- Email: [email protected]
N rats with aging
archives of oral biology 52 (2007) 844–849
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/arob
Occlusal tooth wear in female F344/N rats with aging Kazutoshi Nishijima a,*, Sachi Kuwahara a,b, Tamio Ohno c, Osamu Miyaishi d, Yoshitake Ito e, Yasunori Sumi f, Shin Tanaka a a
Animal Facility for Aging Research, National Institute for Longevity Sciences (NILS), National Center for Geriatrics and Gerontology (NCGG), Obu, Japan b Department of Anatomy, Hyogo College of Medicine, Nishinomiya, Japan c Division of Experimental Animals, Center for Promotion of Medical Research and Education, Graduate School of Nagoya University, Nagoya, Japan d Chubu Rosai Hospital, Nagoya, Japan e Laboratory Animal Research Center, Aichi Medical University, School of Medicine, Nagakute, Aichi, Japan f Division of Oral and Dental Surgery, Department of Advanced Medicine, National Hospital for Geriatric Medicine, NCGG, Obu, Japan
article info
abstract
Article history:
Objectives: This study was conducted to ascertain whether laboratory rats are an adequate
Accepted 6 March 2007
animal model for aging oral cavity research, especially on occlusal tooth wear (OTW), which progresses with aging and causes abnormal occlusions. Mastication has been reported to
Keywords:
relate to cognition in the elderly. Thus, it is important to care for the oral cavity, especially in
Attrition
the frail elderly, for the maintenance of all-round quality of life. Adequate and appropriate
Aging animal model
animal models are essential for basic and clinical research on the oral cavity.
Mastication
Methods: Dried maxilla and mandible specimens from 98 young, aging or aged female F344/
Molar teeth
N rats were used.
Senescence
Results: The levels of OTW of all molars were monitored with aging. The molar tooth began to wear at 1-month old (M) and progressed rapidly till 12 M. Subsequently, OTW progressed slowly till 30 M, and then rapidly again after 35 M. Conclusions: This study showed that progress of OTW is well correlated with the entire life span of the rat, and suggested that the rat aged over 12 M would be an adequate animal model for research on OTW in middle-aged and elderly people. # 2007 Elsevier Ltd. All rights reserved.
1.
Introduction
In developed countries, human life expectancy in recent decades has been greatly prolonged, and the elderly population has been rapidly growing. According to the World Health Report 2006 by WHO, more than 20% of people are over 60 years old in European countries and Japan. The average life expectancy in Japan was 86 years for females and 79 years for males, and in 2002 both male and female reached the longest life expectancy in the world. Japan is becoming an aging society at a globally unprecedented pace. It is widely acknowledged, both within
and outside the government of Japan, that more than one in four Japanese will be over 65 years old by 2015. Health research on senior citizens is needed, especially regarding the frail elderly, for the maintenance of their quality of life (QOL). Aging and/or aged animal models are required in such studies for biological understanding of aging properties. The National Institute for Longevity Sciences (NILS) established an aging farm and succeeded in the production of 30 months old (M) F344/N rats.1 Aging and/or aged rats have been analyzed from various aspects and have revealed the relationship between morphological changes and aging.2–6
* Corresponding author. Tel.: +81 562 446597; fax: +81 562 446597. E-mail address: [email protected] (K. Nishijima). 0003–9969/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2007.03.001
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archives of oral biology 52 (2007) 844–849
Dysfunctions of the oral cavity, caused by various diseases including peridontics, caries and abnormal occlusions, may lead not only to poor nourishment but also to constitutional symptoms like migraine or palsy through temporomandibular disorders.7–10 In addition, mastication has been reported to relate with cognition in both certain young laboratory animals11,12 and elderly humans.13 Since the wellness and maintenance of the oral cavity in the elderly humans have been proposed and approved, a care program as well as animal models are required for the identification of aging changes of oral conditions. Aging changes in the periodontal bone of F344/ N rats have been reported, and it has been suggested that rats could be used as a model for periodontal research.5 However, few precise and systemic aging analysis reports have been available on the teeth of rats. Additionally, the aging characteristics of rats have been reported to be different depending on the strain, substrain and gender.14–16 These are known to differ among laboratory animal species as well. The elucidation and assessment of species-, strains- and genderspecific changes are the key to streamlined, waste-free animal experiments. Survival in male F344/N rats was shorter than in females,17 probably due to a higher incidence and earlier onset of leukemia in males than in females.2 This may indicate that female F344/N rats, without the modification by leukemia, are a better model than males for aging and longevity research. In this study, we focused on and monitored occlusal tooth wear (OTW) of molars, which progresses with aging and causes abnormal occlusions, in female F344/N rats aged from 1 to more than 35 M. Our aim was to clarify aging change of the molar teeth in rats, including the difference in the progress of OTW among quadrants and position of the teeth, and to examine whether rats are adequate laboratory animals to be used for aging tooth research.
2.
Materials and methods
2.1.
