A M . ZOOLOGIST, 10:141-155 (1970)
Induced Regeneration of Hindlimbs in the Newborn Opossum
MERLE MIZELL AND f. JOYCE ISAACS
The Chapman H. Hyams III Laboratory of Tumor Cell Biology,
Department of Biology, Tulane University,
New Orleans, Louisiana 70118
SYNOPSIS. The North American marsupial, Didelphys virginiana, has proved to be
uniquely suited for studies of replacement of mammalian limb. The newborn opossum
provides the rare opportunity to perform chronic experiments on extremely young
mammalian limbs. Since this marsupial is born without lymphocytes, xenoplastic
as well as homoplastic transplants are tolerated; supplementary nerve tissue was
transplanled to newborn hindlimbs and the limbs were amputated immediately above
the ankle. When (he implant remained in place, a regenerative response ensued.
Results of control experiments indicate that neither the trauma of simple amputation,
the trauma of implantation, nor the implantation of other tissues can evoke the
response which results after implanting nerve tissue. These studies demonstrate that
young opossum limbs are capable of regenerating when additional nervous tissue is
supplied.
Tn the spring of 1965, a female opossum
with young in pouch was trapped in a
rural area adjoining New Orleans and
brought into our laboratory. Although we
had not anticipated the introduction of
this marsupial into our amphibian laboratory at just that moment, we were at least
mentally prepared. In fact a plan had been
evolving for several years. In 1961 Simpson, using the skink, Lygosoma, and
Singer, using the chameleon, Anolis, were
successful in inducing a regenerative response in reptilian limbs by augmenting
the nerve supply. Schotte, et al. (1952,
1956) had already shown that regeneration
These investigations were supported by N.S.F.
Grant GB-8575.
The technical assistance of Deborah E. Ramsey,
Grace Bannatyne, and Hawley Martin is gratefully
acknowledged. The following Tulane undergraduate majors in biology were indispensable in the
care and maintenance of our opossum colony over
the past five years: John Butler, Don LaGronc,
Anthony LaXasa, Rand Spencer, and Scott Ziesenis.
Scott Ziesenis was instrumental in the establishment of successful trapping and breeding
procedures. We thank Dr. James D. Clark for veterinary assistance and Dr. James Harkin, B. O.
Spurlock, and T. Trish for the electron microscopy. Dr. Harkin's laboratory is supported by
U.S.P.H.S. Grant XB-04330.
Our laboratory's initial studies were supported
by a giant from the Cancer Association of Greater
Xew Orleans, Inc.
of limbs could be induced in adult anurans (frogs) by implanting additional homologous adrenal tissue, which led to our
using reptilian adrenal implants to induce
regeneration of limbs in these tailless amphibians (Mizell, 1963). Thus, the reptilian nerve-augmentation experiments had
demonstrated that limb regeneration could
be induced in a class of vertebrates higher
than that in which it occurs naturally, and
our experiments indicated that reptilian
adrenal implants are also effective in promoting regeneration of amphibian limbs.
The latter findings suggested that either
the same factor (s) or very similar factors
are operative in these two classes of vertebrates and that the basic requisites for amphibian limb regeneration are probably
similar to those of reptilian regeneration—leading us to state, "Finding
that factors which can support amphibian
regeneration are present in a higher class
of vertebrates also lends encouragement
for future attempts to induce limb regeneration in a still higher class of vertebrates,
namely the mammal" (Mizell, 1963).
Young opossums seemed to be ideally
suited for our projected studies of mammalian limb regeneration. After a short
period of gestation (12.75 days) these
primitive mammals leave the birth canal,
141
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MERLE MIZELL AND J. JOYCE ISAACS
migrate to the pouch, attach to the nipples
and complete their extra-uterine development within this readily accessible maternal environment.
