Effects of prior culture or isoproterenol injections on the regeneration of rat submandibular gland autografts.код для вставкиСкачать
THE ANATOMICAL RECORD 206:11-21(1983) Effects of Prior Culture or lsoproterenol Injections on the Regeneration of Rat Submandibular Gland Autografts NORRIS L. O’DELL, MOHAMED SHARAWY, AND CATHERINE B. PENNINGTON Departments of Oral Biology and Anatomy, Medical College of Georgia, Augusta, G A 30912 ABSTRACT This study compares the acinar cell regenerative response in submandibular gland (SMG) autografts that were cultured before grafting to the rat tongue with the acinar cell regenerative response in direct SMG autografts to the tongue. In addition, the effects of isoproterenol on direct SMG autografts were studied. A portion of the left SMG was excised from each rat and cut into fragments which were autografted either immediately into the middle one-third of the rat’s tongue; or were cultured for 1, 4, or 7 days and then autografted to the donor’s tongue. After 8 weeks the rats were killed and the tongues were removed and processed for light microscopic study. The histologic preparations showed evidence of cytodifferentiationinto acinar cells in four of the previously cultured SMG autografts. Some of the direct SMG autografts did not contain acinar cells, whereas other direct SMG autografts contained numerous acinar cells and even striated ducts. In the SMG autografts that were cultured for 1 day before autografting and in the direct SMG autografts, the most pronounced regenerative responses were seen in autografts that contained ductlike structures that were apparently connected t o the epithelial surface of the tongue. Lastly, isoproterenol appeared to accelerate the regenerative response in some of the direct SMG autografts, and the drug caused acinar cell hypertrophy in two of the direct SMG autografts. Fragments of mature rat submandibular gland (SMG) that were autografted to the tongue showed massive degenerative changes initially, and then began to regenerate during an 8-week period that followed the autografting procedure (Sharawy and O’Dell, 1981). Moreover, the ability of rodent and lagomorph salivary glands to regenerate or recover has been demonstrated following other experimental manipulations such as ductal ligation (Junqueira and Rabinovitch, 1954; Bhaskar et al., 1966; Tamarin, 1971a, b; Shiba et al., 1972), drug intoxication (Ulmansky et al., 1969; Leeb, 19751, irradiation (Cherry and Glucksmann, 1959), partial extirpation (Milstein, 1950; Hanks and Chaudhry, 1971; Boshell and Pennington, 1980), induction of allergic sialadenitis (White and Casarett, 1974; Sharawy and White, 1978), and transplantation of whole glands or glandular fragments to various sites (Hoshino and Lin, 1970, 1971; Sharawy and O’Dell, 1979). In general, the results of these studies indicate that salivary glands have some capacity to recover or regenerate. 0 1983 ALAN R. LISS, INC. Impaired salivary gland function, secondary to radiation therapy, infection, neoplasia, and other causes, may result in significant oral tissue deterioration. Therefore, methods for enhancing the recovery or regeneration of mature salivary gland tissues and their return to normal function would be beneficial. For instance, the ability to remove and culture salivary gland tissues prior to therapeutic radiation of the head and neck, and subsequently autografting these cultured tissues to restore glandular function would be of clinical significance. Previous studies have shown that mature rodent SMG parenchyma undergoes severe degenerative changes in vitro (Tapp, 1967; Lucas, 1969; Wigley and Franks, 1976; O’Dell et al., 1979). With time, cultured mature salivary glands lose their phenotypes and appear undifferentiated. These cells often become arranged in pat- Received May 10,1982; accepted January 27,1983. 