Materials
Aging and aged female F344/N rats were established in the NILS aging farm,1 and younger animals were purchased from Japan SLC, Inc. (Hamamatsu, Japan). All animals were fed with a pellet diet (Labo MR Stock, pH: 6.18, hardness: 31.6 kg/cm2, Nosan Corp., Yokohama) in accordance with the NILS Aging Farm Guide1 (Tanaka et al. 2000). The animals, 98 in all, were divided into 11 groups by their age; 1, 2, 3–4, 6–7, 12, 18, 21, 24, 27, 30 and >35 M, respectively (each group contained 6–12 animals; see Table 1 for details). The animals were sacrificed with an overdose of anesthetic. The head was dissected and autoclaved at 121 8C for 5 min. Major parts of the soft tissue were removed from the skull and mandible which were subsequently incubated in 0.5% papain solution (ED 3.4.22.2, Merck, Darmstadt, Germany) for overnight at 37.0 8C. The dried bone specimens were prepared for measurements of the OTW score. Molar teeth were observed with a stereoscopic microscope, and the OTW level was scored. The experimental protocol was performed with the permission of the Committee for Animal Ethics NILS according to the Guideline of NILS Animal Experimentation with due care for animal rights and welfare.
Table 1 – Age groups of animals used in this study Group
Days old (mean)
1 M 2M 3–4 M 6–7 M 12 M 18 M 21 M 24 M 27 M 30 M >35 M
22–30 (26.3) 42–55 (46.4) 92–136 (111.7) 168–214 (194.5) 350–377 (363.4) 547–554 (548.0) 628–651 (641.9) 726–732 (728.5) 813–828 (821.4) 909–916 (912.5) 1087–1196 (1128.6)
Total
2.2.
No. of animals 7 7 6 8 8 9 10 11 10 12 10 98
Scoring and statistics
The occlusal surface of molar tooth of (especially young) rat has several high cuspis dentis protuberances which made it impossible to distinguish between the enamel and dentin. For this reason, we needed to modify Broca’s index,18 which is based on the degree of exposure of dentin, to be suitable for rats. Scoring was as follows: Score 0, no wear observed; Score 1, wear at apex of cuspis dentis; Score 2, wear of cuspis dentis; Score 3, loss of cuspis dentis; Score 4, loss of corona dentis (Fig. 1). Three molar teeth in each quadrant were numbered M1 to M3 in accordance with anterior to posterior positions. Since Wilcoxon signed-rank test revealed no significant differences in each molar tooth between left side and right side in OTW scores, the scores of bilateral molar teeth were combined. The medians and intraquartile ranges in OTW scores of all molar teeth were calculated in each aging group. Nonparametric Kruskal–Wallis, for multiple comparison, and Wilcoxon rank sum tests were employed to reveal the differences in OTW scores among positions of the teeth (M1– 3) in every aging group with SPSS 13.0. Differences of OTW scores between neighboring aging groups were measured with the same tests. Linear approximation was calculated with Excel (2002, Microsoft) Fseparately, 22–377 (1–12 M), 547–919 (18–30 M) and 1087–1196 (>35 M) days old (D), on scatter plots of OTW scores of each molar tooth (M1–3) in the upper or lower jaw of all individual animals.
3.
Results
The molar teeth of rats are classified as boundont with several cuspis, and three major peaks were prominently observed (Fig. 1A, a). Tooth wear was observed only on the occlusal surface, not on any other aspects of the molar (Fig. 1). OTW was observed in varying degrees in nearly all molar teeth in every aging group. In the youngest group, the cuspis dentis of the molar tooth was highly pointed in shape (Score 1; Fig. 1A, a). The pointed cuspis became more and more blunt with maturation (Score 2; Fig. 1B, b), and almost disappeared (Score 3; Fig. 1C, c) by 12 M (Fig. 2 and Table 2). Between 12 and 30 M, the corona dentis was gradually wearing out (Fig. 2 and Table 2). Eventually, a major part of the corona dentis was lost, and OTW almost reached the cervical line (Score 4: Fig. 1D, d). At this level, the fissures, which had been observed from the
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archives of oral biology 52 (2007) 844–849
Fig. 1 – Scoring of occlusal tooth wear. Photographs of cranial aspect (capital letters) and lingual aspect (lowercase letters) of teeth scored 1 (A, a, 25 D in =1 M group), 2 (B, b, 92 D in 3–4 M group), 3 (C, c, 362 D in 12 M group) and 4 (D, d, 25 1196 D in >35 M group). Arrows: cuspis dentis; arrowheads: cervical line; double-arrowheads: enamel; *: dentin; Bar: 1 mm.
