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Stereological study of acinar growth in the rat parotid gland induced by isoproterenol
Pergamon PII: S0003-9969(97)00025-3
Archs oral Biol. Vol. 42, No. 5, pp. 333-338, 1997 ~,~ 1997 ElsevierScienceLtd. All rights reserved Printed in Great Britain 0003-9969/97 $17.00 + 0.00
S T E R E O L O G I C A L S T U D Y OF A C I N A R G R O W T H I N THE R A T P A R O T I D G L A N D I N D U C E D BY I S O P R O T E R E N O L M. A. O N O F R E , l'* L. B. D E S O U Z A , 2 A. C A M P O S J R 3 and R. T A G A 4 'Department of Diagnosis and Surgery, Dental School of Araraquara, UNESP, SP, Brazil, 2Department of Odontology, Discipline of Oral Pathology, Federal University of RN, RN, Brazil, 3Department of Prothesis Periodontology, Dental School of Bauru, USP, SP, Brazil and 'Department of MorphologyHistology, Dental School of Bauru, USP SP, Brazil (Accepted 14 March 1997)
Summary--The growth of the rat parotid gland induced by daily treatment with isoproterenol (IPR) for 2 weeks was investigated by stereological methods applied to light microscopy. After 7 days of treatment, the glandular mass presented a 286% growth, with the first 3 days being the period of greatest growth. Total acinar volume exhibited a 363% increase during the period from 0 to 7 days, while acinar-cell volume presented a 468% growth from 0 to 5 days of treatment. On the other hand, total acinar-cell number did not increase during the study period. Thus, under the conditions used, IPRstimulated gland growth was essentially hypertrophic. However, a significant increase in the number of bipolar and rnultipolar mitoses was also observed, especially on the third and fifth days of treatment. As no increase in acinar-cell number occurred during growth, the presence of these mitoses suggests that cell death occurred during gland growth. On this basis, bipolar mitoses may occur to replace cells that probably degenerated during treatment, whereas multipolar mitoses may lead to the occurrence of polyploidy. ~) 1997 Elsevier Science Ltd Key words: isoproterenol, hypertrophy, hyperplasia, acini, salivary gland, parotid gland, stereology.
INTRODUCTION
Baserga and Heftier, 1967; Baserga et al., 1969; Novi and Baserga, 1971; Koschel et al. 1976; Durham, 1980). The determination of the percentage of nuclei labelled with [3H]thymidine (labelling index) as an indicator of hyperplastic activity is free from error only when no polyploidy or cell death or blockade of the cell cycle occurs in the tissue under study. As a high rate of polyploidy occurs in isoproterenol-induced salivary gland growth (Radley, 1967; Schneyer et al., 1967) and the literature suggests that cell death (Cataldo et al., 1965; Simson, 1972; Tuch and Matthiesen, 1980) and blockade of the cell cycle (Radley and Hodgson, 1971; Klein et al., 1976) may also occur, in these cases the rate of autoradiographic labelling may not exactly represent a hyperplastic growth rate. Stereological methods for cell number and cell volume determination to evaluate hyperplasia and hypertrophy in the growth of an organ are free of the type of error mentioned above. On this basis, we have now evaluated the participation of hyperplasia and hypertrophy of acinar cells in the marked growth of rat parotid glands by stereological determination of absolute acinar-cell number and volume during the various stages of gland growth.
Daily administration of the synthetic fi-sympathomimetic drug isoproterenol to rats causes a very large growth of the fresh mass of their larger salivary glands, notably the parotid and submandibular glands (Selye et al., 1961; Schneyer, 1962; Barka, 1965a; Sklar and Reid, 1965; Van den Brenk et al., 1970; Wakade, 197!1; Hand and Ho, 1985; Andrade et al., 1994). M a n y studies on the cellular mechanisms that lead to this growth have led to the conclusion that a marked increase in proliferative activity occurs during the first 3-5 days of treatment with the drug, with a consequent elevation in total cell number. This proliferative rate then falls and a significant increase in cell volume occurs simultaneously (Schneyer, 1962; Schneyer et al., 1967; Novi and Baserga, 1971; Wakade, 1979; Matsuura and Hand, 1991). In most studies, the increase in proliferative activity was measured by the autoradiographic method using [3H]thymidine as a marker of the cells that were in tile phase of D N A synthesis, and the increase in cell volume was measured by subjective analysis of cell images in histological sections (Barka, 1965a,b, 1970; Baserga, 1966; *To whom all correspondence should be addressed. 333
M.A. Onofre et al.
