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Use of nude athymic mice for the study of hypertrophic scars and keloidsVascular continuity between mouse and implants.

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THE ANATOMICAL RECORD 225:189-196 (1989)
Use of Nude (Athymic) Mice for the Study of
Hypertrophic Scars and Keloids: Vascular
Continuity Between Mouse and Implants
C. WARD KISCHER, DON SHERIDAN, AND JANA PINDUR
Department of Anatomy, University of Arizona College of Medicine, Tucson, Arizona 85724
ABSTRACT
Hypertrophic scars and keloids appear to be unique to humans
since animals are not known to form these lesions. Therefore, in an effort to develop an experimental model for their study, implants of these human lesions were
made in nude (athymic) mice (ndnu) in suprascapular subcutaneous pockets. The
implants were recovered from 2 to 246 days. By histological and fine structural
parameters all implants remained viable and their morphological character was
maintained. Selected mice were injected with barium to confirm by microangiography vascular flow between mouse and implant. Hoechst stain for DNA, used to
distinguish mouse cells from human cells, confirmed vascular anastamosis between host and implant: barium-filled vessels in the interior of the implant demonstrated human endothelial cells. Peripheral vascularization of the implant with
minimal ingrowth of mouse vessels occurs during the first 8 days. Anastamosis
probably occurs sometime before 16 days postimplantation, or earlier, depending
upon the availability of patent microvessels in the implanted tissue. The presence
of the implant does not appear to prompt a continuing vascular growth into or
throughout the implant. The time frame of 16 days postimplantation should be
taken into account when developing schemata of experimental or therapeutic modalities.
The hypertrophic scar and its closely related lesion,
the keloid, are often the sequelae of deep surface injury. There are reports in the literature claiming that
these lesions have been produced (Silverstein et al.,
1976) or found (Marcenac, 1951) in animals. However,
these claims have not been confirmed and the concensus is that no animal model exists for these lesions.
Studies of hypertrophic scar and keloid implanted into
subcutaneous pockets of the nude mouse1 demonstrated that they survive and retain their character
after both short- and long-term implantation (Shetlar
et al., 1985; Kischer et al., 1988). This permits experimental investigation of these lesions, which otherwise
would be impossible from an ethical or legal point of
view. Because the implants survive unchanged in their
morphology up to 246 days (Kischer et al., 1988) one
could assume vascular anastomosis occurs between
host and implant. Yet, it remained to be demonstrated.
Therefore, the objectives of the present study were 1)
to demonstrate vascular continuity between host and
implant, 2) to establish a time frame during which vascular continuity occurs, 3) to determine if anastomosis
is a continuing event throughout the time of implantation, and 4) to determine if the presence of the im-
‘Nude mouse is the correct designation of this animal, which also
is known genotypically as ndnu. The nude mouse is also always
athymic (Krueger and Briggaman, 1982).
0 1989 ALAN
R. LISS, INC.
plant itself prompts a vascularization by the mouse
into the implant.
MATERIALS AND METHODS
Thirteen samples of hypertrophic scars, six of keloids
and six of Dupuytren’s contracture, were obtained as
excess material from surgical procedures. These samples provided 194 pieces of tissue used as implants into
97 mice. Each piece was trimmed to a standardized size
of 5 x 8 x 5 mm. In every case the donor tissues were
taken from the nodular areas of the lesions.
Nude mice were anesthetized with sodium pentabarbitol (40 mglkg body weight), and full-thickness incisions were made through the skin over each scapular
area. The fine tips of surgical scissors were used to
produce a subcutaneous pocket. A donor tissue was
placed in each pocket and the skin was closed with 12
mm wound clips. The wounds were healed after 10 days
and the clips were removed.
Implants remained in the mice for varying lengths of
time up to 246 days. Seventeen mice with 34 implants
from hypertrophic scar, seven mice with 14 keloid implants, and two mice with four implants from Dupuytren’s contracture were selected for studies of vascular
continuity. Twenty mice with 40 implants were studied
from 2 to 20 days of implantation and six mice with 12
Received November 14, 1988;accepted February 21, 1989.
