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Determination of percent area density of epithelial and stromal components in development of prostatic hyperplasia in spontaneously hypertensive rats
BASIC SCIENCE
DETERMINATION OF PERCENT AREA DENSITY OF EPITHELIAL AND STROMAL COMPONENTS IN DEVELOPMENT OF PROSTATIC HYPERPLASIA IN SPONTANEOUSLY HYPERTENSIVE RATS MOTOKI YAMASHITA, XIANGHUA ZHANG, TAIZO SHIRAISHI, HIROTSUGU UETSUKI, AND YOSHIYUKI KAKEHI
ABSTRACT Objectives. To determine the percent area density of epithelial and stromal components in the development of prostatic hyperplasia in spontaneously hypertensive (SH) rats. Methods. The ventral lobes of prostates obtained from male SH rats and their normotensive counterparts, Wistar Kyoto (WKY) rats, were examined histopathologically at 15, 29, 40, and 54 weeks of age (5 SH and WKY rats each at each age group). The degree of prostatic hyperplasia was evaluated with a score-chart protocol, histoscore. The percent area density of epithelium and stroma of the ventral prostate were determined using a computerized image analysis system. Results. Definite lesions of hyperplastic changes were demonstrated in the ventral prostate of SH rats from 15 to 54 weeks. In comparison with WKY rats, SH rats showed a significantly increased degree of prostatic hyperplasia as reflected by the histoscore values. Furthermore, the histoscore of the ventral prostate of SH rats increased with age (from 21.7 ⫾ 0.7 at 15 weeks to 26.1 ⫾ 0.4 at 54 weeks). The percent area density of epithelium and stroma were significantly increased in SH rats, and the ratio of stroma to epithelium ranged from 1:2.94 to 1:3.50 in SH rats but was maintained at 1:1.15 to 1:1.19 in WKY rats during the observation period. Conclusions. The hyperplastic changes of the ventral prostate may develop with advancing age in SH rats. The development of prostatic hyperplasia may result from both epithelial and stromal proliferation and may be predominantly expressed as a glandular type in SH rats. UROLOGY 61: 484–489, 2003. © 2003, Elsevier Science Inc.
B
enign prostatic hyperplasia (BPH) is the most common nonmalignant condition to affect men of different races.1 The condition usually becomes clinically apparent after the age of 40 years and affects at least 80% of all men at some time thereafter.2 Although the pathogenesis of BPH is poorly understood, there is general agreement that it originates in the transition zone of the prostate and begins with stromal alterations that, second-
From the Department of Urology, Kagawa Medical University, Kagawa; and Second Department of Pathology, Mie University School of Medicine, Mie, Japan Reprint requests: Yoshiyuki Kakehi, M.D., Department of Urology, Kagawa Medical University, 1750-1 Ikenobe, Miki-cho Kita-gun, Kagawa 761-0793, Japan Submitted: April 2, 2002, accepted (with revisions): September 18, 2002
484
© 2003, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
arily, stimulate growth and variably modulate the differentiation of associated epithelial cells.3,4 Hence, the development of BPH has been closely associated with cellular proliferation in epithelial and stromal components. It is well known that BPH is the most commonly occurring neoplastic disease of humans5; however, the study of the spontaneous development of BPH in animal models is limited. Most of the experimental animal models of this disease were induced by excessive exogenous sex hormones or chemical substances.6,7 An ideal animal model for BPH needs to possess such phenotypes as reflecting the aging process and showing both epithelial and stromal changes. Recently, hyperplastic changes of the prostate were reported in spontaneously hypertensive (SH) rats, which have been widely studied as an experimental model of hypertension.8,9 The 0090-4295/03/$30.00 doi:10.1016/S0090-4295(02)02167-2
TABLE I. Histoscore protocol used in this study Variable (score values) Low-power magnification Luminal shape: regular (1), villous (3), papillary (4), cribriform (5) Acinar space: large or moderate (1), branched (3), irregular (5) Interacinar space: large or moderate (1), back-to-back glands (5) Stroma: fine (1), abundant (3), fibrosis/severe smooth muscle hyperplasia (5) High-power magnification Epithelial shape: flattened (1), cuboidal (1), cylindrical (3), hexagonal (5) Number of layers: mono (1); oligo, 2–4 (3); pluri, ⬎5 (5) If ⬎1, add: focal (3), diffuse (5) Alignment: polar (1), apolar (3) If pilling up of epithelial cells add: (3) If budding out of epithelial cells into stroma add: (5) If periacinar clusters of epithelial cells are found add: (3) If isolated clusters of epithelial cells are found outside acini add: (5) Lesion distribution (for apolar or budding out cells, no lesion ⫽ 0) Uniobar: isolated (2), multiple (6) Bilobar: isolated (4), multiple (8) Nuclear shape: round, regular (1); irregular (5) Nuclear size: small (2), large (2), large in same acinus (4) Mitosis per field: absent (0); isolated, 1–2 (2); abundant, 3–5 (5); excesive, ⬎5 (10) Basement membrane: intact (1); interrupted (5), thin (1); thick (5) Total score (arbitrary units).