The eight young found in the pouch of
the female opossum obtained in 1965 became the subjects of the pilot study we had
been considering. Growth curves (Hartman,
1938; Moore and Bodian, 1940) were used
to determine the age of the young, which
was estimated to be 46 days. When the
limbs of these animals were amputated
above the ankle or wrist, typical uneventful mammalian healing ensued with no
indications of regeneration. This might
have been expected, because of the advanced age of the pouch young when captured. However, during this pilot study, we
developed procedures which would be
used in our subsequent experiments. Penthrane (Abbott Laboratories) proved to
be an effective and safe inhalant anesthetizing agent for the female opossum; it
permitted prolonged examination and
manipulation of the young while they remained attached to the mother. Problems
in opossum husbandry were resolved or
minimized during these initial experiments. As the next opossum breeding season approached, our amphibian laboratory
was reasonably competent and fairly confident that we could house, maintain and
perform chronic experiments on very
young opossums.
Trapping commenced at the beginning
of the next opossum breeding season, and
the pouches of many of the captured females contained very young pups. Some of
these litters were young enough to be used
in our regeneration experiments, but most
animals were bred in captivity (when
"older" pups are removed from the pouch,
the female goes into post-lactational estrus and becomes receptive to the male,
and newborns were obtained from females
that gave birth in the laboratory (Fig. 1).
For these, we could be certain of the exact
time of birth and the precise age at the
time of amputation.
Although ATcCrady (1938, p. 203) had
shown that limbs of newborn opossums did
not regenerate after simple amputation,
our first experiments were performed to
determine the histological nature of their
response to amputation.
After anesthetizing the mother, the
pouch was opened and a young pup was
positioned under the dissecting scope; amputations were performed with iridectomy
scissors while the pup remained attached
to the teat. Limbs were amputated directly
above the wrist or ankle and the small
loss of blood made ligatures unnecessary.
Although aseptic techniques were employed during the operation no elaborate
procedures were utilized to maintain sterility in the pouch, nor was it necessary to
employ antibiotics, for in over 250 amputations infection was never noted.
Wound healing and all external changes
in the limb stumps after amputation were
observed; at various intervals, when noteworthy changes occurred, the limbs were
photographed. Periodically some limbs
were fixed, embedded in paraffin, and sectioned at 8 ju. or were prepared for electron
microscopy. Serial sections were stained
with hematoxylin and eosin or Mallory's
polychromatic stain. Tritiated thymidine
was employed in some cases and autoradiographs were prepared to follow cellular division and mitotic activity in the limb.
Also, in some cases the Bodian silver stain
was used to determine the presence and
position of nerve fibers within the limb.
Throughout these experiments, the pups
remained attached to the mother within
the protective and nourishing environment
of the marsupial pouch. The adult opossums had free access to water and were fed
Gaines Meal (General Food Corp.).
Simple Amputation
Figure 2 shows a 12-day-old opossum
from which the left forelimb and left hindlimb were amputated six days after birth.
The stump of the forelimb never showed
any external indication of regeneration, but
the distal portion of the hindlimb appeared somewhat bulbous. However, probing this bulbous structure revealed a fluid
INDUCED LIMB REGENERATION IN THE OPOSSUM
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MERLE MIZELL AND J. JOYCE ISAACS
FIG. 1. Litter of 10 newborn opossums (less than
24 hr old) nursing in pouch of the anesthetized
another. Scale in centimeters.
FIG. 2. Twelve-day-old opossum. Simple amputation of left forelimb and left hindlimb was performed six days after birth. Forelimb shows no
external indication of regenerative reponse but hindlimb has blastema-like (bl) appearance (see text).
Scale in centimeters.
FIG. 3. Photomicrograph of sagittal section of forelimb in Figure 2. Note thickened apical epidermis,
but also note premature redifferentiation of subjacent tissues and the cartilaginous callus (arrows)
forming around the radius. X46.
FIG. 4. Photomicrograph showing close-up of another section of the forelimb seen in Figures 2 and
3. Arrow indicates an osteoclast in the area of
breakdown of bone.
consistency suggestive of edema rather
than the firmer quality which typifies a
blastema. This was confirmed in histological sections of the hindlimb which showed
that the distal portion was edematous.
