Trophic effect of the sympathetic nervous system on the early development of the rat parotid glandA quantitative ultrastructural study.код для вставкиСкачать
THE ANATOMICAL RECORD 201545-654 (1981) Trophic Effect of the Sympathetic Nervous System on the Early Development of the Rat Parotid Gland: A Quantitative Ultrastructural St udy GUNNAR D. BLOOM, BENGT CARLSOO, AKE DANIELSSON, STEN HELLSTROM, A N D ROGER HENRIKSSON Departments o f Anatomy, Histology, Medicine and Otolaryngology, Uniuersity of Umea, and the National Defence Research Institute, Dept. 4, Umea, Sweàen ABSTRACT Rats were sympathetically denervated on one side by avulsion of the superior cervical ganglion either immediately after birth (within 4 hr) or when the salivary glands were fully developed. Nine weeks after ganglionectomy the parotid glands were subjected to microscopical studies. As shown by the lack of specific fluorescence, sympathetic denervation caused an almost total depletion of catecholamines in the acini. This was further substantiated at the electron microscopic level using KMn04 as fixative. No alterations in either gland weight or in acinar cell size were noticeable after adult sympathectomy. On the other hand, neonatal denervation caused a decrease in gland weight as well as acinar cell hypotrophy. The mem volume of individuai acinar cells was reduced by roughiy 25% and the granule volume density by about 50%. Also the mean volume of individual granules was decreased. These findings indicate an important role for the sympathetic nerve system in the maturation of the rat parotid gland. Salivary glands are supplied with both parasympathetic and sympathetic nerves and gland function depends on the integrity of the two nerve systems. The parasympathetic division seems to be of importance for certain gland functions such as secretion of electrolytes and enzymes, and for maintaining gland structure under physiologic conditions (e.g., Schneyer and Hail, 1967, 1976; Emmelin, 1967). In the rat parotid gland adrenergic nerves are intimately associated with the acinar cells (Norberg and Olson, 1965; Bloom et al., 1977). Activation of the adrenergic P,-adrenoceptor gives rise to a pronounced amylase secretion in vitro (Butcher et al., 1975; Carlsöö et al., 1978, 1981) whereas &adrenoceptors or cholinergic receptors appear to play a minor role in this respect (e.g., Bdolah et al., 1964; Bloom et al., 1979). Long-term superstimulation of rodent salivary glands with the 0-adrenoceptor agonist isoprenaline or prolonged electrical stimulation of the superior cervical ganglion induces a tremendous enlargement of the glands (BrownGrant, 1961; Selye et al., 1961; Barka, 1965; Muir et al., 1975; Bloom et al., 1979). This 0003-276X/81/2014-0645$03.00 effect may be blocked by a B-adrenoceptor antagonist such as propranolol, suggesting a direct action of the drug via the P-adrenoceptor (Schneyer, 1969). Repeated administration of isoprenaline to newborn animals, causes not only an enlargement of the acinar cells, but als0 an acceleration of their development (Schneyer and Schackleford, 1963). The cell bodies of sympathetic nerves to the salivary glands are located in the superior cervical ganglion. Extirpation of this structure in adult animals results in an almost complete depletion of noradrenaline and a loss of catecholamine fluorescence in the rat parotid and submandibular glands (e.g., Perec et al., 1975; Alm and Ekström, 1977). Long-term deprivation of adrenergic stimuli caused by surgical or pharmacological denervation induces a slight decrease of acinar cell size as well as of total gland weight (Schneyer and Hall, 1967; Perec et al., 1975). In the present investigation, ganglionectomy was performed in adult as weìl as in newbom Received March 25. 1981; accept4 Mav 27. 1981. Send reprint requests to Dr. R. Henriksson, Department of Histology and Cel1 Biology. University of UmeB. S90187 UmeB. Sweden. O 1981 ALAN R. LISS, INC. G.D. BLOOM E T AL 646 rats. After a period of 9 weeks, quantitative stereological methods at the ultrastructural level were employed to study characteristics of the acinar cells. MATERIALS AND METHODS Animals and tissue preparation Pregnant rats of the Sprague-Dawley strain and adult rats were purchased from Anticimex, Stockholm, Sweden. Food and water were available ad libitum. Within 4 hr after delivery, the newborn rats were anesthetized by hypothermia (Fairfield, 1948)and through a midline incision the left carotid bifurcation was exposed. The left superior cervical ganglion was cut free from pre- and postganglionic fibers and removed. After suturation the newborn rats were resuscitated over a warm plate (37°C)to adapt to normal body temperature. After recovery the animals were returned to their litter and kept there for another 4 weeks. Only female rats were used for this study. In another series of experiments 6-week-oldfemde rats were anesthetized by iv injection of Brietal (a short acting barbiturate) and the left superior cervical ganglion was r e moved as described above. The extirpated tissue was squashed between glass slides, stained witb 1%toluidine blue and examined in a light microscope to verify the success in ganglionectomy. Nine weeks after surgery the animals were starved