studies revealed initial proliferative lesions in the prostate of young SH rats,8 and definite features of benign adenomatous hyperplasia in the ventral prostate of old SH rats9 compared with their normotensive counterparts, Wistar Kyoto (WKY) rats. Additionally, the hyperplastic changes of the prostate were inhibited by administration of alpha-adrenergic blockade in the SH rats.10 We therefore considered that SH rats would serve as a good rodent model for spontaneous BPH. Although induction of atypical prostatic hyperplasia was noted in rats by sympathomimetic stimulation11 and involvement of excessive sympathetic activity in the etiology of BPH was suggested in hypertensive men12 and in SH rats,13,14 the mechanisms of prostatic hyperplasia that spontaneously occurred in SH rats are still unknown. The purpose of this study was to clarify the histologic features of prostatic hyperplasia, particularly alterations in the proportion of epithelial and stromal components in the development of hyperplastic changes, in young and aging SH rats. Using a computerized image analysis system, the percent area density of epithelium, stroma, and luminal plus interacinar parts were quantitatively determined. In addition, the proliferation and apoptotic indexes of the epithelial cells in each group were investigated, and an imbalance (proliferation greater than apoptosis) was observed only in SH rats. UROLOGY 61 (2), 2003
MATERIAL AND METHODS Twenty male SH and 20 male WKY rats (Kurea, Osaka, Japan), 12 weeks of age at the time of beginning the study, were used. Rats were housed two per polycarbonate cage, held in an environmentally controlled room, and illuminated 12 hours each day. Systolic blood pressure was recorded by the tail cuff method. At 15, 29, 40, and 54 weeks of age, the animals (5 SH and 5 WKY rats at each age) were killed, and the prostate was excised. The separated ventral lobes of the prostates were weighed and processed for fixation in 4% neutral buffered formalin, paraffin embedding, and histologic sectioning.
HISTOPATHOLOGIC EXAMINATION AND ASSESSMENT OF HISTOSCORE Through histopathologic examinations, the degree of histologic changes in the ventral prostate was semiquantitatively assessed in the tissue sections stained with hematoxylin-eosin according to the histoscore,9 a score-chart protocol, as shown in Table I. With this approach, various histologic changes were scored for each specimen. The examination, description, and scoring of the slides were performed in a blinded manner.
DETERMINATION OF PERCENT AREA DENSITY To evaluate the percent area density of the epithelium and stroma of the ventral prostates in the SH and WKY rats, a computerized image analysis system, including a BX51 light microscope (Olympus, Tokyo, Japan) equipped with an Olympus DP50 CCD color camera head, a Sony color monitor, and image analysis software of Win Roof (version 3.6, Mitani, Osaka, Japan), was used. The percent area density of the epithelium, stroma, and luminal plus interacinar parts of the prostates were quantitatively determined through the function of color-assisted image analysis of the Win Roof in the tissue sections stained with Masson’s trichrome stain. Ten dif485
TABLE II. Systolic blood pressure, body weight and wet weight of the ventral prostate, and histoscore values in SH and WKY rats of age 15, 29, 40, and 54 weeks Age (wk) Rats SH (n) Mean systolic blood pressure (mm Hg) Body weight (g) Ventral prostate weight (mg) Histoscore* WKY (n) Mean systolic blood pressure (mm Hg) Body weight (g) Ventral prostate weight (mg) Histoscore
15
29
40
5 210 ⫾ 345 ⫾ 456 ⫾ 21.7 ⫾ 5 112 ⫾ 353 ⫾ 525 ⫾ 17.2 ⫾
5 223 ⫾ 395 ⫾ 631 ⫾ 24.4 ⫾ 5 115 ⫾ 431 ⫾ 685 ⫾ 13.3 ⫾
5 ⫾ ⫾ ⫾ ⫾ 5 ⫾ ⫾ ⫾ ⫾
18 12 53 0.7 15 8 64 0.3
24 8 37 0.9
229 424 724 26.1
12 23 83 0.4
120 440 813 12.4
54 31 28 49 0.2
235 446 682 26.1
21 19 114 0.1
128 482 751 12.4
5 ⫾ ⫾ ⫾ ⫾ 5 ⫾ ⫾ ⫾ ⫾
27 16 52 0.2 18 23 131 0.3
KEY: SH ⫽ spontaneously hypertensive; WKY ⫽ Wistar Kyoto. * Statistically significant differences of histoscore between SH and WKY rats from 15 to 54 weeks (P ⬍0.001), and statistically significant difference of histoscore in SH rats between 15 and 54 weeks (P ⫽ 0.015).
ferent fields were examined at a magnification of 100⫻ from each tissue section, and the mean percent area density was determined from the SH and WKY rats (5 animals each at each interval) at 15, 29, 40, and 54 weeks.
ASSESSMENT OF PROLIFERATION INDEX AND APOPTOTIC INDEX To evaluate the relationship between cell proliferation and apoptosis in the SH and WKY rats aged 29 weeks, we performed immunohistochemistry for proliferating cell nuclear antigen (Santa Cruz Biotechnology, Santa Cruz, Calif) and in situ DNA nick end labeling assay. The proliferation or apoptotic index was calculated by dividing the number of positive cells by the total number of counted epithelial cells, multiplied by 100% in five fields per slide.
STATISTICAL ANALYSIS The Student t test and Kruskal-Wallis test were used. A P value less than 0.05 was considered to indicate statistically significant differences.