Simple amputation did not result in regeneration in this 6-day old opossum or any of
its six littermates; nor did amputation of
newborn opossum limbs (less than 24
hours old) give rise to replacement of any
of the lost portion of the limb, confirming
McCrady's (1938) observations.
Nevertheless, the histological study of
these early control limbs did disclose remarkable capacities for dedifferentiation.
Microscopic examination (Fig. 3) revealed
that even the 6-day forelimb underwent
an appreciable amount of dedifferentiation—many osteoclasts were seen in areas
of bone breakdown (Fig. 4); furthermore,
the distal epidermis had thickened. However, these initial regenerative responses
were soon followed by premature rediflerentiation in the forelimb stump; a cartilaginous callus surrounded the shell of bone
(Fig. 3), indicating premature differentiation and cessation of regenerative events.
Thus, simple amputation was followed
by a covert regenerative response which
aborted soon after it was initiated. This
promising responsiveness of the tissues of
the young opossum's limb encouraged us
to attempt inducing regeneration of limbs
in these unique mammals by nerveaugmentation.
IMPLANTATION OF NERVOUS TISSUE
It became apparent during our experiments in 1966 that hindlimb tissues were
more responsive to amputation than forelimb tissues, and redifferentiative phenomena in the hindlimb were delayed and less
pronounced. At birth, the embryo-like opossums display a striking difference in degree
of development of limbs (Fig. 5). Forelimbs
have well-developed digits complete with
claws, which permit the newborn animal
to crawl from the birth canal to the pouch;
but hindlimbs are rather rudimentary
structures, with merely the early external
indications of digits. However, histological
study has shown that cartilaginous models
of all the phalanges are already present in
the hindlimb of the newborn opossum
(Fig. 6).
The initial experiments involving implantation of nervous tissue were performed
on pouch young of varying ages, and most
of our attention was focused on the hindlimb.
Developing forebrain (cerebral cortex)
was chosen as the source of nervous tissue.
The brain was removed from the donor
and placed in chilled normal saline. The
cerebrum was cut into several small pieces
which were then picked up by a previously
prepared fine-drawn glass pipette (inner
diameter approximately 0.5 mm) whose
surface had been lightly coated with silicone (Siliclad, Clay-Adams, Inc.) to facili-
FIG. 5. Newborn opossum in pouch showing dichotomy in limb development: forelimbs welldeveloped with digits and claws; hindlimb (arrow)
with margins of digits just becoming evident.
X 14.
FIG. 6. Histological section of newborn opossum
showing internal structure of hindlimb. Line indicates level of amputation. BW, body wall. X40.
FIG. 7. Fine-drawn glass pipette containing brain
tissue (between arrows) being positioned for insertion into 2-day-old opossum's hindlimb. Tissue has
been stained with vital dye for easier visualization
and positioning in the limb.
FIG. 8. Two-day-old opossum with cylinder of
stained brain tissue implanted along the long axis
of the hindlimb (arrow). Scale in centimeters.
INDUCED LIMB REGENERATION IN THE OPOSSUM
B
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la d
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MERLE MIZELL AND J. JOYCE ISAACS
Late transfer of the tissue (Fig. 7). The
slender point of a watchmaker's forceps
was inserted into the proximal thigh
region of the recipient hindlimb and extended distad, thereby creating a channel
in the 1- or 2-day-old opossum limb. The
pipette was inserted into this channel, and
the small cylinder of nervous tissue (approximately 0.5 by 1.5 mm) was transferred to the hindlimb so that the long
axis of the implant was parallel to the longaxis of the limb (Fig. 8).
Two to four days later the limb was
amputated so that the plane of amputation
transected the implant. All amputations
were attempted through the distal portion
of the tibia and fibula (Fig. 6); but in
some cases, because of the small size, amputation was inadvertently made through
the ankle. When the operation was successful and the implant remained in place, a
regenerative response ensued.
