Download Conjecture: Can Continuous Regeneration Lead to Immortality

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REJUVENATION RESEARCH
Volume 9, Number 1, 2006
© Mary Ann Liebert, Inc.
Conjecture: Can Continuous Regeneration Lead to
Immortality? Studies in the MRL Mouse
ELLEN HEBER-KATZ, JOHN LEFEROVICH, KHAMILIA BEDELBAEVA,
DMITRI GOUREVITCH, and LISE CLARK
ABSTRACT
A particular mouse strain, the MRL mouse, has been shown to have unique healing properties that show normal replacement of tissue without scarring. The serendipitous discovery
that the MRL mouse has a profound capacity for regeneration in some ways rivaling the classic newt and axolotl species raises the possibility that humans, too, may have an innate regenerative ability. We propose this mouse as a model for continuous regeneration with possible life-extending properties. We will use the classical “immortal” organism, the hydra, for
comparison and examine those key phenotypes that contribute to their immortality as they
are expressed in the MRL mouse versus control mouse strains. The phenotypes to be examined include the rate of proliferation and the rate of cell death, which leads to a continual
turnover in cells without an increase in mass.
mouse has a profound capacity for regeneration
in some ways rivaling the classic newt and axolotl species raises the possibility that humans,
too, may have the capacity to regenerate. The
authors propose this mouse as a model for continuous regeneration of tissue with or without
intentional injury and with possible life-extending properties. This paper compares several properties of this mouse to those of the classical “immortal” organism, the hydra.
INTRODUCTION
I
there
could be no life. If everything regenerated there would be no death. All organisms exist between these two extremes.
Other things being equal, they tend toward
the latter end of the spectrum, never quite
achieving immortality because this would
be incompatible with reproduction.
F THERE WERE NO REGENERATION
—Richard J. Goss (1969)1
THE HYDRA
The hydra is a multicellular organism with a
very simple body plan. It is a member of the
Cnidaria, which arose early in metazoan evolution. It has two cell layers, the endoderm and
the ectoderm (which is made up of epithelial
cells interspersed with interstitial cells); these
The MRL mouse has been shown to have
unique healing properties that show epimorphic regeneration with the formation of a
blastema and the replacement of tissue with
normal architecture and little or no scar formation. The serendipitous discovery that the MRL
The Wistar Institute, Philadelphia, Pennsylvania.
3
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two layers are separated by a basement membrane.2 The epithelial cells have been shown to
have a cell cycle time of approximately 3 days
and are continuously cycling so that the tissue
mass of the animal doubles every 3 to 4 days.
These cycling cells are considered to be stem
cells. However, the hydra does not change in
size because there is a dynamic and equivalent
gain and loss of cells.3 The cells that are lost are
either sloughed off from the appendages such
as the head, tentacles, and the foot and make
up around 20% of the tissue, or are lost through
their incorporation into newly forming buds4
making up 80% of the lost cell mass, as can been
seen in Figure 1.
Therefore, it is obvious that “old” cells do not
persist for any length of time in the hydra but are
constantly being replaced. The cells that migrate
to the end of the appendages have already differentiated; these are the cells that are sloughed
off. Thus, the hydra has been considered to be an
immortal organism. However, until recently,
there were little data to prove this claim.
Experimental data on Hydra vulgaris were
collected over a 4-year period and support this
contention.5 Martinez essentially examined
FIG. 1. Cross section of a hydra. The two layers of epithelial cells are seen though the cells of the interstitial cell
lineage are not included. The arrows indicate the movement
of cells to the extremities where they are sloughed off.
Adapted from Ref. 2.
HEBER-KATZ ET AL.
senescence, an increase in age-specific mortality with increasing age, or lack thereof by determining mortality rates and reproductive
rates. He raised the hydra individually and followed multiple animals to determine both mortality and budding rates over 4 years. During
this time period, there was no increase in agespecific mortality. This was compared with
drosophila, annelids, and other organisms,
which clearly show changes over this period of
time. Also, the budding rate showed little or no
decline. Martinez calculated that over the 4
years of observation, the epithelial cells divided
an average of 300 times and the whole body
was replaced approximately 60 times.
The hydra also has a high capacity for cell
regeneration and re-aggregation after amputation, being cut into very small pieces, or separation into cells.6–9 Also, budding in the fully
grown hydra could be considered an example
of the regenerative response.
Hydra is not the only organism that does this.
Planaria as well have been shown to have similar characteristics, with stem cells that have rapid
tissue dynamics, and displays a long or immortal life span. The stem cell population, or
neoblasts, make up 30% of the cellular content of
this flatworm and 99% of those neoblasts proliferate within 3 days, similar to the hydra stem cell
populations.10 It has been shown also that at least
during the regenerative response, the level of
apoptosis is quite high.11 It has been suggested
that planaria are also “almost . . . immortal.”10,12
This paper uses the hydra, which is a regenerating organism, as a model of continuous regeneration leading to immortality, which has
been previously proposed by others. It is proposed here that the key to this, again as noted,
is the cell dynamics in the organism, which include high levels of proliferation and cell death.