RESULTS In comparison with the WKY rats, a significantly higher systolic blood pressure and reduced wet weight of the ventral prostate were recorded in SH rats during the observation period (Table II). HISTOPATHOLOGIC FINDINGS AND HISTOSCORE Our observations showed definite lesions of hyperplastic changes, such as papillary projections of the glandular components into the lumen in the ventral prostate of SH rats from 15 to 54 weeks. The ventral prostate appeared normal in all WKY rats. In addition, more secretory products within the luminal spaces of the ventral prostate were noted in WKY and young SH rats. The degree of prostatic hyperplasia as reflected by the histoscore values was significantly increased in SH rats from 15 to 54 weeks compared with the results in WKY rats. Furthermore, a statistically significant differ486
ence in the histoscore values was noted between 15 and 54 weeks in SH rats. PERCENT AREA DENSITY The percent area density of epithelium, stroma, and luminal plus interacinar parts of the ventral prostates in SH and WKY rats are summarized in Table III. In comparison with the results in WKY rats, the percent area density of both epithelium and stroma was significantly increased and was reduced in the luminal plus interacinar parts with age in the SH rats during the observation period (Fig. 1). The mean ratio of stroma to epithelium was 1:2.94 to 1:3.50 in the ventral prostate of SH rats and 1:1.15 to 1:1.19 in WKY rats during the study period. PROLIFERATION INDEX AND APOPTOTIC INDEX The proliferation index in the SH and WKY rats aged 29 weeks was 14.6 ⫾ 4.7 and 2.4 ⫾ 2.6, respectively. The apoptotic index in the SH and WKY rats aged 29 weeks was 1.2 ⫾ 0.4 and 0.7 ⫾ 0.2, respectively. The proliferation index in the ventral prostate of the SH rats was significantly increased compared with that of the WKY rats (P ⫽ 0.0009). The difference in the apoptotic index between the SH and WKY rats was small (P ⫽ 0.0481). COMMENT Human BPH is the result of proliferation of the mesenchymal-stromal and glandular-epithelial compartments.15 The development of BPH is characterized by slow growth that progresses over decades with a doubling time of at least 10 years.16 Thus, establishing an animal model of spontaneous BPH that develops within months may lead to a clear understanding of the cellular changes. Our UROLOGY 61 (2), 2003
TABLE III. Summarized results of the percent area density, the volume, and the ratios of stroma to epithelium in SH and WKY rat ventral prostate Age (wk) Rats SH Percent area density Epithelium* Stroma† Luminal plus interacinar parts Ratio (stroma/epithelium) Volume (mg)‡ Epithelium Stroma WKY Percent area density Epithelium Stroma Luminal plus interacinar parts Ratio (stroma/epithelium) Volume (mg)‡ Epithelium Stroma
15
29
40
54
29.3 ⫾ 2.1 8.3 ⫾ 1.6 62.4 ⫾ 3.3 1:3.50
34.1 ⫾ 1.8 10.5 ⫾ 3.3 55.4 ⫾ 4.1 1:3.34
39.1 ⫾ 1.3 13.3 ⫾ 1.2 47.6 ⫾ 2.0 1:2.94
40.8 ⫾ 1.3 13.6 ⫾ 1.9 45.6 ⫾ 2.5 1:3.06
134 ⫾ 19 38 ⫾ 5
215 ⫾ 12 67 ⫾ 4
287 ⫾ 16 98 ⫾ 5
278 ⫾ 16 93 ⫾ 6
8.1 ⫾ 0.8 6.8 ⫾ 0.7 85.1 ⫾ 2.4 1:1.19
8.2 ⫾ 0.8 7.1 ⫾ 0.7 84.7 ⫾ 2.7 1:1.15
43 ⫾ 5 36 ⫾ 4
56 ⫾ 4 49 ⫾ 4
8.7 ⫾ 0.5 7.3 ⫾ 1.1 84 ⫾ 2.6 1:1.19 70 ⫾ 6 59 ⫾ 5
8.8 ⫾ 0.6 7.5 ⫾ 0.8 83.7 ⫾ 2.4 1:1.17 66 ⫾ 5 56 ⫾ 4
Abbreviations as in Table II. * Percent area density of epithelium was increased with age (P ⬍0.001). † Percent area density of stroma was increased with age (P ⬍0.05). ‡ Each volume was calculated by the formula: ventral prostate volume (mg) ⫻ percent area density of epithelium or stroma.
data also demonstrated spontaneous occurrence of prostatic hyperplasia from young to aging SH rats. The degree of prostatic hyperplasia, as reflected by the histoscore values, progressed obviously from 15 to 54 weeks in SH rats. The age-specific prevalence of BPH at autopsy investigated by Berry and associates16 showed that the histologic changes of BPH were found in men as young as the late 30s to early 40s, although it was a small population. On the other hand, male rats become reproductive at around 8 to 9 weeks after birth and live on average between 2 and 3 years (longevity of SH rats is less than 1.5 years17). In this study, hyperplastic changes in the ventral prostate were observed as early as 15 weeks of age and became prominent with age. We do not consider that 15 weeks of age for SH rats is physiologically that different from 40 years for the human male. Rather, we believe it is intriguing that the histologic changes occur several weeks after the rats become reproductive. The prostatic growth and function is dependent on both systemic neuroendocrine homeostasis18 –20 and local paracrine stimulation of various growth factors.21,22 SH rats were originally bred for high blood pressure by brother and sister mating of hypertensive rats, and this strain is considered one of the best experimental models of hypertension.23 Excessive sympathetic activity might constitute a pathogenetic link between human BPH and high blood pressure,12 but no consistent suggestion has been found that such an association could be causUROLOGY 61 (2), 2003
al.24 Several studies have provided evidences that may be related to the occurrence of prostatic hyperplasia in SH rats, such as faster growth of primary fibroblast and smooth muscle cells derived from SH rats than those derived from WKY rats25 and excessive basal and environmentally evoked sympathetic activity in SH rats.13,14,26 The mechanisms of spontaneous hyperplastic changes of the prostate occurring in SH rats, however, are still unknown. Comparing the characteristics of prostatic hyperplasia in SH rats with human BPH, which always represents an increased prostate volume with proliferative changes in both stromal and epithelial components, our study showed the coexistence of prostatic hyperplasia with a reduced wet weight of the ventral prostate in SH rats. Similar results have been documented in previous studies,11,27,28 and the adrenergic stimulation that depletes the secretion of the prostate has been suggested to account for this phenomenon.11 The wet weight of prostate may not necessarily reflect the proliferative situations in SH rats. Regarding the histologic features of prostatic hyperplasia in SH rats, the present study first documented alterations in the proportion of both epithelial and stromal components in the development of prostatic hyperplasia from young to aging SH rats. Our study demonstrated a 1.4-fold increase in the percent area density of the epithelium and a 1.6-fold increase in the stroma of the ventral prostate of SH rats between 15 and 54 weeks. BPH 487
FIGURE 1. Determination of percent area density of epithelial and stromal components in Masson’s trichromestained tissue sections of ventral prostate from SH rats (original magnification ⫻100). After Masson’s trichrome staining, ventral prostate epithelial acini were surrounded by collagenous stroma (blue areas) containing smooth muscle cells (red areas) and fibroblasts. (A) Hyperplastic changes showing papillary protrusions in ventral prostate of SH rat at 15 weeks. Note secretory products within all luminal spaces of prostate. (B) Determining process of percent area density of epithelium in part A. (C) Hyperplastic changes showing abundant small acini and dense stroma in the ventral prostate of SH rat at 54 weeks. Note, only a few secretory products scattered in luminal spaces of prostate. (D) Determining process of percent area density of stroma in part C.
