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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 60:1087-1098 (2005)
© 2005 The Gerontological Society of America

Cultured Murine Dermal Fibroblast-Like Cells From Senescence-Accelerated Mice as In Vitro Models for Higher Oxidative Stress Due to Mitochondrial Alterations

Yoichi Chiba1,3, Yoshinori Yamashita2,3, Masaki Ueno4, Hiromi Fujisawa5, Kazunori Hirayoshi6, Ken-ichi Hohmura6, Hidekazu Tomimoto1, Ichiro Akiguchi1, Mamoru Satoh3, Atsuyoshi Shimada3 and Masanori Hosokawa3,

1 Department of Neurology, Graduate School of Medicine
2 Laboratory of Aging Study, Graduate School of Human and Environmental Studies, Kyoto University, Japan.
3 Department of Pathology, Institute for Developmental Research, Aichi Human Service Center, Japan.
4 Department of Inflammation Pathology, Faculty of Medicine, Kagawa University, Japan.
5 Graduate School of Science and Technology, Niigata University, Japan.
6 Department of Ultrastructural Research, Institute for Frontier Medical Sciences, Kyoto University, Japan.

Address correspondence to Masanori Hosokawa MD, PhD, Department of Pathology, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya-cho, Kasugai, Aichi 480-0392, Japan. E-mail: hosokawa{at}inst-hsc.jp


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
The senescence-accelerated mouse is a model for senescence acceleration, a higher oxidative stress status, and age-associated disorders. We studied whether fibroblasts cultured from accelerated senescence-prone SAMP11 mice could be used as in vitro models for oxidative stress in senescence. Dichlorofluorescein and hydroethidine assays demonstrated that cells from SAMP11 mice produced more reactive oxygen species than did cells from accelerated senescence-resistant SAMR1 mice. These differences were not due to the defective induction of antioxidants. Double labeling with hydroethidine and MitoTracker Green revealed that most of the reactive oxygen species were generated within the mitochondria. Nonyl acridine orange and JC-1 assays showed an increase in the mass of the mitochondria, especially those with low membrane potential, in SAMP11 cells. Ultrastructurally, mitochondria with degenerative morphology were increased in SAMP11 cells with longer culture periods. These results suggest that cells from SAMP11 mice are useful models for spontaneous higher oxidative stress in vitro due to dysfunctional mitochondria.

SINCE Harman first proposed the "free-radical theory" of aging (1), oxidative damage to macromolecules has been considered an important factor in the senescence process. As an organism grows older, not only do its physiological functions decline, but it also becomes susceptible to disorders such as Alzheimer's disease, Parkinson's disease, and atherosclerosis. Oxidative damage to cells also plays a pivotal role in the pathogenesis of age-associated neurodegenerative and vascular diseases (2).

The senescence-accelerated mouse (SAM), originally established in our laboratory (3), is a group of related inbred strains consisting of accelerated senescence-prone (SAMP) and accelerated senescence-resistant (SAMR) mice. Each SAMP strain has strain-specific age-associated pathological phenotypes (4). Moreover, all SAMP strains exhibit accelerated senescence when compared with SAMR strains, although both strains show an equal rate of growth and sexual maturation (3,5). We therefore consider a set of SAMP and SAMR strains to represent a murine model for senescence acceleration (6).

There is an increasing amount of in vivo evidence suggesting that a higher oxidative stress status is closely related to degenerative pathologies and senescence acceleration in SAMP mice. Furthermore, several studies have suggested that mitochondrial functions are altered in young and old SAMP mice [for a review, see (6)]. Mitochondria are key organelles in oxidative damage and senescence (7,8), and are the main site for reactive oxygen species (ROS) production as well as targets of ROS attack (9). On the basis of previous studies, we proposed that SAMP mice strains represented spontaneous animal models for a higher oxidative stress status due to mitochondrial dysfunction (6). SAMP mice strains are not only useful in investigating the mechanisms responsible for senescence, but also in elucidating the pathogenesis and treatment of age-dependent disorders (5).

To utilize this animal model to investigate senescence mechanisms in terms of molecular biology and biochemistry, it is necessary to show that the SAMP mice are also under a higher oxidative stress status in vitro. We devised a tissue culture method using murine dermal fibroblast-like (MDF) cells from neonates, and showed that cells from SAMP11 mice exhibited accelerated senescence/crisis in vitro when compared with cells from SAMR1 mice (10). Using this culture system, we demonstrated a higher lipid peroxide (LPO) content in primary cultured cells from SAMP11 mice than in cells from SAMR1 mice (11). We also showed that aminoguanidine, an inhibitor of diamine oxidase (12) and nitric oxide synthase (13), delayed the senescence/crisis and decreased the LPO content of SAMP11 cells to levels found in SAMR1 cells (11). This study suggested that oxidative stress contributes to in vitro senescence acceleration in SAMP11 cells. The assay system had an inherent drawback, however, in that it takes 2–3 months for primary cultured MDF cells to reach senescence/crisis. In addition, the LPO content from SAMP11 cells showed such a large variance that statistically significant differences between the LPO contents from SAMP11 versus SAMR1 cells could not be detected (11).

