The Body Electric
by Robert O. Becker
Nine
The Organ Tree
"I have yet to see any problem, however complicated, which when you
looked at it the right way did not become still more complicated," science fiction writer Paul Anderson once observed. To a certain extent this
is true of regeneration. Intricate nature is still more than a match for our
finest-spun hypotheses. Yet we've now reached the oasis of science that
we call an interim understanding, where the data begin to shake into
place and we can sense the pattern of the rebus from the blanks we've
filled in.
Ultimately we must relate all we learn about regeneration to a general
system of communication among cells, for regrowth is only a special case
of the cooperative cohesion that's the essence of multicellular life. This
communication system includes but extends beyond the gene-protein enzyme subsystems that govern the specialization of cells and unite their
chemical trade routes into smoothly working tissues and organs. During
embryonic development, cells where muscle will appear must receive
instructions from their environment telling them to repress all genes
except the muscle genome, or subcode. In many tissues, perhaps in all,
chemical inductors from previously formed tissue perform this task,
leading embryonic cells step by step through the stages of differentiation. However, chemical reactions and the passage of compounds from
cell to cell can't account for structure, such as the alignment of muscle
fiber bundles, the proper shape of the whole muscle, and its precise
attachment to bones. Molecular dynamics, the simple gradients of diffusion, can't explain anatomy. The control system we're seeking unites all
levels of organization, from the idiosyncratic yet regular outline of the whole organism to the precisely engineered traceries of its microstructure. The DNA-RNA apparatus isn't the whole secret of life, but a sort
of computer program by which the real secret, the control system, expresses its pattern in terms of living cells.
This pattern is part of what many people mean by the soul, which so
many philosophies have tried to explicate. However, most of the proposed answers haven't been connected with the physical world of biology
in a way that offered a toehold for experiment. Like many attempts, the
latest major scientific guess, the morphogenetic field proposed by Paul
Weiss in 1939, was just a restatement of the problem, though a useful
one. Weiss conjectured that development was guided by some sort of
field projected from the fertilized egg. As the dividing cell mass became
an embryo and then an adult, the field changed its shape and somehow
led the cells onward.
The problem was that there were too many "some hows." Even if one
accepted Burr's largely ignored measurements of an electric L-field and
admitted that it might be the morphogenetic field (a possibility Weiss
dogmatically rejected), there was still no way of telling where the L-field
came from or how it acted upon cells. Nor was there an explanation of
how, if the field was an emanation from the cells, it could also guide them
in building an animal or plant. In applying the idea to regeneration,
biologists faced the related and seemingly insurmountable problem of
how a more or less uniform outflow of energy could carry enough information to characterize a limb or organ. Given the complexity of biological structures, this was even harder than imagining how a field could
"somehow" survive when the part it referred to was missing.
However, the morphogenetic field no longer has to account for everything. Acceptance of dedifferentiation lets us divide regrowth into two
phases and better understand each. The first phase begins with the
cleanup of wound debris by phagocytes (the scavenger race of white
blood cells) and culminates in dedifferentiation of tissue to form a
blastema. Redifferentiation and orderly growth of the needed part constitute the second phase.
Simplifying the problem in this way should give biologists an immediate sense of accomplishment, for the first stage is now well understood. After phagocytosis, while the other tissues are dying back a short
distance behind the amputation line, the epidermal cells divide and migrate over the end of the stump. Then, as this epidermis thickens into
an apical cap, nerve fibers grow outward and subdivide to form individual synapse like connections the neuro epidermal junction (NEJ) - with
the cap cells. This connection transmits or generates a simple but highly specific electrical signal in regenerating animals: a few hundred nano amperes of direct current, initially positive, then changing in the course of
a few days to negative.
The pituitary hormone prolactin, the same substance that stimulates
milk flow in nursing mothers, seems to sensitize cells to electric current.
Then the signal causes nearby cells to dedifferentiate and form a
blastema, apparently by changing the way cell membranes pass calcium
ions. After confirming our frog blood-cell work, Art Pilla went on to
produce the same changes by using pulsed DC to make a wave of calcium ions flowing across the culture dish. Steve Smith then confirmed
the importance of calcium by preventing dedifferentiation with a calcium-blocking compound, and restarting it with another substance that
enhanced passage of calcium ions. Working together, Smith and Pilla
next used the same PEMF wave form now in clinical use to nearly double the rate of salamander limb regeneration, while completely preventing it with a different pulse pattern. Widespread recent work on
calcium-binding proteins, such as calmodulin, has made it fairly certain
that electrical control of calcium movement through cell membranes directs the give-and-take among these proteins, which in turn supervises
the cell's entire genetic and metabolic industry.
