Role of thyroid hormones and their receptors in peripheral nerve regeneration.

After peripheral nerve injury in adult mammals, reestablishment of functional connections depends on several parameters including neurotrophic factors, the extracellular matrix, and hormones. However, little is known about the contribution of hormones to peripheral nerve regeneration. Thyroid hormones, which are required for the development and maturation of the central nervous system, are also important for the development of peripheral nerves. The action of triiodothyronine (T3) on responsive cells is mediated through nuclear thyroid hormone receptors (TRs) which modulate the expression of specific genes in target cells. Thus, to study the effect of T3, it is first necessary to know whether the target tissues possess TRs. The fact that sciatic nerve cells possess functional TRs suggests that these cells can respond to T3 and, as a consequence, that thyroid hormone may be involved in peripheral nerve regeneration. The silicone nerve guide model provides an excellent system to study the action of local administration of T3. Evidence from such studies demonstrate that animals treated locally with T3 at the level of transection have more complete regeneration of sciatic nerve and better functional recovery. Among the possible regulatory mechanisms by which T3 enhances peripheral nerve regeneration is rapid action on both axotomized neurons and Schwann cells which, in turn, produce a lasting and stimulatory effect on peripheral nerve regeneration. It is probable that T3 up- or down-regulates gene expression of one or more growth factors, extracellular matrix, or cell adhesion molecules, all of which stimulate peripheral nerve regeneration. This could explain the greater effect of T3 on nerve regeneration compared with the effect of any one growth factor or adhesion molecule.     

Differential Roles for EphA and EphB Signaling in Segregation and Patterning of Central Vestibulocochlear Nerve Projections

Auditory and vestibular afferents enter the brainstem through the VIIIth cranial nerve and find targets in distinct brain regions. We previously reported that the axon guidance molecules EphA4 and EphB2 have largely complementary expression patterns in the developing avian VIIIth nerve. Here, we tested whether inhibition of Eph signaling alters central targeting of VIIIth nerve axons. We first identified the central compartments through which auditory and vestibular axons travel. We then manipulated Eph-ephrin signaling using pharmacological inhibition of Eph receptors and in ovo electroporation to misexpress EphA4 and EphB2. Anterograde labeling of auditory afferents showed that inhibition of Eph signaling did not misroute axons to non-auditory target regions. Similarly, we did not find vestibular axons within auditory projection regions. However, we found that pharmacologic inhibition of Eph receptors reduced the volume of the vestibular projection compartment. Inhibition of EphB signaling alone did not affect auditory or vestibular central projection volumes, but it significantly increased the area of the auditory sensory epithelium. Misexpression of EphA4 and EphB2 in VIIIth nerve axons resulted in a significant shift of dorsoventral spacing between the axon tracts, suggesting a cell-autonomous role for the partitioning of projection areas along this axis. Cochlear ganglion volumes did not differ among treatment groups, indicating the changes seen were not due to a gain or loss of cochlear ganglion cells. These results suggest that Eph-ephrin signaling does not specify auditory versus vestibular targets but rather contributes to formation of boundaries for patterning of inner ear projections in the hindbrain.     

Morphological and physiological regeneration in the auditory system of adult <Emphasis Type="Italic">Mecopoda elongata</Emphasis> (Orthoptera: Tettigoniidae)

Orthopterans are suitable model organisms for investigations of regeneration mechanisms in the auditory system. Regeneration has been described in the auditory systems of locusts (Caelifera) and of crickets (Ensifera). In this study, we comparatively investigate the neural regeneration in the auditory system in the bush cricket Mecopoda elongata. A crushing of the tympanal nerve in the foreleg of M. elongata results in a loss of auditory information transfer. Physiological recordings of the tympanal nerve suggest outgrowing fibers 5 days after crushing. An anatomical regeneration of the fibers within the central nervous system starts 10 days after crushing. The neuronal projection reaches the target area at day 20. Threshold values to low frequency airborne sound remain high after crushing, indicating a lower regeneration capability of this group of fibers. However, within the central target area the low frequency areas are also innervated. Recordings of auditory interneurons show that the regenerating fibers form new functional connections starting at day 20 after crushing.     