In this study, we sought to establish the usefulness of cultured MDF cells from SAMP11 mice as an in vitro model for oxidative stress in senescence by directly demonstrating that cells from SAMP mice produced more ROS starting from the early-passage period. To accomplish this aim, we performed fluorometric assays to evaluate ROS production in the cultured cells. Furthermore, to investigate the cause of the higher oxidative stress status, we evaluated the activity and expression of antioxidant enzymes (AOEs), glutathione (GSH) concentrations, and functional and ultrastructural changes in the mitochondria.


   METHODS
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Animals
SAMP11 and SAMR1 mice strains were used as experimental and control mice, respectively. SAMP11 mice have the shortest life spans among the SAM strains (median survival time: 489 days for males and 514 days for females), whereas SAMR1 mice have a normal life span (median survival time: 554 days for males and 587 days for females) (11). The two strains were reared under conventional conditions at 24 ± 2°C and 45 ± 5% humidity, and were maintained on a commercial diet (CE-2; Nihon CLEA, Tokyo, Japan) and tap water ad libitum. The mice were subjected to a 12-hour light cycle from 7:00 AM to 7:00 PM daily. All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals of Kyoto University, and the Institute for Developmental Research, Aichi Human Service Center.

Chemicals
JC-1 (5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide) and MitoTracker Green FM were purchased from Molecular Probes, Inc. (Eugene, OR). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO), unless otherwise stated. All chemicals were of culture grade or extra pure grade.

Cell Culture
MDF cells were isolated from the dorsal dermis of newborn littermates 18–24 hours after birth, as previously described (10). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM; NISSUI Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). The cells were seeded at a density of 3 x 104 cells/cm2 to 10 x 104 cells/cm2 in 75 cm2 culture flasks (Corning; Acton, MA) and propagated for 72–96 hours after primary seeding. The cells were harvested by trypsinization, counted to determine the population doublings (PDs), and replated onto culture dishes (Becton Dickinson & Co., Franklin Lakes, NJ) or glass bottom dishes (for confocal study; Asahi Technoglass Co., Tokyo, Japan) at a density of 10 x 104 cells/cm2. PDs at this point did not exceed 3. The cells were refed 24 hours after replating, and were maintained on plates until further use.

Evaluation of ROS Production by Fluorescent Probes
We used two fluorescent probes to measure the intracellular oxidative stress: dichlorofluorescein diacetate (DCFH-DA), a marker for the overall oxidative stress (14), and hydroethidine (HEt), or dihydroethidium, a specific probe for intracellular superoxide anions (15). Cultured MDF cells were stained with DCFH-DA (10 µM) or HEt (1 µM) on the 3rd, 5th, 7th, and 11th days after replating. Fluorescence images were obtained using an inverted confocal laser scanning microscope system (LSM410; Carl Zeiss Co. Ltd, Jena, Germany). The intensity of the laser beam and the photodetector sensitivity were kept constant to facilitate quantitative comparisons of the relative fluorescence intensities between experiments. Ten fields were chosen for analysis on a random basis and scanned only once to avoid photo-oxidation by exposure to the laser light. The fluorescence intensity of the oxidized fluorescent derivatives of DCFH-DA and HEt (dichlorofluorescein [DCF] and ethidium, respectively) per field (usually containing about 100 cells) was quantified using Scion Image software for Windows (Scion Corp., Frederick, MD), and was normalized to the cell density. Nuclear regions were excluded from the quantification of HEt-stained images, because the binding of ethidium to polynucleotides leads to extensive fluorescence enhancement (16).

Activity of AOEs and GSH Concentration
The activity of AOEs and the GSH concentration were determined on the 7th day after replating. Cultured MDF cells (approximately 1 x 107 cells for catalase and GSH peroxidase activity assays and 3 x 106 cells for GSH assay) were homogenized with a Potter apparatus and centrifuged for 15 minutes at 3000 g. Catalase activity was measured essentially according to the method of Beers and Sizer (17). Glutathione peroxidase (GPX) activity was measured by the method of Makino and colleagues (18). Cellular GSH concentrations were determined essentially according to the method of Tietze (19). The protein content was determined using Bradford's method (20).