Although not conclusive, the available evidence suggests that the current flows through the perineural cells rather than the neurons themselves (see Chapter 13). These are several types of cells that completely
surround every nerve cell, enclosing all the peripheral fibers in a sheath
and composing 90 percent of the brain. Lizards can replace their tails
without the spinal cord, as long as the ependyma, or perineural cells
surrounding the cord, remains intact, and ependymal tissue transplanted
to leg stumps gives lizards some artificial regeneration there. However,
the circuit may shift tissues near the wound, for Elizabeth Hay's electron
microscope studies clearly show that the peripheral nerve's Schwann cell
sheaths stop just short of the epidermis, and only the naked neuron tips
participate in the NEJ. The exact current pathway in this microscopic
area remains to be charted.
Not all cells can respond, however, as Jim Cullen and I found in one
part of our fortuitous rat-regeneration experiments of 1979. Dedifferentiation occurred only when we passed the deviated sciatic nerve to the
epidermis through the bone marrow. When we led it through the muscle
yet sutured it to the skin in exactly the same way, an NEJ producing the
right current appeared, but no blastema and no regrowth. Muscle cells
apparently weren't competent to differentiate in the adult rat. The
cellular target proved to be just as important as the electrical arrow. There's still some opposition to parts of this scenario. Among some
scientists, the prejudice against electrobiology remains so strong that
one otherwise fine recent review doesn't even mention the NEJ or the
difference between currents of injury in frogs versus salamanders!
Other objections are a little more substantial. A Purdue University
group has measured electrical potentials near the surface of regenerating
limbs underwater, using a vibrating probe. This is an electrode whose
tip, ending in a tiny platinum ball, oscillates rapidly to and fro, giving
the average voltage between the two ends of its motion. These researchers describe an arc of ion flow—they categorically deny the possibility of
electron currents in living tissue—out from the stump and through the
water or, in semiaquatic animals, a film of moisture on the skin. From
the water, they suggest, these ions travel to the limb skin behind the
amputation, then in through all the inner tissues, and finally out of the
stump again to complete the circuit. They believe the epidermis drives
these currents by its normal amphibian function of pumping sodium
(positive) ions from the outside water into the body. They conceive of
this ion flow as the regeneration current itself, because changing the
concentration of sodium in the water directly affects their current measurements, and because certain sodium-blocking techniques have interfered with limb regrowth in about half of their experimental animals.
Of course, the Purdue researchers don't dispute the amply proven
need for nerve and injury currents during regeneration. They've even
confirmed Smith's induction of leg regrowth in frogs with batteries generating electron currents. Nevertheless, they consider nerves the target
rather than the source of current, even though they propose no reason
why their ion flow should be restricted to nerve tissue. In fact, they base
their hypothesis partly on evidence that sodium flows even from denervated limbs.
There are several other problems with this theory: Its proponents' own
measurements show that the sodium ion current almost disappears just
when its supposed effect, blastema formation, is occurring. Moreover, it
fails to explain the easily observed reversal of polarity in injury currents
measured directly on the limb, as well as the crucial role of the NEJ.
The proposed circuit goes right past the NEJ! Finally, it can't account
for the several tests of semiconducting current throughout the nervous
system, or regeneration in dry-skinned animals such as lizards.
To the lay person all this may seem like academic hairsplitting, until
we reflect on the stakes: understanding regeneration well enough to restore it to ourselves. Certainly skin is electrically active. It's piezoelectric
and pyroelectric (turning heat into electricity) as well as a transporter of ions in wet animals. In the last two decades nearly all tissues have been
proven to produce or carry various kinds of electrical charge. Skin may
play an as-yet-unknown role in regeneration besides its part in the NEJ,
or it may merely be producing unrelated electrical effects. In any case,
there are far too many data about the role of nerves to call skin the major
source of the regeneration current. In fact, even the Purdue group has
measured a stump current that's independent of sodium concentration.
A recent experiment by Meryl Rose gave further evidence of neural
DC, without clearing up all aspects of the question. Rose removed the
nerves From larval salamander legs before amputation. Normally such
denervated larval stumps die right back to the body wall, but when Rose
artificially supplied direct currents like those I measured in my first experiment, they regrew normally. This is pretty conclusive proof that the
nerves are the electrical source in phase one. However, since it looks as
though nerves also organize regeneration's second phase (see below), it's
hard to understand how the new legs could have been completely normal
when they were disconnected from the rest of the nervous system. Perhaps salamanders can pinch-hit for nerves at this stage through a tissue
other than nerve. On the other hand, new nerves may simply have regrown into the limbs unbeknownst to Rose by the later stages of the
experiment.
