THE ANATOMICAL RECORD 218:373-379 (1987) Regeneration of Submandibular Gland Autografts in Sympathectomized Rats NORRIS L. O’DELL, MOHAMED SHARAWY, MARY C. RICHARDSON, AND CATHERINE B. PENNINGTON Departments of Oral Biology (N.L. O., M.S., M. C.R., C.B.P.) and Anatomy (N.L. O., M.S.), Medical College of Georgia, Augusta, GA 30912 ABSTRACT This morphologic study compares the regenerative response in submandibular gland (SMG) autografts placed in the tongues of previously sympathectomized rats to autografts placed in tongues of sham-sympathectomized rats. We hypothesized that sympathectomy would alter the process of cellular proliferation and inhibit cytodifferentiation in regenerating SMG autografts. Either 1week, or 8 to 11weeks following the SMG autografting procedure, the rats were sacrificed and their tongues were removed and sectioned in a cryostat. Frozen tissue sections containing the SMG autografts were either reacted for cholinesterase activity, treated with a glyoxylic acid mixture to induce histofluorescence, or stained for histologic examination. In addition, 3H-thymidine labeled and unlabeled cells were counted in autoradiographs of 1-week autografts, and these counts were used to calculate labeling indices. The 1-week SMG autografts from both the sympathectomized and the sham-sympathectomized rats were similar in histologic appearance, and neither group of autografts contained cholinesterase-positive or monoaminergic nerve fibers. The 8- to 11-week autografts from sympathectomized and sham-sympathectomized rats contained cholinesterase-positive fibers, but monoaminergic fibers were present in the autografts only from the sham-operated rats. Acinar cells were observed in one-third of the 8- to 11-weekautografts of both the sympathectomized and the shamsympathectomized rats. This finding suggests that sympathectomy did not preclude cytodifferentiation in the autografts. The autoradiographic data revealed no statistically significant difference between the mean labeling indices of the 1-week autografts from the sympathectomized and sham-sympathectomized rats, which suggests that sympathectomy also did not alter the level of cellular proliferation in the autografts. The present study focuses on the process of regeneration in mature submandibular gland (SMG) fragments that were autografted to the tongues of rats that had been selectively sympathectomized prior to the autografting procedure. In previous studies the morphology of SMG autografts was studied at various times following the grafting procedure at the light microscopic and electron microscopic levels (Sharawy and O’Dell, 1979, 1981; O’Dell et al., 1983). The results of these studies showed that during the first few days after implantation, the autograft was infiltrated with mononuclear cells and exhibited massive necrosis with some parenchyma surviving at the periphery of the autograft (Sharawy and O’Dell, 1979,1981). By one week after implantation, the autograft contained numerous ductlike structures and exhibited some lobular morphogenesis with ductal branching, but the regenerating salivary gland epithelium appeared undifferentiated. However, by 8 weeks after implantation, there was evidence of acinar cell and striated duct cell cytodifferentiation in some of the autografts (Sharawy and O’Dell, 1979,1981; O’Dell et al., 1983). We have inferred that re-establishing the autonomic innervation to SMG autografts that have been totally 0 1987 ALAN R. LISS, INC. separated from their original nerve supply might be critical to the regenerative response seen in these tissues (Sharawy and O’Dell, 1979, 1981; O’Dell et al., 1983). Moreover, previous studies have demonstrated the effects of sympathomimetic agents on salivary gland tissues. For example, isoproterenol, a 0-adrenergic agonist, accelerated cellular differentiation in newborn rat parotid gland and SMG (Schneyer and Shackleford, 19631, mouse salivary gland isografts (Hoshino and Lin, 1970, 19711, and some rat SMG autografts (O’Dell et al., 1983),but retarded mitotic activity in the “reactive zone” of regenerating adult rat SMG (Boshell and Pennington, 1980).Although cholinesterase-positive nerves and monoaminergic nerves have been demonstrated among the ductlike structures in 8-week SMG autografts (O’Dell et al., 19851, the specific roles of these nerves in the processes of initiation, proliferation, morphogenesis, and differentiation within the regenerating SMG autografts are not known. Therefore, to better understand the role Received October 15, 1986; accepted March 23, 1987. Address reprint requests to Dr. Norris L. O’Dell, Department of Oral Biology, Medical College of Georgia, Augusta, GA 30912. 