The brain of young opossums was used
as the source of nervous tissue for transplantation to 83 hindlimbs. In 19 of these
83 cases of homologous implantation, a
positive response was evoked. The development of the best regenerate is shown
in Figures 9, 10, and 11. Figure 9 shows the
animal at 24 days of age (cerebrum implanted at 2 days and limb amputated 4
days later); after 18 days of regeneration, a
recognizable foot-like structure possessing
the first indications of the fourth and fifth
toes could be seen. Development continued, and 14 days later (32 days of regeneration) a heteromorphic foot bearing the
basal jDortions of three toes was evident
(Fig. 10); Figure 11 shows the appear-
ance after 63 days of regeneration. Since
development of the hindlimb at birth (6
days before amputation) had already
leached a state where all the bones were
present as cartilaginous models (Fig. 6),
the replacement of the foot and three toes
must be interpreted as regeneration and
not embryonic regulation. Figures 12 and
13 show the histological appearance of a
similar nerve-implanted limb (in this case
brain stem was used), which was fixed after 5 days of regeneration (at 8 days of
age) to permit study of internal events. A
prominent feature of the internal structure
at this time is breakdown of muscle and
the release of mononucleated muscle cells
(arrows, Fig. 13) from myotubes in the
distal portion of the stump.
In another series of experiments nervous
tissue from the forebrain of young Ran a
pipiens tadpoles (Taylor-Kollros stage VII)
was employed to determine the effects
of xenoplastic nerve transplantation. (Positioning of the implant within the limb
was facilitated by the melanophores which
are present on the external surface of
the tadpole brain). A positive response
was noted in 3 of 14 hindlimbs which
received heterologous transplants of cerebrum. The largest regenerate was a curiously shaped outgrowth (Figs. 14, 15,
16 and 17) consisting of a distal, clubshaped structure which emerged from the
ankle region and possessed a single digitlike protuberance on its medial surface.
This response surpassed that of controls
with simple amputations, but did not approach the extent of development achieved
with homologous nerve-implants.
FIG. 9. Hindlimb of 24-day-old opossum. Nervous
tissue from young opossum's cerebrum was transplanted when the recipient was two days old; limb
was amputated four days later. After 18 days of
regeneration, early indications of digital regrowth
are seen (arrows). Scale in millimeters.
FIG. 10. Same hindlimb seen in Figure 9 and the
unamputated contralateral hindlimb. Animal is
now 38 days old (32 days of regeneration); the
foot and basal portions of three toes are clearly
visible.
FIG. 11. Hindlimbs of animal seen in Figures 9 and
10, pictured at 69 days of age (63 days of
regeneration). Scale in millimeters.
FIG. 12. Photomicrograph of 8-day-old opossum's
limb. Brainstem of opossum was transplanted at
two days of age and the limb amputated one day
later (thus, limb was sacrificed at 5 days of regeneration). Evidence of breakdown of muscle can be
seen in the distal portion of the stump. X'25.
FIG. 13. Higher magnification of area in Figure 12,
showing detail of muscle breakdown. Release
of mononucleated muscle cells from the distal end
of degenerating myotubes can be seen (arrows).
X33O.
INDUCED LIMB REGENERATION IN THE OPOSSUM
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MERLE MIZELL AND J. JOYCE ISAACS
TABLE 1. Results of implantation into ]iinrllimb.
Type of Implant
OPOSSUM
A. Nervous
1. " B r a i n "
2. Brain stem
3. Cerebrum
4. Midbrain
5. Spinal ganglia
B. Controls
1. Kidney
2. Liver
3. Adrenal
FROG
A. Adult
1. Adrenal
2. Lucke tumor
B. Tadpole
1. Cerebrum
2. Spinal cord
OTHEE
Tritunis adrenal
Strict control
(no implants)
TOTALS
Number of
Experimental
Limbs
Regenerative
Response*
22
30
27
4
4
1
6
12
0
2
20.0
44.4
50.0
6
10
15
1
2
11
7
2
0
0
0
0.0
0.0
0.0
11
5
2
100
5
5
0
0
0.0
0.0
5
5
14
3
3
0
21.4
0
11
1
4
18
0
0
0
0
4
15
156 Hindlimbs
%
4.5
0.0
Lack of
Response**
93
24
%
Lost from
Pouch
27.2
33.3
55.5
25.0
50.0
15
14
0
3
0
%
68.3
46.7
0
75.0
0
0
100
0
2
0
28.6
100
100
0
0
0
0
0
2
66.7
0
3
16.7
71.4
78.6
33.3
100
83.3
0
0
0
39
* Gross, external indication of regenerative response.