190
C.W. KISCHER ET AL.
Fig. 1. A macrosectioned 16-day implant (I) with overlying mouse
skin (S), injected with barium. Each section is approximately 1 mm
thick. The bright areas are reinforced areas of barium due to vascular
plexi. ~ 3 . 2 .
Fig, 2. Macrosection of barium-injected 8-day implant with overly-
ing mouse skin. Section shows minimal penetration of barium into
implant. Other sections were negative. x 13.
Fig. 3. Two macrosections of 8-day implant showing barium penetration. x 7.2.
implants were studied from 40 to 106 days of implantation.
For the injection procedure, a n injectate was prepared consisting of 33 g of barium sulphate suspended
in 100 ml of water (Micropaque-Nicholas Labs. Ltd.,
Slough, England) and containing 4 g of gelatin. The
mouse was prepared by injecting 0.1 cc of heparin I.P.
After several minutes to allow circulation of the heparin, the mouse was anesthetized with ether, injected
with a lethal dose of sodium pentabarbitol, and secured
to a dissecting table. The thoracic cavity was opened
and the aorta entered via the left ventricle with a 23-
gauge plastic cannula attached to a 50 cc syringe. The
tip of the cannula was passed into the aorta near the
origin of the right brachiocephalic artery. The cannula
was secured with ligatures around the aorta and the
heart itself. Two volumes of 12 cc each of a dilute heparin solution (approximately 0.2 cc in 30 ml of physiological saline) was then injected into the circulatory
system. The left jugular vein was nicked to allow for
drainage (which also occurred through the intercostal
veins) a s a n indication of perfusion. Three to 5 volumes
(12 cc each) of the barium solution was then injected.
Upon completion of the barium perfusion the whole
191
VASCULARIZATION O F IMPLANTS
mouse was placed in the freezer for 30 minutes t o allow
the barium-gelatin solution to solidify and then was
placed in Karnovsky's fixative overnight. The suprascapular skin with attached implants was removed and
placed in a waxed petri dish for observation and covered with Karnovsky's fixative. One implant, along
with overlying skin, was sectioned with a hand-made,
hand-held macro-tissue slicer (Sheridan et al., 1989),
which assures sections of uniform thickness approximating 1 mm. These sections were laid out in serial
order for microangiography on 2" x 2 Kodak highresolution plates. The sections and plate were wrapped
with a film of clear plastic to hold them in place and
then exposed by using a Faxitron microradiographic
system with 18 kVP for 24 minutes. The developed
x-ray plates were photographed on a Zeiss photoscope
with a 1x objective with a halogen light source and a
2-minute 30-second exposure on Kodak Panatomic-X
film. The other, or companion, implant with overlying
skin was processed for histological evaluation.
To determine if anastomoses occurred between host
and implant, tissue sections from 21 implants were
evaluated by Hoechst dye DNA staining (Grimwood et
al., 1986). Three implants were recovered from 4 to 20
days. Eighteen of the 21 were recovered between 27
and 179 days. Four of the 18 were barium-injected implants. The Hoechst staining procedure distinguishes
mouse cell nuclei from human cell nuclei. Hoechst dye
33258 is a benzimidazole compound which, when applied to cultured mouse cells, induces an enlargement
of the pericentric area of metaphase chromosomes
(Kim and Grzeschik, 1974). There is no similar effect
on human chromosomes. Grimwood et al. (1986) demonstrated in tissue cultures that the same Hoechst dye
caused a bright discrete or punctate staining of mouse
fibroblast nuclei, whereas human fibroblast nuclei
showed a diffuse staining pattern. A similar staining
pattern was seen in frozen sections of basal cell carcinomas grown subcutaneously in nude mice.
Thus, we analyzed cells in the periphery of the implant and within the interior. We especially looked at
the type of endothelial cell in barium-filled vessels.