334 M A T E R I A L AND M E T H O D S
Isoproterenol treatment and gland collection Fifteen male Wistar rats weighing 200 240g were divided into five lots of three rats, each with free access to food and water. The first lot received 1 ml of 154 mm sodium chloride solution by the intraperitoneal route and was used as control. The remaining groups received isoproterenol hydrochloride (N171CF; Winthrop Laboratories, N.Y.) diluted in twice-distilled water at a concentration of 10 mg/ml and administered at the dose of 4 mg/100 g body wt. The drug was given at 24 h intervals for periods of 3, 5, 7 and 14 days. Glands were collected from each group 30 h after the last dose. Rats were weighed and anaesthetized with ethyl ether and the parotid glands were dissected and adipose tissue and adjacent lymph nodes were carefully removed. Fresh gland mass was then measured with a Mettler H-20 precision scale. The material was fixed in Helly's fluid for 3 h and rinsed overnight with running water. On the subsequent day, the glands were submitted to dehydration in a alcohol of increasing concentration (80, 95, 100 and 100%), clearing in xylene, and embedding in paraffin. Semiserial 5 #m sections were cut and stained with haematoxylin and eosin.
Calculation o f processed gland volume Processed gland volume (Vp) was calculated for each animal using the following equation: Vp = m/ d × rf, where m is fresh mass, d is density and r f is the shrinkage caused by histological processing. For these calculations, we used d = 1.089 g/cm 3, a value obtained in our laboratory by the method of Scherle (1970), and rf = 0.7, the value evaluated in our laboratory by the method of Taga and Sesso (1978).
Stereological evaluation o f aeinar volume density (Vvi) and total volume (VTi) For these evaluations we used a 100x oil-immersion objective and an 8x Zeiss Kpl eyepiece containing a Zeiss II integration grid with 10 parallel lines and 100 points symmetrically distributed over a quadrangular area. We selected 40 histological fields per animal by systematic randomization (Weibel, 1969) and counted the points coinciding with the images of acini (Pi) and the total number of points (Pt) on the gland. Volume density (Vvi) was calculated by the following equation: Vvi = Pi/Pt. Having obtained the Vvi and processed gland volume (Vp) values, we calculated total acinar volume (VTi): VTi = Vvi x Vp.
Evaluation o f nuclear (Vni), cytoplasmic (V~sti) and cellular (V~) volume o f acinar cells Nuclear volume was determined from the measurement of the orthogonal diameters of 100
nuclei per animal using a 100x immersion objective and a 10x Ramsden-type Olympus ocular micrometer. We calculated the mean radius of the geometric mean diameter by r = 4~1 × d2 and the nuclear volume by the formula for the volume of a sphere: V = 4/3 x / 7 x r 3. To calculate cytoplasmic volume, we initially determined the volume densities of the nucleus and of the cytoplasm of acinar cells by point volumetry, and corrected the error due to the Holmes effect (Weibel, 1969). In this respect, we counted the points over nuclei (Pn) and over the cytoplasm (Pcyti) in 40 histological fields of the cells under study. The corrected nuclear volume density (P-~or~) was calculated by the equation: Pn,,rr = (Pn/Pn 4- Pcyti)/Ko, where Ko is the correction factor for the overestimate due to the Holmes effect. Ko is calculated by the formula Ko = 1 + 3t/2 d (Weibel, 1969) where d is the mean nuclear diameter and t is section thickness. The corrected cytoplasm volume density is pcyti.... = 1 - Pnoorr"By dividing Pcyti.... by Pn~o.we obtained the cytoplasm/nucleus ratio (Rc/N) of the acinar cells. On the basis of nuclear volume (Vni) and the C/ N ratio, we calculated the cytoplasmic volume (Vcyti) by the equation Vcyti = Vni × RC/N. This then permitted us to calculate the cell volume (V c =
Vni 4- Vcyti ).
Determination of total acinar-cell number (Nvi) Total acinar-cell number was determined by method II of Aherne (1967) and by the method of Floderus (1944). For determination of the total number of cell nuclei by method II of Aherne (1967) we used a Zeiss II integrated grid fitted to a microscope with a 100x immersion objective. In 40 microscopic fields selected at random for each animal, we counted the number of nuclear images (n) and the number of crossings (c) between the margins of the profiles of the nuclear images and the parallel lines of the test system. Knowing the total area examined in mm 2 (A), the distance between the lines of the test system (d), the thickness of the section (t) and gland processed volume (Vp), we obtained the total number of acinar-cell nuclei using the following equation: N = 2n x Vv/A (c/ n x d + 2t). The method of Floderus (1944) permits the determination of the total number of spherical objects per unit volume of the organ. The number of nuclei per unit gland volume (Nvi) is calculated by the following formula: Nvi = n' x t/ (t + 2r - 2k), where Nvi = number of nuclei/mm3, n ' = number of nuclear slices and fragments/mm3, t = section thickness, r = mean radius and k = correction factor for nuclear spherical segments that escape counting and correspond to the vertical length of the smallest fragment observed. Here k is calculated by r 2 = (r - k) 2 4- rf 2, where rf is the radius of the
Induced parotid gland growth
335
Table 1. Morphometric data for the growth of rat parotid gland induced by daily treatment with isoproterenol Period of treatment Morphometric dimensions
Control
Body mass (g) 238.2* + 2.62 Absolute gland (mg) 414.3 _+9.38 Processed gland volume (cm3) 0.27 _+0.006 Acinar volume density (%) 70.9 _+4.97 Acinar total volume (mm 3) 188.3 _+ 11.75 Nuclear volume of acinar cell (/~m3) 80.0 + 9.35 Cellular volume of acinar c e l l (pm 3) 1327.8 + 29.68 Total number of cells x 106 (Aherne) 108.3+13.66 Total number of acinar cells × 10 6 (Floderus) 106.4 _+20.31
3 days
5 days
7 days
14 days
213.0 + 7.68
210.5 + 1.53
213.3 _+ 7.99
210.8 _+ 13.12
1185.0 _+ 9.50
1399.3 _+ 151.58
1600.0+ 127.75
1813.0_ 180.60
0.76 _+ 0.006
0.90 _+0.097
1.03 + 0.082
1.16 _+0.116
68.6 _+ 7.21
84.2 _+ 1.51
84.7 _+ 1.37
92.6 ___0.66
523.0 _+ 57.67
754.8 _+ 71.80
871.8 _+68.22
1079.8 _+ 113.96
92.3 + 9.91
111.8 + 2.94
130.5 + 8.00
139.6 + 6.85
4311.7 + 573.07
7537.9+ 429.76
130.4+22.84
94.5_+7.97
118.1 _+ 31.64
86.7 + 7.79
8153.7+ 1178.29 11099.8 + 1794.60 107.6_+3.32 97.1 + 13.05
101.0_+9.48 89.4 _+ 13.61
*Mean of three animals for experimental group
smallest nuclear spherical fragment observed. By multiplying Nvi by Vp, we obtain the total number (N) of acinar cells in the gland.
