The possible effect of the mechanical factor on the completeness of the regeneration of the spinal skin in mice

In mouse tissues, regenerates forming in the place of full-thickness skin wounds on the back, were exposed to dosed mechanical injuries. As a result the structure of final regenerates in some animals differed from the scar, that is usually formed after healing of this type of wound.     

Reorganization of skin scars on the back of rats]

The method of dosed mechanical injuries (DMI) when applied repeatedly to the mature skin scars in the rat's backs leads to their reduction and transformation into the regenerates having the structure resembling that of the normal skin. The more is the number of applications of DMI--the more is the amount of transformed scars.     

Ability of various injuries to promote resorption of denervated, nerve independent regenerates of adult newts

Young blastemas of the newt resorb if the limb is denervated, and are thus called "nerve dependent". Late bud and later stage regenerates are termed "nerve independent" because, while denervation inhibits their growth, they proceed through differentiation to form a normally patterned regenerate. Schotté and Liversage ('59) found that reamputation of a denervated nerve independent regenerate causes it to resorb. The present study asked whether injuries of varying severity are equally effective at promoting resorption. Newt forelimbs were amputated through the mid-radius/ulna. At nerve independent stages, the regenerates were denervated and injured in one of a variety of ways, then monitored for signs of resorption. Reamputation of the tip or incisions which created large gaps in the wound epidermis promoted resorption in 77-90% of the cases. Histology showed that the tissue removed by tip reamputation was a small proportion of the entire regenerate, suggesting that blastema resorption is not determined simply by the number of cells directly affected by the injury. Pin prick injuries, which created small disruptions of the wound epidermis, never caused resorption. Devascularization, caused by severing the brachial artery, promoted resorption in 17% of cases. These results are not consistent with the hypothesis that avascularity is a major causative factor in nerve dependence.     

Histological and histochemical studies on the nervous influence on minced muscle regeneration of triceps surae of the rat.

The degree of minced rat muscle regeneration in the absence of nerve fibers was compared with that of normal regenerates between one and 270 days postoperatively. Up to around 30 days, the number of muscle fibers and their morphology were comparable in both normal innervated and denervated regenerates; both showed clear cross striations and peripherally located nuclei. Histochemically, SDH and myofibrillar ATPase (pH=9.4) reactions were positive, but there were no typical signs of fiber types in either case of regeneration. The only consistent difference in the early period was the smaller fiber cross sectional areas in denervated regenerates than in innervated ones. Starting about 40 days, the muscle fibers in innervated regenerates became differentiated into different fiber types (fast-twitch-oxidative-glycolytic, FOG., fast-twitch-glycolytic, FG., slow-twitch-oxidative, SO.) but there were no such activities in denervated regenerates, although their SDH and myofibrillar ATPase reactions remained positive for a long time. Degenerating muscle fibers could no longer be identified in innervated regenerates. In the denervated regenerates, however, muscle fibers underwent atrophic or degenerative changes and were replaced by connective tissue. The complete disappearance of muscle fibers varied with individual regenerates. In some cases, it occurred about 90 days and in others, traces of muscle fibers could still be seen as late as 150 days postoperatively. Thus, nerves seem to be important primarily in the late phase of regeneration; namely, differentiation of fiber types and maintenance of the structural integrity of muscle fibers.     

Regeneration of symmetrical forelimbs in the axolotl,Ambystoma mexicanum

Surgically constructed symmetrical double-anterior and double-posterior upper forelimbs of the axolotl were amputated immediately after surgery. Double-anterior limbs either failed to regenerate or formed single digits or spikes. Double-posterior limbs formed symmetrical double-posterior regenerates in 60% of the cases, thus extending the previous finding that the amount of distal transformation in surgically constructed double-half limbs is inversely proportional to the time between grafting and amputation (Tank and Holder, 1978). When these symmetrical regenerates were amputated through the forearm region, all but one formed a symmetrical secondary regenerate. The majority of the secondary regenerates had a larger number of digits than did their corresponding primary regenerates. Reamputation of the secondary regenerates resulted in symmetrical tertiary regenerates, and the majority of these also had a larger number of digits than did their corresponding primary regenerates. The results are compared to those of Slack and Savage (1978a, b) on embryonically derived double-posterior limbs and they are discussed in terms of a formal model for distal transformation (Bryant and Baca, 1978).     

