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Regeneration of submandibular gland autografts in sympathectomized rats.

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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.
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autograft, sympathectomized, submandibular, gland, regenerative, rats
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