Down‐regulation of long non‐coding RNA MEG3 promotes Schwann cell proliferation and migration and repairs sciatic nerve injury in rats

Peripheral nerve injury and regeneration are complex processes and involve multiple molecular and signalling components. However, the involvement of long non‐coding RNA (lncRNA) in this process is not fully clarified. In this study, we evaluated the expression of the lncRNA maternally expressed gene 3 (MEG3) in rats after sciatic nerve transection and explored its potential mechanisms. The expression of lncRNA MEG3 was up‐regulated following sciatic nerve injury and observed in Schwann cells (SCs). The down‐regulation of lncRNA MEG3 in SCs enhanced the proliferation and migration of SCs via the PTEN/PI3K/AKT pathway. The silencing of lncRNA MEG3 promoted the migration of SCs and axon outgrowth in rats after sciatic nerve transection and facilitated rat nerve regeneration and functional recovery. Our findings indicated that lncRNA MEG3 may be involved in nerve injury and injured nerve regeneration in rats with sciatic nerve defects by regulating the proliferation and migration of SCs. This gene may provide a potential therapeutic target for improving peripheral nerve injury.     

Modulation of a neural antigen during metamorphosis in Drosophila melanogaster

Transplantation of the acousticolateral placode to the evacuated eye position in embryos of the frog Rana pipiens has been used to force axons of the VIIIth cranial nerve to penetrate the diencephalon. These ectopic axons establish a growth trajectory that is strikingly similar to their normal growth trajectory within the medulla oblongata despite the fact that no other axons within the diencephalon normally follow this route. The result is discussed in terms of the "blueprint" and substrates pathway hypotheses which have been advanced to explain the initial development of axon tracts within the central nervous system.     

Regeneration of an identifiable motoneuron in the crayfish. I. Patterns of reconnection and synaptic strength established in normal and altered target areas

The superficial flexor muscles of the crayfish are innervated in a position-dependent connectivity pattern, which can be reestablished when the nerve to the muscle is cut. This article deals with the regeneration of the largest excitor motoneuron under three different target scenarios: (1) a normal target with all the muscle fibers present, (2) a reduced target lacking the medial or the lateral muscle fiber population, and (3) when the nerve enters the target in the middle of the muscle field. In scenario 1 the neuron is able to regenerate the normal connectivity pattern within 10 weeks after surgery: all the lateral fibers become innervated, with a linear decline in the probability of connections over the medial fibers. The medial fibers become transiently hyperinnervated before the normal pattern of connections is established. In scenario 2 the normal pattern of connections is established only when the lateral fibers were present; with only medial cells as a target, the transient hyperinnervation stage is stable and no decline in connections was observed. Analysis of regenerated junction potential sizes during the stable hyperinnervation stage show abnormal patterns, suggesting that some aspects of the regeneration program of this neuron can be affected when signals from its prime target cells are missing. In scenario 3 growth begins in both directions until the entire muscle becomes innervated. The normal pattern of connectivity finally emerges after continued lateral growth and diminished medial growth, suggesting that the position of the muscle fibers influences connectivity patterns during the final stages of regeneration.     

Macrophages can modify the nonpermissive nature of the adult mammalian central nervous system

Although astrocytic gliosis has been linked to failure of axonal regeneration in the adult mammalian CNS, its role is not fully established. We used an in vitro assay to investigate the role of reactive astrocytes and macrophages in influencing axonal growth in the lesioned adult rat optic nerve. Soon after optic nerve transection, the nonpermissive nature of the optic nerve is altered to a permissive state near the lesion. This may account for injury-induced axonal sprouting and may contribute to the failure of these sprouts to elongate beyond the site of the lesion in vivo. We provide evidence that this lesion-induced change in the axonal growth-promoting properties of the CNS near the lesion may be produced by mononuclear phagocytes. In addition, several months after optic nerve transection, the degenerated nerves, which consist mainly of astrocytes and lack myelin, i.e., astrocytic "scar" tissue, are a good substrate for neurite growth. Taken together, these results suggest that in this in vitro system, substantial inhibitory effects are not associated with regions of astrocytic gliosis and that the nonpermissive nature of the CNS white matter can be modified by macrophages.     