cranial view (Fig. 1A–C), had disappeared, and the tooth became mortar-like in shape (Fig. 1D, d). Though it was difficult to determine whether the dentin had been exposed to the occlusal surface because of the pointed cuspis dentis, the dentin was clearly observed on the occlusal surface of the teeth scored 3–4 (Fig. 1C and D). M3 teeth in every quadrant of 22 D rats (2 out of 7 rats in group 1 M) had not erupted, and were observed to be buried in the maxilla (data not shown). They were thus excluded from scoring of OTW. OTW scores of M1 in the lower jaw were higher than those in the upper jaw (P < 0.01), and the scores of M3 in the upper jaw were higher than those of M1 (P < 0.01) or M2 (P < 0.05) in the upper jaw (Table 2). Also, it was revealed that the scores of M1 were significantly higher than those of M2 or M3 (P < 0.01) in
the lower jaw and that the scores of M3 were significantly higher than those of M1 or M2 (Table 2). With the observation of individual rats, OTW of M1 in the lower jaw and M3 in the upper jaw tended to progress than others (Fig. 3). Though OTW scores of M2 either in the upper or lower jaw tended to be smaller than others before 30 M, they increased rapidly after 35 M so as to be larger than others (Table 2). Analysis with linear approximations on dispersions of M1–3 in the upper or lower jaw of each individual rat revealed that there were three phases in the increasing rate of the OTW score; the first phase showed an acute increase before 12 M, the second phase a moderate increase between 18 and 30 M, and the third phase an acute increase after 35 M (Fig. 2). However, M1 showed a slower increase than the others in the third phase (Fig. 2).
Table 2 – Occlusal tooth wear score (median/IQR) of each molar (M1–3)
*P < 0.05; **P < 0.01 vs. neighbouring younger aging stage; zP < 0.01 between upper and lower jaws; - - -P < 0.05; —P < 0.01 between positions of molar teeth (Wilcoxon rank sum test).
archives of oral biology 52 (2007) 844–849
Fig. 2 – Relation between age and occlusal tooth wear score. The linear approximation was calculated on a scatter plot of OTW scores of each tooth (M1–3) in the upper (A) or lower (B) jaw of all individual animals. There are three phases in increasing rate of the OTW score; first phase: rapid increase before 12 M, second phase: moderate increase between 18 and 30 M and third phase: rapid increase after 35 M.
4.
Discussion
Tanaka et al.1 reported the survival of female F344/N rats at NILS. The rats started to die after approximately 18 M, but they survived longer if their age exceeded 35 M. The samples used
Fig. 3 – Prominent example of the variation in the occlusal tooth wear score among molar teeth in upper or lower jaw. M1 tooth in lower jaw and M3 tooth in upper jaw were more worn (Score 4) than others (Score 3) in an individual rat of 828 D. Bar: 1 mm.
847
in this study were prepared in light of their study, and the progress of OTW was well correlated with the survival curve as a result. The result revealed that the three phases in progress of OTW corresponded to the respective growing, aging and senescence phase in the whole life span of the rats. OTW has been reported to progress with aging, and is commonly used to estimate the age of humans in anthropology19–21 or animals in zoology.22 This study clearly demonstrates the aging change of OTW in female F344/N rats. OTW originates from mechanical (attrition) and chemical factors (erosion).22,23 With the observation in this study, OTW was not divided into attrition or erosion. However, the animals used in this study were fed on a standard diet (see Section 2), and their oral cavities were maintained in the normal condition. Additionally, since no wear was observed except on the occlusal surface, the erosion would hardly influence the progress of OTW in the rats. The M1 teeth of rats appeared to be inclined forward in the youngest group, and gradually became vertical at the alveolar border when the feeding was changed from liquid to solid diet, and OTW began to progress. The acute increase of OTW before 12 M (Fig. 2) was probably caused by increasing pressure on the tooth in accordance with maturation of developing masticatory muscles and/or the pointed shape of the cuspis dentis (Fig. 1A, a). The acceleration in OTW after 35 M (Fig. 2) should have been caused by the fact that the dentin was softer than enamel. Thus, most of the enamel on the occlusal surface would have worn out by 30 M. Since there were no differences between the right and left quadrants (data not shown), the rats used in this study would have normal occlusion originating in normal mastication at least in the lateral direction. It was interesting that higher OTW progress was observed in the molar teeth at contraposition (lower M1 and upper M3 in Fig. 3 and Table 2). This might be caused by oblique pressure, which possibly reflects some characteristics of rat masticatory patterns. In younger rats, M1 teeth probably played a major role in comminution of alimentary bolus because of its anterior position, larger size and earlier progress of the OTW. However, the imbalanced OTW would cause abnormal occlusion on M1 and M3 teeth by 30 M, and it was speculated that M2 might take on the major role in comminution and thus wear out very rapidly after 35 M (Table 2). Also, the slower increase of the score of M1 than others after 35 M (Fig. 2) might indicate that comminution was scarcely done with M1 teeth. Since the hard enamel surrounded the corona dentis and alimentary bolus between upper and lower molar teeth, the soft dentin wore more than the hard enamel, and every tooth became mortar-like in shape with the progress of OTW (Fig. 1C, c). In human, compared with other animals, OTW increases slowly22–24 along with the length of life span. This might be due to the fact that human mastication puts only a very weak pressure on teeth by having soft diets which need not be torn, crushed or mashed with teeth. Additionally, within clinical dentistry, severe caries or periodontal diseases are cured by tooth extraction and replacement with artificial teeth. Thus, OTW rarely becomes a problem in normal conditions. However, OTW sometimes develops into a problem in case of bruxism or senescence. Recently, therapeutic efforts to retain natural teeth have been widely employed in Japan
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archives of oral biology 52 (2007) 844–849