implies that the cells of the prostate have inordinately multiplied. Our proliferating cell nuclear antigen and in situ DNA nick end labeling staining for 29-week-old SH and WKY rats demonstrated that an imbalance (proliferation greater than apoptosis) was found only in the SH rats. These results suggest that hyperplastic changes occur in both epithelial and stromal components in the SH rats in an age-dependent manner. Various studies have demonstrated close associations between the development and severity of clinical BPH with the cellular composition of the prostate.29,30 Specifically, the stromal/epithelial ratio in men with symptomatic and asymptomatic BPH matched for age and prostate size was 5:1 and 2:1, respectively.30 It is clear that the proportion of stromal components is absolutely higher than that of the epithelial components in human BPH. According to our findings, the ratio of stroma to epithelium in the ventral prostate ranged from 1:2.94 to 1:3.5 in the SH rats but was maintained at 1:1.15 to 1:1.19 in the WKY rats during the observation period. The prostatic hyperplasia that spontane488
ously developed in the SH rats may be predominantly expressed as glandular. Although our study demonstrated a significantly increased percent area density of the ventral prostate of SH rats from 15 to 54 weeks, it is still unclear whether the hyperplastic changes initiate from the stromal components or the epithelial cells. CONCLUSIONS The present study demonstrated the spontaneous occurrence of prostatic hyperplasia from young to aging SH rats and the close associations of the development of prostatic hyperplasia with advancing age in SH rats. The development of prostatic hyperplasia may result from both epithelial proliferation and stromal growth and may be predominantly expressed as glandular in SH rats. REFERENCES 1. Jonler M, Riehmann M, Brinkmann R, et al: Benign prostatic hyperplasia. Endocrinol Metab Clin North Am 23: 795–807, 1994. UROLOGY 61 (2), 2003
2. Appell RA: Pathogenesis and medical management of benign prostatic hyperplasia. Semin Nephrol 14: 531–543, 1994. 3. Steiner MS: Role of peptide growth factors in the prostate—a review. Urology 42: 99 –110, 1993. 4. McNeal JE: Normal histology of the prostate. Am J Surg Pathol 12: 619 –633, 1988. 5. Walsh PC: Benign prostatic hyperplasia: etiological considerations, in Kimball FA, Buhl AE, and Carter DE (Eds): Approaches to the Study of Benign Prostatic Hyperplasia. New York, Liss, 1984, pp 10 –25. 6. Scolnik MD, Servadio C, and Abramovici A: Comparative study of experimentally induced benign and atypical hyperplasia in the ventral prostate of different rat strains. J Androl 15: 287–297, 1994. 7. Ho SM, Leav I, Merk FB, et al: Induction of atypical hyperplasia, apoptosis, and type II estrogen-binding sites in the ventral prostates of Noble rats treated with testosterone and pharmacologic doses of estradiol-17 beta. Lab Invest 73: 356 –365, 1995. 8. Nakamura H, Sugimoto J, Kojima T, et al: Morphological abnormality of the epithelium in the prostate gland of spontaneously hypertensive rats. J Toxicol Pathol 4: 183–193, 1991. 9. Golomb E, Rosenzweig N, Eilam R, et al: Spontaneous hyperplasia of the ventral lobe of the prostate in aging genetically hypertensive rats. J Androl 21: 58 –64, 2000. 10. Nakamura H, and Itakura C: Influence of terazosin on the epithelium of the prostate gland of spontaneously hypertensive rats. J Toxicol Pathol 5: 101–104, 1992. 11. Golomb E, Kruglikova A, Dvir D, et al: Induction of atypical prostatic hyperplasia in rats by sympathomimetic stimulation. Prostate 34: 214 –221, 1998. 12. Pool JL: Role of the sympathetic nervous system in hypertension and benign prostatic hyperplasia. Br J Clin Pract Symp 74: 13–17, 1994. 13. Magee JC, and Schofield GG: Neurotransmission through sympathetic ganglia of spontaneously hypertensive rats. Hypertension 20: 367–373, 1992. 14. Furukawa S, Yamamoto O, Tamura T, et al: Influence of efonidipine hydrochloride, calcium antagonist on the epithelium of prostates in spontaneously hypertensive rats. J Toxicol Sci 19: 213–217, 1994. 15. Foster CS: Pathology of benign prostatic hyperplasia. Prostate Suppl 9: 4 –14, 2000. 16. Berry SJ, Coffey DS, Walsh PC, et al: The development of human benign prostatic hyperplasia with age. J Urol 132: 474 –479, 1984.
