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Caudal autotomy and regeneration in lizards.

Caudal autotomy, or the voluntary self-amputation of the tail, is an anti-predation strategy in lizards that depends on a complex array of environmental, individual, and species-specific characteristics. These factors affect both when and how often caudal autotomy is employed, as well as its overall rate of success. The potential costs of autotomy must be weighed against the benefits of this strategy. Many species have evolved specialized behavioral and physiological adaptations to minimize or compensate for any negative consequences. One of the most important steps following a successful autotomous escape involves regeneration of the lost limb. In some species, regeneration occurs rapidly; such swift regeneration illustrates the importance of an intact, functional tail in everyday experience. In lizards and other vertebrates, regeneration is a highly ordered process utilizing initial developmental programs as well as regeneration-specific mechanisms to produce the correct types and pattern of cells required to sufficiently restore the structure and function of the sacrificed tail. In this review, we discuss the behavioral and physiological features of self-amputation, with particular reference to the costs and benefits of autotomy and the basic mechanisms of regeneration. In the process, we identify how these behaviors could be used to explore the neural regulation of complex behavioral responses within a functional context.

Animals↗

Inhibition and axial deviation of limb regeneration in the newt by means of a digit implanted into the amputated limb.

This research was designed to follow up the observation of Thornton and Kraemer ('51) that regressed, denervated limbs of Ambystoma larvae will not regenerate upon reinnervation if all digits on the limbs were not completely resorbed. The object of this experiment was to determine whether the presence of an apical structure, protruding past the amputation surface, would affect the regenerative process. Both forearms of adult newts were amputated midway between the elbow and the wrist. One limb served as a normal regeneration control, and in the other limb the third digit from the removed hand was implanted in place of the removed radius, so that the three distal phalangeal segments protruded past the plane of amputation. Blastema formation in the experimental limbs was delayed by several weeks as compared with control limbs. Approximately one third of the experimental limbs did not regenerate. The regenerates that did form were strongly deviated (45-90 degrees) radially from the longitudinal axis of the limb. Experimental analysis showed that the delay in regeneration is due largely to the projecting part of the digit. The radial deviation of the regenerates is not due to the digital implant, but rather to the removal of the radius. Trauma alone does not account for this phenomenon.

Ambystoma↗

Poly(L-Lactide) microfilaments enhance peripheral nerve regeneration across extended nerve lesions.

After injury, axonal regeneration occurs across short gaps in the peripheral nervous system, but regeneration across larger gaps remains a challenge. To improve regeneration across extended nerve defects, we have fabricated novel microfilaments with the capability for drug release to support cellular migration and guide axonal growth across a lesion. In this study, we examine the nerve repair parameters of non-loaded filaments. To examine the influence of packing density on nerve repair, wet-spun poly(L-Lactide) (PLLA) microfilaments were bundled at densities of 3.75, 7.5, 15, and 30% to bridge a 1.0-cm gap lesion in the rat sciatic nerve. After 10 weeks, nerve cable formation increased significantly in the filament bundled groups when compared to empty-tube controls. At lower packing densities, the number of myelinated axons was more than twice that of controls or the highest packing density. In a consecutive experiment, PLLA bundles with lower filament-packing density were examined for nerve repair across 1.4- and 1.8-cm gaps. After 10 weeks, the number of successful regenerated nerves receiving filaments was more than twice that of controls. In addition, nerve cable areas for control groups were significantly less than those observed for filament groups. Axonal growth across 1.4- and 1.8-cm gaps was more consistent for the filament groups than for controls. These initial results demonstrate that PLLA microfilaments enhance nerve repair and regeneration across large nerve defects, even in the absence of drug release. Ongoing studies are examining nerve regeneration using microfilaments designed to release neurotrophins or cyclic AMP.

Actin Cytoskeleton↗

Fibroblast growth factor effects on peripheral nerve regeneration in a silicone chamber model.

