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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 57:B415-B421 (2002)
© 2002 The Gerontological Society of America

Age-Related Progressive Neuronal DNA Damage Associated With Cerebral Degeneration in a Mouse Model of Accelerated Senescence

Atsuyoshi Shimadaa, Hiromi Keinoa, Mamoru Satoha, Masao Kishikawaa, Naoyuki Seriub and Masanori Hosokawab

a Department of Pathology, Institute for Developmental Research, Aichi Human Service Center, Japan
b Field of Regeneration Control, Institute for Frontier Medical Sciences, Kyoto University, Japan

Atsuyoshi Shimada, DMedSc, Department of Pathology, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya, Kasugai, Aichi 480-0392, Japan. E-mail:ats7@inst-hsc.pref.aichi.jp

Decision Editor: James R. Smith, PhD


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
The DNA of cerebral neurons in subjects with Alzheimer's disease is extensively damaged, although the morphological features of apoptosis are absent. We investigated whether DNA is damaged in the brains of the SAMP10 strain of mouse, in which accelerated senescence is characterized by age-related cerebral atrophy and cognitive impairment. We performed quantitative terminal deoxynucleotidyl transferase-mediated digoxigenin-labeled dUTP nick end labeling (TUNEL), using paraffin sections. TUNEL positive cells increased in number in the cerebral neurons of SAMP10 mice with aging. TUNEL positive cells were widely distributed in mice at age 13–14 months, and obvious in the olfactory tubercle, anterior cingulate cortex, insular cortex, and amygdala. These TUNEL positive cells did not have the morphological features of apoptosis. Therefore, the DNA became damaged with advancing age through a mechanism other than apoptosis. SAMP10 is a useful mouse model of brain aging that mimics the progressive neuronal DNA damage associated with human neurodegenerative disorders.

SENESCENCE-accelerated mice (SAMs) are established animal models of aging (1)(2)(3). SAMs comprise several inbred strains of mice that are grouped as either accelerated senescence-prone (SAMPs) or accelerated senescence-resistant (SAMRs). SAMP strains are characterized by a short life span and the early onset of systemic senescence (1)(2). Among SAMP strains, the SAMP10 strain of mouse developed by Shimada and colleagues is characterized by age-dependent cognitive impairment, and cerebral atrophy caused by cortical neuronal loss and neuronal shrinkage (4)(5). Atrophy is most marked in the frontal part of the cerebral cortex. The other areas of the cerebral cortex diffusely undergo atrophy but to a lesser extent. Other atrophy-prone regions include the posterior pyriform cortex, the entorhinal cortex, the anterior olfactory nucleus, the nucleus accumbens, the caudate-putamen, and the amygdala (6). The SAMR1 strain of mouse has provided a normal aging control because of its relatively long life span, late onset and mildness of senescence-related physical manifestations, lack of gross cerebral atrophy, and well-preserved cognitive functions (5)(6)(7).

Oxidative stress induced by free radicals plays an important role in aging, stroke, Alzheimer's disease, Parkinson's disease, and Huntington's disease (8)(9)(10)(11)(12). Reactive oxygen species cause oxidative damage in various organs of SAMP strains, including the brains of SAMP1 and SAMP8 mice (13)(14)(15)(16)(17)(18)(19)(20)(21). Although oxidative stress induces age-dependent acceleration of chromosomal abnormality and nuclear DNA strand breaks (22), histomorphological manifestations of such oxidative damage in the brain tissue of SAMP mice have not yet been determined.

The DNA damage that occurs during neuronal apoptosis, necrosis, or degeneration has been examined by using in situ end labeling of nuclear DNA strand breaks, based on the template-independent addition of labeled nucleotides to the newly generated 3'-OH ends of single-strand or double-strand DNA breaks by the enzyme terminal deoxynucleotidyl transferase (TdT) (23). This method has been widely used in studies of human and animal neurodegenerative disorders (11)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37). Such studies have shown that vulnerable brain regions in subjects with Alzheimer's disease contain more cells with DNA damage than those of age-matched controls (26)(27)(31)(33) and that oxidative stress can be a cause of such DNA damage.

In the present study, we examined whether DNA became damaged as the brains of SAMP10 mice aged. We performed in situ TdT-mediated digoxigenin-labeled dUTP nick end labeling (TUNEL) assays, using SAMP10 and SAMR1 mice at various ages to determine which region(s) was vulnerable to DNA damage.