12 N.L. O’DELL, M. SHARAWY, AND C.B. PENNINGTON terns that resemble ducts, and hence have been referred to as ductlike structures (Wigley and Franks, 1976; O’Dell et al., 1979). One of the purposes of the present study was to determine if the ductlike structures seen in cultured rat SMG tissues contain cells that have the potential to differentiate into definitive acinar cells when they are returned to a n in vivo environment. The SMG autograft model (Sharawy and O’Dell, 1981) was used to test this hypothesis. We implanted previously cultured SMG tissues into the rat tongue and compared the regenerative response of these indirect autografts with the regenerative response of SMG tissues that had been autografted directly into the tongue. Henceforth, the latter will be called “direct autografts.” In addition, some of the rats with direct autografts were given isoproterenol injections. Isoproterenol (ISP)produced glandular enlargement by inducing cellular hypertrophy and hyperplasia in rodent salivary glands (Brown-Grant, 1961; Selye et al., 1961; Barka, 1965), and ISP accelerated the postnatal differentiation of rat salivary glands (Schneyer and Shackleford, 1963; Ekfors et al., 1972; Yamashina and Barka, 1972; Bressler, 1973; Srinivasan et al., 1973).Moreover, ISP enhanced acinar cell cytodifferentiation in mouse salivary gland isografts (Hoshino and Lin, 1970, 1971).However, in partial extirpation experiments ISP appeared to inhibit acinar cell mitoses in the “reactive zone” of regenerating rat SMG (Boshell and Pennington, 1980). Therefore, in the present study the effects of multiple ISP injections on regenerating direct autografts were studied. MATERIALS AND METHODS Male Sprague-Dawley rats (Rattus noruegicus) that were at least 12 weeks old a t the beginning of this study were used in each of several experiments. The rats were housed in an animal room with a well-controlled environment and allowed to have food and water ad libitum. For surgical procedures, general anesthesia was obtained by giving intraperitoneal injections of chloral hydrate (250 m g k g body weight) supplemented with ether inhalation. Cultured A utografCs In groups I-A, B, and C (39 rats), each rat was anesthetized, the neck was shaved, and a portion of the left SMG was excised through a small incision in the neck which was closed with silk ligatures. The excised portion of the gland was placed in a watch glass containing Waymouth’s medium MB 752/1 (Waymouth, 1959; GIBCO) and minced with scissors to produce fragments approximately 1.0-2.0 mm3 in size. Approximately 15 of these fragments were explanted onto each Millipore filter (Millipore Corp. type GS, 0.22-pm pores), which was supported by a stainless steel wire grid in a 35 x 10-mm Petri dish (Falcon) containing Waymouth’s medium MB 75211 supplemented with fetal bovine serum (10% V N ) , 1-glutamine, and gentamicin (1 mg%) (Schering Corp.). Three Petri dishes were used to culture the fragments from each excised portion of SMG. The explants were kept in a humidified incubator under a 5% COz/ 95% air atmosphere at 37°C. In cultures maintained longer than 1 day the culture medium was changed every other day. After one day (I-A), 4 days (I-B), or 7 days (I-C) in vitro, approximately 20 explants were selected randomly from the three Petri dishes and were autografted into the original donor rat’s tongue and left in situ for 8 weeks (counted from the day of explantation) before sacrifice. After 8 weeks each rat was anesthetized, the chest was opened, and the animal was perfused through the heart with phosphate-buffered formalin. The tongue and intact SMG were removed and immersed in the same fixative for 5 days. The fixed tissues were processed through alcohols to xylene and embedded in paraffin. The autografts were located by making 6-pm thick sections and staining selected sections with hematoxylin and eosin (H & E). When an autograft was located serial sections containing the autograft were made and stained with H & E, periodic acid Schiff s counterstained with hematoxylin (PASH), or Masson’s. In addition, representative 1-day, 4-day, and 7-day explants were fixed in phosphatebuffered formalin and processed for light microscopy. Moreover, in order to characterize the amount of proliferative activity in the SMG explants a t the time of autografting them back into the donor rats’ tongues, at least two Petri dishes were selected at random for radioautography to represent the explants to be harvested after 1 , 4 , or 7 days in culture. The radioautographic technique used here was described previously (O’Dell et al., 1979).Twenty-four hours prior to harvesting the explants in these selected culture dishes, the explants were fed fresh medium contain- CULTURED SUBMANDIBULAR GLAND AUTOGRAFTS ing 1.0 p Cum1 3H-thymidine (specific activity, 20 Ci/mmole; Amersham Corp.). At the time of harvest these explants were fixed in the Petri dish for 3 days in phosphate-buffered formalin, then processed to p a