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Age-dependent increase of clusterin in the human pituitary gland
Legal Medicine 8 (2006) 28–33 www.elsevier.com/locate/legalmed
Age-dependent increase of clusterin in the human pituitary gland Takaki Ishikawa*, Bao-Li Zhu, Dong-Ri Li, Dong Zhao, Tomomi Michiue, Hitoshi Maeda Department of Legal Medicine, Osaka City University Medical School, Asahi-machi 1-4-3, Abeno, Osaka 545-8585, Japan Received 11 July 2005; received in revised form 9 August 2005; accepted 17 August 2005 Available online 10 October 2005
Abstract Clusterin is a glycoprotein known to play various physiological roles including complement activity, amyloid binding activity in Alzheimer disease, as well as binding with heat shock proteins and abnormal prions. The present study immunohistochemically investigated the expression of clusterin in the human pituitary gland in subjects of 10–88 years of age (nZ173). Causes of death were blunt injury (nZ35), sharp injury (nZ15), poisoning (nZ11), drowning (nZ14), fire fatalities (nZ28), asphyxiation (nZ15), hypothermia (nZ7), hyperthermia (nZ3), and natural diseases (nZ45). Clusterin was detected in mixed cell follicles and the anterior lobar parenchymal cells. The area occupied by cells positive for clusterin were measured, and the ratio to the whole area of the anterior lobe (% clusterin-positive cell area) was estimated. There was a good correlation between the age of the subjects in years and the % clusterin-positive cell area in the anterior lobe of the pituitary gland (rZ0.736, P!0.01). Relationships between % clusterin-positive cell and gender, cause of death, and survival time were insignificant. These findings indicate an age-dependent accumulation of clusterin in the pituitary gland, which may be related to the aging of endocrine systems. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Human pituitary; Clusterin; Aging; Immunohistochemistry
1. Introduction The tasks of forensic pathology involve the investigation of individual predispositions to fatal outcomes due to traumas and diseases, and age estimation for unidentified bodies. For these purposes, evaluation of degenerative changes due to aging is important. Although age estimation is usually performed based on skeletal and dental maturation and degeneration [1–3], histological and immunohistochemical investigations of skeletal muscle, the cerebrum [4], the hippocampus [5] and the pituitary gland [6–8] have been also reported. Previous studies of the pituitary gland showed increases in 8-hydroxy-2 0 -deoxyguanosine (8-OHdG), S-100 protein and follicular structures. Recent anatomical and physiological studies have demonstrated an increase of follicles in the adenohypophysis of aged animals [9,10]. However, various acute stress may also cause such structural changes [11]. Ogawa et al. [10] observed clusterin-containing follicles in * Corresponding author. Tel.: C81 6 6645 3767; fax: 81 6 6634 3871. E-mail address: [email protected] (T. Ishikawa).
1344-6223/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2005.08.009
porcine adenohypophysis and reported the relationship between cells containing clusterin and aging. Clusterin, apolipoprotein J (apoJ), is a circulating glycoprotein produced in various kinds of epithelial cells. This protein is induced in the tissues or organs affected by various diseases, for instance, Alzheimer’s disease, Creutzfeldt– Jakob disease, atherosclerosis, myocardial infarction and acute and chronic renal diseases [12–16]. Previously, clusterin levels were determined by chemical methods [17,18] and by means of the cloning of repair enzymes for clusterin, followed by the production of specific monoclonal antibodies to clusterin [19]. Recently, the immunohistochemical method for detection of clusterin has been established. O’Bryan et al. [20] revealed immunohistochemically the enhanced level of clusterin in mouse brains by aging. The pituitary gland is a central neuroendocrine organ, and it is known that dysfunction or hypofunction occurs in the neuroendocrine system with age [21,22], which may cause susceptibility or lability to diseases and traumas. In the present paper, we report the expression of clusterin in the anterior lobar parenchymal cells of the human
T. Ishikawa et al. / Legal Medicine 8 (2006) 28–33
pituitary gland and discuss the change of clusterin expression during aging as a marker of degeneration of the neuroendocrine systems.