Chronic Functional Consequences of Adult Infraorbital Nerve Transection for Rat Trigeminal Subnucleus Interpolaris

In adult rats, transection of the infraorbital nerve and subsequent regeneration have been shown to result in altered somatotopic organization and changes in response properties of primary afferents within the trigeminal ganglion. The present study examined how these changes affect the postsynaptic targets of these neurons within subnucleus interpolaris of the trigeminal brainstem. Extra-cellular recordings were made from 330 cells in normal rats and 424 cells in rats surviving 57-290 days after transection of the infraorbital nerve in adulthood. Adult infraorbital nerve transection resulted in significant functional reorganization within subnucleus interpolaris. Relative to normal rats, the major changes can be summarized as follows: (1) a decrease in the dorsoventral extent of infraorbital representation; (2) a disruption of inter- and intradivisional somatotopic organization; (3) an increase in the proportion of cells with no discernible receptive field; (4) an increase in receptive field size for cells with infraorbital receptive field components; (5) the appearance of a significant proportion of cells with discontinuous receptive fields; (6) an increase in the proportion of cells exhibiting interdivisional convergence; (7) significant changes in the types of receptor surfaces activating local-circuit neurons with infraorbital receptive field components; (8) the appearance of a significant proportion of cells exhibiting convergence of different receptor surfaces; (9) significant changes in the dynamic response characteristics of cells with infraorbital receptive field components; and (10) an increase in the proportion of spontaneously active infraorbital-responsive cells. The changes observed were quite similar to those reported in adult subnucleus interpolaris following neonatal infraorbital nerve transection. The majority of changes observed in both studies can be most parsimoniously explained by alterations of primary afferents. However, central mechanisms may be more likely substrates for others. Regardless of the mechanism, the mature rodent trigeminal system appears capable of considerable functional reorganization following peripheral nerve damage.     

Chronic functional consequences of adult infraorbital nerve transection for rat trigeminal subnucleus interpolaris

In adult rats, transection of the infraorbital nerve and subsequent regeneration have been shown to result in altered somatotopic organization and changes in response properties of primary afferents within the trigeminal ganglion. The present study examined how these changes affect the postsynaptic targets of these neurons within subnucleus interpolaris of the trigeminal brainstem. Extracellular recordings were made from 330 cells in normal rats and 424 cells in rats surviving 57-290 days after transection of the infraorbital nerve in adulthood. Adult infraorbital nerve transection resulted in significant functional reorganization within subnucleus interpolaris. Relative to normal rats, the major changes can be summarized as follows: (1) a decrease in the dorsoventral extent of infraorbital representation; (2) a disruption of inter- and intradivisional somatotopic organization; (3) an increase in the proportion of cells with no discernible receptive field; (4) an increase in receptive field size for cells with infraorbital receptive field components; (5) the appearance of a significant proportion of cells with discontinuous receptive fields; (6) an increase in the proportion of cells exhibiting interdivisional convergence; (7) significant changes in the types of receptor surfaces activating local-circuit neurons with infraorbital receptive field components; (8) the appearance of a significant proportion of cells exhibiting convergence of different receptor surfaces; (9) significant changes in the dynamic response characteristics of cells with infraorbital receptive field components; and (10) an increase in the proportion of spontaneously active infraorbital-responsive cells. The changes observed were quite similar to those reported in adult subnucleus interpolaris following neonatal infraorbital nerve transection. The majority of changes observed in both studies can be most parsimoniously explained by alterations of primary afferents. However, central mechanisms may be more likely substrates for others. Regardless of the mechanism, the mature rodent trigeminal system appears capable of considerable functional reorganization following peripheral nerve damage.     