r a i n . Sixmicron thick sections were cut and were deparaffinized, hydrated, and processed for radioautography using Kodak NTB2 nuclear track emulsion (Eastman Kodak Co.). These radioautographic preparations were incubated at 4°C for 24 hours, and then developed with Kodak D-19 solution. The developed radioautographic preparations were stained with H & E. Direct Autografts In group I1 (32 rats), each rat was anesthetized, the neck was shaved, and approximately one-half of the left SMG was excised through a small incision in the neck which was closed with silk ligatures. The excised SMG was minced into 2-3 mm3 fragments. Five to ten of these fragments were implanted immediately into the middle onethird of the tongue of the same rat as previously described (Sharawy and O’Dell, 1981). The volume of SMG tissue used for the direct autografts roughly equaled the volume of SMG cultured for each of the indirect autografts. The wound in the tongue was closed with a silk suture. The direct autografts remained in the tongue for 8 weeks, and then the rats were anesthetized and perfused with fixative, and the tongues and intact SMG were removed and processed as described previously under group I. (Note: Eighteen of the rats in group I1 received intraperitoneal injections of saline for 5 days prior to sacrifice and served as controls for the group I11 rats that received ISP injections.) In group I11 (29 rats), the rats received direct autografts, but were also given injections of dl-isoproterenol-HC1(ISP) (Sigma) for 5 days prior to sacrifice. The ISP was dissolved in saline just prior to injecting, and the dosages were 4, 8, or 16 mg of ISP/kg body weight. At the time of sacrifice, these rats were anesthetized and perfused with fixative, and the tongues and intact SMG were removed and processed as described for group I. Sham-Operated Rats In group IV (nine rats), the rats were anesthetized and a portion of the left SMG was excised and discarded. A wound was created 13 in the lateral aspect of the middle one-third of the tongue but no SMG fragments were placed in the tongue. The tongue wound was then closed with a single silk suture. These sham-operated rats were sacrificed after 8 weeks, and the tongue and intact SMG tissues were processed for light microscopy in the same manner as the tissues from group I. RESULTS Cultured AutografCs Appearance of SMG explants before autografting The representative SMG explants that were harvested after 1 day in culture contained some acinar cells at the periphery of the explants, but most of the acinar cells and other parenchymal elements had degenerated. The necrosis was particularly prominent toward the center of the explants which contained mostly stromal elements and cellular debris (Fig. 1).Many of the remaining acinar cells appeared to be rounded in shape, and many of the acini were disorganized. Other parenchymal elements also underwent changes; e.g., the convoluted granular tubule cells became rounded and lost their secretory granules. In addition, the low level of proliferative activity a t this time was evidenced by the paucity of 3H-thymidine-labeled cells (Fig. 1). After 4 or 7 days in vitro the SMG explants had degenerated even further than the 1-day explants (Figs. 2,3). There were no cells present that could be identified unequivocally as acinar cells, convoluted granular tubule cells, or other definitive parenchymal elements. However, the surviving epithelial cells had become more organized and existed in various configurations such as ductlike structures, or as ribbons, islands, and other aggregates of epithelium. The cells lining the ductlike structures were generally cuboidal or flattened in shape and contained a single nucleus. The surrounding acellular areas formed the stroma into which the epithelial A b breuiations Acinar cells D Ductlike structures E Tongue epithelium H & E Hematoxylin and eosin ISP Isoproterenol M Tongue muscle PASH Periodic acid Schiff s and hematoxylin SMG Submandibular gland A 14 N.L. O’DELL, M. SHARAWY, AND C.B. PENNINGTON CULTURED SUBMANDIBULAR GLAND AUTOGRAFTS structures proliferated. In some explants there was a discontinuous layer of epithelial cells around the periphery of the explants. Although the typical SMG morphology was lost, the surviving parenchymal and stromal cells incorporated more 3H-thymidine than the cells seen in radioautographs of the 1-day explants. Cells in the