P. L. Steponkus, Ed.), Vol. 2, pp. 1–52, JAI Press, London, 1993.