for 18 hr with free access to water. They were then anesthetized and the sympathectomized as well as the contralateral nondenervated parotid glands were removed, weighed, and prepared for light and electron microscopy. To verify the degree of denervation of the salivary gland, the tissues were studied by a glyoxylic acid method for visualization of monoamines (De la Torre and Surgeon, 1976). In brief, the method was as follows: The freshly dissected glands were freeze sectioned at -25--30°C. The 15- to 20-pm sections were placed on a glass slide, dipped in a glyoxylic acid solution, and then air dried. When dry, the sections were heated in an oven at 80°C to complete the formation of the fluorophor. They were mounted and studied in a fluorescence microscope. The distribution of autonomic nerves in normal and denervated glands was studied at the electron microscopic level with the aid of the potassium permanganate technique of Richardson (1966)and Hökfelt (1968). For the ultrastructural sterological studies the parotid glands were fixed by vascular per- fusion through the aorta ascendens. As fixative 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.4) was used. After 10 min of perfusion (120 cm H20;20 mlímin) the parotid glands were removed, cut int0 smal1 pieces, and fixed for another 20 min by immersion in the fixative. After a buffer rinse the tissue was postfixed in 1% Os04in the same buffer for 1.5 hr at 4°C. Following dehydration in graded ethanol solutions and propylene oxide the specimens were embeded in Epon 812. Thick (i-pm)as well as thin (70-nm)sections were cut on an LKB Ultrotome. The thin sections were collected on naked copper grids. They were poststained with uranyl acetate andlor lead citrate. The microscope employed was a Philips EM 300 electron microscope. Stereological methods The electron microscopic stereological measurements were carried out on randomly taken micrographs of acinar cells which display their nucleus. Since the acinar celis are polarized, the morphometric measurements were limited to micrographs where both apical and basal cell surfaces were seen (Helander, 1978).From each animal 10 micrographs were chosen als0 at random and paper prints of the cells were enlarged to a total magnification of 6500. The random sampling procedure was used since the parotid gland is homogeneous with the individual components, i.e., acinar cells, not specifically oriented with respect to the plane of section. In such cases no special precautions are required either for sampling tissue blocks or for obtaining sections of micrographs (Weibeland Bolender, 1973; Weibel, 1979). The point counting method was used employing a multipurpose grid (Weibel, 1979). The volume density of the secretory granules was determined in relation to the cytoplasmic volume (not including the nucleus). The nuclear volume density was determined in relation to the total cell volume. All measurements were carried out by the same person and they were repeated some weeks later in order to calculate the personal error in measuring (Eränkö, 1955). This error, expressed as a percentage of the mem values, was 5% for the cell profile area, 5% for nuclear volume density, and 4% for the zymogen granule volume density. The diameters of a l l secretory granules in the electron micrographs were measured in a Zeiss TGZ-3 particle size analyzer. I t can be expected that only a minority of the granules were sectioned through their centers and the others MORPHOMETRY OF DENERVATED PAROTID GLAND were sectioned at various distances from the center. The resulting granule profile diameters were thus smaller than the “true” diameter of the granules. The true granule diameter can be calculated from the size distributions of the slices by several methods. In the present study we used the method described by Bach, since it als0 takes the section thickness int0 consideration (Bach, 1967).Assuming that the nuclei are als0 spherical, their radii were estimated and the true nuclear radius calculated as suggested by Giger and Riedwyl (1970) (Weibel, 1979). This value was then used for calculating the mean nuclear volume, and by dividing with the corrected nuclear volume density (see below), mean cell volume was obtained (Helander, 1978). There are several systemic errors to consider when the above-described methods are used. Since we used the nuclear biased sample, i.e., only cells were measured that displayed their nuclei, we overestimate the nuclear volume density (Konwinski and Koslowski, 1972; Weibel, 1979).Methods for correcting this are only approximations since they assume that the component and its containing volumes are both spheres. Therefore the method by Konwinski and Koslowski (1972) was used as a rough means of correction. However, Helander (1978) has shown that the error in using the correction of Konwinski and Koslowski is negligible when presuming that zymogen cells are spherical, although they have the shape of truncated pyramids, as do rat parotid acinar cells. I t has to be noted that the absolute values obtained by stereological methods and calculations are restricted with different errors due to the preparative procedures such as fixation, dehydration, embedding, and sectioning which cause changes