Statistical analysis The mean and SEM were calculated for each dimension evaluated. The morphometric data for each group were compared to those for the other groups by analysis of variance using the method of Lison (1958), with the level of significance set at p < 0.05 and p < 0.01.
RESULTS The morphometric results obtained are presented in Table 1. Figure 1 is a graphic representation of the evolution of gland mass, total acinar volume, mean acinar-cell volume and total acinar-cell number evaluated by method II of Aherne. Analysis of the data presented in the table and figure showed that gland mass markedly grew from 0 to 7 days of treatment, with an overall increase of 286% (p < 0.01); the increase between 0 and 3 days was 186% (p < 0.01) and the increase between 3 and 7 days was 35% (p < 0.05). A 13% increase occurred from 7 to 14 days but was not statistically significant (p > 0.05). Total acinar volume (mm 3) increased in a highly significant manner (p < 0.01) by 363% from 0 to 7 days of treatment; the increase obtained after this period was no longer significant (p>0.05). The increase from 0 to 3 clays was 178% (p < 0.01) and the increase from 3 to 7 days was 67% (p < 0.05). Mean acinar-cell volume ( ~ m 3) exhibited a huge increase of 468% (p < 0.01) from 0 to 7 days of treatment, i.e. 1327.82-7537.92 m m 3. F r o m 5 to 14
days of treatment, the 47% increase observed was significant only at p = 0.1. The increase from 0 to 3 days was 227% (p < 0.01) and the increase from 3 to 5 days, 75% (p < 0.01). Acinar-cell number, calculated both by method II of Aherne and by the method of Floderus, did not differ significantly between groups (p>0.05), i.e. it was stable throughout the study period.
DISCUSSION We detected a marked increase in parotid gland mass during the period of treatment with isoproterenol, as also reported by others (Selye et al., 1961; Selye et al., 1961; Schneyer, 1962; Chan, 1964; Barka, 1965; Cataldo et al., 1965; Schneyer et al., 1967; Van den Brenk et al., 1970; Novi and Baserga, 1971; Wakade, 1979). Monitoring of gland-mass growth showed that total acinar volume increased substantially during several stages of treatment, i.e. 178% from 0 to 3 days and 67% from 3 to 7 days of treatment, with a mean accumulation of 111.6 and 87.2 mm3/day, respectively. To clarify the processes responsible for this marked increase in total acinar volume, we evaluated stercologically the total number and mean volume of acinar cells. Analysis of the evolution of total cell number obtained by morphometric method II of Aherne and by the method of Floderus, under our experimental conditions, showed no statistically significant difference between methods or between the various experimental groups during the treatment period, indicating that no increase in total acinar-cell number occurred during daily treatment with isoproterenol.
336
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Fig. 1. Evolution of the growth of the rat parotid gland induced by daily treatment with isoproterenol. 3 (c) cell volume (pm); 3 (d) (a) Absolute gland mass (mg); (b) total compartimental acinar volume (mm); total number of acinar cells (×106).
It should be pointed out, however, that, in contrast to our results, many others have reported the occurrence of intense acinar cell proliferative activity based on the visualization of increased numbers of mitotic figures or of [3H]thymidine labelled cells, especially during the initial periods of chronic treatment with isoproterenol (Selye et al., 1961; Schneyer, 1962; Chan, 1964; Barka, 1965a, b, 1970; Cataldo et al., 1965; Baserga, 1966; Baserga and
Heftier, 1967; Schneyer et al., 1967; Van Den Brenk et al., 1970; Novi and Baserga, 1971; Koschel et al., 1976; Durham, 1980; Chapola et al., 1985). In agreement with these observations, we also observed the presence of a significant number of mitotic figures from 3 to 5 days of treatment. As we did not observe an increase in total acinar cell number, we wondered about the meaning of the occurrence of these mitoses.