Complementary DNA Probes
A probe for the18S ribosomal RNA (rRNA) was a gift from Dr. N. Hosokawa (Kyoto University, Japan). Specific AOE complementary DNA (cDNA) fragments were amplified from the total RNA isolated from the cells and tissues of SAM mice strains. The cDNA was synthesized and amplified from 1–1.5 µg of total RNA using Ready-To-Go reverse transcription–polymerase chain reaction beads (Amersham, Buckinghamshire, U.K.). A detailed description of the polymerase chain reaction primers and product sizes is provided in Table 1. The cDNA fragments were cloned into pT7Blue-2 T-vectors (Novagen, Inc., Madison, WI). The cDNA probes were obtained by digestion with the appropriate restriction enzymes, and were labeled with digoxigenin by a random priming technique using DIG-High Prime (Roche Diagnostics GmbH, Mannheim, Germany). We confirmed that these probes recognized a single RNA species (data not shown).


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  		Table 1. PCR Primer Sequences Used for RT-PCR.

 
Messenger RNA Expression of AOEs
Cytoplasmic RNA from cultured MDF cells was isolated using RNeasy Mini Kit (Qiagen, Valencia, CA) on the 3rd, 5th, and 7th days after seeding into the culture dishes according to the manufacturer's protocol. To investigate the changes in the amount of messenger RNA (mRNA) with time in culture, slot blots were prepared using a Bio-Rad apparatus (Bio-Dot SF; Bio-Rad Laboratories, Hercules, CA). Two micrograms of RNA was applied to each slot of the apparatus and blotted onto a nylon membrane (Hybond-N; Amersham). The membranes were hybridized with digoxigenin-labeled probes for 18 hours at 42°C, washed twice with 2 x saline-sodium citrate/0.1% sodium dodecyl sulfate at room temperature, and then twice with 0.5 x saline-sodium citrate/0.1% sodium dodecyl sulfate at 65°C. The hybridized probes were detected with anti-digoxigenin antibodies conjugated to alkaline phosphatase, and visualized with a chemiluminescent substrate, CSPD (Roche Diagnostics). Light emissions were recorded on Fuji SUPER HR-A30 x-ray film (Fujifilm Medical Co., Ltd., Tokyo, Japan), and the signal densities were quantified using Scion Image software. The signals were normalized to the 18S rRNA in each sample.

Double Labeling With HEt and MitoTracker Green FM
Cultured MDF cells were simultaneously stained with HEt and MitoTracker Green FM, a mitochondrial marker independent of the membrane potential. The cells were incubated with a working solution (1 µM HEt + 200 nM MitoTracker Green FM in DMEM) for 30 minutes at 37°C, washed twice, and then observed under an inverted fluorescent microscope system (IX70; Olympus, Tokyo, Japan) equipped with a digital CCD camera (C4742-95; Hamamatsu Photonics, Hamamatsu, Japan). Ethidium was excited at 510–560 nm, and the fluorescence emissions were filtered using a WIG filter (Olympus). MitoTracker Green FM was excited at 470–490 nm, and the fluorescence emissions were filtered using a NIBA filter (Olympus).

Evaluation of Mitochondrial Mass by 10-n-Nonyl Acridine Orange (NAO)
NAO (10-n-nonyl acridine orange) binds specifically to cardiolipin in the inner mitochondrial membrane independent of the membrane potential (21), and is used to monitor mitochondrial mass (22). Cultured MDF cells were incubated with NAO (0.5 µM in DMEM) at 37°C for 5 minutes. After two washes, the cells were observed under a confocal microscope. The area of green fluorescence was measured using Scion Image software.

Evaluation of Mitochondrial Membrane Potentials by JC-1
JC-1 is a fluorescent sensor of the mitochondrial inner membrane potential (23–25). Cultured MDF cells were stained on the 3rd, 5th, 7th, and 11th days after replating by incubating in the staining solution (15 µM in DMEM) for 10 minutes at 37°C. The cells were washed twice, and fluorescent images were obtained using the LSM410. The cells were excited at 488 nm, and the fluorescence emissions were filtered using a 515–560 nm bandpass filter (green fluorescence of the monomers representing mitochondria with low membrane potentials) or a 570 nm longpass filter (red fluorescence of the J-aggregates representing mitochondria with high membrane potentials). The intensity of the laser beam and the photodetector sensitivity were kept constant to facilitate quantitative comparisons of the relative fluorescence intensities between experiments. Ten fields were chosen for analysis on a random basis.

The JC-1 images were analyzed to estimate the mitochondrial function in a cell layer using MATLAB software for Windows (version 6.1.0.450, release 12.1; The MathWorks, Inc., Natick, MA). The color images were divided into red and green channels, and the fluorescence intensities of each pixel in each channel were expressed as values between 0 (dark) and 1 (bright). Pixels that exhibited a red color were defined as R ≥ 0.5 and R ≥ G, where R is the fluorescence intensity in the red channel and G is that in the green channel. Similarly, green pixels were defined as G ≥ 0.2 and G > R. These definitions were determined inductively by the correspondence between the composition of the RGB colors from a given pixel and its appearance on a computer monitor. The number of red or green pixels was counted, and the pixel number was considered to represent an index of the mass of the mitochondria in a given field.