Effects of hypophysectomies and thyroxine replacement upon the initiation of tail regeneration in the lizard, Anolis carolinensis

A histological evaluation of the effects of hypophysectomy and throxine therapy in young tail regenerates was carried out in the small iguanid lizard, Anolis c. carolinensis. Hypophysectomy caused a delay but did not inhibit blastema formation. The growth of the ependyma into the wound region was delayed in hypophysioprivic regenerates by about a week. Growth and differentiation of hypophysioprivic regenerates after blastema formation was variable, ranging from virtually no growth to the formation of a differentiated but very small protuberance. However, actual tail elongation was inhibited by hypophysectomy. In those hypophysioprivic regenerates that did show signs of differentiation, muscle groups were poorly defined, scanty in appearance and not as well differentiated as the cartilage tube. Thyroxine treatment in the young hypophysioprivic regenerates stimulated normal growth and normal appearance and differentiation of promuscle and procartilage aggregates as well as the growth of the ependymal tube into the blastema.     

Interruption of proecdysis by autotomy of partially regenerated limbs in the land crab, Gecarcinus lateralis

In the land crab, Gecarcinus lateralis, autotomy of partially regenerated limbs before a critical stage in the premolt period results in (1) a very rapid decrease in the serum ecdysone titer, (2) a delay in the growth of partial regenerates remaining on the animal, (3) a delay in the deposition of gastroliths, and (4) a delay in cytological changes in the epidermis. Serum ecdysone titers remain low while new limb regenerates form at the sites of those removed. Ecdysone titers rise when these secondary regenerates complete basal regeneration. Premolt events, which had ceased at the time of autotomy of the partial regenerates, resume their normal patterns of development when ecdysone titers reach the level present in the serum at the time of this interruption. We propose that the effect of autotomy before a critical period is to reinitiate a normal proecdysis. The same pattern of events occurs following autotomy of partial regenerates of crabs without eyestalks, suggesting that the decrease of serum ecdysones is brought about by some mechanism other than changes in the titer of the molt inhibitory hormone.     

Regeneration of Haller's sensory organ in two species of hard ticks of the genus Haemaphysalis (Acari: Ixodidae)

A study of Haller's organ regeneration in nymphs and adults of Haemaphysalis turturis and parthenogenetic females of H. longicornis, from which the forelegs had been amputated during the previous instar, revealed structural changes in regenerated organs. The adult regenerates reestablished atavistic structural features, while the nymphal regenerates retained larval features, which is typical of regenerates of two other genera examined previously (Ixodes and Hyalomma). Data on regeneration of Haller's sensory organ testify to an ancient character of the genus, standing closely to the base of the phylogenetic tree of hard ticks.     

Morphogenetic interactions between rotated skin cuffs and underlying stump tissues in regenerating axolotl forelimbs

Rotation of skin cuffs 180° around the longitudinal axis of the underlying tissues in the axolotl forelimb results in a high percentage of multiple regenerates after amputation through the rotated skin. Similar results occur after rotation of only the anteroposterior (A-P) axis of the skin. Rotation of only the proximodistal (Pr-Ds) axis of the skin results in normal regenerates whereas dorsoventral (D-V) axial skin rotation results in single regenerates with some disturbances in symmetry. Rotation of anterior or posterior half cuffs of skin produces results similar to those obtained after A-P rotation of full skin cuffs, and rotation of dorsal or ventral skin halves duplicates the results obtained by rotating full skin cuffs about the D-V axis. Skin cuffs rotated for periods from 6 months to over 2 years before amputation are also capable of causing multiple regenerates to form. No significant difference in the percentage of multiple regenerates was seen after skin rotation and limb amputation through shoulder, upper arm, and forearm levels. X-Radiation (4000 r) of either the skin or underlying tissues before skin rotation resulted in single regenerates after amputation. If a strip of normal skin was turned perpendicularly to the long axis of the irradiated underlying stump tissues, the regenerative response was blocked. In some of the above experiments, regenerates with longitudinally duplicated upper arm and forearm segments appeared. It is postulated that normally both the skin and the underlying limb tissues can influence morphogenesis during regeneration and that they work in harmony. In contrast, rotated skin and the underlying tissues each exert a morphogenetic influence upon the regenerating limb, and the regenerate is not able to integrate these disharmonious influences. This is reflected in the highly abnormal morphology of the regenerates. The nature of the morphogenetic influence disrupted by skin rotation is not yet known.     