337
Induced parotid gland growth In response to this question, we wish to point out that Radley (1967), using cytophotometry, and Schneyer et al. (1967), using chromosome counts, observed that treatment with isoproterenol causes a significant increase in the percentage of polyploid cells. In Radley's study, 36% of the cells were tetraploid and 4% octaploid after 7 days of treatment; in Schneyer's 100% of the mitoses were of polyploid cells after 5 days of treatment. Polyploid cells are known to originate from mitoses called atypical, or incomplete, or multipolar (Brodsky and Uryvaeva, 1977). This, in order to be able to compare our results with those obtained by Schneyer et al., we counted the number of bipolar and multipolar mitoses in the glands of two animals from the groups treated for 3 and 5 days, respectively. A 53% rate of bipolar mitoses and a 47% rate of multipolar mitoses were obtained for the 3day group, and 47 and 53% rates were obtained for the 5-day group. These results confirm those reported by the above authors, i.e. that a significant number of acinar cells entering the replication process under stimulation with isoproterenol may be destined for polyploidy. Polyploidy may occur as a consequence of a forced stimulation of the organ, with a resulting blockade of the normal course of mitosis and an increase in cell size and chromosome number. Cell volume would then increase proportionally to chromosome number (Brodsky and Uryvaeva, 1977). This explanation, however, does not justify the presence of countless bipolar mitotic figures, which have been proven to give origin to two diploid daughter cells. We emphasize that several investigators have observed damage to acinar cells during the gland growth induced by isoproterenol (Selye et al., 1961; Cataldo et al., 1965; Simson, 1969, 1972; Tuch and Matthiesen, 1980). The occurrence of this damage suggests that cell death may take place during the period of most intense gland growth, i.e. during the first few days of treatment. Thus, we propose that the new cells produced by the mitotic process may be destined to replace those that degenerated during growth, with a consequent equilibrium of total cell number and possibly justifying the presence of bipolar mitoses at the beginning of treatment. This would then be a regenerative growth, previously proposed by Tuch and Matthiesen (1980). The data discussed here indicate that the absolute number of acinar cells did not increase during the growth of isoproterenol-stimulated parotid glands. On this basis, the enormous increase in their mass could only have been due to increased cell volume, i.e. hypertrophy of cells already present in the gland, or to new cells produced to replace those that degenerated during growth. Analysis of the present data also permitted us to determine whether acinar cell hypertrophy was sufficient to explain the great increase in mass of the gland.
Cell volume increased in percentage terms only during the first 3 days (224.72%), but continued to grow up to the fifth (74.82%), stabilizing thereafter. It should be pointed out that Bloom et al. (1979), using electron-microscopic stereological analysis, observed in parotid glands of newborn rats submitted to daily administrations of isoproterenol for a period of 9 weeks, an increase on acinar cell volume of 619% and a striking increase in size and number of cytoplasmic secretory granules. From our original data we calculated the mean daily increase in cell volume in/~m3. The mean daily accumulation from 0 to 3 days and from 3 to 5 days of treatment was 994.62/2m3 and 1613.12/tm 3, respectively. We observed that the stage of greatest cell-volume growth in the entire study period was between the third and fifth day of treatment, when both nuclear volume and cytoplasmic volume increased. The higher percentage and rate of cell-volume growth in relation to gland mass unequivocally proves that gland growth was essentially hypertrophic, i.e. the increase in volume of the acinar cells existing in the glands was sufficient to explain the enormous increase in gland mass. It should be pointed out that we obtained similar results in the submandibular gland of rats submitted to chronic treatment with isoproterenol (Andrade et al., 1994). are grateful to Mr Artur Mendonqa for the histological procedures, and to Mrs Thelma Aparecida Gomes, Mr Antonio Medeiros Filho and Mrs Beonildes Teresinha Ruiz Correia for typing the manuscript. Acknowledgements--We
REFERENCES
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analysis and the secretory behaviour of the rat parotid gland after long and short term isoprenaline treatment. Medical Biol. 57, 224-233. Brodsky W. Y. and Uryuaeava I. V. (1977) Cell polyploidy: its relation to tissue growth and function. Int. Rev. CytoL 50, 275-332. Cataldo E., Shklar G. and Reid D. P. (1965) Submaxillary salivary glands treated with isoproterenol. Arch. Pathol.
