Documentation of regeneration dates back to the work of Aristotle (– A study of cell lineages in the regenerating limbs of salamanders has shown Using basic histological and immuno-histochemical techniques, detailed .. Uca pugilator: Histological, physiological and molecular considerations. acterized heart regeneration model system to date. phenomenon of limb regeneration in sala- speed of onset, and memory, the adult frog immune system is systemic analysis of technical considerations in the surgery. Animal regeneration, for example, was discovered in the 18th .. This initiative does not negate the important pathological findings contributed to date, but “ Limb regeneration in amphibians: immunological considerations,”.
Among insects this focus is on genes that are known to be required during the development of legs in embryos. RNA interference-mediated functional studies conducted during regeneration of imaginal discs of Drosophila melanogaster, and nymphal legs of Gryllus bimaculatus reveal that several conserved pathways and transcription factors Wingless, Decapentaplegic, Hedgehog, Dachshund are required for successful regeneration.
Regeneration in decapods, like Uca pugilator and Gecarcinus lateralis, occurs in discrete phases of growth in tandem with the stages of the molt cycle. Recent studies have shown that ecdysteroid hormone signaling is necessary for blastemal proliferation. Although the current research emphases of limb regeneration in insect and crustacean are fairly distinct, the results generated by functional studies of a wide array of regeneration genes will be beneficial for generating testable regeneration models.
Introduction Regeneration is a developmental process that results in the re-growth of a lost or injured body part. Documentation of regeneration dates back to the work of Aristotle — BCwhen he observed re-growth of eyes in swallow-chicks, and re-growth of tails in tadpoles, lizards, and snakes Odelberg Regenerative abilities have been observed in several genera belonging to phyla ranging from cnidarians to chordates Brockes and Kumar Historically, two modes of regeneration have been described: During morphallaxis, the animal regenerates by reorganizing the remaining tissues following an injury or loss of body parts, requiring little or no cell division.
Regeneration in Hydra is an example of this phenomenon, in which a small, dissected part of a hydra has the potential to grow, without cell division, into a new, smaller hydra Galliot Alternatively, epimorphosis occurs when the lost parts are regenerated via cellular proliferation. The mode of epimorphic regeneration involves the formation of a specialized and transient structure called a blastema.
The regenerating blastema is a mass of dedifferentiated cells, obtained through the loss of cellular specialization, with the ability to proliferate and re-differentiate into all cellular components of the lost structure Gilbert Vertebrates like salamanders and tadpoles are capable of regenerating a limb via blastema formation Nacu and Tanaka The cellular organization of this blastema resembles that of limb buds formed during embryogenesis.
In invertebrates, regeneration via blastema formation occurs in planarians Wagner et al. Morgan proposed the two modes of regeneration in ; however, with advances in techniques in regenerative studies it has been shown that not all reported cases of regeneration follow the strict definitions of epimorphosis and morphallaxis. For example, Morgan classified planarian regeneration as morphallaxis, although recent studies have shown presence of a proliferating blastema at the site of the wound Wagner et al.
In addition, it has been observed that a blastema is not a homogeneous population of dedifferentiated cells.
Journal of Immunology Research
A study of cell lineages in the regenerating limbs of salamanders has shown that cells in the blastema retain a memory of their lineage Kragl et al. Although there are common pathways involved in both types of regeneration, there are distinct differences in regenerative processes among various species.
Thus, Agata et al. In the phylum Arthropoda, 35 genera of the sub-phylum Crustacea and 38 genera of the Class Insecta are capable of regenerating limbs Maginnis Within these groups, nymphal legs of hemimetabolous insects, imaginal discs of holometabolus insects, and limbs of decapod crustaceans can regenerate Adiyodi ; Truby ; Skinner ; Hopkins ; Mito et al.
Although there are distinct morphological differences in the regeneration of limbs between insects and crustaceans, two physiological aspects are highly conserved: Regeneration in species from Insecta and Crustacea cannot occur without successful molting. Hence, unlike in crustaceans, regeneration is not observed in adult insects. Regeneration in both insects and in crustaceans requires low levels of circulating arthropod steroid hormones ecdysteroids. Alternatively, regeneration is impeded by high titers of ecdysteroids.
The focus of this review is on studies of the regenerative abilities of limbs across insects and decapod crustaceans and the molecular mechanisms that lead to re-growth of lost limbs. The regeneration studies across Pancrustacea, a phylogenetic group that includes crustaceans and insects, have focused on various biological questions to unravel the hormonal and molecular basis of regeneration.