374 N.L. O’DELL, M. SHARAWY, M.C. RICHARDSON, AND C.B. PENNINGTON of the sympathetic nerves in the process of SMG regeneration, the present study assessed the effects of selective denervation on a n early phase and a later phase of the regenerative response seen in SMG autografts. Specifically, we studied the effects of sympathectomy on the proliferative phase of the regenerative process, which appears to peak about 7 days following the autografting procedure (Sharawy and O’Dell, 1981). We hypothesized that some change in the amount of cell proliferation should occur if the sympathetic nerve supply to the autograft is critical to this early, hyperplastic phase of the regenerative process. Moreover, we hypothesized that there should be no autografts with acinar cells or other differentiated parenchymal cells in 8-week SMG autografts in denervated rats if the sympathetic nerve supply is critical to the process of cell differentiation. MATERIALS AND METHODS Male, Sprague-Dawley rats that were at least 12 weeks old at the beginning of the experiments were used. In the short-term study of the effects of denervation on the hyperplastic regenerative phase, each of 10 rats in sympathectomized group I was anesthetized at the time of surgery with a n intraperitoneal (ip) injection of chloral hydrate (250-500 m g k g b.w.1 supplemented with ether inhalation. A midventral incision was made that extended from the interramal vibrissae to the sternum to provide access to the carotid sheaths and the underlying superior cervical ganglia (Mark, 1980).The ganglia were located and avulsed from the cervical sympathetic chain. Since these ganglia give rise to the postganglionic sympathetic fibers destined for the head region, their removal effected a sympathectomy of the tongue and other structures in the head. Similarly, each of 10 rats in the sham-sympathectomized group I was anesthetized and subjected to the same surgical incision and dissection that were used for the rats in the sympathectomized group I. However, in this sham-operated group, the superior cervical ganglia were located but not excised. Three days after the sympathectomy or sham-sympathectomy, a n SMG autograft was placed in each rat’s tongue. The autografting procedure appears in detail elsewhere (Sharawy and O’Dell, 1981; O’Dell et al., 1983). Briefly, this procedure involves removing approximately one-half of a submandibular gland through a n incision in the neck and closing the incision with silk sutures. The piece of gland is cleaned of sublingual gland and adherent connective tissues and minced into 2-3 mm3 fragments. The mucosa on one side of the middle one-third of the tongue is pierced with a pair of fine, pointed forceps, and 5 to 10 fragments are placed in the tongue. The tongue mucosa is then closed with a single silk suture. In the long-term study, 7 rats were subjected to bilateral superior cervical ganglionectomies (sympathecto- A H&E M SMG uv V Abbreviations acinar cells hematoxylin and eosin tongue muscle submandibular gland ultraviolet illumination blood vessels mized group 11) and received SMG autografts, and 6 rats were sham-operated (sham-sympathectomized group 11) as in the short-term study and then they received SMG autografts. One week following the SMG autografting procedure, the sympathectomized and sham-sympathectomized group I rats were given ip injections of 3H-thymidine at a dose of 1 pCi/gm b.w. (specific activity, 20 Curies/ mMole). One hour later, each rat was sacrificed with a combination of chloral hydrate and ether and the middle one-third of the tongue was removed and placed in a cryostat to freeze (-30°C). Similarly, after 8 to 11weeks, each sympathectomized or sham-sympathectomized group I1 rat was killed with chloral hydrate and ether, and the tongue was removed and placed in the cryostat. In addition, a n intact SMG from each rat was removed from the submandibular region and frozen. Twenty-micrometer-thick cross sections of each tongue were made in the area of the SMG autograft, and sections of the intact SMG were also made. Representative