various aggregates of epithelial cells, as well as individual mononucleated cells were labeled with 3H-thymidine (Figs. 2,3). Appearance of cultured autografts after 8 weeks in tongue - Groups I-A, B, and C The previously cultured SMG autografts i.e., autografts of SMG that had been cultured for l, 4, or 7 days before autografting - also survived in the recipient tongues (Table 1). Ten of 14 autografts were recovered from the 1-day-in-culture-8-weeks-in-tongue group (group I-A). These indirect autografts contained numerous ductlike structures with walls that were generally one cell layer thick. The lumens of the ductlike structures varied in relative diameter (Fig. 4). Occasionally ductlike structures appeared to branch, and some ductlike structures ended in “buds” that contained PAS-positive cells. The ductlike structures of some autografts were well organized in lobules, whereas other autografts were disorganized and contained only epithelial remnants. The autografts were usually infiltrated with numerous mononuclear cells and mast cells. One autograft contained an area of cells that appeared to be acinarlike cells (Fig. 5). A second autograft Fig. 1. Radioautograph of a 1-day SMG explant. Acinar cells (A) and ductal cells (arrowheads) were present at the periphery of this explant. Notice that some of the parenchymal cells were rounded and that many of the acini and ducts appear disorganized. In the center of the explant there was evidence of severe tissue necrosis (*). The paucity of 3H-thymidine-labeledcells indicated a low level of proliferative activity in this explant (arrow). H & E; x 406. Fig. 2. Radioautograph of a 4-day SMG explant. After 4 days differentiated parenchymal elements could no longer be identified. 3H-thymidine-labeled cells were associated with ductlike structures and other epithelial aggregates within the explant (arrows). In addition, labeled mononucleated cells not associated with the epithelial aggregates were present (arrowheads). H & E; x 406. Fig. 3. Radioautograph of a 7-day SMG explant. Several epithelial aggregates (arrowheads) were seen in a connective tissue matrix. Notice that several individual mononucleated cells were labeled heavily with %thymidine (arrows). H & E; x 406. 15 contained obvious acinar cells and striated ducts, as well as a large ductlike structure that appeared to run to the tongue surface epithelium (Fig. 6). Convoluted granular tubules were not seen in any of these autografts. Thirteen of 15 autografts were recovered from the tongues of the 4-days-in-culture-8weeks-in-tongue group (group I-B). These autografts were similar in appearance to the group I-A autografts. In this group, one autograft contained several small acinarlike cells with PAS-positive granules (Fig. 7). In group I-C (7 days in culture-8 weeks in tongue), four of ten autografts were recovered. One autograft was infiltrated heavily with mononuclear cells, and the other three autografts were small and contained ductlike structures with lumens of relatively large diameter in a dense connective tissue matrix. One autograft contained a collection of cells with PAS-positive granules on a n isolated ductlike structure that may be differentiating acinar cells (Fig. 8). Striated ducts and convoluted granular tubules were not found in the autografts of SMG tissues that had been cultured for 4 or 7 days before autografting. Direct AutografCs Appearance after 8 weeks in tongue Group I1 Direct autografts (group 11)that had been in the tongue for 8 weeks were recovered in 29 of the 32 rats (Table 1).These autografts were characterized by the presence of numerous ductlike structures with walls that were generally one cell layer thick (Fig. 9). The cells lining the ductlike structures were generally cuboidal or columnar in shape with a single large nucleus. Many of the autografts were organized into lobules. Some of the ductlike structures branched and had “buds” containing cells that were PAS-positive, whereas other ducts appeared to terminate without branching or budding (Fig. 9). In contrast to the organized autografts, seven of the autografts had ductlike structures that appeared disorganized with little lobular morphogenesis. There was a variable amount of mononuclear cell infiltration of the autografts, and mast cells were observed commonly. Nine autografts (approximately 31% of the recovered autografts) contained cells that were identified as