because of the enjoyment associated with eating and its contribution to the maintenance of QOL. In this case, OTW progresses in natural teeth of the elderly, and serious OTW causes abnormal occlusion. It is shown that abnormal occlusion would influence functional performance of the masticatory muscles25,26 and lead to constitutional symptoms like migraine and palsy.7 To avoid such problems, clinical research on OTW with model animals should be developed. The molar teeth of rats aged over 12 M with low or no pointed cuspis dentis resembled those of humans in shape (Fig. 1C, c) and slow progress of OTW. In this phase, almost all of the occlusal surface of rat molar teeth was worn to the dentin, and this is similar to the profile of the molar teeth occasionally found in humans over 40 years old,27 although it certainly depends on dietary habits or occlusal conditions. This finding suggested that rats aged over 12 M would be an adequate animal model for OTW in middle-aged and elder human beings. In addition, mastication patterns of rats resemble those of human beings with open–close, lateral and back-and-forth movements in comminution of alimentary bolus,28 which might be indicated by the features of the head of the mandible and temporomandibular articulation. This also lends support to the notion that rats could be used as a model animal for research on the oral cavity. However, it is indispensable to consider the differences in dietary patterns between rats and humans when the oral cavity of rats is associated with that of humans. Though it is risky to prepare rats in a senescence phase (>35 M) because of their low survivability,1 findings in rats aged 12–30 M could be extrapolated to humans over 40 years old including the elderly, for tooth research. The resorption of the alveolar bone in the maxilla of F344/N rats reportedly did not progress after 850 D.5 These findings indicate that it is more important to prepare healthy rats aged 12–30 M than to collect rats aged over 35 M for oral cavity studies on the elderly. It is commonly recognized that OTW tends to be more progressive in male than female.21 This is because the masticatory muscles of the male are stronger than those of the female. Additionally, the mineral metabolism probably affects the progress of OTW. Thus, it is important to perform comparative examinations of OTW between genders as well as among strains and substrains in future study.
5.
Conclusions
This study demonstrates that characteristics of OTW change with aging in the molar teeth of female F344/N rats. OTW progressed with aging in three phases, corresponding to growth, aging and senescence stages in the whole life span of rats. Though the rat molar tooth was pointed in shape and readily showed wear, OTW of molar teeth of rats aged over 12 M had a profile similar to that occasionally found in humans over 40 years old. This would suggest that rats aged over 12 M would be an adequate and appropriate animal model for tooth research for middle-aged and elderly people.
references
1. Tanaka S, Segawa T, Tayama N, Ohno T. Establishment of an aging farm of F344/N rats and C57BL/6 mice at the National Institute for Longevity Sciences (NILS). Arch Gerontol Geriatr 2000;30:215–23. 2. Miyaishi O, Tanaka S, Kanawa R, Matsuzawa K, Isobe K. Anisocytosis precedes onset of the large granular lymphocyte leukemia in aged F344/N rats. Arch Gerontol Geriatr 2000;30:161–72. 3. Yamamoto Y, Tanaka A, Kanamaru A, Tanaka S, Tsubone H, Atoji Y, et al. Morphology of aging lung in F344/N rat: alveolar size, connective tissue, and smooth muscle cell markers. Anat Rec A Discov Mol Cell Evol Biol 2003;272:538–47. 4. Yamamoto Y, Tanaka S, Tsubone H, Atoji Y, Suzuki Y. Agerelated changes in sensory and secretomotor nerve endings in the larynx of F344/N rat. Arch Gerontol Geriatr 2003;36: 173–83. 5. Arai K, Tanaka S, Yamamoto-Sawamura T, Sone K, Miyaishi O, Sumi Y. Aging changes in the periodontal bone of F344/N rat. Arch Gerontol Geriatr 2005;40:225–9. 6. Tsukahara S, Tanaka S, Ishida K, Hoshi N, Kitagawa H. Agerelated change and its sex differences in histoarchitecture of the hypothalamic suprachiasmatic nucleus of F344/N rats. Exp Gerontol 2005;40:147–55. 7. Steele JG, Lamey PJ, Sharkey SW, Smith GM. Occlusal abnormalities, pericranial muscle and joint tenderness and tooth wear in a group of migraine patients. J Oral Rehabil 1991;18:453–8. 8. Mongini F, Ciccone G, Ibertis F, Negro C. Personality characteristics and accompanying symptoms in temporomandibular joint dysfunction, headache, and facial pain. J Orofac Pain 2000;14:52–8. 9. Tataroglu C, Kanik A, Sahin G, Ozge A, Yalcinkaya D, Idiman F. Exteroceptive suppression patterns of masseter and temporalis muscles in central and peripheral headache disorders. Cephalalgia 2002;22:444–52. 10. Kim ST, Kim CY. Use of the ID Migraine questionnaire for migraine in TMJ and Orofacial Pain Clinic. Headache 2006;46:253–8. 11. Kato T, Usami T, Noda Y, Hasegawa M, Ueda M, Nabeshima T. The effect of the loss of molar teeth on spatial memory and acetylcholine release from the parietal cortex in aged rats. Behav Brain Res 1997;83:239–42. 12. Onozuka M, Watanabe K, Nagasaki S, Jiang Y, Ozono S, Nishiyama K, et al. Impairment of spatial memory and changes in astroglial responsiveness following loss of molar teeth in aged SAMP8 mice. Behav Brain Res 2000;108:145–55. 13. Miura H, Kariyasu M, Yamasaki K, Arai Y, Sumi Y. Relationship between general health status and the change in chewing ability: a longitudinal study of the frail elderly in Japan over a 3-year period. Gerodontology 2005;22:200–5. 14. Blaszczyk J, Tajchert K. Sex and strain differences of acoustic startle reaction development in adolescent albino Wistar and hooded rats. Acta Neurobiol Exp (Wars) 1996;56:919–25. 15. Snyder DL, Gayheart-Walsten PA, Rhie S, Wang W, Roberts J. Effect of age, gender, rat strain, and dietary restriction, on norepinephrine release from cardiac synaptosomes. J Gerontol 1998;53:B33–41. 16. Goren HJ, Kulkarni RN, Kahn CR. Glucose homeostasis and tissue transcript content of insulin signaling intermediates in four inbred strains of mice: C57BL/6, C57BLKS/6, DBA/2, and 129X1. Endocrinology 2004;145:3307–23. 17. Tanaka S, Segawa T, Tamaya N, Miyaishi O, Ohno T. A group of five parameters as a new biological marker on F344/N rats. Arch Gerontol Geriatr 2001;32:139–50.