tissue sections were stained with hematoxylin and eosin (H & El. Other cryostat sections containing SMG autografts and SMG tissue from groups I and 11rats were collected on glass coverslips and subjected to a cholinesterase localization procedure described by Hanker et al. (1973) as modified by O’Dell et al. (1985). This technique intensifies the cholinesterase reaction product obtained by using the El-Badawi and Schenk (1967) modification of the Karnovsky procedure (Karnovsky and Roots, 1964)by bridging osmium to the Hatchett’s brown deposits through thiocarbohydrazide (Hanker et al., 1966). Cholinesterase activity was localized by placing the cryostat sections in a n incubation medium containing acetylthiocholine iodide as the substrate plus the nonspecific cholinesterase inhibitor, tetraisopropylpyrophosphoramide, at a concentration of approximately 10-4M (Hanker et al., 1973). In addition, some sections were reacted in incubation medium that did not contain substrate as a n additional control on the presence of cholinesterase activity. Other cryostat sections were subjected to a monoamine histofluorescence technique (de la Torre, 1980). This modification of the original technique (de la Torre and Surgeon, 1976) utilizes a glyoxylic acid mixture to induce fluorescence in tissues containing biogenic amines, especially catecholaminergic or adrenergic nerves. Cryostat sections were dipped in a freshly prepared sucrose-potassium phosphate-glyoxylic acid (SPG) solution for 3 seconds, air-dried for 5 minutes, placed in a 95°C oven for 2.5 minutes, and then mounted in mineral oil on a glass slide for microscopic examination. Other sections were dipped in a sucrose-potassium phosphate solution that did not contain glyoxylic acid as a control on the induced histofluorescence. Also, some sections were not treated with any solution to provide a n additional control on the observed pattern of induced histofluorescence. The cholinesterase preparations were studied and photographed on a Zeiss Photomicroscope 11, and the SPG preparations were studied and photographed with the ultraviolet-fluorescence configuration of the same microscope. For the SPG preparations, phase microscopy was used to locate the areas of the tongues that contained the autografts. Then histofluorescence patterns were AUTOGRAFTS IN SYMPATHECTOMIZED RATS 375 Fig. 1. This autoradiographic preparation illustrates a portion of a 1week SMG autograft from a sham-sympathectomized group I rat. Regenerating SMG cells are arranged in various ways including characteristic ductlike structures (arrowheads) in the stroma of the tongue. Some 3H-thymidine-labeledcells (arrows) are shown in the walls of the ductlike structures. H & E; x333. Fig. 2. This low-power photomicrograph shows a 1-week SMG autograft in the tongue of a sham-sympathectomized group I rat. The autograft (arrowheads) is surrounded by muscle (M) and associated connective tissues of the tongue. Although cholinesterase-positive nerve fibers (arrows) are seen around lingual blood vessels (V) and within lingual nerves (asterisks), there are no fibers seen within the SMG autograft. Cholinesterase; x 57. studied using a n exciter filter with a peak transmission of approximately 400 nm and a barrier filter with a cutoff wave length of 530 nm. For the short-term study, tissue sections were subjected to autoradiography in order to obtain labeling indices for these SMG autografts. Cryostat sections were collected on glass coverslips that had been coated with albumin. These sections were fixed in neutral buffered formalin for 1hour and then rinsed in cacodylate buffer. The sections were dipped in Kodak NTB2 nuclear track emulsion, stored in the dark for 3 weeks, and then developed and stained with H & E. These autoradiographic preparations were used to count the number of labeled and unlabeled epithelial cells in each autograft. The autoradiographic preparations were viewed on a n Olympus BH-2 microscope equipped with a n SMI-UNICOMP video camera. The camera was interfaced with the video monitor of a n Apple I1 plus computer and with a Bausch and Lomb HiPad Digitizer. The autograft was located and projected onto the video monitor. The cursor of the digitizer was used to count the number of labeled and unlabeled cells in 7 different fields in each autograft. Two investigators counted the labeled and unlabeled cells