We have developed a silicone nerve regeneration chamber that is partitioned into two compartments by a strip of nitrocellulose paper. The modified two-compartment chamber allows the investigation of the effects on rat sciatic nerve regeneration of trophic or growth factors that are initially bound to the nitrocellulose partition. In this study we compared the effects of untreated nitrocellulose, a siliconized nitrocellulose strip, and a strip that had been soaked in a basic fibroblast growth factor (FGF) solution. FGF is a known angiogenic factor and a mitogen for endothelial cells, fibroblasts, and Schwann cells. All of these cell types are present in the peripheral nerve. In vitro analyses, using 3T3 cells as test cells, showed that some of the bound FGF remained active on the nitrocellulose paper for at least 8-10 days. In vivo experiments, examined at 16 days post-implantation, revealed that spatial migration of all cellular elements (perineurial-like cells, vasculature, and Schwann cells) across the chamber gap was slower with untreated nitrocellulose strips than with siliconized strips but was most advanced with FGF-treated ones. Most striking was the well-developed vascular arborization of the regenerate within the FGF chambers. Histologic sections from the proximal one-half of the chamber revealed that the regenerate in untreated strip chambers consisted of fibrin matrix and erythrocytes, whereas a well-developed structure with all the cellular elements of a regenerating nerve was seen in several of the FGF strip chambers. We conclude that FGF stimulates peripheral nerve regeneration in this model.

Animals↗

Rapid growth of regenerating axons across the segments of sciatic nerve devoid of Schwann cells.

The characteristic response of Schwann cells (SC) accompanies peripheral nerve injury and regeneration. To elucidate their role, the question of whether or not regenerating axons can elongate across the segments of a peripheral nerve devoid of SC was investigated. Rat sciatic nerve was crushed so that the continuity of SC basal laminae was not interrupted. A segment about 15 mm long distal to the crush was either repeatedly frozen/thawed to eliminate SC or scalded by moist heat which, in addition, denatured the proteins in the SC basal laminae, too. Both sensory and motor axons grew rapidly across the frozen/thawed segment of the nerve. Their rate of elongation was reduced by only 30% in comparison to control crushed nerves. SC were not present along the path of growing axons adhering tightly to the bare SC basal laminae. The rate of elongation of regenerating sensory and motor axons in scalded nerve segments was eight times lower than in control crushed nerves. SC were present in that part of the scalded region that had been invaded by the regenerating axons but no further distally. These results suggest that acellular basal laminae of SC provide very good, although not optimal, conditions for elongation of regenerating sensory and motor axons. If biochemical integrity of the basal lamina is destroyed, the regenerating axons must be accompanied or preceded by viable SC. and axon elongation rate is significantly reduced.

Animals↗

Peripheral nerve regeneration is impeded by interleukin-1 receptor antagonist released from a polymeric guidance channel.

Interleukin-1 receptor antagonist (IL-1ra), a true antagonist of the interleukin-1 (IL-1) receptors, is released by activated macrophages and binds specifically to the IL-1 receptors without triggering IL-1 effects. Following peripheral nerve axotomy, activated macrophages release IL-1, which induces the expression of nerve growth factor (NGF) mRNA in Schwann cells. IL-1ra may therefore impede peripheral nerve regeneration by blocking the NGF-mediated effect of IL-1. Peripheral nerve regeneration occurring through polymeric guidance channels releasing IL-1ra was investigated in a 4-mm gap transected mouse sciatic nerve model. Cohorts of five animals were implanted with tubes releasing either bovine serum albumin (BSA), BSA with IL-1ra, or BSA with deactivated IL-1ra (dIL-1ra) for 4 weeks. In vitro release kinetics indicated that after an initial burst, IL-1ra release was linear for the next 3 1/2 weeks. Following implantation of a polymeric guidance channel, a regenerated cable bridged the nerve gap in all animals. The cables were similar in size and were composed of nerve microfascicles containing both unmyelinated and myelinated axons in association with their Schwann cells. Tissue regenerated in tubes releasing BSA-IL-1ra contained, however, significantly fewer myelinated and unmyelinated axons and blood vessels than did tubes releasing BSA alone or BSA-dIL-1ra. We conclude that a naturally occurring antagonist of IL-1 receptors impedes peripheral nerve regeneration, suggesting that macrophages play an essential role in controlling peripheral nerve regeneration through the release of stimulatory and/or inhibitory molecules.

Animals↗

Axonal regeneration of an identified Helisoma neuron depends on the site of axotomy.