2. Materials and methods 2.1. Specimens From serial autopsy cases within 3 days postmortem, adenohypophyses (nZ173: 119 males and 54 females) were obtained. The age of the subjects ranged from 10 to 88 (mean, 48.2) years. The cases were classified according to age into four groups as follows: I, !30 years (nZ27); II, 30–49 years (nZ44); III, 50–69 years (nZ64); IV, O69 years (nZ38). Causes of death were blunt injury (nZ35), sharp injury (nZ15), poisoning (nZ11), drowning (nZ14), fire fatalities (nZ28), asphyxiation (nZ15), hypothermia (nZ7), hyperthermia (nZ3), and natural diseases (nZ45). Of 45 deaths by natural diseases, 32 were acute deaths and 13 were chronic deaths. Of all deaths by traumas, 96 were acute deaths and 32 were delayed deaths.
29
2.4. Statistical analysis Values in the text represent the meanGstandard error of the mean (SEM). Statistical analyses were performed using one factor analysis of variance (ANOVA) to examine the differences in mean values among the groups, followed by Scheffe´ F test to identify the significant difference in means. Simple regression analysis was used to examine the correlation between age and the % clusterin-positive cell area; the correlation coefficient and regression equation were calculated.
3. Results Immunohistochemically positive signals of clusterin were found at the anterior lobar parenchymal cells, mixed cell follicles and stellate cells of the pituitary glands (Figs.1–3). Stellate cells with long cytoplasmic projections were observed enclosing the follicles and cells (Fig. 2c). When the relationship between the % area of clusterinpositive parenchymal cells in the anterior lobe of the pituitary gland and the age of the subjects were examined by grouping, there was an age-dependent increase as shown in
2.2. Immunohistochemistry The pituitary gland specimens were fixed in 4% formaldehyde in phosphate-buffered saline (PBS, pH 7.2) for 12 h, embedded in paraffin and then sectioned at thicknesses of 4 mm. After deparaffinization, the sections were immersed in 0.3% H2O2-methanol for 30 min to inactivate endogenous peroxidase. After washing in PBS (3!5 min), blocking with PBS containing 1% normal goat serum and 1% bovine serum albumin was performed at room temperature for 30 min. Then, rabbit anti-human clusterin-a/b (H-330) polyclonal antibody (Santa Cruz, CA) was reacted at an IgG concentration of 1 mg/ml at 48C for 24 h. After washing in PBS (3!5 min), EnvisionC (Dako, Japan) for rabbit immunoglobulin was reacted at room temperature for 30 min, and positive reactions were then visualized with PBS containing 0.02% diaminobenzidine (Sigma, Japan) at room temperature for 5 min. As negative controls, normal rabbit IgG was added instead of the primary antibody. 2.3. Semi-quantitative measurement of clusterin-containing cells The area of positively stained cells was measured three times using an Olympus Color Image Analyzer (VIP-21CH, Olympus, Tokyo, Japan). The result is expressed as the proportion of the average area of clusterin-containing cells to the whole area of the anterior pituitary (% clusterinpositive cell area).
Fig. 1. (a) H.E. findings of the adenohypophysis from a 15-year-old male. (b) Micrograph showing immunostaining of clusterin-positive cells. Positive signals of clusterin were observed in the follicles and cells of the anterior lobe of the pituitary.
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T. Ishikawa et al. / Legal Medicine 8 (2006) 28–33
Fig. 2. (a) H.E. findings of adenohypophysis from a 55-year-old male. (b) Immunostaining of clusterin-positive cells increases in the adenohypophysis. (c) Micrograph showing a typical picture of clusterin-positive cells engulfing other cells. Bars indicate 30 mm.