in each autograft. These counts were stored in the microcomputer and used to calculate the labeling indices for the autografts from the sympathectomized and sham-sympathectomized group I rats. A labeling index was computed by dividing the number of labeled cells by the total number of labeled plus unlabeled cells in a particular autograft and multiplying that number by 100 to obtain a percentage value. The data from the two investigators were combined to obtain mean labeling indices, which were then compared statistically using a two-tailed, Student’s t-test with pooled variance. (Color Figures 3-5 were prepared and appear elsewhere in this issue. Please see pp. 391-395 for these figures and their accompanying legends.) RESULTS Short-Term Study One-week autografts were located in the tongues of 7 of the 10 sympathectomized group I rats and in the tongues of 8 of the 10 sham-sympathectomized group I rats (Fig. 1).The 1-week autografts from these sympathectomized and sham-operated rats appeared similar histologically. The autografts contained numerous clusters of epithelial cells within a very cellular loose connective tissue matrix. These epithelial cells often formed characteristic ductlike structures (Fig. 1).In some autografts there was evidence of lobular morphogenesis with the ductlike structures branching, whereas other autografts were more disorganized with the ductlike structures unbranched. The 1-week autografts from the sympathectomized and the sham-sympathectomized rats generally did not contain cholinesterase-positive fibers (Fig. 2). Although there were numerous cholinesterase-positive fibers associated with nearby blood vessels and nerves in each rat’s tongue (Fig. 2), only a n occasional cholinesterasepositive fiber was observed within the regenerating SMG autografts, and these fibers were confined to 1autograft in each of these 2 groups. 376 N.L. O’DELL, M. SHARAWY, M.C. RICHARDSON, AND C.B. PENNINGTON TABLE 1. Labeling indices for 1-week SMG autografts from sympathectomized and sham-sympathectomizedgroup I rats No. of Treatment Sympathectomized Sham-sympathectomized labeled autografts counted Labeling index Total No. of cells counteda mean f std. dev. 6 9,410 4.05 f 1.80 7 10,715 3.89 f 1.71 P* > 0.05 aTotal number of cells counted reflects the combined totals of two investigators used to calculate the labeling indices. *Based on a two-tailed, Student’s t-test using pooled variance. When the SPG-induced histofluorescence patterns of the 1-week tissues were examined, the sham-sympathectomized group I rat tongues contained fluorescent fibers associated primarily with branches of the lingual blood vessels. Moreover, fluorescent fibers were seen throughout the stroma of the intact SMG tissues of this group. However, there were no fluorescent fibers seen within the SMG autografts of these sham-operated rats (Fig. 3). Similarly, there were no fluorescent fibers seen within the SMG autografts of the sympathectomized group I rats. In addition, there were no fluorescent fibers seen within the sections of intact SMG tissues or around the blood vessels in the tongue tissues surrounding the SMG autografts from these denervated rats. Six of 7 autoradiographic preparations of autografts recovered from the sympathectomized group I rats, and 7 of 8 autoradiographic preparations of the autografts recovered from the sham-sympathectomized group I rats were suitable for counting labeled and unlabeled parenchymal cells in the 1-week autografts. Figure 1 illustrates a n autoradiographic preparation used to count the labeled and unlabeled cells in the 1-week autografts, and Table 1 summarizes the quantitative data on the labeling indices for the 1-week autografts. The mean labeling index of 4.05 for the sympathectomized group I autografts and a mean labeling index of 3.89 for the sham-sympathectomized group I autografts were not significantly different statistically at the P = 0.05 level. Long-Term Study Autografts were located in the tongues of all the sympathectomized and sham-sympathectomized group I1 rats. The 8-to 11-week autografts from these sympathectomized and sham-operated rats were similar in histologic appearance. Some of the autografts were well organized into lobules with characteristic ductlike structures representing the principal epithelial element. In other autografts, ductlike structures were present but were