UROLOGY 61 (2), 2003
17. Brandle M, al Makdessi S, Weber RK, et al: Prolongation of life span in hypertensive rats by dietary interventions: effects of garlic and linseed oil. Basic Res Cardiol 92: 223–232, 1997. 18. Aumuller G: Morphologic and regulatory aspects of prostatic function. Anat Embryol 179: 519 –531, 1989. 19. Riegle GD, and Miller AE: Age effects on the hypothalamic hypophyseal gonadal control system in the rat. Adv Exp Med Biol 113: 159 –178, 1978. 20. Sheckter CB, Matsumoto M, and Bremner WJ: Testosterone administration inhibits gonadotropin secretion by an effect directly on the human pituitary. J Clin Endocrinol Metab 68: 397–401, 1989. 21. Cunha GR, Sugimura Y, and Bigsby RM: Androgenic response in the prostate: the role of stromal epithelial interactions, in Bruchowsky N, Chapdelain A, and Neumann F (Eds): Regulation of Androgen Action. Berlin, Congressbruck R Bruckner, 1985, pp 191–197. 22. Cunha GR: Role of mesenchymal-epithelial interactions in normal and abnormal development of mammary gland and prostate. Cancer 74: 1030 –1044, 1994. 23. Frohlich ED: Left ventricular hypertrophy: dissociation of structural and functional effects by therapy. Adv Exp Med Biol 308: 175–190, 1991. 24. Boyle P: Epidemiology of benign prostatic hyperplasia: risk factors and concomitance with hypertension. Br J Clin Pract Suppl 74: 18 –22, 1994. 25. Paquet JL, Baudouin-Legros M, Marche P, et al: Enhanced proliferating activity of cultured smooth muscle cells from SHR. Am J Hypertens 2: 108 –110, 1989. 26. Magee JC, and Schofield GG: Alterations of synaptic transmission in sympathetic ganglia of spontaneously hypertensive rats. Am J Physiol 267: 1397–1407, 1994. 27. Aguilar E, Rodriguez-Padilla ML, Bellido C, et al: Changes in follicle-stimulating hormone secretion in spontaneously hypertensive rats. Neuroendocrinology 56: 85–93, 1992. 28. Rodriguez-Padilla M, Bellido C, Pinilla L, et al: Secretion of LH in spontaneously hypertensive rats. J Endocrinol 113: 255–260, 1987. 29. Shapiro E, Becich MJ, Hartanto V, et al: The relative proportion of stromal and epithelial hyperplasia is related to the development of symptomatic benign prostate hyperplasia. J Urol 147: 1293–1297, 1992. 30. Shapiro E, Hartanto V, and Lepor H: The response to alpha blockade in benign prostatic hyperplasia is related to the percent area density of prostate smooth muscle. Prostate 21: 297–307, 1992.
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DETERMINATION OF PERCENT AREA DENSITY OF EPITHELIAL AND STROMAL COMPONENTS IN DEVELOPMENT OF PROSTATIC HYPERPLASIA IN SPONTANEOUSLY HYPERTENSIVE RATS MOTOKI YAMASHITA, XIANGHUA ZHANG, TAIZO SHIRAISHI, HIROTSUGU UETSUKI, AND YOSHIYUKI KAKEHI
ABSTRACT Objectives. To determine the percent area density of epithelial and stromal components in the development of prostatic hyperplasia in spontaneously hypertensive (SH) rats. Methods. The ventral lobes of prostates obtained from male SH rats and their normotensive counterparts, Wistar Kyoto (WKY) rats, were examined histopathologically at 15, 29, 40, and 54 weeks of age (5 SH and WKY rats each at each age group). The degree of prostatic hyperplasia was evaluated with a score-chart protocol, histoscore. The percent area density of epithelium and stroma of the ventral prostate were determined using a computerized image analysis system. Results. Definite lesions of hyperplastic changes were demonstrated in the ventral prostate of SH rats from 15 to 54 weeks. In comparison with WKY rats, SH rats showed a significantly increased degree of prostatic hyperplasia as reflected by the histoscore values. Furthermore, the histoscore of the ventral prostate of SH rats increased with age (from 21.7 ⫾ 0.7 at 15 weeks to 26.1 ⫾ 0.4 at 54 weeks). The percent area density of epithelium and stroma were significantly increased in SH rats, and the ratio of stroma to epithelium ranged from 1:2.94 to 1:3.50 in SH rats but was maintained at 1:1.15 to 1:1.19 in WKY rats during the observation period. Conclusions. The hyperplastic changes of the ventral prostate may develop with advancing age in SH rats. The development of prostatic hyperplasia may result from both epithelial and stromal proliferation and may be predominantly expressed as a glandular type in SH rats. UROLOGY 61: 484–489, 2003. © 2003, Elsevier Science Inc.