The authors propose that these characteristics
are also important in mammalian regeneration.
A regenerating mouse model, the MRL mouse,
is examined to test this proposal.
THE MRL MOUSE RESPONSE
TO INJURY
The MRL mouse13 has been shown to respond
to injury by a regenerative process that replaces
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IMMORTALITY AND CONTINUOUS REGENERATION
injured tissue with new tissue and with the
growth and differentiation of new structures.
This was first shown in the unique closure of ear
holes and the regrowth of cartilage in the ear
pinna.14 Although this is not seen in mice other
than this mouse strain and its parental line, the
LG/J mouse strain, hole closure has been reported in both rabbit ears and bat wings.15,16
The MRL mouse has also been shown to heal
cryoinjuries to the right ventricle of the heart
through cell proliferation, little scar formation,
and functional recovery.17 Recently, it has been
shown also that MRL digit amputation in either
of the two phalanges, shows both cellular proliferation and differentiation through chondrogenesis and osteogenesis. This response is distinct from other mouse studies examining digit
tip regeneration through the nail bed of the
claw,18–20 a response described in humans as
well.21,22
The MRL-type healing has been shown to involve increases in amounts and length of time
expressed in matrix metalloproteinases (MMP),
especially MMP-2 and MMP-9,23 a response
also shown to be important in both vertebrate
and nonvertebrate regeneration.24–28 A key
event in MRL healing is the early breakdown
of the basement membrane leading to epithelial–mesenchymal interactions and blastema
formation,22 again shown to be significant in
the limb regeneration seen in amphibians.29–33
Inhibition of the ear hole closure response in
the MRL has been seen with the use of minocycline, a molecule known to block MMPs.34
Also, reduced levels of MMPs in wounds in the
MRL have been shown to be consistent with reduced regenerative responses, as noted in cortical brain stab injuries in the MRL mouse.35
Here, early elevated MMP responses accompanied significant differences in healing responses between the MRL and the control but
reduction in MMP levels over time accompanied more scar formation in the MRL.
Experiments in the authors’ and other laboratories have examined genetic loci36–39 that
control ear hole closure in these mice using microsatellite mapping and F2 and backcross generation. The results have indicated that ear hole
closure is indeed a complex and sexually dimorphic trait. Gene expression studies also
have revealed significant candidate genes, in-
cluding Pref-1 or dlk-1,40 collagen type I,17
MMP-2,23 and vimentin and keratin.41
The unique type of healing seen in this
mouse provides an opportunity to test the hydra-derived theory of steady-state cell turnover
with increased proliferation and elimination of
cells and enhanced longevity.
What is significant in the MRL? For a proliferative response, examine the incorporation of
the thymidine analogue, bromodeoxyuridine
(BrdU), which is incorporated into DNA and
then detected using specific antibody. Are
there more BrdU cells after BrdU pulsing in
MRL mouse tissue compared with control
mouse tissue? Is this true for both uninjured as
well as injured tissue? For cell elimination or
cell death, the tunnel assay is used to detect
DNA fragmentation or examine the loss of
given cell types in tissue using specific antibody. Again, is there is a difference in uninjured versus injured tissue? Either C57BL/6 or
Swiss Webster mice are used for controls.
Both proliferation and cell death are topics
of interest in the MRL mouse because the
MRL.lpr/lpr mouse has a mutation in the fas
gene, a gene involved in cell death and shown
to be caused by a retrotransposon insertion
into the second intron of the fas gene,42,43 leading to an absence of cell death. Thus, as the
MRL.lpr/lpr mouse ages, lymphocytes in lymphoid organs display unregulated lymphoproliferation. However, it must be pointed out that
8
% BrdU-positive cardiomyocytes
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5
4
3
2
1
0
1
2
3
4
5
6
FIG. 2. BrdU labeling of cardiomyocytes. Mice were
given BrdU in their drinking water over 30 days. Crosssections of hearts were stained with antibody to BrdU and
DAPI and positive cardiomyocytes were identified,
counted, and compared with the total number of cardiomyocytes from sequential H&E sections.
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HEBER-KATZ ET AL.
FIG. 3. Tunnel assays on cardiac tissue. Tunnel assays were carried out on mouse heart tissue using CardioTACS (Trevigen, Inc., Gaithersburg, MD). Normal tissue from MRL (A) and B6 (B) show TdT-directed HRP-nucleotide additions,
which stain the nuclei blue with the tissue being counterstained with nuclear fast red. Heart tissue 2 days after cryoinjury also was examined at and adjacent to the injury site in MRL (C) and B6 (D) tissue and show tunnel-positive cells.
these studies have been done in the MRL/MpJ
mouse, does not have the fas mutation.