   Methods
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 Abstract
 Methods
Results
 Discussion
 References
 
Animals and Tissues
Inbred SAMP10 and SAMR1 strains of mice were examined. SAMP10 mice are characterized by a short life span, early onset of various aspects of systemic senescence, and age-dependent cerebral atrophy. SAMR1 mice are normal aging controls (3). The mice were reared at the Institute for Frontier Medical Sciences of Kyoto University. Housing conditions are described elsewhere (5). Three 3-month-old, four 7-month-old, and five 13- to 14-month-old SAMP10 mice, as well as three 3-month-old, three 13- to 15-month-old, and five 18- to 22-month-old SAMR1 mice, were anesthetized with Nembutal, perfused with 10% neutral buffered formalin, and postfixed in the same fixative for 3 days at 4°C. The brains were removed, embedded in paraffin, and cut into 6-µm-thick coronal sections at eight levels: the anterior olfactory nucleus, nucleus accumbens, anterior commissure, ventral hippocampal commissure, habenular nucleus, substantia nigra, cerebellar flocculus, and C1 spinal cord. Parasagittal sections were cut from the left cerebellar hemisphere. Mouse brain regions were named according to Hof and colleagues (38). All animals were handled in accordance with the Guidelines for Animal Experiments of Kyoto University.

Histochemical Labeling
We detected DNA fragmentation by TUNEL using an Apoptag Peroxidase Kit (Serologicals Corp., Norcross, GA) according to the kit protocol with minor modification. Briefly, sections were deparaffinized and digested with proteinase K (5 µg/ml) for 10 minutes at room temperature. Endogenous peroxidase was quenched in 3% H2O2 for 5 minutes at room temperature. The sections were incubated in 75 µl per section of equilibration buffer for 1 minute at room temperature, followed by 27.5 µl per section of working-strength TdT enzyme for 30 minutes at room temperature. Antidigoxigenin peroxidase conjugate (32.5 µl per section) was applied to the sections; then they were incubated for 15 minutes at room temperature. Color was developed with diaminobenzidine (DAB) (0.1 mg/ml) for 15 minutes and lightly counterstained with cresyl violet. Sections from a 3-month-old SAMR1 mouse (Control 1) and a 14-month-old SAMP10 mouse (Control 2) were always labeled by TUNEL in every set of experiments. The staining pattern of the Controls 1 and 2 was compared between the sets of experiments. When there was a set of experiments in which the pattern of the Controls 1 and 2 deviated from that in the other sets of experiments, such a set was excluded.

Qualitative Microscopy for TUNEL Positive Cells
We examined histological sections by using a light microscope (Optiphot, Nikon, Tokyo, Japan) to determine the distribution of TUNEL positive cells. The staining intensity and frequency of TUNEL positive cells was qualitatively determined in each brain region and scored as marked, moderate, mild, or rare to none.

Counting of TUNEL Positive Cells
TUNEL positive cells were counted in four selected brain regions (layer 2 of the olfactory tubercle, layer 2/3 of the cingulate cortex, layer 2/3 of the agranular insular cortex, and the amygdala) by using a computer-assisted image analyzer, LUZEX 3U (Nikon) connected to a light microscope (Optiphot, Nikon). Three square visual fields (100 µm side length) were randomly sampled from each region. All neuronal cells with a prominent nuclear profile that appeared in the visual fields were examined and divided into two categories: TUNEL positive or negative. In some cells, nuclear staining was very weak and the border between weakly stained cells and unstained cells was obscure. In such a case, TUNEL positivity was measured as follows. The signal intensity of the nucleosome (excluding the nucleolus) of individual neurons stained with DAB was expressed as an 8-bit digital scale (0 to 255, 0 representing the highest intensity or the darkest and 255 representing the lowest intensity or the brightest) by using LUZEX 3U. The signal-intensity scale of the adjacent neuropil was measured in the same manner. TUNEL positivity of a single neuronal nucleus was determined as the difference between the signal-intensity scale of the nucleosome and that of adjacent neuropil. Nuclear staining of the cells with TUNEL positivity in the 30s and 40s was weak, and definitely positive cells had TUNEL positivity equal to or over 50. Therefore, we defined cells with TUNEL positivity less than 50 as TUNEL negative. The number of TUNEL positive neurons was recorded and expressed as percentage of the number of total neurons in each brain region.