Transmission Electron Microscopy
The ultrastructural changes of the mitochondria from cultured MDF cells were evaluated on the 5th, 7th, and 11th days after replating. Confluent cells were fixed with 1% osmium tetroxide in phosphate-buffered saline (PBS) (–), dehydrated with a graded ethanol series, and then embedded in epoxy resin Epon 812 (TAAB Laboratories Equipment Ltd., Berkshire, U.K.).

We also examined several organs from young and old SAMP8 and SAMP11 mice. After perfusion with PBS (pH 7.4), the PBS was replaced by 2.5% glutaraldehyde plus 2% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The brains, hearts, livers, and brown adipose tissues were dissected and immersed in the same fixative at 4°C for 6 hours, placed in a sucrose buffer (pH 7.4) at 4°C for 12–24 hours, and then sectioned. The sections were postfixed in 1% osmium tetroxide at 4°C for 1 hour, dehydrated, and embedded in Epon 812.

Ultrathin sections were cut, placed on coated grids, stained with uranyl acetate and lead nitrate, and then observed under an electron microscope (JEM-200CX or JEM-1200EX; JEOL Ltd., Tokyo, Japan).

Statistical Analyses
The results are expressed as means ± standard error. To compare the AOE activity and GSH concentration between cells from SAMP11 and SAMR1 mice, Student's t tests were used. To analyze differences between the two strains in the time course of ROS production, the expression of AOE mRNA, and the mitochondrial mass and function, a two-way (strain, days in culture) repeated-measures analysis of variance (ANOVA) followed by a Student–Newman–Keuls' post hoc test was used. Differences were considered to be statistically significant when p <.05.


   RESULTS
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Measurements of ROS Production
The time course of ROS production in cultured MDF cells was followed. Representative photographs of DCFH-DA-stained cells are shown in Figure 1A. The fluorescence intensities increased from the 3rd to the 5th day in both cell lines, and differences in the oxidative status became apparent after the 7th day. In the SAMP11 cells, a high fluorescence intensity was maintained until the 11th day. In the SAMR1 cells, in contrast, the fluorescence intensity decreased after the 7th day to a level equivalent to that of the 3rd day. Quantification of the fluorescence intensities showed that the ROS were significantly higher in SAMP11 cells than in SAMR1 cells on the 7th and 11th days (Figure 1B). We also observed similar changes with time in the production of superoxide anions using HEt (Figure 1C).



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  		Figure 1. A, Confocal micrographs of cultured murine dermal fibroblast-like (MDF) cells stained with dichlorofluorescein diacetate (DCFH-DA). Both cell lines showed a similar increase in fluorescence intensity from the 3rd to the 5th day. On the 7th day, cells from the accelerated senescence-prone (SAMP)11 mice exhibited almost the same fluorescence intensity as that observed on the 5th day. In contrast, cells from the accelerated senescence-resistant (SAMR)1 mice showed a remarkable decrease in fluorescence intensity on the 7th and 11th days as compared with the 5th day. PDs: population doublings at the time of replating. Bars = 25 µm. B and C, Change in the reactive oxygen species production of cultured MDF cells. The overall oxidative stress level determined by DCFH-DA staining (B) and the superoxide anion level assessed by hydroethidine staining (C) were represented by fluorescence intensities as described in the text. Closed circles: cells from SAMP11 mice; open circles: cells from SAMR1 mice. Data are expressed as mean ± standard error for six (B) or five (C) independent cell lines from each strain. *p <.05, **p <.01 between the two strains at the same time point, respectively; ap <.01 vs day 3 of the same strain; bp <.05, cp <.01 vs day 5 of the same strain, respectively

 
Activity of AOEs, GSH Concentrations, and Expression of AOE mRNA
We studied the activity of the AOEs and the GSH concentrations on the 7th day after replating, because differences in the ROS were most apparent on the 7th day. Catalase activity did not differ between the two cell lines, whereas GPX activity was significantly higher in the SAMP11 cells than in the SAMR1 cells (Table 2). The GSH concentrations were slightly lower in the SAMP11 cells than in the SAMR1 cells, but there was no statistically significant difference (p =.093) (Table 2). Although we tried to assess the superoxide dismutase (SOD) activity, commercially available kits could not detect any SOD activity from extracts containing 1 x 107 cells (data not shown).


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  		Table 2. Catalase and GPX Activities and the GSH Concentration in Cultured MDF Cells From SAMP11 and SAMR1 Mice.

 
We also examined the differences in the mRNA induction patterns of the AOEs between SAMP11 and SAMR1 cells. We followed the time course of mRNA expression in six independent cell lines from each strain by slot blot analyses. The signal intensity was quantified, and the amount of mRNA on the 5th and 7th days was expressed as a percentage of the amount obtained from the 3rd day samples. As shown in Figure 2, we could not detect any significant difference in the mRNA expression patterns between the two cell lines.