acinar cells, and six contained ducts that appeared to be striated ducts, but none of the autografts contained convoluted granular tubules (Fig. 10). In the 16 N.L. O’DELL, M. SHARAWY, AND C.B. PENNINGTON 17 CULTURED SUBMANDIBULAR GLAND AUTOGRAFTS TABLE 1. Summary of recovery and contents of direct and indirect 8-week submandibular gland autografts to the rat tongue Group I-A I-B I-c I1 I11 Iv Number of: Rats in Autografts group recovered 14 15 10 32 14l 92 Number of autografts exhibiting: Acinar Striated Convoluted cells ducts manular tubules 10 13 4 29 14 0 2 1 1 9 7 - 1 0 0 6 2 - 0 0 0 0 0 - ‘Only the 14 rats that survived five isoproterenol injections are included here 2Sham-operatedtongues without autografting submandibular gland tissue. autografts that contained acinar cells, the number of acinar cells varied. When an autograft contained acinar cells, there were areas within the same autograft that contained numerous ductlike structures but no acinar cells. In three autografts a large ductlike structure appeared to run from the autograft toward the epithelial surface of the tongue, and two of the autografts that contained these structures also contained acinar cells. Appearance after 8 weeks in tongue and after ISP injections - Group I11 Fourteen of the 29 rats in group I11 survived the series of five ISP injections. Only those rats that survived five ISP injections are included in these results. Autografts were located in the tongues of each of the 14 rats in this group (Table 1). The morphology of Fig. 4. Group I-A SMG autograft. After 1 day in culture and 8 weeks in the tongue, this autograft consisted primarily of ductlike structures organized into lobules that did not contain acinar cells. Some of the ductlike structures of larger diameter appeared to branch (arrows). The lobules were delineated by connective tissue (*I, H & E; x 84. Insert: Two ductlike structures with walls that were one cell layer thick. x 328. Fig. 5. Group I-A SMG autograft containing one area of cells that appeared to be acinar cells (arrows). Masson’s; x 328. Fig. 6 . Group I-A SMG autograft. Large ductlike structures (D) ran from the autograft to the tongue epithelium (E). An arrow indicates the area of the acinar cells and striated duct seen in the insert. H & E; x 53. Insert: A striated duct surrounded by acinar cells is shown at higher magnification. H & E; X 210. Fig. 7. Group I-B SMG autograft. After 4 days in culture and 8 weeks in the tongue, this autograft contained several ductlike structures surrounded by connective tissue and muscle (M). Groups of intensely PASpositive cells (arrows) were seen adjacent to ductlike structures (D). PASH; x 328. the autografts in the ISP-treated rats was similar to the morphology of the direct autografts in the group 11 rats. Seven of these autografts (50%of the recovered autografts) contained cells that were identified as acinar cells (Fig. ll),and two of the autografts contained striated ducts. As in group 11, the number of acinar cells in the autografts that contained acini varied, and the autografts that contained acinar cells also had areas that contained the ductlike structures with no acinar cells. Two autografts contained an area or areas of acinar cells that appeared to have responded to the ISP by undergoing cellular hypertrophy Fig. 11).These acinar cells appeared to be larger and to contain more PAS-positive material than the acinar cells in the group I1 autografts. As in group 11,three of the group 111autografts contained large ductlike structures that ran toward the tongue surface, and two of these three autografts had acinar cells present. None of the autografts in group I11 contained convoluted granular tubules. Appearance of Sham-Operated Tongues After 8 Weeks - Group IV After 8 weeks the wounds in the tongues of the sham-operated rats were healed. The muscle, connective tissues, and neurovascular elements in the wound area resembled the tongue tissues in the noninjured areas. As expected, there were no regenerating glandular tissues present in these sham-operated tongues. DISCUSSION The morphology of the rat SMG explants in this study was consistent with the morphology described for mature rat SMG explants (Tapp, 1967) and young adult mouse SMG explants (Wigley and Franks, 1976; O’Dell et al., 1979) maintained in a 5% COz 18 N.L. O’DELL, M. SHARAWY, AND C.B. PENNINGTON CULTURED SUBMANDIBULAR GLAND AUTOGRAFTS in air