Fig. 4. The mean values of % clusterin-positive cell area in groups I, II, III and IV were 25.3G1.8, 32.7G2.8, 36.2G 1.8 and 55.2G3.8%, respectively. There was a significant difference between groups I (!30 years) and IV (O69 years). There were no significant differences in groups I and IV in comparison with groups II (30–49 years) and III (50– 69 years) (PO0.05) or between groups II and III (PO0.05). Regression analysis revealed a significant correlation between the age in years and % clusterin-positive cell area (rZ0.736, P!0.01) (Fig. 5). There was no genderdifference, and the correlation coefficients (r) for males and females were 0.720 (P!0.01) and 0.750 (P!0.01), respectively. In each age group, relationship between
Fig. 3. (a) H.E. findings of adenohypophysis from a 73-year-old male. (b) Numerous clusterin-positive cells occupied the adenohypophysis. (c) A follicle stained by clusterin was surrounded by cells. Bars indicate 60 mm.
% clusterin-positive cell and gender, cause of death, and survival time were insignificant.
4. Discussion In the present study, interestingly, there was a significant difference in pituitary clusterin levels between young (!30 years) and elderly (O69 years) subjects, and individual differences were evident among subjects ranging from 30– 70 years of age. This indicates that the pituitary clusterin level may be available as a marker of aging in individual
T. Ishikawa et al. / Legal Medicine 8 (2006) 28–33
(%)
*
% Clusterin positive cell area
100
* *
75 * 50
25
0 group
I < 30 n=30
II 30 - 49 n=51
III 50 - 69 n=69
IV > 69 n=23
Age (years) Fig. 4. The positive ratios of clusterin-positive cells in each age group. The values are presented as the meanCSEM. The mean positive ratio gradually increased with age. *, P!0.05 by analysis of variance, followed by Scheffe´ F test.
cases to investigate the degeneration of endocrine systems. In addition, in cases involving unidentified bodies, the clusterin level may serve as a marker for age estimation when combined with other morphological, histological, immunohistochemical, and molecular biological markers of aging [5,23–27]. Further investigation is necessary to clarify these findings with regard to susceptibility or lability to diseases and traumas. Recently, clusterin was found in senile plaques in the brains of patients suffering from Alzheimer’s disease (AD)
Age (years)
80
60
40
20
0 0
20
40
60
Clusterin positive ratio
80
(%)
Fig. 5. Linear regression analysis of the positive ratios of clusterin-positive cells in the anterior lobe of the pituitary gland versus age in all 173 samples. There is a significant correlation with age (rZ0.736, P!0.01). The regression equation is YZ1.325XK1.632 (YZage; XZclusterin-positive ratio).
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[28,29], and it was shown that clusterin was a major binding protein of b-amyloid [30]. Furthermore, clusterin mRNA levels were elevated in the AD hippocampus [31]. Although the pathogenetic mechanism for the increased expression of clusterin in AD is unclear, it may be related to a compensation mechanism involving aggregation of soluble b-amyloid, inhibition of complement-mediated cell damage or an adaptive regenerative response following neuronal degeneration [32]. These findings indicate that clusterin may increase in response to cell damage due to degenerative diseases and stress during aging over 30 years, which may subsequently increase individual differences in clusterin level, thus preventing significant age-dependency among subjects 30–70 years of age. Over 70 years of age, retrogressive changes of the pituitary gland may be an inevitable gerontological predisposition in stress responses to attacks of acute diseases and traumas. Previous study showed an increase of pituitary stellate cells in chronic alcohol abusers [33]. Similar increase in clusterin level was observed for liver cirrhosis, although it could not be statistically confirmed (unpublished data). Further investigation is necessary to clarify the cause of individual differences among middle-aged subjects, especially with regard to alcohol or drug abuse, degenerative diseases and chronic metabolic disorders. The observations described above suggest that pituitary clusterin is produced following degenerative changes, although the mechanism has not been established. However, localization of clusterin in the parenchymal cells and stellate cells suggests several hypotheses on the origin of clusterin in follicles of the pituitary gland. One hypothesis is production of clusterin by the stellate cells, which have similar characteristics to those of the astrocytes that produce clusterin [34]. Another possibility is clusterin expression related to apoptosis in the glandular cells adjacent to the stellate cells [35]. When the hormone-producing glandular cells undergo apoptosis involving clusterin production, they may be phagocytosed by the neighboring stellate cells [10, 36–39], and clusterin in the phagocytosed cells may subsequently be accumulated in the follicular colloid. The stellate cells also produce various growth factors and related proteins including basic fibroblast growth factor (bFGF) [40], vascular endothelial growth factor (VEGF) [41], follistatin [41], tissue inhibitor of metalloproteinase-II (TIMP-II) [42], and also regulate the secretion of prolactin [33,43] and cytokines [44,45]. Thus, the clusterin-positive cells can be involved in a spectrum of multifunctional cells in the anterior pituitary gland. Further investigation is necessary to establish the major roles of the clusterinpositive cells. In conclusion, the present study showed that the pituitary clusterin level increased with age, suggesting its usefulness in investigating retrogressive changes in neuroendocrine systems by aging, and its possible application to age estimation.