However, no one system is perfect for understanding the detailed phenomena of regeneration. The limitation in asking probing questions arose due to differences in model systems and in the application of technologies developed to study a particular system. The basic questions asked by scientists in the field of regeneration are: What is the morphological basis of regeneration? What is the molecular basis of regeneration? What is the hormonal basis of regeneration? Researchers have used insects, mainly Drosophila, to study the genetic basis of pattern formation and decapod crustaceans to study the hormonal basis of regeneration.
The tools used to manipulate gene expression spatially and temporally make Drosophila an advantageous model to study gene function in a particular type of cell at any given time during development Jennings In addition, cell death in a specific type of tissue can be induced in a non-invasive manner to study the regeneration in imaginal discs Smith-Bolton et al. Furthermore, using RNA interference RNAi to knockdown genes has been successfully used in hemimetabolous insects to study gene function.
Hence, the tools developed in insect systems to study gene expression and function provide an advantage toward delineating the molecular basis of regeneration. Unlike the situation in insects, in crustacean systems, it is difficult to study transmission genetics and to generate knockout mutants due to longer life cycles.
Inflammation as an Animal Development Phenomenon
Although RNAi has been successfully used in crustaceans, it is difficult to manipulate gene expression in specific tissues Das and Durica However, crustaceans are a well-known model for studying the molt cycle and circulating titers of hormones Soumoff and Skinner ; Hopkins ; Chang et al.
The large size of crustacean models Gecarcinus lateralis and Uca pugilator provides an advantage in that circulating levels of hormones in the hemolymph can be studied at different stages of regeneration without sacrificing the animal. The well-developed methods used to measure hormone titers in crustaceans are radioimmunoassay RIAhigh-performance liquid chromatography, and enzyme-linked immunosorbent assay Soumoff and Skinner ; Hopkins ; Mykles Hence, neither insect nor crustacean models has been able to shed light on the coordinated mechanism of regeneration that includes both molecular and hormonal control together.
This review aims to examine the aspects of research that have been conducted over the past several decades both in crustacean and insect systems, including the divergent directions the research has taken due to differences in the technologies used. Even within a specific group e. For example, a deep cut in the skin of an adult human generally triggers acute inflammation that is followed by a fibroproliferative process and scar formation; the same lesion inflicted on a fetus, however, may result in complete skin regeneration [ 4 ].
Irrespective of the peculiarities of the tissue repair processes in diverse groups of animals, it is rather intuitive to accept that regeneration and inflammation are related processes. Certainly, the processes that underlie the formation of a new salamander tail and the inflammation that occurs in response to a myocardial lesion in a mouse injected with high doses of isoproterenol are similar phenomena. Nonetheless, regeneration is viewed as the building of a structure, whereas inflammation is not recognized as such.
These differences in our perception of these two phenomena are likely due to the history of the characterization of these phenomena, the experimental approaches used to address these topics.
Origins of Inflammatory Certainties The initial framework used to study inflammation, which is widely accepted by the scientific community, is the description of the cardinal sinuses—rubor et calor cum tumor et dolor—performed by the Roman doctor Celsus approximately two thousand years ago [ 5 — 7 ].
This expression is still widely used and is representative of the general perception of inflammation. What is not usually mentioned, however, is that when Celsus proclaimed these words, he viewed inflammation simply as a collection of clinical symptoms and not as a phenomenon in itself.
In contrast, inflammation put in Celsus terms was not considered to be a process, but rather it was defined as a collection of signals resulting from process which remain unknown.
For a long time, pathologists studying inflammation contented themselves with enumerating more and more details of the inflammatory process in organisms becoming ill or injured tissues, without focusing on defining the concept of inflammation [ 89 ]. Even Virchow, the German pathologist who proposed a fifth cardinal inflammatory signal, functio laesa, categorically affirmed that inflammation was not a real entity [ 5 ], but a term that encompassed a series of phenomena so distinct that they should be treated separately.
Even more concerning, the examination of this phenomenon, which was strictly medical, was isolated from other very similar biological processes. Animal regeneration, for example, was discovered in the 18th century and resulted in a period of enriching debates concerning the origin of the animal form [ 11 ]; during the same period, inflammation was reexamined through the lens of cell pathology.
However, there was no attempt to converge these two ideas, as will be discussed later. The comparison of these two phenomena did not occur because regeneration was conceived as a phenomenon of form construction whereas inflammation had not attained the status of a biological phenomenon even by the end of the 19th century.
This perspective was changed by the seminal study described by Julius Cohnheim on the passage of white blood cells through capillaries and into inflamed tissue, known as diapedesis. For Cohnheim, this was not a mere histological description of what occurred in the disease process but the generative mechanism of the cardinal signs of inflammation. In describing changes in capillary structure, with the consequent movement of plasma and the passage of blood cells that compose the pus corpuscles, Cohnheim proposed a mechanism demonstrating how the symptoms described by Celsus were generated [ 12 ].