B
enign prostatic hyperplasia (BPH) is the most common nonmalignant condition to affect men of different races.1 The condition usually becomes clinically apparent after the age of 40 years and affects at least 80% of all men at some time thereafter.2 Although the pathogenesis of BPH is poorly understood, there is general agreement that it originates in the transition zone of the prostate and begins with stromal alterations that, second-
From the Department of Urology, Kagawa Medical University, Kagawa; and Second Department of Pathology, Mie University School of Medicine, Mie, Japan Reprint requests: Yoshiyuki Kakehi, M.D., Department of Urology, Kagawa Medical University, 1750-1 Ikenobe, Miki-cho Kita-gun, Kagawa 761-0793, Japan Submitted: April 2, 2002, accepted (with revisions): September 18, 2002
484
© 2003, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
arily, stimulate growth and variably modulate the differentiation of associated epithelial cells.3,4 Hence, the development of BPH has been closely associated with cellular proliferation in epithelial and stromal components. It is well known that BPH is the most commonly occurring neoplastic disease of humans5; however, the study of the spontaneous development of BPH in animal models is limited. Most of the experimental animal models of this disease were induced by excessive exogenous sex hormones or chemical substances.6,7 An ideal animal model for BPH needs to possess such phenotypes as reflecting the aging process and showing both epithelial and stromal changes. Recently, hyperplastic changes of the prostate were reported in spontaneously hypertensive (SH) rats, which have been widely studied as an experimental model of hypertension.8,9 The 0090-4295/03/$30.00 doi:10.1016/S0090-4295(02)02167-2
TABLE I. Histoscore protocol used in this study Variable (score values) Low-power magnification Luminal shape: regular (1), villous (3), papillary (4), cribriform (5) Acinar space: large or moderate (1), branched (3), irregular (5) Interacinar space: large or moderate (1), back-to-back glands (5) Stroma: fine (1), abundant (3), fibrosis/severe smooth muscle hyperplasia (5) High-power magnification Epithelial shape: flattened (1), cuboidal (1), cylindrical (3), hexagonal (5) Number of layers: mono (1); oligo, 2–4 (3); pluri, ⬎5 (5) If ⬎1, add: focal (3), diffuse (5) Alignment: polar (1), apolar (3) If pilling up of epithelial cells add: (3) If budding out of epithelial cells into stroma add: (5) If periacinar clusters of epithelial cells are found add: (3) If isolated clusters of epithelial cells are found outside acini add: (5) Lesion distribution (for apolar or budding out cells, no lesion ⫽ 0) Uniobar: isolated (2), multiple (6) Bilobar: isolated (4), multiple (8) Nuclear shape: round, regular (1); irregular (5) Nuclear size: small (2), large (2), large in same acinus (4) Mitosis per field: absent (0); isolated, 1–2 (2); abundant, 3–5 (5); excesive, ⬎5 (10) Basement membrane: intact (1); interrupted (5), thin (1); thick (5) Total score (arbitrary units).
studies revealed initial proliferative lesions in the prostate of young SH rats,8 and definite features of benign adenomatous hyperplasia in the ventral prostate of old SH rats9 compared with their normotensive counterparts, Wistar Kyoto (WKY) rats. Additionally, the hyperplastic changes of the prostate were inhibited by administration of alpha-adrenergic blockade in the SH rats.10 We therefore considered that SH rats would serve as a good rodent model for spontaneous BPH. Although induction of atypical prostatic hyperplasia was noted in rats by sympathomimetic stimulation11 and involvement of excessive sympathetic activity in the etiology of BPH was suggested in hypertensive men12 and in SH rats,13,14 the mechanisms of prostatic hyperplasia that spontaneously occurred in SH rats are still unknown. The purpose of this study was to clarify the histologic features of prostatic hyperplasia, particularly alterations in the proportion of epithelial and stromal components in the development of hyperplastic changes, in young and aging SH rats. Using a computerized image analysis system, the percent area density of epithelium, stroma, and luminal plus interacinar parts were quantitatively determined. In addition, the proliferation and apoptotic indexes of the epithelial cells in each group were investigated, and an imbalance (proliferation greater than apoptosis) was observed only in SH rats. UROLOGY 61 (2), 2003
MATERIAL AND METHODS Twenty male SH and 20 male WKY rats (Kurea, Osaka, Japan), 12 weeks of age at the time of beginning the study, were used. Rats were housed two per polycarbonate cage, held in an environmentally controlled room, and illuminated 12 hours each day. Systolic blood pressure was recorded by the tail cuff method. At 15, 29, 40, and 54 weeks of age, the animals (5 SH and 5 WKY rats at each age) were killed, and the prostate was excised. The separated ventral lobes of the prostates were weighed and processed for fixation in 4% neutral buffered formalin, paraffin embedding, and histologic sectioning.
HISTOPATHOLOGIC EXAMINATION AND ASSESSMENT OF HISTOSCORE Through histopathologic examinations, the degree of histologic changes in the ventral prostate was semiquantitatively assessed in the tissue sections stained with hematoxylin-eosin according to the histoscore,9 a score-chart protocol, as shown in Table I. With this approach, various histologic changes were scored for each specimen. The examination, description, and scoring of the slides were performed in a blinded manner.
DETERMINATION OF PERCENT AREA DENSITY To evaluate the percent area density of the epithelium and stroma of the ventral prostates in the SH and WKY rats, a computerized image analysis system, including a BX51 light microscope (Olympus, Tokyo, Japan) equipped with an Olympus DP50 CCD color camera head, a Sony color monitor, and image analysis software of Win Roof (version 3.6, Mitani, Osaka, Japan), was used. The percent area density of the epithelium, stroma, and luminal plus interacinar parts of the prostates were quantitatively determined through the function of color-assisted image analysis of the Win Roof in the tissue sections stained with Masson’s trichrome stain. Ten dif485
TABLE II. Systolic blood pressure, body weight and wet weight of the ventral prostate, and histoscore values in SH and WKY rats of age 15, 29, 40, and 54 weeks Age (wk) Rats SH (n) Mean systolic blood pressure (mm Hg) Body weight (g) Ventral prostate weight (mg) Histoscore* WKY (n) Mean systolic blood pressure (mm Hg) Body weight (g) Ventral prostate weight (mg) Histoscore
15
29
40
5 210 ⫾ 345 ⫾ 456 ⫾ 21.7 ⫾ 5 112 ⫾ 353 ⫾ 525 ⫾ 17.2 ⫾
5 223 ⫾ 395 ⫾ 631 ⫾ 24.4 ⫾ 5 115 ⫾ 431 ⫾ 685 ⫾ 13.3 ⫾
5 ⫾ ⫾ ⫾ ⫾ 5 ⫾ ⫾ ⫾ ⫾
18 12 53 0.7 15 8 64 0.3
24 8 37 0.9
229 424 724 26.1
12 23 83 0.4
120 440 813 12.4
54 31 28 49 0.2
235 446 682 26.1
21 19 114 0.1
128 482 751 12.4
5 ⫾ ⫾ ⫾ ⫾ 5 ⫾ ⫾ ⫾ ⫾
27 16 52 0.2 18 23 131 0.3
KEY: SH ⫽ spontaneously hypertensive; WKY ⫽ Wistar Kyoto. * Statistically significant differences of histoscore between SH and WKY rats from 15 to 54 weeks (P ⬍0.001), and statistically significant difference of histoscore in SH rats between 15 and 54 weeks (P ⫽ 0.015).