Statistics
Cell count data were analyzed by a two-way (Strain x Age) analysis of variance followed by a post hoc test using STATISTICA software (StatSoft, Inc., Tulsa, OK). Mean percentages of TUNEL positive neurons were compared between different age groups in each strain of mouse.


   Results
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 Abstract
 Methods
 Results
Discussion
 References
 
Qualitative Microscopy
TUNEL was not obvious in any brain region in 3-month-old mice in either of the SAMP10 or SAMR1 strains (Fig. 1 and Fig. 1). The number of TUNEL positive cells increased with advancing age in SAMP10 mice (Fig. 1). TUNEL positive cells were distributed in a wide range of the brain in SAMP10 mice at the age of 13–14 months. In these mice, cells intensely labeled by TUNEL were frequently and consistently seen in the neurons of layers 1 and 2 of the olfactory tubercle and layer 2/3 of the anterior cingulate and prelimbic cortex (Fig. 2). TUNEL positive cells with moderate signal intensity were diffusely seen in layer 2/3 of the agranular insular cortex, the rostral part of the caudate-putamen, the nucleus accumbens, the lateral septal nucleus, the amygdala, and the hypothalamus (Fig. 2). Weakly TUNEL positive cells were often seen in the superficial layers of the secondary motor cortex adjacent to the anterior cingulate cortex, the caudal part of the caudate-putamen, the thalamus, the caudal part of the pyriform cortex, and the granule cell layer of the dentate gyrus (Fig. 2). Distribution of the cells with weak TUNEL varied among the mice. Most of the cells labeled by TUNEL had a neuronal morphology, but some were glial. Even intensely stained neurons generally had a loose and finely granular chromatin structure and did not show clear morphological characteristics of apoptosis, namely chromatin condensation, margination of chromatin at the nuclear membrane, nuclear fragmentation, cell shrinkage, and apoptotic bodies (Fig. 1). However, the nuclei of labeled large neurons were sometimes mildly shrunken. The signal intensity of individual cells varied even within an area that contained many intensely TUNEL positive cells (Fig. 1).



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  		Figure 1. Terminal deoxynucleotidyl transferase-mediated digoxigenin-labeled dUTP nick end labeling (TUNEL) assay performed on paraffin sections of anterior cingulate cortex obtained from SAMP10 mice aged 3 (A), 7 (B), and 14 (C) months and from SAMR1 mice aged 3 (D), 13 (E), and 22 (F) months. Numbers of TUNEL positive cells age-dependently increase in SAMP10, but not in SAMR1 mice. G, Higher magnification of anterior cingulate cortex reveals that TUNEL positive cells are mostly neurons with an almost normal nuclear morphology. H, Signal intensity of TUNEL staining among cells in basolateral amygdala varies from very weak to highly intense. Bars in A–F, 50 µm; bars in G and H, 20 µm.

 


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  		Figure 2. Distribution of terminal deoxynucleotidyl transferase-mediated digoxigenin-labeled dUTP nick end labeling (TUNEL) positive cells. Each brain region is indicated by gray levels corresponding to marked, moderate, mild, and rare to none in the signal intensity and frequency of TUNEL positive cells.

 
TUNEL positive cells were also seen in the SAMR1 mice but much less frequently than in the SAMP10 mice at various ages in some regions, including the lateral septal nucleus, anterior cingulate cortex, and olfactory tubercle. TUNEL was sporadic in neurons of the visual cortex but the labeling was not reproducible across the same strain and age. The TUNEL positivity of these cells in SAMR1 mice was generally weaker than that in SAMP10 mice, and the number and staining intensity of these cells appeared not to increase in a consistent age-dependent manner (Fig. 1).

In addition to TUNEL positive cells that lacked the morphological features of apoptosis, there were rare TUNEL positive cells that showed nuclear shrinkage or fragmentation and chromatin condensation with or without degraded cellular processes in 13- to 14-month-old SAMP10 mice (Fig. 3). These cells were distributed chiefly in the midbrain tegmentum and pontine nucleus. Although the origin of these TUNEL positive cells was unknown, we considered that they were undergoing apoptosis.



