The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 58:B291-B296 (2003)
© 2003 The Gerontological Society of America
Delayed Occurrence of Fatal Neoplastic Diseases in Ames Dwarf Mice: Correlation to Extended Longevity
Yuji Ikeno1,2,
Roderick T. Bronson3,
Gene B. Hubbard4,
Shuko Lee1 and
Andrzej Bartke5
1 Research Service, Audie Murphy VA Hospital (STVHCS), San Antonio, Texas.
2 Department of Physiology, The University of Texas Health Science Center at San Antonio.
3 U.S. Department of Agriculture, Human Nutrition Research Center on Aging, Boston, Massachusetts.
4 Department of Laboratory Animal Medicine, Southwest Foundation for Biomedical Research, San Antonio, Texas.
5 Department of Physiology, Southern Illinois University School of Medicine, Carbondale.
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Abstract
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Ames dwarf mice have drawn much attention in aging research because of their marked life extension. Studies demonstrate that some of the physiological characteristics of Ames dwarf mice are similar to those of dietary restricted mice. Because dietary restriction has been shown to suppress and/or delay the occurrence of various age-related diseases, we investigated age-related pathological changes in Ames dwarf mice. We observed Ames dwarf mice to have a delayed occurrence of presumably fatal neoplastic disease compared with their normal siblings. In addition to the delayed occurrence, we found that the incidence of presumably fatal adenocarcinoma in lung was significantly lower in Ames dwarf mice than for their normal siblings. The delayed occurrence of total neoplastic lesions and reduced incidence of adenocarcinoma in lung possibly could be attributed to the retardation of tumor development by changes in the levels of growth hormone and insulin-like growth factor-1, and thereby be a major contributing factor to the extended life span observed in these mice.
SUBSTANTIAL evidence suggests an important role for genetic control in the aging process (1–4). Most of the supportive evidence comes from studies in yeast, Caenorhabditis elegans, and the fruit fly, Drosophila melanogaster (1–4). Other studies suggest that the aging process in higher organisms could be controlled by a certain gene or genes, and there is clear evidence that a single gene mutation can extend longevity in mammals (5–8). Ames dwarf mice are the first mouse line to show a delayed aging process and a remarkable life span extension from a single gene mutation (5). Ames dwarf mice show significant extensions in both median and maximum life spans. These mice show an average life span extension of approximately 50% for male mice and 60% for female mice compared with their normal siblings (5).
The extended life span of Ames dwarf mice has caused them to be extensively investigated and characterized to identify the underlying mechanism(s) of the antiaging action (9). Ames dwarf mice are homozygous for a recessive mutation at a gene named Prophet of Pit-1 (Prop-1) (10), which causes developmental arrest in the pituitary gland of these mice. Further, these mice are deficient in three major hormones (9,10), i.e., growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH) (11).
Interestingly, studies demonstrate that Ames dwarf mice have physiological characteristics that are similar to the effects of dietary restriction (DR). DR is the most established and effective intervention of aging in rodents (12). Some characteristics shared by Ames dwarf mice and DR mice are: a) small body size; b) reduced blood glucose and increased insulin sensitivity; and c) reduced or absent levels of various hormones and growth factors, i.e., GH, TSH, insulin, and insulin-like growth factor-1 (IGF-1) (11,13). DR is beneficially effective not only on the aging process, but also on various age-related disease processes, i.e., delay and/or suppression of the occurrence of both neoplastic and nonneoplastic diseases (14–16). Because Ames dwarf mice show a marked life span extension and similar physiological characteristics to DR mice (11,13), it is of interest to examine the age-related pathological changes in Ames dwarf mice. The data could shed light on the occurrence of various diseases during aging as the major determining factor of an organism's longevity. Initial pathological analysis indicated that Ames dwarf mice and their normal siblings have a similar overall incidence of neoplastic disease (17). Therefore, comprehensive pathological analyses of the Ames dwarf mice was needed, because age-related pathological profiles may show differences between Ames dwarf mice and their normal siblings.