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18. Broca P. Instruction Relatives a` lE´tude Anthropologique du Systeme Dentaire. Bull Soc Anthrop (Paris) 1879;2:149. 19. Seligman DA, Pullinger AG, Solberg WK. The prevalence of dental attrition and its association with factors of age, gender, occlusion, and TMJ symptomatology. J Dent Res 1988;67:1323–33. 20. Li C, Ji G. Age estimation from the permanent molar in northeast China by the method of average stage of attrition. Forensic Sci Int 1995;75:189–96. 21. Kim YK, Kho HS, Lee KH. Age estimation by occlusal tooth wear. J Forensic Sci 2000;45:303–9. 22. Baitchman EJ, Kollias GV. Clinical anatomy of the North American river otter (Lontra canadensis). J Zoo Wildl Med 2000;31:473–83. 23. Ganss C, Klimek J, Starck C. Quantitative analysis of the impact of the organic matrix on the fluoride effect on
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erosion progression in human dentine using longitudinal microradiography. Arch Oral Biol 2004;49:931–5. Ganss C. Definition of erosion and links to tooth wear. Monogr Oral Sci 2006;20:9–16. Bani D, Bani T, Bergamini M. Morphologic and biochemical changes of the masseter muscles induced by occlusal wear: studies in a rat model. J Dent Res 1999;78:1735–44. Bani D, Bergamini M. Dantrolene counteracts the masseter muscle damage induced by artificial occlusal wear: studies in a rat model. J Dent Res 2001;80:1990–4. Butler RJ. Age-related variability in occlusal wear planes. Am J Phys Anthrop 1972;36:381–90. Byrd KE, Chai Y. Three-dimensional movement analysis of lateral pterygoid electromyographic activity during mastication in the rat. Arch Oral Biol 1988;33: 635–40.
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/arob
Occlusal tooth wear in female F344/N rats with aging Kazutoshi Nishijima a,*, Sachi Kuwahara a,b, Tamio Ohno c, Osamu Miyaishi d, Yoshitake Ito e, Yasunori Sumi f, Shin Tanaka a a
Animal Facility for Aging Research, National Institute for Longevity Sciences (NILS), National Center for Geriatrics and Gerontology (NCGG), Obu, Japan b Department of Anatomy, Hyogo College of Medicine, Nishinomiya, Japan c Division of Experimental Animals, Center for Promotion of Medical Research and Education, Graduate School of Nagoya University, Nagoya, Japan d Chubu Rosai Hospital, Nagoya, Japan e Laboratory Animal Research Center, Aichi Medical University, School of Medicine, Nagakute, Aichi, Japan f Division of Oral and Dental Surgery, Department of Advanced Medicine, National Hospital for Geriatric Medicine, NCGG, Obu, Japan
article info
abstract
Article history:
Objectives: This study was conducted to ascertain whether laboratory rats are an adequate
Accepted 6 March 2007
animal model for aging oral cavity research, especially on occlusal tooth wear (OTW), which progresses with aging and causes abnormal occlusions. Mastication has been reported to
Keywords:
relate to cognition in the elderly. Thus, it is important to care for the oral cavity, especially in
Attrition
the frail elderly, for the maintenance of all-round quality of life. Adequate and appropriate
Aging animal model
animal models are essential for basic and clinical research on the oral cavity.
Mastication
Methods: Dried maxilla and mandible specimens from 98 young, aging or aged female F344/
Molar teeth
N rats were used.
Senescence
Results: The levels of OTW of all molars were monitored with aging. The molar tooth began to wear at 1-month old (M) and progressed rapidly till 12 M. Subsequently, OTW progressed slowly till 30 M, and then rapidly again after 35 M. Conclusions: This study showed that progress of OTW is well correlated with the entire life span of the rat, and suggested that the rat aged over 12 M would be an adequate animal model for research on OTW in middle-aged and elderly people. # 2007 Elsevier Ltd. All rights reserved.