80, 3-8. Chan W. C. (1964) Enlargement of the rat's salivary glands and salivary calculus formation induced with isoprenaline. J. Pathol. Bacteriol. 88, 563-574. Chapola A. V., Valle L. B. S., Oliveira-Filho R. M. and Minetti C. (1985) Beta-adrenergic stimulation of mouse parotid gland: amylase activity and secretory granules during isoproterenol-induced mitoses. Gen. Pharmacol. 16, 419-421. Durham J. P. (1980) Effect of ethanol upon isoproterenol stimulation of growth and secretion in the mouse parotid gland. Life Sci. 26, 1423 1430. Floderus S. (1994) Quoted by O. Er~ink6 . In Quantitative Methods in Histology and Microscope Histochemistry, p. 76. S. Karger, Basel. Hand A. R. and Ho B. (1985) Mitosis and hypertrophy of intercalated duct cells and endothelial cells in the isoproterenol-treated rat parotid gland. J. dent. Res. 64, 10311038. Klein R. M., Harrington D. B. and Pilliero S. J. (1976) Isoproterenol-induced changes in cell cycle kinetics of parotid gland acinar cells in 8-day old rats. J. dent. Res. 55, 611-616. Koschel K. W., Hodgson G. S. and Radley J. M. (1976) Characteristics of the isoprenaline stimulated proliferative response of rat submaxillary gland. Cell Tissue Kinet. 9, 157-165. Lison L. (1958) Statistique appliqu~e dt la Biologie Experimentale, p. 304. Gouthiers Villars, Paris. Matsuura S. and Hand A. R. (1991) Quantitative immunocytochemistry of rat submandibular secretory proteins during chronic isoproterenol administration and recovery. J. Histochem. Cytochem. 39, 945 954. Novi A. M. and Baserga R. (1971) Association of hypertrophy and DNA synthesis in mouse salivary glands
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Archs oral Biol. Vol. 42, No. 5, pp. 333-338, 1997 ~,~ 1997 ElsevierScienceLtd. All rights reserved Printed in Great Britain 0003-9969/97 $17.00 + 0.00
S T E R E O L O G I C A L S T U D Y OF A C I N A R G R O W T H I N THE R A T P A R O T I D G L A N D I N D U C E D BY I S O P R O T E R E N O L M. A. O N O F R E , l'* L. B. D E S O U Z A , 2 A. C A M P O S J R 3 and R. T A G A 4 'Department of Diagnosis and Surgery, Dental School of Araraquara, UNESP, SP, Brazil, 2Department of Odontology, Discipline of Oral Pathology, Federal University of RN, RN, Brazil, 3Department of Prothesis Periodontology, Dental School of Bauru, USP, SP, Brazil and 'Department of MorphologyHistology, Dental School of Bauru, USP SP, Brazil (Accepted 14 March 1997)
Summary--The growth of the rat parotid gland induced by daily treatment with isoproterenol (IPR) for 2 weeks was investigated by stereological methods applied to light microscopy. After 7 days of treatment, the glandular mass presented a 286% growth, with the first 3 days being the period of greatest growth. Total acinar volume exhibited a 363% increase during the period from 0 to 7 days, while acinar-cell volume presented a 468% growth from 0 to 5 days of treatment. On the other hand, total acinar-cell number did not increase during the study period. Thus, under the conditions used, IPRstimulated gland growth was essentially hypertrophic. However, a significant increase in the number of bipolar and rnultipolar mitoses was also observed, especially on the third and fifth days of treatment. As no increase in acinar-cell number occurred during growth, the presence of these mitoses suggests that cell death occurred during gland growth. On this basis, bipolar mitoses may occur to replace cells that probably degenerated during treatment, whereas multipolar mitoses may lead to the occurrence of polyploidy. ~) 1997 Elsevier Science Ltd Key words: isoproterenol, hypertrophy, hyperplasia, acini, salivary gland, parotid gland, stereology.
INTRODUCTION
Baserga and Heftier, 1967; Baserga et al., 1969; Novi and Baserga, 1971; Koschel et al. 1976; Durham, 1980). The determination of the percentage of nuclei labelled with [3H]thymidine (labelling index) as an indicator of hyperplastic activity is free from error only when no polyploidy or cell death or blockade of the cell cycle occurs in the tissue under study. As a high rate of polyploidy occurs in isoproterenol-induced salivary gland growth (Radley, 1967; Schneyer et al., 1967) and the literature suggests that cell death (Cataldo et al., 1965; Simson, 1972; Tuch and Matthiesen, 1980) and blockade of the cell cycle (Radley and Hodgson, 1971; Klein et al., 1976) may also occur, in these cases the rate of autoradiographic labelling may not exactly represent a hyperplastic growth rate. Stereological methods for cell number and cell volume determination to evaluate hyperplasia and hypertrophy in the growth of an organ are free of the type of error mentioned above. On this basis, we have now evaluated the participation of hyperplasia and hypertrophy of acinar cells in the marked growth of rat parotid glands by stereological determination of absolute acinar-cell number and volume during the various stages of gland growth.
Daily administration of the synthetic fi-sympathomimetic drug isoproterenol to rats causes a very large growth of the fresh mass of their larger salivary glands, notably the parotid and submandibular glands (Selye et al., 1961; Schneyer, 1962; Barka, 1965a; Sklar and Reid, 1965; Van den Brenk et al., 1970; Wakade, 197!1; Hand and Ho, 1985; Andrade et al., 1994). M a n y studies on the cellular mechanisms that lead to this growth have led to the conclusion that a marked increase in proliferative activity occurs during the first 3-5 days of treatment with the drug, with a consequent elevation in total cell number. This proliferative rate then falls and a significant increase in cell volume occurs simultaneously (Schneyer, 1962; Schneyer et al., 1967; Novi and Baserga, 1971; Wakade, 1979; Matsuura and Hand, 1991). In most studies, the increase in proliferative activity was measured by the autoradiographic method using [3H]thymidine as a marker of the cells that were in tile phase of D N A synthesis, and the increase in cell volume was measured by subjective analysis of cell images in histological sections (Barka, 1965a,b, 1970; Baserga, 1966; *To whom all correspondence should be addressed. 333
M.A. Onofre et al.