The purpose of the present study is to investigate Ames dwarf mice for age-related disease patterns, especially neoplastic disease, and to show a possible correlation between age-related disease profiles and extended life span in these mice.
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Methods
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Animal Maintenance
The Ames dwarf (df/df) mice were produced in a random-bred, closed colony with heterogeneous genetic background, which has been maintained for more than 20 years. The basic operations of the animal colony are previously described (17). The Ames dwarf mice used in this study were derived from mating male dwarfs (df/df) with carrier females (df/+). The animals were maintained in a fully Association for Assessment of Laboratory Animal Care-accredited vivarium at Southern Illinois University (Carbondale, IL). Food (Purina Lab Chow; Purina Mills, St. Louis, MO) and tap water were provided ad libitum. A 12:12 hour light/dark cycle was used. Sentinel mice housed in the same room were sacrificed for the monitoring of viral antibodies (Mouse Level II Complete Antibody Profile; CARB, Ectro, EDIM, GDVII, LCM, M. Ad-FL, M. Ad-K87, MCMV, MHV, M. pul., MPV, MVM, Polyoma, PVM, Reo, Sendai; BioReliance, Rockville, MD). Monitoring was repeated every 6 months. All tests were negative. The animals were monitored daily and weighed weekly. Food consumption was monitored weekly.
Procedures for Pathological Examination of Mice Dying Spontaneously
All mice were inspected at least twice daily (between 7:00 AM and 8:00 AM and between 3:00 PM and 4:00 PM). Mice that died spontaneously were removed from the cage, immediately necropsied for gross pathological examination, and preserved in Bouin's solution. A total of 54 mice were examined: 28 Ames dwarf mice (15 males and 13 females) and 26 normal siblings (wild type [WT]: 18 males and 8 females). Approximately 15% of the mice had severe autolysis, for which we were unable to perform histopathological examination and grade the lesions. Data were obtained from a total of 46 mice (25 Ames dwarf mice [13 males and 12 females] and 21 WT [15 males and 6 females]). Because the sample size was relatively small, and because there were no clear differences in major pathological changes between sexes, we analyzed the pathological data obtained from both the male and female mice.
After the mice were examined for gross pathological lesions, the following organs and tissues were excised: brain, pituitary gland, heart, lung, trachea, thymus, aorta, esophagus, stomach, small intestine, colon, liver, pancreas, spleen, kidneys, urinary bladder, reproductive system (male: prostate, testes, epididymis, seminal vesicles; female: ovaries, uterus), thyroid gland, adrenal glands, parathyroid glands, psoas muscle, knee joint, sternum, and vertebrae. Any other organs or tissues in which lesions were observed by gross inspection were excised. The fixed tissues, which were processed conventionally, were embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin-eosin. All excised organs were examined histologically as described by Bronson and Lipman (14).
Pathological Assessment
We constructed a profile of pathological lesions for each mouse, which included the prevalence of both neoplastic and nonneoplastic diseases. The percentage of tumor-bearing mice, tumor burden, disease burden, overall and age-specific incidence of disease, and severity of each lesion were also examined. The percentage of tumor-bearing mice was determined from the percent of mice in both the dwarf and WT groups possessing neoplastic lesions. In this assessment, all neoplastic lesions were counted regardless of the severity of tumors, i.e., both incidental and presumably fatal tumors were counted. We defined the tumor burden as the number of different types of tumors found in a mouse. For example, a mouse that had lymphoma and pituitary adenoma received a score of 2 in this assessment. The disease burden was also defined as the number of total histopathological changes in the whole body of each mouse in both groups. All pathological analyses were accomplished by a double-blind procedure without knowledge of the animal's identity. Two pathologists made similar, independent diagnoses in most cases.
Grading of Lesions
The severity of neoplastic lesions and glomerulonephritis was determined using the grading systems described below. These lesions were evaluated because they are the most common and prevalent lesions observed in most mice during pathological examination.
Glomerulonephritis was graded in the order of increasing severity: Grade 0: no lesions; Grade 1: minimal change in glomeruli (minimal glomerulosclerosis); Grade 2: Grade 1 with a few (less than 10) casts in renal tubules; Grade 3: Grade 1 with more than 10 casts in renal tubules; and Grade 4: Grade 3 with interstitial fibrosis.