not well organized into lobules. Some of the ductlike structures appeared to be striated ducts, whereas others appeared undifferentiated. Granular convoluted tubules were not seen in any of the autografts. The autografts were infiltrated with numerous mononuclear cells, and mast cells were seen often in the area of the autografts and throughout the surrounding tongue tissues. One autograft in each group contained SMG epithelium that was involved in a granulomatouslike response to pieces of hair that had adhered to the SMG fragments at the time of autografting. The SPG preparations showed that the sham-sympathectomized group I1 SMG autografts and SMG tissues contained numerous yellow-green fluorescent monoaminergic nerve fibers (Fig. 4). These fibers were often associated with larger blood vessels in the tongue as well as small blood vessels around and within the SMG autografts. In addition to these fluorescent monoaminergic fibers, these autografts contained bright yellow fluorescent structures that appeared to be mast cells or their released contents, internal elastic laminae of blood vessels, and a n occasional hair that had adhered to the autograft during implantation. In contrast, the autografts and SMG tissues from 6 of the 7 sympathectomized group I1 rats did not contain fluorescent monoaminergic nerve fibers (Fig. 5). However, the autograft and SMG in the remaining rat in this sympathectomized group did contain some fluorescent fibers, which indicated that the sympathectomy was incomplete or that some nerve fibers had regenerated following the surgery. Therefore, this rat was excluded from the study, which left 6 rats in the sympathectomized group 11. The autografts in the sympathectomized rats did contain collections of bright yellow fluorescent material similar to that seen in the sham-operated group. Acinar cell cytodifferentiation was evident in 2 of the 6 autografts recovered from the sympathectomized group I1 rats, and in 2 of the 6 autografts that were recovered from the sham-sympathectomized group I1 rats (Figs. 6, 7). The most obvious regenerative response was seen in a n autograft from a sham-operated rat. One area of this autograft contained a well-organized lobule of regenerating SMG tissues with numerous acinar cells and an occasional striated duct (Fig. 6). The second autograft that contained acinar cells in this group and the two sympathectomized group I1 autografts that contained acinar cells did not exhibit as remarkable a response as the aforementioned autograft from a sham-sympathectomized rat, but there was still evidence of cytodifferentiation (Fig. 7). The SMG tissues and SMG autografts from the shamsympathectomized group I1 rats contained numerous cholinesterase-positive nerve fibers (Fig. 8). These fibers were associated with the adventitia of the branches of lingual vessels and with adjacent large nerves as well a s with the regenerating SMG parenchyma and stroma. The cholinesterase-positive fibers branched throughout the connective tissues surrounding the numerous ductlike structures within the autografts (Fig. 8).Similarly, the sympathectomized group 11 rat SMG tissues and SMG autografts contained cholinesterase-positive nerve fibers (Fig. 9). However, there appeared to be fewer fibers within the SMG autografts from these sympathectomized rats than there were in the autografts from the sham-sympathectomized rats. AUTOGRAFTS IN SYMPATHECTOMIZED RATS 377 Fig. 6. This photomicrograph shows collections of acinar cells (A) in Fig. 7. Similar to Figure 6, this photomicrograph shows groups of a 9-week SMG autograft from a sham-sympathectornizedgroup I1 rat. acinar cells (A) and ductlike structures (asterisks) among the muscle Two ductlike structures (asterisks) and a muscle fiber (M) are also fibers (MI of the tongue in an 11-week SMG autograft from a sympa. seen. H & E; X230. thectomized group I1 rat. H & E; ~ 2 3 0 . DISCUSSION The 1-week SMG autografts from the sympathectomized and sham-sympathectomized group I rats were similar in histologic appearance to 1-week SMG autografts studied previously (Sharawy and O’Dell, 1981). In the present study there were no morphologic changes in the 1-week autografts to indicate that the bilateral superior ganglionectomies had altered the early stages of the regenerative process. Although the labeling index for the autografts from sham-operated rats in