** No external indication of regenerative response.
Similar experiments were performed
utilizing implants of other elements of the
nervous system. The results of all experiments are summarized in Table 1. Thus
far, spinal ganglia (Fig. 18) of young opossums have been implanted into only one
litter of animals, but the initial results
were significant. The high percentage of
responses noted (50%), albeit based on
only a few animals, is encouraging; more-
over, the use of spinal ganglia allows a
more quantitative approach (ganglia are
readily bisected or cut into quarters; even
after subdivision, the tissue retains its integrity better than other nervous tissues
that have been employed). Furthermore,
the origin of nerve fibers coursing through
the limb stump (arrow, Fig. 19) can be
more readily determined by using spinal
ganglia.
FIG. 14. Hindlimb of 12-day-old opossum. Nervous
tissue from the cerebrum of a Rana pipiens
tadpole was implanted at one day, and the limb
was amputated six days later. Thus, the limb is
pictured at 5 days of regeneration. The early indication of a single digit is seen on the medial aspect
of the limb.
FIG. 15. Hindlimb of the animal in Figure 14
shown after nine days of regeneration. Further
enlargement of the medial digit-like structure can
be seen (open arrow). Note attachment of the
16-day-old opossum to the mother's teat (solid
arrow).
FIG. 16. Medial view of the same hindlimb pictured at 40 days of regeneration, showing further
development of the limb and its heteromorphic
structure. Scale in centimeters.
FIG. 17. Lateral view of the same limb at 44 days
of regeneration. This heteromorphic, club-shaped
regenerate which formed in the ankle region and
bore a single digit-like protuberance represented
the best response shown by a hindlimb which
received a transplant of heterologQUS nerve (issue,
Scale in centimeters.
INDUCED LIMB REGENERATION IN THE OPOSSUM
T'T'T'IS'MIIIUI
150
MERLE MIZELL AND J. JOYCE ISAACS
CONTROL IMPLANTS
Adrenal tissue was also chosen for implantation because of its previously demonstrated
regeneration-inducing
effect
(Scheme, el al., 1952, 1956; Mizell, 1963).
However, in the few limbs studied so far,
no response has been noted. Nevertheless,
additional experiments with adrenal implants are planned, for the limb dichotomy
in the newborn opossum permits simultaneous testing of the effects of systemic
factors on the well-developed forelimb tissues and the relatively undifferentiated
hindlimb tissues.
Opossum kidney and liver were employed as additional, non-nervous implantation-controls for our experiments. Figures
20 and 21 show a control limb 23 days after
receiving an implant of kidney (implanted
at 3 days of age). Two significant features
should be noted in these photomicrographs: (a) recognizable kidney tubules are
still present; (b) there is no lymphocytic infiltration, which would have been elicited
in an older animal. Similar controls with
implanted kidney received injections of 3Hthymidine three hours before sacrifice; autoradiographs prepared from sections of these
limbs indicated that the implants were not
merely viable but were also synthesizing
L)NA and undergoing cellular division.
However, these implants of control tissue
failed to produce a regenerative response
and the healing was similar to that seen in
simple amputational controls.
Thus, the results from control experiments indicated that neither the trauma of
simple amputation, the trauma of implantation, nor implantation of other tissues
could evoke the regenerative response
FFO 18. Photomicrograph of a cross section of
spinal ganglion removed from a GO-day-old. opossum. X125.
F J d t l 9 . Photomicrograph of hindlimb of 8-day-old
opossum which received an implant of opossum brain at two days and was amputated at three
days of age. Bodian's stain was employed to
demonstrate nerve fibers in the distal limb stump.