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  		Figure 2. Quantitative analyses of the messenger RNA (mRNA) expression of antioxidant enzymes (AOEs) by northern slot blots. Closed circles: cells from accelerated senescence-prone (SAMP)11 mice; open circles: cells from accelerated senescence-resistant (SAMR)1 mice. Signal intensities were normalized against levels of 18S ribosomal RNA in each sample, and the relative mRNA quantity on the 5th and 7th days was expressed as a percentage of that observed on the 3rd day. Data are expressed as the mean ± standard error for six independent cell lines from each strain. The AOE mRNA species studied here did not show any significant differences in their induction patterns between the two cell lines. SOD = superoxide dismutase; GPX = glutathione peroxidase

 
Double Labeling With HEt and MitoTracker Green FM
To clarify where the ROS are mainly produced in cultured MDF cells, we performed a double fluorescence labeling experiment. Mitochondria labeled with MitoTracker Green FM had a rod-like appearance (Figure 3A). The sites where the superoxide anions were being produced were labeled with HEt, emitting a dot-like red fluorescence (Figure 3B). The large, round, and bright objects in Figure 3B corresponded to nucleoli-binding ethidium in the nuclei, and were not considered to be ROS production sites. A merged image (Figure 3C) showed that most of the sites labeled with HEt, except for those in the nuclei, were in rod-like mitochondria labeled with MitoTracker Green FM.



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  		Figure 3. Double labeling of cultured murine dermal fibroblast-like (MDF) cells with hydroethidine (HEt) and MitoTracker Green FM. Cells from the accelerated senescence-prone (SAMP)11 mice (1.52 population doublings) were simultaneously stained with HEt and MitoTracker Green FM on the 7th day after replating, and were observed under a fluorescence microscope. Most of the sites stained with HEt, except for intranuclear staining of the nucleoli, overlapped with the rod-like mitochondria visualized by MitoTracker Green FM. A, MitoTracker Green FM; B, HEt; C, a merged image. Bars = 10 µm

 
Evaluation of Mitochondrial Mass and Inner Membrane Potentials
We next studied the changes of the mitochondria in terms of their mass and inner membrane potential using two mitochondria-specific fluorescent probes, NAO and JC-1. The areas stained with NAO increased in the SAMP11 cells over time, whereas the SAMR1 cells did not show such changes (Figure 4A), indicating that the mitochondrial mass increased in SAMP11 cells with a longer culture period. Objects labeled with NAO showed a dot- or rod-like appearance consistent with mitochondria, with little nonspecific diffuse cytosolic staining (data not shown). Representative photographs of JC-1-stained MDF cells from both SAMP11 and SAMR1 strains are shown in Figure 4B. On the 3rd and 5th days, there was no apparent difference in labeling patterns between the SAMP11 and SAMR1 cells. On the 7th and 11th days, the mass of the mitochondria with a low membrane potential (green fluorescence) seemed to increase in the SAMP11 cells, whereas the mass of the mitochondria with a high membrane potential (red fluorescence) remained unchanged. Semiquantitative analyses confirmed that the mitochondria with low membrane potentials increased in the SAMP11 cells after the 5th day (Figure 4D). Mitochondria with high membrane potentials, in contrast, did not show any significant changes with culture period in either strain (Figure 4C).



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  		Figure 4. A, Changes in the mitochondrial mass assessed by nonyl acridine orange (NAO) staining. The area labeled with NAO was measured and normalized against the cell density. Closed circles: cells from accelerated senescence-prone (SAMP)11 mice; open circles: cells from accelerated senescence-resistant (SAMR)1 mice. Data are expressed as mean ± standard error for four independent cell lines from each strain. The total mass of the mitochondria increased in the SAMP11 cells as compared to the SAMR1 cells after the 5th day. *p <.05, **p <.01 between the two strains at the same time point, respectively; ap <.05, bp <.01 vs day 3 of the same strain, respectively; cp <.05, dp <.01 vs day 5 of the same strain, respectively. B, Confocal micrographs of cultured murine dermal fibroblast-like (MDF) cells stained with JC-1. The cells were excited at 488 nm, and two fluorescent images were obtained from the same field using a 515- to 560-nm bandpass filter (green, for monomers) and a 570-nm longpass filter (red, for J-aggregates). Photographs are merged composites of two images. Mitochondria are labeled with green or red. Mitochondria with a low membrane potential seemed to increase in the SAMP11 cells as compared to SAMR1 cells on the 7th and 11th days. The nuclear regions were devoid of any fluorescence. PDs: population doublings at the time of replating. Bars = 25 µm. C and D, Semiquantitative analysis of the JC-1 images. Changes in the mass of the mitochondria were classified according to the JC-1 staining pattern (C, high potential; D, low potential) in cell lines from either SAMP11 (closed circles) or SAMR1 (open circles) mice. Data are expressed as mean ± standard error for six independent cell lines from each strain. SAMP11 cells showed a significant increase in the proportion of the mitochondrial mass with low membrane potentials (D) after the 5th day as compared with SAMR1 mice. The mass of mitochondria with high potentials showed no significant differences (C). ep <.05 vs day 7 of the same strain. Other indications are per the caption for (A)