ferent fields were examined at a magnification of 100⫻ from each tissue section, and the mean percent area density was determined from the SH and WKY rats (5 animals each at each interval) at 15, 29, 40, and 54 weeks.
ASSESSMENT OF PROLIFERATION INDEX AND APOPTOTIC INDEX To evaluate the relationship between cell proliferation and apoptosis in the SH and WKY rats aged 29 weeks, we performed immunohistochemistry for proliferating cell nuclear antigen (Santa Cruz Biotechnology, Santa Cruz, Calif) and in situ DNA nick end labeling assay. The proliferation or apoptotic index was calculated by dividing the number of positive cells by the total number of counted epithelial cells, multiplied by 100% in five fields per slide.
STATISTICAL ANALYSIS The Student t test and Kruskal-Wallis test were used. A P value less than 0.05 was considered to indicate statistically significant differences.
RESULTS In comparison with the WKY rats, a significantly higher systolic blood pressure and reduced wet weight of the ventral prostate were recorded in SH rats during the observation period (Table II). HISTOPATHOLOGIC FINDINGS AND HISTOSCORE Our observations showed definite lesions of hyperplastic changes, such as papillary projections of the glandular components into the lumen in the ventral prostate of SH rats from 15 to 54 weeks. The ventral prostate appeared normal in all WKY rats. In addition, more secretory products within the luminal spaces of the ventral prostate were noted in WKY and young SH rats. The degree of prostatic hyperplasia as reflected by the histoscore values was significantly increased in SH rats from 15 to 54 weeks compared with the results in WKY rats. Furthermore, a statistically significant differ486
ence in the histoscore values was noted between 15 and 54 weeks in SH rats. PERCENT AREA DENSITY The percent area density of epithelium, stroma, and luminal plus interacinar parts of the ventral prostates in SH and WKY rats are summarized in Table III. In comparison with the results in WKY rats, the percent area density of both epithelium and stroma was significantly increased and was reduced in the luminal plus interacinar parts with age in the SH rats during the observation period (Fig. 1). The mean ratio of stroma to epithelium was 1:2.94 to 1:3.50 in the ventral prostate of SH rats and 1:1.15 to 1:1.19 in WKY rats during the study period. PROLIFERATION INDEX AND APOPTOTIC INDEX The proliferation index in the SH and WKY rats aged 29 weeks was 14.6 ⫾ 4.7 and 2.4 ⫾ 2.6, respectively. The apoptotic index in the SH and WKY rats aged 29 weeks was 1.2 ⫾ 0.4 and 0.7 ⫾ 0.2, respectively. The proliferation index in the ventral prostate of the SH rats was significantly increased compared with that of the WKY rats (P ⫽ 0.0009). The difference in the apoptotic index between the SH and WKY rats was small (P ⫽ 0.0481). COMMENT Human BPH is the result of proliferation of the mesenchymal-stromal and glandular-epithelial compartments.15 The development of BPH is characterized by slow growth that progresses over decades with a doubling time of at least 10 years.16 Thus, establishing an animal model of spontaneous BPH that develops within months may lead to a clear understanding of the cellular changes. Our UROLOGY 61 (2), 2003
TABLE III. Summarized results of the percent area density, the volume, and the ratios of stroma to epithelium in SH and WKY rat ventral prostate Age (wk) Rats SH Percent area density Epithelium* Stroma† Luminal plus interacinar parts Ratio (stroma/epithelium) Volume (mg)‡ Epithelium Stroma WKY Percent area density Epithelium Stroma Luminal plus interacinar parts Ratio (stroma/epithelium) Volume (mg)‡ Epithelium Stroma
15
29
40
54
29.3 ⫾ 2.1 8.3 ⫾ 1.6 62.4 ⫾ 3.3 1:3.50
34.1 ⫾ 1.8 10.5 ⫾ 3.3 55.4 ⫾ 4.1 1:3.34
39.1 ⫾ 1.3 13.3 ⫾ 1.2 47.6 ⫾ 2.0 1:2.94
40.8 ⫾ 1.3 13.6 ⫾ 1.9 45.6 ⫾ 2.5 1:3.06
134 ⫾ 19 38 ⫾ 5
215 ⫾ 12 67 ⫾ 4
287 ⫾ 16 98 ⫾ 5
278 ⫾ 16 93 ⫾ 6
8.1 ⫾ 0.8 6.8 ⫾ 0.7 85.1 ⫾ 2.4 1:1.19
8.2 ⫾ 0.8 7.1 ⫾ 0.7 84.7 ⫾ 2.7 1:1.15
43 ⫾ 5 36 ⫾ 4
56 ⫾ 4 49 ⫾ 4
8.7 ⫾ 0.5 7.3 ⫾ 1.1 84 ⫾ 2.6 1:1.19 70 ⫾ 6 59 ⫾ 5
8.8 ⫾ 0.6 7.5 ⫾ 0.8 83.7 ⫾ 2.4 1:1.17 66 ⫾ 5 56 ⫾ 4
Abbreviations as in Table II. * Percent area density of epithelium was increased with age (P ⬍0.001). † Percent area density of stroma was increased with age (P ⬍0.05). ‡ Each volume was calculated by the formula: ventral prostate volume (mg) ⫻ percent area density of epithelium or stroma.
