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Trajectories of regenerating retinal axons in the goldfish tectum: I. A comparison of normal and regenerated axons at late regeneration stages.

To visualize and compare the intratectal path of normal and regenerated retinal axons, HRP was applied to localized sites in the dorsotemporal and dorsonasal retina in normal goldfish and in goldfish at 3-12 months after optic nerve section. The anterogradely labeled axons were traced in tectal whole mounts. In normal animals the axons were confined to the appropriate ventral hemitectum. Therein they ran in very orderly routes (Stuermer and Easter: J. Neurosci. 4:1045-1051, '84) and terminated in regions retinotopic to the labeled ganglion cells in the retina. The terminal arbors of dorsotemporal axons resided in the ventrorostral tectum and those of dorsonasal axons in the ventrocaudal tectum. In regenerating animals the terminal arbors also resided at retinotopic regions, where they sometimes formed two separate clusters. In contrast to normal axons, the regenerating ones traveled in abnormal routes through the appropriate and inappropriate hemitectum. From various ectopic positions, they underwent course corrections to redirect their routes toward the retinotopic target region. In their approach toward their target sites, dorsotemporal and dorsonasal axons behaved differently in that the vast majority of dorsotemporal axons coursed over the more rostral tectum whereas dorsonasal axons progressed into the caudal tectal half. This differential behavior of regenerating dorsonasal and dorsotemporal axons was substantiated by a quantitative evaluation of axon numbers and orientations.

Animals↗

Differential protein profile in the ear-punched tissue of regeneration and non-regeneration strains of mice: a novel approach to explore the candidate genes for soft-tissue regeneration.

Wound repair/regeneration is a genetically controlled, complex process. In order to identify candidate genes regulating fast wound repair/regeneration in soft-tissue, the temporal protein profile of the soft-tissue healing process was analyzed in the ear-punched tissue of regeneration strain MRL/MpJ-Fas(lpr) (MRL) mice and non-regeneration strain C57BL/6J(B6) mice using surface-enhanced laser desorption and ionization (SELDI) ProteinChip technology. Five candidate proteins were identified in which responses of MRL to the ear punch were 2-4-fold different compared to that of B6. Their corresponding genes were predicted using an antigen-antibody assay validated mass-based approach. Most of the predicted genes are known to play a role or are likely to play a role in the wound repair/regeneration. Of the five candidate proteins, the amount of the 23560 Da protein in the ear-punched tissue was significantly correlated with the rate of ear healing in six representative strains of mice, making it a good candidate for fast wound repair/regeneration. We speculate that the increased concentration of the 23560 Da protein in the wound tissue could stimulate the expression of various growth-promoting proteins and consequently speed up the wound repair/regeneration processes. Here, we have shown that examination of protein expression profile using SELDI technology, coupled with database search, is an alternative approach to search for candidate genes for wound repair/regeneration. This novel approach can be implemented in a variety of biological applications.

Animals↗

[Analytical study of Xenopus hindlimb regenerate with special reference to muscle regeneration].

Amputated hindlimbs of Xenopus laevis, develop various types of regenerates in relation with amputation level as well as stage development. The present experiments is an attempt to study the histological characteristics of Xenopus regenerations, i.e., rational changes of tissue components along the length of the regenerated part with special emphasis on the degree of muscle regeneration. Four types of regenerates were studied viz; a 4th toe obtained from a completely restored regenerated limb at 126 days after amputation of limb at base level in stage 51. An amputated limb with no external sign of regeneration of limb at thigh level in stage 60. A spike-shaped regenerate at 96 days after amputation of limb at shank level in stage 63. A spike-shaped regenerate at about 2 years after amputation of limb at shank level in stage 60. Cross sectional areas of muscle, skin gland, epidermis and cartilage in each of the four types of regenerates were measured with Image Analyzing Apparatus (VIP 121 CH, Olympus Co.). The relative area of each tissue was expressed as a percentage of the cross sectional area of the limb. The obtained values were plotted along the length of the regenerate. Digitiform regenerates were found to be more or less similar to the control limbs, i.e., provided joints and muscle, while the heteromorphic spike or rod shaped regenerates were simply provided with cartilaginous axial core without joint formation. Muscle area were reduced rapidly near the amputation area of these heteromorphic regenerates with no more continuation in the regenerated tissue. It is interesting to mention that percentage cartilage area of about 2 years old spike regenerate was higher than that of similar 96 days regenerate. In addition muscle regeneration was completely absent even in such an aged regenerate. The area showed fairly similar ratio irrespective of the external appearance of the regenerate. In 32 regenerates of which limbs were amputated at various developmental stages ranging between stage 51 and adult stage, the histological condition of muscle at the amputation site, were well observed. In all digitated types of regenerates even in those with reduced number of toes, muscles were found grown well in the regenerates. In heteromorphic regenerates without toe formation muscle did not usually regenerate. In few cases, however, a small mass of myoblastic like cells or small aggregation of differentiated muscle cells without any structural continuation with the stump muscles, were seen to develop in the midst of the regenerate.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