The determination of severity of neoplastic lesions was based on previously reported criteria (18), which was further defined by Ikeno and Hubbard based on the histopathological findings of tumor cell involvement as follows: Grade 1: primary site only; Grade 2: primary site and intraorgan or 1 other organ metastasis; Grade 3: metastasis to 2–3 organs; and Grade 4: metastasis to more than 4 organs or Grade 3 + additional pathology, e.g., pleural effusion, ascites, and subcutaneous edema, etc. Hydrothorax, ascites, and subcutaneous edema are the common complications associated with advanced neoplastic disease. Grading of the lesions was accomplished by a double-blind procedure without knowledge of the animal's identity. Two pathologists made similar, independent diagnoses in most cases.
Probable Cause of Death
The probable cause of death for each mouse was determined by the severity of the diseases found by necropsy. Two pathologists made similar, independent diagnoses in most cases, and the determination of probable cause of death was accomplished by a double-blind procedure without knowledge of the animal's identity. When disease was considered not severe enough to terminate the animal's life, the probable cause of death was categorized as "undetermined."
Statistical Analysis
The total frequency of a lesion or grade of lesion was analyzed using a chi-square test for the mice that spontaneously died (19). When the expected frequencies were too small for the chi-square test, the data were analyzed using the Fisher's exact test (19). The Kaplan-Meier survival curve for presumably fatal neoplastic lesions and adenocarcinoma were analyzed using a log-rank test (20).
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Results
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Average Life Span, Tumor-Bearing Mice, and Tumor Burden
The average life span of Ames dwarf mice and WT mice in this study was 1062 days and 781 days, respectively. Since the previous study demonstrated that the Ames dwarf and WT mice have a similar overall incidence of neoplastic disease, we first compared the percentage of tumor-bearing mice in each group that had neoplastic lesions. The percentage of tumor-bearing mice that had neoplastic lesions was similar in both the dwarf and WT groups. More than 90% of the mice in each group had neoplastic lesions. Second, because aging rodents tend to have several different types of tumors at death, we compared the number of different types of tumors (tumor burden) for each mouse in both groups. As the data in Figure 1 show, the tumor burden for the dwarf mice is similar to that of the WT mice (p =.6301), suggesting that the overall tumor incidence is similar in dwarf and WT mice, which supports previous observations (17).
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Figure 1. Tumor burden in wild-type (WT) and Ames dwarf mice. The average number of tumors of any type found in individual mice from each group is shown. The values for the tumor burden represent the mean ± SEM for 25 dwarf and 21 WT mice
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Cause of Death
The probable causes of death in the dwarf and WT groups are shown in Table 1. Approximately 95% of WT mice died from neoplastic diseases, half of which (approximately 48%) died from adenocarcinoma of the lung. Fatal adenocarcinoma of the lung is usually associated with metastasis to the other organs or other pathological lesions, e.g., pleural effusion, ascites, or severe congestion and edema in the lung. The other major, fatal neoplastic diseases that we observed are hepatocellular carcinoma, hemangioma in the liver and spleen, and lymphoma. Dwarf mice showed a slightly lower incidence of fatal neoplasms (28% less; p =.1060); but the incidence of fatal adenocarcinoma of the lung was significantly lower in dwarf mice (88% less; p =.0138). Approximately 25% of the dwarf mice died without obvious evidence of lethal pathological changes.
Age-Specific Distribution of Fatal Neoplasms
Although compared with WT mice the dwarf mice showed a significant reduction in the occurrence of fatal adenocarcinoma in the lung, the change in incidence of fatal disease did not seem to be a major contributing factor to their extended life span. When we compared Kaplan-Meier survival curves for fatal neoplastic disease in both groups, we found that the patterns of the dwarf mice tended to shift toward an older age compared with those of WT mice, as shown in Figure 2 (p <.0001). These data suggest that fatal neoplastic disease occurred at an older age in the dwarf mice than in the WT mice.