334 M A T E R I A L AND M E T H O D S
Isoproterenol treatment and gland collection Fifteen male Wistar rats weighing 200 240g were divided into five lots of three rats, each with free access to food and water. The first lot received 1 ml of 154 mm sodium chloride solution by the intraperitoneal route and was used as control. The remaining groups received isoproterenol hydrochloride (N171CF; Winthrop Laboratories, N.Y.) diluted in twice-distilled water at a concentration of 10 mg/ml and administered at the dose of 4 mg/100 g body wt. The drug was given at 24 h intervals for periods of 3, 5, 7 and 14 days. Glands were collected from each group 30 h after the last dose. Rats were weighed and anaesthetized with ethyl ether and the parotid glands were dissected and adipose tissue and adjacent lymph nodes were carefully removed. Fresh gland mass was then measured with a Mettler H-20 precision scale. The material was fixed in Helly's fluid for 3 h and rinsed overnight with running water. On the subsequent day, the glands were submitted to dehydration in a alcohol of increasing concentration (80, 95, 100 and 100%), clearing in xylene, and embedding in paraffin. Semiserial 5 #m sections were cut and stained with haematoxylin and eosin.
Calculation o f processed gland volume Processed gland volume (Vp) was calculated for each animal using the following equation: Vp = m/ d × rf, where m is fresh mass, d is density and r f is the shrinkage caused by histological processing. For these calculations, we used d = 1.089 g/cm 3, a value obtained in our laboratory by the method of Scherle (1970), and rf = 0.7, the value evaluated in our laboratory by the method of Taga and Sesso (1978).
Stereological evaluation o f aeinar volume density (Vvi) and total volume (VTi) For these evaluations we used a 100x oil-immersion objective and an 8x Zeiss Kpl eyepiece containing a Zeiss II integration grid with 10 parallel lines and 100 points symmetrically distributed over a quadrangular area. We selected 40 histological fields per animal by systematic randomization (Weibel, 1969) and counted the points coinciding with the images of acini (Pi) and the total number of points (Pt) on the gland. Volume density (Vvi) was calculated by the following equation: Vvi = Pi/Pt. Having obtained the Vvi and processed gland volume (Vp) values, we calculated total acinar volume (VTi): VTi = Vvi x Vp.
Evaluation o f nuclear (Vni), cytoplasmic (V~sti) and cellular (V~) volume o f acinar cells Nuclear volume was determined from the measurement of the orthogonal diameters of 100
nuclei per animal using a 100x immersion objective and a 10x Ramsden-type Olympus ocular micrometer. We calculated the mean radius of the geometric mean diameter by r = 4~1 × d2 and the nuclear volume by the formula for the volume of a sphere: V = 4/3 x / 7 x r 3. To calculate cytoplasmic volume, we initially determined the volume densities of the nucleus and of the cytoplasm of acinar cells by point volumetry, and corrected the error due to the Holmes effect (Weibel, 1969). In this respect, we counted the points over nuclei (Pn) and over the cytoplasm (Pcyti) in 40 histological fields of the cells under study. The corrected nuclear volume density (P-~or~) was calculated by the equation: Pn,,rr = (Pn/Pn 4- Pcyti)/Ko, where Ko is the correction factor for the overestimate due to the Holmes effect. Ko is calculated by the formula Ko = 1 + 3t/2 d (Weibel, 1969) where d is the mean nuclear diameter and t is section thickness. The corrected cytoplasm volume density is pcyti.... = 1 - Pnoorr"By dividing Pcyti.... by Pn~o.we obtained the cytoplasm/nucleus ratio (Rc/N) of the acinar cells. On the basis of nuclear volume (Vni) and the C/ N ratio, we calculated the cytoplasmic volume (Vcyti) by the equation Vcyti = Vni × RC/N. This then permitted us to calculate the cell volume (V c =
Vni 4- Vcyti ).