the present study was lower than that found in a previous study (Sharawy and O’Dell, 19811, the present value for the 1week autografts was of the same order of magnitude as the value in the previous study. The lack of a significant difference between the mean labeling indices for the sympathectomized and sham-sympathectomized group I rat autografts suggests that sympathectomy did not effect the early hyperplastic phase of the regenerative process. This apparent lack of a n effect on autograft proliferative activity is consistent with the observation in neonatal rats where the rate of cellular proliferation in sympathectomized SMG was similar to that of nonsympathectomized SMG (Srinivasan and Chang, 1977). However, sympathectomy resulted in a decrease in gland weight and caused acinar cell hypotrophy in neonatal rat parotid gland (Bloom et al., 1981) and SMG (Srinivasan and Chang, 1977), as well as retarded postnatal development of acinar cells and granular convoluted ductal cells in the SMG (Srinivasan and Chang, 1977). In the present study a consistent effect of sympathectomy on the morphology of acinar cells in the autografts was not seen, and granular convoluted ductal cells were not seen in either the sympathectomized or the shamsympathectomized group I1 autografts. The absence of fluorescent fibers in the 1-week autografts of both the sham-sympathectomized and sympathectomized group I rats suggests that there were no functional monoaminergic nerve fibers present during the hyperplastic phase of the regenerative process regardless of whether or not the cervical sympathetic nerve pathways were intact. This lack of fibers in 1-week autografts is reminiscent of the innervation pattern in neonatal rat SMG tissues in which there are no catecholamine-containing nerve fibers present until the fifth to sixth day after birth (Cutler et al., 1981; Bottaro and Cutler, 1984). The 8- to 11-week SMG autografts from the sympathectomized and sham-sympathectomized group I1 rats were similar in histologic appearance to SMG autografts studied previously (Sharawy and O’Dell, 1981; O’Dell et al., 1983,1985). The histofluoresence data indicated that with one exception the sympathectomy procedures were successful in the sympathectomized group I1 rats. Since one-third of these sympathectomized rats had a n SMG autograft that contained acinar cells, the hypothesized elimination of cytodifferentiation following sympathectomy was not found. Although cytodifferentiation was 378 N.L. O’DELL, M. SHARAWY, M.C. RICHARDSON, AND C.B. PENNINGTON Fig. 8. This photomicrograph of a portion of a 9-week SMG autograft from a sham-sympathectomized group I1 rat shows numerous cholinesterase-positive nerve fibers (arrows) among the ductlike structures (asterisks in lumens of three ductlike structures). Some skeletal muscle (M) fibers of the tongue are seen. Cholinesterase; x 118. Fig. 9. This photomicrograph illustrates a portion of an 11-week SMG autograft from a sympathectomized group I1 rat. Although cholinesterase-positive fibers (arrows) are present, they did not appear to be as numerous as those seen in the sham-sympathectomized group I1 autografts. The lumens of three ductlike structures (asterisks) and muscle fibers (M) are indicated. Cholinesterase; x 118. not as remarkable in the two autografts from sympathectomized rats as it was in one of the autografts from a sham-sympathectomized rat, cytodifferentiation did occur in the denervated autografts. These data suggest that functional monoaminergic nerves were not critical to the process of cytodifferentiation in the regenerating SMG autograft. Moreover, the present data suggest that the sympathetic nervous system does not play the critical role in SMG autograft regeneration that general innervation plays in amphibian limb regeneration (e.g., Singer, 1952, 1978; Wallace, 1984). The presence of differentiated cells in the 8-to 11-week SMG autografts of the sympathectomized rats appears to mimic the relationship seen in neonatal rat SMG tissues. Electrophysiologic and histofluorescence data suggest that during the early neonatal period, the adult stimulus-secretion mechanism for protein are evolving in rat SMG cells in the absence of functional adrenergic neural connections (Cutler et al., 1981). In fact, adrenergic neural connections occurred only after the development of the SMG cellular synthetic and