Effect of central axotomy of sensory neurones on the mechanoreception recovery in initial regeneration of the injured sciatic nerve in rats

No apparent effect of lumbar dorsal rhizotomy performed simultaneously with the peripheral nerve injury, has been revealed on the triggering of regeneration of sensitive nerve fibers. Re-innervation of the foot skin by either decentralized regeneration of nerve fibers or those sustaining their connections with the central nervous system (CNS), has been shown to start 30 days after surgery. Using the recording of impulse activity of a single nerve fibre, the mechanical thresholds of decentralized regenerating receptors were found to be significantly higher as compared to the thresholds of the regenerating receptors sustaining their connections with the CNS. The findings suggest that afferent nerve fibers and mechanical receptors formed on the periphery, continue functioning after decentralization and sustain their regenerative capacity after injury. However, in marked contrast, the decrease in sensitivity of regenerating receptors is more pronounced.     

Recovery of CNS Pathway Innervating the Sciatic Nerve Following Transplantation of Human Neural Stem Cells in Rat Spinal Cord Injury

Stem cell research has been attained a greater attention in most fields of medicine due to its potential for many incurable diseases through replacing or helping the regeneration of damaged cells or tissues. Here, we demonstrated the functional recovery and structural connection of the central nervous system pathway innervating the sciatic nerve after total transection of the spinal cord followed by the transplantation of human neural stem cells (hNSC) in the injured rat spinal cord site. The limb function of hNSC-treated group recovered dramatically compared with that in the sham group by Basso–Beattie–Bresnahan (BBB) scores. Transplanted hNSC differentiated into astrocytes and neurons in the injured site. In addition, immunohistochemistry for growth-associated protein 43 showed axonal regeneration in the injured spinal cord site. The pseudorabies viral-Ba (PRV-Ba) tracing method revealed that transplanted hNSC and their differentiated neurons showed positive labeling after sciatic nerve injection. In addition, the PRV-Ba labeling was also observed in several nuclei in the brain innervating the sciatic nerve. This result implies that the rat CNS motor pathway could be reconstructed by hNSC transplantation, and it may contribute to the functional recovery of the limb.     

Ultrastructural evidence of repair and neuronal survival after labyrinthectomy in the squirrel monkey

Although the vestibular and cochlear branches of the VIIIth cranial nerve originate embryologically from the same primordia, results of the present investigation confirm previous findings indicating that the vestibular branch may be more plastic with respect to recovery after surgical insult than the cochlear division. In this report we show ultrastructural details of changes undergone by the vestibular nerve after surgery. Dendrites peripheral to the vestibular nerve ganglion (VNG) were severed by surgically removing the vestibular end organs; the squirrel monkeys were then allowed to recuperate, and tested for their vestibulospinal and vestibulo-oculomotor functions behaviorally. However, behavior deficits resulting from the injury are reported separately. The vestibular nerves excised from the internal acoustic meatus and the temporal bones were examined histologically for changes of VNG and fibers from day 1 to 1,247 days after labyrinthectomy. Light- and electron-microscopic examinations indicated that some perikarya and some fibers of the VNG remained in the ganglionic matrix for up to 1,247 days, the longest period studied, after the operation. Fibers extended toward the remodeled inner ear space in the absence of appropriate sensory cell targets. The surviving neurons and fibers exhibited various degrees of wallerian-like degeneration at first, but many of them retained ultracellular organelles and integrity even after 1,247 days. Since vestibular perikarya are bipolar, the unsevered fibers that project to the brainstem could retain functional synaptic connections, a possibility that is now under investigation. Schwann cells in the ganglionic matrix may also have contributed to vestibular neuron survival by providing the proper nourishment. Morphometric measurements determined that neurons remaining in the ganglion had significantly smaller cross-sectional areas than normal neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a systemic regulation of neurotrophin synthesis in response to peripheral nerve injury