data also demonstrated spontaneous occurrence of prostatic hyperplasia from young to aging SH rats. The degree of prostatic hyperplasia, as reflected by the histoscore values, progressed obviously from 15 to 54 weeks in SH rats. The age-specific prevalence of BPH at autopsy investigated by Berry and associates16 showed that the histologic changes of BPH were found in men as young as the late 30s to early 40s, although it was a small population. On the other hand, male rats become reproductive at around 8 to 9 weeks after birth and live on average between 2 and 3 years (longevity of SH rats is less than 1.5 years17). In this study, hyperplastic changes in the ventral prostate were observed as early as 15 weeks of age and became prominent with age. We do not consider that 15 weeks of age for SH rats is physiologically that different from 40 years for the human male. Rather, we believe it is intriguing that the histologic changes occur several weeks after the rats become reproductive. The prostatic growth and function is dependent on both systemic neuroendocrine homeostasis18 –20 and local paracrine stimulation of various growth factors.21,22 SH rats were originally bred for high blood pressure by brother and sister mating of hypertensive rats, and this strain is considered one of the best experimental models of hypertension.23 Excessive sympathetic activity might constitute a pathogenetic link between human BPH and high blood pressure,12 but no consistent suggestion has been found that such an association could be causUROLOGY 61 (2), 2003
al.24 Several studies have provided evidences that may be related to the occurrence of prostatic hyperplasia in SH rats, such as faster growth of primary fibroblast and smooth muscle cells derived from SH rats than those derived from WKY rats25 and excessive basal and environmentally evoked sympathetic activity in SH rats.13,14,26 The mechanisms of spontaneous hyperplastic changes of the prostate occurring in SH rats, however, are still unknown. Comparing the characteristics of prostatic hyperplasia in SH rats with human BPH, which always represents an increased prostate volume with proliferative changes in both stromal and epithelial components, our study showed the coexistence of prostatic hyperplasia with a reduced wet weight of the ventral prostate in SH rats. Similar results have been documented in previous studies,11,27,28 and the adrenergic stimulation that depletes the secretion of the prostate has been suggested to account for this phenomenon.11 The wet weight of prostate may not necessarily reflect the proliferative situations in SH rats. Regarding the histologic features of prostatic hyperplasia in SH rats, the present study first documented alterations in the proportion of both epithelial and stromal components in the development of prostatic hyperplasia from young to aging SH rats. Our study demonstrated a 1.4-fold increase in the percent area density of the epithelium and a 1.6-fold increase in the stroma of the ventral prostate of SH rats between 15 and 54 weeks. BPH 487
FIGURE 1. Determination of percent area density of epithelial and stromal components in Masson’s trichromestained tissue sections of ventral prostate from SH rats (original magnification ⫻100). After Masson’s trichrome staining, ventral prostate epithelial acini were surrounded by collagenous stroma (blue areas) containing smooth muscle cells (red areas) and fibroblasts. (A) Hyperplastic changes showing papillary protrusions in ventral prostate of SH rat at 15 weeks. Note secretory products within all luminal spaces of prostate. (B) Determining process of percent area density of epithelium in part A. (C) Hyperplastic changes showing abundant small acini and dense stroma in the ventral prostate of SH rat at 54 weeks. Note, only a few secretory products scattered in luminal spaces of prostate. (D) Determining process of percent area density of stroma in part C.
implies that the cells of the prostate have inordinately multiplied. Our proliferating cell nuclear antigen and in situ DNA nick end labeling staining for 29-week-old SH and WKY rats demonstrated that an imbalance (proliferation greater than apoptosis) was found only in the SH rats. These results suggest that hyperplastic changes occur in both epithelial and stromal components in the SH rats in an age-dependent manner. Various studies have demonstrated close associations between the development and severity of clinical BPH with the cellular composition of the prostate.29,30 Specifically, the stromal/epithelial ratio in men with symptomatic and asymptomatic BPH matched for age and prostate size was 5:1 and 2:1, respectively.30 It is clear that the proportion of stromal components is absolutely higher than that of the epithelial components in human BPH. According to our findings, the ratio of stroma to epithelium in the ventral prostate ranged from 1:2.94 to 1:3.5 in the SH rats but was maintained at 1:1.15 to 1:1.19 in the WKY rats during the observation period. The prostatic hyperplasia that spontane488
ously developed in the SH rats may be predominantly expressed as glandular. Although our study demonstrated a significantly increased percent area density of the ventral prostate of SH rats from 15 to 54 weeks, it is still unclear whether the hyperplastic changes initiate from the stromal components or the epithelial cells. CONCLUSIONS The present study demonstrated the spontaneous occurrence of prostatic hyperplasia from young to aging SH rats and the close associations of the development of prostatic hyperplasia with advancing age in SH rats. The development of prostatic hyperplasia may result from both epithelial proliferation and stromal growth and may be predominantly expressed as glandular in SH rats. REFERENCES 1. Jonler M, Riehmann M, Brinkmann R, et al: Benign prostatic hyperplasia. Endocrinol Metab Clin North Am 23: 795–807, 1994. UROLOGY 61 (2), 2003
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