in the tissue. This means that major interest should be focused on comparisons between the different groups of glands, i.e., ganglionectomized glands versus contralateral controls, rather than on the absolute values. RESULTS Upon recovery from anesthesia the adult rats exhibited ptosis on the operated side but not on the contralateral control side. The findings were identical in rats sympathectomized a t birth but they were first observed at 10 days, viz., at the time when the animals generally opened their eyes. The light microscopical technique used in the present study to identify monoaminergic nerves clearly demonstrated lack of such nerve 647 terminals in glands from the ganglionectomized side. The only exceptions to this rule were occasional fluorescent nerve fibers in close association with blood vessels. In nondenervated glands, however, the parenchyma was crisscrossed by fibers with varicosities exhibiting the characteristic fluorescenceof catecholamines (Figs. l a and lb). Employing the KMn04technique for electron microscopic demonstration of adrenergic and cholinergic nerve terminals the findings coincided with the light microscopic observations. Neonatally ganglionectomized glands Sympathetic denervation caused a small but significant reduction in gland weight compared to contralateral glands (0.19 * 0.01 gm vs 0.23 * 0.02 gm; p < 0.05; N = 6). Table 1 summarizes the results of the stereological measurements and it is obvious that the parotid acinar cells in neonatally denervated glands were reduced in volume. The size of the nuclei was, however, unchanged. Consequently the nuclear volume density was increased. The volume of individual zymogen granules was significantly diminished as was the number of granule profiles per cell profile. The size distribution of the secretory granules showed a unimodal distribution in both groups. I t was als0 observed that cellular granule content varied to a greater extent in cells from sympathectomized animals. Furthermore, granule accumulation was frequently noted in the apical portion of the acinar cells whereas the granules were uniformly dispersed throughout the cytoplasm of the normal control cells. A schematic illustration (Fig. 2) depicts in condensed form a comparison between quantitative differences in parotid acinar cells from neonatally sympathectomized glands and nondenervated controls. Figures 3 and 4 show the ultrastructural and light microscopic appearance of sympathectomized and nondenervated glands. Adult ganglionectomized glands Sympathetic denervation of adult parotid glands was not accompanied by any apparent morphological changes in acinar cells when compared to nondenervated glands (Fig. 5). Stereological analysis confirmed these observations (Table 2). A small but not significant weight decrease was noted in denervated glands. DISCUSSION The secretory acini and blood vessels of the rat parotid gland are well supplied with adren- Fig. 1. Histofluorescence of the monoaminergic nerves in parotid gland of neonatauy ganglionectomized rats. (A) Neonatally ganglionectomized parotid gland. Note the paucity of fluorescent nerves in the parenchyma. Occasional fluorescent nerve terminals are observed in the close vicini- t y of b l d vessels. x 250. (B) Nondenervated, control parotid gland. The parenchyma of the gland exhibits numerous fibers and varicosities which are characterized by a yellowgreen fluorescence indicative of catecholamines. "he blood vessels are nchiy innervated. x 250. TABLE I . Stereological data for rat parotid acinar cells of control and neonatal sympathetic denervated glands Control (n=6) Symbols Cel1 profile area Calculated volume of acinar cells Nuclear profile area Nuclear volume density Nuclear volume density corrected according t o (Konwinski and Kozlowski, 1972) Caiculated nuclear volume Granule volume density Number of granule profiles per cel1 profile Granule profile diameter Calculated true granule diameter Volume of one granule Denervated (n= 6) AC VC Km2 An Vvn Km2 21.6 21.6 I 1.1 % I 1.3 21.2 25.2 I 0.7* Vvn % 15.8 f 1.3 19.4 f 0.8* 2.5 125 18.1 f 2.1;' Vn w 3 102.8 f 6.6 859 i 62 firn3 vvg 'în d%! wn 133 33.9 f 37.8 f 4.5 0.81 f 0.02 87.6 649 f 3.8' i 25** 1.2 22.7 f 2.2** 0.67 f 0.02** - D VP Pm lun3 0.97 0.50 f f 0.02 0.04 0.81 f 0.03** 0.29 f 0.03'* MORPHOMETRY OF DENERVATED PAROTID GLAND CONTROL 649 DENERVATED Fig. 2. Schematic illustration of quantitative morphologica1 data from parotid glands of control and neonatally ganglionectomized rats. Mem volume of acinar cells (repre sented by circles) in controls = 859 pm’; in denervated rats = 649 pm’. Sectors represent volume densities of granules (V ), nuclei (Vm) and residual cytoplasm (Vvc). vg TABLE 2. Stereological data for rat parotid acinar cells of control and adult sympathetic deneruated glands Symbols Cel1 profile area Calculated volume of acinar cells Nuclear profile area Nuclear volume density Nuclear volume density corrected according t o (Konwinski and Kozlowski. 