secretory pathways was completed (Cutler et al., 1981). The absence of aminergic nerves in the early regenerative period noted in the present study suggests that the regenerating epithelium may have a trophic effect on the autonomic fibers that eventually reach the autograft. This relationship may be similar to that seen, for example, in developing embryonic mouse tissues in which SMG tissues appeared to stimulate and direct nerve fiber outgrowth from autonomic ganglia (Coughlin, 1975; Coughlin et al., 1978). Although monoaminergic and cholinesterase-positive fibers were found previously in 8-week SMG autografts, the origin of the fibers was not known (O’Dell et al., 1985). However, the absence of numerous cholinesterase-positive fibers in the 1-week autografts of the group I rats indicates that the fibers seen a t later times in the regenerative process (8-11 weeks) represent fibers that grow into the autograft from surrounding nerves in the tongue and do not represent fibers that were transferred to the tongue with the SMG autograft tissues. Also, the use of a butyrylcholinesterase inhibitor in the incubation medium along with other histochemical controls suggests that the cholinesterase-positive fibers found in the SMG autografts are primarily acetylcholinergic fibers. There were no histofluorescent fibers within the sympathectomized or sham-sympathectomized group I autografts and none in the sympathectomized group I1 autografts, but there were histofluorescent fibers in the sham-sympathectomized group I1 autografts. Therefore, these histofluoresence patterns suggest that the monoaminergic fibers seen in the 8- to 11-week SMG autografts in this study and in a previous study (O’Dell et al., 1985)primarily represent monoaminergic fibers that have branched from nerves in the tongue tissues surrounding the autograft and from nerves that accompany blood vessels that are coursing through the autograft area. The observation that there appeared to be less extensive branching of the cholinesterase-positive fibers associated with the sympathectomized group I1 autografts than with the sham-sympathectomized group I1 autografts suggests that some of these fibers may be cholinesterase-positive adrenergic fibers similar to those seen in various other tissues (Eranko et al., 1970; Eranko and AUTOGRAFTS IN SYMPATHECTOMIZED RATS Eranko, 1971; Barajas and Wang, 1975; Tervo, 1977; Burden and Lawrence, 1978). However, confirmation of this observation will require further study. Regardless of the type or types of fibers that are destroyed by superior cervical ganglionectomy, the interruption of these fibers did not appear to effect the course of regeneration in the SMG autografts. In summary, the morphologic and histofluorescence data along with the lack of a statistically significant difference in the mean labeling indices of the sympathectomized and sham-sympathectomized group I autografts indicated that the sympathetic nervous system was not critical for the proliferative activity that characterizes the hyperplastic phase of SMG autograft regeneration. Moreover, these autoradiographic and morphologic data indicated that a n intact sympathetic nervous system was not necessary for the initiation of regeneration in these SMG autografts. Lastly, the presence of differentiated cells in the 8- to 11-week sympathectomized group I1 autografts indicated that a n intact sympathetic innervation was not critical to the process of cytodifferentiation in the SMG autograft model. ACKNOWLEDGMENTS The authors would like to thank Mrs. Linda Cullum for typing this manuscript, and Ms. Vera Larke for preparing the photographic illustrations. This work was supported in part by NIH Biomedical Research Support Grant #SO7-RRO5795. LITERATURE CITED Barajas, L., and P. Wang (1975) Demonstration of acetylcholinesterase in the adrenergic nerves of the renal glomerular arteries. J. Ultrastruct. Res., 53~244-253. Bloom, G.D., B.Carlsoo, A . Danielsson, S. Hellstrom, and R. Henriksson (1981)Trophic effect of the sympathetic nervous system on the early development of the rat parotid gland A quantitative ultrastructural study. Anat. Rec., 201~645-654. Boshell, J.L., and C. Pennington (1980) Histological observations on the effects of isoproterenol on regenerating submandibular glands of the rat. Cell Tiss. Res., 213~411416. Bottaro, B., and L.S. Cutler (1984)An