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  		Figure 3. Rare apoptotic cells in the periaqueductal gray (A) and basis pontis (B). A, Terminal deoxynucleotidyl transferase-mediated digoxigenin-labeled dUTP nick end labeling (TUNEL) of fragmented nucleus is prominent and a faint diaminobenzidine precipitate is in the cytoplasmic processes that indicate properties of glial cells. B, condensed nucleus labeled by TUNEL. Bars are 25 µm.

 
Counting of TUNEL Positive Cells
The mean percentages of TUNEL positive neurons in layer 2 of the olfactory tubercle was very similar between 3-month-old SAMP10 and SAMR1 mice (Fig. 4). The percentage of TUNEL positive neurons increased significantly only in SAMP10 mice over the age of 7 months and reached 78% at the age of 13–14 months. The profile of the age-related increase in the percentage of TUNEL positive neurons was similar but to a lesser extent in the anterior cingulate cortex (Fig. 4), the agranular insular cortex (Fig. 4), and the amygdala (Fig. 4). TUNEL positive neurons of the amygdala of SAMP10 mice increased in number from the first half of life (Fig. 4). In contrast, the mean percentage of TUNEL positive neurons of SAMR1 mice was low at 0–13% and did not change significantly with advancing age up to the age of 22 months in any brain region examined (Fig. 4).



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  		Figure 4. Age-related changes in the percentage of terminal deoxynucleotidyl transferase-mediated digoxigenin-labeled dUTP nick end labeling positive neurons in the olfactory tubercle (A), the anterior cingulate cortex (B), the agranular insular cortex (C), and the amygdala (D) of SAMP10 (•) and SAMR1 ({square}) mice. Values are indicated as means ± standard deviation. *p < .05; **p < .01, compared with 3-month-old mice of the same strain.

 

   Discussion
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 Abstract
 Methods
 Results
 Discussion
References
 
We performed this study to determine whether DNA in the brain was damaged as SAMP10 mice aged. The TUNEL technique is known to label free 3' hydroxyls of DNA that can be present at the ends of single-strand or double-strand DNA breaks. Qualitative microscopy revealed that the TUNEL positive neurons increased in number with advancing age in various brain regions, including the olfactory tubercle, lateral septal nucleus, nucleus accumbens, amygdala, anterior cingulate cortex, agranular insular cortex, caudate-putamen, thalamus, hypothalamus, hippocampus, and pyriform cortex (Fig. 2). Counting the data of the four selected areas revealed a statistically significant increase in the percentage of TUNEL positive neurons in SAMP10 mice at the age of 13–14 months compared with mice at the age of 3 months (Fig. 4).

In addition, rare TUNEL positive cells had the morphological features of apoptosis in the midbrain tegmentum and pontine nucleus (Fig. 3). Besides these apoptotic cells in the brain stem region, widespread TUNEL positive cells distributed in the cerebrum did not have clear apoptotic features, although the nuclei were occasionally somewhat shrunken. The morphology of most nonapoptotic TUNEL positive cells was neuronal (Fig. 1 and Fig. 1).

The SAMP10 mouse develops cerebral atrophy in an age-dependent manner (5), but vulnerability to atrophic changes regionally varies (6). Atrophy-prone brain regions were associated with the distribution of TUNEL positive cells. The regions most vulnerable to atrophy in SAMP10 mice were the olfactory bulb and the rostral part of the cerebral cortex composed of the anterior cingulate cortex, the prelimbic area, the orbital cortex, the agranular insular cortex, and the primary and secondary motor cortex. The percentage of TUNEL positive neurons was high in many of these cortical regions of aged SAMP10 mice. Frequent TUNEL positive neurons in layers 1 and 2 of the olfactory tubercle can be related to degeneration of the olfactory bulb because of the anatomical connections (39). The amygdala, pyriform cortex, nucleus accumbens, septum, and caudate-putamen were vulnerable to age-dependent atrophy in SAMP10 mice as reported (4)(5). The present study revealed many TUNEL positive neurons in the amygdala and some TUNEL positive cells in the pyriform cortex. A moderate increase in the number of TUNEL positive neurons with advancing age was observed in the nucleus accumbens, septum, and caudate-putamen in SAMP10 mice. Atrophy was not obvious in the brain stem or the cerebellum as reported (5), and TUNEL positive cells were rare or absent in these areas. In addition, most brain regions are atrophy resistant in SAMR1 mice throughout life, except for minor atrophy in the parietal cortex (4)(6). TUNEL positive neurons did not increase in number with advancing age in any brain region in SAMR1 mice. Given an association between the distribution of TUNEL positive neurons and atrophy-prone regions, neuronal nuclear DNA damage should represent a manifestation of degenerative changes associated with age.