We have used the Hoechst dye on Karnovsky-fixed
paraffin tissue sections. All of the tissues were originally fixed in Karnovsky's fixative, dehydrated in
graded alcohols, and embedded in paraffin. Tissue sections from injected and noninjected implants were cut
from paraffin blocks a t 6-8 p,m. After deparaffinization, the sections were placed in undiluted Hoescht's
stain (#33258, Flow Labs, Inc.) for 30 minutes. The
slides were drained and then washed 3 x in dHzO. After air drying the slides were coverslipped with Permount. Identification of mouse nuclei is made by observing the specific fluorescing nuclear pattern in the
form of punctate clumps after exposing the stained sections to U.V. light by using a Zeiss 01 filter set (excitation 365 nm, emission 480 nm). Human nuclei show
the diffuse pattern. All exposures on film in the photomicroscope were made under identical conditions.
Likewise processing of the film and printing were performed under identical conditions.
Histological sections of one implant were evaluated
by assigning arbitrary values for magnitude of vessels
present and the extent of microvascular occlusion.
These values were compared with those made from the
TABLE 1. Implant tissues examined by barium
injection for host-implantvascular continuity'
Days of
implantation K1 HS1 HS2 HS3 HS4 HS5 K2 HS6 DC1 DC2
2
- 4
- - _
6
8
+ - -+
9
+
10
11
+ +
+
12
14
16
+ +
20
40
+
62
+
69
70
75
106
+ -
+
+
+
+
+
'Total = 26 mice, 52 implants; + = confirmed barium inflow; 5 =
minimal barium inflow; - = no barium inflow; HS = hypertrophic
scar; K = keloid; DC = Dupuytren's contracture.
histological sections of nonimplanted (zero-time) tissues from the same surgical specimen. This would tell
us if implantation prompted a change in vascular
availability for anastomosis. By evaluating implants of
varying ages and comparing them with their nonimplanted (zero-time) samples, we could determine if
anastomosis was a continuing process.
RESULTS
By examining serial sections completely through the
implant we could distinguish barium-filled vessels
within the implant from those in the periphery (Fig. 1).
All implanted tissues retained their zero-time morphological character with no evidence of implant rejection as evidenced by lymphocyte or small cell infiltration. Gross examination of all implants showed that
most are supplied within the first several days by a
single branch of a subcutaneous artery which then arborizes throughout the periphery of the implant. In a
few cases of the older implants a second branch was
observed.
Table 1 shows the samples of tissues used, the number of mice injected, the number of implants injected,
the days postimplantation studied in each mouse, and
the results for each injection. Of the four lesions implanted into 20 mice from which implants were examined from 2 to 20 days, one shows minimal ingrowth up
to 16 days (Fig. 2). The other three lesions examined
show more extensive barium inflow by 8, 11, and 16
days (Figs. 3-5, respectively). Each of the lesions examined a t 40-106 days demonstrates barium inflow,
which varies in magnitude (Figs. 6,7). Stained sections
from implants not x-rayed show that every implant
demonstrates extensive vascularization in the periphery, extending into the implant from 100 to 300 pm,
most of which is filled with the barium mixture (Fig. 8).
Each of these companion implants was examined for
barium within the vessels deeper into the implant. All
but one sample confirm the presence of internalized
192
C.W. KISCHER ET AL.
Fig. 4. Three macrosections of 11-day implant with barium penetration. x6.3.
Fig. 5. Three macrosections of a 16-day implant with substantial barium penetration. x 7.2.
Fig. 6. Macrosection of 70-day implant showing minimal barium penetration. x 12.2.
Fig. 7. Two macrosections of barium injected implant of 75 days. Extensive barium penetration.
X
15.6.
193
VASCULARIZATION O F IMPLANTS
Fig. 8. Tissue section of implant injected with barium. Periphery of
implant shows extensive vascular arborization with barium filling,
extending into interior of implant. x 90.
Fig. 9. H & E section of companion implant to the one used for
microangiogram in Figure 2 showing minimal penetration of barium.