1.
Introduction
In developed countries, human life expectancy in recent decades has been greatly prolonged, and the elderly population has been rapidly growing. According to the World Health Report 2006 by WHO, more than 20% of people are over 60 years old in European countries and Japan. The average life expectancy in Japan was 86 years for females and 79 years for males, and in 2002 both male and female reached the longest life expectancy in the world. Japan is becoming an aging society at a globally unprecedented pace. It is widely acknowledged, both within
and outside the government of Japan, that more than one in four Japanese will be over 65 years old by 2015. Health research on senior citizens is needed, especially regarding the frail elderly, for the maintenance of their quality of life (QOL). Aging and/or aged animal models are required in such studies for biological understanding of aging properties. The National Institute for Longevity Sciences (NILS) established an aging farm and succeeded in the production of 30 months old (M) F344/N rats.1 Aging and/or aged rats have been analyzed from various aspects and have revealed the relationship between morphological changes and aging.2–6
* Corresponding author. Tel.: +81 562 446597; fax: +81 562 446597. E-mail address: [email protected] (K. Nishijima). 0003–9969/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2007.03.001
845
archives of oral biology 52 (2007) 844–849
Dysfunctions of the oral cavity, caused by various diseases including peridontics, caries and abnormal occlusions, may lead not only to poor nourishment but also to constitutional symptoms like migraine or palsy through temporomandibular disorders.7–10 In addition, mastication has been reported to relate with cognition in both certain young laboratory animals11,12 and elderly humans.13 Since the wellness and maintenance of the oral cavity in the elderly humans have been proposed and approved, a care program as well as animal models are required for the identification of aging changes of oral conditions. Aging changes in the periodontal bone of F344/ N rats have been reported, and it has been suggested that rats could be used as a model for periodontal research.5 However, few precise and systemic aging analysis reports have been available on the teeth of rats. Additionally, the aging characteristics of rats have been reported to be different depending on the strain, substrain and gender.14–16 These are known to differ among laboratory animal species as well. The elucidation and assessment of species-, strains- and genderspecific changes are the key to streamlined, waste-free animal experiments. Survival in male F344/N rats was shorter than in females,17 probably due to a higher incidence and earlier onset of leukemia in males than in females.2 This may indicate that female F344/N rats, without the modification by leukemia, are a better model than males for aging and longevity research. In this study, we focused on and monitored occlusal tooth wear (OTW) of molars, which progresses with aging and causes abnormal occlusions, in female F344/N rats aged from 1 to more than 35 M. Our aim was to clarify aging change of the molar teeth in rats, including the difference in the progress of OTW among quadrants and position of the teeth, and to examine whether rats are adequate laboratory animals to be used for aging tooth research.
2.
Materials and methods
2.1.
Materials
Aging and aged female F344/N rats were established in the NILS aging farm,1 and younger animals were purchased from Japan SLC, Inc. (Hamamatsu, Japan). All animals were fed with a pellet diet (Labo MR Stock, pH: 6.18, hardness: 31.6 kg/cm2, Nosan Corp., Yokohama) in accordance with the NILS Aging Farm Guide1 (Tanaka et al. 2000). The animals, 98 in all, were divided into 11 groups by their age; 1, 2, 3–4, 6–7, 12, 18, 21, 24, 27, 30 and >35 M, respectively (each group contained 6–12 animals; see Table 1 for details). The animals were sacrificed with an overdose of anesthetic. The head was dissected and autoclaved at 121 8C for 5 min. Major parts of the soft tissue were removed from the skull and mandible which were subsequently incubated in 0.5% papain solution (ED 3.4.22.2, Merck, Darmstadt, Germany) for overnight at 37.0 8C. The dried bone specimens were prepared for measurements of the OTW score. Molar teeth were observed with a stereoscopic microscope, and the OTW level was scored. The experimental protocol was performed with the permission of the Committee for Animal Ethics NILS according to the Guideline of NILS Animal Experimentation with due care for animal rights and welfare.
Table 1 – Age groups of animals used in this study Group
Days old (mean)
1 M 2M 3–4 M 6–7 M 12 M 18 M 21 M 24 M 27 M 30 M >35 M
22–30 (26.3) 42–55 (46.4) 92–136 (111.7) 168–214 (194.5) 350–377 (363.4) 547–554 (548.0) 628–651 (641.9) 726–732 (728.5) 813–828 (821.4) 909–916 (912.5) 1087–1196 (1128.6)
Total
2.2.