Phase two begins as the embryonal cells pile up and the blastema
elongates. Early in this stage a sort of spatial memory becomes fixed in the
blastema cells so the limb-to-be will have its proper orientation to the rest
of the body. At the same time or shortly afterward, the cells at the inner
edge of the blastema receive their new marching orders and platoon
assignments. Then they re-differentiate and take their places in the new
structure.
We can infer two things about the control for this part of the process.
Since the blastema forms the right structure in relation to the whole organism, the guidance can't be purely local, but must come from a system
that likewise pervades the whole body. Furthermore, there are no dedifferentiated cells left over when the work is done; there are just enough
and no more. Thus there must be a feedback mechanism between the
redifferentiation controls at the body side of the blastema and the NEJ's
dedifferentiation stimulus at its outer edge.
A large body of earlier work has shown that the redifferentiation instructions are passed along a tissue arc whose main element is the circuit
already established between nerves and epidermis in the first phase. The
electrical component persuasively explains how this arc, an update of the
morphogenetic field, may work. The direction (polarity) plus the magnitude and force (amperage and voltage) of current could serve as a vector
system giving distinct values for every area of the body. The electric
field surrounding continuously charged cells and diminishing with the
distance from the nerve would provide a third coordinate, giving each
cell a slightly different electrical potential.
In addition, a magnetic field
must exist around the current flow, possibly adding a fourth dimension
to the system. Together these values might suffice to pinpoint any cell
in the body. The electric and magnetic fields, varying as the current
varies with the animal's state of consciousness and health, could move
charged molecules wherever they were needed for control of growth or
other processes. Since currents and electromagnetic fields affect the cell
membrane's "choice" of what ions to absorb, reject, or expel, this system—in concert with the chemical code by which neighboring cells
recognize each other—could precisely regulate the activities of every
cell. It could express the exact point along the limb at which new
growth must start; distinguish between right and left, top and bottom;
even explain how totally missing parts, like extirpated bones or all the
little bones of wrist and hand, can reappear.
Furthermore, the difference between electrical values at the inner and
outer edges of the blastema would lessen as a new limb grew behind it.
(Remember that the electrical potential grows increasingly negative
toward the end of an intact limb.) The gradual convergence of these two
values could constitute a feedback signal perfectly reflecting the number
of dedifferentiated cells still needed. Although the results weren't entirely conclusive, perhaps because measurements had to be made under
anesthesia, several experiments in the 1950s suggested that such a voltage differential governed restitution of the proper number of segments in
earthworms. There was even a surge of positive potential that seemed to
indicate when the job was finished.
This is a rich concept, and the details are without doubt more complex than this sketch, but they're all open to experimentation in a way
that Weiss's morphogenetic field and Burr's L-field were not. The best
part of this two-stage analysis is that it gives us a rationale for trying to
foster regeneration after human injuries before we know all the details of
the second phase.
The rat limb experiments strongly suggest that mammals lack two
crucial requirements for the first phase of regeneration: They don't have
the necessary ratio of nerve tissue to total limb tissue, the amount
needed to make the dedifferentiation stimulus strong enough; and they
lack sufficient sensitive cells to respond to the electrical stimulus and
form a big enough blastema. The work on rats pointed the way to defining the proper current, and the ability of electrically injected silver ions
to dedifferentiate fibroblasts now gives us a possible method for producing an adequate blastema. We should now be able to supply the requirements for phase one in humans. Once this is done, the body itself can
probably take care of phase two, even though we don't understand the
process. Fingertip regrowth in children suggests that our bodies still
have the ability to re-differentiate the cells and organize the missing part,
as long as the electrical stimulus and the supply of sensitive cells are
sufficient.
Microsurgeons have performed wonders in reimplanting cleanly severed portions of arms, legs, and fingers, but these limbs are subject to
atrophy and obviously can't be grafted if they're too badly mangled or
riddled with disease. As one who has performed too many amputations
in his time, I find the prospect of being able to give a patient the real
thing instead of a prosthesis tremendously exciting. There's a good
chance that we'll eventually treat some non-genetic birth defects or old
injuries by cutting off the defective part and inducing a normal one to
grow. Perhaps, combined with gene splicing, such techniques could
even rectify genetic birth defects.
Since no one has yet achieved full regeneration in rats or any other
mammal, these dreams won't come true overnight. They aren't
chimerical, however. The remaining problems could probably be solved
in a decade or two of concerted basic research. Meanwhile, human capacities for repair of certain tissues are greater than most people realize, and
there are already promising ways of enhancing some of them.
Cartilage
Fossils show that even the dinosaurs had arthritis, but unfortunately it
outlived them. Many varieties have been described, all of which result in
destruction of the hyaline (glassy) cartilage that lines the ends of the
bones. The remaining cartilage cells try to heal the defect by proliferating and making more cartilage. They're almost never equal to the task,
and scar tissue fills the rest of the hole. The result is pain, for scar tissue
is too spongy to bear much weight or keep the bones from grinding
against each other.