Up-regulation of neurotrophin synthesis is an important mechanism of peripheral nerve regeneration after injury. Neurotrophin expression is regulated by a complex series of events including cell interactions and multiple molecular stimuli. We have studied neurotrophin synthesis at 2?weeks time-point in a transvertebral model of unilateral or bilateral transection of sciatic nerve in rats. We have found that unilateral sciatic nerve transection results in the elevation of nerve growth factor (NGF) and NT-3, but not glial cell-line derived neurotrophic factor or brain-derived neural factor, in the uninjured nerve on the contralateral side, commonly considered as a control. Bilateral transection further increased NGF but not other neurotrophins in the nerve segment distal to the transection site, as compared to the unilateral injury. To further investigate the distinct role of NGF in regeneration and its potential for peripheral nerve repair, we transduced isogeneic Schwann cells with NGF-encoding lentivirus and transplanted the over-expressing cells into the distal segment of a transected nerve. Axonal regeneration was studied at 2?weeks time-point using pan-neuronal marker NF-200 and found to directly correlate with NGF levels in the regenerating nerve.     

Interaction of olfactory ensheathing cells with astrocytes may be the key to repair of tract injuries in the spinal cord: The ‘pathway hypothesis’

Transplantation of cultured adult olfactory ensheathing cells has been shown to induce anatomical and functional repair of lesions of the adult rat spinal cord and spinal roots. Histological analysis of olfactory ensheathing cells, both in their normal location in the olfactory nerves and also after transplantation into spinal cord lesions, shows that they provide channels for the growth of regenerating nerve fibres. These channels have an outer, basal lamina-lined surface apposed by fibroblasts, and an inner, naked surface in contact with the nerve fibres. A crucial property of olfactory ensheathing cells, in which they differ from Schwann cells, is their superior ability to interact with astrocytes. When confronted with olfactory ensheathing cells the superficial astrocytic processes, which form the glial scar after lesions, change their configuration so that their outer pial surfaces are reflected in continuity with the outer surfaces of the olfactory ensheathing cells. The effect is to open a door into the central nervous system. We propose that this formation of a bridging pathway may be the crucial event by which transplanted olfactory ensheathing cells allow the innate growth capacity of severed adult axons to be translated into regeneration across a lesion so that functionally valuable connections can be established.     

Regeneration of the eighth nerve fibers after transection in the red-eared turtle,Chrysemys scripta elegans

The present study examined the time sequence of degeneration and regeneration after transection of the eighth nerve in the red-eared turtle as well as the chromatolytic reaction of the turtle auditory ganglion cells. Horseradish peroxidase (HRP) transport between auditory ganglion cells and the medulla identified eighth nerve connections. The course of eighth nerve degeneration was followed with Fink and Heimer degeneration stain and HRP reaction. Cresyl-violet-stained sections through auditory ganglion cells were observed for chromatolysis. Degeneration by-product was intense in the eighth nerve and primary auditory nuclei in turtles surviving 25 and 32 days after eighth nerve transection. Turtles surviving 45 days or less after eighth nerve transection showed HRP reaction product in the eighth nerve to the point of its dorsolateral penetration into the medulla following cochlear duct injections. Acoustic tubercle injections in 50-day survivors showed HRP filling in eighth nerve and auditory ganglion cells. Cochlear duct injections in 67-day survivors demonstrated HRP filling in the eighth nerve and acoustic tubercle. Sections stained for degeneration in 67-day survivors showed little or no degeneration by-product and 80- and 90-day survivors showed none. The proportion of chromatolytic auditory ganglion cells was greatest in the 50-day postoperative turtles when compared to control turtles and other survival stages. Animals which survived longer than 50 days had reduced numbers of chromatolytic cells. Results suggest that the eighth nerve fibers are regenerated to primary brainstem auditory nuclei in experimental turtles surviving 50 days or more. Regeneration occurs between the 45th and 50th day following transection.     