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Figure 2. Mortality by neoplastic lesions in wild-type (WT) and Ames dwarf mice. Kaplan-Meier survival curves for fatal neoplastic lesions in WT and Ames dwarf mice are shown
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Severity of Diseases
To find a possible explanation for Ames dwarf mice having delayed occurrences of fatal neoplastic diseases, we compared the severity of several neoplastic diseases using the grading system described in Methods. Since adenocarcinoma in the lung is the most common neoplastic disease observed at the time of spontaneous death, we compared the severity of adenocarcinoma (both fatal and incidental) in the dwarf and WT mice (Figure 3). We found that the severity of adenocarcinoma in dwarf mice was significantly less than in the WT mice (p =.0061). For other types of neoplasms, we found that, in general, dwarf mice tended to have less severe neoplastic lesions compared with the WT control, but these apparent differences were not statistically significant.
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Figure 3. Severity of adenocarcinoma in wild-type (WT) and Ames dwarf mice. The severity of the adenocarcinoma was scored using the criteria described in Methods. The average severity of adenocarcinoma in mice from each group is shown. The values for the severity represent the mean ± SEM for 25 dwarf and 21 WT mice. *The value was significantly different (p <.05) from WT mice
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In addition to the severity of neoplastic disease, we compared the severity of nonneoplastic disease. Since the most common nonneoplastic lesion in these mice is glomerulonephritis, we compared the severity of this lesion between the dwarf and WT mice (Figure 4). We found the severity of the lesions was significantly less in dwarf mice than in WT mice (p =.0027).
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Figure 4. Severity of glomerulonephritis in wild-type (WT) and Ames dwarf mice. The severity of the glomerulonephritis was scored using the criteria described in Methods. The average severity of glomerulonephritis in mice from each group is shown. The values for the severity represent the mean ± SEM for 25 dwarf and 21 WT mice. *The value was significantly different (p <.05) from WT mice
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These data indicate that Ames dwarf mice had suppressed development of some neoplastic and nonneoplastic lesions.
Disease Burden
The disease burden, defined as total number of histopathological changes in a body, can serve as a good index of age-related accumulation of tissue and cell injury (14). Figure 5 shows a comparison between the disease burden of the dwarf and WT groups, indicating that the dwarf group had a significantly less (p =.0316) disease burden than the WT group. When the disease burden of each mouse at death was compared, dwarf mice seemed to have slower rates of accumulation of histopathological changes than WT mice (data not shown). Thus, these data show that Ames dwarf mice had a slower rate of tissue injury during aging, which possibly enabled them to maintain a better health status compared with the WT controls.
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Figure 5. Disease burden in wild-type (WT) and Ames dwarf mice. The average disease burden per mouse is the total number of pathological changes of any type found in individual mice from each group. The values for the disease burden represent the mean ± SEM for 25 dwarf and 21 WT mice. *The value was significantly different (p <.05) from WT mice
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Discussion
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Since much of the evidence supports the genetic control of aging and longevity in yeast, C. elegans, and the fruit fly, D. melanogaster (1–4), "longevity assurance genes" have been sought in mammals. In 1996, Brown-Borg and colleagues reported that a single gene mutation delayed aging and extended the life span of Ames dwarf mice (5). Ames dwarf mice are very intriguing animals for aging research not only because of their remarkable life span extension, but also because of the similar physiological characteristics they share with DR mice (13). Therefore, it is important to examine the age-related pathological status of Ames dwarf mice. If the underlying mechanisms of the life-extending effect in Ames dwarf mice are similar to those of DR mice, then the age-related pathologies of both groups should show similar changes.
