Part 5 The Body Electric ...The Organ Tree ...The Lazarus Heart

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. 

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The Self-Mending Net 

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