[Cytochemical analysis of muscle cell histones of Triturus cristatus limbs a) in normal regeneration, b) in regeneration arrested by r-irradiation, and c) in regeneration restored by implants].

The muscular cells of regenerating limbs and of limbs in which regenerative power is restored, show an important decrease in the amount of cytophotometrically detected histones. This decrease is owing to the arginine rich fraction and to the lysine rich fraction. The muscular cells of irradiated limbs show a decrease in the amount of histones. This decrease is owing only to the arginine rich fraction and continues after the thirieth day of irradiation and amputation.

Animals↗

Carcinogens on regeneration. Effects of N-methyl-N'-nitro-N-nitrosoguanidine and 4-nitroquinoline-1-oxide on limb regeneration in adult newts.

A microcrystal (ca 5 micrograms) of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 4-nitroquinoline-1-oxide (4NQO) was directly administered to the regeneration blastema on day 7 after amputation of a forelimb in the newt in order to analyze the effect of such potent carcinogenic substances on regeneration cells. Although neither MNNG nor 4NQO arrested regeneration completely, they caused great retardation of the regeneration cone formation followed by various abnormalities in the bony structures. Abnormal regenerants could be classified into the following four categories; (1) complete absence of both ulna and radius; (2) subregeneration or superregeneration of carpals and digits; (3) multiple disorganization of skeletal elements; (4) arrest of regeneration at the stage of regeneration cone. The polarity of regenerants developed after application of MNNG or 4NQO was very often shifted, during which the regeneration cone was always formed from the site where a microcrystal of the carcinogens was administered. The secondary regeneration initiated by reamputation of the regenerating limb, which had received the carcinogens at the early blastema stage, proceeded in the same way as observed in the case of a simple amputation. This suggested local and temporal effects of the carcinogens applied. Nevertheless, tumor formation has not induced in the newt limb so far. We can learn from these data that both MNNG and 4NQO only alter behavior of the newt regeneration cells without excreting their carcinogenic effects on them, and that the newt cells are highly resistant and stable against the above-mentioned carcinogens.

4-Nitroquinoline-1-oxide↗

Pattern of skeletal muscle regeneration after reautotransplantation of regenerated muscle.

Autotransplantation of rat extensor digitorum longus muscle results in initial myofibre degeneration and subsequent regeneration from precursor myosatellite cells. To determine what effect a reinjury would have on the regenerative response, in the present study, once transplanted and regenerated muscles were reinjured by reautotransplantation. In rats, four weeks after initial transplantation, when the regeneration was complete, the extensor digitorum longus muscle was transplanted again and the pattern of regeneration in reautotransplanted and once autotransplanted muscles was compared. Muscles were analysed 2, 4, 7, 14 and 30 days after autotransplantation and reautotransplantation. Both autotransplanted and reautotransplanted muscles underwent degeneration and regeneration; however, the pattern of regeneration in these two transplants was quite different. In autotransplants, a thin myogenic zone, marked by activated myoblasts, was first seen at 4 days. By 7 days the width of myogenic zone increased but still many degenerating myofibres were present in the central region of the muscle. By 14 days the muscle was filled with regenerated myotubes and myofibres. The reautotransplanted muscles underwent similar regenerative events; however, the rate of regeneration was considerably faster. The myogenic zone was apparent as early as 2 days and was much larger at 4 days, and by 7 days the entire muscle was filled with regenerated myotubes and myofibres which matured at later time intervals. Furthermore, the decrease in muscle weight in reautotransplanted muscles was not as much as that seen after autotransplantation. These findings reveal that not only is skeletal muscle capable of regeneration after a second injury, but the rate of this regeneration is much faster. This increased rate and recovery may be due to a conditioning effect of the first injury.