After our success at getting rat legs to partially regenerate, we studied
this problem in 1973. We reasoned that, suite cartilage was made by
only one kind of cell, getting it to regrow would be easier than working
with a whole limb.
With orthopedic resident surgeon Bruce Baker of the Upstate Medical
Center, we removed the cartilage layer from one side of the femur at the
knee in a series of white rabbits. The operation left a circular hole of
bare bone about 4 millimeters across. In the experimental animals we
implanted silver-platinum couplings like those used on the rats, drilling
the platinum end into the defect and tucking the rest along the bone.
Most of the control animals filled in the defect with scar tissue along
with some inferior fibrous cartilage. About a tenth of them grew a millimeter or two of good hyaline cartilage at the edge of the hole. But sure
enough, the rabbits with the implants showed greatly enhanced repair.
When we used an improved battery implant with silver wires at each
end, we got even better results. Two of the rabbits healed the damage
completely with beautiful hyaline cartilage just like the original material.
A few years later, when we were testing various electrode metals, we
tried a different approach specifically for rheumatoid arthritis, in which
runaway inflammation causes phagocytes to attack healthy cartilage
cells. Gold salts taken orally sometimes control this disease but often
produce toxic side effects. We figured electrical injection of pure gold
directly into the joint with no other ions might work better. To find
out, Joe Spadaro and I produced rheumatoid arthritis in the knees of
both hind legs in forty rabbits, using a standard experimental procedure.
Then we treated one knee in each animal with a positive gold electrode
stuck right into the space between the two bones for two hours. Joe did
the actual treatments. Then we sacrificed the animals gradually over a
period of two months, and I examined both arthritic knees, not knowing
until later which had been given gold. During the first two weeks about
70 percent of the treated knees were markedly better than the untreated
ones. The improvement fell off to about 40 percent thereafter, suggesting that the treatment must be repeated for continued results.
Obviously, these were only preliminary experiments. However, since
an estimated 31 million Americans suffer from arthritis, for which there
is no cure yet, I think both avenues should be thoroughly explored as
soon as possible.
Skull Bones
Lev Polezhaev has spent his career investigating what might be called
the Polezhaev principle - the greater the damage, the better the regrowth. He has found he can often enhance repair by adding homogenates, minces, and extracts of the damaged organs, even though this
doesn't augment the current of injury as his needling procedure did.
Eventually Polezhaev developed a way to induce regeneration of holes
in the skull, which normally heal over with scar tissue. As long as the
dura mater, the cough membrane between skull and brain, is intact, a
paste of blood and fresh (living) powdered bone will induce the bone
cells at the edges to grow and bridge the gap. Microscopic studies have shown that the few live cells remaining in the paste don't survive and
the bone particles themselves soon dissolve. Instead, some substance
from the disintegrated bone stimulates repair. Since its first successful
trial on humans by several Russian surgeons in the mid-1960s, this
method has gradually come into increasing use in the Soviet Union.
Eyes
There is at present no indication whatsoever that humans could ever
regenerate any part of their eyes, but the ability of newts (salamanders of
the genus Triturus) to do so makes this a tantalizing research ideal for the
far future. If the lens in a newt's eye is destroyed, the colored cells of the
top half of the iris extrude their pigment granules, then transform by
direct metaplasia into lens cells. They soon start synthesizing the clear
fibers of which the lens is made, and the whole job is finished in about
forty days. In case the iris is gone, too, a newt can create a new one from
cells of the pigmented retina, and those cells can also transform into the
neural retina layer in front of them. If the optic nerve gets damaged, the
neural retina in turn can regenerate the nerve tract backward and reconnect it properly with the brain.
No one knows why newts are so much more adept at this than all
other creatures; their eyes have no obvious structural or biochemical peculiarities. Steve Smith gave us an important fact to work with when he
found two proteins in the lens that seem to prevent the iris cells from
changing into new lens cells as long as the old lens is in place. Since the
neural retina must be intact for most of the transformations to happen,
it may provide a constant electrical stimulus that goes into effect only
when the inhibitory proteins are removed by injury.
No blastema is formed; instead the cells change costume right
onstage. Furthermore, certain ingenious experiments have shown that a
wound isn't really necessary, only the interruption of the inhibitory
mechanism. Therefore, the stimulus from the neural retina probably
isn't the familiar injury current of limb regrowth. However, despite a
voluminous research literature on newt eye regeneration, no one has yet
studied its electrical aspects. This may be why we're still so far from
understanding the natural process, let alone trying to adapt it to human
eyes.
