Variation in the silver staining of the Golgi complex along the epithelium of the intestinal villi in the adult rat.код для вставкиСкачать
Variation in the Silver Staining of the Golgi Complex Along the Epithelium of the Intestinal Villi in the Adult R a t ' R. L. JOSEPHSON AND G. G . ALTMANN Department of Anatomy, Health Sciences Centre, T h e University of W e s t e r n Ontario, London 7 2 , Ontario, Canada ABSTRACT A light microscopic study was carried out on the Golgi complex of the absorptive epithelial cells of the small intestine of adult male rats. Silver impregnation methods (Da Fano's, Aoyama's, and Elftman's) were used. Deposits of silver in the Golgi complex were heaviest in the lower half of the villi. Thereafter, a progressive diminution in the reaction occurred. In the uppermost quarter of the villus no reaction was visible. Villi of the duodenum, midgut, and terminal ileum all exhibited this phenomenon regardless of variations in fixation method, concentration and pH of the silver solutions, exposure time to the silver solutions, and various dietary regimens (high fat, no fat, and starvation). The change observed in the silver reaction of the Golgi complex probably reflects a structural or functional change of this organelle as the absorptive epithelial cells migrate along the villi toward the extrusion zones. The epithelium of the small intestine of (Da Fano, '20a,b); ( 2 ) Aoyama's silver the rat is a rapidly renewing cell popula- method (Aoyama, '29); ( 3 ) Elftman's dition (Leblond and Stevens, '48). Its cells rect silver method (Elftman, '52). Silver originate in the crypts of Lieberkuhn, mi- impregnation was carried out in the dark. grate upward to the villus to form the ab- In all techniques, the tissues were emsorptive epithelium, and then move toward bedded in paraffin and sectioned longitudithe apex of the villus where they are ex- nally at 3-5 p . Da Fano's method. Pieces of intestine truded. During the migration of these cells morphological and functional transfoma- were fixed in 15% formalin containing 1% lions take place (Palade, '55; Padykula et cobalt nitrate for four to six hours at 24°C. al., '61; Padykula, '62). As a part of a They were then transferred to a bath of study of these transformations, the Golgi 1.5% silver nitrate for 24-36 hours at complex was examined at the light micro- 24°C. Following a wash in distilled water, scopic level using silver impregnation tech- the specimens were placed in Cajal's reniques. ducing solution consisting of 2 % hydroquinone and 0.5% sodium sulphite in 15% MATERIALS AND METHODS formalin (Cajal, '12) for 20 hours at 24°C Altogether 30 adult male Wistar rats ( 2 animals). A few variations were also weighing 300-350 gm were used. Except tried: ( 1 ) the concentration of the silver €or ten animals, all were maintained on solution was doubled to 3% silver nitrate Purina Laboratory Chow. They were sac- while the exposure time of the tissues was 0 and ~ ~ ~P M under held constant (1 animal); ( 2 ) the conrificed between 1 chloroform anaesthesia. Pieces of duode- centration was held at 1.5% while the num (taken next to the pylorus), midpor- exposure time was doubled (1 animal); tion of the small intestine, and terminal (3) both the concentration of the silver ileum (taken next to the caecum), were solution and the exposure time of the tisremoved, cut open longitudinally, flattened sues to it were doubled (1 animal). Peron pieces of cardboard, and placed in the Received Oct. 6, '71. Accepted Jan. 24, '72. appropriate fixative for the following tech1 This work was supported by a grant of the Medical niques: ( 1 ) the Da Fano silver method Research Council of Canada to the second author. ANAT. REC., 1'73: 221-224. 221 222 R. L. JOSEPHSON AND G. G . A L T M A " fusion of fixative solution through the left ventricle followed by immersion in fresh fixative was also carried out ( 1 animal). Aoyamds method. Pieces of intestine were fixed in 15% formalin containing 1% cadmium chloride for six hours at 24°C. The use of cadmium chloride as a source of divalent cations is the only essential difference between this method and that of Da Fano ( 2 animals). Direct silver method. Specimens were fixed and impregnated at the same time in a solution of 15% formalin containing 2% silver nitrate at 24°C. After two hours in this solution, the samples were placed directly into a solution of 2% hydroquinone in 15% formalin for two hours at 24°C. They were then transferred into a 15% formalin solution for 