Animals↗

Expression of regeneration-associated cytoskeletal proteins reveals differences and similarities between regenerating organs.

The unique events which allow regeneration of an entire organ to occur are formation of a specialized wound epidermis and accumulation of progenitor cells (blastemal cells) at the amputated surface to form a blastema. In order to identify some of the molecular events underlying the early stages of the regenerative process which are either common to different systems or specific to one of them, we have investigated whether molecules which are induced in limb blastemas are also expressed in skin repair and during regeneration of other complex body structures (lower jaws, upper jaws, and tails). In addition, we have addressed the issue of the identity of progenitor cells during jaw development and regeneration by analyzing the expression of limb blastemal markers in the developing head and face. We have focused on cytoskeletal components, and particularly on the epidermal keratin NvKII, the simple epithelial keratins 8 and 18 and 22/18, because they are among the few molecules which have been shown to be associated with regeneration in the limb and may play significant roles in various developmental processes. Some important findings emerge from this study: 1) Expression of the epidermal keratin NvKII, unlike that of its mammalian homologue K6, is not simply induced in response to wounding, but is associated with regeneration of specific organs. In fact, NvKII is expressed in regenerating limbs and tails, but not in upper or in lower jaw regenerates, demonstrating the existence of molecular differences in the composition of the wound epidermis in these systems. This, together with the fact that NvKII mRNA is regulated by retinoic acid, which differentially affects patterning of limbs and jaws, argues for distinct inductive abilities of the wound epidermis in different organs. 2) In contrast to the differential expression of the epidermal keratin NvKII, the regeneration-associated cytoskeletal molecules identified in limb blastemal cells are expressed in a similar fashion in jaw and tail blastemas. Therefore, it appears that similar cellular events lead to the establishment of an actively proliferating population of progenitor cells from the stump of different organs. Finally, the mesenchyme of the facial rudiments, unlike that of developing limb buds, expresses simple epithelial keratins. Thus, it appears that mesenchymal progenitor cells of developing and regenerating jaws are alike in regard to their intermediate filament content, and this may be related to nerve-dependent growth control of progenitor cells in different developing and regenerating systems.

Animals↗

Leg regeneration in the cockroach, Blatella germanica. II. Regeneration from a non-congruent tibial graft/host junction.

The interactions occurring between host and graft leg epidermis at a non-congruent junction were studied in the cockroach, Blatella germanica. Graft and host tibia were cut perpendicular to the proximal-distal axis and two heteropleural combinations were used to reverse separately the two transverse axes of the graft relative to the host. Use of dark and light cuticle colour mutants gave a good indication of the graft or host origin of regenerated structures. Graft/host junctions regenerated segmented structures in various spatial arrangements, always comprising two copies of all structures distal to the level of the junction. It is concluded that the categories--two separate laterals, double lateral, completely and partially autonomous regeneration--reflect two processes. (i) If the graft tarsus is removed, graft and host may not heal together and interact, but form autonomous regenerates lying in mirror-image symmetry separating original graft and host levels. (ii) If interaction occurs between graft and host (or their developing autonomous regenerates) two laterals of dual origin are produced, one from each point of transverse axis incongruity. These laterals may secondarily fuse together to form a double structure originating from a point of congruity. The orientation and composition of the component tarsi of the double structure depend on the site of origin and the extent to which the two laterals fuse. It is argued that the four 'faces' and two 'transverse axes' of the leg are merely descriptive terms. A new model is developed whereby lateral regeneration arises directly from the circumferential organisation of the leg epidermis. Previous work has shown that position is specified continuously around the circumference, and that intercalary regeneration occurs by the shortest route between confronted positions. After reversal of one 'transverse axis' the shortest route between confronted graft and host positions is different on the two sides of each of the two points of 'axis' incongruity, and at these points the two halves of a complete circumference are formed. These lateral circumferences, like the terminal circumference exposed by amputation, cannot heal over by intercalary regeneration, and this leads to regeneration of distal structures. The model accounts for lateral regeneration after reversal of both 'transverse axes' by 180 degrees rotation of a homopleural graft. The possibility is discussed that there may be clonal restrictions on the circumferential positions which the progeny of a cell may occupy.