Muscle
Every muscle fiber is a long tube filled with rows of cells (myocytes) laid
end to end with no membranes between them—in effect, one multinucleated cell, called a syncytium. These nuclei direct the manufacture
of contractile proteins, which are lined up side by side and visible, when
stained, as dark bands across the array of myocytes. Each muscle fiber is
surrounded by a sheath, and groups of them are bound together in bundles by thicker sheaths. At the edge of each bundle are long, cylindrical
cells with huge nuclei and very little cytoplasm, called myoblasts or
spindle cells. Also along the edges, between the spindle cells, clusters of
tiny satellite cells can be seen at high magnifications.
After a crushing injury or loss of blood from a deep cut, muscle in the
damaged area degenerates. The myocyte nuclei shrivel up and the cells
die. Soon phagocytes enter to eat the old fibers and cell remnants. Only
the empty sheaths and a few spindle and satellite cells are left.
Now these remaining cells turn into new myocytes, fill up the empty
rubes, and begin secreting new contractile proteins. Although the early
part of this process proceeds without nerves, it can run to completion
only if motor nerve fibers reestablish contact with the terminals, called
end plates, that remain at specific distances along each fiber sheath. If
these end-plate areas are cut out, the nerve endings will enter, sniff around, and then retract. The muscle will atrophy. If the nerves reestablish the connections, new muscle cells will fill most of the original
volume, gradually build up strength, and then completely differentiate
into slow-twitch or fast-twitch fibers.
Attempts to enhance muscle regeneration in humans take two approaches. If available, a graft of a whole muscle from another source is
the more effective. This is actual single-tissue regeneration, because the
original muscle cells die and are replaced after new blood and nerve
connections are made. Since its first clinical use in 1971, this approach
has proven successful in replacing defective small muscles of the face and
also in restoring anal sphincter control. Large limb muscles haven't been
successfully transplanted yet.
Another method may soon be used in humans when grafts aren't possible. Muscle regeneration in birds and laboratory mammals has been
considerably enhanced by inserting muscle tissue minced with fine scissors into pieces of no more than 1 cubic millimeter. Soviet biologist A.
N. Studitsky first devised this method in the 1950s, extending Polezhaev work, but its development has been slow.
Abdominal Organs
Despite over two hundred years of descriptive work, new regenerative
capacities are still discovered in the animal kingdom from time to time.
We've recently learned, for example, that adult frogs can restore their
bile ducts, although for some reason females are better at it than males.
Doctors have long known that the liver can replace most of the mass lost
through injury by compensatory hypertrophy, in which its cells both
enlarge and increase their rate of division so that the organ's chemical processing duties can be maintained even though the ruined architecture
isn't restored. Similarly, damage in one kidney is made up by enlargement of the other without rebuilding the intricate mazes of microtubules
in the glomeruli. Recent studies of rat livers suggest that a combination
of insulin and an epidermal growth factor, modified by at least a dozen
other hormones, enzymes, and food metabolites, control the cell proliferation.
Now it appears that the spleen can make the same kind of comeback,
at least in children. Adults who must have their spleens removed rarely
miss them, but children become more susceptible to meningitis. A few
years ago, medical researchers noted that children whose excised spleens had been damaged by accident were less likely to get meningitis than
those whose spleens had been removed because of disease. Howard Pearson and his colleagues at the Yale University School of Medicine found
that, when ruptured, the delicate spleen left bits of itself scattered in the
abdomen, which grew and gradually resumed the organ's obscure blood cleansing functions. Now when they remove a spleen, many surgeons
wipe it on the peritoneum (the tough membrane that lines the abdominal cavity) to sow replacement seeds.
Another late discovery of regeneration was made in the late 1950s, when
several scientists learned that tadpoles, larval salamanders, and sometimes
adult salamanders could restore up to about four inches of their intestines.
Moreover, all adult amphibians could reconnect the cut ends even if they
couldn't replace a missing section. Allan Dumont, one of my best friends
during medical school and now Jules Leonard Whitehill Professor of Surgery
at NYU-Bellevue, decided to check this potential in mammals after I told him
about my work on rat limbs. He wanted to find out whether regeneration
could be stimulated in mammals to solve one of a general surgeon's most
vexing problems—poor healing of sutured ends of gut after a cancerous or
degenerated segment has been cut out. Even a small opening can spill feces
into the abdominal cavity, with disastrous peritonitis the result.
Like any good scientist, Al started from the basics. After several years
he'd confirmed the earlier reports. When he cut pieces from the gut of
adult frogs and newts and merely put the ends close together in the
abdomen, 40 percent of the animals survived by quickly reconnecting
the two ends and completely healing them in about a month, although
even the newts didn't replace much of the lost length in his experiments. Gut regeneration actually involves several tissues; Al's cell studies showed a blastema quickly forming at the junction and then
differentiating into smooth muscle, mucus cells, and the structural cells
of the villi.