12 hours and processed for paraffin embedding the next day (4 animals). In addition, a number of variations were performed: (1) the concentration of the silver solution was doubled to 4% while exposure time was held constant (1 animal); (2) the concentration was held constant while the exposure time was doubled (1 animal); ( 3 ) both the concentration and exposure time were doubled (1 animal); ( 4 ) the pH of the formalin-silver solution was varied between 4.0 and 7.2 in increments of 0.4 pH (1 animal); (5) 2 cm segments of intestine were tied off, care being taken that the blood supply to the segments remained intact. The formalin-silver solution was then injected into the lumen of the ligated intestinal segments in situ (2 animals); (6) tissue samples were washed for ten minutes in TC Hank's solution (Difco Laboratories, Detroit, Michigan) at room temperature followed by immersion in the formalin-silver solution ( 1 animal). Furthermore, a few experiments were carried out in which the animals were placed on special diets: ( a ) a diet rich in fat ( 3 animals); (b) a diet containing no f a t 3 ( 3 animals); ( c ) starvation for 48 hours prior to sacrifice (2 animals); and (d) the same as in (c) but followed by gastric intubation of 2 ml corn oil one hour prior to sacrifice (as in Cardell et al., '67). Only villi which were sectioned along their longitudinal axis were considered. The conditions for selecting villi cut along their longitudinal axis were reported elsewhere ( Altmann and Leblond, '70). RESULTS All three silver methods revealed that the silver reactivity of the Golgi complex increased as the principal epithelial cells ascended from the crypts of Lieberkuhn to the villus. The epithelial cells on the lower half of the villus showed maximal Golgi impregnation (fig. 1, zone 1). Thereafter, a progressive diminution in the reaction occurred (fig. 1, zone 2). In about the upper quarter of the villus, no Golgiassociated silver deposits could be seen (fig. 1,zone 3). The extent of zone 3 varied slightly from villus to villus. All three intestinal regions exhibited this phenomenon. Aoyama's method appeared to be superior to the Da Fano method in demonstrating the reticular nature of the Golgi complex. However, the direct silver method in the opinion of the authors is by far the best method in both its reliability and its simplicity. Perfusion and intraluminal injection of fixative resulted in better preservation of tissue architecture, although there was no observable difference in the impregnation characteristics of the Golgi complex from those seen with conventional fixation procedures. In all cases, increasing the concentration of the silver solution or the duration of tissue exposure to it, did not change the appearance of the zones shown in figure 1. Varying the pH of the formalin-silver solution in the direct method also resulted in no observable differences. Washing tissue samples in Hank's solution prior to fixation, largely eliminated a layer of debris over the tip area which was otherwise impregnated with silver. Again, no changes in the Golgiassociated silver deposits were observed. The various dietary regimens did not alter the Golgi impregnation characteristics of the absorptive cells. It seemed, however, that the diet had a part in causing an infranuclear region of reduced silver in absorptive cells in the upper one third of the villus (fig. 2). All animals except the ones 2 Ground Purina Laboratory Chow enriched with corn oil (40% by weight). 3 The comaosition of the diet (o/, bv weieht) was as follows: 34% casein; 8% lactalbimin; -30% s< crose; 20% corn starch; .8% ,alphacel; 2% salt mixture 2 U.S.P.:2% vitamin diet fortification mixture. All the materials were obtained from Nutritional Biochemicals Co., Cleveland, Ohio. 223 SILVER STAINING OF THE GOLGI COMPLEX Fig. 1 A whole villus is shown. Composite picture. Zone I shows maximal Golgi reaction. In zone 11, the reaction progressively diminishes. No reaction is visible in zone 111. The extrusion zone ( E l is well delineated by about six darkly stained cells at the villus tip. The change in the silver staining of the Golgi complex is apparent much below the extrusion zone. Da Fano’s method. x 225. Fig. 2 Upper two-thirds of a villus is shown. In the upper third of the absorptive epithelium, the hfranuclear accumulation of silver ( D ) is visible. zones11 and 111 are also visible. Goblet cg) and argentaffin ( a ) cells exhibit dense deposits’ method’ 450’ 224 R. L. JOSEPHSON AND