Animals↗

Detection of insulin receptors in newt liver and forelimb regenerates and the effects of local insulin deprivation on epimorphic regeneration.

Previous in vivo and in vitro studies indicate that insulin is required in adult newt forelimb regeneration. The objectives of the current study were 1) to detect insulin receptors in the liver (a classical target organ for insulin) and once verified, detection of insulin receptors in the adult newt forelimb regenerate; and 2) to determine whether locally implanting insulin antibody-soaked hydrolyzed polyacrylamide beads (hypa beads) into a regenerating forelimb blastema would affect its growth and/or differentiation. The results show that insulin receptors are detectable in the plasma membranes of newt liver and forelimb regenerates. Radioiodinated bovine insulin binding is time-dependent and specific; unlabeled bovine insulin competes with labeled insulin for binding to NLPM more effectively than does insulin-like growth factor-I, guinea pig insulin, and glucagon. The newt hepatic insulin receptor binds insulin with high affinity (1.1 nM-1) and low capacity (63 +/- 8 fmoles/mg). The size of the alpha subunit of the newt insulin receptor is 130 kDA and that of the beta subunit is 95 kDa. The beta subunits undergo insulin-stimulated phosphorylation in response to insulin. An autoantibody against the human insulin receptor recognizes the newt receptor protein. Insulin receptors are also detectable in 15 and 20 day newt forelimb regenerates. Specific immunogold labelling of the receptor-bound antibody appears to be restricted to the cellular processes of the regenerate. Implanting hypa beads soaked with purified insulin antibody into regenerating adult newt forelimbs results in abnormal growth and differentiation of the regenerates, confirming that insulin plays an essential role in adult newt forelimb regeneration.

Affinity Labels↗

Failure of regeneration of the steatotic rat liver: disruption at two different levels in the regeneration pathway.

Hepatic resection or transplantation in patients with fatty liver is associated with increased morbidity and mortality. The regenerative capacity of fatty livers after major tissue loss is unknown. Interleukin 6 (IL-6) is a potent inducer of hepatic regeneration in normal and ischemic livers. Therefore, we studied hepatic regeneration at day 1, day 2, and day 4 in a model of 70% hepatectomy in obese and lean Zucker rats, and obese Zucker rats pretreated with recombinant interleukin 6 (rIL-6). The mitotic cycle in hepatocytes was investigated by 4 different markers of regeneration representing distinct phases of mitosis (proliferating cell nuclear antigen [PCNA] = G(1) phase, bromodeoxy uridine [BrdU] = S phase, mitotic index, and regenerated liver weight = M phase). Obese Zucker rats had significantly decreased regenerative capacity compared with lean Zucker rats (PCNA, BrdU, mitotic index, regenerated liver weight) at days 1 and 2 after surgery. Four days after resection fatty animals showed an increase in the mitotic index indicating a delay of regeneration in steatotic livers. Animal survival after 70% hepatectomy was significantly decreased in obese rats compared with lean animals. Pretreatment of obese animals with rIL-6 normalized PCNA expression (G(1) phase) in steatotic hepatocytes but failed to increase DNA synthesis (BrdU, S phase), mitosis (mitotic index and regenerated liver weight, M phase), and animal survival. These results indicate major impairment of hepatic regeneration in steatotic livers. Two different blockages of regeneration must be present, one rIL-6 sensitive, at the level of IL-6 or upstream, and a second, rIL-6 resistant, at the level of G(1)/S-phase transition.

Animals↗

Peripheral nerve regeneration through alginate gel: analysis of early outgrowth and late increase in diameter of regenerating axons.