Thus, this proposal by Julius Cohnheim, demonstrating that the cause of inflammation resided in the vessels, unified diverse and subsidiary problems around a singular phenomenon and defined inflammation as an organic process. Within this context, inflammation was clearly a pathological event.
There is no doubt that this was a fundamental step in inflammation research. First, he suggested that inflammatory processes are exclusively pathological mechanisms without a physiological counterpart. Placing the vascular lesion as the ontological precedent of the inflammatory responses precludes the possibility of explaining how these processes are involved in the physiology of a healthy organism.
This situation was unusual because we usually seek to understand the physiological role of a process prior to investigating its role in pathology.
For example, we first sought to understand the electrical physiology of cardiac function prior to investigating the pathology of cardiac arrhythmias; however, we do not have the same reservations when studying inflammation in an exclusively pathological context. Except for very few instances, the term physiology is not mentioned in the immune-inflammatory jargon [ 1314 ]. However, if the vascular lesion was a sine qua non condition for the emergence of an inflammatory dynamic, what would occur when an animal lacking a circulatory system suffered a tissue injury?
How do all other animals repair themselves? It was only when this problem was addressed by other disciplines that these limitations were noticed. Metchnikoff, a Russian embryologist, was important for this transformation. Metchnikoff was interested in the formation of new embryonic forms during animal development and was investigating the role of a group of migratory and phagocytic mesenchymal cells in these phenomena.
To Metchnikoff, understanding phagocytosis was important because this event can be observed in all animals, even in the simplest and most primitive forms except in the amphioxus. When comparing phagocytosis among several groups of animals, Metchnikoff observed that phagocytes could ingest not only food particles, but also foreign particles and invading microorganisms reviewed in [ 15 ].
With the proposal that phagocytes were a defense mechanism against the challenges of the environment, Metchnikoff united the most important medical and biological theories of the 19th centuryand because phagocytes are common to all animals, Metchnikoff understood that this would be the primum movens of inflammation.
Thus, inflammation was transformed from a human pathological reaction to an animal health defense response [ 15 ]. When Cohnheim described diapedesis, the defensive aspect was not included in his description.
He had the insight to realize that the same process that the organism was using for defense was also participating in the embryonic and physiological processes of development.
For example, Metchnikoff had already described that phagocytosis was involved in the reabsorption of the tail in one genus of amphibians. Thus, Metchnikoff described a physiological role for inflammation, and for him, the building of an organism was a problem that preceded its defense.
For Metchnikoff, inflammation and immunity were subsidiary conditions of animal development; these situations were particular to the construction of metacellular harmony. Thus, he created the opportunity for studying the physiological aspects of inflammation [ 16 ]. All of his other considerations regarding the physiology of form construction in animals were quickly ignored. The newly founded discipline of immunology grew more concerned with understanding the pathogen-host relationship than any other generative or physiological aspect of the immune system or inflammatory activity.
As a result, two main schools for addressing the inflammatory response were created: A third more recent trend in inflammation research is the pharmacological approach. With the birth of the pharmacological industry, also at the end of the 19th century, the race to develop new methods to intervene in inflammatory processes became important. Thus, while pathologists described the reactions of organisms in response to disease, and the immunologists studied the detection of foreign bodies, the pharmacologists searched for methods to intervene in these events.
In this context, there is one event in inflammation research that deserves to be highlighted: During the first half of the 20th century, the methods for developing anti-inflammatory drugs were laborious and slow. In general, to characterize a potential anti-inflammatory agent, it was necessary to study inflammation as part of the repair of injured tissue.
These models were tiresome, with protocols that lasted several weeks. Ina group of researchers from Merck developed a model that complied with all of the requirements needed by those interested in rapidly developing a product: For heuristic reasons, the industry created a model that separated the cardinal signals of inflammation from the complex processes of tissue repair; as a result, inflammation returned to being a mere clinical symptom.
The practical success of this idea was immediate, and in less than a year, Merck had developed indomethacin, a drug that still is used as a reference drug in the development of new anti-inflammatory agents. Other industries rapidly reproduced this model, and dozens of new anti-inflammatory agents successfully entered the market in that decade. It is interesting that this experimental protocol was widely accepted in the academic sphere and was widely used in basic research, as if it was an adequate model for understanding the inflammatory phenomenon.