Our results demonstrated that the overall pathological profiles were similar for both Ames dwarf mice and their normal siblings in both sexes. The major neoplastic lesions seen in these mice were adenocarcinoma in lung, hemangioma in liver or spleen, lymphoma, and hepatocellular carcinoma. The major nonneoplastic lesion found was glomerulonephritis, which develops as an animal ages. The percentage of tumor-bearing mice (the percent of mice having neoplastic lesions in the group) and the tumor burden (the number of different tumors found in a mouse) were similar in dwarf and WT mice, which is consistent with previous pathological analysis (17). However, Ames dwarf mice showed a slightly lower incidence of fatal neoplasms, with a significantly lower incidence of fatal adenocarcinoma in lung. In addition, fatal neoplastic disease was shown to occur later in life in Ames dwarf mice, and the severity of adenocarcinoma in these mice was significantly less compared with their normal siblings, indicating that the genetic mutation in Ames dwarf mice may delay the progression of fatal neoplastic disease. Ames dwarf mice also showed delayed progression of nonneoplastic lesions. The severity of glomerulonephritis was significantly less in Ames dwarf mice compared with their normal siblings, which also indicates that the progression of glomerulonephritis in Ames dwarf mice is delayed. Furthermore, Ames dwarf mice showed significantly less disease burden and slower age-related accumulation of various pathological changes compared with their normal siblings. These findings indicate that Ames dwarf mice maintain organ and whole-body integrity during aging, thereby delaying the development of both neoplastic and nonneoplastic diseases. This is also supported by evidence showing that approximately 25% of Ames dwarf mice had no obvious evidence of lethal pathology at death, which is one of the characteristic pathological findings in DR C57BL/6 mice (Ikeno and Nelson, unpublished observations).
Deficiency of GH and the resulting suppression of peripheral insulin-like growth factor-1 (IGF-1) levels are assumed to play key roles in the delayed aging of Ames dwarf mice, because a comparable life extension has been seen in animals that have targeted disruption of the GH receptor (GHR), which results in GH resistance and severely suppressed IGF-1 levels (6). Endocrine changes in Ames dwarf mice are found to lead to reductions in plasma IGF-1, insulin and glucose levels, and in somatic growth and adult body size (9). The reduction of plasma IGF-1 and the consequent reduced mitogenic actions of IGF-1 may be related to the delayed occurrence of fatal neoplastic disease, because substantial evidence suggests that GH and IGF-1 levels play important roles in tumor development through their potent mitogenic and antiapoptotic effects (21). Some nonneoplastic diseases also seem to be affected by changes in GH and IGF-1 levels, as was shown by the lessened severity of glomerulonephritis in Ames dwarf mice. Studies of GH transgenic mice, which have pathologically high levels of GH, showed that the early death of these animals appears to be related primarily to pathological changes in their kidneys [glomerulonephritis and glomerulosclerosis (22,23)]; therefore, reduced levels of GH may play an important role in the delayed progression of these lesions in Ames dwarf mice. We suggest that changes in the endocrine system, especially in GH and IGF-1 levels, and subsequent pathophysiological changes, play important roles in the delay of various age-related pathologies and life extension.
We conclude the following from this study: a) Ames dwarf mice and their normal siblings had a similar overall incidence of neoplastic disease; b) Ames dwarf mice had a significantly lower incidence of fatal adenocarcinoma in the lung; c) Ames dwarf mice exhibited a delayed occurrence of presumably fatal neoplastic disease; d) Ames dwarf mice showed less severe lesions (both neoplastic and nonneoplastic) compared with their normal siblings at the time of death; and e) Ames dwarf mice showed a reduced disease burden compared with their normal siblings at the time of death.
All of these changes may be partially, if not completely, similar to the effects of DR on age-related pathology.
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Acknowledgments
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The authors wish to thank Dr. Edward J. Masoro for his critical review of the manuscript. The authors also wish to thank Mr. Chris Wright for his excellent technical support and Ms. Marie Corinne Price for her excellent editorial work. This research was supported by National Institutes of Health Grants AG13319 and AG19899, an American Institute for Cancer Research grant, and a VA VISN 17 grant.
Address correspondence to Yuji Ikeno, MD, PhD, Research Service, Audie L. Murphy VA Hospital (STVHCS), 7400 Merton Mintor Boulevard, San Antonio, TX 78229. E-mail: ikeno{at}uthscsa.edu
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Footnotes
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James R. Smith,, PhD
Received August 9, 2002
Accepted December 9, 2002
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Abstract
Methods