Interior of implant contains very few microvessels. x 90.
barium. In the one exception the microangiogram
shows minimal barium inflow (Fig. 2). The companion
implant demonstrates very few interior vessels (Fig. 9).
In addition to the barium results we examined a minimum of eight sections of every implant (total = 776)
by hematoxylin and eosin staining and Masson’s trichrome method for evidence of microvascular ingrowth. Six of those implants were serially sectioned
and similarly examined. Figure 1Oa-d is representative of the evidence observed by routine tissue section
examination.
Hoechst staining of tissue sections in which barium
was found in the peripheral vascularization demonstrates that these vessels are clearly of mouse origin
(Fig. 1la,b). Punctate patterns of nuclear chromatin
were evident in the peripheral vessels. However, those
vessels within the interior of the implant where barium is found show the endothelial cells to be of human
origin (Fig. 12a,b). Hoeschst-stained sections from all
other implants demonstrate only human endothelial
cells around internal vessels.
In the case in which tissue sections from the implants were arbitarily graded as to the extent of vessels
and magnitude of microvascular occlusion, it was found
that neither the number of vessels nor the level of oc-
clusion in any implants, regardless of length of time,
changed from that in the nonimplanted parent tissue.
DISCUSSION
The combined barium injection, histological section
study, and Hoechst staining demonstrate that vascular
anastomosis occurs between host and implant and is
maintained throughout extended implantation times.
Among the implants in this study, vascular channeling
into the implant occurred by the eighth day in three
cases and by the 1l t h day in the other case. The degree
of barium inflow varied from implant to implant and in
early detection was confined to the outer portions of the
implants. Older implants showed internalized barium
deeper into the implant. The results of Hoechst dye
staining are critical for supporting anastomosis. Barium inflow into the implant may be occurring via arterial side channels growing from the mouse without a
venous return. Consequently, the microangiograms do
not, of themselves, prove anastamosis. Only when we
demonstrate human endothelial nuclei surrounding a
barium-filled lumen do we know anastomosis truly occurred. The histology of the implants complemented
the results with barium in that the degree of internalized barium did not appreciably differ from what was
194
C.W. KISCHER ET AL.
Fig. 10. Set of four serial sections from implant of 111 days, noninjected. Continuity of peripheral
vessel with a deeper vessel shown by arrows. Masson’s trichrome stain. x 200.
VASCULARIZATION OF IMPLANTS
195
Fig. 11. Section of barium-injected implant of 6 days stained only
with Hoechst stain. a: Transmitted light micrograph of periphery of
implant with barium-filled vessels. The endothelial nuclei are identified by light areas (arrows). Barium (B). x 500. b Section observed
under U.V. light only. Fluorescence shows endothelial nuclei with
punctate pattern characteristic for mouse cells (arrows).
Fig. 12. Section of implant of 75 days, stained only with Hoechst
stain. a: Transmitted light micrograph of interior barium-filled vessel
of implant. The endothelial nuclei are identified by the light areas
(arrows). x 500. b Section viewed under U.V. light only. Fluorescence
shows endothelial nuclei around barium-filled vessel with diffuse pattern (arrows).
observed in the microangiograms. The magnitude of
vascular anastomosis appeared to reflect the degree of
patent capillaries in the implant.
The presence of the implant does not appear to
prompt a continuous or extensive neovascularization
into the implant. Several implants showed very little
barium inflow, and these also demonstrated very few
patent microvessels. Vascular growth from the mouse
appears extensive at first only at the periphery but
then must rely on available patent microvessels within
the donor tissue. This seems confirmed by the Hoechst
stain data, which show older implants with interior
barium-filled vessels with only human endothelial
cells.
Blood vessels in the implant which are available for
anastomosis at the time of implantation will determine
the extent of anastomoses and, henceforth, the extent
of anast omoses does not change. This fact is important
196
C.W. KISCHER ET AL.
for characterizing this potential model because it suggests that what is implanted is not complicated by invasive growth from the host.