Naturally, when I was organizing a conference on regeneration in
1979, I invited Al to present his results. About a month before the
meeting, after the program had already gone to the printer, he wanted
to change the title of his paper, for he'd just finished some surprising
work. He asked me, "What would you expect to happen if I took some
adult rats, cut out a centimeter of gut, and dropped the two loose ends
back into the abdomen?" Like any first-year med student, I said they'd
be dead of peritonitis in two or three days. Well, 20 percent of Al's rats
had reconnected then bowels better than surgery could have done, and
were alive and healthy. When Al had given one group of animals a
temporary colostomy above the experimental cut, the survival rate jumped threefold. The perfect healing of the test animals compared with
the controls indicated that sutures actually interfered with regeneration
producing unnecessary scars and adhesions.
No one knows for sure how the two cut ends find each other, but
there's certainly some active search going on, for peritonitis sets in too
quickly for the results to be due to chance. The process resembles a
regrowing nerve fiber's search for its severed part, which may be conducted by electrical factors, a chemical recognition system, or both.
Electrical potentials probably play the most important part, for recent
research has found DC potentials at injuries on the peritoneum, and
experimental changes in the peritoneum's normal bioelectric pattern attract the inner membrane enclosing the bowels, causing it to adhere to
the site of the disturbance. Al has recently learned that, if the ends don't
have to look for each other but instead are connected by a piece of Silastic tubing, rats can, like tadpoles, replace up to 3 centimeters of
missing intestine. There's no reason to believe this technique couldn't be
adapted to humans.
Even though we don't know enough yet to electrically stimulate intestinal healing, Al has proposed a preliminary test of regeneration in large
mammals that could spare some patients a lifetime of misery. It's almost
impossible to surgically rejoin the colon to the anus, and when
The Organ Tree 195
sutures fail, the person ends up with a colostomy. Since the free end of the
colon would be held near its proper position by the local anatomy, Al suggests replacing it without stitches and giving such animals a temporary colostomy upstream from the gap. If X rays later showed regrowth, the
temporary colostomy would be closed, and the animal would have a continuous, healthy intestine. If even a few patients could be spared the indignity of
living attached to a bag, the effort would be well worth the little—yet still
nonexistent—funding required. Intensive study of the electrical details of gut
healing would probably make surgery less devastating for many additional
patients.
Exciting as the prospects in this survey are, they're by no means the
only ones, or even the most spectacular, which are reserved for the following chapters. Many researchers are working to turn the breakthroughs of the last two decades to practical use. Even so, progress isn't
nearly as fast as it could be, perhaps due to disbelief that such widespread self-repair is really possible for us. It's not only possible, it's
nearly certain, given even a modest monetary push,for the "useful dispositions" foreseen by Spallanzani are within our reach.
Ten
The Lazarus Heart
Like Columbus, scientists sometimes stumble upon new continents
when merely seeking a quicker trade route. Our research group had this
good fortune in 1973.
We'd gone back to basics after learning how to dedifferentiate frog
red blood cells and start regeneration in rat limbs. We decided to study
nucleated red cells in a variety of creatures, hoping for leads toward
better regrowth in mammals. Although their circulating erythrocytes
have no nuclei, even mammals have young red cells, with nuclei, forming in the bone marrow. After severe bleeding, up to a fifth of those in
the bloodstream may be immature nucleated types, as the marrow rushes
them into service to make up for the loss. We surveyed the effect of
direct current on red cells from fish, amphibians, reptiles, and birds. All
of the cells responded, but in a different way for each species. We decided to have a more detailed look at the largest and hence most easily
studied red blood cells available, those of our old friend Triturus viridescens, the common green newt.
A newt is so small that you can't just poke a needle into one of its
veins and take a blood sample. The only practical way to get pure blood
is to anesthetize the animal, slice open its chest, cut its heart in two,
extract the blood with a pipette, and throw away the carcass.
As the phrase goes, we "harvested" blood from three newts each week
by this method. One day, when Sharon Chapin had finished the chore,
she asked me, "What would happen if I sewed these animals up?" I
answered that, because their hearts had been destroyed, they would die
within minutes, with or without sutures, from lack of oxygen to the brain, just as all our other amphibian blood donors had. We looked it
up just to make sure. The standard works on regeneration all agreed that
no animal's heart could repair major wounds. Unlike skeletal muscle,
the cardiac variety had no satellite cells to serve as precursors for mature
heart-muscle cells. In any case, the textbooks stated, the animal would
die long before such repair could occur.
Next week Sharon put our three intended sacrifices in a bowl of water
and with a straight face asked me if they looked healthy enough to use. I
told her they looked fine. "Good!" she exclaimed. "These are the same
three we used last week." Score one for the open mind!