Axotomy of molluscan neurons usually results in axonal regeneration. In this study, we tested the axotomy response of an identified neuron of the pond snail Helisoma trivolvis (buccal neuron 4, B4). This neuron has two primary axonal branches, the ipsilateral axon and the contralateral axon, each innervating one of the paired salivary glands. The ipsilateral axon projects via the ipsilateral esophageal nerve trunk whereas the contralateral axon crosses both buccal ganglia and projects via the contralateral esophageal nerve trunk. We tested various procedures of axotomy: injury to one axon or both axons, close to the ganglion or more distal. Surprisingly, we found that proximal axotomy of the ipsilateral axon was not usually followed by axonal regeneration. By contrast, all other procedures of axotomy (e.g., distal ipsilateral, or proximal bilateral) resulted in robust axonal regeneration and target reinnervation. Thus, in this preparation, axotomy may or may not result in axonal regeneration, depending on the site(s) of axotomy. To the best of our knowledge, such a differential result has not yet been found in any other preparation. We conclude that axotomy is not always a sufficient condition for axonal regeneration of molluscan neurons. We hypothesize that a damaged axonal stump may be a necessary condition for the initiation of regeneration. An alternative hypothesis is that neurite outgrowth is inhibited in normal mature neurons by a target-derived factor. This hypothetical factor would be transported retrogradely, but not anterogradely, along axons.

Animals↗

Significance of the relationship between irregular regeneration and two hepatocarcinogenic pathways: "de novo" and so-called "dysplastic nodule-hepatocellular carcinoma" sequence.

BACKGROUND AND OBJECTIVES: Irregular regeneration of hepatocytes has been reported as an important factor in the hepatocarcinogenesis, so we studied the relationship between irregular regeneration and two hepatocarcinogenic pathways, "de novo" and "dysplastic nodule-hepatocellular carcinoma sequence." METHODS: Liver tissue was obtained from surgically resected 112 specimens, early well differentiated hepatocellular carcinomas and non-cancerous tissue. Hepatocellular carcinomas were divided into two groups; carcinoma with dysplastic area (type A) and without dysplastic area (type B) and were compared with irregular regeneration of hepatocytes in the non-cancerous tissue. RESULTS: Eighty-eight of 112 cases were judged to have irregular regeneration, such as anisocytosis, pleomorphism, bulging and map-like distribution. The degree of irregular regeneration was not correlated to the type of early well differentiated hepatocellular carcinomas. However, the existence of pleomorphism, map-like distribution and bulging are significantly correlated with type A. CONCLUSION: Type A might be more frequently occurring in a carcinogenic liver showing some kinds of irregular regeneration.

Carcinoma, Hepatocellular↗

Long-term evaluation of nerve regeneration in a biodegradable nerve guide.

Nerve regeneration using artificial biodegradable conduits is of increasing interest. The aim of this study is to evaluate the regeneration and maturation of a nerve after long-term implantation (2 years) of a biodegradable poly-L-lactide/poly-epsilon-caprolactone (PLLA/PCL) copolymeric nerve guide in the sciatic nerve of the rat. After harvesting, we evaluated both the regenerated nerves and the controls, using light microscopy, transmission electron microscopy, and morphometric techniques. Remnants of biomaterial were still present after 2 years of implantation, but the foreign body reaction was very mild at this stage, due to the rounded shapes of the polymer debris. Morphometric analysis showed significant differences between the regenerated nerve and the normal sciatic nerve: the number of myelinated fibers is higher, and the mean fiber diameter of the myelinated fibers in the regenerated nerve is smaller. In conclusion, the results demonstrate that the new PLLA/PCL nerve guide can provide optimal conditions for regeneration and maturation of damaged nerves.

Animals↗

Phrenic nerve transfer in the treatment of brachial plexus avulsion: an experimental study of nerve regeneration and muscle morphology in rats.

The regeneration of motor and sensory neurons and the morphological changes of the target muscle after phrenic nerve transfer were investigated in adult rats. Six months following nerve transfer, 326.0 +/- 16.31 phrenic motoneurons regenerated into musculocutaneous nerve, which is not different from the normal number of phrenic motoneurons. The regenerated motoneurons exhibited a 14% nonsignificant hypertrophy. Of the dorsal root ganglia (DRG) neurons, 255.8 +/- 45.26 regenerated, which was significantly lower than the number of normal phrenic DRG neurons. The regenerated phrenic DRG neurons showed a 24% close-to-significant atrophy. The target muscle fiber morphology changed considerably after reinnervation. The present results suggest that the phrenic nerve has very good regenerative ability in terms of its motoneurons and a relatively insufficient sensory neuronal regeneration.

Animals↗

Radiation inhibition of mdx mouse muscle regeneration: dose and age factors.

A single hind limb was irradiated with 12, 18, 24, or 30 Gy in mdx and C57 mice aged 12, 21, or 42 days to determine regeneration inhibition dose-response curves in different aged dystrophic mice and to characterize radiation side-effects in normal mice. The anterior tibial muscle mass (8 weeks postirradiation) and percent central nucleated (i.e., regenerated) muscle fibers were measures of regeneration inhibition. Twenty-one-day-old mdx mice irradiated with 18 Gy had complete inhibition of muscle regeneration, but 30 Gy only partially blocked regeneration in mdx mice irradiated at 12 and 42 days in age. In working to produce a clinically relevant model for Duchenne dystrophy, it is crucial to regard mouse age as a major factor in determining radiation effects on mdx muscle regeneration.