 
Ultrastructural Changes of Mitochondria in Cultured MDF Cells
The ultrastructural changes in the mitochondria from MDF cells were followed over time. Upon examination under a phase contrast microscope, the cells appeared almost normal during the culture period up to the 11th (data not shown). Figure 5 shows representative electron micrographs of the mitochondria in the perinuclear regions of the MDF cells from SAMP11 and SAMR1 mice. On the 5th day, the mitochondria in the SAMR1 cells had a normal morphology (Figure 5B). Most of the mitochondria in the SAMP11 cells also exhibited a normal appearance, although a few mitochondria showed swelling with decreased electron densities in their matrices, and a translocation of the cristae to the peripheral regions (Figure 5A). On the 7th day, the mitochondria from the SAMP11 cells showed obvious morphological alterations (Figure 5C). Some of the mitochondria showed an increased density in their matrices and degeneration of the cristae; there were only a few morphologically intact mitochondria remaining. In the SAMR1 cells, most of the mitochondria still had a normal appearance on the 7th day, although an obscuration of the cristae and electron-dense matrices were observed in some of the mitochondria (Figure 5D). The number of altered mitochondria in the SAMR1 cells, however, was much smaller than in the SAMP11 cells. On the 11th day, many of the mitochondria from the SAMP11 cells had an abnormal appearance, demonstrating an increase in the electron densities in the matrices and intermembrane spaces, as well as degeneration of the cristae (Figure 5E). Some mitochondria were extremely condensed in appearance; these could be called residual bodies. Mitochondria with normal configurations in the SAMP11 cells were markedly decreased in number when compared to the SAMR1 cells. In the SAMR1 cells, many mitochondria still showed an almost normal structure, although several mitochondria demonstrated increased electron densities in their matrices (Figure 5F). Membranous whorl bodies, which were probably derived from degenerated mitochondria, were observed in both SAMR1 and SAMP11 cells.



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  		Figure 5. Electron micrographs of mitochondria from perinuclear zones of murine dermal fibroblast-like (MDF) cells. A, Accelerated senescence-prone (SAMP)11 cells, day 5. One mitochondrion showed swelling and decreased electron density in its matrix. The cristae were peripherally displaced (arrow). B, Accelerated senescence-resistant (SAMR)1 cells, day 5. All of the mitochondria in this field appeared normal in morphology. C, SAMP11 cells, day 7. The organelles bound by double unit membranes containing electron-dense material probably represent degenerating mitochondria (*). D, SAMR1 cells, day 7. Most of the mitochondria were normal, although some had an electron-dense matrix (*). E, SAMP11 cells, day 11. Many mitochondria showed an abnormal morphology. The proportion of degenerating mitochondria with high electron densities in their matrices and intermembrane spaces was further increased (*). Some of the degenerating mitochondria appeared as residual bodies (double arrows). There were few mitochondria with a normal appearance. One mitochondrion at the periphery of the field showed direct continuity between a relatively normal part and an abnormal electron-dense part (arrowhead). F, SAMR1 cells, day 11. Many mitochondria still had relatively normal morphologies. A membranous whorl body can be seen (double arrowheads). Bars = 500 nm

 
Ultrastructural Changes of the Mitochondria in Various Tissues From SAMP Mice
Figure 6 shows the ultrastructural changes of the mitochondria in various tissues from young and old SAMP11 and SAMP8 mice. The latter is a strain that exhibits a higher oxidative stress status plus mitochondrial dysfunction in the brain. Swollen mitochondria with decreased cristae were observed in the neuronal axons of the brainstem reticular formation in old SAMP8 mice (Figure 6B). Similar changes in the mitochondria were observed in myocardial cells, hepatocytes, and brown adipose cells from old SAMP11 mice (Figure 6, D, F, and H). Some mitochondria exhibited increased electron densities (in their matrices and intermembrane spaces) and envelope-associated woolly densities. These age-associated morphological changes in the mitochondria in vivo were comparable with those from cultured MDF cells on the 7th and 11th days. In young SAMP mice, these mitochondrial alterations were only seen in a milder form, and mitochondria with a normal appearance comprised the majority (Figure 6, A, C, E, and G).