Cardiac tissue
As stated, after cryoinjury to the right ventricle of the mouse heart, the MRL and the control C57BL/6 (B6) mouse show healing differences.17 Syngeneic bone marrow chimeras
were generated as controls for future adoptive
transfer experiments. Even after x-irradiation
(9 Gy) and bone marrow reconstitution, these
mice could still heal like normal mice. Thus, it
was found that MRL chimeric mice closed ear
holes and healed heart in an MRL-like fashion
and B6 chimeric mice did not close ear holes
and scarred in a B6-like fashion.44 These mice
were examined pre- and post-injury for the degree of cardiomyocyte proliferation as compared with a normal heart from untreated mice.
As shown in the following, differences in BrdU
incorporation were seen in these two mouse
strains (Fig. 2). Normal heart tissue from MRL
(lane 1) showed a greater number of BrdU
positive cardiomyocytes than normal B6 heart
tissue (lane 4). After x-irradiation and syngeneic BM reconstitution, BrdU cardiomyocytes were higher in both MRL (lane 2) and
% of cells that were BrdU+, within 0.3mm
14
MRL
SWISS
12
10
%
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4
2
0
2
4
7
14
Days post lesion
FIG. 4. BrdU-positive cell counts. Mice were injured on
day 0. Four hours before the injury site was examined,
BrdU was injected intraperitoneally. The graph shows the
percentage of cells positive for BrdU and prolonged cell
division up to a distance of 3 mm at 2, 4, 7, and 14 days
post lesioning. Adapted from Ref. 35.
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IMMORTALITY AND CONTINUOUS REGENERATION
FIG. 5. GFAP-positive cells 2 days after cortical stab injury. Frozen sections of the cortical injury were prepared and
stained with anti-GFAP antibody. Figures show the transient increased GFAP-negative zone in the MRL compared
with the Swiss Webster mice adjacent to the injury site. The arrows show where the lesion is and the scale bars 1
mm. Adapted from Ref. 35.
B6 (lane 5), but still showed significant differences between the two strains. Finally, after
cryoinjury of the syngeneic chimeric heart, the
number of BrdU cells almost doubled in the
MRL (lane 3) but did not change in the B6 (lane
6). Thus, in all cases, the degree of proliferation
was at least twice as great in the MRL.
To examine the degree of cell death in heart
tissue, a tunnel assay was used that measures
fragmented 3 DNA ends which react with terminal deoxynucleotide transferase (TdT) and
are end labeled with HRP-coupled nucleotides.
In Figure 3, tunnel was carried out in normal
and 2 day post-cryoinjured cardiac tissue. It can
be seen clearly that there is a vast difference in
the number of tunnel cells in the MRL injured
heart compared with the B6 injured heart.
There also appears to be a difference in uninjured heart, with more tunnel positive cells being present in the MRL. Which cells are undergoing apoptosis was not determined in
these experiments.
Brain cortical stabs
As discussed, brain injuries in MRL versus
Swiss Webster mice showed significant differences early after injury, but the healing seen at
14 days appeared similar in many ways, including scar formation. Two striking differences were seen.35 First, analysis of BrdU cells
after a 4-hour BrdU pulse showed differences
in the MRL, compared with the SW during the
first 7 days (Fig. 4). This response shows
twofold differences, as was seen in the heart.
Second, extreme differences were seen in the
tissue response after injury. In Figure 5, the arrow shows the stab wound in an area of tissue
that is highly rich in astrocytes. In the Swiss
Webster mice, the region around the wound
site is depleted of astrocytes, as measured by
GFAP staining, and appears dark. The area
around the wound in the MRL also is depleted
of astrocytes. However, in this case one cannot
find astrocytes in the field shown, except at the
extreme end distal to the injury site. One presumes that the response to injury in the MRL
involves drastic cell death compared with the
Swiss Webster mice.
CONCLUSION
From the limited data shown in the preceding in two MRL and control mice injury models, the differences seen in increased rates of
proliferation and rates of cell death in the MRL
mouse supports the comparison to the hydra.
This indicates that the MRL mouse has a
higher cell turnover rate and therefore a higher
cell replacement rate with more new cells than
seen in the control mice.
If the hydra is an example of immortality that
is fueled by its high cellular turnover rate, then
one might expect that the MRL mouse also may
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HEBER-KATZ ET AL.
show signs of extended longevity. These experiments are in progress.
ACKNOWLEDGMENTS
This work was generously supported from
its inception by the G. Harold and Leila Y.
Mathers Foundation, F.M. Kirby Foundation,
and the W.W. Smith Foundation, and by a
grant from the National Institutes of Health.
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Address reprint requests to:
Ellen Heber-Katz, Ph.D.
3601 Spruce Street
Philadelphia, PA 19104
E-mail: [email protected]