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[21] Meites J, Goya R, Takahashi S. Why the neuroendocrine system is important in aging processes. Exp Gerontol 1987;22:1–15. [22] Meites J. Importance of the neuroendocrine system in aging processes. Adv Biochem Psychophamacol 1987;43:283–92. [23] Paewinsky E, Pfeiffer H, Brinkmann B. Quantification of secondary dentine formation from orthopantomograms -a contribution to forensic age estimation methods in adults. Int J Leg Med 2005;119:27–30. [24] Olze A, Bilang D, Schmidt S, Wernecke KD, Geserick G, Schmeling A. Validation of common classification systems for assessing the mineralization of third molars. Int J Leg Med 2005; 119:22–6. [25] Ritz-Timme S, Rochholz G, Schutz HW, Collins MJ, Waite Er, Cattaneo C, et al. Quality assurance in age estimation based on aspartic acid racemisation. Int J Leg Med 2000;114:83–6. [26] Ohtani S, Ito R, Arany S, Yamamoto T. Racemization in enamel among different types of teeth from the same individual. Int J leg Med 2005;119:66–9. [27] de la Grandmaison GL, Banasr a, Durigon M. Age estimation using radiographic analysis of laryngeal cartilage. Am J Forensic Med Pathol 2003;24:96–9. [28] Dingemans KP, Feltkamp CA. Nongranulated cells in the mouse adenohypophysis. Z Zellforsch Mikrosk Anat 1972;124:387–405. [29] McGeer PL, Kawamata T, Walker DG. Distribution of clusterin in Alzheimer brain tissue. Brain Res 1992;579:337–41. [30] Choi-Miura NH, Ihara Y, Fukuchi K, Takeda M, Nakano Y, Tobe T, et al. SP-40, 40 is a constituent of Alzheimer’s amyloid. Acta Neuropathol 1992;83:260–4. [31] May PC, Lampert-Etchells M, Johnson SA, Poirier J, Masters JN, Finch CE. Dynamics of gene expression for a hippocampal glycoprotein elevated in Alzheimer’s disease and in response to experimental lesions in rat. Neuron 1990;5:831–9. [32] Lidstrom AM, Bogdanovic N, Hesse C, Volkman I, Davidsson P, Blennow K. Clusterin (apolipoprotein J) protein levels are increased in hippocampus and in frontal cortex in Alzheimer’s disease. Exp Neurol 1998;154:511–21. [33] Ishikawa T, Tachibana T, Ishikawa H, Miyaishi S, Ishizu H. Increase of S-100 protein-positive stellate cells in the anterior pituitary of chronic alcoholic patients with fatty liver or fatty cirrhosis. Acta Med Okayama 2003;57:53–8. [34] Pasinetti GM, Johnson SA, Oda T, Rozovsky I, Finch CE. Clusterin (SGP-2): a multifunctional glycoprotein with regional expression in astrocytes and