Regeneration of Rapid Escape Reflex Pathways in Earthworms

SYNOPSIS. The medial and lateral giant nerve fibers in the earthworm,Eisenia foetida, regenerate cell-specific connections and recoverthrough-conduction capabilities in as little as 1–2 daysafter ventral nerve cord (VNC) transection Similar cell-specificreconnections between giant fibers occur approximately 4–10days after grafting together two posterior pieces of worms ortransplanting lengths of VNC from donor to recipient worms fromwhich a comparable length of VNC has been removed In the lattercase, touch-sensory and giant motor neurons within the transplantedVNC also regenerate, leading to restoration of escape reflexfunction in segments receiving the transplant Results from heterotopicallytransplanted VNC indicate that both central and peripheral regenerationis cell-specific, but specificity is sufficiently broad to includesegmentally homologous target cells from body regions otherthan those of the transplant origin E. foetida and related speciesmay be useful for studying the extent to which differentiatednervous systems, composed of serially homologous neuronal networks,can be remodelled by experimental manipulations such as graftsand transplants.     

Optic nerve regeneration after intravitreal peripheral nerve implants: trajectories of axons regrowing through the optic chiasm into the optic tracts

We have studied axon regeneration through the optic chiasm of adult rats 30 days after prechiasmatic intracranial optic nerve crush and serial intravitreal sciatic nerve grafting on day 0 and 14 post-lesion. The experiments comprised three groups of treated rats and three groups of controls. All treated animals received intravitreal grafts either into the left eye after both left sided (unilateral) and bilateral optic nerve transection, or into both eyes after bilateral optic nerve transection. Control eyes were all sham grafted on day 0 and 14 post-lesion, and the optic nerves either unlesioned, or crushed unilaterally or bilaterally. No regeneration through the chiasm was seen in any of the lesioned control optic nerves. In all experimental groups, large numbers of axons regenerated across the optic nerve lesions ipsilateral to the grafted eyes, traversed the short distal segment of the optic nerve and invaded the chiasm without deflection. Regeneration was correlated with the absence of the mesodermal components in the scar. In all cases, axon regrowth through the chiasm appeared to establish a major crossed and a minor uncrossed projection into both optic tracts, with some aberrant growth into the contralateral optic nerve. Axons preferentially regenerated within the degenerating trajectories from their own eye, through fragmented myelin and axonal debris, and reactive astrocytes, oligodendrocytes, microglia and macrophages. In bilaterally lesioned animals, no regeneration was detected in the optic nerve of the unimplanted eye. Although astrocytes became reactive and their processes proliferated, the architecture of their intrafascicular processes was little perturbed after optic nerve transection within either the distal optic nerve segment or the chiasm. The re-establishment of a comparatively normal pattern of passage through the chiasm by regenerating axons in the adult might therefore be organised by this relatively immutable scaffold of astrocyte processes. Binocular interactions between regenerating axons from both nerves (after bilateral optic nerve transection and intravitreal grafting), and between regenerating axons and the intact transchiasmatic projections from the unlesioned eye (after unilateral optic nerve lesions and after ipsilateral grafting) may not be important in establishing the divergent trajectories, since regenerating axons behave similarly in the presence and absence of an intact projection from the other eye.     

Visualizing Peripheral Nerve Regeneration by Whole Mount Staining

Peripheral nerve trauma triggers a well characterised sequence of events both proximal and distal to the site of injury. Axons distal to the injury degenerate, Schwann cells convert to a repair supportive phenotype and macrophages enter the nerve to clear myelin and axonal debris. Following these events, axons must regrow through the distal part of the nerve, re-innervate and finally are re-myelinated by Schwann cells. For nerve crush injuries (axonotmesis), in which the integrity of the nerve is maintained, repair may be relatively effective whereas for nerve transection (neurotmesis) repair will likely be very poor as few axons may be able to cross between the two parts of the severed nerve, across the newly generated nerve bridge, to enter the distal stump and regenerate. Analysing axon growth and the cell-cell interactions that occur following both nerve crush and cut injuries has largely been carried out by staining sections of nerve tissue, but this has the obvious disadvantage that it is not possible to follow the paths of regenerating axons in three dimensions within the nerve trunk or nerve bridge. To try and solve this problem, we describe the development and use of a novel whole mount staining protocol that allows the analysis of axonal regeneration, Schwann cell-axon interaction and re-vascularisation of the repairing nerve following nerve cut and crush injuries.