Aging↗

Axonal transport of RNA during regeneration of the optic nerves of goldfish.

The distribution of radioactive RNA and RNA precursors in the goldfish optic tecta following intraocular injection of 3H-uridine has been studied during various stages of optic nerve regeneration. 3H-uridine was injected into the posterior chamber of the right eye 17, 30, or 60 days after both optic nerves were crushed. Five were sacrificed at time intervals ranging from 0.5 to 21 days after injection. One day prior to sacrificing, 14C-proline was also injected into the right eye as a marked of fast axonal protein transport. Seventeen to 23 days after crushing, the approximate time of nerve reconnection, the amount of radioactive RNA appearing in the left optic tectum was increased by more than ten times control values. Approximately 30 days after crushing the nerve, when the reconnected nerve is maturing, RNA values were still elevated, but significantly decreased from the earlier stage. By 60 days after crushing the optic nerve, the amounts of RNA in the left tectum was close to normal. Evidence suggesting that, at least, some of the radioactive RNA in the tectum originated from RNA transported along optic axons rather than from RNA synthesized locally in the tectum was provided by autoradiographic experiments. Autoradiograms of paraffin sections taken from the goldfish optic tecta after the intraocular injection of 3H-uridine showed a distribution of grains in a linear pattern, suggesting a distribution over the incoming fibers during the reconnection stage of regeneration. Electron microsocpic autoradiography of glutaraldehyde fixed epoxy sections confirmed that a significant number of grains (shown to be 3H-RNA) were, in fact, over regenerating optic axons. Intracranial injection of 3H-uridine, during the same stage of regeneration, on the other hand, resulted in a distribution of grains, specifically over cell perikaprya. These experiments suggest that during the reconnection phase of nerve regeneration, large amounts of RNA may be carried within regenerating optic axons as they enter the optic tectum.

Animals↗

Regeneration restores some of the altered electrical properties of axotomized bullfrog B-cells.

In bullfrog B-type sympathetic neurones axon injury produces substantial changes in somal membrane properties. These include a shortening of action potential afterhyperpolarization (AHP) and an increase in action potential (AP) duration. In the present experiments we compared two injury situations: nerve crush, which was followed by regeneration, and nerve cut, after which regeneration to the original target was prevented, to investigate whether these electrophysiological changes were related to axon regeneration. Both crush and cut injuries produced a similar maximum decrease in AHP duration (to 33 and 30%) by 14 days after axotomy. After nerve crush, AHP duration recovered to within control values by 42 days, while after cut it remained depressed. AHP amplitude decreased to the same extent after nerve crush or cut (to 62 and 58%), but the rate of decrease was slower following crush when compared with cut, and following both types of injury it still remained depressed at 42 and 49 days. Changes in AP duration also took longer to occur following nerve crush, reaching maximal values at 35-42 days, at which time AHP duration had returned to within the normal range. The early reduction in AHP duration and its rapid recovery in regenerating neurones suggests that the current underlying this membrane property is regulated by events associated with axon outgrowth and peripheral reconnection. In contrast, changes in AHP amplitude and AP repolarization appeared to be independent of the occurrence of axon regeneration and remained abnormal at 49 days despite the recovery of AHP duration. These results imply that the electrophysiological changes seen in B-cells following injury are differentially regulated during subsequent regeneration.

Animals↗

Regeneration of an identifiable motoneuron in the crayfish. II. Patterns of reconnection and synaptic strength established in the presence of an extra nerve.

The regeneration of neuromuscular connections to the superficial flexor muscle system in the crayfish has been studied under a variety of experimental manipulations. These have provided insight into the factors that can influence the regeneration program of neurons. In this work the regeneration of the largest excitor motoneuron was studied under two different conditions: (1) when the original neuron and a transplanted neuron were growing simultaneously into a denervated target, and (2) when a transplanted neuron was growing into a target that had its original nerve supply intact. In condition 1 both the transplanted and the original neuron formed normal patterns of connectivity and synaptic strength in comparable periods of time. In condition 2 the rate of growth of the transplanted neuron is significantly reduced and does not extend into the lateral fibers of the muscle. It is concluded that the regeneration program of this neuron is not affected by the presence of other neurons growing at the same time into a denervated muscle. Since regeneration is seriously affected if growth occurs into a fully innervated target area, it is suggested that lack of growth stimuli from the target or competitive interactions between established and growing synaptic terminals could influence the regeneration program of this neuron.