neurons of the adult rat brain. J Comp Neurol 1994; 339:387–400. [35] Flach R, Cattaruzza M, Koch-Brandt C. Clusterin gene expression in apoptotic MDCK cells is dependent on the apoptosis-inducing stimulus. Biochim Biophys Acta 1995;1268:325–8. [36] Shen ZL, Lassner F, Bader A, Becker M, Walter GF, Berger A. Cellular activity of resident macrophages during Wallerian degeneration. Microsurgery 2000;20:255–61. [37] Giometto B, Miotto D, Botteri M, Alessio L, Scanarini M, An SF. Folliculo-stellate cells of human pituitary adenomas: immunohistochemical study of the monocyte/macrophage phenotype expression. Neuroendocrinology 1997;65:47–52. [38] Kameda Y. Occurrence of colloid-containing follicles in the pars distalis of pituitary glands from aging guinea pigs. Cell Tissue Res 1991;263:115–24. [39] Inoue K, Mogi C, Ogawa S, Tomida M, Miyai S. Are folliculo-stellate cells in the anterior pituitary gland supportive cells or organ-specific stem cells? Arch Physiol Biochem 2002;110:50–3. [40] Ferrara N, Schweigerer L, Neufeld G, Mitchell R, Gospodarowicz D. Pituitary follicular cells produce basic fibroblast growth factor. Proc Natl Acad Sci U S A 1987;84:5773–7. [41] Gospodarowicz D, Lau K. Pituitary follicular cells secrete both vascular endothelial growth factor and follistatin. Biochem Biophys Res Commun 1989;165:292–8. [42] Matsumoto H, Ishibashi Y, Ohtaki T, Hasegawa Y, Koyama C, Inoue K. Newly established murine pituitary folliculo-stellate-like
T. Ishikawa et al. / Legal Medicine 8 (2006) 28–33 cell line (TtT/GF) secretes potent pituitary glandular cell survival factors, one of which corresponds to metalloproteinase inhibitor. Biochem Biophys Res Commun 1993;194:909–15. [43] Ishikawa H, Nogami H, Shirasawa N. Novel clonal strains from adult rat anterior pituitary producing S-100 protein. Nature 1983;303: 711–3. [44] Matsumoto H, Koyama C, Sawada T, Koike K, Hirota K, Miyake A, et al. Pituitary folliculo-stellate-like cell line (TtT/GF) responds to
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novel hypophysiotropic peptide (pituitary adenylate cyclase-activating peptide), showing increased adenosine 3’, 5’-monophosphate and interleukin-6 secretion and cell proliferation. Endocrinology 1993; 133:2150–5. [45] Zhang ZX, Koike K, Sakamoto Y, Jikihara H, Kanda Y, Inoue K, et al. Pituitary folliculo-stellate-like cell line produces a cytokineinduced neutrophil chemoattractant. Neuropeptides 1997;31: 46–51.