Determination of total acinar-cell number (Nvi) Total acinar-cell number was determined by method II of Aherne (1967) and by the method of Floderus (1944). For determination of the total number of cell nuclei by method II of Aherne (1967) we used a Zeiss II integrated grid fitted to a microscope with a 100x immersion objective. In 40 microscopic fields selected at random for each animal, we counted the number of nuclear images (n) and the number of crossings (c) between the margins of the profiles of the nuclear images and the parallel lines of the test system. Knowing the total area examined in mm 2 (A), the distance between the lines of the test system (d), the thickness of the section (t) and gland processed volume (Vp), we obtained the total number of acinar-cell nuclei using the following equation: N = 2n x Vv/A (c/ n x d + 2t). The method of Floderus (1944) permits the determination of the total number of spherical objects per unit volume of the organ. The number of nuclei per unit gland volume (Nvi) is calculated by the following formula: Nvi = n' x t/ (t + 2r - 2k), where Nvi = number of nuclei/mm3, n ' = number of nuclear slices and fragments/mm3, t = section thickness, r = mean radius and k = correction factor for nuclear spherical segments that escape counting and correspond to the vertical length of the smallest fragment observed. Here k is calculated by r 2 = (r - k) 2 4- rf 2, where rf is the radius of the
Induced parotid gland growth
335
Table 1. Morphometric data for the growth of rat parotid gland induced by daily treatment with isoproterenol Period of treatment Morphometric dimensions
Control
Body mass (g) 238.2* + 2.62 Absolute gland (mg) 414.3 _+9.38 Processed gland volume (cm3) 0.27 _+0.006 Acinar volume density (%) 70.9 _+4.97 Acinar total volume (mm 3) 188.3 _+ 11.75 Nuclear volume of acinar cell (/~m3) 80.0 + 9.35 Cellular volume of acinar c e l l (pm 3) 1327.8 + 29.68 Total number of cells x 106 (Aherne) 108.3+13.66 Total number of acinar cells × 10 6 (Floderus) 106.4 _+20.31
3 days
5 days
7 days
14 days
213.0 + 7.68
210.5 + 1.53
213.3 _+ 7.99
210.8 _+ 13.12
1185.0 _+ 9.50
1399.3 _+ 151.58
1600.0+ 127.75
1813.0_ 180.60
0.76 _+ 0.006
0.90 _+0.097
1.03 + 0.082
1.16 _+0.116
68.6 _+ 7.21
84.2 _+ 1.51
84.7 _+ 1.37
92.6 ___0.66
523.0 _+ 57.67
754.8 _+ 71.80
871.8 _+68.22
1079.8 _+ 113.96
92.3 + 9.91
111.8 + 2.94
130.5 + 8.00
139.6 + 6.85
4311.7 + 573.07
7537.9+ 429.76
130.4+22.84
94.5_+7.97
118.1 _+ 31.64
86.7 + 7.79
8153.7+ 1178.29 11099.8 + 1794.60 107.6_+3.32 97.1 + 13.05
101.0_+9.48 89.4 _+ 13.61
*Mean of three animals for experimental group
smallest nuclear spherical fragment observed. By multiplying Nvi by Vp, we obtain the total number (N) of acinar cells in the gland.
Statistical analysis The mean and SEM were calculated for each dimension evaluated. The morphometric data for each group were compared to those for the other groups by analysis of variance using the method of Lison (1958), with the level of significance set at p < 0.05 and p < 0.01.
RESULTS The morphometric results obtained are presented in Table 1. Figure 1 is a graphic representation of the evolution of gland mass, total acinar volume, mean acinar-cell volume and total acinar-cell number evaluated by method II of Aherne. Analysis of the data presented in the table and figure showed that gland mass markedly grew from 0 to 7 days of treatment, with an overall increase of 286% (p < 0.01); the increase between 0 and 3 days was 186% (p < 0.01) and the increase between 3 and 7 days was 35% (p < 0.05). A 13% increase occurred from 7 to 14 days but was not statistically significant (p > 0.05). Total acinar volume (mm 3) increased in a highly significant manner (p < 0.01) by 363% from 0 to 7 days of treatment; the increase obtained after this period was no longer significant (p>0.05). The increase from 0 to 3 clays was 178% (p < 0.01) and the increase from 3 to 7 days was 67% (p < 0.05). Mean acinar-cell volume ( ~ m 3) exhibited a huge increase of 468% (p < 0.01) from 0 to 7 days of treatment, i.e. 1327.82-7537.92 m m 3. F r o m 5 to 14
days of treatment, the 47% increase observed was significant only at p = 0.1. The increase from 0 to 3 days was 227% (p < 0.01) and the increase from 3 to 5 days, 75% (p < 0.01). Acinar-cell number, calculated both by method II of Aherne and by the method of Floderus, did not differ significantly between groups (p>0.05), i.e. it was stable throughout the study period.
DISCUSSION We detected a marked increase in parotid gland mass during the period of treatment with isoproterenol, as also reported by others (Selye et al., 1961; Selye et al., 1961; Schneyer, 1962; Chan, 1964; Barka, 1965; Cataldo et al., 1965; Schneyer et al., 1967; Van den Brenk et al., 1970; Novi and Baserga, 1971; Wakade, 1979). Monitoring of gland-mass growth showed that total acinar volume increased substantially during several stages of treatment, i.e. 178% from 0 to 3 days and 67% from 3 to 7 days of treatment, with a mean accumulation of 111.6 and 87.2 mm3/day, respectively. To clarify the processes responsible for this marked increase in total acinar volume, we evaluated stercologically the total number and mean volume of acinar cells. Analysis of the evolution of total cell number obtained by morphometric method II of Aherne and by the method of Floderus, under our experimental conditions, showed no statistically significant difference between methods or between the various experimental groups during the treatment period, indicating that no increase in total acinar-cell number occurred during daily treatment with isoproterenol.