This had serious consequences because, by adopting a protocol that had been developed to simplify the research model for the pharmaceutical development of anti-inflammatory drugs, inflammation was once again no longer viewed as a physiological phenomenon; instead, it was studied as a cardinal sign of disease, as it had been two thousand years ago in Roman medicine.
Origin of Certainties in Regenerative Biology The initial characterization of the regenerative process in animals is attributed to Adam Trembley for his studies on medusa polyps [ 11 ]. At that time, it was unclear if the medusa polyps were plants or animals. Thus Trembley sectioned them because only plants were thought to be capable of regeneration.
Then after his experiment, it was observed that hydras had the capacity to reconstitute their lost parts, induce complete tissue repair, and rescue the status quo ante as if it was a plant.
However, all other observations on the life cycle of these organisms led to the conclusion that they were indeed animals. Therefore, the possibility of animal regeneration was recognized with great surprise. There is, however, something even more important in this finding by Trembley: Thus, the discovery of regeneration was also the discovery of a new form of asexual reproduction. This exceptional circumstance led to a conception of animal regeneration as not only a repair mechanism for lesions, but also as an event in animal development linked to the problem of reproduction and the generation of form.
Therefore, it is not surprising that embryologists began to study regeneration [ 11 ]. Thus, contrary to inflammation, which in this period was barely treated as a phenomenon in its own right, animal regeneration emerged as part of a framework of well-defined biological ideas and occupied a central position in a world of rich debate.
The theories of regeneration were placed next to those of embryonic development and metamorphosis; that is, regeneration was always perceived as a physiological phenomenon of animal development. Inflammation as an Animal Development Phenomenon Today, research in inflammation has certainly expanded its frontiers into several areas of scientific knowledge and could hardly be addressed within the limits of only one discipline.
This plurality is not only desirable but also necessary. My particular interest in commenting on the emergence of the three main schools of inflammation research—pathological, immunological, and pharmacological—is not merely for the sake of providing historical background, but also to show that it is the context in which we make observations that defines the nature of the phenomenon being studied.
Because of the way it has been perceived throughout history, inflammation emerged as both a symptom and a mechanism. I cannot negate the importance of the medical or industrial perspectives on this topic, but I hope to show that it is also valid to view the topic from a physiological and biological perspective.
For this reason, I will not further discuss the definition of inflammation but show the delineations that it can be acquired when visualized within the context of developmental biology. From the outset, one must perceive that although inflammation and regeneration are phenomena that emerge from very different needs, it makes no sense to study them separately within modern biology.
Albeit legitimate, the medical interest in inflammation should not obscure the fact that, beyond the symptoms and magic bullets, inflammation can be seen in its physiological processes. Thus, there is a need to reconcile this theme with the development and construction of the animal form. More than their role in defense, inflammatory processes are part of the organism construction, and this can be illustrated with innumerable examples. The urodele amphibians are capable of regenerating their eyes, including delicate tissues such as the retina and lenses.
The surgical removal of the lenses from salamander newt eye triggers changes in the pigmented epithelial cells of the pupillary margin of the iris, which are capable of activating the cell cycle and differentiating into a new lens [ 18 ]. In a recently developed experimental model [ 19 ], it was shown that events typically described as immune inflammatory participate in the process of generating a new ocular lens.
When the lenses are pricked with a needle through the cornea, they degenerate by autophagy, a process mediated by dendritic cells, and the elimination of the damaged lenses allows for the regeneration of new tissue from the dorsal edge of the iris. Furthermore, if the animals receiving a transplant of these activated dendritic cells were previously splenectomized, this generative process was inhibited.
Therefore, the formation of new ocular tissues depends on processes that occur in a lymphatic organ, such as the spleen. This is an example of the generation of complex tissues mediated through immune-inflammatory processes; thus, this example illustrates the generative nature of inflammatory activity. The proposal that immune-inflammatory activity is associated with generative phenomena gained even more experimental support when framed within the context of comparative evolution [ 20 ].
The tunicates Urochordates form a sister group of the vertebrates and thus occupy a relevant taxonomic position for understanding the phylogenetic origin of vertebrates and the adaptive immunity as well. In this context, the species Botrylloides leachi is a well-studied animal model for understanding the emergence of immunological activities.
This species is a very common tunicate in the Mediterranean that exhibits the unique capacity of completely regenerating the adult organism from small vascular fragments. This phenomenon has been designated as whole-body regeneration.
Limb regeneration in amphibians: immunological considerations.
In a recent study, Rinkevich et al. However, we need to go further and recognize that the consequences of this admission are not trivial. The genesis of the biological form neither ends at birth nor resumes with disease. To understand this concept, it is necessary to view life as an incessant dynamic of transformation, like a Heraclitian fire [ 21 ].