atmosphere, as well as with the morphology that characterizes rat SMG organ cultures (from 3-month-old rats) that were maintained in a 95% 02/5% C 0 2 atmosphere (Lucas, 1969). In the present study, the use of a 5% C02 in air atmosphere resulted in SMG explants that exhibited extensive cell death and tissue destruction during the various culture periods used. The SMG explants exhibited progressive parenchymal degeneration and necrosis, but with time the epithelial, connective tissue, and other viable elements began to proliferate and became organized into ductlike structures and other epithelial aggregates. Moreover, the radioautographs indicated that cells in the SMG explants were incorporating 3H-thymidine after 1, 4,or 7 days in culture. This suggests that the SMG explants contained viable parenchymal elements at the time of autografting. Moreover, the results showed that in many cases the cultured SMG tissues proliferated and became organized when autografted to the tongue. In addition, two autografts that had been cultured for 1 day before grafting contained acinar cells, which suggests some regenerative potential for these previously cultured SMG tissues. However, the autografts of SMG tissues that had been cultured for 4 or 7 days before grafting to the tongue did not exhibit much evidence of acinar cell cytodifferentiation. Only two autografts of the 17 combined 4-and 7-day autografts contained cells with PAS-positive granules that Fig. 8. Group I-C SMG autograft cultured for 7 days and then placed in the tongue for 8 weeks. PAS-positive cells (arrows) were associated with a ductlike structure surrounded by connective tissue and muscle (MI. PASH; x 480. Fig. 9. Group I1 direct autograft after 8 weeks in the tongue. The autograft consists primarily of ductlike structures that were lined with a single layer of cells. Supporting connective tissue contained numerous mononuclear cells. One ductlike structure is branched (arrow), and another exhibits a “bud” (arrowhead). H & E; x 218. Fig. 10. Another area of the 8-week direct autograft seen in Figure 9. Numerous acinar cells (A)and a striated duct (arrow) are indicative of the extent of regeneration in this area. H & E; x 218. Fig. 11. Direct autograft from a group 111 rat that received five ISP injections. The acinar cells (A) in this area appeared to have responded to the ISP by undergoing hypertrophy (compare to acinar cells in Fig. 10). A ductlike structure (D) was associated with these acinar cells. H & E; x 218. 19 resembled small acinarlike cells. Perhaps the extensive necrosis in the 4- and 7-day cultured SMG tissues adversely affected the ability of the surviving SMG tissues to regenerate during the %week period used in this study. These data suggest that culturing SMG tissues for extended time periods reduced the potential of these tissues to regenerate when they were autografted to the tongue. We are unable to offer a n unequivocal explanation for the inconsistency in the extent of acinar cell and ductal differentiation that occurs in the direct autografts. However, the formation of a ductal connection between the autograft and the epithelium of the tongue might be one explanation for the variable response. The importance of establishing a connection with the tongue epithelial surface is obvious from a functional viewpoint. Moreover, the most pronounced regenerative response was seen in a direct autograft that had a large ductlike structure that ran toward the epithelial surface of the tongue. However, the results suggested that this relationship was neither a prerequisite for acinar cell differentiation nor indicative of the extent of the regenerative response in the autografts, since acinar cells appeared in autografts that did not exhibit a connection to the epithelial surface. Multiple ISP injections initiate cellular hyperplasia in rodent salivary glands in vivo which peaks in a few days, and then in subsequent days the concurrent cellular hypertrophy becomes the predominant response to ISP injections (Schneyer et al., 1967; Schneyer, 1969; Novi and Baserga, 1971). In the present study, some of the acinar cells in two autografts from ISP-injected rats appeared to hypertrophy in response to ISP, whereas the acinar cells in the remaining autografts did not show signs of hypertrophy. Since the SMG autografts were well vasciilarized, it is reasonable to assume that the ISP was available to