X-J25.
FIG. 20. Photomicrograph of control hindlimb of
which resulted after implanting nervous
tissue.
WHY REGENERATION OF OPOSSUM LIMB?
Prior to our experiments with opossums
(Mizell, 1968, 1969«, b), attempts had been
made to uncover a regenerative potential
in other young mammals. Nicholas (1926)
amputated forelimbs of 14-day or older
rat embryos, in utero, and found that these
limbs did not regenerate. After studying
wound healing in young mouse digits
(Schotte and Smith, 1959), Schotte and
Smith (1961) altered the process of wound
healing with ACTH and cortisone in attempts to elicit regeneration. These experiments, nerve-augmentation studies in the
rat by Bar-Moar and Gitlin (1961), and
treatment of young rat limbs with trypsincalcium chloride by Scharf (1961) yielded
unencouraging results. Our own pilot experiments-with newborn mouse digits were
similarly disappointing.
What features of the newborn opossum
render this mammalian system more suitable for studies of regeneration than those
previously investigated? At this point we
cannot arrive at a definitive answer to that
question; however, our results and the
knowledge available from previous studies
of limb regeneration in other vertebrates
suggest some factors which probably play
an important role.
The most strikingly unique feature of
opossums is the early stage of development
of these marsupials at the time of birth;
they leave the womb and migrate to the
pouch after a gestational period of only
12.75 days. The newborn opossum corre26-day-old opossum which received an implant of
opossum kidney at three clays and was amputated
the following day. Implanted tubules can be seen
at upper margin of limb. X^O.
FIG. 21. Close-up of upper portion of Figure 20,
showing cross sections of three intact kidney tubules
23 days after implantation. There is no lymphocytic infiltration into the area of the implant.
X"25.
INDUCED LIMB REGENERATION IN THE OPOSSUM
09b
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MERLE MIZELL AND J. JOYCE ISAACS
sponds to an embryonic stage of eutherian
mammals, and of the mammals Didelphys
undergoes the shortest known period of intrauterine development (Miiller, 1967); at
birth the opossum is about equivalent in
development to a 12-day rat embryo or a
human fetus of two months (Block, 1960).
Thus, opossum "embryos" are available for
repeated handling and experimentation at
a time when it would be impossible to perform chronic experiments on other mammals. The importance of utilizing young
animals was emphasized by the lack of regenerative response in our nerve-implant
experiments when amputation was delayed
until one week after birth or later (Fig.
22).
Another unique feature of newborn
opossums—also related to their immaturity at birth—is the absence of the requisites
of the usual mammalian immune mechanism. Lymphocytes are lacking at birth and
during the first week of life (see Block's
excellent monograph on development of
opossum hemopoietic tissues, 1964). In
fact, the thymus of the newborn opossum
has been described as merely an epithelial
anlage (Rowlands, et al., 1964). Our experience is consistent with these findings
since no lymphocytic infiltration into the
area of transplanted tissue has been seen
during the time-course of our experiments
(Fig. 21). This lack of immune mechanism apparently allows homologous and
even heterologous transplants to persist in
the limbs of these very young animals.
Induced regeneration of limbs in higher
vertebrates has profited by comparisons
with amphibian limb regeneration (Simpson, 1961; Singer, 1961; Hay, 1966; Goss,
1969). Indeed, these comparisons provided
the basis for our studies on the opossum.
However, the regeneration induced in
these young, primitive mammals is better
compared with regenerative phenomena in
another developing limb system rather
than an adult, such as Tritunis. Fortunately, the froglet provides a comparable system for study of regeneration in developing hindlimbs. As the development of
FIG. 22- Seventy-ihrec-day-old opossum. Transplant of cerebrum from opossum to left forclimb
and hindlimb at two days and limbs amputated at
one week of age. Apparently the implants did not
"lake," for no evidence of regeneration was seen
either in the Corelimb or hindlimb.