Our previous study revealed that alginate gel cross-linked with covalent bonds promoted peripheral nerve regeneration in the cat and rat. The present study analyzed nerve regeneration through alginate gel in the early stages within 2 weeks and the late stages up to 21 months after implantation. Four days after surgery, regenerating axons grew without Schwann cell investment through the partially degraded alginate gel, being in direct contact with the alginate without a basal lamina covering. Numerous mast cells infiltrated into the alginate. One to 2 weeks after surgery, regenerating axons were surrounded by common Schwann cells to form small bundles, with some axons at the periphery being partly in direct contact with alginate. At the distal stump, numerous Schwann cells had migrated into the alginate 8-14 days after surgery. They had no basal laminae. The diameter of regenerated myelinated fibers was small (approximately 1 micro m) at 8 weeks, but increased in diameter, having a distribution pattern similar to that of normal nerve 21 months after surgery. Much better nerve regeneration was found in alginate gel-, than collagen sponge-, and fibrin glue-implanted distal stump 12 months after surgery. These results indicate that alginate gel has good biocompatibility for regenerating axon outgrowth and Schwann cell migration, and that regenerated fibers can have a diameter as thick as that of normal fibers in the long term. Alginate gel is a promising material for use as an implant for peripheral nerve regeneration.

Alginates↗

Liver regeneration 4: transcriptional control of liver regeneration.

Determining what factors are responsible for initiating regeneration following partial hepatectomy or toxic damage, and how the liver maintains differentiated functions while the hepatocytes are undergoing cellular proliferation are central issues in understanding the molecular bases of liver regeneration. Examination of the transcriptional milieu in the regenerating liver provides clues to the answers to these questions. Growth factor-generated intracellular signals that trigger liver regeneration result in activation via posttranslational modifications of latent, normally inactive transcription factors that preexist in the liver. Two transcription factors that are activated by this mechanism include posthepatectomy factor/nuclear factor-kappa B) and Stat3. Because cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-l (IL-1), and IL-6 can induce these factors in the liver, the finding of activated Stat3 and PHF/NF-kappa B suggests that these cytokines may play a role in some aspects of growth regulation during liver regeneration. Rapidly induced transcription factors, Stat3, PHF/NF-kappa B, and others are responsible for activation of the primary growth response or immediate-early genes, which play a role in regulating later phases of cell growth in regenerating liver and other mitogen-activated cells. Immediate-early genes encode many members of diverse transcription factor families including the Jun-Fos-LRF-1, nuclear receptor, and myc families to name a few. In this way a transcriptional cascade is established during the G1 phase of liver regeneration. Coexisting with these induced factors are liver-specific transcription factors such as the CAAT enhancer binding proteins and hepatocyte nuclear factors, which may interact with growth-induced factors to help the liver maintain metabolic homeostasis during regeneration. As a result the liver is able to accomplish the goals of reestablishing its mass while it maintains its functional capacity during regeneration.

Animals↗

Insulin receptors in Xenopus laevis liver and forelimb regenerates and the effects of local insulin deprivation on regeneration.

As forelimb regeneration in Xenopus laevis is mainly a cell proliferative event which results in a spike-shaped appendage, we set out to examine the possibility that insulin is a growth-promoting factor in this process. The objectives were 1) to detect the presence of insulin receptors (IRs) in the liver (a specific target organ for insulin) and IRs in the forelimb regenerates of X. laevis, 2) to determine whether the receptor is similar to IRs identified in other organisms, and 3) to absorb insulin locally by implanting anti-insulin antibody-soaked hydrolyzed polyacrylamide beads into regenerating forelimb outgrowths in order to assess the effects of insulin deprivation on regeneration. The results show that IRs are present in Xenopus liver plasma membranes (XLPM) as well as in plasma membranes of 21 day forelimb regenerates. Insulin binding to this receptor is time-dependent and specific, as unlabeled bovine insulin competes with radioiodinated insulin for binding to XLPM more effectively than insulin-like growth factor-I, guinea pig insulin, or glucagon. Scatchard analysis of insulin binding to XLPM describes a two binding site receptor possessing a low affinity (0.16 nM-1), high capacity (3.2 +/- 0.9 pM/mg) binding site and a high affinity (2.7 nM-1), low capacity (0.5 +/- 0.3 pM/mg) binding site. The holoreceptor has a molecular mass of 380 kDa. The reduced receptor has subunits of 130 kDa and 95 kDa. The 95 kDa subunit undergoes autophosphorylation following insulin stimulation. Implantation of hydrolyzed polyacrylamide beads, saturated with anti-insulin antibody, into regenerating Xenopus forelimbs significantly impeded development of the regenerates and, therefore, demonstrates that insulin is required for growth of Xenopus forelimb regenerates.

