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Effects of prior culture or isoproterenol injections on the regeneration of rat submandibular gland autografts.

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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.
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autograft, effect, submandibular, prior, injections, gland, culture, rat, regenerative, isoproterenol
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