No. of animals 7 7 6 8 8 9 10 11 10 12 10 98
Scoring and statistics
The occlusal surface of molar tooth of (especially young) rat has several high cuspis dentis protuberances which made it impossible to distinguish between the enamel and dentin. For this reason, we needed to modify Broca’s index,18 which is based on the degree of exposure of dentin, to be suitable for rats. Scoring was as follows: Score 0, no wear observed; Score 1, wear at apex of cuspis dentis; Score 2, wear of cuspis dentis; Score 3, loss of cuspis dentis; Score 4, loss of corona dentis (Fig. 1). Three molar teeth in each quadrant were numbered M1 to M3 in accordance with anterior to posterior positions. Since Wilcoxon signed-rank test revealed no significant differences in each molar tooth between left side and right side in OTW scores, the scores of bilateral molar teeth were combined. The medians and intraquartile ranges in OTW scores of all molar teeth were calculated in each aging group. Nonparametric Kruskal–Wallis, for multiple comparison, and Wilcoxon rank sum tests were employed to reveal the differences in OTW scores among positions of the teeth (M1– 3) in every aging group with SPSS 13.0. Differences of OTW scores between neighboring aging groups were measured with the same tests. Linear approximation was calculated with Excel (2002, Microsoft) Fseparately, 22–377 (1–12 M), 547–919 (18–30 M) and 1087–1196 (>35 M) days old (D), on scatter plots of OTW scores of each molar tooth (M1–3) in the upper or lower jaw of all individual animals.
3.
Results
The molar teeth of rats are classified as boundont with several cuspis, and three major peaks were prominently observed (Fig. 1A, a). Tooth wear was observed only on the occlusal surface, not on any other aspects of the molar (Fig. 1). OTW was observed in varying degrees in nearly all molar teeth in every aging group. In the youngest group, the cuspis dentis of the molar tooth was highly pointed in shape (Score 1; Fig. 1A, a). The pointed cuspis became more and more blunt with maturation (Score 2; Fig. 1B, b), and almost disappeared (Score 3; Fig. 1C, c) by 12 M (Fig. 2 and Table 2). Between 12 and 30 M, the corona dentis was gradually wearing out (Fig. 2 and Table 2). Eventually, a major part of the corona dentis was lost, and OTW almost reached the cervical line (Score 4: Fig. 1D, d). At this level, the fissures, which had been observed from the
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Fig. 1 – Scoring of occlusal tooth wear. Photographs of cranial aspect (capital letters) and lingual aspect (lowercase letters) of teeth scored 1 (A, a, 25 D in =1 M group), 2 (B, b, 92 D in 3–4 M group), 3 (C, c, 362 D in 12 M group) and 4 (D, d, 25 1196 D in >35 M group). Arrows: cuspis dentis; arrowheads: cervical line; double-arrowheads: enamel; *: dentin; Bar: 1 mm.
cranial view (Fig. 1A–C), had disappeared, and the tooth became mortar-like in shape (Fig. 1D, d). Though it was difficult to determine whether the dentin had been exposed to the occlusal surface because of the pointed cuspis dentis, the dentin was clearly observed on the occlusal surface of the teeth scored 3–4 (Fig. 1C and D). M3 teeth in every quadrant of 22 D rats (2 out of 7 rats in group 1 M) had not erupted, and were observed to be buried in the maxilla (data not shown). They were thus excluded from scoring of OTW. OTW scores of M1 in the lower jaw were higher than those in the upper jaw (P < 0.01), and the scores of M3 in the upper jaw were higher than those of M1 (P < 0.01) or M2 (P < 0.05) in the upper jaw (Table 2). Also, it was revealed that the scores of M1 were significantly higher than those of M2 or M3 (P < 0.01) in
the lower jaw and that the scores of M3 were significantly higher than those of M1 or M2 (Table 2). With the observation of individual rats, OTW of M1 in the lower jaw and M3 in the upper jaw tended to progress than others (Fig. 3). Though OTW scores of M2 either in the upper or lower jaw tended to be smaller than others before 30 M, they increased rapidly after 35 M so as to be larger than others (Table 2). Analysis with linear approximations on dispersions of M1–3 in the upper or lower jaw of each individual rat revealed that there were three phases in the increasing rate of the OTW score; the first phase showed an acute increase before 12 M, the second phase a moderate increase between 18 and 30 M, and the third phase an acute increase after 35 M (Fig. 2). However, M1 showed a slower increase than the others in the third phase (Fig. 2).
Table 2 – Occlusal tooth wear score (median/IQR) of each molar (M1–3)
*P < 0.05; **P < 0.01 vs. neighbouring younger aging stage; zP < 0.01 between upper and lower jaws; - - -P < 0.05; —P < 0.01 between positions of molar teeth (Wilcoxon rank sum test).
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Fig. 2 – Relation between age and occlusal tooth wear score. The linear approximation was calculated on a scatter plot of OTW scores of each tooth (M1–3) in the upper (A) or lower (B) jaw of all individual animals. There are three phases in increasing rate of the OTW score; first phase: rapid increase before 12 M, second phase: moderate increase between 18 and 30 M and third phase: rapid increase after 35 M.
4.
Discussion
Tanaka et al.1 reported the survival of female F344/N rats at NILS. The rats started to die after approximately 18 M, but they survived longer if their age exceeded 35 M. The samples used
Fig. 3 – Prominent example of the variation in the occlusal tooth wear score among molar teeth in upper or lower jaw. M1 tooth in lower jaw and M3 tooth in upper jaw were more worn (Score 4) than others (Score 3) in an individual rat of 828 D. Bar: 1 mm.