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  		Figure 6. Electron micrographs of mitochondria in various tissues from young and old accelerated senescence-prone (SAMP) mice. A, An axon in the brainstem reticular formation of a SAMP8 mouse at 2 months of age. Mitochondria of normal size and structure were scattered. B, Axon in the brainstem reticular formation of a SAMP8 mouse at 23 months of age. Swollen mitochondria with a reduction in the number of cristae were observed. Some mitochondria contained patchy electron-dense woolly densities (arrows). C, Myocardial cells from a SAMP11 mouse at 2 months of age. Normal mitochondria with several dense granules were seen. D, Myocardial cells from a SAMP11 mouse at 16 months of age. Some mitochondria maintained their normal morphologies, but others showed a decreased number of cristae and matrix densification (*). Woolly densities (arrows) can be seen in the mitochondria with dense matrices. Residual bodies, probably derived from the mitochondria, were also observed (double arrows). E, Hepatocytes from a SAMP11 mouse at 2 months of age. Most of the mitochondria appeared normal. F, Hepatocytes from a SAMP11 mouse at 16 months of age. Several degenerating mitochondria were observed. Envelope-associated woolly densities (arrows), in addition to the diffuse densification of the matrix (*), were seen. G, Brown adipose cells with normal mitochondria from a SAMP11 mouse at 2 months of age. H, Brown adipose cells from a SAMP11 mouse at 16 months of age. Electron-dense mitochondria were observed in the center of the field (*), and normal mitochondria were also present. Bars = 200 nm

 

   DISCUSSION
Top
 Abstract
 Methods
 Results
 Discussion
References
 
In this study, we found a characteristic change with time in the ROS production of SAMP11 and SAMR1 cells, as shown in Figure 1. We therefore tested whether the insufficient induction of the antioxidant defense systems could account for the higher oxidative stress in SAMP11 cells. We found no significant differences between the two cell lines in terms of catalase activity, GSH concentration, and mRNA levels for catalase, SODs, and GPXs. These results suggest that a defective antioxidant defense is probably not the cause of the higher oxidative stress in SAMP11 cells. The GPX activities in SAMP11 cells were twice as high as those in SAMR1 cells, suggesting that GPX activities are normally upregulated in response to a higher oxidative stress status. In contrast, there was no significant induction in the mRNA for any of the GPX isozymes. This discrepancy between the mRNA levels and enzymatic activity for the AOEs has been previously reported (26,27), and was attributed to translational or posttranslational regulation, such as an enhanced stabilization of the mRNA (26).

We therefore focused on the mitochondria as a source of ROS. Double labeling assays with HEt and MitoTracker Green FM clearly demonstrated that the mitochondria were the major site of superoxide production in this culture system. ROS are mainly produced in mitochondria with high inner membrane potentials (28), whereas mitochondria injured by ROS showed a decrease in their membrane potential, as demonstrated in H2O2-loaded human lens epithelial cells (29) and Sod2 +/– mice (30). Using JC-1, we observed an increase in the proportion of mitochondria with low potentials but no significant change in high-potential mitochondria in SAMP11 cells. This observation indicates that the number of mitochondria injured by ROS is increased in SAMP11 cells. Although the green fluorescence of JC-1 showed some nonspecific extramitochondrial localization (31), the results of an NAO assay support our hypothesis by demonstrating that the total mitochondrial mass increased with time in SAMP11 cells. NAO staining showed dot- or rod-shaped mitochondria with little diffuse cytosolic fluorescence, thus excluding the possibility of a nonspecific increase in the NAO-stained area induced by mitochondrial depolarization (32). A compensatory increase in mitochondrial mass in response to senescence and oxidative stress in support of this theory has been previously reported (22,33,34).

We demonstrated that ultrastructural changes in the mitochondria from SAMP11 cells were comparable to those seen in various organs from old SAMP mice, suggesting that the mitochondrial changes observed in vitro were homologous to those seen in vivo. The ultrastructural alterations in the mitochondria were also similar to abnormalities that have been observed in aging in vitro (35–38) and in vivo (39–41), including observations in old SAMP8 mice (42). These ultrastructural changes may represent a series of events that eventually lead to mitochondrial degeneration. Woolly densities in the mitochondria are thought to be the most reliable early manifestation of irreversible cell injury (43), and electron-dense matrices and mitochondrial swelling are encountered in the process of mitochondrial involution (43). The condensed mitochondria are incorporated into autolysosomes, and are finally eliminated or transformed into residual bodies. Morphological alterations may also reflect functional changes in the mitochondria. The condensed matrices are believed to indicate altered oxidative phosphorylation (36). Our serial observations of the altered ultrastructures, together with the mitochondrial membrane potentials, suggest that the mass of mitochondria with low membrane potentials increases in parallel with the appearance and propagation of ultrastructurally abnormal mitochondria. Furthermore, the similarity of the ultrastructural changes seen in vivo to those in vitro suggests that mitochondrial dysfunction and the consequent higher oxidative stress may also exist in vivo.