Affinity Labels↗

Spontaneous regeneration of the corticospinal tract after transection in young rats: collagen type IV deposition and astrocytic scar in the lesion site are not the cause but the effect of failure of regeneration.

In young rats the corticospinal tract regenerated after a single transection of the spinal cord with a sharp blade, but regeneration failed if the transection was repeated to make a more traumatic injury. To identify cells and associated molecules that promote or impede regeneration, we compared expression of collagen type IV, glial fibrillary acidic protein (GFAP), and vimentin immunoreactivity (IR) at the lesion sites in combination with anterograde axonal tracing between animals with two types of transection. Axonal regeneration occurred as early as 18 hours after transection; regenerating axons penetrated vessel-like structures with collagen type IV-IR at the lesion site, while reactive astrocytes coexpressing GFAP- and vimentin-IR appeared in the lesioned white matter. In contrast, when regeneration failed astrocytes were absent near the lesion. By 7 days sheet-like structures with collagen type IV-IR and astrocytic scar appeared in the lesioned white matter and persisted until the end of the observation period (31 days). On the basis of their spatiotemporal appearance, collagen type IV-IR sheet-like structures and the astrocytic scar follow, rather than cause, the failure of regeneration. The major sign, and perhaps cause, of failure of axonal regeneration is likely the prolonged disappearance of astrocytes around the lesion site in the early postinjury period.

Animals↗

Implantation of MNNG crystals into a Triturus intact limb affects mitotic and labeling indices, regeneration rate, and morphogenesis in the contralateral, regenerating limb.

Experimental administration of chemical carcinogens to various mammals is highly effective in inducing malignant tumors. In contrast, treatment of regeneration-competent animals even with much higher doses of the same drugs only exceptionally leads to tumor-like growth. Usually, carcinogenic materials implanted or injected into a regenerating limb of urodele amphibia interfere with the regenerative process and frequently lead a). to growth retardation or arrest of regeneration, b). to development of a great variety of abnormal regenerates, and c). to generation of accessory, limb-like structures. Autonomous or experimental incidence of carcinogenesis is extremely low in animals endowed with strong regenerative capabilities. Of exceptional biological significance is the fact that such induced tumors usually regress spontaneously. This unique property of the regeneration-competent animals to resist carcinogenesis provides opportunities to compare non-cancerous alterations in the differentiated state of adult cells to those occurring in neoplasia. The mode of action of the chemical carcinogens on limb regeneration has not yet been clarified with certainty at the cellular and the molecular level. Several scientists claim that the above-mentioned effects might be attributed to local toxic influences of the drugs; therefore the present study was designed to investigate whether the administration of the carcinogen MNNG can affect cell proliferation, histogenesis, and morphogenesis at a region distant from the site of its implantation, even after a relatively long time period. To this end, 40 animals of the species Triturus cristatus had their right hindlimb surgically removed at the distal zeugopod. Then, a small microcrystal (approximately 5 micro g) of MNNG was inserted under the ventral aspect of the skin of the left tarsus in 20 of these animals (groups T and A; see below). Two months later, nine of the MNNG-treated animals were injected intraperitoneally with tritiated thymidine. After 2 h, six of these animals had their right hindlimb amputated at the distal zeugopod, whereas the rest were left to regenerate. The results were evaluated by camera lucida drawings, clearing in methyl benzoate, classical histology, and autoradiography. It was revealed that administration of MNNG at a somatic region (left hindlimb) reduces DNA synthesis and mitosis at a distant place (right hindlimb) even 2 months after MNNG implantation. Despite this, the rate of limb elongation is not substantially reduced. Classical histology revealed normal tissue structure throughout. All regenerated limbs displayed several teratogenic abnormalities.

Amputation, Surgical↗