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Fig. 1. Evolution of the growth of the rat parotid gland induced by daily treatment with isoproterenol. 3 (c) cell volume (pm); 3 (d) (a) Absolute gland mass (mg); (b) total compartimental acinar volume (mm); total number of acinar cells (×106).
It should be pointed out, however, that, in contrast to our results, many others have reported the occurrence of intense acinar cell proliferative activity based on the visualization of increased numbers of mitotic figures or of [3H]thymidine labelled cells, especially during the initial periods of chronic treatment with isoproterenol (Selye et al., 1961; Schneyer, 1962; Chan, 1964; Barka, 1965a, b, 1970; Cataldo et al., 1965; Baserga, 1966; Baserga and
Heftier, 1967; Schneyer et al., 1967; Van Den Brenk et al., 1970; Novi and Baserga, 1971; Koschel et al., 1976; Durham, 1980; Chapola et al., 1985). In agreement with these observations, we also observed the presence of a significant number of mitotic figures from 3 to 5 days of treatment. As we did not observe an increase in total acinar cell number, we wondered about the meaning of the occurrence of these mitoses.
337
Induced parotid gland growth In response to this question, we wish to point out that Radley (1967), using cytophotometry, and Schneyer et al. (1967), using chromosome counts, observed that treatment with isoproterenol causes a significant increase in the percentage of polyploid cells. In Radley's study, 36% of the cells were tetraploid and 4% octaploid after 7 days of treatment; in Schneyer's 100% of the mitoses were of polyploid cells after 5 days of treatment. Polyploid cells are known to originate from mitoses called atypical, or incomplete, or multipolar (Brodsky and Uryvaeva, 1977). This, in order to be able to compare our results with those obtained by Schneyer et al., we counted the number of bipolar and multipolar mitoses in the glands of two animals from the groups treated for 3 and 5 days, respectively. A 53% rate of bipolar mitoses and a 47% rate of multipolar mitoses were obtained for the 3day group, and 47 and 53% rates were obtained for the 5-day group. These results confirm those reported by the above authors, i.e. that a significant number of acinar cells entering the replication process under stimulation with isoproterenol may be destined for polyploidy. Polyploidy may occur as a consequence of a forced stimulation of the organ, with a resulting blockade of the normal course of mitosis and an increase in cell size and chromosome number. Cell volume would then increase proportionally to chromosome number (Brodsky and Uryvaeva, 1977). This explanation, however, does not justify the presence of countless bipolar mitotic figures, which have been proven to give origin to two diploid daughter cells. We emphasize that several investigators have observed damage to acinar cells during the gland growth induced by isoproterenol (Selye et al., 1961; Cataldo et al., 1965; Simson, 1969, 1972; Tuch and Matthiesen, 1980). The occurrence of this damage suggests that cell death may take place during the period of most intense gland growth, i.e. during the first few days of treatment. Thus, we propose that the new cells produced by the mitotic process may be destined to replace those that degenerated during growth, with a consequent equilibrium of total cell number and possibly justifying the presence of bipolar mitoses at the beginning of treatment. This would then be a regenerative growth, previously proposed by Tuch and Matthiesen (1980). The data discussed here indicate that the absolute number of acinar cells did not increase during the growth of isoproterenol-stimulated parotid glands. On this basis, the enormous increase in their mass could only have been due to increased cell volume, i.e. hypertrophy of cells already present in the gland, or to new cells produced to replace those that degenerated during growth. Analysis of the present data also permitted us to determine whether acinar cell hypertrophy was sufficient to explain the great increase in mass of the gland.
Cell volume increased in percentage terms only during the first 3 days (224.72%), but continued to grow up to the fifth (74.82%), stabilizing thereafter. It should be pointed out that Bloom et al. (1979), using electron-microscopic stereological analysis, observed in parotid glands of newborn rats submitted to daily administrations of isoproterenol for a period of 9 weeks, an increase on acinar cell volume of 619% and a striking increase in size and number of cytoplasmic secretory granules. From our original data we calculated the mean daily increase in cell volume in/~m3. The mean daily accumulation from 0 to 3 days and from 3 to 5 days of treatment was 994.62/2m3 and 1613.12/tm 3, respectively. We observed that the stage of greatest cell-volume growth in the entire study period was between the third and fifth day of treatment, when both nuclear volume and cytoplasmic volume increased. The higher percentage and rate of cell-volume growth in relation to gland mass unequivocally proves that gland growth was essentially hypertrophic, i.e. the increase in volume of the acinar cells existing in the glands was sufficient to explain the enormous increase in gland mass. It should be pointed out that we obtained similar results in the submandibular gland of rats submitted to chronic treatment with isoproterenol (Andrade et al., 1994). are grateful to Mr Artur Mendonqa for the histological procedures, and to Mrs Thelma Aparecida Gomes, Mr Antonio Medeiros Filho and Mrs Beonildes Teresinha Ruiz Correia for typing the manuscript. Acknowledgements--We
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