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in this study were prepared in light of their study, and the progress of OTW was well correlated with the survival curve as a result. The result revealed that the three phases in progress of OTW corresponded to the respective growing, aging and senescence phase in the whole life span of the rats. OTW has been reported to progress with aging, and is commonly used to estimate the age of humans in anthropology19–21 or animals in zoology.22 This study clearly demonstrates the aging change of OTW in female F344/N rats. OTW originates from mechanical (attrition) and chemical factors (erosion).22,23 With the observation in this study, OTW was not divided into attrition or erosion. However, the animals used in this study were fed on a standard diet (see Section 2), and their oral cavities were maintained in the normal condition. Additionally, since no wear was observed except on the occlusal surface, the erosion would hardly influence the progress of OTW in the rats. The M1 teeth of rats appeared to be inclined forward in the youngest group, and gradually became vertical at the alveolar border when the feeding was changed from liquid to solid diet, and OTW began to progress. The acute increase of OTW before 12 M (Fig. 2) was probably caused by increasing pressure on the tooth in accordance with maturation of developing masticatory muscles and/or the pointed shape of the cuspis dentis (Fig. 1A, a). The acceleration in OTW after 35 M (Fig. 2) should have been caused by the fact that the dentin was softer than enamel. Thus, most of the enamel on the occlusal surface would have worn out by 30 M. Since there were no differences between the right and left quadrants (data not shown), the rats used in this study would have normal occlusion originating in normal mastication at least in the lateral direction. It was interesting that higher OTW progress was observed in the molar teeth at contraposition (lower M1 and upper M3 in Fig. 3 and Table 2). This might be caused by oblique pressure, which possibly reflects some characteristics of rat masticatory patterns. In younger rats, M1 teeth probably played a major role in comminution of alimentary bolus because of its anterior position, larger size and earlier progress of the OTW. However, the imbalanced OTW would cause abnormal occlusion on M1 and M3 teeth by 30 M, and it was speculated that M2 might take on the major role in comminution and thus wear out very rapidly after 35 M (Table 2). Also, the slower increase of the score of M1 than others after 35 M (Fig. 2) might indicate that comminution was scarcely done with M1 teeth. Since the hard enamel surrounded the corona dentis and alimentary bolus between upper and lower molar teeth, the soft dentin wore more than the hard enamel, and every tooth became mortar-like in shape with the progress of OTW (Fig. 1C, c). In human, compared with other animals, OTW increases slowly22–24 along with the length of life span. This might be due to the fact that human mastication puts only a very weak pressure on teeth by having soft diets which need not be torn, crushed or mashed with teeth. Additionally, within clinical dentistry, severe caries or periodontal diseases are cured by tooth extraction and replacement with artificial teeth. Thus, OTW rarely becomes a problem in normal conditions. However, OTW sometimes develops into a problem in case of bruxism or senescence. Recently, therapeutic efforts to retain natural teeth have been widely employed in Japan
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because of the enjoyment associated with eating and its contribution to the maintenance of QOL. In this case, OTW progresses in natural teeth of the elderly, and serious OTW causes abnormal occlusion. It is shown that abnormal occlusion would influence functional performance of the masticatory muscles25,26 and lead to constitutional symptoms like migraine and palsy.7 To avoid such problems, clinical research on OTW with model animals should be developed. The molar teeth of rats aged over 12 M with low or no pointed cuspis dentis resembled those of humans in shape (Fig. 1C, c) and slow progress of OTW. In this phase, almost all of the occlusal surface of rat molar teeth was worn to the dentin, and this is similar to the profile of the molar teeth occasionally found in humans over 40 years old,27 although it certainly depends on dietary habits or occlusal conditions. This finding suggested that rats aged over 12 M would be an adequate animal model for OTW in middle-aged and elder human beings. In addition, mastication patterns of rats resemble those of human beings with open–close, lateral and back-and-forth movements in comminution of alimentary bolus,28 which might be indicated by the features of the head of the mandible and temporomandibular articulation. This also lends support to the notion that rats could be used as a model animal for research on the oral cavity. However, it is indispensable to consider the differences in dietary patterns between rats and humans when the oral cavity of rats is associated with that of humans. Though it is risky to prepare rats in a senescence phase (>35 M) because of their low survivability,1 findings in rats aged 12–30 M could be extrapolated to humans over 40 years old including the elderly, for tooth research. The resorption of the alveolar bone in the maxilla of F344/N rats reportedly did not progress after 850 D.5 These findings indicate that it is more important to prepare healthy rats aged 12–30 M than to collect rats aged over 35 M for oral cavity studies on the elderly. It is commonly recognized that OTW tends to be more progressive in male than female.21 This is because the masticatory muscles of the male are stronger than those of the female. Additionally, the mineral metabolism probably affects the progress of OTW. Thus, it is important to perform comparative examinations of OTW between genders as well as among strains and substrains in future study.
5.
Conclusions
This study demonstrates that characteristics of OTW change with aging in the molar teeth of female F344/N rats. OTW progressed with aging in three phases, corresponding to growth, aging and senescence stages in the whole life span of rats. Though the rat molar tooth was pointed in shape and readily showed wear, OTW of molar teeth of rats aged over 12 M had a profile similar to that occasionally found in humans over 40 years old. This would suggest that rats aged over 12 M would be an adequate and appropriate animal model for tooth research for middle-aged and elderly people.
references
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