ROS compromises mitochondrial function through a partial inactivation of the Fe-S centers in enzymes from the electron-transport systems, such as complexes I, II, and III, or through the peroxidation of cardiolipin (44,45), a phospholipid required for the normal functioning of respiratory chain complexes (46). An impairment in the electron-transport systems leads to decreases in the rate of state III respiration and increases in state IV respiration (30). Such mitochondria with defective respiratory chain enzymes increase the formation of ROS, thus establishing a vicious cycle (7,8). Although the mechanism responsible for the initial increase in ROS formation in SAMP mice is still unclear, an intrinsic decrease in the activity of complexes I and III in 4-week-old SAMP8 mice has been reported (47). In addition, Nishikawa and colleagues (48) demonstrated a higher redox state and higher mitochondrial respiration activity with a lower respiratory control ratio (RCR) in mitochondria derived from young (2 months old) SAMP8 brains. If these mitochondrial abnormalities are features common to other SAMP strains, they may have a causal relationship to the spontaneous occurrence of higher oxidative stress.

Although the phenotype of senescence acceleration does not become apparent until 6 months of age in SAMP mice, the abnormalities caused by the gene(s) responsible for senescence acceleration are probably set in motion from a younger age. Even a mild deviation from the physiological state can cause cumulative detrimental changes in cell constituents, which result in the phenotype of senescence acceleration. We evaluated the oxidative stress in early-passage (young) cells, not in late-passage (old) cells, to search for such "subclinical" phenomena. We found significant differences in the oxidative stress levels, as well as in mitochondrial function and morphology, even in young cells. There have been several reports demonstrating that young SAMP mice or cells can exhibit abnormal phenotypes relevant to oxidative stress or mitochondrial function, as previously mentioned (11,47,48). Considering our observations and those from previous reports, we expected that intrinsic in vivo and in vitro mechanisms, which are present from a young age, may have caused the differences in oxidative stress status between the SAMP and SAMR mice. One of these mechanisms may be a mild but inefficient hyperactive state with a higher rate of electron leakage in the mitochondrial electron-transport system, leading to an increased production of ROS (48).

Some technical difficulties have been pointed out in the evaluation of ROS production and mitochondrial morphology and function using fluorescent probes. Many researchers have used flow cytometry (22,31,34), because it can analyze a large quantity of cells at a time and can gate the fluorescence easily. In this study, however, we adopted a fluorometric analysis of confocal microscopic images of living cells to assess the ROS production and mitochondrial mass and membrane potentials, thereby attempting to avoid any artifacts induced by manipulating and detaching the adherent cells. Furthermore, we preferred the image analytical method to flow cytometric assay, because the former enabled us to evaluate cellular and mitochondrial morphologies and compare them to ultrastructural changes. DCFH-DA is a widely used marker for cellular oxidative stress (14,49), but is also oxidized to DCF by peroxidase alone or cytochrome c (50,51). The SAMP11 cells exhibited a higher GPX activity, which may have affected DCF formation regardless of ROS. Another fluorescent probe, HEt, has several drawbacks rendering it inappropriate for strict quantitative assays, such as the binding of ethidium to DNA [which causes a disproportional increase in fluorescence (16,52)] and oxidation to oxyethidium, a molecule that is distinct from ethidium in its fluorogenic properties (53,54). Despite these shortcomings, we can conclude that the SAMP11 cells were under a higher oxidative stress status than the SAMR1 cells, because experiments using these two ROS markers yielded consistent results.

When the present and previous observations are taken together, a possible explanation for the higher oxidative stress in SAMP11 cells can be developed as follows. The mitochondria from SAMP11 cells produced a greater amount of ROS as compared to SAMR1 cells, probably because of intrinsic mitochondrial dysfunctions. ROS injure respiratory chain complex proteins and/or mitochondrial membrane phospholipids. These injured mitochondria produce more ROS, and finally become unable to maintain the electrochemical gradients across their inner membranes. In response to the increase in the number of mitochondria with low potentials, the cells try to add mitochondrial mass to compensate for the decrease in the proportion of efficient mitochondria.

Summary
We demonstrated that cultured MDF cells from SAMP11 mice, which show accelerated senescence/crisis in vitro, could be used as an in vitro spontaneous higher oxidative stress model. The cause of the higher oxidative stress was not a decline in antioxidant defense mechanisms, but rather an increased production in ROS from the mitochondria. This in vitro system can be used as a screening tool to evaluate antioxidative substances over a relatively short time, and to develop novel therapeutic and preventive measures against age-dependent disorders. This system can also be a useful model for investigating the mechanisms responsible for oxidative stress-induced senescence in vitro in terms of molecular biology and biochemistry, based on the free radical theory of aging.


   Acknowledgments
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This work was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No.14570186).

We gratefully acknowledge Professor Hiroshi Shibasaki from the Department of Neurology, Graduate School of Medicine, Kyoto University, for critically reading this manuscript. We also thank Mr. Takatoshi Matsushita, Mr. Eishi Deguchi, Ms. Kumiko Kogishi, Ms. Sonoko Matsuda, and Ms. Tomoko Watanabe for their technical assistance.


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Decision Editor: James R. Smith, PhD

Received January 28, 2005

Accepted March 24, 2005


   References
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