Age-dependent increase of clusterin in the human pituitary gland Takaki Ishikawa*, Bao-Li Zhu, Dong-Ri Li, Dong Zhao, Tomomi Michiue, Hitoshi Maeda Department of Legal Medicine, Osaka City University Medical School, Asahi-machi 1-4-3, Abeno, Osaka 545-8585, Japan Received 11 July 2005; received in revised form 9 August 2005; accepted 17 August 2005 Available online 10 October 2005
Abstract Clusterin is a glycoprotein known to play various physiological roles including complement activity, amyloid binding activity in Alzheimer disease, as well as binding with heat shock proteins and abnormal prions. The present study immunohistochemically investigated the expression of clusterin in the human pituitary gland in subjects of 10–88 years of age (nZ173). Causes of death were blunt injury (nZ35), sharp injury (nZ15), poisoning (nZ11), drowning (nZ14), fire fatalities (nZ28), asphyxiation (nZ15), hypothermia (nZ7), hyperthermia (nZ3), and natural diseases (nZ45). Clusterin was detected in mixed cell follicles and the anterior lobar parenchymal cells. The area occupied by cells positive for clusterin were measured, and the ratio to the whole area of the anterior lobe (% clusterin-positive cell area) was estimated. There was a good correlation between the age of the subjects in years and the % clusterin-positive cell area in the anterior lobe of the pituitary gland (rZ0.736, P!0.01). Relationships between % clusterin-positive cell and gender, cause of death, and survival time were insignificant. These findings indicate an age-dependent accumulation of clusterin in the pituitary gland, which may be related to the aging of endocrine systems. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Human pituitary; Clusterin; Aging; Immunohistochemistry
1. Introduction The tasks of forensic pathology involve the investigation of individual predispositions to fatal outcomes due to traumas and diseases, and age estimation for unidentified bodies. For these purposes, evaluation of degenerative changes due to aging is important. Although age estimation is usually performed based on skeletal and dental maturation and degeneration [1–3], histological and immunohistochemical investigations of skeletal muscle, the cerebrum [4], the hippocampus [5] and the pituitary gland [6–8] have been also reported. Previous studies of the pituitary gland showed increases in 8-hydroxy-2 0 -deoxyguanosine (8-OHdG), S-100 protein and follicular structures. Recent anatomical and physiological studies have demonstrated an increase of follicles in the adenohypophysis of aged animals [9,10]. However, various acute stress may also cause such structural changes [11]. Ogawa et al. [10] observed clusterin-containing follicles in * Corresponding author. Tel.: C81 6 6645 3767; fax: 81 6 6634 3871. E-mail address: [email protected] (T. Ishikawa).
1344-6223/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2005.08.009
porcine adenohypophysis and reported the relationship between cells containing clusterin and aging. Clusterin, apolipoprotein J (apoJ), is a circulating glycoprotein produced in various kinds of epithelial cells. This protein is induced in the tissues or organs affected by various diseases, for instance, Alzheimer’s disease, Creutzfeldt– Jakob disease, atherosclerosis, myocardial infarction and acute and chronic renal diseases [12–16]. Previously, clusterin levels were determined by chemical methods [17,18] and by means of the cloning of repair enzymes for clusterin, followed by the production of specific monoclonal antibodies to clusterin [19]. Recently, the immunohistochemical method for detection of clusterin has been established. O’Bryan et al. [20] revealed immunohistochemically the enhanced level of clusterin in mouse brains by aging. The pituitary gland is a central neuroendocrine organ, and it is known that dysfunction or hypofunction occurs in the neuroendocrine system with age [21,22], which may cause susceptibility or lability to diseases and traumas. In the present paper, we report the expression of clusterin in the anterior lobar parenchymal cells of the human
T. Ishikawa et al. / Legal Medicine 8 (2006) 28–33
pituitary gland and discuss the change of clusterin expression during aging as a marker of degeneration of the neuroendocrine systems.
2. Materials and methods 2.1. Specimens From serial autopsy cases within 3 days postmortem, adenohypophyses (nZ173: 119 males and 54 females) were obtained. The age of the subjects ranged from 10 to 88 (mean, 48.2) years. The cases were classified according to age into four groups as follows: I, !30 years (nZ27); II, 30–49 years (nZ44); III, 50–69 years (nZ64); IV, O69 years (nZ38). Causes of death were blunt injury (nZ35), sharp injury (nZ15), poisoning (nZ11), drowning (nZ14), fire fatalities (nZ28), asphyxiation (nZ15), hypothermia (nZ7), hyperthermia (nZ3), and natural diseases (nZ45). Of 45 deaths by natural diseases, 32 were acute deaths and 13 were chronic deaths. Of all deaths by traumas, 96 were acute deaths and 32 were delayed deaths.