Animals↗

Synaptic plasticity in a regenerated crayfish phasic motoneuron.

Crustacean neuromuscular systems provide many advantages for the study of synaptic transmission and plasticity. The present study examines aspects of synaptic transmission in the phasic, fast closer excitor (FCE) motoneuron of regenerated crayfish claws. Excitatory postsynaptic potentials (EPSPs) fatigued rapidly and showed poor long-term facilitation (LTF) in the smallest of regenerating claws. EPSPs in larger regenerating claws fatigued less and showed pronounced facilitation. These observations were not the same as those previously made during primary development of this motoneuron (Lnenicka and Atwood, 1985a, J. Neuroscience 5:459-467). Hence, regeneration is not the recapitulation of primary development. In situ stimulation of the FCE is known to lead to long-lasting adaptation of synaptic performance. This adaptation is age dependent; it is expressed in young but not old animals. In the regenerated FCE of old animals, we observed a novel form of long-lasting adaptation to imposed activity: EPSPs showed large initial EPSPs and did not exhibit resistance to fatigue during maintained stimulation. This indicates that aged motoneurons can express adaptive changes to increased activity following axonal regeneration, but that the adaptive changes are the opposite to what is observed in nonregenerated motoneurons.

Animals↗

Regenerating retinal fibers display error-free homing along undamaged normal fibers.

After crushing one optic nerve in a bony fish, retinal fibers regenerate to both tecta. Anterograde labelling indicates that the ipsilaterally regenerating fibers have a rather straight growth, apparently along the undamaged fibers of the contralateral retina. In contrast, the contralaterally regenerating fibers deviate widely from a straight course. Retrograde labelling shows a mirror-symmetric distribution of regenerated ipsilateral and resident contralateral ganglion cells in a comparable annulus. In contrast, ganglion cells in the regenerated contralateral retina show no topological order after comparable small Dil applications to the ventrolateral tectum. These data suggest that regenerating fibers can orient on the undisturbed, contralateral fibers.

Animals↗

Lectin binding and desmin staining during bupivicaine-induced necrosis and regeneration in rat skeletal muscle.

The processes of bupivicaine-induced necrosis and regeneration in rat skeletal muscle have been studied with a panel of biotinylated lectins and by immunohistochemical staining for desmin. The results indicate that the binding of lectins to the periphery of muscle fibres is not altered in necrotic and regenerating fibres at the light microscopic level although abnormal cytoplasmic staining by lectins occurs early in necrosis and is also present during regeneration. Desmin staining is lost at an early stage of necrosis and is present at an early stage of regeneration. Desmin staining in combination with morphometrical assessment of the rate of fusion and growth of regenerating fibres provides a powerful method for evaluating the processes of muscle regeneration in experimental situations.

Alkaline Phosphatase↗

Human airway surface epithelial regeneration is delayed and abnormal in cystic fibrosis.

Cystic fibrosis (CF) at an advanced stage of the disease is characterized by airway epithelial injury and remodelling. Whether CF remodelling is related to infection and inflammation or due to an abnormal regenerative process is still undecided. We have recently established the expression and secretion profiles of interleukin (IL)-8, matrix metalloproteinase (MMP)-7, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-1 during non-CF airway epithelial regeneration in a humanized nude mouse xenograft model. To enhance our understanding of CF remodelling, we compared the regeneration process of non-infected human CF and non-CF nasal epithelia. In both CF and non-CF situations, epithelial regeneration was characterized by successive steps of cell adhesion and migration, proliferation, pseudostratification, and terminal differentiation. However, histological examination of the grafts showed a delay in differentiation of the CF airway epithelium. Cell proliferation was higher in the regenerating CF epithelium, and the differentiated CF epithelium exhibited a pronounced height increase and basal cell hyperplasia in comparison with non-CF epithelium. In addition, while the number of goblet cells expressing MUC5AC was similar in CF and non-CF regenerated epithelia, the number of MUC5B-immunopositive goblet cells was lower in CF grafts. The expression of human IL-8, MMP-7, MMP-9, and TIMP-1 was enhanced in CF epithelium, especially early in the regenerative process. Together, our data strongly suggest that the regeneration of human CF airway surface epithelium is characterized by remodelling, delayed differentiation, and altered pro-inflammatory and MMP responses.

Adolescent↗