FFG. 23. Close view of left hindlimb of metamorphosing Rana pipiens tadpole shown at TaylorKollros stage XV* just prior to amputating the
hindlimbs at a level above the ankle.
FIG. 24. After metamorphosis, the animal in Figure 23 is shown on the right for comparison with a
control
(unamputated) froglet. The limited
replacement of limbs seen in the photo can be
correlated with the stage of development at the time
of amputation. At an earlier stage, more complete replacement of the limb would have occu ned.
INDUCED LIMB REGENERATION IN THE OPOSSUM
153
the hindlimb proceeds during metamorphosis, the ability to regenerate diminishes
in a proximal to distal fashion (Figs. 23
and 24); in Ranids even the most distal
portion can not regenerate when the animal reaches Taylor-Kollros' stage XVIII
(Schotte and Harland, 1943; Forsyth,
1946; Van Stone, 1955). [During metamorphosis, Xenopus, a primitive anuran,
undergoes a similar loss of regenerative capacity during development of the hindlimb but retains the ability to regenerate a
hypomorphic limb (Dent, 1962)].
The induction of regeneration in limbs
which either lacked the ability to regrow
(reptile) or lost this ability (frog), has
amply demonstrated that a latent "regenerative capacity" exists. Singer's classic study
of nerve-augmentation in the frog (1951)
provided the impetus and protocol for the
induction of regeneration of limbs in reptiles. These studies and the concepts that
emerged from them, e.g., nerve-threshold,
cross-sectional axonal area, etc., provided
the rationale for our experiments on mammalian nerve augmentation.
But why should implants of nerve evoke
a regenerative response in opossum limbs?
An important determinant may be the
relative immaturity of innervation in the
"embryonic" hindlimbs of the newborn
opossum.
A collaborative electron microscopic investigation of innervation in the developing hindlimb has been undertaken
with Dr. James Harkin of the Tulane
Neuropathology Laboratory. The early results of the investigation have revealed
that the nerves of the newborn opossum
are relatively immature in that many axons appear to lack close association with
Schwann cells and most axons which are
associated with Schwann cytoplasm lack
myelination. In fact, as late as three weeks
after birth most of the nerve fibers of the
hindlimb are unmyelinated (Figs. 25 and
26).
We have shown that adding nerve implants to these immaturely innervated tissues yields a regenerative response; as yet
we do not know how these two elements
contribute to give this result. Certainly,
future studies employing implants of
spinal ganglion that will allow quantitation and better visualization of the innervation supplied by an implant should add
significantly to our knowledge of how nervous tissue evokes expression of a regenerative capacity in these limbs.
Thus, although the opossum has afforded
the opportunity to induce regeneration in
young mammals, we are no closer to an
understanding of the mechanism of nerve
action in regeneration than before. However, we are now in a position to compare
regeneration in this mammalian limb with
regeneration in lower vertebrates; as their
similarities and differences become apparent, additional insight into the phenomenon of mammalian cellular differentiation should be gained.
FIG. 25. Electron micrograph showing preponderance of unmyelinated nerve fibers in a 25-day-old
opossum's hindlimb. Note the group of unmyelinated axons surrounded by the cytoplasm of a
single Schwann cell (3); a single axon in an early
stage of myelinization can be seen in the lower
lefthand corner (2). x 10,000.
FIG. 26. Another area of the limb seen in Figure
25, showing two groups of unmyelinated nerve
fibers (3), and a less frequently encountered axis
cylinder (1) in an advanced stage of myelin envelopment. SN = Schwann cell nucleus, x 32,000.
REFERENCES
Bat-Moar, J. A., and G. Gitlin. lOfil. Attempted
induction of forelimb regeneration by augmentation of nerve supply in young rats. Transplantation Bull. 27:460-461.
Block, M. 1960. Wound healing in the new-born
opossum (Didelphis virginianam). Nature 187:
340-341.
Block, M. 1964. The blood forming tissues and
blood of the newborn opossum [Didelphys virginituui). T. Normal development through about
ihe one hundredth day of life. Eigeh. Anal.
Entwickl. 37:237-366.
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