29
2.4. Statistical analysis Values in the text represent the meanGstandard error of the mean (SEM). Statistical analyses were performed using one factor analysis of variance (ANOVA) to examine the differences in mean values among the groups, followed by Scheffe´ F test to identify the significant difference in means. Simple regression analysis was used to examine the correlation between age and the % clusterin-positive cell area; the correlation coefficient and regression equation were calculated.
3. Results Immunohistochemically positive signals of clusterin were found at the anterior lobar parenchymal cells, mixed cell follicles and stellate cells of the pituitary glands (Figs.1–3). Stellate cells with long cytoplasmic projections were observed enclosing the follicles and cells (Fig. 2c). When the relationship between the % area of clusterinpositive parenchymal cells in the anterior lobe of the pituitary gland and the age of the subjects were examined by grouping, there was an age-dependent increase as shown in
2.2. Immunohistochemistry The pituitary gland specimens were fixed in 4% formaldehyde in phosphate-buffered saline (PBS, pH 7.2) for 12 h, embedded in paraffin and then sectioned at thicknesses of 4 mm. After deparaffinization, the sections were immersed in 0.3% H2O2-methanol for 30 min to inactivate endogenous peroxidase. After washing in PBS (3!5 min), blocking with PBS containing 1% normal goat serum and 1% bovine serum albumin was performed at room temperature for 30 min. Then, rabbit anti-human clusterin-a/b (H-330) polyclonal antibody (Santa Cruz, CA) was reacted at an IgG concentration of 1 mg/ml at 48C for 24 h. After washing in PBS (3!5 min), EnvisionC (Dako, Japan) for rabbit immunoglobulin was reacted at room temperature for 30 min, and positive reactions were then visualized with PBS containing 0.02% diaminobenzidine (Sigma, Japan) at room temperature for 5 min. As negative controls, normal rabbit IgG was added instead of the primary antibody. 2.3. Semi-quantitative measurement of clusterin-containing cells The area of positively stained cells was measured three times using an Olympus Color Image Analyzer (VIP-21CH, Olympus, Tokyo, Japan). The result is expressed as the proportion of the average area of clusterin-containing cells to the whole area of the anterior pituitary (% clusterinpositive cell area).
Fig. 1. (a) H.E. findings of the adenohypophysis from a 15-year-old male. (b) Micrograph showing immunostaining of clusterin-positive cells. Positive signals of clusterin were observed in the follicles and cells of the anterior lobe of the pituitary.
30
T. Ishikawa et al. / Legal Medicine 8 (2006) 28–33
Fig. 2. (a) H.E. findings of adenohypophysis from a 55-year-old male. (b) Immunostaining of clusterin-positive cells increases in the adenohypophysis. (c) Micrograph showing a typical picture of clusterin-positive cells engulfing other cells. Bars indicate 30 mm.
Fig. 4. The mean values of % clusterin-positive cell area in groups I, II, III and IV were 25.3G1.8, 32.7G2.8, 36.2G 1.8 and 55.2G3.8%, respectively. There was a significant difference between groups I (!30 years) and IV (O69 years). There were no significant differences in groups I and IV in comparison with groups II (30–49 years) and III (50– 69 years) (PO0.05) or between groups II and III (PO0.05). Regression analysis revealed a significant correlation between the age in years and % clusterin-positive cell area (rZ0.736, P!0.01) (Fig. 5). There was no genderdifference, and the correlation coefficients (r) for males and females were 0.720 (P!0.01) and 0.750 (P!0.01), respectively. In each age group, relationship between
Fig. 3. (a) H.E. findings of adenohypophysis from a 73-year-old male. (b) Numerous clusterin-positive cells occupied the adenohypophysis. (c) A follicle stained by clusterin was surrounded by cells. Bars indicate 60 mm.
% clusterin-positive cell and gender, cause of death, and survival time were insignificant.