the cells in the autografts in high enough concentration to elicit a response. However, in the absence of a consistent response one might speculate that the observed response to ISP may have been due to variations in the number, distribution, or functional status of the adrenergic receptors that are characteristic of SMG acinar cells. This may also explain why even within the same autograft some acinar cells responded t o the ISP whereas others did not. It has also been shown that ISP can accelerate the morphological (Schneyer and Shac- 20 N.L. O’DELL, M. SHARAWY, AND C.B. PENNINGTON kleford, 1963; Bressler, 1973; Yamashina and Barka, 1972; Srinivasan et al., 1973)and biochemical (Ekfors et al., 1972) differentiation of developing rat SMG. If it is assumed that the regeneration seen in the SMG autografts in the tongue involves de novo formation and differentiation of acinar cells, then one would predict that ISP would accelerate the differentiation of the acinar cells in autografts of ISP-treated rats. From a morphologic standpoint one should find either a greater number of autografts with acinar cells or a greater number of acinar cells per autograft in the ISP-treated rats than in the nontreated rats a t any given time after the injections. Our data suggest that the ISP-treated rats (group III) had a higher proportion of autografts containing acinar cells (seven of 14)than did the group I1 rat autografts (nine of 29). These data represent three combined groups of ISPtreated rats, and the suggested enhancement of acinar cell differentiation was apparent in two of the three groups. Therefore, these data suggest that the ISP did enhance the differentiation of acinar cells in the autografts, and these data are consistent with those of Hoshino and Lin (1970, 1971), who showed that ISP enhanced the differentiation of acinar cells in mouse SMG isografts. Although the ISP appeared to increase the proportion of autografts that contained acinar cells, it did not appear to increase consistently the number of acinar cells in the autografts. The same range in the relative number of acinar cells (no acinar cells present to numerous acinar cells present) was found in the autografts from the group I1 and the ISP-treated rats. The apparent lack of a hyperplastic response, and a n inconsistent hypertrophic response to the ISP may have been related to the time period in which the ISP was administered. In earlier studies, Hoshino and Lin (1970, 1971) injected host mice with ISP before and for varying lengths of time after isografting salivary glands to the peritoneal cavity, and found that the ISP injections resulted in more acinar cell regeneration in the isografts. In contrast, we arbitrarily injected rats during the last 5 days of a n %week regeneration period, which may have been too late to initiate a consistent, uniform response - i.e., uniform cellular hypertrophy, hyperplasia, and acceleration of acinar cell differentiation. Moreover, the importance of the stage of development of the salivary glands on the observed response to ISP has been illustrated in studies on neonatal rat salivary glands. For instance, mul- tiple ISP injections produced glandular enlargement and accelerated postnatal differentiation of the submandibular glands in neonatal rats (Schneyer and Shackleford, 1963; Ekfors et al., 1972; Yamashina and Barka, 1972; Srinivasan et al., 1973). However, depending on the age of the rats, multiple ISP injections resulted in either glandular hypertrophy alone or a combination of glandular hypertrophy and hyperplasia (Barka et al., 1973). In summary, our data indicate that SMG explants cultured for 1, 4, or 7 days prior to autografting to the tongue can survive, proliferate, and in some cases differentiate into acinar cells. However, the longer culture periods appear to be detrimental to the regenerative potential. Finally, ISP appeared to accelerate acinar cell differentiation in some direct autografts and induce cellular hypertrophy in two autografts. ACKNOWLEDGMENTS This research was supported by the United States Public Health Service grant 5-SO8RR09043-02. The authors thank Dr. Dale Bockman for his constructive criticism of this manuscript, and Mrs. Maggie Shaw and Mrs. Linda Cullum for typing the manuscript. LITERATURE CITED Barka, T. (1965)Induced cell proliferation: The effect of isoproterenol. Exp. 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