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)     

Pattern-deficient forelimb regeneration in adult bullfrogs

This study was designed to test the ability of adult bullfrogs (Rana catesbeiana) to regenerate forelimbs, both with and without various experimental treatments. Distal humerus-level forelimb amputations provided with additional deviated (sciatic) nerve and/or repeated soft-tissue injury exhibited considerable outgrowth. However, control sham-operated forelimbs also produced regenerates with comparable frequency, size, and morphological complexity. The lengths of the regenerates ranged from 0.4 to 2.6 cm, representing an outgrowth of 10-65% of the portion removed by the distal humerus amputation plane; some regenerates exhibited an external morphology indicative of digitlike structures. Some outgrowths were flexible but only one was capable of independent movement. Victoria Blue staining of whole regenerates revealed a variety of internal cartilage elements. Staining showed a single solid mass of cartilage in some regenerates while others had several individual and variably shaped cartilages projecting distally. Histological analysis also revealed the presence of connective tissue, striated muscle, and abundant nerve fibers in addition to the individual cartilage elements. We have tentatively termed these responses pattern-deficient regeneration.     

Proportion regulation in the planarian,Dugesia tigrina following regeneration of structures

Planarian regenerates with abnormal body proportioning were followed after structure formation in order to determine if body proportion will ‘normalize’ over time. Results show proportioning will occur following structure formation since the pharynx in regenerates with proportionally larger heads and prepharyngeal area moved anteriorly over time. This occurred regardless of whether regenerates were provided with a normal feeding regime which allowed for an increase in body area or if they were on a maintenance diet which did not permit growth. An examination of mitotic indices did not demonstrate significant differences in the level of mitotic activity between pre- and postpharyngeal regions. It is concluded that the ‘normalizing’ of proportion in these abnormal regenerates does occur, but the process by which it occurs is not solely explained through normal growth. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effects of rotation and positional change of stump tissues upon morphogenesis of the regenerating axolotl limb.

Rotation of a skin cuff 180° around the proximodistal axis of the upper arm in the axolotl results in the formation of multiple regenerates in about 80° of cases after amputation of the limb through the rotated skin. Rotation of the dermis or the flexor and extensor muscles folowed by amputation produced similar percentages of multiple regenerates. Rotated bone produced no abnormalities, and rotated stump epidermis was minimally effective in stimulating multiple regeneration. A thin strip of normally oriented skin interposed between a rotated skin cuff and the amputation surface blocks the morphogenetic effect of the rotated stump skin whereas removal of the normal skin between a rotated proximal skin cuff and the amputation surface allows the formation of a low percentage of multiple regenerates. Gross rotation of stump tissue components can be broken down into axial rotation per se and positional dislocation. Experiments conducted upon skin and muscle have shown that positional dislocation along the anteroposterior axis rather than axial rotation is the manipulation that leads to the formation of multiple regenerates. The first morphological indication of multiple regeneration is the appearance of a triaxial apical ridge on the blastema. Subsequently, digits form along the apical ridges.     

Vitamin A induced bilateral asymmetries in triturus forelimb regenerates

After amputation of both forelimb of young, postmetamorphic Triturus alpestris through the middle of the zeugopod, the left and right regenerates grew asynchronously. As a result of oral administration of vitamin A palmitate (250 IU/gbw/day), from the 4th to the 13th dpa, the growth rate of the right regenerates was relatively higher than that of the left ones. Moreover, the percentage of skeletal abnormalities as well as proximodistal duplications (proximalizations), induced by vitamin A, was nearly twice as high in the right regenerates as compared to their left counterparts.     

The relationship of morphogenetic potency of tobacco tissue culture and its cytogenetic features

Two strains of tobacco tissue cultures were investigated cytogenetically, their morphogenetic potency was revealed and the plant regenerates were analyzed. Strain I with less pronounced deficiency of nuclei was more capable to differentiation and organogenesis than the heterogenous strain II. The morphological variability of vegetative and reproductive organs of plant regenerates was described. The variations were characterized by high level of aberations during meiosis and unstable aneuploidy.     

Changes in polyamine content during limb regeneration in adult Xenopus laevis

Polyamine contents in the regenerates were determined at various stages after amputation of the forelimbs of the adult female Xenopus laevis. Putrescine, spermidine, spermine, and sym-homospermidine were detected in all the specimens examined. Cadaverine was detected only in a limited number of samples. At 5 days after amputation of forelimbs, well before the formation of regenerates, the putrescine content in the stump tissues increased, followed by the increase in spermidine content. The putrescine level in the forelimb regenerates was highest between 30 and 50 days after amputation, and then decreased. The spermidine concentration in the regenerates was about 20 times greater than that in intact forelimbs all throughout the experiments. The concentration of spermine was initially lower than that of both putrescine and spermidine and further decreased soon after amputation. The concentration of sym-homospermidine was originally very low and increased slightly during regeneration. The significance of these results, with respect to the function of polyamines in forelimb regeneration of Xenopus laevis, is discussed.     