Flabbergasted, I helped anesthetize and dissect this trio of miracles.
Their hearts were perfectly normal, with no evidence of ever having been
damaged in any way.
The Five-Alarm Blastema
Abruptly I changed my research plans. I asked Sharon to test a series of
newts by cutting away large sections of their hearts and sewing up their
chests, then killing some of the survivors every day and slicing, mounting, and staining the hearts for study under the microscope. Over 90
percent lived through the first operation, and several weeks later we had
hundreds of slides ready for my examination and diagnosis. Unfortunately they all looked the same! Even those from the day after that
horrendous mutilation showed only normal tissues with no sign of injury.
By now we knew we had come upon a first-class mystery and had
better jettison our preconceptions. We reasoned that we could tell when
regeneration was finished by finding out when the blood began flowing
again. Under the microscope we could easily see blood cells streaming
through capillaries in the transparent tail fins of lightly anesthetized
newts. The motion stopped when we cut the heart, and restarted about
four hours later. We sectioned a new series of hearts, this time covering
the first six hours at intervals gradually increasing from fifteen minutes
to one hour.
While waiting for the specimens, we rummaged more thoroughly
through the literature for other reports on heart regeneration. There was
evidence for very limited repair—but no true regeneration—of small
heart wounds in a few animals. The process seemed limited to the very
young. Even then, the results were of poor quality, combining a lot of
scar tissue with only a little proliferation of nearby heart cells, but the mitotic component could be enhanced by various experimental aids.
In 1971, John O. and Jean C. Oberpriller, anatomists at the University of North Dakota School of Medicine in Grand Forks, reported that
small wounds in salamander hearts healed this way but required two
months. A year after that, the English edition of a book by Lev Polezhaev summarized several decades of Russian research, mainly on the
hearts of frogs and lizards. Pavel Rumyantsev, now at the Cytology Institute of the USSR Academy of Sciences in Leningrad, had found in
1954 that newborn mammals (rats and kittens) could repair tiny puncture wounds, and recently he has proved the same capacity in the atria,
or receiving chambers, of adult rat hearts. We even found a German
report of 1914 claiming that human babies had sometimes regenerated
small areas of their hearts damaged by diphtheria.
The Russians claimed some progress in extending this marginal native
healing. In the late 1950s, N. P. Sinitsyn had repaired large holes (up
to 16 square centimeters) in the hearts of dogs by covering the wounds
with patches made of muscle sheath, canvas, suede, or other materials.
Scar tissue still covered the outside, but the patch guided a thin layer of
new muscle fibers forming along its inner surface. Using dogs whose
wounds had already closed with scar tissue, Polezhaev then found he
could induce heart muscle to fill in part of the gap by cutting away the
scar and irritating the edges of the remaining cardiac muscle. Other
Soviet researchers enhanced the muscle cell proliferation a little more
with vitamins B1, B6, and B12, various drugs, extra RNA and DNA,
and heart tissue extracts or minces.
Despite such goads, heart regrowth was limited to very small injuries
or the border zone around larger ones, and it always took several weeks.
No one had even imagined that half a heart could restore its other half,
much less in a matter of hours. I could hardly wait until the next batch
of slides was ready.
They showed us an unprecedented type of regeneration. Where the
missing part of the heart had been, a blastema formed in about two and
a half hours. We saw no evidence of dedifferentiation or mitosis in the
remaining heart-muscle cells, and indeed it would have been impossible
for the processes we'd already studied to make a blastema in such a short
time. Instead, the mass of primitive cells arose dramatically from the
blood.
As soon as the salamander heart is cut open, blood pools around the
wound and clots quickly, usually in about one minute, sealing the hole
like wet plaster. Almost immediately, the nearest red blood cells crack
open like eggs. Their nuclei, surrounded by a thin coating of cytoplasm,
glide by some means yet unknown directly to the raw, frayed edge of the heart muscle and insinuate themselves into the tangle of dying and injured cells. To a biologist this sight is bizarre, uncanny. It's as though
the engine of a passing car could walk up to a stranded truck, climb
under the hood, and drive it away.
Farther away from the wound surface, the red cells also spill out their
nuclei, but these cell yolks clump together, fusing their remaining
cytoplasm to form a syncytium. Still farther away from the center of
action, the red cells undergo the more leisurely dedifferentiation we observed in our frog fractures and DC culture studies. They turn into
primitive ameboid cells that move toward the area of damage and attach
themselves by pseudopods to the injured muscle fibers. In all of biology
there's no precedent for these virtuosic cellular metamorphoses. In fact,
they're so strange that most researchers have simply refused to believe in
their existence or try the experiment for themselves.