Autopsy specimens of brain regions that are vulnerable to Alzheimer's disease (AD) contain more cells with DNA damage than those from age-matched controls (26)(27)(31)(33). Such cells are not considered to die through an apoptotic pathway, because they lack the morphological characteristics of apoptosis such as chromatin condensation (26)(27)(33)(35). The nuclei of these cells labeled by in situ tailing could represent a population of neurons with DNA strand breaks. TUNEL is, in this context, interpreted as a marker of cell injury or degeneration that is independent of the apoptotic, necrotic, or nonapoptotic programmed cell death mechanism (35). The neurons in AD patients are supposed to differ from those of age-matched controls in their susceptibility to death signals, which may result from an accumulation of DNA strand breaks from metabolic disturbances such as excitotoxicity or oxidative stress. Such neurons may represent a struggle between degeneration and repair by upregulating proteins that serve as protective mechanisms (40). Similar metabolic problems are supposed to arise in the neurons of aged SAMP10 mice, which result in DNA strand breaks. Our quantitative data revealed that the mean percentage of TUNEL positive neurons gradually increased with advancing age. This finding indicated that DNA strand breaks gradually accumulate in neurons with advancing age and that such neurons survive for long periods rather than experiencing the relatively fast death and disappearance associated with apoptosis.

Apoptosis is not a main feature of the brains of aged SAMP10 mice, which agrees with the DNA damage–DNA repair theory and can explain the aging process as the result of an accumulation of DNA damage and a decrease in DNA repair mechanisms that causes slow but progressive cell degeneration and eventual death (41)(42)(43)(44). The balance between DNA strand breaks and DNA repair is considered to be disrupted in SAMP10 mice, because the rate of DNA strand breaks is relatively enhanced as a result of being in a hyperoxidative state compared with SAMR mice (13)(14)(15)(16)(17)(18)(19)(20)(21)(22). A synchronous induction and progression of cell death is proposed in AD because TUNEL positive cells in AD have morphological uniformity (33). In contrast to the findings in AD, SAMP10 mice contained TUNEL positive neurons with variable signal intensity. This indicated that DNA damage was initiated in a random, rather than a synchronous, manner in individual cells and that the damage gradually accumulated.

Conclusions
Neuronal DNA damage is a common feature of aging in dogs. Some investigators have claimed that the characteristics of apoptosis are absent in neuronal nuclei labeled with TUNEL (45). The present study detected no age-related increase in the percentage of TUNEL positive neurons in SAMR1 mice. Therefore, neuronal DNA damage is not a common feature of normally aging mice. Because the oldest SAMR1 mice that we examined were 22 months old, which is several months short of the maximal life span, DNA damage might occur in SAMR1 mice that are older than 22 months. Regardless, neuronal degeneration represented by the DNA damage that accumulated with advancing age arose relatively early in the SAMP10 mice. DNA damage that occurred in aging SAMP10 mice was similar to that described in aging dogs and humans as well as in AD, in that most of the TUNEL positive cells lacked the characteristics of apoptosis (31)(33)(45). The SAMP10 strain of mouse, in this sense, is a unique model of age-related neuronal degeneration that mimics aspects of aging and neurodegeneration in humans. The mechanisms underlying DNA damage in SAMP10 mice require further study and will provide important findings that help us to understand neurodegenerative disorders.


   Acknowledgments
 
This work was supported by Grants-in-Aid for Scientific Research (11770128 and 13670238) from the Japan Society for the Promotion of Science.

We thank Noriko Kawamura for expert technical assistance, Mutsumi Matsuu for technical advice on TUNEL staining, Masako Tsuzuki for data analysis, and Dr. Toshio Takeda (SAM Research Council) for critical comments.

Received June 18, 2002

Accepted August 22, 2002


   References
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 Methods
 Results
 Discussion
 References
 

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