Analysis of the progress of hormone-receptor amplification during differentiation in regenerating planarians.

Planarians developing from isolated regenerates, when pretreated with epinephrine at different periods of their second regeneration, show a higher rate of sugar uptake than the controls, which were not treated with epinephrine. The hormone receptor development can be influenced most effectively by pretreatments performed during the 1--3 and 6--9 day periods, though some effect can be evoked at any time in the course of the second regeneration. The head regenerates developing from the anterior end of the original midpiece (and regenerating tails the second time, B1) were found to be most sensitive, while the similar regenerates developing from the hindpiece of the original planarian (C) showed the weakest reactivity. The experiments provide further evidence that the hormone receptors' activity can be amplified in the course of regenerative development, leading thus--at a later stage--to a more intense sugar uptake response of the cells.     

Autoradiographic evaluation of vitamin A effects on Triturus alpestris blastema cells

Autoradiographic studies on Triturus alpestris regenerates treated with vitamin A palmitate for 4, 7, 11 and 14 days after amputation, reveal a considerable diminution in the percentage of cells duplicating DNA, as judged by measurements of labelling indices. Based on previous observations, that vitamin A promotes DNA-synthesis, mitosis and growth of regenerates, we conclude that the lower labelling indices calculated are due to an acceleration of S-phase processes, resulting in a concomitant reduction in its duration.     

Morphogenetic changes during newt tail regeneration under changed gravity conditions

Gravity-dependent shape alterations in newt tail regenerates are described, which were previously noticed in experiments onboard satellites Foton M2, M3 and in corresponding laboratory controls. Laboratory conditions were developed that allow reproducing this phenomenon persistently in the adult newts Pleurodeles waltl (Michahelles, 1830). The newts kept in an aquarium (in partial weightlessness) after 1/3 tail amputation developed normal lanceolate regenerates, while those that stayed on a moist mat (exposed to greater gravity than in aquarium) developed curved tail regenerates. Dynamics of the shape alterations were described using computer morphometric analysis. The curve was shown to develop at stage III of regeneration and to be caused by bending of the developing axial structures: the ependymal tube and the cartilage rode. Cellular processes were described that accompany the tail shape changes, such as cell migration and formation of dense aggregates. Unequal proliferation throughout the wound epidermis and blastema was revealed using BrdU assay. Proliferation increased within dorsal and apical regions of the regenerates in the newts kept on the mat cell compared with the aquarian animals.     

Asymmetric protoplast fusions between wild species and breeding lines of potato – effect of recipients and genome stability

 The objective of this study was to investigate if in asymmetric protoplast fusion experiments the ploidy of the recipient line (di-haploid and tetraploid) has an influence on the extent of the asymmetry of the regenerating fusion products. Nineteen different experiments with the wild species Solanum bulbocastanum and Solanum circaeifolium as donors (irradiated with 210 Gy) and different breeding lines (di-haploid and tetraploid) were carried out. The degree of genome elimination was determined by measuring the relative DNA content using flow cytometry. The data showed that the loss of DNA in hybrid plants was significantly higher for 4x, compared to 2x, plants as recipients. In addition, the stability of asymmetry in the fusion products was studied. For this purpose differences in asymmetry in individual shoots originating from the same callus were analysed. A large variation in the DNA content of individual shoots was detected. Of the 4x to 6x shoots 44% had the same DNA content as another shoot originating from the same callus, 19% had a DNA content between 4x and 6x but different from any other analysed shoot originating from the same callus, 2% were chimeras and 35% had a completely different DNA content (eutetraploid, euhexaploid, eupolyploid or asymmetric with a ploidy level above 6x). RFLP-analysis with single-copy probes of 12 regenerates from six calli (two regenerates per callus) confirmed the assumption that the different regenerates of one callus originate from the same single cell. The analysis of selected regenerates cultivated for a period of more than 1 year demonstrated that the genome of asymmetric regenerates might change during cultivation. Received: 30 April 1998 / Accepted: 24 July 1998