All these changes are well under way within fifteen minutes. Soon
afterward the extruded nuclei, the interconnected syncytial nuclei, and
the ameboid cells are all dividing as fast as they can, building up the
blastema. It's fully formed within three hours after the injury. By then
its cells have already started to re-differentiate into new heart-muscle
cells, synthesizing their orderly arrays of contractile fibers and connecting up with the intact tissue. If the clot contained more blood cells
than were needed, the extras outside of the area now degenerate, apparently so as not to get in the way of the the repair work.
Meanwhile, the newt has survived by absorbing dissolved oxygen from the water through its skin. Now, at about the four-hour mark, there are
enough new muscle cells to withstand contraction, and the heart begins
pumping again, slowly. After five or six hours, most of the blastema cells
have re-differentiated into muscle, which is still somewhat "lacy" or delicate compared with the established tissue. After about eight or ten hours,
however, the heart is virtually normal in appearance and structure, and
after a day it's indistinguishable from an uninjured one.
Why did we see this colossal regeneration, while the Oberprillers
found only a tiny, slow healing response in the salamander heart? Apparently this was another manifestation of the Polezhaev principle. We
made a big wound; they made a small one. Only massive damage unleashed the full power of the cells.
Is this fantastic cellular power forever restricted to salamanders, or
does it reside latent in us, ready at the appropriate impetus to repair
damaged hearts without problem-filled (and frightfully expensive) transplants of donated or artificial pumps? We don't know, but we've found
no other regenerative process that's forever off limits to mammals. At
this point we can only speculate on how such a treatment might be
accomplished, but at least the idea isn't wholly fantasy.
The first job is to identify human target cells able to dedifferentiate
into primitive totipotent cells. Bone marrow cells or immature erythrocytes, the nearest equivalents to amphibian nucleated red blood cells,
are one obvious candidate population, especially since they seem to be
the crucial cells in rat limb regeneration and the inner part of fracture
healing. Fibroblasts despecialized by electrically injected silver ions
might be used. Another possibility is lymphocytes, one class of infection-fighting white blood cells. In our lab we've demonstrated that they,
too, can dedifferentiate in response to appropriate electrical stimuli.
Since newt-type heart regeneration doesn't occur naturally in mammals, we would probably have to grow a large mass of the target cells in
tissue culture. Then, with the patient on a heart-lung machine, the
surgeon could cut away scar tissue and otherwise freshen the wound if it
wasn't recent enough, then apply enough of these ready-made pre-blastema cells to fill the defect. They would be held in place by a blood
clot, sutured pericardium, or some type of patch. Then, assuming we'd
learned the electrical parameters already, electrodes would induce nuclear extrusion, dedifferentiation, consolidation with surrounding muscle, and the final transformation into normal cardiac muscle. The current
would probably have to be adjusted throughout the process to get us
various steps in synchrony, and vitamins or drugs might be used to
enhance mitosis or protein synthesis. Once the scar had been removed, the instructions as to what cells were needed would come from the surrounding healthy heart muscle.
In the salamander this process takes about six hours. Since this exceeds the current limits of "machine time" for artificial circulation in
humans, we would have to extend the capacities of heart-lung devices or
else speed up the cellular processes. Obviously there's a long road of
experiment to travel before we can be more specific about techniques.
One of the things we must learn is whether the newt's electrocardiogram
shuts down during repair. We must know how its presence or absence
relates to the current of injury and other electrical factors in this novel
method of blastema formation.
Personally, I'm sure we can get the human heart to mend itself. As a
result of being confronted by this wonder in newts, I'm convinced that
the potential repertoire of living cells is absolutely enormous, far greater
than the healing powers normally manifested by most animals or even
those dreamed of by doctors. Even in the newt this "superregeneration"
doesn't appear unless 30 to 50 percent of the heart is gone. Something
about the massiveness of the injury or the approach of death then boosts
the healing process into overdrive.
I readily admit that the discovery sounds a bit like science fiction,
even as toned down into the subdued technical prose of our report, published by Nature in 1974. I had trouble believing it myself at first.
Because it seemed so incredible, there was no rush to confirm and extend
our discovery. Today, even though our observations have been corroborated by University of Michigan anatomist Bruce Carlson in 1978 and
by Phil Person in 1979, complete with electron micrographs of the cell
changes, most biologists still don't accept heart restoration as fact. Perhaps because the reality is so outlandish, Carlson wouldn't publish, and
Person has been unable to get his work published in the peer-reviewed
journals. Our original paper of ten years ago is still officially unconfirmed, and the other workers are still puttering around with little
wounds. This attitude must change. Knowledge about the controls of
this process will be of incalculable value to medicine, for this is ideal
healing. Spilled blood closes a wound at the body's center and replaces
the missing part in a few hours. You can't get much more efficient than
that.
next-187s
The Self-Mending Net
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