1972) Calculated nuclear volume Granule volume density Number of granule profiles per cel1 profile Granule profile diameter Calculated true granule diameter Volume of one granule ~~ Control (n= 6) 95.9 f 5.7 660.2 18.4 20.9 I 55.4 I 0.6 I 1.3 15.2 f Denervated (n= 6) 99.1 731.6 h105.8 19.3 i 1.6 20.5 I 2.1 1.3 15.0 2.2 37.7 96.0 34.9 % d f f 4.4 h 2.0 102 I 2.6 35.5 i 3.2 0.84 I 0.02 34.6 i 0.87 I 3.1 0.04 1.01 I 0.03 0.54 I 0.04 1.03 i 0.58 i 0.04 0.07 ~~ Values are means + SEM. ergic nerves (Norberg and Olson, 1965; Bloom et al., 1977). Removal of the superior cervical ganglion led to a pronounced reduction in catecholamine fluorescense in the parenchyma of the glands. However, some sympathetic nerve fibers were still observed in the close vicinity of the blood vessels. These observations are in agreement with previous studies and verify that not al1 synapses between pre- and postgangiionic parotid sympathetic neurons are 10cated in the superior cervical ganglion ( A h and Ekström, 1977).Utilizing an isotope technique (Pimoule et al., 1980) and quantitative analyses of noradrenaline contents (Perec et al., 1975) unilateral ganglionectomy has been proved to effectively diminish the stores of this catecholamine in submandibular glands of the rat. According to de Camplain et al. (1970) and Owman et al. (1971)sympathetic nerve fibers are detectable in rat salivary glands at 1day of age. The activity of choline acetyltransferase and the content of noradrenaline reaches adult levels at Days 15-18 (Ludford and Talamo, 1980) and fi-adrenoceptor density does not reach adult levels before 25 days (Ludford and 650 G.D. BLOOM ET AL Fig. 3. (A) Parotid gland from neonatally ganglionectomized rat. There is variation in grande content of individual ules. Light micrography x 400. (B)Light micrographof contralateral nondenervated gland. x 400. ceìls as weii as pronounced apical localization of the gran- Talamo, 1980). Furthermore, Mangos (1978) and Grand et al., (1975)report that the secretory activity is not mature even at this time and structural development of the parotid gland is reported to continue up to 6 weeks (Redman and Sreebny, 1971; Taga and Sesso, 1979). Thus, in our study the neonatal ganglionectomy was performed in animals with immature parotid glands, and adult denervation was carried out when gland development was completed. The weight of the parotid gland was clearly reduced 9 weeks after neonatal extirpation of the superior cervical ganglion. Morphometric analyses showed a significantly decreased acinar cell size, whereas nuclear size was unchanged. In contrast, Schneyer and Hall (1967) observed no alteration in gland weight or cell size 2 weeks after ganglionectomy performed 8 days after birth. This would seem to indicate either that the induction of hypotrophic changes takes place at a very early stage of gland development, or that the period of 2 weeks is too short to bring about the changes observed in this study. The former hypothesis is further supported by the fact that we did not notice any significant effect on either gland weight or cell size after adult sympathectomy. However, Schneyer and Hall (1966) observed slight and variable decrease in weight and cell size between 2 and 6 weeks after sympathectomy. The dissimilar results could als0 be due to differences in quantitative techniques employed. The contralateral control glands did not display compensatory hypertrophy when compared to age-matched nonoperated rats. MORPHOMETRY OF DENERVATED PAROTID GLAND Fig. 4. Electron micrographs of control and neonatally denervated parotid acinar cells. (A)Denervated gland. Granule profile numbers are decreased as compared to controls. Grandes are preferentially located in the apical cytoplasm. 65 1 x 5,300. (B) Contralateral nondenervated control gland. The acinar cells are densely packed with secretos. grandes. x 5,300. 652 G.D. BLOOM E T AL Fig. 5. Electron micrographs of adult denervated and control parotid acinar cells. (A)Adult denervated gland. x 5.300. (B)Contralateral control gland. x 5,300. There are no major changes in cel1 or grande morphology. MORPHOMETRY OF DENERVATED PAROTID GLAND Stereological measurements in neonatally ganglionectomized glands showed a significant reduction in number of grandes as wel1 as in grande diameters. Adult denervation r e vealed no changes whatever in the grande population. This would seem to indicate the importante of a sympathetic influence for the early development and maturation of the secretory grandes. Wilborn and Schneyer (1972) reported a marked heterogeneity of the acinar cell population 2 weeks after adult ganglionectomy. The morphological features described were considered indicative of either low or high secretory activity. They suggested that the sympathetic nervous system plays an important role in regulating secretory synchrony of acinar cells. In the present study we could not observe the “light” and “ d a r k cells of the above authors although we did notice variations in grande contents between different acinar cells. In conclusion, sympathetic denervation when performed at birth gives rise to hypotrophy of the parotid gland, whereas the same operation performed in adult rats is essentially without effect. The interpretation of this could be that the sympathetic nerve system plays a fundamental role in the development of the parotid gland, which is reinforced by the studies of Srinivasan and Chang (1977)on the submandibular gland. 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