electrophysiological study of the postnatal development of the autonomic innervation of the rat submandibular salivary glands. Archs Oral Biol., 29:237-242. Burden, H.W., and I.E. Lawrence, Jr. (1978) Experimental studies on the acetylcholinesterase-positivenerves in the ovary of the rat. Anat. Rec., 190:233-242. Coughlin, M.D. (1975) Target organ stimulation of parasympathetic nerve growth in the developing mouse submandibular gland. Dev. Biol., 431140-158. Coughlin, M.D., M.D. Dibner, D.M. Boyer, and I.B. Black (1978) Factors regulating development of a n embryonic mouse sympathetic ganglion. Dev. Biol.. 66r513-528. Cutler, L.S., C.P. Christian, and B. Bottaro (1981) Development of stimulus-secretion coupling in salivary glands. In: Methods in Cell Biology. A.R. Hand and C. Oliver, eds. New York, Academic Press, 379 pp. 531-545. de la Torre, J.C. (1980) Standardization of the sucrose-potassium phosphate-glyoxylic acid histofluorescence method for tissue monoamines. Neurosci. Letters, 171339-340. de la Torre, J.C., and J.W. Surgeon (1976) A methodological approach to rapid and sensitive monoamine histofluorescence using a modified glyoxylic acid technique: The SPG method. Histochemistry, 49:81-93. El-Badawi, A., and E.A. Schenk (1967) Histochemical methods for separate, consecutive and simultaneous demonstration of acetylcholinesterase and norepinephrine in cryostat sections. J. Histochem. Cytochem., 15.580-588. Eranko, O., and L. Eranko (1971)Loss of histochemically demonstrable catecholamines and acetylcholinesterase from sympathetic nerve fibres of the pineal body of the rat after chemical sympathectomy with 6-hydroxydopamine. Histochem. J., 3:357-363. Eranko, O., L. Rechardt, L. Eranko and A. Cunningham (1970) Light and electron microscopic histochemical observations on cholinesterasecontaining sympathetic nerve fibers in the pineal body of the rat. Histochem. J., 2 4 7 9 4 8 9 . Hanker, J.S., C. Deb, H.L. Wasserkrug, and A.M. Seligman (1966) Staining tissue for light and electron microscopy by bridging metals with multidentate ligands. Science, 152t1631-1634. Hanker, J.S., L.P. Thornburg, P.E. Yates, and H.G. Moore I11 (1973) The demonstration of cholinesterase by the formation of osmium blacks at the sites of Hatchett’s brown. Histochemie, 37:223-242. Hoshino, K., and C.D. Lin (1970) Selective effects of testosterone and isoproterenol upon regenerating submandibular gland isografts in BALB/c mice. Anat. Rec., 167;489-496. Hoshino, K., and C.D. Lin (1971) Induction of hyperplasia in mouse salivary gland isografts. Eur. J. Cancer, 7:373-376. Karnovsky, M.J., and K. Roots (1964) A “directcoloring” thiocholine method for cholinesterases. J. Histochem. Cytochem., 12:219-221. Mark, M.R. (1980)The role of the autonomic nervous system in regulation of the structural and functional status of the rat parotid gland. Dissertation, The University of Michigan. O’Dell, N.L., M. Sharawy, and J.S. Hanker (1985) Histochemical demonstration of monoaminergic and cholinesterase-positive nerve fibres in regenerating rat submandibular gland autografts. Histochem. J., 17r665-674. O’Dell, N.L., M. Sharawy, and C.B. Pennington (1983) Effects of prior culture or isoproterenol injections on the regeneration of rat submandibular gland autografts. Anat. Rec., 206:ll-21. Schneyer, C.A., and J.M. Shackleford (1963) Accelerated development of salivary glands of early postnatal rats following isoproterenol. Proc. SOC.Exp. Biol. Med., 1121320-324. Sharawy, M., and N.L. O’Dell(1979)Regeneration of acini in submandibular gland autografts. Anat. Rec., 1951431442. Sharawy, M., and N.L. O’Dell (1981) Regeneration of submandibular salivary gland autografted in the rat tongue. Anat. Rec., 201:499511. Singer, M. (1952)The influence of the nerve on the regeneration of the amphibian extremity. Q. Rev. Biol., 27:169-200. Singer, M. (1978) On the nature of the neurotrophic phenomenon in urodele limb regeneration. Am. Zool., 18:829-841. Srinivasan, R., and W.W.L. Chang (1977) Effects of neonatal sympathectomy on the postnatal differentiation of the submandibular gland of the rat. Cell Tiss. Res., 180:99-109. Tervo, T. (1977) Consecutive demonstration of nerves containing catecholamine and acetylcholinesterase in the rat cornea. Histochemistry, 50~291-299. Wallace, H. (1984)The response of denervated axolotl arms to delayed amputation. J. Embryol. Exp. Morph., 84~303-307.