G. G. ALTMANN which received no food 48 hours prior to be changes which indicate a structural or sacrifice, exhibited this infranuclear ac- functional transformation of the organelle cumulation of silver. as the absorptive epithelial cells progress along the villi toward the Extrusion zones. DISCUSSION A progressive differentiation of the normal intestinal epithelium occurs during the migration of the epithelial cells from the crypts of Lieberkuhn to the extrusion zone (Padykula, '62). The changes observed in the silver-reactivity of the Golgi complex may be a morphological expression of these changes. The possibility of the zonation being of artifactual origin is unlikely in view of the fact that the results were not altered by variations in fixation technique or by washing the samples in Hank's solution. Change in the Golgi complex of the intestinal epithelial cells has been observed by a number of authors. For example, it has been shown that the Golgi apparatus becomes more conspicuous as the epithelial cells migrate to the villi (Palade, '55; Padykula, '62). Furthermore, diminution of Golgi-associated substances has been demonstrated by EM radioautography utilizing galactose-3Has a label for glycoprotein synthesis in rat intestinal epithelial cells. All villus columnar cells were seen to incorporate label in the supranuclear Golgi region but the intensity of the reaction decreased gradually from the base to the apex of the villus (Bennett, TO). Since the mechanism of silver impregnation is not well understood, it is not possible at present to state the exact sites or materials which are stained by silver. It is believed by some authors that phospholipids and possibly bound proteins are stained (Elftman, '53; Thompson and Hunt, '66; Pearse, '68). It is therefore possible that the silver deposits were associated with the membranes of the Golgi complex. The exact nature of this reaction, however, is to be clarified by further studies. In conclusion, it appears that changes occur in the Golgi complex as the principal cells migrate along the villus. These changes are reflected in the silver reaction. They do not seem to be dependent on dietary conditions and may therefore LITERATURE CITED Altmann, G. G., and C. P. Leblond 1970 Factors influencing villus size in the small intestine of adult rats as revealed by transposition of intestinal segments. Am. J. Anat., 127: 15-36. Aoyama, F. 1929 Eine Modification der Cajalschen Methode zur Darstellung des Golgischen binnennetz Apparate. Ztschr. Wissen. Mikr., 46: 489-491. Bennett, G. J. 1970 Migration of glycoprotein from Golgi apparatus to cell coat in the columnar cells of the duodenal epithelium. J. Cell Biol., 45: 668-673. Cajal, S. R. Y. 1912 Formula de fijacion para la demonstracion facil del apparato reticolar de Golgi y apuntes sobre la disFosicion de dicho apparata en la retina en 10s nervios y algunos estados patologicos. Trab. del lab. de invest. biol. de la Univ. de Madrid, 10: 209-220. Cardell, R. R., S . Badenhausen and K. R. Porter 1967 Intestinal triglyceride absorption i n the rat. A n electron microscopical study. J. Cell Biol., 34: 123-155. Da Fano, C. 1920a Method for the demonstration of Golgi's internal apparatus. J. Physiol., 53: xcii-xciv. -1920b Method for the demonstration of Golgi's internal apparatus in nervous and other tissues. J. Roy Micr. SOC.,40: 157-161. Elftman, H. 1952 A direct silver method for the Golzi apparatus. Stain Techn., 27: 47-52. 1953 Response of the Golgi material to chemical reagents. J. Histochem. and Cytochem., I: 387-388. Leblond, C. P., and C. E. Stevens 1948 The constant renewal of the intestinal epithelium in the albino rat. Anat. Rec., 100: 357-378. Padykula, H. A. 1962 Recent functional interpretations of intestinal morphology. Fed. Proc., 21: 873-879. Padykula, H. A., E. Strauss, A. J. Ladman and F. H. Gardner 1961 A morphological and histochemical analysis of human jejunal epithelium in nontropical sprue. Gastroent., 40: 735-765. Palade, G . E. 1955 A small particulate component of the cytoplasm. J. Biophys. Biochem. Cytol., I: 59-68. - 1958 A small particulate component of the cytoplasm. In: Frontiers in Cytology. S. L. Palay ed. Yale University Press, New Haven, Conn., pp. 283-304. Pearse, A. G. 1968 Histocheniistry. Theoretical and Applied. Third ed. Vol. 1. J. and A. Churchill Ltd., London W1, pp. 78-79. Thompson, S. W., and R. D. Hunt 1966 Selected Histochemical and Histopathological Methods. Charles C Thomas, Springfield, Ill., pp. 805-809.