4. Discussion In the present study, interestingly, there was a significant difference in pituitary clusterin levels between young (!30 years) and elderly (O69 years) subjects, and individual differences were evident among subjects ranging from 30– 70 years of age. This indicates that the pituitary clusterin level may be available as a marker of aging in individual
T. Ishikawa et al. / Legal Medicine 8 (2006) 28–33
(%)
*
% Clusterin positive cell area
100
* *
75 * 50
25
0 group
I < 30 n=30
II 30 - 49 n=51
III 50 - 69 n=69
IV > 69 n=23
Age (years) Fig. 4. The positive ratios of clusterin-positive cells in each age group. The values are presented as the meanCSEM. The mean positive ratio gradually increased with age. *, P!0.05 by analysis of variance, followed by Scheffe´ F test.
cases to investigate the degeneration of endocrine systems. In addition, in cases involving unidentified bodies, the clusterin level may serve as a marker for age estimation when combined with other morphological, histological, immunohistochemical, and molecular biological markers of aging [5,23–27]. Further investigation is necessary to clarify these findings with regard to susceptibility or lability to diseases and traumas. Recently, clusterin was found in senile plaques in the brains of patients suffering from Alzheimer’s disease (AD)
Age (years)
80
60
40
20
0 0
20
40
60
Clusterin positive ratio
80
(%)
Fig. 5. Linear regression analysis of the positive ratios of clusterin-positive cells in the anterior lobe of the pituitary gland versus age in all 173 samples. There is a significant correlation with age (rZ0.736, P!0.01). The regression equation is YZ1.325XK1.632 (YZage; XZclusterin-positive ratio).
31
[28,29], and it was shown that clusterin was a major binding protein of b-amyloid [30]. Furthermore, clusterin mRNA levels were elevated in the AD hippocampus [31]. Although the pathogenetic mechanism for the increased expression of clusterin in AD is unclear, it may be related to a compensation mechanism involving aggregation of soluble b-amyloid, inhibition of complement-mediated cell damage or an adaptive regenerative response following neuronal degeneration [32]. These findings indicate that clusterin may increase in response to cell damage due to degenerative diseases and stress during aging over 30 years, which may subsequently increase individual differences in clusterin level, thus preventing significant age-dependency among subjects 30–70 years of age. Over 70 years of age, retrogressive changes of the pituitary gland may be an inevitable gerontological predisposition in stress responses to attacks of acute diseases and traumas. Previous study showed an increase of pituitary stellate cells in chronic alcohol abusers [33]. Similar increase in clusterin level was observed for liver cirrhosis, although it could not be statistically confirmed (unpublished data). Further investigation is necessary to clarify the cause of individual differences among middle-aged subjects, especially with regard to alcohol or drug abuse, degenerative diseases and chronic metabolic disorders. The observations described above suggest that pituitary clusterin is produced following degenerative changes, although the mechanism has not been established. However, localization of clusterin in the parenchymal cells and stellate cells suggests several hypotheses on the origin of clusterin in follicles of the pituitary gland. One hypothesis is production of clusterin by the stellate cells, which have similar characteristics to those of the astrocytes that produce clusterin [34]. Another possibility is clusterin expression related to apoptosis in the glandular cells adjacent to the stellate cells [35]. When the hormone-producing glandular cells undergo apoptosis involving clusterin production, they may be phagocytosed by the neighboring stellate cells [10, 36–39], and clusterin in the phagocytosed cells may subsequently be accumulated in the follicular colloid. The stellate cells also produce various growth factors and related proteins including basic fibroblast growth factor (bFGF) [40], vascular endothelial growth factor (VEGF) [41], follistatin [41], tissue inhibitor of metalloproteinase-II (TIMP-II) [42], and also regulate the secretion of prolactin [33,43] and cytokines [44,45]. Thus, the clusterin-positive cells can be involved in a spectrum of multifunctional cells in the anterior pituitary gland. Further investigation is necessary to establish the major roles of the clusterinpositive cells. In conclusion, the present study showed that the pituitary clusterin level increased with age, suggesting its usefulness in investigating retrogressive changes in neuroendocrine systems by aging, and its possible application to age estimation.
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