From the data presented it is suggested that when
considering the use of hypertrophic scar or keloid implants as a potential model for the study of therapeutic
applications, enough time should be given the implant/
host system to initiate internal anastomosis. We judge
this to be at least 8 days and it could reasonably be
about 16 days postimplantation.
A final note: it should be mentioned which system
appears to be anastomosing first, arterial or venous. In
wound-healing systems, a search of the literature reveals no studies indicating which system regenerates
first. Hunt et al. (19781, in a study of vascular outgrowth in healing wounds, showed that capillary budding is prompted by low oxygen tension and proceeds
toward the gradient of high oxygen tension. Capillary
growth, presumably, occurs preemptively from the arterial side. This is reasonable because these microvessels are filled with erythrocytes. Yet, the time when
arterial and venous sides are both reestablished is not
known. Apparently, the delay of a complete vascular
routing within the implant does not affect the viability
of the tissue. The fibroblasts of the hypertrophic scar
and keloid are facultative anaerobes, anyway (Hunt
and Van Winkle, 1979). If nutrients and oxygen are
available only by tissue diffusion from the periphery,
their condition would continue to be sustained. Hypoxia of hypertrophic scars has been demonstrated
(Sloan et al., 1978). Hypoxia also is known to stimulate
collagen synthesis (Chvapil et al., 1970; Chvapil,
1974). This would explain the excess of collagen and,
consequently, the bulk of the hypertrophic scar. The
hypoxia of the scar is explained by widespread microvascular occlusion demonstrated by Kischer et al.
(1982).Thus, the long-term clinical course of this lesion
may be explained.
ACKNOWLEDGMENTS
The authors wish t o express their sincere appreciation t o Andrzej Fryczklowski, M.D., and Bridget Gar-
rity, M.D., for technical assistance with the injection
technique and radiographic procedures, and to Mike
Jaqua for technical assistance.
This study was supported, in part, by NIH research
grant 5R0134928.
REFERENCES
Chvapil, M. 1974 Pharmacology of fibrosis and tissue injury. Environ.
Health Perspect., 9:283-294.
Chvapil, M., J . Jurych, and E. Mirejorska 1970 Effects of long-term
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Grimwood, R.E., C.F. Ferris, L.D. Nielsen, J.C. Huff, and R.A.F.
Clark 1986 Basal cell carcinomas grown in nude mice produce
and deposit fibronectin in the extracellular matrix. J . Invest. Dermatol., 87:42-46.
Hunt, T.K., and W. Van Winkle, J r . 1979 Normal repair. In: Fundamentals of Wound Mangement. T.K. Hunt and J.E. Dunphy, eds.
Appleton-Century-Crofts, New York, pp. 2-62.
Hunt, T.K., W.B. Conolly, S.B. Aronson, and P. Goldstein 1978 Anaerobic metabolism and wound healing: An hypothesis for the initiation and cessation of collagen synthesis in wounds. Am. J. Surg.,
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Kischer, C.W., J. Pindur, M.R. Shetler, and C.L. Shetlar 1988 Implants of hypertrophic scars and keloids into the nude (athymic)
mouse: Viability and morphology. J . Trauma, in press.
Krueger, G.G., and R.A. Briggaman 1982 The nude mouse in the
biology and pathology of skin. Exp. Clin. Res., 2:301-322.
Marcenac, N. 1951 Cheloides cicatricelles du cheval. Rec. Med. Vet.,
127:385-398.
Shetlar, M.R., C.L. Shetlar, L. Hendricks, and C.W. Kischer 1985 The
use of athymic nude mice for the study of human keloids. Proc.
Sac. Exp. Biol. Med., 179:549-552.
Sheridan, D., C.W. Kischer, and J. Pindur 1989 A cutting device to
provide uniform serial macro sections of tissue. Lab. Med., in
press.
Silverstein, P., M.N. Goodwin, G.L. Raulston, and B.A. Pruitt 1976
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the Management of Hypertrophic Scar. J.J. Longacre, ed. Chas C.
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